Impaired proliferative capacity and abnormal cytokine profile of naive and memory CD4 T cells from HIV-seropositive patients.

Purified naive and memory CD4 T cells from healthy donors, HIV+ asymptomatic carriers and AIDS patients were examined for their proliferative activity and their pattern of cytokine secretion (IL-4, IL-6, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha)) upon stimulation with phytohaemagglutinin (PHA), phorbol myristate acetate (PMA) and cross-linked anti-CD3 MoAb, in the presence of recombinant IL-2 (rIL-2). We found a decrease in the proliferative capacity of naive CD4 T cells following stimulation with PHA and PMA, and a sharp decline in this response upon cross-linked anti-CD3 stimulation in both subsets, although it predominated in the naive subpopulation. In AIDS patients, less pronounced impairment of thymidine uptake by the naive subset was found upon PHA and cross-linked anti-CD3 MoAb stimulation. In addition, an altered secretion pattern of the different cytokines was observed, consisting of abnormal secretion of IL-6 by both naive and memory cells, an abnormal pattern of IFN-gamma secretion and frequent loss of detectable IL-4 production by HIV patients. These abnormalities were even more pronounced in AIDS patients than in the asymptomatic carriers. Overall, our results extend previous reports indicating functional impairment of memory CD4 subsets in HIV+ subjects by showing that this impairment involves naive CD4 T cells.     

Lactate dehydrogenase activity and its isoenzymes in concentric layers of adult bovine and calf lenses

The activity of lactate dehydrogenase (LDH) and its isoenzyme pattern were studied in four concentric layers of adult bovine and calf lenses. In both groups the specific activity of the total LDH diminished progressively toward the internal nuclear layer; the decrease was greater in the adult lenses. The enzyme activities in the cortical layers of the calf lens were lower than in the adult lens, but in the inner nuclear layers, the opposite was found. All of the 5 LDH isoenzymes were found in each layer. In both groups of animals the LDH1 isoenzyme prevailed, followed by LDH2. No differences were found in the percentage of each isoenzyme in the different lens layers. The differences in the activitie(s) of LDH found may be due to post-translational or post-synthetic modifications which may occur during the aging process.     

Defective protein tyrosine phosphorylation and altered levels of p59fyn and p56ick in CD4 T cells from HIV-1 infected patients

Early in HIV infection, CD4⁺ lymphocytes exhibit the propertiesof an anergic state characterized by unresponslveness to mltogensor to TCR stimulation and by defective IL-2 production. As tyrosinephosphorylation is the earliest of the biochemical events initiatedby stimulation of CD3-TCR, we studied protein tyrosine phosphorylationin purified CD4⁺ lymphocytes from 25 asymptomatic seropositlvepatients (CD4 T cells >350/mm³) previously stimulated invitro by immobilized antl-CD3 mAb or by co-lmmoblllzed antl-CD3and antl-CD28 mAbs. Purified CD4⁺ lymphocytes from HIV-lnfectedpatients exhibited defective early protein tyrosine phosphorylationin response to CD3 activation when compared with normal subjects.This defect was observed mainly in patients in whom prollferatlveresponses to immobilized antl-CD3 ranged from 2 to 50% of controlvalues obtained in healthy donors, and was frequently associatedwith increased cellular levels of p59^fyn and decreased cellularlevels of p56^ick. Interestingly, these defects appeared to correlatewith the degree of impairment in thymidine incorporation. SinceCD28 mAbs have been reported to enhance prollferatlve responsesto the CD3–TCR pathway in cloned murine or human anergicmodels and to induce tyrosine phosphorylation in human T cells,we studied the role of CD28 mAb as a co-signal. Although antl-CD28co-stlmulatlon augmented the prollferatlve responses in bothcontrols and HIV-lnfected patients, It failed to correct thetyrosine phosphorylation pattern in the latter. Our resultssuggest a relationship between defective early protein tyrosinephosphorylation and impairment of proliferatlve responses inCD4 T cells from HIV-lnfected patients, and evidence is providedthat associated altered cellular levels of the fyn and Ick tyrosinekinases might play an important role in the anergic responseobserved early during HIV infection.     

