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Kenji Inoue,1 Takayuki Fujimoto,1 Risako Higa,1 Ryo Moriyama,1 Hiromi Kohmoto,1 Haruka Nagumo,1 Masato Wakakura,1 Goji Tomita21Inouye Eye Hospital, Chiyoda-ku, Tokyo, 2Second Department of Ophthalmology, Toho University School of Medicine, Meguro-ku, Tokyo, JapanBackground: The purpose of this prospective study was to investigate the intraocular pressure (IOP)-lowering effect and safety of latanoprost 0.005% + timolol maleate 0.5% fixed combination eyedrops, now available in Japan.Materials and methods: Thirty-one patients diagnosed with primary open-angle glaucoma who had an insufficient intraocular pressure (IOP) decrease with latanoprost 0.005% eyedrop monotherapy were enrolled. The latanoprost 0.005% eyedrops were discontinued, and administration of latanoprost 0.005%/timolol maleate 0.5% fixed combination eyedrops was initiated without any washout period. IOP was compared before and at months 1, 3, and 6 after the switch. The incidence of adverse reactions was investigated at every follow-up visit.Results: Mean IOP was 17.3 ± 2.7 mmHg before the switch, 15.5 ± 2.6 mmHg one month after the switch, 14.9 ± 2.4 mmHg 3 months after the switch, and 15.1 ± 2.2 mmHg 6 months after the switch, indicating that IOP decreased significantly after the change. The IOP reduction rate was 9.9% ± 11.5% after one month, 13.1% ± 10.9% after 3 months, and 11.2% ± 11.8% after 6 months. Two patients (6.5%) discontinued therapy due to adverse reactions (one case each of itchiness and bradycardia).Conclusion: When latanoprost 0.005% eyedrop monotherapy was replaced by latanoprost 0.005% + timolol maleate 0.5% fixed combination eyedrops, IOP decreased significantly without increasing the frequency of administration, and safety was satisfactory.Keywords: latanoprost 0.005%, timolol maleate 0.5%, fixed combination, eyedrops, intraocular pressure, switch

Research is presented describing the study of data compression techniques using parallel algorithms.

A 51-year-old man presented with sudden-onset palpitations and dyspnea that had started 8 h earlier. The patient was restless and tachypneic and had persistent vomiting upon arrival. His sensorium and oxygen saturation levels rapidly declined three hours after arrival, and he was placed on a ventilator. On hospitalization day 2, he was removed from the ventilator and claimed that he had consumed a large amount of energy drinks (oral caffeine intake, approximately 1 g). The theophylline level on arrival had been elevated (9.0 μg/mL). Caffeine intoxication should be considered in patients presenting with restlessness, tachypnea, frequent vomiting, lactic acidosis, and electrolyte abnormalities.

[Purpose] When fabricating metal strut ankle-foot orthoses, previous studies have reported that shape acquisition is more efficient with a plastic cast than with a plaster of Paris bandage; however, this has not been quantitatively evaluated. This study aimed to clarify the usefulness of plastic casts by quantitatively evaluating the time spent and body shape accuracy among different methods of shape acquisition. [Participants and Methods] We compared the time spent and acquired shapes among the plaster of Paris bandage, plastic cast, and trace methods in 30 limbs of 15 healthy participants. Three-dimensional scans of the lower legs of participants were used as references to compare the three methods. [Results] The plastic cast method required less time than the plaster of Paris bandage method and provided a more accurate shape than the trace method did. However, the plastic cast method resulted in an increased overall width and circumferential diameter owing to the thickness of the stockinette and plastic cast. [Conclusion] The plastic cast method may be useful in reducing the time and burden of fabricating metal strut ankle-foot orthoses. The use of slightly narrow dimensions during the metal strut bending process can account for the increased width and diameter., Competing Interests: The authors declare no conflicts of interest., (2024©by the Society of Physical Therapy Science. Published by IPEC Inc.)

Video 1How to use an endoscopic hand-suturing device (video version)., Competing Interests: The authors disclosed no financial relationships relevant to this publication., (© 2024 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc.)

