Host Innate Immune Response to <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

This review focuses on recent progress in our understanding of Mycobacterium tuberculosis survival in macrophages, the interaction of M. tuberculosis with Toll-like receptors (TLRs) and the establishment of the link between innate and adaptive immunity, and TLRs and interferon-γ-mediated antimicrobial pathways in macrophages. We also propose a paradigm that TLR2 signaling regulates the magnitude of the host Th1 response leading to either M. tuberculosis persistence and latent infection or replication and disease.     

Immunogenicity and cross-reactivity against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> of proteoliposomes derived from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG

The only currently available vaccine against tuberculosis (TB) is Mycobacterium bovis Bacille Calmette-Guerin (BCG), which has inconsistent efficacy to protect against the disease in adults. M. tuberculosis (MTB) cell wall components have been implicated in the pathogenicity of TB and therefore have been a prime target for the identification and characterization of cell wall proteins with potential application in vaccine development. In this regard, proteoliposomes (PLs) derived from mycobacteria containing lipids and cell wall proteins could be potential vaccine candidates against TB. In the present study PLs derived from BCG were prepared. These homogeneous population of spherical microparticles was then immunized into Balb/c mice. Sera of immunized animals showed high IgG response and strong cross-reactivity against different MTB antigens.These results showed that BCG PLs could be potential vaccine candidates against TB.     

Dominant genotypes of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains isolated from prison inmates in Ozerki

A correlation was observed between the Beijing/W and LAM families of Mycobacterium tuberculosis and drug resistance, and the transmissiveness of multidrug-resistant strains belonging to these two families was also shown.     

The molecular characteristic of multidrug-resistant strains of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolated in Northwestern Russia

The goal of this work was to obtain genotypic characteristics of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (i.e., strains that are resistant at least to rifampicin and isoniazid) isolated from consumptives in Northwestern Russia in 2011–2012. Spoligotyping of 195 strains of Mycobacterium tuberculosis revealed 14 spolingotypes belonging to genetic families Beijing (n = 162), LAM (n = 15), H3/URAL (n = 14), T, Harleem, and X. Spolingotypes SIT1 (Beijing), SIT42 (LAM), and SIT62 (H3/URAL) were predominant. Regardless of genotype, all studied strains were resistant to streptomycin. Multidrug-resistant strains were resistant to ethionamide (56%), amikacin (31%), kanamycin (40%), and capreomycin (33%). The fractions of the strains resistant to ethambutol were 71 (n = 115) and 42% (n = 14) among the Beijing and nonBeijing strains, respectively (p < 0.05). Representatives of the Beijing genetic family remain predominant in Northwestern Russia in the population of multidrug-resistant strains of Mycobacterium tuberculosis (83%).     

Efficient method for mycobacterial DNA extraction in blood cultures aids rapid PCR identification of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> and <Emphasis Type="Italic">Mycobacterium avium</Emphasis>

The study presented here evaluated the utility of several methods of extracting mycobacterial nucleic acids from positive blood culture samples and examined the effect of each method on the performance of an in-house PCR used directly in the peripheral blood of 80 patients with AIDS to identify Mycobacterium spp. The modified Boom method for extracting DNA from blood cultures proved to be the most efficient, with subsequent PCR analysis yielding 100% positivity (7 samples positive for M. avium and 5 for M. tuberculosis). Only three of 12 patients with a positive blood culture had a PCR result positive for M. avium in peripheral blood. The identification of mycobacteria by PCR in blood culture took about 3 days, reducing the time to diagnosis by several weeks. These results demonstrate that PCR is a sensitive and quick method for identifying mycobacteria, especially when a good DNA extraction method is applied.     

Host-directed therapy targeting the <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> granuloma: a review

