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AIDS-Kaposi's Sarcoma-Derived Cells Express Cytokines with Autocrine and Paracrine Growth Effects
Abstract
When grown in vitro, cells from Kaposi's sarcoma lesions of AIDS patients (AIDS-KS cells) constitutively release several growth promoting activities. When inoculated into nude mice, the AIDS-KS cells induce a KS-like lesion of mouse origin. Here it is shown that the AIDS-KS cells express messenger RNA for a complex mixture of cytokines that correlate with several of the biological activities of these cells. Basic fibroblast growth factor, which is a potent angiogenic factor, and interleukin-1 messenger RNAs are expressed at very high levels and seem to account for a large proportion of the activities, since their corresponding proteins are released in biologically active form into the culture media where they induce autocrine and paracrine growth effects.
... Among the chemokines, CCL5, CCL8, and CXCL10 were reported to be upregulated in Kaposi sarcoma versus normal skin cells (24). Furthermore, many cytokines and growth factors involving endothelial development, angiogenesis, and Kaposi sarcoma cell growth (such as ANGPT4, ANGPTL1, ANGPTL4, VEGF-A, -C, and -D), as well as their receptors (VEGFR1 and 3, EDNRA, and EDNRB) are among the DEGs of KSHV-infected PDLSCs, suggesting that KSHV-infected MSCs share the angiogenesis, autocrine, and paracrine growth regulations with Kaposi sarcoma cells (36)(37)(38). The cells from Kaposi sarcoma lesions of AIDS patients constitutively release several growth factors and cytokines including FGF family. ...
... To confirm the RNA-seq data on the cytokine network in KSHVinfected MSCs and determine whether it is a unique feature of infected MSCs, several representative cytokines were analyzed using ELISA for their production upon KSHV infection in MSCs and endothelial cells (lymphatic endothelial cells or LECs). It has been known that the FGF family is strongly expressed in KS, which is 100-fold higher than endothelial cells (37). Our data show that MSCs secreted a high level of bFGF, which was further enhanced by 2.5-fold upon KSHV infection (Fig. 5B). ...
... Finally, KSHV-infected oral MSCs closely resemble Kaposi sarcoma spindle cells in many pathogenic features of Kaposi sarcoma cells. Kaposi sarcoma is considered as a cytokine disease and abundant inflammatory infiltration is a pathogenic feature of Kaposi sarcoma (36,37). In the early stage, Kaposi sarcoma is not a real sarcoma but an angiohyperplastic-inflammatory lesion mediated by inflammatory cytokines and angiogenic factors (36). ...
The major transmission route for Kaposi sarcoma–associated herpesvirus (KSHV) infection is the oral cavity through saliva. Kaposi sarcoma (KS) frequently occurs in the oral cavity in HIV-positive individuals and is often the first presenting sign of AIDS. However, the oral target cells for KSHV infection and the cellular origin of Kaposi sarcoma remain unknown. Here we present clinical and experimental evidences that Kaposi sarcoma spindle cells may originate from virally modified oral mesenchymal stem cells (MSC). AIDS-KS spindle cells expressed neuroectodermal stem cell marker (Nestin) and oral MSC marker CD29, suggesting an oral/craniofacial MSC lineage of AIDS-associated Kaposi sarcoma. Furthermore, oral MSCs were highly susceptible to KSHV infection, and infection promoted multilineage differentiation and mesenchymal-to-endothelial transition (MEndT). KSHV infection of oral MSCs resulted in expression of a large number of cytokines, a characteristic of Kaposi sarcoma, and upregulation of Kaposi sarcoma signature and MEndT-associated genes. These results suggest that Kaposi sarcoma may originate from pluripotent MSC and KSHV infection transforms MSC to Kaposi sarcoma–like cells through MEndT.
Significance: These findings indicate that Kaposi sarcomas, which arise frequently in AIDS patients, originate from neural crest-derived mesenchymal stem cells, with possible implications for improving the clnical treatment of this malignancy. Cancer Res; 78(1); 230–45. ©2017 AACR.
... Of note, spindle cells isolated from ulcerated KS-like skin lesions of BKV/Tat-transgenic mice co-express antigens specific for endothelial, smooth muscle, and antigen-presenting cells, and express a complex mixture of angiogenic factors, including fibroblast growth factor-2 and vascular endothelial growth factor [36], as observed in human KS lesions and primary KS cells isolated from human lesions [1,24,37]. ...
... In addition, similarly to KS cells, endothelial cells activated by IC become angiogenic in nude mice [13,[38][39][40]. This is because IC induce production and release of angiogenic factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), which are highly expressed in all forms of KS and synergize in promoting angiogenesis, matrix-metalloproteases expression and activation, vascular permeability, and edema [13,[37][38][39][40][41][42][43] as observed in human KS lesions [1,15]. ...
Kaposi’s sarcoma (KS) is an angioproliferative tumor showing an increased frequency and aggressiveness in HIV-infected subjects (AIDS-KS), due to the combined effects of inflammatory cytokines (IC), angiogenic factors, and the HIV-1 Tat protein. While the introduction of effective combined antiretroviral regimens greatly improved AIDS-KS incidence and course, it continues to be an incurable disease and the development of new rational targeted therapies is warranted. We used the BKV/Tat transgenic mouse model to evaluate the effects of IC and anti-Tat antibodies (Abs) treatment on KS-like lesions arising in BKV/Tat mice. We demonstrated here that IC-treatment increases the severity and delays the regression of KS-like lesions. Further, anti-Tat Abs reduced KS-like lesion severity developing in IC-treated mice when anti-Tat Abs were administered at an early-stage of lesion development as compared to more advanced lesions. Early anti-Tat Abs treatment also accelerated KS-like lesion regression and reduced the rate of severe-grade lesions. This effect was more evident in the first weeks after Ab treatment, suggesting that a longer treatment with anti-Tat Abs might be even more effective, particularly if administered just after lesion development. Although preliminary, these results are encouraging, and the approach deserves further studies for the development of anti-Tat Ab-based therapies for AIDS-KS. Clinical studies specifically addressing the effect of anti-Tat antibodies in treating AIDS-KS are not yet available. Nevertheless, the effectiveness of anti-Tat antibodies in controlling HIV/AIDS progression, likely due to the neutralization of extracellular Tat activities, is suggested by several cross-sectional and longitudinal clinical studies, indicating that anti-Tat Ab treatment or Tat-based vaccines may be effective to treat AIDS-KS patients or prevent the tumor in individuals at risk.
... In the early stage (patch), KS is not a true sarcoma but an angio-hyperplastic inflammatory lesion with prominent numbers of inflammatory cells and abundant neovascularity (Levy and Ziegler, 1983;Ensoli et al., 2001;Ganem, 2010); In the plaque and nodular stages, spindle-shaped cells proliferate and become dominant but KS exhibits a remarkable heterogeneity in cell types including poorly differentiated spindleshaped KS cells and infiltrating inflammatory cells. The KS spindle cells have few properties in common with typical cancer cellsthey usually lack clonality and fail to display malignant phenotypes such as reduced dependence, malignant growth and forming a tumor in nude mice, unless being incubated in conditioned medium from activated T cells (Ensoli et al., 1989(Ensoli et al., , 2001Ensoli and Sturzl, 1998). The nature and cellular origin of KS spindle cells remain contentious. ...
... Our result that KSHV-infected MSCs can chemotactically migrate to injured and inflammatory sites in mice provided evidence for the hypothesis that KS arises from KSHV-infected MSCs that migrate to inflammatory sites where the inflammatory microenvironment in turn enhances the tumorigenic property of KSHV-infected MSCs. This theory can explain the clinical observation that KS developed in a surgical wound (Webster-Cyriaque, 2002), as well as the experimental finding that proliferation of KS spindle cell is highly dependent on cytokines in conditioned media of T-cell cultures and KS-transplanted cells, did not form a tumor in immunodeficient mice unless being treated with T cell cytokines (Ensoli et al., 1989(Ensoli et al., , 2001Ensoli and Sturzl, 1998). (iii) MSC-mediated tissue regeneration and wound healing hold great promise for restoration of tissue function and treatment of difficultto-heal wounds (Prockop, 1997;Hu et al., 2018). ...
Kaposi's sarcoma (KS) tends to occur in injured or inflamed sites of the body, which is described as the “Koebner phenomenon”. KS is also unique in its extraordinary angio-hyperplastic inflammatory phenotype. Recently, evidence has accrued indicating that KS may derive from KSHV-infected mesenchymal stem cells (MSCs), which possess enhanced migration and homing ability. Inspired by these findings, we hypothesized that KS may arise from KSHV-infected MSCs that chemotactically migrate to preexisting inflammatory or injured sites. Here we report that KSHV infection of human MSCs significantly up-regulated expression of several chemokine receptors and enhanced cell migration ability in vitro. Furthermore, using a wound mouse model, we demonstrated that KSHV infection dramatically promotes MSCs migrating and settling in the wound sites. In addition, two mice in the KSHV-infected group showed purpura and tumors with KS-like features. Taken together, KSHV-enhanced MSC migration ability and inflammatory microenvironment play crucial roles in KS development.
... Spindle cells and CD8-+-infiltrating macrophages or lymphocytes can produce interleukin 6 (IL-6), basic fibroblast growth factor (bFGF), gamma interferon, and a variety of other cytokines in abundance (Ensoli et al, 1989;Miles et al, 1990;Sirianni et al, 1998;Fiorelli et al, 1998). AIDS-KS cell lines have been shown to secrete large amounts of biologically active IL-6, which can promote spindle cell proliferation (Miles et al, 1990). ...
... bFGF is an angiogenic factor, highly expressed in early lesions that promotes spindle cell growth. Gamma-interferon can induce KS-like tumour formation in nude mice and these tumours in turn produce bFGF (Ensoli et al, 1989). ...
The molecular epidemiology of Kaposi's sarcoma-associated herpesvirus (KSHV) was investigated in family groups in Malawi, a region of high KSHV endemicity. It was hypothesised that: KSHV transmission may not only occur along intra-familial routes, as seroepidemiologic studies suggest, but also extra-familially; and that a host living in an endemic region may carry multiple KSHV strains. KSHV DNA in mouth rinse samples was frequently detected in study individuals (n=89 from 22 families) suggesting that saliva was a potential source of infection. Molecular sequences were compared between members of the same family by sequence analysis of hypervariable domains of opening reading frame (ORF) K1 PCR products generated from blood and oral samples. Phylogenetic analysis revealed that in some families (n=5), identical sequences in the variable region 1 were present. In others (n=4), dissimilar sequences were recovered (range of nucleotide sequence divergence: approximately 0.5% to 27%). While sequence similarity between family members is consistent with familial KSHV transmission, sequence dissimilarity and sequence clustering point to extra-familial transmission of closely related viral variants as having taken place. A PCR-based restriction fragment length polymorphism (RFLP) procedure to screen for sequence variation in the internal repeat domain of ORF 73 was then adapted to complement the ORF K1 nucleotide sequencing studies. RFLP patterns were unique for each individual and could be compared between family members. The PCR-RFLP findings broadly corroborated with the sequencing data. Intra-host KSHV sequence variation was investigated using denaturing gel gradient electrophoresis to screen multiple ORF K1 clones derived from oral samples. While intra-sample clonal diversity was observed, further investigation using KSHV infected cell lines revealed that such diversity could be attributed to Taq polymerase nucleotide misincorporation. Therefore, no firm evidence for KSHV viral diversity within single individuals could be found.
... The aetiology of KS, regardless of its setting, remains unclear. The establishment of KS-derived cell cultures has shown that several pro-inflammatory cytokines and growth factors can drive proliferation of the spindle cells (Nakamura et al., 1988;Ensoli et al., 1989). The current hypothesis to explain the formation and development of KS is that of a complex intricate network involving cytokine dysregulation, infection by a herpesvirus, designated human herpesvirus 8 (HHV-8), or KS-associated herpesvirus and modulation of growth by HIV-Tat protein Ensoli et al., 1994;Noel et al., 1996). ...
