Cold Pressor Testing and Sympathetic Nervous System Contribution to Ischemia with No Obstructive Coronary Artery Disease: Results from the Women's Ischemia Syndrome Evaluation-Coronary Vascular Dysfunction Project.

Study Objective: Cold Pressor Testing (CPT) is a known stimulus of the sympathetic nervous system (SNS). To better understand sympathetic contribution to coronary blood flow regulation in women with suspected ischemia and no obstructive coronary arteries (INOCA), we compared myocardial perfusion reserve during CPT stress cardiac magnetic resonance (CMR) imaging between women with suspected INOCA and reference subjects. Design: Prospective cohort. Setting: Academic hospital. Participants: 107 women with suspected INOCA and 21-age-matched reference women. Interventions: CPT stress CMR was performed with measurement of myocardial perfusion reserve index (MPRI), adjusted for rate pressure product (MPRIRPP). Invasive coronary function testing in a subset of INOCA women (n=42) evaluated for endothelial dysfunction in response to acetylcholine, including impaired coronary diameter response ≤0% and coronary blood flow response (ΔCBF) <50%. Main Outcome Measure: MPRIRPP. Results: Compared to reference women, the INOCA group demonstrated higher resting RPP (p=0.005) and CPT MPRIRPP (1.09±0.36 vs 0.83±0.18, p=0.002). Furthermore, INOCA women with impaired ΔCBF (n=23) had higher CPT MPRIRPP (p=0.044) compared to reference women despite lower left ventricular ejection fraction (64±7 % vs 69±2 %, p=0.005) and mass-to-volume ratio (0.79±0.15 vs 0.62±0.09, p<0.0001). These differences in CPT MPRIRPP did not persist after adjusting for age, body mass index, and history of hypertension. CPT MPRIRPP among INOCA women did not differ based on defined acetylcholine responses. Conclusions: Myocardial perfusion reserve to CPT stress is greater among women with INOCA compared to reference subjects. CPT induced a higher MPRIRPP also in women with coronary endothelial dysfunction, suggesting a greater contribution of the SNS to coronary flow than endothelial dysfunction. Further investigation in a larger cohort is needed.

A fragment of the Neurogenin1 gene confers regulated expression of a reporter gene in vitro and in vivo.

The basic helix-loop-helix transcription factor neurogenin1 is required for proper nervous system development in vertebrates. It is expressed in neuronal precursors during embryonic development and is thought to play a role in specifying neuronal fate. To investigate the regulation of neurogenin1 expression, the transcriptional start site of the gene was identified and a 2.7-kb fragment ending in the first exon was shown to possess basal promoter activity. This 2.7-kb fragment was able to promote expression of reporter genes in P19 cells under conditions in which expression of endogenous neurogenin1 was induced. Importantly, the 2.7-kb fragment was able to drive expression of a lacZ reporter gene in transgenic mice in most tissues in which neurogenin1 is normally expressed, including those peripheral ganglia that fail to develop in neurogenin1 "knockout" mice. These findings identify a regulatory region containing elements responsible for appropriate expression of a gene with a crucial role in generating the vertebrate nervous system.

Chlamydia trachomatis major outer membrane protein (MOMP) epitopes that activate HLA class II-restricted T cells from infected humans.

We localized peptide epitopes within the Chlamydia trachomatis (Ct) major outer membrane protein (MOMP) that activate human T cells. T-MOMP' cells were prepared by culturing PBL from 38 humans who had Ct infections in the presence of Ct serovar E MOMP. Some epitopes were first localized by quantifying proliferative responses of T-MOMP' cells to overlapping MOMP segments (sixths) that were produced in Escherichia coli. Further localization was achieved by using overlapping synthetic 16-22 mers that spanned stimulatory MOMP sixths as Ags. The APCs used were human B cell lines (LCL) that were matched or mismatched with respect to HLA class II alleles of the T-MOMP' cells. T cell epitopes were detected in 18 Ct serovar E MOMP 16-22 mers and were presented in association with HLA-DR1, -7, -13, -17, HLA-DRw52, HLA-DQ3 and at least two from among HLA-DR4, -8, -11, -14, -18, in probable addition to HLA-DP4. Peptide 249-265 stimulated T-MOMP' cells from 83% of the subjects; studies with overlapping 11-13 mers spanning peptide 249-265 revealed at least six epitopes presented with different HLA-class II allotypes. Diverse T-MOMP' cultures responded to between 2 and 7 MOMP epitopes. All but 1 of the 23 epitopes localized to date are distributed among four MOMP constant segments. T-MOMP' cells from subjects infected with serovars other than E also responded.

