Do extraglomerular microvasculature and mesenchymal interstitial cell proliferation indicate a stable course of lupus nephritis?

Background: Lupus Nephritis (LN) is a major and frequent manifestation of Systemic Lupus Erythematosus (SLE), an autoimmune disease. Renal biopsy has a pivotal role in the diagnosis, prognosis, and management of the LN. The aim of this study was to count the mesenchymal interstitial cells utilizing CD34 immunohistochemistry (IHC) and morphometric analysis, correlate them with clinical parameters, class, activity, and chronicity indices and see if it can predict the course of the disease. Methods: A total of 32 renal biopsy blocks were analyzed by H&E stain, special stains, and CD34 IHC. Microvasculature density and interstitial stem cells were highlighted by CD34. These were then counted using a previously standardized computerized digital photomicrograph system (Dewinter Optical Inc) and manual count, respectively. Results: Out of the 32 cases, Lupus class 3 comprised of 11 (34.38%) cases, class 4 comprised of 16 (50%) cases, and mixed class 4 + 5 had 5 (15.62%) cases. It was found that CD34 expression in the microvasculature (for both microvascular density and mean vascular lumen diameter) decreased in patients of Lupus Nephritis with higher disease activity (p < 0.05). Although not statistically significant, the number of interstitial stem cells increased with lower disease activity. A statistical significance was found between serum total protein, serum albumin, and serum creatinine among the three groups of LN. Conclusion: Immunohistochemical staining of renal biopsy with CD34 may be used as a surrogate marker of disease activity in Lupus Nephritis patients.

Clinical Profile, Treatment, and Outcome of Patients with Secondary Hemophagocytic Lymphohistiocytosis in Critically Ill Patients: A Prospective Observational Study.

Introduction: The objective of the study was to evaluate the clinical profile and outcome of patients with secondary hemophagocytic lymphohistiocytosis (HLH) in critically ill patients. Materials and methods: A prospective observational study was conducted where critically ill adult patients presenting with fever and bicytopenia were evaluated according to the HLH-2004 diagnostic criteria for the presence of secondary HLH. The underlying trigger, clinical profile, treatment, and outcome of patients with HLH were analyzed. Results: Of the 76 critically ill patients with fever and bicytopenia, 33 (43%) patients were diagnosed with HLH. The following triggers for HLH were identified: bacterial infections (23%), fungal infections (10%), viral infections (10%), parasitic infections (10%), autoimmune diseases (13%), and malignancy (8%). A total of 78% of the HLH cases received steroids, but the use of steroids was not associated with improvement in mortality. Conclusion: There is a high prevalence of HLH in patients presenting with fever and bicytopenia in critically ill adult patients. Infections were identified as the most common trigger of HLH. How to cite this article: Fazal F, Gupta N, Soneja M, Mitra DK, Satpathy G, Panda SK, et al. Clinical Profile, Treatment, and Outcome of Patients with Secondary Hemophagocytic Lymphohistiocytosis in Critically Ill Patients: A Prospective Observational Study. Indian J Crit Care Med 2022;26(5):564-567.

Enhanced exudation of malate in the rhizosphere due to AtALMT1 overexpression in blackgram (Vigna mungo L.) confers increased aluminium tolerance.

Worldwide, 50% of soil is acidic, which induces aluminium (Al) toxicity in plants, as the phyto-availability of Al3+ increases in acidic soil. Plants responds to Al3+ toxicity by exuding organic acids into the rhizosphere. The organic acid responsible for Al3+ stress response varies from species to species, which in the case of blackgram (Vigna mungo L.) is citrate. In blackgram, an Arabidopsis malate transporter, AtALMT1, was overexpressed with the motive of inducing enhanced exudation of malate. Transgenics were generated using cotyledon node explants through Agrobacterium tumefaciens-mediated transformation. The putative transgenics were initially screened by AtALMT1-specific genomic DNA PCR, followed by quantitative PCR. Two independent transgenic events were identified and functionally characterized in the T3 generation. The transgenic lines, Line 1 and 2, showed better root growth, relative water content and chlorophyll content under Al3+ stress. Both lines also accounted for less oxidative damage, due to reduced accumulation of ROS molecules. Photosynthetic efficiency, as measured in terms of Fv /Fm , NPQ and Y(II), increased when compared to the wild type (WT). Relative expression of genes (VmSTOP1, VmALS3, VmMATE) responsible for Al3+ stress response in blackgram showed that overexpression of a malate transporter did not have any effect on their expression. Malate exudation increased whereas citrate exudation did not show any divergence from the WT. A pot stress assay found that the transgenics showed better adaptation to acidic soil. This report demonstrates that the overexpression of a malate transporter in a non-malate exuding species improves adaptation to Al3+ toxicity in acidic soil without effecting its stress response mechanism.

