Detection of mutations in NOD2/CARD15 gene in Arab patients with Crohn's disease.

Background: Mutations in NOD2/CARD15 gene have been linked to an increased risk of Crohn's disease (CD). The objective of this study is to determine NOD2/CARD15 gene mutations, and their association with the risk of CD in Arabs in Kuwait. Methods: Four NOD2 gene mutations, including Pro268Ser (SNP5), Arg702Trp (SNP8), Gly908Arg (SNP12), and Leu1007FsinsC (SNP13) were examined in Arab CD patients (n = 103) and control subjects (n = 100). The genomic DNA was isolated and used in polymerase chain reaction (PCR) with four sets of specific primers. The PCR-amplified DNA fragments were sequenced and analyzed for the NOD2 mutations. Logistic regression was used to estimate the adjusted odds ratios (aOR) and 95% confidence intervals (CI). Results: Of the four genotyped variants, the Arg702Trp (SNP8) and Leu1007FsinsC (SNP13) variants were not informative in our study sample due to minor allele frequency of <1%. The Pro268Ser (SNP5) mutation was detected in 17 (16.5%) CD patients and 32 (32.0%) controls. The Gly908Arg (SNP12) mutation was observed in 24 (23.3%) patients and 10 (10.0%) controls. In the dominant genetic risk model (i.e. carrying at least one minor allele), CD patients compared to controls were less likely to carry either the "CT" or "TT" genotype of variant Pro268Ser (SNP5; aOR = 0.43, 95% CI: 0.22-0.84). In contrast, CD patients compared to controls were more likely to carry the homozygous for the minor allele or the heterozygous genotypes of variant Gly908Arg (SNP12; aOR = 2.67, 95% CI: 1.19-5.97). Conclusions: In this Arab population, carrying at least one copy of the minor allele of Gly908Arg (SNP12) mutation in NOD2 gene was associated with increased susceptibility to CD, while having the heterozygous or homozygous for the minor allele genotype of the Pro268Ser (SNP5) mutation provided protection against CD. Mutations in Arg702Trp (SNP8) and Leu1007FsinsC (SNP13) were not detected in this sample of the Arab population in Kuwait.

Species identification and molecular typing of human Brucella isolates from Kuwait.

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

Development of a potent invigorator of immune responses endowed with both preventive and therapeutic properties.

This article reviews briefly the making of an immunoprophylactic-cum-immunotherapeutic vaccine against leprosy. The vaccine is based on cultivable, heat-killed atypical mycobacteria, whose gene sequence is now known. It has been named Mycobacterium indicus pranii. It has received the approval of the Drug Controller General of India and the US Food and Drug Administration. Besides leprosy, M. indicus pranii has found utility in the treatment of category II ("difficult to treat") tuberculosis. It also heals ugly anogenital warts. It has preventive and therapeutic action against SP2/O myelomas. It is proving to be a potent adjuvant for enhancing antibody titers of a recombinant vaccine against human chorionic gonadotropin, with the potential of preventing pregnancy without derangement of ovulation and menstrual regularity in sexually active women.

Development of Escherichia coli and Mycobacterium smegmatis recombinants expressing major Mycobacterium tuberculosis-specific antigenic proteins.

OBJECTIVE/BACKGROUND: Mycobacterium tuberculosis is an obligate pathogenic bacterial species in the family Mycobacteriaceae and the causative agent of most tuberculosis (TB) cases. Until today, the only approved TB vaccine is Bacille Calmette Guerin (BCG), which has been used since 1921. While BCG provides fairly effective protection for infants and young children, its efficacy in adults is variable around the world. This could be due to several parameters including strains of the vaccine and exposure of individuals to different environmental bacterial infections. The situation is complicated by the emergence of multidrug resistant strains of M. tuberculosis. This urged the demand to develop new improved vaccines and immunotherapies against TB. Development of nonpathogenic recombinant constructs delivering M. tuberculosis-specific antigenic proteins provides the chance to evaluate candidates to be included in diagnostic tools and preventive vaccines. In our study, we are introducing some of the major M. tuberculosis genes in Escherichia coli and Mycobacterium smegmatis. METHODS: DNA corresponding to the genes Rv3891, Rv3020, Rv0287, Rv3875, Rv3874, Rv3872, Rv2346c, and Rv3619 were PCR-amplified from M. tuberculosis genomic DNA and visualized on gel electrophoresis at the expected DNA size. Products were subsequently ligated to the plasmid pGEMTeasy and used to transform TOP10 E. coli. Transformed colonies were selected on appropriate media. At the second stage, genes-DNA were subcultured in expression vectors pDE22 and pGESTH1; the recombinant plasmids were finally used to transform. M. smegmatis and E. coli, respectively. Expression of proteins in E. coli was confirmed by Western blotting and in M. smegmatis by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Amplified genes were successfully cloned and transformed in E. coli and M. smegmatis. Colonies of recombinant bacteria were detected on appropriate media. Western blotting and RT-PCR confirmed the expression of our corresponding proteins in both the bacterial vehicles. CONCLUSION: Positive results of cloning and expression suggest that the constructed clones are ready tools for further assessment of their immunogenicity and can be included in improved diagnostic tools and vaccines against TB.

