Quantification of EGFP expression on Molt-4 T cells using calibration standards.

BACKGROUND: Enhanced green fluorescent protein (EGFP) is used extensively to assess gene expression on cells; however, quantification of this expression by flow cytometry has been limited by the unavailability of calibration standards. Thus, we characterized the response of an experimental set of EGFP calibration standards to environmental changes and then quantitate the expression of EGFP, in molecules of equivalent soluble fluorochrome (MESF) units, of a transfected Molt-4 T cell line by flow cytometry. METHODS: Characterization of the EGFP standards: EGFP standards were equilibrated in suspension solutions having a pH range of 5.0-9.0, temperatures of 37-80 degrees C, and osmolalities of 100-600 mOsm/kg. Quantification of EGFP on cells: For transfections, Molt-4 T cells were incubated with two different concentrations (0.2 microg and 0.4 microg) of pEGFP-N2 vector and the EGFP expression was quantified after 48 h by flow cytometry using the EGFP standards and by the cytofluor technique using a standard curve of known EGFP solutions. RESULTS: The fluorescence intensity of the EGFP standards increased from pH 5.0 to 9.0 and remained relatively constant from 37 degrees C to 65 degrees C, and from 100 to 600 mOsm/kg. After transfection, the expression of the populations with high and low EGFP expression averaged 8,098 +/- 584 MESF and 3,808 +/- 375 MESF respectively. No significant differences were observed after comparing the MESF values obtained by flow cytometry and the values obtained by Cytofluor technique (high: 8,791 +/- 492 MESF; low: 4,082 +/- 398 MESF). CONCLUSIONS: Our data demonstrate the feasibility of using calibration standards to quantify EGFP expression on cells. Our results emphasize the importance of monitoring the effects of environmental changes in the fluorescence intensity of both standards and samples when quantifying the expression of EGFP on living cells.

Formalization of the MESF unit of fluorescence intensity.

This report summarizes the work performed during the past two years at the National Institute of Standards and Technology (NIST) in the refinement and formal definition of the MESF unit of fluorescence intensity. In addition to the theory underlying the MESF unit, considerations of error analysis are also presented. The details of this work may be found in the three publications of the NIST Journal of Research (www.nist.gov) listed as the references 2-4. The use of the fluorescence intensity unit provides a tool to compare quantitative fluorescence intensity measurements over time and across platforms.

Quantitative flow cytometry.

Flow cytometers are instruments that can quantify fluorescence intensity data and provide unique information about cell populations. Significant advances have been made in terms of calibration reagents, evaluation of the quality and limitations of the monoclonal antibodies, standardized sample preparation, and data analysis to ensure interlaboratory comparability and reproducibility. Efforts to standardize quantitative fluorescence intensity measurements impact current clinical flow cytometry applications (i.e., immunophenotyping), but also emerging technologies, such as microarray assays, which also require calibration of fluorescence intensity across different platforms.

Setting a local research agenda for women's health: The National Centers of Excellence in Women's Health.

Although women's health research expanded greatly in the past 10 years, significant gaps in knowledge remain. Prioritization and promotion of research will help assure continuing progress in closing such gaps and improving the health of women. Although a comprehensive agenda for the new millennium has been developed at the national level, the process for establishing a local research agenda is not well defined. The purpose of this study was to describe criteria for and barriers to establishing a local research agenda in women's health. A secondary aim was to describe mechanisms for identifying women's health researchers and for facilitating multidisciplinary research. Directors of Research at National Centers of Excellence in Women's Health (CoEs) (n = 18) were surveyed by mail for this information. The results indicate that the local research agenda should emphasize health issues that are prevalent in women, research that is likely to establish treatment, psychosocial/cultural factors, and quality of life issues. The process of setting a research agenda should include input from the communities served as well as from scientists. Critical evaluation of scientific strengths and weaknesses is an essential preliminary step in prioritizing research opportunities in order to implement and evaluate a research agenda in women's health.

Involving minority and underrepresented women in clinical trials: the National Centers of Excellence in Women's Health.

