Role of tight junction proteins in shaping the immune milieu of malignancies.

INTRODUCTION: Tight junctions (TJs) and their constituent proteins play pivotal roles in cellular physiology and anatomy by establishing functional boundaries within and between neighboring cells. While the involvement of TJ proteins, such as claudins, in cancer is extensively studied, studies highlighting their interaction with immune system are still meager. Studies indicate that alterations in cytokines and immune cell populations can affect TJ proteins, compromising TJ barrier function and exacerbating pro-inflammatory conditions, potentially leading to epithelial cell malignancy. Disrupted TJs in established tumors may foster a pro-tumor immune microenvironment, facilitating tumor progression, invasion, epithelial-to-mesenchymal transition and metastasis. Although previous literature contains many studies describing the involvement of TJs in pathogenesis of malignancies their role in modulating the immune microenvironment of tumors is just beginning to be unleashed. AREAS COVERED: This article for the first time attempts to discern the importance of interaction between TJs and immune microenvironment in malignancies. To achieve the above aim a thorough search of databases like PubMed and Google Scholar was conducted to identify the recent and relevant articles on the topic. EXPERT OPINION: Breaking the vicious cycle of dysbiosis/infections/chemical/carcinogen-induced inflammation-TJ remodeling-malignancy-TJ dysregulation-more inflammation can be used as a strategy to complement the effect of immunotherapies in various malignancies.

Comparative morphology of tumour microenvironment in claudin-low and claudin-high breast cancers.

BACKGROUND: Claudin-low breast cancers (BCs) exhibit more aggressive behaviour compared to claudin-high types. Claudin-low BCs are often characterized by features such as a higher grade, enrichment of stemness characteristics, and a propensity for metastasis. Tumour microenvironment (TME) defined as the intricate network of surrounding cells, blood vessels, and extracellular matrix components influences the behaviour of cancer cells within the breast tissue. Understanding the TME is crucial for comprehending the aggressive characteristics of claudin-low BCs. METHODS: In this study, we have studied the morphology of immune and non-immune TME using Haematoxylin and eosin (H&E)-stained slides of 15 claudin-low and 12 claudin-high tissue samples of BC. RESULTS: TME of claudin-low BCs was observed to have a significantly higher frequency of retraction clefts (66.6 %; n = 10/15), immature desmoplastic response (40 %; n = 6/15), higher stromal cellularity (60 %; n = 9/15); and fibroblastic proliferation (53.3 %; n = 8/15) with a low prevalence of elastosis (66.6 %; n = 10/15). The immune microenvironment revealed a higher frequency of total (80 %; n = 12/15) as well as stromal (86.67 %; n = 13/15) and intra-tumoural TILs (60 %; n = 9/15) in them. CONCLUSION: The above morphology-based study revealed that claudin-low tumours have unique immune and non-immune TME as compared to claudin-high tumours. Future studies exploring the molecular correlates of each of the above morphological features can help in identifying novel therapeutic targets for the treatment of claudin-low BCs.

Unlocking the Therapeutic Potential: Sitagliptin's Multifaceted Approach in Drug-Resistant Epilepsy through a Novel Mechanism Inhibiting Protein Kinase C-γ and a Long-Term Potentiation Pathway.

Drug-resistant epilepsy is a prominent challenge in chronic neurological disorders. Valproate, commonly used to treat epilepsy, can fail due to various side effects and interactions, necessitating the exploration of alternative treatments. Our study primarily investigated sitagliptin's potential as a therapeutic agent for drug-resistant epilepsy. Employing computational modeling and enzyme assay testing, three lead compounds, emixustat, sitagliptin, and distigmine bromide, were evaluated against the target enzyme protein kinase C-γ. In vivo, experiments on a pentylenetetrazolium-induced lamotrigine-resistant epilepsy model were conducted to test sitagliptin's antiseizure effects, compared with the standard phenobarbital treatment. Emixustat and sitagliptin showcased strong inhibitory properties, while distigmine bromide was less effective in the enzyme assay. Mechanistic insights revealed sitagliptin's ability to modulate the seizure grade and first myoclonic jerk latency via oxidative stress markers, like reduced glutathione and glutathione peroxidase emphasizing its antioxidative role in epilepsy. Additionally, it demonstrated anti-inflammatory effects by significantly reducing proinflammatory markers interleukin-1ß and interleukin-6. The modulation of key genes of the long-term potentiation pathway, particularly protein kinase C-γ and metabotropic glutamate receptor 5, was evident through mRNA expression levels. Finally, sitagliptin showed potential neuroprotective properties, limiting pentylenetetrazolium-induced neuronal loss in the hippocampal region. Collectively, our findings suggest sitagliptin's multidimensional therapeutic potential for drug-resistant epilepsy specifically via a long-term potentiation pathway by inhibiting protein kinase C-γ.

