The Effect of Tropical Temperatures on the Quality of RNA Extracted from Stabilized Whole-Blood Samples.

Whole-blood-derived transcriptional profiling is widely used in biomarker discovery, immunological research, and therapeutic development. Traditional molecular and high-throughput transcriptomic platforms, including molecular assays with quantitative PCR (qPCR) and RNA-sequencing (RNA-seq), are dependent upon high-quality and intact RNA. However, collecting high-quality RNA from field studies in remote tropical locations can be challenging due to resource restrictions and logistics of post-collection processing. The current study tested the relative performance of the two most widely used whole-blood RNA collection systems, PAXgene® and Tempus™, in optimal laboratory conditions as well as suboptimal conditions in tropical field sites, including the effects of extended storage times and high storage temperatures. We found that Tempus™ tubes maintained a slightly higher RNA quantity and integrity relative to PAXgene® tubes at suboptimal tropical conditions. Both PAXgene® and Tempus™ tubes gave similar RNA purity (A260/A280). Additionally, Tempus™ tubes preferentially maintained the stability of mRNA transcripts for two reference genes tested, Succinate dehydrogenase complex, subunit A (SDHA) and TATA-box-binding protein (TBP), even when RNA quality decreased with storage length and temperature. Both tube types preserved the rRNA transcript 18S ribosomal RNA (18S) equally. Our results suggest that Tempus™ blood RNA collection tubes are preferable to PAXgene® for whole-blood collection in suboptimal tropical conditions for RNA-based studies in resource-limited settings.

Identification of defective early immune responses to Burkholderia pseudomallei infection in a diet-induced murine model of type 2 diabetes.

Co-occurrence of bacterial infections with type 2 diabetes (T2D) is a global problem. Melioidosis caused by Burkholderia pseudomallei is 10 times more likely to occur in patients with T2D, than in normoglycemic individuals. Using an experimental model of T2D, we observed that greater susceptibility in T2D was due to differences in proportions of infiltrating leucocytes and reduced levels of MCP-1, IFN-γ and IL-12 at sites of infection within 24 h post-infection. However, by 72 h the levels of inflammatory cytokines and bacteria were markedly higher in visceral tissue and blood in T2D mice. In T2D, dysregulated early immune responses are responsible for the greater predisposition to B. pseudomallei infection.

Disparate Effects of Metformin on Mycobacterium tuberculosis Infection in Diabetic and Nondiabetic Mice.

Comorbid type 2 diabetes poses a great challenge to the global control of tuberculosis. Here, we assessed the efficacy of metformin (MET), an antidiabetic drug, in mice infected with a very low dose of Mycobacterium tuberculosis In contrast to diabetic mice, infected nondiabetic mice that received the same therapeutic concentration of MET presented with significantly higher disease burden. This warrants further studies to investigate the disparate efficacy of MET against tuberculosis in diabetic and nondiabetic individuals.

Mucosal delivery of ESX-1-expressing BCG strains provides superior immunity against tuberculosis in murine type 2 diabetes.

Tuberculosis (TB) claims 1.5 million lives per year. This situation is largely due to the low efficacy of the only licensed TB vaccine, Bacillus Calmette-Guérin (BCG) against pulmonary TB. The metabolic disease type 2 diabetes (T2D) is a risk factor for TB and the mechanisms underlying increased TB susceptibility in T2D are not well understood. Furthermore, it is unknown if new TB vaccines will provide protection in the context of T2D. Here we used a diet-induced murine model of T2D to investigate the underlying mechanisms of TB/T2D comorbidity and to evaluate the protective capacity of two experimental TB vaccines in comparison to conventional BCG. Our data reveal a distinct immune dysfunction that is associated with diminished recognition of mycobacterial antigens in T2D. More importantly, we provide compelling evidence that mucosal delivery of recombinant BCG strains expressing the Mycobacterium tuberculosis (Mtb) ESX-1 secretion system (BCG::RD1 and BCG::RD1 ESAT-6 ∆92-95) are safe and confer superior immunity against aerosol Mtb infection in the context of T2D. Our findings suggest that the remarkable anti-TB immunity by these recombinant BCG strains is achieved via augmenting the numbers and functional capacity of antigen presenting cells in the lungs of diabetic mice.

