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Additional file 3. Relative frequency of individual TRBV subgroup usage in perinatally HIV-infected youths (pHIVy), in non-perinatally HIV-infected youths (npHIVy) and in healthy controls (HC). The relative expression of each TRBV transcript was quantified as described by Gorochov G [50].

Purpose of the study : Although HIV infection impacts the proportion and phenotype of regulatory T-cells (Tregs), discrepant results have been reported depending on the surface markers employed to characterize them and on the patient populations. In addition, the effects of a long-term combined antiretroviral therapy (cART) on Treg cells have not been thoroughly documented. Our study investigated the frequency and number of Tregs and their phenotype in two different groups of HIV-infected patients: one aviremic undergoing long-term cART and one viremic naive to cART showing a similar CD4+ cell count. Methods : Thirty-six HIV+ patients with sustained suppression of plasma viremia (

A genotyping assay was setup to assess the prevalence, in the population of a Northern Italian city, of the C/T-13910 single nucleotide polymorphism, closely associated to lactose malabsorption in many world areas including Sardinia. The results were compared to published Italian data, in order to evaluate the worth of a future validation of the assay for use in routine practice. DNA was extracted from blood samples of 123 randomly chosen healthy blood donors coming from the same city area, and was analyzed by a real-time polymerase chain reaction (PCR) genotyping assay; the frequency of the hypolactasia-associated CC-genotype was compared to the weighted average of results extracted from studies reporting the frequency of hypolactasic phenotype or genotype in nearby or distant Italian regions. Sixty-five percent of donors carried the CC-genotype, a percentage similar to other northern Italian cities, but significantly higher than what previously determined in surrounding Italian regions at the phenotype level, i.e. by breath test. This discrepancy parallels recent reports of non concordance between results of genotyping and hypolactasic phenotype in some world areas, including a neighbouring Northern Italian city. A north-south gradient of CC-prevalence was also observed. These results reinforce the notion of wide inter-regional variations in the frequency of C/T-13910 polymorphism and of incostant concordance with hypolactasic phenotype, even in subjects from the same country. Given the unsatisfactory results recently obtained from validation of a related assay in a neighbouring city, the authors decided not to proceed further and keep the assay only as a diagnostic aid in special situations.

Relatively few attempts have been made in the past to isolate and expand lymphatic endothelial cells (LECs). Recently this task has become feasible thanks to the identification of new lymphatic markers such as Podoplanin, Lyve-1, Prox-1 and D2-40. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1-coated beads followed by purification with monoclonal antibody D2-40, we were able to purify and in vitro expand human derived LECs from tissues such as lymph node, spleen, thymus, palatine tonsil and iliac lymphatic vessels. The isolated LECs were expanded on collagen type 1 and fibronectin coated flasks for up to 8-10 passages and then analyzed for phenotypic and functional properties. LECs were able to form a capillary like network, when seeded on Cultrex BME, indicating their capability to form lymphatic vessels in vitro. Comparative studies were performed, and we found that specific lymphatic and vascular markers were differentially expressed by LECs prepared from different sources, clearly demonstrating the phenotypic heterogeneity of LECs from different organs and different segments of the lymphatic vasculature. We here propose a new technique to make available ready sources of abundant well-characterized human LECs to examine normal profiles and behavior to compare with abnormal conditions.

The objectives of the study were to characterize the production, function and survival of T lymphocytes of children with prolonged fetal exposure to dexamethasone for anti-Ro/SS-A antibodies associated congenital complete heart block. The analysis of thymic function, studied by measuring the level of T-cell receptor excision circles, was performed by real time PCR, the composition of T-cell subpopulation was evaluated by flow cytometry and the T-cell diversity was assayed by heteroduplex analysis. T-cell competence was gauged at two functional levels by determining the proliferation and the number of T-cell divisions and by measuring γ-interferon production after mitogenic stimulation. We observed that the thymic output, distribution of T-cell subsets, thymidine incorporation, number of T-cell divisions, and γ-interferon production were comparable to those of age-matched control. On the contrary, heteroduplex analysis demonstrated the presence of both polyclonal and oligoclonal peripheral T-cell repertoires.In conclusion, the analysis of the T-cell compartment in children with prolonged intrauterine exposure to high dose dexamethasone did not disclose any relevant abnormality, except a restriction of T-cell receptor diversity in some patients.

