Experimental Manipulation of Calpain Activity In Vitro.

The calpain activity in cells can be experimentally manipulated in vitro by calpain inhibitors, and various types of calpain inhibitors such as peptide aldehydes and α-mercapto-acrylic acid derivatives are widely used as a valuable tool to elucidate the physiological and pathological roles of calpain. Here I describe the experimental procedures with calpain inhibitors, with human neutrophils being primarily used in this experiment. It should be noted that potent calpain inhibitors not only inhibit the calpain activity but also stimulate cell functions via direct activation of human formyl peptide receptors and/or other G protein-coupled receptors depending on the inhibitors used.

Platelet-Lymphocyte Ratio as a Useful Predictor of the Therapeutic Effect of Neoadjuvant Chemotherapy in Breast Cancer.

Background: The peripheral blood platelet-lymphocyte ratio (PLR) has been proposed as an indicator for evaluating systemic inflammatory responses in cancer-bearing patients. While some reports suggest a correlation between PLR and prognosis, few studies have examined the relationship between PLR and sensitivity to chemotherapy. We conducted a study on whether PLR could serve as a predictor of the therapeutic effects of neoadjuvant chemotherapy (NAC).

Clinical verification of sensitivity to preoperative chemotherapy in cases of androgen receptor-expressing positive breast cancer.

Background: Triple-negative breast cancer (TNBC) patients testing positive for androgen receptor (AR) expression are thought to be chemotherapy resistant, similar to other hormone receptor-positive breast cancers; however, this has not been substantially validated in the clinic. In this study, we investigated the association between chemotherapy sensitivity and AR expression in patients treated with neoadjuvant chemotherapy (NAC) using standardised chemotherapy criteria and regimens.

Methods: A total of 177 patients with resectable early-stage breast cancer were treated with NAC.

Predictive Value of Neutrophil/Lymphocyte Ratio for Efficacy of Preoperative Chemotherapy in Triple-Negative Breast Cancer.

Background: The neutrophil/lymphocyte ratio (NLR) has been reportedly associated with prognosis in cancer patients by influencing both cancer progression and chemosensitivity. However, the correlation between NLR and the outcome of neoadjuvant chemotherapy (NAC) in breast cancer patients remains unclear.

Methods: NLR was evaluated in 177 patients with breast cancer treated with NAC with 5-fluorouracil, epirubicin, and cyclophosphamide, followed by weekly paclitaxel and subsequent curative surgery.

Stress-Induced Glucocorticoid Release Upregulates Uncoupling Protein-2 Expression and Enhances Resistance to Endotoxin-Induced Lethality.

Objective: Although psychological and/or physiological stress has been well documented to influence immune responses, the precise mechanism for immunomodulation remains to be elucidated. The present work describes the role of the hypothalamic-pituitary-adrenal (HPA) axis in the mechanism of stress-mediated enhanced-resistance to lethality after lipopolysaccharide (LPS) injection.

Methods/results: Preconditioning with restraint stress (RS) resulted in enhanced activation of the HPA axis in response to LPS injection and suppressed LPS-induced release of proinflammatory cytokines and nitric oxide metabolites.

Nonfunctional adrenocortical carcinoma initially presenting as retroperitoneal hemorrhage.

Background: Acute adrenal hemorrhage is an uncommon entity. Although trauma is the most common cause of adrenal hemorrhage, non-traumatic etiologies have also been reported. We report an unusual case of a spontaneously ruptured adrenocortical carcinoma that initially presented as a critical massive retroperitoneal hemorrhage.

Ambulatory sentinel lymph node biopsy preceding neoadjuvant therapy in patients with operable breast cancer: a preliminary study.

Background: Sentinel lymph node biopsy (SNB)-oriented stepwise treatment under local anesthesia has been performed in the outpatient-ambulatory setting in patients receiving neoadjuvant therapy (NAT). We retrospectively reviewed our preliminary experience of ambulatory SNB in breast cancer patients scheduled to undergo NAT to evaluate the usefulness and feasibility of this method as a minimally invasive, stepwise treatment protocol.

