Group 11 complexes with a phosphanylphosphaalkene ligand: preparation and stability study.

The reactivities of two selected phosphanylphosphaalkenes, Ph2CP-PtBu2 (1a) and (p-MeO-Ph)2CP-PtBu2 (1b), toward CuCl, AgCl and (tht)AuCl (tht = tetrahydrothiophene) were investigated. As a result, new phosphanylphosphaalkene dimeric and monomeric complexes were formed (Cu and Ag dimeric and Au monomeric). All obtained products were air and moisture stable and light insensitive.

Solvent Impact on the Diversity of Products in the Reaction of Lithium Diphenylphosphide and a Ti(III) Complex Supported by a tBu2P-P(SiMe3) Ligand.

We present two important trends in the reactivity of the titanium complex [MeNacNacTi(Cl){η2-P(SiMe3)-PtBu2}] (MeNacNac- = [Ar]NC(Me)CHC(Me)N[Ar]; Ar = 2,6-iPr2Ph) with nucleophilic reagents RLi (R = Ph2P, tBuO, (Me3Si)2N, and tBu2N) depending on the reaction medium. Reaction in nonpolar solvent (toluene) leads to three main products: via an autoredox process and nucleophilic substitution at the Ti-atom to afford the Ti(IV) complex [MeNacNacTi(R){η2-P-PtBu2}] (1 for R = PPh2), via the elimination of Me3SiR to afford Ti(III) complex [MeNacNacTi(Cl){η2-P-PtBu2}]-[Li(12-crown-4)2]+ (2), and via 2e- reduction process to afford new ionic complex [{ArNC(Me)CHC(Me)}Ti═NAr{η1-P(SiMe3)-PtBu2}]-[Li(12-crown-4)2]+ (3). Quite differently, the complex [MeNacNacTi(Cl){η2-P(SiMe3)-PtBu2}] reacts with Ph2PLi in THF, unexpectedly yielding two new, four-coordinate Ti(IV) imido complexes 4a [{ArNC(Me)═CHC(H)(Me)-P(PtBu2)}Ti═NAr(Cl)]-[Li(12-crown-4)2]+·(toluene)2 and 4b [{ArNC(CH2)CH═C(Me)-P(PtBu2)}Ti═NAr(Cl)]-[Li(12-crown-4)2]+·(Et2O). Complex 2 dissolved in THF converts to 4a and 4b. 1, 2, 3, 4a, and 4b were characterized by X-ray diffraction. 1, 4a, and 4b were also fully characterized by multinuclear NMR spectroscopy.

Reactions of Lithiated Diphosphanes R2P-P(SiMe3)Li (R = tBu and iPr) with [MeNacnacTiCl2·THF] and [MeNacnacTiCl3]. Formation and Structure of TitaniumIII and TitaniumIV ß-Diketiminato Complexes Bearing the Side-on Phosphanylphosphido and Phosphanylphosphinidene Functionalities.

ß-Diketiminate complexes of TiIII-containing phosphanylphosphido ligands [MeNacnacTi(Cl){η2-P(SiMe3)-PR2}] (MeNacnac- = [Ar]NC(Me)CHC(Me)N[Ar]; Ar = 2,6-iPr2C6H3) were prepared by reactions of [MeNacnacTiCl2·THF] with lithium derivatives of diphosphanes R2P-P(SiMe3)Li (R = tBu, iPr) in toluene solutions. Surprisingly, reactions of [MeNacnacTiCl2·THF] with R2P-P(SiMe3)Li in THF solutions led to TiIV complexes containing phosphanylphosphinidene ligands [MeNacnacTi(Cl)(η2-P-PtBu2)] via an autoredox path involving a migration of a nitrene NAr from the Nacnac skeleton to the Ti centers. Solid-state structures of [MeNacnacTi(Cl){η2-P(SiMe3)-PtBu2}] (1a) and [MeNacnacTi(Cl)(η2-P-PtBu2)] (two isomers 2a1, 2a2) together with [MeNacnacTi(Cl){η2-P(SiMe3)-PiPr2}] (1b) and [MeNacnacTi(Cl)(η2-P-PiPr2)] (2b) were established by the single-crystal X-ray diffraction and display clearly side-on geometry of the (Me3Si)P-PR2 and P-PR2 moieties in the solid state. Phosphanylphosphinidene complexes [MeNacnacTi(Cl)(η2-P-PR2)] indicate that the 31P NMR resonances of phosphinidene P atoms appear at a very low field in solution and in the solid state.

