Gene-environment interaction between HLA-DRB1 shared epitope and heavy cigarette smoking in predicting incident rheumatoid arthritis.

BACKGROUND: Previous studies have reported an interaction between ever cigarette smoking and the presence of the human leukocyte antigen (HLA)-DRB1 shared epitope (SE) genotype and rheumatoid arthritis (RA) risk. To address the effect of dosage, a case-control study nested within two prospective cohorts to determine the interaction between heavy smoking and the HLA-SE was conducted. METHODS: Blood was obtained from 32 826 women in the Nurses' Health Study and 29 611 women in the Nurses' Health Study II. Incident RA diagnoses were validated by chart review. Controls were matched for age, menopausal status and postmenopausal hormone use. High-resolution HLA-DRB1 genotyping was performed for SE alleles. HLA-SE, smoking, HLA-SE* smoking interactions and RA risk, were assessed using conditional logistic regression models, adjusted for age and reproductive factors. Additive and multiplicative interactions were tested. RESULTS: In all, 439 Caucasian matched pairs were included. Mean age at RA diagnosis was 55.2 years; 62% of cases were seropositive. A modest additive interaction was observed between ever smoking and HLA-SE in seropositive RA risk. A strong additive interaction (attributable proportion due to interaction (AP) = 0.50; p<0.001) and significant multiplicative interaction (p = 0.05) were found between heavy smoking (>10 pack-years) and any HLA-SE in seropositive RA risk. The highest risk was in heavy smokers with double copy HLA-SE (odds ratio (OR) 7.47, 95% CI 2.77 to 20.11). CONCLUSIONS: A strong gene-environment interaction was observed between HLA-SE and smoking when stratifying by pack-years of smoking rather than by ever smoking. Future studies should assess cumulative exposure to cigarette smoke when testing for gene-smoking interactions.

Hyperglycemia attenuates acute permeability response to advanced glycation end products in retinal microvasculature.

Increased microvascular permeability contributes to the development of diabetic retinopathy and is associated with hyperglycemia and accumulation of advanced glycation end products (AGEs). The isolated perfused retina preparation was used to investigate the effects of hyperglycemia (HG) on the permeability response to AGEs. Retinae were dissected from rats, and the vasculature perfused with sulforhodamine B fluorescent dye and permeability of venular capillaries was determined from the rate of decrease of fluorescence gradient across a vessel during stasis. The resting permeability was very high in streptozotocin treated and some obese Zucker fatty diabetic rats, but low in others. The permeability response to glycated albumin (which is free radical-dependent) in these animals was reduced for a range of concentrations compared to the lean controls. The effects of 15 min 25 mM glucose (HG) superfusion on the retinal microvascular permeability response to 5 microM AGE-BSA was studied in non-diabetic Wistar rats. HG itself had no effect on permeability, but reduced the response to AGE-BSA from 1.02+/-0.08x10(-6) cm s(-1) to 0.31+/-0.07x10(-6) cm s(-1). The response to bradykinin (also free radical-dependent) was not affected by HG. This suggests that chronic exposure to HG down-regulates the signalling pathways activated in response to RAGE stimulation.

Association of serum concentration of soluble class I HLA with HLA allotypes.

We correlated serum concentrations of soluble class I HLA antigens (S-HLA-I) with HLA allotypes in 82 unrelated Caucasian and 58 unrelated African-American putatively normal subjects, as well as in 31 individuals with stable, normally functioning liver transplants. Caucasian and African-American subjects with HLA-A23 or HLA-A24 were high secretors of S-HLA-I. We also observed that some HLA-A allotypes associated with high serum concentrations of S-HLA-I were ethnicity specific. HLA-A33 was associated with high S-HLA-I secretion in African-Americans but not in Caucasians. HLA-A29 was associated with high S-HLA-I secretion in Caucasians but not in African-Americans. All liver transplant recipients studied who were high secretors of S-HLA-I postoperatively carried HLA-A24 or HLA-A29. (There were no HLA-A33 or HLA-A23 allotypes in this group.) The "secretor genes," however, may be autogenous or allogenic (i.e., either donor or recipient HLA-A24 or HLA-A29 resulted in the observed high secretor status in liver transplant recipients after transplantation). It is noteworthy that serum S-HLA-I concentrations were low in all subjects with HLA-A2 regardless of whether the HLA-A2 was of recipient or donor origin. This finding suggests that HLA-A2 could have a suppressive effect on S-HLA-I secretion.