Methods: We retrospectively identified 56 patients with breast cancer without obvious nodal involvement who were scheduled to receive NAT before breast surgery.

Role of adrenocorticotropic hormone in the modulation of pollinosis induced by pollen antigens.

Background: A mild restraint stressor suppressed an increase in the levels of Th2-dependent cytokines and IgE, thereby reducing the symptoms of pollinosis. In the present study, to clarify the mechanism of action of adrenocorticotropic hormone (ACTH) in improving the symptoms of pollinosis, we studied the effects of ACTH on the plasma level of histamine, mast cell number in nasal-associated lymphoid tissue (NALT) and the T cell differentiation in splenocytes.

Methods: The role of ACTH in the development of pollen antigen-induced pollinosis was studied in mice.

Isolation of diploid baker's yeast capable of strongly activating immune cells and analyses of the cell wall structure.

Diploid baker's yeast capable of strongly activating a mouse macrophage was constructed based on haploid mutant AQ-37 obtained previously. The obtained strain BQ-55 activated also human immune cells. To clarify a factor for the activation, the cell wall structure, especially the β-glucan structure, was analyzed, suggesting that the length of branching, β-1,6-glucan, may be one of the factors.

Clinical significance of the sub-classification of 71 cases mucinous breast carcinoma.

Objective: Mucinous breast carcinoma (MBC) is classified into mixed mucinous breast carcinoma (MMBC) and pure mucinous breast carcinoma (PMBC) based on whether the tumor is with or without a component of invasive ductal carcinoma, respectively. PMBC is subtyped into hypocellular PMBC (PMBC-A) and hypercellular PMBC (PMBC-B).

Methods: Of 1,760 primary breast carcinomas, 71 were diagnosed as MBC, and were subtyped for comparison purposes.

Gender difference in tumor necrosis factor-α production in human neutrophils stimulated by lipopolysaccharide and interferon-γ.

The gender difference in tumor necrosis factor-α (TNF-α) production in human neutrophils stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) was explored by using peripheral blood neutrophils from young men and women. As compared with female neutrophils, male neutrophils released greater amounts of TNF-α, and exhibited stronger activation of mitogen-activated protein kinases and phosphatidylinositol 3-kinase in response to LPS stimulation. LPS-induced TNF-α production was markedly enhanced by pretreatment of cells with IFN-γ, and IFN-γ-mediated priming in male neutrophils was significantly greater than that in female neutrophils.

Adjunctive imprint cytology of core needle biopsy specimens improved diagnostic accuracy for breast cancer.

Objective: Recently, therapies targeting the biological characteristics of individual cancers according to markers indicating underlying molecular biological mechanisms have become available. Core needle biopsy (CNB) is widely used, not only to diagnose, but also to determine therapeutic strategies, in patients with breast cancer. Although the diagnostic accuracy of CNB is acceptably high, false-negative results have occasionally been encountered.

Heterogeneous nuclear ribonucleoprotein Q is a novel substrate of SH2 domain-containing phosphatase-2.

SH2 domain-containing phosphatase-2 (SHP2) is a protein-tyrosine phosphatase implicated in activation of cell signalling such as the Ras/extracellular signal-regulated kinase pathway. The substrates of SHP2 and their roles in cell activation are not fully understood. By using the substrate-trapping method with the phosphatase-dead SHP2 mutant, in which C459 was substituted by serine, and the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis, we found that heterogeneous nuclear ribonucleoprotein Q (hnRNP Q), a protein implicated in RNA metabolisms, was a novel substrate of SHP2.

S1-1/RBM10: multiplicity and cooperativity of nuclear localisation domains.

Background Information: S1-1, also called RBM10, is an RNA-binding protein of 852 residues. An alteration of its activity causes TARP syndrome, a severe X-linked disorder with pre- or post-natal lethality in affected males. Its molecular function, although still largely unknown, has been suggested to be transcription and alternative splicing.

