Antimicrobial Resistance and Type III Secretion System Virulotypes of Pseudomonas aeruginosa Isolates from Dogs and Cats in Primary Veterinary Hospitals in Japan: Identification of the International High-Risk Clone Sequence Type 235.

This study aimed to investigate the current trends in antimicrobial resistance among Pseudomonas aeruginosa clinical isolates of canine and feline origin and the prevalence of their sequence types (STs) and type III secretion system (T3SS) virulotypes, which remains unknown in Japan. A total of 240 nonduplicate clinical isolates of P. aeruginosa from dogs (n = 206) and cats (n = 34) collected from 152 primary care animal hospitals between August 2017 and October 2019 were examined. PCR detection of T3SS genes (exoU and exoS) and carbapenemase genes, multilocus sequence typing, and whole-genome sequencing of the representative carbapenem-resistant isolates were performed. Resistance rates to imipenem and meropenem were 6.67% and 2.08%, respectively. A high resistance rate (17.92%) was encountered with ciprofloxacin. The exoU-/exoS+ was the predominant T3SS virulotype (195 isolates, 81.3%), followed by exoU+/exoS- (35 isolates, 14.6%), exoU-/exoS- (7 isolates, 2.9%), and exoU+/exoS+ (3 isolates, 1.3%). A high frequency of the high-risk clones ST235 and clonal complex 235 (CC 235) (28.9%), followed by ST357 (21.1%), were noted among these 38 exoU+ isolates. Seventeen carbapenem-resistant isolates comprising 2 exoU+ isolates, including an ST235 isolate, and 15 exoU-/exoS+ isolates belonging to non-ST235/CC235 were detected, of which all were carbapenemase negative. Different combinations of mutations among oprD, efflux pump regulatory genes, and AmpC ß-lactamase regulatory genes were identified among representative isolates with high-level resistance to imipenem. This study emphasizes the occurrence of ST235 isolates among companion animals, which may represent a threat to public health because of the ability of this clone to acquire and spread resistance elements, including carbapenemase genes. IMPORTANCE Pseudomonas aeruginosa is an environmentally ubiquitous and important opportunistic human pathogen responsible for life-threatening health care-associated infections. Because of its extensive repertoire of virulence determinants and intrinsic and acquired resistance mechanisms, the organism could be one of the most clinically and epidemiologically important causes of morbidity and mortality. In recent years, worldwide spreading of multidrug-resistant high-risk clones, particularly sequence type 235 (ST235), has become a serious public health threat. Companion animals which share much of their living environment with humans could be important reservoirs and spreaders of antimicrobial-resistant bacteria and resistance genes of clinical importance in humans, such as extended-spectrum ß-lactamase-producing Enterobacterales and methicillin-resistant Staphylococcus aureus. However, antimicrobial resistance, virulence, and genotyping of P. aeruginosa in companion animals remain largely unknown. This work sheds light on the potential spread of high-risk clones in companion animals.

Tumor necrosis factor-α-induced apoptosis of gastric cancer MKN28 cells: accelerated degradation of the inhibitor of apoptosis family members.

The role of the inhibitor of apoptosis (IAP) family members in tumor necrosis factor-α (TNF-α)-induced apoptosis of human gastric cancer MKN28 cells was explored. TNF-α induced up-regulation of cIAP2, whereas cycloheximide (CHX) induced down-regulation of XIAP and survivin. Degradation of cIAP1 and XIAP, but not survivin, was accelerated by co-treatment of cells with TNF-α and CHX, and TNF-α-induced up-regulation of cIAP2 was inhibited by BMS-345541 (NF-κB inhibitor). Treatment of MKN28 cells with TNF-α plus CHX induced degradation of survivin and activation of caspase-8 and -3, followed by degradation of cIAP1 and XIAP and apoptosis. Proteasome inhibitors (MG132 and epoxomicin) suppressed TNF-α plus CHX-induced degradation of survivin, cIAP1, and XIAP as well as apoptosis. A caspase inhibitor (z-VAD-fmk) suppressed TNF-α plus CHX-induced apoptosis, but allowed degradation of survivin, cIAP1 and XIAP. TNF-α receptor 1 and 2 were expressed on MKN28 cells. The magnitude of apoptosis induced by TNF-α plus BMS-345541 was much less than that induced by TNF-α plus CHX. These findings suggest that TNF-α plus CHX-induced apoptosis of gastric cancer MKN28 cells may be caused by accelerated degradation of the IAP family members (survivin, cIAP1, and XIAP), in addition to inhibition of NF-κB-dependent synthesis of anti-apoptotic molecules.

