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Background Optimizing management of chronic kidney disease in people with HIV has become a more pressing priority both to minimize morbidity and preserve HIV antiretroviral options. Part of standard care is employing the beneficial effects of angiotensin converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARB) in patients with clinically significant proteinuria. Our analysis looks at the proteinuria outcomes of PWH with CKD co-managed by a multidisciplinary team of infectious disease and nephrology physicians nested in a Ryan White-funded clinic in New Orleans. Methods Patients with HIV and at least grade 3 CKD or with significant proteinuria were referred to the multidisciplinary clinic. Patients were evaluated by an HIV specialist and nephrologist at the initial visit for a jointly developed management plan. Renal function and proteinuria were measured at baseline and longitudinally per standard of care. Change in proteinuria and GFR over time were calculated. Patients were also assessed for whether they were prescribed ACE/ARB therapy, if appropriate. Patients included in this analysis were evaluated between January 1, 2021 and March 31, 2022. Results During the analysis period, 46 patients with at least CKD 3 or proteinuria were evaluated. 35 patients were Black non-Hispanic, 9 were White non-Hispanic, and 2 were Hispanic. 39 were male, and 7 were female. 35 had a previous diagnosis of hypertension, and 10 had diabetes mellitus. Of the 42 with a recorded baseline urine protein measurement, 24 were found to have clinically significant proteinuria. Of those with proteinuria, 16 were on an ACE or ARB at their baseline evaluation. 20 were on ACE/ARB’s the end of the analysis period (p=0.058). Average baseline proteinuria was 1,158 mg/g, while average final was 938 mg/g, which was less but not significantly different (p=0.54). Mean baseline GFR was not significantly different from final GFR (50.8 mL/min vs 50.5 mL/min, p=0.67). Conclusion A multidisciplinary model of managing CKD among people with HIV may improve adherence with management guidelines and limit progression of proteinuria and decrease in GFR. In general, the natural history of kidney disease with proteinuria is to worsen over time, so while not significant, proteinuria in this cohort did decrease over time Disclosures All Authors: No reported disclosures.

Background People with HIV (PWH) who achieve viral suppression have a normal lifespan, however, they now contend with more cardio-metabolic disease and exaggerated rates of malignancies. The etiology of these disparate rates of comorbidities are multifactorial but likely include modifiable behaviors such as smoking, poor diet, and substance use. We created a multidisciplinary team to study health behaviors in PWH. The team included researchers and HIV clinics in three Louisiana cities; New Orleans, Baton Rouge, and Shreveport, forming the Louisiana Translational Collaborative on Health Behaviors [LATCH] network. We hypothesized that patients with low health literacy would have higher BMIs and higher usage of tobacco and illicit drugs. Methods PWH aged ≥ 18 were recruited from three HIV clinics during regular appointments. Participants completed surveys using standardized tools that assessed diet, physical activity, health literacy, tobacco and substance use. Weight and height were also recorded. Results 100 PWH were enrolled. Most were African American (80%), men (57%), and the mean age was 50.72 (range 21-70). Mean BMI was 30.2 (range 18-51). Limited health literacy was found in 50% (REALM-SF < 7). Past month drug use was reported in 24%. Lifetime tobacco use was reported in 59%, with 31% reporting current use. For BMI, the average BMI for those with low health literacy (REALM-SF < 7) was 29.26 compared to 30.62 for those with adequate health literacy (REALM-SF 7) (p< .001). Among patients with limited literacy (REALM-SF < 7) 27.1% reported current tobacco use, compared with 36.7% of those with adequate literacy (REALM-SF 7) (p=0.68). Among patients with limited literacy (REALM-SF < 7) 25% reported past month drug use, compared with 24.5% of those with adequate literacy (REALM-SF 7) (p=0.59). Conclusion We hypothesized that PWH with lower health literacy would have higher BMIs, which was the opposite of what we found. We also found higher rates of smoking among those with higher health literacy although it was not significant. No differences were seen in past month drug use. Health literacy and education to improve health literacy may not directly impact modifiable health behaviors which are likely influenced by many different factors. Disclosures All Authors: No reported disclosures.

