Transcriptase-polymerase Chain Reaction Assay Research Articles

COVID-19 Pneumonia in Patients With HIV: A Case Series

To the Editors: As our understanding of the coronavirus disease 2019 (COVID-19) improves through emerging data from the general population affected with COVID-19, there is a paucity of information regarding the management and prognosis of patients with HIV infection who may be at an increased risk for morbidity and mortality.1 Here, we describe our experience with the management of COVID-19 in patients with HIV at a health care facility in Newark, the center of the HIV epidemic of New Jersey.2 We included all patients with laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection defined as a positive result on a reverse transcriptase polymerase chain reaction assay of a specimen collected on a nasopharyngeal swab. We identified 27 patients who had a known history of HIV at the time of presentation. Of these, 51% (n = 14) were managed in the ambulatory setting, whereas the rest were hospitalized. The median age was 58 years, there were 15 men and 12 women, and 93% (n = 25) were black or African Americans. The top 4 common symptoms at presentation were fever (67%), cough (63%), dyspnea (63%), and fatigue (48%), which had started over a median duration of 3 days before presentation. More than half of the patients had a history of systemic hypertension (59%), and about one-third reported diabetes mellitus (33%) or chronic kidney disease (27%) (Table 1).TABLE 1.: Demographic, Clinical Characteristics, and Outcomes of HIV Patients With COVID-19 (n = 27)TABLE 1-A.: Demographic, Clinical Characteristics, and Outcomes of HIV Patients With COVID-19 (n = 27)Ambulatory patients were hemodynamically stable at the time of presentation and demonstrated an oxygen saturation of 94–100% on ambient air. They were discharged home with no medical therapy but with the following recommendations: (1) self-quarantine for at least 2 weeks, (2) return to the emergency department for management in case of new-onset symptoms such as fever and dyspnea, and (3) adherence to antiretroviral therapy. Ambulatory patients were followed up with phone calls through a telemedicine platform over a median duration of 14 days. Patients were hospitalized and managed in the inpatient setting if they (1) had signs of sepsis or septic shock defined by the 2016 Third International Consensus Definitions for Sepsis and Septic Shock3 and (2) reported dyspnea requiring a step up in oxygenation therapy to maintain an oxygen saturation between 88% and 94%. Major laboratory markers and immunological data of their HIV status are given in Table 1. The median lymphocyte count was 17%, with a serum d-dimer and procalcitonin of 1.9 ng/mL and 0.26 μ/L, respectively. Patients reported a median CD4 count of 551, and the viral load was <120 in 96% (n = 26) of the patients. Data on antiretroviral regimen before presentation were unavailable in 15% (n = 4) of the patients. Among those with full data, integrase-based regimen was used in 33% (n = 9), nonnucleoside reverse transcriptase inhibitor (NNRTI) in 19% (n = 7), protease inhibitor (PI) + integrase in 19% (n = 7), NNRTI + integrase in 7% (n = 3), and PI based in only 1 patient. Of the hospitalized patients, 26% (n = 7) received hydroxychloroquine and 30% (n = 6) were managed with empiric antibiotics for suspected community-acquired pneumonia. Antiretroviral therapy was held during hospitalization. None of the patients had a superimposed infection, defined as an isolated organism in a positive sputum, urine, or blood culture. After a median hospital course of 10 days, 3 patients required intensive unit level of care and 2 of them had died. The deceased subjects were elderly patients, with multiple coexisting conditions whose course was complicated by septic shock and multiorgan dysfunction syndrome. There were no cases of hospital admission for patients who were managed in the ambulatory setting. In this series of HIV patients with COVID-19, we found a similarity in presentation with what has been reported for the general population. Half of the patients were managed in the ambulatory setting with no reported mortality or morbidity. In-hospital survival was 85% and 23% required intensive care unit admission. Of note, all 27 patients had well-controlled HIV infection evidenced by elevated CD4 count levels and low viral loads. As the challenge involving the management of COVID-19 evolves, large-scale studies are necessary to better understand the management and prognosis of patients with HIV who present with COVID-19.

