Parents' and caregivers' role toward childhood vaccination in Albania: assessment of predictors of vaccine hesitancy.

Background: Vaccination has saved millions of lives through the protection of individuals and populations from communicable diseases. Vaccine hesitancy, defined as the delay in acceptance or refusal of vaccines despite the availability of vaccination services, has become a growing global concern. The objective of this study was to investigate parents'/caregivers' hesitancy toward childhood vaccination and its predictors in Albania. Study design: A cross-sectional survey was conducted. Methods: The data comes from a survey conducted on a sample of parents/caregivers (89.6% mothers) of children aged 6 months to 8 years at health care vaccination centers in seven Albanian cities from December 2020 to February 2021. Parents/caregivers (one per child) were interviewed by trained healthcare staff using a standardized questionnaire on six main content domains, including immunization behavior, beliefs about vaccine safety and efficacy, attitudes about vaccines, vaccination confidence, estimation of vaccine delay, and the intention to immunize children against SARS-CoV-2, and a self-reported hesitancy. The Albanian Ministry of Health approved the questionnaire, after it was translated, validated and adapted to the local setting. Statistical analyses included independent sample t-tests (p<0.05) and a logistic regression (OR; 95% C.I.). Results: A total of 475 parents/caregivers of children aged from 6 months to 8 years, attending childhood vaccination in public health services, were interviewed. To the question "how hesitant you are about childhood vaccination", a high number of parents/caregivers (46%) responded that they do not feel hesitant at all, and 32% were not hesitant, a small number of parents/caregivers said they are very hesitant (5%) or somewhat hesitant (12%). A binary logistic model was fitted to the data to test the hypothesis regarding the relationship between parental vaccine hesitancy and possible predictors. A lower parental attitude toward childhood vaccines (OR = 3.7; 95% C.I. 1.102-12.421), a health center with a high vaccine delay (OR = 2.878; C.I. 95% 1.735-4.773), and low confidence in health staff information (OR = 2.042; 95% C.I. 1.156-3.605) were all independent predictors of parental vaccine hesitancy. Regarding intention to vaccinate children against COVID-19, when available, nearly 75% of parents/caregivers showed hesitancy. Conclusions: Our results highlighted the role of positive parents'/caregivers' attitudes toward childhood vaccines followed by high staff confidence and good health center organization in order to deal with vaccine hesitancy, particularly for traditional and well-known childhood vaccines. Nevertheless, the hesitancy can be a critical barrier for childhood vaccination when we have to introduce a new vaccine, as is demonstrated in the recent vaccination campaign against the ongoing pandemic of SARS-CoV2.

Structure-toxicity relationships of saturated and unsaturated free fatty acids for elucidating the lipotoxic effects in human EndoC-ßH1 beta-cells.

Lipotoxicity has been considered a major cause for beta-cell dysfunction in type 2 diabetes mellitus. However, the underlying mechanisms are still unclear. To achieve a better understanding of the toxicity a wide range of structurally different free fatty acids (FFAs) has been analyzed in human EndoC-ßH1 beta-cells. Exposure of human EndoC-ßH1 beta-cells to physiological saturated and monounsaturated long-chain FFAs induced apoptosis. Particularly noteworthy was that the toxicity increased more rapidly with increasing chain length of saturated than of unsaturated FFAs. The highest toxicity was observed in the presence of very long-chain FFAs (C20-C22), whereas polyunsaturated FFAs were not toxic. Long-chain FFAs increased peroxisomal hydrogen peroxide generation slightly, while very long-chain FFAs increased hydrogen peroxide generation more potently in both peroxisomes and mitochondria. The greater toxicity of very long-chain FFAs was accompanied by hydroxyl radical formation, along with cardiolipin peroxidation and ATP depletion. Intriguingly, only saturated very long-chain FFAs activated ER stress. On the other hand saturated very long-chain FFAs did not induce lipid droplet formation in contrast to long-chain FFAs and unsaturated very long-chain FFAs. The present data highlight the importance of structure-activity relationship analyses for the understanding of the mechanisms of lipotoxicity. Chain length and degree of saturation of FFAs are crucial factors for the toxicity of FFAs, with peroxisomal, mitochondrial, and ER stress representing the major pathogenic factors for induction of lipotoxicity. The results might provide a guide for the composition of a healthy beta-cell protective diet.

The monounsaturated fatty acid oleate is the major physiological toxic free fatty acid for human beta cells.

