ELECTRICALLY INDUCED INTRACELLULAR Ca2+ TRANSIENT IN SINGLE VENTRICULAR MYOCYTES: A USEFUL PARAMETER FOR THE STUDY OF CARDIAC DRUGS

1. Fluorescent Ca2+ indicators, such as fura-2/AM and calcium green-1, have become one of the most popular tools for measuring intracellular calcium ([Ca2+]i). 2. Electrical stimulation triggers a cascade of events in the cardiac muscle, which results in a [Ca2+]i transient and, eventually, contraction. The events that occur in electrically induced cardiac myocytes mimic the normal physiological events in vivo. 3. The electrically induced [Ca2+]i transient represents influx of Ca2+ from outside and mobilization of Ca2+ from the intracellular store and is directly related to contraction. Thus, it is more important to determine the electrically induced [Ca2+]i transient than [Ca2+]i. The [Ca2+]i transient can be easily measured with the spectrofluorescence method using fura 2/AM as the Ca2+ indicator in a single ventricular myocyte preparation. 4. We made use of the results of studies on carbachol, tetrandrine and cardiotoxin to illustrate the usefulness of the electrically induced [Ca2+]i transient in the study of actions of cardiac drugs.     

Relative accuracy of two diagnostic schemes for detection of pulmonary embolism by ventilation-perfusion scintigraphy

Two diagnostic schemes for detection of pulmonary embolism by ventilation-perfusion (V-P) scintigraphy were compared for relative accuracy by two groups of observers interpreting 70 V-P scintiscans. Observers in Group B, who used the criteria recently proposed by Biello et al., had a significantly smaller average number of "indeterminate" interpretations (41%) than did the observers in Group A (55%), who used a simpler scheme (p less than 0.05). In addition, Group B showed a slight improvement in positive predictive value without a deterioration in the negative predictive value compared with Group A. Along with this improvement in diagnostic performance, Group B achieved a significant reduction in interobserver variability compared with Group A for patients without pulmonary embolism (p less than 0.05). There was no significant difference in interobserver variability between the two groups for patients with pulmonary embolism. The diagnostic scheme introduced by Biello et al. represents a useful improvement for the diagnosis of pulmonary embolism by V-P imaging.     

Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expanding CAG repeat coding for polyglutamine in the huntingtin protein. Recent data have suggested the possibility that an N-terminal fragment of huntingtin may aggregate in neurons of patients with HD, both in the cytoplasm, forming dystrophic neurites, and in the nucleus, forming intranuclear neuronal inclusion bodies. An animal model of HD using the short N-terminal fragment of huntingtin has also been found to have intranuclear inclusions and this same fragment can aggregate in vitro . We have now developed a cell culture model demonstrating that N-terminal fragments of huntingtin with expanded glutamine repeats aggregate both in the cytoplasm and in the nucleus. Neuroblastoma cells transiently transfected with full-length huntingtin constructs with either a normal or expanded repeat had diffuse cytoplasmic localization of the protein. In contrast, cells transfected with truncated N-terminal fragments showed aggregation only if the glutamine repeat was expanded. The aggregates were often ubiquitinated. The shorter truncated product appeared to form more aggregates in the nucleus. Cells transfected with the expanded repeat construct but not the normal repeat construct showed enhanced toxicity to the apoptosis- inducing agent staurosporine. These data indicate that N-terminal truncated fragments of huntingtin with expanded glutamine repeats can aggregate in cells in culture and that this aggregation can be toxic to cells. This model will be useful for future experiments to test mechanisms of aggregation and toxicity and potentially for testing experimental therapeutic interventions.      

