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This study aimed to set up an experimental model of long bone atrophic nonunion and to explore the potential role of PTH-1–84 (PTH 1–84) and strontium ranelate (SrR). A model of atrophic nonunion was created in Sprague–Dawley rats at the femoral midshaft level. The animals were randomised into four groups. Group A1: control rodents, fracture without bone gap; Group A2: rodents with subtraction osteotomy (non-union model control) treated with saline; Group B: rodents with subtraction osteotomy treated with human-PTH (PTH 1–84); and Group C: rodents with subtraction osteotomy treated with strontium ranelate (SrR). The groups were followed for 12 weeks. X-rays were be obtained at weeks 1, 6 and 12. After sacrificing the animals, we proceeded to the biomechanical study and four point bending tests to evaluate the resistance of the callus and histological study. In second phase, the expression of genes related to osteoblast function was analysed by reverse transcription-quantitative PCR in rats subjected to substraction osteotomy and treated for 2 weeks. The animals were randomised into three groups: Group A2: rodents treated with saline; Group B: rodents treated with PTH 1–84 and Group C: rodents treated with SrR. Results No significant histological differences were found between animals subjected to subtraction osteotomy and treated with either saline or PTH ( p = 0.628), but significant difference existed between animals receiving saline or SrR ( p = 0.005). There were no significant differences in X-ray score between the saline and PTH groups at either 6 or 12 weeks ( p = 0.33 and 0.36, respectively). On the other hand, better X-ray scores were found in the SrR group ( p = 0.047 and 0.006 in comparison with saline, at 6 and 12 weeks, respectively). In line with this, biomechanical tests revealed improved results in the SrR group. Gene expression analysis revealed a slightly decreased levels of DKK1, a Wnt pathway inhibitor, in rats treated with SrR. Conclusions SrR increases has a beneficial effect in this atrophic non-union model in rats. This suggests that it might have a role may have important implications for the potential clinical role in the treatment of fracture nonunion.

The induction of nitric oxide (NO) production and the expression of cytokine-induced neutrophil chemoattractant (CINC-1) were studied in rat peritoneal adherent cells stimulated with insoluble immune complexes containing rabbit IgG Ab and OVA as the cognate Ag (IC). Incubation with IC at concentrations as low as 10 microg/ml induced NO production and the expression of inducible NO synthase (iNOS) protein. This was accompanied by the expression of CINC-1 mRNA and the activation of nuclear factor-kappaB (NF-kappaB). However, the expression of iNOS and CINC-1 mRNA induced by IC showed a different temporal pattern and a different sensitivity to both the antioxidant agent pyrrolidine dithiocarbamate (PDTC) and modulation by NO itself. Whereas iNOS mRNA and protein expression were blunted by PDTC and NO-generating compounds, CINC-1 mRNA expression was either enhanced or not affected by PDTC and NO donors. The time course of NF-kappaB activation was parallel to that of iNOS induction and was influenced in the same sense as iNOS induction by antioxidants, NO donors, the protease inhibitor N-tosyl phenylalanine chloromethyl ketone, and inhibitors of protein tyrosine phosphorylation reactions. These data indicate the existence in rat macrophages of a signaling mechanism triggered by Fc gammaR occupancy that leads to nuclear signaling, is initiated by protein tyrosine phosphorylation reactions, and shows specific sensitivities to antioxidants and NO. Whereas trans-activation of the iNOS gene can be fully explained by the stimulation of NF-kappaB, induction of CINC-1 mRNA expression seems influenced by additional regulatory elements.

Growth. feed consumption, and survival were evaluated for young red grouper Epinephelus morio reared in a recirculating-water system over 283 d. This fish-holding system was chosen because of site constraints and scarcity of seawater. Red grouper were size-graded into four experimental groups and fed chopped frozen clupeids. Water quality was monitored weekly. Daily growth rates of 0.48–0.69 g/fish were lower than those for other grouper species cultured in tanks or floating net cages. Survival ranged from 93.3 to 93.7% for three of the size-groups. Daily feed consumption rates progressively declined during the trial from 3.1–4.7% to 0.9–1.0% of body weight. Some pathological syndromes, such as exophthalmia and abdominal distension, appeared after day 283, resulting in high mortalities in all size-groups. Deaths were probably due to low dissolved oxygen (3.9–4.7 mg/L) during the last 39 d of the grow-out trial. Further studies are needed before the feasibility of red grouper culture can be adequa...

