Enzyme-linked Immunosorbent Assay Research Articles

Recurrent BMP4 variants in exon 4 cause non-HFE-associated hemochromatosis via the BMP/SMAD signaling pathway

BackgroundHereditary hemochromatosis (HH) is an iron overload disorder and can be caused by variants in non-HFE genes in Chinese patients. However, there is still a considerable proportion of patients suffering from unexplained iron overload. In our previous study, we had identified the p.R269Q variant in exon 4 of the Bone morphogenetic protein 4 (BMP4) gene in Chinese patients with unexplained primary iron overload by Whole Exome sequencing, and then the BMP4 p.H251Y variant was identified by Sanger sequencing in a Chinese patient with secondary iron overload. Our study aimed to explore the pathogenicity and underlying mechanism of BMP4 p.H251Y and BMP4 p.R269Q variants in patients with iron overload.MethodsSanger sequencing was conducted to identify the novel variants in the BMP4 gene of patients with unexplained iron overload. MRI and liver biopsy were used to display iron overload in the liver of the patient harboring the BMP4 p.H251Y variant. The BMP4 and hepcidin levels in BMP4 knockdown and BMP4 variant cells were examined by enzyme-linked immunosorbent assay. The effects of BMP4 p.H251Y and BMP4 p.R269Q variants on the hepcidin-regulation pathway were studied.ResultsOne of 54 HH patients (1.85%) harbored the BMP4 p.R269Q variant. One of 148 patients (0.68%) with secondary hemochromatosis harbored the BMP4 p.H251Y variant, and these two variants were not found in 100 Chinese general population. For the patient harboring the BMP4 p.H251Y variant, abdominal MRI and Perl's staining of liver tissue displayed iron overload in the liver. Cells transfected with the BMP4 p.H251Y and p.R269Q variants showed down-regulation of hepcidin level and BMP/SMAD pathway compared with cells transfected with the wild-type BMP4 vector.ConclusionThe BMP4 p.H251Y and p.R269Q variants can downregulate hepcidin levels by inhibiting the BMP/SMAD axis, suggesting they may play pathogenic roles in iron overload.

Transdermal Delivery of Tofacitinib Citraste via Mannose-Decorated Transferosomes Loaded with Tofacitinib Citrate in Arthritic Joints.

Transdermal drug delivery systems are a promising option for the treatment of rheumatoid arthritis (RA) because they can lower systemic adverse effects of immunosuppressants. Janus kinase (JAK) inhibitors were found to be effective for the treatment of RA by inhibiting the JAK-STAT pathway and preventing autoimmune joint destruction. The aim of this study is to deliver tofacitinib (a JAK 1 and 3 inhibitor) through mannose-decorated transferosomes (MDTs) directly to inflamed joints. Transferosomes are composed of phospholipids, Cremophor A25, PEG400, Labrafac lipophile, and oleic acid to enhance the permeation of tofacitinib and control nanovesicle size (∼70-200 nm). Permeation through rat skin was evaluated, where the skin permeation of MDTs (Q24: 38.8 ± 9.82 μg/cm2) and flux (0.5311 ± 0.072 μg/cm2/h) were significantly greater than those of the uncoated transferosomes (Q24 of T1: 1.522 ± 0.329 μg/cm2, Q24 of T2: 3.5002 ± 0.998 μg/cm2, and Q24 of T3: 18.226 ± 5.25 μg/cm2). In addition, MDTs seem to permeate the skin intact, as shown by the transmission electron microscopy (TEM) images of the recipient buffer removed from the Franz diffusion cell. A histopathology assay was performed during the in vivo evaluation of MDTs in an arthritic rat model, in which, significantly less inflammation was observed when MDTs were applied directly to the joint compared to when applied to the dorsal skin and untreated arthritic joints. Furthermore, significantly lower tumor necrosis factor-α (TNFα), IL-6, and IL-1β levels (P < 0.05) were detected by enzyme-linked immunosorbent assay (ELISA) in homogenates of the joints treated with MDTs than in untreated arthritic joints. In conclusion, this study proposed effective MDTs that could deliver tofacitinib directly to inflamed joints possibly by targeting the macrophages circulating in the proximity of the site of inflammation.

Exploring the Role of Adipose Tissue Dysregulation in Vitiligo Pathogenesis: A Body Composition Analysis.

