Search

To analyze immunohematologic reconstitution, particularly of natural killer (NK) cells, we evaluated 13 β-thalassemia patients after 20 and 60 days and 1 year posttransplantation with T cell-depleted HLA-haploidentical stem cells. We assessed lymphocyte and bone marrow (BM) progenitor cell phenotype and differentiation capacity, spontaneous BM cytokine production, stromal cells, and stromal cell interleukin (IL)-7 production. A reduced clonogenic capability manifested at day +20. Patients had significantly lower CD4(+) T cells versus controls, mainly in the CD45RA(+)CD62L(+) subset. NKs were among the first lymphocytes to repopulate the peripheral blood. At day +60, an increase in primitive BM progenitor cells paralleled small increases in CD4(+), naïve CD4(+), and thymic naïve Th cells. A significant increase in CD4(+) and CD8(+) markers paralleled an increase in CD3⁻CD16(+) NKs, especially with full engraftment. In patients with stable mixed chimerism we observed very low levels of CD3(+) donor chimerism early after transplant that increased over time, but a stable population of high donor NK cells, suggesting a role of these cells on donor engraftment. Stromal cells secreted less IL-7 and displayed "macrophage-like" morphology. Patients initially manifested impaired stem/progenitor cell growth and differentiation capacity in parallel with altered T cell homeostasis and a reduced T cell naïve compartment. We hypothesize that T cell compartment damage partly arises from altered new T cell production from the hematopoietic stem/progenitor cells under stromal cytokine influence. NNK subset analysis might be useful for determining transplant outcome.

Hematopoiesis in the bone marrow (BM) is maintained by specific interactions between both hematopoietic and non-hematopoietic stromal cells, which are mesenchymal stem cells (MSCs) capable of giving rise to several cell types. The human periodontal ligament (PDL), a tissue of ectomesenchymal origin, has been shown to also be a source of MSCs. We have investigated whether MSCs expanded from the PDL of healthy volunteers express characteristics similar to BM-derived stem cells using structural, immunocytochemical and molecular approaches. Their ability to support the growth of hematopoietic progenitors was also analyzed. The PDL-MSCs exhibited a fibroblast-like morphology and their chromatin was dispersed, indicating active gene transcription. The mesenchymal-related antigens CD90, CD29, CD166, CD105, and CD44 were homogeneously detected by cytofluorimetric analysis, whereas membrane CXCR4 was expressed only by a minority of cells. The PDL-MSCs differentiated in vitro into osteogenic and adipogenic cells. Immunolocalization of IL-7, IL-7Ralpha, SDF-1alpha, and CXCR4 resulted in a diffuse but specific labeling. RT-PCR analysis confirmed the expression of the above-mentioned transcripts. The cells spontaneously produced high levels of IL-7 and SDF-1alpha and were able to support the development and long-term maintenance of BM precursor cells more efficiently than murine stromal cells and similarly to normal BM human stromal cells. We examined IL-7 and SDF-1alpha secretion pathway during adipogenic and osteogenic differentiation. IL-7 increased during osteogenic and adipogenic differentiation, while the SDF-1alpha secretion was downregulated during osteogenic differentiation but increased during adipogenic induction. Our study provides evidence that in human PDL there is an accessible niche of MSCs showing the features of BM-derived MSCs.

To investigate the pathogenesis of low CD4 T-cell count in subjects who are immunological non responders (InR) to HAART.Thirty-five HIV-positive subjects on HAART for at least 1 year, all with undetectable HIV-1 RNA, were studied. Patients were defined as InR according to a CD4 cell increase20% from CD4 cell baseline or CD4 cell count200/microl; subjects with a CD4 T-cell increase20% from baseline and a CD4 cell count200/microl were defined as immunological responders (IR). We performed a comprehensive study to characterize the immune response of InR.The immunological phenotype of peripheral blood mononuclear cells, thymic naive T cells, T-cell receptor Vbeta repertoire, serum concentration of interleukin (IL)-7, the expression of IL-7Ralpha on naive and memory CD4 and CD8 T cells, and regulatory T cells (Treg) were studied.In InR a significant reduction (P0.0001) of naive and thymic naive CD4 T cells was associated with a reduced expression of IL-7Ralpha in both cell subsets, with an increased serum concentration of IL-7 was observed. Furthermore, an increased immune activation with a reduced Treg frequency and increased number of expansions of Vbeta families was observed.The reduced expression of IL-7Ralpha associated with the persistent immune activation and the alteration of Treg frequencies in part explains the low level of CD4 T cells observed in InR.

Background: Idiopathic CD4+ lymphocytopenia is defined by a stable decrease of CD4+ T cells in the absence of any known cause of immune deficiency. The mechanisms responsible for the immunological impairment are still unknown, but a regenerative failure of hematopoietic stem/progenitor cells has been hypothesized. Methods: We evaluated in the bone marrow (BM) of 5 patients with idiopathic CD4+ lymphocytopenia the phenotype of BM progenitor cells, their differentiation capacity with colony-forming cells and long-term culture-initiating cell assays, in parallel with the spontaneous IL-7 production in the patient sera. Results: Compared with controls, a regenerative failure of hematopoietic stem cells has been observed, both in ‘committed’ and in ‘uncommitted’ progenitor cells, despite high IL-7 serum levels. The percentage of phenotypically primitive CD34+CD38–DR+ cells (this includes the lymphoid precursor cells) was decreased, suggesting an involvement of the more primitive BM compartment in the de novo T cell generation. Conclusions: Despite the low number of patients, due to the low incidence of the disease, the decrease of primitive precursors sustains the possibility that diminished stem cell precursors might contribute to the development of CD4+ T cell depletion.

In most HIV-1-infected patients, highly active antiretroviral therapy (HAART) reduces plasma viral load to

Haematological abnormalities frequently occur in patients infected by human immunodeficiency virus-type 1 (HIV-1). Increasing evidence indicates that bone marrow suppression (BM) results from viral infection of accessory cells, with impaired stromal function and alteration of haematopoietic growth factor network. We have investigated the effects of antiretroviral therapy on cytokine and chemokine production by BM cells and stromal cells in a group of HIV-1-infected subjects before and during treatment. Compared with uninfected controls, an altered cytokine and chemokine production by BM cells was observed before treatment, characterized by decreased interleukin 2 (IL-2) and elevated tumour necrosis factor-alpha, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation, normal T cell-expressed and secreted) levels, along with a defective BM clonogenic activity. Antiretroviral therapy showed increased BM clonogenic capability, associated with normalization of IL-2 production and chemokine receptors expression on CD34+ cells. Pre-therapy, BM accessory cells were represented by macrophage-like cells, in some cases positive for HIV-1 DNA, suggesting that these cells are the main target of HIV-1 infection. During therapy, the stromal cells became predominantly fibroblastoid-like, as observed in normal controls, and were negative for HIV-1 DNA. Controlling HIV-1 replication may produce amelioration of stem cell activity, and restoration of stromal cell pattern and functions, with increased IL-2 production at BM level.

