Role of Geminin as a Tool for Augmenting Accurate Diagnosis of Cervical Neoplasia.

AIM: To determine the role of geminin as a tool for differentiating various types of cervical intraepithelial neoplasia (CIN) and cervical carcinoma (CC). METHODS: Seventy women newly diagnosed with CIN or CC undergoing cervical biopsy were included; their clinical profile, human papilloma virus (HPV) positivity, and colposcopy findings were noted, and biopsy tissue was analyzed for geminin content. RESULTS: On geminin immunohistochemistry, 100% of women with CIN3 and 96.29% of women with CC had geminin two plus or more. When analyzed as ordinal variables, there was a significant correlation (spearman's rho 0.35, p 0.01) between geminin and biopsy results (CIN1, CIN2, CIN3, and CC). CONCLUSIONS: Screening tests for cervical cancer, like conventional pap smears, liquid-based pap smears, and triaging with HPV, have limitations. It is important to be able to differentiate between high-grade lesions, invasive cancer, and low-grade lesions. The detection of geminin in these cells may aid in the confirmation of the diagnosis and ensure adequate treatment. Cervical intraepithelial lesions and carcinoma cervix demonstrated a correlation between increased geminin expression in CIN1 vs. CC and CIN2 vs. CC. Geminin may be a potential surrogate marker for higher-grade cervical lesions, and further research is needed to corroborate evidence in this direction.

Design, Synthesis, and Biological Evaluation of Ferulic Acid Template-Based Novel Multifunctional Ligands Targeting NLRP3 Inflammasome for the Management of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia, which arises due to low levels of acetyl and butyrylcholines, an increase in oxidative stress, inflammation, metal dyshomeostasis, Aß and tau aggregations. The currently available drugs for AD treatment can provide only symptomatic relief without interfering with pathological hallmarks of the disease. In our ongoing efforts to develop naturally inspired novel multifunctional molecules for AD, systematic SAR studies on EJMC-4e were caried out to improve its multifunctional properties. The rigorous medicinal efforts led to the development of 12o, which displayed a 15-fold enhancement in antioxidant properties and a 2-fold increase in the activity against AChE and BChE over EJMC-4e. Molecular docking and dynamics studies revealed the binding sites and stability of the complex of 12o with AChE and BChE. The PAMPA-BBB assay clearly demonstrated that 12o can easily cross the blood-brain barrier. Interestingly, 12o also expresses promising metal chelation activity, while EJMC-4e was found to be devoid of this property. Further, 12o inhibited metal-induced or self Aß1-42 aggregation. Observing the neuroprotection ability of 12o against H2O2-induced oxidative stress in the PC-12 cell line is noteworthy. Furthermore, 12o also inhibited NLRP3 inflammasome activation and attenuated mitochondrial-induced ROS and MMP damage caused by LPS and ATP in HMC-3 cells. In addition, 12o is able to effectively reduce mitochondrial and cellular oxidative stress in the AD Drosophila model. Finally, 12o could reverse memory impairment in the scopolamine-induced AD mice model, as evident through in vivo and ex vivo studies. These findings suggest that this compound may act as a promising candidate for further improvement in the management of AD.

The immunohistochemical biomarker B-cell lymphoma-2 expression in malignant and premalignant lesions of the uterine cervix and its association with human papillomavirus infection.

Introduction: Cervical cancer is one of the leading causes of cancer deaths among women. It results due to human papillomavirus (HPV) infection. Cervical intraepithelial neoplasia (CIN) is the preinvasive condition of cervical cancer. Various objective immunohistochemical (IHC) markers have been studied for cervical cancer. This study is aimed at studying the expression of B-cell lymphoma-2 (Bcl-2) IHC marker among preinvasive and invasive lesions of cervical cancer and its association with HPV infection. Methodology: This prospective study was conducted over a period of 1 year in a tertiary care hospital in central India, included 73 women suffering from CIN and cancer cervix. The expression of Bcl-2 and the presence of HPV genotypes were studied. Results: Out of 73 patients, 34 had cancer cervix, out of which 15 (44%) had Bcl-2 positivity, 24 had CIN 1, out of which 13 (54%) had Bcl-2 positivity, 10 had CIN 2, out of which 4 (40%) had Bcl-2 positivity and 5 had CIN 3, out of which 3 (60%) had Bcl-2 positivity. No significant difference was found in Bcl-2 positivity among CIN-1, CIN-2, CIN-3, and cancer cervix cases with a Chi-square value of 1.116 and P = 0.77. HPV positivity was found in 41 (56%) out of 73 patients where HPV 16 subtype was the most common (31.5%), followed by HPV 18 (13.7%). No significant association between HPV positivity and Bcl-2 positivity was found with P = 0.34. Conclusion: Bcl-2 IHC seems to have variable expression among CIN cases. Although its expression is low among invasive cancer cases when compared with preinvasive lesions, the difference is not significant. Similarly, no significant association was found between Bcl-2 expression and HPV infections.