We have previously proposed the CD20 molecule as a novel suicide gene for T lymphocytes in the context of allogeneic bone marrow transplantation, because CD20 can be used both as a selection marker and as a killer gene after exposure to the anti-CD20 therapeutic antibody rituximab. We now report on preclinical studies using this novel system, in which the best transduction protocol, reproducibility, yield, feasibility, and functionality of the transduced T lymphocytes have been investigated with a large donor series. Wild-type human CD20 cDNA was transduced into human T lymphocytes, using a Moloney-derived retroviral vector. Alternative protocols were tested by employing either one or four spinoculations (in which cells are centrifuged in the presence of retroviral vector supernatant) and stimulating T cells with phytohemagglutinin (PHA) or anti-CD3/CD28. One spinoculation alone was sufficient to obtain approximately 30% CD20-positive cells within four experimental days. Four spinoculations significantly increased transduction to 60%. A small difference in transduction efficiency was observed between the two stimulation methods, with PHA being superior to anti-CD3/CD28. Transduced cells could be purified on immunoaffinity columns, with purity reaching 98% and yield being on average 50%. Finally, 86-97% of immunoselected T lymphocytes could be killed in vitro with rituximab and complement. More importantly, the CD20 transgene did not alter the functionality of T lymphocytes with respect to allogeneic recognition and cytotoxic response, anti-Epstein-Barr virus cytotoxic response, antigenic response to tetanus toxoid antigen, interleukin 2 (IL-2), IL-4, and interferon gamma production; chemotaxis in the presence of stromal cell-derived factor 1, phenotype for several activation markers including HLA-DR, CD25, CD69, and CD95, and T cell repertoire.

Several recent studies suggest that gammadelta T lymphocytes play an important role in immunity against Mycobacterium tuberculosis. However, the dynamics of these cells in the peripheral blood of patients with tuberculosis (TB) with and without HIV infection is not fully understood. A study was undertaken to evaluate the profile of the gammadelta T cell population in patients at the time the diagnosis of TB was established.A cross sectional study was performed in consecutive TB patients from the Department of Infectious Diseases, Spedali Civili, Brescia. CD4+, CD8+ and Vdelta1 and Vdelta2 T cell counts were analysed. Lymphocyte surface membrane expression was evaluated with the FITC-TCRgammadelta, -Vdelta1, -Vdelta2 and PE-Vdelta1 monoclonal antibodies. Blood donors and HIV seropositive asymptomatic individuals acted as controls.Seventy four TB patients were evaluated, 20 of whom (27%) were co-infected with HIV. HIV seronegative TB patients (n=54) had total gammadelta T cells and Vdelta1 subsets comparable to those in blood donors (n=39). However, the percentage with the Vdelta2 subset was significantly lower in patients with TB than in controls (median 1.5 v 2.1; p=0.05). Responsiveness to PPD was not associated with predominance of a specific gammadelta T cell subset. HIV seropositive individuals had a decreased percentage of circulating Vdelta2 cells at a level similar to that in HIV seronegative TB patients, regardless of the presence of active TB.HIV seronegative TB patients and HIV infected individuals (with or without active TB) have a reduced number of circulating Vdelta2 T cells compared with healthy individuals. Whether TB and HIV infection share a common mechanism causing Vdelta2 T cell depletion still needs to be established.

Orofacial granulomatosis (OFG) represents an inflammatory disorder of the facial and oral mucosa, histologically characterized by non-caseating epithelioid cell granulomas. Since other granulomatous diseases have been shown to be characterized by a limited heterogeneity of alpha/beta and gamma/delta T cells, we investigated the T-cell diversity of both types of lymphocytes obtained from the same OFG patient. When we compared the T-cell receptor diversity of the lymphocytes accumulating at the site of the lesions with that of the peripheral blood counterpart, we did not find significant differences. Furthermore, no exclusive expansions of different T-cell clones were seen in the patient. From these data we conclude that, in this OFG patient, the majority of T cells have no specificity for a single or for a few antigens and that tissue accumulation of T lymphocytes is the result of a random influx of cells at the site of inflammation.

