The need for opioid in the postoperative analgesia of dogs undergoing hemilaminectomy due to intervertebral disc extrusion

ABSTRACT: The aim of this study was to evaluate the postoperative analgesic effect of protocols with and without the opioid methadone in dogs with intervertebral disc extrusion undergoing decompressive surgery. Sixteen paraplegic dogs with preserved nociception underwent hemilaminectomy/disc fenestration and were randomly assigned to two groups. The analgesic protocol consisted of methadone, meloxicam and dipyrone in Group I (G1), and meloxicam and dipyrone in Group II (G2). The animals were blindly assessed by two observers, using the visual analogue scale (VAS) and the short-form Glasgow Composite Measure Pain Scale (CMPS-SF). Assessments occurred every 2 hours during first 24 hours post-surgery, and every 4 hours afterwards. There was no statistical difference among groups regarding pain scores or analgesic rescues. Both analgesic protocols provided analgesia in the initial 48 hours postoperatively, demonstrating that opioids are not necessary in the postoperative period of dogs undergoing hemilaminectomy and disc fenestration.

RESUMO: O objetivo deste estudo foi avaliar a analgesia pós-operatória de protocolos com e sem o opioide metadona em cães com extrusão de disco intervertebral submetidos à descompressão cirúrgica. Dezesseis cães paraplégicos com presença de nocicepção foram submetidos à hemilaminectomia/fenestração de disco e distribuídos aleatoriamente em dois grupos. No Grupo I (G1), o protocolo analgésico consistiu em metadona, meloxicam e dipirona e, no Grupo II (G2), por meloxicam e dipirona. Os pacientes foram avaliados de maneira cega por dois avaliadores, com base na escala visual analógica (EVA) e na escala simplificada composta de dor de Glasgow (CMPS-SF). As avaliações ocorreram a cada 2 horas durante as primeiras 24 horas de pós-operatório e, por mais 24 horas, a cada 4 horas. Não houve diferença estatística entre os grupos avaliados em relação à escores de dor e nem a necessidade de resgate analgésico. Ambos os protocolos promoveram analgesia nas 48 horas iniciais de pós-operatório, demonstrando não haver a necessidade do uso de opioide em cães submetidos à hemilaminectomia e fenestração de disco.

The need for opioid in the postoperative analgesia of dogs undergoing hemilaminectomy due to intervertebral disc extrusion / A necessidade de opioide na analgesia pós-operatória de cães submetidos à hemilaminectomia devido à extrusão do disco intervertebral

The aim of this study was to evaluate the postoperative analgesic effect of protocols with and without the opioid methadone in dogs with intervertebral disc extrusion undergoing decompressive surgery. Sixteen paraplegic dogs with preserved nociception underwent hemilaminectomy/disc fenestration and were randomly assigned to two groups. The analgesic protocol consisted of methadone, meloxicam and dipyrone in Group I (G1), and meloxicam and dipyrone in Group II (G2). The animals were blindly assessed by two observers, using the visual analogue scale (VAS) and the short-form Glasgow Composite Measure Pain Scale (CMPS-SF). Assessments occurred every 2 hours during first 24 hours post-surgery, and every 4 hours afterwards. There was no statistical difference among groups regarding pain scores or analgesic rescues. Both analgesic protocols provided analgesia in the initial 48 hours postoperatively, demonstrating that opioids are not necessary in the postoperative period of dogs undergoing hemilaminectomy and disc fenestration.(AU)

O objetivo deste estudo foi avaliar a analgesia pós-operatória de protocolos com e sem o opioide metadona em cães com extrusão de disco intervertebral submetidos à descompressão cirúrgica. Dezesseis cães paraplégicos com presença de nocicepção foram submetidos à hemilaminectomia/fenestração de disco e distribuídos aleatoriamente em dois grupos. No Grupo I (G1), o protocolo analgésico consistiu em metadona, meloxicam e dipirona e, no Grupo II (G2), por meloxicam e dipirona. Os pacientes foram avaliados de maneira cega por dois avaliadores, com base na escala visual analógica (EVA) e na escala simplificada composta de dor de Glasgow (CMPS-SF). As avaliações ocorreram a cada 2 horas durante as primeiras 24 horas de pós-operatório e, por mais 24 horas, a cada 4 horas. Não houve diferença estatística entre os grupos avaliados em relação à escores de dor e nem a necessidade de resgate analgésico. Ambos os protocolos promoveram analgesia nas 48 horas iniciais de pós-operatório, demonstrando não haver a necessidade do uso de opioide em cães submetidos à hemilaminectomia e fenestração de disco.(AU)

