Successful Use of Kidneys from a Deceased Donor with Active Herpes Zoster Infection.

BACKGROUND: The limited donor pool and increasing recipient wait list require a reevaluation of kidney organ suitability for transplantation. Use of higher infectious risk organs that were previously discarded may help improve access to transplantation and reduce patient mortality without placing patients at a higher risk of poor posttransplant outcomes. There is very little data available regarding the safe use of kidney organs from deceased donors with varicella zoster virus infection at the time of organ retrieval. Case Presentation. Here, we report a case of successful transplantation of both kidneys from a deceased donor with active herpes zoster infection at the time of organ retrieval. Recipients were treated preemptively with acyclovir. At 4 months posttransplant, both kidney recipients experienced no infectious complications and were off dialysis with functioning transplant grafts. CONCLUSIONS: The use of kidney organs from donors with active herpes zoster infection appears to be a safe option to expand the kidney donor pool.

COVID-19 Status, Symptom Burden, and Characteristics of Dialysis Patients Residing in Areas of Community Transmission: Research Letter.

BACKGROUND: Routine testing of hemodialysis patients for COVID-19 (outside of those identified as "at risk" based on regional practice) is not universally recommended. However, there is variability in the clinical presentation of COVID-19; patients may experience symptoms that do not meet regional criteria for testing and some patients with active infection may be asymptomatic. To avoid missing individuals who are infected, consideration could be made for regular screening, particularly among those residing in areas with evidence of community spread. OBJECTIVE: To describe the clinical characteristics, symptom burden, and COVID-19 status in a cross-section of hemodialysis patients residing in areas with evidence of community spread. DESIGN: Cross-sectional study. SETTING: Three hemodialysis units in a large tertiary care facility in Nova Scotia, Canada. PATIENTS: In-center hemodialysis patients who resided in areas with evidence of community transmission at the time of the study. METHODS: All dialysis patients (irrespective of whether or not they resided in areas with community spread) completed a standard "at-risk" questionnaire for COVID-19 based on (1) 2 or more of new or worsening cough, fever greater than 38°C, sore throat, headache, runny nose/new or acute respiratory illness consistent with infection or (2) any one of close contact with a known/suspected case, travel outside of the province or residence in a facility with an outbreak prior to entry into the dialysis unit at each treatment. Patients residing in areas with evidence of community spread were swabbed for SARS-CoV-2 over a 1-week period (May 1-7, 2020) using a combined oropharyngeal/nares swab irrespective of whether or not they were identified as "at-risk." MEASUREMENTS: Baseline characteristics of patients were acquired using electronic records. In addition to the "at-risk" questionnaire, patients answered "yes" or "no" to any of the following symptoms at the time of the swab (sneeze, fatigue, myalgia, nausea/vomiting, diarrhea, malaise, abdominal pain, loss of taste, and loss of smell). RESULTS: Of the 334 patients receiving dialysis at the time of the study, 133 resided in areas with evidence of community transmission and 104 consented for the study. No patients met our regional criteria for being "at-risk" and no patients reported cough, sore throat or fever at the time of swab. Many other symptoms were noted, including sneezing (24%), fatigue (16%), myalgias (11%), nausea/vomiting (11%), loss of taste (4%), and loss of smell (4%). Overall, 100% of swabs performed for this study were negative for SARS-CoV-2. LIMITATIONS: Single-center study, and the daily new case rate was exceedingly low (4-14) at the time of the study, emphasizing that the findings are not generalizable to areas of higher prevalence of SARS-CoV-2. CONCLUSIONS: In this study of hemodialysis patients residing in areas with community spread who otherwise did not meet symptom criteria for being "at-risk," we did not identify any individual who tested positive for SARS-CoV-2. Future studies are needed to examine the utility of routine testing for COVID-19 (outside of those who are "at-risk") in areas of higher disease prevalence. TRIAL REGISTRATION: Not applicable as this is not a clinical trial.

Effectiveness of a Web-Based eHealth Portal for Delivery of Care to Home Dialysis Patients: A Single-Arm Pilot Study.

