Eicosanoid pathways regulate adaptive immunity to Mycobacterium tuberculosis.

The fate of infected macrophages has an essential role in protection against Mycobacterium tuberculosis by regulating innate and adaptive immunity. M. tuberculosis exploits cell necrosis to exit from macrophages and spread. In contrast, apoptosis, which is characterized by an intact plasma membrane, is an innate mechanism that results in lower bacterial viability. Virulent M. tuberculosis inhibits apoptosis and promotes necrotic cell death by inhibiting production of prostaglandin E(2). Here we show that by activating the 5-lipoxygenase pathway, M. tuberculosis not only inhibited apoptosis but also prevented cross-presentation of its antigens by dendritic cells, which impeded the initiation of T cell immunity. Our results explain why T cell priming in response to M. tuberculosis is delayed and emphasize the importance of early immunity.

Mycobacterium tuberculosis evades macrophage defenses by inhibiting plasma membrane repair.

Induction of macrophage necrosis is a strategy used by virulent Mycobacterium tuberculosis (Mtb) to avoid innate host defense. In contrast, attenuated Mtb causes apoptosis, which limits bacterial replication and promotes T cell cross-priming by antigen-presenting cells. Here we show that Mtb infection causes plasma membrane microdisruptions. Resealing of these lesions, a process crucial for preventing necrosis and promoting apoptosis, required translocation of lysosomal and Golgi apparatus-derived vesicles to the plasma membrane. Plasma membrane repair depended on prostaglandin E(2) (PGE(2)), which regulates synaptotagmin 7 (Syt-7), the calcium sensor involved in the lysosome-mediated repair mechanism. By inducing production of lipoxin A(4) (LXA(4)), which blocks PGE(2) biosynthesis, virulent Mtb prevented membrane repair and induced necrosis. Thus, virulent Mtb impairs macrophage plasma membrane repair to evade host defenses.

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection.

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRß bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

Receptor mediated disruption of retinal pigment epithelium function in acute glycated-albumin exposure.

Diabetic macular edema (DME) is a major cause of visual impairment. Although DME is generally believed to be a microvascular disease, dysfunction of the retinal pigment epithelium (RPE) can also contribute to its development. Advanced glycation end-products (AGE) are thought to be one of the key factors involved in the pathogenesis of diabetes in the eye, and we have previously demonstrated a rapid breakdown of RPE function following glycated-albumin (Glyc-alb, a common AGE mimetic) administration in monolayer cultures of fetal human RPE cells. Here we present new evidence that this response is attributed to apically oriented AGE receptors (RAGE). Moreover, time-lapse optical coherence tomography in Dutch-belted rabbits 48 h post intravitreal Glyc-alb injections demonstrated a significant decrease in RPE-mediated fluid resorption in vivo. In both the animal and tissue culture models, the response to Glyc-alb was blocked by the relatively selective RAGE antagonist, FPS-ZM1 and was also inhibited by ZM323881, a relatively selective vascular endothelial growth factor receptor 2 (VEGF-R2) antagonist. Our data establish that the Glyc-alb-induced breakdown of RPE function is mediated via specific RAGE and VEGF-R2 signaling both in vitro and in vivo. These results are consistent with the notion that the RPE is a key player in the pathogenesis of DME.

Diagnosis of Retrobulbar Round Cell Neoplasia in a Macaroni Penguin ( Eudyptes chrysolophus ) Through Use of Computed Tomography.

A 25-year-old female macaroni penguin (Eudyptes chrysolophus) was diagnosed with exophthalmos secondary to retrobulbar neoplasia through use of computed tomography (CT). Histopathologic examination of the mass supported a diagnosis of malignant round cell neoplasia. Immunohistochemical (IHC) labeling was applied to determine cell origin; the neoplastic cells did not label with T-cell marker CD3 or B-cell marker BLA.36 and could not be further characterized. The scleral ossicles precluded evaluation of the retrobulbar space by ultrasonography; therefore, CT scanning is recommended for examination of intraorbital structures in penguin and other avian species.

Comparison of hair follicle histology between horses with pituitary pars intermedia dysfunction and excessive hair growth and normal aged horses.

