Keep shocking: Double sequential defibrillation for refractory ventricular fibrillation.

Double sequential defibrillation is proposed as a novel modality of managing refractory ventricular fibrillation (VF). However, existing evidence has not been enough to support this. Here, we report an interesting case of a 54-year-old male who suffered from cardiac arrest with VF rhythm. The patient did not respond to 11 consecutive shocks along with antiarrhythmic medications. However, double sequential defibrillation terminated the VF. He had another episode of VF unresponsive to thirty minutes of standard defibrillation on his way to the catheterization laboratory. Again, the VF was terminated by double sequential defibrillation. Five days later, the patient was discharged home without neurological sequels.

Frequency and safety of switching antithrombin therapy at a regional PCI center.

The impact of switching antithrombin therapy in patients presenting with acute coronary syndromes (ACS) and undergoing percutaneous intervention (PCI) has varied in clinical trials. We sought to assess the incidence and safety of switching antithrombin therapy in ACS patients undergoing PCI at a regional medical center. All patients with ACS undergoing PCI (n = 728) during a specified time period in 2005 and 2007 were identified. Patients who were switched to the antithrombin bivalirudin were defined as the "switch" group (n = 323) and all others were defined as the "consistent" therapy group (n = 405). Primary endpoints were major adverse cardiac event (MACE) (death, MI or urgent revascularization), major bleeding and net adverse clinical event (NACE) (MACE or major bleeding). Multivariate analysis was performed to determine if switching antithrombotic therapy predicted primary outcomes. Among 728 patients undergoing PCI for ACS, 44% were switched to bivalirudin. Switch patient were more likely to be transfers from outside hospitals, older, female, and diabetic. Angiographic characteristics were similar in the two groups. Switch patients had a similar incidence of MACE (7 vs. 8%, P = 0.72), major bleeding (2 vs. 2%) and NACE (9 vs. 10%, P = 0.51) when compared to those who received consistent therapy. On multivariate analysis, switching did not predict MACE (OR = 0.94, 95% CI = 0.53-1.67, P = 0.84) or NACE (OR = 0.82, 95%CI = 0.48-1.41, P = 0.47). In a regional clinical practice of patients presenting with ACS and undergoing PCI, switching of antithrombin therapy to bivalirudin is a common practice and patient who are switched have similar outcomes compared to patients who receive consistent therapy.

Use of an intramedullary nail for correction of femoral deformities combined with total knee arthroplasty: a technical tip.

Simultaneous femoral deformity correction combined with total knee arthroplasty can be a complex procedure, which can be simplified with this described technique. A femoral component that allows access to the intramedullary canal is necessary to be able to use this technique.

Human kallikrein 13 involvement in extracellular matrix degradation.

The human kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4 and shares a high degree of homology. These proteolytic enzymes have diverse physiological functions in many different tissues. Growing evidence suggests that many kallikreins are differentially expressed in cancer and may play a role in metastasis. Human kallikrein gene 13 (KLK13) is a member of this family and codes for a trypsin-like, secreted serine protease (hK13) that is overexpressed in ovarian cancer patients. The aim of this study was to determine if hK13 can degrade extracellular matrix components. Recombinant hK13 was produced in yeast and purified using cation exchange and reverse-phase chromatography. The protein was used as an immunogen to generate mouse monoclonal antibodies. Enzymatic activity of hK13 was verified by using synthetic tri-peptide fluorogenic substrates and gelatin zymography. Active hK13 was incubated with biotinylated extracellular matrix (ECM) proteins and degradation was evaluated by Western blot analysis. hK13-secreting cancer cell lines were treated in a chemotaxis invasion chamber that was coated with various ECM proteins, to determine if hK13 plays a role in tumor cell migration and invasion. Assay with the synthetic substrates and zymography have shown that recombinant hK13 was enzymatically active. The Western blot results showed that hK13 was able to cleave the major components of the extracellular matrix. In the chemotaxis invasion chamber experiment, it was found that ovarian cancer cell lines that secreted hK13 and were treated with an hK13 neutralizing antibody migrated less than untreated cells. Human kallikrein13 may play a role in tissue remodeling and/or tumor invasion and metastasis. Targeting hK13 activity with neutralizing antibodies may have therapeutic applications.

Complex formation between human kallikrein 13 and serum protease inhibitors.

