In vitro human osteoblast and extracellular matrix changes after transforming growth factor beta 1 treatment.

AIMS: Normal bone tissue is characterised by a balancing of osteoblast and osteoclast activity. The activity and differentiation of these cells are regulated by vitamins, hormones and cytokines. The action of these factors on bone tissue cells depends on the composition and mineralisation of extracellular bone matrix. In particular, transforming growth factor beta 1 (TGFbeta1) acts on collagen fibres, glycosaminoglycan secretion and on the enzymes correlated to the turnover of glycosaminoglycans. The normal functions of bone tissue also depend on its mineralisation, which is highly altered in the process of uraemia. METHODS: In this study, we analysed in vitro the effect of transforming growth factor beta on osteoblast proliferation, collagen synthesis and glycosaminoglycan secretion with 3H-thymidine, 3H-proline or 3H-glucosamine incorporation, and on enzymes, such as beta-N-acetyl-D-glucosaminidase and beta-glucuronidase, involved in extracellular matrix turnover. Moreover, phosphatase alkaline activity and osteocalcin related to mineralisation of extracellular matrix were determined. RESULTS: Our data show that TGFbeta1 significantly decreases 3H-thymidine and 3H-proline incorporation and increases (p < or = 0.01) extracellular sulphated glycosaminoglycan synthesis. It also increases osteocalcin levels, phosphatase alkaline, beta-N-acetyl-D-glucosaminidase and beta-glucoronidase activities. CONCLUSION: TGFbeta1 changes the synthesis of extracellular matrix components by osteoblasts. These variations favour the action of cytokine and osteoclasts. Since the TGFbeta1 accumulates in bone tissue and increases during uraemia, with due limitations this action leads to an imbalance between synthesis and degradation and could explain bone alterations in uraemic patients.

Morphometric analysis of primary graft non-function in liver transplantation.

AIMS: Primary graft non-function (PNF) is a life-threatening condition that is thought to be the consequence of microcirculation injury. The aim of the present study was to assess, with a computerized morphometric model, the morphological changes at reperfusion in liver biopsy specimens from patients who developed PNF after liver transplantation. METHODS AND RESULTS: Biopsy specimens were obtained at maximum ischaemia and at the end of reperfusion. Morphology included many stereological parameters, such as volumes of all parenchymal components, surface density, size distribution and mean diameter of hepatocytes. Other variables examined were intensive care unit stay, degree of steatosis, serum liver function tests and ischaemic time. In the postoperative period, the PNF group showed elevated serum levels of alanine transferase, decreased daily rate of bile production and prothrombin activity. Blood lactates were significantly higher in the PNF group than in a control group. When comparing groups, the volumetric parameters related to hepatocytes and sinusoids and the surface densities of the hepatic cells showed an inverse relationship. At the end of reperfusion, in PNF group the volume fraction of hepatocyte cytoplasm was decreased; in contrast, the volume fraction of sinusoidal lumen was markedly increased. The cell profiles showed the same inverse trend: the surface density of the parenchymal border of hepatocytes was decreased in PNF when compared with the control group, while the surface density of the vascular border was increased. In the PNF group, the surface density of the sinusoidal bed was directly correlated with alanine transferase, daily rate of bile production, prothrombin activity and cold ischaemic time. CONCLUSIONS: The alterations in hepatic architecture, as demonstrated by morphometric analysis in liver transplant recipients that developed PNF, provide additional information that may represent useful viability markers of the graft to complement conventional histological analysis.

Ornithine decarboxylase, polyamines and CD11b expression in HL-60 cells during differentiation induced by retinoic acid.

Polyamines (PA) and retinoic acid affect mammalian cell growth, differentiation and apoptosis. Retinoic acid induces granulocytic differentiation of mieloid cell lines and, during this process, is responsible for the expression of CD11b, a surface antigen. In this study we investigate the effects of retinoic acid on HL-60 cells, monitoring ornithine decarboxylase (ODC) activity (enzyme rate of PA), putrescine (PUT), spermidine (SPD), spermine (SPM) levels, CD11b myeloid surface marker differentiation, cell cycle, and apoptosis. ODC activity and PUT levels are correlated with mieloid cell differentiation induced by retinoic acid treatment. Only the ODC/PUT ratio is connected with retinoic acid treated HL-60 cells. Treated cultures show a decrease of proliferation and a cell block in the G0/G1 phase, with consequent diminished S phase. The G0/G1 and S phases are significantly related to ODC activity and to PUT and SPD behavior, whereas in differentiating condition only the decrease of PUT is related to the S phase. CD11b expression, stimulated by retinoic acid treatment, is associated with the SPM trend. Total PA behavior agrees with apoptotic cell increase after 96 h of stimulation. Our data show that retinoic acid treatment modifies ODC activity and the turnover of PA. PUT, SPD and SPM, therefore, have a different role, and may be involved in the differentiative/apoptotic program of retinoic acid treated HL-60 cells.

