Culturomics identified 11 new bacterial species from a single anorexia nervosa stool sample.

The rebirth of bacterial culture has been highlighted successively by environmental microbiologists, the design of axenic culture for intracellular bacteria in clinical microbiology, and, more recently, by human gut microbiota studies. Indeed, microbial culturomics (large scale of culture conditions with the identification of colonies by MALDI-TOF or 16S rRNA) allowed to culture 32 new bacterial species from only four stool samples studied. We performed culturomics in comparison with pyrosequencing 16S rRNA targeting the V6 region on an anorexia nervosa stool sample because this clinical condition has never been explored before by culture, while its composition has been observed to be atypical by metagenomics. We tested 88 culture conditions generating 12,700 different colonies identifying 133 bacterial species, with 19 bacterial species never isolated from the human gut before, including 11 new bacterial species for which the genome has been sequenced. These 11 new bacterial species isolated from a single stool sample allow to extend more significantly the repertoire in comparison to the bacterial species validated by the rest of the world during the last 2 years. Pyrosequencing indicated a dramatic discrepancy with the culturomics results, with only 23 OTUs assigned to the species level overlapping (17 % of the culturomics results). Most of the sequences assigned to bacteria detected only by pyrosequencing belonged to Ruminococcaceae, Lachnospiraceae, and Erysipelotrichaceae constituted by strictly anaerobic species, indicating the future route for culturomics. This study revealed new bacterial species participating significantly to the extension of the gut microbiota repertoire, which is the first step before being able to connect the bacterial composition with the geographic or clinical status.

Destruction of human solid tumors by alkyl lysophospholipids.

Alkyl lysophospholipids (ALP) are synthetic analogs of the naturally occurring 2-lysophosphatidylcholine. The effect of ALP on the proliferation of 22 different gynecologic malignant tumors in humans was studied in vitro. ALP caused progressive death of tumor cells over 24-96 hours. In addition, tumor specimens from the tested human tumors were xenotransplanted into NMRI nude mice. ALP induced a highly significant retardation of the in vivo growing human tumors. Neither oral nor iv administration of these compounds caused any recognizable side effects.

Prognostic relevance of ploidy, proliferation, and resistance-predictive tests in ovarian carcinoma.

In a cooperative study specimens of 37 patients with stage III and IV ovarian carcinomas who had been treated with chemotherapy were investigated utilizing flow cytometry and an in vitro short-term test for predicting resistance. Patients with aneuploid tumors had significantly shorter survival rates than did those with diploid tumors. Patients whose tumors showed a low G0/G1 cell proportion or a high proliferation pool (S- and G2/M cell-proportion) seemed to die earlier. There was also a tendency for patients with in vitro resistant tumors to die earlier under chemotherapy than those with sensitive tumors.

Pneumomediastinum secondary to an apparently trivial stab wound to the neck: the value of the Hamman's sign and thorough radiological investigation.

Perforation of the pharynx and upper oesophagus after stab wounds to the neck is easily overlooked because of the relative lack of symptoms. A case is reported in which pneumomediastinum occurred after an apparently trivial neck wound.

A national survey amongst UK otolaryngologists regarding the treatment of Ménière's disease.

Since Dr Prosper Ménière described the vertiginous syndrome that now bears his name, a large variety of medical and surgical treatments have been introduced. To determine the way in which this condition is currently managed in the United Kingdom, a postal survey amongst consultant otolaryngologists was carried out. It revealed that 52 per cent were actively involved in the treatment of patients with Ménières disease using a wide range of medical and surgical therapies that have little or no evidence base. The survey found that 94 per cent of surgeons prescribe betahistine, 63 per cent diuretics and 71 per cent advise salt restriction to their patients, while 52 per cent of surgeons continue to recommend saccus decompression and 50 per cent are still inserting a grommet. However, two thirds of respondents now advocate the use of gentamicin therapy despite it only being introduced to this country just over 10 years ago. The results of this study and their relevance to the recommended present day management of Meniere's disease are discussed.

