Pericytes may facilitate SARS-CoV-2 entry into the nervous system

Objective: Although the neurological and olfactory symptoms of coronavirus disease 2019 have been identified, the neurotropic properties of the causative virus, severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), remain unknown. We sought to identify the susceptible cell types and potential routes of SARS-CoV-2 entry into the central nervous system, olfactory system, and respiratory system. Method(s): We collected single-cell RNA data from normal brain and nasal epithelium specimens, along with bronchial, tracheal, and lung specimens in public datasets. The susceptible cell types that express SARS-CoV-2 entry genes were identified using single-cell RNA sequencing and the expression of the key genes at protein levels was verified by immunohistochemistry. We compared the coexpression patterns of the entry receptor angiotensin-converting enzyme 2 (ACE2) and the spike protein priming enzyme transmembrane serine protease (TMPRSS)/cathepsin L among the specimens. Result(s): The SARS-CoV-2 entry receptor ACE2 and the spike protein priming enzyme TMPRSS/cathepsin L were coexpressed by pericytes in brain tissue;this coexpression was confirmed by immunohistochemistry. In the nasal epithelium, ciliated cells and sustentacular cells exhibited strong coexpression of ACE2 and TMPRSS. Neurons and glia in the brain and nasal epithelium did not exhibit coexpression of ACE2 and TMPRSS. However, coexpression was present in ciliated cells, vascular smooth muscle cells, and fibroblasts in tracheal tissue;ciliated cells and goblet cells in bronchial tissue;and alveolar epithelium type 1 cells, AT2 cells, and ciliated cells in lung tissue. Conclusion(s): Neurological symptoms in patients with coronavirus disease 2019 could be associated with SARS-CoV-2 invasion across the blood-brain barrier via pericytes. Additionally, SARS-CoV-2-induced olfactory disorders could be the result of localized cell damage in the nasal epithelium.Copyright © Wolters Kluwer Health, Inc. All rights reserved.

Natural Evolution of Porcine Epidemic Diarrhea Viruses Isolated from Maternally Immunized Piglets

Simple SummaryDuring the long-term co-evolution of the virus and the host, even closely related vaccines may emerge with incomplete protective immunity due to the mutations or deletions of amino acids at specific antigenic sites. The mutation of PEDV was accelerated by the recombination of different strains and the mutation of the strains adapting to the environment. These mutations either cause immune escape from conventional vaccines or affect the virulence of the virus. Therefore, researching and developing new vaccines with cross-protection through continuous monitoring, isolation and sequencing are important to determine whether their genetic characteristics are changed and to evaluate the protective efficacy of current vaccines. The porcine epidemic diarrhea virus (PEDV) can cause severe piglet diarrhea or death in some herds. Genetic recombination and mutation facilitate the continuous evolution of the virus (PEDV), posing a great challenge for the prevention and control of porcine epidemic diarrhea (PED). Disease materials of piglets with PEDV vaccination failure in some areas of Shanxi, Henan and Hebei provinces of China were collected and examined to understand the prevalence and evolutionary characteristics of PEDV in these areas. Forty-seven suspicious disease materials from different litters on different farms were tested by multiplex PCR and screened by hematoxylin-eosin staining and immunohistochemistry. PEDV showed a positivity rate of 42.6%, infecting the small and large intestine and mesenteric lymph node tissues. The isolated strains infected Vero, PK-15 and Marc-145 multihost cells and exhibited low viral titers in all three cell types, as indicated by their growth kinetic curves. Possible putative recombination events in the isolates were identified by RDP4.0 software. Sequencing and phylogenetic analysis showed that compared with the classical vaccine strain, PEDV SX6 contains new insertion and mutations in the S region and belongs to genotype GIIa. Meanwhile, ORF3 has the complete amino acid sequence with aa80 mutated wild strains, compared to vaccine strains CV777, AJ1102, AJ1102-R and LW/L. These results will contribute to the development of new PEDV vaccines based on prevalent wild strains for the prevention and control of PED in China.

IDDF2023-ABS-0045 Spatiotemporal immune landscape of Mas-driven pro-inflammatory microenvironment with hyperactivated glycolysis in acetaminophen-induced liver injury

IDDF2023-ABS-0045 Figure 1 IDDF2023-ABS-0045 Figure 2 IDDF2023-ABS-0045 Figure 3 IDDF2023-ABS-0045 Figure 4

Demographic profile and outcome in primary pediatric brain tumors. A single tertiary institute experience in India

