Generation of a novel CD30+ B cell subset producing GM-CSF and its possible link to the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a T helper type 2 (Th2)-associated autoimmune disease characterized by vasculopathy and fibrosis. Efficacy of B cell depletion therapy underscores antibody-independent functions of B cells in SSc. A recent study showed that the Th2 cytokine interleukin (IL)-4 induces granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing effector B cells (GM-Beffs ) in humans. In this study, we sought to elucidate the generation mechanism of GM-Beffs and also determine a role of this subset in SSc. Among Th-associated cytokines, IL-4 most significantly facilitated the generation of GM-Beffs within memory B cells in healthy controls (HCs). In addition, the profibrotic cytokine transforming growth factor (TGF)-ß further potentiated IL-4- and IL-13-induced GM-Beffs . Of note, tofacitinib, a Janus kinase (JAK) inhibitor, inhibited the expression of GM-CSF mRNA and protein in memory B cells induced by IL-4, but not by TGF-ß. GM-Beffs were enriched within CD20+ CD30+ CD38-/low cells, a distinct population from plasmablasts, suggesting that GM-Beffs exert antibody-independent functions. GM-Beffs were also enriched in a CD30+ fraction of freshly isolated B cells. GM-Beffs generated under Th2 conditions facilitated the differentiation from CD14+ monocytes to DC-SIGN+ CD1a+ CD14- CD86+ cells, which significantly promoted the proliferation of naive T cells. CD30+ GM-Beffs were more pronounced in patients with SSc than in HCs. A subpopulation of SSc patients with the diffuse type and concomitant interstitial lung disease exhibited high numbers of GM-Beffs . Together, these findings suggest that human GM-Beffs are enriched in a CD30+ B cell subset and play a role in the pathogenesis of SSc.

Telomere length shortening in patients with dementia with Lewy bodies.

BACKGROUND AND PURPOSE: Shortened telomere length has been considered to be associated with various age-related diseases, especially in dementia such as Alzheimer's disease and vascular dementia. However, changes in telomere length in dementia with Lewy bodies (DLB) remain unclear. To elucidate these changes, we set out to determine telomere length in peripheral leukocytes as well as the level of urinary 8-hydroxy-deoxyguanosine (8-OHdG) as a marker of oxidative stress in DLB. METHODS: Blood samples were obtained from 33 patients with a clinical diagnosis of probable DLB and 35 age-matched, non-demented elderly controls (NEC). Telomere length was assessed by quantitative real-time polymerase chain reaction of genomic DNA extracted from leukocytes, whereas oxidative stress was assessed on the basis of urine 8-OHdG level, which was measured using high-performance liquid chromatography. RESULTS: Telomere length was significantly shorter in the DLB group than in the NEC group. Urinary 8-OHdG levels were significantly higher in the DLB group than in the NEC group. There was a negative correlation between telomere length and age in the DLB group; however, there were no significant relationships between telomere length and clinical findings including disease duration, severity of cognitive decline, presence or absence of fluctuation in cognitive function, visual hallucinations, and Parkinsonism. In both groups, the correlation between telomere length and urinary 8-OHdG levels was not significant. CONCLUSIONS: These findings indicate that the etiopathology of DLB is considered to be an accelerated aging process.

Pancreatic pleural effusion with a pancreaticopleural fistula diagnosed by magnetic resonance cholangiopancreatography and cured by somatostatin analogue treatment.

A 69-year-old man with chronic alcoholic pancreatitis developed a left-sided massive pleural effusion. Magnetic resonance cholangiopancreatography clearly demonstrated the pancreatic cyst and the fistula connecting the cyst with the left pleural cavity, resulting in the diagnosis of pancreatic pleural effusion with a pancreaticopleural fistula. Conservative somatostatin analogue treatment completely eradicated the pancreatic pleural effusion and closed the pancreaticopleural fistula.

Leiomyosarcoma of the larynx: emergency tracheostomy.

Leiomyosarcoma of the larynx is extremely rare. Histological diagnosis for excluding other tumours remains extremely difficult. A rare case of leiomyosarcoma of the larynx, treated by surgery and radiotherapy, is reported. We discuss the clinical presentation, diagnosis, and treatment of leiomysarcoma with a review of the literature.

