Risk stratification of neurological decompression sickness in divers.

INTRODUCTION: Patent foramen ovale (PFO) is a risk factor of decompression sickness (DCS). However, data on risk stratification of divers with a PFO are sparse. This study sought to evaluate the risk of neurological DCS (DCSneuro), based on the presence and grade of a right-to-left shunt (RLS). METHODS: A total of 640 divers were screened for a RLS using TCD between 1/2006 and 4/2017. RLS was graded as low, medium, or high grade with two subgroups - after a Valsalva maneuver or at rest. Divers were questioned about their DCS history. Survival analysis techniques were used to assess risk factors for unprovoked DCS. RESULTS: A RLS was found in 258 divers (40.3 %). 44 (17.1 %) divers with a RLS experienced DCSneuro compared to 5 (1.3 %) divers without a RLS (p <0.001). The proportion of DCSneuro increased from 4.6 % in the low-grade RLS subgroup to 57.1 % in the subgroup with high-grade RLS at rest. The hazard ratio for DCSneuro and RLS was11.806 (p <0.001). CONCLUSIONS: Divers with a RLS had a higher risk of DCSneuro and the risk increased with RLS grade. We suggest that TCD is an appropriate method for RLS screening and risk stratification in divers (Tab. 4, Fig. 2, Ref. 29).

Effect of methylprednisolone on experimental brain edema in magnetic resonance imaging.

Magnetic resonance imaging has been used for evaluating of a brain edema in experimental animals to assess cytotoxic and vasogenic edema by the apparent diffusion coefficient (ADC) and T2 imaging. This paper brings information about the effectiveness of methylprednisolone (MP) on experimental brain edema. A total of 24 rats were divided into three groups of 8 animals each. Rats with cytotoxic/intracellular brain edema induced by water intoxication were assigned to the group WI. These rats also served as the additional control group CG when measured before the induction of edema. A third group (WIMP) was intraperitoneally administered with methylprednisolone 100 mg/kg during water intoxication treatment. The group WI+MP was injected with methylprednisolone 50 mg/kg into the carotid artery within two hours after the water intoxication treatment. We evaluated the results in four groups. Two control groups (CG, WI) and two experimental groups (WIMP, WI+MP). Rats were subjected to MR scanning 24 h after edema induction. We observed significantly increased ADC values in group WI in both evaluated areas - cortex and hippocampus, which proved the occurrence of experimental vasogenic edema, while ADC values in groups WIMP and WI+MP were not increased, indicating that the experimental edema was not developed and thus confirming the protective effect of MP.

The CD34+ Cell Number Alone Predicts Retention of the Human Fat-Graft Volume in a Nude Mouse Model.

Prediction of the final transferred fat volume is essential for the success of fat grafting, but remains elusive. Between 20 and 80 % of the initial transplanted volume can be reabsorbed. Although graft survival has many determinants, CD34+ progenitor cells from the vascular stroma of adipose tissue play a central role by promoting growth of blood vessels and adipocytes. We aimed to verify the hypothesis that a higher proportion of total CD34+ cells in the transplant is associated with better preservation of the graft volume. Human lipoaspirates from 16 patients were processed by centrifugation and two grafts per donor were subcutaneously injected into 32 nude mice in 1 ml volumes in the right upper flank area. The volume of each graft was measured using a preclinical MRI scanner immediately after grafting and at three months. The percentage of CD34+ cells in the graft before implantation was determined by flow cytometry. The final graft volume at three months after implantation directly correlated with the percentage of CD34+ cells in the grafted material (r = 0.637, P = 0.019). The minimum retention of the fat graft was 28 % and the maximum retention was 81 %, with an average of 54 %. Our study found that fat retention after fat transfer directly correlated with the fraction of CD34+ cells in the graft. The simple and fast determination of the CD34+ cell percentage on site can help predicting outcomes of fat transplantation.

[Patent foramen ovale and the risk of paradoxical embolization of venous bubbles in divers - cave for foam sclerotization of varicose veins]. / Perzistence foramen ovale patens a riziko paradoxní embolizace zilních bublin u potápecu - cave pro sklerotizaci varixu penovou metodou.