... Since bFGF is one of the major autocrine growth factors involved in the pathogenesis of all epidemiological forms of KS (Ensoli et al., 1989, we investigated the effect of DFO treatment on bFGF-stimulated KS cells. As expected, exposure of KS cells to bFGF (0.002 µg/ml) resulted in a marked increase in the cell population (285% of control, p Ͻ 0.001) (Fig. 7b). ...
The role of iron in the pathogenesis of several tumours is being increasingly investigated. In particular, its involvement in the pathogenesis of Kaposi's sarcoma (KS) is suggested by the distribution of the endemic form of KS corresponding to continental rifts and associated iron‐oxide‐rich volcanic clays. We investigated in vitro to what extent iron supplementation or withdrawal could affect the growth of KS‐derived cells, by analysing the effects of adding iron salts (iron chloride and ferric nitrilotriacetate) and/or reducing iron by iron chelators (desferrioxamine) on KS‐derived cell cultures. The addition of iron salts strongly stimulated the growth of KS cells, as reflected by increase in thymidine incorporation and cell number. Conversely, desferrioxamine and deferiprone inhibited cell growth. The inhibitory effect of iron chelation was more pronounced on rapidly dividing basic fibroblast‐growth‐factor‐stimulated cells. These results may point to a novel therapeutic approach to KS. Int. J. Cancer 78:720–726, 1998. © 1998 Wiley‐Liss, Inc.
... If the pathogenesis of AIDS-Kaposi's sarcoma is envisioned properly, the first stimulus derives from retro- virus-infected T4 cells (13)(14)(15). The as yet undefined molecular mediators thus generated induce multifocal growth of endothelial cells. ...
... Inducing factor(s) derived from HIV-infected T4 cells (14) bFGF-Iike molecular mediators derived from KS cells (20) IL-6 and IL-6R expression by KS cells for autocrine growth (24) Anti-IFNs and IFN-inactivators (including endogenous acid-labile IFNa) (8) TGF~ production by KS cells (15) antagonizing IL-2 and TNFa, ~; ...
Antiviral and immunomodulatory effects of interferons (IFNs) are well-known. This
communication lists arguments for explicit antiangiogenetic effects exerted by interferons. There is strong
likelihood that IFNs α, β and γ inhibit the int family of genes whose gene product proteins are the acidic
and basic fibroblast growth factors. Some of these growth factors promote the growth of vascular endothelial
cells. IFN-α inhibits basic, and IFN-γ inhibits acidic fibroblast growth factors. Inhibition of Kaposi sarcoma
cell growth is not due to immunological reconstitution of the host. As IFN-α induces clinical remissions
but IFN-γ does not, basic and not acidic fibroblast growth factor should be implied as one of the
proliferation-inducing factors of Kaposi sarcoma cells. lFNs may interfere with the growth promotional
activity of the human immunodeficiency virus Tat protein on Kaposi sarcoma cells. The proliferation of
certain melanoma cell populations requires fibroblast growth factors. If this mechanism is mediated
through amplified int genes, IFNs may be active clinically in this subset of melanomas. Some breast
carcinoma cells induce neovascularization and metastasize. If this activity is mediated through amplified
int genes. IFNs may be active clinically in this subset of breast carcinomas.
... Bu hücre kültüründeki hücre dizilerinin insan umbilikal kord endotelyal hücrelerini (HUVEC) ve KS hücre çoğalmasını indüklediği, bFGF inhibitörlerinin ise bu parakrin sinyal yolağı inhibe ettiği gösterilmiştir. 19 Benzer şekilde AIDS-KS hücre kültürlerinde VEGF, VEGF reseptör 1 (VEGFR1), VEGF reseptör 2 (VEGFR2), VEGF reseptör 3 (VEGFR3) mRNA ekspresyonun artmış olduğu bildirilmiştir. 20 Bu sinyal yolaklarının upregülasyonunda KSHV genlerinden vGPRC, viral interlökin 6, K1 ve LANA genleri sorumlu bulunmuştur. ...
... This plasticity allowed KSHV to reprogram hMSC to upregulate the expression and secretion of inflammatory cytokines like Endoglin (CD105), basic FGF, CXCL8, IL17, CCL2, Osteopontin, Pentraxin 3 (PTX3), Serpin E1/PAI-1 and Tfr (Figure 3). Importantly, it was shown that cytokines have an important role in KS pathogenesis [7,[22][23][24][25]. After KSHV infection in pro-angiogenic conditions, hMSC expresses high levels of PECAM1 (CD31), FLT1 (VEGFR1), ROBO4, XDH, NOX5, ESM1, and HGF implicated in Angiogenesis and endothelial lineage differentiation. ...
Kaposi’s sarcoma (KS) may derive from Kaposi’s Sarcoma Herpesvirus (KSHV)-infected human Mesenchymal Stem Cells (hMSCs) migrating into an inflammatory and angiogenic site, enhancing KS initiation. KSHV infection of primary hMSCs uncovers specific cell subpopulations, mechanisms, and conditions involved in the first steps of the KSHV-induced transformation and reprogramming process toward KS progenitor cells. The pro-angiogenic environmental conditions allowed KSHV to reprogram hMSCs closer to KS gene expression profiles and points to KSHV infection as an important factor inducing the Mesenchymal-to-Endothelial (MEndT) transition of hMSC. Single-cell RNA-sequencing analysis revealed a subpopulation of infected cells growing in a pro-angiogenic environment with both viral and host oncogenes upregulation. This highlights the importance of this condition in boosting the KSHV-induced transformation and reprogramming of hMSC towards MEndT and closer to KS gene expression profiles, reinforcing the notion of these cell subpopulations as KS precursors.
... Infection with KSHV induces high levels of pro-inflammatory cytokines, chemokines and angiogenic factors such as TNF, IL-1α, IL-1β, IL-6, CXCL8, IFN-γ, VEGF, COX-2 and GM-CSF [64][65][66][67][68][69][70]. Although several KSHV proteins promote the transcription of these mRNAs, many harbor AREs in the 3'UTR of their cognate mRNA, meaning their transcripts are subject to additional post-transcriptional regulation by PBs. ...
Kaposi’s sarcoma-associated herpesvirus (KSHV) causes the inflammatory and angiogenic endothelial cell neoplasm, Kaposi’s sarcoma (KS). We previously demonstrated that the KSHV Kaposin B (KapB) protein promotes inflammation via the disassembly of cytoplasmic ribonucleoprotein granules called processing bodies (PBs). PBs modify gene expression by silencing or degrading labile messenger RNAs (mRNAs), including many transcripts that encode inflammatory or angiogenic proteins associated with KS disease. Although our work implicated PB disassembly as one of the causes of inflammation during KSHV infection, the precise mechanism used by KapB to elicit PB disassembly was unclear. Here we reveal a new connection between the degradative process of autophagy and PB disassembly. We show that both latent KSHV infection and KapB expression enhanced autophagic flux via phosphorylation of the autophagy regulatory protein, Beclin. KapB was necessary for this effect, as infection with a recombinant virus that does not express the KapB protein did not induce Beclin phosphorylation or autophagic flux. Moreover, we showed that PB disassembly mediated by KSHV or KapB, depended on autophagy genes and the selective autophagy receptor NDP52/CALCOCO2 and that the PB scaffolding protein, Pat1b, co-immunoprecipitated with NDP52. These studies reveal a new role for autophagy and the selective autophagy receptor NDP52 in promoting PB turnover and the concomitant synthesis of inflammatory molecules during KSHV infection.
... The survival of KS cells has been shown to rely on the production and secretion of various cytokines, with both autocrine and paracrine function, to control cell proliferation (Salahuddin at a/., 1988). These include tumour necrosis factoralpha (TNF-a), and interleukin-1 (IL-1) and -6 (IL-6), which support spindle cell proliferation (Miles at a/., 1990, Corbeil at a/., 1991, and proteins that regulate neovascularisation or angiogenesis such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) (Ensoli at a!., 1989). ...
Human herpesvirus 8 (HHV-8) is the latest member of the Herpesviridae to be described, following its discovery in Kaposi's sarcoma tissue from AIDS patients, in 1994, by Chang et al. using representational difference analysis (RDA). Qualitative and quantitative-competitive PCR (QCPCR) methods were developed for the detection and quantification of HHV-8 DNA in patient samples. In a study looking at various post-mortem tissues of AIDS patients 32/153 (21%) tissues were found to be positive for HHV-8, whereas 0/47 control tissues were positive. HHV-8 viral loads were shown to vary from <10^1 to 10^5.6 genome copies/μg DNA. There was a significant difference in the viral loads between patients with KS and patients without KS, however there was no significant difference in HHV-8 load in tissue samples when correlated with HIV proviral DNA presence or load. In order to gain further insight into the molecular mechanisms that govern the antiviral susceptibility of HHV-8, functional studies of the HHV-8 thymidine kinase (TK) homologue were performed. The recombinant HHV-8 TK was shown to be functional for the phosphorylation of deoxythymidine. Sequence homology of the HHV-8 TK with the HSV TK identified three amino acid residues which may be of importance to the TK function, that were targeted for mutagenesis. These mutants were expressed and shown to have limited or no thymidine kinase activity. The affinity of the HHV-8 TK for known antiviral drugs was examined. Inhibition studies demonstrated that the anti-herpesvirus drugs GCV and ACV were unable to inhibit phosphorylation of dT by HHV-8 TK, whereas the anti-HIV drugs AZT and d4T, and the nucleoside analogue BrdU were competitive inhibitors of dT phosphorylation. In addition, AZT and d4T were shown to be phosphorylated by the HHV-8 TK, however GCV was not. In conclusion, established anti-herpetic agents are extremely poor substrates for the HHV-8 TK, although agents used for the treatment of HIV infection both inhibit the HHV-8 TK and are phosphorylated by its action.
... 9] Over 80% of patients with DKS have a co-infection that further promotes inflammation. Inflammatory cytokines play a major role in the pathogenesis of KS [10][11][12][13][14]. Many of these *Address correspondence to this author at the Av. ...
Background
Disseminated Kaposi sarcoma (DKS) is present in patients with advanced HIV infection in whom co-infection with other opportunistic pathogens can occur. Bone marrow (BM) aspirate and biopsy comprises a robust diagnostic tool in patients with fever, cytopenias, and abnormal liver tests. However, the yield in patients with DKS has not been determined.
Objective
The aim of this study was to evaluate the utility of BM aspirate and biopsy in patients with DKS.
Methods
We included 40 male patients with recent diagnosis of DKS. BM aspirate and biopsy was performed as part of the workup to rule out co-infections.
Results
In four patients, Mycobacterium avium complex (MAC) was recovered from culture. In another four patients, intracellular yeasts were observed in the Grocott stain, diagnosed as Histoplasma. The yield of BM was calculated in 20%. Only 12 patients (30%) had fever and 11 (27.5%) had pancytopenia. Alkaline phosphatase (ALP) above normal values and C-reactive protein (CRP) were higher in patients with positive results for BM than in those with negative results (63% vs. 21.9%, and 3.0 vs. 1.2 mg/L; p = 0.03 in both comparisons). No differences were found on comparing complete blood-count abnormalities.
Conclusion
We recommend performing a BM aspirate for stains, culture, and biopsy in all HIV patients with DKS, as this will permit the early diagnosis of co-infections and prevent further complications in those who receive chemotherapy.
... The main active component of these crude extracts of ECGF is basic fibroblast growth factor (bFGF), which is a very strong mitogen for endothelial cells in culture and an angiogenic growth factor [68,69]. Several lines of evidence suggest that KS is an angiogenic and inflammatory cytokine-mediated driven disease; at least in early stages, and that angiogenic factors and; in particular, bFGF, play a role in lesion development [70,71]. Basic FGF is highly expressed at the RNA and protein level by cultured AIDS-KS cells and in spindle cells from AIDS-KS and classic KS tissue [72,73]. ...
