Matrix metalloproteinase activity assays: Importance of zymography.

INTRODUCTION: Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of degrading extracellular matrix, including the basement membrane. MMPs are associated with various physiological processes such as morphogenesis, angiogenesis, and tissue repair. Moreover, due to the novel non-matrix related intra- and extracellular targets of MMPs, dysregulation of MMP activity has been implicated in a number of acute and chronic pathological processes, such as arthritis, acute myocardial infarction, chronic heart failure, chronic obstructive pulmonary disease, inflammation, and cancer metastasis. MMPs are considered as viable drug targets in the therapy of the above diseases. METHODS: For the development of selective MMP inhibitor molecules, reliable methods are necessary for target validation and lead development. Here, we discuss the major methods used for MMP assays, focusing on substrate zymography. We highlight some problems frequently encountered during sample preparations, electrophoresis, and data analysis of zymograms. RESULTS AND DISCUSSION: Zymography is a widely used technique to study extracellular matrix-degrading enzymes, such as MMPs, from tissue extracts, cell cultures, serum or urine. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and therefore helps to understand the widespread role of MMPs in different pathologies and cellular pathways.

Active management of post-renal transplantation BK virus nephropathy: preliminary report.

OBJECTIVE: To assess the efficacy of leflunomide, intravenous immunoglobulins, and ciprofloxacin as active treatment of postrenal transplant BK virus nephropathy (BKVN) in graft outcome at 1 year. PATIENTS AND METHODS: Renal transplant recipients with positive results of 2 BK virus polymerase chain reaction tests of urine and blood underwent graft biopsy to confirm BKVN. If BKVN was diagnosed, antimetabolite therapy (mycophenolate mofetil or azathioprine) was changed to leflunomide therapy accompanied by a course of immunoglobulin and oral ciproflxacin. RESULTS: Of 18 patients evaluated, 72% were men. Nine patients received cadaveric organs, with a mean of 3.6 HLA mismatches. All patients received induction thereapy (61% thymoglobulin), and 61% received antirejection therapy before BKVN was diagnosed. Maintenance immunosuppression therapy was primarily with prednisolone (94%); mycophenolate mofetil, 2 g/d (94%); and tacrolimus (61%). At baseline, mean (SD) creatinine clearance was 35.6 (11.5) mL/min/1.73(2), which decreased to 29.3 (17.3) mL/min/1.73(2) at 1 year (P = .01). Patients were divided into 2 groups of 9 each according to creatinine clearance values. In group 1, baseline value was 44.5 (6.6) mL/min/1.73(2), compared with 25.36 (7.8) mL/min/1.73(2) in group 2, which decreased to 42.66 (12.8) mL/min/1.73(2) (P = .23) and 16.76 (9.0) mL/min/1.73(2) (P = .009), respectively, at 1 year. Three grafts (16.7%) were lost by the end of the study, all in group 2 (P = .03). CONCLUSION: Late diagnosis and intensive immunosuppression predispose to BKVN. Early active treatment of BKVN may improve graft outcome at 1 year posttransplantation.

Adenovirus gastroenteritis in Hungary, 2003-2006.

The incidence and type distribution of enteric human adenoviruses (HAds) among diarrheic children in south-western Hungary was investigated from 2003 through 2006. Laboratory studies were conducted using commercial antigen detection tests (latex agglutination or immunochromatography), polymerase chain reaction (PCR) amplification, single-strand conformation polymorphism, and sequencing and phylogenetic analysis of a conservative region of the HAd hexon gene. The overall rate of HAd infection in childhood gastroenteritis cases during the 4-year study was 8.1%, with a gradual decrease in detection rates from 11.7% in 2003 to 5.7% in 2006. Molecular studies of a subset of HAd-positive samples found that enteric HAd type 40 strains were identified only in 2003 and 2004, while HAd type 41 strains were identified throughout the 4-year study. Higher detection rates of non-enteric HAds was documented during the first half of the study period when latex agglutination was used in our laboratory for detection. Our study suggests that the choice of diagnostic method may profoundly influence the epidemiologic picture and disease burden attributed to enteric HAd infections.

Survival and causes of death in 366 Hungarian patients with systemic sclerosis.

