CD4+ T cells from preeclamptic patients cause hypertension, autoantibodies and cognitive dysfunction in a pregnant rat model

CD4+ T Cells from Preeclamptic patients with or without a history of COVID-19 during pregnancy cause hypertension, autoantibodies and cognitive dysfunction in a pregnant rat model Objective: Preeclampsia (PE) new onset hypertension (HTN) during pregnancy, is associated with increased autoantibodies, cerebral blood flow (CBF) impaired cognitive function and memory loss. We have shown adoptive transfer of placentalCD4+T cells from PE women into athymic nude pregnant rats causesHTNand autoantibodies associated with PE.COVID-19 (CV) during pregnancy is associated with increased diagnosis of PE. However, we do not know the role of CD4+ T cells stimulated in response to CV in contributing to the PE phenotype seen patients with a Hx of CV during pregnancy. Therefore, we hypothesize that adoptive transfer of placental CD4+ T cells from patients with a CV History (Hx) during pregnancy with PE causes HTN, increased CBF and cognitive dysfunction in pregnant athymic nude recipient rats. Study Design: Placental CD4+ T cells isolated from normotensive (NP), PE, Hx of CV normotensive (CV Hx NT), and Hx of CV with PE (CV Hx+PE) at delivery. One million CD4+ T cells were injected i.p. into nude athymic rats on gestational day (GD) 12. The Barnes maze and the novel object recognition behavioral assays were used to assess cognitive function on GDs 15-19. Blood pressure (MAP) and CBF were measured by carotid catheter and laser Doppler flowmetry on GD19, respectively. A two-way ANOVA was used for statistical analysis. Result(s):MAPincreased inCVHx+PE (111 +/- 4, n = 4) and PE recipient rats (115 +/- 2 mmHg, n = 5) compared to CV Hx NT (100 +/- 4, n = 5) and NP (99 +/- 3 mmHg, n = 4, P < .05). CV Hx+PE and PE exhibited latency with errors navigating in the Barnes maze compared to CV Hx NT and NP groups. Locomotor activity was decreased in CV Hx+PE (P < .05) compared to PE, CV Hx NT, and NP groups. CV Hx+PE and PE spent more time exploring identical objects compared to CV Hx NT and NP groups. PE and CV Hx+ PE had increased CBF compared to CV Hx NT and NP rats. Conclusion(s): Our findings indicate that pregnant recipients of CD4+ T cells from PE with or without a Hx CV during pregnancy cause HTN, increased CBF and cognitive dysfunction compared to recipients of NP or NT Hx COVID-19 CD4+ T cells.

Hyperglycemia and Hypoglycemia Contributions to Acute Respiratory Distress Syndrome

Purpose of Study Non-diabetic COVID-19 patients with elevated admission fasting blood glucose levels ('hyperglycemia') inexplicably have an increased 28 day mortality and higher inhospital complications including the Acute Respiratory Distress Syndrome (ARDS) but potentially contributing blood glucose changes during ARDS development were not reported (Wang S et al: Diabetologia 2020). Our goal was to determine blood glucose alterations before and during acute lung injury development in a rat model used to study ARDS. Methods Used We sequentially evaluated blood glucose levels for 24 hours and lung lavage protein levels (lung permeability) and lung lavage neutrophil numbers (lung inflammation) at 24 hours to assess acute lung injury ('ARDS') in young (~3 month) and old (~12 month) control and a novel strain of hyperoxia surviving 'resistant' rats before and after administering high and low insulin doses and before and after interleukin- 1/lipopolysaccharide (IL-1/LPS) insufflation. Summary of Results Glucose levels increase rapidly and sequentially in young control, but not young resistant, rats peaking ~2 hours after insufflation. Glucose levels also increase in old control and old resistant rats after insufflation compared to young control and young resistant rats after insufflation. The pattern of glucose levels at 2 hours after insufflation resembles lung lavage proteins and neutrophils at 24 h after insufflation (table 1). Administering high insulin (High In) doses decreases glucose levels ('hypoglycemia') and worsens ARDS while administering low insulin (Low In) doses correct glucose levels and improve ARDS. Conclusions Hyperglycemia develops in both young and old rats developing ARDS and high or low glucose levels parallel worse acute lung inflammation and acute lung injury ('ARDS'). Controlling glucose judiciously with insulin may be beneficial in combatting ARDS caused by SARS-CoV-2 infection and other insults.

