Junji Nishioka

Efficacy of procalcitonin in the early diagnosis of bacterial infections in a critical care unit.

Procalcitonin (PCT) is a marker of severe bacterial infections and organ failure due to sepsis. The purpose of the present study was to identify the appropriate cutoff level of PCT based on the findings of a blood culture and polymerase chain reaction (PCR). The PCT levels were measured in 116 patients in an intensive care unit who were suspected of having bacteremia, to examine its relationship with a blood culture or PCR. The PCT levels were significantly high in patients with bacteremia, but they were also moderately high in some patients who were positive for fungus DNA. The area under the curve was significantly higher for PCT than for C-reactive protein. The appropriate cutoff values of PCT for bacteremia were 0.38 &mgr;g/L for the high negative predictive value and 0.83 &mgr;g/L for the high positive predictive value. Procalcitonin was slightly related to mortality, and the combination of a blood culture and PCR was thus found to increase the sensitivity for mortality. These findings suggest that PCT is useful for the diagnosis of bacteremia and that the diagnostic value of PCT in combination a with blood culture and PCR for bacterial infection or mortality further increases.ABBREVIATIONS-PCT-procalcitonin; PCR-polymerase chain reaction; CRP-C-reactive protein; ROC-receiver operating characteristic; MRSA-methicillin-resistant Staphylococcus aureus

A novel role for Notch ligand Delta-1 as a regulator of human Langerhans cell development from blood monocytes.

Human Langerhans cells (LCs) are of hematopoietic origin, but cytokine regulation of their development is not fully understood. Notch ligand Delta‐1 is expressed in a proportion of the skin. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and transforming growth factor‐β1 (TGF‐β1) are also secreted in the skin. We report here that Delta‐1, in concert with GM‐CSF and TGF‐β1, induces the differentiation of human CD14+ blood monocytes into cells that express LC markers: CD1a, Langerin, cutaneous lymphocyte‐associated antigen, CC chemokine receptor 6, E‐cadherin, and Birbeck granules. The resulting cells display phagocytic activity and chemotaxis to macrophage inflammatory protein‐1α (MIP‐1α). In response to CD40 ligand and tumor necrosis factor α, the cells acquire a mature phenotype of dendritic cells that is characterized by up‐regulation of human leukocyte antigen (HLA)‐ABC, HLA‐DR, CD80, CD86, CD40, and CD54 and appearance of CD83. These cells in turn show chemotaxis toward MIP‐1β and elicit activation of CD8+ T cells and T helper cell type 1 polarization of CD4+ T cells. Thus, blood monocytes can give rise to LCs upon exposure to the skin cytokine environment consisting of Delta‐1, GM‐CSF, and TGF‐β1, which may be, in part, relevant to the development of human epidermal LCs. Our results extend the functional scope of Notch ligand δ‐1 in human hematopoiesis.

Mitotic Checkpoint Protein hsMAD2 as a Marker Predicting Liver Metastasis of Human Gastric Cancers

hsMAD2, the human homologue of mitotic arrest deficient 2 (MAD2), is a key component of the mitotic checkpoint system. Recently, mutations and decreased expression of mitotic checkpoint genes including hsMAD2 have been reported in cancer cell lines with defective mitotic checkpoint. However, the genetic alterations in the genomic hsMAD2 gene have not been determined in gastric cancers. Moreover, the biological implications of the overexpressed hsMAD2 in primary cancers are unknown. In this study, we analyzed 32 primary gastric cancers with polymerase chain reaction (PCR) amplification of all exons, including flanking intronic sequences, of the genomic hsMAD2 gene followed by direct DNA sequencing. We also measured the hsMAD2 protein levels in cancer and normal tissues by semi‐quantitative immunoblotting. No mutations were found in the coding sequences, although three single nucleotide polymorphisms (SNPs) were identified in the noncoding sequences in 13 of 32 patients. These SNPs were not associated with either hsMAD2 expression or disease progression. The semi‐quantitative western blot analysis showed hsMAD2 was significantly overexpressed in gastric cancer tissues compared with corresponding normal tissues (P<0.001). The calculated ratio of the hsMAD2 protein in cancer tissue (C) to that in corresponding normal tissue (N) (C/N ratio) was significantly higher in patients with well differentiated adenocarcinoma (P=0.0274) or with synchronous liver metastasis (P=0.0025). A C/N ratio greater than 3 was observed more frequently in patients with synchronous liver metastasis. Therefore, C/N ratio >3 may be clinically important as a predictive indicator for metachronous liver metastasis of gastric cancers.

