Comparison of tests for the detection of circulating filarial antigen (Og4C3-ELISA and AD12-ICT) and ultrasound in diagnosis of lymphatic filariasis in individuals with microfilariae

Significant advances were made in the diagnosis of filariasis in the 1990s with the emergence of three new alternative tools: ultrasound and tests to detect circulating antigen using two monoclonal antibodies, Og4C3 and AD12-ICT-card. This study aimed to identify which of these methods is the most sensitive for diagnosis of infection. A total of 256 individuals, all male and carrying microfilariae (1-15,679 MF/mL), diagnosed by nocturnal venous blood samples, were tested by all three techniques. The tests for circulating filarial antigen concurred 100% and correctly identified 246/256 (96.69%) of the positive individuals, while ultrasound detected only 186/256 (73.44%). Of the circulating antigen tests, ICT-card was the most convenient method for identification of Wuchereria bancrofti carriers. It was easy to perform, practical and quick.

Serological diagnosis of Chagas disease: evaluation and characterisation of a low cost antigen with high sensitivity and specificity

In spite of evident progress in the serology of Chagas disease, the requirement for new diagnostic antigens persists. We have evaluated different antigens obtained from Trypanosoma cruzi grown in medium rich in nutrients or under nutrient stress, autoclaved or sonicated and fractionated by differential centrifugation. The resulting antigens were evaluated for diagnosis of Chagas disease using ELISA. Immunofluorescence of the parasites demonstrated that nutrient stress induced changes in the distribution and density of antigens recognised by a pool of sera from experimentally infected mice. When evaluated using ELISA, it was evident that most fractions had good sensitivity but poor specificity. Surprisingly, the best specificity and sensitivity was observed with parasites cultured under nutrient stress and autoclaved. Furthermore this antigen had low cross reactivity with sera from other parasitic diseases, Leishmaniasis in particular. Western blot analysis demonstrated that autoclaving seems to non-specifically eliminate cross-reactive antigens. In conclusion, autoclaving epimastigotes of T. cruzi, after nutrient stress, allowed us to obtain an antigen that could be used in the serological diagnosis of Chagas disease.

Molecular design of the Calphabeta interface favors specific pairing of introduced TCRalphabeta in human T cells.

A promising approach to adoptive transfer therapy of tumors is to reprogram autologous T lymphocytes by TCR gene transfer of defined Ag specificity. An obstacle, however, is the undesired pairing of introduced TCRalpha- and TCRbeta-chains with the endogenous TCR chains. These events vary depending on the individual endogenous TCR and they not only may reduce the levels of cell surface-introduced TCR but also may generate hybrid TCR with unknown Ag specificities. We show that such hybrid heterodimers can be generated even by the pairing of human and mouse TCRalpha- and TCRbeta-chains. To overcome this hurdle, we have identified a pair of amino acid residues in the crystal structure of a TCR that lie at the interface of associated TCR Calpha and Cbeta domains and are related to each other by both a complementary steric interaction analogous to a "knob-into-hole" configuration and the electrostatic environment. We mutated the two residues so as to invert the sense of this interaction analogous to a charged "hole-into-knob" configuration. We show that this inversion in the CalphaCbeta interface promotes selective assembly of the introduced TCR while preserving its specificity and avidity for Ag ligand. Noteworthily, this TCR modification was equally efficient on both a Mu and a Hu TCR. Our data suggest that this approach is generally applicable to TCR independently of their Ag specificity and affinity, subset distribution, and species of origin. Thus, this strategy may optimize TCR gene transfer to efficiently and safely reprogram random T cells into tumor-reactive T cells.

Lymphoid environment limits superantigen and antigen-induced T cell proliferation at high precursor frequency.