A 66-year-old man presented to the gastroenterology department with anal pain. For >10 years, he had used an electric bidet toilet while defecating for >5 min at a time, because of constipation. Two weeks prior to his visit, he became aware of discomfort in his anal area and had used an enema 1 week previously. He had persistent diarrhea and began to use the electric bidet toilet at the highest water pressure for long periods. As a result, his anal pain worsened. A colonoscopy revealed circumferential inflammation and ulceration extending from the anal canal to the lower rectum. Approximately half of the Japanese population washes their anuses before and after defecation. Cleaning the anus after defecation using a bidet contributes to hand hygiene and local comfort, and may be effective against constipation. However, excessive bidet use may cause rectal disorders, such as rectal mucosal prolapse syndrome and solitary rectal ulcers. Herein, we report a rare case of a patient with advanced rectal ulceration caused by electric bidet toilet usage., Competing Interests: None., (© 2024 The Authors. DEN Open published by John Wiley & Sons Australia, Ltd on behalf of Japan Gastroenterological Endoscopy Society.)

Background: Dyspnea is an unpleasant subjective symptom and is associated with decreased physical activity level (PAL). Effect of blowing air toward the face has received a great deal of attention as a symptomatic therapy for dyspnea. However, little is known about the duration of its effect and its impact on PAL. Therefore, this study aimed to measure dyspnea severity and changes in dyspnea and PALs with air blasts to the face., Methods: The trial conducted was open-label, randomized, and controlled. This study included out-patients with dyspnea caused by chronic respiratory deficiency. Subjects were provided a small fan and instructed to blow air toward their faces either twice a day or when having trouble breathing. Subsequently, severity of dyspnea and PALs was measured using visual analog scale and physical activity scale for the elderly (PASE), respectively, before and after 3-week treatment. Amounts of changes in dyspnea and PALs before and after treatment were compared using analysis of covariance., Results: Overall, 36 subjects were randomized, and 34 were analyzed. Mean age was 75.4 y (26 males [76.5%] and 8 females [23.5%]). Visual analog scale score for dyspnea (SD) before treatment was 33 (13.9) mm and 42 (17.5) mm in the control and intervention groups, respectively. PASE score before treatment was 78.0 (45.1) and 57.7 (38.0) in the control and intervention groups, respectively. No significant difference in changes in dyspnea severity and PAL was observed between the 2 groups., Conclusions: No significant difference was observed for dyspnea and PALs in subjects after blowing air toward their own faces with a small fan for 3 weeks at home. Disease variability and impact of protocol violations were high due to small number of cases. Further studies with a design focused on subject protocol adherence and measurement methods are required to understand impact of air flow on dyspnea and PAL., Competing Interests: The authors have disclosed no conflicts of interest., (Copyright © 2023 by Daedalus Enterprises.)

Objectives: In July 2017, supplementary guidelines on anticoagulants, including direct oral anticoagulants, were published in Japan. We investigated the changes in endoscopic submucosal dissection (ESD) of gastric mucosal lesions after the publication of the supplement, examined the risk factors, and developed a predictive model for post-ESD bleeding., Methods: We included 2272 gastric ESD cases from our hospital between May 2003 and June 2021 and classified them into two groups: 1789 cases before and 483 after the publication of the supplementary guidelines. A predictive model for post-ESD bleeding was developed using the pre-publication cohort data., Results: The proportion of patients receiving warfarin decreased (5.0% vs. 1.4%) and those receiving direct oral anticoagulants increased (1.2% vs. 6.8%) after the publication of the supplementary guidelines. Post-ESD bleeding occurred in 61 patients, but there was no significant difference in the bleeding rate between the groups (50 [2.8%] vs. 11 [2.3%] patients, respectively). Five risk factors (number of antithrombotic agents, dialysis, heparin replacement, resection specimen size, and procedure time) were identified for model development. The C-statistic for the model and post-publication cohorts were 0.83 and 0.72, respectively. In the model, each risk factor for postoperative bleeding was scored, and the risk was classified into three levels according to the total score. Bleeding rates at low, intermediate, and high risks were 1.6%, 10.3%, and 38.9%, respectively., Conclusion: Despite changes in patient characteristics and clinical practice regarding ESD before and after the publication of the supplementary guidelines, we could still develop a simple and useful predictive model., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. DEN Open published by John Wiley & Sons Australia, Ltd on behalf of Japan Gastroenterological Endoscopy Society.)

