Search

The emergency department (ED) is the main port of entry for patients with infectious diseases, the place where a number of diagnostic procedures are performed and treatment is often initiated. The aim of this retrospective study was to estimate the influence of the establishment and introduction of a blood culture standard operating procedure (BC-SOP) and of the subsequent training of microbial diagnostics in an ED.In a before and after study over a study period of 3 months each (November 2017-January 2018 and November 2018-January 2019), the number of blood cultures taken, the rate of blood cultures per 1000 patients, the number of positive blood cultures and the frequency of typical skin pathogens were evaluated. In the interim time between the two study periods, a BC-SOP was developed in collaboration with the hospital's antibiotic stewardship team and subsequently introduced with staff training in the ED. The study was approved by the local ethics committee of the medical faculty of the Heinrich Heine University (2019-392-RetroDEuA).In total 92% of the nursing personnel and 93% of the medical personnel received training. The total number of blood cultures increased from 1757 to 2872 (64% increase) and the rate of blood cultures per 1000 patients from 287 to 481 (68% increase). The number of positive blood cultures decreased from 18.6% to 13.7% (p 0.05). Typical skin pathogens were found in 34.4% and 26.4% of the cases, respectively (p 0.05).The development, introduction and training of a BC-SOP in the ED can make a relevant contribution to the microbial diagnostics and increase the quantity as well as the quality.EINLEITUNG: Zentrale Notaufnahmen stellen die Eintrittspforte für viele stationär aufzunehmende Patienten in einem Krankenhaus dar und sind häufig der Ausgangspunkt für die antiinfektive Diagnostik und Therapie von Notfallpatienten. In dieser retrospektiven Untersuchung soll der Frage nachgegangen werden, wie die Etablierung einer Standard Operating Procedure (SOP) „Blutkulturen“ und deren Schulung die mikrobielle Diagnostik in einer zentralen Notaufnahme verbessern kann.In einer Vorher-und-nachher-Untersuchung wurde über einen jeweils 3‑monatigen Zeitraum (11/2017 bis 01/2018 und 11/2018 bis 01/2019) die Anzahl der abgenommenen Blutkulturen, die Rate an Blutkulturen/1000 Fälle, die Anzahl positiver Blutkulturen und die Häufigkeit typischer Hautkeime analysiert. Im Zeitraum zwischen den evaluierten Zeitabschnitten wurde eine SOP „Blutkulturen“ in Zusammenarbeit mit dem Antibiotic-Stewardship(ABS)-Teams und der zentrale Notaufnahme entwickelt, implementiert und geschult. Ein positives Votum der Ethikkommission der Heinrich-Heine-Universität (2019-392-RetroDEuA) lag vor.Die pflegerischen und ärztlichen Mitarbeiter wurden zu 92 % bzw. 93 % geschult. Die Anzahl der abgenommenen Blutkulturen stieg von 1757 auf 2872 um 64 % ebenso wie die Anzahl der Blutkulturen/1000 Fälle von 287 auf 481 (68 %). Die Anzahl der positiven Blutkulturen reduzierte sich von 18,6 auf 13,7 % (p 0,05). Typische Hautkeime fanden sich in 34,4 % und 26,4 % der Fälle (p 0,05).Die durch Schulungen begleitete Einführung einer SOP „Blutkulturen“ in der zentralen Notaufnahme kann einen relevanten Beitrag zur antimikrobiellen Diagnostik leisten und sowohl die Quantität als auch die Qualität erhöhen.

Background/aim mmune checkpoint inhibition (CPI) has an increasing impact in the multimodal treatment of locally advanced non-small cell lung cancer (LA-NSCLC). Increasing evidence suggests treatment outcome depending on tumor cell PD-L1 expression. The purpose of this retrospective study was to investigate the prognostic value of PD-L1 expression on tumor cells in combination with CD8+ tumor stroma-infiltrating lymphocyte (TIL) density in inoperable LA-NSCLC treated with concurrent chemoradiotherapy (CRT). Patients and method We retrospectively assessed clinical characteristics and initial tumor biopsy samples of 31 inoperable LA-NSCLC patients treated with concurrent CRT. Prognostic impact of tumor cell PD-L1 expression (0% versus ≥1%) and CD8+ TIL density (0–40% vs. 41–100%) for local control, progression-free (PFS) and overall survival (OS) as well as correlations with clinicopathological features were evaluated. Results Median OS was 14 months (range: 3–167 months). The OS rates at 1- and 2 years were 68 and 20%. Local control of the entire cohort at 1 and 2 years were 74 and 61%. Median PFS, 1-year and 2-year PFS were 13 ± 1.4 months, 58 and 19%. PD-L1 expression neg/CD8low) and type IV (PD-L1pos/CD8low) tumors (median OS: 57 ± 37 vs. 10 ± 5 months, p = 0.05), respectively. Conclusion Assessment of PD-L1 expression on tumor cells in combination with CD8+ TIL density can be a predictive biomarker in patients with inoperable LA-NSCLC treated with concurrent CRT.

Background In the last comprehensive review of the literature published in 2002, little information on the prevalence of orofacial clefts was available from low- and middle-income countries (LMICs). Objective To analyze published data on the birth prevalence of cleft lip and/or palate ( CL/P) from LMIC. Design Systematic review of the literature and meta-analysis of data from original papers on the birth prevalence of cleft lip and/or cleft palate (CL/P) in LMICs between 1990 and 2014. Secondary inclusion criteria were developed to analyze lower-quality studies from countries with scarce data. Main Outcome Measure Birth prevalence of undifferentiated CL/P (with or without associated syndrome or other anomaly). Results Twenty-eight studies met strict inclusion criteria. Among 31,475,278 total births, the pooled birth prevalence of undifferentiated CL/P was 1.38 per 1000 births (95% confidence interval [CI]: 1.20 to 1.56). Four studies met criteria for secondary analysis, providing data on 75,627 births, with a pooled prevalence of 0.75 CL/P cases per 1000 births (95% CI: 0.56 to 0.95). Comparison of studies was limited by variable definitions of cases and of the reference population and by inconsistent reporting of outcomes. There is significant heterogeneity in the findings. Conclusions In LMICs, approximately 1 in every 730 children is born with CL/P. To optimize comparability across settings, future research should use a standard classification system and standard criteria for data collection and presentation. As clefting is associated with deprivation, understanding the true scale, risks, and preventive measures for orofacial clefts in LMIC is a matter of both scientific and humanitarian importance.

