Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes.

To demonstrate that antibody-guided targeting of antigenic MHC class I-peptide tetramer on tumor cells can render them susceptible to lysis by relevant cytotoxic T lymphocytes (CTL), biotinylated HLA-A*0201/Flu matrix peptide complexes were tetramerized on streptavidin molecules previously coupled to Fab' fragments from monoclonal antibodies (mAb) specific for cell surface markers such as carcinoembryonic antigen (CEA), ErbB-2 or CD20. Flow cytometry analysis showed that coating of the HLA-A2-peptide complexes on the four HLA-A2-negative human cancer lines tested (including a CEA-positive colon carcinoma, an ErbB-2(+) breast carcinoma and two CD20(+) B lymphomas) was entirely dependent upon the specificity of the conjugated antibody fragments. More importantly, HLA-A2-restricted Flu matrix peptide-specific CTL were then found to lyse specifically and efficiently the MHC-coated target cells. These results open the way to the development of new immunotherapy strategies based on antibody targeting of MHC class I-peptide complexes.

CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display.

Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

AA-amyloidosis caused by visceral leishmaniasis in a human immunodeficiency virus-infected patient.

AA-amyloidosis in the setting of chronic visceral leishmaniasis (VL) has been reported in animal models but documentation in humans is unavailable. Here, we report on a Portuguese man who in 1996 was diagnosed with both human immunodeficiency virus (HIV)-infection and VL. Antiretroviral treatment led to sustained suppression of HIV viremia but CD4+ lymphocytes rose from 8 to only 160 cells/mL. Several courses of antimony treatment did not prevent VL relapses. Renal failure developed in 2006 and renal biopsy revealed AA-amyloidosis. The patient had cryoglobulinemia and serum immune complexes containing antibodies directed against seven leishmanial antigens. Antimony plus amphotericin B, followed by oral miltefosine resulted in a sustained VL treatment response with elimination of circulating Leishmania infantum DNA and CD4+ recovery. The concomitant reduction of serum AA levels and disappearance of circulating leishmanial immune complexes suggests that prolonged VL may lead to AA-amyloidosis in immunocompromised humans.

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells.

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Circulating soluble CR1 (CD35). Serum levels in diseases and evidence for its release by human leukocytes.

C receptor type 1 (CR1, CD35) is present in a soluble form in plasma (sCR1). Soluble CR1 was measured with a specific ELISA assay in normal individuals and in patients with different diseases. The mean serum concentration of sCR1 in 31 normal donors was 31.4 +/- 7.8 ng/ml, and was identical in plasma. An increase in sCR1 was observed in 36 patients with end-stage renal failure on dialysis (54.8 +/- 11.7 ng/ml, p < 0.0001), and in 22 patients with liver cirrhosis (158.3 +/- 49.9 ng/ml, p < 0.0001). The mean sCR1 levels dropped from 181 +/- 62.7 to 52.1 +/- 24.0 ng/ml (p < 0.001) in nine patients who underwent liver transplantation, and was 33.5 +/- 7.3 in 10 patients with functioning renal grafts, indicating that the increase in sCR1 was reversible. Soluble CR1 was elevated in some hematologic malignancies (> 47 ng/ml), which included B cell lymphoma (12/19 patients), Hodgkin's lymphoma (4/4), and chronic myeloproliferative syndromes (4/5). By contrast, no increase was observed in acute myeloid or lymphoblastic leukemia (10) or myeloma (5). In two patients with chronic myeloproliferative syndromes, sCR1 decreased rapidly after chemotherapy. The mean concentration of sCR1 was not significantly modified in 181 HIV-infected patients at various stages of the disease (34.8 +/- 14.4 ng/ml), and in 13 patients with active SLE (38.3 +/- 19.6 ng/ml), although in both groups the number of CR1 was diminished on E. There was a weak but significant correlation between sCR1 and CR1 per E in HIV infection and SLE (r = 0.39, p < 0.0001, and r = 0.60, p < 0.03 respectively). In vitro, monocytes, lymphocytes, and neutrophils were found to release sCR1 into culture supernatants. In vivo, sCR1 was detected in the serum of SCID mice populated with human peripheral blood leukocytes. The sCR1 levels correlated with those of human IgG (r = 0.97, p < 0.0001), suggesting synthesis of sCR1 by the transferred lymphocytes. The mechanisms underlining the increased levels of sCR1 and its biologic consequences remain to be defined.

