IFNγ Response to Mycobacterium tuberculosis,Risk of Infection and Disease in Household Contacts of Tuberculosis Patients in Colombia

Objectives

Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNγ produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNγ levels in HHCs correlate with tuberculosis development.

Methods

A cohort of 2060 HHCs was followed for 2–3 years after exposure to a tuberculosis case. Besides TST, IFNγ responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination.

Results

Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNγ response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNγ responders to CFP-10 (HR 1.82 95% CI 0.79–4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNγ producers was observed (trend Log rank p = 0.007).

Discussion

CFP-10-induced IFNγ production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprohylaxis to child contacts regardless of BCG vaccination.     

A bioanalytical method to determine the cell wall composition of Mycobacterium tuberculosis grown in vivo

Mycobacterium tuberculosis bacilli exhibit cell wall alterations during in vivo growth. Development of ultrasensitive analytical techniques with high specificities is required to analyze the cell wall of M. tuberculosis isolated from experimental animals because of the low amounts of bacteria available and contamination by host tissue. Here we present a novel methodology to analyze all three major components (mycolic acids, arabinogalactan, and peptidoglycan) of the mycobacterial cell wall from mycobacteria isolated from animal tissue. In this procedure, the cell wall carbohydrates are analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) of alditol acetates, the peptidoglycan by GC/MS (mass spectrometry) analysis of the unique amino acid diaminopimelic acid (after derivatization with isopropyl chloroformate), and the mycolic acids by liquid chromatography (LC)/MS (negative ion) without derivatization. The procedure was designed so that all three analyses could be performed starting with a single sample given the difficulty of preparing multiple aliquots in known ratios. Linkage analysis, including an enantiomeric specific procedure, of the arabinogalactan polymer is also presented. These procedures will enable the determination of the cell wall alterations known to occur in the important nongrowing "dormant" M. tuberculosis present during disease. With some adaptations, the methodology is also applicable to the analysis of small amounts of in vivo grown bacteria of other species.     

Genomic Markers for Differentiation of Francisella tularensis subsp. tularensis A.I and A.II Strains

Tularemia is caused by two subspecies of Francisella tularensis, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). F. tularensis subsp. tularensis is further subdivided into two genetically distinct populations (A.I and A.II) that differ with respect to geographical location, anatomical source of recovered isolates, and disease outcome. Using two human clinical isolates, suppression subtractive hybridization was performed to identify 13 genomic regions of difference between A.I and A.II strains. Two PCR assays, one to identify A.I and A.II as well as to discriminate between F. tularensis subsp. holarctica and F. novicida and another specific for A.I, were developed. This is the first report to identify and characterize conserved genomic differences between A.I and A.II.     

A genetic mechanism for deletion of the ser2 gene cluster and formation of rough morphological variants of Mycobacterium avium

A major phenotypic trait of the Mycobacterium avium complex is the ability to produce rough and smooth colony variants. The chemical basis of this morphological variation is the loss of an antigenic surface structure, termed glycopeptidolipid (GPL), by rough variants. Using M. avium serovar 2 strain 2151 as a model system, this laboratory previously reported that rough variants arise via the deletion of large genomic regions encoding GPL biosynthesis. One such deletion encompasses the gene cluster (ser2) responsible for production of the serovar 2 GPL haptenic oligosaccharide. In this study, nucleotide sequencing revealed that both ends of the ser2 gene cluster are flanked by a novel insertion sequence (IS1601) oriented as direct repeats. Detailed analyses of the site of deletion in the genome of M. avium 2151 Rg-1 demonstrated that a single copy of IS1601 remained and that the ser2 gene cluster was deleted by homologous recombination. This same deletion pattern was observed for 10 out of 15 rough colony variants tested. Additionally, these studies revealed that IS1601 contains portions of three independent insertion sequences. This report is the first to define the precise genetic basis of colony variation in Mycobacterium spp. and provides further evidence that homologous recombination between insertion sequence elements can be a primary determinant of genome plasticity in these bacteria.     

Synthesis of New Pyrrolo[1,2-a]quinoxaline Derivatives as Potential Inhibitors of Akt Kinase

Akt kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel Akt kinase inhibitors, based on a quinoxaline or pyrazinone scaffold. A series of new substituted pyrrolo[1,2-a]quinoxaline derivatives, structural analogues of these active quinoxaline or pyrazinone pharmacophores, was synthesized from various substituted 2-nitroanilines or 1,2-phenylenediamine via multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC₅₀ of 4.5 μM, and 1h that inhibited U937 and MCF7 cell lines with IC₅₀ of 5 and 8 μM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730.     

