CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse.

Adenosine deaminase (ADA), a protein whose deficit leads to severe combined immunodeficiency, binds to the cell surface by means of either CD26, A(1) adenosine receptors, or A(2B) adenosine receptors. The physiological role of these interactions is not well understood. Our results show that by a 3-fold reduction in the EC(50) for the antigen, ADA potentiated T cell proliferation in autologous cocultures with antigen-pulsed immature or mature dendritic cells. Costimulation was not due to the enzymatic activity but to the interaction of ADA-CD26 complexes in T cells with an ADA-anchoring protein in dendritic cells. From colocalization studies, it is deduced that ADA colocalizing with adenosine receptors on dendritic cells interact with CD26 expressed on lymphocytes. This costimulatory signal in the immunological synapse leads to a marked increase (3- to 34-fold) in the production of the T helper 1 and proimmflamatory cytokines IFN-gamma, TNF-alpha, and IL-6.

Generation of Human Monoclonal Antibodies from HIV-Infected Individuals / Generación de anticuerpos monoclonales humanos a partir de individuos infectados por VIH

Recently, the protective role of anti-HIV-1 neutralising antibodies has been «re-discovered». Few broadly protective epitopes on the HIV envelope proteins are known, which were defined by few human monoclonal antibodies (mAbs) derived many years ago from non-progressor HIV-infected individuals. It is of interest to try to generate new neutralising human mAbs both to identify new protective epitopes for vaccine design and as potential passive immunotherapy agents. In addition, it is now known that rather than recognising HIV envelope proteins, some neutralising antibodies might be autoantibodies directed to the receptor (CD4) or co-receptors (CCR5 or CXCR4) on the HIV target cells. In that context, we attempted to generate neutralising human mAbs from few HIV-1-infected individuals who, after structured antiretroviral therapy interruptions, showed good virologic and immunologic responses, including serum HIV neutralising activity. We employed the classical heterohybridoma technology and found that the best screening strategy is a primary selection of IgG-producing hybridomas, followed by secondary screenings with different antigens and assays such as HIV neutralisation activity, direct binding to HIV components, binding to HIV target cells, and to cell lines transfected with human HIV receptors and co-receptors. From a single subject, 61 IgG-producing hybridomas out of 5,760 primary wells were obtained, and preliminary data indicate the presence of six (out of 23 tested) neutralising antibodies of the X4 strain, eight anti-p24 antibodies and two antibodies that bind to the MT-2 cell line, the target of X4 strains. None of the 61 mAbs obtained reacted with cells expressing CD4, CXCR4 or CCR5. Other screening tests such as neutralisation assay with the R5 strain, binding to HIV gp120-CD4 covalent complex, and to whole chemically (aldrithiol-2)-inactivated HIV are in progress. The screening strategy is also a means to “open” the repertoire of IgG antibodies of circulating B cells in HIV-infected individuals permitting to assess the frequency of HIV-related IgG antibodies and the IgV genes encoding them, an issue largely unknown (AU)

Recientemente se ha «re-descubierto» el carácter protectivo de los anticuerpos anti-VIH neutralizantes. Se conocen sólo unos pocos epitopos protectivos en las proteínas de la envoltura del VIH definidos gracias a unos pocos anticuerpos monoclonales humanos (mAbs) generados hace ya tiempo en pacientes infectados asintomáticos. Tiene interés intentar generar otros mAbs humanos neutralizantes que puedan definir nuevos epitopos protectivos para el diseño de vacunas, y ser útiles en inmunoterapia pasiva. Además, actualmente está claro que podrían existir anticuerpos neutralizantes que en vez de ir dirigidos contra la envoltura del VIH, reconocieran al receptor (CD4) o coreceptores (CCR5 o CXCR4) en la membrana de las células diana del VIH. En este contexto, hemos intentado obtener mAbs neutralizantes a partir de aquellos pacientes VIH+ que, tras un protocolo de interrupciones estructuradas de la terapia antiretroviral, mostraron buena respuesta virológica e inmunológica, con aumento de actividad sérica neutralizante. Utilizamos la metodología de heterohybridomas convencional y hallamos que la mejor estrategia de escrutinio es la selección primaria de hibridomas secretores de IgG, y luego el escrutinio con distintos ensayos para actividad neutralizante, unión a proteínas víricas, unión a la membrana de las células diana y a células transfectadas con receptores y co-receptores del VIH. A partir de un paciente, de 5.760 microcultivos primarios, se obtuvieron 61 hibridomas productores de IgG entre los que, según datos preliminares, hay seis mAbs (de 23 probados) neutralizantes contra cepas X4, ocho anti-p24 del VIH, y dos que se unen a la componentes de la membrana de la línea MT-2, diana de las cepas X4. Ningún mAb de los 61 obtenidos se unía a CCR5, CD4 o CXCR4. Hay otros escrutinios en curso como neutralización frente a cepa R5, la unión a complejo covalente gp120-CD4 y a VIH total inactivado químicamente con aldritiol-2. El escrutinio utilizado es también un modo de abrir el repertorio de anticuerpos IgG de los linfocitos B circulantes en individuos VIH+ y averiguar la frecuencia de los anticuerpos de especificidad relacionada con el VIH y los genes IgV que los codifican, un aspecto apenas estudiado (AU)

