Involvement of Annexin A2 in Serum-MAF Dependent Phagocytic Activation of Macrophages.

BACKGROUND/AIM: Serum-derived macrophage activating factor (serum-MAF) is expected to have adjuvant effects through rapid phagocytic activation, which depends on F-actin accumulation in multi-layered membrane ruffles induced within 5 min after serum-MAF addition. This study aimed to elucidate the importance of annexin A2, which is a multifunctional Ca2+-binding protein related to cytoskeletal membrane dynamics, in serum-MAF signalling. MATERIALS AND METHODS: Annexin A2 and F-actin localizations were analyzed via immunostaining and confocal microscopy. Using EGTA as chelator, the role of Ca2+ in serum-MAF signalling was examined. RESULTS: Annexin A2 was found to translocate from the cytosol to the cell cortex within 30 s of serum-MAF stimulation. Ca2+ chelation inhibited the translocation of annexin A2, frill-like structure formation, and phagocytic activation by serum-MAF. CONCLUSION: Annexin A2 and Ca2+ were responsible for the rapid phagocytic activation by serum-MAF. This study provides an understanding of phagocytic activation in macrophages, which could be beneficial for cancer immunotherapy.

Alteration of α-N-acetylgalactosaminidase (nagalase) concentration in alcohol-dependent individuals without liver disease, during the detoxification therapy.

BACKGROUND: The present study aimed to investigate for the first time, the alteration of α-N-acetylgalactosaminidase (nagalase) concentration in alcohol-dependent individuals without liver disease, before, during and at the end of the detoxification therapy. METHODS: Forty-eight alcohol-dependent individuals without liver disease who were admitted for alcohol detoxification, and eighty-four healthy controls participated in this study. Patients' blood was obtained upon admission, two weeks later and after the completion of the detoxification period (4-5 weeks). Nagalase concentration in serum was assessed by enzyme-linked immunosorbent assay. RESULTS: Nagalase concentration was significantly elevated in the patient samples in all serum collections as compared to the normal controls, with a progressive fall from admission to discharge (p-value<0.001). Values differed significantly among the three time points, with a net shift to decrease, but remained still high, above normal control level at the end of the therapy. No significant correlations were detected among the nagalase levels and the liver enzymes values. Moreover, no significant correlation was found between the alterations of nagalase concentrations and the amount of consumed alcohol. CONCLUSIONS: The high nagalase concentrations in alcohol abuse might be associated with macrophage impairment through decreasing the endogenous macrophage-activating factor (MAF) production by Gc-protein. The possible pathogenetic association between nagalase activity and alcohol overconsumption remains a matter of further investigation. Nagalase could also serve as a marker of alcohol overconsumption for the evaluation of alcohol-dependent individuals before, as well as during the detoxification therapy.

Effects of body weight reduction on blood adipokines and subcutaneous adipose tissue adipokine mRNA expression profiles in obese ponies.

Fifteen obese ponies were used in a body weight (BW) reduction programme (BWRP, daily energy intake: 7.0-8.4 MJ/100 kg BW). A frequently sampled intravenous glucose tolerance test was used to assess insulin sensitivity. Subcutaneous adipose tissue biopsies of the tail head were obtained for mRNA gene expression profiles of adiponectin, retinol-binding protein 4 (RBP4), interleukin 6 (IL-6) and macrophage activation marker (CD68) before and after BWRP. Blood samples were analysed for serum leptin, serum RBP4 and plasma adiponectin. Significant BW losses occurred with 7 MJ DE/100 kg BW. Serum leptin and RBP4 were initially similar between insulin-resistant (IR) and insulin-sensitive (IS) ponies, and both significantly decreased during BWRP. Compared with IS ponies, IR ponies initially had significantly lower plasma adiponectin levels. At the beginning of BWRP, mRNA expression of RBP4, adiponectin, IL-6 and CD68 was similar between IR and IS ponies. Plasma adiponectin was strongly related to IR, whereas serum leptin and RBP4 were closely linked to adiposity, independent of insulin sensitivity. Adipose tissue mRNA expression profiles did not clearly reflect these differences. However, the role of subcutaneous adipose tissue in IR remains open.

Phenotype of Gc-globulin influences the macrophage activating factor (MAF) levels in serum.

