Fast simultaneous assessment of renal and liver function using polymethine dyes in animal models of chronic and acute organ injury.

Simultaneous assessment of excretory liver and kidney function is still an unmet need in experimental stress models as well as in critical care. The aim of the study was to characterize two polymethine-dyes potentially suitable for this purpose in vivo. Plasma disappearance rate and elimination measurements of simultaneously injected fluorescent dyes DY-780 (hepato-biliary elimination) and DY-654(renal elimination) were conducted using catheter techniques and intravital microscopy in animals subjected to different organ injuries, i.e. polymicrobial sepsis by peritoneal contamination and infection, ischemia-reperfusion-injury and glycerol-induced acute kidney-injury. DY-780 and DY-654 showed organ specific and determined elimination routes in both healthy and diseased animals. They can be measured simultaneously using near-infrared imaging and spectrophotometry. Plasma-disappearance rates of DY-780 and DY-654 are superior to conventional biomarkers in indicating hepatic or kidney dysfunction in different animal models. Greatest impact on liver function was found in animals with polymicrobial sepsis whereas glomerular damage due to glycerol-induced kidney-injury had strongest impact on DY-654 elimination. We therefore conclude that hepatic elimination and renal filtration can be assessed in rodents measuring plasma-disappearance rates of both dyes. Further, assessment of organ dysfunction by polymethine dyes correlates with, but outperforms conventional biomarkers regarding sensitivity and the option of spatial resolution if biophotonic strategies are applied. Polymethine-dye clearance thereby allows sensitive point-of-care assessment of both organ functions simultaneously.

Polymorphism of CC chemokine receptors CCR2 and CCR5 in Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory disease of the intestine that is characterized by mononuclear cell infiltration and a predominant Th1 lymphocyte response. We tested the hypothesis that CC chemokine receptors CCR2 and CCR5 might be important in the regulation of the intestinal immune response in this disease, and we speculated that carriers of a defective 32 base pair deletion mutant of CCR5, CCR5Delta32, which results in a non-functional receptor, might be protected from CD. Using polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) gene frequencies of CCR5Delta32 and of CCR2-641 (replacement of valine-64 by isoleucine in the CCR2 gene) in healthy controls (n=346) and in CD patients (n=235) were determined. In CD patients, subgroup phenotypic analyses were performed according to the Vienna classification. The overall gene frequency of CCR5Delta32 (9.8%) and CCR2-641 (7.6%) in CD patients did not deviate significantly from healthy controls (9.2 and 8.2%, respectively), nor did we observe a significant deviation from the predicted Hardy-Weinberg distribution. No significant differences in the CD phenotype classification for the different CCR5 and CCR2 alleles were observed, except for a trend to disease sparing of the upper gastrointestinal tract (carrier frequency 0 versus 19.6%, Delta=1 9.6%, P=0.079) as well as a more stricturing disease behaviour (23.5 versus 16.2%, Delta=7.3%, P=0.136) in carriers of the mutant CCR5Delta32 allele. These results indicate that the different CCR5 but not CCR2 alleles may influence disease behaviour and thereby contribute to the observed heterogeneity of CD. However, the associations observed are limited and await replication in other datasets. CCR2 and CCR5 polymorphisms are unlikely to be important determinants of overall disease susceptibility.

Inflammation does not decrease intraluminal pH in chronic inflammatory bowel disease.

Intestinal inflammation may influence intraluminal pH. Profiles of the gastrointestinal pH were evaluated in 15 patients with active Crohn's disease of the ileocecal area. In addition, five patients with moderate (1) or severe (4) ulcerative colitis were studied. Fifteen healthy subjects served as controls. Intraluminal pH of the different parts of the gastrointestinal tract was measured by a free-floating pH-sensitive telemetering capsule. A metal sphere was attached to the capsule for exact localization by a metal detector. Physiological patterns of pH were maintained throughout the gastrointestinal tract including the inflamed segments. Median pH in the terminal ileum of the patients with Crohn's disease was 7.5 vs. 7.7 and in the rectum in ulcerative colitis 7.8 vs. 7.2 in the controls. In conclusion, intraluminal pH is not decreased by inflammatory changes in Crohn's disease and ulcerative colitis, allowing eudragit-coated pH-controlled-release formulations of mesalazine to dissolve in diseased areas also.