We report that alpha/beta and gamma/delta T-cell repertoires of three patients with idiopathic CD4+ lymphocytopenia, who showed different clinical manifestations and outcomes over time, were highly restricted. The disruption of T-cell repertoires does not influence the susceptibility to infections: the first patient was unable to attain a protective response to mycobacterium, the second showed clinical improvement and the third did not develop opportunistic infections. These results indicate that idiopathic CD4+ lymphocytopenia could give rise to mono-/oligoclonal T-cell expansions, but the degree of repertoire disturbance is not indicative of the severity of disease progression.

In vertebrates, generation of the T- and B-cell repertoire relies on genomic rearrangement of T-cell receptor and immunoglobulin gene coding segments. This process, known as V(D)J recombination, is initiated by the lymphoid specific proteins Rag1 and Rag2. Both in humans and in animal models, mutations that abrogate expression of either the Rag1 or Rag2 proteins result in severe combined immune deficiency with a complete lack of circulating T and B cells due to an early block in lymphoid development. We have recently shown that mutations that impair, but do not completely abolish the function of Rag1 and Rag2 in humans result in Omenn syndrome, an enigmatic form of combined immune deficiency characterized by oligoclonal, activated T lymphocytes with a skewed Th2 profile.

A novel single-stranded DNA virus named SEN virus (SENV), which was initially cloned from the plasma of an HIV-positive patient in a cohort we previously studied [1], has been observed in patients suffering from acute and chronic liver diseases of unknown etiology [2]. However, the possible role of SENV in hepatic inflammation is not clear [3]. Phylogenetic analysis of SENV has demonstrated the existence of eight subtypes (A–H), with the SENV H and D subtypes being associated with liver disease more frequently [4, 5]. The epidemiology and clinical implications of SENV infection are currently being investigated. Recently, we demonstrated a lack of negative impact of SENV on the clinical progression of HIV infection [6] and the apparent dynamism of SENV infection, with both clearance and reinfection appearing over time [7]. In HIV-infected patients, hepatitis C virus (HCV) has become a frequent cause of morbidity and mortality in the era of highly effective antiretroviral therapy, and hepatic impairment is aggravated by toxicity due to antiretroviral drugs [8]. Thus, it seems necessary to investigate whether SENV has an impact on patients coinfected with HCV and HIV with or without antiretroviral therapy. The goals of the retrospective, cross-sectional study reported here were to investigate the influence that SENV infection (subtypes A, B, D, and H) may exert on the biochemical hepatic features of patients affected by HIV and chronic HCV in the presence or absence of antiretroviral treatment. As a secondary objective, we also studied the associated charac

Background The T-cell compartment of patients with rheumatoid arthritis (RA) is characterized by profound changes, including an acceleration of immune-senescence, reduced diversity of the T-cell repertoire, and expansion of clonal populations. These changes are reflected by the expansion of the CD28 peripheral blood T-cell subpopulations, as consequence of repeated CD28 engagement induced by chronic inflammation. These populations have been reported to have a reduced telomerase activity. Abatacept (CTLA4-Ig; ABA) can prevent the engagement of CD28 and T-cell activation, avoiding CD28 down-modulation and limiting the expansion of CD28 - T cells in RA. Objectives To evaluate the effect of ABA therapy on T-cell repertoire diversity and senescence in patients with RA. Methods In 44 RA patients treated with ABA, peripheral blood T cells were evaluated by flow-cytometry at baseline (T0) and after 12 months (T12). Healthy donors (HD) of comparable sex and age were evaluated. In 17 patients, the composition and diversity of T-cell receptor (TR) repertoire was assessed by spectratyping on mRNA extracted from peripheral blood mononuclear cells (PBMC). Average perturbation within CDR3 region of TR beta chain was calculated by measuring the Hamming distance between the target sample and a control CDR3 distribution profile, obtained from HD (theoretical nonperturbed repertoire). Telomerase reverse transcriptase ( TERT ) expression was assessed by real-time PCR on mRNA extracted from PBMC of 11 patients stimulated with anti-CD3 and expressed, using DDCt method, as normalization rate (NR). Results At T0, CD4+CD28- T cells did not differ between RA patients and HD. At T12, after ABA therapy, the percentage of circulating CD4+CD28- T cells decreased from 4.5% (IQR: 1.0-25.0) to 1.8% (0.2-12.2) of total CD4+ T cells (p:0.016), and their absolute number from 32 cells/microliter (8-152) to 18 (7-121) (p:0.048). The reduction of CD4+CD28- cells correlated with decrease of disease activity (measured by DAS28-CRP; r:0.43; p:0.045). No variation was seen after therapy in the proportion of recent thymic emigrant T cells (RTE; CD45RA+CD31+CD4+ T cells). At T0, RA patients had a higher TRBV average perturbation than HD (17.0% (13.9-25.8) vs.11.6% (10.0-14.1); p TERT expression levels in RA patients did not significantly differ from HD, but was correlated with the average perturbation of TRBV repertoire (r:0.778; p TERT expression did not significantly change during treatment. Conclusions The observed enlargement of TRBV repertoire diversity and the decreased number of CD4+CD28- cell number after ABA treatment are indicative of a renewal of T-cell compartment that may be explained by the block in the peripheral differentiation of CD4+ memory T cells after antigenic presentation and not by a thymic immune reconstitution. Acknowledgements We acknowledge Bristol-Myers-Squibb Italia for the support to our study Disclosure of Interest None Declared

