United States

Background: SARS-CoV-2 or (CoV2) utilizes the receptor binding domain (RBD) of its surface SPIKE protein to infect human cells that express ACE2 receptor. Infected persons can be asymptomatic or progress to a potentially fatal pneumonia-associated illness. Antibodies that prevent SPIKE/RBD/ACE2 interaction can result from infection or vaccination. However, their quantity, function, and cross-reactive protection requires serological testing. We obtained 50 deidentified serum samples, and were blinded to CoV2 vaccination (Vax+; Vax-) or CoV2 positive test (CoV2+; CoV2-). Serum IgG antibodies to SPIKE and RBD were quantified and efficacy in preventing CoV2 infection in vitro determined. We hypothesized that high titer RBD IgG would be efficient at preventing viral infection and be effective against a later CoV2 variant.

Methods: ELISA was used to quantify SPIKE and RBD antibodies in sera. Neutralizing capability of antibodies was determined by mixing sera with a firefly luciferase lentivirus psuedotyped with Wuhan CoV2 SPIKE. The mixture was applied to ACE2 expressing cells and luciferase activity used to calculate infection rates. Samples were unblinded to determine correlation between in vitro results and CoV2 exposure or vaccination. Cross-protection was determined by repeating neutralization assays with the Omicron BA.2 variant.

Results Thirteen participants reported no CoV2 vaccine or CoV2 positive test (Vax-/CoV2-) yet 7 had anti-SPIKE and -RBD titers and neutralizing function suggesting prior asymptomatic CoV2 infection. The remaining 37 participants reported Vax-/CoV2+ (n=8), Vax+/CoV2- (n=27), or Vax+/CoV2+ (n=2). Titers to RBD were comparatively lower than titers to SPIKE in most patient samples. Average RBD antibody titers were higher for the Vax+/CoV2- group (1:2600) versus Vax-/CoV2+ cohort (1:900). Highest RBD antibody titers were found in Vax+/CoV2+ patients (1:5400). Finally, sera with RBD titer ≥1:2700 and neutralization capacity had equal neutralizing function on Omicron BA.2, a variant with 28 amino acid changes in SPIKE and 16 in the critical RBD region.

Conclusion High-titer RBD sera correlated with greater viral neutralization activity highlighting the functional importance of this domain in infection. High-titer anti-RBD IgG to the initial Wuhan CoV2 virus also provided potent neutralization activity against the BA.2 variant despite 16 amino acid changes in its RBD region. This information could be used in future vaccination efforts and passive immunization, as well as provide a greater level of confidence to healthcare workers, first responders, and other high-risk populations regardless of the CoV2 strain. Serial immune studies in high risk populations could provide indications for booster vaccines.

2023 AMA Research Challenge

Functional Serologic Testing and Cross Reactivity of Serum Antibodies to SARS-CoV-2 SPIKE Protein

