The cancer care experiences of gay, lesbian and bisexual patients: A secondary analysis of data from the UK Cancer Patient Experience Survey.

Understanding the effects of population diversity on cancer-related experiences is a priority in oncology care. Previous research demonstrates inequalities arising from variation in age, gender and ethnicity. Inequalities and sexual orientation remain underexplored. Here, we report, for the first time in the UK, a quantitative secondary analysis of the 2013 UK National Cancer Patient Experience Survey which contains 70 questions on specific aspects of care, and six on overall care experiences. 68,737 individuals responded, of whom 0.8% identified as lesbian, gay or bisexual. Controlling for age, gender and concurrent mental health comorbidity, logistic regression models applying post-estimate probability Wald tests explored response differences between heterosexual, bisexual and lesbian/gay respondents. Significant differences were found for 16 questions relating to: (1) a lack of patient-centred care and involvement in decision-making, (2) a need for health professional training and revision of information resources to negate the effects of heteronormativity and (3) evidence of substantial social isolation through cancer. These findings suggest a pattern of inequality, with less positive cancer experiences reported by lesbian, gay and (especially) bisexual respondents. Poor patient-professional communication and heteronormativity in the healthcare setting potentially explain many of the differences found. Social isolation is problematic for this group and warrants further exploration.

Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor.

Interleukin-1 (IL-1) is a proinflammatory cytokine that elicits its pleiotropic effects through activation of the transcription factors NF-kappaB and AP-1. Binding of IL-1 to its receptor results in rapid assembly of a membrane-proximal signalling complex that consists of two different receptor chains (IL-1Rs), IL-1RI and IL-1RAcP, the adaptor protein MyD88, the serine/threonine kinase IRAK and a new protein, which we have named Tollip. Here we show that, before IL-1beta treatment, Tollip is present in a complex with IRAK, and that recruitment of Tollip-IRAK complexes to the activated receptor complex occurs through association of Tollip with IL-1RAcP. Co-recruited MyD88 then triggers IRAK autophosphorylation, which in turn leads to rapid dissociation of IRAK from Tollip (and IL-1Rs). As overexpression of Tollip results in impaired NF-kappaB activation, we conclude that Tollip is an important constituent of the IL-1R signalling pathway.

Sodium channel Nav1.8 immunoreactivity in painful human dental pulp.

BACKGROUND: The tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 (SNS1/PN3) is expressed by nociceptors and may play a role in pain states. METHODS: Using specific antibodies for immunohistochemistry, we studied Nav1.8 immunoreactivity in human dental pulp in relation to the neuronal marker neurofilament. Human tooth pulp was extracted from teeth harvested from a total of twenty-two patients (fourteen without dental pain, eight patients with dental pain). RESULTS: Fibres immunoreactive for Nav1.8, were significantly increased on image analysis in the painful group: median (range) Nav1.8 to Neurofilament % area ratio, non-painful 0.059 (0.006-0.24), painful 0.265 (0.13-0.5), P = 0.0019. CONCLUSION: Nav1.8 sodium channels may thus represent a therapeutic target in trigeminal nerve pain states.

Endothelin receptor antagonism in patients with chronic heart failure.

OBJECTIVE: The relative importance of ETA and ETB receptors in mediating the constrictor effects of endogenous endothelin-1 in patients with chronic heart failure is not known. The primary purpose of this study was to compare the acute effects of selective ETA and ETB receptor antagonists in vivo in healthy subjects and patients with chronic heart failure. Our secondary aim was to examine more closely the effect of chronic heart failure on endothelin biosynthesis. METHODS: We studied the effects of BQ-123 (a selective ETA antagonist) and BQ-788 (a selective ETB antagonist) in ten healthy subjects and ten patients with chronic heart failure. Locally active doses of each antagonist were infused into the non-dominant brachial artery for 90 min on separate days at least 1 week apart. Changes in forearm blood flow were measured by venous occlusion plethysmography. Venous blood samples were obtained prior to antagonist infusion for assay of total endothelin, big endothelin-1 and C-terminal fragment immunoreactivity. RESULTS: BQ-123 (100 nmol/min) increased blood flow by 54+/-10% (P<0.001) and 30+/-5% (P<0.001) in controls and heart failure patients, respectively. BQ-788 (1 nmol/min) reduced blood flow by 15+/-5% (P=0. 036) and 9+/-4% (P=0.001) in controls and heart failure patients, respectively. Total endothelin immunoreactivity was non significantly greater in heart failure patients than controls (6. 8+/-1.4 vs. 4.6+/-0.5 pM; P=0.13). Big endothelin-1 (2.6+/-0.4 vs. 1. 7+/-0.1 pM; P=0.04) and C-terminal fragment immunoreactivity (2. 1+/-0.3 vs. 0.6+/-0.1 pM; P<0.0001) were each significantly greater in heart failure patients than controls. CONCLUSIONS: Selective ETA receptor antagonism caused vasodilatation in the peripheral circulation of healthy subjects and patients with chronic heart failure while selective ETB receptor antagonism caused vasoconstriction in each group. ETB receptor antagonism may therefore cause potentially deleterious vasoconstriction in chronic heart failure. Chronic heart failure is associated with a significant increase in plasma big endothelin-1 and C-terminal fragment immunoreactivity.

Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2.

Remarkable progress has been made recently in identifying a new gene family related to the capsaicin (vanilloid) receptor, VR1. Using a combination of in silico analysis of expressed sequence tag (EST) databases and conventional molecular cloning, we have isolated a novel vanilloid-like receptor, which we call VRL-2, from human kidney. The translated gene shares 46% and 43% identity with VR1 and VRL-1, respectively, and maps to chromosome 12q23-24.1, a locus associated with bipolar affective disorder. VRL-2 mRNA was most strongly expressed in the trachea, kidney, and salivary gland. An affinity-purified antibody against a peptide incorporating the COOH terminal of the receptor localized VRL-2 immunolabel in the distal tubules of the kidney, the epithelial linings of both trachea and lung airways, serous cells of submucosal glands, and mononuclear cells. Unlike VR1 and VRL-1, VRL-2 was not detected in cell bodies of dorsal root ganglia (DRG) or sensory nerve fibers. However, VRL-2 was found on sympathetic and parasympathetic nerve fibers, such as those innervating the arrector pili smooth muscle in skin, sweat glands, intestine, and blood vessels. At least four vanilloid receptor-like genes exist, the newest member, VRL-2 is found in airway and kidney epithelia and in the autonomic nervous system.

Immunolocalization of SNS/PN3 and NaN/SNS2 sodium channels in human pain states.

The tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel SNS/PN3 and the newly discovered NaN/SNS2 are expressed in sensory neurones, particularly in nociceptors. Using specific antibodies, we have studied, for the first time in humans, the presence of SNS/PN3 and NaN/SNS2 in peripheral nerves, including tissues from patients with chronic neurogenic pain. In brachial plexus injury patients, there was an acute decrease of SNS/PN3- and NaN/SNS2-like immunoreactivity in sensory cell bodies of cervical dorsal root ganglia (DRG) whose central axons had been avulsed from spinal cord, with gradual return of the immunoreactivity to control levels over months. In contrast, there was increased intensity of immunoreactivity to both channels in some peripheral nerve fibers just proximal to the site of injury in brachial plexus trunks, and in neuromas. These findings suggest that the expression of these sodium channels in neuronal cell bodies is reduced after spinal cord root avulsion injury in man, but that pre-synthesized channel proteins may undergo translocation with accumulation at sites of nerve injury, as in animal models of peripheral axotomy. The latter may contribute to positive symptoms, as our patients all showed a positive Tinel's sign. Nerve terminals in distal limb neuromas and skin from patients with chronic local hyperalgesia and allodynia all showed marked increases of SNS/PN3-immunoreactive fibers, but little or no NaN/SNS2-immunoreactivity, suggesting that the former may be related to the persistent hypersensitive state. Axonal immunoreactivity to both channels was similar to control nerves in sural nerve biopsies in a selection of neuropathies, irrespective of nerve inflammation, demyelination or spontaneous pain, including a patient with congenital insensitivity to pain. Our studies suggest that the best target for SNS/PN3 blocking agents is likely to be chronic local hypersensitivity.

Anatomical localization and pharmacological activity of mature endothelins and their precursors in human vascular tissue.

