Analysis of collagen fiber domain organization by Fourier second harmonic generation microscopy.

We present an automated and systematic two-dimensional discrete Fourier transform (2D-FFT) approach to analyze collagen fiber organization through the use of second harmonic generation (SHG) microscopy. Average orientations of individual domains and Ising-like order parameters introduced to characterize the correlation between orientations of adjacent domains may be used to quantitatively characterize fibrous tissues. Our approach was applied to analyze tissues including rat tail tendon, mouse skin, bovine corneas, and human corneas. We also show that collagen fiber organization in normal and keratokonus human corneas may be distinguished. The current approach may be used for the quantitative differentiation of SHG collagen fiber morphology in different tissues and may be applied for diagnostic purposes.

Bone material properties in premenopausal women with idiopathic osteoporosis.

Idiopathic osteoporosis (IOP) in premenopausal women is characterized by fragility fractures at low or normal bone mineral density (BMD) in otherwise healthy women with normal gonadal function. Histomorphometric analysis of transiliac bone biopsy samples has revealed microarchitectural deterioration of cancellous bone and thinner cortices. To examine bone material quality, we measured the bone mineralization density distribution (BMDD) in biopsy samples by quantitative backscattered electron imaging (qBEI), and mineral/matrix ratio, mineral crystallinity/maturity, relative proteoglycan content, and collagen cross-link ratio at actively bone forming trabecular surfaces by Raman microspectroscopy and Fourier transform infrared microspectroscopy (FTIRM) techniques. The study groups included: premenopausal women with idiopathic fractures (IOP, n = 45), or idiopathic low BMD (Z-score ≤ -2.0 at spine and/or hip) but no fractures (ILBMD, n = 19), and healthy controls (CONTROL, n = 38). BMDD of cancellous bone showed slightly lower mineral content in IOP (both the average degree of mineralization of cancellous bone [Cn.Ca(Mean) ] and mode calcium concentration [Cn.Ca(Peak) ] are 1.4% lower) and in ILBMD (both are 1.6% lower, p < 0.05) versus CONTROL, but no difference between IOP and ILBMD. Similar differences were found when affected groups were combined versus CONTROL. The differences remained significant after adjustment for cancellous mineralizing surface (MS/BS), suggesting that the reduced mineralization of bone matrix cannot be completely accounted for by differences in bone turnover. Raman microspectroscopy and FTIRM analysis at forming bone surfaces showed no differences between combined IOP/ILBMD groups versus CONTROL, with the exceptions of increased proteoglycan content per mineral content and increased collagen cross-link ratio. When the two affected subgroups were considered individually, mineral/matrix ratio and collagen cross-link ratio were higher in IOP than ILBMD. In conclusion, our findings suggest that bone material properties differ between premenopausal women with IOP/ILBMD and normal controls. In particular, the altered collagen properties at sites of active bone formation support the hypothesis that affected women have osteoblast dysfunction that may play a role in bone fragility.

Bone microarchitecture and stiffness in premenopausal women with idiopathic osteoporosis.

CONTEXT: Idiopathic osteoporosis (IOP) is an uncommon disorder in which low areal bone mineral density (aBMD) and/or fractures occur in otherwise healthy premenopausal women. OBJECTIVES: Our objectives were to characterize bone mass, microarchitecture, and trabecular bone stiffness in premenopausal IOP and to determine whether women with low aBMD who have never fractured have abnormal microarchitecture and stiffness. DESIGN, SETTING, AND PATIENTS: We conducted a prospective cohort study of 27 normal controls and 31 women with IOP defined by low trauma fracture (n = 21) or low BMD (Z score

Prediction of seminal vesicle invasion in prostate cancer: incremental value of adding endorectal MR imaging to the Kattan nomogram.

PURPOSE: To retrospectively determine whether endorectal magnetic resonance (MR) imaging findings contribute incremental value to the Kattan nomogram for predicting seminal vesicle invasion (SVI) in patients with prostate cancer. MATERIALS AND METHODS: The institutional review board issued a waiver of authorization, which included a waiver of informed consent, for this HIPAA-compliant study. From October 2000 through January 2005, 573 patients (mean age, 58.3 years; age range, 36-86 years) underwent endorectal MR imaging before prostate cancer surgery. The endorectal MR imaging results had been prospectively interpreted by seven radiologists, and the likelihood of SVI was retrospectively scored on the basis of radiologists' written reports. MR imaging findings, individual clinical variables (serum prostate-specific antigen [PSA] level, Gleason grade, clinical stage, greatest percentage of cancer in all biopsy cores, percentage of positive cores in all biopsy cores, and perineural invasion), and the Kattan nomogram were evaluated with respect to SVI prediction; surgical pathologic analysis was used as the reference standard. Logistic regression and receiver operating characteristic (ROC) curve analyses were performed. RESULTS: At pathologic analysis, 28 (4.9%) of 573 patients had SVI. At univariate analysis, endorectal MR imaging results and all clinical variables except the percentage of positive biopsy cores were significantly associated with SVI (P<.02); endorectal MR imaging (0.76) had a larger area under the ROC curve (AUC) than any clinical variable (0.62-0.73). At multivariate analysis, endorectal MR imaging results, Gleason grade, PSA level, and the percentage of cancer in all biopsy cores were significantly associated with SVI (P

Endorectal MR imaging in the evaluation of seminal vesicle invasion: diagnostic accuracy and multivariate feature analysis.