There are no officially approved therapies for metastatic pheochromocytomas apart from ultratrace 131I-metaiodbenzylguanidine therapy, which is approved only in the United States. We have, therefore, investigated the antitumor potential of molecular-targeted approaches in murine pheochromocytoma cell lines [monocyte chemoattractant protein (MPC)/monocyte chemoattractant protein/3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], immortalized mouse chromaffin Sdhb−/− cells, three-dimensional pheochromocytoma tumor models (MPC/MTT spheroids), and human pheochromocytoma primary cultures. We identified the specific phosphatidylinositol-3-kinase α inhibitor BYL719 and the mammalian target of rapamycin inhibitor everolimus as the most effective combination in all models. Single treatment with clinically relevant doses of BYL719 and everolimus significantly decreased MPC/MTT and Sdhb−/− cell viability. A targeted combination of both inhibitors synergistically reduced MPC and Sdhb−/− cell viability and showed an additive effect on MTT cells. In MPC/MTT spheroids, treatment with clinically relevant doses of BYL719 alone or in combination with everolimus was highly effective, leading to a significant shrinkage or even a complete collapse of the spheroids. We confirmed the synergism of clinically relevant doses of BYL719 plus everolimus in human pheochromocytoma primary cultures of individual patient tumors with BYL719 attenuating everolimus-induced AKT activation. We have thus established a method to assess molecular-targeted therapies in human pheochromocytoma cultures and identified a highly effective combination therapy. Our data pave the way to customized combination therapy to target individual patient tumors.

Pancreatic ductal adenocarcinoma (PDAC) is a highly devastating disease with poor prognosis and rising incidence. Late detection and a particularly aggressive biology are the major challenges which determine therapeutic failure. In this review, we present the current status and the recent advances in PDAC treatment together with the biological and immunological hallmarks of this cancer entity. On this basis, we discuss new concepts combining distinct treatment modalities in order to improve therapeutic efficacy and clinical outcome – with a specific focus on protocols involving radio(chemo)therapeutic approaches.

Schlaflabore stellen die zentrale Untersuchungseinheit in der Schlafmedizin dar. Eine Qualitatssicherung dieser Untersuchungseinheit ist ein besonders wichtiges Anliegen der klinischen Schlafmedizin. Sie erfolgt durch einen formalen Akkreditierungsprozess. Die Voraussetzungen und die Ablaufe der Akkreditierung werden in diesem Leitfaden der Deutschen Gesellschaft fur Schlafforschung und Schlafmedizin beschrieben und, soweit moglich, mit Begrundung ausgefuhrt. Ein erster Leitfaden zur Akkreditierung wurde im Jahr 2000 veroffentlicht. Aufgrund der aktuellen Veranderungen ist es geboten, den Leitfaden zu aktualisieren, um wissenschaftliche Erkenntnisse und Leitlinien mit zu berucksichtigen. Hierzu gehoren die Einfuhrung neuer Aufzeichnungs- und Auswertungsvorschriften fur die kardiorespiratorische Polysomnographie sowie neue Diagnosemanuale fur Schlafstorungen.

Based on its potent capacity to induce tumor cell death and to abrogate clonogenic survival, radiotherapy is a key part of multimodal cancer treatment approaches. Numerous clinical trials have documented the clear correlation between improved local control and increased overall survival. However, despite all progress, the efficacy of radiation-based treatment approaches is still limited by different technological, biological, and clinical constraints. In principle, the following major issues can be distinguished: (1) The intrinsic radiation resistance of several tumors is higher than that of the surrounding normal tissue, (2) the true patho-anatomical borders of tumors or areas at risk are not perfectly identifiable, (3) the treatment volume cannot be adjusted properly during a given treatment series, and (4) the individual heterogeneity in terms of tumor and normal tissue responses toward irradiation is immense. At present, research efforts in radiation oncology follow three major tracks, in order to address these limitations: (1) implementation of molecularly targeted agents and ‘omics’-based screening and stratification procedures, (2) improvement of treatment planning, imaging, and accuracy of dose application, and (3) clinical implementation of other types of radiation, including protons and heavy ions. Several of these strategies have already revealed promising improvements with regard to clinical outcome. Nevertheless, many open questions remain with individualization of treatment approaches being a key problem. In the present review, the current status of radiation-based cancer treatment with particular focus on novel aspects and developments that will influence the field of radiation oncology in the near future is summarized and discussed.

Schlafbezogene Atmungsstorungen sind eine haufig gestellte Diagnose bei neuromuskularen Erkrankungen. Eine Vielzahl der neuromuskular Erkrankten ist von einem alveolaren Hypoventilationssyndrom im Schlaf betroffen. Der Hauptausloser fur die Hypoventilation ist die Parese und/oder die Dysfunktion des M. diaphragmaticus. Aufgrund der physiologischen Absenkung des Muskeltonus im Rapid-Eye-Movement(REM)-Schlaf tritt die Atmungsstorung initial v. a. in diesem Schlafstadium auf. Haufig wird die nachtliche Hypoventilation erst dann diagnostiziert, wenn es bereits zu einer einschrankenden respiratorischen Storung auch am Tag kommt. Es ist deshalb notwendig, Patienten mit einer neuromuskularen Erkrankung fruhzeitig auf das Vorliegen einer schlafbezogenen Atmungsstorung zu untersuchen. Fur die amyotrophe Lateralsklerose und die Muskeldystrophie Duchenne ist die Lebensverlangerung durch eine adaquate Beatmungstherapie in randomisierten Studien mit kleiner Fallzahl bereits nachgewiesen. Es bleibt zu untersuchen, ob durch eine fruhzeitig eingeleitete adaquate Beatmungstherapie neben einer Verbesserung der Lebensqualitat auch die Krankheitsprogression bei weiteren nicht fatal verlaufenden neuromuskularen Erkrankungen beeinflusst werden kann.