BACKGROUND: Improving a patient's experience with their care through an online interface for communication (an eHealth patient portal) has been shown to be beneficial in some studies of chronic disease populations. However, little is known about the effectiveness of an eHealth portal for delivery of care to home dialysis patients. OBJECTIVES: Primary: To determine whether an eHealth portal is effective at improving a patient's experience with their home dialysis care. Secondary: (1) To determine whether an eHealth portal improves health-related quality of life for home dialysis patients, (2) to assess patient satisfaction with an eHealth portal and perceived impact on aspects of their home dialysis therapy and health, (3) to determine the acceptability of the eHealth portal software, and (4) to determine the change in telephone usage for communication after patient adoption of an eHealth portal. DESIGN: Single-arm pilot trial with recruitment over a 4-month period. SETTING: The multidisciplinary home dialysis clinic in Halifax Nova Scotia Canada. PATIENTS: Adults (>18 years) receiving either home hemodialysis or peritoneal dialysis. MEASUREMENTS: Consumer quality index (CQI), health-related quality of life using the EuroQol Five Dimensions Questionnaire (EQ-5D), acceptability of the eHealth portal software (using the Acceptability E-scale), and satisfaction/perceived impact (using a modified questionnaire). METHODS: A web-based application (McKesson, Canada, RelayHealth®) allowed patients and health care workers to communicate through a secure, password-protected online portal that permitted visualization of the messaging history by patient and provider. Patients and the home dialysis health care team had the ability to send messages related to patient care at any time including proposed changes to medication, instructions after a clinic visit, times of new appointments, upcoming investigations, or questions about care. Patient experience with home dialysis care using the CQI, health-related quality of life using the EQ-5D, acceptability of the eHealth portal software, and satisfaction/perceived impact were assessed at baseline, 6, and 12 months of follow-up (where applicable). Total minutes of telephone communication was assessed prior to and after adoption of the portal. RESULTS: Of the 41 patients who consented to join the portal, 27 (66%) created an online account. At baseline, patients had a positive experience for the care and communication provided by their nephrologist (CQI: 3.63, 95% confidence interval [CI]: 3.50-3.76) and this did not change significantly over the study period. Similar results were observed for the care provided by other nephrology health care team members. Health-related quality of life using the EQ-5D score was 0.80 (interquartile range [IQR]: 0.71-0.83) at baseline and this also did not significantly change over the study period. Patients were satisfied with the eHealth portal (mean Likert scale score of 6.5 ± 0.6 in overall satisfaction, scale ranging from 1 completely dissatisfied to 10 completely satisfied), but only a minority (N = 12) completed a satisfaction questionnaire. Median monthly phone usage decreased from 12.5 to 10 minutes (P = .02) after adoption of the portal. LIMITATIONS: The study is limited by the small sample size, high rate of patient dropout, and limited response rate. CONCLUSIONS: In this study of home dialysis patients, we identified that an eHealth communication did not lead to significant improvements in patient experience with home dialysis care. TRIAL REGISTRATION: ClinicalTrials.gov number NCT02128347.

