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Background: Thrombocytopenia due to hypersplenism is a major complication of hepatitis C virus (HCV)-associated cirrhosis. HCV eradication improves these complications in some patients, but the long-term effects of HCV eradication on these complications remain unclear, especially in patients treated with direct acting antivirals (DAAs). The aim was to evaluate long term changes in thrombocytopenia and leucopenia after HCV eradication with DAAs., Methods: The present multicenter study retrospectively evaluated changes over 5 years in thrombocytopenia and leukocytopenia, as well as changes in liver fibrosis markers and spleen size, in 115 patients with HCV-cirrhosis treated with DAAs., Results: Thrombocytopenia and leukocytopenia were improved 4 weeks after DAA administration, with thrombocytopenia show further gradual improvement over the next year. Fib-4 index was markedly reduced 1 year after DAA, followed by subsequent gradual reduction over the next 4 years. Spleen size showed gradual annual reductions, with patients experiencing spleen size reduction characterized at baseline by bilirubinemia., Conclusions: Rapid DAA-associated HCV eradication might lead to rapid disappearance of liver inflammation and bone marrow suppression due to HCV infection. HCV eradication may gradually improve portal hypertension, reducing spleen size., (© 2023. The Author(s).)

Background/aim: Lenvatinib is a tyrosine kinase inhibitor (TKI) more effective against hepatocellular carcinoma (HCC) than sorafenib, making lenvatinib a first-line treatment option for patients with unresectable HCC. In patients treated with sorafenib, post-progression survival (PPS) rather than progression-free survival (PFS) is essential for overall survival (OS). However, the importance of PPS for OS in patients treated with lenvatinib is uncertain, and optimal treatment after lenvatinib failure has not yet been established., Patients and Methods: The present study investigated the correlations of PFS and PPS with OS in studies of HCC patients treated with lenvatinib by weighted linear regression analysis. Furthermore, the contribution of treatment regimens after lenvatinib failure to OS were evaluated in daily clinical practice., Results: An analysis of 20 studies with 4,054 patients found that PPS had a stronger correlation with OS (r=0.869, p<0.001) than did PFS (r=0.505, p=0.007). Analysis of 79 patients with unresectable HCC treated with first-line lenvatinib showed that subsequent treatment was the most significant contributor to OS. Second-line sorafenib was administered to 25 patients, with late transition to third-line treatment being highest among patients who received second-line treatment., Conclusion: PPS contributes significantly to OS in HCC treatment with TKIs, with multi-sequential treatment being a key determinant of longer OS., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Hepatocellular carcinoma (HCC) in hepatitis C virus (HCV)-infected patients has a high risk of recurrence. Although eradication of HCV is expected to reduce this risk, the risk in patients with a history of HCC may be high after treatment with direct-acting antivirals (DAAs)., Aim: To determine the risk factors for HCC recurrence in patients with HCV and a history of HCC., Methods: The risk of HCC recurrence in patients with a history of HCC and/or of HCC occurrence in patients without a history of HCC after DAA therapy was retrospectively analyzed in 311 HCV patients treated at our institution and several neighboring hospitals. The frequency and predictors of HCC recurrence/ occurrence after DAA treatment were included in these analyses. The clinical course of HCC before and after DAA treatment was also evaluated., Results: HCV patients with a history of HCC were older and had greater progression of liver fibrosis and diabetes than patients without a history of HCC. Median recurrence-free survival (RFS) was 1092 d in patients with a history of HCC, and post-DAA HCC recurrence/occurrence was observed in 29 patients (53.7%) with and 5 (1.9%) without a history of HCC over 6 years ( P < 0.001). RFS in patients with a history of HCC did not differ significantly before and after DAA treatment. The frequency of HCC recurrence/occurrence in patients with a history of HCC was lower after than before DAA treatment. Multivariate analysis showed that the incidence rate of HCC recurrence/occurrence before DAA treatment was the only independent predictor of HCC recurrence/occurrence after DAA treatment. Liver function was well preserved and clinical course was good in patients with HCC recurrence/occurrence after DAA therapy., Conclusion: DAA therapy in patients infected with HCV is also effective in patients with a history of HCC. Curative treatment for HCC is desirable before DAA therapy. The frequency of HCC recurrence/occurrence before DAA therapy was associated with a significantly increased risk of HCC recurrence after DAA therapy. Careful observation after DAA therapy is required in patients with a history of HCC., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