Histopathologic Changes and SARS-CoV-2 Immunostaining in the Lung of a Patient With COVID-19

Histopathologic Changes and SARS-CoV-2 Immunostaining in the Lung of a Patient With COVID-19

Investigating The Alterations of Oxidative Stress Status, Antioxidant Defense Mechanisms, MAP Kinase and Mitochondrial Apoptotic Pathway in Adipose-Derived Mesenchymal Stem Cells from STZ Diabetic Rats.

Objective This study aimed to investigate the reliability of diabetic adipose-derived stem cells (ADSCs) for autologous cell-based therapies by exploring the functionality of signalling pathways involved in regulating oxidative stress and apoptosis. Materials and Methods In this experimental study, ADSCs were isolated from streptozotocin (STZ)-induced diabetic rats (dADSCs) and normal rats (nADSCs). The colonies derived from dADSCs and nADSCs were compared by colony-forming unit (CFU) assay. Reactive oxygen species (ROS) formation and total antioxidant power (TAP) were also measured. Furthermore, the expression of antioxidant enzymes, including catalase (Cat), superoxide dismutase (Sod)-1 and -3, glutathione peroxidase (Gpx)-1, -3 and -4 was measured at mRNA level by semi-quantitative reverse transcriptase polymerase chain reaction assay. The expression of Bax, Bcl2, caspase-3, total and phosphorylated c-Jun N-terminal kinase (JNK) and P38 Mitogen-Activated Protein Kinase (MAPK) at protein level was analyzed by western blotting. Results The results of this study indicated that viability and plating efficiency of dADSCs were significantly lower than those of nADSCs. ROS generation and TAP level were respectively higher and lower in dADSCs. The gene expression of antioxidant enzymes, including Cat, Sod-1, Gpx-3 and Gpx-4 in dADSCs was significantly greater than that in nADSCs. However, Sod-3 and Gpx-1 mRNA levels were decreased in dADSCs. Moreover, Bax/Bcl-2 protein ratio, caspase-3 protein expression and phosphorylation of JNK and P38 proteins were increased in dADSCs compared to nADSCs. ConclusionTaken together, diabetes might impair the cellular functions of dADSCs as candidates for autologous cell- based therapies. This impairment seems to be mediated by JNK, P38 MAPKs, and mitochondria pathway of apoptosis and partly by disruption of antioxidant capacity.

Type A Influenza Virus Detection from Horses by Real-Time RT-qPCR and Insulated Isothermal RT-PCR.

Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the economic impact of the disease. The probe-based real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assays targeting various EIV genes are reported to be highly sensitive and specific compared to the Directigen Flu-A® test and virus isolation in embryonated hens' eggs. Recently, a TaqMan® probe-based insulated isothermal RT-PCR (iiRT-PCR) assay for the detection of EIV H3N8 subtype has been described. These molecular-based diagnostic assays provide a fast and reliable means of EIV detection and disease surveillance.

Severe acute respiratory syndrome coronavirus-2 and tuberculosis coinfection: Double trouble

The ongoing pandemic of novel coronavirus disease 2019 (COVID-19) has received worldwide attention by becoming a major global health threat. We encountered one case with COVID-19 and tuberculosis (TB) coinfection, which has not been frequently reported. A 76-year-old female presented with acute respiratory symptoms superimposed on chronic symptoms, suggestive to have pneumonia. Oropharyngeal throat swab sample for COVID-19 was positive as detected by real-time reverse transcriptase–polymerase chain reaction assay. GeneXpert Ultra detected Mycobacterium tuberculosis complex with rifampicin resistance indeterminate. The patient was treated with appropriate management. Clinicians should suspect coinfection with TB during the ongoing pandemic of COVID-19 as therapeutic strategies need to be determined timely to improve outcomes and prevent transmission in the community.

Aptamer-Functionalized DNA Origami for Targeted Codelivery of Antisense Oligonucleotides and Doxorubicin to Enhance Therapy in Drug-Resistant Cancer Cells.

