Response to Hypoxia and the Ensuing Dysregulation of Inflammation Impacts Mycobacterium tuberculosis Pathogenicity.

Rationale: Different Mycobacterium tuberculosis (Mtb) strains exhibit variable degrees of virulence in humans and animal models. Differing stress response strategies used by different strains of Mtb could influence virulence. Objectives: We compared the virulence of two strains of Mtb with use in animal model research: CDC1551 and Erdman. Methods: Rhesus macaques, which develop human-like tuberculosis attributes and pathology, were infected with a high dose of either strain via aerosol, and virulence was compared by bacterial burden and pathology. Measurements and Main Results: Infection with Erdman resulted in significantly shorter times to euthanasia and higher bacterial burdens and greater systemic inflammation and lung pathology relative to those infected with CDC1551. Macaques infected with Erdman also exhibited significantly higher early inflammatory myeloid cell influx to the lung, greater macrophage and T cell activity, and higher expression of lung remodeling (extracellular matrix) genes, consistent with greater pathology. Expression of NOTCH4 (neurogenic locus notch homolog 4) signaling, which is induced in response to hypoxia and promotes undifferentiated cellular state, was also higher in Erdman-infected lungs. The granulomas generated by Erdman, and not CDC1551, infection appeared to have larger regions of necrosis, which is strongly associated with hypoxia. To better understand the mechanisms of differential hypoxia induction by these strains, we subjected both to hypoxia in vitro. Erdman induced higher concentrations of DosR regulon relative to CDC1551. The DosR regulon is the global regulator of response to hypoxia in Mtb and critical for its persistence in granulomas. Conclusions: Our results show that the response to hypoxia is a critical mediator of virulence determination in Mtb, with potential impacts on bacillary persistence, reactivation, and efficiency of therapeutics.

Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis.

Tuberculosis (TB) is a global pandaemic, partially due to the failure of vaccination approaches. Novel anti-TB vaccines are therefore urgently required. Here we show that aerosol immunization of macaques with the Mtb mutant in SigH (MtbΔsigH) results in significant recruitment of inducible bronchus-associated lymphoid tissue (iBALT) as well as CD4(+) and CD8(+) T cells expressing activation and proliferation markers to the lungs. Further, the findings indicate that pulmonary vaccination with MtbΔsigH elicited strong central memory CD4(+) and CD8(+) T-cell responses in the lung. Vaccination with MtbΔsigH results in significant protection against a lethal TB challenge, as evidenced by an approximately three log reduction in bacterial burdens, significantly diminished clinical manifestations and granulomatous pathology and characterized by the presence of profound iBALT. This highly protective response is virtually absent in unvaccinated and BCG-vaccinated animals after challenge. These results suggest that future TB vaccine candidates can be developed on the basis of MtbΔsigH.

Comparison of the vaginal environment of Macaca mulatta and Macaca nemestrina throughout the menstrual cycle.

PROBLEM: Pigtail macaques, Macaca nemestrina (PT), are more susceptible to vaginal transmission of simian immunodeficiency virus (SIV) and other sexually transmitted diseases (STD) than rhesus macaques (RM). However, comparative studies to explore the reasons for these differences are lacking. METHOD OF STUDY: Here, we compared differences in hormone levels and vaginal mucosal anatomy and thickness of RM and PT through different stages of the menstrual cycle. Concentrations of plasma estradiol (E2) and progesterone (P4) were determined weekly, and vaginal biopsies examined at days 0 and 14 of the menstrual cycle. RESULTS: Consistent changes in vaginal epithelial thickness occurred at different stages of the menstrual cycle. In both species, the vaginal epithelium was significantly thicker in the follicular than in luteal phase. Keratinized epithelium was strikingly much more prominent in RM, especially during the luteal phase. Further, the vaginal epithelium was significantly thinner, and the P4:E2 ratio was higher in PT during luteal phase than RM. CONCLUSIONS: Striking anatomic differences in the vaginal epithelium between rhesus and pigtail macaques combined with differences in P4:E2 ratio support the hypothesis that thinning and less keratinization of the vaginal epithelium may be involved in the greater susceptibility of pigtail macaques to vaginal transmission of SIV or other STD.

Granuloma correlates of protection against tuberculosis and mechanisms of immune modulation by Mycobacterium tuberculosis.