SUMMARYCommon variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by repeated infections and hypogammaglobulinaemia. Additionally, T-cell abnormalities including lymphopenia, decreased proliferation to mitogens and antigens, and the reduced production and expression of cytokines, have also been observed. In this study we have investigated the expression of naive, memory and activation markers in T-cell subpopulations in 17 CVID patients in comparison to age-matched normal controls. The numbers of CD4+ T cells, including CD45RA+CD62L+ and, to a lesser extent, CD45RA–CD62L+/RA+CD62L– were significantly reduced in patients, whereas CD8+ T cells were within normal range. In contrast, HLA-DR+ cells were increased both in CD4+ and CD8+ T cells. To assess the thymic output, we analysed the presence of T-cell receptor excision circles (TRECs) in CD4+ and CD8+ T cells by quantitative PCR. TRECs were decreased significantly in patients and the rate of TREC loss was higher with increasing age. TRECs correlated with naive CD4+ T cells, whereas there was an inverse relationship between TRECs and CD8+HLA–DR+ and CD8+CD45RA–CD62L+/RA+CD62L– T cells. Our results suggest the presence of a defect in the naive T cell compartment with origin at the thymic level in CVID, and indicate that TREC may be a useful marker to monitor thymic function in this primary immunodeficiency.

The effect of highly active antiretroviral therapy (HAART) on the expression of CCR5 and CXCR4 HIV coreceptors and the production of the beta-chemokines regulated upon activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta has been investigated in 30 HIV-1-infected individuals during 12-36 months of therapy. CCR5 expression was increased in both CD4 + and CD8 + subsets, whereas CXCR4 expression was upregulated only in CD4 + cells. CCR5 levels normalized during 36 months of therapy and positively correlated with the levels of memory, CD95 +, and HLA-DR + T cells. In contrast, the frequency of CXCR4-expressing cells was not significantly modified by HAART, although a downregulation was observed early after starting treatment. CXCR4 levels were significantly associated with the frequencies of naive T cells and negatively correlated with plasma viral load, CD95, and HLA-DR expression. An increased production of both spontaneous and lectin-induced RANTES, MIP-1alpha, and MIP-1beta was found at baseline in HIV-infected individuals. The spontaneous beta-chemokines production was not modified by 12 months of HAART, although a significant reduction was seen during the first months of therapy. A transient decrease of lectin-stimulated RANTES production was also observed, whereas the reduction of lectin-induced MIP-1alpha persisted for up to 12 months of therapy. In contrast, MIP-1beta secreted by phytohemagglutinin antigen-stimulated peripheral blood mononuclear cells progressively increased during HAART. In conclusion, our data indicate a normalization of CCR5 but not CXCR4 expression during suppressive therapy and changes in beta-chemokine production that may play a part in dictating the efficiency of viral infection and consequently the disease course.

Long-term non-progressors (LTNP) represent a minority of human immunodeficiency virus (HN) infected individuals characterized by stable or even increasing CD4(+) T-cell count and by stronger immune responses against HIV than progressors. In this study, HIV-specific effector CD8(+) T cells, as detected by both a sensitive ex vivo enzyme-linked immunospot (ELISPOT) assay and specific major histocompatibility complex (MHC) peptide tetramers, were at a low frequency in the peripheral blood of LTNP, and recognized a lower number of HIV peptides than their memory resting cell counterparts, Both factors may account for the lack of complete HIV clearance by LTNP, who could control the viral spread, and displayed a higher magnitude of cytotoxic T lymphocyte (CTL) responses than progressors. By combining cell purification and ELISPOT assays this study demonstrates that both effector and memory resting cells were confined to a CD8(+) population with memory CD45RO(+) phenotype, with the former being CD28(-) and the latter CD28(+) Longitudinal studies highlighted a relatively stable HIV specific effector repertoire, viremia, and CD4(+) T-cell counts, which were all correlated with maintenance of nonprogressor status, In conclusion, the analysis of HIV-specific cellular responses in these individuals may help define clear correlates of protective immunity in HIV infection. Human Immunology 62, 561-576 (2001). (C) American Society for Histocompatibility and Immunogenetics, 2001. Published by Elsevier Science Inc.

The mechanisms responsible for the hematopoietic failure in human immunodeficiency virus type 1 (HIV-1)-infected patients are still unknown. Several findings indicate that the in vitro proliferative potential of precursor cells from AIDS patients is reduced. The changes seen in bone marrow (BM) morphology and the defective BM functions associated with cytopenias have both been proposed as potential explanations. In patients treated with highly active antiretroviral therapy (HAART) an immune reconstitution associated with increased whole blood cell counts has been described. We have investigated the effects of HAART on the number of colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs), using long-term BM cell cultures (LTBMC) in a group of subjects with HIV-1 infection enrolled in an open study to evaluate the mechanisms of immune reconstitution during HAART. In each patient, the increase in colony growth was homogeneous, regardless of the type of hematopoietic progenitor cells assayed; in four subjects an increase in the most primitive progenitor cells (LTC-ICs) was observed. These findings were associated with the in vivo data showing increased numbers of BM mononuclear cells (BMMCs) after HAART and with a rise in peripheral CD4(+) T cell counts and decreased levels of plasma HIV-1 RNA. A decreased number of hematopoietic progenitor cells and/or a defective modulation of progenitor cell growth might be the cause of the hematological abnormalities in AIDS patients. Controlling HIV-1 replication by HAART could determine a restoration of stem cell activity, probably because of the suppression of factors that inhibit normal hematopoiesis.

T cell apoptosis represents a common mechanism of T cell depletion in HIV-1-infected individuals reflecting maturational and functional T cell abnormalities either directly or indirectly induced by the virus. In the present study, the effects of highly active antiretroviral therapy (HAART) on the spontaneous apoptosis of distinct T cell subsets were investigated during a 6-month follow-up in a cohort of HIV-1-infected individuals with CD4 + cell counts between 100 and 500 cells/μl and plasma HIV-1 RNA levels ≥10,000 copies/ml. We determined that the rapid and sustained increase of both naive (CD45RA + CD62L + ) and memory (CD45R0 + and CD45RA + /CD62L − ) CD4 + and, to as lesser extent, CD8 + T cells in peripheral blood was associated with a significant decrease of apoptotic CD4 + and CD8 + as well as CD3 + CD4 − CD8 − T cells. Among CD4 + lymphocytes, at enrollment, the highest frequency of apoptotic cells was observed within the memory compartment, as defined by CD45R0 expression. During HAART, however, the frequency of CD4 + CD45R0 + apoptotic T cells progressively decreased in association with a significant downregulation of surface activation markers that indicated decreased levels of systemic immune stimulation. These results indicate that effective viral suppression can contribute to progressive normalization of maturational and functional T cell abnormalities responsible for the high levels of T cell apoptosis in HIV-1-infected individuals. This, in turn, may contribute to a reduced rate of T cell loss and immune reconstitution during HAART.