Structured interruptions of antiretroviral therapy in HIV infection. Lessons from a therapeutic strategy / Interrupciones estructuradas de la terapia antirretroviral en la infección por el vih. Lecciones de una estrategia terapéutica

Highly active antiretroviral therapy (HAART) does not c u re HIV infection and “HAART for life” would be required to avoid progression to AIDS, a problematic option given the emerging long-term HAART-associated toxicities. Therefore “structured therapy interruptions” (STI) were explored. In patients receiving HAART since the acute infection or since very early stages of chronic infection (CHI), STI was investigated as a “self-vaccination” to increase HIV-specific CD4 and CD8 T-cell responses. These studies showed that in a proportion of cases these responses can be rapidly amplified and be associated with a low viral set point in the absence of HAART at least for significant periods of time, reinforcing the hypothesis that an immune control of HIV is achievable and that combining HAART with appropriate therapeutic vaccine candidates needs to be investigated. Most patients initiate HAART at more advanced stages of CHI, and there are contradictory results on whether STI is of clinical benefit, which might depend on the more preserved immunologic status and low viral load before starting HAART, and not on the immunologic status before starting STI. If there is a history of high viral load and a CD4+ T-cell nadir < 500 cells/ mm3 b e f o re HAART, STI alone would fail to enhance HIV-specific responses and lower the viral set point, regardless of the pre-STI number of CD4+ T-cells. The goal of STI in these patients should be simply to avoid exposure to HAART while controlling viremia. For this important purpose (i) repeated STI with very short off - a n d - on HAART periods are effective; (ii) combining STI with immunosuppressive drugs is being investigated. STI combined with daily low-dose IL-2 to increase HIV-specific T-cell re s p o n s e s is also under investigation and might be an useful approach , that deserves more attention (AU)

La terapia antiretroviral de gran actividad (TARGA) no cura la infección por el VIH-1, por lo que sería necesario administrarla de por vida para evitar la progresión hacia SIDA, una opción inviable dada la crecientemente conocida toxicidad de esa terapia. Por tanto, se ha explorado la estrategia de “interrupciones estructuradas de la TARGA” (IET). En pacientes que recibieron la TARGA desde la infección aguda o desde estadios muy iniciales de la infección crónica, la IET se utilizó como una “auto-vacunación” para intentar incrementar la respuesta de las células T CD4+ y CD8+ contra el virus. Estos estudios mostraron que en una proporción de casos, dichas respuestas pueden amplificarse y asociarse con un control de la viremia en ausencia de TARGA, durante periodos significativos de tiempo, respaldando el concepto de que un control inmune de la infección es factible y que la combinación de la TARGA con vacunas terapéuticas debe investigarse. El mayor contingente de pacientes se halla en distintos estadios de la infección crónica y hay resultados contradictorios sobre si la IET es clínicamente beneficiosa, lo que puede depender de la mayor carga vírica y del menor grado de inmunodeficiencia antes de iniciar la TARGA y no del estado inmunitario antes de iniciar la IET. Cuando antes de la TARGA haya una historia de alta carga vírica y de un nadir de células T CD4+ < 500 células/mm3, la IET sola no será capaz de aumentar las respuesta T y de reducir la viremia basal en ausencia de TARGA, y sólo debería usarse para evitar la exposición a la TARGA manteniendo controlada la viremia. Sobre este importante objetivo (i) repetidas IET de muy cortos periodos con y sin TARGA son efectivas; (ii) se está investigando la combinación de IET con inmunosupresores. La combinación de IET con dosis muy bajas diarias de IL-2 para incrementar la respuesta T VIH-específica se está también investigando, una estrategia que podría ser de utilidad, y que merece ser tenida en cuenta (AU)

Effects of CCR5-Delta32, CCR2-64I, and SDF-1 3'A alleles on HIV-1 disease progression: An international meta-analysis of individual-patient data.