Vitiligo is a commonly acquired pigmentary disease characterized by the progressive destruction of melanocytes. The role of adipose tissue in vitiligo remains unclear. To investigate the role of adipose tissue in the pathogenesis of vitiligo, 50 patients newly diagnosed with vitiligo and 40 age- and sex-matched healthy controls were enrolled, and fat mass and distribution using dual-energy X-ray absorptiometry (DXA) and serum adipokine levels using enzyme-linked immunosorbent assay were assessed. The trunk and whole-body fat mass and the trunk/limb fat mass ratio were significantly higher in patients with vitiligo. Higher leptin and resistin levels and lower adiponectin levels were observed. Positive correlation of the trunk fat mass index with vitiligo area scoring index scores (r = 0.38, p = 0.0071) was found. Additionally, plasma adiponectin levels were negatively correlated with vitiligo disease activity scores (r = -0.307, p < 0.05). Receiver operating characteristic curve analysis showed that the percentage of limb lean tissue mass, trunk/limb fat mass ratio, and serum adipokine levels achieved a high area under the curve score for distinguishing patients with vitiligo from healthy controls. In conclusion, the incidence of central obesity and adipokine dysregulation was higher in patients with vitiligo. The potential role of adipose tissue in the pathogenesis of vitiligo should be emphasized.

Mechanism of vitamin D deficiency involvement in the development of pulmonary arterial hypertension related to monocrotaline-induced connective tissue disease in rats

Objective: To investigating the impact of vitamin D (VitD) deficiency on the jagged 1 protein (Jagged1)/Notch3 signaling pathway in the pulmonary arteries of rats with monocrotaline (MCT)-induced connective tissue disease (CTD)-related pulmonary arterial hypertension (PAH) and to explore the pathological and molecular mechanisms of VitD involvement in the development of CTD-PAH. Methods: Twenty-four 7-week-old male Wistar rats were divided into a normal diet group and a VitD-free diet group using random number table, with 12 rats in each group. After 5 weeks of feeding, the rats were further randomly divided into saline and MCT groups, forming group A (normal diet+saline), group B (normal diet+MCT), group C (VitD-free+saline), and group D (VitD-free+MCT), with 6 rats in each group, and the rats were continued to be fed for another 4 weeks. The MCT group was injected with MCT solution subcutaneously on the back of the neck to construct the CTD-PAH model, and the saline group was injected with an equal amount of saline as a control. At the end of the experiment, blood samples were collected from all rats, and the serum 1, 25-dihydroxy vitamin D levels were determined by enzyme-linked immunosorbent assay. Hemodynamic measurements were performed on all rats, recording and calculating the mean values of right ventricular pressure and pulmonary artery pressure. Lung tissues and pulmonary arteries of all rats were stained with hematoxylin-eosin staining, the inner and outer diameters of pulmonary vessels were measured, and the percentage of pulmonary artery medial layer thickness was calculated. The right ventricular hypertrophy index was determined by weighing the parts of the heart and calculating the ratio. The apoptosis of pulmonary artery smooth muscle cells was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. The expression levels of Jagged1, Notch3, and hairy and enhancer of split-1 (Hes1) mRNA and protein in the pulmonary arteries were analyzed by reverse transcription quantitative polymerase chain reaction and Western blotting. Results: The serum 1, 25-dihydroxy vitamin D levels in group D were significantly lower than those in groups A, B, and C [(51.01±0.96) μg/L, (65.15±1.83), (57.49±0.28), (54.52±2.87) μg/L, respectively, all P<0.05]. The right ventricular pressure, pulmonary artery systolic pressure, pulmonary artery diastolic pressure, and mean pulmonary artery pressure in Group D rats were significantly higher than those in groups A, B and C (all P<0.05). The percentage of pulmonary artery medial layer thickness in group D (56.28%±9.51%) was significantly higher than that in Groups A (21.28%±1.89%), B [22.72% (22.16%, 30.10%)], and C (38.73%±7.34%) (all P<0.05); the right ventricular hypertrophy index in group D (45.74%±12.68%) was significantly higher than that in groups A (21.78%±2.80%), B (40.93%±9.10%), and C (23.71%±1.22%) (all P<0.05). The apoptosis of pulmonary artery smooth muscle cells in group D (0.07±0.01) was significantly lower than that in groups A (1.00±0.08), B (0.17±0.02), and C (0.49±0.07) (all P<0.05). The levels of Jagged1, Notch3, and Hes1 mRNA and protein in Group D were all significantly higher than those in Groups A, B, and C (all P<0.05). Conclusions: VitD deficiency is involved in the pathophysiological mechanisms of CTD-PAH development. This occurs by upregulating the expression of the Jagged1/Notch3/Hes1 pathway in the pulmonary arteries of rats with MCT-induced PAH. The consequences include pulmonary artery thickening, increased pulmonary artery pressure, increased right ventricular pressure, right ventricular hypertrophy, and decreased apoptosis of pulmonary artery cells.

Differential murine responses to Schistosoma mansoni eggs in the liver and small intestine lead to downmodulation of hepatic but not intestinal periovular granulomas.