A systematic approach to simultaneously evaluate safety, immunogenicity, and efficacy of novel tuberculosis vaccination strategies.

Tuberculosis (TB) is the deadliest infectious disease worldwide. Bacille-Calmette-Guérin (BCG), the only licensed TB vaccine, affords variable protection against TB but remains the gold standard. BCG improvement is focused around three strategies: recombinant BCG strains, heterologous routes of administration, and booster vaccination. It is currently unknown whether combining these strategies is beneficial. The preclinical evaluation for new TB vaccines is heavily skewed toward immunogenicity and efficacy; however, safety and efficacy are the dominant considerations in human use. To facilitate stage gating of TB vaccines, we developed a simple empirical model to systematically rank vaccination strategies by integrating multiple measurements of safety, immunogenicity, and efficacy. We assessed 24 vaccination regimens, composed of three BCG strains and eight combinations of delivery. The model presented here highlights that mucosal booster vaccination may cause adverse outcomes and provides a much needed strategy to evaluate and rank data obtained from TB vaccine studies using different routes, strains, or animal models.

Anti-streptococcal antibody and T-cell interactions with vascular endothelial cells initiate the development of rheumatic carditis.

The role of group A streptococcal and Streptococcus dysgalactiae subspecies equisimilis M-protein specific Abs and T-cells in endothelial cell activation was investigated using cultured rat aortic endothelial cells, and in a rat model of autoimmune valvulitis. Heat inactivated serum and mononuclear cells from streptococcal M-protein immunized rats independently induced upregulation of the endothelial cell adhesion molecules, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 in cultured cells. We also observed T-cell migration across endothelial cell monolayers incubated with serum from M-protein-immunized rats. Furthermore, we observed VCAM-1 and ICAM-1 expression in the myocardium of rats injected with M-protein compared to control animals. These observations support the contention that initial interactions between streptococcal M-protein specific Abs and/or T-cells with the heart endothelium lead to endothelial cell activation followed by transmigration of M-protein specific T-cells into heart tissue leading to an inflammatory process that leads to carditis in rheumatic fever and rheumatic heart disease.

Dysregulation of key cytokines may contribute to increased susceptibility of diabetic mice to Mycobacterium bovis BCG infection.

Diabetes is one of the major co-morbidities contributing to the high global burden of tuberculosis (TB). The increased susceptibility of individuals with type 2 diabetes (T2D) to TB is multifactorial and may influence the efficacy of vaccines. This study was undertaken to determine the early immune responses that occur following infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG) in a diet-induced murine model of T2D. The phagocytic capabilities of alveolar (AM) and resident peritoneal macrophages (RPM) were assessed using ex vivo assays. Compared to macrophages from non-diabetic mice, macrophages from diabetic animals showed decreased BCG uptake and killing and inflammatory cytokine production (TNF-α, MCP-1, IL-6, IL-1ß). In vivo susceptibility to BCG was determined following intravenous infection and diabetic mice showed a trend towards increased mortality, higher bacterial burden in the lung, liver and spleen and increased inflammatory lesions compared to controls. Differences between tissue cytokines were observed as early as one day post-infection and by days 14 and 35, lung and liver TNF-α and IFN-γ levels were decreased in diabetic mice compared to controls. These results suggest that early dysregulated immune responses may influence the susceptibility of T2D mice to BCG infection.

Group G Streptococcus Induces an Autoimmune Carditis Mediated by Interleukin 17A and Interferon γ in the Lewis Rat Model of Rheumatic Heart Disease.