Clinical management of dogs with presumptive diagnosis of cervical intervertebral disc disease: 78 cases (2006-2017) / Tratamento clínico de cães com diagnóstico presuntivo de doença do disco intervertebral cervical: 78 casos (2006-2017)

This study aimed to identify dogs with a presumptive diagnosis of cervical intervertebral disc disease (IVDD; C1-C5 or C6-T2) submitted to clinical management and evaluate the outcome. This study also aimed to demonstrate the age, sex, and treatment response according to the neurological degree, and verify whether those factors could potentially influence the outcome. The data were obtained from patients with a neurological dysfunction, admitted at the Veterinary Hospital from January 2006 to March 2017. In addition to patient records, the tutors answered a questionnaire related to the success of therapy. A hundred and seventy-seven neurological records were evaluated, and 78 were included in the study according to the inclusion criteria. The most frequent breeds were Dachshunds, followed by mixed-breed dogs. Regarding the neurological dysfunction degree, 58.97% presented grade I (only neck pain), 25.64% were grade II (ambulatory tetraparesis), and 15.38% grade III (nonambulatory tetraparesis). Absolute and partial space rest were performed in 75.64% and 24.36% of the cases, respectively. The minimum rest time was one week and could come up to four weeks. Most dogs were small-sized (≤15kg). The recovery was satisfactory in 87.17% of dogs and unsatisfactory in 12.83%. Regarding recurrence, we observed that 10.3% of dogs presented satisfactory recovery. The clinical treatment for dogs with cervical IVDD can be indicated with adequate clinical response to dysfunction degrees ranging from I to III, either at rest or in restricted space and with a low rate of relapse.(AU)

O objetivo desse estudo foi identificar cães com diagnóstico presuntivo de doença do disco intervertebral cervical (DDIV; C1-C5 ou C6-T2) submetidos ao tratamento clínico e avaliar a resposta a terapia instituída e o índice de recidiva. Esse estudo também visou demonstrar a idade, o gênero e a resposta ao tratamento de acordo com o grau neurológico, a fim de verificar se esses parâmetros podem ser utilizados como fatores prognósticos para a evolução clínica desses pacientes. Foram revisados os registros neurológicos do Hospital Veterinário Universitário de janeiro de 2006 a março de 2017. Realizaram coleta de dados a partir dos registros e por meio de um questionário respondido pelos tutores. Avaliaram 177 fichas neurológicas de cães e obtidas informações para inclusão no estudo em 78 delas. As raças mais frequentes foram Dachshunds, seguido dos cães sem raça definida. Quanto ao grau de disfunção neurológica, 58,97% apresentavam grau I (somente dor), 25,64% estavam em grau II (tetraparesia ambulatória) e 15,38% em grau III (tetraparesia não ambulatória). O repouso absoluto e em espaço restrito foram realizados em 75,64% e 24,36% dos casos, respectivamente e com duração de no mínimo uma semana, podendo chegar a mais de quatro semanas. A maioria dos animais era de pequeno porte (≤15kg). A recuperação foi satisfatória em 87,17% dos cães e insatisfatória em 12,83%. Quanto à recidiva, esta foi observada em 10,3% dos pacientes com recuperação satisfatória. O tratamento clínico para cães com DDIV cervical pode ser indicado com adequada resposta clínica para graus de disfunção que variam de I a III, seja em repouso absoluto ou em espaço restrito e com baixo índice de recidiva.(AU)

Clinical management of dogs with presumptive diagnosis of thoracolumbar intervertebral disc disease: 164 cases (2006-2017) / Tratamento clínico de cães com diagnóstico presuntivo de doença do disco intervertebral toracolombar: 164 casos (2006-2017)

This study aimed to identify dogs with presumptive diagnosis of cervical intervertebral disc disease (IVDD) submitted to clinical management and to evaluate the outcomes. Data were obtained from the medical records of patients with neurological dysfunction assisted at a University Veterinary Hospital from 2006 to 2017. In addition to the patients' records, dog owners responded to a questionnaire on the success of therapy. Four hundred and thirteen neurological records were evaluated, and 164 met the inclusion criteria of the study. The most common breed was Dachshund, followed by mongrels. Classification of neurological dysfunction in the study sample was as follows: 15.9% with grade I, 25.6% with grade II, 26.8% with grade III, 8.5% with grade IV, and 23.2% with grade V. Outcome was satisfactory in 71.6% of the dogs and unsatisfactory in 28.4% of them. Recurrence was observed in 27.7% of those with satisfactory outcomes. The clinical treatment of dogs with thoracolumbar IVDD is satisfactory, particularly for animals with milder disease grades (I, II, and III). There is possibility of recurrence with conservative therapy and clinical signs may be more severe.(AU)