Background The most important costimulatory signal for T cells is delivered by CD28 molecule. After its engagement, different functional T cell properties are enhanced, such as upregulation of surface activatory molecules (including HLA-DR), cytokines production, and cytotoxic activity. Abatacept (ABA) is a chimeric molecule able to down-modulate T-cell costimulation through the competition with CD28 engagement. Previous studies demonstrated a reduction of pro-inflammatory cytokines synthesis and of macrophage functionality after ABA treatment, but little information is available on the effect on T-cell function. Objectives The aim of the present study is to verify whether ABA might decrease T cell activation and functionality, such as γ-IFN and IL-17 production after T cell in vitro stimulation. Methods 42 consecutive patients (4 males, 38 females; median age: 55) affected from Rheumatoid Arthritis were enrolled. ABA was given for at least 6 consecutive months. DAS28 (CRP) and EULAR Clinical Response Criteria were used to analyze the disease activity and clinical response to treatment. Phenotypical analysis of peripheral primed T lymphocyte was longitudinally evaluable in 24 patients, before treatment and after 6 and 12 months of therapy. In 14 patients, peripheral blood mononuclear cells (PBMCs) were also obtained from blood sample, and stimulated for 5 hours with PMA+Ionomycin (5+500 ng/ml). T cell γ-IFN and IL-17 production was evaluated by flow-cytometry intra-cytoplasmatic staining. Results 22 patients (52%) achieved good clinical response after 6 months of therapy. Phenotypical analysis after ABA treatment demonstrated a significant reduction in the number of CD28-negative CD4+ (35 (10-151) vs 15 (1-113) cells/μl; p:0.043) and CD8+ T cells (112 (37-272) vs 39 (13-219) cells/μl; p:0.049), as well as of primed (HLA-DR+) CD4+ (19 (4-59) vs 10 (2-40) cells/μl; p:0.003) and CD8+ T cells (14 (3-43) vs 8 (1-56) cells/μl; p:0.037) and “terminally differentiated effector memory” (TDEM; CD45RA+CCR7-) CD8+ T cells (69 (22-145) vs 28 (14-207) cells/μl; p:0.041). These phenotypic variations were directly related to the variation of clinical disease activity after 6 months of ABA treatment. Among 14 individuals evaluated as far as cytokine production, 8 patients with good clinical response after 6 months of therapy showed a significant reduction of circulating CD4+ T cells producing IL-17 (19 (12-23) vs 9 (4-16) cells/μl; p:0.017) and of CD8+ T cells producing γ-IFN (70 (45-166) vs 55 (43-82) cells/μl; p:0.036) after in vitro stimulation. On the other hand, no significant variation was seen in non responding patients (n=6). Conclusions Costimulation blockade by ABA may decrease the number of circulating primed and CD28-negative CD4+ and CD8+ T-cells, as well as of TDEM CD8+ T-cells. A reduction in μ-IFN production by CD8+ T cells and of IL-17 production by CD4+ T cells after in vitro stimulation was observed in patients with good clinical response. Disclosure of Interest None Declared

