Non-human primate model of long-COVID identifies immune associates of hyperglycemia.

Hyperglycemia, and exacerbation of pre-existing deficits in glucose metabolism, are manifestations of the post-acute sequelae of SARS-CoV-2. Our understanding of metabolic decline after acute COVID-19 remains unclear due to the lack of animal models. Here, we report a non-human primate model of metabolic post-acute sequelae of SARS-CoV-2 using SARS-CoV-2 infected African green monkeys. Using this model, we identify a dysregulated blood chemokine signature during acute COVID-19 that correlates with elevated and persistent hyperglycemia four months post-infection. Hyperglycemia also correlates with liver glycogen levels, but there is no evidence of substantial long-term SARS-CoV-2 replication in the liver and pancreas. Finally, we report a favorable glycemic effect of the SARS-CoV-2 mRNA vaccine, administered on day 4 post-infection. Together, these data suggest that the African green monkey model exhibits important similarities to humans and can be utilized to assess therapeutic candidates to combat COVID-related metabolic defects.

Recapitulation of pathophysiological features of AD in SARS-CoV-2-infected subjects.

Infection with the etiological agent of COVID-19, SARS-CoV-2, appears capable of impacting cognition in some patients with post-acute sequelae of SARS-CoV-2 (PASC). To evaluate neuropathophysiological consequences of SARS-CoV-2 infection, we examine transcriptional and cellular signatures in the Brodmann area 9 (BA9) of the frontal cortex and the hippocampal formation (HF) in SARS-CoV-2, Alzheimer's disease (AD), and SARS-CoV-2-infected AD individuals compared to age- and gender-matched neurological cases. Here, we show similar alterations of neuroinflammation and blood-brain barrier integrity in SARS-CoV-2, AD, and SARS-CoV-2-infected AD individuals. Distribution of microglial changes reflected by the increase in Iba-1 reveals nodular morphological alterations in SARS-CoV-2-infected AD individuals. Similarly, HIF-1α is significantly upregulated in the context of SARS-CoV-2 infection in the same brain regions regardless of AD status. The finding may help in informing decision-making regarding therapeutic treatments in patients with neuro-PASC, especially those at increased risk of developing AD.

Lupus pathogenesis and autoimmunity are exacerbated by high fat diet-induced obesity in MRL/lpr mice.

OBJECTIVE: SLE is an autoimmune disease characterised by persistent inflammation and autoantibody production. Genetic predisposition and environmental factors such as a high-fat diet (HFD) may contribute to lupus development. However, the immune cell profile and gender difference in response to HFD in lupus have not been reported. Here we investigated the impact of HFD on lupus pathogenesis and autoimmunity using lupus-prone mice. METHODS: Thirty male and 30 female MRL/lymphoproliferation (lpr) mice were fed with regular diet (RD) or HFD. Body weights were recorded weekly. SLE progression was monitored by skin lesion, urine protein, titres of antidouble-strand DNA (dsDNA) and ANA. At week 14, kidney and skin tissue sections were stained with H&E and periodic acid-Schiff to detect histological kidney index and skin score. Splenocytes were identified by immunofluorescence staining and flow cytometry. RESULTS: HFD significantly increased body weight and lipid levels compared with RD (p<0.01). Skin lesions were observed in 55.6% of the HFD group compared with 11.1% of the RD group, with greater histopathological skin scores in the female HFD group (p<0.01). Although both male and female mice had higher serum IgG in the HFD group than in the RD group, only the male HFD group showed an increased trend in anti-dsDNA Ab and ANA titres. Kidney pathological changes in the HFD group were more severe in male mice than in female mice (p<0.05), detected by proteinuria, kidney index and glomerular cell proliferation. Significant increases of germinal centre B cells and T follicular helper cells were observed in the spleens of HFD mice (p<0.05). CONCLUSION: HFD induced an accelerated and exacerbated lupus development and autoimmunity in MRL/lpr mice. Our results parallel many known clinical lupus phenotypes and sexual dimorphism in which male patients are likelier to have a severe disease (nephritis) than female lupus patients who may have a broader range of lupus symptoms.

