Influence of Cryopreservation on Structural, Chemical, and Immunoenzymatic Properties of Aortic Valve Allografts.

OBJECTIVES: The problems in preparing (including cryopreservation) and implanting aortic valve allografts (AVAs) is widely elaborated, but some issues need explanation. MATERIAL AND METHODS: Twenty AVAs cryopreserved in dimethylsulphoxide/RPMI solution under -160°C for 1-15 years and 3 controls stored at +4°C up to 2 weeks, from 19 male and 4 female donors, aged 20-51, ±30.8 years, were examined using light (LM), digital (DM), and scanning electron microscopy (SEM), energy dispersion X-ray spectroscopy (EDS), and enzyme-linked immunosorbent assay immunoenzymatic tests (PECAM1, CD34). RESULTS: All AVAs were macroscopically correct. LM revealed normal structure of leaflets but massive endothelial decellularization (±59 cells remained on the surface of 5 mm scraps). DM and SEM demonstrated generally normal collagen structures, but local alterations, probably influenced by freezing-thawing (gaps, separated plates) or being initial phase of native degeneration (grains). EDS detected a little elevated calcium amount in 1 specimen only. The mean PECAM1 and CD34 concentrations were at similar low level in all probes. CONCLUSIONS: Fresh and cryopreservation technologies did not significantly influence the basic properties of AVA leaflets; however, massive endothelial decellularization was present in both groups. Therefore, no endocardial cell activity nor signs of inflammation were observed. These results were independent of donors' age and sex, processing technology, and time of storage of cryopreserved AVAs.

An immunohistochemical investigation of the expression of somatostatin receptor subtypes - should therapeutic trials be performed to determine the efficacy of somatostatin analogs in treating advanced thyroid malignances?

UNLABELLED: Somatostatin and its analogs through the specific receptor are known to demonstrate antiproliferative, anti-angiogenic and pro-apoptotic actions. The presence of SSTR-1-5 has not been sufficiently explored in poorly differentiated and undifferentiated thyroid tumors. The aim was to investigate the SSTR subtypes expression in these aggressive thyroid tumors. The study also discusses the usefulness of SSTR analogs as an alternative to conventional forms of therapy. METHODS: The analysis was performed by immunohistochemistry on the 14 archived poorly differentiated and 4 anaplastic thyroid carcinomas. A group of benign thyroid pathologies consisting of 11 patients was also included. RESULTS: SSTR-1, 2A, 2B, 3 and 5 were found to be expressed both in benign and malignant thyroid diseases, while SSTR-4 was not. Expression of SSTR-1 and SSTR-5 was found in samples with poorly differentiated thyroid tumors with a score of at least 2.0 being recorded in 10 tumors (71.4%). For SSTR-2A the same or higher score was noted in 5/14 (35.7%), for SSTR-2B in 4/14 (28.6%) and for SSTR-3 in 3/14 (21.4%) samples. SSTR-1, 2B and 5 were found to have a score of at least 2.0 in all undifferentiated thyroid tumors. Immunostaining of SSTR-2A and 3 was observed in 50% of samples. The immunopositive reactions were observed both in the membranes and cytoplasm of the thyroid cancer' cells. In some cases positive immunostaining was localized also in the endothelium of intrathyroidal blood vessels. CONCLUSIONS: The somatostatin multiligand analogs or selective agonists could be considered alternatives to conventional therapeutic agents in aggressive thyroid tumors.

Effects of somatostatin-14 and the receptor-specific somatostatin analogs on chromogranin A and alpha-subunit (alpha-SU) release from "clinically nonfunctioning" pituitary adenoma cells incubated in vitro.