[Pancreas transplantation. Experience in the first 50 patients at the Niguarda Hospital of Milan: clinical outcome and open questions]. / Il trapianto di pancreas. Esperienza dell'Ospedale Niguarda sui primi 50 pazienti: dubbi e certezze.

AIM: Personal experience in 50 patients who underwent combined pancreas-kidney transplantation (PKT), with particular reference to mortality and surgical complications is reported. METHODS: Between October 1993 and December 2001, 50 adult patients (36 males and 14 females), mean age 37 years (range 25-60), with chronic renal failure, and Insulin Dependent Diabetes Mellitus (IDDM), underwent 54 pancreas transplantation (4 patients retransplanted) and 52 kidney transplantation (2 patients retransplanted). Different surgical procedures have been employed during the period of 9 years. All patients underwent the same immunosuppressive regimen; the mean length of follow-up was 49 months. During the follow-up, 30 out of 43 patients who maintained a good graft function fulfilled a questionnaire about their quality of life following the criteria of the Medical Outcome Study (MOS). RESULTS: All patients became euglycemic immediately after the surgical procedure. One patient died post-operatively due to pulmorary thromboembolism after pancreas retransplantation for acute venous thrombosis; 1 other patient died 9 months after the procedure for acute myocardial infarction. Four patients developed acute venous thrombosis. All these patients underwent pancreas retransplantation, but 3 of these patients who survived the procedure lose the graft function for chronic rejection within 1 year. Fourteen patients showed acute rejection, 7 patients CMV infection. Three patients showed hyperchloremic acidosis, 12 patients bronchopulmonar infection and 7 patients urinary infection. Among surgical complications anastomotic fistula in 6 patients was also recorded. Five patients out of 50 lose the pancreatic graft function. After 1 from PKT, 83% of patients who fulfilled a questionnaire were strongly satisfied about their quality of life. No patients developed de novo tumors following chronic immunosuppression. The 5-year survival for patient, kidney and pancreas was 95.6%, 93.4% and 84.7% respectively. CONCLUSIONS: Our experience in 50 patients submitted to PKT shows no graft loss due to acute rejection. Surgical complications (acute venous thrombosis) and chronic rejection are the most important factors leading to graft loss. A graft in "head-up" position, a short portal vein of the graft, a "no-touch technique" during pancreas retrieval can be some of the most important factors which can reduce the rate of surgical complications. Combined kidney-pancreas transplantation showed in our experience a low mortality rate and a moderate incidence of morbidity and should be considered, at the moment, the treatment of choice for patients with renal failure and IDDM.

Massive haemoptysis after living donor liver transplantation.

A 27 year old man with hereditary haemorrhagic telangiectasia who developed progressive liver dysfunction underwent living related right lobe transplantation. Pulmonary arteriography did not reveal arteriovenous malformation or abnormal intrapulmonary venous channels. The postoperative course was characterised by persistent hypoxaemia and respiratory failure developed. On day 6, a massive haemoptysis developed and the patient died shortly thereafter. The native liver showed a nodular pseudocirrhotic transformation, with highly dilated and irregularly interconnected vein-like or arterial-like structures in the fibrous septa. Pathological examination of both lungs showed irregular thickening of the wall of the arteries, secondary to eccentric and/or concentric myointimal hyperplasia. This case suggests that massive haemoptysis can develop even when arteriovenous malformations are undetectable by pulmonary arteriography, and it questions the role and the appropriateness of living donor liver transplantation in high risk patients.

Chick embryo back skin organ and fibroblast cultures. Extracellular matrix changes induced by dialysate fluid and uraemic toxins in relation to proliferation and differentiation processes.

