Antimicrobial photodynamic therapy using a diode laser with a potential new photosensitizer, indocyanine green-loaded nanospheres, may be effective for the clearance of Porphyromonas gingivalis.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) is a new treatment method for the removal of infectious pathogens using a photosensitizer and light of a specific wavelength, e.g., toluidine blue with a wavelength of about 600 nm. We explored a new photosensitizer and focused on indocyanine green (ICG), which has high absorption at a wavelength of 800-805 nm. We investigated the bactericidal effect of PDT on Porphyromonas gingivalis using a new photosensitizer, ICG-loaded nanospheres with an 805 nm wavelength low-level diode laser irradiation. METHODS: We designed ICG-loaded nanospheres coated with chitosan (ICG-Nano/c) as a photosensitizer. A solution containing Porphyromonas gingivalis (10(8)  CFU/mL) with or without ICG-Nano/c (or ICG) was prepared and irradiated with a diode laser or without laser irradiation as a negative control. The irradiation settings were 0.5 W with a duty ratio of 10%, for 3-100 ms in repeated pulse (RPT) or continuous wave mode. CFU were counted after 7 d of anaerobic culture. RESULTS: We observed that ICG-Nano/c could adhere to the surface of P. gingivalis. When ICG-Nano/c was used for aPDT, irradiation with RPT 100 ms mode gave the lowest increase in temperature. Laser irradiation with ICG-Nano/c significantly reduced the number of P. gingivalis (i.e., approximately 2-log10 bacterial killing). The greatest bactericidal effect was found in the RPT 100 ms group. However, laser irradiation (RPT 100 ms) with ICG, as well as without photosensitizer, had no effect on the number of bacteria. CONCLUSIONS: Within the limits of this study, ICG-Nano/c with low-level diode laser (0.5 W; 805 nm) irradiation showed an aPDT-like effect, which might be useful for a potential photodynamic periodontal therapy.

Long-term survival of a patient with amyotrophic lateral sclerosis (ALS) who received autologous adipose-derived mesenchymal stem cells.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a progressive debilitating neurodegenerative disease with a life expectancy of 3-5 years from initial symptoms. We report a case of ALS who received autologous adipose-derived mesenchymal stem cells (ADSC) and was followed up for 7 years. CASE REPORT: A 46-year-old man noticed weakness of his legs, difficulties on going down the stairs and coughing during eating in 2009. After complete workout, a diagnosis of ALS was confirmed. His ALS Functional Rating Scale-R (ALSFS-R) was 43. Symptoms rapidly progressed and he coughed and choked during eating. Starting in 2013, the patient received a total of six intravenous infusions of autologous ADSC. Changes in electromyogram, nerve conduction, and ALSFS-R were assessed. RESULTS: Soon after the administration, he noticed that he did not cough during conversation or eating food. Although he had difficulty in walking down the stairs, he remained well without coughing, dysarthria, or dysphagia. His ALSFS-R increased up to 45. Fascicular potentials were not detected in any muscles examined including trapezius muscle and rectus femoris muscles. The patient was well for 7 years after ADSC therapy by the time of this report and more than 10 years from the time of onset. CONCLUSIONS: The present case suggests that autologous ADSC can be administered safely and may be potentially useful in patients with ALS. Further investigations are warranted in order for the results to be generalized to other ALS patients.

Improving the electron spin properties of nitrogen-vacancy centres in nanodiamonds by near-field etching.

The nitrogen-vacancy (NV) centre in diamond is a promising candidate for quantum computing applications and magnetic sensing applications, because it is an atomic-scale defect with stable coherence time (T2) and reliable accessibility at room temperature. We demonstrated a method for improving the NV spin properties (the full width half maximum (FWHM) value of the magnetic resonance spectrum and T2) through a near-field (NF) etching method under ambient conditions. The NF etching method, based on a He-Cd ultraviolet laser (325 nm), which is longer than the absorption edge of the oxygen molecule, enabled selective removal of defects on the nanodiamond surface. We observed a decrease in the FWHM value close to 15% and an increase in T2 close to 25%. Since our technique can be easily reproduced, a wide range of NV centre applications could be improved, especially magnetic sensing applications. Our results are especially attractive, because they have been obtained under ambient conditions and only require a light source with wavelength slightly above the O2 absorption edge.

