Discovery and Comprehensive Characterization of Novel Circular RNAs of the Apoptosis-Related BOK Gene in Human Ovarian and Prostate Cancer Cells, Using Nanopore Sequencing.

CircRNAs have become a novel scientific research hotspot, and an increasing number of studies have shed light on their involvement in malignant progression. Prompted by the apparent scientific gap in circRNAs from apoptosis-related genes, such as BOK, we focused on the identification of novel BOK circRNAs in human ovarian and prostate cancer cells. Total RNA was extracted from ovarian and prostate cancer cell lines and reversely transcribed using random hexamer primers. A series of PCR assays utilizing gene-specific divergent primers were carried out. Next, third-generation sequencing based on nanopore technology followed by extensive bioinformatics analysis led to the discovery of 23 novel circRNAs. These novel circRNAs consist of both exonic and intronic regions of the BOK gene. Interestingly, the exons that form the back-splice junction were truncated in most circRNAs, and multiple back-splice sites were found for each BOK exon. Moreover, several BOK circRNAs are predicted to sponge microRNAs with a key role in reproductive cancers, while the presence of putative open reading frames indicates their translational potential. Overall, this study suggests that distinct alternative splicing events lead to the production of novel BOK circRNAs, which could come into play in the molecular landscape and clinical investigation of ovarian and prostate cancer.

En-Bloc Resection of Renal Cell Carcinoma With Tumor Thrombus Propagating Into the Intrapericardial Inferior Vena Cava: Efficacy and Safety of Transabdominal Approach.

BACKGROUND: Renal cell carcinoma (RCC) is the most common primary kidney cancer. In up to 4-10% of patients, the tumor is complicated with a malignant thrombus extending to the inferior vena cava (IVC). Complete surgical excision of the RCC and the neoplastic thrombus can be curative. We aim to present a safe and feasible alternative transabdominal operative technique with the omission of thoracotomy, as applied in six patients diagnosed with RCC and IVC thrombus extending over the diaphragm. METHODS: This case series study was conducted in a tertiary university hospital in Athens, Greece. All six patients, who were operated on for RCC and a malignant thrombus exceeding in the intrapericardial IVC in our department from January 2009 until March 2020, were screened. Intraoperatively, the infrarenal and intrapericardial IVC were clamped simultaneously with the renal and liver blood inflow. Access to the intrapericardial IVC was obtained via the central tendon of the diaphragm. Intrathoracic extension of the tumor was confirmed by transesophageal or intraoperative ultrasonography. The intrathoracic IVC was exposed to direct vision and two finger palpation was applied to secure the clamping of the IVC above the tip of the thrombus. The tumor was resected through a longitudinal venotomy and the operation was completed on a standard radical nephrectomy. RESULTS: During the study period six patients presented with RCC and intrapericardial IVC thrombus. All patients, five female and one male, underwent radical nephrectomy combined with IVC thrombectomy, without the need for a thoracotomy. The mean age was 66 years old and the mean operative time was 122.5 minutes. Mean blood loss was 338 ml and only four of the patients were transfused with two units of RBC. Operative and hospital mortality was 0%. The hospital stay was seven (six to nine) days. Only one patient required readmission and reoperation 30 days later, due to intrapericardial herniation. CONCLUSIONS: The proposed surgical technique may be curative in patients with advanced intracaval thrombus and helps reduce the associated morbidity, mortality, and the overall cost of more extended operations.

High Intratumoral i-tRF-GlyGCC Expression Predicts Short-Term Relapse and Poor Overall Survival of Colorectal Cancer Patients, Independent of the TNM Stage.

Colorectal cancer (CRC), one of the most prevalent types of cancer, requires the discovery of new tumor biomarkers for accurate patient prognosis. In this work, the prognostic value of the tRNA fragment i-tRF-GlyGCC in CRC was examined. Total RNA extraction from 211 CRC patient cancer tissue specimens and 83 adjacent normal tissues was conducted. Each RNA extract was subjected to in vitro polyadenylation and reverse transcription. A real-time quantitative PCR assay was used to quantify i-tRF-GlyGCC in all samples. Extensive biostatics analysis showed that i-tRF-GlyGCC levels in CRC tissues were significantly lower than in matched normal colorectal tissues. Additionally, the disease-free survival (DFS) and overall survival (OS) time intervals were considerably shorter in CRC patients with high i-tRF-GlyGCC expression. i-tRF-GlyGCC expression maintained its prognostic value independently of other established prognostic factors, as shown by the multivariate Cox regression analysis. Additionally, survival analysis after TNM stage stratification revealed that higher i-tRF-GlyGCC levels were linked to shorter DFS time intervals in patients with TNM stage II tumors, as well as an increased probability of having a worse OS for patients in TNM stage II. In conclusion, i-tRF-GlyGCC has the potential to be a useful molecular tissue biomarker in CRC, independent of other clinicopathological variables.

