[Antibody levels of measles, rubella and mumps viruses in healthy population in Shanghai from 2010 to 2020].

Objective: To determine IgG antibody levels of measles, rubella, mumps in healthy population in Shanghai from 2010 to 2020 and analyze the trend of antibody changes in different age groups. Methods: 10 828 healthy people without measles, rubella and mumps in Shanghai were included in the study from 2010 to 2020. Serum samples were collected from 12 age groups, and the serum IgG antibody of measles, rubella and mumps were detected by ELISA. The difference of antibody positive rates and antibody levels were analyzed. Results: The median age M (Q1, Q3) of 10 828 objects were 8 years old (9 months old, 20 years old). Males accounted for 48.34% (5 234/10 828) and females accounted for 50.92% (5 514/10 828). Unknown gender information accounted for 0.74% (80/10 828), and 27.03% (2 927/10 828) of participants had unknown MMR immunization history. The total positive rates of measles, rubella and mumps IgG antibody were 76.78%, 64.46% and 64.29% and their GMCs were 541.45 mIU/ml, 31.76 IU/ml and 133.73 U/ml respectively. There were significant differences in serum IgG antibody GMC of measles, rubella and mumps in each year (Fmeasles=180.74, P<0.001; Frubella=189.95, P<0.001; Fmumps=122.40, P<0.001). The positive rate of measles antibody was higher than that of rubella and mumps, and the difference was statistically significant (χ²=518.09, P<0.001). Conclusion: The level of measles IgG antibody in healthy people in Shanghai is higher, while the level of rubella and mumps IgG antibody is slightly lower.

Independent and interactive effects of incremental heat strain, orthostatic stress, and mild hypohydration on cerebral perfusion.

The purpose of this study was to identify the dose-dependent effects of heat strain and orthostasis [via lower body negative pressure (LBNP)], with and without mild hypohydration, on systemic function and cerebral perfusion. Eleven men (means ± SD: 27 ± 7 y; body mass 77 ± 6 kg), resting supine in a water-perfused suit, underwent progressive passive heating [0.5°C increments in core temperature (Tc; esophageal to +2.0°C)] while euhydrated (EUH) or hypohydrated (HYPO; 1.5-2% body mass deficit). At each thermal state, mean cerebral artery blood velocity (MCAvmean; transcranial Doppler), partial pressure of end-tidal carbon dioxide ([Formula: see text]), heart rate (HR) and mean arterial blood pressure (MAP; photoplethysmography) were measured continuously during LBNP (0, -15, -30, and -45 mmHg). Four subjects became intolerant before +2.0°C Tc, unrelated to hydration status. Without LBNP, decreases in [Formula: see text] accounted fully for reductions in MCAvmean across all Tc. With LBNP at heat tolerance (+1.5 or +2.0°C), [Formula: see text] accounted for 69 ± 25% of the change in MCAvmean. The HYPO condition did not affect MCAvmean or any cardiovascular variables during combined LBNP and passive heat stress (all P > 0.13). These findings indicate that hypocapnia accounted fully for the reduction in MCAvmean when passively heat stressed in the absence of LBNP and for two- thirds of the reduction when at heat tolerance combined with LBNP. Furthermore, when elevations in Tc are matched, mild hypohydration does not influence cerebrovascular or cardiovascular responses to LBNP, even when stressed by a combination of hyperthermia and LBNP.

[Phenotype and mechanism of inducible ppp2r1a knockout mouse model].