Efficacy of a hypotonic treatment for peritoneal dissemination from gastric cancer cells: an in vivo evaluation.

The aim of the present study was to determine the efficacy of a hypotonic treatment for peritoneal dissemination from gastric cancer cells using an in vivo model. We firstly evaluated the toxicity of a peritoneal injection of distilled water (DW) (2 mL for 3 days) in mice. Macroscopic and microscopic examinations revealed that the peritoneal injection of DW did not severely damage the abdominal organs of these mice. MKN45 gastric cancer cells preincubated with NaCl buffer or DW for 20 minutes in vitro were then intraperitoneally injected into nude mice, and the development of dissemination nodules was analyzed. The total number, weight, and volume of the dissemination nodules were significantly decreased by the DW preincubation. We then determined whether the peritoneal injection of DW inhibited the establishment of peritoneal dissemination. After a peritoneal injection of MKN45 cells into nude mice, NaCl buffer or DW was injected into the abdominal cavity for 3 days. The total volume of dissemination nodules was significantly lower in DW-injected mice than in NaCl-injected mice. In conclusion, we demonstrated the safeness of a peritoneal injection of DW. Furthermore, the development of dissemination nodules from gastric cancer cells was prevented by a preincubation with or peritoneal injection of DW.

[Pulmonary embolism in esophageal cancer patients receiving neoadjuvant chemotherapy with CDDP and 5-FU].

We report our experience with 2 male patients (71 and 74 years old, respectively) with advanced esophageal cancer who exhibited pulmonary embolism during treatment with neoadjuvant chemotherapy with cisplatin (CDDP) and 5-fluorouracil (5- FU). The patients had no symptoms associated with pulmonary embolism. The pulmonary emboli were incidentally detected on computed tomography performed to assess the effect of chemotherapy. It was found that the pulmonary emboli were associated with the central venous catheter-associated thrombi. The pulmonary emboli were effectively treated with heparin in both patients. A recent meta-analysis showed that the risk of chemotherapy-associated venous thromboembolism was increased by 1.67-fold when CDDP was included in the chemotherapy regimen. The increased risk was proposed to be associated with CDDP-mediated endothelial cell injury, platelet activation, increased levels of coagulation factors, or renal damage. The pulmonary embolism in our cases may be ascribed to the central venous catheterization and the treatment with CDDP. A possible occurrence of venous thromboembolism should be kept in mind when CDDP is used, particularly with central venous catheterization, for the treatment of esophageal cancer.

A regulatory role of K(+)-Cl(-) cotransporter in the cell cycle progression of breast cancer MDA-MB-231 cells.

K(+)-Cl(-) cotransporter (KCC) has been shown to be involved in cell proliferation as well as cell volume regulation. A regulatory role of KCC in cell cycle progression of breast cancer MDA-MB-231 cells was explored by using synchronized MDA-MB-231 cells and dihydro-indenyloxy-alkanoic acid (DIOA), a potent inhibitor of KCC. MDA-MB-231 cells cultured in the presence of DIOA exhibited an increase in cell volume, a decrease in intracellular Cl(-) concentration, and reduction in cell proliferation with the G0/G1 phase arrest, which was accompanied with down-regulation of cyclin D1 and cyclin E2, and up-regulation of p21. Among these molecules, the expression of cyclin E2, a molecule essential for the transition from G1 to S phase, was markedly suppressed by DIOA treatment. DIOA-mediated up- or down-regulation of these molecules occurred at the transcriptional level. These findings suggest that KCC plays an important role in the early phase of cell cycle progression by regulating the expression of cyclin D1, cyclin E2, and p21, the molecules essential for the cell cycle progression.

XB130 as an independent prognostic factor in human esophageal squamous cell carcinoma.