Course, patterns, and risk-factors for chemotherapy-induced emesis in cisplatin-pretreated patients: a study with ondansetron.

Vomiting and nausea are the most distressing side-effects of cancer chemotherapy. With standard antiemetic regimens (e.g. metoclopramide based combinations) sufficient antiemetic control is achieved in 50-70% of cisplatin treated patients. Ondansetron, a selective 5-HT3-receptor antagonist has shown efficacy in cisplatin-induced emesis. In the present study, we evaluated the safety and efficacy of ondansetron in cisplatin pretreated patients who had suffered from severe emesis in spite of antiemetic prophylaxis. Complete antiemetic control was reached in 43.5% on the day of treatment and in 27.2% of the patients regarding a worst day analysis. 25% of the patients suffered from severe cisplatin-induced emesis (greater than 5 emetic episodes per 24 h). We try to characterise risk-factors for cisplatin-induced emesis by performing a multivariate analysis. Sex, cisplatin dose, and combination therapy with cisplatin plus anthracyclines seem to be independent risk-factors for vomiting on day 1 and on worst day. Delayed emesis occurred less often when sufficient antiemetic protection from acute vomiting had been obtained. Female sex, cisplatin dose and recurrent disease seem to influence the probability for occurrence of delayed vomiting.

Diagnosis and staging of ovarian cancer.

The diagnostic process in ovarian carcinoma is divided into the pre- and intraoperative procedures, examinations of tumor tissue, and follow-up. For preoperative diagnosis, the probability of a palpable adnexal mass being a malignant tumor should first be ascertained by sonography. This should be followed by an appropriate general examination, a search for tumor outside the abdominal cavity and in the liver parenchyma as well as by determination of markers. Intraoperative diagnosis determines the tumor stage and must be carried out all the more comprehensively when the ovarian carcinoma is more limited. Histologic subtype and degree of differentiation are in direct relation to the tumor stage, whereas the size of the primary tumor is often indirectly proportional to its extent. Besides the morphological analysis, the determination of possible chemoresistance and chemosensitivity, as well as further investigations on fresh tumor tissue are included in the tissue examination. Follow-up after a curative operation consists of gynecologic examination and Douglas lavages if tumor is still present in CT Scans and sonographs. To verify a relapse, laparoscopy can be used, but to ascertain a complete remission, a laparotomy is necessary.

The occurrence of epidermal growth factor receptors and the characterization of EGF-like factors in human ovarian, endometrial, cervical and breast cancer. EGF receptors and factors in gynecological carcinomas.

In this study we investigated the presence of epidermal growth factor receptors (EGF-R) and the tissue levels of EGF-like factors (EGF-F) in ovarian, endometrial, cervical and breast carcinomas. EGF-R were found in 33/40 (83%) cervical, 15/26 (58%) endometrial, 64/141 (45%) ovarian, and 19/59 (33%) breast carcinomas. The highest number of EGF-R binding sites was detected in cervical carcinomas followed by endometrial, breast and ovarian carcinomas. The tissue concentrations of EGF-like factors, were investigated in extracts of 63 ovarian, 25 breast, 12 cervical, 14 endometrial carcinomas and in 21 biopsies of nonmalignant tissue such as myometrium and ovaries. The extracts of nonmalignant tissues had a mean EGF-F level of 1.5 +/- 0.7 ng/mg with a concentration range from 0 to 4 ng/mg. The mean EGF-F levels of malignant tissues were: ovarian carcinomas 4.2 +/- 1.5 ng/mg (range 0-15 ng), endometrial carcinomas 4.5 +/- 1.7 ng/mg (range 0-12 ng), cervical carcinomas 4.15 +/- 1.1 ng/mg (range 0-8) and breast carcinomas 3.16 +/- 1.1 ng/mg (range 0-10 ng). About 30% ovarian, endometrial and cervical carcinomas and 16% breast carcinomas, respectively, had enhanced EGF levels from 5 ng/mg to 15 ng/mg compared to nonmalignant tissues. The EGF-F of tissue extracts consists of EGF and transforming growth factor TGF alpha) as shown by the results of EGF and TGF alpha radioimmunoassays. It is assumed that in some tumors the EGF-F tissue levels influence the number of biochemically detectable EGF binding sites.

