Development of a Novel SARS-CoV-2 Immune Complex Vaccine Candidate (CRCx) with Broad Immune Responses: A Preclinical Trial in Animal Model

BackgroundThe ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a serious threat to global public health and imposes a severe burden on the entire human population. Faced with a virus that can mutate its structure while immunity is incapacitated, a need to develop a universal vaccine that can boost immunity to coronaviruses is highly needed. DesignFive formulations of two types (CRCx2 and CRCx3) of immune complexes with an immunogen adjuvant were evaluated in a mouse model as candidate SARS CoV-2 vaccines in a pretrial prior to clinical trials in humans. CRCx3 comprises 3 different formulas and CRCx2 comprises 2. Balb/c mice were vaccinated intraperitoneally on days 0/7 with a high or low dose of CRCx2 or on days 0/7/14 with a high, medium, or low dose of CRCx3 series, and their blood was sampled for serum antibody measurements. Mice were challenged with live virus after immunization with either vaccine to evaluate prophylaxis ability or treated with them after challenge to evaluate therapeutic ability on day 15. Immunological markers and histopathological studies as well as titration of neutralizing antibodies to the vaccines were evaluated and analyzed. ResultsCRCx 3 and CRCx 2 vaccine candidates induced elevated levels of positive neutralizing antibodies as well as a cellular immune response with safety, efficient productivity, and good genetic stability for vaccine manufacturing to provide protection against SARS-CoV-2 with relatively higher levels with the high dose CRCx2 candidate combination. ConclusionsHighly efficient protection and therapeutic effect against SARS-CoV-2 were obtained with a double-dose immunization schedule spaced at 7-day intervals using injections 0.25 of or 0.40 ml of CRCx2 vaccine formulations with a 25-mm needle. These results support further evaluation of CRCx in a clinical trial on humans.

PHIDA: A High Throughput Turbidimetric Data Analytic Tool to Compare Host Range Profiles of Bacteriophages Isolated Using Different Enrichment Methods.

Bacteriophages are viruses that infect bacteria and are present in niches where bacteria thrive. In recent years, the suggested application areas of lytic bacteriophage have been expanded to include therapy, biocontrol, detection, sanitation, and remediation. However, phage application is constrained by the phage's host range-the range of bacterial hosts sensitive to the phage and the degree of infection. Even though phage isolation and enrichment techniques are straightforward protocols, the correlation between the enrichment technique and host range profile has not been evaluated. Agar-based methods such as spotting assay and efficiency of plaquing (EOP) are the most used methods to determine the phage host range. These methods, aside from being labor intensive, can lead to subjective and incomplete results as they rely on qualitative observations of the lysis/plaques, do not reflect the lytic activity in liquid culture, and can overestimate the host range. In this study, phages against three bacterial genera were isolated using three different enrichment methods. Host range profiles of the isolated phages were quantitatively determined using a high throughput turbidimetric protocol and the data were analyzed with an accessible analytic tool "PHIDA". Using this tool, the host ranges of 9 Listeria, 14 Salmonella, and 20 Pseudomonas phages isolated with different enrichment methods were quantitatively compared. A high variability in the host range index (HRi) ranging from 0.86-0.63, 0.07-0.24, and 0.00-0.67 for Listeria, Salmonella, and Pseudomonas phages, respectively, was observed. Overall, no direct correlation was found between the phage host range breadth and the enrichment method in any of the three target bacterial genera. The high throughput method and analytics tool developed in this study can be easily adapted to any phage study and can provide a consensus for phage host range determination.

A molecular explanation of cardiovascular protection through abnormal cannabidiol: Involving the dysfunctional ß-adrenergic and ATP-sensitive K+ channel activity in cardiovascular compromised preterm infants.