Daytime sleepiness for any reason leads to impairment of daytime performance and an increased accident rate. The consequences are an increase of illness- and accident-related costs for the health system. Obstructive sleep apnea (OSA) is one of the major reasons for increased daytime sleepiness, especially in professional drivers. The accident frequency in OSA can be significantly reduced by adequate continuous positive airway pressure (CPAP) therapy. Up till now there are no uniform legal regulations about the handling of OSAS patients or patients with daytime sleepiness due to other diseases as far as driving ability is concerned.

Patienten mit erhohter Tagesschlafrigkeit sind in allen beruflichen und sozialen Lebensbereichen beeintrachtigt. Gutachterliche Stellungnahmen werden im Rentenverfahren, zur Beurteilung der Einsatzmoglichkeiten am Arbeitsplatz und zur Fahrtauglichkeit gefordert. Zur Diagnosesicherung stehen nichtapparative und apparative Testverfahren zur Verfugung. Eine Polysomnographie ist zwingend erforderlich. Vor der endgultigen Begutachtung sollten alle schlafmedizinischen Differenzialdiagnosen abgeklart und eine optimale Behandlung angestrebt worden sein. In der aktuellen Fahrerlaubnisverordnung (FeV) wird erstmals von Fahruntauglichkeit bei unbehandelten Schlafstorungen mit Tagesschlafrigkeit ausgegangen. Die Bedeutung der Tagesschlafrigkeit in anderen Berufsgruppen ist arbeitsplatzbezogen zu bewerten. Vor dem Ansprechen einer adaquaten Therapie besteht haufig vorubergehend Arbeitsunfahigkeit. Die arbeitsmedizinische Beurteilung sollte dem Patienten im Hinblick auf Berufswahl und Arbeitsplatzgestaltung mitgeteilt werden. Der Gutachter hat Therapieerfolge zu kontrollieren. Die vorliegende Ubersicht soll gesetzliche Grundlagen und geeignete Untersuchungsverfahren darstellen.

OBJECTIVE. We describe the significance of detecting focal areas of hypermetabolism in the breast in patients undergoing PET/CT for reasons other than for breast cancer detection or staging.CONCLUSION. When evaluated, almost all of the abnormal foci detected in the breast subsequently proved to be breast carcinoma, specifically infiltrating ductal carcinoma.

Contact-mediated interactions between Sertoli cells and gonocytes are important for testicular development. Specifically, down-regulation of neural cell adhesion molecule (NCAM)-based intercellular adhesion during postnatal maturation is likely to be important for appropriate differentiation of testicular cells. Besides NCAM, P-cadherin is also present in neonatal testicular cords, at least in mice, and seems to disappear from the seminiferous epithelium after the first postnatal week. Another factor known to be important in regulating development of the neonatal testis is thyroid hormone (T3). T3 is involved in control of Sertoli cell proliferation and differentiation. Therefore, we examined the effect(s) of T3 on adhesive factors found within the testis using Sertoli cells and gonocytes isolated from neonates and maintained in coculture. T3 (100 nm) down-regulated NCAM expression in vitro, as assessed by Western blotting and immunofluorescent staining. This contrasted with the continued expression of NC...

Di-(2-ethylhexyl) phthalate (DEHP), one of the abundant man-made environmental chemicals, induces testicular damage in both developing and adult animals. However, the nature and mechanism underlying the action of phthalates on testicular development remain largely unexplored. In the present study, we used cocultures of neonatal Sertoli cells and gonocytes (precursors of spermatogonia) to characterize in detail the effects of mono-(2-ethylhexyl) phthalate (MEHP; the active metabolite of DEHP) on these cells and to explore the underlying mechanism(s). Sertoli cells and gonocytes were isolated from rat pups on the 2nd day after birth, cocultured, and exposed to MEHP at concentrations of 0.01, 0.1, or 1.0 microM, or to 0.5% DMSO (vehicle control), or 10 microM DEHP (negative control) for a total of 48 h. We found that exposure to MEHP induced gonocyte detachment from the Sertoli cell monolayers in a time- and dose-dependent manner. When exposed to 1.0 microM MEHP, many gonocytes started to detach after 12 h of exposure and most gonocytes were lost during the media change at 24 h. Gonocyte detachment was also observed in cocultures treated with 0.1 microM MEHP for 24 h of exposure, but not in cultures treated with 0.01 microM MEHP for 48 h. Detached gonocytes were viable as indicated by their ability to exclude trypan blue. Furthermore, when proliferation of cultured Sertoli cells was detected by BrdU labeling and subsequently quantified, we found that exposure to 0.1 or 1.0 microM MEHP for 48 h resulted in a decrease in labeling indices of 33.6 and 83.6%, respectively, compared to the vehicle control (p < 0.01), while the labeling index was unchanged by treatment with 0.01 microM MEHP. In addition, we also tested the potential effect of MEHP on FSH-stimulated Sertoli cell proliferation by simultaneously treating cultures with 200 ng/ml human FSH and different concentrations of MEHP for 48 h. Exposure to 0.1 or 1.0 microM MEHP resulted in decreases of 24.2 and 74.2%, respectively, in FSH-stimulated Sertoli cell proliferation (p < 0. 01). Furthermore, MEHP also inhibited dibutyl cAMP-stimulated Sertoli cell proliferation, regardless of whether dibutyl cAMP was added to the cultures before or at the same time as MEHP. Finally, addition of FSH or dibutyl cAMP had no effect on MEHP-induced gonocyte detachment, and none of the observed effects on either Sertoli cells or gonocytes were detected in control cultures treated with 0.5% DMSO only or with 10 microM DEHP. Therefore, short exposure to low levels of MEHP disrupted adhesion of gonocytes to Sertoli cells and inhibited both basal and FSH-stimulated Sertoli cell proliferation in a dose-dependent manner. The lowest effective dose of MEHP in vitro was 0.1 microM, which is about 10- to 1, 000-fold lower than the dose shown to affect Sertoli cells from prepubertal animals. Moreover, our data indicate that MEHP impairs division of neonatal Sertoli cells by acting at a post-cAMP site in the FSH-response pathway or via a mechanism independent of FSH. These data provide direct new evidence that relatively low levels of MEHP disrupt Sertoli cell-gonocyte physical interactions and suppress Sertoli cell proliferation in neonates via mechanisms specific to neonatal testis where the foundations of adult fertility are established. The results also highlight the neonatal period of testicular development as one particularly sensitive to environmental chemicals.