INTRODUCTION: Foam sclerotization of varicose veins may cause paradoxical embolization through patent foramen ovale (PFO). The aim of our study was to: 1) select an optimal screening method for the detection of PFO; 2) determine the prevalence of PFO in a non-selected population; and 3) test the risk of paradoxical embolization of venous bubbles in patients with PFO. MATERIALS AND METHODS: A diver after decompression is a suitable model for determining the risk of paradoxical embolization of venous gas bubbles. 329 Czech divers were screened for PFO. In a pilot study, we compared Transcranial Doppler Sonography (TCD) with Transesophageal Echocardiography (TEE) in 100 patients. TCD alone was used for further screening. In 31 divers with PFO, nitrogen bubbles were detected after simulated dives. Transthoracic Echocardiography (TTE) was used to detect venous bubbles in right-sided heart chambers; TTE and TCD were used to detect arterial bubbles. The right-to-left shunt was rated as non-significant (<20 arterial bubbles) or significant (20 arterial bubbles). Different decompression regimens were compared. RESULTS: In the pilot study, TCD was compared with the gold standard in PFO detection - TEE. The negative predictive value of TCD was 100%, positive predictive value was 92%. Screening was performed in a total of 329 divers, PFO was detected in 85 (25%), significant R-L shunt in 45 (14%). In simulated dive to 50 m maximum depth, venous nitrogen bubbles were detected in 7/8 (88%) divers. In 6/8 (75%) divers, paradoxical embolization was confirmed - nitrogen bubbles were detected in the systemic circulation. CONCLUSION: PFO prevalence with significant R-L shunt was 14% in the non-selected population of Czech divers. Simulated dives indicate that PFO represents a risk factor for paradoxical embolization of gas bubbles. TCD is a suitable screening method for the detection of PFO and the evaluation of R-L shunt significance. These results are indicative of a possible high risk of paradoxical embolization of gas bubbles and the trombogenic substance in patients with a larger PFO and significant R-L shunt undergoing foam sclerotization of varicose veins.

Effect of PPAR-γ agonist rosiglitazone on bone mineral density and serum adipokines in C57BL/6 male mice.

Thiazolinediones (TZD) are widely used to treat type 2 diabetes, but their mechanism of action still remains only partially understood. Although the in vitro effects of TZD on osteoblastogenesis are well recognized, the in vivo consequences of these compounds on bone turnover are less understood and rather controversial. We demonstrate that a 9-week oral treatment with rosiglitazone in C57BL/6 male mice resulted in significant bone loss that was not dose dependent. The bones of the rosiglitazone-treated mice were characterized by reduction of bone density, and ash, calcium and phosphorus content. Rosiglitazone-treated mice had significantly thinner cortical widths. In contrast to serum TrACP expressed by action of osteoclasts, serum B-ALP activity, which serves as a marker of osteoblastic activity, was significantly lower in the rosiglitazone-fed animals. We did not find any differences in circulating levels of adipokines that could eventually explain rosiglitazone action. As the decrease in osteoblastic activity was demonstrated after rosiglitazone treatment, we anticipated changes in the haematopoietic stem cell pool. These cells seed in endosteal niches which comprise osteoblasts in order to maintain their stem cell function. In our study we did not see any significant influence of rosiglitazone administration on stem cells or any impairment in the lineage restrictions of rosiglitazone-treated stem cells. Our data demonstrate that rosiglitazone administration causes a loss of bone mass in cortical bone, possibly through a decrease in bone formation expressed by decreased B-ALP in male C57BL/6 mice. The levels of adipokines do not play any role.

Adenosine A(1), A(2a), A(2b), and A(3) receptors in hematopoiesis. 1. Expression of receptor mRNA in four mouse hematopoietic precursor cells.