Association between progesterone and estradiol-17beta treatment and protein expression of pgr and PGRMC1 in porcine luminal epithelial cells: a real-time cell proliferation approach.

The correct functionality (sensitivity and receptivity) of endometrial tissue is regulated by paracrine and endocrine pathways that activate several mediators or metabolic pathways and gene cascades. This study aimed to investigate the influence of E2 and P4 on progesterone receptor (PGR) and progesterone receptor membrane component 1 (PGRMC1) protein expression in porcine luminal epithelial cells and their influence on the proliferation of these cells in real-time. Surface uterine luminal epithelial cells were removed using sterile surgical blades from uterine horns of ten crossbred anestrus gilts. Following treatment with collagenase I, cells were separated and transferred into 48-well E-Plates for use in a realtime cell analyzer (RTCA). The luminal epithelial cells were cultured in vitro (IVC) in standard DMEM cell culture medium and incubated with E2 (10 pg/ml, 40 pg/ml, 500 pg/ml) and P4 (10 ng/ml, 40 ng/ ml, 500 ng/ml). The cell proliferation index was analyzed after 0-240 h, 0-120 h, 120-240 h. After using the RTCA analysis we found increased proliferation of luminal epithelial cells after treatment of low doses of P4 (10 and 40 ng/ml), (P < 0.001). Higher doses of P4 led to decrease of proliferation (P < 0.001). Conversely, higher doses of E2 (500 pg/ml) increased the proliferation index as compared to low doses (10 pg/ml) and control (P < 0.001). Confocal microscopic observations revealed that higher concentrations of E2 upregulate the expression of both PGR and PGRMC1. Additionally, P4 used in lower concentrations stimulated the expression of these receptors, too. Our study presents a new influence of E2 and P4 on the expression of PGR and PGRMC1 and on the real-time proliferation of porcine luminal epithelial cells. The relationship between PGR or PGRMC1 expression and the proliferation of luminal epithelial cells may be influenced (up- or down regulated) by E2 or P4 in a steroid type- and dose-dependent manner.

Association between the expression of LHR, FSHR and CYP19 genes, cellular distribution of encoded proteins and proliferation of porcine granulosa cells in real-time.

The process of granulosa cell luteinization is part of the main process determining growth, differentiation and proliferation of these cells. Although the mechanisms underlying the regulation of luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR) and cytochrome P450 aromatase expression in mammalian granulosa cells is well understood, still little is known about the expression of mRNA and encoded proteins in relation to cell proliferation and luteinization in vitro. Porcine granulosa cells were observed in vitro at a168-h period while undergoing real-time proliferation using an RTCA system. Furthermore, LHR, FSHR and CYP19 mRNA expression were detected using RQ-PCR after 168 h of in vitro culture (IVC) at 24-h intervals, and LHR, FSHR and P450arom were examined by confocal microscopic observation at 0 h, 24 h, 48 h, 96 h, and 168 h of IVC. We found increased expression of LHR and CYP19 mRNA at 24 h and 48 h of IVC compared to the other stages (P less than 0.01, P less than 0.001), whereas FSHR mRNA was higher only at 0 h (P less than 0.001). In contrast, LHR, FSHR and P450arom protein expression was significantly higher at the end of the 168-h IVC period compared to 0 h, 24 h, 48 h and 96 h (P less than 0.001). LHR, FSHR and P450arom were distributed in the cytoplasm of porcine GCs at each time point of IVC. When analyzing cell proliferation, differences in cell index were observed (at least P less than 0.05) between the first (0-24 h) and the last period (144-168 h) of IVC; however, soon after 24 h of IVC a logarithmic increase in proliferation was also seen. We assume that the expression of LHR, FSHR and CYP19 mRNAs depends on the period of in vitro cultivation and may be linked with the luteinization process of porcine GCs. Furthermore, the patterns of mRNA and protein expression suggest a post-transcriptional regulation of LHR, FSHR and P450arom. In summary, it can be presumed that mRNA and protein expression and in vitro luteinization and proliferation of porcine GCs are regulated by different mechanisms, because not all of these processes are correlated.