AIM: During uraemia, an increase of middle molecules and acetylpolyamines occurs. In vitro the middle molecules produce cell toxicity, while the acetylpolyamines stimulate cell proliferation and differentiation. These phenomena are related to protein and extracellular glycosaminoglycan production. The aim of the present study was to evaluate the activity of dialysate, dialysate fluid and the chromatographic peaks of dialysate fractionated by G-15 Sephadex column on chick embryo skin development. METHODS: We evaluated the effects of protein and glycosaminoglycan synthesis using monolayer and organotypic cultures. RESULTS: Our data show that dialysate, chromatographic peak II, and 2 x 10(-8)M N1-acetylspermine cause inhibition of chick embryo skin culture development. On the contrary, 10(-8)M N-acetylornithine and dialysate fluid increase protein and extracellular glycosaminoglycan synthesis, whereas chromatographic peak I does not reveal differences when compared to controls. CONCLUSIONS: These extracelluar changes are related to cell proliferation and feather formation in chick embryo organotypic culture. Moreover, the pH changes of culture medium do not influence the biological action of acetylpolyamines and dialysate fluid on protein and extracellular glycosaminoglycan synthesis. Cell death in the presence of N1-acetylspermine, dialysate and peak II appears unrelated to the apoptotic process. The data show that acetylpolyamines, dialysis fluid, dialysate and chromatographic peaks act on fibroblasts, and are able to modify glycosaminoglycan synthesis. The organotypic cultures of chick embryo back skin could represent a model for studying the modifications of the extracellular matrix induced by these substances.

Kaposi's sarcoma in liver transplant recipients: morphological and clinical description.

The clinical course and outcome of 5 adult patients who underwent orthotopic liver transplantation (OLT) and developed Kaposi's sarcoma (KS) is reported. From October 1986 to July 2000, a total of 459 patients underwent 499 OLTs at our hospital. The immunosuppressive regimen consisted of cyclosporine, azathioprine, prednisone, and antithymocyte globulin. Tacrolimus was administered only in selected patients. Five patients developed KS, and the pathological diagnosis was established months 9 to 23 after OLT. Four of 5 patients died of KS, surviving 0 to 6 months after pathological diagnosis. The fifth patient, with KS confined to the skin, is disease free 6 months after diagnosis. All patients were treated with reduction of immunosuppressive therapy and/or chemotherapy. Retrospective molecular investigation by polymerase chain reaction for human herpesvirus type 8 DNA detected specific viral sequences in histological specimens of tissues involved by KS. In our experience with adult liver transplant recipients, we registered a slightly lower prevalence of KS compared with other reported data, but observed a high rate of graft involvement and mortality.

Rapid disappearance of hepatic adenoma after contraceptive withdrawal.

We present the case of a 25-year-old woman who developed a large central liver adenoma after 8 years of continuous oral contraceptive use. The first diagnosis was made by ultrasonography, after a rise in plasmatic gamma-glutamyl-transpeptidase and alkaline phosphatase levels was noted. Withdrawal of the oral contraceptive was followed by shrinkage of the adenoma, with complete disappearance 9 months after the diagnosis. Hepatic adenoma (HA) still presents problems in terms of differential diagnosis and clinical management. There are reports of complete or partial regression of an HA after discontinuation of oral contraceptives, but they are poorly documented. To our knowledge, a patient with such rapid disappearance of a large HA has never been reported.

Liver transplantation in man: morphometric analysis of the parenchymal alterations following cold ischaemia and warm ischaemia/reperfusion.

Ischaemia and reperfusion phases represent critical events during liver transplantation. The purpose of this study was to describe morphological alterations of both vascular and parenchymal compartments after ischaemia and reperfusion and to evaluate the possible relationship between morphometric parameters and biochemical/clinical data. Three needle biopsies were drawn from 20 patients who underwent orthotopic liver transplantation. The first biopsy was taken before flushing with preservation solution, and the second and the third to evaluate respectively the effects of cold ischaemia and of warm ischaemia/reperfusion. Biopsies were examined by an image analyser and morphometric parameters related to the liver parenchyma were evaluated. At the second biopsy we observed a decrease of the endothelium volume fraction while the same parameter referred to the sinusoidal lumen achieved a peak value. The hepatocytes showed a lower surface parenchymal/vascular sides ratio. This parameter was reversed at the end of the reperfusion phase; furthermore the third biopsy revealed endothelial swelling and a decreased volume fraction of the sinusoidal lumen. The results quantify the damage to the sinusoidal bed which, as already known, is one of the main targets of cold ischaemia; warm ischaemia and reperfusion accentuate endothelial damage. The end of transplantation is characterised by damage chiefly to parenchymal cells. Hepatocytes show a rearrangement of their surface sides, probably related to the alterations of the sinusoidal bed. In addition, the fluctuations of morphometric parameters during ischaemia/reperfusion correlate positively with biochemical data and clinical course of the patients.

