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.

Brain neurotransmitters in an animal model with postpartum depressive-like behavior.

Post-Partum Depression (PPD) occurs in 15% of pregnancies and its patho-physiology is not known. We studied female BALB/c ("depressive") and C57BL/6 (control) mice as a model for PPD and assessed their behavior and correlates with brain neurotransmitters (NTs) - norepinephrine, dopamine, serotonin and intermediates, during the pre-pregnancy (PREP), pregnancy (PREG) and post-partum (PP) periods. Depressive-like behavior was evaluated by the Open Field (OFT), Tail Suspension (TST) and Forced Swim (FST) tests. Neurotransmitters (NTs) were determined in the striatum (care-giving), hippocampus (cognitive function) and hypothalamus (maternal care & eating behavior). In the BALB/c mice, while their performance in all behavioral tests was significantly reduced during pregnancy and P-P indicative of the development of depressive-like responses, no changes were observed in the C57BL/6 mice. Changes in NTs in BALB/C were as follows: PREP, all NTs in the three brain areas were decreased, although an increase in dopamine release was observed in the hippocampus. PREG: No changes were observed in the NTs except for a decrease in 5-HT in the striatum. P-P: striatum, low 5-HT, NE and dopamine; Hippocampus: low 5-HT, NE and high Dopamine; hypothalamus: all NTs increased, especially NE. Following pregnancy and delivery, the BALB/c mice developed depressive-like behavior associated with a significant decrease in 5-HT, dopamine and NE in the striatum and 5-HT and NE in the hippocampus. Dopamine increased in the latter together with a significant increase in all NTs in the hypothalamus. These findings suggest that the development of PPD may be associated with NT changes. Normalization of these alterations may have a role in the treatment of PPD.

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.

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 (AU)

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.

Air-stable PbSe/PbS and PbSe/PbSexS1-x core-shell nanocrystal quantum dots and their applications.

The optical properties and functionality of air-stable PbSe/PbS core-shell and PbSe/PbSexS1-x core-alloyed shell nanocrystal quantum dots (NQDs) are presented. These NQDs showed chemical robustness over months and years and band-gap tunability in the near infrared spectral regime, with a reliance on the NQD size and composition. Furthermore, these NQDs exhibit high emission quantum efficiencies of up to 65% and an exciton emission band that is narrower than that of the corresponding PbSe NQDs. In addition, the emission bands showed a peculiar energy shift with respect to the relevant absorption band, changing from a Stokes shift to an anti-Stokes shift, with an increase of the NQD diameter. The described core-shell structures and the corresponding PbSe core NQDs were used as passive Q-switches in eye-safe lasers of Er:glass, where they act as saturable absorbers. The absorber saturation investigations revealed a relatively large ground-state cross-section of absorption (sigma gs = 10(-16) - 10(-15) cm2) and a behavior of a "fast" absorber with an effective lifetime of tau eff approximately 4.0 ps is proposed. This lifetime is associated with the formation of multiple excitons at the measured pumping power. The product of sigma gs and tau eff enables sufficient Q-switching performance and tunability in the near infrared spectral regime. The amplified spontaneous emission properties of PbSe NQDs were examined under continuous illumination by a diode laser at room temperature, suitable for standard device conditions. The results revealed a relatively large gain parameter (g = 2.63 - 6.67 cm-1). The conductivity properties of PbSe NQD self-assembled solids, annealed at 200 degrees C, showed an Ohmic behavior at the measured voltages (up to 30 V), which is governed by a variable-range-hopping charge transport mechanism.

The CCR5Delta32 allele is associated with reduced liver inflammation in hepatitis C virus infection.

CCR5Delta32 is a deletion mutation in the chemokine receptor CCR5. Liver inflammatory activity was found to be significantly reduced (P = 0.005) in Jewish Israeli patients infected with the hepatitis C virus (HCV) carrying the CCR5Delta32 allele. The CCR5Delta32 allele does not alter susceptibility to HCV infection; however, it may play a role in the progression and outcome of the disease.

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.

Hepatitis c virus infection in employees of a large university hospital in Israel.

