Granzyme B in peripheral blood mononuclear cells as a measure of cell-mediated immune response in paraneoplastic neurological syndromes and malignancy.

BACKGROUND: Paraneoplastic neurological syndromes (PNS) may coexist with ovarian or lung cancers. Some tumors coexisting with PNS are smaller and have a better prognosis than tumors without PNS. PNS may constitute an opportunity to observe a natural immune antitumor response. We aimed to investigate a cytotoxic immune response by measuring granzyme B (GrB) in peripheral blood mononuclear cells (PBMC) in patients affected with ovarian or lung malignancy, with and without accompanying PNS. METHODS: We enrolled patients with: nonmalignant lesions (n = 21), ovarian cancer (n = 19), lung cancer (n = 57), and PNS (n = 30). PBMC were isolated by density gradient centrifugation with Ficoll-Paque. We evaluated the expression of GrB in PBMC lysates by ELISA and normalized to protein content as measured by the Lowry method. RESULTS: GrB levels in PBMC in the group with malignant tumors-median 1650 pg/mg protein (interquartile range 663-3260 pg/mg) and in patients with PNS-median 1890 pg/mg protein (range 1290-2640 pg/mg) was lower than in control group with nonmalignant lesions-median 5240 pg/mg protein (range 2160-7440 pg/mg), p = 0.0003 and p = 0.0038, respectively. The differences in GrB levels in PBMC between these groups were independent of epidemiological factors-age, sex, body mass index (BMI), and the number of immune cells, as confirmed by multiple regression analysis. Within the group of patients with malignancy and PNS, GrB levels in PBMC were elevated if onconeural antibodies were detected (2610; 2390-3700 pg/mg protein) as compared to patients without antibodies (1680; 970-1880 pg/mg protein, p = 0.035). GrB in PBMC was higher if the malignancy was diagnosed at the low (3060; 2120-5220 pg/mg protein) as compared to the high stage (1330; 348-2140, p = 0.00048). In patients with lung cancer, the expression of GrB in PBMC was lower (1430; 635-2660 pg/mg protein) than in the group with ovarian cancer (2580; 1730-3730, p = 0.02). CONCLUSION: The cytotoxic response measured in peripheral blood by GrB in PBMC is impaired both in the course of malignancy and PNS. Levels of GrB in PBMC were higher if onconeural antibodies were detected. Tracking reactive immune responses, such as GrB in PBMC may have diagnostic and monitoring value in malignancy and PNS.

Cancer incidence in the Greater Poland region as compared to Europe.

Greater Poland is a region with a high risk of cancer. In terms of age-standardised incidence rate, it is ranked 2nd for men and 3rd for women out of Poland's 16 provinces. Incidence structure in the region of Greater Poland is similar to that in other West European countries. The most common cancers in men are lung, prostate and colorectal, in women: breast, colorectal and lung. In 2016, nearly every third cancer-related death in the region was caused by lung cancer. In women, it was cause no. one. The incidence of chronic diseases, including cancer, is expected to further grow in view of the global ageing of the population. This means that malignancies will remain to be a major challenge for public health care.in the Greater Poland region.

Fractionated dosage of radioiodine for the ablation of low-risk differentiated thyroid cancer has no impact on survival.

INTRODUCTION: Due to a limited number of hospital beds dedicated to radioiodine therapy (RIT) in some countries, a fractionated dose of radioiodine may be considered as the ablation therapy of differentiated thyroid cancer (DTC). The aim of the study was to compare the late effects of ablation therapy with single and fractionated dose of radioiodine in patients with DTC. PATIENTS AND METHODS: Patients with low-risk DTC referred to our institution 5-16 weeks after thyroidectomy, treated with 2.2 GBq of 131I, either in a single dose (2.2 GBq, group 1) or in two fractions (1.1 GBq+1.1 GBq administered with a 24 h interval, group 2) were retrospectively included. Clinical outcome of the treatment and overall survival (OS) was evaluated. RESULTS: 83 patients treated with single dose and 186 patients treated with fractionated dose of radioiodine were included. Mean duration of follow-up was 8.0 vs.7.8 years, respectively (p=ns). There were no significant differences between the groups in male to female ratio, age at the time of the first RIT, proportion of papillary thyroid cancers, volume of the thyroid tissue, thyroid-stimulating hormone and thyroglobulin levels before first RIT. RIT was repeated in 55.4% and 54.8% of patients from group 1 and 2 respectively (p=ns). There were no significant differences including the course and outcomes of the treatment between the groups, measured by: cumulative dose of 131I, mean number of 131I administrations and mean thyreoglobulin concentration at the follow-up. Also the overall survival did not differ significantly between the groups. Probability of 5-year OS was 98.6% for patients treated with single and 99.5% with fractionated dose of 131-I, 10 year OS - 98.6 and 97.1% respectively, 15 year OS - 95.5 and 92.9% respectively (p=ns). CONCLUSIONS: In the long-term follow-up, radioiodine ablation therapy with fractionated doses in low-risk DTC patients is equally effective as with single dose. < p > < /p >.

