Synergistic effects of prostaglandin F2alpha and tumor necrosis factor to induce luteolysis in the pig.

There is ample evidence that prostaglandin F2alpha (PGF2alpha) is a luteolytic substance in sows, however, there is also some evidence that it may stimulate progesterone (P4) secretion in young corpora lutea (CL). In vitro studies also suggested that tumor necrosis factor alpha (TNF) is inhibitory to luteal cell P4 and estradiol-17beta (E2) release. Since E2 is a strong luteotropic substance in porcine CL, we studied the effects of intraluteal application of PGF2alpha and TNF alone and in combination on the secretion of P4 and E2 in freely moving sows. Furthermore, the effects of intraluteal infusion of E2 and its stereoisomer, estradiol-17alpha, on luteal function, were also determined. Microdialysis systems were implanted into CL at Day 10 of the estrous cycle. After a 24-h recovery period, PGF2alpha (10(-6) M) or E2 (10(-6) M) was applied daily for 6 h into the CL. PGF2alpha caused a stimulation of E2 and P4, and E2 also stimulated P4 secretion at Days 11 and 12, but the stimulatory effect of both substances diminished as the CL approached luteolysis. Intraluteal TNF application resulted in a transient increase of P4 secretion, which was followed by a dramatic reduction of P4 release. When TNF-pretreated CL were exposed to PGF2alpha at Day 11 of the estrous cycle, the prostaglandin was no longer able to stimulate but rather inhibited E2 and P4 secretion. Intraluteal application of estradiol-17alpha had no effect on P4 secretion. These results are suggestive that the PGF2alpha-induced E2 secretion in young and middle-aged CL is stimulatory to P4 secretion. Under the influence of macrophage-derived TNF production, E2 secretion is inhibited, and thereby PGF2alpha and TNF cause functional luteolysis.

Ligand blot analysis of insulin-like growth factor-binding proteins using biotinylated insulin-like growth factor-I.

A nonradioactive method for the detection of insulin-like growth factor-binding proteins (IGFBPs) was developed utilizing human recombinant insulin-like growth factor-I (IGF-I) biotinylated with N-hydroxysuccinimido-biotin. Human plasma samples were separated by 15% SDS polyacrylamide gel electrophoresis, and proteins were transferred to nitrocellulose by Western blotting. The nitrocellulose sheets were incubated overnight with IGF-I-biotin at 4 degrees C. The next day streptavidin-peroxidase was added for 1 h, and IGFBPs were visualized by an enhanced chemiluminescence system. Five characteristic bands with molecular weights of 41 and 39 (IGFBP-3), 34 (IGFBP-2), 30 (IGFBP-1) and 24 kD (IGFBP-4) were detected. Binding was specific for IGFs, since unlabeled IGF-I inhibited IGF-I-biotin binding. IGFBP ligand blots with biotinylated IGF-I and (125)I-IGF-I yielded comparable results. The suitability of the new assay for clinical purposes was demonstrated by several clinical examples. In summary, a rapid, reliable, nonradioactive assay for qualitative analysis of IGFBPs has been developed.

Transjugular intrahepatic portosystemic shunt in nonliver transplant candidates: is it indicated?

BACKGROUND: Transjugular intrahepatic portosystemic shunt (TIPS) is an effective treatment of severe portal hypertension complications. Liver transplantation (LT) candidacy has not been a prerequisite to TIPS placement in some medical centers. OBJECTIVES: To investigate the outcome and survival of non-LT candidates after TIPS. METHODS: From November 1991 to February 1994, all patients referred for TIPS placement were evaluated for LT candidacy. Exclusions for LT included: age (> 70 yr), other significant medical conditions, or noncompliance. Indications for TIPS included refractory variceal bleeding during an acute bleed, recurrent bleeding after more than or equal to four sessions of sclerotherapy, or refractory ascites. RESULTS: Sixty patients received TIPS. Nineteen were considered non-LT candidates. Over a 2-yr follow-up, 14 of these non-LT candidates did not survive. Their median age was 63.5 compared with 56.5 yr for LT candidate nonsurvivors (p < 0.05). Among the 14 non-LT candidate nonsurvivors, 10 were Childs C class, and eight had emergent TIPS placement. The 2-year mortality rate was 84% for non-LT candidates versus 24% for LT candidates. Median survival time for non-LT candidates was 2.6 months compared with 20 months in the LT candidates (p < 0.001). Only one death was due to a TIPS-related complication. CONCLUSIONS: TIPS is unquestionably an advancement in the management of patients with portal hypertension complications. Non-LT candidates, compared with LT candidates, tended to be older and of a Child-Pugh C class, and they had survival rates often less than 90 days post-TIPS. Given these high mortality rates, we need to address whether TIPS is indicated in these non-LT candidates.