Centella asiatica extract inhibits Mycobacterium tuberculosis growth in rat tuberculosis models

Background and Aims: The covid-19 pandemic has reversed years of progress in the fight to end tuberculosis. So, the discovery of new drugs as antituberculosis is very much needed. Our previous studies have shown that the extract of Centella asiatica is able to inhibit the growth of Mycobacterium tuberculosis in vitro and requires further research. The aims of this study is to prove the effect of Centella asiatica inhibit Mycobacterium tuberculosis in rat model tuberculosis. Method(s): The protocol in this study was approved by the veterinary ethics committee of Airlangga University. The rat tuberculosis model was induced by intrathecal injection of a suspension of Mycobacterium tuberculosis strain H37 Rv. Twenty-eight tuberculosis rat were randomly divided into four groups. Groups 1,2, and 3 were treated with ethanol extract of Centella asiatica at 375 mg/kgBW, 750 mg/kgBW and 1500 mg/kgBW, and the fourth group was the control group. Centella asiatica extract is administered orally via an intragastric feeding tube for two weeks, once daily At the end of the experimental period, rats were sacrificed by cervical decapitation. The left lung tissue was taken aseptically and cultured on Middlebrook 7H10. Result(s): The results showed that there was no bacterial growth on the culture media in the group that received Centella asiatica extract at a dose of 750 and 1500 mg/kg BW. Conclusion(s): The conclusion in this study, that Centella asiatica extract inhibit the growth of Mycobacterium tuberculosis at doses of 750 and 1500 mg/kg BW. We thank the Directorate of Research and Community Services, the Directorate General of Higher Education, and Ministry of Education and Culture in Indonesia for the financial supportCopyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

HBK-15, a Multimodal Compound, Showed an Anxiolytic-Like Effect in Rats.

Anxiety is a common mental disorder, and its prevalence has lately increased because of the COVID-19 pandemic. Unfortunately, the available anxiolytics are often ineffective, and most possess addictive potential. Thus, searching for novel compounds is essential. In our previous studies, we selected a multimodal compound, HBK-15, which showed a fast antidepressant-like effect in animal models of depression. HBK-15 demonstrated a high affinity for serotonin 5-HT1A receptors and moderate for 5-HT7, dopamine D2, and α1-adrenoceptors. Based on the receptor profile and preliminary studies, we aimed to investigate the anxiolytic potential of HBK-15 using the conditioned-response rat model of anxiety, i.e., the Vogel drinking test. We performed hot plate and free-drinking tests to exclude false positive results in the Vogel test. Using radioligand binding studies, we also investigated the affinity of the compound for the selected biological targets, which play a role in anxiety. Our experiments revealed that HBK-15 showed an anxiolytic-like effect in rats (5 mg/kg) without influencing the pain threshold or the amount of water consumed in the free-drinking test. Furthermore, the tested compound did not show a significant affinity for the selected biological targets, which suggests that its anxiolytic-like mechanism of action could be connected with the interaction with other receptors. This study indicates that multimodal compounds with a receptor profile similar to HBK-15 could be an attractive therapeutic option for patients with a generalized anxiety disorder. However, more studies are required to determine the exact mechanism of action of HBK-15 and its safety profile.

B Cells Induce Hypertension, AT1AA, And Complement Activation In Preeclampsia Or In Response To A Prior Covid-19 Infection During Pregnancy

