An alternative extrinsic pathway of human blood coagulation

To study the interrelationships of the major human coagulation pathways, factor X activation in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin were added to plasma samples containing 3H-labeled factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin the rate of factor X activation in factor VIII or factor IX deficient plasma was only 10% of the activation rate seen for normal or factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, restored normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these experiments, it is inferred that normal activation of factor X in plasma due to dilute thromboplastin requires factors VII, IX and VIII. An alternative extrinsic pathway that involves factors VII, IX, and VIII may be a major physiologic extrinsic pathway, and this pathway may help to explain the clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.     

Minor histocompatibility antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T-cell clones inhibit human hematopoietic progenitor cell growth by a mechanism that is dependent on direct cell-cell contact

HLA-identical bone marrow transplantation (BMT) may be complicated by graft-versus-host disease or graft rejection. Both complications are thought to be initiated by recognition of minor histocompatibility (mH) antigens by HLA-restricted mH-antigen-specific T lymphocytes. Using HLA- A2-restricted mH antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T lymphocyte (CTL) clones, we studied the recognition by these CTL clones of interleukin-2 (IL-2)-stimulated T cells (IL-2 blasts), BM mononuclear cells (BMMNCs), and hematopoietic progenitor cells (HPCs). We showed that, when IL-2 blasts from the BM donors who were investigated were recognized by the HA-1-, -2-, and -4-, and HY- specific CTL clones, their BMMNCs and HPCs were recognized as well by these CTL clones, resulting in antigen-specific growth inhibition of erythrocyte burst-forming units (BFU-E), colony-forming units- granulocyte (CFU-G), and CFU-macrophage (CFU-M). the HA-2-specific CTL clone, however, inhibited BFU-E and CFU-G growth from four donors to a lesser extent than from two other donors. We further investigated whether inhibitory cytokines released into the culture medium by the antigen-specific stimulated CTLs or by stimulated BMMNCs were responsible for suppression of HPC growth or whether this effect was caused by direct cell-cell contact between CTLs and HPCs. HPC growth inhibition was only observed after preincubation of BMMNCs and CTLs together for 4 hours before plating the cells in semisolid HPC culture medium. When no cell-cell contact was permitted before plating, neither antigen-stimulated CTL nor antigen-nonstimulated CTLs provoked HPC growth inhibition. Culturing BMMNCs in the presence of supernatants harvested after incubation of BMMNCs and CTL clones together for 4 or 72 hours did also not result in HPC growth inhibition. Both suppression of HPC growth and lysis of IL-2 blasts and BMMNCs in the 51Cr-release assay appeared to be dependent on direct cell-cell contact between target cells and CTLs and were not caused by the release of inhibitory cytokines into the culture medium by antigen-specific stimulated CTLs or by stimulated BMMNCs. Our results show that mH-antigen-specific CTLs can inhibit HPC growth by a direct cytolytic effect and may therefore be responsible for BM graft rejection after HLA-identical BMT.     

Mechanism underlying bupivacaine inhibition of G protein-gated inwardly rectifying K+ channels

Local anesthetics, commonly used for treating cardiac arrhythmias, pain, and seizures, are best known for their inhibitory effects on voltage-gated Na(+) channels. Cardiovascular and central nervous system toxicity are unwanted side-effects from local anesthetics that cannot be attributed to the inhibition of only Na(+) channels. Here, we report that extracellular application of the membrane-permeant local anesthetic bupivacaine selectively inhibited G protein-gated inwardly rectifying K(+) channels (GIRK:Kir3) but not other families of inwardly rectifying K(+) channels (ROMK:Kir1 and IRK:Kir2). Bupivacaine inhibited GIRK channels within seconds of application, regardless of whether channels were activated through the muscarinic receptor or directly via coexpressed G protein G(beta)gamma subunits. Bupivacaine also inhibited alcohol-induced GIRK currents in the absence of functional pertussis toxin-sensitive G proteins. The mutated GIRK1 and GIRK2 (GIRK1/2) channels containing the high-affinity phosphatidylinositol 4,5-bisphosphate (PIP(2)) domain from IRK1, on the other hand, showed dramatically less inhibition with bupivacaine. Surprisingly, GIRK1/2 channels with high affinity for PIP(2) were inhibited by ethanol, like IRK1 channels. We propose that membrane-permeant local anesthetics inhibit GIRK channels by antagonizing the interaction of PIP(2) with the channel, which is essential for G(beta)gamma and ethanol activation of GIRK channels.     