Regulation of sterol carrier protein 2 (SCP2) gene expression in rat peritoneal macrophages during foam cell formation. A key role for free cholesterol content.

Sterol carrier protein 2 (SCP2) has been shown to be involved in intracellular transport and metabolism of cholesterol. However, there have been no reports concerning SCP2 in macrophages, the major source of atheromatous foam cells. We investigated whether SCP2 is present in rat peritoneal macrophages and determined the changes of SCP2 and its mRNA levels in macrophages during form cell formation induced by acetylated LDL (AcLDL). Immunoblot analysis and Northern blot analysis demonstrated that both SCP2 and its mRNA are expressed in rat peritoneal macrophages. Incubations with AcLDL caused a dose- and time-dependent increase of cellular esterified cholesterol, SCP2 and its mRNA in rat peritoneal macrophages. The inhibitor of acyl-CoA:cholesterol acyltransferase further enhanced AcLDL-induced increase of SCP2 protein and its mRNA. Incubations with 25-hydroxy cholesterol also caused a dose-dependent stimulation of SCP2 gene expression in macrophages, while incubation with maleylated BSA had no effect. These results suggest that the increment of cellular-free cholesterol is responsible for enhanced SCP2 gene expression in macrophages. The enhancement of SCP2 gene expression by AcLDL suggests that SCP2 may play an important role during foam cell formation induced by AcLDL which may be most important step for the atherosclerosis.

Clinical and epidemiological studies of eicosapentaenoic acid (EPA) in Japan.

From 1950 to 1980, the gross alteration in dietary habit in Japan was noted. Intake of total calories has markedly increased. This could be most likely due to a remarkable increase in intake of fat, especially animal fat, egg and milk products. A marked decrease of mortality rate due to cerebral hemorrhage and in contrast a marked increase of mortality rate due to cerebral infarction and ischemic heart disease were noted. An epidemiological study of the intake of fish meat (EPA intake) and the mortality rate of adult diseases was performed in a fishing area and in a farming area in Chiba Prefecture. Intake of fish meat (EPA) by the residents of the fishing area was 2-3 times higher than by the residents of the farming area. The mortality rate due to ischemic heart disease and cerebral vascular diseases tended to be lower in the fishing area than in the farming area. EPA manufactured from sardine oil was orally given to normal subjects and to patients with cerebro- and cardiovascular diseases for 4-16 weeks. Significantly decreased platelet aggregation, decreased platelet retention, lowered whole blood viscosity, prolonged bleeding time, increased erythrocyte deformability, improvement of hyperlipidemia, and clinical improvement in some patients were noted. 12-Lipoxygenase metabolites of EPA (12-HPEPE) and arachidonic acid (12-HPETE) have an equipotent inhibitory action on platelet function.

Acute rejection in allogeneic rodent small bowel transplantation causes smooth muscle dysfunction via an inflammatory response within the intestinal muscularis.