After superantigen challenge a significant proportion of superantigen-reactive T cells remain undivided. We provide evidence that the lymphoid environment limits T cell proliferation in the secondary lymphoid organs when the frequency of superantigen reactive T cells is unusually high. We monitored T cell proliferation and the percentage of undivided cells when the frequency of superantigen-reactive T cells was low (1%), intermediate (15%) or high (30-100%) by transferring fluorescently labeled cells into different recipients. When the frequency was low, practically all the reactive T cells entered cell cycle and proliferated maximally. At intermediate frequencies a large proportion of reactive T cells did not enter cell cycle and the whole population divided less. A further increase in reactive T cells did not alter the percentage of undivided cells but induced a further decrease in the number of cell divisions. Interestingly, the observations made with superantigens were confirmed with peptide antigen and TCR-transgenic mice. Moreover, in vivo and in vitro data suggest that dendritic cells are the most likely candidates in limiting T cell proliferation in the lymphoid environment. In conclusion, we show that the availability of APC in the lymphoid environment can quantitatively limit T cell priming.

Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis

Kinetoplastid membrane protein-11 (KMP-11), a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. A suitable leishmaniasis vaccine candidate molecule must be expressed in amastigotes, the infective stage for mammals. However, the expression of KMP-11 in Leishmania amastigotes has been a subject of controversy. We evaluated the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, of Leishmania amazonensis by immunoblotting, flow cytometry and immunocytochemistry, using a monoclonal antibody against KMP-11. We found that KMP-11 is present in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures (at the cell surface, flagellar pocket and intracellular vesicles). More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. The presence of this molecule in amastigotes is consistent with the previously demonstrated immunoprotective capacity of vaccine prototypes based on the KMP-11-coding gene and the presence of humoral and cellular immune responses to KMP-11 in Leishmania-infected humans and animals.

Immune Microenvironment in Colorectal Cancer: A New Hallmark to Change Old Paradigms

Impact of immune microenvironment in prognosis of solid tumors has been extensively studied in the last few years. Specifically in colorectal carcinoma, increased knowledge of the immune events around these tumors and their relation with clinical outcomes have led to consider immune microenvironment as one of the most important prognostic factors in this disease. In this review we will summarize and update the current knowledge with respect to this intriguing and complex new hallmark of cancer, paying special attention to infiltration by T-infiltrating lymphocytes and their subtypes in colorectal cancer, as well as its eventual clinical translation in terms of long-term prognosis. Finally, we suggest some possible investigational approaches based on combinatorial strategies to trigger and boost immune reaction against tumor cells.

Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen.

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.

Evaluation of local immune response to Fasciola hepatica experimental infection in the liver and hepatic lymph nodes of goats immunized with Sm14 vaccine antigen

Protection against Fasciola hepatica in goats immunized with a synthetic recombinant antigen from Schistosoma mansoni fatty acid-binding protein 14 (rSm14) was investigated by assessing worm burdens, serum levels of hepatic enzymes, faecal egg count and hepatic damage, which was evaluated using gross and microscopic morphometric observation. The nature of the local immune response was assessed by examining the distribution of CD2+, CD4+, CD8+ and γ´+ T lymphocytes along with IgG+, IL-4+ and IFN-γ+ cells in the liver and hepatic lymph nodes (HLN). The goats used consisted of group 1 (unimmunized and uninfected), group 2 [infected control - immunized with Quillaia A (Quil A)] and group 3 (immunized with rSm14 in Quil A and infected), each containing seven animals. Immunization with rSm14 in Quil A adjuvant induced a reduction in gross hepatic lesions of 56.6% (p < 0.001) and reduced hepatic and HLN infiltration of CD2+, CD4+, CD8+ and γ´+ T lymphocytes as well as IL-4+ and IFN-γ+ cells (p < 0.05). This is the first report of caprine immunization against F. hepatica using a complete rSm14 molecule derived from S. mansoni. Immunization reduced hepatic damage and local inflammatory infiltration into the liver and HLN. However, considering that Quil A is not the preferential/first choice adjuvant for Sm14 immunization, further studies will be undertaken using the monophosphoryl lipid A-based family of adjuvants during clinical trials to facilitate anti-Fasciolavaccine development.

