Antimicrobial resistance heterogeneity among multidrug-resistant Gram-negative pathogens: Phenotypic, genotypic, and proteomic analysis.

Microbes evolve rapidly by modifying their genomes through mutations or through the horizontal acquisition of mobile genetic elements (MGEs) linked with fitness traits such as antimicrobial resistance (AMR), virulence, and metabolic functions. We conducted a multicentric study in India and collected different clinical samples for decoding the genome sequences of bacterial pathogens associated with sepsis, urinary tract infections, and respiratory infections to understand the functional potency associated with AMR and its dynamics. Genomic analysis identified several acquired AMR genes (ARGs) that have a pathogen-specific signature. We observed that blaCTX-M-15, blaCMY-42, blaNDM-5, and aadA(2) were prevalent in Escherichia coli, and blaTEM-1B, blaOXA-232, blaNDM-1, rmtB, and rmtC were dominant in Klebsiella pneumoniae. In contrast, Pseudomonas aeruginosa and Acinetobacter baumannii harbored blaVEB, blaVIM-2, aph(3'), strA/B, blaOXA-23, aph(3') variants, and amrA, respectively. Regardless of the type of ARG, the MGEs linked with ARGs were also pathogen-specific. The sequence type of these pathogens was identified as high-risk international clones, with only a few lineages being predominant and region-specific. Whole-cell proteome analysis of extensively drug-resistant K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa strains revealed differential abundances of resistance-associated proteins in the presence and absence of different classes of antibiotics. The pathogen-specific resistance signatures and differential abundance of AMR-associated proteins identified in this study should add value to AMR diagnostics and the choice of appropriate drug combinations for successful antimicrobial therapy.

Metagenomic insights into fungal community composition of the nasopharyngeal region of COVID-19 associated mucormycosis patients from India.

Coronavirus disease 2019 (COVID-19) associated mucormycosis (CAM) was reported predominantly from India during the second wave of COVID-19  and has a high mortality rate. The present study aims to understand the fungal community composition of the nasopharyngeal region of CAM-infected individuals and compare it with severe COVID-19 patients and healthy controls. The fungal community composition was decoded by analyzing the sequence homology of the internal transcribed spacer-2-(ITS-2) region of metagenomic DNA extracted from the upper respiratory samples. The alpha-diversity indices were found to be significantly altered in CAM patients (p < 0.05). Interestingly, a higher abundance of Candida africana, Candida haemuloni, Starmerella floris, and Starmerella lactiscondensi was observed exclusively in CAM patients. The interindividual changes in mycobiome composition were well supported by beta-diversity analysis (p < 0.05). The current study provides insights into the dysbiosis of the nasal mycobiome during CAM infection. In conclusion, our study shows that severe COVID-19 and CAM are associated with alteration in mycobiome as compared to healthy controls. However, the sequential alteration in the fungal flora which ultimately leads to the development of CAM needs to be addressed by future studies.

Correlation of severity & clinical outcomes of COVID-19 with virus variants: A prospective, multicentre hospital network study.

BACKGROUND OBJECTIVES: The clinical course of COVID-19 and its prognosis are influenced by both viral and host factors. The objectives of this study were to develop a nationwide platform to investigate the molecular epidemiology of SARS-CoV-2 (Severe acute respiratory syndrome Corona virus 2) and correlate the severity and clinical outcomes of COVID-19 with virus variants. METHODS: A nationwide, longitudinal, prospective cohort study was conducted from September 2021 to December 2022 at 14 hospitals across the country that were linked to a viral sequencing laboratory under the Indian SARS-CoV-2 Genomics Consortium. All participants (18 yr and above) who attended the hospital with a suspicion of SARS-CoV-2 infection and tested positive by the reverse transcription-PCR method were included. The participant population consisted of both hospitalized as well as outpatients. Their clinical course and outcomes were studied prospectively. Nasopharyngeal samples collected were subjected to whole genome sequencing to detect SARS-CoV-2 variants. RESULTS: Of the 4972 participants enrolled, 3397 provided samples for viral sequencing and 2723 samples were successfully sequenced. From this, the evolution of virus variants of concern including Omicron subvariants which emerged over time was observed and the same reported here. The mean age of the study participants was 41 yr and overall 49.3 per cent were female. The common symptoms were fever and cough and 32.5 per cent had comorbidities. Infection with the Delta variant evidently increased the risk of severe COVID-19 (adjusted odds ratio: 2.53, 95% confidence interval: 1.52, 4.2), while Omicron was milder independent of vaccination status. The independent risk factors for mortality were age >65 yr, presence of comorbidities and no vaccination. INTERPRETATION CONCLUSIONS: The authors believe that this is a first-of-its-kind study in the country that provides real-time data of virus evolution from a pan-India network of hospitals closely linked to the genome sequencing laboratories. The severity of COVID-19 could be correlated with virus variants with Omicron being the milder variant.