Objective: Investigate the effects of inducible ppp2r1a knockout on main physiological function in adult mice and study the mechanism. Methods: Ppp2r1a(flox/flox) mice and CAGG-CreER mice were hybridized to obtain 20 CAGG-CreER ppp2r1a(flox/flox) and 20 mice in homozygous group. Two groups of mice were divided into 4 groups respectively, finally we got 8 groups with 5 mice in each group. Tamoxifen was injected intraperitoneally to acquire inducible ppp2r1a knockout mice. The knockout efficiency of PP2A Aα in vital organs was measured by Western blot. At 0, 2, 4 and 6 days after injection, we measured body weight, histopathological change, peripheral blood cell counts and blood biochemical. Real-time PCR was performed to measure expression of liver glucolipid metabolism genes. Results: After tamoxifen injection for 6 days, the knockout efficiency of PP2A Aα in vital organs was 35%, 12%, 15%, 60%, 69% and 72%, respectively in heart, liver, spleen, lung, kidney and brain. After tamoxifen injection for 6 days, the weight of homozygous mice was lower than that of wild type mice, with values of (17.42±1.76) g and (21.69±1.82) g, respectively (P<0.05). Moreover, the activity level, abdominal and renal fat were significantly decreased in homozygous mice. Homozygous mice survived no more than 7 days. Compared with wild type mice, the organ coefficient of spleen of homozygous mice was decreased at the 6th day, with values of (0.59±0.10)% and (0.36±0.05)% respectively (P<0.05). Obvious spleen atrophy and marked decrease of nucleated cells were showed by performing HE staining. Tunel staining revealed increased apoptosis ratio of splenic lymphocytes in homozygous mice. The levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) of homozygous mice were higher than wild type mice (P<0.05). The values of ALT and AST in homozygous mice were (153.68±62.80) U/L and (193.2±44.28) U/L. The corresponding values in wild type mice were (41.02±12.91) U/L and (69.40±9.55) U/L. The above results indicated that ppp2r1a knockout caused liver damage. Blood sugar level of homozygous mice was lower than in wild type mice (P<0.05), with values of (4.20±1.99) mmol/L and (8.88±0.65) mmol/L respectively. Plasma total cholesterol (TC), high density lipoprotein (HDL) and ß-hydroxybutyric acid (ß-HB) level of homozygous mice were higher than those of wild type mice (P<0.05). The values of TC, HDL and ß-HB in homozygous mice were (3.12±0.39), (1.53±0.38) and (2.49±0.89) mmol/L. The corresponding values in wild type mice were (1.69±0.92), (0.78±0.50) and (0.45±0.30) mmol/L respectively. The above results indicated that ppp2r1a loss interfered glucose and cholesterol metabolism. In addition, we also found that the white blood cell count (WBC) and lymphocyte count (LYM) of homozygous mice were lower than in wild type mice (P<0.05). The values of WBC and LYM in homozygous mice were (1.88±0.89)×10(9)/L and (0.92±0.37)×10(9)/L respectively. The corresponding values in wild type mice were (3.91±0.80)×10(9)/L and (2.74±0.52)×10(9)/L respectively. The mRNA levels of glucose-6-phosphatase (G6P) and phosphoenolpyruvate carboxykinase (PEPCK) of homozygous were lower than wild type mice (P<0.05). The fold change of G6P and PEPCK in homozygous mice was 0.46±0.11 and 0.72±0.07 respectively. The corresponding fold change in wild type mice was 1.02±0.07 and 1.02±0.06 respectively. Conclusion: Whole body ppp2r1a is essential for the survival of adult mice, due to the important role in maintaining the metabolism of glucose and cholesterol of liver.

[The relationship between histone H3Ser10 phosphorylation and DNA damage in periphery blood lymphocytes of polycyclic aromatic hydrocarbons exposed workers].

