Study on Status of Serum Biochemical and Hematological Parameters in COVID-19 Positive Patients Attending a Tertiary Care Hospital.

Background Coronavirus disease 2019 (COVID-19) presents clinically a variety of pathological and clinical organ dysfunctions, ranging in severity from asymptomatic to fatal. The care and monitoring of COVID-19 patients may benefit from the use of biochemical and hematological markers. Objective To observe the alteration of serum biochemical and hematological parameters in COVID-19 positive patients, attending a Tertiary Care Hospital. Method A descriptive cross-sectional study was conducted on all COVID-19 positive patients attending Nobel Medical College Teaching Hospital, Biratnagar, Nepal from 15th December 2021 to 15th February 2022. The test results of different serum biochemical and hematological parameters done for these patients were recorded in clinical laboratory services and obtained retrospectively for the analysis. The data were entered in MS excel and analyzed by SPSS version 20. Result Out of 1537 COVID-11699 declared positive patients, 712 (46.32%) were male and 825 (53.68%) female. Mean age of COVID positive patients was 40.03±20.08 years. The level of serum SGOT, SGPT, ALP and GGT was significantly elevated in 39.9%, 42.8%, 32.3% and 47.2% of COVID positive patients respectively. Blood Urea, creatinine, uric acid and sugar level were significantly elevated in 63%, 56.1%, 33.1% and 47.6% patients respectively. The serum level of LDH, D-dimer, CRP and procalcitonin (PCT) were significantly increased in 52.1%, 75.9%, 71.6% and 61.2% of patients respectively. The serum value of total cholesterol, triglyceride, HDL and LDL were significantly lowered in 52.2%, 43.8%, 70.1% and 60.3% of patients respectively. RBC concentration and level of hemoglobin was reduced in 56.6% and 53.6% of COVID positive patients respectively whereas total leukocyte count was elevated in 80.7% with increase in neutrophil in 87.9% and decrease in lymphocyte in 79.4%. Conclusion A portion of COVID-19 positive patients showed drastically altered test results for various serum biochemical and hematological markers, although many of them had normal findings.

Hepatoprotective potential of ethanolic extract of Pandanus odoratissimus root against paracetamol-induced hepatotoxicity in rats.

BACKGROUND: Pandanus odoratissimus (Pandanaceae) is popular in the indigenous system of medicines like Ayurveda, Siddha, Unani and Homoeopathy. In the traditional system of medicine various plant parts such as leaves, root, flowers, and oils are used as anthelmintic, tonic, stomachic, digestive and in the treatment of jaundice and various liver disorders. OBJECTIVE: The aim was to investigate the hepatoprotective activity of ethanolic extract of the root of P. odoratissimus against paracetamol (PCM) induced hepatotoxicity in rats. MATERIALS AND METHODS: Hepatotoxicity was induced in male Wistar rat by PCM (2 g/kg b.w. p.o. for 7 days). The ethanolic extract of P. odoratissimus root was administered at the dose level of 200 mg/kg and 400 mg/kg b.w. orally for 7 days and silymarin (100 mg/kg b.w. p.o.) as standard drug was administered once daily for a week. The hepatoprotective effect of ethanolic extract was evaluated by assessment of biochemical parameters such as serum glutamic oxaloacetic transaminase, serum glutamic-pyruvic transaminase, serum alkaline phosphatase, total and direct bilirubin and triglycerides. Histopathological study of rat liver was also done. RESULTS: Experimental findings revealed that the extract at dose level of 200 mg/kg and 400 mg/kg of b.w. showed dose dependant hepatoprotective effect against PCM induced hepatotoxicity by significantly restoring the levels of serum enzymes to normal that was comparable to that of silymarin, but the extract at dose level of 400 mg/kg was found to be more potent when compared to that of 200 mg/kg. Besides, the results obtained from histopathological study also support the study. CONCLUSION: From the results, it can be concluded that ethanolic extract of the root of P. odoratissimus afforded significant protection against PCM induced hepatotoxicity in rats.

Antiinflammatory, Analgesic and Antipyretic Activities of Aerial Parts of Costus speciosus Koen.

