Clinically feasible method for assessing leukocyte rheology in whole blood.

This study reports a novel method for assessment of leukocyte rheological activation with a new designed microchannel array chip to mimic the human microvascular network for microchannel array flow analysis (MCFAN). Study subjects were 79 healthy volunteers and 42 patients with type 2 diabetes mellitus (DM) and 36 patients with acute coronary syndrome (ACS). Using the anticoagulants heparin and ethylene-diamine-tetraacetic acid (EDTA)-2Na which inhibits platelets and leukocytes by chelating Ca2+, we were able to quantify leukocyte rheological activation by the subtraction of passage time of blood treated with both heparin and EDTA-2Na from that of blood treated with heparin only. We confirmed that passage times of whole blood with heparin + EDTA-2Na were always shorter than those of whole blood with only heparin in healthy subjects and patients with DM or ACS under suction pressures of - 30 cmH2O. There was a significant correlation between delta whole blood passage time {(heparin tube) - (EDTA-2Na + heparin)} and serum levels of myeloperoxidase and adhesive leukocyte number, respectively, even in blood from patients with DM or ACS, who suffered from inflammation. In conclusion we have developed a clinically feasible method for assessing leukocyte rheological activation in whole blood in ex vivo.

Successful management for repeated bar displacements after Nuss method by two bars connected by a stabilizer.

The Nuss method has become the standard surgery for the treatment of funnel chest, and good therapeutic results have been reported. Among the complications of the Nuss method, displacement of the bar is the most frequent, and there are cases in which reoperation is necessary. In this case report, we have devised a new stabilizer that connects and fixes two bars as bar displacement occurred following each of the two prior Nuss procedures, and the outcome of our procedure was evaluated.

Renin-Angiotensin System Inhibitors Can Prevent Intravenous Lipid Infusion-Induced Myocardial Microvascular Dysfunction and Leukocyte Activation.

BACKGROUND: Levels of triglycerides and free fatty acids (FFAs) are elevated in patients with diabetes and may contribute to endothelial dysfunction through renin-angiotensin system (RAS) activation and oxidative stress. The present study investigated how systemic FFA loading affected myocardial microcirculation during hyperemia via RAS.Methods and Results:Eight healthy men received candesartan, perindopril, or a placebo for 2 days in a double-blind crossover design, and then myocardial microcirculation during hyperemia induced by a 2-h infusion of lipid/heparin was assessed using dipyridamole stress-myocardial contrast echocardiography (MCE). Leukocyte activity and hemorheology were also assessed ex vivo using a microchannel flow analyzer, serum levels of oxidative stress markers, and IκB-α expression in mononuclear cells. Serum FFA elevation by the infusion of lipid/heparin significantly decreased myocardial capillary blood velocity and myocardial blood flow during hyperemia. Both candesartan and perindopril significantly prevented the FFA-induced decrease in capillary blood velocity and myocardial blood flow during hyperemia. Systemic FFA loading also caused an increase in the number of adherent leukocytes and prolonged the whole blood passage time. These effects were blocked completely by candesartan and partially by perindopril. Both agents prevented the FFA-induced enhancement of oxidative stress and IκB-α degradation in mononuclear cells. CONCLUSIONS: Both candesartan and perindopril can prevent FFA-induced myocardial microcirculatory dysfunction during hyperemia via modulation of leukocyte activation and microvascular endothelial function.

Stability of Orbital Floor Fracture Fixation After Endoscope-Assisted Balloon Placement.

In recent years, endoscope-assisted balloon fixation using transantral and endonasal approaches has gained popularity as a minimally invasive treatment for orbital floor fractures. However, the optimal duration for balloon placement and the efficacy of the method have not been fully evaluated. The authors report their assessment of this method using postoperative and chronological measurements of the maxillary sinus volume.Fourteen patients with blowout fracture of the orbital floor who underwent reduction using endoscopic transantral and endonasal approaches followed by 6-week fixation with a balloon were evaluated. The volume of the maxillary sinus was measured for comparison using computed tomography at the time of balloon removal and 6 months after the surgery.The ratio of change in the maxillary sinus volume (maxillary sinus volume 6 months after surgery/maxillary sinus volume at balloon removal) for all subjects was 0.90 to 1.04 (0.96 ±â€Š0.44, mean ±â€ŠSD). No postoperative reduction in volume was detected, indicating satisfactory fixation. Postoperative computed tomography showed bone regeneration in the orbital floor in all patients in whom the fractured bone fragments were removed. No subjects had remaining enophthalmos greater than 2 mm.The postoperative change in the maxillary sinus volume was small, confirming the efficacy of 6-week balloon placement. This method was effective even in patients in whom fractured bone fragments were removed. Therefore, it is advisable to remove the fractured bone fragments if there is concern that the fragments will stray into the orbit due to inflation of the balloon.