Heterogeneous Association of Alzheimer's Disease-Linked Amyloid-ß and Amyloid-ß Protein Precursor with Synapses.

Alzheimer's disease (AD) is increasingly viewed as a disease of synapses. Loss of synapses correlates better with cognitive decline than amyloid plaques and neurofibrillary tangles, the hallmark neuropathological lesions of AD. Soluble forms of amyloid-ß (Aß) have emerged as mediators of synapse dysfunction. Aß binds to, accumulates, and aggregates in synapses. However, the anatomical and neurotransmitter specificity of Aß and the amyloid-ß protein precursor (AßPP) in AD remain poorly understood. In addition, the relative roles of Aß and AßPP in the development of AD, at pre- versus post-synaptic compartments and axons versus dendrites, respectively, remain unclear. Here we use immunogold electron microscopy and confocal microscopy to provide evidence for heterogeneity in the localization of Aß/AßPP. We demonstrate that Aß binds to a subset of synapses in cultured neurons, with preferential binding to glutamatergic compared to GABAergic neurons. We also highlight the challenge of defining pre- versus post-synaptic localization of this binding by confocal microscopy. Further, endogenous Aß42 accumulates in both glutamatergic and GABAergic AßPP/PS1 transgenic primary neurons, but at varying levels. Moreover, upon knock-out of presenilin 1 or inhibition of γ-secretase AßPP C-terminal fragments accumulate both pre- and post-synaptically; however earlier pre-synaptically, consistent with a higher rate of AßPP processing in axons. A better understanding of the synaptic and anatomical selectivity of Aß/AßPP in AD can be important for the development of more effective new therapies for this major disease of aging.

Postvaccination wounds associated predominantly with Arcanobacterium phocae in mink (Neovison vison) at three mink farms.

BACKGROUND: The emerging skin disease fur animal epidemic necrotic pyoderma (FENP) has been attributed to infection with Arcanobacterium phocae (ABP). The exact pathogenesis and risk factors of FENP have yet to be elucidated. ANIMALS: Three mink from each of three different mink farms (A-C) with postvaccination skin wounds at the vaccination site and six mink from an unaffected mink farm (D) that had used the same vaccine batch and vaccination site (hind leg). METHODS AND RESULTS: All mink from farms A-C had severe necrotizing to necropurulent dermatitis where they were vaccinated intramuscularly in the hind leg. ABP was the sole bacterium cultured from six of nine wounds. Using 16S-23S rDNA intergenic spacer region and BOX-PCR, the ABP isolates from these wounds were indistinguishable from isolates originating from several cases of FENP. CONCLUSIONS AND CLINICAL IMPORTANCE: This is the first report of FENP-like lesions at the site of vaccination, in the days following the procedure, associated with ABP. At farms with FENP vaccination, procedures should be considered carefully.

Rapid externalization of 27-kDa heat shock protein (HSP27) and atypical cell death in neutrophils treated with the sphingolipid analog drug FTY720.

The sphingolipid analog fingolimod is known to induce apoptosis of tumor cells and lymphocytes. Its effect on neutrophils has not been investigated so far. Here, we describe a fingolimod-induced atypical cell death mechanism in human neutrophils, characterized by rapid translocation of heat shock protein 27 to the cell surface, extensive cell swelling and vacuolization, atypical chromatin staining and nuclear morphology, and phosphorylation of mixed lineage kinase domain-like protein. Fingolimod also induces typical apoptotic features, including rapid externalization of phosphatidylserine and activation of caspase-8. Fingolimod-induced neutrophil death is independent of sphingosine-1-phosphate receptors and positively regulated by protein phosphatase A. Externalization of phosphatidylserine and heat shock protein 27 can be partially inhibited by inhibitors of caspase-8 [Z-Ile-Glu(O-Me)-Thr-Asp(O-Me)-fluoromethyl ketone], receptor-interacting protein kinase 1 (necrostatin-1), receptor-interacting protein kinase 3 (necrosulfonamide), and heat shock protein 90 [geldanamycin and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin]. Furthermore, NADPH oxidase 1 inhibition with diphenyleneiodonium chloride protects neutrophils against fingolimod-mediated cell death. Overall, these observations suggest that fingolimod acts through a mechanism involving the necrosome signaling complex and the oxidative stress machinery.