Introduction: Pediatric brain tumors are the most common tumor in children after hematological malignancies. There is very few data about the epidemiology of pediatric brain tumors in India. Methods - This was a prospective and retrospective study in pediatric patients who had undergone surgery in our institute (JIPMER,Pondicherry). 80 cases were recruited and followed up for minimum follow up period of 1 year. The demographic profile was analysed and IHC markers were done for embroyonal tumors and glioma. Result(s): Pediatric brain tumors was equally distributed among male and females. (1:1) .Mean age of presentation was 10 years . 27.5 % of our cases were embryonal tumors,low grade glioma (16.25 % ) ,high grade glioma ( 12.5 % ) ,ependymoma and craniopharyngioma comprised 15 % of our cases each. Medulloblastoma comprised 23.75 % of cases Out of which 31.5 % had craniospinal metastasis at time of diagnosis. The most common location of SHH pathway medulloblastoma was cerebellar hemisphere and non WNT/non SHH was fourth ventricle. 45.45 % of patients with high grade glioma had recurrence .50 % of ependymoma cases were infratentorial. we had 2 cases of ganglioglioma ,one in the midbrain and other in temporal lobe .Gross total resection was achieved in 30 % ,Subtotal resection in 46.25 % and partial resection in 20 % of our cases. Outcome of patients at the end of 1 year for low and high grade glioma, ependymoma and craniopharyngioma were similar to western literature. Two patients acquired COVID 19 and died while undergoing treatment. Molecular markers like INI1, LIN28 A was highly sensitive and specific for diagnosing atypical teratoid rhabdoid tumor (ATRT) and embryonal tumor with multilayered rosettes (ETMR )respectively. Conclusion(s): Our study emphasizes the need of standardized and systemic cancer registries in India. (Figure Presented).

COVID-associated pernio: Mechanistic insights to an abortive, seronegative SARS-CoV-2 infection

Introduction: SARS-CoV-2 replicates primarily in the airways but generates a systemic immune response mediated by Type I interferons (IFN-I). Pernio is a rare skin manifestation of disorders characterized by excessive IFN-I signalling. Although pernio increased in incidence during the pandemic, the relationship to SARS-CoV-2 remains controversial. Because of the pivotal nature of interferons in COVID-19 outcomes, pernio offers a window to investigate the biology underlying host resiliency to SARS-CoV-2 infection. Method(s): To further assess COVID-associated pernio, we characterized clinical samples from affected patients across 4 waves of the pandemic and investigated mechanistic feasibility in a rodent model. Patients were followed longitudinally with banking of blood and tissue. Golden hamsters were mock-treated or intra-nasally infected with SARS-CoV-2 and harvested at 3-and 30-days post-infection. Result(s): In affected tissue, immunophenotyping utilizing multiplex immunohistochemistry profiled a robust IFN-1 signature characterized by plasmacytoid dendritic cell activation. Viral RNA was detectable in a subset of cases using in situ hybridization for the SARS-CoV-2 S gene transcript. Profiling of the systemic immune response did not reveal a durable type 1 interferon signature. Consistent with previous literature, antibody and T-cell specific responses to SARS-CoV-2 were not detected. Nasopharyngeal SARS-CoV-2 inoculation in hamsters resulted in rapid dissemination of viral RNA and the generation of an IFN-I response that were both detectable in the paws of infected animals. Conclusion(s): Our data support a durable local IFN signature, with direct evidence of viral SARS-CoV-2 RNA in acral skin and suggest that COVID-associated pernio results from an abortive, seronegative SARS-CoV-2 infection.

Case Report on a Rare Malignancy: Anorectal Melanoma

Introduction: Melanoma of the rectum is an extremely rare disease. The median survival rate is 2-5 years. Current treatment for this aggressive cancer is resection if possible and consider adjuvant or neoadjuvant radiotherapy;immunotherapy in nonresectable cases. Given the rapid spread of disease due to its submucosal growth and metastasis pattern, there is low success rates with treatments. Case Description/Methods: An 84-year-old male presented to the emergency department with an acute COVID-19 infection. The patient was also found to have gram-negative septicemia on blood cultures, so a CT abdomen/pelvis was performed (Figure 1a). The CT showed rectal wall thickening. A flexible sigmoidoscopy was planned for a future outpatient visit after recovering from his acute infection. The patient, however, developed an acute onset of dyspnea and had a high probability V/Q scan while in the hospital. He was started on anticoagulation, and shortly after starting therapy the patient developed bright red rectal bleeding. Due to the new onset of rectal bleeding it was decided to expedite the sigmoidoscopy. The sigmoidoscopy was performed in the hospital showing an ulcerated partially black pigmented non- obstructing medium-sized mass that was partially circumferential involving one-third of the lumen (Figure 1b). A biopsy of the lesion was taken using cold-forceps. The pathology stained positive for S100 consistent with melanoma. The diagnosis of anorectal melanoma was made, and colorectal surgery was consulted. The patient was deemed not to be a surgical candidate secondary to age and active COVID-19 infection. Oncology was consulted, and it was decided to start the patient on radiation and immunotherapy with a PD-1 inhibitor. Discussion(s): The symptoms of anorectal melanoma can be subtle and in this case report completely asymptomatic. Symptoms to be aware of are rectal bleeding and tenesmus. Diagnosing melanoma on sigmoidoscopy can be challenging as most tumors are not pigmented. Biopsies should be taken and sent for immunohistochemical staining for S100, if positive the patient should have a PET scan. Treatment choices for the tumor are based on staging. In a resectable tumor sphincter-saving local excision with radiotherapy to the site of the tumor and the pericolic and inguinal lymphatics is recommended. For unresectable tumors or tumors with distant metastasis, immunotherapy with PD-1 inhibitors (nivolumab and ipilimumab) is an emerging treatment choice.