The tyrosine phosphatase CD45 is a key positive element in multiple lymphocyte signaling pathways. To understand the contribution of CD45 to HIV-1-induced T cell hyporesponsiveness and apoptosis we evaluated the CD45-associated tyrosine phosphatase activity of lymphocytes from patients with different stages of HIV-1 disease and compared it with CD45 expression, spontaneous and Fas-induced apoptosis, anti-CD3-induced T cell proliferation, distribution of CCR5 delta32/wt, and cytokine production. The proliferative response to anti-CD3 as well as the CD45-associated phosphatase activity were significantly reduced in progressors. In long-term nonprogressors (LTNPs) the proliferative response to anti-CD3 was also diminished, although to a lesser extent, while the tyrosine phosphatase activity was not significantly impaired. One-third of LTNPs were found positive for the 32-bp deletion of the CCR5 gene. This mutation had no effects on anti-CD3 proliferative response or CD45 phosphatase activity. A significant reduction in IL-2 and IFN-gamma was observed in both LTNPs and in normal progressors, whereas IL-4 production was significantly decreased only in progressors. Last, we observed a significant correlation between CD45 phosphatase activity and apoptosis. We therefore conclude that the impairment of CD45 tyrosine phosphatase activity correlates with disease progression and the level of T cell apoptosis, but not with anti-CD3-induced T cell proliferation. Moreover, we suggest that evaluation of CD45 tyrosine phosphatase activity may represent an additional tool with which to assess disease progression.

SUMMARYExpression of chemokine receptors and β-chemokine production by peripheral blood mononuclear cells (PBMC) were determined in HIV-1-infected individuals before and after highly active anti-retroviral therapy (HAART) and their relationship to viral load, T cell phenotype and the expression of immunological activation markers was examined. We found that the expression of CCR5 is up-regulated in HIV-1-infected individuals while CXCR4 appears down-regulated on both CD4 and CD8 T cells compared with normal controls. These alterations are associated with the high levels of viral load. In addition, a relationship was observed between the degree of immune activation and chemokine receptor expression on T cells. However, after 3 months of combined anti-retroviral regimen, expression of CXCR4 significantly increased while CCR5 decreased when compared with pretherapy determinations. This was seen in strict association with a dramatic decrease of viral load and an increase of both CD45RA+/CD62L+ (naive) and CD45RA−/CD62L+ or CD45RA+/CD62L− (memory) T cells accompanied by a significant decrease of the expression of immune activation markers such as HLA-DR and CD38. At enrolment, both spontaneous and lectin-induced RANTES, macrophage inflammatory protein-1α (MIP-1α) and MIP-1β production by PBMC were higher in HIV-1-infected individuals compared with normal controls, although differences for MIP-1β were not statistically significant. However, RANTES and MIP-1α production decreased during HAART at levels closer to that determined with normal controls, while MIP-1β production was less consistently modified. These data indicate that the expression of chemokine receptors CCR5 and CXCR4 and the production of β-chemokines are altered in HIV-infected individuals, and suggest that their early modifications during HAART reflect both the peripheral redistribution of naive/memory T cell compartments and the decrease in levels of T cell activation. Such modifications in the expression of host determinants of viral tropism and the production of anti-viral molecules may play a role in the emergence of virus variants when a failure of HAART occurs.

Background The incidence of Kaposi's sarcoma (KS) is increased severalfold in individuals infected with human immunodeficiency virus-1 (HIV). Human herpesvirus 8 (HHV8) has also been implicated in KS. We investigated several factors that may determine the onset of KS, particularly HHV8 infection in individuals after becoming seropositive for HIV. Methods We studied 366 individuals belonging to different HIV-exposure categories (i.e., homosexual activity, intravenous drug use, and heterosexual contact) for whom a negative HIV serologic test and then a positive HIV serologic test were available within a 2-year period. HHV8 antibody testing was performed by use of an immunofluorescence assay on the first serum sample available after the first positive HIV test. Actuarial rates of progression of KS and of other acquired immunodeficiency syndrome (AIDS)-defining diseases were estimated by use of time-to-event statistical methods. All statistical tests were two-sided. Results Twenty-one of the 366 study participants developed AIDS-related KS, and 83 developed AIDS without KS. One hundred forty (38.3%) participants had detectable anti-HHV8 antibodies. The actuarial progression rate to KS among persons co-infected with HIV/HHV8 was nearly 30% by 10 years after HIV seroconversion. Increasing HHV8 antibody titers increased the risk of developing KS (for seronegative versus highest titer [1:125 serum dilution], adjusted relative hazard [RH] = 51.82; 95% confidence interval [CI] = 6.08-441.33) but not of other AIDS-defining diseases (adjusted RH = 1.14; 95% CI = 0.72-1.80). HHV8-seropositive homosexual men compared with HHV8-seropositive participants from other HIV-exposure categories showed an increased risk of KS that approached statistical significance (adjusted RH = 6.93; 95% CI = 0.88-54.84). Conclusions Approximately one third of individuals co-infected with HIV/HHV8 developed KS within 10 years after HIV seroconversion. Progression to KS increased with time after HIV seroconversion. Higher antibody titers to HHV8 appear to be related to faster progression to KS but not to other AIDS-defining diseases.

SUMMARY To investigate whether defective costimulatory signals could be involved in the loss of T lymphocyte functions during HIV-1 infection, we tested the effect of CD28 costimulation on both T cell receptor/CD3 and HIV-1 antigen-induced proliferative responses. Although CD3-mediated responses significantly decreased with more advanced stages of HIV-1 infection, the ability of potentiating the responses through CD28 costimulation was maintained at all stages and did not differ from that of HIV-1− subjects. When CD28 costimulation was studied in lymphocyte cultures stimulated with HIV-1 gp160 or p24, potentiation was seen only when a significant response was present without additional CD28 triggering, namely in subjects receiving active immunization with recombinant gp160. These results confirm the integrity of the CD28 pathway of costimulation during HIV-1 infection, and suggest that lymphocytes responding to soluble HIV-1 antigen are not deleted in HIV-1-infected patients, but do not receive significant priming during the natural course of the infection.

The authors studied CD40 ligant (CD40L) expression and interleukin-10 (IL-10) production in 16 patients with common variable immunodeficiency (CVI). Mean CD40L expression, determined by using cytofluorimetry, and measured as the mean fluorescence intensity following stimulation of peripheral blood mononuclear cells (PBMC) with phorbol myristate acetate (PMA) and calcium ionophore in 12 patients, was comparable to that of controls. However, three CVI patients showed fluorescence intensity in stimulated cells below 2 standard deviations of normal donors' mean and two other patients had only a slight increase of stimulated versus unstimulated cells (< 10 channels). IL-10 production after stimulation of PBMC with both anti-CD3 or anti-CD3 plus PMA gave similar results in CVI patients and normal controls. In vitro stimulation of PBMC with anti-CD40 and various combinations of cytokines (IL-2, IL-4 and IL-10) induced IgG production above 100 ng/ml in one CVI patient out of 13 tested. The data suggest that alterations of IL-10 production are unlikely to play a major role in the pathogenesis of impaired IgG production in most CVI patients. CD40L appears to be normally expressed in two thirds of CVI patients, but it may be functionally defective.

