Exacerbated inflammatory arthritis in response to hyperactive gp130 signalling is independent of IL-17A.

OBJECTIVE: Interleukin (IL)-17A producing CD4 T-cells (TH-17 cells) are implicated in rheumatoid arthritis (RA). IL-6/STAT3 signalling drives TH-17 cell differentiation, and hyperactive gp130/STAT3 signalling in the gp130F/F mouse promotes exacerbated pathology. Conversely, STAT1-activating cytokines (eg, IL-27, IFN-γ) inhibit TH-17 commitment. Here, we evaluate the impact of STAT1 ablation on TH-17 cells during experimental arthritis and relate this to IL-17A-associated pathology. METHODS: Antigen-induced arthritis (AIA) was established in wild type (WT), gp130F/F mice displaying hyperactive gp130-mediated STAT signalling and the compound mutants gp130F/F:Stat1-/- and gp130F/F:Il17a-/- mice. Joint pathology and associated peripheral TH-17 responses were compared. RESULTS: Augmented gp130/STAT3 signalling enhanced TH-17 commitment in vitro and exacerbated joint pathology. Ablation of STAT1 in gp130F/F mice (gp130F/F:Stat1-/-) promoted the hyperexpansion of TH-17 cells in vitro and in vivo during AIA. Despite this heightened peripheral TH-17 cell response, disease severity and the number of joint-infiltrating T-cells were comparable with that of WT mice. Thus, gp130-mediated STAT1 activity within the inflamed synovium controls T-cell trafficking and retention. To determine the contribution of IL-17A, we generated gp130F/F:IL-17a-/- mice. Here, loss of IL-17A had no impact on arthritis severity. CONCLUSIONS: Exacerbated gp130/STAT-driven disease in AIA is associated with an increase in joint infiltrating T-cells but synovial pathology is IL-17A independent.

Role of Rho kinase isoforms in murine allergic airway responses.

Inhibition of Rho-associated coiled-coil forming kinases (ROCKs) reduces allergic airway responses in mice. The purpose of this study was to determine the roles of the two ROCK isoforms, ROCK1 and ROCK2, in these responses. Wildtype (WT) mice and heterozygous ROCK1 and ROCK2 knockout mice (ROCK1(+/-) and ROCK2(+/-), respectively) were sensitised and challenged with ovalbumin. ROCK expression and activation were assessed by western blotting. Airway responsiveness was measured by forced oscillation. Bronchoalveolar lavage was performed and the lungs were fixed for histological assessment. Compared with WT mice, ROCK1 and ROCK2 expression were 50% lower in lungs of ROCK1(+/-) and ROCK2(+/-) mice, respectively, without changes in the other isoform. In WT lungs, ROCK activation increased after ovalbumin challenge and was sustained for several hours. This activation was reduced in ROCK1(+/-) and ROCK2(+/-) lungs. Airway responsiveness was comparable in WT, ROCK1(+/-), and ROCK2(+/-) mice challenged with PBS. Ovalbumin challenge caused airway hyperresponsiveness in WT, but not ROCK1(+/-) or ROCK2(+/-) mice. Lavage eosinophils and goblet cell hyperplasia were significantly reduced in ovalbumin-challenged ROCK1(+/-) and ROCK2(+/-) versus WT mice. Ovalbumin-induced changes in lavage interleukin-13, interleukin-5 and lymphocytes were also reduced in ROCK1(+/-) mice. In conclusion, both ROCK1 and ROCK2 are important in regulating allergic airway responses.

Modulation of ozone-induced airway hyperresponsiveness and inflammation by interleukin-13.

