Neoplastic disorders of prostate glands in the light of synchrotron radiation and multivariate statistical analysis.

The prostate gland is the most common site of neoplastic disorders in men. The pathogenesis of inflammatory cells, prostatic intraepithelial neoplasia (PIN) lesions, and prostate cancer is still under investigation. Inflammatory cells by producing free radicals are considered as major and universal contributors to cancerogenesis. PIN is regarded as a precursor lesion to prostate cancer or a marker signaling the vulnerability of the epithelium to neoplastic transformation [1]. Differentiation markers that are frequently changed in early invasive carcinoma are also changed in PIN lesions. In this study, prostate tissue samples obtained during surgical operation and classified as various disease states (inflammation, PIN lesions, and cancer) were examined. The samples were measured by means of microbeam synchrotron-radiation-induced X-ray emission (micro-SRIXE). Special attention was paid to examine the relationship between the earlier-mentioned disorders and changes in relative concentrations of S, K, Ca, Fe, Cu, and Zn. Applying the image-processing program ImageJ enabled us to select the areas of interest from two-dimensional maps of various prostate samples according to the histopathologist's evaluation. Detailed analysis of micro-SRIXE spectra based on multivariate methods shows significant differences between elemental concentrations in inflammatory cells, PIN lesions, and cancerous tissues, which confirms that this method can be used to distinguish various pathological states in prostate tissues. Information obtained in this way may provide better understanding of the biochemistry of unhealthy prostate tissues, thus opening the way to find new medicines/treatments to prevent or slow down some harmful intracellular processes.

Correlation of concentrations of selected trace elements with Gleason grade of prostate tissues.

The causes of prostate cancer are still obscure but some evidence indicates that there is a close connection between several trace elements and processes which may lead to malignant cells. In our study the microbeam synchrotron radiation X-ray fluorescence emission (micro-SRIXE) technique was applied for quantitative analysis of selected elements. For the first time, we correlate the concentrations of Mn, Fe, Cu, and Zn with the clinical stage of the prostate cancer at the time of operation (described by Gleason grade). Serial sections of prostate tissues were collected from patients undergoing radical prostatectomy. One section, stained with hematoxylin and eosin, was prepared for histopathological analysis; a second, adjacent unstained section was used in micro-SRIXE experiments. All experiments were performed at beamline L at HASYLAB, DESY, Germany. Our results seem to be valuable in light of the determination of the changes in the concentrations of trace elements as a potential diagnostic marker and their etiological involvement in the different stages of prostate diseases.

Calcification of aortic wall in cholesterol-fed rabbits.

Development of the mineralization process in the course of atherogenesis was studied using the cholesterol-fed rabbit model. The aorta samples were investigated by means of proton and electron microprobes, infrared spectroscopy and X-ray diffraction as well as selected histochemical staining. Blood serum was analysed every 2 weeks to determine the content of cholesterol, triglycerides, inorganic phosphorus, ionized calcium, elemental composition as well as activity of alkaline phosphatase. It was found that the administered diet did not disturb the calcium and phosphorus homeostasis. Histochemical findings confirmed the formation of lipid-rich lesions blocking the lumen of the vessel. The dystrophic calcification was observed only in the atheroma, while in the tunica media a slight mineralization similar to that found in controls was observed after 210 days of the diet. In the atheroma the only phase detected was a defective hydroxyapatite. The perfection of the crystals, as well as the diameter of the deposits, increased during the course of the diet reaching about 2 microns after 210 days. The crystals were not contaminated with carbonate groups regardless of the duration of the diet.

Developing an artificial fallopian tube: successful in vitro trials in mice.

This report describes the design and testing of an artificial fallopian tube for the treatment of tubal infertility. Within the device, mouse eggs incubated with sperm were fertilized and a microinfusion pump was used to transport the fertilized ova through the tube. Normal offspring resulted from transfer of the developing embryos into pseudopregnant recipients. These results provide encouraging evidence that an artificial fallopian tube warrants further investigation as a potential alternative to in vitro fertilization.

Effect of the new theophylline derivatives on degranulation of mast cells and phosphodiesterase activity in smooth muscles.

