Alberto Bergamini

Increased hepatitis C virus (HCV)-specific CD4+CD25+ regulatory T lymphocytes and reduced HCV-specific CD4+ T cell response in HCV-infected patients with normal versus abnormal alanine aminotransferase levels.

CD4+CD25+ T regulatory cells may play a role in the different clinical presentations of chronic hepatitis C virus (HCV) infection by suppressing CD4+ T cell responses. Peripheral CD4+CD25+ T cells from chronic HCV carriers with normal and abnormal alanine aminotransferase (ALT) were analysed for specificity and effect on HCV‐specific CD4+ T cell reactivity by flow cytometry for intracellular cytokine production and proliferation assay. HCV‐specific CD4+CD25+high T cells consistently produced transforming growth factor (TGF)‐β but only limited amounts of interleukin (IL)‐10 and no IL‐2 and interferon (IFN)‐γ. The HCV‐specific TGF‐β response by CD4+CD25+high T cells was significantly greater in patients with normal ALT compared to patients with elevated ALT. In addition, a significant inverse correlation was found between the HCV‐specific TGF‐β response by CD4+CD25+high T cells and liver inflammation. In peripheral blood mononuclear cells (PBMC), both HCV antigen‐induced IFN‐γ production and proliferation of CD4+ T cells were greater in patients with elevated ALT compared with patients with normal ALT. Depletion of CD4+CD25+ cells from PBMC resulted in an increase of both IFN‐γ production and proliferation of HCV‐specific CD4+ T cells that was significantly greater in patients with normal ALT levels compared with patients with elevated ALT. In addition, CD4+CD25+ T cells from patients with normal ALT levels proved to be significantly more potent to suppress CD4+ T cell reactivity with respect to those from patients with elevated ALT. In conclusion, these data support the hypothesis that CD4+CD25+ cells may play a role in controlling chronic inflammatory response and hepatic damage in chronic HCV carriers.

1α,25-Dihydroxyvitamin D3 inhibits CD40L-induced pro-inflammatory and immunomodulatory activity in Human Monocytes

CD40 ligand (CD40L) stimulation induces proinflammatory and immunomodulatory activity in monocytes. Here, we report on the effects of the steroid hormone 1alpha,25-dihydroxyvitamin D3 (1,25D3) on human blood monocytes that have been stimulated with the CD40L ligand. Co-treatment of CD40L-stimulated monocytes with 1,25D3 resulted in reduced production and secretion of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, as well as in reduced expression of the surface co-stimulatory molecules CD80 and CD86. In addition, costimulation of CD4+ T lymphocytes by monocytes co-treated with CD40L and 1,25D3 resulted in reduced cell proliferation and diminished interferon (IFN)-gamma but enhanced IL-10 production by CD4+ T cells. Finally, 1,25D3 interfered with the ability of CD40L to rescue monocytes from apoptosis induced by serum withdrawal. These findings suggest that 1,25D3 may regulate the interaction of monocytes with T cells or other cell types that express CD40L, thus influencing the outcome of the immune or inflammatory response.

Proinflammatory modulation of the surface and cytokine phenotype of monocytes in patients with acute Charcot foot.

OBJECTIVE Despite increased information on the importance of an inappropriate inflammatory response in the acute Charcot process, there has been no previous attempt to define the specific pathways that mediate its pathogenesis. Here, the role played by monocytes was analyzed. RESEARCH DESIGN AND METHODS The immune phenotype of peripheral monocytes was studied by fluorescence-activated cell sorter analysis comparing patients with acute Charcot (n = 10) in both the active and recovered phase, diabetic patients with neuropathy (with or without osteomyelitis), and normal control subjects. RESULTS When compared with diabetic control subjects and healthy subjects, monocytes from acute Charcot patients showed a proinflammatory immune phenotype characterized by increased production of proinflammatory cytokines, reduced secretion of anti-inflammatory cytokines, increased expression of surface costimulatory molecules, and increased resistance to serum withdrawal-induced apoptosis. In addition, the pattern of circulating cytokines confirmed activation of proinflammatory cytokines. No modulation of the monocyte phenotype was documented in diabetic control subjects and healthy subjects, thus indicating that the proinflammatory alterations of monocytes are specific and causative of acute Charcot. CONCLUSIONS Together, these data provide evidence for the role of proinflammatory changes in the immune phenotype of monocytes in the pathogenesis of acute Charcot. These alterations may explain the abnormally intense and prolonged inflammatory response that characterizes this disorder and may represent a potential therapeutic target for specific pharmacological interventions.

