Nanoparticle-Modified Apoferritin Nanotransfer for Targeted Cytostatic Transport.

BACKGROUND: Ferritin is a globular intracellular protein that acts as the main reservoir for iron. Malignancies are associated with increased plasma ferritin concentrations. A number of studies show that tumor cells express high levels of transferrin receptors (TfR). Increased TfR expression was observed in prostate carcinoma. Apoferritin (APO) can be used as a protein nanotransporter into which a suitable medicinal substance can be encapsulated. Nanoparticles increase the permeability of tumor cells to nanotransporters and have a photothermal effect. The aim of this study was to encapsulate doxorubicin (DOX) into APO and to modify the resulting APO/DOX with gold (AuNPs) and silver nanoparticles prepared by green synthesis (AgNPsGS). METHODS: APO was characterized using 10% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) - 120 V, 60 min, 24 mM Tris, 0.2 M glycine, 3 mM SDS. DOX fluorescence (Ex 480 nm; Em 650 nm) was observed, with a typical absorption maximum at 560 nm. Electrochemical measurement was performed in Brdicka solution (three-electrode setup). AgNPsGS were prepared by green synthesis using clover (Trifolium pratense L.). RESULTS: An electrophoretic study of APO and APO/DOX (5-100 μg/mL) was performed and the behavior of APO and APO/DOX (10 μM) as a function of pH was monitored. In an acidic environment, APO forms subunits of about 20 kDa; in an alkaline medium, it forms a globular protein of about 450 kDa. A change in APO/DOX mobility (about by 10%) was observed. A film of gold nanoparticles was applied to the APO/DOX surface. APO/DOX-AuNPs were washed with ultra-pure water. pH-dependent release of DOX a was monitored. The amount of DOX analyzed was increased by up to 50%. Furthermore, an AgNPsGS-DOX complex (1 mg AgNPsGS/100 μM DOX) was generated and prepared. Subsequently, the AgNPsGS-DOX complex was encapsulated into APO. To further improve therapeutic efficacy, the APO/AgNPsGS-DOX complex was coated with an Au layer. APO/AgNPsGS-DOX/AuNPs were stable and DOX was released from the complex after physical parameters had changed. CONCLUSION: APO nanocomplexes were prepared and modified to increase therapeutic efficacy against tumors. Tumor cell targeting was achieved by binding to TfR and via increased tumor cell permeability and retention. Release of the drug was made possible due to a pH change and photothermal activation that will now be tested. This work was supported by COST European Cholangiocarcinoma Network CA18122 and International Collaboration Project of The European Technology Platform for Nanomedicine. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 21. 3. 2019 Accepted: 14. 5. 2019.

Chemoembolization for Treatment of Hepatocellular Carcinoma - National Registry-Based Analysis.

BACKGROUND: To evaluate survival benefit in patient undergoing transarterial chemoembolization (TACE) for hepatocellular carcinoma (HCC) by national registry data analysis and comparison of regions with unequal usage of interventional radiology procedures. MATERIALS AND METHODS: A total of 4,343 patients with primary diagnosis of HCC between 2010-2016 were extracted from the databases of The Czech National Cancer Registry. The analysis was supported by data from the National Registry of Paid Health Services and the Death Records Database. Primary treatment option was categorized as liver resection, ablation, TACE and chemotherapy. The regional data analysis provided information of interventional radiology procedures frequency for primary treatment of HCC. The 14 main regions were symmetrically divided to group with well-developed interventional radiology service and low-developed interventional radiology service according the frequency of stage adjusted interventional radiology procedure usage (< 15%, > 15%). Kaplan-Meier and Cox regression were used for survival and hazard ratios (HR) analyses. RESULTS: Only 1,730 patients had assessed any primary treatment option, 16.5 % (285) were treated by TACE. Median of survival were significantly different in regions with well and low developed interventional radiology service for whole study population (13.2 vs. 6.5 months, p < 0.001), patients treated in regions with well-developed interventional radiology service had lower risk of death during treatment (HR 0.73; 0.66-0.81). The patient treated by TACE had median of survival 15.8 months (13.5-18.1), while the survival was not significantly different in region groups. CONCLUSION: The usage of anticancer therapies based on interventional radiology procedures is a huge factor influences the survival of HCC patient according population-based data. Studies gathering data from cancer register databases can provide further information on treatment effectiveness. This work was created at Masaryk University in the project „Oncological radiological interventions and their benefit in complex oncological treatment, comparison of dedicated oncological treatment results data of the Czech republic II“ (MUNI/A/1574/2018), supported by Ministry of Education, Youth and Sports. This publication was additionally suported by Ministry of Health grant No. 15-32484A. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 1. 3. 2019 Accepted: 4. 3. 2019.