Limiting dilution analysis of in vivo-activated (IL-2 responsive) peripheral blood lymphocytes in HIV-1-infected subjects

The progression of infection with human immunodeficiency virus, type 1 (HIV-1), is associated with a loss of helper T cell function, but the mechanism for this loss (e.g., decreased absolute number of helper cells, altered function of helper cells, or both) has not been delineated. Many studies have suggested that T-cell production of and/or responsiveness to the T cell growth factor interleukin-2 (IL-2) declines over the course of HIV-1 infection. Using a highly quantitative 6-day limiting dilution assay (LDA), we investigated whether the number and the proliferative capacity of circulating IL-2 responsive cells in patients with AIDS differ from those in patients in earlier stages of HIV-1 infection (asymptomatic or AIDS-related complex) and healthy seronegative individuals. The frequency of IL-2 responsive cells declined progressively in asymptomatic seropositive subjects, those with ARC, and those with AIDS. In contrast, the proliferative capacity of individual IL-2 responsive cells, as reflected by the magnitude of thymidine uptake per precursor, was reduced only in patients with frank AIDS and was normal in asymptomatic subjects and in those with ARC. These results suggest that the development of AIDS in the setting of HIV-1 infection may reflect a combination of qualitative as well as quantitative changes in lymphocyte function. They also suggest that analysis of lymphocyte responsiveness to IL-2 may provide a useful approach to prediction of the development of AIDS in individuals infected with HIV-1.     

1142280946Effect of estradiol and PAMPs on class II mediated antigen presentation and immunomodulatory molecule expression in the mouse female reproductive tract

Problem:  Antigen presenting cells (APC) in the female reproductive tract play important roles in innate immune defense and activation of the adaptive immune responses. The objective of this study was to examine the effects of estradiol and PAMP on antigen presentation in the female reproductive tract.
Method of Study:  DO11.10 T cell antigen receptor transgenic mice specific for the MHC class II-restricted OVA323–339peptide were used to study the effects of estradiol and PAMP on antigen presentation of OVA by uterine epithelial (EC) and stromal cells as well as vaginal cells to OVA specific memory-T cells.
Results:  Estradiol inhibited antigen presentation of OVA by uterine EC, uterine stromal cells and vaginal cells to OVA specific memory-T cells. When ovariectomized animals were treated with estradiol for 1 or 3 days, antigen presentation decreased by 20–80%. In contrast, incubation with TLR agonists increased antigen presentation by EC (Poly (I:C), Pam3Cys), stromal cells (PGN, Pam3Cys) and vaginal cells (LPS, Pam3Cys). Analysis of mRNA expression by real time RT-PCR indicated that estradiol inhibited CD40, CD80/86 and class II in the uterus and vagina. In contrast, stimulation of antigen presentation by PAMP did not correlate with changes in costimulatory molecule mRNA expression.
Conclusions:  These results indicate that APC in the uterus and vagina are responsive to estradiol, which inhibits antigen presentation and costimulatory molecule expression. These findings suggest that whereas APC in the uterus and vagina respond to TLR agonists with increased antigen presentation, which initiates an adaptive immune response, their effects appear to be at levels other than the expression of costimulatory molecules.
Acknowledgement:  Supported by AI-13541 from NIH.     

Evolution in the management of hepatic trauma: a 25-year perspective

OBJECTIVE: To define the changes in demographics of liver injury during the past 25 years and to document the impact of treatment changes on death rates. SUMMARY BACKGROUND DATA: No study has presented a long-term review of a large series of hepatic injuries, documenting the effect of treatment changes on outcome. A 25-year review from a concurrently collected database of liver injuries documented changes in treatment and outcome. METHODS: A database of hepatic injuries from 1975 to 1999 was studied for changes in demographics, treatment patterns, and outcome. Factors potentially responsible for outcome differences were examined. RESULTS: A total of 1,842 liver injuries were treated. Blunt injuries have dramatically increased; the proportion of major injuries is approximately 16% annually. Nonsurgical therapy is now used in more than 80% of blunt injuries. The death rates from both blunt and penetrating trauma have improved significantly through each successive decade of the study. The improved death rates are due to decreased death from hemorrhage. Factors responsible include fewer major venous injuries requiring surgery, improved outcome with vein injuries, better results with packing, and effective arterial hemorrhage control with arteriographic embolization. CONCLUSIONS: The treatment and outcome of liver injuries have changed dramatically in 25 years. Multiple modes of therapy are available for hemorrhage control, which has improved outcome.