Perceived stigma among Tunisian healthcare workers during the COVID-19 pandemic.

BACKGROUND AND OBJECTIVES: Stigma was a major issue during the COVID-19 pandemic. It posed a serious threat to the lives of healthcare workers (HCWs) who were expected to experience higher levels of stigma and increased psychological distress. This is the first survey to investigate forms and correlates of perceived stigma in Tunisian HCWs during the COVID-19 pandemic. METHODS: A cross-sectional web-based survey was conducted between October 8th and November 10th 2020, among 250 Tunisian HCWs. Data were collected using an online questionnaire using the Google Forms® platform. We used a self-reported instrument measuring COVID-19-related stigma, and the Multidimensional Scale of Perceived Social Support (MSPSS) to measure the perceived adequacy of social support from three sources: family, friends, and significant other. RESULTS: The mean stigma score was 18.6±8. Participants sometimes to often experienced stigma in their relationships with friends (22%), neighbors (27.2%), parents (22,4%), and in social activities (30.8%). This stigma was perceived mainly through avoidance (68.4%), and rarely through verbal (6%) or physical aggression (1.2%). The mean MSPSS total score was 5.26±1.24. In multivariate analysis, depression history (P<0.001), long working experience (P<0.001), having presented ageusia/anosmia (P=0.007) and lower total social support scale (P<0.001) were significantly associated with higher perceived stigma score. CONCLUSION: Our findings showed that HCWs perceived stigma in professional, societal and familial domains. Social support from family, friends and others seemed to protect against perceived stigma. Proper health education targeting the public appears to be an effective method to prevent social harassment of both HCWs and COVID-19 survivors.

Validation of the Hamilton Depression Rating Scale (HDRS) in the Tunisian dialect.

OBJECTIVES: The Hamilton Depression Rating Scale (HDRS) is one of the most frequently used depression assessment scales. In Tunisia, psychiatrists commonly use this scale in a Tunisian dialect. However, to the best of our knowledge, this scale has never been validated in Tunisia. This study aims to investigate the reliability and the validity of the HDRS among Tunisian patients who have been hospitalised for a suicide attempt. A secondary objective is to describe the sociodemographic characteristics of the study population. STUDY DESIGN: This is a cross-sectional study performed in the emergency department. METHODS: Patients who were hospitalised for a suicide attempt were eligible for inclusion in this study. The Tunisian version of the HDRS was developed using a forward-backward translation procedure. Psychometric properties of the Tunisian version of the HDRS were tested, including (i) construct validity with a confirmatory one-factor analysis; (ii) internal validity with Pearson correlations and Cronbach alpha coefficients; and (iii) external validity by correlations with the Patient Health Quality-9 (PHQ-9) scale. We used the Receiver-Operating Characteristic (ROC) curve to analyse the correlation between the total HDRS score and the presence of depression according to the PHQ-9. RESULTS: In total, 101 participants were enrolled in this study. The principal component analysis (PCA) type factor analysis with varimax rotation found a high-grade correlation between HDRS individual items and the total score. The total variance, explained by five factors, was 64.4%. Cronbach's standardised alpha coefficient was 0.86 for the overall scale. Correlations between the total HDRS score and the PHQ-9 score, and its various items, were significant. The ROC curve analysis showed good sensitivity (80.8%) and specificity (91.1%). CONCLUSION: The Tunisian version of the HDRS is an acceptable instrument to screen depression in individuals who have attempted suicide.

Angiotensin II receptor subtypes AT1 and AT2 are down-regulated by angiotensin II through AT1 receptor by different mechanisms.

The regulatory effects of angiotensin II (AngII) on its receptor subtypes, AT1 and AT2, were studied using cultured bovine adrenal cells (BAC), which express both receptor subtypes, and PC12W and R3T3 cells, which express only AT2 receptors. In BAC, AngII caused a decrease in AT1- and AT2-binding sites and their corresponding messenger RNAs (mRNAs), but with different kinetics. AT1-binding sites decreased by more than 50% within the first 3 h, whereas AT1 mRNA started to decline after a lag period of 3 h. Both AT2-binding sites and mRNA remained stable within the first 6 h of AngII treatment. Then, AT2 mRNA decreased rapidly with an apparent half-life of 2-3 h, whereas AT2-binding sites declined with an apparent half-life of about 16 h. Measurement of transcription rate and mRNA half-life by the [3H]uridine-thiouridine method revealed that AngII reduced by 90% the rate of AT1 transcription, but had no effect on AT1 mRNA half-life, whereas it slightly reduced AT2 transcription, but markedly reduced AT2 mRNA stability. All of the effects of AngII on both AT1 and AT2 receptors were blocked by losartan, indicating that they were mediated exclusively through the AT1 receptor. In PC12W cells, AngII was unable to modify AT2-binding sites or mRNA. Moreover, in BAC, [125I]AngII was internalized through the AT1 receptor, whereas occupancy of AT2 receptors in either BAC or PC12W did not produce internalization of the hormone. These results indicate that AngII, through the AT1 receptor, down-regulates both AT1 and AT2, but by different mechanisms; AT1 receptor is regulated through internalization-degradation of the occupied receptor and inhibition of transcription, whereas AT2 receptor is regulated mainly by decreasing the stability of its mRNA. Moreover, the phorbol ester phorbol 12-myristate 13-acetate mimicked most of the effects of AngII in BAC and decreased both AT2-binding sites and mRNA on PC12W cells, indicating that the hormonal regulation of both AT1 and AT2 receptors is mediated through protein kinase C activation.

