Potential biomarkers associated with discrimination between latent and active pulmonary tuberculosis.

SETTING: A third of the world's population has latent tuberculous infection (LTBI). Current TB diagnostics used in developing countries are ineffective and are unable to distinguish LTBI from active TB. Identifying biomarkers that could aid in the early detection of TB and in distinguishing TB states could be a major breakthrough in global TB control. OBJECTIVE: To identify potential immune biomarkers to distinguish active TB from LTBI. DESIGN: A cross-sectional study was conducted among 19 active TB patients, 8 TB-negative individuals (controls) and 16 LTBI non-human immunodeficiency virus infected individuals in Nairobi, Kenya. Excess supernatants from the QuantiFERON®-TB Gold In-Tube test were used to measure immune analytes using a Th17-focused Milliplex® assay. RESULTS: Overall antigen-specific responses were higher in the LTBI group than in active TB patients and controls. Interleukin (IL) 17F, macrophage inflammatory protein 3 alpha (MIP-3α), IL-13, IL-17A, IL-5, interferon-gamma (IFN-γ), IL-9, IL-1ß and IL-2 were significantly differentially produced by individuals with LTBI and active TB patients. Receiver operator curve analysis revealed good discriminative abilities of these analytes. Co-expression analysis highlighted uniquely co-expressed cytokine pairs between TB groups. CONCLUSION: These findings suggest that IL-17F, MIP-3α, IL-13, IL-17A, IL-5, IL-9, IL-1ß and IL-2, in addition to IFN-γ, could identify and uniquely discriminate between TB states.

Latent tuberculosis infection: An overview.

Latent tuberculosis infection (LTBI) is defined as a state of persistent immune response to stimulation by Mycobacterium tuberculosis antigens without evidence of clinically manifested active tuberculosis (TB) disease. Individuals with LTBI represent a reservoir for active TB cases. The detection and management of LTBI is now a key component of the World Health Organization's End TB Strategy and the Government of Canada's federal framework for action on TB prevention and control. This is because people with LTBI can progress to active TB or undergo reactivation, a risk that is greatly increased in those with immunocompromising conditions. This overview provides a summary of LTBI and reactivation risk, as well as the recent advances in the diagnosis and treatment of LTBI.

Automation for clinical CD4 T-cell enumeration, a desirable tool in the hands of skilled operators.

BACKGROUND: Automation in HIV clinical flow cytometry when appropriately applied brings considerable standardisation benefits. The Canadian Immunology Quality Assessment Program (CIQAP) detected situations where operators did not manually override automated software in the event of improper output on the Epics XL and FC500 CD4 immunophenotyping platforms. The automated gating algorithm identifies lymphocytes using a double gate strategy based on CD45 × side scatter (SS) gating and a light scatter FS × SS gate known to fail with sub optimal specimens. METHOD: To generate correct interpretation and results CIQAP introduced a simple protocol modification, bypassing the light scatter gate to include all cells characterized by the CD45 gate. Seventeen problem cases were reanalysed for both absolute and relative T-cell subsets accuracy and compared to the CIQAP group mean values. Results were found to be associated with the percentage of lymphocytes excluded by the automated light scatter gate. RESULTS: The modified manual protocol resolved poor performance in 14 instances out of 17 problem cases. It was found to improve accuracy when the light scatter gate excluded greater than 5% of the cells. The remaining three cases had a lymphocyte recovery of greater than 94.6% in the original automated analysis. CONCLUSION: There is a risk in relying solely on automated gating procedures when using the Epics XL and FC500 CD4 immunophenotyping platforms. Laboratory managers have the responsibility to intervene when required. EQA providers are equally responsible to alert the clinical laboratories of the need to update operator training to deal with stressed specimens. © 2016 International Clinical Cytometry Society.

IFN-γ promoter polymorphisms do not affect QuantiFERON® TB Gold In-Tube test results in a Canadian population.

