Molecular and pathological characterization of natural co-infection of poultry farms with the recently emerged Leucocytozoon caulleryi and chicken anemia virus in Egypt.

In the summers of 2018 and 2019, a disease outbreak stroke 25 broiler chicken farms and 3 broiler breeder farms in different Governorates in Egypt. The disease caused a mortality rate ranging from 3.2 to 9%. Postmortem examination showed petechial hemorrhage in the breast and thigh muscles, thymus gland, and peritoneal cavity and extensive hemorrhages in the kidneys. A total of 140 liver, kidney, lung, skeletal muscles, thymus, and spleen samples were collected. Twenty-eight pooled samples were created and examined by PCR and histopathological examination to identify the causative pathogens. All collected samples were PCR-negative to Newcastle disease virus (NDV), avian influenza viruses (H5, H9, and H7), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and fowl adenovirus (FadV). Leucocytozoon caulleryi (L. caulleryi) genetic material was identified by PCR in 17 out of the 28 collected samples (61%). Five chicken farms (18%) showed positive PCR results for both L. caulleryi and chicken anemia virus (CAV). Histopathological examination revealed unilocular megaloschizonts in thymus, skeletal muscle, and lung as well as massive hemorrhages in parenchymatous organs. Nucleotide sequences of the identified pathogens were compared with other reference sequences available in the GenBank. The identified L. caulleryi has a close relationship with those previously detected in Asia, indicating potential transmission route of the parasite. The CAV has a close genetic relation with CAVs previously identified in Egypt. Furthermore, a real-time PCR for rapid, specific, and quasiquantitative detection of L. caulleryi was developed with a detection limit of 100 genome copies per reaction.

A Highly Sensitive Molecular Technique for RNA Virus Detection.

Zika (ZIKV) and Chikungunya (CHIKV) viruses are mosquito-transmitted infections, or vector-borne pathogens, that emerged a few years ago. Reliable diagnostic tools for ZIKV and CHIKV-inexpensive, multiplexed, rapid, highly sensitive, and specific point-of-care (POC) systems-are vital for appropriate risk management and therapy. We recently studied a detection system with great success in Mexico (Villahermosa, state of Tabasco), working with human sera from patients infected with those viruses. The research conducted in Mexico validated the efficacy of a novel two-step rapid isothermal amplification technique (RAMP). This approach, which encompasses recombinase polymerase amplification (RPA) followed by loop-mediated isothermal amplification (LAMP), had been previously established in the lab using lab-derived Zika (ZIKV) and Chikungunya (CHIKV) viruses. Crucially, our findings confirmed that this technique is also effective when applied to human sera samples collected from locally infected individuals in Mexico.

Collaborative Development and Implementation of a Hybrid Virtual Surgery Clerkship Curriculum in a Vietnamese Medical School.

OBJECTIVE: To collaboratively develop a hybrid virtual curriculum for a medical school surgery clerkship within an emerging medical university in Vietnam. DESIGN: A hybrid virtual surgery clerkship curriculum was collaboratively developed by Vietnamese and American surgeons as part of an international partnership between their respective universities. A set of 25 virtual lectures with associated materials were created and deployed in tandem with live, in-person review and skills sessions. Student quantitative and qualitative evaluation methods were developed and deployed to allow continuous iteration. A 6-month course was deployed to develop surgical faculty into effective teachers. SETTING: The curriculum was deployed at VinUniversity College of Health Sciences, the youngest medical university in Vietnam. It was developed in collaboration with the University of Pennsylvania Perelman School of Medicine. Each cohort of 12 students in the surgical clerkship will participate in the curriculum. CONCLUSIONS: The development of this hybrid surgical clerkship in Vietnam leveraged local resources and expertise with those available remotely. Lessons learned are directly applicable to future collaborative curriculum development efforts at other emerging medical schools.

Providing Remote Aid During a Humanitarian Crisis.

