Article

Pathogenic significance of α-N-acetylgalactosaminidase activity found in the hemagglutinin of influenza virus

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Abstract

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.

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... The activated macrophages develop a large amount of Fc-receptors [Yamamoto and Homma, 1991; Yamamoto, 1996] as well as an enormous variation of receptors [Yamamoto et al., 2005, 2008a] that recognize a variety of antibody-bound and unbound viral and bacterial antigens and cells with surface abnormality such as cancer cells [Yamamoto et al., 2005, 2008a]. Mouse macrophages, by in vitro treatment with 10–50 pg/ml GcMAF for 3 hr, are maximally activated for phagocytosis [Yamamoto, 1996] and superoxide generation [Naraparaju and Yamamoto, 1994; Yamamoto, 1998a]. ...
... The activated macrophages develop a large amount of Fc-receptors [Yamamoto and Homma, 1991; Yamamoto, 1996] as well as an enormous variation of receptors [Yamamoto et al., 2005, 2008a] that recognize a variety of antibody-bound and unbound viral and bacterial antigens and cells with surface abnormality such as cancer cells [Yamamoto et al., 2005, 2008a]. Mouse macrophages, by in vitro treatment with 10–50 pg/ml GcMAF for 3 hr, are maximally activated for phagocytosis [Yamamoto, 1996] and superoxide generation [Naraparaju and Yamamoto, 1994; Yamamoto, 1998a]. ...
... Envelope hemagglutinating protein HA-1 of influenza virus also carries Nagalase activity which is detectable at a level of 1.5–2.0 nmol/min/mg in patient sera immediately after influenza infection [Yamamoto and Urade, 2005]. Upon infection of HIV the hosts develop flu-like symptoms with serum Nagalase activity similar to the influenza acute state. ...
Article
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.
... 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]. ...
Article
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.
... 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. ...
... 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 [21][22][23]. 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]. ...
... The role of GcMAF as a multifunctional immune modulator and possible implications in It is now well known that DBP Gc-globulin plays a crucial role in immune system regulation as a primary defense against infections [14][15][16][17][18][19][20]. In addition to the storage and transport of active vitamin D3, GcMAF's effects include macrophage modulation, osteoclast activation, facilitation of neutrophil chemotaxis mediated by C5 derived peptide, superoxide activity, scavenging of circulating G-actin, anti-angiogenetic and anti-tumor properties [24][25][26][27][28]. Thus, this multifunctional protein, released into the blood stream, acts as a systemic immune modulator without pro-inflammatory activities. ...
... Yamamoto and his collaborators have also shown that both the HIV [24] and influenza [25] 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 [25]; 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. ...
... Yamamoto and his collaborators have also shown that both the HIV [24] and influenza [25] 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 [25]; 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. ...
Article
Full-text available
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. The simplest model developed here depends on only three independent parameters: the number of ordered mutations necessary for a cell to become cancerous, the fraction of the risk population that is immune to developing a specific cancer and the average time between mutations (a time defined as the mutation lifetime ). The values of these independent parameters are determined by fitting the model's cancer incidence function to the cancer incidence data. This model was applied to five widely different cancers: melanoma, pancreatic cancer, female breast cancer, non-Hodgkin lymphoma and prostate cancer. The modeling predicts that all White males in the USA are vulnerable to developing melanoma, five -ordered mutations are required to develop it and the mutation lifetime is 48.3 years. By contrast, the modeling predicts that 80.7% of White females in the USA are immune to developing melanoma, three -ordered mutations are required to develop it and the mutation lifetime is 78.9 years. Remarkably, it was also found that about 70% of females are immune to developing breast cancer and about 70% of males are immune to developing prostate cancer, predictions that fit in with the experimental evidence of cancer immunosurveillance and immunoediting. Clearly, different risk populations can develop the same cancer through different pathways. Delineating the mechanism underlying the prevalence of immunity to specific cancers in specific risk populations should become a research priority. Finding ways of blocking or repairing cellular mutations and/or destroying mutated, potentially cancerous cells would prevent cancers from developing altogether and eliminate a major cause of mortality.