Preeclampsia (PE), new onset hypertension (HTN) during pregnancy, is associated with placental ischemia and chronic inflammation that includes increased CD4+ T cells, B cells secreting agonistic autoantibodies against the angiotensin II type 1 receptor (AT1AA), and activation of the complement system. Previous studies have shown AT1-AA is produced in patients with COVID-19 infection. Interestingly, having had COVID-19 during pregnancy is associated with increased incidence of developing a PE phenotype during pregnancy. We have previously shown an important role for B cell depletion or AT1AA inhibition to attenuate HTN in rat models of PE. Collectively, this data suggests B cells contribute to PE development and that B cells may increase incidence of PE in patients with a history (Hx) of COVID-19 during pregnancy through production of the AT1AA. We hypothesize B cells from PE or CV Hx PE patients produce AT1AA resulting in HTN and complement activation in pregnancy. Placental B cells were isolated from normal pregnant (NP), PE, normotensive (NT) CV Hx, or PE CV Hx patients at delivery. B cells were transferred i.p. into pregnant athymic rats at gestation (GD) 12. On GD18, carotid catheters were inserted. On GD19, blood pressure was measured and tissues collected. PE B cell recipients had increased Mean Arterial Pressure (MAP) (115+/-3 mmHg n=6) compared to NP B cell recipients (97+/-4 mmHg n=6 p<0.05). PE B cell recipients had increased AT1AA (20+/-2 DELTABPM n=4) compared to NP B cell recipients (6+/-1 DELTABPM n=4 p<0.05). PE B cell recipients had increased markers of complement activation such as reduced plasma C4 (1302+/-169 mug/mL n=4) and C3 (516+/-45 mug/mL n=4) compared to recipients of NP B cells (2348+/-338 mug/mL n=4 p<0.05) and (790+/-66 mug/mL n=4 p<0.05) respectively. CV Hx PE B cell recipients had elevated MAP (108+/-3 mmHg n=4) compared to CV Hx NT B cell recipients (101+/-7 mmHg n=4) and increased AT1AA (24+/-3 DELTABPM n=3) compared to CV Hx NT B cell Recipients (4+/-1 DELTABPM n=4 p<0.05). Collectively, this study demonstrates an important role for B cells to cause HTN during pregnancy;and indicates that B cells contribute to a higher incidence of PE in women with a Hx of CV infection during pregnancy possibly by secreting AT1-AA.

Jahreskongress DVO OSTEOLOGIE 2022.

The proceedings contain 79 papers. The topics discussed include: vertebral fractures increase the risk of subsequent vertebral fractures: results from a large German health insurance dataset;analysis of bone architecture using fractal-based TX-Analyzer in adult patients with osteogenesis imperfecta;bone health in nursing home residents in Germany - do we care enough?;types of therapeutic errors in the management of osteoporosis: results of an experimental study;cysteine-rich angiogenic inducer 61 as a tool to efficiently enrich myeloid angiogenic cells from peripheral blood;response of bone tissue to ostarine treatment and/or treadmill exercise in a healthy adult rat model;influence of the cell aggregation technique on the differentiation of human articular chondrocytes in microtissues;changes in dispensing of anti-osteoporotic drugs during COVID-19 pandemic;and loss of adipogenic dickkopf-1 increases trabecular and cortical bone mass by promoting bone formation in male mice.

Mitochondrial enzymes infused into the kidney under hydrodynamic pressures significantly improves creatinine and BUN levels at the onset of moderate and severe acute kidney injuries

Introduction: The COVID-19 pandemic has highlighted the need to address how renal insults are treated. There is an urgent need to better understand the complex relationship between infections and kidney disease and develop safe and effective approaches that can be translated to the clinic. Hydrodynamic fluid delivery has shown promise in influencing renal function in disease models. This technique previously provided preconditioned protection in acute injury models by upregulating the mitochondrial adaptation, while hydrodynamic injections of saline alone have also improved microvascular perfusion. Accordingly, hydrodynamic mitochondrial gene delivery was applied to investigate its ability to halt renal impairment that may occur following episodes of acute moderate and severe injuries in a rat model. Methods: Transgene infusates were prepared by suspending approximately 2 μg of IDH2 (isocitrate dehydrogenase 2 (NADP+) and mitochondrial) plasmid DNA/g of body weight in 0.5 ml of saline. Animals were subjected to moderate (bilateral pedicle clamp 30 mins) or severe (bilateral pedicle clamp 60 mins) forms of ischemia-reperfusion injury (IRI). Infusates were delivered directly into the left renal vein within 5 seconds, roughly 1 hour after IRI was established. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were monitored for 2 weeks. Results: Significant reductions in the levels of both metabolites (p < 0.05 for both cases) were achieved with single transgene treatments administered at both time points. Specifically, the maximal rises in SCr and BUN levels were reduced by at least 50%, which translated the effects of a severe injury to a moderate injury and a moderate injury to a mild injury. Conclusions: Therefore, this study identifies an approach that boosts recovery and halts the progression of ischemia-reperfusion at its inception and can be vital for high-risk conditions and may help devise translation models to address the rising incidence of acute renal diseases. No conflict of interest