Gene required for normal MHC class II expression and function is localized to approximately 45 kb of DNA in the class II region of the MHC.

In certain mutant human B cell lines, MHC-encoded class II molecules displayed at the cell surface have an abnormal conformation and are unstable in the presence of SDS. The mutants cannot present exogenous protein Ags to T cells but elicit responses with exogenous antigenic peptides; thus, formation of intracellular complexes between antigenic peptides and class II molecules is impaired. Previous analysis of LCL deletion mutants, .82, .174, and 5.2.4, showed that genes needed for this function must be present in approximately 230 kb of DNA in the class II region of the MHC. We now describe a new deletion mutant, .61, which has normal class II-mediated Ag processing/presentation. The TAP1, TAP2, LMP2, and LMP7 genes are deleted from .61, demonstrating that those genes are not needed for normal formation of intracellular class II/peptide complexes. The genes in question must be located in DNA that is present in .61 and .82 (both normal) and absent from .174 and 5.2.4. (both defective). Mapping of the deletion breakpoints indicates that genes needed for normal class II-associated Ag processing/presentation are either: 1) in an approximately 40 kb L DNA segment located between the DMB and LMP2 loci or 2) in an R region between the DQA2 and DQB1 loci and are completely included on a 5.1-kb fragment formed by joining of DNA that flanks the deletion in .61. The evidence favors location of the genes in the L DNA segment.

Identification of B-cell epitopes on the S4 subunit of pertussis toxin.

The main purpose of the present study was to identify B-cell epitopes on the S4 subunit of pertussis toxin (PT) by the synthetic peptide approach. Two strategies were followed: (i) screening of two series of overlapping peptides (12- and 25-residue peptides) covering the entire S4 sequence by a panel of murine monoclonal anti-PT antibodies and various polyclonal anti-PT antisera in an enzyme-linked immunosorbent assay (ELISA), and (ii) analysis of the S4 amino acid sequence by a predictive algorithm followed by synthesis and immunization of mice with the predicted peptides coupled to diphtheria toxoid. The anti-peptide conjugate antisera were tested in an ELISA for cross-reactivity with native PT, B oligomer, and S4. Screening of the free peptides in an ELISA by the PT antisera indicated the presence of six B-cell epitope-containing domains covered by residues 18 to 32, 33 to 46, 39 to 52, 51 to 65, 71 to 84, and 91 to 106. None of the peptides, however, were recognized by the monoclonal anti-PT antibodies in an ELISA. Immunization with six computer-predicted peptides (B1 to B6) and three potential T-cell epitopes (T1 to T3) gave rise to very high antibody responses towards the homologous conjugates. With the exception of the anti-T1/diphtheria toxoid antisera, all anti-peptide conjugate antisera cross-reacted with PT in an ELISA at different levels. None of these anti-peptide conjugate antisera, however, showed any PT-neutralizing effect as measured by the Chinese hamster ovary cell assay and the leukocytosis-promoting activity test. The results of the present study suggest that discontinuous epitopes are predominant in the S4 subunit of native PT.

Identification of murine T-cell epitopes on the S4 subunit of pertussis toxin.

