Pathological Study on the Expression of Vasohibins in Peripheral Artery Disease.

Vasohibin-2 (VASH2) is a gene that promotes local angiogenesis. The tubulin carboxypeptidase activity of vasohibin causes detyrosination of alpha-tubulin and may play an important role in the regulation of various phenomena. Pathological and therapeutic angiogenesis are involved in atherosclerotic lesions. This study aimed to investigate whether the expression of VASH2 is associated with peripheral artery disease (PAD) in relation to angiogenesis, tubulin detyrosination, and severity of atherosclerotic lesions. An analysis of femoral and tibial arteries obtained from 86 patients with PAD or abdominal aortic aneurysm (AAA) was performed. The expressions of cluster of differentiation 31, VASH1, VASH2, and detyrosinated alpha-tubulin (DT-tubulin) were examined by immunohistochemistry, and their association with PAD was analyzed. The counts of VASH2 in the tunica media and adventitia in the tibial artery were significantly higher than those in the femoral artery in the PAD (P = 0.005 and P = 0.008, respectively) and AAA (P = 0.002 and P < 0.001, respectively) groups. In the tunica media and adventitia, VASH2 was significantly correlated with DT-tubulin. There was no significant difference in the expression of VASH2 and DT-tubulin in medial smooth muscle cells (McNemar test, P > 0.999). This study revealed the possible involvements of VASH2 in atherosclerosis by two methods-one maybe related to the progression of atherosclerosis by inducing angiogenesis and the second may be related to the decrease in arterial elasticity by increasing DT-tubulin in medial smooth muscle cells.

Bioelectrical impedance analysis for perioperative water management in adult cardiovascular valve disease surgery.

PURPOSE: Bioelectrical impedance analysis (BIA) has been used recently to measure the body water of patients with acute heart failure. We used BIA in this study to better understand, and possibly identify a predictive marker for, perioperative water behavior in cardiac surgery patients. METHODS: We measured body water and studied its behavior in 44 patients undergoing surgery for cardiac valvular disease at our hospital. Measurements included the levels of extracellular water (ECW), intracellular water (ICW), and total body water, the edema index (EI), and the ratio of ECW to total body water. The first measured EI was defined as the "preoperative EI" and the maximum as the "peak EI". RESULTS: A negative correlation was found between the preoperative EI and the preoperative estimated glomerular filtration rate (eGFR) (R = 0.644, p < 0.001). Positive correlations were found between the peak EI and the ICU stay (R = 0.625, p < 0.001), the peak EI and the ventilation time (R = 0.366, p < 0.01), and the preoperative EI and the ICU stay (R = 0.464, p = 0.026). CONCLUSION: The EI is possibly a predictive marker for perioperative water management in cardiac surgery.

Successful palliation for an aged patient with primary pericardial mesothelioma.

An 85-year-old Japanese man with a complaint of exertional dyspnea was admitted to our hospital. Sixty-three years prior to admission at our hospital, he handled asbestos for 2 years in a factory. His chest computed tomography showed a massive pericardial effusion leading to cardiac tamponade and right pleural plaque. After a pericardiocentesis was performed, he recovered from cardiac failure caused by the cardiac tamponade. Pathological examination of the pericardial effusion revealed malignant mesothelial cells. Therefore, he was diagnosed with primary pericardial mesothelioma (PPM) related to asbestos exposure. Although his disease slowly progressed over 18 months, he remained active without any adjuvant treatments such as chemotherapy. Long-term palliation in an aged patient with PPM is rarely obtained using supportive care alone because the prognosis of PPM has been consistently reported to be very poor and almost fatal within a year. Clinical oncologists and thoracic surgeons should be aware of this disease because the accumulation of knowledge on PPM may lead to successful treatment even in aged patients.

TNF-α augmented Porphyromonas gingivalis invasion in human gingival epithelial cells through Rab5 and ICAM-1.

