Soluble and insoluble expression of recombinant human interleukin-2 protein using pET expression vector in Escherichia coli.

Interleukin-2 has emerged as a potent protein-based drug to treat various cancers, AIDS, and autoimmune diseases. Despite its immense requirement, the production procedures are inefficient to meet the demand. Therefore, efficient production procedures must be adopted to improve protein yield and decrease procedural loss. This study analyzed cytoplasmic and periplasmic IL-2 expression for increased protein yield and significant biological activity. The study is focused on cloning IL-2 into a pET-SUMO and pET-28a vector that expresses IL-2 in soluble form and inclusion bodies, respectively. Both constructs were expressed into different E. coli expression strains, but the periplasmic and cytoplasmic expression of IL-2 was highest in overnight culture in Rosetta 2 (DE3). Therefore, E. coli Rosetta 2 (DE3) was selected for large-scale production and purification. Purified IL-2 was characterized by SDS-PAGE and western blotting, while its biological activity was determined using MTT bioassay. The results depict that the periplasmic and cytoplasmic IL-2 achieved adequate purification, yielding 0.86 and 0.51 mg/mL, respectively, with significant cytotoxic activity of periplasmic and cytoplasmic IL-2. Periplasmic IL-2 has shown better yield and significant biological activity in vitro which describes its attainment of native protein structure and function.

Evolution of cell therapy for renal cell carcinoma.

Treatment for renal cell carcinoma (RCC) has improved dramatically over the last decade, shifting from high-dose cytokine therapy in combination with surgical resection of tumors to targeted therapy, immunotherapy, and combination therapies. However, curative treatment, particularly for advanced-stage disease, remains rare. Cell therapy as a "living drug" has achieved hematological malignancy cures with a high response rate, and significant research efforts have been made to facilitate its translation to solid tumors. Herein, we overview the cellular therapies for RCC focusing on allogeneic hematopoietic stem cell transplantation, T cell receptor gene-modified T cells, chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) cells, lymphokine-activated killer (LAK) cells, γδ T cells, and dendritic cell vaccination. We have also included perspectives for using other recent approaches, such as CAR macrophages, dendritic cell-cytokine induced killer cells and regulatory CAR-T cells to shed light on preclinical development of cell therapy and advancing cell therapy into clinic to achieve cures for RCC.

Comparison of Clinical Characteristics and Outcome of COVID-19 Patients Between the 4th and 5th Wave in a Tertiary Care Hospital in Karachi, Pakistan.

ABSTRACT       Objective: To compare the clinical parameters of 4th and 5th COVID waves in Karachi, Pakistan. STUDY DESIGN: Descriptive study. Place and Duration of the Study: PNS Shifa Hospital, Karachi, from July 2021 to February 2022. METHODOLOGY: All patients with COVID-19 in PNS Shifa Hospital were included in the study. The patients were divided into two groups, those who had COVID-19 during the 4th wave, and others who had it during the 5th wave. The patients' demographic details, comorbidities, vaccination status, initial presentation, the severity of disease, clinical progress, and final disposal from the hospital were noted. A comparison of severity, length of hospital stays, and mortality was made between the groups. RESULTS: There were 747 patients admitted during the Delta variant-dominated 4th wave of COVID-19, and 490 patients during the 5th wave, dominated by the Omicron variant. A vast majority of fifth-wave patients had the mild disease (87.35% vs. 49.39%), with significantly lower critical patients (2.04% vs. 7.09%). Vaccination was effective against both the Delta and the Omicron variant of the SARS-CoV-2 virus. The mean length of stay in the hospital for patients was significantly lesser (p < 0.001) during the 5th wave as compared to the 4th wave (6.43 ± 3.37 vs. 9.56 ± 5.45 days). The mortality rate was 7.09% among patients admitted in the 4th wave and 2.04% in the 5th wave. This difference was statistically significant (p < 0.001). CONCLUSION: The severity of disease, length of hospital stays, and mortality were higher in the Delta variant-dominated 4th wave of COVID-19 as compared to the Omicron variant-dominated 5th wave in Pakistan. The vaccination was effective against both the Delta and the Omicron variant of COVID 19, as the mortality rate among the vaccinated patients during the two waves was not significantly different. KEY WORDS: COVID 19, Vaccination, Disease outcome, Mortality.

