Interfering with leukocyte integrin activation--a novel concept in the development of anti-inflammatory drugs

Inflammation is a crucial response against invading pathogens, in which immune cells, including neutrophils and T cells, are recruited into tissue from the bloodstream to help clear infection. However, a prevailing inflammatory response where the immune cells attack healthy tissue is associated with many diseases, including asthma, rheumatoid arthritis, atherosclerosis and multiple sclerosis. Integrins are key players in the recruitment of immune cells from the bloodstream into tissues, and are thus therapeutic targets for intervention with inflammatory responses. Thus far, mainly extracellularly acting therapeutics (monoclonal antibodies) have been developed against integrins, targeting ligand binding sites in these heterodimeric adhesion receptors. However, since these therapeutics nonselectively block all integrin functions, some side effects are expected and have been observed. Therefore, novel concepts need to be developed in the therapeutic targeting of integrins. Recently, major advances have been made in the understanding of integrin biology. Integrin structures have been solved by X-ray crystallography, revealing unexpected data about the activation mechanism of integrins in cells. Additionally, several intracellular factors in the integrin activation process have been identified, providing potential specific targets for therapeutic intervention. Here, we present key events and players in leukocyte integrin activation, and discuss potential new drug targets in the prevention of inflammatory disease.     

Absence of DAZ gene mutations in cases of non-obstructed azoospermia

Sequenced-tagged site (STS) analysis of the Y chromosome long arm (Yq) of azoospermic males has identified a minimum common deleted region of several hundred kilobases in approximately 13% of cases. A candidate azoospermia gene, DAZ (deleted in azoospermia), has been isolated from this region. DAZ has also been shown to be absent in severely oligozoospermic males albeit at a much lower frequency. These data, although highly suggestive, do not constitute formal proof that DAZ actually plays a role in azoospermia, as no small intragenic deletions, rearrangements or point mutations in the gene have been found. In this study we report the screening of DNA from 168 azoospermic/oligospermic males for the presence of the DAZ gene. Deletions involving DAZ were detected in five out of 43 (11.6%) azoospermic males whereas none were found in the remaining 125 oligospermic patients. We present the genomic structure of the 5' end of the DAZ gene together with its sequence analysis in 30 non-obstructed azoospermic males. No mutations in DAZ were found in any of the patients sequenced. These data provide no formal proof that DAZ is AZF. Thus the possibility is still valid that another gene(s) mapping to the deletion interval may be responsible for, or contribute to, the observed phenotypes. Alternatively, if DAZ is AZF, they suggest that the most frequent cause of gene inactivation is via large deletions possibly mobilized by Y chromosome repetitive sequences.      

Personality traits and striatal 6-[18F]fluoro-L-dopa uptake in healthy elderly subjects

In this study, we examined the effects of an intracerebroventricular (i.c.v.) administration of prostaglandin E2 (PGE2) and of selective agonists for PGE2 receptor subtypes, EP1, EP2, EP3 and EP4, on central cardiovascular regulation and renal sympathetic nerve activity (RSNA) in urethane-anesthetized rats. The central administration of PGE2 (0.01-1.0 nmol) resulted in increases in blood pressure, heart rate (HR) and RSNA in a dose-dependent manner. Cardiovascular responses to PGE2 (0.5 nmol, i.c.v.) were attenuated by pretreatment with ganglionic and adrenoceptor blocking agents, but not with SC-19220 (20 nmol, i.c.v.), an EP1 receptor antagonist. An i.c.v. administration of the EP3 agonist ONO-AE-248 (50.0 nmol) resulted in an increase in RSNA with pressor and tachycardia responses, while administration of the EP2 agonist ONO-AE1-259 and the EP4 agonist ONO-AE1-329 caused transient hypotension and slight increases in HR and RSNA. The administration of the selective EP1 agonist ONO-DI-004 showed no effect. These results suggest that the central PGE2-induced activation of the sympathetic nerve activity with hypertension and tachycardia may depend on stimulation of the EP3 receptors in the central nervous system.     

Immunoassay for the discrimination of free prostate-specific antigen (fPSA) forms with internal cleavages at Lys(₁₄₅) or Lys(₁₄₆) from fPSA without internal cleavages at Lys(₁₄₅) or Lys(₁₄₆)

Total levels of circulating prostate-specific antigen (tPSA) are strongly associated with prostate cancer (PCa) risk and outcome but benign prostate disease is the most frequent cause of a moderately elevated PSA level. Free PSA (fPSA) forms are independently associated with PCa risk and contribute modest diagnostic enhancements above and beyond tPSA alone. We developed an immunoassay for fPSA subfractions containing internal cleavages at Lys(145) or Lys(146) (fPSA-N). The assay was based on blocking intact single-chain fPSA (fPSA-I) with antibody 4D4 which does not detect PSA containing internal cleavages at Lys(145) or Lys(146). We also measured fPSA-N in blood from healthy volunteers and in anti-coagulated plasma from 76 men with or without evidence of PCa at biopsy. The analytical and functional detection limits of this assay were 0.016 ng/mL and 0.10 ng/mL, respectively. The median recovery of male fPSA-N from female plasma was 95.0%. All 12 female samples (average age 28 years) had fPSA-N concentrations at or below the analytical detection limit. The median fPSA-N concentration (0.050 ng/mL) in 9 healthy male volunteers (age<40 years) was below the functional detection limit, 0.420 ng/mL in 27 patients with benign prostate conditions and 0.239 ng/mL in 49 patients with PCa. Deming regression analysis of the patient samples showed that the measured fPSA-N concentrations were generally 23% lower than the previously calculated (fPSA minus fPSA-I) concentrations, likely due to differences in the antibody combinations used. In conclusion, we have developed a sensitive, specific and direct immunoassay for fPSA-N which can be used to study the clinical relevance of this PSA isoform.