Humanitarian crises create opportunities for both in-person and remote aid. Durable, complex, and team-based care may leverage a telemedicine approach for comprehensive support within a conflict zone. Barriers and enablers are detailed, as is the need for mission expansion due to initial program success. Adapting a telemedicine program initially designed for critical care during the severe acute respiratory syndrome coronavirus 2 pandemic offers a solution to data transfer and data analysis issues. Staffing efforts and grouped elements of patient care detail the kinds of remote aid that are achievable. A multiprofessional team-based approach (clinical, administrative, nongovernmental organization, government) can provide comprehensive consultation addressing surgical planning, critical care management, infection and infection control management, and patient transfer for complex care. Operational and network security create parallel concerns relevant to avoid geolocation and network intrusion during consultation. Deliberate approaches to address cultural differences that influence relational dynamics are also essential for mission success.

Design of comprehensive Alzheimer's disease centers to address unmet national needs.

The problem of Alzheimer's disease (AD) exemplifies the challenges of dealing with a broad range of aging-related chronic disorders that require long-term, labor-intensive, and expensive care. As the baby boom generation ages and brain diseases become more prevalent, the need to confront the pending health care crisis is more urgent than ever before. Indeed, there is now a critical need to expand significantly the national effort to solve the problem of AD, with special focus on prevention. The Campaign to Prevent Alzheimer's Disease by 2020 (PAD2020) aims to create a new paradigm for planning and supporting the organization of worldwide cooperative research networks to develop new technologies for early detection and treatments of aging-related memory and motor impairments. PAD 2020 is developing an implementation plan to justify (1) increasing the federal budget for research, (2) developing novel national resources to discover new interventions for memory and motor disorders, and (3) creating innovative and streamlined decision-making processes for selecting and supporting new ideas. Since 1978 the National Institute on Aging or National Institute of Health (NIH) established an extensive national network of AD research facilities at academic institutions including AD Centers (ADCs), Consortium to Establish a Registry for AD, AD Cooperative Study (ADCS), AD Drug Discovery Program, National Alzheimer's Coordinating Center, National Cell Repository for AD, and AD Neuroimaging Initiative. However, despite the success of these programs and their critical contributions, they are no longer adequate to meet the challenges presented by AD. PAD 2020 is designed to address these changes by improving the efficiency and effectiveness of these programs. For example, the ADCs (P30s and P50s) can be enhanced by converting some into Comprehensive Alzheimer's Disease Centers (CADCs) to support not only research, but also by being demonstration projects on care/treatment, clinical trials, and education as well as by seamlessly integrating multisite collaborative studies (ADCS, AD Neuroimaging Initiative, Patient Registries, Clinical Data Banks, etc) into a cohesive structure that further enhances the original mission of the National Institute on Aging ADCs. Regional CADCs offer greater efficiency and cost savings while serving as coordinating hubs of existing ADCs, thereby offering greater economies of scale and programmatic integration. The CADCs also broaden the scope of ADC activities to include research on interventions, diagnosis, imaging, prevention trials, and other longitudinal studies that require long-term support. Thus, CADCs can address the urgent need to identify subjects at high risk of AD for prevention trials and very early in the course of AD for clinical trials of disease modification. The enhanced CADCs will allow more flexibility among ADCs by supporting collaborative linkages with other institutions and drawing on a wider expertise from different locations. This perspective article describes the University of Pennsylvania (Penn) CADC Model as an illustrative example of how an existing ADC can be converted into a CADC by better utilization of Penn academic resources to address the wide range of problems concerning AD. The intent of this position paper is to stimulate thinking and foster the development of other or alternative models for a systematic approach to the study of dementia and movement disorders.

Sub-Saharan Africa Tackles COVID-19: Challenges and Opportunities.

As of May 2020, the global COVID-19 pandemic had reached 187 countries with more than 3.7 million confirmed cases and 263,000 deaths. While sub-Saharan Africa (SSA) has not been spared, the extent of disease is currently far less than in Europe or North America leading some to posit that climatic, genetic or other conditions will self-limit disease in this location. Nonetheless, infections in tropical Africa continue to rise at an alarming pace with the potential to soon exceed health resource availability and to exhaust a health care workforce that is already grossly under supported and ill-equipped. This perspective outlines the context of COVID-19 disease in Africa with a focus on the distinctive challenges faced by African nations and a potential best path forward.