The impact of cancer incidence and stage on optimal utilization of radiotherapy: Methodology of a population based analysis by the ESTRO-HERO project.

BACKGROUND AND PURPOSE: The impact of differences in the distribution of major cancer sites and stages at diagnosis among 4 European countries on the optimal utilization proportion (OUP) of patients who should receive external beam radiotherapy was assessed within the framework of the ESTRO-HERO project. MATERIALS AND METHODS: Data from Australian Collaboration for Cancer Outcomes Research and Evaluation (CCORE) were used. Population based stages at diagnosis from the cancer registries of Belgium, Slovenia, the Greater Poland region of Poland, and The Netherlands were used to assess the OUP for each country. A sensitivity analysis was carried out. RESULTS: The overall OUP by country varied from the lowest of 48.3% in Australia to the highest of 53.4% in Poland; among European countries the variation was limited to 3%. Cancer site specific OUPs showed differences according to the variability in stage at diagnosis across countries. The most important impact on the OUP by country was due to changes in relative frequency of tumours rather than stage at diagnosis. CONCLUSIONS: This methodology can be adapted using European data, thus facilitating the planning of resources required to cope with the demand for radiotherapy in Europe, taking into account the national variability in cancer incidence.

Quantitative evaluation of fungi of the genus Candida in the feces of adult patients with type 1 and 2 diabetes - a pilot study.

BACKGROUND: Gastrointestinal tract microbiota, particularly bacterial microflora, seem to have a different qualitative and quantitative composition in both type 1 (T1DM) and type 2 diabetes (T2DM) mellitus cases as compared to non-diabetic individuals. So far, there are no data from diabetes research concerning the prevalence of fungi, particularly the most common genus, i.e. Candida, which are important components of human colon microflora. We aimed to examine whether there are quantitative changes of Candida fungi in the feces of patients with T1DM and T2DM as compared to healthy controls. FINDINGS: Overall, we included 44 diabetic patients (27 patients with T1DM and 17 with T2DM) as well as 17 healthy, non-diabetic controls. Feces and blood samples were collected from all study individuals. DNA was isolated from fecal samples and quantitative real time PCR (qPCR) was applied in order to determine the number of fungal cells. Statistical association with selected clinical and biochemical features was examined. There was a difference in the amount of Candida in the feces among the three examined groups (p = 0.007). Candida spp. populations in T1DM and T2DM subjects were larger as compared to controls (p = 0.017 and p = 0.037, respectively). However, no difference was found between T1DM and T2DM. No association was identified between the quantity of fungi and examined patients' characteristics, except for negative correlation with blood lipid parameters in T2DM group. CONCLUSIONS: Candida fungi appear to be more prevalent in the feces of patients with T1DM and T2DM. Their amount seems to be associated with serum lipids in T2DM patients. This initial finding requires further confirmation.

Cancer incidence and mortality in the Greater Poland Region-Analysis of the year 2010 and future trends.

BACKGROUND AND AIM: The Greater Poland Region is one of the most industrialised areas of Poland, with a high rate of cancer incidence and mortality. The present report estimated incidence and mortality data for Greater Poland in the year 2010. METHODS: Statistical reports in this study include absolute number of cases and crude incidence rates. The derived age-, sex-, and site specific rates were age-standardised (ASRs per 100,000 person-years) using the European (ASRE) standard population. RESULTS: In 2010, a total 13,581 new cancer cases were reported to the Greater Poland Cancer Registry. The number of new cases increased by 24% compared to 2001. Greater Poland has the second-highest ASR for both females and males among the 16 regions in Poland. The most common cancers are similar to those in other Western European countries. Among men, the most common cancers are lung (C34), colorectal (C18-C21), and prostate (C61) cancer. In women, breast cancer is the most common (C50), followed by colon (C18-C21) and lung (C34) cancer. Lung cancer in males accounts for more than one-third of all cancer-related deaths in Greater Poland. As in 2009, lung cancer is the leading cause of death in women. CONCLUSIONS: Given the ageing of the population, the incidence of chronic diseases, including cancer, is expected to grow. These data indicate that cancer will continue to represent an important challenge both to local health authorities and the National Health Fund, which will need to meet the growing demand for cancer care.

