Category Archives: Original Research

The utility of MLPA in Familial Hypercholesterolemia diagnosis

George Valeriu Moldovan1, Adina Huțanu2, Liliana Demian3, Laszlo Hadadi4, Bogdan Mănescu2, Minodora Dobreanu2

1. Microbiology Department, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania
2. Laboratory Medicine Department, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania
3. Center for Advanced Medical and Pharmaceutical Research, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania
4. Emergency Institute for Cardiovascular Diseases and Transplantation Targu Mures, Romania

DOI: 10.2478/amma-2021-0021

Background: Familial Hypercholesterolemia (FH) is an inherited disease, associated with an increased risk of atherosclerosis, manifested clinically as premature coronary heart disease. FH is biochemically characterized by increased Cholesterol and Low-density Lipoprotein Cholesterol serum levels. The diagnosis is often made using clinical scores however, the definitive FH diagnosis should point out the underlying molecular change, which can be: a point mutation within the three major genes, a number of single nucleotide polymorphisms determining the polygenic etiology, or copy number variations in the Low-density lipoprotein receptor gene.
Objective: In the present study we investigated copy number variations as a possible etiological factor for FH in a cohort of patients with documented premature coronary heart disease.
Methods: The study population consisted of 150 patients with premature coronary heart disease documented by angiography, all being under lipid-lowering therapy, and 20 apparently healthy controls. Serum lipids were assessed using the Cobas Integra 400 plus and commercial reagents. Copy number variations were evaluated with the SALSA MLPA Probemix P062 LDLR kit.
Results: Cholesterol, Triglycerides, Low-density Lipoprotein Cholesterol and High-density Lipoprotein Cholesterol showed no difference between patients and controls. No copy number variations were detected in the investigated regions, namely all 18 exons and the promoter region of the Low-density lipoprotein receptor gene.
Conclusions: Even in the presence of negative results, the Familial Hypercholesterolemia genetic diagnosis has to be further pursued in the presence of a clinical diagnosis, as the identification of the molecular etiology may bring additional clinical and therapeutical benefits, as well as open the possibility for “cascade screening”.

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Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

Monica Vuță1, Ionela-Maria Cotoi1, Ion Bogdan Mănescu2, Doina Ramona Manu3, Minodora Dobreanu1,2,3

1. George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania
2. County Emergency Clinical Hospital of Targu Mureș, Romania
3. Center for Advanced Medical and Pharmaceutical Research, Targu-Mures, Romania

DOI: 10.2478/amma-2021-0012

Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be stimulated directly after isolation or frozen for later use. However, cryopreservation may affect cell recovery, viability and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells.
Methods: PBMCs were obtained by Ficoll gradient centrifugation from heparinized peripheral blood of 6 middle-aged clinically healthy subjects. Half of these cells (labeled “Fresh”) was further processed and the other half (labeled “Cryo”) was cryopreserved at -140°C for up to 3 months. Fresh-PBMCs were activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin for 5 hours immediately after isolation while Cryo-PBMCs were identically activated after thawing and cell resting. Activated cells were fixed, permeabilized and intracellular cytokine staining was performed using Phycoerythrin (PE)-conjugated antibodies for Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), and Interferon-gamma (IFN-g). All samples were analyzed within 24 hours by flow cytometry.
Results: Both Fresh and Cryo CD3+CD4+/CD3+CD4- subpopulations partially produced each of the three cytokines. A higher percentage of CD4+ T cells produced IL-2 and TNF-a and a greater percentage of CD4- T cells were found to produce IFN-g. A significantly higher percentage of Cryo-lymphocytes was shown to produce TNF-a in both CD3+CD4+ (31.4% vs 24.9%, p=0.031) and CD3+CD4- (22.7% vs 17.9%, p=0.031) subpopulations. No notable difference was found for IL-2 and IFN-g production between Fresh and Cryo T cells.
Conclusion: Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

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Evaluation of arterial stiffness in systolic heart failure