CONTEXTE: Le recours à un outil de communication en ligne (un portail santé destiné aux patients) pour améliorer l'expérience des patients en regard de leurs soins s'est avéré bénéfique dans quelques études sur des populations de patients atteints de maladies chroniques. Cependant, on en sait peu sur l'efficacité d'un tel portail pour la prestation de soins aux patients dialysés à domicile. OBJECTIFS: Principal ­ Déterminer si un portail de santé en ligne se montre efficace pour améliorer l'expérience des patients en matière de soins de dialyse à domicile. Secondaires ­ a. Déterminer si ce portail améliore la qualité de vie liée à la santé des patients dialysés à domicile; b. évaluer la satisfaction des patients à l'égard de l'outil en ligne et connaître son incidence sur certains aspects de leur santé générale et de leurs traitements; c. avoir une idée du niveau d'acceptation du logiciel du portail; d. évaluer les changements dans l'usage du téléphone comme outil de communication, une fois le portail en ligne adopté par le patient. TYPE D'ÉTUDE: Un essai pilote à un seul bras pour lequel le recrutement s'est étalé sur une période de quatre mois. CADRE: La clinique multidisciplinaire de dialyse à domicile d'Halifax (Nouvelle-Écosse) au Canada. SUJETS: Des patients adultes recevant des traitements de dialyse à domicile (hémodialyse ou dialyse péritonéale). MESURES: La qualité de l'expérience des patients à l'égard de leurs soins a été évaluée avec le Consumer Quality Index ou CQI (norme néerlandaise mesurant l'expérience des patients à l'égard des soins de santé). On a mesuré le score de qualité de vie liée à la santé à l'aide du questionnaire EQ-5D (EuroQol Five Dimensions Questionnaire); et l'acceptation du logiciel du portail de santé par l'entremise de l'AES (Acceptability E-scale). Enfin, la satisfaction des patients et l'incidence perçue sur la santé et les traitements ont été évaluées à l'aide d'un questionnaire modifié. MÉTHODOLOGIE: Une application sur le Web (McKesson, Canada, RelayHealth®) a permis aux patients et aux professionnels de la santé de communiquer par le biais d'un portail en ligne sécurisé et protégé par un mot de passe. L'historique des messages envoyés par le patient et le fournisseur de soins était visible sur le portail. Les patients et les membres de l'équipe de soins avaient en tout temps la possibilité d'envoyer des messages liés aux soins du patient. Les messages concernaient notamment des changements proposés dans la médication, des instructions à la suite d'une visite en clinique, les dates et heures de rendez-vous, les enquêtes à venir ou des questions générales relatives aux soins. L'expérience du patient en regard de la dialyse à domicile, évaluée par le CQI; la qualité de vie liée à la santé, évaluée par le questionnaire EQ-5D; l'acceptation du logiciel, de même que la satisfaction générale et l'incidence perçue sur la santé et les traitements ont été mesurées au début de l'étude et après six mois et douze mois de suivi (lorsque possible). La durée des communications téléphoniques a été évaluée avant et après l'adoption du portail. RÉSULTATS: Des 41 patients ayant accepté de joindre le portail, 27 (66 %) ont créé un compte en ligne. Au début de l'étude, les patients disaient avoir une expérience positive en regard des soins offerts et de la communication avec leur néphrologue (CQI : 3,63; IC 95 % : 3,50-3,76) et cette perception est demeurée sensiblement la même tout au long de l'étude. Des résultats similaires ont été observés pour les soins offerts par les autres membres de l'équipe de soins en néphrologie. Le score de la qualité de vie relative à la santé, mesuré par le questionnaire EQ-5D, était de 0,80 (ÉIQ : 0,71-0,83) au début de l'étude et n'a pas varié de façon significative au cours de la période de l'étude. De manière générale, les répondants se sont dits satisfaits du portail de santé en ligne, avec un score moyen de 6,5 ±0,6 sur l'échelle de Likert pour la satisfaction générale (échelle allant de 1, pour « pas du tout satisfait ¼, à 10, pour « entièrement satisfait ¼). Par contre, seule une minorité de patients (n=12) a rempli le questionnaire évaluant la satisfaction. L'utilisation mensuelle médiane du téléphone pour les communications avait diminué à la suite de l'adoption du portail, passant de 12,5 minutes initialement, à 10 minutes après l'adoption. LIMITES: Les constatations de cette étude sont limitées par le très faible échantillon de sujets, par le faible taux de réponse aux questionnaires et par le taux élevé d'abandon des patients. CONCLUSION: Dans cette étude, menée auprès de patients dialysés à domicile, nous avons constaté que le recours à un outil de communication en ligne n'a pas amélioré de façon significative l'expérience des patients en matière de soins de dialyse à domicile.

Macrocytosis may be associated with mortality in chronic hemodialysis patients: a prospective study.