Functional deficiency of antigen-presenting cells in the synovial fluid of rheumatoid arthritis.

In our study of rheumatoid arthritis (RA) patients, we observed a decrease of tetanus toxoid antigen-presenting capacity of synovial fluid (SF) adherent cells to autologous T cells of either SF or peripheral blood. Additionally, we found a higher capacity of adherent synovial cells to stimulate autologous T-lymphocytes. Our results suggest that antigen-presenting cells of the SF of RA patients have defects that may play a role in defective presentation of antigens in joints and may account for other abnormal functions important in the pathogenesis of RA.

Antigen specificity of mixed lymphocyte response-induced suppressor cells.

The nature of the antigens recognized by mixed lymphocyte response-generated suppressor cells is currently unknown. Previous investigations have yielded conflicting results, with different studies finding that suppressor cells recognize HLA class I antigens, class II antigens, or neither. To characterize the antigens recognized by suppressor cells (modulators) further, we generated 36 different modulators and assayed them for suppressor activity against a random 48-member HLA-typed panel in a total of 473 assays. Logistic regression analysis of the data revealed that suppression was correlated with B and DQ antigenic sharing between the original stimulator (used to generate the suppressor cells) and the test culture stimulator (p = 0.0043 and 0.0277, respectively). A role for DR antigen sharing could not be excluded. Overall, 35% of all suppressed assays could not be accounted for by the sharing of either any classical private HLA antigens, or of HLA-A or B locus cross-reactive group specificities. Suppression in these instances may involve the sharing of minor antigenic determinants, unidentified private HLA epitopes, or possibly another gene related to suppression that exists in linkage disequilibrium with the HLA-B locus or the DQ subregion.

Soluble HLA-I in rheumatic diseases.

OBJECTIVE: To study serum levels of Class I soluble HLA (sHLA-I) in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis or dermatomyositis (PM/DM) or scleroderma and to assess the possible influence of ethnic factors on concentration in each disease group. METHODS: Solid-phase enzyme linked immunoassay was used to measure sHLA-I in the serum of 385 patients with varied ethnic backgrounds (American-Caucasians, African-Americans, Georgian-Caucasians) with rheumatic diseases. Studies on patients were compared to similar measurements of 189 healthy individuals. RESULTS: Mean sHLA-I levels were significantly higher in patients with SLE than those observed in healthy individuals or other rheumatic diseases. Highest concentrations were present in Georgian-Caucasian patients with SLE. American-Caucasian patients with RA or scleroderma had higher sHLA-I levels than normal Caucasian individuals. The majority of patients with PM/DM in all ethnic subgroups were low secretors of sHLA-I. CONCLUSION: Mechanisms underlying the secretion of sHLA-I appear to differ among the rheumatic diseases studied and various ethnic groups. These genetic differences in sHLA-I secretion could be associated with ethnic and pathophysiologic differences among these rheumatic diseases.

Distribution of extracellular tracers in perivascular spaces of the rat brain.

Large molecular weight tracers (india ink or albumin labeled with colloidal gold, Evans blue or rhodamine) were micro-injected into the perivascular space of an artery or vein on the brain surface, or within the cerebral cortex or the subarachnoid space of anesthetized rats. The subsequent distribution was followed both under intravital microscopy, in order to outline the pathways and direction of tracer movement, and in histological section, in order to describe the pathways of flow at the light and electron microscopic level. The tracers remained largely in the perivascular spaces and in the interconnecting network of extracellular channels, including the subpial space and the core of subarachnoid trabeculae. Tracer also leaked across the pia into subarachnoid CSF. Bulk flow of fluid within the perivascular space, around both arteries and veins, was suggested from video-densitometric measurements of fluorescently labeled albumin. However, this flow was slow, and its direction varied in an unpredictable way. These results confirm that perivascular spaces may serve as channels for fluid exchange between brain and CSF, but do not support the idea that CSF circulates rapidly through brain tissue via perivascular spaces.

Audits of the treatment of genital warts: closing the feedback loop.