First-Trimester Reference Intervals for Thyroid Function Testing among Women Screened at a Tertiary Care Hospital in India.

Objectives Due to differences in the method of assay and population-specific factors, each laboratory needs to establish its own gestation-specific reference intervals (GRIs) for thyroid hormones. Materials and Methods Three-hundred forty-one women with less than 14 weeks gestation were screened at a tertiary care hospital in Chhattisgarh, India. Serum levels of thyroid-stimulating hormone (TSH), free thyroxine (fT4), and thyroid peroxidase antibody (anti-TPO) were measured using an ADVIA Centaur XP immunoassay. GRIs (2.5th and 97.5th percentiles) were determined for TSH and fT4. TSH and fT4 concentrations were converted to multiples of the median (MoM) values. Effect of maternal age, gestational age, and maternal weight was analyzed. Statistical Analysis Quantitative variables were expressed as means and standard deviations (SD), and qualitative variables were expressed as frequencies and percentages. Normality of the data was checked using the Kolmogorov-Smirnov test. Values that were normally distributed were expressed only as means and SD. Those that were not normally distributed were expressed as medians and interquartile range. For all statistical analysis, p < 0.05 was considered as statistically significant. Results First-trimester GRI was 0.245 to 4.971 mIU/L for TSH, 10.2 to 18.9 pmol/L for fT4, and 27.0 to 56.89 kIU/L for anti-TPO. There was no significant difference in the mean serum TSH ( p = 0.920), fT4 ( p = 0.714), or anti-TPO ( p = 0.754) values among women in 4 to 7th week and 7 to 14th week of gestation. The 1st and 99th centile MoMs were 0.03 and 4.09 for TSH and 0.66 and 1.39 for fT4. There was a significant positive correlation between the maternal weight and TSH MoM values ( p = 0.027, r = 0.120). Conclusion These laboratory- and first-trimester-specific GRI for TSH and fT4 shall help in proper diagnosis and treatment of subclinical thyroid dysfunctions. TSH and fT4 MoM values can be used to indicate high or low values in a quantitative manner independent of the reference ranges and may be used by other laboratories.

Transcription factor 4 expression and correlation with tumor progression in gallbladder cancer.

Background: Dysregulation in Wnt/ß-catenin signaling has been associated with the initiation and metastasis of cancer cells. Transcription factor 4 (TCF4) (also named as transcription factor 7-like 2) is a key transcriptional factor of the Wnt signaling pathway, which, when interact with ß-catenin activates Wnt genes which plays an essential role in tumor development. The expression pattern and clinical significance of TCF4 in gallbladder cancer (GBC) are not yet established. Aims: This study was performed to assess the expression pattern of TCF4 in GBC tissue and attempted to correlate its expression with different clinicopathological parameters. Materials and Methods: The study was conducted on 33 surgically resected specimens of gallbladder carcinoma (GBC) and 12 cases of chronic cholecystitis (CC) as control, which had been confirmed from histology. The expression of TCF4 was performed by the reverse transcription polymerase chain reaction and immunohistochemistry. Results: Relative mRNA expression levels of ß-catenin and TCF4 in GBC tissues were significantly (P < 0.05) higher than in CC samples. TCF4 protein expression was observed in 81.82% (27/33) GBC cases. Specifically, among GBC samples, 21.21% (7/33) was graded as strongly positive, 60.61% (20/33) graded as moderately positive, whereas 18.18% (6/33) graded as negative. All 12 CC samples graded as negative. Overall, TCF4 expression in GBC tissues was statistically significant over CC samples (P < 0.05). Moreover, we observed that TCF4 expression was significantly higher (P < 0.05) in high tumor grades than low grade, higher (P < 0.05) in Stage 2 and Stage 3 than Stage 1. Conclusion: The present study suggests that TCF4 may exert an oncogenic role in the progression of GBC and may serve as a new potential candidate biomarker for tumor progression, and it might be a potential therapeutic target against GBC.