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Background:
Atherosclerosis is a vascular inflammatory disease that is the leading cause of morbidities and mortalities worldwide, with late-stage plaque instability as the major pathology in myocardial infarction. The pathogenesis of atherosclerosis consists of lipid deposits in the inner wall of vasculature and chronic inflammation induced by oxidized lipoproteins. Efferocytosis is a process that contributes to the clearance of apoptotic cells in the plaque but is impaired in the late stage of the disease. Mitogen-activated protein kinase (MAPK) phosphatase-5 (MKP-5) is known to regulate tissue fibrosis and the dampening of inflammatory responses. MKP-5 deficiency in late-stage atherosclerosis contributes to greater plaque instability through thinner fibrous caps and increased necrosis in the atherosclerotic plaque by impaired efferocytosis. This project aimed to conduct an RNA sequencing (RNAseq) analysis and to identify pathways and genes of interest, highlighting the regulatory role of MKP-5 in late-stage atherosclerotic plaque stability. 
Methods:
Bone-marrow-derived macrophages (BMDMs) isolated from wild-type (WT) and MKP-5 knockout (MKP-5-/-) mice were stimulated with ox-LDL for varying durations (0 hours, 6 hours, and 24 hours). Bulk RNAseq was performed on these cells generating a binary alignment mapping (BAM) and subjected to principal component analysis (PCA) to validate the data. Volcano maps were then generated for each marker of stimulation, highlighting genes of interest. Gene set enrichment analysis (GSEA) was then conducted to identify the most enriched gene sets. A heatmap was used to visualize the expression levels of several enriched gene sets and known gene pathways, namely efferocytosis. To study efferocytosis capability directly, WT and MKP-5-/- BDMDs were incubated with apoptotic Jurkat cells and subsequently analyzed with confocal microscopy and flow cytometry at different time points (0 hours, 30 minutes, 1 hour, 2 hours). 
Results: 
Compared to WT BMDMs, we identified 40720 differential expressed genes (p < 0.05) in MKP-5-/- cells, including 1267 upregulated genes (FC > 2), and 1460 downregulated genes (FC < 0.5) at basal conditions. In 6 hours of ox-LDL stimulation, we found 4050 differential expressed genes including 303 upregulated and 484 downregulated genes in MKP-5-/- BMDMs. In 24 hours of stimulation, we found 37195 differential expressed genes including 892 upregulated and 477 downregulated genes. Through GSEA analysis on these significant genes, we found the top 15 WT enriched and MKP-5-/- enriched gene sets (q > 0.25). The MKP-5-/- enriched gene sets include protein tyrosine kinase binding and small GTPase-mediated signal transduction. The WT enriched gene sets include intracellular transport and stress-activated protein kinase signaling cascade. The efferocytosis pathways were a particular focus of our research, and corresponding heat maps show that efferocytosis was significantly inhibited in MKP-5-/- BMDMs. Furthermore, in-vitro assays indicate that the loss of MKP-5-/- impairs efferocytosis. 
Conclusion: 
Through bioinformatics analysis and biological assays, our results show that MKP-5 is essential for efferocytosis in atherogenic conditions.

Bioinformatics analysis identifies the crucial role of MAP Kinase Phosphatase-5 in regulating macrophage functions in atherogenesis

Background:
Intrahepatic cholangiocarcinoma (iCCA) is an aggressive cancer with few treatment options. Oncolytic viruses (OV) can induce anti-tumor immunity by exploiting immuno-inflammatory responses. Emerging concepts suggest cooperative immune evasion between dsDNA Vaccinia (VACV) and other OVs, like ssRNA Vesicular Stomatitis Virus (VSV) can enhance anti-tumor immunity. This project tests the hypothesis that VACV attenuation of interferon-α/β (IFN) enhances VSV oncolysis and immunogenicity.
Methods: 
To understand iCCA susceptibility to select OVs, we infected iCCA cells with VACV +/- VSV. To elucidate OV efficacy as a function of IFNα/β stimulation, we measured cytotoxicity and viral replication via MTT assay and 50% tissue culture infectious dose (TCID50) +/- IFNα stimulation. To study cooperative immune evasion (VACV-induced IFNα/β suppression enhancing VSV oncolysis), we quantified proteins from pathways that activate IFNα/β. Western blots (WB) of STING (DNA pathogen sensing) & RIG1 (RNA pathogen sensing) were performed to evaluate the impact of infection on IFN-stimulating cell-signaling. 
To assess efficacy of VACV+VSV in vivo as a function of IFNα/β response, we infected patient-derived iCCA xenograft (PDX) with PBS, VACV, VSV, or VACV+VSV. Tumor size and IFNα/β signaling (RIG1/STING) were assessed. Tumor growth abrogation was evaluated with repeated measures, one-way ANOVA, and post hoc multiple comparison was made with Dunnett’s test between groups.
Results: 
In vitro, VACV & VSV both show dose-dependent viral replication and cytotoxicity against iCCA measured through TCID50 and MTT cell viability assays. IFNα pretreated iCCA cells showed significant and robust upregulation of both RIG1/STING following viral infection with VACV (p=0.003/0.03, respectively), slightly less intense upregulation was seen in VACV+VSV combination (p=0.02/0.03, respectively), and no change was seen with VSV infection (p=0.89/0.78, respectively).
In vivo, iCCA PDX growth was significantly abrogated in the VACV group (p=0.007) & combination group (p=0.04) compared to control (figure 1). ANOVA results showed significant effect of treatment on tumor volume changes [F(1.385, 12.46)=11.97, p=0.0025, η²=0.5708]. No significant differences observed between the PBS & VSV groups (p=0.5452). WB quantification showed upregulated RIG1 in the combination group (p=0.03) and no significant difference between groups for STING expression (p=.4304).
Conclusions: 
The VACV and VACV + VSV combination treatments resulted in significant tumor growth abrogation compared to VSV treatment and PBS control. However, the RIG1 and STING signaling suggests that this does not occur via immune evasion or rescue of VSV by VACV in this model. These findings underscore the therapeutic potential of combination OV in iCCA and warrant further investigation to elucidate the underlying mechanism.


