Time and Temperature Stability of TGF-ß1, EGF and IGF-1 in 20% and 100% Human Serum.

Autologous serum eye drops (ASEDs) are used as a treatment for severe dry eye disease. The concentration and stability of various growth factors in ASEDs is determinative for their efficiency. We therefore assessed the concentrations of transforming growth factor beta 1 (TGF-ß1), epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1) in ASEDs following storage at 4-8, -20, -80 and -156 °C. Twenty % and 100% sera from eight healthy volunteers were analysed by the sandwich enzyme immunoassay at different time intervals up to seven months. The mean levels of TGF-ß1 and EGF in undiluted and 20% serum did not differ significantly from the baseline levels in fresh serum for any storage conditions after 7 days at 4-8 °C, as well as after 4- and 7-month preservation at sub-zero temperatures. In 20% serum, no IGF-1 concentration decrease was found following 7 days of preservation at 4-8 °C. However, a decrease to 78 % and 81 % (P < 0.01) of baseline values was found in 20% serum after 4-month storage at -20 °C and 7-month storage at -156 °C, respectively. A more pronounced decrease in IGF-1 was observed in undiluted serum. All assessed growth factors present in 20% frozen serum remained stable for up to 7 months. The highest stability was achieved at -80 °C. At -20 and -156 °C, some decrease in IGF-1 occurred. Our results indicate that 20% ASEDs can be stored frozen up to 7 months under proper conditions.

Circulating Cell-Free DNA Extraction from Liquid Biopsy for Cancer Research.

As the number of cancer patients globally increases, a need for reliable biomarkers including circulating tumour DNA from liquid biopsy for diagnosis, prognosis and monitoring of the disease is rising. Currently, mainly tissue samples from biopsy are used, but there are certain limitations: firstly, it is an invasive technique, and secondly, in some cases it is almost impossible to obtain an acceptable tissue sample. This could be changed by using circulating cell-free DNA from liquid biopsy, which also gives the possibility of repeated examination. Here, we focus on the options of isolating circulating cell-free DNA from plasma samples using two isolation techniques: precision manual QIAamp Circulating Nucleic Acid Kit and automatic MagNA Pure Compact (MPC) using Nucleic Acid Isolation Kit I. Manual extraction gave significantly better yields of circulating tumour DNA (P < 0.05). This DNA also had less contaminants (organic compounds or proteins). DNA obtained by both tested methods of isolation is suitable for subsequent molecular genetic methods.

The Effects of Different Storage Conditions and Repeated Freeze/Thaw Cycles on the Concentration, Purity and Integrity of Genomic DNA.

The crucial requirement of molecular genetic methods is high-quality input material. The key question is "how to preserve DNA during long-term storage." Biobanks are recommended to aliquot isolated DNA into provided volumes. The aim of this study was to analyse the effect of repeated freezing and thawing on the genomic DNA integrity, quality and concentration. The aliquoted DNA isolated from blood cells using the automatic MagNA system and manual salting out method underwent freeze/thaw cycles at different storage conditions (-20 °C, -80 °C and liquid nitrogen). The average initial concentrations were 270.6 ng/µl (salting out method) and 125.0 ng/µl (MagNA). All concentration deviations relative to the concentration after the first freeze/ thaw cycle were less than 5 % for -20 °C and -80 °C cycling with both isolation methods. The average percentage differences of liquid nitrogen samples were higher, and the MagNA isolation method showed significant differences. There were no significant changes in the DNA purity or quality. The repeating freeze/ thaw up to 100 cycles (through -20 °C and -80 °C, respectively) did not significantly influence the integrity, concentration, or purity of genomic DNA, suggesting that storage of samples in high-volume pools without multiple aliquoting is possible. Storage in a freezer seems to be the most suitable way of long-term DNA preservation, because liquid nitrogen storage leads to formation of DNA clumps.

[The interpreter role of clinical geneticist in the process of genetic testing]. / Interpret--jedna z rolí klinického genetika.