O objetivo desse estudo foi identificar cães com diagnóstico presuntivo de DDIV toracolombar submetidos ao tratamento clínico, a fim de avaliar a resposta à terapia instituída. Foram revisados os registros neurológicos de cães atendidos pelo Serviço de Neurologia e Neurocirurgia Veterinária no período de 2006 a 2017 de um Hospital Veterinário Universitário. Foi realizada coleta de dados a partir dos registros e por meio de um questionário respondido pelos tutores. Foram avaliadas 413 fichas neurológicas de cães e obtidas informações para inclusão no estudo em 164 delas. As raças mais frequentes foram dachshunds, seguido de cães sem raça definida. Quanto ao grau de disfunção neurológica foi definido como grau I para 15,9% dos cães, grau II para 25,6%, grau III para 26,8%, grau IV para 8,5% e grau V para 23,2%. A recuperação foi satisfatória em 71,6% dos cães e insatisfatória em 28,4%. Dos que se recuperaram satisfatoriamente, 27,7% tiveram recidivas. Com base nos resultados obtidos pode-se concluir que o tratamento clínico em repouso absoluto e administração de anti-inflamatórios e analgésicos opióides para cães com DDIV toracolombar é efetivo, principalmente para cães em graus mais leves da doença (grau I, II e III). Há possibilidade de recidiva com esse tipo de terapia cujos sinais clínicos poderão ser mais graves.(AU)

Development and Validation of an Analytical Method to Quantitate Tris(chloroisopropyl)phosphate in Rat and Mouse Plasma using Gas Chromatography with Flame Photometric Detection.

Tris(chloropropyl)phosphate (TCPP) is an organophosphorus flame retardant (OPFR) and plasticizer increasingly used in consumer products and as a replacement for brominated flame retardants. Commercially available TCPP is a mixture of four structural isomers the most abundant of which is tris(1-chloro-2-propyl)phosphate (TCPP-1). Although there is a widespread use of TCPP and potential for human exposure, there is limited data on the safety or toxicity of TCPP. The National Toxicology Program is conducting long-term studies to examine the toxicity of the TCPP in rats after lifetime exposure, including perinatal oral exposure. Quantitative estimates of internal dose are essential to interpret toxicological findings in rodents. To aid in this, a method was fully validated to quantitate the most abundant isomer, TCPP-1, in female Harlan Sprague Dawley (HSD) rat and B6C3F1 mouse plasma with partial validation in male rat plasma, and male and female mouse plasma. The method used protein precipitation using trichloroacetic acid followed by the extraction with toluene, and analysis by gas chromatography with flame photometric detection. The performance of the method was evaluated over 5-70 ng TCPP-1/mL plasma. The method was linear (r ≥ 0.99), accurate (inter-day relative error: ≤ ± -7.2) and precise (inter-batch relative standard deviation: ≤27.5%). The validated method has lower limits of quantitation and detection of ~5 and 0.9 ng/mL, respectively, in female HSD rat plasma and can be used on samples as small as 50 µL demonstrating the applicability to plasma samples from toxicology studies.

Análise do líquido cérebro-espinhal de três doenças do sistema nervoso central de cães / Analysis of cerebral-spinal fluid of three central nervous system diseases of dogs