Growing cannabis efficacy, usage frequency, legal supply, and declining awareness of danger recently led to expanded United States cannabis exposure. In turn, cannabis use among elderly people over 50 has more than tripled in a decade and has contributed toward a positive association of cannabis use with pathological conditions, which include type II diabetes, metabolic syndrome, neurovascular and cardiovascular disease. Remarkably, all these outcome results are mediated by the involvement of the ATP-sensitive K+ channel. Cardiovascular compromise is a common syndrome in preterm infants that leads to incidence and death and has been distinguished by poor systemic flow or hypotension. Conditions of cardiovascular compromise include vasodysregulation and myocardial malfunction through dysfunctional ß-adrenergic activity. To avoid organ hypoperfusion progressing to tissue hypoxia-ischemia, inotropic drugs are used. Many premature children, however, respond insufficiently to inotropic activity with adrenergic agonists. The clinical disturbance including myocardial dysfunction through the activation of the ATP-sensitive K+ channel is often involved and the comparative efficacy of the nonpsychotropic cannabinoid, abnormal cannabidiol (Abn-CBD) is not yet known. Therefore, our primary aim was to investigate the molecular exploration of the cannabinoid system specifically Abn-CBD in cardiovascular protection involving dysregulated KATP.

Plantar fasciitis: Talonavicular instability/spring ligament failure as the driving force behind its histological pathogenesis.

The aetiology of plantar fasciitis (PF) remains uncertain and to date, it is not known if there is an association with spring ligament laxity. In this study, 28 patients with unilateral plantar fasciitis were evaluated. A digital Klaumeter was used to assess first ray for instability and lateral plane translation was used as a measure of spring ligament laxity in the affected vs unaffected foot (internal control). Retromalleolar tenderness as a sign of a reactive tibialis posterior tendon was also assessed. The mean lateral translation score for symptomatic feet was 67.2 (95% CI [63.26-71.14]), compared to asymptomatic feet mean of 33.0 (95% CI [27.35-38.65] p < 0.05). The mean TMT instability score for symptomatic feet was 11.3 (95% CI [10.29-12.3]), compared to the asymptomatic feet mean of 5.9 (95% CI [4.49-7.31] p < 0.05). 100% of symptomatic feet had a retromalleolar tenderness over the tibialis posterior compared to 14% of asymptomatic feet. This is the first study to demonstrate a statistically significant increase in spring ligament strain in feet affected with PF using internal controls. The study postulates that tensile overload at the medial plantar fascia develops secondary to spring ligament failure regardless of foot shape. Furthermore, this condition can be regarded as an early warning sign of adult acquired flat foot disorder (AAFD). Future treatments for PF should not further destabilise the medial arch. This understanding may allow development of new treatment strategies in restoring spring ligament integrity to offload the plantar fascia strain.

The emerging potential of SIRT-3 in oxidative stress-inflammatory axis associated increased neuroinflammatory component for metabolically impaired neural cell.

People suffering from conditions like epilepsy, where there is an excess of neuron excitement, stroke, and cardiac arrest, where there are oxygen and glucose deprivation, Alzheimer, Parkinson, and Huntington's disease that causes metabolic and also oxidative stress-inflammatory axis; are known to be more vulnerable to disturbances in the metabolism, and there is a lot of inadequacy in defining the inflammation's mechanistic connections, as well as neurodegeneration and the bioenergetic deficiencies in the CNS. We retrieved relevant studies from PubMed/ScienceDirect/Medline/Public library of science/Mendeley/Springer link as well as Google Scholar. We used various keywords both individually and in combination with the literature search. 'Epidemiology of neurodegenerative disorders', 'neurodegenerative diseases associated hyper inflammation', 'Mechanism of inflammation in neuronal cell', 'Involvement of SIRTin inflammation', 'Pathogenesis of mitochondrial associated metabolic impairment in neurons', 'Reactive oxygen species-mediated mitochondrial dysfunction' were a few of the keywords used for the search. PINCH, which is a chronic neuro-inflammatory component that cannot be detected in matured neurons which are healthy, though expressed in oxidative stress inflammatory axis related tauopathy and diseases that cause neurodegeneration. We attempted to study the regulatory mechanisms that cause changes in the bioenergetics and its neuronal defects and mitochondrial subcellular localization that are PINCH protein-mediated on the other handSIRT1, the most intensively studied sirtuin, in oxidative stress-mediated inflammatory consequence for many diseases but very few research data explore the role of SIRT-3 for correction of the chronic neuroinflammatory component. Thus, in this review, we investigate the very recently identified molecules involving in the pathogenesis during stimulated oxidative stress-inflammatory axis in the excitatory neuronal cell which changes brain metabolism. Simultaneously, in CNS neurons of diseases with a component of chronic neuroinflammation which exhibit neuroprotective response, the consequences (mechanistic and biological) of SIRT-3, could be emerging future targets for neurodegenerative disorder treatment with impaired metabolisms.