Free fatty acids (FFAs) can cause glucose intolerance and diabetes. Lipotoxicity to the pancreatic beta cells is considered to be a major underlying cause for this phenomenon. The aim of this study was to analyse the toxicity profile of FFAs in the human EndoC-ßH1 beta-cell line and to compare the results with isolated rat and human islets with special reference to the physiologically most prevalent FFAs palmitic acid (PA) and oleic acid (OA). Toxicity after a 2-day incubation with the different FFAs was analysed by the caspase-3 assay and confirmed by the propidium iodide and annexin V staining tests. The long-chain saturated PA (C16:0) and the monounsaturated OA (C18:1) were both toxic to human EndoC-ßH1 beta cells and pseudoislets, as well as to rat islets, and, as confirmed in a pilot experiment, also to human islets. Furthermore, OA provided no protection against the toxicity of PA. Likewise, elaidic acid (EA, the trans isomer of OA; trans-OA) was significantly toxic, in contrast to the non-metabolisable analogues methylated PA (MePA) and methylated OA (MeOA). Fatty acids with a chain length < C16 were not toxic in EndoC-ßH1 beta cells. Caspase-3 was also activated by linoleic acid (LA)(C18:2) but not by γ-linolenic acid (γ-LNA)(C18:3). Overall, only long-chain FFAs with chain lengths > C14, which generate hydrogen peroxide in the peroxisomal beta-oxidation, were toxic. This conclusion is also supported by the toxicity of the branched-chain FFA pristanic acid, which is exclusively metabolised in the peroxisomal beta-oxidation. The lack of a protective effect of the monounsaturated fatty acid OA has important consequences for a beta-cell protective lipid composition of a diet. A cardioprotective diet with a high OA content does not fulfil this requirement.

Impact of scavenging hydrogen peroxide in the endoplasmic reticulum for ß cell function.

Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase family and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is accompanied by generation of hydrogen peroxide (H2O2). Because of the high rate of insulin biosynthesis and the low expression of H2O2-inactivating enzymes in pancreatic ß cells, it has been proposed that the luminal H2O2 concentration might be very high. As the role of this H2O2 in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells, the influence of ER-specific H2O2 removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H2O2 significantly with a threefold increase of the EC50 value for H2O2. However, the expression of cytokine-induced ER stress genes and viability after incubation with ß cell toxic cytokines (IL1ß alone or together with TNFα+IFNγ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells, insulin secretion and proinsulin content was identical, while removal of luminal H2O2 reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H2O2 toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H2O2 in the ER in spite of a reduced insulin transcription and processing.

N-glycosylation-negative catalase: a useful tool for exploring the role of hydrogen peroxide in the endoplasmic reticulum.

Disulfide bond formation during protein folding of nascent proteins is associated with the generation of H2O2 in the endoplasmic reticulum (ER). Approaches to quantifying H2O2 directly within the ER failed because of the oxidative environment in the ER lumen, and ER-specific catalase expression to detoxify high H2O2 concentrations resulted in an inactive protein owing to N-glycosylation. Therefore, the N-glycosylation motifs at asparagine-244 and -439 of the human catalase protein were deleted by site-directed mutagenesis. The ER-targeted expression of these variants revealed that the deletion of the N-glycosylation motif only at asparagine-244 (N244) was associated with the maintenance of full enzymatic activity in the ER. Expression of catalase N244 in the ER (ER-Catalase N244) was ER-specific and protected the cells significantly against exogenously added H2O2. With the expression of ER-Catalase N244, a highly effective H2O2 inactivation within the ER was achieved for the first time. Catalase has a high H2O2-inactivation capacity without the need of reducing cofactors, which might interfere with the ER redox homeostasis, and is not involved in protein folding. With these characteristics ER-Catalase N244 is an ideal tool to explore the impact of ER-generated H2O2 on the generation of disulfide bonds or to study the induction of ER-stress pathways through protein folding overload and accumulation of H2O2.

Influence of cytokines on Dmt1 iron transporter and ferritin expression in insulin-secreting cells.

Free intracellular ferrous iron (Fe(2+)) is essential for the generation of the extremely toxic hydroxyl radicals, which contribute to ß-cell destruction by cytokines. Therefore the expression of the different divalent metal transporter 1 (Dmt1) isoforms and ferritin (Ft) subunits, responsible for iron import and chelation, was analyzed under pro-inflammatory conditions (IL1ß alone or together with TNFα+IFNγ). The Dmt1 isoforms (1A/1B and +IRE/-IRE) and the total Dmt1 expression in insulin-producing cells (RINm5F and INS-1E), in primary rat islets and, for comparison, in the neuroendocrine PC12 cell line were quantified by qRT-PCR. In addition, the expression of the light (L-Ft) and heavy Ft (H-Ft) subunits and the mitochondrial Ft isoform (Mtft) in insulin-producing cells under control conditions and after cytokine treatment was estimated. The 1B isoform was the predominant Dmt1 mRNA in all insulin-producing cells, accounting for almost 100% of the 1A/1B isoform expression. For the IRE variants, +IRE expression was higher than -IRE expression. Pro-inflammatory cytokines accelerated the expression of Dmt1 isoforms significantly with an overall 2.5- to 3-fold increase in the total Dmt1 expression. In contrast, the expression of the iron-buffering ferritin subunits L- and H-Ft was unaffected by IL1ß and only slightly induced by the cytokine mixture. Mtft expression was also not increased. Dmt1 expression was significantly elevated through pro-inflammatory cytokines, whereas Ft expression was marginally increased. This imbalance between the increased iron transport capacity and the almost unaffected iron storage capacity can foster cytokine-mediated formation of hydroxyl radicals and thus pro-inflammatory cytokine toxicity through elevated free iron concentrations.