Vladimir V. Yurovsky

Production of type 2 cytokines by CD8+ lung cells is associated with greater decline in pulmonary function in patients with systemic sclerosis

OBJECTIVE This study addresses the hypothesis that a profibrotic pattern of cytokines is produced in the lungs of patients with systemic sclerosis (SSc) and causes fibrosis. METHODS Using a reverse transcriptase-polymerase chain reaction technique, interleukin-4 (IL-4), IL-5, and interferon-gamma (IFNgamma) messenger RNA (mRNA) were measured in unseparated CD8+ and CD4+ bronchoalveolar lavage (BAL) cells from SSc patients and healthy controls. To confirm the results, CD8+ T cells were cloned from BAL fluids, and the pattern of cytokine mRNA made by these cells was determined. Serial pulmonary function tests were done. RESULTS BAL cells from healthy controls made IFNgamma mRNA, with no or little IL-4 or IL-5 mRNA. In contrast, BAL cells from the majority of SSc patients made IL-4 and/or IL-5 mRNA, with or without approximately equal amounts of IFNgamma mRNA. This pattern of cytokines was made by CD8+ T cells, which were increased in the lungs of these SSc patients. Patients whose BAL cells made this type 2 pattern of cytokine mRNA had a significant decline in forced vital capacity over time after the BAL, whereas patients whose BAL cells made IFNgamma mRNA alone did not. Both wild-type and an alternative splice variant of IL-4 mRNA were increased in BAL cells from SSc patients. Both forms of IL-4 stimulated alpha2(I) collagen mRNA in human dermal and lung fibroblasts. CONCLUSION The type 2 pattern of cytokine mRNA produced by BAL cells from SSc patients differs from unopposed IFNgamma production found in healthy BAL cells. This production of type 2 cytokine mRNA by CD8+ T cells is associated with a significant decline in lung function over time, which suggests a pathologic role for these T cells in interstitial fibrosis in SSc.

Protective Effect of Delayed Treatment With Low-Dose Glibenclamide in Three Models of Ischemic Stroke

Background and Purpose— Ischemia/hypoxia induces de novo expression of the sulfonylurea receptor 1-regulated NC(Ca-ATP) channel. In rodent models of ischemic stroke, early postevent administration of the sulfonylurea, glibenclamide, is highly effective in reducing edema, mortality, and lesion volume, and in patients with diabetes presenting with ischemic stroke, pre-event plus postevent use of sulfonylureas is associated with better neurological outcome. However, the therapeutic window for treatment with glibenclamide has not been studied. Methods— We examined the effect of low-dose (nonhypoglycemogenic) glibenclamide in 3 rat models of ischemic stroke, all involving proximal middle cerebral artery occlusion (MCAo): a thromboembolic model, a permanent suture occlusion model, and a temporary suture occlusion model with reperfusion (105 minutes occlusion, 2-day reperfusion). Treatment was started at various times up to 6 hours post-MCAo. Lesion volumes were measured 48 hours post-MCAo using 2,3,5-triphenyltetrazolium chloride. Results— Glibenclamide reduced total lesion volume by 53% in the thromboembolic MCAo model at 6 hours, reduced corrected cortical lesion volume by 51% in the permanent MCAo model at 4 hours, and reduced corrected cortical lesion volume by 41% in the temporary MCAo model at 5.75 hours (P<0.05 for all 3). Analysis of pooled data from the permanent MCAo and temporary MCAo series indicated a sigmoidal relationship between hemispheric swelling and corrected cortical lesion volume with the half-maximum cortical lesion volume being observed with 10% hemispheric swelling. Conclusions— Low-dose glibenclamide has a strong beneficial effect on lesion volume and has a highly favorable therapeutic window in several models of ischemic stroke.

Glibenclamide Is Superior to Decompressive Craniectomy in a Rat Model of Malignant Stroke

Background and Purpose— Treating patients with malignant cerebral infarctions remains a major unsolved problem in medicine. Decompressive craniectomy (DC) improves the bleak outlook but is suboptimal. Using a rat model of severe ischemia/reperfusion with very high mortality due to malignant cerebral edema, we tested the hypothesis that blocking of sulfonylurea receptor 1–regulated NCCa-ATP channels with glibenclamide would compare favorably to DC when reperfusion and treatment were begun 6 hours after onset of ischemia. Methods— Male Wistar rats underwent filament occlusion of the middle cerebral artery to reduce laser Doppler flowmetry perfusion signals by >75%, with filament removal plus treatment 6 hours later. In rats treated with vehicle versus glibenclamide (10 &mgr;g/kg IP plus 200 ng/h SC), we compared mortality, neurologic function, and brain swelling at 24 hours. In rats treated with DC versus glibenclamide, we compared neurologic function for 2 weeks and histologic outcomes. Results— Compared with vehicle, glibenclamide treatment reduced 24-hour mortality from 67% to 5% and reduced hemispheric swelling at 24 hours from 21% to 8%. DC eliminated 24-hour mortality, but neurologic function during the next 2 weeks was significantly better with glibenclamide compared with DC. Watershed cortex and deep white matter were significantly better preserved with glibenclamide compared with DC. Conclusions— In a rat model of severe ischemia/reperfusion, with reperfusion and treatment beginning 6 hours after onset of ischemia, glibenclamide is as effective as DC in preventing death from malignant cerebral edema but is superior to DC in preserving neurologic function and the integrity of watershed cortex and deep white matter.