Four mouse bone marrow or thymus cell populations, namely granulopoietic/monocytopoietic, erythropoietic, B-lymphopoietic, and T-lymphopoietic precursor cells have been assayed by RT-PCR technique for the presence and relative amounts of adenosine A(1), A(2a), A(2b), and A(3) receptor mRNA. It has been found that (i) all four populations studied express all four adenosine receptor subtypes, (ii) the A(1), receptor is the least expressed in all populations studied, (iii) the A(3) receptor is markedly expressed in the populations of granulopoietic/monocytopoietic and erythropoietic cells, (iv) the A(2a) receptor is markedly expressed in the populations of B-lymphopoietic and T-lymphopoietic cells, and v) the A(2b) receptor does not predominate in any of the precursor cells studied. Our data offer a new possibility for the assessment of the readiness of these cells to respond, by receptor-mediated mechanisms, to adenosine or its analogs present in the tissues as a result of endogenous processes and/or following their administration.

Hepcidin downregulation by repeated bleeding is not mediated by soluble hemojuvelin.

Hepcidin is a key regulator of iron homeostasis, while hemojuvelin is an important component of the hepcidin regulation pathway. It has been recently proposed that soluble hemojuvelin, produced from hemojuvelin by the protease furin, decreases hepcidin expression. The aim of the presented study was to examine the downregulation of hepcidin by chronic bleeding in hemojuvelin-mutant mice. Male mice with targeted disruption of the hemojuvelin gene (Hjv-/- mice) and wild-type littermates were maintained on an iron-deficient diet and subjected to weekly phlebotomies for 7 weeks. Gene expression was examined by real-time PCR. In wild type mice, repeated bleeding decreased hepcidin mRNA by two orders of magnitude. In Hjv-/- mice, basal hepcidin expression was low; however, repeated bleeding also decreased hepcidin mRNA content by an order of magnitude. Phlebotomies reduced hepatic iron overload in Hjv-/- mice by 80 %. Liver and muscle furin mRNA content was not significantly changed. No effect on hepatic Tmprss6 mRNA content was observed. Results from the study indicate that soluble hemojuvelin is not the sole factor responsible for hepcidin downregulation. In addition, the presented data suggest that, under in vivo conditions, tissue hypoxia does not transcriptionally regulate the activity of furin or TMPRSS6 proteases.

The late-stage foetal liver microenvironment is essential for later sensitivity of B-lymphopoiesis to suppression by oestrogens.

B-lymphopoiesis in FL differs notably from that of adult B-lymphopoiesis in being resistant to suppression by oestrogens due to the lack of expression of oestrogen receptors in B-cell progenitors and precursors. We have transplanted middle-stage FL cells (E14.5) to adult male mice and demonstrated that B-lymphopoiesis derived from FL cells remained resistant to suppression by exogenous oestrogen for several months compared to adult BM cells. This significant difference strongly suggests that the latestage FL environment exerts an inductive action on the haematopoietic stem cells and is mandatory for later sensitivity of B-lymphopoiesis to suppression by oestrogens. The results also provide the first in vivo functional confirmation of a differential responsiveness of FL- and adult BM-derived B-lymphopoiesis to suppression by oestrogens.

Risedronate has no adverse effects on mouse haematopoiesis.

Bisphosphonates are commonly used for treatment of osteoporosis. They inhibit osteoclast activity and thus bone resorption. It was shown that they also affect some other cell types including tumour and endothelial cells. The effects of risedronate on bone marrow microenvironment were not studied yet. As endothelial cells are integral part of bone marrow microenvironment, it is important to know whether prolonged administration of bisphosphonates does not affect haematopoietic stem cells and bone marrow haematopoiesis. We fed mice two weeks with risedronate. We found no effect of risedronate treatment on bone marrow stem cells using the method of congenic bone marrow repopulation. Risedronate administration in the dose which is considered to be comparable to a dose of risedronate used for treatment of osteoporosis in women seems to be safe in terms of effects on mouse haematopoiesis.

[Paradoxical embolization and patent foramen ovale in scuba divers: screening possibilities]. / Paradoxní embolizace pri foramen ovale patens u potápecu: moznosti screeningu.