Foi realizado um estudo retrospectivo do líquido cérebro-espinhal de cães (LCE), atendidos pelo Serviço de Neurologia do Hospital Veterinário da Instituição, de 2004 a 2015, com o objetivo de analisar os resultados de cães com sinais neurológicos, comparar as alterações encontradas em dois locais de colheita no mesmo paciente e verificar se esse exame auxiliou o clínico em reforçar a suspeita clínica das principais doenças do sistema nervoso central. A pleocitose linfocítica esteve presente em 78,3% (29/37) das amostras de cães com cinomose e em 23,2% (10/43) de cães com DDIV. Houve dissociação albuminocitológica (DAC) em 73% (19/26) das amostras de cães com tumores IC e em 64,3% (9/14) de cães com tumores envolvendo a ME. Em cães com DDIV, houve significância estatística (p<0,05) entre o grau de disfunção neurológica e o total de células nucleadas (TCN) e total de proteínas (TP). Em 29 cães, houve a colheita do LCE da cisterna magna e da cisterna lombar e em 12 (41,4%) os resultados foram diferentes entre as duas amostras colhidas do mesmo cão, onde dois (6,9%) apresentaram alteração na amostra colhida cranial à lesão. Pode-se concluir que a pleocitose linfocítica foi a principal alteração encontrada no LCE de cães com cinomose e DDIV e DAC nas neoplasias, IC e ME, cães acometidos pela DDIV apresentaram sinais neurológicos mais severos conforme o TCN e o TP aumentaram e o LCE sofreu alteração, mesmo colhido cranial ao local da lesão e auxiliou o clínico em reforçar a suspeita clínica, mas não confirmou, as principais doenças neurológicas em cães.(AU)

A retrospective study including the analysis of the cerebrospinal fluid (CSF) of dogs neurologically affected was conducted by the Neurology Service of the Veterinary Hospital at the Institution, between 2004 and 2015. The aim of this study was to analyze the results of the CSF of dogs with neurological signs, and compare the changes in the CSF in two sampling sites in the same patient and see if this test helped the clinician to strengthen clinical suspicion of the major diseases of the central nervous system. Lymphocytic pleocytosis was present in 78.3% (29/37) of samples from dogs with distemper and in 23.2% (10/43) of samples from dogs with IVDD. The albumin cytologic dissociation (ACD) was found in 73% (19/26) of samples from dogs with IC tumors and in 64.3% (9/14) from dogs with tumors involving the SC. For dogs with IVDD, there was statistical significance (p<0.05) between the degree of neurological dysfunction and the total nucleated cells (TNC) and total protein (TP). In 29 dogs, CSF was collected from the cistern magna and the lumbar and in 12 (41.4%) the results were different between the samples of the same dog, where two cases (6,9%) showed alterations in the sample collected cranial to the injury. It can be concluded that the lymphocytic pleocytosis was the main alteration found in the CSF of dogs with distemper and IVDD and ACD in tumors. Dogs affected by IVDD had more severe neurological signs as TNC and TP increased and the CSF was altered even collected cranial to the lesion site and helped the clinician to strengthen the clinical suspicion, but not confirm, the major neurological diseases in dogs.(AU)

A peripheral blood diagnostic test for acute rejection in renal transplantation.

Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross-validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q-PCR analysis of a five gene-set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training-set (n = 47) and validated on independent test-set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non-AR phenotypes with 91% sensitivity and 90% specificity. The 5-gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.

Complete steroid avoidance is effective and safe in children with renal transplants: a multicenter randomized trial with three-year follow-up.

To determine whether steroid avoidance in pediatric kidney transplantation is safe and efficacious, a randomized, multicenter trial was performed in 12 pediatric kidney transplant centers. One hundred thirty children receiving primary kidney transplants were randomized to steroid-free (SF) or steroid-based (SB) immunosuppression, with concomitant tacrolimus, mycophenolate and standard dose daclizumab (SB group) or extended dose daclizumab (SF group). Follow-up was 3 years posttransplant. Standardized height Z-score change after 3 years follow-up was -0.99 ± 2.20 in SF versus -0.93 ± 1.11 in SB; p = 0.825. In subgroup analysis, recipients under 5 years of age showed improved linear growth with SF compared to SB treatment (change in standardized height Z-score at 3 years -0.43 ± 1.15 vs. -1.07 ± 1.14; p = 0.019). There were no differences in the rates of biopsy-proven acute rejection at 3 years after transplantation (16.7% in SF vs. 17.1% in SB; p = 0.94). Patient survival was 100% in both arms; graft survival was 95% in the SF and 90% in the SB arms (p = 0.30) at 3 years follow-up. Over the 3 year follow-up period, the SF group showed lower systolic BP (p = 0.017) and lower cholesterol levels (p = 0.034). In conclusion, complete steroid avoidance is safe and effective in unsensitized children receiving primary kidney transplants.

CD14, a key candidate gene associated with a specific immune response to cockroach.

