Estudio retrospectivo en pacientes con Cáncer de Colon etapa III. ¿El tratamiento adyuvante es iniciado de forma oportuna?

Introducción: En Chile, el Cáncer colorrectal (CC) abarca el 11,5% de todas las neoplasias malignas. La cirugía es la piedra angular del tratamiento del cáncer de colon, y en pacientes en etapa III, la quimioterapia adyuvante forma parte del tratamiento estándar. Materiales y Métodos: Estudio descriptivo retrospectivo transversal, de centro único, de pacientes con cáncer de colon en estadio III patológico. Objetivo principal de este estudio es conocer si, en pacientes con cáncer de colon etapa III, la quimioterapia adyuvante se entrega de manera oportuna. Resultados: En el período comprendido entre abril de 2016 y abril de 2021 se operaron 35 pacientes con cáncer de colon en estadio III patológico. Se realizó quimioterapia adyuvante en un 80%, y en siete pacientes durante las primeras ocho semanas poscirugía. La dehiscencia de anastomosis ocurrió en un 11,4%, aumentando la mediana de hospitalización en 2,2 veces. Discusión: En nuestro estudio, la adyuvancia en cáncer de colon etapa III se administró a un alto porcentaje de los pacientes (80%), pero observamos un retraso importante, ya que sólo en un 25% se inició el tratamiento durante las primeras 8 semanas poscirugía, lo cual puede ser explicado por múltiples factores, siendo la dehiscencia de anastomosis un punto importante a considerar. Conclusión: En estadio III de CC la adyuvancia puede verse retrasada por múltiples factores, lo que puede repercutir en la sobrevida de los pacientes, por lo tanto, conocer las causas de este retraso podría ayudar a instaurar nuevas estrategias, como la neoadyuvancia, para mejorar los resultados oncológicos.

Introduction: In Chile, colorectal cancer covers 11.5% of all malignant neoplasms. Surgery is the cor- nerstone of colon cancer treatment and in stage III patients adjuvant chemotherapy is part of standard treatment. Materials and Methods: A descriptive, retrospective, cross-sectional study, single center, of patients with pathological stage III colon cancer. Main objective of this study is to know if in patients with stage III colon cancer adjuvant chemotherapy is delivered in a timely manner. Results: Between April 2016 and April 2021, 35 patients with pathological stage III colon cancer were operated on. Adjuvant chemotherapy was performed in 80%, and in seven patient during the first eight weeks after surgery. Anastomotic dehiscence occurred in 11.4%, the median hospitalization increased by 2.2 times. Discussion: In this study, adjuvant stage III colon cancer was administered to a high percentage of patients (80%), but we observed a significant delay, since only 25% began treatment during the first 8 weeks post-surgery, which can be explained by multiple factors, with anastomotic dehiscence being an important point to consider. Conclusion: In stage III CC, adjuvant treatment can be delayed by multiple factors, which may affect patient survival; therefore, knowing the causes of this delay could help to establish new strategies, such as neoadjuvant therapy, to improve oncological results.

Versatility of Tunneled Island Flaps for the Reconstruction of Facial Defects. / Versatilidad del «colgajo en isla tunelizado¼ para la reconstrucción de defectos faciales.

BACKGROUND AND OBJECTIVE: Tunneled island flaps that transfer skin from a site adjacent to the wound are an effective way to reconstruct defects. These flaps provide good aesthetic and functional results and can be completed during a single surgical session, thus reducing complications. The procedure consists of taking an island of skin about the size of the surgical defect and moving it through a subcutaneous tunnel to cover the wound. We aimed to exemplify the use of these flaps in different regions of the face. MATERIAL AND METHODS: Retrospective descriptive study of 12 cases in which tunneled island flaps were used after removal of malignant tumors in different facial regions. RESULTS: The tumors, all of which were basal cell carcinomas, were removed by means of conventional surgery from 9 patients and Mohs micrographic surgery from 3 patients. Histology demonstrated tumor-free margins after excision in all cases. None of the tumors recurred during a mean follow-up period of 25 months. All complications were minor and transient. There were 3 cases of trapdoor effect and 2 each of postsurgical bleeding, transient superficial flap necrosis, and slight protrusion of the pedicle. The final cosmetic and functional outcome was satisfactory in all cases. CONCLUSIONS: Tunneled island flaps are particularly useful for repairing defects in the center of the face, where several anatomical structures converge. These flaps, which use donor skin that is similar to skin in the receptor site, can be used to close large defects in one-step procedures with only slight alteration of the facial architecture.

Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry.

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

Reconstructing Ear Defects Using the Revolving Door Island Flap: A Case Series. / Reconstrucción de defectos auriculares mediante el «colgajo en isla en puerta giratoria¼. Serie de casos.

BACKGROUND AND OBJECTIVES: Defects of the anterior region of the pinna pose a reconstruction challenge owing to the complex topography and difficult access. We report our experience using the revolving door island flap (RDIF) to reconstruct pinna defects and present the surgical results. MATERIAL AND METHODS: Retrospective descriptive study of patients undergoing ear reconstruction using RDIF at different sites of the anterior region after resection of malignant tumors. RESULTS: Seventeen patients underwent surgery between 2011 and 2019 for squamous cell carcinoma (n=7) and basal cell carcinoma (n=10). Conventional histology showed disease-free surgical margins in all the resected tissue specimens. The mean follow-up period was 40 months. One local recurrence of squamous cell carcinoma was observed, which responded well to radiation therapy; all patients were free of disease at the end of follow-up. Only 3 patients presented minor complications (depression of the flap surface in 1 case and retraction of the ear toward the mastoid process in 2 cases). Subjective rating scales were used to evaluate cosmetic and functional outcomes as assessed by both the dermatologist and the patient. The normal contour of the pinna was conserved in all patients and no patients required additional corrective surgery. CONCLUSIONS: Reconstruction of the pinna using RDIF allowed for satisfactory repair following complete resection of the tumors, while conserving structure and functionality. RDIF has been reported to be a very good option for reconstruction of the pinna. It is a simple technique that makes it possible to repair large defects with minimal risk of necrosis. In our series, the cosmetic results were excellent and recurrence rates were very low. RDIF provided optimal results in repairing lesions in the concha and in other regions, such as the antihelix, triangular fossa, and scapha.

Epidemiología, tratamiento y mortalidad en pacientes infectados por enterobacterias productoras de carbapenemasas: estudio retrospectivo / Epidemiology, treatment and mortality in infection by carbapenemase-producing Enterobacteriaceae: retrospective study

Resumen Introducción: Las enterobacterias productoras de carbapenemasas (EPC) suponen un reto para la salud pública y la práctica clínica. Objetivo: Analizar la epidemiología, el tratamiento y la mortalidad en pacientes infectados por EPC. Material y Métodos: Análisis retrospectivo de 163 pacientes infectados por EPC en un hospital universitario desde julio de 2013 a octubre de 2015. Resultados: Klebsiella pneumoniae fue aislada en 95,1% de los casos, y la mayoría de las carbapenemasas pertenecían al grupo OXA-48 (93%). La adquisición fue nosocomial en 124 casos (77%), asociada a cuidados sanitarios en 30 (18,6%), y 7 (4,3%) fueron de adquisición comunitaria. Las infecciones más frecuentes fueron las del tracto urinario (48,4%) y las respiratorias (19,5%). Aproximadamente, la mitad de los pacientes recibieron monoterapia antimicrobiana. La tasa de mortalidad a los 30 días fue de 23,3%. El análisis multivariante identificó que la presencia de shock séptico al diagnóstico (OR 4,2; IC 95% 1,5-11) estaba asociada de manera independiente con mayor mortalidad en el primer mes, sin lograr identificar asociación con el tratamiento antimicrobiano inapropiado. Discusión: Son necesarios más estudios para aclarar si el tratamiento antimicrobiano de las infecciones por EPC debe ser combinado o si podría ser suficiente la monoterapia en infecciones leves.

