Development, Histological, and Ultrastructural Evaluation of Sheep Hyperimmune Serum Therapy for COVID-19 / Desarrollo, Evaluación Histológica y Ultraestructural de la Terapia con Suero Hiperinmune de Oveja para COVID-19

SUMMARY: In response to the threat posed by new variants of SARS-CoV-2 and the urgent need for effective treatments in the absence of vaccines, the aim of this study was to develop a rapid and cost-effective hyperimmune serum (HS) derived from sheep and assess its efficacy. The utilization of a halal-certified, easily maintained in certain geographic regions, easy-to-handle animal such as sheep could provide a viable alternative to the expensive option of horses. Sheep were immunized with a whole inactivated SARS-CoV- 2 antigen to produce HS, which was evaluated for neutralizing potency using the PRNT50 assay. K18-hACE2 transgenic mice (n=35) were divided into three groups: control, SARS-CoV-2 exposure through inhalation, and SARS-CoV-2 exposed mice treated with HS. HS efficacy was assessed through serum proinflammatory cytokine levels, qRT-PCR analysis, histopathological examination of lungs and hearts, and transmission electron microscopy. Purified HS exhibited significant neutralizing activity (1/24,576). The SARS-CoV-2+HS group showed lower levels of TNF-α, IL-10, and IL-6 (P<0.01) and relatively lower levels of MCP-1 compared to the SARS-CoV-2 group. HS prevented death, reduced viral RNA levels in the lungs and hearts, protected against severe interstitial pneumonia, preserved lung tissue integrity, and prevented myocyte damage, while the SARS-CoV-2 group exhibited viral presence in the lungs. This study successfully developed a sheep-derived HS against the entire SARS-CoV-2 virus, resulting in a significant reduction in infection severity, inflammation, and systemic cytokine production. The findings hold promise for treating severe COVID-19 cases, including emerging viral variants, and immunocompromised patients.

En respuesta a la amenaza que suponen las nuevas variantes del SARS-CoV-2 y la urgente necesidad de tratamientos eficaces en ausencia de vacunas, el objetivo de este estudio fue desarrollar un suero hiperinmune (HS) rápido y rentable derivado de ovejas. y evaluar su eficacia. La utilización de un animal con certificación halal, de fácil mantenimiento en determinadas regiones geográficas y de fácil manejo, como las ovejas, podría proporcionar una alternativa viable a la costosa opción de los caballos. Las ovejas fueron inmunizadas con un antígeno de SARS-CoV-2 completamente inactivado para producir HS, cuya potencia neutralizante se evaluó mediante el ensayo PRNT50. Los ratones transgénicos K18-hACE2 (n = 35) se dividieron en tres grupos: control, exposición al SARS-CoV-2 mediante inhalación y ratones expuestos al SARS-CoV-2 tratados con HS. La eficacia de HS se evaluó mediante niveles de citoquinas proinflamatorias en suero, análisis qRT-PCR, examen histopatológico de pulmones y corazones y microscopía electrónica de transmisión. El HS purificado exhibió una actividad neutralizante significativa (1/24,576). El grupo SARS-CoV-2+HS mostró niveles más bajos de TNF-α, IL-10 e IL-6 (P<0,01) y niveles relativamente más bajos de MCP-1 en comparación con el grupo SARS-CoV-2. HS evitó la muerte, redujo los niveles de ARN viral en los pulmones y el corazón, protegió contra la neumonía intersticial grave, preservó la integridad del tejido pulmonar y evitó el daño de los miocitos, mientras que el grupo SARS-CoV-2 exhibió presencia viral en los pulmones. Este estudio desarrolló con éxito un HS derivado de ovejas contra todo el virus SARS-CoV-2, lo que resultó en una reducción significativa de la gravedad de la infección, la inflamación y la producción sistémica de citocinas. Los hallazgos son prometedores para el tratamiento de casos graves de COVID- 19, incluidas las variantes virales emergentes y los pacientes inmunocomprometidos.

