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OBJECTIVE: To study the relationship between the boundary-lubricating ability of synovial fluid (SF) and articular cartilage damage in a rabbit knee injury model, to correlate collagen markers of such damage with SF boundary-lubricating ability and elastase activity, and to examine the lubricating ability of SF, together with collagen markers of articular cartilage damage, under the inflammatory conditions of knee joint synovitis (KJS) and rheumatoid arthritis (RA). METHODS: SF was aspirated weekly from the affected knee joints of 10 adult rabbits following transection of the anterior and posterior cruciate ligaments. The boundary-lubricating ability of SF was determined in vitro using a previously described friction apparatus. Lubricin concentrations and type II collagen (CII) peptides were quantified by sandwich enzyme-linked immunosorbent assays (ELISAs). Levels of the C-terminal neoepitope 9A4 (derived from collagenase degradation of CI, CII, and CIII) and of epitope 5-D-4 of keratan sulfate (a marker of proteoglycan depletion) were quantified by inhibition ELISAs. Elastase activity was measured spectrophotometrically. The sensitivity of purified human lubricin to digestion by neutrophil elastase (NE) was examined by Western blotting. RESULTS: The lubricating ability of SF from injured rabbit knees was significantly decreased at weeks 2 and 3 compared with week 1 after injury. Lubricin concentrations were significantly higher at week 1 than at weeks 2 and 3. CII peptide concentrations increased significantly at weeks 2 and 3 compared with week 1, while 9A4 neoepitope concentrations increased significantly at week 3 compared with weeks 1 and 2. There were no significant differences in epitope 5-D-4 concentrations among the 3 weeks. Elastase activity in SF increased significantly at weeks 2 and 3 compared with week 1. Elastase activity correlated significantly with diminishing lubrication at weeks 1, 2, and 3. SF from patients with KJS or RA exhibited deficient lubrication and elevated levels of CII peptides compared with SF from normal controls. NE was shown to completely degrade purified human lubricin in vitro. CONCLUSION: Loss of boundary-lubricating ability of SF after injury is associated with damage to the articular cartilage matrix. This can be attributed to inflammatory processes resulting from the injury, particularly in the early phases. This association also exists in patients with acute knee injuries or progressive chronic inflammatory arthritis. [ABSTRACT FROM AUTHOR]

Background and purposeInterprofessional education between bachelor of science pharmaceutical science (BSPS) students and Doctor of Pharmacy (PharmD) students is rare. According to the Association of American Medical Colleges, more than 80% of medical schools incorporate simulation based teaching within all four years of the curriculum. Educational activity and setting: The University of Rhode Island College of Pharmacy healthcare simulation lab has developed integrated educational opportunities for both groups of students by offering independent study opportunities that allow BSPS students to collaborate with PharmD students. A recent example of this model includes BSPS student development of patient cases which are integrated into high-fidelity human patient simulators with faculty assistance. A senior BSPS student researched and designed four clinical patient cases which were presented to P3 pharmacy students. Findings: In all four cases, there was an increase in knowledge and attitudes following the simulation. Qualitative comments from students noted the importance of patient education and an enhanced ability to manage disease and side effects. Summary: The nature of the simulation lab at the University of Rhode Island is a platform that can be modeled by other institutions with both PharmD and BSPS programs.

Dual specificity phosphatase 6 (DUSP6) is a protein phosphatase that deactivates extracellular-signal-regulated kinase (ERK). Since the ovarian cancer biomarker human epididymis protein 4 (HE4) interacts with the ERK pathway, we sought to determine the relationship between DUSP6 and HE4 and elucidate DUSP6's role in epithelial ovarian cancer (EOC). Viability assays revealed a significant decrease in cell viability with pharmacological inhibition of DUSP6 using (E/Z)-BCI hydrochloride in ovarian cancer cells treated with carboplatin or paclitaxel, compared to treatment with either agent alone. Quantitative PCR was used to evaluate levels of ERK pathway response genes to BCI in combination with recombinant HE4 (rHE4), carboplatin, and paclitaxel. Expression of EGR1, a promoter of apoptosis, was higher in cells co-treated with BCI and paclitaxel or carboplatin than in cells treated with chemotherapeutic agents alone, while expression of the proto-oncogene c-JUN was decreased with co-treatment. The effect of BCI on the expression of these two genes opposed that of rHE4. Pathway focused quantitative PCR also revealed suppression of ERBB3 in cells co-treated with BCI plus carboplatin or paclitaxel. Finally, expression levels of DUSP6 in EOC tissue were evaluated by immunohistochemistry, revealing significantly increased levels of DUSP6 in serous EOC tissue compared to adjacent normal tissue. A positive correlation between HE4 and DUSP6 levels was determined by Spearman Rank correlation. In conclusion, DUSP6 inhibition sensitizes ovarian cancer cells to chemotherapeutic agents and alters gene expression of ERK response genes, suggesting that DUSP6 could plausibly function as a novel therapeutic target to reduce chemoresistance in EOC., Competing Interests: CONFLICTS OF INTEREST The authors declare no commercial or financial conflicts of interest.

Epithelial Ovarian Cancer (EOC) is associated with dismal survival rates due to the fact that patients are frequently diagnosed at an advanced stage and eventually become resistant to traditional chemotherapeutics. Hence, there is a crucial need for new and innovative therapies. Septin-2, a member of the septin family of GTP binding proteins, has been characterized in EOC for the first time and represents a potential future target. Septin-2 was found to be overexpressed in serous and clear cell human patient tissue compared to benign disease. Stable septin-2 knockdown clones developed in an ovarian cancer cell line exhibited a significant decrease in proliferation rates. Comparative label-free proteomic analysis of septin-2 knockdown cells revealed differential protein expression of pathways associated with the TCA cycle, acetyl CoA, proteasome and spliceosome. Further validation of target proteins indicated that septin-2 plays a predominant role in post-transcriptional and translational modifications as well as cellular metabolism, and suggested the potential novel role of septin-2 in promoting EOC tumorigenesis through these mechanisms., Competing Interests: CONFLICTS OF INTEREST RGM is listed on a patent application relating to this study. Other authors have no competing interests to declare.