Lajos Fehérvári1, István Adorján Szabó2, Lóránd Kocsis1, Attila Frigy3

1. Department of Cardiology, Clinical County Hospital Mures, Targu Mures, Romania
2. Department of Pathophysiology, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania
3. Department of Internal Medicine IV, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania

DOI: 10.2478/amma-2021-0009

Objective: Micro- and macrovascular changes can occur in heart failure, and could influence its prognosis and management. In a prospective study, we proposed the evaluation of arterial stiffness (macrovascular function) and its correlations in patients with systolic heart failure.
Methods: 40 patients (32 men, 8 women, mean age 63±2.9 years), with hemodynamically stable systolic heart failure (left ventricular ejection fraction, EF<40%) were enrolled in the study. In every patient, beyond routine explorations (ECG, cardiac and carotid ultrasound, laboratory measurements), arterial stiffness was assessed by measuring pulse wave velocity (PWV). The correlations of PWV with clinical and echocardiographic characteristics were studied using t-test and chi-square test (p<0.05 being considered for statistical significance).
Results: The average PWV was 8.55±2.2 m/s, and 16 patients had increased PWV (>10 m/s). We found significantly higher PWV values in patients older than 65 years (p<0.001), in patients with eGFR <60 ml/min/1.73 m2 (p<0.001), hypertension (p=0.006), and increased (>1 mm) carotid intima-media thickness (p=0.016). PWV was found to be significantly lower when EF was <30% (p=0.049). Furthermore, the presence of an increased PWV was correlated significantly with age (p<0.001), and (with borderline significance) with eGFR <60 ml/min/1.73 m2 and, inversely, with EF<30%.
Conclusions: Increased arterial stiffness reflected by high PWV is frequently present in patients with systolic heart failure, and is mainly correlated with general risk factors of arterial involvement. Low EF, due to low stroke volume and decreased systolic arterial wall tension can influence the values and the interpretation of PWV.

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Formulation and Evaluation of Sustained Release Ibuprofen Matrix Tablets Using Starch from Maize Genotypes as Polymer

Lateef Gbenga Bakre1, Emmanuel Akinyele1, Oluyemisi Bamiro1, Olutayo Adeleye1, Olobayo Kunle2

1. Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Olabisi Onabanjo University, Nigeria
2. Department of Pharmaceutical Technology and Raw Materials Development, National Institute for Pharmaceutical Research and Development, Nigeria

DOI: 10.2478/amma-2021-0018

Objective: Maize plants have been genetically engineered to produce genotypes with agriculturally desirable traits such as high starch content, pest resistance and increased nutritional value. Maize starch has been widely used as an excipient in pharmaceutical formulations. This study aims to produce sustained release ibuprofen tablets using starch obtained from different maize genotypes as polymers.
Methods: Ibuprofen matrix tablets were prepared with the starches isolated from the maize genotypes and the unmodified plant. The mechanical properties of the tablets were evaluated using the crushing strength (CS), friability (FR) and CSFR. A 32 factorial design was applied using the time taken for 50 % (T50) and 90 % (T90) drug release as dependent variables while the polymer-drug ratio and polymer types were the independent variables.
Results: The CSFR was significantly higher (p<0.05) in tablets formulated with the starches obtained from the modified cultivars. Drug release for all the formulations fitted the Higuchi model while the mechanism of release was generally by super case transport. The polymer-drug ratio and polymer type strongly interacted to increase the dissolution times (T50 and T90) and CSFR. Starches isolated from the genetically modified cultivars provided a more sustained release of ibuprofen from the tablet matrix through erosion and polymer relaxation.
Conclusion: The results indicate that the genetic modification of maize can quantitatively affect the drug release modifying effects of maize starch in drug formulation.