[Management of care of HIV-positive pregnant women in the period 1996-2014]. / Management péce o HIV pozitivní tehotné v období let 1996-2014.

OBJECTIVE: The evaluation of multidisplinary care about HIV positive pregnant women in the Czech Republic. DESIGN: Review. SETTINGS: Gynekologicko-porodnická klinika 1. LF UK a Nemocnice na Bulovce, Praha. METHODS: The vertical transmission of HIV infection from mother to fetus occurs most often during birth, still 1-2% of HIV-positive pregnant women will transfer the virus transplacenta. Due to careful screening for HIV during pregnancy, counselling, combination antiretroviral (cART) therapy, childbirth planning and its performance by C-section there appears a significant decrease of the virus transmission to the fetus, its occurrence is around 2%. If the HIV infection is detected in the context of screening for sexually transmitted infections (STIs), we begin with combined antiretroviral therapy (cART) depending on the level of viremia and CD4 as soon as possible. All HIV-positive pregnancies are tested for possible coinfection with hepatitis C. Since the first application of the antiretroviral treatment, the therapy is applies throughout the duration of the pregnancy. The labours of the HIV- positive women in the Czech Republic are scheduled. The primary choice is a caesarean section during the 38th week of pregnancy. CONCLUSION: From 1996-2014 the HIV positive status at 18 months of child age was confirmed in 4 cases in the Czech Republic. Three children were born to mothers whose HIV status was unknown at the time of the birth. Thanks to strict adherence to the interdisciplinary care, HIV positive woman have a chance to deliver a HIV-negative newborn and the risk of the transmission of the virus is significantly low.

[Cereblon -  a new target of therapy in the treatment of multiple myeloma]. / Cereblon -  nový terapeutický ciel v liecbe mnohopocetného myelómu.

Treatment of multiple myeloma (MM), currently an incurable disease, aims to achieve complete remission. Immunomodulatory drugs (IMiDs), represented by thalidomide, are one class of very effective drugs. However, the mechanism of IMiDs action is not yet completely understood. Recent research suggests that cereblon (CRBN) plays an important role in mediating anti-tumor effects of IMiDs; therefore, our review focuses on this protein. CRBN is a substrate receptor of Cul4- E3 ubiquitin ligase complex, and thus recognizes proteins destined for degradation. Bind-ing of CRBN and IMiDs inhibits function of the entire ubiquitin proteasome complex which partly explains their anti-tumor effects. In addition, a correlation between CRBN gene expression and effectiveness of treatment in MM patients treated with IMiDs was confirmed. These findings suggest that CRBN expression could possibly serve as a bio-marker to predict response to IMiD in MM patients.

[Guidelines for caring for HIV-infected adults and postexposure prophylaxis for HIV infection]. / Doporuceny postup pece o dospele infikovane HIV a postexpozicni profylaxe infekce HIV.

