Blood, sweat, and tears: developing clinically relevant protein biosensors for integrated body fluid analysis.

Biosensors are being developed to provide rapid, quantitative, diagnostic information to clinicians in order to help guide patient treatment, without the need for centralised laboratory assays. The success of glucose monitoring is a key example of where technology innovation has met a clinical need at multiple levels ­ from the pathology laboratory all the way to the patient's home. However, few other biosensor devices are currently in routine use. Here we review the challenges and opportunities regarding the integration of biosensor techniques into body fluid sampling approaches, with emphasis on the point-of-care setting.

Characterization of glucokinase regulatory protein-deficient mice.

The glucokinase regulatory protein (GKRP) inhibits glucokinase competitively with respect to glucose by forming a protein-protein complex with this enzyme. The physiological role of GKRP in controlling hepatic glucokinase activity was addressed using gene targeting to disrupt GKRP gene expression. Heterozygote and homozygote knockout mice have a substantial decrease in hepatic glucokinase expression and enzymatic activity as measured at saturating glucose concentrations when compared with wild-type mice, with no change in basal blood glucose levels. Interestingly, when assayed under conditions to promote the association between glucokinase and GKRP, liver glucokinase activity in wild-type and null mice displayed comparable glucose phosphorylation capacities at physiological glucose concentrations (5 mM). Thus, despite reduced hepatic glucokinase expression levels in the null mice, glucokinase activity in the liver homogenates was maintained at nearly normal levels due to the absence of the inhibitory effects of GKRP. However, following a glucose tolerance test, the homozygote knockout mice show impaired glucose clearance, indicating that they cannot recruit sufficient glucokinase due to the absence of a nuclear reserve. These data suggest both a regulatory and a stabilizing role for GKRP in maintaining adequate glucokinase in the liver. Furthermore, this study provides evidence for the important role GKRP plays in acutely regulating of hepatic glucose metabolism.

Comparison of the nitric oxide synthase inhibitors methylarginine and aminoguanidine as prophylactic and therapeutic agents in rat adjuvant arthritis.

OBJECTIVE: To compare the nitric oxide synthase (NOS) inhibitors NG-methyl-L-arginine (L-NMA) and aminoguanidine (AG) as prophylactic and therapeutic agents in rat adjuvant induced arthritis (AIA). METHODS: Arthritis was induced in male Lewis rats by the injection of adjuvant into the base of the tail. L-NMA or AG was administered twice daily by gastric intubation starting at the time of adjuvant injection, just before the onset of clinical symptoms, or after the onset of clinical symptoms. Paw swelling, plasma fibrinogen levels and urinary NO2-/NO3- excretion were measured to assess the effect of the inhibitors on the arthritis response and whole body NO biosynthesis. Selected joints were also evaluated histopathologically. The abilities of L-NMA, AG and another NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), to inhibit NO production by chondrocytes and synoviocytes were also compared. RESULTS: Treatment with L-NMA (400 mg/kg/day) or AG (500 mg/kg/day) reduced the urinary excretion of NO2-/NO3- to the control level. L-NMA suppressed the development of AIA when administered prophylactically; however, its antiarthritic properties declined with increasing delay of application. It was only weakly effective against established AIA. AG had neither a prophylactic nor a therapeutic antiarthritic effect. AG and L-NAME were much weaker inhibitors of NO production by chondrocytes and synoviocytes than L-NMA. CONCLUSION: Although L-NMA completely suppresses the development of AIA when administered prophylactically, it is much less effective when administered therapeutically. Furthermore, not all inhibitors of NOS show equal prophylactic activity against AIA. In addition, NOS inhibitors may be only weakly therapeutic, or even detrimental, in established disease. These findings should be considered when evaluating NOS inhibitors as potential therapeutic agents for the treatment of established human arthritis.

Potent peptide inhibitors of stromelysin based on the prodomain region of matrix metalloproteinases.

