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Purpose: Major histocompatibility complex class II (MHC-II) deficiency is a rare inborn error of immunity (IEI). Impaired antigen presentation to CD4+ T-cells results in combined immunodeficiency. Patients typically present with severe respiratory and gastrointestinal tract infections at early ages. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy. Methods: We describe the clinical, immunologic, and genetic features of eighteen unrelated Iranian patients with MHC-II deficiency. Results: Consanguinity was present in all affected families. The median age at the initial presentation was 5.5 months (range seven days to 18 years). The main symptoms included failure to thrive, persistent diarrhea, and pneumonia. Autoimmune and neurologic features were documented in 30% of the patients, respectively. Thirteen patients carried RFXANK gene mutations, two carried RFX5 gene mutations, and three carried a RFXAP gene mutation. Six patients shared the same RFXANK founder mutation (c.162delG); limited to the Iranian population and dated to approximately 1,296 years ago. Four of the patients underwent HSCT; three of them are alive. On the other hand, nine of the fourteen patients who did not undergo HSCT had a poor prognosis and died. Conclusion: MHC-II deficiency is not rare in Iran, with a high rate of consanguinity. It should be considered in the differential diagnosis of combined immunodeficiency (CID) at any age. With the limited access to HSCT and its variable results in MHC-II deficiency, implementing genetic counseling and family planning for the affected families are mandatory. We better determined the c.162delG RFXANKheterozygous mutation frequency in the Iranian population.

For diagnosis of CMV infection in colon biopsies, immunohistochemistry (IHC) and polymerase chain reaction (PCR) can be helpful besides pathologic examination. We performed CMV IHC and PCR on formalin–fixed paraffin-embedded (FFPE) and fresh tissue (FT) in clinically suspicious children for colitis and compared the results with the patients’ clinical course. In a prospective cohort study, among 153 candidate children for the colonoscopy due to colitis, 50 showed positive CMV PCR on FT. PCR on FFPE tissue was positive in 17/40 (42.5 %) cases, of which 2/40 (5%) had positive IHC. Low titer plasma CMV DNA level was seen in 7/17 FFPE-PCR positive cases. Two patients had a high plasma CMV DNA level, who were the two IHC-positive cases. Fecal calprotectin level showed an association with disease histologic activity, but not with CMV positivity on either of the methods of detection. IHC positivity was more likely to be associated with a high plasma CMV DNA level. There was a high rate of false positivity by CMV PCR test on fresh tissue that was neither associated with histopathology nor with the CMV DNA plasma level. Antiviral treatment was correlated with a poorer outcome in patients with inflammatory bowel disease (P=0.035). Conclusion: The PCR result of FT is correlated neither to histology nor to the serum CMV-DNA level. High fecal calprotectin level is associated with active lesions in histopathology. Large randomized clinical trials are recommended to assess the efficacy of antiviral treatment in acute colitis cases that have a high copy number of CMV in the blood or positive results on IHC.

Background Evidence shows the increase of anti-tissue transglutaminase (tTG) levels in various conditions, including infectious agents, independently of celiac disease (CD). The aim of this study was to investigate the effect of helicobacter pylori (H.pylori) infection eradication on serum level of tTG in children with CD. Methods This study was conducted on children aged 2 to 18 who referred to reference hospitals for diagnosis of CD. After upper endoscopy and biopsy to confirm CD and H.pylori infection, the children were divided into three groups (including group one: 16 CD patients with positive H. pylori; group two: 16 non-CD patients with positive H. pylori; and group three: 56 CD patients with negative H. pylori), respectively. The tTG level in study groups were compared after the eradication of H.pylori. Results The mean age of the subjects in the group one, two, and three was 9.7 ± 3.33, 11.8 ± 3.14, and 7.6 ± 3.32 years, respectively. Our results showed that in group one, mean tTG increased after eradication of H.pylori infection, however, these changes were not significant (182.43 vs. 157.18, P = 0.121). In the second group, although unlike the first group, mean tTG decreased after eradication of the infection, but still these changes were not significant (9.56 vs. 22.18, P = 0.449). Furthermore, at the baseline level, the mean tTG in the group three was closer to the mean tTG in the first group. Conclusion Our findings showed that the eradication of H.pylori infection does not have a significant effect on tTG levels in children with and without CD.