Analysis of the origin and importance of acetone and isopropanol levels in the blood of the deceased for medico-legal testimony.

UNLABELLED: The aim of the study was to analyze the incidence of acetone and isopropanol in the blood of the deceased, and to assess cases in which the compounds have been detected with a focus on their origin and usefulness for medico-legal testimony. MATERIAL AND METHODS: The study material consisted of results of tests detecting ethyl alcohol and reports of autopsies performed at the Department of Forensic Medicine, Medical University of Warsaw, from January 2008 to April 2009 - a total of 2,475 cases. The test group proper (group B) comprised only those cases in which acetone was detected in blood, either with or without isopropanol [n = 202 (8.2%)]. The blood levels of isopropanol varied depending on the cause of death. The need for differentiating the origin of isopropanol in the case of its presence in the blood of the deceased was pointed out. RESULTS: The results of the present study show that the differentiation should be based on the isopropanol and acetone concentration ratio, as isopropanol concentration alone is not sufficient for preparing expert opinions. Even high concentrations of isopropanol, when accompanied by even higher concentrations of acetone, imply that isopropanol could have been formed as a result of acetone transformations. Isopropanol concentrations exceeding acetone levels strongly point to the exogenous origin of isopropanol, particularly when high levels of ethanol are concurrently detected.

Study on connexin gene and protein expression and cellular distribution in relation to real-time proliferation of porcine granulosa cells.

Granulosa cells (GCs) play an important role during follicle growth and development in preovulatory stage. Moreover, the proteins such as connexins are responsible for formation of protein channel between follicular-cumulus cells and oocyte. This study was aimed to investigate the role of connexin expression in porcine GCs in relation to their cellular distribution and real-time cell proliferation. In the present study, porcine GCs were isolated from the follicles of puberal gilts and then cultured in a real-time cellular analyzer (RTCA) system for 168 h. The expression levels of connexins (Cxs) Cx36, Cx37, Cx40 and Cx43 mRNA were measured by RQ-PCR analysis, and differences in the expression and distribution of Cx30, Cx31, Cx37, Cx43 and Cx45 proteins were analyzed by confocal microscopic visualization. We found higher level of Cx36, Cx37, and Cx43 mRNA expression in GCs at recovery (at 0 h of in vitro culture, IVC) compared to all analyzed time periods of IVC (24, 48, 72, 96, 120, 144 and 168 h; P<0.001). On the other hand, the expression level of Cx40 transcripts was higher after 24 h of IVC compared to 0 h and the other times of IVC (P<0.001). Similarly to mRNAs, the expression levels of Cx31, Cx37 and Cx45 proteins were higher before (0 h) compared to after 168 h of IVC. The expression of Cx30 and Cx43, however, did not vary between the groups. In all, the proteins were distributed throughout the cell membrane rather than in the cytoplasm both before and after IVC. After 24 h of IVC, we observed a significant increase in the proliferation of GCs (log phase). We found differences in the proliferation index between 72-96 and 96- 140 h within the same population of GCs. In conclusion, the decrease in the expression of Cx mRNAs and proteins following IVC could be associated with a breakdown in gap-junction connections (GJCs), and leads to the decreased of their activity, which may be a reason of non-functional existence of connexon in follicular granulosa cells. These data indicated that the differentiation and proliferation of GCs and lutein cells are regulated by distinct mechanisms in pigs.

Involvement of the orphan nuclear receptor SF-1 in the effect of PCBs, DDT and DDE on the secretion of steroid hormones and oxytocin from bovine granulosa cells.