The present study aimed to determine whether the T-helper cell type 2-derived cytokines, interleukin (IL)-4 and -13, can modulate the lung response to ozone exposure. IL-13(-/-), IL-4/13(-/-) and IL-13-overexpressing transgenic (Tg) mice were exposed to ozone (3 ppm; 3 h) or air. Wild-type (Wt) Balb/c mice and transgenic-negative littermates (IL-13Wt) were used as controls for gene-deficient and IL-13Tg mice, respectively. IL-4/13(-/-) and IL-13(-/-) mice developed a lesser degree of ozone-induced airway hyperresponsiveness (AHR) while IL-13Tg mice developed a greater degree of AHR compared with ozone-exposed wild-type or IL-13Wt mice, respectively. Ozone caused a time-dependent increase of bronchoalveolar lavage (BAL) neutrophils and macrophages in wild-type mice, maximal at 20-24 h, which was attenuated in the IL-13(-/-) and IL-4/13(-/-) mice. In IL-13Tg mice, there was a greater increase in BAL neutrophils after ozone exposure compared with IL-13Wt mice. Using quantitative real-time PCR, ozone-induced mRNA expression for IL-6 and keratinocyte chemokine was further enhanced in IL-13(-/-) and IL-4/13(-/-) mice, and was inhibited in IL-13Tg mice. Macrophage inflammatory protein (MIP)-3alpha/CCL20 expression was enhanced after ozone exposure in wild-type mice, inhibited in IL-13(-/-) and IL-4/13(-/-) mice, while in IL-13Tg mice it was enhanced. A similar pattern of expression was observed with lipopolysaccharide-induced cytokine (LIX/CXCL5/ENA-78) expression. In conclusion, interleukin-13 augments ozone-induced airway hyperresponsiveness and neutrophilic inflammation, possibly through modulation of certain cytokines induced by ozone exposure.

Point mutations in the stem-loop at the 3' end of mouse histone mRNA reduce expression by reducing the efficiency of 3' end formation.

Mammalian histone mRNAs end in a highly conserved stem-loop structure, with a six-base stem and a four-base loop. We have examined the effect of mutating the stem-loop on the expression of the histone mRNA in vivo by introducing the mutated histone genes into CHO cells by stable transfection. Point mutations have been introduced into the loop sequence and into the UA base pair at the top of the stem. Changing either the first or the third base of the conserved UYUN sequence in the loop to a purine greatly reduced expression, while changing both U's to purines abolished expression. A number of alterations in the stem sequence, including reversing the stem sequence, reversing the two base pairs at the base of the stem, or destroying the UA base pair at the top of the stem, also abolished expression. Changing the UA base pair to a CG or a UG base pair also reduced expression. The loss of expression is due to inefficient processing of the pre-mRNA, as judged by the efficiency of processing in vitro. Addition of a polyadenylation site or the wild-type histone processing signal downstream of a mutant stem-loop resulted in rescuing the processing of the mutant pre-histone mRNA. These results suggest that if the histone pre-mRNA is not rapidly processed, then it is degraded.

Effect of liposomally encapsulated MTX-DMPE conjugates upon TNF alpha and PGE2 release by lipopolysaccharide stimulated rat peritoneal macrophages.

The ability of liposomally encapsulated preparations of methotrexate (MTX) and three of its lipophilic derivatives (MTX-gamma-DMPE, MTX-alpha-DMPE and MTX-alpha,gamma-diDMPE) to alter mediator release by lipopolysaccharide (LPS)-stimulated rat peritoneal macrophages (PM theta) was investigated. The viability of these macrophages when incubated with approximately 6.0 nmol/10(5) cells of the respective liposomal preparations (MTX-LIPO, MTX-gamma-LIPO, MTX-alpha-LIPO and MTX-di-LIPO) for 20 h was greater than 80%. Treatment of macrophages, which had been incubated with MTX-alpha-LIPO (5.5 nmol/10(5) cells), MTX-gamma-LIPO (6.9 nmol/10(5) cells) and MTX-di-LIPO (4.5 nmol/10(5) cells) for 20 h, with antibody-coated sheep red blood cells resulted in 105 +/- 9.6%, 80.6 +/- 5.6% and 91 +/- 11.4% phagocytosis respectively (mean +/- S.E.M.). At similar concentrations of MTX-alpha-LIPO, MTX-gamma-LIPO and MTX-di-LIPO (6.5 nmol/10(5) cells), PGE2 release from LPS-stimulated rat peritoneal macrophages was inhibited by 85.3 +/- 3.7%, 68.7 +/- 0.6% and 88.8 +/- 2.2%, respectively (mean +/- S.E.M., n = 4). Incubation of these macrophages with 12, 10 and 9.4 nmol/10(5) cells of the respective liposomal preparations resulted in 89 +/- 3.3%, 62 +/- 5.5% and 85 +/- 3.9% inhibition of TNF alpha release (mean +/- S.E.M., n = 4). However, at this concentration MTX-di-LIPO was toxic. Neither MTX (20-2.5 nmol/10(5) cells) nor MTX-LIPO (5.6 nmol/10(5) cells) affected TNF alpha release from LPS-stimulated macrophages. Whilst free MTX was also ineffective at inhibiting PGE2 from these cells, incubation with MTX-LIPO at the above concentration resulted in 76.9 +/- 2.6% inhibition of the prostaglandins release.