A gut-homing, oligoclonal CD4+ T cell population in severe-combined immunodeficient mice expressing a rearranged, transgenic class I-restricted alpha beta T cell receptor.

We studied the peripheral T cell compartment of H-2b severe combined immunodeficient (scid) mice that express a transgenic (tg) alpha beta T cell receptor (TcR) specific for the H-Y (male) epitope presented by the H-2 class I Db molecule. Large populations of CD3+ NK1.1-TCR beta T+ T cells were present in spleen, mesenteric lymph nodes, peritoneal cavity, lamina propria and epithelial layer of the small and large intestine of 6- to 10-month-old, male and female tg scid mice. Only low numbers of CD3+ T cells were recovered from inguinal, popliteal, or axillary lymph nodes. We studied CD4+ T cells in these tg scid mice. CD4+ T cells were found in the peritoneal cavity, in the mesenteric lymph nodes and in the intraepithelial layer and lamina propria of the gut. All CD4+ T cells were CD44+ (i.e. showed evidence of antigen-driven differentiation) and expressed the tg V beta 8.2 TcR beta-chain (TcR beta T+). Only few CD4+ T cells expressed the tg V alpha 3+ TcR alpha-chain (TcR alpha T). cDNA was prepared from CD4+ T cells from spleen or mesenteric lymph nodes of individual male and female tg scid mice; sequence analyses of polymerase chain reaction-amplified, endogenous TcR alpha-chain (TcR alpha E) transcripts indicated that > 90% of the TcR alpha E-chain transcripts were in-frame, that the TcR alpha E repertoire in CD4+ T cell populations was oligoclonal, and that the TcR alpha E repertoire was different in individual tg scid mice. Hence, an oligoclonal, leaky CD4+ T cell population is selected in tg scid mice that apparently responds to gut-derived antigens. No inflammatory bowel disease (IBD) was evident in the small or large intestine of 6- to 10-month old tg scid mice. After adoptive transfer of purified CD4+ T cells (10(5) cells per mouse) from tg scid mice into non-tg H-2b scid mice, CD4+ TcR alpha T-beta T+ cells were found in gut tissues of the immunodeficient host. Transplanted scid mice developed clinical and histological signs of IBD. An oligoclonal, gut-homing, memory/effector CD4+ CD44+ TcR beta T+ TcR alpha T-T cell subset from leaky tg scid mice thus has a pathogenic potential when released from the control of TcR beta T+ TcR alpha T+ T cells.

Regional specialization of intraepithelial T cells in the murine small and large intestine.

