In vitro substance P-dependent induction of bone marrow cells in common (CD10) acute lymphoblastic leukaemia.

The aim of the present research was to investigate the possible in vitro stimulatory effect of substance P (SP) on blasts induction in childhood common acute lymphoblastic leukaemia (ALL). Bone marrow aspirates were incubated with SP receptor agonist or antagonist (spantide) and subsequently assayed for the presence of human interleukin (IL)-1b using ELISA kit. Blast cells incubated with SP receptor agonist were found to result in a significant increase of IL-1b concentration while incubated with spantide resulted in control levels of IL-1b. These findings suggest the novel possible role of SP in blasts proliferation in childhood ALL of common (CD10) origin.

The significance of VEGF-C/VEGFR-2 interaction in the neovascularization and prognosis of nephroblastoma (Wilms' tumour).

AIM: To investigate the immunohistochemical expression of vascular endothelial growth factor (VEGF)-C and VEGFR-2 in nephroblastoma tissue and correlate their presence with the survival rate of children diagnosed with stage III Wilms' tumour. METHODS AND RESULTS: The material included nephroblastoma tissue obtained from 25 children hospitalized in the Department of Paediatric Oncology, Haematology and Transplantology between 1997 and 2003. VEGF-C and VEGFR-2 expression was evaluated by immunohistochemical assay. VEGF-C was expressed in all cells of the blastemal component and in 30% of tumour cells in the stromal part. It was absent from epithelial elements. VEGFR-2 expression was spread over the surface of numerous stromal cells as well as all the epithelial cells forming dysplastic tubules. The blastemal component of Wilms' tumour was VEGFR-2-negative. VEGF-C-immunopositive stromal cells were situated in the closest proximity to VEGF-C-immunonegative but VEGFR-2-immunoreactive tubules. VEGF-C expression was of prognostic value for both clinical progression (P = 0.0005) and tumour-related death (P = 0.0365). CONCLUSIONS: VEGF-C expression in Wilms' tumour constitutes a potent unfavourable risk factor and may direct future antiangiogenic treatment strategies. The proximity of VEGF-C and VEGFR-2 in the stromal and epithelial components of nephroblastoma could be the neoplastic equivalent of the binary VEGF-C function observed in epithelial and endothelial morphogenesis.

Vascular endothelial growth factor C--a potent risk factor in childhood acute lymphoblastic leukaemia: an immunocytochemical approach.

AIMS: To investigate the immunocytochemical expression of vascular endothelial growth factor C (VEGF-C) and its receptors (VEGFR-2 and VEGFR-3) in childhood acute lymphoblastic leukaemia (ALL) blasts and to determine the possible role of this complex in the pathogenesis and prognosis of ALL. METHODS AND RESULTS: Bone marrow samples were taken from 120 children diagnosed with ALL. An indirect immunocytochemical procedure was performed with the use of monoclonal mouse anti-human antibodies against VEGF-C, VEGFR-2 and VEGFR-3 (diluted 1 : 100). The immunocytochemical expression of VEGF-C was confirmed exclusively in the cytoplasm of ALL lymphoblasts (the mean percentage was 36.4 +/- 7.2). It was absent from the cytoplasm of normal haematopoietic cells in the control group. No VEGFR-2 or VEGFR-3 expression was detected in the children of either the study or control groups. The risk of induction failure or leukaemic relapse was found to be significant in all VEGF-C+ patients (P < 0.0001 and P < 0.02, respectively; Fisher's exact test). CONCLUSIONS: The absence of VEGF-C in blast cells predicts long-lasting remission in all leukaemic children. Our findings also suggest that leukaemic cell invasion, following VEGF-C-driven lymphangiogenesis, could be related to a mediating role of this peptide produced by blast cells themselves.

The predicting role of substance P in the neoplastic transformation of the hypoplastic bone marrow.

AIMS: To estimate the expression of substance P in the haematopoietic cells of hypoplastic bone marrow and define its relationship with the course of bone marrow hypoplasia. METHODS: Bone marrow specimens were obtained from 42 children with bone marrow hypoplasia who were hospitalised in the Department of Pediatric Oncology, Hematology and Transplantology, Poznan University of Medical Sciences, Poznan, Poland, between 1996 and 2003. Substance P and Ki-67 expression were evaluated using immunochemical and hybridocytochemical assays. RESULTS: The expression of substance P (as evidenced by both immunocytochemical and hybridisation techniques) was confirmed in the cytoplasm of B lymphocytes in 8 of 11 children who developed acute leukaemia in 45 (SD 12) days. The percentage of substance P-positive cells ranged from 67.6 to 95.8 (mean of 81.5% cells with immunocytochemistry and 84.3% with in situ hybridisation). The risk of development of leukaemia secondary to bone marrow hypoplasia was found to be significant (p<0.001) in those children who expressed substance P in normal-looking lymphocytes at the initial bone marrow evaluation. CONCLUSIONS: The presence of substance P in B lymphocytes of hypoplastic bone marrow may predict its neoplastic transformation. A marked correlation between substance P-positive bone marrow pattern and the expansion of tumour cells may prove the potential value of this oligopeptide in the pathogenesis of leukaemia.

