Alveolar and blood T lymphocyte profiles in Pneumocystis jirovecii-positive patients: effects of HIV status.

BACKGROUND: There are substantial differences in the risk evaluation, clinical presentation, and outcome of Pneumocystis pneumonia between human immunodeficiency virus (HIV)-positive and HIV-negative immunocompromised patients. To compare the host immune defenses against Pneumocystis jirovecii, the blood and alveolar lymphocyte profile was explored in these 2 populations. METHODS: The total, CD3(+), CD4(+), and CD8(+) T-lymphocyte counts were measured in the blood and alveoli of immunocompromised patients with a P. jirovecii DNA detected in their bronchoalveolar lavage samples, according to their HIV status. RESULTS: In blood and alveoli, the CD4(+) and CD8(+) T-lymphocyte counts were higher and lower, respectively, in the HIV-negative group. The threshold for initiating prophylaxis in HIV-positive persons, 200 CD4(+) T cells/µL, was not pertinent for HIV-negative patients. The P. jirovecii burden correlated with the blood CD4(+) T-cell counts in the HIV-positive but not in the HIV-negative group. Nevertheless, whatever the HIV status, a correlation was observed between alveolar CD4(+) T cells and the P. jirovecii burden. CONCLUSIONS: The T-lymphocyte profile was different between HIV-positive and HIV-negative patients with P. jirovecii, suggesting a distinct pathogenesis. Alveolar CD4(+) T cells could be critical to explain the development of Pneumocystis pneumonia but may also be important for evaluation of disease risk, mostly among HIV-negative immunocompromised patients.

Cellular and cytokine changes in the alveolar environment among immunocompromised patients during Pneumocystis jirovecii infection.

Limited data exist on the cytokine and cellular changes in the alveolar environment in immunocompromised patients during Pneumocystis jirovecii infection. A cellular and a cytokine analysis were performed on bronchoalveolar lavage (BAL) samples from three groups of patients, i.e., an initial study group of 64 immunocompromised P. jirovecii-positive individuals and two control groups of P. jirovecii-negative patients who had been or not immunosuppressed (65 patients). The results were related to alveolar dilution as determined by urea measurement. Compared with non-infected groups, P. jirovecii-infected patients had a lower level of alveolar macrophages (AM), particularly those with high burdens of P. jirovecii. Alveolar macrophages over-expressed the Dectin-1 receptor, which was largely implicated in P. jirovecii clearance. The alveolar CD8+T and CD4+T lymphocyte counts were increased and an inverse correlation was observed between the alveolar CD4+ cell count and the P. jirovecii burden. Although the alveolar IL-6 level was considerably increased, alveolar IL-17, IL-10, TNF-α, TGF-ß concentrations of P. jirovecii patients were not different from the control groups. Changes in the pulmonary environment were also highlighted during P. jirovecii colonization. Our study suggests that there is a correlation between the P. jirovecii burden in the alveolus (from colonization to a high P. jirovecii burden), and the degree of impairment of the alveolar immune response.

HIV-1 Tat protein induces IL-10 production by an alternative TNF-alpha-independent pathway in monocytes: role of PKC-delta and p38 MAP kinase.

HIV-1 Tat protein stimulates the production of both TNF-alpha and IL-10 in human monocytes. Taking into account the ability of TNF-alpha to induce IL-10 production, we evaluated the link between Tat, TNF-alpha and IL-10 and the implication of PKC and p38 MAP kinase pathways. Our data showed that (i) in the presence of neutralizing anti-TNF-alpha antibodies, IL-10 production is only partially inhibited; (ii) in a calcium-free medium, while TNF-alpha production is totally inhibited, Tat continues to induce IL-10; (iii) under these conditions, Tat-mediated IL-10 production is associated with PKC-delta activation; and (iv) downstream of PKC, p38 MAP kinase is crucial for TNF-alpha independent IL10 production. Overall, our data suggest a new mechanism, implicating Tat protein, by which HIV-1 may maintain a constant production of the immunosuppressive IL-10 cytokine, even in the absence of TNF-alpha production. In consequence, HIV-1 may escape immune surveillance and thus promote the establishment of an immunosuppressive state.