Drug resistance is a major obstacle to the efficient therapy of drug-resistant cancer. To overcome this problem, we constructed a multifunctional DNA origami-based nanocarrier for codelivery of a chemotherapeutic drug (doxorubicin, Dox) and two different antisense oligonucleotides (ASOs; B-cell lymphoma 2 (Bcl2) and P-glycoprotein (P-gp)) into drug-resistant cancer cells for enhanced therapy. To increase the targeting ability of origami, staple strands with 5'-end extended MUC1 sequences were used in the preparation of aptamer-functionalized origami carrying ASOs (Apt-origami-ASO). Dox-loaded Apt-origami-ASO (Apt-Dox-origami-ASO) was prepared by electrostatic adsorption of Dox in origami. Atomic force microscopy (AFM) images demonstrated the successful preparation of Apt-origami-ASO. In vitro studies showed that the Apt-Dox-origami-ASO (Apt-DOA) could controllably release Dox in pH 5.0 phosphate-buffered saline (PBS) buffer and release ASOs in response to glutathione. Further experiments revealed that the origami could protect ASOs against nuclease degradation in 10% FBS. Confocal imaging showed that the Apt-DOA nanocarrier could efficiently enter the Hela/adriamycin (ADR) cells and escape from lysosomes for codelivery of Dox and ASOs into the cytoplasm. The quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot assays testified the efficient silencing of Bcl2 and P-gp mRNA and downregulation of the corresponding protein expressions by Apt-DOA in Hela/ADR cells. Moreover, with the synergetic effect by codelivery of multi-ASOs and Dox, the anticancer assay showed that Apt-DOA could circumvent multidrug resistance and significantly enhance cancer therapy in Hela/ADR and MCF-7/ADR cells. Hence, this multifunctional origami-based codelivery nanocarrier presents a new strategy for efficient therapy of drug-resistant cancer.

Angiotensin II Type I Receptor Blockade Is Associated with Decreased Cutaneous Scar Formation in a Rat Model

Angiotensin II engagement of angiotensin II type 1 receptor (AT1R) is implicated in fibrogenesis, with AT1R blockers used clinically to attenuate cardiac and renal fibrosis. The authors tested the hypothesis that the AT1R blocker losartan could reduce postsurgical cutaneous scarring in rats. Human dermal fibroblasts were treated with losartan and assessed for viability, contractile activity, migration, and profibrotic gene transcription by means of calcein, collagen gel, scratch, and quantitative reverse transcriptase polymerase chain reaction assays, respectively. Monocyte migration and adhesion to losartan-treated and control fibroblasts were examined. Losartan effects in vivo were assessed using a mechanical distraction hypertrophic scar model. Three days after incisions were made and closed on their backs, rats were assigned randomly to receive drinking water with or without losartan (1 mg/kg per day; n = 6 per group). Distraction devices were applied and activated up to day 14. On day 28, scars underwent cross-sectional area and elevation index analyses, and α-SMA (alpha-smooth muscle actin) and CD68 (monocyte/macrophage marker) immunostaining. Losartan-treated human dermal fibroblasts displayed decreased contractile activity, migration, and gene expression of transforming growth factor-β1, collagen I, and monocyte chemoattractant protein-1 relative to controls (p < 0.05). Monocyte migration and adhesion to losartan-treated fibroblasts were reduced (p < 0.01). Compared to controls, scars from losartan-treated rats demonstrated decreased cross-sectional area (19.4 ± 3.1 mm versus 45.0 ± 5.2 mm; p = 0.002), elevation index (1.5 ± 0.1 versus 2.6 ± 0.3; p = 0.003), and α-SMA and CD68 immunostaining (p < 0.001). Losartan decreases myofibroblast activity and reduces monocyte trafficking to cutaneous scar. These findings support losartan as a potential novel therapy for the prevention of hypertrophic scars.