Objective: To investigate the effect of polycyclic aromatic hydrocarbons (PAHs) exposure on the level of histone H3Ser10 phosphorylation (p-H3S10) and DNA damage degree in peripheral blood lymphocyte (PBLCs). Method: 75 coke oven workers from Benxi steel plant in Liaoning Province of China (PAHs-exposed group) and local 50 hot rolling workers (control group) were recruited in this study with age, working years, labor intensity and high temperature for matching factors using cluster sampling method in 2014. HPLC-fluorescence was performed to determine the level of urinary 1-hydroxypyrene (1-OHP), DNA damage and specific histone modification were measured in PBLCs of the subjects through comet assay and ELISA assay, respectively. Linear regression model analysis was used to analyze the differences among PAHs exposure, DNA damage and p-H3S10 level in two groups. The Mediation analysis was used to analyze the regulated relationships between urinary 1-OHP, DNA damage and histone modification through the bootstrap method. Results: Age of the control and the exposed group were (45.32±8.32) and (43.87±5.67) years old (P=0.284). The concentration of urinary 1-OHP, OTM value, Tail DNA% and p-H3S10 level in exposure group were higher than that in control group, while the M (P(5)-P(95)) of p-H3S10 levels in control and exposed group were 2.21 (0.68-4.71), 4.54 (1.85-23.91) (P<0.001). The degree p-H3S10 level was increased after the subgroups which were (2.59±1.19)%, (3.24±2.81)%, (5.55±3.25)%, (8.77±7.84)%, respectively, divided by quantitated 1-OHP concentration as P(0)-P(25), P(26)-P(50), P(51)-P(75) and P(76)-P(100) (P<0.001). We also found the correlations between urinary 1-OHP and p-H3S10 level or OTM value or Tail DNA%, ß (95%CI) were 0.264 (0.167-0.360), 0.500 (0.299-0.702), and 0.510 (0.384-0.671), respectively (P<0.001). Similar result was also observed between p-H3S10 level and OTM value or Tail DNA%, ß (95%CI) were 0.149 (0.073-0.226) and 0.220 (0.132-0.308) (P<0.001). Moreover, the mediation effect value of DNA damage on PAHs induced p-H3S10 alteration was 0.054(P=0.040). Conclusion: The results suggested that PAHs exposure could induce DNA damage and an increase in histone H3Ser10 phosphorylation in PBLCs. Particularly, the alteration of H3S10 phosphorylation may play an important role in regulating cell DNA damage repair.

Atomic structure, tensile property, and dislocation behavior of Fe-W interfaces from molecular dynamics simulation.

Molecular dynamic simulations based on a recently constructed potential reveal that quasi-repeating patterns could appear in both Fe(110)/W(110) and W(110)/Fe(110) interfaces, and that three kinds of atomic displacements of Fe atoms because of the Fe-W interaction intrinsically bring about the interesting quasi-repeating patterns of the Fe-W interfaces. It is also found that the Fe-W interface becomes more brittle with less critical strains under tensile loading than pure Fe or W, which is fundamentally attributed to the movement of the interface dislocations as a result of the lattice mismatch between Fe and W. Interestingly, the dislocation loops could be formed in the Fe-W interface under tensile loading due to the pinning of the100edge dislocations by the edge dislocations of1/2111, whereas no dislocation loop would be generated in pure Fe or W.

MiR-126 on mice with coronary artery disease by targeting S1PR2.