INTRODUCTION: The cause of decompression sickness (DCS) in scuba-divers is bubble formation in tissues and in venous blood during ascent. Divers with patent foramen ovale (PFO) have an increased risk of paradoxical embolization to the brain or other vital organs. The aim of our study was to assess the incidence of PFO in scuba-divers with DCS, to compare the group with asymptomatic controls, and to evaluate ultrasound contrast methods suitable for screening. METHODOLOGY: We examined 28 scuba-divers (more than 100 dives). The right-to-left shunt detection was performed by bubble contrast transthoracic echocardiographic examination (TTE) and transcranial Doppler sonography over arteria cerebri media (TCD) in all divers. In divers with shunting, transoesophageal echocardiography (TEE) was performed to prove PFO. RESULTS: 15 divers had DCS associated with the ascent. In this group, PFO was diagnosed in 53% (8/15). The symptoms of all of them retrospectively were of paradoxical embolization (neurological form of DCS). In the group of asymptomatic divers, PFO was proven on the basis of right-to-left shunt screening in 1 diver (8% 1/13). TCD proved right-to-left shunt in all divers with PFO. CONCLUSION: DCS can unmask a so far asymptomatic intracardiac right-to-left shunting. PFO is a risk factor for paradoxical embolization in divers. TCD is suitable for screening; TEE is a gold standard in PFO detection. Our results showed that PFO detection is a useful clinical tool after repeated DCS and in all frequent divers and instructors.

Protein kinase inhibitors.

Since protein kinases have been found to be implicated in many diseases, first of all malignancies, they are considered as promising therapeutic targets. Many protein kinase inhibitors have been designed by now. These molecules have a low molecular weight and most of them bind to protein kinases competing with ATP for the ATP-binding site. Some protein kinase inhibitors currently undergo clinical trials or have already been successfully introduced into treatment as exemplified by Bcr-Abl, c-kit and PDGFR inhibitor imatinib mesylate (Gleevec), flavopiridol and roscovitine, inhibitors of cyclin-dependent kinases, or erlotinib and gefitinib inhibiting EGFR. Discovery of these molecules seems to begin a new era in medicine, especially oncology. Targeting protein kinases represents a promising approach and gives us new hopes of effective non-invasive cancer treatment.

Protein kinases, their function and implication in cancer and other diseases.

Protein phosphorylation is known to play an important role in various cellular processes such as cell division, metabolism, survival and apoptosis. It is driven by specific enzymes, tyrosine and serine-threonine protein kinases. Human protein kinases constitute a complicated system with intricate internal and external interactions. The complexity and sophistication of the system implies its vulnerability. Alterations in functions of these enzymes may launch series of pathological changes within the cell and as a result cause diseases. Protein kinases have been shown to be involved in various pathological processes, first of all malignancies. Deregulation of different protein kinases has been found in chronic myelogenous leukaemia, gastrointestinal stromal tumours, various other sarcomas and cancers as well as non-malignant disorders. Therefore, they are regarded as important effectors in human pathology and represent prospective therapeutic targets.

Effect of lipopolysaccharide and bleeding on the expression of intestinal proteins involved in iron and haem transport.

Haem carrier protein 1 (Hcpl) is a component of the haem-iron uptake pathway in the small intestine. Using quantitative real-time PCR, we examined the expression of Hcp1 and other intestinal iron-transporting proteins in male C57BL/6 mice with experimentally altered iron homeostasis. Intestinal Hcp1 mRNA content was not significantly changed by iron overload (600 mg/kg); however, it was increased to 170 % of controls 72 h after withdrawal of 0.7 ml of blood; the same treatment increased intestinal Cybrd1 mRNA to 900 % of controls. LPS treatment (1 mg/kg, 6 h) decreased intestinal Hcp1 mRNA content to 66 % of controls and Flvcr mRNA content to 65 % of controls, while Cybrd1 mRNA, Dmt1 mRNA and Fpn1 mRNA decreased to 6 %, 43 % and 32 %, respectively. In 129SvJ mice with targeted disruption of the hemojuvelin (Hfe2) gene, which display very low expression of liver hepcidin, Cybrd1 mRNA content increased to 1040 %, Dmt1 mRNA content to 200 % and Fpn1 mRNA to 150 % when compared to wild-type mice; changes in Hcp1, Abcg2 and Flver mRNA content were only minor. Overall, these results suggest that, during inflammation, the intestinal haem-iron uptake pathway is not as strongly transcriptionally downregulated as the non-haem iron uptake pathway. A decrease in circulating hepcidin increases the expression of proteins participating in non-haem iron uptake, but has no significant effect on Hcp1 mRNA content.