Proton pump inhibitors (PPIs) have been widely used in the treatment of gastroesophageal reflux disease and peptic ulcer disease. Although they have a potent acid suppressive effect and excellent efficacy in acid-related diseases, PPI-induced rhabdomyolysis has been reported. Here, we report the case of a patient with reflux esophagitis who developed rhabdomyolysis after esomeprazole treatment. A 67-year-old man with reflux esophagitis who had started esomeprazole treatment for the preceding 10 months complained of back and limb fatigue and myalgia. His serum creatinine kinase (CK) level was markedly elevated, and CK isozyme exhibited an MM pattern. He was diagnosed with rhabdomyolysis induced by esomeprazole. The cessation of esomeprazole rapidly improved his symptoms, and the serum CK level was normalized within 16 days. PPI-induced rhabdomyolysis is a rare complication. In most cases, PPI-induced rhabdomyolysis occurs within 3 months after starting PPIs. However, rhabdomyolysis occurred at 10 months after starting esomeprazole treatment in our patient. Early diagnosis of PPI-induced rhabdomyolysis is required even in long-term PPI users.

Unlabelled: Adrenocortical oncocytomas are rarely reported, occur almost exclusively in adults, and are mostly nonfunctional. Here, we report an interleukin-6 (IL-6)-producing adrenocortical oncocytoma in an 11-year-old girl presenting with fever, body weight loss, and increased levels of inflammatory markers and serum IL-6. Imaging studies revealed a 4-cm mass in the left adrenal gland. After complete resection, laboratory findings returned to normal. Histology was consistent with adrenocortical oncocytoma, and the tumor cells stained positive for IL-6., Conclusion: IL-6-producing adrenocortical oncocytoma should be included in the differential diagnosis and imaging studies should be performed in patients presenting with persistent fever of unknown origin and high levels of inflammatory markers.

A patient with acute lymphoblastic leukemia who was hypersensitive to native Escherichia coli L-asparaginase (L-asp) underwent readministration of the L-asp without serious adverse effects for 11 doses using a desensitization protocol every time. Monitoring of anti-L-asp antibody and L-asp activity levels revealed that the serum L-asp activity was below the effective levels during the administration of first 6 to 7 doses because of extremely high levels of anti-L-asp IgG. Sustained L-asp activity was attained since the eighth dose was administered, when the antibody levels were <5 U/mL. L-asp activity levels in patients with L-asp hypersensitivity should be monitored during the desensitization courses to ensure a sufficient L-asp activity.

A 13-yr-old boy with recurrent acute myeloid leukemia underwent HSCT using cells from an unrelated donor who matched all HLA antigens except one. Forty-two days later, the patient developed a steroid-refractory hepatitic variant of liver GVHD with peak ALT and T.Bil values of 1406 mU/mL and 10.4 mg/dL, respectively. He was successfully treated with pulse Cy (1000 mg/dose × one day) without a change in chimerism being observed or acquiring an infection. All immunosuppressant therapies could be discontinued 12 months after HSCT. Two yr after HSCT, the patient remains in CR without chronic GVHD. This single case report suggests that pulse Cy may be a promising therapy for steroid-refractory GVHD, especially hepatitic GVHD, but needs to be further tested in clinical trials., (© 2012 John Wiley & Sons A/S.)

A 10-year-old Japanese boy developed acute hepatitis with high levels of serum Torque teno virus DNA and marked lymphocytopenia, especially CD4 T-lymphocytopenia. Although the total lymphocyte counts rose as the patient recovered from hepatitis, this was largely because of a marked rise in CD8 cells. In contrast, CD4 cells recovered poorly, resulting in a further striking fall in the CD4/8 ratio. Two months later, the patient developed hepatitis-associated aplastic anemia. He was successfully treated with immunosuppressive therapy, which normalized the lymphocyte subset proportions. T-cell subsets analysis at the onset of hepatitis might be useful for predicting development of hepatitis-associated aplastic anemia.

Patients with hemophilia and high titers of inhibitors are hard to treat during bleeding events and consequently are more likely to incur high treatment costs and to experience deterioration in quality of life. We report here the case of a boy with hemophilia A and high titers of inhibitors who responded well to prophylactic activated prothrombin complex concentrate (APCC) treatment. Previously, he had to be hospitalized frequently because of painful bleeding of target joints of the knee and ankle. At the age of 4 years and 3 months, APCC prophylaxis at a dose of 60 U/kg, three times a week, was initiated together with on-demand therapy with recombinant factor VIIa. This reduced the frequency and severity of bleeding and ended the need for hospitalization. This, together with a decreased requirement for bypass agents, APCC treatment significantly reduced the cost of treatment for this patient.

