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Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage-activating factor (MAF). The precursor activity of serum Gc protein was lost or reduced in HIV-infected patients. These patient sera contained alpha-N-acetylgalactosaminidase (Nagalase), which deglycosylates serum Gc protein. Deglycosylated Gc protein cannot be converted to MAF and thus loses MAF precursor activity, leading to immunosuppression. Nagalase in the blood stream of HIV-infected patients was complexed with patient immunoglobulin G, suggesting that this enzyme is immunogenic, seemingly a viral gene product. In fact, Nagalase was inducible by treatment of cultures of HIV-infected patient peripheral blood mononuclear cells with a provirus-inducing agent. This enzyme was immunoprecipitable with polyclonal anti-HIV but not with anticellular constitutive enzyme or with antitumor Nagalase. The kinetic parameters (km value of 1.27 mM and pH optimum of 6.1), of the patient serum Nagalase were distinct from those of constitutive enzyme (km value of 4.83 mM and pH optimum of 4.3). This glycosidase should reside on an envelope protein capable of interacting with cellular membranous O-glycans. Although cloned gp160 exhibited no Nagalase activity, treatment of gp160 with trypsin expressed Nagalase activity, suggesting that proteolytic cleavage of gp160 to generate gp120 and gp41 is required for Nagalase activity. Cloned gp120 exhibited Nagalase activity while cloned gp41 showed no Nagalase activity. Since proteolytic cleavage of protein gp160 is required for expression of both fusion capacity and Nagalase activity, Nagalase seems to be an enzymatic basis for fusion in the infectious process. Therefore, Nagalase appears to play dual roles in viral infectivity and immunosuppression.
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... Based on the aforementioned findings and on documented analogies between SARS-CoV-2 and HIV , we hypothesized that the reduced conversion activity of the Gc protein (human group-specific component (Gc)) into the macrophage activating factor (MAF) could have a key role in the dysregulate immune response induced by SARS-CoV-2, just like for HIV infected patients [14,15]. If this hypothesis is correct, it might help to set a valid strategy of immunotherapy also based on an off-label use of GcMAF in critically ill COVID-19 patients. ...
... Stepwise hydrolysis of Gc protein by the inducible membranous β-galactosidase of stimulated B-lymphocytes, and by the Neu-1 sialidase of T-lymphocytes converts it into the active GcMAF . On the contrary, deglycosilation of Gc protein by action of the enzyme alpha-N-acetylgalactosaminidase, named nagalase, secreted from HIV-infected cells leads to lack of macrophage activation and to immunosuppression, as a consequence [14,15]. It is remarkable that nagalase was demonstrated to be an intrinsic component not only of the envelope glycoproteins gp120 and gp160 of HIV but also of the hemagglutinin (HE) of influenza virus [15,20] and even produced by neoplastic cells . ...
... On the contrary, deglycosilation of Gc protein by action of the enzyme alpha-N-acetylgalactosaminidase, named nagalase, secreted from HIV-infected cells leads to lack of macrophage activation and to immunosuppression, as a consequence [14,15]. It is remarkable that nagalase was demonstrated to be an intrinsic component not only of the envelope glycoproteins gp120 and gp160 of HIV but also of the hemagglutinin (HE) of influenza virus [15,20] and even produced by neoplastic cells . Indeed, flu-like symptoms with serum nagalase activity similar to the influenza acute state were reported in the early stage of HIV-infection, so that the serum enzyme activity may be detectable at all phases of HIV-infection [14,15]. ...
... Since macrophage activation for phagocytosis and antigen-presentation to B and T lymphocytes is the first indispensable step in development of both humoral and cellular immunity [Yamamoto et al., 1995; Yamamoto and Naraparaju, 1998], lack of macrophage activation leads to immunosuppression. In fact, immunosuppression in AIDS [Yamamoto et al., 1995; Yamamoto, 2006] and advanced cancer patients is caused by lack of macrophage activation. Microbial infection induces inflammation that results in macrophage activation as for innate defense. ...
... However, cultivation of lyso-Pc-treated patient PBMCs in medium containing patient own serum (0.1%) resulted in lack or reduced levels of macrophage activation [Yamamoto et al., 1995; Yamamoto, 1997 Yamamoto, , 1999, as a consequence of lost or decreased MAF precursor activity of the patient serum Gc protein [Yamamoto et al., 1995; Yamamoto, 1997 Yamamoto, , 1999. Loss of MAF precursor activity is due to deglycosylation of serum Gc protein by a-N-acetylgalactosaminidase (termed Nagalase) secreted from HIV-infected cells (Fig. 1b) [Yamamoto et al., 1995; Yamamoto, 1997 Yamamoto, , 1998b Yamamoto, , 2006. Since Nagalase is an intrinsic component of the envelope gp120 [Yamamoto, 1999 [Yamamoto, , 2006, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and unassembled envelope proteins (gp160 and gp120) released from HIV-infected cells. ...
... Loss of MAF precursor activity is due to deglycosylation of serum Gc protein by a-N-acetylgalactosaminidase (termed Nagalase) secreted from HIV-infected cells (Fig. 1b) [Yamamoto et al., 1995; Yamamoto, 1997 Yamamoto, , 1998b Yamamoto, , 2006. Since Nagalase is an intrinsic component of the envelope gp120 [Yamamoto, 1999 [Yamamoto, , 2006, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and unassembled envelope proteins (gp160 and gp120) released from HIV-infected cells. Because of Nagalase being an HIV viral component, serum Nagalase is already complexed with patient own immunoglobulin G (anti-HIV IgG) [Yamamoto, 1999 [Yamamoto, , 2006. ...
Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by alpha-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV-infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years.
... The modeling also leads to the prediction that only 0.40% of White Males are born with the 10 th ordered mutation and require only 33 ...
... Moreover, Yamamoto and Urade found that administering vitamin D-binding protein (Gc protein)derived macrophage activating factor (GcMAF) at the rate of 100 ng once a week over a period of no more than 6 months can overcome the effect of Nagalase and reduce Nagalase activity in breast and prostate cancer cohorts to within normal levels [30,31,32,33,34,35]. Since the number of cancerous cells in a patient is proportional to the patients Nagalase activity, reducing the Nagalase activity to normal levels implies that all cancerous cells have been eliminated from the patient by an activated immune system, a conclusion that was confirmed by CAT-scans [30,31,32,33,34,35]. ...
... Moreover, Yamamoto and Urade found that administering vitamin D-binding protein (Gc protein)derived macrophage activating factor (GcMAF) at the rate of 100 ng once a week over a period of no more than 6 months can overcome the effect of Nagalase and reduce Nagalase activity in breast and prostate cancer cohorts to within normal levels [30,31,32,33,34,35]. Since the number of cancerous cells in a patient is proportional to the patients Nagalase activity, reducing the Nagalase activity to normal levels implies that all cancerous cells have been eliminated from the patient by an activated immune system, a conclusion that was confirmed by CAT-scans [30,31,32,33,34,35]. ...
Natural immunity to breast and prostate cancers is predicted by a novel, saturated ordered mutation model fitted to USA (SEER) incidence data, a prediction consistent with the latest ideas in immunosurveillance. For example, the prevalence of natural immunity to breast cancer in the white female risk population is predicted to be 76.5%; this immunity may be genetic and, therefore, inherited. The modeling also predicts that 6.9% of white females are born with a mutation necessary to cause breast cancer (the hereditary form) and, therefore, are at the highest risk of developing it. By contrast, 16.6% of white females are born without any such mutation but are nonetheless susceptible to developing breast cancer (the sporadic form). The modeling determines the required number of ordered mutations for a cell to become cancerous as well as the mean time between consecutive mutations for both the sporadic and hereditary forms of the disease. The mean time between consecutive breast cancer mutations was found to vary between 2.59 - 2.97 years, suggesting that such mutations are rare events and establishing an upper bound on the lifetime of a breast cell. The prevalence of immunity to breast cancer is predicted to be 79.7% in Blacks, 86.5% in Asians, and 85.8% in Indians. Similarly, the prevalence of immunity to prostate cancer is predicted to be 67.4% for Whites, 50.5% for Blacks, 77.7% for Asians, and 78.6% for Indians. It is of paramount importance to delineate the mechanism underlying immunity to these cancers.
... Macrophages are the major phagocytic and antigen-presenting cells. Because macrophage activation for phagocytosis and antigen presentation to B and T lymphocytes is the first indispensable step in the development of both humoral and cellular immunity, lack of macrophage activation leads to immunosuppression252627282930 . Advanced cancer patients have high serum Nagalase activities , resulting in no macrophage activation and severe immunosuppression that explain why cancer patients die with overwhelming infection (e.g., pneumonia) [25,26]. ...