BACKGROUND: Macrocytosis occurs in chronic hemodialysis (CHD) patients; however, its significance is unknown. The purpose of this study was to establish the prevalence and distribution of macrocytosis, to identify its clinical associations and to determine if macrocytosis is associated with mortality in stable, chronic hemodialysis patients. METHODS: We conducted a single-centre prospective cohort study of 150 stable, adult CHD patients followed for nine months. Macrocytosis was defined as a mean corpuscular volume (MCV) > 97 fl. We analyzed MCV as a continuous variable, in tertiles and using a cutoff point of 102 fl. RESULTS: The mean MCV was 99.1 ± 6.4 fl, (range 66-120 fl). MCV was normally distributed. 92 (61%) of patients had an MCV > 97 fl and 45 (30%) > 102 fl. Patients were not B12 or folate deficient in those with available data and three patients with an MCV > 102 fl had hypothyroidism. In a logistic regression analysis, an MCV > 102 fl was associated with a higher Charlson-Age Comorbidity Index (CACI) and higher ratios of darbepoetin alfa to hemoglobin (Hb), [(weekly darbepoetin alfa dose in micrograms per kg body weight / Hb in g/L)*1000]. There were 23 deaths at nine months in this study. Unadjusted MCV > 102 fl was associated with mortality (HR 3.24, 95% CI 1.42-7.39, P = 0.005). Adjusting for the CACI, an MCV > 102 fl was still associated with mortality (HR 2.47, 95% CI 1.07-5.71, P = 0.035). CONCLUSIONS: Macrocytosis may be associated with mortality in stable, chronic hemodialysis patients. Future studies will need to be conducted to confirm this finding.

The Ca(2+) channel blocker flunarizine induces caspase-10-dependent apoptosis in Jurkat T-leukemia cells.

Flunarizine is a Ca(2+) channel blocker that can be either cytoprotective or cytotoxic, depending on the cell type that is being examined. We show here that flunarizine was cytotoxic for Jurkat T-leukemia cells, as well as for other hematological maligancies, but not for breast or colon carcinoma cells. Treatment of Jurkat cells with flunarizine resulted in caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and laddering of DNA fragments, all of which are hallmarks of apoptosis. Flunarizine-induced DNA fragmentation was inhibited by the caspase-3 inhibitor z-DEVD-fmk, the caspase-8/caspase-10 inhibitor z-IETD-fmk, and the caspase-10 inhibitor z-AEVD-fmk, but was not reduced in caspase-8-deficient Jurkat cells, indicating the involvement of caspase-10 upstream of caspase-3 activation. Interestingly, FADD recruitment to a death receptor was not involved since flunarizine caused DNA fragmentation in FADD-deficient Jurkat cells. Flunarizine treatment of Jurkat cells also resulted in reactive oxygen species production, dissipation of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, and caspase-9 activation, although none of these events were necessary for apoptosis induction. Collectively, these findings indicate that flunarizine triggers apoptosis in Jurkat cells via FADD-independent activation of caspase-10. Flunarizine warrants further investigation as a potential anti-cancer agent for the treatment of hematological malignancies.

Vav1 regulates the migration and adhesion of dendritic cells.

Dendritic cells (DCs) are the most potent APCs for activating naive T cells, a process facilitated by the ability of immature DCs to mature and home to lymph nodes after encountering an inflammatory stimulus. Proteins involved in cytoskeletal rearrangement play an important role in regulating the adherence and motility of DCs. Vav1, a guanine nucleotide exchange factor for Rho family GTPases, mediates cytoskeletal rearrangement in hematopoietic cells following integrin ligation. We show that Vav1 is not required for the normal maturation of DCs in vitro; however, it is critical for DC binding to fibronectin and regulates the distribution but not the formation of podosomes. We also found that DC Vav1 was an important component of a signaling pathway involving focal adhesion kinase, phospholipase C-gamma2, and ERK1/2 following integrin ligation. Surprisingly, Vav1(-/-) DCs had increased rates of migration in vivo compared with wild-type control DCs. In vitro findings show that the presence of adhesive substrates such as fibronectin resulted in inhibition of migration. However, there was less inhibition in the absence of Vav1. These findings suggest that DC migration is negatively regulated by adhesion and integrin-mediated signaling and that Vav1 has a central role in this process.