We investigated intraepithelial T cells from the small intestine, SI (jejunum, ileum) and the large intestine, LI (colon) of euthymic (BALB/c, H-2d; C.B-17+/+, H-2d; C57BL/6, H-2b) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/xid) mice. From individual euthymic and athymic mice, 7 x 10(6) intraepithelial lymphocytes (IEL) per mouse were isolated from the SI. Ten-fold lower numbers of IEL were obtained from the LI epithelium (4 x 10(5) IEL per mouse). Thymus-dependent and -independent T cells represented > 80% of SI-IEL but the fraction of T cells was reduced from 20% to 40% in LI-IEL. In euthymic mice, alpha beta T cells predominated in SI-IEL and in particular in LI-IEL populations, while SI-IEL and LI-IEL populations of athymic mice contained predominantly gamma delta T cells. The intraepithelial T cell subset distribution was different in SI versus LI: mainly CD8+ T cells were present in the SI, but a large CD4+ T cell subset was present in the LI. 'Double positive' CD4+ CD8 alpha+ T cells were present mainly in the SI epithelium but were rare in the LI epithelium. In euthymic as well as athymic mice, T cells expressing the homodimeric CD8 alpha alpha isoform predominated in the SI epithelium, while T cells expressing the heterodimeric CD8 alpha beta isoform predominated in the LI epithelium. LI-derived TCR alpha beta+ IEL displayed the CD2+ CD28+ LPAM-1/2- M290+ phenotype, and a fraction of them expressed the L-selectin LECAM-1. In contrast, a large fraction of TCR alpha beta+ SI-IEL was CD2- CD28- LPAM-1/2- M290+ and LECAM-1-. RAG-1/2 expression was detectable by RT-PCR in IEL from the SI but not the LI. Striking differences in phenotype were thus apparent between thymus-dependent and thymus-independent T cells in the epithelial layer of the jejunum/ileum and the colon of the mouse.

Luteotropic and luteolytic effects of oxytocin in the porcine corpus luteum.

The presence and the release of oxytocin (OT) by corpora lutea (CL) of a number of species (Wathes et al. 1986, Watkins and Choy 1988) including ruminants (Ivell and Richter 1984, Hirst et al. 1986, Rodgers et al. 1983, Sawyer et al. 1986), primates (Dawood and Khan-Dawood 1986, Khan-Dawood 1987, Maas et al. 1992, Khan-Dawood et al. 1993), and the pig (Pitzel et al. 1984, Einspanier et al. 1991, Jarry et al. 1992) have been amply verified. Conflicting results concerning the effects of OT on steroidogenesis have been published; the peptide has been shown to be luteotrophic (Sawyer et al. 1986, Maas et al. 1992, Jarry et al. 1990), to have no effects (Rodgers et al. 1985) or to be luteolytic (Auletta et al. 1984, Auletta et al. 1988, Pitzel et al. 1988) and it appears that this confusion is only in part due to species differences but also the age of the luteal tissue seems to be of crucial importance for the understanding of the effects of OT (Schams et al. 1983, Wuttke et al. 1993, 1994). In the present contribution we will focus largely on our results obtained in the pig and where applicable, compare them with those obtained in other species. We will thus demonstrate that OT is released by luteal cells (Jarry et al. 1990, Einspanier et al. 1991, Jarry et al. 1992) and that luteal cells have OT receptors (Sernia et al. 1989, Pitzel et al. 1993a) which mediate the effects of the peptide on steroidogenesis. Finally, we will address the question whether OT is inhibitory or stimulatory to progesterone (P) and estradiol (E2) release, and we will come to the conclusion that OT is both luteotropic and luteolytic (Wuttke et al. 1993, 1994). The CL of all species investigated so far consists of two steroidogenic cell types. The so-called large luteal cells stem from follicular granulosa cells and they appear to be barely responsive to luteinizing hormone (LH)/human chorionic gonadotrophin (hCG) but they are highly receptive to prostaglandin F2 alpha (PGF2 alpha) (Hansel and Dowd 1986, Pitzel et al. 1990). Furthermore, they appear to produce OT (Rodgers et al. 1983, Theodosis et al. 1986). The small luteal cells are believed to derive from the follicular theca cells (Hansel and Dowd 1986, Pitzel et al. 1990). They are LH-receptive but synthesize few, if any, regulatory peptides. In the last few years it has become increasingly evident that cells deriving from the white blood cell line are involved in processes such as ovulation and luteolysis. Of crucial importance for the understanding of luteolysis is the morphological observation that macrophages invade the CL at the time of luteal regression (Adashi 1990, Paavola 1977, Kirsch et al. 1981).

Luteotrophic and luteolytic actions of ovarian peptides.

