Clinical significance of plasma vascular endothelial growth factor in gastrointestinal cancer.

Circulating vascular endothelial growth factor (VEGF) was measured in gastric and colorectal cancer patients using an enzyme-linked immunosorbent assay (ELISA). Firstly, serum and plasma samples were collected from 20 normal controls to compare the values of VEGF and to determine which specimen type was most suitable for detecting circulating VEGF. Seventeen of 20 normal controls had plasma VEGF levels under the limit of detection (15 pg/ml) and the levels of the remaining three controls were 21, 22 and 38 pg/ml. In contrast, all serum samples indicated high levels of VEGF (mean 238 pg/ml), ranging from 44 to 450 pg/ml. In a time-course test of two normal controls serum VEGF values increased markedly between 30 and 60 min and remained high, whilst plasma VEGF values were low up to 480 min. Thus, plasma samples are more suitable for the measurement of circulating VEGF. Next, plasma VEGF levels were examined in 44 patients with gastric cancer (8 early, 7 advanced without remote metastasis and 29 metastatic), 13 with colorectal adenoma (2 with focal cancer) and 26 with colorectal carcinoma (8 advanced without metastasis and 18 metastatic) before treatment. An extremely high plasma concentration of VEGF was seen in some cancer patients with metastasis. To discriminate these patients, a cut-off level was determined, considering both the distribution of the sample concentration and the upper limit of 95% confidence area of VEGF in the cancer patients without metastasis. The cut-off value was 108 pg/ml and most cancer patients without metastases had VEGF levels below the cut-off value. In 11 of 29 metastatic gastric cancer patients (38%) and 9 of 18 metastatic colorectal cancer patients (50%), plasma VEGF levels were higher than the cut-off value. Survival was also analysed in the patients with metastasis. It was significantly longer in the patients with low VEGF levels (below the cut-off) than in those with high VEGF levels (logrank test, P = 0.042). 34 patients with metastasis (19 gastric cancer and 15 colorectal cancer) were treated with systemic chemotherapy, and their pretreatment levels of plasma VEGF and conventional tumour markers (CEA and CA19-9) were evaluated in relation to response. The response to chemotherapy was significantly higher in patients with low VEGF levels (< or = 108 pg/ml) than in those with high VEGF levels (P = 0.047). Such a difference was not observed with CEA/CA19-9. In conclusion, plasma VEGF is a useful marker for tumour metastasis and patient survival, and a possible predictive factor for the response of patients with gastrointestinal cancer to chemotherapy.

Total necrosis of hepatocellular carcinoma with a combination therapy of arterial infusion of chemotherapeutic lipiodol and transcatheter arterial embolization: report of 14 cases.

Combination therapy consisting of Lipiodol (Laboratoire Guerbet, Villepinte, France) containing styrene maleic acid neocarzinostatin and transcatheter arterial embolization (L-TAE) has been an important conservative therapy for hepatocellular carcinoma (HCC). We examined the clinical and pathologic characteristics of 14 HCC cases that achieved total tumor necrosis in response to L-TAE. The HCCs of all cases were resected 45 +/- 17 days after L-TAE and were confirmed to be totally necrotic. Ultrasonography showed a mean tumor size of 2.5 +/- 1.0 cm, often with a halo formation around the tumor. Angiographically, neovascularity and clear tumor stains were observed in all cases. Computed tomography portography showed nodular perfusion defects in all the cases examined. There were portal invasions in two cases. On Lipiodol-computed tomography, Lipiodol was densely and homogeneously retained within the whole tumor. The number of tumors was single in all diagnostic images. Macroscopic view of HCCs were single nodular type in nine cases and single nodular type with extra growth in four cases. Clear capsular formation was seen in each HCC nodule. Soft x-rays were taken to observe the exact distribution of Lipiodol in the operative specimens. Microscopic intrahepatic metastases were found histologically in four cases. Histologic examination showed the trabecular pattern with broad blood spaces in which Lipiodol was positive with Sudan III staining. Necrosis was seen not only in the main tumor, but also in the capsular invasions and microscopic metastases with Lipiodol deposition. The characteristics of the cases with total tumor necrosis were as follows. Deposition of Lipiodol throughout the tumor was essential, and clinically the cases showed a single HCC tumor with a diameter of more than 5 cm and arterial hypervascularity. The pathologic findings included expansive growth with capsular formation and trabecular-type HCC with abundant blood spaces. These findings are important for evaluating the radical efficacy of L-TAE.