Introduction: Heterosexual couples are at high risk for HIV acquisition in sub-Saharan Africa, and HIV self-testing (HST) is an additional approach to expand access to HIV testing services. However, it is not well known how gender equality is associated with HST.Methods: We used intervention-arm data from a cluster-randomised controlled HST intervention trial (N = 1 618) conducted in Uganda to determine the association between attitudes towards intimate partner violence (IPV), decision-making power and male partner’s uptake of HST among heterosexual couples expecting a child in south-central Uganda. The original study question was to assess the impact of providing pregnant women with HST kits to improve male partner’s HIV testing rates. For this analysis, the primary exposures were gender equality (measured by male partner’s and female partner’s attitudes towards IPV and the female partner’s household decision-making power), and the primary outcome was the male partner’s uptake of HST. Multivariate logistic regression was used for analysis.Results: We found that male partner HST uptake did not vary depending on male partner’s attitudes towards IPV or decision-making power; however, male partner HST uptake did depend on the female partner’s attitude towards IPV, with 1.76 times more testing (95% CI 1.06–2.92) in couples where the woman had “medium” versus “high” acceptance of IPV, and 1.82 times more testing (95% CI 1.08–3.08) in couples where the woman had “low” versus “high” acceptance of IPV.Conclusions: This study shows the importance of appropriate negative attitudes by women to IPV in increasing male partner’s HST uptake to integrate HST into national health care policies.

Ophthalmic study of collagen CVII hypomorphic mice is uniquely challenging due to the strain's published survival rate to weaning of 24%. Because chronic ocular fibrosis requires time to develop, optimizing the survival rate is of critical importance. In this study, standard husbandry practices were enhanced by the addition of sterilized diet and drug delivery gels, acidified water, irradiated food pellets, cellulose fiber bedding, minimal handling, removal of siblings within 2-3 wk from birth, and a preferred housing location. Survival rates per breeding cycle, sex, weight, and cause of early euthanasia were recorded and analyzed over 43 mo. Overall, 49% of mice survived to weaning and 76% of weaned mice survived to 20 wk of age. Corneal opacities were seen in 65% of mice by 20 wk, but only 10% of eyes showed the sustained opacification that was indicative of fibrosis. Corneal opacities occurred at the same rate as in humans with epidermolysis bullosa. 66% of the mice showed weight loss at 11 wk. Males required early euthanasia 4 times more often than did females. Euthanasia was required for urinary obstruction due to penile prolapse in 88% of males. With our enhanced care protocol, hypomorphic mice in our colony survived at twice the published rate. With this revised husbandry standard, experiments planned with termination endpoints of 14 wk for males and 17 wk for females are more likely to reach completion.

Congestion is a major determinant of clinical outcomes in heart failure (HF). We compared the acute hemodynamic effects of occlusion of the superior (SVC) versus the inferior vena cava (IVC) and tested a novel SVC occlusion system in swine models of HF. IVC occlusion acutely reduced left ventricular (LV) systolic and diastolic pressures, LV volumes, cardiac output (CO), and mean arterial pressure (MAP). SVC occlusion reduced LV diastolic pressure and volumes without affecting CO or MAP. The preCARDIA system is a balloon occlusion catheter and pump console which enables controlled delivery and removal of fluid into the occlusion balloon. At 6, 12, and 18 h, SVC therapy with the system provided a sustained reduction in cardiac filling pressures with stable CO and MAP. Intermittent SVC occlusion is a novel approach to reduce biventricular filling pressures in HF. The VENUS-HF trial will test the safety and feasibility of SVC therapy in HF.

