Payá, Carlos V.

Three multicenter studies have shown that the addition of mycophenolate mofetil (MMF) to an immunosuppressive regime consisting of cyclosporin A (CSA) and prednisone (PRED) decreases the incidence of acute rejection episodes when compared with azathioprine (AZA) or placebo (1–3). In those patients receiving 3 g/d of MMF, the highest dose used in the studies, there was a trend towards an increased incidence of cytomegaloviral sepsis (CMV). We postulated therefore that MMF may represent an independent risk factor for the development of CMV infection in patients receiving renal allografts and MMF at our institution. Having altered the triple drug regime from CSA, AZA (2‐2.5 mg/kg/d) and PRED to CSA. MMF (2 g/d) and PRED in July 1995, we elected to study all patients undergoing kidney transplantation for the 33‐month period January 1994‐September 1996, by undertaking a case control analysis to determine independent risk factors for the development of CMV infection, as defined by CMV viremia or tissue‐invasive CMV. Three CMV disease‐free control patients were matched to each case, these patients having been randomly selected from the entire pool of patients in the observation period. There were 31 CMV case patients and 102 control patients. Univariate analysis indicated that gender, a concomitant pancreas transplant, acute rejection and CMV seropositivity in the donor were risk factors. However, multivariate analysis indicated that only acute rejection and donor CMV seropositivity were independently linked (p<0.05) to CMV disease in this sample. Specifically, the odds ratio (OR) for CMV disease between MMF and AZA was 1.0 (95% confidence interval (CI): 0.46‐2.18). Therefore, in this case control study we find no evidence that MMF at a dose of 2 g/d is an independent risk factor for primary CMV viremia or tissue invasion in renal allograft recipients.

Abstract Alveolar macrophages play a crucial role in initiating the inflammatory response in allergic asthma through the cross-linking of the low affinity IgE receptors (FcεRIIb or CD23) by IgE-allergen immunocomplexes. We have previously shown that CD23 cross-linking in monocytes and U937 cells targets IκBα, leading to the activation of the transcription factor NF-κB. We demonstrate in this paper that CD23-initiated signaling in U937 cells leads to hyperphosphorylation of IκBα at Ser32/Ser36 residues. Overexpression of a dominant-negative IκBα transgene containing mutations at Ser32/Ser36 completely inhibits degradation of IκBα, NF-κB activation, and gene transcription that follows CD23 cross-linking. Investigation of the second messengers mediating the CD23-dependent activation of NFκB demonstrates that IκB kinases (IKKs) but not p90rsk are selectively activated following CD23 cross-linking and mediates the phosphorylation of IκBα. Cotransfection experiments with an IKKβ negative dominant completely inhibit CD23 induced NFκB activation. Furthermore, the activation of tyrosine kinase(s) by CD23 is required for the induction of IKK activity, IκBα degradation, and NF-κB nuclear translocation. Taken together, our results show that CD23 cross-linking in the monocytic lineage induces tyrosine kinase activation followed by activation of IKK, which phosphorylates IκBα at the N-terminal domain (Ser32/Ser36), inducing its degradation, NF-κB activation and gene transcription.

Abstract Activation-induced cell death of peripheral T cells results from the interaction between Fas and Fas ligand. Resting peripheral T cells are resistant to Fas-induced apoptosis and become susceptible only after their activation. We have investigated the molecular mechanism mediating the sensitization of resting peripheral T cells to Fas-mediated apoptosis following TCR stimulation. TCR activation decreases the steady state protein levels of FLIP (FLICE-like inhibitory protein), an inhibitor of the Fas signaling pathway. Reconstitution of intracellular FLIP levels by the addition of a soluble HIV transactivator protein-FLIP chimera completely restores resistance to Fas-mediated apoptosis in TCR primary T cells. Inhibition of IL-2 production by cyclosporin A, or inhibition of IL-2 signaling by rapamycin or anti-IL-2 neutralizing Abs prevents the decrease in FLIP levels and confers resistance to Fas-mediated apoptosis following T cell activation. Using cell cycle-blocking agents, we demonstrate that activated T cells arrested in G1 phase contain high levels of FLIP protein, whereas activated T cells arrested in S phase have decreased FLIP protein levels. These findings link regulation of FLIP protein levels with cell cycle progression and provide an explanation for the increase in TCR-induced apoptosis observed during the S phase of the cell cycle.

