Coagulation inhibitor elicited by thrombin.

A protein fraction present in bovine prothrombin complex, or in prothrombin-free preparations obtained from serum, is converted by thrombin into an anticoagulant that strongly interferes with the intrinsic pathway of blood coagulation and with thrombin formation, initiated also by the extrinsic mechanism. If one assumes that a similar phenomenon takes place in vivo, the association between thrombosis and hemorrhage may be better understood.

Salsolinol-a potential inhibitor of the gonadotropic axis in sheep during lactation.

This study tested the hypothesis that salsolinol, a derivative of dopamine, affects GnRH and LH secretion in lactating sheep. In the in vivo experiment, the structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1-MeDIQ), was infused into the infundibular nucleus-median eminence of sheep at the fifth wk of lactation to antagonize salsolinol's action. Simultaneously, cerebrospinal fluid from the third brain ventricle, to determine GnRH concentration, and plasma samples, to measure LH concentration, were collected. In the in vitro experiment, the anterior pituitary (AP) explants from weaned sheep were incubated in culture medium containing 2 doses of salsolinol, 20 and 100 µg/mL (S20 and S100, respectively). The concentration of LH in the collected media and relative expression of LHß subunit messenger RNA in the AP explants were determined. No significant difference was found in mean GnRH concentration in response to 1-MeDIQ infusion, but both mean plasma LH concentration and LH pulse frequency increased significantly (P < 0.001 and P < 0.05, respectively) compared with those in controls. Significantly higher LH concentrations occurred during the first (P < 0.001), second (P < 0.001), and fourth (P < 0.05) h of 1-MeDIQ infusion. In the in vitro study, both the S20 and S100 doses of salsolinol caused a significant decrease in the mean medium LH concentration compared with that in the control (P < 0.01 and P < 0.001, respectively). Salsolinol had no effect on the relative LHß subunit messenger RNA expression in the incubated tissue. In conclusion, salsolinol is a potential inhibitor of the secretory activity of the gonadotropic axis in lactating sheep, at least at the AP level. Although no significant changes in GnRH release were directly confirmed, an increase in the frequency of LH pulses does not allow to exclude the central action of salsolinol.

Involvement of salsolinol in the suckling-induced oxytocin surge in sheep.

During lactation, the main surge of oxytocin is induced by a suckling stimulus. Previous studies have shown that salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline), a dopamine-derived compound, stimulates both the synthesis and the release of oxytocin in lactating sheep. The objective of the present study was to verify the hypothesis that salsolinol is involved in the mechanism that generates the oxytocin surge that occurs during suckling. Thus, a structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), known to antagonize some of its actions, was infused into the third ventricle of the brain of lactating sheep nursing their offspring. Serial 30-min infusion of 1MeDIQ (4 × 60 µg/60 µL) or vehicle were administered at 30-min interval from 10 AM to 2 PM. The experimental period in every ewe consisted of a nonsuckling period (10 AM-12 PM) and a suckling period (12 PM-2 PM). Blood samples were collected every 10 min, to measure plasma oxytocin concentration by RIA. In control sheep, oxytocin surges of high amplitude were observed during the suckling period. The oxytocin surges induced by suckling were significantly (P < 0.01) diminished in sheep receiving 1MeDIQ infusions as compared to those that received control infusions. However, no significant effect of 1MeDIQ was observed on basal oxytocin release, before suckling. Furthermore, oxytocin release, as measured by the area under the hormone response curve (AUC), was significantly decreased by the administration of 1MeDIQ during the suckling period. This study shows that elimination of the effect of salsolinol within the central nervous system of lactating sheep attenuates the oxytocin surge induced by suckling. Therefore, salsolinol may be an important factor in the oxytocin-stimulating pathway in lactating mammals.

Salsolinol Up-Regulates Oxytocin Expression and Release During Lactation in Sheep.

