Chemical and Microbiological Changes of Expired Platelet Concentrate.

Background: Platelets are a commonly used blood component to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. They are stored at room temperature (22-24°C) for five days unless specific measures are taken to extend the shelf life to seven days or more. After five days, this study evaluated platelet units' biochemical changes and bacterial growth. Study Design and Methods: Platelet concentrate was collected from 30 random donors: 8 females and 22 males. The collected samples were then placed on an agitator at room temperature and tested for their pH, protein content, and glucose levels using Roche Combur 100 Test® Strips. The Haemonetics eBDS™ System was used for bacterial detection. The measurements were taken on day five as the control and then repeated on days 7, 9, and 11 to observe any changes. On days 5 and 7, all parameters remained unchanged. However, glucose levels significantly changed (p=<0.0001) on days 9 and 11. Regarding pH, a significant change was observed on day 9 (p=0.033) and day 11 (p=0.0002). Results: There were no significant changes in all parameters on days 5 and 7. However, glucose was substantially changed (p=<0.0001) on days 9 and 11. For pH, there was a significant change in pH on day 9 (p=0.033) and day 11 (p=0.0002). Discussions: Our study found that platelet concentrate extension is possible for up to seven days. However, further studies are needed to evaluate platelet function during expiry time and to assess the stability of platelet morphology and function.

Detection of human platelets antigen polymorphism (HPA-1 and HPA-3) and human factor XIII mutation in Sudanese women with recurrent pregnancy loss.

BACKGROUND: Recurrent pregnancy Loss (RPL) is common problem affecting many couples. A certain genetic variants link to increase the danger of this condition particularly HPA-1, HPA-3 and Human Factor XIII Val34Leu Mutation. The present study aims to find an association between RPL and the Factor XIII Val34Leu polymorphism, as well as HPA-1 and HPA-3 in Sudanese women with RPL. METHODS: This case-control study conducted between June 2022 and December 2022 included 216 women, with 103 cases having minimum three abortions in the past, and 113 healthy controls with at least two full-term births and no abortion history. DNA was isolated from whole blood and the status of three genetic polymorphisms (HPA-1, HPA-3, and factor XIII) was done using a polymerase chain reaction (PCR). Data was analysed using the SPSS version 24 software. RESULTS: The A/A genotype was found to be more prevalent in cases (79.6%) and controls (96.5%) regarding HPA-1. A significant difference was observed in overall allele frequency for B allele (97.0%) and expected frequency of A allele was (81.1%) using the Hardy-Weinberg distribution (p < 0.001). The genotype A/A was most common in these patients (90.3%) and controls (100%), while B/B genotype was only (9.7%) in patients regarding HPA-3. Furthermore, the frequency of Val/Val genotype was higher in cases (88.3%) as compared with controls (90.3%). The risk of RPL in patients was nearly the same in Val/Leu individuals and controls group but all these differences were not statistically significant (p > 0.05). CONCLUSION: Our results indicate a link between Human Platelet Antigen-1 (HPA-1), Human Platelet Antigen-3 (HPA-3) and Factor XIII gene polymorphism with RPL.

Antibiotic Resistance, Virulence Gene Detection, and Biofilm Formation in Aeromonas spp. Isolated from Fish and Humans in Egypt.

The genus Aeromonas is widely distributed in aquatic environments and is recognized as a potential human pathogen. Some Aeromonas species are able to cause a wide spectrum of diseases, mainly gastroenteritis, skin and soft-tissue infections, bacteremia, and sepsis. The aim of the current study was to determine the prevalence of Aeromonas spp. in raw fish markets and humans in Zagazig, Egypt; identify the factors that contribute to virulence; determine the isolates' profile of antibiotic resistance; and to elucidate the ability of Aeromonas spp. to form biofilms. The examined samples included fish tissues and organs from tilapia (Oreochromis niloticus, n = 160) and mugil (Mugil cephalus, n = 105), and human skin swabs (n = 51) and fecal samples (n = 27). Based on biochemical and PCR assays, 11 isolates (3.2%) were confirmed as Aeromonas spp. and four isolates (1.2%) were confirmed as A. hydrophila. The virulence genes including haemolysin (hyl A) and aerolysin (aer) were detected using PCR in A. hydrophila in percentages of 25% and 50%, respectively. The antimicrobial resistance of Aeromonas spp. was assessed against 14 antibiotics comprising six classes. The resistance to cefixime (81.8%) and tobramycin (45.4%) was observed. The multiple antibiotic resistance (MAR) index ranged between 0.142-0.642 with 64.2% of the isolates having MAR values equal to 0.642. Biofilm formation capacity was assessed using a microtiter plate assay, and two isolates (18.1%) were classified as biofilm producers. This study establishes a baseline for monitoring and controlling the multidrug-resistant Aeromonas spp. and especially A. hydrophila in marine foods consumed in our country to protect humans and animals.