Cloning ovarian carcinoma cells in an agar double layer versus a methylcellulose monolayer system. A comparison of two methods.

Human ovarian cancer cells from ten patients were cultured in the agar double layer assay as described by Hamburger and Salmon and in a methylcellulose monolayer system. The assays were compared under the same experimental conditions. The rate of positives (defined as greater than 30 colonies/dish) was 75% in the methylcellulose assay and 69% in the agar double layer. Plating efficiency ranged in the methylcellulose assay between 0.021% and 0.089% and in the agar double layer from 0.015% to 0.094%. Cytological and cytochemical staining of cells obtained from colonies in both test systems and of the tumour cells prior to plating revealed the same morphology. The methylcellulose monolayer system requires less additives than necessary in the agar double layer system. Furthermore, it is easier to handle with respect to the plating procedure and less time consuming. In addition, the effect of the anti-oestrogen tamoxifen on colony formation was tested. The dose response curves for colony formation with tamoxifen proved to be identical in both systems. At a concentration of 10(-6) M an inhibition of colony formation of more than 70% of controls was observed in the agar and in the methylcellulose system.

Human papillomavirus DNA in invasive carcinoma of the vagina.

The presence of human papillomavirus (HPV) DNA in invasive carcinomas of the vagina, in their lymph node metastases, and in corresponding normal tissue was investigated by Southern blot hybridization with 32P-labeled HPV DNA. Tumor tissue from ten of 18 patients with vaginal carcinoma contained HPV DNA. Three of the 18 patients had a history of cervical neoplasia more than 14 years before the diagnosis of vaginal carcinoma. Five of 15 primary squamous cell carcinomas, one primary adenocarcinoma, and a vulvar recurrence of a vaginal squamous carcinoma contained HPV 16. A primary squamous carcinoma yielded HPV-related sequences. The HPV copy number varied from 0.5 to 50 per cellular genome. Four histologically positive inguinal lymph nodes from three patients contained HPV DNA. In six tumor-free control tissues from four patients, no HPV DNA was detected. No relationship was established between HPV positivity, HPV type, or copy number of the tumor and the grade of differentiation or keratinization or the clinical stage. After a median follow-up of 13 months, five of nine HPV-positive patients were alive without recurrence, whereas all seven HPV-negative patients had died because of disease. The results of this study indicate a possible major role of HPV in the development of vaginal cancer.

Diagnostic formula for the differentiation of adnexal tumors by transvaginal sonography.

OBJECTIVE: To create a strategy for sonographic differentiation of benign and malignant adnexal tumors in premenopausal and postmenopausal patients. METHODS: Multiple sonomorphologic criteria were analyzed prospectively in 754 tumors. Four hundred were found in premenopausal and 354 in postmenopausal women. In a logistic regression model, relevant criteria were selected, and a diagnostic formula for tumor differentiation was derived. RESULTS: There were 165 malignant tumors, of which 37 (9.2%) were found in premenopausal and 128 (36.2%) in postmenopausal women. In both groups, the criteria of solid phase and ascites were the most significant. Further important diagnostic criteria were structure and tumor size in premenopausal women and cyst architecture and tumor surface in postmenopausal women. These results allowed an estimation of the probability of malignancy. Using a cutoff point of 10% for the probability to classify tumors as malignant, the sensitivity and specificity in premenopausal patients were 86.5% and 92.6%, respectively, with an accuracy of 92%. In postmenopausal women, the sensitivity, specificity, and accuracy were 93%, 82.7%, and 86.6%, respectively. Assuming a prevalence as given in the study, the positive and negative predictive values were 54.4% and 98.5% in premenopausal and 75.3% and 95.4% in postmenopausal women. CONCLUSIONS: With four binary criteria, a useful diagnostic formula for tumor differentiation was obtained. However, estimates for sensitivity, specificity, and accuracy may be too optimistic because they were derived from the same data that were already used for model selection.