A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood.

BACKGROUND: The study describes the application of the PCR method for the simultaneous detection of DNA of Gram-negative bacteria, Gram-positive bacteria, yeast fungi and filamentous fungi in blood and, thus, a whole range of microbial etiological agents that may cause sepsis. Material for the study was sterile blood inoculated with four species of microorganisms (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus) and blood collected from patients with clinical symptoms of sepsis. The developed method is based on nested-multiplex real-time PCR . RESULTS: Analysis of the obtained data shows that sensitivity of nested-multiplex real-time PCR remained at the level of 10(1) CFU/ml for each of the four studied species of microorganisms and the percentage of positive results of the examined blood samples from the patients was 70% and 19% for the microbiological culture method. The designed primers correctly typed the studied species as belonging to the groups of Gram-positive bacteria, Gram-negative bacteria, yeast fungi, or filamentous fungi. CONCLUSIONS: Results obtained by us indicated that the designed PCR methods: (1) allow to detect bacteria in whole blood samples, (2) are much more sensitive than culture method, (3) allow differentiation of the main groups of microorganisms within a few hours.

Comparison of nested, multiplex, qPCR; FISH; SeptiFast and blood culture methods in detection and identification of bacteria and fungi in blood of patients with sepsis.

BACKGROUND: Microbiological diagnosis of sepsis relies primarily on blood culture data. This study compares four diagnostic methods, i.e. those developed by us: nested, multiplex, qPCR (qPCR) and FISH with commercial methods: SeptiFast (Roche) (SF) and BacT/ALERT® 3D blood culture system (bioMérieux). Blood samples were derived from adult patients with clinical symptoms of sepsis, according to SIRS criteria, hospitalized in the Intensive Care Unit. RESULTS: Using qPCR, FISH, SF, and culture, microbial presence was found in 71.8%, 29.6%, 25.3%, and 36.6% of samples, respectively. It was demonstrated that qPCR was significantly more likely to detect microorganisms than the remaining methods; qPCR confirmed the results obtained with the SF kit in all cases wherein bacteria were detected with simultaneous confirmation of Gram-typing. All data collected through the FISH method were corroborated by qPCR. CONCLUSIONS: The qPCR and FISH methods described in this study may constitute alternatives to blood culture and to the few existing commercial molecular assays since they enable the detection of the majority of microbial species, and the qPCR method allows their identification in a higher number of samples than the SF test. FISH made it possible to show the presence of microbes in a blood sample even before its culture.

Evaluation of the activity of thermostable DNA polymerases in the presence of heme, as a key inhibitor in the real time PCR method in diagnostics of sepsis.

The study aim was evaluation of the usefulness of several thermostable DNA polymerases in real time PCR conducted in the presence of the heme. Our study had the advantage of testing several different polymerases, one of which proved to be the least sensitive to heme activity. We also found that there is no need of supplementing the reaction mixture with protective substances like BSA. Selection of the appropriate polymerase can increase the efficiency of the PCR reaction which is very important for diagnosis of sepsis and for other analyses performed on DNA template isolated from the blood.

Novel left ventricular assist systems I and II for cardiac recovery: the driver.

We have recently described the Novel Left Ventricular Assist Systems (Novel LVAS) I and II, which avoid cannulation of cardiac chambers and synchronize pumping with the patient's electrocardiogram. We now describe the drive system in more detail. The drive unit is an air-driven pulsatile system. The driver's parameters can be programmed. This electro-pneumatic unit contains 3 modules. A remarkable feature of the driver system is that it contains 2 pneumatic units that alternate in their function every 15 minutes. This prevents overheating and component fatigue or failure, and it enables the use of smaller units. If one of the units fails, an alarm will warn of the problem, and the other will continue indefinitely. This LVAS is synchronized with the patient's ECG, which enables it to eject the stroke volume during diastole and in this way to act as a chronic counterpulsator. We have designed the Novel LVAS to operate at a low-frequency rate. This fact, together with the electrocardiographic synchronization, offers the best prospect for myocardial recovery in patients who are also receiving beta-adrenergic blocking agents. This dual therapy will help adjust heart rate to pump frequency.