The Combined Effect of Licorice Extract and Bone Marrow Mesenchymal Stem Cells on Cisplatin-Induced Hepatocellular Damage in Rats.

Drug-induced liver damage is a life-threatening disorder, and one major form of it is the hepatotoxicity induced by the drug cisplatin. In folk medicine, Licorice (Glycyrrhiza glabra (is used for detoxification and is believed to be a potent antioxidant. Currently, the magically self-renewable potential of bone marrow mesenchymal stem cells (BM-MSCs) has prompted us to explore their hepatoregenerative capability. The impact of G. glabra extract (GGE) and BM-MSCs alone and, in combination, on protecting against hepatotoxicity was tested on cisplatin-induced liver injury in rats. Hepatic damage, as revealed by liver histopathology and increased levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and malondialdehyde (MDA), was elevated in rats by received 7 mg/kg of cisplatin intraperitoneally. The combination of GGE and BM-MSCs returned the enzyme levels to near the normal range. It also improved levels of liver superoxide dismutase (SOD) and glutathione (GSH) and reduced MDA levels. Additionally, it was found that when GGE and BM-MSCs were used together, they significantly downregulated caspase9 (Casp9), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and interleukin-1ß (IL-1ß), which are involved in severe proinflammatory and apoptotic signaling cascades in the liver. Moreover, combining GGE and BM-MSCs led to the normal result of hepatocytes in several examined liver histological sections. Therefore, our findings suggest that GGE may have protective effects against oxidative liver damage and the promising regenerative potential of BM-MSCs.

Molecular Determination of Vascular Endothelial Growth Factor, miRNA-423 Gene Abnormalities by Utilizing ARMS-PCR and Their Association with Fetal Hemoglobin Expression in the Patients with Sickle Cell Disease.

Recent studies have indicated that microRNA and VEGF are considered to be genetic modifiers and are associated with elevated levels of fetal haemoglobin HbF, and thus they reduce the clinical impact of sickle haemoglobin (HbS) patients. This cross-sectional study was performed on clinical confirmed subjects of SCD cases. miR-423-rs6505162 C>T and VEGF-2578 C>A genotyping was conducted by ARMS-PCR in SCD and healthy controls. A strong clinical significance was reported while comparing the association of miR-423 C>T genotypes between SCD patients and controls (p = 0.031). The microRNA-423 AA genotype was associated with an increased severity of SCD in codominant model with odd ratio (OR = 2.36, 95% CI, (1.15-4.84), p = 0.018) and similarly a significant association was observed in recessive inheritance model for microRNA-423 AA vs (CC+CA) genotypes (OR = 2.19, 95% CI, (1.32-3.62), p < 0.002). The A allele was associated with SCD severity (OR = 1.57, 95% CI, (1.13-2.19), p < 0.007). The distribution of VEGF-2578 C>A genotypes between SCD patients and healthy controls was significant (p < 0.013). Our results indicated that in the codominant model, the VEGF-2578-CA genotype was strongly associated with increased SCD severity with OR = 2.56, 95% CI, (1.36-4.82), p < 0.003. The higher expression of HbA1 (65.9%), HbA2 (4.40%), was reported in SCD patients carrying miR-423-AA genotype than miR-423 CA genotype in SCD patients carrying miR-423 CA genotype HbA1 (59.98%), HbA2 (3.74%) whereas SCD patients carrying miR-423 CA genotype has higher expression of HbF (0.98%) and HbS (38.1%) than in the patients carrying AA genotype HbF (0.60%), HbS (36.1%). ARMS-PCR has been proven to be rapid, inexpensive and is highly applicable to gene mutation screening in laboratories and clinical practices. This research highlights the significance of elucidating genetic determinants that play roles in the amelioration of the HbF levels that is used as an indicator of severity of clinical complications of the monogenic disease. Further well-designed studies with larger sample sizes are necessary to confirm our findings.