Prognostic value of stage IV neuroblastoma metastatic immunophenotype in the bone marrow: preliminary report.

AIM: To correlate the immunophenotype of metastatic cells in the bone marrow of patients with neuroblastoma with early treatment failure. METHODS: The studies were performed on bone marrow material obtained from children treated in the department of paediatric oncology, haematology, and transplantology, Poznan University of Medical Sciences, Poland from 1996 to 2003. Immunocytochemical analysis of nervous tissue markers (using the immunomax technique) was performed on 108 bone marrow preparations obtained from 36 children diagnosed with neuroblastoma (stage IV with bone marrow metastases). The analysis included expression of PGP 9.5 protein, substance P, chromogranin A, bombesin, galanin, neuropeptide Y (NPY), and vasoactive intestinal peptide in neuroblastoma metastatic cells defined by the expression of neurone specific enolase. RESULTS: Nineteen relapses occurred within 12 months of the end of treatment. Correlation between the various markers studied and early treatment failure, using Fisher's exact test, revealed that chromogranin A and NPY are strong indicators of an unfavourable prognosis in patients with stage IV neuroblastoma (p < 0.001 and p < 0.0002, respectively). CONCLUSION: Determination of metastatic cell immunophenotypes in bone marrow (particularly chromogranin A and NPY) may help establish the short term prognosis in children with neuroblastoma.

Pneumadin in the rat ventral prostate and its hormonal regulation.

Pneumadin (PNM) is a decapeptide originally isolated from mammalian lungs, and exerts a potent antidiuretic action by stimulating arginine-vasopressin release. We have recently developed a sensitive and specific radioimmunoassay (RIA) for rat PNM and detected high concentrations of PNM--not only in the rat lungs, but also in the prostate. Hence, we investigated whether prostate PNM content is regulated by sex hormones. Male adult rats were orchidectomized or sham-operated and given a subcutaneous injection of testosterone or estradiol (40 and 5 mg/kg), respectively. The animals were decapitated one week after surgery, and their ventral prostates were promptly removed and weighed. PNM concentration and localization in the prostate were investigated by RIA and immunocytochemistry (ICC). Orchidectomy resulted in significant decreases in the prostate weight and PNM concentration, and testosterone administration prevented these effects. Estradiol administration to sham-operated rats caused prostate atrophy without changing PNM concentration. ICC localized PNM immunoreactivity (IR) exclusively in the epithelial cells of the ventral prostate. Orchidectomy markedly reduced PNM-IR concentration, while testosterone abolished this effect. Estradiol did not modify PNM-IR concentration in the atrophic prostate of sham-operated rats. We conclude that PNM content of rat prostate is dependent on the presence of adequate levels of circulating testosterone. The possibility that PNM plays a key role in the maintenance of the prostate growth is unlikely since estradiol-induced gland atrophy is not associated with any decrease in PNM concentration. The localization of PNM in the epithelial cells could suggest that this peptide may be involved in the regulation of some testosterone-dependent secretory functions of the rat prostate.

Immunocytochemical study on endothelial integrity of saphenous vein grafts harvested by minimally invasive surgery with the use of vascular mayo stripers. A randomized controlled trial.

OBJECTIVES: The aim of this study is to compare the endothelial integrity of saphenous vein grafts harvested by minimally invasive surgery and veins harvested conventionally for coronary artery bypass surgery in 200 participants who were assigned to interventions by using random allocation. DESIGN: Randomized controlled trial. Methods. Immunocytochemistry with anti-CD 31 antibodies and anti-nitric oxide synthase (NOS) antibodies were employed to identify the endothelial integrity. RESULTS: The CD 31 immunostaining showed that the endothelial cell integrity of the minimally invasive harvested veins was preserved in 82+/-13% of the circumference of luminal endothelium, while in conventionally harvested grafts it was reduced to 64+/-15% (p=0.05).> This was associated with the lack of CD 31 expression in vasa vasorum (10 and 18%) in both groups, respectively, (p=0.02). The NOS immunostaining revealed that the endothelial integrity of the minimally invasive harvested grafts was preserved in 96+/-4% of the luminal endothelium circumference as compared to 74+/-10% in conventionally harvested grafts (p=0.05). The percentage of cases with the lack of NOS expression in all vasa vasorum was 12 and 21%, in G1 and G2, respectively, (p=0.02). CONCLUSION: The endothelial integrity of saphenous vein grafts harvested by minimally invasive surgery is better preserved than with the grafts obtained by the conventional manner. This could play an important role in improving vein graft patency rates.