BACKGROUND: Adaptor proteins, with multimodular structures, can participate in the regulation of various cellular functions. A novel adaptor protein XB130 has been implicated as a substrate and regulator of tyrosine kinase-mediated signaling and in controlling cell proliferation and apoptosis in thyroid and lung cancer cells. However, its expression and role in gastrointestinal cancer have not been investigated. We sought to determine the role of XB130 in cell cycle progression of esophageal squamous cell carcinoma (ESCC) cells and to examine its expression and effects on the prognosis of patients with ESCC. METHODS: Expression of XB130 in human ESCC cell lines was analyzed by Western blot testing and immunofluorescent staining. Knockdown experiments with XB130 small interfering RNA (siRNA) were conducted, and the effect on cell cycle progression was analyzed. Immunohistochemistry of XB130 for 52 primary tumor samples obtained from patients with ESCC undergoing esophagectomy was performed. RESULTS: XB130 was highly expressed in TE2, TE5, and TE9 cells. In these cells, knockdown of XB130 with siRNA inhibited G1-S phase progression and increased the expression of p21, the cyclin-dependent kinase inhibitor. Immunohistochemistry showed that 71.2% of the patients expressed XB130 in the nuclei and/or cytoplasm of the ESCC cells. Further, nuclear expression of XB130 was an independent prognostic factor of postoperative survival. CONCLUSIONS: These observations suggest that the expression of XB130 in ESCC cells may affect cell cycle progression and impact prognosis of patients with ESCC. A deeper understanding of XB130 as a mediator and/or biomarker in ESCC is needed.

Evaluation of lymph node metastasis in patients with gastric cancer: a comparison of the directionality of lymph node metastasis and the total number of metastatic lymph nodes.

PURPOSE: The clinical impact of the directionality of lymph node (LN) metastasis was assessed in comparison with the staging by the Japanese Classification of Gastric Carcinoma (JCGC), a numerical LN staging system. METHODS: Two hundred forty-one gastric cancer patients who were diagnosed pathologically to have LN metastasis, and 54 patients who underwent preoperative multidetector-row computed tomography (MDCT) with an image thickness of 1 mm were classified into three groups (unidirectional [Uni-], bidirectional [Bi-], and tridirectional [Tri-] groups) depending on the directionality of their LN metastasis. RESULTS: The prognosis of the Uni-group was better than that of the Bi- or the Tri-group when assessed on the basis of the pathological findings of metastatic LN and also the preoperative MDCT findings. The exact preoperative evaluation was 70.2 % for the directionality system and 61.7 % for the JCGC system, respectively. The stages were less frequently underestimated by the directionality system than the JCGC system (P < 0.02, 19.1 vs. 34.0 %), and the staging could be more precisely performed by both systems in combination. CONCLUSIONS: More precise preoperative evaluation of disease stage could be obtained by the directionality system and the JCGC system in combination.

Total pancreatectomy for pancreatic carcinoma: evaluation of safety and efficacy.

BACKGROUND/AIMS: To determine the safety and the efficacy of total pancreatectomy for the curative treatment of pancreatic carcinoma. METHODOLOGY: Retrospective analysis was performed using 10 patients receiving total pancreatectomy. RESULTS: The median duration of the operative procedure was 8.7 hours and the median estimated blood loss was 2,700mL. Seven patients developed postoperative complications, including infections in 5 cases. There was no death associated with the operative procedure itself. Median period of postoperative hospital stay was 55 days. Anastomotic ulcer was prevented by administration of proton- pump inhibitors. Blood glucose level was well controlled by subcutaneous injection of sliding scale insulin during the postoperative period and the dosage of insulin required was 0.45±0.13units/kg body weight/ day at the time of discharge. The mean HbA1c level at 3 months after the operation was 6.1%. Four patients needed medication with anti-diarrheal drugs. CONCLUSIONS: Total pancreatectomy could be performed safely and postoperative daily performance was reasonable with effective medication. We suggest that total pancreatectomy should be considered for the treatment of pancreatic carcinoma when the patient status is appropriate for this procedure.

The directionality of lymphatic spreading as a prognostic factor in gastric cancer: uni- or multidirectional lymphatic metastases.

BACKGROUND/AIMS: The most critical problem of the numerical lymph node staging system is difficulty in evaluating preoperative staging. We investigated the directionality of metastatic lymph nodes as an alternative prognostic indicator of gastric cancer. METHODOLOGY: One hundred and seventy two patients who had undergone curative gastrectomy were examined. We divided the stations of lymph nodes into three zones according to the anatomical site. Then patients were classified into two groups according to the number of positive zones, unidirectional or multidirectional lymphatic spreading. RESULTS: Seventy-five patients showed unidirectional lymphatic spreading, whereas 97 patients showed multidirectional. Logistic regression analysis showed that directionality correlated with the number of metastatic nodes. Directionality was found to be a significant prognostic factor on univariate analysis, but not on multivariate analysis. CONCLUSIONS: The directionality could be a surrogate marker of total number of metastatic lymph nodes as a prognostic factor in patients with gastric cancer.

