Expression of SV2 isoforms during rodent brain development.

BACKGROUND: SV2A, SV2B and SV2C are synaptic vesicle proteins that are structurally related to members of the major facilitator superfamily (MFS). The function and transported substrate of the SV2 proteins is not clearly defined although they are linked to neurotransmitters release in a presynaptic calcium concentration-dependent manner. SV2A and SV2B exhibit broad expression in the central nervous system while SV2C appears to be more restricted in defined areas such as striatum. SV2A knockout mice start to display generalized seizures at a late developmental stage, around post-natal day 7 (P7), and die around P15. More recently, SV2A was demonstrated to be the molecular target of levetiracetam, an approved anti-epileptic drug (AED). The purpose of this work was to precisely analyze and quantify the SV2A, SV2B and SV2C expression during brain development to understand the contribution of these proteins in brain development and their impact on epileptic seizures. RESULTS: First, we systematically analyzed by immunohistofluorescence, the SV2A, SV2B and SV2C expression during mouse brain development, from embryonic day 12 (E12) to P30. This semi-quantitative approach suggests a modulation of SV2A and SV2B expression in hippocampus around P7. This is the reason why we used various quantitative approaches (laser microdissection of whole hippocampus followed by qRT-PCR and western blot analysis) indicating that SV2A and SV2B expression increased between P5 and P7 and remained stable between P7 and P10. Moreover, the increase of SV2A expression in the hippocampus at P7 was mainly observed in the CA1 region while SV2B expression in this region remains stable. CONCLUSIONS: The observed alterations of SV2A expression in hippocampus are consistent with the appearance of seizures in SV2A-/- animals at early postnatal age and the hypothesis that SV2A absence favors epileptic seizures around P7.

An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγnull mice allows HIV replication and development of anti-HIV immune responses.

Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγ(null) (NSG) and NOD/SCID/IL2Rγ(null) (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.

Lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice.

The respiratory tract is continuously exposed to both innocuous airborne antigens and immunostimulatory molecules of microbial origin, such as LPS. At low concentrations, airborne LPS can induce a lung DC-driven Th2 cell response to harmless inhaled antigens, thereby promoting allergic asthma. However, only a small fraction of people exposed to environmental LPS develop allergic asthma. What prevents most people from mounting a lung DC-driven Th2 response upon exposure to LPS is not understood. Here we have shown that lung interstitial macrophages (IMs), a cell population with no previously described in vivo function, prevent induction of a Th2 response in mice challenged with LPS and an experimental harmless airborne antigen. IMs, but not alveolar macrophages, were found to produce high levels of IL-10 and to inhibit LPS-induced maturation and migration of DCs loaded with the experimental harmless airborne antigen in an IL-10-dependent manner. We further demonstrated that specific in vivo elimination of IMs led to overt asthmatic reactions to innocuous airborne antigens inhaled with low doses of LPS. This study has revealed a crucial role for IMs in maintaining immune homeostasis in the respiratory tract and provides an explanation for the paradox that although airborne LPS has the ability to promote the induction of Th2 responses by lung DCs, it does not provoke airway allergy under normal conditions.

Molecular classification of T-cell lymphomas.

T-cell neoplasms encompass a heterogeneous group of relatively rare disease entities. This review, focused on lymphoblastic tumors (T-ALL/LBL) and nodal-based peripheral T-cell lymphomas (PTCL), summarizes recent advances in the molecular characterization of these diseases. In T-ALL/LBL, molecular subgroups delineated by gene expression profiling correlate with leukemic arrest at specific stages of normal thymocyte development and different oncogenic pathways, and seem to be of interest for prognosis prediction. Angioimmunoblastic T-cell lymphoma (AITL), one of the most common PTCL entities, comprises neoplastic cells with a molecular signature similar to normal follicular helper T cells, and this cellular derivation might account for several of the peculiar aspects of this disease. Except in ALK-positive anaplastic large cell lymphoma, defined by ALK gene fusions, chromosomal translocations are otherwise rare in PTCLs, but some recurrent rearrangements might be associated with distinct lymphoma subtypes. In PTCL, not otherwise specified (PTCL, NOS), novel molecular biomarkers of potential therapeutic interest have been recently identified.

