[Changing clinical image of celiac sprue in childhood]. / Menící se klinický obraz celiakální sprue v detském veku.

BACKGROUND: Celiac sprue is considered to be the second most common chronic disease in childhood after allergic diseases. At present, the prevalence of this disease is stated as high as approximately 1% in inhabitants of the North America and Western Europe. Aetiology of celiac sprue is multifactorial as it is in other chronic diseases. Pathogenetically, it is an autoimmune disease whose main autoantigene is the tissue transglutaminase. It affects those individuals carrying HLA-DO2 or HLA-DO8 gene and those who were exposed to wheat gliadine or similar amino acids (prolamines) in rye and barley. The purpose of the present study was to find out whether clinical manifestation of celiac sprue changed in our group of patients in course of 23 years. METHODS AND RESULTS: In forty-eight children celiac sprue was diagnosed according to histopathological and histochemical findings in the small intestine mucosa. In the children examined within 5 years in 1982-1987, main clinical symptom of celiac sprue was diarrhoea. In the control group of children examined within 7 months in 2004-2005, intestinal and extraintestinal symptoms in celiac sprue were equally distributed; the so-called sleeping forms occurred too. CONCLUSIONS: It is discussed what are the trigger mechanisms and the possible danger of celiac sprue manifestations and why celiac sprue is diagnosed in the older children at present time compared with the past years.

Changes of E-cadherin and beta-catenin in human and mouse intestinal tumours.

An immunohistochemical analysis of E-cadherin and beta-catenin was performed in human colorectal cancer as well as in surrounding normal intestinal tissue. We also analysed the expression of these two cell adhesion proteins in transgenic Apc1638N mice as a model of human familial adenometous polyposis syndrome. In the normal intestinal mucosa of both species, E-cadherin and beta-catenin were localized along the lateral plasma membranes of epithelial cells. In intestinal tumour cells, however, they were also present in the cytoplasm. The expression of both proteins was reduced in human and mouse tumours. The pattern of their distribution was frequently heterogenous with strongly positive cells in a mosaic of negative ones. Further, E-cadherin and beta-catenin expression did not correlate to the Duke's staging of tumours and therefore neither can be used as prognostic criteria.

[Factors which affect cytodiagnosis in gynecology]. / Faktory, které ovlivnují gynekologickou cytodiagnostiku.

The incidence of invasive carcinoma of the cervix in the Czech Republic is higher than in Western Europe. The only effective test for early discovery of the precancerous lesions is cytodiagnostics--"PAP" smears. The study points to preventing mistakes in the cytodiagnostic process and stresses quality control according to the International Academy of Cytology (I.A.C.) and the European Federation of Cytological Societies (E.F.C.S.).

Aspiration cytology from the pouch of Douglas at hysteroscopy.

Eighty-seven fine needle aspiration cytological samples from 29 patients suffering from irregular perimenopausal uterine bleeding were evaluated. Aspiration cytology was performed prior to hysteroscopy, after distension of the uterine cavity and finally after uterine curettage. In this paper, cytological examination of fluid from the pelvic content in women with benign endometrial findings is compared with that of patients with adenocarcinoma. Endometrial curettage influenced the cell content of the cytologica specimens.

Different expression of some molecular markers in sporadic cancer of the left and right colon.

The expression of cytoplasmic c-erbB2, epidermal growth factor receptor (EGFR), proliferating cell nuclear antigen (PCNA) and dipeptidylpeptidase IV (DPP IV) was significantly higher in sporadic cancer of the right than of the left colon. In addition, cytoplasmic c-erbB2 displayed the same difference in the adjacent (less than 2 cm) and distant (more than 5 cm from the tumour margin) mucosa. The findings cannot be related to Dukes staging. It is suggested that different ontogenic development of the right (from the midgut) and the left (from the hindgut) colon may be a possible explanation. Therefore, data on the expression of different molecular markers in colorectal cancer and surrounding mucosa should always be supplemented by data on tumour location.

Cytologic detection of cervical and endometrial carcinoma with other genital tract involvement.