Hepatocellular Carcinoma Presenting as a Clival Mass

Introduction: Hepatocellular carcinoma (HCC) comprises the majority of primary liver cancer and has a poor prognosis. Clivus metastasis is rare with only a few reported cases in the medical literature. We report a case of a patient who presented with clival mass found to have metastatic HCC. Case Description/Methods: A 63-year-old woman presented for neurosurgical evaluation after she was found to have a skull base mass on computerized tomography (CT) of the head at an outside hospital. She endorsed dysphagia for three months, however denied headaches or visual disturbances. A magnetic resonance imaging (MRI) revealed a 5.4 cm by 2.9 cm by 3.6 cm mass in the clivus, which was deemed as the cause of dysphagia (Figure 1a). The patient subsequently underwent an endoscopic transsphenoidal resection of the clival mass. Histopathology from the tissue revealed a hepatoid carcinoma, concerning for metastatic HCC (Figure 1b and 2c). Immunohistochemical strains were positive for hepatocytic marker arginase-1 (Figure 1d). Laboratory studies revealed alpha fetoprotein (AFP) of 56,344 ng/mL, CA-125 of 376 ng/mL, normal B-HCG and carcinoembryonic antigen (CEA). Thereafter, a triple phase CT of the liver revealed two LI-RADS 5 lesions suggestive of HCC as the primary malignancy. Patient's case was discussed at multidisciplinary tumor board with recommendations for systemic immunotherapy with atezolimumab plus bevacizumab and radiation therapy to the clivus. Discussion(s): The incidence of HCC has almost tripled since the 1980s making it the fastest rising cause of cancer related deaths. Metastasis to the brain comprises 0.26% to 2.2% of cases and the skull base is the most rarely affected anatomical site. Although CNS presentation is rare, we may see more neurological manifestations of metastatic HCC with the persistence of chronic hepatitis infections, the rise of metabolic diseases such as NASH, and an increase in alcohol-related liver disease during the COVID-19 pandemic. Although exceedingly rare, metastasis to the clivus should be considered in the differential diagnosis of skull base masses. Despite detection and treatment, prognosis remains poor and emphasis should be placed on consistent HCC surveillance. This case emphasizes that skull masses must be evaluated diligently as they can be the first sign of underlying liver malignancy. Given the morbidity and mortality associated with HCC, recognition of atypical manifestations of HCC can lead to a prompt diagnosis and initiation of life-saving treatment. (Figure Presented).

Esophageal Mucosal Calcinosis: A Rare Site of Calcium Deposition in End-Stage Renal Disease

Introduction: Calciphylaxis, otherwise known as calcium uremic arteriolopathy, is defined as calcium deposition around blood vessels in skin and fat tissue which occurs in 1-4% of patients with end-stage renal disease (ESRD). Calcium deposition in the esophagus is extremely rare;to date, there have been only 4 cases reported worldwide. We report the fifth case of esophageal mucosal calcinosis occurring in a young male with ESRD. Case Description/Methods: A 37-year-old Thai man with ESRD on peritoneal dialysis since 2005 presented with generalized weakness and odynophagia due to oral ulcers, resulting in poor PO intake. He denied drinking alcohol, illicit drug use, or smoking. On exam his abdomen was soft, non-distended, non-tender, without any guarding. Past medical history included hypertension and COVID-19 in January 2022. Laboratory tests revealed neutropenia and pancytopenia, hyperphosphatemia, and hypocalcemia. EGD revealed distal esophageal esophagitis and hemorrhagic erosive gastropathy. Biopsy showed ulcerative esophagitis with dystrophic calcification, consistent with esophageal mucosal calcinosis .No intestinal metaplasia was noted. Immunohistochemistry was negative for CMV, HSV1, and HSV2. The patient was treated with pantoprazole 40mg IV every 12 hours, Magic Mouthwash 5ml qid, and Carafate 10mg qid. He was transferred to a cancer center where he had a bone marrow biopsy formed which was negative. His symptoms resolved and the patient was discharged to home (Figure). Discussion(s): Esophageal mucosal calcinosis is extremely rare. It is due to a combination of factors involving acidosis and the phenotypic differentiation (and apoptosis) of vascular smooth muscle cells (VSMC) into chondrocytes or osteoblast-like cells. These changes, along with the passive accumulation of calcium and phosphate, induce calcification. Acidosis is well-known to promote inflammation of the arterial walls, releasing cytokines that induce vascular calcification. The benefits of treatment with sodium thiosulfate remain unclear. An ample collection of cases should help devise standardized treatment options and establish management guidelines for this condition.

