Nucleoside triphosphate diphosphohydrolase-1 ectonucleotidase is required for normal vas deferens contraction and male fertility through maintaining P2X1 receptor function.

In this work, we report that Entpd1(-/-) mice, deficient for the ectonucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), produce smaller litters (27% reduction) compared with wild-type C57BL6 animals. This deficit is linked to reduced in vivo oocyte fertilization by Entpd1(-/-) males (61 ± 11% versus 88 ± 7% for Entpd1(+/+)). Normal epididymal sperm count, spermatozoa morphology, capacitation, and motility and reduced ejaculated sperm number (2.4 ± 0.5 versus 3.7 ± 0.4 million for Entpd1(+/+)) pointed to vas deferens dysfunction. NTPDase1 was localized by immunofluorescence in the tunica muscularis of the vas deferens. Its absence resulted in a major ATP hydrolysis deficiency, as observed in situ by histochemistry and in primary smooth muscle cell cultures. In vitro, Entpd1(-/-) vas deferens displayed an exacerbated contraction to ATP, a diminished response to its non-hydrolysable analog αßMeATP, and a reduced contraction to electrical field stimulation, suggesting altered P2X1 receptor function with a propensity to desensitize. This functional alteration was accompanied by a 3-fold decrease in P2X1 protein expression in Entpd1(-/-) vas deferens with no variation in mRNA levels. Accordingly, exogenous nucleotidase activity was required to fully preserve P2X1 receptor activation by ATP in vitro. Our study demonstrates that NTPDase1 is required to maintain normal P2X1 receptor functionality in the vas deferens and that its absence leads to impaired peristalsis, reduced spermatozoa concentration in the semen, and, eventually, reduced fertility. This suggests that alteration of NTPDase1 activity affects ejaculation efficacy and male fertility. This work may contribute to unveil a cause of infertility and open new therapeutic potentials.

Adenosine potentiates human lung mast cell tissue plasminogen activator activity.

We investigated whether adenosine, a potent contributor to the regulation of pulmonary function, can modulate human lung mast cell (HLMC) fibrinolytic activity. Tissue plasminogen activator (tPA) activity and tPA transcript expression levels from a human mast cell line (HMC-1) and HLMC were monitored following adenosine application. Adenosine potentiated mast cell tPA activity and tPA gene expression in a dose-dependent manner. Adenosine effects were abolished in the presence of adenosine deaminase. HMC-1 cells and HLMC predominantly expressed adenosine A(2A) and A(2B) receptor transcripts (A(2B) ≈ A(2A) > A(3) >> A(1)). Pharmacological and signaling studies suggest that the A(2A) receptor is the major subtype accounting for adenosine-induced mast cell tPA activity. Finally, the supernatant from HMC-1 cells and HLMC treated with adenosine (for 24 h) significantly increased fibrin clot lysis, whereas ZM241385, an A(2A) receptor antagonist, abolished this effect. To our knowledge, this study provides the first data to demonstrate the potentiating effect of adenosine on mast cell tPA activity and fibrin clot lysis.

Purines 2010: Adenine Nucleosides and Nucleotides in Biomedicine.

The Purines 2010: Adenine Nucleosides and Nucleotides in Biomedicine meeting, held in Tarragona, Spain, included topics covering new findings in the field of purinergic signaling and the development of purine-based drugs. This conference report highlights selected presentations on developments in purinerigic signaling, medicinal chemistry, the therapeutic potential of purine-based drugs, and the role of purines and adenosine receptors in neurodegenerative disorders, sickle cell disease, bone homeostasis, pulmonary fibrosis and pain. Investigational drugs discussed include CF-101 (Can-Fite BioPharma Ltd/NIH/Kwang Dong Pharmaceutical Co Ltd/Seikagaku Corp) and denufosol tetrasodium (Cystic Fibrosis Foundation Therapeutics Inc/Inspire Pharmaceuticals Inc).

Characterization of the allergen filarial tropomyosin with an invertebrate specific monoclonal antibody.

Tropomyosins of invertebrates are pan-allergens responsible for wide spread allergic reactions against seafood and arthropods. As invertebrate tropomyosins are highly conserved, helminth tropomyosins are likely to show properties similar to these medically important allergens. Studies with a monoclonal antibody, NR1, raised against tropomyosin of the rodent filarial nematode Acanthocheilonema viteae revealed a B cell epitope common to helminths and marine mollusks, which does not occur in vertebrate tropomyosin. This antibody detected tropomyosin of A. viteae, other filariids, nematodes, trematodes and a cestode, and recognized as well tropomyosin of oyster, squid and octopus, but not of arthropods and vertebrates. Immunohistological analyses of A. viteae, Onchocerca volvulus and other nematodes using NR1 showed that tropomyosin is located in the fibrillar part of the body wall muscles and the uterus, and is also conspicuous in muscles of the pharynx, the vagina and other organs of the nematodes. The abundance of a pan-allergen like tropomyosin in parasitic worms and the counterintuitive, but well documented protection against allergic reactivity by some chronic helminth infections is discussed.

Helminths and allergy: the example of tropomyosin.

Parasitic worms contain potent allergens, but epidemiological and experimental studies suggest that infections with certain helminths are negatively associated with the prevalence of allergic diseases. This seeming contradiction can be addressed by using filarial tropomyosin as an example. This protein shares structural features and crossreacting B-cell epitopes with other highly allergenic invertebrate tropomyosins. Nevertheless, it usually does not provoke allergic disease in infected individuals. In addition, it is one of the most prominent candidates for an anti-nematode vaccine. Recent data suggest mechanisms that might prevent hosts from developing allergic reactions against allergens of their parasites, such as filarial tropomyosin.

A nematode allergen elicits protection against challenge infection under specific conditions.

We describe tropomyosin of the filarial nematode Acanthocheilonema viteae as an allergen and study its protective potential in the natural rodent host Meriones unguiculatus (jird). Jirds immunized with recombinant E. coli-expressed A. viteae tropomyosin emulsified in alum were not protected, while immunization with recombinant A. viteae tropomyosin or with protein purified from worms together with the adjuvant STP led to reduction of adult worm burdens by 30%. Vaccination with cDNA induced protection of about 30%, while application of cDNA together with aluminium phosphate increased the protection to >40%. Our data suggest that vaccination with tropomyosin under Th1 conditions, which are atypical for nematode infections, induces protection via an antibody independent effector mechanism.