Structure of a cluster of mouse histone genes.

The structure of a 25 kilobase region of mouse DNA containing 6 functional histone genes and an H2a pseudogene has been determined. The sequences and levels of expression of the H3 and H2b gene as well as the sequence of the H2a pseudogene have been determined.

7-Oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridines as novel inhibitors of human eosinophil phosphodiesterase.

High-throughput file screening against inhibition of human lung PDE4 led to the discovery of 3-ethyl-1-(4-fluorophenyl)-6-phenyl-7-oxo-4, 5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine (11) as a novel PDE4 inhibitor. Subsequent SAR development, using an eosinophil PDE assay, led to analogues up to 50-fold more potent than 11 with IC50 values of 0.03-1.6 microM. One such compound, CP-220,629 (22) (IC50 = 0.44 microM), was efficacious in the guinea pig aerosolized antigen induced airway obstruction assay (ED50 2.0 mg/kg, po) and demonstrated a significant reduction in eosinophil (55%), neutrophil (65%), and IL-1beta (82%) responses to antigen challenge in atopic monkeys (10 mg/kg, po).

Interleukin-1beta (IL-1beta) inhibition: a possible mechanism for the anti-inflammatory potency of liposomally conjugated methotrexate formulations in arthritis.

1. Liposomes with conventional and long-circulation times were employed as carriers for the methotrexate derivative MTX-gamma-DMPE (MTX-EPC and MTX-PEG respectively), their mechanism of action was investigated in vitro and in vivo and their therapeutic efficacy assessed using the rat collagen-induced arthritis (CIA) model. 2. At non-toxic dose, both MTX-EPC and MTX-PEG inhibited the lipopolysaccharide (LPS) induced release of IL-1beta from activated rat peritoneal macrophages (rPMPhi) in a dose and time dependent manner. Free methotrexate (MTX) was not active in this respect. After a single intravenous injection (i.v.), and at equivalent doses, both free MTX (500 microg) and MTX-EPC inhibited the LPS induced rise in plasma IL-1beta levels observed in MTX-PEG and saline treated rats. 3. When used to treat established CIA, MTX-EPC resulted in significantly lower clinical score (CS) (1.0+/-0.42 (P<0.001)) and hind paw diameter (HPD) (6.5+/-0.34 mm (P<0.001)) measurements than controls (3.0+/-0.26; 7.33+/-0.41 mm), after only two i.v. doses, and remained significantly lower for the entire experimental period. By day 24 both CS (2+/-0.61 (P<0.001)) and HPD (6.97+/-0.25 mm (P<0.002)) measurements had also become significantly lower in MTX-PEG treated rats than in saline treated controls (3.62+/-0.17, 7. 92+/-0.38 mm) and remained lower until day 30. Joint inflammation in MTX treated rats was completely ameliorated by day 20 but the health and well being of the animals was compromised and the experiment terminated at this time-point. 4. Our results clearly demonstrate that both MTX-EPC and MTX-PEG liposomes have potential for development into therapeutic modalities for the treatment of inflammatory joint disease in man.