Syphilis: "The Great Imitator" Presenting as Acute Hepatitis

Introduction: Syphilis is a multi-systemic disease caused by spirochete Treponema pallidum. Very rarely, it can affect the liver and cause hepatitis. Since most cases of hepatitis are caused by viral illnesses, syphilitic hepatitis can be missed. Here, we present a case of syphilitic hepatitis in a 35-year-old male. Case Description/Methods: Patient was a 35-year-old male who presented to the hospital for jaundice and mild intermittent right upper quadrant abdominal pain. His medical history was only significant for alcohol abuse. His last drink was 4 weeks ago. He was sexually active with men. On exam, hepatomegaly, mild tenderness in the right upper quadrant, jaundice, and fine macular rash on both hands and feet were noted. Lab tests revealed an ALT of 965 U/L, AST of 404 U/L, ALP of 1056 U/L, total bilirubin of 9.5 mg/dL, direct bilirubin of 6.5 mg/dL, INR of 0.96, and albumin of 2.0 g/dL. Right upper quadrant ultrasound showed an enlarged liver but was negative for gallstones and hepatic vein thrombosis. MRI of the abdomen showed periportal edema consistent with hepatitis without any gallstones, masses, or common bile duct dilation. HIV viral load and Hepatitis C viral RNA were undetectable. Hepatitis A & B serologies were indicative of prior immunization. Hepatitis E serology and SARS-CoV-2 PCR were negative. Ferritin level was 177 ng/mL. Alpha-1-antitrypsin levels and ceruloplasmin levels were normal. Anti-Smooth muscle antibody titers were slightly elevated at 1:80 (Normal < 1:20). Anti-Mitochondrial antibody levels were also slightly elevated at 47.9 units (Normal < 25 units). RPR titer was 1:32 and fluorescent treponemal antibody test was reactive which confirmed the diagnosis of syphilis. Liver biopsy was then performed which showed presence of mixed inflammatory cells without any granulomas which is consistent with other cases of syphilitic hepatitis. Immunohistochemical stain was negative for treponemes. Patient was treated with penicillin and did have Jarisch-Herxheimer reaction. ALT, AST, ALP, and total bilirubin down trended after treatment. Repeat tests drawn exactly 1 month post treatment showed normal levels of ALT, AST, ALP, and total bilirubin (Figure). Discussion(s): Liver damage can occur in syphilis and can easily be missed because of the non-specific nature of presenting symptoms. In our patient, the fine macular rash on both hands and feet along with history of sexual activity with men prompted us to test for syphilis which ultimately led to diagnosis and treatment in a timely manner. (Figure Presented).

Anca-Associated but Not Vasculitis

Background/Aims A 72-year-old lady presented in primary care with complaints of generalised body aches, bilateral leg weakness and constitutional symptoms following a first dose of COVID-19 vaccine. Blood tests showed slightly raised inflammatory markers. She was initially diagnosed with polymyalgia rheumatica and was started on 40mg prednisolone with minimal improvement. Methods The examination in the rheumatology clinic was unremarkable. Investigations revealed raised white cell count, consistent with high dose steroid treatment, and elevated monocytes. There was mild improvement in inflammatory markers. The working diagnosis was of self-limiting viral illness. Further testing discovered strongly positive MPO ANCA (115 IU/ml), and the patient received three pulses of 500mg methylprednisolone for suspected vasculitis arranged by the medical team. There was no evidence of renal involvement. The diagnosis made at this point was autoimmune inflammatory disorder with unclear aetiology. At the subsequent clinic visit she reported mild shortness of breath, but no other symptoms suggestive of either vasculitis or connective tissue disease. Repeat ANCA showed significant reduction in MPO titre following pulse steroid treatment. CT of chest, abdomen and pelvis demonstrated a localised lobular/ nodular deformity of the liver. Viral hepatitis screen was negative. CA19-9 was raised at 100 U/ml. Liver biopsy was reported as poorly differentiated carcinoma without specific localising immunohistochemical features. Results The patient underwent hemi-hepatectomy for histologically confirmed pT2pNXM0R0 liver cholangiocarcinoma in a tertiary centre followed by adjuvant chemotherapy with capecitabine. With treatment, her MPO ANCA and CA19-9 levels declined. An interval CT scan of chest, abdomen and pelvis performed ten months after the surgery, showed no recurrence of malignancy. Given the fact that the patient's MPO ANCA fell following the treatment of cholangiocarcinoma, it is likely that positive MPO ANCA is associated with underlying malignancy rather than an active vasculitis. Conclusion This unusual case describes an evolution of the diagnostic process guided by non-specific symptoms and ANCA positivity, arriving at an unexpected diagnosis of malignancy. Although ANCA is a sensitive and specific marker of vasculitides, it can be positive in other conditions particularly hepatitis B, inflammatory bowel disease and autoimmune liver disorders. Malignancy can also be associated with ANCA in the absence of vasculitis. In one study, of 118 ANCA positive patients without ANCA-associated vasculitis, four were found to have malignancy. In a study of 1024 patients who had ANCA tested, 61 patients were found to have malignancy, predominantly haematological and lung cancers. However, after adjustment for sex, age and time of blood draw, no association was found between ANCA status and incidence of cancer. Interestingly, paraneoplastic vasculitis such as polyarteritis nodosa (PAN) has been described in the context of underlying cholangiocarcinoma, and is associated with ANCA rise. Moreover, patients with raised ANCA and PAN also have raised CA 19- 9.