The BacT/Alert system has been used for detecting the presence of bacteria in various clinical settings as well as in blood services, but it is associated with a relatively high incidence of false-positive results. We analyzed the results of our quality control sterility testing of blood products by BacT/Alert 3D to understand the mechanism of false-positive results. Anaerobic and aerobic bottles were inoculated with 10 mL of samples and cultured in BacT/Alert 3D for 10 days. Positive-reaction cases were classified as true positive if any bacterium was identified or false positive if the identification test had a negative result. The detection algorithm and the bottle graph pattern of the positive reaction cases were investigated. Among the 43,374 samples, 25 true positives (0.06%) and 29 false positives (0.07%) were observed. Although the detection algorithm of all true positives and 25 of 29 false positives was accelerating production of CO 2 , a steep rise in the bottle graph was observed only in the true positives, and it was not observed in either of the false positives. Four of 29 false positives were dependent on high baseline scatter reflections. Furthermore, evaluating the bottle graph pattern of Streptococcus pneumoniae, a bacterium known to autolyze, we confirmed that no viable bacterium was detected even if a steep rise was observed. In conclusion, the bottle graph pattern of positive reactions allows the differentiation between true positives and false positives. In case of a steep rise without bacterium detection, the bacterium might have autolyzed. Moreover, positive reactions with high baseline scatter reflections, despite immediate loading of bottles after sampling, are potentially false positive. IMPORTANCE In clinical settings, false-positive results are treated as positive until bacterial identification. It may result in the discarding of blood products in blood centers or affect clinical decisions in hospitals or testing facilities. Moreover, the management of these samples is usually time- and labor-consuming. The results of our study may help clinicians and laboratory staff in making a more precise evaluation of positive reactions in BacT/Alert.

Case : A 66-year-old man started carboplatin + etoposide + atezolizumab therapy for advanced small cell lung cancer. Seventeen days after the start of treatment, the patient presented with hematemesis and underwent emergency endoscopy, which revealed multiple erosions and ulcers in the duodenum. Some ulcers showed pulsating bleeding, which was stopped by clipping and cauterization using hemostats. Biopsy of the mucosal peri-ulcer showed lymphocyte, eosinophil, and plasma cell infiltration. The patient was suggested to have acute hemorrhagic duodenitis, which was associated with immune checkpoint inhibitors (ICIs), and conservative treatment with blood transfusion and antacids was continued. However, 11 days after hemostasis, bleeding from a new ulcer was observed. Hemostasis was achieved by coagulation and clipping again, but the general condition of the patient deteriorated owing to the rapid progression of the primary disease, and he died 8 weeks after the start of treatment. Discussion : Although there have been several reports of colitis and other adverse events caused by ICIs, there have been very few reports of duodenitis. Endoscopic findings include diffuse erythema, erosions/ulcerations, and villous atrophy, and pathological findings include eosinophilic infiltration and increased levels of CD8-positive T cells. However, there have been no reports of duodenal mucosal damage caused after administration of atezolizumab nor of severe cases of massive bleeding requiring endoscopic hemostasis and blood transfusion, as in this case., Competing Interests: Authors declare no conflict of interests for this article., (© 2021 The Authors. DEN Open published by John Wiley & Sons Australia, Ltd on behalf of Japan Gastroenterological Endoscopy Society.)

To enable the release of the encapsulated nucleic acids into the cytosol of targeted cells, the interaction of lipid nanoparticles (LNPs) with endosomes is critical. We investigated changes in the physicochemical properties of LNPs containing ionizable cationic lipids that were induced by acidic pH, which reflects the conditions in the maturation of endosomes. We prepared a LNP containing an ionizable cationic lipid. The laurdan generalized polarization values, which are related to the hydration degree of the lipid membrane interface and are often used as an indicator of membrane packing, decreased with a decrease in pH value, showing that the membrane packing was decreased under acidic conditions. Furthermore, the pH-induced variation increased with an increasing percentage of ionizable cationic lipids in the LNPs. These results indicated that electrostatic repulsion between lipid molecules at acidic pH decreased the packing density of the lipids in the LNP membrane. Reducing the order of lipids could be a trigger to form a non-bilayer structure and allow fusion of the LNPs with the membrane of maturing endosomes in an acidic environment. The LNPs were used to incorporate and transport small interfering RNA (siRNA) into cells for knockdown of the expression of β-galactosidase. The knockdown efficiency of siRNA encapsulated in LNPs tended to increase with the ratio of KC2. These results, which demonstrate the underlying phenomena for the fusion of membranes, will help clarify the mechanism of the release of encapsulated nucleic acids., (Copyright © 2021 Elsevier B.V. All rights reserved.)