PURPOSE: To retrospectively determine the accuracy of endorectal magnetic resonance (MR) imaging in demonstrating seminal vesicle invasion (SVI) and to investigate the MR imaging features that can predict SVI. MATERIALS AND METHODS: The Institutional Review Board granted exempt status for this retrospective study, with waiver of informed consent; patient data were collected and handled in accordance with HIPAA regulations. Fifty-one men (age range, 44-73 years) with SVI and 303 men (age range, 40-76 years) without SVI who underwent endorectal MR imaging before radical prostatectomy between January 2000 and October 2004 were included in the study. Endorectal MR images were retrospectively and independently analyzed by two radiologists for SVI, tumor at prostate base, extracapsular extension, and other features considered indicative of SVI. Areas under the receiver operating characteristic curves (AUCs) were used to assess the accuracy of detecting SVI at endorectal MR imaging. A multiple logistic regression was used to explore the combinations of MR imaging features that might facilitate the detection of SVI. RESULTS: Readers 1 and 2 had an AUC of 0.93 and 0.81, respectively, for the detection of SVI. For both readers, the features that had the highest sensitivity and specificity were low signal intensity within the seminal vesicle and lack of preservation of seminal vesicle architecture. At multiple regression analysis, tumor at the prostate base that extended beyond the capsule and low signal intensity within a seminal vesicle that has lost its normal architecture were highly predictive of SVI. CONCLUSION: Endorectal MR imaging is accurate in demonstrating SVI prior to radical prostatectomy, and recognition of the most predictive features may facilitate the use of this modality.

RPR203494 a pyrimidine analogue of the p38 inhibitor RPR200765A with an improved in vitro potency.

Following the discovery of RPR200765, a series of pyrimidine analogues have been prepared as backups. Amongst them, RPR203494 was identified with a better in vitro profile than RPR200765A.

The discovery of RPR 200765A, a p38 MAP kinase inhibitor displaying a good oral anti-arthritic efficacy.

RPR132331, a 2-(2-dioxanyl)imidazole, was identified as an inhibitor of tumour necrosis factor (TNF)alpha release from lipopolysaccharide (LPS)-stimulated human monocytes. An intensive programme of work exploring the biology, toxicity and physical chemistry of a novel series of inhibitors, derived from RPR132331, has led to the identification of RPR200765A, a development candidate for the treatment of rheumatoid arthritis (RA). RPR200765A is a potent and selective inhibitor of p38 MAP kinase (IC50 = 50 nM). It inhibits LPS-stimulated TNFalpha release both in vitro, from human monocytes (EC50 = 110 nM), and in vivo in Balb/c mice (ED50 = 6 mg/kg). At oral doses between 10 and 30 mg/kg/day it reduces the incidence and progression in the rat streptococcal cell wall (SCW) arthritis model when administered in either prophylactic or therapeutic dosing regimens. The compound, which is a mesylate salt and exists as a stable monohydrate, shows good oral bioavailabiltiy (F = 50% in the rat) and excellent chemical stability. The data from the SCW disease model suggests that RPR200765A could exhibit a profile of disease modifying activity in rheumatoid arthritis (RA) patients which is not observed with current drug therapies.

Selective ET(A) antagonists. 5. Discovery and structure-activity relationships of phenoxyphenylacetic acid derivatives.

The fifth paper in this series describes the culmination of our investigations into the development of a potent and selective ET(A) receptor antagonist for the treatment of diseases mediated by ET-1. Receptor site mapping of several ET(A) antagonists prepared previously identified a common cationic binding site which prompted synthesis of phenoxyphenylacetic acid derivative 13a, which showed good in vitro activity (IC(50) 59 nM, rat aortic ET(A)). Optimization of 13a led to the identification of 27b, which exhibited an IC(50) of 4 nM. Although this did not translate into the expected in vivo potency, a compound of comparable in vitro activity, 27a (RPR118031A), showed a far better pharmacokinetic profile and in vivo potency (75 micromol/kg) and was duly proposed and accepted as a development candidate.

