The CXCR4 inhibitor BL-8040 induces the apoptosis of AML blasts by downregulating ERK, BCL-2, MCL-1 and cyclin-D1 via altered miR-15a/16-1 expression.

CXCR4 is a key player in the retention and survival of human acute myeloid leukemia (AML) blasts in the bone marrow (BM) microenvironment. We studied the effects of the CXCR4 antagonist BL-8040 on the survival of AML blasts, and investigated the molecular mechanisms by which CXCR4 signaling inhibition leads to leukemic cell death. Treatment with BL-8040 induced the robust mobilization of AML blasts from the BM. In addition, AML cells exposed to BL-8040 underwent differentiation. Furthermore, BL-8040 induced the apoptosis of AML cells in vitro and in vivo. This apoptosis was mediated by the upregulation of miR-15a/miR-16-1, resulting in downregulation of the target genes BCL-2, MCL-1 and cyclin-D1. Overexpression of miR-15a/miR-16-1 directly induced leukemic cell death. BL-8040-induced apoptosis was also mediated by the inhibition of survival signals via the AKT/ERK pathways. Importantly, treatment with a BCL-2 inhibitor induced apoptosis and act together with BL-8040 to enhance cell death. BL-8040 also synergized with FLT3 inhibitors to induce AML cell death. Importantly, this combined treatment prolonged the survival of tumor-bearing mice and reduced minimal residual disease in vivo. Our results provide a rationale to test combination therapies employing BL-8040 and BCL-2 or FLT3 inhibitors to achieve increased efficacy of these agents.

Safety and efficacy of 188-rhenium-labeled antibody to melanin in patients with metastatic melanoma.

There is a need for effective "broad spectrum" therapies for metastatic melanoma which would be suitable for all patients. The objectives of Phase Ia/Ib studies were to evaluate the safety, pharmacokinetics, dosimetry, and antitumor activity of (188)Re-6D2, a 188-Rhenium-labeled antibody to melanin. Stage IIIC/IV metastatic melanoma (MM) patients who failed standard therapies were enrolled in both studies. In Phase Ia, 10 mCi (188)Re-6D2 were given while unlabeled antibody preload was escalated. In Phase Ib, the dose of (188)Re-6D2 was escalated to 54 mCi. SPECT/CT revealed (188)Re-6D2 uptake in melanoma metastases. The mean effective half-life of (188)Re-6D2 was 12.4 h. Transient HAMA was observed in 9 patients. Six patients met the RECIST criteria for stable disease at 6 weeks. Two patients had durable disease stabilization for 14 weeks and one for 22 weeks. Median overall survival was 13 months with no dose-limiting toxicities. The data demonstrate that (188)Re-6D2 was well tolerated, localized in melanoma metastases, and had antitumor activity, thus warranting its further investigation in patients with metastatic melanoma.

Tissue microarray-based study of patients with lymph node-positive breast cancer shows tyrosine phosphorylation of signal transducer and activator of transcription 3 (tyrosine705-STAT3) is a marker of good prognosis.

BACKGROUND: Although lymph node-positive breast cancers are associated with poorer prognosis, individual patients may have different clinical outcomes. Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signalling pathways. The goal of this study was to determine the prognostic value of phosphorylated (tyrosine705)-STAT3 in node-positive breast cancer patients. METHODS: Immunohistochemical analysis of Phospho- STAT3 was performed on a tissue microarray of breast cancer specimens. The expression pattern of Phospho-STAT3 was correlated with survival outcome, and clinical and pathological parameters. RESULTS: Out of 125 interpretable tumours, positive Phospho- STAT3 nuclear expression was seen in 35 (28%) of tumours. There was no significant relationship between Phospho-STAT3 expression and clinical-pathological parameters including age, hormonal receptor status, grade and tumour size. Interestingly positive tumours had a significantly improved disease-free survival at 5 years (p=0.035). Additionally, positive Phospho-STAT3 nuclear expression was correlated with significantly improved survival at both 5 years (p=0.023) and 10 years (p=0.026). Finally, in multivariate analyses Phospho-STAT3 was found to be an independent prognostic marker of overall survival in node-positive breast cancer patients. CONCLUSION: These findings support the role of Phospho- STAT3 as an important independent prognostic marker in node-positive breast cancer patients.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

The CXCR4 antagonist 4F-benzoyl-TN14003 stimulates the recovery of the bone marrow after transplantation.