... The data were expressed as nanomoles of superoxide produced per minute per 10 6 cells (macrophages). These values represent the MAF precursor activity of patient serum Gc protein [28,29]. Lost or reduced MAF precursor activity of patient serum Gc protein is expressed as a decrease in superoxide generation compared with the healthy human Gc protein control. ...
... In contrast, Nagalase is secreted exclusively from cancerous cells but not from normal tissues (even inflamed noncancerous tissues). Thus, the level of Nagalase activity in blood stream is proportional to the tumor burden in the hosts [25,28,29] and has been used as a prognostic index for GcMAF therapy for preclinical and clinical cancer models [14,15,25,32333436] . Serum Nagalase deglycosylates serum Gc protein. ...
Serum Gc protein (known as vitamin D(3)-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years.
... nMol/mL/min). Nagalase activity fell 27.8% after the three-month experience, Fig. 2. Nagalase is a marker for inflammation often found elevated in viral infections, cancer and other chronic conditions (Carter et al., 2020;Yamamoto and Urade, 2005;Yamamoto, 2006). These results are consistent with previous observations (Blythe et al., 2017;Carter et al., 2020); we hypothesize that decrease of serum nagalase activity is due to the presence in the probiotic yogurt of high levels of naturally formed Gc protein-derived Macrophage Activating Factor that binds and neutralizes nagalase (Carter et al., 2020;Pacini et al., 2011). ...
... Nagalase is considered an indicator of cancer cell proliferation, viral infections as well as a marker of systemic inflammation, (Carter et al., 2020;Yamamoto and Urade, 2005;Yamamoto, 2006;Thyer et al., 2013). Since many of the toxicants described in this article are associated with increased cancer risk, immune dysfunction and chronic inflammation, it is tempting to speculate that the decrease of nagalase activity represented a reduction of the risks associated with exposure to those toxicants. ...
... This activity was measured as a function of reduced serum levels of the enzyme a-N-acetylgalactosaminidase (nagalase). Nagalase deglycosylation of DBP prevents it from activating macrophages and therefore suppresses the immune response . The basic mechanism proposed in all cases is largely the activation of macrophages and the ensuing immune responses. ...
... The presence of elevated nagalase in the plasma of HIV patients suggests that macrophage activation may be inhibited in these patients . In addition, nagalase has been shown to be an intrinsic component of an envelope protein promoting fusion for the initiation of infection . The plasma concentration of nagalase in patients with systemic lupus erythematosus was also found to be elevated. ...
Vitamin D binding protein-macrophage activating factor (DBP-maf) is a potent inhibitor of tumor growth. Its activity, however, has been attributed to indirect mechanisms such as boosting the immune response by activating macrophages and inhibiting the blood vessel growth necessary for the growth of tumors.
In this study we show for the first time that DBP-maf exhibits a direct and potent effect on prostate tumor cells in the absence of macrophages. DBP-maf demonstrated inhibitory activity in proliferation studies of both LNCaP and PC3 prostate cancer cell lines as well as metastatic clones of these cells. Flow cytometry studies with annexin V and propidium iodide showed that this inhibitory activity is not due to apoptosis or cell death. DBP-maf also had the ability to inhibit migration of prostate cancer cells in vitro. Finally, DBP-maf was shown to cause a reduction in urokinase plasminogen activator receptor (uPAR) expression in prostate tumor cells. There is evidence that activation of this receptor correlates with tumor metastasis.
These studies show strong inhibitory activity of DBP-maf on prostate tumor cells independent of its macrophage activation.
... Macrophages are the major phagocytic and antigen-presenting cells. Since macrophage activation for phagocytosis and antigen presentation to B and T lymphocytes are the Wrst indispensable steps in the development of both humoral and cellular immunities, lack of macrophage activation leads to immu- nosuppression [32, 34, 35, 37, 39, 42, 44, 45]. Advanced cancer patients have high serum Nagalase activity, resulting in no macrophage activation and severe immunosuppression that explain why cancer patients die with overwhelming infection (e.g., pneumonia) . ...
... Because the fate and staging of the malignant disease correlate with tumor burden and the degree of immunosuppression [11, 37], the potency of macrophages and tumor burden index for each patient must be determined, regardless the lapse of time after tumor resection and adjuvant therapy. Since macrophage activation for phagocytosis and subsequent antigen presentation is the Wrst indispensable step for immune development [37, 41, 43, 45] , the lack of macrophage activation leads to immunosuppression. Because serum Gc protein is the precursor for the principal MAF, the MAF precursor activity of patient serum Gc protein was Wrst to be examined. ...
Serum vitamin D binding protein (Gc protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of colorectal cancer patients was lost or reduced because Gc protein is deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Deglycosylated Gc protein cannot be converted to MAF, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage-activating factor (GcMAF) ever discovered, but it produces no side effect in humans. Macrophages treated with GcMAF (100 microg/ml) develop an enormous variation of receptors and are highly tumoricidal to a variety of cancers indiscriminately. Administration of 100 nanogram (ng)/ human maximally activates systemic macrophages that can kill cancerous cells. Since the half-life of the activated macrophages is approximately 6 days, 100 ng GcMAF was administered weekly to eight nonanemic colorectal cancer patients who had previously received tumor-resection but still carried significant amounts of metastatic tumor cells. As GcMAF therapy progressed, the MAF precursor activities of all patients increased and conversely their serum Nagalase activities decreased. Since serum Nagalase is proportional to tumor burden, serum Nagalase activity was used as a prognostic index for time course analysis of GcMAF therapy. After 32-50 weekly administrations of 100 ng GcMAF, all colorectal cancer patients exhibited healthy control levels of the serum Nagalase activity, indicating eradication of metastatic tumor cells. During 7 years after the completion of GcMAF therapy, their serum Nagalase activity did not increase, indicating no recurrence of cancer, which was also supported by the annual CT scans of these patients.
... According to Yamamoto et al. [7,8], the inhibitor of the Naga6 activity could be removed by precipitation of the serum protein with 70% saturated (NH 4 ) 2 SO 4 in 50 mM Na-citrate buffer (pH 6) and subsequent dialysis of the re-dissolved pellet against this buffer for ca. 2 h . ...
... (NH 4 ) 2 SO 4 precipitation plus subsequent removal of this salt did not change this behaviour (Fig. S3, trace b), although a slight decrease of all activities was observed. Again, this experiment did not confirm the reports of Yamamoto et al. [7,8] that (NH 4 ) 2 SO 4 precipitation plus dialysis results in removal of a Naga6 inhibitor. ...
With the substrate DNP-α-GalNAc (2,4-dinitrophenyl-N-acetyl-α-d-galactosaminide) three α-N-acetylgalactosaminidase-like activities could be distinguished in serum, in addition to the classical lysosomal enzyme (Naga, EC 22.214.171.124, pH optimum at 4). Two activities had optima in the pH 5 to 6 region and one peaked around pH 8. Like the Naga activity at pH 4, the activity at pH 8 was detectable under standard assay conditions. However, the two activities in the pH 5 to 6 range were not readily apparent in such assays. They could be unmasked as separate activities only when low serum concentrations were used. Addition of 1% saturated ammonium sulphate to the assay medium stimulated these activities. All activities in the pH 5 to 8 range decreased with increasing serum concentration in the assay, suggesting the presence of endogenous inhibitors. The activities between pH 5 and 6 might be similar to an activity described in 1996, which was considerably elevated in serum of patients with great variety of cancers (N. Yamamoto, V.R. Naraparaju, and S.O. Asbell (1996). Deglycosylation of serum vitamin D3-binding protein leads to immunosuppression in cancer patients. Cancer Res. 56, 2827–2811).
... Evidence also shows changes in the GalNAc components of the extracellular matrixIran J Basic Med Sci, Vol. 20, No. 9, Sep 20172 and extracellular membrane attachment component such as integrins in cancer tissues or cells (16, 17). These findings confirm that the presence of Nagalase in many pathogens such as bacteria, parasites and viruses may be effective in their spreading in the body (15,18,19). One of the most important role of Nagalase has been investigated in macrophage suppression (20). ...
Extracellular matrix (ECM) is composed of many kinds of glycoproteins containing glycosaminoglycans (GAGs) moiety. The research was conducted based on the N-Acetylgalactosamine (GalNAc) degradation of ECM components by α-N-acetylgalactosaminidase (Nagalase) which facilitates migration and invasion of cancer cells. This study aims to investigate the effects of Naga-shRNA downregulation on migration and invasion of cancer cell lines.
