Diagnosis and management of refractoriness to platelet transfusion

Improvements in the availability and quality of platelet transfusions have markedly reduced the morbidity and mortality associated with intensive myelosuppressive therapy. Alloimmunization and refractoriness to platelet transfusion remains a significant clinical problem, although the incidence of alloimmunization may be declining due to more widespread use of leucocyte depleted products. Alloimmunization can be distinguished from other causes of poor post transfusion increments by the measurement of lymphocytotoxic or antiplatelet antibodies. In addition to medical approaches to reduce the risk of bleeding in individual patients, identification of histocompatible donors can usually be accomplished by HLA matching of donor and recipient, platelet cross matching or a combination of both techniques. There are a number of selection strategies which can be utilized and optimal patient management requires close cooperation and communication between clinicians and blood centers.     

The changes of IGF binding proteins after rhGH administration to patients totally dependent on parenteral nutrition

The objective was to study the effect of recombinant human growth hormone (rhGH) administration to patients with chronic malnutrition maintained on total parenteral nutrition (TPN) on the levels of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) during a double-blind trial. After 1 week of TPN the patients were randomized into group I (placebo) or group II (rhGH). Samples were collected on the first day (start of the TPN) to measure basal values, the seventh day to study the effect of TPN and the 10th, 14th and 21st days to evaluate the rhGH effect. Basal laboratory evaluation, nutritional status and glucose tolerance were assessed using standard laboratory techniques. Radioimmunoassays were used to analyse IGF-I, free IGF-I (fIGF-I) and IGFBP1-3. Electrophoresis with Western ligand blotting and Western immunoblotting was applied to find the pattern of IGFBPs. TPN had no effect on the circulating IGF-I concentration and the pattern of IGFBPs present in the studied groups of patients. The rhGH administration led to significant increases of IGF-I, total IGFBP-3, glycosylated IGFBP-3 (39, 42 kDa) and the 29 kDa fragment of IGFBP-3 and the decrease of IGFBP-2 during the trial (P<0.05). The mean levels of IGFBP-1, fIGF-I and the parameters of nutritional status in group II during the trial were not significantly influenced by rhGH. However, it has been found that IGFBP-1 and fIGF-I levels were correlated with the levels of the weekly nitrogen balance of each patient in group II at the end of the trial. In spite of the significant changes of IGF-I, IGFBP-2, total IGFBP-3 and IGFBP-3 (29 kDa proteolytic fragment) after rhGH administration to patients with malnutrition, maintained on parenteral nutrition, the increase of nitrogen balance was seen only in patients who decreased their IGFBP-1 and increased bioavailable IGF-I as reflected by measurement of fIGF-I. The levels of IGFBP-1 may provide a useful marker of IGF-I bioavailability in monitoring the efficiency of the rhGH therapy in malnourished patients.     

Maternal nutrition affects the ability of treatment with IGF-I and IGF-II to increase growth of the placenta and fetus, in guinea pigs

The aim of this study was to investigate how administration of IGF-I and IGF-II, during early to mid pregnancy, affects maternal growth and body composition as well as fetal and placental growth, in ad libitum fed, and in moderately, chronically food restricted guinea pigs. From day 20 of gestation, mothers (3-4 months old) were infused with IGF-I, IGF-II (565 microg/day) or vehicle for 17 days and then killed on day 40 of gestation. Maternal organ weights, fetal and placental weights were assessed. Treatment with IGFs did not alter body weight gain and had small effects on body composition in the mothers. Both IGF-I and IGF-II increased fetal and placental weights in ad libitum fed dams and IGF-I increased placental weight in food restricted dams. In conclusion, treatment with IGF-I during the first half of pregnancy stimulates placental growth in both ad libitum fed and food restricted guinea pigs without affecting maternal growth while fetal growth is stimulated by IGF treatment only in ad libitum fed animals.     