Exploring the effect of 6-BIO and sulindac in modulation of Wnt/ß-catenin signaling pathway in chronic phase of temporal lobe epilepsy.

The prospective involvement of the Wnt/ß-catenin signaling pathway in epilepsy, with the proposed therapeutic uses of its modulators, has been suggested; however, comprehensive knowledge in this regard is currently limited. Despite postulations about the pathway's significance and treatment potential, a systematic investigation is required to better understand its implications in chronic epilepsy. We investigated the role of key proteins like ß-catenin, GSK-3ß, and their modulators sulindac and 6-BIO, in Wnt/ß-catenin pathway during chronic phase of temporal lobe epilepsy. We also evaluated the role of modulators in seizure score, seizure frequency and neurobehavioral parameters in temporal lobe epilepsy. We developed status epilepticus model using lithium-pilocarpine. The assessment of neurobehavioral parameters was done followed by histopathological examination and immunohistochemistry staining of hippocampus as well as RT-qPCR and western blotting to analyse gene and protein expression. In SE rats, seizure score and frequency were significantly high compared to control rats, with notable changes in neurobehavioral parameters and neuronal damage observed in hippocampus. Our study also revealed a substantial upregulation of the Wnt/ß-catenin pathway in chronic epilepsy, as evidenced by gene and protein expression studies. Sulindac emerged as a potent modulator, reducing seizure score, frequency, neuronal damage, apoptosis, and downregulating the Wnt/ß-catenin pathway when compared to 6-BIO. Our findings emphasize the potential of GSK-3ß and ß-catenin as promising drug targets for chronic temporal lobe epilepsy, offering valuable treatment options for chronic epilepsy. The promising outcomes with sulindac encourages further exploration in clinical trials to assess its therapeutic potential.

SEW2871 reduces seizures via the sphingosine 1-phosphate receptor-1 pathway in the pentylenetetrazol and phenobarbitone kindling model of drug-refractory epilepsy.

Epilepsy is a prevalent neurological disorder characterized by neuronal hypersynchronous discharge in the brain, leading to central nervous system (CNS) dysfunction. Despite the availability of anti-epileptic drugs (AEDs), resistance to AEDs is the greatest challenge in treating epilepsy. The role of sphingosine-1-phosphate-receptor 1 (S1PR1) in drug-resistant epilepsy is unexplored. This study investigated the effects of SEW2871, a potent S1PR1 agonist, on a phenobarbitone (PHB)-resistant pentylenetetrazol (PTZ)-kindled Wistar rat model. We measured the messenger ribonucleic acid (mRNA) expression of multi-drug resistance 1 (MDR1) and multi-drug resistance protein 5 (MRP5) as indicators for drug resistance. Rats received PHB + PTZ for 62 days to develop a drug-resistant epilepsy model. From day 48, SEW2871 (0.25, 0.5, 0.75 mg/kg, intraperitoneally [i.p.]) was administered for 14 days. Seizure scoring, behaviour, oxidative markers like reduced glutathione, catalase, superoxide dismutase, inflammatory markers like interleukin 1 beta tumour necrosis factor alpha, interferon gamma and mRNA expression (MDR1 and MRP5) were assessed, and histopathological assessments were conducted. SEW2871 demonstrated dose-dependent improvements in seizure scoring and neurobehavioral parameters with a reduction in oxidative and inflammation-induced neuronal damage. The S1PR1 agonist also downregulated MDR1 and MRP5 gene expression and significantly decreased the number of dark-stained pyknotic nuclei and increased cell density with neuronal rearrangement in the rat brain hippocampus. These findings suggest that SEW2871 might ameliorate epileptic symptoms by modulating drug resistance through downregulation of MDR1 and MRP5 gene expression.

Ameliorating effect of pioglitazone on prenatal valproic acid-induced behavioral and neurobiological abnormalities in autism spectrum disorder in rats.