Study on Effects of Probiotics on Gut Microbiome and Clinical Course in Patients with Critical Care Illnesses.

Ventilator-associated pneumonia (VAP) is a nosocomial infection contracted by ventilator patients in which bacteria colonize the upper digestive tract and contaminated secretions are released into the lower airway. This nosocomial infection increases the morbidity and mortality of the patients as well as the cost of treatment. Probiotic formulations have recently been proposed to prevent the colonization of these pathogenic bacteria. In this prospective observational study, we aimed to investigate the effects of probiotics on gut microbiota and their relation to clinical outcomes in mechanically ventilated patients. For this study, 35 patients were recruited (22 probiotic-treated and 13 without probiotic treatment) from a cohort of 169 patients. Patients in the probiotic group were given a dose of 6 capsules of a commercially available probiotic (VSL#3®:112.5 billion CFU/cap) in three divided doses for 10 days. Sampling was carried out after each dose to monitor the temporal change in the gut microbiota composition. To profile the microbiota, we used a 16S rRNA metagenomic approach, and differences among the groups were computed using multivariate statistical analyses. Differences in gut microbial diversity (Bray Curtis and Jaccard distance, p-value > 0.05) between the probiotic-treated group and the control group were not observed. Furthermore, treatment with probiotics resulted in the enrichment of Lactobacillus and Streptococcus in the gut microbiota of the probiotic-treated groups. Our results demonstrated that probiotics might lead to favorable alterations in gut microbiome characteristics. Future studies should focus on the appropriate dosages and frequency of probiotics, which can lead to improved clinical outcomes.

The diagnosis of high altitude illness by the determination of plasma dermcidin isoform 2 levels by enzyme linked immunosorbent assay.

BACKGROUND: High altitude illness (HAI) is a cluster of syndromes which develops due to the injury of the central nervous system produced by the reduction of the partial pressure of O2 in the atmosphere which disappears on decent. The HAI also results in a prothrombotic condition leading to acute coronary syndrome (ACS), which cannot be controlled on descent to the ground level. There is no diagnosis in HAI to forewarn of the impending ACS. A protein identified to be dermcidin isoform 2 (dermcidin), produced in the system due to environmental stresses, has been reported to be a potent diabetogenic agent. Investigation was carried out to determine the systemic stimulation of dermcidin synthesis at different levels of altitudes in normal adult male volunteers to assess the feasibility of developing a diagnosis for ACS in HAI due to dermcidin synthesis. METHODS: Normal, nondiabetic, normotensive male volunteers (25 - 35 years old, n = 16) participated in the study. The plasma dermcidin level was determined by enzyme linked immunosorbent assay (ELISA) and by in vitro translation of dermcidin mRNA. The plasma insulin level was determined by ELISA and blood glucose level was determined in a glucometer (Behringer). RESULTS: The plasma dermcidin level in the volunteers at ground level was 10 +/- 2.10 nM and increased to 80 +/- 4.62 nM at 15000 feet altitude. For each 1000 feet increase of altitude, the dermcidin level increased by 5.83 +/- 0.21 nM with a Coefficient of Correlation "r" = +0.9405. The increase of plasma dermcidin level was found to be inversely related to the decrease of plasma insulin level from 23 microunit/mL to 5 microunit/mL from sea level to 15000 feet height ("r" = -0.9951) with concomitant increase of blood sugar level from 80 +/- 3.6 mg/dL to 135 +/- 2.01 mg/dL. CONCLUSIONS: These results suggest the feasibility of a diagnosis of a prediabetic condition by determining the plasma dermcidin level in HAI by simple ELISA which may also be useful to forewarn of the possibility of developing an impending prothrombotic condition in HAI.