Increased Fas ligand expression of peripheral B-1 cells correlated with CD4+ T-cell apoptosis in filarial-infected patients.

Cellular hyporesponsiveness observed during helminth infections is attributed to factors such as antigen-presenting cells (APC) dysfunction, increased interleukin-10(IL-10), regulatory T cells and induction of CD4+ T (Th)-cell apoptosis. Increased Fas ligand (FasL) expression on the surface of B-1 cells and induction of apoptosis of Th cells by FasL-expressing B-1 cells due to helminth infection were demonstrated in murine model of helminth infection where as profile of FasL expression, Th-cell apoptosis and correlation between these two populations of cells in clinical filariasis remain unknown. In this study, we have scored the profile of apoptotic Th-cell population and FasL-expressing B-1 cells in different clinical categories of filariasis. The peripheral apoptotic T-helper cells were significantly increased in filarial patients compared to endemic controls. Expression of FasL on the surface of peripheral B-1 cells increased in filarial patients and positively correlated with peripheral apoptotic T-helper cells indicating FasL-expressing B-1 cells may be one of the important mediators of Th-cell apoptosis and immune anergy during filarial pathology.

Development and validation of a stability-indicating RP-HPLC method for estimation of atazanavir sulfate in bulk.

A stability-indicating reverse phase-high performance liquid chromatography (RP-HPLC) method was developed and validated for the determination of atazanavir sulfate in tablet dosage forms using C18 column Phenomenix (250 mm×4.6 mm, 5 µm) with a mobile phase consisting of 900 mL of HPLC grade methanol and 100 mL of water of HPLC grade. The pH was adjusted to 3.55 with acetic acid. The mobile phase was sonicated for 10 min and filtered through a 0.45 µm membrane filter at a flow rate of 0.5 mL/min. The detection was carried out at 249 nm and retention time of atazanavir sulfate was found to be 8.323 min. Linearity was observed from 10 to 90 µg/mL (coefficient of determination R2 was 0.999) with equation, y=23.427x+37.732. Atazanavir sulfate was subjected to stress conditions including acidic, alkaline, oxidation, photolysis and thermal degradation, and the results showed that it was more sensitive towards acidic degradation. The method was validated as per ICH guidelines.

Assessment of Her-2/neu status using immunocytochemistry and fluorescence in situ hybridization on fine-needle aspiration cytology smears: experience from a tertiary care centre in India.

Breast carcinoma shows amplification/overexpression of Her-2/neu in ∼20-30% of cases. The determination of Her-2/neu expression accurately is vital in clinical practice as it has significant predictive value and eligibility for anti Her-2/neu therapy. Amplification and overexpression of Her-2/neu gene is traditionally identified by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) on tissue sections; only a few studies have evaluated feasibility of these techniques on cytological smears. One hundred cases of breast cancer with fine-needle aspiration cytology (FNAC) samples and corresponding surgically resected specimen were selected. Immunocytochemistry (ICC) and FISH for Her-2/neu was done on FNA smears, whereas IHC was performed on corresponding tissue sections. Diagnostic accuracy of ICC was 99% when compared with IHC. Comparison of FISH results with IHC showed 100% concordance. Unlike many centers in West, FNAC is still routinely performed in developing countries like India where vast majority of breast cancer cases present as palpable lumps. The high rates of accuracy of ICC and FISH for Her-2/neu detection can make FNAC a relevant first line of investigation as a cost effective model with a rapid turn-around time, providing complete information necessary for initial management of breast cancer patients.

