CD81 expression in peripheral blood lymphocytes before and after treatment with interferon and ribavirin in HIV/HCV coinfected patients.

BACKGROUND: CD81 is expressed on lymphocytes and confers HCV viral infectivity support. The aim of our study was to quantify CD81 expression in peripheral blood B- and T-cells of HCV/HIV-coinfected patients and healthy subjects to examine its association with several HCV virological characteristics and the therapeutic responsiveness to HCV antiviral treatment. METHODS: We carried out a cross-sectional study on 122 naïve patients. For a duration of 48 weeks, 24 out of 122 patients underwent HCV antiviral therapy with interferon (IFN)-alpha and ribavirin. T- and B-cell subsets were analysed by flow cytometry. RESULTS: We found that HIV/HCV coinfected patients with HCV-RNA > or =850 000 IU/mL had lower values of %CD19+CD81-CD62L+ and %CD19+CD62L+; and higher values of CD19+CD81+CD62L- and CD19+CD81+ percentages and absolute counts than patients with HCV-RNA <850 000 IU/mL. Similarly, HIV/HCV coinfected patients with the genotype 1 had lower values of %CD19+CD81-CD62L+ and higher values of CD3+CD81+CD62L- and CD3+CD81+ percentages and absolute counts than patients without genotype 1. Moreover, we found that HIV/HCV coinfected patients had higher values of %CD19+HLA-DR+CD25+, %CD19+CD40+CD25+ and %CD19+CD25+ than healthy control patients. When we studied the B- and T-cell subset kinetics of 24 HIV/HCV coinfected patients on HCV antiviral therapy, we found a significant decrease in CD3+CD81+and CD3+CD81+CD62L- subsets and a significant increase in CD3+CD62L+ and CD3+CD81+CD62L+ percentages and absolute counts, but the variation in these markers disappeared several months after stopping the treatment. CONCLUSIONS: We observed a different pattern of CD81 T-cell and B-cell levels in naïve HIV/HCV coinfected patients according to HCV virological status and their subsequent variations during HCV antiviral treatment. CD81 expression might influence HCV pathogenesis and response to HCV antiviral treatment.

Tumor necrosis factor-alpha and nitric oxide in vertically HIV-1-infected children: implications for pathogenesis.

We performed a cross-sectional study to investigate the plasma TNF-alpha and nitric oxide (NO) production in 44 vertically HIV-1-infected children, and the relationship with immunological status and viral replication. As a control group, 36 healthy, uninfected children were studied. Plasma TNF-alpha and NO levels were determined by ELISA. Viral load was quantified using standard assays. Cell proliferation, apoptosis and viral replication were evaluated in vitro by incorporation of (3H)-thymidine, flow cytometry and p24 antigen, respectively. Higher plasma TNF-alpha and NO levels were observed in HIV-1-infected children compared with healthy controls. We found a very strong correlation between plasma TNF-alpha and NO levels in HIV-1-infected children (r = 0.98; p < 0.001). Moreover, HIV-1-infected children with higher viral load (> 4.7 log10) showed higher TNF-alpha and NO levels than those with viral load below this threshold. Interestingly, we detected inducible nitric oxide synthase (iNOS) mRNA in T-lymphocytes from HIV-1-infected children. To address their possible patho-physiological significance, we tested the in vitro effects of NO and TNF-alpha in HIV-1 replication. Addition of TNF-alpha and NO donors to mitogen-activated, HIV-1-infected PBMC cultures produced a significant increase in viral replication. Moreover, HIV-1 replication in mitogen-stimulated, PBMC cultures was partially inhibited by iNOS specific inhibitors, and a neutralising, anti-TNF-alpha monoclonal antibody. Our results indicate that TNF-alpha and NO correlated with high viral load in HIV-1-infected children and favoured HIV-1 in vitro replication. These data suggest a detrimental role of NO in HIV-1 infection, and that NOS inhibitors may have some therapeutic benefit in HIV-1-infection.

[Role of tumor necrosis factor-a, nitric oxide and markers of clinical progression in the nutritional status of children with vertically-acquired hiv-1infection]. / Papel del TNF-a, óxido nítrico ymarcadores de progresión en el estado nutricional de niños con infección vertical por VIH-1.