Enhanced Production of Tumor Necrosis Factor-α and Interleukin-6 Due to Prolonged Response to Lipopolysaccharide in Human Macrophages Infected In Vitro with Human Immunodeficiency Virus Type 1

Elevated levels of circulating tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 have been detected in human immunodeficiency virus (HIV) type 1 infection. The overproduction of these cytokines could contribute to AIDS pathogenesis. Thus, the expression of TNF-alpha and IL-6 in human macrophages infected with HIV-1 was investigated. HIV-1 infection, per se, did not induce any TNF-alpha or IL-6 production or cytokine-specific mRNA expression. In contrast, HIV-1 primed macrophages to a prolonged TNF-alpha and IL-6 response to lipopolysaccharide (LPS) stimulation with respect to uninfected cells. Time-course analysis and flow cytometry demonstrated that cytokine production stopped at 6 h in uninfected macrophages but continued up to 24 h in HIV-1-infected cells. RNA studies suggested that HIV-1 interfered with late steps of cytokine synthesis. No modulation of membrane CD14 was found to account for the enhanced response to LPS. Finally, the effect of HIV-1 on cytokine response could not be abolished by the antiviral compound U75875.

Clinical analysis of human urine by means of potentiometric Electronic tongue.

The Electronic tongue (ET) composed of different kind of potentiometric chemical sensors has been applied for the detection of urinary system dysfunctions and creatinine levels. The creatinine contents evaluated by ET were compared with those obtained by automated Jaffes method and GC-MS, obtaining a satisfying agreement for both methods. Partial least square regression discriminate analysis (PLS-DA) and feed forward back-propagation neural network (FFBP NN) classified 51 urine specimens from healthy volunteers in four classes, according to the creatinine content, showing that both techniques can satisfactorily differentiate urines according to this parameter. The best accuracy result of 92.2% correct classification of unknown samples was achieved with FFBP NN. Moreover, the possibility of ET system to distinguish between urine samples of healthy patients, and those with malignant and non-malignant tumor diagnosis of bladder has been shown.

Downregulation of CD40 ligand response in monocytes from sepsis patients.

ABSTRACT It has been suggested that a defective adaptive immune response contributes to septic immunosuppression. Here, the response of monocytes to CD40 ligand (CD40L) for patients with sepsis due to infection with gram-negative organisms has been analyzed. Compared to cells from controls, monocytes from septic patients showed significantly reduced production of tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-12 and were unable to acquire high levels of CD80 and CD86 molecules. These alterations were observed at the onset of sepsis and persisted at day 7. However, the ability of monocytes to respond to CD40L stimulation was partially but significantly restored in cells from patients who recovered from sepsis. In addition, costimulation of autologous CD4+ T lymphocytes by CD40L-activated monocytes from septic patients failed to induce cell proliferation and gamma interferon production. Finally, the ability of CD40L to rescue monocytes from apoptosis was severely impaired. We conclude that downregulation of the CD40L response may be an appropriate model for the monocyte alteration observed during septic immunosuppression and may help in the development of novel therapeutic strategies.

Different pattern of activity of inhibitors of the human immunodeficiency virus in lymphocytes and monocyte/macrophages.

Monocyte/macrophages (M/M) are important targets for HIV in the body, and represent the majority of cells infected by the virus in some body compartments such as the central nervous system (CNS). M/M can be different from T-lymphocytes in terms of surface antigens, cell replication and drug metabolism. Thus, we evaluated, in M/M and in T-lymphocytes, the pattern of viral inhibition induced by various anti-HIV drugs, and assessed some of the mechanisms of action related to such antiviral activity. Inhibitors of HIV binding on CD4 receptors have similar activity in M/M and T-lymphocytes, while AZT and other dideoxynucleosides (ddN) are in general more active against HIV in M/M than in T-lymphocytes. This phenomenon can be related to the increased ratio in M/M of ddN-triphosphate/deoxynucleoside-triphosphate, and can at least in part explain the ability of zidovudine and didanosine in improving neurological dysfunctions in AIDS patients. Moreover, the antiviral activity of AZT (but not of other ddN- or HIV-binding inhibitors) is potently enhanced by cytokines like granulocyte-macrophage colony stimulating factor (GM-CSF) in M/M, while anti-HIV activity of TIBO compounds in M/M is not down-modulated by GM-CSF and other cytokines. Finally, non-toxic concentrations of adriamycin, an anticancer drug reported to be active against DNA viruses, can inhibit HIV replication in M/M (but not in T-lymphocytes). Taken together, these results suggest that M/M are selective targets for HIV with peculiarities different from those of T-lymphocytes. Thus, promising anti-HIV compounds should be evaluated both in T-cells and in M/M before reaching clinical trials. This may help in selecting drugs with good chances of being effective in patients with HIV-related disease.