Autism spectrum disorder (ASD) is a neurodevelopment disorder that mainly arises due to abnormalities in different brain regions, resulting in behavioral deficits. Besides its diverse phenotypical features, ASD is associated with complex and varied etiology, presenting challenges in understanding its precise neuro-pathophysiology. Pioglitazone was reported to have a fundamental role in neuroprotection in various other neurological disorders. The present study aimed to investigate the therapeutic potential of pioglitazone in the prenatal valproic acid (VPA)-model of ASD in Wistar rats. Pregnant female Wistar rats received VPA on Embryonic day (E.D12.5) to induce autistic-like-behavioral and neurobiological alterations in their offspring. VPA-exposed rats presented core behavioral symptoms of ASD such as deficits in social interaction, poor spatial and learning behavior, increased anxiety, locomotory and repetitive activity, and decreased exploratory activity. Apart from these, VPA exposure also stimulated neurochemical and histopathological neurodegeneration in various brain regions. We administered three different doses of pioglitazone i.e., 2.5, 5, and 10 mg/kg in rats to assess various parameters. Of all the doses, our study highlighted that 10 mg/kg pioglitazone efficiently attenuated the autistic symptoms along with other neurochemical alterations such as oxidative stress, neuroinflammation, and apoptosis. Moreover, pioglitazone significantly attenuated the neurodegeneration by restoring the neuronal loss in the hippocampus and cerebellum. Taken together, our study suggests that pioglitazone exhibits therapeutic potential in alleviating behavioral abnormalities induced by prenatal VPA exposure in rats. However, further research is needed to fully understand and establish pioglitazone's effectiveness in treating ASD.

Evaluation of Wnt/ß-catenin signaling and its modulators in repeated dose lithium-pilocarpine rat model of status epilepticus: An acute phase study.

The role of the Wnt/ß-catenin signaling pathway in epilepsy and the effects of its modulators as efficacious treatment options, though postulated, has not been sufficiently investigated. We evaluated the involvement of ß-catenin and GSK-3ß, the significant proteins in this pathway, in the lithium chloride-pilocarpine-induced status epilepticus model in rodents to study acute phase of temporal lobe epilepsy (TLE). The modulators studied were 6-BIO, a GSK-3ß inhibitor and Sulindac, a Dvl protein inhibitor. The disease group exhibited increased seizure score and seizure frequency, and the assessment of neurobehavioral parameters indicated notable alterations. Furthermore, histopathological examination of hippocampal brain tissues revealed significant neurodegeneration. Immunohistochemical study of hippocampus revealed neurogenesis in 6-BIO and sulindac groups. The gene and protein expression by RT-qPCR and western blotting studies indicated Wnt/ß-catenin pathway downregulation and increased apoptosis in the acute phase of TLE. 6-BIO was very efficient in upregulating the Wnt pathway, decreasing neuronal damage, increasing neurogenesis in hippocampus and decreasing seizure score and frequency in comparison to sulindac. This suggests that both GSK-3ß and ß-catenin are potential and novel drug targets for acute phase of TLE, and treatment options targeting these proteins could be beneficial in successfully managing acute epilepsy. Further evaluation of 6-BIO to explore its therapeutic potential in other models of epilepsy should be conducted.

An image analysis approach to characterize micronuclei differences in different subtypes of breast cancer.

BACKGROUND: Micronuclei (MN) have been used as screening, diagnostic and prognostic markers in multiple cancer types, including breast cancer (BC). However, the question that the MN present in all subtypes of BC are similar or different remains unanswered. We thus hypothesized that MN present in different subtypes of BC may differ in their contents which may be visible as differences in their morphologic and morphometric features. This study was thus carried out with the aim to identify the differences between MN morphometry, complexity, and texture in different subtypes of BC, such as estrogen and progesterone receptor-positive (ER+/PR+; MCF-7, T-47D), human epidermal growth factor receptor-positive (Her2 +;SKBR3) and triple-negative BC (TNBC; MDA-MB-231, MDA-MB-468) cell lines (CLs) by ImageJ software. METHODS: For analysis of MN dimensions, MN irregularity, and texture, we used morphometry and two mathematical computer-assisted algorithms, i.e., fractal dimension (FD) and grey level co-occurrence matrix (GLCM) of ImageJ software. RESULTS: MN area and perimeter values showed differences in the size of MN in different subtypes of BC, with the largest MN in TNBC CLs. GLCM parameters (entropy, angular second moment, inverse difference moment, contrast, and correlation) showed highly heterogenous texture in case of TNBC MN as compared to the others. FD analysis also revealed more complexity and irregularity in MN found in TNBC cells. CONCLUSION: The study for the first time showed morphometric, architectural and texture related differences amongst MN present in different subtypes of BC. The above may reflect differences in their composition and contents. Further, these differences may point towards the distinct mechanisms involved in the formation of MN in different subtypes of BC that need to be explored further.

Aspirin as a potential drug repurposing candidate targeting estrogen receptor alpha in breast cancer: a molecular dynamics and in-vitro study.