Recent attention to reducing health disparities among population groups has focused on the need to include in clinical studies, especially clinical trials, participants who represent the diversity of the populations to which study results will be applied. While scientists generally applaud the goal of broadening the characteristics of participants in clinical trials, they are faced with multiple challenges as they seek to include historically underrepresented populations in their research. This article examines the historical and sociocultural context of participation by underrepresented groups, especially women and minorities, in clinical trials, identifies major barriers and challenges facing researchers, and suggests strategies for meeting these challenges. The article draws upon the experiences of the investigators affiliated with the National Centers of Excellence of Women's Health (CoEs).

Standardizing flow cytometry: a classification system of fluorescence standards used for flow cytometry.

The growing number of standards commercially available in the field of flow cytometry makes it difficult to know which standards to use to obtain a desired level of quality assurance. A classification system of fluorescence standards has been developed on the basis of their physical characteristics. In turn, these physical characteristics determine the ability of the specific standards to perform selected functions, such as alignment, target referencing, compensation, and calibration. Knowing the properties and limitations of specific standards will help flow cytometer users to select the appropriate standard for the application that they will be performing, especially in regard to intra- and interlaboratory quality assurance. Common protocols used in conjunction with specific classifications of reference standards can provide unified analysis regions or window of analysis across different instruments and/or laboratories. In addition, specific classifications of calibration standards can help select those standards that will provide independent and direct comparison of instrument performance parameters, especially in studies involving multiple laboratories. Knowledge and understanding of the classification system can guide flow cytometer users in more efficient and accurate instrument setup and quality control when conducting research, as well as clinical applications.

Standardizing flow cytometry: construction of a standardized fluorescence calibration plot using matching spectral calibrators.

Calibration of flow cytometers is becoming an increasingly important issue for both quality control of instrument performance and quantitation of antibody binding capacity of cells. Due to the numerous different instruments and analysis software currently available, a standardized method of calibration is necessary if interlaboratory comparison of instrument performance and antibody binding is to be achieved. This report describes a new methodology to obtain a standard calibration plot that can be derived from all instruments and from which specific instrument-independent performance parameters may be calculated that can be used to directly compare the performance and setup of these instruments. The requirements that the calibrated standards must meet are discussed, as well as the acceptable ranges proposed for the instrument-independent performance parameters. In addition, data are presented from standard calibration plots generated by different flow cytometers in numerous laboratories. The corresponding Primary Performance Parameters calculated from these plots are presented and compared. It is expected that the use of this calibration method may help standardize flow cytometric measurements and will provide instrument-independent performance parameters to monitor quality control of instruments and reagents.

Effects of gentamicin on glomerular renin release.

Gentamicin nephrotoxicity is associated with impaired glomerular function. To examine whether the effects of gentamicin on glomerular function are mediated through alterations in the renal-angiotensin system, basal and stimulated glomerular renin release was assessed in isolated glomeruli from control and gentamicin-treated rats. Male Sprague-Dawley rats (220 +/- 20 g) were studied immediately after treatment with gentamicin sulfate (4 mg/kg BW/day, n = 6) for 1 or 2 consecutive weeks and after 1 week of recovery from 2 weeks of treatment. Control rats received an equivalent volume of saline (n = 9). After the respective treatment, renal renin content was measured. In addition, glomeruli from control and gentamicin-treated rats were isolated and glomerular renin release was measured under basal conditions and after stimulation with the calmodulin inhibitor trifluoperazine (1 x 10(-4) M). Renin concentration was determined in aliquots of the supernatant by measuring the generation of angiotensin I using radioimmunoassay techniques at 15-min intervals. Renal renin content was significantly increased after 2 weeks of gentamicin treatment (+47%) and remained elevated (+62%) 1 week after discontinuing a 2-week gentamicin treatment. Both basal and stimulated glomerular renin release were lower in glomeruli isolated from gentamicin-treated rats. The effect of gentamicin added in vitro to glomeruli isolated from untreated rats was also evaluated. Exposure of normal glomeruli to in vitro gentamicin (1 mM) resulted in a significant inhibition of both basal (-47%, p < .05) and stimulated (-84%, p < .05) glomerular renin release. To determine whether the inhibitory action of gentamicin on glomerular renin release was dependent on extracellular calcium concentration, the effects of gentamicin on glomerular renin release were also assessed in the absence of extracellular calcium. Our data revealed that in the absence of extracellular calcium, the inhibitory effect of gentamicin on both basal and stimulated glomerular renin release was abolished. Taken together, these findings strongly suggest an inhibitory effect of gentamicin on glomerular renin release. Furthermore, the inhibition of glomerular renin release induced by gentamicin appears to be dependent on extracellular calcium.