OBJECTIVES: We developed four selective rabbit antisera in order to compare the distribution of immunoreactive mature endothelins and their precursors, proendothelin-1, proendothelin-2 and proendothelin-3, in the endothelium from human vascular tissue. Our second aim was to use in vitro pharmacological assays to test the vasoconstrictor actions of the mature endothelin and proendothelin peptides. METHODS: The antisera were shown to be selective by enzyme-linked immunosorbent assays. With these antisera, we detected immunoreactivity in serial cryostat sections from saphenous and mesenteric veins, and mesenteric and internal mammary arteries, using a peroxidase-antiperoxidase technique. In pharmacological experiments, segments of human coronary and mesenteric arteries were exposed to cumulative (0.06-60 nmol/l) concentrations of the endothelins and their precursors. RESULTS: Antisera directed against mature endothelin stained the cytoplasm of endothelial cells in all vessels tested. Immunoreactive proendothelin-1 and proendothelin-2 were also detected, but not proendothelin-3. Endothelin-1 and endothelin-2 were strongly vasoactive, with similar molar potencies, and caused a dose-related increase in contractile force in human coronary arteries (0.06-60 nmol/l). However, proendothelin-1 and proendothelin-2 were 100-fold and 1000-fold less vasoactive than their respective mature peptides. No contractile effect was seen with proendothelin-3 or endothelin-3 at the concentrations tested in human coronary arteries, and similar results were obtained with human mesenteric arteries. CONCLUSIONS: These results suggest that proendothelin-1 and proendothelin-2 must be converted to their corresponding mature peptides to produce vasoconstrictor activity in human vessels. Immunoreactive mature endothelin is widely distributed in human vascular endothelial cells and, if released, may produce endothelin-mediated vasoconstriction.

Distribution of the messenger RNA for the small conductance calcium-activated potassium channel SK3 in the adult rat brain and correlation with immunoreactivity.

Small conductance calcium-activated potassium channels are voltage independent potassium channels which modulate the firing patterns of neurons by activating the slow component of the afterhyperpolarization. The genes encoding a family of small conductance calcium-activated potassium channels have been cloned and up to now three known members have been described and named small conductance calcium-activated potassium channel type 1, small conductance calcium-activated potassium channel type 2 and small conductance calcium-activated potassium channel type 3; the distribution of their messenger RNA in the rat CNS has already been performed but only in a limited detail. The present study represents the first detailed analysis of small conductance calcium-activated potassium channel type 3 mRNA distribution in the adult rat brain and resulted in a strong to moderate expression of signal in medial habenular nucleus, substantia nigra compact part, suprachiasmatic nucleus, ventral tegmental area, lateral septum, dorsal raphe and locus coeruleus. Immunohistological experiments were also performed and confirmed the presence of small conductance calcium-activated potassium channel type 3 protein in medial habenular nucleus, locus coeruleus and dorsal raphe. Given the importance of dorsal raphe, locus coeruleus and substantia nigra/ventral tegmental area for serotonergic, noradrenergic and dopaminergic transmission respectively, our results pose the morphological basis for further studies on the action of small conductance calcium-activated potassium channel type 3 in serotonergic, noradrenergic and dopaminergic transmission.

Selective beta1-adrenoceptor blockade enhances the activity of the stimulatory G-protein in human atrial myocardium.

1. Chronic selective beta1-adrenoceptor (beta1AR) blocker treatment enhances the sensitivity of beta2-adrenoceptor (beta2AR) in human heart (Hall et al., 1990; 1991). To clarify the mechanism of the cross-sensitization between beta1AR and beta2AR, we determined whether the stimulatory G-protein (G(s)alpha) function is increased in atria from beta1AR-blocker treated patients compared with non-beta-blocked patients, and investigated whether this change is caused by an alteration of post-translational modification of Gsalpha protein. 2. G(s)alpha function was determined by reconstitution of human atrial G(s)alpha into S49 cyc- cell membranes. In the reconstitution system, GTPgammaS stimulated cyclic AMP generation in a dose-dependent manner. Upon 10(-4) M GTPgammaS stimulation, G(s)alpha activity in the beta1AR-blocker, atenolol, treated group (78.2+/-10. 3 pmol cyclic AMP mg(-1) min(-1) 10(-3)) was 65% higher than that in non-beta-blocked patients (47.3+/-6.3 pmol cyclic AMP mg(-1) min(-1) 10(-3), n=15, P=0.02). 3. Isoelectric point (pI) valu G(s)alpha were measured by two dimensional gel electrophoresis (2D-E) and the amount of each isoform quantified by image analysis of a Western blot of the gel using specific antibody. Multiple isoforms of G(s)alpha were detected by 2D-E with different pI values. There were no significant differences between the groups of patients in either pI values or the proportions of the acidic isoforms of G(s)alpha to the main basic form (n=12, P>0.05). 4. The results suggest that chronic beta1AR-blockade enhances Gsalpha function in human atrium, and this may account in part for the hypersensitivity of beta2AR and other Gs-coupled receptors during beta1AR-blockade. The increased G(s)alpha function is unlikely to be caused directly by blockade of protein kinase A phosphorylation of G(s)alpha protein.