OBJECTIVE: To investigate the possibility of a correct cytologic diagnosis of cervical and endometrial carcinoma with other genital organ involvement. STUDY DESIGN: From uteri removed during hysterectomy due to cervical (33 cases) and endometrial (44 cases) cancer, samples were taken by cytobrush or spatula from the ectocervix, endocervix and endometrium of uteri opened longitudinally. Smears and cytosediments were stained by the Papanicolaou polychrome method. Moreover, acid beta-galactosidase activity was demonstrated in serial cytosediments by the indigogenic method of Lojda. From quenched tissue samples taken from the same sites as those for cytology, a series of cryostat sections was prepared and stained by hematoxylin and eosin or azure A, or subjected to the reaction for acid beta-galactosidase. RESULTS: In 17 of 33 patients with cervical cancer, the same type of cancer was also found in smears of the endocervix and endometrium. In six patients the type of cancer was different. Of 44 patients with endometrial cancer, 16 had an endocervical malignancy of the same type. In seven cases the type of cancer was different. The reaction for acid beta-galactosidase helped in the differentiation between squamous (negative reaction in cancer cells) and cylindrocellular (positive reaction) cancer in cytologic preparations. CONCLUSION: Before treatment, it is necessary to determine if there is involvement of the endocervix in endometrial cancer and of the endometrium in cervical cancer. Routine cytologic examination supplemented by the reaction for acid beta-galactosidase proved to be useful for this purpose.

Expression of beta-catenins and cadherins by follicular dendritic cells in human lymph nodes.

In lymph nodes, dendritic cells form a complex meshwork and are linked by intercellular junctions. Intercellular junctions contribute to the integrity of lymphatic follicles and can potentially be affected by malignant processes in neighbouring B cells. We examined whether transmembrane molecules that constitute "adherens junctions" are present in follicular dendritic cells of normal human lymph nodes. We found that follicular dendritic cells but not interdigitating dendritic cells or sinus lining cells expressed cadherin molecules. Follicular dendritic cells also expressed beta-catenin but not vinculin. The cadherin molecules, which were identified in situ with the use of a monoclonal pan-cadherin antibody, were not recognized by antibodies to E-cadherin, N-cadherin or P-cadherin. Intrafollicularly, cadherins were clearly colocalized with beta-catenins, in a dot-like fashion. We also detected intrafollicular expression of desmogleins and desmosomal plaque proteins. These findings indicate the presence of desmosomes within the dendritic meshwork. However, pan-cadherin reactivity was not only colocalized with desmoglein immunoreactivity that was abundantly present. Immunoprecipitation showed that pan-cadherin reactivity was absent in fractions of desmosomal plaque proteins or pan-desmogleins. We speculate that complexes of cadherins of an unknown subclass and beta-catenins form non-desmosomal intercellular junctions in the intrafollicular dendritic meshwork.

Are intestinal tumours in Apc+/-mice a suitable model of colorectal carcinoma in man?

In snap frozen sections of the duodenum, jejunum, ileum, the right and left colon of APC+/-mice mucosubstances, activities of brush border glycosidases and proteases, immunoreactivity of sucrase and activities of some enzymes of pericellular proteolysis were studied. Multiple adenomas (tubular or tubulovillous) the numbers of which decreased in the aboral direction occurred in the small intestine. Two tubulovillous adenomas with dysplastic nuclei but with no invasion were found in the right colon. The morphological and histochemical findings resembled those of human colorectal tumours. Activities of brush border enzymes and sucrase immunoreactivity were decreased to various extent or were not present at all. The findings fluctuated even within the same section. Activities of enzymes of pericellular proteolysis were slightly increased in comparison with non affected mucosa. This model is suitable and deserves further studies.

Processing of free cells for electron microscopy using a fibrin clot.

A method of fibrin clot preembedding permitting the simple and gentle handling of free cells to be processed for electron microscopy is described. This technique is particularly useful for immunocytochemical techniques such as Lowicryl and thawed croysection approaches and represents a convenient alternative to procedures such as gelatine or agar preembeddings.

Long-term action of potassium bromide on the rat thyroid gland.

Male rats fed by a standard diet with determined of bromine and iodine content were exposed to a 133-day oral administration of KBr (100, 200, 400 mg Br-/l drinking water). Their thyroid glands showed increased growth of the epithelial cells reflected by a microfollicular rearrangement of the parenchyma due to proliferation of very small follicles with a low or zero content of colloid. Morphometric analysis of thyroids of Br(-)-exposed animals revealed a significant decrease in the volume of intrafollicular colloid and marked increase in the number of the smallest follicles (areas up to 100 and 100-300 micron 2). In addition, the nuclei of thyrocytes showed an increased number of mitoses. The vascularization was increased as well. In the blood plasma of the Br(-)-exposed animals the T4 concentration was significantly decreased in dependence on the bromine concentrations. Thyroglobulin immunoreactivity in the colloid of Br(-)-exposed animals decreased after administration of 400 mg Br-/l drinking water. Increasing concentrations of Br- in the drinking water caused an increased bromine concentration in the thyroid, a decreased iodine content and a decreased I/Br molar ratio. The changes in the rat thyroid caused by long-term administration of 100 mg Br-/l were similar to hyperplastic parenchymal goitre and were comparable to those induced in previous experiments by the same bromine concentration administered over a 16- and 66-day period respectively.