BACKGROUND: Sensitization to cockroach allergen is one of the strongest predictors of asthma morbidity, especially among African Americans. OBJECTIVE: Our aims were to determine the genomic basis of cockroach sensitization and the specific response to cockroach antigen. METHODS: We investigated the Th1/Th2 cytokine profile of co-cultured plasmacytoid dendritic cells (pDCs) and CD4+ T cells and the 'transcript signature' of the immune response to cockroach antigen using high-throughput expression profiling of co-cultured cells. RESULTS: We observed significantly elevated levels of IL-13, IL-10, and TNF-alpha, but undetectable levels of IL-12p70 and IFN-alpha, when cultures were exposed to crude cockroach antigen. A significant difference was observed for IL-13 between cockroach-allergic and non-allergic individuals (P=0.039). Microarray analyses demonstrated a greater response at 48 h compared with 4 h, with 50 genes being uniquely expressed in cockroach antigen-treated cells, including CD14, S100A8, CCL8, and IFI44L. The increased CD14 expression was further observed in purified pDCs, human monocytic THP-1 cells, and the supernatant of co-cultured pDCs and CD4+ T cells on exposure to cockroach extract. Furthermore, the most differential expression of CD14 between cockroach allergy and non-cockroach allergy was only observed among individuals with the CC 'high-risk' genotype of the CD14-260C/T. Ingenuity Pathways Analysis analyses suggested the IFN signalling as the most significant canonical pathway. CONCLUSION: Our results suggest that these differentially expressed genes, particularly CD14, and genes in the IFN signalling pathway may be important candidates for further investigation of their role in the immune response to cockroach allergen.

Transgenic expression of AQP1 in the fiber cells of AQP0 knockout mouse: effects on lens transparency.

Mutations and knockout of aquaporin 0 (AQP0) result in dominant lens cataract. To date, several functions have been proposed for AQP0; however, two functions, water permeability and cell-to-cell adhesion have been supported by several investigators and only water channel function has been readily authenticated by in vitro and ex vivo studies. Lens shifts protein expression from the more efficient AQP1 in the equatorial epithelial cells to the less efficient water channel, AQP0, in the differentiating secondary fiber cells; perhaps, AQP0 performs a distinctive function. If AQP0 has only water permeability function, can the more efficient water channel AQP1 transgenically expressed in the fiber cells compensate and restore lens transparency in the AQP0 knockout (AQP0(-/-)) mouse? To investigate, we generated a transgenic wild-type mouse line expressing AQP1 in the fiber cells using alphaA-crystallin promoter. These transgenic mice (TgAQP1(+/+)) showed increase in fiber cell membrane water permeability without any morphological, anatomical or physiological defects compared to the wild type indicating that the main purpose of the shift in expression from AQP1 to AQP0 may not be to lessen the membrane water permeability. Further, we transgenically expressed AQP1 in the lens fiber cells of AQP0 knockout mouse (TgAQP1(+/+)/AQP0(-/-)) to determine whether AQP1 could restore AQP0 water channel function and regain lens transparency. Fiber cells of these mice showed 2.6 times more water permeability than the wild type. Transgene AQP1 reduced the severity of lens cataract and prevented dramatic acceleration of cataractogenesis. However, lens fiber cells showed deformities and lack of compact cellular architecture. Loss of lens transparency due to the absence of AQP0 was not completely restored indicating an additional function for AQP0. In vitro studies showed that AQP0 is capable of cell-to-cell adhesion while AQP1 is not. To our knowledge, this is the first report which uses an animal model to demonstrate that AQP0 may have an additional function, possibly cell-to-cell adhesion.

Heritability of platelet function in families with premature coronary artery disease.

BACKGROUND: Variations in platelet function among individuals may be related to differences in platelet-related genes. The major goal of our study was to estimate the contribution of inheritance to the variability in platelet function in unaffected individuals from white and African American families with premature coronary artery disease. METHODS: Platelet reactivity, in the absence of antiplatelet agents, was assessed by in vitro aggregation and the platelet function analyzer closure time. Heritability was estimated using a variance components model. RESULTS: Both white (n = 687) and African American (n = 321) subjects exhibited moderate to strong heritability (h(2)) for epinephrine- and adenosine diphosphate-induced aggregation (0.36-0.42 for white and >0.71 for African American subjects), but heritability for collagen-induced platelet aggregation in platelet-rich plasma was prominent only in African American subjects. Platelet lag phase after collagen stimulation was heritable in both groups (0.47-0.50). A limited genotype analysis demonstrated that the C825T polymorphism of GNB3 was associated with the platelet aggregation response to 2 muM epinephrine, but the effect differed by race. CONCLUSIONS: Considering the few and modest genetic effects reported to affect platelet function, our findings suggest the likely existence of undiscovered important genes that modify platelet reactivity, some of which affect multiple aspects of platelet biology.

