High colonisation by probiotic Escherichia coli A0 34/86 strain is associated with a less diverse microbiome related to children's age.

Probiotic supplementation in childhood serves as an additional source of bacterial colonisers and represents an opportunity to beneficially manipulate the intestinal microbiome. Differences in the ability of probiotic strains to colonise the gut may be related to the variously diversified gut microbiome. We report the results of the association between composition of the gut microbiome and the colonisation capacity of the probiotic strain Escherichia coli A0 34/86 (CNB - Colinfant New Born supplement) in the cases of three healthy children in different development stages (infant, toddler, and pre-school), as a preliminary insight to possible future prospective studies of this subject. Microbiome composition was estimated by 16S rRNA gene sequencing of 55 stool samples collected during approximately 3.5-13 months long periods. Detailed characterisation of the E. coli population was performed using colony PCR to detect 33 E. coli genetic determinants. In all children, genetic determinants typical for the probiotic E. coli A0 34/86 strain were detected immediately after administration of the probiotics. Analysis of the initial sample composition (the last sample taken before the probiotic administration) showed that the gut microbiome of infant and toddler with lower bacterial diversity was more successfully colonised by the probiotic strain. In our case report of three children, we showed for the first that supplementation with CNB probiotics in early infancy and toddlerhood was associated with high E. coli A0 34/86 colonisation and a significant change in the composition of the gut microbiome. Our results indicate that administration of CNB for its recommended duration might be efficient only in very early childhood.

[Molecular Pathology of Colorectal Cancer, Microsatellite Instability - the Detection, the Relationship to the Pathophysiology and Prognosis]. / Molekulární patologie kolorektálního karcinomu, mikrosatelitová nestabilita - zpusob detekce, vztah k patofyziologii a prognóze.

BACKGROUND: Colorectal carcinoma (CRC) is third most common cancer worldwide with very heterogenous character. In most cases, it is caused by sporadic events leading to disruption of epithelial cells of the colon. The minority evolves from germline mutations associated with hereditary cancer syndromes. Mechanisms leading to mutations of oncogenes, tumour suppressors and genes of DNA repair mechanisms include: 1. chromosomal instability, 2. microsatellite instability and 3. CpG island methylator phenotype. Microsatellite instability (MSI) usually arises from a germline mutation of the component of mismatch repair machinery (MMR) or somatic hypermethylation of the MLH1 promoter. The diagnostic approaches include PCR methods and immunohistochemistry for the detection of the loss of MMR part. The aim of our study was to characterise the cohort of ongoing study of gut microbiome in CRC patients considering MSI. MATERIAL AND METHODS: The consecutive study group consisted of 103 patients diagnosed with CRC. The cohort consisted of 45 women (43.7%) and 58 men (56.3%). Patient age at the time of diagnosis was within the range of 31-83 years (median 66 years). The expression of MLH1, MSH2, MSH6 and PMS2 proteins was detected by immunohistochemical method and the positivity was correlated with the stage and the localization of the primary tumour. RESULTS: The MMR status was determined by immunohistochemical method in 43 (41.7%) from the existing total of 103 patients. MSI was detected in 11 (25.6%) cases while 32 (74.4%) were microsatellite stabile. With the respect to cancer clasification the most cases of MSI was detected in stage II (8 cases; 22.2%). In regard to localization of primary tumour, MSI rather correlates to right site CRC, while microsatellite stable tumours do not show any site preferences. CONCLUSION: Considering low number of MMR status determination in study group, statistic evaluation is inaccurate so far. However there is a trend in our cohort in relation to determination of the portion of MSI in CRC population and also in localization of primary tumour according to literature.Key words: colorectal carcinoma - microsatellite instability - Lynch syndrome The work was supported by the project MEYS - NPS I - LO1413 and AZV 16-31966A. 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: 13. 3. 2017Accepted: 26. 3. 2017.