Treating intrahepatic cholangiocarcinoma with combination viroimunnotherapy

Background: Cholangiocarcinoma (CCA) is a malignancy of the bile ducts with five-year survival rates of less than 10%. For those with early-stage disease, resection or liver transplantation can be curative. Unfortunately, existing diagnostic tools fail to enable definitive diagnosis, as CCA is notoriously difficult to distinguish from fibrotic or inflammatory lesions. Therefore, novel tools are needed to enable early and accurate diagnosis of CCA, especially in those with indeterminate lesions. Proteases are enzymes that are frequently dysregulated in cancer, including CCA, playing diverse roles in angiogenesis, invasion, and metastasis. We have previously developed a class of peptide-based diagnostic agents, activatable zymography probes (AZPs), which bind to cell membranes after proteolytic cleavage and can be used for ex vivo and in vivo cancer imaging. In this work, we sought to develop AZPs to distinguish CCA from fibrosis.

Methods: We induced CCA in the Mdr2-/- mouse model of biliary fibrosis via hydrodynamic injection of plasmids encoding for AKT, YAP1, and the Sleeping Beauty (SB) transposase. We synthesized a panel of 26 AZPs, applied them to liver tissue sections from CCA and Mdr2-/- mice, and performed whole-slide fluorescence imaging. Select AZPs were co-stained with antibodies against vimentin and cytokeratin 7 and validated in a mouse model of FBXW7/AKT-mutant CCA. Finally, AZPs were PEGylated to increase solubility, intravenously injected into CCA and Mdr2-/- mice, and livers were imaged with in vivo imaging system (IVIS) to assess tumor uptake in vivo. 

Results: We found that 14 AZPs, most notably AZP6, exhibited significantly increased binding to CCA tumors (padj¬ < 0.05, fold change > 1.5) relative to fibrotic bile ducts. In CCA tumors, AZP6 bound significantly more strongly to cells with higher expression of vimentin (p < 0.01) and lower expression of cytokeratin 7 (p < 0.001), suggesting that AZP6 is cleaved by mesenchymal cells. AZP6 also specifically labeled tumors from an FBXW7/AKT-mutant CCA model in a pattern similar to that seen in the YAP1/AKT model, demonstrating its generalizability. Finally, after in vivo administration, AZP6 demonstrated significantly more fluorescence in explanted livers from mice with CCA than mice with fibrosis (p < 0.01), enabling perfect discrimination of malignant and benign disease (AUC = 1, p = 0.009).

Conclusion: AZPs detect and localize dysregulated protease activity ex vivo and in vivo in mouse models of CCA, without false positives from benign biliary fibrosis. With further refinement, AZPs may aid in the diagnosis of CCA.