Susceptibility to ozone-induced airway inflammation is associated with decreased levels of surfactant protein D.

BACKGROUND: Ozone (O3), a common air pollutant, induces exacerbation of asthma and chronic obstructive pulmonary disease. Pulmonary surfactant protein (SP)-D modulates immune and inflammatory responses in the lung. We have shown previously that SP-D plays a protective role in a mouse model of allergic airway inflammation. Here we studied the role and regulation of SP-D in O3-induced inflammatory changes in the lung. METHODS: To evaluate the effects of O3 exposure in mouse strains with genetically different expression levels of SP-D we exposed Balb/c, C57BL/6 and SP-D knockout mice to O3 or air. BAL cellular and cytokine content and SP-D levels were evaluated and compared between the different strains. The kinetics of SP-D production and inflammatory parameters were studied at 0, 2, 6, 12, 24, 48, and 72 hrs after O3 exposure. The effect of IL-6, an O3-inducible cytokine, on the expression of SP-D was investigated in vitro using a primary alveolar type II cell culture. RESULTS: Ozone-exposed Balb/c mice demonstrated significantly enhanced acute inflammatory changes including recruitment of inflammatory cells and release of KC and IL-12p70 when compared with age- and sex-matched C57BL/6 mice. On the other hand, C57BL/6 mice had significantly higher levels of SP-D and released more IL-10 and IL-6. Increase in SP-D production coincided with the resolution of inflammatory changes. Mice deficient in SP-D had significantly higher numbers of inflammatory cells when compared to controls supporting the notion that SP-D has an anti-inflammatory function in our model of O3 exposure. IL-6, which was highly up-regulated in O3 exposed mice, was capable of inducing the expression of SP-D in vitro in a dose dependent manner. CONCLUSION: Our data suggest that IL-6 contributes to the up-regulation of SP-D after acute O3 exposure and elevation of SP-D in the lung is associated with the resolution of inflammation. Absence or low levels of SP-D predispose to enhanced inflammatory changes following acute oxidative stress.

Transmural gradients in Na/K pump activity and [Na+]I in canine ventricle.

There are well-documented differences in ion channel activity and action potential shape between epicardial (EPI), midmyocardial (MID), and endocardial (ENDO) ventricular myocytes. The purpose of this study was to determine if differences exist in Na/K pump activity. The whole cell patch-clamp was used to measure Na/K pump current (I(P)) and inward background Na(+)-current (I(inb)) in cells isolated from canine left ventricle. All currents were normalized to membrane capacitance. I(P) was measured as the current blocked by a saturating concentration of dihydro-ouabain. [Na(+)](i) was measured using SBFI-AM. I(P)(ENDO) (0.34 +/- 0.04 pA/pF, n = 17) was smaller than I(P)(EPI) (0.68 +/- 0.09 pA/pF, n = 38); the ratio was 0.50 with I(P)(MID) being intermediate (0.53 +/- 0.13 pA/pF, n = 19). The dependence of I(P) on [Na(+)](i) or voltage was essentially identical in EPI and ENDO (half-maximal activation at 9-10 mM [Na(+)](i) or approximately -90 mV). Increasing [K(+)](o) from 5.4 to 15 mM caused both I(P)(ENDO) and I(P)(EPI) to increase, but the ratio remained approximately 0.5. I(inb) in EPI and ENDO were nearly identical ( approximately 0.6 pA/pF). Physiological [Na(+)](i) was lower in EPI (7 +/- 2 mM, n = 31) than ENDO (12 +/- 3 mM, n = 29), with MID being intermediate (9 +/- 3 mM, n = 22). When cells were paced at 2 Hz, [Na(+)](i) increased but the differences persisted (ENDO 14 +/- 3 mM, n = 10; EPI 9 +/- 2 mM, n = 10; and MID intermediate, 11 +/- 2 mM, n = 9). Based on these results, the larger I(P) in EPI appears to reflect a higher maximum turnover rate, which implies either a larger number of active pumps or a higher turnover rate per pump protein. The transmural gradient in [Na(+)](i) means physiological I(P) is approximately uniform across the ventricular wall, whereas transporters that utilize the transmembrane electrochemical gradient for Na(+), such as Na/Ca exchange, have a larger driving force in EPI than ENDO.