Immunohistochemical localization of membrane and alpha-granule proteins in human megakaryocytes: application to plastic-embedded bone marrow biopsy specimens

Using a new technique for antigen localization, we have demonstrated platelet proteins in megakaryocytes in plastic-embedded biopsy specimens of normal human bone marrow. In a series of 25 specimens, megakaryocytes showed labeling with antibodies to the integral membrane glycoproteins IIIa, IIb, and the IIb-IIIa complex; granule membrane protein 140; and five alpha-granule matrix proteins: thrombospondin, factor VIII-related antigen, beta-thromboglobulin, platelet factor 4, and fibrinogen. The antibodies to the membrane glycoproteins IIIa, IIb, and IIb-IIIa produced diffuse cytoplasmic staining and heavier staining on the plasma membrane, whereas the antibodies to the alpha-granule matrix proteins produced a distinct granular staining within the cytoplasm. Staining for granule membrane protein 140 was also granular in distribution. Rare mononuclear cells consistent with megakaryocyte precursors were labeled with these markers. Other enzyme histochemical and lectin-binding studies showed that the enzyme alpha-naphthyl acetate esterase, the lectin Ulex europaeus I, and the periodic-acid Schiff reaction were consistent, but not specific, markers of megakaryocytes. This immunohistochemical technique should facilitate the examination of qualitative and quantitative changes in megakaryocytes in a variety of physiologic and pathologic processes.     

Improved Survival with Anti-VEGF Therapy in the Treatment of Unresectable Appendiceal Epithelial Neoplasms

Annals of Surgical Oncology - Currently, cytoreductive surgery and hyperthermic intraperitoneal chemotherapy are accepted treatments for surgically resectable appendiceal epithelial neoplasms....     

Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V

Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity. In purified reaction mixtures, Gln506-FVa in comparison to normal FVa showed reduced susceptibility to APC, because it was inactivated approximately 10-fold slower than normal Arg506-FVa. It was previously reported that inactivation of normal FVa by APC involves an initial cleavage at Arg506 followed by phospholipid- dependent cleavage at Arg306. Immunoblot and amino acid sequence analyses showed that the 102-kD heavy chain of Gln506-FVa was cleaved at Arg306 during inactivation by APC in a phospholipid-dependent reaction. This reduced but measurable susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance is a mild risk factor for thrombosis because APC can inactivate both normal FVa and variant Gln506-FVa. In summary, this study shows that purified Gln506- FV can account for APC resistance of plasma because Gln506-FVa, whether generated by thrombin or FXa, is relatively resistant to APC.     

Monoclonal antibodies to human prostatic acid phosphatase: probes for antigenic study.

Hybrid cell lines producing monoclonal antibodies against human prostatic acid phosphatase [PAPase; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) were prepared by the fusion of mouse myeloma cells with the spleen cells of PAPase-immunized BALB/c mice. Approximately 23% of the hybrid cells initially plated after cell fusion produced specific antibodies: 34 microcultures were cloned, and 8 eventually yielded stable cell lines. The monoclonal antibodies produced by these eight hybridomas were characterized for their isotypes, isoelectric points, concentrations, and affinities. All of the eight monoclonal antibodies exhibited strict specificity for PAPase as determined by radioimmunoassay and immunohistochemical methods. These antibodies were used as probes for the antigenic mapping of this enzyme, and three nonoverlapping determinants were recognized. Further binding studies with PAPase fragments, generated by cleavage with a submaxillaris protease, showed that those three determinants are clustered on one fragment of PAPase. These monoclonal antibodies may be useful in refinement of clinical immunoassays of PAPase or immunohistological study of PAPase-synthesizing cells.     

Monitoring Thiopurine Metabolites in Korean Pediatric Patients with Inflammatory Bowel Disease

Purpose

This study aimed to assess the role of thiopurine S-methyltransferase (TPMT) and 6-thioguanine nucleotide (6-TGN) as predictors of clinical response and side effects to azathioprine (AZA), and estimate the optimal AZA dose in Korean pediatric inflammatory bowel disease (IBD) patients.

Materials and Methods

One hundred and nine pediatric IBD patients in whom AZA treatment was required were enrolled. Thiopurine metabolites were monitored since September 2010. Among them, 83 patients who had prescribed AZA for at least 3 months prior to September 2010 were enrolled and followed until October 2011 to evaluate optimal AZA dose, adverse effects and disease activity before and after thiopurine metabolite monitoring.