Characterization of a cross-reactive, immunodominant and HLA-promiscuous epitope of Mycobacterium tuberculosis-specific major antigenic protein PPE68.

PPE68 (Rv3873), a major antignic protein encoded by Mycobacteriun tuberculosis-specific genomic region of difference (RD)1, is a strong stimulator of peripheral blood mononuclear cells (PBMCs) obtained from tuberculosis patients and Mycobacterium bovis bacillus Calmette Guerin (BCG)-vaccianted healthy subjects in T helper (Th)1 cell assays, i.e. antigen-induced proliferation and interferon-gamma (IFN-γ) secretion. To confirm the antigen-specific recognition of PPE68 by T cells in IFN-γ assays, antigen-induced human T-cell lines were established from PBMCs of M. Bovis BCG-vaccinated and HLA-heterogeneous healthy subjects and tested with peptide pools of RD1 proteins. The results showed that PPE68 was recognized by antigen-specific T-cell lines from HLA-heteregeneous subjects. To further identify the immunodominant and HLA-promiscuous Th1-1 cell epitopes present in PPE68, 24 synthetic peptides covering the sequence of PPE68 were indivdually analyzed for HLA-DR binding prediction analysis and tested with PBMCs from M. bovis BCG-vaccinated and HLA-heterogeuous healthy subjects in IFN-γ assays. The results identified the peptide P9, i.e. aa 121-VLTATNFFGINTIPIALTEMDYFIR-145, as an immunodominant and HLA-DR promiscuous peptide of PPE68. Furthermore, by using deletion peptides, the immunodominant and HLA-DR promiscuous core sequence was mapped to aa 127-FFGINTIPIA-136. Interestingly, the core sequence is present in several PPE proteins of M. tuberculosis, and conserved in all sequenced strains/species of M. tuberculosis and M. tuberculosis complex, and several other pathogenic mycobacterial species, including M. leprae and M. avium-intracellulalae complex. These results suggest that the peptide aa 121-145 may be exploited as a peptide-based vaccine candidate against tuberculosis and other mycobacterial diseases.

Immune responses against Mycobacterium tuberculosis-specific proteins PE35 and CFP10 in mice immunized with recombinant Mycobacterium vaccae.

OBJECTIVE: To clone and express Mycobacterium tuberculosis (M. tuberculosis) proteins PE35 and culture filtrate protein (CFP)10 in Mycobacterium vaccae (M. vaccae), and subsequently, evaluate the humoral and cellular immunity responses against these recombinant constructs in mice. METHODS: The DNA of PE35 and CFP 10 genes were cloned into the shuttle plasmid pDE22, and the recombinant plasmids were electroporated into M. vaccae. The recombinant constructs were then tested for expression of PE35 and CFP10 by Western immunoblotting using rabbit anti-sera. Furthermore, splenocytes and sera from groups of 5 mice immunized with recombinant M. vaccae (rVaccae) were tested for cellular and humoral responses in proliferation, and antibody assays. Experiments were carried out in the laboratory of the Faculty of Medicine, Kuwait University, Safat, Kuwait between 2009 and 2011. RESULTS: The results of Western immunoblot suggested the expression of only PE35. However, splenocyte assays showed positive proliferation in response to peptide pools, and 4 and 5 of the 6 overlapping synthetic peptides covering the sequence of PE35 and CFP10. In addition, positive antibody reactivity was detected with PE35 peptide pool and a single peptide, namely, P2. CONCLUSION: The expression of PE35 and CFP10 proteins in rVaccae constructs led to the induction of cellular immune responses to multiple epitopes.