Substance P--a potent risk factor in childhood lymphoblastic leukaemia.

The study focused on determining the expression of substance P (SP) in neoplastic bone marrow cells in childhood acute lymphoblastic leukaemia (ALL) in terms of its mRNA and the level of protein production. An attempt has also been made to demonstrate a correlation of SP with leukaemia risk factors and treatment failure. The study group comprised 120 children treated for ALL. Expression of SP was examined by in situ hybridisation with a 5'-biotinylated probe and by immunocytochemistry with specific anti-human SP antibody. Out of 80 patients with common ALL, the expression of SP was demonstrated in 33 cases (41.2%). In the group of 24 children with pre-B ALL, the presence of SP was noted in six cases (25.0%). Of 16 patients with T-cell leukaemia, SP expression was demonstrated in 13 cases (81.2%). The percentage of immunopositive cells in the SP-positive cases ranged from 79.8 to 97.3. Treatment failure in the children with ALL was closely related to the expression of SP observed at the beginning of treatment. The results showed a connection between the presence of SP-positive blasts and leukaemia relapse. This may indicate that SP expression, involved in the proliferation of the tumour cells, may represent a novel risk factor in ALL.

Morphometric studies on rat epididymis in the course of postnatal development (computerised image analysis).

The studies were aimed at histological and morphometric analysis of postnatal development of epididymis in rats aged 1, 5, 10, 20, 28, 35, 45 and 59 days. Diameter of ductuli efferentes and of ductus epididymidis, height of epithelium, section area fractions occupied by the epithelium and by the sublayer were estimated (using MultiScan software) and serum testosterone levels were measured. The results documented a stepwise development of the epididymis, in three distinguishable stages.

Neonatal treatment with monosodium glutamate (MSG): structure of the TSH-immunoreactive pituitary cells.

Glutamic acid represents the most abundant stimulatory neurotransmitter in the central nervous system. Monosodium glutamate (MSG), subcutaneously administered to newborn rats in the perinatal period, induces lesions in 80 to 90% of the neurocytes of arcuate nuclei in the hypothalamus. These nuclei are the site of production of numerous stimulatory and inhibitory hormones including growth hormone releasing hormone (GHRH). The present studies were performed on male Wistar strain rats, subcutaneously injected on days 2, 4, 6, 8, and 10 of postnatal life with MSG at a dose of 4 mg/g body weight. Eighteen-month-old rats were additionally treated with Ambinon. When the animals reached the ages of 6 or 12 months, their body weight, body length and weight of pituitary were determined. On paraffin sections, using immunohistochemical techniques, TSH-immunoreactive cells were detected and characterised by computerised image analysis. The results were subjected to statistical analysis using Student's t test. The rats which were perinatally treated with MSG and examined after 6 or 12 months of life were obese and shorter than control rats by 7% and 10% respectively. They also exhibited a reduction in the weight of the pituitary of 30% and 40% respectively in the two age groups. The proportion of TSH-immunoreactive cells in the pituitary remained unchanged and amounted to 4.5% in the 6-month-old and 5.4% in the 12-month-old rats, respectively. The number of TSH-positive cells per mm2 area remained unchanged. The area and circumference of the cells in the 12-month-old rats were reduced by 22% and 18%, respectively. Perinatal injury to hypophyseal arcuate nuclei induced by monosodium glutamate injection, was not associated with any significant alterations in pituitary structure, as defined by the proportion of pituitary volume occupied by TSH-immunoreactive cells.

Long-term effect of neonatal monosodium glutamate (MSG) treatment on reproductive system of the female rat.

The study aimed at determining effects of monosodium glutamate (MSG), introduced in the perinatal period, on the reproductive system of sexually mature female rats. In days 2, 4, 6, 8, 10 the newborns received s.c. injections of MSG (4 mg/g body weight) or 2% NaCl solution. When the animals reached the age of 6, 12 or 18 months, their ovaries and uteri were isolated for histological and morphometric studies while in their sera estradiol level was estimated by the RIA technique. The perinatal injection of MSG was found to decrease relative weights of ovaries and uteri. In the ovaries increased numbers of primordial follicles and decreased numbers of graafian follicles were detected. Also the thickness of endometrium and of the epithelium, which lined the endometrium, were lowered in females, which received perinatal injections of MSG, as compared to the controls. Serum estradiol level in MSG injected females was lowered at the age of 12 and 18 months. In 12 and 18 month old females the alterations were accompanied by obesity and a decreased body length.

