A two-step, test-guided Mokken scale analysis, for nonclustered and clustered data.

PURPOSE: Mokken scale analysis (MSA) is an attractive scaling procedure for ordinal data. MSA is frequently used in health-related quality of life research. Two of MSA's prime features are the scalability coefficients and the automated item selection procedure (AISP). The AISP partitions a (large) set of items into scales based on the observed item scores; the resulting scales can be used as measurement instruments. There exist two issues in MSA: First, point estimates, standard errors, and test statistics for scalability coefficients are inappropriate for clustered item scores, which are omnipresent in quality of life research data. Second, the AISP insufficiently takes sampling fluctuation of Mokken's scalability coefficients into account. METHODS: We solved both issues by providing point estimates and standard errors for the scalability coefficients for clustered data and by implementing a Wald-based significance test in the AISP algorithm, resulting in a test-guided AISP (T-AISP), that is available for both nonclustered and clustered test scores. RESULTS: We integrated the T-AISP into a two-step, test-guided MSA for scale construction, to guide the analysis for nonclustered and clustered data. The first step is performing a T-AISP and select the final scale(s). For clustered data, within-group dependency is investigated on the final scale(s). In the second step, the strength of the scale(s) is determined and further analyses are performed. The procedure was demonstrated on clustered item scores obtained from administering a questionnaire on quality of life in schools to 639 students nested in 30 classrooms. CONCLUSIONS: We developed a two-step, test-guided MSA for scale construction that takes into account sample fluctuation of all scalability coefficients and that can be applied to item scores obtained by a nonclustered or clustered sampling design.

Is the "Brainwork Intervention" effective in reducing sick leave for non-permanent workers with psychological problems? Results of a controlled clinical trial.

BACKGROUND: Both the presence of psychological problems and the absence of an employment contract are related to long-term sickness absence, prolonged work disability and unemployment. Studies researching the effectiveness of return-to-work interventions on these non-permanent workers, including unemployed and temporary agency workers and workers with an expired fixed-term contract, are lagging behind. Therefore, a return-to-work intervention called "Brainwork" was developed. The aim of this study was to assess the effectiveness of the 'Brainwork Intervention' in reducing the duration of sick leave compared to usual care over a 12-month follow-up. METHODS: In a multicenter controlled clinical trial, using a quasi-randomization procedure, we compared the Brainwork Intervention (n = 164) to usual care (n = 156). The primary outcome was the duration of sick leave. Secondary outcomes were the duration of sick leave starting from Social Security Agency transfer; the proportion of workers returned to work; the number of hours of paid employment during the follow-up period; the degree of worker participation; the level of psychological complaints; and the self-efficacy for return to work. Protocol adherence (Brainwork Intervention) was considered sufficient when at least three of the five protocol steps were followed. Cox regressions, linear and ordinal regression, and Mixed Model analyses were performed. RESULTS: All 320 participants were analyzed. The Brainwork Intervention resulted in a non-significant reduction of the duration of sick leave compared to usual care (269 days versus 296 days; HR = 1.29; 95% CI 0.94-1.76; p = 0.11). For those working (46%) during the 12-month follow-up, the mean number of hours of paid employment was non-significantly higher in the usual care group (682 h versus 493 h; p = 0.053). No significant differences were found for other secondary outcomes. Protocol adherence was 10%. CONCLUSIONS: The Brainwork Intervention as performed with a low protocol adherence did not result in a significant reduction of the duration of sick leave compared to usual care. It remains unclear what the results would have been if the Brainwork Intervention had been executed according to protocol. TRIAL REGISTRATION: The Netherlands Trial Register (NTR); NTR3976 (old registration number NTR4190). Registered September 27th 2013.

An Open Label Phase Ib Dose Escalation Study of TRC105 (Anti-Endoglin Antibody) with Axitinib in Patients with Metastatic Renal Cell Carcinoma.