Activated protein C improves ischemic flap survival and modulates proangiogenic and antiinflammatory gene expression.

BACKGROUND: Flap necrosis remains a major complication in reconstructive surgery. The authors evaluated whether systemic activated protein C, a natural serum anticoagulant with anti-inflammatory, proangiogenic, and cytoprotective properties, can improve ischemic skin flap survival. METHODS: Cranially based dorsal cutaneous flaps were elevated on 44 rats. Animals received intravenous injections of activated protein C (25 microg/kg) or saline. Rats were divided into three groups depending on the timing of the first injection: postoperative (45 minutes postoperatively, n = 12), late preoperative (45 minutes preoperatively, n = 5), and early preoperative (3 hours preoperatively, n = 5). In all groups, second and third injections were performed at 3 and 24 hours postoperatively. Flap survival was measured on day 7. Histological and real-time polymerase chain reaction specimens were collected on days 2 and 7 and at 3 and 24 hours, respectively. RESULTS: Postoperative activated protein C improved flap survival (68.9 +/- 4.3 percent) compared with control treatment (39.3 +/- 1.5 percent; p < 0.001). Late preoperative treatment produced diffuse flap hemorrhage. Early preoperative activated protein C injection produced near-complete flap survival (96.1 +/- 1.1 percent for activated protein C versus 50.1 +/- 3.3 percent for control; p < 0.001). Significantly fewer inflammatory cells, improved muscle viability, and increased blood vessel density were observed in activated protein C-treated versus control rats. Activated protein C treatment significantly reduced mRNA levels of intercellular adhesion molecule-1 and tumor necrosis factor-alpha, while increasing levels of Egr-1, vascular endothelial growth factor receptor 2, and Bcl-2. CONCLUSIONS: Systemic activated protein C modulates genes involved in angiogenesis, inflammation and apoptosis and improves ischemic flap survival.

Role of activated protein C and its receptor in inhibition of tumor metastasis.

Engagement of endothelial protein C receptor (EPCR) by activated protein C (aPC) decreases expression of endothelial adhesion molecules implicated in tumor-endothelium interactions. We examined the role of the aPC/EPCR pathway on tumor migration and metastasis. In vitro, B16-F10 melanoma cells showed decreased adhesion to and transmigration through endothelium treated with recombinant human aPC (rhaPC). In murine B16-F10 metastasis models, transgenic EPCR overexpressing (Tie2-EPCR) mice exhibited marked reductions in liver (50%) and lung (92%) metastases compared with wild-type (WT) animals. Intravital imaging showed reduced B16-F10 entrapment within livers of Tie2-EPCR compared with WT mice. A similar reduction was observed in WT mice treated with rhaPC. Strikingly, rhaPC treatment resulted in a 44% reduction in lung metastases. This was associated with decreased lung P-selectin and TNF-alpha mRNA levels. These findings support an important role for the aPC/EPCR pathway in reducing metastasis via inhibition of tumor cell adhesion and transmigration.

Activated protein C: an emerging therapeutic agent in the prevention of ischemia-reperfusion injury.

Free flap necrosis continues to be a significant problem in microvascular surgery. Despite improved microsurgical techniques and equipment, flap loss remains the major operative complication. Although ischemia-induced reperfusion injury remains a significant etiologic factor in flap loss, there is continued interest in endothelial mechanisms that regulate microvascular injury and thrombosis. In recent years, activated protein C (APC) has emerged as a promising therapy in counteracting microcirculatory injury. APC is an anticoagulant that is also involved in signaling pathways that modulate inflammation, apoptosis, and vascular permeability. This article presents the mechanism of action of APC and the benefits of this therapeutic agent, including a possible role in the prevention of free flap ischemia-reperfusion injury.

Expression of a functional single-chain variable-fragment antibody against complement receptor 1 in Streptococcus gordonii.