Cellular immune responses to recombinant Mycobacterium bovis BCG constructs expressing major antigens of region of difference 1 of Mycobacterium tuberculosis.

Besides being the most widely used vaccine directed against tuberculosis (TB) worldwide, Mycobacterium bovis BCG is also the most controversial vaccine in current use. Its protective efficacy varies widely in different parts of the world. One approach to improving the current BCG vaccine might be to produce recombinant BCG strains that express major antigens encoded by genes that are present in the M. tuberculosis-specific region of difference 1 (RD1), such as pe35, cfp10, and esat6. In this study, pe35, cfp10, and esat6 genes were cloned into shuttle plasmid pDE22 to generate the recombinant plasmids PDE22-PE35, PDE22-CFP10, and PDE22-ESAT6, which were electroporated into BCG to generate recombinant BCGs (rBCGs). The cellular immune responses (antigen-induced proliferation and secretion of selected T helper 1 [Th1], Th2, and anti-inflammatory cytokines, i.e., gamma interferon [IFN-γ], interleukin 5 [IL-5], and IL-10, respectively) that are specific to the proteins of cloned genes were studied by using spleen cells from mice immunized with native BCGs and rBCGs and synthetic peptides covering the protein sequence of the cloned genes. The results showed that the spleen cells did not secrete IL-5, whereas IL-10 was secreted in response to peptides of all three proteins from mice immunized with rBCGs only, suggesting expression of the cloned genes and in vivo priming of spleen cells to the expressed proteins. However, in Th1 cell assays that correlate with protective cellular immune responses, i.e., antigen-induced proliferation and IFN-γ secretion, only mice immunized with rBCG-pDE22-PE35 yielded positive responses to the peptides of PE35. These results suggest that rBCG-PDE22-PE35 is the only one of the three vaccines used in this work that is worthy of consideration as a new vaccine candidate against TB.

Comparative evaluation of MPT83 (Rv2873) for T helper-1 cell reactivity and identification of HLA-promiscuous peptides in Mycobacterium bovis BCG-vaccinated healthy subjects.

MPT83 (Rv2873), a surface lipoprotein excreted in the culture of Mycobacterium tuberculosis, is immunoreactive in antibody assays in humans and animals and provides protection as a combined DNA vaccine in mice and cattle. This study was undertaken to determine the reactivity of MPT83 in T helper 1 (Th1)-cell assays, i.e., antigen-induced proliferation and gamma interferon (IFN-γ) secretion, using peripheral blood mononuclear cells (PBMCs) obtained from Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated and/or M. tuberculosis-infected healthy subjects. PBMCs were tested with complex mycobacterial antigens and pools of synthetic peptides corresponding to MPT63, MPT83, MPB70, LppX, PPE68, CFP10, and ESAT-6. The results showed that MPT83 is among the strongest Th1 cell antigens of M. tuberculosis, and it was recognized equally strongly by BCG-vaccinated and by BCG-vaccinated and M. tuberculosis-infected healthy subjects. Furthermore, HLA heterogeneity of the responding donors suggested that MPT83 was presented to Th1 cells by several HLA-DR molecules. The analysis of the mature MPT83 sequence (amino acids [aa] 1 to 220) and its 14 overlapping synthetic peptides for binding prediction to HLA class II molecules and actual recognition of the peptides by PBMCs from HLA-DR-typed subjects in antigen-induced proliferation and IFN-γ assays suggested that Th1 cell epitopes were scattered throughout the sequence of MPT83. In addition, the HLA-promiscuous nature of at least three peptides, i.e., P11 (aa 151 to 175), P12 (aa 166 to 190), and P14 (aa 196 to 220), was suggested by HLA-DR binding predictions and recognition by HLA-DR heterogeneous donors in Th1 cell assays. These results support the inclusion of MPT83 in an antigen cocktail to develop a new antituberculosis vaccine.

Identification, diagnostic potential, and natural expression of immunodominant seroreactive peptides encoded by five Mycobacterium tuberculosis-specific genomic regions.