OBJECTIVE: To screen the differentially expressed micro ribonucleic acids (miRNAs) in the serum of coronary atherosclerosis patients, and to investigate their possible mechanisms of action. PATIENTS AND METHODS: The differentially expressed serum miRNAs were screened from 3 coronary artery disease (CAD) patients and 3 healthy controls using miRNA expression profiles, which were verified using low-throughput quantitative Reverse Transcription-Polymerase Chain Reaction (RT-qPCR) assay. 60 apolipoprotein E (ApoE)-/- mice were divided into model group, agomir-126 group, agomir-control (con) group, and antagomir-126 group using a random number table. They were fed with high-fat diets (21% fat and 0.15% cholesterol) ad libitum for 15 weeks to establish the mouse model of CAD. Then, hematoxylin and eosin (HE) staining was applied to detect the impact of miR-126 expression level on the tissue morphology in the thoracic aortic region. The influences of miR-126 expression level on the secretion levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-10 were determined via enzyme-linked immunosorbent assay (ELISA). Western blotting assay was performed to examine the effects of miR-126 expression level on the expression levels of nuclear factor-kappa B (NF-κB) and vascular cell adhesion molecule-1 (VACM-1) in the tissues of the thoracic aortic region of the mice. The correlation between miR-126 expression level and sphingosine-1-phosphate receptor 2 (S1PR2) in the serum of CAD patients and animal models was analyzed by the Pearson correlation coefficient method. The targets of miR-126 were predicted using the bioinformatics method, and the direct targets were verified through investigations. Western blotting assay and ELISA were adopted to detect the impacts of miR-126 expression level on the expression and secretion levels of TNF-α, IL-1ß, and IL-10 in S1P + oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs). Lentivirus-small hairpin RNA (shRNA) was utilized to knock down the expression level of S1RP2 to determine whether miR-126 affected the increase in the inflammation level in S1P + ox-LDL-induced HUVECs by targeting S1RP2. RESULTS: Compared with those in control group, 4 miRNAs (miR-126, miR-206, miR-4297, and miR-3646) in the serum of CAD patients exhibited the most significant expression differences, which increased by 6.72, 7.11, 13.57, and 21.22 times, respectively. The verification results of low-throughput RT-qPCR assay indicated that there were remarkable changes in the expression levels of the 4 selected miRNAs with differential expressions in comparison with those in control group, displaying statistically significant differences (p<0.01). The results of HE staining manifested that the coronary atherosclerotic plaques were reduced markedly in agomir-126 group, while notably more coronary atherosclerotic plaques were formed in the thoracic aortic region in antagomir-126 group. Meanwhile, the elevated expression level of miR-126 evidently lowered the expressions of serum TNF-α and IL-1ß, but significantly increased the expression of IL-10 in the mouse model of CAD. According to the analysis results of the Pearson correlation coefficient method, the miR-126 expression level was negatively correlated with S1PR2 expression level in the serum of both CAD patients and animal models (r=-0.6123, r=-5.37). It was shown in bioinformatics prediction and luciferase reporter gene assay that miR-126 negatively regulated the S1PR2 expression by targeting the 3' untranslated region (UTR) of S1PR2 messenger RNA (mRNA). In the in vitro inflammation model, the increased expression level of miR-126 could relieve the inflammation in cells induced by S1P + ox-LDL. Based on the results of both Western blotting assay and ELISA, the differences in the expression and secretion levels of TNF-α, IL-1ß, and IL-10, as well as the expression levels of signaling molecules of the NF-κB signaling pathway, in the cells were not statistically significant among miR-126 mimic treatment group, sh-S1PR2 group, and miR-126 mimic + sh-S1PR2 group, indicating that miR-126 affects the inflammation level in HUVECs by targeting S1PR2. CONCLUSIONS: MiR-126 represses the progression of coronary atherosclerosis in the mice by binding to S1PR2. The results of this research may propose a new mechanism of miR-126 in exerting its therapeutic effects and possess potential value for the treatment of CAD in the future.

Strain-stress relationship and dislocation evolution of W-Cu bilayers from a constructed n-body W-Cu potential.

An n-body W-Cu potential is constructed under the framework of the embedded-atom method by means of a proposed function of the cross potential. This W-Cu potential is realistic to reproduce mechanical property and structural stability of WCu solid solutions within the entire composition range, and has better performances than the three W-Cu potentials already published in the literature. Based on this W-Cu potential, molecular dynamics simulation is conducted to reveal the mechanical property and dislocation evolution of the bilayer structure between pure W and W0.7Cu0.3 solid solution. It is found that the formation of the interface improves the strength of the W0.7Cu0.3 solid solutions along tensile loading perpendicular to the interface, as the interface impedes the evolution of the dislocation lines from the W0.7Cu0.3 solid solutions to the W part. Simulation also reveals that the interface has an important effect to significantly reduce the tensile strength and critical strain of W along the tensile loading parallel to the interface, which is intrinsically due to the slip of the edge or screw dislocations at low strains as a result of the lattice mismatch.

A fluorescent probe for H2S in vivo with fast response and high sensitivity.

In this work, we design and synthesize a new near-infrared (NIR) ratiometric fluorescent probe FD-H2S for the highly sensitive (DL 68.2 nM) detection of H2S with fast response (15 s), large emission shift (220 nm) and excellent enhancement (168-fold in ratiometric value). The probe could be applied for monitoring and imaging of exogenous or endogenous H2S in live MCF-7 cells and in live mice with the fastest response.