A one-year-old boy with neuroblastoma (NBoma)-associated opsoclonus-myoclonus syndrome (OMS) was treated by oral high-dose dexamethasone (DEX) pulses (20 mg/m(2)/day of DEX for three consecutive days) every 28 days for 6 months after resection of the tumor. All OMS symptoms improved after the first course of DEX pulse therapy and disappeared after the last course. No adverse effects were observed. Minor deterioration of his developmental quotient was noted 33 months after the onset of the disease. NBoma remission has been maintained since treatment. Before DEX pulse therapy, frequency of T lymphocyte, in particular CD4-positive cell decreased markedly resulted in low CD4/8 ratio in the peripheral blood (PB). The frequency of B lymphocyte increased, especially in cerebrospinal fluid. These aberrant values in PB were reversed by DEX pulse therapy and correlated well with the neurological symptoms. A prospective study that assesses the efficacy of this promising and inexpensive treatment for OMS is warranted., (Copyright © 2011 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

A 5-year-old girl presented with abdominal pain and bloody stools 2 weeks after suffering from influenza A infection. Enhanced computed tomographic scan showed widespread splanchnic venous thrombosis and small intestine necrosis. She recovered after the necrotic bowel was resected. The patient continues to receive anticoagulant therapy. Thrombophilia screening after the complete resolution consistently showed mildly decreased protein S (PS) activity with normal PS antigen levels. Sequence analysis detected a heterozygous K196E mutation in the PROS1 gene. Type 2 PS deficiency was diagnosed. This is the first report of mesenteric vein thrombosis in a child with a type 2 PS deficiency.

This report describes the case of a 10-month-old boy who was diagnosed to have kaposiform hemangioendothelioma (KHE) with Kasabach-Merritt syndrome (KMS), which is a rare pediatric vascular tumor with a high mortality rate. Although both KHE with KMS were resistant to various therapies, such as oral prednisolone, sclerotherapy, and chemotherapy, repeated radiation therapy with methylprednisolone pulse therapy did reduce the volume of KHE and improved the symptoms of KMS. Unfortunately, a regrowth of KHE with KMS was observed 4 months after the cessation of treatment and the patient thereafter died from an intracranial hemorrhage and Pneumocystis carinii pneumonia, which is a complication related to repetitive radiation and steroid therapy. A histopathologic examination of autopsy specimens confirmed a diagnosis of KHE and immunohistologic staining was positive for vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3. These findings may provide the rationale to further investigate the role of VEGFRs in the pathogenesis of KHE and also to elucidate its prognostic value, along with the application of inhibitors for VEGFRs for the treatment of refractory KHE.

The clinical outcome of childhood aplastic anaemia (AA) with aberrant cytogenetic clones at diagnosis was surveyed. Among 198 children with newly diagnosed AA registered with the AA Committee of the Japanese Society of Paediatric Hematology between 1994 and 1998, cytogenetic studies of bone marrow (BM) cells were completed in 159 patients. Apart from one Robertsonian translocation, seven patients (4.4%) showed clonal chromosomal abnormalities in hypoplastic BM without myelodysplastic features. The patients included six girls and one boy with a median age of 11 years (range 5-14 years). Six patients had del(6), del(5), del(13), del(20), or -7, and one showed add(9). Four patients responded to the first immunosuppressive therapy (IST: cyclosporin A plus anti-thymocyte globulin) and one obtained a spontaneous remission. Cytogenetic abnormalities remained in two patients with an IST response. On the other hand, two patients showed no IST response. One did not respond to repeat IST and died of acute graft-versus-host disease after an unrelated-BM transplant. Another obtained a complete response after a successful BM transplant. No haematological findings at diagnosis predicted the treatment response. No significant morphological changes developed during the course of the illness. A literature review revealed that half of 24 AA patients with chromosomal abnormalities responded to the first IST, and that +6 was the sole predictable marker for IST unresponsiveness. These results suggest that IST can be applied as the initial therapy for AA with cytogenetic abnormalities in the absence of completely matched donors.

