Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function

Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly61Ala, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced expression compared to wild-type TM (100%), Arg385Ser (50.2% +/- 5%, P <.001), Pro477Ser (76.8% +/- 1%, P <.001), Pro483Leu (82.1% +/- 8%, P <.007). No TM protein expression could be detected on the cell surface for mutation del791-801. The cofactor activity of TM in protein C activation was also evaluated. The Michaelis constant (K(m)) for wild-type thrombin-TM complex was 634 +/- 6 nmol/L. Two mutants, with Arg385Ser and Pro477Ser, had increased (P <.0001) K(m), 2967 +/- 283 nM, and 2342 +/- 219 nM, respectively, demonstrating impaired function of the thrombin-TM complex. This work presents biochemical evidence that certain (but not all) natural mutations in the TM gene reduce expression and impair function of the protein on the cell surface, and helps clarify the suggested contribution that these mutations might make to the risk of thromboembolic disease.     

Elevated temporal lability of myocardial repolarization after coronary artery bypass grafting

Introduction

Ventricular arrhythmias are uncommon after coronary artery bypass grafting (CABG), but the incidence and mortality are high in certain subsets of patients during the early recovery after surgery. Elevated temporal lability of myocardial repolarization has been associated with sudden cardiac death. The aim of the current study was to explore temporal variability of myocardial repolarization during both early and longtime follow-up after CABG.

Methods and Results

Patients (n = 61) who had undergone CABG and healthy subjects (HS, n = 33) were examined. Electrocardiogram and beat-to-beat blood pressure were recorded at 5 weeks and 5 months after surgery. The QT variability index (QTVI) was calculated as the log ratio between the temporal variabilities of the QT and RR intervals. The QTVI and QT variances were elevated by 40% and 44%, whereas RR variances were reduced by 40% among patients 5 weeks after CABG compared to HS (−0.90 ± 0.59, 29 ± 30, and 1223 ± 1895 ms² vs −1.50 ± 0.29, 15 ± 16, and 2200 ± 2877 ms² for HS; P < .01 for all). The QTVI and QT variances decreased by 38% and 31% between 5 weeks and 5 months after CABG, whereas the RR variances increased by 51% (P < .01 for all). The QTVI values remained elevated among patients compared to HS at 5 months after CABG (P < .01), whereas QT and RR variances did not differ.

Conclusion

Elevated temporal lability of myocardial repolarization prevails particularly during the early recovery phase after CABG and may reflect increased susceptibility to ventricular arrhythmia.     

Analysis of vascular gene expression in arthritic synovium by laser-mediated microdissection

OBJECTIVE: In rheumatoid arthritis (RA), formation of new blood vessels is necessary to meet the nutritional and oxygen requirements of actively proliferating synovial tissue. The aim of this study was to analyze the specific synovial vascular expression profiles of several angiogenesis-related genes as well as CD82 in RA compared with osteoarthritis (OA), using laser-mediated microdissection (LMM). METHODS: LMM and subsequent real-time polymerase chain reaction were used in combination with immunohistochemical analysis for area-specific analysis of messenger RNA (mRNA) and protein expression of vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR-1), VEGFR-2, hypoxia-inducible factor 1alpha (HIF-1alpha), HIF-2alpha, platelet-derived growth factor receptor alpha (PDGFRalpha), PDGFRbeta, inhibitor of DNA binding/differentiation 2 (Id2), and CD82 in RA and OA synovial microvasculature and synovial lining. RESULTS: Expression of Id2 mRNA was significantly lower in RA synovial vessels compared with OA synovial vessels (P=0.0011), whereas expression of VEGFR-1 was significantly higher in RA (P=0.0433). No differences were observed for the other parameters. At the protein level, no statistically significant differences were observed for any parameter, although Id2 levels were 2.5-fold lower in RA (P=0.0952). However, the number of synovial blood vessels and the number of VEGFR-2-expressing blood vessels were significantly higher in RA compared with OA. CONCLUSION: Our results underscore the importance of area-specific gene expression analysis in studying the pathogenesis of RA and support LMM as a robust tool for this purpose. Of note, our results indicate that previously described differences between RA and OA in the expression of angiogenic molecules are attributable to higher total numbers of synovial and vascular cells expressing these molecules in RA rather than higher expression levels in the individual cells.     