In the present study, methanol extracts of Costus speciosus Koen. aerial parts were assessed for antiinflammatory, analgesic and antipyretic activities in experimental animals. The antiinflammatory activity of methanol extract of Costus speciosus (400 and 800 mg/kg, p.o.) was evaluated using carrageenan-induced paw oedema test. Analgesic effect was evaluated using acetic acid-induced writhing and Eddy's hot-plate models and antipyretic activity was assessed by Brewer's yeast-induced pyrexia in rats. The methanol extract of aerial parts of Costus speciosus in a dose of 400 and 800 mg/kg showed significant antiinflammatory activity (19.36 and 40.05% reduction) at 5 h postmedication. In analgesic models extract treated animals at (400 and 800 mg/kg) inhibited writhing's caused by acetic acid by 14.24 and 31.90%, respectively, and it also increased the latency period at both high and low doses which showed the mean reaction time at 16.60±0.355 s and 14.12±0.355 s, respectively, when compared to control in hot-plate test. It also reduces the rectal temperature of the animals at low and high doses significantly 37.03±0.108° and 36.63±0.098°, respectively, in Brewer's yeast induced pyrexia. The obtained results of the present investigation revealed that methanol extract of Costus speciosus has significant antiinflammatory, analgesic and antipyretic activities.

Changing tuberculosis trends in Nepal in the period 2001-2008.

To assess the trends on indicators of TB control in Nepal over a period from 2001-2008. Retrospective analysis of information from Annual Reports of NTP, Nepal from 2001-2008. The incidence of New Smear Positive (NSP) TB declined from 58.9 in 2001 to 53.4 in 2006 per 100000 populations then reversed in the period 2006-2008. This TB incidence decreased in males and the age group <45 years (except 0-14 years). The notification rate of all cases of TB declined by 3 % overall over the entire period from 2001 to 2008. Mortality among smear negative and extra pulmonary declined significantly. The failure rate and defaulter rate were declined significantly and the case detection rate (CDR) was increased significantly within the study period. Increasing trend in CDR, Treatment success rate and decreasing trend in failure rate, defaulter rate are the evidence of progress of NTP, in Nepal. Since there is reversal of incidence of NSP from 2006, a detailed analysis of existing TB control measures is required. If the success is continued and quality care is provided as per International Standard of TB Care, the Millennium Development Goals will be an achievable target.

First national tuberculin survey in Nepal.

SETTING: The size of the tuberculosis (TB) problem in Nepal is unknown, as no national tuberculin or TB prevalence survey has yet been performed. OBJECTIVE: To assess the prevalence of TB infection and the annual risk of TB infection (ARTI) in primary schoolchildren in the three ecological zones (mountains, hills and terai) and Kathmandu valley. DESIGN: A representative sample of primary schoolchildren were tuberculin skin tested using the Mantoux method. The data were analysed using cut-off levels to define infection and by the mirror method. RESULTS: Of 19577 children registered, 17260 (88.2%) were available for analysis. Seventy-eight per cent had a visible bacille Calmette-Guérin scar. The best estimate of the prevalence of TB infection was 7.0% (95%CI 4.2-9.7), with an ARTI of 0.86% (95%CI 0.49-1.23) using the mirror method, with a mode at 16 mm. Although the ARTI was higher in Kathmandu and the mountains compared to the hills and terai, the difference between the areas was not significant. CONCLUSION: The ARTI in Nepal is lower than previous estimates, indicating a decrease in transmission or overestimation of previous estimates. To obtain information about the trend of the ARTI in Nepal, the survey needs to be repeated in 5 to 7 years.

SV40-Mediated immortalization.

Human diploid cells have a limited life span, ending in replicative senescence, in contrast to cell lines derived from tumors, which show an indefinite life span and are immortal, suggesting that replicative senescence is a tumor suppression mechanism. We have utilized introduction of SV40 sequences to develop matched sets of nonimmortal and immortal cell lines to help dissect the mechanism of immortalization and have found that it has multiple facets, involving both SV40-dependent and -independent aspects. These studies have led to the identification of a novel growth suppressor gene (SEN6) as well as providing a model system for the study of cellular aging, apoptosis, and telomere stabilization among other things. It is anticipated that SV40-transformed cells will continue to provide a very useful experimental system leading to insights into the behavior of cells with altered expression of oncogenes and growth suppressor gene products.