Phosphoramidon inhibition of the in vivo conversion of big endothelin-1 to endothelin-1 in the human forearm.

1. The vasoconstrictor peptide, endothelin-1 (ET-1) and a biologically inactive C-terminal fragment (CTF) are generated from an intermediate big ET-1 by a putative ET converting enzyme, sensitive to phosphoramidon. We have developed a procedure using selective solid-phase extraction and specific radioimmunoassays to measure the levels of immunoreactive (IR) big ET-1 and the products of conversion (ET-1 and CTF) in human plasma. These techniques have been used to determine the levels of the three peptides in venous plasma following local infusions of ET-1 and big ET-1, both alone and together with phosphoramidon. 2. Infusion of ET-1 into the brachial artery (5 pmol min-1) significantly increased (P < 0.05) IR ET levels from a basal level of 2.3 pM to 55.2 pM in plasma from the infused arm after 60 min of infusion. This corresponded with a marked decrease in forearm blood flow from a basal level of 2.6 ml dl-1 min-1 to 1.7 ml dl-1 min-1. The levels of IR big ET-1 and CTF were unchanged. Co-infusion of phosphoramidon (30 nmol min-1) with ET-1 had no significant effect on the plasma IR levels of ET, big ET-1, CTF, or blood flow. 3. Big ET-1 (50 pmol min-1) significantly increased (P < 0.05) venous concentrations of all three IR peptides after 60 min compared to basal (ET: from 2.2 to 7.7 pM, big ET-1; from 0 to 386.0 pM, CTF: from 0.2 to 37.0 pM). Forearm blood flow decreased significantly (P<0.05) from a basal level of 3.0 ml dl-1 min-1 to 1.6 ml dl-1 min-1.4. When phosphoramidon was co-infused with big ET-1, both the rise in IR ET and associated vasoconstriction were abolished. However, IR CTF was still detected, suggesting that either some conversion by phosphoramidon-insensitive enzyme(s) was occurring, and/or that CTF was being protected from further degradation by phosphoramidon.5. These data show that in the human forearm the activity of a phosphoramidon-sensitive ET converting enzyme is at least in part responsible for the vasoconstrictor properties of exogenous big ET-1. Furthermore, because measurable levels of newly synthesized ET-1 are likely to be rapidly reduced in the blood/plasma through receptor binding, assay of IR big ET-1 and CTF may be a more sensitive measure of ET-1 generation in disease.

The increase in human plasma immunoreactive endothelin but not big endothelin-1 or its C-terminal fragment induced by systemic administration of the endothelin antagonist TAK-044.

1. We examined the effects of systemic infusion, in healthy human volunteers, of the endothelin antagonist TAK-044 on the plasma concentrations of mature endothelin, big endothelin-1 and the C-terminal fragment of big endothelin-1, by selective solid-phase extraction and specific radioimmunoassays. 2. Unlabelled TAK-044 competed with specific [125I]-endothelin-1 binding to human left ventricle tissue in a biphasic manner giving KD values of 0.11 nM and 26.8 nM at the ETA and ETB receptor subtypes, respectively, indicating a 244 fold selectivity for the ETA receptor subtype. 3. A 15 min intravenous infusion of placebo or 30 mg TAK-044 (giving a serum concentration of 2 nM, calculated to block > 95% of ETA but < 5% ETB receptors) had no effect on the immunoreactive plasma concentrations of the three peptides. 4. At the higher dose of 750 mg TAK-044 (giving a serum concentration of 80 nM, calculated to block > 99% of ETA and > 75% ETB receptors), the immunoreactive plasma endothelin concentrations were increased 3.3 fold over basal levels (P < 0.01). The concentrations of big endothelin-1 or C-terminal fragment of big endothelin-1 were unchanged. 5. At both doses of TAK-044, there were significant decreases in diastolic blood pressure, and peripheral vascular resistance, with corresponding increases in cardiac index and stroke index. There were no changes in systolic or mean arterial blood pressures or heart rate. 6. Since only the concentrations of the mature peptide were increased, we conclude that the most likely sources of endothelin contributing to the observed rise were displacement of receptor-bound peptide and reduction in plasma clearance rather than peptide synthesis.

