Rashmi Kaul

BACKGROUND Hepatocellular carcinoma (HCC) is a global health concern, representing the second most common cause of malignancy-related mortality in the world. The primary cause of HCC in the United States is chronic infection with the hepatitis C virus (HCV). Clinical observations have established sex-based differences in HCV infection with the disease progressing more severely and more rapidly in males and postmenopausal females compared to premenopausal females, suggesting that estrogens and their receptors may play an important role in hepatic defenses and development of HCV-mediated HCC. However, the precise mechanism of estrogen protection and their effects on inflammation is poorly understood. AIM To determine whether estrogen receptor (ER) expression is correlated with the expression of tumor necrosis factor-alpha (TNF-α) in males and females with HCV-associated diseases. METHODS The role of ERs in modulating innate immune responses was investigated using human liver tissues with HCV/cirrhosis and HCV/HCC. Messenger RNA (mRNA) and protein (nuclear and cytoplasmic) expression were measured for all markers of interest and compared to normal human liver tissue samples. RESULTS ERβ was reported for the first time to have a greater mRNA expression than ERα in normal liver (P ≤ 0.001). In addition, ERβ mRNA expression was found to be decreased in diseased livers (P ≤ 0.05), while TNF-α expression was increased (P ≤ 0.0001). Upon stratifying by sex within each disease group, ESR1 was found to be negatively correlated with ESR2 in females with HCV/cirrhosis (r = -0.84, P ≤ 0.001), whereas males with HCV/cirrhosis were found to have a significant positive correlation (r = 0.57, P ≤ 0.05). ESR2 mRNA expression had a significant positive correlation with TNF-α in both HCV/cirrhosis (r = 0.61, P ≤ 0.001) and HCV/HCC patients (r = 0.45, P ≤ 0.05). CONCLUSION All together, these findings indicate that changes in ERβ and TNF-α expression are associated with worsening disease, and may be part of the sex-dependent factors in HCC pathogenesis.

The bladder epithelial cells elicit robust innate immune responses against urinary tract infections (UTIs) for preventing the bacterial colonization. Physiological fluctuations in circulating estrogen levels in women increase the susceptibility to UTI pathogenesis, often resulting in adverse health outcomes. Dr adhesin bearing Escherichia coli (Dr E. coli ) cause recurrent UTIs in menopausal women and acute pyelonephritis in pregnant women. Dr E. coli bind to epithelial cells via host innate immune receptor CD55, under hormonal influence. The role of estrogens or estrogen receptors (ERs) in regulating the innate immune responses in the bladder are poorly understood. In the current study, we investigated the role of ERα, ERβ and GPR30 in modulating the innate immune responses against Dr E. coli induced UTI using human bladder epithelial carcinoma 5637 cells (HBEC). Both ERα and ERβ agonist treatment in bladder cells induced a protection against Dr E. coli invasion via upregulation of TNFα and downregulation of CD55 and IL10, and these effects were reversed by action of ERα and ERβ antagoinsts. In contrast, the agonist-mediated activation of GPR30 led to an increased bacterial colonization due to suppression of innate immune factors in the bladder cells, and these effects were reversed by the antagonist-mediated suppression of GPR30. Further, siRNA-mediated ERα knockdown in the bladder cells reversed the protection against bacterial invasion observed in the ERα positive bladder cells, by modulating the gene expression of TNFα, CD55 and IL10, thus confirming the protective role of ERα. We demonstrate for the first time a protective role of nuclear ERs, ERα and ERβ but not of membrane ER, GPR30 against Dr E. coli invasion in HBEC 5637 cells. These findings have many clinical implications and suggest that ERs may serve as potential drug targets towards developing novel therapeutics for regulating local innate immunity and treating UTIs. Highlights: Estrogen receptor (ER) subtypes regulate the gene expression of innate immune molecules, CD55, TNFα and IL10 in human bladder epithelial cells impacting the bacterial colonization by Dr E. coli . Activation of nuclear ER subtypes, ERα and ERβ, upregulate the gene expression of proinflammatory cytokine, TNFα, but downregulate the gene expression of anti-inflammatory cytokine, IL10, and Dr E. coli colonization receptor, CD55, thus leading to efficient bacterial clearance in human bladder cells. In contrast, activation of the membrane ER subtype, GPR30, shows opposite effects to ERα and ERβ that were mediated on TNFα, IL10 and CD55 gene expression, thus leading to impaired bacterial clearance of Dr E. coli in human bladder cells. ER subtypes can serve as potential drug candidates for designing new therapies to boost or modulate the local immunity in the human bladder preventing the establishment of E. coli infections.

Transcription factor NAM-B1 has a major role in the process of senescence, which results in higher Fe and Zn concentrations in grains of wild wheat (T. durum; Td). The absence of the wild type NAMB1 in T. aestivum (Ta), one of the cardinal crops essential for more than 1/3^rd of the global population, affects Fe and Zn remobilisation to the maturing grain from the flag leaf resulting in lesser micronutrient bioavailability. The cardinal difference in the NAMB1 gene between the two species is the absence of +1bp allele in Ta. Insilico studies using NAMB1 from Td and Ta was performed to explore the variation in the interaction with the conserved cis-element DNA motif (CATGTG) as both the proteins share the same domain, but there are no in silico studies reported of these proteins. The secondary structure, 3d-modelling of the proteins, DNA-protein docking and dynamics have computed by Schrodinger Prime Suite. Predicted secondary structures were energy minimised using Macromodel and docking was performed based on binding energy and hydrogen bonds. Molecular dynamics simulation of NAMB1-Ta and NAMB1-Td individually and with the cis-element motif, performed for 100ns, revealed significant variations in the protein-DNA interaction in Ta. This work provides the modelled 3d-interaction profile caused by a single bp frameshift mutation in understanding the difference in function between NAMB1 orthologs due to lack of NAC domain. The overall computational analysis reveals that NAMB1-Ta and NAMB1-Td proteins display a good amount of dissimilarity in their structure, dynamics and DNA-binding characteristics.