An initial audit of the treatment of patients presenting to the GUM Department at Leeds General Infirmary with a first episode of anogenital warts was reported in 1993. Treatment was found to be unselective and poorly monitored and the results of treatment were disappointing. As a consequence, guidelines for the management of new patients presenting with genital warts were devised. In order to establish whether these guidelines had produced any improvements in outcome, a second audit was performed looking at the results of treatment in patients with new genital warts who attended 6 months or more after the new guidelines were introduced. Progress was documented for 6 months after presentation. There was a significant fall in the numbers of patients receiving podophyllin 25% solution as first-line treatment, and corresponding increases in the initial use of cryotherapy, trichloracetic acid and, in men, podophyllotoxin solution. (Podophyllotoxin was not licensed for use in women at the time of the second audit.) There were significant improvements in the outcome of treatment. Originally 44% of men had warts despite receiving treatment for 3 months, and 32% were still attending for treatment 6 months after presentation. After the introduction of treatment guidelines, these figures had fallen to 8% and 3% respectively. In the first audit 38% of women still had warts after 3 months' treatment but in the second audit this figure was reduced to 18%. At 6 months, the percentage of women still attending for treatment was halved from 12% in the first audit to 6% in the second audit. The mean number of clinic visits fell from 5 to 3 in men and from 9 to 6 in women. The treatment protocols have been modified and now include the use of podophyllotoxin cream and solution in both men and women.

The effect of cyclosporine on urinary kallikrein excretion in patients with rheumatoid arthritis.

The effect of cyclosporine (6 to 8 mg/kg/24 hr) on urinary kallikrein excretion is summarized for 9 patients with rheumatoid arthritis using a specific kallikrein radioimmunoassay. Baseline serum creatinine and BUN values were within the normal range for all patients, and baseline kallikrein excretion rates were either normal (n = 5) or more than two S.D. below the mean of the normal group (n = 4). The patients with normal baseline values excreted significantly less urinary kallikrein three and six months after cyclosporine therapy was started, but all of them completed the six-month protocol. Patients in the subgroup with low baseline values also decreased their kallikrein excretion in response to cyclosporine therapy, and two of the four in this group experienced elevations of BUN such that therapy was terminated. In a low-dose (3 mg/kg/24 hr) open extension that followed the initial trial, kallikrein excretion decreased by almost 50% at least one month before any change in serum creatinine was observed. The data suggest that changes in urinary kallikrein excretion rates may be an indicator or predictor of cyclosporine nephrotoxicity. Decreased kallikrein excretion rates could also be a factor in the diminished renal blood flow reported in patients treated with cyclosporine.

Immunogenetic and racial determinants of gold toxicity in rheumatoid arthritis.

Typing for antigens HLA-A,B,C and DR was performed on 165 rheumatoid arthritis patients (14 black, 151 white) who had received gold therapy to determine the relationship between HLA antigens and gold dermatitis, stomatitis, thrombocytopenia, and proteinuria. Dermatitis and stomatitis occurred in both black and white patients. Thrombocytopenia and proteinuria occurred only among the white patients studied. The absence of thrombocytopenia and proteinuria among the black patients was not statistically significant. Antigen HLA-DR7 was uncommon among black and white subjects with dermatitis (0 of 6 blacks, 4 of 48 whites), but this decrease in frequency was not statistically significant. Antigen HLA-DR3 was an important risk factor for thrombocytopenia (relative risk = 11.8, P = .0043) and proteinuria (RR = 5.8, P = .032). These results are consistent with previous studies of HLA-DR3 and gold toxicity. The only black patient with stomatitis possessed the A1B8DR3 phenotype. Future studies should examine whether the same HLA antigen confers risk of different gold toxicities in different racial groups, and whether there are HLA antigens that provide a protective effect.

The medical treatment of secretory otitis media. A clinical trial of three commonly used regimes.

A clinical trial was undertaken to evaluate three medical treatments commonly used for chronic secretory otitis media. The treatments compared were Ephedrine nose drops, an oral antihistamine and decongestant (Dimotapp) and autoinflation of the middle ear. Changes in middle ear compliance and pressure were used as objective criteria of the efficacy of treatment in addition to changes in pure-tone threshold and to clinical assessment. Symptoms and abnormal signs tended to remit during the trial but there was no evidence from pure-tone audiometry and tympanometry that any of the treatments was beneficial. The period of observation enabled 28% of the children to avoid surgical treatment. Good and bad prognostic features are described which should help in deciding whether to manage a case conservatively or whether to proceed directly to surgery.

Novel technique for estimating cerebrovascular permeability demonstrates capsazepine protection following ischemia-reperfusion.