Infection by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb) is a major cause of morbidity and mortality worldwide. Slow progress has been made in lessening the impact of tuberculosis (TB) on human health, especially in parts of the world where Mtb is endemic. Due to the complexity of TB disease, there is still an urgent need to improve diagnosis, prevention, and treatment strategies to control global spread of disease. Active research targeting avenues to prevent infection or transmission through vaccination, to diagnose asymptomatic carriers of Mtb, and to improve antimicrobial drug treatment responses is ongoing. However, this research is hampered by a relatively poor understanding of the pathogenesis of early infection and the factors that contribute to host susceptibility, protection, and the development of active disease. There is increasing interest in the development of adjunctive therapy that will aid the host in responding to Mtb infection appropriately thereby improving the effectiveness of current and future drug treatments. In this review, we summarize what is known about the host response to Mtb infection in humans and animal models and highlight potential therapeutic targets involved in TB granuloma formation and resolution. Strategies designed to shift the balance of TB granuloma formation toward protective rather than destructive processes are discussed based on our current knowledge. These therapeutic strategies are based on the assumption that granuloma formation, although thought to prevent the spread of the tubercle bacillus within and between individuals contributes to manifestations of active TB disease in human patients when left unchecked. This effect of granuloma formation favors the spread of infection and impairs antimicrobial drug treatment. By gaining a better understanding of the mechanisms by which Mtb infection contributes to irreversible tissue damage, down regulates protective immune responses, and delays tissue healing, new treatment strategies can be rationally designed. Granuloma-targeted therapy is advantageous because it allows for the repurpose of existing drugs used to treat other communicable and non-communicable diseases as adjunctive therapies combined with existing and future anti-TB drugs. Thus, the development of adjunctive, granuloma-targeted therapy, like other host-directed therapies, may benefit from the availability of approved drugs to aid in treatment and prevention of TB. In this review, we have attempted to summarize the results of published studies in the context of new innovative approaches to host-directed therapy that need to be more thoroughly explored in pre-clinical animal studies and in human clinical trials.     

Concurrent <Emphasis Type="Italic">M. tuberculosis</Emphasis>, <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>, and <Emphasis Type="Italic">Candida albicans</Emphasis> infection in liver metastasis of bowel carcinoma

We describe the case of a 62-year-old patient with fever and abdominal pain of the right upper quadrant with a known history of adenocarcinoma of the colon and presence of hepatic metastases. One of the liver lesions underwent aspiration, and pus was sent for microbiological testing. Cultures of the pus were positive for Klebsiella pneumoniae and Candida albicans and polymerase chain reaction for Mycobacterium tuberculosis was concurrently positive; the patient received treatment for all three pathogens and improved clinically. One may consider searching not only for the usual pathogens of liver abscesses (gram-negative bacteria, anaerobic bacteria and gram-positive bacteria), but in special cases might consider pursuing a more detailed search for coexistence of fungi and mycobacteria in patients with cancer.     

The Phenomenon of the Formation of Biofilms by <Emphasis Type="Italic">Brevibacillus</Emphasis> spp. and <Emphasis Type="Italic">Bacillus</Emphasis> spp. in the Presence of Clinical Strains of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The formation of biofilms by M. tuberculosis on Shkolnikova’s medium (synthetic medium, analogue of Sauton’s medium) has been researched. We studied 150 clinical and 20 laboratory strains of M. tuberculosis. None of the 150 strains isolated from human beings produced biofilms (pellicle), but all yielded abundant planktonic growth. Twenty reference strains of M. tuberculosis produced both biofilms (pellicle) and planktonic growth. The phenomenon of biofilm formation by mixed cultures was observed when inoculating sputum treated with NALC-NaOH from patients with tuberculosis. We obtained 63 mixed biofilms. In 30.2% (19/63) of cases, biofilms contained the DNA of the causative agent of tuberculosis. The RV-PCR method was used to select six samples with the highest concentration of mycobacterial DNA. Molecular cloning and sequencing of a fragment of the 16S rRNA gene from one of the biofilms was carried out. The nucleotide sequence had 99% homology with the Bacillus thermoamylovorans species. From the mixed biofilms obtained, three strains of spore-forming bacilli were isolated. Strains are identified by Sanger’s sequencing of the 16S rRNA gene, one as Bacillus licheniformis, and the other two as Brevibacillus spp. A study of the resistance of isolated strains of spore bacilli against 12 antituberculosis drugs of the first and second series was carried out. All three strains were resistant to maximum concentrations of isoniazid, streptomycin, ethionamide, and ethambutol. Strains of Brevibacillus spp. were additionally resistant to para-aminosalicylic acid (PAS) and kanamycin. In a model experiment, the possibility of cogrowth of clinical strains of M. tuberculosis and B. licheniformis was demonstrated with prolonged co-incubation in Shkolnikova’s medium. In the first few days of growth, B. licheniformis produced a biofilm that remained stable for the entire observation period of 45 days. The hypothesis suggesting the possibility of a short-term persistence of some “saprophytic” species of bacilli in the caseous contents of necrosis foci in the late stages of pulmonary tuberculosis has been postulated.     