Estrogen receptor alpha (ERα) is expressed by 70% of breast cancers (BCs). Any deregulation in ERα signaling is crucial for the initiation and progression of BC. Because of development of resistance to anti-estrogenic compounds, repurposing existing drugs is an apt strategy to avoid a long drug-discovery process. Substantial epidemiologic evidence suggests that Aspirin use reduces the risk of different cancers including BC, while its role as an adjuvant or a possible antineoplastic agent in cancer treatment is being investigated. In this study, we attempted to explore possibilities of ERα inhibition by Aspirin which may act through competitive binding to the ligand binding domain (LBD) of ERα. A list of 48 ERα-LBD crystal structures bound with agonists, antagonists, and selective ER modulators (SERMs) was thoroughly analysed to determine interaction patterns specific to each ligand category. Exhaustive docking and 500 ns molecular dynamics (MD) studies were performed on three ERα - Aspirin complexes generated using agonist, antagonist, and SERM-bound crystal structures. Besides, three ERα crystal structures bound to agonist, antagonist, and SERM respectively were also subjected to MD simulations. Aspirin showed good affinity to LBD of ERα. Comparative analyses of binding patterns, conformational changes and molecular interaction profiles from the docking results and MD trajectories suggests that Aspirin was most stable in complex generated using SERM bound crystal structure of ERα and showed interactions with Gly-521, Ala-350, Leu-525 and Thr-347 like SERMs. In addition, in-vitro assays, qPCR, and immunofluorescent assay demonstrated the decline in the expression of ERα in MCF-7 upon treatment with Aspirin. These preliminary bioinformatical and in-vitro findings may form the basis to consider Aspirin as a potential candidate for targeting ERα, especially in tamoxifen-resistant cancers.Communicated by Ramaswamy H. Sarma.

Pharmacokinetic assessment of rifampicin and des-acetyl rifampicin in carbon tetrachloride induced liver injury model in Wistar rats.

OBJECTIVES: Preclinical evidence is needed to assess drug-metabolite behaviour in compromised liver function for developing the best antitubercular treatment (ATT) re-introduction regimen in drug-induced liver injury (DILI). The pharmacokinetic behavior of rifampicin (RMP) and its active metabolite des-acetyl-rifampicin (DARP) in DILI's presence is unknown. To study the pharmacokinetic behavior of RMP and DARP in the presence of carbon tetrachloride (CCl4) plus ATT-DILI in rats. METHODS: Thirty rats used in the experiment were divided equally into six groups. We administered a single 0.5 mL/kg CCl4 intraperitoneal injection in all rats. Groups II, III, IV, and V were started on daily oral RMP alone, RMP plus isoniazid (INH), RMP plus pyrazinamide (PZA), and the three drugs INH, RMP, and PZA together, respectively, for 21-days subsequently. Pharmacokinetic (PK) sampling was performed at 0, 0.5, 1, 3, 6, 12, and 24 h post-dosing on day 20. We monitored LFT at baseline on days-1, 7, and 21 and sacrificed the rats on the last day of the experiment. RESULTS: ATT treatment sustained the CCl4-induced liver injury changes. A significant rise in mean total bilirubin levels was observed in groups administered rifampicin. The triple drug combination group demonstrated 1.43- and 1.84-times higher area-under-the-curve values of RMP (234.56±30.66 vs. 163.55±36.14 µg h/mL) and DARP (16.15±4.50 vs. 8.75±2.79 µg h/mL) compared to RMP alone group. Histological and oxidative stress changes supported underlying liver injury and PK alterations. CONCLUSIONS: RMP metabolism inhibition by PZA, more than isoniazid, was well preserved in the presence of underlying liver injury.

"Comparative evaluation of different chemical agents induced Autism Spectrum Disorder in experimental Wistar rats".

Autism Spectrum Disorder (ASD) is a complex neurodevelopmental condition with uncertain etiology and pathophysiology. Several studies revealed that the commonly used animal models like Valproic Acid (VPA) and Propionic Acid (PPA) do not precisely represent the disease as the human patient does. The current study was conducted on different chemically (VPA, PPA, Poly I:C, Dioxin (2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)) & Chlorpyrifos (CPF)) induced ASD-like animal models and validated the best suitable experimental animal model, which would closely resemble with clinical features of the ASD. This validated model might help to explore the pathophysiology of ASD. This study included rat pups prenatally exposed to VPA, PPA, Poly I:C, Dioxin & CPF within GD9 to GD15 doses. The model groups were validated through developmental and behavioral parameters, Gene Expressions, Oxidative Stress, and Pro-inflammatory and Anti-inflammatory cytokines levels. Developmental and neurobehavioral parameters showed significant changes in model groups compared to the control. In oxidative stress parameters and neuro-inflammatory cytokines levels, model groups exhibited high oxidative stress and neuro-inflammation compared to control groups. Gene expression profile of ASD-related genes showed significant downregulation in model groups compared to the control group. Moreover, the Poly I:C group showed more significant results than other model groups. The comparison of available ASD-like experimental animal models showed that the Poly I:C induced model represented the exact pathophysiology of ASD as the human patient does. Poly I:C was reported in the maternal immune system activation via the inflammatory cytokines pathway, altering embryonic development and causing ASD in neonates.