Diversity and plasticity of the anti–DNA topoisomerase I autoantibody response in scleroderma

OBJECTIVE To examine domain recognition by anti-DNA topoisomerase I (anti-DNA topo I, or anti-topo I) antibodies over time in scleroderma patients. METHODS Serial serum samples from scleroderma patients with known reactivity to Scl-70, a 70-kd topo I breakdown product, were tested by immunoblot for IgM, IgG, IgA, kappa, and lambda reactivity to Scl-70 and 8 overlapping recombinant peptide fragments (F1-F8) that span the human topo I molecule. RESULTS IgM, IgG, kappa, and lambda anti-topo I antibodies in both early-disease and late-disease serum samples preferentially recognized the Scl-70 molecule rather than the F1-F8 peptides, suggesting preferential recognition of conformational determinants on Scl-70 throughout the disease course. Amounts of both primary and secondary anti-topo I antibodies to Scl-70 varied over time, including increases in primary antibody responses late in the disease course. Striking variability in recognition of the F1-F8 peptides by IgM, IgG, IgA, kappa, and lambda anti-topo I antibodies was seen in serial samples. Most often, the change in FI-F8 recognition from one sample to the next was unpredictable, although occasionally patterns of antibody recognition were reciprocal in serial samples. Of note, in several patients, what could have been interpreted as domain spreading among F1-F8 in 2 successive samples was just a part of changing antibody reactivity to these peptides that again became more restricted in a third sample. CONCLUSION Titers and immunodominant domains recognized by both primary and secondary anti-topo I antibodies are highly variable over time. This suggests continual antigen presentation and regulation of the anti-topo I antibody response in scleroderma, even late in the disease course.

Characteristics of non-opioid β-endorphin receptor

Tritium-labeled selective agonist of non-opioid β-endorphin receptor, the decapeptide immunorphine ([2H]SLTCLVKGFY) with specific activity of 24 Ci/mmol has been prepared. By its use, non-opioid β-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. The non-opioid β-endorphin receptor of macrophages has in addition to immunorphine (Kd of the [2H]immunorphinereceptor complex was 2.4 ± 0.1 nM) and β-endorphin (Ki of the [2H]immunorphine specific binding was 2.9 ± 0.2 nM) a high affinity for Fc-fragment of human IgG1, pentarphine (VKGFY), cyclopentarphine [cyclo(VKGFY)], and [Pro2]pentarphine (VKPFY) (Ki values were 0.0060 ± 0.0004, 2.7 ± 0.2, 2.6 ± 0.2, and 2.8 ± 0.2 nM, respectively) and is insensitive to naloxone and [Met5]enkephalin (Ki > 100 μM). Treatment of macrophages with trypsin resulted in the loss of their ability for the specific binding of [2H]immunorphine. Values of the specific binding of 8.4 nM [2H]immunorphine to rat adrenal, spleen, myocardium, and brain membranes were determined to be 1146.0 ± 44.7, 698.6 ± 28.1, 279.1 ± 15.4, and 172.2 ± 1.8 fmol/mg protein, respectively. Unlabeled β-endorphin, pentarphine, [Pro2]pentarphine, cyclopentarphine, cyclodipentarphine [cyclo(VKGFYVKGFY)], and Fc-fragment of IgG1 inhibited the binding of [2H]immunorphine to membranes from these organs. No specific binding of [2H]immunorphine to rat liver, lung, kidney, and intestine membranes was found.

Oligoclonal Expansion of Vδ1+γ/δ T-Cells in Systemic Sclerosis Patientsa

Systemic sclerosis (SSc) is a multisystem disease characterized by T-cell infiltration of involved tissues, fibrosis, and small vessel vasculopathy. Using flow cytometric analyses, we found an increased percentage of gamma/delta T-cells expressing the T-cell antigen receptor variable (V) delta 1 gene segment in the peripheral blood and bronchoalveolar lavage fluid of patients with SSc. To estimate clonality of these V delta 1+ T-cells, the diversity of V delta 1 junctional regions (V-Diversity-Joining gene segments) was examined using a reverse transcriptase-polymerase chain reaction to amplify T-cell antigen receptor delta chain transcripts isolated from peripheral blood mononuclear cells, lung, esophagus, stomach, or skin of patients and controls. Limited diversity of V delta 1-J delta junctional regions in SSc patients was demonstrated by the finding of greater restriction in the nucleotide lengths of junctional region cDNAs in individual SSc patients than in controls. Sequence analyses confirmed that V delta 1-J delta junctional regions from the blood of SSc patients had less diversity than those from controls, in that a significantly higher proportion of sequences were repeated in patients (54.4% vs. 19.4% in controls). Evidence for selection of the V delta 1+ T-cells in tissues of individual SSc patients came from the findings that the same V delta 1-J delta junctional sequences could be isolated from the same tissue over time and that identical V delta 1-J delta junctional sequences could be isolated from multiple tissues. These data suggest that expansion of V delta 1+ gamma/delta T cells may be antigen driven in SSc patients.

