Prospects for reducing and refining the use of dogs in the regulatory toxicity testing of pharmaceuticals

A workshop was held to critically discuss the need for a nonrodent species and the role of the dog in regulatory toxicity testing of pharmaceuticals; to discuss opportunities to reduce and refine the use of dogs in preclinical toxicology; and to identify a number of specific recommendations which could be feasibly achieved to move the process forward. To facilitate a preliminary evaluation of the contribution of dog studies to the risk assessment process, anonymised, unpublished data were provided from fully evaluated, repeat-dose toxicity studies in the rat and dog. Results of the International Life Sciences Institute (ILSI) Human Toxicity Project were also presented and discussed. Analysis of the data demonstrated that the dog can provide additional toxicity information, which, in some cases, was shown to be predictive for humans. Discussions indicated that there is potential for achieving a reduction in dog use and several possible approaches were identified. To further the progress of this initiative, there is a need to collate the results of pharmacology, toxicology, and clinical studies to address some of the proposed approaches. One of the outcomes of the workshop will be the establishment of a steering group to co-ordinate data collation for further analysis.     

Serum anion gap in the differential diagnosis of metabolic acidosis in critically ill newborns

OBJECTIVES: To determine in critically ill newborn infants (1) the range of the serum anion gap without metabolic acidosis and (2) whether the serum anion gap can be used to distinguish newborns with lactic acidosis from those with hyperchloremic metabolic acidosis. STUDY DESIGN: Umbilical arterial blood gases and serum electrolyte and lactate concentrations were measured simultaneously in 210 samples from 63 infants over the first week of life. Metabolic acidosis was defined as a blood base deficit (BD) >4 mmol/L. The anion gap was calculated as [Na(+)] - [C1(-)] - [TCO (2)]. Lactic acidosis was defined as a serum lactate concentration >2 SD above the mean serum lactate concentration in samples without metabolic acidosis. RESULTS: In 89 blood samples with BD <4 mmol/L, serum lactate concentration decreased with postnatal age (r = 0.51). The upper limit of serum lactate concentration was 3.8 mmol/L at less than 48 hours, 2.4 mmol/L between 48 and 96 hours, and 1.5 mmol/L for infants greater than 96 hours of age. The mean serum anion gap +/- 2 SD in 174 samples without lactic acidosis was 8 +/- 4 mmol/L; in 36 samples with lactic acidosis it was 16 +/- 9 mmol/L (P <.0001). Serum anion gap and lactate concentration were poorly correlated for samples without lactic acidosis (r = 0.04) but highly correlated in those with lactic acidosis (r = 0.81, P <.0001). None of the 85 samples with metabolic acidosis but without lactic acidosis had an anion gap >16 mmol/L; only 4 of 36 samples with lactic acidosis had an anion gap <8 meq/L. However, 25 of 36 samples with lactic acidosis had serum anion gaps of 8 to 16 mmol/L. CONCLUSION: In the presence of metabolic acidosis, a serum anion gap >16 mmol/L is highly predictive of lactic acidosis; a serum anion gap <8 is highly predictive of the absence of lactic acidosis; an anion gap = 8 - 16 mmol/L has no use in the differential diagnosis of metabolic acidosis in the critically ill newborn.     

Antigen phenotype of cultured decidual stromal cells of human term decidua

We previously reported that decidual stromal cells (DSC) from early human decidua express antigens associated with hematopoietic cells and develop different immune functions. Here we study the antigenic phenotype of DSC from term decidua and compare it with the phenotype reported for DSC from early decidua. Decidual stromal cells were isolated from human term deciduas and maintained in culture until highly purified DSC cultures were obtained. Most term DSC, like most early DSC, expressed CD10. Term DSC expressed antigens specific for follicular dendritic cells (FDC), such as DRC-1 (CD21L) and HJ2, together with CD21, CD23 and CD80, which are detected on FDC as well. Also like early DSC, term DSC were negative for CD3, CD14, CD15 and CD45. Although early DSC were reported to be HLA-DR-positive and CD86-positive, these antigens were not expressed by term DSC. These discrepant results suggest that two types of cells, or cells at different stages of differentiation (decidualization) were selected during culture of decidual cells from different periods of gestation. This possibility was further supported by the finding that term DSC expressed desmin and prolactin, two markers of decidualization, whereas these molecules have not previously been detected in early DSC.     

Bovine mammary epithelial cell invasion by Streptococcus uberis.

