Prospects and limitations of cumate-inducible lentivirus as a tool for investigating VEGF-A-mediated pathology in diabetic retinopathy.

Diabetic retinopathy (DR) is a multifactorial disease displaying vascular-associated pathologies, including vascular leakage and neovascularization, ultimately leading to visual impairment. However, animal models accurately reflecting these pathologies are lacking. Vascular endothelial growth factor A (VEGF-A) is an important factor in the development of micro- and macro-vascular pathology in DR. In this study, we evaluated the feasibility of using a cumate-inducible lentivirus (LV) mediated expression of vegf-a to understand DR pathology in vitro and in vivo. Retinal pigment epithelial cells (ARPE-19) were transduced with cumate-inducible LV expressing vegf-a, with subsequent analysis of vegf-a expression and its impact on cell proliferation, viability, motility, and permeability. Cumate tolerability in adult Wistar rat eyes was assessed as an initial step towards a potential DR animal model development, by administering cumate via intravitreal injections (IVT) and evaluating consequent effects by spectral domain optical coherence tomography (SD-OCT), flash electroretinography (fERG), ophthalmic examination (OE), and immunohistochemistry. Transduction of ARPE-19 cells with cumate-inducible LV resulted in ~ 2.5-fold increase in vegf-a mRNA and ~ threefold increase in VEGF-A protein secretion. Transduced cells displayed enhanced cell proliferation, viability, permeability, and migration in tube-like structures. However, IVT cumate injections led to apparent retinal toxicity, manifesting as retinal layer abnormalities, haemorrhage, vitreous opacities, and significant reductions in a- and b-wave amplitudes, along with increased microglial activation and reactive gliosis. In summary, while cumate-inducible LV-mediated vegf-a expression is valuable for in vitro mechanistic studies in cellular drug discovery, its use is not a feasible approach to model DR in in vivo studies due to cumate-induced retinal toxicity.

DKCNN: Improving deep kernel convolutional neural network-based covid-19 identification from CT images of the chest.

BACKGROUND: An efficient deep convolutional neural network (DeepCNN) is proposed in this article for the classification of Covid-19 disease. OBJECTIVE: A novel structure known as the Pointwise-Temporal-pointwise convolution unit is developed incorporated with the varying kernel-based depth wise temporal convolution before and after the pointwise convolution operations. METHODS: The outcome is optimized by the Slap Swarm algorithm (SSA). The proposed Deep CNN is composed of depth wise temporal convolution and end-to-end automatic detection of disease. First, the datasets SARS-COV-2 Ct-Scan Dataset and CT scan COVID Prediction dataset are preprocessed using the min-max approach and the features are extracted for further processing. RESULTS: The experimental analysis is conducted between the proposed and some state-of-art works and stated that the proposed work effectively classifies the disease than the other approaches. CONCLUSION: The proposed structural unit is used to design the deep CNN with the increasing kernel sizes. The classification process is improved by the inclusion of depth wise temporal convolutions along with the kernel variation. The computational complexity is reduced by the introduction of stride convolutions are used in the residual linkage among the adjacent structural units.

Lysyl oxidase like-1 deficiency in optic nerve head astrocytes elicits reactive astrocytosis and alters functional effects of astrocyte derived exosomes.

