Analytical validation of anti-toxoplasma IgG immunoassays

There are often discrepancies when using different methods to measure anti-Toxoplasma gondii IgG levels in patient samples. The diagnostic performance of a chemiluminescent immunoassay (CLIA) and an enzyme-linked fluorescent assay (ELFA) used as confirmatory tests for samples identified as positive or equivocal by an electrochemiluminescent immunoassay (ECLIA) were examined. Cut-off values were those stated by the manufacturer, and Western blot was used to confirm the results of all methods. All samples identified as positive by ECLIA (n = 93) were confirmed as positive by Western blot, as were 14 of the 28 samples identified as equivocal. When these 121 samples were retested, the sensitivities of CLIA and ELFA were 64.4% and 73.8%, respectively. Both methods exhibited a specificity of 100%. This study confirms that the results obtained from the different immunoassays are not comparable, and neither CLIA nor ELFA should be used to confirm ECLIA results, which should instead be confirmed by methods such as Western blot or Sabin-Feldman dye test.     

Interleukin-3, GM-CSF, and TPA induce distinct phosphorylation events in an interleukin 3-dependent multipotential cell line

The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hematopoietic cell line, B6SUtA1. Murine granulocyte- macrophage colony-stimulating factor (mGM-CSF) was as potent as mIL-3 in stimulating these cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-O-tetradecanoyl- phorbol-13-acetate (TPA), a known activator of protein kinase C, also stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA1 cells exposed for brief periods to mIL-3, mGM-CSF, and TPA were analyzed for changes in phosphorylation patterns using metabolic 32P-labeling and antibodies to phosphotyrosine. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68-Kd cytosolic protein, whereas all three agents stimulated the serine-specific phosphorylation of a 68-Kd membrane protein. Furthermore, mIL-3 stimulated tyrosine phosphorylation of the 68-Kd membrane protein, as well as of 140-, 90-, 55, and 40-Kd proteins. The 90-Kd protein was also tyrosine phosphorylated in response to mGM-CSF. These phosphotyrosine containing proteins were not detected in TPA-treated cells. These results indicate that protein phosphorylations on tyrosine and serine residues occur in B6SUtA1 cells following short-term incubation with mIL-3 or mGM-CSF and that most of these phosphorylation events are mediated by kinases other than protein kinase C (PkC).     

Vimentin expression and myofibroblast infiltration are early markers of renal dysfunction in kidney transplantation: an early stage of chronic allograft dysfunction?

Introduction

The objective of this study was to show the morphologic characteristics of allograft renal biopsies in renal transplant patients with stable renal function, which can potentially be early markers of allograft dysfunction, after 5 years of follow-up.

Methods

Forty-nine renal transplant patients with stable renal function were submitted to renal biopsies and simultaneous measurement of serum creatinine (Cr). Histology was evaluated using Banff scores, determination of interstitial fibrosis by Sirius red staining and immunohistochemical study of proximal tubule and interstitial compartment (using cytokeratin, vimentin, and myofibroblasts as markers). Biopsies were evaluated according to the presence or absence of the epitheliomesenchymal transition (EMT). The interstitial presence of myofibroblasts and tubular presence of vimentin was also analyzed simultaneously. Renal function was measured over the follow-up period to estimate the reduction of graft function.

Results

Median posttransplant time at enrollment was 105 days. Patients were followed for 64.3 ± 8.5 months. The mean Cr at biopsy time was 1.44 ± 0.33 mg/dL, and after the follow-up it was 1.29 ± 0.27 mg/dL. Nine patients (19%) had a reduction of their graft function. Eleven biopsies (22%) had tubulointerstitial alterations according to Banff score. Seventeen biopsies (34%) presented EMT. Fifteen biopsies (32%) had high interstitial expression of myofibroblasts and tubular vimentin. Using Cox multivariate analysis, HLA and high expression of interstitial myofibroblasts and tubular vimentin were associated with reduction of graft function, yielding a risk of 3.3 (P = .033) and 9.8 (P = .015), respectively.

Conclusion

Fibrogenesis mechanisms occur very early after transplantation and are risk factors for long-term renal function deterioration.     