BACKGROUND: The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Human kallikrein gene 13 (KLK13) is a member of this family and encodes for a trypsin-like, secreted serine protease (hK13). Given that other kallikreins are sequestered by serum protease inhibitors, we hypothesized that hK13 may also interact with similar inhibitors. Our objective was to identify serum protease inhibitors that interact with human hK13. METHODS: Recombinant hK13 produced in yeast was added to male and female sera and various biological fluids and the spiked samples were analyzed with an hK13 ELISA assay. Enzymatically active hK13 was 125I-labeled and used in in vitro reactions with candidate protease inhibitors and serum samples. The mixtures were then subjected to gel filtration and SDS-PAGE analysis. Candidate inhibitors were also tested in enzymatic assays of hK13 activity. RESULTS: The recovery of recombinant hK13 from male and female sera, measured by three versions of the hK13-ELISA, ranged from 5% to 10%. The same recovery was obtained when serum samples from males and females were spiked with hK13 from amniotic fluid and seminal plasma. However, when hK13 was added to other biological fluids, such as amniotic fluid and breast milk, recovery ranged from 70% to 98%. In vitro analysis indicated that enzymatically active 125I-labeled hK13 forms SDS-stable complexes with alpha2-antiplasmin, alpha2-macroglobulin and alpha1-antichymotrypsin. When added to serum, active hK13 formed stable complexes with molecular masses corresponding to hK13 and the inhibitors mentioned above. CONCLUSIONS: hK13 interacts and forms complexes with serum protease inhibitors, including alpha2-macroglobulin, alpha1-antichymotrypsin and alpha2-antiplasmin.

The human kallikrein protein 5 (hK5) is enzymatically active, glycosylated and forms complexes with two protease inhibitors in ovarian cancer fluids.

The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Binding of kallikreins to protease inhibitors is an important mechanism for regulating their enzymatic activity and may have potential clinical applications. Human kallikrein gene 5 (KLK5) is a member of this family and encodes for a secreted serine protease (hK5). This kallikrein was shown to be differentially expressed at the mRNA and protein levels in diverse malignancies. Our objective was to study the enzymatic activity and the interaction of recombinant hK5 protein with protease inhibitors. Recombinant hK5 protein was produced in yeast and mammalian expression systems and purified by chromatography. HPLC fractionation, followed by ELISA-type assays, immunoblotting and radiolabeling experiments were performed to detect the possible interactions between hK5 and proteinase inhibitors in serum. Enzymatic deglycosylation was performed to examine the glycosylation pattern of the protein. The enzymatic activity of hK5 was tested using trypsin and chymotrypsin-specific synthetic fluorogenic substrates. In serum and ascites fluid, in addition to the free ( approximately 40 kDa) form, hK5 forms complexes with alpha(1)-antitrypsin and alpha(2)-macroglobulin. These complexes were detected by hybrid ELISA-type assays using hK5-specific coating antibodies and inhibitor detection antibodies. The ability of hK5 to bind to these inhibitors was further verified in vitro. Spiking of serum samples with 125I-labeled hK5 results in the distribution of the protein in two higher molecular mass (bound) forms, in addition to the unbound form. The hK5 mature enzyme is active and shows trypsin, but not chymotrypsin-like, activity. The pro-form of hK5 is not active. Recombinant hK5 shows a higher than predicted molecular mass due to glycosylation. hK5 is partially complexed with alpha(1)-antitrypsin and alpha(2)-macroglobulin in serum and ascites fluid of ovarian cancer patients. The recombinant protein is glycosylated and its mature form shows trypsin-like activity.

Human kallikrein 13: production and purification of recombinant protein and monoclonal and polyclonal antibodies, and development of a sensitive and specific immunofluorometric assay.

BACKGROUND: The aims of this study were to develop immunologic reagents and a sensitive and specific immunoassay for human kallikrein 13 (hK13) and to examine the presence of hK13 in human tissues and biological fluids. METHODS: Recombinant hK13 protein was produced and purified with use of a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse monoclonal and rabbit polyclonal anti-hK13 antibodies. A sandwich-type immunoassay was developed with these antibodies. The assay was used to measure hK13 in various biological fluids and tissue extracts. Immunohistochemical analysis was also performed on nondiseased and cancerous prostatic sections. RESULTS: The hK13 immunoassay had a detection limit of 0.05 micro g/L and showed no cross-reactivity with homologous kallikreins. The assay was linear at 0-20 micro g/L, and within-and between-run CVs were <10% (n = 12). hK13 was detected in tissues, including esophagus, tonsil, trachea, lung, cervix, and prostate. hK13 was also found in seminal plasma, amniotic fluid, follicular fluid, ascites of ovarian cancer patients, breast milk, and cytosolic extracts of ovarian cancer tissues. hK13 was immunohistochemically localized in epithelial cells of both nondiseased and cancerous prostate. hK13 appears to be overexpressed in 50% of ovarian cancer tissues compared with healthy ovarian tissues. Recovery of active enzyme added to milk or amniotic fluid was 70-98%, but was <20% when added to serum, suggesting rapid sequestration by protease inhibitors. In fluids and tissue extracts, hK13 was found in its free (approximately 30 kDa) form. CONCLUSIONS: This immunofluorometric assay for hK13 may be used to examine the value of hK13 as a disease biomarker and to further explore the physiologic and pathobiologic role of this enzyme in human disease.