SAFE USE OF INTRA-OPERATIVE TOURNIQUETS IN A DISTRICT HOSPITAL IN THE UK-AN AUDIT STUDY IN ORTHOPAEDIC THEATRES AND REVIEW OF CURRENT LITERATURE.

The use of tourniquet is common in orthopaedic surgeries as it reduces blood loss, enhances visualization of the operating field, and leads to quicker procedures. However, the use of tourniquet has certain risks which can be avoided by following guidelines like British Orthopaedic Association Standards for Trauma (BOAST) guidelines for safe use of tourniquet. This audit study was done in a District general hospital to check the compliance of two trauma theatres with BOAST guidelines. The audit found that there was poor documentation of tourniquet details in the operation notes (10%). Regarding tourniquet time and pressure, the compliance in the two theatres was 95 % & 97.5 %. The recommendations of this audit were to use a template to improve documentation of tourniquet details in the operation notes and training of theatre staff on BOAST guidelines for safe use of tourniquet.

THE ROLE OF DISTAL LOCKING IN INTRAMEDULLARY NAILS FOR HIP FRACTURE FIXATION: A REVIEW OF CURRENT LITERATURE.

Traditionally, it was believed that both proximal and distal locking are essential for achieving stability during intra-medullary fixation for extra-capsular hip fractures. However, recent literature has presented varying perspectives on the necessity of distal locking. Distal locking plays a significant role in managing hip fractures with uncertainties regarding longitudinal and rotational stability. This includes cases of comminuted intertrochanteric fractures with subtrochanteric extension, reverse oblique and high oblique fractures, broad medullary canals, comminution of the lateral wall, diaphyseal fractures, and large posteromedial fragments extending below the level of the lesser trochanter. In stable pertrochanteric fractures, with the lag screw passing through the lateral cortex of the distal fragment, may not require a distal locking screw. Distal locking has been associated with potential complications, including irritation of the fascia lata, prolonged operation time, increased radiation exposure, greater blood loss, implant loosening, secondary femoral stress fractures, and damage to the femoral artery. Thus, although distal locking is of doubtful significance in stable pertrochanteric fractures it is essential in unstable fracture patterns.

Associations of CYP1A2 Polymorphisms with the Risk Haplotypes in Lung Cancer in the Slovak Population.

Phase I enzymes, including cytochrome P450, family 1, subfamily A, and polypeptide 2 (CYP1A2), are involved in the activation of carcinogens to reactive intermediates that are capable of binding covalently to DNA to form DNA adducts, potentially initiating the carcinogenic process. The aim of present study was to investigate the association of CYP1A2 gene polymorphisms and haplotypes with lung cancer risk. A case-control study was carried out on 105 lung cancer patients and 189 controls. To investigate three CYP1A2 polymorphisms: rs2472299, rs2470890, rs11072508 we used a high resolution melting analysis. We found significant allele associations (rs2470890 and rs2422299) with lung cancer risk. We searched for meaningful associations for all variants in the dominant, recessive, and additive genetic models. Genotype associations in the recessive model were of marginal significance for the same single nucleotide polymorphisms. A haplotype analysis included five variants with the frequency higher than 1 %. The haplotype "acc", present with the highest frequency, was associated with increased lung cancer risk (38.7 % vs. 31.5 %; OR 1.38; 95 %CI 0.95-2.01). On the contrary, rare haplotype "gtc" was significantly associated with decreased lung cancer risk in the Slovak population. In conclusion, the present study identified the risk alleles and haploid genotype associations of the CYP1A2 gene in lung cancer.

Mixed chimerism induction influences cytokine release from chimeric mice cells.