BACKGROUND: Studies relating certain chemokine and chemokine receptor gene alleles with the outcome of HIV-1 infection have yielded inconsistent results. OBJECTIVE: To examine postulated associations of genetic alleles with HIV-1 disease progression. DESIGN: Meta-analysis of individual-patient data. SETTING: 19 prospective cohort studies and case-control studies from the United States, Europe, and Australia. PATIENTS: Patients with HIV-1 infection who were of European or African descent. MEASUREMENTS: Time to AIDS, death, and death after AIDS and HIV-1 RNA level at study entry or soon after seroconversion. Data were combined with fixed-effects and random-effects models. RESULTS: Both the CCR5-Delta32 and CCR2-64I alleles were associated with a decreased risk for progression to AIDS (relative hazard among seroconverters, 0.74 and 0.76, respectively; P = 0.01 for both), a decreased risk for death (relative hazard among seroconverters, 0.64 and 0.74; P < 0.05 for both), and lower HIV-1 RNA levels after seroconversion (difference, -0.18 log(10) copies/mL and -0.14 log(10) copies/mL; P < 0.05 for both). Having the CCR5-Delta32 or CCR2-64I allele had no clear protective effect on the risk for death after development of AIDS. The results were consistent between seroconverters and seroprevalent patients. In contrast, SDF-1 3'A homozygotes showed no decreased risk for AIDS (relative hazard for seroconverters and seroprevalent patients, 0.99 and 1.03, respectively), death (relative hazard, 0.97 and 1.00), or death after development of AIDS (relative hazard, 0.81 and 0.97; P > 0.5 for all). CONCLUSIONS: The CCR5-Delta32 and CCR2-64I alleles had a strong protective effect on progression of HIV-1 infection, but SDF-1 3'A homozygosity carried no such protection.

Thyroid autoimmune disease: demonstration of thyroid antigen-specific B cells and recombination-activating gene expression in chemokine-containing active intrathyroidal germinal centers.

Autoimmune thyroid disease--Hashimoto thyroiditis and Graves' disease--patients produce high levels of thyroid autoantibodies and contain lymphoid tissue that resembles secondary lymphoid follicles (LFs). We compared the specificity, structure, and function of tonsil and lymph node LFs with those of the intrathyroidal LFs to assess the latter's capability to contribute to autoimmune response. Thyroglobulin and thyroperoxidase binding to LFs indicated that most intrathyroidal LFs were committed to response to thyroid self-antigens and were associated to higher levels of antibodies to thyroglobulin, thyroperoxidase, and thyroid-stimulating hormone receptor. Intrathyroidal LFs were microanatomically very similar to canonical LFs, ie, they had well-developed germinal centers with mantle, light, and dark zones and each of these zones contained B and T lymphocytes, follicular dendritic and interdigitating dendritic cells with typical phenotypes. Careful assessment of proliferation (Ki67) and apoptosis (terminal dUTP nick-end labeling) indicators and of the occurrence of secondary immunoglobulin gene rearrangements (RAG1 and RAG2) confirmed the parallelism. Unexpected high levels of RAG expression suggested that receptor revision occurs in intrathyroidal LFs and may contribute to generate high-affinity thyroid autoantibodies. Well-formed high endothelial venules and a congruent pattern of adhesion molecules and chemokine expression in intrathyroidal LFs were also detected. These data suggest that ectopic intrathyroidal LFs contain all of the elements needed to drive the autoimmune response and also that their microenvironment may favor the expansion and perpetuation of autoimmune response.

The virological and immunological consequences of structured treatment interruptions in chronic HIV-1 infection.

BACKGROUND: Some individuals with chronic HIV-1 infection have discontinued their drug therapy with consequent plasma virus rebound. In a small number of patients, a delayed or absent rebound in plasma virus load has been noted after drug cessation, apparently associated with prior drug interruptions and autologous boosting of HIV-1 specific immune responses. We hypothesized that cyclic structured treatment interruptions structured treatment interruptions (STI) could augment HIV-1 specific immune responses in chronic HIV-1 infection, which might help to control HIV-1 replication off therapy. METHODS: We initiated an STI pilot study in 10 antiretroviral treatment-naive HIV-1 chronically infected subjects with baseline CD4 T-cell counts > 500 x 10(6) cells/l and plasma viral load > 5000 copies/ml who received highly active antiretroviral therapy (HAART) for 1 year with good response (plasma viral load < 20 copies/ml for at least 32 weeks). Three cycles of HAART interruption were performed. RESULTS: In all of the patients viral load rebounded, but doubling times increased significantly between the first and third stops (P = 0.008), and by the third stop, six out of nine subjects had a virological set-point after a median 12 months off therapy that was lower than baseline before starting HAART (ranging from 0.6 log(10) to 1.3 log(10) lower than baseline) and in four it remained stable below 5000 copies/ml. Those subjects who controlled viral replication developed significantly stronger HIV-1 specific cellular immune responses than subjects lacking spontaneous decline (P < 0.05). During viral rebounds no genotypic or phenotypic changes conferring resistance to reverse trancriptase inhibitors or protease inhibitors was detected, but mean absolute CD4 T-cell counts declined significantly, although never below 450 x 10(6)/l and the mean value at 12 months off therapy was significantly higher than the pre-treatment level (P = 0.004). CONCLUSIONS: Our findings suggest that STI in chronic HIV-1 infection might augment HIV-1-specific cellular immune responses associated with a spontaneous and sustained drop in plasma viral load in some subjects but at the potential cost of lower CD4 T-cell counts.