[Circulating Myeloid Suppressor Cells and Their Role in Tumour Immunology]. / Cirkulující myeloidní supresorové bunky a jejich úloha v nádorové imunologii.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are heterogenic population of multipotent progenitors of myeloid lineage. For their immunosuppressive effect, MDSC are responsible for tumour escape from the host immune surveillance. Furthermore, MDSCs support tumour by promotion of angiogenesis and metastasis. Membrane markers of human MDSCs are myeloid markers CD11b and CD13, these cells are HLA-Drlow/- and expression of CD15 or CD14 differentiate them into granulocytic (Gr-MDSCs) and monocytic (Mo-MDSCs), resp. PATIENTS AND METHODS: Using flow cytometry, we investigated Mo-MDSC counts in peripheral blood of non-cancer individuals - control group (n = 61), breast (n = 39) and colorectal (n = 52) cancer patients. These cells were detected as CD45+CD11b+CD33+CD14+HLA-Drlow/- and quantified as percentage of total white blood cells and as absolute count. RESULTS: In control group, circulating Mo-MDSCs was gender-and age-independent and the average value was 1.09% and 0.073 × 109/l. Breast cancer patients had higher circulating Mo-MDSCs compared to control group with average values: 3.57% and 0.229 × 109/l (p < 0.001) and we also observed increase in Mo-MDSC number after granulopoietic growth factors administration (p = 0.043). Colorectal cancer patients had higher average number of circulating Mo-MDSCs compared to control group: 1.71% a 0.125 × 109/l (p = 0.003) and its number did not correlate with tumour clinicopathological stage, localization of primary tumour (colon vs. rectum), site (left vs. right) and microsatellite instability. CONCLUSION: Increased number of MDSCs in circulation and within tumour microenvironment has been associated with immune suppression and tumour progression. Colorectal cancer patients at diagnosis showed higher circulating Mo-MDSCs possibly reflecting immunosuppressive effect of tumour microenvironment. Change of Mo-MDSC number from baseline level need to be evaluated in the context of CRC patients outcome. Recombinant granulopoietic growth factors increase number of circulating Mo-MDSCs and the effect of this phenomenon on cancer prognosis remains to be elucidated.Key words: myeloid-derived suppressor cells - colorectal cancer - breast cancer - immunology - immunosuppression - G-CSF This work was supported by MEYS by NPU I (LO1413), grant AZV 16-31966A and MH DRO 00209805. 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: 11. 3. 2017Accepted: 26. 3. 2017.

Distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features.

BACKGROUND: Differences exist between the proximal and distal colon in terms of developmental origin, exposure to patterning genes, environmental mutagens, and gut flora. Little is known on how these differences may affect mechanisms of tumorigenesis, side-specific therapy response or prognosis. We explored systematic differences in pathway activation and their clinical implications. MATERIALS AND METHODS: Detailed clinicopathological data for 3045 colon carcinoma patients enrolled in the PETACC3 adjuvant chemotherapy trial were available for analysis. A subset of 1404 samples had molecular data, including gene expression and DNA copy number profiles for 589 and 199 samples, respectively. In addition, 413 colon adenocarcinoma from TCGA collection were also analyzed. Tumor side-effect on anti-epidermal growth factor receptor (EGFR) therapy was assessed in a cohort of 325 metastatic patients. Outcome variables considered were relapse-free survival and survival after relapse (SAR). RESULTS: Proximal carcinomas were more often mucinous, microsatellite instable (MSI)-high, mutated in key tumorigenic pathways, expressed a B-Raf proto-oncogene, serine/threonine kinase (BRAF)-like and a serrated pathway signature, regardless of histological type. Distal carcinomas were more often chromosome instable and EGFR or human epidermal growth factor receptor 2 (HER2) amplified, and more frequently overexpressed epiregulin. While risk of relapse was not different per side, SAR was much poorer for proximal than for distal stage III carcinomas in a multivariable model including BRAF mutation status [N = 285; HR 1.95, 95% CI (1.6-2.4), P < 0.001]. Only patients with metastases from a distal carcinoma responded to anti-EGFR therapy, in line with the predictions of our pathway enrichment analysis. CONCLUSIONS: Colorectal carcinoma side is associated with differences in key molecular features, some immediately druggable, with important prognostic effects which are maintained in metastatic lesions. Although within side significant molecular heterogeneity remains, our findings justify stratification of patients by side for retrospective and prospective analyses of drug efficacy and prognosis.

[Comparison of laparotomic and laparoscopic techniques for implantation of the peritoneal part of the shunt in the treatment of hydrocephalus]. / Srovnání laparotomické a laparoskopické techniky pri implantaci peritoneální cásti shuntu v terapii hydrocefalu.