The immune system has experienced major changes in both its organization and function during the evolution of tetrapod animals. Still, the ancestry of specific cell types and the historical relationships between modern immune cells has received little attention. While all tetrapods possess mononuclear blood cells and monocytes, vasculature-patrolling nonclassical monocytes (CD16+CD14-in humans, Ly6C-in mice) have so far only been described in mammals. The question of whether nonclassical monocytes are specific to the mammalian lineage has not been answered. Cell types can be described as traits that persist via the inheritance of core regulatory complexes of transcription factors. We utilized transcriptional network analyses on a human monocyte single-cell RNA-sequencing dataset to highlight the core regulatory complex associated with nonclassical monocyte production (nCoRC). We then applied BLAST-based analyses to quantify the conservation of human nCoRC members amongst mammals and nonmammals. We determined that the average sequence similarity of nCoRC members is highest amongst mammals and that such conservation is not observed in non-mammalian tetrapods. We also discovered that this sequence similarity is specifically driven by boreoeutherian placental mammals. Finally, we found the receptor proteins upstream of nonclassical monocyte assembly are also more homologous in mammals than non-mammals. This work provides an evolutionary model in which the capacity for nonclassical monocyte production is unique to the mammalian lineage.

The authors have requested that this preprint be removed from Research Square.

JavaScript is an increasingly popular language for server-side development, thanks in part to the Node.js runtime environment and its vast ecosystem of modules. With the Node.js package manager npm, users are able to easily include external modules as dependencies in their projects. However, npm installs modules with all of their functionality, even if only a fraction is needed, which causes an undue increase in code size. Eliminating this unused functionality from distributions is desirable, but the sound analysis required to find unused code is difficult due to JavaScript's extreme dynamicity. We present a fully automatic technique that identifies unused code by constructing static or dynamic call graphs from the application's tests, and replacing code deemed unreachable with either file- or function-level stubs. If a stub is called, it will fetch and execute the original code on-demand, thus relaxing the requirement that the call graph be sound. The technique also provides an optional guarded execution mode to guard application against injection vulnerabilities in untested code that resulted from stub expansion. This technique is implemented in an open source tool called Stubbifier, which supports the ECMAScript 2019 standard. In an empirical evaluation on 15 Node.js applications and 75 clients of these applications, Stubbifier reduced application size by 56% on average while incurring only minor performance overhead. The evaluation also shows that Stubbifier's guarded execution mode is capable of preventing several known injection vulnerabilities that are manifested in stubbed-out code. Finally, Stubbifier can work alongside bundlers, popular JavaScript tools for bundling an application with its dependencies. For the considered subject applications, we measured an average size reduction of 37% in bundled distributions., 29 pages. This work has been submitted to the Journal on Empirical Software Engineering