Metabolic syndrome components and disease disability in egyptian multiple sclerosis patients.

BACKGROUND: There is limited and inconsistent data on metabolic syndrome (MetS) in multiple sclerosis (MS) patients. The aim of this study was to estimate the frequency of MetS and its components in MS patients and to evaluate their association with disease disability in Egyptian MS patients. METHODS: A cross-section study was carried out on 60 patients (19 males and 41 females) with relapsing remitting MS. All patients were subjected to full general and neurological examination, laboratory and radiological investigations. Assessment of disease disability was performed using Expanded Disability Status Scale (EDSS) and MetS was diagnosed according to National Cholesterol Education Program Adult Treatment Panel Ш (NCEP-ATP III). RESULTS: The frequency of MetS in MS patients was 36.7%. Our findings show that 53.3% of MS patients had abdominal obesity, 21.7% had hypertension, 38.3% had diabetes mellitus, 43.3% had elevated triglycerides level and 56.7% had dyslipidemia. Linear regression analysis revealed that body mass index (BMI), dyslipidemia, current medication and disease duration were significantly associated with disease disability. CONCLUSION: High frequency of MetS and its component was observed in MS patients. Disease duration and current medication as well as some MetS component such as BMI, dyslipidemia, were significantly associated with disability in MS patients.

Vardenafil and cilostazol can improve vascular reactivity in rats with diabetes mellitus and rheumatoid arthritis co-morbidity.

Endothelial dysfunction and vascular reactivity defects secondary to metabolic and immunological disorders carry risk of serious cardiovascular complications. Here, the effects of the phosphodiesterase (PDE) inhibitors vardenafil and cilostazol were examined against rheumatoid arthritis (RA)/diabetes mellitus (DM)-co-morbidity-induced endothelial dysfunction and vascular reactivity defects. After setting of RA/DM-co-morbidity model, rats were divided into a normal control group, an RA/DM-co-morbidity group, and two treatment groups receiving oral vardenafil (10 mg/kg/day) and cilostazol (30 mg/kg/day) for 21 days after RA/DM-co-morbidity induction. Aorta was isolated for biochemical estimations of the pro-inflammatory vasoconstrictor molecules angiotensin-II (Ang-II) and endothelin-1 (ET-1), the adhesion molecules P-selectin and vascular cell adhesion molecule-1 (VCAM-1), the energy sensor adenosine-5'-monophosphate-activated protein kinase (AMPK), and the vasodilator anti-inflammatory molecule vasoactive intestinal peptide (VIP) using enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Immunohistochemical estimations of endothelial nitric oxide synthase (eNOS) and matrix metalloproteinase (MMP)-2 were performed coupled with histopathological examination using routine hematoxylin and eosin (H&E) and special Masson trichrome staining. The in vitro study was conducted using aortic strips where cumulative concentration response curves were done for the endothelium-dependent relaxing factor acetylcholine and the endothelium-independent relaxing factor sodium nitroprusside after submaximal contraction with phenylephrine. Vardenafil and cilostazol significantly improved endothelial integrity biomarkers in vivo supported with histopathological findings in addition to improved vasorelaxation in vitro. Apart from their known PDE inhibition, up-regulation of vascular AMPK and eNOS coupled with down-regulation of Ang-II, ET-1, P-selectin, VCAM-1 and MMP-2 may explain vardenafil and cilostazol protective effect against RA/DM-co-morbidity-induced endothelial dysfunction and vascular reactivity defects.

