Efficacy, usability and sequence of operations of a workflow-integrated algorithm for basal-bolus insulin therapy in hospitalized type 2 diabetes patients.

AIMS: To evaluate glycaemic control and usability of a workflow-integrated algorithm for basal-bolus insulin therapy in a proof-of-concept study to develop a decision support system in hospitalized patients with type 2 diabetes. METHODS: In this ward-controlled study, 74 type 2 diabetes patients (24 female, age 68 ± 11 years, HbA1c 8.7 ± 2.4% and body mass index 30 ± 7) were assigned to either algorithm-based treatment with a basal-bolus insulin therapy or to standard glycaemic management. Algorithm performance was assessed by continuous glucose monitoring and staff's adherence to algorithm-calculated insulin dose. RESULTS: Average blood glucose levels (mmol/l) in the algorithm group were significantly reduced from 11.3 ± 3.6 (baseline) to 8.2 ± 1.8 (last 24 h) over a period of 7.5 ± 4.6 days (p < 0.001). The algorithm group had a significantly higher percentage of glucose levels in the ranges from 5.6 to 7.8 mmol/l (target range) and 3.9 to 10.0 mmol/l compared with the standard group (33 vs. 23% and 73 vs. 53%, both p < 0.001). Physicians' adherence to the algorithm-calculated total daily insulin dose was 95% and nurses' adherence to inject the algorithm-calculated basal and bolus insulin doses was high (98 and 93%, respectively). In the algorithm group, significantly more glucose values <3.9 mmol/l were detected in the afternoon relative to other times (p < 0.05), a finding mainly related to pronounced morning glucose excursions and requirements for corrective bolus insulin at lunch. CONCLUSIONS: The workflow-integrated algorithm for basal-bolus therapy was effective in establishing glycaemic control and was well accepted by medical staff. Our findings support the implementation of the algorithm in an electronic decision support system.

Episodes of hypocarbia and early-onset sepsis are risk factors for cystic periventricular leukomalacia in the preterm infant.

BACKGROUND: Septic episodes in preterm infants recently have been reported to be associated with periventricular leukomalacia (PVL). The role of hypocarbia as an independent risk factor for PVL in clinical studies raises many questions without conclusive answers. AIMS: To evaluate risk factors for cystic PVL focussing on the influence of hypocarbia. STUDY DESIGN: Retrospective single centre case-control study. SUBJECTS: Preterm infants 24 to 35 weeks of gestational age and matched (1:2 for gender, birth year, gestational age and birth weight) controls. OUTCOME MEASURES: Multivariate analysis of perinatal factors being associated with cystic PVL diagnosed by serial ultrasound examinations. RESULTS: Univariate analysis of risk factors revealed lower 5 and 10 min Apgar scores, and higher rates of neonatal seizures, early-onset sepsis, neonatal steroids, respiratory distress syndrome with surfactant replacement therapy, and episodes of hypocarbia significantly being associated with PVL. Multivariate analysis using a logistic regression model revealed early-onset sepsis and hypocarbia being significantly associated with PVL (p=.022 and .024, respectively). Lowest PaCO(2) values did not differ as did not the duration of hypocarbia, but the onset of hypocarbia was significantly later in PVL cases compared to controls (mean 26 vs. 15 h, p=.033). Neurodevelopmental follow-up at a median time of 46 months was poor showing 88% of the cases having an adverse neurological outcome. CONCLUSION: We found early-onset sepsis and episodes of hypocarbia within the first days of life being independently associated with PVL.

Respiratory insufficiency related to COPD accelerates systemic inflammation, under-nutrition, and angiogenesis in esophageal malignancies.