Gastrointestinal Stromal Tumor in a Patient with Neurofibromatosis: Abscess Formation in the Tumor Leading to Bacteremia and Seizure.

A 66-year-old woman with neurofibromatosis type 1 (NF1) was brought to the emergency room with seizures and high-grade fever. Seizure in adult NF1 patients raises concern for intracranial lesions. However, neurological examination and central nervous system imaging failed to detect any causative intracranial lesions for her seizure. Gram-positive cocci, Streptococcus anginosus, were detected by blood cultures. Abdominal computed tomography revealed a well-defined round mass 7 cm in diameter, which was found to be a small intestinal gastrointestinal stromal tumor (GIST) containing an abscess. There was fistula formation between the intestinal lumen and the abscess, in which there were numerous Gram-positive cocci. The seizure may have been caused by hypoosmolality (hyponatremia and hypoproteinemia), which may result from decreased food intake associated with high-grade fever and general malaise. In this case GIST originating from the small intestine was invaded by S. anginosus through a fistula, leading to abscess formation, bacteremia, high-grade fever, and seizure, which was the first clinical manifestation.

[The role of adjuvant chemotherapy with S-1 for gastric cancer patients].

A randomized controlled trial of adjuvant chemotherapy with S-1 for gastric cancer (ACTS-GC) demonstrated that the chemotherapy with S-1 improved the relapse-free survival and overall survival rates of patients with locally advanced gastric cancer. We examined retrospectively 47 patients with locally advanced gastric cancer, who received adjuvant chemotherapy with S-1 after curative gastrectomy. Patients who received more than 50% of the total scheduled dose of S-1 during the first 1 year after starting the chemotherapy showed a tendency to favorable outcome in terms of relapse-free survival compared with patients who received less than 50% of the dose, although no statistical significance was detected. Age did not show a significant correlation with tolerability. The number of elderly people with gastric cancer will increase in the next decade. They should be candidates for adjuvant chemotherapy on the basis of a geriatric assessment.

[A case of S-1-resistant gastric cancer with multiple bone metastases and disseminated intravascular coagulation--effective treatment by combined chemotherapy with CDDP and CPT-11].

A 49-year-old female with advanced gastric cancer complicated with peritoneal dissemination underwent distal gastrectomy, and thereafter she was treated with a combined chemotherapy of S-1 and paclitaxel for 5 months, followed by treatment with S-1 alone. A year after the gastrectomy, she developed disseminated intravascular coagulation (DIC) with multiple bone metastases despite the continuous treatment with S-1, indicating that S-1 was no longer effective. She was then effectively treated by a combined chemotherapy with cisplatin(CDDP)and irinotecan hydrochloride (CPT-11), and DIC subsided within 7 days after the treatment. These findings suggest that combined chemotherapy with CDDP and CPT-11 is a useful regimen for the treatment of certain patients with DIC associated with S-1-resistant advanced gastric cancer.

Appendiceal ascariasis in children.

BACKGROUND: The propensity of Ascaris lumbricoides to wander leads to varied surgical complications in the abdomen. Wandering A lumbricoides may sometimes reach the vermiform appendix and its presence there may remain silent or incite pathology. Our aim was to study ascariadial appendicitis. METHODS: Over a period of 3 years, we identified children who were found to have appendiceal ascariasis during surgery for different intestinal complications due to ascariasis. We studied the relationship between ascariasis and its lodgement inside the vermiform appendix in these patients. No preoperative diagnosis was made in this series. RESULTS: We found 11 patients with appendiceal ascariasis. It was incidentally found that 8/11 (72.7%) patients had worms inside their vermiform appendix but not appendicitis, whereas the remaining three patients (27.2%) were found to have Ascaris-associated appendicitis. The characteristic finding in Ascaris-infested vermiform appendix was that the worm is positioned with its head at the base and its tail at the tip of the appendix. CONCLUSION: Migration of A lumbrocoides inside the vermiform appendix is an incidental finding and tends to pursue a silent course in most patients. Only rarely does the presence of Ascaris inside the vermiform appendix cause appendicitis.

Calpain inhibition induces activation of the distinct signalling pathways and cell migration in human monocytes.