Background: The role of dairy foods in inflammatory bowel disease (IBD) has been controversial and it is debatable if patients with IBD should avoid milk and dairy products or not, as well as the relationship between these foods and symptoms among those population. Objective: This multi centric cross-sectional study designed to evaluate if it is really necessary to deprive IBD patients from consumption of dairy foods. Methods: A multicenter study with 12 gastroenterology referral centers in four countries was designed to evaluate gastrointestinal (GI) symptoms after consumption of dairy foods from all outpatients with IBD during 6 months and to compare patients treated at the same centers without IBD (non IBD cases). Results: Overall 1888 cases included (872 IBD patients and 1016 non IBD cases). 56.6% of participants were female with average age of 40.1 years. Racially 79.8% participants were Caucasians and originally they were citizens of 10 countries. Relative prevalence of IBD was higher in Africans and Indians and the most frequent prevalence of dairy foods intolerance was seen in Asians. Among IBD patients, 571 cases diagnosed as ulcerative colitis and 189 participants as Crohn’s disease. Average duration of diagnosis as IBD was 6.8 years (from 2 months to 35 years). The most prevalent GI symptoms after consumption of all the dairy foods were bloating and abdominal pain. Totally, intolerance of dairy foods and lactase deficiency was more prevalent among IBD patients in comparison with non IBD cases (65.5% vs 46.1%, P=0.0001). But the rate of GI complains among IBD patients who had not any family history of lactase deficiency, history of food sensitivity or both were 59.91%, 52.87% & 50.33% respectively and similar to non IBD cases (P=0.68, 0.98 & 0.99 respectively). Conclusion: The rate of dairy foods intolerance among IBD patients without family history of lactase deficiency or history of food sensitivity is similar to non IBD cases and probably there is no reason to deprive them from this important source of dietary calcium, vitamin D and other nutrients.

Background Cryopreserved peripheral blood mononuclear cells (PBMCs) are frequently collected and provide disease- and treatment-relevant data in clinical studies. Here, we developed combined protein (40 antibodies) and transcript single-cell (sc)RNA sequencing (scRNA-seq) in PBMCs. Results Among 31 participants in the Women’s Interagency HIV Study (WIHS), we sequenced 41,611 cells. Using Boolean gating followed by Seurat UMAPs (tool for visualizing high-dimensional data) and Louvain clustering, we identified 50 subsets among CD4+ T, CD8+ T, B, NK cells, and monocytes. This resolution was superior to flow cytometry, mass cytometry, or scRNA-seq without antibodies. Combined protein and transcript scRNA-seq allowed for the assessment of disease-related changes in transcriptomes and cell type proportions. As a proof-of-concept, we showed such differences between healthy and matched individuals living with HIV with and without cardiovascular disease. Conclusions In conclusion, combined protein and transcript scRNA sequencing is a suitable and powerful method for clinical investigations using PBMCs.

Background: Majority of the patients affected by Kaposi sarcoma (KS) have human immunodeficiency virus (HIV). Highly active antiretroviral therapy (HAART) lessened the incidence of AIDS (acquired immunodeficiency syndrome)- related KS. Cutaneous signs are the most common, but involving the gastrointestinal (GI) tract is very important and dangerous because of serious complications including perforation and bleeding. Case Presentation: This report is a rare case of gastric-KS presenting as melena in a non-AIDS 67-year-old woman. We describe the diagnosis and management of this rare complication. Conclusion: GI-KS is often asymptomatic with different endoscopic appearances, and maybe present without cutaneous lesions. Thus, a high diagnostic suspicion is needed and we should attend these GI complications.

To compare the effects of functional electrical stimulation (FES) with conventional therapy and conventional therapy alone on improvement of faecal incontinence (FI) symptoms in a number of children with functional non-retentive FI.Data of 28 children with FI were accepted for this retrospective study. The case group (n = 14) underwent FES + conventional therapy and the control group (n = 14) received conventional therapy only. Data of children with faecal retention, inflammatory, anatomic, metabolic and neurological disorders were excluded. Children were assessed with a paediatric FI score questionnaire, and a bowel habit diary both before treatment sessions, after they ended, and after 6 months. A FI quality of life questionnaire was completed before and after treatment for all children.Full response to the treatment (100% reduction in FI episodes) was significantly observed in 8/14 (57.1%) of children in the case group compared to 2/14 (14.2%) of children in the control group after the ending of treatment sessions (P = 0.005). The baseline mean ± SD of FI episodes per week was 3.7 ± 2.1 among both groups which significantly decreased after the ending of treatment sessions in the case group compared to the control group (1.4 ± 2.1 vs 3 ± 2.7; P = 0.05). Mean ± SD of FI score was significantly reduced in the case group compared to the controls after the ending of treatment sessions (3.9 ± 4.5 vs 8 ± 4.7; P = 0.02) and maintained after 6 months (P = 0.05).Functional electrical stimulation is a safe, effective, non-invasive, inexpensive, reproducible and easy-to-use modality for treatment of functional non-retentive faecal incintinence in children.