Trachea, lung, and tracheobronchial lymph nodes are the major sites where antigen-presenting cells are detected after nasal vaccination of mice with human papillomavirus type 16 virus-like particles.

Vaccination by the nasal route has been successfully used for the induction of immune responses. Either the nasal-associated lymphoid tissue (NALT), the bronchus-associated lymphoid tissue, or lung dendritic cells have been mainly involved. Following nasal vaccination of mice with human papillomavirus type 16 (HPV16) virus-like-particles (VLPs), we have previously shown that interaction of the antigen with the lower respiratory tract was necessary to induce high titers of neutralizing antibodies in genital secretions. However, following a parenteral priming, nasal vaccination with HPV16 VLPs did not require interaction with the lung to induce a mucosal immune response. To evaluate the contribution of the upper and lower respiratory tissues and associated lymph nodes (LN) in the induction of humoral responses against HPV16 VLPs after nasal vaccination, we localized the immune inductive sites and identified the antigen-presenting cells involved using a specific CD4(+) T-cell hybridoma. Our results show that the trachea, the lung, and the tracheobronchial LN were the major sites responsible for the induction of the immune response against HPV16 VLP, while the NALT only played a minor role. Altogether, our data suggest that vaccination strategies aiming to induce efficient immune responses against HPV16 VLP in the female genital tract should target the lower respiratory tract.

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.

Human CD8(+) T cells engineered with supraphysiological TCRs are functionally inhibited via PD-1 and SHP-1 phosphatase

Purpose/Objective: Protective CD8+ T cell responses rely on TCRdependent recognition of immunogenic peptides presented by MHC I. Cytolytic T lymphocytes directed against self/tumor antigens express TCRs of lower affinity/avidity than pathogen-derived T lymphocytes and elicit less protective immune responses due to mechanisms of central and peripheral tolerance. Anti-tumor T cell reactivity can be improved by increasing the TCR-pMHC affinity within physiological limits, while intriguingly further increase in the supraphysiological range (KD < 1 lM) leads to drastic functional declines. We aim at identifying the molecular mechanisms underlying the loss of T cell responsiveness associated with supraphysiological TCRpMHC affinities in order to improve effectiveness of TCR-engineered T cells used in adoptive cell transfer (ACT) cancer immunotherapy. Materials and methods: Using a panel of human CD8+ T cells engineered with TCRs of incremental affinity for the HLA-A2-resticted tumor cancer testis antigen NY-ESO-1, we performed comparative gene expression microarray and TCR-mediated signaling analysis together with membrane receptors level analysis. Results: As compared to cells expressing TCR affinities generating optimal function (KD from 5to 1 lM), those with supraphysiological affinity (KD from 1 lM to 15 nM) had an overall reduced expression of genes implied in signaling, cell activation and proliferation, and showed impaired proximal and distal TCR signaling capacity. This correlated with a decline in surface expression of CD8b, CD28 and activatory TNFR superfamily members. Importantly, expression of inhibitory receptor PD-1 and SHP-1 phosphatase was upregulated in a TCR affinity-dependent manner. Consequently, PD-L1 and SHP-1 blockade restored the function of T cells with high TCRs affinity. Moreover, SHP-1 inhibition also augmented functional efficacy of T cells with TCRs of optimal affinity. Conclusions: Our findings indicate that TCR affinity-associated regulatory mechanisms control T cells responsiveness at various levels to limit potential auto-reactive cytotoxic effects. They also support the development of ACT therapies combined with blockade of inhibitory molecules such as SHP-1 to enhance effectiveness of T cell immunotherapy.

Marked increase in the secretion of a fully antigenic recombinant carcinoembryonic antigen obtained by deletion of its hydrophobic tail.