... 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) [37]. ...
Article
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.
... 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 [20], HIV [21] or Herpes Simplex Virus-HSV [22] 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. ...
Article
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.
... 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]. ...
Article
Full-text available
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.
... 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. ...
Article
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.
... Evidence also shows changes in the GalNAc components of the extracellular matrix 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). ...
Article
Full-text available
Objective(s) 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. Results Response to transduction by shRNA compared to the control group, migrative and invasive properties of the transfected cells were significantly inhibited. Conclusion 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.
... 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 [25]. 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]. ...
Article
Full-text available
α-N-acetylgalactosaminidase (EC 3.2.1.49) (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.
... 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 [23][24][25]. 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). ...
Article
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Therapeutic potentials of immunotherapy in cancer treatment and a discussion of a breast cancer case managed with complementary immunotherapy
... (28,29) Beyond considering the potential impacts of increased GC production on the development of the immunosuppressed phenotype we characterized in the vast majority of COVID-19 patients in this study, we must also consider the possibility that GCs modulate the immune microenvironment differently in the context of distinct viral encounters. There are known but underappreciated immunosuppressive features of influenza (30)(31)(32); coronaviruses are similarly known to utilize a number of nonstructural proteins to disrupt host gene expression and protein synthesis (33,34), with the potential for profound impacts on various immune signaling pathways (35). Although the details of these mechanisms in the context of COVID-19 immune dysregulation remain to be fully elucidated, our own analyses demonstrate enhanced GC signaling in cells from COVID-19 patients, consistent with an enhanced production of GCs or enhanced sensitivity to GCs after infection. ...
Preprint
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Coronavirus disease 2019 (COVID-19) is characterized by a high incidence of acute respiratory failure. The underlying immunopathology of that failure and how it compares to other causes of severe respiratory distress, such as influenza virus infection, are not fully understood. Here we addressed this by developing a prospective observational cohort of COVID-19 and influenza subjects with varying degrees of disease severity and assessing the quality and magnitude of their immune responses at the cellular and protein level. Additionally, we performed single-cell RNA transcriptional profiling of peripheral blood mononuclear cells from select subjects. The cohort consists of 79 COVID-19 subjects, 26 influenza subjects, and 15 control subjects, including 35 COVID-19 and 7 influenza subjects with acute respiratory failure. While COVID-19 subjects exhibited largely equivalent or greater activated lymphocyte counts compared to influenza subjects, they had fewer monocytes and lower surface HLA-class II expression on monocytes compared to influenza subjects and controls. At least two distinct immune profiles were observed by cytokine levels in severe COVID-19 patients: 3 of 71 patients were characterized by extreme inflammation, with greater than or equal to ~50% of the 35 cytokines measured greater than 2 standard deviations from the mean level of other severe patients (both influenza and COVID-19); the other immune profile, which characterized 68 of 71 subjects, had a mixed inflammatory signature, where 28 of 35 cytokines in COVID-19 patients had lower mean cytokine levels, though not all were statistically significant. Only 2 cytokines were higher in COVID-19 subjects compared to influenza subjects (IL-6 and IL-8). Influenza and COVID-19 patients could be distinguished statistically based on cytokine module expression, particularly after controlling for the significant effects of age on cytokine expression, but again with lower levels of most cytokines in COVID-19 subjects. Further, high circulating levels of IL-1RA and IL-6 were associated with increased odds of intubation in the combined influenza and COVID-19 cohort [OR = 3.93 and 4.30, respectively] as well as among only COVID-19 patients. Single cell transcriptional profiling of COVID-19 and influenza subjects with respiratory failure identified profound suppression in type I and type II interferon signaling in COVID-19 patients across multiple clusters. In contrast, COVID-19 cell clusters were enriched for alterations in metabolic, stress, and apoptotic pathways. These alterations were consistent with an increased glucocorticoid response in COVID-19 patients compared to influenza. When considered across the spectrum of innate and adaptive immune profiles, the immune pathologies underlying severe influenza and COVID-19 are substantially distinct. The majority of COVID-19 patients with acute respiratory failure do not have a cytokine storm phenotype but instead exhibit profound type I and type II IFN immunosuppression when compared to patients with acute influenza. Upregulation of a small number of inflammatory mediators, including IL-6, predicts acute respiratory failure in both COVID-19 and influenza patients.