Cutaneous leiomyosarcomas are malignant mesenchymal tumors of smooth muscle origin and are reported occasionally in avian species. A 14-y-old male laboratory White Carneau pigeon ( Columba livia) was presented for surgical excision of a cervical soft tissue mass. Ultrasonography with color flow Doppler imaging revealed multiple cavitations of mixed echogenicity within the mass and vascularization. Histologically, the dermis and subcutis were expanded by a densely cellular multinodular mass comprised of fusiform cells forming haphazardly arranged broad streams and short interwoven bundles, often surrounding blood vessels and variably sized cavitations. Neoplastic cells were strongly immunopositive for desmin and α–smooth muscle actin, and negative for pancytokeratin, S100, and von Willebrand factor. Based on histopathology and IHC findings, the cutaneous mass was diagnosed as leiomyosarcoma (LMS). The pigeon died 312 d post-operatively. Postmortem examination revealed masses infiltrating the left and right pulmonary airways and one hepatic nodule, but no regrowth at the surgical site. Histologic and IHC evaluation of the pulmonary and hepatic masses were consistent with LMS, representing metastatic foci from the primary cutaneous LMS. Our case highlights the malignant behavior and histomorphologic features of cutaneous LMS in an avian species.

Purpose. Group B Streptococcus (S. agalactiae, GBS) is a Gram-positive opportunistic pathogen that inhabits the respiratory, urogenital and gastrointestinal tracts of humans and animals. Maternal colonization of GBS is a risk factor for a spectrum of clinical diseases in humans and a principle cause of neonatal meningitis and septicaemia.Methodology. We describe polymicrobial sepsis including GBS in two gravid adult female Long-Evans rats experiencing acute mortality from a colony of long-term breeding pairs. Fluorescent in situ hybridization confirmed GBS association with pathological changes in affected tissues, including the heart and uterus.Results. Characterization of seven GBS strains obtained from clinically affected and non-affected animals indicated similar antibiotic resistance and susceptibility patterns to that of human strains of GBS. The rat strains have virulence factors known to contribute to pathogenicity, and shared serotypes with human invasive isolates. Phylogenetic analyses revealed that one rat-derived GBS strain was more closely related to human-derived strains than other rat-derived strains, strengthening the notion that interspecies transmission is possible.Conclusions. To our knowledge, this is the first investigation of genotypic and phenotypic features of rat-derived GBS strains and their comparison to human- and other animal-derived GBS strains. Since GBS commonly colonizes commercially available rats, its exclusion as a potential pathogen for immunocompromised or stressed animals is recommended.

Individuals suffering from autoimmune disorders possess a hyperactive cellular phenotype where tolerance to self-antigens is lost. Autophagy has been implicated in both the induction and prevention of autoimmunity, and modulators of this cellular recycling process hold high potential for the treatment of autoimmune diseases. In this study, we determine the effects of a loss of autophagy in dendritic cells (DCs), as well as both B cells and DCs, in a TLR7-mediated model of autoimmunity, similar to systemic lupus erythematosus, where both cell types are critical for disease. Although a loss of DC autophagy slowed disease, the combined loss of autophagy in both cell types resulted in a lethal sepsis-like environment, which included tissue inflammation and hyperproduction of inflammasome-associated cytokines. Ablation of B cell signaling reversed this phenotype, indicating that activation of these cells is an essential step in disease induction. Thus, autophagy plays a dichotomous role in this model of disease.

Barth syndrome (BTHS) is an X-linked recessive disease where patients most commonly die from cardiomyopathy-induced heart failure before middle age. BTHS is caused by mutations in the tafazzin (TAZ) gene, resulting in defective TAZ protein. TAZ is an enzyme that generates mature cardiolipin (CL) from monolysocardiolipin (MLCL) in the mitochondrial membrane, a reaction essential for normal mitochondrial function. Current therapies can only treat the symptoms of BTHS. In this study, we propose an enzyme replacement therapy for BTHS which utilizes recombinant human TAZ fused to a cell penetrating peptide (hTAZ-CTP) to facilitate tissue uptake. The efficacy of this protein was tested in vitro on C2C12 TAZ-knockout (TAZ-KO) skeletal myoblasts and in vivo on a myocardial-specific TAZ conditional knockout mouse, modelling the cardiomyopathy associated with BTHS. In vitro tests of TAZ-KO cells, using oxygen consumption rate as a measure of mitochondrial activity, showed treatment of the cells with hTAZ-CTP effected a partial rescue of the fatty acid oxidation capabilities of the TAZ-KO cells. In vivo tests showed that BTHS mice display increasing septal wall thickness over time, an effect halted upon treatment with hTAZ-CTP. Pressure-volume (PV) loop analysis indicated that heart function, impaired in the vehicle-treated BTHS mouse, was similar between treated mice and normal mice. The ratio of MLCL/CL, a direct measure of TAZ enzymatic activity, was measured in heart mitochondria isolated from BTHS and control mice after treatment. The vehicle treated BTHS mouse showed the high MLCL/CL ratio typical of BTHS patients, whereas the MLCL/CL ratios in protein-treated mice matched the much lower ratio of the control mice. Similarly, oxygen consumption rate measurements of these isolated heart mitochondria demonstrated partial rescue by hTAZ-CTP treatment. Coupled with the lack of toxicity observed in the liver, spleen, kidney, and heart due to hTAZ-CTP injection, these results indicate that TAZ enzyme replacement therapy has great potential as a future treatment for BTHS.