La respuesta de células t frente a antígenos de toxoplasma gondii (tg) discrimina los individuos sanos con y sin infección crónica por tg. su interés para evaluar la reconstitución de células t cd4+ específicas de tg en pacientes con sida co-infectados por tg que reciben tratamiento antirretroviral / T-cell response to Toxoplasma gondii (Tg) antigens discriminates healthy individuals with and without chronic infection. Its interest to evaluate the reconstitution of Tg-specific CD4+ T cells in Tgcoinfected AIDS patients receiving antiretroviral therapy

La demostración de que entre las nuevas células T CD4 circulantes que aparecen tras la terapia antirretrovial de alta actividad (TARGA) en los pacientes con SIDA co-infectados con Toxoplasma gondii (Tg), se hallan también aquéllas específicas de antígenos solubles del Tg, es una información que sería de gran importancia para decidir la seguridad de interrumpir la profilaxis primaria y sobre todo la secundaria de la Encefalitis Toxoplásmica (ET). No está claro si la respuesta de células T frente a extractos de antígenos solubles de Tg (SATg) puede discriminar entre las células T específicas y las activadas policlonalmente por el SATg. Este estudio ha abordado esta cuestión comparando la respuesta linfoproliferativa y la producción de citocinas de células mononucleares sanguíneas frente a SATg y a proteínas recombinantes de Tg (rTg), SAG-1, SAG3, ROP-2, de individuos sanos Tg-seropositivos (n=12) y Tgs e ronegativos (n=12).La respuesta linfoproliferativa frente a S ATg fue claramente superior en los individuos Tg-seropositivos (p=0,003), con índices de estimulación (SI) 10 en el 92 por ciento de estos casos, y 50 UI/ml. La producción de IFN- , pero no la de IL-12p40, frente a SATg (p=0,05), así como la de ambas citocinas (p=0,02, p=0,03, respectivamente ) frente a las rTg fue superior en los Tg-seropositivos. Estos datos indican que la respuesta de células T frente a antígenos solubles de Tg permite identificar a la mayoría de individuos normales que controlan la infección crónica por Tg, y sugiere que estas pruebas podrían ser útiles para evaluar si, tras la TARGA, se restauran las células T CD4+ anti-Tg en individuos con SIDA que reciben profilaxis contra la ET (AU)

Comparison of T-cell subsets' reconstitution after 12 months of highly active antiretroviral therapy initiated during early versus advanced states of HIV disease.

UNLABELLED: This research comprised a pilot open prospective clinical study comparing T-cell subset reconstitution in antiretroviral-naive patients, after 12 months of HAART when treatment was initiated in early stages (ES; n = 18) of infection versus advanced stages (AS; n = 20). CONTROL GROUP: 10 healthy HIV-negative individuals. Immunophenotypic markers were evaluated before and after 6-and 12-months' therapy. Functional assays were performed in one subset. RESULTS: Viral load (VL) was < 200 copies/ml in all patients. Median percentages of CD4+ pretherapy were 33% and 6%, respectively, in the ES and AS groups, increment after 12 months of therapy was +15% and +13% respectively. Only the ES group achieved normal values. Declines of CD8+ percentage were significantly higher in the ES (-18%) than in the AS group (-2%). CD4+ memory and naive cells in the ES group were similar to those of controls before treatment and did not change after therapy. In contrast, CD4+ memory and naive cells in the AS group did not reach normal levels despite treatment. In the ES group, there was a significant increment in CD8+ naive cells (+8%) and a decrement in CD8+CD38+ cells (-17%), both populations reached values close to normal, whereas these subsets remained far from normal in the AS group. Improvement of lymphoproliferative response after therapy was observed in both groups. One patient in the ES group showed positive LPR against p24 after treatment. After 12 months' highly active antiretroviral therapy, only those who began such therapy in ES disease reached values within the range of healthy controls. To achieve a more complete immunologic reconstitution, early initiation of potent antiretroviral therapy should be recommended.

Comparison of immunologic restoration and virologic response in plasma, tonsillar tissue, and cerebrospinal fluid in HIV-1-infected patients treated with double versus triple antiretroviral therapy in very early stages: The Spanish EARTH-2 Study. Early Anti-Retroviral Therapy Study.