Regulation of primary response and specific genes in adrenal cells by peptide hormones and growth factors.

Using cultured bovine adrenal fasciculata cells (BAC), we investigated the effects of two hormones, corticotropin (ACTH) and angiotensin II (Ang-II) and two growth factors, insulin-like growth factors I (IGF-I) and transforming growth factor beta 1 (TGF beta 1), on the mRNA levels of nuclear proto-oncogenes of the Fos and Jun families and on the mRNA levels of genes expressed in BAC coding for ACTH and AT1 receptors, cytochrome P450scc and P450 17 alpha and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). ACTH and IGF-1 increased c-fos and jun-B mRNA levels early with later increases in the levels of mRNA for the ACTH receptor and the three steroidogenic enzymes, and enhanced steroidogenic responses to both ACTH and Ang-II. In contrast, Ang-II increased mRNA coding for the three proto-oncogenes (cfos, c-jun, and jun-B), decreased those for P450 17 alpha and 3 beta-HSD, and caused marked homologous and heterologous steroidogenic desensitization. TGF beta 1 increased only jun-B mRNA and markedly reduced BAC-differentiated functions and steroidogenic responsiveness to both ACTH and Ang-II. The long-term effects of ACTH on human adrenal fasciculata cells were comparable with those observed in BAC, whereas the long term effects of Ang-II and TGF beta 1 were different from those observed in BAC. Whether these species-specific differences are related to a different effect of these factors on proto-oncogene expression is not yet known.

Peptide hormone and growth factor regulation of nuclear proto-oncogenes and specific functions in adrenal cells.

Among the large number of immediate early genes, nuclear proto-oncogenes of the Fos and Jun families, have been postulated to be involved in the long-term effects of several growth factors on cell differentiation and/or multiplication. Since adrenal cell differentiated functions appear to be regulated by specific hormones and growth factors, the effects of these factors on proto-oncogene mRNA levels were analysed in bovine adrenal fasciculata cells (BAC) in culture. Corticotropin (ACTH) and insulin-like growth factor I increased c-fos and jun-B mRNA, but had no effect on c-jun mRNA and these early changes were associated with a later increase in BAC specific function [ACTH receptors, cytochrome P450 17 alpha) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)] and an enhanced steroidogenic responsiveness to both ACTH and angiotensin-II (A-II). On the other hand, A-II increased the three proto-oncogene (c-fos, c-jun and jun-B) mRNAs, induced a decrease of P450 17 alpha and 3 beta-HSD and caused a marked homologous and heterologous (ACTH) densitization. Transforming growth factor beta 1 which only increased jun-B mRNA, markedly reduced BAC differentiated functions and the steroidogenic responsiveness to both ACTH and A-II. Thus, it is postulated that the proto-oncoproteins encoded by the immediate early genes may play a role in the long-term effects of peptide hormones and growth factors on BAC differentiated functions.

Regulation by growth factors of angiotensin II type-1 receptor and the alpha subunit of Gq and G11 in bovine adrenal cells.

In the present work we have investigated the effects of several growth factors on the expression of Angiotensin II (A-II) receptors subtype AT1 and their pertussis toxin-insensitive coupling to G-proteins in bovine adrenal fasciculata-reticularis cells (BAC). Insulin, Insulin-like growth factor and basic Fibroblast growth factor increased AT1 receptors (mRNA and binding sites) as well as the alpha subunit of Gq (mRNA and protein) and G11 (protein). These changes were associated with an enhanced A-II-induced inositol phosphate accumulation and cortisol production. In contrast, Transforming growth factor beta 1, which reduced slightly AT1 binding sites, but not the level of alpha q or alpha 11 proteins, did not change the A-II-induced inositol phosphate accumulation. However, this factor, as previously reported, markedly reduced cortisol production.

Identification and characterization of angiotensin-II receptor subtypes in cultured bovine and human adrenal fasciculata cells and PC12W cells.