Effect of the new theophylline derivative (beta-hydroxy-benzylopiperazinopropyl-theophylline), designed as R6 on degranulation of the mast cells from rat mesenteries, was tested in vitro and in vivo. In vitro it was found that R6 significantly inhibited mast cell degranulation induced both by the histamine liberator C 48/80 and by rabbit globulin directed against rat serum proteins. The effect was comparable to that revealed by mepyramine, but much stronger than the effects of DSCG and aminophylline. The influence of R6 on mast cells degranulation was less pronounced in the in vivo systems. It was demonstrated by histochemical methods that R6 did not change activity of lactic and succinic dehydrogenases, neither activity of lysosomal hydrolases. In separate system including small intestine from rat it was shown that R6 inhibited activity of the phosphodeisterase in smooth muscles. It was suggested that the effect of R6 on mast cells degranulation is mediated by an increase in cAMP as well as by indirect influence on Ca++ influx.

Dialysis for psoriasis - preliminary remarks concerning mode of action.

In one patient treated by peritoneal dialysis for psoriasis the skin lesions cleared completely. In two persons hemodialysis gave unsatisfactory results. In treated and another three untreated patients cellular immunity was evidently suppressed. This phenomenon is similar to the immunologic changes in persons with uremia. Solutes in middle molecular weight range (SMMWR) suppress cellular immunity. These compounds were therefore investigated in psoriatics. The level of SMMWR was lower before than after each dialysis, although their concentration increased slightly during the dialytic treatment. This suggests that solutes in middle molecular weight are fixed in the epidermis and/or in the walls of the skin capillaries and that dialysis liberates them from these places. Peritoneal dialysis seems to be a more effective method of treating psoriasis than hemodialysis. Solutes in middle molecular weight range might play an important role in the pathomechanisms of this multifactorial disease.

Development of the innervation of long bones: expression of the growth-associated protein 43.

It has been known from clinical and experimental observations that the peripheral nervous system is involved in the development of long bones. Expression of growth-associated protein 43 (GAP-43/B-50) was found in axonal growth cones during embryonic and postnatal ontogeny as well as in regenerating axons after nerve injury. The aim of the present study was to examine the occurrence of growing nerve fibers in rat tibia from gestational day 16 (GD 16) to postnatal day 28 (PD28). An indirect immunoenzymatic reaction using antibodies raised against GAP-43 was applied to detect outgrowing nerve fibers penetrating into the developing bone. On GD 16 and GD 17 no GAP-43-immunoreactive (IR) fibers were observed in the close vicinity of bone rudiments. On GD19 GAP-43-IR fibers were scarcely present within the periosteum of the central portion of the diaphysis. In the perichondrium surrounding the proximal epiphysis, nerve fibers were first detected around birth. From PD1 onward, numerous fibers were seen in the fibrous buds of the perichondrium at the epi-metaphyseal junction (Ranvier's grooves), some of them being adjacent to the blood vessels. Nerve fibers penetrating into the bone and located in the bone marrow, predominantly associated with blood vessels, were first observed on GD21 and their number increased with further development. They were initially located in the central portion of the diaphysis and later extended towards the metaphyses. On PD4 an increased number of GAP-43-IR fibers appeared in the perichondrium of proximal and distal epiphyses. In the fibrous strands penetrating into the epiphyses and in the secondary ossification centers, nerve fibers were first observed on PD10. From PD14 onward the pattern of tibial innervation remained unchanged but the intensity of GAP-43 immunostaining visibly decreased. The present study demonstrates that developing long bones of rat hindlimbs are supplied by growing nerve fibers immunoreactive for GAP-43 from GD 19 onward. Time and location of their appearance were at least partially correlated with known events taking place during long bone development, e.g. formation of primary and secondary ossification centers. Decreased expression of GAP-43 immunoreactivity in later developmental stages is believed to reflect nerve fiber maturation.

Investigation of inorganic deposits in selected organic matrices.

Inorganic deposits in the wall of human and animal arteries and in experimental tumor (Morris hepatoma 7777) were examined using proton induced X-ray emission (PIXE) and PIXE in combination with proton microprobe (micro-PIXE) techniques. The sections adjacent to the irradiated ones part were submitted to histological investigations and one part of the material was additionally investigated by infrared (IR) spectroscopy. For identification of mineral deposits, the micro-PIXE method appeared the most sensitive. The mineral deposits were detected in the artery samples, even in those without visible morphological changes, as well as in tumor samples. The deposites showed different localization and composition, depending on age and type of vessel. There were also differences between human and animal arteries. IR spectroscopy revealed the presence of carbonate apatite within the artery samples from old individuals. Matching of histological observations with data obtained by micro-PIXE method allows a better correlation of morphological and analytical results.