Abstract Background: Carbapenemase-producing Enterobacteriaceae (CPE) has become a significant problem in terms of public health and clinical outcome. Objective: To assess the epidemiology, treatment and mortality in patients with infection due to CPE. Material and Methods: A retrospective analysis of 163 patients with CPE infection was carried out in a university hospital from July 2013 to October 2015. Results: A total of 163 patients were included over the study period. Klebsiella pneumoniae was isolated in 95.1% of cases, and most of carbapenemases belonged to the OXA-48 group (93.0%). Acquisition was nosocomial in 124 cases (77.0%), healthcare-associated in 30 (18.6%), and 7 cases (4.3%) were community-acquired. The most frequent infections identified in this study were urinary tract (48.4%) and respiratory (19.5%) infections. Approximately half of the patients received antibiotic monotherapy. The 30-day mortality rate was 23.3%. Multivariate analysis revealed that the presence of septic shock at diagnosis (OR 4.2; IC 95% 1.5-11) was independently associated with an increase in death during the first month, unable to identify association with inappropriate antibiotic treatment. Discussion: Further studies are needed to clarify whether antibiotic treatment of EPC infections should be combined or if monotherapy might be sufficient in mild infections.

Functional Surgery for Malignant Subungual Tumors: A Case Series and Literature Review. / Cirugía funcional en tumores malignos subungueales. Serie de casos y revisión de la literatura.

BACKGROUND AND OBJECTIVES: Amputation is the conventional treatment for malignant subungual tumors (MSUTs), namely, subungual squamous cell carcinoma (SUSCC) and subungual melanoma (SUM). Functional surgery consisting of wide local excision (WLE) of the nail unit can preserve function without modifying prognosis in such cases. We present a series of MSUTs treated with WLE of the nail unit, describe the technique, and review its indications. MATERIAL AND METHODS: Retrospective observational study of MSUTs treated with WLE of the nail unit between 2008 and 2017. The technique consisted of en bloc supraperiosteal excision of the nail unit with a margin of 5mm followed by repair with a full-thickness graft. RESULTS: Eleven MSUTs were treated in the study period: 7 SUMs (4 in situ; mean thickness, 1.17mm; range, 0-4mm) and 4 SUSCCs (mean thickness, 3.4mm; range, 1.6-6mm). WLE of the nail unit was performed in 9 patients and amputation in 2 patients with invasive SUM. Mean follow-up was 39 months (range, 12-96 months) and no local or regional recurrences were detected. One of the 2 patients who underwent amputation developed metastasis to the brain and died. In our review of the literature, we identified 5 series of patients with SUSCC treated with WLE of the nail unit (105 patients) and 14 series of patients with SUM (243 patients). Based on an analysis of these cases and ours, it would appear that WLE of the nail unit is associated with a very low rate of local recurrence (<7%) and offers better functional and cosmetic outcomes than amputation. CONCLUSIONS: WLE of the nail unit is the treatment of choice for SUSCC without bone involvement and for thin noninvasive SUM (Breslow depth <1mm). It is also feasible in intermediate-thickness SUMs when detailed histologic examination of the margins confirms complete resection. Amputation, by contrast, is the treatment of choice for SUSCCs with bone involvement, very thick SUMs (>4mm), and recurrent tumors.

Progesterone suppresses the lipopolysaccharide-induced pro-inflammatory response in primary mononuclear cells isolated from human placental blood.

Progesterone is an essential hormone that induces deep immune adaptations favoring pregnancy maintenance. We aimed at evaluating the effects of progesterone on the synthesis of pro- and anti-inflammatory cytokines by mononuclear cells isolated from human placental blood stimulated with lipopolysaccharide, emulating an infection-inflammation environment. Mononuclear cells isolated form human placental blood were obtained from nine women undergoing elective cesarean delivery at term (not in labor), isolated by density gradient sedimentation, cultured and co-stimulated with lipopolysaccharide (500 ng/ml) from Escherichia coli in the presence or not of progesterone (0.01, 0.1, or 1.0 µM) for 24 h. Culture supernatants were assayed for pro-inflammatory (IL-1ß, TNFα, IL-6), anti-inflammatory (IL-10) cytokines, chemokines (IL-8, MIP-1α) and total MMP-9 by ELISA. In comparison with basal conditions, lipopolysaccharide treatment induced IL-1ß, TNFα, IL-6, IL-8, MIP-1α, and MMP-9 synthesis. lipopolysaccharide co-treatment with progesterone significantly decreased the bacterial endotoxin-induced IL-1ß, TNF-α, IL-6, IL-8, and MIP-1α secretion. In contrast, co-treatment with progesterone increased the level of IL-10 secreted to the culture medium. The present results support the concept that progesterone can modulate--partially--the inflammatory response of professional immune cells isolated from placental blood. Therefore, progesterone might be part of the natural compensatory mechanism that limits the cytotoxic effects associated with an intrauterine infection process during gestation.