UNLABELLED: A number of esophageal cancer patients suffer from respiratory insufficiency due to the coexistence of chronic obstructive pulmonary disease (COPD). AIM: To test the hypothesis that COPD-related systemic hypoxemia may result in accelerated inflammation, malnutrition, and angiogenesis in esophageal cancer patients. METHODS: Serum levels of C-reactive protein (CRP), albumin, transferrin, interleukin-1, interleukin-6, interleukin-8, TNF-alpha, platelet-derived growth factor (PDGF-BB), and midkine and patient BMI and weight-loss rate were determined and compared with blood oxygenation status (pO(2), SaO(2)) in 35 esophageal cancer patients and 42 controls. RESULTS: The incidence of cachexia tended to be higher in patients with systemic hypoxemia (67% vs 40%, p = 0.169). Mean SaO(2) level was also significantly decreased in cachectic patients (90.3 vs 93.3%, p = 0.026) and pO(2) exhibited a similar trend (58.0 vs 63.4 mmHg, p = 0.120). Transferrin (234 vs 316 mg/dl, p = 0.005) and albumin (31.9 vs 37.1 mg/dl, p= 0.002) concentrations were reduced and CRP was elevated (129.9 vs 54.7 mg/l, p = 0.004) in hypoxemic patients and correlated with pO(2) (r = 0.47, p = 0.016; r= 0.48, p = 0.012; r = -0.37, p = 0.064) and SaO(2) (r = 0.52, p = 0.006; r = 0.53, p = 0.006; r = -0.40, p= 0.042). Interleukin-6 (9.97 vs 2.21 pg/ml, p = 0.005) and midkine (2101 vs 944 pg/ml, p < 0.001) were elevated and PDGF-BB was decreased (12.2 vs 17.3 pg x 10(-6)/PLT, p = 0.014) in hypoxemic compared with normoxemic patients. Interleukin-6 and midkine negatively correlated with pO(2) (r = -0.44, p = 0.016; r = -0.42, p = 0.011) and SaO(2) (r = -0.54, p = 0.003; r = -0.57, p < 0.0001) and PDGF-BB correlated positively (r = 0.53, p = 0.003; r = 0.44, p = 0.020). Interleukin-8 level was affected by pO(2) (r = -0.55, p = 0.015) and SaO(2) (r= -0.55, p = 0.018) only in hypoxemic patients. CONCLUSIONS: COPD-related systemic hypoxemia negatively affects the status of esophageal cancer patients by accelerating inflammation, under-nutrition, and angiogenesis.

Von Willebrand gene expression in damaged human liver.

BACKGROUND/AIMS: von Willebrand factor (vWf) is an adhesive glycoprotein known to play a role in hemostasis and in tissue injury. It is found in high levels in plasma of patients with acute hepatic failure and chronic liver disease. The aim of this study was to investigate the pattern of tissue vWf in acute liver failure in humans. METHODOLOGY: We studied vWf immunostaining and mRNA expression in the liver of three patients with fulminant liver failure, two patients with chronic liver disease, and two controls. PECAM-1 (CD31) immunostaining and mRNA expression were used as an additional endothelial marker. RESULTS: In chronic liver cirrhosis, vWf deposits were strongly detected at the scar-parenchyma interface. In fulminant hepatic failure, intense deposits were seen in tissue sections in the area of necrosis. A similar pattern of immunostaining was seen with PECAM-1. vWf transcripts were abundant in the liver of patients with chronic disease and minimally expressed in patients with acute hepatic failure and in controls. CONCLUSIONS: vWf is deposited within the liver sinusoids early after liver damage. The factor is only partially produced locally during the acute phase of the disease, but is overproduced in chronic disease states. These changes may suggest a role for vWf in liver injury and repair.

The bcl, NFkappaB and p53/p21WAF1 systems are involved in spontaneous apoptosis and in the anti-apoptotic effect of TGF-beta or TNF-alpha on activated hepatic stellate cells.

Activated hepatic stellate cells (HSC) are thought to play a pivotal role in development of liver fibrosis which takes place in chronic liver diseases. Previous studies have shown that "activated" rat HSC undergo spontaneous apoptosis probably through the CD95/CD95L pathway. TGF-beta as well as TNF-alpha reduced spontaneous apoptosis and CD95L expression. The aim of this study was to investigate the possible mechanisms responsible for the spontaneous apoptosis and for the anti-apoptotic effect of TGF-beta and TNF-alpha on activated HSC. While bcl-2, bax, NFkappaB and p53 gene expression were spontaneously upregulated, bcl-xL and p21WAF1 gene expression decreased and IkappaB remained unchanged during the activation process in vitro. TGF-beta as well as TNF-alpha induced activation of NFKB and upregulated bcl-xL. The latter was inhibited by overexpression of IkappaB. By suppressing spontaneous apoptosis TGF-beta as well as TNF-alpha inhibited p53 gene expression while that of the p21WAF1 gene was increased. We conclude that TGF-beta as well as TNF-alpha may act as surviving factors for activated rat HSC not only through reduction of CD95L gene expression but also by upregulating the anti-apoptotic factors NFKB, bcl-xL and p21WAF1 and by downregulating the proapoptotic factor p53. The interaction with these factors may lead to the generation of new antifibrotic drugs.