Production of Sars-Cov-2 Membrane Protein as Better Target Protein for Diagnostic Application

Intro: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, an enveloped RNA of the coronavirus family. The advancement in molecular technology and biochemistry has accelerated the development of diagnostic reagents and assays. Much attention has been focused on the S protein, but the high mutation rate in this region could lead to false negative results. Thus, a better target protein for diagnostic application is needed for accurate detection. Method(s): Nucleotide sequences encoded for membrane (M) glycoprotein gene region of SARS-CoV-2 from Malaysian isolates were extracted from GISAID, aligned, and selected accordingly. The DNA plasmid was commercially synthesized with codon optimization for Escherichia coli (E. coli), and the presence of the M gene was confirmed by PCR. The plasmid was then transformed into E. coli. Later, the expression of M glycoprotein was induced, separated on an SDS-PAGE gel, and transferred onto a nitrocellulose membrane, followed by immunostaining. Finding(s): The analysis of the M glycoprotein against the Omicron strains demonstrated that the amino acid is conserved (99.5%). The M glycoprotein was successfully expressed and detected with antibodies from SARS-CoV-2 infected patients at ~26 kDa. The protein is currently upscale for the generation of monoclonal Ab (Mab). Discussion(s): The M protein of SARS-CoV-2 is more conserved among the virus and also has been reported to confer antigenic properties. Selection of M protein perhaps a better option compared to current detection assays that use spike (S) protein, which could lead to false negative results, as this gene region particularly the ribosome-binding domain (RBD) rapidly undergoes mutations. The utilization of M protein potentially improves negative predictive value (NPV) of the diagnostic test. Conclusion(s): Further development of diagnostic reagents is needed to improve the assay's specificity. The newly developed M protein and the MAb can be used to generate a more accurate viral detection assay.Copyright © 2023

Gastrointestinal stromal tumors in elderly patients

Background: Gastrointestinal stromal tumors (GISTs) account for 1 to 3% of all primary malignant tumors of the gastrointestinal tract. The global incidence of GISTs varies in the range of 7-15 cases per 1 million people per year. In about 95% of cases, the incidence is sporadic. Despite the fact that some success has been achieved in the treatment of this pathology, the problem of GISTs treatment is urgent, especially in elderly and senile patients in particular. The aim of the study: To study the age-related characteristics of GISTs development in patients of older age groups. Material(s) and Method(s): A retrospective analysis of 56 clinical cases of GISTs in patients of different age groups according to the WHO classification was carried out in the study. Result(s): The most common variant of the immunohistochemical structure was the spindle cell one 62.5%. In most cases, tumors were localized in the stomach 82.2%. Elderly patients had larger tumor sizes compared with young and middle-Aged patients. In patients of older age groups, the disease was most often detected at stage II. In most cases, a comorbid pathology was detected, most often a combination of several diseases of the cardiovascular system. Conclusion(s): In patients of older age groups, the spindle cell structure of the GISTs is most common, the tumor was most often localized in the stomach (77.4%), most often the tumor was localized along the lesser curvature. In most cases, the tumor was up to 10.0 cm in diameter. On average, the disease was detected at stage II. Comorbid pathology occurred in 87.3% of cases. In 2020-2021, the disease was detected more often, the of tumors sizes were smaller, due to an increase in the number of CT scans of the chest for the diagnosis of the new coronavirus infection.Copyright © 2022 AME Publishing Company. All rights reserved.

Mucosa-Associated Lymphoid Tissue Lymphoma of the Rectum Incidentally Found in a Patient with Ulcerative Colitis

Introduction: Mucosa-associated lymphoid tissue (MALT) lymphomas are extra-nodal marginal zone B-cell lymphomas, most commonly found in the stomach, associated with Helicobacter pylori infections, and generally not linked with inflammatory bowel disease. Rectal MALT lymphoma is very uncommon and often associated with painful defecation, change in bowel habits, or rectal pressure/prolapse. Here, we present a rare case of an asymptomatic female with ulcerative colitis (UC) found to have benign-appearing rectal polyps during a routine screening colonoscopy. Case Description/Methods: The patient is a 56-year-old female with a history of left-sided UC, diagnosed in 1993, with one flare after receiving the 2nd dose of the Moderna COVID-19 vaccine, taking oral Olsalazine 500 mg twice daily, low-dose Prednisone, and mesalamine suppositories as needed presenting for screening colonoscopy. The patient was asymptomatic, citing regular non-bloody bowel movements and normal stool consistency. Colonoscopy revealed two 7 mm sessile, non-bleeding rectal polyps, surrounded by congested, erythematous, friable, and ulcerated mucosa in the rectosigmoid colon. Cold forceps biopsies were taken. Hematopathology evaluation of the routine colon biopsy samples revealed chronic nonspecific colitis while pathology of the rectal polyps showed marked lymphoplasmacytic infiltrate and extra-nodal marginal zone lymphoma of MALT. Ancillary studies, immunohistochemistry, and molecular studies for B-cell gene rearrangement confirmed extra-nodal marginal zone lymphoma of MALT with prominent plasmacytic differentiation. The patient was informed and close follow-up in Gastroenterology clinic was arranged. (Figure) Discussion: Rectal MALT lymphoma is rare with unclear management options. Treatments of UC include watchful waiting, surgical resection, endoscopic mucosal resection, radiation, and/or chemotherapy. Helicobacter pylori infections, though strongly linked with gastric MALT lymphoma, have not been shown to be strongly correlated with rectal MALT lymphoma. Given that patients with UC have chronic UCassociated colonic inflammation, lymphoma is often difficult to distinguish visually during colonoscopy, frequently masked by ulcerations and pseudo-polyps. In cases like these, more definitive treatments such as surgical resection could therefore be warranted. Long-term follow-up data is sparse and definitive management remains a clinical conundrum, thus these patients require reliable long-term multidisciplinary close follow-up. (Figure Presented).