BACKGROUND: TRC105 is an IgG1 endoglin monoclonal antibody that potentiates VEGF inhibitors in preclinical models. We assessed safety, pharmacokinetics, and antitumor activity of TRC105 in combination with axitinib in patients with metastatic renal cell carcinoma (mRCC). SUBJECTS, MATERIALS, AND METHODS: Heavily pretreated mRCC patients were treated with TRC105 weekly (8 mg/kg and then 10 mg/kg) in combination with axitinib (initially at 5 mg b.i.d. and then escalated per patient tolerance to a maximum of 10 mg b.i.d.) until disease progression or unacceptable toxicity using a standard 3 + 3 phase I design. RESULTS: Eighteen patients (median number of prior therapies = 3) were treated. TRC105 dose escalation proceeded to 10 mg/kg weekly without dose-limiting toxicity. Adverse event characteristics of each drug were not increased in frequency or severity when the two drugs were administered concurrently. TRC105 and axitinib demonstrated preliminary evidence of activity, including partial responses (PR) by RECIST in 29% of patients, and median progression-free survival (11.3 months). None of the patients with PR had PR to prior first-line treatment. Lower baseline levels of osteopontin and higher baseline levels of TGF-ß receptor 3 correlated with overall response rate. CONCLUSION: TRC105 at 8 and 10 mg/kg weekly was well tolerated in combination with axitinib, with encouraging evidence of activity in patients with mRCC. A multicenter, randomized phase II trial of TRC105 and axitinib has recently completed enrollment (NCT01806064). IMPLICATIONS FOR PRACTICE: TRC105 is a monoclonal antibody to endoglin (CD105), a receptor densely expressed on proliferating endothelial cells and also on renal cancer stem cells that is implicated as a mediator of resistance to inhibitors of the VEGF pathway. In this Phase I trial, TRC105 combined safely with axitinib at the recommended single agent doses of each drug in patients with renal cell carcinoma. The combination demonstrated durable activity in a VEGF inhibitor-refractory population and modulated several angiogenic biomarkers. A randomized Phase II trial testing TRC105 in combination with axitinib in clear cell renal cell carcinoma has completed accrual.

Chronic sleep reduction in adolescents-clinical cut-off scores for the Chronic Sleep Reduction Questionnaire (CSRQ).

The Chronic Sleep Reduction Questionnaire is a validated questionnaire that measures symptoms of prolonged insufficient and/or poor sleep and therefore accounts for individuals' sleep need and sleep debt. This study extends its psychometric properties by providing cut-off scores, using a matched sample of 298 healthy adolescents (15.38 ± 1.63 years, 37.9% male, mean Chronic Sleep Reduction Questionnaire score: 32.98 ± 6.51) and 298 adolescents with insomnia/delayed sleep-wake phase disorder (15.48 ± 1.62 years; 37.9% male, mean Chronic Sleep Reduction Questionnaire score: 42.59 ± 7.06). We found an area under the curve of 0.84 (95% confidence interval: 0.81-0.87). Cut-off scores for optimal sensitivity, optimal specificity and based on Youden's criterion are provided. These cut-off scores are highly relevant for use of the Chronic Sleep Reduction Questionnaire in future studies and clinical practice.

Pediatric epilepsy and comorbid reading disorders, math disorders, or autism spectrum disorders: Impact of epilepsy on cognitive patterns.