For quantitative analysis, data should be obtained at a sample concentration that is within the range of linearity. We examined the effect of sample concentration on nanoparticle tracking analysis (NTA) of small extracellular vesicles (sEVs), including exosomes, by comparing NTA results of sEVs with those obtained for polystyrene nanoparticles (PSN) and liposomes, which mimic lipid composition and physicochemical properties of exosomes. Initially, NTA of PSN at different concentrations was performed and the particle sizes determined were validated by dynamic light scattering. The major peak maxima for PSN mixtures of different sizes at the higher particle numbers were similar, with some fluctuation of the minor peak maxima observed at the lower particle number, which was also observed for sEVs. Sample concentration is critical for obtaining reproducible data for liposomes and exosomes and increasing the sample concentration caused an increase in data variability because of particle interactions. The inter-day repeatability of particles sizes and concentration for sEVs were 7.47 and 4.51%, respectively. Analysis of the linearity range revealed that this was narrower for sEVs when compared with that of liposomes. Owing to the use of liposomes that mimic the lipid composition and physicochemical properties of exosomes and proteinase-treated sEVs, it was demonstrated that these different analytical results could be possibly caused by the protein corona of sEVs. Consideration of the sample concentration and linearity range is important for obtaining reproducible and reliable data of sEVs.

Immunodeficient mice engrafted with human peripheral blood cells are promising tools for in vivo analysis of human patient individual immune responses. However, when human peripheral blood mononuclear cells (PBMCs) are transferred into NOG (NOD/Shi-scid, IL-2rg null ) mice, severe graft versus host disease (GVHD) hinders long term detailed analysis. Administration of human PBMCs into newly developed murine MHC class I- and class II-deficient NOG (NOG-dKO; NOG- Iab, B2m-double-knockout) mice showed sufficient engraftment of human immune cells with little sign of GVHD. Immunization with influenza vaccine resulted in an increase in influenza-specific human IgG Ab, indicating induction of antigen-specific B cells in the NOG-dKO mice. Immunization with human dendritic cells pulsed with HLA-A2 restricted cytomegalovirus peptide induced specific cytotoxic T cells, indicating the induction of antigen-specific T cells in the NOG-dKO mice. Adoptive cell therapies (ACTs) using melanoma antigen recognized by T cells (MART-1)-specific TCR-transduced activated T cells showed strong tumor growth inhibition in NOG-dKO mice without any sign of GVHD accompanied by preferential expansion of the transferred MART-1-specific T cells. ACTs using cultured human melanoma infiltrating T cells also showed anti-tumor effects against autologous melanoma cells in NOG-dKO mice, in which changes in human cancer phenotypes by immune intervention, such as increased CD271 expression, could be evaluated. Therefore, NOG-dKO mice are useful tools for more detailed analysis of both the induction and effector phases of T-cell and B-cell responses for a longer period than regular NOG mice.

P2X receptors are non-selective cation channels gated by extracellular ATP, and the P2X7 receptor subtype plays a crucial role in the immune and nervous systems. Altered expression and dysfunctions of P2X7 receptors caused by genetic deletions, mutations, and polymorphic variations have been linked to various diseases, such as rheumatoid arthritis and hypertension. Despite the availability of crystal structures of P2X receptors, the mechanism of competitive antagonist action for P2X receptors remains controversial. Here, we determine the crystal structure of the chicken P2X7 receptor in complex with the competitive P2X antagonist, TNP-ATP. The structure reveals an expanded, incompletely activated conformation of the channel, and identified the unique recognition manner of TNP-ATP, which is distinct from that observed in the previously determined human P2X3 receptor structure. A structure-based computational analysis furnishes mechanistic insights into the TNP-ATP-dependent inhibition. Our work provides structural insights into the functional mechanism of the P2X competitive antagonist.P2X receptors are nonselective cation channels that are gated by extracellular ATP. Here the authors present the crystal structure of chicken P2X7 with its bound competitive antagonist TNP-ATP and give mechanistic insights into TNP-ATP dependent inhibition through further computational analysis and electrophysiology measurements.