Materials and Methods
In this study, MCF-7 cell line (human mammary carcinoma cell line) and A2780 (human ovarian carcinoma cell line) were used. The level of normalized Naga expression and Nagalase protein were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay/western blotting, respectively. Migration and invasion were determined using transwell assays, and statistical analysis was carried out by ANOVA test.
Response to transduction by shRNA compared to the control group, migrative and invasive properties of the transfected cells were significantly inhibited.
These results indicate that Nagalase may have an important role in migration and invasion of cancer cells and can be considered as a candidate for further studies.
... Another point that requires reinterpretation is the role of nagalase in autism; thus, it is difficult to envisage the source of nagalase in those patients since autism is not associated with viruses known to produce the enzyme such as influenza virus (Yamamoto and Urade, 2005) or HIV-1 (Yamamoto, 2006). If, however, we consider that autism is associated with widespread inflammation and with higher concentration of proinflammatory cytokines (Masi et al., 2015), then it can be speculated that nagalase, being a lysosomal enzyme (Suh et al., 2015), may be interpreted as a marker of chronic inflammation rather than a marker of immunodeficiency. ...
In this Editorial I reinterpret the results observed with the Gc protein-derived Macrophage Activating Factor (GcMAF) at the light of two recent papers published on this topic. According to the results and the hypothesis emerging from these papers, the biological and clinical effects thus far attributed to GcMAF are indeed to be ascribed to a glycosaminoglycan, chondroitin sulfate, that binds both the precursor and the active form of GcMAF. Such an interpretation has the advantage of solving all the contradictions and inconsistencies that have recently characterized the field of GcMAF-based immunotherapy. This novel interpretation is particularly apt at explaining the results observed in vitro and in vivo concerning the administration of GcMAF to autistic subjects.
... Indeed, it is possible that GcMAF therapy may be effective against all cancers. Yamamoto and his collaborators have also shown that both the HIV  and influenza  virions contain Nagalase and that patients infected with these virions exhibit elevated Nagalase activity. Yamamoto also found that GcMAF therapy (100 ng once a week) was effective in reducing the elevated Nagalase activity in three HIV-infected patients with acquired immunodeficiency syndrome (AIDS) to the level of healthy controls ; the eradication of HIV and HIV-infected cells by GcMAF therapy was confirmed by the complete clearance of viral antigens (p24 and gp120) in the patients' blood. ...
The series of ordered mutations that cause a specific cell to become cancerous is modeled so that the fraction of a risk population (e.g. White men) that has developed a specific cancer (e.g. melanoma) at any age can be calculated. The saturated model constructed and solved here is isomorphic to the physical model describing an ordered chain of radioactive nuclei decays with the exception that it allows for the possibility that a fraction of a risk population may be immune to developing a specific cancer.
... There is abundant evidence in favor of the presence and role of the nagalase in different pathogens. The pathogenesis of some viruses such as Influenza , HIV  or Herpes Simplex Virus-HSV  is attributed to nagalase. There is evidence of this enzyme playing a crucial role in various bacteria, especially in Intestinal Microbiota such as Bifidobacterium spp. ...
In the constant battle against cancer cells, macrophages are of great importance. Their activation is achieved through various mechanisms such as Vitamin D binding protein (VDBP or Gc). After undergoing modifications via enzymes secreted by stimulated lymphocytes, VDBP is modified into Macrophages Activator Form/Factor (Gc-MAF). Some studies (particularly those focusing on cancer) have reported that an enzyme known as α-N-acetylgalactosaminidase (nagalase) facilitates the deglycosylation of Gc-MAF, which in turn inhibits the activation of macrophages. The aim of this review was to evaluate studies associated with nagalase and its escalation in various diseases and to propose hypothetical solutions in order to neutralize the effects of nagalase in cancer patients.
... It was found that the deficiency of lysosomal alpha-NaGalase in the human body causes a dangerous hereditary Schindler/Kanzaki disease, which stimulated an intensive, comprehensive study of this enzyme . alpha-NaGalase activity is an enzymatic basis for the fusion process and play dual roles in viral infectivity of influenza and human immunodeficiency Type I virus, as well as in immunosuppression [26,27]. ...
α-N-acetylgalactosaminidase (EC 126.96.36.199) (alpha-NaGalase) catalyzes the hydrolysis of N-acetamido-2-deoxy-α-d-galactoside residues from non-reducing ends of various complex carbohydrates and glycoconjugates. It is known that human cancer cells express an alpha-NaGalase, which accumulates in the blood plasma of patients. The enzyme deglycosylates the Gc protein-derived macrophage activating factor (GcMAF) and inhibits macrophage activity acting as an immunosuppressor. The high specific activity 0.033 ± 0.002 μmol mg−1 min−1 of the enzyme was found in human colon carcinoma cells DLD-1. The alpha-NaGalase of DLD-1 cells was isolated and biochemical characterized. The enzyme exhibits maximum activity at pH 5.2 and temperature 55 °C. The Km is 2.15 mM, Vmax–0.021 μmol min−1 mL−1, kcat–1.55 min−1 and kcat/Km–0.72 min−1 mM−1 at 37 °C, pH 5.2. The effects of fucoidan from the brown alga Fucus evanescence on the activity of alpha-NaGalase in human colon carcinoma DLD-1 cells and on the biosynthesis of this enzyme were investigated. It was shown that fucoidan did not inhibit free alpha-NaGalase, however, it reduced the expression of the enzyme in the DLD-1 cells at IC50 73 ± 4 μg mL−1.
... If an effective co-receptor blocker could be discovered, then administered as a prophylactic to uninfected people engaged in high-risk behavior, it would prevent infection, and administered to those already infected with HIV would prevent the transition to AIDS. However, recent experiments by Nobuto Yamamoto and his research team on HIV-infected patients using an enzymatically modified serum vitamin D 3 -binding protein called Gc macrophage activating factor (GcMAF) stimulated macrophage action against HIV and eradicated all traces of viral antigens in the patients' blood for 2 years after the completion of therapy . Thus, hope of finding a way to strengthen the immune response against HIV infection so that the fatal nature of the disease is eliminated should not be abandoned. ...
Novel dynamical models are introduced demonstrating that the T helper cell (THC) density drops in the acute infection phase of HIV infection, sometimes causing transient AIDS, and at the end of the incubation period causing chronic AIDS have a common dynamical cause. The immune system's inability to produce enough uninfected THCs to replace the infected ones it is destroying causes a drop in the THC density at any stage of HIV infection. Increases in viral infectivity, probably caused by random mutation of HIV, are shown to drive the progression of the infection. The minimum incubation period for the long term non-progressors (LTNPs) was calculated from a novel physical model: 0.3% of infecteds have incubation periods of 23.1 years or more, and there is no biomedical difference between LTNPs and progressors. Chronic AIDS is shown to result from three random transitions linking four clinically-distinct stages of HIV infection following seroconversion.
... Alpha-Nacetylgalactominidase a.k.a. Nagalase is a matrix-degrading enzyme secreted by cancer cells during tumor invasion and a component of the envelope protein of several virus, such as HIV and HSV-1 and 2 . Nagalase deglycosylates the vitamin D3-binding protein DBP (also known as Gc-protein). Gc-protein contains three sugars and is the precursor for the major macrophage-activating factor (MAF). ...
Therapeutic potentials of immunotherapy in cancer treatment and a discussion of a breast cancer case managed with complementary immunotherapy
... Serum alpha-N-acetylgalactosaminidase (nagalase) and C-reactive Protein (CRP) are two markers of chronic conditions that have been used to monitor a variety of diseases as well as to predict successful aging and healthy life expectancy. Nagalase, an enzyme, was first proposed as a marker for cancer and viral infections and its increased serum activity in these conditions was associated with immune system deficiency since nagalase prevents the formation of Gc protein-derived Macrophage Activating Factor (GcMAF), an immune stimulant cytokine (Yamamoto and Naraparaju, 1997;Yamamoto and Urade, 2005;Yamamoto, 2006). This occurs because nagalase breaks the bond between alpha-Nacetylgalactosamine and the amino acid threonine situated in the third domain of Gc protein (the precursor of GcMAF), thus effectively removing alpha-Nacetylgalactosamine that is considered the active site of GcMAF. ...
... Since macrophage activation for phagocytosis and subsequent antigen presentation to B and T cells is the first indispensable step for humoral and cellular immunity development, 28,29,35,39,40 lack of macrophage activation leads to immunosuppression. Thus, the MAF precursor activity of patient serum Gc protein was first to be examined. ...
Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of breast cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Patient serum Nagalase activity is proportional to tumor burden. The deglycosylated Gc protein cannot be converted to MAF, resulting in no macrophage activation and immunosuppression. Stepwise incubation of purified Gc protein with immobilized beta-galactosidase and sialidase generated probably the most potent macrophage activating factor (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages treated in vitro with GcMAF (100 pg/ml) are highly tumoricidal to mammary adenocarcinomas. Efficacy of GcMAF for treatment of metastatic breast cancer was investigated with 16 nonanemic patients who received weekly administration of GcMAF (100 ng). As GcMAF therapy progresses, the MAF precursor activity of patient Gc protein increased with a concomitant decrease in serum Nagalase. Because of proportionality of serum Nagalase activity to tumor burden, the time course progress of GcMAF therapy was assessed by serum Nagalase activity as a prognostic index. These patients had the initial Nagalase activities ranging from 2.32 to 6.28 nmole/min/mg protein. After about 16-22 administrations (approximately 3.5-5 months) of GcMAF, these patients had insignificantly low serum enzyme levels equivalent to healthy control enzyme levels, ranging from 0.38 to 0.63 nmole/min/mg protein, indicating eradication of the tumors. This therapeutic procedure resulted in no recurrence for more than 4 years.
... This glycosidase helps in the degradation of mucin carbohydrates and removes terminal Alpha-N-acetylgalactosamine residues from glycolipids and glycoproteins (69). When NAGA deglycosylates Gc protein (serum vitamin D3-binding protein) its conversion to the precursor of a principal macrophage-activating factor, MAF is not possible, and immunosuppression will be the net result (70). The underexpression of Naga in Atlantic salmon could be suggesting that probiotic feeding may not cause immunosuppression. ...
The efficacy of a microbial feed additive (Bactocell(®)) in countering intestinal inflammation in Atlantic salmon was examined in this study. Fish were fed either the additive-coated feed (probiotic) or feed without it (control). After an initial 3-week feeding, an inflammatory condition was induced by anally intubating all the fish with oxazolone. The fish were offered the feeds for 3 more weeks. Distal intestine from the groups was obtained at 4 h, 24 h, and 3 weeks, after oxazolone treatment. Inflammatory responses were prominent in both groups at 24 h, documented by changes in intestinal micromorphology, expression of inflammation-related genes, and intestinal proteome. The control group was characterized by edema, widening of intestinal villi and lamina propria, infiltration of granulocytes and lymphocytes, and higher expression of genes related to inflammatory responses, mul1b, il1b, tnfa, ifng, compared to the probiotic group or other time points of the control group. Further, the protein expression in the probiotic group at 24 h after inducing inflammation revealed five differentially regulated proteins - Calr, Psma5, Trp1, Ctsb, and Naga. At 3 weeks after intubation, the inflammatory responses subsided in the probiotic group. The findings provide evidence that the microbial additive contributes to intestinal homeostasis in Atlantic salmon.
While the development of combined active antiretroviral therapy (cART) has dramatically improved life expectancies and quality of life in HIV-infected individuals, long-term clinical problems, such as metabolic complications, remain important constraints of life-long cART. Complete immune restoration using only cART is normally unattainable even in cases of sufficient plasma viral suppression. The need for immunologic adjuncts that complement cART remains, because while cART alone may result in the complete recovery of peripheral net CD4+ T lymphocytes, it may not affect the reservoir of HIV-infected cells. Here, we review current immunotherapies for HIV infection, with a particular emphasis on recent advances in cytokine therapies, therapeutic immunization, monoclonal antibodies, immune-modulating drugs, nanotechnology-based approaches and radioimmunotherapy.
Acquired immune deficiency syndrome (AIDS) is characterized by opportunistic infections and by 'opportunistic neoplasms' (for example, Kaposi's sarcoma). Persistent generalized lymphadenopathy (PGL) is epidemiologically associated with AIDS, especially in male homosexuals. A subset of T lymphocytes positive for the CD4 antigen (also termed T4 antigen), is depleted in AIDS and PGL patients. A retrovirus found in T-cell cultures from these patients is strongly implicated in the aetiology of AIDS because of the high frequency of isolation and the prevalence of specific antibodies in the patients. Here we have detected cell-surface receptors for the AIDS retrovirus (human T-cell leukaemia virus-III (HTLV-III) and lymphadenopathy-associated virus-1 (LAV-1) isolates) by testing the susceptibility of cells to infection with pseudotypes of vesicular stomatitis virus bearing retroviral envelope antigens, and by the formation of multinucleated syncytia on mixing virus-producing cells with receptor-bearing cells. Receptors were present only on cells expressing CD4 antigen; among 155 monoclonal antibodies tested, each of the 14 anti-CD4 antibodies inhibited formation of syncytia and blocked pseudotypes. Productive infection of CD4+ cells with HTLV-III or LAV-1 markedly reduced cell-surface expression of CD4. In contrast, receptors for HTLV-I and HTLV-II were not restricted to CD4+ cells, were not blocked by anti-CD4 antibodies; cells productively infected with HTLV-I and HTLV-II expressed surface CD4. Hence, we conclude that the CD4 antigen is an essential and specific component of the receptor for the causative agent of AIDS.
A brief (30 min) treatment of mouse peritoneal cells (mixture of nonadherent lymphocytes and adherent macrophages) with 1-20 micrograms of lysophosphatidylcholine (lyso-PC) per ml in serum-supplemented RPMI medium 1640, followed by a 3-hr cultivation of the adherent cells alone, results in a greatly enhanced Fc receptor-mediated phagocytic activity of macrophages. This rapid process of macrophage activation was found to require a serum factor, the vitamin D3 binding protein (the human protein is known as group-specific component; Gc). Efficient activation of macrophages was achieved by using medium containing purified human Gc protein. Analysis of intercellular signal transmission among nonadherent (B and T) cells revealed that lyso-PC-treated B cells modify Gc protein to yield a proactivating factor, which can be converted by T cells to the macrophage-activating factor. This rapid generation process of the macrophage-activating factor was also demonstrated by stepwise incubation of Gc protein with lyso-PC-treated B-cell ghosts and untreated T-cell ghosts, suggesting that Gc protein is modified by preexisting membranous enzymes to yield the macrophage-activating factor. Incubation of Gc protein with a mixture of beta-galactosidase and sialidase efficiently generated the macrophage-activating factor. Stepwise incubation of Gc protein with B- or T-cell ghosts and sialidase or beta-galactosidase revealed that Gc protein is modified by beta-galactosidase of B cells and sialidase of T cells to yield the macrophage-activating factor. Administration to mice of a minute amount (4-10 pg per mouse) of in vitro, enzymatically generated macrophage-activating factor resulted in a greatly enhanced (3- to 7-fold) ingestion activity of macrophages.
Infection with human immunodeficiency virus-type 1 (HIV-1) depletes T cells expressing CD4 and B cells expressing immunoglobulin (Ig) VH3 gene products. A subpopulation of normal B cells from non-HIV-infected individuals was shown to bind to HIV gp120 by means of membrane Ig; most of these B cells expressed VH3 family Ig. Serum VH3 IgM from uninfected individuals also avidly bound gp120. Finally, gp120 selectively induced Ig secretion by VH3 B cells, indicating that the binding of gp120 functionally activated these cells. These results indicate that naturally occurring VH3 Ig is a second ligand for gp120 and a candidate superantigen for VH3 B cells.
Vitamin D3-binding protein (Gc protein), a serum glycoprotein, is the precursor for the macrophage activating factor. Cancer patient sera contain alpha-N-acetylgalactosaminidase that deglycosylates Gc protein. Deglycosylated Gc protein cannot be converted to macrophage activating factor, leading to immunosuppression. Of 46 oral cancer patients with squamous cell carcinoma, approximately 22% had greatly reduced precursor activities. The precursor activity of approximately 61% of these patients was moderately reduced. The remaining patients (17%) had precursor activities equivalent to those of healthy humans. Patients with low precursor activity of serum Gc protein had high serum alpha-N-acetylgalactosaminidase activity. In contrast, patients with high precursor activity had low serum alpha-N-acetylgalactosaminidase activity. Thus, levels of serum alpha-N-acetylgalactosaminidase of individual patients have an inverse correlation with precursor activities of their serum Gc protein. Surgical removal of tumors resulted in a subtle decrease in serum alpha-N-acetylgalactosaminidase activity with concomitant increase in the precursor activity of serum Gc protein. Serum enzyme analysis of nude mice transplanted with a human oral squamous carcinoma cell line revealed that serum alpha-N-acetylgalactosaminidase activity is directly proportional to tumor burden. Thus, alpha-N-acetylgalactosaminidase activity in patient bloodstream can serve as a diagnostic/prognostic index.