Prophylactic administration of rosmarinic acid ameliorates depression-associated cardiac abnormalities in Wistar rats: Evidence of serotonergic, oxidative, and inflammatory pathways.

Psychiatric disorders and associated cardiac comorbidities have increased the risk of mortality worldwide. Researchers reported that depression increases the possibility of future cardiac abnormalities by approximately 30%. Therefore, there is an unmet need to develop therapeutic interventions to treat depression and associated cardiac abnormalities. The present study was conducted to evaluate the prophylactic effect of rosmarinic acid (RA) against chronic unpredictable stress (CUS)-induced depression associated cardiac abnormalities in Wistar rats. The CUS paradigm, which comprised several stressors, was employed for 40 days to induce depressive-like behavior and associated cardiac abnormalities in rats. Along with CUS, RA at a dose of 25 and 50 mg/kg was administered orally to two groups of animals for 40 days. Behavioral tests (forced swim test and sucrose consumption test) and molecular biomarkers (corticosterone and serotonin) were performed. Electrocardiography was performed before CUS (Day 0), Day 20, and Day 40 to study electrocardiogram parameters. Furthermore, changes in body weight, organ weight, tissue lipid peroxidation, glutathione, catalase, cTn-I, MMP-2, and proinflammatory cytokines (TNF-α and IL-6) were estimated. Our results showed that RA treatment caused a reduction in immobility period, adrenal hyperplasia, corticosterone level, tissue lipid peroxidation, cTn-I, MMP-2, proinflammatory cytokines, and QRS complex duration, while an increase in sucrose consumption, brain serotonin level, T-wave width, glutathione, and catalase activity as compared with the CUS-control group. The results of our study proved that RA administration ameliorates CUS-induced depression-associated cardiac abnormalities in rats via serotonergic, oxidative, and inflammatory pathways.

Evaluation of rosmarinic acid against myocardial infarction in maternally separated rats.

Depression and coronary heart diseases are the common comorbid disorder affecting humans globally. The present study evaluated the effectiveness of rosmarinic acid (RA) against myocardial infarction (MI) in comorbid depression induced by maternal separation in rats. Maternal stress is one of the childhood crises that may be a potential risk factor for coronary heart disease in later part of life. As per protocol, 70-80% of pups were separated daily for 3 h between postnatal day 1 (PND1) and postnatal day 21 (PND21). Forced-swim test, sucrose preference test, and electrocardiography were performed during the experiment. Body weight was measured on PND0, PND35, and PND55. Orally rosmarinic acid (25 mg/kg and 50 mg/kg) and fluoxetine (10 mg/kg) was done from PND35 to PND55. On PND53 and PND54, isoproterenol (100 mg/kg, subcutaneously) was administered to induce myocardial infarction. On PND55, blood was collected and animals sacrificed, and plasma corticosterone, brain-derived neurotrophic factor, cardiac biomarkers, interleukine-10, and anti-oxidant parameters were measured. Rosmarinic acid and fluoxetine ameliorated the maternal separation-induced increase in immobility period, anhedonia, body weight, ST elevation, corticosterone, creatine kinase-MB (CK-MB), and lactate dehydrogenase (LDH). At the same time, both drugs elevated the tissue levels of BDNF, IL-10, glutathione, and superoxide dismutase activity. This study provides the first experimental evidence that maternal stress is an independent risk factor of cardiac abnormalities in rats. Moreover, maternal stress synergistically increases the severity of cardiac abnormalities induced by isoproterenol. Interestingly, fluoxetine and rosmarinic acid effectively ameliorated behavioral anomalies and myocardial infarction in maternally separated rats. Schematic representation of possible molecular mechanism of action of rosmarinic acid against MS-induced myocardial infarction. RA, rosmarinic acid; MS, maternal separation; PND, postnatal days; ISO, isoproterenol; BDNF, brain-derived neurotrophic factor; GSH, glutathione; SOD, superoxide dismutase; IL-10, interleukin-10; MI, myocardial infarction.

Design, synthesis and evaluation of 4,7-disubstituted 8-methoxyquinazoline derivatives as potential cytotoxic agents targeting ß-catenin/TCF4 signaling pathway.