Glaucoma is a multifactorial progressive ocular pathology that manifests clinically with damage to the optic nerve (ON) and the retina, ultimately leading to blindness. The optic nerve head (ONH) shows the earliest signs of glaucoma pathology, and therefore, is an attractive target for drug discovery. The goal of this study was to elucidate the effects of reactive astrocytosis on the elastin metabolism pathway in primary rat optic nerve head astrocytes (ONHA), the primary glial cell type in the unmyelinated ONH. Following exposure to static equibiaxial mechanical strain, we observed prototypic molecular and biochemical signatures of reactive astrocytosis that were associated with a decrease in lysyl oxidase like 1 (Loxl1) expression and a concomitant decrease in elastin (Eln) gene expression. We subsequently investigated the role of Loxl1 in reactive astrocytosis by generating primary rat ONHA cultures with ∼50% decreased Loxl1 expression. Our results suggest that reduced Loxl1 expression is sufficient to elicit molecular signatures of elastinopathy in ONHA. Astrocyte derived exosomes (ADE) significantly increased the length of primary neurites of primary neurons in vitro. In contrast, ADE from Loxl1-deficient ONHA were deficient of trophic effects on neurite outgrowth in vitro, positing that Loxl1 dysfunction and the ensuing impaired elastin synthesis during reactive astrocytosis in the ONH may contribute to impaired neuron-glia signaling in glaucoma. Our data support a role of dysregulated Loxl1 function in eliciting reactive astrocytosis in glaucoma subtypes associated with increased IOP, even in the absence of genetic polymorphisms in LOXL1 typically associated with exfoliation glaucoma. This suggests the need for a paradigm shift toward considering lysyl oxidase activity and elastin metabolism and signaling as contributors to an altered secretome of the ONH that may lead to the progression of glaucomatous changes. Future research is needed to investigate cargo of exosomes in the context of reactive astrocytosis and identify the pathways leading to the observed transcriptome changes during reactive astrocytosis.

Utilizing variable auto encoder-based TDO optimization algorithm for predicting loneliness from electrocardiogram signals.

Several seniors and a substantial part of the general population are living in social isolation. This frequently occurs in vulnerability, isolation, and depression, which then have a poor impact on other health-related factors. A number of health problems, including a higher risk of cardio problems, are brought on by social isolation and loneliness. Electrocardiogram (ECG) usage for mental condition recognition enables accurate determination of a person's internal representation. The electrocardiogram (ECG) signals can be thoroughly analyzed to uncover hidden data that may be helpful for the precise identification of cardiac problems. ECG time-series information typically have great dimensions and complicated componentry. Using relevant information to guide training is among the main achievements of this type of learning. An ECG signal plays a significant part in the individual body's ability to manage behavior. Furthermore, loneliness identification is crucial since it has the worse effect on the circumstances that afflict persons. This study suggested an approach for detecting loneliness from an ECG signal to use a variable auto encoder-based optimization algorithm for ESN technique. The suggested approach consists of three phases for identifying a person's loneliness. Firstly, undecimated discrete wavelet transform is used to preprocess the acquired ECG data. Next, further characteristics are extracted from the precompiled signals using a variable auto encoder. For the precise categorization of loneliness in the ECG signal, a metaheuristic optimized ESN is, therefore, presented. The outcomes of the tests demonstrate that the suggested system with suitable ECG representations produces improved accuracy as well as performance.

Effectiveness of multi-disciplinary structured training program on mentoring and mental well-being for officers and instructors in the Indian Air Force.

Background: The military environment is characterized by unpredictable situations, intensive training, demanding workload, and job-associated stressors, which make it highly stressful. Mentorship and mental well-being training could be beneficial to both officers and the new adolescent recruits of the Indian Air Force (IAF). Aim: This study aimed at evaluating the effect of a multi-disciplinary structured training on mentoring and mental well-being among officers and instructors in the IAF. Methods: Seventy IAF officers/instructors underwent a week-long multi-disciplinary structured training program, which was conducted at a tertiary care neuro-psychiatric hospital in South India. A quasi-experimental design with a single-group pre- and post-test was adopted. Outcome measures included a) knowledge on mentorship and mental health and b) self-perceived competence in addressing mental health distress. Results: Post training, there was a statistically significant improvement in scores on mentorship/mental health knowledge and a significant increase in self-perceived competence in addressing mental distress. Conclusion: Mentorship and mental well-being training for officers and instructors in the IAF improved mental health knowledge and self-perceived competence. Therefore, administration of regular and in-depth structured mental health-related training interventions could be beneficial not only to the officers but also to the new recruits/mentees in the IAF.

Endocrine assessment, chemotherapy, nonsurgical treatment, and rehabilitation for osteoporotic spine fractures: WFNS spine committee recommendations.