INTRODUCTION: In pediatric epilepsy, comorbidities are reported to be frequent. The present study focusedon the cognitive patterns of children with isolated epilepsy, children with isolated neurodevelopmental disorders (reading disorders, math disorders, autism spectrum disorders), and children with epilepsy and these neurodevelopmental disorders as comorbidities. METHODS: Based on two samples of referred children, one with epilepsy, reading disorders, math disorders, or ASDs occurring in "isolation" (n=117) and one with reading disorders, math disorders, and ASDs occurring comorbid with epilepsy (n=171), cognitive patterns were compared. The patterns displayed by verbal and nonverbal abilities from the WISC series were studied with repeated measures ANOVA. Thereafter, an exploratory 2∗3∗2 factorial analysis was done to study the independent contribution of the type of comorbidity and of the presence or absence of epilepsy to the VIQ-PIQ pattern. RESULTS: In isolated epilepsy, a VIQ>PIQ pattern was found, which was not seen in the other disorders. When epilepsy and another disorder co-occurred, patterns were altered. They resembled partly the pattern seen in isolated epilepsy and partly the pattern seen in the isolated neurodevelopmental disorder. In comorbid reading disorders, the VIQ>PIQ pattern was mitigated; in comorbid math disorders, it was exacerbated. In comorbid ASDs, no clear pattern emerged. In the presence of epilepsy, patterns characteristic of isolated disorders appeared systematically shifted toward relatively lowered performance abilities or relatively spared verbal abilities. The similar "impact" exerted by epilepsy on the patterns of the various conditions suggested shared mechanisms.

The impact of education and globalization on sexual and reproductive health: retrospective evidence from eastern and southern Africa.

The objective of this study is to qualify the relationship between sexual and reproductive health (SRH) and educational attainment in eastern and southern Africa (ESA). We hypothesize that the regional level of globalization is a moderating factor in the relationship between SRH and educational attainment. Using retrospective data from Kenya, Malawi, Tanzania, and Zambia, the associations between SRH (eight indicators), educational attainment, and globalization were examined using multilevel logistic regression analysis. It was found that the model fit for every SRH outcome indicator increased significantly after including the interaction between globalization and educational attainment, supporting the hypothesis. Depending on the level of globalization, three types of relationships between education and SRH were found: (1) for the indicators "more than four children," "intercourse before 17 years," "first child before 20 years," and "one or more child died" education is risk-decreasing, and the reduction is stronger in more globalized regions; (2) for the indicators "condom use at last intercourse" and "current contraceptive use" education is risk-decreasing, and the reduction is stronger in less globalized regions; (3) for the indicators "HIV positive" and "more than four lifetime sexual partners" education is risk increasing, but only in less globalized regions. In conclusion, these effects are related to three types of access: (1) access to services, (2) access to information, and (3) access to sexual networks. The findings highlight the relevance of globalization when analyzing the association between SRH and education, and the importance of structural factors in the development of effective SRH promotion interventions.

Moral orientation and relationships in school and adolescent pro- and antisocial behaviors: a multilevel study.

This multilevel study examined the relationships between moral climate factors and prosocial as well as antisocial behaviors inside and outside the school (school misconduct, delinquent behavior, and vandalism). The moral climate factors were punishment- and victim-based moral orientation, relationships among students, and teacher-student relationships. The analyses of data from 670 students in 69 classes showed that the classroom-level variables only had a significant impact on misconduct at school of students aged 12 to 20. For the other outcome variables, the student-level variables (student and teacher-student relationships, but especially students' moral orientation) were significant. A novel finding was that a positive teacher-student relationship not only proved to be related to less misconduct inside the school but also to less delinquent behavior and vandalism outside the school. This indicates that the teacher is an important socializing agent for adolescent behavior in general.

A phase 1 study of TRC102, an inhibitor of base excision repair, and pemetrexed in patients with advanced solid tumors.

INTRODUCTION: TRC102 potentiates the activity of cancer therapies that induce base excision repair (BER) including antimetabolite and alkylating agents. TRC102 rapidly and covalently binds to apurinic/apyrimidinic (AP) sites generated during BER, and TRC102-bound DNA causes topoisomerase II-dependent irreversible strand breaks and apoptosis. This study assessed the safety, maximum-tolerated dose (MTD), pharmacokinetics and pharmacodynamics of TRC102 alone and in combination with pemetrexed. PURPOSE: Patients with advanced solid tumors received oral TRC102 daily for 4 days. Two weeks later, patients began standard-dose pemetrexed on day 1 in combination with oral TRC102 on days 1 to 4. The pemetrexed-TRC102 combination was repeated every 3 weeks until disease progression. METHODS: Twenty-eight patients were treated with TRC102 at 15, 30, 60 or 100 mg/m(2)/d. The MTD was exceeded at 100 mg/m(2)/d due to grade 3 anemia in 50 % of patients. TRC102 exposure increased in proportion to dose with a mean t1/2 of 28 h. A pharmacodynamic assay confirmed that TRC102 binds to pemetrexed-induced AP sites at all doses studied. Stable disease or better was achieved in 15 of 25 patients evaluable for response (60 %), including one patient with recurrent metastatic oropharyngeal carcinoma that expressed high levels of thymidylate synthase, who achieved a partial response and was progression free for 14 months. CONCLUSIONS: When administered with pemetrexed, the maximum tolerated dose of oral TRC102 is 60 mg/m(2)/d for 4 days. Randomized controlled studies are planned to evaluate the clinical benefit of adding TRC102 to pemetrexed and other agents that induce BER.