Comparison of Reverse Transcriptase Loop-Mediated Isothermal Amplification and Reverse Transcriptase Polymerase Chain Reaction for Detection of Prostate Specific Antigen

Background: Research shows that prostate cancer ranks second among the top five most common cancers in men. It has been confirmed that when circulating Prostate Specific Antigen (PSA) transcripts are successfully detected, prostate cancer cells can be diagnosed at an early stage. A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was developed and compared to reverse transcriptase polymerase chain reaction (RT-PCR) assay for detection of PSA. Methods: 47 patients, including 30 patients with prostate cancer, 15 with Benign Prostate Hyperplasia (BPH) and 2 healthy subjects as negative controls were included in this study. The prostate cancer cell lines (PC3 and LNCaP) of two patients were included in the study as positive controls. Next, RNA was extracted from fresh samples and a first strand cDNA synthesis kit was applied for the synthesis of cDNA. The human prostate specific antigen gene was used to design specific primers. Results: The results indicated that the control subjects and participants suffering from BPH were not positive. 13 out of 15 (86.6%) patients suffering from localized cancer were PSA positive. PSA positive results were observed among all 15 metastatic patients and positive controls (100%). RT-LAMP is an advantageous method because it is highly sensitive (1000-fold), quite cheap, user-friendly, and safe; in addition, it can be quickly performed by visual detection using GineFinderTM dye in a water bath. Conclusion: RT-LAMP technique can be simply and reliably applied with the aid of basic instruments, and its results can be visually inspected in laboratory studies.

Middle East Respiratory Syndrome: Its Occurrence, Aetiology, Epidemiology, Associated Clinicopathological Findings, Diagnosis, Prevention and Control in Humans and Animals: An Overview

Middle East respiratory syndrome (MERS) is a serious zoonotic disease. It is characterized by severe infection of the lower respiratory pathway in humans. Dromedary camels are considered to be the likeliest source of the very pathogenic aetiologic agent, Middle East respiratory syndrome coronavirus (MERS-CoV). Although, the first case of MERS-CoV was initially identified in Kingdom of Saudi Arabia (KSA) in September 2012, most of the reported cases have been detected in Arabian Peninsula but worthy of note is that travel-associated MERS cases have been reported around the world especially in Europe, Asia, Africa and North-America. This virus is very endemic in camel populations of East Africa and the Arabian Peninsula but worryingly, the zoonotic transmission of MERS-CoV are now well reported. It is diagnosed in camels using IgG immunofluorescence assay, protein microarray and virus neutralisation assay Diagnosis in humans is based on chest radiographs and computed tomographic scans but confirmatory diagnosis of suspected MERS patients could be done by taking a intratracheal aspirate for detection test of MERS-CoV RNA positive real-time reverse transcriptase polymerase chain reaction assays. The major control measures of MERS-CoV spread include strict regulation of camel movement, regular herd screening and culling of infected camels.

HOXC10 promotes cell migration, invasion, and tumor growth in gastric carcinoma cells through upregulating proinflammatory cytokines

HOXC10 plays a critical role in many cellular processes, such as proliferation, migration, and invasion, but the function of HOXC10 in gastric carcinoma is not clear. In this study, we aimed to investigate the expression profile of HOXC10 and its role in gastric carcinoma cells and in vivo experiments. HOXC10 expression patterns were detected in clinical samples and gastric cancer cells lines by reverse transcriptase polymerase chain reaction assays, and then, we focused on its role in regulating cell proliferation, cell cycle, migration, and invasion after transfection of silencing and overexpression plasmids in vitro and in vivo. Finally, we confirmed the correlation between HOXC10 and nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and epidermal growth factor receptor expression. We found that HOXC10 expression increased in clinical samples, especially in poorly differentiated (PD) gastric cancer cells. Silencing HOXC10 suppressed proliferation, migration, and invasion in vitro, and inhibited tumor growth and induced apoptosis in vivo. Overexpression of HOXC10 showed the opposite effect on PD gastric cancer cells. In addition, silencing HOXC10 inhibited the expression of interleukin-6, TNF-α, TGF-β, and epidermal growth factor, and overexpressing HOXC10 induced their expression both in vitro and in vivo. Luciferase reporter assays and chromatin immunoprecipitation indicated that HOXC10 may activate the NF-κB signaling pathway through regulation of P65 transcriptional activity by binding to the P65 promoter. HOXC10 may play an important role in PD gastric carcinoma cell proliferation, cell cycle, migration, invasion, and metastasis through upregulating proinflammatory cytokines via NF-κB pathway, suggesting HOXC10 may serve as a novel therapeutic target for PD gastric cancer.