Due to the progress in genetic research, development and rapid introduction of new genetic tests into clinical practise can be expected. This is raising many ethical issues which need to be carefully considered. First, genetic information is a familial. Thus, the test result of one person may have direct health implications for others who are genetically related. Second, the risks of genetic testing are also psychological, social and financial. Third, due to complex ways of genes interactions, genetic information often has limited predictive power. Finally, many genetic conditions remain difficult to treat or prevent, meaning the value of genetic information may be limited for altering the clinical care for the person. Given these concerns, detailed counselling and informed consent should be key aspects of genetic testing process. Genetic counselling in Czech Republic is provided by clinical geneticist. Therefore he is playing a key role in addressing these issues to patients. His second role is to interpret the genetic information revealed in genetic testing into the language understandable for patient, which means translation of genetic data into diagnosis and clinical management of individual, a transformation from statistics to physical persons. This interpretation is determining many aspects of patient's future life (future planning, reproductive decisions, prevention, health behaviour, etc.) and also family attitudes towards testing. The importance of genetic counselling, informed consent process and precise interpretation of results will be increasing over the time when new generation of genetic technologies for detecting the common conditions will be introduced into the practise.

[Psychosocial factors associated with genetic testing for certain hereditary types of neoplasms]. / Psychosociální faktory spojené s genetickým testováním nekterých dedicne podmínených nádorových onemocnení.

Mutations in predisposing genes for some of the hereditary forms of cancer exhibit autosomal dominant mode of inheritance. Introduction of genetic tests for these mutations to the clinical practice initiated studies focused on the psychosocial factors associated with genetic testing. Undergoing the genetic testing is a stressful experience for both the healthy individuals in risk and the patients already affected with cancer. The psychosocial characteristics of the tested individual influence not only the psychological functioning during the testing but also the acceptance of the test, and generally his life style and health practices. Psychological support during the genetic testing process is mostly provided by the genetic counsellor. The findings of psychosocial studies might be therefore helpful for the focusing of the genetic consultation, and fulfilling the client needs and expectations towards testing. Factors of motivation, psychological state, influence of family situation and support, and optionally the involvement of a psychologist into the process of genetic testing are observed.

[Perspective and present possibilities of chemotherapy in viral respiratory infections]. / Perspektivy a soucasné moznosti chemoterapie virových respiracních nákaz.

The author presents a brief history and contemporary trend of research and trends of antiviral chemotherapy with special attention to possible prophylaxis and treatment of viral respiratory infections. For substances which are at present available the author describes the mechanism of action, mode of administration, pharmacokinetics and undesirable side-effects.

The effect of cyclophosphamide and X-irradiation on experimental influenza in mice.

Mice treated with Cyclophosphamide (Cy) shortly before inoculation of influenza A virus exhibited increased mortality and delayed mean time of death. The extrapulmonary dissemination of the infection was observed more often in Cy-treated animals with the titres of virus in different organs substantially higher than in equally infected immunocompetent controls. Although the humoral antibody response was not impaired in Cy-treated mice, they were more susceptible to challenge with a lethal dose of virus than normal animals. In X-irradiated mice, the increased multiplication of virus in lungs and spread of the infection to other organs was observed, with prolonged persistence of virus in lungs and brains as compared to adequate controls, reminding the author's previous observation in immunocompromised persons, who died in the course of influenza.

Contribution to laboratory diagnosis of mumps and parainfluenza.

Specific IgM and IgG antibodies to mumps virus (MV) were detected in sera of mumps-patients by ELISA in agreement with the results obtained by indirect immunofluorescence (IF). Of given sera 37.5% contained IgM reacting in indirect ELISA also with the antigens of parainfluenza virus (PiV) T3. In all patients with respiratory illness over 2 years of age, the significant increase of antibodies to PiV in haemagglutination inhibition (HI) test was in good correlation with serum IgM and IgG antibody levels to PiV T3 determined by ELISA; but, in addition, 30.7% of these sera cross-reacted with MV antigens. The cross-reactions were eliminated by using MV-nucleocapsid antigen in indirect ELISA, or in direct ELISA using the peroxidase-labelled whole virion antigen. In some children under two years of age a discrepancy was observed between the significant increase of serum antibodies in HI and the inability to detect specific IgM antibodies by means of ELISA in their sera. The low-avidity antibodies appearing after primary PiV infection were probably washed off during the ELISA procedure.

Natural killer cells and cytomegalovirus and Epstein-Barr virus infection in cardiac graft recipients.

The serum of 10 male patients undergoing heart transplantation was found to contain anamnestic antibodies (IgG) against cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Patients with decreased natural killer (NK) cell activity displayed specific IgM against these viruses, a fact suggesting their activation and reproduction. Patients whose NK activity was within the range of that of healthy blood donors did not have serum IgM against CMV and EBV. Decreased NK activity and presence of specific IgM are associated with an increased number of LGL and monocytes, determined both by a monoclonal antibody (MEM-18) against their surface antigen, and morphologically.