Cytogenetic analysis of an adenoid cystic carcinoma of the Bartholin's gland. A rare, semimalignant tumor of the female genitourinary tract.

Cytogenetic analysis has been performed on short-term cultures from a 56-year-old woman suffering from an adenoid cystic carcinoma of Bartholin's gland. Beside a normal female karyotype, the tumor revealed an abnormal cell line with complex chromosome changes involving the chromosomes 1, 4, 6, 11, 22, and 14. The mainly structural and nonbalanced rearrangements led to the loss of the chromosome segments 1p31----qter, 4q22----q28, 6p12----qter, 11p11.2----pter, 14q24----qter, and 22q13----qter. Clonal numerical aberrations were not observed. To our knowledge, such a tumor has to-date not been cytogenetically investigated.

In vitro responsiveness of ovarian epithelial carcinomas to endocrine therapy.

As previously reported, ovarian epithelial carcinomas may respond to endocrine therapy. We examined the direct effect of progesterone, medroxyprogesteroneacetate, gestoneron, 17-beta-estradiol, tamoxifen, 4-OH-tamoxifen, or N-desmethyltamoxifen on the proliferative capacity of ovarian carcinoma cells by means of the colony assay described by Hamburger and Salmon. The growth rate of 25 tested tumors (ascitic fluid, primary tumor, metastases) was 68%. The plating efficiency was 0.078%. Beside the drug testing estrogen and progesterone receptor levels were determined. The inhibition of colony survival was slightest with 17-beta-estradiol, more pronounced with medroxyprogesteroneacetate, gestoneron, N-desmethyltamoxifen, and progesterone, and greatest with 4-OH-tamoxifen and tamoxifen. Significant and dose-dependent inhibition of greater than 70% was observed with tamoxifen and 4-OH-tamoxifen in 80% of the tested tumors. There was no significant correlation between the in vitro responsiveness and the level of hormonal act not only via an estrogen receptor but also via an antiestrogen-binding site.

Evaluation of cytokine levels in whole cell cultures of patients with gynaecological tumors as diagnostic parameters.

The measurement of the cytokine production of activated lymphocytes and monocytes supposedly provides a tool for evaluating the actual status of the cellular immunity potential of a person. We measured the levels of IFN-gamma and IL-1-alpha in the 4 day post-induction supernatants of mitogen-stimulated unseparated blood cell cultures by a rapid and sensitive immunoassay using various monoclonal and polyclonal antibodies. With this reproducible and easy-to-handle system we tested 90 patients with primary gynaecological tumors and the same number of age-matched female controls. In the blood cell cultures of the tumor patients significantly lower values of IFN-gamma and IL-1-alpha were observed, although lymphocyte and monocyte counts were almost normal. There was also a difference in the IFN-gamma-levels between the four tumors, in as much as patients with ovarian and endometrial carcinomas had significantly lower values than patients with breast and cervical carcinomas.

Expression analysis of EGF-R and TGFa in human ovarian carcinomas.

