Abnormal deposition of collagen around hepatocytes in Wilson's disease is associated with hepatocyte specific expression of lysyl oxidase and lysyl oxidase like protein-2

BACKGROUND/AIMS: Lysyl-oxidases catalyze the oxidation of lysine residues in collagen and elastin thereby promoting their polymerization. We have studied here the expression of four lysyl-oxidases in normal and diseased human liver. METHODS: The expression of the different lysyl-oxidases in paraffin embedded liver sections was studied using in-situ hybridization and immunohistochemistry. The enzymatic activity of lysyl-oxidase like protein-2 (Loxl2 or LOR-1) using a previously described lysyl-oxidase assay. RESULTS: We have found that the four lysyl-oxidases which we examined are not significantly expressed in the normal liver. By contrast, Wilson's disease and primary biliary cirrhosis (PBC) patients express lysyl-oxidase (Lox) and lysyl-oxidase like protein-2 (Loxl2 or LOR-1) in hepatocytes, and the expression is accompanied by collagen deposition around the hepatocytes. Lysyl-oxidases are also expressed in additional fibrotic liver diseases such as hepatitis B and C but in these diseases the expression is confined to the fibrotic lesions and collagen does not accumulate around hepatocytes. We have found that Loxl2 is able to oxidize lysine residues of collagen, and behaves in that respect similarly to Lox. The copper chelator D-penicillamine inhibits Loxl2 induced oxidation of collagen but the Lox inhibitor beta-aminopropionitrile did not inhibit the oxidation using a BAPN concentration at which Lox activity was completely inhibited. Loxl2 also catalyzed the oxidation of cell surface proteins on HepG2 hepatoblastoma cells and inhibited their proliferation. CONCLUSIONS: Upregulation of Lox and Loxl2 in hepatocytes of Wilson's disease and PBC patients may contribute to liver damage by various mechanisms. The upregulation of Lox and Loxl2 in Wilson's disease could perhaps be utilized for diagnostic purposes since their expression is up-regulated in hepatocytes even before the onset of fibrosis.     

Development of the Croatian model of organ donation and transplantation

During the past ten years, the efforts to improve and organize the national transplantation system in Croatia have resulted in a steadily growing donor rate, which reached its highest level in 2011, with 33.6 utilized donors per million population (p.m.p.). Nowadays, Croatia is one of the leading countries in the world according to deceased donation and transplantation rates. Between 2008 and 2011, the waiting list for kidney transplantation decreased by 37.2% (from 430 to 270 persons waiting for a transplant) and the median waiting time decreased from 46 to 24 months. The Croatian model has been internationally recognized as successful and there are plans for its implementation in other countries. We analyzed the key factors that contributed to the development of this successful model for organ donation and transplantation. These are primarily the appointment of hospital and national transplant coordinators, implementation of a new financial model with donor hospital reimbursement, public awareness campaign, international cooperation, adoption of new legislation, and implementation of a donor quality assurance program. The selection of key factors is based on the authors'' opinions; we are open for further discussion and propose systematic research into the issue.Transplantation is a widely accepted and successful life-saving treatment providing the best therapeutic benefit for hundreds of thousands of patients (1). Unfortunately, many people die while awaiting an organ transplant. A global shortage of organs available for transplantation raises many bioethical concerns, including the dilemma how to allocate limited resources to an unlimited number of needs and thus offer a fair and equal access to organ transplantation to all patients. Great efforts have been made to increase organ donation worldwide, but with only a moderate success in most of the countries. In contrast with this general trend, Croatia has recently experienced a boom in organ donation and transplantation. In 2011, Croatia had the highest rates of utilized cadaveric donors, kidney transplantations, and liver transplantations in the world (2-5) (Figure 1).Open in a separate windowFigure 1The number of kidney transplantations and the number of patients on the waiting list in Croatia between 2008 and 2011.Remarkably, only one decade ago, Croatia was lagging far behind other European countries with a low donation rate (2.7 donors per million population [p.m.p.] in 2000). The continuous improvement of the organization of the Croatian transplant program resulted in a steadily growing donor rate, which reached the highest level in 2011, with 33.6 utilized donors p.m.p (2). We analyzed the factors that might have contributed mostly to this great success.     