Plasma level of basic fibroblast growth factor increases with progression of chronic liver disease.

Basic fibroblast growth factor (FGF) is thought to be involved in carcinogenesis and, to clarify its clinical significance, the study of its blood level in cancer patients is important. Plasma levels of basic FGF are reported to be elevated in some cancers. However, little is known of basic FGF levels in plasma in hepatocellular carcinoma (HCC). In this study, we measured basic FGF plasma levels in patients with chronic liver disease and compared the levels in chronic hepatitis (CH), liver cirrhosis (LC), and HCC. We also examined whether these levels were related to serum levels of asparate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, alkaline phosphatase, leucine aminopeptidase, total bilirubin, total protein, and albumin, and to the indocyanine green test (i.e., liver function tests) and to type III procollagen. 7S domain of IV type collagen, and hyaluronic acid (i.e., markers of liver fibrosis). Levels of basic FGF, determined by a quantitative "sandwich" enzyme immunoassay, were significantly elevated with the progression of liver disease; being 3.67 +/- 2.37 (mean +/- SD). 7.78 +/- 6.61, and 12.37 +/- 7.67 pg/ml in the CH, LC, and HCC groups, respectively. FGF levels were elevated to a greater extent in the HCC patients than in the CH (P < 0.0001) and LC patients (P = 0.0117). Levels were higher in LC than in CH (P = 0.0204). None of the liver function test findings or levels of markers of liver fibrosis were correlated with levels of basic FGF. These results suggest that circulating basic FGF could serve as a new indicator of the progression of chronic liver disease. The extremely elevated plasma of level basic FGF in the HCC group suggests that basic FGF may be related to the development of HCC.

Evaluation of endoscopic injection sclerotherapy with and without simultaneous ligation for the treatment of esophageal varices.

For more effective and simple endoscopic injection sclerotherapy (EIS) for esophageal varices, we developed an EIS procedure with ligation (EISL) that is non-invasive, in which EIS and endoscopic variceal ligation (EVL) are performed simultaneously. In this study, we compared EISL and EIS in a randomized sample of patients (n = 14 for each procedure). For EISL, EVL was performed, including the injection site, after the injection of 5% ethanolamine oleate with iopamidol (EOI) into a varix. The mean number of treatment sessions required for eradication of esophageal varices was 2.3+/-0.5 for EISL and 3.9+/-0.8 for EIS (P < 0.001); the mean number of treatment sites was 6.2+/-2.2 for EISL and 14.0+/-5.0 for EIS (P < 0.001); the mean total amount of EOI used was 13.8+/-5.2ml for EISL and 26.3+/-9.8ml for EIS (P < 0.001). There were no significant differences in rates of recurrence of varices or in bleeding between the two groups. For EISL, fewer treatment sessions and less sclerosant were sufficient, probably because the sclerosants were more effective due to the blockage of variceal blood flow by the ligation. This method should provide a novel modification of EIS.

Circulating vascular endothelial growth factor (VEGF) is a possible tumor marker for metastasis in human hepatocellular carcinoma.

Vascular endothelial growth factor (VEGF) is closely related to angiogenesis in various human cancers. However, little is known of its circulating levels in hepatocellular carcinoma (HCC). We examined circulating VEGF levels in chronic liver disease to assess their clinical significance. Plasma VEGF concentrations were determined, by enzyme immunoassay, in patients with chronic hepatitis (CH; n = 36), liver cirrhosis (LC; n = 77), and HCC (n = 86) for a cross-sectional study. Plasma VEGF levels in healthy controls (n = 20) and CH, LC, and HCC patients were 17.7 +/- 5.4 (mean +/- SD), 30.6 +/- 22.8, 34.4 +/- 27.0, and 51.1 +/- 71.9 pg/ml, respectively. The levels were significantly elevated in the HCC group, compared with the control, CH, and LC groups. Plasma VEGF levels in stage I, II, III, IVA, and IVB HCC patients were 27.6 +/- 16.1, 26.5 +/- 13.7, 35.8 +/- 15.3, 45.4 +/- 39.4, and 103.1 +/- 123.2 pg/ml, respectively. The stage IVB patients with remote metastasis showed significantly marked elevation compared with the patients at the other stages. Platelet numbers were weakly correlated with plasma VEGF levels in the HCC group. Plasma VEGF level was highly elevated in patients with HCC, particularly those with metastatic disease. We consider that plasma VEGF is a possible tumor marker for metastasis of HCC. Circulating VEGF may be derived mainly from the large burden of tumor cells, and partly from platelets activated by the vascular invasion of HCC cells.