Acute rheumatic fever and rheumatic heart disease (ARF/RHD) have long been described as autoimmune sequelae of Streptococcus pyogenes or group A streptococcal (GAS) infection. Both antibody and T-cell responses against immunodominant GAS virulence factors, including M protein, cross-react with host tissue proteins, triggering an inflammatory response leading to permanent heart damage. However, in some ARF/RHD-endemic regions, throat carriage of GAS is low. Because Streptococcus dysgalactiae subspecies equisimilis organisms, also known as ß-hemolytic group C streptococci and group G streptococci (GGS), also express M protein, we postulated that streptococci other than GAS may have the potential to initiate or exacerbate ARF/RHD. Using a model initially developed to investigate the uniquely human disease of ARF/RHD, we have discovered that GGS causes interleukin 17A/interferon γ-induced myocarditis and valvulitis, hallmarks of ARF/RHD. Remarkably the histological, immunological, and functional changes in the hearts of rats exposed to GGS are identical to those exposed to GAS. Furthermore, antibody cross-reactivity to cardiac myosin was comparable in both GGS- and GAS-exposed animals, providing additional evidence that GGS can induce and/or exacerbate ARF/RHD.

Increased Neurotropic Threat from Burkholderia pseudomallei Strains with a B. mallei-like Variation in the bimA Motility Gene, Australia.

Neurologic melioidosis is a serious, potentially fatal form of Burkholderia pseudomallei infection. Recently, we reported that a subset of clinical isolates of B. pseudomallei from Australia have heightened virulence and potential for dissemination to the central nervous system. In this study, we demonstrate that this subset has a B. mallei-like sequence variation of the actin-based motility gene, bimA. Compared with B. pseudomallei isolates having typical bimA alleles, isolates that contain the B. mallei-like variation demonstrate increased persistence in phagocytic cells and increased virulence with rapid systemic dissemination and replication within multiple tissues, including the brain and spinal cord, in an experimental model. These findings highlight the implications of bimA variation on disease progression of B. pseudomallei infection and have considerable clinical and public health implications with respect to the degree of neurotropic threat posed to human health.

Anti-mycobacterial function of macrophages is impaired in a diet induced model of type 2 diabetes.

Type 2 diabetes (T2D) is one of the major risk factors for tuberculosis (TB). In this study, a diet induced murine model of T2D (DIMT2D) was developed and characterized in the context of metabolic, biochemical and histopathological features following diet intervention. Mycobacterial susceptibility was investigated using Mycobacterium fortuitum as a surrogate. Phagocytic capability of alveolar macrophages and resident peritoneal macrophages were determined by in vitro assays using mycolic acid coated beads and M. fortuitum. Results demonstrated that bacillary loads were significantly higher in liver, spleen, and lungs of diabetic mice compared to controls. Higher inflammatory lesions and impaired cytokine kinetics (TNF-α, MCP-1, IL-12, IFN-γ) were also observed in diabetic mice. Macrophages isolated from diabetic mice had lower uptake of mycolic acid coated beads, reduced bacterial internalization and killing and altered cytokine responses (TNF-α, IL-6, MCP-1). This model will be useful to further investigate different facets of host-pathogen interactions in TB-T2D.

Wnt Signaling Pathway Inhibitor Sclerostin Inhibits Angiotensin II-Induced Aortic Aneurysm and Atherosclerosis.

