Mortality of Roma population in Serbia, 2002-2005

Aim

To describe and compare mortality and population changes in the Roma and non-Roma population in Serbia in 2002 and 2005.

Methods

The number of cases of death were obtained from the 2002 and 2005 Mortality Database and population data from the Population Census 2002. Standardized sex specific rates of non-traumatic and traumatic mortality in 2002 and 2005 were calculated in relation to the European standard population. We presented population pyramid and aging index for both populations in 2002 and compared sex specific standardized traumatic and non-traumatic mortality rates and the average age of death for 2002 and 2005. The causes of death were coded according to the 10th revision of the International Classification of Diseases (ICD-10) groups, and the proportional mortalities in the year 2002 and 2005 were compared between the Roma and non-Roma population using χ² test.

Results

Standardized mortality rates were higher in the Roma than in the general population. Non-traumatic mortality rate in Roma men in 2002 was 18.2 per 1000 and in slightly decreased to 18.0 per 1000 in 2005; it was significantly higher than in non-Roma men in both years (11.9 per 1000 in 2002 and 12.5 per 1000 in 2005; P<0.001). Standardized non-traumatic mortality rate in Roma women decreased significantly from 16.78 per 1000 in 2002 to 14.89 per 1000 in 2005 (P=0.014), but it was still significantly higher than in non-Roma women (8.46 per 1000 in 2002 and 8.84 per 1000 in 2005; P<0.001). Morbidity structure indicated that the most common causes of death in the Roma population were cardiovascular diseases, neoplasms, and respiratory system diseases. In relation to the general population respiratory system diseases were denoted as main causes of deaths in significantly higher percent (6% vs 3% in 2002 and 7% vs 4% in 2005; P<0.001) and cardiovascular diseases in significantly lower percent (44%:55% in 2002 and 46%:57%; P<0.001).

Conclusions

Our data show that mortality rates in the Roma population are significantly higher than in the general population, and morbidity structure of the most common causes of death significantly different from that of general population.According to the most recent population census in the Serbia from 2002, 108 193 or 1.44% residents declared themselves as Romani. The Roma population is among the most imperiled and potentially most vulnerable groups. Living Standards Measurement Survey (LSMS) conducted in Serbia in 2002-2003 indicated significant differences in living conditions between the Roma and non-Roma population (1). The prevalence of poverty, defined as an average total consumption below the poverty line of 57 euros per month/consumption unit, in the Roma population was as high as 64.4%, which is 6.1 times more frequent than in the general population (10.5%) (1). Extremely low level of education and very high level of unemployment characterize the social status of the Romani people. Almost two thirds (62%) of the Roma older than 15 years did not finish primary school, as opposed to 19% of the general population (1). The unemployment rate is also high, reaching 45%, while in the rest of the Serbian population it amounts to 9% (1). Such findings are not unexpected since these characteristics are highly associated with poverty (1).Although the range of vaccination coverage in Serbian general population is 98%-100%, many Roma children are not included in vaccination programs (2). Twenty nine percent of Roma children aged 18-29 months do not even have vaccination cards. Data regarding the education of Roma children are also discouraging: 62% of them attended pre-school programs one year prior to the first grade, as opposed to 89% children in the general population. Also, the vast majority (98%) of children of primary school age in general population attends school, while the attendance rate among Roma children is significantly lower (74%). The difference between the proportions of Roma children and children in general population who attend secondary schools is even more drastic (only 10% vs 85%).During the last decade, several studies dealing with diverse problems of the Roma population have been conducted in South-Eastern Europe (3-10). Compared with the general populations in Eastern and Central Europe, life expectancy of the Roma population is 10-year shorter because of poor living conditions and poverty (11). A study conducted in the Czechoslovakia in 1989 compared census data and pointed out that life expectancy of the Roma men and women were 12.1 and 14.4 years, respectively, shorter than in the general population (12).The aim of this study was to describe and compare mortality and population changes in the Roma and non-Roma population in Serbia in 2002 and 2005. We present population pyramid and aging index for both populations for the year 2002 and compared sex specific standardized traumatic and non-traumatic mortality rates and the average age of death for 2002 and 2005.     