Cytopenia represents a significant complication after chemotherapy, irradiation before bone marrow (BM) transplantation or as a therapy for cancer. The mechanisms that determine the pace of BM recovery are not fully understood. During the recovery phase after chemotherapy or irradiation, the signals for retention of white blood cells within the BM increase significantly. This leads to a delay in the release of WBC, which can be overcome by targeting the CXCR4 axis with the antagonist 4F-benzoyl-TN14003 (T140). The delay in the release of WBC is also accompanied by suppression in the production of progenitor cells and mature cells by the BM stroma. Administration of T140 to mice transplanted with BM cells stimulates the production of all types of progenitors and mature cells, and increases the exit of mature cells to the periphery. Moreover, addition of T140, but not AMD3100, to BM stromal cultures stimulates the production of mature cells and progenitors from all lineages. The unique ability of the CXCR4 antagonist, T140 to stimulate the production and exit of WBC cells may be used as a novel therapeutic approach to overcome cytopenia associated with treatments for cancer and BM transplantation.

Imaging transgene expression in live animals.

Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assessing the effectiveness of systems of gene therapy delivery. We applied a new light-detection cooled charged-coupled device (CCCD) camera for continuous in vivo assessment of commonly used gene therapy delivery systems (such as ex vivo manipulated cells, viral vectors, and naked DNA), without the need to kill animals. We examined a variety of criteria related to real-time monitoring of luciferase (luc) gene expression in tissues including bone, muscle, salivary glands, dermis, liver, peritoneum, testis, teeth, prostate, and bladder in living mice and rats. These criteria included determination of the efficiency of infection/transfection of various viral and nonviral delivery systems, promoter specificity, and visualization of luciferase activity, and of the ability of luciferin to reach various organs. The exposure time for detection of luc activity by the CCCD camera is relatively short (approximately 2 minutes) compared with the intensified CCD camera photon-counting method (approximately 15 minutes). Here we transduce a variety of vectors (such as viruses, transfected cells, and naked DNA) by various delivery methods, including electroporation, systemic injection of viruses, and tail-vein, high-velocity-high-volume administration of DNA plasmids. The location, intensity, and duration of luc expression in different organs were determined. The distribution of luciferin is most probably not a barrier for the detection of in vivo luciferase activity. We showed that the CCCD photon detection system is a simple, reproducible, and applicable method that enables the continuous monitoring of a gene delivery system in living animals.

Hyper-IL-6 gene therapy reverses fulminant hepatic failure.

Fulminant hepatic failure is a catastrophic condition caused by massive hepatocellular apoptosis and necrosis. Inhibition of hepatocyte apoptosis and the enhancement of the endogenous potential for liver regeneration could potentially form an effective basis for treatment of this condition. In response to injury in the liver, IL-6 mediates the acute-phase response and induces both cytoprotective and mitogenic functions. Hyper-IL-6 is a superagonistic designer cytokine consisting of human IL-6 linked by a flexible peptide chain to the secreted form of the IL-6 receptor. In a mouse model of acute liver failure induced by d-galactosamine administration, a single low dose of a hyper-IL-6-encoding adenoviral vector, in contrast to an adeno-IL-6 vector, maintained liver function, prevented the progression of liver necrosis, and induced liver regeneration, leading to dramatically enhanced survival. Thus, hyper-IL-6 gene therapy may be useful for the treatment of fulminant hepatic failure, which is often fatal even following treatment by transplantation.

Lack of known hepatitis virus in hepatitis-associated aplastic anemia and outcome after bone marrow transplantation.