INTRODUCTION: We reviewed the endocrine assessment, chemotherapy, and rehabilitation of the osteoporotic vertebral fractures (OVF) to create recommendations for the disease. EVIDENCE ACQUISITION: A PubMed and Medline search between 2011 and 2021 was conducted using key words. Case reports, experimental studies, papers other than the English language, and unrelated studies were excluded. Up-to-date information on endocrine assessment, medical and nonsurgical treatment, and rehabilitation for osteoporotic spine fractures were reviewed, and statements were produced to reach a consensus in two separate virtual consensus meetings of the World Federation of Neurosurgical Societies (WFNS) Spine Committee. The statements were voted and achieved a positive or negative consensus using Delphi method. EVIDENCE SYNTHESIS: Endocrine assessment of osteoporosis is necessary if it is diagnosed in premenopausal women or men less than 50 years of age. Dual X-Ray absorptiometry (DXA) alone may not be predictive of fracture. Endocrine causes of osteoporosis must also be evaluated if the Z-score ≤-2.0. In case of a vertebral fracture in postmenopausal women and men aged 50 years and above, pharmacologic treatment for osteoporosis must be started. Strengthening exercise, balance and gait training, and assistive devices are recommended to prevent and reduce falls in the elderly. In addition, rehabilitation must be a part of the overall treatment to help patients return to function. CONCLUSIONS: Nonsurgical management helps treat OVF. Although there is weak evidence on the type of medical treatment and usage of braces, bed rest, and other measures, current literature supports endocrinological management, medical treatment, and rehabilitation after osteoporotic vertebral fractures.

TGF-ß2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression.

Purpose: The multifunctional profibrotic cytokine TGF-ß2 is implicated in the pathophysiology of primary open angle glaucoma (POAG). While the underlying cause of POAG remains unclear, TGF-ß2 dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Mitochondrial-targeted antioxidants have been recently shown by our group to markedly attenuate TGF-ß2 profibrotic responses, strongly implicating oxidative stress as a key facilitator of TGF-ß2 signaling in human TM cells. In this study, we determined the mechanism by which oxidative stress facilitates TGF-ß2 profibrotic responses in cultured primary human TM cells. Methods: Semiconfluent cultures of primary or transformed human TM cells were conditioned overnight in serum-free media and subsequently challenged without or with TGF-ß2 (5 ng/mL). Relative changes in the mRNA content of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) isoforms, connective tissue growth factor (CTGF), collagen 1α1 and 4α1 isoforms or relative changes in the protein content of Nox4, phospho- and total-Smad2 and -Smad3, collagens I and IV were determined in the absence or presence of GKT137831, a Nox1-Nox4 dual enzyme inhibitor, and quantified by real-time qPCR or by immunoblot, respectively. Relative in situ changes in collagens I and IV and in alpha smooth muscle actin (αSMA) were semiquantified by immunocytochemistry, whereas relative changes in filamentous actin stress fiber formation was semiquantified by phalloidin staining. Results: Quiescent primary human TM cells cultured in the presence of TGF-ß2 exhibited a marked selective increase in endogenous Nox4 mRNA and Nox4 protein expression. Actinomycin D prevented TGF-ß2 mediated increases in Nox4 mRNA expression. TM cells reverse transfected with siRNA against Smad3 prevented TGF-ß2 mediated increases in Nox4 mRNA expression. Pre-incubating TM cells with GKT137831 attenuated TGF-ß2 mediated increases in intracellular reactive oxygen species (ROS), in COL1A1, COL4A1, and CTGF mRNA expression, in Smad3 protein phosphorylation, in collagens I, collagens IV, and αSMA protein expression, and in filamentous actin stress fiber formation. Conclusions: TGF-ß2 promotes oxidative stress in primary human TM cells by selectively increasing expression of NADPH oxidase 4. Dysregulation of redox equilibrium by induction of NADPH oxidase 4 expression appears to be a key early event involved in the pathologic profibrotic responses elicited by TGF-ß2 canonical signaling, including ECM remodeling, filamentous actin stress fiber formation, and αSMA expression. Selective inhibition of Nox4 expression/activation, in combination with mitochondrial-targeted antioxidants, represents a novel strategy by which to slow the progression of TGF-ß2 elicited profibrotic responses within the TM.

An assessment of the efficacy of clinical skills simulation using standardized patient in teaching behavior management and modification skills in Pediatric Dentistry to dental undergraduate students: A double-blinded, randomized, controlled trial.