Endoglin inhibitor TRC105 with or without bevacizumab for bevacizumab-refractory glioblastoma (ENDOT): a multicenter phase II trial.

BACKGROUND: Glioblastoma (GBM), the most lethal primary brain tumor, has limited treatment options upon recurrence after chemoradiation and bevacizumab. TRC105 (carotuximab), a chimeric anti-endoglin (CD105) antibody, inhibits angiogenesis and potentiates activity of VEGF inhibitor bevacizumab in preclinical models. This study sought to assess safety, pharmacokinetics, and efficacy of TRC105 for bevacizumab-refractory GBM. METHODS: We conducted a pre-registered (NCT01564914), multicenter, open-label phase II clinical trial (ENDOT). We administered 10 mg/kg TRC105 monotherapy (first cohort) in adults with GBM and radiographic progression following radiation, temozolomide and bevacizumab therapy. Primary outcome was median time-to-progression (TTP), amended after first cohort's enrollment to median overall survival (mOS). Secondary outcomes were objective response rate, safety and tolerability, and progression-free survival (PFS). RESULTS: 6 patients were enrolled in TRC105 monotherapy cohort. Median TTP and PFS of 5 evaluable patients receiving monotherapy was 1.4 months, in whom plasma VEGF-A levels were elevated post-therapy. Lack of response led to protocol amendment, and second cohort's addition of bevacizumab+TRC105 with primary endpoint of mOS. 16 patients were enrolled in bevacizumab+TRC105 cohort. mOS of 15 evaluable patients was 5.7 (95%CI: 4.2-9.8) months. All 22 patients had measurable disease at baseline. Median PFS of 14 evaluable patients receiving bevacizumab+TRC105 was 1.8 months (95%CI 1.2-2.1). Serum TRC105 was measurable above target concentration of 25 ug/mL in all evaluable patients. Study medications were well-tolerated in both cohorts. Combined administration did not potentiate known toxicities of either medication, with cerebral hemorrhage not observed. CONCLUSIONS: Single-agent TRC105 lacks activity in bevacizumab-refractory GBM, possibly secondary to upregulated VEGF-A expression. Meaningful mOS in bevacizumab+TRC105 cohort warrants further trials to investigate efficacy of combination therapy.

Glioblastoma is an aggressive and lethal brain tumor, with patients typically expected to survive for 14 to 16 months after diagnosis. Nearly all patients experience tumor recurrence once conventional treatment strategies fail, after which a drug called bevacizumab is used. However, subsequent treatment options are extremely limited. We performed a clinical trial in which we investigated how safe and effective a new drug called TRC105 (carotuximab) is in patients who no longer respond to chemotherapy, radiotherapy or bevacizumab. We tested TRC105 both with and without bevacizumab, since TRC105 might enhance the activity of bevacizumab. We found that patients survived for an average of 5.7 months when given TRC105 and bevacizumab in combination. These findings suggest that further clinical trials are needed to confirm whether or not this combination therapy is a useful approach in patients with glioblastoma recurrence.

Promoting physical distancing during COVID-19: a systematic approach to compare behavioral interventions.

