Single-cell adhesivity distribution of glycocalyx digested cancer cells from high spatial resolution label-free biosensor measurements.

The glycocalyx is a cell surface sugar layer of most cell types that greatly influences the interaction of cells with their environment. Its components are glycolipids, glycoproteins, and oligosaccharides. Interestingly, cancer cells have a thicker glycocalyx layer compared to healthy cells, but to date, there has been no consensus in the literature on the exact role of cell surface polysaccharides and their derivatives in cellular adhesion and signaling. In our previous work we discovered that specific glycocalyx components of cancer cells regulate the kinetics and strength of adhesion on RGD (arginine-glycine-aspartic acid) peptide-coated surfaces [1]. Depending on the employed enzyme concentration digesting specific components both adhesion strengthening and weakening could be observed by monitoring the averaged behavior of thousands of cells. The enzyme chondroitinase ABC (ChrABC) was used to digest the chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate components in the glycocalyx of cancer cells. In the present work, a high spatial resolution label-free optical biosensor was employed to monitor the adhesivity of cancer cells both at the single-cell and population level. Population-level distributions of single-cell adhesivity were first recorded and analyzed when ChrABC was added to the adhering cells. At relatively low and high ChrABC concentrations subpopulations with remarkably large and weak adhesivity were identified. The changes in the adhesivity distribution due to the enzyme treatment were analyzed and the subpopulations most affected by the enzyme treatment were highlighted. The presented results open up new directions in glycocalyx related cell adhesion research and in the development of more meaningful targeted cancer treatments affecting adhesion.

Relationship between natural and infection-induced antibodies in systemic autoimmune diseases (SAD): SLE, SSc and RA.

Infection or vaccine-induced T cell-dependent immune response and the subsequent high-affinity neutralizing antibody production have been extensively studied, while the connection between natural autoantibodies (nAAbs) and disease-specific antibodies has not been thoroughly investigated. Our goal was to find the relationship between immunoglobulin (Ig)M and IgG isotype nAAbs and infection or vaccine-induced and disease-related autoantibody levels in systemic autoimmune diseases (SAD). A previously described indirect enzyme-linked immunosorbent assay (ELISA) test was used for detection of IgM/IgG nAAbs against citrate synthase (anti-CS) and F4 fragment (anti-F4) of DNA topoisomerase I in 374 SAD samples, with a special focus on systemic lupus erythematosus (SLE) (n = 92), rheumatoid arthritis (n = 73) and systemic sclerosis (n = 157) disease groups. Anti-measles IgG and anti-dsDNA IgG/IgM autoantibodies were measured using commercial and in-house indirect ELISA tests. In all SAD groups the anti-measles IgG-seropositive cases showed significantly higher anti-CS IgG titers (P = 0·011). In anti-dsDNA IgG-positive SLE patients, we detected significantly higher levels of anti-CS and anti-F4 IgG nAAbs (P = 0·001 and < 0·001, respectively). Additionally, we found increased levels of IgM isotypes of anti-CS and anti-F4 nAAbs in anti-dsDNA IgM-positive SLE patients (P = 0·002 and 0·016, respectively). The association between IgG isotypes of pathogen- or autoimmune disease-related antibodies and the IgG nAAbs may underscore the immune response-inducible nature of the diseases investigated. The relationship between protective anti-dsDNA IgM and the IgM isotype of anti-F4 and anti-CS may provide immunoserological evidence for the beneficial roles of nAAbs in SLE patients.

Male Antarctic fur seals: neglected food competitors of bioindicator species in the context of an increasing Antarctic krill fishery.

The fishery for Antarctic krill is currently managed using a precautionary, ecosystem-based approach to limiting catch, with performance indices from a long-term monitoring program focused on several krill-dependent predators that are used to track ecosystem health. Concerns over increased fishing in concentrated areas and ongoing efforts to establish a Marine Protected Area along the Peninsula, a key fishing region, is driving the development of an adaptive management system for the fishery. The cumulative effects of fishing effort and interactions among krill-dependent predators and their performance is at present neglected in the CCAMLR Ecosystem Monitoring Program. However, we show considerable overlap between male Antarctic fur seals and the krill fishery in a complex mosaic, suggesting potential for cumulative impacts on other krill dependent predators. A holistic view is required as part of future efforts to manage the krill fishery that incorporates various sources of potential impacts on the performance of bioindicator species, including the fishery and its interactions with various krill dependent predators.

