Arbovírus Morumbi (Phlebovirus : Bunnyaviridae) - estudo histopatológico e imuno-histoquímico do fígado na infecção experimental em camundongos: comparação entre as vias cerebral, intraperitoneal e subcutânea

O vírus Morumbi é membro do sorogrupo Phlebotomus fever (família Bunyavírídae: gênero Phlebovírus) nativo da Região Amazônica. Seu vetor é desconhecido, mas supõem-se ser transmitido por flebotomíneos. Foi isolado em 1988 de ser humano apresentando quadro febril agudo. Este arbovírus, quando inoculado em camundongo por via cerebral, demonstrou viscerotropismo, induzindo inclusive lesões no fígado do animal inoculado. Com os objetivos de: i) estabelecer as características anátomo-patológicas e imuno-histoquímicas em fígado de camundongos albinos Swíss recém-nascidos experimentalmente infectados pelo vírus Morumbi; ii) verificar se o vírus apresenta hepatotropismo diferenciado na dependência de inoculação pelas vias cerebral, peritoneal ou subcutânea; iii) caracterizar detalhadamente os padrões anátomo-patológicos sequenciais no fígado; iv) demonstrar a localização do antígeno viral no tecido hepático ao longo da infecção experimental; v) estudar possíveis inter-relações entre os achados anátomo-patológicos e os imuno-histoquímicos. Foram estudados experimentalmente 71 camundongos Swíss recém-nascidos (dois e três dias), distribuídos ao final do experimento como segue: 21 animais inoculados por via intracerebral (IC), 21 por via intraperitoneal (IP) e 29 animais inoculados por via subcutânea (SC). Utilizou-se a dose infectante 5,0DL 50 /0,02ml de suspensão de vírus. Outros trinta, animais que não receberam inóculos, foram utilizados como grupo controle. Subgrupos de oito animais (seis inoculados e dois do grupo controle) foram sacrificados diariamente a intervalos de 24 em 24 horas, até 96 horas para os grupos IC e IP e até 120 horas para o grupo SC. Fragmentos de fígado de todos os animais foram fixados em solução de formalina neutra a 10%, incluídos em parafina, de onde foram obtidos cortes de 5 mm que foram corados pela técnica de hematoxilina-eosina para análise morfológica e, cortes adicionais, foram submetidos à técnica de imuno-histoquímica (Sistema Envision, DAKO, USA), utilizando a fosfatase alcalina e soro hiperimune do vírus Morumbi preparado em camundongos jovens, para detecção de antígeno viral. Foram estudados seis parâmetros de lesão em áreas portais e nove outros nos lóbulos, que foram semiquantificados numa escala que variou de zero (0) a três cruzes (+++), onde zero significou ausência de lesão e três cruzes lesão intensa. À microscopia óptica, ficou evidente que o vírus Morumbi inoculado em camundongos por três diferentes vias induz lesões em áreas portais e lobulares, caracterizando uma hepatite aguda com presença de corpúsculos acidófilos, semelhantes aos corpúsculos de Councilman -Rocha Lima, de distribuição irregular nos lóbulos, cujo aparecimento foi observado 24 horas pós-inoculação (p.i.) e atingiu o máximo de intensidade às 72 horas p.i. em animais inoculados por via IP. O exame imuno-histoquímico mostrou presença leve de antígeno viral a partir de 24 horas p.i. no grupo IC e a partir de 48 horas p.i. nos grupos IP e SC, havendo certo paralelismo em relação a intensidade de lesão morfológica, tendo- se observado o máximo de detecção de antígeno viral em animais inoculados por via IP e sacrificados às 72 horas p.i. A distribuição geral de antígeno foi observada especificamente nos lóbulos hepáticos, no citoplasma de hepatócitos íntegros e necrosados e no interior de células de Kupffer, não havendo preferência por nenhuma das três zonas do lóbulo. Concluiu-se que: i) o modelo de infecção experimental em camundongos foi excelente para o estudo das lesões causadas pelo vírus Morumbi, podendo ser selecionada a via IP como referencial; ii) em todas as vias utilizadas (IP, IC e SC) se confirmou a infecção pelo vírus Morumbi com marcante detecção de seu antígeno, no tecido hepático de camundongos Swiss; iii) a presença de antígeno do vírus Morumbi no fígado desses camundongos associou-se ao aparecimento de hepatite aguda, com necrose focal; iv)hepatite intensa pôde ser observada em fígado de camundongos sacrificados 72 h p.i. com o vírus Morumbi por via IP, o que não foi verificado com as outras duas vias; v) a hepatite aguda mostrou-se limitada, neste experimento, tendendo a desaparecer na maioria dos camundongos inoculados, com avançar das horas; vi) colestase não alteração freqüente na hepatite experimental pelo vírus Morumbi, quando inoculada por via IC, IP e SC; vii) o antígeno do vírus Morumbi teve predominância pela localização intracitoplasmática, padrão granular, nos hepatócitos e células de Kupffer; viii) antígeno viral foi detectado em fragmento hepático de animais experimentalmente inoculados com o vírus Morumbi, a partir das 24 horas via IC e a partir de 48 horas nas vias IP e SC.