BACKGROUND: Clinical skills simulation (CSS) is an important tool in teaching and learning. The literature review showed a scarcity of research data regarding the use of CSS,in teaching, especially in dentistry. The use of CSS in dental teaching was found restricted to the use of low fidelity typhodonts fitted to phantom heads used in teaching cavity preparation and crown cutting. AIM: The aim of the study was to determine the efficacy of CSS using standardized patient in teaching behavior management and modification skills to dental undergraduate students. SETTINGS AND DESIGN: This double-blinded, randomized controlled trial was undertaken among 3rd year dental undergraduate students, and the study was undertaken at the Department of Pediatric and Preventive Dentistry. MATERIALS AND METHODS: Fifty, 3rd year BDS students were randomly allotted to simulation and nonsimulation groups. Baseline data regarding their knowledge in the behavior management of child patients were assessed. Simulation group was further divided into group of six students and underwent CSS with standardized patient. Pretest and posttest knowledge regarding behavior management was assessed in the simulation group using questionnaires approved by an expert committee. The results were analyzed to see if there is any improvement in their knowledge after CSS. Students in simulation and nonsimulation groups were assessed for their behavior management skills during patient management, by an independent observer, using a checklist. STATISTICAL ANALYSIS: Mean, standard deviation (SD), and unpaired student t-test were done to assess the baseline knowledge of students who participated in the study. Mean, SD, and paired t-test were used to compare the pretest and posttest score of students who underwent simulation. Mean, SD, and unpaired t-test were used to compare the behavior management skills of both groups of students. RESULTS AND CONCLUSIONS: The knowledge of students in both groups before the study was comparable with no statistically significant differences. There was a statistically significant improvement in the knowledge of students who underwent CSS regarding behavior management of child patients. The unpaired Student's t-test showed a significant difference in the behavior management skill of dental undergraduate students when treating a child patient. The students who underwent CSS fared better compared to students who were taught behavior management methods by traditional methods only. Clinical skill simulation using standardized patient is an effective adjunct to be used along with traditional method of teaching while teaching behavior management and modification skills to dental undergraduate students.

Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants.

Optic nerve head astrocytes are the specialized glia cells that provide structural and trophic support to the optic nerve head. In response to cellular injury, optic nerve head astrocytes undergo reactive astrocytosis, the process of cellular activation associated with cytoskeletal remodeling, increases in the rate of proliferation and motility, and the generation of Reactive Oxygen Species. Antioxidant intervention has previously been proposed as a therapeutic approach for glaucomatous optic neuropathy, however, little is known regarding the response of optic nerve head astrocytes to antioxidants under physiological versus pathological conditions. The goal of this study was to determine the effects of three different antioxidants, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), resveratrol and xanthohumol in primary optic nerve head astrocytes. Effects on the expression of the master regulator nuclear factor erythroid 2-related factor 2 (Nrf2), the antioxidant enzyme, manganese-dependent superoxide dismutase 2 (SOD2), and the pro-oxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), were determined by quantitative immunoblotting. Furthermore, efficacy in preventing chemically and reactive astrocytosis-induced increases in cellular oxidative stress was quantified using cell viability assays. The results were compared to the effects of the prototypic antioxidant, Trolox. Antioxidants elicited highly differential changes in the expression levels of Nrf2, SOD2, and NOX4. Notably, Mn-TM-2-PyP increased SOD2 expression eight-fold, while resveratrol increased Nrf2 expression three-fold. In contrast, xanthohumol exerted no statistically significant changes in expression levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and lactate dehydrogenase (LDH) release assays were performed to assess cell viability after chemically and reactive astrocytosis-induced oxidative stress. Mn-TM-2-PyP exerted the most potent glioprotection by fully preventing the loss of cell viability, whereas resveratrol and xanthohumol partially restored cell viability. Our data provide the first evidence for a well-developed antioxidant defense system in optic nerve head astrocytes, which can be pharmacologically targeted by different classes of antioxidants.

