Development of an immunochromatographic lateral flow dipstick for the detection of Mycobacterium tuberculosis 16 kDa antigen (Mtb-strip).

Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that causes tuberculosis (TB). This contagious disease remains a severe health problem in the world. The disease is transmitted via inhalation of airborne droplets carrying Mtb from TB patients. Early detection of the disease is vital to prevent transmission of the infection to people in close contact with the patients. To date, there is a need of a simple, rapid, sensitive and specific diagnostic test for TB. Previous studies showed the potential of Mtb 16 kDa antigen (Ag16) in TB diagnosis. In this study, lateral flow immunoassay, also called simple strip immunoassay or immunochromatographic test (ICT) for detection of Ag16 was developed (Mtb-strip) and assessed as a potential rapid TB diagnosis method. A monoclonal antibody against Ag16 was optimized as the capturing and detection antibody on the Mtb-strip. Parameters affecting the performance of the Mtb-strip were also optimized before a complete prototype was developed. Analytical sensitivity showed that Mtb-strip was capable to detect as low as 125 ng of purified Ag16. The analytical sensitivity of Mtb-strip suggests its potential usefulness in different clinical applications.

Tuberculosis vaccine candidates based on mycobacterial cell envelope components.

Even after decades searching for a new and more effective vaccine against tuberculosis, the scientific community is still pursuing this goal due to the complexity of its causative agent, Mycobacterium tuberculosis (Mtb). Mtb is a microorganism with a robust variety of survival mechanisms that allow it to remain in the host for years. The structure and nature of the Mtb envelope play a leading role in its resistance and survival. Mtb has a perfect machinery that allows it to modulate the immune response in its favor and to adapt to the host's environmental conditions in order to remain alive until the moment to reactivate its normal growing state. Mtb cell envelope protein, carbohydrate and lipid components have been the subject of interest for developing new vaccines because most of them are responsible for the pathogenicity and virulence of the bacteria. Many indirect evidences, mainly derived from the use of monoclonal antibodies, support the potential protective role of Mtb envelope components. Subunit and DNA vaccines, lipid extracts, liposomes and membrane vesicle formulations are some examples of technologies used, with encouraging results, to evaluate the potential of these antigens in the protective response against Mtb.

Allele frequencies of human platelet antigens in Banjar, Bugis, Champa, Jawa and Kelantan Malays in Peninsular Malaysia.

OBJECTIVES: The major aims of this study are to characterise and compile allelic data of human platelet antigen (HPA)-1 to -6 and -15 systems in five Malay sub-ethnic groups in Peninsular Malaysia. BACKGROUND: HPAs are polymorphic glycoproteins expressed on the surface of platelet membranes and are genetically differentiated across ethnogeographically unrelated populations. METHODS: Blood samples were obtained with informed consent from 192 volunteers: Banjar (n = 30), Bugis (n = 37), Champa (n = 51), Jawa (n = 39) and Kelantan (n = 35). Genotyping was done using polymerase chain reaction-sequence specific primer method. RESULTS: In general, frequencies of HPAs in the Malay sub-ethnic groups are more similar to those in Asian populations compared with other more distinct populations such as Indians, Australian Aborigines and Europeans. CONCLUSIONS: This study provides the first HPA datasets for the selected Malay sub-ethnic groups. Subsequent analyses including previously reported HPA data of Malays, Chinese and Indians revealed details of the genetic relationships and ancestry of various sub-populations in Peninsular Malaysia. Furthermore, the comprehensive HPA allele frequency information from Peninsular Malaysia provided in this report has potential applications for future study of diseases, estimating risks associated with HPA alloimmunization and for developing an efficient HPA-typed donor recruitment strategy.

Distribution of cytokine gene polymorphisms in five Malay subethnic groups in Peninsular Malaysia.

In this survey, we have successfully genotyped 22 single nucleotide polymorphisms in the 13 cytokine genes for five Malay subethnic groups (Kelantan, Acheh, Mandailing, Minangkabau and Patani Malays) using polymerase chain reaction-sequence-specific primer cytokine genotyping kit (Invitrogen, Carlsbad, CA, USA). Most of the cytokine genes showed similar pattern of allelic spectra with wild-type alleles (e.g. ILIa-889/C, ILIB+3962/C and IL6 nt565/G) that represent more than 80% in the studied Malay subethnic groups. These newly observed cytokine alleles and subsequent analyses clearly indicate genetic contribution from Asia in the studied Malay subethnic groups with evidence of admixture from neighbouring populations in Patani Malays. The cytokine data sets for the five Malay subethnic groups deposited in this report can also be used as reference standard for searching suitable donor for allograft transplant and diseases association study. This is particularly relevance as our analyses showed differences between the Malay subethnic groups and other populations screened for cytokine genes.

Sequence comparison of six human microRNAs genes between tuberculosis patients and healthy individuals.