While selective overexpression of serum clinical biomarker Human epididymis secretory protein 4 (HE4) is indicative of ovarian cancer tumorigenesis, much is still known about the mechanistic role of the HE4 gene or gene product. Here, we examine the role of the secretory glycoprotein HE4 in ovarian cancer immune evasion. Through modified subtractive hybridization analyses of human peripheral blood mononuclear cells (PBMCs), we have characterized gene targets of HE4 and established a preliminary mechanism of HE4-mediated immune failure in ovarian tumors. Dual specificity phosphatase 6 (DUSP6) emerged as the most upregulated gene in PBMCs upon in vitro exposure to HE4. DUSP6 was found to be upregulated in CD8 + cells and CD56 + cells. HE4 exposure reduced Erk1/2 phosphorylation specifically in these cell populations and the effect was erased by co-incubation with a DUSP6 inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI). In co-culture with PBMCs, HE4-silenced SKOV3 human ovarian cancer cells exhibited enhanced proliferation upon exposure to external HE4, while this effect was partially attenuated by adding BCI to the culture. Additionally, the reversal effects of BCI were erased in the co-culture with CD8 + / CD56 + cell deprived PBMCs. Taken together, these findings show that HE4 enhances tumorigenesis of ovarian cancer by compromising cytotoxic CD8 + and CD56 + cells through upregulation of self-produced DUSP6.

Ovarian cancers are known to evade immunosurveillance and to orchestrate a suppressive immune microenvironment. Here we examine the role of human epididymis protein 4 (HE4), an ovarian cancer biomarker, in immune evasion. Through modified subtractive hybridization analyses we have characterized the gene targets of HE4 in human peripheral blood mononuclear cells (PBMCs), and established a preliminary mechanism for HE4-mediated immune failure in ovarian tumours. Upon exposure of purified PMBCs to HE4, osteopontin (OPN) and dual-specificity phosphatase 6 (DUSP6) emerged as the most suppressed and up-regulated genes, respectively. SKOV3 and OVCAR8, human ovarian carcinoma cell lines, exhibited enhanced proliferation in conditioned media from HE4-exposed PBMCs, an effect that was attenuated by the addition of recombinant OPN or OPN-inducible cytokines [interleukin (IL)-12 and interferon (IFN)-Ɣ]. Additionally, upon co-culture with PBMCs, HE4-silenced SKOV3 cells were found to be more susceptible to cytotoxic cell death. The relationship between HE4 and OPN was reinforced further through the analysis of serous ovarian cancer patient samples. In these biopsy specimens, the number of OPN + T cells correlated positively with progression free survival (PFS) and inversely with serum HE4 level. Taken together, these findings show that HE4 enhances ovarian cancer tumorigenesis by compromising OPN-mediated T cell activation., (© 2018 British Society for Immunology.)

Cranberry flavonoids (flavonols and flavan-3-ols), in addition to their antioxidant properties, have been shown to possess potential in vitro activity against several cancers. However, the difficulty of isolating cranberry compounds has largely limited anticancer research to crude fractions without well-defined compound composition. In this study, individual cranberry flavonoids were isolated to the highest purity achieved so far using gravity and high performance column chromatography and LC-MS characterization. MTS assay indicated differential cell viability reduction of SKOV-3 and OVCAR-8 ovarian cancer cells treated with individual cranberry flavonoids. Treatment with quercetin aglycone and PAC DP-9, which exhibited the strongest activity, induced apoptosis, led to caspase-3 activation and PARP deactivation, and increased sensitivity to cisplatin. Furthermore, immunofluorescence microscopy and western blot study revealed reduced expression and activation of epidermal growth factor receptor (EGFR) in PAC DP-9 treated SKOV-3 cells. In addition, quercetin aglycone and PAC DP-9 deactivated MAPK-ERK pathway, induced downregulation of cyclin D1, DNA-PK, phospho-histone H3 and upregulation of p21, and arrested cell cycle progression. Overall, this study demonstrates promising in vitro cytotoxic and anti-proliferative properties of two newly characterized cranberry flavonoids, quercetin aglycone and PAC DP-9, against ovarian cancer cells.