... 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. ...
... chDBP 1차적으로 간에서 분비되며, 대식세포 활성화에 관여하는 단백질 전구체로서 혈청에 풍부하다고 알려져 있 다 (White et al., 2000;Fang et al., 2009). chDBP는 인체에서 인플루엔자 감염 시 탈글리코실화 되어 대식세포의 활성화 능력을 잃게 하여 면역 결핍을 유도한다고 보고되었다 (Yamamoto et al., 2005). 본 실험 결과에서도 비교적 어린 1 주령에 조류인플루엔자 감염 시 음성대조군에 비하여 이 단 백질이 낮은 수치를 나타냈으나, 이후의 주령에 감염될 시 에는 특이적인 변화양상이 확인되지 않았으며, 4주령에 이 르러서는 모든 그룹이 비슷한 결과 값을 보였다. ...
... As their serum consists of α-N-acetylgalactosaminidase (Nagalase), it deglycosylates Gc protein and thus prevents MAF activity, contributing for immunosuppression. [64] Considering the outbreaks of H 1 N 1 influenza in 2009, Edlich et al. strongly recommended to test and treat Vitamin D deficiency among health-care workers and patients as a preventive measure. [65] Pro-inflammatory cytokine production is also regulated by Vitamin D, thus playing a crucial role in recovery from cytokine storm in H 1 N 1 infection. ...
Article
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Vitamin D has potential antimicrobial activity, the deficiency of which has deleterious effects on the general well‑being and longevity, predisposing major public health problem worldwide. About 1 billion people have Vitamin D deficiency, which is prevalent among all ethnicities and age groups throughout the world. In addition, the incidence of antimicrobial resistance has emerged as a major threat to public health, and it is estimated to cause 10 million deaths annually by 2050 throughout the world. Vitamin D, as a mighty antimicrobial agent, may decrease the occurrence of infection through numerous pathways. Vitamin D strengthens innate immunity by modulating the production of various anti‑microbial peptide (AMPs), cytokine, chemokines and interleukin responses. Vitamin D is responsible for the regulation of >200 genes, including cell proliferation, differentiation, and apoptotic genes. It acts as the key holder for modulating systemic inflammation, oxidative stress, and mitochondrial respiratory functions. Thus, a Vitamin D replete state appears to benefit most infections. As an antiviral agent, Vitamin D may constitute an inexpensive prophylactic option either by itself or as a synergistic agent during the treatment of different viral infections. The present review stipulates the importance of Vitamin D and its possible mechanisms against treating any kind of viruses. Relevant published articles were summarized by performing computerized literature searches (searches were made in PubMed/Medline, EMBASE, ScienceDirect, and Scirus) of different authentic databases using the following keywords: Vitamin D, VDR, infections, antimicrobial peptides, viruses, and COVID‑19. The future for the sunshine vitamin as an antiviral agent looks brighter. More scientific proposition entailing in vitro, in vivo, or genomic studies are required to understand how important Vitamin D is against viral infections.