Stromelysin is secreted as an inactive zymogen that is activated in the extracellular space by cleavage of the His81-Phe82 bond with the release of the 81-amino acid propeptide domain. This segment contains a 12-amino acid sequence (MRKPRC75GVPDVG) that is highly conserved in all matrix metalloproteinases. Previous studies have shown that the hexapeptide, Ac-RCGVPD-NH2, and the pentapeptide, Ac-RCGVP-NH2, based on this region retain significant inhibitory activity. This new structure-activity relationship study of both peptides has shown that only Cys75 and Val77 are essential for inhibitory activity. Peptides based on this series inhibited stromelysin and collagenase with equal potency. Additional peptides spanning this region were synthesized in order to focus on these two sites. Significantly, isocysteine was substituted for Cys75 without significant loss of inhibitory activity. Tyr-(2,6-dichlorobenzyl) was substituted for Val77. The introduction of these 2 new residues into Ac-CGVP-NH2 produced a very potent inhibitor, Ac-isoCGY-(2,6 dichlorobenzyl)-P-NH2 with an IC50 of 3 microM. The following factors, acting in combination, determine the inhibitory activity of peptides in this series: distance between the sulfur atom and the peptide backbone, coordination geometry of the thiol side chain with the active-site zinc, and conformational flexibility of the side-chain.

Inhibition of human stromelysin by peptides based on the N-terminal domain of tissue inhibitor of metalloproteinases-1.

The tissue inhibitors of metalloproteinases (TIMPs) represent a family of naturally occurring protein inhibitors of stromelysin and other members of the family of matrix metalloproteinases. A series of peptides based on the N-terminal sequence of natural TIMP-1 was synthesized and assessed for inhibitory activity against purified human stromelysin. Inhibitor peptides were identified in the loop (bounded by the disulfide bonds [C3-C99] and [C13-C124]), e.g., [C3(Acm)-C13], (IC50, 42 microM). It was established that inhibition was due to the free sulfhydryl group of either C13 or C124. However, peptides within [C70(Acm)-C98(Acm)] inhibited stromelysin independently of zinc co-ordination by cysteine. The binding epitope in TIMP-1 may be discontinuous and comprised of sequences from at least 2 loops.

N-monomethyl arginine, an inhibitor of nitric oxide synthase, suppresses the development of adjuvant arthritis in rats.

OBJECTIVE: To test the hypothesis that nitric oxide (NO) is involved in the pathophysiology of arthritis. METHODS: Arthritis was induced in male Lewis rats by the injection of adjuvant into the base of the tail. The NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine (L-NMA), was administered daily by the oral route for 19 days. Paw swelling, plasma fibrinogen levels, and urinary NO2/NO3 levels were measured to assess the effect of L-NMA on the arthritic response and whole-body NO production, respectively. On day 20, the ankle joints were processed for histopathologic evaluation. RESULTS: The onset of clinical symptoms was preceded by elevated biosynthesis of NO. In a dose-dependent manner, L-NMA inhibited both NO biosynthesis and paw swelling; histopathologic changes in the ankle joints were also prevented. D-NMA had no effect on the development of arthritis, while L-arginine reversed the effects of L-NMA. Fibrinogen levels in rats with arthritis were unaffected by L-NMA. CONCLUSION: NO is critical to the development of both the inflammatory and erosive components of adjuvant arthritis in rats. There may be a future clinical role for suitable inhibitors of NO production or activity.

Interleukin-1 beta induces cytosolic phospholipase A2 and prostaglandin H synthase in rheumatoid synovial fibroblasts. Evidence for their roles in the production of prostaglandin E2.

