Oncolytic viruses and cancer treatment.

BACKGROUND: The fundamental difference between tumor and normal tissue growth is the emergence of the microenvironment with diminished or extinguished immunogenicity. One of the main functions of oncolytic viruses is the formation of such a microenvironment, which leads to a revival of immunological processes and loss of viability of cancer cells. Oncolytic viruses are being continuously improved and should be considered as a possible adjuvant immunomodulatory cancer treatment. A key requirement for the success of this cancer therapy is the specificity of the oncolytic viruses, which replicate only in tumor cells but do not affect normal cells. In this review, optimization strategies to achieve cancer specificity with increased efficacy are discussed and the most interesting results from preclinical and clinical trials are presented. PURPOSE: This review provides information on the current status of the development and use of oncolytic viruses as part of the bio-logical treatment of cancer.

[The substance of genetic information--nucleic acids]. / Hmotná podstata genetických informací - nukleové kyseliny.

If we look at the history of our knowledge of nucleic acids, we would see in the distant past of 140 years Friedrich Miescher who had identified the acidic substance within the cell nucleus, which he called nuclein. About 70 years after his initial observation, this substance was connected with genetic information. This very substantial finding happened during the World War II. This was the impulse that research of nucleic acids received to speed up continuously growing mountain of information, which is more and more difficult to understand. Another eruption of new information about our genome was the result of ten years of intensive cooperation of many manufacturers divided into two competitive blocks which offered us knowledge of nucleotide sequence of all 46 DNA molecules. The year 2000 became the landmark marking the start of the postgenomic era. It did not mean that human genome was totally explored, but the cornerstone has been settled. Since then, we could concentrate our efforts on variability; use of the project of 1,000 genomes brought many important findings, eg. copy number variability (CNV) exceeds the single nucleotide polymophisms (SNP). Also intergenomic relationships, studies on function and pathways began to be much more understandable by elucidation of the genome primary structure. NGS as a tool also accelerated the epigenetic research. All this improved molecular diagnostics by discovering many new markers playing their role in disease and treatment and allowed us to enter the field of multifactorial illnesses including cancer. The progress in diagnostic technologies which has happened during the last decade forced our research teams to include other professions - eg. bioinformatics.

HVRII of mtDNA in cord blood cells of newborn children and in their saliva 10 years later.

Comparison of hypervariable region II nucleotide sequences of mitochondrial DNA obtained from cord blood cells and saliva cells of the same individual at birth and after ten years revealed a few differences at the so-called mutation hot spots (three transitions and three indels within the C-tract). The personal identity of samples was proved by short tandem repeat profiling. Comparison of individuals living in two regions that differ by air pollution, however, did not reveal statistically significantly increased number of mutations in the population from the region of poorer environmental conditions, although indicating such tendency.

Genetic variability of HVRII mtDNA in cord blood and respiratory morbidity in children.

Genetic polymorphisms were examined using direct sequencing of the hypervariable region II (HVRII) in the D-loop of mtDNA in the cord blood of 355 children living in two areas of the Czech Republic - the industrial district of Teplice and the agricultural district of Prachatice. The incidence of the most frequent nucleotide variants of HVRII, C150T (10.1%), T152C (19.7%), T195C (19.7%) and 309.nC (41.4% for 309.2C and 13.8% for 309.3C), and the respiratory morbidity at the ages of 0-2 years and 2-6 years were investigated, considering many other factors such as locality, gender, ethnicity, heating by coal in household, maternal age, asthma bronchiale, allergic rhinitis, pollinosis, conjunctivitis and maternal tobacco exposure during and after pregnancy. We found that the T195C transversion in HVRII is connected with an increased risk of early childhood (0-2 years) bronchitis (RR 1.38, p=0.034, 95% CI 1.04-1.85) and with increased risk of otitis media in children aged 2-6 years (RR 1.62, p=0.032, 95% CI 1.04-2.53). Another polymorphism, 309.nC, is associated with an increased risk of bronchitis in children aged 2-6 years (RR 1.46, p=0.030, 95% CI 1.04-2.06). The results indicate that genetic polymorphisms in mtDNA may be an important factor not only for various types of cancers and neurodegenerative diseases, but also for respiratory morbidity in children.