Angiotensin-II (Ang II) receptor subtypes (AT1 and AT2) were analyzed in bovine adrenal cells (BAC) by binding and cross-linking experiments using [125I]Ang II and [125I]CGP42112, a specific ligand of AT2 receptors. [125I]Ang II binding was reduced by 80% and 20% in the presence of maximal concentrations of the AT1 antagonist losartan (DuP 753) and CGP42112, respectively, whereas [125I]CGP42112 binding was inhibited by Ang II or CGP42112, but not by losartan. In the presence of the reducing agent dithio-1,4-erythritol, the binding of [125I] CGP42112 was increased 2-fold; this was due to an increase in the binding affinity (Kd, 8 +/- 4 x 10(-10) vs. 4.8 +/- 1.2 x 10(-10) M). Cross-linking of [125I]Ang II to BAC in the presence of disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed a band of 70,000 +/- 8,000 mol wt (M(r)) under both reducing and nonreducing conditions. This band disappeared when the incubation was performed in the presence of 10(-6) M Ang II or 5 x 10(-8) M CGP42112, but not in the presence of 10(-5) M losartan. Dithio-1,4-erythritol (10 mM) markedly enhanced the band. After cross-linking with 1,5-difluoro-2,4-dinitrobenzene and solubilization of the cells in the presence of protease inhibitors, two radioactive bands were observed with M(r) of 70,000 and 50,000. The first disappeared after the addition of Ang II or CGP42112, whereas the second disappeared in the presence of Ang II or losartan, but not in the presence of CGP42112. Cross-linking of [125I]AngII to either human adrenal fasciculata-reticularis cells, which contain only AT1 sites, or COS-7 cells transfected with human AT1 cDNA revealed a major band of 50,000 M(r) that was blunted by Ang II or losartan, but not by CGP42112. Moreover, cross-linking of [125I]Ang II to PC12W cells, which contain only the AT2 receptor subtype, revealed a single radioactive band of 70,000 Mr that was blunted by CGP42112 but not by losartan. Thus, in both BAC and PC12W cells, the AT2 receptor has a M(r) of 70,000, whereas the AT1 receptor in BAC, human adrenal cells, and cells transfected with human AT1 receptor cDNA has a Mr of 50,000. Therefore, the heterogeneity of the size of the Ang II receptor previously reported after photoaffinity or cross-linking was probably due to only to a variation in the degree of glycosylation between tissues and species, but also to the presence of two different receptor subtypes.

Angiotensin-II-induced expression of proto-oncogene (c-fos, jun-B and c-jun) mRNA in bovine adrenocortical fasciculata cells (BAC) is mediated by AT-1 receptors.

We have shown previously that angiotensin-II (A-II) controls proto-oncogene (c-fos, jun-B and c-jun) mRNA accumulation in bovine adrenal fasciculata cells (BAC). Since BAC contain both subtypes (AT-1 and AT-2) of the A-II receptor, we have investigated which subtype was involved in the effect of A-II on proto-oncogene mRNA by using a selective antagonist for AT-1 (DUP 753) and for AT-2 (CGP 42112A). DUP 753, but not CGP 42112A, inhibited the stimulatory effect of A-II on proto-oncogene mRNA, with ID50s of 4 x 10(-7) M, 7 x 10(-7) M and 2 x 10(-6) M for c-fos, jun-B and c-jun, respectively. Neither of the two antagonists by themselves had a direct effect on proto-oncogene mRNA. As the A-II AT-1 receptors are coupled to the phospholipase C system in BAC, we have investigated whether the A-II effects on the proto-oncogenes were mediated by protein kinase C (PKC) or by Ca2+ calmodulin. First, activation of PKC by the phorbol ester, PMA, increased the level of three proto-oncogene mRNAs, whereas calcium ionophore had no effect. Second, staurosporine, a specific inhibitor of PKC, reduced the stimulatory action of A-II on proto-oncogene mRNA by 80-90%, whereas trifluoroperazine, an inhibitor of calmodulin, had no significant effect. These results demonstrate that the effects of A-II on proto-oncogene mRNA are mediated by AT1 receptor subtypes, mainly through activation of the PKC pathway.

Characterization and coupling of angiotensin-II receptor subtypes in cultured bovine adrenal fasciculata cells.