Acute comitant esotropia in children with brain tumors.

If acute onset of esotropia is comitant, its cause is generally believed to be benign. Although this is, by and large, true, it is now clear that acute comitant esotropia may be associated infrequently with central nervous system illness. We describe six children who presented with acute onset of comitant esotropia, and who were found to have tumors of the brain stem or cerebellum. Four of the patients underwent strabismus surgery after appropriate neurologic and neurosurgical treatment was completed. In none of these patients was ocular motor fusion reestablished.

The isolation and characterization of a novel G protein-coupled receptor regulated by immunologic challenge.

Using a degenerate PCR based approach, a fragment of the novel G protein-coupled receptor, VTR 15-20, was identified from the rat ventral tegmentum. Hybridization screening and RACE PCR were employed to isolate the full length clone. The cDNA encodes a protein of 305 amino acids which shares homology to several orphan as well as known G protein-coupled receptors. Amino acid analysis demonstrates the VTR 15-20 contains specific regions conserved among the G protein-coupled receptor superfamily. Messenger RNA encoding VTR 15-20 is expressed throughout the mammalian nervous system. Using primary rat culture systems we have demonstrated the expression of VTR 15-20 mRNA in both microglia and astrocytes. The highest levels of VTR 15-20 mRNA expression are detected in peripheral tissues including the spleen. Moreover, we have found that the expression of VTR 15-20 mRNA in brain and spleen is regulated by immunologic challenge. Based on the cellular distribution and regulation by immune challenge and neuronal insult, we hypothesize that VTR 15-20 plays a role in neuroimmune function.

Histologic study of healing after ab interno laser sclerostomy.

We examined the histologic characteristics of healing after ab interno laser sclerostomy in a human eye. A KTP 532 green laser coupled to a 300-microns quartz fiberoptic probe was used to create an ab interno sclerostomy in a terminally ill patient with pigmentary glaucoma. The intraocular pressure increased five days postoperatively and did not respond to medical treatment. The patient died six weeks postoperatively of metastatic lung cancer. Histopathologic analysis showed a patent 150-microns scleral lumen from the anterior chamber to the episclera, surrounded by a 300-microns zone of acellular thermal damage. There was no healing of the lumen. The subconjunctival end of the lumen was capped with a thick episcleral scar, which caused the failure of the operation.

A prospective trial of intraoperative fluorouracil during trabeculectomy in a black population.

In a study population of black Africans with advanced glaucoma in Ghana we conducted a prospective study of intraoperative 5-fluorouracil alone. Eyes undergoing trabeculectomy were randomly selected either to receive or not receive a single intraoperative application of 5-fluorouracil (50 mg/ml for five minutes). Fifty-five eyes had a mean follow-up of 282 days (minimum, 92 days). Twenty of 24 eyes (83%) in the 5-fluorouracil group vs 12 of 31 eyes (39%) in the control group had postoperative intraocular pressure of 20 mm Hg or less with or without medical therapy (P = .01). Eleven of 24 eyes (46%) in the 5-fluorouracil group and five of 31 eyes (16%) in the control group had intraocular pressure of 15 mm Hg or less (P = .02). Without medical therapy, 17 of 24 eyes (71%) in the 5-fluorouracil group and ten of 31 eyes (32%) in the control group had intraocular pressure of 20 mm Hg or less (P = .02). The overall complications were similar in the two groups. In this population, intraoperative 5-fluorouracil markedly improved the ability of trabeculectomy to lower intraocular pressure. We recommend that intraoperative 5-fluorouracil be considered in glaucoma surgery with poor prognosis as an alternative to postoperative subconjunctival injections when multiple injections are not feasible.

Trabeculectomy with intraoperative 5-fluorouracil vs mitomycin C.