BACKGROUND: Tumor necrosis factor alpha (TNF-α) plays a central role in the initiation and maintenance of immune responses to periodontopathic bacteria. However, excess TNF-α leads to dysregulated immune responses and progression of periodontitis. Porphyromonas gingivalis (P. gingivalis) invades gingival epithelial cells and then multiplies and survives for a long period. Additionally, increment of TNF-α in periodontal sites is associated with a high prevalence of gram-negative anaerobes such as P. gingivalis. However, it has not been determined whether TNF-α affects invasion of P. gingivalis in periodontal tissues. RESULTS: We examined the effect of TNF-α on invasion of P. gingivalis in gingival epithelial cells and clarified the mechanism by which TNF-α augments invasion of P. gingivalis. Invasion of P. gingivalis into Ca9-22 cells was augmented by stimulation with TNF-α and it was inhibited by treatment with an antibody to TNF receptor-1. TNF-α increased production of ICAM-1, and P. gingivalis invasion was inhibited by an antibody to ICAM-1 in Ca9-22 cells. Silencing of Rab5 mRNA inhibited P. gingivalis invasion. Furthermore, the JNK inhibitor SP600125 inhibited invasion of P. gingivalis and also decreased the active form of Rab5 in Ca9-22 cells. CONCLUSION: TNF-α augments invasion of P. gingivalis in human gingival epithelial cells through increment of ICAM-1 and activation of Rab5. These phenomena may contribute to persistent infection of P. ginigvalis and prolongation of immune responses in periodontal tissues.

POT1b regulates phagocytosis and NO production by modulating activity of the small GTPase Rab5.

The protection of telomeres 1 (POT1) protein is a 75-kDa protein that plays an important role in telomere protection, which is related to telomere elongation. Although POT1 is present in and acts in the nuclei, little is known about the functions of POT1 in the cytosol. We here examined the role of POT1b in phagocytosis in a macrophage-like RAW 264 cell line. We found that POT1 was present in the cytosol, where it was bound to Rab5, which is a protein important for endocytosis. POT1b knockdown in RAW 264 cells increased Rab5 activity and facilitated the phagocytosis of whole cells of Escherichia coli and Staphylococcus aureus. Furthermore, POT1b knockdown enhanced the expression of inducible nitric oxide synthase (iNOS), followed by the promotion of nitric oxide (NO) generation in response to stimulation by bacterial whole cells. These results suggest that POT1b negatively regulates phagocytosis by controlling Rab5 activity and thereby modulates bacteria-induced NO generation. These findings suggest that POT1b participates in innate immune responses.

Assembly mechanism of FCT region type 1 pili in serotype M6 Streptococcus pyogenes.

The human pathogen Streptococcus pyogenes produces diverse pili depending on the serotype. We investigated the assembly mechanism of FCT type 1 pili in a serotype M6 strain. The pili were found to be assembled from two precursor proteins, the backbone protein T6 and ancillary protein FctX, and anchored to the cell wall in a manner that requires both a housekeeping sortase enzyme (SrtA) and pilus-associated sortase enzyme (SrtB). SrtB is primarily required for efficient formation of the T6 and FctX complex and subsequent polymerization of T6, whereas proper anchoring of the pili to the cell wall is mainly mediated by SrtA. Because motifs essential for polymerization of pilus backbone proteins in other Gram-positive bacteria are not present in T6, we sought to identify the functional residues involved in this process. Our results showed that T6 encompasses the novel VAKS pilin motif conserved in streptococcal T6 homologues and that the lysine residue (Lys-175) within the motif and cell wall sorting signal of T6 are prerequisites for isopeptide linkage of T6 molecules. Because Lys-175 and the cell wall sorting signal of FctX are indispensable for substantial incorporation of FctX into the T6 pilus shaft, FctX is suggested to be located at the pilus tip, which was also implied by immunogold electron microscopy findings. Thus, the elaborate assembly of FCT type 1 pili is potentially organized by sortase-mediated cross-linking between sorting signals and the amino group of Lys-175 positioned in the VAKS motif of T6, thereby displaying T6 and FctX in a temporospatial manner.

Pili of oral Streptococcus sanguinis bind to salivary amylase and promote the biofilm formation.

Streptococcus sanguinis is a member of oral streptococci and one of the most abundant species found in oral biofilm called dental plaque. Colonization of the oral streptococci on the tooth surface depends on the adhesion of bacteria to salivary components adsorbed to the tooth surface. Recently, we identified unique cell surface long filamentous structures named pili in this species. Herein, we investigated the role of S. sanguinis pili in biofilm formation. We found that pili-deficient mutant, in which the genes encoding the three pilus proteins PilA, PilB and PilC have been deleted, showed an impaired bacterial accumulation on saliva-coated surfaces. Confocal microscopic observations suggested that the mutant was incapable of producing typical three-dimensional layer of biofilm. Ligand blot analysis showed that the ancillary pilus proteins PilB and PilC bound to human whole saliva. Additional analysis demonstrated that PilC bound to multiple salivary components, and one of which was found to be salivary α-amylase. These results indicate that pilus proteins are members of saliva-binding proteins of oral S. sanguinis, and suggest the involvement of pili in its colonization on saliva-coated tooth surfaces and in the human oral cavity.

Total Debranching Plus Antegrade Thoracic Endovascular Aortic Repair without Side Clamping in a Patient with Arch Aneurysm and Ascending Aorta Calcification.