Acquired type 3-like von Willebrand syndrome preceded full-blown systemic lupus erythematosus.

We report a quite rare case of acquired type 3-like von Willebrand syndrome (vWS) that preceded full-blown systemic lupus erythematosus (SLE). A 16-year-old woman with no previous disease history and no family history of hemorrhagic diathesis was referred to our hospital because of recurrent epistaxis and gingival bleeding. She was diagnosed as having atypical type 3 von Willebrand disease because of prolonged bleeding time with normal platelet count and prolonged activated partial thromboplastin time (aPTT), and an almost complete absence of von Willebrand factor (vWF) antigen, ristocetin cofactor activity (vWF:RCo) and ristocetin-induced platelet agglutination (RIPA). Furthermore, electrophoretic analysis of plasma vWF revealed a trace amount of vWF and an absence of the multimeric form of vWF. Infusions of either vasopressin or factor VIII/vWF concentrates improved bleeding symptoms and corrected the aPTT and RIPA. However, she complained of low-grade fever, general fatigue and polyarthralgia 5 months later, and leukocytepenia and hypo-complementemia developed. Anti-double-stranded DNA antibodies and lupus erythematosus cells became positive. These findings were compatible with SLE. Mixing the patient's platelet-poor plasma (PPP) with normal platelet-rich plasma (PRP) (PPP/PRP = 2/1) resulted in a complete inhibition of RIPA, suggesting the presence of vWF inhibitor in her plasma. Treatment with prednisolone (40 mg/day) started and the bleeding tendency gradually improved. One month later, all of the laboratory data including aPTT, bleeding time, RIPA and vWF:RCo became normal. These findings indicate that she has an acquired type 3-like vWS associated with SLE.

Serum soluble Fas antigen in gastric MALT (mucosa-associated lymphoid tissue) lymphoma patients.

We measured the serum level of soluble Fas (sFas) antigen in gastric MALT (mucosa-associated lymphoid tissue) lymphoma patients to investigate immune response of B lymphoma cells in gastric MALT lymphoma. The serum sFas antigen levels in 21 patients with Helicobacter pylori (HP)-positive MALT lymphoma were 4.51+/-0.25 (mean+/-SD) in the high grade malignant group (n=9) that demonstrated diffuse large cell (DL) components and 2.53+/-0.46 (mean+/-SD) in the low grade malignant group (n=12) that did not demonstrate DL components. Among 12 patients who underwent HP eradication therapy, the blood sFas antigen level lowered after eradication in 7 out of 10 patients who showed complete response or partial response, while sFas antigen level did not decrease in the 2 patients who showed no change or progression of the disease. In the high grade malignant group, 2 of 3 patients who underwent HP eradication treatment showed a mild decrease in the sFas antigen level as well as an improvement on endoscopic and histological examinations, but these sFas level were not normalized. The blood sFas antigen level tended to coincide with pathological activity and malignancy, suggesting that the sFas antigen level may be a prognostic indicator and useful index for the response of HP eradication treatment in gastric MALT lymphoma.

Survival simulation of hepatocellular carcinoma derived from follow-up studies of 450 patients.

A simulation model to predict the survival probability of individual patients with hepatocellular carcinoma (HCC) after therapy was derived from the results of various therapies and follow-up studies of 450 HCC patients. Twenty-two prognostically important variables were analyzed by Cox's proportional hazards model. The 9 significant variables that were extracted were used to build the simulation. In this model, S(t), the expected estimated survival rate for individual patient at time t (month), is calculated by the following equation: S(t) = (exp (-0.03655t) (exp [0.9479 ([portal vein invasion]-0.222) + 0.3846 ([tumor number]-2.00) + 0.2578 ([tumor size]-3.231) + 0.0742 ([loge AFP]-5.647) + 0.8184 ([metastasis]-0.036) + 0.2810 ([Child's class]-1.689)-0.7088 ([transcatheter arterial embolization]-0.578)-0.9746 ([percutaneous ethanol injection]-0.153)-0.5377 ([hepatectomy]-0.109)]) The validity of the model was assessed using a split-sample technique. This paper does not discuss the superiority or inferiority of the therapies, because some selection bias for prognostic factors among the therapies can not be completely excluded. But this model is proposed as a practical model to predict the survival of patients with HCC.