A rapid point-of-care population-scale dipstick assay to identify and differentiate SARS-CoV-2 variants in COVID-19-positive patients.

Delta and Omicron variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are remarkably contagious, and have been recognized as variants of concern (VOC). The acquisition of spontaneous substitutions or insertion-deletion mutations (indels) in the spike protein-encoding gene substantially increases the binding affinity of the receptor binding domain (RBD)-hACE2 complex and upsurges the transmission of both variants. In this study, we analyzed thousands of genome sequences from 30 distinct SARS-CoV-2 variants, focusing on the unique nucleic acid signatures in the spike gene specific to the Delta and Omicron variants. Using these variant-specific sequences, we synthesized a range of oligonucleotides and optimized a multiplex PCR (mPCR) assay capable of accurately identifying and differentiating between the Delta and Omicron variants. Building on this mPCR assay, we developed a dipstick format by incorporating a tag linker sequence at the 5' end of the forward primer and adding biotin to the 3' end of the oligonucleotides, enhancing the assay's usability and accessibility. Streptavidin-coated latex beads and the dipstick imprinted with a probe for the tag linker sequence in the test strips were used for the detection assay. Our dipstick-based assay, developed as a rapid point-of-care test for identifying and differentiating SARS-CoV-2 variants has the potential to be used in low-resource settings and scaled up to the population level.

Diagnosis of essential hypertension in humans by the determination of plasma renal cortexin using enzyme-linked immunosorbent assay.

BACKGROUND: More than 90% of all hypertensive persons are reported to have essential hypertension (EH), a particular form of elevated blood pressure, for which no diagnostic test is currently available. The level of plasma renal (R) cortexin (PRC), a hypotensive protein produced in the kidney cortex cells, was reported to be reduced from 218 nM in the plasma of normotensive persons (NP) to 0 nM in the plasma of patients with EH. The feasibility of using the determination of PRC by enzyme-linked immunosorbent assay (ELISA) as a diagnostic test for EH was investigated. METHODS: The PRC was determined by ELISA using electrophoretically pure cortexin as the antigen. A total of 344 persons (male and female) with EH, with or without diabetes mellitus (DM), and receiving or not receiving any anti-hypertensive and/or anti-diabetic medication at presentation, as well as an equal number of age- and gender-matched NP participated in the study. RESULTS: All persons with EH, with or without co-existing DM, were found to have 0 nM PRC, regardless of whether they were receiving anti-hypertensive and anti-diabetic drugs, including those who had been taking these medications over an extended period of time (3 months). CONCLUSIONS: The determination of PRC as a marker protein by ELISA, a rapid method that can be carried out in any diagnostic laboratory, was shown to be suitable for the diagnosis of EH, even in those subjects who had co-existing DM and were receiving both anti-hypertensive and anti-diabetic medication.

Effect of herbal extracts and Saroglitazar on high-fat diet-induced obesity, insulin resistance, dyslipidemia, and hepatic lipidome in C57BL/6J mice.

We evaluated the effects of select herbal extracts (Tinospora cordifolia [TC], Tinospora cordifolia with Piper longum [TC + PL], Withania somnifera [WS], Glycyrrhiza glabra [GG], AYUSH-64 [AY-64], and Saroglitazar [S]) on various parameters in a diet-induced obesity mouse model. After 12 weeks of oral administration of the herbal extracts in high-fat diet (HFD)-fed C57BL/6J mice, we analyzed plasma biochemical parameters, insulin resistance (IR), liver histology, and the expression of inflammatory and fibrosis markers, along with hepatic lipidome. We also used a 3D hepatic spheroid model to assess their impact on profibrotic gene expression. Among the extracts, TC + PL showed a significant reduction in IR, liver weight, TNF-α, IL4, IL10 expression, and hepatic lipid levels (saturated triglycerides, ceramides, lysophosphocholines, acylcarnitines, diglycerides, and phosphatidylinositol levels). Saroglitazar reversed changes in body weight, IR, plasma triglycerides, glucose, insulin, and various hepatic lipid species (fatty acids, phospholipids, glycerophospholipids, sphingolipids, and triglycerides). With the exception of GG, Saroglitazar, and other extracts protected against palmitic acid-induced fibrosis marker gene expression in the 3D spheroids. TC + PL and Saroglitazar also effectively prevented HFD-induced insulin resistance, inflammation, and specific harmful lipid species in the liver.