Toxicity of pamam‐coated gold nanoparticles in different unicellular models

Polyamidoamine (PAMAM) dendrimers are used for many pharmaceutical and biomedical applications. However, the toxicological risks of several PAMAM‐based compounds are still not fully evaluated, despite evidences of PAMAM deleterious effects on biological membranes, leading to toxicity. In this report, we investigated the toxicity of generation 0 PAMAM‐coated gold nanoparticles (AuG0 NPs) in four different models to determine how different cellular systems are affected by PAMAM‐coated NPs. Toxicity was evaluated in two mammalian cell lines, Neuro 2A and Vero, in the green alga Chlamydomonas reinhardtii and the bacteria Vibrio fischeri. AuG0 NP treatments reduced cell metabolic activity in algal and bacterial cells, measured by esterase enzymatic activity (C. reinhardtii) and luminescence emission (V. fischeri). EC50 value after 30 min of treatment was similar in both organisms, with 0.114 and 0.167 mg mL^?1 for C. reinhardtii and V. fischeri, respectively. On the other hand, AuG0 NPs induced no change of mitochondrial activity in mammalian cells after 24 h of treatment to up to 0.4 mg mL^?1 AuG0 NPs. Change in the absorption spectra of AuG0 NP in the mammalian cell culture media may indicate an alteration of NP properties that contributed to the low toxicity of AuG0 NPs in mammalian cells. For a safe development of PAMAM‐based nanomaterials, the difference of sensitivity between mammalian and microbial cells, as well as the modulation of NPs toxicity by medium properties, should be taken into account when designing PAMAM NPs for applications that may lead to their introduction in the environment. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 328–336, 2014.     

Lipid,blood pressure and kidney update 2013

The year 2013 proved to be very exciting as far as landmark trials and new guidelines in the field of lipid disorders, blood pressure and kidney diseases. Among these are the International Atherosclerosis Society Global Recommendations for the Management of Dyslipidemia, European Society of Cardiology (ESC)/European Society of Hypertension Guidelines for the Management of Arterial Hypertension, American Diabetes Association Clinical Practice Recommendations, the Kidney Disease: Improving Global Outcomes Clinical Practice Guidelines for Managing Dyslipidemias in Chronic Kidney Disease (CKD) Patients, the American College of Cardiology/American Heart Association Guideline on the Treatment of Blood Cholesterol to Reduce Atherosclerotic Cardiovascular Risk in Adults, the Joint National Committee Expert Panel (JNC 8) Evidence-Based Guideline for the Management of High Blood Pressure in Adults, the American Society of Hypertension/International Society of Hypertension Clinical Practice Guidelines for the Management of Hypertension in the Community, the American College of Physicians Clinical Practice Guideline on Screening, Monitoring, and Treatment of Stage 1–3 CKD and many important trials presented among others during the ESC Annual Congress in Amsterdam and the American Society of Nephrology Annual Meeting—Kidney Week in Atlanta, GA. The paper is an attempt to summarize the most important events and reports in the mentioned areas in the passing year.     

Clinical characteristics and functional outcome of patients with West Nile neuroinvasive disease in Serbia

Neurologic manifestations are prominent characteristic of West Nile virus (WNV) infection. The aim of this article was to describe neurological manifestations in patients with WNV neuroinvasive disease and their functional outcome at discharge in the first human outbreak of WNV infection in Serbia. The study enrolled patients treated in the Clinic for Infectious and Tropical Diseases, Clinical Center Serbia in Belgrade, with serological evidence of acute WNV infection who presented with meningitis, encephalitis and/or acute flaccid paralyses (AFP). Functional outcome at discharge was assessed using modified Rankin Scale (mRS) and Barthel index. Fifty-two patients were analysed. Forty-four (84.6 %) patients had encephalitis, eight (15.4 %) had meningitis, and 13 (25 %) had AFP. Among patients with AFP, 12 resembled poliomyelitis and one had clinical and electrodiagnostic findings consistent with polyradiculoneuritis. Among patients with encephalitis, 17 (32.7 %) had clinical signs of rhombencephalitis, and eight (15.4 %) presented with cerebellitis. Respiratory failure with subsequent mechanical ventilation developed in 13 patients with WNE (29.5 %). Nine (17.3 %) patients died, five (9.6 %) were functionally dependent (mRS 3–5), and 38 (73.1 %) were functionally independent at discharge (mRS 0–2). In univariate analysis, the presence of AFP, respiratory failure and consciousness impairment were found to be predictors of fatal outcome in patients with WNV neuroinvasive disease (p < 0.001, p < 0.001, p = 0.018, respectively). The outbreak of human WNV infection in Serbia caused a notable case fatality ratio, especially in patients with AFP, respiratory failure and consciousness impairment. Rhombencephalitis and cerebellitis could be underestimated presentations of WNV neuroinvasive disease.     

Muscle activity,cross-sectional area,and density following passive standing and whole body vibration: A case series

Objective

To investigate the effects of intermittent passive standing (PS) and whole body vibration (WBV) on the electromyography (EMG) activity, cross-sectional area, and density of lower extremity muscles in individuals with chronic motor complete spinal cord injury (SCI).

Design

Case series.