How Should Schools Respond to Learners' Demands for Global Health Training?

In the past decade, more students than ever entered medical school with the desire, if not the expectation, of participating in meaningful global health experiences. Schools must now weigh benefits to students of global experiences against burdens of students' learning experiences on institutions and individuals with whom schools partner. Most often, global health training is done as offsite immersion rotations in research or clinical settings. This article explores ethical dimensions of expanding global health offerings while respecting local partners' goals by focusing on the experience of the University of Pennsylvania's global health training programs.

Modification of a viral envelope glycoprotein cell-cell fusion assay by utilizing plasmid encoded bacteriophage RNA polymerase.

Many viruses enter cells via an interaction of the viral envelope glycoprotein (Env) with receptor inducing fusion of viral and cellular membranes. These interactions are often evaluated in cell-cell fusion, gene-reporting systems with effector cells expressing Env and target cells expressing receptors. A common system utilizes vaccinia virus encoding T7 RNA polymerase (RNAP) in effector cells and a T7 promoted reporter plasmid in target cells. Fusion is quantified with expression of the reporter plasmid. However, direct activation of reporter plasmid from vaccinia virus can occur increasing background activity. We report here a modification of this assay in which T7 RNAP is expressed from a plasmid rather than vaccinia. This modification increased sensitivity with a ten-fold reduction in background. A novel dual T7/SP6 RNAP fusion assay was also developed to allow rapid screening for functional Env clones. Using these assays, we show that Envs from two CD4-independent HIV-2 isolates (VCP and ROD/B), which are able to fuse with chemokine receptor CXCR4 in a CD4-independent manner, are also able to fuse with alternative coreceptors GPR1 and GPR15 in the absence of CD4. The assay could also detect fusion of murine leukemia virus on target cells expressing the ecotropic MCAT-1 receptor showing its broad utility in other viral systems.

A Novel Point-of-Care BioNanoSensor for Rapid HIV Detection and Treatment Monitoring.

We report here a new diagnostic approach to the direct detection of HIV in blood or other body fluids that is rapid, sensitive and potentially applicable in a point-of-care setting. The approach follows on the development of a novel BioNanoSensor (BNS) device that utilizes piezoelectric technology to detect the presence of the HIV surface glycoprotein gp120 in a nanoscale format. The detection range of the BNS device for the biomarker gp120 displayed a low-end sensitivity of 6.5×104 HIV viral particles/ml, while using a small fluid sample (5 µl) and with a reaction time of less then 30 seconds. Performance of this device indicated that the BNS has utility for direct detection of HIV particles prior to, and independent from, antibody formation. Accordingly, this device holds utility to monitor the status of HIV infection both early after exposure to virus as well as during chronic HIV infection. The BNS parameters of small sample volume, compact device size, and detection sensitivity indicate that the BNS is potentially useful in the point-of-care and/or home setting for monitoring decisions regarding HIV treatment on a real-time basis.

Platelet- and megakaryocyte-derived microparticles transfer CXCR4 receptor to CXCR4-null cells and make them susceptible to infection by X4-HIV.

OBJECTIVE: Under some circumstances the HIV virus may infect cells that do not express receptors essential to HIV-entry. We hypothesized that platelet- and megakaryocyte-derived microparticles (MP) could play a role in such infections. MP are circular membrane fragments shed from the surface of eukaryotic cells. After adhesion to target cells, MP may transfer membrane-associated proteins to these cells. We found that peripheral blood platelet- (PMP) and megakaryocyte-derived MP (MegaMP) that highly express CXCR4 may transfer this receptor from the surface of platelets or megakaryocytes to the surface of CXCR4-null cells. DESIGN: Since this mechanism could potentially allow CD4+/CXCR4-null cells to become infected by T-tropic HIV, we incubated several human CD4+/CXCR4-null cells such as normal erythroblasts, glioblastomas U87, MAGI and hematopoietic cell lines UT-7, HEL and TF-1 with PMP or MegaMP. We found that these cells became CXCR4+. We next exposed these cells to X4-HIV (IIIB) and evaluated their susceptibility to infection by PCR, ELISA, and morphological analysis. RESULTS: We observed in all instances that after CD4+/CXCR4-null cell lines 'acquired' CXCR4 from PMP or MegaMP, they could became infected by X4 HIV. CONCLUSIONS: We postulate that both PMP and MegaMP may play a novel and important role in spreading HIV-1 infection by transferring the CXCR4 co-receptor to CD4+/CXCR4-null cells.

