The Impact of Vitamin A Deficiency on Tuberculosis Progression.

BACKGROUND: Although previous studies have shown that vitamin A deficiency is associated with incident tuberculosis (TB) disease, the direction of the association has not been established. We investigated the impact of vitamin A deficiency on TB disease progression. METHODS: We conducted a longitudinal cohort study nested within a randomized clinical trial among HIV-infected patients in Haiti. We compared serial vitamin A levels in individuals who developed TB disease to controls matched on age, gender, follow-up time, and time to antiretroviral therapy initiation. We also evaluated histopathology, bacterial load, and immune outcomes in TB infection in a guinea pig model of dietary vitamin A deficiency. RESULTS: Among 773 participants, 96 developed incident TB during follow-up, 62.5% (60) of whom had stored serum samples obtained 90-365 days before TB diagnosis. In age- and sex- adjusted and multivariate analyses, respectively, incident TB cases were 3.99 times (95% confidence interval [CI], 2.41 to 6.60) and 3.59 times (95% CI, 2.05 to 6.29) more likely to have been vitamin A deficient than matched controls. Vitamin A-deficient guinea pigs manifested more extensive pulmonary pathology, atypical granuloma morphology, and increased bacterial growth after experimental TB infection. Reintroduction of dietary vitamin A to deficient guinea pigs after established TB disease successfully abrogated severe disease manifestations and altered cellular immune profiles. CONCLUSIONS: Human and animal studies support the role of baseline vitamin A deficiency as a determinant of future TB disease progression.

Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens.

Toll-like receptor (TLR) signaling in macrophages is required for antipathogen responses, including the biosynthesis of nitric oxide from arginine, and is essential for immunity to Mycobacterium tuberculosis, Toxoplasma gondii and other intracellular pathogens. Here we report a 'loophole' in the TLR pathway that is advantageous to these pathogens. Intracellular pathogens induced expression of the arginine hydrolytic enzyme arginase 1 (Arg1) in mouse macrophages through the TLR pathway. In contrast to diseases dominated by T helper type 2 responses in which Arg1 expression is greatly increased by interleukin 4 and 13 signaling through the transcription factor STAT6, TLR-mediated Arg1 induction was independent of the STAT6 pathway. Specific elimination of Arg1 in macrophages favored host survival during T. gondii infection and decreased lung bacterial load during tuberculosis infection.

The formation of the granuloma in tuberculosis infection.

The development of the granuloma and its subsequent degeneration and necrosis, is the hallmark of infection caused by Mycobacterium tuberculosis. These structures probably evolved as primitive particle responses, but in mammals they are facilitated by the emerging acquired immune response, in which cytokines and chemokines help control their formation and integrity. In this brief review we discuss the pathology of these lesions in the two most widely used animal models (mice and guinea pigs). In addition, we argue against the idea that there is a balance between host immunity and bacterial survival, and that the latter possess mechanisms that control this, as some currently believe, and moreover discuss newer information regarding the ability of bacilli to persist in these structures long enough to eventually escape and become retransmitted.

Human IL-32 expression protects mice against a hypervirulent strain of Mycobacterium tuberculosis.

Silencing of interleukin-32 (IL-32) in a differentiated human promonocytic cell line impairs killing of Mycobacterium tuberculosis (MTB) but the role of IL-32 in vivo against MTB remains unknown. To study the effects of IL-32 in vivo, a transgenic mouse was generated in which the human IL-32γ gene is expressed using the surfactant protein C promoter (SPC-IL-32γTg). Wild-type and SPC-IL-32γTg mice were infected with a low-dose aerosol of a hypervirulent strain of MTB (W-Beijing HN878). At 30 and 60 d after infection, the transgenic mice had 66% and 85% fewer MTB in the lungs and 49% and 68% fewer MTB in the spleens, respectively; the transgenic mice also exhibited greater survival. Increased numbers of host-protective innate and adaptive immune cells were present in SPC-IL-32γTg mice, including tumor necrosis factor-alpha (TNFα) positive lung macrophages and dendritic cells, and IFN-gamma (IFNγ) and TNFα positive CD4(+) and CD8(+) T cells in the lungs and mediastinal lymph nodes. Alveolar macrophages from transgenic mice infected with MTB ex vivo had reduced bacterial burden and increased colocalization of green fluorescent protein-labeled MTB with lysosomes. Furthermore, mouse macrophages made to express IL-32γ but not the splice variant IL-32ß were better able to limit MTB growth than macrophages capable of producing both. The lungs of patients with tuberculosis showed increased IL-32 expression, particularly in macrophages of granulomas and airway epithelial cells but also B cells and T cells. We conclude that IL-32γ enhances host immunity to MTB.