The aim of the study was to examine the effect of somatostatin (SST) and its analogs on the release of chromogranin A (CgA) and alpha-subunit (alpha-SU) from clinically non-functioning pituitary adenomas incubated in vitro. Seven pituitary macroadenomas surgically removed were investigated. All of the tumors were diagnosed before surgery as non-functioning, but they expressed either gonadotropins or their subunits as detected by immunohistochemistry. Two tumors additionally expressed prolactin and growth hormone. All adenomas also expressed chromogranin A (CgA) and at least 3 of 5 subtypes of somatostatin receptors. The cells isolated from the examined tumors were exposed in vitro to either native SST-14 or the following receptor-specific SST analogs: BIM-23926 (agonist of sst1 receptor), BIM-23120 (agonist of sst2 receptor), BIM-23206 (agonist of sst5 receptor) and BIM23A387 (somatostatin/dopamine chimera). The concentration of CgA was measured by means of ELISA method and of alpha-SU was measured by an immunoradiometric method. It was found that the exposure on SST-14 resulted in the decrease of CgA and alpha-SU release from tumor cells in majority of samples, and the effect on CgA was positively correlated with the expression of sst3 and also with the sst2A/sst2B expressions ratio. The inhibitory effect of SST-14 on CgA and alpha-SU seems also to correlate negatively with the expression of sst2B. CgA inhibition also correlates positively with sst5 expression. Among the other compounds studied, only the sst2 agonist decreased the release in all the investigated samples. The remaining substances (agonists of sst1 and sst5 and SST/DA chimera) produced the divergent changes (increased or decreased release, depending on the sample). The data suggest that the inhibition of CgA (and possibly of alpha-SU) release by SST is mediated via subtypes sst2A, sst3 and sst5, whereas sst2B subtype may induce the opposite effect.

Angiotensins II and IV modulate adrenocortical cell proliferation in ovariectomized rats.

Effects of angiotensins II (AngII), angiotensin IV (AngIV, 3-8 fragment of angiotensin II) and losartan (an antagonist of angiotensin receptor type 1) on the proliferation of adrenocortical cells in ovariectomized rats have been studied. The incorporation of bromodeoxyuridine (BrdU) into cell nuclei was used as an index of cell proliferation. AngIV decreased BrdU labeling index mainly in the reticularis zone and losartan (Los) was able to partially reverse this inhibitory effect of AngIV. AngII had no effect on the adrenocortical cell proliferation when given alone, however Los given simultaneously diminished BrdU incorporation into nuclei of glomerulosa and reticularis zones as compared with AngII. These findings suggest that AngII and AngIV modulate adrenocortical cell proliferation in ovariectomized rats.

The effect of selective sst1, sst2, sst5 somatostatin receptors agonists, a somatostatin/dopamine (SST/DA) chimera and bromocriptine on the "clinically non-functioning" pituitary adenomas in vitro.

The aim of the work was to investigate the effects of somatostatin analogs acting selectively on sst1 (BIM-23926), sst2 (BIM-23120) and sst5 (BIM-23206) receptor subtypes on the viability of "clinically non-functioning" pituitary adenomas in vitro. The effects of native SST (SST-14), a SST/DA chimera (BIM-23A387) and a D(2)-dopamine receptor agonist bromocriptine (BC) were also examined. The study was performed on 10 surgically removed pituitary macroadenomas, diagnosed before surgery as "non-functioning". A part of each tumor was mechanically dispersed and digested with collagenase to isolate the tumoral cells. Another part of each tumor was fixed, embedded in paraffin and immunostained to reveal the pituitary hormones and SST receptor subtypes (sst1, sst2A, sst2B, sst3, sst4, sst5). The tumoral cell suspensions were incubated for 24 h with the substances mentioned above. The quantity of viable cells was estimated using the EZ4U system. The results were compared with the immunohistochemical evaluation of the hormonal profile of adenoma and the sst receptor subtype immunoreactivities present. The findings indicate that selective sst1, sst2 and sst5 receptors agonists, SST/DA chimera and D(2)-dopamine receptor agonist bromocriptine affect the viability of some, but not all, "clinically non-functioning" pituitary adenomas in vitro. The most effective was bromocriptine. The investigated somatostatin analogs including SST/DA chimera exerted roughly similar inhibitory effects. Further studies are needed to fully evaluate the potential usefulness of these compounds in the pharmacological treatment of "non-functioning" pituitary tumors.

Angiotensin IV stimulates the activity of tyrosine kinases in rat anterior pituitary gland acting via AT1-like receptors?