Polychlorinated biphenyls (PCBs), DDT and its metabolite (DDE) belong to estrogen-like endocrine disruptors. However, though their activity is approximately 1000-fold lower than the activity of estradiol (E2), this steroid's high concentration in follicular fluid and incubation media does not inhibit the influence of these xenobiotics. It was hypothesized that these xenobiotics might affect Steroidogenic Factor-1 (SF-1) and impair ovary function. To test this hypothesis, granulosa cells were obtained from ovarian follicles >1 or <1cm in diameter, which were treated with PCB-77, PCB-153, DDT or DDE (each at 10ng/ml), alone or jointly with an SF-1 antagonist (F0160). Treatment with the SF-1 antagonist inhibited (P<0.05) the secretion of P4 from cells of both sizes of follicles, as induced (P<0.05) by an SF-1 activator (HxP), DDE or PCB-153. All xenobiotics and HxP stimulated (P<0.05) the synthesis and secretion of oxytocin (OT). However, the effect on mRNA expression for NP-I/OT, which is OT precursor, was inhibited (P<0.05) by F0160 in all cultures treated with PCB-77, except for granulosa cells derived from follicles <1cm. Moreover, F0160 inhibited the effect on OT secretion of HxP, as well as all xenobiotics except for PCB-77 and DDE, in granulosa cells derived from follicles <1cm. Xenobiotic treatment did not affect (P>0.05) the expression for SF-1 mRNA. It is suggested that the SF-1 receptor may be involved in the adverse effects of xenobiotics on P4 secretion as well as the synthesis and secretion of OT.

Effect of cortisol on neurophysin I/oxytocin and peptidyl glycine-alpha-amidating mono-oxygenase mRNA expression in bovine luteal and granulosa cells.

Cortisol stimulates the synthesis and secretion of oxytocin (OT) from bovine granulosa and luteal cells, but the molecular mechanisms of cortisol action remain unknown. In this study, granulosa cells or luteal cells from days 1-5 and 11-15 of the oestrous cycle were incubated for 4 or 8 h with cortisol (1 x 10(-5), 1 x 10(-7) M). After testing cell viability and hormone secretion (OT, progesterone, estradiol), we studied the effect of cortisol on mRNA expression for precursor of OT (NP-I/OT) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA). The influence of RU 486 (1 x 10(-5) M), a progesterone receptor blocker and inhibitor of the glucocorticosteroid receptor (GR), on the expression for both genes was tested. Cortisol increased the mRNA expression for NP-I/OT and PGA in granulosa cells and stimulated the expression for NP-I/OT mRNA in luteal cells obtained from days 1-5 and days 11-15 of the oestrous cycle. Expression for PGA mRNA was increased only in luteal cells from days 11-15 of the oestrous cycle. In addition, RU 486 blocked the cortisol-stimulated mRNA expression for NP-I/OT and PGA in both types of cells. These data suggest that cortisol affects OT synthesis and secretion in bovine ovarian cells, by acting on the expression of key genes, that may impair ovary

Elevated blood plasma concentrations of active ghrelin and obestatin in benign ovarian neoplasms and ovarian cancers.

Both ghrelin and obestatin are derived from preproghrelin by post-translational processing. The two peptides are secreted into the blood but circulating levels of these peptides have not been assessed in women with ovarian tumours. Therefore, the purpose of this study was to evaluate peripheral blood concentrations of active and total ghrelin and obestatin in patients with benign ovarian tumours and those with ovarian cancer. The studies were conducted on 22 patients operated due to benign ovarian tumours, and 31 patients operated due to ovarian cancer. A control group consisted of 32 women, 24 to 65 years of age. Both in women with benign ovarian tumours and those with ovarian cancer blood concentrations of active ghrelin and obestatin were higher than in the control group (active ghrelin: 90 +/- 4, 84 +/- 4 and 56 +/- 9 pg/ml, respectively, obestatin: 660 +/- 36; 630 +/- 30 and 538 +/- 31 ng/ml (x +/- SE), respectively). In contrast, total ghrelin concentrations in blood were similar in the studied groups. The alterations resulted in increased values of active to total ghrelin concentration ratio in the peripheral blood of patients with benign ovarian tumours or with ovarian cancer (0.79 +/- 0.02 and 0.93 +/- 0.05, respectively vs 0.58 +/- 0.02 in the control group). Due to the absence of any convincing proof for the presence of a functional GHS-R-1a receptor for ghrelin in human ovaries it did not seem probable that the observed elevated levels of active ghrelin and obestatin were directly linked to development of ovarian tumours.