Background Abatacept (ABA) is a fusion molecule used in the treatment of Rheumatoid Arthritis (RA), which is able to compete with the engagement of CD28, the receptor for most important costimulatory signal for T cells. CD28-mediated signaling regulate several T-cell functions, including inflammatory cytokine production and Treg differentiation. Objectives The aim of our study was to evaluate the effect of therapy with ABA on peripheral blood T lymphocyte cytokine production and on the number of circulating Treg. Methods In 24 RA patients treated with ABA for at least 6 months the proportion and absolute number of peripheral blood T cells producing γ-interferon (γ-IFN) and interleukin-17 (IL-17) after in vitro stimulation, as well as those of Treg were longitudinally evaluated by flow-cytometry at baseline and after 6 and 12 months. The EULAR response criteria were applied to evaluate the clinical response to treatment. Sixteen healthy donors (HD) of comparable age were used as controls. Results At baseline, compared with HD, RA patients had a higher percentage of circulating Treg (median percentage of CD4+ T cells 6.4 (25th-75th percentile: 4.8-7.5) vs 4.7 (4.0-4.8); p: 0.041), as well as of CD4+ and CD8+ T-cells producing IL-17 (1.1% of CD4+ T cells (0.4-2.0) vs 0.5 (0.3-0.8); p: 0.021; 1.2% of CD8+ T cells (0.7-1.8) vs 0.7 (0.5-0.8); p:0.006). After 6 months of therapy with ABA, there was a decrease of the percentage of Treg (from 6.4% of CD4+ T cells (4.8-7.5) to 4.8 (3.0-6.3); p: 0.008), of γ-IFN- and IL-17- producing CD8+ T cells (from 25.6% (12.9-34.4) to 17.2 (11.9-28.7), p:0.033; and from 1.2% (0.7-1.8) to 1.0 (0.6-1.3), p:0.035, respectively), while that of IL-17-producing CD4+ T cells decreased at 12 months (from 1.1% of CD4+ T cells (0.4-2.0) to 0.3 (0.2-0.5); p:0.005). The number of Treg and of IL-17 producing T-cells that were higher than in HD at baseline normalized after ABA therapy. All these variations were statistically significant only in RA patients with good clinical response (n:17), whereas the variations were milder and not significant in the other group of patients (n:7). The reduction of IL-17 producing CD4+T cells absolute number was correlated with the reduction of disease activity, evaluated with the DAS28-CRP (r: 0.477; p: 0.039). Conclusions These findings enhance our understanding of the mechanism of action of ABA underlying the relevance of the CD28-mediated signaling as pharmacological target for RA. Acknowledgements We acknowledge Bristol-Myers-Squibb Italia for the support to our study Disclosure of Interest None Declared

We have recently developed a new colorimetric method, DNA enzyme immunoassay (DEIA), for detecting specific hybrids of complementary nucleic acids. This technology is based on an antibody that selectively recognizes double, but not single-stranded DNA. The molecule does not react with a specific probe immobilized on microwells through an avidin-biotin bridge, nor with non-specific amplified sequences, since they are removed by washing. The antibody reveals the hybridization event, independently of the DNA sequences and, for this reason, the method is broadly applicable and extremely versatile. Although we chose a format based on the immobilization of the probe through an avidin-biotin interaction, DEIA assay can also be applied to other analytical schemes that do not require any modification of the probe. Most importantly, the test has an ELISA format and is rapid and convenient for processing large numbers of samples. This technology has been applied, in our laboratory, to different areas, including virology, genetic and basic immunology. The DEIA assay has been successfully used to detect the presence of hepatitis B (HBV), hepatitis C (HCV) and hepatitis delta virus (HDV) sequences in serum of patients, to discriminate different HLA alleles, to identify mutations in the Cystic Fibrosis gene, and to investigate the role of the T cell receptor in some immunological diseases. The results obtained in all our experiments demonstrated that the advantages offered by the assay do not penalize its analytical performance as compared to conventional Southern blot.

NZW rabbits with acute serum sickness given Cyclosporin A (CyA) 25 mg/kg/day develop glomerular capillary thrombosis similar to that seem in the haemolytic uraemic syndrome (HUS). Bone marrow recipients treated with CyA may also develop a similar renal lesion associated with a haemolytic uraemic-like syndrome. In the HUS, impaired production of prostacyclin by vascular tissue may be found and has been associated with a lack of a plasma factor which stimulates prostacyclin synthesis. We therefore examined, in six normal rabbits, treated with CyA 25 mg/kg for five days, the ability of plasma from treated and untreated rabbits to stimulate prostacyclin synthesis from normal rabbit aortic rings. Plasma from untreated rabbits produced 21.5 +/- 6.9 ng 6-keto PGF1 alpha/ml/mg wet weight aorta (mean +/- SEM). However, the ability of plasma from CyA-treated rabbits to stimulate prostacyclin production was profoundly reduced. This was apparent within 24 hours of starting and persisted for seven days after therapy was stopped: mean of values from all rabbits bled from start of therapy until seven days after therapy stopped was 3.7 +/- 0.5 ng/ml/mg. We suggest that the renal complications of CyA therapy are related to a failure of normal vascular prostacyclin synthesis due to lack of a prostacyclin-stimulating plasma factor.