Angiotensin-II (A-II) receptor subtypes and their potential coupling mechanisms were investigated in bovine adrenal fasciculata cells (BAC) in culture, by the use of selective antagonists for AT1 (DUP 753 or Losartan) and AT2 (PD 123177 and CGP 42112A) sites. Competition for [125I]A-II specific binding with AT1 or AT2 selective ligands produced a biphasic displacement curve, suggesting two distinct A-II binding sites. In the presence of PD 123177 (10(-5) M), a concentration at which most of the AT2 sites were saturated, DUP 753 displaced [125I]A-II specific binding in a monophasic manner with an IC50 of 6.2 +/- 1.4 x 10(-7) M. In the presence of DUP 753 (10(-5) M), the displacement produced by CGP 42112A and PD 123177 was also monophasic, with IC50s of 8 +/- 3 x 10(-10) and 4.6 +/- 2.1 x 10(-7) M, respectively. The reducing agent dithio-1,4-erythritol inhibited the binding of [125I]A-II to AT1 (DUP 753 sensitive) sites, but increased its binding to AT2 sites 2-fold. The IC50 values for these two effects were about 0.5 and 3 mM, respectively. The biological effects of A-II in BAC, phosphoinositide hydrolysis and cortisol production, were inhibited in a dose-dependent manner by DUP 753, but not by AT2 antagonists. Similarly, the potentiating action of A-II on corticotropin-induced cAMP production was blocked by DUP 753, but not by AT2 antagonists. These data indicate that BAC contain both receptor subtypes, but that all the known effects of A-II in BAC were induced via the AT1 receptor subtype.

Opposite effects of angiotensin-II and corticotropin on bovine adrenocortical cell steroidogenic responsiveness.

The long-term effects of angiotensin-II (A-II) and corticotropin (ACTH) on bovine adrenal fasciculata cells (BAC) were studied. Cells were pretreated for 3 days with either A-II or ACTH followed by an examination of the acute steroidogenic response to both hormones as well as the ability to convert several steroid precursors to cortisol and corticosterone. ACTH pretreatment caused a marked increase in cortisol output associated with a decrease in corticosterone secretion in response to both hormones leading to a 50-fold decrease in the corticosterone/cortisol ratio compared to control cells. After incubation with saturating concentrations (5 X 10(-5) M) of 22 R-hydroxycholesterol, pregnenolone or progesterone, ACTH-pretreated cells produced more cortisol than corticosterone whereas the contrary was observed in control cells. However, the conversion of 17 alpha-hydroxyprogesterone and 11-deoxycortisol to cortisol by ACTH-pretreated cells was lower than by control cells. Thus, the main effects of ACTH were a marked increase of 17 alpha-hydroxylase and a small but significant decrease of 21-hydroxylase and 11 beta-hydroxylase activities. A-II pretreatment produced, in a concentration-dependent manner, a down-regulation of its own receptors and homologous and heterologous steroidogenic desensitization. At maximal concentrations (10(-6) M) A-II reduced by 70% its own receptors while the steroidogenic response to A-II and ACTH was reduced by 95% and 75%, respectively. However, the coupling of A-II receptors to phosphoinositide pathway and to Ca2+ influx, as well as its potentiation effect on ACTH-induced cAMP production were similar in control and A-II pretreated cells. Moreover, the conversion of several steroid precursors to corticosterone was similar in control cells and A-II-pretreated cells, whereas the conversion to cortisol was reduced by approximately 30% due mainly to a decrease of 17 alpha-hydroxylase activity. Thus, the marked steroidogenic desensitization induced by A-II is most likely related to some alteration located beyond the activation of the two branches of the phosphoinositide pathway and before the first steps of steroidogenesis.

Characterization and regulation of angiotensin and corticotropin receptors on cultured bovine adrenal cells.

Cultured bovine adrenal fasciculata cells were used to characterize angiotensin II (A-II) and corticotropin (ACTH) receptors and to study their homologous and heterologous regulation. These cells contain one type of high affinity binding sites for A-II (KD congruent to 2.4 +/- 0.3 10(-9) M) and about 100000 sites/cell. Photoaffinity labeling followed by SDS-PAGE under reducing conditions revealed a single macromolecule of apparent MR 65,000. Treatment of cells with increasing concentrations of A-II produced down-regulation of its own receptors and marked homologous and heterologous (ACTH) steroidogenic desensitization. However, the desensitization was not correlated with receptor loss and was mainly due to alterations of the steroidogenic pathway. Pretreatment of cells with ACTH also reduced A-II receptors, but this was not associated with steroidogenic desensitization. Bovine fasciculata cells contain two binding sites for ACTH: one of high affinity (KD congruent to 2.6 +/- 0.4 10(-10) M) and low capacity (2030 +/- 390 sites/cell) and the other of low affinity and high capacity. Affinity cross-linking of ACTH to plasma membranes prepared from adrenal cells revealed a labeled macromolecule of apparent MR 43000. However, cross-linking experiments to intact cells revealed, both under reducing and non-reducing conditions, two labeled macromolecules of apparent MR of 123000 and 43000. Pretreatment of cells with ACTH enhanced its receptor and the cAMP and cortisol responses to further ACTH stimulation. These effects were time- and dose-dependent. The maximal effects were observed at 10(-10) to 10(-9) M. A-II alone had no effect but it blocked partially the stimulatory action of ACTH.