Imbalance of power and equality in sexual relationships is linked to health in various ways, including (1) reduced ability to get information or take action, (2) increased violence between partners, and (3) influence on the reduced use of health services. While there has been research assessing multiple social and economic variables related to gender inequality, studies have used many different definitions of gender inequality, and there is a lack of this research within a pregnancy context. Here, we attempt to identify social and economic predictors of gender inequality (measured by decision-making power and acceptance of intimate partner violence) within heterosexual couples expecting a child in central Kenya. We ran a secondary data analysis using data from a three-arm individually randomized controlled HIV self-testing intervention trial conducted in 14 antenatal clinics in central and eastern Kenya among 1410 women and their male partners. The analysis included Cochran Mantel-Haenszel, logistic regression, proportional odds models, and generalized linear mixed model (GLMM) framework to account for site-level clustering. Overall, we show that there are significant social and economic variables associated with acceptance of intimate partner violence including higher age, being married, "other" religion, lower partner education, higher wealth status, and variables associated with decision-making power including lower partner education and lack of equality in earnings. This study contributes to the literature on the influence of social and economic factors on gender inequality, especially in Kenya which has a high burden of HIV/AIDS. Our results show some areas to improve these specific factors (including education and employment opportunities) or create interventions for targeted populations to potentially improve gender equality in heterosexual pregnant couples in Kenya.

Background Similar to other sub-Saharan countries, Ethiopia suffers from a severe shortage of adequately trained health professionals. Academic partnerships can support sustainable training programs and build capacity for low-resource settings. 3D modeling and simulation-based training provide necessary tools, especially for rarely-encountered clinical situations, such as needle cricothyroidotomy. Methods Departments of Anesthesiology, Otolaryngology, and Learning Health Sciences collaborated to develop a low-cost, high-fidelity simulator and Cricothryoidotomy Skills Maintenance Program (CSMP). Twelve anesthesia residents at St. Paul's Hospital Medical Millennium College in Addis Ababa, Ethiopia participated in CSMP. The program consisted of a didactic session with presentation and demonstration and an immersive CICO scenario. Program evaluation was performed using pre/post-training knowledge and 2 procedural performance assessments-the CSMP Global Rating Scale and the Checklist. With consent, performances were videotaped and rated independently by 3 University of Michigan faculty. Results Improvements were identified in all areas, including residents' knowledge, measured by mean summed test scores (Mpre = 3.31,Mpost = 4.46,p = 0.003), time to perform cricothyroidotomy (Mpre = 96.64,Mpost = 72.82,p = 0.12), residents' performance quality, measured by overall mean Global ratings, (Mpre = 0.20; Mpost = 0.70) with improvements identified at the item-level, p = 0.001 with moderate-large effect sizes, and residents' ability to complete tasks, measured by mean Checklist ratings (Mpre = 0.51,Mpost = 0.90, with item-level improvements observed, p ≤ 0.01, with small-large effect sizes. Residents' self-reported confidence also improved (Mpre = 1.69, Mpost = 3.08,p = 0.001). Conclusion Our work shows that cricothyroidotomy skills taught to anesthesia residents at SPHMMC with a 3D printed laryngotracheal model improves knowledge, skills, and confidence. The creation of a low-cost, high-fidelity simulator and a CSMP has the potential to impact patient care and safety world-wide.