[On human ageing and longevity--2. Internal determinants]. / O lidském stárnutí a dlouhovekosti--2. Vnitrní podmínky.

Longevity as a result of prolonged or postponed ageing is substantially influenced by genetic determinants. Most of them were analyzed by studying different progeria syndromes and their genetic control. From this point of view the use of experimental animals, because it enables to identify genes involved in shortening or prolongation of life-span by changing experimental conditions, has been also very effective. Expression of ageing processes at cellular as well on organism level as a biological unit revealed sets of genetic pathways involved in longevity. Several theories which elicit different aspects have been constructed to explain the ageing process.

[On human longevity--1. external influences]. / O lidské dlouhovekosti--1. vnejsí vlivy.

Human life-span is controlled by many factors, both internal (genomic) and external. In this paper only impact of external conditions is to be discussed. External factors are very variable, sometimes difficult to ascertain, but their control is relatively easier to address than the internal ones. The effect of human activities on human lifespan can be positive or negative. Experimental results have shown that one of the ways leading to prolongation of life-span is caloric restriction (CR). There are, of course, many other factors, not only of nurture character, inflicting on quality and length of human life. Some of them are human products including stress with different levels of inherited ability to resist it, but also possibility to improve individual capacity to control its effect due to hormesis. Prolongation of life expectancy--ageing of the population has become an economic burden in many countries, especially for social and health services.

[Clinical validity and clinical utility of molecular-genetic laboratory testing]. / Klinická validita a uzitecnost molekulárne genetických laboratorních zkousek.

Intensity of genetic determination of our phenotypes differs both quantitatively and qualitatively and concerns not only physiological, but also pathological features. Clinical diagnoses are more and more dependent on laboratory findings, whose quality and utility have to be objectively estimated and assesed. For medical practice the knowledge of genetic determination is substantial and for this reason several approaches have beeen adopted. Genetic determination can be measured and expressed by several parameters, of which heritability and genetic risk are discussed in this paper to elucidate their properties, importace, significance, and limitations.

[Personalized medicine--genomic phase]. / Personalizovaná medicína--genomická fáze. Proc a zac.

Personalized medicine has become declared to be a strategic goal of nearly all medical disciplines, a modern fashion of medicine, which corresponds to unexpected progress in knowledge of structure and sometimes also understanding the function of our genome. This was made possible by development of new technologies and methodological approaches, which led to improved and more detailed diagnostics and categorization of diseases. Also pharmaceutical industry is producing many new drugs, but not all are effective as supposed, and sometimes also cause adverse reactions (ADR). To prevent uneffectiveness and ADR, preliminary genetic testing of patients seems to be necessary. Utility of personalized medicine is sometimes discussed from the point of view of supposed inevitable increase of cost and problems with its adoption by existing system of healthcare.

[Direct-to-consumer genetic testing also in our country]. / Genetické testování zpusobem DTC i v nasich krajích.

The main features of direct-to-consumer (DTC) genetic testing suffers from the loss of personal contact between a client and a genetic consultant, who is apt to assess the usefulness of genetic testing and to interpret results of genetic tests and who is also sufficiently trained to provide professional way of consultation. Laboratories offering these services often inform clients by phone, and neglect their identification - the samples examined may be anonymous. Also samples from children are accepted for testing. Quality control is not mandatory and laboratory genetic testing is not a licenced business in the Czech Republic. Because of these problems, which are not specific to our situation, but rather global, the possibility of the ban concerning these activities is being discussed in several European countries.

JNK2 and p38 MAPK over-expressions do not represent key events in chronic myeloid leukemia transformation.