OBJECTIVE: To assess whether hospital work constitutes a risk factor for hepatitis C virus (HCV) infection among employees of a large hospital in Israel. DESIGN: Seroprevalence survey. SETTING: A 1,006-bed, tertiary-care university hospital in Jerusalem. PARTICIPANTS: All 5,444 employees (18-65 years old) were eligible; 4,287 (79%) participated in the survey. METHODS: Sera were tested for antibodies to HCV (anti-HCV) using a third-generation enzyme immunoassay. A third-generation strip immunoblot assay was used for confirmation. Participants were interviewed regarding their occupational history, and they completed a self-administered questionnaire covering history of non-occupational exposure to blood and country of birth. Other demographic information was obtained from the personnel department. Rates and odds ratios (ORs) were calculated, and multivariate logistic-regression analyses were performed to adjust for potential confounding variables. RESULTS: Anti-HCV was found in 0.9% of employees (37/4,287; 95% confidence interval, 0.6-1.1), ranging from 0.1% among those born in Israel to 5.7% among those born in Central Asia. After age, gender, social status, country of birth, and history of blood transfusion were controlled for in a logistic regression, occupational exposure to blood > or = 10 years was significantly associated with the presence of antibodies (OR, 2.6; P=.01). Presence of anti-HCV also was associated with country of birth (range: Israel OR, 1; West OR, 3.8 [P=.1]; Central Asia OR, 48.6 [P<.0001]) and history of blood transfusion (OR, 2.7; P=.01). No significant associations were found between anti-HCV and age, gender, social status, history of tattoo, acupuncture, current occupation, department, exposure to blood in current occupation, adherence to safety precautions, or history of percutaneous injury. The association with length of exposure was stronger (OR, 3.6; P=.01) when the same logistic regression was run excluding the outlier ethnic group of Central Asia. CONCLUSIONS: Hospital work does not seem to constitute a major risk factor for HCV infection in Israel today. A higher prevalence of anti-HCV among employees with longer versus shorter lengths of occupational exposure may be due to a cumulative effect of exposure over the years. Infection control efforts in recent years may have contributed to this association.

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.

Preclinical evaluation of two human anti-hepatitis B virus (HBV) monoclonal antibodies in the HBV-trimera mouse model and in HBV chronic carrier chimpanzees.

Two human monoclonal antibodies (mAbs) against hepatitis B surface antigen (HBsAg) generated in the Trimera mouse system are described. Both mAbs 17.1.41 and 19.79.5 are of the IgG1 isotype and have high affinity constants for HBsAg binding in the range of 10(-10) mol/L. Monoclonal antibody 17.1.41 recognizes a conformational epitope on the a determinant of HBsAg whereas mAb 19.79.5 recognizes a linear one. The 2 mAbs bind to a panel of hepatitis B virus (HBV) subtypes with distinct patterns. The neutralizing activity of these antibodies was tested in 2 different animal model systems. Administration of each mAb to HBV-Trimera mice, a system that provides a mouse model for human hepatitis B infection, reduced the viral load and the percentage of HBV-DNA-positive mice in a dose-dependent manner. These 2 mAbs were more effective than a polyclonal antibody preparation (Hepatect; Biotest Pharma, Dreieich, Germany) in both inhibition of HBV liver infection and reduction of viral load. A single administration of a mixture of these mAbs into HBV chronic carrier chimpanzees resulted in immediate reduction in HBsAg levels followed by recurrence to initial levels within few days. Thus, these mAbs may be potential candidates for preventive therapy or in combination with other antiviral agents against HBV. Further studies in humans are needed to assess these mAbs in various clinical indications.

Transient transmission of hepatitis C virus from mothers to newborns.

A study was performed in Israel to determine the rate of mother-to-infant HCV transmission in newborns at risk. A group of 22 HCV-infected mothers and their 23 newborns were followed up from early after birth by testing their serum for the presence of HCV antibodies and HCV-RNA. Antibody against HCV was detected in the blood of all newborns immediately after birth, but dropped to low or undetectable levels by 7 months of age. HCV-RNA was detected 2 days after birth in the blood of five infants (22%) but fell to undetectable levels by 6 months. HCV-HVR1 sequence analysis performed in one mother-infant pair on the second day after birth revealed two nucleotide changes. Two months later the same sequence was detected again in the HVR1, suggesting a very low replication rate. Thus, the study showed that vertically transmitted HCV was eliminated in all newborn infants by 6 months after delivery, with concomitant disappearance of HCV antibodies. The mechanism of HCV elimination in newborns at risk remains to be elucidated.