It is not fully understood how antigen-specific or-nonspecific T cells migrate into the liver in various liver diseases. Macrophage inflammatory protein (MIP)-3alpha is a chemokine that is expressed mostly in the liver, and the receptor CCR6 is reported to be expressed on memory T cells. In the present study, we focused on the expression of CCR6 on T cells from peripheral blood (PB) and inflamed liver, and analyzed the involvement of MIP-3alpha and CCR6 in the immunopathogenesis of liver diseases. Intrahepatic T cells showed significantly (P <.0001) higher percentages of CCR6(+)CD4(+) T cells compared with PB, which is significantly (P =.037) correlated with the necroinflammatory response in the liver. Most of intrahepatic CCR6(+)CD4(+) cells were also positive for CCR5, which is known to express on T-helper 1 cells. MIP-3alpha was stained in the cells located near piecemeal necrosis, where dendritic cells (DCs) are often observed, and coculture of activated DCs with apoptotic cells induced MIP-3alpha production from the DCs. These data suggest that MIP-3alpha is produced by periportal DCs and/or macrophages after necroinflammatory response, leading to the recruitment of activated T cells into the liver. This process could be important to augment the local immune response in the livers of various liver diseases. The finding might be important not only for the understanding of immunopathogenesis of liver diseases but also for the therapeutic strategy to control the local immune response in the liver.

FKHRL1, a member of the Forkhead transcription factor family, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt. This molecule is a mammalian homolog of DAF-16, which plays an important role in the longevity of Caenorhabditis elegans. In this study we found that Akt and FKHRL1 proteins were detectable in highly purified normal human megakaryocytes and that these molecules were actually phosphorylated by thrombopoietin (TPO). To clarify the functional role of FKHRL1 in TPO signaling, we established a tetracycline-inducible system in the human TPO-dependent leukemia cell line UT-7/TPO. Induced expression of active FKHRL1 led to cell cycle arrest at G0/G1 phase in this cell line. These results suggest that FKHRL1 plays an important role in the cell cycle of megakaryocytic cells as one of the downstream target molecules of phosphatidylinositol 3-kinase-Akt, presumably mediated through the activation or inactivation of cell cycle-associated gene(s).

Background: The liver is the most frequent site of tumor metastasis. It has been suggested that partial hepatectomy promotes liver metastasis of malignant disease and that expression of E-selectin, a cell adhesion molecule, plays roles in tumor metastasis. However, no reports are available concerning the expression of E-selectin after hepatectomy., Methods: In the present study, we used BALB/c mice subjected to 30% partial hepatectomy after injection of 1 x 10(4) colon 26 cells to determine the effects of partial hepatectomy on tumor metastasis to liver. E-Selectin expression within the liver after partial hepatectomy was evaluated using reverse transcription polymerase chain reaction and Western blotting. In addition, we injected polyclonal antibody to E-selectin into mice in which partial hepatectomy had augmented liver metastasis., Results: Mice subjected to partial hepatectomy had significantly increased numbers of liver metastases (sham operation, 1.5 +/- 2.0, vs partial hepatectomy, 35.5 +/- 19.3; P < 0.001). Expression of E-selectin mRNA within the liver was markedly increased 4 h after partial hepatectomy, but subsequently decreased at 24 h. E-Selectin protein was detected 8 h after hepatectomy, but subsequently decreased at 24 h as measured by Western blotting. Mice subjected to intraperitoneal injection of neutralizing antibody after operation had significantly decreased numbers of liver metastases (phosphate-buffered saline, 20.6 +/- 9.2, P < 0.05, and normal IgG, 18.0 +/- 8.0, P < 0.05, compared with polyclonal antibody to E-selectin, 5.6 +/- 4.8)., Conclusion: Induction of E-selectin by partial hepatectomy promotes hematogenous liver metastasis. Our findings can be applied to surgical treatment of liver tumor to reduce the recurrence of liver metastasis after hepatectomy by inhibiting E-selectin-mediated adhesion using reagents to E-selectin., (Copyright 2001 Academic Press.)

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 micromol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.