Detection of cholangiocarcinoma with protease activity probes

Background
Leukemia, lymphoma and myeloma accounted for nearly 10% of new cancer cases and more than 50,000 deaths in 2022. Although multiple treatment modalities exist, Chimeric Antigen Receptor (CAR) T cells have been particularly useful, using genetic modification of a patient’s harvested T cells to better target cancer cells. Current methods of altering antigen specificity and targeting, however, have proven to be inadequate over time, requiring additional genes to boost the immune response. One such gene is OX40, a co-stimulatory cassette member of the Tumor Necrosis Factor Receptor (TNFR) superfamily that is thought to boost T cell migration, proliferation and survival. Given these inherent qualities of OX40, we hypothesized that incorporating OX40 variants into CAR T cells could enhance their duration of response and tumor-targeting capabilities.

Methods
We combined variants of the OX40 gene with our 2nd generation CAR T cells, adding a costimulatory cassette to CD19 CARs. We started with transformations of plasmid cloning vectors containing OX40 genes, using these CD19 CAR/OX40 constructs to transiently transfect retroviral cell lines. The transient virus produced was used to transduce donor human T cells, inducing uptake of the CD19 CAR/OX40 vectors. Once transduced, T cell reactivity and longevity were assessed using ELISA assays, flow cytometry and cell counts.

Results
We successfully used our CD19 CAR/OX40 T cells to transduce various human donor T cells, demonstrating a 1:1 linear relationship between OX40 and CAR T cell expression via flow cytometry. We also found that the constructs produced markedly higher levels of OX40+ expression compared to endogenous levels, particularly in regulatory T cells. When tested against a Burkitt’s lymphoma line, our
constructs showed reactivity above the standard deviation. With regards to longevity, our highest proliferating construct was a truncated version that survived over 60 days in culture.

Conclusion
We were able to produce CD19 CAR/OX40 donor T cells that had increased longevity and enhanced hematologic tumor targeting capabilities over time, likely due to an increased regulatory T cell response. Surprisingly, our truncated construct survived and proliferated the most, suggesting that the missing signaling domain could be key to balancing OX40’s enhanced growth effects versus over-proliferation.
With the endogenous signaling domain in place, OX40 is a promising addition to our CAR repertoire, boosting the in vitro antitumor response while balancing sustained proliferation and longevity. Our next steps will include translation into in vivo mouse models before bringing it to patients’ bedsides as a novel immunotherapy treatment.

Driving CARs Forward: An OX40 Tale of Immunotherapy

The role of Lcn2 in Triple Negative Breast Cancer Organoids

The kappa-opioid receptor (KOR) has been a target of recent interest in the treatment of alcohol use disorder, due to its role in negatively reinforced ethanol drinking, whereby aversive stimuli can promote ethanol intake. The KOR is densely expressed in the nucleus accumbens (NAc) on terminals of afferent neurons from the ventral tegmental area (VTA), where their activation has been found to suppress dopamine release such as is observed during alcohol withdrawal. We have previously found that KOR agonism differs along the rostro-caudal axis of the NAc shell, and that there may be a sex-related difference in baseline levels of ethanol intake. In the present study, we sought to determine if KOR stimulation in the rostral NAc shell could reverse effects of dopamine afferent activation on ethanol drinking. In Experiments 1 and 2, we used Long-Evans rats, as well as female transgenic tyrosine hydroxylase (TH)-Cre rats on a Long-Evans background in Experiment 3, which have a TH promoter directing expression of Cre recombinase in dopamine-containing cells, and we allowed them to drink under an intermittent access model, which establishes binge-like ethanol drinking. After the establishment of stable drinking, we bilaterally infused an excitatory designer receptors exclusively activated by designer drugs (DREADD) virus in the VTA of Experiment 2 groups, and a Cre-dependent excitatory DREADD in the VTA of the Experiment 3 group. After time for transduction, we injected into the NAc shell (1) the DREADD agonist, clozapine-N-oxide (CNO) (Experiments 2-3), (2) the KOR agonist, U50,488H (Experiments 1-3), (3) CNO + U50,488H (Experiments 2-3), and (4) saline vehicle (Experiments 1-3), counterbalanced in a Latin-Square design, and measured subsequent ethanol drinking. Surprisingly, we found that both U50,488H and CNO, independently and combined, enhanced ethanol drinking in high-level drinkers, and that these effects were not additive. Furthermore, the opposite trend was seen in low-level drinkers, suggesting a phenotype-dependent effect. These results suggest that both the DREADD activation and the KOR agonist microinjection have a similar net effect on dopamine release into the NAc shell, and that these effects depended on the phenotype of the drinker. The findings support the use of KOR antagonism as a therapy for alcohol use disorder, despite its potential unconventional effects on the dopamine system, and the overall use of individualized medication that considers an individual’s sex and phenotype.