Comparative genomic studies have identified several Mycobacterium tuberculosis-specific genomic regions of difference (RDs) which are absent in the vaccine strains of Mycobacterium bovis BCG and which may be useful in the specific diagnosis of tuberculosis (TB). In this study, a total of 775 synthetic peptides covering the sequences of 39 open reading frame (ORF) proteins encoded by genes predicted in five RDs of M. tuberculosis, i.e., RD1, RD4, RD5, RD6, and RD7, were tested by enzyme-linked immunosorbent assays for antibody reactivity with sera from HIV-negative pulmonary TB patients (n = 100) and M. bovis BCG-vaccinated healthy subjects (n = 100). The results identified three immunodominant peptides reactive with TB sera, i.e., amino acids (aa) 346 to 370 of RD1ORF Rv3876, aa 241 to 265 of RD6ORF Rv1508c, and aa 325 to 336 of RD6ORF Rv1516c. These peptides had significantly stronger antibody reactivity with sera from TB patients than with sera from healthy subjects (P < 0.05) and significantly higher rates of positivity with TB sera (positives = 66 to 93%) than sera from healthy subjects (positives = 10 to 28%). Antipeptide antibodies were raised in rabbits after immunization with pools of 11 peptides corresponding to each protein. Probing of culture filtrates and whole-cell lysates of M. tuberculosis with antipeptide antibodies suggested the natural expression of Rv1516c in whole-cell lysates of M. tuberculosis. The results suggest the potential of the identified immunodominant RD peptides in the serodiagnosis of TB.

Amplification of six putative RD1 genes of Mycobacterium tuberculosis for cloning and expression in Escherichia coli and purification of expressed proteins.

OBJECTIVES: To amplify, clone and express in Escherichia coli six open reading frames (ORFs) predicted in the RD1 DNA segment of Mycobacterium tuberculosis and purify the expressed proteins to homogeneity. MATERIALS AND METHODS: DNA corresponding to the coding regions of six RD1 ORFs, i.e. ORF10 to ORF15, was amplified from genomic DNA of M. tuberculosis, cloned in the plasmid vector pPCR-Script and subcloned in expression plasmid vectors pET29a and/or pGEX-4T for expression in E. coli as fusion proteins. The recombinant fusion proteins were identified by sodium dodecyl polyacrylamide gel electrophoresis and Western immunoblotting. Attempts were made to obtain purified proteins, free of the fusion partner, using affinity and fast protein liquid chromatography. RESULTS: DNA corresponding to all six targeted RD1 ORFs was amplified from the genomic DNA of M. tuberculosis and five of the six ORFs, with the exception of ORF13, were cloned in the plasmid vectors and expressed in E. coli. Because of extensive degradation of ORF10 and ORF12 fusion proteins or nonbinding to the affinity columns of ORF15 fusion proteins, only ORF11 and ORF14 proteins were purified, free of the fusion partner, to homogeneity. CONCLUSION: All of the six targeted RD1 genes were amplified and five expressed using E. coli hosts, but only two of the expressed proteins were purified to homogeneity. Alternative expression systems are required to obtain all RD1 proteins for functional characterization.

Cell-mediated immune responses to complex and single mycobacterial antigens in tuberculosis patients with diabetes.

OBJECTIVE: To evaluate cell-mediated immune (CMI) response in diabetic and non-diabetic tuberculosis (TB) patients and healthy subjects in response to complex, fractionated and single antigens of Mycobacteriumtuberculosis. MATERIAL AND METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from patients suffering from pulmonary TB and type II diabetes (n = 7), pulmonary TB without diabetes (n = 10) and healthy subjects without TB and diabetes (n = 10). PBMC were assessed for CMI responses in antigen-induced proliferation assays in response to complex mycobacterial antigens (whole cells, cell walls and culture filtrate of M. tuberculosis), a battery of naturally purified or recombinant produced secreted (ESAT6, MPT59, MPT64 and MTB38) and cytosolic (MTB10, MTB70, ML10, ML28, ML36, ML65 and MB65) mycobacterial antigens and fractionated culture filtrate proteins (fractions F1-F10) of M. tuberculosis. RESULTS: The majority (>70%) of diabetic and non-diabetic TB patients and healthy subjects responded to the complex antigens of M. tuberculosis. However, among the single antigens, ESAT6 was most frequently recognized by TB patients with and without diabetes, but least recognized by healthy subjects. The secreted antigens MPT59 and MPT64 were recognized by all the groups, whereas the cytosolic antigens were recognized best by healthy subjects. When tested with fractionated secreted proteins present in the culture filtrate of M. tuberculosis, the best responses in both diabetic and non-diabetic TB patients were obtained with fractions containing low-molecular-weight proteins. CONCLUSIONS: Diabetic and non-diabetic TB patients respond frequently to secreted low-molecular-weight ESAT6 antigen of M. tuberculosis, indicating that this antigen may be useful in the diagnosis of TB in both the groups.