Differences in the tumor biology of ovarian carcinomas probably influence operability and response to chemotherapy which are the most relevant prognostic factors. The phenotype of different malignant epithelial tumors including ovarian carcinomas is obviously associated with an activation of the EGF/TGFa signal pathway. When we analysed the expression of EGF-R and TGFa with biochemical, molecular-chemical and immunohistochemical methods in 29 different ovarian carcinomas, we found a correlation between the mRNA and protein levels of EGF-R as well as TGFa for tumors with low or high expressing rates. However, the concentration of measurable free EGF-Rs seems to depend on the amount of TGFa expression by the tumors. The EGF-R binding ligand TGFa is produced by the tumor cells; stromal cells are TGFa negative as shown by immunohistochemistry. By the use of an immunostaining index the TGFa protein concentration was measured semiquantitatively, classifying tumors according to their TGFa production rate. The comparison of TGFa mRNA amounts and staining index supports the hypothesis that TGFa is modified posttranslationally. EGF-R or TGFa expressing ovarian carcinomas had a high response rate to chemotherapy, whereas the EGF-R or TGFa negative tumors mostly exhibit a no change or progressive disease behaviour. These findings are the basis for our assumption that ovarian carcinomas with the basis for our assumption that ovarian carcinomas with an activated EGF-TGFa system are tumor biologically different compared to the EGF-R/TGFa negative tumors.

Relationship between epidermal growth factor receptor (EGF-R) and various prognostic factors in human endometrial carcinoma.

Eighty primary endometrial carcinomas were analyzed for the presence of epidermal growth factor receptors (EGF-R) by the use of a single point (1ng 125I-EGF) EGF-R assay. Fifty percent of the analyzed specimens were EGF-R positive (EGF-R(+)) with binding capacities > 1 fmol mg-1 and 15% bound > 7 fmol mg-1. The EGF-R status was correlated with different clinically relevant prognostic factors and the survival rates were analyzed. The correlations revealed no significant differences in the grade of tumor differentiation and in the depth of myometrial invasion. The advanced tumor stages III and IV and tumors with a squamous cell component in the histologic examination expressed EGF-R in a higher percentage. Between the EGF-R and the steroid receptor status existed a weak negative correlation. Estrogen receptor positive tumors were in 47% and progesterone receptor positive tumors in 45% EGF-R (+). The corresponding number for the receptor negative tumors were 57 and 65%. Life table analyses were performed with different cut-off limits for specific EGF binding ranging from > 1, > 3, > 5 and > 7 fmol mg-1. Dependent on the cut-off limits the analysis demonstrates a reduced survival probability for patients with EGF-R+ tumors. These differences were mainly found in the small group with a high number of EGF-R's (> 7 fmol). The analysis of tissue extracts for the presence of factors binding to the EGF-R revealed in some specimens high concentrations of EGF-like factors. By the use of TGFa (transforming growth factor alpha) immunohistochemistry we were able to demonstrate that the tumor cells produce TGFa, whereas the stroma is TGFa negative. We assume that the EGF-like factors consist mainly of TGFa.

Co-cultivation of ovarian carcinoma cells with dermal fibroblasts induces fibroblast expression of sex steroid receptor transcripts and protein.

We have previously reported that stromal fibroblasts of ovarian carcinoma specimens may express estrogen (ER) and progesterone receptors (PR) when the malignant epithelial cells do not, and even when the specimens have been obtained from such non-Müllerian structures as the omentum whose fibroblasts normally express neither ER nor PR. In an attempt to investigate whether our observations of the expression of ER and PR in fibroblasts surrounding metastatic invasive epithelial ovarian carcinoma cells might result from an interaction involving malignant epithelial cells and stromal fibroblasts, we co-cultivated in vitro BG1 ovarian carcinoma cells with sex steroid receptor-negative dermal fibroblasts to determine whether carcinoma cells might induce the latter to express ER or PR protein and transcripts at levels detectable by standard immunocytochemical (ICC) and in situ hybridization (ISH) techniques. We report the in vitro induction of ER and PR transcripts and protein in previously steroid receptor-negative skin fibroblasts after co-cultivation with BG1 ovarian adenocarcinoma cells. Such observations suggest that a juxtacrine mechanism is responsible for the observed phenomenon, possibly involving ER- and PR-inducing factors (ER-IF and PR-IF).