Kaposi’s sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). Unanswered questions regarding KS are its cellular ontology and the conditions conducive to viral oncogenesis. We identify PDGFRA(+)/SCA-1(+) bone marrow-derived mesenchymal stem cells (Pα(+)S MSCs) as KS spindle-cell progenitors and found that pro-angiogenic environmental conditions typical of KS are critical for KSHV sarcomagenesis. This is because growth in KS-like conditions generates a de-repressed KSHV epigenome allowing oncogenic KSHV gene expression in infected Pα(+)S MSCs. Furthermore, these growth conditions allow KSHV-infected Pα(+)S MSCs to overcome KSHV-driven oncogene-induced senescence and cell cycle arrest via a PDGFRA-signaling mechanism; thus identifying PDGFRA not only as a phenotypic determinant for KS-progenitors but also as a critical enabler for viral oncogenesis.
... The first key factor shown to induce angiogenesis in KS is basic fibroblast growth factor (bFGF) [167][168][169][170], which is highly expressed in KS tissues. Since then, increasing evidence supports that KSHV plays an essential role in altering the phenotype of infected cells. ...
Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as Human herpesvirus 8 (HHV-8), is a member of the lymphotropic gammaherpesvirus subfamily and a human oncogenic virus. Since its discovery in AIDS-associated KS tissues by Drs. Yuan Chang and Patrick Moore, much progress has been made in the past two decades. There are four types of KS including classic KS, endemic KS, immunosuppressive therapy-related KS, and AIDS-associated KS. In addition to KS, KSHV is also involved in the development of primary effusion lymphoma (PEL) and certain types of multicentric Castleman’s disease. KSHV manipulates numerous viral proteins to promote the progression of angiogenesis and tumorigenesis. In this chapter, we review the epidemiology and molecular biology of KSHV and the mechanisms underlying KSHV-induced diseases.
... In 1981 it became one of the signs that marked the beginning of the AIDS epidemic. This enigmatic disease is now recognized as a cytokine-mediated angioproliferative disease [1,2] caused by human herpesvirus 8 (HHV-8), also referred as Kaposi sarcoma-associated herpesvirus (KSHV) [3,4] indeed HHV-8/KSHV fulfilled Koch principles for an infectious disease pathogen. KS course is unpredictable even in the combined antiretroviral therapy (cART) era, and it is still associated with significant mortality [5,6]. ...
Objective
To investigate the predictive factors for the development of Kaposi sarcoma-related immune reconstitution inflammatory syndrome (KS-IRIS) and long-term prognosis in patients starting combined antiretroviral therapy (cART). Methods
We studied a retrospective-cohort of consecutive antiretroviral-naïve patients with KS initiating cART from January 2005 to December 2011 and followed through June 2013. KS-IRIS was defined as ≥2 of the following: abrupt increase in number of KS lesions, appearance or exacerbation of lung-opacities or lymphedema, concomitantly with an increase in CD4+ cell-count ≥50 cells/mm3 and a decrease of >1 log in viral-load once started cART. We compared individuals who met KS-IRIS criteria with those that did not and described the long-term follow-up. ResultsWe included 89 patients, 88 males; 35 (39%) developed KS-IRIS at a median of 10 weeks (IQR 4–16). KS-IRIS patients had more pulmonary-involvement (60% vs. 16.6% of patients; p < 0.0001), eight died attributed to pulmonary-KS. Thrombocytopenia <100,000/mm3 at follow-up occurred in 36% of KS-IRIS vs. 4% in non-KS-IRIS patients (p = 0.0002), 45% KS-IRIS patients with thrombocytopenia died, non without KS-IRIS. Chemotherapy (bleomicyn–vincristine) was more frequently prescribed in KS-IRIS patients (88.6% vs. 29.6%) with no differences in outcome; 80% of all patients achieve KS complete remission, 52% of them never received chemotherapy. No difference between groups in the long-term follow-up (mean 52.4 ± 27.4 months) was found, only one patient developed a secondary malignancy (1.12%). Conclusions
Lung-involvement was predictive of IRIS development. Thrombocytopenia in KS-IRIS patients at week 12 follow-up after cART initiation was associated with high mortality. Over a third of patients with KS achieve remission without chemotherapy. Individuals that survive the initial period of KS-IRIS adhere to cART had a good long-term prognosis.
... KS has been considered as a cytokine disease as KS lesions over-produce cytokines, especially angiogenic growth factors, that contribute to the major pathogenic features of KS [53,54]. In the early stage, KS is not a real sarcoma but an angiohyperplastic-inflammatory lesion mediated by inflammatory cytokines and angiogenic factors [55]. ...
APE1 is a multifunctional protein with a DNA base excision repair function in its C-terminal domain and a redox activity in its N-terminal domain. The redox function of APE1 converts certain transcription factors from inactive oxidized to active reduced forms. Given that among the APE1-regulated transcription factors many are critical for KSHV replication and pathogenesis, we investigated whether inhibition of APE1 redox function blocks KSHV replication and Kaposi’s sarcoma (KS) phenotypes. With an shRNA-mediated silencing approach and a known APE-1 redox inhibitor, we demonstrated that APE1 redox function is indeed required for KSHV replication as well as KSHV-induced angiogenesis, validating APE1 as a therapeutic target for KSHV-associated diseases. A ligand-based virtual screening yielded a small molecular compound, C10, which is proven to bind to APE1. C10 exhibits low cytotoxicity but efficiently inhibits KSHV lytic replication (EC50 of 0.16 μM and selective index of 165) and KSHV-mediated pathogenic phenotypes including cytokine production, angiogenesis and cell invasion, demonstrating its potential to become an effective drug for treatment of KS.
Acquired immunodeficiency syndrome (AIDS)-associated Kaposi sarcoma (KS) is an angioproliferative neoplasia caused by infection with human herpesvirus 8 (HHV-8). It typically presents with mucocutaneous involvement, but it can be disseminated. Initial presentation with primarily pulmonary KS is rare. We present a case of a 32-year-old male with untreated human immunodeficiency virus (HIV) diagnosed 1 year before presentation who developed progressively worsening cough and shortness of breath for 6 months. He was hospitalized twice and treated for unresolved pneumonia in an outside hospital. The patient concomitantly developed purplish nodules on his face, then the upper trunk, back, chest, and thighs bilaterally that gradually increased in size and number. Histopathology findings from skin lesions were consistent for KS. Bronchoscopy found multiple erythematous plaques throughout the tracheobronchial tree with telangiectasias and inflammation suggestive of pulmonary KS. His imaging findings and positive serum HHV-8 polymerase chain reaction (PCR) were consistent with disseminated KS. He started antiretroviral therapy (ART) to treat his HIV infection, followed by liposomal doxorubicin chemotherapy. But both ART and chemotherapy were interrupted due to adherence and insurance issues. The patient was readmitted with acute respiratory failure requiring mechanical ventilation with multiple vasopressors that led to the patient’s demise. The late recognition of KS diagnosis and delayed treatment can lead to worse outcomes.
Endothelial-to-mesenchymal transition has been described in tumors as a source of mesenchymal stroma, while the reverse process has been proposed in tumor vasculogenesis and angiogenesis. A human oncogenic virus, Kaposi’s sarcoma herpes virus (KSHV), can regulate both processes in order to transit through this transition ‘boulevard’ when infecting KS oncogenic progenitor cells. Endothelial or mesenchymal circulating progenitor cells can serve as KS oncogenic progenitors recruited by inflammatory cytokines because KSHV can reprogram one into the other through endothelial-to-mesenchymal and mesenchymal-to-endothelial transitions. Through these novel insights, the identity of the potential oncogenic progenitor of KS is revealed while gaining knowledge of the biology of the mesenchymal-endothelial differentiation axis and pointing to this axis as a therapeutic target in KS.
Kaposi sarcoma herpesvirus (KSHV)-associated diseases (Kaposi sarcoma, multicentric Castleman disease, primary effusion lymphoma, and KSHV inflammatory cytokine syndrome) are associated with immune suppression and dysregulation and loss of KSHV-specific immunity. These diseases are most frequent in people living with HIV as well as those with primary or iatrogenic immune deficiencies. KSHV itself can modulate the immune system via viral homologs of host cytokines or downregulation of immune-surface markers altering host immune surveillance. These factors make KSHV-associated diseases prime targets for immunotherapy approaches. Several agents have been studied or are under investigation in KSHV-associated diseases, including monoclonal antibodies, immunomodulatory agents, and therapeutic cytokines. Here, we review the role of immunotherapies in KSHV-associated diseases.
We have previously shown that the signal peptideless cytokine interleukin 1 alpha (IL-1 alpha) may play a role as an intracellular regulator of human endothelial cell senescence (J. A. M. Maier, P. Voulalas, D. Roeder, and T. Maciag, Science 249:1570-1574, 1990). To investigate the potential intracellular function of IL-1 alpha, transformed endothelial cells were transfected with the human cDNAs that code for the two forms of IL-1 alpha, the precursor molecule IL-1(1-271) and the mature protein IL-1(113-271). The subcellular localization of the two different polypeptides was investigated directly or by using chimeric genes constructed by fusion of different fragments of the IL-1 alpha gene and the beta-galactosidase open reading frames. The IL-1(113-271) protein was cytoplasmic, while IL-1(1-271) was nuclear. The basic cluster at the NH2 terminus of IL-1, KVLKKRR, has been shown to mediate IL-1 alpha nuclear targeting. Moreover, nuclear localization of IL-1 alpha correlates with impaired cell growth and expression of some IL-1 alpha-inducible genes. These results suggest that transport of endogenous IL-1(1-271) into the nucleus is required for it to modulate endothelial cell function.
One hundred percent of primary effusion lymphoma (PEL) cases are associated with Kaposi sarcoma-associated herpesvirus (KSHV). PEL cell lines, such as BCBL-1, are the workhorse for understanding this human oncovirus and the host pathways that KSHV dysregulates. Understanding their function is important for developing new therapies as well as identifying high-risk patient groups. The myeloid differentiation primary response 88 (MYD88)/interleukin-1 receptor associated kinase (IRAK) pathway, which has progrowth functions in other B cell lymphomas, has not been fully explored in PEL. By performing CRISPR/Cas9 knockout (KO) studies targeting the IRAK pathway in PEL, we were able to determine that established PEL cell lines can circumvent the loss of IRAK1, IRAK4, and MYD88; however, the deletion clones are deficient in interleukin-10 (IL-10) production. Since IL-10 suppresses T cell function, this suggests that the IRAK pathway may serve a function in vivo and during early-stage development of PEL.
Evidence indicates that, at least in the early stage, Kaposi's sarcoma (KS) is a cytokine-mediated disease and that it is consistently associated with a novel herpesvirus termed human herpesvirus-8 (HHV-8). To gain insights into the mechanisms by which cytokines and HHV-8 may cooperate in disease pathogenesis, we examined the phenotype, the Th1 (γ-interferon [γIFN]) and Th2 (interleukin-4 [IL-4]) cytokine profile and the presence of HHV-8 in peripheral blood mononuclear cells (PBMC), tumor-infiltrating lymphocytes (TIL), and spindle cell cultures derived from skin lesions of patients affected by classical KS (C-KS) and acquired immunodeficiency syndrome (AIDS)-associated KS (AIDS-KS). TIL and spindle cell cultures were examined at day 0 or after culture in conditioned media from activated T cells (TCM) that contain the same cytokines increased in KS tissues. No differences were found in the immunophenotype of PBMC from C-KS patients versus controls, except for AIDS-KS patients who showed a T-CD8+ expansion. However, a preferential infiltration of T-CD8+ cells was found in all KS lesions examined, which was maintained after culture of TIL in TCM. γIFN production was found in both PBMC and cultures derived from all KS examined; some IL-4 positive supernatants were found only in three AIDS-KS cases. Uninvolved skin did not show appreciable lymphocyte infiltration or cytokine production. The culture conditions of the lesional skin allowed also the appearance of adherent, spindle-like cells bearing markers of tissue macrophages. Finally, most or all of the PBMC, lesions, and macrophagic cell cultures from the skin lesions were found to be positive for HHV-8 infection by nested polymerase chain reaction (PCR). These findings indicate that patients with KS express a Th1 phenotype with a prevalent γIFN production, likely accounted for by the local T-CD8+ infiltration. By analogy with other viral infections (ie, Epstein-Barr virus), this suggests that in loco recruitment of lymphoid cells and the subsequent γIFN production may be in response to or elicited by HHV-8 that was found in both PBMC and macrophagic cell cultures from the lesions of the same patients.
