Challenges of laboratory diagnosis of Lyme disease ‒ Questionnaire survey results.

Lyme disease. Our second goal was to identify bacterial and viral co-infections occurring concurrently with Lyme disease. Furthermore, it was our intention to also analyze the correlation of laboratory testing with the occurrence of erythema migrans (EM). BACKGROUND: The accuracy in diagnostic testing for Lyme disease in the early stages of infection is an important factor necessary for delivering proper treatment to patients. METHODS: A total of 173 individuals with confirmed Lyme disease or with laboratory testing underway participated in the quantitative survey. RESULTS: ELISA was the first test conducted in 51% of the respondents, 28% of whom yielded positive findings of both IgM and IgG antibody classes. The positivity of ELISA test findings was confirmed by Western blot in 100% of results. Negative results of ELISA were consistent with Western blot only in less than half of the patients. More than half of the respondents had not been tested for any bacterial or viral co-infections. The results of serological testing were not consistent with clinical findings in all cases, including those with clinically discernible skin manifestation of erythema migrans. CONCLUSION: The comparison of results obtained by ELISA and Western blot revealed significant discrepancies. Simultaneous infections by vectors with several pathogens were detected (Tab. 3, Fig. 2, Ref. 15).

Extracellular vesicles derived from dental mesenchymal stem/stromal cells with gemcitabine as a cargo have an inhibitory effect on the growth of pancreatic carcinoma cell lines in vitro.

Extracellular vesicles (EVs) are nowadays a target of interest in cancer therapy as a successful drug delivering tool. Based on their many beneficial biocompatible properties are designed to transport nucleic acids, proteins, various nanomaterials or chemotherapeutics. Extracellular vesicles derived from mesenchymal stem/stromal cells (MSCs) possess their tumor-homing abilities. This inspired us to engineer the MSC's EVs to be packed with chemotherapeutic agents and deliver it as a Trojan horse directly into tumor cells. In our study, human dental pulp MSCs (DP-MSCs) were cultivated with gemcitabine (GCB), which led to its absorption by the cells and subsequent secretion of the drug out into conditioned media in EVs. Concentrated conditioned media containing small EVs (potentially exosomes) significantly inhibited the cell growth of pancreatic carcinoma cell lines in vitro. DP-MSCs were simultaneously engineered to express a suicide gene fused yeast cytosinedeaminase:uracilphosphoribosyltransferase (yCD::UPRT). The product of the suicide gene converts non-toxic prodrug 5-fluorocytosine (5-FC) to highly cytotoxic chemotherapeutic drug 5-fluorouracil (5-FU) in the recipient cancer cells. Conversion of 5-FC to 5-FU had an additional effect on cancer cell's growth inhibition. Our results showed a therapeutic potential for DP-MSC-EVs to be designed for successful delivering of chemotherapeutic drugs, together with prodrug suicide gene therapy system.

The Potential of Liquid Biopsy in Detection of Endometrial Cancer Biomarkers: A Pilot Study.

Endometrial cancer belongs to the most common gynecologic cancer types globally, with increasing incidence. There are numerous ways of classifying different cases. The most recent decade has brought advances in molecular classification, which show more accurate prognostic factors and the possibility of personalised adjuvant treatment. In addition, diagnostic approaches lag behind these advances, with methods causing patients discomfort while lacking the reproducibility of tissue sampling for biopsy. Minimally invasive liquid biopsies could therefore represent an alternative screening and diagnostic approach in patients with endometrial cancer. The method could potentially detect molecular changes in this cancer type and identify patients at early stages. In this pilot study, we tested such a detection method based on circulating tumour DNA isolated from the peripheral blood plasma of 21 Slovak endometrial cancer patients. We successfully detected oncomutations in the circulating DNA of every single patient, although the prognostic value of the detected mutations failed to offer certainty. Furthermore, we detected changes associated with clonal hematopoiesis, including DNMT3A mutations, which were present in the majority of circulating tumour DNA samples.