BACKGROUND: Gc-globulin is a polymorphic protein with three phenotypes: Gc 1-1, Gc 2-1 and Gc 2-2. Deglycosylation of Gc-globulin results in a Gc-macrophage activating factor (Gc-MAF). This study investigated the potential of MAF as a tumour marker and the influence of Gc-phenotypes on serum MAF-concentrations. METHODS: Gc-phenotype of 98 healthy individuals and 60 cancer patients was determined. MAF-levels of healthy individuals and cancer patients were analysed according to their Gc-phenotype using a Helix pomatia agglutinin-based ELISA. ROC curves analysed the efficiency of MAF as a tumour marker. RESULTS: MAF-levels between controls and patients were significantly different (p<0.001). No phenotypic differences were found in the patients. In comparison with the controls, MAF-values were significantly lower in cancer patients carrying Gc 1-1 (p<0.01) and Gc 2-1 (p<0.001). No difference was observed in Gc 2-2 phenotype. Diagnostic accuracy of MAF as a tumour marker also demonstrated pronounced differences between Gc-phenotypes. CONCLUSIONS: Gc-phenotype is a confounding factor when interpreting MAF-data. The value of MAF as a tumour marker varies according to phenotype. Future studies on MAF will have to consider the effect of Gc-phenotype.

Glycosylation status of vitamin D binding protein in cancer patients.

On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.

[Impact of highly active antiretroviral therapy on plasma MCP-1 and MSP in AIDS patients].

OBJECTIVE: To study the effect of highly active antiretroviral therapy (HAART) on plasma levels of MSP and MCP-1 in AIDS patients. METHODS: Forty Chinese AIDS patients were treated with HAART for 3 months and 84 German AIDS patients with HAART for 3 to 6 years. The pre-treatment and post-treatment plasma levels of MSP and MCP-1 were measured by enzyme-linked immunosorbent assay (ELISA), and their correlations with CD4+ cell counts and viral loads were analyzed. RESULT: The mean levels of MCP-1 were significantly higher and MSP were significantly lower in HIV-infected patients compared with the HIV-negative controls (P <0.01). After HAART for three months, there were no significant changes in the levels of these cytokines. But after long-term HAART (for 3 to 6 y), the level of MCP-1 was increased and that of MSP decreased significantly (P<0.01). There was a negative correlation between MSP and MCP-1 levels, and the same for MSP level and CD4+ cell counts; while there was a positive correlation between MCP-1 levels and CD4+ cell counts. CONCLUSION: The changed plasma levels of MSP and MCP-1 are associated with HIV-1 infection and HAART may reverse the levels of these two cytokines.

Prognostic usefulness of plasma monocyte/macrophage and T-lymphocyte activation markers in patients with acute coronary syndromes.

Macrophages and T lymphocytes accumulate and are activated in atherosclerotic plaques. We tested the hypothesis that plasma levels of the monocyte/macrophage and T-lymphocyte activation markers, monocyte chemoattractant protein-1 (MCP-1) and soluble interleukin-2 receptor (sIL-2r), respectively, can be used in acute coronary syndrome classification and risk prediction. Blood samples were collected at hospital admissions of 183 patients who had ischemic chest pain. Of these, 59 had acute myocardial infarction, 60 had unstable angina, and 64 had angina pectoris. No significant differences in the levels or proportions of subjects with increased levels of MCP-1 or sIL-2r were found across groups. During a mean follow-up of 13 months, 117 patients (64%) had a study end point (i.e., cardiac death, recurrent myocardial infarction, unstable angina, or revascularization). Increased levels (above median) of MCP-1 and sIL-2r were associated with increased risk, with odds ratios of 1.85 (95% confidence interval 0.92 to 3.73, p = 0.08) and 2.34 (95% confidence interval 1.16 to 4.71, p <0.02), respectively. In summary, in this unselected patient population with a very high rate of coronary events during follow-up, increased plasma levels of MCP-1 and sIL-2r were helpful for predicting new coronary events.

Multiple regulation of carp (Cyprinus carpio L.) macrophages and neutrophilic granulocytes by serum factors: influence of infection with atypical Aeromonas salmonicida.

Normal carp serum contains inhibitory and stimulatory factors for macrophage and neutrophilic granulocyte respiratory burst activity. As stimulatory factors were only effective in combination with phorbol myristate actetate (PMA) activation, it is concluded that they are probably linked to protein kinase C activation. Both the stimulatory and inhibitory factors are heat stable. Macrophage- and neutrophilic granulocyte-enriched cell fractions from the pronephros of carp had high respiratory burst- and high bactericidal in vitro responses to virulent atypical Aeromonas salmonicida bacteria. Serum factors were inhibitory for the A. salmonicida induced respiratory burst activity. No change in inhibitory or stimulatory serum factors could be observed during a 12-day challenge experiment with A. salmonicida, or during a rechallenge of survivors from a previous sub-lethal infection. The sensitivity of macrophages and neutrophilic granulocytes to stimulation of respiratory burst activity by PMA was not significantly altered. Culture supernatants from PHA pre-treated lymphocytes stimulated the respiratory burst activity of macrophages and neutrophilic granulocytes suggesting that serum factors may partially be lymphocyte derived.

Structural modification of serum vitamin D3-binding protein and immunosuppression in AIDS patients.

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.