The objective of antiretroviral therapy is to obtain an almost complete and durable suppression of viral replication in all compartments to facilitate recovery of the immune system. We assessed the virologic effect in plasma, tonsillar tissue, and cerebrospinal fluid (CSF) in 94 HIV-1-infected patients with CD4 counts >500 x 106 cells per liter and viral load >5000 copies/ml randomly assigned to triple antiretroviral therapy (two nucleoside reverse transcriptase inhibitors (NRTIs) plus one protease inhibitor) versus double therapy (two NRTIs). We also analyzed the immunologic recovery in this cohort of patients. Lymphoid tissue and cerebrospinal fluid viral load, development of genotypic resistance, proliferative responses to HIV-1 specific antigens, and other immunophenotypic markers were analyzed. The proportion of patients who achieved a decrease in HIV RNA levels to <200 copies/ml was significantly greater in the triple therapy group than in the two drug groups (p =.0002 for each pair-wise difference). At week 52, tonsillar tissue HIV RNA from 5 patients treated with triple therapy was lower than the limit of detection, whereas the mean +/- standard error in patients with double therapy (n = 5) was 5.03 +/- 0.34 copies/mg/tissue. In all 10 patients, CSF viral load (VL) was <20 HIV-1 RNA copies/ml at week 52. CSF cell counts and protein levels tended to decrease after 52 weeks of antiretroviral therapy. After 1 year of therapy, 13 of 21 patients (62%) in the double-therapy groups (zidovudine plus lamivudine [n = 9] and stavudine plus lamivudine [n = 12]) had evidence of M184V mutation. None of the 10 samples of patients receiving triple therapy could be amplified because of low HIV RNA levels. The mean increase in CD4 cells at week 52 was greater in the stavudine and lamivudine and indinavir group than in the double-treatment arms (186 versus 67 and 102, respectively; p =.03). In patients treated with triple therapy, the increase in naive T cells (CD4 and CD8) was greater than in patients treated with double therapy. Markers of activation decreased further in patients treated with the regimen that included protease inhibitors. Proliferative responses to HIV-1 p24 antigen were never recovered after double or triple therapy. Our study suggests that even in very early stages of HIV-1 disease only therapy with two NRTIs and one protease inhibitor reduces plasma, lymphoid tissue, and CSF VL to undetectable levels. HIV-1-related immune system abnormalities improved but were still defective after 1 year of antiretroviral therapy.

Immunological benefits of antiretroviral therapy in very early stages of asymptomatic chronic HIV-1 infection.

OBJECTIVES: To assess whether an almost complete restoration of immune system can be achieved when antiretroviral therapy is initiated at very early stages of asymptomatic chronic HIV-1 infection. DESIGN: T cell subsets and cell-mediated responses were analysed at baseline and after 12 months of either a double or a triple antiretroviral therapy in 26 asymptomatic HIV-1-infected patients with CD4 T cell counts > 500 x 10(6) cells/l and a baseline plasma viral load > 10000 copies/ml. RESULTS: Triple therapy was significantly more effective in reducing plasma HIV RNA to undetectable levels, in returning CD4:CD8 ratio to nearly normal levels, in reducing activated cells (CD38) and in increasing naive (CD45RA+CD45RO-) and memory (CD45RA-CD45RO+) CD4 cells. Both double and triple therapies caused a clear decrease in memory (CD45RA-CD45RO+) CD8 cells as well as a significant increase in the CD28 subset of CD8 cells. At baseline, there was an important increase in cells producing interferon-gamma (IFNgamma) with no significant abnormalities in T lymphocytes producing interleukin 2 (IL-2), tumour necrosis factor alpha and interleukin 4. Both types of therapy reduced IFNgamma- and IL2-producing CD4 T lymphocytes while IFNgamma-producing CD8 cells remained increased. Even before therapy, these HIV-1-positive patients lacked significant abnormalities in the T cell responsiveness to polyclonal stimuli as well as in the secretion of CCR5 chemokines by peripheral blood mononuclear cells. CONCLUSIONS: Initiating highly active antiretroviral therapy at very early stages of chronic HIV-1 infection allows rapid and almost complete normalization of T cell subsets and preservation of T cell functions. These early-treated patients could be excellent candidates for receiving additional HIV-specific immune-based therapies, which might be essential for the control of HIV infection.

Comparison of twice-daily stavudine plus once- or twice-daily didanosine and nevirapine in early stages of HIV infection: the scan study.

OBJECTIVES: To evaluate the safety and effectiveness of once-daily didanosine and nevirapine plus twice-daily stavudine versus twice-daily administration of all three drugs. METHODS: This open-label, randomized, multicentre study enrolled 94 antiretroviral-naive patients with chronic HIV infection, CD4+ cell counts > 500 x 10(6) cells/l, and viral loads > 5000 copies/ml. Patients were treated with either 40 mg stavudine (twice daily) plus 400 mg didanosine (once daily) and 400 mg nevirapine (once daily) or 40 mg stavudine (twice daily) plus 200 mg didanosine (twice daily) and 200 mg nevirapine (twice daily). RESULTS: After 12 months, 68% of patients who received twice-daily didanosine and nevirapine had viral loads < 200 copies/ml in the intention-to-treat and 79% in the on-treatment analysis, respectively. The corresponding values for patients treated with didanosine and nevirapine, taken once-daily, were 73 and 85%. The percentages of patients in each group with viral loads < 5 copies/ml at 12 months were 40% (once daily ) and 45% (twice daily) for the intention-to-treat analysis. Five of 11 patients (45%) with plasma viral loads < 5 copies/ml at 12 months had detectable virus in tonsillar tissue. Genotypic resistance to nevirapine was noted in seven of the 14 patients with detectable viral load at month 12. Mean changes in CD4+ cell counts for patients treated with stavudine plus once- or twice-daily didanosine and nevirapine were 154 and 132 x 10(6) cells/l, respectively. Treatment was interrupted due to adverse events in seven patients (8%) (four who received once-daily didanosine and nevirapine and three treated with twice-daily doses). CONCLUSIONS: The combination of twice-daily stavudine plus once-daily didanosine and nevirapine was as safe and well tolerated as twice-daily administration of all three agents. Both regimens were equally effective in reducing viral loads and in increasing CD4+ cell counts.