Corpora lutea of all species investigated so far, including the human, produce oxytocin and a variety of other regulatory peptides. The role of these peptides is largely unknown. The subtypes of large luteal cells are able to produce tumour necrosis factor (TNF) and at the end of the luteal phase TNF-producing macrophages invade the aged corpus luteum, indicating that this cytokine may be involved in the process of luteolysis. The present contribution reviews briefly the known functions of oxytocin and substance P in the corpus luteum and then elaborates the possible involvement of luteal and macrophage TNF during luteolysis. Oxytocin applied to intact corpus luteum stimulates the secretion of progesterone and oestradiol. The stimulation of progesterone secretion by oxytocin is due to the stimulated oestrogen production. TNF, when tested in vitro, inhibits both luteal cell progesterone and oestradiol production. The TNF-mediated inhibition of aromatase activity therefore prevents the luteotrophic effects of a variety of peptides including oxytocin. This appears to be the mechanism by which TNF induces luteolysis.

The diversity of in-frame TCR delta chain transcripts in aging SCID mice.

From spleen, liver, bone marrow, and gut of old C.B-17 scid/scid (SCID) mice, TCR delta chain transcripts were amplified by the polymerase chain reaction (PCR) using V delta 1-, V delta 2-, V delta 3-, V delta 4-, V delta 5-, V delta 6-, and C delta-specific primers. Selectively amplified, TCR delta-chain-encoding cDNA from these organs of five old SCID mice was cloned, and 175 randomly selected clones were sequenced. In this panel, 44 distinct rearrangement events were detected, 82% (36/44) of which were in-frame. For V delta 2, V delta 4, V delta 5, and V delta 6, 34 in-frame transcripts were found in five old SCID mice. No potentially functional transcript containing V delta 3 was found and an unusual type of transcript containing the V delta 1 gene segment spliced directly in-frame to C delta was the predominant type of V delta 1 expression. Junctional diversity was evident in most sequenced clones indicating an extensive potential diversity of SCID-derived TCR delta chains. Identical transcripts were amplified from different organs of the same old SCID mouse. No organ-specific expression of V delta genes was evident. TCR delta chain transcripts could be neither amplified from young SCID mice nor from young SCID mice nor from young or old scid/scid nu/nu mice (bred on a BALB/c background). Hence, an apparently thymus-dependent development of oligoclonal populations of TCR delta+ cells is observed in old SCID mice.

Effects of anemia on morphology of rat renal cortex.

Information on morphological and functional effects of anemia in kidney is scarce, although this organ plays a major role in erythropoietin production, which is strongly stimulated in anemia. We undertook a morphological study of kidneys of anemic rats. Anemia was induced by X-irradiation and subsequent injection of a hemolytic drug. The most striking effects of anemia on renal morphology were damages in the proximal tubule and a volume increase of the peritubular space. These effects were evident only in the cortical labyrinth. Morphometry showed that the enlargement of the peritubular space reflected an increase of the volumes of both capillaries and interstitium. The structural changes in the cortical interstitium were associated with increased activity of the ecto-5'-nucleotidase in the fibroblasts. We suggest that hypoxia accounts for most of the observed alterations. The hypoxic proximal tubule might release the nucleotide AMP, which would be hydrolyzed to adenosine by the ecto-5'-nucleotidase in the interstitium. Adenosine has been reported to trigger the synthesis of erythropoietin and the growth of blood capillaries.

Diversity of functional T-cell receptor delta-chain transcripts from bone marrow cells of athymic nude mice.

CD3+ cells are detectable in bone marrow of athymic mice homozygous for the nude mutation. As previously shown, cells expressing the gamma delta T-cell receptor (TcR) represent 30-40% of this T-cell population. Using V delta-specific, V alpha 4-specific, and C delta-specific primers, TcR delta-chain transcripts were reverse transcribed and polymerase chain reaction (PCR)-amplified from total RNA prepared from bone marrow cells (BMC) of 6-month-old NMRI nu/nu mice. Amplified TcR delta-chain cDNA was cloned, and 49 randomly selected clones derived from seven amplification reactions were sequenced. Sequence analyses showed: (1) more than 80% of the sequenced clones represented in-frame transcripts of the TcR delta-chain; (2) in-frame transcripts containing V delta 1-, V delta 2-, V delta 3-, V delta 4-, V delta 5-, V delta 6- and V alpha 4-gene segments were detectable in nude BMC; (3) V delta 2-, V delta 4- and V delta 5-containing transcripts were more abundant and more diverse than V delta 1- and V delta 3-containing transcripts; (4) extensive N-region diversity was present in the V delta-D delta 2 (N1), D delta 2-D delta 1 (N2) and D delta 1-J delta 1 (N3) junctional regions; (5) P nucleotide additions were present in many transcripts; and (6) unusual truncated, in-frame transcripts with deleted D- and J-region genes were detected. A large potential TcR delta-chain repertoire is thus present in nude BMC.