BACKGROUND: Several studies have shown polymorphisms within the interferon-gamma (IFN-γ) promoter influence cytokine expression. The interferon-gamma release assay (IGRA) relies on the ability to produce IFN-γ in response to tuberculosis (TB) specific antigens. This study determined the relationship between the IFN-γ +874 A/T promoter polymorphism and the performance of the QuantiFERON®-TB Gold In-Tube (QFT-GIT) test in an ethnically diverse Canadian population. METHODS: A total of 190 participants were categorised into three groups based on history of and exposure to TB: active TB (n = 55), TB exposed (n = 55) and presumably TB unexposed controls (n = 80). All participants underwent QFT-GIT testing, and DNA was extracted from whole blood and probed for polymorphism at position +874 (T/A) of intron 1 of IFN-γ. Statistical relationships between the QFT-GIT results, polymorphisms and demographic data were evaluated. RESULTS: IFN-γ +874 genotype frequencies among the entire study population (n = 190) were A/A (45.8%), T/A (39.5%), and T/T (14.7%). Among the three study groups, there was no correlation between QFT-GIT results and the IFN-γ +874 A/T genotype, and no correlation of genotype with IFN-γ production in response to either Mycobacterium tuberculosis antigens or mitogenic stimulation. CONCLUSION: Our results indicate that the IFN-γ +874 promoter polymorphism does not influence QFT-GIT performance in this study population.

Cataloguing of Potential HIV Susceptibility Factors during the Menstrual Cycle of Pig-Tailed Macaques by Using a Systems Biology Approach.

UNLABELLED: Our earlier studies with pig-tailed macaques demonstrated various simian-human immunodeficiency virus (SHIV) susceptibilities during the menstrual cycle, likely caused by cyclic variations in immune responses in the female genital tract. There is concern that high-dose, long-lasting, injectable progestin-based contraception could mimic the high-progesterone luteal phase and predispose women to human immunodeficiency type 1 (HIV-1) acquisition and transmission. In this study, we adopted a systems biology approach employing proteomics (tandem mass spectrometry), transcriptomics (RNA microarray hybridization), and other specific protein assays (enzyme-linked immunosorbent assays and multiplex chemokine and cytokine measurements) to characterize the effects of hormonal changes on the expression of innate factors and secreted proteins in the macaque vagina. Several antiviral factors and pathways (including acute-phase response signaling and complement system) were overexpressed in the follicular phase. Conversely, during the luteal phase there were factors overexpressed (including moesins, syndecans, and integrins, among others) that could play direct or indirect roles in enhancing HIV-1 infection. Thus, our study showed that specific pathways and proteins or genes might work in tandem to regulate innate immunity, thus fostering further investigation and future design of approaches to help counter HIV-1 acquisition in the female genital tract. IMPORTANCE: HIV infection in women is poorly understood. High levels of the hormone progesterone may make women more vulnerable to infection. This could be the case during the menstrual cycle, when using hormone-based birth control, or during pregnancy. The biological basis for increased HIV vulnerability is not known. We used an animal model with high risk for infection during periods of high progesterone. Genital secretions and tissues during the menstrual cycle were studied. Our goal was to identify biological factors upregulated at high progesterone levels, and we indeed show an upregulation of genes and proteins which enhance the ability of HIV to infect when progesterone is high. In contrast, during low-progesterone periods, we found more HIV inhibitory factors. This study contributes to our understanding of mechanisms that may regulate HIV infection in females under hormonal influences. Such knowledge is needed for the development of novel prevention strategies.

Acting locally: innate mucosal immunity in resistance to HIV-1 infection in Kenyan commercial sex workers.

Cohort studies of female commercial sex workers (CSWs) in Kenya were among the first to identify highly HIV-1-exposed seronegative (HESN) individuals. As natural resistance is usually mediated by innate immune mechanisms, we focused on determining whether expression and function of innate signaling pathways were altered locally in the genital mucosa of HESN CSWs. Our results demonstrated that selected pattern-recognition receptors (PRRs) were significantly reduced in expression in cervical mononuclear cells (CMCs) from HESN compared with the new HIV-negative (HIV-N) and HIV-positive (HIV-P) groups. Although baseline levels of secreted cytokines were reduced in CMCs of HESN, they were highly stimulated following exposure to ssRNA40 in vitro. Importantly, cervical epithelial cells from HESN also expressed reduced levels of PRRs, but Toll-like receptor 3 (TLR3) and TLR7 as well as nuclear factor-κB and activator protein 1 were highly expressed and activated. Lastly, inflammatory cytokines interleukin (IL)-1ß, IL-8, and RANTES (regulated and normal T cell expressed and secreted) were detected at lower levels in cervicovaginal lavage of HESN compared with the HIV-N and HIV-P groups. Overall, our study reveals a local microenvironment of HIV resistance in the genital mucosa consisting of a finely controlled balance of basal immune quiescence with a focused and potent innate anti-viral response critical to resistance to sexual transmission of HIV-1.