A randomized study comparing triple versus double antiretroviral therapy or no treatment in HIV-1-infected patients in very early stage disease: the Spanish Earth-1 study.

BACKGROUND: Most current guidelines state that antiretroviral therapy should be considered for HIV-infected patients with plasma HIV RNA > 5000-10000 copies/ml and CD4 cells > 500 x 10(6) cells/l. However, there is increasing concern about whether this is the optimal point to begin treatment or whether it is better to delay the initiation to more advanced stages. OBJECTIVE: To study the immunological and virological benefits of starting antiretroviral therapy at these early stages. METHODS: A total of 161 HIV-infected asymptomatic patients with CD4 cell count > 500 x 10(6) cells/l and viral load > 10000 copies/ml were randomly assigned to one of five treatment groups: no treatment, twice daily zidovudine and thrice daily zalcitabine (ZDV-ddC), twice daily zidovudine and didanosine (ZDV-ddI), twice daily stavudine and didanosine (D4T-ddI), or a twice daily three-drug regimen with stavudine and lamivudine and ritonavir. The endpoints were progression to < 350 x 10(6) cells/l CD4 cells, to < 500 x 10(6) cells/l with either two Centers for Disease Control class B symptoms or an increase of viral load > 0.5 log10 copies/ml above baseline, or to AIDS or death. In various substudies, the lymphoid tissue and cerebrospinal fluid viral load, development of genotypic resistance, proliferative responses to mitogens and cytomegalovirus, and HIV-1 specific antigens and other immunophenotypic markers were also analysed. RESULTS: Progression rates to study endpoints within 1 year were greater in the control group (31%) than in all groups receiving antiretroviral therapy pooled together (5%; estimated hazard ratio 7.41; 95% confidence interval 5.72-74.55; P < 0.001). The peak mean viral load decrease was greater in the three-drug group when compared with any of the three groups with a two-drug regimen (2.32, 1.65, 1.72 and 1.84, respectively; P < or = 0.001). At 1 year, viral load remained below 20 copies/ml in 30 out of 33 patients in the three-drug group (91%) and in only eight out of 94 patients (9%) in two-drug groups (P = 0.001). The peak mean increase in CD4 cells was also greater in the three-drug group than in the double treatment arms (259 versus 85, 144 and 145 x 10(6) cells/l, respectively; P = 0.001). By comparison, 36% of patients in the three-drug group regimen had to change the therapy as a result of adverse events. Substudies were performed in 60 patients recruited at two sites. Tonsillar tissue HIV RNA was measured in seven patients (two in the two-drug groups and five in the three-drug group) in whom plasma HIV RNA was < 20 copies/ml at 1 year. It was 15151 and 133333 copies/mg tissue in the two patients from the two-drug group, < 40 copies/mg tissue in four patients in the three-drug group, and 485 copies/mg in one patient in the three-drug group. At 1 year there was a mean increase of 4.21+/-2.94% in CD8+CD38+ cells in the control group and a decrease of 9.48+/-3.36% in the two-drug groups (P = 0.01), and 19.87+/-3.64 in the three-drug group (P = 0.001 and P = 0.05, for comparisons with control group and two-drug groups, respectively). Although proliferative responses to cytomegalovirus antigens were significantly greater in those receiving antiretroviral therapy, response to HIV-1 p24 antigen was not detected in any patient in either treatment group. CONCLUSIONS: This study supports the recommendation to start antiretroviral therapy with a three-drug combination during very early stages of HIV-1 disease, at least if viral load is above a cut-off point (10000 copies/ml in our study). The risk of progression was sevenfold higher in non-treated patients at 8 months of follow-up. Some immune system parameters improved toward normal values after 1 year of antiretroviral therapy, but the proliferative response of CD4 T lymphocytes against the p24 HIV-1 antigen was not recovered. Therapeutic approaches with more potent, better-tolerated and more convenient regimens will increasingly favour early intervention with antiretroviral t

Identification of a natural soluble form of human CD5.