Is lymphocele in renal transplantation an avoidable complication?

BACKGROUND: This study evaluated the impact of surgery in the incidence of lymphocele after kidney transplantation (KTx). METHODS: A prospective randomized study was conducted during a 6-year period on a group of patients undergoing KTx and operated on by the same surgeon (CVS). A total of 280 patients undergoing KTx were randomly allocated into two groups: (1) group C (control group) was 140 patients who were submitted to KTx with standard technique: implantation of the kidney in the controlateral iliac fossa with vascular anastomoses on the external iliac vessels; and (2) group M (modified technique group) was 140 patients who underwent a modified technique with a cephalad implantation of the graft in the ipsilateral iliac fossa and vascular anastomoses in the common iliac vessels. Both groups were comparable for age, cold ischemia time, incidence of rejection episodes, presence of adult polycystic kidney disease, and source of donor graft. RESULTS: Group M showed an incidence of lymphocele production (3 patients, 2.1%) significantly lower than group C (12 patients, 8.5%). Eight patients (1 in group M and 7 in group C) required surgical treatment by peritoneal fenestration. No allograft or recipient was lost as a result of fluid collection but the hospitalization was shorter in group M than in group C. CONCLUSIONS: A cephalad implantation of the renal graft in the ipsilateral iliac fossa has been associated with a lower incidence of lymphocele, probably because vascular anastomoses on the common iliac vessels cause less lymphatic derangement than those performed on the external iliac vessels.

Is thrombocytopenia in liver failure dependent on an inadequate synthesis of thrombopoietic stimulating factor by the liver?

Thrombocytopenia in liver diseases has been considered secondary to portal hypertension or to a consumption mechanism associated with fibrinolytic disorders. Several conflicting clinical reports and evidence from experimental models justify the above mentioned mechanisms only in part. We propose that thrombocytopenia may be consequent to an inadequate synthesis of a factor stimulating thrombopoiesis produced by the liver.

Effect of superoxide dismutase on liver ischemia-reperfusion injury in the rat: a biochemical monitoring.

Oxygen free radicals have been implicated in the pathogenesis of postischemic liver injury. High-dose superoxide dismutase (SOD), a radical scavenging enzyme, has been investigated in a rat model of liver ischemia reperfusion by biochemical monitoring. Blood vessels to the median and left lobe were clamped for 1 h and then reperfusion was allowed. The indices used were serial venous blood levels of AST, ALT, calcium, and ATP determination in liver tissue. In SOD-treated animals (7,5000 U i.v.) a significant attenuation of the rise in enzyme levels was observed as well as the absence of the decrease in calcium level in the early phase after reperfusion as compared with control rats, and furthermore ATP restoration was significantly increased.

Liver arterialization prevents thrombocytopenia after portacaval shunt in rats.

A low platelet count is a common finding in liver cirrhosis. Clinical practice has shown that a variable number of cirrhotic patients in whom portasystemic shunting procedures have been performed does not recover from thrombocytopenia: this observation questions the role that portal hypertension may have in maintaining the low platelet count. We have previously described the appearance of thrombocytopenia in rats submitted to portacaval shunt 1 month after the operation. In the present study we have investigated a supposed protective influence of a good liver function in maintaining a normal thrombocytopenia: 56 male Wistar rats were divided into 4 groups: group A (15 rats) sham-operated; group B (16 rats) submitted to portacaval shunt (PCS); group C (17 rats) submitted to PCS plus arterialization of the portal stump by the right renal artery, and group D (8 rats) submitted to PCS plus right nephrectomy. Group B (PCS) and D (PCS plus right nephrectomy) showed a marked thrombocytopenia, whereas group A (sham-operated) and C (PCS plus liver arterialization) evidenced a normal platelet count. These results strongly support the hypothesis that a low platelet count can ensue during a chronic liver disease in the absence of portal hypertension and that restoration of the hepatic blood flow can prevent thrombocytopenia.