Fluoxetine induces direct inhibitory effects on mesenchymal stem cell­derived osteoprogenitor cells independent of serotonin concentration.

Selective serotonin reuptake inhibitors are the most commonly prescribed antidepressants worldwide, which have been reported to exert potential detrimental effects on bone mineral density and increase the risk of developing fractures. The present study aimed to investigate the pathways underlying the negative effects of fluoxetine on bone using mesenchymal stem cells (MSCs) derived from rat adipose tissue as a source of osteoprogenitor cells. MSCs were harvested from adipose tissue using a collagenase enzyme digestion method and were allowed to differentiate into osteoprogenitor cells. Various concentrations of fluoxetine were added to the cells, which were harvested and analyzed by flow cytometry to detect apoptotic markers Annexin V and caspase­3, in order to assess the levels of apoptosis. The levels of endogenous serotonin released in the extracellular matrix were measured using a serotonin ELISA kit. The underlying molecular pathways associated with the effects of fluoxetine on bone were investigated with reverse transcription­quantitative polymerase chain reaction. The results of the present study revealed a significant dose­dependent increase in apoptosis in response to increasing doses of fluoxetine, which was independent of serotonin levels in the culture supernatant. These findings indicated that fluoxetine exerted a direct inhibitory effect on bone cells via an apoptosis­dependent pathway. Furthermore, the expression levels of serotonergic genes, including serotonin 1B receptor, serotonin 2A receptor (HTR2A), serotonin 2B receptor and serotonin transporter, were down regulated; of these genes, HTR2A exhibited the highest expression levels. Further in vitro and in vivo studies are required to verify this association and to determine the molecular pathways involved in fluoxetine­induced bone loss. Fluoxetine­induced apoptosis of osteoprogenitor cells may be the mechanism underlying the increased incidence of bone loss observed in patients treated with fluoxetine.

More than one disease process in chronic sinusitis based on mucin fragmentation patterns and amino Acid analysis.

Objective. To characterise fragmentation patterns and amino acid composition of MUC2 and MUC5AC in chronic sinusitis. Methods. Antigenic identity of purified sinus mucins was determined by ELISA. Fragmentation patterns of a MUC5AC rich sample mucin were analysed by Sepharose CL-2B gel chromatography. Samples, divided into one MUC2 rich and one MUC5AC rich group, were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their amino acid contents were analysed. Results. Reduction, trypsin digestion, and papain digestion produced progressively smaller mucin species. On SDS-PAGE, digested MUC5AC rich mucin produced four distinct products. Amino acid analysis was characteristic of mucins with high serine, threonine, and proline contents and reduction and proteolysis increased relative proportions of these amino acids. MUC5AC rich mucins contained more protein than MUC2 rich mucins. Conclusion. Sinus mucin fragmentation produced mucin subunits and glycopeptide units of smaller molecular sizes which are likely to have lower viscoelastic properties. Applying this in vivo could alter mucus physical properties and biologic functions. Amino acid contents of MUC2 and MUC5AC mucins are different. This could be contributing to biological properties and functions of sinus mucins. These data suggest that there may be different pathological processes occurring at the cellular level on chronic sinusitis.

Synchronous spectrofluorimetric determination of naphthalene in water samples using mixed micellar medium.

A mixed micellar medium of sodium dodecyl sulfate and Pluronic F-127 was used to enhance the fluorescence of naphthalene and to obtain lower LODs. The method was based upon measuring the first-derivative synchronous fluorescence spectrum of naphthalene by using a mixed micellar medium at a constant wavelength difference delta lambda = 60 nm, where a greater fluorescence enhancement was observed if compared to using a single surfactant separately. A linear fluorimetric calibration curve was obtained for naphthalene in a concentration range of 10-200 ng/mL. The LOD was 7.13 ng/mL, which is well below the health advisory limit for naphthalene in drinking water as suggested by the U.S. Environmental Protection Agency. The method can be easily adopted for determination of naphthalene in aqueous media including tap water and river water. The recoveries obtained were 95.979-115.645%. The proposed method was validated according to International Conference on Harmonization guidelines and successfully applied to determine naphthalene in real life water samples from different sources.

