Analysis by enzyme-linked immunosorbent assay and immunoblot of the antibody responses of patients infected with Mycobacterium marinum

The serum antibody responses of a total of 14 patients with active or recently cured Mycobacterium marinum infections were analysed via a combination of enzyme-linked immunosorbent assay (ELISA) and the immunodevelopment of Western blots of M. marinum antigen. Normal human sera and sera from patients with active pulmonary tuberculosis were also analysed as controls. The detectable IgG response of M. marinum patients, as demonstrated by ELISA, was highly variable and did not differ significantly from normal controls. IgA and IgM levels were generally low in the M. marinum patients and were not significantly different from normal controls. Immunodevelopment of Western blots of M. marinum antigen with the sera of patients with M. marinum infections revealed that a number of antigens were recognised. Of particular note was an 18-kDa species that was recognised by 11 out of 14 patients (and by none of the normal controls). The 18-kDa antigen may be a useful serodiagnostic marker in the identification of M. marinum infections.     

Impacts of climate, host and landscape factors on Culicoides species in Scotland

Culicoides biting midges (Diptera: Ceratopogonidae) vector a wide variety of internationally important arboviral pathogens of livestock and represent a widespread biting nuisance. This study investigated the influence of landscape, host and remotely-sensed climate factors on local abundance of livestock-associated species in Scotland, within a hierarchical generalized linear model framework. The Culicoides obsoletus group and the Culicoides pulicaris group accounted for 56% and 41%, respectively, of adult females trapped. Culicoides impunctatus Goetghebuer and C. pulicaris s.s. Linnaeus were the most abundant and widespread species in the C. pulicaris group (accounting for 29% and 10%, respectively, of females trapped). Abundance models performed well for C. impunctatus, Culicoides deltus Edwards and Culicoides punctatus Meigen (adjusted R(2) : 0.59-0.70), but not for C. pulicaris s.s. (adjusted R(2) : 0.36) and the C. obsoletus group (adjusted R(2) : 0.08). Local-scale abundance patterns were best explained by models combining host, landscape and climate factors. The abundance of C. impunctatus was negatively associated with cattle density, but positively associated with pasture cover, consistent with this species' preference in the larval stage for lightly grazed, wet rush pasture. Predicted abundances of this species varied widely among farms even over short distances (less than a few km). Modelling approaches that may facilitate the more accurate prediction of local abundance patterns for a wider range of Culicoides species are discussed.     

Cytokine Responses to Novel Antigens in an Indian Population Living in an Area Endemic for Visceral Leishmaniasis

Background

There are no effective vaccines for visceral leishmaniasis (VL), a neglected parasitic disease second only to malaria in global mortality. We previously identified 14 protective candidates in a screen of 100 Leishmania antigens as DNA vaccines in mice. Here we employ whole blood assays to evaluate human cytokine responses to 11 of these antigens, in comparison to known defined and crude antigen preparations.

Methods

Whole blood assays were employed to measure IFN-γ, TNF-α and IL-10 responses to peptide pools of the novel antigens R71, Q51, L37, N52, L302.06, J89, M18, J41, M22, M63, M57, as well as to recombinant proteins of tryparedoxin peroxidase (TRYP), Leishmania homolog of the receptor for activated C kinase (LACK) and to crude soluble Leishmania antigen (SLA), in Indian patients with active (n = 8) or cured (n = 16) VL, and in modified Quantiferon positive (EHC^+ve, n = 20) or modified Quantiferon negative (EHC^−ve, n = 9) endemic healthy controls (EHC).

Results

Active VL, cured VL and EHC^+ve groups showed elevated SLA-specific IFN-γ, but only active VL patients produced IL-10 and EHC^+ve did not make TNF-α. IFN-γ to IL-10 and TNF-α to IL-10 ratios in response to TRYP and LACK antigens were higher in cured VL and EHC^+ve exposed individuals compared to active VL. Five of the eleven novel candidates (R71, L37, N52, J41, and M22) elicited IFN-γ and TNF-α, but not IL-10, responses in cured VL (55–87.5% responders) and EHC^+ve (40–65% responders) subjects.

Conclusions

Our results are consistent with an important balance between pro-inflammatory IFNγ and TNFγ cytokine responses and anti-inflammatory IL-10 in determining outcome of VL in India, as highlighted by response to both crude and defined protein antigens. Importantly, cured VL patients and endemic Quantiferon positive individuals recognise 5 novel vaccine candidate antigens, confirming our recent data for L. chagasi in Brazil, and their potential as cross-species vaccine candidates.     