Primary effusion lymphoma (PEL) is a distinct clinicopathologic entity associated with Kaposi's sarcoma-associated herpes virus (KSHV). Several cytokines, including interleukin-6 (IL-6), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) may be important for survival of KS cells. However, little is known about the interaction of cytokines with KSHV-infected lymphocytes from PEL. Therefore, we investigated what cytokines were produced by KSHV-infected PEL cell lines (KS-1, BC-1, BC-2), what cytokine receptors were expressed by these cells, what response these cells had to selected cytokines, and what was the effect of IL-6 antisense phosphorothioated oligonucleotides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and protein studies showed that these three cell lines produced IL-10, IL-6, and the receptors for IL-6. The granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-8, IL-12, bFGF, PDGF, and c-kit transcripts were not detected in the cell lines. High levels (0.7 to 5 ng/mL/106cells/48 hours) of IL-6 protein were consistently detected in supernatants of the cell lines by enzyme-linked immunosorbent assay (ELISA) tests. In clonogenic assays, interferon-α (IFN-α) and IFN-γ suppressed the clonal growth of the PEL cells, but GM-CSF, IL-4, IL-6, IL-8, IL-10, and oncostatin M did not change it. We examined for several autocrine loops that have been suggested to occur in KS. Experiments using antisense oligonucleotides showed that the clonal growth of KS-1 and BC-1 was nearly 100% inhibited by IL-6 antisense oligonucleotides (10 μmol/L), but not at all by either oligonucleotides (≤10 μmol/L) to IL-6 sense, IL-6 scrambled, viral IL-6 (vIL-6) antisense, or IL-10 antisense. Furthermore, the IL-6 antisense oligonucleotides had no effect on two B-cell lymphoma cell lines, which were not infected with KSHV. Addition of IL-6 antibody did not inhibit clonal growth of any of the cell lines. Taken together, we have defined the cytokines and their receptors expressed on PEL cells and have found that these cells synthesized IL-6 and IL-6 receptors; interruption of this pathway by IL-6 antisense oligonucleotides specifically prevented the growth of these cells. These findings will offer potential new therapeutic strategies for PEL.
Glucocorticoid (GC) use is known to induce or enhance the growth of Kaposi's sarcoma (KS) in many clinical settings including human immunodeficiency virus infection, collagen vascular disease, lymphoproliferative disorders, and renal transplantation. Because GCs may induce immune suppression and thus tumor growth, we determined whether GCs had a direct effect on KS growth. We found that GCs directly induce the growth of KS cell lines. In examining the mechanism of action of GCs, we did not observe induction of known autocrine growth factors for KS including interleukin-1 (IL-1), IL-6, oncostatin-M, basic fibroblast growth factor (bFGF ), and vascular endothelial growth factor (VEGF ). We thus examined factor(s) that inhibit KS growth. Transforming growth factor-β (TGF-β) is produced by KS cells and has pleiotropic effects, including inhibiting the growth of hematopoietic and endothelial cells. We show that TGF-β is produced by KS cells in both the latent and active forms, and that TGF-β is an autocrine growth inhibitory factor. We then studied the effects of GCs on the regulation of TGF-β and found that GCs do not inhibit TGF-β transcription, but significantly inhibit TGF-β activation. This effect is mediated through regulation of the TGF-β activation pathway. TGF-β is activated by plasmin which is positively regulated by plasminogen activator (PA) and PA receptor (PAR), and negatively regulated by plasminogen activator inhibitor (PAI). GCs downregulated PAR and upregulated PAI. Thus, glucocorticoids enhance KS cell growth through the regulation of TGF-β activation.
Kaposi’s sarcoma (KS) is the most common tumor in human immunodeficiency virus infection and acquired immune deficiency syndrome. Recent clinical trials with human chorionic gonadotropin (hCG) prepared from early pregnancy urine have shown encouraging results in the resolution of KS lesions. A urinary protein with antitumor activity, ANUP (antineoplastic urinary protein), a dimer of 32 kD, has previously been shown to inhibit the growth of various tumor cell lines in vivo. It was thus studied for its activity in KS cell lines in vitro and in vivo to determine whether it could be a source of the anti-KS activity observed in hCG preparations. ANUP is a strong growth inhibitor for KS cell lines, but has little or no effect on fibroblast, aortic smooth muscle, T- and B-lymphocyte, and monocyte cell lines. ANUP also inhibited the proliferation of endothelial cell lines, suggesting that the in vitro effects were endothelial cell lineage–specific. However, ANUP antibodies did not block the inhibitory effect of certain commercial preparations of hCG, previously shown to be active in KS. Thus, the active protein in these commercial preparations of hCG may be distinct from ANUP. The antitumor activity of ANUP was further confirmed in a chicken allantoic membrane (CAM) assay in which vascular endothelial growth factor (VEGF) and beta fibroblast growth factor (bFGF)-induced angiogenesis was inhibited by ANUP in a dose-dependent manner. In vivo activity of ANUP was demonstrated in the murine model of KS, where ANUP inhibited tumor growth. ANUP is thus a potential candidate for development in the treatment of KS and other diseases in which angiogenesis plays an important role.
We examined 26 patients with human immunodeficiency virus-1 (HIV-1)- associated Kaposi′s sarcoma (KS), and 76 HIV-1-infected (HIV-1+) people without KS or uninfected (HIV-1-) controls for the presence of circulating KS-like spindle cells. Adherent cells that had spindle morphology and several characteristics of spindle cells of KS lesions (KS cells) were identified in the peripheral blood mononuclear cell fraction only after culture in the presence of conditioned medium (CM) from activated lymphocytes. The peripheral blood-derived spindle cells (PBsc) expressed a variety of endothelial cell markers, such as Ulex europaeus I lectin, EN4, EN2/3, EN7/44, CD13, CD34, CD36, CD54, ELAM-1, and HLA-DR. However, they were negative for CD2, CD19, PaIE, and factor VIII-related antigen. The PBsc produced angiogenic factors as evidenced by the ability of CM from these cells to promote growth of normal vascular endothelial cells. In addition, subcutaneously injected PBsc stimulated angiogenesis in vivo in athymic nude mice. We determined that the number of PBsc grown from the peripheral blood of HIV-1+ patients with KS or at high risk to develop KS were increased by 78- fold (P = .0001) and 18-fold (P = .005), respectively, when compared with HIV-1- controls. The number of spindle cells cultured from the HIV- 1+ patients at low risk for developing KS, eg, HIV-1+ injection drug users, showed no statistical increase when compared with HIV-1- controls. The presence of increased PBsc with characteristics of KS cells in HIV-1+ KS patients or patients at high risk for developing KS gives insights into the origin of KS cells and may explain the multifocal nature of the disease. In addition, this may be useful in predicting the risk of KS development.
Clinically advanced and rapidly progressive AIDS-associated Kaposi sarcoma (AIDS-KS) tumors require an aggressive tumor-directed therapy. We have observed that AIDS-KS cells express high levels of receptors for immune regulatory cytokine, interleukin-13 (IL-13). Two tumorigenic AIDS-KS cell lines, KS Y-1 and KS-imm, expressed 4560 and 9480 IL-13 binding sites per cell with an affinity (kd) of ∼0.9 and 3.7 nmol/L, respectively. IL-13 cytotoxin IL13-PE38QQR, consisting of human IL-13 and a derivative of Pseudomonas exotoxin, is specifically cytotoxic to KS tumor cells. Systemic and loco regional administration of IL13-PE38QQR in immunodeficient mice with established human KS tumors produced remarkable antitumor activity. Three intratumoral (IT) injections of IL-13 toxin (250 μg/kg per dose) on alternate days (qod) or 5 daily (qd) IT injections with lower doses (50 or 100 μg/kg per dose) resulted in a complete regression of established subcutaneous tumors in most animals. Daily IT treatment with 250 μg/kg of IL-13 toxin in another KS-derived cell line also produced complete responses. Twice daily intraperitoneal injections of IL13-PE38QQR (25 or 50 μg/kg per dose) for 10 days (total injections = 20) also completely eradicated KS Y-1 tumors. Intravenous administration of IL13-PE38QQR also suppressed tumor growth; however, complete responses were not observed. All animals tolerated the therapeutic doses of IL-13 toxin without any visible signs of toxicity. The efficacy of receptor-directed IL13-PE38QQR therapy in mice warrants further exploration of this drug for AIDS-KS treatment.
Kaposi sarcoma (KS) is responsive to a number of different steroid hormones, such as glucocorticoids and retinoids. An active metabolite of vitamin D, 1α,25 dihydroxyvitamin D3, was used to study the effect of this steroid hormone in KS. Steroid hormones exert their effect through their cognate nuclear receptors, which for vitamin D metabolites is the vitamin D receptor (VDR). It was first shown that KS cell lines and primary tumor tissue express high levels of VDR, whereas endothelial cells had minimal expression and fibroblasts had no expression. Second, KS cell growth was inhibited by VDR agonist 1α,25 dihydroxyvitamin D3 with a 50% inhibitory concentration of 5 × 10 −8 mol/L, whereas endothelial cells and fibroblast cells showed no response. Studies on the mechanism of KS tumor growth inhibition by 1α,25 dihydroxyvitamin D3 showed that production of autocrine growth factors interleukin (IL)-6 and IL-8 was reduced in a dose-dependent manner, whereas no effect was observed on vascular endothelial growth factor and basic fibroblast growth factor. Transcription initiated at the IL-6 promoter was repressed by VDR agonist. The DNA sequences required to mediate this repression were localized to nucleotides −225/−110 in the 5′-flanking region. The antitumor activity of VDR agonists was also confirmed in KS tumor xenograft and after topical application in patients with KS. 1α,25 Dihydroxyvitamin D3 and its analogs may thus be candidates for clinical development in KS.
Kaposi sarcoma (KS) is an angioproliferative inflammatory condition that occurs commonly in patients infected with human immunodeficiency virus (HIV). Inflammatory cytokines and growth factors promote the development of KS. Because physiologically important cytokine polymorphisms modulate host inflammatory responses, we investigated the association between KS and common regulatory polymorphisms in 5 proinflammatory cytokine genes encoding interleukin (IL) IL-1α, IL-1β, tumor necrosis factor (TNF) α, TNF-β, and IL-6 and in the IL-1 receptor antagonist (IL1RN). We also examined the contribution of stromal-derived factor 1 and chemokine receptor 5 (Δ32) polymorphisms to KS development. The population consisted of 115 HIV-infected men with KS and 126 deceased HIV-infected men without KS. The only strong association was observed between an IL6promoter polymorphism (G-174C) and susceptibility to KS in HIV-infected men (P = .0035). Homozygotes for IL6 allele G, associated with increased IL6 production, were overrepresented among patients with KS (P = .0046), whereas allele C homozygotes were underrepresented (P = .0062). Substantial in vitro evidence indicates that IL-6 contributes to the pathogenesis of KS. Our results show thatIL6 promoter genotypes associated with altered gene expression are risk factors for development of KS. Identification of a genetic risk factor for development of KS has important clinical implications for prevention and therapy.