Cartilage destruction is a crucial process in arthritis and is characterized by the degradation of cartilage proteins, proteoglycans, and type II collagen (CII), which are embedded within the extracellular matrix. While proteoglycan loss can be reversed, the degradation of CII is irreversible and has been correlated with an over-expression and over-activation of matrix metalloproteinases (MMPs). Among the various MMPs, the collagenase MMP-13 possesses the greatest catalytic activity for CII degradation. Here we show that the pomegranate-derived polyphenols, punicalagin (PA) and ellagic acid (EA), inhibit MMP-13-mediated degradation of CII in vitro. Surface plasmon resonance studies and molecular docking simulations suggested multiple binding interactions of PA and EA with CII. The effects of PA on bovine cartilage degradation (stimulated with IL-1β) were investigated by assaying proteoglycan and CII release into cartilage culture media. PA inhibited the degradation of both proteins in a concentration-dependent manner. Finally, the anti-inflammatory effects of PA (daily IP delivery at 10 and 50mg/kg for 14days) were tested in an adjuvant-induced arthritis rat model. Disease development was assessed by daily measurements of body weight and paw volume (using the water displacement method). PA had no effect on disease development at the lower dose but inhibited paw volume (P<0.05) at the higher dose., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Cocrystals of tenoxicam, a non-steroidal anti-inflammatory drug, are screened, prepared, and characterized in this study. Nine tenoxicam cocrystals were identified using solvent-drop grinding (SDG) techniques. Structural characterization was performed using powder X-ray diffraction (PXRD), differential scanning calorimetry, and multinuclear solid-state NMR (SSNMR). Thermal analysis, PXRD, and 1D SSNMR are used to detect solvates and phase mixtures encountered in SDG cocrystal screening. 2D SSNMR methods are then used to confirm cocrystal formation and determine structural aspects for selected cocrystals formed with saccharin, salicylic acid, succinic acid, and glycolic acid in comparison to Forms I and III of tenoxicam. Molecular association is demonstrated using cross-polarization heteronuclear dipolar correlation (CP-HETCOR) methods involving (1)H and (13)C nuclei. Short-range (1)H-(13)C CP-HETCOR and (1)H-(1)H double-quantum interactions between atoms of interest, including those engaged in hydrogen bonding, are used to reveal local aspects of the cocrystal structure. (15)N SSNMR is used to assess ionization state and the potential for zwitterionization in the selected cocrystals. The tenoxicam saccharin cocrystal was found to be similar in structure to a previously-reported cocrystal of piroxicam and saccharin. The four selected cocrystals yielded intrinsic dissolution rates that were similar or reduced relative to tenoxicam Form III., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The red raspberry ( Rubus idaeus ) fruit contains bioactive polyphenols including anthocyanins and ellagitannins with reported anti-inflammatory properties. This study sought to investigate the cartilage-protecting and anti-inflammatory effects of a polyphenolic-enriched red raspberry extract (RRE; standardized to total polyphenol, anthocyanin, and ellagitannin contents) using (1) an in vitro bovine nasal explant cell culture model and (2) an in vivo adjuvant-induced arthritis rat model. RRE contained 20% total polyphenols (as gallic acid equivalents), 5% anthocyanins (as cyanidin-3-glucoside equivalents), and 9.25% ellagitannins (as ellagic acid equivalents). In the in vitro studies, bovine nasal explants were stimulated with 10 ng/mL IL-1β to induce the release of proteoglycan and type II collagen. On treatment with RRE (50 μg/mL), there was a decrease in the rate of degradation of both proteoglycan and type II collagen. In the in vivo antigen-induced arthritis rat model, animals were gavaged daily with RRE (at doses of 30 and 120 mg/kg, respectively) for 30 days after adjuvant injection (750 μg of Mycobacterium tuberculosis suspension in squalene). At the higher dose, animals treated with RRE had a lower incidence and severity of arthritis compared to control animals. Also, histological analyses revealed significant inhibition of inflammation, pannus formation, cartilage damage, and bone resorption by RRE. This study suggests that red raspberry polyphenols may afford cartilage protection and/or modulate the onset and severity of arthritis.

Objective: To determine the prevalence, characteristics, and use of professional technical standards among colleges and schools of pharmacy accredited by the Accreditation Council for Pharmacy Education (ACPE)., Methods: The Web site of every college and school of pharmacy accredited by ACPE was searched to identify information regarding the availability, content, and use of technical standards and to obtain demographic information., Results: Information was obtained from all of the 114 colleges and schools of pharmacy and 67 (59%) had technical standards in place. Common themes for technical standards were: observation; communication; motor; intellectual, conceptual, integrative and quantitative abilities; and behavioral and social attributes. Of those colleges and schools with technical standards, 61 (91%) had standards that addressed all 5 of these themes and 34 (51%) specified that the technical standards were used in their admission, progression, and graduation procedures., Conclusion: More than half of the colleges and schools of pharmacy examined in this study have technical standards; however, 41% have yet to develop and implement them. Colleges and schools of pharmacy looking for guidance in technical standards development could use the technical standards themes identified in this study.

The immune tolerance and de novo vascularization are two highly intriguing processes at the maternal-fetal interface that appear to be central to normal pregnancy outcome. Immune tolerance occurs despite the local presence of an active maternal immune system including macrophages, dendritic cells and specialized CD56(bright)CD16(-) uterine natural killer (uNK) cells (65-70%). Recent observations indicate that the phenotypic and functional repertoire of uNK cells is distinct from peripheral blood NK and endometrial NK cells, challenging the understanding of their temporal occurrence and function. Origin and specialized programming of uNK cells continue to be debated. uNK cells, replete with an armamentarium to kill the foreign, tolerate the conceptus and facilitate pregnancy. Why do these uNK cells remain non-cytotoxic? Are these NK cells 'multitasking' in nature harboring beneficial and detrimental roles in pregnancy? Are there distinct subpopulations of NK cells that may populate the decidua? We propose that the endometrium/decidua functions as an 'inducible tertiary lymphoid tissue' that supports the recruitment and expansion of CD56(bright)CD16(-) NK cells and induces transcriptional up-regulation of angiogenic machinery in response to exposure to local hormonal factors, cytokine milieu and perhaps hypoxia. The angiogenic features of uNK cells could further result in a 'multitasking' phenotype that still remains to be characterized. This article discusses the factors and pathways that bridge the angiogenic and non-cytotoxic response machineries at the maternal-fetal interface.

The pH-selective insertion and folding of a membrane peptide, pHLIP [pH (low) insertion peptide], can be used to target acidic tissue in vivo, including acidic foci in tumors, kidneys, and inflammatory sites. In a mouse breast adenocarcinoma model, fluorescently labeled pHLIP finds solid acidic tumors with high accuracy and accumulates in them even at a very early stage of tumor development. The fluorescence signal is stable for >4 days and is approximately five times higher in tumors than in healthy counterpart tissue. In a rat antigen-induced arthritis model, pHLIP preferentially accumulates in inflammatory foci. pHLIP also maps the renal cortical interstitium; however, kidney accumulation can be reduced significantly by providing mice with bicarbonate-containing drinking water. The peptide has three states: soluble in water, bound to the surface of a membrane, and inserted across the membrane as an alpha-helix. At physiological pH, the equilibrium is toward water, which explains its low affinity for cells in healthy tissue; at acidic pH, titration of Asp residues shifts the equilibrium toward membrane insertion and tissue accumulation. The replacement of two key Asp residues located in the transmembrane part of pHLIP by Lys or Asn led to the loss of pH-sensitive insertion into membranes of liposomes, red blood cells, and cancer cells in vivo, as well as to the loss of specific accumulation in tumors. pHLIP nanotechnology introduces a new method of detecting, targeting, and possibly treating acidic diseased tissue by using the selective insertion and folding of membrane peptides.