Potential risk of cartilage damage in double bundle ACL reconstruction: impact of knee flexion angle and portal location on the femoral PL bundle tunnel

The aim of this study was to compare the impact of knee flexion angle and the level of the medial drilling portal on a potential damage to the subchondral bone in double bundle ACL reconstruction, drilling the femoral PL tunnel through an accessory medial portal. We hypothesized that a knee flexion angle of 70° and 90° or a high accessory medial portal will result in a potential damage to the subchondral bone of the lateral femoral condyle. In a sawbone knee model, the medial portal location was standardized as 0 mm above the meniscus (low portal) and 10 mm above the meniscus (high portal). Femoral PL bundle tunnels were drilled at three different knee flexion angels: 70°, 90°, and 110° of knee flexion. For each portal, ten specimens were used for every flexion angle. Drilling the PL tunnel through the high medial portal at a knee flexion angle of 70° resulted in damage of the subchondral bone plate in all specimens. At 110° of flexion the distance of the tunnel exit to the subchondral bone plate was significantly higher than at 70° of flexion for both the groups, drilling through the high and low medial portal (P < 0.05). Drilling through the low portal did not result in bone plate damage at 90 and 110° of knee flexion angle. Drilling of the femoral PL bundle tunnel through a high medial portal at low knee flexion angles may damage the subchondral bone of the lateral compartment. In ACL reconstruction restoring the AM and PL bundle separately, high medial portal drilling should be avoided. We recommend drilling of the femoral PL bundle tunnel through a low medial portal in high knee flexion.     

Adverse effects by artificial grapefruit seed extract products in patients on warfarin therapy

Objective Grapefruit seed extract (GSE) is promoted as a natural product with antibacterial and antiviral properties. The aim of this study was to investigate the composition of some commercially available GSE products and evaluate their effect in vitro on two cytochrome P450 enzymes, CYP2C9 and CYP3A4. Methods A couple on lifelong treatment with warfarin and continuous regular follow-ups took some drops of a GSE product for 3 days. The female patient experienced a minor subcutaneous haematoma 3 days later, and her international normalised ratio (INR) value was 7.9. This was reported to the Swedish Medical Products Agency (MPA) as a spontaneous post-marketing report concerning adverse drug reactions/interactions. The composition of the GSE products was determined by proton and carbon-13 nuclear magnetic resonance spectroscopy (NMR). The inhibitory effect of the GSE products on the cytochrome P450 enzymes was tested in an in vitro baculosome assay. Results The NMR analysis showed that all three investigated GSE products contained the synthetic preservative benzethonium chloride (BTC) in addition to glycerol and water. No authentic GSE extract was found in any of the three GSE products analysed. Furthermore, BTC was found to be a potent inhibitor of CYP3A4 and CYP2C9 activity in vitro. Conclusion Our results suggest that BTC in the GSE products is responsible for the increase in the INR value in a patient on warfarin treatment.     

Inflammatory cell and cytokine patterns in patients with chronic polyarthritis and temporomandibular joint involvement

OBJECTIVE: To investigate the occurrence of selected markers for inflammatory cells and cytokines in patients with chronic polyarthritis (CPA) and temporomandibular joint (TMJ) involvement. MATERIAL AND METHODS: Eleven patients (11 joints) with CPA and TMJ disorder were included in the study. Synovial specimens were obtained during TMJ open surgery and these were subjected to immunohistochemistry on frozen sections post-fixed with paraformaldehyde and with the cell membranes permeabilized by saponin. In all patients, the cytokines IL-1alpha, IL-1beta, IL-1ra, TNFalpha, IFNgamma, IL2, and TGFbeta were investigated using specific antibodies. The occurrence of macrophages and T-lymphocytes was investigated using immunohistochemistry with monoclonal antibodies against antigens CD68 and CD45RO, respectively. In addition, PCNA was used as a marker for cell proliferation. RESULTS: Staining of IL-1alpha, IL-1, and TGF was seen in all 11 specimens, IFN? in 1, TNFalpha in 4, and IL-2 in none. CD45RO-positive T cells were detected in 7 specimens, CD68-positive macrophages in 6, and cell proliferation seen with PCNA was noted in 8. CONCLUSIONS: The predominant cytokines of TMJ CPA were IL-1alpha, IL-1beta, and TGFbeta, and there appeared to be no differences between the subgroups (rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis) involved. Moreover, the cytokine pattern of TMJ CPA patients seemed to differ from patients with osteoarthritis, as shown in our previous study. The main difference was the absence of IFNgamma and TNFalpha in TMJ CPA patients and a stronger TGFbeta and IL-1alpha expression.     

Histone deacetylase inhibitors promote the tumoricidal effect of HAMLET

Histone deacetylase inhibitors (HDIs) and HAMLET (human alpha-lactalbumin made lethal to tumor cells) interact with histones, modify the structure of chromatin, and trigger tumor cell death. This study investigated how the combination of HDIs and HAMLET influences cell viability, histone acetylation, and DNA integrity. The pretreatment of tumor cells with HDIs was shown to enhance the lethal effect of HAMLET and the histone hyperacetylation response to HDIs increased even further after HAMLET treatment. HDIs and HAMLET were shown to target different histone domains as HAMLET bound tailless core histones, whereas HDIs modify the acetylation of the histone tail. DNA damage in response to HAMLET was increased by HDIs. The DNA repair response (p21WAFI expression) was induced by both agonists but abolished when the two agonists were combined. The results suggest that the synergy of HDIs and HAMLET is based on different but converging death pathways, both involving chromatin alterations. We speculate that HAMLET and HDIs might be combined to promote tumor cell death in vivo.