Cross-Kingdom Interaction of miRNAs and Gut Microbiota with Non-Invasive Diagnostic and Therapeutic Implications in Colorectal Cancer.

Colorectal cancer (CRC) has one of the highest incidences among all types of malignant diseases, affecting millions of people worldwide. It shows slow progression, making it preventable. However, this is not the case due to shortcomings in its diagnostic and management procedure and a lack of effective non-invasive biomarkers for screening. Here, we discuss CRC-associated microRNAs (miRNAs) and gut microbial species with potential as CRC diagnostic and therapy biomarkers. We provide rich evidence of cross-kingdom miRNA-mediated interactions between the host and gut microbiome. miRNAs have emerged with the ability to shape the composition and dynamics of gut microbiota. Intestinal microbes can uptake miRNAs, which in turn influence microbial growth and provide the ability to regulate the abundance of various microbial species. In the context of CRC, targeting miRNAs could aid in manipulating the balance of the microbiota. Our findings suggest the need for correlation analysis between the composition of the gut microbiome and the miRNA expression profile.

DNA analysis of partial hydatidiform mole revealing triandric monogynic tetraploidy.

The authors present a case of a partial hydatidiform mole where DNA analysis (STR - short tandem repeat genotyping) showed a triandric monogynic tetraploid genome composition with a XXXY gonosomal complement. This genetic finding clinicopathologically correlates with a partial hydatidiform mole, although it is rare in comparison with the typical, diandric monogynic triploid partial moles. The genetic analysis definitively confirmed the suspected diagnosis of a partial mole. To exclude the possibility that molar pregnancy represented retained products of conception after elective pregnancy termination, STR profiles from molar pregnancy and previous products of conception were compared. Short tandem repeats genotyping is a useful molecular genetic method in the differential diagnosis of partial hydatidiform moles, where clinical-pathological findings are frequently ambiguous.

The effects of local anesthetics on cancer cells.

During cancer surgery, the perioperative period is characterized by stress response and immunosuppression that can lead to further worsening of the disease and metastatic spread. Local anesthetics have antiproliferative, cytotoxic and antimetastatic effects on cancer cells in vitro. There is scientific evidence that local anesthetics possess anti-inflammatory effects, help to preserve normal immune function and reduce the possibility of metastatic spread. Anesthetic care affects pain, inflammation, and immunosuppression, which may have a great impact on the outcome of oncological patients. The use of local anesthetics during the perioperative period in oncological patients may have a beneficial effect on their survival and cancer recurrence. This article summarizes the effects of local anesthetics in vitro (Tab. 1, Fig. 1, Ref. 36). Keywords: local anesthetics, cancer cells.

Liquid Biopsy as a Source of Nucleic Acid Biomarkers in the Diagnosis and Management of Lynch Syndrome.

Lynch syndrome (LS) is an autosomal dominant inherited cancer predisposition disorder, which may manifest as colorectal cancer (CRC), endometrial cancer (EC) or other malignancies of the gastrointestinal and genitourinary tract as well as the skin and brain. Its genetic cause is a defect in one of the four key DNA mismatch repair (MMR) loci. Testing of patients at risk is currently based on the absence of MMR protein staining and detection of mutations in cancer tissue and the germline, microsatellite instability (MSI) and the hypermethylated state of the MLH1 promoter. If LS is shown to have caused CRC, lifetime follow-up with regular screening (most importantly, colonoscopy) is required. In recent years, DNA and RNA markers extracted from liquid biopsies have found some use in the clinical diagnosis of LS. They have the potential to greatly enhance the efficiency of the follow-up process by making it minimally invasive, reproducible, and time effective. Here, we review markers reported in the literature and their current clinical applications, and we comment on possible future directions.

Risk genetic polymorphism and haplotype associated with papillary thyroid cancer and their relation to associated diseases in Slovak population.