Reports of soft tissue injuries from orthodontic face-bows have continued to appear in the dental and medical publications since the American Association of Orthodontists reported problems in 1975. In 1994 the results of a preliminary survey of face-bow injuries of the orthodontic societies and dental schools in Europe recorded nine serious injuries. The results of a recent face-bow injury survey of orthodontic practitioners in the United Kingdom and Eire recorded 33. The relative effectiveness of current extraoral traction safety products is discussed in relation to the cause of these continuing injuries.

Objective In 1998 the delivery of cleft care in the United Kingdom was examined by the Clinical Standards Advisory Group, the outcomes of which led to the wide restructuring of cleft services in the United Kingdom. We present a retrospective study evaluating the radiographic outcome of 53 consecutively performed alveolar bone grafts following the regional centralization of secondary alveolar bone grafting to the South West Cleft Centre between 2004 and 2006. Methods A retrospective audit of one surgeon's outcome of 53 consecutively performed alveolar bone grafts, assessed radiographically using the Kindelan method. Results That 94% of bone-grafted sites achieved a successful radiographic outcome compares favorably with the previously published Clinical Standards Advisory Group data of 58% with Bergland scores. Conclusion The radiographic outcome for alveolar bone grafting has improved with centralization. A small number of patients are being grafted after the ideal chronological age, and this needs to be addressed throughout the region. The Kindelan assessment provides a reliable method of early assessment for alveolar bone grafting.

Movement of postnatal gonocytes to the periphery of the seminiferous cord, where they contact the basement membrane, and resumption of mitosis by these previously quiescent cells are likely to be critically important in establishing spermatogenesis in neonatal rats. We used several approaches both in vivo and in vitro to determine precisely when each of these two events begins, to study their temporal relationship to each other, to determine whether gonocyte division is a prerequisite for relocation or vice versa, and to probe the source of factors initiating and/or regulating these events. Both light and electron microscopy were used to determine that the first gonocytes make contact with the basement membrane on postnatal day 4, while quantitative autoradiography following 3H-thymidine administration in vivo indicated that the first gonocytes to re-initiate cell division do so one day earlier, on day 3, and that the percentage of gonocytes dividing remains at a stable level through day 5. Moreover, we organ-cultured neonatal testes from birth onwards in the presence of defined, serum- and hormone-free medium and determined that both proliferation and relocation of gonocytes begin and continue in vitro as in vivo. This observation argues against involvement of extratesticular factors in stimulating gonocyte relocation and division, and points to the testis itself as the most likely source of agent(s) regulating postnatal maturation of these cells. In other, similar incubations we included 3H-thymidine for varying periods of time to label either those gonocytes that are the first to divide or all gonocytes that divide during the first 48 hr of culture. From these studies, we confirmed that the first gonocytes to divide do so while separated from the basement membrane and found that, although some cells divide before moving peripherally, others do not.

Interaction between Sertoli cells and germ cells is likely to be critical for normal development of the testis. We have established and characterized cocultures of neonatal Sertoli cells and gonocytes and have begun to study the physical and functional relationship between these cells in vitro. Cells were isolated from rat pups by sequential enzymatic treatment and cultured in serum-free medium. When plated on Matrigel, Sertoli cells rapidly attach, and gonocytes adhere to the underlying Sertoli cells shortly thereafter. We observed that some of these germ cells develop cytoplasmic processes and elongate during the first day of culture, essentially mimicking their behavior in vivo. Electron microscopic examination of typical cultures revealed the presence of desmosome-like adhesion sites and apparent gap junctions between Sertoli cells and gonocytes. To determine whether Sertoli cells and gonocytes are functionally coupled in the cocultures, we used the glass bead-loading technique of McNeil and Warder to introduce Lucifer yellow (LY), a gap junction-permeant probe, and Rhodamine-dextran (RD), a larger marker excluded by gap junctions, simultaneously into cultures 24 h after plating. Immediate fixation and viewing of cultures with fluorescence microscopy indicated that all bead-loaded cells received both probes. We studied other living cultures 10 min after bead-loading and located RD-negative (i.e. nonbead-loaded) gonocytes that were in obvious contact with RD- and LY-positive bead-loaded Sertoli cells; these gonocytes were scored for the presence or absence of cytoplasmic LY. This analysis revealed that many gonocytes were able to obtain LY from adjacent Sertoli cells, presumably via gap junctions maintained with these cells. In addition, we quantified the percentage of gonocytes that elongated with increasing time in vitro and correlated the morphology of these cells with their ability to acquire LY from adjacent Sertoli cells. Our findings indicate that although the absolute numbers of gonocytes present decreases, more of those remaining elongate as time in vitro increases. We can also conclude from our data that gonocytes with and without processes are equally likely to be coupled with Sertoli cells under these conditions. These observations provide the first demonstration of functional coupling between Sertoli cells and premeiotic germ cells. Together with our morphological observations, they suggest that gap junction-mediated communication between these cells may be involved in stimulating or regulating changes in the gonocyte population during postnatal development of the testis.