The aim of the present study, a multicentre trial of didanosine (ddI) compassionate use, was to identify factors associated with a better outcome in patients given ddI monotherapy. Enrolled were 1047 HIV-positive patients intolerant of and/or unresponsive to zidovudine (ZDV) therapy, with CD4+ cell counts of < 200/μl or AIDS. Didanosine was given at a dose of 250 mg b.i.d. (patients ≥ 60 kg) or 167 mg b.i.d. (patients < 60 kg). Clinical examinations and laboratory tests were performed every two months. Endpoints included death, the occurrence of a new AIDS-defining disease, or permanent discontinuation of ddI for a severe adverse event. At entry, the median CD41 cell count was 47/μl and the median duration of prior ZDV treatment 19 months; 446 patients (43%) were classified as having AIDS. Severe toxicity occurred in 143 subjects (14%); the frequency of pancreatitis was very low (0.2%), The benefit in terms of CD4+ cell counts was greater for patients whose counts exceeded 100/μl at entry and remained at this level until month 12 in those patients still receiving treatment. Death and/or new AIDS-defining events were observed in 374 cases (36%) over a median follow-up of eight months. AIDS dementia was observed in 11 patients (1 %). Multivariate analysis of survival without disease progression showed that the factors associated with a worse outcome include the severity of immunodepression, a diagnosis of AIDS at entry, and a history of both intolerance of and clinical resistance to ZDV. Surprisingly, the patients who had received previous prolonged treatment with ZDV had a better outcome. In conclusion, severely immunodepressed patients previously administered long-term monotherapy may receive a short-term benefit from being switched to another antiretroviral drug.

In this paper we investigated the lymphoproliferative and cytotoxic responses of peripheral blood mononuclear cells (PBMC) of patients affected with lymphoproliferative disease of granular lymphocytes

The main purpose of this research was (i) to perform a comparative study of sperm parameters in human immunodeficiency virus (HIV) seropositive and high risk subjects in order to identify any possible alterations in the semen which specifically result from HIV infection and (ii) to study the p24 antigen as an early diagnostic marker of infection in high risk subjects. HIV seropositive subjects showed no significant variations regarding sperm densities, motility and viscosity compared to high risk subjects and controls. On the other hand, these HIV seropositive subjects showed (a) a significantly higher percentage of cytoplasmic droplet forms and immature germ cells, perhaps caused by an early failure of epididymal function and/or by a condition of stress affecting spermatogenesis after HIV infection and (b) a significantly higher level of spermiophage cells, suggesting that HIV activates mechanisms that increase spermiophagy. In addition, HIV seropositive men showed a significant positive correlation between blood CD4+ and sperm motility as well as a significant inverse correlation between CD4+ and sperm abnormalities. This is perhaps due to a decrease in testosteronaemia leading to defective epididymal sperm maturation. To date, p24 has not been found in the serum or seminal plasma of high risk subjects. The longitudinal study in progress should provide further information on this point.

Impaired haematopoiesis during HIV-1 infection may be caused by the overproduction of inflammatory cytokines by immune cells at the bone marrow level inducing Fas-mediated apoptosis of stem progenitors. In this study, we evaluated the effects of highly active antiretroviral therapy on apoptosis of CD34+ stem cells derived from the bone marrow of HIV-1-infected patients, and observed decreased Fas expression on progenitor cells, in parallel with the diminution of TNF-alpha levels and the amelioration of clonogenic parameters.

The prevalence of human herpesvirus 6 (HHV-6) infection and the course of human immunodeficiency virus (HIV) disease were investigated in 25 Romanian children with nosocomial HIV-1 infection. HHV-6 IgM and IgG antibodies were detected by enzyme immunoassay (EIA) and immunofluorescence assay (IFA) at the beginning of the study and after 18 months, concomitantly with collection of virologic, immunologic and clinical data. The initial HHV-6 seropositivity was 92% by EIA and 76% by IFA, whereas final testing showed 100% positivity by EIA and 84% by IFA. Positive HHV-6 IgM antibodies were detected in 10 children (40%) by EIA and IFA. Of these 9 children (36%) by EIA and 6 (24%) by IFA had both initial and final IgM antibodies. Children with HHV-6 IgM antibodies had a higher prevalence of pneumonitis than those without (100% vs. 53.3%; P < 0.01). In addition they more frequently showed positive p24 antigen detection (67% vs. 40%) and positive HIV-1 culture (80% vs. 69%). Nevertheless the patients with HHV-6 IgM antibodies showed a slight increase in the final mean CD4+ T cell count (from 1.140 to 1.185 x 10(6)/liter), whereas those with HHV-6 IgG alone showed a statistically significant (P = 0.01) decrease (from 1.395 to 968 CD4+ T-cells x 10(6)/liter). Therefore current or recent HHV-6 infection, as revealed by positive HHV-6 IgM antibodies, appeared to be associated with the development of pneumonitis but not with progression of HIV disease. A possible competitive inhibition of HIV-1 by HHV-6 or a stimulating effect of HHV-6 on CD4+ T-cell production may be suggested.

SUMMARY We tested for infection with hepatitis C virus (HCV) in 58 patients affected by humoral immunodeficiencies: 43 common variable immunodeficiency (CVI), two hyper IgM syndrome (HIM), two IgG subclass deficiency, four ataxia-telangiectasia (AT), and seven X-linked agamma-globulinaemia (XLA). While the assessment of serum specific HCV antibodies in some of these patients was not informative because of the impairment in specific antibody production, the reverse transcriptase polymerase chain reaction (RT-PCR) assay used to detect serum HCV RNA was a useful method for diagnosing infection. We found that 38% of late onset hypogamma-globulinaemic patients (CVI, HIM or IgG subclass deficiency) had evidence of HCV infection. HCV infection was not detectable in patients with XLA or AT. The majority of our pa hems had persistent viraemia. and those who underwent liver biopsy showed histological findings of chronic hepatitis. Moreover, we could demonstrate in vitro that eight of 18 HCV-infected patients were actively producing anti-HCV antibodies, despite their impaired antibody production. The high rate of HCV infection in hypogammaglobulinaemic patients could be related to several nosoeomial routes of transmission, including intravenous immune globulin administration. Despite the persistent viremia only two patients had cirrhosis and none had hepatocarcinoma.

SUMMARYIn vitro lymphoproliferative responses to HIV-1 recombinant antigens (gp160, p24, and Rev protein) were studied in 83 patients with asymptomatic HIV-1 infection (CDC groups 11 and III) and circulating CD4 lymphocyte numbers > 400/mm3. Significant response to at least one of the three antigens was detected in 52.4% of the subjects, but the responses were weak, and concordance of the response to the three antigens was rare, the frequency of individuals responding to each antigen not exceeding 22.4%. Increasing frequencies of response were observed when recall antigens (tetanus toxoid and Candida albicans glycomannoprotein) (65.5%) and anti-CD3 MoAb (76.6%) were used as stimuli. Although a significant association between lymphocyte response to p24, but not gp160, and steadiness of CD4 lymphocyte numbers before the assay was observed, no predictive value for lack of CD4 cell decrease was confirmed for either antigen, and fluctuation of the responses to HIV antigens was seen during subsequent follow up. The panel of T cell assays used could be regarded as appropriate for monitoring both HIV-specific responses and T lymphocyte function during immunotherapy with soluble HIV antigens.