OBJECTIVE: In order to investigate potential regulatory mechanisms for the increased production of prostaglandin E2 (PGE2) in interleukin-1 beta (IL-1 beta)-stimulated rheumatoid synovial fibroblasts (RSF), this study examined the induction of phospholipase A2 (PLA2) and prostaglandin H synthase (PGHS) enzymes and the correlation of these events with PGE2 production in IL-1 beta-stimulated RSF. METHODS: Protein and messenger RNA (mRNA) levels of cytosolic PLA2 (cPLA2) and PGHS-2 enzymes in IL-1 beta-stimulated RSF were measured by Western and Northern blotting, respectively, using specific antisera and complementary DNA probes. Enzymatic activity of cPLA2 was determined in cell-free reaction mixtures utilizing mixed micelles of 14C-phosphatidylcholine and Triton X-100 as the substrate. PGE2 levels were quantitated using a commercial enzyme immunoassay kit. RESULTS: Incubation of RSF with IL-1 beta increased the mRNA and protein levels for the high molecular weight cPLA2 as well as for the mitogen/growth factor-responsive PGHS (PGHS-2). The IL-1 receptor antagonist completely abolished the induction of these two enzymes and the stimulation of PGE2 production by IL-1 beta in RSF. In contrast, levels of the other known forms of these enzymes, i.e., the 14-kd secretory group II PLA2 (sPLA2) and the constitutive form of PGHS (PGHS-1), were unaffected by IL-1 beta treatment. CONCLUSION: These are the first data to demonstrate the coordinate induction by IL-1 of cPLA2 and PGHS-2 in RSF. The time-course for the induction of these enzymes suggests that their increase contributes to the increased production of PGE2 in IL-1-treated RSF, and may help explain the capacity of RSF to produce large amounts of PGE2.

Effect of treatment with interleukin-1 receptor antagonist on the development of carrageenan-induced pleurisy in the rat.

Pretreatment of rats with the human interleukin-1 receptor antagonist (IL-1ra) by the subcutaneous route at -0.5 h, relative to the intrapleural injection of carrageenan (CG), suppressed the infiltration of cells into the pleural cavity of intact and adrenalectomized rats at 5 h (28 and 74% reduction in intact animals at 0.3 and 10 mg/kg, respectively). Exudate volume at 5 h was also suppressed at dosages of IL-1ra > or = 3 mg/kg. IL-1ra was still effective as an antiinflammatory agent in the 5-h pleurisy model when administered 1 or 2 h, but not 3 h, after CG. The addition of IL-1ra to a maximally effective antiinflammatory dosage of indomethacin (5 mg/kg) resulted in further reductions of cell number and exudate volume, suggesting that the antiinflammatory effects of IL-1ra in the 5-h model were not due solely to inhibition of IL-1-induced prostaglandin biosynthesis. The antiinflammatory effects of suboptimal dosages of IL-1ra and dexamethasone, administered in combination, were essentially additive. In 24-h pleurisy, IL-1ra reduced exudate volume and PMN number after a single dosage of 10 mg/kg, subcutaneously at -0.5 h and after dosages of 3 mg/kg at -0.5 and 5 h. Additional dosages of IL-1ra (3 mg/kg) at 10 and 15 h did not further inhibit PMN accumulation. Treatment with IL-1ra did not affect macrophage accumulation in 24-h CG-induced pleurisy. IL-1ra was not active as an antiinflammatory agent in the 24-h pleurisy model after three dosages of IL-1ra (3 mg/kg) at 5, 10 and 15 h.

Interleukin-1 beta induces cytosolic PLA2 in parallel with prostaglandin E2 in rheumatoid synovial fibroblasts.

We investigated the temporal relationship between the increase in enzymatic activity and protein of a high molecular weight (100 kDa), cytosolic PLA2 (cPLA2) in interleukin-1 beta (IL-1 beta)-treated rheumatoid synovial fibroblasts (RSF). Both of these responses increased according to a similar time-course which correlates with PGE2 production by these cells. In contrast, 14 kDa, secreted PLA2 (sPLA2), which was also produced by RSF, was not affected by IL-1 beta treatment. These findings support that an augmentation of CPLA2 activity, caused by an induction of cPLA2 protein, rather than sPLA2, is temporally associated with increased PGE2 production in IL-1 beta-treated RSF.

