Pathogenesis of diabetes-tuberculosis comorbidity, The

2014 Summer.Exposure to the bacterium, Mycobacterium tuberculosis, only leads to the active form of tuberculosis disease (TB) in 5-10% of infected individuals. The development of active TB, at any stage of infection, is often the result of a known TB risk factor, either intrinsic to the individual or acquired as a communicable or non-communicable disease. An association between diabetes and TB has long been recognized, but only recently was diabetes confirmed to increase the risk of developing active TB disease. The convergence of a growing diabetes epidemic on regions with endemic TB has positioned diabetes as an emerging global threat to TB control. Of particular importance is the rapidly growing incidence of type 2 diabetes, which accounts for up to 95% of the global diabetic population. Since the potential impact of this growing comorbidity has only been recently emphasized, little is known regarding the mechanisms of dysregulated immune function and metabolism by which diabetes predisposes to active TB disease. The current understanding of this comorbidity is further limited by the lack of appropriate animal models that replicate the pathogenesis of both human type 2 diabetes and TB. The guinea pig is a well-established model of TB that replicates human pathology and disease progression. This species was emphasized in this series of studies with the goal of better understanding the impact of type 2 diabetes on TB progression and the mechanisms that may change the host response to M. tuberculosis infection. In Chapter 2, we investigated the impact of hyperglycemia alone, induced as post-prandial hyperglycemia through daily administration of sucrose, on TB disease progression in non-diabetic guinea pigs. Guinea pigs receiving daily sucrose developed both higher bacterial burdens in pulmonary and extrapulmonary tissue and also more severe pathology by day 60 of infection. This exacerbated disease manifestation was accompanied by the accumulation of advanced glycation end-products, which are inflammatory by-products of chronic hyperglycemia with known involvement in the development of diabetes-related complications. Interestingly, by monitoring glucose and lipid metabolism in these guinea pigs, we learned that TB alone leads to severe metabolic disturbances, manifesting as hyperglycemia and accumulation of circulating total free fatty acids. From this study, we were able to conclude that not only does mild post-prandial hyperglycemia worsen the course of TB disease in guinea pigs, but also, infection with M. tuberculosis alone induces metabolic disease resembling diabetes, similar to what has been previously reported in human TB. These conclusions rationalize the investigation of novel adjunctive therapies to restore metabolic homeostasis, which may improve the host response to infection, limit bacterial growth, and increase the efficacy of frontline antimicrobial drugs. In Chapter 3, we developed a novel model of type 2 diabetes in the guinea pig to be used in future investigations of type 2 diabetes-TB comorbidity. Previously, the guinea pig as a diabetic model has been described only in the context of β-cell cytotoxicity with the drug, streptozotocin (STZ), but with variable efficacy. In this study, we initially optimized the dose response and STZ preparation to achieve an induction of hyperglycemia that was uniform with limited mortality. This hyperglycemic response was transient but could be stabilized through continued β-cell stress, in the form of a high fat, high sugar diet. Feeding of this modified diet led to impaired glucose tolerance and a compensatory β-cell response that could be abrogated with the use of a single optimized dose of STZ. This novel model of type 2 diabetes develops both insulin resistance and β-cell failure, which replicate the typical progression of type 2 diabetes in humans, all within a reasonable experimental timeframe. From this study, two models emerged, a type 2 diabetic guinea pig as well as a model of impaired glucose tolerance, or prediabetes, that would be used to investigate the mechanisms of diabetes-TB comorbidity. In Chapter 4, the newly developed guinea pig models were used to investigate the overall impact of type 2 diabetes and impaired glucose tolerance on TB progression and the host immune response to M. tuberculosis infection. Although impaired glucose tolerance alone had limited impact on TB progression with exacerbation of disease only at chronic end points, M. tuberculosis infected type 2 diabetic guinea pigs closely resembled the reported manifestations of human diabetes-TB comorbidity including more severe TB disease, higher bacterial burdens, and a robust innate and cell-mediated immune response. Despite evidence of strong Th1 cell-mediated immunity, which is known to be critical for limiting bacterial growth and disease progression, diabetic guinea pigs were unable to control bacterial growth and developed damaging neutrophilic inflammation. To better understand the immune mechanisms leading to uncontrolled bacterial growth and severe disease, in Chapter 5, we investigated the innate and adaptive immune response over the course of early infection in type 2 diabetic guinea pigs. Diabetic guinea pigs were slow to develop early lesions with delayed bacterial transport to the lung draining lymph node, and a corresponding delay in antigen-specific Th1 immunity. Early alterations in cytokine expression were identified that may explain the delayed development of cell-mediated immunity and allow for substantial growth of M. tuberculosis in the lung of infected diabetic guinea pigs. These data indicate that not only does type 2 diabetes increase the severity of TB but also that the chronic inflammatory process associated with TB itself may worsen diabetes. This has important implications worthy of further investigation revolving around the diagnostic criteria for diabetes when associated with TB, the impact of active TB on medical management of diabetes, and the investigation of novel therapeutic targets, both metabolic and immunological, to enhance the host immune response to infection and limit TB disease severity in diabetics