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is a dopamine-derived compound present in the central nervous system and pituitary gland. Several previous studies on lactating sheep and rats have reported that salsolinol plays a crucial role in the regulation of prolactin secretion. The present study investigated the effects of salsolinol, which was infused into the third ventricle of the brain, on oxytocin expression and release in lactating sheep, 48 h after weaning of 8-week-old lambs. Serial 30-min infusions of salsolinol and vehicle were performed at 30-min intervals from 10.00 to 15.00 h. Blood samples were collected every 10 min. The supraoptic nucleus (SON), paraventricular nucleus (PVN) and posterior pituitary were collected immediately after the experiment. Expression levels of mRNAs for oxytocin and peptidylglycine α-amidating monooxygenase (PAM), the terminal enzyme in the oxytocin synthesis pathway, were measured using a real-time polymerase chain reaction. Oxytocin peptide content in the posterior pituitary was measured by an enzyme-linked immunosorbent assay, and plasma oxytocin concentration was measured by radioimmunoassay. Salsolinol treatment significantly up-regulated oxytocin and PAM gene expression in the SON (P < 0.01 and P < 0.05, respectively), PVN (P < 0.01 and P < 0.05, respectively) and posterior pituitary (P < 0.05 and P < 0.05, respectively). Oxytocin peptide content in the posterior pituitary and the area under the response curve of plasma oxytocin were significantly (P < 0.05 and P < 0.01, respectively) higher in salsolinol-treated sheep than in control animals. The present study shows for the first time that salsolinol stimulates oxytocin secretion during lactation in sheep.

Interleukin-1 ß Modulates Melatonin Secretion in Ovine Pineal Gland: Ex Vivo Study.

The study was designed to determine the effect of proinflammatory cytokine, interleukin- (IL-) 1ß, on melatonin release and expression enzymes essential for this hormone synthesis: arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) in ovine pineal gland, taking into account the immune status of animals before sacrificing. Ewes were injected by lipopolysaccharide (LPS; 400 ng/kg) or saline, two hours after sunset during short day period (December). Animals were euthanized three hours after the injection. Next, the pineal glands were collected and divided into four explants. The explants were incubated with (1) medium 199 (control explants), (2) norepinephrine (NE; 10 µM), (3) IL-1ß (75 pg/mL), or (4) NE + IL-1ß. It was found that IL-1ß abolished (P < 0.05) NE-induced increase in melatonin release. Treatment with IL-1ß also reduced (P < 0.05) expression of AA-NAT enzyme compared to NE-treated explants. There was no effect of NE or IL-1ß treatment on gene expression of HIOMT; however, the pineal fragments isolated from LPS-treated animals were characterized by elevated (P < 0.05) expression of HIOMT mRNA and protein compared to the explants from saline-treated ewes. Our study proves that IL-1ß suppresses melatonin secretion and its action seems to be targeted on the reduction of pineal AA-NAT protein expression.

Combined in vitro and in vivo T lymphocyte depletion for the control of graft-versus-host disease following haploidentical marrow transplant.

Most patients requiring allogeneic bone marrow transplantation (BMT) lack a human leukocyte antigen genotypically identical sibling and require an alternative donor. This carries an increased risk of graft failure and acute graft-versus-host disease (GVHD). We sought to overcome these problems with transplants by using grafts obtained from the most readily available source: the haploidentical, partially mismatched, related donor. This study of 40 patients used a novel approach combining in vitro and in vivo T cell depletion with T lymphocyte targeted monoclonal antibodies (mAb) and intensified conditioning therapy, including fractionated total body irradiation before etoposide, cytoside arabinoside, cyclophosphamide, and methylprednisolone. Grafts were treated with T10B9.1A-31 mAb, directed against the alpha-beta heterodimer of the T cell receptor, and rabbit complement. In vivo depletion was attempted with an anti-CD5 mAb-Ricin A-chain (H65-RTA) immunotoxin (IT). Study patients were compared with a historical control group of 17 patients not given H65-RTA. Rates of engraftment were not significantly different (93% vs. 100%, P=0.12), although patients receiving IT engrafted more rapidly. The incidence of > grade I GVHD was significantly lower in the study group (36% vs. 100%, P=0.0001), as well as for severe grade III-IV GVHD (19% vs. 92%, P=0.0001). Five-year survival tended to be improved in the study group (40% vs. 18%, P=0.21). Transplant from haploidentical family members is indicated for patients without a matched sibling in whom allogeneic BMT offers the best opportunity to achieve cure.