Cytokine gene expression in regenerating haematopoietic tissues of mice after cyclophosphamide treatment.

The goal of the study was to investigate changes in expression of selected growth factors tentatively involved in regeneration of haematopoietic tissues (bone marrow and spleen) following cyclophosphamide (CY) damage in the mouse. The bone marrow (BM) and spleen were examined separately, since the regenerating pattern for haematopoietic progenitor cells (HPC) markedly differs in these two haematopoietically active organs after CY. Cytokines assumed to have a stimulatory effect on HPC - stem cell factor (SCF), fetal liver tyrosine kinase 3-ligand (flt3-ligand), thrombopoietin (TPO), stromal cell-derived factor 1 (SDF-1), oncostatin M (OSM) -, a suppressive effect on HPC proliferation - macrophage inflammatory protein-1alpha (MIP-1alpha), transforming growth factor-beta1 (TGFbeta1), tumour necrosis factor-alpha (TNFalpha) - and to be involved in migration of HPC (SCF, flt3-ligand, MIP1alpha, SDF-1) were examined at the level of mRNA expression by means of real-time RT-PCR. The expression of a particular cytokine appears to be similar in both BM and spleen of untreated mice. CY administration changed the expression pattern of the studied genes in BM and spleen. In BM, the levels of mRNAs for SCF and SDF-1 were increased and that for TGFbeta1 decreased at time intervals at which HPC are known to proliferate intensively during BM regeneration. In contrast, stimulated proliferation of HPC in spleen was accompanied by increased expression of flt3-ligand and oncostatin M. Upon mobilization of HPC from BM into blood after CY, the expression of SCF, TPO, SDF-1 and TGFbeta1 tends to decrease in BM. Accumulation of HPC in spleen is accompanied by increased mRNA for flt3-ligand and OSM. Our findings demonstrate that different cytokines may be involved in the proliferation and mobilization/homing of HPC during recovery after CY damage in BM and spleen.

Development of hydrogel implants for urinary incontinence treatment.

Hydrogel implants for urinary incontinence treatment based on HEMA supplemented with 10% methacrylic acid have been developed. The swelling properties of implants were tested in vitro and in vivo after implantation to laboratory mice. Biocompatibility has been determined by incubation of implants in tissue culture, by histological examination of adjacent tissues after subcutaneous application of implants to laboratory mouse in a long-term experiment, and by flow cytometry examination of blood cells. The swelling of hydrogel implants was completed in 6-24 h. There was no effect on in vitro growth of cells incubated with implants. In mice, implants were well tolerated without any sign of inflammatory reaction. The material allows an elastic compression of urethra compensating a damaged sphincter after trans-urethral sub-mucosal implantation of hydrogel cylinders.

[Methods used in the detection of apoptosis]. / Soucasné metody pouzívané k detekci apoptózy.

Apoptosis is a physiological form of cell death, whose dysregulation may lead to cancer or to autoimmune and degenerative diseases. In this review article, distinctive biochemical, molecular and morphological features of apoptosis are being discussed, as well as the principles of the elementary detection methods. Apoptosis is characterized by nuclear, plasma membrane, and mitochondrial changes. For reliable detection of apoptosis, it is generally recommended to employ at least two different methods together with microscopic verification of the distinctive morphological alterations.

Estimation of extent of cell death in different stages of normal murine hematopoiesis.