Expression of decorin, transforming growth factor-beta 1, tissue inhibitor metalloproteinase 1 and 2, and type IV collagenases in chronic hepatitis.

Decorin is a small extracellular matrix proteoglycan. It binds and modulates transforming growth factor (TGF)-beta 1 action, the major stimulator of fibrogenesis. Its role in the pathogenesis of human liver cirrhosis is unknown. Therefore, we studied the relationship of the 2 proteins in normal human liver and in 43 chronic hepatitis and liver cirrhosis specimens. To understand the mechanism that maintains matrix deposition in stage IV hepatitis, we studied expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, as well as the activities of type IV collagenases. Gene expression was analyzed on messenger RNA and protein level by morphologic and biochemical approaches. Decorin proved to be an early marker of fibrogenesis, and its deposition increased parallel to that of TGF-beta 1 and to inflammatory activity. Liver fibrosis progressed despite high temporospatial expression of decorin with TGF-beta 1. Neither decorin nor TGF-beta 1 protein deposition increased further in cirrhosis with low inflammatory activity, suggesting that impaired extracellular matrix catabolism rather than active production plays a role in this stage. This possibility was supported by high message levels of metalloproteinase inhibitors, no 72-kd collagenase activities, and low 92-kd collagenase activities.

Liver fibrosis and altered matrix synthesis.

Liver fibrosis represents the uniform response of liver to toxic, infectious or metabolic agents. The process leading to liver fibrosis resembles the process of wound healing, including the three phases following tissue injury: inflammation, synthesis of collagenous and noncollagenous extracellular matrix components, and tissue remodelling (scar formation). While a single liver tissue injury can be followed by an almost complete restitution ad integrum, the persistence of the original damaging noxa results in tissue damage. During the establishment of liver fibrosis, the basement membrane components collagen type IV, entactin and laminin increase and form a basement membrane-like structure within the space of Disse. The number of endothelial fenestrae of the sinusoids decreases. These changes of the sinusoids are called 'capillarization' because the altered structure of the sinusoids resembles that of capillaries. At the cellular level, origin of liver fibrogenesis is initiated by the damage of hepatocytes, resulting in the recruitment of inflammatory cells and platelets, and activation of Kupffer cells, with subsequent release of cytokines and growth factors. The hepatic stellate cells seem to be the primary target cells for these inflammatory stimuli, because during fibrogenesis, they undergo an activation process to a myofibroblast-like cell, which represents the major matrix-producing cell. Based on this pathophysiological mechanism, therapeutic methods are developed to inhibit matrix synthesis or stimulate matrix degradation. A number of substances are currently being tested that either neutralize fibrogenic stimuli and prevent the activation of hepatic stellate cells, or directly modulate the matrix metabolism. However, until now, the elimination of the hepatotoxins has been the sole therapeutic concept available for the treatment of liver fibrogenesis in humans.

Trypanosoma cruzi: presence of the two major phylogenetic lineages and of several lesser discrete typing units (DTUs) in Chile and Paraguay.