Impact of COVID-19 on Blood Donation and Supply: A Multicenter Cross-Sectional Study from Saudi Arabia.

BACKGROUND: The coronavirus disease-19 (COVID-19) pandemic caused a major impact on blood donation process and supply globally. A lockdown management procedure was launched nationally in Saudi Arabia to manage this global health crisis. The main aim of this study was to determine the effect of COVID-19 lockdown on blood donation services and supply in different regions of Saudi Arabia. Study Design and Methods. A retrospective cross-sectional study was conducted in the blood bank centers of 5 major cities including Riyadh, Jeddah, Dammam, Hail, and Jizan in Saudi Arabia. Demographic and blood characteristics were retrieved from the first 6 months of 2019 (January-June) and compared to the same period of 2020. RESULTS: Our findings showed variation in the characteristics of blood donation and supply among the centers surveyed, as some of these centers were adversely affected, while others showed an increase in the availability of blood products during the pandemic. For example, Jeddah's center was significantly affected by COVID-19 lockdown whereas Hail's center showed a significant increase in the analyzed characteristics of blood donation services in 2020 compared to 2019. Overall, there was no major difference among the surveyed centers between 2020 and 2019, and this might be due to the effective management of blood supply and transfusion. Discussion. Although blood supply and transfusion practice was slightly affected at various degree among the surveyed centers, the whole process did not show a significant effect on the overall outcome. This is in fact due to the proper preparedness, management of blood requirements and supplies, and efficient response of the surveyed centers in Saudi Arabia.

The association between different blood group systems and susceptibility to COVID-19: a single center cross-sectional study from Saudi Arabia.

Background: Since the beginning of COVID-19 pandemic, many associated factors have been investigated to clarify the susceptibility and severity among the affected individuals. Biological markers can play an important role in identification of individual susceptibility to such pandemic. Growing evidence suggest the influence of different blood group systems on susceptibility to COVID-19 virus, with a particular blood type conferring selection advantage. Objectives: The study aimed to determine the association of ABO, Rhesus (D) and P1 blood groups with COVID-19 susceptibility in Taif city, Western Saudi Arabia. Methods: ABO, D and P1 blood antigens were determined in 104 blood samples of COVID-19 patients versus 100 control samples using either automated immunohematology analyser or test tube method. Statistical differences between patients and control samples were calculated based on p-value where results of ≤ 0.05 were considered significant. Results: O+ve blood group constituted the predominant type among the studied samples. Determination of P1 antigen showed significant association where Anti-P1 was positive in 76.9% of patients compared to 61.0% of controls with a P value of 0.01 conferring the susceptibility of P1+ve patients to COVID-19. Conclusion: Although our study showed no significant association between ABO and D, and susceptibility to COVID-19, there was a significant association between P1+ve and COVID-19. P1+ve participants were 2.131 times more associated with the risk of COVID-19 infection than those with Anti P1-ve. Thus, P1 antigen can be used as a biological marker for identification of individuals susceptibility to COVID-19. It is strongly advised that such individuals should consider extra protective measures. Further studies on other contributing factors should also be considered for more scientific clarity.

Wound Healing After Alkali Burn Injury of the Cornea Involves Nox4-Type NADPH Oxidase.

Purpose: Corneal injury that occurs after burning with alkali initiates wound-healing processes, including inflammation, neovascularization, and fibrosis. Excessive reactions to injury can reduce corneal transparency and thereby compromise vision. The NADPH oxidase (Nox) enzyme complex is known to be involved in cell signaling for wound-healing angiogenesis, but its role in corneal neovascularization has been little studied. Methods: The center corneas of wild-type and Nox4 knockout (KO) mice were injured with 3 µL 1 M NaOH, while the contralateral corneas remained untouched. On day 7, mRNA expression levels of NADPH oxidase isoforms, the proangiogenic factors VEGF-A and TGFß1, and proinflammatory genes ICAM-1 and VCAM-1 were determined. Corneal neovascularization and fibrosis were visualized using PECAM-1 antibody and picrosirius red staining, respectively, on the same day. Results: Expressions of both Nox2 and Nox4 gene isoforms as well as the above genes were markedly increased in the injured corneas at 7 days. Injured corneas showed neovascularization and fibrosis as well as an increase in clinical opacity score. All responses stimulated by alkali burn were abrogated in Nox4 KO mice. Conclusions: Nox4 could be a new target to treat pathologic corneal wound-healing responses and such targeting might prevent blindness caused by burn injuries.