Plasma endotoxin and serum cytokine levels in patients with alcoholic hepatitis: relation to severity of liver disturbance.

BACKGROUND: Endotoxin plays an important role in the initiation and aggravation of alcoholic liver disease. In this study, we evaluated plasma endotoxin levels and serum concentrations of cytokines and lipopolysaccharide binding protein (LBP) during the acute and recovery phase of patients with alcoholic hepatitis; we also explored the prognostic factors associated with a fatal outcome. METHODS: Fourteen patients, consisting of eight patients with alcoholic hepatitis (AH), five cirrhotics with superimposed AH (LC+AH), and one patient with severe alcoholic hepatitis (SAH), were studied. Among these, two with LC+AH died of hepatic failure. RESULTS: Plasma endotoxin levels in the acute phase were higher in patients with AH (184.4 +/- 159.4 pg/ml) and LC+AH (206.9 +/- 174.9 pg/ml) than in healthy subjects (10.4 +/- 5.5 pg/ml, p < 0.001). In particular, in one patient with SAH and one of two nonsurvivors, plasma endotoxin levels were markedly high relative to the other cases. In most survivors, plasma endotoxin levels decreased in the recovery phase, whereas they further increased at the terminal stage in one of two nonsurvivors. Serum interleukin (IL)-6 and IL-8 levels in the acute phase were significantly higher in patients with AH and LC+AH as compared with healthy subjects. These levels were especially high in nonsurvivors and in one patient with SAH. IL-10 increased in two nonsurvivors, one patient with SAH, and one with LC+AH. In the recovery phase, these cytokine levels in survivors tended to decrease, but in nonsurvivors, IL-6 remained high, and IL-8 and IL-10 further increased. Tumor necrosis factor-alpha levels were below the detection limit throughout the course in all patients. Serum lipopolysaccharide binding protein (LBP) generally was elevated in the acute phase and decreased in the recovery phase in all survivors, but in one of the nonsurvivors, LBP was elevated markedly at the terminal stage. In the acute phase, plasma endotoxin levels were correlated positively with white blood cell counts, neutrophil counts, and serum IL-8. IL-8 was correlated positively with neutrophil counts and negatively with serum cholinesterase, hepaplastin test, and serum albumin levels. IL-6 was correlated positively with white blood cell and neutrophil counts, C-reactive protein, and serum total bilirubin and negatively with hepaplastin test and serum total protein levels. Serum LBP was correlated positively with white blood cell and neutrophil counts. CONCLUSIONS: Endotoxemia and related elevation of IL-8 may play an important role in the activation and migration of neutrophils in patients with alcoholic hepatitis. Marked elevation of inflammatory cytokines, IL-6 and IL-8, are related to severity and poor prognosis of alcoholic hepatitis. Serum LBP may serve as an index of inflammatory reaction in alcoholics.

Transient cyclophosphamide treatment before intraportal readministration of an adenoviral vector can induce re-expression of the original gene construct in rat liver.

Although adenovirus is an attractive vehicle for transferring therapeutic genes in vivo, animal studies have indicated that the clinical usefulness of adenoviruses may be limited by their immunogenicity. Although immunosuppressive strategies around the time of initial exposure of adenoviruses have been shown to prevent the formation of neutralizing antibodies and permit the successful readministration of adenoviruses in animals, the practicality of the approaches remains questionable. Because the majority of prospective gene therapy patients have already been infected with wild-type adenoviruses, initial treatment with adenoviruses in humans may correspond to readministration of adenoviruses into animals. It is shown here that although intraportal infusion of adenoviruses carrying a reporter lacZ gene resulted in transient high levels of transgene expression in the rat liver, intraportal readministration of adenoviruses failed to induce detectable levels of transgene expression. Conversely, when animals were treated transiently with cyclophosphamide before the intraportal readministration of adenoviruses, development of neutralizing antibodies and antigen-specific T cell proliferation in response to adenoviral readministration was significantly suppressed and successful re-expression of the transgene was achievable. These results may have important implications for efficacy considerations when adenoviral vectors are employed in clinical settings.