Usefulness of indocyanine green injection during ultrasound-guided liver biopsy for the diagnosis of small hepatocellular carcinoma.

To diagnose hepatocellular carcinoma (HCC) functionally and immediately, we examined the usefulness of indocyanine green (ICG) injection during ultrasound-guided liver biopsy. Liver specimens were obtained after intravenous ICG injection by ultrasound-guided biopsy from 251 space-occupying lesions (SOL) in 136 patients. The tissues were immediately examined for ICG uptake using an infrared Vidicon camera and were also subjected to histopathological examinations. Of the 112 ICG-negative biopsy specimens, 105 were histologically diagnosed as HCC, 6 as dysplastic nodules (DN) and 1 as a regenerative nodule (RN). Of the 139 ICG-positive specimens, 18 were diagnosed as HCC, 1 as DN and 120 as RN. The sensitivity of the absence of ICG uptake (SEAIU), the specificity of the absence of ICG uptake (SPAIU), and the positive predictive value of the absence of ICG uptake (PPAIU) for the diagnosis of HCC were 85.3%, 94.5% and 93.8%, respectively. Of the 251 SOLs, 184 were less than 2 cm. SEAIU, SPAIU and PPAIU for the diagnosis of these small HCC were 85.3%, 94.5% and 91.4%, respectively. These results support the reliability of ICG injection during ultrasound-guided liver biopsy to diagnose even small HCC.

Asthma classification by a score calculated from clinical findings and examinations in subjects sensitive to inhalant allergens.

Twenty-one patients with atopic asthma were classified into three types according to their symptoms (clinical diagnosis): Ia, simple bronchoconstriction; Ib, bronchoconstriction + hypersecretion; and II, bronchiolar obstruction, and this classification was compared with a classification made according to clinical findings and examinations (score diagnosis). Type Ib asthma was characterized by the increased incidence of eosinophils in bronchoalveolar lavage fluid (BALF), while type II was characterized by ventilatory dysfunction in small airways and the increased incidence of neutrophils in BALF. Four patients, whose expectoration was between 50 and 99ml/day, of the 12 with type Ia assessed by clinical diagnosis were evaluated as type Ib by score diagnosis. One patient with type II by clinical diagnosis was assessed as questionable type II by score diagnosis. In the other 16 patients, the clinical and score diagnoses were the same.

[Studies of human bone marrow stromal cells--effects of antimetabolites on the growing dynamics of human bone marrow cells and their support of hemopoietic cells].

The effects of antimetabolites on the growing dynamics of human bone marrow stromal cells and their support of hemopoietic cells were tested by using a modified version of Dexter's culture system. Cytosine arabinoside (ara-C) was found to suppress neither the growing dynamics nor the supportability. On the other hand, methotrexate (MTX) suppressed the supportiveness, even though it hardly suppressed the growing dynamics. The recognition of injury to marrow cells could be of potential importance in cancer chemotherapy. Our in vitro evidence may provide clinical insights for cancer chemotherapy including prolonged marrow suppression or pretreatment of bone marrow transplantation.

[Successful treatment of refractory anemia with excess of blasts in transformation with cytarabine ocfosfate].

A 58-year-old female was diagnosed as myelodysplastic syndrome (MDS) [refractory anemia with excess of blasts (RAEB)]. Although melphalan was administered, no response was obtained in the peripheral blood. Sixteen months after diagnosis, she developed RAEB in transformation (RAEB-t), and then overt leukemia. White blood cell (WBC) count elevated to 28,600/microliters with 34% of blasts. She was administered cytarabine ocfosfate (200 mg-->300 mg/day) orally, resulting in decrease of WBC count and blasts in peripheral blood. The drug has been given for 11 months, and her hematological data have now remained stable in RAEB. Cytarabine ocfosfate might be a useful drug for the treatment of high risk MDS such as RAEB and RAEB-t.