Effects of intraoperative versus postoperative administration of rabbit antithymocyte antibodies on 1-year renal function in renal transplant patients.

OBJECTIVES: The aim of our study was to prospectively assess 1-year allograft outcomes and the evolution of lymphocyte subsets in a group of renal transplant patients who had received intraoperative rabbit antithymocyte antibodies (RATG). MATERIALS AND METHODS: We compared 1-year allograft transplant outcomes in renal transplant recipients who had received intraoperative RATG (group 1, n=53) with the outcomes observed in patients in a historical control group who had received postoperative RATG (group 2, n=49). RATG were given at the same dosage (1 mg/kg) during the first 3 days, and then the dosage was adapted according to CD2 count, until calcineurin inhibitors were started. RESULTS: The overall dosage of RATG administered was significantly lower in group 1. At day 4, CD2, CD3, and CD19 T-cell subset counts were significantly higher in patients in group 1. From 3 months after transplantation, CD4/CD8 ratios were significantly lower in patients in group 1 because of a rapid regeneration of CD8 T cells. One-year total lymphocyte counts were significantly higher in patients in group 1. There were fewer severe infectious complications in patients in group 1. One-year renal function was better in patients in group 2. Donor age was the only independent factor associated with renal function at both 1 month and 1 year after transplantation. CONCLUSIONS: When RATG are infused intraoperatively, a lower total amount of RATG is required to prevent acute rejection as compared with postoperative RATG infusion. Consequently, fewer serious lymphopenia-associated complications are observed during the first year after transplantation.

Methodological aspects of minimal residual disease assessment by flow cytometry in acute lymphoblastic leukemia: A French multicenter study.

BACKGROUND: Minimal residual disease (MRD) assessment provides a powerful prognostic factor for therapeutic stratification in acute lymphoblastic leukemia (ALL). Multiparameter flow cytometry (MFC) has the potential for a rapid and sensitive identification of high risk patients. Our group has previously published that MRD levels analyzed by clone specific Ig/TcR-QPCR and MFC were concordant at a sensitivity of 10(-4) . Here we report the MFC methodological aspects from this multi-center experience. METHODS: MRD was assessed by MFC in 1030 follow-up samples from 265 pediatric and adult patients with de novo ALL treated in the FRALLE, EORTC, or GRALL clinical trials. MRD assessment as applied by the eight participating MFC laboratories is described in detail regarding cell preparation, leukemia-associated immunophenotype (LAIP) markers and data analysis. Samples were obtained from bone marrow (BM) and peripheral blood (PB). Immunostaining was performed after erythrocyte lysis or Ficoll enrichment. RESULTS: This study confirms the applicability of MFC-based MRD assessment in 97% of patients with ALL at the 10(-4) cut-off. MRD values after Ficoll enrichment and erythrocyte lysis were found comparable. Higher MRD values were obtained in BM than in PB, especially for B-lineage ALL. CONCLUSIONS: Measurement of MRD by MFC at the 10(-4) cut-off is applicable within a few hours for almost all patients and using a comparable analytical strategy allows for multicenter collaborative studies. The method can be introduced in a strategy aimed at defining the risk of failure of patients with childhood or adult ALL.

Human chromaffin cell graft into the CSF for cancer pain management: a prospective phase II clinical study.

A number of pre-clinical studies have demonstrated the value of adrenal medullary allografts in the management of chronic pain. The present longitudinal survey studied 15 patients transplanted for intractable cancer pain after failure of systemic opioids due to the persistence of undesirable side-effects. Before inclusion, all the patients had their pain controlled by daily intrathecal (I-Th) morphine administration. The main evaluation criteria of analgesic activity of the chromaffin cell allograft was the complementary requirement of analgesics and in particular the consumption of I-Th morphine required to maintain effective pain control. Out of the 12 patients who profited from enhanced analgesia with long-term follow-up (average 4.5 months), five no longer required the I-Th morphine (with prolonged interruption of systemic opioids as well), two durably decreased I-Th morphine intake and five were stabilized until the end of their follow-up. Durable decline and stabilization were interpreted as indicative of analgesic activity by comparison with the usual dose escalation observed during disease progression. In most cases, we noted a relationship between analgesic responses and CSF met-enkephalin levels. The results of this phase II open study demonstrate the feasibility and the safety of this approach using chromaffin cell grafts for long-term relief of intractable cancer pain. However, while analgesic efficacy was indicated by the reduction or stabilization in complementary opioid intake, these observations will need to be confirmed in a controlled trial in a larger series of patients.