Effects of Dopamine and Kappa Opioid Receptor Activity in the Rostral Nucleus Accumbens Shell on Ethanol Drinking in Rats

Background

Numerous studies have explored COVID-19 in adults and pediatric patients with chronic illnesses, but limited research has focused on previously healthy children. This study reports results from a COVID-19 screening program implemented at two pediatric clinics in central California, focusing on the clinical presentation of COVID-19 in this population.

Methods

Patients seeking care at the clinics completed a screening questionnaire to identify symptoms consistent with COVID-19. Temperature and oxygen saturation were measured, and nasal swabs were collected for antigen testing. Chief complaints, final diagnoses, and test results were recorded. The severity of COVID-19 illness was categorized using the CDC's severity index.
1) Asymptomatic/ pre-symptomatic - patient reported no symptoms and vital signs were normal.
2) Mild disease - respiratory or GI symptoms were present with or without fever.
3) Moderate disease; evidence of lower respiratory tract involvement was present.
4) Severe disease; oxygen saturation was < 94%.
5) Critical disease; the patient was transferred to the ER for further treatment.

Results

Between 11/2020 and 12/2022, 11,649 COVID-19 antigen screening tests were performed. Patients ranged in age from 0.1 to 17.0 years (mean 8.7, SD 4.5). 47.7% of patients identified as female, 50.5% as male, and 1.8% as other. In total, 1,560 pts. (13.4%) tested positive. Among these, 665 (43%) were asymptomatic/pre-symptomatic, 560 (36%) had mild disease, 318 (20%) had moderate disease, and 17 (1%) had severe disease. No critical cases or transfers to the emergency room were reported. A higher body mass index (BMI) was associated with increased severity of illness. 4,446 patients reported no symptoms at the time of screening. Among these, 85% tested negative, and 15% tested positive. Demographics were similar between these 2 groups. 7,203 patients report symptoms consistent with COVID-19. Among these, 87.6% tested negative and were presumed to have an infection other than COVID-19, while 12.4% tested positive. Median severity of illness did not differ significantly between the 2 groups.

Conclusion

The screening program effectively detected pediatric COVID-19 cases, including asymptomatic patients. In healthy children, COVID-19 was mild, with severity similar to other viral infections. Higher BMI is linked to greater illness severity. These findings enhance understanding of acute COVID-19 in outpatient pediatric populations, emphasizing the need for proper screening to ensure safe delivery of care.

Clinical Manifestations of COVID-19 in Previously Healthy Pediatric Patients Presenting to a Primary Care Office.

Background
Over 500 clinical trials combining radiation (RT) and immune checkpoint blockade (ICB) have been initiated based on preclinical evidence that RT can augment local immunity and improve the efficacy of ICB. However, the majority of clinical trials have found no benefit of combining RT and ICB, raising questions about whether a synergy exists. We examined whether RT and ICB interact to beneficially stimulate the immune response in patients and explored genomic biomarkers of response to combination RT and ICB.