Identification of scavenger receptor B1 as the airway microfold cell receptor for Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) can enter the body through multiple routes, including via specialized transcytotic cells called microfold cells (M cell). However, the mechanistic basis for M cell entry remains undefined. Here, we show that M cell transcytosis depends on the Mtb Type VII secretion machine and its major virulence factor EsxA. We identify scavenger receptor B1 (SR-B1) as an EsxA receptor on airway M cells. SR-B1 is required for Mtb binding to and translocation across M cells in mouse and human tissue. Together, our data demonstrate a previously undescribed role for Mtb EsxA in mucosal invasion and identify SR-B1 as the airway M cell receptor for Mtb.

Mitochondrial-Targeted Antioxidants Attenuate TGF-ß2 Signaling in Human Trabecular Meshwork Cells.

Purpose: POAG is a progressive optic neuropathy that is currently the leading cause of irreversible blindness worldwide. While the underlying cause of POAG remains unclear, TGF-ß2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Early studies have also demonstrated markedly elevated levels of oxidative stress markers in AH from POAG patients along with altered expression of antioxidant defenses. Here, using cultured primary or transformed human TM cells, we investigated the role oxidative stress plays at regulating TGF-ß2-mediated remodeling of the ECM. Methods: Primary or transformed (GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-ß2 and relative changes in intracellular reactive oxygen species (ROS) were measured using oxidation-sensitive fluorogenic dyes CellROX green or 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). TGF-ß2-mediated changes in the content of connective tissue growth factor (CTGF) and collagen types 1α1 (COL1A1) and 4α1 (COL4A1) mRNA or collagens I and IV isoform proteins were determined in the absence or presence of mitochondrial-targeted antioxidants (XJB-5-131 or MitoQ) and quantified by quantitative PCR or by immunoblot and immunocytochemistry. Smad-dependent canonic signaling was determined by immunoblot, whereas Smad-dependent transcriptional activity was quantified using a Smad2/3-responsive SBE-luciferase reporter assay. Results: Primary or transformed human TM cells cultured in the presence of TGF-ß2 (5 ng/mL; 2 hours) exhibited marked increases in CellROX or fluorescein fluorescence. Consistent with previous reports, challenging cultured human TM cells with TGF-ß2 elicited measurable increases in regulated Smad2/3 signaling as well as increases in CTGF, COL1A1, and COL4A1 mRNA and collagen protein content. Pretreating human TM cells with mitochondrial-targeted antioxidants XJB-5-131 (10 µM) or MitoQ (10 nM) attenuated TGF-ß2-mediated changes in Smad-dependent transcriptional activity. Conclusions: The multifunctional profibrotic cytokine TGF-ß2 elicits a marked increase in oxidative stress in human TM cells. Mitochondrial-targeted antioxidants attenuate TGF-ß2-mediated changes in Smad-dependent transcriptional activity, including marked reductions in CTGF and collagen isoform gene and protein expression. These findings suggest that mitochondrial-targeted antioxidants, when delivered directly to the TM, exhibit potential as a novel strategy by which to slow the progression of TGF-ß2-mediated remodeling of the ECM within the TM.

Screening Mycobacterium tuberculosis Secreted Proteins Identifies Mpt64 as a Eukaryotic Membrane-Binding Bacterial Effector.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.IMPORTANCE Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.

Indirubin-3'-monoxime prevents aberrant activation of GSK-3ß/NF-κB and alleviates high fat-high fructose induced Aß-aggregation, gliosis and apoptosis in mice brain.

Deciphering the molecular mechanisms of amyloid pathology and glial cell-mediated neuroinflammation, offers a novel avenue for therapeutic intervention against neurodegeneration. Recent findings demonstrate a crucial link between activation of glycogen synthase kinase-3ß (GSK-3ß), amyloid deposition and a neuroinflammatory state. However, studies demonstrating the pharmacological effects of GSK-3ß inhibition and the interlinked molecular mechanisms still remain elusive. The present study explores whether high fat-high fructose diet (HFFD)-induced neuropathological changes could be alleviated by indirubin-3'-monoxime (IMX), a GSK-3ß inhibitor. Male Swiss albino mice (8 weeks old) were fed with normal pellet or HFFD for 60 days. HFFD mice were treated with IMX once daily for last 7 days of the experimental period. HFFD fed-mice had significant amyloid deposits in cerebral cortex and hippocampus, and protein expression analyses showed activation of GSK-3ß, nuclear translocation of NF-κB p65 and upregulation of inflammatory (TNF-α, IL-6, COX-2), astrocytic (GFAP), glial surface (CD-68) and pro-apoptotic markers (Bax and caspase-3). IMX treatment promotes the inhibitory phosphorylation of GSK-3ß at Ser9 and moreover, a marked reduction in the phosphorylation of IKK-ß, which prevents translocation and activation of NF-κB. Protein expression studies in IMX-treated brain tissues positively correlate with the anti-neuroinflammatory effects of GSK-3ß inhibition. Taken together, our results provide substantial evidence that IMX could potentially attenuate neuroinflammation in coordination with the master transcription factor-NF-κB.