One of the major contributors to sickle cell disease (SCD) pathobiology is the hemolysis of sickle red blood cells (RBCs), which release free hemoglobin and platelet agonists including adenosine 5’-diphosphate (ADP) into the plasma. While platelet activation/aggregation may promote tissue ischemia and pulmonary hypertension in SCD, modulation of sickle platelet dysfunction remains poorly understood. Calpain-1, a ubiquitous calcium-activated cysteine protease expressed in the hematopoietic cells, mediates aggregation of platelets in healthy mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice to investigate the role of calpain-1 in the steady state and hypoxia/reoxygenation (H/R)-induced sickle platelet activation and aggregation, clot retraction, and pulmonary arterial hypertension. Using multi-electrode aggregometry, which measures platelet adhesion and aggregation in whole blood, we determined that steady state SSCKO mice exhibit significantly impaired PAR4-TRAP-stimulated platelet aggregation as compared to Townes sickle (SS) and humanized control (AA) mice. Interestingly, the H/R injury induced platelet hyperactivity in SS and SSCKO, but not AA mice, partially rescued the aggregation defect in SSCKO mice. The PAR4-TRAP-stimulated GPIIb-IIIa/αIIbβ3 integrin activation was normal in SSCKO platelets suggesting that an alternate mechanism mediates the impaired platelet aggregation in steady state SSCKO mice. Taken together, we provide the first evidence that calpain-1 regulates platelet hyperactivity in sickle mice, and may offer a viable pharmacological target to reduce platelet hyperactivity in SCD.

To evaluate the biocompatibility of silk gel for cervical injection.Silk gel was injected into the cervix of pregnant rats on day 13 (n = 11) and harvested at day 17. Histology of silk gel was compared with suture controls. Also, human cervical fibroblasts were cultured on silk gel and tissue culture plastic (TCP) in vitro. Cell viability, proliferation, metabolic activity, gene expression (COL1A1, COL3A1, and COX2), and release of proinflammatory mediators (interleukin [IL] 6 and IL-8) were evaluated.In vivo, a mild foreign body response was seen surrounding the silk gel and suture controls. In vitro, cervical fibroblasts were viable, metabolically active, and proliferating at 72 hours. Release of IL-6 and IL-8 was similar on silk gel and TCP. Collagen and COX2 gene expression was similar or slightly decreased compared with TCP.Silk gel was well tolerated in vivo and in vitro, which supports continuing efforts to develop silk gels as an alternative to cervical cerclage.

Background Oxidative stress and mitochondrial dysfunction are central mediators of cardiac dysfunction after ischemia/reperfusion. ATP binding cassette mitochondrial erythroid (ABC-me; ABCB10; mABC2) is a mitochondrial transporter highly induced during erythroid differentiation and predominantly expressed in bone marrow, liver, and heart. Until now, ABC-me function in heart was unknown. Several lines of evidence demonstrate that the yeast ortholog of ABC-me protects against increased oxidative stress. Therefore, ABC-me is a potential modulator of the outcome of ischemia/reperfusion in the heart. Methods and Results Mice harboring 1 functional allele of ABC-me (ABC-me +/− ) were generated by replacing ABC-me exons 2 and 3 with a neomycin resistance cassette. Cardiac function was assessed with Langendorff perfusion and echocardiography. Under basal conditions, ABC-me +/− mice had normal heart structure, hemodynamic function, mitochondrial respiration, and oxidative status. However, after ischemia/reperfusion, the recovery of hemodynamic function was reduced by 50% in ABC-me +/− hearts as a result of impairments in both systolic and diastolic function. This reduction was associated with impaired mitochondrial bioenergetic function and with oxidative damage to both mitochondrial lipids and sarcoplasmic reticulum calcium ATPase after reperfusion. Treatment of ABC-me +/− hearts with the superoxide dismutase/catalase mimetic EUK-207 prevented oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and restored mitochondrial and cardiac function to wild-type levels after reperfusion. Conclusions Inactivation of 1 allele of ABC-me increases the susceptibility to oxidative stress induced by ischemia/reperfusion, leading to increased oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and to impaired functional recovery. Thus, ABC-me is a novel gene that determines the ability to tolerate cardiac ischemia/reperfusion.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, defined by loss of B cell self-tolerance that results in production of antinuclear antibodies (ANA) and chronic inflammation. While the initiating events in lupus development are not well defined, overexpression of the RNA-recognizing toll-like receptor (TLR)7 has been linked to SLE in humans and mice. We postulated that autophagy plays an essential role in TLR7 activation of B cells for the induction of SLE by delivering RNA ligands to the endosomes, where this innate immune receptor resides. To test this hypothesis, we compared SLE development in Tlr7 transgenic (Tg) mice with or without B cell-specific ablation of autophagy (Cd19-Cre Atg5f/f). We observed that in the absence of B cell autophagy the 2 hallmarks of SLE, ANA and inflammation, were eliminated, thus curing these mice of lupus. This was also evident in the significantly extended survival of the autophagy-deficient mice compared to Tlr7.1 Tg mice. Furthermore, glomerulonephritis was ameliorated, and the serum levels of inflammatory cytokines in the knockout (KO) mice were indistinguishable from those of control mice. These data provide direct evidence that B cells require TLR7-dependent priming through an autophagy-dependent mechanism before autoimmunity is induced, thereafter involving many cell types. Surprisingly, hyper-IgM production persisted in Tlr7.1 Tg mice in the absence of autophagy, likely involving a different activation pathway than the production of autoantibodies. Furthermore, these mice still presented with anemia, but responded with a striking increase in extramedullary hematopoiesis (EMH), possibly due to the absence of pro-inflammatory cytokines.

