Genetic Evaluation of the Patients with Clinically Diagnosed Inborn Errors of Immunity by Whole Exome Sequencing: Results from a Specialized Research Center for Immunodeficiency in Türkiye.

Molecular diagnosis of inborn errors of immunity (IEI) plays a critical role in determining patients' long-term prognosis, treatment options, and genetic counseling. Over the past decade, the broader utilization of next-generation sequencing (NGS) techniques in both research and clinical settings has facilitated the evaluation of a significant proportion of patients for gene variants associated with IEI. In addition to its role in diagnosing known gene defects, the application of high-throughput techniques such as targeted, exome, and genome sequencing has led to the identification of novel disease-causing genes. However, the results obtained from these different methods can vary depending on disease phenotypes or patient characteristics. In this study, we conducted whole-exome sequencing (WES) in a sizable cohort of IEI patients, consisting of 303 individuals from 21 different clinical immunology centers in Türkiye. Our analysis resulted in likely genetic diagnoses for 41.1% of the patients (122 out of 297), revealing 52 novel variants and uncovering potential new IEI genes in six patients. The significance of understanding outcomes across various IEI cohorts cannot be overstated, and we believe that our findings will make a valuable contribution to the existing literature and foster collaborative research between clinicians and basic science researchers.

Clinical characteristics of children with asthma exacerbations: a cross-sectional descriptive study.

AIM: In this cross-sectional descriptive study, we aimed to determine the clinical characteristics of children admitted to a tertiary hospital with asthma exacerbations in a city in southern Turkey where aeroallergens are common and to determine how these characteristics affect the severity of exacerbations. METHODS: Data from a cross-sectional analysis of children with asthma exacerbations who were followed up at the Cukurova University Medical Faculty Pediatric Emergency Department (ED) and Pediatric Allergy & Immunology inpatient clinic were retrospectively analyzed. The study included 106 children who were diagnosed with asthma and did not have any additional comorbidities. In a comparative analysis, the clinical characteristics and laboratory parameters of children with mild/moderate and severe exacerbations were examined. RESULTS: While 81.1% of the patients had mild/moderate exacerbation, 18.8% had severe exacerbation. Additional atopic disease, Alternaria positivity in the skin prick test, the frequency of exacerbations in the previous year, Streptococcus pneumoniae infection, and the rate of noncompliance with treatment were significantly higher in children with severe asthma exacerbations. PEF, FEV1, and FEV1/FVC values were considerably lower in patients with severe exacerbations. CONCLUSIONS: Bacterial infections, presence of atopic disease, Alternaria exposure, low spirometric measures, number of exacerbations in the previous year, and low rate of treatment adherence may be relevant in predicting the severity of asthma exacerbations.

Therapeutic modalities and clinical outcomes in a large cohort with LRBA deficiency and CTLA4 insufficiency.

BACKGROUND: LPS-responsive beige-like anchor (LRBA) deficiency (LRBA-/-) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) insufficiency (CTLA4+/-) are mechanistically overlapped diseases presenting with recurrent infections and autoimmunity. The effectiveness of different treatment regimens remains unknown. OBJECTIVE: Our aim was to determine the comparative efficacy and long-term outcome of therapy with immunosuppressants, CTLA4-immunoglobulin (abatacept), and hematopoietic stem cell transplantation (HSCT) in a single-country multicenter cohort of 98 patients with a 5-year median follow-up. METHODS: The 98 patients (63 LRBA-/- and 35 CTLA4+/-) were followed and evaluated at baseline and every 6 months for clinical manifestations and response to the respective therapies. RESULTS: The LRBA-/- patients exhibited a more severe disease course than did the CTLA4+/- patients, requiring more immunosuppressants, abatacept, and HSCT to control their symptoms. Among the 58 patients who received abatacept as either a primary or rescue therapy, sustained complete control was achieved in 46 (79.3%) without severe side effects. In contrast, most patients who received immunosuppressants as primary therapy (n = 61) showed either partial or no disease control (72.1%), necessitating additional immunosuppressants, abatacept, or transplantation. Patients with partial or no response to abatacept (n = 12) had longer disease activity before abatacept therapy, with higher organ involvement and poorer disease outcomes than those with a complete response. HSCT was performed in 14 LRBA-/- patients; 9 patients (64.2%) showed complete remission, and 3 (21.3%) continued to receive immunosuppressants after transplantation. HSCT and abatacept therapy gave rise to similar probabilities of survival. CONCLUSIONS: Abatacept is superior to immunosuppressants in controlling disease manifestations over the long term, especially when started early, and it may provide a safe and effective therapeutic alternative to transplantation.