In the wake of the COVID-19 pandemic, physical distancing behavior turned out to be key to mitigating the virus spread. Therefore, it is crucial that we understand how we can successfully alter our behavior and promote physical distancing. We present a framework to systematically assess the effectiveness of behavioral interventions to stimulate physical distancing. In addition, we demonstrate the feasibility of this framework in a large-scale natural experiment (N = 639) conducted during an art fair. In an experimental design, we varied interventions to evaluate the effect of face masks, walking directions, and immediate feedback on visitors' contacts. We represent visitors as nodes, and their contacts as links in a contact network. Subsequently, we used network modelling to test for differences in these contact networks. We find no evidence that face masks influence physical distancing, while unidirectional walking directions and buzzer feedback do positively impact physical distancing. This study offers a feasible way to optimize physical distancing interventions through scientific research. As such, the presented framework provides society with the means to directly evaluate interventions, so that policy can be based on evidence rather than conjecture.

Bias of Two-Level Scalability Coefficients and Their Standard Errors.

Two-level Mokken scale analysis is a generalization of Mokken scale analysis for multi-rater data. The bias of estimated scalability coefficients for two-level Mokken scale analysis, the bias of their estimated standard errors, and the coverage of the confidence intervals has been investigated, under various testing conditions. It was found that the estimated scalability coefficients were unbiased in all tested conditions. For estimating standard errors, the delta method and the cluster bootstrap were compared. The cluster bootstrap structurally underestimated the standard errors of the scalability coefficients, with low coverage values. Except for unequal numbers of raters across subjects and small sets of items, the delta method standard error estimates had negligible bias and good coverage. Post hoc simulations showed that the cluster bootstrap does not correctly reproduce the sampling distribution of the scalability coefficients, and an adapted procedure was suggested. In addition, the delta method standard errors can be slightly improved if the harmonic mean is used for unequal numbers of raters per subject rather than the arithmetic mean.

Standard errors of two-level scalability coefficients.

For the construction of tests and questionnaires that require multiple raters (e.g., a child behaviour checklist completed by both parents) a novel ordinal scaling technique is currently being further developed, called two-level Mokken scale analysis. The technique uses within-rater and between-rater coefficients to assess the scalability of the test. These coefficients are generalizations of Mokken's scalability coefficients. In this paper we derived standard errors for the two-level coefficients and for their ratios. The coefficients, the estimates, the estimated standard errors and the software implementation are discussed and illustrated using a real-data example, and a small-scale simulation study demonstrates the accuracy of the estimates.

MCMC estimation for the p(2) network regression model with crossed random effects.

The p(2) model is a statistical model for the analysis of binary relational data with covariates, as occur in social network studies. It can be characterized as a multinomial regression model with crossed random effects that reflect actor heterogeneity and dependence between the ties from and to the same actor in the network. Three Markov chain Monte Carlo (MCMC) estimation methods for the p(2) model are presented to improve iterative generalized least squares (IGLS) estimation developed earlier, two of which use random walk proposals. The third method, an independence chain sampler, and one of the random walk algorithms use normal approximations of the binary network data to generate proposals in the MCMC algorithms. A large-scale simulation study compares MCMC estimates with IGLS estimates for networks with 20 and 40 actors. It was found that the IGLS estimates have a smaller variance but are severely biased, while the MCMC estimates have a larger variance with a small bias. For networks with 20 actors, mean squared errors are generally comparable or smaller for the IGLS estimates. For networks with 40 actors, mean squared errors are the smallest for the MCMC estimates. Coverage rates of confidence intervals are good for the MCMC estimates but not for the IGLS estimates.

Exposure to virtual social interactions in the treatment of social anxiety disorder: A randomized controlled trial.