A population-based epidemiological study of neuromyelitis optica spectrum disorder in Hungary.

BACKGROUND AND PURPOSE: The goal of this study was to determine the prevalence and incidence of neuromyelitis optica spectrum disorder (NMOSD) in Hungary based on the 2015 International Panel of NMO Diagnosis (IPND) criteria. METHODS: A retrospective population-based cohort study was conducted of 6.4 million Hungarians (age ≥ 16 years) between 1 January 2006 and 31 December 2016. Possible NMOSD patients were selected via multistage re-evaluation from multiple sources. Crude and sex- and serostatus-specific prevalence (per 100 000 persons) and incidence rates (per 1 000 000 person-years) from 2006 to 2015 were estimated and age-adjusted rates were determined. RESULTS: Of 2262 study candidates, 154 NMOSD patients (age ≥ 16 years) with onset until 31 December 2016 were identified based on 2015 IPND criteria. The prevalence analysis on 1 January 2016 included 123 NMOSD living cases, resulting in a prevalence of 1.91 [95% confidence interval (CI) 1.52-2.28] per 100 000 persons. The 101 incident cases emerging from the observed 76 394 288 person-years provided an incidence rate of 1.32 (95% CI 1.08-1.61) per 1 000 000 person-years. Age-adjusted prevalence was 1.87 (95% CI 1.56-2.23) per 100 000 persons and incidence was 1.20 (95% CI 0.98-1.46) per 1 000 000 person-years. CONCLUSIONS: In this first report of a large population-based epidemiological study from an Eastern European Caucasian population using robust case validation, a greater prevalence and incidence of NMOSD was found compared to previous large studies in Caucasian populations.

A global cline in a colour polymorphism suggests a limited contribution of gene flow towards the recovery of a heavily exploited marine mammal.

Evaluating how populations are connected by migration is important for understanding species resilience because gene flow can facilitate recovery from demographic declines. We therefore investigated the extent to which migration may have contributed to the global recovery of the Antarctic fur seal (Arctocephalus gazella), a circumpolar distributed marine mammal that was brought to the brink of extinction by the sealing industry in the eighteenth and nineteenth centuries. It is widely believed that animals emigrating from South Georgia, where a relict population escaped sealing, contributed to the re-establishment of formerly occupied breeding colonies across the geographical range of the species. To investigate this, we interrogated a genetic polymorphism (S291F) in the melanocortin 1 receptor gene, which is responsible for a cream-coloured phenotype that is relatively abundant at South Georgia and which appears to have recently spread to localities as far afield as Marion Island in the sub-Antarctic Indian Ocean. By sequencing a short region of this gene in 1492 pups from eight breeding colonies, we showed that S291F frequency rapidly declines with increasing geographical distance from South Georgia, consistent with locally restricted gene flow from South Georgia mainly to the South Shetland Islands and Bouvetøya. The S291F allele was not detected farther afield, suggesting that although emigrants from South Georgia may have been locally important, they are unlikely to have played a major role in the recovery of geographically more distant populations.

Extreme diversity in the songs of Spitsbergen's bowhead whales.

Almost all mammals communicate using sound, but few species produce complex songs. Two baleen whales sing complex songs that change annually, though only the humpback whale (Megaptera novaeangliae) has received much research attention. This study focuses on the other baleen whale singer, the bowhead whale (Balaena mysticetus). Members of the Spitsbergen bowhead whale population produced 184 different song types over a 3-year period, based on duty-cycled recordings from a site in Fram Strait in the northeast Atlantic. Distinct song types were recorded over short periods, lasting at most some months. This song diversity could be the result of population expansion, or immigration of animals from other populations that are no longer isolated from each other by heavy sea ice. However, this explanation does not account for the within season and annual shifting of song types. Other possible explanations for the extraordinary diversity in songs could be that it results either from weak selection pressure for interspecific identification or for maintenance of song characteristics or, alternatively, from strong pressure for novelty in a small population.