We report the clinical-radiological case of a 25 year-old female patient who developed reversible posterior leukoencephalopathy syndrome (RPLS) in the postpartum period, without evidence of preeclampsia-eclampsia or chronic arterial hypertension. RPLS is associated with diverse clinical entities including eclampsia. Ten days after giving birth, the patient presented with clinical symptoms of headache, elevated blood pressure and seizures. Reversible vasogenic oedema affecting the white matter in the posterior regions was the characteristic finding in magnetic resonance imaging (MRI) of the brain. Although the prognosis is favourable, treatment needs to be early and aggressive, with rapid control of the convulsions and arterial hypertension, with the aim of preventing ischemia and cerebral infarct from developing. There is a need to be highly alert and to consider the diagnosis of RPLS in women presenting with convulsions and other neurological symptoms in postpartum.

Deposition of immune complexes in tissues is the pathogenic mechanism underlying tissue injury in a number of diverse clinical conditions affecting the skin, joints, blood vessels and renal glomeruli. Initial approaches to the understanding of these conditions have stressed the roles of both the activation of the complement system and the accumulation of polymorphonuclear leukocytes as the main molecular and cellular mechanisms explaining the sequence of events leading to tissue damage. Recent findings on (i) the molecular biology of the leukocyte chemoattractants, (ii) the chemical structure and function of receptors for the Fc portion of the antibody molecule and (iii) the signaling events coupled to the engagement of these receptors have led to an understanding of the biochemical events involved in immune-complex injury and have provided a promising avenue for the development of therapeutic approaches. This review will focus on our current understanding of signal transduction events in the effector phase of immune-complex-mediated tissue injury.

The purpose of this study is to assess the role of nitric oxide (NO) in the intestinal lesions of passive anaphylaxis, since this experimental model resembles necrotizing enterocolitis. Sprague-Dawley rats were sensitized with IgE anti-dinitrophenol monoclonal antibody. Extravasation of protein-rich plasma and haemorrhagia were measured in the small intestine. Plasma histamine was measured to assess mast cell activation. The effect of exogenous NO on the lesions was assessed by using two structurally unrelated NO-donors: sodium nitroprusside and S-nitroso-Nacetyl-penicillamine (SNAP). An increased basal production of NO was observed in cells taken after anaphylaxis, associated with a reduced response to platelet-activating factor, interleukin 1beta, and IgE/DNP-bovine serum albumin complexes. The response to bacterial lipopolysaccharide and dibutyryl cyclic adenosine monophosphate (AMP) was enhanced 24 h after challenge, but at earlier times was not significantly different from that observed in controls. Treatment with either sodium nitroprusside or SNAP produced a significant reduction of the haemorrhagic lesions, which are a hallmark of rat anaphylaxis. The extravasation of protein-rich plasma was not influenced by NO-donors. The increase of plasma histamine elicited by the anaphylactic challenge was not influenced by SNAP treatment. NO-donors protect intestinal haemorrhagic lesions of rat anaphylaxis by a mechanism apparently independent of mast cell histamine release.

The production of nitric oxide was studied in rat adherent peritoneal cells stimulated with preformed IgE/DNP-BSA complexes made of IgE obtained from a secreting hybridoma. Stimulation with complexes at equivalence induced both the production of NO and an increased expression of the mRNA of the inducible isoform of NO synthase (iNOS). Production of NO was also elicited by a rabbit polyclonal F(ab')2 anti-CD23 cross-reacting with rat CD23. Because IgE/DNP-BSA complexes did not elicit Ca2+ mobilization and genistein did not influence the production of NO, cyclic AMP was considered as an alternative signaling molecule. Combination of a suboptimal concentration of dibutyryl cyclic AMP and IgE/DNP-BSA complexes showed an additive effect on NO production, whereas this was not observed when the agonists were used at supraoptimal doses. The inhibitor of cyclic AMP-specific phosphodiesterase IV, rolipram, which acts on the enzyme isoform predominantly expressed in inflammatory cells, also induced the production of NO. Furthermore, IgE/DNP-BSA complexes increased intracellular levels of cyclic AMP. Taken together, these data indicate that stimulation of mononuclear phagocytes via the low-affinity receptor Fc epsilon RII or rising intracellular concentrations of cyclic AMP leads to an enhanced expression of iNOS. Evidence in favor of the involvement of cyclic AMP in the signaling pathway linked to Fc epsilon RII is provided by the effect of IgE/DNP-BSA complexes on intracellular cyclic AMP levels and by the additive effect produced by dibutyryl cyclic AMP on NO production elicited by IgE/DNP-BSA complexes.