Morphometric studies on the testes of rats treated neonatally with oestrogen and subsequently with gonadotrophins and testosterone.

The experiments involved male rats, which were given a single subcutaneous dose of 1 mg stilboestrol on the first day of life. Beginning on day 28, subgroups of the rats received either gonadotrophins or testosterone for 39 days. The weight of the testes, serum luteinizing hormone and testosterone levels were determined while sections of the testes were subjected to morphological analysis and morphometric measurements, based on computerized techniques. The results demonstrated that a single dose of oestrogen caused a reduction in the cross-sectional area of the seminiferous tubules and a reduction in the thickness of the seminiferous epithelium, accompanied by inhibition of spermatogenesis. The number of and area occupied by Leydig cells, as well as the size of their cell nuclei, were also diminished, and the levels of serum testosterone decreased by 73%. All the experimental animals manifested significantly increased serum luteinizing hormone levels. Stimulation with gonadotrophins markedly increased the number of Leydig cells, their size and the size of their cell nuclei. This was associated with significantly increased levels of serum testosterone. Under these conditions, the cross-sectional area of the seminiferous tubules and the thickness of seminiferous epithelium remained less than those in the untreated controls. Following stimulation with testosterone the pattern of the seminiferous tubules resembled that noted after stimulation with gonadotrophins; the number of Leydig cells was markedly reduced but the size of both the cells themselves and of their nuclei approached normal values.

Bradykinin and pituitary-adrenocortical function in the rabbit: in vitro and in vivo studies.

Bradykinin (BK) is a 9-amino acid peptide, which has been found to affect adrenocortical secretion in the calf and rat. We investigated the in vitro and in vivo effects of BK and its receptor antagonist [D-Arg, (Hyp3,D-Phe7)]-BK (BK-A) on pituitary-adrenocortical function in the rabbit. BK and BK-A raised basal release of aldosterone, but not of corticosterone by dispersed zona glomeralosa and zona fasciculata-reficularis cells, respectively. Both peptides did not affect ACTH-stimulated aldosterone secretion. Conversely, BK concentration-dependently decreased ACTH-stimulated corticosterone production, and BK-A annulled this effect. The bolus intravenous injection of BK did not alter plasma ACTH concentration. However, BK lowered the blood concentration of both aldosterone and corticosterone, as well as the overall production of the two hormones over a period of 90 min after its administration. The simultaneous injection of BK-A blocked these effects of BK. BK-A alone did not evoke any sizeable change in blood hormonal levels. Collectively, these findings allow us to conclude that in rabbits (i) exogenous BK depresses adrenocortical secretion, through a receptor-mediated mechanism, which does not involve the inhibition of pituitary ACTH release-, and (ii) endogenous BK-like peptides do not play a relevant role in the functional regulation of the pituitary-adrenal axis, at least under basal conditions.

Effect of neonatal treatment with MSG (monosodium glutamate) on thyroid of the adult male rats.

Monosodium glutamate (MSG), administered to newborn rats produces extensive lesions in neurons of the arcuate nuclei of the hypothalamus. The cells represent the site of neurohormone production, including the production of both growth-hormone releasing hormone (GHRH) and somatostatin. Studies were performed on male Wistar strain rats, subcutaneously injected with MSG, at 4 mg/g body weight, on days 2, 4, 6, 8 and 10 of life. When the rats reached the age of 18 months, they were additionally stimulated with a single dose of TSH (Ambinon). When the rats reached the age of 6, 12, or 18 months, their thyroids were isolated and fixed in Bouin's solution. In HE-stained preparations, planimetric and volumetric proportions occupied by the epithelial fraction, colloid and stroma, the number of thyroid follicles per mm2 and the thickness of epithelium were determined. Serum T3 and T4 levels were quantified by RIA. Significance of differences was tested using Student's t test. The weight of experimental rat thyroids showed no significant variations as compared to the controls but were greatest in the group of 12-month-old rats. The same was noted for the volumetric fractions of epithelium, colloid and stroma. The planimetric fractions occupied by epithelium, colloid and stroma in the thyroid remained unchanged and amounted to 60%, 31% and 9%, respectively. The number of follicles per mm2 thyroid cross-section in the MSG-treated 6-, 12- or 18-month-old rats was 131.3, 116.2 and 130.4, respectively, and did not differ from control values. Thickness of follicular epithelium showed no significant variations. Serum T3 levels in the rats examined after 6, 12 or 18 months were increased by 67%, 89% and 33%, respectively, as compared to serum T4 levels. When compared to the controls, the serum T4 level was lower only in the 12-month-old MSG-treated rats and the serum T3 level was higher in 18-month-old MSG-treated rats. The ability of the thyroid to respond to Ambinon stimulation was preserved. The results of our investigations suggest that the rat hypothalamic centers involved in regulation of the pituitary-thyroid axis are slightly affected by neonatal administration of MSG.