Cross-reactive T cell responses in HIV CRF01_AE and B'-infected intravenous drug users: implications for superinfection and vaccines.

Abstract We previously observed limited cross-reactive T cell responses in two HIV-1-superinfected injection drug users (IDUs) before superinfection [Ramos A, et al.: J Virol 2002;76(15):7444-7452]. To elucidate the role of such responses in superinfection we examined cross-reactive T cell responses in IDUs infected with a single HIV-1 subtype. In this study, IFN-gamma ELISPOT assays were performed using recombinant vaccinia constructs and peripheral blood mononuclear cells (PBMCs) from 43 IDUs singly infected with CRF01_AE or B' from the same cohort as the superinfected IDUs. PBMCs were from time points corresponding to pre- (early) or post- (late) superinfection in the superinfected IDUs. We observed that most singly infected IDUs had cross-reactivity in samples from early (84% of CRF01_AE and 78% of B'-infected IDUs) and late (96% of CRF_01AE and 77% of B'-infected IDUs) time points. Frequent homologous reactivity at early (67% of CRF-01AE and 100% of B') and late (84% of CRF01_AE-infected and 100% of B'-infected IDUs) time points was also observed. Cross-reactive responses were predominantly to Pol and were broader and higher in CRF01_AE than in B'-infected IDUs (medians of 825 vs. 90 and 585 vs. 60 spot-forming units/10(6) PBMCs at early and late time points, respectively). Our results show that cross-reactive responses were more prevalent with greater height and breadth in singly infected IDUs than previously observed in corresponding collection time points of superinfected IDU. Thus, low or absent cross-reactivity may have contributed to the previously observed superinfections. These data are relevant for understanding superinfection and improving vaccine design.

Characterization of an antibody panel for immunohistochemical analysis of canine muscle cells.

Immunohistochemistry is an indispensable tool in the assessment and characterization of lineage-specific differentiation of grafted cells in cell-based-therapy. This strategy is under investigation for the treatment of many muscle disorders and different animals such as dogs are used as models to study the tissue regeneration. The aim of the present study was to characterize an antibody panel for the analysis of canine muscle cells, useful in routinely processed formalin-fixed paraffin-embedded tissues. Overall, 12 antibodies (8 mouse monoclonal and 4 goat polyclonal), validated for use on human tissues tested for cross-reactivity on canine smooth muscle (bladder, intestine, and uterus), skeletal muscle and heart. Specific staining was achieved with eight antibodies, of which six were cytoplasmic markers (desmin, HDAC8, MHC, SMA, Troponin I and Troponin T) and two were cardiac nuclear markers (GATA-4 and Nkx-2.5). This antibody panel may be useful not only for the evaluation of cell-based therapies in muscle disorders, but also for the evaluation of canine soft tissue neoplasms in veterinary pathology.

The prevention of spontaneous apoptosis of follicular lymphoma B cells by a follicular dendritic cell line: involvement of caspase-3, caspase-8 and c-FLIP.

BACKGROUND: Follicular lymphoma, the neoplastic counterpart of germinal center B cells, typically recapitulates a follicular architecture. Several observations point to the crucial role of the cellular microenvironment in the development and/or progression of follicular lymphoma cells in vivo. The aim of our study was to characterize the spontaneous apoptosis of follicular lymphoma cells in vitro, and the modulation of this apoptosis by follicular dendritic cells. DESIGN AND METHODS: We used a cell line derived from follicular dendritic cells to model the functional interactions of these cells and lymphoma cells in co-culture. Follicular lymphoma cells were isolated from tissue biopsies. Apoptosis was quantified by flow cytometry and apoptotic pathways were investigated by western blotting. RESULTS: The spontaneous apoptosis of follicular lymphoma cells in vitro involves the activation of caspases-3 and -8 but not of caspase-9, occurs despite persistent high levels of BCL-2 and MCL-1, and is associated with down-regulation of c-FLIP(L). Spontaneous apoptosis of follicular lymphoma cells is partially prevented by co-culture with the follicular dendritic cells, which prevents activation of caspase-8, caspase-3 and induces an upregulation of c-FLIP(L). Using neutralizing antibodies, we demonstrated that interactions involving CD54 (ICAM-1), CD106 (VCAM-1) and CD40 are implicated in this biological process. CONCLUSIONS: Follicular dendritic cells constitute a useful tool to study the functional interactions between follicular lymphoma cells and follicular dendritic cells in vitro. Understanding the molecular mechanisms involved in these protective interactions may lead to the identification of therapeutic agents that might suppress the survival and growth of follicular lymphoma cells.