Von Willebrand factor and the thrombophilia of severe COVID-19: in situ evidence from autopsies.

Background: COVID-19 is accompanied by a hypercoagulable state and characterized by microvascular and macrovascular thrombotic complications. In plasma samples from patients with COVID-19, von Willebrand factor (VWF) levels are highly elevated and predictive of adverse outcomes, especially mortality. Yet, VWF is usually not included in routine coagulation analyses, and histologic evidence of its involvement in thrombus formation is lacking. Objectives: To determine whether VWF, an acute-phase protein, is a bystander, ie, a biomarker of endothelial dysfunction, or a causal factor in the pathogenesis of COVID-19. Methods: We compared autopsy samples from 28 patients with lethal COVID-19 to those from matched controls and systematically assessed for VWF and platelets by immunohistochemistry. The control group comprised 24 lungs, 23 lymph nodes, and 9 hearts and did not differ significantly from the COVID-19 group in age, sex, body mass index (BMI), blood group, or anticoagulant use. Results: In lungs, assessed for platelets by immunohistochemistry for CD42b, microthrombi were more frequent in patients with COVID-19 (10/28 [36%] vs 2/24 [8%]; P = .02). A completely normal pattern of VWF was rare in both groups. Accentuated endothelial staining was found in controls, while VWF-rich thrombi were only found in patients with COVID-19 (11/28 [39%] vs 0/24 [0%], respectively; P < .01), as were NETosis thrombi enriched with VWF (7/28 [25%] vs 0/24 [0%], respectively; P < .01). Forty-six percent of the patients with COVID-19 had VWF-rich thrombi, NETosis thrombi, or both. Trends were also seen in pulmonary draining lymph nodes (7/20 [35%] vs 4/24 [17%]; P = .147), where the overall presence of VWF was very high. Conclusion: We provide in situ evidence of VWF-rich thrombi, likely attributable to COVID-19, and suggest that VWF may be a therapeutic target in severe COVID-19.

MECHANISM AND RESISTANCE STUDIES OF SARS-CoV-2 MPRO INHIBITOR POMOTRELVIR (PBI-0451)

Background: The unprecedented scale of the COVID-19 pandemic and rapid evolution of SARS-CoV-2 variants underscores the need for broadly active inhibitors with a high barrier to resistance. The coronavirus main protease (Mpro) is an essential viral enzyme required for viral polyprotein processing and is highly conserved across human coronaviruses. Pomotrelvir (PBI-0451) is a novel Mpro inhibitor currently completing phase 2 clinical trial. Here we describe the mechanism of action, broad activity against SARS-CoV-2 clinical isolates, combination studies with other SARS-CoV-2 inhibitors and favorable resistance profile of pomotrelvir. Method(s): The kinetic parameters of pomotrelvir Mpro inhibition and its interaction with nirmaltrevir were determined in a kinetic protease assay. The IC50s of pomotrelvir on mutant Mpro proteins were measured in an endpoint Mpro assay. Combination studies of pomotrelvir with remdesivir and molnupiravir were carried out in A549-hACE2 cells infected with SARS-CoV-2 NLuc virus. Activity against SARS-CoV-2 clinical variants was assessed by infection of A549-ACE2-TMPRSS2 cells followed by immunostaining of the viral nucleocapsid protein. Result(s): Pomotrelvir is a potent competitive inhibitor of SARS-CoV-2 Mpro (Ki =2.7 nM). Binding of pomotrelvir and the Mpro inhibitor nirmatrelvir to the active site is mutually exclusive. In the SARS-CoV-2 NLuc assay, pomotrelvir is additive when combined with remdesivir or molnupiravir, two nucleoside analogs targeting viral RNA synthesis. When the effect of Mpro substitutions previously selected in a resistance study of pomotrelvir were analyzed in an enzyme assay, only Mpro-N133H showed a significant increase in IC50 (45-fold). The catalytic efficiency of Mpro-N133H is reduced by 10-fold and the recombinant virus SARSCoV-2 (WA1) -N133H is not viable, suggesting that N133H has lower replicative fitness. Lastly, pomotrelvir exhibits broad activity against all SARS-CoV-2 clinical isolates tested to date, including five omicron variants. Conclusion(s): PBI-0451 is a potent competitive inhibitor of SARS-CoV-2 Mpro and is broadly active against SARS-CoV-2 clinical isolates including omicron variants. Results from inhibitor interaction studies support the potential combination of pomotrelvir with remdesivir and molnupiravir but not nirmatrelvir. Enzymatic characterization of in vitro selected pomotrelvir resistant variants indicates they either confer no resistance or have reduced fitness.