Crohn's disease of the duodenum complicated by pancreatitis and common bile duct obstruction.

It is well known that Crohn's disease can involve the duodenum, but isolated secondary complications such as pancreatitis or common bile duct obstruction have only rarely been reported, and never in the same patient. Herein, we describe a patient with duodenal Crohn's disease and both pancreatitis and calculous common bile obstruction. This unusual constellation of findings was managed with percutaneous techniques in which transhepatic catheterization of the bile duct permitted balloon dilatation of the ampulla of Vater, as well as a duodenal stricture. These maneuvers resulted in passage of the biliary stone and relief of the patient's symptoms. The management of this patient may serve as a guide possibly to delay or even prevent surgical intervention in similar cases of benign enteric strictures.

Bone marrow cells of athymic nude mice express functional T cell receptor alpha chain transcripts rearranged to V delta 2, 3, 4, 5, 6 genes.

From bone marrow cells (BMC) of athymic nude mice, T cell receptor (TcR) alpha chain transcripts were selectively amplified by polymerase chain reaction (PCR) using V delta 2-, V delta 3-, V delta 4-, V delta 5-, V delta 6- and C alpha-specific primers. Amplified DNA fragments were cloned, and 32 randomly selected clones from 5 PCR were sequenced. Twenty-three distinct rearrangement events were detected, of which 87% (20/23) were in-frame. All five tested V delta genes (V delta 2, 3, 4, 5, 6) rearranged in-frame to J alpha-C alpha. N-region diversity in V delta-J alpha junctions present in most clones was limited to two to five nucleotides. P-nucleotide additions in this region were also detected. The V delta 5 gene located 3' of C delta in reversed transcriptional orientation was rearranged to J alpha by inversion. The J alpha usage pattern of the sequenced clones was strongly biased towards rearrangement of the most 5' genes (located nearest to C delta) of the J alpha cluster: the most 5' J alpha (J alpha TA1) was used by 30% of all clones, and 78% of all J alpha rearranged to V delta were located in the 5' 12 kb of the 60-kb J alpha cluster. As distinct V delta/C delta and V alpha/C alpha TcR usage patterns are prevalent in peripheral T cell populations, our data suggest that these TcR usage patterns results from repertoire selections operating in alpha beta and gamma delta T cell lineages, but not from preferential V delta-C delta and V alpha-C alpha rearrangement patterns.

CD3+ T cells in severe combined immunodeficiency (scid) mice. II. Transplantation of dm2 lymphoid cells into semi-allogeneic scid mice.