Control of the reversibility during boronic ester formation: application to the construction of ferrocene dimers and trimers.

Control of the reversibility during boronic ester formation from boronic acids and diols was found to be possible by choosing an appropriate solvent. As an example, ferrocene dimers and trimers were constructed by using tetrol 1 with an indacene framework, 1,1'-ferrocenediboronic acid 2, and ferrocenemonoboronic acid 4. When equimolar amounts of 1 and 2 were mixed in methanol under equilibrating conditions, two kinds of stacked ferrocene dimers homo- and hetero-3 were selectively obtained depending on the reaction time and both structures were determined by X-ray crystallographic analysis. On the other hand, the ferrocene trimer 7 was successfully constructed by stepwise assembly in the presence of anhydrous magnesium sulfate in acetone where the equilibration of boronic esters was suppressed, while no formation of ferrocene trimer 7 was detected when all components 1, 2 and 4 (2 : 1 : 2 ratio) for trimer 7 were mixed at a time in methanol under equilibrating conditions.

Coombs-negative Autoimmune Hemolytic Anemia Followed by Anti-erythropoetin Receptor Antibody-associated Pure Red Cell Aplasia: A Case Report and Review of Literature.

A 76-year-old woman was referred to our hospital because of anemia. The laboratory findings revealed hemolysis. Although a direct Coombs test was negative, a high titer of RBC-bound IgG was detected, and a diagnosis of Coombs-negative autoimmune hemolytic anemia was made. She was successfully treated with prednisolone. One year and five months later, she again presented anemia and was diagnosed with pure red cell aplasia. Anti-erythropoietin receptor antibody was detected in the serum. She was treated with cyclosporine and obtained prompt recovery. We herein report this rare case and review the pertinent literature.

Inclusion of two different guest molecules within a rationally designed macrocyclic boronic ester in organic solvent.

The inclusion of two different guest molecules in a macrocyclic boronic ester in organic solvent utilizing only π-stacking interactions has been successfully realized. For this purpose, a new tetrol which has an appropriate distance between two 1,2-diol units for the inclusion of two aromatic molecules is designed and synthesized. Simple mixing of the new tetrol with 2,7-pyrenediboronic acid in the presence of pyrene-4,5-quinone efficiently affords the desired macrocyclic boronic ester, which is found by (1)H NMR spectroscopy, ESI-MS, and isothermal titration calorimetry studies to include one molecule each of a dinitronaphthalimide derivative and pyrene. Furthermore, inclusion of two planar molecules within the macrocyclic boronic ester is revealed by X-ray analysis.

O6-carboxymethylguanine in DNA forms a sequence context-dependent wobble base-pair structure with thymine.

N-Nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O(6)-carboxymethylguanine (O(6)-CMG) in DNA. O(6)-CMG has been identified in DNA derived from human colon tissue and its occurrence has been linked to diets high in red and processed meats, implying an association with the induction of colorectal cancer. By analogy to O(6)-methylguanine, O(6)-CMG is expected to be mutagenic, inducing G-to-A mutations that may be the molecular basis of increased cancer risk. Previously, the crystal structure of the DNA dodecamer d(CGCG[O(6)-CMG]ATTCGCG) has been reported, in which O(6)-CMG forms a Watson-Crick-type pair with thymine similar to the canonical A:T pair. In order to further investigate the versatility of O(6)-CMG in base-pair formation, the structure of the DNA dodecamer d(CGC[O(6)-CMG]AATTTGCG) containing O(6)-CMG at a different position has been determined by X-ray crystallography using four crystal forms obtained under conditions containing different solvent ions (Sr(2+), Ba(2+), Mg(2+), K(+) or Na(+)) with and without Hoechst 33258. The most striking finding is that the pairing modes of O(6)-CMG with T are quite different from those previously reported. In the present dodecamer, the T bases are displaced (wobbled) into the major groove to form a hydrogen bond between the thymine N(3) N-H and the carboxyl group of O(6)-CMG. In addition, a water molecule is bridged through two hydrogen bonds between the thymine O(2) atom and the 2-amino group of O(6)-CMG to stabilize the pairing. These interaction modes commonly occur in the four crystal forms, regardless of the differences in crystallization conditions. The previous and the present results show that O(6)-CMG can form a base pair with T in two alternative modes: the Watson-Crick type and a high-wobble type, the nature of which may depend on the DNA-sequence context.