Stimulation of human formyl peptide receptors by calpain inhibitors: homology modeling of receptors and ligand docking simulation.

Calpain inhibitors, including peptide aldehydes (N-acetyl-Leu-Leu-Nle-CHO and N-acetyl-Leu-Leu-Met-CHO) and α-mercapto-acrylic acid derivatives (PD150606 and PD151746), have been shown to stimulate phagocyte functions via activation of human formyl peptide receptor (hFPR) and/or hFPR-like 1 (hFPRL1). Using the homology modeling of the receptors and the ligand docking simulation, here we show that these calpain inhibitors could bind to the putative N-formyl-Met-Leu-Phe (fMLF) binding site on hFPR and/or hFPRL1. The studies with HEK-293 cells stably expressing hFPR or hFPRL1 showed that the concentrations of calpain inhibitors required to induce an increase in cytoplasmic free Ca(2+) ([Ca(2+)](i)) was much higher (>100 folds) than those of fMLF and Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm).

Shared and distinct functions of the transcription factors IRF4 and IRF8 in myeloid cell development.

Interferon regulatory factor (IRF) 8 and IRF4 are structurally-related, hematopoietic cell-specific transcription factors that cooperatively regulate the differentiation of dendritic cells and B cells. Whilst in myeloid cells IRF8 is known to modulate growth and differentiation, the role of IRF4 is poorly understood. In this study, we show that IRF4 has activities similar to IRF8 in regulating myeloid cell development.

Calpain inhibitors stimulate phagocyte functions via activation of human formyl peptide receptors.

Calpain inhibitors induce pertussis toxin (PTx)-sensitive chemotaxis in human neutrophils and monocytes. Here, we show that various calpain inhibitors (PD150606, PD151746, N-acetyl-Leu-Leu-Nle-CHO [ALLN], N-acetyl-Leu-Leu-Met-CHO [ALLM], and calpeptin) and γ-secretase inhibitor I induced PTx-sensitive increase in cytoplasmic free Ca(2+) ([Ca(2+)](i)) in human neutrophils and neutrophil migration. HEK-293 cells stably expressing human formyl peptide receptor (hFPR) or hFPR-like 1 (hFPRL1) displayed stimulus-specific increase in [Ca(2+)](i) in response to calpain inhibitors (PD150606, PD151746, ALLN, ALLM, MG-132, and calpeptin), γ-secretase inhibitor I, and N-formyl-Met-Leu-Phe.

Granulocyte colony-stimulating factor negatively regulates Toll-like receptor agonist-induced cytokine production in human neutrophils.

We studied the effect of G-CSF on TLR agonist-induced cytokine production in human neutrophils. Human neutrophils produced IL-8 and TNF-alpha in response to stimulation with TLR agonists such as LPS and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine. This response was dependent on activation of ERK, p38, and PI3K, but not JNK.

Granule swelling and cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis.

Extracellular signal-regulated kinase and p38 have been shown to be cleaved in human neutrophils undergoing apoptosis induced by tumor necrosis factor-alpha and cycloheximide. However, the cleavage products of these molecules were undetected when apoptotic neutrophils were pretreated with phenylmethylsulfonyl fluoride or disrupted by nitrogen cavitation before preparation of cell lysates. The electron microscopy revealed that granules in apoptotic neutrophils were significantly swollen than those in control cells.

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.

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.

Expression and tyrosine phosphorylation of Crk-associated substrate lymphocyte type (Cas-L) protein in human neutrophils.

Crk-associated substrate lymphocyte type (Cas-L) protein, also known as human enhancer of filamentation 1 (Hef1) or neural precursor cell-expressed, developmentally down-regulated gene 9 (Nedd9), belongs to the Cas family of adapter proteins, which are involved in integrin signaling. Previous reports showed that Cas-L is expressed preferentially in lymphocytes and epithelial cells. Cas-L mediates signals from integrins, T-cell receptors, B-cells receptors, and transforming growth factor beta, leading to cell movement and cell division.

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.

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.