INTRODUCTION: Isogeneic intestinal transplantation elicits an inflammatory response within the intestinal muscularis that is associated with dysmotility. Usually the inflammation and the postoperative motor dysfunction resolve within a few days after small bowel transplantation (SBTx). However, the onset of acute rejection in allogeneic SBTx is again associated with dysmotility. We hypothesized that dysmotility during acute rejection is based on coexpression of kinetically active mediators by the alloreactive leucocyte infiltrate. MATERIALS AND METHODS: Rat SBTx (BN to Lew and BN to BN) was performed without immunosuppression. Animals were sacrificed at 1, 4, and 7 days after SBTx. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry. Mediator mRNA expression was determined by reverse transcriptase polymerase chain reaction. Muscle contractility was assessed in a standard organ bath. RESULTS: Transplanted animals showed a significant inflammatory response within the muscularis at day 1 after SBTx. However, allogeneic transplanted animals exhibited a significant second inflammatory peak at day 7 (mRNA induction: iNOS 150-fold; tumor necrosis factor-alpha 18-fold; interferon-gamma 397-fold), parallel to the onset of rejection. This change was associated with a significant leukocyte infiltration. Compared to controls, allogeneic transplanted animals showed a 29% decrease in smooth muscle contractility on days 1 and 4 and a 71% decrease of contractility on postoperative day 7. CONCLUSIONS: The motor dysfunction of transplanted small bowel during acute rejection is associated with an inflammatory reaction in the intestinal muscularis. The initial unspecific inflammation process immediately after transplantation is reactivated and intensified during acute rejection.

Mechanism and impact of organ harvesting and ischemia-reperfusion injury within the graft muscularis in rat small bowel transplantation.

INTRODUCTION: Inflammatory events within the gut muscularis contribute to dysmotility. We hypothesized that manipulation during organ harvesting initiated an inflammatory response via muscularis macrophages and that this cascade was amplified during reperfusion. METHODS: Small bowel transplantation was performed in Lewis rats. To investigate the impact of organ harvesting on muscularis inflammation, cold whole-body perfusion was performed after versus prior to organ harvesting. The role of macrophages was investigated by transplantation of the macrophage-depleted gut. Leukocyte infiltration was investigated in muscularis whole mounts. Mediator mRNA expression was determined by real-time reverse transcriptase polymerase chain reaction. Contractility was assessed in a standard organ bath. RESULTS: Organ harvesting and ischemia-reperfusion induced leukocyte recruitment and mRNA upregulation in the muscularis: interleukin-6 12217-fold, monocyte chemoattractant protein-1 62-fold, ICAM-1 12-fold, cyclooxygenase-2: 8-fold, iNOS: 150-fold. Although organ harvesting with cold ischemia prevented early gene expression, peak expression at 3-hour reperfusion was not changed by modification of the harvesting technique. Compared to controls, transplanted animals showed a 63% decrease in smooth muscle contractility. In contrast, transplanted macrophage-depleted gut exhibited significantly fewer leukocytes and only a 16% decrease in contractility. CONCLUSIONS: Gut manipulation during organ harvesting initiates an inflammatory response within the muscularis that is massively intensified during reperfusion. This change contributes to muscular dysfunction. Furthermore, the results suggested that resident macrophages play a key role in initiating this process.

Induction of antitumor cytotoxic T lymphocytes with a MAGE-3-encoded synthetic peptide presented by human leukocytes antigen-A24.

For the development of immunotherapy using MAGE peptides, the identification of additional tumor antigens is required. Because HLA-A24 is the most common allele in Japanese and is also frequently present in Caucasians, MAGE-3-encoded synthetic peptides with binding affinity for HLA-A24 were thus tested for the induction of specific CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA-A24 healthy donors using a simplified method. By using a peptide with a sequence of IMPKAGLLI (amino acid position in MAGE-3 195-203), the CTL responses could thus be induced from unseparated PBMCs by stimulation with freshly isolated, peptide-pulsed PBMCs as antigen-presenting cells (APCs) and by also using interleukin 7 and keyhole limpet hemocyanin for a primary culture. The induced CTLs could lyse HLA-A24 carcinoma cells expressing MAGE-3, as well as the peptide-pulsed target cells, in an HLA class-I restricted manner. The identification of the MAGE-3/HLA-A24 peptide, IMPKAGLLI, may thus potentially offer the opportunities to design peptide-based immunotherapeutic approaches that might prove to be effective in treating patients with MAGE-3-positive malignant tumors.

MR Imaging Findings of a Leiomyosarcoma of the Thoracic Spine: A Case Report.

