Rapid-onset clinical and mechanistic effects of anti-C5aR treatment in the mouse collagen-induced arthritis model.

Preclinical evidence supports targeting the C5a receptor (C5aR) in rheumatoid arthritis (RA). To support ongoing clinical development of an anti-C5aR monoclonal antibody, we have investigated for the first time the mechanism of action and the pharmacodynamics of a blocking anti-murine C5aR (anti-mC5aR) surrogate antibody in mouse collagen-induced arthritis (CIA). First, efficacy was demonstrated in a multiple-dose treatment study. Almost complete inhibition of clinical disease progression was obtained, including reduced bone and cartilage destruction in anti-mC5aR-treated mice. Then, the mechanism of action was examined by looking for early effects of anti-mC5aR treatment in single-dose treatment studies. We found that 48 h after single-dose treatment with anti-mC5aR, the neutrophil and macrophage infiltration into the paws was already reduced. In addition, several inflammatory markers, including tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-17A were reduced locally in the paws, indicating reduction of local inflammation. Furthermore, dose-setting experiments supported a beneficial clinical effect of dosing above the C5aR saturation level. In conclusion, these preclinical data demonstrated rapid onset effects of antibody blockade of C5aR. The data have translational value in supporting the Novo Nordisk clinical trials of an anti-C5aR antibody in rheumatoid arthritis patients, by identifying potential biomarkers of treatment effects as well as by providing information on pharmacodynamics and novel insights into the mechanism of action of monoclonal antibody blockade of C5aR.

Characterization of the interleukin-17 isoforms and receptors in lesional psoriatic skin.

BACKGROUND: Th17 cells are a lineage of proinflammatory T helper cells producing interleukin (IL)-17. The importance of Th17 cells in inflammation and autoimmunity has now been recognized. The IL-17 cytokine family consists of six isoforms (IL-17A-IL-17F) whereas five members of the IL-17 receptor (IL-17R) family have been identified (IL-17RA-IL-17RE). OBJECTIVES: To characterize the expression of the IL-17 isoforms and receptors in lesional and nonlesional psoriatic skin. Methods Keratome and punch biopsies taken from patients with psoriasis were examined by enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction in order to measure the IL-17 isoforms and receptors. RESULTS: We demonstrated significantly increased mRNA expression of IL-17A, IL-17C and IL-17F in psoriatic skin. In contrast, the mRNA expression of IL-17B and IL-17D was significantly decreased in lesional compared with nonlesional skin, while IL-17E mRNA was undetectable. The increased mRNA expression of IL-17A, IL-17C and IL-17F was paralleled by an increased protein accumulation of these cytokines in psoriatic skin. Analysis of the IL-17R mRNA expression revealed significantly impaired mRNA expression of IL-17RB, IL-17RC, IL-17RD and IL-17RE in lesional psoriatic skin, whereas the mRNA expression of IL-17RA was similar in lesional and nonlesional psoriatic skin. CONCLUSIONS: This study characterizes the mRNA profile of the IL-17 isoforms and receptors in psoriatic skin lesions. Furthermore, we demonstrate for the first time augmented protein levels of IL-17A, IL-17C and IL-17F in psoriatic skin lesions, indicating a possible role for IL-17C in addition to IL-17A and IL-17F in the pathogenesis of psoriasis.

Identification of alternatively spliced TIMP-1 mRNA in cancer cell lines and colon cancer tissue.

TIMP-1 is a promising new candidate as a prognostic marker in colorectal and breast cancer. We now describe the discovery of two alternatively spliced variants of TIMP-1 mRNA. The two variants lacking exon 2 (del-2) and 5 (del-5), respectively, were identified in human cancer cell lines by RT-PCR. The del-2 variant was, furthermore, detected in extracts from 12 colorectal cancer tissue samples. By western blotting additional bands of lower molecular mass than full-length TIMP-1 were identified in tumor tissue, but not in plasma samples obtained from cancer patients. The two splice variants of TIMP-1 may hold important clinical information, and either alone or in combination with measurement of full-length TIMP-1 they may improve the prognostic and/or predictive value of TIMP-1 analyses.

A precise and efficient stereological method for determining murine lung metastasis volumes.