Most people have witnessed mirages such as the distant “puddles” that appear on a highway when the pavement is warmed by the Sun. The warmed surface heats the nearby air creating a temperature gradient with the cooler (and more dense) air above. The apparent displacement of distant objects occurs as light refracts through the different air densities. Rays of light from the sky that are originally directed toward the ground can be bent upward, appearing to a viewer as though coming from the ground. This effect is known as an inferior mirage; a superior mirage occurs when cooler air is underneath.1,2 In this paper, a mirage is created indoors using an electric hotplate and a saucepan filled with ice water.

CCN proteins play crucial roles in development, angiogenesis, cell motility, matrix turnover, proliferation, and other fundamental cell processes. Early embryonic lethality in CCN5 knockout and over-expressing mice led us to characterize CCN5 distribution in early development. Previous papers in this series showed that CCN5 is expressed widely in mice from E9.5 to adult; however, its distribution before E9.5 has not been studied. To fill this gap in our knowledge of CCN5 expression in mammals, RT-PCR was performed on preimplantation murine embryos: 1 cell, 2 cell, 4 cell, early morula, late morula, and blastocyst. CCN5 mRNA was not detected in 1, 2, or 4 cell embryos. It was first detected at the early morula stage and persisted to the preimplantation blastocyst stage. Immunohistochemical staining showed widespread CCN5 expression in post-implantation blastocysts (E4.5), E5.5, E6.5, and E7.5 stage embryos. Consistent with our previous study on E9.5 embryos, this expression was not limited to a particular germ layer or cell type. The widespread distribution of CCN5 in early embryos suggests a crucial role in development.