Comparing the levels of CTLA-4-dependent biological defects in patients with LRBA deficiency and CTLA-4 insufficiency.

BACKGROUND: Lipopolysaccharide-responsive beige-like anchor protein (LRBA) deficiency and cytotoxic T-lymphocyte protein-4 (CTLA-4) insufficiency are recently described disorders that present with susceptibility to infections, autoimmunity, and lymphoproliferation. Clinical and immunological comparisons of the diseases with long-term follow-up have not been previously reported. We sought to compare the clinical and laboratory manifestations of both diseases and investigate the role of flow cytometry in predicting the genetic defect in patients with LRBA deficiency and CTLA-4 insufficiency. METHODS: Patients were evaluated clinically with laboratory assessments for lymphocyte subsets, T follicular helper cells (TFH ), LRBA expression, and expression of CD25, FOXP3, and CTLA4 in regulatory T cells (Tregs) at baseline and 16 h post-stimulation. RESULTS: LRBA-deficient patients (n = 29) showed significantly early age of symptom onset, higher rates of pneumonia, autoimmunity, chronic diarrhea, and failure to thrive compared to CTLA-4 insufficiency (n = 12). In total, 29 patients received abatacept with favorable responses and the overall survival probability was not different between transplanted versus non-transplanted patients in LRBA deficiency. Meanwhile, higher probability of survival was observed in CTLA-4-insufficient patients (p = 0.04). The T-cell subsets showed more deviation to memory cells in CTLA-4-insufficiency, accompanied by low percentages of Treg and dysregulated cTFH cells response in both diseases. Cumulative numbers of autoimmunities positively correlated with cTFH frequencies. Baseline CTLA-4 expression was significantly diminished in LRBA deficiency and CTLA-4 insufficiency, but significant induction in CTLA-4 was observed after short-term T-cell stimulation in LRBA deficiency and controls, while this elevation was less in CTLA-4 insufficiency, allowing to differentiate this disease from LRBA deficiency with high sensitivity (87.5%) and specificity (90%). CONCLUSION: This cohort provided detailed clinical and laboratory comparisons for LRBA deficiency and CTLA-4 insufficiency. The flow cytometric approach is useful in predicting the defective gene; thus, targeted sequencing can be conducted to provide rapid diagnosis and treatment for these diseases impacting the CTLA-4 pathway.

Clinical features of anaphylaxis in children.