Differential Regulation of Wnt/ß-catenin Signaling in Acute and Chronic Epilepsy in Repeated Low Dose Lithium-Pilocarpine Rat Model of Status Epilepticus.

Epilepsy is a chronic neurological complication characterized by unprovoked seizure episodes due to the imbalance between excitatory and inhibitory neurons. The epileptogenesis process has been reported to be involved in chronic epilepsy however, the mechanism underlying epileptogenesis remains unclear. Recent studies have shown the possible involvement of Wnt/ß-catenin signaling in the neurogenesis and neuronal reorganization in epileptogenesis. In this study, we used repeated low dose lithium-pilocarpine model of status epilepsy (SE) to study the involvement of Wnt/ß-catenin signaling at acute and chronic stages post SE induction. The acute study ranged from day 0 to day 28 post SE induction and the chronic study ranged from day 0 to day 56 post SE induction. Several neurobehavioral parameters and seizure score and seizure frequency was analysed until the end of the study. The proteins involved in the regulation of Wnt/ß-catenin signaling and downstream cascading were analysed using western blot and quantitative real-time PCR analysis. The Wnt/ß-catenin pathway was found inactive in acute SE, while the same was found activated at the chronic stage. Our findings suggest that the activated Wnt/ß-catenin signaling in chronic epilepsy might be the possible mechanism underlying epileptogenesis as indicated by increased neuronal count, increased synaptic density, astrogliosis and apoptosis in chronic epilepsy. These findings can help target the Wnt/ß-catenin pathway differentially depending upon the type of epilepsy. The acute stage characterized by SE can be improved by targeting GSK-3ß levels and the chronic stage characterized by temporal lobe epilepsy can be improved by targeting ß-catenin and disheveled proteins.

Study of fingolimod, nitric oxide inhibitor, and P-glycoprotein inhibitor in modulating the P-glycoprotein expression via an endothelin-sphingolipid pathway in an animal model of pharmacoresistant epilepsy.

BACKGROUND: The overexpression of P-glycoprotein (P-gp) contributes to drug resistance in patients with epilepsy, and the change of P-gp expression located at the blood-brain barrier alienates the anti-seizure effects of P-gp substrates. Thus, the present study explored the effect of fingolimod (FTY720) acting through an endothelin-sphingolipid pathway on P-gp-induced pentylenetetrazol (PTZ)-kindled phenobarbital (PB)-resistant rats. MATERIALS AND METHODS: PTZ kindling (30 mg/kg; i.p.) and PB (40 mg/kg; orally) were used to develop an animal model of refractory epilepsy. The effect of Fingolimod on seizure score (Racine scale), plasma and brain levels of PB (high-performance liquid chromatography), and blood-brain barrier permeability (Evans blue dye) was determined. Further, Fingolimod's neuroprotective effect was determined by measuring the levels of various inflammatory cytokines, oxidative stress parameters, and neurotrophic factors in rat brain homogenate. The Fingolimod's effect on P-gp expression was estimated by reverse transcriptase-polymerase chain reaction and immunohistochemistry in rat brain. The H and E staining was done to determine the neuronal injury. RESULTS: Fingolimod significantly (P < 0.001) reduced the seizure score in a dose-dependent manner and alleviated the blood-brain barrier permeability. It decreased the P-gp expression, which further increased the brain PB concentration. Fingolimod significantly (P < 0.01) reduced oxidative stress as well as inflammation. Moreover, it attenuated the raised neuronal injury score in a resistant model of epilepsy. CONCLUSION: The modulation of the P-gp expression by Fingolimod improved drug delivery to the brain in an animal model of refractory epilepsy. Therefore, S1P signaling could serve as an additional therapeutic target to overcome refractoriness.

The gene expression and proteomic profiling of Acanthamoeba isolates.

