Functional characterization of HUVEC-CS: Ca2+ signaling, ERK 1/2 activation, mitogenesis and vasodilator production.

While many endothelial cell lines exist, few are of human origin with characteristics close to the parent endothelial cell. We derived a subline (HUVEC-CS) of immortalized human umbilical vein endothelial cells (HUVEC-C) that proliferate in standard growth media and exhibit positive acetylated low-density lipoprotein (AcLDL) uptake, express eNOS, CD31 and ve-cadherin, and spontaneously form capillary-like structures when grown on Matrigel. HUVEC-CS also maintain endothelial cell characteristics at the level of mitogenesis, kinase activation and vasodilator production. Like primary HUVEC cells, HUVEC-CS express many of the key proteins necessary for vasodilator production, including epithelial nitric oxide synthase (eNOS), HSP 90, cav-1 and -2, cPLA2, and COX-1 and -2. Prostaglandin I synthase (PGIS) was not detectable by Western blot analysis, consistent with primary HUVEC in which PGI2 production is minimal. Receptors were detected for angiotensin II (AII), bradykinin, ATP and growth factors. ATP induced a dose- and time-dependent rise in the intracellular free Ca2+ concentration ([Ca2+]i). Initially, ATP stimulates P2Y receptors rather than P2X receptors, as demonstrated by the inability of ATP to initiate a Ca2+ response subsequent to emptying of the internal Ca2+ stores by thapsigargin. AII, bradykinin, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) also caused a rise in [Ca2+]i in a subset of the cells. ATP, basic fibroblastic growth factor (bFGF), EGF and VEGF induced mitogenesis and caused a rise in ERK 2 activation within 10 min. L-Arginine to L-citrulline conversion assays showed that ATP, EGF and VEGF induced a significant rise in eNOS activity, and this correlates with an ability to induce Ca2+ mobilization and ERK 2 activation. In conclusion, HUVEC-CS are indeed endothelial cells and appear to be functionally very similar to primary HUVEC. These cells will prove a valuable tool for future studies in both basic and therapeutic sciences.

BAALC, the human member of a novel mammalian neuroectoderm gene lineage, is implicated in hematopoiesis and acute leukemia.

The molecular basis of human leukemia is heterogeneous. Cytogenetic findings are increasingly associated with molecular abnormalities, some of which are being understood at the functional level. Specific therapies can be developed based on such knowledge. To search for new genes in the acute leukemias, we performed a representational difference analysis. We describe a human gene in chromosome 8q22.3, BAALC (brain and acute leukemia, cytoplasmic), that is highly conserved among mammals but evidently absent from lower organisms. We characterized BAALC on the genomic level and investigated its expression pattern in human and mouse, as well as its complex splicing behavior. In vitro studies of the protein showing its subcellular localization suggest a function in the cytoskeleton network. Two isoforms are specifically expressed in neuroectoderm-derived tissues, but not in tumors or cancer cell lines of nonneural tissue origin. We show that blasts from a subset of patients with acute leukemia greatly overexpress eight different BAALC transcripts, resulting in five protein isoforms. Among patients with acute myeloid leukemia, those overexpressing BAALC show distinctly poor prognosis, pointing to a key role of the BAALC products in leukemia. Our data suggest that BAALC is a gene implicated in both neuroectodermal and hematopoietic cell functions.

Inhibited breathing decreases renal sodium excretion.

This study tested the hypothesis that breathing at the upper end of the normal range of end tidal CO2 decreases renal sodium excretion. Normotensive human subjects learned to self-regulate end tidal CO2 using a respiratory gas monitor and feedback procedure. Urine flow rates were increased by a standardized water drinking regimen. Urinary volume and sodium and potassium excretion were decreased during 30 minutes of inhibited (i.e. high normal end tidal CO2) breathing, compared with levels preceding and after task performance. Blood pressure, but not heart rate, increased during task performance. Plasma volume increase under these conditions is indicated by the observation that urinary excretion of an endogenous digoxin-like factor was increased. The physiological mechanism by which inhibited breathing elicits renal sodium retention remains to be determined. This breathing pattern could mediate the role of behavioral stress in some forms of hypertension.