Molecular and cellular similarities in the brain of SARS-CoV-2 and Alzheimer's disease individuals.

Infection with the etiological agent of COVID-19, SARS-CoV-2, appears capable of impacting cognition, which some patients with Post-acute Sequelae of SARS-CoV-2 (PASC). To evaluate neuro-pathophysiological consequences of SARS-CoV-2 infection, we examine transcriptional and cellular signatures in the Broadman area 9 (BA9) of the frontal cortex and the hippocampal formation (HF) in SARS-CoV-2, Alzheimer's disease (AD) and SARS-CoV-2 infected AD individuals, compared to age- and gender-matched neurological cases. Here we show similar alterations of neuroinflammation and blood-brain barrier integrity in SARS-CoV-2, AD, and SARS-CoV-2 infected AD individuals. Distribution of microglial changes reflected by the increase of Iba-1 reveal nodular morphological alterations in SARS-CoV-2 infected AD individuals. Similarly, HIF-1α is significantly upregulated in the context of SARS-CoV-2 infection in the same brain regions regardless of AD status. The finding may help to inform decision-making regarding therapeutic treatments in patients with neuro-PASC, especially those at increased risk of developing AD. Teaser: SARS-CoV-2 and Alzheimer's disease share similar neuroinflammatory processes, which may help explain neuro-PASC.

Neuropathology and virus in brain of SARS-CoV-2 infected non-human primates.

Neurological manifestations are a significant complication of coronavirus disease (COVID-19), but underlying mechanisms aren't well understood. The development of animal models that recapitulate the neuropathological findings of autopsied brain tissue from patients who died from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are critical for elucidating the neuropathogenesis of infection and disease. Here, we show neuroinflammation, microhemorrhages, brain hypoxia, and neuropathology that is consistent with hypoxic-ischemic injury in SARS-CoV-2 infected non-human primates (NHPs), including evidence of neuron degeneration and apoptosis. Importantly, this is seen among infected animals that do not develop severe respiratory disease, which may provide insight into neurological symptoms associated with "long COVID". Sparse virus is detected in brain endothelial cells but does not associate with the severity of central nervous system (CNS) injury. We anticipate our findings will advance our current understanding of the neuropathogenesis of SARS-CoV-2 infection and demonstrate SARS-CoV-2 infected NHPs are a highly relevant animal model for investigating COVID-19 neuropathogenesis among human subjects.

CDCP1 enhances Wnt signaling in colorectal cancer promoting nuclear localization of ß-catenin and E-cadherin.

Elevated CUB-domain containing protein 1 (CDCP1) is predictive of colorectal cancer (CRC) recurrence and poor patient survival. While CDCP1 expression identifies stem cell populations that mediate lung metastasis, mechanisms underlying the role of this cell surface receptor in CRC have not been defined. We sought to identify CDCP1 regulated processes in CRC using stem cell populations, enriched from primary cells and cell lines, in extensive in vitro and in vivo assays. These experiments, demonstrating that CDCP1 is functionally important in CRC tumor initiation, growth and metastasis, identified CDCP1 as a positive regulator of Wnt signaling. Detailed cell fractionation, immunoprecipitation, microscopy, and immunohistochemical analyses demonstrated that CDCP1 promotes translocation of the key regulators of Wnt signaling, ß-catenin, and E-cadherin, to the nucleus. Of functional importance, disruption of CDCP1 reduces nuclear localized, chromatin-associated ß-catenin and nuclear localized E-cadherin, increases sequestration of these proteins in cell membranes, disrupts regulation of CRC promoting genes, and reduces CRC tumor burden. Thus, disruption of CDCP1 perturbs pro-cancerous Wnt signaling including nuclear localization of ß-catenin and E-cadherin.

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

A patient-derived orthotopic xenograft model enabling human high-grade urothelial cell carcinoma of the bladder tumor implantation, growth, angiogenesis, and metastasis.