Expression of the proliferating cell nuclear antigen (PCNA) in the rat thyroid gland after exposure to bromide.

Analysis of expression of the proliferating cell nuclear antigen (PCNA) was used to determine the presumed hyperplastic character of morphological changes in the rat thyroid evoked by bromide administration. Male rats fed by a standard diet with determined iodine and bromine content were given potassium bromide. Control animals received no bromide. Experimental animals were given 10, 50 or 100 mg Br- per 11 drinking water for 16 and 66 days, or 100, 200, 400 mg Br-/l drinking water for 133 days. The thyroids of treated animals showed activation of growth of the epithelial follicular component as well as diffuse and focal microfollicular rearrangement of the parenchyma with higher follicular cells accompanied by a decrease of the amount of colloid even at low bromine concentrations (10-100 mg Br-/l drinking water). Using the PCNA-LI index (PCNA-positive nuclei.100/total number of follicular cell nuclei in the section), immunohistochemical analysis of PCNA in the nuclei of the follicular cells was carried out in parrafin sections. The index was significantly higher in bromide exposed animals (P < 0.01) and correlated well with the histological changes, with bromide concentration and with a increased mitotic activity of the follicular cells. PCNA analysis showed that morphological changes resembling a parenchymatic goitre reflect a microfollicular rearrangement of the thyroid of rats exposed to bromide and have the character of hyperplasia owing to the increased mitotic activity of the follicular epithelium.

[Difficulties in cytological diagnosis of changes in the endocervix]. / Obtíze cytologické diagnostiky zmen v endocervixu.

1. Cytological diagnosis of changes of the cylindrical epithelium in the endocervix has not been elaborated in such detail as the cytodiagnosis of squamous epithelium of the ectocervix. 2. The original proposal and experience of Vooijs of 1995 for the classification of incipient endocervical lesions corresponds best to histological practice. 3. Cytodiagnosis of endocervical changes will be more accurate if we use for evaluation the cellular pattern from the endocervix, demonstrated on the plates. 4. If we find any cellular abnormalities of the cylindrical epithelium, we recommend a procedure as used in lesions of squamous epithelium a) Low-grade abnormalities should be followed up primarily by cytological methods. If the lesion persists or progresses, histological examination is indicated. Examination of an endocervical lesion is usually not accessible to colposcopy. b) High-grade abnormalities and in particular those with the above described architecture, should be subjected to histological examination. 5. In mixed cylindrical and squamous lesions the procedure depends on the more serious lesion. In mixed lesions or suspected mixed lesions it is an advantage to use histochemical examination for betagalactosidase, the method described by Lojda, which differentiates safely the squamous and cylindrical component and in particular its tendency of malignant reversal.

Sucrase-isomaltase and other brush border glycosidases in colorectal tumors.

Sucrase-isomaltase (SI), trehalase (T) and lactase-beta-glucosidase (LG) activities were assessed histochemically in samples of colorectal adenomas (11 tubular, 12 tubulovillous, 10 villous) and 30 adenocarcinomas obtained by biopsy during colonoscopy or from specimens removed by surgical intervention. Small samples of tumor tissue, tissue of the transitional zone and of macroscopically normal mucosa were quenched in heptan cooled in an acetone-dry ice mixture. Cryostat sections, transferred to non-precooled slides and in some cases to semipermeable membranes, were dried and subjected to the histochemical reactions for SI, T and LG. Sucrose, 2-naphthyl, 6-Br-2-naphthyl, and 5-Br-4-Cl-3-indoxyl alpha-D-glucosides, trehalose, and 5-Br-4-Cl-3-indoxyl-beta-D-fucoside were used as substrates. Sections of jejunal biopsies with normal activities of brush border glycosidases were used as controls. From samples of 5 adenomas, 5 adenocarcinomas and collected rests of jejunal biopsies with a normal finding 10% (w/vol) homogenates in 2% Triton X-100 were prepared. Homogenates were frozen and thawed 3 times and their supernatants subjected to isoelectric focusing on polyacrylamide gel plates. Zymograms were developed with the same methods as for the detection of alpha-glucosidases in sections. In no colorectal tumor LG was present. SI was found in 70% adenocarcinomas, 50% villous, 25% tubulovillous and 19% tubular adenomas when the method with sucrose, glucose oxidase-peroxidase and 3,3'-diaminobenzidine was used. Hardly discernible traces of activity were found in tumors with azo-coupling reactions applied at pH 5, 6 and 6.5. No reaction was detected with the indigogenic method applied at pH above 6.0. However, jejunal biopsies displayed very strong reactions confined to the brush border of enterocytes under the same conditions. A strongly positive reaction was seen in 7 of 12 tumors investigated recently when the indigogenic reaction was applied at pH below 6.0 (particularly at pH 5.0). In this case the deposition of indigo was due to membrane and lysosomal alpha-glucosidases of the tumor cells and lysosomal alpha-glucosidase of macrophages and leukocytes. These findings were corroborated by zymograms. T was detected in the same tumors as SI; its activity was lower, however. SI activity in colorectal tumors is a useful, but not general marker of these tumors.