OBJECTIVE: Survival analysis of a series of 366 consecutive patients with systemic sclerosis (SSc). METHODS: Clinical and laboratory data were evaluated from 1983 until 2005 using a standard protocol. The female/male ratio was 315/51. The mean (SD) age of the patients was 56.8 (12.2) years. The duration of disease was 12 (5-19) years with a median follow-up of 6 (3-12) years. RESULTS: Kaplan-Meier univariate analysis showed that renal, cardiac involvement, pigmentation disturbances, malabsorption, a forced vital capacity <50%, diffuse scleroderma, presence of early malignancy, anaemia, and increased erythrocyte sedimentation rate (ESR) were signs of unfavourable prognosis, whereas anti-centromere antibodies were indicators of a good survival. In the multivariate Cox proportional hazards model the presence of diffuse scleroderma, renal involvement, coexistence of a malignant disease, and increased ESR were poor independent prognostic signs. Elderly age at the onset of disease also caused an unfavourable outcome. A total of 86 SSc-related deaths were recorded during the follow-up. Of them, 65% were attributed to cardiorespiratory manifestation of disease. Tumour associated early death was found in 12 cases (14%). CONCLUSIONS: In addition to the well-known factors influencing the outcome (diffuse subset, internal organ involvements, and inflammatory signs), the coexistence of scleroderma with a malignancy also causes a poor outcome.

The dangers of intraosseous fibrosing agent injection in the treatment of bone cysts. The origin of major complications shown in a rabbit model.

Direct intraosseous injection of fibrosing agent is widely used in the treatment of aneurysmal bone cysts. The purpose of this study was to evaluate the consequences of fibrosing agent penetrating the medulla of bones. This may be the case when, by mistake, the fibrosing agent is administered into the medulla or when the wall of the cyst ruptures and fibrosing agent is able to drift into the medulla. Twelve rabbits were injected transcutaneously with a fibrosing agent directly into the proximal metaphysis of the tibia. Prior to injection 0.5 ml of liquid-like, bloody, intraosseal tissue was aspirated, then 0.5 ml of fibrosing agent was administered. Fibrosing agent was introduced slowly (20 s) to avoid overpressure. Nine rabbits (75%) died within minutes after the introduction of fibrosing agent. A full body roentgenogram was taken of each rabbit and the animals that died underwent autopsy to find the exact cause of death. Roentgenograms of the chest showed massive multiple pulmonary emboli confirmed in all lethal cases by the autopsy. This animal model was created to draw attention to the dangers of any leakage of the fibrosing agent into the medulla of bones.

Melanoma cells exhibit strong intracellular TASK-3-specific immunopositivity in both tissue sections and cell culture.

Amplification of the kcnk9 gene and overexpression of the encoded channel protein (TASK-3) seems to be involved in carcinogenesis. In the present work, TASK-3 expression of melanoma cells has been studied. For the investigation of TASK-3-specific immunolabelling, a monoclonal antibody has been developed and applied along with two, commercially available polyclonal antibodies targeting different epitopes of the channel protein. Both primary and metastatic melanoma cells proved to be TASK-3 positive, showing prominent intracellular TASK-3-specific labelling; mostly concentrating around or in the proximity of the nuclei. The immunoreaction was associated with the nuclear envelope, and with the processes of the cells and it was also present in the cell surface membrane. Specificity of the immunolabelling was confirmed by Western blot and transfection experiments. As TASK-3 immunopositivity of benign melanocytes could also be demonstrated, the presence or absence of TASK-3 channels cannot differentiate between malignant and non-malignant melanocytic tumours.

BK virus nephropathy in renal transplant recipients in Kuwait: a preliminary report.