Recombinant desmoglein 3 has the necessary epitopes to adsorb and induce blister-causing antibodies.

The development of an animal model for studying the pathogenesis of pemphigus vulgaris (PV) has been hampered by the unavailability of the purified full-length autoantigen desmoglein 3 (Dsg 3).Therefore, we expressed Dsg 3 using a baculovirus expressed system. The expressed protein was identified as Dgs 3 by its reactivity with a pan-cadherin anti-serum, an anti-serum to a Dsg 3 synthetic peptide, or patient serum, and by amino-terminal sequencing. Carbohydrate analysis showed that recombinant Dsg 3 was glycosylated. While a majority of the recombinant protein was cell associated, by immunoprecipitation, some Dsg 3 was demonstrated in the medium. The Dgs 3 could adsorb out blister-causing antibodies from patient sera. Rabbit anti- Dsg 3 antibodies induced by the recombinant Dsg 3 showed specific binding to intercellular spaces of monkeys esophagus by indirect immunofluorescence. Moreover, these antibodies induced PV-like blisters in neonatal mice and weakly bound perilesional epidermis. Availability of large quantities of relatively pure Dsg 3 should now facilitate studies aimed at understanding Dsg 3 structure and pathogenesis of PV, with implications for developing specific immunotherapies.

Dual mechanism of perturbation of thyrotropin-mediated activation of thyroid cells by antibodies to the thyrotropin receptor (TSHR) and TSHR-derived peptides.

To further define the epitopes with which anti-TSH receptor (anti-TSHR) antibodies react and mediate their biological effects, we used antibodies against the extracellular domain of TSHR (ETSHR) protein and nine peptides derived from the ETSHR. Peptides were chosen based on their predicted immunogenicity as well as their uniqueness to the TSHR. Antipeptide antibodies showed varying degrees of reactivity against ETSHR, with antipeptide-2-(352-366) and -3A-(357-372) showing relatively stronger reactivity with the receptor. Antibodies were tested for their ability to stimulate thyroid cells and were found to be ineffective in causing both cAMP release and iodide uptake. However, anti-3A and anti-ETSHR showed blocking TSHR antibody (TSHRAb) activities of 76.9% and 79.7%, respectively, which were significantly different (P < 0.005) compared to that of preimmune serum. Anti-2 and -91 (AA 32-46) also showed blocking TSHRAb activities of 37.5% and 35.6%, respectively (P < 0.05). Antisera were also tested for their ability to block TSH binding to thyroid membranes in a RRA. Anti-ETSHR, but not any of the antipeptide antibodies, displayed TSH binding inhibitory immunoglobulin activity. These findings suggest that there might be different mechanisms that mediate blocking TSHR antibody activity. One mechanism involves the inhibition of TSH binding to the receptor, and the other probably involves a step subsequent to TSH binding.

Drug-induced in vitro tolerance to allogeneic antigens. II. Further analysis of in vitro-tolerized spleen cells in a fully allogeneic murine combination.

C3H/HeSlc (C3H, H-2k) spleen cells were made tolerant in vitro to C57BL/6CrSlc (B6, H-2b) at the cell-mediated cytotoxicity (CMC) level by in vitro stimulation for 48 hr with mitomycin C (MMC)-treated B6 spleen cells, and treatment with 5 micrograms/ml of 5-fluorouracil for a further 9 hr. These cells were given intraperitoneally to neonate (C3HxB6) F1 mice to examine whether these tolerized spleen cells would cause lethal graft-versus-host disease (GVHD). Despite the lack of CMC, the tolerized C3H spleen cells caused lethal GVHD in most of the neonate F1 mice. Evaluating from various immune parameters, it was evident that T cell populations responsible for IL-2 production, cytostasis, and delayed footpad reaction (DFR) were retained intact after in vitro tolerance induction, probably because of their less-proliferative characteristics in response to fully allogeneic antigen stimulation, and were considered to be responsible for lethal GVHD. Contribution of natural killer (NK) cells to lethal GVHD was not ruled out.

