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.

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.

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.

MICA, HLA-B haplotypic variation in five population groups of sub-Saharan African ancestry.

The human major histocompatibility complex (MHC) class I chain-related gene A (MICA), located 46 kb centromeric to HLA-B, encodes a stress-inducible protein, which is a ligand for the NKG2D receptor. In addition to its primary role in immune surveillance, data suggest that MICA is involved in the immune response to transplants and in susceptibility to some diseases. In this study, 152 subjects from the Yoruba (n=74), Efik (n=32), and Igbo (n=46) tribes of southern Nigeria, 39 nationwide African-American stem cell donors, and 60 African-American individuals residing in the metropolitan Boston area were studied for MICA, HLA-B allelic variation, haplotypic diversity, and linkage disequilibrium (LD). MICA and HLA-B exhibited a high degree of genetic diversity among the populations studied. In particular, MICA allele and HLA-B-MICA haplotype frequencies and LD in the Efik and Igbo tribes were significantly different from the other study groups. HLA-B and MICA loci demonstrated significant global LD in all five populations (P-values &<0.00001). LD also varied in a haplotype-specific manner. A novel MICA allele was detected in the Boston population. These findings are important from an anthropologic perspective, and will inform future HLA-linked disease association studies in related ethnic groups of African-derived ancestry.

TNF-alpha promoter single nucleotide polymorphisms are markers of human ancestry.

We present a map of single nucleotide polymorphisms (SNPs) in the human tumor necrosis factor (TNF)-alpha promoter based upon exploratory sequencing of 333 human TNF-alpha gene promoters from individuals of distinct ancestral backgrounds. We detect 10 TNF-alpha promoter SNPs that occur with distinct frequencies in populations of different ancestry. Consistent with these findings, we show that two TNF-alpha SNPs, the -243 SNP and the -856 SNP, are the first SNP markers of a sub-Saharan African-derived extended haplotype and an Amerindian HLA haplotype, respectively. Comparisons of TNF-alpha promoter SNP allele frequencies can thus help elucidate variation of HLA haplotypes and their distribution among existing ethnic groups and shed light into the history of human populations.

Soluble HLA-II expression in African-Americans.

OBJECTIVE: In this study we evaluated the contribution of major histocompatibility complex (MHC) genes to soluble histocompatibility antigen class II (sHLA-II) secretion in African American patients with rheumatoid arthritis (RA). METHODS: A sensitive enzyme-linked immunoassay was used to quantitate sHLA-II in the serum of 7 patients with RA, as well as 28 of their kinships and 49 HLA typed normal African American individuals. RESULTS: Mean sHLA-II values were higher in patients with RA than those in healthy African American individuals (p < 0.05). There were variations in concentrations in individual patients but these were unrelated to any apparent clinical event. The proportion of unaffected family members with detectable levels of sHLA-II was not significantly different than those in normal controls. Neither specific HLA-haplotype, or HLA-allele(s) correlated with high or low sHLA-II secretion. CONCLUSIONS: Our data suggest that sHLA-II molecules are not regulated by MHC linked genes but may be regulated by non-MHC linked genes and racial background may reflect genetic heterogeneity of the expression of this soluble HLA material. These observations contrast with previous observations concerning soluble HLA class I (sHLA-I) molecules in a described population sample which were almost the precise reverse.

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.

Polymorphism of the human retinoid X receptor beta and linkage disequilibrium with HLA-DPB1.

The human retinoid X receptor beta (RXRB) gene is localized in the major histocompatibility complex (MHC) region between DPB1 and RING2. The RXRB gene sequence reported by different investigators suggests that the gene may be polymorphic. In this study, we confirmed one polymorphism by sequencing genomic DNA from four Caucasian individuals. We also developed a restriction fragment length polymorphism (RFLP) analysis to detect this specific polymorphism. Linkage analysis studies between RXRB alleles and a number of HLA markers showed significant linkage disequilibrium between RXRB*T and HLA-DPB1*0401.