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Injection of Gross's passage A leukemia virus in newborn C3Hf/Bi mice interferes with the development of their responsiveness to bacteriophage T2. This deficiency was present in mice antigenically stimulated at 3 weeks and 9 weeks of age, and, notably, before obvious malignant cells are present.
Persistent generalized lymphadenopathy (PGL) is observed predominantly in subjects at risk of developing AIDS. Twenty-seven individuals belonging to such groups: twelve homosexual males and fifteen intravenous drug users, were investigated for immunological abnormalities with particular attention to monocyte functions. They were compared with five AIDS patients. Twenty out of twenty-two individuals had anti-LAV/HTLV-III antibodies and most had abnormalities characteristic of AIDS: polyclonal hypergammaglobulinemia, decreased cell-mediated immunity, inverted T-cell helper/suppressor ratio and histological alterations of lymph nodes. As for peripheral blood monocyte functions, phagocytic capacity and production of O2- were normal and bactericidal capacity was decreased. Monocytes cultured in the presence of concanavalin A produced less PGE2 and more IL-1/MCF than normal monocytes. Similar abnormalities were found using monocytes from AIDS patients. These data suggest that (i) monocytes from patients with PGL have functional alterations that may be either intrinsic or secondary to lymphocyte dysfunction(s); (ii) these alterations do not account for the decreased capacity of lymphocytes to respond to mitogens but (iii) may explain the uncontrolled activation of B cells.
Persistently infected cell lines of BHK21/WI-2 cells have been established by infection with the wild type rubella virus strain M-33. These cell lines, BHK-MP1 and BHK-MP2, showed immunity-like resistance to superinfection with M-33 virus at both 34° and 39.5° C. They also showed intrinsic interference with the replication of Newcastle Disease Virus at 34° C but not at 39.5° C. They released a small number of infectious virus particles which were temperature sensitive variants, being able to form plaques at 34° C, but not at 39.5° C on BHK21/WI-2 and on its derivative, BSR.
When BHK-MP1 cells were cultured at 34° C in growth medium containing 10–20 µg/ml of 5-bromodeoxyuridine (BudR) there was a 5- to 10-fold increase in infectious virus in the medium as compared with the untreated controls. Mitomycin C (0.5 µg/ml) treatment for 7 hours likewise stimulated the release of virus from these cells. The enhancement of viral release by BudR was completely blocked by pretreatment with actinomycin D (5 µg/ml) for 3 hours prior to BudR treatment. Since the variant can be induced by these prophage inducers and inhibited by actinomycin D it is suggested that the viral genome is converted to a DNA provirus which is analogous to the lysogenic state of bacteriophage.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Our objective was to provide information on survival and disease progression in human immunodeficiency virus antibody-positive pregnant women undergoing prospective evaluation.
After an index delivery, 103 human immunodeficiency virus antibody-positive pregnant women were identified and underwent follow-up for 3 years. The patients were assessed medically and/or gynecologically when hospitalized for a human immunodeficiency virus-related illness or at each follow-up visit. The life-table method was used to estimate the cumulative probabilities of survival and remaining free of acquired immunodeficiency syndrome. Cox's proportional-hazards analyses were used to identify prognostic factors for survival and progression to acquired immunodeficiency syndrome.
The majority of human immunodeficiency virus-infected pregnant women were alive 3 years later. Lymphadenopathy syndrome or herpes genitalis was significantly associated with a subsequent diagnosis of acquired immunodeficiency syndrome. Of the 103 original patients, six had acquired immunodeficiency syndrome at the index delivery and acquired immunodeficiency syndrome developed in 24. Approximately 94% of evaluable patients with development of acquired immunodeficiency syndrome had CD4-lymphocyte counts < 200/mm3. The most common opportunistic infection was Pneumocystis carinii pneumonia. Acquired immunodeficiency syndrome and postpartum zidovudine therapy were independent prognostic factors affecting survival.
Survival was affected by Centers for Disease Control group status of human immunodeficiency virus infection at the index delivery.
Pneumocystis carinii pneumonia (PCP) was reported to be the predominant cause of human immunodeficiency virus (HIV)-related deaths prior to 1988, the year that effective prophylaxis against PCP entered routine use. Our study was performed to study the causes of HIV-related death since January 1988 in a region where patient tracking is virtually complete.
We surveyed physicians associated with the Brown University Acquired Immunodeficiency Syndrome (AIDS) Program who cared for greater than 95% of known HIV-positive patients in Rhode Island. These physicians identified all those HIV-infected persons who had died under their care between January 1988 and July 1990, and determined these patients' causes of death by chart review. For comparison, death certificates of identified persons were also reviewed at the Rhode Island Department of Vital Statistics.
Among 126 deaths since January 1988, bacterial infections were the most common cause of death (30%), whereas PCP was responsible for only 16% of deaths. Persons not receiving any form of PCP prophylaxis were more likely to die from PCP than were those who received prophylaxis (26% versus 11% [p = 0.04]). Cause of death as recorded on actual death certificates was imprecise, although bacterial infections were again the most common cause indicated. Only one death occurred in a patient with a CD4 count greater than 200/mL, and this was not HIV-related.
PCP has not been the leading cause of death in our region since January 1988. Bacterial infections contribute substantially to mortality, and this may influence future prophylactic regimens. HIV-related deaths in patients with CD4 counts greater than 200/mL are unusual.
In the present study the microbicidal activities of granulocytes and monocytes from AIDS patients (CDC group IV) were assessed and compared with those of healthy controls. The phagocytosis and intracellular killing of Staphylococcus aureus by patient and control cells were measured using a method in which the rate of intracellular killing can be assessed independently of the rate of phagocytosis. Both granulocytes and monocytes of AIDS patients showed a decreased phagocytosis of S. aureus in comparison to phagocytes of healthy individuals. The rates of intracellular killing of S. aureus by granulocytes and monocytes did not differ significantly between these patients with late-stage HIV infection and controls.
Oxidative burst responses of monocytes and monocyte-derived macrophages (MDM) were studied in 40 subjects with HIV infection of different clinical stages. Oxidative burst was assessed as reduction of nitroblue tetrazolium (NBT) with or without stimulants. Results were determined as oxidative burst responses per cell and as a stimulatory ratio between stimulated and unstimulated NBT reduction. Cells from 12 HIV-seronegative homosexual men and 38 blood donors served as control groups. In patients with asymptomatic HIV infection, monocyte oxidative burst responses were reduced compared with the blood donors. In MDM from the same patients, stimulatory ratios were reduced. In AIDS patients, stimulatory ratios of both monocytes and MDM were reduced compared with controls. In contrast to the progressive deterioration of CD4+ lymphocyte counts as well as other immune functions in HIV infection, monocyte oxidative burst responses are impaired already in the asymptomatic phase of the infection, almost to the same extent as in patients with AIDS.
In vitro treatment of mouse peritoneal cells (mixture of adherent and nonadherent cells) with lysophosphatidylcholine (lyso-Pc) in 10% FCS supplemented medium RPMI 1640 results in a greatly enhanced FcR-mediated phagocytic activity of macrophages. This macrophage-activation process requires a serum factor. Fractionation studies with starch block electrophoresis of fetal calf and human sera revealed that alpha 2-globulin fraction contains a serum factor essential for macrophage activation. To identify the serum factor, human serum was precipitated with 50% saturated ammonium sulfate and fractionated on a Sephadex G-100 column. A protein fraction with a lower m.w. than albumin had the capacity to support activation of macrophages. The active serum factor in this protein fraction was analyzed by immunoabsorption by using rabbit antisera against three major proteins of human alpha 2-globulin. This active serum factor was shown to be a vitamin D3-binding protein (group specific component, Gc). By using a monoclonal anti-Gc-absorbed active column fraction of human serum, we observed no enhanced macrophage activation over the results with serum fraction-free cultivation of lyso-Pc-treated peritoneal cells. Cultivation of lyso-Pc-treated peritoneal cells in a medium containing a low concentration of purified human Gc protein (0.1 to 2.6 ng/ml) produced a greatly enhanced phagocytic activity of macrophages. When purified human Gc protein was used in a serum-free medium for stepwise cultivation of lyso-Pc-treated nonadherent cell types, a macrophage-activating factor was efficiently generated. Therefore, it is concluded that the vitamin D3-binding protein is the essential serum factor for the lyso-Pc-primed activation of macrophages.