Influence of ionizing radiation on the antimicrobial activity of the antibiotics sulfamethoxazole and trimethoprim.

The response of the antimicrobial compounds sulfamethoxazole (SMX) and trimethoprim (TMP) - individually and in mixtures - to ionizing radiation was investigated using laboratory prepared mixtures and a commercial pharmaceutical formulation. The residual antibacterial activity of the solutions was monitored using Staphylococcus aureus and Escherichia coli test strains. Based on antibacterial activity, SMX was more susceptible to ionizing radiation as compared to TMP. The antibacterial activity of SMX and TMP was completely eliminated at 0.2 kGy and 0.8 kGy, respectively. However, when SMX and TMP were in a mixture, the dose required to eliminate the antibacterial activity was 10 kGy, implying a synergistic antibacterial activity when these are present in mixtures. Only when the antibiotic concentration was below the Minimum Inhibitory Concentration of TMP (i.e., 2 µmol dm-3) did the antibacterial activity of the SMX and TMP mixture disappear. These results imply that the synergistic antimicrobial activity of antimicrobial compounds in pharmaceutical waste streams is a strong possibility. Therefore, antimicrobial activity assays should be included when evaluating the use of ionizing radiation technology for the remediation of pharmaceutical or municipal waste streams.

Estimating the upper limit of prehistoric peak ground acceleration using an in situ, intact and vulnerable stalagmite from Plavecká priepast cave (Detrekoi-zsomboly), Little Carpathians, Slovakia-first results.

Earthquakes hit urban centres in Europe infrequently, but occasionally with disastrous effects. Obtaining an unbiased view of seismic hazard (and risk) is therefore very important. In principle, the best way to test probabilistic seismic hazard assessments (PSHAs) is to compare them with observations that are entirely independent of the procedure used to produce PSHA models. Arguably, the most valuable information in this context should be information on long-term hazard, namely maximum intensities (or magnitudes) occurring over time intervals that are at least as long as a seismic cycle. The new observations can provide information of maximum intensity (or magnitude) for long timescale as an input data for PSHA studies as well. Long-term information can be gained from intact stalagmites in natural caves. These formations survived all earthquakes that have occurred over thousands of years, depending on the age of the stalagmite. Their 'survival' requires that the horizontal ground acceleration (HGA) has never exceeded a certain critical value within that time period. Here, we present such a stalagmite-based case study from the Little Carpathians of Slovakia. A specially shaped, intact and vulnerable stalagmite in the Plavecká priepast cave was examined in 2013. This stalagmite is suitable for estimating the upper limit of horizontal peak ground acceleration generated by prehistoric earthquakes. The critical HGA values as a function of time going back into the past determined from the stalagmite that we investigated are presented. For example, at the time of Jóko event (1906), the critical HGA value cannot have been higher than 1 and 1.3 m/s2 at the time of the assumed Carnuntum event (∼340 AD), and 3000 years ago, it must have been lower than 1.7 m/s2. We claimed that the effect of Jóko earthquake (1906) on the location of the Plavecká priepast cave is consistent with the critical HGA value provided by the stalagmite we investigated. The approach used in this study yields significant new constraints on the seismic hazard, as tectonic structures close to Plavecká priepast cave did not generate strong earthquakes in the last few thousand years. The results of this study are highly relevant given that the two capitals, Vienna and Bratislava, are located within 40 and 70 km of the cave, respectively.

Evidence for the modulation of nociception in mice by central mast cells.