We report a case of leiomyosarcoma of the thoracic spine. Primary leiomyosarcoma is a malignant connective tissue tumor originating from smooth muscle cells. Leiomyosarcoma frequently occurs in the uterus, retroperitoneal space, gastrointestinal tract, and deep soft tissues; primary leiomyosarcoma of the bone is rare. The MR imaging including intravoxel incoherent motion (IVIM) imaging findings of the current case indicated a low diffusion coefficient and high blood flow, which were in concurrence with high cell density on histology and increased vascularity by angiography. Although some benign tumors such as osteoblastoma and giant cell tumor would show similar findings on IVIM imaging, these additional imaging features may narrow the differential diagnosis of spinal tumors.

DeltaFosB, but not FosB, induces delayed apoptosis independent of cell proliferation in the Rat1a embryo cell line.

The fates of Rat1a cells expressing FosB and DeltaFosB as fusion proteins (ER-FosB, ER-DeltaFosB) with the ligand binding domain of human estrogen receptor were examined. The binding of estrogen to the fusion proteins resulted in their nuclear translocation and triggered cell proliferation, and thereafter delayed cell death was observed only in cells expressing ER-DeltaFosB. The proliferation of Rat1a cells, but not cell death triggered by ER-DeltaFosB, was completely abolished by butyrolactone I, an inhibitor of cycline-dependent kinases, and was partly suppressed by antisense oligonucleotides against galectin-1, whose expression is induced after estrogen administration. The cell death was accompanied by the activation of caspase-3 and -9, the fragmentation of the nuclear genome and cytochrome c release from the mitochondria, and was suppressed by zDEVD-fmk and zLEHD-fmk but not zIETD-fmk. The cell death was not suppressed by exogenous His-PTD-Bcl-x(L) at all, suggesting involvement of a Bcl-x(L)-resistant pathway for cytochrome c release.

Identification of a MAGE-2-encoded human leukocyte antigen-A24-binding synthetic peptide that induces specific antitumor cytotoxic T lymphocytes.

Because MAGE-2 gene is expressed in a wide variety of malignant tumors and HLA-A24 is the most common allele in the Japanese population and is also frequently present in Caucasians, the identification of MAGE-2-encoded peptide presented by HLA-A24 is, therefore, considered to be important in order to develop specific immunotherapy for malignant tumors using peptides as a vaccine. By using a MHC-binding assay, eight peptides derived from MAGE-2 were found to bind with sufficient affinity to the HLA-A24 molecule. When the induction of specific cytotoxic T lymphocytes (CTLs) was examined using a simplified method, the highest human lymphocyte antigen (HLA) binder (EYLQLVFGI) in these peptides was able to elicit CTLs from unseparated peripheral blood mononuclear cells in HLA-A24 healthy donors by stimulation with freshly isolated, peptide-pulsed peripheral blood mononuclear cells as antigen-presenting cells and also by using interleukin 7 and keyhole-limpet hemocyanin in a primary culture. The induced CTL could, thus, lyse HLA-A24 tumor cells expressing MAGE-2, as well as the peptide-pulsed target cells, with antigen specificity in a HLA class I-restricted manner. The identification of this peptide may, thus, be of therapeutic value in peptide-based vaccines for the treatment of several types of malignant tumors expressing MAGE-2.

Dendritic cell vaccination with MAGE peptide is a novel therapeutic approach for gastrointestinal carcinomas.

