[Disseminated cryptococcosis as inaugural manifestation of AIDS]. / Cryptococcose disseminée: Manifestation inaugurale d'un SIDA.

Disseminated cryptococcal disease is typically seen in patients with HIV infection. We report here the evolution of a patient with disseminated cryptococcosis whose treatment failed after ten weeks of induction therapy with amphotericin B. This case illustrates the importance of careful initial evaluation, and close clinical follow-up of these patients who are at risk of developing other opportunistic infections and drug-related complications.

HIV-1 Nef associated PAK and PI3-kinases stimulate Akt-independent Bad-phosphorylation to induce anti-apoptotic signals.

A highly conserved signaling property of Nef proteins encoded by human or simian immunodeficiency virus is the binding and activation of a PAK kinase whose function is unclear. Here we show that Nef-mediated p21-activated kinase (PAK) activation involves phosphatidylinositol 3-kinase, which acts upstream of PAK and is bound and activated by Nef similar to the manner of Polyoma virus middle T antigen. The Nef-associated phosphatidylinositol-3-PAK complex phosphorylated the pro-apoptotic Bad protein without involving the protein kinase B-Akt kinase, which is generally believed to inactivate Bad by serine phosphorylation. Consequently, Nef, but not a Nef mutant incapable of activating PAK, blocked apoptosis in T cells induced by serum starvation or HIV replication. Nef anti-apoptotic effects are likely a crucial mechanism for viral replication in the host and thus in AIDS pathogenesis.

A natural variability in the proline-rich motif of Nef modulates HIV-1 replication in primary T cells.

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC). The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.

Common germinal-center B-cell origin of the malignant cells in two composite lymphomas, involving classical Hodgkin's disease and either follicular lymphoma or B-CLL.

BACKGROUND: Classical Hodgkin's disease (HD) and B-cell non-Hodgkin lymphoma (NHL) occasionally occur in the same patient. Such composite lymphomas represent interesting models to study the pathogenesis of B-cell lymphomas and the relationship between HD and B-cell NHL. MATERIALS AND METHODS: We analyzed two composite lymphomas (a combination of classical HD with follicular lymphoma [FL] and a combination of classical HD with B-cell chronic lymphocytic leukemia [B-CLL]) by micromanipulation of single cells from tissue sections and amplification of immunoglobulin V region genes for the clonal relationship of the tumor cells. RESULTS: In both cases, clonally related variable (V) genes with both shared as well as distinct somatic mutations were obtained from the two lymphomas, showing that in each of the cases the distinct tumor cells were members of a common germinal center (GC) B-cell clone. FL cells from two different lymph nodes of patient 1 showed a similar mutation pattern, suggesting that infiltration of these lymph nodes by tumor cells was not restricted to a particular FL cell or subclone. In the FL, a single cell was identified with a mutation signature indicating that premalignant cells can persist in the tissue. CONCLUSIONS: The cases presented here further underline the close relationship between HD and B-cell NHL and the role of the GC in lymphomagenesis. Whereas the latter was already suggested for FL and HD, the present study indicates that also in the B-CLL subset characterized by mutated Ig genes, important steps in malignant transformation happen in the GC, and that HRS cells can derive from CD5-positive B cells.

Dynamic Nef and Nef dynamics: how structure could explain the complex activities of this small HIV protein.

The Nef protein of the human immunodeficiency virus is as important for disease progression as it is perplexing in its plethora of target molecules and functions. In this article, it is proposed that the complex biology of Nef is regulated through conformational changes of the protein that are triggered by cellular location and specific interactions as Nef traffics through the infected cell.

Nef from human immunodeficiency virus type 1(F12) inhibits viral production and infectivity.

Human immunodeficiency virus type 1(F12) (HIV-1(F12)) interferes with the replication of other strains of HIV. Its accessory protein, Nef, is sufficient for this phenotype, where the production and infectivity of HIV are impaired significantly. The analysis of three rare mutations in this Nef protein revealed that these effects could be separated genetically. Moreover, the defect in virus production correlated with the lack of processing of the p55(Gag) precursor in the presence of Nef from HIV-1(F12). Importantly, the introduction of one of these mutations (E177G) into Nef from HIV-1(NL4-3) also created a dominant-negative Nef protein. Effects of Nef from HIV-1(F12) on virus production and Gag processing correlated with its altered subcellular distribution. Moreover, the association with two new cellular proteins with molecular masses of 74 and 75 kDa, which do not interact with other Nef proteins, correlated with the decreased virion infectivity. The identification of a dominant-negative protein for the production and infectivity of HIV suggests that Nef plays an active role at this stage of the viral replicative cycle.