The use of substrates with 7-amino-3-trifluoromethylcoumarine (AFC) leaving group in the localization of protease activities in situ.

A method for the localization of activities of proteases using substrates with 7-amino-3-trifluoromethylcoumarine (AFC) leaving group is described. 0.1 ml of 5-20 mMol solution of the respective substrate (Gly-Pro-AFC, Ala-Pro-AFC, Z-Ala-Arg-Arg-AFC, Z-Gly-Arg-Arg-AFC, Z-Gly-Gly-Arg-AFC, D-Val-Leu-Lys-AFC) in dimethylsulfoxide or dimethylformamide was added to 0.9 ml of 0.1 M Tris-HCl buffer, pH 7.4-7.8 or 0.1 M cacodylate buffer, pH 5-5.5. In the case of Z-Ala-Arg-Arg-AFC (cathepsin B substrate) 15 mM EDTA and 12 mM dithiothreitol were added. 7 mM amiloride or 2 mg/1 ml aprotinin were used as inhibitors with Z-Gly-Gly-Arg-AFC (urokinase substrate) and with D-Val-Leu-Lys-AFC (plasmin substrate). Substrate solutions were mixed with an equal amount of 2% agar solution in distilled water or in the respective buffer the pH of which was adjusted according to the pH optimum of the enzyme to be demonstrated. The agar solution was kept in a water bath at a temperature of 50-60 degrees C. After careful mixing, the substrate solution in agar was poured into a cylindrical vessel closed with a semipermeable membrane (Nephrophan) on which unfixed cryostat sections were mounted. 1-5 mM AFC solution in dimethylsulfoxide or dimethylformamide instead of the substrate was used as the control. Quenched samples of rat kidney and jejunum, biopsies of human jejunal mucosa, and of colorectal and uterine tumors were employed for the preparation of sections. After gelification of the medium in a refrigerator the vessels with sections were incubated in the dark at 37 degrees C for 0.5-several h. The reaction was controlled in a fluorescence microscope with an epiillumination adjusted to the FITC fluorescence and documented. A yellowish green fluorescence depicts sites where AFC was set free (sites with enzyme activity). When the reaction reached the required intensity the membranes were cut off, transferred to glass slides, mounted in glycerol, observed and photographed immediately (due to the solubility of AFC in glycerol). An acceptable cellular localization was achieved. The method with AFC substrates can be recommended for comparative biochemical and histochemical studies of proteases using the same substrate and for cases in which no other reliable procedure for the localization of the respective enzyme activity is available (e.g. urokinase, plasmin).

Histochemical study on xanthine oxidase activity in the normal rabbit cornea and lens and after repeated irradiation of the eye with UVB rays.

In the normal rabbit cornea and lens the activity of xanthine oxidase, an enzyme belonging to oxidases generating reactive oxygen species (ROS), is present in the corneal epithelium as well as endothelium and lens epithelium. Repeated irradiation of the eyes with UVB rays (5 min 1 x daily, for 1 to 4 days) caused a gradual increase of xanthine oxidase activity, particularly in the corneal epithelium. Application of catalase, a scavenger of hydrogen peroxide, to the eye surface during the irradiation diminished the increase of xanthine oxidase activity. On the contrary, the pretreatment of the rabbit eyes with 3-aminotriazole, an inhibitor of catalase, for 3 days before the irradiation enhanced the increase of xanthine oxidase activity. In comparison to untreated eyes, protracted irradiation of the eyes with UVB rays (up to 10 days) caused a decrease of xanthine oxidase activity in the same cell layers of the cornea and lens. It is suggested that xanthine oxidase is involved in the generation of ROS in the anterior eye segment during early irradiation of the eyes with UVB rays and participates in its damage. Prolonged repeated irradiation of the eye (5 min 1 x daily for 5 to 10 days) caused a decrease of xanthine oxidase activity in the cornea and lens which is attributed to profound damage of the whole anterior eye segment.