Antithrombin III and heparin inactivation in thrombin involving reactions.

The inhibitory capacity of antithrombin III (AT III) was measured by a quantitative method independent of the velocity of inhibition. When AT III was in excess of thrombin in plasma or in purified system the capacity of inhibitor decreased quantitatively in proportion to the amount of thrombin neutralized. Heparin present in reaction together with thrombin invariably induced a more extensive utilization of inhibitor than thrombin alone. The extent of this additional loss of inhibitory capacity was to a limited degree related to the concentration of heparin. Heparin itself was neutralized in thrombin-AT III reaction losing its anticoagulant property in proportion to the amount of thrombin bound by inhibitor. This quantitative neutralization of heparin occurred not only when the anticoagulant participated in thrombin-AT III binding but also when heparin was added to a medium containing a preformed thrombin-AT III complex. These results suggest that acceleration of binding and increased utilization of binding capacity are the two regular effects of heparin on thrombin-involving reactions of AT III. Both of these effects may be abolished by quantitative binding of heparin to thrombin-AT III complex.

The interaction of immobilized thrombin with human antithrombin III.

Interaction of human antithrombin III (AT III) with human alpha-thrombin coupled to Sepharose 4B was investigated. Despite markedly reduced esterolytic, amidolytic and especially coagulant activity, more than 90% of immobilized thrombin formed stable complexes with purified AT III. Presence of high affinity heparin did not facilitate the inhibition to the degree seen in reactions conducted with soluble thrombin. Instead, heparin induced proteolysis of up to 66% of the inhibitor that remained in solution. This led to the isolation of a homogeneous protein fragment which migrated in SDS-gel electrophoresis as a band of 50,000 Mr, cross-reacted with antibodies to human AT III but showed no biologic activity nor sufficient affinity for heparin. Out of the three major inhibitors capable of binding soluble thrombin in human plasma, only AT III reacted with immobilized thrombin. However, Sepharose-coupled thrombin mixed with plasma in the presence of heparin produced outstanding quantities of residual immunoreactive AT III devoid of inhibitory activity. These data suggest that presence of high affinity heparin in the environment of thrombin attached to a solid support may dramatically decrease the efficiency of enzyme inhibition.

High molecular weight forms of antithrombin III complexes in blood.

A double antibody competition radioimmunoassay was developed that allowed to detect specifically as little as 15 ng antithrombin III (AT III) per ml of the assayed material. In normal plasma examined by this assay, AT III concentration averaged 199 +/- 21 micrograms/ml. Complexes of AT III with thrombin or factor Xa crossreacted with free AT III in 87% and 95%, respectively. Molecular forms of AT III produced in plasma treated with coagulation enzymes, or in serum, were assessed by measuring immunoreactive AT III in fractions obtained by gel filtration chromatography on Sephadex G-200. AT III bound by thrombin in fibrinogen free-plasma ranged in molecular size from 160,000 to above 250,000. Similar aggregation occurred when monomeric complex of purified AT III and thrombin, of 90,000 Mr, was added to plasma. Presence of heparin intensified the degree of aggregation. In factor Xa-treated plasma AT III was converted into components with 160,000 Mr, or less. No complexes below 200,000 Mr were present in serum. They decreased in size to 160,000 Mr after affinity chromatography on heparin-Sepharose. These results indicated that blood represents a unique milieu conducive to aggregation of bound AT III. It appears, however, that AT II complexes present in blood may not only aggregate, but also associate with other serum proteins through unstable binding most likely caused by the enzyme component of the complex.

Laboratory control in predicting clinical efficacy of T cell-depletion procedures used for prevention of graft-versus-host disease: importance of limiting dilution analysis.