Kaposi's sarcoma (KS) developed in a patient with human T-cell leukemia virus type I (HTLV-I)-associated adult T-cell leukemia who was treated with a short-term course of monoclonal antibody immunotherapy. The presentation was transient and temporally related to the underlying clinical course. The association of KS in an HTLV-I infected, but not human immunodeficiency virus (HIV)-infected, individual should alert investigators to the occurrence of KS in retroviral-associated diseases other than acquired immunodeficiency disease syndrome. Recognition of the similarities and differences between HTLV-I and HIV infections may provide insights concerning the angiopathogenesis of KS.
We examined 26 patients with human immunodeficiency virus-1 (HIV-1)- associated Kaposi′s sarcoma (KS), and 76 HIV-1-infected (HIV-1+) people without KS or uninfected (HIV-1-) controls for the presence of circulating KS-like spindle cells. Adherent cells that had spindle morphology and several characteristics of spindle cells of KS lesions (KS cells) were identified in the peripheral blood mononuclear cell fraction only after culture in the presence of conditioned medium (CM) from activated lymphocytes. The peripheral blood-derived spindle cells (PBsc) expressed a variety of endothelial cell markers, such as Ulex europaeus I lectin, EN4, EN2/3, EN7/44, CD13, CD34, CD36, CD54, ELAM-1, and HLA-DR. However, they were negative for CD2, CD19, PaIE, and factor VIII-related antigen. The PBsc produced angiogenic factors as evidenced by the ability of CM from these cells to promote growth of normal vascular endothelial cells. In addition, subcutaneously injected PBsc stimulated angiogenesis in vivo in athymic nude mice. We determined that the number of PBsc grown from the peripheral blood of HIV-1+ patients with KS or at high risk to develop KS were increased by 78- fold (P = .0001) and 18-fold (P = .005), respectively, when compared with HIV-1- controls. The number of spindle cells cultured from the HIV- 1+ patients at low risk for developing KS, eg, HIV-1+ injection drug users, showed no statistical increase when compared with HIV-1- controls. The presence of increased PBsc with characteristics of KS cells in HIV-1+ KS patients or patients at high risk for developing KS gives insights into the origin of KS cells and may explain the multifocal nature of the disease. In addition, this may be useful in predicting the risk of KS development.
Multicentric Castleman's disease (MCD) is an atypical lymphoproliferative disorder defined using clinical and pathologic criteria. A characteristic of the MCD is a close association with Kaposi's sarcoma (KS), which occurs during the clinical course of most human immunodeficiency virus (HIV)-associated MCD cases and also, but less frequently, in HIV-negative patients. Recently, sequences of a putative new Herpesvirus (KSHV) have been isolated and further detected in almost all the acquired immunodeficiency syndrome (AIDS) KS and in most of the non-AIDS KS samples. In this study, we searched for these Herpesvirus-like sequences in MCD samples of 31 patients. KSHV sequences were detected in 14 of 14 cases of HIV-associated MCD, including 5 cases without detectable KS. Moreover, KSHV was detected in 7 of 17 MCD cases in HIV-negative patients, including 1 case associated with a cutaneous KS. In 34 non-MCD reactive lymph nodes (follicular and/or interfollicular hyperplasia) in HIV-negative patients, KSHV was detected in only 1 case. In 1 HIV-negative case of MCD, KSHV was found in both the lymph node and peripheral blood samples. These data suggest that KSHV could play a role in the pathogenesis of MCD, especially in HIV-infected patients.
Kaposi’s sarcoma-associated herpesvirus (KSHV) is causally associated with several malignant tumors: Kaposi’s sarcoma (KS), multicentric Castleman’s disease (MCD), and primary effusion lymphoma (PEL). KS remains the most common AIDS-related malignancy since the AIDS epidemic and thus has been extensively studied. KS is characterized as an angioproliferative disease with massive immune cell infiltration at the early stage. High levels of proinflammatory cytokines and growth factors are found in KS lesions, and their involvement in the survival and growth of tumor cells has been well characterized. However, little is known about the role of the inflammatory microenvironment in the regulation of KSHV gene expression and/or viral replication. In the present study, we demonstrated that IFN-γ and TNF-α profoundly inhibited KSHV progeny production in primary human lymphatic endothelial cells (LECs) as well as induced KSHV-producer cells (iSLK.219) with doxycycline. Of note, IFN-γ inhibited overall KSHV gene expression, while the effects of TNF-α were confined to a selected set of genes, which were also downregulated by IFN-γ. The addition of IFN-γ up to 36 hr after induction of viral lytic replication was effective in terms of the inhibition of infectious virion production, suggesting that its inhibitory effect is exerted at the early stages of KSHV life cycle. We believe these data have potentially important implications for rationalizing a therapeutic agent to treat KSHV-induced tumors in which lytic replication plays a critical role in their pathogenesis: KS and MCD.
This book describes cellular level sensors that act as switches, turning on gene expression and other metabolic processes necessary for cell survival and differentiation. These responses can also initiate programmed cell death or activate latent human immunodeficiency virus or animal leukemia viruses. These redox sensors are nonspecific in sensitivity but specific in response. Unlike ligand/antiligand-type specific sensors, they respond to ionizing and ultraviolet radiations, pH gradients, heat, light, electric and magnetic fields, redox chemicals, mechanical stress, and other nonspecific stressors. The sensors are type-b cytochromes, including NADPH oxidases, NO synthases, and nitrogen oxide reductases. The intense radiation of early pre-biotic earth may have been the evolutionary driving force for the development of their common ancestor.
The recently identified human herpesvirus 8 (HHV-8, or Kaposi’s sarcoma-associated herpesvirus) has been implicated in the etiology of both Kaposi’s sarcoma (KS) and primary effusion (body cavity-based) lymphoma (PEL) (Y. Chang et al., Science 266:1865–1869, 1994; P. S. Moore et al., J. Virol. 70:549–558, 1996). An important feature of the association of HHV-8 with these malignancies is the expression of an abundant, latency-associated 0.7-kb transcript, T0.7 (W. Zhong et al., Proc. Natl. Acad. Sci. USA 93:6641–6646, 1996). T0.7 is found in all stages in nearly all KS tumors of different epidemiologic origin, including AIDS-associated, African endemic, and classical KS (K. A. Staskus et al., J. Virol. 71:715–719, 1997), as well as in a body cavity-based lymphoma-derived cell line, BCBL-1, that is latently infected with HHV-8 (R. Renne et al., Nat. Med. 2:342–346, 1996). T0.7 encodes a unique HHV-8 open reading frame, K12, also known as kaposin. In this study, we report that the kaposin gene induced tumorigenic transformation. Constructs with kaposin expressed either from its endogenous promoter or from a heterologous promoter induced focal transformation upon transfection into Rat-3 cells. All transformed Rat-3 cell lines containing kaposin sequences produced high-grade, highly vascular, undifferentiated sarcomas upon subcutaneous injection of athymic nu/nu mice. Tumor-derived cell lines expressed kaposin mRNA, suggesting a role in the maintenance of the transformed phenotype. Furthermore, kaposin protein was detected in transformed and tumor-derived cells by immunofluorescence and localized to the cytoplasm. More importantly, expression of kaposin protein was also detected in the PEL cell lines BCBL-1 and KS-1. These findings demonstrate the oncogenic potential of kaposin and suggest its possible role in the development of KS and other HHV-8-associated malignancies.
In this paper some global aspects of the intraseasonal oscillations on the time scale of 30 to 50 days are explored. Noting that the variability of zonal flow of the monsoon, the atmospheric angular momentum and the length of day are strongly correlated on this time scale, we have made an effort to examine the global variability using the length of day as a point of reference. The scenario of this cycle is presented starting from a super cloud cluster at the near equatorial latitudes. This seems to be accompanied with an acceleration of zonal flows, an increase of the atmospheric angular momentum and an increase in the length of day. The transfer of westerly angular momentum from the earth to the atmosphere occurs over regions of the surface easterlies to the east of the super cloud clusters resulting in an increase in the length of day. During this transition from a mean length of day to a maximum length of day, an active phase of the Indian summer monsoon is noted. The interesting aspect of the length of day transition occurs on its return cycle when the near equatorial cloud cover eases or moves away from the equator with a decrease in the monsoonal zonal flows and a reduction of this component of atmospheric angular momentum. The length of day does not simply go back to an equilibrium value, but the long term data from the laser ranger shows an overshooting beyond that to a minimum value. This transition is characterized in general by monsoon break-like conditions, counter monsoon flows in the low levels and by a transition from high index to low index conditions in the upper troposphere of the middle latitudes. Phenomenologically, some blocking situations have been noted over the higher middle latitudes during this transition. The reduction of the angular momentum is attributed to the transfer of the westerly angular momentum from the atmosphere to the earth via frictional and mountain torques. These torques exhibit a clear relationship to the changes in the atmospheric angular momentum on this time scale. The behavior of the middle latitude low frequency variability is also in part explained by the meridional wave energy flux. That problem is examined in this context with the full non-linear equations in the frequency domain. It is shown that unlike the linear problems where such fluxes are inhibited beyond the critical latitude, the nonlinear problem permits the temporal oscillations of zonal flows on this time scale. As a consequence, a significant tropical-middle latitude coupling is noted by this process.
A simple mathematical model of the oscillation is also presented. It is a local theory in which ocean and atmosphere interact. Initially, the atmosphere is stably stratified with weak winds at the sea surface and stronger winds aloft; the ocean has a surface mixed layer of temperature Ts lying over deep cold water. Solar heating gradually increases Ts which leads to atmospheric convection with associated transport of horizontal momentum and increased winds at the sea surface. Increased winds lead to deepening of the mixed layer and a drop in Ts because of mixing of deep cold water with surface waters. Convection ceases, winds decay, and the cycle repeats only after solar heating has once more increased Ts. The period of this oscillation is shown to be on the order of 30 days.
First described by Rous in 1911 as filterable agents capable of inducing sarcomas in chickens, retroviruses (initially called RNA tumor viruses) are among the earliest known viruses. In the 1950s and -60s these agents were found to cause a variety of mammalian cancers and leukemias and were shown to be RNA viruses. Human retroviruses were first described beginning in 1979, and their link etiologically to human leukemia (HTLV) and subsequently to Kaposi’s Sarcoma and Non-Hodgkins Lymphoma (HIV) represents some of the most important advances in biologic research in decades. In this chapter, we will summarize the available knowledge regarding the oncologic potential of the HTLV family (onco-retroviruses), and the HIV family (lente retroviruses).
1981 wurde das klinische Bild des sog. „erworbenen Immunmangelsyndroms“ („Aquired Immune Deficiency Syndrome“, AIDS) erstmals beschrieben [28]. Dieses Krankheitsbild ist charakterisiert durch einen komplexen erworbenen Immundefekt, der sich klinisch in der Entwicklung von opportunistischen Infektionen und Tumoren manifestiert [19]. Mittlerweile wurde das „Humane Immundefizienz Virus“ (HIV), ein Retrovirus aus der Gruppe der Lentiviren, als kausales Agens identifiziert [7, 67, 89] und gezeigt, daß das AIDS-Vollbild lediglich ein fortgeschrittenes Stadium eines breiten klinischen Spektrums der HIV-Infektion darstellt [9, 19]. 1988 waren über 3000 AIDS- Fälle in der Bundesrepublik gemeldet bei einer geschätzten Gesamtzahl von 50 000-100 000 HIV-infizierten Personen [81].
Kaposi’s sarcoma (KS) is a mesenchymal tumor of blood and lymph vessels associated with infection of human herpesvirus type 8 (HHV-8). Four distinct clinical variants are found including (1) classic, (2) African or endemic, (3) AIDS related or epidemic, and (4) iatrogenic. Commonly affected sites include mucocutaneous surfaces, the oropharynx, genitals, lower extremities, and, in advanced stages, viscera.