Increased levels of serum IgE and eosinophilia have been described in human immunodeficiency virus (HIV) infection, almost exclusively in patients with CD4+ cell count < 200 cells/microliters. IgE production is regulated by CD4+ T helper type 2 (Th-2) lymphocytes, producing interleukin 4 (IL-4) and expressing a ligand for the B cell-specific CD40 molecule (CD40 ligand [L]). A shift to a Th-2-like pattern of cytokine secretion has been postulated to be associated with progression toward acquired immunodeficiency syndrome (AIDS). We studied three AIDS patients with very high levels of IgE and almost complete depletion of CD4+ lymphocytes, suggesting that IgE synthesis could not be driven by CD4+ cells. IgE in vitro synthesis by cells from such patients was, however, inhibited by anti-IL-4. We show that both CD8+ T cell lines and the majority of CD8+ T cells clones derived from these patients produce IL-4, IL-5, and IL-6 in half of the cases together with interferon gamma (IFN-gamma). 44% of CD8+ T cell clones expressed a CD40L, and the supernatants of the clones were capable of inducing IgE synthesis by normal B cells costimulated with anti-CD40. CD8+ T cells in these patients therefore functionally mimic Th-2 type cells and may account for hyper-IgE and eosinophilia in the absence of CD4+ cells. The presence of such CD8+ cells may also provide a source of IL-4 directing the development of predominant Th-2 responses in HIV infection.

The aim of this study was to assess the effect of natural killer stimulatory factor (NKSF)/IL-12) on the cytolytic potential of natural killer (NK) cells from normal donors or HIV-1 infected individuals, at different stages of disease. The results obtained confirm the powerful ability of IL-12 to enhance PBLs' cytotoxic activity to tumour cells in normals and in those HIV-1 infected patients, with maintained cytotoxic activity. IL-12 was however poorly effective in enhancing the cytolytic potential of any donors. Similar results were obtained adding IL-2, even at doses as high as 100 IU/ml in patients with CD4 number below 200/mm3. These results should be taken into account when designing therapeutic trials of HIV-1 infection based on cytokines.

Objective Investigation of the prevalence and pathogenic role of parvovirus B19 infection in Italian and Rumanian children with AIDS, compared with age-matched HIV-negative children (controls) with various recurrent infections of unknown aetiology. Design Detection of B19-specific immunoglobulin (Ig) M and IgG antibodies as the most indicative markers of past or current B19 infection. Methods B19 antibodies were detected by two enzyme immunoassays using synthetic peptide or recombinant protein, which corresponded to different B19 epitopes, as coating antigens. Results B19 IgM and IgG were seen in 10 out of 20 (50%) Italian and in 20 out of 51 (39.2%) Rumanian children with AIDS, in contrast to none out of 17 Italian and one out of 22 Rumanian controls (P less than 0.001). In addition, two Italian controls (11.8%), two Rumanian children with AIDS (3.9%), and two Rumanian controls (9.1%) had B19 IgM alone. Specific IgG alone was detected in eight (40%) Italian and 14 (27.5%) Rumanian children with AIDS, and in seven (41.2%) Italian and four (10.2%) Rumanian controls. Conclusions While it is possible to attribute some B19 infections in Rumanian children to blood transfusion, the source was unknown for Italian children. However, in three of the Italian children who had B19 IgM and IgG persistently for 15-22 months, and in a 2-month-old Italian infant with B19 IgM and IgG, HIV might have activated a congenital or perinatally-acquired B19 infection.

Several immunological abnormalities have been observed in ataxia-telangiectasia (AT), the most consistent being defects of immunoglobulin isotypes, decreased T-cell numbers, and reduced proliferative responses to mitogens. We examined the distribution of T lymphocytes expressing distinctive surface Ag characteristic of "naive" (CD45RA+) and "memory" (CD29+, CD45RO+) T cells, in both CD4+ and CD8+ (bright and dim) lymphocytes from 13 AT patients, compared with healthy age-matched controls. We found that, irrespective of age, patients with AT had a severe deficiency of CD4+/CD45RA+ lymphocytes. This decrease accounted for the reduction of total CD4+ cells, since the absolute numbers of memory CD4+ cells were not significantly different in AT and in controls. Functional tests revealed poor proliferative responses to phytohemagglutinin and normal responses to soluble Ag (tetanus toxoid) in AT patients. These data fit with the distribution of naive and memory cells, which are known to respond predominantly to mitogens or to recall Ag, respectively. CD45RA molecules were normally expressed on CD8+ lymphocytes. This rules out a generalized defect of regulation or differential splicing as the cause of defective expression of CD45RA on CD4+ cells. The selective deficiency of CD4+CD45RA+ may provide a cellular basis for some functional T-cell abnormalities of AT patients. Furthermore, it might practically serve for an early, or even prenatal, diagnosis of this disease.

Summary The anti-IgE autoantibody was detected, using a radioimmunoassay, in 17 out of 35 (48.6%) of patients with atopic dermatitis. Significant increased levels of IgG-anti-IgE were seen in the patients studied compared with the control group. The specificity of the anti-IgE autoantibody was confirmed by competitive inhibition assay using IgG, IgM, IgE myeloma. A correlation was observed between the levels of IgG -anti-IgE and serum IgE but not between the IgG subclasses with anti-IgE activity and the clinical status. These data demonstrate that the IgG subclass distribution with anti-IgE activity belongs mostly to the IgG1 and IgG4 subclasses compared with the controls. Moreover, ultracentrifugation analysis indicated that the IgG-anti-IgE in the serum samples from the patients with atopic dermatitis was present in the form of an immune complex with self-IgE. These observations may suggest that the anti-IgE complexes may play a broader role in the modulation of the immune response and that this autoantibody may mask recognition of IgE in conventional assays.

Background: Evans syndrome (ES) is a rare disorder characterized by combined autoimmune thrombocytopenia and autoimmune hemolytic anemia. Several studies have documented a number of B cell defects, whereas only limited information is currently available about the T cell subset. Methods: A wide panel of immunological analyses aiming specifically at a quantitative and qualitative evaluation of the T cell compartment was performed in an unusual case of ES. The peripheral distribution of the T cell subsets, the diversity of the T cell receptor (TCR) repertoires, the cytokine profile and the T cell apoptosis have been longitudinally evaluated. Results: On first investigation, flow-cytometric immunophenotyping showed a remarkable alteration of T cell homeostasis with deeply reduced CD4+ naive T cells and recent thymic emigrants. This was seen in association with increased levels of T cell activation and apoptosis. Consistently with these data the cytokine profile was characterized by high interferon-γ and low interleukin-2 levels. Staining for CD4 and CD25 molecules showed decreased percentages of circulating regulatory T cells according to the autoimmune nature of ES. Finally, restricted TCR repertoires were demonstrated by a skewed TCR β chain variable (TCRBV) gene usage as well as oligoclonal third complementarity- determining region (CDR3) profiles. A deterioration of the above-mentioned parameters and a worsening of the clinical condition were observed during the follow-up requiring more intensive treatments. Conclusion: The demonstration of multiple T cell defects, in addition to providing pathogenetic information, is likely to alter both acute treatment and outcome of ES.