Treatment with ribavirin and interferon-α reduces interferon-γ expression in patients with chronic hepatitis C

Recent studies in vitro and in animals have suggested that ribavirin may potentiate the antihepatitis C virus (HCV) activity of interferon‐α (IFN‐α) by up‐modulating the production of T cell‐derived cytokines, such as interleukin (IL)‐2 and IFN‐γ, which play a key role in the cellular immune response against HCV. To study the immune‐modulatory mechanisms of ribavirin further, cytokine production by activated T cells and circulating cytokine levels were studied by FACS analysis and ELISA testing in 25 patients with chronic hepatitis C unresponsive to IFN‐α, before and after treatment with either ribavirin plus IFN‐α or IFN‐α alone. After 16 weeks of treatment, both the expression of IFN‐γ by activated T cells and the blood levels of IFN‐γ, were significantly reduced with respect to pretreatment values in patients treated with ribavirin and IFN‐α but not in those undergoing treatment with IFN‐α alone. The expression of IFN‐γ was significantly lower in patients that gained normal ALT levels with respect to those that did not. No modification of the expression of IL‐2, IL‐4 and IL‐10 was found before and after treatment in either group of patients. In conclusion, the results of this study do not support up‐modulation of IFN‐γ and IL‐2 production as the mechanism by which ribavirin potentiates IFN‐α anti HCV activity. In addition, our findings suggest that ribavirin may exert an anti‐inflammatory effect and may help reducing IFN‐γ‐driven T cell activation and liver damage.

A tetrazolium-based colorimetric assay for quantification of HIV-1-induced cytopathogenicity in monocyte-macrophages exposed to macrophage-colony-stimulating factor

A sensitive assay was developed for in vitro evaluation of anti-HIV agents in monocyte-macrophage cells (M/M) (a crucial target of HIV in the body). Monocyte-macrophage cells are usually poorly sensitive to the cytopathic effect induced by HIV. However, when fresh adherent monocyte-macrophage cells are cultured at relatively high density in the presence of macrophage-colony stimulating factor (M-CSF), they undergo cytolysis and die in 2-3 weeks. HIV-mediated cell-killing can thus be assessed with a method based on the reduction of the yellow colored 3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by metabolically active cells to a blue formazan, which can be measured spectrophotometrically. HIV-mediated cytopathic effect of M-CSF-exposed monocyte-macrophage cells was consistently achieved in all experiments performed under the conditions described herein. Anti-HIV activity of zidovudine (AZT) was also comparatively evaluated in M-CSF- and normal monocyte-macrophage cells both using the MTT assay and by measuring HIV-p24 antigen production in supernatants of monocyte-macrophage cells cultures, and similar results obtained with both methods. These results support the use of this colorimetric assay for broad screening of anti-HIV agents in monocyte-macrophage cells.

Evaluation of the Severity of Chronic Hepatitis C with 3-T1H-MR Spectroscopy

OBJECTIVE The purpose of this study was to compare the spectral characteristics of lipids, choline-containing compounds, and glutamine-glutamate complex assessed with (1)H-MR spectroscopy with the histologic findings in patients with chronic hepatitis C. SUBJECTS AND METHODS Nine healthy controls and 30 patients with biopsy-proven hepatitis C virus-related liver disease participated in this prospective study. Degree of fibrosis and histologic activity were scored according to the METAVIR classification. The percentage of involved hepatocytes was used to grade steatosis. Hepatic spectra were obtained with a 3-T spectroscopic system. Tenfold cross-validated stepwise discriminant analysis was performed to classify disease severity on the basis of the spectroscopic findings. RESULTS There was a strong correlation between (1)H-MR spectroscopically measured lipid concentration and the degree of steatosis at histologic examination (r = 0.9236, p < 0.0001). This finding enabled clear separation of groups according to degree of histologically determined steatosis. Variation in lipid concentration was consistent with the degree of steatosis (r = 0.7265, p < 0.0001) and stage of fibrosis (r = 0.8156, p < 0.0001). In univariate analysis, concentrations of both choline-containing compounds and glutamine-glutamate complex had a direct correlation with histologic grade (p < 0.0001) and degree of steatosis (p < 0.0001) but not with stage of fibrosis (p > 0.05). In multivariate analysis, the only factor independently associated with concentrations of choline-containing compounds and glutamine-glutamate complex was histologic grade. In cross-validated discriminant analysis based on choline-containing compound, glutamine-glutamate complex, and lipid resonance, 70% (21 of 30) of the histologic grade groups and 73% (22 of 30) of the steatosis groups were correctly classified. CONCLUSION Hydrogen-1 MR spectroscopy can be an alternative to liver biopsy in the evaluation of steatosis and necroinflammatory activity in liver disease but is not useful for complete evaluation of hepatic fibrosis.