Interest in mixed chimerism has evolved from its role in the induction of alloantigen tolerance. However, its precise impact on the host organism remains to be elucidated. In the present work, we analyzed cytokine secretion from chimeric mice cells to assess the influence of different mixed chimerism induction protocols on immune system function in recipient mice. To our knowledge, there have been no reports on using this parameter for the optimization of the mixed chimerism induction method. B6.SJL-PtprcaPep3b or C57BL/6J mice were used as recipients and Balb/c as donors. We utilized four protocols which consisted of: 3Gy total body irradiation (day -1), the injection of 20-30×10(6) bone marrow cells (day 0), and a combination of CD40L (days 0 and 4), CD8 (day -2), and NK1.1 (day -3) blocking antibodies and cyclophosphamide (175mg/kg - day 2). The concentrations of cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF) were evaluated in the supernatants of unstimulated or phytohemagglutinin-stimulated chimeric spleen, bone marrow and peripheral blood cells in the 8th week of experiment. The induction of tolerance to Balb/c mouse antigens was initially tested in chimeric mice by assessing the presence of Vß5 and Vß11 TCR-expressing lymphocytes. The cytokine production was considerably increased, especially in chimeric mice treated by cyclophosphamide. Also the mixed chimerism itself seems to affect IFN-γ, IL-2, IL-4, IL-6, IL-17A, and TNF secretion. Using the optimized induction protocol, we established that chimeric mice cells secreted lower IFN-γ, IL-2, IL-4 and higher IL-6, IL-17A, and TNF levels as compared to control animals. We found that both donor and recipient cells markedly participated in the cytokine production. In conclusion, our optimization study based on cytokine assessment contributes to establishing an effective protocol of mixed chimerism induction with no cyclophosphamide use and better understanding of the influence of this phenomenon on the recipient organism.

Induction of mixed chimerism in mice by employing different conditioning protocols and bone marrow cell transplantation.

Mixed chimerism has been suggested to produce allograft tolerance. Since this phenomenon is not fully understood, the aim of our study was to evaluate various protocols for chimerism induction in a mouse model. B6.SJL-Ptprc(a)Pep3(b) mice were injected with 20 to 30 x 10(6) bone marrow cells from Balb C mice. Conditioning consisted of total body gamma irradiation with 9.5, 5, and 3 Gy on "-1 day" of the experiment, with 200 mg/kg cyclophosphamide (CP) ("+2 day"). Additionally, one group of mice received blocking antibody against CD40L on days 0, 1, 4, and 7. The presence of mixed chimerism in peripheral blood was assessed at 1, 2, 3, 4, 6, and 8 weeks using flow cytometry to detect CD45.1 or CD45.2 antigen expression. Moreover, the chimerism was examined in CD4, CD8, CD45/B220, Mac-1alpha subpopulations in peripheral blood and bone marrow cells at 8 weeks. We also compared chimerism in peripheral blood, bone marrow, and spleen leukocyte populations. We observed that the most effective conditioning method with relatively low toxicity was based on concomitant use of 5 Gy total body irradiation and CP. The percentage of donor cells differed among peripheral blood subpopulations and bone marrow cells, but was similar in leukocyte populations derived from various sources. Our experiments sought to optimize the induction of stable mixed chimerism.

An optimization of protocol for mixed chimerism induction in mice model.

Studies on mixed chimerism are currently focused primarily on obtaining less toxic conditioning protocols. With these issues in mind, we have undertaken the attempt to optimize the procedure of mixed chimerism induction in mice. In order to reduce toxicity, we used decreasing doses of total body irradiation (TBI) together with combination of blocking antibodies. We also tried to eliminate immunosuppression (cyclophosphamide - CP) treatment after bone marrow transplantation. B6.SJL-PtprcaPep3b mice were injected with 20-30 x 106 bone marrow cells from Balb C mice. Mice were treated with TBI (3 - 1.5 - 0 Gy) on "-1" day of the experiment and blocking antibodies against CD40L ("0", and "4" days) and additionally anti-CD8 ("-2" day) and/or anti-NK1.1 ("-3" day). Mice in certain groups also received CP (175 mg/kg) on "2" day. Presence of mixed chimerism was assessed in peripheral blood cells by flow cytometry on the 1st, 2nd, 3rd, 4th, 6th and 8th weeks of the experiment by detecting of CD45.1 (characteristic for B6.SJL-PtprcaPep3b strain) and CD45.2 (characteristic for Balb C strain) antigens expression. We also analyzed the percentage of peripheral blood CD8 T-cells (CD3e/CD8a) and NK cells (Ly-49D/NK1.1). We found that reduction of TBI dose and elimination of CP decrease the rate of mixed chimerism formation. The highest percentage of donor cells was obtained in the group of animals treated with 3 Gy of TBI, CP and combination of anti-CD40L, anti-CD8, and anti-NK1.1 antibodies. The 3 Gy TBI was necessary to induce stable mixed chimerism, but it could be obtained without the CP use. The percentage of CD3e/CD8a and Ly-49D/NK1.1 cells was significantly lower in the groups of mice treated by corresponding antibodies. Moreover, we observed the lowest number of peripheral blood Ly-49D/NK1.1 cells in the group of animals with highest mixed chimerism. Our experiments in mice model can help in better understanding of mixed chimerism phenomenon and in selecting the method of mixed chimerism induction with lowest possible toxicity. This also might improve the protocols of stable mixed chimerism induction in humans, and in the future, the effectiveness of vascularized organ transplantation.