Over-expression of two members of MAP kinase family (JNK2 and p38) has been already observed in chronic myeloid leukemia (CML). In the present study, significance of this deregulation was investigated. Impacts of JNK2/p38 suppression on gene expression profile of CML cell lines (K562/KU-812) were studied using an experimental approach that combines siRNA-mediated specific inhibition of the genes and array-based expression analyses. After JNK2 depletion, 27 out of 588 tested genes showed significant expression changes, with 13 down-regulated genes and 14 up-regulated genes. Among others, expression of MSH2 and MSH6, mdm2, and caspase-2 was reduced and, on the other hand, MKK1 and MKK6, RFC2, cytokeratins K18 and K19, BAD, and DR5 expression was up-regulated. In the case of p38 silencing, 20 genes were considered as significantly deregulated (7 genes reduced, 13 over-expressed). These genes included caspase-10, SOD1, and Notch4 (down-regulation) and caspase-2 and caspase-3, CDC2, CDK4, and c-kit (up-regulation). In conclusion, comparison of expression profiles after JNK2 or p38 gene silencing revealed distinct sets of affected genes. The results implied an unequal impact of the MAPK deregulation on the CML cells. Further, we demonstrated that neither JNK2 nor p38 siRNAmediated inhibition led to significant change of CML cell proliferation. It suggests that there are other important, likely upstream regulators essential for CML malignant cell growth/transformation; therefore, separate inhibition of JNK2 or p38 MAPK gene is not sufficient for a proliferation arrest.

[Homage to McKusick's "Mendelian inheritance in man"]. / Hold McKusickove "Mendelian inheritance in man".

McKusick's database MIM has grown since its early beginning in sixties to 1985 when the online version (OMIM) appeared. The last edition of three volumes was printed in 1998. It has become a very valuable tool for all geneticists, and also clinicians of other disciplines started using it as a source of important information. The original limitation to disorders with mendelian inheritance has been step by step broken down, all components of human genome and also genes without known function and their epigenetic changes have been included. It was a pleasure for all of us to congratulate to McKusick's honorary degree obtained this year by the oldest European university in Bologna (with a short biography).

[Availability of molecular genetic diagnostic services in the Czech Republic. Is it too little, too much or sufficient?]. / Nabídka diagnostických sluzeb v oblasti molekulární genetiky v Ceské republice. Je to málo, moc, nebo akorát?

Review of diagnostic services offered by molecular genetic laboratories classified according the number of laboratories dealing with individual diagnoses is presented. Three different categories were distinguished: frequently, medium and rarely offered diagnostic service. Services that concerned trombophilic factors F2 a F5 and methylentetrahydrofolatreductase, cystic fibrosis, paternity testing, gender determination, molecular testing for hemochromatose--HFE, detection of BCR/ABL fused gene, and Y-chromosomal loci DAZ and AYF examination were most frequently offered.

Hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy is a multigenetic cardiac disease with autosomal dominant pattern of inheritance and incomplete penetrance, with the exclusion of those cases caused by mutations in the mitochondrial genome. The disease is usually caused by mutations in several sarcomeric contractile protein genes. Mutations have been found in four genes that encode components of the thick filament: beta myosin heavy chain (5), essential myosin light chains (6), regulatory myosin light chains (6), and cardiac myosin binding protein -C (7), (8); in five genes that encode thin filament proteins: cardiac actin (9), cardiac troponin T (10), cardiac troponin C (11), cardiac troponin I (12), and alpha-tropomyosin (10); and in the sarcomeric cytoskeletal protein titin (13). In addition to mutations in contractile sarcomeric proteins, mutations in other genes encoding for non-sarcomeric proteins also have been identified in patients with-non pure form of hypertrophic cardiomyopathy. As a complex cardiac disease, hypertrophic cardiomyopathy has unique pathophysiological characteristics and a various morphological, functional, and clinical features.

[What is the future of our genome?]. / Kam smeruje nase genetická výbava?