Hematological abnormalities frequently occur in patients infected with HIV-1. Increasing evidence indicates that bone marrow (BM) suppression results from viral infection of accessory cells, with impaired stromal function and alteration of hematopoietic growth factor network. We investigated the effects of antiretroviral therapy on cytokine and chemokine production by BM cells and stromal cells, in a group of HIV-1-infected subjects before and during treatment. Compared with uninfected controls, an altered cytokine and chemokine production by BM cells has been observed before treatment, characterised by decreased IL-2 and elevated TNF-alpha, MIP-1alpha, MIP-1beta, and RANTES levels, along with a defective BM clonogenic activity. Antiretroviral therapy determined an amelioration of stem cell activity, a restoration of stromal cell pattern and functions, and an increased IL-2 production at BM level and a decrease of Fas expression on progenitor cells, in parallel with the diminution of TNF-alpha levels. HIV-1 protease inhibitors (PIs) may improve hematopoietic functions owing to their direct effects on the BM progenitor cells. Ritonavir and indinavir increased the colony growth of BM obtained either from HIV-1-infected patients or from normal individuals, in parallel with the normalization of functional and morphologic characteristics of stromal cells.

Background Recurrent lower respiratory tract infections caused by encapsulated bacteria might cause permanent organ damage in patients with common variable immunodeficiency (CVID). Despite the profound hypogammaglobulinemia, some patients do not experience bacterial pneumonia. We have shown that IgM memory B cells and natural antibodies play an important role in the defense against encapsulated bacteria. Objective In this study we addressed the question of whether the apparent paradox of patients with severe hypogammaglobulinemia but no increased frequency of respiratory infections can be explained by the presence of IgM memory B cells and anti-pneumococcal polysaccharide (anti-PnPS) IgM. Methods We measured the frequency of memory B cells and the levels of anti-PnPS IgM antibodies in 26 patients with CVID with recurrent bacterial pneumonia and bronchiectasis (group 1) and 22 who never had pneumonia and showed no lung lesions (group 2). An additional 6 patients had a clinical history of recurrent pneumonia without lung abnormalities at computed tomographic scanning. Results Patients of group 1 lacked IgM memory B cells and failed to produce anti-PnPS IgM antibodies, and those of group 2 had a normal frequency of IgM memory B cells and produced anti-PnPS IgM antibodies. Conclusions IgM memory B cells and anti-PnPS IgM antibodies protect patients with CVID from bacterial pneumonia. Evaluation of these 2 parameters discriminates patients with low or high risk of recurrent infections caused by encapsulated bacteria and low or high risk of bronchiectasis. Identification of high-risk individuals at diagnosis might help in the planning of a more effective therapeutic strategy and prevent permanent organ damage

In patients with primary Ab deficiencies, hematological and immunological abnormalities are frequently observed. A regenerative failure of hemopoietic stem/progenitor cells has been hypothesized. We evaluated in the bone marrow (BM) of 11 patients with common variable immunodeficiency, the phenotype of BM progenitors and their in vitro growth by colony-forming cell (CFC) and long-term culture (LTC) assays. A significant decrease in erythroid and mixed CFC and, to a greater extent, in primitive LTC-CFC progenitors was observed in patients compared with healthy controls. The frequency of BM pre-B and pro-B cells correlated directly with the absolute number of CD19+ lymphocytes. BM cells cultured in vitro produced spontaneously lower amounts of IL-2 and elevated levels of TNF-α compared with controls, indicating a skewing toward a proapoptotic cytokine pattern. In addition, stromal cells generated after BM LTC secreted less IL-7 and displayed by immunohistochemistry an altered phenotype. These findings were associated with a significant decrease in naive Th cells coexpressing CD31 in the peripheral blood. These results indicate an impaired growth and differentiation capacity of progenitor cells in patients with common variable immunodeficiency.

Proinflammatory cytokines are deemed to play a pivotal role in the pathogenesis of multiple sderosis (MS). They provide signals for T-cell activation and inflammatory cell recruitment in the brain and might directly alter neuroglial and neuronal cell survival and function. We found that peripheral blood mononuclear cells (PBMCs) from MS patents spontaneously produce high levels of TNFalpha, TNFbeta, IFNgamma, and oncostatin M (oncM), a proinflammatory cytokine actng on cells of neural, vascular, hematopoietic, and lymphoid origin. Spontaneous production of these cytokines was significantly higher (p < 0.01) in PBMC short-term culture supernatants from MS patients than in blood donors (HC). On average, lectin-induced production of these cytokines by PBMC was higher in MS patents than in HC significantly so only for TNFalpha (p = 0.013). Determination of TNFalpha, TNFbeta IFNgamma, and oncM in corresponding sera showed that on average, oncM levels were higher in MS patients than in HC, though the results were not statistically significant whereas levels of TNFalpha, TNFbeta and IFNgamma were below the assay threshold in most patients. The finding that MS PBMCs are primed in vivo to produce and release high levels of proinflammatory cytokines suggests the presence of a basal activation of the immune system which, in turn, may play a role in the complex circuitry of molecular and cellular interactions responsible for neurologic damage in MS.

Receptors interacting with Major Histocompatibility Complex class I molecules have been initially found on the surface of human natural killer (NK) cells, where they deliver inhibitory signals to the lysis, being thus defined killer inhibitory receptors (KIR). Subsequently, they were detected also on the surface of T-CD8+ lymphocytes and are particularly expanded during human immunodeficiency virus (HIV) infection, where they downregulate HIV-specific cytolysis. The expression of KIR recognizing human leukocyte antigen-C alleles was assessed in HIV-infected patients, undergoing highly active antiretroviral therapy (HAART). To this end, the combined expression of CD16/CD56, of CD3 and CD8 as well as of KIR (CD158a and CD158b) surface molecules was analyzed on peripheral blood mononuclear cells by monoclonal antibodies, and flow cytometry. An increase of CD3+CD8+CD158b+ cells was found after 6 months of HAART. This finding may have implications for the regulation of T-cell mediated cytolysis during HAART.

SUMMARYDiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) are associated with chromosome 22q11.2 deletion. Limited information is available on the T cell receptor (TCR) Vβ repertoire. We therefore investigated TCR Vβ families in lymphocytes isolated from blood and thymic samples of seven patients with DGS and seven patients with VCFS, all with 22q11.2 deletion. We also studied activities related to TCR signalling including in vitro proliferation, anti-CD3-induced protein tyrosine phosphorylation, and susceptibility to apoptosis. Reduced CD3+ T cells were observed in most patients. Spontaneous improvement of T cell numbers was detected in patients, 3 years after the first study. Analysis of CD4+ and CD8+ TCR Vβ repertoire in peripheral and thymic cells showed a normal distribution of populations even if occasional deletions were observed. Lymphoproliferative responses to mitogens were comparable to controls as well as anti-CD3-induced protein tyrosine phosphorylation. Increased anti-CD3-mediated apoptosis was observed in thymic cells. Our data support the idea that in patients surviving the correction of cardiac anomalies, the immune defect appears milder than originally thought, suggesting development of a normal repertoire of mature T cells.

During HIV disease an increased in vitro apoptosis of peripheral blood mononuclear cells has been demonstrated. This can be reversed in vitro by interleukin (IL)-2. Recent trials with highly active antiretroviral therapy (HAART) and IL-2 in HIV-1-infected patients showed promising immunological and clinical results. Here we investigated the effects of subcutaneous low-dose IL-2 administration in combination with HAART on in vitro apoptosis and the relationship between apoptosis, CD4 + counts, and HIV replication. Twenty-two asymptomatic HIV patients were randomized for HAART (arm I) and HAART plus IL-2 (arm II). Spontaneous apoptosis was decreased in both arms after 28 weeks of therapy but the reduction was highly significant only in arm II ( P = 0.05 vs P = 0.001). As the percentage of apoptosis decreased, there was a significantly higher increase of both CD4 + and CD4 + naive T cells in arm II vs arm I. HIV plasma viremia was reduced in all patients after therapy. Our data suggest that intermittent therapy with low-dose subcutaneous IL-2 in addition to HAART induces a positive immunomodulation in asymptomatic HIV-infected patients.