Angiotensin II (Ang II) is known to modulate tyrosine kinases (PTKs) activity in pituitary tumor cells. It is known that AngII is acting via AT1 receptors in many tissues. The aim of this study was to see whether 3-8 fragment of AngII, called angiotensin IV (AngIV) has a similar effect on tyrosine kinase activity in normal pituitary gland and what type of angiotensin receptor is involved in this process. The homogenates of normal rat pituitaries was a source of enzymes. The PTKs activity was determined using the synthetic substrate poly GluTyr and gamma-(32)P-ATP. Ang IV was found to increase the PTK activity in the rat anterior pituitary tissue, with the maximal effect at concentration of 10(-10) M. AngII was ineffective at all concentrations studied. Losartan, a selective AT1 receptor blocker, added together with Ang IV abolished the effect of the latter, however losartan diminished also the PTK activity when applied together with Ang II, but only when it was added immediately before, but not after, the addition of Ang II. The involvement of a non-classic AT1-like receptor is suggested.

Effect of thyroliberin on the course of experimental candidosis in mice.

Thyroliberin (TRH) is one of the hormones, which affect immunologic processes. This hormone was studied in experimental subacute candidosis in mice. BALB/c males were given intraperitoneally single dose of Candida albicans cells (1 x 10(7) to 1 x 10(9) cfu of strain no. 244-33 ATCC). The animals from the experimental groups were injected subcutaneously, after 24 h from inoculation, dose of 10 microg TRH in 0.2 ml 0.9% NaCl, seven times at 24 h intervals. The control animals were given respectively 0.2 ml of physiologic NaCl solution. We have found that the examined hormone significantly decreases mortality in these animals (LD50 C. albicans for mice treated with TRH was three times higher than that in the control groups), prolongs mean survival time for mice and decreases the intensity of fungal invasion of the animal organs.

Anti-inflammatory effects of somatostatin analogs on zymosan-induced earlobe inflammation in mice: comparison with dexamethasone and ketoprofen.

The aim of the present study was the estimation of the anti-inflammatory effects of the somatostatin analogs, octreotide (OCT) and vapreotide (RC-160), in zymosan-induced mice ear inflammation and to compare their effects with those of the glucocorticoid dexamethasone (DX) and the non-steroid anti-inflammatory drug ketoprofen (KP), which are the well-known and potent suppressors of the inflammatory reaction. The inflammation was induced by injecting 20 microl of 1% suspension of zymosan intradermally into one of the earlobes of the mouse. The control animals received a vehicle 0.9% NaCl. The zymosan-treated animals were injected subcutaneously with one of the following substances: 0.9% NaCl, OCT, RC-160, DX, KP or with OCT plus DX and OCT plus KP. The edema of earlobes, the area of inflammatory focus and the area of vascular profiles in the inflamed tissues were estimated. A reduction of the ear edema in the mice treated with OCT, DX, KP, OCT + DX and OCT + KP was observed. The administration of all drugs caused the decrease of the area of the inflammatory focus and of the area of vascular profiles. The antiphlogistic activity was more pronounced in the OCT-treated animals in comparison to those treated with RC-160. The joint treatment with either OCT plus DX or OCT plus KP almost totally inhibited the zymosan-induced inflammatory reaction. In summary, the somatostatin analog OCT possesses antiphlogistic activity roughly comparable with classical anti-inflammatory drugs such as DX and KP. The somatostatin analogs may constitute a new promising group of anti-inflammatory agents.

Correlation between PCNA expression and AgNOR dots in pituitary adenomas.

Nucleolar organizer regions are segments of DNA associated with argyrophilic proteins (AgNORs). Our previous findings showed that the number, the area, and the intranuclear localization of AgNOR dots differ according to tumor aggressiveness and to the hormone-immunopositivity of pituitary adenomas. Proliferating cell nuclear antigen (PCNA) is a nuclear protein, whose expression is correlated with cell proliferation. The aim of the present paper was to examine PCNA-labeling indexes in pituitary adenomas and to correlate them with AgNOR dots in various immunohistochemical types of the tumors. Histological slides from 32 pituitary tumors and one normal pituitary were silver-stained and analyzed with a computerized system for microscopic image analysis. We found that the percentage of PCNA-positive cells did not differ significantly among examined groups of monohormonal adenomas. However, tumors immunopositive for alpha-subunit (alpha-SU) showed a significantly higher (p < 0.05) PCNA index than adenomas immunonegative for that unit. PCNA index in recurrent tumors was significantly higher than in primary adenomas. There was a moderate positive correlation between the PCNA index and the mean area of AgNOR dots and a similar correlation between the PCNA index and the area of the biggest dot in the nucleus. The obtained results reveal that the PCNA indexes and estimated parameters of AgNOR dots differ according to tumor aggressiveness.