This randomized controlled trial investigated the efficacy of a stand-alone virtual reality exposure intervention comprising verbal interaction with virtual humans to target heterogeneous social fears in participants with social anxiety disorder. Sixty participants (Mage = 36.9 years; 63.3% women) diagnosed with social anxiety disorder were randomly assigned to individual virtual reality exposure therapy (VRET), individual in vivo exposure therapy (iVET), or waiting-list. Multilevel regression analyses revealed that both treatment groups improved from pre-to postassessment on social anxiety symptoms, speech duration, perceived stress, and avoidant personality disorder related beliefs when compared to the waiting-list. Participants receiving iVET, but not VRET, improved on fear of negative evaluation, speech performance, general anxiety, depression, and quality of life relative to those on waiting-list. The iVET condition was further superior to the VRET condition regarding decreases in social anxiety symptoms at post- and follow-up assessments, and avoidant personality disorder related beliefs at follow-up. At follow-up, all improvements were significant for iVET. For VRET, only the effect for perceived stress was significant. VRET containing extensive verbal interaction without any cognitive components can effectively reduce complaints of generalized social anxiety disorder. Future technological and psychological improvements of virtual social interactions might further enhance the efficacy of VRET for social anxiety disorder.

Increase of adipose differentiation by hypolipidemic fibrate drugs in Ob 17 preadipocytes: requirement for thyroid hormones.

The action of hypolipidemic fibrate drugs (HFD) (clofibrate, bezafibrate, fenofibrate) was studied in relation to thyroid hormone (TH) action in the TH-sensitive Ob 17 preadipocyte cells which require an early presence of TH for terminal differentiation. HFD markedly amplified the adipose differentiation and the development of several lipogenic enzymes, thus accelerating their appearance after cell growth arrest. This amplifying action could be obtained whatever the time of drug addition to the cells and required the continuous presence of the drug. HFD action was strictly dependent on the presence of TH. Within the active concentration range (0.01-0.25 mM) in serum-containing medium, HFD moderately down-modulated the nuclear TH receptor level (and c-erb alpha mRNA abundance), this being additive to the known maximal but partial down-regulation provoked by TH. The results strengthen the TH obligatory role for Ob 17 cell differentiation and give arguments against a TH-like role of HFD. In this cell line, HFD mainly behave as amplifiers for the expression of lipogenic phenotypes in already committed cells.

Retinoic acid decreases nuclear triiodothyronine receptor expression and impairs an early step of adipose differentiation in the thyroid hormone-sensitive mouse Ob 17 preadipocyte cell line.

In the murine preadipocyte cell line Ob 17, T3 is known to be necessary at an early step of adipose differentiation for the expression of late phenotypes [lipogenic enzymes such as malic enzyme, glycerol-3-phosphate dehydrogenase (GPDH), etc.] and not necessary for the expression of lipoprotein lipase (LPL), which emerges earlier, at growth arrest. These cells contain nuclear T3 receptors, which mainly belong to products of the c-erbA alpha gene and are down-regulated by T3. In this work, retinoic acid (RA) added to Ob 17 cells at growth arrest impaired morphological differentiation and the development of both late (malic enzyme and GPDH) and early (LPL) phenotypes regardless of whether T3 was added. T3 sensitized the cells to the inhibitory action of RA; the ED50 for GPDH activity was shifted from 0.5 microM to 3 nM in cells cultured with 1.5 nM T3. Later addition of RA (6 days after growth arrest) did not inhibit the differentiation. RA also brought out a marked and fast decrease in nuclear T3 receptors. This was observed whatever the stage of cell development and related to both a rapid decrease in the relative abundance of c-erbA alpha-related mRNA species and an increased disappearance rate, suggesting the involvement of pre- and posttranslational events. RA and T3 acted additively in decreasing the T3 receptor and c-erbA alpha mRNA levels. The effects of RA on T3 receptors were rapidly reversed after RA withdrawal; the reversal was large (75%) when RA was introduced at growth arrest and total when introduced later. The cell sensitivity to RA, considering the T3 receptors, was higher at growth arrest (ED50 for RA, 0.2 and 1.5 microM in assays with RA added at growth arrest and 5 days later, respectively). The results suggest intricated regulatory pathways between RA and T3 at an early step of adipose differentiation and also suggest that among different mechanisms through which RA may impair this differentiation, a decreased level of nuclear T3 receptors at an early period should play a role.

