Higher COVID-19 pneumonia risk associated with anti-IFN-α than with anti-IFN-ω auto-Abs in children.

We found that 19 (10.4%) of 183 unvaccinated children hospitalized for COVID-19 pneumonia had autoantibodies (auto-Abs) neutralizing type I IFNs (IFN-α2 in 10 patients: IFN-α2 only in three, IFN-α2 plus IFN-ω in five, and IFN-α2, IFN-ω plus IFN-ß in two; IFN-ω only in nine patients). Seven children (3.8%) had Abs neutralizing at least 10 ng/ml of one IFN, whereas the other 12 (6.6%) had Abs neutralizing only 100 pg/ml. The auto-Abs neutralized both unglycosylated and glycosylated IFNs. We also detected auto-Abs neutralizing 100 pg/ml IFN-α2 in 4 of 2,267 uninfected children (0.2%) and auto-Abs neutralizing IFN-ω in 45 children (2%). The odds ratios (ORs) for life-threatening COVID-19 pneumonia were, therefore, higher for auto-Abs neutralizing IFN-α2 only (OR [95% CI] = 67.6 [5.7-9,196.6]) than for auto-Abs neutralizing IFN-ω only (OR [95% CI] = 2.6 [1.2-5.3]). ORs were also higher for auto-Abs neutralizing high concentrations (OR [95% CI] = 12.9 [4.6-35.9]) than for those neutralizing low concentrations (OR [95% CI] = 5.5 [3.1-9.6]) of IFN-ω and/or IFN-α2.

TULP3 NLS inhibition: an in silico study to hamper cargo transport to nucleus.

TULP3 is involved in cell regulation pathways including transcription and signal transduction. In some pathological states like in cancers, increased level of TULP3 has been observed so it can serve as a potential target to hamper the activation of those pathways. We propose a novel idea of inhibiting nuclear localization signal (NLS) to interrupt nuclear translocation of TULP3 so that the downstream activations of pathways are blocked. In current in silico study, 3D structure of TULP3 was modeled using 8 different tools including I-TASSER, CABS-FOLD, Phyre2, PSIPRED, RaptorX, Robetta, Rosetta and Prime by Schrödinger. Best structure was selected after quality evaluation by SAVES and implied for the investigation of NLS sequence. Mapped NLS sequence was further used to dock with natural ligand importin-α as control docking to validate the NLS sequence as binding site. After docking and molecular dynamics (MD) simulation validation, these residues were used as binding side for subsequent docking studies. 70 alkaloids were selected after intensive literature survey and were virtually docked with NLS sequence where natural ligand importin-α is supposed to be bound. This study demonstrates the virtual inhibition of NLS sequence so that it paves a way for future in-vivo studies to use NLS as a new drug target for cancer therapeutics.Communicated by Ramaswamy H. Sarma.

Antibiotic Resistance Pattern Of Acinetobacter Baumannii Isolated From Bacteremia Patients In Pakistan.

BACKGROUND: Acinetobacter baumannii causes a number of life-threatening infections in Hospitalized patients attributed to its ability to develop resistance against multiple antibiotics. The current scrutinisation is aimed to observe the prevalence and antibiotic resistance profile of A. baumannii strains isolated from blood of tertiary care Hospitalized patients in Lahore, Pakistan. METHODS: This research is a retrospective study conducted over a period of one year where 1864 blood samples were collected from both male and female patients with septicaemia. Total 156 A. baumannii species were identified by conventional method and their antimicrobial resistance pattern against 22 antimicrobials (representing all known classes of antibiotics) was evaluated by Kirby Bauer disc diffusion method. MICs of colistin, polymyxin B and vancomycin against A. baumannii were calculated by E test and broth dilution method. RESULTS: More males (n=97, 62%) were found infected than females (n=59, 38%). The spreading rate of A. baumannii was highest (n=101, 65%) in patients of age ≤20 years, and lowest (n=12, 7%) in the patients with the age of 41-60 years. Most of the strains of A. baumannii (n=118, 75.6%) were found to be MDR (multi drug resistant), 37 (23.7%) strains were XDR (Extensively drug-resistant) and only 1 (0.05%) strain was PDR (pandrug resistant). All the strains were sensitive to minocycline and tigecycline whereas highest non-susceptibility (n=144, 92%) was seen against Ampicillin-Sulbactam. Most of the strains demonstrated resistance against carbapenem and cephalosporin beckoning that A. baumannii can no longer be considered for salvage therapy by carbapenem. MICs of colistin, polymyxin B and vancomycin against A. baumannii divulged polymixin B as the most effective drug. CONCLUSIONS: Use of wide range of drugs has made A. baumannii multidrug resistant. Colistin, polymyxin B and vancomycin are the preferable drugs for the treatment of A. baumannii infections.

Interferon α2-Thymosin α1 Fusion Protein (IFNα2-Tα1): A Genetically Engineered Fusion Protein with Enhanced Anticancer and Antiviral Effect.