Interobserver reliability of store-and-forward teledermatology in a clinical practice setting.

INTRODUCTION AND OBJECTIVES: Although many studies have evaluated the diagnostic reliability of store-and-forward (SF) teledermatology, the reliability of the technique for the diagnosis of general skin conditions in a clinical practice setting has never been demonstrated. We evaluated the reliability of SF teledermatology in clinical practice by analyzing the diagnostic agreement achieved in a subgroup of patients from the DERMATEL-2 study. MATERIAL AND METHODS: Patients referred from primary care settings were randomized to 3 groups: SF, a combination of videoconferencing and SF technology (VC-SF), and a control group. This article focuses on the SF group. Clinical data were recorded and photographs taken by primary care physicians, who forwarded the data electronically. Each SF consultation package was assessed by 3 dermatologists (D1,D2,D3). Subsequently all the patients were assessed by a single dermatologist (D1) in a face-to-face consultation. Finally, 2 other dermatologists (D4,D5) assessed the agreement between the diagnoses obtained by SF and FF. RESULTS: In total, 457 patients (200 males and 257 females) aged between 2 months and 86 years were randomized (192 to SF, 176 to VC-SF, and 89 to the control group). The diagnostic categories were as follows: tumors (49.4%), inflammatory (25.7%), adnexal (11%), infectious (9.4%) and other processes (4.4%) Since 170 patients had consultations deemed valid for analysis, the study included a total of 510 SF assessments. Most of the images and clinical records were of high quality (71.2% and 91.2% respectively), and diagnostic confidence was high in 81.4% of the cases studied. In 58.4% of cases the condition was managed exclusively by teledermatology. Levels of complete and aggregate interobserver agreement between SF and face-to-face evaluators were 0,72 and 0.90, respectively, for diagnosis and 0.61 and 0.80 for treatment. Diagnostic agreement correlated with the image quality (P < .001), diagnostic confidence (P<.001), felt need for conventional consultation (P<.001), and the quality of the clinical record (P=.013). CONCLUSION: The interobserver reliability of SF diagnosis in clinical practice is good. Dermatologists are able to predict errors in diagnosis by analyzing their own diagnostic confidence and evaluating the quality of the images.

Aspergilosis nasosinusal con extensión intracraneal en una paciente inmunocompetente: una entidad infrecuente con características radiológicas típicas / Sinonasal aspergillosis with intracranial extension in an immunocompetent patient: a rare disease with typical imaging features

El aspergilo es un hongo ubicuo. Las localizaciones de infección primaria más comunes son el tracto respiratorio y los senos paranasales. La afectación intracraneal es rara y conlleva una alta mortalidad. Ocurre mayoritariamente por extensión hematógena desde el pulmón, pero en pacientes inmunocompetentes, la extensión directa desde los senos paranasales es más común. Describimos el caso de una mujer de 25 años originaria de India que se presentó en el servicio de urgencia de nuestro centro hospitalario con cefalea frontal crónica y progresiva. Los hallazgos en los estudios de imágenes sugirieron el diagnóstico de sinusitis fúngica con extensión intracraneal, siendo el patógeno más frecuente el aspergilo. El diagnóstico fue confirmado anátomo-patológicamente. Revisamos los hallazgos radiológicos típicos que deben ayudar al diagnóstico precoz de esta entidad, rara, pero potencialmente mortal.