Intravenous injection of nonfractionated BALB/c-H-2dm2 (dm2) (Ld-) spleen cells into 4-week-old, semi-allogeneic (H-2d, Ld+) C.B-17 scid/scid severe combined immunodeficient (scid) mice (2 x 10(7) cells/mouse) reconstituted T lymphopoiesis in thymi and repopulated the lymphoid white pulp in spleens of these immunodeficient recipients. Transplantation of dm2 thymocytes into young scid mice (5 x 10(7) cells/mouse) established a donor-derived CD3+ T cell population in spleens of recipient scid mice, in which CD4+T cells predominated. This was demonstrated by marker analyses of thymocytes and splenocytes, and determinations of serum immunoglobulin levels in transplanted scid mice. Transfer of splenocytes from young primary scid recipients into young secondary or tertiary recipients (3 x 10(6) cells/mouse) engrafted preferentially dm2-derived CD3+CD4+CD8- T cells in spleens of scid mice despite the strong selective Ld-associated alloantigenic stimulus for CD8+ T cells. Intravenous injections of nonfractionated dm2 spleen cells (2 x 10(7) cells/mouse) or thymocytes 5 x 10(7) cells/mouse) into 10- to 12-month-old, "leaky" scid mice induced severe clinical signs of graft-vs.-host disease (GVHD) in all scid recipients. Lymphoid repopulation of spleen and thymus in old scid recipients was incomplete. This GVHD was not transferrable by injecting 3 x 10(6) spleen cells from old diseased primary scid recipients into secondary or tertiary young scid recipient mice. In these serial transfers, dm2-derived CD3+CD4+CD8- T cells were again preferentially engrafted in spleens of scid recipients. Transfer of purified CD4+ dm2 T cells into young scid mice (2 x 10(5) to 5 x 10(5) cells/mouse) engrafted this T cell subset into the spleen of semi-allogeneic scid recipients. This was revealed by histological examinations, surface marker analyses, in vitro isolation of donor-type CD3+CD4+ T cell lines from spleens of transplanted scid mice, and serial transfer experiments. These data indicated that the CD4+ T cell compartment of scid mice can be selectively repopulated by semi-allogeneic T cells. Injections of purified CD8+ dm2 T-cells into young scid mice (2 x 10(5) cells/mouse) did not establish a CD8+ T cell graft in spleens of recipients. It was necessary to inject transplanted scid mice biweekly with 10(4) units recombinant interleukin 2 to establish and/or maintain transferred dm2 CD8+ T cells in spleens of these recipients, dm2 CD8+ T cell-transplanted and interleukin 2-treated scid mice did not develop any evidence of GVHD over the 9-week observation period.

CD3+ T cells in severe combined immune deficiency (scid) mice. I. Transferred purified CD4+ T cells, but not CD8+ T cells are engrafted in the spleen of congenic scid mice.

Young (less than 3 months of age) and old (greater than 1 year of age) C.B-17 scid/scid mice were tested for the presence of immunoglobulin in serum and CD3+ T cells in spleen and peritoneal cavity. In all old severe combined immune deficiency (scid) mice tested we found detectable, but very variable levels of serum immunoglobulin as well as splenic and peritoneal CD3+ T cells comprising 3% to 10% of the nonfractionated cell populations of these organs (n = 10). In contrast, none of the analyzed young scid mice showed any evidence of peripheral lymphocytes. Low numbers (2 x 10(5) to 5 x 10(5) cells/mouse) of highly purified CD4+ cells from congenic C.B-17 or BALB/c donor mice were injected intravenously into young scid recipient mice. A CD4+ T cell population was clearly engrafted when transplanted scid mice were analyzed 8 to 13 weeks after T cell transfer: (a) a CD3+CD4+CD8- T cell population was detectable in the spleens of all recipient scid mice by flow microfluorometry analyses; (b) CD3+CD4+CD8 T cell lines could be grown out of these spleens in vitro; (c) the histological examination revealed evidence of lymphoid cell repopulation in the spleens of all transplanted scid mice and (d) transplanted CD4+ T cell populations could be serially transferred into secondary and tertiary recipient scid mice. These data indicate that scid mice can be constructed in which only the CD4+ T cell compartment is selectively reconstituted. In contrast to the successful engraftment of CD4+ T cell, highly purified congenic CD8+ T cells could not be engrafted into the spleen of scid mice.

Correlation of metabolic stability and altered quaternary structure of oncoprotein p53 with cell transformation.

The phosphoprotein p53 seems to be implicated in various processes connected with cell transformation and in particular with the regulation of cell cycle and probably DNA replication. In the present paper we have analyzed two sets of closely related cell lines expressing the same p53 which exhibited either a nontransformed or a transformed phenotype. These cell lines were used to study biochemical properties of the p53 protein which might be correlated with cell transformation. We found a positive correlation among an elevated stability of p53, the formation of high-molecular-weight forms of p53, and the transformed phenotype of the corresponding cell lines. Furthermore, these data indicate that self-aggregation prevents p53 from rapid degradation. By a comparative analysis of the stability and oligomerization properties of mutant p53 and wild-type p53, we could demonstrate that elevated stability and self-aggregation of p53 are correlated with the transformed phenotype of the cells and independent of a particular mutation in the p53 gene.