HLA class I associations with rates of HIV-1 seroconversion and disease progression in the Pumwani Sex Worker Cohort.

Class I human leukocyte antigens (HLA) play an important role in the adaptive immune response by presenting antigens to CD8+ T cells. Studies have reported that several HLA class I alleles are associated with differential disease progression in human immunodeficiency virus (HIV)-infected individuals, however, few class I associations with resistance or susceptibility to HIV-1 infection have been reported. We typed HLA-A, -B and -C of >1000 women enrolled in the Pumwani Sex Worker Cohort using a sequence-based typing method. Kaplan-Meier analysis was used to identify alleles influencing seroconversion and disease progression to acquired immune deficiency syndrome (CD4 < 200/mm³). A*01 (P = 0.020), C*06:02 (P = 0.042) and C*07:01 (P = 0.050) are independently associated with protection from seroconversion. Women with any of these alleles are less likely to seroconvert [P = 0.00001, odds ratio (OR): 0.503, 95% confidence interval (CI): 0.320-0.790]. Conversely, A*23:01 (P = 0.004), B*07:02 (P = 0.003) and B*42:01 (P = 0.025) are independently associated with rapid seroconversion. Women with any of these alleles are twice as likely to seroconvert (P = 0.002, OR: 2.059, 95% CI: 1.290-3.285). The beneficial alleles confer threefold protection from seroconversion when compared with the susceptible alleles (P = 0.000001, OR: 0.268, 95% CI: 0.132-0.544). B*07:02 is the contributing allele, within the B7 supertype, to the rapid seroconversion. A*74:01 (P = 0.04/P = 0.006), B*14 (P = 0.003/P = 0.003) and B*57:03 (P = 0.012/P = 0.038) are independently associated with slower CD4+ decline and LTNP phenotype, while B*07:02 (P = 0.020), B*15:10 (P = 0.022) and B*53:01 (P = 0.007) are independently associated with rapid CD4+ T-cell decline. B7 supertype (P = 0.00006), B*35*-Py (P = 0.028) and B*35-Px (P = 0.001) were also significantly associated with rapid CD4+ T-cell decline. Understanding why these HLA class I alleles are associated with protection/susceptibility to HIV-1 acquisition and disease progression could contribute to the development of effective prophylactic and therapeutic vaccines for HIV-1.

A distinct cytokine and chemokine profile at the genital mucosa is associated with HIV-1 protection among HIV-exposed seronegative commercial sex workers.

The predominance of HIV-1 sexual transmission requires a greater understanding of the interaction between HIV-1 and the mucosal immune system. The study of HIV-1-exposed seronegative (HESN) individuals serves as a model to identify the correlates of protection and to aid in microbicide development. A total of 22 cytokines/chemokines were analyzed at the systemic and mucosal compartments in 57 HESN, 51 HIV-1-negative, and 67 HIV-1-infected commercial sex workers from Nairobi, Kenya. HESN individuals had significantly lower expression of monokine induced by interferon-γ (MIG), interferon-γ-induced protein 10 (IP-10), and interleukin-1α (IL-1α) in their genital mucosa compared with controls. HESN cytokine expression also distinctly correlates with mucosal antiproteases, suggesting that HESN individuals have a unique pattern of mucosal chemokine/cytokine expression, which may result in reduced trafficking at the mucosa. These data support the immune quiescence model of protection, whereby lower T-cell activation/recruitment at the mucosal compartment reduces HIV-1 target cell numbers and is an important component of natural protection from HIV-1.

Comprehensive proteomic study identifies serpin and cystatin antiproteases as novel correlates of HIV-1 resistance in the cervicovaginal mucosa of female sex workers.