INTRODUCTION: The free-living protozoan Acanthamoeba can cause severe keratitis known as Acanthamoeba Keratitis (AK) and granulomatous amoebic encephalitis (GAE). The pathogenesis of Acanthamoeba includes intricate interactions between the organism and the host's immune system. The downstream analysis of a well-annotated genome assembly along with proteomic analysis can unravel several biological processes and aid in the identification of potential genes involved in pathogenicity. METHODS: Based on the next-generation sequencing data analysis, genes including lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein were selected as probable pathogenic targets that were validated by conventional PCR in a total of 30 Acanthamoeba isolates. This was followed by real-time PCR for the evaluation of relative gene expression in the keratitis and amoebic encephalitis animal model induced using keratitis (CHA5), encephalitis (CHA24) and non-pathogenic environmental isolate (CHA36). In addition, liquid chromatography-mass spectrometry (LC-MS/MS) was performed for keratitis, encephalitis, and non-pathogenic environmental isolate before and after treatment with polyhexamethylene biguanide (PHMB). RESULTS: The conventional PCR demonstrated the successful amplification of lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein genes in clinical and environmental isolates. The expression analysis revealed phospholipase, lysophospholipase, and mannose-binding genes to be significantly upregulated in the keratitis isolate (CHA 5) during AK in the animal model. In the case of the amoebic encephalitis model, phospholipase, lysophospholipase, S8/S53 peptidase, and carboxylesterase were significantly upregulated in the encephalitis isolate compared to the keratitis isolate. The proteomic data revealed differential protein expression in pathogenic versus non-pathogenic isolates in the pre and post-treatment with PHMB. CONCLUSION: The gene expression data suggests that lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein (MBP) could play a role in the contact-dependent and independent mechanisms of Acanthamoeba pathogenesis. In addition, the proteomic profiling of the 3 isolates revealed differential protein expression crucial for parasite growth, survival, and virulence. Our results provide baseline data for selecting possible pathogenic targets that could be utilized for designing knockout experiments in the future.

Pharmacokinetic Assessment of Pyrazinamide and Pyrazinoic Acid in Carbon tetrachloride-induced Liver Injury Model in Wistar Rats.

Background: We investigated the pharmacokinetic behavior of pyrazinamide (PZA) and pyrazinoic acid (PA) in the presence of carbon-tetrachloride (CCl4) plus antitubercular treatment (ATT) drug-induced liver injury (DILI) in rats. Methods: Thirty rats utilized in the experiment were separated equally into five groups. Each rat was injected with 0.5 ml/kg CCl4 intra-peritoneal injection on day zero. Group, I rats did receive only CCl4 (single i.p. injection, 0.5 ml/Kg in olive oil in a 1:1 ratio). Groups II, III, IV, and V did receive daily oral PZA, PZA plus isoniazid (INH), rifampicin (RMP) plus pyrazinamide (PZA), and three drugs together, respectively, for 21-days. Pharmacokinetic sampling was performed at 0, 0.5,1,3,6,12 and 24 hours post-dosing on day-20. Liver function test (LFT) was assessed at days 0,1,7, and 21 days after CCl4 and ATT administration, and rats were sacrificed on the last experiment day. Results: ATT treatment maintained the liver function changes initiated by CCl4 administration. An evidential LFT rise was observed in groups administered with pyrazinamide. Co-administration of Isoniazid caused a 2.02 and 1.78 times increase in Area-under-the-curve (AUC) values of PZA and PA, respectively (p < 0.05). Histological and oxidative-stress changes supported the biochemical and pharmacokinetic observations. Conclusion: The enzyme inhibitory capacity of isoniazid is well-preservd in CCl4-induced liver injury.

Pharmacokinetic Assessment of Isoniazid and Acetylisoniazid in Carbon Tetrachloride-Induced Liver Injury Model in Wistar Rats.

