Downregulation of major histocompatibility class I on human dendritic cells by HIV Nef impairs antigen presentation to HIV-specific CD8+ T lymphocytes.

The HIV early regulatory Nef protein downregulates surface expression of major histocompatibility class I (MHC I) molecules on various immortalized cell lines and on T lymphocytes. MHC I-restricted presentation induces CD8+ T cell responses, which have a major role in limiting HIV infection. Induction of primary immune responses requires dendritic cells, which are major candidates as the first cells that can internalize the virus and present it to T cells in mucosal contamination. To test the effect of Nef on MHC I-restricted antigen presentation by dendritic cells, we used recombinant vaccinia viruses. Flow cytometric analysis of double labeling for a vaccinia protein and MHC I showed that HIV-1 Lai Nef indeed downregulated MHC I surface expression on dendritic cells. MHC I-restricted presentation to a Nef-specific CD8+ cell clone from an infected patient was decreased in an interferon gamma ELISpot assay. Presentation of a reverse transcriptase epitopic peptide on sorted Nef-infected cells was decreased in a peptide concentration-dependent way, confirming the role of MHC I downregulation in the impairment of the CD8+ cell-specific response. Therefore, Nef downregulates MHC I surface expression on human dendritic cells, impairing presentation to HIV-specific CD8+ cells. This action of Nef probably induces a deleterious delay in the early CD8+ responses during the first days of infection and at the onset of new viral mutants.

Mitochondrial membrane potential and apoptosis peripheral blood monocytes in severe human sepsis.

UNLABELLED: Reduced mitochondrial membrane potential (Delta(Psi)m), which is considered as an initial and irreversible step towards apoptosis, as well as cell death regulating proteins, such as Fas, Hsp70, or Bcl-2, may play an important role in sepsis. We studied the relationship between sepsis severity and peripheral blood monocyte Delta(Psi)m, cell death (necrosis and apoptosis), soluble Fas ligand, Hsp70, and Bcl-2 expression over time in 18 patients with sepsis, and compared these data with those of a group of 17 healthy control subjects. All measurements were performed within 3 d of the onset of severe sepsis (T1), then 7 to 10 d later (T2), and finally at hospital discharge (T3). Delta(Psi)m was expressed as the percent monocytes with altered Delta(Psi)m (%Delta(Psi)m). Patients with sepsis had greater %Delta(Psi)m at T1 and T2 but not at T3 (14.6 +/- 2.6% and 15.9 +/- 2%, respectively, versus control 6.6 +/- 0.2%, p < 0.01). Septic patients exhibited greater cell death in their monocytes and had greater Hsp70 expression only at T1. Bcl-2 levels were similar in septic and control subjects. Comparing survivors with non-survivors of sepsis, nonsurvivors had a greater %Delta(Psi)m at T1 (26.4 +/- 5.3% versus 10.1 +/- 2.7%, p < 0.01) and a significant decrease in Bcl-2 expression, whereas no difference was found in Hsp70 levels. These results indicate that mitochondrial dysfunction and subsequent cell death occur in severe sepsis and suggest that %Delta(Psi)m is a marker of severity in human sepsis. KEYWORDS: mitochondria; apoptosis; sepsis; heat-shock protein 70; proto-oncogene protein c-Bcl-2

Constitutively active STAT5 variants induce growth and survival of hematopoietic cells through a PI 3-kinase/Akt dependent pathway.

Signal Transducer and Activator of Transcription (STATs) are important mediators of cytokine and growth factor-induced signal transduction. STAT5A and STAT5B have been shown to play a role in survival and proliferation of hematopoietic cells both in vitro and in vivo and to contribute to the growth and viability of cells transformed by the TEL-JAK2 oncoprotein. In this study, we investigated the molecular mechanisms by which constitutively active STAT5 proteins induce cell proliferation and survival of Ba/F3 cell lines expressing either dominant positive STAT5A or STAT5B variants or TEL-JAK2 or TEL-ABL fusion proteins. Our results showed that active STAT5 constitutively interacted with p85, the regulatory subunit of the PI 3-kinase. A constitutive activity of the PI 3-kinase/Akt pathway was observed in these cells and required for their cell cycle progression. In contrast, while activity of the PI 3-kinase/Akt pathway was required for survival of Ba/F3 cells expressing the constitutively active forms of STAT5A or STAT5B, it was dispensable for cells transformed by TEL-JAK2 or TEL-ABL fusion proteins, suggesting that additional survival pathways take place in these transformed cells.