The Discovery of 2-Aminobenzimidazoles That Sensitize Mycobacterium smegmatis and M.†tuberculosis to ß-Lactam Antibiotics in a Pattern Distinct from ß-Lactamase Inhibitors.

A library of 2-aminobenzimidazole derivatives was screened for the ability to suppress ß-lactam resistance in Mycobacterium smegmatis. Several non-bactericidal compounds were identified that reversed intrinsic resistance to ß-lactam antibiotics in a manner distinct from ß-lactamase inhibitors. Activity also translates to M. tuberculosis, with a lead compound from this study potently suppressing carbenicillin resistance in multiple M. tuberculosis strains (including multidrug-resistant strains). Preliminary mechanistic studies revealed that the lead compounds act through a mechanism distinct from that of traditional ß-lactamase inhibitors.

Increased severity of tuberculosis in Guinea pigs with type 2 diabetes: a model of diabetes-tuberculosis comorbidity.

Impaired glucose tolerance and type 2 diabetes were induced in guinea pigs to model the emerging comorbidity of Mycobacterium tuberculosis infection in diabetic patients. Type 2 diabetes mellitus was induced by low-dose streptozotocin in guinea pigs rendered glucose intolerant by first feeding a high-fat, high-carbohydrate diet before M. tuberculosis exposure. M. tuberculosis infection of diabetic guinea pigs resulted in severe and rapidly progressive tuberculosis (TB) with a shortened survival interval, more severe pulmonary and extrapulmonary pathology, and a higher bacterial burden compared with glucose-intolerant and nondiabetic controls. Compared with nondiabetics, diabetic guinea pigs with TB had an exacerbated proinflammatory response with more severe granulocytic inflammation and higher gene expression for the cytokines/chemokines interferon-γ, IL-17A, IL-8, and IL-10 in the lung and for interferon-γ, tumor necrosis factor-α, IL-8, and monocyte chemoattractant protein-1 in the spleen. TB disease progression in guinea pigs with impaired glucose tolerance was similar to that of nondiabetic controls in the early stages of infection but was more severe by day 90. The guinea pig model of type 2 diabetes-TB comorbidity mimics important features of the naturally occurring disease in humans. This model will be beneficial in understanding the complex pathogenesis of TB in diabetic patients and to test new strategies to improve TB and diabetes control when the two diseases occur together.

Second generation 2-aminoimidazole based advanced glycation end product inhibitors and breakers.

The formation of advanced glycation end-products (AGE) as a result of the action of reducing sugars on host macromolecules plays a role in increased morbidity of diabetic patients. There are currently no clinically available therapeutics for the prevention or eradication of AGEs. Following our previous identification of 2-aminoimidazole (2-AI) based AGE inhibitors and breakers, we now report the use of a rapid, scalable, two-step procedure to access a second generation of 2-AI based anti-AGE compounds from commercially available amino acids. Several second generation compounds exhibit increased AGE inhibition and breaking activty compared to the first generation compounds and to the known AGE inhibitor aminoguanidine.

Inhibition and breaking of advanced glycation end-products (AGEs) with bis-2-aminoimidazole derivatives.

Advanced glycation end-products (AGEs), unregulated modifications to host macromolecules that occur as a result of metabolic dysregulation, play a role in many diabetes related complications, inflammation and aging, and may lead to increased cardiovascular risk. Small molecules that have the ability to inhibit AGE formation, and even break preformed AGEs have enormous therapeutic potential in the treatment of these disease states. We report the screening of a series of 2-aminoimidazloles for anti-AGE activity, and the identification of a bis-2-aminoimidazole lead compound that possesses superior AGE inhibition and breaking activity compared to the known AGE inhibitor aminoguanidine.