Phylogenetic Analysis of Infectious Bronchitis Viruses Currently Circulating in the Egyptian Field

One of the major problems of infectious bronchitis virus (IBV) is the frequent emergence of new variants in the Egyptian field. In the present study, 42 broiler chicken flocks suffered from respiratory troubles were investigated for infectious bronchitis virus in 10 Governorates in Egypt during 2016-2018. Ten out of 42 examined flocks revealed variable degrees of embryo lesions after 3-5 passages with negative heamagglutination (HA) activity. From these 10 flocks, IBV was confirmed in six flocks using reverse transcriptase polymerase chain reaction (RT-PCR) assay. Four IBV-positive isolates were selected for further sequence analysis. Partial sequencing of S1 gene revealed four IBV variant-2 isolates circulating among chickens in Egypt. These isolates are IB-Beh-Ch-F2-2016, IB-Sh-Ch-F25-2017, IB-Sh-Ch-F41-2018 and IB-Sh-Ch-F42-2018 and submitted on Gen Bank with accession numbers MH460643, MH460644, MK408615 and MK408616, respectively. The IB-Sh-Ch-F25-2017 isolate had only one amino acid substitution while IB-Beh-Ch-F2-2016, IB-Sh-Ch-F41-2018 and IB-Sh-Ch-F42-2018 isolates had much higher genetic diversity. The similarity between classic viruses of vaccine origin used in Egypt (H120, Ma5) and our four filed isolates ranged between 75.7 and 80.4%. It could be concluded that IB variant-2 strains still circulate in the Egyptian field in spite of vaccination. Therefore we need to revise the IBV vaccines used in Egypt and try to prepare local vaccines with periodic evaluation of cross protection of such vaccines.

Regulation of iRhom-2/Tumor Necrosis Factor-α Converting Enzyme Pathway and Oxidative Stress Protects the Renal Injury with Anemonin in Streptozotocin-Induced Diabetic Nephropathy Neonatal Rat Model

Diabetic nephropathy (DN) is a chronic complication of diabetes, and thus the present investigation evaluates the nephroprotective effect of anemonin against streptozotocin (STZ)-induced DN rats. Diabetes was induced by intraperitoneal administration of STZ (50 mg/kg) on day 2 and 3 postnatal, and rats were kept as such for the duration of 12 weeks. Thereafter, rats were treated with anemonin 75 and 150 mg/kg per oral for the period 4 week which means between the period of 12–16 weeks. Effect of anemonin was estimated by determining the blood glucose, markers of nephropathy, and mediators of inflammation in the serum and activity of tumor necrosis factor-α (TNF-α)converting enzyme (TACE) in the kidney tissue of DN rats. Moreover, reverse transcriptase polymerase chain reaction and western blot assay were determined in the kidney tissue homogenate of DN rats. Histopathology study was done by Periodic acid-Schiff’s and masson staining for the pathological changes and apoptosis of podocytes in the kidney tissue of DN rats. Moreover, production of reactive oxygen species (ROS) was estimated in the kidney tissue by 2’,7’-dichlorofluorescein staining. Data of study reveal that anemonin significantly reduces (p < 0.01) the blood glucose and markers of renal injury in the serum and urine of DN rats. There was a reduction in the level of cytokines in the serum, and production of ROS and activity of TACE were reduced in the kidney tissue of the anemonin-treated group than in the DN group. Expression of iRhom-2, TACE, TNF-α, and inducible nitric oxide synthase protein and histopathology of kidney tissue were attenuated in the anemonin-treated group in DN rats. In conclusion, data of study reveal that treatment with anemonin ameliorates progression of renal injury by regulating TACE/iRhom-2 signaling pathway.