Factors contributing to the antioxidant power of plasma may play a role in the control of arachidonic acid (AA) metabolism. The purpose of this study was to evaluate the possible effects of vitamin E deficiency on platelet and vascular prostaglandin synthesis. CD-COBS male rats were fed for 7 mo a diet containing either 1 or 75 mg/kg vitamin E. At the end of this period, serum levels of vitamin E were 0.4 +/- 0.1 and 10 +/- 0.6 micrograms/ml in the two groups, respectively. Malondialdehyde (MDA) generation by AA was significantly higher in platelet-rich plasma from vitamin E-deficient rats. Thromboxane B2 (TxB2) in serum from vitamin E-deficient rats increased about 16 times compared with controls, whereas 6-keto-PGF1 alpha levels increased only 3 times. The ratio between TxB2 and 6-keto-PGF1 alpha, increased therefore manyfold in vitamin E-deficient animals. MDA formation and [14C]AA metabolism in washed platelets were similar in the two groups of animals. No significant difference was found in the PGI2 (prostacyclin) antiaggregating activity released from the aortic rings resuspended in buffer. In contrast, the capacity of plasma to stimulate PGI2 activity from "exhausted" aortic rings (prostacyclin-stimulating factor) was significantly reduced in vitamin E-deficient animals. In conclusion, vitamin E deficiency induces an unbalanced plasma regulation of AA metabolism. This results in an excessive production of platelet TxA2 compared with vascular PGI2 generation. On the other hand, vitamin E deficiency does not seem to affect directly the enzymatic pathways of AA metabolism.

By culture of human melanoma Colo 38 cells in the presence of increasing concentrations of recombinant immune interferon (IFN-gamma), the clone RZ gamma-4G.1 resistant to the antiproliferative action of IFN-gamma (3 X 10(4) units/ml) was isolated. This clone was cultured for 6 weeks in the absence of IFN-gamma and was subsequently treated with increasing concentrations of IFN-gamma. Contrary to the response of its parental cell line, treatment of this clone with IFN-gamma did not significantly alter the rate of protein or DNA synthesis and did not markedly modulate the cell surface expression of HLA Class I antigens, of the high molecular weight melanoma associated antigen, and of a Mr 100,000 melanoma associated antigen. IFN-gamma caused an increase in the cell surface expression and in the shedding of HLA Class II antigens from IFN-gamma resistant cells. Four proteins with molecular weights of 32,000, 38,000, 46,000, and 50,000 were induced by IFN-gamma in the parental melanoma cells but not in the resistant clone. Both cell lines bound equivalent amounts of 125I-IFN-gamma to their surface, indicating that the lack of specific surface receptors was not the cause of insensitivity to IFN-gamma. These results indicate that at the cellular level IFN-gamma modulates the expression and shedding of HLA Class II antigens through different mechanisms to those responsible for the antiproliferative action, modulation of the cell surface expression of melanoma associated antigen and of HLA Class I antigens, and induction of new proteins in cultured melanoma cells. Since the success of therapy of malignant diseases with IFN-gamma depends on the extent of resistance of individual tumor cells, the present study may provide a better understanding of the biology of IFN-gamma insensitive tumor cells and particularly the malignant melanoma.