Cancer gene therapy with HSV-tk/GCV system depends on T-cell-mediated immune responses and causes apoptotic death of tumor cells in vivo.

To examine the immunological mechanisms involved in cancer gene therapy using the herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV), murine hepatocellular carcinoma (HCC) cells, BNL1ME A.7R.1, were transduced retrovirally with the HSV-tk gene. HSV-tk-transduced cells exhibited a more than 2,000-fold higher sensitivity to GCV compared with untransduced parental cells. When HSV-tk-transduced HCC cells were mixed with parental cells at a 50% ratio and implanted subcutaneously into immunocompetent syngeneic mice, complete inhibition of tumor formation was achieved by GCV treatment. Conversely, no significant inhibitory effects on tumor formation were observed in athymic nude mice. When established solid tumors in immunocompetent mice containing HSV-tk-transduced cells at an only 5% ratio were treated with GCV, marked infiltration by lymphocytes including CD4(+) and CD8(+) ones, and apoptotic death of tumor cells were induced, and significant reduction or even complete regression of tumors was achieved. Furthermore, such cured mice rejected rechallenge with parental HCC cells into the contraflank regions. Our results indicate that cancer gene therapy with the HSV-tk/GCV system can indeed induce efficient antitumor effects and protective immunity in immunocompetent mice but not in nude mice, indicating that T-cell-mediated immune responses may be a critical factor for achieving successful gene therapy against cancer using the HSV-tk/GCV system.

Study of p53 in elderly patients with myelodysplastic syndromes by immunohistochemistry and DNA analysis.

We analyzed the tumor suppressor gene product, p53, in elderly patients with myelodysplastic syndromes (MDS) and in overt leukemia patients after transformation from MDS using immunohistochemical techniques. We examined 52 MDS patients (mean age 79 years, range 68 to 96) from the time of initial diagnosis to death or development of overt leukemia. p53 protein was detected by immunohistochemistry (IHC) in 8/52 patients (15%) at initial diagnosis: 1/26 with refractory anemia (RA), 0/4 with RA with ringed sideroblasts, 3/11 with RA with an excess of blasts (RAEB), 3/8 with RAEB in transformation, and 1/3 with chronic myelomonocytic leukemia. We also analyzed gene mutations in patients with positive IHC. p53 mutations were detected in 3/8 (38%) patients. IHC-positive patients had a significantly higher incidence of leukemic transformation and the presence of a complex karyotype with monosomy 17. IHC-positive cells included blasts as well as mature myeloid cells, erythroblasts, and megakaryocytes. Scrutiny of our data in combination with previous data revealed that patients with positive IHC in multilineage cells were older than those in whom positivity was noted mostly in myeloblasts. This suggests that p53 IHC positivity with a multilineage pattern may be a characteristic of MDS in older patients.

Complete cure of established murine hepatocellular carcinoma is achievable by repeated injections of retroviruses carrying the herpes simplex virus thymidine kinase gene.

Although xenotransplantation of retrovirus-producing cells into a tumor has been shown to be effective for the treatment of cancer, injections of recombinant retroviruses are much more feasible for clinical applications. We established a clone producing retroviruses carrying the herpes simplex virus thymidine kinase (HSVtk) gene with titers of up to 4 x 10(7) colony-forming units/ml, and examined the effectiveness of in vivo gene therapy against cancer. Syngeneic mice were inoculated subcutaneously with murine hepatocellular carcinoma (HCC) cells, BNL1ME A.7R.1, and the treatment was initiated after tumors were established. When mice were given an intratumoral injection of HSVtk-carrying retroviruses or their producing cells followed by ganciclovir (GCV) treatment, significantly prolonged survival periods were observed. When mice were treated with repeated intratumoral injections of HSVtk-carrying retrovirus-producing cells, significant antitumor responses and some cures were induced by GCV treatment. Furthermore, repeated intratumoral injections of HSVtk-carrying retroviruses and GCV treatment resulted in complete regression of established HCC tumors in all animals used in the experiment. Mice that completely eradicated tumors exhibited protective immunity against wild-type HCC tumors. These results suggest that repeated injections of HSVtk-carrying retroviruses followed by GCV treatment is a potent modality for the treatment of solid tumors.