Enhancement of host-guest interactions using rationally designed macrocyclic boronic esters with a naphthalene core.

Efficient inclusion of electron-deficient aromatic guest molecules in an organic solvent utilizing π-stacking interactions was achieved by using two kinds of macrocyclic boronic esters, 1,4-naph-[2+2] and 1,5-naph-[2+2], which were easily prepared by self-assembly of 1,4-naphthalenediboronic acid (3) or 1,5-naphthalenediboronic acid (4) and racemic tetrol 1 with an indacene framework in a protic solvent. The X-ray crystallographic analyses revealed that the tilt angles of the two naphthalene rings are different: that of 1,4-naph-[2+2] is about 15° and that of 1,5-naph-[2+2] is about 0°. Owing to the parallel alignment of two aromatic rings, 1,5-naph-[2+2] has a much higher binding ability than 1,4-naph-[2+2]. This knowledge could be useful for the design of the new host molecules in organic solvents.

[Myeloid/natural killer cell precursor acute leukemia diagnosed by cell marker analysis].

We report a case of myeloid/natural killer cell precursor acute leukemia. A 68-year-old man was diagnosed as having lymphoma in his neck, and was referred to our department for further examination and treatment. After admission, blastoid-cells appeared and increased rapidly in his peripheral blood. Cell marker analysis revealed that the blastoid-cells expressed CD7, CD56, CD33, and CD34. He was then diagnosed with myeloid/natural killer cell precursor leukemia. This form of leukemia was recently established as a distinct disease entity. Further clinicopathological evaluation and the establishment of treatment are necessary.

Interleukin-5 plays a key role in mouse strain- dependent susceptibility to contact hypersensitivity through its effects on initiator B cells.

BACKGROUND: Elicitation of contact hypersensitivity requires antigen-specific immunoglobulin M (IgM) antibodies that trigger recruitment of effector T cells to the local tissue. These antigen-specific IgM antibodies are produced by B-1-like 'initiator B cells'. In this study, we compared susceptibility to hapten-induced contact hypersensitivity between BALB/c and C57BL/6 mice. METHODS: BALB/c and C57BL/6 mice were sensitized by painting oxazolone onto the skin and were challenged on the ears with the same hapten on day 4. Ear thickness and serum hapten-specific IgM levels were measured at 24 h post-challenge. Peritoneal cells were harvested and the numbers of B cell subpopulations were counted. Interleukin (IL)-5 was intraperitoneally injected into BALB/c and C57BL/6 mice, and the change in numbers of B cell subpopulations and serum IgM levels was monitored. RESULTS: Oxazolone induced stronger ear swelling and specific IgM responses in BALB/c mice than in C57BL/6 mice. BALB/c mice had higher numbers of peritoneal B-1 cells than C57BL/6 mice at steady state. IL-5 injection increased the number of peritoneal B-1 cells and serum IgM levels more significantly in BALB/ mice than in C57BL/6 mice. CONCLUSIONS: BALB/c mice exhibit higher susceptibility to hapten-induced contact hypersensitivity than C57BL/6 mice, most likely because they have a higher number of B-1 cells, leading to massive production of hapten-specific IgM antibodies upon contact sensitization. The differences in the number of B-1 cells and IgM responses between the two strains of mice may be attributed to the difference in responsiveness of B-1 cells to IL-5.

Structures of DNA duplexes containing O6-carboxymethylguanine, a lesion associated with gastrointestinal cancer, reveal a mechanism for inducing pyrimidine transition mutations.

N-nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O(6)-carboxymethylguanine (O(6)-CMG) in DNA. O(6)-CMG has been identified in DNA derived from human colon tissue, and its occurrence has been linked to diets high in red and processed meats. By analogy to O(6)-methylguanine, O(6)-CMG is expected to be highly mutagenic, inducing G to A mutations during DNA replication that can increase the risk of gastrointestinal and other cancers. Two crystal structures of DNA dodecamers d(CGCG[O(6)-CMG]ATTCGCG) and d(CGC[O(6)-CMG]AATTCGCG) in complex with Hoechst33258 reveal that each can form a self-complementary duplex to retain the B-form conformation. Electron density maps clearly show that O(6)-CMG forms a Watson-Crick-type pair with thymine similar to the canonical A:T pair, and it forms a reversed wobble pair with cytosine. In situ structural modeling suggests that a DNA polymerase can accept the Watson-Crick-type pair of O(6)-CMG with thymine, but might also accept the reversed wobble pair of O(6)-CMG with cytosine. Thus, O(6)-CMG would permit the mis-incorporation of dTTP during DNA replication. Alternatively, the triphosphate that would be formed by carboxymethylation of the nucleotide triphosphate pool d[O(6)-CMG]TP might compete with dATP incorporation opposite thymine in a DNA template.