Objective: To investigate the effect of arthritis development and progression on the integrity and function of lubricin and the relationship of lubricin to cartilage damage in a rat antigen-induced arthritis model., Methods: Arthritis was induced in the right knee joints, using methylated bovine serum albumin and Freund's complete adjuvant. Whole joint friction measurements were performed ex vivo with a modified Stanton pendulum configuration, and coefficients of friction (mu) were determined. Levels of messenger RNA (mRNA) for lubricin, cathepsin B, and interleukin-1beta (IL-1beta) in synovial tissue from control and affected joints were determined by quantitative polymerase chain reaction. Lubricin staining in cartilage was performed using a lubricin-specific monoclonal antibody., Results: The mu values in excised right joints following arthritis induction were significantly (P < 0.001) higher than those in excised left joints. Lubricin mRNA expression levels in synovial tissue on days 4 and 7 after arthritis induction were significantly (P < 0.001) lower in the right joints compared with the left joints, whereas levels of cathepsin B and IL-1beta mRNA expression on days 4, 7, and 14 were significantly (P < 0.001) higher in the right joints than the left joints. Lubricin staining was diminished in cartilage from the right joints compared with the left joints., Conclusion: Elevated coefficients of friction in arthritic joints occur concurrently with enhanced proteolytic degradation by up-regulated cathepsin B and other proteases, as well as decreased lubricin synthesis by synovial fibroblasts and superficial zone chondrocytes. Loss of joint lubrication is an early event in inflammatory arthropathy. Restoring chondroprotection and preventing potential wear-induced cartilage degradation may require lubricin supplementation in synovial fluid.

In arthritic diseases e.g. osteoarthritis (OA) and rheumatoid arthritis (RA), the stability of the collagen type II (CII) fibers, a major component of articular cartilage, is compromised with extensive proteolytic breakdown leading to cartilage erosion and joint deterioration. A clinical need for molecular markers that give instantaneous measure of rate of joint deterioration has developed, as other measurements e.g. arthroscopy, and joint space narrowing are insensitive to small changes in disease status over short periods of time. Owing to its exclusive presence in cartilaginous tissues, markers of CII synthesis and degradation have been extensively studied. Assays that measure these markers in biological fluids e.g. synovial fluid (SF), serum, and urine have been developed and applied to detect early disease onset, monitor disease progression, and response to anti-arthritic drugs. CII synthesis markers include the procollagen type II C-propeptide (PIICP) and the procollagen type IIA N-propeptide (PIIANP). CII degradation markers include CII C-telopeptide (CII-X), CII neoepitope (TIINE), helix II, C2C, CNBr 9.7, Coll 2-1, and Coll 2-1 NO(2). Most of these markers differentiate between early stages of OA, RA and reference controls. The best correlations with structural changes occur when measurements are made in SF while serum measurement frequently did not correlate with structural changes. Although the selection of an optimal marker or a set of markers is still problematic, few markers are of considerable utility in early detection and monitoring of arthritic diseases. The current challenge is to improve the discriminatory power of these markers so they can be used to guide therapeutic decisions.

The pregnane X receptor (PXR) interacts with a vast array of structurally dissimilar chemicals and confers induction of several major types of drug metabolizing enzymes such as cytochrome P450s (CYP). We previously reported that the expression of PXR was markedly increased in rats treated with clofibrate and perfluorodecanoic acid (PFDA). The present study was undertaken to test the hypothesis that induced expression of PXR increases PXR ligand-dependent induction on CYP3A23. Rat hepatocytes were treated with clofibrate or PFDA individually, or along with PXR ligand pregnenolone 16alpha-carbonitrile (PCN), and the levels of PXR and CYP3A23 were determined by Western blots. Both clofibrate and PFDA markedly increased the expression of PXR with PFDA being more potent, and the induction was abolished by actinomycin D, an inhibitor for mRNA synthesis. As expected, PCN alone markedly induced the expression of CYP3A23. Interestingly, co-treatment with clofibrate enhanced the induction, whereas co-treatment with PFDA suppressed it. Clofibrate and PFDA represent multi-classes of chemicals called peroxisome proliferators including many therapeutic agents and industrial pollutants. The opposing effects of clofibrate and PFDA on the PCN-induced expression of CYP3A23 suggest that peroxisome proliferators likely increase the expression of PXR but differentially alter its ligand-dependent induction. The interaction between PXR inducer and ligand provides a novel mechanism on how functionally and structurally distinct chemicals cooperatively regulate the expression of xenobiotic-metabolizing enzymes and transporters.

Kava kava (Piper methysticum), an herbal remedy, is widely used for the treatment of mild to moderate cases of anxiety. The therapeutic activity is presumably achieved through multiple constituents called kavalactones. Recently, kava extracts were shown to induce CYP3A4 and activate human pregnane X receptor (PXR). This study was undertaken to test the ability of purified kavalactones to induce CYP3A23 and activate PXR. Rat hepatocytes were treated with desmethoxyyangonin, dihydrokawain, dihydromethysticin, kawain, methysticin, or yangonin, and the expression of CYP3A23 was monitored. Among the kavalactones, only desmethoxyyangonin and dihydromethysticin markedly induced the expression of CYP3A23 (approximately 7-fold). A similar magnitude of induction was detected with combined six kavalactones at a noninductive concentration when individually used. The induced expression, however, was markedly reduced or completely abolished if dihydromethysticin, desmethoxyyangonin, or both were excluded from the mixtures. Interestingly, regardless of whether dihydromethysticin or desmethoxyyangonin was used alone or together with other kavalactones, similar amounts of total kavalactones were needed to produce comparable induction, suggesting that the inductive activity of dihydromethysticin and desmethoxyyangonin is additively/synergistically enhanced by other kavalactones. In addition, treatment with dihydromethysticin, desmethoxyyangonin, or pregnenolone 16alpha-carbonitrile (PCN) markedly increased the levels of CYP3A23 mRNA, and inhibition of mRNA synthesis abolished the induction. In contrast to PCN, dihydromethysticin and desmethoxyyangonin only slightly activated rat or human PXR. These findings suggest that the induction of CYP3A23 by dihydromethysticin and desmethoxyyangonin involves transcription activation, probably through a PXR-independent or PXR-involved indirect mechanism., (Copyright 2004 The American Society for Pharmacology and Experimental Therapeutics)