Overactivation of Wnt/ß-catenin signaling by accumulated ß-catenin in the nucleus has been shown to play a crucial role in the etiology of cancer. Interaction of ß-catenin with Transcription factor 4 (TCF4) is a key step for the activation of Wnt genes in response to upstream signals of the Wnt/ß-catenin pathway. Hence, down regulation of Wnt/ß-catenin signaling or targeting downstream events by selective ß-catenin/TCF4 protein-protein interaction inhibitors could be a potential therapeutic strategy against such cancers. In this study structure-based drug design approach was followed to design novel 4,7-disubstituted 8-methoxyquinazoline-based derivatives which could act as potential cytotoxic agents inhibiting the ß-catenin/TCF4 protein-protein interactions. Fifteen compounds possessing 4,7-disubstituted 8-methoxyquinazoline scaffold were synthesized. Cytotoxic potential of the synthesised derivatives were determined against constitutively activated ß-catenin/TCF4 signaling pathway cancer cells (HCT116 and HepG2) using the sulforhodamine B assay. The most potent compound (18B) was selected for detailed biological evaluation. Cell morphology, Hoechst 33342 and Annexin V/PI staining were used to detect apoptosis, while inhibition of cell migration was assessed by in vitro wound healing assay against HCT116 and HepG2 cells. Effect on ß-catenin/TCF mediated transcriptional activity was assessed by TOPFlash/FOPFlash assay, TCF4 and ß-catenin protein expression by immunocytofluorescence, and Wnt target genes (like c-MYC and Cyclin D1) mRNA levels by RT-PCR against HCT116 cells. Cytotoxic potency of the most potential compound (18B) against primary human gallbladder cancer cells was also evaluated. The derivatives showed interactions with active site residues of ß-catenin and were capable of hindering the TCF4 binding, thereby disrupting ß-catenin/TCF4 interactions. Cytotoxic potencies (IC50) of these derivatives ranged from 5.64 ± 0.68 to 23.18 ± 0.45 µM against HCT116 and HepG2 cells respectively. Compound (18B), the most potent compound among the series, induced apoptosis and inhibited cell migration against HCT116 and HepG2 cells. Mechanistic studies indicated that compound (18B) downregulated ß-catenin/TCF4 signaling pathway, ß-catenin and TCF4 protein expression, and mRNA levels of c-MYC andCyclin D1 in HCT116 cells and showed cytotoxicity against primary human gallbladder cancer cells with IC50 value of 8.50 ± 1.44 µM. Thus, novel 4,7-disubstituted 8-methoxyquinazoline derivatives were identified as potential cytotoxic agents with potencies comparable to that of imatinib mesylate. Compound (18B) represents a promising lead molecule as anticancer agent against colon, hepatocellular and gallbladder cancers targeting ß-catenin/TCF4 signaling pathway.

Effect of Exogenous Progesterone on Fetal Nuchal Translucency: An Observational Study.

Introduction Nuchal translucency is a reliable first trimester screening test for fetal structural and chromosomal defects. Neonates with increased nuchal thickness are at greater risk for anomalies. Exogenous progesterone supplementation may affect nuchal translucency and thus the first trimester anomaly screening. We aimed to study if there was a difference in nuchal thickness between women receiving progesterone in the first trimester compared to those who were not supplemented with progesterone. Material and methods Forty-seven women with documented progesterone intake in the first trimester for at least 10 continuous days before the day of the nuchal translucency scan served as the study group compared to 47 other women who did not receive progesterone. Nuchal translucency was measured between 11 and 13 weeks and six days of gestation. Results The mean nuchal translucency increased with increasing gestation in both groups. Maximum mean nuchal translucency was greatest in the age group 18-20 years (1.35 + 0.1 millimeters) in women receiving progesterone compared to 36-40 years (1.65 + 0.49 millimeters) in controls. The mean nuchal translucency in women receiving progesterone was 1.15 + 0.26 millimeters, and in those that did not receive progesterone, it was 1.23 + 0.35 millimeters (p = 0.314). Conclusions Nuchal translucency increased with increasing gestation in both groups, irrespective of progesterone supplementation. There was no significant difference in mean nuchal translucency in women supplemented with progesterone compared to those that did not receive progesterone in the first trimester.

Consumption of Ashtanga Ghrita (clarified cow butter added with herb extracts) improves cognitive dysfunction induced by scopolamine in rats via regulation of acetylcholinesterase activity and oxidative stress.