BACKGROUND: Pituitary pars intermedia dysfunction (PPID) in older equids is commonly recognized by a long hair coat that fails to shed. OBJECTIVE: The aim of this study was to compare hair follicle stages in PPID-affected horses with excessively long hair coats with the stages of normal aged horses (controls) and to compare hair follicle stages in PPID-affected horses after 6 months of treatment with pergolide mesylate with those of control horses. ANIMALS: Eight PPID-affected horses and four normal, age-matched, control horses. METHODS: Skin biopsies were collected from the neck and rump of PPID-affected and control horses. A diagnosis of PPID was established based on hair coat changes and supportive overnight dexamethasone suppression test results. Skin biopsies were repeated after 6 months of treatment with pergolide. The number of hair follicles in anagen (A) or telogen (T) was counted for each skin biopsy using transverse sections. RESULTS: Pretreatment biopsies had a greater percentage of A follicles (neck 96%, rump 95%) and a lower percentage of T follicles (neck 4%, rump 5%) in PPID-affected horses than in control horses (A, neck 15%, rump 25%; and T, neck 85%, rump 75%). After treatment with pergolide, all PPID-affected horses had improved shedding, and the percentages of A follicles (neck 69%, rump 70%) and T follicles (neck 31%, rump 30%) were not different from untreated control horses (A, neck 68%, rump 82%; and T, neck 32%, rump 18%). CONCLUSIONS: These findings document that excessive hair growth (hypertrichosis) in PPID-affected horses is due to persistence of hair follicles in A. Furthermore, treatment with pergolide improved shedding and reduced the percentage of A follicles in PPID-affected horses.

The Mef2A transcription factor coordinately regulates a costamere gene program in cardiac muscle.

The Mef2 family of transcription factors regulates muscle differentiation, but the specific gene programs controlled by each member remain unknown. Characterization of Mef2A knock-out mice has revealed severe myofibrillar defects in cardiac muscle indicating a requirement for Mef2A in cytoarchitectural integrity. Through comprehensive expression analysis of Mef2A-deficient hearts, we identified a cohort of dysregulated genes whose products localize to the peripheral Z-disc/costamere region. Many of these genes are essential for costamere integrity and function. Here we demonstrate that these genes are directly regulated by Mef2A, establishing a mechanism by which Mef2A controls the costamere. In an independent model system, acute knockdown of Mef2A in primary neonatal cardiomyocytes resulted in profound malformations of myofibrils and focal adhesions accompanied by adhesion-dependent programmed cell death. These findings indicate a role for Mef2A in cardiomyocyte survival through regulation of costamere integrity. Finally, bioinformatics analysis identified over-represented transcription factor-binding sites in this network of costamere promoters that may provide insight into the mechanism by which costamere genes are regulated by Mef2A. The global control of costamere gene expression adds another dimension by which this essential macromolecular complex may be regulated in health and disease.

Mycoplasmosis in ferrets.

We report an outbreak of severe respiratory disease associated with a novel Mycoplasma species in ferrets. During 2009-2012, a respiratory disease characterized by nonproductive coughing affected ≈8,000 ferrets, 6-8 weeks of age, which had been imported from a breeding facility in Canada. Almost 95% became ill, but almost none died. Treatments temporarily decreased all clinical signs except cough. Postmortem examinations of euthanized ferrets revealed bronchointerstitial pneumonia with prominent hyperplasia of bronchiole-associated lymphoid tissue. Immunohistochemical analysis with polyclonal antibody against Mycoplasma bovis demonstrated intense staining along the bronchiolar brush border. Bronchoalveolar lavage samples from 12 affected ferrets yielded fast-growing, glucose-fermenting mycoplasmas. Nucleic acid sequence analysis of PCR-derived amplicons from portions of the 16S rDNA and RNA polymerase B genes failed to identify the mycoplasmas but showed that they were most similar to M. molare and M. lagogenitalium. These findings indicate a causal association between the novel Mycoplasma species and the newly recognized pulmonary disease.

Modulation of angiotensin II-mediated cardiac remodeling by the MEF2A target gene Xirp2.