Resistance training decreases matrix metalloproteinase-2 activity in quadriceps tendon in a rat model of osteoarthritis

BackgroundOsteoarthritis (OA) is a degenerative disease that induces peri-articular tissue degradation. OA induces an imbalance between synthesis and degradation of the extracellular matrix components in favor of catabolic events, promoting pathological remodeling and involving degradative enzymes, such as matrix metalloproteinases (MMPs).ObjectiveThis study aimed to investigate the effects of 8-weeks resistance training (RT) on MMP-2 activity in the quadriceps tendon and patellar tendon in an OA model.MethodsTwenty-four Wistar rats were randomly divided into six groups: Control, Exercise, Sham, Sham with Exercise, OA, and OA with Exercise (OAE). The OA model was performed by anterior cruciate ligament transection surgery on the left knee. The 8-week RT consisted of climbing a 1.1-m vertical ladder three times per week with progressive weights secured to the animals’ tails. MMP-2 activity was analyzed by zymography.ResultsThe OAE group displayed lower pro, intermediate, and active MMP-2 activity in the quadriceps tendon compared with the OA group (p < 0.05). For the patellar tendon, there was no significant difference between the OAE group compared with the other groups (p > 0.05) for pro, intermediate, and active MMP-2 activity. Moreover, MMP-2 activity differed between tissues, the OA and OAE groups presented lower pro, intermediate, and active MMP-2 activity in the quadriceps tendon compared to the patellar tendon.ConclusionRT induced down-regulated MMP-2 activity in the quadriceps tendon. RT is a potential therapeutic approach to minimize the deleterious effects of extracellular matrix degeneration.     

Hydroxychloroquine decreases Th17-related cytokines in systemic lupus erythematosus and rheumatoid arthritis patients

OBJECTIVES:

Hydroxychloroquine is an antimalarial agent that has been used in systemic lupus erythematosus and rheumatoid arthritis treatment for many years. Recently, novel mechanisms of action have been proposed, thereby broadening the therapeutic perspective of this medication. The purpose of this study was to evaluate the immunomodulatory activity of hydroxychloroquine in T helper 17 (Th17) cytokines in healthy individuals and patients.

METHODS:

Eighteen female patients with systemic lupus erythematosus (mean age 39.0±12.9 years) and 13 female patients with rheumatoid arthritis (mean age 51.5±7.7 years) were recruited from Universidade Federal de Pernambuco-Brazil. The patients were included after fulfilling four classification criteria for systemic lupus erythematosus or rheumatoid arthritis from the American College of Rheumatology. After being stimulated with phorbol 12-myristate 13-acetate and ionomycin in the absence or presence of different concentrations of hydroxychloroquine, the interleukin 6, 17 and 22 levels were quantified with an enzyme-linked immunosorbent assay in culture supernatants of peripheral blood mononuclear cells from healthy individuals and patients.

RESULTS:

We demonstrated that in peripheral blood mononuclear cells from healthy volunteers and in systemic lupus erythematosus and rheumatoid arthritis patients, there was a significant reduction in the IL-6, IL-17 and IL-22 supernatant levels after adding hydroxychloroquine.

CONCLUSIONS

Our in vitro results demonstrated that hydroxychloroquine inhibits IL-6, IL-17 and IL-22 production and contributes to a better understanding of the mechanism of action of this medication.     

Urothelial Cancer in Patients with Endemic Balkan Nephropathy (EN) after Renal Transplantation