Role of NADPH Oxidase-4 in Human Endothelial Progenitor Cells.

Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization.

Trichostatin A, a histone deacetylase inhibitor suppresses NADPH Oxidase 4-Derived Redox Signalling and Angiogenesis.

Histone deacetylase (HDAC) inhibitors are known to suppress abnormal development of blood vessels. Angiogenic activity in endothelial cells depends upon NADPH oxidase 4 (Nox4)-dependent redox signalling. We set out to study whether the HDAC inhibitor trichostatin A (TSA) affects Nox4 expression and angiogenesis. Nox4 expression was measured by real time PCR and Western blot analysis in endothelial cells. Hydrogen peroxide (H2 O2 ) was measured by amplex(®) red assay in endothelial cells. Nox4 was knocked down by Nox4 shRNA. In vitro angiogenic activities such migration and tubulogenesis were assessed using wound healing and Matrigel assays, respectively. In vivo angiogenic activity was assessed using subcutaneous sponge assay in C57Bl/6 and Nox4-deficient mice. Trichostatin A reduced Nox4 expression in a time- and concentration-dependent manner. Both TSA and Nox4 silencing decreased Nox4 protein and H2 O2 . Mechanistically, TSA reduced expression of Nox4 via ubiquitination of p300- histone acetyltransferase (p300-HAT). Thus, blocking of the ubiquitination pathway using an inhibitor of ubiquitin-activating enzyme E1 (PYR-41) prevented TSA inhibition of Nox4 expression. Trichostatin A also reduced migration and tube formation, and these effects were not observed in Nox4-deficient endothelial cells. Finally, transforming growth factor beta1 (TGFß1) enhanced angiogenesis in sponge model in C57BL/6 mice. This response to TGFß1 was substantially reduced in Nox4-deficient mice. Similarly intraperitoneal infusion of TSA (1 mg/kg) also suppressed TGFß1-induced angiogenesis in C57BL/6 mice. Trichostatin A reduces Nox4 expression and angiogenesis via inhibition of the p300-HAT-dependent pathway. This mechanism might be exploited to prevent aberrant angiogenesis in diabetic retinopathy, complicated vascular tumours and malformations.

Smad-independent pathway involved in transforming growth factor ß1-induced Nox4 expression and proliferation of endothelial cells.

NADPH oxidase-derived reactive oxygen species are important for various cellular functions, including proliferation. Endothelial cells predominantly express the Nox4 isoform of NADPH oxidase, but it is not entirely clear how it is regulated. In this study, we investigated the signalling pathways involved in transforming growth factor-ß1 (TGF-ß1)-induced Nox4 expression and the proliferation of human microvascular endothelial cells (HMECs). TGF-ß1 stimulated Nox4 messenger RNA and protein expression in HMECs. TGF-ß1-induced Nox4 also increased hydrogen peroxide production, which was inhibited by diphenyleneiodonium and EUK134. The acute treatment of HMECs with TGF-ß1 enhanced the phosphorylation of Smad2 and extracellular signal-regulated kinase (ERK) 1/2, without affecting p38MAPK, Akt, or Jun N-terminal kinase 1/2 (JNK1/2) pathways. Further, inhibition of Smad2 signalling using an inhibitor of activin receptor-linked kinase 5 SB431542 reduced TGF-ß1-induced Nox4 expression, while inhibition of ERK1/2 with the inhibitor of mitogen-activated protein kinase kinase 1/2 U0126 decreased both basal and TGF-ß1-induced Nox4 expression. Inhibition of ERK1/2 phosphorylation with U0126 did not affect Smad2 phosphorylation. Finally, TGF-ß1 enhanced endothelial cell proliferation, which was reduced by U0126 but not by SB431542. These findings suggest that the non-canonical pathway ERK1/2 regulates Nox4 expression and may be involved in TGF-ß1-induced proliferation of endothelial cells, which is vital during angiogenesis and vascular development.