Not all individuals exposed to HIV-1 become infected, and evidence from HIV-1 highly exposed seronegative women (HIV-1-resistant) suggests that mucosal factors in the female genital tract, the first site of contact for the virus, are playing a role. To better understand factors mediating protection from HIV-1, we performed a large clinical study using the tools of systems biology to fully characterize the cervicovaginal mucosa proteome in HIV-1-resistant women. Cervicovaginal lavage fluid was collected from 293 HIV-1-resistant, uninfected, and infected sex workers and analyzed by 2D-LC LTQ-FT-MS. Of the more than 360 unique proteins identified, 41 were differentially abundant (>3-fold cutoff) in HIV-1-resistant women. The majority of over-abundant proteins were antiproteases (>40%), some with described anti-inflammatory and anti-HIV-1 activity. Quantification of specific anti-HIV-1 antiproteases Serpin A1, Serpin A3, and Cystatin B and an epithelial antiprotease A2ML1 found them to be significantly over-abundant in HIV-1-resistant women (p = 0.004; p = 0.046; p = 0.0003; and p = 0.04, respectively). Expression levels were not correlated to sexual practices or other epidemiological factors. Mucosal antiprotease levels correlated with pro-inflammatory cytokine concentration (p = <0.0001), but independently of pro-inflammatory cytokine levels in HIV-1-resistant women including TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, and IL-8. This comprehensive systems biology approach identifies mucosal serpins and cystatins as novel correlates of HIV-1-resistance. This represents the first study characterizing these factors in the female genital tract.

Defining the genital immune correlates of protection against HIV acquisition: co-infections and other potential confounders.

The sexual transmission of HIV is very inefficient, presumably because mucosal immune defences prevent infection after most exposures. Since numerous genital immune factors have antiviral effects in vitro, their elucidation might greatly inform the microbicide and HIV prevention fields, particularly in the context of HIV-exposed but persistently seronegative (ESN) individuals. However, several important confounders must be considered in such research. First, sound epidemiological criteria are needed to define individuals as ESN. Then, since high-risk sexual activity is commonly one of these criteria, its potential impact on genital immunology must be carefully considered, both the direct effects of sex and the secondary immune effects of genital co-infections. This means that it may be very difficult to determine whether differences in genital immunology between ESN and control groups are responsible for HIV protection, or are a consequence of high-risk sexual activity. To overcome this confounding, the demographics and epidemiology of ESN cohorts must be described very carefully, thorough co-infection diagnostics must be performed and, if possible, prospective studies with an endpoint of HIV acquisition should be performed to define the direction of causality.

Improved mRNA quantitation in LightCycler RT-PCR.

BACKGROUND: Real-time polymerase chain reaction (PCR) utilizing the LightCycler and similar systems is an increasingly used technique for quantitative reverse transcription (RT)-PCR of mRNA levels from genes of immunologic interest. A commonly encountered limitation with these systems is that the fluorescence induced by SYBR Green (a fluorophore that binds double-stranded DNA) can result from primer dimers (PDs) as well as the PCR product of interest, thus interfering with the ability to reproducibly quantitate mRNA levels. METHODS: We use a modification of the LightCycler PCR strategy to overcome this problem by altering the PCR strategy to take advantage of the LightCycler's ability to measure fluorescence at a temperature greater than the melting point of PDs. The resulting measurements determine fluorescence of only the desired PCR product. RESULTS: We demonstrate that by using this modified PCR strategy, one can eliminate the fluorescence induced by PDs and obtain accurate product quantitation. CONCLUSIONS: This simple modification allows more precise quantitation of sample mRNA levels by eliminating the contaminating fluorescence induced by the formation of PCR PDs. This modification obviates the need to redesign PCR primers in RT-PCR experiments where this is impractical or impossible.

HIV-1-specific cellular immune responses among HIV-1-resistant sex workers.