For the period 1971–2014, we identified 44 reported cases of penile KS: 6 in HIV-seropositive patients, 29 in HIV-seronegative patients, and 9 patients with unspecified HIV status. Excisional biopsy and histology including intralesional HHV-8 testing were performed in all cases to establish the diagnosis. HIV-seropositive patients had more severe presentations including multiple lesions, ulceration, lesions extending to the groin, and non-discolored nodules. Treatment for these patients included maximizing highly active antiretroviral therapy (HAART). Patients with other clinical variants (HIV negative or unknown) had local treatment performed in 55.3 % (21 out of 38) of cases. Average follow-up was 14.2 months with local recurrence in two cases (2 out of 38, 5.3 %) and distant recurrence in five cases (5 out of 38, 13.2 %). In addition, six case reports of genitourinary KS in HIV-seropositive patients were reported. These cases had more severe presentations including nonpigmented nodules, multiple ulcerated lesions, lesions extending to groin area, deformity of the penis, and urethral stricture. More invasive treatment was attempted in addition to maximizing HAART.
Penile KS is a virus-induced neoplasm affecting primarily men who are HIV positive, have other forms of immunosuppression, or are of Mediterranean or African origin. These subgroups may have different patterns of presentation and require different treatment strategies.
bFGF belongs to the family of the heparin-binding growth factors (Basilico and Moscatelli, 1992). The single copy human bFGF gene encodes multiple bFGF isoforms with molecular weights ranging from 24 kD to 18 kD. High molecular weight isoforms (HMW-bFGFs) are colinear NH2-terminal extensions of the better characterized 18 kD protein (Florkiewicz and Sommer, 1989). Both low and high molecular weight bFGFs exert angiogenic activity in vivo and induce cell proliferation, protease production, and Chemotaxis in cultured endothelial cells (Gualandris et al., 1994). Also, bFGF has been shown to stimulate endothelial cells to form capillary-like structures in collagen gels (Montesano et al., 1986) and to invade the amniotic membrane in vitro (Mignatti et al, 1989). The phenotype induced in vitro by bFGF in endothelial cells includes also modulation of integrin expression (Klein et al., 1993), gap-junctional intercellular communication (Pepper and Meda, 1992) and urokinase receptor upregulation (Mignatti et al., 1991). Experiments performed with neutralizing anti-bFGF antibodies have implicated endogenous bFGF in wound repair (Broadley et al., 1989), vascularization of the chorioallantoic membrane during chick embryo development (Ribatti et al., 1995), and tumor growth (Baird et al., 1986; Gross et al., 1993).
Kaposi’s sarcoma (KS: initially called idiopathisches multiples Pigmentsarkom der Haut, later called multiple idiopathic haemorrhagic sarcoma; Kaposi 1872) was first described in eastern Europe. A sudden increase in the number of KS has been observed along with the epidemic of AIDS. Since the occurrence of the first case of AIDS (CENTERS FOR DISEASE CONTROL 1981), KS has been considered to be one of its main complications. The aetiology, pathogenesis and disease entity of KS are currently topics of interest.
Kaposi's sarcoma accounts for more than 3 % of neoplasms occurring in patients who have undergone atransplant. An epidemiologic study showed that in renal transplanted patients, the incidence of Kaposi's sarcomawas 400 to 500 times higher than in controls of the same ethnic origin. We report a case of Kaposi's sarcomainvolving the lung and skin after immunosuppressive therapy in a patient with renal transplant. A plain chestradiograph showed diffusely increased interstitial opacity with multiple, ill-defined small nodules in both lungfields. HRCT revealed multiple small nodules, predominantly in the peribronchovascular regions, and ill-definedareas of ground-glass opacity and consolidation in both lungs.
Das Kaposi-Sarkom (KS) ist eine multifokale, von mesenchymalen Zellen ausgehende Neoplasie unklarer Genese an Haut und inneren Organen, deren Erstbeschreibung „Sarcoma idiopathicum multiplex haemorrhagicum“ durch Moritz Kaposi 1872 erfolgte [78]. Es wird auch als europäisches oder klassisches KS bezeichnet. Daneben finden sich das afrikanische KS und das KS bei iatrogener Immunsuppression. Vielfältiges wissenschaftliches Interesse erweckte dieser Tumor, nachdem Friedman-Kien et al. 1981 [49] über das gehäufte Auftreten von KS bei jungen homosexuellen Männern in New York berichteten, die gleichzeitig an einer erworbenen Immunschwäche erkrankt waren. Dieser Zusammenhang war so charakteristisch, daß ein Zusammenhang schon vor der Entdeckung des „human immunodeficiency virus“ (HIV) offensichtlich schien und das KS, verbunden mit einer nicht erklärbaren Immunschwäche, als AIDS-definierend eingestuft und diese Variante des KS als epidemisches KS bezeichnet wurde [50,92,104].
In einer Diskussion der Ätiologie des Kaposi-Sarkoms (KS) kann heute davon ausgegangen werden, daß sämtliche klinischen und epidemiologischen Erscheinungsformen des KS im Prinzip ein und dieselbe Krankheit darstellen, allerdings unterschiedliche Positionen innerhalb eines Krankheitsspektrums einnehmen:
Das KS stellt kein echtes Sarkom dar, sondern einen zumindest potentiell reversiblen proliferativen Prozeß
Die KS-Zellen stammen von Endothelzellen ab, wobei allerdings nicht endgültig geklärt ist, ob es sich um Blut- oder Lymphgefäßendothelzellen handelt
Eine Reihe von Zytokinen und Wachstumsfaktoren induzieren oder unterhalten das Wachstum und die Proliferation der KS-Zellen, zu denen IL6, basischer Fibroblastenwachstumsfaktor (bFGF), plättchenabstammender Wachstumsfaktor (PDGF), Onkostatin M, das hauptsächlich indirekt über eine Erhöhung der IL6-Expression von KS-Zellen wirkt, sowie das HIV-1-tat-Protein gehören
KS-Zellen produzieren selbst angiogene Faktoren, wie bFGF, IL6, IL1β, TNPα und GM-CSF, die sowohl auf autokrinem Weg das eigene Wachstum fördern als auch durch parakrine Aktivierung benachbarter ortsständiger Zellen zur Neoangiogenese führen können [54, 55].
This volume focuses on microbes that cause disease but not by an infectious process, meaning that the effects of the microbe are very delayed, indirect, or both. Strictly speaking, the microbe is infectious but when the disease presents itself the microbe is not present or “inactive”. I will describe microbes which fit the general theme but not precisely, because during disease the microbe may still be transmitted (hence “infectious”) albeit often with difficulty and not in the acute fashion we usually think of an infectious process.
Kaposi’s sarcoma (KS) is a hallmark of the AIDS pandemic. Originally described over ioo years ago, the increased incidence of this tumor in a nontypical population led to the original hypothesis of AIDS in young, homosexual men. One of the three AIDS defining malignancies, KS is the most common tumor to affect HIV positive individuals. Within the AIDS population the disease is much more common amongst homosexual and bisexual men than heterosexual men. Tumor incidence, initially affecting up to 50% of HIV positive individuals at the start of the AIDS pandemic, now affects between so and 20% of HIV infected patients. This is thought to be due to changes in sexual practice, implying some form of sexual transmission of the agent that causes KS. Despite this decrease in incidence within HIV infected individuals, the increase in the worldwide incidence of HIV infection implies an increasing public health problem with advanced KS. For individuals affected, the average age at first diagnosis of KS is between 30 and 40 and the median survival period following diagnosis is 12 months.1–3
The purification of the fibroblast growth factor (FGF) from bovine brain has led to the isolation of two peptides. FGF-1 with 128 amino acids and FGF-2 with 107 amino acids. The biological activity of these two peptides is acid- and heat-labile. As indicated by the amino acid composition of FGF-1 and -2, these two peptides are derived from a common precursor and bear no resemblance to pituitary FGF. The brain FGF peptides, like pituitary FGF, are mitogenic in vitro for the same wide variety of mesoderm-derived cells. Since their mitogenic activity is acid- and heat-labile, they are thereby distinguished from the platelet factor isolated from platelets and from the cationic peptide isolated from serum which has been shown to have the same molecular weight and isoelectric point as brain and pituitary FGF.
Transforming growth factor type beta (TGF-beta), when injected subcutaneously in newborn mice, causes formation of granulation tissue (induction of angiogenesis and activation of fibroblasts to produce collagen) at the site of injection. These effects occur within 2-3 days at dose levels than 1 microgram. Parallel in vitro studies show that TGF-beta causes marked increase of either proline or leucine incorporation into collagen in either an NRK rat fibroblast cell line or early passage human dermal fibroblasts. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) do not cause these same in vivo and in vitro effects; in both rat and human fibroblast cultures, EGF antagonizes the effects of TGF-beta on collagen formation. We have obtained further data to support a role for TGF-beta as an intrinsic mediator of collagen formation: conditioned media obtained from activated human tonsillar T lymphocytes contain greatly elevated levels of TGF-beta compared to media obtained from unactivated lymphocytes. These activated media markedly stimulate proline incorporation into collagen in NRK cells; this effect is blocked by a specific antibody to TGF-beta. The data are all compatible with the hypothesis that TGF-beta is an important mediator of tissue repair.
Fibroblast growth factors (FGFs) are potent mitogens for vascular and capillary endothelial cells in vitro and can stimulate the formation of blood capillaries (angiogenesis) in vivo. A crucial event in this process is the invasion of the perivascular extracellular matrix by sprouting endothelial cells. Using a recently developed in vitro model of angiogenesis, we show here that highly purified basic pituitary FGF can induce capillary endothelial cells to invade a three-dimensional collagen matrix and to organize themselves to form characteristic tubules that resemble blood capillaries. We also show that basic FGF concomitantly stimulates endothelial cells to produce a urokinase-type plasminogen activator, a protease that has been implicated in the neovascular response. The results demonstrate that basic FGF can stimulate processes that are characteristic of angiogenesis in vivo, including endothelial cell migration, invasion, and production of plasminogen activator.
Cultured human embryonal rhabdomyosarcoma cells express the basic fibroblast growth factor (bFGF) gene and they produce bFGF, which is apparently composed of two microheterogenous forms with Mrs of 16,500 and 17,200, respectively. bFGF derived from the rhabdomyosarcoma cells stimulates their own proliferation and that of human or bovine vascular endothelial cells. It is conceivable that the rhabdomyosarcoma-derived bFGF stimulates the growth and neovascularization of human rhabdomyosarcomas and that it may thereby contribute to the development of these tumors.
Cultured monolayers of bovine pituitary follicular cells, which transport ions, contain high amounts of mitogenic activity for endothelial cells which, on the basis of gene expression analysis, heparin-Sepharose elution profile, bioassay, immunoblotting, radioimmunoassay, and radioreceptor assay, has been identified as basic fibroblast growth factor (bFGF). These data indicate that follicular cells may be a major source of bFGF in the pituitary gland. Considering that bFGF has been proposed to play a role in paracrine regulation of pituitary hormone secretion, the data also suggest that these cells may exert important local regulatory functions.
Thirteen endothelial cell growth factors have been purified to homogeneity by heparin affinity and reversed-phase high performance liquid chromatography, and their chromatographic and electrophoretic properties were compared. The amino acid compositions of 10 of these mitogens have also been determined. The results indicate that these heparin-binding growth factors (HBGFs) can be subdivided into two classes. Class 1 HBGFs are anionic mitogens of molecular weight 15,000-17,000 found in high levels in neural tissue and include acidic brain fibroblast growth factor and retina-derived growth factor. Class 2 HBGFs are cationic mitogens of molecular weight 18,000-20,000 found in a variety of normal tissues and are typified by pituitary fibroblast growth factor and cartilage-derived growth factor. Typical class 2 HBGFs have also been isolated from a rat chondrosarcoma, a human melanoma, and a human hepatoma, suggesting that tumors do not make a structurally distinct HBGF class. These results provide a sound basis for the evaluation of the HBGFs purified from a variety of tissues and species and for the delineation of their normal and pathological functions in vivo.