Formulation, in vitro drug release study and anticancer activity of 5-fluorouracil loaded gellan gum microbeads.

5-Fluorouracil loaded calcium-zinc-gellan and calcium-zinc-gellan-ethyl cellulose microbeads were successfully prepared by simple ionotropic gelation and oil in water ionotropic gelation technique, respectively. Prepared microbeads were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and evaluated for particle size, drug content, encapsulation efficiency, drug release and cell cytotoxicity study. Microbeads formed were spherical with rough surface. As concentration of gellan and ethyl cellulose has increased encapsulation efficiency, particle size and sustained drug release effect also increased. The release of 5-fluorouracil from microbeads has followed Hixson Crowell model suggesting the mechanism of drug release as dissolution controlled. Cytotoxicity analysis on HT-29 human colon cancer cell lines indicated that 5-FU loaded gellan gum/gellan in combination with ethyl cellulose microbeads leads to sustained releases of drug and thus delayed apoptosis over a long period of time. The formulation with drug:gellan:ethyl cellulose ratio 2.5:7.5:1 was found to be more effectual in terms of sustained drug release activity in addition to anti-cancer activity.

Extraction of nickel by microbial reduction of lateritic chromite overburden of Sukinda, India.

Microbial extraction of nickel from lateritic chromite overburden (COB), Sukinda by Acidithiobacillus ferrooxidans has been investigated in this work. In anoxic environment, A. ferrooxidans reduced the ferric iron in goethite [Fe(O)OH] mineral of COB by using elemental sulphur as electron donor. Nickel embedded in the complex goethite matrix of COB was successfully recovered by cumulative action of sulphuric acid, generated by oxidation of elemental sulphur and reduction of ferric iron in goethite matrix by A. ferrooxidans. Forty one percent of the nickel present in COB was extracted in a 3 L scale bioreactor (pH of 1.8 ± 0.05, temperature of 28 ± 2°C) maintained in anoxic environment. In contrast, only 11% of the nickel present in COB was extracted with continuous supply of air to the bioreactor keeping all the parameters unchanged. Kinetics study of anoxic microbial processing of COB revealed that the chemical reaction rate control model fits to the rate of nickel dissolution (R(2)=0.975).

Infantile systemic hyalinosis associated with a putative splice-site mutation in the ANTXR2 gene.

Infantile systemic hyalinosis (ISH) is a rare autosomal recessive genetic disorder characterized by dermal and subcutaneous fibromatosis, joint contractures and bone deformities. The condition usually presents at birth, resulting in death in infancy. ISH is caused by mutations in the anthrax toxin receptor 2 gene, ANTXR2, also known as CMG2. We report an Indian child with ISH in whom we identified a homozygous acceptor splice site mutation, IVS2-4G>A. In silico analysis of this sequence showed that it changed predicted cryptic splicing, leading to out-of-frame transcripts and little, if any, functional protein. Mutations in the ANTXR2 gene can also cause juvenile hyaline fibromatosis (JHF). Although there are currently no effective treatments for ISH or JHF, identification of pathogenetic mutations in the ANTXR2 gene makes DNA-based prenatal diagnosis feasible for subsequent pregnancies.

Excess copper induced oxidative stress and response of antioxidants in rice.

To investigate the effects of copper (Cu), rice plant (Oryza sativa. L. var. MSE-9) was treated with different Cu concentrations (0, 10, 50 and 100 µM) for 5 days in hydroponic condition. Gradual decrease in shoot and root growth was observed with the increase of Cu concentration and duration of treatment where maximum inhibition was recorded in root growth. Cu was readily absorbed by the plant though the maximum accumulation was found in root than shoot. Hydrogen peroxide (H(2)O(2)) production and lipid peroxidation were found increased with the elevated Cu concentration indicating excess Cu induced oxidative stress. Antioxidant enzymes superoxide dismutase (SOD), guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) and glutathione reductase (GR) were effectively generated at the elevated concentrations of Cu though catalase (CAT) did not show significant variation with respect to control. Ascorbate (ASH), glutathione (GSH) and proline contents were also increased in all the Cu treated plants compared with the control. SOD isoenzyme was greatly affected by higher concentration of Cu and it was consistent with the changes of the activity assayed in solution. The present study confirmed that excess Cu inhibits growth, induced oxidative stress by inducing ROS formation while the stimulated antioxidative system appears adaptive response of rice plant against Cu induced oxidative stress. Moreover proline accumulation in Cu stress plant seems to provide additional defense against the oxidative stress.