Multilocus enzyme electrophoresis (MLEE) of 99 Chilean and 11 Paraguayan stocks of Trypanosoma cruzi, the agent of Chagas disease, was performed for 22 variable genetic loci. As previously shown for this parasite in other geographic areas, a pattern of long-term clonal evolution of T. cruzi genotypes was inferred, both by strong departures of Hardy-Weinberg expectations and high linkage disequilibrium. The presence of the two major phylogenetic lineages that subdivide the species T. cruzi [Tibayrenc, M., 1995. Population genetics of parasitic protozoa and other microorganisms. In: Baker, J.R., Muller, R., Rollinson, D. (Eds.), Advances in Parasitology, vol. 36, Academic Press, New York, pp. 47-115; Souto, R.P., Fernandes, O., Macedo, A.M., Campbell, D.A., Zingales, B., 1996. DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Mol. Biochem. Parasitol. 83, 141-152], and of several lesser genetic subdivisions ('discrete typing units' or DTUs; Tibayrenc, M., 1998a. Genetic epidemiology of parasitic protozoa and other infectious agents: the need for an integrated approach. Int. J. Parasitol. 28 (1), 85-104; Tibayrenc, M., 1998b. Beyond strain typing and molecular epidemiology: integrated genetic epidemiology of infectious diseases. Parasitol. Today 14, 323-329; Tibayrenc, M., 1998c. Integrated genetic epidemiology of infectious diseases: the Chagas model. Mem. Inst. Oswaldo Cruz 93 (5), 577-580), was recorded in this region. Comparison between clonal populations in sylvatic and domestic transmission cycles of the disease in Chile strongly suggests that these two cycles are at least partially separated from one another.

Decrease of platelet-endothelial cell adhesion molecule 1-gene-expression in inflammatory cells and in endothelial cells in the rat liver following CCl(4)-administration and in vitro after treatment with TNFalpha.

UNLABELLED: Platelet-endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily is strongly expressed at endothelial cell-cell junctions, on most leukocytes and on monocytes. PECAM-1 has been implicated as a key mediator of the transendothelial migration of leukocytes and monocytes. To further define the physiological role of PECAM-1, we studied the modulation of PECAM-1-expression in a model of liver inflammation in both mononuclear cells (MCs) and sinusoidal endothelial cells (SECs). In normal rat liver sections, PECAM-1 immunohistology indicated a sinusoidal pattern similar to the ICAM-1 staining. Both, SECs, small and large MCs isolated from control rats express PECAM-1 as demonstrated by immunocytochemistry, flow cytometry, and Northern blot analysis. Immunohistochemical studies on liver sections from CCl(4)-treated animals indicated, that in the areas of necrosis 24-48 h after a single administration of the toxin, there was an accumulation of LFA-1-, ED1- (marker for rat monocytes) and ICAM-1-positive, but ED2-(marker for tissue macrophages)-negative inflammatory cells. Most of these cells were PECAM-1-negative. In situ hybridization indicated that there is no accumulation of PECAM-1 specific transcripts after CCl(4) treatment within the pericentral region. Immunocytology, flow cytometry, and Northern blot analysis of MCs and SECs isolated at different times after the administration of CCl(4) revealed a decrease of PECAM-1 gene expression in MCs and in SECs, whereas ICAM-1 expression increased. As TNFalpha has been shown to be upregulated early after CCl(4) administration, the influence of TNFalpha on PECAM-gene-expression was analyzed. TNFalpha treatment of cultured rat SECs and of small and large MCs from normal liver decreased PECAM-1 specific transcript level in parallel to the increase of ICAM-1 transcript level. CONCLUSIONS: Early production of TNFalpha after liver injury could induce an increased ICAM-1-expression and a decreased PECAM-1 expression, which might be essential for the transmigration of inflammatory cells into the parenchyma.

Effect of heparin and liver heparan sulphate on interaction of HepG2-derived transcription factors and their cis-acting elements: altered potential of hepatocellular carcinoma heparan sulphate.

Proteoglycan assembly in malignant tumours is subject to profound changes. The significance of these alterations is not well understood; especially, their role in nuclear regulation is a topic for debate. The capacity of heparin and liver carcinoma heparan sulphate (HS) to alter DNA-transcription factor interactions has been studied to provide further evidence concerning the regulatory potential of glycosaminoglycan (GAG) in the nucleus. Experiments both in vitro and in vivo indicated that heparin and HS are capable of inhibiting the interaction of transcription factors with their consensus oligonucleotide elements. Among five transcription factors studied, AP-1, SP-1, ETS-1 and nuclear factor kappaB proved to be sensitive to heparin and heparan sulphate, whereas TFIID was hardly inhibited in either in vitro or in vivo systems. Interestingly, HS from peritumoral liver was five times more effective than heparin. Liver carcinoma HS was less effective than liver HS, but its activity was comparable with that of heparin. These results indicate that the structural differences of GAG chains strongly influence their biological behaviour. The loss of their recognized functional activity in malignant tumours might promote the development of uncontrolled growth and gene expression favouring the neoplastic process.