Fatigue in mothers of infants discharged to the home on apnea monitors.

A comparative study was done to determine differences in caregiver fatigue between two groups of mothers of preterm infants at baseline, in the hospital (Time 1), 1 week postdischarge (Time 2), and 1 month postdischarge (Time 3). Group 1 infants were discharged home on apnea monitors (AM) (n = 28), and Group 2 infants were not on apnea monitors (nonAM)(n = 46). Measured by the Multidimensional Assessment of Fatigue (MAF) scale, mean fatigue scores from Time 1 to Time 3 markedly increased for the monitor group and decreased for the nonmonitor group. The scores were significantly different between the two groups at Times 2 and 3 but not at baseline. Two-way analysis of variance (ANOVA) with repeated measures showed group by time interaction effects on fatigue. Monitoring and alleviation of fatigue in home caregivers of preterm infants on apnea monitors are necessary.

Defining and measuring fatigue.

In response to a long history of problems with defining and measuring fatigue, the University of Kansas School of Nursing established a Center for Biobehavioral Studies of Fatigue Management to facilitate the study of fatigue in diverse populations. The purpose of this article is to review past efforts to define and measure fatigue and the conceptual problems relevant to currently used measures of fatigue. Several distinct characteristics and corresponding measures of fatigue are identified and a definition and framework for the study of fatigue are discussed. Future research on fatigue must attend to the conceptual distinctions among various measures and the measures of fatigue most appropriate to the goals of a study.

Fatigue among elders in caregiving and noncaregiving roles.

Fatigue is a complex symptom prevalent in informal caregiving. When role demands exceed caregiver resources, fatigue ensues and caregiving can be compromised. The purpose of this study was to compare perceptions of fatigue among older adults (N = 92) caring for spouses with Alzheimer's disease, Parkinson's disease, or cancer with a control group of older adults (N = 33) whose spouses required no extra care. Caregiving elders reported more fatigue, less energy, and more sleep difficulty than did control participants. All caregiving groups reported similar levels of fatigue, energy, sleep, and self-reported health even though there were marked differences regarding spousal status. Health care providers can support older caregivers in monitoring their own health and in recognizing the need for services that support the caregiving role.

Gastrointestinal pH profiles in patients with inflammatory bowel disease.

BACKGROUND: 5-Amino salicylic acid preparations are used in therapy for patients with inflammatory bowel diseases. The bioavailability of these drugs depends on their coating. AIM: To determine whether intraluminal pH is decreased by the presence of inflammation, thereby altering the release of 5-amino salicylic acid in the intestinal lumen. METHODS: Intraluminal gastrointestinal pH was measured by means of a radiotelemetry capsule in 12 healthy controls, in 12 patients with Crohn's disease (five with active disease), and in 11 patients with ulcerative colitis (seven with active disease). RESULTS: The median gastric pH values in the patient groups (Crohn's disease 2.4, range 1.5-4.1; ulcerative colitis 1.95, range 1.55-4.4) were significantly higher than those observed in the controls (1.55, range 0.95-2.6). In the small bowel and colonic segments, all the pH values of Crohn's disease patients were comparable to those of the controls, as were the pH values in the proximal small intestine and in the left colon in patients with ulcerative colitis. However, the latter group had higher pH values in the terminal ileum, the caecum and the right colon. Patients with active disease had comparable median gastrointestinal pH values to patients in remission. CONCLUSIONS: The luminal release of 5-amino salicylic acid might not be inhibited by low pH in patients with active inflammatory bowel diseases. This supports a safe disintegration of the slow release mesalazine preparations even in the presence of severe disease.

The cyclic adenosine monophosphate-dependent protein kinase (PKA) is required for the sustained activation of mitogen-activated kinases and gene expression by nerve growth factor.