Effective and safe gene therapy for colorectal carcinoma using the cytosine deaminase gene directed by the carcinoembryonic antigen promoter.

We have recently isolated carcinoembryonic antigen (CEA) promoter regions consisting of 419 bp and 204 bp from CEA-producing human colorectal carcinoma (CRC). We constructed CEA419/CD and CEA204/CD retroviruses carrying the bacterial cytosine deaminase (CD) gene directed by the CEA promoter regions. pCD2 retroviruses carrying the CD gene directed by the retrovirus long terminal repeat promoter were also used. CEA419/CD or CEA204/CD retrovirus-infected CRC cells were found to be susceptible to 5-fluorocytosine (5-FC), while non-CRC cells infected with the same retroviruses were not. CD-transduced CRC xenografts in nude mice were sensitive to 5-FC treatment, resulting in arrest of tumor growth. When mice with intraperitoneally disseminated CRCs were given intraperitoneal injections of CEA419/CD retrovirus-producing cells followed by 5-FC treatment, significantly prolonged survival rates were observed compared with animals injected with pCD2 retrovirus-producing cells followed by 5-FC treatment. Importantly, bone marrow suppression was not observed in animals injected with CEA419/CD retrovirus-producing cells and 5-FC, while profound bone marrow suppression was observed in those injected with pCD2 retrovirus-producing cells and 5-FC. These results indicate that effective and safe in vivo gene therapy for advanced CRC may be feasible by transferring the CD gene controlled by the CEA promoter followed by 5-FC treatment.

Cytosine deaminase/5-fluorocytosine gene therapy can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic nude mice.

Murine hepatocellular carcinoma cells were retrovirally transduced with the bacterial cytosine deaminase (CD) gene. CD-transduced cells exhibited more than 120-fold higher sensitivity to 5-fluorocytosine (5-FC) compared with parental cells. When syngeneic immunocompetent mice were inoculated s.c. with parental hepatocellular carcinoma cells containing as little as 5% CD-transduced cells, significant inhibition of tumor formation was induced by 5-FC treatment. Furthermore, established solid tumors in immunocompetent mice containing only 5% CD-transduced cells were infiltrated markedly with CD4- and CD8+ T lymphocytes and macrophages by 5-FC treatment, such that significant reduction or even complete regression of tumors was observed. These tumor-free mice resisted subsequent rechallenge with wild-type tumor. Conversely, when athymic nude mice were inoculated with a cell mixture containing CD-transduced cells and parental cells at a ratio of 40:60, all developed tumors despite 5-FC treatment. Our results indicate that gene therapy using the CD/5-FC system can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic immunodeficient mice, suggesting that the host's immunocompetence may be a critical factor for achieving successful gene therapy against cancer.

Hepatocellular carcinoma in an orthotopic mouse model metastasizes intrahepatically in cirrhotic but not in normal liver.

Prognosis of hepatocellular carcinoma (HCC) still remains poor mainly because of intrahepatic metastasis. In the majority of cases, HCC is found in conjunction with liver cirrhosis. It is, therefore, of great importance to investigate the invasive and metastatic behavior of HCC in cirrhotic liver. To examine this, a liver cirrhosis model was produced by injecting thioacetamide i.p. into mice. Murine HCC cells were labeled with the fluorescent carbocyanine dye, DiI, and implanted directly under the capsule of cirrhotic and normal livers of syngeneic mice. DiI-labeled HCC cells in the liver were observed under fluorescent and confocal microscopy. Histological analysis of cirrhotic and normal livers revealed that implanted HCC cells migrated to and invaded the adjacent periportal regions, but not the adjacent centrolobular areas. This characteristic behavior of HCC was more evident in cirrhotic liver than in normal liver. Furthermore, intrahepatic metastasis to unimplanted hepatic lobes was observed in cirrhotic liver as early as 7 days after implantation, while it was not detected in normal liver even 4 weeks later. Thus, an orthotopic animal model for HCC with cirrhosis described here may be suitable for investigating the invasive and metastatic behavior of HCC. Importantly, labeling tumor cells with a fluorescent dye before orthotopic implantation may be a convenient and useful method to investigate the invasive and metastatic behavior of various types of cancer.