Angiotensin II and catecholamines interaction in short-term low protein feeding.

Renal and systemic hemodynamic responses to an alpha-adrenergic agonist (norepinephrine, NE) and an alpha-adrenergic antagonist (phentolamine, PHEN) were studied in weanling rats pair-fed isocaloric diets containing either normal (NP, 23%) or low (LP, 6%) protein. Mean arterial pressure (MAP) rose less with NE and fell more with PHEN in LP than in NP. Plasma NE and epinephrine (E; 46 +/- 5 and 51 +/- 4 ng/ml) were higher in LP than in NP (26 +/- 3 and 39 +/- 3 ng/ml). These could not be attributed to changes in red cell mass nor the volumes of plasma, extracellular, or interstitial fluid in LP versus NP. Plasma angiotensin II (Ang II), renin (PRA), and aldosterone (PA) were lower in LP than in NP. An increased number without changes in affinity of glomerular Ang II receptors was found in LP compared to NP, while alpha 1- and alpha 2-adrenergic receptors were down-regulated in LP as compared to NP without changes in affinity for the alpha 1 receptor but with an increase in renal alpha 2 receptor affinity. LP (vs. NP) decreased GFR and RPF, and increased renal vascular resistance (RVR). NE decreased RPF equally in NP versus LP but raised RVR approximately twofold in NP versus LP. PHEN decreased RPF and increased RVR less in LP than in NP. Moreover, PHEN increased renal renin content approximately seven-fold over the basal NP values. Exogenous Ang II increased RVR and lowered RPF more in LP than in NP. Enalapril abolished all the hemodynamic changes of LP and restored the systemic response to NE.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of clinical standards for flow cytometry.

Flow cytometers are instruments that can determine multiparameter data simultaneously and have a great potential in providing unique information about cells. To transfer this potential to the clinical laboratory, the development and the proper use of standards and calibrators are required to ensure comparability and reproducibility of data from different flow cytometers. Although there are a number of types of standards for flow cytometry, each has its specifically designed purpose to ensure that data from this complex instrument are accurate, precise, and reproducible among instruments over time.

Renal hemodynamics and urinary concentrating capacity in protein deprivation: role of antidiuretic hormone.

The role of antidiuretic hormone (ADH) in the renal concentration defect and hemodynamic changes in protein malnutrition was evaluated in rats with diabetes insipidus (DI) after 2 weeks of low protein feeding. Free water reabsorptive capacity (TcH2O), glomerular filtration rate (GFR), and renal plasma flow (RPF) were measured in the protein deprived rats and in DI rats fed a normal protein diet. The effect of urea supplementation of the low protein diet on renal concentrating capacity was also evaluated. In addition, the renal hemodynamic response to acute administration of ADH was measured and correlated with changes in plasma renin concentration and renal renin content (RRC). Protein deprivation in DI rats resulted in reduced urine osmolality and urea excretion, differences which were reversed by urea supplementation. Protein deprivation did not affect free water reabsorptive capacity but did reduce GFR and RPF. Acute ADH administration significantly increased GFR and RPF in protein-deprived rats; these changes were associated with a reduction in RRC and release. These results suggest that dietary protein restriction does not directly affect the tubular capacity to generate and reabsorb free water. The hemodynamic changes seen in protein deprivation are not mediated by ADH and may be secondary to increased intrarenal angiotensin II.

Functional studies in experimental renal cortical necrosis in the rat.