Background: Two current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP1(19)) and apical membrane antigen 1 (AMA1), have been prioritized because of outstanding protective efficacies achieved in a rodent malaria Plasmodium yoelii model. However, P. falciparum vaccines based on these antigens have had disappointing outcomes in clinical trials. Discrepancies in the vaccine efficacies observed between the P. yoelii model and human clinical trials still remain problematic., Methodology and Results: In this study, we assessed the protective efficacies of a series of MSP1(19)- and AMA1-based vaccines using the P. berghei rodent malarial parasite and its transgenic models. Immunization of mice with a baculoviral-based vaccine (BBV) expressing P. falciparum MSP1(19) induced high titers of PfMSP1(19)-specific antibodies that strongly reacted with P. falciparum blood-stage parasites. However, no protection was achieved following lethal challenge with transgenic P. berghei expressing PfMSP1(19) in place of native PbMSP1(19). Similarly, neither P. berghei MSP1(19)- nor AMA1-BBV was effective against P. berghei. In contrast, immunization with P. yoelii MSP1(19)- and AMA1-BBVs provided 100% and 40% protection, respectively, against P. yoelii lethal challenge. Mice that naturally acquired sterile immunity against P. berghei became cross-resistant to P. yoelii, but not vice versa., Conclusion: This is the first study to address blood-stage vaccine efficacies using both P. berghei and P. yoelii models at the same time. P. berghei completely circumvents immune responses induced by MSP1(19)- and AMA1-based vaccines, suggesting that P. berghei possesses additional molecules and/or mechanisms that circumvent the host's immune responses to MSP1(19) and AMA1, which are lacking in P. yoelii. Although it is not known whether P. falciparum shares these escape mechanisms with P. berghei, P. berghei and its transgenic models may have potential as useful tools for identifying and evaluating new blood-stage vaccine candidate antigens for P. falciparum.

The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. Here we showed that TIM-4 is expressed on human and mouse macrophages and dendritic cells, and both TIM-4 and TIM-1 specifically bound phosphatidylserine (PS) on the surface of apoptotic cells but not any other phospholipid tested. TIM-4(+) peritoneal macrophages, TIM-1(+) kidney cells, and TIM-4- or TIM-1-transfected cells efficiently phagocytosed apoptotic cells, and phagocytosis could be blocked by TIM-4 or TIM-1 monoclonal antibodies. Mutations in the unique cavity of TIM-4 eliminated PS binding and phagocytosis. TIM-4 mAbs that blocked PS binding and phagocytosis mapped to epitopes in this binding cavity. These results show that TIM-4 and TIM-1 are immunologically restricted members of the group of receptors whose recognition of PS is critical for the efficient clearance of apoptotic cells and prevention of autoimmunity.

Inhibition of dephosphorylation of the 20 kDa myosin light chain (MLC(20)) is an important mechanism for the Ca(2+)-induced sensitization of vascular smooth muscle contraction. We investigated whether this mechanism operates in prostaglandin F(2alpha) (PGF(2alpha))-induced contraction of rabbit aortic smooth muscle and, if so, whether protein kinase C (PKC) or rho-associated kinase (rho kinase) contribute to the inhibition of dephosphorylation. In normal medium, PGF(2alpha) (10 microM) increased the phosphorylation of MLC(20) and developed tension. The rho-kinase inhibitors fasudil and hydroxyfasudil inhibited these changes, despite having no effect on a phorbol-ester-induced MLC(20) phosphorylation. After treatment with verapamil or chelation of external Ca(2+) with EGTA, PGF(2alpha) increased the MLC(20) phosphorylation and tension without an increase in [Ca(2+)](i), all of which were sensitive to fasudil and hydroxyfasudil. ML-9, a MLC kinase inhibitor, quickly reversed the KCl-induced MLC(20) phosphorylation and contraction to the resting level. However, fractions of PGF(2alpha)-induced contraction and MLC(20) phosphorylation were resistant to ML-9 but were sensitive to fasudil. Ro31-8220 (10 microM), a PKC inhibitor, did not affect the phosphorylation of MLC(20) and the tension caused by PGF(2alpha), thus excluding the possibility of the involvement of PKC in the PGF(2alpha)-induced MLC(20) phosphorylation. PGF(2alpha) increased phosphorylation at Thr654 of the myosin binding subunit (MBS) of myosin phosphatase, which is a target of rho kinase, and fasudil decreased the phosphorylation. These data suggest that the PGF(2alpha)-induced contraction is accompanied by the inhibition of MLC(20) dephosphorylation through rho kinase-induced MBS phosphorylation, leading to Ca(2+) sensitization of contraction. An actin-associated mechanism may also be involved in the PGF(2alpha)-induced sensitization.