Introduction: With an estimated 15 million patients and no drug that addresses its etiology, sickle cell disease (SCD) remains an area of unmet need. Vaso-occlusive pain crisis (VOPC), the hallmark of SCD, is initiated by sickle RBCs (sRBCs) recruiting leukocytes and platelets to potentiate vessel occlusion. ADP released by sRBCs is a potent activator of platelets, and sickle cell patients are known to have activated platelets in circulation both at steady state and during VOPC. However, the mechanism underlying platelet dysfunction in SCD is not fully understood. Platelet activation mediated by the protease activated receptors (PAR1 and PAR4 in humans, PAR3 and PAR4 in mice), triggers PLC-β activation resulting in calcium mobilization. The increased calcium flux leads to activation of GPIIbIIIa/aIIbb3, GP1b, and P-selectin involved in platelet aggregation, adhesion, and rolling. Prior evidence has established a role of the calcium-activated cysteine protease, calpain-1 in platelet activation. Washed platelets from calpain-1 knockout C57BL/6 mice demonstrated impaired platelet aggregation. However, due to the critical contribution of sRBCs to platelet dysfunction in SCD, whole blood (impedance) aggregometry represents a physiological assessment of platelet aggregation. Methods: Townes SCD mice (SS) were backcrossed with calpain-1 knockout (CKO) mice to generate SCD mice lacking calpain-1 (SSCKO). Humanized mice (AA) were used as controls. Using flow cytometry, we evaluated in vivo platelet activation following stimulation with ADP and Thrombin GPRP. Platelet counts were obtained via ADVIA 120 and flow cytometry. For platelet aggregation, 500 μL of blood was harvested from the vena cava of AA, SS, SSCKO, and CKO mice. Whole blood aggregation in response to PAR4 stimulation was assessed using the Roche Multiplate Analyzer. A separate group of mice were challenged with hypoxia/reoxygenation (H/R) treatment (3 hours of 10.5% O2, followed by 4 hours of 21% O2) prior to platelet aggregation testing. SCD mice are characterized by tissue infarcts suggestive of thrombus formation. To examine whether H/R treatment induces formation of fibrin thrombi, we harvested brain, lungs, heart, kidneys, liver, and spleen following blood collection, and performed histology. Results: Compared to AA, SS and SSCKO mice are thrombocytopenic. Similar to Berkeley, Townes mouse platelets are activated in vivo, demonstrated by activated GPIIbIIIa on circulating platelets. At steady state, PAR4 agonist-induced platelet aggregation is similar in AA and SS mice (64 U v. 53 U, n = 6-10/group, p = 0.3). As depicted in Fig 1., SSCKO mice show significantly reduced platelet aggregation compared to SS mice (13 U v 53 U, p Figure 1. Impaired steady state whole blood platelet aggregation in SSCKO mice (panel 3). Figure 1. Impaired steady state whole blood platelet aggregation in SSCKO mice (panel 3). Figure 2. H/R relieves calpain-1-mediated inhibition of platelet aggregation in SS-CKO mice (panel 3). Figure 2. H/R relieves calpain-1-mediated inhibition of platelet aggregation in SS-CKO mice (panel 3). Disclosures Jakubowski: Eli Lilly and Company: Employment, Equity Ownership. Frelinger:GL Synthesis: Research Funding; Celerion: Research Funding; Megakaryon: Research Funding; Bristol-Myers Squibb: Research Funding; Sysmex: Research Funding; Eisai: Research Funding; Baxalta: Research Funding; Pfizer: Research Funding; GE Global Research: Research Funding; NIH: Research Funding.

The transfer of fetal cells to maternal organs occurs in mouse and human pregnancy. Techniques such as polymerase chain reaction and flow cytometry do not permit study of fetal cell morphology or anatomic location. Using a green fluorescent protein (GFP) transgenic mouse model, our objective was to determine whether GFP+ signal emanates from intact or degraded fetal cells, and whether they have a characteristic appearance and location within maternal lung. Four wild-type female mice were mated to males homozygous for the Gfp transgene and studied at days e16–18. Controls were 2 females mated to wild-type males. Morphologic appearance and anatomic position of each GFP+ object within maternal lung was recorded. GFP signals were sufficiently bright to be visualized without anti-GFP antibody and were confirmed by confocal microscopy to be separate from fluorescent artifact. Of 438 GFP+ objects detected, 375 (85.6%) were from intact cells, and 63 (14.4%) were acellular. Four distinct categories of intact cells were observed. Of these, 23.2% had mononuclear morphology with a relatively large nucleus and GFP+ cytoplasm (Group A). An additional group of cells (10.1%) had mononuclear morphology and podocyte extensions (Group B). The remainder of cells had fragmented nuclei or cytoplasm. Both intact cells and acellular fragments were predominantly localized to the maternal alveolar septum (P

To determine whether chemically induced miscarriage affects fetomaternal trafficking in a mouse model, we measured the amount of fetal DNA present in various maternal organs by polymerase chain reaction amplification following exposure to lipopolysaccharide (LPS). As the frequency of fetal cells and the number of animals with detectable microchimerism following LPS injection were significantly increased, particularly in lung tissue compared to controls, with no signs of an inflammatory response, we conclude that LPS-induced miscarriage results in increased murine fetomaternal cell trafficking, supporting a relationship between fetal loss and the establishment of fetal cell microchimerism.