Human carcinomas were shown to express mRNA and protein for IL-2R alpha, beta and gamma chains. Recently, human carcinomas were also shown to constitutively express protein and mRNA for IL-2 in vivo and in vitro. Here we report that the expression levels of cytoplasmic IL-2 as well as IL-2Rbeta- and gamma-chain in human carcinoma cells change during the cell cycle progression. Carcinoma cells synchronized in the G2/M phase of the cell cycle expressed significantly more intracytoplasmic IL-2 as well as IL-2Rbeta and gamma proteins than tumor cells in the G0/G1 phase. The level of mRNA for IL-2 was 5-10-fold higher in the M phase than in the G0/G1-phase, as shown by quantitative competitive RT-PCR. Expression of the cyclin-dependent kinase (CDK) inhibitor p27kip1 in these carcinoma cells was found to be high in the G0/G1 phase, nearly absent in the S phase, and it increased again in the G2/M phase of the cell cycle. In synchronized cells, the decrease in p27 expression coincided with high levels of expression of IL-2. Using the IL-2 specific antisense oligonucleotide to block synthesis of endogenous IL-2 in tumor cells, we observed increased levels of p27 as well as p21. The antisense oligonucleotides specific for p27 or p21 blocked expression of these proteins but not of IL-2. Thus, endogenous IL-2 is important in regulating expression of p27 as well as p21 and, therefore, in controlling cell cycle progression of tumor cells, while its own expression remains independent of the CDK inhibitors.

We have previously reported that immune anti-tumor effector cells, both cytotoxic T lymphocytes (CTLs) and IL-2-activated natural killer (A-NK) cells, are effective at eliminating human head-and-neck cancer (HNC) targets in vitro and in vivo in xenograft models. In this study, these 2 types of human effector cell were compared for the ability to prevent the development of lymph node metastases in a metastasis model of human squamous-cell carcinoma of the head and neck (SCCHN) established in nude mice. A tumor cell line, OSC-19, was injected into the floor of the mouth in nude mice, and the tumor grew progressively and metastasized to cervical lymph nodes by day 21. As effector cells, a human HLA-A2-restricted CTL line recognizing a shared antigen on OSC-19 and human non-MHC-restricted A-NK cells were used. Both types of effector cell mediated high levels of lysis against OSC-19 targets in 4-hr (51)Cr-release assays. Administration of human CTLs or A-NK cells and IL-2 to the site of tumor growth in mice with 7-day OSC-19 tumors resulted in significant reduction of the number of lymph node metastases relative to untreated or sham-operated controls or to mice treated with IL-2 without the effector cells. Our results suggest that in a xenograft model of human SCCHN implanted in the oral cavity of nude mice, the development of lymph node metastases can be successfully controlled by adoptive transfer of human SCCHN-specific CTLs or SCCHN-reactive A-NK cells plus IL-2., (Copyright 1999 Wiley-Liss, Inc.)

Intrahepatic lymphoid follicle (ILF) formation is one of the most characteristic and commonly observed histological features in patients with chronic hepatitis C. However, little is known regarding whether follicles in the liver belong to functional lymphoid tissues, where B cells are activated, differentiated, and proliferated, or if the lymphocytes are merely infiltrated after recruitment from the secondary lymphoid organs. To ascertain this possibility, we examined the expression of markers for B-cell activation, differentiation, and proliferation in ILFs in patients with chronic hepatitis C using surgically resected specimens, and compared them with specimens of perihepatic lymph nodes by an immunohistochemical technique. Germinal center (GC) formation in the ILFs was frequently found in HCV-positive cases. The distribution of immunoglobulin M (IgM)-, IgD-, and IgG-positive cells and the expression patterns of Ki-67, CD23, or bcl-2 and bcl-6 gene products in the follicles with GC formation in the liver of patients with chronic hepatitis C were similar to those of lymph nodes, indicating that B cells are activated, proliferated, and differentiated in the ILFs with GC formation in patients with chronic hepatitis C. Oligoclonal expansion of B cells in the livers with ILFs was confirmed by an analysis of immunoglobulin heavy chain (IgH) gene rearrangement determined by polymerase chain reaction (PCR). These data strongly suggest that ILFs with GC formation, which are frequently found in patients with chronic hepatitis C, may functionally be the same as those found in lymph nodes with respect to B-cell expansion and maturation.