BACKGROUND: Hyperalgesia that develops following nerve ligation corresponds temporally and in magnitude with the number of thalamic mast cells located contralateral to the ligature. We tested the possibility that mast cells modulate nociception centrally, similar to their role in the periphery. METHODS: We examined the central effect of two hyperalgesic compounds that induce mast cell degranulation and of stabilized mast cells using cromolyn. RESULTS: Thermal hyperalgesia (tail flick) induced by nerve growth factor (NGF, a neurotrophic compound) and mechanical hyperalgesia (von Frey) induced by dynorphin A (1-17) (opioid compound) each correlated with the per cent of thalamic mast cells that were degranulated. Degranulation of these mast cells by the central injection of compound 48/80, devoid of neurotrophic or opioid activity, was sufficient to recapitulate thermal hyperalgesia. Stabilization of mast cells by central injections of cromolyn produced no analgesic effect on baseline tail flick or von Frey fibre sensitivity, but inhibited thermal hyperalgesia produced by compound 48/80 and tactile hyperalgesia induced by dynorphin and by Freund's complete adjuvant. Finally, chemical nociception produced by the direct activation of nociceptors by formalin (phase I) was not inhibited by centrally injected cromolyn whereas chemical nociception dependent on central sensitization (formalin-phase II and acetic acid-induced abdominal stretches) was. CONCLUSIONS: These convergent lines of evidence suggest that degranulation of centrally located mast cells sensitizes central nociceptive pathways leading to hyperalgesia and tonic chemical sensitivity. SIGNIFICANCE: Hyperalgesia induced by spinal nerve ligation corresponds temporally and in magnitude with degranulation of thalamic mast cells. Here, we provide evidence that hyperalgesia induced by NGF, formalin and dynorphin also may depend on mast cell degranulation in the CNS whereas cromolyn, a mast cell stabilizer, blocks these effects in mice.

The turn of the gradient? Educational differences in breast cancer mortality in 18 European populations during the 2000s.

This study aims to investigate the association between educational level and breast cancer mortality in Europe in the 2000s. Unlike most other causes of death, breast cancer mortality tends to be positively related to education, with higher educated women showing higher mortality rates. Research has however shown that the association is changing from being positive over non-existent to negative in some countries. To investigate these patterns, data from national mortality registers and censuses were collected and harmonized for 18 European populations. The study population included all women aged 30-74. Age-standardized mortality rates, mortality rate ratios, and slope and relative indexes of inequality were computed by education. The population was stratified according to age (women aged 30-49 and women aged 50-74). The relation between educational level and breast cancer mortality was predominantly negative in women aged 30-49, mortality rates being lower among highly educated women and higher among low educated women, although few outcomes were statistically significant. Among women aged 50-74, the association was mostly positive and statistically significant in some populations. A comparison with earlier research in the 1990s revealed a changing pattern of breast cancer mortality. Positive educational differences that used to be significant in the 1990s were no longer significant in the 2000s, indicating that inequalities have decreased or disappeared. This evolution is in line with the "fundamental causes" theory which stipulates that whenever medical insights and treatment become available to combat a disease, a negative association with socio-economic position will arise, independently of the underlying risk factors.

Contribution of smoking to socioeconomic inequalities in mortality: a study of 14 European countries, 1990-2004.

BACKGROUND: Smoking contributes to socioeconomic inequalities in mortality, but the extent to which this contribution has changed over time and driven widening or narrowing inequalities in total mortality remains unknown. We studied socioeconomic inequalities in smoking-attributable mortality and their contribution to inequalities in total mortality in 1990-1994 and 2000-2004 in 14 European countries. METHODS: We collected, harmonised and standardised population-wide data on all-cause and lung-cancer mortality by age, gender, educational and occupational level in 14 European populations in 1990-1994 and 2000-2004. Smoking-attributable mortality was indirectly estimated using the Preston-Glei-Wilmoth method. RESULTS: In 2000-2004, smoking-attributable mortality was higher in lower socioeconomic groups in all countries among men, and in all countries except Spain, Italy and Slovenia, among women, and the contribution of smoking to socioeconomic inequalities in mortality varied between 19% and 55% among men, and between -1% and 56% among women. Since 1990-1994, absolute inequalities in smoking-attributable mortality and the contribution of smoking to inequalities in total mortality have decreased in most countries among men, but increased among women. CONCLUSIONS: In many European countries, smoking has become less important as a determinant of socioeconomic inequalities in mortality among men, but not among women. Inequalities in smoking remain one of the most important entry points for reducing inequalities in mortality.