Necrosis of the outer two-thirds of the cortex (CN) was induced with boiling water in the left kidney of rats. Two days afterward, morphological damage was shown to be limited to the superficial cortex; deep nephron population was well-preserved. Glucose reabsorption under basal and glucose loading conditions, and extraction of p-aminohippurate, used as indices of proximal tubule integrity, were normal in control and experimental kidneys 48 h after cortical necrosis. Basal fractional water and electrolyte excretion did not differ between control and experimental kidneys. Calculated mean single-nephron glomerular filtration rate (GFR) and plasma flow for superficial (SupGFR and SupNPF) and juxtamedullary nephrons (JMGFR and JMPF) were similar to those obtained by micropuncture and Hanssen's technique for SupGFR, and for JMGFR by Hanssen's. Volume expansion led to a 27% increase in calculated SupGFR, but no change in JMGFR. The JMPF increased by 81%, whereas SupNPF increased by only 23%, suggesting that, in this model, GFR of deep nephrons may be independent of plasma flow. The results indicate that deep nephrons retain their functional integrity 48 h after cortical necrosis. After volume expansion fractional excretion of sodium was greater, and fractional water reabsorption less, in CN than in control kidneys. Thus handling of sodium and water by superficial and deep nephrons under basal conditions was similar, but reabsorptive capacity for deep nephrons of CN was lower during volume expansion. The present studies suggest that deep nephrons can maintain relatively normal function in cortical necrosis.

Renal and systemic effects of short-term high protein feeding in normal rats.

Various studies have shown that a high protein (HP) diet, compared to a low protein (LP) diet, leads to hypercalciuria and alterations in renal and systemic hemodynamics. The authors compared the effects of HP diet to those of normal protein diet (NP) to determine the possible mechanisms by which changes in systemic hemodynamics and hypercalciuria occurred. The studies were conducted in awake rats; the effects of dietary sodium content on the changes induced by HP also were evaluated. The relationship of prostaglandins (PG), renin (PRA), and aldosterone (PA) to changes in blood pressure (BP) was assessed. Two weeks after HP and normal sodium feeding (40%), glomerular filtration rate (GFR) and urine flow (V) were not different from the same values in a group on an NP diet (23%). When HP was fed with low sodium, there was a rise in V as a consequence of greater fluid intake. Although plasma calcium remained constant, the hypercalciuria correlated with high protein and sodium content. Alterations in 1,25(OH)2 vitamin D3 or PTH (cyclic AMP excretion) function did not explain the hypercalciuria induced by HP. This suggests that HP leads to inhibition of tubular calcium reabsorption by mechanism(s) yet to be elucidated. Although HP did not alter GFR, it led to an increase in BP, a fall in renal vascular resistance, and an increase in RPF, regardless of sodium intake. PRA and urine PGE2 excretion were significantly higher in the rats on HP diet, whereas PA remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

A new model of genetic hypertension in rats with superficial glomeruli.

Despite renal involvement in the genesis of hypertension, the precise renal hemodynamic events prior to and during development of hypertension have not been obtainable by direct study in the available rat models of genetic hypertension. We have developed a model of genetic salt-sensitive hypertension in rats with superficial glomeruli, using the protocol described by Dahl to develop the Brookhaven model. Adult Munich Wistar rats were purchased from Charles River and bred in our laboratory. Offspring were allowed to mature without intervention. Systolic blood pressure and body weight were measured weekly to determine the normal longitudinal changes with increasing age for this strain. Balance studies were also carried out longitudinally, and during these studies plasma renin activity (PRA) was determined. When the rats were 16 weeks old, the availability of superficial glomeruli was assessed under microscopic examination and only those rats with numerous superficial glomeruli/field were mated. The offspring of these breeders were used as the zero generation (F0) of salt-sensitive rats, and were treated as described by Dahl. Systolic blood pressure, body weights and balance studies were carried out in this and in all subsequent generations. Sibling breeding was maintained and rats were bred for salt-sensitivity (systolic blood pressure greater than 155 mmHg at the age of 2 months) and availability of superficial glomeruli. Those rats with systolic blood pressure of less than 120 mmHg were bred in an attempt to develop a salt-resistant strain, and untreated Munich Wistar rats were bred to provide another control.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of angiotensin-converting enzyme inhibition on altered renal hemodynamics induced by low protein diet in the rat.