The MAGE gene is selectively expressed in cancer tissues such as melanoma or gastrointestinal carcinomas, whereas no expression is observed in normal tissues except testis. There are several reports of successful induction of HLA class I-restricted antitumor CTLs using MAGE peptides, and some clinical trials with these immunogenic peptides were reported as effective for some patients with malignant melanoma. However, there are no similar studies in gastrointestinal carcinomas, which are important neoplasms. Autologous dendritic cells (DCs) were generated ex vivo and were pulsed with MAGE-3 peptide, depending on the patient's HLA haplotype (HLA-A2 or A24). Patients were immunized with DC pulsed with MAGE-3 peptide every 3 weeks at four times. Twelve patients with advanced gastrointestinal carcinoma (six stomach, three esophagus, and three colon) were treated, and no toxic side effects were observed. Peptide-specific CTL responses after vaccination were observed in four of eight patients. Improvement in performance status was recognized in four patients. Tumor markers decreased in seven patients. In addition, minor tumor regressions evidenced by imaging studies were seen in three patients. These results suggested that DC vaccination with MAGE-3 peptide is a safe and promising approach in the treatment of gastrointestinal carcinomas.

Repeatedly passed FRTL-5 rat thyroid cells can develop insulin and insulin-like growth factor-I-sensitive cyclooxygenase and prostaglandin E2 isomerase-like activities together with altered basal and thyrotropin-responsive thymidine incorporation into DNA.

Repeatedly passed or aged rat FRTL-5 thyroid cells develop a high level of basal [3H]thymidine incorporation into DNA and a reduced response to TSH in medium containing 5% serum and insulin (5H medium). The basal [3H]thymidine incorporation into DNA of aged cells can exceed the TSH-induced increase in earlier passages of the same cell line (fresh cells) and the TSH response decreases from more than 10-fold above basal in fresh cells to less than 2-fold in aged cells. This change is not associated with a loss of the diploid karyotype, a change in basal cAMP levels, or a change in dependence on TSH for cell growth. Attenuation of the TSH response in the [3H]thymidine incorporation assay is more evident than the reduced effect of TSH on cAMP levels or iodide transport; moreover, the TSH effect on cAMP levels does not correlate with that on [3H] thymidine incorporation as a function of hormone concentration. The high basal activity in [3H]thymidine incorporation into DNA in aged cells is due to an increased responsiveness to insulin, insulin-like growth factor-I (IGF-I), or serum. Thus, removal of serum and insulin from the medium eliminates the high basal [3H]thymidine incorporation into DNA, and this activity is restored by insulin or IGF-I in a concentration-dependent manner. The increased responsiveness of aged cells to insulin or IGF-I is inhibited by indomethacin or hydrocortisone and is associated with insulin or IGF-I, but not TSH, stimulation of cyclooxygenase and prostaglandin E2 (PGE2) isomerase-like activity. Fresh cells, in contrast, require TSH plus insulin or IGF-I to increase these activities. Increased responsiveness of cyclooxygenase activity to insulin or IGF-I in aged cells reflects at least in part an increase in cyclooxygenase mRNA levels. We suggest that insulin/IGF-I stimulation of PGE2 production leads to the high basal thymidine incorporation into DNA in aged cells maintained in TSH-depleted (5H) medium; the reduced stimulation by TSH of cAMP content or iodide uptake may reflect PG inhibition (negative feedback regulation) of cAMP production.

Thyrotropin (TSH) receptor antibodies (TSHrAb) can inhibit TSH-mediated cyclic adenosine 3',5'- monophosphate production in thyroid cells by either blocking TSH binding or affecting a step subsequent to TSH binding.