Let's Look at Cannabis from This Angle: COVID-19

Cannabis sativa (Can nabis=hemp) contains cannabinoid-derived main components. Cannabinoids exert biological effects by stimulating cannabinoid receptors distributed throughout the body. Cannabis products affect various systems in the body such as the cardiovascular, respiratory, nervous and immune systems. They show analgesic, antiemetic, anxiolytic, antidepressant, anti-inflammatory, antimicrobial, antihypertensive, antitussive and cardioprotective effects. COVID-19 affects different organ systems in the body and causes symptoms depending on the mutation, such as dry cough, shortness of breath, nausea, vomiting, loss of appetite and sense of smell. In this article, effectiveness of C. sativa and its components on COVID-19 have been evaluated, based on the fact that components or the extracts can affect the whole body due to cannabinoid receptors. Although cannabis and its components have potential beneficial effects for the entry of COVID-19 through the receptors into the body and the symptoms, further studies are needed for their safe use. Appropriate formulation and administration routes should be determined to eliminate the side effects and risk of addiction.

POS-906 TREATMENT WITH RECOMBINANT MUTATED HUMAN ANGIOPOIETIN-LIKE 4 AMELIORATES GLOMERULAR INJURY IN A MODEL OF THE COVID-19 CYTOKINE STORM

Introduction: Some patients with COVID-19 infection develop proteinuria or Acute Kidney Injury, and cases of COVID-19 related Collapsing Glomerulopathy have also been reported. Three published human kidney biopsy studies did not find viral particles in the glomeruli or tubules. This raises the possibility of glomerular and kidney injury induced by the extensive cytokine storm documented early on in the pandemic in hospitalized COVID-19 patients. Mutated forms of Human Angiopoietin-like 4 (protein 8520) were previously shown to reduce proteinuria in rat models of FSGS and diabetic nephropathy (Clement LC, Mace C et al. Nature Medicine 2014). Using different components or innate and adaptive immunity triggered during infection, we developed cytokine cocktails that mimic the COVID-19 cytokine storm, and tested the ability of protein 8520 to reduce glomerular injury in this model. Methods: After baseline urine and blood collections, two groups of BALB/c mice (mean n = 5 mice per group) were injected intravenously with dose 1.8 X of COVID Cocktail D. One hour later, they received 10 μg of His-Tag purified protein 8520 or control human albumin. Overall activity status was documented at 6 and 24 hours. At 24 hours after model induction, blood and urine samples were collected, and the mice euthanized, followed by harvesting of multiple organs including kidneys. Urine albumin was measured by ELISA (Bethyl Labs), and urine and serum creatinine by Mass Spectrometry. Results: Baseline urine albumin to creatinine ratio was similar between the two groups. At 24 hours, urine albumin to creatinine ratio was significantly lower in the 8520 treated compared to control treated group (P < 0.05). Serum creatinine on Day 1 was similar between both groups, and not significantly elevated compared to baseline. Kidney sections were processed for light and electron microscopy, and analysis of these studies is in progress. Conclusions: Treatment with recombinant mutated human Angiopoietin-like 4 reduces albuminuria in a mouse model of the COVID-19 cytokine storm. Further studies will be conducted to establish a potential therapeutic role in viral cytokine storms. No conflict of interest

Lipopolysaccharide induces acute lung injury and alveolar haemorrhage in association with the cytokine storm, coagulopathy and AT1R/JAK/STAT augmentation in a rat model that mimics moderate and severe Covid-19 pathology.

Progress in the study of Covid-19 disease in rodents has been hampered by the lack of angiotensin-converting enzyme 2 (ACE2; virus entry route to the target cell) affinities for the virus spike proteins across species. Therefore, we sought to determine whether a modified protocol of lipopolysaccharide (LPS)-induced acute respiratory distress syndrome in rats can mimic both cell signalling pathways as well as severe disease phenotypes of Covid-19 disease. Rats were injected via intratracheal (IT) instillation with either 15 mg/kg of LPS (model group) or saline (control group) before being killed after 3 days. A severe acute respiratory syndrome (SARS)-like effect was observed in the model group as demonstrated by the development of a "cytokine storm" (>2.7 fold increase in blood levels of IL-6, IL-17A, GM-CSF, and TNF-α), high blood ferritin, demonstrable coagulopathy, including elevated D-dimer (approximately 10-fold increase), PAI-1, PT, and APTT (p < 0.0001). In addition, LPS increased the expression of lung angiotensin II type I receptor (AT1R)-JAK-STAT axis (>4 fold increase). Chest imaging revealed bilateral small patchy opacities of the lungs. Severe lung injury was noted by the presence of both, alveolar collapse and haemorrhage, desquamation of epithelial cells in the airway lumen, infiltration of inflammatory cells (CD45+ leukocytes), widespread thickening of the interalveolar septa, and ultrastructural alterations similar to Covid-19. Thus, these findings demonstrate that IT injection of 15 mg/kg LPS into rats, induced an AT1R/JAK/STAT-mediated cytokine storm with resultant pneumonia and coagulopathy that was commensurate with moderate and severe Covid-19 disease noted in humans.