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.

The spleen is an important site of T cell activation during human hepatosplenic schistosomiasis

We have undertaken a comparative immunephenotypic study of spleen cells from hepatosplenic patients (HS) and uninfected individuals (NOR) using flow cytometry. Our data did not show any significant differences in the mean percentage of T-cells and B-cells between the two groups. Analysis of activated T-cells demonstrated that HS present an increased percentage of CD3+HLA-DR+ splenocytes in comparison to NOR. Analysis of T-cell subsets demonstrated a significant increase on the percentage of both activated CD4+ T-splenocytes and CD8+ cells in HS. We did not find any difference in the mean percentage of CD28+ T-cells. Analysis of the B-cell compartment did not show any difference on the percentage of B1-splenocytes. However, the spleen seems to be an important reservoir/source for B1 lymphocytes during hepatosplenic disease, since after splenectomy we found a decreased the percentage of circulating B1-lymphocytes. We observed an increase on the percentage of CD2+CD3- lymphocytes in the spleen of HS suggesting that the loss of CD3 by activated T-cells or the expansion of NK-cells might play a role in the development/maintenance of splenomegaly.

Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909.

The induction of potent CD8+ T cell responses by vaccines to fight microbes or tumors remains a major challenge, as many candidates for human vaccines have proved to be poorly immunogenic. Deoxycytidyl-deoxyguanosin oligodeoxynucleotides (CpG ODNs) trigger Toll-like receptor 9, resulting in dendritic cell maturation that can enhance immunogenicity of peptide-based vaccines in mice. We tested whether a synthetic ODN, CpG 7909, could improve human tumor antigen-specific CD8+ T cell responses. Eight HLA-A2+ melanoma patients received 4 monthly vaccinations of low-dose CpG 7909 mixed with melanoma antigen A (Melan-A; identical to MART-1) analog peptide and incomplete Freund's adjuvant. All patients exhibited rapid and strong antigen-specific T cell responses: the frequency of Melan-A-specific T cells reached over 3% of circulating CD8+ T cells. This was one order of magnitude higher than the frequency seen in 8 control patients treated similarly but without CpG and 1-3 orders of magnitude higher than that seen in previous studies with synthetic vaccines. The enhanced T cell populations consisted primarily of effector memory cells, which in part secreted IFN- and expressed granzyme B and perforin ex vivo. In vitro, T cell clones recognized and killed melanoma cells in an antigen-specific manner. Thus, CpG 7909 is an efficient vaccine adjuvant that promotes strong antigen-specific CD8+ T cell responses in humans.

Human chronic chagasic cardiopathy: participation of parasite antigens, subsets of lymphocytes, cytokines and microvascular abnormalities

This article tries to demonstrate by new pathological findings (with the use of immunohistochemical technique and confocal laser microscopy) that chronic chagasic cardiomyopathy is a result of multiple factors involving myocarditis, immunodepression, severe fibrosis and microvessels dilatation and that all of these alterations are probably directly related with the presence of Trypanosoma cruzi parasites in the host associated with inadequate immunological response of the host.

Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes.

We have recently cloned the human homologue of the murine pT49 cDNA (hpT49h), a transcript encoding a protein homologous to the beta- and gamma-chains of fibrinogen. Here, we report the identification of the hpT49h gene product using mAbs generated against a peptide corresponding to the carboxyl-terminal end of the deduced protein and a recombinant protein fragment expressed in Escherichia coli. mAbs 23A6, 7B12, and 3F4 specifically recognized a protein of 70 kDa in reducing SDS-PAGE in the culture supernatant of 293T cells transiently transfected with the full length hpT49h cDNA and freshly isolated PBMC. Under nonreducing conditions, the material migrated with a molecular mass of 250 to 300 kDa, indicating that the 70-kDa protein forms a disulfide bonded complex. Because of its homology with fibrinogen, we have termed this protein fibroleukin. Fibroleukin is spontaneously secreted in vitro by freshly isolated CD4+ and CD8+ T lymphocytes. RT-PCR analysis revealed preferential expression of fibroleukin mRNA in memory T lymphocytes (CD3+/CD45R0+) compared with naive T lymphocytes (CD3+/CD45RA+). Fibroleukin production by PBMC was rapidly lost in culture. Production could be partially maintained in the presence of IFN-gamma, while T lymphocyte activation had no effect. To demonstrate fibroleukin production in vivo, we analyzed colon mucosa by immunohistology. Fibroleukin staining was detected in the extracellular matrix of the T lymphocyte-rich upper portion of the lamina propria mucosa. While the exact function of fibroleukin remains to be defined, these data suggest that fibroleukin may play a role in physiologic lymphocyte functions at mucosal sites.