Human interferon α2 (IFNα2) and thymosin α1 (Tα1) are therapeutic proteins used for the treatment of viral infections and different types of cancer. Both IFNα2 and Tα1 show a synergic effect in their activities when used in combination. Furthermore, the therapeutic fusion proteins produced through the genetic fusion of two genes can exhibit several therapeutic functions in one molecule. In this study, we determined the anticancer and antiviral effect of human interferon α2-thymosin α1 fusion protein (IFNα2-Tα1) produced in our laboratory for the first time. The cytotoxic and genotoxic effect of IFNα2-Tα1 was evaluated in HepG2 and MDA-MB-231 cells. The in vitro assays confirmed that IFNα2-Tα1 inhibited the growth of cells more effectively than IFNα2 alone and showed an elevated genotoxic effect. The expression of proapoptotic genes was also significantly enhanced in IFNα2-Tα1-treated cells compared to IFNα2-treated cells. Furthermore, the HCV RNA level was significantly reduced in IFNα2-Tα1-treated HCV-infected Huh7 cells compared to IFNα2-treated cells. The quantitative PCR analysis showed that the expression of various genes, the products of which inhibit HCV replication, was significantly enhanced in IFNα2-Tα1-treated cells compared to IFNα2-treated cells. Our findings demonstrate that IFNα2-Tα1 is more effective than single IFNα2 as an anticancer and antiviral agent.

High yield expression, characterization, and biological activity of IFNα2-Tα1 fusion protein.

The use of interferon α-2 in combination with thymosin α-1 shows higher anti-cancer effect in comparison when both are used individually because of their synergistic effects. In this study we produced an important human interferon α-2-thymosin α-1 (IFNα2-Tα1) fusion protein with probable pharmaceutical properties coupled to its high-level expression, characterization, and study of its biological activity. The IFNα2-Tα1 fusion gene was constructed by over-lap extension PCR and expressed in Escherichia coli expression system. The expression of IFNα2-Tα1 fusion protein was optimized to higher level and its maximum expression was obtained in modified terrific broth medium when lactose was used as inducer. The fusion protein was refolded into its native biologically active form with maximum yield of 83.14% followed by purification with ∼98% purity and 69% final yield. A band of purified IFNα2-Tα1 fusion protein equal to ∼23 kDa was observed on 12 % SDS-PAGE gel. The integrity of IFNα2-Tα1 fusion protein was confirmed by western blot analysis and secondary structure was assessed by CD spectroscopy. When IFNα2-Tα1 fusion protein was subjected to its biological activity analysis it was observed that it exhibits both IFNα2 & Tα1 activities as well as significantly higher anticancer activity as compared to IFNα-2 alone.

Development of latent Interferon alpha 2b as a safe therapeutic for treatment of Hepatitis C virus infection.

Interferon therapy for the treatment of hepatitis C virus infection has very limited clinical application due to short serum half-life and side effects of therapy in systemic route of administration. In the present study, we have focused to improve the interferon therapy by overcoming the limitation of side effects. We hypothesized that latent interferon alpha 2b (IFNα2b) produced by fusion of Latency associated protein (LAP) domain of TGFß and IFNα2b having HCV NS3 protease cleavage site as linker that will be activated only at target site (liver) by viral protease (HCV NS3 protease) present on the surface of infected cells. The fusion proteins were expressed in pichia pastoris as homodimer and cleaved by recombinant HCV NS3 protease in vitro into two fragments corresponding to the IFNα-2b and LAP respectively. The latency of chimeric proteins and biological activity after treatment with HCV NS3 protease was assessed by cytopathic effect inhibition assay in A594 cells infected with encephalomyocarditis virus (EMCV) and reduction in HCV viral load in Huh7 cells. The HCV NS3 protease was present on the surface of HCV replicating Huh7 cells in amount that activated half of the effective concentration (EC50) of latent IFNα2b fusion protein. As free circulating HCV NS3 protease was not detected in sera from chronic HCV patients and in vitro cleavage of intact latent IFNα2b fusion protein was not observed with peripheral blood mononuclear cells (PBMCs) isolated from chronic HCV patients, thus there are less likely chances of activation and off target binding of latent IFNα2b to show side effects during systemic route of administration. Therefore, most of the side effects of interferon can be overwhelmed at the cost of 50% reduced biological activity. Thus, the use of latent IFNα2b can be considered again as an option for treatment of HCV infection in combination with direct acting antivirals rather than alone with improved safety profile.

The IFN-λ Genetic Polymorphism Association With the Viral Clearance Induced by Hepatitis C Virus Treatment in Pakistani Patients.

BACKGROUND: Polymorphisms in the interferon λ (INF λ) genes on chromosome 19 have been associated with clearance of hepatitis C virus (HCV) induced by interferon and ribavirin therapy however there is no such data available for Pakistani patients with HCV infection. OBJECTIVES: In this study, the effects of single nucleotide polymorphisms (SNPs) have been investigated in response to treatment with interferon-α and ribavirin in a cohort of 75 HCV genotype 3a patients. PATIENTS AND METHODS: A total number of 50 SNPs from the Interferon λ region on chromosome 19 were genotyped to investigate allelic associations with the treatment response in HCV type 3a patients. Thirteen SNPs were associated with HCV clearance, with the most significant alleles being RS8109886 (Fisher's P = 0.0001), RS8113007 (Fisher's P = 0.0001) and RS12979860 (Fisher's P = 0.0002). RESULTS: These SNPs were found to be the most suitable SNPs for predicting treatment response in the present study. These findings support those reported previously. This could be used to improve HCV treatment strategies and suggest that Pakistani patients should be genotyped for the relevant SNPs to identify the patients who are more likely to respond to interferon and ribavirin therapy. CONCLUSIONS: This therapy is costly and can be accompanied by several adverse side-effects, hence pre-treatment prediction of patients who are most likely to benefit would have both economic and patient benefits in the long term.