Safety and Immunogenicity Analysis of a Newcastle Disease Virus (NDV-HXP-S) Expressing the Spike Protein of SARS-CoV-2 in Sprague Dawley Rats.

Despite global vaccination efforts, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and spread globally. Relatively high vaccination rates have been achieved in most regions of the United States and several countries worldwide. However, access to vaccines in low- and mid-income countries (LMICs) is still suboptimal. Second generation vaccines that are universally affordable and induce systemic and mucosal immunity are needed. Here we performed an extended safety and immunogenicity analysis of a second-generation SARS-CoV-2 vaccine consisting of a live Newcastle disease virus vector expressing a pre-fusion stabilized version of the spike protein (NDV-HXP-S) administered intranasally (IN), intramuscularly (IM), or IN followed by IM in Sprague Dawley rats. Local reactogenicity, systemic toxicity, and post-mortem histopathology were assessed after the vaccine administration, with no indication of severe local or systemic reactions. Immunogenicity studies showed that the three vaccination regimens tested elicited high antibody titers against the wild type SARS-CoV-2 spike protein and the NDV vector. Moreover, high antibody titers were induced against the spike of B.1.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta) variants of concern (VOCs). Importantly, robust levels of serum antibodies with neutralizing activity against the authentic SARS-CoV-2 USA-WA1/2020 isolate were detected after the boost. Overall, our study expands the pre-clinical safety and immunogenicity characterization of NDV-HXP-S and reinforces previous findings in other animal models about its high immunogenicity. Clinical testing of this vaccination approach is ongoing in different countries including Thailand, Vietnam, Brazil and Mexico.

Active-site molecular docking of nigellidine with nucleocapsid-NSP2-MPro of COVID-19 and to human IL1R-IL6R and strong antioxidant role of Nigella sativa in experimental rats.

The recent outbreak of SARS CoV-2 has changed the global scenario of human lives/economy. A significant number of the non-survivors showed cardiac renal vasculature dysfunction. A 'cytokine storm' namely, interleukin IL6-IL1 receptors, i.e. IL6R-IL1R over-functioning was reported. Here, nigellidine, an indazole alkaloid and key component of Nigella sativa L. (NS) commonly known as black cumin seed was analysed for COVID-19 protein targeting and IL1R-IL6R inhibition through molecular docking study and biochemical study in experimental rat to evaluate antioxidative capacity. The NMR/X-ray crystallographic/electron microscopic structures of COVID-19 main protease (6LU7)/spike glycoprotein (6vsb)/NSP2 (QHD43415_2)/nucleocapsid (QHD43423), human IL1R (1itb)-IL6R (1pm9) from PDB were retrieved analysed for receptor-ligand interaction. Then, those structures were docked with nigellidine using AutoDock and PatchDock server. A brief comparison was made with nigellicine thymoquinone from N. sativa. Where nigellidine showed highest binding energy of -6.6 kcal/mol, ligand efficiency of -0.3 with COVID19 Nsp2 forming bonds with amino acid CYS240 present in binding pocket. Nigellidine showed strong interaction with main protease (BE: -6.38/LE: -0.29). Nigellidine showed affinity to IL1R (-6.23). The NS treated rat showed marked decline in ALP-SGPT-SGOT-malondialdehyde (MDA) than the basal levels. From the Western blot and activity analysis, it was observed that Nigellidine (sulphuryl group drug) showed no impact on phenol-catalysing ASTIV and steroid-catalysing oestrogen-sulphotransferase expressions and activities in liver tissue and thus has no influence in sulphation-mediated adverse metabolic processes. Conclusively, nigellidine has hepato-reno-protective/antioxidant-immunomodulatory/anti-inflammatory activities with inhibit potentials of COVID-19 proteins. Further validation is necessary.