OBJECTIVE: Sclerostin (SOST) has been identified as an important regulator of bone formation; however, it has not been previously implicated in arterial disease. The aim of this study was to assess the role of SOST in aortic aneurysm (AA) and atherosclerosis using human samples, a mouse model, and in vitro investigations. APPROACH AND RESULTS: SOST protein was downregulated in human and mouse AA samples compared with controls. Transgenic introduction of human SOST in apolipoprotein E-deficient (ApoE-/-) mice (SOSTTg .ApoE-/-) and administration of recombinant mouse Sost inhibited angiotensin II-induced AA and atherosclerosis. Serum concentrations of several proinflammatory cytokines were significantly reduced in SOSTTg .ApoE-/- mice. Compared with controls, the aortas of mice receiving recombinant mouse Sost and SOSTTg .ApoE-/- mice showed reduced matrix degradation, reduced elastin breaks, and preserved collagen. Decreased inflammatory cell infiltration and a reduction in the expression of wingless-type mouse mammary virus integration site/ß-catenin responsive genes, including matrix metalloproteinase-9, osteoprotegerin, and osteopontin, were observed in the aortas of SOSTTg .ApoE-/- mice. SOST expression was downregulated and the wingless-type mouse mammary virus integration site/ß-catenin pathway was activated in human AA samples. The cytosine-phosphate-guanine islands in the SOST gene promoter showed significantly higher methylation in human AA samples compared with controls. Incubation of vascular smooth muscle cells with the demethylating agent 5-azacytidine resulted in upregulation of SOST, suggesting that SOST is epigenetically regulated. CONCLUSIONS: This study identifies that SOST is expressed in the aorta and downregulated in human AA possibly because of epigenetic silencing. Upregulating SOST inhibits AA and atherosclerosis development, with potential important implications for treating these vascular diseases.

Angiopoietin-2 attenuates angiotensin II-induced aortic aneurysm and atherosclerosis in apolipoprotein E-deficient mice.

Angiogenesis and inflammation are implicated in aortic aneurysm and atherosclerosis and regulated by angiopoietin-2 (Angpt2). The effect of Angpt2 administration on experimental aortic aneurysm and atherosclerosis was examined. Six-month-old male apolipoprotein E deficient (ApoE-/-) mice were infused with angiotensin II (AngII) and administered subcutaneous human Fc-protein (control) or recombinant Angpt2 (rAngpt2) over 14 days. Administration of rAngpt2 significantly inhibited AngII-induced aortic dilatation and rupture of the suprarenal aorta (SRA), and development of atherosclerosis within the aortic arch. These effects were blood pressure and plasma lipoprotein independent and associated with Tie2 activation and down-regulation of monocyte chemotactic protein-1 (MCP-1) within the SRA. Plasma concentrations of MCP-1 and interleukin-6 were significantly lower in mice receiving rAngpt2. Immunostaining for the monocyte/macrophage marker MOMA-2 and the angiogenesis marker CD31 within the SRA were less in mice receiving rAngpt2 than controls. The percentage of inflammatory (Ly6Chi) monocytes within the bone marrow was increased while that in peripheral blood was decreased by rAngpt2 administration. In conclusion, administration of rAngpt2 attenuated angiotensin II-induced aortic aneurysm and atherosclerosis in ApoE-/- mice associated with reduced aortic inflammation and angiogenesis. Up-regulation of Angpt2 may have potential therapeutic value in patients with aortic aneurysm and atherosclerosis.

Repeat exposure to group A streptococcal M protein exacerbates cardiac damage in a rat model of rheumatic heart disease.

Rheumatic fever and rheumatic heart disease (RF/RHD) develop following repeated infection with group A streptococci (GAS). We used the Rat Autoimmune Valvulitis (RAV) model of RF/RHD to demonstrate that repetitive booster immunization with GAS-derived recombinant M protein (rM5) resulted in an enhanced anti-cardiac myosin antibody response that may contribute to the breaking of immune tolerance leading to RF/RHD and increased infiltration of heart valves by mononuclear cells. With each boost, more inflammatory cells were observed infiltrating heart tissue which could lead to severe cardiac damage. We also found evidence that both complement and anti-M protein antibodies in serum from rM5-immunized rats have the potential to contribute to inflammation in heart valves by activating cardiac endothelium. More importantly, we have demonstrated by electrocardiography for the first time in the RAV model that elongation of P-R interval follows repetitive boost with rM5. Our observations provide experimental evidence for cardiac alterations following repeated exposure to GAS M protein with immunological and electrophysiological features resembling that seen in humans following recurrent GAS infection.

Preclinical immunogenicity and safety of a Group A streptococcal M protein-based vaccine candidate.