Rat fetuses from mothers stressed chronically by immobilization and high intensity illumination beginning on day 14 of gestation have higher than normal levels of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity in Leydig cells on day 17 of gestation and lower than normal levels on days 18 and 19. Plasma testosterone titers in normal and stressed male fetuses closely parallel the activity of 3 beta HSD in fetal Leydig cells. In the present study quantitative cytochemistry was used to determine whether the stress-induced alterations in 3 beta HSD activity could be prevented by treating the mother with naltrexone, an opioid receptor blocker, before each stress session. Naltrexone normalized 3 beta HSD activity on days 18 and 19 of gestation, suggesting that the stress-induced suppression involves the endogenous opioid system. In contrast, naltrexone did not prevent the elevation in enzyme activity seen on day 17 in stressed fetuses. The persistence of a stress-induced surge on day 17, in spite of naltrexone therapy, suggests that some nonopioid mechanism is operational at that time.

During perinatal development, when the size of the Sertoli cell population is determined, Leydig cells produce beta-endorphin, a peptide which may interact with Sertoli cells to modify their FSH-responsiveness, as suggested by our previous work. The goal of the present study was first, to test directly the possibility that beta-endorphin modifies the proliferative response of neonatal Sertoli cells to FSH, and second, to gain information on a mechanism(s) involved in any observed effect. We treated isolated 6-day-old Sertoli cells with FSH or vehicle in vitro and measured their incorporation of exogenous, radiolabeled thymidine with quantitative autoradiography. After 2 days in culture with FSH, we detected a 10-fold increase in the rate of Sertoli cell proliferation. The level of cell division in these FSH-treated cultures was identical to that in other cultures exposed to cAMP under similar conditions. In addition, inclusion of beta-endorphin 3 hr prior to FSH or cAMP decreased the effect of the hormone by 50% but left the cAMP response unchanged. Thus, beta-endorphin acts on isolated, neonatal Sertoli cells at a point prior to intracellular production of cAMP to suppress their response to FSH. When other cultures were treated with pertussis toxin, a blocker of intracellular GTP-binding proteins such as Gi, before sequential addition of endorphin and FSH, the effect of beta-endorphin on FSH-responsiveness was abolished. Moreover, when other cultures were exposed to pertussis toxin in the absence of endorphin, followed by FSH, their response to the hormone was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

In this study, we explored the impact on both Sertoli cells and gonocytes of a single, relatively low dose of di-(2-ethylhexyl) phthalate (DEHP; 20-500 mg/kg) administered in vivo to 3-day-old rat pups. In parallel, we assessed the potential for two immediate metabolites of DEHP to produce similar testicular changes and began to explore the possible mechanisms involved. Morphological examination revealed the presence of many abnormally large, multi-nucleated germ cells by 24 h posttreatment with DEHP and with its metabolite, mono-ethylhexyl phthalate (MEHP), but not with another metabolite, 2-ethylhexanol (2-EH; all at 1.28 mmol/kg) or with vehicle alone. These cells persisted through 48 h posttreatment, the longest time point examined in our study. We also assessed the rate of Sertoli cell proliferation in pups at intervals after dosage with either chemical or vehicle by administering bromodeoxy uridine (BrdU) 3 h before euthanasia. By 24 h after treatment with DEHP or MEHP, but not 2-EH or vehicle, the number of BrdU-labeled Sertoli cells was obviously diminished in testicular sections. Quantitation of DEHP-treated pups and controls indicates that a dose-response relationship exists between chemical treatment and labeling index (LI) of Sertoli cells, with a LI at the highest DEHP dose tested that was only 20% of that in controls. In addition, when we examined the time course of the effect of an intermediate dose of DEHP, we found that there the LI of Sertoli cells rebounds by 48 h after dosage, when we found the rate of proliferation in treated pups to be significantly higher than in controls. We also explored the potential mechanism involved in the response to DEHP and found serum levels of FSH to be unaffected by the chemical. In addition, study of cell cycle-related proteins including p27kip1 and cyclins D1, D2, and D3 with Western and Northern analysis indicated that cyclin D2 mRNA is specifically down-regulated by DEHP in a dose-dependent manner, and this decrease is manifest as a small, transient but reproducible reduction in the amount of cyclin D2 protein detectable in samples from treated pups compared to controls. Our findings characterize the changes in neonatal Sertoli cells and gonocytes that follow in vivo to low levels of DEHP and its metabolite, MEHP, as well as providing new information on the underlying mechanism and highlighting the extreme sensitivity of the neonatal testis to injury by this toxicant.

During neonatal testicular development in the rat, events critical for subsequent germ cell development occur that set the stage for fertility later in life. Some gonocytes resume mitotic activity and/or migrate to the surrounding basal lamina, and use of a carefully defined Sertoli cell-gonocyte coculture system indicates that these crucial events occur without added factors or hormones and are hence likely to depend on interaction with adjacent Sertoli cells. Coupling of the Kit receptor protein on gonocytes to stem cell factor from Sertoli cells is vital for successful migration by gonocytes, as antagonism of the former suppresses and addition of the latter stimulates gonocyte migration. During the neonatal period, intercellular adhesion is modified in a developmental manner such that neural cell adhesion molecule (NCAM) is the main adhesive molecule expressed and functioning at birth, with a progressive decline as development proceeds. This decline in NCAM expression is supported by the addition of exogenous 3,3',5-triiodothyronine in vitro, and because this factor is recognized as supporting Sertoli cell differentiation, it seems likely that changing intercellular adhesion is a function of progressive development of Sertoli cells. Other avenues whereby maturing testicular cells influence each other doubtless exist, including secretion of growth factors and other peptides and developmentally important changes in the makeup of the extracellular matrix, which Sertoli cells and gonocytes contact. Continued investigation in these areas will be very valuable in enlarging our understanding of how neonatal testicular development provides the basis for successful spermatogenesis.