We have previously reported that human liver cancer cell lines produce interleukin-8 (IL-8) at high levels. Those tumor cells appeared to express two kinds of IL-8 receptor on their surface. In order to analyze the role of IL-8 on the biological characteristics of those tumor cells, we suppressed IL-8 production from human liver (HuH-7 and HuCC-T1) and pancreatic cancer cell lines (HuP-T4) by treatment with IL-8 antisense oligonucleotides. Suppression of IL-8 production resulted not only in inhibition of cell growth, but also in an increase in the concentrations of some tumor-associated substances such as carbohydrate antigen 19-9 (CA19-9) in the medium. These data indicate that IL-8 produced by human liver and pancreatic tumors may act as an autocrine growth factor and may control the production of some tumor-associated substances. Furthermore, surface expression of sialyl-Lewis(a), which is a ligand for ELAM-1 on human umbilical vein endothelial cells (HUVEC), HuCC-T1 and HuP-T4 cells was decreased and the attachment of these tumor cells to HUVEC was inhibited by treatment with IL-8 antisense oligonucleotide. Since the soluble form of CA19-9 (sialyl-Lewis(a)) was shown to inhibit the tumor cell binding to HUVEC, the decrease in release of CA19-9 into the medium and increase in the expression of sialyl-Lewis(a) on the cell surface may suggest that IL-8 production from the tumor cells enhances metastatic potential by augmenting the binding activity of the tumor cells to HUVEC. These data demonstrate that a cytokine produced by tumor cells may function as an autocrine growth factor and affect tumor cell dissemination.

Thrombopoietin (TPO) acts on megakaryopoiesis and erythropoiesis in vitro and in vivo. We isolated a novel subline, UT-7/GMT, from the human leukemia cell line UT-7/GM (N. Komatsu, et al., Blood, 89: 4021-4033, 1997). A small population of UT-7/GM cells positively stained for hemoglobin (Hb) after a 7-day exposure to TPO. More than 50% of TPO-treated UT-7/GMT cells positively stained for Hb. Using UT-7/GMT cells, we examined how TPO promotes hemoglobinization. TPO induced tyrosine phosphorylation of the TPO receptor but not the erythropoietin (EPO) receptor. There was no competition between TPO and EPO for binding to EPO receptor. These findings suggest that TPO has a direct effect on hemoglobinization via a specific receptor on UT-7/GMT cells. Isoelectric focusing demonstrated that TPO induced fetal and adult Hb synthesis, whereas EPO induced embryonic, fetal, and adult Hb synthesis. Thus, our data suggest that TPO has a distinct action on erythropoiesis.

A variety of strategies have been attempted in the past to stably transduce natural killer (NK) cells with cytokine or other cellular genes. Here, we demonstrate the successful delivery of the interleukin-2 (IL-2) gene into two human NK cell lines, IL-2-dependent NK-92 and IL-2-independent YT, by retroviral transduction. An MuLV-based retroviral vector expressing human IL-2 and neor markers from a polycistronic message was constructed and transduced into a CRIP packaging cell line. By coincubation of NK cells with monolayers of CRIP cells or by using retrovirus-containing supernatants in a flow-through method, 10% to 20% of NK cells were stably transduced. Upon selection in the presence of increasing G418 concentrations, transduced NK cells were able to proliferate independently of IL-2 for more than 5 months and to secrete up to 5.5 ng/10(6) cells/24 h of IL-2. IL-2 gene-transduced NK-92 cells had an in vitro cytotoxicity against tumor targets that was significantly higher than that of parental cells and secreted interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) in addition to IL-2. Moreover, the in vivo antitumor activity of IL-2 gene-transduced NK-92 cells against established 3-day liver metastases in mice was greater than that of parental nontransduced NK cells. Stable expression of the IL-2 transgene in NK cells improved their therapeutic potential in tumor-bearing hosts. Thus, transduced NK cells secreted sufficient quantities of bioactive IL-2 to proliferate in vitro and mediated the antitumor effects both in vitro and in vivo in the absence of exogenous IL-2. These results suggest that genetic modification of NK cells ex vivo could be useful for clinical cancer therapy in the future.