OBJECTIVES: Ashtanga Ghrita (AG), an Indian traditional formulation, has been used to promote neuropharmacological activities. AG is made up of clarified cow butter (ghee) and eight different herbs. METHODS: To test whether scopolamine (SCP)-induced dementia and brain oxidative stress can be counteracted by AG, rats were separated into five groups (n=6/group): group one control, group two SCP (1 mg/kg b.w., i.p.) treated and group three to five were co-treated with different doses of AG (1.25, 2.5 and 5 g/kg b.w., orally) and SCP. After the treatment regimen, behavioral (Y-maze test) and brain biochemical changes were measured in all groups. RESULTS: Microbial load and heavy metals were found within permissible limits. Results from attenuated total reflection Fourier-transform infrared spectroscopy demonstrated the complexation/interaction of herbal phytoconstituents with the functional groups of Ghrita. Preliminary phytochemical analysis of AG exhibited the occurrence of flavonoids, phenolics, glycosides, steroids, triterpenes, tannins, and amino acids. Findings of the experimental study exhibited that AG significantly protected the rats from SCP-induced behavioral dysfunction and brain biochemical alterations. CONCLUSIONS: This study demonstrates that AG protects the brain from SCP-induced dementia by promoting brain antioxidant activity and thus could be a promising drug for the treatment of neurodegenerative disease.

Role of various screening techniques in detecting preinvasive lesions of the cervix among symptomatic women and women having unhealthy cervix.

INTRODUCTION: Cervical cancer which is preventable, occurs due to humanpapiloma virus infection and results in a preinvasive condition called cervical intraepithelial neoplasm (CIN) before the development of cancer. Majority of the patients with CIN or early stage of cervical cancer present with symptoms such as abnormal vaginal discharge or bleeding, and unhealthy looking cervix. Selectively screening these symptomatic patients, can detect more number of positive cases and also most effective screening technique for these selective patients can be advocated. MATERIALS AND METHODS: All married women between 21 and 65 years attending gynecology outpatient department of a tertiary care health center in Central India and having unhealthy cervix or abnormal vaginal discharge were included. All women were subjected to Pap smear, visual inspection under acetic acid (VIA), visual inspection under Lugol's iodine (VILI) and colposcopy. Biopsy was taken in all cases. Diagnostic value of each screening method was determined in terms of sensitivity, specificity, positive predictive value and negative predictive value. RESULTS: Out of 352 patients, around 20% of them were found to have abnormal cytology. The sensitivity and specificity of Pap smear was found to be 34% and 94%. But colposcopy has high sensitivity and low specificity, i.e., 99% and 31%, respectively. On the other hand the sensitivity and specificity of VIA and VILI are comparable i.e., 65% and 45% and 64% and 48% respectively. Pap smear shows high positive predictive value among all, i.e., 85% and colposcopy shows 58% for the same. CONCLUSION: Pap smear carries low sensitivity but high positive predictive value. As compared to Pap smear, VIA and VILI are more sensitive and are of low cost. Colposcopy can be considered as a preferred method of screening due to its extremely high sensitivity.

Pre-Ovulatory Hormones on Day of Human Chorionic Gonadotropin Trigger and Assisted Reproductive Technique Outcomes in Different Ovarian Response Groups.

BACKGROUND: Evidence regarding impact of pre-ovulatory hormone levels on assisted reproductive technique (ART) outcomes in different ovarian response groups is sparse. AIMS: The objective of this study was to evaluate and compare the association between pre-ovulatory hormonal profile and ART outcomes in different ovarian responses. SETTING AND DESIGN: This is a single-centre retrospective cohort study of 273 non-donor fresh ART cycles between January 2013 and June 2016. MATERIALS AND METHODS: Data on clinical profile, basal and peak hormonal levels, characteristics of controlled ovarian stimulation and ART outcomes were collected. Progesterone elevation (PE) was defined as pre-ovulatory serum progesterone >1.5 ng/mL or progesterone to oestradiol ratio >1. The association between peak hormonal levels and ART outcomes in poor (≤4 oocytes retrieved), intermediate (5-13 oocytes retrieved) and high (≥14 oocytes retrieved) ovarian responders was analysed and compared. STATISTICAL ANALYSIS: Continuous and categorical variables were summarised as median (interquartile range) and percentages, respectively, and compared using Kruskal-Wallis H-test or Mann-Whitney U-test and Chi-square test or Fisher's exact test, respectively. RESULTS: The incidence of PE, by both criteria and clinical pregnancy rates (35.7%, 36.8% and 18.6% in high, normal and poor responders, respectively; P = 0.073), was similar among the three response groups. Except fertilisation rates in normo-responders, PE did not influence ART outcomes in any response group. Furthermore, there were no differences between peak hormone concentrations or incidence of PE between those who conceived and those who did not. CONCLUSION: Pre-ovulatory sex steroid levels do not seem to be the primary determinant of ART outcomes in any ovarian response category; hence, decision to freeze all embryos in the event of PE should be tailored.