Monoclonal antibodies (MoAbs) 225.28, 657.9, and 902.5 recognizing distinct epitopes of the human high molecular weight melanoma associated antigen (HMW-MAA) were used to investigate the molecular and cellular heterogeneity of the HMW-MAA synthesized by human melanoma cells. Sequential immunodepletion and immunoprecipitation experiments showed that not all HMW-MAA molecules synthesized by a melanoma cell line express the antigenic determinants recognized by the three monoclonal antibodies. The majority of the HMW-MAA molecules expressed the epitope defined by MoAb 657.9 since this monoclonal antibody depleted the melanoma cell lysate of all antigen molecules recognized by the other two monoclonal antibodies. Depletion with MoAb 902.5 resulted in the removal of a large proportion of the HMW-MAA molecules precipitated by MoAb 657.9. The MoAb 225.28 depleted the cell lysate of only a fraction of the HMW-MAA molecules recognized by MoAb 657.9 and 902.5. Two-dimensional gel electrophoresis and peptide mapping analysis did not detect any significant difference among the HMW-MAA immunoprecipitated by the three monoclonal antibodies. The heterogeneity of the epitopes recognized by the three monoclonal antibodies is, at least partly, due to glycosylation of the antigen molecule, since treatment of melanoma cells with glycosidases differentially affects their ability to bind the three anti-HMW-MAA monoclonal antibodies. Fluorescent activated cell sorting analysis of the melanoma cells showed that the heterogeneity exhibited by the HMW-MAA is not due to the presence of different cell clones in the culture but reflects a differential distribution of epitopes on the HMW-MAA expressed on the surface of individual cells. Immunohistochemical staining of surgically removed benign and malignant lesions of melanocytic origin, of normal tissues, and of malignant lesions has shown a differential tissue distribution of the determinants recognized by the three monoclonal antibodies. Staining of melanoma cell lines and of surgically removed melanoma lesions with combinations of the three monoclonal antibodies did not cause any significant change of the percentage of stained cells but markedly increased the intensity of staining. These results indicate that combinations of monoclonal antibodies to distinct determinants of HMW-MAA can markedly increase the sensitivity of immunohistochemical techniques to detect melanoma cells.

Altered glomerular metabolism of arachidonic acid (AA) has already been demonstrated in experimental nephrotoxic nephritis. The enhanced synthesis of thromboxane A2 (TxA2) in isolated glomeruli that has been found may mediate changes in renal hemodynamics. The objectives of this investigation were: to check whether glomerular AA metabolism is also altered in a model of glomerulopathy in which no leukocyte infiltration or platelet deposition could be demonstrated; to establish a correlation between the altered AA metabolism and proteinuria; and to explore whether the alteration of the prostaglandin (PG) pathway found in isolated glomeruli is an in vitro artifact or reflects a modification in vivo. We used a model of glomerular damage characterized by heavy and persistent proteinuria, which was induced in the rat by a single intravenous injection of adriamycin. At light microscopy, minimal glomerular abnormalities were found in this model. Electron microscopy showed profound alterations of glomerular epithelial cells with extensive fusion of foot processes and signs of epithelial cell activation. Electron microscopy of numerous glomeruli showed no platelet deposition or macrophage and leukocyte infiltration in this model. Isolated glomeruli from nephrotic rats studied 14 or 30 d after a single intravenous injection of adriamycin (7.5 mg/kg) when animals were heavily proteinuric generated significantly more TxB2, the stable breakdown product of TxA2, than normal glomeruli. No significant changes were found in the other major AA metabolites formed through cyclooxygenase. Urinary excretion of immunoreactive TxB2 was also significantly higher in nephrotic than in normal animals. Administration of a selective Tx synthetase inhibitor, UK-38,485, from day 14 to day 18 after adriamycin resulted in a significant reduction of proteinuria compared with pretreatment values. Glomerular synthesis and urinary excretion of TxB2 were normal during the UK-38,485 treatment. Additional experiments showed that elevated glomerular synthesis and urinary excretion of TxB2 were not a consequence of increased substrate availability. Maximal stimulation of the renin-angiotensin axis with furosemide increased glomerular TxB2 synthesis in normal rats, which was significantly lower than in nephrotic animals. Finally, experiments using a unilateral model of adriamycin nephrosis indicated that the enhancement of glomerular TxB2 synthesis is not simply a consequence of the nephrotic syndrome. We conclude that: there is an abnormality of glomerular AA metabolism in nephritic syndrome, which leads to increased TxA2 production; the increased Tx generation correlates with protein excretion and might be responsible for altering the glomerular basement membrane permeability to protein; and the alteration found in isolated glomeruli probably reflects a modification in vivo, in that urinary excretion of immunoreactive TxB2 is also consistently increased in adriamycin nephrosis.