High mobility group box 1 acts as an adjuvant for tuberculosis subunit vaccines.

In order to ensure an ample supply of quality candidate tuberculosis (TB) subunit vaccines for clinical trials, it is imperative to develop new immunostimulatory adjuvants. High Mobility Box Group 1 (HMGB1), a member of the alarmin group of immunostimulatory proteins, is released by antigen-presenting cells under various conditions and has been shown to induce T helper type 1 cytokines. We report that HMGB1 is effective as an adjuvant to enhance the protective efficacy and cellular immune response of TB subunit vaccines and that it is not dependent on the interaction between HMGB1 and receptor for advanced glycation end products, a major receptor for HMGB1. In the mouse model of TB, HMGB1 protein, when formulated with dioctadecylammonium bromide and 6000 MW early secretory antigenic target (ESAT-6), was protective as a subunit vaccine but did not protect as molecular adjuvant in an ESAT-6-based DNA formulation. We then evaluated the immunoprophylactic and protective potential of a fusion protein of HMGB1 and ESAT-6. The HMGB1-ESAT-6 fusion protein induced strong antigen-specific T helper type 1 cytokines at 30 days post-immunization. The fusion protein vaccine enhanced activated and effector memory CD4 and CD8 T-cell responses in the lungs and spleens of mice at 80 days post vaccination. Vaccination with the HMGB1-ESAT-6 fusion protein also resulted in elevated numbers of poly-functional CD4 T cells co-expressing interleukin-2, interferon-γ and tumour necrosis factor-α. The potent cell-mediated immune response generated by the fusion protein correlated with protection against subsequent challenge with Mycobacterium tuberculosis in the mouse TB model.

GM-CSF knockout mice for preclinical testing of agents with antimicrobial activity against Mycobacterium abscessus.

OBJECTIVES: Of the non-tuberculous mycobacteria, Mycobacterium abscessus is particularly refractory to antimicrobial therapy and new agents with activity against these pathogens are urgently needed. The screening of candidate antimicrobial agents against M. abscessus requires a relevant and reproducible animal model of chronic infection. Granulocyte-macrophage colony-stimulating factor knockout (GM-CSF KO) mice were used to develop a new animal model of chronic pulmonary M. abscessus infection that can be used for preclinical efficacy testing of antimicrobial drugs. METHODS: GM-CSF KO mice were infected with a clinical isolate of M. abscessus via intrapulmonary aerosol delivery using a microsprayer device. The clinical condition, histology and cfu of M. abscessus-infected GM-CSF KO mice were evaluated over a period of 4 months. Mice were treated with azithromycin (100 mg/kg) by oral gavage and the clinical condition, histology and bacterial burden was determined after 2 weeks of treatment. RESULTS: We show that pulmonary infection of GM-CSF KO mice with M. abscessus results in a chronic pulmonary infection that lends itself to preclinical testing of new antimicrobial drugs against this bacterium. Azithromycin treatment of M. abscessus-infected GM-CSF KO mice resulted in a lower bacterial burden in the lungs and spleen, weight gain and significant improvement in lung pathology. CONCLUSIONS: Intrapulmonary aerosol infection of GM-CSF KO mice with M. abscessus is a useful animal model for studying pathogenesis as well as pre-clinical testing of new compounds against M. abscessus in acute or chronic phases of infection.

Microhemorrhage is an early event in the pulmonary fibrotic disease of PECAM-1 deficient FVB/n mice.

