Human papillomavirus-mediated expression of complement regulatory proteins in human cervical cancer cells.

OBJECTIVES: This study aimed to evaluate the expression pattern of complement regulatory proteins (CRPs) CD46, CD59, and CD55 in HPV-positive (HPV+) & negative (HPV-) cervical cancer cell lines in search of a reliable differential biomarker. STUDY DESIGN: We analysed the expression of CRPs in HPV 16-positive SiHa cell line, HPV 18-positive HeLa cell line, and HPV-negative cell line C33a using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. RESULTS: We observed a differential expression profile of CRPs in HPV+ and HPV- cervical cancer cell lines. The mRNA level of CD59 & CD55 showed a higher expression pattern in HPV+ cells when compared to HPV- cancer cells. However, flow cytometry-based experiments revealed that CD46 was preferentially expressed more in HPV 16-positive SiHa cells followed by HPV 18-positive HeLa cells when compared to HPV- C33a cells. Interestingly, confocal microscopy revealed a high level of CD59 expression in Hela cells and SiHa cells but low expression in HPV- C33a cells. In addition, HPV 18-positive HeLa cells expressed more CD55, which was lower in SiHa cells and very weak in C33a cells. CONCLUSION: The study demonstrates the differential expression of CRPs in both HPV+ and HPV- cervical cancer cells for the first time, and their potential to serve as an early diagnostic marker for cervical carcinogenesis.

Reducing waiting lists for laparoscopic cholecystectomy: An intensive approach to aid COVID-19 recovery.

Background: Laparoscopic cholecystectomy is one of the most frequently performed operations in the United Kingdom, commonly due to symptomatic gallstones. Delay between diagnosis and definitive surgical intervention often leads to a significant readmission rate, growing financial burden and increased complexity of the ultimate surgical intervention. Resource reallocation and reduced operational capacity during the coronavirus disease 2019 (COVID-19) pandemic has led to an impending waiting list crisis. Methods: In an attempt to address the backlog of cases, five intensive dedicated operating lists were allocated for laparoscopic cholecystectomies across a weekend in October 2020 at a single Trust. Prospective data were collected to include baseline demographics, operative procedure, 30-day post-operative outcomes and financial implications. Results: A total of 21 cholecystectomies were performed in total, with a majority ASA 2 (American Society of Anaesthesiologists) for predominantly biliary colic indication. All were completed laparoscopically, with a 90.5% rate for complete resection. There were no reported on-table complications and 81.0% of patients discharged as a day case. Thirty day follow-up revealed a complication rate of 9.5%, with 2 patients requiring oral antibiotics for a superficial wound infection. The 30 day COVID-19 rate was 14.3%. Compared to completion on an average weekday list, the total weekend was estimated to have saved over £70,000 in overall costs. Conclusion: Our study showed that weekend focused operating, with a caveat of careful patient selection and high-quality multidisciplinary working, can be a feasible solution to long waiting lists due to COVID-19 pandemic. It was safe, with avoidance of increased burden on emergency resources, and significantly increased theatre efficiency.

Pooling of Nasopharyngeal Swab Samples To Overcome a Global Shortage of Real-Time Reverse Transcription-PCR COVID-19 Test Kits.

The global outbreak and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have created an urgent need for large-scale testing of populations. There is a demand for high-throughput testing protocols that can be used for efficient and rapid testing of clinical specimens. We evaluated a pooled PCR protocol for testing nasopharyngeal (NP) swabs using known positive/negative and untested clinical samples that were assigned to pools of 5 or 10. In total, 630 samples were used in this study. Individual positive samples with cycle threshold (CT ) values as high as 33 could be consistently detected when pooled with 4 negative samples (pool of 5), and individual positive samples with CT values up to 31 could be consistently detected when pooled with 9 negative samples (pool of 10). Pooling of up to 5 samples can be employed in laboratories for the diagnosis of COVID-19 for efficient utilization of resources, rapid screening of a greater number of people, and faster reporting of test results.

Estrogen receptors in human bladder cells regulate innate cytokine responses to differentially modulate uropathogenic E. coli colonization.

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, ERß 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 ERß 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 ERß 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 ERß 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.

SARS-CoV-2 Genomes From Oklahoma, United States.