SARS-CoV-2 Causes Brain Damage: Therapeutic Intervention with AZD8797.

Elevated CX3CL1 is associated with severe COVID-19 and neurologic symptoms. We aimed to investigate the potential protective effects of selective CX3CR1 antagonist AZD8797 on SARS-CoV-2-induced neuronal damage, and to identify the underlying mechanisms. K18-hACE2 transgenic mice (n = 37) were randomly divided into control groups and SARS-CoV-2 groups, with and without intraperitoneal administration of vehicle or AZD8797 (2.5 mg/mL/day), following exposure to either a single dose of SARS-CoV-2 inhalation or no exposure. Object recognition and hole board tests were performed to assess memory function. Postinfection 8 days, brain tissues were analyzed for histopathological changes, viral, glial, apoptotic, and other immunohistochemical markers, along with measuring malondialdehyde, glutathione, and myeloperoxidase activities. Serum samples were analyzed for proinflammatory cytokines. The SARS-CoV-2 group showed significant weight loss, neuronal damage, oxidative stress, and impaired object recognition memory, while AZD8797 treatment mitigated some of these effects, especially in weight, apoptosis, glutathione, and MCP-1. Histopathological analyses supported the protective effects of AZD8797 against SARS-CoV-2-induced damage. The CX3CL1-CX3CR1 signaling pathway could offer a promising target for reducing SARS-CoV-2's neurological impact, but additional research is needed to confirm these findings in combination with other therapies and assess the clinical significance.

Evidence for residual SARS-CoV-2 in corpus cavernosum of patients who recovered from COVID-19 infection.

BACKGROUND: The COVID-19 is an ongoing health problem with millions of cases and deaths worldwide. Although the virus is transmitted with droplets through the respiratory system, the involvement of different organs has been reported. OBJECTIVES: The pandemic caused urological procedures to be postponed when patient is infected with SARS-CoV-2. However, the reliability of 1 month postpone period and long-term complications of the virus, such as a possible erectile dysfunction (ED) is not clarified. We aimed to compare the corpus cavernosum of patients 1 and 7 months after COVID-19 infection with control patients who had not COVID-19 and search for SARS-CoV-2 in tissues using immunohistochemistry and electron microscopy. MATERIALS AND METHODS: Three groups of subjects underwent penile prosthesis implantation and Nesbit procedure for Peyronie's disease 1 and 7 months after COVID-19 infection and control group without previous COVID-19 infection. We searched for SARS-CoV-2 in penile tissue using RT-PCR, electron microscopy and immunohistochemistry. RESULTS: Electron microscopy and immunohistochemical staining showed SARS CoV-2 virus in the penile corpus cavernosum of patients 1 month after COVID-19 recovery. Immunohistochemical staining intensity correlated with the severity of previous infection. Transmission electron microscopy revealed intracellular virtual particles of about 80 nm with a typical morphology of prominent spikes and electron-dense dots of nucleocapsid in addition to vesicles filled with virus-like particles. Cells showed increased membrane trafficking. The 1 month after COVID-19 group showed an increased number of fibroblasts. The 7 months after COVID-19 group had similar morphology and immunoreactivity as control group. DISCUSSION: This is the first study of late post-COVID examination of penis and the second study of early post-COVID examination of corpus cavernosum. For 1 month post-COVID patients, the aetiology of ED could be the viral infection that is also affecting corpora cavernosa. We hypothesize that viral infection affects the endocytic and exocytic pathways, hence the metabolic activity of cells that can be the reason of altered functions in some post-COVID patients. CONCLUSION: This study is important because it did not detect any virus residue in the tissue samples at the seventh month. In addition, we can say that the penile surgeries should be postponed more than 1 month after the COVID infection according to this study. But, there is a need for new studies with large series and high levels of evidence that can show how long the virus remains in the corpus cavernosum. Patients should be followed in this respect.