Murine hematopoiesis has been analyzed by many authors, and available data allow for quantitative evaluation of this dynamic process. In this study, the capacity of several populations of the bone marrow clonogenic cells (progenitors) to produce blood cells was compared with their actual production. The cell cycle progression rate was directly measured in the following types of hematopoietic progenitors: day 8 colony-forming units-spleen, GM-colony-forming cells, BFU-E, and CFU-E in normal mice. The cell cycle progression rates of the individual progenitors, together with their numbers in the whole hematopoietic tissue, were used to calculate the absolute numbers produced daily in each population. The data reviewed from literature were analyzed in parallel. The capacity of the progenitors to produce mature blood cells were derived from the daily production of progenitors multiplied by their clonogenic potential. This theoretical capacity to produce blood cells was compared to the actual blood cell production determined from the turnover of circulating blood elements. The comparison strongly suggested an intensive cell death rate occurring at the early stages of differentiation and its decline as the hematopoietic cells become more differentiated and mature.

Hematopoietic reserve provided by spleen colony-forming units (CFU-S).

Using quantitative data available from the literature on murine hematopoiesis, the functional reserve of multipotential stem cells was calculated by comparing daily blood cell production with the potential of clonogenic spleen colony-forming cells (CFU-S) to generate blood cells. The potential of the day-8 CFU-S (CFU-S-8) population is estimated to be from 4000 to 37,000 times greater than needed in steady-state hematopoiesis. The CFU-S population may thus serve to provide a functional reserve to supply large numbers of peripheral blood cells via committed lineage-specific cells within days, whenever needed.

On control of the hematopoietic cell proliferation.

Hematopoietic cells, detected as spleen colony forming units (CFU-S), vary in their proliferation rate, and this can be investigated in vivo by determining the fraction of CFU-S synthesizing DNA. A large number of measurements of CFU-S number and the fraction synthesizing DNA has been obtained under standardized conditions in normal mice and after a single dose of arabinosyl cytosine (ara-C). These data are analyzed with respect to the correlation between the number of CFU-S and their DNA-synthesizing fraction since, in the past, it has been maintained that the CFU-S proliferation rate responds sensitively to changes in CFU-S numbers. The present analysis does not support this traditional view--it instead demonstrates that natural variations in CFU-S numbers occurring in the same range as those following ara-C do not trigger CFU-S proliferation. On the other hand, administration of ara-C triggers most of the surviving CFU-S into the cell cycle, while decreasing CFU-S numbers by only about 40%. Data are also presented showing changes of CFU-S numbers and their DNA-synthesizing fraction in mice given a single dose of cyclophosphamide (CY). CY reduces CFU-S numbers, but the fraction synthesizing DNA can be either increased or decreased depending on time after treatment. Both these experimental situations argue against a close relationship between CFU-S numbers and their proliferation rate and suggest a different or a more complex regulation.

Haemopoietic stem cells: spleen colony-forming cells are normally actively proliferating.

The haemopoietic stem cells forming spleen colonies (CFU-S) had on average 30 to 40% of cells engaged in the DNA synthesis in normal mice continuously over 4 years. A majority of experiments aimed at the suppression of the CFU-S proliferation, which included suppression of the T-lymphocytes by means of cyclosporin A or by adult thymectomy, administration of antibacterial and antifungal agents and maintainance of mice in a sterile environment, suppression of antibody-producing cells by a successive administration of the bacterial lipopolysaccharide and cyclophosphamide and attempts to increase the total number of CFU-S in the body through massive transfusions of bone marrow cells or by grafting plugs of the bone marrow under the kidney capsulae, have not been sufficiently effective. A transient suppression of CFU-S proliferation occurred during recovery of the haemopoietic tissue from damage caused by cyclophosphamide. The results support the view that changes in CFU-S numbers and in the proportion of them in DNA synthesis may be positively correlated when CFU-S numbers fluctuate physiologically about their normal values. The failure to manipulate the CFU-S proliferation rate easily suggests that proliferation of these cells may not be under a strong 'switch on - switch off' control.