Where do we go and what will happen with our genomes? Viewed as a reflection of what we know about our origin as Homo sapiens, history and forecast. Archeogenetics tries to find our sourcing place and to explain how humans occupied the globe. One of the first theories based on mtDNA polymorphisms@ studies stressed on our African origin followed by stepwise dispersal all over the world which took approximately 80,000 years. But not all findings agreed with this assumption if based on nuclear loci and supported multifocal origin of humans. The process of establishing a new species--Homo sapiens, is still not fully understood and many questions remained unanswered. From the point of view of population genetics we can assume that: 1. Mutability (natural or from internal causes) does not change though we cannot neglect suspicion that environment could influence its increase. The content of harming mutations in our genome, due to the protective effect of health care, which blocks natural selection, is increasing and moreover changes of our life style opened the door for manifestation of week deleterious alleles accumulated during foregoing period of evolution. Also prolongation of our life span is accompanied with effects of genotypes positively selected because of their positive effect on our reproductive period but which could be harmful during postreproductive stage--antagonistic pleiotropy. 2. According traditional assumption on the quality of new mutations is that they are either neutral or harming. Changes which are drift-dependent are becoming reduced. 3. Effective population size (steady state could be supposed or some increase due to more intensive local migration). 4. Migration (In spite of absence of corresponding demographic data) seems to be nowadays more intensive than it was in the past.

[Significance of confirmatory testing of HLA system in unrelated haematopoietic stem cell donors]. / Význam konfirmacních testu HLA systému nepríbuzných dárcu hematopoetických kmenových bunek.

BACKGROUND: Haematopoietic stem cell transplantation is a standard curative therapy for some acquired haematological diseases and inherited metabolic and immunological disorders. The HLA compatibility in five loci (HLA class I -A, -B and -C and HLA class II -DRB1 and -DQB 1) of the donor/recipient pair is a prerequisite for the success of haematopoietic stem cell transplantation which represents a process of adoption donors immunity. METHODS AND RESULTS: HLA is the most polymorphic system in the human genome and this polymorphism is exactly detected by molecular genetics methods on DNA level only. In period of 2001-2004 we performed confirmatory testing of 366 unrelated haematopoietic stem cells donors from Czech and foreign registers for 256 patients. Only 16% of the donors completely matched the patients in all HLA loci. We detected HLA mismatches in the samples of 81% patient/donor pairs but these results were consonant with previous results from registers. 3% of confirmatory samples were discrepant with previous registry data. CONCLUSIONS: Despite of increasing number of available unrelated haematopoietic stem cell donors and the quality of registry HLA typing the possibility of finding the completely match donor is still limited.

[Genetic databases. Though we were not the first ones, at least let us not to be the last]. / Genetické databáze. Kdyz uz nemuzeme být první, at' nejsme alespon poslední.

One of the most necessary tools of today's medical care is the well working database of personal genotypes. It could effectively reduce the increasing expenses because many of the genetic testing could be done only once a life. Any delay in establishing such database would bring not only internal, but also across-border complications due to internationalization of the genetic services. The most modem approach has been applied by the laws which put stress more against abuse than on collecting data. European laws including the Czech Republic seem to be much less progressive. General rules for data storage, and gene banking are still missing in the Czech Republic.

[Frequency view on genome changes testing]. / Frekvencní pohled na vysetrení odchylek genomu.

Laboratories dealing with human genome, both inherited and acquired changes, dispose with similar methods and technology. The spectrum of genetic tests is relatively broad and the number of mutations or variants tested differs substantially. Also the number of examinations carried out in individual laboratories varies. Data presented in the tables come from the year 2004 and indicate the number of examinations requested and number of positive results. Many laboratories mentioned in the registry CZDDNAL (http://www.uhkt.cz/lab_a_vysetreni/nr lab_dna_diag/dna_lab_db) perform the same tests but there is also a great number of tests carried out by only one laboratory. Reasons of the request, cost-effectiveness and clinical utility of genetic testing is being discussed.

[Ten years of molecular monitoring of chronic myeloid leukemia by quantitative RT-PCR]. / Deset let molekulárního monitorování chronické myeloidní leukémie metodou kvantitativní RT-PCR.