To control schistosomiasis mansoni, it is important to attempt preventing the worms' egg-induced pathology in the liver and limiting pathogen transmission following egg exit from the intestines to the exterior. Therefore, the present study aimed to clarify the reasons behind the decades-long riddle of periovular granulomas downmodulation in the liver, but not the small intestine, with the progression of murine schistosomiasis mansoni. Outbred female CD-1 mice were percutaneously exposed to 15 Schistosoma mansoni cercariae. The liver and small intestine were collected from mice harboring a minimum of a worm couple at 8, 12, 16, and 20 weeks post-infection, assessed for egg counts/g and histopathological changes, and used to prepare Triton X-100 extracts. Content of cytokines, saturated and unsaturated fatty acids, triglycerides, cholesterol, reactive oxygen species, and uric acid per mg tissue extract proteins were evaluated using capture enzyme-linked immunosorbent assays, gas chromatography-flame ionization detector, and standard commercially available reagents, respectively. Examination of hematoxylin-eosin-stained tissue sections confirmed the decrease in size and changes in cellular composition of periovular granulomas in the liver but not the small intestine, associated with wide differences in released cytokines types and amounts, and content of the bioactive lipids, arachidonic and docosahexaenoic acids, reactive oxygen species, and uric acid. The results together disclosed that the downmodulation of hepatic, but not the small intestine, circumoval granulomas with the progression of murine S. mansoni naturally results from site- and tissue- specific immunological and biochemical responses to the egg-derived antigens and molecules and suggested that the intestines appear to harbor immune-privileged sites.

Association of Chronic Urticaria With Hepatitis C Infection In Female Patients Visiting Tertiary Care Hospital In Pakistan

Objective: To evaluate association of chronic urticaria with hepatitis C infection in female patients. Study Design: comparative cross-sectional study. Place and Duration of Study: Fauji Foundation Hospital Rawalpindi. One-year duration (Jan 2020-Jan 2021). Methodology: One Hundred female patients (group 1) of age 60 years or less with chronic urticaria were enrolled in this study. Patients with chronic liver disease or signs of hepatic decompensation were excluded. Equal number of age and gender matched patients not having urticarial symptoms were selected as group 2. Anti-Hepatitis C Virus Antibody (HCV ab) was evaluated by Enzyme linked immunosorbent assay (ELISA). Disease severity was calculated by Urticaria activity score 7(UAS 7). Association of HCV infection with chronic urticaria and its severity was analyzed by applying appropriate statistical tests. Results: Anti HCV antibody positivity frequency was 14/100(14%) in group 1 and 4/100(4%) in group 2 (p value: 0.013; significant). Mean ages in group 1 and 2 were 39.88+1.38 and 38.79+1.35 years respectively (p value: 0.57; insignificant). Mean UAS 7 calculated in the HCV negative Urticaria cases was 6.73+2.94 while it was significantly higher in HCV positive group i.e. 8.78+6.459 (p value 0.01). Frequency of HCV positivity in patients with well controlled urticaria and mild urticaria was 6/59(10.0%) and 5/36(13.8%) respectively while in moderate urticaria was 3/5(60%) which was significantly higher (p value 0.04). Conclusion: There is a significant association of chronic urticaria with anti HCV antibody positivity. HCV positive patients have more severe disease as compared to HCV negative patients.

Inhibition of iron-induced cofilin activation and inflammation in microglia by a novel cofilin inhibitor.

Neuroinflammatory conditions linked to iron dysregulation pose significant challenges in neurodegenerative diseases. Iron-loaded microglia are observed in the brains of patients with various neuroinflammatory conditions, yet how iron overload affects microglial function and contributes to various neuroinflammatory processes is poorly understood. This invitro study elucidates the relationship between excess iron, cofilin activation, and microglial function, shedding light on potential therapeutic avenues. Iron overload was induced in Human Microglial Clone 3 cells using ferrous sulfate, and the expressions of ferritin heavy chain, ferritin light chain, divalent metal transporter 1, cofilin, p-cofilin, nuclear factor-κB (NF-κB), and various inflammatory cytokines were analyzed using real-time quantitative polymerase chain reaction, immunocytochemistry, Western blotting, and enzyme-linked immunosorbent assay. Results revealed a notable increase in cofilin, NF-κB, and inflammatory cytokine expression levels following excess iron exposure. Moreover, treatment with deferoxamine (DFX), a known iron chelator, and a novel cofilin inhibitor (CI) synthesized in our laboratory demonstrate a mitigating effect on iron-induced cofilin expression. Furthermore, both DFX and CI exhibit promising outcomes in mitigating the inflammatory consequences of excess iron, including the expression of pro-inflammatory cytokines and NF-κB activation. These findings suggest that both DFX and CI can potentially alleviate microglia-induced neuroinflammation by targeting both iron dysregulation and cofilin-mediated pathways. Overall, this study provides valuable insights into iron-induced cofilin activation and microglial activation, offering avenues for potential targeted therapies for neuroinflammatory conditions associated with iron and cofilin dysregulation in neurodegenerative diseases.