BACKGROUND: Risk for developing papillary thyroid carcinoma (PTC), the most common endocrine malignancy, is thought to be mediated by lifestyle, environmental exposures and genetic factors. Recent progress in the genome-wide association studies of thyroid cancer leads to the identification of several genetic variants conferring risk to this malignancy across different ethnicities. METHODS AND RESULTS: We set out to elucidate the impact of selected single nucleotide polymorphisms (SNPs) on papillary thyroid carcinoma risk and to evaluate the interactions of these genetic variants with associated diseases for the first time in the Slovak population. Six SNPs (rs966423, rs2439302, rs965513, rs116909374, rs1537424 and rs944289) were genotyped in 86 patients with PTC and 99 healthy control subjects. The association analysis and multivariable modelling of PTC risk by the genetic factors, supplemented with a rigorous statistical validation, were performed. One of the six SNPs rs966423 (DIRC3, OR=1.51, p=0.03) was significantly associated with PTC. Next two SNPs rs965513 (PTCSC2, OR=1.34) and rs116909374 (MBIP, OR=0.44) showed a suggestive association. Haplotype TTC (SNPs located on chromosome 14q13) showed a suggestive association with PTC (p=0.07, OR=1.55). In the PTC group, significant associations were observed between rs966423 (DIRC3) and ischemic heart diseases (p=0.009), rs965513 (PTCSC2) and diabetes mellitus (p=0.04) and haplotype 14q13 and musculoskeletal diseases. Next three associations rs966423 (DIRC3) and arterial hypertension; rs116909374 (MBIP) and other benign diseases; rs1537424 (MBIP) and disorder lipid metabolism, rs965513 (PTCSC2) and anti-Tg (thyroglobulin antibody) showed suggestive associations. CONCLUSION: These results indicate that germline variants not only predispose to PTC, but may also be related to other risk factors, including associated diseases. However, these associations were only moderate, and further multi-ethnic studies are required to evaluate the usefulness of these germline variants in the clinical stratification of PTC patients (Tab. 8, Ref. 37).

Intracellular prodrug gene therapy for cancer mediated by tumor cell suicide gene exosomes.

Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene-fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT-MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5-fluorocytosine (5-FC) to cytotoxic drug 5-fluorouracil (5-FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT-MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT-transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5-FC to 5-FU and to 5-FUMP in a dose-dependent manner. Most of tumor cell-derived suicide gene exosomes were tumor tropic, in 5-FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP-MSCs. Tumor cell-derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.

Selective Apoptotic Effect of Plasma Activated Liquids on Human Cancer Cell Lines.

Plasma medicine is a new field focusing on biomedical and clinical applications of cold gas plasmas, including their anticancer effects. Cold plasmas can be applied directly or indirectly as plasma-activated liquids (PAL). The effects of plasma-activated cell growth medium (PAM) and plasma-activated phosphate buffered saline (PAPBS) were tested, using a plasma pen generating streamer corona discharge in ambient air, on different cancer cell lines (melanoma A375, glioblastoma LN229 and pancreatic cancer MiaPaCa-2) and normal cells (human dermal fibroblasts HDFa). The viability reduction and apoptosis induction were detected in all cancer cells after incubation in PAL. In melanoma cells we focused on detailed insights to the apoptotic pathways. The anticancer effects depend on the plasma treatment time or PAL concentration. The first 30 min of incubation in PAL were enough to start processes leading to cell death. In fibroblasts, no apoptosis induction was observed, and only PAPBS, activated for a longer time, slightly decreased their viability. Effects of PAM and PAPBS on cancer cells showed selectivity compared to normal fibroblasts, depending on correctly chosen activation time and PAL concentration, which is very promising for potential clinical applications. This selectivity effect of PAL is conceivably induced by plasma-generated hydrogen peroxide.

Peptaibol-Containing Extracts of Trichoderma atroviride and the Fight against Resistant Microorganisms and Cancer Cells.