Effects of pneumadin (PNM) on the adrenal glands. 6. Further studies on the inhibitory effect of PNM on dexamethasone-induced atrophy of the rat adrenal cortex.

Pneumadin (PNM) is a biologically active decapeptide, which has previously been found to enhanced rat adrenal growth; the mechanism is indirect and probably involves the stimulation of both arginine-vasopressin (AVP) and ACTH release. The effects of 2- and 6-day PNM administration on the atrophic adrenal cortices of rats treated for 8 and 12 days, respectively, with daily subcutaneous injections of 15 or 40 g/100 g body weight of dexamethasone (Dx) were investigated. Morphometry showed that PNM counteracted Dx-induced adrenal atrophy, by preventing the decrease in volume and number of the parenchymal cells. PNM raised aldosterone and corticosterone production of adrenal quarters from Dx-treated rats, but it did not evoke significant changes in the plasma concentrations of the two hormones. The preventive effect of PNM was only partial and almost exclusively evident in rats administered the lower dose of Dx. In light of these findings the following conclusions are drawn: (i) PNM is able to partially overcome the Dx-induced inhibition of the rat hypothalamo-pituitary-adrenal axis, probably by stimulating the pituitary release of AVP and ACTH, that in turn enhance adrenocortical growth; (ii) the PNM-induced improvement of the secretory capacity of atrophic adrenocortical cells is not sufficient to raise the blood level of corticosteroid hormones; and (iii) Dx exerts a direct inhibitory action on adrenocortical cells, which is not counteracted by PNM.

Effects of melatonin, testosterone and the two hormones administered in parallel on epididymis of the rat estrogenized with stilbestrol in the first day of life.

Effect of melatonin, testosterone and of both hormones given in parallel on rat epididymis was tested in rats given a single dose of 1 mg stilbestrol on the first day of the life. The hormones were given daily for 39 days, beginning from the 20th or 28th day of life. The single dose of estrogen treatment resulted in epididymis atrophy, accompanied by changes in glandular epithelium and in its stroma, when the rats reached mature age (59 or 67 days of life). In such rats, LH gonadotropin level was elevated and testosterone level was decreased. Administration of melatonin failed to affect the changes induced by estrogen treatment. Administration of testosterone alone or of testosterone in parallel with melatonin caused the epididymis status to resemble more closely that seen in control animals. Efferent ductules of the testis (head of epididymis) were also demonstrated to be more sensitive to the performed experimental procedures than the duct of the epididymis (body and tail of the epididymis).

Effects of melatonin, testosterone and the two hormones administered in parallel on ventral prostate of the rat treated with stilbestrol in the first day of life.

Effects of melatonin, testosterone and the two hormones administered in parallel on ventral prostate were examined in the rats treated with estrogens in the first day of life. Thirty-nine-day long hormonal stimulation with melatonin, testosterone or the two hormones in parallel was started in rats aged 20, 28, 35 or 45 days. A single dose of estrogens led to atrophy of ventral prostate when the animals reached maturity, which was associated with high LH levels and low testosterone levels in the serum. Melatonin accentuated estrogen-induced changes in prostate morphology of ventral prostate while initemporal administration of melatonin and testosterone led to a resultant effect of testosterone-induced stimulation and melatonin-induced inhibition.

Effects of bombesin on the morphology and function of the rat adrenal cortex: comparison of the acute and chronic responses.

The acute and chronic effects of bombesin (BM) on the structure and function of rat adrenal cortex were investigated by morphometric and radioimmunological techniques. An intraperitoneal bolus injection of 2 micrograms/rat BM markedly raised plasma corticosterone (B) concentration (PBC). The intraperitoneal BM infusion (1 microgram/rat.h-1) for 1, 2 or 4 days evoked a notable increase in the number of adrenocortical cells, without inducing apparent changes in either PBC or B output by adrenal quarters. Since proliferation and expression of specialized functions are mutually exclusive states of cells, our findings suggest that the conspicuous stimulation of adrenocortical-cell proliferation evoked by BM infusion may be responsible for the apparent lack of effect of this treatment on B secretion.