Avaliação do estado emocional associado à catalepsia causada pela administração intraperitoneal de haloperidol

Haloperidol is a dopamine receptor antagonist used to treat schizophrenia. When systemically administered in rodents, haloperidol induces catalepsy, a state of immobility very similar to that seen in Parkinson's disease. It is known that many of Parkinson's disease symptoms are dependent on the emotional state since patients are still able to respond to external triggers such as loud noise or visual signaling. Recent data highlighted the importance of glutamatergic neurotransmission in the inferior colliculus (IC) on the cataleptic state induced by haloperidol in rats. Given the importance of IC in the brain aversion system and its connections to motor pathways, and based on the clinical reports of the emotional influence on the motor aspect of Parkinson's disease, the objective of the present study was to evaluate the emotional aspect related to catalepsy induced by intraperitoneal administration of haloperidol. To this end, we analysed ultrasonic vocalizations (UVs) of 22 kHz (indicative of aversion) in rats during the tests of catalepsy, open field and contextual conditioned fear. Systemic administration of haloperidol affected the motor activity, inducing catalepsy and decreasing exploratory activity in the open field. There were no UVs of 22 kHz resulting from treatment with haloperidol in catalepsy or open field tests. In the contextual conditioned fear test, haloperidol increased freezing when administered before the test, but decreased freezing on test day when administered before training. In this same test, haloperidol decreased the UVs on the day it was administered (training or test). The catalepsy induced by systemic administration of haloperidol seems to have also affected the motor aspect of UVs. In this way, it was not possible to clarify the existence of an aversive emotional state associated haloperidol induced catalepsy

Avaliação da proliferação celular na próstata de gerbilos vasectomizados, após indução química pela injeção intraperitoneal do carcinógeno N-metil-N-nitrosuréia (MNU)

Prostate cancer is the second leading cause of cancer death in the country. Due to this factor, the interest of showing what relationship exists between vasectomy surgery and the incidence of prostate carcinoma has started. Some epidemiological studies showed an increased risk of prostate cancer in vasectomized men (Emard et al., 2001). On the other hand other authors have argued that there is no correlation (Patel et al., 2005) and there are those who said that vasectomy is linked to reduced risk of prostate cancer (Ross, 1983). Faced with an analysis of published works, such discussion remains today. The vasectomy, or deferentectomia, is a contraceptive male method, which is the section of the vas deferens of man by preventing the sperm from being expelled along with the seminal fluid during ejaculation, and is one of the most simple, economical, uncomplicated post- operative. This study aims to evaluate the process of cell proliferation and to evaluate immuno-histochemically the expression of specific markers of PCNA (Proliferating Cell Nuclear Antigen) and Ki-67, in the prostate of the gerbil after chemical induction by intraperitoneal injection of the carcinogen N-methyl -N-nitrosourea (MNU), because the regulation of the functional balance between cell proliferation and apoptosis is associated with hyperplasia and prostatic carcinoma. The experimental procedure was performed at the Laboratory of the Department of Anatomy, along with collecting the data for later analysis

Intraperitoneal murine Echinococcus multilocularis infection induces differentiation of TGF-beta-expressing DCs that remain immature