Carcinoembryonic antigen (CEA) is a well-known tumor marker, consisting of a single heavily glycosylated polypeptide chain (mol. wt 200 kD), bound to the cell surface by a phosphatidylinositol-glycan anchor. The hydrophobic domain, encoded by the 3' end of the open reading frame of the CEA gene is not present in the mature protein. This domain is assumed to play an important role in the targeting and attachment of CEA to the cell surface. To verify this hypothesis, a recombinant CEA cDNA lacking the 78 b.p. of the 3' region, encoding the 26 a.a. hydrophobic domain, was prepared in a Rc/CMV expression vector containing a neomycin resistance gene. The construct was transfected by the calcium phosphate technique into CEA-negative human and rat colon carcinoma cell lines. Geneticin-resistant transfectants were screened for the presence of CEA in the supernatant and positive clones were isolated. As determined by ELISA, up to 13 micrograms of recombinant CEA per 10(6) cells was secreted within 72 hr by the human transfected cells and about 1 microgram by the rat cells. For comparison, two human carcinoma cell lines, CO112 and LS174T, selected for high CEA expression, shed about 45 and 128 ng per 10(6) cells within 72 hr, respectively. Western blot analysis showed that the size of the recombinant CEA secreted by the transfected human cells is identical to that of reference CEA purified from human colon carcinomas metastases (about 200 kD). The recombinant CEA synthesized by the transfected rat carcinoma cells has a smaller size (about 144 kD, possibly due to incomplete glycosylation), as has already been observed for CEA produced by rat colon carcinoma cells transfected with full-length CEA cDNA. The 100-fold increase in secretion of rCEA encoded by truncated CEA cDNA transfected in human cells confirms the essential role of this domain in the targeting and anchoring of the glycoprotein. These results suggest a new approach for the in vitro production of large amounts of CEA needed in research laboratories and for immunoassay kits.

Kallikrein genes are associated with lupus and glomerular basement membrane-specific antibody-induced nephritis in mice and humans.

Immune-mediated nephritis contributes to disease in systemic lupus erythematosus, Goodpasture syndrome (caused by antibodies specific for glomerular basement membrane [anti-GBM antibodies]), and spontaneous lupus nephritis. Inbred mouse strains differ in susceptibility to anti-GBM antibody-induced and spontaneous lupus nephritis. This study sought to clarify the genetic and molecular factors that maybe responsible for enhanced immune-mediated renal disease in these models. When the kidneys of 3 mouse strains sensitive to anti-GBM antibody-induced nephritis were compared with those of 2 control strains using microarray analysis, one-fifth of the underexpressed genes belonged to the kallikrein gene family,which encodes serine esterases. Mouse strains that upregulated renal and urinary kallikreins exhibited less evidence of disease. Antagonizing the kallikrein pathway augmented disease, while agonists dampened the severity of anti-GBM antibody-induced nephritis. In addition, nephritis-sensitive mouse strains had kallikrein haplotypes that were distinct from those of control strains, including several regulatory polymorphisms,some of which were associated with functional consequences. Indeed, increased susceptibility to anti-GBM antibody-induced nephritis and spontaneous lupus nephritis was achieved by breeding mice with a genetic interval harboring the kallikrein genes onto a disease-resistant background. Finally, both human SLE and spontaneous lupus nephritis were found to be associated with kallikrein genes, particularly KLK1 and the KLK3 promoter, when DNA SNPs from independent cohorts of SLE patients and controls were compared. Collectively, these studies suggest that kallikreins are protective disease-associated genes in anti-GBM antibody-induced nephritis and lupus.

Passive lipoidal diffusion and carrier-mediated cell uptake are both important mechanisms of membrane permeation in drug disposition.

Recently, it has been proposed that drug permeation is essentially carrier-mediated only and that passive lipoidal diffusion is negligible. This opposes the prevailing hypothesis of drug permeation through biological membranes, which integrates the contribution of multiple permeation mechanisms, including both carrier-mediated and passive lipoidal diffusion, depending on the compound's properties, membrane properties, and solution properties. The prevailing hypothesis of drug permeation continues to be successful for application and prediction in drug development. Proponents of the carrier-mediated only concept argue against passive lipoidal diffusion. However, the arguments are not supported by broad pharmaceutics literature. The carrier-mediated only concept lacks substantial supporting evidence and successful applications in drug development.

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells.

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.