Angiotensins II and IV stimulate the rat adrenocortical cell proliferation acting via different receptors.

OBJECTIVE: The effects of angiotensins II (Ang II) and IV (Ang IV,3-8 fragment of angiotensin II) on the adrenocortical cell proliferation have been investigated in the rat. METHODS: The male adult Wistar rats were injected subcutaneously with saline, captopril or captopril together with either Ang II or Ang IV. A part of animals received additionally losartan - an antagonist of AT1 subtype of angiotensin receptors. Bromodeoxyuridine (BrDU) incorporation into cell nuclei was used as the index of cell proliferation. RESULTS: It was found that both Ang II and Ang IV increased the BrDU labeling in the adrenal cortex of captopril-pretreated rats. This effect involved mainly the zona glomerulosa cells. The proliferogenic effect of Ang II was blocked by AT1 receptor antagonist losartan. In contrast, losartan did not block the effect of Ang IV. CONCLUSION: Both Ang II and Ang IV stimulate the adrenocortical cell proliferation in the rat, but they act via different receptors - AT1 in the case of Ang II and non-AT1 (probably AT4) in the case of Ang IV.

Anti-tumoral action of octreotide and bromocriptine on the experimental rat prolactinoma: anti-proliferative and pro-apoptotic effects.

OBJECTIVES: The purpose of the study was to compare the effects of bromocriptine (BC) - D-2 receptor agonist and octreotide (OCT) - somatostatin analog on the tumor weight, prolactin (PRL) secretion, cell proliferation and apoptosis in the diethylstilboestrol (DES)-induced rat prolactinoma. MATERIAL AND METHODS: Male four-week Fisher 344 rats were used in the experiment. The animals were implanted subcutaneously (s.c.) with capsules containing DES. Six weeks after the implantation the rats were given OCT (2 x 25 microg/animal/24 h s.c.) or BC (3 mg/kg b.w./24 h s.c.) for 10 days. The incorporation of bromodeoxyuridine (BrDU) into the tumor cell nuclei was used as an index of cell proliferation (labeling index - LI). The labeling of nuclear DNA fragmentation according to the TUNEL method was considered as an index of apoptosis (AI). PRL was measured by radioimmunoassay (RIA). RESULTS: It has been found that OCT and BC significantly decreased the tumor weight and LI of tumor cells to the same extent. Both OCT and BC suppressed the PRL levels, but the inhibitory effect of BC was stronger than that of OCT. BC and OCT significantly enhanced the number of apoptotic cells in the tumor, but the pro-apoptotic effect of BC was more pronounced. The joint treatment exerted additive effects on tumor mass reduction, PRL secretion and cell proliferation, but OCT attenuated the pro-apoptotic effect of BC. CONCLUSIONS: Summing up, both OCT and BC inhibit PRL secretion and cell proliferation. The anti-tumoral action of BC, and to some extent the action of OCT, is also connected with induction of apoptosis.

Melatonin and RZR/ROR receptor ligand CGP 52608 induce apoptosis in the murine colonic cancer.

The effects of melatonin and the thiazolinidinedione derivative CGP 52608 on apoptosis of Colon 38 cancer cells were investigated. Male mice were implanted subcutaneously with a suspension of Colon 38 cells. Ten days after induction of tumors, the animals were treated with melatonin or CGP 52608. Both substances were given in subcutaneous injections in daily doses of 10 or 100 microg in the evening for 6 days. The control group received solvent. The apoptotic cells were visualized in paraffin sections by means of the transferase-mediated dUTPnick end-labeling method. Both treatments increased significantly and to the same degree the number of apoptotic cells in tumors. This finding confirms our earlier observation that melatonin exerts a pro-apoptotic effect on murine colonic cancer cells. Moreover, because CGP 52608 is a ligand of RZR/ROR receptors and the latter are considered by some investigators as nuclear binding sites for melatonin, our data suggest the involvement of these receptors in the pro-apoptotic effect of melatonin.