Elevated blood active ghrelin and normal total ghrelin and obestatin concentrations in uterine leiomyoma.

Ghrelin and obestatin originate from the same peptide precursor, preproghrelin. Both peptides are secreted in the blood. We investigated serum active and total ghrelin and obestatin concentrations in women with uterine myomatosis. Serum concentrations of active ghrelin in uterine leiomyoma were significantly higher compared to women in the control group (86 +/- 3 vs 56 +/- 9 pg/ml, respectively; p < 0.02). On the other hand, serum concentrations of total ghrelin and obestatin in uterine leiomyoma did not differ from those in the control group. In the control group the ratio of active to total ghrelin concentrations amounted to 0.62, while in women with uterine myoma it was 0.95, pointing to a prevalence of the active form of ghrelin in women with uterine myoma. Also the ratio of active ghrelin concentration to obestatin concentration was higher in the latter group while the ratio of total circulating ghrelin to obestatin concentrations was similar in the two groups. The data may suggest a role of active ghrelin in the development of a myoma. Moreover, the results indicate that increased blood ratios of active to total ghrelin and to obestatin concentrations are not specific for cachexia.

G protein receptors 7 and 8 are expressed in human adrenocortical cells, and their endogenous ligands neuropeptides B and w enhance cortisol secretion by activating adenylate cyclase- and phospholipase C-dependent signaling cascades.

Neuropeptides B and W (NPB and NPW) are regulatory peptides that act via two subtypes of G protein-coupled receptors, named GPR7 and GPR8. RT-PCR demonstrated the expression of these receptors in both zona glomerulosa and zona fasciculata-reticularis (ZF/R) cells of the human adrenal cortex. NPB and NPW did not affect aldosterone secretion from dispersed zona glomerulosa cells but enhanced cortisol production from ZF/R cells, NPB being more effective than NPW. NPB evoked sizable cAMP and inositol triphosphate responses from ZF/R cells, which were abrogated by the adenylate cyclase inhibitor SQ-22536 and the phospholipase C inhibitor U-73122, respectively. Cortisol response to NPB was lowered by either SQ-22536 and the protein kinase (PK) A inhibitor H-89 or U-73122 and the PKC inhibitor calphostin-C and abolished by the simultaneous exposure to H-89 and calphostin-C. NPW elicited only a rise in cAMP production from dispersed ZF/R cells, and its cortisol response was suppressed by both SQ-22536 and H-89. PreproNPB and preproNPW mRNAs were detected in human adrenal cortexes. We conclude that: 1) NPB and NPW exert a secretagogue action on human ZF/R cells, probably acting in an autocrine-paracrine manner; and 2) the effect of NPB is mediated by both the adenylate cyclase/PKA and the phospholipase C/PKC cascades, whereas that of NPW involves only the activation of the former signaling pathway.

Prolonged cerebellin administration inhibits the growth, but enhances steroidogenic capacity of rat adrenal cortex.

Cerebellin is a 16-amino acid peptide, that has been previously found to acutely stimulate steroid secretion from rat adrenal cortex in vivo and in vitro. We have investigated the effects of a prolonged cerebellin treatment (daily injections of 15 nmoles/kg for 6 consecutive days) on the growth and secretion of rat adrenal cortex. Cerebellin lowered adrenal weight, and morphometry showed that this was due to the decrease in the volume of each adrenocortical zone exclusively ensuing from the reduction in the number of its parenchymal cells. Cerebellin did not alter plasma concentration of ACTH, but it raised the levels of circulating aldosterone and corticosterone. The conclusion is drawn that cerebellin chronic administration evokes a marked hypoplastic atrophy of rat adrenocortical cells, that is coupled with an enhanced ACTH-independent steroidogenic capacity of the remaining parenchymal cells.