Efficient testing of large pools of Mycobacterium tuberculosis RD1 peptides and identification of major antigens and immunodominant peptides recognized by human Th1 cells.

Comparative genomics has identified several regions of difference (RDs) of Mycobacterium tuberculosis that are deleted or absent in Mycobacterium bovis BCG vaccines. To determine their relevance for diagnostic and vaccine applications, it is imperative that efficient methods are developed to test the encoded proteins for immunological reactivity. In this study, we have used 220 synthetic peptides covering sequences of 12 open reading frames (ORFs) of RD1 and tested them as a single pool (RD1(pool)) with peripheral blood mononuclear cells obtained from pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects in Th1 cell assays that measure antigen-induced proliferation and IFN-gamma secretion. The results showed that RD1(pool) induced strong responses in both TB patients and BCG-vaccinated healthy subjects. The subsequent testing of peptide pools of individual ORFs revealed that all ORFs induced positive responses in a portion of donors, but PPE68, CFP10, and ESAT6 induced strong responses in TB patients and PPE68 induced strong responses in BCG-vaccinated healthy subjects. In addition, HLA-DR and -DQ typing of donors and HLA-DR binding prediction analysis of proteins suggested HLA-promiscuous presentation of PPE68, CFP10, and ESAT6. Further testing of individual peptides showed that a single peptide of PPE68 (121-VLTATNFFGINTIPIALTEMDYFIR-145) was immunodominant. The search for sequence homology revealed that a part of this peptide, 124-ATNFFGINTIPIAL-137, was present in several PPE family proteins of M. tuberculosis and M. bovis BCG vaccines. Further experiments limited the promiscuous and immunodominant epitope region to the 10-amino-acid cross-reactive sequence 127-FFGINTIPIA-136.

Whole blood assays to identify Th1 cell antigens and peptides encoded by Mycobacterium tuberculosis-specific RD1 genes.

OBJECTIVE: To identify Th1 cell-stimulating antigens/peptides encoded by the genes predicted in the Mycobacterium tuberculosis-specific genomic region of difference (RD)1, deleted in Mycobacterium bovis Bacille Calmette-Guérin(BCG), by using synthetic peptides and whole blood from tuberculosis (TB) patients. MATERIALS AND METHODS: Heparinized peripheral blood was obtained from culture-proven pulmonary TB patients (n = 16) attending the Chest Disease Hospital, Kuwait. Whole blood was diluted with tissue culture medium RPMI-1640 and tested for Th1 cell stimulation using antigen-induced proliferation and interferon-gamma (IFN-gamma) secretion assays. The antigens included a peptide pool of 220 peptides covering the sequence of 12 open reading frames (ORFs) of RD1 (RD1(mix)), peptide pools of RD1 ORF5 (ORF5(mix)), ORF6 (ORF6(mix)) and ORF7 (ORF7(mix)), and individual peptides of ORF6 (P6.1-P6.6) and ORF7 (P7.1-P7.6). M. tuberculosis culture filtrate, cell walls and whole-cell M. bovis BCG were used as complex mycobacterial antigens. The results obtained with different antigens and peptides were statistically analyzed for significant differences using Z test. RESULTS: The complex mycobacterial antigens (culture filtrate, cell walls and M.bovis BCG) and RD1(mix) induced comparable (p > 0.05) positive antigen-induced proliferation and IFN-gamma responses with whole blood from TB patients. However, the positive IFN-gamma responses induced by ORF6(mix) and ORF7(mix) were higher than ORF5(mix). Among the individual peptides, P6.4 and P7.1 of ORF6 and ORF7, respectively, induced the highest IFN-gamma responses, suggesting that these peptides represented the immunodominant Th1 cell epitopes of RD1 ORF6 and ORF7 in the patients tested. CONCLUSION: The whole blood assays with synthetic peptides are useful to identify Th1 cell antigens/peptides encoded by genes located in M. tuberculosis-specific genomic regions.