Chronic myeloid leukemia (CML) is characterized by the presence of BCR-ABL fusion gene resulting from the reciprocal chromosome translocation t(9;22)(q34;q 11), karyotypically detected as Ph chromosome. BCR-ABL gene was proved to play the principal role in CML pathogenesis. It is a hallmark of CML used in diagnostics and monitoring of the response to the therapy. The most sensitive method of detecting BCR-ABL aberration is RT-PCR which is able to find a single in leukemic cell between 10(6) normal leukocytes. Monitoring of BCR-ABL transcript level by quantitative RT-PCR is of the high prognostic value. High or increasing BCR-ABL transcript number signalizes bad response to treatment and a bad prognosis. On the contrary RT-PCR negativity, low level, or decreasing BCR-ABL transcript number denotes good response to treatment and good prognosis. Q-RT-PCR can detect changes in disease status several weeks or even months earlier than other methods. In 1994 the Q-RT-PCR was introduced at the Institute of Hematology and Blood Transfusion in Prague and was used for early detection of relapse after transplantation. At present it is used in all patients with CML for monitoring of response to the treatment. We have confirmed that this precise, sensitive and non-invasive method is of the principal importance for monitoring of disease status in CML patients.

[Specific silencing of PCNA gene expression in leukemic cell lines using siRNA]. / Cílené tlumení exprese genu PCNA pomocí siRNA v leukemických bunecných liniích.

BACKGROUND: Increased expression of PCNA gene was detected in chronic myeloid leukemia (CML) patients in our laboratory. The gene may participate in the disease development. The aim of the study was to develop appropriate conditions for PCNA-siRNA transfection into K562 and MOLM-7 cell lines (which both have the up-regulated PCNA gene) and to silence the increased expression. METHODS AND RESULTS: Key parameters of successful siRNA delivery into the cells are type and quantity of transfection reagent, cells and siRNA concentration or cultivation time before an expression analysis. Transfection reagents ExGene 500 (Fermentas), Metafectene (Biontex), Oligofectamine (Qiagen) and siPORT Amine (Ambion) were tested. Transfection efficiency was monitored by fluorescence microscopy of fluorescein labeled siRNA. Gene silencing was determined at mRNA level by real-time PCR and at protein level by western blots. As the most suitable reagent was chosen Oligofectamine, which achieved 70% decrease of PCNA mRNA level. Further, 50 nM siRNA concentration, 1 x 10(6) cells/ml and amount of Oligofectamine 4 microl per 1 ml of transfected cells were selected. The best cultivation time after siRNA delivery was 48 h. CONCLUSIONS: Based on the results of this study, transfection method for siRNA delivery into the K562 and MOLM-7 cell lines was proposed. The procedure can be transferred also on further selected genes potentially involved in CML and afterwards it will be possible to monitor the impact of siRNA-inhibition on expression profile. In the future siRNAs against some over-expressed genes would be used for gene therapy of CML.

Prediction and reversion of post-transplant relapse in patients with chronic myeloid leukemia using mixed chimerism and residual disease detection and adoptive immunotherapy.

In the prospective study, we examined hematopoietic mixed chimerism (using polymerase chain reaction (PCR) of variable number of tandem repeat-VNTR sequences) and minimal residual disease (MRD) status (using qualitative and in the case of positivity quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for the BCR/ABL fusion mRNA) in serial peripheral blood samples taken from 25 patients after bone marrow transplantation (BMT) for chronic myeloid leukemia (CML). Increasing mixed chimerism in correlation with increasing signal of MRD was detected in 10 patients. In two patients mixed chimera status and BCR/ABL rearrangement led to hematologic relapse, in five patients molecular relapse was followed by reappearance of Ph chromosome and three patients developed molecular relapse only. Adoptive immunotherapy-donor lymphocyte infusion (DLI), interferon (INF) and discontinuation of post-transplant immunosupression-separately or in different combinations was used in nine patients with molecular, cytogenetic or hematologic relapse of CML. The results demonstrate that significant response at the molecular level can be achieved for a majority of CML patients and that using of all forms of adoptive immunotherapy controlled by MC and MRD is more efficient in patients treated in early molecular relapse-with minimal disease burdens.