In the present study, rabbit antibodies that possess thyroid stimulation-blocking activity were used to investigate potential mechanisms by which TSH receptor antibodies can inhibit thyroid cell function. The antibodies were produced against two synthetic peptides corresponding to amino acids 357-372 (p357) and 367-386 (p367) of the human TSHr (hTSHr). By enzyme-linked immunosorbent assay, both antisera (alpha 357 and alpha 367) had high titers ( > 1:100,000) of IgG against their respective peptides and recombinant extracellular TSHr protein (ETSHr); alpha 357 had a low IgG titer to p367 (1:800), and alpha 367 had a low IgG titer to p357 ( < 1:200). Based on competitive inhibition studies, alpha 357 and alpha 367 displayed similar relative binding affinities for their respective peptides and for recombinant ETSHr. When tested by commercial RRA, alpha 357 did not block (TSH binding inhibition index, -3.7%), whereas alpha 367 blocked TSH binding to TSHr (TSH binding inhibition index, 53.9%). The blocking effect of alpha 367 could be reversed by incubating the antiserum with p367 before assay. When applied alone to FRTL-5 cells, IgG from alpha 357 inhibited [compared to normal rabbit IgG (NRI); P < 0.01] based cAMP production by the cells, whereas IgG from alpha 367 did not. IgG from both alpha 357 and alpha 367, however, were able to inhibit (P < 0.001) TSH-mediated cAMP production by FRTL-5 cells [bovine (b) TSH, 2.5 x 10(-10) M; cAMP (mean +/- SD; picomoles per ml): NRI, 62.5 +/- 6.1; alpha 357, 12.2 +/- 2.4; alpha 367, 36.2 +/- 3.5]. Alpha 357 continued to inhibit (P < 0.05) cAMP production by FRTL-5 cells in 10(-8) M bTSH, whereas alpha 367 no longer inhibited cAMP production at bTSH concentrations above 5 x 10(-10) M. Compared to NRI, both alpha 357 and alpha 367 were also able to inhibit (P < 0.001) Graves' IgG-mediated cAMP production by FRTL-5 cells. When IgG were tested on FRTL-5 cells in the presence of 10(-7) M forskolin, only alpha 357 inhibited (P < 0.001) cAMP production (NRI, 75.1 +/- 4.8; alpha 357, 52.3 +/- 4.5; alpha 367, 77.2 +/- 1.4). To determine whether the inhibitory effect of alpha 357 on forskolin-mediated stimulation was thyroid cell dependent, IgG were tested on Chinese hamster ovary (CHO) cells transfected with the complementary DNA of the hTSHr (CHO-R). Again, alpha 357 inhibited (P < 0.005) cAMP production mediated by forskolin (at 10(-7) M; NRI, 68.7 +/- 4.4; alpha 357, 36.8 +/- 5.7; alpha 367, 64.6 +/- 8.5). alpha 357 did not inhibit forskolin-mediated cAMP production by untransfected CHO cells (CHO-N), indicating that the inhibitory effect of alpha 357 on forskolin stimulation was TSHr dependent. In addition, alpha 357 inhibited (P < 0.01) basal cAMP production by CHO-R cells, but not by CHO-N cells. alpha 367 had no effect on the basal cAMP production in either CHO-R or CHO-N cells. Neither alpha 357 nor alpha 367 inhibited cholera toxin-mediated cAMP production in FRTL-5 cells. In all relevant bioassays, the inhibitory effects of alpha 357 and alpha 367 could be reversed by preincubating the IgG with the respective peptides. From these data, we conclude that 1) alpha 367 binds to the ETSHr and blocks TSH-mediated cAMP production by inhibiting TSH from binding to its receptor; 2) alpha 357 binds to the TSHr and, without blocking TSH binding, inhibits TSH-mediated cAMP production at a step(s) subsequent to ligand binding that affects adenylate cyclase activity; and 3) forskolin-mediated cAMP production by thyroid cells can be inhibited by IgG that bind directly to the TSHr.

The formation of thyrotropin receptor (TSHR) antibodies in a Graves' animal model requires the N-terminal segment of the TSHR extracellular domain.