Despite the decades-old knowledge that males and people with diabetes mellitus (DM) are at increased risk for coronary artery disease (CAD), the reasons for this association are only partially understood. Among the immune cells involved, recent evidence supports a critical role of T cells as drivers and modifiers of CAD. CD4+ T cells are commonly found in atherosclerotic plaques. We aimed to understand the relationship of CAD with sex and DM by single-cell RNA (scRNA-Seq) and antibody sequencing (CITE-Seq) of CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) of 61 men and women who underwent cardiac catheterization were interrogated by scRNA-Seq combined with 49 surface markers (CITE-Seq). CAD severity was quantified using Gensini scores, with scores above 30 considered CAD+ and below 6 considered CAD−. Four pairs of groups were matched for clinical and demographic parameters. To test how sex and DM changed cell proportions and gene expression, we compared matched groups of men and women, as well as diabetic and non-diabetic subjects. We analyzed 41,782 single CD4+ T cell transcriptomes for sex differences in 16 women and 45 men with and without coronary artery disease and with and without DM. We identified 16 clusters in CD4+ T cells. The proportion of cells in CD4+ effector memory cluster 8 (CD4T8, CCR2+ Em) was significantly decreased in CAD+, especially among DM+ participants. This same cluster, CD4T8, was significantly decreased in female participants, along with two other CD4+ T cell clusters. In CD4+ T cells, 31 genes showed significant and coordinated upregulation in both CAD and DM. The DM gene signature was partially additive to the CAD gene signature. We conclude that (1) CAD and DM are clearly reflected in PBMC transcriptomes, and (2) significant differences exist between women and men and (3) between subjects with DM and non-DM.

Milk kefir is a fermented dairy product with numerous attributed health benefits due to the presence of a complex eukaryotic and prokaryotic microbiota. In this study, a total number of 26 yeast isolates were obtained from eight kefir samples from three different cities of Iran. The isolates belonged to Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia fermentans and P. kudriavzevii. The potential probiotic characteristics of the isolates were evaluated based on their ability to tolerate the stimulated condition of the gastrointestinal tract. In addition, hemolytic activity, adherence to different solvents, auto-aggregation, adhesion to the epithelial intestine-derived cells and antimicrobial activity of the selected isolates were evaluated. Overall, four yeast strains (three strains of S. cerevisiae and one strain of P. fermentans) showed resistance and survival ability against the gastrointestinal physiological conditions including acidic pH, presence of bile salt and digestive enzymes. They were able to grow at 37°C and had the capacity to adhere to epithelial intestine-derived cells. These results suggest that the selected strains can be proper candidates as probiotic yeast strains for the development of novel functional foods.

Introduction: According to the World Health Organization, the incidence of severe acute hepatitis of unknown origin in children is increasing worldwide. However, there are no known causes and treatments to deal with this disease. This study presents three children with severe acute hepatitis of unknown origin in a hospital medical center in Iran. Case Presentation: Two 12-year-old children and a six-year-old child were admitted to the hospital with jaundice. Laboratory tests showed markedly elevated liver enzymes and total and direct bilirubin in these patients. However, hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) antibodies (Ab), hepatitis A virus (HAV) IgM, HEV IgM, Epstein-Barr virus (EBV) IgM, anti-microsomal Ab, anti-liver kidney (LKM Ab), anti-smooth muscle antibodies (ASMA), anti-nuclear antibodies (ANA), and total IgG were all negative. After excluding the probable infectious causes of acute hepatitis and per the progressive course of fulminant hepatic failure, intravenous methylprednisolone was started for two of three cases during hospitalization. These children were discharged from the hospital in good general condition, and the liver function tests gradually decreased and approximated to near normal during the follow-up visits. Conclusions: General practitioners and pediatricians are advised to examine the underlying causes and consider severe acute hepatitis of unknown origin when visiting children under 16 years with severe jaundice and very high transaminases.