TARC and IL-5 expression correlates with tissue eosinophilia in peripheral T-cell lymphomas.

The current study attempts to characterize the eosinophilia associated with T-cell lymphomas and to investigate its possible relationship with the secretion of eosinophil-stimulating factors by lymphoma cells and/or intra-tumoral surrounding cells. Paraffin-embedded specimens from 50 patients diagnosed with peripheral T-cell lymphomas, either unspecified (PTCL-U, n=30) or angioimmunoblastic (AITL, n=20) were morphologically assessed for intra-tumoral eosinophilia and analyzed by immunohistochemistry using specific antibodies directed against TARC, IL-5, RANTES, and eotaxin. The AITL and PTCL-U cases contained a mean of 147+/-41 and 102+/-37 eosinophils per 10 high power fields, respectively. Thirty-two of 47 cases (68%) showed IL-5-positive lymphoma cells while 15/50 (30%) tumors showed variable staining for TARC in scattered non-lymphoid cells with dendritic morphology. TARC and IL-5-positive cases possessed significantly more eosinophils. Our data indicate that IL-5 and TARC expression highly correlate with eosinophilia in T-cell lymphomas, suggesting that these chemokines are involved in the recruitment of eosinophils into the tumors.

The gene expression profile of nodal peripheral T-cell lymphoma demonstrates a molecular link between angioimmunoblastic T-cell lymphoma (AITL) and follicular helper T (TFH) cells.

The molecular alterations underlying the pathogenesis of angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, unspecified (PTCL-u) are largely unknown. In order to characterize the ontogeny and molecular differences between both entities, a series of AITLs (n = 18) and PTCLs-u (n = 16) was analyzed using gene expression profiling. Unsupervised clustering correlated with the pathological classification and with CD30 expression in PTCL-u. The molecular profile of AITLs was characterized by a strong microenvironment imprint (overexpression of B-cell- and follicular dendritic cell-related genes, chemokines, and genes related to extracellular matrix and vascular biology), and overexpression of several genes characteristic of normal follicular helper T (T(FH)) cells (CXCL13, BCL6, PDCD1, CD40L, NFATC1). By gene set enrichment analysis, the AITL molecular signature was significantly enriched in published T(FH)-specific genes. The enrichment was higher for sorted AITL cells than for tissue samples. Overexpression of several T(FH) genes was validated by immunohistochemistry in AITLs. A few cases with molecular T(FH)-like features were identified among CD30(-) PTCLs-u. Our findings strongly support that T(FH) cells represent the normal counterpart of AITL, and suggest that the AITL spectrum may be wider than suspected, as a subset of CD30(-) PTCLs-u may derive from or be related to AITL.

Establishment and characterisation of two novel human KSHV- and EBV-negative Burkitt cell lines, GAL-01 and GAL-02, from a primary lymphomatous effusion.

OBJECTIVES: Burkitt's lymphoma (BL) is a highly aggressive mature B-cell neoplasm comprising endemic, sporadic and immunodeficiency-associated variants. Human cell lines constitute a very useful tool to investigate the biology of lymphoid neoplasia. In this study, we succeeded in establishing two human cell lines, GAL-01 and GAL-02, from a HIV-negative patient with Epstein-Barr virus (EBV) -negative sporadic BL presenting as an effusion. GAL-01 and GAL-02 were established at diagnosis and after one course of polychemotherapy, respectively. The in vivo effusion occurred in a very peculiar clinical setting; the patient having a previous history of intestinal diffuse large B-cell lymphoma. METHODS: The morphologic, immunophenotypic and molecular genetic features of GAL cell lines are reported and compared with those of the parental tumour. The findings clearly demonstrated that the Burkitt effusion did not represent disease progression of the intestinal tumour, but represented a second primary haematological malignancy. The in vivo tumorigenic properties of the cells were tested by subcutaneous injection to NOD/SCID mice. RESULTS: Both cell lines were composed of medium-sized lymphoid cells with clumped chromatin, multiple medium-sized nucleoli and moderate amounts of vacuolated cytoplasm. GAL cells display the phenotype and genotype of a B-cell lineage (positive for CD20, CD79a and clonal rearrangement of Ig heavy chain), carry the c-MYC rearrangement by t(8;22)(q24;q11) translocation and are characterised by the expression of the germinal centre-associated antigens CD10, BCL6, CD38 and absent to low BCL2 expression. EBV and HHV8 were not identified within parental tumour or in cultured cells. Subcutaneous injection of both cell lines to NOD/SCID mice induced tumour formation. CONCLUSIONS: GAL-01 and GAL-02, two novel EBV-negative human BL cell lines represent a potentially useful experimental model to study the biology of BL possibly including the resistance to chemotherapy.