We co-cultured gonocytes and Sertoli cells isolated on the day of birth and observed the appearance, 1 and 3 days after the start of culture, of gonocytes that had developed cellular processes and that were labeled by [3H]thymidine, respectively. These events occurred in the absence of hormones, etc. and with a time course very similar to that seen in vivo. In other incubations, we found that the presence of laminin in the underlying substrate was critical in promoting proliferation and elongation of gonocytes. These observations strongly suggest that interactions between gonocytes and other testicular cells/factors in the co-cultures promote maturation of these germ cells in vitro, and thus provide new evidence to support the concept that paracrine mechanisms are important during testicular development as well as in adults.

The Myb gene family currently consists of three members, named A-, B- and c-myb1,2. These genes encode nuclear proteins that bind DNA in a sequence-specific manner and function as regulators of transcription. In adult male mice, A-myb is expressed predominantly in male germ cells2,3. In female mice, A-myb is expressed in breast ductal epithelium, mainly during pregnancy-induced ductal branching and alveolar development. We report here that mice homozygous for a germline mutation in A-myb develop to term but show defects in growth after birth and male infertility due to a block in spermatogenesis. Morphological examination of the testes of A-myb−/− males revealed that the germ cells enter meiotic prophase and arrest at pachytene. In adult homozygous null A-myb female mice, the breast epithelial compartment showed underdevelopment of breast tissue following pregnancy and the female mice were unable to nurse their newborn pups. These results demonstrate that A-myb plays a critical role in spermatogenesis and mammary gland development.

Introduction: Studies of FNP in the diagnosis of focal lesions of the pancreas have reported a sensitivity of 57-93% and a specificity of 95-100% for the method. The objective of the present investigation was to evaluate the sensitivity and specificity of FNP in the diagnosis of focal lesions of the pancreas. Patients and Methods: Between May, 1995 and June, 1996 we performed FNP on 25 patients (14 females, 11 males; average age 61.4 + 10.91 years, range 31-77 years). In nine cases, confirmation of findings was surgical, in the remaining 16 patients based on a one-year clinical follow-up period. Results: Aspiration of diagnostically adequate material was possible in 18 cases (72%) (malignancy, 10; chronic pancreatitis, 4; benign lesions, 4). For the other seven patients (28%), clinical follow-up revealed pancreatic carcinoma in two patients, pancreatitis in four patients, while in one case even the surgical specimen obtained for histologic examination failed to provide a diagnosis. For patients with adequate cytology findings, one-year follow-up revealed false-negative findings in three cases: three of four patients with chronic pancreatitis developed carcinoma. Discussion.: FNP in the diagnosis of focal pancreatic lesions showed a sensitivity of 77% and a specificity of 100%. There were no complications observed in our group. Conclusion: Negative findings at FNP in chronic pancreatitis does not exclude the possibility of carcinoma. In the case of equivocal findings, criteria for surgery should be broad.

Quantitative cytochemistry was used to determine the effect of subjecting pregnant rats to environmental stress on the activity of delta 5-3 beta hydroxysteroid dehydrogenase (3 beta-HSD) in Leydig cells of their fetuses. Enzyme activity was measured by microspectrophotometry in individual Leydig cells in cryostat sections of fetal testes on Days 16-21 postconception. Fetuses of stressed mothers lacked the peak of enzyme activity on Days 18 and 19 of gestation that is characteristic of Leydig cells of normal fetuses at this time. In addition, both before and after these 2 days, 3 beta-HSD activity in Leydig cells of stressed fetuses was significantly higher than normal. The altered developmental pattern of 3 beta-HSD activity in the stressed fetuses largely corresponds to the changes in plasma testosterone found previously in male fetuses of mothers exposed to the same regimen of stress. Thus, in the fetal Leydig cell, the activity of 3 beta-HSD, a key steroidogenic enzyme, can be modified by environmental stress, and provides an index of steroidogenic activity of the fetal testes and of the titers of circulating testosterone.

Proliferation of Sertoli cells in the rat testis occurs only during the perinatal period and is maximal during fetal life. This interval is thus of critical importance in establishing the complement of Sertoli cells that populates the adult testis. FSH has been implicated in this process, but direct evidence in support of its involvement is lacking. In the present study, we have used in vivo and in vitro approaches to determine whether FSH produced by the fetal pituitary has a role in regulating Sertoli cell division in the fetal testis of the rat. On day 18 of gestation, just before the onset of maximal Sertoli cell proliferation, fetuses were either decapitated in utero or given antiserum to FSH. Light microscope autoradiography was then used to compare uptake of [3H]thymidine by Sertoli cell nuclei in testes from decapitated or antiserum-treated fetuses to that in corresponding controls on the following day. Both treatments produced dramatic and equal reductions in the percentages of Sertoli cells preparing to divide on day 19, suggesting that FSH from the fetal pituitary stimulates Sertoli cell proliferation in fetal testes. The effect of FSH or (Bu)2cAMP on Sertoli cell proliferation was also studied in vitro by placing testes from intact or decapitated fetuses into organ culture, with or without exogenous hormone or cyclic nucleotide. In all cases, [3H]thymidine was present for the final 4 h of culture. When testes were placed into medium containing isotope immediately after their removal from the fetus, the difference in labeling between testes from intact and decapitated fetuses was similar to that measured in vivo. After testes from decapitated fetuses were cultured for 8 h with or without FSH or (Bu)2cAMP, labeling of Sertoli cells in the treated group increased markedly over that in untreated cultures. After 28 h of exposure to FSH or (Bu)2cAMP, labeling in testes from decapitated fetuses remained significantly higher than that in corresponding untreated controls. In contrast, when testes from intact rats were cultured for 8 h in the presence of either cAMP or FSH, (Bu)2cAMP, but not FSH, brought about an increase in the percentage of Sertoli cells labeled compared to the control value. However, after exposing these testes to either FSH or (Bu)2cAMP for 28 h, the percentage of Sertoli cells labeled was greatly enhanced. Taken together, the data obtained from these experiments identify FSH as a major factor in controlling expansion of the Sertoli cell population during fetal development of the rat.(ABSTRACT TRUNCATED AT 400 WORDS)