An optimization of hematopoietic stem and progenitor cell isolation for scientific and clinical purposes by the application of a new parameter determining the hematopoietic graft efficacy.

The transplantation of hematopoietic stem and progenitor cells (HSPC) is an established lifesaving therapy. Bone marrow (BM), harvested from heparinized cadaveric organ donors, peripheral blood (PB) and cord blood (CB), are important sources of hematopoietic stem cells. HSPCs, which are used for transplantation purposes, are routinely evaluated in terms of number of mononuclear cells (MNCs), CD34+ MNCs count and viability. The efficacy of grafting is determined additionally in clonogenic tests in vitro. These tests deliver important information about the number of HSPCs and their proliferative potential. Unfortunately, they do not give a possibility to evaluate the functional HSPC chemotactic reactivity in the SDF-1 gradient, which is probably the key phenomenon for HSPC homing after transplantation procedure. Thus, the aim of our study was to optimize HSPC isolation according to their chemotactic reactivity in SDF-1 gradient. Using multiparameter cell sorter (FACS Aria, BD) we examined the HSPCs attracted by SDF-1 on a single cell level. The population of cells which participated in the chemotactic process was highly enriched in CXCR4+lin-AC133+CD45+ cells (referred as hematopoietic stem cells) and to our surprise in CXCR4+lin-AC133+CD45- cells (referred as pluripotent stem cells) in quantitative amounts. Since reactivity of HSPCs may depend on various factors involved in the protocol of their isolation and short-term storage, we tested the most commonly used anticoagulants (ACD, CPDA-1, EDTA and Heparin) and culture media (DME, IMDM, RPMI). HSPCs, harvested from CB, PB and BM, were subsequently investigated for clonogenic growth of CFU-GM in methylcellulose cultures and for the level of apoptosis by employing annexin V staining. Evaluating clonogenic potential, ability of chemotactic reactivity in SDF-1 gradient and intensification of apoptosis of HSPC as the most safe anticoagulant and medium were selected. This study has proved that chemotactic reactivity of HSPCs is a new but very important parameter which should be included in the procedure of their isolation.

Morphological and molecular characterization of novel population of CXCR4+ SSEA-4+ Oct-4+ very small embryonic-like cells purified from human cord blood: preliminary report.

Recently, we purified from adult murine bone marrow (BM) a population of CXCR4(+), Oct-4(+) SSEA-1(+), Sca-1(+) lin(-) CD45(-) very small embryonic-like (VSEL) stem cells and hypothesized that similar cells could be also present in human cord blood (CB). Here, we report that by employing a novel two-step isolation procedure -- removal of erythrocytes by hypotonic lysis combined with multiparameter sorting -- we could isolate from CB a population of human cells that are similar to murine BM-derived VSELs, described previously by us. These CB-isolated VSELs (CB-VSEL) are very small (3-5 micro m) and highly enriched in a population of CXCR4(+)AC133(+)CD34(+)lin(-) CD45(-) CB mononuclear cells, possess large nuclei containing unorganized euchromatin and express nuclear embryonic transcription factors Oct-4 and Nanog and surface embryonic antigen SSEA-4. Further studies are needed to see if human CB-isolated VSELs similar to their murine BM-derived counterparts are endowed with pluripotent stem cell properties.

Expression of stem cell markers on mononuclear cells derived from heparinized cadaveric organ donors before and after disconnection from the respirator.

We have attempted to evaluate the level of the earliest human hematopoietic cell marker expression (CD34, CD117, CD133, CD184) on cells obtained from heparinized cadaveric organ donors before and after disconnection from the respirator. Moreover, we compared various cell populations: (1) coexpressing CD34/CD117; (2) CD34/CD133; (3) highly enriched hematopoietic stem cells (CD34+CXCR4+CD45+); and (4) highly enriched tissue-committed stem cells (CD34+CXCR4+CD45-). Finally, we analyzed whether the level of hematopoietic stem cell marker expression depended on the age of the donor. The expression of the membrane receptors (CD34, CD45, CD117, CD133, CD184) was studied by flow cytometry. We observed that the proportion of mononuclear cells expressing these markers slightly decreased in bone marrow harvested after disconnection from the respirator compared with the samples obtained before disconnection. Moreover, the proportion of cells expressing CD117 antigen depended on age of the donor.

Lung cancer in relation to occupational and environmental chromium exposure and smoking.