Human immunodeficiency virus type 1 Tat protein induces an intracellular calcium increase in human monocytes that requires DHP receptors: involvement in TNF-alpha production.

HIV-1 Tat protein, acting at the cell membrane, stimulates the production by human monocytes of TNF-alpha, a cytokine implicated in both HIV-1 replication and pathogenesis. Here, we analyze, in primary human monocytes, the mechanisms involved in Tat-stimulated calcium mobilization and its relationship with TNF-alpha production. We show that the Tat protein induces a calcium signal by mobilizing calcium from extracellular stores. This calcium signal is totally blocked when cells are stimulated in the presence of DHP receptor inhibitors such as nimodipine or calcicludine, thus suggesting the implication of this L-type calcium channel. By using RT-PCR amplification, Western blot with antibodies directed against the alpha1D subunit, binding assays with specific agonists or antagonists, and inhibition with specific antisense oligonucleotides, we show that DHP receptors are expressed and functional in primary human monocytes. Interestingly, we demonstrate that Tat-induced calcium mobilization is tightly linked to TNF-alpha production, thus indicating that Tat-induced mobilization and TNF-alpha production are entirely mediated by DHP receptors, as shown by their total inhibition by nimodipine, calcicludine, or anti-alpha1D antisense oligonucleotides.

Signaling pathways triggered by HIV-1 Tat in human monocytes to induce TNF-alpha.

In this study we investigated the signaling pathways triggered by Tat in human monocyte to induce TNF-alpha. In monocytes, the calcium, the PKA, and the PKC pathways are highly implicated in the expression of cytokine genes. Thus, these three major signaling pathways were investigated. Our data show that (i) PKC and calcium pathways are required for TNF-alpha production, whereas the PKA pathway seems to be not involved; (ii) downstream from PKC, activation of NFkappaB is essential while ERK1/2 MAP kinases, even though activated by Tat, are not directly involved in the pathway signaling leading to TNF-alpha production.

Effect of anti-IL-2Ralpha antibody on IL-2-induced Jak/STAT signaling.

Acute allograft rejection is driven by production of cytokines such as interleukin-2 (IL-2) that activate and expand alloreactive T cells by ligating high-affinity IL-2 receptors composed of three subunit chains: alpha, beta, gamma The alpha chain, expressed only on activated T cells, has become an important therapeutic target. Monoclonal antibodies (mAbs) that bind IL-2Ralpha chains significantly decrease transplant rejection. We examined the ability of the humanized anti-IL-2Ralpha antibody daclizumab to block high-affinity IL-2Rs and interrupt T-lymphocyte signaling. Our evaluation focused on a pathway critical for T-cell proliferation, the Jak/STAT pathway. Daclizumab markedly inhibited phosphorylation of the Jak1, Jak3 and STAT5a/b components of the IL-2R-dependent pathway. Suppression by daclizumab was associated with internalization of IL-2Ralpha but not IL-2Rbetagamma chains. High IL-2 doses overcame daclizumab-induced blockade of Jak/STAT phosphorylation despite absent cell surface highaffinity IL-2Rs. Under these circumstances, IL-2-mediated Jak/STAT pathway activation might be generated through residual intermediate affinity IL-2Rbetagamma receptors, and this was demonstrated by complete blockade of signaling when anti-IL-2Rbeta monoclonal antibody was added. Humanized antibodies are an important part of strategies to induce alloantigen tolerance. Understanding the molecular events associated with their beneficial clinical effect is critical to design of future immunosuppressive strategies.