Comparative evaluation of (1, 3)-beta-D-glucan, mannan and anti-mannan antibodies, and Candida species-specific snPCR in patients with candidemia.

BACKGROUND: Candidemia is a major infectious complication of seriously immunocompromised patients. In the absence of specific signs and symptoms, there is a need to evolve an appropriate diagnostic approach. A number of methods based on the detection of Candida mannan, nucleic acid and (1,3)-beta- D- glucan (BDG) have been used with varying specificities and sensitivities. In this retrospective study, attention has been focused to evaluate the usefulness of two or more disease markers in the diagnosis of candidemia. METHODS: Diagnostic usefulness of Platelia Candida Ag for the detection of mannan, Platelia Candida Ab for the detection of anti-mannan antibodies, Fungitell for the detection of BDG, and of a semi-nested PCR (snPCR) for the detection Candida species-specific DNA have been retrospectively evaluated using 32 sera from 27 patients with culture-proven candidemia, 51 sera from 39 patients with clinically suspected candidemia, sera of 10 women with C. albicans vaginitis, and sera of 16 healthy controls. RESULTS: Using cut-off values recommended by the manufacturers, the sensitivity of the assays for candidemia patients were as follows: Candida snPCR 88%, BDG 47%, mannan 41%, anti-mannan antibodies 47%, respectively. snPCR detected 5 patients who had candidemia due to more than one Candida species. The sensitivities of the combined tests were as follows: Candida mannan and anti-mannan antibodies 75%, and Candida mannan and BDG 56%. Addition of snPCR data improved the sensitivity further to 88%, thus adding 10 sera that were negative by BDG and/or mannan. In clinically suspected, blood culture negative patients; the positivities of the tests were as follows: Candida DNA was positive in 53%, BDG in 29%, mannan in 16%, and anti-mannan antibodies in 29%. The combined detection of mannan and BDG, and mannan, BDG and Candida DNA enhanced the positivity to 36% and 54%, respectively. None of the sera from Candida vaginitis patients and healthy subjects were positive for Candida DNA and mannan. CONCLUSION: The observations made in this study reinforce the diagnostic value of snPCR in the sensitive and specific diagnosis of candidemia and detection of more than one Candida species in a given patient. Additionally, in the absence of a positive blood culture, snPCR detected Candida DNA in sera of more than half of the clinically suspected patients. While detection of BDG, mannan and anti-mannan antibodies singly or in combination could help enhancing sensitivity and eliminating false positive tests, a more extensive evaluation of these assays in sequentially collected serum samples is required to assess their value in the early diagnosis of candidemia.

Internalization by HeLa cells of latex beads coated with mammalian cell entry (Mce) proteins encoded by the mce3 operon of Mycobacterium tuberculosis.

The mammalian cell entry (Mce) operon 3 (mce3) is one of four homologous mce operons of Mycobacterium tuberculosis, encoding six (Mce3A-F) invasin-like membrane-associated proteins. Previous studies have shown that recombinant expression of Mce1A encoded by the mce1 operon in Escherichia coli allows this non-pathogenic bacterium to invade and survive inside macrophages, and latex beads coated with Mce1A are internalized by non-phagocytic HeLa cells. However, the role of other mce1 operon proteins (Mce1B-F) and proteins encoded by the operons mce2-4 in facilitating the internalization of M. tuberculosis in mammalian cells has not been studied. This study was carried out to determine whether Mce proteins encoded by the mce3 operon also facilitated the internalization of latex beads by HeLa cells. Recombinant pure Mce3A and lipoprotein LprM (Mce3E) were expressed and purified from E. coli cells. Mce1A expressed as a fusion protein with glutathione S-transferase (GST-Mce1A) and GST alone, purified similarly from E. coli cells, were used as control proteins. Fluorescent latex beads coated with purified proteins were used to study their uptake by HeLa cells using fluorescence microscopy, flow cytometry and electron microscopy. Fluorescence microscopy and flow cytometry showed an association of HeLa cells with beads coated with both Mce3A and LprM, whilst GST-Mce1A and GST yielded the expected results. Transmission electron microscopy confirmed the uptake of beads coated with Mce3A or LprM by HeLa cells. The data showed that Mce3A encoded by the mce3 operon facilitated the uptake and internalization of latex beads by HeLa cells. The data also showed, for the first time, the role of another Mce protein (LprM/Mce3E) in facilitating the interaction and internalization of M. tuberculosis by mammalian cells.