Ancestral SARS-CoV-2-Driven Antibody Repertoire Diversity in an Unvaccinated Individual Correlates with Expanded Neutralization Breadth.

Understanding the quality of immune repertoire triggered during natural infection can provide vital clues that form the basis for development of a humoral immune response in some individuals capable of broadly neutralizing pan-SARS-CoV-2 variants. In the present study, we report variations in neutralization potential against Omicron variants of two novel neutralizing monoclonal antibodies (MAbs), THSC20.HVTR11 and THSC20.HVTR55, isolated from an unvaccinated convalescent individual that represent distinct B cell lineage origins and epitope specificity compared to five MAbs we previously reported that were isolated from the same individual. In addition, we observed neutralization of Omicron variants by plasma antibodies obtained from this particular individual postvaccination with increased magnitude. Interestingly, this observation was found to be comparable with six additional individuals who initially were also infected with ancestral SARS-CoV-2 and then received vaccines, indicating that hybrid immunity can provide robust humoral immunity likely by antibody affinity maturation. Development of a distinct antigen-specific B cell repertoire capable of producing polyclonal antibodies with distinct affinity and specificities offers the highest probability of protecting against evolving SARS-CoV-2 variants. IMPORTANCE Development of robust neutralizing antibodies in SARS-CoV-2 convalescent individuals is known; however, it varies at the population level. We isolated monoclonal antibodies from an individual infected with ancestral SARS-CoV-2 in early 2020 that not only varied in their B cell lineage origin but also varied in their capability and potency to neutralize all the known variants of concern (VOCs) and currently circulating Omicron variants. This indicated establishment of unique lineages that contributed in forming a B cell repertoire in this particular individual immediately following infection, giving rise to diverse antibody responses that could complement each other in providing a broadly neutralizing polyclonal antibody response. Individuals who were able to produce polyclonal antibody responses with higher magnitude have a higher chance of being protected from evolving SARS-CoV-2 variants.

Role of aspirin activated nitric oxide synthase in controlling DOCA-salt-induced hypertension in rats through the stimulation of renal r-cortexin in kidney cortex cells.

Objectives: Because the damage of kidney tissue is associated with hypertension and impaired nitric oxide (NO) synthesis, and as aspirin is reported to stimulate the synthesis of renal r-cortexin, an anti-hypertensive protein, we investigated the role of aspirin as bolus dose on elevated blood pressure induced by deoxycorticosterone acetate (DOCA)-salt in animal model. Methods: The chronic antihypertensive effect of aspirin on DOCA treated with ASA group of rats (n = 6) was evaluated after ingestion of 0.35 µM aspirin as a bolus dose in every 24 h using tail cuff methods. The plasma aspirin, NO, and r-cortexin levels were determined by spectrophotometric, methemoglobin, and ELISA methods, respectively. Synthesis of r-cortexin mRNA was determined. Aspirin activated nitric oxide synthase (AANOS) was purified by chromatographic methods. Results: Our results showed after 3 h of administration of aspirin (0.35 µM) to the DOCA treated with ASA group of rats decreased the systolic blood pressure from 139.39 ± 7.36 mm of Hg to 116.57 ± 6.89 mm of Hg and diastolic blood pressure from 110.4 ± 7 mm of Hg to 86.4 ± 2.76 mm of Hg. The reduction of BPs was found to be related to the increased plasma aspirin from 0.00 µM to 0.042 µM, plasma NO from 0.4 ± 0.19 nM to 1.9 ± 0.5 nM, and cortexin levels from 64.36 ± 12.6 nM to 216.7 ± 21.3 nM. The molecular weight of purified AANOS is 18 kDa. Conclusion: It can be concluded that aspirin possesses antihypertensive effect on blood pressure in chronic administration. Aspirin can stimulate NO synthesis through the activation of AANOS, which stimulated the production of r-cortexin in kidney cortex cells and thereby reducing elevated BP in hypertensive rats.