The ability of PCA 4248 [2-(phenylthio)ethyl-5-methoxycarbonyl- 2,4,6-trimethyl-1,4-dihydropyridine-3-carboxylate] to block PAF-induced systemic hypotension and protein-rich plasma extravasation in rats, and PAF-induced death in mice, was tested. These studies were complemented with experiments using soluble aggregates of immunoglobulin G (A-IgG), bacterial endotoxin and the cytokine tumor necrosis factor as putative inducers of the generation of endogenous PAF. Significant inhibition of PAF-induced systemic hypotension was observed with i.v. PCA 4248 at doses of 0.3 to 1 mg/kg (IC50 value, 0.45 mg/kg, with PAF 0.33 micrograms/kg). Reversal of the hypotension was rapidly observed when PCA 4248 was administered after PAF. The extravasation induced by 1 microgram/kg PAF was also blocked by PCA 4248 (IC50 value, 0.36 mg/kg). Inhibition of the extravasation induced by A-IgG and endotoxin was also provided by PCA 4248 at the dose of 1 mg/kg, and lasted for at least 1 hr in the experiments carried out with endotoxin, which caused extravasation with a temporal pattern more protracted than that of PAF and A-IgG. Intradermal extravasation induced by PAF reached a maximum at 30 min after injection, and was also inhibited by PCA 4248. In contrast, PCA 4248 caused a less remarkable, but statistically significant reduction of the intradermal extravasation caused by tumor necrosis factor. Pretreatment of mice with an oral dose of 30 mg/kg PCA 4248, 5 min before challenge with PAF (LD84 = 80 micrograms/kg PAF, i.v.) increased the survival rate from 16% to 68%. These data indicate that compounds containing a 1,4-dihydropyridine structure can antagonize PAF effects on experimental animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of free serum DNA and/or antibodies against nuclear antigens was studied in 65 patients in chronic hemodialysis. Most of patients showed increased levels of both free single-stranded DNA (ss DNA) (28 out of 29) and native (double-stranded) DNA (n DNA) (25 our of 28) at the beginning of each dialysis session (69 +/- 26 vs. 119 +/- 14, p less than 0.05) suggesting a rapid in vivo degradation of the n DNA released. 15 out of 65 patients (23%) developed low anti-ss DNA antibody titers by Millipore filtration assay. 2 of them presented anti-n DNA antibodies simultaneously. Isolated anti-n DNA, anti-Sm and anti-RNP antibodies were systematically negative in all patients. No relationship was found between the presence of anti-ss DNA antibodies and the type of nephropathy, the duration of treatment and the method of dialysis. The fate of a renal allograft was studied in 12 patients after having measured serum-free DNA and anti-DNA antibodies. The 4 patients with good graft function after 2 years of transplantation had significantly higher circulating ss-DNA levels than the other 8 patients whose grafts were rejected.

Zymosan coated with complement (Zc) was observed to induce a transient elevation of the intracellular cyclic AMP in human polymorphonuclear cells: a two- to three-fold increase was observed within 1 min after stimulation and approached prestimulation levels by 2 min incubation. These changes in cyclic AMP were not associated with significant changes in cyclic GMP levels. Zymosan caused the release of PAF and beta-glucuronidase and particle uptake, which was initiated about 5 min after stimulation. These results suggest that the transient increase in cyclic AMP content might regulate an early event during mediator release. In an attempt to study further the significance of this rise in cyclic AMP, cells were preincubated with various phosphodiesterase inhibitors. Preincubation of the cells with methylisobutylxanthine (MIX, 10(-6) M to 5 X 10(-5) M), theophylline (3 X 10(-5) to 3 X 10(-3) M) or dipyridamole (10(-6) M to 10(-4) M) enhanced the increase in cyclic AMP levels, but resulted in dose-dependent inhibition of Zc-induced mediator release. Particle uptake and beta-glucuronidase release were less sensitive than PAF release to phosphodiesterase inhibitors, which argues in favour of the independence of both phenomena. Synergistic experiments with MIX and cyclic AMP indicate that the effect of this drug is through its action on cyclic AMP levels. These results suggest that while Zc-induced cyclic AMP elevation might occur in an intracellular place critical to its effect; phosphodiesterase inhibitors may elevate cyclic AMP levels throughout the cell and therefore inhibit the biological response.