Involvement of the NF-kappaB pathway in the transforming properties of the TEL-Jak2 leukemogenic fusion protein.

Constitutively active tyrosine kinases are frequently expressed in various types of human leukemias as the result of chromosomal translocations. The TEL-Jak2 fusion oncoprotein possesses transforming properties in both animal and cellular models, that are tightly dependent on Stat5 activation. In the IL-3-independent TEL-Jak2-transformed Ba/F3 cells, activation of the PI-3K/Akt pathway appears essential to cell proliferation. Here we report a sustained activation of NF-kappaB factors in Ba/F3 cells, which inhibition dramatically impairs cell viability, indicating that NF-kappaB signaling exerts a major role in the anti-apoptotic activities of TEL-Jak2 oncoprotein.

Identification of two intracellular mechanisms leading to reduced expression of oncoretrovirus envelope glycoproteins at the cell surface.

All retrovirus glycoproteins have a cytoplasmic domain that plays several roles in virus replication. We have determined whether and how the cytoplasmic domains of oncoretrovirus glycoproteins modulate their intracellular trafficking, by using chimeric proteins that combined the alpha-chain of the interleukin-2 receptor with the glycoprotein cytoplasmic domains of five oncoretroviruses: human T-cell leukemia virus type 1 (HTLV-1), Rous sarcoma virus (RSV), bovine leukemia virus (BLV), murine leukemia virus (MuLV), and Mason-Pfizer monkey virus (MPMV). All of these proteins were synthesized and matured in the same way as a control protein with no retrovirus cytoplasmic domain. However, the amounts of all chimeric proteins at the cell surface were smaller than that of the control protein. The protein appearing at and leaving the cell surface and endocytosis were measured in stable transfectants expressing the chimera. We identified two groups of proteins which followed distinct intracellular pathways. Group 1 included chimeric proteins that reached the cell surface normally but were rapidly endocytosed afterwards. This group included the chimeric proteins with HTLV-1, RSV, and BLV cytoplasmic domains. Group 2 included chimeric proteins that were not detected at the cell surface, despite normal intracellular concentrations, and were accumulated in the Golgi complex. This group included the chimeric proteins with MuLV and MPMV cytoplasmic domains. Finally, we verified that the MuLV envelope glycoproteins behaved in the same way as the corresponding chimeras. These results indicate that retroviruses have evolved two distinct mechanisms to ensure a similar biological feature: low concentrations of their glycoproteins at the cell surface.

Interactions of the cytoplasmic domains of human and simian retroviral transmembrane proteins with components of the clathrin adaptor complexes modulate intracellular and cell surface expression of envelope glycoproteins.

The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXO, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXO motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXO motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXO motifs interact with the micro1 and micro2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the beta2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXO-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.

Flow cytometry is a rapid and reliable method for evaluating heat shock protein 70 expression in human monocytes.

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37 degrees C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.

Embryonic implantation in galectin 1/galectin 3 double mutant mice.

Galectin 1 and galectin 3 are first expressed in the trophectoderm cells of the implanting embryo and have been implicated in the process of implantation. However, we had previously shown that the lack of galectin 1 in galectin 1 null mutant mice is compatible with implantation. In this study, we describe the generation of galectin 3 null mutant mice and show that they are viable and have no overt abnormalities. The importance of galectin 1 and galectin 3 in implantation was assessed by obtaining double mutant mice [gal1 -/-; gal3 -/-]. We find that implantation can still occur in the absence of both galectin 1 and galectin 3. However, we show that galectin 5, a third member of this gene family, is also present in the blastocyst at the time of implantation.