In an attempt to assess the usefulness of partial elimination of T lymphocytes from histocompatible donor bone marrow as a sole method of graft-versus-host disease prophylaxis in patients undergoing bone marrow transplantation, we compared in vitro and clinically the efficacy of two depletion protocols, each resulting in a different degree of T cell reduction. The protocols were based on complement-dependent T cell lysis induced either by one (T10B9) or two (T10B9 and T12A10) monoclonal antibodies, contributing respectively to 95% and 99% depletion of T cells defined functionally by limiting dilution analysis. Phenotypic analysis was unsuitable for detecting differences in the efficiency of the two depletion modalities due to very low numbers of residual OKT3-positive cells generally present. Of the 34 patients with advanced leukemias transplanted with marrow from HLA-matched sibling donors, 26 (ages 15-52) received two-antibody depleted grafts (group I) with subsequent incidence of severe acute graft-versus-host disease of 8% (in two of 24 engrafted). The T cell dose for two patients of this group whose grafts were directly evaluated by limiting dilution analysis was less than 2 x 10(5)/kg and presumably did not exceed 3.2 x 10(5)/kg for others, based on the depletion efficiency of the protocol estimated in additional small marrow samples. The remaining eight patients (ages 8-55) received one-antibody depleted grafts (group II) with 5-18 x 10(5) T cells/kg defined functionally in five grafts. Severe acute graft-versus-host disease (grade 3-4) developed in all seven engrafted subjects. Although with phenotypic analysis several grafts in this group revealed no residual T lymphocytes, they apparently carried doses of donor T cells not tolerable in a HLA-identical host unprotected by additional immunosuppression. Careful evaluation by functional T cell analysis should be considered in choosing a depletion protocol as a method for reducing the risk of graft-versus-host disease in allogeneic bone marrow transplantation.

Long-term survival in advanced chronic myelogenous leukemia following bone marrow transplantation from haploidentical related donors.

From 1987 to 1991, 26 patients with CML and a median age of 31 years received allogeneic BMT from a partially mismatched related donor (PMRD) who shared at least one haplotype with the recipient. Nine patients were in accelerated phase (AP), and 11 patients were in blast crisis (BC) at the time of BMT. Patients were mismatched either in graft-versus-host or host-versus-graft directions for one antigen in 3 patients, two antigens in 14 patients, and three antigens in 9 patients. All patients were prepared with a regimen consisting of total body irradiation, etoposide, cytosine arabinoside, cyclophosphamide and methylprednisolone. All marrows were treated ex vivo with T10B91.A-31, a monoclonal antibody directed toward the alpha beta heterodimer of the CD3 receptor, and rabbit complement. Additional GVHD prophylaxis included either the anti-CD5 immunoconjugate XomaZyme-H65, cyclosporine, or both in combination with methylprednisolone. Eight patients did not have sustained engraftment. The 100-day survival was 42%. The incidence of > or = grade II acute GVHD was 29%. The incidence of chronic GVHD was 50% and was limited in all cases. The median survival at 4 years for all 26 patients was 27%. Seven patients (CP 1, AP 3, BC 3) remain in hematologic remission 1297-2241+ days after transplantation. AlloBMT from a PMRD may be considered for patients with advanced CML who lack a matched sibling or unrelated donor.

Allogeneic bone marrow transplantation with T cell-depleted partially matched related donors for advanced acute lymphoblastic leukemia in children and adults: a comparative matched cohort study.

Allogeneic BMT provides the best treatment currently available for long-term disease-free survival in patients with recurrent ALL. Historically, partially matched related donors provided the opportunity for treatment to a greater number of patients than matched related donors at the expense of decreased overall survival. In this study we compare the results in recurrent ALL patients transplanted with either HLA identical sibling bone marrow or partially matched related bone marrow. Thirty-two patients with relapsed ALL received partially matched bone marrows from a relative with one to three HLA, A, B and Dr antigen mismatches. Bone marrow was partially T cell-depleted with murine T10B9.1A-31 moAb. Sixteen patients with relapsed ALL received HLA-matched sibling bone marrows. All partially matched patients received additional GVHD prophylaxis with methylprednisolone in addition to anti-CD5 immunotoxin and/or CYA. All matched patients in addition to methylprednisolone received MTX and/or CYA. We observed no difference in disease-free survival between patients transplanted with partially matched bone marrow (median follow-up 1252 days, range 778-2035 days) vs those transplanted with HLA-matched bone marrow (median follow-up 1472 days, range 1165-2800 days; P = 0.48). Median survival for all patients is 38% (95% CI 24-52%) at 6 years. Patients transplanted in remission had a significant increase in disease-free survival when compared to those in relapse (P = 0.007). Our data suggest that partially matched BMTs from related donors are a comparable alternative to fully matched transplants in patients with ALL.