Comparison of immunophenotypes of primary breast carcinomas and multiple corresponding distant metastases: an autopsy study of 25 patients.

Phenotypical change in metastatic breast carcinoma has widely been accepted as an inherent biological feature rather than technical fault. We analyzed the immunohistochemical phenotype and histopathological features of 25 primary breast carcinomas and 90 corresponding distant metastases in 23 organs retrospectively. Histological slides were reviewed for prognostic and predictive factors. Overall, metastases were more similar to each other and often differed from the primary tumor. We created a 3-step grouping system based on the localization of metastases. Regions: tumors metastasizing to the abdominal region were likely to lose ER (p = 0.002); we detected loss of PR in metastases to the thorax (p = 0.039) and abdomen (p < 0.001). Organ systems: loss of ER and PR was observed in metastases to the gastrointestinal system (p = 0.026 and p = 0.001, respectively), in the respiratory system only the loss of PR was significant (p = 0.05). Individual organs: the primaries were likely to lose the hormone receptors in liver metastases (ER p = 0.026; PR p = 0.004). In lung metastases only loss of PR was apparent (p = 0.049). We did not observe significant change in HER2 status, regarding Ki67 change occurred only in bone metastases compared to the primary (p = 0.048). 7/25 patients' distant metastases had heterogeneous immunoprofiles. The later the metastasis was discovered the more likely it had a differing IHC profile compared to the primary tumor, patients who had longer OS had a higher chance to develop a discordant metastasis. Immunoprofile of metastases may differ from primary breast cancer and from each other, probably resulting in different response to therapy.

The Protective Role of PAC1-Receptor Agonist Maxadilan in BCCAO-Induced Retinal Degeneration.

A number of studies have proven that pituitary adenylate cyclase activating polypeptide (PACAP) is protective in neurodegenerative diseases. Permanent bilateral common carotid artery occlusion (BCCAO) causes severe degeneration in the rat retina. In our previous studies, protective effects were observed with PACAP1-38, PACAP1-27, and VIP but not with their related peptides, glucagon, or secretin in BCCAO. All three PACAP receptors (PAC1, VPAC1, VPAC2) appear in the retina. Molecular and immunohistochemical analysis demonstrated that the retinoprotective effects are most probably mainly mediated by the PAC1 receptor. The aim of the present study was to investigate the retinoprotective effects of a selective PAC1-receptor agonist maxadilan in BCCAO-induced retinopathy. Wistar rats were used in the experiment. After performing BCCAO, the right eye was treated with intravitreal maxadilan (0.1 or 1 µM), while the left eye was injected with vehicle. Sham-operated rats received the same treatment. Two weeks after the operation, retinas were processed for standard morphometric and molecular analysis. Intravitreal injection of 0.1 or 1 µM maxadilan caused significant protection in the thickness of most retinal layers and the number of cells in the GCL compared to the BCCAO-operated eyes. In addition, 1 µM maxadilan application was more effective than 0.1 µM maxadilan treatment in the ONL, INL, IPL, and the entire retina (OLM-ILM). Maxadilan treatment significantly decreased cytokine expression (CINC-1, IL-1α, and L-selectin) in ischemia. In summary, our histological and molecular analysis showed that maxadilan, a selective PAC1 receptor agonist, has a protective role in BCCAO-induced retinal degeneration, further supporting the role of PAC1 receptor conveying the retinoprotective effects of PACAP.

Endometrioid adenocarcinoma arising from adenomyosis: a case report.