Cloning and expression of an anti-cancerous cytokine: human IL-29 gene in Chlamydomonas reinhardtii.

Green algae, Chlamydomonas reinhardtii, with low cultivation cost, absence of endotoxins and insusceptibility to human pathogens is emerging as a potential system for the future production of recombinant proteins. The recent development of molecular tools enabling recombinant protein expression in algae chloroplast has provided new research and advance opportunities for developing low-cost therapeutic proteins. In the present study, algae chloroplast expression system was evaluated for the recombinant production of an anti-cancerous therapeutic protein, Interleukin 29 (IL29). The IL29 gene was cloned into algae chloroplast expression vector (pSRSapI). After the transformation, the positive clones were screened for homoplasmy and the presence of the IL29 gene by spot test and PCR analysis, respectively. The expressed SDS-PAGE and western blotting assay characterized IL-29. The algae expressed IL-29 was biologically active in an anti-proliferating bioassay using HepG2 cells. The results suggest that the Chlamydomonas reinhardtii expression system is convenient, low-cost, eco-friendly, and safe to express IL29.

Genetic and expression deregulation of immunoregulatory genes in rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases globally, and is an important public health concern, associating with early death and systemic complications. Although key development in RA treatment has already been made still RA affected individuals face comorbidity and disability. Therefore, there is a need to discover new risk factors in helping early diagnosis and treatment of RA. The present study is designed to assess the variations of Vitamin D receptor (VDR) and interleukin -6 (IL-6) in RA patients. Polymorphisms of said genes were calculated in 300 RA patients and 300 controls, using Tetra-ARMS polymerase chain reaction. Secondly, expression levels of selected genes were checked using the quantitative PCR (qPCR) and obtained results were evaluated using a different statistical test. Logistic regression analysis showed that frequency of mutant allele of VDR gene polymorphisms (rs11168268, OR = 4.84; 95% CI = 2.94-7.97; p = 0.0001; rs2248098, OR = 1.65; 95% CI = 1.07-2.54; p = 0.02) and IL-6 gene polymorphisms (rs184229712, OR = 2.47; 95% CI = 1.56-3.92, p = 0.0001; rs36215814, OR = 2.14; 95% CI = 1.30-3.53; p = 0.002) was observed significantly higher in RA patients vs controls. Expression analysis showed the significant upregulation of IL-6 (p < 0.0001) and downregulation of VDR gene (p < 0.0001) in RA cases vs controls. ROC curve analysis showed that downregulation of IL-6 (AUC = 0.86, p < 0.001) and upregulation of VDR (AUC = 0.77, p < 0.001) was act as the good diagnostic marker for detection/diagnosis of arthritis. In conclusions, data from the present study showed the significant involvement of VDR and IL-6 gene variations in RA pathogenesis.

Method for efficient soluble expression and purification of recombinant human interleukin-15.

Periplasmic expression of recombinant proteins ensures the production of biologically active proteins in a correctly folded state with several key advantages. This research focused on the in-frame cloning of rhIL-15 in pET-20 (+) vector with pelB-leader sequence to direct the protein to the bacterial periplasm. The target construct periplasmic expression was evaluated in four strains, BL21 (DE3), BL21 (DE3) pLysS, Rosetta 2 (DE3) and Rosetta-gami 2 (DE3). Soluble periplasmic expression of IL-15 was highest in Rosetta-gami 2 (DE3) followed by Rossetta 2 (DE3) whereas negligible expression was observed with rest of two expression host. Best expression clone was selected for purification by dye ligand affinity chromatography. Purified rhIL-15 was characterized by SDS-PAGE, Western blotting and SEC-HPLC. This is the first report of functional recombinant human interleukin-15 being expressed and purified with yield of 120 mg/L in the periplasmic space of E. coli.