Insulin resistance in prostate cancer patients and predisposing them to acute ischemic heart disease.

Lack of insulin or insulin resistance (IR) plays a central role in diabetes mellitus and makes diabetics prone to acute ischemic heart disease (AIHD). It has likewise been found that many cancer patients, including prostate cancer patients die of AIHD. Previously it has been delineated from our laboratory that dermcidin could induce anomalous platelet aggregation in AIHD and also impaired nitric oxide and insulin activity and furthermore dermcidin was also found in a few types of cancer patients. To determine the role of this protein in prostatic malignancy, a retrospective case-control study was conducted and blood was collected from prostate cancer patients and healthy normal volunteers. So, we measured the level of dermcidin protein and analyzed the IR by Homeostasis Model Assessment (HOMA) score calculation. Nitric oxide was measured by methemoglobin method. HDL, glycated hemoglobin (HbA1c), BMI, hs-cTroponin-T were measured for the validation of the patients' status in the presence of Dermcidin isoform-2 (DCN-2). Multiple logistic regression model adjusted for age and BMI identified that the HOMA score was significantly elevated in prostate cancer patients (OR = 7.19, P<0.001). Prostate cancer patients are associated with lower level of NO and higher level of both proteins dermcidin (OR = 1.12, P<0.001) and hs-TroponinT (OR = 1.76, P<0.001). From the results, it can be interpreted that IR plays a key role in the pathophysiology of prostate cancer where dermcidin was the cause of IR through NO inhibition leading to AIHD was also explained by high-sensitive fifth generation cTroponin-T (hs-cTroponinT) and HbA1c level which are associated with endothelial dysfunction.

Estriol Inhibits Dermcidin Isoform-2 Induced Inflammatory Cytokine Expression Via Nitric Oxide Synthesis in Human Neutrophil.

BACKGROUND: An increase in the level of cytokines like TNF-α and IL-6 causes the inflammatory surge in acute ischemic heart disease (AIHD). OBJECTIVE: A high-level dermcidin isoform-2 (DCN-2) occurrence in AIHD was subjected to determine a possible regulation of cytokines expression. The effect of estrogen to counteract the inflammatory response was determined. METHODS: Blood was collected from AIHD patients and normal volunteers with consent. Nitric oxide (NO) synthesis was done with methemoglobin method.TNF-α and IL-6 expression were determined by ELISA and Western blot. RESULTS: (DCN-2) incubation with 120nM to the normal neutrophil solution for 2h resulted in the increase of TNF-α from 3.82±1.53pg/ml to 20.7±6.9pg/ml and IL-6 from 3.27±1.52pg/ml to 47.07±3.4pg/ml. In AIHD patients, the cytokine level was18.3- 27.3pg/ml, with a median value 21.86pg/ml (TNF-α) and IL-6 level was 23.54- 52.73pg/ml, with a median value 42.16pg/ml. Treatment with 0.6nM estriol, a kind of female steroid hormone estrogen for 45min decreased the elevated cytokine level in 120nM DCN-2 treated normal neutrophils. DCN-2 induced TNF-α synthesis in neutrophils was further determined by Western blot technique with a thickened band intensity of TNF-α. Estriol (0.6nM) treatment also influenced the DCN-2 induced inhibition of nitric oxide (NO) synthesis from 0nmol NO/ml to 0.56nmol/ml. The subsequent reduction of TNF-α level correlates the increase of NO level. CONCLUSION: In conclusion, the stress-induced DCN-2 production in AIHD propagates the inflammatory response. Steroid molecule like estriol plays a protective role by reducing DCN-2 responses in the NO synthesis.

Role of Acetyl Salicylic Acid in Controlling the DOCA-Salt Induced Hypertension in Rats by Stimulating the Synthesis of r-Cortexin in the Kidney.