Drug-induced tolerance to allografts in mice. XII. The relationships between tolerance, chimerism, and graft-versus-host disease.

When AKR/J Sea (AKR, H-2k) mice were primed i.v. with 1 X 10(8) viable spleen cells from naive C3H/He Slc (C3H, H-2k) mice and treated i.p. with 200 mg/kg cyclophosphamide (CP) 2 days later, a minimal degree of mixed chimerism associated with tolerance to C3H skin was established without graft-versus-host disease (GVHD) and maintained for at least one month. When AKR mice were primed i.v. with 1 X 10(8) viable spleen cells from C3H mice preimmunized i.v. 7 days earlier with 5 X 10(7) viable AKR spleen cells, and treated with 200 mg/kg CP, chimerism became exclusive, but lethal GVHD occurred in the AKR mice. Moreover, most of normal AKR mice primed with the preimmunized C3H spleen cells without CP died of GVHD. In contrast, in a major histocompatibility complex (MHC)-incompatible combination of AKR (H-2k)-C57BL/6 Cr Slc (B6, H-2b), mixed chimerism, tolerance to skin allografts, and GVHD were not observed, whether or not the mice had been treated with naive or preimmunized B6 spleen cells with or without CP.

Drug-induced tolerance to allografts in mice. IX. Establishment of complete chimerism by allogeneic spleen cell transplantation from donors made tolerant to H-2-identical recipients.

Graft-versus-host reaction (GVH) after allogeneic spleen cell transplantation was completely suppressed in an H-2-matched murine combination (AKR/J Sea [H-2k]----lethally irradiated C3H/He Slc [H-2k]) by pretreatment of the donors with recipient spleen cell antigen plus cyclophosphamide (CP). Irradiated recipients receiving cells became chimeric. In contrast to the H-2 matched combination, lethal GVH reaction could not be prevented in an H-2-mismatched fully allogeneic combination (C57BL/6 Cr Slc [H-2b]----lethally irradiated C3H/He Slc [H-2k]) by pretreatment of the donors. The results suggest that the effectors responsible for the GVH reaction were abrogated by pretreatment of the donors with allogeneic recipient spleen cells plus CP in the H-2-matched combination, but donor pretreatment failed to abrogate GVH reaction in the H-2-mismatched combination.

Drug-induced tolerance to allografts in mice. X. Augmentation of split tolerance in murine combinations disparate at both H-2 and non-H-2 antigens by the use of spleen cells from donors preimmunized with recipient antigens.

In a fully allogeneic murine combination of C3H/HeSlc (C3H) (H-2k) and C57BL/6CrSlc (B6) (H-2b), C3H mice were primed i.v. with 1 X 10(8) spleen cells from B6 mice preimmunized i.v. with 5 X 10(7) C3H spleen cells and then were given i.p. 200 mg/kg cyclophosphamide (CP) 2 days later (Im-B6-Sc plus CP group). The tolerant state in those recipient mice was compared with that in mice made tolerant conventionally with 1 X 10(8) naive B6 spleen cells plus 200 mg/kg CP (naive-B6-Sc plus CP group). B6 skin was rejected in an almost normal fashion in both the naive-B6-Sc plus CP group and the Im-B6-Sc plus CP group. However, EL4 tumor allografts (B6 origin) inoculated after complete rejection of B6 skin grafts were specifically accepted in both groups. Moreover, the tumor growth in the Im-B6-Sc plus CP group was faster than that in the naive-B6-Sc plus CP group. Mixed lymphocyte reaction, cytotoxic T lymphocyte activity, and antibody production against the tolerogen were depressed more profoundly in the Im-B6-Sc plus CP group than in the naive-B6-Sc plus CP group. These observations were consistent with the results from tumor allografting. The other immunological parameters examined in the present study, including helper T cell activity and delayed foot-pad reaction, were retained in the Im-B6-Sc plus CP group at the same levels as in the naive-B6-Sc plus CP group. These observations were consistent with the results from skin allografting. In conclusion, tumor allograft tolerance was made more profound by the use of spleen cells from donors preimmunized with recipient antigens as the tolerogen than by the use of naive spleen cells. However, skin allograft tolerance was not achieved at all by these same treatments. The contribution of graft-versus-host disease to this phenomenon was excluded by the chimeric analysis in AKR/JSea (H-2k) mice given the preimmunized (with AKR antigens) B6 spleen cells plus CP. These results strongly support the existence of a less proliferative lymphocyte population which does not evoke cell divisions to mature even after the strong stimulation with the preimmunized spleen cells and is resistant to tolerance induction.