Respiratory virus associated with surgery in children patients

BackgroundViral respiratory infection (VRI) is a common contraindication to elective surgery. Asymptomatic shedding among pediatric surgery patients (PSPs) could potentially lead to progression of symptomatic diseases and cause outbreaks of respiratory diseases. The aim of this study is to investigate the incidence of infection among mild symptomatic PSP group and asymptomatic PSP group after surgical procedure.MethodsWe collected the induced sputum from enrolled 1629 children (under 18 years of age) with no respiratory symptom prior to pediatric surgery between March 2017 and February 2019. We tested 16 different respiratory virus infections in post-surgery mild symptomatic PSP group and asymptomatic PSP group using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assay panel. We analyzed symptom data and quantitative viral load to investigate the association between viruses, symptoms and viral quantity in qRT-PCR-positive PSPs.ResultsOut of 1629 children enrolled, a total of 204 respiratory viruses were present in 171 (10.50%) PSPs including 47 patients with mild symptoms and 124 with no symptoms after surgery. Commonly detected viruses were human rhino/enterovirus (HRV/EV, 42.19%), parainfluenza virus 3 (PIV3, 24.48%), coronavirus (CoV NL63, OC43, HKU1, 11.46%), and respiratory syncytial virus (RSV, 9.9%). PIV3 infection with a higher viral load was frequently found in PSPs presenting with mild symptoms, progressing to pneumonia with radiographic evidence after surgery. HRV/EV were the most commonly detected pathogens in both asymptomatic and mild symptomatic PSPs. CoV (OC43, HKU1) infections with a higher viral load were mostly observed in asymptomatic PSPs progressing to alveolar or interstitial infiltration.ConclusionsOur study suggested that PIV3 is a new risk factor for VRI in PSPs. Employing a more comprehensive, sensitive and quantitative method should be considered for preoperative testing of respiratory viruses in order to guide optimal surgical timing.

Seroepidemiology of dengue virus infection among adults during the ending phase of a severe dengue epidemic in southern Taiwan, 2015

BackgroundA severe dengue epidemic occurred in 2015 which resulted in over 22,000 laboratory-confirmed cases. A cross-sectional seroprevalence study was conducted during the ending phase of this epidemic to evaluate the true incidence of dengue virus (DENV) infection and the level of herd immunity.MethodsAdult residents in three administrative districts with high dengue incidence were recruited; workers in two districts with intermediate dengue incidence were also recruited for comparison. DENV-specific IgM and IgG were tested using commercial enzyme-linked immunosorbent assays. DENV RNA was detected using commercial quantitative real-time reverse transcriptase polymerase chain reaction assay. Univariate and multivariate logistic regressions were performed to identify risk factors for recent and past DENV infection.ResultsThe overall seroprevalence of anti-DENV IgM and IgG in 1391 participants was 6.8 and 17.4%, respectively. The risk of recent DENV infection increased with age, with the elderly having the highest risk of infection. Living in areas with high incidence of reported dengue cases and having family members being diagnosed with dengue in 2015 were also independent risk factors for recent DENV infection. One sample was found to have asymptomatic viremia with viral load as high as 105 PFU/ml.ConclusionsComparing the seroprevalence of anti-DENV IgM with the incidence of reported dengue cases in 2015, we estimated that 1 out of 3.7 dengue infections were reported to the surveillance system; widespread use of rapid diagnostic tests might contribute to this high reporting rate. The results also indicate that the overall herd immunity remains low and the current approved Dengvaxia® is not quite suitable for vaccination in Taiwan.

IGF2BP1 promotes LPS-induced NFκB activation and pro-inflammatory cytokines production in human macrophages and monocytes

BackgroundLipopolysaccharide (LPS)-induced macrophage/monocyte activation and pro-inflammatory cytokines production are important mediators for periodontitis progression. The current study tested the potential role of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in the process. MethodsTHP-1 human macrophages and primary human peripheral blood mononuclear cells (PBMCs) were treated with LPS. mRNA and protein expression of IGF2BP1 were tested by qPCR and Western blotting assay. IGF2BP1 expression was altered by shRNAs or CRISPR/Cas-9 gene editing methods. LPS-induced cytokine production was tested by ELISA assay. Cytokine mRNA expression was tested by the quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay. ResultsIn THP-1 human macrophages and PBMCs, treatment with LPS induced mRNA and protein expression of IGF2BP1. IGF2BP1 silencing (by targeted shRNAs) or CRISPR/Cas-9 knockout largely inhibited LPS-induced production of multiple pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Conversely, forced over-expression of IGF2BP1 facilitated LPS-induced pro-inflammatory cytokines production in THP-1 cells. For the mechanism study, we show that IGF2BP1 co-immunoprecipitated with p65-p52 nuclear factor kappa B (NFκB) complex in nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced p65-p52 nuclear translocation and NFκB activation were inhibited by IGF2BP1 silencing or CRISPR/Cas-9 knockout. ConclusionIGF2BP1 promotes LPS-induced NFκB signalling and transcriptional activation in human macrophages and monocytes.