The overexpression of eIF4E decreases oxytocin levels and induces social-cognitive behavioral disorders in mice.

Overexpression of the eukaryotic initiation factor 4E (eIF4E) gene has been associated with excessive stereotypic behaviors and reduced sociability, which manifest as autism-like social cognitive deficits. However, the precise mechanisms by which eIF4E overexpression induces insufficiently these autism-like behaviors and the specific brain regions implicated remain insufficiently understood . Oxytocin, a neurotransmitter known for its role in social behavior, has been proposed to modulate certain autism-related symptoms by influencing microglial function and attenuating neuroinflammation. Nonetheless , the contributions of the hippocampus and oxytocin in the content of eIF4E overexpression-induced autistic behaviors remain elucidated . To investigate this issue,esearchers utilized the three-chamber social interaction test, the open field test, and the Morris water maze to evaluate the social cognitive behaviors of the two groups of mice. Additionally, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blotting, and RT-qPCR were employed to quantify oxytocin levels and assess hippocampal microglial activation.The results indicate that overexpression of eIF4E in mice is associated with significant impairments in social cognition, alongside pronounced marked hyperactivation of hippocampal microglia.Significance statement Autism spectrum disorder (ASD) encompasses a range of neurodevelopmental disorders characterized by social cognitive impairment.Research has indicated a correlation between the overexpression of the eukaryotic initiation factor 4E (eIF4E) gene and autism-like social cognitive impairment.Oxytocin (OXT), a neurotransmitter, plays a role in regulating hippocampal microglial activity and attenuating neuroinflammation. This modulation may impact social cognition in individuals with autism.Nevertheless,it remains unclear whether there is an involvement of the hippocampus and oxytocin in autism-like social cognitive impairments due to eIF4E overexpression. The present study suggests that overexpression of eIF4E may induce hyperactivation of microglia and contribute to social cognitive impairment by decreasing oxytocin levels in the hippocampus.These findings offer molecular insights into the manifestation of autism-like behavior resulting from eIF4E overexpression and may guide future clinical interventions.

COVID-19 vaccination survey and anti-SARS-CoV-2 IgG responses in a human cohort from Schistosoma mansoni-endemic villages in Mayuge District, Uganda: a cross-sectional study.

The coronavirus disease 2019 (COVID-19) pandemic has resulted in devastating health and economic consequences worldwide. Vaccination has been a central pillar for COVID-19 prevention and control. Understanding the immunomodulatory effects of helminth infections on COVID-19 vaccine-induced immune responses and vaccine efficacy is crucial to the development and deployment of effective vaccination strategies in low- and middle-income countries with a high prevalence of worms. In September 2022, we conducted a cross-sectional, population-based survey in five Schistosoma mansoni endemic villages in Mayuge District, Uganda (n = 450). The prevalence of schistosomiasis and soil-transmitted helminths was determined by the Kato-Katz (KK) technique on two stool samples collected from each participant. A subset of individuals (n = 204) were interviewed in a COVID-19 vaccination survey. IgG levels against the SARS-CoV-2 spike S1 subunit (anti-S1 IgG) were measured by enzyme-linked immunosorbent assay (ELISA) in collected serum samples. The overall schistosomiasis and hookworm prevalence rates in the five villages were 36.4% (166/450) and 36.9% (168/450), respectively. Within the cohort, 69.78% (314/450) of the subjects had a positive anti-S1 IgG response. COVID-19 vaccination coverage among the interviewed participants was 93.14% (190/204; 95% CI, 88.8% - 95.9%). However, 81% (154/190) of COVID-19 vaccinees had an anti-S1 IgG titre ≤200. In an adolescent group receiving a single dose of the BNT162b2 mRNA vaccine (n = 23), an inverse correlation was observed between anti-S1 IgG antibody level/titre and faecal egg count. Within the above group, anti-S1 IgG levels/titres were significantly lower in subjects with moderate or heavy S. mansoni infection (n = 5) than those in KK-negative individuals (n = 9). Although the acceptance rate of COVID-19 vaccination was high, the majority of participants received only a single vaccine dose and the overall anti-S1 IgG titres in confirmed vaccinees were low. Moderate-to-heavy schistosome infections blunted the antibody responses following vaccination with a single dose of BNT162b2. These observations confirm the necessity for a second COVID-19 vaccine dose for two-dose primary immunization series and call for implementation research that may inform the development of a 'treat and vaccinate' policy during vaccination roll-out in regions with heavy worm burdens.