[Histological effect of arterial embolization chemotherapy for metastatic liver tumors from colorectal cancer--report of cases of hepatectomy after arterial embolization chemotherapy].

To evaluate the histological effect of arterial embolization chemotherapy (AEC) for the metastatic liver tumors from colorectal cancer, 5 lesions were examined in 4 patients who were treated with surgery after AEC. In addition, to compare the histological changes of the metastatic liver tumors, we histologically examined 7 lesions in 5 patients who were treated with surgery alone as a historical control. For the patients with AEC, anti-cancer agents and lipiodol were administered by selected hepatic arterial infusion with fragments of sponzel. In a group of AEC, all metastatic liver tumors were detected after resection of the primary tumors. The range of tumor size in these lesions was from 2.5 to 4.2 cm in diameter. Otherwise, in a group of non-AEC, the size of tumors ranged from 2 to 5 cm in diameter. In angiography, tumor stainings were detected in 2 lesions, and in all lesions no accumulation of lipiodol was detected by CT scan. The overall response rate at the surgery was 7 to 33% (average 23%) in a group of AEC. In addition, in 2 lesions, about 60% of the lesions were necrotic tissues, and more than 90% of the tumor lesions in 3 lesions were histologically necrosis. Within these lesions, encapsulation of the metastatic tumors was observed in 3 lesions. Otherwise, in a group of non-AEC, 50-70% of the tumor lesions were histologically detected as necrosis, and in one lesion, encapsulation was observed. These evidences suggest the histological effect of AEC may be evaluated when more than 70% of tumor necrosis in lesions is observed. In conclusion, the treatment was effective in 3 cases and not effective in one case.

Intercellular adhesion molecule-1 release from human hepatocellular carcinoma.

Serum levels of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) were measured in 38 patients with chronic hepatitis (CH), 29 with liver cirrhosis (LC), and 43 with hepatocellular carcinoma (HCC) using enzyme-linked immunosorbent assays. All the patients showed significantly higher serum levels of these circulating adhesion molecules than 40 normal controls. The serum E-selectin level showed no relationship to the levels of the other adhesion molecules. Serum VCAM-1 levels were well correlated with serum ICAM-1 levels in CH and LC patients. In HCC patients, however, the close correlation between VCAM-1 and ICAM-1 was lost, because the patients with large tumors (100 cm2) showed relatively high ICAM-1 levels. The amount of ICAM-1 shed from the tumor cells was calculated in the HCC patients as follows: actual serum ICAM-1 level minus basal ICAM-1 level released from the noncancerous liver (obtained from the regression line between ICAM-1 and VCAM-1 in CH and LC patients). Although there was no relationship between the actual ICAM-1 level and the tumor size in HCC patients, the predicted ICAM-1 shedding was closely correlated with tumor size. When in situ expression of these adhesion molecules was evaluated in 10 HCC tissues using immunohistochemistry, the tumor cells exhibited enhanced ICAM-1 expression but did not express E-selectin and VCAM-1. These results suggest that the elevated serum levels of adhesion molecules in HCC patients are mainly attributable to the associated liver inflammation, although some ICAM-1 is shed into the circulation by tumor cells.

Detection of circulating intercellular adhesion molecule-1 in hepatocellular carcinoma.

Serum levels of circulating intercellular adhesion molecule-1 (cICAM-1) were measured in 23 patients with chronic hepatitis (CH), 22 with liver cirrhosis (LC) and 45 with hepatocellular carcinoma (HCC) using an ELISA. Serum samples from all patients showed significantly higher cICAM-1 levels than serum from 50 normal controls. The cICAM-1 level was significantly increased in LC or HCC when compared with CH, but no differences were noted between LC and HCC. Levels of cICAM-1 correlated well with serum bilirubin, retention rate of indocyanine green, hyaluronic acid, type IV collagen 7-S and type III procollagen peptide levels but not with tumor size or circulating tumor markers (alpha-fetoprotein and des-gamma-carboxyprothrombin). Our findings indicate that the measurement of cICAM-1 is useful for the determination of the severity of liver disease and hepatic fibrosis. HCC tissues obtained from 10 patients were immunohistochemically stained for ICAM-1. Enhanced ICAM-1 expression was found on the tumor cell membranes. Sequential measurements of cICAM-1 levels showed that they changed in a similar manner to those of alpha-fetoprotein during the course of treatment of HCC in a patient with very high pretreatment levels of both markers. These results suggest that HCC cells shed ICAM-1 into the circulation. We conclude that cICAM-1 is not a diagnostic marker for HCC, but may be useful for monitoring the response to treatment.