INTRODUCTION: Implantation of a ventriculoperitoneal shunt is a standard procedure in the treatment of hydrocephalus. Shunt malfunction can be due to various causes, such as failure of the peritoneal (distal) part of the shunt with a frequency of 5% to 47%. OBJECTIVE: The aim of this study was to compare laparoscopic and laparotomic techniques for implantation of a ventriculoperitoneal shunt. MATERIAL AND METHODS: We considered a cohort of 304 patients with hydrocephalus, acquired during a 10-year period, who underwent surgical intervention at the Neurosurgical and Surgical Clinics of the University Hospital Brno. RESULTS: The 304 patients underwent a total of 392 operations, of which 67 (17.1%) were performed using a laparoscopic approach and 325 (82.9%) using a laparotomic approach. In the laparotomy group, 59 (18.2%) interventions were repeated due to complications of the peritoneal part of the shunt, while in the laparoscopy group revisions accounted for only 3 cases (4.5%). CONCLUSIONS: The laparoscopic technique significantly reduces the risk of complications of the peritoneal part of the shunt, and thus the overall complications associated with the implantation of the ventriculoperitoneal shunt. Laparoscopy is indicated in the case of migration of the peritoneal catheters into the abdominal cavity and is also very helpful in revisions in the case of malfunction of the peritoneal part of the shunt or in the case of previous abdominal surgery. It can explain the anatomical conditions in the abdominal cavity and it is able to treat any incidental pathology.

NKT-like cells are expanded in solid tumour patients.

CD3+ CD56+ NKT-like cells have been shown to produce substantial amounts of pro-inflammatory cytokines and to mediate lysis of malignant cells. Using flow cytometry, we evaluated the absolute NKT-like cell count in peripheral blood from individuals in a reference population and the median number was 0.085 x 10(9)/l. The average number of NKT-like cells in patients with disseminated cancer was 2.65 fold higher than in the reference population. The number of CD3+ CD56+ cells in solid tumour patients who achieved complete remission was comparable to the reference population. In breast cancer patients with initially (prior to therapy) increased number of NKT-like cells, we observed a trend toward longer disease-free survival. Thus we conclude that CD3+ CD56+ NKT-like cells have potential to suppress tumour evasion and are expanded in peripheral blood of some epithelial tumour patients.

[Profiles of low-molecular proteome spectrum obtained through SELDI-TOF mass spectrometry in the sera of patients with metastatic malignant melanoma: pilot study]. / Profily nízkomolekulárniho proteomového spektra získané pomocí hmotnostní spektrometrie SELDI-TOF v sérech pacientus diseminovaným maligním melanomem: pilotní studie.

BACKGROUNDS: Recently, research at genetic and molecular levels has extensively accelerated due to advances in new technologies. Since the mid-90s, a relatively new discipline--clinical proteomics, has evolved, which focuses on studying gene products--proteins. The evaluation of protein profiles may contribute to the more accurate stratification of patients in the future, in terms of both prediction of treatment results and prognosis. In pursuing this objective, proteomic approaches are currently used for the identification of new biomarkers. This is also the case with malignant melanoma, a disease without typical serum marker possessing high sensitivity and high specificity. METHODS: We analyzed human blood serum samples from 25 patients with metastatic malignant melanoma treated with palliative chemotherapy at the Masaryk Memorial Cancer Institute, Brno, in 2004-2006. The analysis was performed by Surface Enhanced Laser Desorption/lonisation Time of Flight Mass Spectrometry (SELDI-TOF-MS). Our patients were divided into two subgroups: a group relatively resistant to chemotherapy--14 patients--and a group with certain clinical benefit from the treatment (complete and partial remission, stabilized disease)--11 patients. We were searching for a new biomarker or typical protein profile in the selected two subgroups. Then, we recategorized our patients into three groups according to the similarity of their protein profiles regardless of sensitivity to chemotherapy. Finally, we evaluated differences in laboratory and clinical parameters, between both the groups of chemo-resistant and chemo-sensitive patients, and newly defined subgroups with similar protein profiles. CONCLUSION: We did not identify any significant differences in protein profiles or laboratory parameters in the predefined chemo-sensitive or chemo-resistant groups of patients. However, with regard to the new groups with similar protein profiles, we identified a subgroup of patients with different laboratory and clinical parameters. The results are very interesting and merit further research.

[Functional integrity of neural retina in 2. type diabetics]. / Funkcná integrita neurosenzorickej sietnice u diabetikov 2. typu.