CD5 is a 67 kDa type I glycoprotein which belongs to the Scavenger Receptor Cysteine-Rich (SRCR) family of receptors. This family includes either cell-surface (e.g. CD6) or secreted (e.g. Spalpha) proteins implicated in the development of the immune system and the regulation of immune responses. In this study, we purified and characterised a circulating natural soluble CD5 form (nsCD5) which is indistinguishable (in apparent molecular mass, glycosylation pattern, and antibody reactivity) from a recombinant soluble CD5 form (rsCD5) composed of the three extracellular SCRC domains. The nsCD5 is a N-glycosylated 52 kDa molecule present in normal human serum and in supernatants of in vitro phorbol ester- and CD3-stimulated peripheral blood mononuclear cells. The nsCD5 concentration in sera from healthy donors is relatively low (median 1.75 ng/ml, rn=166) and is similar to that found in sera from patients suffering of various autoimmune (systemic lupus erythematosus, primary Sjogren syndrome, rheumatoid arthritis) and non-autoimmune (chronic renal failure, B-cell chronic lymphocytic leukemia) disorders. In vitro experiments indicate that nsCD5 is released by proteolytic cleavage of the membrane form. These results represent the first evidence of proteolytic release of a transmembrane SRCR family member following cell activation.

Cerebrospinal fluid HIV-1 RNA levels in asymptomatic patients with early stage chronic HIV-1 infection: support for the hypothesis of local virus replication.

OBJECTIVE: To assess HIV-1 RNA levels in cerebrospinal fluid (CSF) and their potential correlation with plasma viral load and central nervous system (CNS) HIV-1 infection markers in stable asymptomatic patients with a CD4 T cell count >500x10(6) cells/l. PATIENTS AND METHODS: Consecutive patients screened for two trials were eligible for lumbar puncture assessment. At day 0, simultaneous samples of CSF and plasma were obtained and levels of total proteins, albumin, IgG, antibodies against HIV-1 p24 antigen, HIV-1 RNA (using the polymerase chain technique) and white cells were measured. RESULTS: The integrity of the blood-brain barrier was preserved (albumin index > or =7) in 59 out of 70 patients (84%). Intrathecal production of antibodies against HIV-1 p24 antigen was demonstrated in 55 out of 70 individuals (78%). Viral load in CSF was significantly lower than plasma values (3.13+/-0.95 versus 4.53+/-0.53, P = 0.0001). HIV-1 RNA was not detected in CSF in only three of the 70 patients (4%). Overall, there was a significant correlation between plasma and CSF HIV-1 RNA levels (r = 0.43, P = 0.0001); however, in 29 patients (41%) there were significant differences (>1.5 log10 copies/ml) between the viral loads in plasma and CSF. In the multivariate analysis, a high level of protein and white cells in CSF, but not the HIV-1 RNA plasma level, were factors independently associated with a higher level of HIV-1 RNA in CSF (P = 0.0001). CONCLUSIONS: HIV-1 RNA can be detected almost always in CSF of asymptomatic patients in early stages of HIV-1 infection including those with a preserved integrity of the blood-brain barrier. The important discrepancies between plasma and CSF viral load, and the independent association between CSF abnormalities and CSF viral load, support the hypothesis of local production of HIV-1.

Dynamics of viral load rebound and immunological changes after stopping effective antiretroviral therapy.

BACKGROUND: This study addresses the dynamic of viral load rebound and immune system changes in a cohort of eight consecutive HIV-1-infected patients in very early stages [all the patients were taking highly active antiretroviral therapy (HAART} and were recruited in the coordinating center from a larger study] who decided to discontinue HAART after 1 year of treatment and effective virologic response. The safety of this procedure and the outcome with reintroduction of the same treatment was also investigated. METHODS: Plasma, cerebrospinal fluid (CSF), and lymphatic tissue viral loads were measured at baseline; lymphocyte immunophenotyping and CD4 lymphocyte proliferative responses to mitogens and specific antigens were assessed. The same antiretroviral therapy was reintroduced as soon as plasma viral load became detectable (above 200 copies/ml). RESULTS: At day 0, plasma viral load was below 20 copies/ml in all eight patients (and below 5 copies/ml in five of eight patients). A rebound in plasma viral load was detected in all patients from day 3 to day 31 with a mean doubling time of 2.01 (SE 0.29) days. Three out of eight patients achieved a peak plasma viral load at least 0.5 log10 above baseline, pretreatment values. Mutations associated with resistance to reverse transcriptase or protease inhibitors were not detected. After 31 days off therapy, CD4 lymphocytes decreased [mean 45% (SE 4) to 37% (SE 3); P = 0.04], CD8+CD28+ lymphocytes decreased [mean 59% (SE 5) to 43% (SE 4); P = 0.03], and CD8+CD38+ lymphocytes increased [mean 55% (SE 3) to 66% (SE 4); P = 0.009]. Mean stimulation indices of lymphocytes treated with phytohemagglutinin (PHA) and CD3 decreased from day 0 to day 31 from 34% (SE 8) to 17% (SE 9) (P = 0.06) and from 24% (SE 8) to 5% (SE 2) (P = 0.02), respectively. These changes were mainly contributed by the group of five patients with plasma viral load below 5 copies/ml at day 0. Viral load dropped below 20 copies/ml in all patients after 1 month of restarting the same antiretroviral regimen. CONCLUSIONS: Discontinuation of HAART after 1 year of successful treatment is followed by a rapid rebound of viral load; this rapidly returns to undetectable levels following reintroduction of the same treatment. In patients with more effective control of virus replication (viremia below 5 copise/ml), discontinuation of treatment was associated with more severe impairment of immunologic parameters.