Treatment of leukemia with partially matched related bone marrow transplantation.

The results of partially matched related donor (PMRD) marrow transplantation for 82 patients with leukemia are reported, including 45 who received two antigen disparate grafts. Following intensive radiochemotherapy, patients received grafts which were partially depleted of T cells by the monoclonal antibody T10B9 and complement. Actuarial probability of engraftment was 86% (95% CI = 78-93%). The median day to engraftment was similar among recipients of grafts disparate at one, two or three antigen loci. The incidence of severe (grades III and IV) acute graft-versus-host disease and extensive chronic graft-versus-host disease was 13% and 6%, respectively. The probability of disease-free survival for the entire cohort of patients is 31% at 3 years. Age < or = 30 years, early or intermediate stage disease and a graft disparate at one or two loci predicted longer disease-free survival in multivariant analysis. Moreover, 47% of patients receiving PMRD grafts disparate at two loci who had both these favorable pretransplant characteristics were alive and free of disease 3 years after transplantation. We believe that the utilization of PMRDs, especially those with two antigen disparate grafts, can extend allogeneic transplantation to additional leukemic patients lacking a histocompatible donor, with acceptable results.

Enhancement by heparin of thrombin-induced antithrombin III proteolysis: its relation to the molecular weight and anticoagulant activity of heparin.

Previous findings indicated that binding of heparin to antithrombin III (AT III) facilitates thrombin-induced proteolysis of the inhibitor. We now studied this property of heparin in regard to its molecular weight and anticoagulant activity. Commercial heparin was resolved on Sephadex G-200 into six fractions of decreasing molecular weight. From each fraction high affinity (HA) heparin was isolated by chromatography on AT III-Sepharose and examined in reaction of alpha-thrombin with a molar excess of 125I AT III. Proteolysis of the inhibitor was assessed by SDS polyacrylamide gel electrophoresis. In the presence of the HA heparin from 18% to 38% of AT III participating in reaction appeared in the form of inactive 50,000-dalton fragment, as opposed to 7% of AT III fragmented in the absence of heparin. Although the ability to potentiate proteolysis was at its peak in the medium-molecular-size heparin fraction, the amount of degraded inhibitor relative to anticoagulant activity increased with decreasing molecular weight of the polysaccharide. These findings are consistent with the possibility that the ability of bound heparin to facilitate the cleavage of AT III by thrombin is generally less contingent upon secondary characteristics of the polysaccharide than the anticoagulant activity.

[Therapeutic effect of zuclopenthixol acetate on positive and negative symptoms in schizophrenia]. / Ocena wplywu terapeutycznego octanu zuklopentyksolu na objawy pozytywne i negatywne w schizofrenii.

Fifty schizophrenic in-patients (DSM-IV) were treated in an open study with zuclopenthixol acetate. Mental status, improvement and side-effects were measured before administration of the drug as well as after the 1st, 2nd and 3rd injection. Positive and negative symptoms were evaluated with the use of PANSS. 60% of patients received three injections. Usually the intervals between injections lasted 48 hours. The improvement after the 3rd injection of zuclopenthixol acetate was found in 80% of patients. All positive symptoms improved after the treatment (p < 0.001), among them excitement (54% reduction vs. baseline), hostility (49%) suspiciousness/persecution (45%). The study revealed that parallel to the decrease of positive symptoms, the severity of negative symptoms also decreased, in particular: difficulty in abstract thinking (28%) and stereotyped thinking (27%) (p < 0.001). Passive/apathetic social withdrawal and lack of spontaneity as well as flow of conversation only slightly improved (p < 0.05). 50% of patients experienced side-effects--usually extrapyramidal reactions.