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During a search for glycosidase inhibitors among marine natural products, we applied an integrated in vitro and in silico approach to evaluate the potency of some aaptamines and makaluvamines isolated from marine sponges on the hydrolyzing activity of α-N-acetylgalactosaminidase (α-NaGalase) from human cancer cells and the recombinant α-D-galactosidase (α-PsGal) from a marine bacterium Pseudoalteromonas sp. KMM 701. These alkaloids showed no direct inhibitory effect on the cancer α-NaGalase; but isoaaptamine (2), 9-demethylaaptamine (3), damirone B (6), and makaluvamine H (7) reduced the expression of the enzyme in the human colorectal adenocarcinoma cell line DLD-1 at 5 μM. Isoaaptamine (2), 9-demethylaaptamine (3), makaluvamine G (6), and zyzzyanone A (7) are slow-binding irreversible inhibitors of the bacterial α-PsGal with the inactivation rate constants (kinact) 0.12 min-1, 0.092 min-1, 0.079 min-1, and 0.037 min-1, as well as equilibrium inhibition constants (Ki) 2.70 µM, 300 µM, 411 µM, and 105 µM, respectively. Docking analysis revealed that these alkaloids bind in a pocket close to the catalytic amino acid residues Asp451 and Asp516 and form complexes, due to π-π interactions with the Trp308 residue and hydrogen bonds with the Lys449 residue. None of the studied alkaloids formed complexes with the active site of the human α-NaGalase.
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Evidence exists that vitamin D has a potential antimicrobial activity and its deficiency has deleterious effects on general well-being and longevity. Vitamin D may reduce the risk of infection through multiple mechanisms. Vitamin D boosts innate immunity by modulating production of anti-microbial peptides (AMPs) and cytokine response. Vitamin D and its analogues via these mechanisms are playing an increasing role in the management of atopic dermatitis, psoriasis, vitiligo, acne and rosacea. Vitamin D may reduce susceptibility to infection in patients with atopic dermatitis and the ability to regulate local immune and inflammatory responses offers exciting potential for understanding and treating chronic inflammatory dermatitides. Moreover, B and T cell activation as well as boosting the activity of monocytes and macrophages also contribute to a potent systemic anti-microbial effect. The direct invasion by pathogenic organisms may be minimized at sites such as the respiratory tract by enhancing clearance of invading organisms. A vitamin D replete state appears to benefit most infections, with the possible noteworthy exception of Leishmaniasis. Antibiotics remain an expensive option and misuse of these agents results in significant antibiotic resistance and contributes to escalating health care costs. Vitamin D constitutes an inexpensive prophylactic option and possibly therapeutic product either by itself or as a synergistic agent to traditional antimicrobial agents. This review outlines the specific antimicrobial properties of vitamin D in combating a wide range of organisms. We discuss the possible mechanisms by which vitamin D may have a therapeutic role in managing a variety of infections.
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There is evidence that the vast majority of hospitalized patients have vitamin D deficiency. Vitamin D deficiency is a poorly recognized pandemic with evidence to indicate inadequate testing and monitoring of response to treatment in high-risk populations. Vitamin D receptors are ubiquitous in the human body and while the endocrine effects of vitamin D are well recognized, the autocrine and paracrine effects of this steroid hormone are less well appreciated. These functions include antimicrobial and immunomodulation effects as well benefits on cardiovascular health, autoimmune disease, cancer and metabolism. Vitamin D deficiency increases mortality and even a modest amount of vitamin D may enhance longevity. Emerging evidence suggests that a vitamin D replete state carries significant health benefits in acute illness. In this review, we discuss the role of vitamin D deficiency and potential benefits in treating this deficiency focusing on the implications for managing acute illness in elderly patients and those with an underlying chronic illness.
Article
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.
Article
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.