Calcitriol down-modulates the 3,5,3' triiodothyronine (T3) receptors and affects, in a biphasic manner, the T3-dependent adipose differentiation of Ob 17 preadipocytes.

In previous reports, we showed that T3 is required for terminal differentiation of the murine Ob 17 preadipocytes, and that it partially down-modulates the abundance of its own nuclear receptor sites (T3R). We also reported that a profound depletion of the T3R was produced by all-trans-retinoic acid at concentrations that inhibit adipose differentiation. Here, we report that calcitriol (VD), which activates a nuclear receptor (VDR) closely related to the T3R and retinoid receptors, also markedly affects nuclear T3 binding and T3-induced differentiation of Ob 17 cells. Within a nearly physiological concentration range (0.1-2.5 nM), calcitriol profoundly down-modulated T3R abundance without altering the affinity for T3. The T3R depletion was a fast event, sustained under VD and reversed within 48 h of VD withdrawal. The order of efficient concentration ranges of VD and analogs suggests an involvement of the VDR. The T3R-depleting effect of VD was observed at every stage of adipose differentiation and was additive to the depleting effect of T3. Within the 0.1-2.5 nM VD concentration range, the c-erbA alpha and -alpha 1 messenger RNA levels (only c-erbA alpha gene products were detected in these cells) were poorly decreased; VD also did not alter a protein band specifically detected with specific anti-c-erbA alpha 1 antibodies in Western blots of nuclear extracts. VD accelerated the T3R disappearance rate; the results suggest that this would probably involve sequestration, rather than degradation, events. Interestingly, calcitriol added to the culture medium of Ob 17 preadipocytes markedly influenced the adipose differentiation, exerting a clear-cut stimulation at levels of 0.25 nM or less and profound inhibition at concentrations above 0.25 nM. Both effects were observed provided that VD was added within an early critical period of the differentiation process, as we previously reported for T3. The stimulations caused by low concentrations of VD and 1.5 nM T3 were additive. Increasing the VD concentration produced a progressive attenuation, then a suppression, of the stimulating effect of T3. Comparative analyses of VD-related changes in adipose differentiation and T3R abundance suggest that a correlation may exist between optimal differentiation and a partial depletion of the T3R, whereas a profound depletion of the T3R occurred at inhibitory concentrations of VD. The present results sustain the concept that T3R play a role in the differentiation of Ob 17 preadipocytes. Moreover, the results suggest that there may be a T3 receptor site concentration optimal for efficient differentiation. A regulation of this concentration involves ligands of other closely related receptors and, thus, probably the interplays that exist between these receptors.

Transient expression of c-erbAbeta1 messenger ribonucleic acid and beta1 thyroid hormone receptor early in adipogenesis of Ob 17 cells.

In the murine Ob 17 preadipocyte cell line, the thyroid hormone T3 is an adipogenic factor necessary at an early stage for differentiation into adipocyte. We demonstrate here that this T3 dependence may involve a transient expression (at both the messenger RNA and the protein levels) of c-ErbA beta-type receptors (T3R), although a large body of T3R remained the product of the c-erbAalpha gene, as previously described. c-ErbAbeta1 (and not beta2) expression emerged significantly at growth arrest, peaked 2 days later, and almost disappeared in maturing adipocytes. This expression is related to the presence of T3, as total deprivation of culture medium from T3 prevented it, and the addition of 1.5 nM T3 to preconfluent cultures was able to restore it. When cells were cultured in the presence of T3 and thus were able to differentiate, the c-erbAbeta peak was accompanied by sequential rapid increases in CAAT/enhancer-binding protein-delta(C/EBPdelta), peroxisome proliferator-activated-gamma receptor (PPARgamma), and C/EBPalpha gene expressions. On the contrary, under thyroid hormone-deprived culture conditions that result in nondifferentiation of the preadipocytes, c-erbAbeta1, PPARgamma, and the large C/EBPalpha expressions were blunted, and a moderate early increase in c-erbAalpha1 transcripts was sustained for a longer period. Addition of T3 to T3-deprived preconfluent cells restored PPARgamma and C/EBPalpha expressions. Taken together, the results highlight the important role of T3 in the adipogenesis of Ob 17 cells through the involvement of both beta1 and alpha1 T3R subtypes.