Hepatitis B virus exposure in human immunodeficiency virus seropositive Cuban patients

In order to estimate the prevalence of serological markers of exposure to Hepatitis B Virus (HBV), 295 subjects were selected at random from the National Registry of human immunodeficiency virus positive subjects. Evidence of exposure to HBV was defined as: testing Hepatitis B surface antigen (HBsAg) and anti-Hepatitis B core antigen (anti-HBc) positive or anti-HBc positive only. Overall, 133 (45.5%) were positive for anti-HBc and 15 (5.1%) resulted positive to HBsAg. Significant statistical association was found between male sex and exposure to HBV (p<0.01). Homosexual or bisexual behavior was found to be strongly associated to HBV exposure (p<0.001). In conclusion, the prevalence of HBV serological markers is higher in Cuban HIV positive subjects compared to the Cuban general population.

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.

Human immunodeficiency virus type 1 Brazilian subtype B variant showed an increasing avidity of the anti-V3 antibodies over time compared to the subtype B US/European strain in São Paulo, Brazil

The Brazilian variant of human immunodeficiency virus type 1 (HIV-1) subtype B, (serotype B"-GWGR), has a tryptophan replacing the proline in position 328 the HIV-1 envelope. A longer median time period from infection to acquired immunodeficiency syndrome (AIDS) for serotype B (B"-GWGR) infected subjects compared to the B-GPGR US/European strain was reported. In a cohort study, in São Paulo city, 10 B"-GWGR patients had a statistically significant increased avidity of the anti-V3 antibodies, from 79% ± 33% to 85% ± 75%, versus from 48% ± 59% to 32% ± 17% for the 10 B-GPGR subjects (p = 0.02). The T CD4+ cells showed a mean increase of + 0.45 cells/month for the B-GPGR subjects and for B"-GWGR the slope was + 1.24 cells/month (p = 0.06), for 62 and 55 months of follow up, respectively. RNA plasma viral load decreased from 3.98 ± 1.75 to 2.16 ± 1.54 log10 in the B"-GWGR group while B-GPGR patients showed one log10 reduction in viral load from 4.09 ± 0.38 to 3.17 ± 1.47 log10 over time (p = 0.23), with a decreasing slope of 0.0042 ± log10,/month and 0.0080 ± log10/month, for B-GPGR and B"-GWGR patients, respectively (p = 0.53). Neither group presented any AIDS defining events during the study, according to Center for Diseases Control criteria. Although the sample size is small, these results may indicate that differences in the pathogenicity of the 2 HIV-1 B serotypes which co-circulate in Brazil may be correlated to the avidity of anti-V3 antibodies.

Implication of DNA methylation and PAX5 factor in the transcriptional regulation of hTERT, the human telomerase reverse transcriptase gene