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Beneficial effects of metformin on haloperidol-induced motor deficits in rats. A behavioral assessment

George Jîtcă1,2, Zsolt Gáll1, Camil E Vari1, Bianca E Ősz1, Amelia Tero-Vescan3, Alexandra Groșan1, Maria T. Dogaru1

1. Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania
2. Doctoral School of Medicine and Pharmacy, I.O.S.U.D., George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania
3. Department of Biochemistry, Faculty of Pharmacy, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania

DOI: 10.2478/amma-2021-0017

Objective: One of the most common side effects of haloperidol is the extrapyramidal syndrome, resulting from inhibition of nigrostriatal dopaminergic circuits and mitochondrial dysfunction due to structural similarities to pyridinium derivative, MPP+ that induce oxidative stress. In exchange, the use of metformin appears to enhance neurogenesis, energy metabolism, and oxidative status, so these properties can be speculated in the context of drug-induced pseudoparkinsonism by haloperidol.
Methods: To assess motor coordination and activity, rodents were divided into four groups: CTR (n = 10) – animals that received distilled water, METF (n = 10) – animals that received metformin 500 mg / kgbw, HAL (n = 10) – animals that received haloperidol 2mg / kgbw, HALMETF (n = 10) – haloperidol 2mg / kgbw and metformin 500 mg / kgbw. The treatment was administered for 34 days at the same time by gastric gavage, during which time behavioral tests, rotarod (days 7, 14, 21, 28), catalepsy (day 30), open field (day 32) and novel object recognition (day 34) were performed.
Results: The monitored parameters, showed significant differences between the groups of interest (HAL and HALMETF respectively), so that the administration of metformin at the beginning of treatment reduces the cataleptic behavior. The HALMETF group shows an attenuation of the motor deficit during the rotarod test and the freezing period from the Open Field test, is diminished.
Conclusions: Metformin treatment has a beneficial effect in haloperidol-treated rats, demonstrated by decreased cataleptic behavior, improved motor performance and reduced haloperidol-induced anxiety behavior.

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High-risk morphological features are less prevalent among small (<5mm) papillary thyroid microcarcinomas compared to larger (≥5mm) tumors: a study of 206 cases

Emőke – Andrea Szász1, Adela Nechifor-Boilă1,2, Ancuța Zahan1, Angela Borda1,3

1. Department of Histology, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania
2. Department of Pathology, Targu Mures County Hospital, Targu­ Mures, Romania
3. Department of Pathology, Targu Mures Emergency County Hospital, Targu Mures, Romania

DOI: 10.2478/amma-2021-0016

Introduction: Papillary thyroid microcarcinoma (PTMC) is defined as a PTC measuring 1 cm or less, incidentally discovered. The aim of this study was to determine whether small (<5mm) tumors by contrast with large (≥5mm) ones are less frequently associated with high-risk morphological features, predictive of tumor aggressiveness.
Materials and methods: All consecutive PTMC cases registered at the Department of Pathology, Târgu-Mureş Emergency County Hospital between 2003-2014 were reviewed. The following have been assessed: tumor size, subcapsular versus nonsubcapsular location, extrathyroidal extension/invasion into the perithyroidal adipose tissue, multifocality, resection margins, lymph node involvement, histological variant, tumor border, stromal reaction (fibrosis/desmoplasia/sclerosis), presence of plump pink cells, nuclear features of the tumor cells, intratumoral lymphocytic infiltrate, multinucleated giant cells, psammoma bodies and stromal calcification. The cases were split in two categories: small (< 5mm) and large (≥ 5mm) PTMCs and the pathological features were evaluated in comparison.
Results: Our study included 206 cases, 91 large and 115 small PTMCs, respectively. Large PTMCs were significantly associated with the presence of plump pink cells (p=0.002), well developed PTC nuclear features (p=0.003), stromal reaction (fibrosis/desmoplasia/sclerosis) (p<0.001), infiltrative tumor border (p=0.011), subcapsular location (p<0.001), positive resection margins (p=0.022), stromal calcifications (p<0.001) and intratumoral multinucleated giant cells (p<0.001). Small PTMCs were generally well circumscribed and nonsubcapsular.
Conclusions: Our results have shown that small (<5mm) PTMCs are less frequently associated with high-risk morphological features, predictive of tumor aggressiveness compared with large (≥5mm) tumors and could thus be considered as low-risk cancers.