Mucin gene expression in reflux laryngeal mucosa: histological and in situ hybridization observations.

Objectives/Hypothesis. To determine if laryngopharyngeal reflux alters mucin gene expression in laryngeal mucosa. Methods. In situ hybridization was employed to study the expression of the 8 well-characterised mucin genes MUC1-4, 5AC, 5B, 6, and 7 in reflux laryngeal mucosa from laryngeal ventricles, posterior commissures, and vocal folds compared to control/normal laryngeal mucosa. Results. MUC1-5 genes are expressed in normal and reflux laryngeal mucosa. MUC1, 3 and 4 are expressed in respiratory and squamous mucosa whereas MUC2 and 5AC are expressed in respiratory mucosa only. MUC3, 4 and 5AC are downregulated in reflux mucosa. MUC5AC expression is significantly reduced in the 3 mucosal sites and when mucosal type was taken into account, this remains significant in combined laryngeal and ventricular mucosa only. Conclusions. MUC3, 4 and 5AC expression is downregulated in laryngopharyngeal reflux. This may be due to laryngeal mucosal metaplasia and/or alteration of mucin gene expression in the preexisting mucosa. Altered mucin gene expression might predispose laryngeal mucosa to the damaging effect of reflux.

Nasosinus mucin expression in normal and inflammatory conditions.

PURPOSE OF REVIEW: Mucin expression in normal nasosinus mucosa is upregulated in inflammatory conditions. However, as the first nine mucin genes are expressed, and as more mucins are expected to be expressed in nasosinus mucosa, the range and pattern of variations in mucin expression in nasosinus mucosa would be wide and complicated. This review discusses current knowledge on mucin expression in normal and pathologic nasosinus mucosa. RECENT FINDINGS: Studies on nasosinus mucin expression indicate that mucin genes upregulated in inflamed nasosinus mucosa include MUCs1, 2, 4, 5AC, 5B and 8 in different combinations and to various degrees. Nasosinus submucosal glands play a more important role in mucin expression than surface epithelium. However, most of the studies on nasosinus mucin expression are focused on basic science and as yet the clinical implications of nasosinus mucin gene expression are still unclear. SUMMARY: Nasosinus mucin expression is very complicated. Studies including large numbers of samples are needed to investigate the role of different physiological and pathological variables, including inflammatory mediators, in the control of nasosinus mucin expression. This is essential for better understanding of clinical implications of altered nasosinus mucin expression and for future therapeutic applications.

Association of beta-defensin 1 single nucleotide polymorphism with atopic dermatitis.