Prolonged orexin administration stimulates steroid-hormone secretion, acting directly on the rat adrenal gland.

Orexins A and B are two hypothalamic peptides, that play a role in the central control of food intake. Orexins act via two subtypes of receptors: OX1R which is selective for orexin A, and OX2R which binds both orexins. Reverse transcription-polymerase chain reaction demonstrated the expression of both OX1R and OX2R gene in the adrenal cortex of adult female rats. The prolonged systemic administration of orexins A and B (20 ng/kg x day, for 7 days) affected neither adrenal weight and the morphology of adrenocortical zones (as evaluated by morphometric techniques) nor ACTH plasma concentration in rats. In contrast, the treatment with both orexins increased plasma concentration of both aldosterone and corticosterone. Taken together, these findings indicate that orexins exert a marked direct chronic secretagogue action on adrenocortical cells, acting through both OX1R and OX2R.

Anterior pituitary corticotropes of adrenalectomized, leptin-administered rats.

Leptin (LEP), the product of the ob gene is an adipose-tissue secreted hormone that acts to decrease caloric intake and increase energy expenditure. Some observations suggest the mutual relationship between leptin and blood ACTH levels. In the rat acute LEP administration enhances blood ACTH levels while prolonged treatment lowers blood corticotropin concentrations. Since the pituitary-derived ACTH is an important element in functioning of that loop, studies were undertaken to investigate the effect of prolonged LEP administration on anterior pituitary ACTH cells of adrenalectomized rats. Studies were performed on bilaterally adrenalectomized adult female rats. They were administered for 3 or 6 days with 2.7 nmol/rat/day LEP (recombinant human leptin) or with the vehicle (0.9% NaCl). LEP administration did not affect the body weight of bilaterally adrenalectomized rats. During the whole experiment the average increase in body weight was 3.9-4.3 g/day. LEP administration into adrenalectomized rats had no effect on anterior pituitary weight. This treatment resulted in a significant increase in pituitary ACTH concentration and content, and these changes were accompanied by a potent decrease in blood corticotropin level. LEP administration into adrenalectomized rats only insignificantly lowered the quantity of anterior pituitary ACTH-immunoreactive cells and their average area. On the opposite, the average volume of pituitary corticotropes of LEP-treated rats was notably lower than that in adrenalectomized-vehicle administered ones. Results of performed experiments clearly demonstrate that prolonged LEP administration results in a notable inhibition of the growth and secretory activity of anterior pituitary corticotropes of the adrenalectomized rats.

Vascularized bone marrow transplanted in orthotopic hind-limb stimulates hematopoietic recovery in total-body-irradiated rats.

Hematopoietic recovery after bone marrow transplantation (BMT) is reported to be slow with long-lasting immune deficiency. This may be attributable to lack of a proper microenvironment for hematopoietic cell proliferation and differentiation. We have designed a model in which complete hematopoietic reconstitution of lethally irradiated rats can be achieved by vascularized bone marrow transplantation (VBMT) in an orthotopic hind-limb graft. The aim of the study was to investigate the process of repopulation of bone marrow cavities and peripheral blood of irradiated rats after VBMT and, in particular, to follow the contribution of grafted BM cells and residual recipient BM cells in hematopoietic regeneration. Lewis hind-limbs were transplanted orthotopically to totally irradiated (8 Gy) syngeneic sex-mismatched recipients (VBMT). In the control group 8 x 10(7) BM cells in suspension were injected intravenously (BMCT). After 10 days BM and peripheral blood (PB) cells were collected from the recipient. For cell subset analysis cytomorphological evaluation of BM smears and flow cytometry of PB cells were performed. Additionally, PCR was performed using specific primers for rat Y chromosome (sex-determining region Y-Sry) to detect male (donor or recipient) cells in sex-mismatched BM graft recipients and the products were analyzed by electrophoresis. VBMT brought about much faster replenishment of nucleated cells in BM and PB than did BMCT. Cytometry analysis of PB cells revealed more lymphocytes in VBMT than in BMCT recipients. The amount of donor DNA of bands corresponding to Y-Sry was also higher in PB cells of VBMT than of BMCT recipients. The presence of host DNA was observed in PB cells of VBMT rats but was not detected in PB population of BMCT recipients. VBMT is highly effective in hematopoietic reconstitution of irradiated recipients. The fast cell maturation and repopulation may be due to the presence of stromal cells transplanted in a normal spatial relationship with donor hematopoietic cells in hind-limb graft. Self renewal of radioresistant host cells was seen after VBMT but not after BMCT.