Immunohistochemistry for the diagnosis of hepatitis E virus infection.

Hepatitis E virus (HEV) is an emerging pathogen and the most common cause of acute viral hepatitis all over the world. We describe here an immunohistochemical method for the detection of HEV antigens (pORF2 and pORF3) in formalin-fixed, paraffin-embedded liver tissues using monoclonal antibodies raised against two of the virus proteins (pORF2 and pORF3). We analysed their specificity and sensitivity in comparison with serology and nucleic acid detection in cases of acute liver failure (ALF). We used this test on 30 liver biopsies collected post-mortem from the patients of ALF caused by HEV infection. These cases were selected on the basis of positive results for enzyme immunoassay (IgM anti-HEV). Of the 30 cases taken from the archives of the Department of Pathology, the antibodies successfully stained all. However, only 25 serum samples (83.3%) of these were positive for HEV RNA. Fifteen controls used (Five noninfected liver tissues, five HBV- and five hepatitis C virus-infected liver tissues) were all negative. The immunohistochemical assay described here may prove a valuable tool for the detection of HEV infection in biopsy, autopsy and explant liver tissues and can serve as a link along with other available tests to delineate the extent of HEV-associated problem worldwide.

Study of cellular immune response against Hepatitis E virus (HEV).

Hepatitis E virus infection (HEV) is a major cause of acute viral hepatitis in the developing world. The immunopathology of HEV infections has not yet been elucidated. The virus is noncytopathic, and therefore, liver injury may be attributed to immune-mediated damage by cytotoxic T cells and natural killer cells. Therefore, we studied the nature of immune cells involved in HEV-induced liver damage using immunohistochemistry in liver biopsies taken from patients with HEV-induced acute liver failure and demonstrated a significant infiltration of activated CD8(+) T cells containing granzymes. These findings suggest the possible involvement of cytotoxic T cells in disease pathogenesis during HEV infection.

Clinical relevance of HBV DNA load in patients with chronic hepatitis B infection.

BACKGROUND: Hepatitis B virus (HBV) DNA detection and quantification are now playing an increasing role in the assessment of disease activity and response to therapy. However, viraemia levels which define various stages of HBV infection have not yet been established. AIM: To define viraemia levels which describe various stages of chronic hepatitis B virus infection. METHODS: In a retrospective study, stored sera samples of chronic hepatitis B virus (CHB) infected patients registered at AIIMS liver clinic, from January 1996 to June 2005 were subjected to competitive, quantitative PCR analysis. RESULTS: The median HBV DNA load was lowest among carriers and highest among patients with chronic hepatitis B [0 (0-8) vs. 7 (0-12) log10 copies/ml, respectively; p<0.05]. As compared to chronic hepatitis patients the DNA load was also lower among cirrhotics [7 (0-12) vs. 4.5 (0-8) log10 copies/ml, respectively; p<0.05] and hepatocellular cancer patients [ 7(0-12) vs. 0 (0-8) log10 copies/ml, respectively; p<0.05]. Patients with carriers had a DNA load which was significantly lower than e antigen negative CHB [0 (0-8) vs. 6 (0-10) log10 copies/ml; p<0.05] or e antigen positive CHB [0 (0-8) vs 8 (0-12) log10 copies/ml; p<0.05]. A threshold of 3.5 log10 copies/ml had sensitivity and specificity of 83% and 58% respectively in differentiating carriers from e antigen negative CHB. There was a strong positive correlation of HBV DNA load with inflammatory grade (R=0.334; p=0.0001), fibrosis stage (R=0.276; p=0.001) and ALT levels (R=0.378; p=0.0001). 82% (9/11) of those who lost e antigen had a decline in HBV DNA levels to <5 log10 copies/ml, whereas only 12.5% (1/8) of those who did not lose e antigen had a decline in DNA load below this level. CONCLUSIONS: HBV DNA viraemia levels correlate positively with the inflammatory grade, fibrosis stage and ALT levels. Most patients who loose e antigen have a decline in DNA load to below 5 log10 copies/ml. Further prospective studies employing repeated measurements are required to define a threshold to differentiate between HBV carriers and e antigen negative CHB.