CMV infection in patients with chronic renal failure and in those following transplantation.

Serum anti-CMV IgG was found in 62% of healthy blood donors. Patients in a chronic haemodialysis programme were seropositive in 80%, and kidney graft recipients in 93 to 100%, depending on the post-transplantation period. Median of the IgG titre was 1:10 in healthy persons, 1:80 in dialysed patients, and 1:40 in graft recipients. Anti-CMV IgG was never detected in healthy blood donors. IgM positivity was found in one of 30 dialysed patients, and in four of 27 kidney graft recipients. Compared with healthy blood donors, a higher percentage of CD 8 lymphocytes and LGL was found in dialysed patients exhibiting an anti-CMV IgG above 1:40. NK activity was impaired in all patients in the chronic haemodialysis programme.

Specific IgM antibodies in the saliva of mumps patients.

Antibodies to mumps virus were detected in 63.5% of saliva samples from mumps patients. The secretory antibody response was of primary type. Specific IgM antibodies were found in some samples collected early after the onset of disease. Specific IgA were detected in later obtained samples. Persons over 15 years of age reacted more often and more promptly than the children. The authors discuss the possible significance of prior antigenic stimulation by related paramyxoviruses (namely parainfluenzaviruses) for the intensity of local antibody response to mumps virus infection.

Contribution to rapid diagnosis of infectious mononucleosis.

By indirect immunofluorescence (IF) technique humoral antibodies to Epstein-Barr virus capsid antigen (EB-VCA) and to cytomegalovirus (CMV) were detected in 47% and 9% of persons with infectious mononucleosis (IM), respectively. In 23% of the patients examined, IgM antibodies to both viruses were detected, while in 8% of them high titres of IgG only were found in the absence of IgM class antibodies to EB-VCA or to CMV. The finding of IgM antibody to EB-VCA was in good correlation with the persisting symptoms of the disease. Discrepancy between the presence of specific IgM and the absence of heterophilic antibodies was observed in some children and in all persons with persistent or recurrent signs of IM. In the latter, specific IgM was found only during exacerbation of the disease, but during remissions IgG antibodies persisted in high levels. Antibodies to Epstein-Barr virus nuclear antigen (EBNA) were detected in all chronically ill persons and antibodies to the R-component of Epstein-Barr virus early antigen (EA) were present in the majority of them.

Studies on prophylactic efficacy of N-2-hydroxyethyl palmitamide (Impulsin) in acute respiratory infections. Serologically controlled field trials.

The results of three serologically controlled double blind field trials in army units are presented. The evaluation of results according to morbidity, regardless of aetiology, showed a significant reduction in acute respiratory diseases (ARD) after administration of Impulsin. In the 1973 trial (901 volunteers), 22.7% of ARD cases were found in the Impulsin group contrary to 34.4% in the placebo group (P less than 0.0002). The relevant values in the 1974 trial (610 volunteers) were 19.7% and 40.7% (P less than 0.002) and in the 1975 trial (353 volunteers) 10.6% and 28.8% (P less than 0.004). The study of the immunological background in representative sets of volunteers allowed determination of the aetiology, the proportion of asymptomatic infections and possible deformation of results due to preexisting protective antibodies. Manifestation rate (MR) expressing the proportion of sick persons out of all sensitive subjects with serologically proved infection was found useful. This indicator is relatively independent of randomization and is more sensitive as compared to the incidence rate. In the 1973 trial, influenza A 2 England was prevalent, the MR of infection being 15.4% in the Impulsin group and 44.9% in the placebo group (P less than 0.0002). After elimination of persons with preinfection antibodies greater than or equal to 1:256 the corresponding values of MR were 17.6% and 46.6% (P less than 0.005), reflecting the "relatively clean effect" of Impulsin. In the 1974 trial, where influenza B Hong-Kong was prevalent, MR was 14.3% and 57.1%, respectively (P less than 0.001). Preinfection antibodies were negligible. The preliminary prophylactic index of the drug seemed to be 4.3 for combined adenoviral infections (trials 1973 and 1974 taken together). In the 1975 trial, the results of serological examination were unsatisfactory. Antibodies vs. influenza A Port Chalmers were found in 24.5% of ARD only. The differnce is aetiologically unclarified ARD was statistically significant. Although displaying a significant limitation of clinical infections, the administration of Impulsin did not seem to have any influence on the formation of antibodies.