INTRODUCTION: BK virus nephropathy (BKVN) is a significant cause of graft loss among renal transplant recipients. The treatment outcomes of BKVN have been variably reported in the literature. PATIENTS AND METHODS: We prospectively investigated BKV infection and BKVN among a population of renal transplant recipients with suspected BKV infection. The 42 subjects who all had acute allograft dysfunction, were categorized in three groups: those with clinical, laboratory, and histological findings that did not suggest acute rejection, drug toxicity, or obstruction (group 1, n = 24); those with findings that suggested probable acute cellular rejection but did not respond to antirejection treatment (group 2, n = 10); and those whose renal histology suggested BKVN (group 3, n = 8). Polymerase chain reaction analysis was done to detect BKV DNA in urine and blood samples from each subject. BKV DNA was detected in 19 (45%) urine samples with 11 of these subjects (26.1% of total) having BK viremia as well. RESULTS: No evidence of BKVN was detected histologically in seven subjects with isolated BK viruria, while the others proved to be JC virus infections. Among the 11 subjects with BK viremia, eight had BKVN based on renal histology at the time of diagnosis with BKV infection, while the other three subsequently developed histological features of BKVN. BKVN developed after 5.3 +/- 2.5 (2 to 44) months after transplantation. The serum creatinine at time of BKVN diagnosis was 158.9 +/- 58 (87 to 285) micromol/L. All subjects were initially treated with a 50% reduction in immunosuppressive drug doses. Further decreases in immunosuppression were performed in all patients with close monitoring of renal function. All subjects were followed up for a of 18.2 +/- 5 (12 to 26) months. Two grafts were lost not due to BKVN, and one patient was lost to follow-up during this period. The latest serum creatinine in eight recipients is 113 + 20 (81 to 138) micromol/L, which is better than the renal function at diagnosis. CONCLUSION: The prevalence of BKVN in suspected BKV infection was 26%. Although the study period was short (30 months), BK viremia strongly correlated with BKVN, which seemed to be successfully treated with reduction in immunosuppression.

The most severe forms of Perthes' disease associated with the homozygous Factor V Leiden mutation.

It has recently been postulated that thrombophilia may have a role in the aetiology of Perthes' disease. The published reports, however, remain conflicting. In this study a retrospective analysis of the coagulation parameters was made in 47 patients with Perthes' disease and the results compared with the clinical data. Five patients with Factor V Leiden mutation were found (10.6%) and surprisingly four of them had a homozygous pattern. These four patients showed the most severe form of the disease, Catterall group IV, with flattening of the entire epiphysis, involvement of the metaphysis, shortening and broadening of the femoral neck, trochanteric overgrowth and developed mushroom-shaped aspherical laterally displaced femoral heads in dysplastic acetabula. We would like to suggest that the homozygous form of Factor V Leiden mutation has some role in the clinical course of Perthes' disease and particularly its most severe form.

PM-SCL autoantibody positive scleroderma with polymyositis (mechanic's hand: clinical aid in the diagnosis).

This report concerns a 56-year-old-woman presenting PM-SCL autoantibody positive scleroderma with dermatomyositis (scleromyositis) and concomitant interstitial lung fibrosis. The recently observed overlapping syndrome is characterized by the presence of specific autoantibodies, HLA-type association and benign course. A new skin symptom ("mechanic's hands") predicts the disease, in particular the interstitial lung pathology, which is its most relevant internal manifestation.

Telescopic esophageal anastomosis: operative technique, clinical experiences.

The authors reconstructed the continuity of the alimentary tract by performing telescopic esophagogastrostoma in 208 patients who underwent either esophageal resection or total gastrectomy. The substance of the telescopic technique is to invaginate the distal section of any oral tubular organ to the lumen of an aboral tubular one and to fix it there. In case of telescopic esophageal anastomosis a 10-15 mm long esophageal segment is invaginated into the gastric tube or jejunum. A 3-4 mm wide serosal surface of the wall of the distal anastomosing organ straps the esophagus circularly. Ninety-six transthoracic and 12 transhiatal esophagectomies, 19 partial esophageal resections, four esophageal bypasses, and 77 total or extended total gastrectomies were reconstructed using telescopic anastomosis. Undisturbed healing could be observed in 67 patients after esophageal operations and in 46 patients of total gastrectomies. Anastomosis leakage occurred in 12 of 108 patients (11.1%) after cervical esophagogastrostomy. Leakage could be observed in 7 of 44 patients (15.9%) after end to side and in 5 of 64 patients (7.8%) in case of end to end esophago gastrostoma. There were no failures after two cases of cervical esophago-ileocolostoma and 21 of esophagogastrostomas in the thoracic position. All of the 59 intra-abdominal anastomoses healed without complication. Thirteen of 131 patients (9.9%) died after esophageal operations and four of 77 (5.2%) after gastrectomies. There were no mortal complications due to anastomotic leakage. The telescopic anastomosis is a safe alternative method in cases of total gastrectomy or esophageal operation.

[Experiences with an Invagination Technique for Esophagogastric End-to-End Anastomoses]. / Erfahrungen mit der Invaginationstechnik bei ösophago-gastrischen End-zu-End- Anastomosen.

