A simple, sensitive method for the determination of extracellular catecholamines in the rat hypothalamus using in vivo dialysis

The design, construction and characterisation of a dialysis probe suitable for perfusing any deep brain structures is described. Using high-performance liquid chromatography with electrochemical detection (HPLC-ECD) the effects of flow rate, concentration, dialysate composition and temperature on the recovery within the dialysate of authentic catecholamines are detailed. The dialysis probe was used to collect endogenous catecholamines from the anterior hypothalamus of urethane-anaesthetised rats. Following organic phase extraction of the in vivo samples, a small basal release of noradrenaline (NA) of 37 +/- 4 pg/30 min sample was found. Potassium stimulation markedly elevated the release of NA and dopamine from the anterior hypothalamus in a calcium ion and dose dependent manner. It appears therefore that the dialysis probe described here, in conjunction with HPLC-ECD, can be used to follow changes in neuronally released catecholamines within the anterior hypothalamus, providing a valuable tool to study the role of these neurotransmitters in physiological and pharmacological function.     

Plasma cell granuloma of jaw A case report

A 65 year old Punjabi male presented with an exophytic lesion, 3 Cm. in diameter, which was pedunculated, on the attached gingiva. It was situated on the right side, near the motars. On histological examination, it was diagnosed as plasma cell granuloma.     

Prevention of renovascular and cardiac pathophysiological changes in hypertension by angiotensin II type 1 receptor antisense gene therapy

Hypertension produces pathophysiological changes that are often responsible for the mortality associated with the disease. However, it is unclear whether normalizing blood pressure (BP) with conventional therapy is effective in reversing the pathophysiological damage. The duration and initiation of treatment, site of administration, and agent used all appear to influence the reversal of the pathophysiological alterations associated with hypertension. We have previously established that retrovirally mediated delivery of angiotensin II type 1 receptor antisense (AT₁R-AS) attenuates the development of high BP in the spontaneously hypertensive (SH) rat model of human essential hypertension. Our objective was to determine whether this attenuation of high BP is associated with prevention of other pathophysiological changes induced by the hypertensive state. Intracardiac delivery of AT₁R-AS in neonates prevented the development of hypertension in SH rats for at least 120 days. Contractile experiments demonstrated an impaired endothelium-dependent vascular relaxation (acetylcholine) and an enhanced contractile response to vasoactive agents (phenylephrine and KCl) in the SH rat renal vasculature. In addition, the voltage-dependent K⁺ current density, which is believed to contribute to smooth muscle resting membrane potential and basal tone, was decreased in renal resistance artery cells of the SH rat. AT₁R-AS treatment prevented each of these renal vascular alterations. Finally, AT₁R-AS delivery prevented the pathological alterations observed in the SH rat myocardium, including left ventricular hypertrophy, multifocal fibrosis, and perivascular fibrosis. These observations demonstrate that viral-mediated delivery of AT₁R-AS attenuates the development of hypertension on a long term basis, and this is associated with prevention of pathophysiological changes in SH rats.     

Is the diaphragm motion probability density function normally distributed?

During radiotherapy treatment planning, the margins given to the clinical target volume to form the planning target volume accounts for internal motion and set-up error. Most margin formulas assume that the underlying distributions are independent and normal. Clinical data suggests that the set-up error probability density function (pdf) can be considered to have an approximately normal distribution. However, there is evidence that internal motion does not have a normal distribution. Thus, in general, a convolution of the two pdfs should be performed to determine the total geometric error. The goals of this article were to (1) determine if the internal motion pdf due to respiration can be characterized using a normal distribution, and (2) if not, determine if the total geometric uncertainty for combining internal motion and set-up error can be characterized by a normal distribution. Sixty fluoroscopy diaphragm motion data sets were obtained using three breathing training types: free breathing, audio instruction, and visual feedback. Diaphragm motion was used as a surrogate for liver and lung cancer motion. The data were analyzed with normality tests in the following groups: (1) single motion measurements, (2) combined motion measurements for each patient, and (3) combined motion measurements for all patients. Following this analysis, the diaphragm motion pdfs were convolved with a set-up error pdf, and the standard deviation of the set-up error pdf at which the total geometric error pdf became normal was determined. At set-up error standard deviation values of at least 0.27 and 0.1 cm for free breathing, 0.57 and 0.42 cm for audio instruction, and 0.55 and 0 cm for visual feedback, for single motion measurements and combined motion measurements for each patient, respectively, total geometric error pdfs became approximately normal. When the motion measurements for all the patients were combined, diaphragm motion pdfs were approximately normal for all feedback types. Therefore, for treatment planning purposes in the absence of individual patient measurements, the diaphragm motion pdf can be considered an approximately normal distribution. However, care should be taken when determining a margin based on individual patients measurements as the total geometric error will, in general, not be normally distributed.     