Mouse Models to Study Natural Killer Cell-Mediated Immunosurveillance and Metastatic Latency.

Metastatic latency is a major concern in the clinic, yet how these disseminated cancer cells survive and initiate metastases is unknown (Massagué and Obenauf, Nature 529:298-306, 2016). Here, we describe an approach to isolate latency competent cancer (LCC) cells from early stage human lung and breast carcinoma cell lines using mouse xenograft models (Malladi, Cell 165:45-60, 2016). Cancer cell lines labeled with GFP-luciferase and antibiotic selection markers were injected intracardially into athymic mice. Three months, post-injection, LCC cells were identified in situ and isolated. Upon reinjection, LCC cells retain their tumorigenic potential, enter a slow-cycling or quiescent state, and evade NK cell-mediated innate immune surveillance.

Critical role of endoplasmic reticulum stress in chronic endothelial activation-induced visual deficits in tie2-tumor necrosis factor mice.

Diabetic retinopathy (DR) is the leading cause of vision loss among working-age adults. The interplay between hyperglycemia and endothelial activation in inducing endoplasmic reticulum (ER) stress pathways and visual deficits in DR is not fully understood. To address this, we used a mouse model of chronic vascular activation using endothelial-specific tumor necrosis factor-α (TNF-α)-expressing (tie2-TNF) mice to induce diabetes with streptozotocin. At 4 weeks post streptozotocin, a significant 2-fold to 10-fold increase in retinal neurovascular inflammatory gene transcript response in tie2-TNF mice was further increased in diabetic tie2-TNF mice. A decrease in visual acuity and scotopic b-wave amplitude in tie2-TNF mice was further accentuated in diabetic tie2-TNF mice and these changes correlated with a multi-fold increase in retinal ER stress markers and a reduction in adherens junctions. Cultured retinal endothelial cells showed a significant decrease in trans-endothelial resistance as well as VE-cadherin expression under TNF-α and high glucose stress. These changes were partly rescued by tauroursodeoxycholic acid, a potent ER stress inhibitor. Taken together, constant endothelial activation induced by TNF-α further exacerbated by hyperglycemia results in activation of ER stress and chronic proinflammation in a feed forward loop ultimately resulting in endothelial junction protein alterations leading to visual deficits in the retina. Inhibition of ER stress and endothelial activation may prove to be a novel therapeutic target in DR.

A baculovirus-conjugated mimotope vaccine targeting Mycobacterium tuberculosis lipoarabinomannan.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a major cause of morbidity and mortality worldwide. However, an effective vaccine for M. tuberculosis is lacking. We panned a phage display library using monoclonal antibodies against M. tuberculosis liporabinomannan (LAM), an important component of the M. tuberculosis cell wall, and identified two peptide sequences, HSFKWLDSPRLR or SGVYKVAYDWQH, with high antibody affinity after multiple rounds of panning. Only the HSFKWLDSPRLR peptide induced an anti-LAM response when conjugated to either keyhole limpet hemocyanin (KLH) or to the baculovirus Autographa californica multicapsid nucleopolyherovirus (AcMNPV) when introduced into mice by injection or via intranasal inoculation, respectively. Vaccination with AcMNPV conjugated HSFKWLDSPRLR peptide delayed mortality in a mouse model of tuberculosis. Thus, we report a proof of principle M. tuberculosis vaccination strategy combining an anti-LAM mimotope with a baculovirus delivery system.

The Ubiquitin Ligase Smurf1 Functions in Selective Autophagy of Mycobacterium tuberculosis and Anti-tuberculous Host Defense.