BACKGROUND: The BCG vaccine is ineffective against adult tuberculosis. Hence, new antituberculosis vaccines are needed. Correlates of protection against tuberculosis are not known. We studied the effects of BCG vaccination on gene expression in tuberculosis granulomas using macaques. METHODS: Macaques were BCG-vaccinated or sham-vaccinated and then challenged with virulent Mycobacterium tuberculosis. Lung lesions were used for comparative transcriptomics. RESULTS: Vaccinated macaques were protected with lower bacterial burden and immunopathology. Lesions from BCG-vaccinated nonhuman primates (NHPs) showed a better balance of α- and ß-chemokine gene expression with higher levels of ß-chemokine expression relative to nonvaccinated animals. Consistent with this, sham-vaccinated macaques recruited fewer macrophages relative to neutrophils in their lungs. The expression of indoleamine 2,3-dioxygenase (IDO), a known immunosuppressor, was significantly higher in both week 5 and 10 lesions from sham-vaccinated, relative to BCG-vaccinated, NHPs. IDO expression was primarily limited to the nonlymphocytic region of the lesions, within the inner ring structure surrounding the central necrosis. CONCLUSIONS: Our study defines lung gene expression correlates of protective response against tuberculosis, relative to disease, which can potentially be employed to assess the efficacy of candidate antituberculosis vaccines. Mycobacterium tuberculosis may modulate protective immune responses using diverse mechanisms, including increased recruitment of inflammatory neutrophils and the concomitant use of IDO to modulate inflammation.

Locally infiltrative ameloblastic fibroma in a rhesus macaque (Macaca mulatta) with characterizations of its proliferating activity and biological behavior.

An 8-year-old male rhesus macaque (Macaca mulatta) presented with unilateral enlargement of the left mandible. Radiographs revealed a marked expansion of the left mandible with a multilocular radiolucent mass with abundant osteolysis. The mass was grossly firm, fleshy, and gelatinous on the cut surface. Histologically, the mass was locally infiltrative and composed of neoplastic epithelial and mesenchymal components that stained positive for cytokeratin and vimentin, respectively. Occasional densely spherical condensations of fibroblasts resembling the cap stage of odontogenesis were present in the mesenchyma. Immunohistochemical staining with Ki-67, S-100, and CD34 indicated that both epithelial and mesenchymal components of the neoplasm had low proliferation. Alcian blue, periodic acid-Schiff, and trichrome stains showed an immature stromal component with no collagen formation. Based on the clinical, histologic, and immunophenotypic features, the tumor was identified as a locally infiltrative ameloblastic fibroma.

The Mycobacterium tuberculosis stress response factor SigH is required for bacterial burden as well as immunopathology in primate lungs.

BACKGROUND: Sigma H (sigH) is a major Mycobacterium tuberculosis (Mtb) stress response factor. It is induced in response to heat, oxidative stress, cell wall damage, and hypoxia. Infection of macrophages with the Δ-sigH mutant generates more potent innate immune response than does infection with Mtb. The mutant is attenuated for pathology in mice. METHODS: We used a nonhuman primate (NHP) model of acute tuberculosis, to better understand the phenotype of the Δ-sigH mutant in vivo. NHPs were infected with high doses of Mtb or the mutant, and the progression of tuberculosis was analyzed in both groups using clinical, pathological, microbiological, and immunological parameters. RESULTS: Animals exposed to Mtb rapidly progressed to acute pulmonary tuberculosis as indicated by worsening clinical correlates, high lung bacterial burden, and granulomatous immunopathology. All the animals rapidly succumbed to tuberculosis. On the other hand, the NHPs exposed to the Mtb:Δ-sigH mutant did not exhibit acute tuberculosis, instead showing significantly blunted disease. These NHPs survived the entire duration of the study. CONCLUSIONS: The Mtb:Δ-sigH mutant is completely attenuated for bacterial burden as well as immunopathology in NHPs. SigH and its regulon are required for complete virulence in primates. Further studies are needed to identify the molecular mechanism of this attenuation.

Sustained release of the CCR5 inhibitors CMPD167 and maraviroc from vaginal rings in rhesus macaques.

Antiretroviral entry inhibitors are now being considered as vaginally administered microbicide candidates for the prevention of the sexual transmission of human immunodeficiency virus. Previous studies testing the entry inhibitors maraviroc and CMPD167 in aqueous gel formulations showed efficacy in the macaque challenge model, although protection was highly dependent on the time period between initial gel application and subsequent challenge. In this paper, we describe the sustained release of maraviroc and CMPD167 from matrix-type silicone elastomer vaginal rings both in vitro and in vivo. Both inhibitors were released continuously during 28 days from rings in vitro at rates of 100 to 2,500 µg/day. In 28-day pharmacokinetic studies in rhesus macaques, the compounds were measured in the vaginal fluid and vaginal tissue; steady-state fluid concentrations were ~10(6)-fold greater than the 50% inhibitory concentrations (IC(50)s) for simian human immunodeficiency virus 162P3 inhibition in macaque lymphocytes in vitro. Plasma concentrations for both compounds were very low. The pretreatment of macaques with Depo-Provera (DP), which is commonly used in macaque challenge studies, was shown to significantly modify the biodistribution of the inhibitors but not the overall amount released. Vaginal fluid and tissue concentrations were significantly decreased while plasma levels increased with DP pretreatment. These observations have implications for designing macaque challenge experiments and also for ring performance during the human female menstrual cycle.

Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection.

The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates. Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4-6 months later. Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnostic peptide were measured longitudinally and declined in all treated animals. B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals. Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys. These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered post-dissemination, in a primate host. Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) in vitro, it appears to become tolerant post-dissemination in the primate host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not they can contribute to symptoms post-treatment.

Limited or no protection by weakly or nonneutralizing antibodies against vaginal SHIV challenge of macaques compared with a strongly neutralizing antibody.

To guide vaccine design, we assessed whether human monoclonal antibodies (MAbs) b12 and b6 against the CD4 binding site (CD4bs) on HIV-1 gp120 and F240 against an immundominant epitope on gp41 could prevent vaginal transmission of simian HIV (SHIV)-162P4 to macaques. The two anti-gp120 MAbs have similar monomeric gp120-binding properties, measured in vitro, but b12 is strongly neutralizing and b6 is not. F240 is nonneutralizing. Applied vaginally at a high dose, the strongly neutralizing MAb b12 provided sterilizing immunity in seven of seven animals, b6 in zero of five animals, and F240 in two of five animals. Compared with control animals, the protection by b12 achieved statistical significance, whereas that caused by F240 did not. For two of three unprotected F240-treated animals there was a trend toward lowered viremia. The potential protective effect of F240 may relate to the relatively strong ability of this antibody to capture infectious virions. Additional passive transfer experiments also indicated that the ability of the administered anti-gp120 MAbs to neutralize the challenge virus was a critical influence on protection. Furthermore, when data from all of the experiments were combined, there was a significant increase in the number of founder viruses establishing infection in animals receiving MAb b6, compared with other nonprotected macaques. Thus, a gp120-binding, weakly neutralizing MAb to the CD4bs was, at best, completely ineffective at protection. A nonneutralizing antibody to gp41 may have a limited capacity to protect, but the results suggest that the central focus of HIV-1 vaccine research should be on the induction of potently neutralizing antibodies.

Noninflammatory gluten peptide analogs as biomarkers for celiac sprue.

New tools are needed for managing celiac sprue, a lifelong immune disease of the small intestine. Ongoing drug trials are also prompting a search for noninvasive biomarkers of gluten-induced intestinal change. We have synthesized and characterized noninflammatory gluten peptide analogs in which key Gln residues are replaced by Asn or His. Like their proinflammatory counterparts, these biomarkers are resistant to gastrointestinal proteases, susceptible to glutenases, and permeable across enterocyte barriers. Unlike gluten peptides, however, they are not appreciably recognized by transglutaminase, HLA-DQ2, or disease-specific T cells. In vitro and animal studies show that the biomarkers can detect intestinal permeability changes as well as glutenase-catalyzed gastric detoxification of gluten. Accordingly, controlled clinical studies are warranted to evaluate the use of these peptides as probes for abnormal intestinal permeability in celiac patients and for glutenase efficacy in clinical trials and practice.

Complications of gastric catheters implanted in rhesus macaques (Macaca mulatta).

As part of a study addressing chronic alcohol consumption and simian immunodeficiency virus, 31 rhesus macaques (Macaca mulatta) were implanted with gastric catheters used to deliver alcohol or isocaloric sucrose (control). Once implanted, the animals wore jackets and were housed in specialized cages modified with swivels and tethers. During the course of the study, 3 animals developed clinical signs indicating possible instability of the implanted gastric catheter. All 3 animals were found to have a string foreign body wrapped around the distal end of the catheter, with 2 of the catheters perforating the intestinal wall. Gastroscopy was used to screen remaining animals to determine catheter position and the presence of a foreign body attached to the end of the catheter. Results of the screening revealed that of the 28 remaining animals, 9 had malpositioned catheters; string foreign bodies were associated with 3 of the 9 malpositioned catheters. We initially hypothesized that the peristaltic motion of the stomach, combined with the attachment of string, which was probably ingested by subjects after manipulating their jackets, led to eventual catheter displacement. We later concluded that the string may have played a secondary role but was not the primary cause of catheter instability, because several malpositioned catheters had no string attached at the time of diagnosis. Subsequent modifications were instituted, including modifying the surgical technique, altering the type of gastric catheter used, and increasing environmental enrichment for animals with known tendency to manipulate their jackets.