We have recently reported that constitutively active calpain negatively regulates activation of the distinct signalling pathways and cell migration in human neutrophils. Here, we report that a similar regulatory system is also functioning in human monocytes, but not lymphocytes. Calpain was constitutively active in resting human monocytes, but not lymphocytes. Mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI3K)/Akt and p21-activated kinase (PAK, an effector molecule of Rac) were rapidly (within 1 min) activated in monocytes, but not lymphocytes, upon exposure to calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO), but not PD145305 (the inactive analogue of PD150606). Following activation of these signalling pathways, monocytes displayed active migration within 5 min after exposure to calpain inhibitors, and active migration was sustained for more than 45 min. The micropipette method revealed that calpain inhibition-mediated monocyte migration was chemotaxis, not random migration. The studies with pharmacological inhibitors suggest that calpain inhibition-mediated monocyte migration is mediated by activation of ERK, p38, JNK, PI3K/Akt and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor-induced phosphorylation of distinct signalling molecules (PAK, ERK, p38, JNK and Akt) as well as cell migration, suggesting that the PTX-sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signalling pathways and cell migration in resting monocytes, but not lymphocytes.

Noncontrast and contrast enhanced computed tomography for diagnosing acute appendicitis: A retrospective study for the usefulness.

Abdominal computed tomography (CT) provides great benefits for the differential diagnosis in patients complaining of acute abdominal pain. However, the use of diagnostic X-rays is associated with the cumulative risk of cancer development. In order to determine the relative usefulness of noncontrast and enhanced CT with intravenous contrast material for diagnosing acute appendicitis, the retrospective analysis was performed using 247 patients (46 children and 201 adults) with clinically suspected appendicitis, who were admitted to our hospital from 2002 to 2006 and underwent noncontrast or combined noncontrast and enhanced CT examination. Of 185 patients who were diagnosed to have acute appendicitis with appendiceal thickening (167 cases) or normal-sized appendix (18 cases), 73 cases underwent noncontrast CT alone and these 73 cases could be retrospectively diagnosed to have appendicitis on noncontrast CT. On the other hand, 112 cases of these 185 patients underwent noncontrast CT followed by enhanced CT, and vermiform appendix was detected in 86 cases of them (86/112, 76.8%) on noncontrast CT. These 86 cases could be retrospectively diagnosed to have acute appendicitis on noncontrast CT, whereas enhanced CT was required to detect vermiform appendix and to obtain the final diagnosis of appendicitis in the remaining 26 cases (26/112, 23.2%). Enhanced CT was superior to noncontrast CT in diagnosing appendicitis in all age and any gender groups. We suggest that enhanced, but not noncontrast, CT should be primarily performed for diagnosing acute appendicitis in all patients to minimize the radiation exposure unless intravenous administration of contrast material is contraindicated.

Calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of protein kinase A and protein kinase A-mediated stabilization of Mcl-1 and X-linked inhibitor of apoptosis (XIAP).

Human neutrophils underwent spontaneous apoptosis, which was accompanied with proteasome-mediated degradation of Mcl-1 and X-linked inhibitor of apoptosis (XIAP). Calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO) prevented spontaneous neutrophil apoptosis and degradation of Mcl-1 and XIAP, and the effects of calpain inhibitors on neutrophils were resistant to cycloheximide. Calpain inhibitors induced protein kinase A (PKA) activation, which was unaccompanied with an increase in intracellular cyclic AMP. Calpain inhibition-mediated delayed neutrophil apoptosis, stabilization of Mcl-1 and XIAP, and phosphorylation of PKA substrates were suppressed by H-89 (specific PKA inhibitor). These findings suggest that calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of PKA and PKA-mediated stabilization of Mcl-1 and XIAP.

Torsion of vermiform appendix with fecalith: a case report.

INTRODUCTION: Torsion of the vermiform appendix is a rare disorder, which causes abdominal symptoms indistinguishable from acute appendicitis and is found by chance during the laparotomy. CASE PRESENTATION: We report a case (a 76-year-old male) suffering of torsion of the vermiform appendix with fecalith. It was twisted 540 degrees in an anti-clockwise direction. Appendectomy was done. CONCLUSION: Appendiceal torsion may be assocated with the presence of fecalith. This case is the oldest one among the patients with appendiceal torsion reported to literature.

Distinct role of c-Jun N-terminal kinase isoforms in human neutrophil apoptosis regulated by tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor.