Analysis of diversity of T cell antigen receptor genes using polymerase chain reaction and sequencing gel electrophoresis

A sensitive, highly resolvable, and quantitative method was designed to analyse the diversity of polymerase chain reaction (PCR)-amplified transcripts which possess length polymorphism. A reverse transcriptase-PCR technique was used to amplify rearranged T cell antigen receptor (TCR) transcripts isolated from human blood. Oligonucleotide primers specific for conserved TCR V and C region sequences were used in PCR, with one of the primers end-labeled with 32P. Amplified cDNA products were analysed by polyacrylamide sequencing gel electrophoresis with an M13mp18 sequencing ladder as a size marker. 32P-labeled products were detected by either autoradiography or PhosphorImager. The method allowed determination of the sizes of PCR products with the precision of one nucleotide. The resolution using this technique was much higher than by electrophoresis in agarose gel with ethidium bromide staining. The sizes of PCR products determined by sequencing gel electrophoresis were consistent with the lengths of nucleotide sequences obtained after subcloning PCR products in competent bacterial cells. Analysis of PCR products by sequencing gel electrophoresis was more rapid and as accurate as nucleotide sequence analysis in determining the relative ratios of TCR mRNA in mixtures of T cell clones. The method is applicable for analysis of both rearranged TCR and immunoglobulin genes.

Elucidation and Characteristics of Non-opioid β-Endorphin Receptors in Rat Adrenal Cortex

Abstractβ-Endorphin-like decapeptide immunorphin (SLTCLVKGFY), a selective agonist of non-opioid β-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid β-endorphin receptors on rat adrenal cortex membranes (Kd1 = 39.6 ± 2.0 nM, Bmax1 = 40.7 ± 2.3 pmol/mg protein; Kd2 = 0.25 ± 0.01 μM, Bmax2 = 187.8 ± 9.4 pmol/mg protein). β-Endorphin was found to inhibit the [3H]immunorphin specific binding to membranes (Ki = 70.0 ± 9.2 nM); naloxone, [Met5]enkephalin, and α- and γ-endorphins tested in parallel were inactive. Immunorphin at concentrations of 10–9-10–6 M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, while intramuscular injection of immunorphin at doses of 10-100 μg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.

Synthetic peptide immunocortin stimulates the production of 11-oxycorticosteroides by rat adrenal cortex through ACTH receptors

Adrenocorticotropic hormone (ACTH)-like peptide immunocortin (IMC) VKKPGSSVKV, corresponding to the amino acid sequence 11-20 of the variable part of human immunoglobulin G (IgG) 1 heavy chain, at concentrations of 10(-9)-10(-6) I was found to increase the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunocortin at doses of 10-100 microg/kg was found to stimulate the secretion of 11-oxycorticosteroids (CS) from the adrenals to the bloodstream. Immunocortin was labeled with tritium to specific activity of 22 Ci/mmol. Receptor binding studies revealed that [(3)H]immunocortin ([(3)H]IMC) bound with high affinity and specificity to ACTH receptors on rat adrenal cortex membranes (K(d)=2.1+/-0.2 nM, B(max)=1.1+/-0.1 pmol/mg protein).

Simultaneous quantitation of cytokine mRNAs by reverse transcription-polymerase chain reaction using multiple internal standard cRNAs.

Cytokines produced in abnormal amounts or patterns contribute to many immunologically mediated human diseases. We describe a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay to measure interleukin (IL)1-2, IL-4, and interferon-γ (IFN-γ) mRNAs within the sample. Internal standard cRNAs and native cytokine mRNAs are reverse transcribed and then amplified by PCR in the same reaction tubes to control for tube-to-tube variability in these reactions. In contrast to systems that use a single multigene internal standard cRNA, this method uses separate internal standard cRNAs for IL-2, IL-4, and IFN-γ, allowing independent dosing of the internal standards, which reduces the number of tubes processed and the amount of starting mRNA required. Internal standards are produced from cytokine cDNAs by the insertion of short segments of DNA. The same oligonucleotide primers are used to amplify internal standard and native cytokine cDNAs. Each internal standard cDNA and its matching native cytokine cDNA are amplified with equal efficiency. The RT-PCR products of the internal standards and native cytokines are distinguished by size. This technique can detect a twofold difference in mRNA levels. Examples of using this technique to measure cytokine mRNAs in peripheral blood mononuclear cells and in bronchoalveolar lavage cells are given.