Category Archives: AMM 2021, Volume 67, Number 2

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

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

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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Three-dimensional technologies used for patient specific applications in orthopedics

DOI: 10.2478/amma-2021-0020

Background: Three-dimensional (3D) technologies have numerous medical applications and have gained a lot of interest in medical world. After the advent of three-dimensional printing technology, and especially in last decade, orthopedic surgeons began to apply this innovative technology in almost all areas of orthopedic traumatic surgery.
Objective: The aim of this paper is to give an overview of 3D technologies current usage in orthopedic surgery for patient specific applications. Methods: Two major databases PubMed and Web of Science were explored for content description and applications of 3D technologies in orthopedic surgery. It was considered papers presenting controlled studies and series of cases that include descriptions of 3D technologies compatible with applications to human medical purposes.
Results: First it is presented the available three-dimensional technologies that can be used in orthopedic surgery as well as methods of integration in order to achieve the desired medical application for patient specific orthopedics. Technology starts with medical images acquisition, followed by design, numerical simulation, and printing. Then it is described the state of the art clinical applications of 3D technologies in orthopedics, by selecting the latest reported articles in medical literature. It is focused on preoperative visualization and planning, trauma, injuries, elective orthopedic surgery, guides and customized surgical instrumentation, implants, orthopedic fixators, orthoses and prostheses.
Conclusion: The new 3D digital technologies are revolutionizing orthopedic clinical practices. The vast potential of 3D technologies is increasingly used in clinical practice. These technologies provide useful tools for clinical environment: accurate preoperative planning for cases of complex trauma and elective cases, personalized surgical instruments and personalized implants. There is a need to further explore the vast potential of 3D technologies in many other areas of orthopedics and to accommodate healthcare professionals with these technologies, as well as to study their effectiveness compared to conventional methods.

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Advantages of lung ultrasound in triage, diagnosis and monitoring COVID-19 patients: review

DOI: 10.2478/amma-2021-0019

Over the last decades, especially during the COVID-19 pandemic period, lung ultrasound (LUS) gained interest due to multiple advantages: radiation-free, repeatable, cost-effective, portable devices with a bedside approach. These advantages can help clinicians in triage, in positive diagnostic, stratification of disease forms according to severity and prognosis, evaluation of mechanically ventilated patients from Intensive Care Units, as well as monitoring the progress of COVID-19 lesions, thus reducing the health care contamination. LUS should be performed by standard protocol examination. The characteristic lesions from COVID-19 pneumonia are the abolished lung sliding, presence of multiple and coalescent B-lines, disruption and thickening of pleural line with subpleural consolidations. LUS is a useful method for post-COVID-19 lesions evaluation, highlight the remaining fibrotic lesions in some patients with moderate or severe forms of pneumonia.

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

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

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

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

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

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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