Assessing cytokines' talking patterns following experimental myocardial damage by applying Shannon's information theory.

BACKGROUND: The simultaneous measurement of multiple cytokines in parallel by using multiplex proteome arrays (MPA) is of great interest to understanding the inflammatory response following myocardial infarction; however, since cytokines are pleiotropic and redundant, increase of information throughput (IT) attained by measuring multiple cytokines remain to be determined. We aimed this study to assess the IT of an MPA system designed to assess 8 cytokines - commercially available at the time of the study - serum levels, before (control state) and after experimental myocardial cryoinjury (activated state) in rats. METHODS: By assuming that redundant information do not generally increase the IT, we derived Entropy (H) and Redundancy (R) of information by using formulas of Shannon modified accordingly, where a high IT (high H and low R) corresponds to a low level of correlation between cytokines and vice versa for a low IT. The maximum theoretical level of IT and the contribution of each cytokine were also estimated. RESULTS: In control state, no significant correlations were found between cytokines showing high IT; on the contrary, in activated state, several significant correlations were found supporting a complex cross-talk pattern between cytokines with low IT. Using as reference the maximum theoretical level of IT, in activated state, H was reduced of 67.0% and R was increased of 77.4% supporting a reduction of IT. Furthermore, the contribution of individual cytokines to H value of MPA was variable: in control state, IL-2 gave the most contribution to H value, conversely during activated state IL-10 gave most contribution. Finally during activated state, IL-1ß was the only cytokine strongly correlated with values of all other cytokines, suggesting a crucial role in the inflammatory cascade. CONCLUSIONS: Paradoxically, by analyzing an MPA system designed for redundant analytes such as cytokines, translating the Shannon's information theory from the field of communication to biology, the IT system in our model deteriorates during the activation state by increasing its redundancy, showing maximum value of entropy in the control conditions. Finally, the study of the mutual interdependence between cytokines by the contribution to the IT may allow formulating alternative models to describe the inflammatory cascade after myocardial infarction.

Fine-tuning of membrane permeability by reversible photoisomerization of aryl-azo derivatives of thymol embedded in lipid nanoparticles.

Responsiveness of liposomes to external stimuli, such as light, should allow a precise spatial and temporal control of release of therapeutic agents or ion transmembrane transport. Here, some aryl-azo derivatives of thymol are synthesized and embedded into liposomes from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine to obtain light-sensitive membranes whose photo-responsiveness, release behaviour, and permeability towards Cl- ions are investigated. The hybrid systems are in-depth characterized by dynamic light scattering, atomic force microscopy and Raman spectroscopy. In liposomal bilayer the selected guests undergo reversible photoinduced isomerization upon irradiation with UV and visible light, alternately. Non-irradiated hybrid liposomes retain entrapped 5(6)-carboxyfluorescein (CF), slowing its spontaneous leakage, whereas UV-irradiation promotes CF release, due to guest trans-to-cis isomerization. Photoisomerization also influences membrane permeability towards Cl- ions. Data processing, according to first-order kinetics, demonstrates that Cl- transmembrane transport is enhanced by switching the guest from trans to cis but restored by back-switching the guest from cis to trans upon illumination with blue light. Finally, the passage of Cl- ions across the bilayer can be fine-tuned by irradiation with light of longer λ and different light-exposure times. Fine-tuning the photo-induced structural response of the liposomal membrane upon isomerization is a promising step towards effective photo-dynamic therapy.

Extracellular matrix proteins and displacement of cultured fibroblasts from duodenal biopsies in celiac patients and controls.