Pregnenolone sulfate modulates angiotensin II-induced inositol 1,4,5-trisphosphate changes in the anterior pituitary of female rat.

The present study was designed to test whether pregnenolone sulfate can influence the angiotensin II (AngII) action in the anterior pituitary. Female rats were ovariectomized and 10 days after operation were treated with either 50 or 250 microg per animal in one of the following substances: oil (control), pregnenolone sulfate (PREG-S), estradiol benzoate (E(2),) progesterone (P), or dehydroepiandrosterone sulfate (DHEA-S), given in single intraperitoneal injection. Because AngII is known to act in the anterior pituitary through the phosphatidiloinositol breakdown, thus increasing the level of inositol-1,4,5-trisphosphate (IP(3)), the IP(3) concentration was determined 24 h after the injection in the anterior pituitary homogenate after in vitro exposure to AngII. Among the tested steroids only PREG-S enhanced the basal (without AngII) and AngII-induced level of IP(3) in both doses used. There was no difference between the effect of a high and low dose of PREG-S. These results show that PREG-S may modulate AngII action in the anterior pituitary, regardless of the presence of ovary.

Calreticulin affects beta-catenin-associated pathways.

Calreticulin, a Ca(2+) storage protein and chaperone in the endoplasmic reticulum, also modulates cell adhesiveness. Overexpression of calreticulin correlates with (i) increased cell adhesiveness, (ii) increased expression of N-cadherin and vinculin, and (iii) decreased protein phosphorylation on tyrosine. Among proteins that are dephosphorylated in cells that overexpress calreticulin is beta-catenin, a structural component of cadherin-dependent adhesion complexes, a member of the armadillo family of proteins and a part of the Wnt signaling pathway. We postulate that the changes in cell adhesiveness may be due to calreticulin-mediated effects on a signaling pathway from the endoplasmic reticulum, which impinges on the Wnt signaling pathway via the cadherin/catenin protein system and involves changes in the activity of protein-tyrosine kinases and/or phosphatases.

Immunocytochemical prognostic markers in pituitary tumors.

The value of different immunocytochemical markers in the evaluation of the pituitary tumor aggressiveness is discussed on the basis of the data from the literature and the author's personal experience. The usefulness of the proliferation markers (PCNA, Ki-67), prothymosin-alpha, the endothelial markers revealing the intratumoral vascularization and of the expression or co-expression of glycoprotein hormones in predicting the aggressive behaviour of the tumor is likely, but still needs further studies.

The involvement of angiotensins in the control of prostatic epithelial cell proliferation in the rat.

The effects of captopril (the inhibitor of the angiotensin-converting enzyme) and of angiotensins II and IV (3-8 fragment of angiotensin II) on cell proliferation of the prostatic epithelium was investigated in the rat. The incorporation of bromodeoxyuridine into cell nuclei was used as an index of cell proliferation. It was found that the treatment with captopril resulted in the suppression of prostatic epithelial cell proliferation. The antiproliferative effect of captopril was reversed (at least partially) by a simultaneous treatment with either angiotensin II or angiotensin IV. The effects of angiotensins were not blocked by the administration of losartan--AT1 angiotensin receptor blocker. These findings suggest the involvement of angiotensins in the control of prostatic growth, acting via the receptors different from the AT1-subtype (presumably via AT4 receptors).

[Pituitary adenoma penetrating the sphenoidal sinus and nasal cavity: a case report]. / Gruczolak przysadki penetrujacy do zatoki klinowej i jamy nosa--opis przypadku.