Relationship Between Serum Levels of Oxidized Lipoproteins, Circulating Levels of Myeloperoxidase and Paraoxonase 1, and Diet in Young Subjects with Insulin Resistance.

Oxidized low-density lipoproteins (ox-LDLs) are involved in atherosclerotic plaque formation and progression and have been linked to insulin resistance (IR). Myeloperoxidase is a potent oxidant of lipoproteins related to atherogenic risk. High-density lipoproteins (HDLs) are considered antioxidants due to their association with paraoxonase 1 (PON1). However, HDL can also be oxidized (ox-HDL), and its relationship with IR has not been described. This study evaluated the relationship between circulating levels of myeloperoxidase and paraoxonase 1, diet, and serum levels of ox-LDL and ox-HDL in young people with IR. This cross-sectional study examined 136 young subjects (67 and 69 with and without insulin resistance, respectively). Serum levels of ox-LDL, ox-HDL, myeloperoxidase, and PON1 were quantified using an enzyme-linked immunosorbent assay. The nutritional dietary content of the foods was determined with a food frequency questionnaire, which was analyzed with Nutrimind 2013 software. Serum ox-HDL levels were higher in young subjects without IR than those with IR (p = 0.031). Women with IR presented increased ox-LDL levels compared with women without IR (p = 0.012) and men with IR (p < 0.001). In the IR group, serum ox-LDL levels were negatively correlated with total cholesterol, triglycerides, and LDL-C, whereas the correlation was positive in the insulin-sensitive group. Consumption of vitamins B1 and B2 was related to increased HDL-C levels, while higher ox-LDL levels were related to vitamin K intake. In addition, low energy consumption and phosphorus increased PON1 levels. The results suggest that insulin resistance in young women may promote lipoprotein oxidation, and the intake of B complex vitamins may have an antiatherogenic effect.

Neuroprotective and inflammatory biomarkers in pediatric drug-resistant epilepsy: Interplay between GDNF, IL-1β and vitamin D 25-OH

Drug-resistant epilepsy in pediatric patients is associated with neuroinflammation and neurodegeneration. Vitamin D 25-OH exerts neuroprotective effects, while glial cell line-derived neurotrophic factor (GDNF) and the proinflammatory cytokine interleukin-1β (IL-1β) are implicated in the mechanisms of neuroinflammation and epileptogenesis. The aim of this study was to investigate the relationship between vitamin D 25-OH, IL-1β, and GDNF levels with seizure severity and frequency in children with drug-resistant epilepsy. A cross-sectional study was conducted at Adam Malik Hospital, Medan, Indonesia, among children with drug-resistant epilepsy. Vitamin D 25-OH, IL-1β and GDNF levels were measured using enzyme-linked immunosorbent assay (ELISA). Epilepsy severity was assessed using the Hague Seizure Severity Scale (HASS), while seizure frequency was assessed using the Global Assessment of Severity of Epilepsy (GASE). The present study identified a significant correlation between GDNF levels and epilepsy severity, as measured by the HASS score (r=0.318; p=0.006). However, no significant correlation was observed between vitamin D 25-OH or IL-1β levels and epilepsy severity or seizure frequency (p&gt;0.05). IL-1β levels correlated significantly with GDNF levels (r=0.525; p=0.001), but IL-1β did not directly correlate with seizure frequency or epilepsy severity. In conclusion, GDNF levels significantly correlated with epilepsy severity, suggesting that GDNF may serve as a potential biomarker for assessing epilepsy severity. However, further studies investigating the role of GDNF as a potential neurotrophic factor in the pathophysiology of epilepsy and its possible application as a therapeutic target are important.

Evidence of Site-Specific Mucosal Autoantibody Secretion in Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPA) have been detected in sputum and saliva, indicating that anti-modified protein antibodies (AMPA) can be produced at mucosal sites in patients with rheumatoid arthritis (RA). However, the body's largest mucosal compartment, the gut, has not yet been examined. We therefore investigated the presence of several AMPA(ACPA, anti-carbamylated protein antibodies [anti-CarP], and anti-acetylated protein antibodies [AAPA]) at different mucosal sites, including the intestinal tract. Paired fecal/ileal wash, saliva, and serum samples of patients with RA and healthy volunteers were collected in two independent cohorts. Data involving feces were replicated in a third cohort. In these secretions, AMPA were analyzed using in-house enzyme-linked immunosorbent assay with unmodified peptides as control. In fecal samples, total IgA and anti-Escherichia coli IgA were measured. ACPA, anti-CarP, and AAPA IgA were measurable in saliva of seropositive patients with RA (prevalence 9%-40%). No AMPA could be detected in feces. IgA was present because total IgA and anti-E. coli IgA were detectable in feces of ACPA-positive patients with RA and healthy donors. Results were confirmed in another cohort using colonoscopically collected ileal wash samples. Our study shows the presence of ACPA, anti-CarP, and AAPA IgA in saliva of ACPA-seropositive patients with RA. However, no AMPA could be detected in feces/ileal wash samples of these patients, although our assays were able to measure other antigen-specific antibodies. These data suggest that mucosal autoantibody secretion may occur in the oral mucosa of patients with RA, whereas no evidence could be found for this process in the lower gastrointestinal tract.