Alkylglycerols, inflammation products of cancerous membrane lipids, efficiently activate macrophages. A brief in vitro treatment (30 min) of peritoneal cells (mixture of non-adherent and adherent cells) with a small amount (50 ng/ml) of synthetic dodecylglycerol (DDG) resulted in greatly enhanced Fc-receptor-mediated ingestion activity of macrophages. However, treatment of adherent cells (macrophages) alone with DDG produced no significant enhancement of macrophage ingestion activity, implying that macrophage activation requires a contribution of non-adherent cells. DDG-treated non-adherent cells were found to generate a macrophage-activating signal factor. Studies with a serum free-0.1% egg albumin-supplemented RPMI 1640 medium revealed that a serum factor is essential for macrophage activation process. Time course analysis of stepwise transfers of conditioned media of DDG-treated or untreated B cells and T cells revealed that DDG-treated B cells rapidly transmit a factor to untreated T cells which yield the ultimate macrophage-activating factor. This signal transmission among these cells for the macrophage activation process is too rapid to allow time for synthesis of inducible gene products. Thus, we hypothesized that a serum factor is modified by the pre-existing function of DDG-treated B cells and further modified by the pre-existing function of untreated T cells to yield macrophage-activating factor. This hypothesis was confirmed by the demonstration that DDG-treated splenic non-adherent cell ghosts modify a serum factor to yield macrophage-activating factor.
To perform a human immunodeficiency virus (HIV) plaque assay in nonadherent host cells, we developed a novel technique in which HIV-infected MT-2 cells were formed into monolayers by centrifugation through molten agarose. Infection, formation of cell monolayers, and enumeration of plaques all took place in 96-well microtiter plates. When this process was preceded by 18 h of incubation of HIV with patient serum samples, neutralizing antibody titers between 1:10 and 1:5,000 could be accurately determined in patient serum samples. In addition to the determination of neutralizing antibody titers (with the use of various serum dilutions and a constant virus concentration), neutralization indices could also be determined with different virus dilutions and a single dilution of patient serum.
The envelope protein of human immunodeficiency virus (HIV) is synthesized as a polyprotein (gp160) and cleaved intracellularly to a gp120-gp41 heterodimer. In this study, the tryptic-like endoproteolytic cleavage site was removed by site-directed mutagenesis and replaced with a chymotryptic-like site. The resultant mutant, RIP7/mut10, was found to be indistinguishable from wild-type HIV when analyzed at the level of proviral replication, RNA processing, protein expression, and viral assembly. However, the gp160 polyprotein was not cleaved and the mutated virions were biologically inactive, until and unless they were exposed to limiting concentrations of chymotrypsin. As is the case for other enveloped mammalian viruses, endoproteolytic cleavage of the HIV envelope protein and release of a unique hydrophobic domain appear to be necessary for the full expression of viral infectivity.
Human immunodeficiency virus type 1 (HIV-1) selectively infects cells expressing the CD4 molecule, resulting in substantial quantitative and qualitative defects in CD4+ T lymphocyte function in patients with acquired immunodeficiency syndrome (AIDS). However, only a very small number of cells in the peripheral blood of HIV-1-infected individuals are expressing virus at any given time. Previous studies have demonstrated that in vitro infection of CD4+ T cells with HIV-1 results in downregulation of CD4 expression such that CD4 protein is no longer detectable on the surface of the infected cells. In the present study, highly purified subpopulations of peripheral blood mononuclear cells (PBMCs) from AIDS patients were obtained and purified by fluorescence-automated cell sorting. They were examined with the methodologies of virus isolation by limiting dilution analysis, in situ hybridization, immunofluorescence, and gene amplification. Within PBMCs, HIV-1 was expressed in vivo predominantly in the T cell subpopulation which, in contrast to the in vitro observations, continued to express CD4. The precursor frequency of these HIV-1-expressing cells was about 1/1000 CD4+ T cells. The CD4+ T cell population contained HIV-1 DNA in all HIV-1-infected individuals studied and the frequency in AIDS patients was at least 1/100 cells. This high level of infection may be the primary cause for the progressive decline in number and function of CD4+ T cells in patients with AIDS.
The envelope glycoproteins of the human immunodeficiency virus (HIV) type 1 are synthesized as a precursor molecule, gp160, which is cleaved to generate the two mature envelope glycoproteins, gp120 and gp41. The cleavage reaction, which is mediated by a host protease, occurs at a sequence highly conserved in retroviral envelope glycoprotein precursors. We have investigated the sequence requirements for this cleavage reaction by introducing four single-amino-acid changes into the glutamic acid-lysine-arginine sequence immediately amino terminal to the site of cleavage. We have also examined the effects of these mutations on the syncytium formation induced by HIV envelope glycoproteins. Our results indicate that a glutamic acid to glycine change at gp120 amino acid 516, a lysine to isoleucine change at amino acid 517, and an arginine to lysine change at amino acid 518 affect neither gp160 cleavage nor syncytium formation. The results obtained with the arginine to lysine change at amino acid 518 differ significantly from the results obtained with the same mutation at the envelope precursor cleavage site of a murine leukemia virus (E. O. Freed, and R. Risser, J. Virol. 61:2852-2856, 1987). An arginine to threonine mutation at gp120 amino acid 518, the terminal residue of gp120, abolishes both gp160 cleavage and syncytium formation. These findings demonstrate that despite its highly conserved nature, the basic pair of amino acids at the site of gp160 cleavage is not absolutely required for proper envelope glycoprotein processing. This report also supports the idea that cleavage of gp160 is required for activation of the HIV envelope fusion function.
Since the acquired immunodeficiency syndrome (AIDS) is characterized by opportunistic infections and malignancies indicative of a profound suppression in cell-mediated immunity, we investigated the antibody-dependent cell-mediated cytotoxicity (ADCC) of peripheral blood mononuclear cells of patients with AIDS against chicken red blood cells (CRBC). A marked decrease in ADCC-CRBC activity was observed from patients with AIDS as compared to healthy controls. Furthermore, suppression in ADCC activity was seen when mononuclear cells from healthy subjects were assayed using media containing 25% or 40% sera from AIDS patients. Two of two patients with AIDS and impaired ADCC-CRBC activity were also found to have in vivo impaired reticuloendothelial system Fc-specific clearance of 51Cr-labelled, anti-Rho (D) IgG-sensitized autologous erythrocytes. These data provide further evidence of monocyte-macrophage dysfunction in AIDS and help explain the widespread occurrence of opportunistic pathogens in AIDS.
Cellular damage and inflammatory processes cause activation of phospholipase A in plasma membranes resulting in the production of various lysophospholipids. Treatment of mice with L-alpha-lysophosphatidylcholine, a decomposition product of phosphatidylcholine, greatly stimulates mouse peritoneal macrophages to ingest target cells via the Fc receptors. Similarly, treatment of mice with L-alpha-lysophosphatidylethanolamine and L-alpha-lysophosphatidyl-L-serine resulted in an enhanced ingestion activity of macrophages. Cancer cell membranes contain alkyl ether derivatives of phospholipids and neutral lipids. Inflamed cancer cells release decomposition products of alkyl ether phospholipids and neutral lipids, alkyl-lysophospholipids and alkylglycerols, respectively. Administration of alkyl ether analogues of lysophospholipids into mice were able to induce stimulation of macrophages for ingestion with Fc receptor preference. Two synthetic alkylglycerols, dodecylglycerol and tridecylglycerol, were tested. Dodecylglycerol induced an efficient stimulation of macrophages for Fc-mediated ingestion whereas tridecylglycerol induced a minimal level of activation. Therefore, in vivo effect of dodecylglycerol on macrophage stimulation is similar to that of lysophospholipids and their alkyl analogues. These in vivo stimulations of macrophages for Fc receptor-mediated ingestion activity were reproduced in in vitro activation of macrophages by treatment of peritoneal cells with the alkyl lipid derivatives. Among these compounds, dodecylglycerol was found to be the most potent agent for macrophage stimulation. Since macrophages are antigen-presenting cells, the degradation products of cancer cell membrane lipids may have immune potentiating capacity.