Hepatitis B surface antigen- and tetanus toxoid-specific clonal expansion of CD4+ cells in vitro determined by TCRBV CDR3 length and nucleotide sequence.

We demonstrate activation of primary human TCRBV-specific CD4+ cells in vitro towards hepatitis B surface antigen (HBsAg) and tetanus toxoid (TT) without the use of cell lines, clones or added cytokines. By multiplex PCR analysis and spectratyping, antigen-activated cells exhibited clonal T cell receptor expansion within specific and limited TCRBV families. The expanded CD4+ T cells were CD45RO. Three of four unrelated HBsAg responders showed CD4+ expansion within the TCRBV16 family. The response comprised predominantly single CDR3 sequences in all three donors and was completely monoclonal in one of them. However, the CDR3 lengths and sequences differed among the responders. Clonality induced by HBsAg in TCRBV16 was specific, reproducible and distinct from that induced by TT in terms of sequence, nucleotide addition and diversity (BD) or junctional (BJ) element usage. Thus, for the first time, we show monoclonal or oligoclonal expansion of primary human CD4- peripheral blood mononuclear cells (PBMC) in vitro in response to nominal protein antigen without manipulations utilizing exogenous IL-2. The ability to induce monoclonal/ oligoclonal responses to HBsAg now permits motif identification studies for determining the T cell role in nonresponsiveness to the HBsAg vaccine.

MICA genetic polymorphism and linkage disequilibrium with HLA-B in 29 African-American families.

The human major histocompatibility complex (MHC) class I chain-related gene A ( MICA) is located 46 kb upstream of HLA-B and encodes a stress-inducible protein which displays a restricted pattern of tissue expression. MICA molecules interact with NKG2D, augmenting the activation of natural killer cells, CD8(+) alpha beta T cells, and gamma delta T cells. MICA allelic variation is thought to be associated with disease susceptibility and immune response to transplants. We investigated MICA allelic variations and linkage disequilibrium with HLA-A, B, and DRB1 loci on 110 parental haplotypes from 29 African-American families. PCR/sequence-specific oligonucleotide probing (SSOP) was used to define MICA polymorphisms in exons 2, 3, and 4. Ambiguous allelic combinations were resolved by sequencing exons 2, 3, and 4. Exon 5 polymorphisms were analyzed by size sequencing. For HLA-A, B and DRB1 typing, low-resolution PCR/SSOP and allelic PCR/sequence-specific priming techniques were used. Twelve MICA alleles were observed, the most frequent of which were MICA*008, MICA*004, and MICA*002, with gene frequencies of 28.2, 26.4, and 25.5%, respectively. Thirty-eight HLA-B- MICA haplotypic combinations were uncovered, 22 of which have not been reported in the HLA homozygous typing cell lines from the 10th International Histocompatibility Workshop. Significant positive linkage disequilibria were found in 8 HLA-B- MICA haplotypes. Furthermore, haplotypes bearing HLA-B*1503, *1801, *4901, *5201, *5301, and *5703 were found to segregate with at least two different MICA alleles. Our results provide new data about MICA genetic polymorphisms in African-Americans, which will form the basis for future studies of MICA alleles in allogeneic stem cell transplantation outcome.

Acute effects of bradykinin on cerebral microvascular permeability in the anaesthetized rat.