Activation of upper respiratory tract mucosal innate immune responses in cats by liposomal toll‐like receptor ligand complexes delivered topically

BackgroundNonspecific induction of local innate immune responses by mucosally administered immunotherapy is a new approach to protection from upper respiratory tract infections. Therefore, a new liposome‐toll‐like receptor complex (LTC) immune stimulant was developed and investigated for its ability to activate innate immune responses in cats, both in vitro and in vivo, as part of an initial evaluation of LTC for use as an immunotherapeutic agent in cats.ObjectivesWe hypothesized that LTC could activate innate immune responses in cats after topical application to nasal and oropharyngeal mucosal surfaces.AnimalsMucosal immune responses to topical administration of LTC were assessed in 7 healthy, purpose‐bred cats, and in vitro responses were assessed using blood samples from healthy cats.MethodsCytokine and cellular immune responses to LTC were evaluated in blood samples, nasal lavage specimens, and pharyngeal swabs from cats, using reverse transcriptase polymerase chain reaction assays, ELISA assays, and flow cytometry.ResultsLiposome‐TLR complexes rapidly activated leukocytes in vitro, including upregulation of costimulatory molecule expression and cytokine production. Topical administration of LTC in healthy cats triggered rapid recruitment of monocytes to the nasal and oropharyngeal mucosa.Conclusions and Clinical ImportanceLiposome‐TLR complexes were found to effectively activate innate immune responses in cats after mucosal administration. These findings suggest that LTC have potential for use as a new mucosally administered immunotherapy for nonspecific protection from viral and bacterial respiratory tract infections.

English

Watermelon plants can be naturally infected by several viruses in single or mixed infections, of which the diagnosis is difficult and require specific techniques. This study aims to detect and to verify the presence of Cucumber mosaic virus (CMV), Papaya ringspot virus type W (PRSV-W), Watermelon mosaic virus (WMV), Zucchini lethal chlorosis virus (ZLCV), Zucchini yellow mosaic virus (ZYMV), and the main cucurbit viruses in Brazil using multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay. Oligonucleotides were designed towards the conserved regions of the virus genomes. In the duplex-PCR, it was possible to detect all the virus combinations, except ZLCV with ZYMV. The amplified product sizes were 644 bp (CMV), 535 bp (WMV-2), 398 bp (PRSV-W), 244 bp (ZLCV), and 214 bp (ZYMV). To test the efficacy of the methodology, we analyzed plants with virus symptoms from four municipalities in the state of Tocantins, located at Brazilian Cerrado. Mixed infections were detected in 80% of samples in all the municipalities. The multiplex RT-PCR assay can be used to detect and differentiate watermelon viruses that affect crops in the state of Tocantins. &nbsp; Key words: Cucumovirus, multiplex RT-PCR, Orthotospovirus, potyvirus, watermelon.

Chitosan Membrane Modified With a New Zinc(II)-Vanillin Complex Improves Skin Wound Healing in Diabetic Rats.