Somatic mutations can lead to a loss of superantigenic and polyreactive binding

Although antibodies have been assumed to bind a specific antigen, evidence exists showing that a single antibody can bind to multiple unrelated antigens. We previously studied a human monoclonal antibody expressing a mutated form of the V(H)3-73 gene and displaying anti-tubulin activity in a patient suffering from an immunocytic lymphoma. Despite its expression of a V(H)3 family member, this immunoglobulin failed to react with protein A (SpA), suggesting that somatic mutations could account for its change in specificity. To examine this possibility, we produced recombinant Ig expressing germ-line (IgM kappa-Germ) or the mutated form (IgM kappa-PER) of the V(H)3-73 fragment. Comparison of the respective affinities of the two Ig demonstrated that IgM kappa-Germ restores its SpA-binding capacity, and shows a moderate decrease in its affinity for tubulin. Interestingly, IgM kappa-Germ displayed polyreactive specificity for different autoantigens, which contrasted to the monospecific binding of IgM kappa-PER to tubulin. These results suggest that the monoreactive IgM kappa-PER antibody may be derived from a natural polyreactive antibody through somatic mutation. In addition, both temperature modification and mild denaturation succeeded in recovering the polyreactivity of IgM kappa-PER, which favors the view that conformational modifications of the tertiary structure of antibodies may play a key role in the genesis of polyreactivity.     

Production and functional characterization of two mouse/human chimeric antibodies with specificity for the tumor-associated Tn-antigen

In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.     

In vitro antioxidant treatment recovers proliferative responses of anergic CD4+ lymphocytes from human immunodeficiency virus-infected individuals

Oxidative stress has been proposed to be involved in the immunologic defeat observed in effector calls of the immune system as well as in lymphocyte cell death and viral replication in human immunodeficiency virus (HIV)-infected patients. Because thiol-containing antioxidants such as N-acetyl-L-cysteine have been shown to have beneficial effects on CD4+ lymphocyte survival and to inhibit programmed cell death and HIV-1 replication, they may play a role in therapeutic strategies of this disease. In this work we have studied the cellular thiol levels and the affect of in vitro antioxidant treatment of purified CD4+ lymphocytes from HIV-infected patients, and correlated these parameters to proliferative responses and programmed cell death. We show that CD4+ lymphocytes from HIV-infected patients display impaired proliferative responses and a significant decrease in cellular thiol levels, indicating a disturbed redox status. Interestingly, antioxidant treatment succeeded to restore defective proliferative responses to CD3- mediated activation in 8 of 11 patients (high antioxidant responders). In contrast to high responders, patients failing to respond to antioxidant treatment (low antioxidant responders), were characterized by an abnormal ratio of apoptotic cells, which was not affected by N- acetyl-L-cysteine and/or 2-beta-mercaptoethanol preincubation. These results demonstrate for the first time that antioxidant treatment is able to revert the impaired proliferative activity of CD4 cells from HIV-infected patients and could help designing therapeutic strategies with antioxidant drugs. However, this action is not observed in cells undergoing programmed cell death.     

Natural killer (NK) cell activity during HIV infection: a decrease in NK activity is observed at the clonal level and is not restored after in vitro long-term culture of NK cells.

NK cell activity is impaired in HIV-infected patients. The mechanisms behind the altered NK functions are not clear, and conflicting data concerning NK and antibody-dependent cellular cytotoxicity (ADCC) activity have been reported. In order to investigate whether this impairment is also observed at the clonal level and whether it is related to a defect at the target cell binding and/or the post-binding level, we evaluated highly purified NK cell lines and cloned NK cells obtained from 22 HIV-infected patients at different stages of disease and compared them with normal controls for their ability to: (i) kill K-562 and U-937 cell lines using a 51Cr release assay; (ii) bind and kill K-562 and U-937 cells at the single cell binding level; (iii) release NK cytotoxic factor (NKCF), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma); (iv) kill anti-IgM preincubated Daudi cell line (ADCC activity). This study with cloned NK cells or NK cell lines from HIV-infected individuals showed: (i) a decrease in their lytic capability against target cell lines; (ii) a low ability to form conjugates with K-562 and U-937 cell lines with respect to controls; (iii) a decreased ability to kill bound target cells; (iv) low levels of released NKCF, TNF-alpha and IFN-gamma after incubation with U-937 cells. Taken together, these findings suggest that the impaired NK cell function during HIV infection is also observed at the clonal level and is related to defects both at the target and post-binding levels. However, the precise mechanisms remain to be determined. The inability to restore normal NK activity after long-term culture in the presence of high levels of recombinant IL-2 is in agreement with the hypothesis of a 'general anergic process' during HIV infection.