HIV infection is immunologically characterized by progressive decrease of CD4’ lymphocytes and hypergammaglobulinemia. 1 Clinically, it spans from asymptomatic infection to full-blown AIDS, divided into definite stages by the Center for Disease Control classification.* A high incidence of atopic manifestations has been reported in HIV seropositive individuals3* ’ with increased serum levels of IgE.5 Recently, the rise of serum IgE has been related to the decrease of CD4+ cells.6 We selected 22 HIV seropositive subjects at different stages of disease, who presented allergic symptoms either for the first time or as a reexacerbation of previous atopic disease. In their sera, we measured the levels of total IgE, allergen-specific IgE, and ECP, a basic protein with cytotoxic properties released by EOSs, which is increased in sera and secretions of atopic patients.‘.’ As control subjects, we chose 25 healthy nonatopic subjects, 20 patients with acute recurrent infection (both bacterial and viral), and 29 HIV seropositive cases without history or present symptoms of atopy. Care was taken to match the HIV groups for age, sex, and disease stages. Detailed data on the composition of the groups are reported in Table I. Additional tests in HIV-positive subjects were white blood cell count, EOS count, both by standard methods, CD4’ lymphocyte count, with direct immunofluorescence with monoclonal anti-CD4 (OKT4, Ortho Diagnostic Systems, Raritan, N . J .) and read by flow cytometry (Cytoron, Ortho Diagnostic Systems), and serum levels of IgG, IgA, and IgM by nephelometry with a TDX analyzer (Abbott Diagnostics, Irving, Texas). Results of serum levels of IgE, mea

Objectives: Evaluation of immunological reconstitution after 2 years of highly active antiretroviral therapy (HAART) in AIDS patients. Design: Previous data showed the effectiveness of HAART but conflicting evidence of immune reconstitution has been found in severely immunocompromised patients. Therefore, T-cell subsets and functions were analysed during 24 months of HAART in 21 AIDS patients (mean baseline CD4 cell count, 20¥106/l). Methods: Subjects were tested at baseline and after 4, 12 and 24 months of therapy for clinical symptoms and the following investigations were carried out: plasma HIV RNA, T-cell subsets and lymphoproliferative responses to mitogens (phytohaemagglutinin, anti-CD3), and recall antigens (Candida mannoprotein, tetanus toxoid and recombinant glycoprotein 160). Results: Increase in body weight, improvement of Karnofsky‚s score and reduction of opportunistic infections were observed. All patients showed an initial increase in the CD4 memory subset, whereas naive CD4 cells consistently increased only after 1 year. The magnitude of immune recovery was stronger in patients showing a significant reduction in viral load. However seven out of 21 patients who did not reach a sustained suppression of viral load showed also an increase in T-cell subsets. The majority of patients recovered lymphoproliferative responses to mitogens, whereas only four subjects showed a functional response to Candida mannoprotein. No patients showed a response to HIV recombinant glycoprotein 160 or tetanus toxoid. Conclusions: The immune recovery observed is slower and not complete in severely immunocompromised patients. Our data suggest that HAART may be continued also in the absence of a significant HIV RNA decrease if alternative drugs are not available.

This study reports an analysis of clinical, virological, and immunologic outcomes in a cohort of 77 multidrug-experienced AIDS patients during 24 months of highly active antiretroviral therapy (HAART). Our results have shown a reduced risk of AIDS complications, prolonged survival, and immunologic benefit even in the absence of sustained virus suppression. The degree of immunodepression, the risk factors for HIV-1 infection, the use of 2 drugs instead of 3, and a change in protease inhibitor were independently correlated with virological failure. In the majority of studied patients, an increase in CD4 + T cells was observed after HAART. However, the increase was more pronounced in patients who showed a decrease in virus load than in those who did not. Moreover, we observed an absence of relapses among patients who permanently discontinued prophylaxis for Cytomegalovirus retinitis and atypical mycobacterial infections. Peripheral lipodystrophy developed in the majority of patients, regardless of treatment used and virological outcome. Antiretroviral therapies based on 2 nucleoside reverse transcriptase inhibitors (NRTIs) in combination with a protease inhibitor (PI) have been shown to cause profound and sustained virus suppression in HIV-1‐infected patients [1, 2]. Plasma HIV1 RNA levels fall below detection limits in the majority of patients who remain under treatment. This is associated with an increase in CD4 1 T-cell counts [3, 4] and is accompanied

We investigated the presence of hepatitis C virus (HCV) infection in 58 patients with humoral immunodeficiencies. Forty-three of these patients had common variable immunodeficiency (CVI), 2 had sporadic hyperimmunoglobulin M (HIM) syndrome, 2 had immunoglobulin G subclass deficiency, 4 had ataxia-telangiectasia (AT), and 7 had X-linked agammaglobulinemia (XLA). Patients with late-onset hypogammaglobulinemia (those with CVI, HIM, or immunoglobulin G subclass deficiency) had a 38.2% prevalence of HCV infection. In patients with XLA or AT, HCV infection was not detectable. Most of the HCV-infected patients had persistent viremia, with histologic findings of chronic hepatitis. Although patients positive for HCV ribonucleic acid (RNA) had received several lots of immunoglobulin, we were unable to detect any correlation between the time that alanine aminotransferase levels increased and the time that intravenous immunoglobulin therapy was given, except in one patient with CVI. Moreover, we found no differences in the number of blood transfusions, surgical procedures, or administrations of intravenous or intramuscular immunoglobulin between HCV RNA-positive and HCV RNA-negative groups. We concluded that: (1) the incidence of HCV infection in patients with hypogammaglobulinemia is much higher than that reported in the Italian general population; (2) although patients with hypogammaglobulinemia have persistent viremia, they do not show an aggressive course of HCV disease, nor does hepatocarcinoma develop; and (3) intravenous immunoglobulins are only one of several possible causes of HCV transmission in patients with humoral immunodeficiencies.