The function of synapsin I is regulated by phosphorylation of the molecule at multiple sites; among them, the Ser(603) residue (site 3) is considered to be a pivotal site targeted by Ca(2+)/calmodulin-dependent kinase II (CaMKII). Although phosphorylation of the Ser(603) residue responds to several kinds of stimuli, it is unlikely that many or all of the stimuli activate the CaMKII-involved pathway. Among the several stimulants tested in PC12 cells, bradykinin evoked the phosphorylation of Ser(603) without inducing the autophosphorylation of CaMKII, which was determined using phosphorylation site-specific antibodies against phospho-Ser(603)-synapsin I (pS603-Syn I-Ab) and phospho-Thr(286/287)-CaMKII. The bradykinin-evoked phosphorylation of Ser(603) was not suppressed by the CaMKII inhibitor KN62, whereas high KCl-evoked phosphorylation was accompanied by CaMKII autophosphorylation and inhibited by KN62. Thus, we attempted to identify Ser(603) kinase(s) besides CaMKII. We consequently detected four and three fractions with Ca(2+)/calmodulin-independent Ser(603) kinase activity on the DEAE column chromatography of bovine brain homogenate and PC12 cell lysate, respectively, two of which were purified and identified by amino acid sequence of proteolytic fragments as p21-activated kinase (PAK) 1 and PAK3. The immunoprecipitants from bovine brain homogenate with anti-PAK1 and PAK3 antibodies incorporated (32)P into synapsin I in a Cdc42/GTPgammaS-dependent manner, and its phosphorylation site was confirmed as Ser(603) using pS603-Syn I-Ab. Additionally, recombinant GST-PAK2 could phosphorylate the Ser(603) residue in the presence of Cdc42/GTPgammaS. Finally, we confirmed by immunocytochemical analysis that the transfection of constitutively active rat alphaPAK (PAK1) in PC12 cells evokes the phosphorylation of Ser(603) even in the resting mutant cells and enhances it in the bradykinin-stimulated cells, whereas that of dominant-negative alphaPAK quenches the phosphorylation. These results raise the possibility that Ser(603) on synapsin I is alternatively phosphorylated by PAKs, not only by CaMKII, in neuronal cells in response to some stimulants.

Phosphoinositide 3-kinases (PI3Ks) constitute a family of lipid kinases that regulate an array of fundamental cellular responses by neutrophils [polymorphonuclear leukocytes (PMN)]. p85alpha Gene-disrupted mice were used to help accurately identify the physiological role of the PI3K isoform in PMN activation in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF). PMN from the p85alpha-/- mice showed normal cellular motility, and the quantity of superoxide anion (O(2(-))) produced by PMN upon stimulation with formyl-Met-Leu-Phe did not significantly differ between p85alpha-/- and wild-type mice under controlled conditions. In p85alpha-/- mice, the O(2(-)) production by PMN was enhanced (primed) by GM-CSF when stimulated with the chemotactic peptide but to a significantly lesser extent than in wild-type mice. In addition, no major GM-CSF-dependent delay in apoptosis or activation of Akt protein phosphorylation by GM-CSF was observed in the p85alpha-/- mice. In terms of targeting strategy, however, the mutation actually expressed a small amount of Ia-type (p85alpha-regulated) PI3K activity (partially abrogated) in the mice. These results demonstrate that Ia-type PI3K plays a critical role in the enhancement of the GM-CSF-modulated function of PMN and in the PI3K/Akt pathway-dependent delay of PMN apoptosis.