High-resolution mapping of fluoroquinolones in TB rabbit lesions reveals specific distribution in immune cell types.

Understanding the distribution patterns of antibiotics at the site of infection is paramount to selecting adequate drug regimens and developing new antibiotics. Tuberculosis (TB) lung lesions are made of various immune cell types, some of which harbor persistent forms of the pathogen, Mycobacterium tuberculosis. By combining high resolution MALDI MSI with histology staining and quantitative image analysis in rabbits with active TB, we have mapped the distribution of a fluoroquinolone at high resolution, and identified the immune-pathological factors driving its heterogeneous penetration within TB lesions, in relation to where bacteria reside. We find that macrophage content, distance from lesion border and extent of necrosis drive the uneven fluoroquinolone penetration. Preferential uptake in macrophages and foamy macrophages, where persistent bacilli reside, compared to other immune cells present in TB granulomas, was recapitulated in vitro using primary human cells. A nonlinear modeling approach was developed to help predict the observed drug behavior in TB lesions. This work constitutes a methodological advance for the co-localization of drugs and infectious agents at high spatial resolution in diseased tissues, which can be applied to other diseases with complex immunopathology

A model of type 2 diabetes in the guinea pig using sequential diet-induced glucose intolerance and streptozotocin treatment

Type 2 diabetes is a leading cause of morbidity and mortality among noncommunicable diseases, and additional animal models that more closely replicate the pathogenesis of human type 2 diabetes are needed. The goal of this study was to develop a model of type 2 diabetes in guinea pigs, in which diet-induced glucose intolerance precedes β-cell cytotoxicity, two processes that are crucial to the development of human type 2 diabetes. Guinea pigs developed impaired glucose tolerance after 8 weeks of feeding on a high-fat, high-carbohydrate diet, as determined by oral glucose challenge. Diet-induced glucose intolerance was accompanied by β-cell hyperplasia, compensatory hyperinsulinemia, and dyslipidemia with hepatocellular steatosis. Streptozotocin (STZ) treatment alone was ineffective at inducing diabetic hyperglycemia in guinea pigs, which failed to develop sustained glucose intolerance or fasting hyperglycemia and returned to euglycemia within 21 days after treatment. However, when high-fat, high-carbohydrate diet-fed guinea pigs were treated with STZ, glucose intolerance and fasting hyperglycemia persisted beyond 21 days post-STZ treatment. Guinea pigs with diet-induced glucose intolerance subsequently treated with STZ demonstrated an insulin-secretory capacity consistent with insulin-independent diabetes. This insulin-independent state was confirmed by response to oral antihyperglycemic drugs, metformin and glipizide, which resolved glucose intolerance and extended survival compared with guinea pigs with uncontrolled diabetes. In this study, we have developed a model of sequential glucose intolerance and β-cell loss, through high-fat, high-carbohydrate diet and extensive optimization of STZ treatment in the guinea pig, which closely resembles human type 2 diabetes. This model will prove useful in the study of insulin-independent diabetes pathogenesis with or without comorbidities, where the guinea pig serves as a relevant model species

High-resolution mapping of fluoroquinolones in TB rabbit lesions reveals specific distribution in immune cell types.

