ST18 affects cell-cell adhesion in pemphigus vulgaris in a tumour necrosis factor-α-dependent fashion.

BACKGROUND: Pemphigus vulgaris (PV) is a life-threatening mucocutaneous autoimmune blistering disease. We previously showed that genetic variants within the ST18 gene promoter area confer a sixfold increase in the propensity to develop PV. ST18, a transcription factor, was found to be overexpressed in the epidermis of patients with PV. In addition, it was found to promote autoantibody-mediated abnormal epidermal cell-cell adhesion and secretion of proinflammatory mediators by keratinocytes. OBJECTIVES: To delineate the mechanism through which ST18 contributes to destabilization of cell-cell adhesion. METHODS: We used quantitative reverse-transcriptase polymerase chain reaction, immunofluorescence microscopy, a luciferase reporter system, site-directed mutagenesis, chromatin immunoprecipitation (ChIP) and the dispase dissociation assay. RESULTS: The ChIP and luciferase reporter assays showed that ST18 directly binds and activates the TNF promoter. Accordingly, increased ST18 expression contributes to PV pathogenesis by destabilizing cell-cell adhesion in a tumour necrosis factor (TNF)-α-dependent fashion. In addition, dual immunofluorescence staining showed increased expression of both ST18 and TNF-α in the skin of patients with PV carrying an ST18-associated PV risk variant, which was found to be associated with a more extensive PV phenotype. CONCLUSIONS: Our findings suggest a role for TNF-α in mediating the deleterious effect of increased ST18 expression in PV skin.

Laryngeal mucous membrane pemphigoid serves as a prognostic factor for poor response to treatment with rituximab.

Mucous membrane pemphigoid (MMP) is an autoimmune blistering, scarring and occasionally mutilating disease that may progress to blindness or airway obstruction. Over the past few years, rituximab (RTX) has emerged as a potential therapeutic solution for MMP; however, the literature regarding its efficacy in the treatment of severe MMP is sparse. We studied four patients with recalcitrant MMP who were treated with RTX. Three of these had recalcitrant laryngeal disease; two were unresponsive to RTX, while the third patient achieved complete remission (CR) but relapsed twice. The fourth patient, who had oral and ocular disease, also achieved CR. In addition, we reviewed 143 cases of MMP treated with RTX reported in the literature to date. Of these, 120 had late observation endpoints, of whom 81 (67.5%) achieved CR, 24 (20%) received partial remission and 15 (12.5%) had no remission. Based on this study, the presence of laryngeal MMP seems to predict refractoriness to RTX treatment. In conclusion, we found that RTX can ameliorate the MMP course and that laryngeal involvement, which is known to be a prognostic factor for severe MMP, may also predict poor response to RTX.

Palmoplantar keratoderma caused by a missense variant in CTSB encoding cathepsin B.

BACKGROUND: Palmoplantar keratoderma (PPK) refers to a large group of disorders characterized by extensive genetic and phenotypic heterogeneity. PPK diagnosis therefore increasingly relies upon genetic analysis. AIM: To delineate the genetic defect underlying a case of diffuse erythematous PPK associated with peeling of the skin. METHODS: Whole exome and direct sequencing, real-time quantitative PCR, protein modelling and a cathepsin B enzymatic assay were used. RESULTS: The patient studied had severe diffuse erythematous PPK transgrediens. Pedigree analysis suggested an autosomal dominant mode of inheritance. Whole exome sequencing revealed a heterozygous missense mutation in the CTSB gene, encoding the cysteine protease cathepsin B. Genomic duplications in a noncoding region, which regulates the expression of CTSB, were recently found to cause erythrokeratolysis hiemalis, a rare autosomal dominant disorder of cornification. This mutation affects a highly conserved residue, and is predicted to be pathogenic. Protein modelling indicated that the mutation is likely to lead to increased endopeptidase cathepsin B activity. Accordingly, the CTSB variant was found to result in increased cathepsin B proteolytic activity. CONCLUSION: In summary, we report the identification of the first gain-of-function missense mutation in CTSB, which was found to be associated in one individual with a dominant form of diffuse PPK.