Viral infection has been shown to induce aplastic anemia, unidentified types of hepatitis being the most common cause for aplastic anemia-associated viral hepatitis. The survival rate for this group of patients after bone marrow transplantation with stem cells from an HLA-matched sibling is not well known. The aim of this study was to determine the prevalence of hepatitis G virus (HGV) and transfusion transmitted virus (TTV) infection in non-A, non-B, non-C hepatitis associated-aplastic anemia (HAAA) patients, and to define the role of bone marrow transplantation (BMT) as a therapeutic modality for this disease. Sixty-eight patients (43 males and 25 females) with aplastic anemia, underwent allogeneic BMT at the Hadassah University Hospital between 1981 and 1997. Onset of hepatitis was defined as jaundice and elevated alanine aminotransaminase (ALT) levels. Onset of aplastic anemia was defined as the first date on which varying degrees of pancytopenia occurred: hemoglobin level below 10 g/dl, WBC below 2 x 10(9)/l and low platelet count 10 x 10(10)/l. Serial serum samples from HAAA patients were assayed for virological and/or serological markers of hepatitis A, B, C, D, E, G viruses, TTV and parvovirus B19. Seventeen of the 68 patients with aplastic anemia (25%) suffered from hepatitis, 12 males and five females, ages 5 to 36 years. The mean interval between onset of hepatitis and first indication of aplastic anemia was 62 days (range 14-225 days). The development of aplastic anemia was unrelated to age, sex or severity of hepatitis. Ten of the 17 patients (59%) achieved complete ALT recovery prior to the diagnosis of aplastic anemia. Serum samples were available for 15 patients; none had evidence of acute or active hepatitis A, B, C, D, E, G and TTV virus infection at the time of diagnosis. Parvovirus B19 DNA sequences were not detectable in 10 of 12 tested cases; two positive results were detected in serum samples obtained after blood transfusion, making the analysis of these positive results difficult. All 17 patients underwent BMT. The mean post-BMT follow-up period was 38 months (range 1 day-123 months), five patients (30%) died 1 to 160 days post BMT, and 12 (70%) are alive 31 to 123 months after BMT. Relapsing hepatitis was not observed in any of the patients. In conclusion, HAAA is a disease of the young and the etiologic agent associated with HAAA remains unknown. HGV, TTV and parvovirus B19 sequences were not detected in any of the HAAA cases. The survival rate after BMT with stem cells from an HLA-matched sibling is similar to that for patients with non-hepatitis-associated aplastic anemia.

Liver regeneration induced by a designer human IL-6/sIL-6R fusion protein reverses severe hepatocellular injury.

The cytokine IL-6 plays a significant role in liver regeneration in conjunction with additional growth factors (HGF, TNF-alpha, and TGF-alpha). Many IL-6 effects depend on a naturally occurring soluble IL-6 receptor (sIL-6R). Here, the chimeric protein hyper-IL-6, constructed from the human IL-6 protein fused to a truncated form of its receptor, was found to have superagonistic IL-6 properties, and as such, enhanced liver cell regeneration. Hyper-IL-6 reversed the state of hepatotoxicity and enhanced the survival rates of rats suffering from fulminant hepatic failure after D-galactosamine administration. The hyper-IL-6 protein has a significant potential for use in the treatment of severe human liver diseases.

Human interleukin-6 facilitates hepatitis B virus infection in vitro and in vivo.

BACKGROUND AND AIM: Research on hepatitis B virus (HBV) infection in vivo has been limited due to the absence of a suitable animal model. We have developed a human-mouse radiation chimera in which normal mice, preconditioned by lethal total body irradiation and radioprotected with SCID mouse bone marrow cells, are permissive for engraftment of human hematopoietic cells and solid tissues. This resulting human-mouse model, which comprises three genetically disparate sources of tissue, is therefore termed Trimera. This study was aimed at assessing the effect of human IL-6 on HBV infection in vivo in Trimera mice. METHODS: Trimera mice were transplanted with human liver tissue fragments or with HepG2-derived cell lines, which had been previously infected ex vivo with HBV in the presence or absence of human interleukin-6 (hIL-6) and in the presence of anti-IL-6-neutralizing antibodies. RESULTS: HBV sequences appeared in the sera of animals in which the liver tissue was incubated with both HBV and hIL-6 prior to transplantation. A similar result was obtained when a human hepatoblastoma cell line (HepG2), expressing the hIL-6 receptor, was infected ex vivo with HBV in the presence of hIL-6 prior to their injection into spleens of Trimera mice. However, when liver fragments were infected ex vivo and simultaneously treated with neutralizing antibodies against hIL-6 or were incubated with HBV prior to transplantation without hIL-6, the rate of mice positive for HBV DNA in their sera was lower. Human mononuclear cells are also permissive for HBV infection in vitro: in the presence of hIL-6 the infection of these cells is enhanced; and this infection is suppressed by the chimeric protein named Hyper-IL-6, generated by the fusion of hIL-6 to the soluble hIL-6 receptor (sIL-6Ralpha, gp80). CONCLUSION: hIL-6 facilitates HBV infection in vitro and in vivo.

High susceptibility to bacterial infection, but no liver dysfunction, in mice compromised for hepatocyte NF-kappaB activation.