We describe a case of pituitary adenoma penetrating to the sphenoidal sinus and nasal cavity in a patient with recurrent nasal polyps. Histopathological examination of the removed polyps revealed transitional carcinoma. CT and MRI of the head showed a solid tumour filling the spheniodal sinus and the sella, penetrating to posterior ethmoid cells and superior nasal duct. In hormonal investigations increased concentration of prolactin (PRL) was found. Histopathological examination performed after neurosurgical operation revealed pituitary adenoma, and the diagnosis of pituitary adenoma was established. About 30% of tumour cells gave positive reaction with anti-PRL antibody in the immunocytochemical investigation. The immunocytochemical investigation of the nasal polips was also done--similarly about 30% of cells showed strong positive reaction with anti-PRL antibody. The investigations indicate the presence of pituitary macroadenoma (prolactinoma), manifesting initially as nose tumour. Considering cases of ectopic pituitary adenomas covered with transitional epithelium (for example some nasal tumours and paranasal sinuses tumours) immunocytochemical investigations are recommended in such cases.

Female house mice develop a unique ovarian lesion in colonies that are at maximum population density.

Colonies of house mice reach maximum population density in 120-180 days, irrespective of cage size and initial number of colonizing animals. Reproduction ceases because the females become aggressive and unreceptive to mating. The aggressive behavior is correlated with elevated levels of testosterone (T) and corticosterone (B) (Chapman et al., Phys Behav 64:529-533, 1998). In two of seven strains of mice, females developed ovarian lesions. The occurrence of the lesion in one strain was correlated with the age of the animal and duration of the study. In the second strain, cage size was the determining factor. Lesioned ovaries weighed significantly more than nonlesioned ovaries. The lesion consisted of accumulations of luteal membrane and organelle fragments, and other cellular debris, suggestive of incomplete and prolonged luteolysis. Electron microscopic (EM) analyses revealed the presence of deposits of permanganate-resistant congophilic amyloid fibrils in the intima and smooth muscle cells of luteal thecal arteries. Population females had thymus glands and uteri that weighed significantly less than the same organs from females housed in the breeding colony, whereas the adrenal glands from the population females weighed significantly more. It is proposed that the female aggression is due to high levels of T. It is also proposed that the high levels of B suppress the immune cells involved in normal luteolysis and contribute to the incomplete and prolonged luteolysis.

Effects of somatostatin and its analogues on tyrosine kinase activity in rodent tumors.

The effects of native somatostatin-14 (SS-14) and of its two analogues, octreotide and CH-275, on the activity of tyrosine kinases (TK) in two rodent tumors (rat pituitary tumor and murine colonic cancer) were studied in vitro. The activity of TK was measured in tissue homogenates using gamma[(32)P]ATP as the donor of the phosphoryl group and poly(Glu(80), Tyr(20)) as a substrate. It was found that native SS-14 inhibited TK activity in both investigated tumors. Octreotide, which acts preferentially via somatostatin receptor subtype 2 (SSTR2), was very effective in inhibiting TK activity in the rat pituitary tumor, but it is a rather weak inhibitor of TK activity in murine colonic cancer. CH-275, a selective ligand of the SSTR1 subtype of SS receptors, suppressed TK activity in the pituitary tumor but was ineffective in the colonic cancer. It is hypothesized that the effect of neuropeptide somatostatin (SS-14) on murine colonic cancer is exerted via the subtype of receptor which does not interact with CH-275 and has no or low affinity for octreotide (SSTR 4, 3 or 5?).

Effect of 17-beta-estradiol and progesterone on angiotensin II-induced changes in inositol-1,4,5-trisphosphate content and protein kinase C activity in anterior pituitary.

Angiotensin II (AngII) is known to act in the anteriorpituitary through phosphatidiloinositol breakdown, increasing the level of inositol-1,4,5-trisphosphate (IP(3)) and diacyloglycerol (DAG), a potential activator of protein kinase C (PKC). We examined the effect of estradiol and progesterone treatment in vivo on IP(3) levels and activity of PKC under the influence of AngII. Three groups of intact female rats received in vivo injections of 17-beta-estradiol, progesterone, and oil (control) for five days, and then the in vitro effect of AngII was examined using homogenate of the anterior pituitary. AngII increased either the IP(3) concentration or the synapsin I phosphorylation catalyzed by PKC. Estradiol enhanced the basal (without AngII) IP(3) level and PKC activity induced by AngII. Progesterone did not change the basal and AngII-induced IP(3) concentrations. On the other hand, it decreased the basal PKC activity and blocked the effect of AngII. Our data suggest that ovarian steroids can modulate the effect of AngII on the anterior pituitary gland.