BACKGROUND: Endometriosis is most commonly found in the peritoneum of the lesser pelvis and in the genital tract (in the ovaries). Its malignant transformation is quite rare, which usually appears in patients who previously underwent surgical procedures aimed at treating endometriosis. Years of hormone substitution (unopposed estrogen therapy) is also considered to have a role. According to the present authors' current knowledge, these are mostly well-differentiated tumors with low malignancy, which are primarily treated surgically. CASE: In the present case the authors present a 73-year-old female patient who underwent a laparotomy due to abdominal pain and a mass in the lesser pelvis. The authors performed hysterectomy along with bilateral adnexectomy and omental resection. The histological examination of the specimens verified an endometrial adenocarcinoma formed on the ground of adenomyosis and the endometrial adenocarcinoma of the left ovary. CONCLUSION: The malignant transformation of endometriosis is rare, and the mechanisms how it develops on the grounds of adenomyosis is currently unclear.

Ischemia/reperfusion-induced Kidney Injury in Heterozygous PACAP-deficient Mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with very diverse distribution and functions. Among others, PACAP is a potent cytoprotective peptide due to its antiapoptotic, anti-inflammatory, and antioxidant actions. This also has been shown in different kidney pathologies, including ischemia/reperfusion-induced kidney injury. Similar protective effects of the endogenous PACAP are confirmed by the increased vulnerability of PACAP-deficient mice to different harmful stimuli. Kidneys of homozygous PACAP-deficient mice have more severe damages in renal ischemia/reperfusion and kidney cell cultures isolated from these mice show increased sensitivity to renal oxidative stress. In our present study we raised the question of whether the partial lack of the PACAP gene is also deleterious, i.e. whether heterozygous PACAP-deficient mice also display more severe damage after renal ischemia/reperfusion. Mice underwent 45 or 60 minutes of ischemia followed by 2 weeks reperfusion. Histological evaluation of the kidneys was performed and individual histopathological parameters were graded. Furthermore, we investigated apoptotic markers, cytokine expression, and the activity of superoxide dismutase (SOD) enzyme 24 hours after 60 minutes of renal ischemia/reperfusion. We found no difference between the intact kidneys of wild-type and heterozygous mice, but marked differences could be observed following ischemia/reperfusion. Heterozygous PACAP-deficient mice had more severe histological alterations, with significantly higher histopathological scores for most of the tested parameters. Higher level of the proapoptotic pp38 MAPK and of some proinflammatory cytokines, as well as lower activity of the antioxidant SOD could be found in these mice. In conclusion, the partial lack of the PACAP gene results in worse outcomes in cases of renal ischemia/reperfusion, confirming that PACAP functions as an endogenous protective factor in the kidney.

Mechanism of Procedural Stroke Following Carotid Endarterectomy or Carotid Artery Stenting Within the International Carotid Stenting Study (ICSS) Randomised Trial.

OBJECTIVE: To decrease the procedural risk of carotid revascularisation it is crucial to understand the mechanisms of procedural stroke. This study analysed the features of procedural strokes associated with carotid artery stenting (CAS) and carotid endarterectomy (CEA) within the International Carotid Stenting Study (ICSS) to identify the underlying pathophysiological mechanism. MATERIALS AND METHODS: Patients with recently symptomatic carotid stenosis (1,713) were randomly allocated to CAS or CEA. Procedural strokes were classified by type (ischaemic or haemorrhagic), time of onset (intraprocedural or after the procedure), side (ipsilateral or contralateral), severity (disabling or non-disabling), and patency of the treated artery. Only patients in whom the allocated treatment was initiated were included. The most likely pathophysiological mechanism was determined using the following classification system: (1) carotid-embolic, (2) haemodynamic, (3) thrombosis or occlusion of the revascularised carotid artery, (4) hyperperfusion, (5) cardio-embolic, (6) multiple, and (7) undetermined. RESULTS: Procedural stroke occurred within 30 days of revascularisation in 85 patients (CAS 58 out of 791 and CEA 27 out of 819). Strokes were predominately ischaemic (77; 56 CAS and 21 CEA), after the procedure (57; 37 CAS and 20 CEA), ipsilateral to the treated artery (77; 52 CAS and 25 CEA), and non-disabling (47; 36 CAS and 11 CEA). Mechanisms of stroke were carotid-embolic (14; 10 CAS and 4 CEA), haemodynamic (20; 15 CAS and 5 CEA), thrombosis or occlusion of the carotid artery (15; 11 CAS and 4 CEA), hyperperfusion (9; 3 CAS and 6 CEA), cardio-embolic (5; 2 CAS and 3 CEA) and multiple causes (3; 3 CAS). In 19 patients (14 CAS and 5 CEA) the cause of stroke remained undetermined. CONCLUSION: Although the mechanism of procedural stroke in both CAS and CEA is diverse, haemodynamic disturbance is an important mechanism. Careful attention to blood pressure control could lower the incidence of procedural stroke.