Design, synthesis and biological evaluation of novel naturally-inspired multifunctional molecules for the management of Alzheimer's disease.

In our overall goal to overcome the limitations associated with natural products for the management of Alzheimer's disease and to develop in-vivo active multifunctional cholinergic inhibitors, we embarked on the development of ferulic acid analogs. A systematic SAR study to improve upon the cholinesterase inhibition of ferulic acid with analogs that also had lower logP was carried out. Enzyme inhibition and kinetic studies identified compound 7a as a lead molecule with preferential acetylcholinesterase inhibition (AChE IC50 = 5.74 ± 0.13 µM; BChE IC50 = 14.05 ± 0.10 µM) compared to the parent molecule ferulic acid (% inhibition of AChE and BChE at 20 µM, 15.19 ± 0.59 and 19.73 ± 0.91, respectively). Molecular docking and dynamics studies revealed that 7a fits well into the active sites of AChE and BChE, forming stable and strong interactions with key residues Asp74, Trp286, and Tyr337 in AChE and with Tyr128, Trp231, Leu286, Ala328, Phe329, and Tyr341 in BChE. Compound 7a was found to be an efficacious antioxidant in a DPPH assay (IC50 = 57.35 ± 0.27 µM), and it also was able to chelate iron. Data from atomic force microscopy images demonstrated that 7a was able to modulate aggregation of amyloid ß1-42. Upon oral administration, 7a exhibited promising in-vivo activity in the scopolamine-induced AD animal model and was able to improve spatial memory in cognitive deficit mice in the Y-maze model. Analog 7a could effectively reverse the increased levels of AChE and BChE in scopolamine-treated animals and exhibited potent ex-vivo antioxidant properties. These findings suggest that 7a can act as a lead molecule for the development of naturally-inspired multifunctional molecules for the management of Alzheimer's and other neurodegenerative disorders.

Relationship of subclinical hypothyroidism and obesity in polycystic ovarian syndrome patients.

OBJECTIVES: To determine the prevalence of obesity and its relationship with subclinical hypothyroidism in women with polycystic ovarian syndrome (PCOS). To compare the clinico-biochemical parameters of obese and lean PCOS patients. MATERIALS AND METHODS: A total of 287 women with PCOS were included in this study after consent. The demographic, anthropometry, clinical, and hormonal (thyroid-stimulating hormone [TSH] and total testosterone) parameters were recorded along with pelvic ultrasonography (USG) for all PCOS subjects. They were divided into lean (body mass index [BMI] between 18.5 and 22.9) and overweight (BMI ≥23), and the number of subclinical hypothyroid patients were calculated in each group. The clinico-biochemical parameters of both groups were compared. RESULTS: The majority (61%) of our patients were overweight. There was no significant difference in the prevalence of subclinical hypothyroidism between overweight and lean PCOS patients. The obese PCOS patients were older than lean PCOS patients, and they had higher serum testosterone with elevated systolic and diastolic blood pressure (BP). CONCLUSION: The majority of our patients were found to be overweight and there was no association between obesity and subclinical hypothyroidism among PCOS patients.

Caffeine-enhanced anti-tumor activity of anti-PD1 monoclonal antibody.