Immunization of AKR/N mice with murine fibroblasts, transfected with the TSH receptor (TSHR) and a murine major histocompatibility complex class II molecule having the same H-2k haplotype (but not either alone), induces immune thyroid disease with the humoral and histological features of human Graves', including the presence of two different TSHR antibodies (TSHRAbs): stimulating TSHRAbs, which cause hyperthyroidism; and TSH-binding-inhibiting immunoglobulins. The primary functional epitope for both types of antibodies in Graves' patients is on the N-terminal portion of the extracellular domain of the TSHR, residues 25 to 165; most require residues 90-165 to express TSHRAb activity, as evidenced in studies using chimeras of the TSHR and lutropin-choriogonadotropin receptor (LH-CGR). To evaluate the role of this region of the TSHR in the formation of Graves' TSHRAbs, we immunized AKR/N mice with fibroblasts transfected with three human TSHR chimeras with residues 9-165 (Mc1+2), 90-165 (Mc2), or 261-370 (Mc4) substituted by equivalent residues of the rat LH-CGR. Mice immunized with the Mc1+2 and Mc2 chimeras, with the N-terminal portion of the extracellular domain of the TSHR substituted by LH-CGR residues, did not develop TSHRAbs. Mice immunized with the Mc4 chimera, having a major portion of the C-terminal portion of the extracellular domain of the TSHR replaced by comparable LH-CGR residues, can develop TSHRAbs. The results suggest that the N-terminal segment of the TSHR extracellular domain is not only a critical functional epitope for Graves' TSHRAbs, but it is important also in their formation in a mouse model of Graves' disease.

Thyrotropin induces G1 cyclin expression and accelerates G1 phase after insulin-like growth factor I stimulation in FRTL-5 cells.

We have investigated the mechanism by which TSH pretreatment potentiates insulin-like growth factor I (IGF-I)-induced DNA synthesis in FRTL-5 cells. As previously described, pretreatment with TSH increased IGF-I-induced DNA synthesis, suggesting that the effect of TSH is mediated through the cAMP pathway. TSH and A kinase activators required at least 12 h to precondition cells to respond to IGF-I stimulation. The presence of cycloheximide abolished the effect of TSH to increase IGF-I-induced DNA synthesis. When the time course of thymidine uptake after IGF-I addition was studied, TSH pretreatment increased the maximum DNA incorporation and shortened the G1 phase interval. These results indicated that some proteins induced by TSH are required for the effect of TSH on IGF-I activity, and the proteins are important for cell cycle progression. Cyclins are key regulators of the cell cycle; therefore, we investigated the expression of cyclins D1 and E after TSH stimulation. TSH- and A kinase-activating agents increased the expression of cyclins D1 and E after 24 h. The same amounts of cyclins D1 and E induced by IGF-I were increased after TSH pretreatment. TSH pretreatment induced the expression of G1 cyclin in FRTL-5 cells, and IGF-I caused the accumulation of enough G1 cyclins to drive the cell cycle from G1 to S phase in a short time, which accounts for the effect of TSH on IGF-I induced DNA synthesis.

Thyrotropin receptor epitopes recognized by graves' autoantibodies developing under immunosuppressive therapy.

Abnormal modulation of the immune system is a prerequisite for the expression of Graves' disease. Thus, when hyperthyroidism developed in a renal transplant recipient under long term immunosuppression with cyclosporine A and prednisone, we carefully evaluated the basis for her hyperthyroidism and her state of immunosuppression. Immunosuppression was confirmed by finding markedly deficient lymphocyte responses to common mitogens. Lymphocyte phenotype frequencies were those previously found in Graves', i.e. elevated frequencies of CD3/DR, CD5/26, and CD3/25 lymphocytes. There was also reversal of the CD4/CD8 ratio due to increased CD8 frequency; this is not a typical finding in autoimmune hyperthyroidism, but has been seen in the intrathyroidal lymphocyte populations of some Graves' patients and is associated with other forms of autoimmunity. The patient's serum contained a broad spectrum of TSH receptor autoantibodies (TSHRAbs) characteristic of Graves' disease. To determine whether these were an unusual population of autoantibodies, we determined their functional epitopes before and for nearly 1 yr after radioiodine therapy. Stimulating TSHRAbs that increase cAMP levels were human receptor (TSHR) specific and consistently recognized functional epitopes located on TSHR residues 90-165. Stimulating TSHRAbs that increased arachidonate release and inositol phosphate levels recognized residues 25-90, as did TSH binding inhibitory Igs present in the patient. These data demonstrate that Graves' disease with a wide array of TSHRAbs can develop in a patient despite adequate immunosuppression. More importantly, they show that the cAMP-stimulating TSHRAb associated with disease expression in this patient had a homogeneous subtype dependent on TSHR residues 90-165. As persistence of this type of TSHRAb over time has been associated with resistance to methimazole therapy in Graves' patients, we speculate that the development and persistence of TSHRAb with this homogeneous epitope may be linked to resistance to immunosuppressive therapy.