The authors present instructions for providing antiretroviral therapy in the Czech health care system, based partly on recommendations from abroad and partly on their own experiences of caring for HIV /AIDS patients. The structure and content are similar to those in the 2010 edition, with new study outcomes and modern trends in treatment strategy being taken into consideration. The guidelines are based on systematic patient assessment and aimed at making an accurate diagnosis and formulating recommendations according to individual criteria. The document provides specific instructions for decisions on initiating antiretroviral therapy, selection of individual drugs, monitoring of treatment effect and adverse reactions, and reaction to potential therapy failure. Special attention is paid to administration of antiretroviral drugs to pregnant women and patients with comorbidities, especially tuberculosis, hepatitis or renal insufficiency. The new version includes procedures for postexposure prophylaxis for HIV infection. The guidelines are supplemented by a table summary of antiretroviral drugs. The presented document is to be used in negotiations between the association,state authorities and health care payers.

Monitoring of methylation changes in 9p21 region in patients with myelodysplastic syndromes and acute myeloid leukemia.

Epigenetic de novo methylation of CpG islands is an important event in malignant transformation. Two genes are frequently methylated: cyclin-dependent kinase inhibitor 2B (CDKN2B) and cyclin-dependent kinase inhibitor 2A (CDKN2A). In our study methylation of these genes was studied in 63 patients with myelodysplastic syndromes (MDS), 2 with myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and 13 with acute myeloid leukemia (AML). Five patients were monitored during 5-azacytidine treatment. Twenty-six healthy donors were tested in a control group. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) method with all associated techniques was used for detection. Aberrant methylation was present in the CDKN2A gene in 38% and in the CDKN2B gene in 77% of the patients in MDS group. The level of methylation was higher in the group of AML patients - 77% in CDKN2A gene and 100% in CDKN2B gene. In MDS patients, an aberrant methylation was associated with a tendency to disease progression towards more advanced forms according to the World Health Organization (WHO) classification and the International Prognostic Scoring System (IPSS). Significant differences in methylation level were observed between early and advanced forms of MDS in CDKN2B gene (P value < 0.05) but not for CDKN2A gene. The trend of methylation in patients treated with azacitidine was analyzed in CDKN2B gene and correlated with the course of the disease. Increased methylation was connected with disease progression. We concluded that the methylation level of CDKN2B gene might be used as a marker of leukemic transformation in MDS. Our study indicates the role of hypermethylation as an important event in the progression of MDS to AML.

First detection of Chlamydia trachomatis LGV biovar in the Czech Republic, 2010-2011.

We present four cases of proctitis in HIV-infected men having sex with men (MSM) living in the Czech Republic. The causative agent in all cases was the lymphogranuloma venereum (LGV) biovar of Chlamydia trachomatis. The spread of proctitis caused by C. trachomatis serovars L1­3 among MSM has been observed in several European countries, the United States and Canada since 2003. To our knowledge, no LGV cases in eastern Europe have been published to date.

[Taxus baccata poisoning]. / Smrtelná otrava tisem cerveným.

Common yew poisoning occurs by per oral application of needles or extracts of needles usually. The determination of the cause of death is more difficult in the latter case. In our article, we advise of circumstances which could be helpful in diagnostic. In addition, we describe the substances contained in yew, their effect, importance and toxicological detection.

Patients with special needs within treatment under general anesthesia - meta-analysis.

The study evaluates results obtained from the documentation of patients with special needs, who have undergone treatment under general anesthesia at the pediatric dentistry department. DMFT (decayed, missing, filled teeth) was used for comparison. A sample of 1,836 children from our dental clinic was evaluated. The sample was divided into two groups - special patient and disabled patient populations. A group of 5 years old children was chosen on the bases of WHO guidelines for the setting of the oral health status. A descriptive statistical analysis of the mean standard deviation was conducted with a focus on three factors: sex, age and year. A questionnaire was prepared to compare the oral health habits in the families of the patients. A literary review was conducted to compare the results with other studies from various countries. The meta-analysis using the software MedCalc was done. The DMFT of the patient was counted (disabled DMFT 11.05 - SD 4.82, special patient population 8.8 - SD 3.7) and the descriptive statistics mean standard deviation was calculated. The significant difference between the DMFT of disabled patient and special patient population was exerted (p<0.00003). It was statistically evaluated that the data gained from the studies and from the WHO as population average for 5 years old children are situated under the minimum limit of the confidence interval for our study. The meta-analysis confirmed the hypothesis that oral health status of the special needs children is worse than in other countries.