Antibodies targeting PD1 receptor have emerged as a promising therapeutic strategy against multiple types of solid cancers. However, relatively low complete response rates observed with anti-PD1 mAb monotherapy emphasizes the importance of testing new immunotherapeutic combinations. The production of extracellular adenosine in solid tumors was recently identified as a major immunosuppressive pathway, targeting this pathway would enhance the therapeutic activity of anti-PD1 mAbs. In this study, we evaluated the anti-tumor activity and mechanism of action of caffeine and anti-PD1 mAb combination therapy against carcinogen- and cell line-induced tumors. Our results demonstrate that combination therapy enhanced the anti-tumor activity and prolonged overall survival period against 3-MCA-induced tumors. In addition, combination therapy showed a significant anti-tumor activity against B16F10 melanoma tumors. We found that combination therapy showed additive increase in infiltration of CD4+ and CD8+ T lymphocytes into the B16F10 melanoma tumors. On the other hand, combination therapy showed significant decrease in infiltration of CD4+CD25+ T regulatory cells. We further investigated whether the observed anti-tumor effect of caffeine and anti-PD1 mAb combination therapy is mediated through the release of cytokines. We found that caffeine and anti-PD1 mAb combination therapy significantly increased intra-tumoral TNF-α and IFN-γ levels. Our work suggests that administration of caffeine and anti-PD1 mAb harness the therapeutic potential of effector T cells in vivo possibly due to combined blockade of PD1 and adenosine-A2A receptor pathway. This study provides the scientific basis for testing combination regimens of caffeine and anti-PD1 mAbs for sustained tumor control in cancer patients.

Caffeine-enhanced anti-tumor immune response through decreased expression of PD1 on infiltrated cytotoxic T lymphocytes.

Caffeine (1,3,7-trimethylxanthine) is a naturally occurring, habitually consumed food constituent throughout the world. Prospective cohort studies revealed that caffeine consumption reduces the relative risk of various cancers. Avoiding immune destruction is one of the emerging hallmarks of cancer. One of the immunosuppressive pathways that contribute in avoiding immune destruction by cancer cells is adenosine-A2A receptor pathway. Based on prospective epidemiological and mechanistic preclinical studies of caffeine and its predominant antagonistic effect on A2A receptor, we aimed to investigate the effect of caffeine on T cell infiltration into the tumor and expression of PD-1 receptor on T lymphocytes during tumor initiation and progression in a carcinogen-induced tumor model. Our results demonstrate that caffeine treatment significantly lowered tumor incidence and tumor growth rate. We found that the total T-lymphocyte infiltration and CD8+ T lymphocyte infiltration was significantly increased in caffeine-treated groups. On the other hand, the infiltration of CD4+CD25+ regulatory T lymphocyte was significantly decreased in caffeine-treated groups. In addition, the PD-1 expression on CD8+ T lymphocytes and CD4+CD25+ regulatory T lymphocytes was significantly reduced in caffeine-treated groups. We further investigated whether the observed anti-tumor effect of caffeine is mediated through the release of cytokines. We found that TNF-α and IFN-γ levels were significantly higher in caffeine-treated groups. The findings of the present study unraveled the immune-related mechanisms behind the caffeinated coffee consumption and lower tumor incidence in humans. In conclusion, the blockade of adenosine pathway by caffeine may constitute an effective means to enhance anti-tumor immune response.

Ascorbic acid therapy: A potential strategy against comorbid depression-like behavior in streptozotocin-nicotinamide-induced diabetic rats.

This study examined the potency and efficacy of ascorbic acid (AA) in the management of depression-like behavior in diabetic rats. Diabetes mellitus was induced by single intraperitoneal injections of nicotinamide (120 mg/kg) and streptozotocin (65 mg/kg) administered 15 min apart. Diabetic (blood glucose ≥250 mg/dL) rats were subjected to intermittent foot-shocks to induce comorbid depression. Seven groups of diabetes comorbid depressed rats received vehicle (1 mL/kg) or AA (10, 25, 50, 100, 200, or 400 mg/kg) orally for eleven days. Three control groups namely- nondiabetic, diabetic, and depressed rats received the vehicles only. The potency (ED50) and efficacy (Emax) of AA against immobility period, hypercorticosteronemia, adrenal hyperplasia, hyperglycemia, hypoinsulinemia, oxidative stress, and inflammatory response were estimated. AA administration caused a dose-dependent decrease (P < 0.05) in immobility period with maximum inhibition of 69% (efficacy) at 200 mg/kg and ED50 of 14 mg/kg (potency). AA at 200 mg/kg produced the maximal reduction in hypercorticosteronemia (55.1%) and adrenal hyperplasia (52.6%) with ED50 of 9.8 and 14.4 mg/kg, respectively. AA at 400 mg/kg produced the maximal reduction in hyperglycemia (35.5%), hypoinsulinemia (32.7%), and lipid peroxidation (82%) with ED50 of 18.6, 13.7, and 20.7 mg/kg, respectively. Moreover, AA at 400 mg/kg produced the maximal increase in SOD content (83%), CAT activity (77.9%), and IL-10 level (63%) with ED50 of 21.5, 21, and 21 mg/kg, respectively. In conclusion, the present results suggest that AA has therapeutic potential against diabetes comorbid depression but better regulation of hyperglycemia and hypoinsulinemia is required to achieve maximal benefits.