Article
This overview describes the research of Nobutu Yamamoto (Philadelphia) concerning immunotherapy with GcMAF for patients with cancer and for patients infected with pathogenic envelope viruses. GcMAF (Group-specific component Macrophage-Activating Factor) is a mammalian protein with an incredible potency to directly activate macrophages. Since the late 1980s Yamamoto's investigations were published in numerous journals but in order to understand the details of his research, a minute survey of many of his patents was required. But even then, regrettably, a precise description of his experiments was sometimes lacking. This overview tries to summarize all of Yamamoto's research on GcMAF, as well as some selected more recent papers from other investigators, who tried to verify and/or reproduce Yamamoto's reports. In my opinion the most important result of the GcMAF research deserves widespread renewed attention: human GcMAF injections (100 ng per week, intramuscular or intravenous) can help to cure patients with a great variety of cancers as well as patients infected with pathogenic envelope viruses like the human immunodeficiency virus 1 (HIV-1), influenza, measles and rubella (and maybe also SARS-CoV-2). From Yamamoto's data it can be calculated that GcMAF is a near-stoichiometric activator of macrophages. Yamamoto monitored the progress of his immunotherapy via the serum level of an enzyme called nagalase (α-N-acetylgalactosaminidase activity at pH 6). I have extensively discussed the properties and potential catalytic site of this enzyme activity in an Appendix entitled: "Search for the potential active site of the latent α-N-acetylgalactosaminidase activity in the glycoproteins of some envelope viruses".
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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.
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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.
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Ehrlich ascites tumor cells and ascitic fluid were assayed for glycosidase activity. alpha-Galactosidase and beta-galactosidase, alpha- and beta-mannosidase, alpha-N-acetylgalactosaminidase, and beta-N-acetylglucosaminidase activities were detected using p-nitrophenyl glycosides as substrates. alpha-Galactosidase and alpha-N-acetylgalactosaminidase were isolated from Ehrlich ascites tumor cells on epsilon-aminocaproylgalactosylamine-Sepharose. alpha-Galactosidase was purified 160,000-fold and was free of other glycosidase activities. alpha-N-Acetylgalactosaminidase was also purified 160,000-fold but exhibited a weak alpha-galactosidase activity which appears to be inherent in this enzyme. Substrate specificity of the alpha-galactosidase was investigated with 12 substrates and compared with that of the corresponding coffee bean enzyme. The pH optimum of the Ehrlich cell alpha-galactosidase centered near 4.5, irrespective of substrate, whereas the pH optimum of the coffee bean enzyme for PNP-alpha-Gal was 6.0, which is 1.5 pH units higher than that for other substrates of the coffee bean enzyme. The reverse was found for alpha-N-acetylgalactosaminidase: the pH optimum for the hydrolysis of PNP-alpha-GalNAc was 3.6, lower than the pH 4.5 required for the hydrolysis of GalNAc alpha 1,3Gal. Coffee bean alpha-galactosidase showed a relatively broad substrate specificity, suggesting that it is suited for cleaving many kinds of terminal alpha-galactosyl linkages. On the other hand, the substrate specificity of Ehrlich alpha-galactosidase appears to be quite narrow. This enzyme was highly active toward the terminal alpha-galactosyl linkages of Ehrlich glycoproteins and laminin, both of which possess Gal alpha 1, 3Gal beta 1,4GlcNAc beta-trisaccharide sequences. The alpha-N-acetylgalactosaminidase was found to be active toward the blood group type A disaccharide, and trisaccharide, and glycoproteins with type A-active carbohydrate chains.
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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.
Article
25-Hydroxyvitamin D3-Sepharose was prepared by coupling 25-hydroxyvitamin D3-3β-(1,2-epoxypropyl)-ether to thio-activated Sepharose CL-6B, forming a protease-resistant linkage between the sterol and the matrix. Vitamin D-binding protein from human plasma was obtained 85–92% pure after ligand affinity chromatography. Subsequent hydroxylapatite chromatography provided homogeneous protein. The purified vitamin D-binding protein was fully active in regard to 25-hydroxyvitamin D3 and actin binding cappabilities.