The treatment of chronic wounds is considered a public health problem. When the condition affects at-risk groups such as those with diabetics, it becomes a great clinical challenge. In this work, we evaluated the healing effects of a new zinc complex, [Zn(phen)(van)2], identified as ZPV, which was synthesized, characterized and associated with chitosan (CS) membranes and tested on cutaneous wounds of diabetic rats. Chitosan membranes were modified by Schiff base reaction with the complex under two experimental conditions (14 and 21 days), resulting in membranes with concentrations of complex equal to 0.736 μmol cm-2 (CS-ZPV1) and 1.22 μmol cm-2 (CS-ZPV2). Release assays in aqueous medium indicated that the membranes release the complex gradually when exposed to an aqueous medium. Diabetes was inducted in Wistar rats using 40 mg/kg (i.v.) streptozotocin. On the 7th day after diabetic induction, a circular excision on the skin (1.0 cm) was performed with a punch. The lesions were treated with the pure chitosan membrane and the membrane associated with the zinc-vanillin complex in two different doses. Skin samples were subjected to macroscopic and histopathological analyses, cytokine (TNF-α, IL-1β, and IL-10) quantification and reverse transcriptase polymerase chain reaction (TGF-β and VEGF) assays. The analyses showed a decrease in wound size, reepithelialization, angiogenic stimulus, collagen deposition, and reduced levels of TNF-α and IL-1β as well as increased IL-10 and gene expression of TGF-β and VEGF. The evaluated parameters suggest that CS-ZPV in the two concentrations tested may be effective in the treatment of chronic wounds.

Lactobacillus rhamnosus biosurfactant inhibits biofilm formation and gene expression of caries-inducing Streptococcus mutans

It is cleared that some probiotic strains inhibit biofilm formation of oral bacteria, but its mechanisms are not clearly understood yet. It is proposed that one of the mechanisms can be biosurfactant production, a structurally diverse group of surface-active compounds synthesized by microorganisms. Hence, this study focused on the evaluation of the anti-biofilm and antiadhesive activities of the L. rhamnosus derived-biosurfactant against Streptococcus mutans and its effect on gtfB/C and ftf genes expression level. In this in vitro study Lactobacillus rhamnosus ATCC7469 overnight culture was used for biosurfactant production. The biosurfactant effect on the surface tension reduction was confirmed by drop collapse method. Chemical bonds in the biosurfactant were identified by Fourier transform infrared (FTIR). Anti-biofilm and antiadhesive activities of the biosurfactant were determined on glass slides and in 96-well culture plates, respectively. The effect of the biosurfactant on gtfB/C and ftf genes expression level was also investigated after biofilm formation, total RNA extraction, and reverse transcription by quantitative real-time reverse transcriptase polymerase chain reaction (PCR) assay (quantitative PCR). The data were assessed by one-way analysis of variance in the Tukey-Kramer postdeviation test for all pairs. P < 0.05 was considered statistically significant. The FTIR results of biosurfactant showed that it was protein rich. It also showed anti-biofilm formation activity on the glass slide and antiadhesive activity till 40% on microtiter plate wells. It also showed a significant reduction (P < 0.05) in gtfB/C and ftf genes expression level. L. rhamnosus-derived biosurfactant exhibits a significant inhibitory effect on biofilm formation ability of S. mutans due to downregulation of biofilm formation associated genes, gtfB/C and ftf. L. rhamnosus-derived biosurfactant with substantial antiadhesive activity is suitable candidates for use in new generations of microbial antiadhesive agents.

Report of a fatal case of Lassa fever in Parakou in 2018: clinical, therapeutic and diagnostic aspects

BackgroundLassa fever is one of the most lethal neglected tropical diseases in West Africa. It is a serious public health problem in this region of Africa where it is endemic in several countries. However, it remains a very little known disease by healthcare workers. The lack of specificity of its clinical manifestations makes its diagnosis difficult even in an epidemic context.Case presentationWe report here a confirmed case of Lassa fever whose diagnosis could not be suspected until 11 days after the symptomatology began. This case was recognized as a suspected case of Lassa fever in the Internal Medicine Department of the Regional and Teaching Hospital of Borgou due to the persistence of the fever and the worsening of the patient’s clinical condition despite triple antibiotic therapy in general and especially due to the appearance of hemorrhages. Confirmation of the presence of Lassa fever virus by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) assay on blood sample was obtained after his death despite late initiation of Ribavirin treatment.ConclusionThis case challenges Benin’s health authorities on the need to facilitate access to diagnosis of viral hemorrhagic fevers and to train caregivers at all levels of the health system for better management of these diseases.