Objective: To determine if joint effusions encountered in the emergency department (ED) requiring arthrocentesis possess normal lubricating ability or evidence cartilage degeneration. Chondroprotection of articulating joint surfaces is provided by lubricin, a mucinous glycoprotein that is a product of megakaryocyte-stimulating factor gene (GenBank U70136) expression. Loss of synovial fluid's (SF) lubricating ability has been implicated in the pathogenesis of degenerative joint disease., Methods: A retrospective ED observational study from May 1, 1999, to October 1, 2000, of adult and pediatric patients presenting with radiographically negative knee joint complaints and clinical evidence of joint effusion. Knee joints were aspirated by the emergency physician and the synovial fluid tested for lubricating ability and collagen type II degeneration. Lubricating ability was assayed in vitro in an arthrotripsometer oscillating latex apposed to polished glass under a load of 0.35 106 N/m2. Results were reported as the coefficient of friction ( micro ) relative to that of a 0.9% NaCl control; negative deltamicro value indicates lubrication. Comparisons of deltamicro were made to normal SF and aspirates from patients with osteoarthritis (OA) and rheumatoid arthritis. Collagen type II fragments were measured by a novel sandwich ELISA., Results: Synovial fluid aliquots (n = 57) lubricated poorly with deltamicro = -0.045 (95% confidence interval = -0.006, -0.083) compared to normal SF with D micro = -0.095 (95% CI = -0.088, -0.101). Only 20.6% of knee joint aspirates possessed normal lubricating ability. An association exists between nucleated cell count and deltamicro described by a logarithmic function. Collagen type II fragments were present in aspirates at a concentration of 0.636 microg/ml (95% CI 0.495-0.777 microg/ml), significantly higher than 0.173 microg/ml (95% CI 0.154-0.193 microg/ml) in the OA comparison group., Conclusion: Knee joints with synovitis, commonly encountered in the ED, are frequently nonlubricated bearings and display catabolism of collagen type II. This may play a role in acute cartilage destruction ultimately resulting in posttraumatic OA.

Objective: We have sought to determine if markers of proteoglycans and collagen type II (CII) degradation can be detected at an early stage following acute knee injury in the synovial fluid (SF) from a group of patients diagnosed with non-infectious knee joint synovitis (KJS). CII, proteoglycans and elastase activity in the SF from patients with KJS were compared to SF from patients with two chronic arthritis conditions: osteoarthritis (OA) and rheumatoid arthritis (RA) as well as normal SF controls., Methods: CII peptides were measured by sandwich ELISA using two monoclonal antibodies: 8:6:D8, a CII-specific antibody, and 14:7:D8 which binds to an amino acid sequence on CII as well as collagens type I, III and V. Epitope 9A4, a neo-epitope resulting from collagenase digestion of CI, CII, and CIII was measured by inhibition ELISA. Proteoglycans measurement included total sulfated glycosaminoglycans (sGAG) by dye-binding assay and 5-D-4 epitope, a keratan sulfate epitope, by inhibition ELISA. Elastase activity was measured colorimetircally using N-succinyl trialanine p-nitroanilide (SANA) substrate., Results: The quantified CII peptide concentrations by sandwich and inhibition ELISA were significantly higher in SF from patients with KJS (P<0.05) compared to SF from patients with OA, RA and normal aspirates. 5-D-4 and sGAG concentrations were significantly lower (P<0.05) in SF from patients with KJS compared to SF from patients with OA and RA. Elastase activity in SF from patients with KJS and RA were significantly higher (P<0.05) than SF from patients with OA. A significant correlation exists between elastase activity and 9A4 epitope concentration in SF from patients with KJS., Conclusion: The elevated CII peptides concentrations in KJS SF compared to normal and OA aspirates indicate early signs of cartilage network damage. The low proteoglycans concentrations in SF from patients with KJS may indicate that injury is limited to the superficial zone of cartilage in the patient population studied. The high elastase activity in SF from patients with KJS and RA are linked to the high CII peptides concentration. The elastase activity in the SF from patients with KJS is due to the action of neutrophil elastase (NE) and collagenases, where both contribute to the destruction of the articular cartilage.

The repressed expression of cytochrome P450 (CYP) enzymes in septic patients contributes significantly to therapeutic failures. Mice treated with sepsis-inducing agent lipopolysaccharide (LPS) sequentially express reduced mRNA levels of the pregnane X receptor (PXR) and its target genes Cyp3a(s), suggesting that reduction of Cyp expression is associated with the repression of PXR. The present study was undertaken to determine whether septic rats induced by LPS and cecal ligation/puncture (CLP) express reduced levels of rat PXR protein and whether the subcellular distribution of PXR is altered in septic conditions. Rats were treated with LPS (55 vs. 1 mg/kg) or underwent CLP, and the expression of CYP3A and PXR was determined. In LPS-treated rats, the expression of CYP3A enzymes was consistently decreased regardless of the doses used. In contrast, high dose and repeated low dose of LPS caused significant decreases on the nuclear PXR, whereas the opposite was true with the cytosolic PXR. When rats were administered with only a single low dose of LPS, both nuclear and cytosolic PXR levels were significantly increased. In the CLP model, rats undergoing CLP for 30 h expressed significantly lower levels of CYP3A but the PXR levels were not significantly altered. In addition, when rats were treated with dexamethasone, a significant induction of CYP3A was detected. However, such an induction was markedly antagonized by the treatment with LPS. The differential changes on the levels of the nuclear PXR and CYP3A between LPS and CLP models suggest that PXR plays negligible roles in the constitutive expression of CYP3A. The antagonism of LPS against dexamethasone-mediated CYP3A induction suggests that endotoxemia minimizes the inducibility of PXR target genes.