Based on the essential involvement of NF-kappaB in immune and inflammatory responses and its apoptosis-rescue function in normal and malignant cells, inhibitors of this transcription factor are potential therapeutics for the treatment of a wide range of diseases, from bronchial asthma to cancer. Yet, given the essential function of NF-kappaB in the embryonic liver, it is important to determine its necessity in the liver beyond embryogenesis. NF-kappaB is normally retained in the cytoplasm by its inhibitor IkappaB, which is eliminated upon cell stimulation through phosphorylation-dependent ubiquitin degradation. Here, we directed a degradation-resistant IkappaBalpha transgene to mouse hepatocytes in an inducible manner and showed substantial tissue specificity using various means, including a new method for live-animal imaging. Transgene expression resulted in obstruction of NF-kappaB activation, yet produced no signs of liver dysfunction, even when implemented over 15 months. However, the transgene-expressing mice were very vulnerable both to a severe immune challenge and to a systemic bacterial infection. Despite having intact immunocytes and inflammatory cells, these mice were unable to clear Listeria monocytogenes from the liver and succumbed to sepsis. These findings indicate the essential function of the hepatocyte through NF-kappaB activation in certain systemic infections, possibly by coordinating innate immunity in the liver.

Antigen-specific B and T cells in human/mouse radiation chimera following immunization in vivo.

Adoptive transfer of human peripheral blood mononuclear cells (PBMC) into mice with severe combined immunodeficiency (SCID) or into lethally irradiated BALB/c mice radioprotected with SCID bone marrow, leads to marked engraftment of human T and B cells. In such chimeras, human serum antibody responses can be stimulated readily by vaccination with recall antigens, but the detection of antigen-specific functional T or B cells has been extremely difficult. In the present study, we were able to detect by Elispot analysis high frequencies of immunoglobulin G (IgG)-secreting B cells and mitogen-responsive interferon-gamma (IFN-gamma) or interleukin-4 (IL-4)-secreting T cells in peritoneum and spleen of human/BALB/c chimeric mice during the first 3 weeks after PBMC transfer. Moreover, specific memory responses were elicited by vaccination with tetanus toxoid (TT) or hepatitis B virus (HBV) surface (HBs) antigen of chimeric mice transplanted with PBMC derived from TT- or HBV-immune donors. Substantially higher TT-specific B-cell frequencies were found during the first 3 weeks after vaccination in mice challenged with the specific antigen compared to the levels found in control animals. High numbers of TT-specific IFN-gamma-secreting T cells persisted in the peritoneum of vaccinated, but not of unvaccinated, animals during the entire observation period, but only low numbers of specific IL-4-secreting T cells were found in vaccinated mice. Similar results were achieved following vaccination with HBs antigen of chimeric mice, transplanted with PBMC of HBV immunized donors. Thus, TT or HBsAg-specific antibody responses in our model correlate closely with the existence of specific IFN-gamma-secreting T helper 1/0 cells. Furthermore, these results show that adoptive transfer of human PBMC into lethally irradiated mice provides an efficient approach to generate specific B-cell fusion partners for the production of human monoclonal antibodies and specific T-cell lines for adoptive cell therapy of malignant or infectious diseases.

Human monoclonal antibodies specific to hepatitis B virus generated in a human/mouse radiation chimera: the Trimera system.

An approach to develop fully human monoclonal antibodies in a human/mouse radiation chimera, the Trimera system, is described. In this system, functional human lymphocytes are engrafted in normal strains of mice which are rendered immuno-incompetent by lethal total body irradiation followed by radioprotection with severe combined immunodeficient (SCID) mouse bone marrow. Following transplantation, human lymphocytes colonize murine lymphatic organs and secrete human immunoglobulins. We have established this system as a tool to develop fully human monoclonal antibodies, and applied it for the generation of monoclonal antibodies specific for hepatitis B virus surface antigen. A strong memory response to hepatitis B surface antigen was elicited in Trimera engrafted with lymphocytes from human donors positive for antibodies to hepatitis B surface antigen. The human specific antibody fraction in the Trimera was 10(2)-10(3)-fold higher as compared with that found in the donors. Spleens were harvested from Trimera mice showing high specific-antibody titres and cells were fused to a human-mouse heteromyeloma fusion partner. Several stable hybridoma clones were isolated and characterized. These hybridomas produce high-affinity, IgG, anti-hepatitis B surface antigen antibodies demonstrating the potential of the Trimera system for generating fully human monoclonal antibodies. The biological function and the neutralizing activity of these antibodies are currently being tested.

Suppression of human hepatoma in mice through adoptive transfer of immunity to the hepatitis B surface antigen.