Platelet-endothelial cell adhesion molecule-1 gene expression in liver sinusoidal endothelial cells during liver injury and repair.

BACKGROUND/AIMS: Platelet-endothelial cell adhesion molecule (PECAM)-1 is suggested to be critical for transmigration processes. It is a matter of debate whether PECAM-1 is expressed in liver sinusoids and whether it is involved in liver inflammation. METHODS: Indirect immunostaining and in situ hybridization was used to analyze PECAM-1 gene expression in normal and diseased rat and human livers as well as in isolated rat sinusoidal endothelial cells (SECs), hepatic stellate cells and hepatocytes. At various time points after the administration of CCl4 (6 h, 24 h, 48 h, and 72 h), PECAM-1 gene expression was analyzed in livers and in SECs by immunostaining, and Northern blot analysis. RESULTS: In normal rat or human livers PECAM-1 immunoreactivity was detected along the sinusoids in a pattern similar to ICAM-1 staining. PECAM-1 specific transcripts were detected in freshly isolated and cultured SECs. After a single CCl4-administration, PECAM-1 immunoreactivity did not increase along the sinusoids in contrast to the early increase of ICAM-1. Northern blot analysis indicated that PECAM-1 expression in liver tissue and in isolated SECs does not increase after a single administration of CCl4, whereas ICAM-1 steady-state level increased after 6 h. In diseased human livers PECAM-1 was detectable along the sinusoids, within inflammatory infiltrates and within fibrotic septa. Neither in acutely nor chronically diseased human livers was an obvious increase of PECAM-1 immunoreactivity detectable. CONCLUSIONS: PECAM-1 is expressed by SECs. In contrast to ICAM-1, PECAM-1 transcript level is not enhanced during liver damage.

Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.

Previous in vitro studies indicated that hepatic stellate cells (HSC) and rat liver myofibroblasts (rMF) have to be regarded as different cell populations of the myofibroblastic lineage with fibrogenic potential. Employing the discrimination features defined by these studies the localization of HSC and rMF was analyzed in diseased livers. Normal and acutely as well as chronically carbon tetrachloride-injured livers were analyzed by immunohistochemistry and by in situ hybridization. In normal livers HSC [desmin/glial fibrillary acid protein (GFAP)-positive cells] were distributed in the hepatic parenchyma, while rMF (desmin/smooth muscle alpha actin-positive, GFAP-negative cells colocalized with fibulin-2) were located in the portal field, the walls of central veins, and only occasionally in the parenchyma. Acute liver injury was characterized almost exclusively by an increase in the number of HSC, while the amount of rMF was nearly unchanged. In early stages of fibrosis, HSC and rMF were detected within the developing scars. In advanced stages of fibrosis, HSC were mainly present at the scar-parenchymal interface, while rMF accounted for the majority of the cells located within the scar. At every stage of fibrogenesis, rMF, in contrast to HSC, were only occasionally detected in the hepatic parenchyma. HSC and rMF are present in normal and diseased livers in distinct compartments and respond differentially to tissue injury. Acute liver injury is followed by an almost exclusive increase in the number of HSC, while in chronically injured livers not only HSC but also rMF are involved in scar formation.

Transforming growth factor-beta1 stimulates the synthesis of basement membrane proteins laminin, collagen type IV and entactin in rat liver sinusoidal endothelial cells.