Sports-related spinal injuries can be catastrophic in nature. Athletes competing in collision sports (eg, football) may be particularly prone to injury given the high-impact nature of these activities. Due to the oftentimes profound impact of sports-related spinal injuries on health and quality-of-life, they are also associated with a substantial risk of litigation. However, no study to date has assessed litigation risks associated with sports-related spinal injuries. A better understanding of the risk factors surrounding these legal claims may provide insights into injury prevention and other strategies to minimize litigation risks. In addition, it may allow the spine surgeon to better recognize the health, socioeconomic, and legal challenges faced by this patient population.To provide a comprehensive assessment of reported legal claims involving sports-related spinal injuries, including a comparative analysis of legal outcomes between collision and non-collision sports. To discuss strategies to prevent sports-related spinal injuries and minimize litigation risks.Retrospective review.Athletes experiencing spinal injuries during sports.Outcomes included verdict outcome (defendant vs. plaintiff), legal claims, injuries sustained, clinical symptoms, and award payouts.The legal research database Westlaw Edge (Thomson Reuters) was queried for legal claims brought in the United States from 1950 to 2021 involving sports-related spinal injuries. Verdict or settlement outcomes were collected as well as award payouts, time to case closure, case year, and case location. Demographic data, including type of sport (ie collision vs. non-collision sport) and level of play were obtained. Legal claims, spinal injuries sustained, and clinical symptoms were also extracted. Furthermore, the nature of injury, injured spinal region, and treatment pursued were collected. Descriptive statistics were reported for all cases and independent-samples t-tests and chi-square tests were used to compare differences between collision and non-collision sports.Of the 840 cases identified on initial search, 78 met our criteria for in-depth analysis. This yielded 62% (n=48) defendant verdicts, 32% (n=25) plaintiff verdicts, and 6% (n=5) settlements, with a median inflation-adjusted award of $780,000 (range: $5,480-$21,585,000) for all cases. The most common legal claim was negligent supervision (n=38, 46%), followed by premises liability (n=23, 28%), and workers' compensation/no fault litigation (n=10, 12%). The most common injuries sustained were vertebral fractures (n=34, 44%) followed by disc herniation (n=14, 18%). Most cases resulted in catastrophic neurological injury (n=37, 49%), either paraplegia (n=6, 8%) or quadriplegia (n=31, 41%), followed by chronic/refractory pain (n=32, 43%). Non-collision sport cases had a higher percentage of premises liability claims (41% vs. 11%, p=.006) and alleged chronic/refractory pain (53% vs. 28%, p=.04). Conversely, collision sport cases had a higher proportion of workers' compensation/no fault litigation (23% vs. 4%, p=.03) and cases involving disc herniation (29% vs. 9%, respectively; p=.04).Sports-related spinal injuries are associated with multiple and complex health, socioeconomic, and legal consequences, with median inflation-adjusted award payouts nearing $800,000 per case. In our cohort, the most commonly cited legal claims were negligent supervision and premises liability, emphasizing the need for prevention guidelines for safe sports practice, especially in non-professional settings. Cases involving athletes participating in non-collision sports were significantly associated with claims citing chronic/refractory pain, highlighting the importance of long-term care in severely injured athletes.