Protein C activation in NIDDM patients

Summary Enhanced activation of the clotting system has been recently implicated in the pathogenesis of vascular complications in patients with diabetes mellitus. Abnormalities of the anticoagulant system may constitute a potential trigger factor for the haemostatic activation observed in diabetic subjects. The current study aimed to evaluate anticoagulant activity in diabetic patients by assessing the plasma levels of activated protein C-protein C inhibitor complex; and by measuring the anticoagulant response to exogenous thrombomodulin. This study comprised 61 patients (34 men, 27 women) with non-insulin-dependent diabetes mellitus (NIDDM) of whom 22 showed microalbuminuria and 39 normoalbuminuria. Data obtained in 31 non-obese and non-diabetic subjects were available for comparison. The plasma levels of fibrinogen (p < 0.02), prothrombin fragment 1 + 2 (p < 0.05), fibrin monomer (p < 0.0001), protein C antigen (p < 0.005), total protein S antigen (p < 0.02), soluble thrombomodulin (p < 0.005) and soluble E-selectin (p < 0.005) were significantly higher in diabetic patients than in healthy subjects. The plasma level of activated protein C-protein C inhibitor complex (7.4 ± 3.8 vs 3.0 ± 0.4 pmol/l) was significantly higher (p < 0.0001) and the anticoagulant response to exogenous thrombomodulin (23.4 ± 2.6 vs 35.3 ± 3.0 ng/ml) was markedly lower (p = 0.005) in all diabetic patients than in healthy subjects. Cases with microalbuminuria presented low plasma levels of activated protein C-protein C inhibitor complex (5.5 ± 0.6 vs 8.6 ± 0.7 pmol/l, p < 0.05) and significantly decreased values of the anticoagulant response to exogenous thrombomodulin (16.5 ± 2.9 vs 23.4 ± 2.6 %, p = 0.03) as compared to those with normoalbuminuria. The present study suggests that the hyper-coagulable state in NIDDM is associated with an increased activation of protein C but with a poor plasma reactivity to the anticoagulant effect of thrombomodulin. [Diabetologia (1996) 39: 1455–1461]

Protein C inhibitor secreted from activated platelets efficiently inhibits activated protein C on phosphatidylethanolamine of platelet membrane and microvesicles.

Protein C inhibitor (PCI) was detected in human platelets (2.9 ng/109 cells) and megakaryocytic cells (1.5 ng/106 cells). PCI mRNA was also detected in both platelets and megakaryocytic cells using nested polymerase chain reaction. PCI was found to be located in the α-granules of resting platelets. Approximately 30% of the total amount of PCI in platelets was released after stimulation with ADP, collagen, adrenalin, thrombin, or thrombin receptor-activating peptide. Secreted PCI was detected on the surface of activated platelets and phospholipid microvesicles. PCI secreted from thrombin receptor-activating peptide-stimulated platelets inhibited activated protein C (APC) efficiently. PCI significantly inhibited APC in the presence of phospholipid vesicles prepared using rabbit brain cephalin (RBC) or a mixture of 40% phosphatidylethanolamine (PE), 20% phosphatidylserine (PS), and 40% phosphatidylcholine (PC) with a second order rate constant of 1.0 × 106 m −1·min−1. Of these phospholipids, PE was critical for this inhibition. The dissociation constants of the binding of APC or PCI to solid phase phospholipids showed that APC binds more preferably to PE than to RBC or PS, and PCI to PE or RBC than to PS or PC. PCI binding to solid phase phospholipids depended on the presence of PE. RBC- or PE-bound PCI inhibited APC significantly but only weakly the γ-carboxyglutamic acid domainless APC. The γ-carboxyglutamic acid fragment of protein C suppressed the PCI-mediated inhibition of APC on solid phase RBC or PE. Most of the APC·PCI complex formed on solid phase RBC or PE was released into the soluble phase. These findings suggest that PCI secreted from activated platelets binds preferably to PE of platelet membrane and microvesicles and that it inhibits phospholipid-bound APC efficiently.