Mechanistic considerations in chemotherapeutic activity of caffeine.

Caffeine (1,3,7-trimethylxanthine) is one of the most commonly consumed food ingredient throughout the world for thousands of years. It is naturally present in beans, leaves, seeds, and fruits of more than 60 plants and the most common sources are cocoa beans, coffee beans, tea leaves, kola nuts, and guarana berries. Several epidemiological studies suggested that consumption of coffee reduces the risk of different types of cancers. Understanding the chemotherapeutic molecular mechanisms of caffeine would facilitate in designing efficacious combination strategies with other anticancer drugs. Therefore, the aim of this review is to identify and highlight all the mechanisms involved in chemotherapeutic activity of caffeine. Mechanistically, caffeine has been shown to affect cell cycle progression at checkpoints, induce apoptosis, and inhibit drug efflux from cells. Caffeine at lower (micromolar) and relatively non-toxic concentrations has been shown to promote anti-tumor immune response (A2 A) and to inhibit tumor angiogenesis (A3) and migration (A2B) by antagonizing adenosine receptors. Considering the fact that a lower (micromolar) and relatively non-toxic concentrations of caffeine are required for its anti-tumor immune response and antiangiogenic activity, developing a combination strategy with immune checkpoint (PD-1 or CTLA-4) inhibitors would represent a novel approach to treat cancer.

Psychological sequelae of myocardial infarction.

Patient with myocardial infarction (MI) are often affected by psychological disorders such as depression, anxiety, and post-traumatic stress disorder. Psychological disorders are disabling and have a negative influence on recovery, reduce the quality of life and causes high mortality rate in MI patients. Despite tremendous advancement in technologies, screening scales, and treatment strategies, psychological sequelae of MI are currently understudied, underestimated, underdiagnosed, and undertreated. Depression is highly prevalent in MI patients followed by anxiety and post-traumatic stress disorder. Pathophysiological factors involved in psychopathologies observed in patients with MI are sympathetic over-activity, hypothalamic-pituitary-adrenal axis dysfunction, and inflammation. Numerous preclinical and clinical studies evidenced a positive association between MI and psychopathologies with a common molecular pathophysiology. This review provides an update on diagnostic feature, prevalence, pathophysiology, clinical outcomes, and management strategies of psychopathologies associated with MI. Moreover, preclinical research findings on molecular mechanisms involved in post-MI psychopathologies and future therapeutic strategies have been outlined in the review.

Metformin and ascorbic acid combination therapy ameliorates type 2 diabetes mellitus and comorbid depression in rats.

Diabetes mellitus and depression are the common comorbid disorders affecting humans worldwide. There is an unmet need to develop therapeutic strategies to treat both diabetes mellitus and comorbid depression. The present study evaluated the effectiveness of metformin and ascorbic acid against type 2 diabetes mellitus and comorbid depression in rats. Four groups of diabetic rats were orally administered with vehicle (1mL/kg), metformin (25mg/kg), ascorbic acid (25mg/kg), or combination of metformin (25mg/kg) and ascorbic acid (25mg/kg) for 11 consecutive days. Diabetes was induced by single-dose administration of streptozotocin (65mg/kg, i.p.) with nicotinamide (120mg/kg, i.p.). Comorbid depression was induced by five inescapable foot-shocks (2mA, 2ms duration) at 10s intervals on days 1, 5, 7, and 10. One group of healthy rats received only vehicles to serve as nondiabetic control group. On day 11, animals were sacrificed, and blood and brain samples were collected from each rat following forced swim test. Plasma glucose, insulin, and corticosterone levels were estimated in plasma. The levels of monoamines, proinflammatory cytokines, and oxidative stress were measured in prefrontal cortex. The combination therapy significantly reduced immobility period, glucose, and corticosterone levels relative to diabetes with comorbid depression group. Furthermore, the combination therapy increased the levels of insulin and monoamines, and caused a significant reductions in oxidative stress and proinflammatory cytokines. In conclusion, the present study revealed that metformin and ascorbic acid combination therapy could be a potential strategy to treat type 2 diabetes mellitus and comorbid depression.