Detection of immunoglobulin G in the lung and liver of hamsters with visceral leishmaniasis

Several organs are affected in visceral leishmaniasis, not only those rich in mononuclear phagocytes. Hypergammaglobulinemia occurs during visceral leishmaniasis; anti-Leishmania antibodies are not primarily important for protection but might be involved in the pathogenesis of tissue lesions. The glomerulonephritis occurring in visceral leishmaniasis has been attributed to immune complex deposition but in other organs the mechanism has not been studied. In the current study we demonstrated the presence of IgG in the lung and liver of hamsters with visceral leishmaniasis. Hamsters were injected intraperitoneally with 2 x 10(7) amastigotes of Leishmania (Leishmania) chagasi and the presence of IgG in the liver and lung was evaluated at 7, 15, 30, 45, 80 and 102 days postinfection (PI) by immunohistochemistry. The parasite burden in the spleen and liver increased progressively during infection. We observed a deposit of IgG from day 7 PI that increased progressively until it reached highest intensity around 30 and 45 days PI, declining at later times. The IgG deposits outlined the sinusoids. In the lung a deposit of IgG was observed in the capillary walls that was moderate at day 7 PI, but the intensity increased remarkably at day 30 PI and declined at later times of infection. No significant C3 deposits were observed in the lung or in the liver. We conclude that IgG may participate in the pathogenesis of the inflammatory process of the lung and liver occurring in experimental visceral leishmaniasis and we discuss an alternative mechanism other than immune complex deposition

Isoform-specific function and distribution of Na/K pumps in the frog lens epithelium.

Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies of the Na/K ATPase. RNase protection assays showed that all cells express alpha(1)- and alpha(2)-isoforms of the Na/K pump but not the alpha(3)-isoform, however the alpha(2)-isoform dominates in anterior cells whereas the alpha(1)-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the Na/K pump current (I(P)), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I(P) blockade data indicate the alpha(1)-isoform has a dissociation constant of 100 microm DHO whereas for the alpha(2)-isoform it is 0.75 microm DHO. Both alpha(1)- and alpha(2)-isoforms are half maximally activated at an intracellular Na(+)-concentration of 9 mm. The alpha(1)-isoform is half maximally activated at an extracellular K(+)-concentration of 3.9 mm whereas for the alpha(2)-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the alpha(1)-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the alpha(2)-isoform. Under normal physiological conditions, I(P) in equatorial cells is approximately 0.23 microA/microF, and in anterior cells it is about 0.14 microA/microF. These current densities refer to the area of cell membrane assuming a capacitance of around 1 microF/cm(2). Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 microA/cm(2) at the equator vs. 0.5 microA/cm(2) at the anterior pole.

Proportion positive for Epstein-Barr virus, cytomegalovirus, human herpesvirus 6, Toxoplasma, and human immunodeficiency virus types 1 and 2 in heterophile-negative patients with an absolute lymphocytosis or an instrument-generated atypical lymphocyte flag.

OBJECTIVES: To determine the proportion of patients with evidence of an acute infection due to Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), Toxoplasma, or human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) in heterophile-negative patients with an absolute lymphocytosis or an instrument-generated atypical lymphocyte flag, and to develop a cost-effective testing algorithm for managing such heterophile-negative patients. DESIGN: We conducted a prospective investigation of 70 selected outpatients who tested negative for heterophile antibody in association with an absolute lymphocytosis or instrument-generated atypical lymphocyte flag. The control population consisted of 50 patients who were heterophile negative and had a normal absolute lymphocyte count and no instrument-generated atypical lymphocyte flag. SETTING: A large outpatient laboratory system. INTERVENTION: Viral serology for HHV-6 was performed by immunofluorescence, and all other serologies were performed by enzyme-linked immunoassay. All testing was for immunoglobulin (Ig) M antibodies, except in the case of HIV. RESULTS: The proportion of study patients positive for EBV was 40% (28/70); for CMV, 39% (27/70); for HHV-6, 25% (16/65); for Toxoplasma, 3% (2/70); and for HIV, 0% (0/70). All 50 control patients were negative for EBV IgM antibodies. When patients with more than 1 positive viral test were excluded from analysis, positivity was 20% (9/45) for EBV, 22% (10/45) for CMV, 9% (4/45) for HHV-6, and 2% (1/45) for Toxoplasma. Utilizing hypothesis-generating logistic regression models, Downey type II atypical lymphocytes were significantly associated with EBV positivity (P =.006), while Downey type III lymphocytes were significantly associated with HHV-6 positivity (P =.016), and there was a trend for the association of Downey type I lymphocytes with CMV positivity (P =.097). CONCLUSIONS: A positive viral serology was identified in 70% of study patients. Multiple positive serologies complicate establishing a definitive diagnosis. Potential cost savings may be associated with the use of an appropriate testing algorithm.