BACKGROUND: Celiac disease (CD) is mainly characterised by villous atrophy and mucosal architectural rearrangement. The fibroblasts (FBs) are the most abundant mesenchymal cell type in the intestinal mucosa and are responsible for both the architectural arrangement of the villi and the formation of the extracellular matrix (ECM). This study aimed at the evaluation of both the intracellular distribution of different proteins involved in ECM and FBs characterisation, and the cellular displacement of primary FBs obtained from duodenal endoscopic biopsies of healthy subjects and celiac patients. METHODS: Primary healthy and celiac duodenal FBs were evaluated by means of immuno-fluorescence assay for collagen type I and IV, fibronectin, actin, alpha-Smooth Muscle Actin (alpha-SMA), Fibroblast Surface Protein (FSP) and transglutaminase type 2 (TG2). The geometric indexes of the fluorescence signals were investigated by image analysis software (Image J, NIH). Both morphology and kinetic were evaluated during a 72 hours time course movie. TG2 medium activity was evaluated by means of ELISA. RESULTS: All the cells examined were immunopositive for FSP, alpha-SMA, actin, collagen I, collagen IV and TG2. CD cells showed a signet collagen-I and collagen-IV pattern, as compared to the controls being characterised by a spindle geometry. Moreover, the collagen signals in CD FBs showed a significantly higher circularity index (major orthogonal diameter ratio) than the controls (p<0.0001), whereas the perimeter and area ratio were significantly lower (p<0.0001). The TG2 signal had a decreased area (p<0.05), but a two-fold increased medium activity. The time course highlighted a reduction of the displacement of CD FBs. CONCLUSIONS: The isolated primary CD FBs showed a different collagen and TG2 pattern of distribution associated with a different cellular displacement. The reasons for such CD cell peculiar characteristics are yet unknown but they might represent a factor in the progression of the intestinal damage.

Healthcare Systems across Europe and the US: The Managed Entry Agreements Experience.

This systematic study aims at analyzing the differences between the approach of the European healthcare systems to the pharmaceutical market and the American one. This paper highlights the opportunities and the limitations given by the application of managed entry agreements (MEAs) in European countries as opposed to the American market, which does not regulate pharmaceutical prices. Data were collected from the Organisation for Economic Co-operation and Development (OECD), the European Medicines Agency, and the national healthcare agencies of US and European countries. A literature review was undertaken in PubMed, Scopus, MEDLINE, and Google for a period ten years (2010-2019). The period 2020-2021 was considered to compare health expenditure before and after the SARS-CoV-2 pandemic. Scarce information from national agencies has been given in terms of MEAs related to the COVID-19 pandemic. The comparison between the United States approach and the European one shows the importance of a market access regulation to reduce the cost of therapies, increasing the efficiency of national healthcare systems and the advantages in terms of quality and accessibility to the final users: patients. Nevertheless, it seems that the golden age of MEAs for Europe was during the examined period. Except for Italy, countries will move to other forms of reimbursements to obtain higher benefits, reducing the costs of an inefficient implementation and outcomes in the medium term.

The prognostic value of heart rate variability in the elderly, changing the perspective: from sympathovagal balance to chaos theory.

Heart rate variability (HRV) is the temporal beat-to-beat variation in successive RR intervals on an electrocardiographic (ECG) recording and it reflects the regulation of the heart rate (HR) by the autonomic nervous system (ANS). HRV analysis is a noninvasive tool for the assessment of autonomic function that gained momentum in the late 1980s when its clinical relevance as a predictor of mortality was established by a milestone study by Kleiger et al. in patients with postacute myocardial infarction. In the last few decades, the increasing availability of commercial ECG devices offering HRV analysis has made HRV a favorite marker for risk stratification in the setting of cardiovascular disease. The rapid aging of the world population and the growing popularity of HRV have also fueled interest for the prognostic value of HRV in the elderly, outside a specific cardiological context. However, the discussion of HRV measures in the elderly is still very much centered on the rather reductionistic model of sympathovagal balance, with the orthosympathetic and parasympathetic limbs of the ANS exercising opposing effects on the heart via autonomic tone. The expanding application of nonlinear dynamics to medicine has brought to the forefront the notion of system complexity, embedded in the mathematical concepts of chaos theory and fractals, and provides an opportunity to suggest a broader interpretation for the prognostic significance of HRV, especially in the elderly. Although the use of novel indices of HRV may be hampered by practical issues, a more holistic approach to HRV may still be safeguarded if traditional time- and frequency-domain measures are viewed in terms of autonomic modulation. This review focuses on HRV in geriatric populations. It considers studies on the prognostic value of HRV in elderly subjects, discussing the potential confounding effect of erratic rhythm, and concentrates on the conceptual distinction between autonomic tone and autonomic modulation. It also briefly addresses the question of the practicality of ECG recordings and identifies a promising area for future research in the effects of common noncardioactive drugs on HRV.

Preparation, Characterization, and Biological Evaluation of a Hydrophilic Peptide Loaded on PEG-PLGA Nanoparticles.