Fast lymphoid reconstitution after vascularized bone marrow transplantation in lethally irradiated rats.

BACKGROUND: Bone marrow (BM) transplantation for treatment of hematological and solid malignancies is routinely carried out in conjunction with radio- and chemotherapy. Many patients achieve complete remission of the malignant process; however, their lymphohematopoietic recovery remains in most cases incomplete. This is probably due to the functional changes in the recipient BM stromal cells subsequent to myeloablative therapy. Transplantation of BM hematopoietic cells in a spatial relationship with stromal cells would give an insight into the kinetics of hematological repopulation of the recipient. The aim of this study was to investigate the lymphopoietic reconstitution of irradiated rats after vascularized bone marrow transplantation (VBMT) in comparison with i.v. bone marrow cell (BMC) infusion. METHODS: Lewis rats were totally irradiated with 8Gy and repopulated with syngeneic BMC introduced i.v. or in orthotopic hind limb graft. Ten days after irradiation and BMC graft BM, peripheral blood (PB) and mesenteric lymph nodes (MLN) were collected. The yield and the phenotype of cells were analyzed. RESULTS: VBMT brings much higher cell repopulation of BM cavities of lethally irradiated rats than BMC infusion. Orthotopic hind limb graft promotes also rapid lymphocyte replenishment of PB and MLN of lethally irradiated syngeneic recipients. The population rate of BMC, PB lymphocytes, and MLN lymphocytes was higher after VBMT than BMC injection in suspension. The percentage of T and B lymphocytes in PB and MLN on day 10 after VBMT was comparable with control values. Reconstituted PB lymphocytes showed two subsets of CD4+ cells: "bright" and "dull." All CD4+ cells in PB lymphocytes of i.v. BMC infused recipients expressed low level of these molecules ("dull" subset). CONCLUSIONS: The results of our studies indicate that the presence of stromal cells in their close relationship with stem cells is essential for the fast lyphohematopoietic repopulation of irradiated recipients. The population of CD4+dull cells may represent immature cells. These cells were not found in MLN of VBMT rats. All MLN CD4+ cells represented the "bright" subset, what suggests that the process of cell maturation may occur in the lymphoid organs.

Skin allografts--lymph veiled (dendritic) cells are responsible for initiation of rejection in canine skin/SCID mouse chimera model.

Skin allografts are acutely rejected despite of intensive immunosuppressive therapy. Resistance of skin dendritic cells to immunosuppressive drugs and irradiation may be responsible for this phenomenon. Skin allograft is a site of interaction between the dendritic cells and lymphocytes of the donor and host origin and "direct" and "indirect" pathway of antigen presentation. Increasing evidence supports the significant role for the "indirect" allorecognition in graft rejection. To investigate a critical role of skin dendritic cells in the "indirect" allorecognition and graft destruction we have used a canine skin to severe-combined-immunodeficient (SCID)-mice transplantation model. At the time the skin grafts were deprived of own dendritic (Langerhans) cells, SCID mice were reconstituted with allogeneic canine whole lymph leukocytes, lymph lymphocytes, lymph veiled (dendritic) cells or peripheral blood mononuclear cells, and an early phase of skin rejection was evaluated in histopathological studies. We demonstrated that circulating canine allogeneic veiled cells facilitate recruitment of T lymphocytes into skin graft and promote an extensive graft destruction, compared to the less expressed effect of allogeneic peripheral lymph lymphocytes or blood mononuclear cells. These drug and radiation-resistant dendritic cells may be responsible for initiation of the difficult to control rejection process.