Intrathymic expression of neuromuscular acetylcholine receptors and the immunpathogenesis of myasthenia gravis.

The thymus has been considered to play an important role in the pathogenesis of myasthenia gravis (MG), an autoimmune disease characterized by skeletal muscle weakness. However, the pathogenic role of the thymus still remains a mystery. The neuromuscular type of acetylcholine receptor (AChR) was the first self-protein associated with a defined autoimmune disease that was found to be expressed by thymic stromal populations. The studies described herein represent our efforts to determine how this "promiscuous" autoantigen expression may be involved in the immunopathogenesis of MG. We review our work, characterizating the expression of the alpha subunit of AChR (AChRalpha) in the thymus, and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.

A new model linking intrathymic acetylcholine receptor expression and the pathogenesis of myasthenia gravis.

The thymus is thought to play an important role in the pathogenesis of myasthenia gravis (MG), an autoimmune disease characterized by skeletal muscle weakness. However, its role remains a mystery. The studies described represent our efforts to determine how intrathymic expression of the neuromuscular type of acetylcholine receptors (nAChRs) is involved in the immunopathogenesis of MG. We review our work characterizing the expression of the alpha subunit of nAChR (nAChRalpha) in the thymus and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.

A decrease in the cellular phosphodiester to phosphomonoester lipid ratio is characteristic of HIV-1 infection.

The ability to detect HIV-1 in tissues that are not readily amenable to biopsy greatly limits the diagnosis and control of HIV infection, and ultimately, our ability to understand HIV-induced disease pathology. In view of this, we explored the utility of diagnostically measuring HIV-1 infection using (31)P nuclear magnetic resonance ((31)P-NMR). (31)P-NMR enables the correlation of infection to changes in the concentration of specific intracellular metabolites, macromolecules and of bioenergetic parameters that are key to mammalian cell physiology. Examples include primary components of biological membranes such as phosphomonoester (PME) and phosphodiester (PDE) lipids. Using (31)P-NMR we found that changes in the ratio of PDE/PME in human cell lines and primary isolates were significantly altered following HIV-1 infection. Our findings showed that the ratio of cellular PDE/PME uniformly decreased 2.00-2.26 fold in HIV-1 infected cells. Using the altered PDE/PME ratio as a selection criterion, we next assessed HIV-1 infection in lymphocytes isolated from both HIV-1 seropositive and non-infected human subjects. A decreased PDE/PME ratio was characteristic of HIV-1 infection in each instance. These results demonstrate that changes in cellular phospholipids induced during HIV-1 infection may be used to uncover basic mechanisms of HIV-1 pathology, and potentially, may be extrapolated to explore the application of NMR analysis as a technique for imaging infected organs and tissues in situ.

HIV-1 infection inhibits cytokine production in human thymic macrophages.