Human polymorphonuclears release a platelet-activating factor (PAF-acether) when challenged with various stimuli. PAF-acether is a mediator that is synthesized during cell activation in a process in which a phospholipase A2 and an acetyltransferase take part. These enzymes are finely regulated and accordingly PAF-acether release may be modulated. The authors have studied some of the transductory mechanisms which are triggered during cell stimulation and the effect of their pharmacological modulation on PAF-acether release. Theophylline, methylisobutylxanthine and dipyridamole, which block phosphodiesterase of cyclic nucleotides, induce a dose-dependent inhibition of PAF-acether release without affecting phagocytic uptake. Polyamines (dansylcadaverine, rimantadine and amantadine) reduced PAF-acether release and the phagocytic process in an order of potency similar to their ability to inhibit phospholipid methylation and the cholinephosphotransferase pathway. The calmodulin antagonist trifluoperazine induced a dose-dependent inhibition of PAF-acether release and acetyltransferase at concentrations from 10(-4) to 10(-5) M. Hence it appears that modulation of PAF-acether release can be obtained by different pharmacological blockades: phosphodiesterase of cyclic nucleotides, phospholipid metabolism and calcium-calmodulin.

The levels of platelet-activating factor (paf-acether) were measured in blood and ascitic fluid from cirrhotic patients and in blood from a group of controls, using a recently described technique for extraction and measurement. In addition, activity of acetylhydrolase, the main catabolic enzyme for paf-acether, was also measured. The highest levels of paf-acether in blood were found in decompensated cirrhotics (1.78 +/- 0.62 ng ml-1; mean +/- SD, n = 8). Compensated cirrhotics showed lower blood values (0.79 +/- 0.21, n = 4), but higher than controls (0.20 +/- 0.04, n = 12). Paf-acether levels in ascitic fluid were similar to those of blood. Values of acetylhydrolase in serum were similar in all the groups studied (3.0 +/- 0.4 in cirrhotics vs. 2.3 +/- 0.4 nmol min-1 mg-1 of protein in controls). These data suggest an enhanced production of paf-acether in cirrhotic patients rather than a decreased catabolism. High levels of paf-acether in blood could be involved in the impaired haemodynamics of cirrhotic patients and in their renal function alterations.

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine can be synthesized either by acetylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine or by transcholination of 1-alkyl-2-acetyl-sn-glycerol. Addition of opsonized zymosan to isolated human polymorphonuclear leukocytes induces a fast, transient, up to 10-fold activation of the acetyltransferase reaction without affecting the cholinephosphotransferase pathway. Based on the following results, we propose that the generation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by human polymorphonuclear leukocytes during phagocytosis is mediated by a stimulation of the acetyltransferase reaction: 1) the time course of acetyltransferase activation agrees with it being the enzyme responsible for the synthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; and 2) a close correlation exists (r = 0.91) between the generation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine in response to various doses of zymosan and the magnitude of acetyltransferase activation.

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine can be synthesized either by acetylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine or by transcholination of 1-alkyl-2-acetyl-sn-glycerol. Addition of opsonized zymosan to isolated human polymorphonuclear leukocytes induces a fast, transient, up to 10-fold activation of the acetyltransferase reaction without affecting the cholinephosphotransferase pathway. Based on the following results, we propose that the generation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by human polymorphonuclear leukocytes during phagocytosis is mediated by a stimulation of the acetyltransferase reaction: 1) the time course of acetyltransferase activation agrees with it being the enzyme responsible for the synthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; and 2) a close correlation exists (r = 0.91) between the generation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine in response to various doses of zymosan and the magnitude of acetyltransferase activation.