Aspergillus is a ubiquitous fungus. The most common primary sites of infection are the respiratory tract and sinuses. Intracranial infection is rare and implies a high mortality. It occurs mainly by hematogenous extension from the lung, but in immunocompetent patients, direct extension from the sinuses is more common. We describe the case of a 25 year old woman from India who consulted in the emergency room of our hospital with chronic and progressive frontal headache. The findings in imaging studies suggested the diagnosis of fungal sinusitis with intracranial extension, being the most common pathogen of Aspergillus. The diagnosis was anatomically-pathologically confirmed. We review the typical radiological findings which should help in the early diagnosis of this rare but potentially fatal disease.

Pituitary adenylate cyclase-activating polypeptide controls stimulus-transcription coupling in the hypothalamic-pituitary-adrenal axis to mediate sustained hormone secretion during stress.

External and internal stimuli that threaten homeostasis trigger coordinated stress responses through activation of specialised neuroendocrine circuits. In mammals, the hypothalamic-pituitary-adrenal (HPA) axis mediates responses to stressors such as restraint, ultimately enhancing adrenocortical hormone secretion. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in central control of the HPA axis, and we have recently shown PACAP-dependent expression of corticotropin-releasing hormone (CRH) and secretion of corticosterone in response to restraint. We now provide a more detailed analysis of PACAP-dependent HPA axis stimulation in the mouse, indicating that the hypothalamic paraventricular nucleus (PVN) is the primary site of action. We demonstrate by quantitative polymerase chain reaction and in situ hybridisation that up-regulation of mRNAs encoding CRH and inducible transcription factors, from the Nr4a family (Nur77, Nor1) in the PVN is PACAP-dependent. Furthermore, CRH hnRNA is rapidly up-regulated in cultured hypothalamic neurones after treatment with PACAP. Induction of Nr4a factors (Nur77, Nurr1) in response to restraint is attenuated in the pituitary gland of PACAP-deficient mice. In the adrenal glands, restraint elicits a marked PACAP-dependent increase in adrenocortical mRNA levels of all three Nr4a transcription factors, steroidogenic factor 1 (Nr5a1), steroidogenic acute regulatory protein and steroid 21-hydroxylase. Taken together, our results show that PACAP controls HPA responses to restraint primarily at the level of the hypothalamus by up-regulating CRH, possibly involving transcription factors such as Nur77 and Nor1. Subsequent adrenocortical steroidogenesis also appears to involve PACAP-dependent stimulus-transcription coupling, suggesting a mechanism by which PACAP exerts control over HPA axis function during stress.

Vasopressin protects hippocampal neurones in culture against nutrient deprivation or glutamate-induced apoptosis.

Vasopressin (VP) secreted within the brain modulates neuronal function by acting as a neurotransmitter. Recent studies show that VP prevents serum deprivation-induced apoptosis in the neuronal cell line, H32. To determine whether VP is anti-apoptotic in hippocampal neurones, primary cultures of these neurones were used to examine the effect of VP on neuronal culture supplement (B27) deprivation-, or glutamate-induced apoptosis, and the signalling pathways mediating the effects. Removal of B27 supplement from the culture medium for 24 h or the addition of glutamate (3-10µm) decreased neuronal viability (P<0.05) and increased Tdt-mediated dUTP nick-end labelling (TUNEL) staining and caspase-3 activity (P<0.05), which is consistent with apoptotic cell death. VP (10 nm) reduced B27 deprivation- or glutamate-induced cell death (P<0.05). These anti-apoptotic effects of VP were completely blocked by a V1 but not a V2 receptor antagonist, indicating that they are mediated via V1 VP receptors. The anti-apoptotic effect of VP in neurones involves activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and inositol trisphosphate/protein kinase B (Akt) signalling pathways. This was shown by the transient increases in phospho-ERK and phospho-Akt after incubation with VP revealed by western blot analyses, and the ability of specific inhibitors to reduce the inhibitory effect of VP on caspase-3 activity and TUNEL staining by 70% and 35%, respectively (P<0.05). These studies demonstrate that VP has anti-apoptotic actions in hippocampal neurones, an effect that is mediated by the MAPK/ERK and phosphatidylinositol-3 kinase/Akt signalling pathways. The ability of VP to reduce nutrient deprivation or glutamate overstimulation-induced neuronal death suggests that VP acts as a neuroprotective agent within the brain.