Intraperitoneal larval infection (alveolar echinococcosis, AE) with Echinococcus multilocularis in mice impairs host immunity. Metacestode metabolites may modulate immunity putatively via dendritic cells. During murine AE, a relative increase of peritoneal DCs (pe-DCs) in infected mice (AE-pe-DCs; 4% of total peritoneal cells) as compared to control mice (naive pe-DCs; 2%) became apparent in our study. The differentiation of AE-pe-DCs into TGF-beta-expressing cells and the higher level of IL-4 than IFN-gamma/IL-2 mRNA expression in AE-CD4+pe-T cells indicated a Th2 orientation. Analysis of major accessory molecule expression on pe-DCs from AE-infected mice revealed that CD80 and CD86 were down-regulated on AE-pe-DCs, while ICAM-1(CD54) remained practically unchanged. Moreover, AE-pe-DCs had a weaker surface expression of MHC class II (Ia) molecules as compared to naive pe-DCs. The gene expression level of molecules involved in MHC class II (Ia) synthesis and formation of MHC class II (Ia)-peptide complexes were down-regulated. In addition, metacestodes excreted/secreted (E/S) or vesicle-fluid (V/F) antigens were found to alter MHC class II molecule expression on the surface of BMDCs. Finally, conversely to naive pe-DCs, an increasing number of AE-pe-DCs down-regulated Con A-induced proliferation of naive CD4+pe-T cells. These findings altogether suggested that TGF-beta-expressing immature AE-pe-DCs might play a significant role in the generation of a regulatory immune response within the peritoneal cavity of AE-infected mice.

Intraperitoneal Echinococcus multilocularis infection in mice modulates peritoneal CD4+ and CD8+ regulatory T cell development

Intraperitoneal proliferation of the metacestode stage of Echinococcus multilocularis in experimentally infected mice is followed by an impaired host immune response favoring parasite survival. We here demonstrate that infection in chronically infected mice was associated with a 3-fold increase of the percentages of CD4+ and CD8+ peritoneal T (pT) cells compared to uninfected controls. pT cells of infected mice expressed high levels of IL-4 mRNA, while only low amounts of IFN-gamma mRNA were detected, suggesting that a Th2-biased immune response predominated the late stage of disease. Peritoneal dendritic cells from infected mice (AE-pDCs) expressed high levels of TGF-beta mRNA and very low levels of IL-10 and IL-12 (p40) mRNA, and the expression of surface markers for DC-maturation such as MHC class II (Ia) molecules, CD80, CD86 and CD40 was down-regulated. In contrast to pDCs from non-infected mice, AE-pDCs did not enhance Concanavalin A (ConA)-induced proliferation when added to CD4+ pT and CD8+ pT cells of infected and non-infected mice, respectively. In addition, in the presence of a constant number of pDCs from non-infected mice, the proliferation of CD4+ pT cells obtained from infected animals to stimulation with ConA was lower when compared to the responses of CD4+ pT cells obtained from non-infected mice. This indicated that regulatory T cells (Treg) may interfere in the complex immunological host response to infection. Indeed, a subpopulation of regulatory CD4+ CD25+ pT cells isolated from E. multilocularis-infected mice reduced ConA-driven proliferation of CD4+ pT cells. The high expression levels of Foxp3 mRNA by CD4+ and CD8+ pT cells suggested that subpopulations of regulatory CD4+ Foxp3+ and CD8+ Foxp3+ T cells were involved in modulating the immune responses within the peritoneal cavity of E. multilocularis-infected mice.