The aim of the present study was to identify murine T-cell epitopes on pertussis toxin subunit S4. Six mouse strains with five different haplotypes at the H-2 locus were immunized with the pertussis toxin B oligomer. Lymph node lymphocytes were isolated and stimulated in an in vitro proliferation assay with pertussis toxin components and 11 overlapping synthetic peptides synthesized on the basis of the primary sequence of S4. In vitro proliferative responses to the synthetic peptides revealed the presence of four distinct murine T-cell epitopes on subunit S4. The recognition of the peptides was major histocompatibility complex restricted. Immunizing four of the six mouse strains with the synthetic peptides showed that the peptides which were demonstrated to contain T-cell epitopes following immunization with the B oligomer were able to induce proliferative responses to detoxified pertussis toxin and pertussis toxin components containing subunit S4. One of the identified murine T-cell epitopes corresponded to one of the major human T-cell epitopes previously identified on subunit S4. It is hoped that this murine model system will facilitate the development of a synthetic immunogen mimicking the protective properties of pertussis toxin.

Quantification of pertussis toxin, filamentous haemagglutinin, 69 kDa outer membrane protein, agglutinogens 2 and 3 and lipopolysaccharide in the Danish whole-cell pertussis vaccine.

The amounts of pertussis toxin (PT), filamentous haemagglutinin (FHA), 69 kDa outer membrane protein (69 kDa OMP) and agglutinogens (AGG) 2 and 3 in extracts from the Danish whole-cell pertussis vaccine were studied in quantitative capture ELISA. With the exception of PT, the most effective extraction of these antigens was by heating the bacteria at 60 degrees C for 30 min in 2 M urea followed by sonication for 45 s. Extraction by 1 M sodium chloride prior to sonication resulted in higher levels of antigenic and biologically active PT. On average, a single human dose of pertussis vaccine (approximately 16 opacity units) was found to contain 5520 ng FHA, 63 ng PT, 1061 ng 69 kDa OMP, 397 ng AGG 2, 534 ng AGG 3 and 4840 ng lipopolysaccharide (LPS). The antigen content of one dose of the Danish pertussis vaccine appears to be low compared with the amounts found in the acellular vaccines currently in use. These findings may have important implications for the evaluation of the protective substances and the immunogenicity of whole-cell as opposed to acellular pertussis vaccines.

Proliferative responses to purified and fractionated Bordetella pertussis antigens in mice immunized with whole-cell pertussis vaccine.

The specificity of the cell-mediated immune response to Bordetella pertussis following immunization of C57B1 mice with a whole-cell pertussis vaccine was assessed in a proliferation assay. A proliferative response of lymph node lymphocytes to the filamentous haemagglutinin, the 69 kDa outer membrane protein and the agglutinogens 2 and 3 was demonstrated. The proliferative cells were T cells of the CD4+ phenotype. In addition, several as yet uncharacterized antigens expressed by B. pertussis were shown to induce a proliferative response, also mediated by T cells of the CD4+ phenotype. Although a range of different immunization schedules and preparations of pertussis toxin were used, no specific proliferative responses to pertussis toxin, which is regarded as a protective antigen of major importance from B. pertussis, were found.

Proliferative responses and gamma interferon and tumor necrosis factor production by lymphocytes isolated from tracheobroncheal lymph nodes and spleen of mice aerosol infected with Bordetella pertussis.

A group of mice was aerosol infected with live, virulent Bordetella pertussis bacteria. During a period of 7 weeks following the infection, with intervals of 1 week, lymphocytes were isolated from the tracheobroncheal lymph nodes (TBL) and the spleens (SPL) of the infected mice. The in vitro proliferative responses as well as the gamma interferon and tumor necrosis factor production levels of the isolated lymphocytes in response to stimulation with whole killed B. pertussis bacteria were measured as parameters for cell-mediated immunity (CMI). The course of the infection was monitored by counting of CFU in the lungs of the mice. Moreover, antibody responses in serum against a range of B. pertussis antigens were assessed. The results showed that a vigorous proliferative response of the TBL and SPL to stimulation with whole killed B. pertussis bacteria was induced by the infection. The proliferative response of the TBL was significantly higher than the response of the SPL. The proliferative responses were maximal 3 to 4 weeks after the infection and were paralleled by in vitro gamma interferon and tumor necrosis factor production upon specific stimulation. The development of the CMI was observed simultaneously with the clearance of the infection from the lungs. Antibody responses became measurable in the sera only after the infection was cleared. A specific CMI against pertussis toxin, the filamentous hemagglutinin, the 69-kDa outer membrane protein, and the agglutinogens 2 and 3, antigens which are under consideration for inclusion in future acellular pertussis vaccines, was successfully demonstrated in mice 3 weeks after the infection.