The goal of the present study was to determine whether there were HIV-1 specific cellular immune responses among a subgroup of women within a cohort of Nairobi prostitutes (n = 1800) who, despite their intense sexual exposure to HIV-1, are epidemiologically resistant to HIV-1 infection. Of the 80 women defined to be resistant, 24 were recruited for immunological evaluation. The HIV-1-specific T-helper responses were determined by IL-2 production following stimulation with HIV-1 envelope peptides and soluble gp120. Cytotoxic T lymphocyte responses were determined by lysis of autologous EBV-transformed B cell lines infected with control vaccinia virus or recombinant vaccinia viruses containing the HIV-1 structural genes env, gag and pol. Resistant women had significantly increased HIV-1 specific T-helper responses, as determined by in vitro IL-2 production to HIV-1 envelope peptides and soluble glycoprotein 120, compared with low-risk seronegative and HIV-1-infected controls (P < or = 0.01, Student's t-test). Seven of the 17 (41%) resistant women showed IL-2 stimulation indices > or = 2.0. HIV-1-specific CTL responses were detected among 15/22 (68.2%) resistant women compared with 0/12 low-risk controls (Chi-squared test, P < 0.001). In the two resistant individuals tested, the CTL activity was mediated by CD8+ effectors. Many HIV-1-resistant women show evidence of HIV-1-specific T-helper and cytotoxic responses. These data support the suggestion that HIV-1-specific T-cell responses contribute to protection against HIV-1 infection.

Acute sexually transmitted infections increase human immunodeficiency virus type 1 plasma viremia, increase plasma type 2 cytokines, and decrease CD4 cell counts.

In Kenya, the median incubation time to AIDS in seroconverting sex workers is 4 years; this incubation time is specific to female sex workers. We studied the influence of acute sexually transmitted infections (STIs) on several immunologic parameters in 32 human immunodeficiency virus type 1 (HIV-1)-positive and 10 HIV-1-negative women sex workers who were followed for 1-5 months. Plasma cytokines, soluble cytokine receptors, CD4 and CD8 T cell counts, and HIV-1 plasma viremia were quantitated before, during, and after episodes of STI. Increases in interleukin (IL)-4, IL-6, IL-10, soluble tumor necrosis factor (TNF)-alpha, and viremia and a decline in CD4(+) T cell counts occurred during gonococcal cervicitis and returned to baseline after treatment. Increases in viremia correlated with increased IL-4 and decreased IL-6 concentrations. Similar changes were seen among women with acute pelvic inflammatory disease. Acute bacterial STI resulted in increased HIV-1 viremia. This may be mediated through increased inflammatory cytokines or through modulation of immune responses that control HIV-1 viremia.

Influence of HLA supertypes on susceptibility and resistance to human immunodeficiency virus type 1 infection.

Certain human leukocyte antigens, by presenting conserved immunogenic epitopes for T cell recognition, may, in part, account for the observed differences in human immunodeficiency virus type 1 (HIV-1) susceptibility. To determine whether HLA polymorphism influences HIV-1 susceptibility, a longitudinal cohort of highly HIV-1-exposed female sex workers based in Nairobi, Kenya, was prospectively analyzed. Decreased HIV-1 infection risk was strongly associated with possession of a cluster of closely related HLA alleles (A2/6802 supertype; incidence rate ratio [IRR], 0.45; 95% confidence interval [CI], 0.27-0.72; P=.0003). The alleles in this supertype are known in some cases to present the same peptide epitopes for T cell recognition. In addition, resistance to HIV-1 infection was independently associated with HLA DRB1*01 (IRR, 0.22; 95% CI, 0.06-0.60; P=.0003), which suggests that anti-HIV-1 class II restricted CD4 effector mechanisms may play an important role in protecting against viral challenge. These data provide further evidence that resistance to HIV-1 infection in this cohort of sex workers is immunologically mediated.

Identifying inflorescence phytoliths from selected species of wheat (Triticum monococcum, T. dicoccon, T. dicoccoides, and T. aestivum) and barley (Hordeum vulgare and H. spontaneum) (Gramineae).