Article
The infectivity of virions of the WSN strain of influenza A ( ) and the 1760 strain of influenza B (B/1760) which possess their hemagglutinin in the form of the uncleaved HA polypeptide can be enhanced as much as 100-fold by proteolytic cleavage of HA to yield HA1 and HA2. Hemagglutinating activity is unaffected by cleavage. The HA polypeptide of virions is susceptible to cleavage by trypsin and plasmin, whereas that of B/1760 virions is resistant to plasmin. The increase in infectivity can be demonstrated in different cell types, i.e., MDBK, BHK21-F, CEF, or HKCC cells, as well as in the chick embryo, and in titrations in both liquid medium and plaque assays under agar. Chymotrypsin cleaves the HA polypeptide of WSN virions in the same general region of the molecule as trypsin and plasmin, but without the enhancement of infectivity produced by the latter enzymes. Incubation of WSN virions with chymotrypsin prevents the enhancement by subsequent treatment with trypsin, but the increase in infectivity caused by an initial treatment with trypsin is not abolished by subsequent treatment with chymotrypsin. These results indicate that enhancement of infectivity of influenza virions results from proteolytic cleavage at a specific site on the HA molecule; however an increase in infectivity is not associated with cleavage at a nearby but different site.
Article
A comparative analysis has been carried out on the infectivity of virus of several influenza A strains grown in different host systems. Strains A/swine/Shope/31 (Hsw1N1), A/PR/8/34 (HON1), A/FM/1 (H1N1), A/Singapore/1/57 (H2N2), A/equine/Miami/1/63 (Heq2Neq2), and A/chick/Germany/49 (Hav2Neq1) exhibit host-dependent differences in infectivity. Virions grown in embryonated eggs and cultures of chorioallantoic membrane cells are highly infectious, whereas virions grown in cultures of chick embryo cells have a low infectivity that significantly increases after treatment in vitro with trypsin. In contrast, fowl plague viruses do not show host-dependent variations in infectivity. Virions grown in all host systems tested are highly infectious, and the infectivity of virions grown in chick embryo cells cannot be enhanced by trypsin treatment.The activation of virus particles appears to be based on the cleavage of hemagglutinin glycoprotein HA. This concept is supported by the following observations: (i) In virions of low infectivity only uncleaved glycoprotein HA can be detected. Virions of high infectivity exhibit complete or at least partial cleavage of the hemagglutinin. (ii) The activation of virions by trypsin treatment is always paralleled by cleavage of HA. (iii) Cleavage of HA is the only effect which can be detected after trypsin treatment. The neuraminidase is neither inactivated nor removed from the virion. (iv) Studies on recombinants of virus N and fowl plague virus (Rostock) show that host-dependent variation of infectivity and activation by trypsin, features specific for parent virus N, are found only with recombinant N(H)-FPV/Ro(N) but not with recombinant FPV/Ro(H)-N(N).Efficient plaque formation and serial passages are possible only if highly infectious particles are formed in a given host system. Thus, all strains analyzed undergo, in the absence of trypsin, successive growth cycles in eggs and chorioallantoic membrane cells and form plaques in chorioallantoic membrane cells. In contrast, in chick embryo cells only viruses containing the fowl plague virus hemagglutinin produce plaques and replicate under multiple cycle conditions without the addition of trypsin.The data show that cleavage of HA is not a precondition for virus assembly and hemagglutinating activity, but that it is necessary for infectivity. These findings are compatible with the hypothesis that, in addition to its role in adsorption, the hemagglutinin has another function in the infection process and cleavage is required for this function.
Article
Influenza persists in this last quarter of the 20th century as an important cause of wholesale human incapacity and death. Paradoxically, it does so in the face of recent improvements in inactivated vaccines developed 30 years ago and striking advances in the understanding of the structure, replication, and genetics of the influenza viruses during the past decade. Neither prior infection nor artificial immunization provides lasting immunity against the constantly changing virus of influenza. In part, the transient protection induced by presently available inactivated viral vaccines is related to rapid decline of vaccine-induced homologous antibody; in part, vaccine obsolescence is the result of viral mutation to antigens unlike or less like those contained in the vaccine. The threat of pandemic influenza is so awesome that major efforts are required for its curtailment upon even the hint of its inception, as was the case with swine influenza during 1976 (1).