Streptococcus pyogenes (group A streptococcus, GAS) causes a wide range of clinical manifestations ranging from mild self-limiting pyoderma to invasive diseases such as sepsis. Also of concern are the post-infectious immune-mediated diseases including rheumatic heart disease. The development of a vaccine against GAS would have a large health impact on populations at risk of these diseases. However, there is a lack of suitable models for the safety evaluation of vaccines with respect to post-infectious complications. We have utilized the Lewis Rat model for cardiac valvulitis to evaluate the safety of the J8-DT vaccine formulation in parallel with a rabbit toxicology study. These studies demonstrated that the vaccine did not induce abnormal pathology. We also show that in mice the vaccine is highly immunogenic but that 3 doses are required to induce protection from a GAS skin challenge even though 2 doses are sufficient to induce a high antibody titer.

Development of a diet-induced murine model of diabetes featuring cardinal metabolic and pathophysiological abnormalities of type 2 diabetes.

The persistent rise in global incidence of type 2 diabetes (T2D) continues to have significant public health and economic implications. The availability of relevant animal models of T2D is critical to elucidating the complexity of the pathogenic mechanisms underlying this disease and the implications this has on susceptibility to T2D complications. Whilst many high-fat diet-induced rodent models of obesity and diabetes exist, growing appreciation of the contribution of high glycaemic index diets on the development of hyperglycaemia and insulin resistance highlight the requirement for animal models that more closely represent global dietary patterns reflective of modern society. To that end, we sought to develop and validate a murine model of T2D based on consumption of an energy-dense diet containing moderate levels of fat and a high glycaemic index to better reflect the aetiopathogenesis of T2D. Male C57BL/6 mice were fed an energy-dense (ED) diet and the development of pathological features used in the clinical diagnosis of T2D was assessed over a 30-week period. Compared with control mice, 87% of mice fed an ED diet developed pathognomonic signs of T2D including glucose intolerance, hyperglycaemia, glycosylated haemoglobin (HbA1c) and glycosuria within 30 weeks. Furthermore, dyslipidaemia, chronic inflammation, alterations in circulating leucocytes and renal impairment were also evident in ED diet-fed mice compared with mice receiving standard rodent chow. Longitudinal profiling of metabolic and biochemical parameters provide support of an aetiologically and clinically relevant model of T2D that will serve as a valuable tool for mechanistic and therapeutic studies investigating the pathogenic complications of T2D.

Predicted Coverage and Immuno-Safety of a Recombinant C-Repeat Region Based Streptococcus pyogenes Vaccine Candidate.

The C-terminal region of the M-protein of Streptococcus pyogenes is a major target for vaccine development. The major feature is the C-repeat region, consisting of 35-42 amino acid repeat units that display high but not perfect identity. SV1 is a S. pyogenes vaccine candidate that incorporates five 14mer amino acid sequences (called J14i variants) from differing C-repeat units in a single recombinant construct. Here we show that the J14i variants chosen for inclusion in SV1 are the most common variants in a dataset of 176 unique M-proteins. Murine antibodies raised against SV1 were shown to bind to each of the J14i variants present in SV1, as well as variants not present in the vaccine. Antibodies raised to the individual J14i variants were also shown to bind to multiple but different combinations of J14i variants, supporting the underlying rationale for the design of SV1. A Lewis Rat Model of valvulitis was then used to assess the capacity of SV1 to induce deleterious immune response associated with rheumatic heart disease. In this model, both SV1 and the M5 positive control protein were immunogenic. Neither of these antibodies were cross-reactive with cardiac myosin or collagen. Splenic T cells from SV1/CFA and SV1/alum immunized rats did not proliferate in response to cardiac myosin or collagen. Subsequent histological examination of heart tissue showed that 4 of 5 mice from the M5/CFA group had valvulitis and inflammatory cell infiltration into valvular tissue, whereas mice immunised with SV1/CFA, SV1/alum showed no sign of valvulitis. These results suggest that SV1 is a safe vaccine candidate that will elicit antibodies that recognise the vast majority of circulating GAS M-types.