80 patients with esophageal cancer underwent esophagectomy with the stomach as an esophageal substitute. For end-to-end esophagogastrostomy an invagination technique was used. We describe the technique and the results. An anastomotic leak was observed in 5 of 80 patients (6.3 %) in the total, in 5 of 64 cases with cervical anastomosis (7.8 %) and in none of the 16 patients with thoracic anastomosis. 6 of 80 patients (6.5 %) died, 5 due to nonsurgical complications and one patient due to a necrosis of the esophageal substitute. On the basis of these results we recommend the described anastomotic technique due to its simplicity and low anastomotic leakage rate.

Screening adenoviruses in stool samples: evaluation of a genus-specific monoclonal antibody based enzyme immunoassay.

To screen fecal samples for adenovirus antigens a genus-specific monoclonal antibody based enzyme immunoassay was developed. In a comparative analysis with commercial latex agglutination test, high sensitivity was demonstrated. The assay did not detect other viruses usually found in faeces suggesting its specificity. One hundred and eighty stool samples collected in Baranya County were tested and 13 (7.2%) of them showed reactivity. The application of our immunoassay combined with other, more sophisticated methods may help us to determine the serotype specificity of these adenovirus isolates and assess the importance of adenoviruses in viral gastroenteritis.

Detection of antibodies to polyomavirus SV40 in two central European countries.

Simian virus 40 (SV40) is significantly associated with some human cancers. However, the frequency of tumor-associated virus detection differs by geographic regions, so it is important to understand the status of SV40 infections in different populations. Poliovaccines potentially containing live SV40 were used in well-documented nationwide vaccination programs in Hungary and the Czech Republic that are reported here. We analyzed serum samples from periodic surveillance programs in those two countries for antibodies to SV40 using a specific plaque reduction neutralization assay. The prevalence of antibodies was between 1.3 and 8.7% in Hungary and from 1.0 to 4.0% in the Czech Republic. Females had a higher rate of antibodies than males, reaching in certain age groups 15.6% in Hungary and 8.3% in the Czech Republic. Antibodies to SV40 were found in similar proportions in both countries among persons not directly exposed to poliovaccines and subjects vaccinated in the era of SV40-free vaccines. Complexities and limitations of current serological approaches to epidemiological studies of SV40 in humans are discussed. These data suggest that SV40 may be present in these populations and emphasize the importance of follow-up studies to determine the pathogenesis of infections by this emerging human agent.

[Ischaemic stump necrosis after subtotal gastric resection - a rare complication]. / Eine seltene Komplikation - ischämische Stumpfnekrose - nach subtotaler Magenresektion.

The development of gastric stump necrosis after subtotal gastrectomy with Roux-en-Y loop reconstruction and splenectomy in a patient with gastric cancer is described. The patient's life could be saved by re-operations on the 12th and 50th postoperative day. Due to the patient's poor general condition and local peritonitis the gastric stump was removed and a catheter-jejunostomy was prepared by using the Roux-en-Y loop. The aboral end of the oesophagus was closed. To relieve the oesophageal stump a cervical mucoso-oesophagostoma was carried out. As the patient's general condition improved the reconstruction of the alimentary tract was performed by preparing a new Roux-en-Y loop oesophago-jejunostomy and by closing the mucoso-oesophagostoma.9 years after the operation the patient died of pulmonary metastases. The reasons for the delayed re-operation and the advantages of the cervical mucoso-oesophagostoma are explained.

[Role of surgery, its results and complications, in the combined treatment of primary gastric non-Hodgkin lymphoma]. / A mútét helye--eredményei és szövódményei--a primer non-Hodgkin gyomor lymphoma kezelésében.

During ten years 580 patients have been treated for gastric tumour in our department, 510 of them were operated on. Resection could be performed in 296 cases. 17 resections, 5.7 per cent of all were performed because of primary non-Hodgkin gastric lymphoma. No gastric lymphoma was found among the non-resected patients. The preoperative histological diagnosis was correct only in 8 cases. MALT origin could be proved in 5 patients. Synchronous adenocarcinoma and lymphoma was diagnosed in 2 patients. Staging was decided according to Lugano classification. There were six stage I, four stage II, and seven stage IV patients. 8 subtotal and 9 total gastrectomy was performed, 5 were extended and 2 were combined. R0 resection could be carried out in five stage I, two stage II and in one stage IV patient. We lost 2 patients in the postoperative period. Patients were treated with adjuvant chemotherapy (VEP, CHOP) except for 2 patients with low grade MALT lymphoma. The likelihood of one-year survival is 73 per cent, average two-year survival is 63 per cent. When the tumour is operable by total gastrectomy we suggest to perform splenectomy as well, despite of the fact that some postoperative complications can be related to it. We think it is reasonable to perform palliative resection in cases of locally extended stage IV tumours, which affect the patient's quality of life: to cease the pain, passage troubles, bleeding and to improve the conditions for adjuvant treatment.