Emerging evidence for endoplasmic reticulum stress in the pathogenesis of idiopathic pulmonary fibrosis

While the factors that regulate the onset and progression of idiopathic pulmonary fibrosis (IPF) are incompletely understood, recent investigations have revealed that endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are prominent in alveolar epithelial cells in this disease. Initial observations linking ER stress and IPF were made in cases of familial interstitial pneumonia (FIP), the familial form of IPF, in a family with a mutation in surfactant protein C (SFTPC). Subsequent studies involving lung biopsy specimens revealed that ER stress markers are highly expressed in the alveolar epithelium in IPF and FIP. Recent mouse modeling has revealed that induction of ER stress in the alveolar epithelium predisposed to enhanced lung fibrosis after treatment with bleomycin, which is mediated at least in part by increased alveolar epithelial cell (AEC) apoptosis. Emerging data also indicate that ER stress in AECs could impact fibrotic remodeling by altering inflammatory responses and inducing epithelial-mesenchymal transition. Although the cause of ER stress in IPF remains unknown, common environmental exposures such as herpesviruses, inhaled particulates, and cigarette smoke induce ER stress and are candidates for contributing to AEC dysfunction by this mechanism. Together, investigations to date suggest that ER stress predisposes to AEC dysfunction and subsequent lung fibrosis. However, many questions remain regarding the role of ER stress in initiation and progression of lung fibrosis, including whether ER stress or the UPR could be targeted for therapeutic benefit.     

Right place, right time: the evolving role of herpesvirus infection as a "second hit" in idiopathic pulmonary fibrosis

Over the course of the past decade, increasing evidence has implicated alveolar epithelial cell injury and dysfunction in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Genetic factors, cigarette smoking, and other environmental exposures have been identified as potential factors leading to a population of vulnerable alveolar epithelial cells. In addition, molecular techniques have demonstrated herpesviruses are commonly detectable in the lungs of patients with IPF, raising suspicion that, in the setting of a vulnerable alveolar epithelium, lytic (or latent) herpesvirus infection may act as a "second hit" leading to the development of pulmonary fibrosis. Intriguingly, in vivo modeling has shown that herpesvirus infection induces or worsens lung fibrosis when combined with immunodeficiency or other injurious stimuli. Here, we discuss potential mechanisms through which herpesvirus infection may contribute to the pathogenesis of IPF. Ultimately, antiviral therapy may hold promise for halting the progression of this deadly disease.     

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.     

Role of MicroRNA Processing in Adipose Tissue in Stress Defense and Longevity

The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner?mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S.?cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of?a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.     

Subcellular location of PKA controls striatal plasticity: stochastic simulations in spiny dendrites

Dopamine release in the striatum has been implicated in various forms of reward dependent learning. Dopamine leads to production of cAMP and activation of protein kinase A (PKA), which are involved in striatal synaptic plasticity and learning. PKA and its protein targets are not diffusely located throughout the neuron, but are confined to various subcellular compartments by anchoring molecules such as A-Kinase Anchoring Proteins (AKAPs). Experiments have shown that blocking the interaction of PKA with AKAPs disrupts its subcellular location and prevents LTP in the hippocampus and striatum; however, these experiments have not revealed whether the critical function of anchoring is to locate PKA near the cAMP that activates it or near its targets, such as AMPA receptors located in the post-synaptic density. We have developed a large scale stochastic reaction-diffusion model of signaling pathways in a medium spiny projection neuron dendrite with spines, based on published biochemical measurements, to investigate this question and to evaluate whether dopamine signaling exhibits spatial specificity post-synaptically. The model was stimulated with dopamine pulses mimicking those recorded in response to reward. Simulations show that PKA colocalization with adenylate cyclase, either in the spine head or in the dendrite, leads to greater phosphorylation of DARPP-32 Thr34 and AMPA receptor GluA1 Ser845 than when PKA is anchored away from adenylate cyclase. Simulations further demonstrate that though cAMP exhibits a strong spatial gradient, diffusible DARPP-32 facilitates the spread of PKA activity, suggesting that additional inactivation mechanisms are required to produce spatial specificity of PKA activity.