Influenza is an important cause of hospitalization due to lower respiratory tract involvement for which there is no specific antiviral treatment with proven efficacy. We conducted a double-blind, randomized, placebo-controlled trial to assess the tolerability and efficacy of nebulized zanamivir (16 mg four times a day) in combination with rimantadine compared to rimantadine with nebulized saline for treating influenza in adults hospitalized with influenza. Twenty patients tolerated the inhaled zanamivir (ZNV) plus rimantadine without decline in peak expiratory flow rates compared to the 21 who received inhaled saline. The study was terminated early because the approval of ZNV made further enrolment untenable. No significant differences were observed in the proportion of patients shedding virus by treatment day 3 (57% ZNV plus rimantadine, 67% placebo plus rimantadine), or in the durations of hospitalization and supplemental oxygen use. More ZNV plus rimantadine recipients exhibited no or mild cough on day 3 of treatment (94 vs 55%, P=0.01). Two rimantadine-resistant viruses emerged during rimantadine monotherapy; no ZNV resistance was observed. Nebulized ZNV appears to be well tolerated in this hospitalized population but further studies are needed to assess its efficacy.

ABSTRACTHuman monocytes and macrophages are persistent reservoirs of human immunodeficiency virus (HIV) type-1. Persistent HIV infection of these cells results in increased levels of NF-κB in the nucleus secondary to increased IκBα, IκBβ, and IκBɛ degradation, a mechanism postulated to regulate viral persistence. To characterize the molecular mechanisms regulating HIV-mediated degradation of IκB, we have sought to identify the regulatory domains of IκBα targeted by HIV infection. Using monocytic cells stably expressing different transdominant molecules of IκBα, we determined that persistent HIV infection of these cells targets the NH2but not the COOH terminus of IκBα. Further analysis demonstrated that phosphorylation at S32and S36is necessary for HIV-dependent IκBα degradation and NF-κB activation. Of the putative N-terminal IκBα kinases, we demonstrated that the Iκκ complex, but not p90rsk, is activated by HIV infection and mediates HIV-dependent NF-κB activation. Analysis of viral replication in cells that constitutively express IκBα negative transdominant molecules demonstrated a lack of correlation between virus-induced NF-κB (p65/p50) nuclear translocation and degree of viral persistence in human monocytes.

ABSTRACTThis work aims at characterizing the interplay between human immunodeficiency virus type 1 (HIV-1) and the antiapoptotic cellular protein Bcl-2 responsible for a persistent infection in lymphoblastoid T (J.Jhan) or monocytic (U937) cells. We report that the kinetics of Bcl-2 protein level during the establishment of a chronic infection is biphasic, characterized by a transient decrease followed by restoration to the initial level. The extent and duration of this transient decrease were inversely correlated with the basal level of Bcl-2 as shown by kinetics of Bcl-2 levels in J.Jhan or U937 clones exhibiting different levels of Bcl-2. Using these clones, we also showed that Bcl-2 downregulates HIV-1 replication. Therefore, the cells overexpressing Bcl-2 are characterized by a low viral burden which, in turn, has little effect on the level of this protein. The observed bipasic kinetics is the result of a dual regulation of Bcl-2 induced by HIV-1 infection itself: an upregulation at the transcriptional level of thebcl-2gene concomitant with a downregulation at the protein level. Convergent data suggest that this downregulation is caused by the oxidative stress induced by the infection itself as shown by the associated modulations of glutathione and thioredoxin levels and by the prevention of these dysregulations byN-acetylcysteine. Altogether, these data indicate that infection first results in a decrease of Bcl-2, permitting an initial boost of replication. Then, as the synthesis at the transcriptional level proceeds, the replication is negatively controlled by Bcl-2 to reach a balance characterized by low virus production and a level of Bcl-2 compatible with cell survival. We suggest that the basal level of Bcl-2, together with infection-inducible transcription factors able to activatebcl-2gene transcription, is a critical cellular determinant in the tendency toward an acute or a persistent infection.