Results

The result of the TPMT genotype was that 102 patients were *1/*1 (wild type), four were *1/*3C, one was *1/*6, one was *1/*16 (heterozygote) and one was *3C/*3C (homozygote). Adverse effects happened in 31 patients pre-metabolite monitoring and in only nine patients post-metabolite monitoring. AZA dose was 1.4±0.31 mg/kg/day before monitoring and 1.1±0.46 mg/kg/day after monitoring (p<0.001). However, there were no statistical differences in disease activity during metabolite monitoring period (p=0.34). Adverse effects noticeably decreased although reduction of the AZA dose since monitoring.

Conclusion

TPMT genotype and thiopurine metabolite monitoring could be helpful to examine TPMT genotypes before administering AZA and to measure 6-TGN concentrations during prescribing AZA in IBD patients.     

Application of Lidocaine Jelly on Chest Tubes to Reduce Pain Caused by Drainage Catheter after Coronary Artery Bypass Surgery

The objective of this study was to assess the effect of lidocaine jelly application to chest tubes on the intensity and duration of overall pain, chest tube site pain and the required analgesics for postoperative pain relief in coronary artery bypass graft (CABG) patients. For patients in group L, we applied sterile 2% lidocaine jelly on the chest tubes just before insertion, and for patients in group C, we applied normal saline. Overall visual analogue scale (VAS), maximal pain area with their VAS were documented postoperatively, and the frequency that button of patient-controlled analgesia was pressed (FPB) and total fentanyl consumption were assessed. The number of patients who complained that tube site was the most painful site was significantly higher in group C than in group L (85% vs. 30% at extubation, P<0.001). The overall VAS score was significantly higher in group C than in group L (39.14±12.49 vs. 27.74±13.76 at extubation, P=0.006). After all of the tubes were removed, the VAS score decreased more in group C (5.74±4.77, P<0.001) than in group L (3.05±2.48, P<0.001). FPB and total fentanyl consumption were significantly higher in group C than in group L (73.00, 59.00-78.00 vs. 34.00, 31.00-39.25, P<0.001; 2,214.65±37.01 vs. 1,720.19±361.63, P<0.001, respectively). Lidocaine jelly application is a very simple way to reduce postoperative pain by reducing chest tube site pain after CABG. (Clinical Trials Registry No. ACTRN 12611001215910)     

Clinical outcomes of spontaneous bacterial peritonitis due to extended-spectrum beta-lactamase-producing Escherichia coli or Klebsiella pneumoniae: a retrospective cohort study

Purpose

The aim of this study was to (1) evaluate the clinical outcomes of spontaneous bacterial peritonitis (SBP) due to extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli or Klebsiella pneumoniae (EK) and (2) investigate the relationship between the adequacy of initial antibiotic treatments and patient outcomes.

Methods

We conducted a retrospective cohort study of cirrhotic patients with SBP caused by EK. We evaluated the 30-day mortality rate and used Cox proportional hazard models to identify risk factors for mortality.

Results

Between January 2006 and December 2012, a total of 231 episodes of SBP due to EK were recorded. Among them, 52 were caused by ESBL-producing EK (ESBL-EK). The 30-day mortality rate was significantly higher in patients with SBP due to ESBL-EK than in those with non-ESBL-producing EK (non-ESBL-EK) (34.6 vs. 18.4 %, respectively; p = 0.013). Multivariate analysis revealed that ESBL production [adjusted HR (aHR) 1.82, 95 % confidence interval (CI) 1.00–3.31], nosocomial infection (aHR 2.24, 95 % CI 1.26–3.95), septic shock (aHR 4.84, 95 % CI 2.70–8.65), higher Child-Pugh score (aHR 1.57, 95 % CI 1.28–1.92), and higher Charlson comorbidity index (aHR 1.37, 95 % CI 1.15–1.64) were independent risk factors for 30-day mortality in the total cohort. When we analyzed patients with SBP due to ESBL-EK separately, septic shock (aHR 3.64, 95 % CI 1.40–9.77), accompanying bacteremia (aHR 3.71, 95 % CI 1.37–10.08), and hepatocellular carcinoma (aHR 3.21, 95 % CI 1.20–8.56) were independent risk factors.

Conclusions

Both 7- and 30-day mortalities for SBP due to ESBL-EK were significantly higher than for SBP due to non-ESBL-EK. Initial antibiotic choice was not associated with poor clinical outcomes in patients with SBP due to ESBL-EK.