Progression to increased malignancy frequently occurs in human brain tumors of glial origin and usually involves neovascularization--a massive proliferation of endothelial cells into the tumor tissue. We have shown previously that subversion of a normal growth factor-related pathway is frequently associated with human gliomas. Here we show that human glioma cell lines express the gene encoding the angiogenic peptide endothelial cell growth factor (ECGF) or acidic fibroblast growth factor (a-FGF) and that an ECGF-like polypeptide is produced by these cells. The glioma-derived growth factor was partially purified from cell extracts by heparin-Sepharose affinity chromatography where it eluted at 1.5 M sodium chloride. On reversed-phase h.p.l.c., growth factor activity for endothelial cells was eluted at the same concentration of acetonitrile as found for bovine brain-ECGF, also a potent mitogen for endothelial cells. Moreover, human glioma cells possess specific cell surface receptors for ECGF and are mitogenically stimulated by exogenous addition of this growth factor. Glioma derived-ECGF may therefore have a dual influence: first, by autocrine growth-stimulation of human gliomas and, second, by paracrine-stimulation of endothelial cell proliferation which results in neovascularization of the tumor tissue.
Clones encoding the angiogenic endothelial cell mitogen, basic fibroblast growth factor (FGF), have been isolated from human cDNA libraries made from kidney, fetal heart, fetal liver, term placenta, and a breast carcinoma. Basic FGF cDNA clones are present in these libraries at very low levels when compared to the quantity of the growth factor in the tissues. This observation, combined with the fact that several of the clones represent unspliced transcripts, suggests that cytoplasmic basic FGF mRNA is unstable and that the protein is stored in tissues. The amino acid sequence of human basic FGF, deduced from the sequence of these cDNAs and from genomic clones, is 99% homologous to that of bovine basic FGF, implying a strong selection pressure for maintenance of function and structure. As with the bovine factor, human basic FGF does not appear to have a signal peptide sequence. Southern blot analysis of human genomic DNA and mapping of the cloned gene shows that there is only one basic FGF gene. All of the basic, heparin-binding endothelial cell mitogens of similar amino acid composition that have been described must therefore be products of this single gene.
Clones of complementary DNA encoding the human lymphokine known as granulocyte-macrophage colony-stimulating factor (GM-CSF)
were isolated by means of a mammalian cell (monkey COS cell) expression screening system. One of these clones was used to
produce recombinant GM-CSF in mammalian cells. The recombinant hematopoietin was similar to the natural product that was purified
to apparent homogeneity from medium conditioned by a human T-cell line. The human T-cell GM-CSF was found to be 60 percent
homologous with the GM-CSF recently cloned from murine lung messenger RNA.
• Immunosuppressed patients are at risk of acquiring Kaposi's sarcoma. We describe here a 66-year-old man with bronchial asthma who was receiving immunosuppressive medication (prednisone given for systemic effect) and in whom Kaposi's sarcoma developed. The literature on this subject is reviewed.
(Arch Dermatol 116:1280-1282, 1980)
Substantially pure basic fibroblast growth factor (bFGF), a 146 amino acid residue polypeptide, is produced. The amino acid residue sequence of bFGF is disclosed as well as a DNA chain encoding the polypeptide. By appropriately inserting a synthesized DNA chain into a cloning vector and using the cloning vector to transform cells, synthetic bFGF can be obtained from transformed cell lines, both prokaryotic and eukaryotic.
We recently reported the cloning of a rearranged human oncogene following transfection of DNA from Kaposi's sarcoma into NIH 3T3 cells. To identify the protein(s) encoded in two novel mRNAs of 3.5 and 1.2 kb expressed in NIH 3T3 transformants, we constructed a cDNA library. One of the cDNA clones isolated (KS3) corresponded to the 1.2 kb mRNA and transformed NIH 3T3 cell when inserted into a mammalian expression vector. The 1152 nucleotide KS3 cDNA encodes a protein of 206 amino acids with significant homology to the growth factors basic FGF and acidic FGF. Expression of the KS3 product as a bacterial fusion protein or in COS cells allowed us to determine that both proteins had significant growth-promoting activity and that the COS cell protein was glycosylated. Thus one of the mRNAs transcribed from the KS oncogene encodes a growth factor that could transform cells by an autocrine mechanism and appears to represent a new member of the FGF family.
Colony-stimulating factors (CSFs) are glycoproteins that stimulate the growth of hematopoietic progenitors and enhance the functional activity of mature effector cells. Human granulocyte/macrophage colony-stimulating factor (GM-CSF) is a 22-kDa glycoprotein that stimulates the growth of myeloid and erythroid progenitors in vitro and increases the responsiveness of neutrophils, monocytes, and eosinophils to physiologic stimuli. Elucidation of the cell and tissue sources of CSFs, as well as study of their regulation of expression, is required to understand their role in physiologic and pathophysiologic states. An extensive survey of normal and neoplastic human tissues did not reveal constitutive production of detectable levels of GM-CSF mRNA in any of the 64 samples studied. Antigen- or lectin-activated T lymphocytes have been shown to produce GM-CSF; therefore, to elucidate the genetic sequences required, we constructed recombinant plasmids containing 5' flanking DNA of the GM-CSF gene linked to the marker chloramphenicol acetyltransferase gene. The recombinant constructs were transfected into a human T-cell leukemia virus type I (HTLV)-infected T-lymphoblast cell line that can be stimulated to produce high levels of GM-CSF. We show here that the 5' flanking sequences of the GM-CSF gene can direct increased expression of the chloramphenicol acetyltransferase gene in activated T-lymphoblast cells.
The purpose of this study was threefold; to ascertain if immunosuppression may be a factor in the causation of Kaposi's sarcoma, to find out if there is a correlation between abnormalities in in vitro studies of cellular immune function and the extent of the disease, and to analyze the literature on Kaposi's sarcoma arising in renal transplant recipients to determine a management policy for these patients.The charts of 44 patients with Kaposi's sarcoma seen in a 20 year period at The Princess Margaret Hospital were reviewed in a search for evidence of immunosuppression as a possible risk factor. Such evidence was found in seven patients. Four of these seven patients were renal transplant recipients, two had generalized lymphomas and were receiving chemotherapy whereas one had a glioblastoma multiforme and was receiving chemotherapy.Studies of cellular immunity using phytohemagglutinin, conconavalin A, pokeweed mitogen, the mixed leukocyte reaction and dinitrochlorobenzene skin testing, in eight patients with Kaposi's sarcoma, three of whom had previous renal transplants, indicate that a correlation exists between the degree of immunologic deficiency and the extent of the Kaposi's sarcoma.Our seven patients were all of Jewish or Mediterranean ancestry. The four cases of Kaposi's sarcoma arising in renal transplant recipients developed in a population of 100 renal transplant recipients of similar ethnic background (4 per cent). This incidence, when compared with our experience of 40 cases arising in 500,000 people of similar ancestry in the Toronto area, represents a 400 to 500 fold greater incidence in renal transplant recipients than in the control population. A literature review has yielded 12 additional cases of Kaposi's sarcoma developing in renal transplant recipients. On the basis of this review and our own experience we have proposed a management policy for these patients.It is proposed that the etiology of Kaposi's sarcoma is multifactorial and that a combination of immunosuppression and/or immunologic stimulation combined with a hereditary predisposition to the disease are responsible for the major increase in its incidence.
Extracts of bovine hypothalamus were found to contain a significant level of mitogenic activity when tested in a Swiss 3T3 cell [3H]dThd incorporation assay and in a human umbilical vein endothelial cell growth assay. The mitogenic activity responsible for 3T3 cell activity was purified and characterized as a fibroblast growth factor (FGF)-like mitogen. Neither the biologically active FGF-like mitogen purified from the hypothalamus extracts nor FGF purified from bovine pituitary glands was mitogenic when added to human endothelial cells in vitro, suggesting the presence of more than one mitogen in the hypothalamic extracts. The 3T3 and endothelial cell biological activities of hypothalamic extracts were both found to be inactivated by trypsin, subtilisin, and heat treatment, but were stable to dialysis. The endothelial cell growth factor activity could be efficiently separated from the FGF activity by gel exclusion chromatography. The endothelial cell mitogen possessed a molecular weight of approximately 75,000, whereas that of FGF was approximately 15,000. The endothelial cell growth factor activity was found to be inactivated with reducing agents whereas the 3T3 cell mitogenic activity was stable after incubation with 2-mercaptoethanol. Significant levels of endothelial cell mitogenic activity were also found in extracts of bovine brain and pituitary glands.
Basic and acidic fibroblast growth factors (FGF's) are potent mitogens for capillary endothelial cells in vitro, stimulate angiogenesis in vivo, and may participate in tissue repair. An oligonucleotide probe for bovine basic FGF was designed from the nucleotide sequence of the amino-terminal exon of bovine acidic FGF, taking into account the 55 percent amino acid sequence homology between the two factors. With this oligonucleotide probe, a full length complementary DNA for basic FGF was isolated from bovine pituitary. Basic FGF in bovine hypothalamus was shown to be encoded by a single 5.0-kilobase messenger RNA; in a human hepatoma cell line, both 4.6- and 2.2-kilobase basic FGF messenger RNA's were present. Both growth factors seem to be synthesized with short amino-terminal extensions that are not found on the isolated forms for which the amino acid sequences have been determined. Neither basic nor acidic FGF has a classic signal peptide.
Within the past 2 years, several angiogenic factors have been fully purified, their amino acid sequences determined, and their
genes cloned. These polypeptides include acidic and basic fibroblast growth factor, angiogenin, and transforming growth factors
alpha and beta. Other less well characterized angiogenesis factors have also been isolated, some of which are lipids. This
article traces the discovery of the angiogenic factors and describes their possible significance in understanding growth regulation
of the vascular system. When evaluated according to their putative targets, they appear to fall into two groups: those that
act directly on vascular endothelial cells to stimulate locomotion or mitosis, and those that act indirectly by mobilizing
host cells (for example, macrophages) to release endothelial growth factors. In addition to their presence in tumors undergoing
neovascularization, the same angiogenic peptides are found in many normal tissues where neovascularization is not occurring.
This suggests that physiological expression of angiogenic factors is tightly regulated. In addition to the persistent angiogenesis
induced by tumors, it now appears that a variety of nonneoplastic diseases, previously thought to be unrelated, can be considered
as "angiogenic diseases" because they are dominated by the pathologic growth of capillary blood vessels.
The role of polypeptide growth factors in the processes of inflammation and repair was investigated by analyzing the influence of transforming growth factor-beta (TGF-beta), applied directly to linear incisions made through rat dorsal skin. A dose-dependent, direct stimulatory effect of a single application of TGF-beta on the breaking strength of healing incisional wounds was demonstrated. An increase in maximum wound strength of 220 percent of control was observed at 5 days; the healing rate was accelerated by approximately 3 days for at least 14 days after production of the wound and application of TGF-beta. These increases in wound strength were accompanied by an increased influx of mononuclear cells and fibroblasts and by marked increases in collagen deposition at the site of application of TGF-beta. TGF-beta is thus a potent pharmacologic agent that can accelerate wound healing in rats.
Cells derived from lung biopsies and pleural effusions from AIDS patients with Kaposi's sarcoma (KS) of the lungs were established in long-term culture with the aid of conditioned medium from HTLV-II-transformed T cells (HTLV-II CM). These AIDS-KS cells were similar to the so-called spindle cells in KS lesions and had some of their features. They produced factors that supported their own growth (autocrine) and the growth of other cells (paracrine), including umbilical vein endothelium and fibroblasts. That the AIDS-KS cells also expressed potent angiogenic activity was demonstrated by the chorioallantoic membrane assay and by subcutaneous inoculation of AIDS-KS cells into nude mice, which resulted in the development of angiogenic lesions composed of mouse cells and showing histological features similar to those of human KS lesions. These data suggest that AIDS-associated KS and possibly other types of KS may be initiated by signals that induce the growth of particular cells (spindle cells of lymphatic or vascular origin) and the expression of autocrine and paracrine activities.