Intraperitoneal and intra-nasal vaccination of mice with three distinct recombinant Neospora caninum antigens results in differential effects with regard to protection against experimental challenge with Neospora caninum tachyzoites

Recombinant NcPDI(recNcPDI), NcROP2(recNcROP2), and NcMAG1(recNcMAG1) were expressed in Escherichia coli and purified, and evaluated as potential vaccine candidates by employing the C57Bl/6 mouse cerebral infection model. Intraperitoneal application of these proteins suspended in saponin adjuvants lead to protection against disease in 50% and 70% of mice vaccinated with recNcMAG1 and recNcROP2, respectively, while only 20% of mice vaccinated with recNcPDI remained without clinical signs. In contrast, a 90% protection rate was achieved following intra-nasal vaccination with recNcPDI emulsified in cholera toxin. Only 1 mouse vaccinated intra-nasally with recNcMAG1 survived the challenge infection, and protection achieved with intra-nasally applied recNcROP2 was at 60%. Determination of cerebral parasite burdens by real-time PCR showed that these were significantly reduced only in recNcROP2-vaccinated animals (following intraperitoneal and intra-nasal application) and in recNcPDI-vaccinated mice (intra-nasal application only). Quantification of viable tachyzoites in brain tissue of intra-nasally vaccinated mice showed that immunization with recNcPDI resulted in significantly decreased numbers of live parasites. These data show that, besides the nature of the antigen, the protective effect of vaccination also depends largely on the route of antigen delivery. In the case of recNcPDI, the intra-nasal route provides a platform to generate a highly protective immune response.

Intraperitoneal mesh implantation for fascial dehiscence and open abdomen

Postoperative fascial dehiscence and open abdomen are severe postoperative complications and are associated with surgical site infections, fistula, and hernia formation at long-term follow-up. This study was designed to investigate whether intraperitoneal implantation of a composite prosthetic mesh is feasible and safe.

Intraperitoneal Echinococcus multilocularis infection in C57BL/6 mice affects CD40 and B7 costimulator expression on peritoneal macrophages and impairs peritoneal T cell activation

One of the most important immunopathological consequence of intraperitoneal alveolar echinococcosis (AE) in the mouse is suppression of T cell-mediated immune responses. We investigated whether and how intraperitoneal macrophages (MØs) are, respectively, implicated as antigen-presenting cells (APCs). In a first step we showed that peritoneal MØs from infected mice (AE-MØs) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells. In a subsequent step, AE-MØs as well as naïve MØs (positive control) proved their ability to uptake and process C-Ova fluorescein isthiocyanate (FITC). Furthermore, in comparison with naïve MØs, the surface expression of Ia molecules was up-regulated on AE-MØs at the early stage of infection, suggesting that AE-MØs provide the first signal via the antigen-Ia complex. To study the accessory activity of MØs, AE-MØs obtained at the early and late stages of infection were found to decrease Con A-induced proliferation of peritoneal naïve T cells as well as of AE-sensitized peritoneal T cells, in contrast to stimulation with naïve MØs. The status of accessory molecules was assessed by analysing the expression level of costimulatory molecules on AE-MØs, with naïve MØs as controls. It was found that B7-1 (CD80) and B7-2 (CD86) expression remained unchanged, whereas CD40 was down-regulated and CD54 (= ICAM-1) was slightly up-regulated. In a leucocyte reaction of AE-MØs with naïve or AE-T cells, both types of T cells increased their proliferative response when CD28 - the ligand of B7 receptors - was exposed to anti-CD28 in cultures. Conversely to naïve MØs, pulsing of AE-MØs with agonistic anti-CD40 did not even partially restore their costimulatory activity and failed to increase naïve or AE-T cell proliferation. Neutralizing anti-B7-1, in combination with anti-B7-2, reduced naïve and AE-T cell proliferation, whereas anti-CD40 treatment of naïve MØs increased their proliferative response to Con A. These results point at the key role of B7 receptors as accessory molecules and the necessity of the integrity of CD40-expression by naïve MØs to improve their accessory activity. Taken together, the obstructed presenting-activity of AE-MØs appeared to trigger an unresponsiveness of T cells, contributing to the suppression of their clonal expansion during the chronic phase of AE-infection.

A reduction in severe hypoglycaemia in type 1 diabetes in a randomized crossover study of continuous intraperitoneal compared with subcutaneous insulin infusion

Continuous intraperitoneal insulin infusion (CIPII) with the DiaPort system using regular insulin was compared to continuous subcutaneous insulin infusion (CSII) using insulin Lispro, to investigate the frequency of hypoglycemia, blood glucose control, quality of life, and safety.