Background The aim of this study was to evaluate the serum level of matrix metalloproteinase 7 (MMP7) in infants with cholestasis and the diagnostic values of this biomarker to differentiate biliary atresia (BA) from other causes of cholestasis. Methods This multi-center study is conducted during 2 years in Mofid children’s hospital and Children’s Medical Center, Pediatrics Center of Excellence Tehran, Iran. 54 infants with cholestasis were enrolled in this study with a control group consists of 41 healthy infants with the same age. Serum samples were taken from all these patients to assess serum levels of MMP7, Gamma-glutamyl Transferase (GGT). For each biomarker, we calculated the sensitivity and specificity and other statistical characteristics. Results There were 89 subjects, 22 patients with BA, 32 patients with non-BA cholestasis and 41 subjects as control group. The mean serum MMP7 levels in BA, non-BA cholestasis and control group was 15.91 ng/ml ± 6.64, 4.73 ng/ml ± 2.59 and 0.49 ng/ml ± 0.33, respectively. The best cut-off point is calculated 7.8 ng/ml for MMP7 and 434.5 U/L for GGT. The area under curve (AUC) for these two markers are 0.988 ± 0.008 and 0.854 ± 0.052, respectively. The sensitivity and specificity of MMP7 to differentiate biliary atresia from nonbiliary atresia cholestasis in our study was 95.5% and 94.5%, respectively. The sensitivity and specificity of GGT was 77.3% and 77.8%, respectively. These results show that the MMP7 has more sensitivity and specificity in differentiation. Conclusion MMP7 demonstrated good accuracy to differentiate biliary atresia from other causes of cholestasis.

We compared the effects of transcutaneous functional electrical stimulation (TFES) and biofeedback therapy with TFES alone in a cohort of children with functional non-retentive fecal incontinence (FNRFI). This prospective, single-center randomized clinical trial was performed on 40 children with FNRFI. Patients were randomly allocated into two equal treatment groups. Group A (n = 20) underwent TFES + biofeedback therapy, and group B (n = 20) received TFES alone. All patients were assessed with a pediatric fecal incontinence (FI) score questionnaire, a visual pain score, and a bowel habit diary both before and at the end of treatment sessions and also at 6 months of follow-up. In addition, a FI quality-of-life (QoL) questionnaire was recorded for all patients before and 6 months after the treatment. FI significantly improved in 13/20 (65%) patients in group A and 11/20 (55%) patients in group B (P

Despite the availability of various diagnostic tests for inflammatory bowel diseases (IBD), misdiagnosis of IBD occurs frequently, and thus, there is a clinical need to further improve the diagnosis of IBD. As gut dysbiosis is reported in patients with IBD, we hypothesized that supervised machine learning (ML) could be used to analyze gut microbiome data for predictive diagnostics of IBD. To test our hypothesis, fecal 16S metagenomic data of 729 subjects with IBD and 700 subjects without IBD from the American Gut Project were analyzed using five different ML algorithms. Fifty differential bacterial taxa were identified [linear discriminant analysis effect size (LEfSe): linear discriminant analysis (LDA) score > 3] between the IBD and non-IBD groups, and ML classifications trained with these taxonomic features using random forest (RF) achieved a testing area under the receiver operating characteristic curves (AUC) of ∼0.80. Next, we tested if operational taxonomic units (OTUs), instead of bacterial taxa, could be used as ML features for diagnostic classification of IBD. Top 500 high-variance OTUs were used for ML training, and an improved testing AUC of ∼0.82 (RF) was achieved. Lastly, we tested if supervised ML could be used for differentiating Crohn’s disease (CD) and ulcerative colitis (UC). Using 331 CD and 141 UC samples, 117 differential bacterial taxa (LEfSe: LDA score > 3) were identified, and the RF model trained with differential taxonomic features or high-variance OTU features achieved a testing AUC > 0.90. In summary, our study demonstrates the promising potential of artificial intelligence via supervised ML modeling for predictive diagnostics of IBD using gut microbiome data. NEW & NOTEWORTHY Our study demonstrates the promising potential of artificial intelligence via supervised machine learning modeling for predictive diagnostics of different types of inflammatory bowel diseases using fecal gut microbiome data.