[Diagnostic pitfalls of HIV-associated Kaposi's sarcoma]. / Uskalí diagnostiky Kaposiho sarkomu sdruzeného s HIV infekcí.

Kaposi's sarcoma was one of the very first diseases which indicated the advent of the AIDS pandemic. Despite the marked fall in its occurrence thanks to the introduction of the cART, Kaposi's sarcoma remains the most frequent tumour in HIV-positive patients and still represents a major diagnostic and therapeutic problem. Particularly in the early stages both the macroscopic and histopathological picture of Kaposi's sarcoma may be very atypical, which can cause diagnostic difficulties right at the time when an early therapy may be most successful. In order to improve both the diagnostics and therapy of Kaposi's sarcoma, close collaboration between physicians taking care of HIV-positive patients--mainly infectologists, dermatologists and pathologists, is necessary.

[EGFR tyrosine kinase inhibitors as a targeted therapy for bronchioloalveolar carcinoma of the lung: a case report of a clinically prompt and intensive response and literature review]. / Cílená lécba bronchioloalveolárního plicního adenokarcinomu inhibitory tyrozinkinázové aktivity EGFR: kazuistika klinicky promptní a výrazné odpovedi a prehled literatury.

INTRODUCTION: Bronchioloalveolar carcinoma (BAC) is an adenocarcinoma belonging to non-small cell lung carcinomas (NSCLC) that, in addition to its morphology and endobronchial spread, presents with certain specific clinical characteristics: greater incidence in women, non-smokers and younger patients, presence of malignant bronchorrhea and lower susceptibility to conventional cytostatic therapies in comparison to other subtypes of NSCLC. On the other hand, nonmucinous type of BAC may show better therapeutic response to targeted therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) erlotinib or gefitinib, as it is 5 times more frequently a carrier of EGFR gene mutations compared to conventional lung adenocarcinomas. CASE DESCRIPTION: We present a case of a 41 years old man, non-smoker for the last 5 years, who was diagnosed with a pneumonic form of nonmucinous bronchioloalveolar carcinoma. Metastases to regional and distant lymph nodes and massive involvement of skeleton with infiltrations in the bone marrow were present at the diagnosis. During the first line palliative chemotherapy with combination regimen of carboplatin and paclitaxel, the disease progressed significantly and the patient's condition deteriorated (performance status (PS) 3, severe dyspnoea at rest, malignant bronchorrhea). Subsequently, administration of erlotinib was initiated based on a series of case studies describing good response of BAC to treatment with EGFR TKI. An evident improvement of the patient's condition was observed as early as 4 days of administration, together with regression of peripheral lymphadenopathy. Nearly complete disappearance of pulmonary infiltrates was observed after 30 days of therapy, with the patient becoming asymptomatic, PS 0. Molecular genetics confirmed the tumour phenotype to be highly responsive to EGFR TKI therapy. The tumour contained EGFR mutation in exon 19 (in-frame L747-753insS deletion) and wild-type K-ras. Disease relapse in the liver occurred 6 months later confirming disease progression. Further treatment remained ineffective despite brief stabilisations of liver enzyme progression following repeated administration of pemetrexed and gefitinib. The patient died 12 months after the diagnosis. CONCLUSIONS: Our case confirms the importance of targeted therapy when treating tumours of an appropriate phenotype. Such treatment may have prompt and intensive effect that may reverse the course of the disease even in patients with poor overall health status.

CCAAT/enhancer-binding protein alpha (CEBPA) polymorphisms and mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia.