RATIONALE: The vasoactive peptide angiotensin II (Ang II) is a potent cardiotoxic hormone whose actions have been well studied, yet questions remain pertaining to the downstream factors that mediate its effects in cardiomyocytes. OBJECTIVE: The in vivo role of the myocyte enhancer factor (MEF)2A target gene Xirp2 in Ang II-mediated cardiac remodeling was investigated. METHODS AND RESULTS: Here we demonstrate that the MEF2A target gene Xirp2 (also known as cardiomyopathy associated gene 3 [CMYA3]) is an important effector of the Ang II signaling pathway in the heart. Xirp2 belongs to the evolutionarily conserved, muscle-specific, actin-binding Xin gene family and is significantly induced in the heart in response to systemic administration of Ang II. Initially, we characterized the Xirp2 promoter and demonstrate that Ang II activates Xirp2 expression by stimulating MEF2A transcriptional activity. To further characterize the role of Xirp2 downstream of Ang II signaling we generated mice harboring a hypomorphic allele of the Xirp2 gene that resulted in a marked reduction in its expression in the heart. In the absence of Ang II, adult Xirp2 hypomorphic mice displayed cardiac hypertrophy and increased beta myosin heavy chain expression. Strikingly, Xirp2 hypomorphic mice chronically infused with Ang II exhibited altered pathological cardiac remodeling including an attenuated hypertrophic response, as well as diminished fibrosis and apoptosis. CONCLUSIONS: These findings reveal a novel MEF2A-Xirp2 pathway that functions downstream of Ang II signaling to modulate its pathological effects in the heart.

Histone Deacetylase Inhibition Restores Retinal Pigment Epithelium Function in Hyperglycemia.

In diabetic individuals, macular edema is a major cause of vision loss. This condition is refractory to insulin therapy and has been attributed to metabolic memory. The retinal pigment epithelium (RPE) is central to maintaining fluid balance in the retina, and this function is compromised by the activation of advanced glycation end-product receptors (RAGE). Here we provide evidence that acute administration of the RAGE agonist, glycated-albumin (gAlb) or vascular endothelial growth factor (VEGF), increased histone deacetylase (HDAC) activity in RPE cells. The administration of the class I/II HDAC inhibitor, trichostatin-A (TSA), suppressed gAlb-induced reductions in RPE transepithelial resistance (in vitro) and fluid transport (in vivo). Systemic TSA also restored normal RPE fluid transport in rats with subchronic hyperglycemia. Both gAlb and VEGF increased HDAC activity and reduced acetyl-α-tubulin levels. Tubastatin-A, a relatively specific antagonist of HDAC6, inhibited gAlb-induced changes in RPE cell resistance. These data are consistent with the idea that RPE dysfunction following exposure to gAlb, VEGF, or hyperglycemia is associated with increased HDAC6 activity and decreased acetyl-α-tubulin. Therefore, we propose inhibiting HDAC6 in the RPE as a potential therapy for preserving normal fluid homeostasis in the hyperglycemic retina.

Progressive Early Breakdown of Retinal Pigment Epithelium Function in Hyperglycemic Rats.

PURPOSE: Diabetic macular edema (DME), an accumulation of fluid in the subretinal space, is a significant cause of vision loss. The impact of diabetes on the breakdown of the inner blood-retina barrier (BRB) is an established event that leads to DME. However, the role of the outer BRB in ocular diabetes has received limited attention. We present evidence that the breakdown of normal RPE function in hyperglycemia facilitates conditions conducive to DME pathogenesis. METHODS: Brown Norway rats (130-150 g) were injected intraperitoneally with streptozotocin (STZ; 60 mg/kg) to induce hyperglycemia. After 4 weeks, Evans blue (EB) dye was injected intravenously to determine whether there was leakage of albumin into the retina. Subretinal saline blebs (0.5-1 µL) were placed 4 and 9 weeks after STZ injection, and time-lapse optical coherence tomography tracked the resorption rate. In a subset of rats, intravitreal bevacizumab, a humanized monoclonal antibody targeted to VEGF, was given at 5 weeks and resorption was measured at 9 weeks. RESULTS: The ability of the RPE to transport fluid was reduced significantly after 4 and 9 weeks of hyperglycemia with a reduction of over 67% at 9 weeks. No EB dye leakage from inner retinal vessels was measured in hyperglycemic animals compared to control. The intravitreal administration of bevacizumab at week 5 significantly increased the rate of fluid transport in rats subjected to hyperglycemia for 9 weeks. CONCLUSIONS: These results demonstrate that chronic hyperglycemia altered RPE fluid transport, in part dependent on the actions of VEGF. These results support the idea that RPE dysfunction is an early event associated with hyperglycemia that contributes to fluid accumulation in DME.