Nuclear factors specifically favor thyroid hormone binding to c-ErbA alpha 1 protein (thyroid hormone receptor alpha) over-expressed in E. coli.

A recombinant rat thyroid hormone receptor alpha (TR alpha or c-ErbA alpha 1) was produced in E. coli as a non-mutated, nonfusioned protein and obtained as an efficient DNA and T3 binding protein that could be easily handled in a buffer-soluble state (rec-TR alpha). It was found that nuclear extracts (NE) added to rec-TR alpha markedly amplified not only DNA binding, which has been well documented, but also T3 binding (increased binding site concentration), which has not yet been reported. This T3 binding amplifying effect on rec-TR alpha occurs at low NE protein concentrations that produce no or minimal endogenous TR with respect to rec-TR, while similar concentrations of other proteins (e.g. ovalbumin or cytosol) only moderately enhanced T3 binding. The T3 binding amplifying nuclear factors, which are partly heat-labile, appeared as necessary auxiliaries in the analyses of partially purified rec-TR alpha. A protective effect of NE against a loss of affinity for T3 under the action of antibodies directed to certain sequences in the TR alpha D domain suggests that nuclear factors help rec-TR alpha to acquire and/or stabilize a conformation that allows the high affinity T3 binding. The nature of this nuclear amplifying factor is still unknown: RXR alpha which, produced in vitro, could amplify binding of the rec-TR alpha to a DNA thyroid response element, was unable to display such a rescue of high affinity binding sites.

Antibodies against restricted sequences in human c-ErbA hinge domain recognize differentially natural mammalian alpha- or beta-type triiodothyronine receptors and interfere differently with hormone binding.

In our first report, rabbit antibodies directed to recombinant polypeptides of human alpha-type c-ErbA sequences recognized natural triiodothyronine (T3) receptors (TR) in adipocytes (mouse Ob 17 cell line) but not in liver (mouse, rat). Moreover, some of them, directed to the sequence 150-228, markedly interfered with hormone binding to adipocyte T3 receptors. We now raised antibodies against shorter synthetic peptides within this alpha-type 150-228 c-ErbA sequence, which encompasses part of the hinge (D) domain and N-terminus of the E domain (alpha-150-166 and alpha 172-191) and against a beta-type c-ErbA sequence (beta 204-220 aligned on alpha 150-166, and differing by eight amino acids). Our present antibodies, which bear the expected c-ErbA alpha- or beta-type specificity, immunoprecipitated the TR in nuclear extracts, with a different pattern between tissues: exclusive precipitation by anti-c-ErbA alpha antibodies in Ob 17 adipocytes; large but non-exclusive precipitation by anti-cErbA beta antibodies in rat or mouse liver, which also expresses some alpha-type TR. This pattern of discriminative immunoprecipitation, also obtained in parallel analysis using our previously described antibodies to other c-ErbA alpha or beta sequences (anti-alpha 144-162, anti-alpha 1 403-410 and anti-beta 62-82), roughly verifies results of c-erbA mRNA expression in these tissues. Slight differences appeared in the extent of alpha-type TR recognition by antibodies directed to alpha 172-191, whether TR were liganded or not to T3 before antibody addition. This evokes a different conformation of this region after hormone binding. Most interestingly, these anti-alpha 172-191 antibodies lowered the Ka for T3 and extensively dissociated the adipocyte T3-TR complexes; they interfered poorly with the binding of T3 in liver nuclear extracts. This strongly supports the concept that internal sequences in c-ErbA alpha, more precisely in a restricted C-terminal part of the D domain, are necessary for efficient T3 binding, which also need the C-terminal part of domain E.