Identification of human T-cell epitopes on the S4 subunit of pertussis toxin.

Ten adult humans were vaccinated with the Japanese acellular pertussis vaccine JNIH-3, containing detoxified pertussis toxin (PT), formaldehyde, and filamentous hemagglutinin. The vaccination induced a specific antibody response to PT and filamentous hemagglutinin, and a Western blot (immunoblot) analysis of the antibody response to PT revealed antibodies to PT subunits S1, S2, S3, S4 and S5. The response of peripheral lymphocytes to PT was assessed in an in vitro proliferation assay. A proliferative response to detoxified PT and PT dimers S2-S4 and S3-S4 was found, and it was further demonstrated that the proliferative response to detoxified PT and dimer S2-S4 was mediated by T cells of the CD4+ phenotype. The specificity of the proliferative response to subunit S4 was analyzed with a range of synthetic peptides synthesized on the basis of the primary sequence of subunit S4. The proliferative response to the peptides revealed two major and one minor T-cell epitope located in the NH2-terminal end of subunit S4.

Induction of polyclonal antibodies to the S1 subunit of pertussis toxin by synthetic peptides coupled to PPD: effect of conjugation method, adjuvant, priming and animal species.

Two peptides, designated L and K, covering a sequence near the NH-terminal end of the S1 subunit of pertussis toxin (PT) were conjugated to the PPD (purified protein derivative) of M. tuberculosis by either glutaraldehyde (GLUT) or succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and injected into groups of mice and guinea pigs. Initially, the effect of priming the animals with BCG vaccine and the use of aluminium hydroxide as adjuvant for the anti-peptide antibody response was studied. The group of BCG-primed mice immunized with adsorbed peptide conjugates showed the highest anti-peptide conjugate antibody response. Based on this finding, groups of BCG-primed mice were immunized four times with either adsorbed peptide L-GLUT, peptide L-SMCC/SPDP or peptide K-SMCC/SPDP conjugates and the fine peptide specificity as well as the PT and S1 cross-reactivity was investigated in ELISA. Mice immunized with peptide L-GLUT showed a significant antibody response to the homologous conjugate, only, whereas the group injected with the peptide L-SMCC/SPDP conjugate gave a significant response to both peptide K and L conjugated by the SMCC-SPDP method. Likewise, mice immunized with the peptide K-SMCC/SPDP conjugate reacted with the homologous and peptide L-SMCC/SPDP conjugate, although only the response to the former conjugate was significantly greater than the response to PPD. All groups showed a strong anti-PPD response. The anti-PT/S1 cross-reactivity of the antisera varied considerably within each group but was found to be highest in the peptide L-GLUT-immunized animals. The results of the present study not only stress the importance of BCG priming and use of aluminium hydroxide adjuvants for the immunogenicity of the peptides in question but also point to the specificity of the conjugation methods employed as low cross-reactivity between the anti-peptide L-GLUT and L-SMCC/SPDP antisera was noted. Moreover, it appeared that the choice of conjugation method may have an effect on the ability of the peptide conjugates to induce an antibody response cross-reacting with the native protein.

The cell mediated and humoral immune response to vaccination with acellular and whole cell pertussis vaccine in adult humans.