The encapsulation of peptides and proteins in nanosystems has been extensively investigated for masking unfavorable biopharmaceutical properties, including short half-life and poor permeation through biological membranes. Therefore, the aim of this work was to encapsulate a small antimicrobial hydrophilic peptide (H-Ser-Pro-Trp-Thr-NH2, FS10) in PEG-PLGA (polyethylene glycol-poly lactic acid-co-glycolic acid) nanoparticles (Nps) and thereby overcome the common limitations of hydrophilic drugs, which because they facilitate water absorption suffer from rapid degradation. FS10 is structurally related to the well-known RNAIII inhibiting peptide (RIP) and inhibits S. aureus biofilm formation. Various parameters, including different method (double emulsion and nanoprecipitation), pH of the aqueous phase and polymeric composition, were investigated to load FS10 into PEG-PLGA nanoparticles. The combination of different strategies resulted in an encapsulation efficiency of around 25% for both the double emulsion and the nanoprecipitation method. It was found that the most influential parameters were the pH­which tailors the peptides charge­and the polymeric composition. FS10-PEG-PLGA nanoparticles, obtained under optimized parameters, showed size lower than 180 nm with zeta potential values ranging from −11 to −21 mV. In vitro release studies showed that the Nps had an initial burst release of 48−63%, followed by a continuous drug release up to 21 h, probably caused by the porous character of the Nps. Furthermore, transmission electron microscopy (TEM) analysis revealed particles with a spherical morphology and size of around 100 nm. Antimicrobial assay showed that the minimum inhibitory concentration (MIC) of the FS10-loaded Nps, against S. aureus strains, was lower (>128 µg/mL) than that of the free FS10 (>256 µg/mL). The main goal of this work was to develop polymeric drug delivery systems aiming at protecting the peptide from a fast degradation, thus improving its accumulation in the target site and increasing the drug-bacterial membrane interactions.

Beneficial effects of treatment with transglutaminase inhibitor cystamine on the severity of inflammation in a rat model of inflammatory bowel disease.

Inflammatory bowel disease (IBD) represents a socially and clinically relevant disorder, characterized by intestinal chronic inflammation. Cystamine (CysN) is a multipotent molecule with healthy effects and, moreover, it is an inhibitor of transglutaminases (TGs), including the TG type 2 (TG2), an enzyme with pleiotropic functions, involved in different pathways of inflammation and central in the pathogenesis of some human disorders as the IBD. Our aim was to evaluate the effect of CysN in an IBD rat model. A total of 30 rats were divided into 4 groups: controls without treatment (CTR; n=7); receiving the 2,4,6-trinitrobenzene sulfonic acid enema (TNBS group; n=8); treated with TNBS enema plus oral CysN (TNBS-CysN group; n=8); treated with CysN (CysN group; n=7). After killing, bowel inflammation was evaluated applying specific scores. TG activity, TG2 and isopeptide bond immunohistochemical expression, and tumor necrosis factor-α (TNF-α) were evaluated in the colonic tissue, such as interleukin-6 (IL-6) serological levels (ELISA). TG2 was also evaluated on the luminal side of the colon by immunoautoradiography. Colonic samples from IBD patients were compared with animal results. TNBS-CysN group developed a less severe colitis compared with the TNBS group (macroscopic score 0.43±0.78 vs 3.28±0.95, microscopic score 6.62±12.01 vs 19.25±6.04, P<0.05, respectively) associated with a decrease of TG activity, TG2 and isopeptide bond immunohistochemical expression, TNF-α and IL-6 levels. No statistically significant differences were found between CysN and CTR groups. The colonic immunolocalization of TG2 was comparable in humans affected by IBD and TNBS-administered animals. This is the first demonstration that treatment with a CysN has an anti-inflammatory effect, reducing severity of colitis in a rat model. CysN could be tested as a possible treatment or co-treatment in IBD therapeutic trials.

Absence of mucosal inflammation in uncomplicated diverticular disease.