BACKGROUND/AIMS: It is suggested that during fibrogenesis as well as during carcinogenesis of the liver, the hepatic microvascular phenotype is transformed from sinusoids - which lack a basement membrane--into continuous capillaries which rest on a basement membrane. As transforming growth factor (TGF)-beta1 seems to be the most effective mediator in the stimulation of matrix protein synthesis, we were interested in the modulation of basement membrane proteins collagen type IV, laminin, and entactin expression by TGF-beta1 in liver sinusoidal endothelial cells (SECs), especially since a stimulation of the synthesis of collagen type IV but not of entactin and laminin by TGF-beta1 has been demonstrated in a fibrosarcoma cell line. METHODS: The synthesis of the basement membrane (BM) proteins entactin, laminin, and collagen type IV and of the extracellular matrix (ECM) proteins tenascin and fibronectin with or without TGF-beta1--stimulation was analyzed by immunostaining, immunoprecipitation of endogenously labeled proteins and Northern blot analysis of total RNA extracted from freshly isolated or cultured SECs from rat or guinea pig livers. Furthermore, SECs were isolated from acutely and chronically CCl4-damaged rat livers and were analyzed for matrix protein expression. RESULTS: SECs were adherent 24 h after isolation and formed confluent monolayers on day 4 of primary culture. Specific immunoprecipitates and specific transcripts for the BM proteins entactin, laminin, and collagen type IV and for ECM proteins tenascin and fibronectin were detectable in freshly isolated or cultured SECs. The synthesis of all tested BM proteins and ECM proteins was stimulated at least 3-fold by TGF-beta1. In SECs isolated after CCl4-induced acute and chronic liver damage, increased levels of matrix protein transcripts were detectable. CONCLUSIONS: The stimulation of the synthesis of all BM-proteins by TGF-beta1 in vitro and the accumulation of ECM transcripts in SECs isolated from CCl4-treated livers, suggests that SECs are involved in the formation of a basement membrane during the "capillarization" of the sinusoids during liver disease.

Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are considered the principal matrix-producing cells of the damaged liver. However, other cell types of the fibroblast lineage that have not yet been characterized are also involved in liver tissue repair and fibrogenesis. METHODS: We established cultures of cells of the fibroblast lineage, termed rat liver myofibroblasts, and analyzed their phenotypical and functional properties in comparison with HSCs. RESULTS: HSCs and rat liver myofibroblasts were discernible by morphological criteria and growth behavior. Prolonged subcultivation of rat liver myofibroblasts was achieved, but HSCs were maintained in culture at maximum until second passage. HSCs were characterized by expression of glial fibrillary acidic protein, desmin, and vascular cell adhesion molecule 1, which were almost completely absent in rat liver myofibroblasts. For synthetic properties, HSCs and rat liver myofibroblasts displayed mostly overlapping properties with 4 striking differences. The complement-activating protease P100 and the protease inhibitor alpha(2)-macroglobulin were preferentially expressed by HSCs, whereas interleukin 6-coding messenger RNAs and the extracellular matrix protein fibulin 2 were almost exclusively detectable in rat liver myofibroblasts. CONCLUSIONS: The data show that morphologically and functionally different fibroblastic populations, HSCs and rat liver myofibroblasts, can be derived from liver tissue. HSCs may not represent the single matrix-producing cell type of the fibroblast lineage in the liver.

Expression and regulation of cell adhesion molecules by hepatic stellate cells (HSC) of rat liver: involvement of HSC in recruitment of inflammatory cells during hepatic tissue repair.

Hepatic stellate cells (HSC), a pericyte-like nonparenchymal liver cell population, are regarded as the principal matrix-synthesizing cells of fibrotic liver. They might also play a role during liver inflammation. The present study analyzed (i) expression of cell adhesion molecules (CAMs) mediating cell infiltration, like intercellular adhesion molecule-1 (I-CAM-1) and vascular cell adhesion molecule-1 (V-CAM-1), by HSC, (ii) CAM regulation in HSC by growth factors and inflammatory cytokines, and (iii) CAM expression in situ during liver inflammation, using immunochemistry and Northern blot analysis. I-CAM-1 and V-CAM-1 expression was present in HSC in vitro and in cells located in the sinusoidal/perisinusoidal area of normal liver. Growth factors, eg, transforming growth factor-beta1, down-regulated I-CAM-1- and V-CAM-1-coding mRNAs and stimulated N-CAM expression of HSC. In contrast, inflammatory cytokines like tumor necrosis factor-alpha reduced N-CAM-coding mRNAs, whereas induction of I-CAM-1- and V-CAM-1-specific transcripts increased several fold. In situ, messengers specific for I-CAM-1 and V-CAM-1 were induced 3 hours after CCl4 treatment (thereby preceding mononuclear cell infiltration starting at 12 hours), were expressed at maximal levels 9-12 hours after CCl4 application, and decreased afterwards. I-CAM-1 and V-CAM-1 immunoreactivity increased in a linear fashion starting 3 hours after CCl4-induced liver injury, was detected in highest amounts at 24-48 hours characterized by maximal cell infiltration, and returned to baseline values at 96 hours. Interestingly, the induction/repression of CAM-specific messengers paralleled the time kinetics of tumor necrosis factor-alpha transforming growth factor-beta1 expression in injured liver. HSC might be important during the onset of hepatic tissue injury as proinflammatory elements and might interact with I-CAM-1 and V-CAM-1 ligand-bearing cells, namely lymphocyte function-associated antigen-1- or Mac-1/very late activation antigen-4-positive inflammatory cells, thereby modulating the recruitment and migration of mononuclear cells within the perisinusoidal space of diseased livers.