Genomic sequencing has played a major role in understanding the pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the current pandemic, it is essential that SARS-CoV-2 viruses are sequenced regularly to determine mutations and genomic modifications in different geographical locations. In this study, we sequenced SARS-CoV-2 from five clinical samples obtained in Oklahoma, United States during different time points of pandemic presence in the state. One sample from the initial days of the pandemic in the state and four during the peak in Oklahoma were sequenced. Previously reported mutations including D614G in S gene, P4715L in ORF1ab, S194L, R203K, and G204R in N gene were identified in the genomes sequenced in this study. Possible novel mutations were also detected in the S gene (G1167V), ORF1ab (A6269S and P3371S), ORF7b (T28I), and ORF8 (G96R). Phylogenetic analysis of the genomes showed similarity to other SARS-CoV-2 viruses reported from across the globe. Structural characterization indicates that the mutations in S gene possibly influences conformational flexibility and motion of the spike protein, and the mutations in N gene are associated with disordered linker region within the nucleocapsid protein.

Estrogen receptor alpha differentially modulates host immunity in the bladder and kidney in response to urinary tract infection.

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 ERα in modulating immune responses and UTI outcome. Mice were pre-treated with either ERα agonist, propyl-pyrazole-triol (PPT), or ERα antagonist, methyl-piperidino-pyrazole (MPP), before experimental UTI. The UTI outcome was determined by checking the bacterial load, CD55 and TNFα 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 TNFα levels. In contrast, MPP showed remarkable bacterial clearance only in the bladder that corresponded with reduced CD55 and TNFα expression. MPP treatment in uninfected state induced a significant increase in TNFα production (P < 0.05) in the bladder, but not in the kidney. Our results suggest a protective role of ERα 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.

Nanodiamonds facilitate killing of intracellular uropathogenic E. coli in an in vitro model of urinary tract infection pathogenesis.

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<0.001). 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<0.01). 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.

AIM: To investigate gender-specific liver estrogen receptor (ER) expression in normal subjects and patients with hepatitis C virus (HCV)-related cirrhosis and hepatocellular carcinoma (HCC). METHODS: Liver tissues from normal donors and patients diagnosed with HCV-related cirrhosis and HCV-related HCC were obtained from the NIH Liver Tissue and Cell Distribution System. The expression of ER subtypes, ERα and ERß, were evaluated by Western blotting and real-time RT-PCR. The subcellular distribution of ERα and ERß was further determined in nuclear and cytoplasmic tissue lysates along with the expression of inflammatory [activated NF-κB and IκB-kinase (IKK)] and oncogenic (cyclin D1) markers by Western blotting and immunohistochemistry. The expression of ERα and ERß was correlated with the expression of activated NF-κB, activated IKK and cyclin D1 by Spearman's correlation. RESULTS: Both ER subtypes were expressed in normal livers but male livers showed significantly higher expression of ERα than females (P < 0.05). We observed significantly higher mRNA expression of ERα in HCV-related HCC liver tissues as compared to normals (P < 0.05) and ERß in livers of HCV-related cirrhosis and HCV-related HCC subjects (P < 0.05). At the protein level, there was a significantly higher expression of nuclear ERα in livers of HCV-related HCC patients and nuclear ERß in HCV-related cirrhosis patients as compared to normals (P < 0.05). Furthermore, we observed a significantly higher expression of phosphorylated NF-κB and cyclin D1 in diseased livers (P < 0.05). There was a positive correlation between the expression of nuclear ER subtypes and nuclear cyclin D1 and a negative correlation between cytoplasmic ER subtypes and cytoplasmic phosphorylated IKK in HCV-related HCC livers. These findings suggest that dysregulated expression of ER subtypes following chronic HCV-infection may contribute to the progression of HCV-related cirrhosis to HCV-related HCC. CONCLUSION: Gender differences were observed in ERα expression in normal livers. Alterations in ER subtype expression observed in diseased livers may influence gender-related disparity in HCV-related pathogenesis.

Purification and functionalization of nanodiamond to serve as a platform for amoxicillin delivery.

Urinary tract infections (UTIs) cost $0.4-0.5 billion a year in the US and is the second most common disease affecting millions of people. As resistance to antibiotics becomes more common, a greater need for alternative treatments is needed. Nanodiamond particles (NDPs) are actively researched as drug delivery platforms due to their biocompatibility, particle size, and stable inert core. This research is aimed at developing NDPs as antibiotic drug delivery platforms for treating UTIs. To this end, 100 nm, 75 nm, 25 nm and 6 nm size NDPs are purified with acid and heat treatment techniques. Raman spectra of the NDPs showed that the acid treatment method resulted in higher diamond yield. Fourier transform infrared spectroscopy (FTIR) studies showed that both purification techniques result in oxygen terminated surface groups. Efficiency of loading amoxicillin on 25 nm NDPs based on electrostatic interaction of NDPs, functionalizing surfaces of NDPs with hydrogen, and polyethylenimine (PEI) are investigated. It is found that the electrostatic and surface hydrogenation approaches are not efficient in loading amoxicillin on the NDPs. On the other hand, PEI functionalized NDPs produced successful loading with amoxicillin as indicated by the presence of the ß-lactam peak at 1770 cm(-1), amide peak at 1680 cm(-1), and bond between PEI NH stretching and amoxicillin -COOH group at 3650 cm(-1) by the FTIR spectra. These results are expected to lay the foundation for developing NDP based targeted drug delivery treatment techniques for treating UTIs and other infectious diseases.