Direct in vitro binding of full-length human immunodeficiency virus type 1 Nef protein to CD4 cytoplasmic domain.

The Nef protein of the simian and human immunodeficiency viruses is known to directly bind and downregulate the CD4 receptor. Although the molecular mechanism is well understood, direct binding of Nef and CD4 is difficult to demonstrate and is believed to be of low affinity. Applying nuclear magnetic resonance and fluorescence spectroscopy, we biophysically reevaluated the CD4-Nef complex and found the dissociation constant to be in the submicromolar range. We conclude that additional, so far disregarded residues in the N terminus of Nef are important for interaction with CD4.

Functional and structural defects in HIV type 1 nef genes derived from pediatric long-term survivors.

DNA sequences and three distinct in vitro functions of Nef were evaluated in a group of seven perinatally infected children. nef gene sequences obtained before and after virus culture showed that one of the five non-/slow progressors harbored a virus with large deletions. nef genes from the remaining four children were full length but contained discrete changes at a higher frequency than the rapid progressors. In functional studies, 40 of 44 Nef proteins derived from the whole study group were capable of binding the cellular serine kinase p62, indicating that this function is well conserved among naturally occurring viruses. In contrast, representative Nef proteins derived from the long-term non-/slow progressors were found to be defective or far less capable of enhancing viral replication and/or viral infectivity in herpesvirus saimiri-transformed human T cells and peripheral blood mononuclear cells. On reversion of highly prevalent point mutations in the defective proteins, viral replication could be restored to wild-type levels. Our results suggest that nef genes derived from pediatric long-term nonprogressors have gross deletions in isolated cases but a higher prevalence of discrete changes that may impair Nef function in primary T cell assays, but not all functions reported for Nef.

CD34/QBEND10 immunostaining in bone marrow biopsies: an additional parameter for the diagnosis and classification of myelodysplastic syndromes.

CD34/QBEND10 immunostaining has been assessed in 150 bone marrow biopsies (BMB) including 91 myelodysplastic syndromes (MDS), 16 MDS-related AML, 25 reactive BMB, and 18 cases where RA could neither be established nor ruled out. All cases were reviewed and classified according to the clinical and morphological FAB criteria. The percentage of CD34-positive (CD34 +) hematopoietic cells and the number of clusters of CD34+ cells in 10 HPF were determined. In most cases the CD34+ cell count was similar to the blast percentage determined morphologically. In RA, however, not only typical blasts but also less immature hemopoietic cells lying morphologically between blasts and promyelocytes were stained with CD34. The CD34+ cell count and cluster values were significantly higher in RA than in BMB with reactive changes (p<0.0001 for both), in RAEB than in RA (p=0.0006 and p=0.0189, respectively), in RAEBt than in RAEB (p=0.0001 and p=0.0038), and in MDS-AML than in RAEBt (p<0.0001 and p=0.0007). Presence of CD34+ cell clusters in RA correlated with increased risk of progression of the disease. We conclude that CD34 immunostaining in BMB is a useful tool for distinguishing RA from other anemias, assessing blast percentage in MDS cases, classifying them according to FAB, and following their evolution.

Rare occurrence of classical Hodgkin's disease as a T cell lymphoma.

Recent work identified Hodgkin and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease (cHD) as clonal progeny of mature B cells. Therefore, it is generally assumed that cHD homogenously represents a B cell lymphoma. In a subset of cHD, however, H/RS cells expressing T cell-associated proteins may be candidates for alternative lineage derivation. Single H/RS cells with cytotoxic T cell phenotype were micromanipulated from three cases of cHD and analyzed by single cell polymerase chain reaction for immunoglobulin heavy (IgH) and light chain (IgL) gene rearrangements, T cell receptor (TCR)-beta gene rearrangements, and germline configuration of the IgH and TCR-beta loci. H/RS cells from two cases of cHD harbored clonal, somatically mutated Ig gene rearrangements, whereas TCR-beta loci were in germline configuration. In contrast, H/RS cells from an additional case harbored clonal TCR-beta variable/diversity/joining (VDJ) and DJ gene rearrangements, whereas the IgH locus was in germline configuration on both alleles. Thus, in two cases of cHD with H/RS cells expressing cytotoxic T cell molecules, the tumor cells are derived from mature B cells that aberrantly express T cell markers. In a third case, however, H/RS cells were derived from a T cell, demonstrating that cHD can also occur as a T cell lymphoma.