Background: Despite the considerable increase in anaphylaxis frequency, there are limited studies on clinical features of anaphylaxis in children in developing countries. Objective: We aimed to analyze the demographic and clinical features of anaphylaxis in children in Turkey by comparing different age groups and triggers. Methods: Medical records of 147 children, ages 0-18 years, diagnosed with anaphylaxis between 2010 and 2019 were retrospectively analyzed. Results: The mean ± standard deviation age at first anaphylaxis episode was 5.9 ± 5.2 years, with a male predominance (63.9%); 25.2% were infants and 52.4% were < 6 years of age at their first anaphylaxis episode; 78.2% were atopic, with the highest frequency in children with food-induced anaphylaxis (FIA). The home (51.7%) was the most frequent setting. The overall leading cause of anaphylaxis was food (44.2%), which was more frequent at < 6 years of age, followed by drugs (28.6%) and bee venom (22.4%), both were more frequent among older children (>6 years). The patients with venom allergy had the highest rate of rapid onset of symptoms (p < 0.001). Gastrointestinal symptoms were observed significantly more in infants (48.6%) and in children with FIA (38.5%); cardiovascular symptoms were more frequently observed in children > 6 years of age (48.6%) and in children with drug-induced anaphylaxis (64.3%). Although recurrent anaphylaxis was reported for 23.1% of the patients, it was highest in the patients with FIA (35.9%). Overall, only 47.6% of the patients received epinephrine in the emergency department (ED) and 27.3% were referred to an allergy specialist, with the patients with FIA having the lowest rate for both, 32.3% and 10.8%, respectively. Children with drug-induced anaphylaxis had the highest rate of severe anaphylaxis (57.1%). Conclusion: There is a need to improve anaphylaxis recognition and management in all children regardless of age and trigger. Inadequate treatment was most evident in infants and patients with FIA.

Experience of Autoimmune and autoinflammatory diseases in a Turkish pediatric cohort with primary immunodeficiencies.

BACKGROUND: Primary immunodeficiency diseases (PID) are the diseases characterized by a dysfunction of the immune system. Affected patients share a different phenotype such as chronic infections, allergy, autoimmunity, and autoinflammation. METHODS: In all, 433 children with PID were enrolled in this study. Clinical, laboratory, and demographic data of patients were reviewed retrospectively to investigate autoimmune and autoinflammatory complications. Autoinflammation in all patients with inflammation was confirmed by genetic analysis after excluding infectious etiology. RESULTS: Clinical features of 433 PID patients were evaluated retrospectively with long-term follow-up. Autoimmune disorders were identified in 69 (15.9%) patients with PID; 31 (45%) patients had a history of autoimmune disease before diagnosis of PID. The frequency of autoimmunity in immune dysregulation subgroup (76.6%) was higher than other forms of PID. The most common autoimmune manifestations were reported to be Addison's disease, hypoparathyroidism, and autoimmune hemolytic anemia. Autoinflammation were identified in 22 of the 433 (5.1%) patients with PID, including hyper immunoglobulin D syndrome (n = 9), Aicardi-Goutieres syndrome 1 (n = 6), adenosine deaminase 2 deficiency (n = 3), Blau syndrome (n = 2), tumor necrosis factor (TNF) receptor-associated periodic syndrome (n = 1), and auto-inflammation and phospholipase Cγ2-associated antibody deficiency and immune dysregulation syndrome (n = 1). CONCLUSIONS: It is important to recognize association between autoimmunity, autoinflammation, and PID, which in the future could be useful for increased awareness and early diagnosis for these diseases.

MHC Class II Deficiency: Clinical, Immunological, and Genetic Insights in a Large Multicenter Cohort.

BACKGROUND: Major histocompatibility complex class II deficiency, a combined immunodeficiency, results from loss of HLA class II expression on antigen-presenting cells. Currently, hematopoietic stem cell transplantation stands as the sole curative approach, although factors influencing patient outcomes remain insufficiently explored. OBJECTIVES: To elucidate the clinical, immunologic, and genetic profiles associated with MHC-II deficiency and identify prognostic indicators that affect survival rates. METHODS: In this multicenter retrospective analysis, we gathered data from 35 patients with a diagnosis of MHC-II deficiency across 12 centers in Turkey. We recorded infection histories, gene mutations, immune cell subsets, and surface MHC-II expression on blood cells. We conducted survival analyses to evaluate the impact of various factors on patient outcomes. RESULTS: Predominant symptoms observed were pneumonia (n = 29; 82.9%), persistent diarrhea (n = 26; 74.3%), and severe infections (n = 26; 74.3%). The RFXANK gene mutation (n = 9) was the most frequent, followed by mutations in RFX5 (n = 8), CIITA (n = 4), and RFXAP (n = 2) genes. Patients with RFXANK mutations presented with later onset and diagnosis compared with those with RFX5 mutations (P =.0008 and .0006, respectively), alongside a more significant diagnostic delay (P = .020). A notable founder effect was observed in five patients with a specific RFX5 mutation (c.616G>C). The overall survival rate for patients was 28.6% (n = 10), showing a significantly higher proportion in individuals with hematopoietic stem cell transplantation (n = 8; 80%). Early death and higher CD8+ T-cell counts were observed in patients with the RFX5 mutations compared with RFXANK-mutant patients (P = .006 and .009, respectively). CONCLUSIONS: This study delineates the genetic and clinical panorama of MHC-II deficiency, emphasizing the prevalence of specific gene mutations such as RFXANK and RFX5. These insights facilitate early diagnosis and prognosis refinement, significantly contributing to the management of MHC-II deficiency.