Streptococcus gordonii, an oral commensal organism, is a candidate vector for oral-vaccine development. Previous studies have shown that recombinant S. gordonii expressing heterologous antigens was weakly immunogenic when delivered intranasally. In this study, antigen was specifically targeted to antigen-presenting cells (APC) in order to potentiate antigen-APC interactions and increase the humoral immune response to the antigen. To achieve this goal, a single-chain variable-fragment (scFv) antibody against complement receptor 1 (CR1) was constructed. Anti-CR1 scFv purified from Escherichia coli was able to bind to mouse mixed lymphocytes and bone marrow-derived dendritic cells. The in vivo function of the anti-CR1 scFv protein was assessed by immunizing mice intranasally with soluble scFv and determining the immune response against the hemagglutinin (HA) peptide located on the carboxy terminus of the scFv. The serum anti-HA immunoglobulin G (IgG) immune response was dose dependent; as little as 100 ng of anti-CR1 scFv induced a significant IgG immune response, while such a response was minimal when the animals were given an unrelated scFv. The anti-CR1 scFv was expressed in S. gordonii as a secreted protein, which was functional, as it bound to dendritic cells. Mice orally colonized by the anti-CR1-secreting S. gordonii produced an anti-HA IgG immune response, indicating that such an approach can be used to increase the immune response to antigens produced by this bacterium.

Prolonged costimulation is required for naive T cell activation.

Costimulation by members of the B7 family of molecules is critical for the activation of naive CD4+ T cells. While prolonged TCR signaling is necessary for T cell activation, the duration of costimulatory signals required has not been established. In this study, murine bone marrow-derived dendritic cells (DC) and naïve CD4+ T cells were used to determine the temporal costimulatory requirements for naive T cell activation. By blocking CD80/CD86 costimulation at various time points during DC-T cell interaction and using the CFSE technique to assess the dynamics of T cell proliferation, we found that prolonged costimulation was required for naive T cells to enter and progress through the cell cycle over a wide range of peptide concentrations. Prolonged costimulation was also important for IL-2 production and CD25/CD69 expression by naive T cells. Video microscopy demonstrated that DC and naive T cells formed stable conjugates that persisted for more than 6 h. Thus, persistent CD80/CD86 signaling during prolonged interactions with DC allows naive T cells to enter the cell cycle and programs the daughter cells to undergo subsequent divisions.

Cutting edge: dendritic cell actin cytoskeletal polarization during immunological synapse formation is highly antigen-dependent.

Dendritic cells (DC) actively rearrange their actin cytoskeleton to participate in formation of the immunological synapse (IS). In this study, we evaluated the requirements for DC participation in the IS. DC rearrange their actin cytoskeleton toward naive CD4(+) T cells only in the presence of specific MHC-peptide complexes. In contrast, naive CD4(+) T cells polarized their cytoskeletal proteins in the absence of Ag. DC cytoskeletal rearrangement occurred at the same threshold of peptide-MHC complexes as that required for T cell activation. Furthermore, T cell activation was inhibited by specific blockade of DC cytoskeletal rearrangement. When TCR-MHC interaction was bypassed by using Con A-activated T cells, DC polarization was abrogated. In addition, directional ligation of MHC class II resulted in DC cytoskeletal polarization. Our findings suggest that a high Ag specificity is required for DC IS formation and that MHC class II signaling plays a central role in this process.

Thy-1 signaling in the context of costimulation provided by dendritic cells provides signal 1 for T cell proliferation and cytotoxic effector molecule expression, but fails to trigger delivery of the lethal hit.