Potential of p38 inhibitors in the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic debilitating disease estimated to afflict 13% of the world population. Although palliative treatments (nonsteroidal antiinflammatory drugs or NSAIDs) are widely prescribed, there are currently only a few treatments that can modify the insidious progression of the disease (disease-modifying antirheumatic drugs or DMARDs), which frequently leads to physical incapacitation and, on occasion, death. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are implicated in the disease onset and in the progression of bone and joint destruction that characterizes chronic RA. p38 is an intracellular mitogen/stress-activated protein kinase (MAPK/SAPK) that regulates both the release and the actions of TNF-alpha and IL-1 beta. Inhibition of p38 kinase is thus an important potential target for novel DMARDs. Following the pioneering work conducted at SmithKline Beecham and elucidation of the roles of p38 with potent and selective inhibitors such as SB-203580, many pharmaceutical companies have embarked upon p38 synthetic programs, as indicated by the ever-increasing number of patents in this domain. At Aventis, a rapid parallel synthesis project led to the identification of RPR-200765A, a potent and selective p38 inhibitor of the lipopolysaccharide-induced release of TNF-alpha in vitro in mononuclear phagocytes and in vivo in the rat. It also reduces disease incidence and progression in the rat streptococcal cell wall arthritis model when administered orally in either a prophylactic or a therapeutic dosing regimen. Development of p38 inhibitors has been slow, probably because of toxicological problems, which might explain why only two oral p38 inhibitors, SB-242235 and VX-745, have advanced into clinical development. In the present article, the preclinical data exemplified in studies on RPR-200765A indicating why p38 inhibitors are attracting so much attention as potential novel anti-RA drugs are reviewed. Current information on the different structural classes of p38 inhibitors is presented and possible reasons for the delays in their development are critically discussed.

Recent advances in non-peptidic RANTES antagonists.

RANTES is a member of a large family of cytokines, called chemokines, which are thought to play a regulatory role in leukocyte recruitment and inflammatory processes. The involvement of RANTES in diseases is now well documented and proved using antibody and peptide inhibitors. Non-peptidic RANTES antagonists, which have recently started to appear in the literature, will be reviewed.

Selective endothelin A receptor antagonists. 4. Discovery and structure-activity relationships of stilbene acid and alcohol derivatives.

This publication describes the synthesis and optimization of a novel series of stilbene endothelin antagonists. Analysis of the SAR established for previous papers in this series prompted the design and synthesis of (Z)-4-phenyl-5-(3-benzyloxyphenyl)pent-4-enoic acid 3 which was found to be a moderately active inhibitor of the binding of [125I]ET-1 to ETA receptors with an IC50 of 6 microM. More interestingly, the intermediate compound (E)-2-phenyl-3-(3-benzyloxyphenyl)propenoic acid 5 was equiactive with 3. Optimization of 5 resulted in the preparation of (E)-2-phenyl-3-(2-cyano-5-(thien-3-ylmethoxy))phenylprope noic acid 18 (RPR111723) which had an IC50 in the binding assay of 80 nM on the ETA receptor and a pKB of 6.5 in the functional assay, measured on rat aortic strips. Reduction of the acid group of 5 gave the first nonacidic ETA antagonist in our series, (E)-2-phenyl-3-(3-benzyloxyphenoxy)prop2-enol 6 with an IC50 of 20 microM. Optimization of 6 resulted in the preparation of 2-(2-methylphenyl)-3-(2-cyano-5-(thien-3-ylmethyl)phenyl)pro p-2-enol 33 with an IC50 of 300 nM on the ETA receptor.

Identification of a non peptidic RANTES antagonist.

A series of phenothiazines demonstrating inhibition of RANTES binding to THP-1 cell membranes has been identified. The lead compound RP23618 (IC50 = 3 microM) was found to inhibit specific binding of 125I-RANTES, but not 125I-MCP-1 to THP-1 cell membranes and furthermore to antagonize RANTES, but not MCP-1-induced chemotaxis of THP-1 cells.

New low-density lipoprotein receptor upregulators acting via a novel mechanism.

The synthesis and biological activity of a new series of benzamides and related compounds that upregulate the expression of the low-density lipoprotein (LDL) receptor in human hepatocytes (HepG2 cells) by a novel mechanism are described. The lead compound, N-[5-[(3-cyclohexylpropionyl)amino]-2-methylphenyl]-4-hydroxybe nzamide (1, RPR102359), increased the expression of the LDL receptors in HepG2 cells by 80% when tested at a concentration of 3 microM. Mevinolin (lovastatin) was found to increase the LDL receptor expression by 70% at the same concentration. In contrast to mevinolin, 1 was found to have no effect on cholesterol biosynthesis in liver homogenates or in HepG2 cells at doses where substantial upregulation of the LDL receptor was observed and thus stimulated LDL receptor expression by a novel mechanism.

Self-regulation of the immune system through biobehavioral strategies.

Increasing scientific study and attention is being directed to mind-body interactions, particularly to the interrelationships between the brain and the immune system. This effort has created a new interdisciplinary field, psychoneuroimmunology. An overview of this new field is provided, followed by a comprehensive survey of the growing evidence suggesting that measurable immune system parameters can be influenced by biobehavioral strategies involving self-regulation. Modalities included are relaxation and imagery techniques, biofeedback-assisted strategies, the use of humor, and affiliation to influence emotional states, hypnosis, and conditioning paradigms. The majority of studies have reported significant immune parameter changes. Additional well-designed research is needed to clarify and confirm these initial findings. Future research would benefit from investigating the clinical implications of these immune parameter changes. Knowledge of effects of the mind on the body is rapidly expanding and may be important for achieving improved immune system functioning and health.