CD8+CD45RA+ T lymphocytes present two distinct subpopulations expressing the CD11a molecule (LFA-1 alpha chain) with different intensity. CD11adim cells represent the unprimed population within the CD8+CD45RA+ subset, whereas CD11abright cells are activated and may be considered as memory lymphocytes. The aim of this study was to analyze the expression of CD11a and CD18 within the CD8+CD45RA+ population in young HIV-infected individuals at different stages of disease as a marker of activation and of disease progression. Blood cells from 82 HIV-infected individuals and 23 age-matched healthy controls were stained with unconjugated CD11a, CD18, PE-goat F(ab')2 anti-mouse, FITC-CD45RA, and TRI-Color CD8. Quantitative analysis for three-color immunofluorescence was carried out by flow cytometry. HIV+ subjects were subdivided into three groups according to their CD4+ cell number (group A, CD4+ cells500/microliters [20 subjects], group B, CD4+ cells between 500 and 200/microliters [36 subjects], and group C, CD4+ lymphocytes200/microliters [26 subjects]). We found a significant increase of CD11abright in the CD8+CD45RA+ subpopulation throughout the progression of the disease. The CD11abright percentage of positivity (mean) within the CD8+CD45RA+ subpopulation was 31% in healthy donors, 51% in group A, 52% in group B, and 68% in group C. CD11abright expression was closely related to CD18bright (p0.001), but not to CD38. The relative increase of CD11a and CD18 expression in CD8+CD45RA+ T lymphocytes parallels the decrease of CD4+ cells and the progression of disease: an inverse correlation between the percentages of CD4+ cells/microliter and CD8+CD45RA+CD11abright cells (p0.001) and a direct correlation between the number of CD4+ lymphocytes per microliter and both the number of CD8+CD45RA+CD11adim cells (p0.001) and the number of CD8+CD45RA+CD11abright (p = 0.002) was observed. The relative increase of CD8+CD45RA+CD11abright cells may represent an additional marker for monitoring HIV-induced immunodeficiency.

The immunologic defect in X-linked immunodeficiency and hyperimmunoglobulinemia M (HIM) are related to defective expression of the CD40 ligand (CD40L). We have studied two female patients with HIM to evaluate the role of CD40/CD40L in the pathogenesis of impaired immunoglobulin switching. In addition to recurrent infections characteristic of humoral immunodeficiencies, the two patients had chronic hepatitis caused by type C virus. Phenotypic characterization of peripheral blood mononuclear cells showed a similar picture in both patients, with a reduction in the absolute numbers of CD4 cells and increased numbers of CD8 and CD3/DR cells. B cells (CD19+) were reduced in one patient, but CD40 was expressed on all CD19+ cells in both patients. The expression of CD40L was normal on peripheral blood mononuclear cells from the two patients with HIM on both resting and stimulated cells. The combination of anti-CD40 and cytokines (interleukin-2, interleukin-4, and interleukin-10) was able to restore proliferative capacity to anti-IgM. Peripheral blood mononuclear cells from the two patients with HIM showed a high spontaneous production of IgM in vitro and no production of IgG or IgE. Our data suggest that the defect of isotype switching in female patients with HIM is not related to defective expression of the CD40/CD40L receptor system. A possible role for chronic hepatitis C virus infection in the pathogenesis of the disease is suggested by the detection of specific production of anti-hepatitis C virus IgM.

SUMMARYThe molecular mechanisms regulating IFN-γ production have yet to be well characterized. We describe here how treatment of activated cultures of peripheral blood mononuclear cells (PBMC) with the phosphotyrosine phosphatases (PTP) inhibitor sodium ortho vanadate results in greatly enhanced IFN-γ production. Conversely, cellular proliferation of the same cultures is profoundly inhibited by treatment with vanadate, while the expression of IL-2R and DR molecules on activated lymphocytes remains substantially unmodified. Increased IFN-γ production, but not inhibition of cellular proliferation, was also observed in mitogen-activated vanadate-treated Jurkat cells. On the other hand, IFN-γ production induced in cultures of PBMC treated or not with vanadate, was strongly inhibited by incubation with the protein tyrosine kinase (PTK) inhibitor herbimycin A. As a result of the inhibited phosphatase activity, substrates for PTK become hyperphosphorylated on tyrosine residues, as shown by Western blot analysis of cell lysates from cultures of PBMC treated with vanadate. We suggest that the tyrosine phosphorylation pathway plays a role in regulating IFN-γ production.

In a cohort of 1000 Gambian children immunized with four doses of 10 micrograms of plasma-derived hepatitis B virus vaccine, 44 subjects (4.4%) showed no response (< 10 mIU/ml; 6 subjects) or low specific antibody response (10 to 99 mIU/ml; 38 subjects) to hepatitis B surface antigen. Serologic indices, potentially correlated with low immunologic response, were investigated in sera obtained from these children and in sex-, age- and village-matched controls who showed a normal response. The presence of circulating immune complexes in similar proportion of responding and poorly responding children together with a low prevalence of rheumatoid factors suggested that polyclonal B cell activation was not correlated with the subnormal humoral response. Concentrations of serum immunoglobulin (Ig) and IgG subclasses did not differ in the two groups. Some of the African prevalent Ig allotypes were determined, but no significant differences in the two groups were found. The humoral response to hepatitis B surface antigen did not correlate with the response to tetanus toxoid.

SUMMARYWe have studied IL-6 gene expression and production by in vitro stimulated peripheral blood mononuclear cells (PBMC) isolated from common variable immunodeficiency (CVI) patients. A strong hybridization signal for the IL-6 probe was observed in mRNA extracted from phyto-haemagglutinin (PHA)- and PHA/phorbol myristate acetate (PMA)-stimulated PBMC from most of 12 CVI patients analysed. IL-6 production by PHA-stimulated PBMC from 28 CVI patients was evaluated in ELISA and found to be significantly (P < 0.0001) higher than in normal controls. IL-6 production, however, did not correlate with the lymphocyte populations examined, nor with the absolute number of monocytes. We have also showed that IL-6 was able to increase IgM secretion by several Epstein Barr virus (EBV)-transformcd cell lines derived from both normal donors and CVI patients, but it failed to modify substantially the amounts of IgM and IgG produced in vitro by PBMC derived from CVI patients and activated with pokeweed mitogen (PWM) or anti-IgM. Our data indicate that IL-6 gene expression and production is increased in CVI, but CVI cells do not respond to IL-6 with increased production of immunoglobulin.

Peripheral blood mononuclear cells (PBMC) from HIV-infected subjects have an increased mortality rate (MR) when incubated in vitro for 3 days in a culture medium. We have previously shown that fibroblast-conditioned medium (FCM) can preserve viability, without significant activation, of human lymphocytes in vitro. We therefore tested the ability of two FCM and other factors to reduce spontaneous MR in HIV-positive PBMC.PBMC were cultured for 3 days in control medium and medium supplemented with FCM or recombinant cytokines [interleukin (IL)-2, IL-6, IL-7, granulocyte macrophage colony-stimulating factor]. Cells viable at day 3 were counted in a cytofluorimeter after staining with ethidium bromide. DNA was extracted from the cultures and evaluated for the presence of low molecular weight fragmentation.The MR of PBMC from 51 HIV-positive subjects and from 21 healthy controls were 30.1 and 9.5%, respectively (P0.0001). The MR was higher in 40 patients with a CD4+ lymphocyte count400 x 10(6)/l than in subjects with a count400 x 10(6)/l (32.84 versus 20.96%; P = 0.047). IL-2 and FCM significantly reduced MR in HIV-positive subjects (MR: 17.8 and 20.4%; P:0.001 and 0.005, respectively). This effect was more evident in subjects with a CD4+ lymphocyte count400 x 10(6)/l and in subjects with negative p24 antigenaemia. Cellular proliferation accounts for increased survival in IL-2-supplemented cultures but not in those with FCM. DNA was extracted from fresh PBMC and cells cultured for 3 days for 22 HIV-positive cases. DNA degradation was documented and bands related to an apoptotic mechanism of death observed, especially in subjects with more advanced disease.Our data suggest that FCM inhibits accelerated cell death in vitro of PBMC isolated from HIV-positive patients.