Investigation of the alveolar macrophages and T lymphocytes in 15 patients with systemic sclerosis.

The cell distribution and function of alveolar macrophages and T lymphocytes were investigated in the bronchoalveolar lavage (BAL) of 15 patients with systemic sclerosis (SSc). In alveolar macrophages, both spontaneous and PMA-stimulated TNF-alpha production were increased in SSc. PMA-induced IL-6 production was also elevated. Spontaneous IL-6 excretion of scleroderma alveolar macrophages was similar to the controls. Yeast and C3b-coated yeast (opsonised yeast) phagocytosis, chemotaxis and Fc receptor activity of alveolar macrophages were normal. The proportion of CD3, CD4 and CD8 T-lymphocyte subsets in the BAL was similar to the control values. The lymphocyte blast transformation index of the non-adherent cells deriving from the BAL fluid was markedly decreased.

[Telescopic anastomosis in esophageal and gastric surgery]. / Süllyesztett anasztomózis a nyelócsó és gyomorsebészetben.

In 1991 we tried to adopt the telescopic anastomosis technique which had been used in bowel surgery made with the esophagus. In our method a 1.5-2 cm long stump of the distal esophagus is laid in the lumen end-to-side of the other organ (stomach, jejunum, large bowel). The oral the worst blood supplied part of the stomach has also removed and an end-to-end anastomosis has been made. We used this method in 125 patients until the end of 1997. In 89 cases during esophageal resections, and from it in 36 cases the anastomoses were made and-to-end, in 36 cases during total gastrectomies were made telescopic anastomosis leakage in the neck that healed after conservative treatment. We noticed advanced ischemic necrosis of the stomach used for replacement in 4 cases. Two of these patients healed after operative treatment. After gastrectomies all anastomosis healed without any complication. In 6 cases occurred anastomotic stenosis always after anastomotic leakage. 3 patients could be treated with dilatation, in 3 cases had to make reoperations due to stenosis. The authors emphasize that the method is simple, made in single lawyer, the sutures are not drained the lumen. They have not got strong opinion about the propensity for stenosis due to few cases.

First isolation of enteric adenoviruses type 41 from children with acute gastroenteritis in Hungary.

In a retrospective study, 122 stool specimens, collected 1989-1990, from children with gastroenteritis were found to contain adenoviruses. Twenty-nine adenoviruses of 50 randomly selected adenovirus-positive fecal samples were successfully propagated in Graham 293 cells and were typed by DNA restriction enzyme analysis with SmaI. Six strains were typed as adenovirus 41 (Ad41), and 23 strains as non-enteric adenovirus. All six adenoviruses type 41 had the same DNA pattern identical with that of the variant Ad41/D12. Ad40 viruses were not detected. Enteric Ad41 infections were observed only in males and were found throughout the year. This is the first report on isolation and typing of enteric adenoviruses 41 in Hungary.

Functional improvement after knee arthroplasty without resurfacing of patella.

There has been no universal agreement so far regarding the necessity of patellar resurfacing in total knee arthroplasty. As resurfacing has been reported to be associated with high incidence of complications, this practice has been avoided in our Department. A report is given on the analysis of the functional outcome of 60 knee arthroplasties without patellar resurfacing in 53 patients (7 bilateral) followed up for twelve to thirty months, with special regard to the functions closely related to patelloformal articulation. The underlying diagnosis was osteoarthritis in 78.3%, rheumatoid arthritis in 13.3%, and posttraumatic arthritis in 8.3% of the patients. Graded according to the modified knee-rating system of the Hospital for Special Surgery, excellent or good results were obtained in the case of 55 knees (91.6%) and the mean score improved from 53.6 points preoperatively to 82.6 points following arthroplasty. Subjective and objective functional assessment of stair climbing and transfer activities have shown no functional deficit attributed to the patellofemoral joint of the replaced knee.