High-grade urothelial cell carcinoma of the bladder has a poor prognosis when lymph nodes are involved. Despite curative therapy for clinically-localized disease, over half of the muscle-invasive urothelial cell carcinoma patients will develop metastases and die within 5 years. There are currently no described xenograft models that consistently mimic urothelial cell carcinoma metastasis. To develop a patient-derived orthotopic xenograft model to mimic clinical urothelial cell carcinoma progression to metastatic disease, the urothelial cell carcinoma cell line UM-UC-3 and two urothelial cell carcinoma patient specimens were doubly tagged with Luciferase/RFP and were intra-vesically (IB) instilled into NOD/SCID mice with or without lymph node stromal cells (HK cells). Mice were monitored weekly with bioluminescence imaging to assess tumor growth and metastasis. Primary tumors and organs were harvested for bioluminescence imaging, weight, and formalin-fixed for hematoxylin and eosin and immunohistochemistry staining. In this patient-derived orthotopic xenograft model, xenograft tumors showed better implantation rates than currently reported using other models. Xenograft tumors histologically resembled pre-implanted primary specimens from patients, presenting muscle-invasive growth patterns. In the presence of HK cells, tumor formation, tumor angiogenesis, and distant organ metastasis were significantly enhanced in both UM-UC-3 cells and patient-derived specimens. Thus, we established a unique, reproducible patient-derived orthotopic xenograft model using human high-grade urothelial cell carcinoma cells and lymph node stromal cells. It allows for investigating the mechanism involved in tumor formation and metastasis, and therefore it is useful for future testing the optimal sequence of conventional drugs or the efficacy of novel therapeutic drugs.

An Optimal Orthotopic Mouse Model for Human Colorectal Cancer Primary Tumor Growth and Spontaneous Metastasis.

BACKGROUND: Colorectal cancer is a leading cause of cancer-related death. Small animal models allow for the study of different metastatic patterns, but an optimal model for metastatic colorectal cancer has not been established. OBJECTIVE: The purpose of this study was to determine which orthotopic model most accurately emulates the patterns of primary tumor growth and spontaneous liver and lung metastases seen in patients with colorectal cancer. DESIGN: Using luciferase-tagged HT-29 cells coinoculated with lymph node stromal analog HK cells, 3 tumor cell delivery models were compared: intrarectal injection, intracecal injection, and acid enema followed by cancer cell instillation. Tumor growth was monitored weekly by bioluminescent imaging, and mice were sacrificed based on primary tumor size or signs of systemic decline. Liver and lungs were evaluated for metastases via bioluminescent imaging and histology. SETTINGS: The study was conducted at a single university center. MAIN OUTCOME MEASURES: Primary tumor and metastasis bioluminescent imaging were measured. RESULTS: Intrarectal injection had the lowest mortality at 4.0% (1/25) compared with the intracecal group at 17.4% (4/23) and the acid enema followed by cancer cell instillation group at 15.0% (3/20).The primary tumors in intrarectal mice had the highest average bioluminescence (3.78 × 10 ± 4.94 × 10 photons) compared with the mice in the intracecal (9.52 × 10 ± 1.92 × 10 photons; p = 0.012) and acid enema followed by cancer cell instillation groups (6.23 × 10 ± 1.23 × 10 photons; p = 0.0016). A total of 100% of intrarectal and intracecal mice but only 35% of mice in the acid enema followed by cancer cell instillation group had positive bioluminescent imaging before necropsy. Sixty percent of intrarectal mice had liver metastases, and 56% had lung metastases. In the intracecal group, 39% of mice had liver metastases, and 35% had lung metastases. Only 2 acid enema followed by cancer cell instillation mice developed metastases. LIMITATIONS: Tumor injections were performed by multiple investigators. Distant metastases were confirmed, but local lymph node status was not evaluated. CONCLUSIONS: Intrarectal injection is the safest, most reproducible, and successful orthotopic mouse model for human colorectal cancer primary tumor growth and spontaneous metastasis.