A high-risk patient with aortic arch aneurysm, associated with severe calcification of the ascending aorta and iliac arteries, was treated with total debranching and antegrade thoracic endovascular aortic repair (TEVAR) via the ascending aorta. Proximal anastomosis for a triple-branched graft to the ascending aorta was performed without side clamping using the "real chimney technique." After bypassing the supra-aortic branches, a TEVAR was performed in an antegrade fashion through the ascending aorta. This case suggests that the approaches mentioned above should be considered in patients with arch aneurysms and severe calcified degeneration.

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion.

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.

Balloon-expandable stent graft for coral reef aorta.

Coral reef aorta (CRA) is a rare aortic occlusive disease with calcified intraluminal lesions. We report the case of a patient with CRA who underwent endovascular treatment (EVT). A 78-year-old woman presented with intermittent claudication. A computed tomography scan showed a preocclusive calcified lesion in the infrarenal aorta. EVT with a balloon-expandable stent graft was successfully performed, and her intermittent claudication improved. Compared with the conventional surgical treatments for CRA, EVT with a balloon-expandable stent graft is less invasive. This procedure is an effective option for the treatment of CRA.

Iatrogenic femoral vein pseudoaneurysm.

Femoral vein pseudoaneurysm is a rare complication during percutaneous interventions. We report the case of a patient with common femoral vein pseudoaneurysm caused by unsuccessful manual compression. A 68-year-old woman underwent catheter ablation for atrial fibrillation at another institution. Postoperatively, she experienced right groin pain and leg edema. Doppler ultrasound examination revealed a 2-cm venous pseudoaneurysm as a compressible and hypoechoic lesion. We successfully performed venous aneurysmectomy after failed ultrasound-guided compression therapy. Appropriate procedures must be selected for patients with femoral pseudoaneurysm. The efficacy of hemostatic techniques for preventing vascular complications after venous sheath removal should not be underestimated.

Nrc of Streptococcus pneumoniae suppresses capsule expression and enhances anti-phagocytosis.

Streptococcus pneumoniae is a major pathogen of community-acquired pneumonia and one of its major virulence factors is pneumolysin, which functions as a cholesterol-dependent cytolytic pore-forming toxin. In this study, we identified the ply-like gene spd0729 in a BLAST search. Unexpectedly, hemolytic and cytotoxic assays showed no significant differences between a Deltaspd0729 mutant strain and the wild-type strain, whereas the mutant strain exhibited weaker anti-phagocytic activity in human peripheral blood. In addition, real-time RT-PCR analysis revealed that four capsular biosynthesis genes in the mutant strain had expressions 7- to 432-fold greater than those of the wild type, while an enzyme-linked immunoassay showed a mean 3-fold greater amount of total capsular polysaccharide in the mutant strain. These results suggest that Spd0729 is not a cytolysin, though it plays crucial roles in anti-phagocytosis and regulation of capsule expression. Thus, we named Spd0729 as a negative regulator of capsular polysaccharide synthesis (Nrc).

Requirements for surface expression and function of adhesin P1 from Streptococcus mutans.

In this report, we define requirements for the successful translocation and functional maturation of the adhesin P1 of Streptococcus mutans. Conformational epitopes recognized by anti-P1 monoclonal antibodies (MAbs) were further characterized, thus facilitating the use of particular MAbs as tools to monitor the locations of various forms of the protein. We show that correct localization of P1 is dependent on structural features of the molecule itself, including a requisite A region-P region intramolecular interaction that occurs within the cell prior to secretion. P1 also was shown to be affected by several members of the protein-folding-secretion-turnover apparatus. It does not achieve a fully functional form in the absence of the trigger factor PPIase homolog RopA, and its translocation is delayed when DnaK levels are limited. In addition, dnaK message levels are differentially altered in the presence of P1 lacking the alanine-rich compared to the proline-rich repeat domains. Lastly, nonsecreted P1 lacking the P region accumulates within the cell in the absence of htrA, implying an intracellular HtrA protease function in the degradation and turnover of this particular internal-deletion polypeptide. However, the opposite effect is seen for full-length P1, suggesting a sensing mechanism and substrate-dependent alteration in HtrA's function and effect that is consistent with its known ability to switch between chaperone and protease, depending on environmental perturbations.

Basis of beneficial immunomodulation by monoclonal antibodies against Streptococcus mutans adhesin P1.