Crimean-Congo haemorrhagic fever among healthcare workers in Iran 2000-2023, a report of National Reference Laboratory.

Crimean-Congo haemorrhagic fever (CCHF) is a lethal acute viral zoonosis with a case fatality rate of 5-50%. Due to the potential of human-to human transmission of the disease, healthcare workers (HCWs) are at risk of occupational exposure to CCHF virus. Little is known about CCHF virus route of transmission and risks in Iranian HCWs. Therefore this study was designed to identify the routes of exposure to the CCHF virus among Iranian HCWs. From Oct 2000 to Feb 2023, 96 CCHF suspected healthcare workers referred to national reference laboratory were tested for CCHF virus infection by the use of RT-PCR and IgM Capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) and exposure history of cases were investigated to determine the CCHF virus routes of transmission in nosocomial settings. Twelve CCHF confirmed cases were identified including seven nurses and five physicians, with the median age of 32.5 years (range 23-53 years) and the median incubation period of 6.8 days (range from 1 to 22 days). None of the cases reported a history of tick bite or close contact with tissues or animal blood. The cases were from Razavi Khorasan (seven cases), Sistan and Baluchistan (two cases), Isfahan (one case), South Khorasan (one case) and Fars (one case). Percutaneous exposure (needle stick) (three cases), mucosal exposure (blood splash in to face) (three cases) and skin contact with blood (three cases) constituted the most prevalent routes of transmission. Since 2013, no CCHF cases have been identified among Iranian HCWs. In healthcare settings, physicians and nurses are at risk of nosocomial CCHF virus infection. The routes of transmission mainly include direct exposures via needle-stick, mucosal or direct contact with the skin to infected blood. Continuous education and implementation of infection prevention and control measures are key factors to minimize the incidence of healthcare related CCHF.

Evaluation of the Hypothalamic-Pituitary-Adrenal Axis in Patients with Primary Sjögren's Disease.

Background and Objectives: Patients with primary Sjögren's disease (pSjD) show contradictory results regarding the activity of the hypothalamic-pituitary-adrenal (HPA) axis. The aim of this study was to determine the salivary cortisol concentration to evaluate the function of the HPA axis (hypoactive/hyperactive) between patients with pSjD and control subjects. Materials and Methods: A total of 34 subjects participated in the cross-sectional study: 17 patients with pSjD and 17 control subjects. Stimulated whole saliva (SWS) was used to determine salivary cortisol concentration using an enzyme-linked immunosorbent assay (ELISA). Results: The salivary cortisol concentration showed a statistically significant difference between patients with pSjD and control subjects (4.69 ± 2.88 vs. 0.49 ± 0.37; p < 0.001; Student t-test). The area under the curve (AUC) was 100.0% in patients with pSjD (p < 0.001). The cut-off point was set to >1.454. The patients with pSjD had four times higher scores for depression and stress and six times higher scores for anxiety compared to the control subjects (p = 0.048, p < 0.001, p = 0.038; Mann-Whitney U test). The patients with pSjD had a statistically significantly higher total Oral Health Impact Profile (OHIP) score compared to the control subjects (p < 0.001, Mann-Whitney U test). Conclusions: The patients with pSjD showed short-term hyperactivity of the HPA axis compared to the control subjects.

ADAMTS16 drives epithelial-mesenchymal transition and metastasis through a feedback loop upon TGF-β1 activation in lung adenocarcinoma