In experimental animals, influenza prediposes the lung to superinfection by reducing the antibacterial efficiency of the alveolar macrophage system. Because such defects may represent abnormalities in ingestion or inactivation of inhaled bacteria, these subcomponents of phagocytosis were tested in mice infected 5 days previously with influenza A virus (NWS or WSN). The mice were exposed to aerosols of Staphylococcus epidermidis and then the rates of bacterial inactivation and percentages of intracellularly located staphylococci were measured. Rates of bacterial inactivation were determined for the left lung by pour-plate enumeration methods. The percentage of ingested bacteria was determined in the in situ perfused right lung by histologically determining the intra- or extracellular location of 100 or more staphylococci. Rates of inactivation of S. epidermidis at 4 hours after bacterial challenge were: control, 90.1 per cent; WSN, 73.0 per cent; NWS, 68.6 per cent, P less than 0.01. The percentage of intracellular staphylococci at 4 hours were: control, 90.9 per cent; WSN, 69.9 per cent; and NWS, 73.8 per cent, P less than 0.01. Microcolonies of proliferating staphylococci were also observed within macrophages of mice infected with each strain of influenza. These experiments demonstrated that in this experimental model, influenzal infection impairs the inactivation of inhaled bacteria by retarding the ingestion of bacteria and by allowing bacteria to proliferate within macrophages.
Article
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.
Article
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.
Article
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.
Article
Lysophosphatidylcholine (lyso-PC), a product of inflammation, stimulates (in vivo) mouse peritoneal macrophages to ingest target cells via Fc receptors. In vitro treatment of macrophages with lyso-Pc was unable to enhance ingestion activity. When a mixture of macrophages and nonadherent (B and T) cells was treated with 20 micrograms of lyso-Pc/ml for 30 min, a greatly enhanced Fc-mediated ingestion was observed at about 3 hr after treatment, suggesting that nonadherent cells contributed to activation mechanism of macrophages. The accumulated evidence suggests that treated B cells collaborated with untreated T cells in a stepwise fashion for the exchange of a signaling factor(s) for macrophage activation. When conditioned medium prepared by stepwise cultivation from treated B cells to untreated T cells was used for cultivation of untreated macrophages, a markedly enhanced Fc-mediated ingestion was observed. However, cultivation of macrophages with stepwise conditioned medium of treated T cells and untreated B cells produced no significant enhancement of phagocytic activity. Therefore, we concluded that lyso-Pc-treated B cells initiated the macrophage activation process by releasing and transmitting a signaling factor to T cells, and, in turn, the T cells modified the factor or supplied a new factor capable of the ultimate activation of macrophages for ingestion capacity. This lyso-Pc-induced factor(s) appears to be distinct from the established interleukins 1 and 2.
Article
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.
Article
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.
Article
In the present review we have identified how viruses can alter the host's susceptibility to bacterial infections by altering both environmental conditions in the lung which favor bacterial replication as well as by suppressing the host's defense mechanisms which prevent clearance of the bacteria. In many instances, these interactions are extremely complex but similar for many viruses. If the virus can overcome the initial host defense mechanisms, which include local antibody and mucus, the virus initiates tissue damage as a result of direct replication within the epithelial cells lining the mucosal surfaces of the respiratory tract. As a result of virus infection, the host cells respond by producing a variety of mediators including various types of interferons, which can alter both virus replication and host response. Replication also produces by-products of virus infection capable of initiating an inflammatory process, which in turn, through release of other mediators, can further modify lung defense mechanisms and encourage bacterial adherence and growth. The bacterium, in turn, releases chemotactic factors which encourage infiltration of specific effector cells into the lung. These effector cells can cause tissue damage and immunopathology, which encourage rapid bacterial growth and may result in death of the animal. In order to be able to control this complicated scenario, it is important either to prevent the initial infection with viruses or to reduce the degree of immunosuppression, so that bacterial clearance can occur rapidly before microcolony formation and extensive lung damage occur. Once a large amount of bacterial replication and lung damage is present, the use of antibiotics is generally of limited value. A schematic illustration of the complexity of the various interactions and counteractions occurring during virus--bacterial synergistic interactions is presented in Fig. 1.