OBJECTIVE: To study the correlation between the immunologic and virologic markers of clinical progression and the nutritional status of children with vertically acquired HIV-1infection. MATERIAL AND METHODS: We performed an anthropometric study in 34 HIV-1infected children. T cell subpopulations were analyzed by flow cytometry. Viral load (VL) was quantified by a standard commercial molecular assay. Tumor necrosis factor (TNF-) and nitric oxide (NO) concentrations were quantified by enzyme immunoassay. RESULTS: Z-weight, Z-height, Z-Quetelet index, Z-tricipital pleat, Z-arm perimeter, Z-cephalic perimeter, and McLaren's nutritional index were negatively correlated with VL. These anthropometric parameters were positively correlated with the percentage of CD4T lymphocytes but the correlation between these parameters and VL was lower. HIV-1infected children with a VL> 5 log 10 showed higher TNF- and NO concentrations and lower anthropometric scores. TNF- and NO concentrations were significantly higher in HIV-1infected children with a VL>5 log 10 than in those with a VL<5 log 10. TNF- and NO concentrations were significantly lower in HIV-1infected children undergoing antiretroviral treatment than in untreated HIV-1infected children. TNF- and NO concentrations were significantly higher in the HIV-1infected children than in the control group. CONCLUSIONS: Our data suggest an association between anthropometric characteristics indicating the nutritional status of HIV-1infected children and immunologic and virologic markers of clinical progression (percentage of CD 4T lymphocytes and VL). Moreover, TNF- and NO play a significant role in the nutritional status and neurological alterations of these children.

Papel del TNF-alfa, óxido nítrico ymarcadores de progresión en el estado nutricional de niños con infección vertical por VIH-1 / Role of tumor necrosis factor-alpha, nitric oxide and markers of clinical progression in the nutritional status of children with vertically-acquired hiv-1infection

Objetivo: Evaluar la asociación entre los marcadores de progresión clínica y el estado nutricional de niños infectados verticalmente por el virus de la inmunodeficiencia humana (VIH). Material y métodos: Se realizó un estudio antropométrico en 34 niños infectados por el VIH. Las subpoblaciones celulares se realizaron por citometría de flujo. La carga vírica (CV) se cuantificó mediante un ensayo molecular estándar comercial. El factor de necrosis tumoral alfa (TNF-alfa) y el óxido nítrico (NO) se cuantificaron por técnicas de enzimoinmunoanálisis (ELISA). Resultados: ZP (Z peso), ZT (Z talla), ZIQ (Z índice de Quetelet), ZPT (Z pliegue tricipital), ZPB (Z perímetro del brazo), ZPC (Z perímetro cefálico) e índice nutricional de McLaren tuvieron una asociación negativa con la CV. La asociación del porcentaje CD 41 con estos parámetros antropométricos fue de signo positivo, pero más débil que para la CV. Los niños con CV>5 log 10 obtuvieron valores más altos de TNF- alfa, además de los valores antropométricos más bajos. Los niños infectados por el VIH con CV> 5 log 10 tuvieron valores significativamente más altos de TNF- alfa y NO que los niños CV< 5 log 10. Los niños infectados por el VIH con tratamiento antirretrovírico tuvieron valores de TNF- alfa y NO inferiores a los niños no tratados. Los valores de TNF- alfa y NO fueron significativamente más altos en los niños infectados por el VIH que en los niños del grupo control. Conclusiones: Nuestros datos muestran una asociación entre las variables antropométricas que indican el estado nutricional del niño infectado por el VIH y los marcadores de progresión de la enfermedad utilizados por lo habitual en la práctica clínica (porcentaje CD 41 y CV). Además el TNF- alfa y el NO desempeñan un papel muy importante en el estado nutricional y en las alteraciones neurológicas de estos niños (AU)

Regulation of human immunodeficiency virus type 1 replication in human T lymphocytes by nitric oxide.

Addition of nitric oxide (NO) donors to mitogen-activated human immunodeficiency virus type 1 (HIV-1)-infected peripheral blood mononuclear cultures produced a significant increase in virus replication, and this effect was not associated with a change in cell proliferation. This effect was only observed with T-tropic X4 or X4R5 virus but not with R5 virus. Moreover, HIV-1 replication in mitogen-stimulated cultures was partially prevented by the specific inhibitors of the inducible nitric oxide synthase (iNOS). NO donors also enhanced HIV-1 infection of the human T-cell lines, Jurkat and MT-2. We have also observed that NO leads to an enhancement of HIV-1 replication in resting human T cells transfected with a plasmid carrying the entire HIV-1 genome and activated with phorbol ester plus ionomycin. Thus, in those cultures NO donors strongly potentiated HIV-1 replication in a dose-dependent manner, up to levels comparable to those with tumor necrosis factor alpha (TNF-alpha) stimulation. Furthermore, iNOS inhibitors decreased HIV-1 replication in HIV-1-transfected T cells to levels similar to those obtained with neutralizing anti-TNF-alpha antibodies. Moreover, HIV-1 replication induced iNOS and TNF-alpha transcription in T cells and T-cell lines. Interestingly, NO donors also stimulated long terminal repeat (LTR)-driven transcription whereas iNOS inhibitors partially blocked TNF-alpha-induced LTR transcription. Therefore, our results suggest that NO is involved in HIV-1 replication, especially that induced by TNF-alpha.

Induction of adhesion/differentiation of human neuroblastoma cells by tumour necrosis factor-alpha requires the expression of an inducible nitric oxide synthase.

