OX40L blockade and allergen-induced airway responses in subjects with mild asthma.

BACKGROUND: The OX40/OX40L interaction contributes to an optimal T cell response following allergic stimuli and plays an important role in the maintenance and reactivation of memory T effector cells. OBJECTIVE: We tested whether treatment with an anti-OX40L monoclonal antibody (MAb) would inhibit allergen-induced responses in subjects with asthma. METHODS: Twenty-eight mild, atopic asthmatic subjects were recruited for a double-blind, randomized, placebo-controlled, parallel-group trial (ClinicalTrials.gov identifier NCT00983658) to compare blockade of OX40L using a humanized anti-OX40L MAb to placebo-administered intravenously in 4 doses over 3 months. Allergen inhalation challenges were carried out 56 and 113 days after the first dose of study drug. The primary outcome variable was the late-phase asthmatic response. Other outcomes included the early-phase asthmatic response, airway hyperresponsiveness, serum IgE levels, blood and sputum eosinophils, safety and tolerability. RESULTS: Treatment with anti-OX40L MAb did not attenuate the early- or late-phase asthmatic responses at days 56 or 113 compared with placebo. In the anti-OX40L MAb treatment group, total IgE was reduced 17% from pre-dosing levels, and sputum eosinophils decreased 75% by day 113 (both P = 0.04). There was no effect of anti-OX40L MAb on airway hyperresponsiveness or blood eosinophils. The frequency of AEs was similar in both groups. CONCLUSION AND CLINICAL RELEVANCE: Pharmacological activity of anti-OX40L MAb was observed by decreases in serum total IgE and airway eosinophils at 16 weeks post-dosing, but there was no effect on allergen-induced airway responses. It is possible that the treatment duration or dose of antibody was insufficient to impact the airway responses.

PTEN/MMAC1 is infrequently mutated in pT2 and pT3 carcinomas of the prostate.

Deletion of the q23-24 region of human chromosome 10 is one of the most frequent genetic alterations in prostate cancer, suggesting that inactivation of a tumor suppressor gene in this region is involved in the development or progression of this carcinoma. A candidate gene, PTEN/MMAC1, has been identified from this chromosomal region; mutations of this gene have been found in various advanced tumors and cell lines including those of prostate cancer. To further define the role of PTEN/MMAC1 in the development of prostate cancer and its spectrum of genetic alterations, we analysed 40 pT2 or pT3 prostate tumors for allelic loss, mutations, and homozygous deletions using PCR-based methods. Six tumors showed loss of heterozygosity for one of the ten markers analysed, while one tumor showed loss of two markers. None of the markers within PTEN/MMAC1 was lost. Direct sequencing of PCR amplified exons and intron/exon junctions of all 40 tumors revealed three sequence variants, one of which was a point mutation in exon 9, while the other two were polymorphisms. Using multiplex PCR, no homozygous deletions were detected in any of the neoplasms. Our results showing a low frequency of alterations of PTEN/MMAC1 in pT2 and pT3 prostate cancers suggest that this gene plays an insignificant role in the development of most low stage carcinomas of the prostate.

Role of oxidants in NF-kappa B activation and TNF-alpha gene transcription induced by hypoxia and endotoxin.

The transcription factor NF-kappa B stimulates the transcription of proinflammatory cytokines including TNF-alpha. LPS (endotoxin) and hypoxia both induce NF-kappa B activation and TNF-alpha gene transcription. Furthermore, hypoxia augments LPS induction of TNF-alpha mRNA. Previous reports have indicated that antioxidants abolish NF-kappa B activation in response to LPS or hypoxia, which suggests that reactive oxygen species (ROS) are involved in NF-kappa B activation. This study tested whether mitochondrial ROS are required for both NF-kappaB activation and the increase in TNF-alpha mRNA levels during hypoxia and LPS. Our results indicate that hypoxia (1.5% O2) stimulates NF-kappa B and TNF-alpha gene transcription and increases ROS generation as measured by the oxidant sensitive dye 2',7'-dichlorofluorescein diacetate in murine macrophage J774.1 cells. The antioxidants N-acetylcysteine and pyrrolidinedithiocarbamic acid abolished the hypoxic activation of NF-kappa B, TNF-alpha gene transcription, and increases in ROS levels. Rotenone, an inhibitor of mitochondrial complex I, abolished the increase in ROS signal, the activation of NF-kappa B, and TNF-alpha gene transcription during hypoxia. LPS stimulated NF-kappa B and TNF-alpha gene transcription but not ROS generation in J774.1 cells. Rotenone, pyrrolidinedithiocarbamic acid, and N-acetylcysteine had no effect on the LPS stimulation of NF-kappa B and TNF-alpha gene transcription, indicating that LPS activates NF-kappa B and TNF-alpha gene transcription through a ROS-independent mechanism. These results indicate that mitochondrial ROS are required for the hypoxic activation of NF-kappa B and TNF-alpha gene transcription, but not for the LPS activation of NF-kappa B.

Effect of low dose methotrexate on neutrophil chemotaxis induced by leukotriene B4 and complement C5a.

When mediators of inflammation such as complement component C5a or leukotriene B4 are introduced into an air pouch created in mice, these mediators induce the migration of neutrophils into the air pouch. Pretreatment of mice with low doses of methotrexate inhibits leukotriene B4 or C5a induced neutrophil migration into the air pouch. Inhibition of neutrophil chemotaxis by methotrexate may, at least in part, account for the rapid onset of antiinflammatory activity that was observed in clinical trials with methotrexate in rheumatoid arthritis.

Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing.

During hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1alpha protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (rho(0) cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H(2)O(2) stabilizes HIF-1alpha protein during normoxia and activates luciferase expression in wild-type and rho(0) cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1alpha stabilization during hypoxia.