Factors affecting formula compliance of infants with IgE mediated cow's milk protein allergy during the pandemic.

Introduction: Cow's milk protein allergy (CMPA) is the most commonly encountered food allergy in the world, usually seen in infants under the age of 2 years. This study aims to determine the factors including COVID-19 affecting formula compliance of CMPA patients. Methods: This study is a prospective, observational study based on 10 different Paediatric Allergy-Immunology clinics in Turkey. Patients aged between 6 months and 2 years, who were followed up with IgE-mediated CMPA treatment or newly diagnosed and using breast milk and/or formula were included in the study. The sociodemographic characteristics of the patients, their symptoms, the treatments they received, and the effects of the COVID-19 pandemic on adherence to formula were evaluated with a questionnaire administered to the parents. Results: The compliance rate for formula-based treatment was 30.8% (IQR: 28.3, SD: 21.86). The number of patients with a single and multiple food allergy was 127 (51.6%) and 71 (28.9%), respectively. Breastfeeding duration, daily amount of prescribed formula and addition of sweetener to the formula were found to reduce compliance (p = 0.010, p = 0.003, and p = 0.004, respectively). However, it was determined that the patient's height, weight, age at diagnosis, and age of formula onset did not have a significant effect on compliance. Conclusion: It was found that the duration of breastfeeding, the increase in the daily amount of formula requirement, and the addition of sweeteners had adverse effects on formula compliance. There was no significant correlation between the formula adherence of CMPA patients and the pandemic.

Hemophagocytic lymphohistiocytosis in children with Griscelli syndrome type 2: genetics, laboratory findings and treatment.

Griscelli syndrome is a rare inherited autosomal recessive syndrome that causes immunodeficiency. Hemophagocytic lymphohistiocytosis (HLH), which is characterized by a high mortality rate, may develop because of Griscelli syndrome type 2 (GS2). We aimed to share our experience with the diagnosis and treatment methods of patients who developed HLH secondary to GS2. Patients with GS2 who were diagnosed and treated for HLH between 2017 and 2022 at the Cukurova University Division of Pediatric Allergy & Immunology and Division of Pediatric Hematology were included in the study. Microscopic examination of the hair shaft and next-generation sequencing for molecular genetic testing of RAB27A helped in the diagnosis of GS2. The first clinical presentation of 8 patients was HLH. One patient presented with CNS involvement and two patients presented with recurrent fever. Over 5 years, GS2 was diagnosed in 15 patients, of whom 11 (73.3%) developed HLH. The HLH-2004 protocol was used to treat these patients. Hematopoietic stem cell transplantation (HSCT) was performed in five patients who were matched with suitable donors. While all patients who underwent HSCT were alive, three patients who could not undergo HSCT because no donor could be found died. Deletion of CAAGC at nucleotides 514_518 in GS2 patients is associated with CNS involvement and a poor prognosis. HLH may be the first sign of presentation in patients with GS2. Although further research is needed, regardless of the conditioning regimen utilized, early HSCT remains the primary therapy option for preventing GS2-induced mortality in HLH.