MicroRNA-675-5p Overexpression Is an Independent Prognostic Molecular Biomarker of Short-Term Relapse and Poor Overall Survival in Colorectal Cancer.

Colorectal cancer (CRC) is the main cause of cancer-related deaths globally, highlighting the importance of accurate biomarkers for early detection and accurate prognosis. MicroRNAs (miRNAs) have emerged as effective cancer biomarkers. The aim of this study was to investigate the prognostic potential of miR-675-5p as a molecular prognostic biomarker in CRC. For this reason, a quantitative PCR assay was developed and applied to determine miR-675-5p expression in cDNAs from 218 primary CRC and 90 paired normal colorectal tissue samples. To assess the significance of miR-675-5p expression and its association with patient outcome, extensive biostatistical analysis was performed. miR-675-5p expression was found to be significantly downregulated in CRC tissue samples compared to that in adjacent normal colorectal tissues. Moreover, high miR-675-5p expression was associated with shorter disease-free (DFS) and overall survival (OS) in CRC patients, while it maintained its unfavorable prognostic value independently of other established prognostic factors. Furthermore, TNM stage stratification demonstrated that higher miR-675-5p levels were associated with shorter DFS and OS intervals, particularly in patients with CRC of TNM stage II or III. In conclusion, our findings suggest that miR-675-5p overexpression constitutes a promising molecular biomarker of unfavorable prognosis in CRC, independent of other established prognostic factors, including TNM staging.

Identification of Two Novel Circular RNAs Deriving from BCL2L12 and Investigation of Their Potential Value as a Molecular Signature in Colorectal Cancer.

The utility of circular RNAs (circRNAs) as molecular biomarkers has recently emerged. However, only a handful of them have already been studied in colorectal cancer (CRC). The purpose of this study was to identify new circRNAs deriving from BCL2L12, a member of the BCL2 apoptosis-related family, and investigate their potential as biomarkers in CRC. Total RNA extracts from CRC cell lines and tissue samples were reversely transcribed. By combining PCR with divergent primers and nested PCR followed by Sanger sequencing, we were able to discover two BCL2L12 circRNAs. Subsequently, bioinformatical tools were used to predict the interactions of these circRNAs with microRNAs (miRNAs) and RNA-binding proteins (RBPs). Following a PCR-based pre-amplification, real-time qPCR was carried out for the quantification of each circRNA in CRC samples and cell lines. Biostatistical analysis was used to assess their potential prognostic value in CRC. Both novel BCL2L12 circRNAs likely interact with particular miRNAs and RBPs. Interestingly, circ-BCL2L12-2 expression is inversely associated with TNM stage, while circ-BCL2L12-1 overexpression is associated with shorter overall survival in CRC, particularly among TNM stage II patients. Overall, we identified two novel BCL2L12 circRNAs, one of which can further stratify TNM stage II patients into two subgroups with substantially distinct prognosis.

Revised Exon Structure of l-DOPA Decarboxylase (DDC) Reveals Novel Splice Variants Associated with Colorectal Cancer Progression.

Colorectal cancer (CRC) is a highly heterogenous malignancy with an increased mortality rate. Aberrant splicing is a typical characteristic of CRC, and several studies support the prognostic value of particular transcripts in this malignancy. l-DOPA decarboxylase (DDC) and its derivative neurotransmitters play a multifaceted role in physiological and pathological states. Our recent data support the existence of 6 DDC novel exons. In this study, we investigated the existence of additional DDC novel exons and transcripts, and their potential value as biomarkers in CRC. Next-generation sequencing (NGS) in 55 human cell lines coupled with Sanger sequencing uncovered 3 additional DDC novel exons and 20 splice variants, 7 of which likely encode new protein isoforms. Eight of these transcripts were detected in CRC. An in-house qPCR assay was developed and performed in TNM II and III CRC samples for the quantification of transcripts bearing novel exons. Extensive biostatistical analysis uncovered the prognostic value of specific DDC novel exons for patients' disease-free and overall survival. The revised DDC exon structure, the putative protein isoforms with distinct functions, and the prognostic value of novel exons highlight the pivotal role of DDC in CRC progression, indicating its potential utility as a molecular biomarker in CRC.