OBJECTIVE: There has been some discussion as to whether the pial vasculature behaves in the same way as the blood-brain barrier as a whole. Recent studies have shown that capsazepine protects these vessels from the effects of ischemia-reperfusion. We have now used a new method to examine this protection in the whole brain. METHODS: Horseradish peroxidase concentrations were measured in brain sections and plasma, following starch microsphere induced ischemia, which lasted from 20 to 60 minutes, with 30 minutes reperfusion. The PS product was calculated from the Crone-Renkin equation. RESULTS: Permeability increase, which depended on duration of ischemia, was considerably greater in the pia than the parenchyma. The increase was also greater in tissue surrounding large radial venules of the cortex. Single vessel studies showed that these differences mirror those between small and large pial venules. Capsazepine treatment protected the parenchymal blood-brain barrier by limiting the post-ischemic permeability increase to about one third, but had no effect on the pia or radial vessel permeability. CONCLUSIONS: Permeability has been estimated in tissue sections with good spatial resolution using this new technique, which has demonstrated that the TRPV1 receptor plays an important role in the whole brain, not confined to small pial venules.

High sensitivity, specificity and predictive value of the Connective Tissue Disease Screening Questionnaire among urban African-American women.

The Connective Tissue Disease Screening Questionnaire (CSQ), developed to screen populations for SLE and other CTDs, has been validated in a predominantly Caucasian population with hospital-based controls. We aimed to test the performance characteristics of the CSQ in an urban, predominantly African-American population. The CSQ was administered by interview to women recruited for a study of environmental risk factors and SLE, including 99 cases with SLE validated by medical record review and 202 healthy controls recruited from the community. Overall, 88% of subjects had African heritage, 6% were Hispanic and 4% were non-Hispanic Caucasian. Controls were more likely to report African heritage than cases (91% versus 82%, P = 0.001). Sensitivity for detecting SLE was 88% and specificity was 91%. In this study, where the prevalence of SLE was 33%, predictive value of a positive CSQ was 82% and predictive value of a negative CSQ was 94%. The CSQ has slightly lower sensitivity but greater specificity for SLE in an urban, predominantly African-American population with community-based controls compared with a Caucasian population with hospital-based controls. These results suggest that the CSQ has adequate sensitivity and specificity and could be used in population studies to screen African-American women for SLE.

The arrangement of endocrine and exocrine pancreatic microcirculation observed in the living rabbit.

1. The microcirculation of the pancreas in the anaesthetized rabbit was examined by in vivo microscopy and micro-cinephotography. 2. The islets of Langerhans were identified by their appearance following the systemic injection of diphenylthiocarbazone. 3. Arterial injection of Evans blue demonstrated the arrangement of vessels within the pancreas. Blood leaving the islets passed via capillaries into the capillary meshwork; no islet was ever seen to be drained directly by a vein. 4. This significance of this portal arrangement is discussed.

Permeability of disrupted cerebral microvessels in the frog.

1. This study reports the results of varying the hydrostatic pressure on measurements of permeability coefficient to the low molecular weight impermeant dye carboxyfluorescein (MW = 376) in single leaky cerebral microvessels. A mathematical model, that solved the convective diffusion equations used to analyse the measurements, showed that the measurements were consistent with leakiness being due to 22 nm wide parallel-sided slits between endothelial cells. 2. Microvessels on the surface of the frog's brain were cannulated with a micropipette and perfused with an artificial cerebrospinal fluid containing the dye. Vessels were occluded with a glass microneedle and the rate of change in dye concentration in a 12 microns length section was measured using video-intensified microscopy. 3. It was found that the rate of dye loss at all points along the occluded microvessel segment could be accounted for by a model for convection and diffusion, and that changes in dye concentration at a point remote from the segment entrance can give a good measure of diffusive permeability. 4. When series of measurements were carried out on a single vessel, permeability rose over the course of 20 min. Mean permeability for all measurements was 3.01 x 10(-5) cm sec-1, n = 64 (mode, 2.0; range, 0.48-9.6). The hydrostatic pressure applied during the perfusion had no effect on the measured permeability. 5. The dye concentration along the vessel axis was measured at the steady state and was shown to respond to changes in hydrostatic perfusion pressure in a way predicted by the model. This indicates that hydrostatically driven bulk flow can be important, and thus convection may account for effects previously ascribed to vesicular transcytosis. 6. The possible anatomical basis for the porous pathway is discussed in the light of recent observations on the presence of 0.5 microns perijunctional gaps, the possibility of transendothelial channels, and the unzipping of tight junctions to leave a 22 nm wide slit.

Variable restriction of albumin diffusion across inflamed cerebral microvessels of the anaesthetized rat.