Methods
We analyzed 2,046 patients from 4 cohorts. The COSINR dataset is a randomized clinical trial of 22 patients with non-small cell lung cancer (NSCLC) treated with concurrent or sequential RT and ICB. Paired pre/on-treatment biopsies of an irradiated metastasis underwent exome and RNA-seq. On-treatment biopsies were obtained after RT and prior to ICB (sequential) or after RT and one cycle of ICB (concurrent). The UC cohort contains targeted sequencing of 58 NSCLCs treated with ICB alone, sequential RT and ICB, or concurrent RT and ICB. The SU2C cohort comprises targeted sequencing of 306 ICB-treated NSCLCs from 11 institutions. The pan-cancer MSKCC cohort contains targeted sequencing of 1,660 patients treated with ICB. 

Aneuploidy score (AS) was defined as the fraction of chromosome arms with arm-level copy number alterations. Single-sample gene set enrichment analysis was used to compute Hallmark immune signaling pathway scores. Survival analyses utilized the Kaplan-Meier method and Cox proportional hazards models. 

Results
In the COSINR trial, RT and ICB increased, whereas RT alone decreased, expression of effector T-cell IFN-gamma and adaptive immune signatures (P<0.05). Baseline AS was negatively associated with expression of interferon, interleukin, and inflammatory signaling pathways (P<0.05). High AS was independently associated with poor OS following ICB after controlling for TMB and ICB paradigm in the MSKCC cohort (hazard ratio[95% CI]: 1.44[1.07–1.94], P=0.014) and controlling for TMB in the SU2C cohort (hazard ratio[95% CI]: 3.06[1.58–5.94], P=9.5e-4). In the UC cohort, high (≥median) AS tumors treated with concurrent RT and ICB had superior 1-year OS vs. sequential RT and ICB or ICB alone (76% vs. 38% vs. 31%, P=0.044). RT did not improve OS in low AS patients. Our findings were corroborated in the COSINR cohort: patients whose tumors exhibited high, but not low, AS had improved survival when treated with concurrent vs. sequential RT and ICB (1-year overall survival [OS] 100% vs. 17%, P=0.025).

Conclusions
Our findings distinguish the genomic and transcriptomic effects of RT versus RT and ICB and challenge the prevailing paradigm that local ablative RT alone positively stimulates the immune response. We propose the use of aneuploidy as a biomarker in personalizing immunotherapeutic treatment approaches. Concurrent RT and ICB may augment survival in patients with highly aneuploid, immunologically cold tumors.

Concurrent radiation and immunotherapy augments immunity and survival in aneuploid lung cancer

Background: Recurrent urinary tract infections (rUTI) are common in postmenopausal women with type 2 diabetes mellitus (T2DM). However, little is known about the composition of the urinary microbiome in this population and how it contributes to rUTI susceptibility. We aimed to identify key differences in urinary microbiome composition and function between women with and without T2DM through shotgun metagenomic sequencing of their urine.

Methods: Following IRB approval, urine samples were collected from consenting postmenopausal women with T2DM across 3 groups: rUTI remission (rUTI history, no active infection), rUTI relapse (active infection), and no history of UTI. Groups were age and estrogen-matched and DNA was purified from 20 mL of urine per patient. Sample concentrations were standardized and 20 pg of spike-in DNA of a rare bacterial genome was added. Shotgun metagenomic sequencing was performed using the Illumina NextSeq2000 platform. Reads were quality controlled and purified of human-mapping sequences using CLC Genomics Workbench. All microbial reads were taxonomically analyzed using MetaPhlan3. For further controls, data for non-diabetic women in the same groups were obtained.