Article
The vitamin D-binding protein in human serum (the group-specific component) is an alpha 2-globulin which is genetically polymorphic in all populations studied. Previous work (J. Svasti and B. H. Bowman (1978) J. Biol. Chem. 253, 5188-5194, and J. Svasti, A. Kurosky, A. Bennett, and B. H. Bowman (1979) Biochemistry 18, 1611-1617) has shown that the electrophoretic variations of the proteins controlled by two allelic genes, Gc1 and Gc2, are due to at least three amino acid substitutions between Gc1 and Gc2 (Svasti et al. (1979] and to heterogeneity in the Gc1 phenotype arising from carbohydrate dissimilarities. Gc1 migrates electrophoretically as two protein bands, while Gc2 migrates cathodally as a single band. This study demonstrates a post-translational glycosylation difference occurring in a single area of the Gc1 sequence which accounts for the heterogeneity observed previously. The glycosylation site, a threonine residue, appears to be in a sequence which differs between Gc1 and Gc2. The O-glycosidic bond, which is typical of mucins, is rare in plasma proteins. The cyanogen bromide fragment containing the galactosamine-containing carbohydrate in Gc1 was partially sequenced through 20 residues from the amino terminus. No detectable galactosamine could be found in the homologous cyanogen bromide fragment in Gc2. A new purification procedure for the vitamin D-binding protein in human plasma has been developed. Three chromatographic steps provide purified protein.
Article
On a highly purified preparation, the structure of the carbohydrate chain of the human vitamin D-binding protein was investigated and two genetic forms of this protein were considered (Gc 2 and Gc 1 proteins). It was found that only the Gc 1 protein (Gc1a isoform) was glycosylated, the glycan moiety representing about 1% of the protein. The structure of this O-glycosidically linked glycan was determined to be: Neu Ac alpha (2 leads to 3) Gal beta (1 leads to 3) GaINAc alpha (1 leads to 0) Ser (or Thr). A tetrasaccharidic O-glycan with two N-acetylneuraminic residues was also characterized. The vitamin D-binding protein is a rare example of a serum protein O-glycosylated only on some genetic forms.
Article
A conformational change in the hemagglutinin glycoprotein of influenza virus has been observed to occur to pH values corresponding to those optimal for the membrane fusion activity of the virus. CD, electron microscopic, and sedimentation analyses show that, in the pH range 5.2-4.9, bromelain-solubilized hemagglutinin (BHA) aggregates as protein-protein rosettes and acquires the ability to bind both lipid vesicles and nonionic detergent. Trypsin treatment of BHA in the pH 5.0-induced conformation indicates that aggregation is a property of the BHA2 component and that the conformation change also involves BHA1. The implications of these observations for the role of the glycoprotein in membrane fusion are discussed.
Article
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.
Article
A very small amount (0.0005 to 0.001%) of an ammonium sulfate [50% saturated (NH4)2SO4]-precipitable protein fraction of alpha 2-globulin efficiently supported inflammation-primed activation of macrophages. This fraction contains vitamin D3-binding protein essential for macrophage activation. Comparative macrophage activation studies with fetal calf serum, alpha 2-globulin fraction, 50% (NH4)2SO4 precipitate, and purified bovine vitamin D3-binding protein revealed that fetal calf serum and alpha 2-globulin fraction appear to contain an inhibitor for macrophage activation while ammonium sulfate precipitate contains no inhibitor. This inhibitor was found to be serum albumin. When bovine serum albumin (25 micrograms/ml) was added to a medium supplemented with 0.0005 to 0.05% (NH4)2SO4 precipitate or 1 to 10 ng of vitamin D3-binding protein per ml, activation of macrophages was inhibited.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Diagnostic and prognostic ELISA assays of serum α-N-acetylgalactosaminidase for AIDS
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