PURPOSE: To compare the effectiveness of intraoperative 5-fluorouracil (5-FU) and mitomycin C used adjunctively with trabeculectomy in a black West African population. METHODS: Eighty-five consecutive eyes of 85 black patients undergoing primary trabeculectomy for open-angle glaucoma were prospectively randomly assigned to receive either 5-FU (50 mg/ml for 5 minutes) or mitomycin C (0.5 mg/ml for 3 1/2 minutes) intraoperatively by soaked sponge. RESULTS: Of the 81 eyes with at least a 3-month postoperative follow-up, 41 of 44 (93.2%) in the mitomycin C group and 27 of 37 (73.0%) in the 5-FU group had a final intraocular pressure of less than 21 mm Hg (P = .01). Twenty-eight of 44 eyes (63.6%) in the mitomycin C group and 18 of 37 (51.4%) in the 5-FU group had a final intraocular pressure of less than 15 mm Hg (P = .26). Mean postoperative intraocular pressure was 13.7 mm Hg in the mitomycin C group and 16.3 mm Hg in the 5-FU group (P = .05). There were no differences between the two groups in mean age, preoperative intraocular pressure, postoperative visual acuity, and complications. Mean follow-up was 10.0 +/- 4.41 months (range, 4 to 19 months). CONCLUSIONS: The adjunctive use of mitomycin C with trabeculectomy is equally safe and more efficacious compared to 5-FU in this West African population. Use of mitomycin C in this study was not associated with a statistically significantly greater proportion of patients achieving low intraocular pressure (less than 15 mm Hg) compared to 5-FU.

Prostaglandin and tumor necrosis factor secretion by peritoneal macrophages isolated from normal and arthritic rats treated with liposomal methotrexate.

The effect of a novel liposomal preparation containing a phospholipid conjugate of methotrexate (MTX-LIPO) upon macrophage mediator release was investigated in normal and arthritic rats ex vivo. Peritoneal macrophages isolated from MTX-LIPO-treated arthritic rats and stimulated with lipopolysaccharide produced significantly less tumor necrosis factor (TNF) and prostaglandin (PGE2) than did macrophages isolated from saline-treated controls. In the same experimental system, free methotrexate only inhibited prostaglandin release, but it was more potent than MTX-LIPO in this respect. Additional studies are presently underway to investigate the effect of MTX-LIPO and MTX treatment upon the lipopolysaccharide-induced rise in plasma levels of various proinflammatory mediators in vivo. Haematopoietic toxicity was demonstrated in blood isolated from rats treated with free MTX, and this was as characterized by a significant reduction in reticulocyte count compared with MTX-LIPO and saline-treated rats.

Effect of three lipophilic methotrexate derivatives upon mediator release by lipopolysaccharide-stimulated rat peritoneal macrophages.

The ability of methotrexate and three lipophilic derivatives (methotrexate-gamma-dimyristoylphosphatidylethanolamine (M gamma D), methotrexate-alpha-dimyristoylphosphatidylethanolamine (M alpha D) and methotrexate-alpha-gamma-di-dimyristoylphosphatidylethanolamine (M alpha gamma D) to modulate mediator release by lipopolysaccharide-stimulated rat peritoneal macrophages was investigated. At nontoxic concentrations, approximately 10 nmol/10(5) cells, M alpha D and M gamma D produced 11.06 +/- 1.0 and 75.6 +/- 5.2%, respectively, inhibition of tumour necrosis factor (TNF) release (mean +/- s.e.m., n = 4). At this same dose M alpha gamma D resulted in 68.8 +/- 2.1% inhibition of TNF but cellular ATP levels were reduced by 80%. The inhibitory activity of all three derivatives was dose-dependent. Non-derivatized methotrexate at a concentration of 25 nmol/10(5) cells had no inhibitory effect upon TNF release (14.7 +/- 0.8%, n = 3). Determination of prostaglandin E2 (PGE2) levels in the same samples demonstrated that all three conjugates were powerful inhibitors of prostaglandin release. At a quarter of the conjugate concentrations described above the monoamides M alpha (3.1 nmol/10(5) cells) and M gamma D (2.5 nmol/10(5) cells) maintained their effects on PGE2 production with 73 +/- 2.3 and 71 +/- 2.0% (n = 4) inhibition, respectively. At this lower concentration, however, the diamide M alpha gamma D (3.1 nmol/10(5) cells) was less effective in reducing the amount of PGE2 released from the macrophages (29 +/- 18%, n = 4). Maximal PGE2 inhibition by each of the conjugates was attained at approximately 5 nmol/10(5) cells. Unconjugated methotrexate (range of 2.5-20 nmol/10(5) cells) did not inhibit the release of PGE2 from lipopolysaccharide-stimulated macrophages.