Methods

Seven adult men with chronic (≥2 years), thoracic motor complete (AIS A–B) SCI completed a 40-week course of thrice-weekly intermittent PS-WBV therapy, in a flexed knee posture (160°), for 45 minutes per session at a frequency of 45 Hz and 0.6–0.7 mm displacement using the WAVE^® Pro Plate, with an integrated EasyStand™ standing frame. EMG was measured in major lower extremity muscles to represent muscle activity during PS-WBV. The cross-sectional area and density of the calf muscles were measured using peripheral quantitative computed tomography at the widest calf cross-section (66% of the tibia length) at pre- and post-intervention. All measured variables were compared between the pre- and post-intervention measurements to assess change after the PS-WBV intervention.

Results

PS-WBV acutely induced EMG activity in lower extremity muscles of SCI subjects. No significant changes in lower extremity EMG activity, muscle cross-sectional area, or density were observed following the 40-week intervention.

Conclusions

Although acute exposure to PS-WBV can induce electrophysiological activity of lower extremity muscles during PS in men with motor complete SCI, the PS-WBV intervention for 40 weeks was not sufficient to result in enhanced muscle activity, or to increase calf muscle cross-sectional area or density.     

Comparative Investigation of the Efficacy of Three Different Adsorbents against OTA-Induced Toxicity in Broiler Chickens

The aim of our study was to determine the efficacy of three different adsorbents, inorganic (modified zeolite), organic (esterified glucomannans) and mixed (inorganic and organic components, with the addition of enzymes), in protecting broilers from the toxic effects of ochratoxin A in feed. Broilers were fed diets containing 2 mg/kg of ochratoxin A (OTA) and supplemented with adsorbents at the recommended concentration of 2 g/kg for 21 days. The presence of OTA led to a notable reduction in body weight, lower weight gain, increased feed conversion and induced histopathological changes in the liver and kidneys. The presence of inorganic, organic and mixed adsorbents in contaminated feed only partially reduced the negative effects of OTA on the broiler performances. Broilers that were fed with adsorbent-supplemented feed reached higher body weight (17.96%, 19.09% and 13.59%), compared to the group that received only OTA. The presence of adsorbents partially alleviated the reduction in feed consumption (22.68%, 12.91% and 10.59%), and a similar effect was observed with feed conversion. The applied adsorbents have also reduced the intensity of histopathological changes caused by OTA; however, they were not able to prevent their onset. After the withdrawal of the toxin and adsorbents from the feed (21–42 days), all previously observed disturbances in broilers were reduced, but more remarkably in broilers fed with adsorbents.     

In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region

We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti–IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.