Adolescencia prevenida: pérfil sociopsicológico del adolescente rural medido a través del inventario autodescriptivo del adolescente / Prevented adolescence: socio psychological profile of rural adolescent measured by the adolescent self-descriptive inventory

Esta investigación parte y suma sus resultados a una base de datos de estudios sobre el perfil positivo y negativo del adolescente rural en cada una de las áreas evaluadas, establecidas en la macro investigación “Adolescencia Prevenida Rural” que lleva a cabo como área de estudio la Cátedra de Psicología Experimental I y II de la UCA desde 2001.En este caso el objetivo general es describir y comparar el perfil sociopsicológico del adolescente paraguayo rural de la Zona de Chaco (Villa Hayes-Cerrito) y sus variantes en relación al sexo y edad, mediante la utilización del Inventario Autodescriptivo. La muestra seleccionada estuvo compuesta por n=89 participantes de ambos sexos siendo la misma no probabilística, intencionada y autoseleccionada. Las variables comparadas de las áreas del “Inventario Autodescriptivo” evalúan los factores de riesgo presentes en las mismas y del Perfil sociodemográfico. El diseño empleado es de tipo transversal, descriptivo-exploratorio-comparativo.Los resultados concluyen que el perfil sociopsicológico del Adolescente rural presenta factores de riesgo tanto en varones y mujeres, lo cual se debe probablemente a sus características sociodemográficas e individuales.

[Focal epithelial hyperplasia]. / Hiperplasia epitelial focal.

Focal epithelial hyperplasia is a rare disease of the oral mucosa caused by the human papilloma virus (HPV). It appears as a benign epithelial growth, usually in the mucosa of the lower lip. It is mainly associated with HPV serotypes 13 and 32 and there is a clear racial predilection for the disease in Native Americans and Eskimos. We describe the case of a 17-year-old girl from Ecuador with multiple papular lesions in both lips that were clinically and histologically consistent with focal epithelial hyperplasia. Analysis by polymerase chain reaction detected HPV serotype 13.

Interaction between oestrogen and oxytocin on hypothalamic-pituitary-adrenal axis activity.

In addition to its role in reproduction, oxytocin has central actions modulating behavioural and hypothalamic-pituitary-adrenal (HPA) axis responses during late pregnancy and lactation. The hypothesis that ovarian hormones modulate the effects of oxytocin on HPA axis activity was studied in 7-day ovariectomised rats receiving oestradiol with or without progesterone replacement and intracerebroventricular (i.c.v) minipump infusion of oxytocin (100 ng/h). In an initial experiment, i.c.v. oxytocin had no effect on basal or restraint-stimulated plasma adrenocorticotrophic hormone (ACTH) and corticosterone concentrations or hypothalamic corticotrophin-releasing factor (CRF) mRNA expression with low oestradiol replacement alone but it had a stimulatory effect in the presence of low oestradiol and progesterone. To investigate further whether oestradiol modulates central actions of oxytocin, rats received low dioestrous (low), pro-oestrous (medium) or pregnancy (high) oestradiol replacement levels, yielding plasma concentrations of < 5, 17.3 +/- 4.5 and 258 +/- 32 pg/ml, respectively, with or without i.c.v. oxytocin. Oestradiol caused dose-dependent increases in basal plasma ACTH and corticosterone concentrations but decreased the ACTH response to restraint stress. In parallel to the changes in basal plasma ACTH, high oestrogen increased basal CRF hnRNA, CRF mRNA in the paraventricular nucleus and pro-opiomelanocortin (POMC) mRNA in the pituitary gland, while decreasing restraint stress-stimulated levels. Intracerebroventricular administration of oxytocin reduced basal and stress-stimulated plasma ACTH, hypothalamic CRF hnRNA (30 min), CRF mRNA and pituitary POMC mRNA (4 h) levels parallel to the increases induced by elevating plasma oestradiol. The present study demonstrates the converse effects of oestradiol on basal and restraint stress-stimulated basal HPA axis activity, and that the ability of central oxytocin to inhibit HPA axis activity depends on the levels of circulating oestradiol.