A newly developed carrier for the vitrification of prepubertal testicular tissue and its comparison with four different carriers.

RESEARCH QUESTION: The cryopreservation of prepubertal testicular tissue is important for children who are to undergo gonadotoxic treatment. There is ongoing debate around the optimal carrier for an inexpensive and rapid vitrification technique. How efficient would a novel, practical and sterilizable metal brush be when compared with previously used carriers? DESIGN: The testicular tissues of prepubertal rats were vitrified using four different carriers and evaluated by light microscopy and transmission electron microscopy. RESULTS: Nuclei were easily discriminated in the metal brush, aluminium foil and high-security straw groups, but there was decreased discrimination of structures in the metal wire group. Minimal cytoplasmic degeneration, vacuolization and mild reversible degenerative effects were seen in spermatogonial stem cells and Sertoli cells in the metal brush group. Mild to moderate structural changes were found in the aluminium foil group. Severe pyknosis of the nuclei, a high degree of swelling, expansion of the endoplasmic reticulum, and swelling and blurring of the mitochondria were seen in the metal wire and high-security straw groups. The cell viabilities in the metal brush, aluminium foil, metal wire and high-security straw groups were 91.6 ± 3.85%, 83.0 ± 4.06%, 76.0 ± 3.16% and 68.6 ± 4.93%, respectively. CONCLUSIONS: The metal brush is a promising new carrier for prepubertal testis vitrification.

Histopathologic evaluation of saphenous vein grafts in patients with type II diabetes mellitus undergoing coronary artery bypass grafting.

INTRODUCTION: Diabetes Mellitus (DM) has been known to be a risk factor for the development of more severe form of saphenous vein graft disease after coronary artery bypass grafting (CABG). We aimed to evaluate the impact of type II-DM on histopathological features of great saphenous vein grafts of patients undergoing CABG. PATIENTS AND METHODS: Forty consecutive patients undergoing elective CABG were enrolled into the study. Patients were grouped into two; Diabetic group (n = 20); includes patients with preoperative diagnosis of type II-DM and Nondiabetic group (n = 20): those without type II-DM. In all patients, a short segment of the great saphenous vein graft at the level of medial malleolus was taken for light microscopy and transmission electron microscopy (TEM) evaluation. Moreover, immunoexpressions of Caveolin-1, Vascular cell adhesion protein 1 (VCAM-1) and endothelial nitric oxide synthase (eNOS) were studied. RESULTS: There were no differences in the demographics of patients between two groups. The magnitude of intimal fibrosis in diabetic group was slightly higher than in nondiabetics (1.95 ± 0.99 versus 1.3 ± 0.8, P = .04). In TEM, vacuolization in endothelial cells, substance accumulation along with coarse collagen fibers and cytoplasmic degeneration with vacuolization in muscle cells were detected in diabetic group. While there were no differences in Caveolin-1 and VCAM-1 immunostaining, the intensity of positive eNOS immunostaining was significantly higher in endothelium (2.10 ± 0.64 versus 1.55 ± 0.68, P = .01) and tunica media 1.75 ± 0.63 versus 1.2 ± 0.52, P = .007) in nondiabetic group, respectively) compared with diabetic group. CONCLUSION: Type II DM might be a reason for decreased expression of eNOS and increased intimal fibrosis, vacuolization of endothelial and smooth muscle cells in saphenous vein grafts. The clinical implications of these alterations on the graft patency need to be evaluated.

Milrinone Attenuates Heart and Lung Remote Injury after Abdominal Aortic Cross-Clamping.