Demonstration of the neurotransmitter role of calcitonin gene-related peptides (CGRP) by immunoblockade with anti-CGRP monoclonal antibodies.

1. Monoclonal antibodies (MAbs) against rat alpha-calcitonin gene-related peptide (alpha CGRP) were produced. Those which bound CGRP in a radioimmunoassay and inhibited the binding of 2-[125I]-iodohistidyl10-CGRP in a receptor binding assay were selected for immunoblockade experiments. 2. The effect of MAbs on CGRP inhibition of electrically stimulated contractions of the rat isolated vas deferens was characterized. Four out of 11 MAbs tested shifted the concentration-response curve of CGRP to the right compared with vehicle or irrelevant MAb control. MAb C4.19 produced equipotent blockade of rat alpha CGRP and rat beta CGRP and was chosen for further studies. MAb C4.19 had no pharmacologically significant effect on the concentration-response relationship of isoprenaline, rat beta-endorphin or somatostatin. 3. We demonstrated that the pharmacological response to CGRP in the presence of MAb C4.19 could be predicted when the dissociation constant and concentration of binding sites of the antibody were known. Comparison of experimental and computer simulated data showed good agreement for EC50 and maximum effect of CGRP in the presence of MAb C4.19. 4. Capsaicin at 1 microM inhibited the electrically stimulated contractions by 60.8% (95% confidence interval 51.8% to 69.9%). This effect was significantly attenuated by MAb C4.19 to 26.0% (95% confidence interval 15.2% to 36.8%; P < 0.003). 5. The immunoblockade of exogenous and endogenous CGRP described here, together with complementary evidence from other studies, strongly suggest that CGRP has a major neurotransmitter role at the neuroeffector junction of the rat vas deferens.

Comparative distribution of voltage-gated sodium channel proteins in human brain.

Antisera directed against unique peptide regions from each of the human brain voltage-gated sodium channel alpha subunits were generated. In immunoblots these were found to be highly specific for the corresponding recombinant polypeptides and to recognise the native holoprotein in human brain membrane preparations. These antisera were used to perform a comparative immunohistochemical distribution analysis of all four brain sodium channel subtypes in selected human CNS regions. Distinct but heterogeneous distribution patterns were observed for each of the alpha subunits. In general, these were complimentary to that previously shown for the corresponding human mRNAs. A high degree of conservation with respect to the distribution found in rat was also evident. The human alpha subunit proteins exhibited distinct subcellular localisation patterns. Types I, III and VI immunoreactivity was predominantly in neuronal cell bodies and proximal processes, whereas type II was concentrated along axons. This is similar to rat brain and suggests the different the sodium channel subtypes have distinct functions which are highly conserved between human and rodents. A notable difference was that the type III protein was detected in all human brain regions examined, unlike in rat brain where expression in adults is very restricted. Also in contrast to rat brain, the human type VI protein was not detected in axons of unmyelinated neurons. These differences may reflect true species variation and could have important implications for understanding the function of the sodium channel subtypes and their roles in human disease.

Localization of the type VI voltage-gated sodium channel protein in human CNS.

The cellular distribution of the type VI human voltage-gated sodium channel (Type VI) was examined in selected human brain regions. Antibodies designed to be specific to rat and human Type VI were raised against a synthetic peptide from the predicted NH2-terminal of the protein, and used for an immunohistochemical investigation. Immunoblot experiments showed that purified antibodies specifically detected the presence of Type VI in transfected cells and human brain membrane preparations. Immunohistochemistry on perfusion fixed human tissue revealed a predominantly somato-dendritic distribution of Type VI in major output neurons of the cerebellum, cerebral cortex and hippocampus. The observed localisation of this channel may reflect an important role in the integration of synaptic input in the human CNS.

Sodium channel beta1 and beta2 subunits parallel SNS/PN3 alpha-subunit changes in injured human sensory neurons.