The relationship between class switch recombination (CSR) and somatic hypermutation has been unclear. By using human CD27(-) naive B cells, we investigated the somatic hypermutation and producibility of immunoglobulins (Igs) that occur after CSR. Although neither adult CD27(-) nor cord blood B cells, which showed the unmutated Ig V-region genes, produced IgG, IgM, or IgA in response to conventional stimuli, they produced IgG and IgM but not IgA in the presence of Staphylococcus aureus Cowan strain (SAC) + interleukin-2 (IL-2) + IL-10 + anti-CD40 mAb + CD32 transfectants (CD40/CD32T). The naive B cells also produced IgE when combined with IL-4 + CD40/CD32T. In parallel with IgG production, the expression of mature gamma1 and gamma 2 transcripts was induced from naive B cells by the stimuli. The CD27 expression on human naive B cells was induced remarkably by CD40 signaling or B-cell receptor engagement, but somatic hypermutation could not be induced. The proliferation and differentiation into plasma cells were induced from naive B cells, whereas most of the plasma cells displayed very low levels of mutations in Ig V-region genes. CD27(-) naive B cells expressed activation-induced cytidine deaminase messenger RNA by the stimuli later than CD27(+) memory B cells. Our results demonstrate that CSR, but not noticeable somatic hypermutation, can be induced from CD27(-) naive B cells upon B-cell receptor engagement and CD40 signaling in cooperation with cytokines, suggesting that CSR and somatic hypermutation processes can occur independently, and the antibodies produced in this in vitro system are low-affinity antibodies.

The understanding of theophylline as a bronchodilator has been reconsidered in recent years. We undertook to determine its immunomodulatory actions in granulocytes and elucidate their mechanism. Preincubation of neutrophils with theophylline (10(-5) to 5 x 10(-3) M) had a biphasic effect on O2(-) production stimulated with N-formyl-methionyl-leucyl-phenylalanine or C5a. Theophylline potentiates O2(-) production via adenosine A(2A) receptor antagonism induced by receptor-linked agonists from neutrophils, but not from eosinophils. The addition of theophylline caused a significant decline in neutrophil chemotaxis at lower concentrations than those for eosinophil motility. Theophylline reduces neutrophil chemotaxis via adenosine A1 receptor antagonism. At high concentrations, with an intracellular cAMP accumulation as a result of phosphodiesterase (PDE) inhibition, theophylline also exerts an inhibitory effect on the O2(-) production and chemotaxis of both types of cells. The difference in theophylline's effect on neutrophils and eosinophils appears to depend on the existence of specific adenosine receptors. Theophylline thus modulates granulocyte functions in association with specific adenosine receptor antagonism and cAMP-PDE inhibition.

This study was designed to determine whether theophylline would augment granulocyte apoptosis via a mechanism of adenosine A2A receptor antagonism. A selective adenosine A2 receptor agonist (CGS-21680, 1 microM) exhibited the most efficient potency for decreasing neutrophil apoptosis for 16 h from 63+/-5 to 19+/-4% (P < 0.001); it exerted poor and adverse effects on eosinophil survival. A selective protein kinase A inhibitor KT-5720 (10 microM) reversed the capacity of dibutyryl cAMP but not CGS-21680 to induce an inhibitory effect on neutrophil apoptosis, suggesting that occupancy of adenosine A2 receptors inhibit neutrophil apoptosis by a cAMP-independent mechanism. Theophylline derivatives show the following pattern of potency for inducing neutrophil apoptosis competing with CGS-21680: 8-phenyltheophylline = 8-p-sulfophenyltheophylline > theophylline >> enprofylline. This pattern is consistent with the affinity established for A2A receptors. Theophylline demonstrated an additive effect to that of anti-Fas antibody (CH11, 1 microg/mL) in inducing neutrophil apoptosis, but not to that of adenosine deaminase or KF-17837 (a selective A2 receptor antagonist; 1 microM), suggesting conflicting effects on the receptor antagonism. These findings suggest that theophylline has an immunomodulatory action on neutrophil apoptosis via a mechanism of A2A antagonism.