The purpose of this prospective longitudinal study was to investigate early defects in functional integrity of neural retina in 2. type diabetic patients without or with mild diabetic retinopathy (DR) since there is an evidence of early functional changes in neural retina before occurrence of clinical manifestation of DR. Psychophysical test of contrast sensitivity (CS) was used for the detection of these changes. Relation between CS and systemic risk factors (HbAlc, blood pressure (BP), serum lipids and BMI) were also evaluated during a follow-up time. There were 48 recent diabetics without DR included in this study that were examined 3 times and compared to 23 diabetics with mild DR. The CS tests were performed using both Sine Wave Contrast Test (SWCT) and Pelli-Robson (PR) test. The reference values for CS threshold were derived from a CS of a control group of 52 healthy individuals. Abnormal CS ascertained by both methods, SWCT and PR, was observed in diabetics with mild NPDR. In comparison to the control group, there was a statistically significant difference of CS in spatial frequencies (SF) of 1.5, 6, 12, 18 cycles per degree (cy/deg). In comparison to diabetics without DR there was a significant difference of CS in SF of 6, 12 and 18 cy/deg in diabetics with mild NPDR. Abnormal CS was noticed in 47.8% (SWCT) or 21.7% (PR) of diabetics with DR. Statistically significant influence of high systolic BP on CS values and visual acuity was noticed. There were no abnormalities in CS in patients without DR comparing to control group during the whole follow-up. However, there was an improvement of CS in SF of 18 cy/deg observed between 1. and 3. evaluation of CS. Interaction of change in values of HbAlc and total cholesterol to HDL ratio had significant influence on CS improvement. Diabetics without DR had significantly better diabetes and blood pressure control in comparison to the diabetics with DR. In conclusion, it was not proved in this study that CS test is suitable for the screening for DR or early functional defects in neural retina before clinical manifestation of DR. Early diagnosis of DM and good compensation of diabetes, blood pressure and serum lipids level can postpone the onset of DR as well as the visual functions impairments.

Differential gene expression of bone marrow CD34+ cells in early and advanced myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder characterized by ineffective hematopoiesis and dysplasia in one or more blood cell lines. Because it often progress to poor outcome stages or acute leukemia we searched for candidate genes associated with disease progression. Using microarrays we performed gene expression profiling in CD34+ cells of 4 early and 4 advanced MDS patients and identified 286 significantly differentially expressed genes between these two categories. Out of these, 136 genes were up-regulated and 150 down-regulated in early MDS compared to advanced MDS. Using clustering analysis those two patient categories were clearly differentiated. Further, we selected three genes (ADAM8, BIRC5, MPL) for gene expression validation by qRT-PCR in an additional set of 29 MDS and sAML patients. We confirmed decreasing trend for BIRC5 expression from early to advanced stages of MDS, with the lowest levels in sAML patients. On the contrary, higher ADAM8 and MPL expression was observed in most advanced MDS patients compared to the early MDS patients. Association between gene expression levels and bone marrow blast proportion was tested, but only BIRC5 expression showed negative correlation (r=-0.83 at p<0.001). This study demonstrates stage-specific expression of some genes that may have potential prognostic significance.

Identification of putative ancestors of the multidrug-resistant Salmonella enterica serovar typhimurium DT104 clone harboring the Salmonella genomic island 1.

The origin of multidrug-resistant Salmonella enterica serovar typhimurium (S. typhimurium) harboring the Salmonella Genomic Island 1 (SGI1), which was detected for the first time in the mid-1980s is unknown. In this study, we performed microarray genomotyping of four multidrug-resistant SGI1 positive strains and found that unlike the S. typhimurium LT2 strain, the multidrug-resistant strains lacked genes STM0517-0529 allowing the utilization of allantoin as a sole nitrogen source. We extended this observation by PCR screening of additional 120 S. typhimurium field strains and found that this locus was absent in all SGI1 positive and also in 24% of SGI1 negative strains, which were proposed to be the original recipients of SGI1. To prove this hypothesis, we compared the STM0517-0529 negative strains (with or without the SGI1) by PFGE and PCR prophage typing and found that 8 out of 11 of the SGI1 negative strains and 17 out of 22 SGI1 positive strains were of identical PFGE pattern and PCR prophage pattern, while this specific pattern was never observed among STM0517-0529 positive strains. We therefore propose that a lineage of the S. typhimurium DT104 sensitive strain first lost the ability to metabolize allantoin and then acquired SGI1.