Induction of neuronal differentiation of the rat pheochromocytoma cell line, PC12 cells, by nerve growth factor (NGF) requires activation of the mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK). cAMP-dependent protein kinase (protein kinase A (PKA)) also can induce differentiation of these cells. Like NGF, the ability of PKA to differentiate PC12 cells is associated with a sustained activation of ERKs. Here we show that maximal sustained activation of ERK1 by NGF requires PKA. Inhibitors of PKA partially blocked activation of ERK1 by NGF but had no effect on activation of ERK1 by EGF. Inhibition of PKA also reduced the ability of NGF and cAMP, but not EGF, to activate the transcription factor Elk-1, reduced the induction of both immediate early and late genes after NGF treatment, and blocked the nuclear translocation of ERK1 induced by NGF. We propose that PKA is an important contributor to the activation of ERK1 by NGF and is required for maximal induction of gene expression by NGF.

Effects of exercise on fatigue, aerobic fitness, and disease activity measures in persons with rheumatoid arthritis.

The effects of 12 weeks of low-impact aerobic exercise on fatigue, aerobic fitness, and disease activity were examined in a quasi-experimental time series study of 25 adults with rheumatoid arthritis (RA). Measures were obtained preintervention, midtreatment (after 6 weeks of exercise), end of treatment (after 12 weeks of exercise), and at a 15-week follow-up. ANOVAS for repeated measures showed that those subjects who participated more frequently reported decreased fatigue, while those who participated less frequently reported an increase in fatigue. All subjects, on average, showed increased aerobic fitness and increased right and left hand grip strength, decreased pain, and decreased walk time. There were no significant increases in joint count or sedimentation rate. Significant improvements in measures at the 15-week follow-up also were found. Findings indicate that persons with RA who participate in appropriate exercises may lessen fatigue levels and experience other positive effects without worsening their arthritis.

A role for mitogen-activated protein kinase in mediating activation of the glycoprotein hormone alpha-subunit promoter by gonadotropin-releasing hormone.

Gonadotropin-releasing hormone (GnRH) interacts with a G protein-coupled receptor and increases the transcription of the glycoprotein hormone alpha-subunit gene. We have explored the possibility that mitogen-activated protein kinase (MAPK) plays a role in mediating GnRH effects on transcription. Activation of the MAPK cascade by an expression vector for a constitutively active form of the Raf-1 kinase led to stimulation of the alpha-subunit promoter in a concentration-dependent manner. GnRH treatment was found to increase the phosphorylation of tyrosine residues of MAPK and to increase MAPK activity, as determined by an immune complex kinase assay. A reporter gene assay using the MAPK-responsive, carboxy-terminal domain of the Elk1 transcription factor was also consistent with GnRH-induced activation of MAPK. Interference with the MAPK pathway by expression vectors for kinase-defective MAPKs or vectors encoding MAPK phosphatases reduced the transcription-stimulating effects of GnRH. The DNA sequences which are required for responses to GnRH include an Ets factor-binding site. An expression vector for a dominant negative form of Ets-2 was able to reduce GnRH effects on expression of the alpha-subunit gene. These findings provide evidence that GnRH treatment leads to activation of the MAPK cascade in gonadotropes and that activation of MAPK contributes to stimulation of the alpha-subunit promoter. It is likely that an Ets factor serves as a downstream transcriptional effector of MAPK in this system.

A novel mitogen-activated protein kinase phosphatase. Structure, expression, and regulation.

Mitogen-activated protein (MAP) kinase lies at the convergence of various extracellular ligand-mediated signaling pathways. It is activated by the dual-specificity kinase, MAP kinase kinase or MEK. MAP kinase inactivation is mediated by dephosphorylation via specific MAP kinase phosphatases (MKPs). One MKP (MKP-1 (also known as 3CH134, Erp, or CL100)) has been reported to be expressed in a wide range of tissues and cells. We report the identification of a second widely expressed MKP, termed MKP-2, isolated from PC12 cells. MKP-2 showed significant homology with MKP-1 (58.8% at the amino acid level) and, like MKP-1, displayed vanadate-sensitive phosphatase activity against MAP kinase in vitro. Overexpression of MKP-2 in vivo inhibited MAP kinase-dependent gene transcription in PC12 cells. MKP-2 differed from MKP-1 in its tissue distribution and in its extent of induction by growth factors and agents that induce cellular stress, suggesting that these MKPs may have distinct physiological functions.