Atopic dermatitis (AD) is a chronic relapsing, pruritic, inflammatory skin disease, which results from a complex interplay between genetic and environmental factors. Defensins are broadly dispersed family of antimicrobial peptides which are classified into 2 distinct families: the alpha-defensins and the beta-defensins. The primary function of defensins is to protect the skin from invasion by foreign pathogens. Previous studies suggested that single nucleotide polymorphisms (SNPs) of the beta-defensin 1 gene (DEFB1) could be involved in the development of AD. The Aim of the study is to examine DEFB1 gene to gain a better understanding of their role in the pathophysiology of AD patients and their involvement in AD susceptibility and severity. 35 atopic patients and 10 healthy volunteers as controls were investigated. They were subjected to analysis of absolute eosinophil count, total and specific IgE and detection of Beta-defensin-1 gene polymorphism at position 692 and 1654 using PCR amplification and restriction analysis. We observed significant difference in the distribution of the DEFB1 AIG polymorphism at 692 (P<0.01) in AD patients compared to controls, but not at 1654. A statistical significant association between DEFB1 692 GG genotype and elevated total serum IgE level (P<0.01), and between DEFB1 692 GG and AG genotypes & 1654 AA genotype and high absolute eosinophil count (P<0.05) were found. Concerning Specific IgE there was significant association between DEFB1 692 GG genotype and positive specific IgE to dermatophytes and HDM (House Dust Mite) (P1<0.01) while DEFB1 1654AA genotype shows significant association with positive specific IgE to cockroaches (P<0.05). Regarding SCORAD severity index, there was significant statistical association between DEFB1 692 GG and AG & DEFB1 1654 AA and AG genotype with severe AD disease (P<0.05). The correlation between atopic markers and SCORAD severity index shows that there was a significant statistical relationship between serum levels of total IgE (P<0.01), absolute eosinophil count (P<0.01), specific IgE to cat (P<0.05), HDM (P<0.01) and cockroaches (P<0.01) and SCORAD. Our findings support previously studies suggesting that DEFB1 gene is one of the candidate genes for atopy. G allele at site 692& AA genotype at site 1654 may be useful as markers for AD susceptibility and severity

Laryngopharyngeal reflux: diagnosis and treatment of a controversial disease.

PURPOSE OF REVIEW: Laryngopharyngeal reflux is a well-recognized and widely used term in ear, nose and throat practice. However, the symptoms and signs attributed to laryngopharyngeal reflux are non-specific and treatment is usually empirical. This review discusses current knowledge on diagnosis and treatment of laryngopharyngeal reflux. RECENT FINDINGS: Information is evolving regarding the implications of laryngopharyngeal reflux in the development of pathological conditions affecting the upper aerodigestive tract epithelium such as chronic laryngitis, otitis media with effusion and chronic sinusitis. However, there is still much to learn about the pathophysiologic mechanisms of laryngopharyngeal reflux and their role in its related disease conditions and there is still considerable controversy on diagnostic as well as therapeutic parameters for this condition. There is no consensus on the diagnosis and treatment of laryngopharyngeal reflux and the majority of clinicians depend mainly on clinical findings and empirical therapeutic tests rather than more specific investigations. SUMMARY: The concept of laryngopharyngeal reflux is still controversial. The current practice of empirical treatment with proton-pump inhibitors is based on weak evidence. However, this practice seems to be widely accepted and will not change until further clinical and laboratory studies improve our understanding of this common and well-recognized condition.

Microscopic transtracheal repair of migrating tracheoesophageal fistula.

OBJECTIVE: The repair of a persistently leaking migrating tracheoesophageal fistula (TEF) represents a particular challenge owing to the low site of the fistula down to the tracheoesophageal septum (TES). A simple microscopic approach to repair a migrating TEF is described. DESIGN: A description of five cases of migrating TEF. The repair technique and surgical outcome are described in detail. SETTING: Tertiary care referral hospital. METHODS: Excision of the fistula tract was done under local anesthesia and microscopic vision using microlaryngoscopic instruments followed by one-layer repair without soft tissue interposition. This technique was used in one patient with a leaking migrating TEF when planned dissection through the TES was abandoned. Subsequently, the technique was employed in four other patients with a similar TEF. MAIN OUTCOME MEASURES: Evidence of complete closure of the fistula was assessed clinically 1 week postoperatively. This was followed by methylene blue and Gastrografin swallowing tests. The methylene blue test was repeated after 6 months to exclude recurrence of the fistula and confirm persistent closure. RESULT: Complete closure of the fistulae was achieved when assessed clinically and by methylene blue and Gastrografin tests. All patients were discharged on a normal diet. Stable closure was confirmed by the methylene blue test after 6 months. The microlaryngoscopic instruments and surgical microscope have greatly facilitated access and dissection of the migrating fistula with minimum soft tissue loss. CONCLUSION: The described technique is simple, relatively safe, and reproducible for closure of a small migrating TEF. It can also be used to repair small, nonmigrating TEF.