INTRODUCTION: Hypertension is a metabolic disease which is caused by vasoconstriction and that results into elevated blood pressure. A chronic hypertensive condition affects and even damages to various systems in the body. Presence of renal cortexin (r-cortexin), an antihypertensive protein, which is released from the kidney cortex controls the blood pressure. The effect of r-cortexin was mediated through nitric oxide (NO), a universal vasodilating agent. AIM: In our study, acetyl salicylic acid (aspirin), a well-known activator of the endothelial nitric oxide synthase (eNOS) induced r-cortexin synthesis. METHODS: The hypertensive rat model was prepared by injecting deoxy corticosterone acetate (DOCA). Synthesis of r-cortexin was measured by the anti-r-cortexin antibody which was raised in adult white Wister albino rat model. NO level was determined by using methemoglobin method and later confirmed by chemiluminescence method. Change in blood pressure was determined indirectly by using NIBP monitoring system. RESULTS: Aspirin increased the r-cortexin expression from 64.36 ± 12.6 nM to 216.7 ± 21.31 nM in DOCA induced hypertensive rats. The mechanism was proved with the findings of increased level of NO from 0.4 to 1.9 µM. The DOCA induced blood pressure was also decreased from 139.39 ± 7.36 mm of Hg to 116.57 ± 6.89 mm of Hg in case of systolic blood pressure and in case of diastolic pressure from 110.41 ± 7 mm of Hg to 86.4 ± 2.76 mm of Hg that are quite approximate. CONCLUSION: So, from this study it has been found that aspirin induces the r-cortexin synthesis in kidney cortex through the activation of eNOS in DOCA induced hypertensive rats.

Expression of a Nitric Oxide Synthesizing Protein in Arterial Endothelial Cells in Response to Different Anti-Anginal Agents Used in Acute Coronary Syndromes.

BACKGROUND: Organic "nitro" compounds such as nitroglycerine, isosorbide dinitrate are useful in the control of chest pain in acute coronary syndrome. But the mechanism of it in pain regulation remains speculative. Here, increase of NO production was investigated by the possible regulation of constitutive nitric oxide synthase (cNOS) function from goat arterial endothelial cells. This protein was purified and sequence wise characterized as protein disulfide isomerase (PDI) in response to different nitro compounds. METHOD: The NO generating protein was isolated from arterial endothelial cells and prepared to homogeneity. NO was determined by methemoglobin method. Protein sequence was analyzed by (µLC/MS/MS). RESULTS: A protein of Mr. ~57 kDa was isolated and found to be activated by not only "nitro" compounds but also by acetyl salicylic acid, insulin and glucose. The global BLAST of the protein sequence showed a significant alignment of the protein sequence with PDI. This protein trivially called pluri activator stimulated endothelial NOS (PLASENOS). The enzyme was stimulated by the above-mentioned activators in the presence of Ca2+. Lineweaver-Burk plot of this NOS like activities were demonstrated with its specific substrate l-arginine as Vmax = 5(nmol NO/mg of protein/hr) and Km≈ 0.5µM by the above activators. The enzyme activity was inhibited by the l-NAME, the specific inhibitor of NOS. CONCLUSION: The organic nitro compounds, acetyl salicylic acid, insulin and glucose were found to activate PLASENOS in the arterial endothelial cells for a continuous supply of NO to control the chest pain in acute coronary syndrome.

Estriol-induced fibrinolysis due to the activation of plasminogen to plasmin by nitric oxide synthesis in platelets.

Estriol, an oestrogen, at 0.6 nmol/l was reported to inhibit ADP-induced platelet aggregation through nitric oxide synthesis. As nitric oxide has been reported to cause fibrinolysis due to the activation of plasminogen to plasmin, the role of estriol as a fibrinolytic agent was investigated. Also, the mechanism of estriol-induced nitric oxide synthesis in anucleated platelets was investigated. The estriol-induced lysis of platelet-rich plasma (PRP) clot was determined by photography of the clot lysis and by the assay of fibrin degradation products in the lysate and was obtained by SDS-PAGE. Nitric oxide was determined by methemoglobin method. The platelet membrane protein was isolated from the platelets by using Triton X-100 (0.05% v/v). The binding of estriol to the protein was determined by Scatchard plot by using an ELISA for estriol. Estriol at 0.6 nmol/l was found to lyse the clotted PRP due to fibrinolysis that produced fibrin degradation products in the lysate. The amino acid analysis of the platelet membrane protein, which resembles with nitric oxide synthase (NOS) activity, was activated nearly 10-fold over the control in the presence of estriol and was identified to be a human serum albumin precursor (Mr. 69 kDa) that binds to estriol with Kd1 of 6.0 × 10 mol/l and 39 ±â€Š2 molecules of estriol bound the NOS molecule. The estriol-induced nitric oxide is capable of inducing fibrinolysis of the clotted PRP. The binding of estriol to platelet membrane NOS activated the enzyme in the absence of DNA in the platelet.