Native New Zealand plants with inhibitory activity towards <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Plants have long been investigated as a source of antibiotics and other bioactives for the treatment of human disease. New Zealand contains a diverse and unique flora, however, few of its endemic plants have been used to treat tuberculosis. One plant, Laurelia novae-zelandiae, was reportedly used by indigenous Maori for the treatment of tubercular lesions.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Comparison of the BDProbeTec ET System with the Cobas Amplicor PCR for Direct Detection of <Emphasis Type="Italic">Mycobacterium </Emphasis><Emphasis Type="Italic">tuberculosis</Emphasis> in Respiratory Samples

In the study presented here, the performance of the BDProbeTec ET system (Becton Dickinson, USA) was compared with the Roche Cobas Amplicor-PCR (Roche, Switzerland) to detect Mycobacterium tuberculosis complex (MTB) in clinical respiratory samples. The Bactec MGIT 960 liquid culture system (Becton Dickinson) was used as a reference method. A total of 411 samples were tested. Of the 93 culture-positive samples, both the BDProbeTec ET system and the Cobas Amplicor-PCR detected 87 (sensitivity, 93.5%). When only smear-negative samples were considered, the BDProbeTec ET exhibited a sensitivity of 50% and the Cobas Amplicor-PCR 60%. Specificity was 99.7% for the BDProbeTec ET system and 100% for the Cobas Amplicor-PCR. Percent agreement between the two nucleic amplification methods was 98.7%. Inhibition occurred in three (0.7%) samples in the BDProbeTec ET system. The high sensitivity and specificity of the BDProbeTec ET system suggest it is a useful method for the rapid and direct detection of MTB in smear-positive respiratory samples.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Using a multiplex polymerase chain reaction for the identification of Beijing strains of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The genotype of a Beijing strain of Mycobacterium tuberculosis (MTB) is usually determined by spoligotyping. However, this technique requires special equipment and is time-consuming. In this study, we developed a new multiplex polymerase chain reaction (PCR) to differentiate between Beijing and non-Beijing strains of MTB. A total of 323 MTB isolates were genotyped by both spoligotyping and the novel multiplex PCR. By spoligotyping, 169 (52.3%) isolates were determined to be Beijing strains and the remaining 154 (47.7%) isolates were non-Beijing strains. The multiplex PCR method produced results identical to those of spoligotyping in the identification of Beijing strains of MTB. This method is highly sensitive, specific, and fast. It is also cost-effective and suitable for screening large numbers of samples.     

A first insight into the genotypic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> from Rwanda

Background

Mycobacterium tuberculosis complex (MTC) is the causative agent of tuberculosis (TB). Globally, increasing evidence shows that in M. tuberculosis, transmission varies from strain to strain and that different strains exhibit a range of geographical and host specificities, pathogenicity, and drug susceptibility. Therefore rapid and accurate differentiation of the members of MTC is critical in guiding treatment and public health decisions. We carried out a study at different health units and the National Reference Laboratory in Rwanda identify Mycobacterium tuberculosis complex species prevalent in TB patients in Rwanda. We further characterized the isolates using spoligotyping in order to gain an insight into the strain diversity of drug resistant and susceptible isolates of M. tuberculosis in this setting.

Methods

A total of 151 isolates from culture positive sputum samples were harvested, heat killed at 80°C for two hours, and then shipped to Makerere University College of Health Sciences, Uganda, for speciation and typing. Species identification was achieved by regions of difference (RD) analysis, while Spoligotyping was done to identify strain types.

Results

Region of difference analysis identified all the 151 isolates as M. tuberculosis. Spoligotyping revealed predominance of the T2 family (58.3%, 88/151), with SIT 52 being the most prevalent strain (31.8%, 48/151). Among the 151 isolates, 64 (42.4%) were multidrug resistant (MDR) with 3 cases on mono-resistance. Of 94 retreatment cases, 48 (51.1%) were MDR and of 46 newly presenting cases 14 (30.4%) were MDR. There was a significant difference (p=0.01) in anti-TB drug resistance between new and retreatment cases in the sample. However, there was no significant relationship between HIV serostatus and the two major strain types SIT 52 (p =0.15and SIT 152 (p = 0.41).

Conclusion

Mycobacterium tuberculosis is the most prevalent species of Mycobacterium tuberculosis complex in Rwanda, and SIT 52 (T2) the predominant strain. There is significantly more MDR in the retreatment cases but no significant difference was observed by HIV status in relation to any spoligotypes.