A murine model of human cold agglutinin disease.

Progress has been limited in the treatment of cold agglutinin (CA) disease by the absence of an animal model. We have recently studied at the molecular level one CA displaying the rare anti-Sia-1b specificity (CAGAS), CAGAS displays strong CA activity and is able to haemolyse mouse RBC in the presence of complement, thus constituting a suitable Ab for creating a murine model of CA disease. In the present work we introduced CAGAS VH and VL domains into eukaryotic expression vectors and transfected them into the non-secreting mouse myeloma X63 cell line. Clones expressing complete engineered pentameric IgM kappa CAGAS (eCAGAS) recapitulating the characteristics of serum CA (sCAGAS) could be obtained. The i.p. injection of eCAGAS to normal BALB/c mice induced a typical haemolytic anaemia, as demonstrated by the presence of spontaneous cold agglutination of RBC, induction of anaemia and significant reticulocytosis. Of interest, conspicuous bilateral ear loss was observed in one of these animals. In addition, i.p. injection of X63 transfected line into BALB/c nude mice induced ascites, typical haemolytic anaemia, and shortening of the mean RBC survival. These findings validate the practical interest of constructing a transgenic mouse model expressing eCAGAS.

Anti-Sia-lb (anti-Gd) cold agglutinins bind the domain NeuNAc alpha2-3Gal in sialyl Lewis(x), sialyl Lewis(a), and related carbohydrates on nucleated cells and in soluble cancer-associated mucins.

Anti-Sia-lb (formerly anti-Gd) cold agglutinins (CAs) recognize sialylated carbohydrates on both adult and neonate red blood cells (RBCs). RBC CA activity inhibition experiments reported here indicate that the domain NeuNAc alpha2-3Gal, as found in sialyllactose, synthetic sialyl(s) Lewis(Le)(x) and sLe(a), sialyllactosamine, sialyl-fucosyllactose, and nonfucosylated sLe(a), constitutes the minimal epitope for these CAs, implicating that these autoantibodies could be able to bind this domain in sLe(x) and sLe(a) and related carbohydrates expressed on nucleated cells and in soluble cancer-related mucins. The following data obtained with the previously characterized monoclonal IgMk anti-Sia-lb CA, GAS, show that this is the case. GAS epitope expression among leukocytes that lack sLe(a) parallels that of sLe(x) determinant as detected by mouse monoclonal antibodies (MoAbs), especially MoAb KM-93. It is also found on epithelial malignant cells bearing both sLe(x) and sLe(a). GAS epitope on these nucleated cells, (1) like that present on RBC, is abolished by sialidase, unaffected by proteases, and inhibited by sialyllactose; and (2) is overlapping and/or proximal to that recognized by anti-sLe(x) MoAb, CSLEX-1, and KM-93. Moreover, CAGAS binds soluble cancer-associated mucins bearing sLe(x) and sLe(a) determinants. This binding is inhibited by sialyllactose and these mucins inhibit the RBC CA activity of CAGAS. The possible significance of anti-Sia-lb (anti-Gd) CAs as autoantibodies directed to carbohydrate ligands of host adhesion molecules that might be receptors of microbial adhesins of some CA-inducing pathogens is discussed.

Desialylation of T lymphocytes overcomes the monocyte dependency of pokeweed mitogen-induced T-cell activation.

Activation of T lymphocytes by pokeweed mitogen (PWM) is strictly monocyte (Mo)-dependent and results in T-cell mitogenesis and interleukin-2 (IL-2) secretion, coupled with an inability to utilize IL-2 due to an impaired expression of functional IL-2 receptor (IL-2R). Such IL-2R impairment could arise in PWM-activated T cells themselves or, alternatively, be the result of Mo-derived influences, as it is known that PWM binds Mo strongly and does not or poorly binds lymphocytes, and Mo becomes rapidly destroyed in PWM-stimulated cultures of blood mononuclear cells or T cells plus Mo. The present study investigated these possibilities. The results show for the first time that desialylation of T lymphocytes strongly increases their PWM-binding capacity and, in addition, overcomes the Mo requirement for PWM to induce T-cell mitogenesis and IL-2 secretion. Such secreted IL-2 levels were even higher that those found in cultures of Mo-dependent PWM-activated T lymphocytes but similarly to the latter, PWM-activated desialylated purified T lymphocytes exhibited negligible high-affinity IL-2 binding capacity and an inability to utilize the IL-2 they produced. These effects were not due to desialylation itself, as indicated by data obtained with peanut agglutinin, a lectin that becomes strongly reactive with desialylated T lymphocytes. The data clearly indicate the existence of PWM-related events capable of impairing the expression of functional IL-2R without affecting IL-2 secretion, and indicate that such events are due to mechanisms arising at the level of PWM-activated T cells themselves.