Neutralization by Acetyl Salicylic Acid of the Testosterone induced Impaired Maspin Synthesis Stimulated by Estriol in Neutrophils through Nitric Oxide Synthesis.

PURPOSE: Maspin, an anti breast cancer protein in the mammary cell and normal neutrophil has been reported to be synthesised by the stimulation of NO production induced by estriol. The role of testosterone was investigated in the synthesis of maspin in relation to that of estriol. METHODS: Fifty normal female between the ages of 25-65 years old participated in the study. Maspin synthesis was demonstrated by in vitro translation of maspin mRNA, followed by the quantification of maspin by enzyme linked immune absorbent assay. NO was determined by methomoglobin method. RESULTS: Incubation of the neutrophils in HBSS both with 30 nM estriol resulted in the synthesis of 1.8 ngm maspin with simultaneous increase of NO synthesis. In contrast incubating neutrophils with 20 nM testosterone in the presence of estriol inhibited maspin synthesis to 0.33 nM with simultaneous inhibition of NO synthesis from 1.89 nM to 0 nM at the same time. Addition of 0.2µM flutamide, a testosterone receptor blocker to the incubation mixture restored the synthesis of maspin by 60.64 %. Incubation of 25µM aspirin that stimulated NO synthesis restored the inhibition of maspin synthesis by testosterone by 79.1%. I-NAME, an inhibitor of nitric oxide synthase, abolished both maspin and NO synthesis. Scatchard plot of estriol binding in the presence of testosterone demonstrated that the male sex hormone inhibited the female sex hormone binding to its receptor by "cross talk" between the receptors. It was found that while 1.02 × 10(3) molecules of estriol bind each neutrophil at equilibrium, in the presence of testosterone (20nM) in the binding mixture decreases the binding of estriol to 0.5 × 10(3) with little change in the dissociation constant compared to controls. CONCLUTION: Estriol was found to stimulate maspin synthesis through the stimulation of NO, testosterone inhibited maspin synthesis through the inhibition of NO synthesis.

Dermcidin isoform-2 induced nullification of the effect of acetyl salicylic acid in platelet aggregation in acute myocardial infarction.

The aggregation of platelets on the plaque rupture site on the coronary artery is reported to cause both acute coronary syndromes (ACS) and acute myocardial infarction (AMI). While the inhibition of platelet aggregation by acetyl salicylic acid was reported to produce beneficial effects in ACS, it failed to do in AMI. The concentration of a stress induced protein (dermcidin isoform-2) was much higher in AMI than that in ACS. Incubation of normal platelet rich plasma (PRP) with dermcidin showed one high affinity (Kd = 40 nM) and one low affinity binding sites (Kd = 333 nM). When normal PRP was incubated with 0.4 µM dermcidin, the platelets became resistant to the inhibitory effect of aspirin similar to that in the case of AMI. Incubation of PRP from AMI with dermcidin antibody restored the sensitivity of the platelets to the aspirin effect. Incubation of AMI PRP pretreated with 15 µM aspirin, a stimulator of the NO synthesis, resulted in the increased production of NO in the platelets that removed the bound dermcidin by 40% from the high affinity binding sites of AMI platelets. When the same AMI PRP was retreated with 10 µM aspirin, the aggregation of platelets was completely inhibited by NO synthesis.

Effect of progesterone receptor status on maspin synthesis via nitric oxide production in neutrophils in human breast cancer.