Role of delayed feeding and of feeding increments in necrotizing enterocolitis.

In spite of a number of studies on necrotizing enterocolitis, there remains controversy concerning prevention regimens, especially with regard to enteral alimentation. This report is of a matched case-control study of the relationship of necrotizing enterocolitis to timing of first feeding, size of feeding volumes and increments, and a risk factor index in 59 case patients with necrotizing enterocolitis and 59 matched control patients. Comparison with control patients showed that case patients were fed earlier, received full-strength formula sooner, and received larger feeding volumes and increments. More highly stressed infants, as measured by the risk index, were more vulnerable to larger feeding increments. Among case patients there was significant correlation of age at first feeding and age at diagnosis (p < 0.0001) even after control for birth weight and risk index score, indicating that delayed feeding was related to delayed onset of disease. These analyses support the theory that earlier, more rapid feeding places stressed infants at greater risk for the development of necrotizing enterocolitis, and that infants with more severe respiratory problems are more vulnerable to such feeding practices.

Diarrhea in tube-fed patients: feeding formula not necessarily the cause.

PURPOSE: This study of diarrhea in tube-fed patients was undertaken to determine the proportion of cases in which feeding formula is not responsible for the diarrhea, the causes other than the feeding formula, and the diagnostic approach to diarrhea in tube-fed patients. PATIENTS AND METHODS: Inpatients at the Truman Memorial Veterans Hospital who received nasoenteric feeding during the time period from October 1986 through May 1988 were eligible for this study. Of 123 patients who received nasoenteric feeding, 32 patients had documented diarrhea (greater than 500 mL per day for at least two consecutive days) and were enrolled. Three of these patients received hypertonic feeding formula, whereas the remaining 29 received isotonic feeding formula. Prospective determinations of the causes of diarrhea were performed. Laboratory tests included fecal leukocytes, stool osmolality, stool electrolytes, and Clostridium difficile toxin assay. Diarrhea was considered osmotic if the stool osmotic gap was greater than 100 mmol/L. Clinical management involved reducing or stopping the feeding formula, stopping suspected medications, or administering appropriate antibiotics. RESULTS: There were 32 episodes of diarrhea in tube-fed patients during the study period. A single cause could be specified in 29 cases. The tube feeding formula was responsible for diarrhea in only 21% of these cases. Medications were directly responsible in 61% and C. difficile in 17% of cases. Stool osmotic gap correctly distinguished osmotic from non-osmotic diarrhea in all cases. CONCLUSION: When diarrhea develops in properly tube-fed patients, the feeding formula is usually not responsible for the diarrhea. Patients receiving nasoenteric tube feeding are frequently placed on liquid forms of medications. Many medicinal elixirs contain sorbitol, which is often the cause of diarrhea in tube-fed patients. Review of the medications and determination of the stool osmotic gap are the initial diagnostic steps of highest yield.

Effect of chronic partial pancreatic duct obstruction on pancreatic duct secretory pressure and permeability in cats.

Pancreatic secretion during pancreatic duct obstruction results in increased duct pressure. The normally impermeable pancreatic duct becomes permeable to macromolecules the size of pancreatic enzymes after secretion against obstruction. Permeability and morphologic changes may be related to increased secretory pressure during obstruction. We obstructed the main pancreatic duct of cats by 25-100% of its luminal diameter in different groups for 2, 7, or 28 days. Permeability to macromolecules of fluoresceinated dextran (FD) was greatest in cats with less than 75% obstruction compared with cats with greater than 75% obstruction regardless of the duration of obstruction. The frequency of permeability to FD decreased significantly as both the degree and duration of obstruction increased. Secretory pressure also changed according to degree and duration of obstruction. The highest pressures were in cats with complete obstruction at 2 days. Pressure decreased as the degree and duration of obstruction increased. Histologic changes such as acinar lobular atrophy and interstitial fibrosis were most severe in cats with the greatest degree and duration of obstruction. Pressure and permeability changes indicate a greater sensitivity to increased duct pressure than previously thought. These observations may clarify the role of pancreatic duct obstruction in pancreatic disease.