Streptococcus uberis commonly causes bovine mastitis; however, the pathogenesis of this infection is poorly understood. In this study, the ability of S. uberis to invade mammary epithelial cells in culture was investigated. Two strains of S. uberis isolated from bovine mammary secretions were capable of invading bovine mammary epithelial cells in vitro at different levels, suggesting strain differences in invasiveness. Invasion required microfilaments but not microtubular cytoskeletal elements. No morphological changes in epithelial cells were observed for up to 24 h postinfection, suggesting no cellular injury. Strains of S. uberis evaluated were genetically distinct and differed phenotypically in expression of potential virulence factors. Whether a single factor or combination of factors was responsible for differences in invasiveness was not addressed in this study. These data provide a foundation for a better understanding of the processes used by S. uberis to invade epithelial cells. Epithelial cell invasion may be a potentially important mechanism in the pathogenesis of S. uberis mastitis.     

Left ventricular mass and diastolic function in normotensive young adults with autosomal dominant polycystic kidney disease

Left ventricular hypertrophy is often found very early in the course of autosomal dominant polycystic kidney disease (ADPKD). Diastolic dysfunction has been shown in hypertensive adult patients with ADPKD with increased left ventricular mass (LVM), but there are no data about diastolic function in the young ADPKD population without hypertension and with normal renal function. To evaluate very early alterations in cardiac structure and diastolic function in young normotensive patients with ADPKD, color Doppler echocardiography was performed in 46 young normotensive patients with ADPKD and 35 healthy subjects. LVM, transmitral pulsed Doppler flow (diastolic function), and valvular abnormalities were studied. Patients with ADPKD showed higher LVM indices (LVMIs) than controls (89.7+/-17.3 v 68.5+/-17.2 g/m2; P < 0.0001). Peak early diastolic velocity (E wave) deceleration time and isovolumic relaxation time were significantly prolonged in patients with ADPKD compared with controls (E wave deceleration time, 182.5+/-51.3 v 149.4+/-34 msec; P=0.002; isovolumic relaxation time, 97.7+/-17.5 v 79+/-15 msec; P=0.0001). No differences were found in valvular abnormalities in the two groups. In conclusion, young normotensive patients with ADPKD showed increased LVMIs and Doppler abnormalities consistent with early diastolic dysfunction.     

Is image subtraction necessary in the clinical interpretation of single-day split-dose stress cerebral perfusion single-photon emission tomography using technetium-99m compounds?

The aim of this study was to validate a simplified semiquantitative method of evaluating a single-day stress cerebral perfusion test to obtain cerebrovascular reserve capacity (CVRC) for routine clinical uses. A split-dose protocol was tested in 36 pairs of technetium-99m hexamethylpropylene amino oxime baseline (low dose) and acetazolamide (high dose) stress brain single-photon emission tomographic (SPET) studies from 16 patients with cerebrovascular disease. The images were displayed on a semiquantitative color scale with (corrected) and without (uncorrected) image subtraction, dose adjustment, and decay correction. The representative CVRC was determined by placing 3×3 pixel regions of interest on midthalamic and midcerebellar slices. The corrected and uncorrected relative changes in CVRC were correlated using linear regression. The relative changes of corrected (x) and uncorrected (y) CVRC by quantitative analysis were highly correlated in a linear fashion (y=0.67x+0.002,r=0.998,P<0.0005). As predicted by theory, the slope was related to the ratio of split dose and independent of ROI sampling. Single-day split-dose stress brain SPET can be accurately performed without image subtraction and complicated dose adjustment or decay correction for clinical studies.     

SecA suppresses the temperature-sensitive SecY24 defect in protein translocation in Escherichia coli membrane vesicles.

Genetic analysis of protein secretion in Escherichia coli has identified secY/prlA and secA as components of the secretory apparatus. We have examined the roles of the secY(prlA) gene product (an integral membrane protein) and the soluble secA gene product in translocation of OmpA and alkaline phosphatase precursors in an in vitro system. The protein translocation defect of the secY24 mutation was recently demonstrated in vitro as was its suppression by an S300 extract. We show here that the extract was essentially inactive in SecY24 suppression when SecA protein was removed from it by immunoaffinity chromatography. Furthermore, purified SecA protein suppressed the SecY24 defect. Preincubation of the inactivated SecY24 membrane vesicles either with S300 containing SecA or with purified SecA protein reconstituted the membranes and restored the translocation activity when assayed in the absence of additional soluble proteins. These results suggest that the SecY24 translocation defect is suppressed by SecA interacting, directly or indirectly, with SecY24 on the cytoplasmic membrane.     

Retroperitoneal malignant fibrous histiocytoma

Five cases of malignant fibrous histiocytoma located in the retroperitoneum are reported. Paraneoplastic syndrome, abdominal mass, tendency to local recurrence, laborious therapy and poor prognosis are the clinical characteristics of this tumor. Accuracy of actual diagnosis is evaluated and pathological features are analyzed.