1. The possibility of restricted diffusion of macromolecules in single cerebral venular capillaries that have become leaky due to inflammation was investigated by comparing the permeabilities to Lucifer Yellow (457 Da; PLY) and rhodamine-labelled albumin (69 kDa; PRh-A). 2. The dyes were trapped between two micro-occlusion probes and the permeabilities were measured from the rates of decrease in dye fluorescence at low intraluminal hydrostatic pressure. 3. Removal of one probe had little effect on PLY but did reduce PRh-A, consistent with the influence of convection on diffusion through 22 nm wide transendothelial slits 1 micron deep. 4. Direct comparisons were made over time between PLY and PRh-A in six vessels while hydrostatic pressure effects were controlled. In all vessels PRh-A:PLY varied from being similar to the ratio of the free diffusion coefficients to virtually zero even though PLY remained high. The question of the source of this variable restriction to albumin is discussed in terms of the secretion and sloughing of glycosaminoglycans and the possible role of transient formation of transendothelial gaps.

The role of guanylyl cyclases in the permeability response to inflammatory mediators in pial venular capillaries in the rat.

Inflammatory mediators have a role in the formation of cerebral oedema and there is evidence that cGMP is an important signal in vascular permeability increase. We have investigated the role and the source of cGMP in mediating the permeability response to acutely applied bradykinin and the histamine H(2) agonist dimaprit on single cerebral venular capillaries, by using the single vessel occlusion technique. We found that 8-bromo-cGMP applied acutely resulted in a small and reversible permeability increase with a log EC(50) -7.2 +/- 0.15 M. KT 5823, the inhibitor of cGMP-dependent protein kinase, abolished the permeability responses to both bradykinin and dimaprit, while zaprinast, an inhibitor of type 5 phosphodiesterase, potentiated the response to bradykinin. On the other hand, L-NMMA blocked the response to dimaprit, but not that to bradykinin. Inhibitors of soluble guanylyl cyclase, LY 85353 and methylene blue, also inhibited the permeability response to dimaprit, but not bradykinin. The permeability responses to the natriuretic peptides ANP and CNP were of similar magnitude to that of bradykinin with log EC(50) -10.0 +/- 0.33 M and -8.7 +/- 0.23 M, respectively. The natriuretic peptide receptor antagonist HS-142-1 blocked permeability responses to bradykinin as well as to ANP, and leukotriene D(4) blocked the responses to CNP and bradykinin, but not to dimaprit. In conclusion, the histamine H(2) receptor appears to signal via cGMP that is generated by a NO and soluble guanylyl cyclase, while bradykinin B(2) receptor also signals via cGMP but through particulate guanylyl cyclase.

The organization of the salivary gland microcirculation.

1. The microvasculature of the rabbit submandibular salivary gland has been investigated employing in vivo microscopy, blood flow measurements, latex casts, microsphere injections and examination of fixed sections of the gland.2. Two principal microcirculations were distinguished in the living gland, one supplying the acini and the other the intralobular ducts. Parasympathetic nerve stimulation (2, 5 or 10 sec(-1)) elicited different responses in each of the two microcirculations. Flow in the capillaries around the acini slowed initially before increasing. In contrast, flow in the intralobular duct capillaries increased soon after beginning stimulation.3. In some experiments both whole gland flow and microvascular flow were monitored simultaneously. Whole gland flow increased at the same time as flow in the acinar capillaries was decreasing and as flow in the intralobular duct capillaries increased. Flow in acinar capillaries increased about 5 sec after glandular flow started to increase.4. These observations could be explained if either the vascular beds of the acini and the intralobular ducts were arranged in parallel or if arteriovenous anastomoses were to shunt the acinar circulation. No such anastomoses were found in latex casts made of the gland vasculature, and microspheres injected into the artery supplying the gland were not found in the venous effluent.5. The intraglandular distribution of microspheres was measured in histological sections of the injected glands to give an estimate of the distribution of blood flow between the duct and acinar microcirculations. At rest and during maintained stimulation about 55% of the blood flow passed through the intralobular duct microcirculations, whilst during this initial 15 sec of stimulation this proportion was increased to over 70%. This finding is consistent with a parallel arrangement of the two microcirculations.6. The conclusions drawn from these observations are that the duct and acinar microcirculations are arranged in parallel, that there are differences in the way the vasodilatation is mediated in these circulations, and that arterio-venous anastomoses play no significant role in this gland.