Health-Related Quality of Life and Cosmesis After Thyroidectomy: Long-Term Outcomes.

BACKGROUND: Health-related quality of life (HRQoL) after thyroidectomy has been recently reported with conflicting conclusions. In this study, we assess HRQoL and neck scar cosmesis of thyroid patients several years after thyroidectomy for benign and malignant pathology. METHODS: Three hundred and thirty patients who underwent thyroidectomy between 2000 and 2010 answered the SF-36 Health Survey and Patient Scar Assessment Questionnaire (PSAQ) in 2010 and at the end of 2018. Changes in the SF-36 and PSAQ scores were analyzed taking into account various demographic, surgical and medical characteristics of the patients. RESULTS: Patients reported worse SF-36 scores after 8.5 years, in scales of physical functioning (p < 0.001), social role functioning (p = 0.002), bodily pain (p = 0.001) and general health perceptions (p < 0.001). Interestingly enough, there were no significant changes in scales of physical role functioning (p = 0.304), mental health (p = 0.681), emotional role functioning (p = 0.903) and vitality (p = 0.121). Multivariate analysis showed that several chronic diseases were related to worse HRQoL scores. On the other hand, PSAQ appearance, symptoms and consciousness scores improved during this period (p < 0.001). CONCLUSIONS: In the long term, patients undergoing thyroidectomy do not show worse HRQoL outcomes in terms of mental health, emotional functioning and vitality, whereas scar cosmesis perception is improved. They show deteriorated outcomes in terms of physical, social functioning and bodily pain, which is mainly related to specific chronic diseases that are common to the aging person.

miR-34a overexpression predicts poor prognostic outcome in colorectal adenocarcinoma, independently of clinicopathological factors with established prognostic value.

OBJECTIVES: MicroRNA-34a (miR-34a) is regulated by TP53 and, in response, downregulates the expression of a gamut of protein-coding genes, including apoptosis regulators, transcription factors, cyclins, and cyclin-dependent kinases. Its upregulation initiates a reprogramming of gene expression and promotes apoptosis. The purpose of this study was the investigation of the potential clinical significance of miR-34a as a molecular prognostic biomarker in colorectal adenocarcinoma using an in-house real-time quantitative PCR (qPCR) methodology. DESIGN AND METHODS: Total RNA was extracted from 113 primary colorectal adenocarcinoma specimens and 61 paired non-cancerous colorectal tissue samples. After polyadenylation and reverse transcription, miR-34a molecules were determined using qPCR based on SYBR Green chemistry. Calculations were performed using the comparative CT method. Finally, extensive biostatistical analysis was performed. RESULTS: miR-34a expression does not significantly differ between colorectal adenocarcinoma tissue specimens and adjacent non-cancerous mucosae. However, miR-34a expression increases progressively as colorectal adenocarcinoma loses its differentiation, being highest in grade III tumors (P=0.010). Moreover, miR-34a expression is a potential unfavorable prognostic biomarker in colorectal adenocarcinoma, predicting poor disease-free and overall survival (P=0.002 and P=0.019, respectively), independently of classical clinicopathological parameters. Most importantly, miR-34a expression stratifies patients without local (N0) and/or distant metastasis (M0) at the time of diagnosis into two groups with substantially different prognosis (P=0.013 and P=0.002, respectively). CONCLUSIONS: High miR-34a levels in colorectal adenocarcinoma predict a rather increased risk for disease recurrence and poor overall survival, particularly in patients at an early TNM stage. The unfavorable prognostic potential of miR-34a expression is independent of established prognostic features of colorectal adenocarcinoma.

Elevated expression of miR-24-3p is a potentially adverse prognostic factor in colorectal adenocarcinoma.