The hst gene was originally identified as a transforming gene in DNAs from human stomach cancers and from a noncancerous portion of stomach mucosa by DNA-mediated transfection assay using NIH3T3 cells. cDNA clones of hst were isolated from the cDNA library constructed from poly(A)+ RNA of a secondary transformant induced by the DNA from a stomach cancer. The sequence analysis of the hst cDNA revealed the presence of two open reading frames. When this cDNA was inserted into an expression vector containing the simian virus 40 promoter, it efficiently induced the transformation of NIH3T3 cells upon transfection. It was found that one of the reading frames, which coded for 206 amino acids, was responsible for the transforming activity.
Two distinct but distantly related complementary DNAs encoding proteins sharing human interleukin-1 (IL-1) activity (termed IL-1 alpha and IL-1 beta), were isolated from a macrophage cDNA library. The primary translation products of the genes are 271 and 269 amino acids long, although expression in Escherichia coli of the carboxy-terminal 159 and 153 amino acids produces IL-1 biological activity.
Immunohistochemical studies were performed in thirty skin biopsies from patients with Kaposi's sarcoma, who did and did not have the acquired immune deficiency syndrome (AIDS). Tumour histogenesis was rigorously tested using a battery of endothelial cell markers, which included two new monoclonal antibodies, EN4 and PAL E. These are both specific for endothelial cells and can be visualised in appropriately fixed paraffin embedded tissue. Whereas EN4 labels all endothelial cells, PAL E is negative in endothelium of lymphatic derivation. Lectin binding with Ulex europaeus agglutinin 1 (UEA-1) and the presence of factor VIII related antigen (FVIIIRA) and laminin were also examined. In nodular lesions of Kaposi's sarcoma the spindle cell areas were positive with EN4 and UEA-1, negative with PAL E, and showed focal staining for FVIIIRA and laminin. These results confirm that the tumour is of endothelial cell origin. Six patch stage lesions showed a network of angulated spaces, lined by cells that were positive with EN4 and UEA-1, negative with PAL E and anti-FVIIIRA, and showed only weak staining for laminin. This pattern was observed in both AIDS and non-AIDS related cases and strongly favours a lymphatic derivation for the tumour. This has important implications as it suggests that lymphatic endothelium may have special characteristics that lead to neoplastic transformation in patients with retrovirus infection.
Studies of the biology and pathogenesis of Kaposi's sarcoma (KS) have been hampered by the inability to maintain long-term cultures of KS cells in vitro. In this study AIDS-KS-derived cells with characteristic spindle-like morphology were cultured with a growth factor (or factors) released by CD4+ T lymphocytes infected with human T-lymphotropic virus type I or II (HTLV-I or HTLV-II) or with human immunodeficiency virus type 1 or 2 (HIV-1 or HIV-2). Medium conditioned by HTLV-II-infected, transformed lines of T cells (HTLV-II CM) contained large amounts of this growth activity and also supported the temporary growth of normal vascular endothelial cells, but not fibroblasts. Interleukin-1 and tumor necrosis factor-alpha stimulated the growth of the KS-derived cells, but the growth was only transient and these could be distinguished from that in HTLV-II CM. Other known endothelial cell growth promoting factors, such as acidic and basic fibroblast growth factors and epidermal growth factor, did not support the long-term growth of the AIDS-KS cells. The factor released by CD4+ T cells infected with human retroviruses should prove useful in studies of the pathogenesis of KS.
The complementary DNAs and genes encoding the four major human myeloid growth factors--granulocyte colony-stimulating factor,
macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3--have all been molecularly
cloned. These DNA clones have proved valuable for studying the molecular biology of these important regulatory molecules as
well as for the large-scale production of the recombinant growth factor proteins. These advances have led to a much better
understanding of the role of the myeloid growth factors in regulating hematopoiesis in vivo that should soon find practical
application in clinical medicine.
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Bovine aortic and corneal endothelial cells synthesize a growth factor that remains mostly cell-associated but can also be extracted from the subendothelial extracellular matrix (ECM) deposited by these cells. The endothelial cell-derived growth factors extracted from cell lysates and from the extracellular matrix appear to be structurally related to basic fibroblast growth factor by the criteria that they bind to heparin-Sepharose and are eluted at 1.4-1.6 M NaCl, have a molecular weight of about 18,400, cross-react with anti-basic fibroblast growth factor antibodies when analyzed by electrophoretic blotting and immunoprecipitation, and are potent mitogens for bovine aortic and capillary endothelial cells. It is suggested that endothelium can store growth factors capable of autocrine growth promotion in two ways: by sequestering growth factor within the cell and by incorporating it into the underlying extracellular matrix.
A Mr 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig brain along with the typical Mr 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The Mr 25,000 form, like the Mr 18,000 form, was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The Mr 25,000 guinea pig protein stimulated plasminogen activator production by cultured bovine capillary endothelial cells in a dose-dependent manner at concentrations of 0.1-10 ng/ml, the same range that was effective for guinea pig and human Mr 18,000 bFGFs. The binding of human 125I-labeled bFGF to baby hamster kidney cells is inhibited equally by the Mr 25,000 guinea pig protein and the Mr 18,000 guinea pig and human bFGFs. Polyclonal antibodies raised against human bFGF recognize both the Mr 25,000 and 18,000 guinea pig proteins in an immunoblot analysis. In a radioimmunoassay, both the Mr 25,000 and Mr 18,000 guinea pig proteins compete equally well with iodinated human bFGF for binding to the anti-human bFGF antibodies. When treated with low concentrations of trypsin, the Mr 25,000 guinea pig bFGF was converted to a Mr 18,000 protein. These results show that the two molecules are closely related and suggest that the Mr 25,000 protein shares substantial homology with the Mr 18,000 bFGF.
Adherent human blood monocytes were stimulated with heat-killed Staphylococcus albus or Escherichia coli lipopolysaccharide in the presence of 35S-methionine-, [3H]leucine-, or 14C-labeled amino acids. After incubation, interleukin 1 (IL 1) activity in the supernatant medium was purified over an anti-human IL 1 immunoadsorbent followed by gel filtration and chromatofocusing. The purity of the IL 1 was assessed by fluorography of one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Isoelectric and chromatofocusing of low m.w. proteins (less than 20,000 m.w.) revealed three charged 18,000 m.w. species of IL 1 with approximate pI's of 7, 6, and 5, with the most abundant form at pI 7. During the purification procedures, lymphocyte co-mitogenic activity, fever in rabbits, and prostaglandin E2 release from dermal fibroblasts co-eluted in the same fractions. In addition, these fractions were active when injected into endotoxin-resistant C3H/HeJ mice for the production of fever, the induction of serum amyloid A protein, a decrease in serum iron concentration, and an increase in the number of circulating neutrophils. Fluorography revealed homogeneous bands with an m.w. of about 18,000 which correlated with these biological activities. The specific activity of the pI 6 or 5 IL 1, as judged by the ratio of T cell co-mitogenic activity to incorporated radiolabeled amino acid, was at least 10-fold greater than that observed for the pI 7 form. This result suggests that the amino acid compositions of the two 18,000 m.w. acidic forms are unrelated to the pI 7 species. These results also demonstrate that the pI 7 human monocyte IL 1 is the predominant 18,000 m.w. form synthesized and, furthermore, that homogeneous pI 7 IL 1 exhibits multiple biological properties on various tissues by modulating immunologic, inflammatory, metabolic, and neurologic functions. Data are also presented for the existence of a high m.w. (32,000) human pro-IL 1 molecule as the predominant monocytic intracellular form. This pro-IL 1 is degraded artifactually during isolation to lower m.w. forms in the presence of an extracellular serine protease activity. These data are consistent with a model for IL 1 secretion in which pro-IL 1 is first synthesized within the cell and is processed during or after extracellular transport.
Transforming growth factor-beta (TGF-beta) inhibits the growth of endothelial cells derived from various sources, including human umbilical vein, bovine aorta, and rat heart. Long-term exposure of rat heart endothelial cells to TGF-beta also induces dramatic changes in morphology that are characteristic of senescent cells. These changes are accompanied by a decrease in the number of high-affinity receptors for epidermal growth factor (EGF), with almost no change in total receptor number. Additionally, the EGF-induced expression of specific competence genes (c-myc, JE, KC) is decreased, whereas the induction of c-fos gene expression by EGF is unaltered by TGF-beta treatment. These data suggest that growth inhibitors such as TGF-beta may act by altering the cell's response to growth-stimulatory factors.
Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.
Kaposi's sarcoma is a multifocal systemic neoplasm histologically characterized by proliferating fibroblastic and microvascular elements. Initial signs include macules, papules, or nodules on the skin or mucosal surface. Lesions are frequently found on the trunk, arms, and head and neck. In general, sites of involvement and tumor load do not correlate with prognosis. A general decrease in the functional capacities of T and B cells is found in most patients. Kaposi's sarcoma is reported as the initial manifestation of the acquired immunodeficiency syndrome (AIDS) in approximately 30% of cases. Most cases are in men, although it has been reported in all risk groups. Kaposi's sarcoma in AIDS is more frequent among whites and homosexuals than blacks and intravenous drug abusers. Overall mortality is approximately 41%, with over 60% of patients alive at 1 year and 50% at 22 months. Overall survival is 18 months; however, some patients who have had the disease for 3 to 4 years are still doing well.
Vascular endothelial cells have a central role in various pathophysiological responses such as acute inflammation, wound healing and atherogenesis. The anatomical position of endothelial cells between blood leukocytes and the surrounding vascular smooth muscle cells or stromal fibroblasts may intensify and focus the effects of released endothelial cell products. Endothelial cells in culture produce a platelet-derived growth factor (PDGF)-like mitogen. PDGF purified from platelets is a basic protein with an apparent relative molecular mass (Mr) of approximately 30,000 (reviewed in refs 2, 3) and is believed to comprise two polypeptide chains, PDGF-A and PDGF-B (also referred to as PDGF-1 and PDGF-2; refs 5, 6). Sequence analysis of PDGF B chain has revealed a striking homology with the predicted sequence of p28sis, the transforming protein of simian sarcoma virus. sis-Homologous transcripts have been detected by Northern blot analysis of RNA from cultured endothelial cells. However, there are no structural data available on either the protein product or the messenger RNA to establish the identity of the endothelial-derived mitogen with either chain of PDGF. Here we report the isolation and complete sequence analysis of a sis-homologous complementary DNA clone from human endothelial cells, providing an opportunity to study the structure of sis as transcribed by a normal (untransformed) cell. Our results establish that normal human endothelial cells in culture express the B chain of PDGF, and that endothelial-derived PDGF B chain is synthesized as a predicted precursor polypeptide of Mr 27,281.
Six hundred twenty-four cases of Kaposi's sarcoma occurring in Ugandan Africans have been reviewed. Thirty-four patients came to necropsy, and almost all were found to have visceral lesions. Patients with nodular cutaneous disease usually had a long history of disease and often had small numbers of visceral nodules which only very seldom produced symptoms. Locally aggressive tumors occurred more commonly in Ugandans than in whites; in spite of locally destructive growth, they seldom metastasized widely and were often present for many years. Young people with generalized involvement were seen rarely and most of these patients had had malaria. The pattern of disease was of two types: in African children, tumor was found predominantly in nodes, whereas in young adults involvement of skin nodes and viscera was seen. The prognosis in such cases was poor. Africans seem to be less likely to develop a second neoplasm than whites. Females suffer from Kaposi's sarcoma less frequently than males but are relatively more likely to develop rapidly fatal generalized forms of the disease.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.