In a previous study, we used autoradiography to demonstrate specific FSH-binding sites on Sertoli plasma membranes in the basal compartment of rat seminiferous tubules. The present study was undertaken to determine whether spermatogonia in the rat testis also possess FSH-binding sites. Approximately 1 Mg [l25I]hFSH was administered as an intratesticular injection to 40-day-old rats that had been hypophysectomized 10 days earlier; a second group of rats received a 500-fold excess of cold hormone along with the [I25I]- hFSH, and served as a control for specificity of binding. Subsequently, all testes were perfused with a hyperosmotic fixative, which produces preferential shrinkage of spermatogonia and Sertoli cells in the basal compartment. The resulting separation of the cells from one another allowed us to determine whether spermatogonia were the source of any autoradiographic grains. In the absence of excess cold hormone, some grains were clearly localized to spermatogonial surfaces, although the majorit...

Diabetes which occurs spontaneously in the BB Wistar rat is associated with reduced fertility, predominantly in breeding males. In the first month of diabetes, there is a significant (p less than 0.05) reduction in serum testosterone associated with a transient decrease of serum LH and the accumulation of lipid in Leydig cells. Between one and three months of diabetes, there is an increase in both serum testosterone and LH and a further deposition of lipid droplets in Leydig cells. From three to six months of diabetes, there is a reduction of serum testosterone similar to age-matched controls, but high serum LH levels persist. Similar levels of LH and testosterone are noted after six months of diabetes, and all BB rats show marked changes in seminiferous tubules. These morphological changes in tubules consist of increased tubular wall thickness, severe germ-cell depletion, and Sertoli-cell vacuolization. Similar morphological changes of testes associated with generalized atrophy are noted in all control rats after 16 months of age. Decreased fertility in the BB rat appears to be associated with a primary disorder of Leydig cells, which precedes changes in seminiferous tubules consistent with accelerated aging. Preliminary data in impotent diabetic men suggest that the BB rat may be a valuable model for investigating human diabetic impotence and infertility.

Previous evidence has indicated that the testicular receptors for FSH reside on plasma membranes in seminiferous tubules, and that the Sertoli cell is a primary target of the hormone. Since the Sertoli cell is large and elaborately structured, it is important to know whether there is a regional distribution of receptors over its extensive surface. In the present study, the specific binding of 25I-labeled hFSH to seminiferous tubules of hypophysectomized rats was examined, utilizing autoradiography at both the light and electron microscope levels. Purified hFSH (LER-1575C) was iodinated to an average specific activity of 29 μCi/μg, and approximately 1 μg was administered intratesticularly in 40 day old rats which had been hypophysectomized 10 days earlier. In some cases, testosterone was administered after hypophysectomy in order to maintain spermatogenesis. Controls were of two types: 40 day old intact littermates in which endogenous FSH presumably occupied most of the receptors, and hypophysectomized rat...

The urine of patients with proteinuria of various etiologies was examined to determine if proteinuria alone was associated with significant glycosphingolipiduria. In all cases of proteinuria examined, the level of glycosphingolipids in the urine was found to be markedly elevated. There was no evidence of a glycosphingolipid storage disorder in any case. It was concluded that significant glycosphingolipiduria may occur in proteinuria as well as in the glycosphingolipid storage disorders.

Rat pups were hemicastrated and uptake of [3 H] thymidmne by Sertoli cells in the remaining testis was compared to that in testes of sham-operated pups at intervals of from 8 h to 21 days after surgery. Labeled thymidine was administered subcutaneously 2 h before sacrifice. Testes were processed for light microscope autoradiography and the percent of Sertoli cell nuclei that had incorporated [3 H] thymidmne was determined by scoring nuclei in tissue sections as labeled or unlabeled. The percentage of cells labeled was increased in hemicastrates over intact controls by 8 h after surgery and testicular hypertrophy became apparent in hemicastrates by the following day. Labeling of Sertoli cells in hemicastrates remained elevated for 4 days and then returned to normal. When plasma levels of gonadotropmns were measured in both groups 4 days after surgery, follicle-stimulating hormone (FSH) was found to be more than twice normal in hemicastrates while luteinizing hormone (LH) was unchanged. The effect of testosterone on the response of Sertoli cells to hemicastration was also examined. In hemicastrates, 2 days of androgen therapy depressed, and an additional 2 days abolished, the proliferative response of the Sertoli cells. Our findings suggest that increased proliferation of Sertoli cells within the remaining testis is involved in the enlargement of the testis that follows hemicastration. They also imply that prevention of compensatory hypertrophy by testosterone involves interference with this response of Sertoli cells in some way. Finally, our data implicate FSH in control of Sertoli cell proliferation in vivo in immature rats.

The pituitary-testicular axis was investigated in the streptozotocin diabetic male rat to determine the relationship between hormonal alterations and Leydig cell morphology. Male Wistar rats (60 days of age) were made diabetic with a single intravenous injection (65 mg/kg body weight) of streptozotocin and were studied with non-diabetic controls at 7–11, 14, 21, and 28 days after treatment. The observations on these animals were compared to those from diabetic rats (28 days) treated with 1–2 U protamine zinc insulin/100 mg body weight. Rats with untreated diabetes had hyperglycaemia and weighed less than controls. Untreated rats also had low plasma testosterone levels associated with decreased prostate and seminal vesicle weights. Insulin treatment partially or completely prevented these abnormalities. Studies of testosterone production y perfused testis suggested Leydig cell insensitivity to LH as one possible cause of decreased androgen blood levels in diabetic rats. Lipid droplets not found in Leydig cells of normal rats were seen in diabetic animals. Plasma FSH levels were also low in untreated diabetic rats but were normal in insulin