BACKGROUND: Phosphodiesterase enzymes play a pivotal role in the pathogenesis of ischemia/reperfusion (IR). We examined the role of milrinone (MIL), a phosphodiesterase 3 inhibitor, on remote injury of the heart and lung after abdominal aortic cross-clamping. DESIGN: Experimental study. METHODS: Twenty-one Wistar rats were divided into 3 groups: (1) control (C, n = 7), underwent laparotomy and exploration of abdominal aorta only; (2) IR (n = 7), normal saline was applied intraperitoneally (i.p) before IR induced by clamping of the abdominal aorta for 1 hr and then allowing reperfusion for 1 hr; and (3) MIL + IR (n = 7), MIL was given (0.5 mg/kg, i.p) before IR. After sacrification, the lungs and hearts were taken out for analyses and the tissue malondialdehyde (MDA) and glutathione (GSH) were studied. All tissues were examined under light microscopy and transmission electron microscopy (TEM). Expressions of caveolin (Cav)-1 in the lung and Cav-1 and Cav-3 in the heart were examined immunohistochemically. RESULTS: The MIL + IR group had significantly a lower magnitude of oxidative stress than the IR group both in the lung and heart (lung: P = 0.03 for MDA and 0.001 for GSH and heart: P = 0.002 for MDA and 0.000 for GSH). In light microscopy, the MIL + IR group had statistically a lower total injury score than the IR group for both the lung and heart tissue (P = 0.03 and P = 0.04, respectively). In TEM, regression of mitochondrial degeneration and lamellar bodies in type II pneumocytes in the lungs and obvious improvements in disruption at the intercalated discs and mitochondrial degeneration in the hearts in the MIL + IR group were detected compared with the IR group. The expression of both Cav-1 and Cav-3 in the MIL + IR group was improved compared with the IR group (P = 0.03 for both). CONCLUSIONS: MIL attenuates remote injury of heart and lung in lower body IR by inhibiting oxidative stress. Moreover, Cav-1 and Cav-3 might have a potential role in MIL-induced cardioprotection.

Bacterial cellulose tubes as a nerve conduit for repairing complete facial nerve transection in a rat model.

PURPOSE: Functionality of the facial nerve is cosmetically important. While many techniques have been investigated, early and effective treatment for traumatic facial nerve paralysis remains challenging. Here, we aim to examine bacterial cellulose (BC) as a new tubularization material for improving facial nerve regeneration. METHODS: Our study was performed on 40 female Sprague Dawley rats. Rats were randomly divided into four groups, with 10 rats per group. In all rats, the main trunk of the facial nerve was completely cut 8 mm before the branching point. For repairing the facial nerve, in group 1, the nerve was left to recover spontaneously (control group); in group 2, it was repaired by primary suturing (8.0 Ethilon sutures, Ethicon); in group 3, BC tubes alone were used to aid nerve repair; and in group 4, both BC tubes and primary sutures (8.0 Ethilon sutures) were used. After 10 weeks, the facial nerve regeneration was evaluated by the whisker movement test and electrophysiologically (nerve stimulation threshold and compound muscle action potential). Nerve regeneration was assessed by calculating the number of myelinated nerve fibers, and by microscopically evaluating the amount of regeneration and fibrosis. RESULTS: No significant difference was observed among the groups in terms of whisker movement and electrophysiological parameters (P > 0.05). We found that the numbers of regenerating myelinated fibers were significantly increased (P < 0.05) when BC tubes were used as a nerve conduit. CONCLUSIONS: BC can be easily shaped into a hollow tube that guides nerve axons, resulting in better nerve regeneration after transection.

Obestatin improves oxidative brain damage and memory dysfunction in rats induced with an epileptic seizure.