Changes in activity of selected lysosomal enzymes in peritoneal macrophages of renal failure patients on peritoneal dialysis.

Activity of acid phosphatase (AP), beta-glucuronidase (GR), N-acetyl-beta-D-glucosaminidase (GZ), and peroxidase (P) was assessed using a semiquantitative cytochemical method in peritoneal macrophages of 30 patients with end-stage renal failure treated by intermittent peritoneal dialysis and of 30 control patients with normal renal function. The dialysed patients showed a significantly higher activity of GR and P at the beginning of the treatment as compared with the respective activities observed in the control group and a further significant rise of these activities after 4 months of dialysis. Activity of AP at the beginning of the treatment was insignificantly lower than in the control group and the difference became significant at the end of the investigated period. There was no significant difference between the dialysed patients and the control group in the activity of GZ assessed at the beginning of the dialytic treatment and after 4 months of dialysis. A significant decrease in that activity was, however, observed in the course of dialysis.

Light microscopic differential staining of epoxy-embedded adenohypophysis.

Light microscopic histochemical methods for differential staining of adenohypophysis glandular cells were applied to Epon-embedded tissue, after prior softening of resin with saturated solution of NaOH in absolute ethanol. All investigated staining procedures, i.e. carmoisin L-orange G, PAS-orange G, PAS-range G-methyl blue, Gabe's aldehyde fuchsin-Halmi counterstain, aldehyde thionin-PAS-orange G and performic acid-alcian blue-PAS-orange G were found to give satisfactory results and tissue structure showed no signs of damage caused by the Epon-dissolving reagent. The features of light microscopic image given by plastic-embedded semithin sections: nearly 2-dimensional picture as well as the highest possible resolving power provide the opportunity for much more precise and detailed examination of adenohypophysis prepared that way, as compared with conventionally paraffin-embedded gland. Moreover, the described technique permits to establish a direct connection between light microscopic differential histochemistry of adenohypophysis and the electron microscopy by means of adjacent sections.

The analysis of mineral deposits and proteoglycans content in the cartilage of mouse trachea using PIXE in combination with proton microprobe.

The tracheal cartilage of mature mice have been investigated using PIXE (proton induced X-ray emission) in combination with a proton microprobe on snap frozen cryosectioned material. The localization and quantitative measurements of P, S, Cl, K, Ca, Fe, and Zn concentrations as well as direct and indirect assessment of glycosaminoglycans by measurement of S content and measurement of bound colloidal iron at pH = 1.8 has been performed. Adjacent sections were stained with the Hale method in Müller modifications and the v. Koss method for sulphated mucins and inorganic deposits respectively. It has been found that hyaline cartilage in trachea contains mineral deposits and that P + Ca amounts up to 22% of cartilage dry mass. The Ca/P ratio approaches 2 what indicates hydroxyapatite type crystals. The cartilage contains substantial amounts of S reflecting the presence of sulphate groups. It was found that the cartilage binds also colloidal iron at low pH. There is a good correlation between places with high amount of bound colloidal iron assessed by PIXE and places showing strong Prussian Blue staining. The Fe/S ratio was, however, much lower in the cartilage than in other tissues what indicates that the colloidal iron method does not give quantitative results. There were no regions showing substantial decrease in Fe/S ratio which we found previously as typical for degenerating and calcifying growth plate cartilage. This may be connected with a relatively low degree of calcification degree of the tracheal cartilage.

Calcification of the aortic wall in hypercalcemic rabbits.

The mineralization process was investigated in the aortic wall of hypercalcemic rabbits. The elevated calcium level in serum was induced by intramuscular injection of vitamin D3. The animals were killed at different times of the experiment (max. 246 d). The freeze-dried tissue homogenates were used for elemental composition studies by means of proton induced X-ray emission (PIXE) and atomic absorption spectroscopy. The structural information was obtained from infrared (IR) and X-ray diffraction (XRD) spectra. Moreover, the ascending part of the aortic arch was separated and used for micro-PIXE (PIXE in combination with proton microprobe) and histochemical examinations. It was found that hypercalcemia (blood serum Ca content elevated by about 20%) induced calcification of the aortic wall. The mineral phase within the aortic wall consisted of Ca-P salts. The Ca/P ratio continuously increased during the experiment and approached 2 after 246 d of the vitamin D3 treatment. The IR and XRD studies made possible the identification of the complex phase composition of the samples. The hydroxyapatite crystals were detected after 196 days, however, in earlier phases of the experiment, amorphous calcium phosphate, dicalcium phosphate dihydrate and octacalcium phosphate were also observed. On the basis of the data obtained, the mechanism of the precipitation and growth of inorganic deposits in the tunica media of the aortic wall was discussed.