BACKGROUND: Although progesterone receptor (PR) status, similarly to estrogen receptor status, is of prognostic importance in breast cancer, the involvement of the PR in breast cancer remains obscure. Studies were conducted to determine the function of the PR in neutrophils in the nitric oxide-induced synthesis of maspin, an anti-breast-cancer protein produced in nonmalignant mammary cells and in neutrophils in the circulation. METHODS: PR status was determined by immunohistochemistry. Maspin synthesis was determined by in-vitro translation of messenger RNA and quantified by enzyme-linked immunosorbent assay. Nitric oxide was determined by the methemoglobin method. RESULTS: It was found that PR status in neutrophils was identical with that in malignant breast tissues. A Scatchard plot for progesterone binding to normal and PR-positive (PR+) neutrophils revealed that whereas normal neutrophils had 11.5 × 10(10) PR sites/cell with K d = 47.619 nM, PR+ neutrophils had 6.6 × 10(10) PR sites/cell with K d = 47.619 nM. The progesterone negative (PR-) neutrophils failed to bind to progesterone. Incubation of normal and PR+ neutrophils with 25 nM progesterone produced 1.317 µM NO and 2.329 nM maspin; the PR+ neutrophils produced 0.72 µM NO and 1.138 nM maspin. The PR- neutrophils failed to produce any NO or maspin in the presence of progesterone. Inhibition of progesterone-induced NO synthesis led to complete inhibition of maspin synthesis in all neutrophils. CONCLUSION: These results suggest that estrogen and progesterone complement each other in NO-induced maspin synthesis, and do not necessarily antagonize in the synthesis of the anti-breast-cancer protein.

Reduction of death rate due to acute myocardial infarction in subjects with cancers through systemic restoration of impaired nitric oxide.

INTRODUCTION: Excessive aggregation of platelets at the site of plaque rupture on the coronary artery led to the formation of thrombus which is reported to precipitate acute myocardial infarction (AMI). Nitric oxide (NO) has been reported to inhibit platelet aggregation and induce thrombolysis through the in situ formation of plasmin. As the plasma NO level in AMI patients from two different ethnic groups was reduced to 0 µM (median) compared to 4.0 µM (median) in normal controls, the effect of restoration of the NO level to normal ranges on the rate of death due to AMI was determined. METHODS AND RESULTS: The restoration of plasma NO level was achieved by a sticking small cotton pad (10×25 mm) containing 0.28 mmol sodium nitroprusside (SNP) in 0.9% NaCl to the abdominal skin of the participants using non-toxic adhesive tape which was reported to normalize the plasma NO level. The participants (8,283) were volunteers in an independent study who had different kinds of cancers and did not wish to use any conventional therapy for their condition but opted to receive SNP "pad" for their condition for 3 years. The use of SNP "pad" which normalized (≈4.0 µM) the plasma NO level that in consequence reduced the death rate due to AMI, among the participants, was found to be significantly reduced compared to the death due to AMI in normal population. CONCLUSION: Our data suggested that the use of SNP "pad" significantly reduced the death due to AMI. TRIAL REGISTRATION: www.ctri.nic.in CTRI/2013/12/004236.

The Role of Neutrophil Estrogen Receptor Status on Maspin Synthesis via Nitric Oxide Production in Human Breast Cancer.

PURPOSE: Estrogen, through its binding to nuclear estrogen receptor (ER), has been implicated in the development of human breast cancer. The presence or absence of ER in breast lesions has been used to classify breast cancer into ER+ or ER- type. Maspin, an anti-breast cancer protein produced in normal mammary cells, has also been reported to control the condition. Studies have been conducted to determine the role of ER+ and ER- status in neutrophils in the synthesis of maspin in human breast cancer. METHODS: Maspin presence was determined by enzyme linked immunosorbent assay, while nitric oxide (NO) level was determined using the methemoglobin method. RESULTS: Scatchard plots of the equilibrium binding of estrogen demonstrated the presence of 4.18×10(7) receptors per normal neutrophil and 2.46×10(7) receptors per ER+ neutrophil with a similar dissociation constant (0.926 nM). The ER- type showed nonspecific estrogen binding only. At 0.6 nM estrogen, NO synthesis was maximally increased to 1.829 and 0.887 µM NO/10(9) cells at 4 hours in normal and ER+ neutrophils respectively, with synthesis of 2.383 and 1.422 nM maspin in normal and ER+ neutrophils respectively. Estrogen failed to produce these effects in ER- neutrophils. CONCLUSION: ER status in neutrophils determined maspin synthesis in breast cancer through the stimulation of NO synthesis. Neutrophils with ER- status which do not produce any maspin when treated with estrogen, might imply a worse prognostic outcome in ER- breast cancer due to the lack of anti-breast cancer protein synthesis.