Hashimoto's thyroiditis with heterogeneous antithyrotropin receptor antibodies: unique epitopes may contribute to the regulation of thyroid function by the antibodies.

Blocking-type TSH-binding inhibitor Igs (TBIIs) are known to cause hypothyroidism and an atrophic thyroid gland in patients with primary myxedema. They can block the activity of thyroid-stimulating antibodies (TSAbs) in Graves' patients as well as the activity of TSH. The majority of the epitopes for these blocking-type TBIIs have been, and are shown herein, to be present on the C-terminal region of the extracellular domain of the human TSH receptor (TSHR), whereas those for Graves' TSAbs are on the N-terminus. We report on a patient with Hashimoto's thyroiditis who suffered from mild hypothyroidism and a moderately sized goiter. Her serum had a potent blocking-type TBII and a weak TSAb in human and porcine TSHR systems. Using human TSHR/lutropin-CG receptor chimeras, we determined that the functional epitope of her blocking-type TBII was uniquely present on the N-terminal, rather than the C-terminal, region of the extracellular domain of the TSHR, unlike the case for blocking-type TBIIs in primary myxedema patients. The epitope of her TSAb was also unusual. Although the functional epitopes of most TSAbs are known to involve the N-terminal region of the receptor, her TSAb epitope did not seem to be present solely on the N- or C-terminus of the extracellular domain of the receptor. Blocking-type TBIIs from patients with primary myxedema blocked her TSAb activity as well as stimulation by TSH; her blocking-type TBII was able to only partially block her TSAb. In contrast, her blocking-type TBII almost completely blocked TSAbs from Graves' patients. Thus, we suggest that the unique epitopes of this patient's heterogeneous population of TSH receptor antibodies, at least in part, contribute to regulation of her thyroid function.

Epitopes for thyroid-stimulating antibodies in Graves' sera: a possible link of heterogeneity to differences in response to antithyroid drug treatment.

To evaluate the extent and clinical relevance of epitope heterogeneity for stimulating TSH receptor antibodies (TSHRAbs), we measured the activity of IgG preparations from 66 untreated patients with Graves' disease using Chinese hamster ovary (CHO) cells transfected with wild-type human TSHR and two TSHR chimeras with residues 9-165 (Mc1 + 2) or 90-165 (Mc2) substituted by equivalent residues of the LH/CG receptor. IgG from 68% of patients lose all of the stimulating TSHRAb activity with the chimeras; IgG from 27% lose most of the activity. Thus, we show that 95% of patients have stimulating TSHRAbs that require epitopes on the N-terminal portion of the extracellular domain of the TSHR and demonstrate the importance of epitopes within residues 90-165 for the first time. Heterogeneous epitope distribution, residual activity with one or both chimeras, i.e. with epitopes other than on the N-terminus of the TSHR, occurred in 21 patients (group A). Forty-five patients with homogeneous epitope distribution (group B) had stimulating TSHRAbs that depended only on epitopes on the N-terminus of the TSHR. Patients in group A were more likely to become euthyroid during antithyroid drug therapy and to do so more quickly than group B patients. The CHO-human TSHR cell system described herein appears to be as effective as the FRTL-5 rat thyroid system in stimulating TSHRAb detection; however, the two systems appear to measure different antibody populations in about 30% of cases. Further, stimulating TSHRAb activities measured in the FRTL-5 system tend to correlate better with goiter size and 99mTc pertechnetate uptake, whereas stimulating activities measured in the CHO-human TSHR/chimera system correlate better with free T4 and T3 levels.

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.