BACKGROUND: Uncomplicated diverticular disease is a common condition in patients older than 50 years. Symptoms are aspecific and overlapping with those of irritable bowel syndrome. Nowadays, patients are often treated with antinflammatory drugs (5-aminosalicilic acid). AIM: Our purpose was to evaluate the presence of inflammation in the colonic mucosa of patients with symptomatic uncomplicated diverticular disease compared with subjects without diverticula. METHODS: Endoscopic biopsies of colon from 10 patients with symptomatic uncomplicated diverticular disease and 10 from subjects without diverticula (controls) were taken. Specimens were homogenised and IL2, IL4, IL5, IL8, IL10, IL12p70, IL13, IFN gamma, TNF alfa (searchlight multiplex technique), TGF beta, transglutaminase type 2 and caspase 9 were measured. Histochemistry for transglutaminase type 2 and TUNEL were performed on the histological sections, in addition to morphologic evaluation, as markers of tissue remodelling and apoptosis. For statistical analysis Student's t test and Spearman correlation test were used. RESULTS: No histological differences were detected between the patients with an uncomplicated diverticular disease and controls. Mean values of mucosal cytokines and of the other tested parameters did not show statistically significant differences between patients with uncomplicated diverticular disease and controls. CONCLUSIONS: Even if based on a small number of patients, the study demonstrates the absence of inflammation in the mucosa of subjects affected by uncomplicated diverticular disease.

Predictability in Contemporary Medicine.

Medical practice is increasingly coming under the guidance of statistical-mathematical models that are, undoubtedly, valuable tools but are also only a partial representation of reality. Indeed, given that statistics may be more or less adequate, a model is still a subjective interpretation of the researcher and is also influenced by the historical context in which it operates. From this opinion, I will provide a short historical excursus that retraces the advent of probabilistic medicine as a long process that has a beginning that should be sought in the discovery of the complexity of disease. By supporting the belonging of this evolution to the scientific domain it is also acknowledged that the underlying model can be imperfect or fallible and, therefore, confutable as any product of science. Indeed, it seems non-trivial here to recover these concepts, especially today where clinical decisions are entrusted to practical guidelines, which are a hybrid product resulting from the aggregation of multiple perspectives, including the probabilistic approach, to disease. Finally, before the advent of precision medicine, by limiting the use of guidelines to the original consultative context, an aged approach is supported, namely, a relationship with the individual patient.

Boron-based hybrids as novel scaffolds for the development of drugs with neuroprotective properties.

Novel boron-based compounds (BBCs) were synthesized and evaluated as potential candidates for the development of novel drugs against Alzheimer's disease (AD). The neuroprotective profile of novel BBCs was evaluated using Aß1-42-treated-SH-SY5Y cells while their antioxidant activity was evaluated by total antioxidant capacity (TAC) and total oxidative status (TOS) assays. Results showed that BLA (a novel boron-based hybrid containing an antioxidant portion) inhibited cell death induced by Aß1-42-exposure in differentiated SH-SY5Y cells, resulting in an increase in cell viability by 25-33% (MTT assay) and by 63-71% (LDH assay) in a concentration range of 25-100 µM. Antioxidant assays demonstrated a good capability of BLA to counteract the oxidative status. Moreover, BLA possessed a significant ability to inhibit acetylcholinesterase (AChE) (22.96% at 50 µM), an enzyme whose enzymatic activity is increased in AD patients. In the present work, absorption and distribution properties of boron-based hybrids were predicted using Pre-ADMET software. In vitro preliminary results suggested that boron-based hybrids could be new structural scaffolds for the development of novel drugs for the management of AD.

Anticancer effects of novel NSAIDs derivatives on cultured human glioblastoma cells.

Several epidemiologic, clinical and experimental reports indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) could have a potential as anticancer agents. The aim of this study was the evaluation of cytotoxic potential in human glioblastoma cells of novel synthesized NSAID derivatives, obtained by linking, through a spacer, α-lipoic acid (ALA) to anti-inflammatory drugs, such as naproxen (AL-3, 11 and 17), flurbiprofen (AL-6, 13 and 19) and ibuprofen (AL-9, 15 and 21). The effects on the level of gene expression were also determined using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. According to our results, NSAID derivatives exhibited concentration dependent cytotoxic effects on U87-MG cell line when compared with the control group. Moreover, treatment of the most active compounds (AL-3, AL-6 and AL-9) caused upregulation of tumor suppressor gene PTEN and downregulation of some oncogenes such as AKT1, RAF1 and EGFR. In conclusion, our results revealed that AL-3, AL-6 and AL-9 could be suitable candidates for further investigation to develop new pharmacological strategies for the prevention of cancer.