The site-specific delivery of ursodeoxycholic acid to the rat colon by sulfate conjugation

Because ursodeoxycholate has been shown to act as a tumor-suppressive agent in the colon, the absorption and metabolism of its sulfate conjugates were examined in rats to show that sulfation would facilitate the site-specific delivery of ursodeoxycholate to the colon. Bile acids were measured in intestinal contents, feces, urine, plasma, and liver tissue after oral administration of ursodeoxycholate and its C-3, C-7, and C-3,7 sulfate derivatives. Ursodeoxycholate was found in the jejunum after administration of all bile acids, but the mass was greatest for ursodeoxycholic acid administration. In the colon, lithocholic acid, normally found in negligible amounts, became the major bile acid after ursodeoxycholate administration. In contrast, reductions in mass and proportions of lithocholate and deoxycholate occurred after administering the C-7 sulfates. The fecal lithocholate/deoxycholate ratio, a risk marker for colon cancer, increased markedly after administration of ursodeoxycholate and its C-3 sulfate, but did not change after administering the C-7 sulfates. Unlike ursodeoxycholate or its C-3 sulfate, which increased liver concentrations of lithocholate and ursodeoxycholate, the C-7 sulfates had the opposite effect, which was consistent with poor absorption. Sulfation of ursodeoxycholate, specifically at the C-7 position, protects the molecule from bacterial degradation and inhibits its intestinal absorption, thereby facilitating delivery to the colon.     

An intravenous loading dose of azathioprine decreases the time to response in patients with Crohn's disease

Azathioprine, an effective therapy for Crohn's disease, is limited by a prolonged time to response. The aim of this study was to determine the safety and utility of a loading dose of azathioprine to decrease the time to response in patients with Crohn's disease. Twelve patients were studied: 6 with 13 fistulae and 6 with inflammatory disease. All patients received an intravenous infusion of azathioprine (50 mg/h for 36 hours). Response was determined by physical and radiographic examination for fistulae and by the Crohn's Disease Activity Index for inflammatory disease. Erythrocyte concentrations of azathioprine metabolites were measured by chromatography. Seven of 13 fistulae closed by week 4, and three had a temporary decrease in drainage. One fistula improved at week 16. Two fistulae failed to improve. Four of 6 patients with inflammatory disease achieved remission, and 1 improved temporarily. Improvement was rapid (≤4 weeks). Peak concentrations of azathioprine metabolites occurred within 3 days. Clinical response did not correlate with azathioprine metabolite concentrations at the azathioprine dose studied. No adverse events occurred. An 1800-mg intravenous loading dose of azathioprine is safe and may decrease the time to response to ≤4 weeks in patients with Crohn's disease. Correlation between clinical response and azathioprine metabolite concentrations at larger azathioprine doses should be determined.     

Subcellular localization of hepatitis B core antigen in relation to hepatocyte regeneration in chronic hepatitis B

To test whether the dominant cytoplasmic expression of hepatitis B core antigen (HBcAg) in active chronic hepatitis B is secondary to liver damage and regeneration, the relationship between subcellular localization of HBcAg, liver inflammatory activity, and hepatocyte regeneration in chronic hepatitis B was studied. Correlation of the clinical and laboratory data with the topographical distribution of HBcAg was studied in 30 patients. The subcellular localization of HBcAg in relation to hepatocyte cell cycles was studied by double immunostaining of HBcAg and proliferating cell nuclear antigen. Patients with predominant cytoplasmic HBcAg had significantly higher levels of biochemical and histological activities and proliferating cell nuclear antigen expression than patients with predominant nuclear HBcAg. The levels of proliferating cell nuclear antigen expression correlated positively with biochemical and histological activities and degrees of cytoplasmic HBcAg expression but negatively with degrees of nuclear HBcAg expression. Proliferating cell nuclear antigen expression was shown in 49% of hepatocytes with cytoplasmic HBcAg but in only 2% of hepatocytes with nuclear HBcAg. These findings suggested that, following liver damage, the regeneration of surviving hepatocytes might cause the shift of intracellular HBcAg from nucleus to cytoplasm. As a result, the extent of nuclear HBcAg expression reduces with concomitant increase in cytoplasmic HBcAg expression.     