SARS-CoV-2 BA.5 CAUSES HIGHER REPLICATION AND LETHAL INFECTION IN K18-hACE2 MICE

Background: The continuous evolution of SARS-CoV-2 in the diverse immune landscape (natural, vaccine, hybrid) is giving rise to novel immune escape mutations. So far, the resulting new variants (BA.1, BA.2, BA.2.12.1) were observed to cause mild infections, however, BA.5 infections are associated with an increased risk of hospitalization.1 Therefore it is essential to investigate the pathogenesis of BA.5. Method(s): Here we compared the pathogenicity of Pre-Omicron (B.1.351) and Omicron (BA.1, BA.2.12.1, and BA.5) variants in wild-type C57BL/6J mice and K18-hACE2 mice. The virus replication kinetics was also studied in human Calu3, pulmonary alveolar type 2 (AT2) cells, and airway organoids (HAO). Cell-to-cell spread of virus was measured by syncytia formation assay and immunohistochemistry (IHC) of infected lungs. Result(s): In the results, infection in C57BL/6J mice showed severe weight loss ( >15%) for B.1.351 infected mice and moderate ( >5%) for BA.5 infected. C57BL/6J mice showed higher virus replication of B.1.351 followed by BA.5, BA.1, and BA.2.12.1. At the peak of virus replication (2 days) plaque-forming units from lung extract of BA.5 infected mice were two, and three logs higher compared to BA.1 and BA.2.12.1 respectively. BA.5 infection was lethal to 80% of infected K18-hACE2 mice, whereas the mice looked normal after infection with BA.1 and BA.2.12.1. BA.5 infected mice showed high virus replication in brain tissue. Surprisingly the syncytia formation assay and IHC for BA.5 was comparable to that of B.1.351, indicating the higher cell-to-cell spread of BA.5 and B.1.351 compared to BA.1 and BA.2.12.1, which is one of the measures of pathogenicity. Calu3 and HAO showed the same trend of virus replication as was observed in-vivo experiments however AT2 cells were found to be resistant to BA.5 replication. Conclusion(s): These results suggest that the BA.5 variant (lineage) of Omicron has the potential to regain the pathogenicity as it shows increased virulence compared to other Omicron sub-variants. Lethal infection of BA.5 in K18-hACE2 mice may be attributed to catastrophic encephalitis and increased cell-to-cell spread.

Cellular tropism of SARS-CoV-2 in various organs - A Histopathology and Immunohistochemistry study on Covid-19 Deaths

Background and objectives: SARS-C0V-2 infections have varied manifestations among individuals ranging from asymptomatic or mild symptoms to severe disease and death. This study is done to look into various histopathological changes in lung, liver, and kidney tissues among Covid19 positive autopsies with cellular tropism and viral load among various organs by immunohistochemistry (IHC) for the SARS-C0V-2 viral marker. Method(s): A prospective descriptive study of core biopsies from covid19 positive autopsies from the lung, liver, and kidneys were taken from 20 cases. A routine histopathological examination of the tissues with IHC staining for SARS-CoV-2 cocktail antibodies was performed and assessed. Result(s): Histopathological changes in the lung, liver, and kidney tissues showed changes of varying severity. On IHC, in the lung, the tropism for SARS-CoV-2 was seen in pneumocytes, bronchial epithelial cells, endothelial cells, and macrophages. In the kidney, tropism was seen towards tubular epithelial cells and endothelial cells. In the liver, hepatocytes and bile duct epithelial cells were positive. Variable viral density was seen in different organs which varied from case to case. The density of the viral load was highest in the lung and lower in the kidney and least in the liver. Conclusion(s): In this study the various histopathological changes and cellular tropism of the SARS-CoV-2 among Lung, liver, and kidney tissues have been described and compared with various similar studies across the globe.Copyright © 2023 International Journal of Life Sciences Biotechnology and Pharma Research. All rights reserved.

Preparation and identification of broad-spectrum monoclonal antibodies against N protein of avian infectious bronchitis virus

[Objective] To prepare broad-spectrum monoclonal antibody against N protein of avian infectious bronchitis virus (IBV), so as to lay a foundation for identifying conservative domain epitope of N protein and establish a universal IBV detection method. [Method] N protein of GX-YL5, a representative strain of IBV dominant serotype in Guangxi, was expressed in prokaryote. BALB/c mice were immunized with the purified protein. After the serum titer of the immunized mice reached 104 or more, the splenocytes were fused with SP2/0 myeloma cells. After screening by indirect ELISA, monoclonal antibody was prepared by ascites-induced method. Western blotting, IFA and indirect ELISA were used to identify the titer, subtype, reaction specificity and cross-reaction spectrum. And the prepared monoclonal antibody was used for immunohistochemical detection. And the prepared monoclonal antibody was used to detect the IBV in the trachea and kidney tissues of SPF chickens artificially infected with 4 representative IBV variants (GX-N130048, GX-N160421, GX-QZ171023 and GX-QZ170728). [Result] The prepared monoclonal antibody N2D5 had a titer greater than 217 and its subtype was IgG2b. The Western blotting and IFA results showed that the monoclonal antibody N2D5 only reacted with IBV, and were negative with Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (aMPV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV) and Marek's disease virus (MDV). Monoclonal antibody N2D5 reacted with many genotypes in China and all 7 serotypes of IBV currently prevalent in Guangxi, including commonly used standard strains, vaccine strains and field strains. Immunohistochemistry showed that the virus signals could be detected in the trachea and kidney tissues of SPF chickens at different time after artificial infection of 3 representative IBV strains from chicken and 1 isolated strain from duck, which further proved its broad spectrum. [Conclusion] The monoclonal antibody N2D5 of IBV prepared based on hybridoma technology belongs to the IgG2b subtype. It has the characteristics of high specificity, wide response spectrum and strong binding ability with IBV. It can be used as a specific diagnostic antibody for clinical diagnosis of IBV and the study of virus distribution.