Viral hepatitis in India.

Viral hepatitis is a major public health problem in India, which is hyperendemic for HAV and HEV. Seroprevalence studies reveal that 90%-100% of the population acquires anti-HAV antibody and becomes immune by adolescence. Many epidemics of HEV have been reported from India. HAV related liver disease is uncommon in India and occurs mainly in children. HEV is also the major cause of sporadic adult acute viral hepatitis and ALF. Pregnant women and patients with CLD constitute the high risk groups to contract HEV infection, and HEV-induced mortality among them is substantial, which underlines the need for preventive measures for such groups. Children with HAV and HEV coinfection are prone to develop ALF. India has intermediate HBV endemicity, with a carrier frequency of 2%-4%. HBV is the major cause of CLD and HCC. Chronic HBV infection in India is acquired in childhood, presumably before 5 years of age, through horizontal transmission. Vertical transmission of HBV in India is considered to be infrequent. Inclusion of HBV vaccination in the expanded programme of immunization is essential to reduce the HBV carrier frequency and disease burden. HBV genotypes A and D are prevalent in India, which are similar to the HBV genotypes in the West. HCV infection in India has a population prevalence of around 1%, and occurs predominantly through transfusion and the use of unsterile glass syringes. HCV genotypes 3 and 2 are prevalent in 60%-80% of the population and they respond well to a combination of interferon and ribavirin. About 10%-15% of CLD and HCC are associated with HCV infection in India. HCV infection is also a major cause of post-transfusion hepatitis. HDV infection is infrequent in India and is present about 5%-10% of patients with HBV-related liver disease. HCC appears to be less common in India than would be expected from the prevalence rates of HBV and HCV. The high disease burden of viral hepatitis and related CLD in India, calls for the setting up of a hepatitis registry and formulation of government-supported prevention and control strategies.

Non-alcoholic fatty liver disease may not be a severe disease at presentation among Asian Indians.

AIM: To evaluate the clinical and biochemical profile of patients with non alcoholic fatty liver disease (NAFLD) and to assess their histological severity at presentation. METHODS: Consecutive patients presenting to the liver clinic of All India Institute of Medical Sciences (AIIMS) with raised transaminases to at least 1.5 times upper limit of normal, and histologically confirmed non-alcoholic fatty liver disease were included. Patients who had significant alcohol intake or positive markers of other liver diseases or who were taking drugs known to produce fatty liver were excluded. The clinical, biochemical and histological profile of this group was studied. RESULTS: Fifty-one patients with NAFLD formed the study population. Their median age and BMI were 34(17-58) years and 26.7(21.3-32.5) kg/m(2) respectively and 46 (90.1%) were males. The majority of the patients had mild inflammation, either grade 1 [32 (63%)] or grade 2 [16 (31%)] and only 3 (6%) patients had severe (grade 3) inflammation. Twenty-three (45%), 19 (37%), 8(16%) and 1(2%) patient had stage 0, 1, 2 and 3 fibrosis respectively on index biopsy and none had cirrhosis. On univariate analysis, triglyceride levels more than 150 mg % (OR = 7.1; 95% CI: 1.6-31.5, P = 0.002) and AST/ALT ratio>1 (OR = 14.3; 95% CI: 1.4-678.5, P = 0.008) were associated with high grades of inflammation and none was associated with advanced fibrosis. On multivariate logistic regression analysis, hypertriglyceridemia >150 mg% was the only factor independently associated with presence of high grade of inflammation (OR = 1.6; 95% CI: 1.3-22.7, P = 0.02), while none was associated with advanced fibrosis. Triglyceride levels correlated positively with inflammatory grade (r = 0.412; P = 0.003). CONCLUSION: NAFLD in North Indian patients is a disease of young over-weight males, most of whom are insulin resistant and they tend to have a mild histological disease at presentation.