Clinical studies indicate gender bias in cutaneous squamous cell carcinoma (cSCC) incidence with worse prognosis observed in males than in females, suggesting estrogen-mediated protection. In contrast, recent clinical population studies show risk of cSCC by use of oral contraceptives, thus raising controversy. However, animal studies indicate a protective role of estrogen and estrogen receptor (ER)s in cSCC. Currently we have a poor understanding of ERs that are expressed in human cSCC cells and their possible role in malignant transformation. The focus of current study was to determine ER subtype specific expression on cSCC A431 cells and investigate if ER agonist based activation modulates tumor markers CD55 and Cyclin D1 in the cells. ERα, ERβ and G protein-coupled receptor (GPR30) subtype expression at mRNA and protein level was determined in human cSCC A431 cells by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting, respectively. The localization of ER subtypes was determined by confocal microscopy. ER subtype agonist based activation on A431 cells was performed to investigate their role in modulating mRNA and protein expression of tumor markers CD55 and Cyclin D1. A431 cells differentially expressed all three ER subtypes- ERα, ERβ and GPR30 with GPR30 expression being the highest. Confocal studies confirmed that all three ER subtypes were expressed in the cytoplasm and ERα and ERβ lacked nuclear expression. Agonist based activation of both ERα and GPR30 significantly upregulated Cyclin D1 and CD55 expression. Blocking of GPR30 led to significantly downregulation of both Cyclin D1 and CD55 expression. In contrast to ERα and GPR30, ERβ activation significantly downregulated CD55 expression. Taken together, here we demonstrate for the first time that all three ERs- ERα, ERβ and GPR30 are expressed in human A431 cSCC cells and further ER agonist based activation modulates the expression of tumor markers CD55 and Cyclin D1.

The protective role of endogenous estrogen against Urinary Tract Infection (UTI) is well recognized, but the involvement of estrogen receptors (ERs) in modulating immunity in the urinary tract during UTI pathogenesis has not been investigated. The current study investigates the role of ER alpha in modulating immune responses and UTI outcome. Mice were pre-treated with either ER alpha agonist, propyl-pyrazole-triol (PPT), or ER alpha antagonist, methyl-piperidino- pyrazole (MPP), before experimental UTI. The UTI outcome was determined by checking the bacterial load, CD55 and TNF alpha expression in the bladder and kidney tissues. We observed opposite effects of PPT and MPP treatment on bacterial clearance in bladder versus kidney. PPT significantly reduced bacterial load (P < 0.05) only in the kidney, with minimal changes in CD55 and TNF alpha levels. In contrast, MPP showed remarkable bacterial clearance only in the bladder that corresponded with reduced CD55 and TNF alpha expression. MPP treatment in uninfected state induced a significant increase in TNF alpha production (P < 0.05) in the bladder, but not in the kidney. Our results suggest a protective role of ER alpha in the kidney. However, protection in the bladder may be mediated via other ER subtypes that may be involved in boosting the local immune responses. Drugs targeting specific ERs in bladder may serve as an adjunct treatment for boosting immune responses in the urogenital tract for efficient bacterial clearance.

About 25–44% of women will experience at least one episode of recurrent UTI and the causative agent in over 70% of UTI cases is uropathogenic Escherichia coli (UPEC). UPEC cause recurrent UTI by evading the bladder’s innate immune system through internalization into the bladder epithelium where antibiotics cannot reach or be effective. Thus, it is important to develop novel therapeutics to eliminate these intracellular pathogens. Nanodiamonds (NDs) are biocompatible nanomaterials that serve as promising candidates for targeted therapeutic applications. The objective of the current study was to investigate if 6 or 25 nm NDs can kill extracellular and intracellular UPEC in infected bladder cells. We utilized the human bladder epithelial cell line, T24, and an invasive strain of UPEC that causes recurrent UTI. We found that acid-purified 6 nm NDs displayed greater antibacterial properties towards UPEC than 25 nm NDs (11.5% vs 94.2% CFU/mL at 100 μg/mL of 6 and 25 nm, respectively; P.001). Furthermore, 6 nm NDs were better than 25 nm NDs in reducing the number of UPEC internalized in T24 bladder cells (46.1% vs 81.1% CFU/mL at 100 μg/mL of 6 and 25 nm, respectively; P.01). Our studies demonstrate that 6 nm NDs interacted with T24 bladder cells in a dose-dependent manner and were internalized in 2 hours through an actin-dependent mechanism. Finally, internalization of NDs was required for reducing the number of intracellular UPEC in T24 bladder cells. These findings suggest that 6 nm NDs are promising candidates to treat recurrent UTIs.

Estrogen receptor expression in chronic hepatitis C and hepatocellular carcinoma pathogenesis