Spontaneous Atraumatic Mediastinal Hemorrhage: Challenging Management of a Life-Threatening Condition and Literature Review.

Spontaneous atraumatic mediastinal hematomas are rare. We present a case of a previously fit and well middle-aged lady who presented with acute breathlessness and an increasing neck swelling and spontaneous neck bruising. On plain chest radiograph, widening of the mediastinum was noted. The bruising was later confirmed to be secondary to mediastinal hematoma. This life-threatening diagnostic conundrum was managed conservatively with a multidisciplinary team approach involving upper gastrointestinal and thoracic surgeons, gastroenterologists, radiologists, intensivists, and hematologists along with a variety of diagnostic modalities. A review of literature is also presented to help surgeons manage such challenging and complicated cases.

Status of bacterial colonization, Toll-like receptor expression and nuclear factor-kappa B activation in normal and diseased human livers.

Epidemiological data on bacterial translocation (BT), colonization and inflammation in normal human livers is lacking. In this study we investigated the status of bacterial colonization and inflammation in the normal, cirrhotic primary biliary cirrhosis (PBC), and nonalcoholic steatohepatitis (NASH) human liver tissues. Comparatively normal livers showed increased bacterial colonization than PBC and NASH. We analyzed mRNA levels of Toll-like receptors (TLR) 2 and TLR4, and protein levels of TLR4. Phosphorylated IKKα (pIKKα) protein estimation served as a marker for nuclear factor-kappa B (NF-κB) activation. In spite of the increased bacterial colonization in normal liver tissues, lower levels of TLR2/4 mRNA and TLR4 and pIKKα proteins were found compared to PBC and NASH indicating the maintenance of suppressed inflammation and immune tolerance in normal livers. To our knowledge, this is the first clinical evidence showing suppressed inflammation despite bacterial colonization in normal human livers thus maintaining liver immune homeostasis.

Role of sex steroid receptors in pathobiology of hepatocellular carcinoma.

The striking gender disparity observed in the incidence of hepatocellular carcinoma (HCC) suggests an important role of sex hormones in HCC pathogenesis. Though the studies began as early as in 1980s, the precise role of sex hormones and the significance of their receptors in HCC still remain poorly understood and perhaps contribute to current controversies about the potential use of hormonal therapy in HCC. A comprehensive review of the existing literature revealed several shortcomings associated with the studies on estrogen receptor (ER) and androgen receptor (AR) in normal liver and HCC. These shortcomings include the use of less sensitive receptor ligand binding assays and immunohistochemistry studies for ERalpha alone until 1996 when ERbeta isoform was identified. The animal models of HCC utilized for studies were primarily based on chemical-induced hepatocarcinogenesis with less similarity to virus-induced HCC pathogenesis. However, recent in vitro studies in hepatoma cells provide newer insights for hormonal regulation of key cellular processes including interaction of ER and AR with viral proteins. In light of the above facts, there is an urgent need for a detailed investigation of sex hormones and their receptors in normal liver and HCC. In this review, we systematically present the information currently available on androgens, estrogens and their receptors in normal liver and HCC obtained from in vitro, in vivo experimental models and clinical studies. This information will direct future basic and clinical research to bridge the gap in knowledge to explore the therapeutic potential of hormonal therapy in HCC.

Quantification of penicillin G during labor and delivery by capillary electrophoresis.