Regulation of pituitary corticotropin releasing factor type-1 receptor mRNA binding proteins by modulation of the hypothalamic-pituitary-adrenal axis.

Long-term adrenalectomy (ADX) is associated with marked down-regulation of pituitary corticotropin releasing factor type-1 receptors (CRF-R1) but normal CRF-R1 mRNA levels, suggesting that regulation of receptor levels occurs at post-transcriptional sites. We have reported that adrenal cytosolic proteins, which bind to cis elements in the 5' leader sequence (5'LS) of the rat angiotensin II type 1a receptor (AT(1a)R) mRNA, participate in the regulation of AT(1a)R density by inhibiting AT(1a)R mRNA translation. In this study, we examined anterior pituitary cytosolic proteins that form RNA protein complexes (RPC) with the 5'LS of the CRF-R1 and the AT(1a)R. Competition studies and ultraviolet-crosslinking analysis suggest that formation of CRF-R1 and AT(1a)R 5'LS RPC require at least some proteins that are common to both receptor mRNAs. Pituitaries isolated from male Sprague-Dawley rats six days after ADX showed significant (P < 0.05) increases of 2.9-fold in CRF-R1 5'LS RNA binding protein (BP) activity compared to pituitaries from sham-operated rats; this effect of ADX was prevented by glucocorticoid replacement. By contrast, no differences in the number of pituitary AT(1a)R binding sites or pituitary AT(1a)R 5'LS BP activity were observed between sham and ADX rats, indicating that the effect of ADX on RPC formation was specific for CRF-R1 mRNA. Addition of pituitary cytosolic extracts inhibited in vitro translation of CRF-R1 mRNA by 79% under conditions that had no significant effects on the translation of a control mRNA. The data suggest that CRF-R1 translation is regulated by modulation of the HPA axis through pituitary cytosolic proteins binding to the CRF-R1 5'LS.

Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signalling in an immortalized hypothalamic cell line.

Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.

Lipopolysaccharide endotoxin potentiates the effect of osmotic stimulation on vasopressin synthesis and secretion in the rat hypothalamus.

Vasopressin secreted by magnocellular neurones of the hypothalamic supraoptic and paraventricular nuclei is essential for water balance. In this study, we examined magnocellular neurone responses to osmotic stimulation in vehicle-injected controls or rats receiving an intraperitoneal (i.p.) injection of 250 microg/100 g of lipopolysaccharide (LPS), 3 h or 6 h earlier. LPS injection had no effect on plasma vasopressin concentrations in control rats but it caused marked and transient potentiation of the responses to a single i.p. injection of hypertonic saline (five- and two-fold, 3 and 6 h after LPS, respectively). The enhancement of plasma vasopressin responses was independent of plasma sodium concentrations or changes in blood pressure. Basal vasopressin mRNA expression in the paraventricular and supraoptic nuclei decreased slightly 6 h after LPS injection, without changes in vasopressin transcription as indicated by vasopressin heteronuclear (hn) RNA levels. Parvocellular neurones showed expected increases in vasopressin hnRNA expression following LPS injection and a further increase after i.p. hypertonic saline injection (due to the painful component). In contrast to magnocellular vasopressin mRNA expression, the effects of LPS and hypertonic saline injections in parvocellular neurones were additive and not synergistic. Light microscopic immunohistochemical examination revealed an increase in size of vasopressin but not oxytocin axonal terminals in the neural lobe 3 h after LPS injection. Osmotic stimulation caused marked depletion of vasopressin immunoreactivity in axonal terminals of the neural lobe in both control and LPS-pretreated rats. The changes in vasopressin axon terminals were accompanied by induction of interleukin (IL)-1 beta and IL-6 in the posterior pituitary. The data show that endotoxemia causes morphological and functional alterations of the hypothalamic neurohypophyseal system, resulting in facilitation rather than inhibition of vasopressin synthesis, and secretion in response to osmotic stimulation.