Regulation of carcinoma cell invasion by protein C inhibitor whose expression is decreased in renal cell carcinoma

Protein C inhibitor (PCI), a member of the serine protease inhibitor family, is produced in various human tissues, including the liver, kidney and testis. In addition to inhibiting the anticoagulant protein C pathway, PCI also inhibits urinary plasminogen activator (uPA), which is a well‐known mediator of tumor cell invasion. In the present study, to clarify the biologic significance of PCI in the kidney, we compared the expression of PCI between human renal cell carcinoma (RCC) tissue and nontumor kidney tissue. The PCI antigen level in RCC tissue was found to be significantly lower than in nontumor kidney tissue, and expression of PCI mRNA was detected in normal renal proximal tubular epithelial cells (RPTEC), but not in RCC or in an RCC cell line (Caki‐1 cells). No differences were detected between the nucleotide sequence of the major cis‐elements in the promoter region of the PCI gene from nontumor kidney and RCC tissues, RPTEC and Caki‐1 cells, an RPTEC‐derived RCC cell line. The in vitro invasiveness of Caki‐1 cells transfected with a PCI expression vector was significantly decreased compared to mock‐transfected Caki‐1 cells, and it was blocked in the presence of anti‐PCI antibody. Since PCI itself did not affect the proliferation rate of Caki‐1 cells or cell expression of uPA in vitro, the effect of uPA, PCI, heat‐inactivated PCI and plasminogen activator inhibitor (PAI)‐1 on the invasive potential of cultured RCC cells was evaluated. The in vitro invasiveness of Caki‐1 cells, which express uPA, was significantly enhanced by the addition of uPA, and it was inhibited by anti‐uPA antibody, PCI and PAI‐1, but not by heat‐inactivated PCI. In addition, uPA activity was significantly decreased and uPA‐PCI complex level was significantly increased in the culture medium of PCI expression vector‐transfected Caki‐1 cells as compared to mock‐transfected Caki‐1 cells. These findings strongly suggest that PCI regulates the invasive potential of RCC cells by inhibiting uPA secreted by these cells. The results of our study suggest that PCI might be a potential therapeutic agent for inhibiting renal tumor invasion.

Frequency of Abnormal Biphasic aPTT Clot Waveforms in Patients with Underlying Disorders Associated with Disseminated Intravascular Coagulation

Abnormal biphasic waveform (BPW) patterns were previously reported when the activated partial thromboplastin time (aPTT) was performed in plasma from patients with disseminated intravascular coagulation (DIC). In this study, the prevalence of the BPW was examined in a cohort of 508 hospitalized patients with elevated fibrinogen degradation products (FDP) levels (>10 μg/mL). The presence of a BPW was automatically flagged by the MDA® analyzer when the slope of the precoagulation phase in the waveform exceeded a threshold value of -0.25%T/sec. In our cohort, 76 patients (15%) were diagnosed with overt DIC according to the criteria recently proposed by the International Society of Thrombosis and Haemostasis (ISTH), whereas 96 patients (18.9%) were diagnosed with DIC following the criteria of the Japanese Ministry of Health and Welfare (JMHW). The JMHW and ISTH criteria agreed in 93% of cases (kappa coefficient 0.76). The concordance between both scoring systems was high in patients with infection but low in solid cancer. The BPW appeared in 65 patients (12.8%), with the highest prevalence (23.6%) in patients with infection. The BPW was more prevalent in the subgroup of patients with DIC: 59.2% and 47.9% for DIC diagnosed by ISTH and JMWH scores, respectively. The prevalence of the BPW was particularly high in patients with DIC and infection: 86.4% and 75.0% for DIC diagnosed by ISTH and JMWH scores, respectively. For the total cohort, the presence of the BPW was significantly associated with DIC. Odds ratios were 29.9 and 19.0 for ISTH and JMWH scores, respectively (p<0.0001). The BPW showed a moderate sensitivity (59.2% for the ISTH score; 47.9% for the JMWH score), but a high specificity (95.4% for both scores). Waveform analysis of the aPTT potentially provides a practical tool in risk assessment of critical care patients, in whom development of DIC is known to worsen the prognosis.

Plasma levels of activated protein C-protein C inhibitor complex in patients with hypercoagulable states