Frequent methylation silencing of p15(INK4b) (MTS2) and p16(INK4a) (MTS1) in B-cell and T-cell lymphomas.

The methylation status of p15(INK4b) (MTS2), p16(INK4a) (MTS1) and p14(ARF) (p16beta) was analyzed in 56 lymphomas by restriction-enzyme related polymerase chain reaction (PCR) (REP), methylation-specific PCR (MSP), and bisulfite genomic sequencing (BGS). Methylation of the p15 and p16 genes was detected, respectively, in 64% and 32% of the B-cell lymphomas, in 44% and 22% of the T-cell lymphomas, and in none of the 5 reactive lymph nodes analyzed. Both p15 and p16 genes were methylated more often in the high-grade (78% and 50%, respectively) than in the low-grade B-cell lymphomas (55% and 21%, respectively). For 5 cases, mapping of the methylated CpGs of the p16 promoter region confirmed the results of REP and MSP. In addition, a large variation in the methylation patterns of p16 exon 1 was observed, not only from one lymphoma to another, but also within a given tumor. Methylation of p15 and p16 was associated with an absence of gene expression, as assessed by reverse transcription-PCR. The p14 gene was unmethylated and normally expressed in all 56 tumors. We found no mutations of p15, p16, or p14 in any of the 56 lymphomas. Our results suggest a role for p15 and p16 gene methylation during lymphomagenesis and a possible association between p15 and p16 inactivation and aggressive transformation in B-cell and T-cell lymphomas.

Chromosomal abnormalities in renal cell neoplasms associated with acquired renal cystic disease. A series studied by comparative genomic hybridization and fluorescence in situ hybridization.

Sporadic renal cell carcinomas (RCCs) display different chromosomal abnormalities according to their morphology; gains of chromosomes 7 and 17 and loss of Y are commonly observed in papillary lesions, whereas loss of 3p sequences and multiple losses of specific chromosomes are found in non-papillary and chromophobe cell carcinomas, respectively. Acquired renal cystic disease (ARCD) is associated with an increased incidence of renal cell tumours, especially papillary lesions. The aim of this study was to examine a series of ARCD-related tumours for chromosomal abnormalities and to compare the findings with those abnormalities commonly observed in sporadic RCCs. Nine tumours from four patients with ARCD were examined using comparative genomic hybridization (CGH) and interphase cytogenetics. Gain of chromosomes 7 and 17 was observed in all four papillary lesions and loss of Y in three. In addition, gain of chromosome 16 was observed in three papillary tumours. Three chromophobe RCCs originating from the same kidney showed different genomic profiles; two had no abnormalities, whereas one showed loss of chromosome 17p. Two non-papillary RCCs failed to show chromosome 3p alterations. In conclusion, renal cell tumours developing in ARCD may show chromosomal abnormalities both similar to and different from those seen in sporadic tumours.

Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain.

During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4(+) and CD8(+) T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the zeta chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain. Conformation for the importance of zeta for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.

Prognostic value of follicular dendritic cells in nodular sclerosing Hodgkin's disease.

AIMS: In nodular sclerosing Hodgkin's disease (NSHD), the prognostic relevance of the histopathological grading in two subtypes NSI (low-grade) and NSII (high-grade) remains controversial. Analysis of follicular dendritic cells (FDC) may provide new prognostic parameters. METHODS AND RESULTS: Tumours from 59 patients with NSHD were studied. Mean follow-up time was 8 years. Forty-one cases were classified as NSI and 18 as NSII. FDC were immunostained with the paraffin-resistant monoclonal antibodies CD21 and CNA.42. We distinguished three patterns in the neoplastic tissue: FDC1, the presence of well-defined follicle-like structures (n = 20); FDC2, the presence of largely destroyed FDC networks (n = 25); and FDC3, no or a few isolated FDC (n = 14). The three groups differed clearly regarding the frequency of relapse and the survival. The longest survival was seen in the FDC1 group, the shortest in the FDC3 group, the FDC2 group being intermediate (P = 0.0025). FDC status was a discriminating prognostic factor for all patients, and within various age and stage categories. Combining the FDC status and the NSI-NSII grading defined the best survival group as FDC1-NSI. CONCLUSIONS: Assessment of FDC pattern, associated with histological subtyping, brings valuable data for predicting survival and outcome in NSHD.

The N-terminus of Nef from HIV-1/SIV associates with a protein complex containing Lck and a serine kinase.