We studied the functions of peripheral blood monocytes and polymorphonuclear cells in 15 apparently healthy homosexual men, eight homosexual or bisexual subjects with unexplained generalized lymphadenopathies (pre-AIDS), four homosexual men with acquired immunodeficiency syndrome (AIDS), and 15 heterosexual men. In comparison with normal controls, the homosexual groups studied presented a decreased monocyte candidacidal activity for Candida pseudotropicalis that gradually deteriorates as the clinical symptoms progress towards AIDS. The monocyte phagocytic function was retained. Although the phagocytic and candidacidal activities of the polymorphonuclear cells did not differ from those of the normal controls, the candidacidal activity in some of the cases studied was unusually enhanced, indicating that the cells were in an activated state. In addition, only two of nine sera tested from asymptomatic homosexual males were positive for antibodies to HTLV-III/LAV, while six out of eight pre-AIDS and both of the two AIDS patients tested had antibodies to AIDS-associated retrovirus. We suggest that in AIDS the phagocytic system is already involved, together with B and T lymphocyte abnormalities, during the early events of the syndrome, even without the detection of AIDS-associated retrovirus antibodies.
CD4 functions as the cell-surface receptor for human immunodeficiency virus (HIV); however, the mechanism of virus entry into susceptible cells is unknown. To explore this question we used a human T lymphoblastic cell line (VB) expressing high levels of surface CD4. Neutralization of endosomal compartments (pH greater than 6.4) with lysosomotropic agents did not effectively inhibit HIV nucleocapsid entry into the cytoplasm, and virus treated at low pH (5.5) failed to induce rapid cell-to-cell fusion in uninfected cells. Electron microscopy of VB cells acutely exposed to HIV at neutral pH revealed direct fusion of the virus envelope with the plasma membrane within minutes at 4 degrees C. No endocytosed virions were visualized upon rewarming the HIV-exposed cells to 37 degrees C for as long as 60 min. These results indicate that HIV penetrates CD4-positive T cells via pH-independent membrane fusion.
The acquired immunodeficiency syndrome (AIDS) is an immunoregulatory disorder characterized by the presence of Kaposi's sarcoma or opportunistic infections suggestive of an underlying cellular immunodeficiency. Other documented immune abnormalities occurring with AIDS include polyclonal hypergammaglobulinemia, circulating immune complexes, and abnormal B cell function with decreased proliferative response to T cell-independent mitogens of B cells.
The acquired immunodeficiency syndrome (AIDS) is a novel, epidemic form of immunodeficiency that has been widely recognized within the past three years. Full-blown AIDS, as defined clinically by the Centers for Disease Control surveillance definition, is characterized by opportunistic infections and malignant diseases in patients without a known cause for immunodeficiency.
The immunologic abnormalities in AIDS are on the one hand selective and distinctive and on the other hand broad and heterogeneous. The common denominator of the immune defects in AIDS patients is a quantitative and qualitative defect in the T4 inducer/helper subset of T cells. However, AIDS patients also manifest a remarkable abnormality of B cell function characterized by a polyclonal activation of B cells with spontaneous secretion of Ig and increased spontaneous proliferation. This likely explains the hypergammaglobulinemia that is consistently seen in AIDS patients. Furthermore, the spontaneous polyclonal activation of the B cells is accompanied by a refractoriness to be novo stimuli rendering them defective in their ability to mount a humoral response to a new antigen. AIDS patients also manifest defects in T cell and NK cell-mediated cytotoxicity, both of which are at least partially corrcted by IL-2 in vitro. Finally, monocyte funection is also abnormal in AIDS. The recent demonstration that the T4 lymphocytotropic retrovirus HTLV-III is likely the underlying and primary etiologic agent in AIDS is entirely compatible with the immunologic defects observed in this syndrome.
97 symptom-free homosexuals were studied clinically, serologically, and with in-vivo and in-vitro tests of cellular immune function in the context of the acquired immunodeficiency syndrome (AIDS). A high proportion of these men showed abnormalities: lymphopenia (33%), decreased T-helper/T-suppressor (Th/Ts) cell ratios (43%), both these abnormalities (12%), decreased total T-helper cells (15%), monocyte chemotactic (10%) and phagocytic (27%) defects, anergy to three recall antigens (32%), and anergy to purified protein derivative despite BCG inoculation (55%). The lymphocyte abnormalities and anergy characteristic of AIDS were seen in 5%. No clear clinical or serological associations were seen for the AIDS-like defects. Trends of association were seen between higher lymphocyte counts, lower Th/Ts ratios, more T-suppressor cells and serological evidence of previous virus infection. The combination of lymphocyte abnormalities and anergy observed in these symptom-free homosexuals may represent a latent phase of AIDS.
The simultaneous availability of a virus that routinely transforms T cells (HTLV-I) and of a related virus that kills T cells (HTLV-III) might offer a unique opportunity to analyze the mechanisms critical to human cell growth and destruction.
The outer surface of mouse B lymphocytes carries constitutive and inducible beta-galactosidase isozymes. A brief (30 min) treatment of B lymphocytes with lysophosphatidylcholine (lyso-Pc) immediately induced an approximate 3-fold higher beta-galactosidase activity than the constitutive isozyme of untreated B lymphocytes. Thus, the lyso-Pc-inducible isozyme is not a de novo enzyme. Outer surface of mouse T lymphocytes carries constitutive (non-Neu-1) and inducible (Neu-1) sialidase isozymes. The lyso-Pc-inducible beta-galactosidase of B lymphocytes and the Neu-1 sialidase of T lymphocytes were required for conversion of vitamin D3-binding protein (Gc protein) to a potent macrophage activating factor. This enzymatic generation of the macrophage activating factor was mediated via enzyme-associated receptors.
Inflamed lesions release degradation products of membrane lipids, lysophospholipids, and inflamed tumor tissues release alkylglycerols. Macrophages were activated by administration of lysophosphatidylcholine (lyso-Pc) or dodecylglycerol (DDG) to mice. In vitro treatment of mouse peritoneal cells (mixture of nonadherent and adherent cells) with lyso-Pc or DDG in fetal calf serum supplemented medium for 30 min, followed by 3-h cultivation of adherent cells (macrophages) alone, resulted in greatly enhanced Fc-receptor mediated phagocytic activity and superoxide generating capacity of macrophages. The tumor lipid metabolite, DDG, is far more potent (400-fold) than lyso-Pc in terms of doses required for the maximal levels of macrophage activation. The inflammation-primed macrophage activation required a serum factor, vitamin D binding protein, as a precursor for the macrophage activating factor. Treatment of mouse peritoneal cells with 1 microgram lyso-Pc/ml or 50 ng DDG/ml in a serum-free 0.1% egg albumin supplemented medium for 30 min, followed by 3-h cultivation of the treated peritoneal cells in a medium supplemented with a very small amount (0.0005-0.05%) of ammonium sulfate [20-50% saturated (NH4)2SO4] precipitable protein fraction of FCS, resulted in greatly enhanced superoxide generating capacity of macrophages. The ammonium sulfate precipitable fraction was found to contain vitamin D binding protein.
Highly conserved DBP (human DBP is known as Gc) of serum alpha 2-globulin fraction can be converted to a potent macrophage activating factor by stepwise modification of Gc glycoprotein with beta-galactosidase of B cells and sialidase of T cells. These glycosidases, beta-galactosidase and sialidase, are membrane bound and not soluble in culture medium. Thus, consecutive contact of Gc protein with B cells and T cells, presumably via specific receptors, is required for conversion of Gc glycoprotein to the macrophage activating factor. The essential role of T cell sialidase in macrophage activation was confirmed by the finding that peritoneal nonadherent cells of SM/J mouse, whose T cells are deficient in sialidase activity, were unable to convert Gc protein to the macrophage activating factor and thus did not activate macrophages. Treatment with sialidase of a conditioned medium of lipid metabolite-treated SM/J mouse nonadherent cells efficiently generated the macrophage activating factor. When Gc protein was first treated with soluble or immobilized sialidase and used in a medium for 2 h cultivation of lipid metabolite-treated SM/J mouse nonadherent cells or BALB/c mouse B cells, the resultant conditioned media contained a large amount of the macrophage activating factor. These results support the hypothesis that Gc protein carries a dibranched trisaccharide with galactose and sialic acid termini.
The concept that HIV causes AIDS only by directly killing CD4 cells has been questioned by a number of investigators. There has been experimental support for a number of indirect mechanisms such as apoptosis, anergy, superantigen-induced cell proliferation and depletion, defective signaling, molecular mimicry, and autoimmunity. In this article we review the available evidence in support of these theories and suggest that in spite of their apparent differences, signaling by HIV through the T cell receptor could initiate the markedly different responses of activation, anergy, and apoptosis. However, the unifying mechanism as to how this is achieved remains unclear. It is likely that more than one of these mechanisms are involved in CD4 cell depletion during different phases of the disease. Understanding these mechanisms and their role in HIV pathogenesis would be important in new vaccine and therapeutic approaches.