Analysis of pacemaker malfunction and complications of temporary pacing in the coronary care unit.

The medical records of 100 patients who received 113 temporary transvenous pacemakers were reviewed to determine the incidence of complications and malfunction. Malfunction, defined as failure to capture or sense, or both, occurred in 42 (37 percent) of 113 temporary pacemakers. The initial malfunction occurred within 24 hours in 21 (50 percent) and within 48 hours in 36 (86 percent) of the 42 pacemakers. Although the incidence of malfunction was not significantly different for brachial and femoral venous pacing catheters, 7 (37 percent) of 19 brachial venous pacemakers required repositioning or replacement compared with 8 (9 percent) of 91 femoral venous catheters (p = 0.005). Thirty-seven complications occurred in 23 (20 percent) of 113 episodes of pacing; ventricular tachycardia during catheter insertion, fever and phlebitis were the most common complications. No complication resulted in death. The incidence of complications and perforation was greater for brachial than for femoral venous pacemakers (p less than 0.05). Sepsis, local infection and pulmonary embolus occurred only with femoral venous pacemakers. Sepsis, phlebitis and pulmonary embolus were more common with temporary pacemakers in place for 7 hours or longer (p = 0.04). Recognition to the problems peculiar to each pacing catheter site and shortening the duration of pacing should help minimize problems with temporary pacing.

Pancreatic secretions as a clue to the presence of pancreatic cancer.

Pancreatic secretory function is abnormal in at least 90% of patients with pancreatic cancer. These abnormalities may be due to direct involvement of the secretory cells by the malignant process and/or the effects of pancreatic duct obstruction. There is no specific stimulus (secretin and/or cholecystokinin, CCK, or CCK-like hormones) of pancreatic secretion that is clearly superior to any other as a test of pancreatic function. Pancreatic secretion is abnormal in animal models of pancreatic cancer and secretory abnormalities antedate the histologic appearance of the cancer. A decrease in protein secretion after CCK stimulation is the most significant finding in experimental partial pancreatic duct obstruction (the condition most commonly seen in pancreatic cancer). In the absence of any identifiable high-risk group within the population, it is unlikely that the testing of pancreatic function provides a means for the earlier diagnosis of pancreatic cancer.

Effect of 16, 16 dimethyl prostaglandin E2 on aspirin-induced permeability changes in the pancreatic duct.

Perfusion of the pancreatic duct with acidified aspirin in cats increased the permeability of the duct to HCO3-. Intravenous administration of the synthetic prostaglandin analog 16,16 dimethyl prostaglandin E2 (PGE2) prevented this permeability change. The effect was dose-related and at the highest dose (50 microgram/kg/hour) was essentially complete. The beneficial effect of the PGE2 was apparent even when it was given after the duct had been exposed to aspirin. PGE2 had no effect on pancreatic water or electrolyte secretion in the unstimulated nonsecreting gland. No histologic changes in the pancreatic ducts were seen after exposure to aspirin, PGE2 or both. The effectiveness of PGE2 in restoring membrane permeability towards normal suggests the possibility of its therapeutic role in pancreatitis.

Pancreatic cancer: diagnostic value of pancreatic function tests.

Pancreatic secretion is abnormal in at least 90% of patients with pancreatic cancer. Any of the standard tests of pancreatic function that employ an exogenous secretin or CCK-like stimulus effectively reveal those abnormalities. However, despite a high sensitivity, the tests lack the specificity necessary to distinguish reliably between pancreatic cancer and chronic pancreatitis. With the current practice of testing pancreatic function in symptomatic patients and in the absence of an identifiable high-risk group within the population, it is unlikely that the established pancreatic function tests will provide a means for the earlier detection of pancreatic cancer.

Pancreatic function in domestic cats with pancreatic fluke infection.

Thirty-one of 290 cats (10.7%) from the area around St Louis, Mo, were infected with Eurytrema procyonis. In some cats, the pancreas was severely affected, with almost complete atrophy and fibrous replacement of the gland. Both bicarbonate and protein secretions were impaired, although clinically evident pancreatic insufficiency was not seen.