Interfaces between dendritic cells, other immune cells, and nerve fibres in mouse Peyer's patches: potential sites for neuroinvasion in prion diseases.

In this study, we examined where immune cells and nerve fibres are located in mouse Peyer's patches, with a view to identifying potential sites for neuroinvasion by prions. Special attention was paid to dendritic cells, viewed as candidate transporters of infectious prion. Double immunofluorescence labellings with anti-CD11c antibody and marker for other immune cells (B cells, T cells, follicular dendritic cells) were carried out and analysed by confocal microscopy on Peyer's patch cryosections. To reveal the extensive ganglionated networks of the myenteric and submucosal plexi and the sparse meshworks of nerve strands, we used antibodies directed against different neurofilament subunits or against glial fibrillary acidic protein. In the suprafollicular dome, dendritic cells connect, via their cytoplasmic extensions, enterocytes with M cells of the follicle-associated epithelium. They are also close to B and T cells. Nerve fibres are detected in the suprafollicular dome, notably in contact with dendritic cells. Similar connections between dendritic cells, T cells, and nerve fibres are seen in the interfollicular region. Germinal centres are not innervated; inside them dendritic cells establish contacts with follicular dendritic cells and with B cells. After immunolabelling of normal prion protein, dendritic cells of the suprafollicular dome are intensely positive labelled.

Lymphotoxin-beta receptor-dependent genes in lymph node and follicular dendritic cell transcriptomes.

Affinity maturation and Ab class switches occur in lymphoid germinal centers (GCs), in which differentiation and maintenance depend on lymphotoxin (LT) signaling and include differentiation of follicular dendritic cells (FDCs). The events leading to FDC and GC maturation are poorly defined. Using several approaches of functional genomics, we enumerated transcripts affected in mice by suppressing LT beta receptor (LTbetaR) signaling and/or overrepresented in FDC-enriched GC isolates. Protein expression analysis of 3 of 12 genes both enriched in FDCs and down-regulated by LTbetaR signaling suppression validated them as FDC markers. Functional analysis of one of these three, clusterin, suggests a role as an FDC-derived trophic factor for GC B cells. Hence, the set of genes presented in this study includes markers emanating from LTbetaR signaling and transcripts relevant to GC and FDC function.

Do bovine lymphocytes express a peculiar prion protein?

The cellular prion protein (PrPc) is a glycolipid-anchored cell surface protein that usually exhibits three glycosylation states. Its post-translationally modified isoform, PrPsc, is involved in the pathogenesis of various transmissible spongiform encephalopathies (TSEs). In bovine species, BSE infectivity appears to be restricted to the central nervous system; few or no detectable infectivity is found in lymphoid tissues in contrast to scrapie or variant CJD. Since expression of PrPc is a prerequisite for prion replication, we have investigated PrPc expression by bovine immune cells. Lymphocytes from blood and five different lymph organs were isolated from the same animal to assess intra- and interindividual variability of PrPc expression, considering six individuals. As shown by flow cytometry, this expression is absent or weak on granulocytes but is measurable on monocytes, B and T cells from blood and lymph organs. The activation of the bovine cells produces an upregulation of PrPc. The results of our in vitro study of PrPc biosynthesis are consistent with previous studies in other species. Interestingly, western blotting experiments showed only one form of the protein, the diglycosylated band. We propose that the glycosylation state could explain the lack of infectivity of the bovine immune cells.