Molecular epidemiology of pachyonychia congenita in the Israeli population.

BACKGROUND: Pachyonychia congenita (PC) is a rare autosomal dominant disorder featuring palmoplantar keratoderma, nail dystrophy, oral leucokeratosis, pilosebaceous cysts and natal teeth. PC results from dominant mutations in one of five genes (KRT6A, KRT6B, KRT6C, KRT16, KRT17) encoding keratin proteins. AIM: To delineate the clinical and genetic features of PC in a series of Israeli patients. METHODS: We used direct sequencing of genomic DNA, and also used cDNA sequencing where applicable. RESULTS: We collected clinical information and molecular data in a cohort of Israeli families diagnosed with PC (n = 16). Most of the patients were Ashkenazi Jews and had a family history of PC. The most common clinical findings were painful focal plantar keratoderma (94%) accompanied by nail dystrophy (81%), pilosebaceous cysts (31%) and prenatal/natal teeth (13%). In contrast to the high prevalence of KRT6A mutations in other populations, we found that KRT16 mutations were the most common type among Israeli patients with PC (56%). Most (77%) of the Israeli patients with PC with KRT16 mutation carried the same variant (c.380G>A; p.R127H) and shared the same haplotype around the KRT16 locus, suggestive of a founder effect. CONCLUSION: The data gleaned from this study emphasizes the importance of population-specific tailored diagnostic strategies.

Treatment of hereditary hypotrichosis simplex of the scalp with topical gentamicin.

BACKGROUND: Hypotrichosis simplex of the scalp (HSS) is characterized by progressive loss of scalp hair that results in almost complete baldness at a young age. HSS is often caused by dominant nonsense mutations in CDSN encoding corneodesmosin, leading to the formation of an amyloid-like material, which interferes with normal hair follicle cycle. OBJECTIVES: As gentamicin has been shown to mediate ribosomal read-through, we aimed to ascertain its therapeutic efficacy in a small series of patients carrying a recurrent mutation in CDSN . METHODS: We used a green fluorescence reporter assay system, confocal microscopy and Western blot analysis to ascertain in vitro the ability of gentamicin to induce translational read-through across a causative CDSN mutation. RESULTS: Using a reporter assay, we initially showed that gentamicin induces read-through activity across an HSS-causing nonsense mutation. Gentamicin was further shown to rescue corneodesmosin translation in primary keratinocytes obtained from a patient with HSS. To validate the in vitro data, we conducted a pilot clinical trial where the scalp of four patients was treated topically with gentamicin for 6 months, demonstrating significant improvement as ascertained by the Severity of Alopecia Tool score. CONCLUSIONS: Our findings indicate that topical gentamicin should be considered as a potential therapeutic modality in HSS. What's already known about this topic? Hypotrichosis simplex of the scalp (HSS) is caused by nonsense mutations in CDSN encoding corneodesmosin. The mutant corneodesmosin has been hypothesized to be toxic to the hair follicles, leading to hypotrichosis. Disorders caused by nonsense mutations are amenable to ribosomal read-through using gentamicin. What does this study add? Gentamicin enhanced read-through activity and promoted full-length corneodesmosin synthesis in primary keratinocytes derived from patients carrying a nonsense mutation in CDSN. Topical treatment with gentamicin was found to rescue the hypotrichosis phenotype partially in four patients with HSS. What is the translational message? Topical gentamicin should be considered as a potential treatment for HSS.

A phenotype combining hidradenitis suppurativa with Dowling-Degos disease caused by a founder mutation in PSENEN.