SV40-mediated transformation and immortalization of human cells.

SV40 infection of human cells results in both transformation and lytic infection. We have used origin-defective viral mutants which are unable to replicate in permissive cells to help analysis of transformation. Expression of large T antigen (T ag) and small t antigen results in the altered growth phenotypes characteristic of transformation in other species. Human diploid fibroblasts (HF) have a limited lifespan and undergo senescence; T ag results in extension of lifespan but only in rare cases are the cells capable of continuous growth and are immortal. We have developed matched sets of non-immortal and immortal transformed HF for assessment of the steps required for immortalization. Results are summarized to characterize both T-dependent and T-independent functions. A novel growth suppressor gene SEN6 has been identified, the inactivation of which is required for immortalization; it may also serve as a marker to distinguish cells in which SV40 is replicating from those in which it is responsible for tumorigenesis.

SEN6, a locus for SV40-mediated immortalization of human cells, maps to 6q26-27.

Normal cells show a limited lifespan in culture and the phenotype of cellular senescence. Tumors and tumor cell lines have typically overcome this form of growth suppression and grow continuously as immortal cell lines in culture. We have exploited the DNA virus SV40 to study the mechanism by which human fibroblasts overcome senescence and become immortal. Multiple steps have now been identified, including inactivation of cellular growth suppressors through direct interaction with SV40 large T antigen and through mutation of a gene on chromosome 6 (designated SEN6). In this study, we sublocalize the site of SEN6 to 6q26-27 based on molecular genetic analysis. Twelve SV40-immortalized fibroblast cell lines share a deletion in this area based on assessment for loss of heterozygostiy (LOH) for seven informative markers on 6q. Two immortal cell lines (AR5 and HALneo) appeared to have retained separate single copies of chromosome 6 despite the fact that they are both derived from the same preimmortal SV40-transformant and should share the same mutated allele of SEN6 (Hubbard-Smith et al., 1992). Detailed analysis by polymerase chain reaction, restriction fragment length polymorphism and fluorescence in situ hybridization shows, however, that although they differ for 17 markers from the centromere to 6q26, they share AR5 derived sequences (eight markers) distal to 6q26 including the minimal deletion region, further supporting the assignment of SEN6 to this region. Since human tumors including non-Hodgkins lymphoma, mammary carcinoma and ovarian carcinoma show LOH in 6q26-27, inactivation of SEN6 may be responsible for immortalization of these tumors as well.

SV40-mediated immortalization of human fibroblasts.

We have identified a multistep mechanism by which the DNA virus SV40 overcomes cellular senescence. Expression of SV40 T antigen is required for both transient extension of life span and unlimited life span or immortalization. These effects are mediated through inactivation of function of growth suppressors pRB and p53 via complex formation with T antigen. However, immortalization additionally requires inactivation of a novel growth suppressor gene, which has recently been identified to be on the distal portion of the long arm of chromosome 6, designated SEN6. We propose that SEN6 is responsible for cellular senescence in fibroblasts and other cells.

Senescence of immortal human fibroblasts by the introduction of normal human chromosome 6.

In these studies we show that introduction of a normal human chromosome 6 or 6q can suppress the immortal phenotype of simian virus 40-transformed human fibroblasts (SV/HF). Normal human fibroblasts have a limited life span in culture. Immortal clones of SV/HF displayed nonrandom rearrangements in chromosome 6. Single human chromosomes present in mouse/human monochromosomal hybrids were introduced into SV/HF via microcell fusion and maintained by selection for a dominant selectable marker gpt, previously integrated into the human chromosome. Clones of SV/HF cells bearing chromosome 6 displayed limited potential for cell division and morphological characteristics of senescent cells. The loss of chromosome 6 from the suppressed clones correlated with the reappearance of immortal clones. Introduced chromosome 6 in the senescing cells was distinguished from those of parental cells by the analysis for DNA sequences specific for the donor chromosome. Our results further show that suppression of immortal phenotype in SV/HF is specific to chromosome 6. Introduction of individual human chromosomes 2, 8, or 19 did not impart cellular senescence in SV/HF. In addition, introduction of chromosome 6 into human glioblastoma cells did not lead to senescence. Based upon these results we propose that at least one of the genes (SEN6) for cellular senescence in human fibroblasts is present on the long arm of chromosome 6.

Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway.

The wild-type p53 gene product plays an important role in the control of cell proliferation, differentiation, and survival. Altered function is frequently associated with changes in p53 stability. We have studied the role of the ubiquitination pathway in the degradation of p53, utilizing a temperature-sensitive mutant, ts20, derived from the mouse cell line BALB/c 3T3. We found that wild-type p53 accumulates markedly because of decreased breakdown when cells are shifted to the restrictive temperature. Introduction of sequences encoding the human ubiquitin-activating enzyme E1 corrects the temperature sensitivity defect in ts20 and prevents accumulation of p53. The data therefore strongly indicate that wild-type p53 is degraded intracellularly by the ubiquitin-mediated proteolytic pathway.

Altered chromosome 6 in immortal human fibroblasts.

Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene.

Retinoblastoma protein and simian virus 40-dependent immortalization of human fibroblasts.

Transformation and immortalization of human diploid fibroblasts by simian virus 40 (SV40) is at least a two-stage process, since transformants have a limited lifespan in culture. We have isolated immortalized derivatives (AR5 and HAL) from transformants generated with an origin-defective SV40 genome encoding a heat-labile large T protein (T antigen) and reported that both preimmortal and immortal transformants are continuously dependent on T antigen function for growth as determined by temperature shift experiments. In this study, we demonstrate complex formation between T antigen and the retinoblastoma susceptibility gene product (Rb) at 35 degrees C and observed a reduction in complexes under conditions of loss of T antigen function and growth inhibition at 39 degrees C. Viral oncogenes (polyomavirus large T protein and adenovirus E1A 12S protein) known to bind Rb were introduced into AR5 and HAL cells, both stably by gene transfer and transiently by virus vectors. Such double transformants are still unable to proliferate at 39 degrees C, although complex formation with the newly introduced oncogenes was demonstrated. We suggest that T antigen interacts with other cellular processes in addition to Rb to transform and immortalize human cells in culture. Our finding that p53-T antigen complexes are also temperature dependent in AR5 and HAL cells could provide such an additional mechanism.

Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.

Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension.

A method for fusion of protoplasts bearing amplified plasmids and human diploid fibroblasts or other cell types in suspension is described. Transient expression of plasmid-encoded proteins occurs in up to 50% of the human cells, as demonstrated for simian virus 40 T antigen by immunofluorescence and the Escherichia coli xanthine-guanine phosphoribosyl transferase by autoradiography. In contrast, frequencies of stable transformants were similar to those obtained by the CaPO4 coprecipitation technique. However, experiments with both methods involving the recombinant pRSVneo (in which the Rous sarcoma virus long terminal repeat regulates expression of the antibiotic-inactivating aminoglycoside phosphotransferase) revealed a much higher frequency of colonies in G418 selective medium with constructions in which the early region of simian virus 40 DNA was present as well. We propose a role for the simian virus 40 T antigen in enhancing stable transformation in this system.

Temperature-sensitive mutants of BALB/3T3 cells. III. Hybrids between ts2 and other mouse mutant cells affected in DNA synthesis and correction of ts2 defect by human X chromosome.

Complementation studies were performed with ts2, a mouse 3T3 cell mutant temperature sensitive (ts) for cell and viral DNA synthesis. The ts phenotype is corrected by non-ts mouse or human cells and a non-DNA ts mutant. This gene had been localized to a region on the human X chromosome near the HPRT locus based on isozyme and karyotype analysis of hybrids. Unusually rapid loss and fragmentation of human chromosomes occurs in hybrids with ts2. Hybrids between ts2 and other DNA- ts mutants of mouse cells did not show complementation of the growth phenotype.

Expression of transformation in cell hybrids. II. Nonsuppression of the transformed phenotype in hybrids between a chemically transformed and nontransformed derivatives of Balb/3T3.

Hybrid clones derived from a nitrosocarbaryl-transformed Balb/3T3 cell line, Clone H, and a nontransformed cell line THO2 resemble the transformed parent in the clone morphology, higher saturation density, colony formation in medium with reduced serum concentration, growth in agarose and ability to form clones on Balb/3T3 monolayer. Results are discussed in the framework of genetic models which permit or require dominant mutations for the expression of transformed phenotype.