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) deficient mice in the FVB/n strain exhibit fatal chronic pulmonary fibrotic disease. The illness occurs in the absence of a detectable pro-inflammatory event. PECAM-1 is vital to the stability of vascular permeability, leukocyte extravasation, clotting of platelets, and clearance of apoptotic cells. We show here that the spontaneous development of fibrotic disease in PECAM-1 deficient FVB/n mice is characterized by early loss of vascular integrity in pulmonary capillaries, resulting in spontaneous microbleeds. Hemosiderin-positive macrophages were found in interstitial spaces and bronchoalveolar lavage (BAL) fluid in relatively healthy animals. We also observed a gradually increasing presence of hemosiderin-positive macrophages and fibrin deposition in the advanced stages of disease, corresponding to the accumulation of collagen, IL-10 expression, and myofibroblasts expressing alpha smooth muscle actin (SMA). Together with the growing evidence that pulmonary microbleeds and coagulation play an active part in human pulmonary fibrosis, this data further supports our hypothesis that PECAM-1 expression is necessary for vascular barrier function control and regulation of homeostasis specifically, in the pulmonary environment.

Copper resistance is essential for virulence of Mycobacterium tuberculosis.

Copper (Cu) is essential for many biological processes, but is toxic when present in excessive amounts. In this study, we provide evidence that Cu plays a crucial role in controlling tuberculosis. A Mycobacterium tuberculosis (Mtb) mutant lacking the outer membrane channel protein Rv1698 accumulated 100-fold more Cu and was more susceptible to Cu toxicity than WT Mtb. Similar phenotypes were observed for a M. smegmatis mutant lacking the homolog Ms3747, demonstrating that these mycobacterial copper transport proteins B (MctB) are essential for Cu resistance and maintenance of low intracellular Cu levels. Guinea pigs responded to infection with Mtb by increasing the Cu concentration in lung lesions. Loss of MctB resulted in a 1,000- and 100-fold reduced bacterial burden in lungs and lymph nodes, respectively, in guinea pigs infected with Mtb. In mice, the persistence defect of the Mtb mctB mutant was exacerbated by the addition of Cu to the diet. These experiments provide evidence that Cu is used by the mammalian host to control Mtb infection and that Cu resistance mechanisms are crucial for Mtb virulence. Importantly, Mtb is much more susceptible to Cu than other bacteria and is killed in vitro by Cu concentrations lower than those found in phagosomes of macrophages. Hence, this study reveals an Achilles heel of Mtb that might be a promising target for tuberculosis chemotherapy.

Mucosal expression of aquaporin 5 and epithelial barrier proteins in chronic rhinosinusitis with and without nasal polyps.

OBJECTIVES: The purpose of this study is to characterize the association between altered epithelial barrier function, represented by changes in histology and differential expression of the mucosal water membrane permeability protein aquaporin 5 (AQP5), and the pathophysiology of chronic refractory sinusitis (CRS) in patients with and without nasal polyposis. STUDY DESIGN: Prospective clinical study. SETTING: Tertiary rhinology referral center. PARTICIPANTS: Sinonasal samples were obtained from seven CRS subjects with nasal polyps (CRSwNP), seven CRS without nasal polyposis (CRSsNP), and five control healthy patients. METHODS: Mucosal membrane changes were evaluated through hematoxylin and eosin staining of the membrane barrier and immunohistochemical staining of AQP5 expression, a membrane channel protein that affects trans-epithelial water permeability and tissue edema. AQP5 expression was confirmed by real-time PCR (rt-PCR) and western blot. Levels of other membrane proteins, including E-cadherin and Septin-2, were also assessed. RESULTS: CRSwNP patients showed substantial histologic evidence of membrane remodeling with increased edema and glandular hyperplasia. The epithelial expression of AQP5 was significantly lower in CRSwNP as compared to CRSsNP or control. There was no significant difference in the expression of E-cadherin and Septin-2. CONCLUSIONS: Collectively, these data suggest that the mucosal epithelial barrier is compromised in the context of CRS (predominantly in CRSwNP) when compared to control and that AQP5 acts as a key tight junction protein in the maintenance of mucosal water homeostasis. We hypothesize that AQP5 plays a possible role in the pathophysiology of mucosal edema and polyp formation.

Copper-boosting compounds: a novel concept for antimycobacterial drug discovery.