Epitope mapping of neutralizing monoclonal antibody in avian influenza A H5N1 virus hemagglutinin.

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.

Rituximab plus 70% cyclophosphamide, doxorubicin, vincristine and prednisone for Japanese patients with diffuse large B-cell lymphoma aged 70 years and older.

In the rituximab era, several large studies have suggested that full-dose rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) might be the best treatment for patients with diffuse large B-cell lymphoma (DLBCL) aged 60 years and older. However, it remains unclear whether this is also the case for those aged 70 years and older. Previously untreated patients with DLBCL aged 70 years and older (elderly) were treated with R-70%CHOP, and patients younger than 70 years (younger) were treated with full-dose R-CHOP every 3 weeks, for a total of 6-8 cycles. Complete remission (CR) rates in elderly versus younger patients were 75 vs. 78% (p = 0.7), respectively. The 3-year overall survival, event-free survival and progression-free survival of elderly versus younger patients were 58 vs. 78% (p < 0.05), 45 vs. 70% (p < 0.05) and 64 vs. 72% (p = 0.43), respectively. Severe adverse events were more frequent in the elderly, even with the dose reduction in that age group. Three-year PFS with R-70%CHOP for patients aged 70 years and older was not significantly worse than that with full-dose R-CHOP for younger patients, suggesting that R-70% CHOP might be a reasonable choice for patients with DLBCL aged 70 years and older, especially for those with comorbidities.

Evaluation of Salmonella enterica serovar Typhimurium and Choleraesuis slyA mutant strains for use in live attenuated oral vaccines.

SlyA protein plays a key role in virulence in Salmonella enterica. In this study, we evaluated the ability of the slyA mutant strain of S. enterica serovar Choleraesuis (S. choleraesuis) to protect against swine salmonellosis. Using a murine model infected with S. enterica serovar Typhimurium (S. typhimurium), we showed that the Salmonella strain with a deletion of slyA could be used as a highly immunogenic, effective and safe vaccine in mice. Based on these data, a slyA mutant of S. enterica serovar Choleraesuis strain RF-1 was constructed, and the ability of this mutant to protect immunized pigs from S. choleraesuis infection was examined. As with the S. typhimurium slyA mutant, immunization of pigs with the S. choleraesuis slyA mutant strain provided significant protection against subsequent challenge by the wild-type RF-1. These results demonstrate that SlyA is a potential target in the development of a novel live attenuated vaccine against S. enterica.

Mesenchymal stromal cells inhibit Th17 but not regulatory T-cell differentiation.

BACKGROUND AIMS: A previous study has demonstrated that mouse mesenchymal stromal cells (MSC) produce nitric oxide (NO), which suppresses signal transducer and activator of transcription (STAT) 5 phosphorylation and T-cell proliferation under neutral and T helper 1 cells (Th1) conditions. We aimed to determine the effects of MSC on T helper 17 cells (Th17) and regulatory T-cell (T-reg) differentiation. METHODS: CD4 T cells obtained from mouse spleen were cultured in conditions for Th17 or Treg differentiation with or without mouse MSC. Th17 and Treg differentiation was assessed by flow cytometry using antibodies against interleukin (IL)-17 and forkhead box P3 (Foxp3), a master regulator of Treg cells. RESULTS: MSC inhibited Th17 but not Treg differentiation. Under Th17 conditions, MSC did not produce NO, and inhibitors of indoleamine-2,3-dioxygenase (IDO) and prostaglandin E(2) (PGE2) both restored MSC suppression of differentiation, suggesting that MSC suppress Th17 differentiation at least in part through PGE2 and IDO. CONCLUSIONS: Our results suggest that MSC regulate CD4 differentiation through different mechanisms depending on the culture conditions.

Split-face comparison study of cryogen spray cooling versus pneumatic skin flattening in skin tightening treatments using a long-pulsed Nd:YAG laser.