Lung adenocarcinoma (LUAD) is the major subtype of lung cancer. The poor prognosis of LUAD patients is attributed primarily to metastasis. ADAMTS16 is a crucial member of the ADAMTS family and is involved in tumor progression. However, its role and regulatory mechanism in LUAD remain unexplored. In this study, ADAMTS16 was identified as a crucial oncogene and survival predictor in LUAD via analyses of public datasets. Clinical specimens and tissue microarrays confirmed the differential expression and prognostic value of ADAMTS16 in LUAD patients. Transcriptome data and in vitro experiments demonstrated that ADAMTS16 was positively associated with epithelial-mesenchymal transition (EMT) and the migration abilities of LUAD cells. Knockdown of ADAMTS16 attenuated lung and pleural metastasis in an animal model. Mechanistically, the results of the enzyme-linked immunosorbent assay (ELISA) and western blot (WB) suggested that ADAMTS16 activated the TGF-β signaling pathway by facilitating the conversion of LAP-TGF-β1 to active TGF-β1. Co-Immunoprecipitation (co-IP) indicated an interaction between ADAMTS16 and LAP-TGF-β1. Inhibition of ADAMTS16 impaired EMT and aggressiveness of LUAD cells, while treatment with recombinant TGF-β1 reversed this inhibition. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays indicated that SOX4 acted as a transcriptional activator of ADAMTS16 and that TGF-β1 regulated the expression of ADAMTS16 by increasing the binding of SOX4 to the promoter of ADAMTS16. Suppressing the TGF-β signaling pathway inhibited ADAMTS16 expression, EMT, and lung metastasis, whereas overexpressing SOX4 reversed this inhibition. Therefore, ADAMTS16 forms a positive feedback loop with the TGF-β1/SOX4 axis to regulate EMT and metastasis, and disruption of this feedback loop inhibits tumor progression. These findings underscore the potential of ADAMTS16 as a prognostic biomarker and therapeutic target in LUAD and offer novel insight into the mechanism of EMT and metastasis.

Proposal of a Rapid Detection System Using Image Analysis for ELISA with an Autonomous Centrifugal Microfluidic System.

In this study, with the aim of adapting an enzyme-linked immunosorbent assay (ELISA) system for point-of-care testing (POCT), we propose an image analysis method for ELISAs using a centrifugal microfluidic device that automatically executes the assay. The developed image analysis method can be used to quantify the color development reaction on a TMB (3,3',5,5'-tetramethylbenzidine) substrate. In a conventional ELISA, reaction stopping reagents are required at the end of the TMB reaction. In contrast, the developed image analysis method can analyze color in the color-developing reaction without a reaction stopping reagent. This contributes to a reduction in total assay time. The microfluidic devices used in this study could execute reagent control for ELISAs by steady rotation. In the demonstration of the assay and image analysis, a calibration curve for mouse IgG detection was successfully prepared, and it was confirmed that the image analysis method had the same performance as the conventional analysis method. Moreover, the changes in the amount of color over time confirmed that a calibration curve equal to the endpoint analysis was obtained within 2 min from the start of the TMB reaction. As the assay time before the TMB reaction was approximately 7.5 min, the developed ELISA system could detect TMB in just 10 min. In conventional methods using a plate reader, the assay required a time of 90 min for manual handling using microwell plates, and in the case of using automatic microfluidic devices, 30 min were required. The time of 10 min realized by this proposed method is equal to the time required for detection in an immunochromatographic assay with a lateral flow assay; therefore, it is expected that ELISAs can be performed sufficiently to adapt to POCT.

Anti-integrin αvβ6 autoantibody in primary sclerosing cholangitis: a Japanese nationwide study.

Although specific biomarkers for primary sclerosing cholangitis (PSC) are required, no such biomarkers have been identified. We previously reported that patients with PSC had anti-integrin αvβ6 autoantibodies at only two hospitals. In this study, we aimed to validate the accuracy of the autoantibodies in diagnosing PSC using the newly developed Anti-integrin αvβ6 enzyme-linked immunosorbent assay (ELISA) Kit, which enables quantitation and comparison of antibodies among different facilities. Overall, 81 patients with PSC in a Japanese PSC registry recruited from 17 medical centers and hospitals, and 358 controls were enrolled. We retrospectively assessed anti-integrin αvβ6 autoantibodies using the Anti-integrin αvβ6 ELISA Kit and in-house ELISA. Anti-Integrin αvβ6 ELISA Kit and in-house ELISA exhibited a significant correlation (r = 0.97, P < 0.001). Anti-integrin αvβ6 autoantibodies were detected in 67 of 81 (82.7%) patients with PSC and 20 of 358 (5.6%) controls, resulting in a sensitivity of 82.7% and specificity of 94.4% for PSC, using the anti-integrin αvβ6 ELISA Kit. When focusing on the presence or absence of inflammatory bowel disease (IBD), the sensitivities for PSC with ulcerative colitis, Crohn's disease, unclassified-IBD, and without IBD were 97.8% (43/44), 100% (1/1), 80.0% (8/10), and 53.8% (7/13), respectively. Antibody concentrations were significantly higher in PSC patients without IBD than in controls (P < 0.001). We validated that anti-integrin αvβ6 autoantibodies have high sensitivity and specificity for diagnosing PSC. This study provides further evidence that anti-integrin αvβ6 autoantibodies are a useful biomarker for diagnosing PSC.

Sennoside A represses the malignant phenotype and tumor immune microenvironment of non-small cell lung cancer cells by inhibiting the TRAF6/NF-κB pathway.