The increased occurrence of lung cancer in residents of Dolny Kubin, the North-Slovakia district with ferrochromium industry, compared to the general population of Slovakia, led us to the study assessing influence of the occupational and environmantal exposure to chromium on the lung cancer incidence, respecting also the risk coming from cigarette smoking. Residents of Dolny Kubin district with the diagnosed lung cancer in 1984-1999 were involved in the study. The occurrence of lung cancer was significantly higher in people working in ferrochromium industry. The age at the onset of the disease in people exposed to chromium was by 5.5 years lower than in non-exposed. Smoking was an important risk factor, which has been proved particularly in non-exposed group where 62% were smokers and the onset of the lung cancer in them occured about 3.4 years earlier than in non-smokers. In exposed groups, no significant effect of smoking was found. We can conclude, that occupational exposure to chromium was identified as the main risk factor of lung cancer in Dolny Kubin district even overlaying effect of smoking.

Influence of leukemia inhibitory factor (LIF) on the survival, proliferation and differentiation of human erythroid progenitor cells. In vitro studies under serum free conditions.

Different inflammatory cytokines are involved in the pathogenesis of the anemia of chronic disease (ACD), by inhibiting the proliferation of erythroid progenitors. Leukemia inhibitory factor (LIF), is an important inflammatory cytokine, and the purpose of this study was to evaluate its effect on the proliferation and viability of human erythroid progenitors. We have found that LIF slightly increased the survival of human early progenitor cells cultured under serum free conditions. LIF also slightly costimulated in vitro growth of human erythroid colonies. This last effect seems to be a direct one, because we found that LIF receptor (LIF-R) is expressed in cells isolated from growing in vitro human erythroid BFU-E colonies. These data, and the data reported by others in in vivo models, where LIF administration to experimental animals did not change the values of erythropoietic parameters, demonstrate that this inflammatory cytokine itself is not involved in the pathogenesis of ACD.

[Effect of macrophage inflammatory protein-1 alpha (MIP-1 alpha) on human erythropoiesis in vitro--clinical implications]. / Wplyw makrofagalnego bialka zapalnego -1 alpha (MIP-1 alpha) na ludzka erytropoeze in vitro--implikacje kliniczne.

The influence of macrophage inflammatory protein-1 alpha (MIP-1 alpha) on cloning efficiency of human erythroid progenitors was evaluated. In our hands, in performed experiments in serum supplemented as in serum free cloning culture systems we did not observe any effect of MIP-1 alpha on erythroid colony formation by human CD34+ bone marrow cells. Therefore, our data did not confirm the possibility of the direct role of MIP-1 alpha in the pathogenesis of the anaemia of chronic disease.

A comparative study of the alkaline disassembly of two strains of tobacco mosaic virus.

Exposure of tobacco (Tb) and tomato (Tm) isolates of tobacco mosaic virus (TMV) to dilute alkaline solutions (pH > 8.0) at 0 degrees results in disassembly of the virus particles. Over the range of pH and NaCl concentration studied, Tm-TMV was more sensitive to alkaline conditions than was Tb-TMV. Kinetic analysis demonstrates that both isolates disassemble in a stepwise manner and that each produces six major intermediate-size particles.

Reassembly of particles using the RNA from partially disassembled tobacco mosaic virus.

Controlled alkaline disassembly of tobacco mosaic virus (TMV) results in the production of two populations of partially stripped virus. These particles have been designated PSV5 and 6 and are the two smallest particles produced during alkaline disassembly of TMV. The RNA from these particles was able to reassemble with TMV coat protein. The reassembled particles were resistant to ribonuclease treatment and sedimented upon sucrose density gradient centrifugation to the same position as authentic PSV5 and 6 particles.

Factors Influencing the Production of Intermediate Particles During Alkaline Degradation of Tobacco Mosaic Virus: Time, pH, Salt Concentration, and Temperature.

Kinetic analysis of the alkaline degradation of tobacco mosaic virus revealed degradation to be a stepwise process during which five distinct intermediate nucleoprotein particles were formed. Each intermediate accumulated before being degraded to the next smaller particle. After prolonged exposure to alkali, a stable nucleoprotein particle accumulated. The rate of alkaline degradation of tobacco mosaic virus was retarded by lowering pH (10.3 to 9.0), increasing salt concentration (0 to 100 mM), or increasing incubation temperature over the range of 0 to 22 degrees C. A fraction (15 to 25%) of the total virus population appeared to be completely resistant to alkaline degradation; however, the progeny of this virus fraction was sensitive to alkaline degradation.