Effect of germfree state on the capacities of isolated rat colonocytes to metabolize n-Butyrate, glucose, and glutamine

Among substrates available to the colonic mucosa, n-butyrate from bacterial origin represents a major fuel. The present work investigated possible modifications of energy substrate metabolism in colonocytes isolated from germfree rats. Colonocytes isolated from germfree vs. conventional rats were incubated (30 minutes at 37°C) in the presence of ¹⁴C-labeled n-butyrate (10 mmol/L), glucose (5 mmol/L), or glutamine (5 mmol/L). ¹⁴CO₂ and metabolites generated were measured. Possible regulatory steps were also investigated. Glucose use rate was 25% lower in germfree rat colonocytes due to a reduced glycolytic capacity in these cells. Differences in 6-phosphofructo-1-kinase activity could account for this decrease. In contrast, glutamine use rate was 45% higher, and this was correlated with a higher maximum velocity of glutaminase in these cells. Nevertheless, the capacities to oxidize glucose and glutamine remained unchanged. Although the capacity to use n-butyrate was maintained in colonocytes of germfree rats, the ketogenic capacity was lower, whereas the capacity to oxidize n-butyrate was higher. The mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase protein was identified in the colonic mucosa. Moreover, the messenger RNA and amount of protein were 75% lower in the germfree state. The absence of an intestinal microflora induces specific changes in the metabolic capacities of colonocytes.     

Body temperature circadian rhythm variability corresponds to left ventricular systolic dysfunction in decompensated cardiomyopathic hamsters

Background

A declining amplitude of body temperature circadian rhythm (BTCR) predicts decompensation or death in cardiomyopathic hamsters. We tested the hypothesis that changes in BTCR amplitude accompany significant changes in left ventricular (LV) size and function.

Methods and Results

Using intraperitoneal transmitters, we continuously monitored the temperature of 30 male BIO TO-2 Syrian dilated cardiomyopathic hamsters. Cosinor analysis was used to detect significant changes—defined as changes >1 standard deviation from the baseline amplitude for 3 consecutive days—in BTCR amplitude over each hamster’s lifespan. The Student t-test was used to compare BTCR variability and LV size and function (as assessed by 2D echocardiography) between baseline and the time that BTCR amplitude declined. All hamsters received 10 mg/kg furosemide daily. At the time of BTCR amplitude decline, functional parameters had changed significantly (P < .0001) from baseline: ejection fraction (0.31 ± 0.09% vs. 0.52 ± 0.08%), LV end-systolic volume (0.11 ± 0.03 vs. 0.05 ± 0.02 cm³), and LV end-diastolic volume (0.16 ± 0.04 vs. 0.10 ± 0.03 cm³).

Conclusions

In decompensated cardiomyopathic hamsters, a decline in BTCR amplitude was associated with progression of heart failure and cardiac decompensation. Variation in BTCR warrants further investigation because of its potential implications for the diagnosis and treatment of cardiovascular disorders.     

Improving central venous catheter insertion documentation to facilitate electronic surveillance of bundle compliance

We developed an electronic surveillance system and modified electronic medical record and documentation practices to allow automated tracking of the central line bundle documentation with the insertion of central venous catheters. Despite significant improvements in documented use of the central line bundle in >80% of insertions, our central line-associated bloodstream infection rate remained unchanged, suggesting the need to further improve compliance toward a goal of 100% of central line insertions, as well as training and education on proper line maintenance for catheters that are medically required for prolonged periods.     