Reasons Of Self-Discharge From Nursery Of A Tertiary Care Hospital.

BACKGROUND: Patients who leave against medical advice (LAMA) from a health facility is a recognized problem. In neonatology practice this issue is particularly sensitive as repercussions can be severe. The purpose of this study was to evaluate the factors influencing the decision of parents to self-discharge their babies against medical advice. METHODS: This descriptive case series was conducted in the Department of Neonatology, of the Children's Hospital and the Institute of Child Health, Lahore from January to June 2015. A total of 240 patients who self-discharged/were included. RESULTS: There were (59.6%) males and (40.4%) females with a male to female ratio of 2:1.5. Term babies constituted (67.9%), spontaneous vaginal deliveries (59.1%) and (55.8%) were delivered at hospitals. Seventy seven new-borns (32.2%) had birth asphyxia followed by neonatal sepsis (27.9%). Sixty four (64.5%) self-discharged within first week of admission. More babies were signed LAMA at week end (32.1%). Likewise (53.1%) babies were self- discharged during the night shift. Highest rate of LAMA was seen in parents belonging to low socioeconomic class (72.1%). Ninety eight parents (40.8%) had no formal education while well-educated parents were found to be 35 (14.6%). The commonest reason for selfdischarge was "perceived poor clinical outcome" (36.7%) by parents. CONCLUSIONS: Multiple factors were implicated in self-discharges from neonatology unit. Commonest reasons cited by parents were perception of poor clinical outcome and family pressures. Other contributory factors were male gender; those delivered vaginally, diagnosis of birth asphyxia, first week of life, at weekends and night hours. Low socioeconomic class and education of parents was also a major causative factor.

A 7-month-old infant with Cushing's disease.

Cushing's disease in children is not rare but in infants it is quite rare and an important medical condition needing proper line of investigations and management options. Craniopharyngioma as a cause of Cushing's disease is well reported and practical inference of the condition is of clinical importance. Craniopharyngioma generally affects children at 5 - 10 years of age and is rarely seen in infancy. It usually manifests as endocrinological deficits such as short stature, delayed puberty, and obesity. We report the case of a 7 months old infant who presented with obesity and Cushing's disease associated with craniopharyngioma.

A neonate with Poland-Mobius syndrome.

Poland-Mobius syndrome is a combination of two rare congenital syndromes characterized by congenital facial weakness, loss of abduction of eyes, limb defects, and absence or hypoplasia of the pectoral muscles. A patient with features of both the Mobius syndrome and the Poland syndrome is presented in this case report.

A child with Roberts syndrome.

Roberts syndrome is a genetically determined rare birth defect causing, skeletal deformities, particularly symmetrical limb reduction and craniofacial anomalies. For any child with limb and craniofacial bony malformations, this syndrome should be considered in the differentials. Although this syndrome represents only a small proportion of the total number of individuals with limb deficiency, it is important to be identified in order to give accurate genetic counselling including recurrence risk in siblings and possible prenatal diagnosis. This is the case report of a 22 days old male infant who presented with defective development of all four extremities and craniofacial abnormalities. The overall clinical and radiological features were suggestive of Roberts syndrome.

A case of polyglandular autoimmune syndrome type I with unusual presentation.

Eight years old girl presented with mucocutaneous candidiasis, nail dystrophy, twitching left half of face, progressively increasing generalized skin hyperpigmentation and hypopigmented patches over both shins. Her investigations revealed low intact PTH level, low serum cortisol, high ACTH, impaired glucose tolerance test and candidal onycomycosis. She was diagnosed as Polyglandular Autoimmune Syndrome (PGA) type I. She also developed idiopathic generalized epileptiform seizures and Alcaligenes faecalis septicemia not previously reported with PGA type I. The patient responded well to alphacalcidol, hydrocortisone, valproate sodium, topical antifungal and systemic antibiotics.