The cell mediated immune response (CMI) against pertussis antigens following vaccination with the traditional Danish whole cell pertussis vaccine (WC-P) and the Japanese acellular pertussis vaccine (A-PV) JNIH-3 was studied in four adult human volunteers. Vaccination with the A-PV induced an in vitro proliferative response of peripheral blood lymphocytes to pertussis toxin (PT) subunits S2-S4, S3-S4 and S5 and the filamentous hemagglutinin (FHA), and a better serological response to native PT, detoxified PT (dPT) and FHA than the WC-PV. The induced CMI and serological response were followed over a period of 17 weeks, and were not seen to decline during this period. Further, an in vitro proliferative response to Bordetella pertussis agglutinogen 2 and 3 were demonstrated using lymphocytes from recently and not-so-recently pertussis-vaccinated adults.

Adjuvanticity of pertussis toxin is mediated by differential effects on the activity of T suppressor, T amplifier and T helper cells.

The immunomodulatory effects of pertussis toxin (Ptx) were studied in BALB/c mice immunized with type III pneumococcal polysaccharide (SSS-III). The antibody response to SSS-III is predominantly of the IgM class and is regulated by two opposing types of T cells, a T suppressor (TS) and a T amplifier (TA) cell. Treatment of mice with Ptx at the time of immunization with SSS-III results in an enhancement of the splenic antigen-specific IgM response, which was estimated by a plaque-forming cell assay. Numbers of non-antigen-specific IgM producing cells were not significantly affected. This adjuvant effect occurred in nu/+ but not in T cell deficient nu/nu mice. Such adjuvanticity appears to be due in part to the neutralization of TS, since (a) Ptx-treatment was found to reverse TS-mediated low-dose immunological tolerance induced in mice previously exposed to a subimmunogenic dose of SSS-III, and (b) in vitro Ptx-treatment of a cell population enriched in SSS-III-specific TS, activity abolished the capacity of such cells to transfer suppression when given to recipients at the time of immunization with SSS-III. In another type of cell transfer experiment, it was shown that Ptx treatment resulted in an increased frequency of TA in mice immunized with SSS-III. We conclude that Ptx differentially affects at least two populations of regulatory T cells, and that these effects are not directly related to the mitogenicity of Ptx for T cells or to a direct effect upon B cells.

Differences in specificity of "DPw2-specific" cytotoxic T cell clones revealed with HLA-mutant lines: evidence that non-DP HLA genes influence recognition by some clones.

Seven allospecific cytotoxic T lymphocyte (CTL) clones derived from DPw2-specific bulk populations were characterized by three approaches to obtain a more detailed understanding of the T cell recognition of the HLA-DPw2 molecule. All seven of the clones were DPw2 specific and indistinguishable in specificity by three approaches: (a) patterns of lysis of panels of targets from normal donors; (b) inhibition of lysis by anti-class II monoclonal antibodies (mAb); (c) lysis of mutant lymphoblastoid B cell lines (LCL) with isolated loss of DP2 alpha or DP2 beta gene expression (as a result of selection for resistance to DPw2-specific CTL). However, only four of the seven CTL clones (which we designate "orthodox") lysed all mutant DPw2+ LCL tested; the other three ("heterodox") CTL clones showed reduced or no lysis of particular LCL which expressed DPw2 but had been mutagenized and selected for loss of DR expression. Cold target blocking experiments with the mutant LCL confirmed differences in: (a) specificity among CTL clones and (b) CTL-defined phenotype among serologically indistinguishable DR-DPw2+ mutant LCL. Differences were not explained by different levels of DP expression by the mutant LCL. Given the nature of the mutagens and mutations, it is highly improbable that point mutations in DP account for differences in recognition. These data suggest that non-DP HLA genes influence recognition by some DP-specific clones, potentially due to corecognition of HLA-DR alpha or another non-DP HLA product in the context of a DPw2 alpha/beta dimer.

Mapping of prolactin and tumor necrosis factor-beta genes on human chromosome 6p using lymphoblastoid cell deletion mutants.