To probe the possible role of endogenous opiates in Sertoli cell proliferation during testicular development, the effect of interfering with beta-endorphin action either in vivo or in vitro was determined. The percent of Sertoli cells dividing was measured with quantitative autoradiography in [methyl 3H]-thymidine-exposed fetal testes maintained in organ culture with or without FSH, in the presence or absence of the opiate blocker naloxone. After 1 or 2 days in culture, naloxone enhanced the rise in Sertoli cell proliferation seen with FSH alone, while 2 days of incubation with naloxone alone markedly raised the percent of Sertoli cells dividing above that in untreated cultures. Moreover, when endorphin antiserum was injected directly into testes of pups and Sertoli cell proliferation in vivo measured 8 or 19 h later, there was a dramatic increase in the percent of Sertoli nuclei labeled by [methyl 3H]-thymidine compared to controls. These findings suggest that beta-endorphin produced within the testis is a paracrine modifier of the proliferative response of Sertoli cells to FSH. This implies that communication occurs between Leydig and Sertoli cells during development via endogenous testicular opiates.

To probe the relationship between the size of the Sertoli cell population, established during perinatal development, and production of germ cells in the adult testis, a Sertoli cell-depleted rat model was developed. This was accomplished by delivering an antimitotic drug, cytosine arabinoside (araC), directly to the testis of newborn pups. Initial studies of these araC-treated neonates indicated that 1) the drug is cleared rapidly from the testis; 2) it substantially reduces the level of Sertoli cell proliferation; 3) Sertoli cell division ceases at a normal time in spite of the previous drug treatment; and 4) araC itself has no residual effect on germ cell proliferation, which begins several days after the injection. Pups given araC were allowed to reach maturity, and their testes were perfuse-fixed for light microscopic morphometry. When the numbers of Sertoli cells in adult rats given araC as were compared with those in normal littermates, a 54% decrease in the size of the Sertoli cell population was detected in treated rats, now referred to as Sertoli cell-depleted. Moreover, when round spermatids were quantified and compared in normal and Sertoli cell-depleted adults, testes of the latter were found to contain 55% fewer round spermatids. Since, in the araC-treated group, the decrease in Sertoli cell population size was paralleled by a reduction in spermatid production of equal magnitude, the number of round spermatids per Sertoli cell was essentially identical in normal and Sertoli cell-depleted animals. Measurements of serum androgen-binding protein (ABP) and FSH in both groups indicated that the circulating level of ABP in Sertoli cell-depleted rats was approximately half, and the concentration of FSH approximately twice, that in normal animals. Thus, even though FSH is elevated in Sertoli cell-depleted rats, the production of ABP per Sertoli cell is unchanged. In addition, collective volume of Leydig cells and ventral prostate weights were normal in the Sertoli cell-depleted group, suggesting that Leydig cell function in these rats is normal. In summary, a Sertoli cell-depleted rat model has been produced by interfering specifically with Sertoli cell proliferation early in postnatal life, before onset of germ cell division. Moreover, our findings with this model indicate that production of normal numbers of germ cells in adults depends, at least in part, on the size of the Sertoli cell population. Thus, our observations identify the perinatal period, when the Sertoli cell population is established, as critical for development of quantitatively normal spermatogenesis in the adult.

Proliferation of Sertoli cells during fetal and postnatal development of the rat was examined and quantified with light microscope autoradiography. Fetuses in utero were injected subcutaneously with 3H-thymidine. The percentages of Sertoli nuclei that had incorporated label were determined in autoradiographs from fetuses aged 16 through 21 days of gestation. To compare the degree of Sertoli cell proliferation during fetal development with that occurring after birth, pups were also studied at intervals between the day of birth and 3 weeks of age. For each fetus or pup, at least 500 Sertoli cell nuclei in each of three sections were scored as labeled or unlabeled. These data were subjected to analysis of variance and the Newman-Keuls test. The percentage of Sertoli cells incorporating 3H-thymidine increased progressively from day 16 of gestation onward, to a maximum of 26.8% on day 20, two days before birth. Thereafter, this percentage dropped steadily until, in pups 21 days after birth, no labeled Sertoli cells were detected. These findings highlight the fetal period as the time of greatest expansion of the Sertoli cell population and indicate that, at birth, proliferation of these cells is already on the decline.

Hyperglycemia (experimental diabetes) was induced in adult male rats by destruction of the pancreatic beta cells with a single intravenous injection of streptozotocin (STZ). Testes from diabetic, from insulin-treated diabetic, and from sham-injected normal rats were fixed by vascular perfusion. The fine structure of Leydig cells was examined at two, three, and four weeks after the STZ injection in the untreated diabetic animals, and at four weeks in the controls and insulin-treated diabetic rats. A number of morphological changes was observed in Leydig cells of untreated diabetic animals. Most obvious of these was an accumulation of lipid droplets, not normally present in Leydig cells in adults of this species. Smooth endoplasmic reticulum (SER) was markedly reduced in Leydig cells of the hyperglycemic rats. Several types of intracellular bodies were seen exclusively in Leydig cells of the untreated diabetic animals. Many resembled secondary lysosomes or dense bodies, while others appeared to be autophagic vacuoles. In addition, a small, granule-containing lamellar structure was seen either within a typical dense body or free in the cytoplasm. Myelin-like structures were commonly observed within the cytoplasm of the Leydig cell or within mitochondria. The appearance of the mitochondria in diabetic rats was otherwise normal. The extracellular spaces surrounding Leydig cells from untreated hyperglycemic rats also contained large accumulations of myelin-like material. These structural changes appear to be direct consequences of the diabetic state of the animals, since the ultrastructure of insulin-treated diabetic rats did not differ from that of the controls. These findings may reflect an alteration or breakdown of Leydig cell components normally involved in the synthesis of androgen, and correlate with previous reports of lowered circulating levels of testosterone in diabetic rats.