Effect of lactose, lactulose and bisacodyl on gastrointestinal transit studied by metal detector.

AIM AND METHODS: To study the effect of 45 g lactose, 30 g lactulose and 10 mg bisacodyl on gastrointestinal transit in 30 healthy volunteers by metal detector and Hinton marker method. The first set of measurements were performed under standard conditions. In a second stage, transit was slowed to twice the original value by loperamide to simulate constipation conditions. RESULTS: Bisacodyl drastically accelerated small and large intestinal transit. Colonic transit was shortened to 23% and to 31% of control values, without and with loperamide. Bisacodyl increased stool weight and decreased stool consistency in all persons. Lactulose marginally shortened small intestinal transit (P = 0.08) but significantly increased stool weight and decreased stool consistency. The accelerating effect of lactose on small intestinal transit was abolished by loperamide. Lactose did not influence colonic transit, stool weight or stool consistency. Results of metal detector and Hinton marker method corresponded well (r = 0.75), the metal detector method measuring slightly shorter transit times than the Hinton marker method. CONCLUSIONS: With the dose chosen in this trial, lactose did not have any laxative effect in lactose tolerant persons. Laxative effect was mild with lactulose and most pronounced with bisacodyl.

Complex alternative splicing partially inactivates the human chorionic somatomammotropin-like (hCS-L) gene.

The human growth hormone/human chorionic somatomammotropin (hGH/hCS) gene cluster contains five genes: hGH-N, hGH-V, hCS-A, hCS-B, and hCS-L. The expression of the first four genes has been well documented. In contrast, the hCS-L gene has been considered a pseudogene inactivated by loss of the normal intron 2 splice donor site. Previously our laboratory has shown that hCS-L transcripts are present in human placenta and that their levels are induced during the second trimester. More detailed studies of hCS-L transcript processing and mRNA structure are hindered by overwhelming levels of the structurally similar hCS-A and hCS-B transcripts in the placenta. To circumvent this problem, we have established stably transfected cell lines selectively expressing the hCS-L gene. Analysis of hCS-L mRNA from these cell lines demonstrates at least five major alternative splicing pathways, four of which could be confirmed qualitatively by parallel analysis of placental RNA. This analysis reveals an unexpectedly high frequency of exon 2 skipping (73%) as well as utilization of three competing exon 3 splice acceptor sites. Since exon 2 encodes the signal peptide, the majority of hCS-L transcripts are unable to express a secreted protein. Three of the defined hCS-L mRNAs contain an extended open reading frame similar to that present in the functional GH and CS genes. All three of these hCS-L transcripts are of minor abundance and only two, hCS-L(L) and hCS-L(L'), contain exon 2. In vitro translation and signal peptide processing of hCS-L(L) mRNA yields a 20-kDa hCS-L isoform in vitro. These data confirm the placental expression of the hCS-L gene, demonstrate surprising complexity in the splicing of its transcripts, predict that the majority of processed hCS-L mRNAs are nonfunctional, and identify specific, low abundance mRNAs that may encode novel gestational hormones.

Influence of senna, fibre, and fibre + senna on colonic transit in loperamide-induced constipation.