Neuroblastoma cell lines (SK-N-SH and SK-N-MC) were induced to differentiate, as detected by the expression of neurofilament proteins of 68 and 200 kDa, and to express adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule) after stimulation with tumour necrosis factor-alpha (TNF-alpha). This induction was accompanied by the arrest of cell growth. The induction of neuroblastoma adhesion by TNF-alpha could be inhibited by the nitric oxide synthase inhibitors, L-N-monomethyl arginine (L-NMMA) and L-N6-(1-imidoethyl)-lysine (highly specific for the inducible enzyme), but not by the inactive enantiomer D-NMMA. These results indicate that TNF-alpha induces the adhesion of neuroblastoma cells via nitric oxide. This was confirmed by the finding that the adhesion/differentiation of SK-N-SH and SK-N-MC cells can be directly induced by the addition of nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetyl-penicillamine, into the culture medium. The isoform of the nitric oxide synthase induced in human neuroblastoma cells by TNF-alpha treatment was identified enzymatically as isoform II by Western blotting and by the polymerase chain reaction. Thus TNF-alpha induces the in vitro adhesion/differentiation of human neuroblastoma cells through nitric oxide synthesized by a calcium-independent inducible form of nitric oxide synthase, clearly indicating that isoform II of nitric oxide synthase can be expressed in human neuronal cell types.

Casein kinase 2 inactivation by Mg2+, Mn2+ and Co2+ ions.

Mg2+ as well as Mn2+, and Co2+, which may substitute Mg2+ in the mental ion requirement of casein kinase 2 (Gatica et al., FEBS Lett: 315:173-173, 1993), have been repeatedly reported to display an optimal concentration at which activity of casein kinase 2 is maximal. As far as we know this intriguing property has always been observed with casein as substrate. This phosphoprotein is not the natural substrate of the enzyme, and it is well known that it binds divalent metal ions, which provoke the aggregation and precipitation of the protein. Since an optimal concentration of metal ion might have a regulatory role, we have examined if it is a consequence of the particular properties of casein, or it is an inherent property of the enzyme, extensive to other substrates. We have used the type II regulatory subunit of protein kinase A which is a physiological substrate of the enzyme, and the peptide RRREEETEEE as a specific substrate. No optimal concentration of Mg2+ is observed when these two substrates are used. The results explain, however, why that optimum is observed with casein. Although low concentration of Mn2+, and Co2+ render about 25% of the maximal activity found with Mg2+, they inactivate the enzyme almost fully at concentrations at which Mg2+ yield the maximal activity.

Different phosphorylation behaviour of regulatory subunit isoforms of type II cAMP-dependent protein kinase from bovine heart.

Two forms of the regulatory subunit of the type II cAMP-dependent protein kinase (RII55 and RII52) were identified from bovine heart by gel electrophoretic behaviour. After autophosphorylation the RII55 isoform migrated more slowly (RII55/57) while the migration of RII52 isoform did not shift. Both isoforms showed different affinity for cAMP. The RII55/57 isoform was eluted from a cAMP-agarose column at 10 mM cAMP at low ionic strength whereas the RII52 isoform required cAMP, plus 2M NaCl. Partial proteolysis, using trypsin or formic acid, of autophosphorylated regulatory subunit isoforms resulted in different cleavage pattern as determined by peptide mapping. However, the V8 125I-peptides patterns of both isoforms are quite similar. Incubation of partially purified holoenzyme with 10 nM [gamma-32P]ATP (low ATP concentration) yielded a single band of Mr = 57,000 which corresponds to the RII55/57 isoform. The incubation, however, at 20 microM [gamma-32P]ATP yielded two phosphobands corresponding to both RII55/57 and RII52 isoforms. The phosphorylation of RII52 took place with a lower efficiency and was more sensitive to the cAMP than the corresponding phosphorylation of the RII55/57.

Regulation of lipid metabolism by dipyridamole and adenosine antagonists in rat adipocytes.

1. Acute effects of dipyridamole, an inhibitor of adenosine transport, direct activators of adenylate cyclase and thirteen adenosine antagonist analogs on fatty acid synthesis have been examined in terms of the control of [1-14C]acetate incorporation into labeled fatty acids in the presence of glucose. 2. This monosaccharide acts as a stimulator of lipogenesis by generating NADPH for the lipid synthesis. 3. The relationship between lipogenesis and lipolysis was compared with a variety of adenylate cyclase stimulators. 4. The data obtained reveals that dipyridamole potentiated the inhibitory or stimulatory effects of isoproterenol and forskolin on lipogenesis and on lipolysis, respectively. 5. In these cases the data show that it exists an inverse relationship between lipogenesis and lipolysis. 6. Dipyridamole and methylxanthine analogs only moderately affect the rate of lipolysis whereas its effects are more potent on lipogenesis and lend further support to the hypothesis that dipyridamole antagonize adenosine actions as well as methyl xanthines. 7. These results suggest that dipyridamole and adenosine antagonists alter lipogenesis independently of the lipolytic process and that it exists an inverse relationship between lipogenesis and lipolysis under some conditions whereas there are not under others.