Antitumor effect of electrochemotherapy on colorectal carcinoma in an orthotopic mouse model.

Electropermeabilization was shown to markedly increase the sensitivity of murine colorectal carcinoma (CRC) cells to bleomycin (BLM), resulting in more than 2,500-fold higher susceptibility to BLM. Subsequent in vivo electrochemotherapy with BLM revealed profound antitumor effects on subcutaneous CRC tumors. Furthermore, when electrochemotherapy with BLM was employed for the treatment of orthotopic CRC tumors in mice, significantly prolonged survival priods were observed. These results indicate the feasibility of electrochemotherapy with BLM for the treatment of CRC and demonstrate that electrochemotherapy can be translated from the treatment of cutaneous and subcutaneous tumors to the treatment of internal cancers including CRC.

Bystander effect caused by cytosine deaminase gene and 5-fluorocytosine in vitro is substantially mediated by generated 5-fluorouracil.

Because it appears impossible to transfer toxic genes to all the cells of a cancer, the bystander effect is critical to induce effective antitumor effects. In the present study, possible in vitro mechanisms of the bystander effect by the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) were investigated. CD-transduced cancer cells exhibited much higher sensitivity to 5-FC compared to parental cells. CD-transduced cells caused killing of neighboring parental cells in the presence of 5-FC, irrespective of direct cell-to-cell contact. Media conditioned by CD-transduced cells and 5-FC contained considerable amounts of 5-fluorouracil (5-FU) and exhibited profound cytotoxicity on parental cells. Furthermore, this killing ability of conditioned media correlated well with 5-FU levels converted from 5-FC by CD-transduced cells. CD was shown not to be secreted into media from cells. These results indicate that diffusible 5-FU plays the substantially causative role in the in vitro bystander effect caused by the CD/5-FC system.

Vascular endothelial growth factor tightly regulates in vivo development of murine hepatocellular carcinoma cells.

Angiogenesis is essential for the development of a solid tumor, including hepatocellular carcinoma (HCC). HCC is a well-known hypervascular tumor. Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors. Its role has not been clarified in vivo in HCC development. We used a self-contained, tetracycline-regulated retroviral vector system to elucidate the effect of VEGF on murine HCC development in a xenograft experimental model. By delivering the VEGF gene within the retroviral vector and under the control of a tetracycline-regulated promoter, we were able to manipulate VEGF expression in vivo tumor by providing tetracycline in the drinking water. Overexpression of VEGF showed a marked increase in tumor development accompanied by augmentation of neovascularization. The degree of tumor enlargement corresponded to the level of VEGF gene expression. Suppression of VEGF led to a decrease in tumor growth at the established tumor size, whether relatively small or large. The level of VEGF expression did not alter the proliferation of HCC cells in vitro. In a double-chamber chemoinvasion assay, the in vitro invasion activity of VEGF-transduced cells was not changed. In the presence of endothelial cells (EC), however, VEGF-transduced cells showed a marked increase in their in vitro invasion activity. These results suggested that VEGF plays a critical role in the development of HCC in cooperation with EC

Comparison of carcinoembryonic antigen promoter regions isolated from human colorectal carcinoma and normal adjacent mucosa to induce strong tumor-selective gene expression.

To establish in vivo gene therapy against cancer, it is requisite to induce strong, cancer cell-selective expression of a therapeutic gene. Comparison of the promoter activity of 5' flanking regions of the carcinoembryonic antigen (CEA) gene isolated from various origins is therefore of considerable interest. The 5' flanking region of the CEA gene between -135 and +69 bp upstream from the transcriptional start site, which is recognized as the core promoter region, was isolated from CEA-producing human colorectal carcinoma (CRC), normal adjacent mucosa, CEA-producing cell lines and CEA-non-producing cell lines. No mutations were observed by single-strand conformation polymorphism in the CEA promoter regions. Subsequent sequence analysis revealed that there were no mutations in the CEA promoter regions isolated from CEA-producing CRC and normal adjacent mucosa. Furthermore, nuclear extracts prepared from CEA-producing human CRC cells could equally bind to both the CEA promoter fragments isolated from CEA-producing CRC and normal mucosa. Both CEA promoter regions could direct 5- to 20-fold higher expression of a luciferase reporter gene in CEA-producing cells than in CEA-non-producing cells. Therefore, we suggest that the use of either CEA promoter region isolated from CRC or normal mucosa is equally effective to induce strong, CEA-producing cancer-selective expression of a therapeutic gene.