Interleukin-1 beta stimulates cytosolic phospholipase A2 in rheumatoid synovial fibroblasts.

Phospholipase A2 (PLA2) activities in rheumatoid synovial fibroblasts (RSF) stimulated with interleukin-1 beta (IL-1 beta) were investigated. RSF incubated in the presence of IL-1 beta (120 pg/ml) for 18 h secreted 35 fold more PGE2 than did those incubated without IL-1 beta. IL-1 beta treatment did not increase the level of secretory PLA2 (sPLA2) activity or sPLA2 protein in the conditioned medium or subcellular fractions of lysed RSF. In contrast, the cell-associated PLA2 activity increased 3 to 4 fold in IL-1 beta stimulated RSF when compared with the control. The IL-1 beta stimulated, cell-associated PLA2 required submicromolar concentrations of calcium for activity, a characteristic consistent with the calcium sensitivity of cytosolic PLA2 (cPLA2) activity reported in other cell types, such as U937 cells. These findings demonstrate that an elevation in a cytosolic PLA2, rather than a sPLA2, is associated with increased PGE2 production in IL-1 beta stimulated RSF.

Macrophage-mediated killing of splenic lymphocytes in adjuvant-induced arthritis.

After 72 hrs in culture, unseparated spleen cells from rats with adjuvant-induced arthritis (AA) stimulated with high concentrations of Con A (greater than 0.63 micrograms/ml) leaked more lactate dehydrogenase (LDH) than did either unstimulated cultures or cultures stimulated with lower concentrations of Con A. This reflects an increase in dead or dying cells at concentrations of Con A greater than 0.63 micrograms/ml. Since Con A did not increase LDH leakage in macrophage-depleted cultures of AA splenocytes, the decreased viability in unseparated, Con A-stimulated cultures appears to be a macrophage-dependent phenomenon. Since the increase in LDH release at high concentrations of Con A paralleled the decrease in [3H]-thymidine incorporation, these results suggest that Con A (greater than 0.63 micrograms/ml) induces macrophages from AA rats to kill splenic lymphocytes in culture.

Effects of a novel anti-inflammatory retinoid-like 2,4,6,8-nonatetraenoic acid on the immunological changes associated with adjuvant-induced arthritis.

Proliferative responses to the T-cell mitogen, Con A, were markedly suppressed in spleen cells isolated from rats 12-16 days following induction of adjuvant-induced arthritis (AA). These responses were only partially restored following removal of plastic-adherent cells (AC-depleted). Prophylactic treatment of AA rats with a novel anti-inflammatory retinoid-like 2,4,6,8-nonatetraenoic acid, Ro 23-6457, increased mitogen-induced proliferative responses in spleen cells, particularly in AC-depleted cultures. Treatment of AA rats with Ro 23-6457 significantly increased Con A-induced IL-2 production by both unseparated and AC-depleted spleen cells. Although exogenous IL-2 did not restore proliferative responses to Con A-stimulated spleen cells from vehicle-treated AA rats, responses in AC-depleted cells from Ro 23-6457-treated AA rats were further enhanced by the addition of IL-2. Following stimulation with LPS, supernatants from cultures of adherent spleen cells isolated from AA rats contained more IL-1 (expressed as units/ml) than cultures from normal rats. Treatment of AA rats with a high dose of Ro 23-6457 normalised IL-1 levels in these cultures. Treatment of normal rats with Ro 23-6457 had no significant effects on any parameter tested. These data suggest that Ro 23-6457's modulation of certain disease-associated alterations in immune function in AA rats may contribute to its anti-inflammatory activity.

Anti-inflammatory and immuno-modulatory effects of novel retinoid-like 2,4,6,8-nonatetraenoic acids (NTA) in adjuvant-induced arthritis.