Non-small cell lung cancer (NSCLC) is a prominent cause of cancer death worldwide. Sennoside A (SA) is the primary anthraquinone active component from Rheum officinale Baill and exerts antitumor functions in multiple human tumors. This research aimed to elucidate the function and mechanism of SA in NSCLC. SA functions in NSCLC were determined using Cell Counting Kit-8 (CCK-8) assay, Terminal deoxynucleotidyl transferase dUTP nick-end labeling analysis, Transwell assay, Enzyme-Linked Immunosorbent Assay (ELISA), and Western blot. Meanwhile, the SA mechanism in NSCLC was examined with Western blot, immunofluorescence assay, CCK-8 assay, Transwell analysis, and ELISA. Furthermore, SA functions in NSCLC growth in vivo were assessed by the establishment of a tumor xenograft model, hematoxylin-eosin staining, analysis of NSCLC tissue apoptosis, and immunohistochemistry assays. Functionally, less than 200µM SA had no significant effect on normal human bronchial epithelial cell BEAS-2B cell viability. Furthermore, H460 cell viability was decreased when the SA dose was greater than or equal to 25µM (IC50 = 53.34µM). A549 cell viability was reduced when the SA dose was greater than or equal to 12.5µM (IC50 = 48.21µM). Also, SA repressed NSCLC cell proliferation and boosted cell apoptosis. SA restrained NSCLC cell invasion and tumor microenvironment. Mechanically, SA weakened NSCLC cell proliferation, invasion, and tumor microenvironment, yet this impact was abolished after transfecting pcDNA3.1-TRAF6, which indicated that SA repressed NSCLC cell proliferation, invasion, and tumor microenvironment through inactivating TRAF6/NF-κB. Moreover, SA reduced subcutaneous tumor volume and promoted NSCLC tissue apoptosis in vivo. SA repressed NSCLC cell proliferation, invasion, and tumor microenvironment through inactivating TRAF6/NF-κB.

Comparison of RT.PCR and ELISA Methods in the Diagnosis of Human Cytomegalovirus in Kidney Transplant Patients

Background: In kidney transplant recipients prone to infections like cytomegalovirus (CMV), a vital need for an accurate diagnostic method is evident. This study at Khorshid Laboratory in Tehran rigorously compares real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) for CMV detection in transplant patients. Methods: In January to March 1400, 70 kidney transplant recipients were assessed for CMV DNA using RT-PCR and concurrent ELISA tests, with statistical analysis aided by SPSS. Results: In kidney transplant patients (average age: 49.40 ± 13.64 years), 4.3% tested positive for CMV via PCR. Strong correlation between serological and molecular methods. IgG and IgM antibody detection both showed high sensitivity and specificity, advocating for efficient CMV diagnosis at Khorshid Laboratory, Tehran. Conclusion: This study emphasizes the need for a quick and efficient CMV diagnostic approach in kidney transplant patients. The strong correlation between molecular and serological methods favors using the faster RT-PCR method, crucial for timely management, especially with the increasing age- related CMV incidence. The findings strongly recommend RT-PCR integration for enhanced sensitivity in kidney transplant patients at Khorshid Laboratory, Tehran.

Expression of TSPAN1 and its link to thyroid nodules: one step forward on the path to thyroid tumorigenesis biomarkers

BackgroundThyroid cancer is ranked as the most common malignancy within the endocrine system and the seventh most prevalent cancer in women globally. Thyroid malignancies require evaluating biomarkers capable of distinguishing between them for accurate diagnosis. We examined both mRNA and protein levels of TSPAN1 in plasma and tissue samples from individuals with thyroid nodules to aid this endeavour.MethodsIn this case-control study, TSPAN1 was assessed at both protein and mRNA levels in 90 subjects, including papillary thyroid cancer (PTC; N = 60), benign (N = 30), and healthy subjects (N = 26) using enzyme-linked immunosorbent assay (ELISA) and SYBR-Green Real-Time PCR, respectively.ResultsTSPAN1 plasma levels were decreased in PTC and benign compared to healthy subjects (P = 0.002). TSPAN1 mRNA levels were also decremented in the tumoral compared to the paired normal tissues (P = 0.012); this drop was also observed in PTC patients compared to benign patients (P = 0.001). Further, TSPAN1 had an appropriate diagnostic value for detecting PTC patients from healthy plasma samples with a sensitivity of 76.7% and specificity of 65.4% at the cutoff value < 2.7 (ng/ml).ConclusionTSPAN1 levels are significantly reduced in patients with benign and PTC, demonstrating its potential value as a diagnostic biomarker. Additionally, the significant reduction in TSPAN1 mRNA expression within PTC tumor tissues may suggest its involvement in tumor progression and development. Further studies, including larger-scale validation studies and mechanistic investigations, are imperative to clarify the molecular mechanisms behind TSPAN1 and, ultimately, its clinical utility for treating thyroid disorders.