Goal. Analysis of the incidence of urothelial cancer and outcome of treatment in patients with Endemic Balkan Nephropathy (EN) after renal transplantation. Methods. From January 1985 until October 2006, 550 kidney transplantations (389 cadaveric) and 5 combined kidney and pancreas transplantations were performed in University Hospital Center Rijeka. In only 6 (1.1%) of 555 transplant recipients, EN was diagnosed as the original kidney disease, based on medical history, clinical findings, and laboratory results, but without pathohistologic verification. All patients with EN received the first renal transplant from a cadaver. Patients' mean age at transplantation was 50.3 ±15.9 yrs, five patients (83.3%) were male. The incidence of malignant tumors in all 555 transplant recipients was analyzed, with an emphasis on the incidence of urothelial cancer and outcome of treatment in the group of patients with EN. Results. During posttransplant follow-up period, malignancy was diagnosed in 27 (4.9%) out of 555 transplant recipients. Skin cancer was diagnosed in 7 patients (1.3%), followed by cancer of the urinary tract in 6 patients (1.1%) and breast cancer in 3 patients (0.5%). In 3 of 6 patients with EN, urothelial cancer was diagnosed, resulting in the death in two patients. In the third patient, urothelial cancer showed a high affinity for recurrence, and besides the strong reduction of immunosuppressive therapy, repeated surgical treatment was needed. Conclusions. Patients with EN show a high incidence of urothelial cancer after renal transplantation. A thorough nephro-urological evaluation is needed before transplantation, and a careful follow-up is required afterward to ensure an early diagnosis of malignancy. Preventive nephroureterectomy is recommended.     

Population Pharmacokinetic Modeling to Describe the Total Plasma and Free Brain Levels of Fluconazole in Healthy and <Emphasis Type="BoldItalic">Cryptococcus neoformans</Emphasis> Infected Rats: How Does the Infection Impact the Drug’s Levels on Biophase?

Purpose

The present work aimed to evaluate the influence of experimental meningitis caused by C. neoformans on total plasma and free brain concentrations of fluconazole (FLC) in Wistar rats.

Method

The infection was induced by the administration of 100 μL of inoculum (1.10⁵ CFU) through the tail vein. Free drug in the brain was assessed by microdialisys (μD). Blood and μD samples were collected at pre-determined time points up to 12 h after intravenous administration of FLC (20 mg/kg) to healthy and infected rats. The concentration-time profiles were analyzed by non-compartmental and population pharmacokinetics approaches.

Results

A two-compartmental popPK model was able to simultaneously describe plasma and free drug concentrations in the brain for both groups investigated. Analysis of plasma and μD samples showed a better FLC distribution on the brain of infected than healthy animals (1.04?±?0.31 vs 0.69?±?0.14, respectively). The probability of target attainment was calculated by Monte Carlo simulations based on the developed popPK model for 125 mg/kg dose for rats and 400–2000 mg for humans.

Conclusions

FLC showed a limited use in monotherapy to the treatment of criptoccocosis in rats and humans to value of MIC >8 μg/mL.

     

In vivo monosomy 3 detection of posterior uveal melanoma: 3-year follow-up

Background Monosomy 3 is a highly specific marker for poor prognosis in posterior uveal melanoma. Unfortunately, cytogenetic prognostication is limited to enucleated eyes or resected tumors. The aim of this study was to evaluate mid-term natural history and safety of in vivo detection of chromosome 3 status in posterior uveal melanomas undergoing plaque brachytherapy. Methods A 25-gauge transscleral fine needle aspiration biopsy (FNAB) was performed in 32 eyes affected by posterior uveal melanoma undergoing plaque brachytherapy, just before applying the radioactive plaque. Sampled material underwent fluorescence in situ hybridization (FISH) with centromeric probes for chromosome 3. All patients had a follow-up of at least 36 months. Results Mean follow-up was 47.1 ± 8.5 months. Mean largest basal diameter and mean thickness of the tumors were 12.5 ± 2.7 mm and 8 ± 2.3 mm respectively. FNAB yielded sufficient material in 26 of 32 cases (81.2%). Adequacy of the sample ranged from 91.1% (ciliary body tumors) to 76.8% (choroidal tumors). Seventeen cases had monosomy 3 (65.3%). No correlation was found between monosomy 3 and tumor dimensions or location (ciliary body vs choroidal tumors). No early and mid-term local complications were documented. Seven patients (21.8%) died during follow-up: five (15.6%) of them died due to metastatic disease (all had monosomy 3 tumors). Conclusions Posterior uveal melanomas may be adequately and safely sampled, by intra-operative transscleral FNAB, to detect in vivo monosomy 3. The authors have no financial interest in the subject of this paper. The authors have full control of all primary data and they agree to allow Graefe’s Archive for Clinical and Experimental Ophthalmology to review their data if requested.