Analysis of microfossil silica phytoliths is becoming an increasingly important research tool for taxonomists, archaeobotanists, and paleoecologists. Expanded use of phytolith analysis by researchers is dependent upon development of phytolith systematics. In this study phytoliths produced by the inflorescence bracts from four species of wheat, Triticum monoccocum, T. dicoccon, T. dicoccoides, and T. aestivum, and two species of barley, Hordeum vulgare, and H. spontaneum, were analyzed using computer-assisted image and statistical analysis with the intent to develop taxonomic tools to distinguish among the taxa. A classification key based on significant differences among the mean morphometries of the inflorescence phytoliths produced by each species was created and tested. Discriminant analysis of the morphometries of several morphotypes of phytoliths was also conducted to determine whether this computer-assisted statistical procedure could be used as another method to classify the taxa and to determine which morphotypes have measurements that can best be used in discriminant functions. Test results indicated that, at the genus level, both the classification key and discriminant analysis of certain morphotypes of phytoliths were relatively reliable tools for distinguishing among phytoliths produced in the inflorescence bracts of the taxa considered. For distinguishing among the taxa at the species level, the classification key was most reliable. Of the discriminant analyses tested, that based on all the phytolith morphotypes combined was more reliable than those based on only one morphotype.

HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan sex workers.

OBJECTIVES: Most HIV-1 transmission is sexual; therefore, immune responses in the genital mucosa may be important in mediating protection against HIV infection. This study examined HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan female sex workers. METHODS: HIV-1-specific immune responses were compared in HIV-1-resistant and HIV-1-infected sex workers, and in lower risk uninfected women. Cervical and vaginal samples from each group were tested for HIV-1-specific IgA and IgG by enzyme immunoassay. Systemic T-helper lymphocyte cell responses to HIV-1 envelope peptide epitopes were assayed using an interleukin 2 bioassay. HIV-1 risk-taking behaviours were assessed using standardized questionnaires. RESULTS: HIV-1-specific IgA was present in the genital tract of 16 out of 21 (76%) HIV-1-resistant sex workers, five out of 19 (26%) infected women, and three out of 28 (11%) lower risk women (P < 0.0001). Among lower risk women, the presence of HIV-1-specific IgA was associated with HIV-1 risk-taking behaviour. Systemic T-helper lymphocyte responses to HIV-1 envelope peptides were present in 11 out of 20 (55%) HIV-1-resistant women, four out of 18 (22%) infected women, and one out of 25 (4%) lower risk women (P < 0.001). T-helper lymphocyte responses did not correlate with the presence or titre of virus-specific mucosal IgA in any study group. CONCLUSIONS: HIV-1-specific IgA is present in the genital tract of most HIV-1-resistant Kenyan sex workers, and of a minority of lower risk uninfected women, where it is associated with risk-taking behaviour. These data suggest a role for mucosal HIV-1-specific IgA responses in HIV-1 resistance, independent of host cellular responses.

Resistance to HIV-1 infection among highly exposed sex workers in Nairobi: what mediates protection and why does it develop?

Variability in susceptibility to infection and disease caused by infectious agents is a characteristic of all populations. Among susceptible individuals exposed to an infection, not all become infected and among infected individuals, not all develop disease. It seems logical that variability in susceptibility to infection and disease would apply to infection and disease with human immunodeficiency viruses. However, until recently, it has been generally held that there is no natural immunity to HIV-1 and that once infected, all individuals would ultimately succumb to AIDS.

Naturally occurring IgG anti-HLA alloantibody does not correlate with HIV type 1 resistance in Nairobi prostitutes.

In an effort to identify an immunological basis for natural resistance to HIV-1 infection, we have examined serum antibody responses to HLA class I antigens in female prostitutes of the Nairobi Sex Workers Study. Anti-HLA antibodies are known to block HIV infectivity in vitro and can be protective against SIV challenge in macaques immunized with purified class I HLA. Thus, it was postulated that broadly cross-reactive alloantibodies recognizing common HLA alleles in the client population might contribute to the prevention of heterosexual transmission of HIV. In fact, 12% of the women were found to have serum IgG antibodies against class I alloantigens. However, this alloantibody did not correlate with the HIV status of the women and was found in a similar proportion of HIV-positive and HIV-resistant women. The observed levels of alloantibody did not increase with HIV infection in susceptible individuals, suggesting that potential antigenic mimicry between HIV and host HLA class I antigens does not significantly increase levels of anti-class I antibodies. The lack of correlation between serum anti-allo-class I HLA antibodies and the risk of sexual transmission indicates that this humoral immune response is unlikely to be the natural mechanism behind the HIV-resistance phenotype of persistently HIV-seronegative women. This result, however, does not preclude the further investigation of alloimmunization as an artificial HIV immunization strategy.