Retarded colonic transit and disturbed defecation are the most prominent pathophysiological mechanisms in constipation. Both may be influenced by bulking agents and by laxatives such as senna. Direct measurements of the influence of such substances on colonic transit are rare mainly because of technical problems. We measured gastric emptying, small and large intestinal transit in 24 healthy volunteers by a newly developed method employing a metal detector. Twelve persons taking a normal diet received loperamide in a dose sufficient to double the individual transit time. All subjects measured gastrointestinal transit time under normal conditions and with Sennatin containing purified sennosides 20 mg, Agiocur (30 g) as a fibre product containing 20 g Plantago ovata seeds/husks, or Agiolax (10 g) as a combination of 5.4 g P. ovata seeds/husks + 1.2 g senna pod with a sennoside content of 30 mg. Colonic transit was reduced by Sennatin and by Agiolax from 39 +/- 4 h to 17 +/- 3 h (p < 0.005). Agiocur did not influence colonic transit (39 +/- 3 h). Loperamide prolonged colonic transit from 27 +/- 0.7 to 72 +/- 12 h. This effect was abolished by Sennatin (30 +/- 5 h) and Agiolax (27 +/- 1 h) (p < 0.005), but not by Agiocur (64 +/- 13 h). The same effects were seen when right and left colonic transit were analyzed separately. Neither gastric emptying nor small intestinal transit were affected by either substance. All of the three study drugs increased stool weight significantly (p < 0.05). When stool frequency and consistency were compared, the effects were less clear.(ABSTRACT TRUNCATED AT 250 WORDS)

Azathioprine combined with prednisolone or monotherapy with prednisolone in active Crohn's disease.

BACKGROUND: The role of azathioprine (AZA) in the treatment of active Crohn's disease (CD) is still controversial. This study examined whether AZA combined with standard prednisolone therapy improved the therapeutic outcome compared with monotherapy with prednisolone. METHODS: Forty-two patients with a Crohn's Disease Activity Index (CDAI) of > 150 were randomized into two groups. Both received 60 mg of prednisolone daily in a tapering regimen to a maintenance dose of 10 mg. In addition, group 1 received 2.5 mg AZA/kg body wt and group 2 received a placebo over the whole study period of 4 months. RESULTS: At the end of the trial, 16 of 21 patients (76%) in group 1 were in remission (CDAI < 150), compared with 8 of 21 (38%) in group 2 (P = 0.03). The CDAI in group 1 dropped from 290 +/- 97 (SD) to 72 +/- 84 and from 285 +/- 110 to 155 +/- 105 in group 2. The differences between activity indices in groups 1 and 2 became statistically significant after 8 weeks. The average prednisolone dose per day was 20.9 mg in group 1 and 26.7 mg in group 2 (P = 0.02). No major side effects were observed in this study. CONCLUSION: The combination of prednisolone and AZA was superior to the treatment with prednisolone alone in active CD. Patients receiving AZA showed remission more frequently, more quickly, and with lower doses of prednisolone.

[Spontaneous bacterial peritonitis]. / Die spontane bakterielle Peritonitis.

Patients with liver cirrhosis and ascites suffer from spontaneous bacterial peritonitis (SBP) in up to 25%. The typical clinical signs are abdominal pain with tenderness and fever. 30% have no signs of peritonitis. Then clinical worsening, encephalopathy, rising serum creatinine levels, and therapy resistant ascites may be the only clinical features. SBP must be differentiated from bacterascites and culture negative neutrocytic ascites by the polymorphonuclear neutrophil (PMN) count in the ascites and the presence of positive culture results, which has prognostic implications. Gram negative rods from the colon play an important etiological role in SBP. Gastrointestinal bleeding, lack of serum complement, a low ascites protein and the extent of intrahepatic shunts predispose to SBP. Then, prophylaxis with the comparable drugs neomycin and norfloxacin is indicated. Coexisting encephalopathy has to be treated by the therefore effective neomycin. Otherwise, norfloxacin is the drug of choice because of better acceptance and lower costs. Chemical parameters of the ascites (pH value less than 7.4; LDH and lactate greater than serum levels; glucose less than 50 mg%) help to assess the severity of peritonitis. The course of ascitic PMN under therapy and the time of persisting positive cultures can discriminate SBP from secondary peritonitis. Antibiotics of choice are amoxicillin-clavulanic acid and cefotaxime. Short course therapy (5 days) is a effective as long course therapy (10 days). Today SBP is no more life-threatening because diagnosis, prophylaxis and therapy have improved. However, complication rate of patients with liver cirrhosis and ascites has not changed.

[The effect of food intake on the gastric emptying of gastric juice-resistant tablets and capsules]. / Einfluss der Nahrungsaufnahme auf die Magenentleerung magensaftresistenter Tabletten und Kapseln.