The CCAAT/enhancer-binding protein alpha, encoded by the intronless CEBPA gene, is a transcription factor that induces expression of genes involved in differentiation of granulocytes, monocytes, adipocytes and hepatocytes. Both mono- and bi-allelic CEBPA mutations were detected in acute myeloid leukaemia and myelodysplastic syndrome. In this study we also identified CEBPA mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia. We found 16 various deletions with the presence of two direct repeats in CEBPA by analysis of 431 individuals. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493- 498_865-870), GG (486-487_885-886), and GCCAAGCAGC (508-517_907-916), all according to GenBank Accession No. NM_004364.2. In one case we identified that a father with ischaemic heart disease and his healthy son had two identical deletions (493_864del and 508_906del, both according to GenBank Accession No. NM_004364.2) in CEBPA. The occurrence of deletions between two repetitive sequences may be caused by recombination events in the repair process. A double-stranded cut in DNA may initiate these recombination events in adjacent DNA sequences. Four types of polymorphisms in the CEBPA gene were also detected in the screened individuals. Polymorphism in CEBPA gene 690 G>T according to GenBank Accession No. NM_004364.2 is the most frequent type in our analysis. Statistical analysis did not find significant differences in the frequency of polymorphisms in CEBPA in patients and in healthy individuals with the exception of P4 polymorphism (580_585dup according to GenBank Accesion No. NM_004364.2). P4 polymorphism was significantly increased in ischaemic heart disease patients.

[The correlation of results of breath analysers and laboratory blood examinations of blood-ethanol concentration (Czech and Slovak study)]. / Mezirepubliková Cesko-Slovenská studie korelace výsledku dechových analyzátoru a výsledku laboratorní analýzy pri zjistování hladiny alkoholu v krvi.

The target of this study was to compare the results of breath analysers and "lege artis" laboratory blood examinations when determining alcohol levels. This was then used to determine whether any differences exist between the two methods, and how large these differences are. 610 cases from 11 workplaces in the Czech Republic and Slovakia were analysed. The type of breath analyser was not taken into consideration. All cases had to be in the elimination phase. Difference of time between breath test and blood test were rectified through the use of reverse recomputation. It was detected that only 20.8% of the results of respiratory analyser tests correspond to the detected real alcohol level in blood. The maximum difference when a respiratory analyser measured more than a blood test was 1.34 g x kg(-1). and the maximum difference when the analyse measured less was 1.86 g x kg(-1).

Antibody response of HIV-infected patients to latent, cerebral and recently acquired toxoplasmosis.

The aim of this longitudinal study with 626 HIV-infected patients was to evaluate the capability of serological tests in diagnosing the presence of Toxoplasma gondii infection in HIV-infected patients, as well as the potential impact of various treatment regimes on serological results. Low IgG antibody levels and stable or declining titres predominated. IgM positivity occurred in ten patients (one seroconversion, seven latent, two cerebral toxoplasmosis). Complement fixation test (CFT) titres >or=1:32 imply that the relative risk of cerebral toxoplasmosis is 6.84 (95% confidence interval [CI] 1.44-32.5) but with a predictive value of only 14.0% (95% CI 5.3-27.9). Values of specific antibodies are not biassed by antiretroviral treatment and/or prophylaxis for toxoplasmosis, and the detection of specific antibodies is very useful in the identification of T. gondii infection in the HIV-infected population, but the role of serology in predicting the clinical manifestation of T. gondii infection is limited.

[Prevalence of glycoprotein B (gB) genotypes in the patients with high risk of symptomatic cytomegalovirus infection]. / Zastoupení ruzných genotypu glykoproteinu gB u pacientu s vysokým rizikem symptomatické infekce lidským cytomegalovirem (CMV).