Understanding the distribution patterns of antibiotics at the site of infection is paramount to selecting adequate drug regimens and developing new antibiotics. Tuberculosis (TB) lung lesions are made of various immune cell types, some of which harbor persistent forms of the pathogen, Mycobacterium tuberculosis. By combining high resolution MALDI MSI with histology staining and quantitative image analysis in rabbits with active TB, we have mapped the distribution of a fluoroquinolone at high resolution, and identified the immune-pathological factors driving its heterogeneous penetration within TB lesions, in relation to where bacteria reside. We find that macrophage content, distance from lesion border and extent of necrosis drive the uneven fluoroquinolone penetration. Preferential uptake in macrophages and foamy macrophages, where persistent bacilli reside, compared to other immune cells present in TB granulomas, was recapitulated in vitro using primary human cells. A nonlinear modeling approach was developed to help predict the observed drug behavior in TB lesions. This work constitutes a methodological advance for the co-localization of drugs and infectious agents at high spatial resolution in diseased tissues, which can be applied to other diseases with complex immunopathology

Standardized guinea pig model for Q fever vaccine reactogenicity.

Historically, vaccination with Coxiella burnetii whole cell vaccines has induced hypersensitivity reactions in humans and animals that have had prior exposure to the pathogen as a result of infection or vaccination. Intradermal skin testing is routinely used to evaluate exposure in humans, and guinea pig hypersensitivity models have been developed to characterize the potential for reactogenicity in vaccine candidates. Here we describe a refinement of the guinea pig model using an alternate vaccine for positive controls. An initial comparative study used viable C. burnetii to compare the routes of sensitizing exposure of guinea pigs (intranasal vs intraperitoneal), evaluation of two time points for antigen challenge (21 and 42 days) and an assessment of two routes (intradermal and subcutaneous) of challenge using the ruminant vaccine Coxevac as the antigenic control. Animals sensitized by intraperitoneal exposure exhibited slightly larger gross reactions than did those sensitized by intranasal exposure, and reactions were more pronounced when skin challenge was performed at 42 days compared to 21 days post-sensitization. The intradermal route proved to be the optimal route of reactogenicity challenge. Histopathological changes at injection sites were similar to those previously reported and a scoring system was developed to compare reactions between groups receiving vaccine by intradermal versus subcutaneous routes. Based on the comparative study, a standardized protocol for assessment of vaccine reactogenicity in intranasally-sensitized animals was tested in a larger confirmatory study. Results suggest that screens utilizing a group size of n = 3 would achieve 90% power for detecting exposure-related reactogenic responses of the magnitude induced by Coxevac using either of two outcome measures

The Efficacy of the BCG Vaccine against Newly Emerging Clinical Strains of Mycobacterium tuberculosis.

To date, most new vaccines against Mycobacterium tuberculosis, including new recombinant versions of the current BCG vaccine, have usually been screened against the laboratory strains H37Rv or Erdman. In this study we took advantage of our recent work in characterizing an increasingly large panel of newly emerging clinical isolates [from the United States or from the Western Cape region of South Africa], to determine to what extent vaccines would protect against these [mostly high virulence] strains. We show here that both BCG Pasteur and recombinant BCG Aeras-422 [used here as a good example of the new generation BCG vaccines] protected well in both mouse and guinea pig low dose aerosol infection models against the majority of clinical isolates tested. However, Aeras-422 was not effective in a long term survival assay compared to BCG Pasteur. Protection was very strongly expressed against all of the Western Cape strains tested, reinforcing our viewpoint that any attempt at boosting BCG would be very difficult to achieve statistically. This observation is discussed in the context of the growing argument made by others that the failure of a recent vaccine trial disqualifies the further use of animal models to predict vaccine efficacy. This viewpoint is in our opinion completely erroneous, and that it is the fitness of prevalent strains in the trial site area that is the centrally important factor, an issue that is not being addressed by the field