We and others recently identified copper resistance as important for virulence of Mycobacterium tuberculosis. Here, we introduce a high-throughput screening assay for agents that induce a copper hypersensitivity phenotype in M. tuberculosis and demonstrate that such copper-boosting compounds are effective against replicating and nonreplicating M. tuberculosis strains.

Opposing biological functions of tryptophan catabolizing enzymes during intracellular infection.

Recent studies have underscored physiological and pathophysiological roles for the tryptophan-degrading enzyme indolamine 2,3-dioxygenase (IDO) in immune counterregulation. However, IDO was first recognized as an antimicrobial effector, restricting tryptophan availability to Toxoplasma gondii and other pathogens in vitro. The biological relevance of these findings came under question when infectious phenotypes were not forthcoming in IDO-deficient mice. The recent discovery of an IDO homolog, IDO-2, suggested that the issue deserved reexamination. IDO inhibition during murine toxoplasmosis led to 100% mortality, with increased parasite burdens and no evident effects on the immune response. Similar studies revealed a counterregulatory role for IDO during leishmaniasis (restraining effector immune responses and parasite clearance), and no evident role for IDO in herpes simplex virus type 1 (HSV-1) infection. Thus, IDO plays biologically important roles in the host response to diverse intracellular infections, but the dominant nature of this role--antimicrobial or immunoregulatory--is pathogen-specific.

Gliosis, misfolded protein aggregation, and neuronal loss in a guinea pig model of pulmonary tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis infection, is an ongoing epidemic with an estimated ten million active cases of the disease worldwide. Pulmonary tuberculosis is associated with cognitive and memory deficits, and patients with this disease are at an increased risk for Parkinson's disease and dementia. Although epidemiological data correlates neurological effects with peripheral disease, the pathology in the central nervous system is unknown. In an established guinea pig model of low-dose, aerosolized Mycobacterium tuberculosis infection, we see behavior changes and memory loss in infected animals. We correlate these findings with pathological changes within brain regions related to motor, cognition, and sensation across disease progression. This includes microglial and astrocytic proliferation and reactivity. These cellular changes are followed by the aggregation of neurotoxic amyloid ß and phosphorylated tau and, ultimately, neuronal degeneration in the hippocampus. Through these data, we have obtained a greater understanding of the neuropathological effects of a peripheral disease that affects millions of persons worldwide.

Establishment of CD1b-restricted immunity to lipid antigens in the pulmonary response to Mycobacterium tuberculosis infection.

CD1 is an antigen presenting glycoprotein homologous to MHC I; however, CD1 proteins present lipid rather than peptide antigen. CD1 proteins are well established to present lipid antigens of Mycobacterium tuberculosis (Mtb) to T cells, but understanding the role of CD1-restricted immunity in vivo in response to Mtb infection has been limited by availability of animal models naturally expressing the CD1 proteins implicated in human response: CD1a, CD1b and CD1c. Guinea pigs, in contrast to other rodent models, express four CD1b orthologs, and here we utilize the guinea pig to establish the kinetics of gene and protein expression of CD1b orthologs, as well as the Mtb lipid-antigen and CD1b-restricted immune response at the tissue level over the course of Mtb infection. Our results indicate transient upregulation of CD1b expression during the effector phase of adaptive immunity that wanes with disease chronicity. Gene expression indicates that upregulation of CD1b is the result of transcriptional induction across all CD1b orthologs. We show high CD1b3 expression on B cells, and identify CD1b3 as the predominant CD1b ortholog in pulmonary granuloma lesions. We identify ex vivo cytotoxic activity directed against CD1b that closely paralleled the kinetic changes in CD1b expression in Mtb infected lung and spleen. This study confirms that CD1b expression is modulated by Mtb infection in lung and spleen, leading to pulmonary and extrapulmonary CD1b-restricted immunity as a component of the antigen-specific response to Mtb infection.

Metabolomic signatures in guinea pigs infected with epidemic-associated W-Beijing strains of Mycobacterium tuberculosis.