Disease state differentiation and identification of tuberculosis biomarkers via native antigen array profiling

A critical element of tuberculosis control is early and sensitive diagnosis of infection and disease. Our laboratories recently showed that different stages of disease were distinguishable via two-dimensional Western blot analyses of Mycobacterium tuberculosis culture filtrate proteins. However, this methodology is not suitable for high throughput testing. Advances in protein microarray technology provide a realistic mechanism to screen a large number of serum samples against thousands of proteins to identify biomarkers of disease states. Techniques were established for separation of native M. tuberculosis cytosol and culture filtrate proteins, resulting in 960 unique protein fractions that were used to generate protein microarrays. Evaluation of serological reactivity from 42 patients in three tuberculosis disease states and healthy purified protein derivative-positive individuals demonstrated that human immunodeficiency virus (HIV)-negative cavitary and noncavitary tuberculosis (TB) patients' sera recognized 126 and 59 fractions, respectively. Sera from HIV patients coinfected with TB recognized 20 fractions of which five overlapped with those recognized by non-HIV TB patients' sera and 15 were unique to the HIV+TB+ disease state. Identification of antigens within the reactive fractions yielded 11 products recognized by both cavitary and noncavitary TB patients' sera and four proteins (HspX, MPT64, PstS1, and TrxC) specific to cavitary TB patients. Moreover four novel B cell antigens (BfrB, LppZ, SodC, and TrxC) of human tuberculosis were identified.     

IFN-gamma-mediated inhibition of human IgE synthesis by IL-21 is associated with a polymorphism in the IL-21R gene

IL-21 is a cytokine produced by CD4+ T cells that has been reported to regulate human, as well as, mouse T and NK cell function and to inhibit Ag-induced IgE production by mouse B cells. In the present study, we show that human rIL-21 strongly enhances IgE production by both CD19+ CD27- naive, and CD19+ CD27+ memory B cells, stimulated with anti-CD40 mAb and rIL-4 and that it promotes the proliferative responses of these cells. However, rIL-21 does not significantly affect anti-CD40 mAb and rIL-4-induced Cepsilon promoter activation in a gene reporter assay, nor germline Cepsilon mRNA expression in purified human spleen or peripheral blood B cells. In contrast, rIL-21 inhibits rIL-4-induced IgE production in cultures of PBMC or total splenocytes by an IFN-gamma-dependent mechanism. The presence of a polymorphism (T-83C), in donors heterozygous for this mutation was found to be associated not only with lower rIL-21-induced IFN-gamma production levels, but also with a lower sensitivity to the inhibitory effects of IL-21 on the production of IgE, compared with those in donors expressing the wild-type IL-21R. Taken together, these results show that IL-21 differentially regulates IL-4-induced human IgE production, via its growth- and differentiation-promoting capacities on isotype-, including IgE-, committed B cells, as well as via its ability to induce IFN-gamma production, most likely by T and NK cells, whereas the outcome of these IL-21-mediated effects is dependent on the presence of a polymorphism in the IL-21R.     

Hormonal Therapy and Risk of Breast Cancer in Mexican Women

The use of hormonal therapies, including hormonal contraceptives (HC) and postmenopausal hormone replacement therapy (HRT) have been shown to influence breast cancer (BC) risk. However, the variations of these effects among populations and ethnic groups are not completely documented, especially among Hispanic women. We evaluated the association between HC and premenopausal BC risk, and between HRT and postmenopausal BC risk in Mexican women. Data from a Mexican multi-center population-based case–control study ofwomen aged 35 to 69 years were analysed. A total of 1000 cases and 1074 matched controls were recruited between 2004 and 2007. Information on hormonal therapy was collected through a structured questionnaire. Results were analysed using conditional logistic regression models. Overall, HC were used by 422/891 (47.3%) premenopausal women and HRT was used by 220/1117 (19.7%) postmenopausal women. For HC, odds ratios (ORs) for BC were 1.11 (95% confidence interval (CI): 0.82, 1.49) for current users and 1.68 (95% CI: 0.67, 4.21) for ever-users. No clear effect of duration of use was observed. For HRT, the OR for BC was significantly increased in ever users (OR: 1.45; 95% CI: 1.01, 2.08). A non-significant increased risk was observed for combined estrogen/progestin, (OR =  1.85; 95% CI: 0.84, 4.07) whereas no effect was observed for the use of estrogen alone (OR = 1.14; 95% CI: 0.68, 1.91). Our results indicate that, HC had a non-significant effect on the risk of pre-menopausal BC, but suggested that injected contraceptives may slightly increase the risk, whereas HRT had a significant effect on post-menopausal BC in this population. This study provides new information about the effects of HC and HRT on BC risk in a Mexican population, which may be of relevance for the population of Latin America as a whole.