OBJECTIVE: The thymus serves as a critical site of T-lymphocyte ontogeny and selection. Thymic infection by HIV-1 is known to disrupt thymocyte maturation by both direct and indirect means; however, the mechanism behind these effects remains poorly defined. Macrophages represent one of the most important peripheral targets of HIV-1 infection, are resident in the thymic stroma, and play a central role in thymocyte maturation. MATERIALS AND METHODS: Studies presented here define three primary features and outcomes of thymic macrophages (TM) and HIV-1 infection: (1) The distinctive TM phenotype (surface markers and cytokine production measured by immunofluorescence, fluorescence-activated cell sorting, and reverse transcriptase polymerase chain reaction) relative to macrophages from other sources (blood [monocyte-derived macrophages] and bone marrow); (2) infection of TM by different HIV-1 subtypes (X4, R5, and X4/R5) measured by enzyme-linked immunosorbent assay and polymerase chain reaction; and (3) consequences of HIV-1 infection on cytokine production by TM measured by reverse transcriptase polymerase chain reaction. RESULTS: The results demonstrate that TM display a distinctive phenotype of HIV-1 receptors (CD4(lo), CXCR4(lo), CCR5(med), CCR3(hi)), chemokine production (macrophage inflammatory protein-1α(+); regulated on activation, normal T expressed and secreted(+); macrophage inflammatory protein-1b(-); stromal cell-derived factor -1(-)); and cytokine production (tumor necrosis factor-α(+), interleukin-8(+), macrophage colony-stimulating factor(+), interleukin-6(-)) relative to either monocyte-derived macrophages or bone marrow. TM were infected in vitro with R5 and X4/R5-tropic HIV-1 subtypes, and developed syncytia formation during long-term X4/R5 culture. In contrast, TM supported only transient replication of X4-tropic HIV-1. Lastly, infection of TM with HIV-1 abolished the production of all cytokines tested in long-term in vitro cultures. CONCLUSIONS: Taken together, these results indicate that TM are a potential direct target of in situ HIV-1 infection, and that this infection may result in the disruption of macrophage functions that govern normal thymocyte maturation.

Facilitating emergency care research networks: integration into the Clinical Translational and Science Award (CTSA) infrastructure.

Emergency care research (ECR) does not fit neatly into the traditional National Institutes of Health (NIH) funding model, because emergency research involves undifferentiated disease presentations involving multiple disciplines and time-sensitive interventions. A task force of emergency care researchers and other stakeholders was convened to discuss the present and future state of clinical research networks. Integration of ECR with the Clinical Translational and Science Award (CTSA) program through a multidisciplinary emergency care research network (ECRN) would obviate the duplication of research efforts by disease-specific or institute-specific multicenter networks and reduce startup and maintenance costs. Strategies to enhance integration must include the training of emergency physician investigators in biostatistical and epidemiologic methods, as well as educating collaborative investigators in emergency care-related methodologies. Thus, an ECRN would be of great benefit to CTSA awardees and applicants and should be considered a priority.

TR1.3 viral pathogenesis and syncytium formation are linked to Env-Gag cooperation.

Infection with murine leukemia virus (MLV) TR1.3 or the related molecular construct W102G causes severe neuropathology in vivo. Infection is causally linked to the development of extensive syncytia in brain capillary endothelial cells (BCEC). These viruses also induce cell fusion of murine cell lines, such as SC-1 and NIH 3T3, which are otherwise resistant to MLV-induced syncytium formation. Although the virulence of these viruses maps within the env gene, the mechanism of fusion enhancement is not fully determined. To this end, we examined the capacity of the syncytium-inducing (SI) TR1.3 and W102G MLVs to overcome the fusion inhibitory activity inherent in the full-length Env cytoplasmic tail. These studies showed that the TR1.3 and W102G Envs did not induce premature cleavage of p2E, nor did they override p2E fusion inhibition. Indeed, in the presence of mutations that disrupt p2E function, the TR1.3 and W102G Envs significantly increased the extent of cell fusion compared to that with the non-syncytium-inducing MLV FB29. Surprisingly, we also observed that TR1.3 and W102G Envs failed to elicit syncytium formation in these in vitro assays. Coexpression of gag-pol with env restored syncytium formation, and accordingly, mutations within gag-pol were used to examine the minimal functional requirements for the SI phenotype. The results indicate that both gag-dependent particle budding and cleavage of p2E are required to activate the SI phenotype of TR1.3 and W102G viruses. Collectively, these data suggest that the TR1.3 and W102G viruses induce cell fusion by the fusion-from-without pathway.

Linkage of reduced receptor affinity and superinfection to pathogenesis of TR1.3 murine leukemia virus.