OBJECTIVES: Several miRNAs are aberrantly expressed in cancer. miR-24-3p is involved in cancer-related cellular processes, including cell cycle control, cell growth, proliferation, and apoptosis. In this study, we examined the potential diagnostic and prognostic significance of miR-24-3p expression in colorectal adenocarcinoma. DESIGN AND METHODS: Total RNA was isolated from 182 colorectal adenocarcinoma specimens and 86 paired non-cancerous colorectal mucosae. After polyadenylation of 2µg total RNA and reverse transcription into first-strand cDNA using an oligo-dT-adapter primer, miR-24-3p expression was quantified using an in-house-developed reverse-transcription real-time quantitative PCR method, based on the SYBR Green chemistry. SNORD43 (RNU43) was used as a reference gene. RESULTS: miR-24-3p levels do not significantly differ between colorectal adenocarcinoma and non-cancerous colorectal mucosae. Thus, miR-24-3p expression cannot be used for diagnostic purposes. However, high miR-24-3p expression predicts poor disease-free survival (DFS) and overall survival (OS) of colorectal adenocarcinoma patients. Multivariate Cox regression analysis confirmed that miR-24-3p overexpression is a significant predictor of relapse in colorectal adenocarcinoma and that its prognostic significance is independent of other established prognostic factors and treatment of patients. Of note, miR-24-3p overexpression retains its rather unfavorable prognostic value in the subgroup of patients with advanced yet locally restricted colorectal adenocarcinoma (T3) and in those without distant metastasis (M0). Moreover, miR-24-3p overexpression is a potentially unfavorable prognosticator for patients who were not treated with radiotherapy. CONCLUSIONS: Strong expression of miR-24-3p predicts poor DFS and OS of colorectal adenocarcinoma patients, independently of clinicopathological parameters that are currently used for prognosis in this human malignancy.

KLK11 mRNA expression predicts poor disease-free and overall survival in colorectal adenocarcinoma patients.

BACKGROUND: Dysregulated expression of several KLK family members has been observed in colorectal adenocarcinoma. In the present study, the prognostic value of KLK11 mRNA expression as a molecular tissue biomarker in colorectal adenocarcinoma was examined. MATERIALS & METHODS: Using quantitative real-time PCR, KLK11 mRNA expression was studied in 120 cancerous and 41 paired noncancerous colorectal specimens obtained from 120 patients with primary colorectal adenocarcinoma. RESULTS: A significant upregulation of KLK11 transcripts in colorectal tumors was observed. KLK11 mRNA expression was associated with the depth of tumor invasion and the histological grade. Furthermore, KLK11 mRNA expression predicted poor disease-free and overall survival, independently of patient gender, age, tumor size, location, histological subtype, grade, venous invasion, lymphatic invasion, TNM stage, radiotherapy and chemotherapy treatment. CONCLUSION: KLK11 mRNA expression could be considered as a new molecular prognostic biomarker in colorectal adenocarcinoma, with additional prognostic value in patients with highly invasive tumors and/or positive lymph nodes.

Kallikrein-related peptidase-6 (KLK6) mRNA expression is an independent prognostic tissue biomarker of poor disease-free and overall survival in colorectal adenocarcinoma.

Members of the family of tissue kallikrein and kallikrein-related peptidases possess important prognostic value in cancer. Moreover, the oncogenic role of kallikrein-related peptidase-6 (KLK6) in colorectal cancer has been well documented so far. This study investigated the prognostic value of KLK6 mRNA expression as a molecular tissue biomarker in colorectal adenocarcinoma. For this purpose, KLK6 mRNA expression was studied in 110 primary colorectal adenocarcinomas and 39 paired noncancerous colorectal specimens. A dramatic upregulation of KLK6 mRNA expression was observed in colorectal tumors. KLK6 mRNA overexpression was associated with high depth of tumor invasion, presence of distant metastases, and tumor-node-metastasis (TNM) stage of patients. Furthermore, KLK6 mRNA expression was shown to predict poor disease-free and overall survival independently of patient gender, age, tumor size, location, histological subtype, grade, venous invasion, lymphatic invasion, TNM stage, radiotherapy, and chemotherapy treatment. Moreover, Kaplan-Meier survival analysis revealed that colorectal adenocarcinoma patients with negative regional lymph nodes (N0) and those without distant metastases (M0) harboring KLK6 mRNA-positive colorectal tumors tended to relapse and die earlier than N0 and M0 patients with KLK6 mRNA-negative colorectal adenocarcinoma. Thus, KLK6 mRNA expression could be considered as an independent, unfavorable molecular prognostic biomarker in colorectal adenocarcinoma, with additional prognostic value in patients without regional or distant metastases.