     

Evaluation of the humoral immune response and cross reactivity against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> of mice immunized with liposomes containing glycolipids of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

Mycobacterium smegmati s (Ms) is a nonpathogenic mycobacteria of rapid growth, which shares many characteristics with Mycobacterium tuberculosis (MTB), the major causative agent of tuberculosis. MTB has several cell wall glycolipids in common with Ms, which play an important role in the pathogenesis of tuberculosis and the induction of a protective immune response against MTB infection in some animal models. In this study, the humoral immune response and cross reactivity against MTB, of liposomes containing a mixture of cell wall glycolipids of Ms and commercial lipids was evaluated, in order to study its possible use as a component of a vaccine candidate against tuberculosis. Liposomes containing total lipids extracted from Ms, distearoyl phosphatidyl choline and cholesterol were prepared by the dehydration-rehydration technique. Balb/c mice were immunized with the liposomes obtained and the antibody response and cross reactivity against MTB were tested by ELISA. Total lipids extract from Ms showed the presence of several polar glycolipids in common with MTB, such as phosphatidylinositol mannosides. Liposomes that contained glycolipids of Ms were capable of inducing a specific IgG antibody response that allowed the recognition of surface antigens of MTB. The results of this study demonstrated the presence of immunogenic glycolipids in Ms, which could be included to enhance the protective effects of subunit vaccine formulations against tuberculosis.     

Functional polymorphisms in <Emphasis Type="Italic">XRCC</Emphasis>-<Emphasis Type="Italic">1</Emphasis> and <Emphasis Type="Italic">APE</Emphasis>-<Emphasis Type="Italic">1</Emphasis> contribute to increased apoptosis and risk of ulcerative colitis

Objective  

The present study was designed to investigate the role of X-ray cross-complementing group 1 (XRCC1) and apurinic/apyrimidinic endonuclease 1 (APE1) polymorphisms in apoptosis and the risk of ulcerative colitis (UC).     

Immune activation and regulatory T cells in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> infected lymph nodes

Background

Lymph node tuberculosis (LNTB) is the most frequent extrapulmonary form of tuberculosis (TB). Studies of human tuberculosis at sites of disease are limited. LNTB provides a unique opportunity to compare local in situ and peripheral blood immune response in active Mycobacterium tuberculosis (Mtb) disease. The present study analysed T regulatory cells (Treg) frequency and activation along with CD4+ T cell function in lymph nodes from LNTB patients.

Results

Lymph node mononuclear cells (LNMC) were compared to autologous peripheral blood mononuclear cells (PBMC). LNMC were enriched for CD4+ T cells with a late differentiated effector memory phenotype. No differences were noted in the frequency and mutifunctional profile of memory CD4+ T cells specific for Mtb. The proportion of activated CD4+ and Tregs in LNMC was increased compared to PBMC. The correlation between Tregs and activated CD4+ T cells was stronger in LNMC than PBMC. Tregs in LNMC showed a strong positive correlation with Th1 cytokine production (IL2, IFNγ and TNFα) as well as MIP-1α after Mtb antigen stimulation. A subset of Tregs in LNMC co-expressed HLA-DR and CD38, markers of activation.

Conclusion

Further research will determine the functional relationship between Treg and activated CD4+ T cells at lymph node sites of Mtb infection.

     

Candidate biomarkers for discrimination between infection and disease caused by <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Infection with Mycobacterium tuberculosis is controlled by an efficacious immune response in about 90% of infected individuals who do not develop disease. Although essential mediators of protection, e.g., interferon-gamma, have been identified, these factors are insufficient to predict the outcome of M. tuberculosis infection. As a first step to determine additional biomarkers, we compared gene expression profiles of peripheral blood mononuclear cells from tuberculosis patients and M. tuberculosis-infected healthy donors by microarray analysis. Differentially expressed candidate genes were predominantly derived from monocytes and comprised molecules involved in the antimicrobial defense, inflammation, chemotaxis, and intracellular trafficking. We verified differential expression for alpha-defensin 1, alpha-defensin 4, lactoferrin, Fcgamma receptor 1A (cluster of differentiation 64 [CD64]), bactericidal permeability-increasing protein, and formyl peptide receptor 1 by quantitative polymerase chain reaction analysis. Moreover, we identified increased protein expression of CD64 on monocytes from tuberculosis patients. Candidate biomarkers were then assessed for optimal study group discrimination. Using a linear discriminant analysis, a minimal group of genes comprising lactoferrin, CD64, and the Ras-associated GTPase 33A was sufficient for classification of (1) tuberculosis patients, (2) M. tuberculosis-infected healthy donors, and (3) noninfected healthy donors.     

Antitrichomonal strains of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Parasporal inclusion proteins from a total of 816 Bacillus thuringiensis strains isolated in Japan were examined for antitrichomonal activity against Trichomonas vaginalis. Ten strains of B. thuringiensis inhibited the growth of T. vaginalis in 48 h cultures at 37 degrees C. Moreover, the B622 and B626 strains clearly showed trichomonacidal effects against T. vaginalis. The H antigen serotypes of both strains were identified as H13/29 (pakistani/amagiensis). The parasporal inclusion protein from both strains consisted of three major polypeptides of 77, 45 and 25 kDa.