We studied the role of c-Jun N-terminal kinase (JNK) in human neutrophils stimulated by tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Stimulation of neutrophils with TNF-alpha and GM-CSF caused phosphorylation of p54 or p46 JNK or both. The phosphorylated p46 JNK band in TNF-alpha-stimulated neutrophils mobilized faster than that in GM-CSF-stimulated cells. The JNK isoform transcripts expressed in neutrophils were JNK1beta1, JNK1beta2, JNK2alpha1, and JNK2alpha2. The JNK isoforms phosphorylated by TNF-alpha and GM-CSF stimulation were found to be JNK1 and JNK2, respectively, on the basis of the molecular mass and the capture assay. TNF-alpha-induced JNK phosphorylation was sustained in the presence of cycloheximide, which was accompanied by accelerated neutrophil apoptosis. The JNK inhibitors (SP600125 and TAT-TI-JIP(153163)) suppressed neutrophil apoptosis induced by TNF-alpha plus cycloheximide, whereas they attenuated the GM-CSF-mediated antiapoptotic effect on neutrophils. The JNK inhibitor did not affect the levels of Mcl-1 and XIAP (antiapoptotic molecules), which were regulated by TNF-alpha plus cycloheximide and GM-CSF. The JNK inhibitor markedly suppressed TNF-alpha-induced and GM-CSF-induced superoxide release. These findings suggest that JNK1 and JNK2 are involved in TNF-alpha-induced neutrophil apoptosis and GM-CSF-mediated antiapoptotic effect on neutrophils, respectively, and both JNK isoforms are involved in TNF-alpha-induced and GM-CSF-induced superoxide release.

Calpain-mediated regulation of the distinct signaling pathways and cell migration in human neutrophils.

We studied the mechanisms underlying calpain inhibition-mediated human neutrophil migration. MAPKs, including ERK, p38, and JNK, MEK1/2, MAPK kinase 3/6 (MKK3/6), PI-3K/Akt, c-Raf, and p21-activated kinase (PAK; an effector molecule of Rac) were rapidly (within 30 s) activated in neutrophils upon exposure to calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO) but not PD145305 (inactive analog of PD150606). Following activation of these pathways, neutrophils displayed active migration (chemotaxis), which was sustained for more than 45 min. The studies with pharmacological inhibitors suggest that calpain inhibition-mediated neutrophil migration is mediated by activation of MEK/ERK, p38, JNK, PI-3K/Akt, and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor-induced phosphorylation of distinct signaling molecules (PAK, c-Raf, MEK1/2, ERK, MKK3/6, p38, JNK, and Akt) as well as cell migration, suggesting that the PTX-sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. Differentiated neutrophil-like HL-60 cells but not undifferentiated cells displayed cell migration and activation of MAPKs and PI-3K/Akt on calpain inhibition. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signaling pathways and cell migration in resting neutrophils, and this regulatory system develops during differentiation into mature neutrophils.

Calcium channel blockers suppress cytokine-induced activation of human neutrophils.

BACKGROUND: Neutrophils, in concert with proinflammatory cytokines, play an important role in the progression of atherosclerosis. Calcium channel blockers are commonly used in the treatment of hypertension, and their pleiotropic effects, other than the lowering of blood pressure, have been recently recognized. METHODS: We studied the effects of various calcium channel blockers (amlodipine, nicardipine, cilnidipine, benidipine, efonidipine, nifedipine, azelnidipine, verapamil, and diltiazem; each being used at 5 and 10 micromol/l) on superoxide (O(2)(-)) release, migration, and signaling pathways in human neutrophils stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor-alpha (TNF-alpha). RESULTS: GM-CSF-induced O(2)(-) release was suppressed by amlodipine, nicardipine, and cilnidipine, whereas TNF-alpha-induced O(2)(-) release was suppressed by amlodipine, nicardipine, cilnidipine, benidipine, efonidipine, nifedipine, and azelnidipine. TNF-alpha-induced phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, but not p38 mitogen-activated protein kinase (MAPK), was attenuated by nicardipine, cilnidipine, benidipine, efonidipine, and azelnidipine. By contrast, GM-CSF-induced phosphorylation of ERK, p38, and Akt was affected by none of the blockers. GM-CSF-induced neutrophil migration was also suppressed by amlodipine and nicardipine, but not by azelnidipine, when these blockers were assessed for their effect on neutrophil migration. CONCLUSIONS: These findings suggest that (i) some calcium channel blockers can suppress cytokine-induced neutrophil activation, leading to possible prevention of the progression of atherosclerosis; and (ii) that activation of the ERK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways, induced by TNF-alpha but not by GM-CSF, is selectively affected by some blockers.