A structured autopsy-based audit of 370 firearm fatalities: Contribution to inform policy decisions and the probability of the injured arriving alive at a hospital and receiving definitive care.

The objectives of this autopsy-based audit of firearm-related fatalities were to acquire data to inform policy decisions and to assess the probability of the injured arriving alive at a hospital and receiving definitive care. EVALUATED VARIABLES: Demographics; co-morbidities; location and intention of the injury; toxicology; types of firearms; Abbreviated Injury Scale; Injury Severity Score (ISS); transfer means and time; and location of death. RESULTS: Of a total of 370 fatalities, 85.7% were male. The median age was 38 (9-95) years. Suicides (47%) and assaults (45.1%) were the most common underlying intentions. The most seriously injured regions were the head (44.5%), thorax (25.7%), abdomen (10.7%), and spine (5.7%). Of the 370 total subjects, 4.9% had an ISS<16 and 59.5% had an ISS≤74; both groups were classified as potentially preventable deaths. The majority (84%) died at the scene, and only 9.8% left the emergency department alive for further treatment. Multivariate analyses documented that postmortem ISS is an independent factor that predicts the probability of the injured reaching a hospital alive and receiving definitive care. Individuals injured in greater Athens and those most seriously injured in the face, abdomen or spine had significantly greater chances of reaching a hospital alive and receiving definitive care, whereas those injured by a shotgun and the positive toxicology group were significantly less likely to. In conclusion, this study provides data to inform policy decisions, calls for a surveillance network and establishes a baseline for estimating the probability regarding the location of firearm-related deaths.

Evaluation of carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule EpCAM (GA733-2), and carbohydrate antigen 19-9 (CA 19-9) levels in colorectal cancer patients and correlation with clinicopathological characteristics.

BACKGROUND: Colorectal cancer (CRC) is a major public health problem and one of the leading causes of death worldwide. The aim of our study was: a) to determine the CEA, CA 19-9, EGFR, and EpCAM (GA733-2) levels both in healthy volunteers and in colorectal cancer patients, b) to evaluate the ELISA method for EGFR and EpCAM (GA733-2) measurement, and c) to correlate the tumor marker levels with clinicopathological findings in the CRC patients group. METHODS: Our study was conducted on 50 blood samples obtained from CRC patients and 40 blood samples from healthy individuals. CEA and CA 19-9 measurements were performed using electrochemiluminescence immune-assay technology, while EGFR and EpCAM (GA733-2) measurements were performed by an in-house enzyme immunoassay. RESULTS: CEA, CA 19-9, and EpCAM (GA733-2) levels were higher in the CRC patients group than in the control group. EGFR levels were lower in the patients group than in the control group. The mean levels of CA 19-9 and EpCAM (GA733-2) vary at different colon cancer stages. CEA, CA19-9, and EpCAM (GA733-2) vary according to performance status. CONCLUSIONS: CEA, CA 19-9, and EpCAM (GA733-2) showed similar specificity (80%, 80% and 84%, respectively). EGFR showed the lowest sensitivity and specificity. CA 19-9 was the marker with the highest sensitivity. The need for convenient tumour marker tests with high sensitivity is of great importance for early diagnosis and monitoring of CRC.

Asymptomatic omental granuloma following spillage of gallstones during laparoscopic cholecystectomy protects patients and influences surgeons' decisions: a review.

Spillage of gallstones in the peritoneal cavity during laparoscopic cholecystectomy (LC) occurs at rates varying from 5.7% to 16%. These gallstones often cannot be retrieved and can cause early and late abscesses at rates ranging from 0.08% to 1.4%. The case of an 86-year-old woman with colon cancer is described because during an elective right hemicolectomy a granuloma of the omentum with retained gallstones from LC performed 8 years earlier was unexpectedly found. Importantly, the gallstones were found high up in the abdominal cavity. Moreover, this report reaffirms the excellent response of the peritoneal cavity defence mechanisms for protecting patients against gallstones through asymptomatic omental granuloma. Current data indicate that every effort should be made to retrieve spilled gallstones, but routine conversion to an open cholecystectomy is not recommended. Identifying factors that impair host defence mechanisms should help surgeons' decision-making.

Patients with pelvic fractures due to falls: A paradigm that contributed to autopsy-based audit of trauma in Greece.