With the understanding that the laboratory propagated strain of Mycobacterium tuberculosis H37Rv is of modest virulence and is drug susceptible, in the present study, we performed a nuclear magnetic resonance-based metabolomic analysis of lung tissues and serum obtained from guinea pigs infected by low dose aerosol exposure to clinical isolates of Mycobacterium tuberculosis. High Resolution Magic Angle Spinning NMR coupled with multivariate statistical analysis of 159 lung tissues obtained from multiple locations of age-matched naïve and 30 and 60 days of infected guinea pig lungs revealed a wide dispersal of metabolic patterns, but within these, distinct clusters of signatures could be seen that differentiated between naive control and infected animals. Several metabolites were identified that changed in concert with the progression of each infection. Major metabolites that could be interpreted as indicating host glutaminolysis were consistent with activated host immune cells encountering increasingly hypoxic conditions in the necrotic lung lesions. Moreover, glutathione levels were constantly elevated, probably in response to oxygen radical production in these lesions. Additional distinct signatures were also seen in infected serum, with altered levels of several metabolites. Multivariate statistical analysis clearly differentiated the infected from the uninfected sera; in addition, Receiver Operator Characteristic curve generated with principal component 1 scores showed an area under the curve of 0.908. These data raise optimism that discrete metabolomic signatures can be defined that can predict the progression of the tuberculosis disease process, and form the basis of an innovative and rapid diagnostic process.

Evaluation of a mouse model of necrotic granuloma formation using C3HeB/FeJ mice for testing of drugs against Mycobacterium tuberculosis.

Persistence of Mycobacterium tuberculosis remains a significant challenge for the effective treatment of tuberculosis in humans. In animals that develop necrotic lung lesions following infection with M. tuberculosis, drug-tolerant bacilli are present and persist in an extracellular microenvironment within the necrotic cores. In this study, we examined the efficacy of drug treatment in C3HeB/FeJ (Kramnik) mice that develop lesions with liquefactive necrosis, in comparison to BALB/c mice that develop nonnecrotic lesions following aerosol challenge. To accomplish this, Kramnik and BALB/c mice were infected by aerosol with M. tuberculosis and treated for 7 to 8 weeks with monotherapy using drugs with different modes of action. The efficacy of drug therapy was quantified by enumeration of bacterial load. The progression of disease and location and distribution of bacilli within lesions were visualized using various staining techniques. In the late stages of infection, Kramnik mice developed fibrous encapsulated lung lesions with central liquefactive necrosis containing abundant extracellular bacilli, whereas BALB/c mice formed nonnecrotic lesions with primarily intracellular bacilli. Necrotic lesions in Kramnik mice showed evidence of hypoxia by pimonidazole staining. Kramnik mice were significantly more refractory to drug therapy, especially for pyrazinamide. Metronidazole showed no bactericidal activity in either model. There were significantly higher numbers of drug-resistant colonies isolated from the Kramnik mice compared to BALB/c mice. These results suggest that the Kramnik mouse model will be a valuable model to test antituberculosis drugs, especially against bacilli that persist within necrotic lesions.

Defined tuberculosis vaccine, Mtb72F/AS02A, evidence of protection in cynomolgus monkeys.

The development of a vaccine for tuberculosis requires a combination of antigens and adjuvants capable of inducing appropriate and long-lasting T cell immunity. We evaluated Mtb72F formulated in AS02A in the cynomolgus monkey model. The vaccine was immunogenic and caused no adverse reactions. When monkeys were immunized with bacillus Calmette-Guérin (BCG) and then boosted with Mtb72F in AS02A, protection superior to that afforded by using BCG alone was achieved, as measured by clinical parameters, pathology, and survival. We observed long-term survival and evidence of reversal of disease progression in monkeys immunized with the prime-boost regimen. Antigen-specific responses from protected monkeys receiving BCG and Mtb72F/AS02A had a distinctive cytokine profile characterized by an increased ratio between 3 Th1 cytokines, IFN-gamma, TNF, and IL-2 and an innate cytokine, IL-6. To our knowledge, this is an initial report of a vaccine capable of inducing long-term protection against tuberculosis in a nonhuman primate model, as determined by protection against severe disease and death, and by other clinical and histopathological parameters.