Glioblastoma Susceptibility to SARS-CoV-2: Analysis of Cancer Stem Cells and Immune Tumor Microenvironment on Human Brain, Patient-Derived Tumors, and Organoid Models

INTRODUCTION: Glioblastoma (GBM) is the most common and deadliest primary brain tumor, characterized by chemoradiation resistance and an immunosuppressive tumor microenvironment (TME). SARS-CoV-2, the COVID-19 virus, produces a significant proinflammatory response and a spectrum of clinical presentations after central nervous system infection. METHOD(S): Patient-derived GBM tissue, primary cell lines, and organoids were analyzed with immunohistochemistry and pixel-line intensity quantification. Data from tumor-bulk and single-cell transcriptomics served to describe the cell-specific expression of SARS-CoV-2 receptors in GBM and its association with the immune TME phenotype. Normal brain and iPSC-derived organoids served as controls. RESULT(S): We demonstrate that patient-derivedGBMtissue and cell cultures express SARS-CoV2 entry factors such as ACE2, TMPRSS2, and NRP1. NRP1 expression was higher in GBM than in normal brains (p<0.05), where it plays a crucial role in SARS-CoV-2 infection. NRP1 was expressed in a cell-type and phenotype-specific manner and correlated with TME infiltration of immunosuppressive cells: M2 macrophages (r = 0.229), regulatory T cells (r = 0.459), NK cells (r = -0.346), and endothelial cells (r = 0.288) (p < 0.05). Furthermore, gene ontology enrichment analysis showed that leukocyte migration and chemotaxis are among the top 5 biological functions mediated by NRP1 (p < 0.05). We found our GBM organoids recapitulate tumoral expression of SARSCoV- 2 entry factors, which varies based on distance from surface as surrogate of TME oxygenation (p < 0.05). CONCLUSION(S): GBM cancer cells and immune TME cells express SARS-CoV-2 entry factors. Glioblastoma organoids recapitulate this expression and allow for currently undergoing studies analyzing the effect of SARS-CoV-2 infection in GBM. Our findings suggest that SARSCoV- 2 could potentially target GBM, opening the door to future studies evaluating SARS-CoV-2-driven immune modulation.

Pathogenicity analysis of one avian infectious bronchitis virus stain isolated from Shanxi

In order to perform the isolation of avian infectious bronchitis virus (IBV) and study the pathogenicity of IBV isolate, the RT-PCR was used to detect nucleic acid extracted from a clinical sample of chickens, which were suspected to be infected with infectious bronchitis virus (IBV) and provided by a farmer in Yuncheng, Shanxi province. And the sample was detected as IBV positive by RT-PCR. Then 9-11-day-old SPF chicken embryonated eggs were inoculated with the sample filtered from the grinding fluid, and the obtained allantoic fluid was blindly passed by three generations (F3) and was also tested as IBV positive;The F11 generation passaged in embryonated eggs caused typical "dwarf embryo" lesions to SPF chicken embryonated eggs, and induced the loss of cilia in tracheal rings. The results showed that an IBV strain was isolated and named as YC181031. The S1 gene amplification and sequencing analysis showed that YC181031 strain belonged to IBV GI-22 genotype, which is also nephropathogenic type IBV. Seven-day-old SPF chicks were used to test the pathogenicity of the isolate. The results showed that several clinical symptoms were showed in chicks infected with YC181031, such as breathing with difficulty, depression, excreting watery droppings and death. The mortality of infected chicks was 20%. Typical pathological changes such as enlargement of kidney and urate deposition in the kidney were observed in infected chicks. The immunohistochemical assay and viral load detection were performed for the tissue samples from infected and dead chicks. The tissue lesions and distribution of virus were observed in the kidney, trachea, lung, glandular stomach, spleen and liver samples of infected chicks. RT-PCR detection of pharyngeal anal swabs showed that the virus shedding by infected chicks could be continuously detected within 14 days of the test period;The viral loads of various tissues were detected by RT-qPCR and the results showed that the viral load from high to low was kidney, trachea, lung, stomach, spleen and liver. The viral load of kidney was significantly higher than that of other tissues (P < 0.05).In this study, the pathogenicity characteristics of GI-22 genotype strain were systematically studied for the first time, providing a reference for the prevention and treatment of the disease.