OBJECTIVE/BACKGROUND: MicroRNAs (miRNAs) play an important role in diseases development. Therefore, human miRNAs may be able to inhibit the survival of Mycobacterium tuberculosis (Mtb) in the human host by targeting critical genes of the pathogen. Mutations within miRNAs can alter their target selection, thereby preventing them from inhibiting Mtb genes, thus increasing host susceptibility to the disease. METHODS: This study was undertaken to investigate the genetic association of pulmonary tuberculosis (TB) with six human miRNAs genes, namely, hsa-miR-370, hsa-miR-520d, hsa-miR-154, hsa-miR-497, hsa-miR-758, and hsa-miR-593, which have been predicted to interact with Mtb genes. The objective of the study was to determine the possible sequence variation of selected miRNA genes that are potentially associated with the inhibition of critical Mtb genes in TB patients. RESULTS: The study did not show differences in the sequences compared with healthy individuals without antecedents of TB. CONCLUSION: This result could have been influenced by the sample size and the selection of miRNA genes, which need to be addressed in future studies.

Killer immunoglobulin-like receptor diversity in Malay subethnic groups of Peninsular Malaysia.

The KIR system shows variation at both gene content and allelic level across individual genome and populations. This variation reflects its role in immunity and has become a significant tool for population comparisons. In this study, we investigate KIR gene content in 120 unrelated individuals from the four Malay subethnic groups (Kelantan, Jawa, Banjar and Pattani Malays). Genotyping using commercial polymerase chain reaction-sequence-specific primer (PCR-SSP) kits revealed a total of 34 different KIR genotypes; 17 for Kelantan, 15 for Banjar, 14 for Jawa and 13 for Pattani Malays. Two new variants observed in Banjar Malays have not previously been reported. Genotype AA and haplotype A were the most common in Jawa (0.47 and 0.65, respectively), Banjar (0.37 and 0.52, respectively) and Pattani (0.40 and 0.60, respectively) Malays. In contrast, Kelantan Malays were observed to have slightly higher frequency (0.43) of genotype BB as compared with the others. Based on the KIR genes distribution, Jawa, Pattani and Banjar subethnic groups showed greater similarity and are discrete from Kelantan Malays. A principal component plot carried out using KIR gene carrier frequency shows that the four Malay subethnic groups are clustered together with other South-East Asian populations. Overall, our observation on prevalence of KIR gene content demonstrates genetic affinities between the four Malay subethnic groups and supports the common origins of the Austronesian-speaking people.

Draft Genome Sequence of a Clinical Isolate of Mycobacterium tuberculosis Strain PR05.

We report the annotated genome sequence of a clinical isolate, Mycobacterium tuberculosis strain PR05, which was isolated from the human cerebrospinal fluid of a patient diagnosed with tuberculosis.

Immunogenicity of recombinant BCG-based vaccine expressing the 22 kDa of serine repeat antigen (SE22) of Plasmodium falciparum.

The Plasmodium falciparum serine repeat antigen (SERA) is one of the promising blood-stage malarial vaccine candidates. In this study, recombinant Mycobacterium bovis bacille Calmette-Guerin (rBCG) expressing the 22 kDa protein (SE22) from the 47 kDa Nterminal domain of serine repeat antigen (SERA), generated in favour of mycobacterium codon usage, elicited specific immune response in BALB/c mice with a mixed Th1/Th2 profile. Immunized sera containing high levels of specific IgG1 and IgG2a against the epitope (as determined by ELISA) were reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA). Furthermore, the lymphocyte proliferative response to SE22 antigen from rBCG-immunized mice was higher than that of controls. The expression of intracellular cytokines (IL-2, IL-4 and IFNγ) in CD4+- and CD8+-cells was also enhanced following in-vitro stimulation with SE22. These findings indicate that a rBCG-based vaccine candidate expressing a blood-stage antigen of P. falciparum could enhance both humoral and cellular immune responses, thus paving the way for the rational use of rBCG as a vaccine candidate against malaria.

Plasmodium falciparum 19 kDa of merozoite surface protein-1 (MSP-1(19)) expressed in Mycobacterium bovis bacille Calmette Guerin (BCG) is reactive with an inhibitory but not a blocking monoclonal antibody.

Proteins on the surface of Plasmodium falciparum merozoites are good targets for vaccine development against malaria because they are accessible to antibodies in the plasma. The 19 kDa C-terminus of merozoite surface protein-1 (MSP-1(19)) has been shown to induce both inhibitory as well as blocking antibodies, the latter blocking the protective effects of the former. Inhibitory antibodies bind to MSP-1(19) and inhibit merozoite invasion of red blood cells (RBC) but the binding of blocking antibodies can prevent binding of inhibitory antibodies thereby allowing the parasite to invade RBC. We constructed a synthetic version of the MSP-1(19) of the P. falciparum using mycobacterium codon usage by assembly PCR. The synthetic MSP-1(19) was mutated at various sites to promote the production of inhibitory but not blocking antibodies as previously reported. The native and mutated MSP-1(19) were cloned and expressed in Mycobacterium bovis bacille Calmette-Guerin (BCG) and the expressions of the recombinant proteins were detected by specific monoclonal antibodies (mAbs) namely, 12.10 and 1E1 against MSP-1(19) using Western blotting. The mutated MSP-1(19) protein reacted with the inhibitory mAb, 12.10, but not the blocking mAb, 1E1, paving the way for the construction of a potential recombinant BCG (rBCG) blood stage vaccine against malaria.