Cross-linking of the GPI-anchored protein Thy-1 results in T cell proliferation and IL-2 synthesis. However, the exact function of Thy-1 in the process of T cell activation remains unknown, as does the effect of costimulation on Thy-1-driven T cell responses. In this study, we have investigated the ability of Thy-1 to substitute for traditional signal 1 in the context of costimulation provided by dendritic cells. Dendritic cells dramatically enhanced T cell proliferation and IL-2 synthesis in response to Thy-1 triggering by anti-Thy-1 mAb. This effect was not dependent on dendritic cell Fcgamma receptors, but was a result of B7-mediated costimulation (signal 2). T cells were also activated when microbeads coated with a combination of anti-Thy-1 and anti-CD28 mAbs were used to supply signals 1 and 2, respectively. Thy-1-stimulated T cells adhere to target cells and express perforin, granzyme B, and Fas ligand, but fail to kill target cells due to an inability to reorganize their secretion machinery. Moreover, in contrast to TCR signaling, Thy-1 triggering failed to induce cytotoxicity in redirected lysis assays. We conclude that Thy-1 triggering can partially substitute for signal 1, which, in combination with a strong signal 2, leads to robust T cell proliferation, IL-2 synthesis, and cytotoxic effector molecule expression, but does not induce cytolytic function. The block at the level of cytotoxic effector function that results when T cells are activated in the absence of a classical, Ag-specific signal 1 may constitute a mechanism to ensure the specificity of CTL responses and prevent potentially harmful promiscuous cytotoxicity.

Experimental model for ophthalmopathy in BALB/c and outbred (CD-1) mice genetically immunized with G2s and the thyrotropin receptor.

In an attempt to develop an animal model for thyroid-associated ophthalmopathy (TAO) we have genetically immunized BALB/c and outbred (CD-1) mice with cDNAs encoding the thyroid and eye muscle shared protein G2s and full length human thyrotropin receptor (TSHr). Firstly, BALB/c mice were immunized with cDNAs for G2s and the TSHr, alone or in tandem with cDNAs for interleukin (IL)4 or IL12. Control mice were immunized with empty vehicle only. Sera from the great majority of experimental mice contained antibodies against a G2s fusion protein and the flavoprotein (Fp) subunit of mitochondrial succinate dehydrogenase, the "64 kDa protein", with the greatest levels being found at sacrifice (17 wk). Antibody levels in mice immunized with G2s + TSHr or G2s + IL12 were generally higher than those in mice immunized with G2s only. TSHr antibodies (TRAb), measured as TSH binding inhibition, were detected in only two mice. On histological examination of the orbits, mild edema, eye muscle fiber separation and mast cell infiltration in and around the eye muscles were found in the majority of experimental mice, but not in control mice. Splenocytes were transferred from selected G2s-immunized mice to normal syngeneic litter mates. None of the transfer mice had serum antibodies against G2s, Fp or TSHr but their orbital tissue showed the same degree of mast cell infiltration as primary mice. No major histological changes were observed in the thyroid or other skeletal muscle in either primary or transfer mice. Similar results were observed in CD-1 mice although, overall, the model was better expressed than in BALB/c mice. In these mice, serum anti-G2s antibody levels were not significantly different between the various experimental groups except at 16 wk, when they were slightly greater than in control animals. Anti-Fp antibodies were detected at 12, 14 and 16 wk, in all experimental groups, including those immunized with G2s only, and were greatest in mice immunized with TSHr alone. TRAb levels were greatest in mice immunized with both G2s and the TSHr in the presence of TL4, but not IL12. The finding of negative anti-G2s but positive anti-Fp antibodies in some CD-1 mice suggests that eye muscle damage and Fp release must have been mediated by T lymphocytes, rather than antibodies, targeting G2s or some other as yet unidentified cell membrane antigen. Histological changes in the orbit were similar to those observed in BALB/c mice although mast cell numbers were greater, in both primary and transfer mice. Overall, the greatest histological changes were observed in CD-1 mice immunized with both G2s + TSHr + IL4. None of the animals became overtly hyperthyroid or hypothyroid during the course of the study although several of the CD-1 mice had abnormal TSH or T4 levels. These results indicate that we have established a valid model for human ophthalmopathy using the novel thyroid and eye muscle expressed protein G2s, now recognized as a fragment of the winged-helix transcription factor Foxp1, and TSHr, and that G2s and the TSHr are both primary antigens in TAO. Reactivity against a TSHr-like protein may be the first event leading to ophthalmopathy in humans with TAO and experimental mice and eye muscle damage may result from autoimmunity against G2s and Fp as a result of "antigen spreading".