Two monoclonal antibodies directed against distinct epitopes of recombinant human immune interferon (rIFN-gamma) were used to investigate the relationship between the molecular organization of IFN-gamma and its various biological activities on cultured human melanoma cells. Both monoclonal antibodies inhibited the increase in the expression of cell surface human lymphocyte antigens Class I and II antigens and the antiproliferative and antiviral actions of rIFN-gamma. On the other hand neither monoclonal antibody affected the binding of rIFN-gamma to melanoma cells and its ability to reduce the expression of a high molecular weight-melanoma associated antigen. These data indicate that the functional domains of IFN-gamma responsible for antiviral activity, increased human lymphocyte antigen expression and antiproliferative effects on human melanoma cells may be distinct from that (those) involved in reduced expression of the high molecular weight-melanoma associated antigen and in IFN-gamma binding to cell receptors.

T cell clones were generated from human T cells stimulated with autologous phytohemagglutinin (PHA)-activated T (TPHA) cells. Characterization of three T cell clones originated from donor SF and one from donor JM showed that they proliferated when stimulated with autologous TPHA cells, non-T cells, and peripheral blood mononuclear cells, but did not proliferate when stimulated with allogeneic TPHA cells, non-T cells, and mononuclear cells, with autologous and allogeneic resting T cells, and with PHA. These results in conjunction with the blocking of the proliferation by anti-histocompatibility leukocyte antigen class II monoclonal antibodies indicate that these class II antigens are involved in the proliferation of T cell clones stimulated with autologous lymphoid cells. The four T cell clones are cytotoxic neither to autologous lymphoid cells nor to a panel of cultured human cell lines. The four T cell clones display immunosuppressive activity, since they inhibit the proliferation of autologous and allogeneic cells stimulated with antigens and mitogens and the secretion of immunoglobulin by B cells stimulated with pokeweed mitogen in presence of T cells. Furthermore, the four T cell clones display differential inhibitory activity on the proliferation of cultured human cell lines. The immunosuppressive activity is species-specific, since the T cell clones do not inhibit the proliferation of murine cells. The suppression is mediated by a factor(s) with an apparent m.w. of 13,000 to 16,000. The suppressor activity is labile at alkaline pH and is lost following incubation with pronase (100 U/ml) for 30 min at 37 degrees C.

Additional file 4. Correlation between the percentage of TRBV perturbations and CD4+ (A), CD8+ lymphocytes (B) and CD4/CD8 ratio (C) in perinatally HIV-infected youths (pHIVy) and in non-perinatally HIV-infected youths (npHIVy).

According to CD28 molecule expression, CD8+ T cells can be classed as CD28bright, CD28dim, and CD28−. The CD28dim T cells were found to derive from mitogenic stimulated CD28− T cells but also from CD28bright T cells through a mechanism of CD28 down-modulation. Moreover, after prolonged in vitro interleukin-2 stimulation, clonal CD28bright cells showed a CD28dim expression before further evolution to a stable CD28− phenotype. This loss was concomitant with the disappearance of CD28 mRNA. A study of the cytokine production pattern revealed that CD28dim and CD28−T cell clones produced similar levels of type 1 and type 2 cytokines, which differed from those produced by the CD28bnght T cell clones. A high percentage of CD28dim and CD28− cells, with similarities in their cytokine production pattern, were found in the blood samples of HIV-infected patients, as compared to healthy donors. The CD28 down-modulation may account for the increased number of CD8+CD28− T cells in HIV-infected patients. J. Leukoc. Biol. 65: 641–648; 1999.

Additional file 4. Correlation between the percentage of TRBV perturbations and CD4+ (A), CD8+ lymphocytes (B) and CD4/CD8 ratio (C) in perinatally HIV-infected youths (pHIVy) and in non-perinatally HIV-infected youths (npHIVy).

Additional file 2. Correlation between number of (A) TRECs and (B) telomere length (expressed as T/S ratio) with CD4+, CD8+ cells and CD4/CD8 ratio in perinatally HIV-infected youths (pHIVy), in non-perinatally HIV-infected youths (npHIVy) and in healthy controls (HC).

Additional file 2. Correlation between number of (A) TRECs and (B) telomere length (expressed as T/S ratio) with CD4+, CD8+ cells and CD4/CD8 ratio in perinatally HIV-infected youths (pHIVy), in non-perinatally HIV-infected youths (npHIVy) and in healthy controls (HC).