A collection of human B lymphoblastoid cell lines (LCLs) was used to map two genetic sequences for which polymorphism had not been identified: human prolactin (PRL) and tumor necrosis factor-beta (TNFB). The LCLs have overlapping deletions on chromosome 6p produced by gamma-irradiation of LCL 721. After using two chromosome 6p sequences for which LCL 721 is heterozygous to validate our scanning densitometry (SD) method for inferring gene copy number, SD was used to map TNFB and PRL. TNFB maps to the interval between the C4 complement and HLA-B loci within the MHC on chromosome 6p. PRL lies within the 6p21.3-6p22.2 interval distal to HLA-C. We found that LCL 721 is heterozygous for PRL DNA fragment lengths generated by HpaII but not MspI digestion, indicating that the two copies of PRL in LCL 721 are differentially methylated. This novel methylation RFLP was used to corroborate the region PRL assignment.

Derepression of HPRT locus on inactive X chromosome of human lymphoblastoid cell line.

Human XX lymphoblastoid cells with a deletion in the HPRT locus on the active X were exposed to HPRT clone pHPT32. HPRT+ isolates GPT3 and GPT5 lacked pHPT32 DNA, suggesting that their HPRT+ phenotype resulted from expression of a cellular gene. GPT3 mutated to thioguanine resistance at least 100 times more frequently than cells in which the expressed HPRT locus was on the active X. Most GPT3-derived HPRT- had lost one entire X chromosome, indicating that the HPRT+ phenotype of GPT3 resulted from derepression of the HPRT locus on its inactive X. Virtually unchanged G6PD and PGK activities and the presence of a late-replicating X in GPT3 suggest that derepression of the inactive X was not general. Eleven of the GPT3-derived mutants had a tiny centric remnant that may result from a frequently operative mechanism of X chromosome loss. The detection of partial or complete loss of an X by direct selection presents unusual opportunities for genotoxicity detection with human cells.

Molecular analysis of HLA class I and class II antigen loss mutants reveals a homozygous deletion of the DR, DQ, and part of the DP region: implications for class II gene order.

The mutant human B-lymphoblastoid cell lines, 721.174 and 721.180, previously reported to exhibit greatly reduced expression of human HLA class I and II antigens (DeMars et al., Hum Immunol 11:77, 1984), were analyzed by Southern blotting using class II cDNA and genomic clones as hybridization probes. All genomic sequences complementary to DR alpha, DR beta, DQ alpha, and DQ beta probes were absent from these mutants. DZ alpha genomic sequences were deleted as were the DP alpha 1 and DP beta 1 loci but the DP beta 2 and most, if not all, of the DP alpha 2 locus were retained. However, no RNA transcripts for either DP alpha 2 or DP beta 2 could be detected. The mapping of the deletion breakpoint within the DP cluster allows the orientation of the loci in the DP region with respect to the centromere as follows: centromere, DP beta 2, DP beta 1, DP alpha 1, (DQ, DR). In addition, the analysis of a set of DR-, DQ-, DP+ homozygous deletion mutants (721.82, 721.84, and 721.101) reveals a deletion breakpoint between the DQ alpha 1/DQ beta 1 loci and the DQ alpha 2/DQ beta 2 loci. These mutants retain DZ alpha genomic sequences, tentatively mapping the DZ alpha locus between the DQ and the DP region. The residual ability of the DR-, DQ-, DP- mutants (174 and 180)* to stimulate allogeneic and autologous lymphoproliferative responses must be attributed to expression of as yet unidentified class II antigens, or to non-class II antigens.

Repair of a subglottic stenosis by submucosal resection.

A subglottic, intralaryngeal stenosis in a 5-year-old boy was successfully removed by microsurgical submucosal resection. The method reported provides adequate subglottic augmentation and interferes with the laryngeal cartilage only minimally. The use of small split-thickness skin grafts for lining material is advocated, as they take immediately and reduce the time for stenting. At followup, no recurrent stenosis has developed and the site of the skin grafts has been lined with ciliated mucosa. Post-operative hospitalization was considered to be acceptably short.