Simplifying procedure for prediction of resistance risk in CML patients - Test of sensitivity to TKI ex vivo.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL have dramatically improved chronic myeloid leukemia therapy. While imatinib remains to be the first line therapy, about 30% of patients develop resistance or intolerance to this drug and are recommended to switch to other TKIs. Nilotinib and dasatinib are currently implemented into the first line therapy and other inhibitors have already entered the clinical practice. This opens further questions on how to select the best TKI for each patient not only during the therapy but also at diagnosis. The individualized therapy concept requires a reliable establishment of prognosis and prediction of response to the available TKIs. We tested the ex vivo sensitivity of patient primary leukocytes to imatinib, nilotinib and dasatinib - two concentrations of each inhibitor for 48h incubation - and we evaluated the usefulness of such tests for the clinical practice. Besides reflecting the actual sensitivity to the therapy, our optimized simple tests were able to predict the outcome in 90/87% of patients, for the next 12/24months, respectively. According to these results, the presented ex vivo testing could help clinicians to select the appropriate drug for each patient at diagnosis and also at any time of the therapy.

The Lesser Egyptian Gerbil (Gerbillus gerbillus) is a suitable host for the long-term propagation of Cryptosporidium andersoni.

We describe the course of infection of Cryptosporidium andersoni LI03, originally isolated from cattle, in outbred Gerbillus gerbillus (Lesser Egyptian Gerbil), Meriones unguiculatus (Mongolian gerbil), and Meriones tristrami (Tristram's jird). While both Meriones spp. partially cleared the infection and shed a low number of oocysts (less than 15,000 oocysts per gram (OPG)), chronic infection with a mean infection intensity reaching 200,000 OPG was observed in G. gerbillus. These data suggest that G. gerbillus can be used as a laboratory model for the long-term maintenance and study of C. andersoni without the need for host immunosuppression.

Hsp90 - a potential prognostic marker in CML.

Heat shock proteins (Hsp) are important for the stability and function of cell proteins and thus for cell survival under physiological as well as stress conditions. Hsps were also reported to play an important role in tumorogenesis including leukemias. In this study we followed up Hsp70 and 90 protein levels in samples from patients with chronic myeloid leukemia (CML) to evaluate these Hsps with regard to their ability to characterize the disease status and disease prognosis. We analyzed 68 samples of total leukocytes of CML patients with different response to therapy with tyrosine kinase inhibitors. The results of Western blot analyses showed that the level of Hsp70 did not change in the course of the disease and did not correlate with response to therapy. In contrast, Hsp90 levels showed good correlation with the disease state. Patients with good response to therapy (major molecular response-MMR, molecular remission-CMR) had low expression levels of Hsp90, similar to those in healthy individuals. High Hsp90 levels were found in patients with resistance to therapy (hematological relapse-HR, accelerated phase or blast crisis), and in leukemic cell line K562. The results of the study suggested that not the kinetics but the particular level of Hsp90 at any time point since therapy start is of prognostic value: Hsp90 level above 0.27 significantly predicted poor response to TKI therapy (relapse, progression) and the level below 0.085 good response (MMR, CMR). In conclusion, Hsp90 level in total leukocytes could serve as a risk factor at diagnosis as well as during therapy and might help in clinical decision making especially in cases where BCR-ABL monitoring is of low predictive value. Our data suggest that high expression of HSP90 contributes to the aggressivity of the disease and should be considered as an important target for specialized CML therapy.

Prevalence and molecular characterization of Cryptosporidium spp. in dairy cattle in South Bohemia, the Czech Republic.

A total of 995 samples from slaughtered dairy cattle (6 months and older) were collected during two consecutive years (from 2007 to 2008), stained by aniline-carbol-methyl violet and examined microscopically. DNA was extracted from Cryptosporidium positive samples and from 200 randomly selected microscopically negative samples. Nested PCR was performed to amplify the partial SSU rRNA and GP60 genes of Cryptosporidium that were subsequently digested by SspI, VspI and MboII restriction enzymes to determine the Cryptosporidium species and genotype present. The highest prevalence of infection (18.2%) was in the animals in age group of 12-18 months. The sequence analyses of PCR-positive specimens identified 41 samples as Cryptosporidium andersoni (4.1%), 2 samples as Cryptosporidium bovis (0.2%), and 1 sample as Cryptosporidium parvum (0.1%). C. bovis was detected only in a group of cattle older than 18 months and C. parvum in heifer which was older than 14 months. Seasonal dependency of Cryptosporidium spp. prevalence was not proven to any significant degree. Infection intensity was generally low. Sequence analysis of the glycoprotein (GP60) gene showed that detected C. parvum belonged to the IIaA16G1R1 subtype. This is the first published report about subtyping of the Cryptosporidium GP60 gene from cattle in the Czech Republic.

Infectivity and pathogenicity of Cryptosporidium andersoni to a novel host, southern multimammate mouse (Mastomys coucha).

The infectivity and pathogenicity of Cryptosporidium andersoni (bovine isolate) for neonatal and adult southern multimammate mice (Mastomys coucha) was studied using transmission experiments. C. andersoni isolate used in this study was not infective for BALB/c mice, but experimental infection proved susceptibility of neonatal and adult M. coucha to the infection. The prepatent period was 20-24 days, the patent period varied between 46 and 59 days. No signs of clinical illness or macroscopic findings were detected in infected animals. Cryptosporidium developmental stages were detected only in the glandular part of the stomach of M. coucha in histological sections stained with Wolbach's modification of Giemsa and using immunofluorecence. Histopathological changes were characterized by dilatation and epithelial metaplasia of infected gastric glands without inflammatory response in the lamina propria. Neonatal M. coucha were more susceptible to C. andersoni infection than adults. M. coucha seems to be a useful laboratory model for study of C. andersoni infection.