Clinical characteristics of patients requiring lung transplantation referral in national cystic fibrosis registry data.

BACKGROUND: We aimed to determine the number of cystic fibrosis (CF) patients recorded in the Cystic Fibrosis Registry of Türkiye (CFRT) who were in need of lung transplantation (LT) referral and examine clinical differences between patients who were LT candidates due to rapid forced expiratory volume in one second (FEV1) decline and LT candidates without rapid FEV1 decline in the last year to identify a preventable cause in patients with such rapid FEV1 decline. METHODS: All CF patients recorded in the CFRT in 2018 were evaluated in terms of LT. Patients were divided into those with FEV1 below 50% and in need of LT due to a decrease of 20% or more in the previous year (Group 1) and those who did not have FEV1 decline of more than 20% in the previous year but had other indications for LT (Group 2). Demographic and clinical features were compared between the two groups. RESULTS: Of 1488 patients registered in CFRT, 58 had a need for LT. Twenty patients were included in Group 1 and others in Group 2. Our findings did not reveal any significant variations in treatment, chronic infection status, or complications between the two groups. The average weight z-score was significantly higher in Group 1. Positive correlations were detected between weight z-score and FEV1 in 2017 in Group 1 and between FEV1 values in 2017 and 2018 in Group 2. CONCLUSIONS: There appears to be a relationship between the nutritional status and weight z-scores of CF patients and pulmonary function, which may indirectly affect the need for lung transplantation referral.

First Documented Case of Pulmonary Alveolar Proteinosis with Atopy Presenting Secondary to CSFR2B Mutation.

Pulmonary alveolar proteinosis (PAP) is a rare lung disorder in which surfactant-derived lipoproteins accumulate excessively within pulmonary alveoli, causing severe respiratory distress. It is essential to gain a better understanding of the signs to clinically diagnose PAP and include PAP among the differential diagnoses of interstitial pulmonary diseases or other diseases with similar manifestations. We describe a 2.5-year patient with atopy who presented with pulmonary infiltration, recurrent wheezing, and cough despite steroid and salbutamol administration via inhalation. High-resolution computed tomography revealed crazy-paving patterns in both lungs, suggesting PAP. An open lung biopsy revealed intra-alveolar granular amphophilic material, which was strongly positive on periodic acid-Schiff staining. The results of pulmonary-associated surfactant protein B and C gene analyses were normal. However, granulocyte-macrophage colony-stimulating factor receptor beta-protein was not detected in leucocytes, and a novel mutation was identified in the CSF2RB gene. The patient was diagnosed with PAP and treated with whole-lung lavage. Key Words: Pulmonary alveolar proteinosis, Child, Atopy, Wheezing.

The success of the Cystic Fibrosis Registry of Turkey for improvement of patient care.

BACKGROUND: Cystic fibrosis (CF) registries play an essential role in improving disease outcomes of people with CF. This study aimed to evaluate the association of newly established CF registry system in Turkey on follow-up, clinical, growth, treatment, and complications of people with this disease. METHODS: Age at diagnosis, current age, sex, z-scores of weight, height and body mass index (BMI), neonatal screening results, pulmonary function tests, history of meconium ileus, medications, presence of microorganisms, and follow-up were evaluated and compared to data of people with CF represented in both 2017 and 2019 registry data. RESULTS: There were 1170 people with CF in 2017 and 1637 in 2019 CF registry. Eight hundred and fourteen people were registered in both 2017 and 2019 of whom z-scores of heights and BMI were significantly higher in 2019 (p = 0.002, p =0.039, respectively). Inhaled hypertonic saline, bronchodilator, and azithromycin usages were significantly higher in 2019 (p =0.001, p = 0.001, p = 0.003, respectively). The percent predicted of forced expiratory volume in 1 sec and forced vital capacity were similar in 2017 and 2019 (88% and 89.5%, p = 0.248 and 84.5% and 87%, p =0.332, respectively). Liver diseases and osteoporosis were significantly higher, and pseudo-Bartter syndrome (PBS) was significantly lower in 2019 (p = 0.011, p = 0.001, p = 0.001, respectively). CONCLUSIONS: The z-scores of height and BMI were higher, the use of medications that protect and improve lung functions was higher and incidence of PBS was lower in 2019. It was predicted that registry system increased the care of people with CF regarding their follow-up. The widespread use of national CF registry system across the country may be beneficial for the follow-up of people with CF.