Implantation of Prophylactic Nonabsorbable Intraperitoneal Mesh in Patients With Peritonitis Is Safe and Feasible

BACKGROUND: Patients with peritonitis undergoing emergency laparotomy are at increased risk for postoperative open abdomen and incisional hernia. This study aimed to evaluate the outcome of prophylactic intraperitoneal mesh implantation compared with conventional abdominal wall closure in patients with peritonitis undergoing emergency laparotomy. METHOD: A matched case-control study was performed. To analyze a high-risk population for incisional hernia formation, only patients with at least two of the following risk factors were included: male sex, body mass index (BMI) >25 kg/m(2), malignant tumor, or previous abdominal incision. In 63 patients with peritonitis, a prophylactic nonabsorbable mesh was implanted intraperitoneally between 2005 and 2010. These patients were compared with 70 patients with the same risk factors and peritonitis undergoing emergency laparotomy over a 1-year period (2008) who underwent conventional abdominal closure without mesh implantation. RESULTS: Demographic parameters, including sex, age, BMI, grade of intraabdominal infection, and operating time were comparable in the two groups. Incidence of surgical site infections (SSIs) was not different between groups (61.9 vs. 60.3 %; p = 0.603). Enterocutaneous fistula occurred in three patients in the mesh group (4.8 %) and in two patients in the control group (2.9 %; p = 0.667). The incidence of incisional hernia was significantly lower in the mesh group (2/63 patients) than in the control group (20/70 patients) (3.2 vs. 28.6 %; p < 0.001). CONCLUSIONS: Prophylactic intraperitoneal mesh can be safely implanted in patients with peritonitis. It significantly reduces the incidence of incisional hernia. The incidences of SSI and enterocutaneous fistula formation were similar to those seen with conventional abdominal closure.

Intraperitoneal bevacizumab for control of malignant ascites due to advanced-stage gastrointestinal cancers: A multicentre double-blind, placebo-controlled phase II study - AIO SUP-0108

PURPOSE: Malignant ascites is debilitating for patients with advanced cancer. As shown previously, tumour cell production of vascular endothelial growth factor might be a major cause of the formation of malignant ascites. Intraperitoneal bevacizumab could therefore be an option for symptom control in refractory ascites. PATIENTS AND METHODS: Patients with advanced gastrointestinal cancer and malignant ascites who had undergone paracentesis at least twice within the past 4 weeks were randomly assigned in a 2:1 ratio to intraperitoneal bevacizumab (400 mg absolute) or placebo after paracentesis. During the 8-week treatment period, a minimum interval of 14 d was kept between the applications of the study drug. Primary end-point was paracentesis-free survival (ParFS). RESULTS: Fifty-three patients (median age 63 years) were randomised. Forty-nine patients received at least one study drug application and qualified for the main analysis. The proportion of patients with at least one common toxicity criteria grade III-V event was similar with 20/33 (61%) on bevacizumab and 11/16 (69%) on placebo. Median ParFS was 14 d (95% confidence interval [CI]: 11-17) in the bevacizumab arm and 10.5 d (95% CI: 7-21) on placebo (hazard ratio 0.74, 95% CI: 0.40-1.37; P = 0.16). The longest paracentesis-free period was 19 d on bevacizumab (range 6-66 d) and 17.5 d in the placebo arm (range 4-42) (P = 0.85). Median overall survival was 64 d (95% CI: 45-103) on bevacizumab compared to 31.5 d (95% CI: 20-117) on placebo (P = 0.31). CONCLUSION: Intraperitoneal bevacizumab was well tolerated. Overall, treatment did not result in a significantly better symptom control of malignant ascites. However, patients defined by specific immune characteristics may benefit.

Individual monitoring of immune responses in rainbow trout after cohabitation and intraperitoneal injection challenge with Yersinia ruckeri

Acknowledgements This work was funded by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs, grant G1100675). The authors are grateful to the aquarium staff at the University of Aberdeen (Karen Massie) and Dr David Smail at Marine Scotland for valuable discussion during the establishment of the experimental design.