Carotid artery: overview on current trials--selecting the low-risk patient.

The ultimate goal of carotid stenosis treatment is the long-term prevention of stroke. While a large number of studies focusing on patients with symptomatic carotid stenosis have been carried out, fewer data are available from trials on asymptomatic and low-risk patients. Currently existing information on the optimal management of these patients is inconclusive and contradictory. Our aim was to review previous major trials conducted on carotid disease with a main focus on asymptomatic patients with carotid stenosis. Efforts to present currently ongoing trials involving asymptomatic carotid patients, to survey recent studies determining patients' risk for future stroke or periprocedural events, as well as to summarize data on promising structural and functional variables and biomarkers predicting future stroke risk have been made.

Emergence of VIM-4 metallo-ß-lactamase-producing Klebsiella pneumoniae ST15 clone in the Clinical Centre University of Pécs, Hungary.

Since November 2009 carbapenemase-producing Klebsiella pneumoniae isolates have been detected in increasing numbers at the Clinical Centre University of Pécs. Molecular typing was performed for 102 clinical isolates originating from different time periods and various departments of the Clinical Centre. Pulsed-field gel electrophoresis revealed the predominance of a single clone (101/102), identified as sequence type ST15. PCR and sequencing showed the presence of blaCTX-M-15 and blaVIM-4 genes. The blaVIM-4 was located on a class 1 integron designated In238b. To our knowledge, this is the first description of a blaVIM-4 gene in the predominant CTX-M-15 extended spectrum ß-lactamase-producing Hungarian Epidemic Clone/ST15.

Factor XIII deficiency: complete phenotypic characterization of two cases with novel causative mutations.

Coagulation factor XIII (FXIII) exists as heterotetramer (FXIII-A2B2) in the plasma and as dimer (FXIII-A2) in cells. Activated FXIII mechanically stabilizes fibrin and protects it from fibrinolysis by cross-linking fibrin chains and α2-plasmin inhibitor to fibrin. FXIII is essential to maintaining haemostasis, and its deficiency causes severe bleeding diathesis. Due to improper laboratory practices, FXIII deficiency is considered the most under-diagnosed bleeding disorder. The aim of this study was to demonstrate in two cases how FXIII deficiency is properly diagnosed and classified, and to compare results of laboratory analysis and clinical symptoms. FXIII activity from plasma and platelets was measured by a modified ammonia release assay, while FXIII-A2B2, FXIII-A and FXIII-B antigens were determined by ELISA. The exon-intron boundaries and the promoter region of F13A1 gene were amplified by PCR and the amplified products were analysed by direct fluorescent sequencing. FXIII-A mRNA in platelets was determined by RT-qPCR. Two children with severe bleeding symptoms were investigated. In both cases FXIII activity and FXIII-A antigen were undetectable in the plasma and platelet lysate. In the plasma no FXIII-A2B2 antigen was found, while FXIII-B antigen was >30% in both cases. Proband1 was a compound heterozygote possessing a known missense mutation (c.980G>A, p.Arg326Gln) and a novel splice-site mutation (c.1112+2T>C). Proband2 was homozygote for a novel single nucleotide deletion (c.212delA) leading to early stop codon. The discovered mutations explain the severity of clinical symptoms and the laboratory data. Methods precise in the low activity/antigen range are required to draw valid conclusion on phenotype-genotype relationship.