Diffuse lamellar keratitis related to endotoxins released from sterilizer reservoir biofilms.

OBJECTIVE: To investigate the risk factors and control mechanisms used to control the outbreak of diffuse lamellar keratitis (DLK) associated with laser in situ keratomileusis (LASIK) and examine the relationship between DLK and endotoxins released from sterilizer biofilm reservoirs. DESIGN: Clinic-based cohort and laboratory study. PARTICIPANTS: All patients undergoing LASIK at our clinic from October 7, 1998 through August 31, 1999. The case definition was a diffuse infiltrate in the interface developing within the first week after surgery. INTERVENTIONS: Biofilm control in the sterilizer, changes in sterilizer, distilled water, instruments, and irrigating fluids. MAIN OUTCOME MEASURES: The incidence of DLK after LASIK surgery. RESULTS: There were 983 evaluable patients, with three whose DLK status was not recorded. There were 52 cases of DLK. Burkholderia pickettii was isolated from the sterilizer reservoir. Potential risk factors and associations, for which there was no significant difference, included age and sex of the patients, surgeon, operating suite temperature or humidity, drapes used, saline solutions used, time of day the surgery was performed, and microkeratome use. Sterilizers 1 and 2, before biofilm control, were compared with sterilizer 3, after control. The relative risk was 9.4 (confidence limits [CL], 7.5-11.8) for sterilizer 1 versus 3 and 18. 7 (CL, 11-32) for sterilizer 2 versus 3. Three cases occurred after biofilm control, but were sporadic in nature and associated with epithelial defects. CONCLUSIONS: Clusters of DLK may be related to endotoxins released from gram-negative biofilms in sterilizer reservoirs. We experienced an outbreak of DLK affecting 52 patients and isolated B. pickettii from the sterilizer reservoir. Epidemiologic investigation showed that biofilm control in the sterilizer reservoirs was associated with a significant reduction in the development of DLK. We encourage any clinics that experience a cluster of DLK to consider microbiologic and epidemiologic investigation for the effectiveness of sterilizer biofilm control.

The role of MIP in lens fiber cell membrane transport.

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in CatFr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (pH2O) on the order of 1 micron/sec whereas normal fiber cell membrane pH2O was 17 micron/sec frog, 32 micron/sec rabbit and 43 micron/sec mouse. CatFr mouse lens fiber cell pH2O was reduced by 13 micron/sec for heterozygous and 30 micron/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the pH2O conferred by MIP is not sensitive to Hg2+ whereas that of CHIP28 (AQP1) is blocked by Hg2+. The fiber cell membrane pH2O was also not sensitive to Hg2+ whereas lens epithelial cell pH2O (136 micron/sec in rabbit) was blocked by Hg2+. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous CatFr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses.

Isoform-specific regulation of the sodium pump by alpha- and beta-adrenergic agonists in the guinea-pig ventricle.

1. Guinea-pig ventricle was used in the RNase protection assays to determine which alpha-isoforms of the Na+-K+ pumps are present, and ventricular myocytes were used in whole cell patch clamp studies to investigate the actions of alpha- and beta-adrenergic agonists on Na+-K+ pump current. 2. RNase protection assays showed that two isoforms of the alpha-subunit of the Na+-K+-ATPase are present in guinea-pig ventricle. The mRNA for the alpha1-isoform comprises 82 % of the total pump message, the rest being the alpha2-isoform. 3. We have previously shown that beta-adrenergic agonists affect Na+-K+ pump current (Ip) through a protein kinase A (PKA)-dependent pathway. We now show that these beta-effects are targeted to the alpha1-isoform of the Na+-K+ pumps. 4. We have also previously shown that alpha-adrenergic agonists increase Ip through a protein kinase C (PKC)-dependent pathway. We now show that these alpha-isoform effects are targeted to the alpha2-isoform of the Na+-K+ pumps. 5. These results suggest the effects of adrenergic activation on Na+-K+ pump activity in the heart can be regionally specific, depending on which alpha-isoform of the Na+-K+ pump is expressed.