Asthma and risk factors in the first 6 years of life in a population-based cohort.

BACKGROUND: The frequency of asthma increases in childhood and asthma is associated with risk factors varying across age groups. The aim of our study was to assess the prevalence of asthma and its associated risk factors in the first six years of life. METHODS: Within the scope of the Adana Pediatric Allergy and Risk Factor (ADAPAR) birth cohort study, 203 infants that had experienced at least one wheezing attack during the first year of life were followed for asthma development until the age of six years. Additionally, 223 infants that were followed within the scope of the same study and had no wheezing attacks in the first year of life were assigned to the control group. RESULTS: At the end of the sixth year, 46 (22.7%) infants were diagnosed with asthma and the use of antibiotics of the mother during pregnancy (OR: 2.98), the presence of allergic diseases in the mother (OR: 4.70) and sibling (OR:2.11), the presence of atopy (OR:4.76), and recurrence of wheezing in the first age (OR:17.35) were identified as risk factors for asthma. CONCLUSIONS: The prevalence of asthma at six years of age was higher than that of other studies. Prevention of infections at an early age and during pregnancy can reduce the prevalence of asthma.

Monogenic lupus due to DNASE1L3 deficiency in a pediatric patient with urticarial rash, hypocomplementemia, pulmonary hemorrhage, and immune-complex glomerulonephritis.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease and usually involves the skin, musculoskeletal system, and kidneys. More than 30 genes have been to monogenic lupus, so far. Monogenic lupus is often characterized by an early-onset, similar family history, and syndromic appearance. Herein we present a pediatric patient with DNASE1L3 deficiency, suffering from both urticarial skin lesions, recurrent hemoptysis, and renal involvement, eventually diagnosed as this rare monogenic lupus. The patient suffered from recurrent urticarial rash and hemoptysis since the age of 15 months of age. He had microscopic hematuria, mild proteinuria, hypocomplementemia, and positive antinuclear antibody, anti-dsDNA, and antineutrophil cytoplasmic antibodies. Renal biopsy yielded immunocomplex glomerulonephritis. Due to early-onset, similar sibling history and consanguineous parents, we suspected monogenic lupus and performed whole-exome sequencing, which further revealed a homozygous T97Ifs*2 mutation (NM_004944.4: c.290_291delCA/p.Thr97Ilefs*2) in DNASE1L3 gene. In conclusion, DNASE1L3 deficiency should be thought when juvenile SLE occurs with early disease-onset, pulmonary hemorrhage, glomerulonephritis, and recurrent urticarial rash along with ANCA positivity.

Current Status of Genetic Diagnosis Laboratories and Frequency of Genetic Variants Associated with Cystic Fibrosis through a Newborn-Screening Program in Turkey.