Identification of transcriptionally active open reading frames within the RD1 genomic segment of Mycobacterium tuberculosis.

OBJECTIVE: To identify transcriptionally active open reading frames (ORFs), predicted by bioinformatics, within RD1 genomic segment of Mycobacterium tuberculosis using reverse transcription-polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: M. tuberculosis H37Rv was grown in Middlebrook 7H9 medium for 8 weeks and total RNA was isolated using standard procedures. The cDNA was synthesized using first-strand cDNA synthesis kit and general primers provided in the kit [pd (N)6, and/or Not I-d(T)18] as well as forward primers specific for each predicted RD1 ORF. Specific forward and reverse primers in PCR were used to amplify ORF-specific cDNA. The amplified products were identified on the basis of size using agarose gel electrophoresis, and their identity was confirmed by DNA sequencing. RESULTS: RT-PCR demonstrated expression of 13 of the 14 bioinformatics-predicted ORFs within RD1 genomic segment of M. tuberculosis. However, cDNA synthesis and PCR amplifications of specific products varied with respect to primer requirement and reaction conditions, respectively. All ORFs of <1.5 kb were amplified in standard RT-PCR, whereas several large-size ORFs (>1.5 kb) required internal primers for amplification in semi-nested RT-PCR. The sequencing of RT-PCR-amplified products of ORFs confirmed their identity. CONCLUSION: Bioinformatics analysis of DNA can accurately predict ORFs within M. tuberculosis-specific genomic regions, and RT-PCR is a suitable technique to confirm their expression in bacteria.

ProPred analysis and experimental evaluation of promiscuous T-cell epitopes of three major secreted antigens of Mycobacterium tuberculosis.

In the search for safe vaccine candidates against tuberculosis (TB), subunit vaccines including peptide-based candidates deserve consideration. However, an important requirement for such vaccine candidates is their promiscuous presentation to Th1 cells mediating protective immunity against TB, i.e. Th1 cells secreting IFN-gamma. The aim of the present study was to identify promiscuous Th1 cell epitopes of three major secreted antigens of Mycobacterium tuberculosis, i.e. ESAT-6, CFP10 and MPT70 by using a virtual matrix-based prediction program (ProPred) for peptide binding to 51 HLA-DR alleles. The ProPred analysis of these proteins was performed using the server (http:www.imtech.res.in/raghava/ProPed/). The peptides predicted to bind > 50% HLA-DR alleles included in the ProPred were considered promiscuous for binding predictions. Based on this criteria, one region in ESAT-6 (aa 69-77), two regions in CFP10 (aa 55-66 and aa 76-84) and four regions in MPT70 (aa 1-11, aa 81-95, aa 124-140 and aa 182-191) were considered promiscuous HLA-DR binders. The experimental evaluation of these regions, by using overlapping synthetic peptides for presentation to T-cells, confirmed the promiscuous nature of peptides covering the regions aa 69-77, aa 76-84 and aa 182-191 of ESAT-6, CFP10 and MPT70, respectively. These results demonstrate that the ProPred analysis can facilitate the selection of promiscuous peptides recognized by Th1 cells, and thus it can be useful in the identification of peptide-based vaccine candidates against TB.

HLA-DR binding prediction and experimental evaluation of T-cell epitopes of mycolyl transferase 85B (Ag85B), a major secreted antigen of Mycobacterium tuberculosis.