Influence of adjuvants on the induction of autoantibodies to the thyrotropin receptor.

To determine the influence of adjuvant on the induction of antibodies to thyrotropin receptor (TSHR), we immunized BALB/c mice with a extracellular domain of the TSHR (ETSHR) protein in complete Freund's adjuvant (CFA), Titer Max (TM) and Gerbu. Similarly, control groups of mice were immunized with bovine serum albumin (BSA) in each of the different adjuvants. As determined by ELISA, ETSHR given along with CFA elicited high titers of antibodies to ETSHR which were mainly restricted to the IgG1 subclass. Mice immunized with ETSHR in TM also developed high titers of anti-ETSHR antibodies but had higher levels of both IgG1 and IgG2a. However, immunization with ETSHR in Gerbu resulted in low titers of antibodies, restricted to IgG1 subclass. Immunization of mice with BSA in each of the three adjuvants induced higher antibody titers to BSA. The subclass of antibodies in mice immunized with BSA in CFA and TM were predominantly IgG1 and IgG2a with lower levels of IgG2b, whereas in Gerbu treated group, antibody to BSA was restricted to IgG1 subclass. Analysis of specificity of antibodies against ETSHR, in mice immunized with ETSHR, revealed that irrespective of the adjuvant used, the dominant reactivity was against peptide 1 (AA 22-41) with weaker reactivity against several other. peptides. The only exception was in mice immunized with ETSHR in TM which also showed significant reactivity against peptide 23 (AA 352-371). Mice immunized with the ETSHR in CFA or in TM showed elevated levels of serum TSH binding inhibitory immunoglobulins (TBII). However, mice immunized with ETSHR in Gerbu, which had lower titers of antibodies to ETSHR, showed normal TBII levels. These studies showed that adjuvant composition could influence the titer, subclass and fine specificity of antibodies to ETSHR which in turn could affect the development of TBII activity.

Analysis of autoantibody reactivity in patients with Graves' disease using recombinant extracellular domain of the human thyrotropin receptor and synthetic peptides.

Graves' disease is characterized by hyperthyroidism leading to enhanced production of thyroid hormones. Hyperthyroidism is primarily mediated by the binding of autoantibodies to the thyrotropin receptor (TSHr). In the past, either thyroid cells or thyroid membranes were used as a source of TSHr to detect anti-TSHr antibodies. Recently, we expressed the extracellular domain of the human TSHr (ETSHr) using the baculovirus expression system. In this study, we used ETSHr protein in an ELISA to detect anti-TSHr antibodies. Our data show that this assay can be used to analyze and quantitate isotype specific antibodies against the TSHr. To map immunogenic epitopes on the TSHr, we tested patients sera against synthetic peptides derived from two highly immunogenic regions (amino acid, AA 12-46 and 316-397) of the receptor. Although sera from patients with Graves' disease reacted with several peptides, they showed particularly strong reactivity against peptides from a relatively narrow region (i.e. AA 352-394) of the TSHr. The present study demonstrates the usefulness of the recombinant ETSHr to detect and characterize anti-TSHr antibodies in a simple and sensitive ELISA, and has lead to the identification of some of the immunoreactive epitopes on the TSHr.

Induction of hyperthyroxinemia in BALB/C but not in several other strains of mice.