Pustular psoriasis complicated with acute generalized exanthematous pustulosis.

BACKGROUND: Pustular psoriasis of the digits (acrodermatitis continua of Hallopeau) may be localized to one or more digits for over an extended period of time. Characteristic presentation is that of tender, diffusely eroded, and fissured pustular plaques on one or more digits. Transition to other forms of psoriasis and to generalized pustular psoriasis is known to occur. These patients have an increased risk of acute generalized exanthematous pustulosis (AGEP) compared to the general population. Pustular psoriasis is often therapy resistant. MAIN OBSERVATIONS: We report the case of a 54-year-old Caucasian woman who presented with a pustular psoriasis flare complicated by AGEP. Treatment course included hospital admission, cyclosporine, acitretin, and discontinuation of cephalexin. CONCLUSION: The precipitating factor in the course of treatment is thought to be cephalexin. When treating patients with pustular psoriasis the occurrence of druginduced complications should be carefully examined. Our case suggests that avoidance of ß-lactam antibiotics in these patients is warranted unless absolutely indicated.

Efferocytosis is an innate antibacterial mechanism.

Mycobacterium tuberculosis persists within macrophages in an arrested phagosome and depends upon necrosis to elude immunity and disseminate. Although apoptosis of M. tuberculosis-infected macrophages is associated with reduced bacterial growth, the bacteria are relatively resistant to other forms of death, leaving the mechanism underlying this observation unresolved. We find that after apoptosis, M. tuberculosis-infected macrophages are rapidly taken up by uninfected macrophages through efferocytosis, a dedicated apoptotic cell engulfment process. Efferocytosis of M. tuberculosis sequestered within an apoptotic macrophage further compartmentalizes the bacterium and delivers it along with the apoptotic cell debris to the lysosomal compartment. M. tuberculosis is killed only after efferocytosis, indicating that apoptosis itself is not intrinsically bactericidal but requires subsequent phagocytic uptake and lysosomal fusion of the apoptotic body harboring the bacterium. While efferocytosis is recognized as a constitutive housekeeping function of macrophages, these data indicate that it can also function as an antimicrobial effector mechanism.

Wolbachia enhance Drosophila stem cell proliferation and target the germline stem cell niche.

Wolbachia are widespread maternally transmitted intracellular bacteria that infect most insect species and are able to alter the reproduction of innumerous hosts. The cellular bases of these alterations remain largely unknown. Here, we report that Drosophila mauritiana infected with a native Wolbachia wMau strain produces about four times more eggs than the noninfected counterpart. Wolbachia infection leads to an increase in the mitotic activity of germline stem cells (GSCs), as well as a decrease in programmed cell death in the germarium. Our results suggest that up-regulation of GSC division is mediated by a tropism of Wolbachia for the GSC niche, the cellular microenvironment that supports GSCs.

A comparative lipidomics platform for chemotaxonomic analysis of Mycobacterium tuberculosis.

The lipidic envelope of Mycobacterium tuberculosis promotes virulence in many ways, so we developed a lipidomics platform for a broad survey of cell walls. Here we report two new databases (MycoMass, MycoMap), 30 lipid fine maps, and mass spectrometry datasets that comprise a static lipidome. Further, by rapidly regenerating lipidomic datasets during biological processes, comparative lipidomics provides statistically valid, organism-wide comparisons that broadly assess lipid changes during infection or among clinical strains of mycobacteria. Using stringent data filters, we tracked more than 5,000 molecular features in parallel with few or no false-positive molecular discoveries. The low error rates allowed chemotaxonomic analyses of mycobacteria, which describe the extent of chemical change in each strain and identified particular strain-specific molecules for use as biomarkers.