ABSTRACT Detection of cytomegalovirus (CMV) DNA in blood by PCR is a sensitive method for the detection of infection in patients posttransplantation. The test, however, has low specificity for the identification of overt CMV disease. Quantitative CMV PCR has been shown to overcome this shortcoming. The COBAS AMPLICOR CMV MONITOR test was evaluated by using consecutive serum and peripheral blood mononuclear cell (PBMN) samples from liver transplant patients. Twenty-five patients had CMV viremia (by shell vial cell culture assay) and/or tissue-invasive disease (by biopsy); 20 had no active infection. A total of 262 serum and 62 PBMN specimens were tested. Of 159 serum specimens from patients with overt CMV infection, the COBAS assay detected CMV DNA in 21 patients (sensitivity, 84%). Only 1 of 103 samples from patients with no evidence of active infection had detectable CMV DNA (341 copies/ml). By comparison of 62 matching serum and PBMN samples by the same assay, 12 PBMN samples were exclusively positive, whereas only 2 serum samples were exclusively positive (P < 0.05). At the time of clinical CMV infection, viral copy numbers were higher in PBMNs than serum from four of five patients. The COBAS AMPLICOR CMV MONITOR test is a sensitive and specific test for the quantitative detection of CMV DNA in blood. Clinical applications of the assay will require further validation with samples from a larger population of transplant patients.

ABSTRACT A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants. A total of 95% (50 of 51) of CMV-positive (cell culture- and antigenemia immunofluorescence [AG-IFA]-positive) clinical specimens were positive by the NASBA assay. Results from different operators over multiple testing days were consistent for each of four panel members containing different concentrations of CMV mRNA, indicating the reproducibility of the assay. The estimated 95% reliable upper detection limit of the assay was 600 mRNA copies; the lower limit of detection was less than 25 mRNA copies. The clinical utility of the assay was evaluated with longitudinally collected specimens from solid-organ transplant patients ( n = 21). A total of 98% (81 of 83) of the specimens from CMV-negative patients were negative by the NASBA assay, while 90% (10 of 11) of patient specimens that were positive by cell culture or AG-IFA were positive by the NASBA assay. Positive NASBA assay results were obtained earlier than AG-IFA or cell culture results for 55% of the patients and at the same time for the remainder of the patients (45%). The overall agreement between the NASBA assay and current reference tests was 86% when active CMV infection was present. These studies indicate that the CMV pp67 mRNA NASBA assay has reproducible and sensitive performance characteristics that should enable more rapid diagnosis of CMV infection.

ABSTRACT The availability of microbiologic methods that detect early replication of cytomegalovirus (CMV) posttransplantation will enhance the process of initiating preemptive antiviral therapy prior to the appearance of CMV disease. Using PCR techniques we sought to determine which region of the CMV genome present in peripheral blood leukocytes (PBLs) or serum provides the highest sensitivity for the detection of CMV posttransplantation. Blood samples were prospectively collected weekly for at least 8 weeks from a cohort of 21 consecutive liver transplant recipients not receiving anti-CMV prophylaxis. Results of PCR assays were correlated with recovery of CMV in cell cultures and histopathological findings from biopsy specimens of infected organs to assess clinical symptomatic infection. Of 148 specimens, primer pairs directed to the Hin dIII-X fragment region of CMV detected target DNA with a 94% sensitivity, compared to an 87% sensitivity with primer pairs directed to Eco RI fragment D, 32% sensitivity with primer pairs directed to the immediate-early antigen 1 gene (IEA1 gene), and 20% sensitivity with primer pairs directed to the major immediate-early (MIE) gene. The performance characteristics in terms of the sensitivity of primers for amplifying CMV DNA associated with symptomatic infection ranged from 100% ( Hin dIII-X) to 20% (MIE gene); however, specificity was inversely related ( Hin dIII-X, 45%; MIE gene, 91%) to primers directed to these gene targets. When Hin dIII-X and Eco RI-D primer sets were used, CMV DNA from PBLs was a more sensitive target than CMV DNA from serum for the early detection of symptomatic CMV infection (17 versus 12 days). Importantly, CMV DNA was not detected in five patients with no evidence of this viral infection. In conclusion, primers directed to the Hin dIII-X fragment region were the most optimal for the early detection of CMV DNA in PBLs and sera from symptomatic liver transplant recipients.