Sinusoidal intercellular adhesion molecule-1 up-regulation precedes the accumulation of leukocyte function antigen-1-positive cells and tissue necrosis in a model of carbontetrachloride-induced acute rat liver injury.

The mechanisms leading to the infiltration of inflammatory cells into the liver and to liver cell necrosis remain undefined. To elucidate this process, the present work analyzes the kinetics of the expression of intercellular adhesion molecule-1 (ICAM-1) and the accumulation of inflammatory leukocyte function antigen-1 (LFA-1)-positive cells in relation to the appearance of hepatocellular necrosis in the model of acute carbontetrachloride (CCl4)-induced liver injury. ICAM-1- and LFA-1-immunoreactivity was analyzed in normal livers and in livers obtained 3, 6, 9, 12, 18, 24, 48, and 72 hours after CCl4-administration, as well as in liver cells isolated 3, 6, 9, 12, 18, and 24 hours after CCl4-administration. Total RNA extracted from livers and cells was used for Northern blot analysis. ICAM-1-positivity, which was detected along the sinusoids in normal rat livers, increased 3 to 6 hours after CCl4-administration and finally accumulated in the necrotic areas (24 to 48 hours post-administration). ICAM-1 steady-state mRNA levels in liver tissue increased 3 to 6 hours after CCl4-treatment and returned to normal levels at 48 hours after treatment. Increased amounts of ICAM-1-specific transcripts could be observed in isolated sinusoidal endothelial cells and in hepatocytes as early as 3 to 6 hours after CCl4-administration. In normal rat livers, a few LFA-1-immunoreactive cells were present around the vessel walls. Starting 12 hours after CCl4-administration, the number of LFA-1-immunoreactive cells increased around the vessel walls and along the sinusoids, accumulating later in the necrotic areas. In accordance, the number of mononuclear phagocytes isolated from the liver increased 12 hours after CCl4-treatment. These data demonstrate an early up-regulation of ICAM-1 in liver cells and the accumulation of LFA-1-expressing cells prior to the development of necrotic areas. The up-regulation of ICAM-1 and accumulation of inflammatory cells seem to be critical for the induction of CCl4-induced hepatotoxicity.

The complement-activating protease P100 is expressed by hepatocytes and is induced by IL-6 in vitro and during the acute phase reaction in vivo.

Ra reactive factor, a lectin present in the sera of a wide variety of vertebrates, is composed of mannan-binding proteins and a serine protease termed P100, which is known to activate complement. Using differential mRNA display technology to study the "activation"-dependent gene expression of hepatic stellate cells (HSC), we partially cloned a cDNA encoding the rat homolog of P100, which displayed 94% and 88% homology to mouse and human P100 cDNA, respectively. In the rat P100, specific transcripts 5.4, 4.0, and 3.3 kb in size were detected in major amounts in normal liver, but were absent or near the detection limit in other organs. Among the different liver cell populations studied during primary culture, P100-specific transcripts of 4.0 kb were prominent in HSC and present in hepatocytes and hepatoma cells, whereas Kupffer cells and sinusoidal endothelial cells were P100-negative. In addition to 4.0-kb mRNA, freshly isolated hepatocytes also contained transcripts of 5.4 and 3.3 kb, which were down-regulated during primary culture. In situ hybridization of normal liver tissue confirmed the in vitro data in that P100 was expressed by hepatocytes and nonparenchymal liver cells, which probably represent HSC. In vitro P100 steady-state mRNA levels of hepatocytes were stimulated by IL-6 and/or dexamethasone. During the acute phase reaction induced by turpentine injection, P100 steady-state mRNA levels were up-regulated in rat liver. The data demonstrate that: (a) the liver is the primary site for P100 expression in the rat; (b) HSC and hepatocytes appear to represent the cellular sources; and (c) P100 steady-state mRNA levels are up-regulated by the acute phase mediators IL-6 and dexamethasone in vitro and during the acute phase reaction in vivo, suggesting that P100 represents a novel, positive acute-phase gene in the rat.