Objective: This study employed immunohistochemistry to investigate the pattern of type II collagen (CII) degradation in an acute injury model of osteoarthritis. It was hypothesized, based on previous studies of primary osteoarthritis (OA), that the worst areas of CII degradation would be located in the superficial and upper middle zones of the articular cartilage, with staining extending into the deep zone as the OA became more severe., Design: In this model of secondary OA, rabbits were made osteoarthritic by transecting the anterior and posterior cruciate ligaments and removing the meniscus. At various times post surgery, articular cartilage was examined for CII degradation using monoclonal antibody 18:6:D6. This antibody reacts to an epitope that is exposed when CII is degraded as the result of protease cleavage. Proteoglycans (PG) were localized using Safranin-O/Fast Green. Staining intensities were quantitated using image analysis software., Results: In the joints with surgically induced OA, degradation of CII was seen as early as 3 weeks with the majority of the degradation localized in zones I and III. At 14 weeks the destruction of CII was more pronounced, but there was a surprising lack of degradation in zone II. There were also several other unexpected findings. The sham-operated joints, for example, were intended to serve as controls yet CII degradation was observed in rabbits killed 3 weeks after surgery. It was also expected that greater CII degradation would occur in cartilage from medial condyles, but after 14 weeks there was no significant difference between medial and lateral condyles. Finally, the loss of staining for PG correlated with the degradation of CII except in zone III where limited PG loss was observed., Conclusion: Differences were observed between the pattern of articular cartilage destruction in this model of secondary OA and that of primary OA. Further investigation of the mechanical and biochemical processes underlying the development of both types of OA needs to be conducted., (Copyright 1999 OsteoArthritis Research Society International.)

In the adult human, mesenchymal stem cells (MSCs) resident in bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages, including cartilage. In this study, culture-expanded human MSCs (hMSCs) of 60 human donors were induced to express the morphology and gene products of chondrocytes. Chondrogenesis was induced by culturing hMSCs in micromass pellets in the presence of a defined medium that included 100 nM dexamethasone and 10 ng/ml transforming growth factor-beta(3) (TGF-beta(3)). Within 14 days, cells secreted an extracellular matrix incorporating type II collagen, aggrecan, and anionic proteoglycans. hMSCs could be further differentiated to the hypertrophic state by the addition of 50 nM thyroxine, the withdrawal of TGF-beta(3), and the reduction of dexamethasone concentration to 1 nM. Increased understanding of the induction of chondrogenic differentiation should lead to further progress in defining the mechanisms responsible for the generation of cartilaginous tissues, their maintenance, and their regeneration.

Propagated Swaram rat chondrosarcoma cells, rabbit chrondrocytes (from articular cartilage of knee, shoulder and hip joints), and bovine nasal cartilage explant cultures were studied. Type II collagen (CII) and its peptide fragments were quantitated in cell medium and cell layer separately, using two previously developed assays; one assay employed a monoclonal antibody, C4F6, that reacts specifically with triple helical CII and the other assay used an antibody, EIE5, that reacts specifically with a peptide of CII. A time-dependent increase in the content of CII and CII-derived peptides was observed in both rat and rabbit cultures. In both culture systems the majority of the native type II collagen is found associated with the cell layer (97% in rat cultures and 73% in rabbit cultures), while the major part of the CII peptides is found in the media (73% in rat cultures, 88% in the rabbit cultures). The concentration of peptides in the media reaches approximately 2 micrograms mL-1 in both chondrocyte monolayer cultures after 4 days. The CII peptide assay employing E1E5 was well suited to quantitate articular cartilage collagen degradation in explant culture. thus it can be used to evaluate potential therapeutic agents that can modify or inhibit cartilage degradation. The assay has the added potential that it could be used in-vivo to evaluate the effectiveness of potential metalloproteinase inhibitors in animal models of osteoarthritis or in clinical trials.

Cartilage destruction is a characteristic feature of osteoarthritis. Treatment with certain nonsteroidal anti-inflammatory drugs could exacerbate cartilage destruction by impairing the synthesis of cartilage matrix proteins, type II collagen and proteoglycan. In order to monitor the changes occurring in cartilage collagen synthesis, we developed a type II collagen specific ELISA. The effects of antiarthritic agents on type II collagen and glycosaminoglycan synthesis were examined in rat chondrosarcoma cultures. Drugs were added to the monolayer cultures and 4 days later the total type II collagen, as determined by the type II collagen ELISA, and glycosaminoglycan content, as measured by dimethyl-methylene blue dye binding assay, was measured. All drugs except tiaprofenic acid decreased type II collagen synthesis by at least 40% at 100 micrograms/ml. Tiaprofenic acid at 1 microgram/ml increased type II collagen content by 54% of the controls. Glycosaminoglycan synthesis was decreased by acetylsalicylic acid, diclofenac and tiaprofenac acid, at 50 micrograms/ml or above. Indomethacin, naproxen and dexamethasone had no effect. Interestingly, tenidap stimulated the glycosaminoglycan synthesis by 32% at 100 micrograms/ml. We show that the combination of chondrosarcoma cultures, type II collagen specific ELISA and dimethylmethylene blue dye binding assay serves as a useful model for screening the effects of agents capable of modulating type II collagen and glycosaminoglycan synthesis.

