Identification and preclinical development of an anti-proteolytic uPA antibody for rheumatoid arthritis.

Blocking the proteolytic capacity of urokinase-type plasminogen activator (uPA) with a monoclonal antibody (mAb) reduces arthritis progression in the collagen-induced mouse arthritis model to an extent that is on par with the effect of blocking tumor necrosis factor-alpha by etanercept. Seeking to develop a novel therapy for rheumatoid arthritis, a humanized mAb, NNC0266-0043, was selected for its dual inhibition of both the zymogen activation and the proteolytic capacity of human uPA. The antibody revealed nonlinear elimination kinetics in cynomolgus monkeys consistent with binding to and turnover of endogenous uPA. At a dose level of 20.6 mg kg-1, the antibody had a plasma half-life of 210 h. Plasma uPA activity, a pharmacodynamic marker of anti-uPA therapy, was reduced to below the detection limit during treatment, indicating that an efficacious plasma concentration was reached. Pharmacokinetic modeling predicted that sufficient antibody levels can be sustained in arthritis patients dosed subcutaneously once weekly. The anti-uPA mAb was also well tolerated in cynomolgus monkeys at weekly doses up to 200 mg kg-1 over 4 weeks. The data from cynomolgus monkeys and from human material presented here indicates that anti-uPA mAb NNC0266-0043 is suitable for clinical testing as a novel therapeutic for rheumatic diseases. KEY MESSAGES: Background: Anti-uPA therapy is on par with etanercept in a mouse arthritis model. A new humanized antibody blocks activation and proteolytic activity of human uPA. The antibody represents a radically novel mode-of-action in anti-rheumatic therapy. The antibody has PK/PD properties in primates consistent with QW clinical dosing.

Stellate cells and mesenchymal stem cells in benign mammary stroma are associated with risk factors for breast cancer - an observational study.

BACKGROUND: It is not known whether stromal cells in benign breast tissue can mediate risk of breast cancer. We recently described aldehyde dehydrogenase 1 A1 (ALDH1) positive (+) cells in morphologically normal breast stroma of premenopausal women, and the data indicated that their distribution is associated with clinical risk factors for breast cancer. The aim of the present study was to define the identities of these cells using histologic and immunohistologic methods, and to investigate associations between those cells and hormonal and genetic risk factors in pre- and postmenopausal women. METHODS: Stroma of morphologically normal tissue was analyzed in samples from 101 well-characterized women whose breasts had been operated. Morphology and immunolabeling were applied to determine cell identities based on the putative stem cell markers ALDH1 and stage-specific embryonic antigen-3 (SSEA3), and immunophenotypes indicating mast cells or stellate cells. The results were compared with the patients' risk factors using regression analysis (two-tailed). RESULTS: ALDH1+ round/oval cells were associated with low parity in BRCA1/2 carriers (p = 0.022), while in non-BRCA1/2-carriers they were negatively associated with nulliparity (p = 0.057). In premenopausal women ALDH1+ round/oval cells were associated with family history (p = 0.058). SSEA3+ round/oval cells were morphologically and immunohistologically consistent with multilineage stress-enduring (Muse) cells, and these cells were independently associated with the breast cancer risk factors low parity (p = 0.015), family history (p = 0.021), and hormone use after menopause (p = 0.032). ALDH1+ spindle-shaped/polygonal cells were immunohistologically consistent with stellate cells, and were negatively associated with family history of breast cancer (p = 0.001). CONCLUSION: This study identified novel stromal cell types in benign breast tissue that have a potential for stratifying women for breast cancer risk.

Expression of nitric oxide synthase isoforms in the mouse kidney: cellular localization and influence by lipopolysaccharide and Toll-like receptor 4.

We determined the cellular mRNA expression of all intrarenal nitric oxide (NO)-producing NO synthase (NOS) isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS) and inducible NOS (iNOS) in kidneys from wild-type mice (WT) and immune deficient Toll-like receptor 4 (TLR4) mutant mice, during normal physiological conditions and during a short-term (6-16 h) endotoxic condition caused by systemically administered lipopolysaccaride (LPS). Investigations were performed by means of in situ hybridization and polymerase chain reaction amplification techniques. In WT, LPS altered the expression rate of all intrarenal NOS isoforms in a differentiated but NOS-isoform coupled expression pattern, with iNOS induction, and up- and down-regulation of the otherwise constitutively expressed NOS isoforms, e.g. eNOS and nNOS and an iNOS isotype. In TLR4 mutants, LPS caused none or a lowered iNOS induction, but altered the expression rate of the constitutive NOS isoforms. It is concluded that the intrarenal spatial relation of individual NOS-isoforms and their alteration in expression provide the basis for versatile NO-mediated renal actions that may include local interactions between NOS isoforms and their individual NO-target sites, and that the NOS-isoform dependent events are regulated by TLR4 during endotoxic processes. These regulatory mechanisms are likely to participate in different pathophysiological conditions affecting NO-mediated renal functions.

Molecular identification and developmental expression of UV and green opsin mRNAs in the pineal organ of the Atlantic halibut.

The pineal organ is the only differentiated photoreceptor organ present in embryos and early larvae of the Atlantic halibut (Hippoglossus hippoglossus). We investigated the molecular identity of opsins in the pineal organ, and their expression during different life stages. Using RT-PCR we identified two 681-bp gene sequences, named HPO1 and HPO4, in cDNA from adult pineal and whole embryos. The predicted amino acid sequences showed highest identity to the transmembrane regions of teleostean RH2 green cone opsins (HPO1, 72-91%) and SWS-1 UV cone opsins (HPO4, 71-83%). In situ hybridization revealed expression of HPO1 and HPO4 mRNA transcripts in photoreceptors in the pineal organ of embryos, larvae and adults. HPO1 and HPO4 mRNA transcripts were also expressed in the larval retina. Our study provides molecular evidence for short and middle wavelength light sensitive photoreceptors in the pineal organ of Atlantic halibut throughout life, and suggests that pineal photoreception may play an important role during embryonic and larval life stages, especially at the time when the retina does not possesses corresponding photoreceptor capacity.