In smooth muscle, a Rho-regulated system of myosin phosphatase exists; however, it has yet to be established whether Rho kinase, one of the downstream effectors of Rho, mediates the regulation of myosin phosphatase activity in vivo. In the present study, we demonstrate in permeabilized vascular smooth muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca(2+)-induced 20-kDa myosin light chain (MLC(20)) phosphorylation due to abrogating Rho-mediated inhibition of MLC(20) dephosphorylation. By an immune complex phosphatase assay, we found that guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) stimulation of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-binding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent manner. HA-1077 abolished both of the Rho-mediated events. Moreover, we observed that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTPgammaS-induced phosphorylation of MBS. These results provide direct in vivo evidence that Rho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC(20) phosphorylation in smooth muscle and reveal the usefulness of HA-1077 as a Rho kinase inhibitor.

B cells can differentiate into the antibody-secreting cells, plasma cells, whereas the crucial signals that positively control the entry into the pathway to plasma cells have been unclear. Triggering via CD27 by CD27 ligand (CD70) on purified peripheral blood B cells yielded an increase in the number of plasma cells in the presence of interleukin-10 (IL-10). Differentiation into plasma cells by a combination of IL-10 and CD70 transfectants occurred in CD27+ B cells but not in CD27- B cells. Moreover, addition of IL-2 to the IL-10 and CD70-transfect activation system greatly induced differentiation into plasma cells. In the presence of only IL-2, IL-4, or IL-6, CD70 transfectants did not promote differentiation into plasma cells. On the other hand, CD40 signaling increased the expansion of a B-cell pool from peripheral blood B cells primarily activated by IL-2, IL-10, and anti-CD40 monoclonal antibody (MoAb). Finally, CD27 signaling also rescued B cells from IL-10-mediated apoptosis. These data demonstrate that CD27 ligand (CD70) is a key molecule to prevent the IL-10-mediated promotion of apoptosis and to direct the differentiation of CD27+ memory B cells toward plasma cells in cooperation with IL-10.

In primary cultured mouse epidermal cells, protein kinase C (PKC) zeta consists of multiple forms: a low salt-eluted PKC zeta (1-PKC zeta, 79 and 85 kDa) and a high salt-eluted PKC zeta (h-PKC zeta, 79 and 85 kDa) by anion-exchange column chromatography (K. Nishikawa et al., Cell. Signal. 7, 491-504, 1995). In the present study, PKC isozyme-specific responses during terminal differentiation of epidermal cells, which were induced by the increase of Ca(2+)-concentration in culture medium, were examined. After 24 hr-treatment with 1.8 mM Ca2+, 79-kDa 1-PKC zeta in the particulate fraction was apparently shifted to the 85-kDa form. The phosphatidylserine-dependent kinase activity of this 1-PKC zeta was increased in association with the shift. These results suggest the pivotal role of 1-PKC zeta in the particulate fraction in the Ca(2+)-induced epidermal cell differentiation processes.

A new Epstein-Barr virus nuclear antigen (EBNA)-positive B-cell line, designated BALL-2, was spontaneously established from the peripheral blood of a 14-year-old boy with an EBNA-negative B-cell acute lymphoblastic leukemia (B-ALL), L2 in the French-American-British classification. The BALL-2 cell line grew in suspension with or without forming clumps of cells. The cultured cells exhibited lymphoid morphology with indented or lobulated nuclei, prominent nucleoli, and relatively abundant cytoplasm. Immunologic and cytogenetic studies showed that the BALL-2 cell line expressed the B-cell phenotype, CpIg+, SmIg+, CD19+, CD20+, CD38-, Ia+, and had chromosome translocation, t(8;14) (q24;q32). The same phenotypic and chromosome markers were present in original leukemia cells. These results indicated that the cell line was derived from the patient's leukemia cells. Unexpectedly, however, BALL-2 cells were positive for EBNA and EB virus DNA. Gene analysis of the BALL-2 cell line showed biallelic rearrangements in the JH locus. One of the JH rearrangement comigrated with a rearranged c-myc gene, indicating the translocation had occurred between JH and c-myc loci. The t(8;14) abnormality is a known chromosome marker of Burkitt lymphoma and L3 type ALL. Our studies revealed that this translocation and myc gene rearrangement can also be found in L2 type B-ALL.