BACKGROUND: Evaluation of the pelvic fractures (PFx) population in auditing effective components of trauma care is the subject of this study. METHODS: A retrospective, case-control, autopsy-based study compared a population with PFx to a control-group using a template with trauma outcome variables, which included demographics, ICD-9, intention, mechanisms, toxicology, Abbreviated Injury Scale (AIS-90), Injury Severity Score (ISS), causes of haemorrhage, comorbidity, survival time, pre-hospital response, in hospital data, location of death, and preventable deaths. RESULTS: Of 970 consecutive patients with fatal falls, 209 (21.5%) had PFx and constituted the PFx-group while 761 (78.5%) formed the control-group.Multivariate analysis showed that gender, age, intention, and height of fall were risk factors for PFx. A 300% higher odds of a psychiatric history was found in the PFx-group compared to the control-group (p < 0.001).The median ISS was 50 (17-75) for the PFx-group and 26 (1-75) for the control-group (p < 0.0001). There were no patients with an ISS less than 16 in the PFx group.Associated injuries were significantly more common in the PFx-group than in the control-group. Potentially preventable deaths (ISS < 75) constituted 78% (n = 163) of the PFx-group. The most common AIS3-5 injuries in the potentially preventable subset of patients were the lower extremities in 133 (81.6%), thorax in 130 (79.7%), abdomen/pelvic contents in 99 (60.7%), head in 95 (58.3%) and the spine in 26 (15.9%) patients.A subset of 126 (60.3%) potentially preventable deaths in the PFx-group had at least one AIS-90 code other than the PFx, denoting major haemorrhage. Deaths directly attributed to PFx were limited to 6 (2.9%).The median survival time was 30 minutes for the PFx-group and 20 hours for the control-group (p < 0.001). For a one-group increment in the ISS-groups, the survival rates over the post-traumatic time intervals were reduced by 57% (p < 0.0001).Pre-hospital mortality was significantly higher in the PFx-group i.e. 70.3% of the PFx-group versus 42.7% of the control-group (p < 0.001). CONCLUSIONS: The PFx-group shared common causative risk factors, high severity and multiplicity of injuries that define the PFx-group as a paradigm of injury for audit. This reduced sample of autopsies substantially contributed to the audit of functional, infrastructural, management and prevention issues requiring transformation to reduce mortality.

Perforated mucinous cystadenoma of the vermiform appendix: an overview in reasoning clinical decisions.

Recent advances in the management of appendiceal mucinous neoplasms (AMN) such as peritonectomy combined with hyperthermic intraperitoneal chemotherapy have introduced new standards of care. However, many dilemmas are encountered in decision making as in the following patient. A 74-year-old woman was admitted with an appendiceal cystadenoma found in a preadmission CT scan. However, the tumour was not documented by the in hospital investigation due to its perforation and its reduction in size. Consequently, a series of management dilemmas were encountered that were solved by cautious evaluation of the pre and peroperative findings. She was submitted to a right hemicolectomy. A spontaneous perforation was suspected, but the accurate diagnosis was documented postoperatively by histopathology. This paradigm motivated this review which concluded that reasoning clinical decisions in the light of recent advances and appropriate care based on the disease-stage are essential for an optimal outcome in the management of AMN.

Flavonols inhibit proinflammatory mediator release, intracellular calcium ion levels and protein kinase C theta phosphorylation in human mast cells.

Mast cells participate in allergies, and also in immunity and inflammation by secreting proinflammatory cytokines. Flavonoids are naturally occurring polyphenolic plant compounds, one group of which -- the flavonols, inhibits histamine and some cytokine release from rodent basophils and mast cells. However, the effect of flavonols on proinflammatory mediator release and their possible mechanism of action in human mast cells is not well defined. Human umbilical cord blood-derived cultured mast cells (hCBMCs) grown in the presence of stem cell factor (SCF) and interleukin (IL)-6 were preincubated for 15 min with the flavonols quercetin, kaempferol, myricetin and morin (0.01, 0.1, 1, 10 or 100 microM), followed by activation with anti-IgE. Secretion was quantitated for IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), histamine and tryptase levels. Release of IL-6, IL-8 and TNF-alpha was inhibited by 82-93% at 100 microM quercetin and kaempferol, and 31-70% by myricetin and morin. Tryptase release was inhibited by 79-96% at 100 microM quercetin, kampferol and myricetin, but only 39% by morin; histamine release was inhibited 52-77% by the first three flavonols, but only 28% by morin. These flavonols suppressed intracellular calcium ion elevations in a dose-response manner, with morin being the weakest; they also inhibited phosphorylation of the calcium-insensitive protein kinase C theta (PKC theta). Flavonol inhibition of IgE-mediated proinflammatory mediator release from hCBMCs may be due to inhibition of intracellular calcium influx and PKC theta signaling. Flavonols may therefore be suitable for the treatment of allergic and inflammatory diseases.