Color Doppler flow criteria of breast lesions.

Color Doppler technique has been available for several years. The sensitivity of the equipment has improved and allows for assessment of tumor vascularity. We investigated multiple parameters in 258 patients, with 176 benign and 82 malignant lesions to define characteristic flow criteria. Median (25-75% quartiles) and p-values are given for benign vs. malignant lesions. Number of tumor vessels: 2 (1-2) vs. 8 (5-14), p < 0.0001; mean peak systolic flow velocity: 11.1 cm/s (6.4-14.9) vs. 18.8 cm/s (13.7-25.1), p < 0.0001; maximum flow velocity: 12.5 cm/s (6.7-18) vs. 32.5 cm/s (22.5-47.3), p < 0.0001; sum of all systolic flow velocities: 18.9 cm/s (7-34.2) vs. 147 cm/s (71.3-266.7), p < 0.0001; minimum systolic flow velocity: 8.9 cm/s (5.4-12.1) vs. 9 cm/s (6.3-11.3), p > 0.05; average resistance index (RI): 0.68 (0.58-0.72) vs. 0.75 (0.67-0.81), p > 0.05; maximum RI: 0.71 (0.65-0.78) vs. 0.88 (0.78-0.99), p < 0.0001; minimum RI: 0.64 (0.57-0.68) vs. 0.64 (0.53-0.71), p > 0.05; average A/B ratio: 3.1 (2.7-3.7) vs. 4.3 (3.2-7.7), p < 0.0001; maximum A/B ratio: 3.4 (2.9-4.6) vs. 8.4 (4.5-9.9), p < 0.0001; minimum A/B ratio: 2.8 (2.3-3.2) vs. 2.9 (2.2-3.5), p > 0.05. The data analysis shows that flow resistance in malignancies is increased. This is in contrast to gynecological malignancies, where an increased diastolic flow indicates that flow resistance is decreased.

Assessment of the otoscopic skills of general practitioners and medical students: is there room for improvement?

Ear, nose and throat problems are common in general practice, yet undergraduate and postgraduate teaching in the subject is variable and often sparse. The assumption that direct experience in otoscopy in practice will compensate for inadequate previous tuition was tested by assessing a group of 53 general practitioners and 59 medical students. Confidence in otoscopy was assessed using a visual analogue scale and skill was assessed by clinical examination of four ears. Otoscopy was divided into identifying the tympanic membrane, distinguishing a normal from an abnormal membrane and identifying specific features of the membrane. The medical students and general practitioners were comparable in both confidence and skill for all parameters except skill in identification of specific features of the tympanic membrane, in which the students' ability was greater (Student's t-test, P < 0.01). In both groups the percentage of false negative observations was reassuringly low--for students the mean was 3.0%; and for general practitioners 4.3%. There is room for improvement in general practitioner's training in otoscopy. Supervised tuition is essential and cannot be compensated for by unsupervised experience. More involvement with ear, nose and throat problems in vocational training or attendance at continuing education courses is suggested.

[Sarcomas of the uterus: morphological criteria and clinical course (author's transl)]. / Sarkome des Uterus: Morphologische Kriterien und klinischer Verlauf.

Sixty-nine cases of uterine sarcoma were reviewed histologically withhh respect to their grade of malignancy. Mitotic activity is the most important criterion of malignancy. A histological grading was performed on the basis of mitotic counts per high power field. Clinical follow-up showed that except for the local extension of the tumor, the prognosis of sarcomas depends greatly on mitotic activity. There is a good correlation in the lower stages I and II between number of mitoses and survival rate. The 5 year survival rate of patients with grades I or II is 77% compared to 41% for grades III and IV. Vascular invasion is not evident in distant metastases in our material. Adjuvant chemotherapy is recommended in clinical stages II-IV and in histological grades III and IV.