Fighting resistance to antibiotics and chemotherapeutics has brought bioactive peptides to the fore. Peptaibols are short α-aminoisobutyric acid-containing peptides produced by Trichoderma species. Here, we studied the production of peptaibols by Trichoderma atroviride O1 and evaluated their antibacterial and anticancer activity against drug-sensitive and multidrug-resistant bacterium and cancer cell lines. This was substantiated by an analysis of the activity of the peptaibol synthetase-encoding gene. Atroviridins, 20-residue peptaibols were detected using MALDI-TOF mass spectrometry. Gram-positive bacteria were susceptible to peptaibol-containing extracts of T. atroviride O1. A synergic effect of extract constituents was possible, and the biolo-gical activity of extracts was pronounced in/after the peak of peptaibol synthetase activity. The growth of methicillin-resistant Staphylococcus aureus was reduced to just under 10% compared to the control. The effect of peptaibol-containing extracts was strongly modulated by the lipoteichoic acid and only slightly by the horse blood serum present in the cultivation medium. Peptaibol-containing extracts affected the proliferation of human breast cancer and human ovarian cancer cell lines in a 2D model, including the multidrug-resistant sublines. The peptaibols influenced the size and compactness of the cell lines in a 3D model. Our findings indicate the molecular basis of peptaibol production in T. atroviride O1 and the potential of its peptaibol-containing extracts as antimicrobial/anticancer agents.

Partial hydatidiform moles with unclear histopathological profile - importance of genetic diagnostics.

OBJECTIVE: The aim of the study was the genetic characterization of a set of cases with an unclear morphological profile of the placental tissue suspected of a partial hydatidiform mole. PATIENTS AND METHODS: This work presents the results of a genetic analysis of a group of 10 patients with various clinical manifestations of reproductive loss, where a partial hydatidiform mole was suspected on the basis of a histopathological examination. The composition of the genome of the products of conception was determined by short tandem repeats (STR) genotyping using a commercial kit;Devyser Compact v3 (Devyser). RESULTS AND CONCLUSIONS: Out of 10 analyzed cases, five had diandric monogynic triploid genome, characteristic for a partial mole. Aneuploidies of chromosomes 13, 18, 21, X and Y were excluded in four cases and Pataus syndrome was dia-gnosed in one case. In the case of an unclear histopathological profile, consultative DNA analysis (ideally STR genotyping) can significantly help the pathologist in the differential dia-gnosis of a partial mole. The histopathological profile of a partial hydatidiform mole may be in some cases incomplete and unclear, especially in the early weeks of gestation, which can lead to false negativity of the examination. On the other hand, other pathologies, for example aneuploides or digynic triploidy, may produce a histopathological profile similar to a partial mole, which leads to false positivity. Accurate dia-gnosis of a partial hydatidiform mole using molecular genetic methods contributes to the determination of adequate dispensary care for patients.

Preleukemic fusion genes typical for acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly heterogeneous subtype of leukemia, accounting for 25 % of childhood leukemias. By the presence of genetic mutations in hematopoietic/ progenitor stem cells, the bone marrow produces a large number of abnormal undifferentiated leukocytes (blasts), which significantly impairs the proper differentiation of cells. AML is induced by two interventions. Chromosomal translocation during hematopoiesis of intrauterine development is the first intervention. This creates preleukemic fusion genes (PFG), which can later be transformed by a second intervention (point genetic mutation - deletion, insertion ) into a functional malignant clone. Characteristic AML fusion genes include AML1-ETO, PML-RARA or MLL-AF9, which in turn produce hybrid proteins with altered function. Several studies suggest that these PFGs are considered an important prognostic tool in disease assessment. While the incidence of PFG characteristic of acute lymphoblastic leukemia (ALL) has been relatively well studied by several research groups and has been estimated at 1 to 5% in the umbilical cord blood of healthy neonates, PFG relevant to AML are still not sufficiently clarified.

Prodrug suicide gene therapy for cancer targeted intracellular by mesenchymal stem cell exosomes.

