Development of a new method for the preparation of an acellular allodermis, quality control and cytotoxicity testing.

Demand for use of acellular allodermis is high but commercially appropriate products are not used routinely because of very high price and limited availability. These facts did motivate us to prepare acellular allodermis using a new, simple and less expensive method. We have developed a original method for preparation of acellular allogeneic dermis based on action of a proteolytic enzyme in combination with distilled water. Hypotonic environment in comparison with SDS or Triton ansure no toxicity of the final product. Trials for determination of optimal trypsin concentrations, temperature and time of action were performed. According to our results, the use of 2.5% trypsin/EDTA solution overnight at +4 °C was proving to be optimal. The histology confirmed absence of cells in the prepared dermis. No toxicity of final acellular dermis was confirmed by three independent tests (agar diffusion test contact cytotoxicity test and grow curve). The prepared acellular dermis seems to be suitable not only for direct clinical use, but it can be used as a scaffold for cell cultivation as well.

Telocytes (interstitial Cajal-like cells) in human Fallopian tubes.

BACKGROUND: Telocytes represent a relatively newly discovered population of cells found within the various tissues and organs, including Fallopian tubes. It is presumed that telocytes could serve as a sensor of hormone levels or regulate activity of muscle peristaltic movement. METHODS: Tissue sections from anatomically different parts of Fallopian tubes of 48 women (age 48.8±9.1) were stained for the expression of five different antigens: c-kit (CD117), CD34, vimentin, podoplanin (D2-40) and Dog-1. RESULTS: Telocytes form a network associated with the smooth muscle cells. From the mentioned antibodies, only anti-c-kit (CD117) seems to be relatively selective specific to the telocytes, others react also with numerous other cells and tissue structures. Our results when using antibodies against podoplanin and Dog-1 are in dissonance with recent literature - with regards to our results, they are not suitable for detection of telocytes. CONCLUSION: Methods of immunohistochemistry are suitable for identification of telocytes in Fallopian tubes. C-kit (CD117) antigens are useful for routine identification of telocytes in histological sections. This antigen can be combined with CD34 or vimentin in cases of double staining immunohistochemistry.

Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization.

Sterilization is an important step in the preparation of biological material for transplantation. The aim of the study is to compare morphological changes in three types of biological tissues induced by different doses of gamma and electron beam radiation. Frozen biological tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10 % formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, especially in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradiation by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.

Structural changes of skin and amnion grafts for transplantation purposes following different doses of irradiation.

An important part of the preparation of biological material for transplantation is sterilization. The aim of our study was to assess the impact of ionizing radiation on three types of biological tissues and the impact of different doses on cells and extracellular matrix. Three types of frozen tissues (porcine skin xenografts, human skin allografts and human amnion) were divided into five groups, control and groups according to the dose of radiation to which these samples were exposed (12.5, 25, 35 and 50 kGy). The tissue samples were fixed by formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid schiff reaction and silver impregnation. The staining with hematoxylin and eosin showed hydropic degeneration of the cells of epidermis in xenografts by the dose of 12.5 kGy, in human skin it was observed by the dose of 35 kGy. The staining for elastic fibers revealed damage of fine elastic fibers in the xenografts dermis by the dose of 12.5 kGy, in the allografts by 35 kGy. Another change was the disintegration of basement membrane of epithelium, especially in the human amnion at the dose of 50 kGy. The silver impregnation visualized nuclear chromatin condensation mainly in human amnion at the dose of 12.5 kGy. Our results have shown that the porcine xenografts and human amnion were more sensitive to irradiation than the human skin. In the next phase of the project we will focus at more detailed changes in the tissues using immunohistochemical techniques.

[Acute coronary syndrome imitated by malignant melanoma or real acute coronary syndrome?]. / Malígnym melanómom imitovaný akútny koronárny syndróm alebo reálny akútny koronárny syndróm?

The article describes the case of 67 year  old woman, hospitalized at our department due to history of syncopes. At the time of admission the patient complained of back pain in the area of exophytic growing tumour. During the diagnostic process routine laboratory tests revealed severe normocytic hypochromic anemia, hypoproteinemia, elevation of inflammatory parameters (leukocytes, C reactive protein) and imaging methods (ultrasonography, computer  aided tomography) helped to formulate suspicion of disseminated cancerous disease (metastases in spleen, skeleton and lymph nodes). Patient was subsequently operated at the surgical ward, where she had removed the tumour on the back. After the surgery patients condition was complicated by chest pain accompanied by changes on electrocardiogram and dynamics in cardiospecific markers. We evaluated patients condition as developing acute coronary syndrome. The patient received conservative treatment of acute coronary syndrome, but despite the provided care she died. However, the autopsy revealed another immediate cause of death -  metastases of malignant melanoma of back.

[Parameters of non-specific immunity in umbilical cord blood in response to the stress of labor and surgery in healthy mothers and patients with late gestoses]. / Nekteré parametry nespecifické imunitní odpovedi na porodní a operacní stres v pupecníkové krvi matek zdravých a nemocných pozdní gestózou.

The authors investigated six proteins of the acute phase in the serum of mothers and in the umbilical blood of the foetus during spontaneous delivery and after Caesarean section in 40 women and their neonates. Half the women were in good health, half suffered from late gestoses. The patients' age, weeks of delivery and operation, the mean body weight of the neonates and evaluation of their condition after delivery by the Agpar score were comparable. The results were processed statistically. Mean levels of six proteins of the acute phase were assessed in the serum of the mother and the umbilical blood in all four groups of women and their neonates and percentage ratios of proteins in mother and foetus were assessed. On evaluation by the two-sample t-test in the umbilical blood of spontaneous deliveries in late gestoses significantly lower prealbumin values were found, and the same applied to Caesarean sections. Conversely a significant increase of A2MG levels and orosomucoid was found in the umbilical blood of children from mothers with late gestoses after Caesarean section. The values of the orosomucoid/prealbumin index according to Hollinshead were markedly elevated in late gestoses in the maternal blood as well as in the umbilical blood of the foetus.

[Acute phase proteins during labor and surgical stress in women with normal pregnancy and late gestoses]. / Bílkoviny akutní fáze pri porodním a operacním stresu u zen s normálním tehotenstvím a s pozdní gestózou.

The authors investigated within the framework of non-specific immunity reactions proteins of the acute phase during spontaneous delivery and Caesarean section up to the 5th day after delivery and on the 1st, 5th and 10th day after delivery in forty women, half of them healthy and half suffering from late gestoses. Half the women had spontaneous deliveries, half had deliveries by Caesarean section. The proteins of the acute phase reacted sensitively to the stress of childbirth during spontaneous delivery and their activation was inhibited by general anaesthesia in deliveries by Caesarean section. After spontaneous delivery the elevated proteins of the acute phase returned to baseline values by the 5th day, after operation their levels rose further up to the 5th day, orosomucoid to the 10th day after operation. In late gestoses with spontaneous delivery or by Caesarean section there were significantly elevated alpha-1-antitrypsin and alpha-2-macroglobulin and orosomucoid levels, whole in patients with late gestoses in the prealbumin levels were reduced. The elevated levels of proteinase inhibitors in late gestoses support the view on a possibly impaired coagulation equilibrium in late gestoses, and the high ceruloplasmin levels suggest an enhanced catecholamine breakdown during the stress alarm reaction.