To test the effect of food intake on gastric emptying of gastric juice-resistant drugs, emptying time of a 11 x 6 mm tablet and a 20 x 7 mm capsule was measured by means of a metal detector in 10 healthy persons (5 men and 5 women; mean age 25 [18-30] years) after fasting and after eating three main and three in-between meals. After fasting the tablets left the stomach after 78 +/- 18 min, the capsules after 60 +/- 16 min, while meal intake delayed emptying by a factor of 10 to 12 +/- 1.3 hours and 10 +/- 1.8 hours, respectively. The slightly shorter emptying time of capsules was statistically not significant. The results indicate that gastric juice-resistant tablets taken during day-time may, if several meals are eaten, accumulate in the stomach and then be emptied together at night. It is recommended that such drugs be taken in the fasting state in the morning and between meals, while avoiding in-between meals.

Protein kinase C isoforms in human glioblastoma cells.

Protein kinase C (PKC), an enzyme involved in signal transduction, responds to diacyl glycerol and also to phorbol ester, a ligand analogous to diacyl glycerol. We have studied the expression of the major isoforms (alpha, beta I, beta II, and gamma) in eight human glioblastoma cell lines. In all eight lines, PKC-alpha mRNA and protein were expressed. In none of the eight did a probe for PKC-beta I and -beta II mRNA give positive results nor were Western blots for PKC-beta II positive. The half-life for PKC alpha mRNA was approximately 16 h and levels of the mRNA were increased slightly following addition of phorbol myristate acetate (PMA) or transforming growth factor-beta (TGF beta). PKC-gamma was present in most of the glioblastomas. In cell line A172, 82% of the PKC-alpha was present in the cytosol with the remainder evenly divided between plasma membrane and nucleus. Thirty minutes after addition of PMA, 33% of the total original protein was in the plasma membrane and 48% in the nuclear fraction. By 21 h, no PKC-alpha was recovered from any fraction. PKC-gamma was also down-regulated in the presence of PMA, but there was no evidence for translocation to the plasma membrane or nuclear fraction. In a more detailed study, translocation of PKC-alpha in the presence of PMA was complete by 10 min, and a major decrease in the PKC translocated to the plasma-membrane fraction occurred some time between 2 and 4 h after PMA addition, while a major decrease in the translocated nuclear fraction occurred some time after 6 h. cAMP alone had no effect on the PKC alpha protein level or distribution, nor did it alter the translocation and down-regulation due to PMA exposure. In these studies the level of PKC-alpha mRNA in tumors was similar to that in normal glial cells.

Gastric emptying of indigestible tablets in relation to composition and time of ingestion of meals studied by metal detector.

Enteric-coated tablets leave the stomach mainly during the interdigestive phase. Composition as well as time of ingestion of meals may influence their gastric emptying considerably. In 12 normal volunteers gastric emptying of a plastic tablet with a metal core was followed by a metal detector in relation to different compositions and various times of ingestion of meals. With an empty stomach and after ingestion of 250 ml water, the mean time for gastric emptying of the tablet was 38 +/- 11 min (mean +/- SEM) and 38 +/- 8 min. Two hundred fifty milliliters of milk (652 kJ) and a formula diet (1000 kJ) delayed gastric emptying time to 128 +/- 14 and 152 +/- 6 min, respectively (P less than 0.05). Breakfast (2200 kJ) further retarded gastric emptying compared with both liquids to 249 +/- 24 min (P less than 0.05). There was a close correlation between nutritive density and gastric emptying of the tablet (r = 0.92; P less than 0.001). Main meals also delayed gastric emptying of tablets when compared to empty stomach (P less than 0.05). A snack after breakfast further delayed gastric emptying from 201 +/- 10 to 278 +/- 19 min (P less than 0.05). The largest delay was observed following ingestion of breakfast, lunch, dinner, and additional snacks (509 +/- 220 min). We conclude that the delay of gastric emptying of enteric-coated tablets by food is related to its nutritive density and eating habits. The gastric emptying of an enteric coated tablet that is ingested early in the morning may be delayed until late at night when several meals and snacks are ingested during the day, leading to unwanted alterations in bioavailability and to possible adverse effects.