Circulating platelet-derived endothelial cell growth factor increases in hepatocellular carcinoma patients.

BACKGROUND: Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic factor that is expressed in various cancer tissues. Little is known regarding plasma PD-ECGF levels in patients with chronic liver disease such as chronic hepatitis (CH), cirrhosis, and hepatocellular carcinoma (HCC) with cirrhosis. The expression of PD-ECGF in HCC tissues also remains to be clarified. METHODS: Plasma PD-ECGF levels in patients with chronic liver disease were determined with an enzyme-linked immunoadsorbent assay system using the mouse monoclonal antibodies specific to PD-ECGF. These were cross-sectionally compared among groups of normal persons, CH, cirrhosis, and HCC patients. The HCC patients were classified into two groups based on TNM stage: early and advanced stage disease groups. PD-ECGF expressions in HCC tissues were immunohistologically examined. RESULTS: The plasma PD-ECGF levels from the normal individuals and those with CH, cirrhosis, and HCC specimens were 4.2+/-0.5, 4.3+/-0.6, 4.6+/-1.1, and 6.0 +/-2.5 U/mL, respectively. The plasma PD-ECGF concentration was highest in HCC (P < 0.05). No significant difference was found among the normal subjects, CH, and cirrhosis specimens. Plasma PD-ECGF concentrations were significantly higher in the advanced stage disease HCC group compared with the early stage disease group (6.75+/-2.62 U/mL vs. 4.19+/-0.34 U/mL) (P < 0.05). Immunohistochemical expression of PD-ECGF in HCC cells increased significantly compared with normal liver cells (P < 0.05). CONCLUSIONS: Circulating PD-ECGF plasma level might be a new tumor marker for progression in patients with HCC. Immunohistological findings correspond to elevation of the plasma PD-ECGF in HCC patients. It is possible that increased production of PD-ECGF in HCC cells causes abundant neovascularization.

Laparoscopic features of primary biliary cirrhosis in AMA-positive and AMA-negative patients.

BACKGROUND AND STUDY AIMS: Antimitochondrial antibody (AMA)-negative primary biliary cirrhosis (PBC) has been difficult to diagnose. Laparoscopic features of AMA-negative PBC were evaluated in comparison with those of AMA-positive PBC and autoimmune hepatitis. PATIENTS AND METHODS: 71 patients who fulfilled the diagnostic criteria for PBC were enrolled in the study; 48 were AMA-positive and 23 were AMA-negative. As a disease control, 46 autoimmune hepatitis patients were included. Both the frequency and specificity of each laparoscopic finding were evaluated. A laparoscopic scoring system was introduced, which used, common and uncommon laparoscopic findings, and was evaluated for the diagnosis of AMA-negative PBC. RESULTS: The characteristic laparoscopic findings for AMA-positive PBC were yellowish-white marking (92 %), dark-brown discoloration (73 %), gentle undulation (67 %), reddish patch (38 %), and yellowish-white nodules (32 %). On the other hand, laparoscopic findings such as trench-like depression, reddish markings, and wide and small depressions were uncommon in PBC compared with autoimmune hepatitis. The frequencies of characteristic and uncommon laparoscopic findings did not differ statistically between AMA-positive and AMA-negative PBC, but were different between AMA-positive or AMA-negative PBC and autoimmune hepatitis. Scores based on common and uncommon laparoscopic findings were 5.5 +/- 1.5 (mean +/- SD) in AMA-positive PBC, 5.6 +/- 2.0 in AMA-negative PBC, and - 0.30 +/- 0.5 in autoimmune hepatitis. CONCLUSION: The laparoscopic findings in AMA-negative PBC did not differ from those of AMA-positive PBC. A laparoscopic scoring system may be helpful in the diagnosis of AMA-negative PBC.