BACKGROUND/AIMS: Adoptive transfer of immunity against hepatitis B surface antigen (HBsAg) has previously been shown to occur in mice and humans through transplantation of bone marrow cells from donors immunized against HBsAg (anti-HBs) to non-immune recipients. In the present study we evaluated the effect of adoptive transfer of immunity to HBsAg on the growth of HbsAg-secreting hepatocellular carcinoma (HCC) xenografts in athymic mice. METHODS: Immunocompetent mice were immunized with recombinant HBsAg. Bone marrow cells from anti-HBs+ mice were injected intravenously to irradiated athymic Balb/c mice which had been previously transplanted subcutaneously with Hep3B human hepatoma cells. Treatment groups included mice receiving bone marrow transplantation from HBV-immunized (anti-HBs positive) and non-immunized (anti-HBs negative) donors. RESULTS: At 9 weeks post bone marrow transplantation, tumor volume and serum alpha-fetoprotein levels in athymic mice receiving HBV-immune bone marrow cells were 11.5 mm3 and 363 ng/ml, respectively, as compared to 1579 mm3 and 19,000 ng/ml, in recipients of non-immune bone marrow transplantation (p<0.005). T-cell depletion of antiHBs+ immune bone marrow prior to transplantation decreased the anti-tumor effect but did not abolish it. A mild nonspecific, bone marrow-derived, graft versus tumor effect was observed in mice transplanted with human hepatoma cells that do not express HBsAg. CONCLUSIONS: Adoptive transfer of immunity to HBV facilitates suppression of experimental human HCC expressing HBsAg. This effect is the result of a combination of specific anti-viral surface antigen effect and a nonspecific graft versus tumor effect.

Pregnancy associated with colon carcinoma overexpressing p53.

The development of colon carcinoma during pregnancy is a rare event. However, when colon carcinoma develops during pregnancy it is considered a lethal coincidence due to rapid progression. We report two rare cases of colon adenocarcinoma diagnosed during gestation. Both tumors displayed increased nuclear immunostaining for p53. The increased expression of p53 in tumor cells could indicate that the p53 gene is either mutated or stabilized or alternatively overexpressed as a responses to DNA damage. It is hypothesized that the development of colon carcinoma during pregnancy can be attributed to alterations of the p53 tumor suppressor gene or gene product on one hand and a maternal immune-tolerant state on the other.

Tissue-Specific Expression of the Tobacco Mosaic Virus Movement Protein in Transgenic Potato Plants Alters Plasmodesmal Function and Carbohydrate Partitioning.

Transgenic potato (Solanum tuberosum) plants expressing the movement protein (MP) of tobacco mosaic virus (TMV) under the control of the promoters from the class I patatin gene (B33) or the nuclear photosynthesis gene (ST-LS1) were employed to further explore the mode by which this viral protein interacts with cellular metabolism to change carbohydrate allocation. Dye-coupling experiments established that expression of the TMV-MP alters plasmodesmal function in both potato leaves and tubers when expressed in the respective tissues. However, whereas the size-exclusion limit of mesophyll plasmodesmata was increased to a value greater than 9.4 kD, this size limit was smaller for plasmodesmata interconnecting tuber parenchyma cells. Starch and sugars accumulated in potato leaves to significantly lower levels in plants expressing the TMV-MP under the ST-LS1 promoter, and rate of sucrose efflux from petioles of the latter was higher compared to controls. It is interesting that this effect was expressed only in mature plants after tuber initiation. No effect on carbohydrate levels was found in plants expressing this protein under the B33 promoter. These results are discussed in terms of the mode by which the TMV-MP exerts its influence over carbon metabolism and photoassimilate translocation, and the possible role of plasmodesmal function in controlling these processes.

Sanctuary of hepatitis B virus in bone-marrow cells of patients undergoing liver transplantation.

Hepatitis B virus (HBV) reinfection after liver transplantation is a major problem. HBV is mainly a hepatotrophic virus but replicates in many extrahepatic tissues. We present here two cases of infected patients who underwent liver transplantation. Both underwent bone marrow (BM) and liver biopsies after transplantation. Biopsy specimens were stained for hepatitis B surface antigen (HB-sAg), and bone marrow aspirates and were separated for all subsets of cells. In both cases, HBV DNA analysis detected DNA in all BM fractions after transplantation, but HBV recurrence was found only in one case. We suggest that graft reinfection after liver transplantation may be caused by active replication of HBV in extrahepatic tissues and that BM cells are probably one of the major sanctuaries. The use of immunoprophylaxis based on BM-HBV studies is discussed.

Esophageal malignancy after liver transplantation in a patient with Barrett's esophagus.

Patients undergoing liver transplantation are predisposed to develop extrahepatic malignancies. It is also known that patients with predisposed conditions, such as Barrett's esophagus, have higher rates of esophageal carcinoma. We present here a patient who underwent liver transplantation, had Barrett's esophagus, and developed esophageal malignancy a short time after transplantation. Liver transplantation may be associated with acceleration of the precancerous condition and the development of malignancies.