Immunoassays are used for the specific measurement of type II collagen, a major cartilage protein, which is lost in osteoarthritic joints. Poor immunogenicity and species dependent immune response to type II collagen make it difficult to obtain specific antibodies required for immunoassay development. In addition, type II collagen antibodies exhibit reactivity to structurally dissimilar antigens such as actin, myoglobin, thyroglobulin and ssDNA, complicating the isolation of specific antibodies. It is therefore necessary to characterize the antibody reactivity against both noncollagenous antigens and different collagen types. In this study, immune response to type II collagen was improved by conjugation to carrier proteins, KLH and BSA. Hybridomas were generated by fusions of lymphocytes derived from lymph nodes or spleens with X63-653-Ag8 myeloma cells. Compared to spleens, the utilization of lymph nodes as a source of lymphocytes resulted in a 23% higher number of hybridomas secreting type II collagen antibodies. Hybridomas secreting polyreactive antibodies were identified based on their reactivity to thyroglobulin and eliminated. Extensive testing of the remaining monoclonal antibodies with other structurally dissimilar antigens and various types of collagen for reactivity, allowed us to isolate specific monoclonal antibodies to type II collagen. We emphasize the importance of characterization of the reactivity of type II collagen monoclonal antibodies before employing them for immunoassays.

In the marrow space hemopoietic progenitors interact with stromal cells allowing homing, migration and the expression of developmental programs. Some of these interactions are mediated by extracellular matrix molecules (ECM) which are produced by marrow stromal cells. In the murine system the nature of these interactions and its biological significance have been studied in vitro by using indistinctly, either whole stroma (obtained from long-term marrow cultures) or isolated stromal (fibroblast-like) cells. In an attempt to analyze whether in the human system both sources of stromal cells are equivalent in terms of production of ECM components, we measured the expression of genes encoding for several ECM proteins. The results obtained show that whole stroma as well as marrow fibroblasts express genes for collagen types I, III, IV, V and VI as well as for fibronectin and laminin. However, the relative abundance of mRNA transcripts for some of these proteins was higher in marrow fibroblasts as compared to whole stroma. The latter was further supported by an increased collagen synthesis observed in marrow fibroblasts as compared to whole stroma.

Doxazosin was administered to rabbits fed diets enriched in cholesterol and peanut oil for 7.5 or 12 weeks, in 2 separate experiments. Doxazosin suppressed the accumulation of cholesterol and formation of atherosclerotic plaques in the aortas of treated rabbits and prevented a diet-induced increase in aortic collagen and wall mass. Doxazosin was more effective in the thoracic and abdominal segments of the aorta than in the aortic arch. Pharmacokinetic analysis indicated that treated rabbits were exposed to concentrations of doxazosin, integrated over 24 h, which were consistent with the therapeutic range of doxazosin measured in patients treated for hypertension. Doxazosin did not alter serum levels of cholesterol or triglycerides, nor were there any consistent effects on glucose, free fatty acid or ketone levels. Hypotheses of the mechanism of action of doxazosin are discussed, including the possible involvement of alpha 1-adrenergic receptors in recruitment of smooth muscle cells by subintimal macrophages and nonadrenergic mechanisms of inhibition of lipid infiltration.

This paper describes the development of quantitative immunoassays utilizing mouse monoclonal antibodies which are monospecific to type II collagen. The monoclonal antibodies were characterized and tested extensively for reactivity against a panel of antigens including actin, myoglobin, thyroglobulin, ssDNA, tetanus toxin and different types of collagens including their CnBr-derived peptides. Four monoclonal antibodies having strict monospecificity to type II collagen were selected. Quantitative immunoassays developed with these antibodies can measure either type II collagen in its native conformation or type II collagen-derived cyanogen bromide peptides. The assay conditions such as coating concentration of antigen, monoclonal antibody concentration, second antibody concentration and incubation times were optimized to obtain maximum possible sensitivity. These quantitative immunoassays can be employed to measure type II collagen or type II collagen-derived peptides in low amounts ranging from 20 to 100 ng/ml. The assays can be applied to chondrocyte cultures without interference from serum components or other collagen types.

Streptozotocin (STZ)-induced diabetes depresses the rate of vascular collagen synthesis in the spontaneously hypertensive rat (SHR), but it also reduces arterial pressure (SAP) in this strain. We investigated this phenomenon further by comparing the SHR with the renovascular hypertensive (RVH) rat, because diabetes does not affect SAP in the latter model of hypertension. Renovascular hypertension was induced by clipping the left renal artery of Wistar-Kyoto (WKY) rats; sham-operated WKY were included as normotensive controls. Collagen synthesis of arterial tissue in vitro was quantified as prolyl hydroxylase activity and the rate of radioactive proline incorporation into collagen. Arterial collagen synthesis of nondiabetic SHR and RVH animals was elevated compared to that of the nonhypertensive WKY controls. STZ-induced diabetes (8 weeks) reduced SAP of SHR, but had no effect on SAP of either RVH or normotensive WKY rats. However, diabetes significantly depressed vascular collagen synthesis of both SHR and RVH rats, and, less consistently, of the WKY. The results strongly suggest that STZ-induced diabetes in SHR impairs arterial collagen synthesis independent of associated changes in arterial pressure.

Hybridoma antibodies against human lysyl oxidase were produced by fusing Sp. 2.0-Ag 14 myeloma cells with spleen cells from mice hyperimmunized with lysyl oxidase isolated from umbilical cords. Hybridomas positive by enzyme-linked immunosorbent assay (ELISA) for human lysyl oxidase were cloned by the dilution method. Eight hybridomas producing antibodies were isolated, three of which also recognized purified bovine aortic lysyl oxidase in an ELISA. One of these antibodies, monoclonal antibody I, was purified and attached to Sepharose CL-4B. The immobilized antibody was effective in binding an enzymatically active 30,000 dalton species. Immunoblot analysis of four of these antibodies showed reactivity against the 30,000 dalton catalytically active enzyme and the 24,000 dalton fragment of lysyl oxidase. These monoclonal antibodies should be useful tools for studying the localization and biosynthesis of lysyl oxidase.