In this study, a capillary electrophoresis (CE) method was developed as a means to measure levels of penicillin G (PCN G) in Group B Streptococcus (GBS) positive pregnant women during labor and delivery. Volunteers for this developmental study were administered five million units of PCN G at the onset of labor. Urine, blood, and amniotic fluid samples were collected during labor and post delivery. Samples were semi-purified by solid-phase extraction (SPE) using Waters tC18 SepPak 3cc cartridges with a sodium phosphate/methanol step gradient for elution. Capillary electrophoresis or reversed-phase high-performance liquid chromatography (RP-HPLC) with diode-array absorbance detection were used to separate the samples in less than 30 min. Quantification was accomplished by establishing a calibration curve with a linear dynamic range. The tC18 SPE methodology provided substantial sample clean-up with high recovery yields of PCN G ( approximately 90%). It was found that SPE was critical for maintaining the integrity of the separation column when using RP-HPLC, but was not necessary for sample analysis by CE where no stationary phase is present. Quantification results ranged from millimolar concentrations of PCN G in maternal urine to micromolar concentrations in amniotic fluid. Serum and cord blood levels of PCN G were below quantification limits, which is likely due to the prolonged delay in sample collection after antibiotic administration. These results show that CE can serve as a simple and effective means to characterize the pharmacokinetic distribution of PCN G from mother to unborn fetus during labor and delivery. It is anticipated that similar methodologies have the potential to provide a quick, simple, and cost-effective means of monitoring the clinical efficacy of PCN G and other drugs during pregnancy.

A comparative review of HLA associations with hepatitis B and C viral infections across global populations.

Hepatitis B (HBV) and hepatitis C (HCV) viral infection or co-infection leads to risk of development of chronic infection, cirrhosis and hepatocellular carcinoma (HCC). Immigration and globalization have added to the challenges of public health concerns regarding chronic HBV and HCV infections worldwide. The aim of this study is to review existing global literature across ethnic populations on HBV and HCV related human leukocyte antigen (HLA) associations in relation to susceptibility, viral persistence and treatment. Extensive literature search was conducted to explore the HLA associations in HBV and HCV infections reported across global populations over the past decade to understand the knowledge status, weaknesses and strengths of this information in different ethnic populations. HLA DR13 is consistently associated with HBV clearance globally. HLADRB1*11/*12 alleles and DQB1*0301 are associated with HBV persistence but with HCV clearance worldwide. Consistent association of DRB1*03 and *07 is observed with HCV susceptibility and non-responsiveness to HBV vaccination across the population. HLA DR13 is protective for vertical HBV and HCV transmission in Chinese and Italian neonates, but different alleles are associated with their susceptibility in these populations. HLA class I molecule interactions with Killer cell immunoglobulin like receptors (KIR) of natural killer (NK) cells modulate HCV infection outcome via regulating immune regulatory cells and molecules. HLA associations with HBV vaccination, interferon therapy in HBV and HCV, and with extra hepatic manifestations of viral hepatitis are also discussed. Systematic studies in compliance with global regulatory standards are required to identify the HLA specific viral epitope, stage specific T cell populations interacting with different HLA alleles during disease progression and viral clearance of chronic HBV or HCV infections among different ethnic populations. These studies would facilitate stage specific therapeutic strategies for clearance of HBV and HCV infections or co-infections across global populations and aid in identification of HBV-HCV combined vaccine. HLA associations of chronic HBV or HCV development with confounding host factors including alcohol, drug abuse, insulin resistance, age and gender are lacking and warrant detailed investigation across global populations.

Global molecular epidemiology of the O15:K52:H1 extraintestinal pathogenic Escherichia coli clonal group: evidence of distribution beyond Europe.

Escherichia coli O15:K52:H1 is a significant extraintestinal pathogen in Europe (G. Prats et al., J. Clin. Microbiol. 38:201-209, 2000). To search for evidence of this clonal group outside of Europe, 75 non-European E. coli isolates of serogroup O15 were compared with five members of the O15:K52:H1 clonal group from Barcelona, Spain, according to genomic background, virulence genotypes, and antimicrobial resistance profiles. Amplification phylotyping showed that 16 (21%) of the 75 non-European O15 isolates corresponded with the O15:K52:H1 clonal group. The 16 non-European O15:K52:H1 clonal group members represented diverse geographic locales. They were isolated almost exclusively from humans with extraintestinal infections and accounted for 50% of all O15 isolates from five human clinical collections studied. Most non-European clonal group members exhibited a consensus virulence factor profile that included the F16 or F7-2 papA alleles (P fimbrial structural subunit), papG allele II (P fimbrial adhesin), iha (putative adhesin siderophore), and iutA (aerobactin receptor). This resembles the virulence profiles of (i) European representatives of the O15:K52:H1 clonal group and (ii) phylogenetically related "clonal group A," a recently recognized significant contributor to trimethoprim-sulfamethoxazole resistance in the United States (A. R. Manges et al., N. Engl. J. Med. 345:1007-1013, 2001). Antimicrobial resistance profiles were variable, and resistance was inconsistently transferred by conjugation. These findings indicate that the O15:K52:H1 clonal group is broadly distributed beyond Europe, exhibits previously unrecognized phenotypic and genotypic diversity, and contributes significantly to extraintestinal infections in humans.