Coexpression of 5-HT2A and 5-HT4 receptors coupled to distinct signaling pathways in human intestinal muscle cells

The type and function of 5-hydroxytryptamine (5-HT) receptors on intestinal muscle cells in humans are not known. 5-HT receptors were characterized pharmacologically and by radioligand binding. Contraction, relaxation, inositol 1,4,5-triphosphate (IP₃) and adenosine 3′,5′-cyclic monophosphate (cAMP) formation, and 5-HT binding were measured in dispersed muscle cells and in cells in which only one receptor type was preserved by selective receptor protection. 5-HT binding was completely inhibited by 5-HT and partially by 5-HT_2A (ketanserin), 5-HT₄ (SDZ-205,557), and 5-HT_1p (N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide; 5-HTP-DP) receptor antagonists. 5-HT caused contraction that was inhibited by ketanserin and augmented by SDZ-205,557 and 5-HTP-DP. In the presence of ketanserin, 5-HT caused relaxation of cholecystokinin-contracted cells that was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT increased IP₃, which was inhibited by ketanserin, and cAMP, which was inhibited by SDZ-205,557 and 5-HTP-DP. In cells with only 5-HT_2A receptors, 5-HT caused contraction only, and residual binding was inhibited by ketanserin. In cells with only receptors, 5-HT caused only relaxation and residual binding was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT_2A receptors mediating contraction and 5-HT₄ receptors mediating relaxation coexist on human intestinal muscle cells. The 5-HT₄ receptors are closely similar or identical to 5-HT_1p receptors.     

Bacterial translocation to mesenteric lymph nodes is increased in cirrhotic rats with ascites

Cirrhotic patients are predisposed to develop spontaneous bacteremias and/or peritonitis, mainly caused by enteric bacteria. The aim of this study was to investigate if bacterial translocation, which is the passage of bacteria from the intestinal lumen to regional lymph nodes and/or the systemic circulation, is increased in a rat model of cirrhosis. Rats were studied after 12–16 weeks of CCl₄ inhalation, when samples of mesenteric lymph nodes, blood, liver, and spleen for standard bacteriologic cultures and a fragment of colon and liver for histology were obtained. Immunostaining of the cecum was performed using a polyclonal anti-Escherichia coli antibody. A significantly greater proportion of rats with cirrhosis and ascites (5 of 9; 56%) had positive mesenteric lymph node cultures compared with cirrhotics without ascites (0 of 9) and normal controls (0 of 12) (P < 0.01). In one cirrhotic rat, E. coli was isolated from both mesenteric lymph nodes and ascites. Rats with cirrhosis and ascites had significantly greater cecal submucosal edema and inflammation than rats with no ascites and controls. Immunoreactivity with E. coli was present in the cecal wall in 3 of 5 animals with E. coli translocation to mesenteric lymph nodes. In cirrhotic rats, bacterial translocation is increased after the development of ascites and may be a major factor in the development of spontaneous infections in cirrhosis.     

Depolarization induces insulin-like growth factor binding protein-2 expression in vivo via NMDA receptor stimulation

The effect of depolarization and N-methyl-D-aspartate (NMDA) receptor blockade on insulin-like growth factor-I (IGF-I), IGF binding protein-2 (IGFBP-2) and IGFBP-4 expression was analysed in vivo. Depolarization was induced in adult rat brains by applying 3 M KCl to the exposed cortex for 10 min. A subgroup of animals also received daily injections of MK-801. Four days after KCl exposure, the brains were analysed by in situ hybridization, immunohistochemistry and TUNEL. A significant upregulation of IGFBP-2 mRNA and protein was detected in astrocytes after KCl exposure This upregulation was reduced by MK-801 treatment. No alterations in IGF-I or IGFBP-4 mRNA levels were noted. We did not detect TUNEL positive cells, morphological signs of necrosis or apoptosis, or neuronal loss in the depolarized zone. Taken together, these findings indicate that upregulation of IGFBP-2 by depolarization is mediated by NMDA receptors, and, as no neuronal damage was detected, astrocytic NMDA receptors may be responsible for this upregulation.