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How we did it – an easy and feasible experimental rat model of protective role of Lipid Emulsion in Ropivacaine induced Local Anesthetic Systemic Toxicity – technique presentation and preliminary results

Alexandra Lazar, Marcel Perian, Bogdan Cordoș, Mircea Gherghinescu, Bianca Liana Grigorescu

George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania

DOI: 10.2478/amma-2021-0014

Introduction: Local Anesthetic Systemic Toxicity (LAST) is the most feared local anesthesia accident. As the cardiac arrest determined by LAST is mostly refractory to known resuscitation protocols, due local anesthetic blockade produced in the cardiac cells, the Lipid Emulsion (L.E) has been proved to be beneficial in resuscitating the cardiac arrest determined by local anesthetic. The aim for this presentation is to ease future studies on this topic, to ensure a starting point for next related research on LAST and LE mechanism of action.
Method: Under genaral anesthesia we induced Local Anesthetic Systemic Toxicity  to a rat model, by injecting Ropivacaine into the inferior vena cava. We monitored the cardiac activity of the subjects during the experiment. We used 4 groups of rats, control group- no intervention, lipid group- lipid emulsion was adminsitered, local anesthetic group- local anesthetic was administered and local anesthetic and lipid emulsion group- a dose of lipid emulsion was adminsitered before administering the local anesthetic.
Results: After a few attemps to incannulate teh peripheral veins we tried the more complex approach of inferior vena cava, which ensured a secure access which allowed us to repetedly adminster the local anesthetic and the lipid emulsion.
Conclusion: The presented experimental animal model of induced LAST and the protective effects of LE is one of the few described in the literature, is a reproducible model, feasible, simple, low cost and can be used as starting point in future LAST research.

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A brief reflection on the role of cholesterol in psychopathology among female psychiatric patients

Edina Dimeny1, Erika Bán2, Attila Brassai2

1. George Emil Palade University of Medicine, Pharmacy, Science and Technology, Department of Pharmacology
2. Department of Pharmacology, Faculty of Medicine in English, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania

DOI: 10.2478/amma-2021-0015

Objective: Cholesterol is one of the cardiovascular risk factors, but also a core component of the central nervous system. Moreover, hypercholesterolemia and hypocholesterolemia are directly related to numerous mental illnesses too. This study intends to examine the association between cholesterol level and autolytic behavior among female psychiatric patients.
Methods: The present study involves 123 female subjects, who suffered from suicidal thoughts at the moment of hospitalization. The risk of suicidal intentions was assessed by the Modified Scale for Suicide Ideation (Miller et al) and their total serum cholesterol levels were measured. We performed a case-control, analytical, randomized, observational study at the Clinical Hospital of Neurology and Psychiatry Brasov among adult female psychiatric patients admitted during 2014.
Results: By our results we distinguished 3 categories: 38 patients with low suicide risk, 32 with moderate risk and 53 with high suicide risk. Significant difference can be noticed in the higher suicide risk patients’ blood cholesterol levels: 44 patients having under 4,5mmol/L total cholesterol level (83%). Although, in other two categories, this proportion is minimal: in the moderate-risk category were 8 patients, representing just 25 %, and in the low-risk category only 1 patient had her cholesterol level under 4,5mmol/L (2,6%).
Conclusions: According to our results, proposing cholesterol-level as a biomarker for the determination of high-risk suicide behavior can be important. The presence of other important risk factors (sociodemographic and psychiatric variables) can increase exponentially the suicide behavior. The limitations of this study are the relatively small number of cases and the lack of longitudinal subsequent follow-up. Further investigations are needed on a larger and more heterogenous sample of patients in order to clarify this suggestive correlation.