The natural behavior of mesenchymal stem cells (MSCs) and their exosomes in targeting tumors is a promising approach for curative therapy. Human tumor tropic mesenchymal stem cells (MSCs) isolated from various tissues and MSCs engineered to express the yeast cytosine deaminase::uracil phosphoribosyl transferase suicide fusion gene (yCD::UPRT-MSCs) released exosomes in conditional medium (CM). Exosomes from all tissue specific yCD::UPRT-MSCs contained mRNA of the suicide gene in the exosome's cargo. When the CM was applied to tumor cells, the exosomes were internalized by recipient tumor cells and in the presence of the prodrug 5-fluorocytosine (5-FC) effectively triggered dose-dependent tumor cell death by endocytosed exosomes via an intracellular conversion of the prodrug 5-FC to 5-fluorouracil. Exosomes were found to be responsible for the tumor inhibitory activity. The presence of microRNAs in exosomes produced from naive MSCs and from suicide gene transduced MSCs did not differ significantly. MicroRNAs from yCD::UPRT-MSCs were not associated with therapeutic effect. MSC suicide gene exosomes represent a new class of tumor cell targeting drug acting intracellular with curative potential.

Isoprenoids responsible for protein prenylation modulate the biological effects of statins on pancreatic cancer cells.

BACKGROUND: Statin treatment of hypercholesterolemia is accompanied also with depletion of the mevalonate intermediates, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) necessary for proper function of small GTPases. These include Ras proteins, prevalently mutated in pancreatic cancer. In our study, we evaluated the effect of three key intermediates of the mevalonate pathway on GFP-K-Ras protein localization and the gene expression profile in pancreatic cancer cells after exposure to individual statins. METHODS: These effects were tested on MiaPaCa-2 human pancreatic cancer cells carrying a K-Ras activating mutation (G12C) after exposure to individual statins (20 µM). The effect of statins (atorvastatin, lovastatin, simvastatin, fluvastatin, cerivastatin, rosuvastatin, and pitavastatin) and mevalonate intermediates on GFP-K-Ras protein translocation was analyzed using fluorescence microscopy. The changes in gene expression induced in MiaPaCa-2 cells treated with simvastatin, FPP, GGPP, and their combinations with simvastatin were examined by whole genome DNA microarray analysis. RESULTS: All tested statins efficiently inhibited K-Ras protein trafficking from cytoplasm to the cell membrane of the MiaPaCa-2 cells. The inhibitory effect of statins on GFP-K-Ras protein trafficking was partially prevented by addition of any of the mevalonate pathway's intermediates tested. Expressions of genes involved in metabolic and signaling pathways modulated by simvastatin treatment was normalized by the concurrent addition of FPP or GGPP. K-Ras protein trafficking within the pancreatic cancer cells is effectively inhibited by the majority of statins; the inhibition is eliminated by isoprenoid intermediates of the mevalonate pathway. CONCLUSIONS: Our data indicate that the anticancer effects of statins observed in numerous studies to a large extent are mediated through isoprenoid intermediates of the mevalonate pathway, as they influence expression of genes involved in multiple intracellular pathways.

Candidate gene studies of diabetic retinopathy in human.

Diabetic retinopathy (DR) is a multifactorial disease with complex pathophysiology. It is the main cause of blindness among the people in productive age. The purpose of this literature review is to highlight recent achievements in the genetics of diabetic retinopathy with particular focus on candidate gene studies. We summarized most of the available published data about candidate genes for diabetic retinopathy with the goal to identify main genetic aspects. We conclude that genetic studies reported contradictory findings and no genetic variants meet criteria of a diagnostic marker, or significantly elucidate the root of DR development. Based on these findings it is important to continue with the research in the field of DR genetics, mainly due to the fact that currently new possibilities and approaches associated with utilization of next-generation sequencing are available.

Current Challenges of Methylation-Based Liquid Biopsies in Cancer Diagnostics.