Eficacia de la ropivacaína 0,1% intraperitoneal en el control del dolor postoperatorio en cirugía ginecológica laparoscópica

Introducción: Determinar la eficacia de la administración preincisional de ropivacaína al 0,1 % intraperitoneal en el control del dolor abdominal y/o de hombro, durante la primera semana de la cirugía laparoscópica ginecológica benigna. Diseño: Ensayo clínico aleatorizado y doble ciego. Material y métodos: Se realizó un ensayo clínico aleatorizado y doble ciego donde participaron 64 pacientes ASA I-III sometidas a cirugía laparoscópica ginecológica por patología benigna. Tras la realización del neumoperitoneo, se administraron 100 ml de ropivacaína 0,1 % o suero fisiológico intraperitoneal, dependiendo del grupo al que pertenecieran. Las pacientes recibieron, además, AINE junto con una bomba de PCA con opción de morfina de rescate como analgesia multimodal asociada. Se evaluó el dolor abdominal y/o de hombro al despertar, en reposo y en movimiento, a los 5, 30, 60 y 120 minutos, así como a las 24 horas. Se registró el consumo de morfina en las primeras 24 horas y la incidencia de náuseas y/o vómitos postoperatorios. A la semana, mediante encuesta telefónica, se registró la presencia de dolor de hombro a partir de las 24 horas, así como de dolor abdominal persistente al 7º día. Resultados: No se observaron diferencias significativas en el ENV durante las primeras 24 horas. Tampoco se observaron diferencias en el consumo de morfina, en la incidencia de náuseas y/o vómitos o en el dolor de hombro. Se evidenciaron diferencias estadísticamente significativas en la incidencia de dolor abdominal persistente al 7º día (18,52 % en grupo tratamiento vs. 57,58 % en grupo control con p = 0,04). Conclusiones: La administración intraperitoneal preincisional de 100 ml de ropivacaína 0,1 % en comparación con la administración de suero fisiológico, en el contexto de una técnica anestésica y analgésica multimodal, no ha demostrado reducir el dolor postoperatorio, el consumo de opioides ni la incidencia de náuseas y vómitos postoperatorios en las primeras 24 horas. Tampoco ha demostrado reducción del dolor de hombro a partir del primer día tras cirugía laparoscópica ginecológica. El uso de ropivacaína al 0,1 % intraperitoneal preincisional presenta una disminución estadísticamente significativa en la incidencia de dolor abdominal persistente al séptimo día de postoperatorio.

Pautas de protección frente al riesgo de exposición a citostáticos en quimioterapia intraperitoneal hipertérmica

Objetivo: El objetivo del trabajo realizado es identificar las tareas de riesgo asociadas a la manipulación de citostáticos, detectadas en la aplicación de Quimioterapia Intraperitoneal con Hipertermia (HIPEC) aplicada según la técnica de Sugarbaker y las diferentes soluciones llevadas a cabo para su control. Método: La detección y evaluación de los riesgos existentes, así como la propuesta y selección de las diferentes medidas de prevención implantadas se realizaron siguiendo las pautas de: entrevista y estudio de la actividad con el personal especialista implicado, estudio bibliográfico, identificación de requerimientos legales y normativos aplicables, para finalizar estableciendo las medidas de control. Por último se comprobó mediante consulta a las partes involucradas el grado de satisfacción con las medidas adoptadas (adherencia a las medidas) y la eficacia de las medidas adoptadas (número de accidentes e incidentes). Resultados: Se identificaron las tareas de mayor riesgo y las principales vías de exposición. Se determinaron medidas de control de la exposición por vía inhalatoria y dérmica, acordes a los requisitos establecidos para la protección de los trabajadores y para uso en campo quirúrgico, minimizando el disconfor que pudieran provocar al personal. Conclusiones: Se pone de manifiesto la posibilidad de conjugar protección del personal, cumplimiento normativo, preservación del campo quirúrgico y confort del personal. A través de acciones formativas e informativas y el trabajo cooperativo de los diferentes servicios participantes, se consiguió un elevado grado de aceptación por parte del personal involucrado de las medidas de prevención establecidas. Se minimizó la inquietud del personal frente a los riesgos laborales afrontados en HIPEC. Como cierre de la acción preventiva, se considera recomendable la comprobación de la eficacia de las medidas adoptadas a través de evaluación cuantitativa.