[Histochemical differentiation of lymphatic organs during intrauterine development]. / Histochemische Differenzierung der lymphatischen Organe während der intrauterinen Entwicklung.

The activities of some enzymes (phosphatases, esterases peptidases, and dehydrogenases) were studied. The activities of the enzymes in the youngest embryos (4-8 weeks) were relatively low (ALP-activity on membranes of the primitive capillary endothelium, ACP and UE activities in the cytoplasm of the epithelial reticulum cells). The DPP IV activity was observed on membranes of the epithelial reticulum cells. The activities of GPDH and SDH were relatively strong in the epithelial cells of the primitive cytoreticulum. In the fetal period, the activities of the studied enzymes are gradually increasing. The immunohistochemical findings demonstrate that 95% of the lymphocytes in the thymus anlage (in the fetal period) are T-lymphocytes. The enzyme activities in other lymphatic organs (spleen, lymph nodes) were significantly lower.

The appearance of active plasminogen activator of urokinase type (u-PA) in the rabbit anterior eye segment irradiated by UVB rays. A histochemical and biochemical study.

Repeated irradiation of the rabbit eye with UV rays of 312 nm wavelength (UVB) evoked the appearance of active plasminogen activator of urokinase type (u-PA) in the anterior eye segment. Using histochemistry, active u-PA appeared first in the corneal epithelium followed by the corneal endothelium, inflammatory cells in the corneal stroma and the lens epithelium. With a semiquantitative fluorescent method active u-PA was also found in the tear fluid and aqueous humour. UV rays of 365 nm wavelength (UVA) under the same conditions did not cause the appearance of active u-PA in the anterior eye segment.

The damaging effect of UV rays below 320 nm on the rabbit anterior eye segment. II. Enzyme histochemical changes and plasmin activity after prolonged irradiation.

Prolonged irradiation of the rabbit eyes with UVB rays (312 nm) caused serious enzymatic disturbances in the cornea and lens and the development of an inflammatory reaction in the whole anterior eye segment, particularly in the cornea. In the corneal stroma many inflammatory cells with high activities of acid glycosidases and lysosomal proteases were present. This was accompanied with significantly elevated plasmin activity in the tear fluid (1.6 IU/ml). Plasmin appeared also in the aqueous humour (0.8 IU/ml). For the treatment of these changes catalase (1 mg/1 ml saline), aprotinin (100 micrograms/1 ml saline) and catalase-aprotinin mixture (1:1) were applied on the eye surface during irradiation. The catalase-aprotinin mixture was most efficient and decreased plasmin activity in the tear fluid and diminished disturbances of the anterior eye segment. Obviously both, active oxygen species and elevated plasmin activity in the tear fluid contribute to the damage of the anterior eye segment and development of intracorneal inflammation after irradiation of the eye with UVB rays.

Effect of domperidone-induced hyperprolactinemia on selected immune parameters in healthy women.

Domperidone, anti-emetic drug, given to healthy female volunteers, induced an elevation of plasma prolactin (PRL) concentration with the peak in 1-4 h. The release of prolactin had a transient stimulating effect on theophylline sensitive T lymphocytes and on concanavalin A induced mitogenic activity, suggesting an enhanced activity of T suppressor lymphocytes. The relative number of CD4+ lymphocytes decreased markedly one hour after domperidone administration and returned to normal values within 2 h (that means 3 h after taking the drug). The number of lymphocytes positive for dipeptidyl peptidase IV exhibited similar transient increase and normalization of activity. No change was observed in the number of CD8+ lymphocytes. The production of interferon by leukocytes treated with Newcastle disease virus was found to be significantly increased 2 h after domperidone administration. The results suggest that prolactin can selectively stimulate some functions of cellular immunity as well as the release of cytokines (IFN). The present study may contribute to the understanding of the role of the immune system in endogenous hyperprolactinemia.