We previously identified five monoclonal antibodies (MAbs) against Streptococcus mutans adhesin P1 that modulate the humoral response when bound to whole bacteria and immune complexes (ICs) are administered to BALB/c mice. The two MAbs that redirected the response towards increased efficacy recognize discontinuous epitopes involving pre-alanine-rich domain sequence; therefore, to evaluate whether epitope specificity contributes to a desirable outcome a further MAb with this characteristic was tested. A beneficial immune response was promoted. None of the three MAbs that promoted a beneficial response was opsonic, suggesting that increased uptake of ICs by phagocytes does not mediate the improvement of the IC-elicited antibodies to inhibit bacterial adherence. Finally, two of the six anti-P1 MAbs activated complement but did not partition according to desirable vs. nondesirable effects.

Vinculin and Rab5 complex is required [correction of requited]for uptake of Staphylococcus aureus and interleukin-6 expression.

Vinculin, a 116-kDa membrane cytoskeletal protein, is an important molecule for cell adhesion; however, little is known about its other cellular functions. Here, we demonstrated that vinculin binds to Rab5 and is required for Staphylococcus aureus (S. aureus) uptake in cells. Viunculin directly bound to Rab5 and enhanced the activation of S. aureus uptake. Over-expression of active vinculin mutants enhanced S. aureus uptake, whereas over-expression of an inactive vinculin mutant decreased S. aureus uptake. Vinculin bound to Rab5 at the N-terminal region (1-258) of vinculin. Vinculin and Rab5 were involved in the S. aureus-induced phosphorylation of MAP kinases (p38, Erk, and JNK) and IL-6 expression. Finally, vinculin and Rab5 knockdown reduced infection of S. aureus, phosphorylation of MAPKs and IL-6 expression in murine lungs. Our results suggest that vinculin binds to Rab5 and that these two molecules cooperatively enhance bacterial infection and the inflammatory response.

Effect of expression level on immune responses to recombinant oral Salmonella enterica serovar Typhimurium vaccines.

Live, attenuated Salmonella has been used to express heterologous antigens for development of oral vaccines. Often, expression must be regulated because of deleterious effects on the Salmonella vector. The effect of varying expression levels on immune response parameters has not been well defined. In this study we introduced mutations in the -10 region of the trc promoter in the expression plasmid to generate series of vaccine strains with different levels of expression of a model antigen, the hemagglutinin HagB from Porphyromonas gingivalis. There was no difference in growth rates of the Salmonella vaccine strains containing the wild-type, the mutant plasmids or the empty expression vector. The primary IgG response in serum in mice orally immunized with the wild-type strain peaked 3-4 weeks earlier than the intermediate expression level strains, suggesting that high expression levels may favor an earlier response. While there was a trend for anti-HagB recall responses to correlate with higher expression level, the peak levels were not significantly different even for expression levels as low as 33% of wild-type. A similar trend in terms of response level was seen with serum and salivary IgA. The subclass of the IgG response was predominately IgG2a regardless of expression level, consistent with a Th1 response. These data suggest that isotype distribution, immune response level and T helper cell profile are largely unaffected over a wide range of expression levels.

Expression of a Porphyromonas gingivalis hemagglutinin on the surface of a Salmonella vaccine vector.

Live, attenuated Salmonella strains can serve as vectors for the delivery of recombinant vaccine antigens for development of oral mucosal vaccines. Various vaccine parameters can affect the immune responses elicited by Salmonella vectors, including the expression level, location and timing of expressed antigens. We have previously established immunogenic Salmonella enterica serovar Typhimurium strains which cytoplasmically express hemagglutinin B (HagB) of Porphyromonas gingivalis, a putative periodontal pathogen. In this study, we sought to determine whether the 39 kDa HagB protein could be stably expressed on the surface of an avirulent Salmonella vaccine strain. The hagB gene was cloned into an expression plasmid as a C-terminal fusion with Lpp-OmpA, a hybrid surface display system. High expression of Lpp-OmpA-HagB proved to be toxic to the vaccine strain, and it was necessary to introduce attenuating mutations in the trc promoter. Stable expression was obtained in transformants with promoter mutations that resulted in low levels of expression. The expression of Lpp-OmpA-HagB was confirmed by ELISA and Western blot. Localization to the outer membrane/periplasm was confirmed by transmission electron microscopy using immunogold labeling, surface labeling of whole mounts using electron microscopy, flow cytometry, and by quantitation of HagB in cytoplasmic, as well as inner and outer cell membrane fractions. When delivered orally in mice, the surface-expressing strain induced higher serum IgG and IgA responses to HagB than a cytoplasmic expressing strain, while responses in secretions were comparable. These results suggest that surface localization may differentially enhance the immunogenicity of antigens expressed by live, avirulent Salmonella vaccine vectors.