Phagocytic activity and pro-inflammatory cytokines production by the murine macrophage cell line J774A.1 stimulated by a recombinant BCG (rBCG) expressing the MSP1-C of Plasmodium falciparum.

Macrophages are involved in innate immunity against malaria due to their ability to phagocytose infected erythrocytes and produce inflammatory cytokines, which are important for controlling parasite growth during malaria infection. In this study, the ability of a recombinant BCG (rBCG) vaccine expressing the 19-kDa C-terminus of merozoite surface protein-1 (MSP1-C) of Plasmodium falciparum, to stimulate the phagocytic activity and secretion of pro-inflammatory cytokines by the macrophage cell line J774A.1 was measured at varying times. The results demonstrate the ability of the rBCG construct to activate the inflammatory action of macrophages, which is important as a first-line of defence in clearing malaria infections.

Roxithromycin potentiates the effects of chloroquine and mefloquine on multidrug-resistant Plasmodium falciparum in vitro.

Chloroquine (CQ) and mefloquine (MQ) are no longer potent antimalarial drugs due to the emergence of resistant Plasmodium falciparum. Combination therapy has become the standard for many regimes in overcoming drug resistance. Roxithromycin (ROM), a known p-glycoprotein inhibitor, is reported to have antimalarial activity and it is hoped it will potentiate the effects of both CQ/MQ and reverse CQ/MQ-resistance. We assayed the effects of CQ and MQ individually and in combination with ROM on synchronized P. falciparum (Dd2 strain) cultures. The IC(50) values of CQ and MQ were 60.0+/-5.0 and 16.0+/-3.0 ng/ml; these were decreased substantially when combined with ROM. Isobolograms indicate that CQ-ROM combinations were relatively more synergistic (mean FICI 0.70) than MQ-ROM (mean FICI 0.85) with their synergistic effect at par with CQ-verapamil (VRP) (mean FICI 0.64) and MQ-VRP (mean FICI 0.60) combinations. We conclude that ROM potentiates the CQ/MQ response on multidrug-resistant P. falciparum.

Immunogenicity of a recombinant Mycobacterium bovis bacille Calmette-Guèrin expressing malarial and tuberculosis epitopes.

Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing the malarial epitopes F2R(II)EBA and (NANP)3 as well as two T cell epitopes of the M. tuberculosis ESAT-6 antigen, generated in favour of mycobacterium codon usage elicited specific immune response against these epitopes. Immunised Balb/c mice demonstrated an increase in almost all of the IgG subclasses against both malarial epitopes and enhanced splenocyte proliferative response against the malarial epitopes as well as selected peptides of ESAT-6. Furthermore, flow cytometric analyses showed elevated numbers of CD4+ lymphocytes expressing IFN-gamma and IL-2 against the ESAT-6 peptides, suggesting a specific Th1-mediated response. This study demonstrated that expressing malarial and TB epitopes in a single rBCG construct induced appropriate humoral and cellular immune response against immunogenic epitopes from both organisms.

Forensic DNA profiling and database.

The incredible power of DNA technology as an identification tool had brought a tremendous change in crimnal justice . DNA data base is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. This article discusses the essential steps in compilation of COmbined DNA Index System (CODIS) on validated polymerase chain amplified STRs and their use in crime detection.

The transcription of the peroxisome proliferator-activated receptor alpha gene is regulated by protein kinase C.

The transcriptional regulation of peroxisome proliferator-activated receptor alpha (PPARalpha) by a variety of peroxisome proliferators was investigated. The treatment of primary cultures of rat hepatocytes with Wy14,643 or clofibrate increased mRNA steady state levels of both PPARalpha and acyl coenzyme A oxidase (ACOX). In contrast, fenofibrate and ciprofibrate increased the expression of ACOX without affecting that of PPARalpha. Inhibition of protein kinase C (PKC) activity using bisindolylmaleimide or calphostin C abolished the increased PPARalpha expression by the peroxisome proliferators whereas the expression of the ACOX gene remained unaffected. Phorbol-12-myristate-13-acetate increased PPARalpha mRNA levels without altering ACOX mRNA levels. It can thus be concluded that a number of peroxisome proliferators activate a PKC-dependent signalling pathway in addition to the PPARalpha pathway. The PKC signal transduction pathway contributes to the regulation of PPARalpha expression but does not influence the transcriptional activity of PPARalpha.