We recently expressed the extracellular domain of the human TSHR (ETSHR) protein using a baculovirus expression system and purified it to homogeneity. The ETSHR specifically binds both TSH and antibodies to TSHR. In the present study, C57BL/6J, SJL/J, BALB/cJ and B10BR.SgSnJ mice were immunized with the recombinant ETSHR or an equivalent amount of control antigen. All strains of mice produced high titers of antibody against the TSHR protein which were capable of blocking the binding of TSH to native TSHR. However, only BALB/cJ mice showed significantly elevated levels of thyroxine in their sera compared to the control mice. Similarly, BALB/cJ mice primed with ETSHR and then challenged with thyroid membranes showed significantly elevated levels of thyroxine. In addition, histopathological examination of thyroid glands from affected mice showed morphological changes characterized by hydropic and subnuclear vacuolar changes and focal scalloping, with no apparent inflammation or glandular destruction. Moreover, mice with elevated thyroxine levels showed increased in vivo thyroidal uptake of 131Iodine. Together, these data suggest that BALB/cJ mice are susceptible to the induction of hyperthyroxinemia.

Experimental autoimmunity to thyrotropin receptor.

Since the cloning of a full length cDNA encoding the thyrotropin receptor (TSHr), several laboratories have been actively trying to develop an optimal animal model to understand the pathogenesis of TSHr mediated autoimmune diseases and have made considerable progress. To date, results from our laboratory have indicated that the nature of the antigen, and the adjuvant used for immunization, immunogenetic background of the animal and fine specificities of antibodies elicited might play an important role in determining the qualitative nature of the antibody response. Although an ideal animal model for either Graves' disease or primary myxedema is not yet available, ongoing studies in our laboratory and elsewhere hold promise for establishing animal models for various TSHr mediated autoimmune diseases in the near future.

Abnormalities in platelets and vascular endothelial cells induced by glycated lipoproteins.

We studied the effects of glycated lipoproteins of low- and high-density (LDL and HDL) on platelets and vascular endothelial cells. After pretreatment for 5 minutes at 37 degrees C, the thrombin-induced synthesis of thromboxane B2 in washed platelets was significantly increased by glycated LDL as compared with native LDL (198.9 +/- 16.2 vs 90.3 +/- 29.4 ng/10(9) platelets, n = 8, p less than 0.01). Platelet aggregation was also increased by glycated LDL as compared with native LDL. After treatment with platelet-rich plasma for 5 hours at 37 degrees C, these values were suppressed by native HDL vs the control (buffer), but not by glycated HDL. Abnormalities in the release of 6-keto prostaglandin F1 alpha and lactate dehydrogenase from vascular endothelial cells were also induced by glycated LDL and/or HDL. These observations suggest that abnormalities induced in platelets and vascular endothelial cells by glycated lipoproteins may play an important role in the development of atherosclerosis in patients with diabetes mellitus.

[Establishment of the vascular intimal model in vitro and its application in studying atherosclerosis].

An endothelial culture model in vitro mimicking the vascular intima in vivo was designed which was composed of an upper and a lower well separated by a layer of amnion membrane, and upon which endothelial cells (ECs) isolated from the human umbilical vein were cultured. The upper well, the subendothelial amnion, and the lower well were analogical to the vascular lumen, the subendothelial tissue and the extravascular space, respectively. In comparison with the ECs cultured on the plastic, dishes, ECs cultured on the amnion membrane maintained more morphologic features as in vivo and could be cultured for up to 15 days without apparent detachment. Monocytes, loaded in the upper wells, were able to adhere to the cytoplasmic membrane of ECs. Furthermore, with the presence of the chemotactic factor (fMLP) in the lower well, monocytes showed active migrating ability passing through the EC junctions. If LDL (100 micrograms/ml) was added in the media simultaneously, monocytes might be aggregated beneath the subendothelial space and some of them took an foamy appearance. In conclusion, the culture model is of value in the study of experimental inflammation and atherosclerosis.