PHoral: Effects of carnosine supplementation on quantity/quality of oral salivae in healthy volunteer and in subjects affected by common oral pathologies.

BACKGROUND: Diseases of the oral cavity (OC) with an infectious trigger such as caries and periodontal disease are extremely common in the general population and can also have effects at the cardiovascular level. The oral salivary flow, with its buffering capacity, is able to regulate the pH of the OC and, therefore, significantly contribute to the ecological balance of the microenvironment in which the oral microbiome (OM) develops. On the other side, when the quality/quantity of salivary flow is altered it is supposed the disruption of this balance with the potential increase in oral pathogens and triggered diseases. Among the endogenous substances able to exert a significant effect on the salivary flow and its characteristics, carnosine (Car), a dipeptide originally isolated in skeletal muscle, represents, thanks to the known buffering properties, a promising principle. METHODS: We aimed this protocol to evaluate the quantitative/qualitative characteristics of the salivary flow in healthy volunteer subjects (n = 20) and in subjects suffering from common OC pathologies (n = 40), before and after 7 days of supplementation with SaliflussTM (Metis Healthcare srl, Milan, Italy), a Class I medical device on the market as 400 mg mucoadhesive oral tablets that has Car as the main ingredient. DISCUSSION: Combining the characteristics of saliva with the OM and comparing them with OC pathologies, we expect to clarify their reciprocal relationship and, using quantitative proteomics techniques, to help clarify the mechanism of action of Car.

Calixarene-based artificial ionophores for chloride transport across natural liposomal bilayer: Synthesis, structure-function relationships, and computational study.

An amphiphilic calix[6]arene, alone or complexed with an axle to form a pseudo-rotaxane, has been embedded into liposomes prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the permeability of the membrane-doped liposomes towards Cl- ions has been evaluated by using lucigenin as the fluorescent probe. The pseudo-rotaxane promotes transmembrane transport of Cl- ions more than calix[6]arene does. Surprisingly, the quenching of lucigenin was very fast for liposomes doped with the positively charged axle alone. Molecular dynamics (MD) simulations and quantum-chemical calculations were also carried out for providing a semi-quantitative support to the experimental results.

History repeating. The plague of 1630 in Milan and the COVID-19 pandemia.

In western democracies, individual behaviour will be crucial to control the spread of COVID-19, as well as government actions [1] that unfortunately, except China, South Korea and Italy, followed by others,  seems to be generally unconvinced and, speculatively, late. Indeed human history has been marked by epidemics/pandemics which have affected, more or less, large geographical areas [2]. Italy, as well as the rest of Europe, has often been affected by these phenomena and, Lombardy, due to his position, was, as today by COVID-19, severely stroked in Italy that is, after China, the second most affected country [3]. This is also linked to the position of Lombardy and its capital, Milan, but this is beyond this brief comment. There are several differences between the past plagues and the actual COVID-19 pandemic and these must be sought in the increased ability to transmit diseases at-distance through the mobility of humans and goods [4], and in the catastrophic consequences of the breakdown of ecosystems, as told, a few years ago, by David Quammen in the book Spillover [5].

Umbelliprenin as a novel component of the phytochemical pool from Artemisia spp.

Plants belonging to Artemisia spp. are known to biosynthesize a wide panel of 3,3-dimethylallyl- and sesquiterpenyl- substituted coumarins. In this short communication we applied a novel extraction methodology based on the use of subcritical butane under a counter-current mode to further characterize the presence of selected biologically active oxyprenylated phenylpropanoids, namely coumarins and ferulic acid derivatives, in extracts deriving from aerial parts of Artemisia vulgaris L. (commonly known as "common mugwort") (Asteraceae). In the mean time, we assessed the efficiency of the above mentioned extractive methodology with other routes like maceration and ultrasounds and microwaves-based methods using absolute EtOH as the solvents. UHPLC analysis coupled to UV/Vis detection revealed that, among the 5 pure chemical standard assayed, only umbelliprenin (7-farnesyloxycoumarin) was recorded, while boropinic acid, 4'-geranyloxyferulic acid, 7-isopentenyloxycoumarin, and auraptene were not detected. The best extractive yield (0.18 %) was obtained after extaction with subcritical butane. The presence of umbelliprenin in Artemisia plant species has been reported herein for the first time. This coumarin may represent the biosynthetic precursors of sesquiterpenyloxycoumarins with more complex structures typically found in this genus.