1. The permeability response to acutely applied bradykinin and [des-Arg9]-bradykinin on single cerebral venular capillaries has been investigated using the low molecular mass fluorescent dyes Lucifer Yellow and Sulforhodamine B with the single vessel occlusion technique. 2. When bradykinin was applied repeatedly for up to 2 h, the permeability increase was small and reversible for concentrations that ranged from 5 nM to 50 microM. 3. The logEC50 of the permeability response to bradykinin was -5.3 +/- 0.15 (logM; mean +/- s.e.m.). This was reduced to -6.37 +/- 0.24 with the angiotensin-converting enzyme inhibitor captopril, to -6.33 +/- 0.19 with the neutral endopeptidase inhibitor phosphoramidon and to -7.3 +/- 0.20 with captopril and phosphoramidon combined. 4. The permeability response to bradykinin was blocked by the bradykinin B2 receptor antagonist HOE 140, by inhibition of the Ca2+-independent phospholipase A2, by the scavenging of free radicals, or by inhibition of both cyclo-oxygenase and lipoxygenase in combination. Block of Ca2+ entry channels with SKF 96365 had no effect on the response. 5. Application of [des-Arg9]-bradykinin also increased permeability over the concentration range 5 nM to 50 microM, with a logEC50 of -5.6 +/- 0. 37. This response was not affected by free radical scavenging, but was completely blocked by the histamine H2 receptor blocker cimetidine. 6. These results imply that the acute permeability response to bradykinin is mediated via the release of arachidonic acid, which is acted on by cyclo-oxygenase and lipoxygenase resulting in the formation of free radicals, and that the response to [des-Arg9]-bradykinin is mediated via histamine.

HLA-Cw*1701 is associated with two sub-Saharan African-derived HLA haplotypes: HLA-B*4201, DRB1*03 and HLA-B*4202 without DRB1*03.

Different extended haplotypes have been described for many ethnic groups, such as African-Americans. The complotype FC(1,90)0 is in linkage disequilibrium with HLA-B42, DRB1*0302 in African-Americans and Southern African Xhosa individuals, suggesting a common ancestry. In order to analyze the distribution of Cw*17 alleles (Cw*1701, 1702) in relation to this African-derived extended haplotype, we studied a large panel of samples from African-American individuals and additionally a group of selected samples carrying HLA-B42, DR3 and HLA-B42, non-DR3 antigens. HLA alleles were assigned using sequence-specific amplification (SSP) and sequence-specific oligonucleotide probe hybridization (SSOP). We have found that all haplotypes (10 in total) carrying the extended haplotypes [HLA-B42, FC(1,90)0, DRB1*0302] were positive for HLA-Cw*1701. Interestingly, HLA B*4201 was found in all samples (17 in total) carrying HLA-B42, DR3, Cw*1701, whereas HLA-B*4202 was found in 10 out of 13 samples from individuals carrying HLA B42, Cw*1701 non-DR3. These findings suggest that HLA-Cw*17 polymorphism is conserved in different ethnic populations and that HLA-B42 alleles seem to separate at least different African-derived haplotypes. The historical context of these findings are important for the study of human evolution and they may be useful for the development of strategies in the search for possible donors in organ transplantation for African-derived populations.

HLA-DRB1 leprogenic motifs in nigerian population groups.

Amino acid residues involved in the peptide binding groove of HLA-DRB1 alleles were examined in three Nigerian ethnic groups with leprosy (n = 287) and 170 controls to determine the role of DRB1 alleles in disease outcome with Mycobacterium leprae. Nine positively charged motifs and two others with neutral charge to the binding groove were detected. These motifs occurred more frequently in leprosy (leprogenic) than was expected by chance (P < 0.0001). In contrast, five motifs with net negative or 'modified' neutral charges to the pocket were negatively associated with leprosy. We conclude that clinical outcome of infection with M. leprae is largely determined by a shared epitope in DRB1 alleles marked by several motifs. These motifs occur in otherwise normal DRB1 alleles, characterized by net positive or neutral charges in the binding groove. We hypothesize that these polarities cause poor binding of DRB1 to M. leprae. On presentation, the signal via the T cell receptor results in muted cell-mediated immunity. The resulting response translates to various forms of leprosy depending on degree of charge consonance between M. leprae and host DRB1 allele. Other factors within or without the HLA complex, such as the T cell receptor repertoire, may also influence the resulting disease.

CD4 TCRBV CDR3 analysis in prevalent SLE cases from two ethnic groups.