A novel Pasteurella-like organism was recovered postmortem from lung tissue of two captive Wahlberg's epauleted fruit bats (Epomophorus wahlbergi), with severe, unilateral pneumonia. The bats had been recently shipped and died shortly after release from a 30-day quarantine. One presented with clinical signs of anorexia and lethargy before death; the other died without prior clinical symptoms. The same Pasteurella-like organism was recovered antemortem from subcutaneous abscesses in two captive little golden mantled flying foxes (Pteropus pumilus) housed with additional E. wahlbergi. The organism was also cultured on tracheal wash from one Malaysian flying fox (Pteropus vampyrus) and another E. wahlbergi, both demonstrating clinical signs of pneumonia. All recovered isolates appeared morphologically and biochemically similar to the initial isolates and were further characterized as either a Pasteurella or Actinobacillus organism on the basis of biochemical and cellular fatty acid profiles. Screening of the current collection using pharyngeal swabs isolated this organism from 12 of 15 E. wahlbergi, two of three P. vampyrus, one of 26 island flying foxes (Pteropus hypomelanus), and one of nine Rodrigues fruit bats (Pteropus rodricensis). The organism was not identified in pharyngeal culture from eight Indian flying foxes (Pteropus giganteus), nine Egyptian fruit bats (Rousettus aegypticus), or an additional 16 P. pumilus.

Systemic Lupus Erythematosus (SLE) is a heterogeneous disease, defined by a loss of B cell self-tolerance resulting in the production of autoantibodies. One of the many mechanisms described resulting in a break in B cell self-tolerance is overexpression of the endosomal toll-like receptor, TLR7. We hypothesized that autophagy plays a crucial role in TLR7 activation in B cells by delivering ligand to endosomes. To test this model, we produced TLR7 transgenic (tg) mice with a B cell-specific (CD19-cre) ablation of autophagy (ATG5 ko), and compared SLE development to that of TLR7 tg mice with intact autophagy machinery. We observed that loss of ATG5 in B cells significantly increased the survival of TLR7 tg mice, reduced serum IgG and anti-nuclear autoantibodies, thereby preventing glomerulonephritis. These data suggest that B cell autophagy is required for TLR7-mediated SLE disease progression. However, while symptoms of SLE were ameliorated, extramedullary hematopoiesis (EMH) was dramatically increased with a splenic red pulp expansion in these mice. Thus, autophagy in B cells plays an important role in controlling lineage determination in hematopoiesis, likely by regulating chemokine and cytokine production, the mechanism by which is currently under investigation. A better understanding of the control steps involved in TLR7 activation and cytokine production could lead to improved therapeutics for treatment of SLE.

The endosomal Toll-like receptor, TLR7, acts as critical line of defense against viral pathogens; however, overexpression of TLR7 has been shown to lead to spontaneous lupus-like disease (SLE). Thus, tight regulation of this pattern recognition receptor is crucial in preventing a hyperactive cellular state. We hypothesized that autophagy plays a role in TLR7 activation by delivering ligand to endosomes and by controlling subsequent inflammatory responses. To test this model, we crossed TLR7 transgenic (tg) mice that succumb to SLE with mice where ATG5, a critical autophagy gene, was deleted in dendritic cells (DCs) or B cells or both. These mice were compared to TLR7 tg mice with intact autophagy for morbidity and the inflammatory response. We observed that loss of ATG5 in either DCs or B cells significantly increased the survival of TLR7 tg mice, while ATG5 deletion in both cell types only modestly improved survival. On the other hand, loss of autophagy led to an elevated inflammatory response compared to TLR7 tg mice with intact autophagy, as evidenced by vastly increased lymphoid organ mass and elevated numbers of granulocytes and macrophages. The inflammatory mediators involved in this process are currently under investigation. These data suggest that autophagy plays an important and dichotomous role in TLR7-mediated autoimmunity, both enabling and ameliorating symptoms of SLE. It is likely that autophagy is affecting multiple TLR7-dependent signaling processes.