OBJECTIVE: To identify T-cell epitopes of Ag85B by analysis of its sequence for prediction to bind HLA-DR alleles and evaluate the predicted peptides for recognition by T cells in antigen-induced proliferation assays. MATERIALS/SUBJECTS AND METHODS: The complete sequence of Ag85B was analyzed for HLA-DR binding prediction to 51 HLA-DR alleles by using a virtual matrix-based prediction program (ProPred). Synthetic peptides covering the sequence of mature Ag85B were also analyzed for binding to HLA-DR alleles, and evaluated for recognition in antigen-induced proliferation assays with Ag85B-specific T-cell lines established from the peripheral blood mononuclear cells of 10 HLA-DR-heterogeneous tuberculosis patients. RESULTS: The ProPred analysis of the full-length Ag85B (325 aa), signal peptide (40 aa) and the mature protein (285 aa) predicted their binding to 100, 76 and 98% of the 51 HLA-DR alleles, respectively. The analysis of 31 synthetic peptides for binding to HLA-DR alleles showed that 4 of them could bind >50% HLA-DR alleles, and were considered promiscuous. Testing of Ag85B-specific T-cell lines with synthetic peptides showed that all of the T-cell lines responded to one or more peptides of Ag85B, and 9 of the 10 cell lines responded to one or more of the four peptides considered promiscuous for binding to HLA-DR alleles. CONCLUSION: The ProPred program was useful in predicting the HLA-DR alleles binding regions of Ag85B and identifying the promiscuous peptides recognized by T cells.

PCR-enzyme immunoassay of rDNA in the diagnosis of candidemia and comparison with amplicon detection by agarose gel electrophoresis.

We have developed a semi-nested PCR-enzyme immunoassay (snPCR-EIA) for the detection of Candida species in serum specimens, and the sensitivity of amplicon detection was compared with the detection of amplified product by agarose gel electrophoresis (AGE). The universal outer primers amplified the 3' end of 5.8S and the 5' end of 28S rDNA including the internally transcribed spacer 2 (ITS2) in PCR with genomic DNA as template from all the tested Candida species. The biotin-labeled species-specific primers derived from ITS2 from the four commonly encountered Candida species, viz. C. albicans, C. tropicalis, C. parapsilosis and C. glabrata, together with digoxigenin-labeled reverse primer amplified species-specific DNA in the reamplification step of the snPCR. The snPCR-EIA was positive for genomic DNA recovered from 0.06 Candida cells in culture and one organism/ml in spiked serum specimens. Evaluation of snPCR-EIA and snPCR-AGE for specific identification of Candida species with 26 clinical Candida isolates showed 100% concordant results with Vitek and ID32C yeast identification systems. Further evaluation of snPCR-EIA and snPCR-AGE for detection of Candida species in serum samples from culture proven (n = 6) and suspected (n = 10) patients showed concordance with the corresponding species isolated in culture. The serum samples from none of the healthy volunteers (n = 10) were positive for the presence of Candida DNA by snPCR-EIA or snPCR-AGE. Our results show that the snPCR-EIA has the same sensitivity as snPCR-AGE, however, it offers additional advantages of simultaneous testing of a large number of serum samples and avoids the use of ethidium bromide, a potent mutagen. The snPCR-EIA could, therefore, be a method of choice for the diagnosis of candidemia.

Tuberculosis: looking beyond BCG vaccines.

Tuberculosis (TB) is an infectious disease of international importance and ranks among the top 10 causes of death in the World. About one-third of the world's population is infected with Mycobacterium tuberculosis. Every year, approximately eight million people develop active disease and two million die of TB. The currently used BCG vaccines have shown variable protective efficacies against TB in different parts of the world. Moreover, being a live vaccine, BCG can be pathogenic in immunocompromised recipients. Therefore, there is an urgent need to develop new vaccines against TB. The comparative genome analysis has revealed the existence of several M. tuberculosis-specific regions that are deleted in BCG. The work carried out to determine the immunological reactivity of proteins encoded by genes located in these regions revealed several major antigens of M. tuberculosis, including the 6 kDa early secreted antigen target (ESAT6). Immunization with ESAT6 and its peptide (aa51-70) protects mice challenged with M. tuberculosis. The protective efficacy of immunization further improves when ESAT6 is recombinantly fused with M. tuberculosis antigen 85B. In addition, ESAT6 delivered as a DNA vaccine is also protective in mice. Whether these vaccines would be safe or not cannot be speculated. The answer regarding the safety and efficacy of these vaccines has to await human trials in different parts of the world.