Interleukin-2 (IL-2) is recognized as a T cell growth factor. We have previously reported that human carcinoma cell lines are inhibited in growth by exogenous IL-2, which binds to the IL-2 receptor beta (IL-2Rbeta) chain ubiquitously expressed on the surface of tumor cells. A possibility was considered that IL-2Rbeta on carcinomas responsible for negative signaling was different from that expressed on hematopoietic cells. To investigate this possibility, mRNA for the IL-2Rbeta chain was amplified and compared in carcinoma and lymphoid cells. Using RT-PCR with pairs of sense-antisense oligonucleotide primers specific for the various regions of extracellular, transmembrane and intracellular domains of the IL-2Rbeta chain, we amplified mRNA obtained from three human carcinoma cell lines and human lymphoid cells as controls. The identity of the amplicons was confirmed by Southern analysis with the 32P-labeled cDNA probe coding for the entire span of the IL-2Rbeta chain. In addition, genomic DNA obtained from the tumor cell lines was sequenced to examine the possibility that a mutation is present in the gene coding for the intracellular IL-2Rbeta chain domain. No mutations or deletions were detected. The message for all three domains of the beta chain was identical in tumor cells and in normal lymphoid cells used as controls. Also, by Western blot and northern analyses no differences between IL-2Rbeta chain in tumors vs that expressed in lymphoid cells were demonstrable. The IL-2Rgamma chain, which participates in IL-2/IL-2R signaling pathway, was expressed in tumor cells. Expression of JAK1 transcripts in these cells was comparable to that in lymphocytes. However, RT-PCR analysis identified differences in expression of JAK3 splice variants (B and M) in tumor cells. These differences may be responsible for altered downstream signaling by IL-2. Overall, our data indicate that the same IL-2/IL-2R pathway is operative in human carcinomas and in normal epithelial or lymphoid cells.

Two human squamous cell carcinoma of the head and neck (SCCHN) cell lines, PCI-13 and PCI-52, were transduced with the retroviral construct containing human interleukin-2 (IL-2) cDNA and selected for neomycin resistance in G418 medium. Stably transduced SCCHN cells produced and secreted IL-2, which was shown to have biologic activity in a bioassay, using an IL-2-dependent CTLL-2 cell line. By immunohistochemistry, IL-2 gene-transduced PCI-13 cells were strongly positive for IL-2, and by flow cytometry showed both cell surface and intracytoplasmic expression of IL-2 protein. Expression of IL-2 mRNA was measured by quantitative RT-PCR and found to be considerably increased in transduced SCCHN relative to that in parental cells. There was no difference in expression of IL-2R between the parental and IL-2 gene-transduced cells. In vitro proliferation of IL-2 gene-transduced tumor cells was consistently more rapid than that of parental cells. Sensitivity of the parental and IL-2 gene-transduced targets to lysis or apoptosis mediated by purified human natural killer (NK) cells or IL-2-activated NK (A-NK) cells was comparable as measured in 4-hour 51Cr-release and 1-hour [3H]thymidine-release assays, respectively. However, transduced cells were significantly more sensitive than parental cells to these effectors in 24-hour MTT assays, most likely due to IL-2 production by the transduced targets. PCI-52 cells selected for in vivo experiments formed large subcutaneous tumors in immunosuppressed nude mice. Tumors established by subcutaneous injections of 1 x 10(7) IL-2 gene-transduced cells regressed completely by day 25, while those formed by parental or LacZ gene-transduced tumor cells grew progressively. Tumor regression was mediated by numerous mononuclear cells, identified as murine NK cells and macrophages by immunohistochemistry, which accumulated around the IL-2-secreting, but not parental, tumors within 5-6 days after tumor cell injections. Thus, IL-2 gene-transduced SCCHN cells produce functional IL-2 in vivo in amounts sufficient to support the recruitment to the tumor site and antitumor activity of cytotoxic effector cells. IL-2-secreting SCCHN cells may be a useful component of vaccines designed to induce and sustain effector cell activation at the tumor site.

We transduced a human gastric carcinoma cell line, HR, with the interleukin 2 (IL-2) gene. Stable HR transfectants secreted nanogram quantities of biologically active IL-2 and had significantly increased expression of IL-2 mRNA relative to that in parental cells. Expression of intracellular IL-2 protein was not quantitatively different in the parental and IL-2 gene-transduced cells, although the former did not secrete IL-2. Surface expression of IL-2 receptors was comparable in the parental and transduced cells at the mRNA or protein levels. Nevertheless, in vitro proliferation of IL-2 gene-transduced HR cells was significantly more rapid than that of parental cells. Both parental and IL-2 gene-transduced HR cells were equally sensitive to lysis by IL-2-activated natural killer (A-NK) cells, as measured in 4 hr 51Cr-release assasys or to apoptosis induced by NK or A-NK cells, assessed in 1 hr 3H-TdR-release assays. In 24 hr MTT assays, however, the IL-2 gene-transduced cells were significantly more sensitive to these effector cells than were parental cells. Upon intrasplenic injection of 5 x 10(6) parental or transduced HR cells into nude mice, liver metastases developed. Metastases of parental HR cells killed the animals in 24 days. In contrast, metastases of the IL-2 gene-transduced HR cells became necrotic by day 14 and were found to be surrounded by murine NK cells and macrophages. Survival of nude mice injected with IL-2 gene-transduced HR cells was significantly prolonged (>50 days) relative to that of mice injected with parental HR. Thus, IL-2 gene-transduced HR cells produced sufficient amounts of functional IL-2 in vivo to be able to recruit to the tumor site and support functions of endogenous cytotoxic immune effector cells, which were responsible for regression of hepatic metastases and significant improvement of survival in these mice.