Enhanced expression of stem cell antigen-1 (Ly-6A/E) in lymphocytes from lupus prone mice correlates with disease severity

B6.Sle1 mice, congenic for the NZM2410-derived lupus susceptibility locus, Sle1 on chromosome 1 exhibit many of the features seen in human lupus including activated lymphocytes and high titers of antinuclear autoantibodies. Among the different surface molecules that were aberrantly expressed on the B6.Sle1 lymphocytes was Ly-6A/E. Splenic B- and T-lymphocytes but not myeloid cells from B6.Sle1 mice exhibited enhanced levels of Ly-6A/E compared to B6 controls. In particular, MZ B cells, GC B cells and B-cell blasts expressed the highest levels of Ly-6A/E in both strains, with the levels being even higher on B6.Sle1 derived cells. Following stimulation with LPS or anti-IgM, there was a profound up-regulation in Ly-6A/E, particularly on MZ B cells and B-cell blasts. CD4 and CD8 T cells also up-regulated Ly-6A/E after stimulation with anti-CD3 and anti-CD28. These studies were extended to additional autoimmune strains including B6.Sle3, B6.Sle1.lpr and BXSB. Importantly, Ly-6A/E levels on lymphocytes were commensurate with the degree of disease exhibited by these lupus strains. Finally, it appears that increased interferon levels, in addition to antigen receptor stimulation, may also be a factor accounting for elevated Ly-6A/E in lupus. Given these observations it is important to elucidate the functional role of Ly-6A/E in lupus in future studies.     

Comparative evaluation of commonly used laboratory tests for post-mortem diagnosis of rabies

Animal brain samples received at WHO Collaborating Centre laboratory at National Institute of Communicable Diseases (NICD) during the years 1991-2002 were tested by Seller's stain, Fluorescent Antibody Test (FAT) and Mouse Innoculation Test (MIT) as methods of rabies diagnosis. Negri bodies on Seller's staining could be detected in 52.5% of MIT positive brains, the concordance of this test with MIT was found to be 77.8%. FAT was positive in 91.5% of MIT positive brains, though it showed concordance of 95.7% with MIT results in the total samples. 12.2% of the samples were found positive by FAT of which 1/3rd also showed the presence of Negri bodies when MIT was negative i.e. showing that the virus is present in inactivated form. Thus emphasizing the need for timely and proper collection and transportation of specimens for testing. Seller's stain and FAT give reliable diagnosis of rabies in the brain samples in majority of the cases. MIT being time-intensive test, is of academic value only in decision making as regards initiation of Post Exposure Treatment (PET), it is recommended that in cases where Seller's stain and FAT have yielded negative results the decision to initiate PET should give due consideration to the nature and circumstances of the animal bite and other epidemiological features.     

Role of Periductal and Ductular Epithelial Cells of the Adult Rat Pancreas in Pancreatic Hepatocyte Lineage: A Change in the Differentiation Commitment

Development of pancreatic hepatocytes in adult rats maintained on copper deficient diet containing 0.6% trien (CuDT) has been reported recently. To elucidate the histogenesis of hepatocytes a sequential study was undertaken using morphologic, histochemical, immunochemical, in situ hybridization, and Northern blot analysis. Male F-344 rats weighing 80 to 90 g were fed CuDT for 8 weeks and returned to normal rat chow. Beginning from 4 weeks of copper depletion, there was a progressive loss of acinar cells and by 8 weeks more than 90% of the acinar tissue was lost. During this period, there was an increase in the number of adipocytes in the interstitium, and in the number of interstitial and ductular cells. Morphologic observations were confirmed by immunoblot and Northern blot analysis, in which the amount of pancreatic proteins and their mRNAs decreased between 5 and 8 weeks. During this period, a progressive increase in the level of albumin mRNA was observed. In situ hybridization, performed at 7 weeks of copper deficiency, showed localization of albumin mRNA over interstitial and ductular cells. Pancreatic hepatocytes were identified immediately after the rats were returned to a normal diet and gradually increased in number. The hepatocytes occupied almost 60% of the pancreatic volume by 8 weeks. During the early recovery phase, hepatocytes were identified in ductules as well as in the interstitium. Based on these studies, it is concluded that both the ductular cells and interstitial cells, which resemble oval cells of liver, are capable of transforming into pancreatic hepatocytes and these cells may be considered stem-cell equivalent.     