[Ranking of color-coded duplex ultrasonography (CCDU) in the staging of breast tumors]. / Stellenwert der farbkodierten Duplexsonographie (FKDS) bei der Dignitätsbestimmung von Mammatumoren.

PURPOSE: of the study was to detect vessels indirectly in conspicuous focal findings of the breast by means of measurement of blood flow by using colour-coded duplex sonography (CCDS) and to examine whether additional criteria can be defined to determine the pathological relevance of breast tumours. MATERIALS AND METHODS: In a prospective study 149 patients were investigated, in whom either palpable breast lesions had been noticed or focal findings in imaging diagnostics. The perfusion in the focus and its surroundings (if present) was demonstrated and documented by means of CCDS. The investigation results were compared and contrasted with the histological findings after the operation. RESULTS: Indirect detection of the vessels depended on the size of the malignant tumours and was successful in 41.7% of cases with a tumour diameter of < or = 1 cm, in 90% of cases with a diameter of 1-2 cm and in 100% of cases with a diameter of > 2 cm. Carcinoma and metastases could be detected with a sensitivity of 87.5% and overall specificity of 56.9%. No typical perfusion pattern allowing appraisal of the pathologic relevance was seen. Postoperative scars did not show perfusion in any case. CONCLUSIONS: CCDS is of limited suitability only for determining the relevance of breast tumours, but provides additional diagnostic information especially on T1 tumours having a diameter between 1 and 2 cm. According to our results obtained so far, CCDS appears to be reliable and informative in differential diagnosis of tumour recurrence and an older scar.

Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair.

Ito cells (lipocytes, stellate cells) are regarded as the principle matrix-producing cell of the liver and have been shown recently to express glial fibrillary acidic protein, an intermediate filament typically found in glia cells of the nervous system. The present study examines 1) whether Ito cells of rat liver express central nervous system typical adhesion molecules, namely, neural cell adhesion molecule (N-CAM), in a cell-type-specific manner and 2) whether N-CAM expression is affected by activation of Ito cells in vitro and during rat liver injury in vivo. As assessed by reverse transcriptase polymerase chain reaction, Northern blotting, Western blotting, and immunocytochemistry of freshly isolated and cultivated hepatic cells, N-CAM expression was restricted to Ito cells and was absent in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Ito cells expressed predominantly N-CAM-coding transcripts of 6.1 and 4.8 kb in size and 140-kd isoforms of the N-CAM protein, which was localized on the cell surface membrane of Ito cells. In parallel to glial fibrillary acidic protein down-regulation and smooth muscle alpha-actin up-regulation, N-CAM expression was increased during in vitro transformation of Ito cells from resting to activated (myofibroblast-like) cells and by the fibrogenic mediator transforming growth factor-beta 1. By immunohistochemistry, N-CAM was detected in normal rat liver in the portal field as densely packed material and in a spot as well as fiber-like pattern probably representing nerve structures. However, after liver injury, N-CAM expression became detectable in mesenchymal cells within and around the necrotic area and within fibrotic septae. In serially cut tissue sections, N-CAM-positive cells were predominantly co-distributed with smooth muscle alpha-actin-positive cells rather than glial fibrillary acidic protein-positive cells, especially in fibrotic livers. The experimental results illustrate that N-CAM positivity in the liver cannot be solely ascribed to nerve endings as, among the different types of resident liver cells, Ito cells specifically express N-CAM in vitro and presumably in vivo. In addition to its role as potential cell-type-specific marker protein for activated Ito cells, the induction of N-CAM expression might illustrate a mechanism by which mesenchymal cell proliferation might be inhibited when tissue repair is concluded.