We describe a 43-year-old woman with clinical features compatible with autoimmune cholangiopathy recently reported by Ben Ari et al. She was negative for anti-mitochondrial antibody, positive for high titer anti-nuclear antibody with homogeneous pattern, high levels of serum immunoglobulin G and nearly normal levels of serum immunoglobulin M for more than five years. In the early stages of the disease, the elevations of serum transaminase, alkaline phosphatase and gamma-glutamyl transpeptidase were well controlled by the administration of ursodeoxycholic acid. After five years of follow-up, she showed the second exacerbation of liver function tests, which then rapidly improved by prednisone administration. To analyze the antigen diversity recognized by T-cells in the liver, T-cell receptor repertoire was examined by immuno-histochemistry. The liver biopsy obtained in the early stage showed clonal accumulation of V beta 5.1-positive cells in portal areas, which was found in patients neither with primary biliary cirrhosis nor autoimmune hepatitis. In conclusion, these data suggest that T-cell response in autoimmune cholangiopathy is different from those two autoimmune liver diseases, which may imply a distinct entity of the disease.

Serum levels of soluble forms of intracellular adhesion molecule-1 (sICAM-1) and lymphocyte function-associated antigen-3 (sLFA-3) in 122 patients with chronic liver disease including hepatocellular carcinoma (HCC) were measured by enzyme-linked immunosorbent assays. Serum levels of sICAM-1 in patients with HCC were significantly higher than those of chronic hepatitis (CH) and cirrhosis. On the other hand, serum levels of sLFA-3 in patients with HCC were almost the same as those of cirrhosis. Western blot analyses showed that molecular sizes of sICAM-1 and sLFA-3 detected in the sera were 90 kd and 50 kd, respectively, indicating that both molecules include whole extracellular domains. In patients with HCC, circulating sICAM-1 levels were significantly (P < .001) correlated with tumor volume (r = .50), total bilirubin (r = .38), serum aspartate aminotransferase levels (r = .51), and gamma-globulin (r = .63). Furthermore, serum sICAM-1 levels were significantly elevated in patients with multiple HCC (tumor number > 3) or HCC with tumor embolus in the first branch or trunk of portal vein. Survival periods were analyzed in relation to serum sICAM-1 levels in patients with HCC who had been treated by transcatheter arterial chemoembolization. The HCC patients with < 1,000 ng/mL of serum ICAM-1 showed significantly (P = .0005) longer survival than those with higher levels of the molecule. The same results were obtained when only patients with moderately differentiated HCC were analyzed (P = .02). Analyses by Cox's proportional hazard model showed that sICAM-1 is a significant (P = .032) prognostic factor for patients with HCC.(ABSTRACT TRUNCATED AT 250 WORDS)

The patient first noticed general muscle stiffness at the age of 36. Two years later, she suffered from a tonic-clonic seizure which brought her to a hospital for the first time. Choreoathetoid movement, ataxia and cognitive deficit were apparent. At the age of 44, tonic-clonic seizures became more frequent and she was admitted to our hospital as being status epilepticus. After the cessation of clinical seizures, she became appllic. Gradual increase of atrophic changes in cerebrum, cerebellum and brain stem were observed by MRI and CT. Hematological study showed that she had abnormal hemoglobin, Hb Takamatsu. Four of her five children were clinically examined; all of them showed abnormal EEG findings; three being mentally retarded and had clinical generalized convulsive seizures; two had hemoglobinopathy (Hb Takamatsu). The patient died from sepsis at the age of 50 and the autopsy was carried out. The brain weighed 930 gram. Histological findings confirmed the diagnosis of dentato-rubro-pallido-luysian atrophy; neuronal loss accompanied by gliosis in dentate nuclei, red nuclei, lateral part of globus pallidus, and subthalamic nuclei. The coincidence of the hereditary traits of two independent diseases, DRPLA and familial hemoglobinopathy (Hb Takamatsu) suggests closeness of their genetic loci.