Prolyl hydroxylase and an immunologically related protein (CRP) were purified from neonatal rabbit skin at various time periods following administration of 3H-leucine. The peak incorporation of label into prolyl hydroxylase was found to be 12 hours, while peak incorporation into CRP occurred within 2 hours. Semi-log plots of the loss of radioactivity from these protein pools against time indicated an apparent T 1/2 for prolyl hydroxylase of 78 hours, and a T 1/2 of CRP of 44 hours. Calculated Kd values indicate that that breakdown of active enzyme does not account for the amount of CRP found in tissues.

Stereospecifically labeled mevalonic acid was incorporated into the carotenoids of Rhodotorula. The randomized results are discussed in relation to mechanisms proposed for the conversion of isopentenyl pyrophosphate to dimethylallyl pyrophosphate and the prenol transferase enzyme system to Rhondotorula rubra.

A sanitary microbiological survey of fresh and processed fish on sale in Guatemalan markets and supermarkets was carried out. Samples of tables, floor and freezer surfaces, and of the water, ice and liquid drippings of the different stands were also taken and analyzed. Aerobic counts exceeded 10(6)/g in 72% of the samples, and 16% had more than 40 fecal coliforms/g. Coagulase-positive Staphylococcus aureus were never higher than 100/g in fish. The bacterial counts were higher in the ice and liquid drippings.

Human monoclonal antibodies reactive with type II collagen were obtained from patients with rheumatoid arthritis and relapsing polychondritis by fusion of cells with the human-mouse myeloma analogue HMMA2.11 TG/0. Direct fusion of peripheral blood or bone marrow mononuclear cells was unsuccessful in obtaining antibody producing hybridomas although fusion efficiency was high (1 hybridoma per 25,400 mononuclear cells fused). Polyclonal, Epstein Barr Virus transformed B cell lines derived from peripheral blood and bone marrow mononuclear cells after direct transformation, in vitro secondary immunization, and/or CD8 depletion were found to produce antibodies that reacted with type II collagen. Antibody producing EBV transformed B cells were fused with the human-mouse myeloma analogue HMMA2.11 TG/0 and six separate, stable IgM antibody producing hybridomas obtained. These results demonstrate the difficulty in obtaining human monoclonal autoantibodies, particularly of IgG isotypes, using readily accessible sources of cells in patients with chronic autoimmune diseases without expansion and preselection.

Relative bioavailability of two iron fortificants, electrolytic Fe and ferric orthophosphate, was related to that of the reference ferrous sulfate with in vitro and rat model depletion-repletion methods in four laboratories to compare values directly with those obtained in a parallel human study. In vitro testing was performed on Fe compounds with both solubility and dialysis in a simulated in vitro gastrointestinal digestion system. Two depletion-repletion techniques, hemoglobin-regeneration efficiency (HRE) and an official method of the Association of Official Analytical Chemists (AOAC), were examined. AOAC relative biological values (RBV) of electrolytic Fe were 0.66 and 0.78 and of FePO4 were 0.25 and 0.34. HRE values were 0.78 and 0.58 for electrolytic Fe and FePO4, respectively. When compared with FeSO4 in a radiolabeled farina-based meal fed to humans, the RBV of FePO4 was 0.25 and electrolytic Fe 0.75. Results obtained with the AOAC method serve as the most reliable prediction of Fe bioavailability in the human although in vitro dialysis is a promising screening technique.

The effects of diphenylamine, dimethyl sulfoxide, streptomycin, AMO-1618, and beta-ionone on the carotene composition of a wild-type and three mutant strains of Phycomyces blakesleeanus have been examined. Diphenylamine increased the phytoene and phytofluene concentrations of all strains while reducing the levels of the color carotenes. Dimethyl sulfoxide reduced the concentration of both cyclic and acyclic carotenes, whereas AMO-1618 increased the levels of all carotenes in all the strains. The wild type and mutants responded differently to the presence of streptomycin and beta-ionone. The possible mode of action of the above agents on carotenoid biosynthesis is discussed.

In this paper we have described all the steps needed to develop an efficient program for distributing children's fortified formulas. The easiest steps are to develop the formulas on an experimental and laboratory basis. The real obstacles lie in implementation at the national level. All that has been described applies to experience in Chile, which we consider very successful because of the acceptance of the program as well as spectacular advances in preventing malnutrition. The figures for 1977 show that combining all degrees of malnutrition in the under six age group, the prevalence is now 12.2% compared to nearly 60% found 10 years ago. This progress is due, not only to the specific program, but also to the many others that constitute the national food and nutrition policy (CONPAN, 1976). This success is due to the persistent, skilled effort of many professional and technical staff members. It is impractical to acknowledge all individual contributions here. The National Food and Nutrition Council, the National Health Service, Universities, Research Bodies, and private enterprizes have all contributed to this joint effort. The purpose of this presentation is that this experience may benefit other countries with similar problems.

Prolyl hydroxylase [EC 1.14.11.2] was shown to be inhibited by an ultrafiltrate (less than 30,000 molecular weight) fraction isolated from skin and blood of neonatal and adult rabbits. This fraction also inhibited two other alpha-ketoglutarate requiring mixed function oxidases, lysyl hydroxylase [EC 1.14.11.4] and alpha-butyrobetaine hydroxylase [EC 1.14.11.1] but not the amine oxidase, lysyl oxidase. Purification of the skin ultrafiltrate on Sephadex G-25 demonstrated a peak of prolyl hydroxylase inhibitory activity which chromatographed at a molecular weight corresponding to approximately 3,000. Chromatography of a blood ultrafiltrate separated a similar peak of material which was inhibitory for prolyl hydroxylase.