We assessed the role of angiotensin II in mediating the alterations in renal hemodynamics known to result from low protein feeding to normal rats by examining the effect of the angiotensin-converting enzyme (ACE) inhibitor captopril. 2 wk of low protein (6% casein) diet resulted in decreased glomerular filtration rate (normal protein [NP], 1.82 +/- 0.17 vs. low protein [LP], 0.76 +/- 0.01 ml/min; P less than 0.05) and renal plasma flow (NP, 6.7 +/- 0.2 vs. LP, 3.3 +/- 0.3 ml/min; P less than 0.05); renal vascular resistance rose (NP, 8.7 +/- 0.4 vs. LP, 19.8 +/- 1.4 dyn . s per cm5; P less than 0.05). These changes were accompanied by a significant decrease in plasma renin activity (NP, 7.0 +/- 0.7 vs. LP, 4.4 +/- 0.8 ng A I/ml per h; P less than 0.05), plasma aldosterone concentration (NP, 7.0 +/- 0.6 vs. LP, 4.1 +/- 0.7 ng/dl; P less than 0.05), and urinary PGE2 excretion (NP, 3,120 +/- 511 vs. LP, 648 +/- 95 pg/mgCr; P less than 0.05); by contrast renal renin content was significantly increased (NP, 2,587 +/- 273 vs. LP, 7,032 +/- 654 ng A I/mg protein; P less than 0.05). Treatment with captopril (30 mg/kg per d) raised glomerular filtration rate (GFR; LP + capt, 1.6 +/- 0.2 ml/min) and renal plasma flow (RPF; LP + capt, 6.7 +/- 0.7 ml/min), and reduced renal vascular resistance (LP + capt, 9.2 +/- 0.5 dyn/s per cm5) in low protein-fed animals. These values were not different from those measured in untreated and captopril-treated rats fed a normal (23%) protein diet. There were no changes in systemic mean arterial pressure in any group of rats. These data provide evidence that intrarenal angiotensin II mediates the changes in intrarenal hemodynamics induced by protein deprivation. The effects of low protein feeding may be partly potentiated by the reduction in PGE2 synthesis. However, the normalization of GFR and RPF in view of only modest increases in PGE2 excretion after captopril (LP, 648 +/- 95 vs. LP + capt, 1,131 +/- 82 pg/mgCr; P less than 0.05) suggests that if PGE2 is involved in these changes, it plays a permissive but not essential role in the increased renovascular resistance.

Effects of extracellular volume expansion on plasma renin concentration in rats with diabetes insipidus.

The effect of acute and chronic expansion of the extracellular fluid volume on plasma renin concentration (PRC) was studied in normal Long-Evans rats (LE rats) and in rats with hereditary hypothalamic diabetes insipidus (DI rats). Chronic deoxycorticosterone acetate (DOCA) treatment, combined with a high sodium intake, significantly reduced PRC of both DI and LE rats. PRC of DI rats, however, remained higher than that of LE rats. Acute volume expansion, either alone or with DOCA treatment, also significantly diminished PRC of both DI and LE rats. PRC of untreated and DOCA-treated DI rats again remained significantly higher than that of LE rats after acute volume expansion. These findings suggest that the elevated PRC normally observed in DI rats is not due solely to diminished volume of extracellular fluid. Instead, the absence of ADH per se may directly alter renin secretion or the sensitivity of the granular cell to other stimuli.

Role of renal nerves in maintaining sodium balance in unrestrained conscious rats.

This study was designed to investigate the effects of bilateral renal denervation on sodium and water balance, the renin-angiotensin system, and systemic blood pressure in unrestrained conscious rats maintained on a normal- or low-sodium diet. Renal denervation was proven by chemical and functional tests. Both bilaterally denervated rats (n = 18) and sham-denervated rats (n = 15) maintained positive sodium balance while on a normal sodium intake. Both groups were in negative sodium balance for 1 day after dietary sodium restriction was instituted but were in positive sodium balance for the following 9 days. Systolic blood pressure was higher in sham-denervated (115 +/- 3 mmHg) than in denervated rats (102 +/- 3 mmHg) while on a normal diet (P less than 0.05) and remained so during sodium restriction. Plasma renin concentration (PRC) and plasma aldosterone concentration (PAC) were significantly diminished in the denervated rats during normal sodium intake (P less than 0.05). After dietary sodium restriction, PRC increased in both groups but remained significantly lower in the denervated rats (P less than 0.05). Following dietary sodium restriction, PAC also increased significantly to levels that were similar in both groups of rats. These results demonstrate that awake unrestrained growing rats can maintain positive sodium balance on a low sodium intake even in the absence of the renal nerves. However, efferent renal nerve activity influenced plasma renin activity in these animals.