OBJECTIVES: Based on genetic variability of the dominant envelope glycoprotein (gB), human cytomegalovirus is classified into four major genotypes. The aims were to determine the prevalence of particular gB genotypes in Czech CMV-infected patients and to compare three groups of the patients with high risk of symptomatic CMV infection, i.e., haematopoietic stem cell transplant (HSCT) recipients, HIV-positive persons and infants. MATERIAL AND METHODS: The study was performed on 134 archived CMV-positive DNA isolates from the patients tested in the National Reference Laboratory for Herpesviruses in 2004-2007. For genotyping, the variable part of gB was amplified and analysed using the restriction fragment length polymorphism (RFLP) method. RESULTS: The most frequently detected genotype was gB1 (33%), followed by gB2 (29%), gB3 (18%) and gB4 (7%). However, the distribution of gB genotypes varied between groups of high-risk patients: gB2 dominated in HIV-positives (55%, p = 0.004), while gB3 was most common in HSCT recipients (26%, p = 0.03) and gB4 was relatively more frequent in infants (20%, p = 0.03). In HSCT recipients, we found increased frequency of gB3 (26%, p = 0.03) and co-infection with two or more gB genotypes (17%, p = 0.016). CONCLUSIONS: The distribution of CMV gB genotypes from Czech CMV-infected patients is similar to that reported in other European countries or in the United States. Differences in the prevalence of CMV gB genotypes between groups of high-risk patients indicate variation in biological properties of particular gB genotypes, possibly resulting in distinct virulence, immunogenicity or epidemiological characteristics of circulating strains.

[Analysis of a group of HIV positive women who gave birth in the Czech Republic]. / Analýza souboru HIV pozitivních rodicek v Ceské republice.

OBJECTIVE: An analysis of HIV positive women who gave birth between 1st January 1985 to 31st December 2006 in the Czech Republic. SUBJECT: A retrospective descriptive analysis. SETTING: Teaching Hospital Bulovka, 1st Faculty of Medicine, Charles University, Prague. SUBJECT AND METHODS: The study included HIV positive women that gave birth between 1st January 1985 to 31st December 2006 at Bulovka hospital. The group of 62 HIV positive women (including 7 secundiparae) gave birth to 71 new-borns (twice twins). The deliveries were performed by C-section. We interrupted breast-feeding by all these women. RESULTS: All new-borns were born alive, no one had Apgar score less than 7 at five minutes. No congenital disorders were found. Three new-borns were transfered to Intensive care unit for new-born babies, two due to dysmaturity and one due to abstinence syndrome. 3 new-borns out of total 71 new-borns were HIV positive (4.2%). CONCLUSION: Routine prenatal screening for HIV and high-quality cooperation between obstetricians and infection control doctors are the basic condition of low rate of vertical trasmission HIV infection in the Czech Republic.

[Recommendation for diagnostics and therapy of toxoplasmosis in persons with HIV infection]. / Doporucený postup diagnostiky a terapie toxoplazmózy u osob s HIV infekcí.

Reactivation of latent toxoplasmosis is a serious complication in patients with deep immunodeficiency, but the disease has a good prognosis if early diagnosed and effectively treated. Definitive etiologic proof of the reactivation may be difficult and thus an empiric method (therapeutic trial) is used for confirmation of the diagnosis in clinical practice. The preferred therapy is a combination of pyrimethamine + sulfadiazine.

Long-term analysis of the resistance development in HIV-1 positive patients treated with protease and reverse transcriptase inhibitors: correlation of the genotype and disease progression.

In this study, 27 HIV-1-positive patients on long-term highly active antiretroviral therapy (HAART) in the Czech Republic were followed for a period of up to 7 years. Variability of the HIV-1 protease (PR) sequence common in the Czech Republic was observed. Under the pressure of inhibitors of protease (PRIs) and reverse transcriptase (RTIs) mutations in PR were detected. Development of resistance to PRIs was followed by a decrease in CD4 count and increase in viral load. The dynamics of viral load closely corresponded to the accumulation of specific primary mutations in PR and RT. Out of 27 patients 18 developed resistance to PRIs and the prolonged therapy led to the accumulation of a higher number of amino acid changes associated with the resistance and, consequently, cross-resistance to several PRIs was observed. These multi-resistant variants of HIV-1 with mutations in PR could not be inhibited sufficiently with PRIs that are currently available in clinical practice. Efficient yet temporary suppression of viral replication was achieved by a lopinavir (LPV) treatment.