Inhibitory effects of human sera on adenovirus-mediated gene transfer into rat liver.

Recent advances in molecular biology have made gene therapy for cancer feasible in clinical trials. Although recombinant adenovirus is an attractive vehicle for transferring therapeutic genes in vivo, animal studies have indicated that the clinical usefulness of adenovirus vectors may be limited by their immunogenicity. It has been shown that neutralizing antibodies against adenoviruses reduce the efficiency of vector readministration. It is of great importance to examine the effects of human sera on adenovirus-mediated gene transfer, because the majority of prospective gene therapy patients are likely to have been exposed to wild-type adenoviruses. In the present study, it was shown that anti-adenovirus antibody-positive human sera with the lowest positive titer substantially inhibit the adenovirus-mediated gene transfer not only in vitro but also in vivo. These results may have important implications for efficacy considerations when adenovirus vectors are employed in the clinical setting.

Effect of transient cyclic AMP elevation on DNA synthesis in rat hepatocytes at G1 phase.

Maintaining high levels of intracellular cyclic AMP (cAMP) is known to inhibit the growth of various proliferating cells including hepatocytes. We show here that transient (30 min) elevations of cAMP induced by addition of 8-bromo-cAMP (1 mmol/L) to rat hepatocytes in primary culture at three time points (12 h, 16 h and 20 h) after seeding stimulated DNA synthesis. Sustained levels of cAMP stimulated DNA synthesis to a lesser degree at a lower concentration (1 mumol/L), but inhibited it at concentrations higher than 100 mumol/L. We also determined cyclin-dependent kinase 2 (cdk2) activity in the hepatocytes during this incubation period. The transient addition of 8-bromo-cAMP at the late G1 phase increased cdk2 activity. This suggests that transient cAMP elevation in hepatocytes at the late G1 phase has a growth stimulation effect. Up-regulation of cdk2 activity may have a role in this process.

A case of myelodysplastic syndrome with an intronic point mutation of the p53 tumour suppressor gene at the splice donor site.

We analysed genomic DNA and mRNA of the p53 gene in a case of myelodysplastic syndrome (MDS) with monosomy of chromosome 17. DNA analysis revealed a mutation at the splice donor site (GT to GC) of intron 5. mRNA analysis revealed the presence of abnormal splicing with 46 nucleotide deletion in exon 5, producing a downstream frame shift and a predicted truncated protein which lacked normal function. The p53 gene mutation at the splice donor site contributes to the inactivation of the p53 gene function and may play an important role in the pathogenesis, progression and therapeutic responsiveness of MDS.

Expression of a retrovirally transduced gene under control of an internal housekeeping gene promoter does not persist due to methylation and is restored partially by 5-azacytidine treatment.

Although expression of transgenes under the control of a retroviral long terminal repeat (LTR) promoter has been shown not to persist due to methylation, it has been observed that internal promoter may be active even if expression from the LTR promoter is silent. We constructed a retroviral vector carrying the herpes simplex virus thymidine kinase (HSVtk) gene under the control of the albumin gene promoter and transduced the HSVtk gene into hepatocellular carcinoma cells. Three of 14 mice, however, could not eradicate HSVtk-transduced grafts completely despite ganciclovir (GCV) treatment. These GCV-refractory cell lines exhibited resistance to GCV after recultivation. Subsequent Southern blot analysis revealed that the HSVtk gene was not deleted but extensively or completely methylated in GCV-refractory lines. Treatment with 5-azacytidine, a demethylating agent, partially restored the sensitivity of GCV-refractory lines to GCV. These results indicate that expression of retrovirally transduced gene may not persist in vivo due to methylation even when the gene is directed by an internal housekeeping gene promoter. These observations may also have important implications for future clinical applications of retrovirus-mediated gene therapy.