Voltage-gated sodium channels consist of a pore-containing alpha-subunit and one or more auxiliary beta-subunits, which may modulate channel function. We previously demonstrated that sodium channel SNS/PN3 alpha-subunits were decreased in human sensory cell bodies after spinal root avulsion injury, and accumulated at injured nerve terminals in pain states. Using specific antibodies for immunohistochemistry, we have now detected sodium channel beta1 and beta2 subunits in sensory cell bodies within control human postmortem sensory ganglia (78% of small/medium (< or = 50 microm) and 68% of large (> or = 50 microm) cells); their changes in cervical sensory ganglia after avulsion injury paralleled those described for SNS/PN3 alpha-subunits. Our results suggest that alpha- and beta-subunits share common regulatory mechanisms, but present distinct targets for novel analgesics.

Plasticity of TTX-sensitive sodium channels PN1 and brain III in injured human nerves.

Sensory neurones co-express voltage-gated sodium channels that mediate TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) currents, which may contribute to chronic pain after nerve injury. We previously demonstrated that TTX-R channels were decreased acutely in human sensory cell bodies after central axotomy, but accumulated in nerve terminals after peripheral axotomy. We have now studied the TTX-S channels PN1 and Brain III, using specific antibodies for immunohistochemistry, in dorsal root ganglia (DRG) from 10 patients with traumatic central axotomy, nerves from 16 patients with peripheral axotomy, and controls. PN1 showed temporal changes similar to the TTX-R channels in sensory cell bodies of injured DRG. In contrast, Brain III was found only in injured nerves (not control nerves, or control/central axotomy DRG). PNI and Brain III are distinct targets for novel analgesics.

In vitro expression of calcitonin gene-related peptide in human endothelial cells transfected with plasmid and retroviral vectors.

Introduction of cDNA encoding human alpha-calcitonin gene-related peptide (CGRP), a potent vasodilator, into cultured human umbilical vein endothelial cells (HUVEC) was undertaken by using plasmids and retroviral vectors. In order to improve expression, modification of context coding sequence for the initiation of CGRP translation and deletion of nontranslated regions of CGRP cDNA in the transfection vectors were tested. Stable transfer of neo in the HUVEC was achieved with both plasmid and retroviral vectors. Integration rates obtained by using retrovirus (approximately 1%), where higher than those achieved with plasmid-mediated transfection (< 1/1000). CGRP expressed in the transfected HUVEC was secreted into culture medium when a leading sequence was included in the expression vectors. CGRP was detected by enzyme-linked immunosorbent assay (ELISA) in the supernatants of both transiently transfected and stably transfected/infected HUVEC. Higher levels of expression were achieved by using plasmid (giving a maximum CGRP concentration of 6.5 +/- 0.5 pM in the supernatant) than retroviruses. Lipofectin-mediated transfer of CGRP cDNA also resulted in transient expression of CGRP in the HUVEC.

Development of an enzyme-linked immunosorbent assay to measure total TIMP-1 (free TIMP-1 and TIMP-1 in combination with matrix-metalloproteinases) and measurement of TIMP 1 and CRP in serum.

A panel of six monoclonal antibodies (MAbs) was raised against purified human fibroblast tissue inhibitor of metalloproteinase-1 (TIMP-1) and characterised. All possible antibody pairs were tested for their suitability as capture and revealing antibodies in a two-site enzyme-linked immunosorbent assay (ELISA) to measure total TIMP-1 (both free TIMP-1 and TIMP-1 together with matrix metalloproteinases (MMPs)). Using the best combination of MAbs the assay was optimised. The sensitivity of detection of the assay was 1.4 ng/ml, and inter- and intra-assay coefficients of variation were between 10.4-13.7% and 8.8-9.7%, respectively. Dilution series of human cerebrospinal and synovial fluids, plasma and sera paralleled those of the TIMP-1 standard curve indicating that the immunoreactivity detected in these samples was authentic TIMP-1. TIMP-2 shows no detectable cross reactivity in this assay confirming that this ELISA is specific for TIMP-1. The levels of total TIMP-1 and collagenase were measured in conditioned medium from A2058 human melanoma cells cultured in the absence or presence of human recombinant interleukin-1 alpha (hrIL-1 alpha). Total TIMP-1 was also measured in serum samples with known C-reactive protein (CRP) (n = 100) and alpha 1 antichymotrypsin (ACT) (n = 52) concentrations; no correlation was found between TIMP-1 levels and either of these acute phase reactants although the levels of TIMP-1 were raised when compared to normal sera. This ELISA provides a rapid and convenient procedure for the quantitation of total TIMP-1 in human biological fluids and supernatants from cultured cell lines.