A serum glycoprotein, vitamin D3-binding protein (Gc protein), can be converted by beta-galactosidase of stimulated B lymphocytes and sialidase of T lymphocytes to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is a precursor for MAF. Treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high-titered MAF (GcMAF). When peripheral blood monocytes/macrophages of 46 HIV-infected patients were treated with GcMAF (100 pg/ml), the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of plasma Gc protein was low in 16 (35%) of of these patients. Loss of the MAF precursor activity appeared to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase found in the patient blood stream. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Thus, precursor activity of Gc protein and alpha-N-acetylgalactosaminidase activity in patient blood can serve as diagnostic and prognostic indices.
Serum vitamin D3-binding protein (Gc protein) can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor of the macrophage activating factor (MAF). Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high titered MAF, Gc-MAF. When peripheral blood monocytes/macrophages of 52 patients bearing various types of cancer were incubated with 100 pg/ml of GcMAF, the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of patient plasma Gc protein was found to be severely reduced in about 25% of this patient population. About 45% of the patients had moderately reduced MAF precursor activities. Loss of the precursor activity was found to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase detected in the patient's bloodstream. The source of the enzyme appeared to be cancerous cells. Radiation therapy decreased plasma alpha-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting alpha-N-acetylgalactosaminidase. Both alpha-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient bloodstream can serve as diagnostic and prognostic indices.
Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days.
Vitamin D3-binding protein (DBP; human DBP is known as Gc protein) is the precursor of macrophage activating factor (MAF). Treatment of mouse DBP with immobilized beta-galactosidase or treatment of human Gc protein with immobilized beta-galactosidase and sialidase generated a remarkably potent MAF, termed DBPMAF or GcMAF, respectively. The domain of Gc protein responsible for macrophage activation was cloned and enzymatically converted to the cloned MAF, designated CdMAF. In Ehrlich ascites tumor-bearing mice, tumor-specific serum alpha-N-acetylgalactosaminidase (NaGalase) activity increased linearly with time as the transplanted tumor cells grew in the peritoneal cavity. Therapeutic effects of DBPMAF, GcMAF, and CdMAF on mice bearing Ehrlich ascites tumor were assessed by survival time, the total tumor cell count in the peritoneal cavity, and serum NaGalase activity. Mice that received a single administration of DBPMAF or GcMAF (100 pg/mouse) on the same day after transplantation of tumor (1 x 10(5) cells) showed a mean survival time of 35 +/- 4 days, whereas tumor-bearing controls had a mean survival time of 16 +/- 2 days. When mice received the second DBPMAF or GcMAF administration at day 4, they survived more than 50 days. Mice that received two DBPMAF administrations, at days 4 and 8 after transplantation of 1 x 10(5) tumor cells, survived up to 32 +/- 4 days. At day 4 posttransplantation, the total tumor cell count in the peritoneal cavity was approximately 5 x 10(5) cells. Mice that received two DBPMAF administrations, at days 0 and 4 after transplantation of 5 x 10(5) tumor cells, also survived up to 32 +/- 4 days, while control mice that received the 5 x 10(5) ascites tumor cells only survived for 14 +/- 2 days. Four DBPMAF, GcMAF, or CdMAF administrations to mice transplanted with 5 x 10(5) Ehrlich ascites tumor cells with 4-day intervals showed an extended survival of at least 90 days and an insignificantly low serum NaGalase level between days 30 and 90.
Freund's adjuvant produced severe inflammation that augments development of antibodies. Thus, mixed administration of antigens with adjuvant was not required as long as inflammation was induced in the hosts. Since macrophage activation for phagocytosis and antigen processing is the first step of antibody development, inflammation-primed macrophage activation plays a major role in immune development. Therefore, macrophage activating factor should act as an adjuvant for immunization. The inflammation-primed macrophage activation process is the major macrophage activating cascade that requires participation of serum vitamin D3-binding protein (DBP; human DBP is known as Gc protein) and glycosidases of B and T lymphocytes. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase efficiently generated the most potent macrophage activating factor (designated GcMAF) we have ever encountered. Administration of GcMAF (20 or 100 pg/mouse) resulted in stimulation of the progenitor cells for extensive mitogenesis and activation of macrophages. Administration of GcMAF (100 pg/mouse) along with immunization of mice with sheep red blood cells (SRBC) produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. Thus, GcMAF has a potent adjuvant activity for immunization. Although malignant tumours are poorly immunogenic, 4 days after GcMAF-primed immunization of mice with heat-killed Ehrlich ascites tumour cells, the ascites tumour was no longer transplantable in these mice.
Cancerous cells secrete alpha-N-acetylgalactosaminidase (NaGalase) into the blood stream, resulting in deglycosylation of serum vitamin D3-binding protein (known as Gc protein), which is a precursor for macrophage activating factor (MAF). Incubation of Gc protein with immobilized beta-galactosidase and sialidase generates the most potent macrophage activating factor (designated GcMAF). Administration of GcMAF to cancer-bearing hosts can bypass the inactivated MAF precursor and act directly on macrophages for efficient activation. Therapeutic effects of GcMAF on Ehrlich ascites tumor-bearing mice were assessed by survival time and serum NaGalase activity, because serum NaGalase activity was proportional to tumor burden. A single administration of GcMAF (100 pg/mouse) to eight mice on the same day after transplantation of the tumor (5 x 10(5) cells) showed a mean survival time of 21 +/- 3 days for seven mice, with one mouse surviving more than 60 days, whereas tumor-bearing controls had a mean survival time of 13 +/- 2 days. Six of the eight mice that received two GcMAF administrations, at Day 0 and Day 4 after transplantation, survived up to 31 +/- 4 days whereas, the remaining two mice survived for more than 60 days. Further, six of the eight mice that received three GcMAF administrations with 4-day intervals showed an extended survival of at least 60 days, and serum NaGalase levels were as low as those of control mice throughout the survival period. The cure with subthreshold GcMAF-treatments (administered once or twice) of tumor-bearing mice appeared to be a consequence of sustained macrophage activation by inflammation resulting from the macrophage-mediated tumoricidal process. Therefore, a protracted macrophage activation induced by a few administrations of minute amounts of GcMAF eradicated the murine ascites tumor.
Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The precursor activity of serum Gc protein was reduced in all influenza virus-infected patients. These patient sera contained alpha-N-acetylgalactosaminidase (Nagalase) that deglycosylates Gc protein. Deglycosylated Gc protein cannot be converted to MAF, thus it loses the MAF precursor activity, leading to immunosuppression. An influenza virus stock contained a large amount of Nagalase activity. A sucrose gradient centrifugation analysis of the virus stock showed that the profile of Nagalase activity corresponds to that of hemagglutinating activity. When these gradient fractions were treated with 0.01% trypsin for 30 min, the Nagalase activity of each fraction increased significantly, suggesting that the Nagalase activity resides on an outer envelope protein of the influenza virion and is enhanced by the proteolytic process. After disruption of influenza virions with sodium deoxycholate, fractionation of the envelope proteins with mannose-specific lectin affinity column along with electrophoretic analysis of the Nagalase peak fraction revealed that Nagalase is the intrinsic component of the hemagglutinin (HA). Cloned HA protein exhibited Nagalase activity only if treated with trypsin. Since both fusion capacity and Nagalase activity of HA protein are expressed by proteolytic cleavage, Nagalase activity appears to be an enzymatic basis for the fusion process. Thus, Nagalase plays dual roles in regulating both infectivity and immunosuppression.
Diagnostic and prognostic ELISA assays of serum or plasma ␣-N-acetylgalactosaminidase for cancer
Yamamoto N. Diagnostic and prognostic ELISA assays of serum or plasma ␣-N-acetylgalactosaminidase for cancer. U.S. patent number 5,712,104, 1998.
Cloning of malignant cell-derived serum ␣-N-acetylgalactosa-minidase that plays a role in immunosuppression
Uematsu T, Fukunaga J, Naraparaju VR, and Yamamoto N. Cloning of malignant cell-derived serum ␣-N-acetylgalactosa-minidase that plays a role in immunosuppression. Proc Am Assoc Cancer Res 1999;40:376–377.
Diagnostic and prognostic ELISA assays of serum ␣-N-acetylgalactosaminidase for AIDS
Yamamoto N: Diagnostic and prognostic ELISA assays of serum ␣-N-acetylgalactosaminidase for AIDS. U.S. patent number: 5,985,545, 1999.