Distribution of elements in rabbit liver lobule.

The elemental composition of rabbit liver was determined by the PIXE and micro-PIXE methods. The mean concentrations of P, S, Cl, K, Fe, Cu, Zn, and Rb measured by both methods were similar. The latter method also allowed for localization of elements within lobule territory. It has been found that some elements are more prevalent in the veins (Cl, Fe) and others in the liver parenchyma (P, Cu, Zn). Moreover, Zn showed the characteristic intralobular distribution. Some methodological aspects of microbeam application to biological materials were also discussed.

[Calcification of allogenic aortic heart valves]. / Mineralizacja allogennych zastawek aortalnych serca.

The material used for the studies consisted of allogenic aortic valves (AAV) collected from 14 individuals. The necessity of AAV replacement arose from growing circulation insufficiency and AAV dysfunction. The aim the study was the determination of the elemental composition and crystallographic structure of the inorganic deposits in AAV. Moreover, the results of the physicochemical investigations were correlated with clinical data (age of the patient, time between valve replacement surgeries, endomyocarditis, number of infections during last 12 months, arterial hypertension and disturbance of the lipid balance) and with echocardiographic examinations (cusp mineralization and perforation, vegetation, systolic and diastolic dimensions of the left ventricle, maximal and average gradient through allograft valve as well as range of the recoil wave to left ventricle). It was found that mineralization of the AAV cusps was a time-dependent process and took place predominantly at the surface of the cusp. The elemental composition and crystallographic data revealed that the inorganic deposits in AAV were composed of hydroxyapatite crystals. However, the presence of other calcium salts was also found. The development of the mineralization process in AAV does not correlate with endomyocarditis, arterial hypertension and the disturbance of the lipid balance. Probably, endomyocarditis and arterial hypertension induce the pathologic alternations of AAV independently from the mineralization process. The echocardiographic estimations of the pathomorphologic changes of the aortic valve cups are not always consistent with the results of the physicochemical studies.

[Effect of calf thymus extracts on the surface structure and the lysosomal apparatus of lymphocytes in patients with chronic lymphatic leukemia]. / Wplyw wyciagu grasicy cielecej na strukture powierzchniowa i aparat lizosomalny limfocytów u chorych z bialaczka limfocytowa przewlekla

In 6 patients with chronic lymphatic leukaemia receiving subcutaneously purified calf thymus extract a greater frequency of the following processes was observed: 1) increase in the number of smooth-surface lymphocytes (T cells) in scanning electron microscopy, 2) increased number of lymphocytes forming rosettes with uncoated sheep erythrocytes, 3) increased number of lymphocytes showing high activity of acid phosphatase in cytochemical investigations.

Effect of cultured autologous oral keratinocyte suspension in fibrin glue on oral wound healing in rabbits.

The effect of cultured autologous oral keratinocyte suspension in fibrin glue on the healing of surgically produced oral mucosal wounds was assessed in the rabbit model. Using the light microscope and a digital image analysis system, the epithelization parameters (marginal epithelization and percentage of wound re-epithelization) were measured in haematoxylin-eosin stained sections of the wound area and compared with those of wounds treated with fibrin glue alone and untreated ones. The epithelization was significantly higher in keratinocytes plus fibrin glue-treated wounds on postoperative days 3 and 7. No significant differences were observed on postoperative day 1, when the healing process had just begun, and on postoperative day 14, when re-epithelization was completed or nearly completed in all groups. The inflammatory infiltration of the wounded mucosa was weakest in keratinocyte-treated wounds and strongest in untreated wounds. In conclusion, suspension of cultured autologous oral keratinocytes in fibrin glue significantly accelerates oral wound healing in the rabbit model and could be beneficial in the treatment of oral wounds in patients.