During antibacterial autophagy, ubiquitination of intracellular bacteria recruits proteins that mediate bacterial delivery to the lysosome for degradation. Smurf1 is an E3 ubiquitin ligase whose role in selective bacterial autophagy is unknown. We show that Smurf1 facilitates selective autophagy of the human pathogen Mycobacterium tuberculosis (Mtb). Smurf1-/- macrophages are defective in recruiting polyubiquitin, the proteasome, the ubiquitin-binding autophagy adaptor NBR1, the autophagy protein LC3, and the lysosomal marker LAMP1 to Mtb-associated structures and are more permissive for Mtb growth. This function of Smurf1 requires both its ubiquitin-ligase and C2 phospholipid-binding domains, and involves K48- rather than K63-linked ubiquitination. Chronically infected Smurf1-/- mice have increased bacterial load, increased lung inflammation, and accelerated mortality. SMURF1 controls Mtb replication in human macrophages and associates with bacteria in lungs of patients with pulmonary tuberculosis. Thus, Smurf1 is required for selective autophagy of Mtb and host defense against tuberculosis infection.

Lymphoblastoid Cell Lines as a Tool to Study Inter-Individual Differences in the Response to Glucose.

BACKGROUND: White blood cells have been shown in animal studies to play a central role in the pathogenesis of diabetic retinopathy. Lymphoblastoid cells are immortalized EBV-transformed primary B-cell leukocytes that have been extensively used as a model for conditions in which white blood cells play a primary role. The purpose of this study was to investigate whether lymphoblastoid cell lines, by retaining many of the key features of primary leukocytes, can be induced with glucose to demonstrate relevant biological responses to those found in diabetic retinopathy. METHODS: Lymphoblastoid cell lines were obtained from twenty-three human subjects. Differences between high and standard glucose conditions were assessed for expression, endothelial adhesion, and reactive oxygen species. RESULTS: Collectively, stimulation of the lymphoblastoid cell lines with high glucose demonstrated corresponding changes on molecular, cellular and functional levels. Lymphoblastoid cell lines up-regulated expression of a panel of genes associated with the leukocyte-mediated inflammation found in diabetic retinopathy that include: a cytokine (IL-1B fold change = 2.11, p-value = 0.02), an enzyme (PKCB fold change = 2.30, p-value = 0.01), transcription factors (NFKB-p50 fold change = 2.05, p-value = 0.01), (NFKB-p65 fold change = 2.82, p-value = 0.003), and an adhesion molecule (CD18 fold change = 2.59, 0.02). Protein expression of CD18 was also increased (p-value = 2.14x10-5). The lymphoblastoid cell lines demonstrated increased adhesiveness to endothelial cells (p = 1.28x10-5). Reactive oxygen species were increased (p = 2.56x10-6). Significant inter-individual variation among the lymphoblastoid cell lines in these responses was evident (F = 18.70, p < 0.0001). CONCLUSIONS: Exposure of lymphoblastoid cell lines derived from different human subjects to high glucose demonstrated differential and heterogeneous gene expression, adhesion, and cellular effects that recapitulated features found in the diabetic state. Lymphoblastoid cells may represent a useful tool to guide an individualized understanding of the development and potential treatment of diabetic complications like retinopathy.

Microfold Cells Actively Translocate Mycobacterium tuberculosis to Initiate Infection.

The prevailing paradigm is that tuberculosis infection is initiated when patrolling alveolar macrophages and dendritic cells within the terminal alveolus ingest inhaled Mycobacterium tuberculosis (Mtb). However, definitive data for this model are lacking. Among the epithelial cells of the upper airway, a specialized epithelial cell known as a microfold cell (M cell) overlies various components of mucosa-associated lymphatic tissue. Here, using multiple mouse models, we show that Mtb invades via M cells to initiate infection. Intranasal Mtb infection in mice lacking M cells either genetically or by antibody depletion resulted in reduced invasion and dissemination to draining lymph nodes. M cell-depleted mice infected via aerosol also had delayed dissemination to lymph nodes and reduced mortality. Translocation of Mtb across two M cell transwell models was rapid and transcellular. Thus, M cell translocation is a vital entry mechanism that contributes to the pathogenesis of Mtb.