Paradoxical sleep deprivation and sleep recovery: effects on the hypothalamic-pituitary-adrenal axis activity, energy balance and body composition of rats

Numerous studies indicate that sleep deprivation alters energy expenditure. However, this conclusion is drawn from indirect measurements. In the present study, we investigated alterations of energy expenditure, body composition, blood glucose levels, plasma insulin, adrenocorticotropic hormone (ACTH) and corticosterone levels immediately after 4 days of sleep deprivation or after 4 days of sleep recovery. Rats were sleep deprived or maintained in a control environment (groups sleep-deprived/deprivation and control/deprivation). One half of these animals were sacrificed at the end of the deprivation period and the other half was transported to metabolic cages, where they were allowed to sleep freely (groups sleep-deprived/recovery and control/recovery). At the end of the sleep recovery period, these rats were sacrificed. After sleep deprivation, sleep-deprived rats exhibited loss of body weight, augmented energy expenditure and reduced metabolic efficiency compared to control rats. These alterations were normalised during the sleep recovery period. The body composition of sleep-deprived rats was altered insofar as there was a loss of fat content and gain of protein content in the carcass compared to control rats. However, these alterations were not reversed by sleep recovery. Finally, plasma levels of insulin were reduced during the sleep deprivation period in both control and sleep deprived groups compared to the recovery period. After the deprivation period, plasma ACTH and corticosterone levels were increased in sleep-deprived rats compared to control rats, and although ACTH levels were similar between the groups after the sleep recovery period, corticosterone levels remained elevated in sleep-deprived rats after this period. By means of direct measurements of metabolism, our results showed that sleep deprivation produces increased energy expenditure and loss of fat content. Most of the alterations were reversed by sleep recovery, except for corticosterone levels and body composition.     

Knowledge and behavior among health professionals in relation to cadaveric organ donation and transplantation: a questionnaire-based analysis in Portuguese hospitals

Objective

To assess knowledge and behavior relative to cadaveric organ donation and transplantation among physicians and nurses working at nine Portuguese hospitals.

Methods

This prospective study between May and October 2009 included 495 questionnaires that were returned among 840 that were delivered, a 59% response rate. The transplant questionnaire comprised three main sections: section 1, sociodemographic-professional variables of the participants with nine questions; section 2, generic questions on organ donation and transplantation with 12 questions; and section 3 evaluated knowledge with 20 specific questions on organ donation and transplantation.

Results

Sixty-one percent of the participants were females, with 40% of the overall cohort between 31 and 40 years old; 62% married; 63% working in the emergency department; and 78% nurses. Although 78% of the participants stated that they had undergone specific training on organ donation and transplantation, 62% felt that they needed more training. The reasons evoked for the low rate of donation included difficulties in initiating the process of organ donation, in diagnosing cerebral death and in obtaining necessary human resources. There were no significant differences in knowledge between physicians and nurses concerning specific organ donation and transplantation issues. Having had specific training on organ donation and transplantation was significantly associated with correct answers to several questions, including those pertaining to knowledge of the national legislation on organ donation and transplantation.

Conclusions

The present study showed a lack of specific knowledge and training on organ donation and transplantation issue. It also suggested that for hospital staff to successfully act as initiators of the organ procurement process, more information and education are necessary. This effort could significantly increase the rate of organ donation and transplantation in Portugal.     

Non-Invasive Monitoring Based on Bioreactance Reveals Significant Hemodynamic Instability during Elective Cesarean Delivery under Spinal Anesthesia

Background and objectives

Blood pressure monitoring offers a limited understanding of the hemodynamic consequences of spinal anesthesia for cesarean delivery. The purpose of this study was to assess, with the aid of a non-invasive cardiac output monitor based on bioreactance, the hemodynamic changes during elective cesarean delivery under spinal anesthesia in which intermittent boluses of phenylephrine were used to prevent and treat hypotension.

Methods

This observational study was conducted with the Research Ethics Board approval, and all participants provided written informed consent. Healthy patients undergoing elective cesarean delivery under spinal anesthesia were enrolled. Intermittent boluses of phenylephrine were administered in an attempt to maintain systolic blood pressure at baseline levels, and patients were assessed with a non-invasive cardiac output monitor based on bioreactance. Hemodynamic data was collected continuously at baseline, and during the postspinal and postdelivery periods. Data was analyzed using a mixed model ANOVA, and a p < 0.05 was considered significant.

Results

Systolic blood pressure was maintained within 79.2 ± 14.2 and 105.8 ± 10.0 percent of baseline during the postspinal period, and 78.4 ± 11.3 and 100.9 ± 10.7 percent of baseline in the postdelivery period (mean ± SD) There were significant fluctuations in systolic blood pressure, heart rate, and cardiac output during the postspinal period, and significant fluctuations in systolic blood pressure and cardiac output in the postdelivery period.

Conclusions

A new non-invasive monitor based on bioreactance reveals significant hemodynamic fluctuations during cesarean delivery under spinal anesthesia, despite attempts to maintain blood pressure at baseline levels with intermittent boluses of phenylephrine.