TR1.3 is a Friend murine leukemia virus (MLV) that induces selective syncytium induction (SI) of brain capillary endothelial cells (BCEC), intracerebral hemorrhage, and death. Syncytium induction by TR1.3 has been mapped to a single tryptophan-to-glycine conversion at position 102 of the envelope glycoprotein (Env102). The mechanism of SI by TR1.3 was examined here in comparison to the non-syncytium-inducing, nonpathogenic MLV FB29, which displays an identical BCEC tropism. Envelope protein expression and stability on both infected cells and viral particles were not statistically different for TR1.3 and FB29. However, affinity measurements derived using purified envelope receptor binding domain (RBD) revealed a reduction of >1 log in the K(D) of TR1.3 RBD relative to FB29 RBD. Whole-virus particles pseudotyped with TR1.3 Env similarly displayed a markedly reduced binding avidity compared to FB29-pseudotyped viral particles. Lastly, decreased receptor affinity of TR1.3 Env correlated with the failure to block superinfection following acute and chronic infection by TR1.3. These results definitively show that acquisition of a SI phenotype can be directly linked to amino acid changes in retroviral Env that decrease receptor affinity, thereby emphasizing the importance of events downstream of receptor binding in the cell fusion process and pathology.

The intrathymic pathogenesis of myasthenia gravis.

The thymus is considered to play an important role in the pathogenesis of Myasthenia gravis, an autoimmune disease characterized by antibody-mediated skeletal muscle weakness. However, its role is yet to be defined. The studies described herein summarize our efforts to determine how intrathymic expression of the neuromuscular type of acetylcholine (ACh) receptors is involved in the immunopathogenesis of this autoimmune disease. We review the work characterizing the expression of neuromuscular ACh receptors in the thymus and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.

Neuropathogenic murine leukemia virus TR1.3 induces selective syncytia formation of brain capillary endothelium.

Exposure of newborn BALB/c mice to murine leukemia virus (MLV) TR1.3 induces fusion of brain capillary endothelial cells (BCEC), loss of cerebral vessel integrity, hemorrhagic stroke, and death. Although TR1.3 infects endothelial cells in multiple organs, syncytia are only observed in BCEC. To determine if viral and cellular factors are responsible for selective syncytia formation, capillary endothelial cells (CEC) from multiple organs were assayed in vitro for MLV infection and cell fusion. Following incubation with virus, all CEC were infected to an equal extent as determined by expression of MLV envelope and infectious virus production; however, MLV-induced syncytia were only observed in TR1.3-infected BCEC cultures. These in vitro results mirror the in vivo pattern of TR1.3 MLV infection and neuropathology, and definitively show that selective fusion and pathology of BCEC by MLV is determined by properties unique to BCEC as contrasted to other endothelial cell types.

Disparate regions of envelope protein regulate syncytium formation versus spongiform encephalopathy in neurological disease induced by murine leukemia virus TR.

The murine leukemia virus (MLV) TR1.3 provides an excellent model to study the wide range of retrovirus-induced central nervous system (CNS) pathology and disease. TR1.3 rapidly induces thrombotic events in brain microvessels and causes cell-specific syncytium formation of brain capillary endothelial cells (BCEC). A single amino acid substitution, W102G, in the MLV envelope protein (Env) regulates the pathogenic effects. The role of Env in determining this disease phenotype compared to the induction of spongiform encephalomyelitis with a longer latency, as seen in several other MLV and in human retroviruses, was determined by studying in vitro-attenuated TR1.3. Virus cloned from this selection, termed TRM, induced progressive neurological disease characterized by ataxia and paralysis and the appearance of spongiform neurodegeneration throughout the brain stem and spinal cord. This disease was associated with virus replication in both BCEC and highly ramified glial cells. TRM did not induce syncytium formation, either in vivo or in vitro. Sequence and mutational analyses demonstrated that TRM contained a reversion of Env G102W but that neurological disease mapped to the single amino acid substitution Env S159P. The results demonstrate that single nucleotide changes within disparate regions of Env control dramatically different CNS disease patterns.