Plasma levels of activated protein C (APC)–protein C inhibitor (PCI) were significantly increased in patients with disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP), acute myocardial infarction (AMI), pulmonary embolism (PE), or deep vein thrombosis (DVT) and in patients undergoing hemodialysis (HD). Plasma levels of APC–α1‐antitrypsin (AT) complex were significantly increased in patients with DIC and in those with TTP. Plasma levels of PCI were significantly decreased in patients with DIC, non‐DIC, or TTP and in those undergoing HD. In the pre‐DIC stage, the plasma levels of APC–PCI complex were significantly increased but not those of APC–α1‐AT complex. These data suggest that measurements of APC–PCI complex and APC–α1‐AT complex may be useful for the diagnosis of DIC. After treatment of DIC, the plasma levels of APC–PCI complex and APC–α1‐AT complex were significantly decreased, but not those of PCI. Plasma levels of thrombin–antithrombin complex (TAT), plasmin–α2‐plasmin complex (PPIC), D‐dimer, and soluble fibrin monomer (SFM) were markedly increased in patients with DIC or pre‐DIC and were moderately increased in patients with non‐DIC, TTP, AMI, PE, or DVT and in those undergoing HD. The receiving operating characteristic (ROC) analysis showed that SFM and the APC–PCT complex are useful markers for diagnosis of DIC. The specificity of plasma TAT and PPIC levels was low. The positive rate of APC–PCI complex was higher than 90% with DIC, TTP, AMI, PE, and it was higher than 60% with DVT and HD. Since the APC–PCI complex was elevated not only in patients with venous thrombosis but also in those with arterial thrombosis, components of the protein C pathway might be useful markers for the diagnosis of arterial thrombosis. Am. J. Hematol. 65:35–40, 2000.

Protein C inhibitor inhibits breast cancer cell growth, metastasis and angiogenesis independently of its protease inhibitory activity

Protein C inhibitor (PCI) regulates the anticoagulant protein C pathway and also inhibits urinary plasminogen activator (uPA), a mediator of tumor cell invasion. In the present study, we evaluated the effect of human PCI and its inactive derivatives on tumor growth and metastasis of human breast cancer (MDA‐231) cells, and on angiogenesis in vivo. The invasiveness of MDA‐231 cells was inhibited by recombinant intact PCI, but not by reactive site‐modified PCI (R354APCI) or by the N‐terminal fragment of protease‐cleaved PCI (NTPCI). The in vitro invasiveness of MDA‐231 cells expressing intact PCI (MDA‐PCI) was significantly decreased as compared to MDA‐231 cells expressing R354APCI (MDA‐R354APCI) or NTPCI (MDA‐NTPCI). Further, in vivo growth and metastatic potential of MDA‐PCI, MDA‐R354APCI and MDA‐NTPCI cells in severe combined immunodeficient (SCID) mice were significantly decreased as compared to MDA‐Mock cells. Angiogenesis was also significantly decreased in Matrigel implant containing MDA‐PCI, MDA‐R354APCI or MDA‐NTPCI cells as compared to that containing MDA‐Mock cells. In vivo angiogenesis in rat cornea and in vitro tube formation were also inhibited by recombinant intact PCI, R354APCI and NTPCI. Furthermore, the anti‐angiogenic activity of PCI was strong as cleaved antithrombin (AT), and slightly stronger than that of plasminogen activator inhibitor (PAI)‐1 and pigment epithelium‐derived factor (PEDF). Overall, this study showed that, in addition to a reactive site‐dependent mechanism, PCI may also regulate tumor growth and metastasis independently of its protease inhibitory activity by inhibiting angiogenesis.

Bacterial lipopolysaccharide decreases thrombomodulin expression in the sinusoidal endothelial cells of rats – a possible mechanism of intrasinusoidal microthrombus formation and liver dysfunction

BACKGROUND/AIMS To elucidate the mechanism of liver dysfunction occurring in patients with sepsis, we evaluated the effect of bacterial lipopolysaccharide (LPS) on the expression of thrombomodulin (TM) in rat sinusoidal endothelial cells (SECs) and the therapeutic efficacy of exogenous recombinant TM. METHODS We induced endotoxemia in rats by bolus intraperitoneal injection of LPS. TM antigen levels within tissues were assessed by immunohistochemistry. We measured TM in cultured SECs by enzyme immunoassay, functional analysis and real-time polymerase chain reaction (PCR). RESULTS TM antigen and activity levels were significantly decreased in SECs isolated from LPS-treated rats after 3 and 6 h treatment, and recovered after 12 h treatment, correlating with immunohistochemical observations. In contrast, TM messenger RNA was decreased after 6 and 12 h treatment, and slightly recovered after 24 h treatment. TM expression in cultured SECs isolated from normal rats was also reduced after treatment with LPS and tumor necrosis factor (TNF)-alpha in vitro. The increased levels of serum fibrin degradation products (FDP), fibrin deposition within liver sinusoids, injury of SECs and liver dysfunction induced by LPS in our rat model was improved by recombinant TM treatment. CONCLUSIONS Decreased TM expression in SECs of LPS-treated rats may result in intrasinusoidal microthrombus formation and subsequent liver dysfunction during sepsis.