Motivation dependence of brain self-stimulation in the pigeon.

Several brain sites in the pigeon were identified as maintaining electrical brain self-stimulation. Depending on the site, stimulus currents yielding maximal responding varied from 20 to 160 µA. A high proportion of the sites only yielded self-stimulation behaviour if the subjects were deprived of food; when the birds were at full weight there was only one site at which the stimulation continued to be rewarding. Some, but weak, evidence of stimulus satiation was found. Overt behaviour elicited by non-contingent stimulation did not correlate with the reinforcing or neutral nature of the sites tested. While some positive sites were associated with structures known to be involved in the control of feeding, others were not. The hypothesis that stimulation at the hunger-dependent sites might have elicited temporary satiation signals is considered.

Population Behavior of Meloidogyne graminis in Field-grown 'Tifgreen' Bermudagrass.

The vertical distribution and overwintering potential of Meloidogyne graminis on field-grown Cynodon sp (var. 'Tifgreen' bermudagrass) was measured. Total populations of M. graminis were found to be lowest in March and highest in May. Larvae were most abundant in the top 5-cm of soil during periods favoring bermudagrass growth and least numerous during periods of host dormancy. Throughout the year, more t h a n 50% of the nematodes recovered each month were in roots within the top 5-cm of the soil profile. Both eggs and larvae of M. graminis overwinter in eastern Virginia.

The Influence of Temperature on Development and Sex Differentiation of Meloidogyne graminis.

Meloidogyne grarninis (Sledge and Golden) Whitehead on Cynodon sp. (var. 'Tifgreen' bermudagrass) was studied at four temperatures; 16, 21, 27, and 32 C. Both mode and rate of development were temperature dependent. Females developed more rapidly and in greater numbers at 27 C: saccate females exuding matrices were present 14 days following inoculation, eggs were laid after 21 days and newly-hatched larvae were present in the matrix at 25 days. Sex differentiation to males was 80% at 32 C and 4% at 27 C. No males were observed at 21 or 16 C. Developing males were present 14 days following inoculation and emerged from roots after 21 days at 32 C. In populations pre-exposed to 27 C then transferred to 32 C, the percentage of males ranged from 0 for 1 day exposure at the initial temperature to 45.5% after 5 days. After 11 days pre-exposure the recovery of males was 4.3%. Individuals interpreted to be male sex reversals and male intersexes were noted. Pre-exposure at 32 C for 1 or 2 days followed by 27 C produced 1-2% males, while exposure for 3 or more days at 32 C followed by 27 C produced 90% males.

Pectolytic and Cellulolytic Enzymes of Two Populations of Ditylenchus dipsaci on 'Wando' Pea (Pisum sativum L.).

'Wando' pea is susceptible to Ditylenchus dipsaci from Raleigh, N. C. (RNC) but resistant to the same species from Waynesville, N. C. (WNC). Homogenates of RNC and WNC were analyzed for pectolytic and cellulolytic enzyme activity; both had high C(x) activity with WNC two to three times more active than RNC. Polymethylglacturonase activity was three to five times higher in RNC, but polygalacturonase was up to 100 times higher in WNC. Polygalacturonate-trans-eliminase was not detected although a Ca(++)-stimulated pectin methyl-trans-eliminase was present. Enzyme analyses of healthy and infected pea tissue showed only slight enzyme activity unrelated to that in nematode homogenates. No correlation between enzyme activity and the differing pathogenicities could be detected.