BACKGROUND: Nd:YAG pulsed lasers are used for non-ablative skin tightening treatment. A common drawback of this procedure is pain, which is often intolerable. Pneumatic skin flattening (PSF) is a new technology which alleviates pain in laser treatments. The purpose of this study is to assess the clinical efficacy and pain reduction in Nd:YAG laser skin tightening treatments with PSF compared to Nd:YAG laser skin tightening treatments with cryogen spray cooling (CSC). METHODS: Eleven patients were treated on the face with an Nd:YAG laser operated at an energy setting of 30 J/cm(2) and a pulse duration of 50 ms. Half of the face was treated with the Nd:YAG using CSC and the other side was treated with the Nd:YAG using PSF. A comparison of pain elicited from each treatment was done by comparing each patient's VAS pain scoring of the two sides. RESULTS: Treatment efficacy with CSC and with PSF was identical. The study confirmed significant pain reduction with PSF (p < 0.01). The average pain scores for PSF and CSC were 2.4 and 6.9, respectively. Mild ecchymosis was observed in two patients on the PSF side. CONCLUSION: Nd:YAG laser skin tightening treatments with PSF are less painful than Nd:YAG laser skin tightening treatments with CSC; the PSF treatments can be done with minimum side effects.

Binding of Salmonella-specific antibody facilitates specific T cell responses via augmentation of bacterial uptake and induction of apoptosis in macrophages.

BACKGROUND: Most antigens from intracellular bacteria or vaccines induce both humoral and cell-mediated immune responses, but interactions between these responses are not fully understood. This study aims to resolve how specific antibodies participate in the activation of specific T cells in protecting hosts against Salmonella enterica serotype Typhimurium (S. typhimurium) infection. METHODS: Mice were administered anti-Salmonella immunoglobulin G (IgG) 1 day before Salmonella infection, and survival rate was observed. For in vitro assay, Salmonella bacteria were treated with anti-Salmonella IgG or control IgG before infection of the RAW264.7 or HEp2 cells. After infection, cell-associated bacteria number, induction of apoptosis, and production of nitric oxide were examined. In addition, antigen presentation assays using Salmonella-primed T cells were performed. RESULTS: Treatment of S. typhimurium with anti-Salmonella IgG enhanced the macrophages' uptake of bacteria and induced high-frequency apoptotic cell death. In vitro antigen presentation assay revealed that the extracellular vesicles isolated from apoptotic cells caused by infection with anti-Salmonella IgG-treated S. typhimurium facilitated the responses of Salmonella-specific T cells. CONCLUSION: Our findings suggest that humoral immunity cooperates with cell-mediated immunity upon induction of apoptosis in host cells to establish protective immunity against Salmonella infection, even if it does not directly eliminate intracellular microorganisms.

Combined use of pretest clinical probability score and latex agglutination D-dimer testing for excluding acute deep vein thrombosis.

OBJECTIVE: Currently, the latex agglutination D-dimer assay is widely used for excluding deep vein thrombosis (DVT) but is considered less sensitive than the enzyme-linked immunosorbent assay-based D-dimer test. The purpose of the present study was to determine if a combination of different cutoff points, rather than a single cutoff point of 1.0 microg/mL, on the latex agglutination D-dimer assay and the pretest clinical probability (PTP) score would be able to reduce the use of venous duplex ultrasound (DU) scanning in patients with suspected DVT. METHODS: The PTP score and D-dimer testing were used to evaluate 989 consecutive patients with suspected DVT before venous DU scanning. After calculating the clinical probability scores, patients were divided into low-risk (< or =0 points), moderate-risk (1-2 points), and high-risk (> or =3 points) pretest clinical probability groups. Receiver operating characteristic (ROC) curve analysis was used to determine the appropriate D-dimer cutoff point for each PTP with a negative predictive value of >98% for a positive DU scan. RESULTS: There were 886 patients enrolled. The study group included 609 inpatients (68.7%) and 277 outpatients (31.3%). The prevalence of DVT in this series was 28.9%. There were 508 patients (57.3%) classified as low-risk, 237 (26.8%) as moderate-risk, and 141 (14.9%) as high-risk PTP. DVT was identified in 29 patients (5.7%) with low-risk, 118 (49.8%) with moderate-risk, and 109 (77.3%) with high-risk PTP scores. ROC curve analysis was used to select D-dimer cutoff points of 2.6, 1.1, and 1.1 microg/mL for the low-, moderate- and high-risk PTP groups, respectively. In the low-risk PTP group, specificity increased from 48.9% to 78.2% (P < .0001) with use of the different D-dimer cutoff value. In the moderate- and high-risk PTP groups, however, the different D-dimer levels did not achieve substantial improvement. Despite this, the overall use of venous DU scanning could have been reduced by 43.0% (381 of 886) if the different D-dimer cutoff points had been used. CONCLUSIONS: Combination of a specific D-dimer level with the clinical probability score is most effective in low-risk PTP patients for excluding DVT. In moderate- and high-risk PTP patients, however, the recommended cutoff points of 1.0 microg/mL may be preferable. These results show that different D-dimer levels for patients differing in risk is feasible for excluding DVT using the latex agglutination D-dimer assay.