Background: N-acetyl transferase 2 (NAT2) polymorphism testing could not see the light of success as a biomarker tool in tuberculosis management. Additionally, the antitubercular treatment (ATT) drug's reintroduction regimen variations exist because of the scarcity of robust preclinical evidence on ATT drug metabolism. Objective: The experiment was planned to understand the pharmacokinetic (PK) behavior of isoniazid and acetylisoniazid (AcINH) in a Wistar rat model of acute liver injury induced by carbon tetrachloride (CCl4) and preclinical drug-induced liver injury (DILI) model induced with CCl4 + anti-Tuberculosis (TB) drugs together. Materials and Methods: Thirty rats were used for the experiment and were divided into five groups. All rats were administered a single 0.5 ml/kg CCl4 intraperitoneal injection on day 0 to induce an animal model of DILI. Group I rats received CCl4 alone. Groups II-V were started on additional gavage feedings of isoniazid (H) alone, H plus rifampicin (R), H plus pyrazinamide (Z), and H, R, and Z together, respectively, daily for 21 days subsequently. Isoniazid and AcINH PK assessment was accomplished on day 20 of continuous once-daily dosing. Liver function test (LFT) monitoring was done at baseline on days 1, 7, and 21. On the last day of experiments, all experimental rats were sacrificed. Results: Three-week ATT administration sustained the CCl4-induced LFT changes. Area under the curve (AUC) values for isoniazid and AcINH were found to be 2.24 and 1.69 times higher in the H + R group compared with the CCl4 + H group, respectively (P < 0.05). Isoniazid and AcINH maximum concentration (Cmax) reached the highest, while isoniazid clearance reached the lowest in the H + R group. AcINH AUC increased by double in the CCl4 + Isoniazid+Rifampicin+Pyrazinamide (HRZ) group compared with the CCl4 + H group (P < 0.05). Biochemical, histological, and antioxidant changes were consistent with the new liver injury model's development. Conclusion: Rifampicin almost doubles up the isoniazid and AcINH exposure, in presence if DILI.

Polymeric immunoglobulin receptor and galectin-3-binding protein are raised in biliary atresia: Reveals a proteomic-based study.

To identify and evaluate differentially expressed plasma proteins in biliary atresia (BA), we performed plasma proteome profiling using liquid chromatography with tandem mass spectrometry (LC-MS/MS) in 20 patients with BA and 10 control children. Serological assays validated the most significant and highly upregulated proteins in a cohort of 45 patients and 15 controls. Bioinformatics tools were used for functional classification and protein-protein interactions of differentially expressed proteins (DEPs). Of 405 proteins detected in patients and 360 in controls, 242 proteins, each with ≥2 unique peptides (total of 3230 peptides), were common in both groups. Compared to controls, 90 proteins in patients were differentially expressed and were dysregulated. Twenty-five were significantly upregulated with polymeric immunoglobulin receptor (PIgR), galectin-3-binding protein (Gal-3BP), complement C2, the most prominent, and 15 had low expression. The bioinformatic analysis revealed functional interaction between DEPs and their role in an inflammatory immune response. Enzyme immunoassay for PIgR and Gal-3BP in patients' plasma showed their levels raised significantly (p = 0.0021 and p = 0.0369, respectively). The PIgR and Gal-3BP are novel proteins upregulated in BA and may be tested further for their utility as potential circulating disease biomarker(s). SIGNIFICANCE: The study shows that plasma PIgR and GAL-3BP levels are significantly raised in infants with BA within the first 3 months of life. If tested in a larger cohort, these proteins may be found to have their diagnostic potential and utility as disease biomarkers. The study also provides valuable information on the involvement of several DEPs in innate immune response, chronic inflammation, and fibrosis. This strengthens the hypothesis that the immune-mediated inflammatory processes are responsible for the progressive nature of BA.

Assessment of pathogenic potential of Acanthamoeba isolates by in vitro and in vivo tests.

Acanthamoeba are free-living protozoa present ubiquitously in numerous environmental reservoirs that exist as an actively feeding trophozoite or a dormant cyst stage. The pathogenic Acanthamoeba are known to cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Despite their omnipresence, the number of infections is quite low. The reason behind this low frequency of Acanthamoeba infections could be the existence of many non-pathogenic strains or a successful host immune response to these infections. Studies in the past have proposed a few physiological parameters for the differentiation of pathogenic and non-pathogenic strains. Additionally, in vivo experiments are known to play an essential role in understanding the virulence of parasites, immunological aspects, and disease pathogenesis. The thermotolerance (30 °C, 37 °C, and 40 °C) and osmotolerance (0.5 M, 1 M, and 1.5 M) tests were performed on 43 Acanthamoeba isolates from patients with keratitis (n = 22), encephalitis (n = 5), and water samples (n = 16). In addition, the genotype of 10 Acanthamoeba isolates (keratitis (n = 2), encephalitis (n = 2), water (n = 6)) was determined and were then evaluated for pathogenicity on mouse model by inducing Acanthamoeba keratitis and amoebic encephalitis. The results of the thermotolerance and osmotolerance assays categorized 29/43 (67.4%) isolates as pathogenic, 8 as low pathogenic (18.6%), and the remaining 6 (13.9%) as non-pathogenic. The 10 Acanthamoeba isolates were categorized as T11 (5 isolates), T5 (2 isolates), T4 (2 isolates), and T10 (1 isolate) genotypes. Out of 10 Acanthamoeba isolates, 9 were successful in establishing AK, amoebic encephalitis, or both in the mice model, and a single isolate was found non-pathogenic. Two isolates from water samples were non-pathogenic in the physiological tests but successfully established Acanthamoeba infection in the mice model. The results of the physiological assays and in vivo experiments were analogous for 7 isolates while 1 isolate from the water was low pathogenic in the physiological assays but failed to produce pathogenicity during in vivo experiments. The physiological parameters are not very dependable to test the pathogenic potential of Acanthamoeba isolates, and thus results must always be validated by in vivo experiments. There is no infallible approach for determining the potential pathogenicity of environmental isolates of Acanthamoeba because several parameters regulate the pathogenic potential.