Increased numbers of activated mast cells in endometriosis lesions positive for corticotropin-releasing hormone and urocortin.

PROBLEM: Mast cells are critical in allergic and inflammatory diseases such as interstitial cystitis, which is often clinically associated with or mistaken as endometriosis. Mast cells had previously been reported to be increased at sites of endometriosis, and tryptase may contribute to the fibrosis and inflammation characterizing endometriosis. METHOD OF STUDY: This is a pilot study of mast cell numbers and its activation in endometriosis biopsies (n = 10) by immunostaining for mast cell tryptase, corticotropin-releasing hormone (CRH) and urocortin (Ucn). RESULTS: This is the first report that tryptase positive mast cells were not only increased (64-157 mast cells/mm(2)) in human endometriosis, but also highly activated (89%) in areas strongly stained positive for CRH/Ucn. Normal endometrium was weakly positive for both CRH/Ucn. CONCLUSION: High numbers of activated mast cells are present in endometriosis sites that were strongly positive for CRH/Ucn. CRH and Ucn may activate mast cells and contribute to the fibrosis and inflammation in endometriosis.

Corticotropin-releasing hormone and its structurally related urocortin are synthesized and secreted by human mast cells.

Stress activates the hypothalamic-pituitary-adrenal axis through CRH, leading to production of glucocorticoids that down-regulate immune responses. However, acute stress also has proinflammatory effects. We previously showed that restraint stress, as well as CRH and its structurally related urocortin (Ucn), could activate mast cells and trigger mast cell-dependent vascular permeability. Here we show for the first time that human cord blood-derived cultured mast cells (hCBMC) at 10 wk, but not at 2 wk, are immunocytochemically positive for CRH and Ucn; human leukemic mast cells are weakly positive for both peptides. The ability of these mast cells to synthesize CRH and Ucn was confirmed by showing mRNA expression with RT-PCR. hCBMC (8-14 wk) synthesize and store 1-10 ng/106 cells (10-20 microg/g) of both CRH and Ucn detected by ELISA of cell homogenates. Stimulation of IgE-sensitized hCBMC with anti-IgE results in secretion of most CRH and Ucn. These findings indicate that mast cells are not only the target, but also a potential source of CRH and Ucn that could have both autocrine and paracrine functions, especially in allergic inflammatory disorders exacerbated by stress.

High levels of intrauterine corticotropin-releasing hormone, urocortin, tryptase, and interleukin-8 in spontaneous abortions.

Stress induces CRH secretion that activates hypothalamic-pituitary-adrenal axis and is also abortogenic. In addition to hypothalamus, CRH and its analog urocortin (Ucn) are also secreted locally outside the brain where they activate mast cells leading to inflammation; however, the level of CRH and Ucn or mast cell mediators has not been examined in products of conception (POC). CRH and Ucn were measured by enzyme immunoassay, tryptase by fluoroenzyme immunoassay, and IL-8 by ELISA in POC of 7-9 wk gestation from Caucasian women; they were divided into group I with elective abortions (n = 4), group II with one spontaneous abortion (n = 12), and group III with at least two spontaneous abortions (n = 7). CRH, Ucn, tryptase, and IL-8 levels were higher (P < 0.05) in group III (8683 +/- 1201 pg/g, 7961 +/- 1499 pg/g, 1553 +/- 572 ng/g, and 8317 +/- 1874 pg/g, respectively) than group II (2561 +/- 314 pg/g, 2349 +/- 394 pg/g, 403 +/- 97 ng/g, and 3199 +/- 449 pg/g, respectively) and group I (163 +/- 162 pg/g, 328 +/- 327 pg/g, 72 +/- 31 ng/g, and 3681 +/- 931 pg/g, respectively). Immunostaining of POC showed significantly more tryptase in group III women. High POC levels of CRH and Ucn under stress in habitual spontaneous abortions may activate uterine mast cells to secrete abortogenic tryptase and IL-8.