The Nef protein of human and primate lentiviruses is a key factor in HIV/SIV pathogenesis. Here we report that Nef associates with two different kinases, forming a multiprotein complex at the far N-terminus of the viral protein. One of the kinases was identified as Lck, whereas the second protein was found to be a serine kinase that phosphorylated Nef and Lck in vitro and could be discriminated from the serine kinase identified previously. The Nef-associated kinase complex (NAKC) was demonstrated in COS cells, in HIV-infected cells, and in vitro using recombinant Lck and Nef proteins. Deletion of a short amphipathic alpha-helix in the N-terminus, which was found to be conserved in all Nef proteins, inhibited association of the NAKC and significantly reduced virion infectivity.

Follicular dendritic cells in bone marrow lymphoproliferative diseases: an immunohistochemical study including a new paraffin-resistant monoclonal antibody, DR53.

Two-hundred and twenty-one bone marrow biopsies with lymphoid infiltrates were studied histologically and immunohistochemically, to assess the incidence and the pattern of follicular dendritic cells. Three monoclonal antibodies selective for follicular dendritic cells were used: CD21, CD35 and DR53, all reactive on paraffin-embedded material. Follicular dendritic cells were present in two of 38 benign lymphoid aggregates, 92 of 134 low grade B-cell lymphomas (45 of 62 lymphocytic, 16 of 27 lymphoplasmacytoid, 0 of six hairy cell leukaemias, five of six centrocytic, 19 of 21 centroblastic-centrocytic, seven of 12 low grade NOS), one of 23 high grade B-cell lymphomas, 0 of 10 T-cell lymphomas, 0 of three Hodgkin's disease and four of 13 suspicious infiltrates. Follicular dendritic cells were found in lymphomatous involvement with nodular, patchy and massive growth pattern, but not in interstitial ones. They formed follicle-like networks, whose number and size were directly correlated to the tumour mass. The origin and frequency distribution of follicular dendritic cells in bone marrow biopsy lymphomas is discussed and the diagnostic relevance of follicular dendritic cell immunostaining in routine bone marrow biopsy lymphoid infiltrates is assessed.

A conserved domain and membrane targeting of Nef from HIV and SIV are required for association with a cellular serine kinase activity.

Among the primate lentiviruses (human immunodeficiency virus (HIV) -1, HIV-2, and simian immunodeficiency virus (SIV), the nef gene is highly conserved and encodes a myristylated protein of approximately 27 kDa (HIV-1) or approximately 34 kDa (HIV-2, SIV). Previously, we found Nef expressed either as a CD8-Nef fusion protein or as a native protein in virally infected T cell lines associates with a cellular serine kinase. This kinase activity phosphorylated two proteins of 62 and 72 kDa that coimmunoprecipitate with Nef in in vitro kinase assays. Using transient expression, various Nef alleles and mutants have been analyzed for association with the cellular kinase activity. The ability of Nef to associate with the kinase activity is conserved among several alleles of HIV-1 as well as SIVmac239 and is observed in non-lymphoid cell lines of simian and murine origins. Two separate regions of HIV-1SF2 Nef are critical for the associated kinase activity. One domain overlaps with a central highly conserved region found in all primate lentivirus nef genes and has been provisionally mapped to amino acids 45-127. Because membrane localization of Nef is important for the associated cellular kinase activity, the second domain represents a membrane targeting signal. Moreover, point mutations within the central region that abrogate the Nef-associated kinase activity in HIV-1SF2 Nef have the same effect when introduced into SIVmac239open Nef.

HIV-1 Nef leads to inhibition or activation of T cells depending on its intracellular localization.

Nef of primate lentiviruses is required for viremia and progression to AIDS in monkeys. Negative, positive, and no effects of Nef have also been reported on viral replication in cells. To reconcile these observations, we expressed a hybrid CD8-Nef protein in Jurkat cells. Two opposite phenotypes were found, which depended on the intracellular localization of Nef. Expressed in the cytoplasm or on the cell surface, the chimera inhibited or activated early signaling events from the T cell antigen receptor. Activated Jurkat cells died by apoptosis, and only cells with mutated nef genes expressing truncated Nefs survived, which rendered Nef nonfunctional. These mutations paralleled those in other viral strains passaged in vitro. Not only do these positional effects of Nef reconcile diverse phenotypes of Nef and suggest a role for its N-terminal myristylation, but they also explain effects of Nef in HIV infection and progression to AIDS.