We examined CD4+ T cell TCRBV-CDR3 transcripts from 19 lupus patients and 16 controls to test the hypothesis that CD4+ TCRBV-CDR3 expression in SLE differs from normals. Within the disease group we also performed exploratory analyses to determine the association between risk of oligoclonality and HLA-DRB specificities and the duration of the CDR3 patterns. Oligoclonal patterns consistent with CDR3 restriction were three times more likely in SLE than in controls (OR = 3.7). TCRBV1, BV4, BV5.1, BV7, BV9, BV18 and BV22 gene segment CDR3 patterns of oligoclonality were seen exclusively among lupus patients. HLA-DRB3 increased the risk of oligoclonal expression in SLE. In four patients studied over time, the pattern of TCRBV-CDR3 expression was stable in a second sample obtained 6-14 months later. The increased frequency of CD4+ T cell TCRBV-CDR3 oligoclonal expression in SLE when compared to controls and the persistence of these patterns are consistent with an expanded pool of autoreactive CD4 T cells in SLE which recognize peptides derived from autoantigens. The association of HLA-DRB3 genes with increased risk of CDR3 oligoclonality among the SLE subjects is compatible with the hypothesis that molecules encoded by HLA-DRB3 may facilitate autoantigen recognition by CD4 T cells.

Association of IL-6 gene alleles with systemic lupus erythematosus (SLE) and with elevated IL-6 expression.

To evaluate the association of alleles of regions having regulatory potential in the IL-6 gene, with SLE, the AT-rich minisatellite in the 3' flanking region and the 5' promoter-enhancer of the IL-6 gene were genotyped by PCR- and RFLP-based methods. The AT-rich minisatellite allele distribution pattern was significantly different in SLE (n = 146) as compared to 139 controls (chi 2(7) = 48.97, P = 0.001, Caucasians; and chi 2(7) = 19.93, P = 0.006, African-Americans). In either race, short allele sizes (< or = 792 bp) were seen exclusively in SLE patients (P = 0.001), whereas the 828-bp allele was over-represented in controls (P = 0.015 and 0.002). In contrast, there was no preferential association of SLE with G/C alleles in the 5' region of the IL-6 gene. Furthermore, our results suggest that the 3' minisatellite alleles have biological significance: (1) B lymphoblastoid cells of patients having one or two SLE-associated alleles secreted IL-6 in 3- to 4-fold higher levels than non-allelic cells (P < 0.05); (2) higher percentages (approximately 4-fold) of IL-6 positive monocytes were observed in individuals having SLE-associated IL-6 alleles; (3) in lupus patients having SLE-associated minisatellite alleles, IL-6 mRNA stability was significantly enhanced.

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.

Regulation of cerebral microvascular permeability by histamine in the anaesthetized rat.

1. The permeability response of slightly leaky pial venular capillaries to histamine was investigated using the single microvessel occlusion technique. 2. Histamine dose-response curves showed that concentrations between 5 nm and 5 microM increased permeability, while concentrations from 50 microM to 5 mM reduced it. 3. The H2 receptor antagonist cimetidine (2 microM) blocked the effects of lower concentrations of histamine, while the H1 receptor antagonist mepyramine (3 nM) blocked those of higher concentrations of histamine. 4. The effects of lower doses of histamine were mimicked by the H2 receptor agonist dimaprit, and the effects of higher doses of histamine were mimicked by the H1 receptor agonist alpha-2-(2-aminoethyl)pyridine (AEP). 5. Low concentrations of histamine, which normally increase the permeability of Lucifer Yellow (PLY), reduced it when co-applied with the phosphodiesterase 4 (PDE4) inhibitor rolipram. Rolipram also potentiated the response to AEP, but had no effect on that to dimaprit. 6. The effects of dimaprit were blocked by reducing extracellular Ca2+ from 2.5 mM to nominally Ca2+ free, or by applying the calcium entry blocker SKF 96365.