The Clinical Health Economics System Simulation (CHESS): a teaching tool for systems- and practice-based learning.

Academic medical centers face barriers to training physicians in systems- and practice-based learning competencies needed to function in the changing health care environment. To address these problems, at the University of Virginia School of Medicine the authors developed the Clinical Health Economics System Simulation (CHESS), a computerized team-based quasi-competitive simulator to teach the principles and practical application of health economics. CHESS simulates treatment costs to patients and society as well as physician reimbursement. It is scenario based with residents grouped into three teams, each team playing CHESS using differing (fee-for-service or capitated) reimbursement models. Teams view scenarios and select from two or three treatment options that are medically justifiable yet have different potential cost implications. CHESS displays physician reimbursement and patient and societal costs for each scenario as well as costs and income summarized across all scenarios extrapolated to a physician's entire patient panel. The learners are asked to explain these findings and may change treatment options and other variables such as panel size and case mix to conduct sensitivity analyses in real time. Evaluations completed in 2003 by 68 (94%) CHESS resident and faculty participants at 19 U.S. residency programs preferred CHESS to a traditional lecture-and-discussion format to learn about medical decision making, physician reimbursement, patient costs, and societal costs. Ninety-eight percent reported increased knowledge of health economics after viewing the simulation. CHESS demonstrates the potential of computer simulation to teach health economics and other key elements of practice- and systems-based competencies.     

Microdissection genotyping of mixed glial and primitive neuroectodermal central nervous system neoplasm

A 22-year-old man with previous radiation treatment for childhood astrocytoma underwent resection of a right parietooccipital lesion. Histopathology revealed a malignant neoplasm with areas of astrocytic and primitive neuroectodermal components. To resolve the relationship and cellular origin, representative tissue was microdissected from several targets, obtaining a balanced mixture of each element. Nonneoplastic brain parenchyma was separately microdissected to determine polymorphic marker informativeness and to serve as an internal negative control. Despite the relatively small quantity of tissue removed for each microdissection target, sufficient material was available for reliable, balanced, polymerase chain reaction-format genotyping encompassing a panel of tumor suppressor genes and genetic loci associated with these forms of neoplasia. The findings revealed distinct discordant genotypic profiles for each of the neoplastic components. The efficacy of the approach used for molecular analysis of this complex neoplasm and the implication of the genotypic findings are discussed.     

Development and validation of a simple sensitive enzyme immunoassay (EIA) for GH determination in buffalo plasma

A simple and highly sensitive enzymeimmunoassay (EIA) for GH determination in buffalo plasma on microtitreplates using biotin-streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to GH and used to bridge between streptavidin-peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 100 microL buffalo plasma. The GH standards ranging from 0.05 ng/well/100 microL to 12.8 ng/well/100 microL were prepared in hormone free plasma collected from an aged (> 15 years) senile buffalo. The sensitivity of the EIA procedure was 50 pg/well GH. which corresponded to 0.50 ng/mL plasma; the 50% relative binding sensitivity was seen at 800 pg/well/100 microL. Plasma volumes for the EIA, viz., 25, 50, and 100 microL did not influence the shape of standard curve, even though a slight drop in the OD450 was seen with higher plasma volumes. For the biological validation of the assay, 12 Murrah buffalo calves were used. Six of these were administered synthetic bovine growth hormone-releasing factor (10 microg/100 kg body weight, i.v., and the remaining six animals were administered sterile normal saline and kept as controls. Jugular blood samples were collected at -60, -45, -30, -15, -10, -5, 5, 10, 15, and 30 min and, thereafter, at an interval of 15 min using an indwelling jugular catheter, beginning 1 h prior to GRF injection up to 8 h post treatment. In all animals, a peak of GH was recorded within 5 to 20 min of GRF administration, which confirms the biological validation of the EIA. To confirm homogeneity of buffalo GH with bovine GH, a parallelism test was conducted between the buffer standard curve of bovine GH and GH measured from serial dilution of buffalo plasma containing a high level of endogenous growth hormone.