In current clinical practice, effective cancer testing and screening paradigms are limited to specific types of cancer, exhibiting varying efficiency, acceptance, and adherence. Cell-free DNA (cfDNA) methylation profiling holds promise in providing information about the presence of malignity regardless of its type and location while leveraging blood-based liquid biopsies as a method to obtain analytical samples. However, technical difficulties, costs and challenges resulting from biological variations, tumor heterogeneity, and exogenous factors persist. This method exploits the mechanisms behind cfDNA release but faces issues like fragmentation, low concentrations, and high background noise. This review explores cfDNA methylation's origins, means of detection, and profiling for cancer diagnostics. The critical evaluation of currently available multi-cancer early detection methods (MCEDs) as well as tests targeting single genes, emphasizing their potential and limits to refine strategies for early cancer detection, are explained. The current methodology limitations, workflows, comparisons of clinically approved liquid biopsy-based methylation tests for cancer, their utilization in companion diagnostics as well as the biological limitations of the epigenetics approach are discussed, aiming to help healthcare providers as well as researchers to orient themselves in this increasingly complex and evolving field of diagnostics.

Post-mortem rapid aneuploidy testing for holoprosencephaly.

BACKGROUND: Abortion and fetal death are common in fetuses with holoprosencephaly, so genetic examinations often have to be made in a post-mortem setting. The efficiency of the conventional karyotyping using cultured fibroblasts in these situations is limited due to frequent culture failure. In the current study, archived cases of holoprosencephaly, where post-mortem genetic evaluation was requested and sufficient frozen material was available, were reevaluated using the quantitative fluorescence polymerase chain reaction (QF-PCR) technique. METHODS: Testing for aneuploidies of chromosomes 13, 15, 16, 18, 21, 22, X, and Y with the QF-PCR technique was carried out on DNA isolated from archived frozen chorionic villi in seven cases of holoprosencephaly. RESULTS: QF-PCR was successful in all seven cases. Two cases of trisomy 13, two cases of triploidy, and one case of trisomy 18 was found meaning a 71% diagnostic yield. The success rate of QF-PCR (100%, 7/7) was superior compared to conventional karyotyping (43%, 3/7). CONCLUSIONS: Rapid aneuploidy testing using the QF-PCR technique is a simple, reliable, time- and cost-effective method sufficient to conclude the etiologic investigation in the majority of holoprosencephaly cases post-mortem.

Short tandem repeats genotyping of gestational choriocarcinoma - our experiences.

OBJECTIVE: This short communication demonstrates how short tandem repeat genotyping can identify the origin of gestational choriocarcinoma. MATERIALS AND METHODS: The origin of gestational choriocarcinoma in our three cases was determined using the short tandem repeats genotyping technique, which involved quantitative fluorescent PCR and fragmentation analysis. RESULTS: In Case 1 despite no medical history of molar pregnancy, DNA analysis indicated that the choriocarcinoma originated from a homozygous complete hydatidiform mole. We conclude, that the patient's complete abortion 10 years prior to the choriocarcinoma diagnosis was an undiagnosed complete hydatidiform mole. In Case 2 and Case 3 the clinically presumed origin of choriocarcinoma was confirmed. CONCLUSION: Determining the origin of choriocarcinoma is essential for clinical application, as it affects the FIGO scoring system for gestational trophoblastic neoplasia, which determines the patient's prognosis and treatment approach.

Determination of the Prevalence of Microsatellite Instability, BRAF and KRAS/NRAS Mutation Status in Patients with Colorectal Cancer in Slovakia.

Slovakia has one of the highest rates of colorectal cancer among the developed countries, ranking as the second highest in the incidence of this disease for men worldwide. Despite the significant burden on both quality of life and the healthcare system this disease imposes, data on molecular analysis of biomarkers in CRC-diagnosed patients is scarce. In our study, we analyzed confirmed CRC patients from the database of the National Cancer Institute (NCI) and evaluated the presence of 4 biomarkers in tumor tissues. Altogether, 83 FFPE tumor tissues from CRC patients listed in the NCI database were analyzed for microsatellite instability status, presence of BRAF and KRAS/NRAS mutations, and neoplastic cell percentage in tissue samples. We identified 4 MSI-high samples, 39 KRAS/NRAS mutations, and 5 BRAF p.V600E mutations, with one case of coexistence of all three markers in a single tumor sample. We also evaluated possible relationships between biomarkers, their coexistence, and the age and sex of the studied population.