Prophylactic treatment (p.o.) of rats with adjuvant-induced arthritis (AA) with two retinoid-like 2,4,6,8-nonatetraenoic acids (NTA), Ro 23-6457 and Ro 23-2895, significantly reduced hind paw swelling between days 10-23 and the level of plasma fibrinogen (MED approximately 25 mumoles/kg). When given therapeutically (75 mumoles/kg between day 21 and 28) either NTA arrested the progression of the disease (MED, 25-75 mumoles/kg). Unseparated and adherent cell (AC) depleted spleen cells from rats with AA (day 12-15) responded poorly to the T cell mitogen, Con A (2.5 micrograms/ml) and the B cell mitogen, LPS (10 micrograms/ml). The responses were partially restored (approximately 30% of normal responses) in AC-depleted (but not unseparated) spleen cells from Ro 23-6457 treated rats (75 and 250 mumoles/kg/day). These data demonstrate an immunomodulatory effect of Ro 23-6457 in the adjuvant rat which may contribute to its anti-inflammatory activity in AA.

Comparative study of natural and synthetic retinoids as inhibitors of arachidonic acid release and metabolism in rat peritoneal macrophages.

The effect of several natural and synthetic retinoids on the release and metabolism of arachidonic acid (20:4) in rat peritoneal macrophages (M phi), stimulated in vitro by either Ca2+ ionophore A23187 (A23187), opsonized zymosan (OZ) or 12-O-tetradecanoylphorbol-13-acetate (TPA), was investigated. With the exception of Ro 10-1670, the retinoids containing a free carboxylic acid group [i.e., all-trans-retinoic acid (all-trans-RA), 13-cis-RA, Ro 13-7652, Ro 12-7310 and Ro 13-7410] inhibited 20:4 metabolite formation in A23187- and OZ-stimulated Mø at 1-33 microM. However, only all-trans-RA, Ro 12-7310 and Ro 13-7410 inhibited the formation of 20:4 metabolites in TPA-stimulated Mø. These data suggest that part of the therapeutic effect of retinoids in inflammatory, hyperproliferative dermatologic conditions might be attributed to reduced 20:4 metabolite production.

Analogs of arachidonic acid methylated at C-7 and C-10 as inhibitors of leukotriene biosynthesis.

The syntheses and biological activity of (all Z)-7,7-dimethyl-5,8,11,14- eicosatetraenoic acid, (all Z)-7,7,-dimethyl-5,8,11-eicosatrienoic acid, (Z,Z)-7,7-dimethyl-5,8-eicosadienoic acid, (all Z)-10,10-dimethyl-5,8,11,14-eicosatetraenoic acid, (all Z)-10,10-dimethyl-5,8,11-eicosatrienoic acid, and rac.-(Z,Z)-15-hydroxy-7,7-dimethyl-5,8-eicosadienoic acid are described. These arachidonic acid analogs are all inhibitors of ionophore-induced SRS-A biosynthesis in rat peritoneal cells. Their mode of action may involve inhibition of phospholipase A2 rather than delta 5-lipoxygenase. These compounds failed to exhibit significant activity in an in vivo model designed to detect inhibitors of antigen-induced, leukotriene-mediated bronchoconstriction in sensitized guinea pigs.

Inflammation and collagenase production in rats with adjuvant arthritis reduced with 13-cis-retinoic acid.

Oral administration of 13-cis-retinoic acid (40 or 160 milligrams per kilogram of body weight daily) significantly reduced the inflammation associated with developing and established adjuvant arthritis, an experimentally induced arthritis in rats that resembles human rheumatoid arthritis. The amount of collagenase secreted in tissue culture by adherent cells isolated from the inflamed joints of adjuvant rats treated with 13-cis-retinoic acid also decreased as compared to the amount secreted by cells from vehicle-treated adjuvant rats. Collagenase is important in the joint destruction accompanying rheumatoid arthritis. The successful use of retinoids in the treatment of this proliferative but nonmalignant disorder demonstrates a new application of these compounds.