We have developed a computer-assisted stereological method based on unbiased principles for estimating metastasis volumes in mouse lungs. We evaluated this method using the transplantable Lewis lung carcinoma. Twenty-one days after subcutaneous inoculation of 10(6) Lewis lung cells into C57BL/6J mice, the mice had primary tumors with an average volume of 2300 mm(3). After perfusion fixation, the lungs were removed, embedded in OCT compound, snap-frozen, and processed for stereology. The metastasis volumes were estimated by application of the Cavalieri principle after evaluation of single sections from several evenly distributed tissue levels. The metastasis volume in a group of nine mice varied between 0.01 and 14.4 mm(3), with an average of 6.1 mm(3). The coefficient of variation was 0.9. The coefficient of error of the volume estimation was determined in five cases and varied from 0.08 to 0.23. Thus, the variation on the metastasis volumes that is achieved by this method contributes very little, 2.5%, to the total variance within the group of mice. In conclusion, we have developed an efficient and unbiased method to determine the metastasis burden in mouse lungs.

Lactational competence and involution of the mouse mammary gland require plasminogen.

Urokinase-type plasminogen activator expression is induced in the mouse mammary gland during development and post-lactational involution. We now show that primiparous plasminogen-deficient (Plg(-/-)) mice have seriously compromised mammary gland development and involution. All mammary glands were underdeveloped and one-quarter of the mice failed to lactate. Although the glands from lactating Plg(-/-) mice were initially smaller, they failed to involute after weaning, and in most cases they failed to support a second litter. Alveolar regression was markedly reduced and a fibrotic stroma accumulated in Plg(-/-) mice. Nevertheless, urokinase and matrix metalloproteinases (MMPs) were upregulated normally in involuting glands of Plg(-/-) mice, and fibrin did not accumulate in the glands. Heterozygous Plg(+/-) mice exhibited haploinsufficiency, with a definite, but less severe mammary phenotype. These data demonstrate a critical, dose-dependent requirement for Plg in lactational differentiation and mammary gland remodeling during involution.

Poly(ADP-ribose) polymerase is found in both the nucleus and cytoplasm of human CNS neurons.

There is evidence that inhibitors of poly(ADP-ribose) polymerase (PARP) may be therapeutically useful in neurodegenerative diseases. Using immunocytochemistry, we have investigated the distribution of PARP in the human CNS. Some neuronal groups showed cytoplasmic staining in addition to the expected staining of nuclei. Considerable variation between different neuronal groups was noted: motor neurons in the spinal cord showed greatest cytoplasmic staining, whereas staining was virtually absent in other neurons, notably in the hippocampus. These results indicate that PARP can be associated with sub-cellular components other than the nucleus, and may indicate additional roles for this enzyme.

The expression of the glial glutamate transporter protein EAAT2 in motor neuron disease: an immunohistochemical study.

Emerging evidence suggests that a disturbance of the glutamate neurotransmitter system may be a contributory factor to motor neuron injury in motor neuron disease. Previous autoradiographic and immunoblotting studies have suggested that there may be reduced expression of glutamate transporter proteins in pathologically affected areas of the CNS in motor neuron disease. This study further explores the possible alteration in expression of the excitatory amino acid transporter protein EAAT2 in MND, by examining the protein expression in situ, in frozen sections, using immunohistochemistry. The aim of the study was to compare the distribution and density of EAAT2 in the motor cortex and spinal cord of MND cases (n = 16) compared with neurologically normal controls (n = 12), matched for relevant parameters. A novel, previously characterized, monoclonal antibody to EAAT2 was employed. EAAT2 immunoreactivity in motor neuron disease and control cases was compared using relative optical density measurements generated by computerized image analysis. In the motor cortex, EAAT2 immunoreactivity was laminated comprising a superficial intense band (corresponding to layers 1 and 2); a paler middle band (layer 3 and part of 5) and a more intense deep layer (layers 5 and 6). In the spinal cord, the ventral horn showed strong immunoreactivity with dense perisomatic staining around motor neuron cell bodies, the substantia gelatinosa showed moderate diffuse staining and the intermediate spinal laminae showed weak staining. This general pattern of immunoreactivity was preserved in the motor neuron disease cases. However, in the motor neuron disease cases compared with controls, the optical density values for EAAT2 immunoreactivity were significantly reduced in all grey matter regions of the lumbar spinal cord (P < 0.001) and were increased in the middle laminae of the motor cortex (P < 0.05). This study indicates that glutamate transporter pathology in motor neuron disease may be a more complex phenomenon than previously recognized.