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Protocol modification proposed for congenital hypothyroidism screening programme in Romania

Imola Gyorfi1, Stefan Barbu1, Oana Oprea1,2, Minodora Dobreanu1,2

1. Emergency Clinical County Hospital Targu Mures, Romania
2. Department of Laboratory Medicine, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania

DOI: 10.2478/amma-2021-0011

Objective: In Romania, congenital hypothyroidism screening is performed by measuring thyroid-stimulating-hormone levels from dried blood samples. If the initial value is above the recommended cut-off value (10 mUI/L), the newborn is recalled for a second blood collection. The aim of this study was to investigate and report potential improvements on the screening protocol that is currently applied in our country in order to reduce the time between birth and treatment initiation in newborns positive to congenital hypothyroidism screening.
Methods: Blood samples were collected from 41 full-term newborns between February and March 2019 at the maternity ward from Targu Mures Emergency County Hospital. Thyroid-stimulating-hormone values were measured with a chemiluminescent microparticle immunoassay in serum samples from cord blood collected at birth, and with a fluorometric enzyme-linked immunoassay in dried blood spots collected at day 3-5 after birth. To obtain whole blood values, serum values were transformed using a formula supplied by the kit manufacturer. Calculated cord blood values were compared with dried blood spots values using the Wilcoxon test.
Results: After serum-to-whole-blood conversion,  cord blood values ranged from 2.58 to 3.66 mUI/L (95% CI). Dried blood spot values ranged from 6.70 to 7.50 mUI/L (95% CI). The Wilcoxon test p value between cord blood and dried blood spots thyroid-stimulating-hormone levels was statistically significant (p<0.01).
Conclusions: Thyroid-stimulating-hormone levels above the cut-off value were flagged by both techniques. An improvement to the existing protocol is proposed that may reduce time from positive screening results to confirmation of congenital hypothyroidism and treatment initiation.

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Romanian adaptation of Centrality of Religiosity Scale and the utility of this scale in cognitive-behavioral therapy for religious populations

Adrian V. Rus1, Alexander Clark2, Aurelian Petrus Plopeanu3, Elena Adriana Tomuletiu4, Anca Bejenaru5, Ecaterina Stativa6, Wesley C. Lee1, Sheri R. Parris7, Cosmin Octavian Popa8, Simona Muresan8

1. Department of Social and Behavioral Sciences, Southwestern Christian University, Bethany, OK, USA
2. College of Education and Professional Studies, University of Central Oklahoma, Edmond, OK, USA
3. Institute of Interdisciplinary Research, Department of Social Sciences and Humanities, Alexandru Ioan Cuza University of Iasi, Romania
4. “Dimitrie Cantemir” University of Tîrgu-Mures, Romania
5. Lucian Blaga University of Sibiu, Faculty of Social Sciences and Humanities, Department of Social Work, Journalism, Public Relations, and Sociology, Romania
6. The National Institute for Mother and Child Health “Alessandrescu-Rusescu”, Bucharest, Romania
7. Brite Divinity School, Fort Worth, Texas, USA
8. George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania

DOI: 10.2478/amma-2021-0010

Background: Cognitive-Behavioral Therapy is a psychotherapeutic intervention that proved to be an efficient treatment for a variety of psychiatric disorders. Religious features can be efficiently integrated within cognitive-behavioral approaches both during the assessment and treatment processes. The use of a specific instrument for measuring religious beliefs and attitudes may improve psychological intervention for a widely spectrum of mental disorders, including anxiety and psychosis.
Objective: The present study aims to establish a Romanian version of the Centrality of Religiosity Scale (CRS).
Method: The sample of this study included 134 undergraduate students from Romania (112 women and 22 men), aged between 18 and 46 years. The questionnaire was translated from English into Romanian by three independent translators, and then from Romanian into English by other three independent translators.
Results: Our findings indicate that the Romanian version of CRS demonstrated high internal consistency for all scales, with Cronbach alpha coefficients ranging from 0.749 to 0.881 for the individual subscales of the instrument.
Conclusion: The CRS is a valid instrument that can be used for assessing religious beliefs in Romanian population.

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