Powerful selection technologies have made in vitro evolution of protein binders more efficient and paved the way for the use of tailor-made antibodies in therapy. After initial selections of antibody candidates with desired specificity, lead antibodies are typically improved by affinity maturation in multiple rounds of randomization and selection (1) to reach the subnanomolar affinities ideally required for targeting soluble ligands (2–4). This is usually attempted by introduction of point substitutions, either at random positions across the entire V-gene (5, 6) or in the complementary-determining regions (CDRs; e.g., by CDR walking mutagenesis) (7).In Nature, diversification of the primary antibody repertoire occurs by several mechanisms that generate variation in the regions forming the antigen-binding site, the CDRs, including considerable length variation (8–11) that is initially introduced by recombination of V(D)J gene segments. Length variations are concentrated in the CDR3 region (12), at the junctions of the joined segments, where additional diversity is produced by N- or P-nucleotide additions that can further extend the CDR3. The length of the CDRs considerably affects the topography of the combining site, as different shapes brought about by extension or shortening can form pockets, grooves, or fill space (13, 14).Following B cell stimulation by the antigen, further diversification of the antigen-binding interface is generated through somatic hypermutation (SHM) (15), involving mainly point mutagenesis that preferentially targets hotspots in the CDRs (16, 17). This process is initiated through deamination of cytosine to uracil by activation-induced cytidine deaminase (AID), leading to uracil:guanine mismatches (16). Upon removal of these uracil bases by base excision-repair enzymes, error-prone DNA polymerases are then recruited to fill in the gaps and introduce mutations around the position of the deaminated cytosines. Interestingly, up to 6% of the mutations generated by SHM are insertions and deletions (InDels) (18), which occur due to misalignment of repeated DNA sequences (19, 20). Thus, insertions occur by duplication, while deletions are brought about by removal of repeated sequences (21, 22).A small percentage of antibodies selected by in vivo SHM contain InDels in the CDRs 1 and 2 (1.6 to 6.5%) (21–24), while junctional diversity by N- or P-nucleotide additions in the CDR3 confounds the analysis of SHM-derived InDels, leading to an underestimation of the total percentage of affinity-improving InDels. In vitro-directed evolution has been unsuitable for introduction of InDels at random positions into an antibody gene, because of restrictions in the diversity of InDels that could be introduced (i.e., insertions by duplication in in vitro SHM) (22, 25). Rational (26) or computational (27) strategies have been successful at introducing InDels in a few, carefully chosen positions instead of random sampling. In contrast, an unusually high percentage of InDels with a functional role among in vivo affinity matured broadly neutralizing antibodies (bnAbs) to HIV-1 (28–30): ∼40% of the reported anti–HIV-1 bnAbs contain InDels that accumulate during in vivo SHM (28). Based on the frequent occurrence of InDels among multispecific, cross-reactive antibodies, one could infer that they provide a molecular solution for recognizing multiple targets by providing an altered interface (enlarged or tightened), possibly even involving conformational diversity (31). The accumulation of InDels in bnAbs has been attributed to extensive in vivo SHM, so that even positions that are rarely modified by SHM are also altered (17, 28).Insertions in the V-genes occur only by duplication of adjacent sequences (21, 22), so that the actual sequence diversity of the resulting insertions is limited because they repeat existing modules. To introduce more diversity in the inserted sequences, point mutations are required in subsequent rounds of SHM. However, since the CDRs can tolerate considerable length variation, it is likely that the antibody fold can accommodate a larger number of affinity-enhancing InDels compared to those observed in antibodies affinity-matured by SHM.Affinity gains by introduction of InDels have indeed been recognized (22, 25, 26, 32, 33) in in vitro-directed evolution, but often were by-products of campaigns focused on point mutations and not elicited systematically (32, 33). Only in mammalian cell surface display does the action of AID lead to InDels, just as AID brings about InDels in SHM in vivo (22, 25). In a seminal study by Bowers et al. (22), overexpression of AID enabled in vitro SHM of 53 antibodies against 21 antigens to identify InDels in multiple regions likely to improve binding, in particular to variable heavy domain (V_H) and variable light domain (V_L) CDR1, where 9 of 53 antibodies contained InDels. Despite the comprehensive nature of this study, AID-enabled insertions mirrored in vivo SHM and were therefore limited to direct duplication of adjacent sequences, not allowing the full exploration of length and sequence diversity in the insertions, and the low frequency of incorporation of in-frame InDels by AID (<0.1%) limited the combinatorial diversity explored. Finally, InDels have been introduced rationally based on structural analysis and natural length variation (26, 27). Taken together, only limited diversity of InDels in terms of length, position, and insert sequence across the variable domains has been explored thus far.Here we address this omission and explore libraries with in-frame InDels of different lengths and high diversity of inserted sequences at random positions across the entire antibody variable regions (Fig. 1). We applied a new transposon-based mutagenesis approach, dubbed TRIAD (transposition-based random insertion and deletion mutagenesis) (34) that introduces short in-frame insertions and deletions randomly across a gene (in sequences of steps following transposition that excise the transposon, religate the plasmid, and insert designed cassettes) (SI Appendix, Figs. S1 and S2). TRIAD was used here to build libraries with InDels at random positions across an entire single-chain variable fragment (scFv) gene. The antibody chosen for this campaign was the anti–IL-13 antibody BAK1 (35), a derivative of which, tralokinumab, is under clinical investigation for asthma (36). In addition, we built libraries that explore diversity in the different lengths of insertions in a semirandom approach, insertional-scanning mutagenesis (InScaM). These InDel libraries were starting points for antibody affinity evolution in vitro, leading to insertions in two loops that, together with two previously known point mutations, brought about a 256-fold affinity improvement. The observation of alternative routes to affinity maturation validate our strategy and suggest that InDel mutagenesis can complement existing approaches.Open in a separate windowFig. 1.Overview of the affinity maturation of the antibody BAK1 by transposon-based TRIAD and subsequent insertional scanning mutagenesis. TRIAD (Left) was applied to make libraries with deletions of one to three amino acids (step 1a) or single amino acid insertions (step 1b) at random positions across the scFv gene. These libraries were recombined (step 2) and four rounds of ribosome display selections for improved affinity to IL-13 were carried out by panning (step 3). The best binder was carrying an insertion in the V_L FWR3 (BAK1-INS1). Scanning (Right) was used to guide the design of libraries with different lengths of insertions at targeted positions. A fraction of the insertion library generated in step 1b (5,632 variants) was screened by HTRF to identify variants with insertions that retained binding to IL-13 (step 4). Based on sequencing analysis, regions able to tolerate single amino acid insertions were identified (Fig. 4) and the V_L CDR3 was chosen for targeted insertional mutagenesis. Libraries with zero to five amino acid insertions in targeted positions in the V_L CDR3 were constructed (step 5), followed by four rounds of phage display selections for improved affinity to IL-13 (step 6).