Modulation of Kinin B2 Receptor Signaling Controls Aortic Dilatation and Rupture in the Angiotensin II-Infused Apolipoprotein E-Deficient Mouse.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. Activity of the local kallikrein-kinin system may be important in cardiovascular disease. The effect of kinin B2 receptor (B2R) agonist and antagonist peptides on experimental AAA was investigated. APPROACH AND RESULTS: AAA was induced in apolipoprotein E-deficient mice via infusion of angiotensin II (1.0 µg/kg per minute SC). B2R agonists or antagonists were given via injection (2 mg/kg IP) every other day. The B2R agonist (B9772) promoted aortic rupture in response to angiotensin II associated with an increase in neutrophil infiltration of the aorta in comparison to controls. Mice receiving a B2R/kinin B1 receptor antagonist (B9430) were relatively protected from aortic rupture. Neutrophil depletion abrogated the ability of the B2R agonist to promote aortic rupture. Progression of angiotensin II-induced aortic dilatation was inhibited in mice receiving a B2R antagonist (B9330). Secretion of metalloproteinase-2 and -9, osteoprotegerin, and osteopontin by human AAA explant was reduced in the presence of the B2R antagonist (B9330). B2R agonist and antagonist peptides enhanced and inhibited, respectively, angiotensin II-induced neutrophil activation and aortic smooth muscle cell inflammatory phenotype. The B2R antagonist (B9330; 5 µg) delivered directly to the aortic wall 1 week post-AAA induction with calcium phosphate in a rat model reduced aneurysm growth associated with downregulation of aortic metalloproteinase-9. CONCLUSIONS: B2R signaling promotes aortic rupture within a mouse model associated with the ability to stimulate inflammatory phenotypes of neutrophils and vascular smooth muscle cells. B2R antagonism could be a potential therapy for AAA.

Plasmacytoid dendritic cell bactericidal activity against Burkholderia pseudomallei.

Melioidosis sepsis, caused by Burkholderia pseudomallei, is associated with high mortality due to an overwhelming inflammatory response. Plasmacytoid dendritic cells (pDC) are potent producers of type I interferons (IFN). This study investigated whether pDC and type I IFN play a role during the early stages of B. pseudomallei infection. Human and murine pDC internalised and killed B. pseudomallei as efficiently as murine conventional DC (cDC). pDC derived from B. pseudomallei-susceptible (BALB/c) mice demonstrated poor intracellular killing and increased IFN-alpha compared to pDC derived from B. pseudomallei-resistant (C57BL/6) mice. This is the first evidence of pDC bactericidal activity against B. pseudomallei infection.

Animal models to investigate the pathogenesis of rheumatic heart disease.

Rheumatic fever (RF) and rheumatic heart disease (RHD) are sequelae of group A streptococcal (GAS) infection. Although an autoimmune process has long been considered to be responsible for the initiation of RF/RHD, it is only in the last few decades that the mechanisms involved in the pathogenesis of the inflammatory condition have been unraveled partly due to experimentation on animal models. RF/RHD is a uniquely human condition and modeling this disease in animals is challenging. Antibody and T cell responses to recombinant GAS M protein (rM) and the subsequent interactions with cardiac tissue have been predominantly investigated using a rat autoimmune valvulitis model. In Lewis rats immunized with rM, the development of hallmark histological features akin to RF/RHD, both in the myocardial and in valvular tissue have been reported, with the generation of heart tissue cross-reactive antibodies and T cells. Recently, a Lewis rat model of Sydenham's chorea and related neuropsychiatric disorders has also been described. Rodent models are very useful for assessing disease mechanisms due to the availability of reagents to precisely determine sequential events following infection with GAS or post-challenge with specific proteins and or carbohydrate preparations from GAS. However, studies of cardiac function are more problematic in such models. In this review, a historical overview of animal models previously used and those that are currently available will be discussed in terms of their usefulness in modeling different aspects of the disease process. Ultimately, cardiologists, microbiologists, immunologists, and physiologists may have to resort to diverse models to investigate different aspects of RF/RHD.