Obestatin was shown to alleviate renal, gastrointestinal and haemorrhage-induced brain injury in rats. In order to investigate the neuroprotective effects of obestatin on seizure-induced oxidative brain injury, an epileptic seizure was induced with a single intraperitoneal (i.p.) dose of pentylenetetrazole (PTZ, 45mg/kg) in male Wistar rats. Thirty minutes before the PTZ injection, rats were treated with either saline or obestatin (1µg/kg, i.p.). Seizure was video-taped and then evaluated by using Racine's scoring (0-5). For the assessment of memory function, passive-avoidance test was performed before seizure induction, which was repeated on the 3rd day of seizure. The rats were decapitated at the 24th or 72nd hour of seizures and brain tissues were obtained for histopathological examination and for measuring levels of malondialdehyde (MDA), glutathione (GSH), reactive oxygen radicals and myeloperoxidase (MPO) activity. Obestatin treatment reduced the average seizure score, decreased the occurrence and duration of generalized tonic-clonic seizures, presenting with a shorter latency to their onset. Increased lipid peroxidation and enhanced generation of oxygen-derived radicals detected at the post-seizure 72nd h were suppressed by the consecutive treatments of obestatin, but no changes were observed by the single obestatin treatment in the 24-h seizure group. Neuronal damage and increased GFAP immunoreactivity, observed in the hippocampal areas and cortex of PTZ-induced rats were alleviated in 3-day obestatin-treated PTZ group. PTZ-induced memory dysfunction was significantly improved in obestatin-treated PTZ group as compared to saline-treated rats. The present data indicate that obestatin ameliorated the severity of PTZ-induced seizures, improved memory dysfunction and reduced neuronal damage by limiting oxidative damage.

Effects of Myrtus communis extract treatment in bile duct ligated rats.

BACKGROUND: The aim of our study was to investigate the antifibrotic and antioxidant effects of Myrtus communis subsp. communis (MC) extract against liver injury and fibrosis occurring in rats with biliary obstruction. MATERIALS AND METHODS: The rats were randomized into four groups (n = 8). Control group (C), MC-administrated group (MC), the bile duct ligation (BDL), and BDL + MC groups. MC was administered at a dose of 50 mg/kg a day orally for 28 days. In blood samples, total bilirubin, direct bilirubin, alanine aminotransferase, aspartate aminotransferase levels, tumor necrosis factor-α, and interleukin-1ß measurement were measured. Oxidative injury was examined by measuring luminol and lucigenin chemiluminescence, malondialdehyde and glutathione levels, superoxide dismutase and myeloperoxidase activities. Transforming growth factor-beta and hydroxyproline levels were measured for analyzing fibrosis. The hepatic injury was also analyzed microscopically. RESULTS: Plasma total bilirubin, direct bilirubin, alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-α, and interleukin-1ß levels were found significantly high in the BDL group, while these values significantly decreased in the BDL group treated with MC. On the other hand, the glutathione and superoxide dismutase values significantly decreased in the BDL group compared to the control group but increased markedly in BDL + MC group compared to the BDL group. Malondialdehyde levels, myeloperoxidase activity, tissue luminol, lucigenin, transforming growth factor-beta, and hydroxyproline levels when compared with the control group increased dramatically in the BDL group and reduced the MC + BDL group. CONCLUSIONS: Our results suggest that MC protects the liver tissues against oxidative damage following BDL via its radical scavenging and antioxidant activities, which appear to involve the inhibition of tissue neutrophil infiltration.

The role of CX3CL1 in fetal-maternal interaction during human gestation.

Embryo implantation and subsequent placentation require a fine balanced fetal-maternal cross-talk of hormones, cytokines and chemokines. Amongst the group of chemokines, CX3CL1 (also known as fractalkine) has recently attracted attention in the field of reproductive research. It exists both as membrane-bound and soluble isoforms. On the basis of current experimental evidence, fractalkine is suggested to regulate adhesion and migration processes in fetal-maternal interaction at different stages of human pregnancy. Expressed by uterine glandular epithelial cells, predominantly during the mid-secretory phase of the menstrual cycle, fractalkine appears to prime the blastocyst for forthcoming implantation. After implantation, fractalkine is suggested to regulate invasion of extravillous trophoblasts by altering their expression profile of adhesion molecules. With onset of perfusion of the intervillous space at the end of first trimester, fractalkine present at the apical microvillous plasma membrane of the syncytiotrophoblast may mediate close interaction of placental villi with circulating maternal blood cells.