BACKGROUND: Cystic fibrosis (CF) is the most common worldwide, life-shortening multisystem hereditary disease, with an autosomal recessive inheritance pattern caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The national newborn screening (NBS) program for CF has been initiated in Turkey since 2015. If the immunoreactive trypsinogen (IRT) is elevated (higher than 70 µg/L in the second control) and confirmed by sweat test or clinical findings, genetic testing is performed. The aims of this study are to emphasize the effect of NBS on the status of genetic diagnosis centers with the increasing numbers of molecular testing methods, and to determine the numbers and types of CFTR mutations in Turkey. METHODS: The next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) results of 1595 newborns, who were referred to Cukurova University Adana Genetic Diseases Diagnosis and Treatment Center (AGENTEM) for molecular genetic testing, were evaluated with positive CF NBS program results since 2017. RESULTS: According to the results; 560 (35.1%) of the 1595 patients carried at least 1 (one) CF-related variant, while 1035 patients (64.9%) had no mutation. Compound heterozygosity for two mutations was the most common in patients, while two detected variants were homozygote in 14 patients. A total of 161 variants were detected in 561 patients with mutations. Fifteen novel variants that have not been previously reported were found. Moreover, p.L997F was identified as the most frequent pathogenic mutation that might affect the IRT measurements used for the NBS. The distribution of mutation frequencies in our study showed a difference from those previously reported; for example, the well-known p.F508del was the third most common (n = 42 alleles), rather than the first. The most striking finding is that 313 cases had a pathogenic variant together with the V470M variant, which might have a cumulative effect on CF perpetuation. CONCLUSION: This study is the first to determine the mutational spectrum of CFTR in correlation with the NBS program in the Turkish population. NBS for CF raises issues regarding screening in diverse populations, both medical and non-medical benefits, and carrier identification. Through the lens of NBS, we focused on the integrated diagnostic algorithms and their effect on the results of genetic testing.

Simultaneous production of alpha and beta amylase enzymes using separate gene bearing recombinant vectors in the same Escherichia coli cells.

The present study describes the simultaneous expression of thermostable industrial alpha (α) and beta (ß) amylase enzymes that have been used widely in starch industry. Genomic DNA of Bacillus stearothermophilus DSM 22 strain for α amylase and, Thermoanaerobacterium (Clostridium) thermosulfurogenes DSM 2229 strain for ß amylase were used as gene sources. Both genes were ligated into pETDuet-1 expression vector separately and resulting recombinant vectors were transformed into Escherichia coli BL21 competent cells by electroporation. The cells were first transformed by pETDuet-1/ αAmy recombinant plasmid, then the competent cells carrying this plasmid were prepared for the transformation of pETDuet-1/ ßAmy plasmid. Enzymatic activities of bacterial colonies were detected on LB agar staining with iodide. Both enzymes were more produced by IPTG induction in BL21 cells and were purified using Ni-NTA agarose column. SDS-PAGE and western blot analyses showed that the molecular weight of purified α and ß amylase to be approximately 60 kDa and 55kDa, respectively. The concentration of the purified α and ß amylase were calculated as 4.59 µg/mL and 3.17 µg/mL with IPTG as an inducer in LB medium. The present study proposes a novel and efficient method for the production of thermostable α and ß amylases at the same E coli cells containing separate engineered plasmid vectors.

Does naloxone prevent seizure in tramadol intoxicated patients?

BACKGROUND: Tramadol poisoning has increased in recent years. Seizure is one of the side-effects of tramadol toxicity. There is a controversy about possible preventive effect of naloxone in tramadol poisoning induced seizure. Therefore, this study was performed to compare seizure incidence in tramadol poisoning patients who received and did not receive naloxone, as an opioid antagonist. METHODS: This study involved prospective data collection followed by retrospective analysis on 104 tramadol poisoning patients who were admitted in a referral poisoning center. The incidences of seizure were compared between patients received naloxone and those did not. Outcome was considered as survived without or with complications and death. Logistic Regression analysis was used to determine the effects of different variables on seizure incidence. RESULTS: 70 (67.3%) of the patients were men. The mean age of the patients was 26.3 ± 9 years old. 18.3% of the patients received naloxone in their treatment period. Seizure incidence was significantly higher among tramadol poisoning patients who did not receive naloxone compare with those received naloxone (14.1% vs. 5.1%). Among different variable studied, age had a significant effect on predicting of seizure (odds ratio = 2.09; 95% of confidence interval: 1.82-2.26; P value, 0.004). CONCLUSIONS: Although the seizure incidence was lower in patients with tramadol poisoning who received naloxone, the logistic regression did not support the preventive effect of naloxone on seizure in tramadol poisoning cases.