Urinary excretion pattern of catecholestrogens in preovulatory LH surge during the 4-day estrous cycle of rats.

The formation of catecholestrogens represents a major pathway of estrogen metabolism and catecholestrogens are regarded as the main estrogen metabolite in non-pregnant state of various mammalian systems. In the present investigation, level of 2-hydroxyestrone, the major catecholestrogen excreted in rat urine, was measured by radioimmunoassay following acid hydrolysis and column chromatography of the 24-h urine samples of female Sprague Dawley non-pregnant rats during their 4-day estrous cycle. Urinary levels of estrone, estradiol and estriol were measured. Unlike the plasma level, urinary 2-hydroxyestrone showed a marked increase during the pre-ovulatory LH surge suggesting a plausible role of catecholestrogen in the mid-cycle elevation of the gonadotropin level in normal cycling rats.

The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious.

Hepatitis E virus (HEV) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the Indian subcontinent and prevalent in most of the developing parts of the world. The infection is often associated with acute liver failure and high mortality, particularly in pregnant women. In order to develop methods of intervention, it is essential to understand the biology of the virus. This is particularly important as no reliable in vitro culture system is available. We have constructed a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of an Indian epidemic isolate. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells by strand-specific reverse transcription-PCR and slot blot hybridization. The viral proteins pORF2 and pORF3 and processed components of the pORF1 polyprotein (putative methyltransferase, helicase, and RNA-dependent RNA polymerase) were identified in the transfected cells by metabolic pulse-labeling with [(35)S]methionine-cysteine, followed by immunoprecipitation with respective antibodies. The expression of viral proteins in the transfected cells was also demonstrated by immunofluorescence microscopy. Viral replication was detected in the transfected cells up to 33 days posttransfection (six passages). The culture supernatant from the transfected cells was able to produce HEV infection in a rhesus monkey (Macaca mulatta) following intravenous injection, indicating the generation of viable HEV particles following transfection of cells with in vitro-synthesized genomic RNA. This transient cell culture model using in vitro-transcribed RNA should facilitate our understanding of HEV biology.

Cloning, sequencing, and expression of the hepatitis E virus (HEV) nonstructural open reading frame 1 (ORF1).

Hepatitis E virus (HEV) causes enterically transmitted epidemic and sporadic viral hepatitis affecting millions of people in the developing world. Different geographical isolates of HEV show a high degree of homology at the nucleotide and amino acid levels. The approximately 7.2 kb RNA genome has three open reading frames of which ORF1 is predicted to code for the viral nonstructural polyprotein. The expression, processing and properties of the nonstructural ORF1 polyprotein have not been reported so far. In this study, the complete HEV ORF1 was reconstructed from overlapping fragments amplified by polymerase chain reaction (PCR) of total RNA isolated from the bile fluid of a rhesus monkey experimentally infected with HEV isolate from an epidemic. The complete assembled ORF1 was sequenced using HEV specific primers. The ORF1 polyprotein was expressed in E. coli, in a cell free translation system and in HepG2 cells, and was characterized by western blotting and immunoprecipitation using acute phase patient serum as well as polyclonal antibodies raised against defined parts of the ORF1 polyprotein. The nonstructural polyprotein of HEV was expressed as a 186 kDa protein. No processing was observed into discrete units, either in-vitro based on a kinetic analysis, or in HepG2 cells based on immunoprecipitation.

Fecal excretion pattern of bile acids in rats fed high fat diets and neomycin in induced colon tumorigenesis.

Neomycin augments colon tumorigenesis in 1,2 - dimethylhydrazine treated rats fed polyunsaturated fat diet and decreases fecal cholic acid excretion, while it inhibits tumorigenesis with increased cholic acid and decreased deoxycholic acid excretions in rats fed high cholesterol diet. Participation of other fecal bile acids seems to be insignificant in relation to colon carcinogenesis.