RESUME La télomérase est une enzyme dite "d'immortalité" qui permet aux cellules de maintenir la longueur de leurs télomères, ce qui confère une capacité de réplication illimitée aux cellules reproductrices et cancéreuses. A l'inverse, les cellules somatiques normales, qui n'expriment pas la télomérase, ont une capacité de réplication limitée. La sous-unité catalytique de la télomérase, hTERT, est définie comme le facteur limitant l'activité télomérasique. Entre activateurs et répresseurs, le rôle de la méthylation de l'ADN et de l'acétylation des histones, de nombreux modèles ont été suggérés. La découverte de l'implication de CTCF dans la régulation transcriptionnelle de hTERT explique en partie le mécanisme de répression de la télomérase dans la plupart des cellules somatiques et sa réactivation dans les cellules tumorales. Dans les cellules télomérase-positives, l'activité inhibitrice de CTCF est bloquée par un mécanisme dépendent ou non de la méthylation. Dans la plupart des carcinomes, une hyperméthylation de la région 5' de hTERT bloque l'effet inhibiteur de CTCF, alors qu'une petite région hypométhylée permet un faible niveau de transcription du gène. Nous avons démontré que la protéine MBD2 se lie spécifiquement sur la région 5' méthylée de hTERT dans différentes lignées cellulaires et qu'elle est impliquée dans la répression partielle de la transcription de hTERT dans les cellules tumorales méthylées. Par contre, nous avons montré que dans les lymphocytes B normaux et néoplasiques, la régulation de hTERT est indépendante de la méthylation. Dans ces cellules, le facteur PAX5 se lie sur la région 5' de hTERT en aval du site d'initiation de la traduction (ATG). L'expression exogène de PAX5 dans les cellules télomérase-négatives active la transcription de hTERT, alors que la répression de PAX5 dans les cellules lymphomateuses inhibe la transcription du gène. PAX5 est donc directement impliqué dans l'activation de l'expression de hTERT dans les lymphocytes B exprimant la télomérase. Ces résultats révèlent des différences entre les niveaux de méthylation de hTERT dans les cellules de carcinomes et les lymphocytes B exprimant la télomérase. La méthylation de hTERT en tant que biomarqueur de cancer a été évaluée, puis appliquée à la détection de métastases. Nous avons ainsi montré que la méthylation de hTERT est positivement corrélée au diagnostic cytologique dans les liquides céphalorachidiens. Nos résultats conduisent à un modèle de régulation de hTERT, qui aide à comprendre comment la transcription de ce gène est régulée par CTCF, avec un mécanisme lié ou non à la méthylation du gène hTERT. La méthylation de hTERT s'est aussi révélée être un nouveau et prometteur biomarqueur de cancer. SUMMARY Human telomerase is an "immortalizing" enzyme that enables cells to maintain telomere length, allowing unlimited replicative capacity to reproductive and cancer cells. Conversely, normal somatic cells that do not express telomerase have a finite replicative capacity. The catalytic subunit of telomerase, hTERT, is defined as the limiting factor for telomerase activity. Between activators and repressors, and the role of DNA methylation and histone acetylation, an abundance of hTERT regulatory models have been suggested. The discovery of the implication of CTCF in the transcriptional regulation of hTERT in part explained the mechanism of silencing of telomerase in most somatic cells and its reactivation in neoplastic cells. In telomerase-positive cells, the inhibitory activity of CTCF is blocked by methylation-dependent and -independent mechanisms. In most carcinoma cells, hypermethylation of the hTERT 5' region has been shown to block the inhibitory effect of CTCF, while a short hypomethylated region allows a low transcription level of the gene. We have demonstrated that MBD2 protein specifically binds the methylated 5' region of hTERT in different cell lines and is therefore involved in the partial repression of hTERT transcription in methylated tumor cells. In contrast, we have shown that in normal and neoplastic B cells, hTERT regulation is methylation-independent. The PAX5 factor has been shown to bind to the hTERT 5'region downstream of the ATG translational start site. Ectopic expression of PAX5 in telomerase-negative cells or repression of PAX5 expression in B lymphoma cells respectively activated and repressed hTERT transcription. Thus, PAX5 is strongly implicated in hTERT expression activation in telomerase-positive B cells. These results reveal differences between the hTERT methylation patterns in telomerase-positive carcinoma cells and telomerase-positive normal B cells. The potential of hTERT methylation as a cancer biomarker was evaluated and applied to the detection of metastasis. We have shown that hTERT methylation correlates with the cytological diagnosis in cerebrospinal fluids. Our results suggest a model of hTERT gene regulation, which helps us to better understand how hTERT transcription is regulated by CTCF in methylation-dependant and independent mechanisms. Our data also indicate that hTERT methylation is a promising new cancer biomarker.