Novel anti-Alzheimer phenol-lipoyl hybrids: Synthesis, physico-chemical characterization, and biological evaluation.

To date, drugs that hit a single target are inadequate for the treatment of neurodegenerative diseases, such as Alzheimer's or Parkinson's diseases. The development of multitarget ligands, able to interact with the different pathways involved in the progession of these disorders, represents a great challenge for medicinal chemists. In this context, we report here the synthesis and biological evaluation of phenol-lipoyl hybrids (SV1-13), obtained via a linking strategy, to take advantage of the synergistic effect due to the antioxidant portions and anti-amyloid properties of the single constituents present in the hybrid molecule. Biological results showed that SV5 and SV10 possessed the best protective activity against Aß1-42 induced neurotoxicity in differentiated SH-SY5Y cells. SV9 and SV10 showed remarkable antioxidant properties due to their ability to counteract the damage caused by H2O2 in SHSY-5Y-treated cells. Hovewer, SV5, showing moderate antioxidant and good neuroprotective activities, resulted the best candidate for further experiments since it also resulted stable both simulated and plasma fluids.

Isolation and culture of fibroblasts from endoscopic duodenal biopsies of celiac patients.

BACKGROUND: Fibroblasts are actually considered pivotal in inflammation and tissue remodelling process and for these reasons they are involved in the pathogenesis of autoimmune disorders such as celiac disease. Investigations to define the role of fibroblasts in celiac diseases are obstructed by the absence of specific models. Our objective is to isolate and culture primary fibroblasts from endoscopic duodenal biopsies of celiac and non-celiac subjects, to analyze their growth patterns and the morphometric characteristics. METHODS: 60 duodenal bioptic specimens from 20 celiac patients and 114 from 38 non-celiac subjects were mechanically chopped and enzymatically digested in order to obtain primary cell cultures. Growth patterns, karyotype (Q-banding analysis), expression of typing proteins (fibroblast surface protein and cytokeratin 20) and morphometric parameters (diameters and their ratio, perimeter, area and perimeter/area ratio at computerised image analysis) were investigated on cultured cells. RESULTS: Primary cells were successfully cultured in 78% of the collected duodenal biopsies. Cultured cells, expressing the fibroblast surface protein, were negative for cytokeratine 20 and maintained a normal kariotype. Cells grew slowly without differences between the celiac and the non celiac group. Morphometric analysis of celiac fibroblasts revealed significantly increased dimensions, with a preserved diameters ratio, and a reduced perimeter/area ratio. CONCLUSION: For the first time this study demonstrates the feasibility of culturing primary fibroblast cell from endoscopic duodenal biopsies in celiac and non-celiac subjects, opening a new window of opportunity in studies intended to establish the role of fibroblasts as a possible partaker in the pathogenesis of the celiac mucosal damage.

Time arrow in published clinical studies/trials indexed in MEDLINE: a systematic analysis of retrospective vs. prospective study design, from 1960 to 2017.

Clinical studies/trials are experiments or observations on human subjects considered by the scientific community the most appropriate instrument to answer specific research questions on interventions on health outcomes. The time-line of the observations might be focused on a single time point or to follow time, backward or forward, in the so called, respectively, retrospective and prospective study design. Since the retrospective approach has been criticized for the possible sources of errors due to bias and confounding, we aimed this study to assess if there is a prevalence of retrospective vs. prospective design in the clinical studies/trials by querying MEDLINE. Our results on a sample of 1,438,872 studies/trials, (yrs 1960-2017), support a prevalence of retrospective, respectively 55% vs. 45%. To explain this result, a random sub-sample of studies where the country of origin was reported (n = 1,576) was categorized in high and low-income based onthe nominal Gross Domestic Product (GDP) and matched with the topic of the research. As expected, the absolute majority of studies/trials are carried on by high-income countries, respectively 86% vs. 14%; even if a slight prevalence of retrospective was recorded in both income groups, for the most part prospective studies are carried out by high-GDP countries, 85% vs. 15%. Finally, the differences in the design of the study are understandable when considering the topic of the research.