Neuroprotective Effect of Celastrus Paniculatus Seed Extract on Epilepsy and Epilepsy-associated Cognitive Deficits.

Introduction: Cognitive deficit is one of the common comorbidity accompanying epilepsy. The present study evaluated the effect of Celastrus paniculatus seed extract on seizure severity and cognitive deficit following the pentylenetetrazole (PTZ)-induced chemical kindling model. Methods: PTZ kindling model was developed by daily administration of the sub-convulsive dose of PTZ 30 mg/kg for four weeks. After four weeks of induction, the following treatment, namely sodium valproic acid (SVA) 200 mg/kg, C. paniculatus 500 mg\kg, pergolide 2 mg/kg, C. paniculatus (250 mg\kg)+ Pergolide (1 mg/kg), and C. paniculatus (250 mg\kg)+ SVA (100 mg/kg) were administered 30 minutes prior to PTZ (30 mg/kg) injection for a period of next 14 days. Neurobehavioral parameters, including superoxide dismutase (SOD), Catalase (CAT), glutathione (GSH), and dopamine levels were assessed and the Morris water maze test (MWM) and Grip strength test (GPS) were performed. Hematoxylin & Eosin (H&E) staining of hippocampal cornu ammonis (CA1), CA2, CA3, dentate gyrus (DG), and frontal cortex was performed. Results: C. paniculatus (500 mg/kg) alone and in combination (C. paniculatus (250 mg\ kg)+ pergolide (1 mg/kg) and C. paniculatus (250 mg\kg)+ SVA (100 mg/kg)) significantly (P<0.05) reduced the seizure score, mean latency time, and distance traveled in the MWM. However, no significant effect was seen in GPS. Biochemical analysis showed elevated antioxidant markers, namely GSH, CAT, and SOD, and also elevated dopamine levels. C. paniculatus and its combination also significantly (P<0.05) protected against neuronal loss in the hippocampus and frontal cortex evidenced by H&E staining. Conclusion: C. paniculatus alone and in combination with other agents may have the potential to treat epilepsy and associated cognitive deficits.

Vibrio Phage VMJ710 Can Prevent and Treat Disease Caused by Pathogenic MDR V. cholerae O1 in an Infant Mouse Model.

Cholera, a disease of antiquity, is still festering in developing countries that lack safe drinking water and sewage disposal. Vibrio cholerae, the causative agent of cholera, has developed multi-drug resistance to many antimicrobial agents. In aquatic habitats, phages are known to influence the occurrence and dispersion of pathogenic V. cholerae. We isolated Vibrio phage VMJ710 from a community sewage water sample of Manimajra, Chandigarh, in 2015 during an outbreak of cholera. It lysed 46% of multidrug-resistant V. cholerae O1 strains. It had significantly reduced the bacterial density within the first 4-6 h of treatment at the three multiplicity of infection (MOI 0.01, 0.1, and 1.0) values used. No bacterial resistance was observed against phage VMJ710 for 20 h in the time-kill assay. It is nearest to an ICP1 phage, i.e., Vibrio phage ICP1_2012 (MH310936.1), belonging to the class Caudoviricetes. ICP1 phages have been the dominant bacteriophages found in cholera patients' stools since 2001. Comparative genome analysis of phage VMJ710 and related phages indicated a high level of genetic conservation. The phage was stable over a wide range of temperatures and pH, which will be an advantage for applications in different environmental settings. The phage VMJ710 showed a reduction in biofilm mass growth, bacterial dispersal, and a clear disruption of bacterial biofilm structure. We further tested the phage VMJ710 for its potential therapeutic and prophylactic properties using infant BALB/c mice. Bacterial counts were reduced significantly when phages were administered before and after the challenge of orogastric inoculation with V. cholerae serotype O1. A comprehensive whole genome study revealed no indication of lysogenic genes, genes associated with possible virulence factors, or antibiotic resistance. Based on all these properties, phage VMJ710 can be a suitable candidate for oral phage administration and could be a viable method of combatting cholera infection caused by MDR V. cholerae pathogenic strains.