Results: Fifteen TD2M and 15 non-T2DM samples were compared for this study. The mean genomic DNA yield across diabetic samples was 18.5 ng/uL. We observed an average of 75.8% human mapping reads, which were censored from further analysis. The remaining dataset contained an average of 5.1 million microbial reads. In the No UTI group, common urogenital flora microbes dominated across all samples in both T2DM and non-T2DM groups. In the rUTI Remission group, urogenital flora microbes were the most common again in non-diabetics, while common UTI pathogens such as Escherichia coli, Staphylococcus spp. and Pseudomonas aeruginosa were present in diabetic samples. For the rUTI Relapse group, both T2DM and non-T2DM were infected with common UTI pathogens (Escherichia coli, Klebsiella pneumoniae, Anaerococcus vaginalis, Enterococcus faecalis), however, 5/5 diabetic samples showed multi-strain infections while only 2/5 non-diabetic samples did.

Conclusion: We aim to identify differences in urinary microbiomes between women with T2DM with rUTI relapse, rUTI remission, and no history of UTI. By comparing our results with a published study on women without T2DM, we found that our cohort had ~10% more human mapping reads. Our pilot study suggest that T2DM patients may have higher rates of multi-strain infections compared to their non-T2DM counterparts. An expanded study (n = 60) is currently in progress to further understand the differences in urinary microbiomes in women with and without T2DM. 


Metagenomic Sequencing of the Urinary Microbiome of Postmenopausal Women with Recurrent Urinary Tract Infection and Type 2 Diabetes

Background
Obstructive sleep apnea (OSA) is a breathing disorder characterized by recurrent relaxation of the tongue and soft palate during sleep. The genioglossus (GG) muscle, the largest extrinsic tongue muscle, maintains upper airway patency. Obesity-induced OSA decreases muscular tone in the GG, causing airway obstruction. Other muscles impacted by obesity, such as the diaphragm (DIA) and the sternocleidomastoid, may contribute to OSA complications. Leptin partakes in central regulation of upper airway patency and diaphragmatic control. Leptin-
deficient obese (ob/ob) mice exhibit pharyngeal collapsibility and provide an effective model of OSA effects. By using ob/ob mice, we study the impact of obesity on GG, DIA, and sternomastoid (ST) muscles with a focus on the neuromuscular junction (NMJ), the best indicator of muscle function. 

Methods
GG, ST, and DIA muscles were extracted from 16 and 20-week wild-type (WT) and ob/ob male and female mice. Some samples were fixed and incubated with Alexa594TM alpha-bungarotoxin to visualize nicotinic acetylcholine receptors (nAChRs), and Alexa488TM Cholera toxin to label lipid rafts. They were imaged with a NikonTM inverted fluorescence microscope and a Confocal Microscope. Other GG and ST samples were “snap frozen,” sectioned in a cryostat, and stained
with H&E and NADH.

Results
GG muscles of ob/ob male mice showed a significant decrease in postsynaptic nAChRs and lipid rafts density compared to age-matched controls. In contrast to WT, GG of ob/ob male mice displayed multiply-innervated NMJs, nerve sprouting, and multiple Schwann cells engulfing nerves. Furthermore, the GG of ob/ob males exhibited central nuclei, intermuscular fat deposition, and hypertrophy. ST muscles in 16-week male WT and ob/ob mice displayed no differences in nAChR density, but nAChR density decreased in 20-week ob/ob male mice. The DIA showed higher nAChR density in ob/ob mice compared to controls. Female WT and ob/ob
mice showed insignificant changes in nAChR density in GG, ST, and Dia. 

Conclusion
The reduction in postsynaptic nAChRs and lipid rafts densities, and presynaptic remodeling of NMJs of GG observed in male ob/ob mice suggest synaptic disassembly. NMJ dismantlement, not observed in female ob/ob mice, may suggest female sex hormones contribute to synapse preservation through a neuroprotective pathway. Central nuclei in the GG indicate hypertrophic regeneration. Increased nAChR density in the male ob/ob DIA suggests a compensatory mechanism. Therefore, obesity-induced OSA-associated pathology exclusive to male GG, ST, and DIA affects the synaptic strength of these muscles. The observed sexual dimorphism and the possibility of reversing these observed changes should be further explored.

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