BACKGROUND: Dowling-Degos disease (DDD), featuring reticulate pigmentation, and familial hidradenitis suppurativa (HS) share many clinical features including autosomal dominant inheritance, flexural location and follicular defects. The coexistence of the two disorders was recently found to result from mutations in PSENEN, encoding the γ-secretase subunit protein presenilin enhancer. OBJECTIVES: To investigate PSENEN mutations in a series of four unrelated patients who presented with combined DDD and HS. METHODS: Mutation and haplotype analysis of PSENEN by polymerase chain reaction, and cellular assays investigating the Notch signalling pathway. RESULTS: Here we report four families of Jewish Ashkenazi origin who presented with clinical features characteristic of both disorders. All patients were found to carry the same, heterozygous mutation in PSENEN (c.168T>G, p.Y56X). Haplotype analysis revealed that the mutation originated from a common ancestor. Genes associated with DDD, as well as HS, have been shown to encode important regulators of Notch signalling. Accordingly, using a reporter assay, we demonstrated decreased Notch activity in a patient's keratinocytes. CONCLUSIONS: The present data confirm the genetic basis of the combined DDD-HS phenotype and suggest that Notch signalling may play a central role in the pathogenesis of this rare condition.

Recessive epidermolytic ichthyosis results from loss of keratin 10 expression, regardless of the mutation location.

Epidermolytic ichthyosis (EI) is a rare skin disorder caused by mutations in the genes KRT1 and KRT10, and is usually inherited in an autosomal dominant fashion. Only five recessive mutations causing EI have been described, all of which are located in the central region of the KRT10 gene. In the current study, we aimed to identify the genetic defect underlying EI in a 12-year-old patient. Direct sequencing of the patient's genomic DNA revealed a novel homozygous nonsense mutation residing within the proximal part KRT10 first exon. The mutation was found to co-segregate with the disease phenotype in an autosomal recessive fashion. Using real-time quantitative PCR, we found an almost two-fold decrease in KRT10 expression in the patient's skin compared with the skin of healthy controls. Western blot analysis showed complete absence of keratin 10 protein in the patient's skin, suggesting early protein degradation.

Ambient temperature and congenital heart defects.

STUDY QUESTION: Does maternal exposure during pregnancy to higher ambient temperature increase the risk of congenital heart defects (CHDs)? SUMMARY ANSWER: Significant associations were found between maternal exposure during pregnancy to higher ambient temperature and CHDs risk especially during the cold season. WHAT IS KNOWN ALREADY: From rodents to non-human primates, a teratogenic effect of hyperthermic insult has been demonstrated. There are fewer data regarding the effect on the human fetus and specifically the association between maternal exposures during pregnancy to higher ambient temperature and CHDs. STUDY DESIGN, SIZE, DURATION: This population registry-based cohort study included 135 527 live and stillbirths in the Tel-Aviv region of Israel in 2000-2006. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two clinical diagnostic groups of isolated cardiac defects (atrial septal defects and ventricular septal defects: n = 542 and 481, respectively) and one group of multiple cardiac defects (defined by the presence of two or more cardiac malformations, n = 607) were studied. Temperature measurements were constructed from ambient stations and used to assess the impact of maternal exposure to average ambient temperature and extreme heat events (daily average temperature above the 90th percentile) during Weeks 3-8 of pregnancy on risk of CHDs. Logistic models, adjusted for sociodemographic covariates, were used to evaluate the associations between temperature and CHDs. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, we found no significant associations between ambient temperature and CHDs throughout the year, with one exception for multiple CHDs. After stratifying by season of conception, continuous exposure to average ambient temperature and maximum peak temperature (1°C increase) during the cold season increased the risk for multiple CHDs [odds ratio (OR) 1.05, 95% confidence interval (CI): 1.00, 1.10 and OR 1.03, 95% CI: 1.01, 1.05, respectively]. A 1-day increase in extreme heat events increased the risk for multiple CHDs (OR 1.13, 95% CI: 1.06, 1.21) and also for isolated atrial septal defects (OR 1.10, 95% CI: 1.02, 1.19). LIMITATIONS, REASONS FOR CAUTION: Information both on CHD cases and on ambient temperature was based on registries and it is possible that this may cause some misclassification. In urban areas, pregnant women may be exposed to higher temperatures than recorded by ambient monitors because of the 'heat island effect'. Furthermore, data for the amount of time spent indoors were unavailable and this could have resulted in exposure misclassification. WIDER IMPLICATIONS OF THE FINDINGS: The findings are important within the context of global climate change, which may have implications for public health in countries with mild winters and hot summers. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Israeli Ministry of Environmental Protection (research grant-7-2-7) and by the Environment and Health Fund (PhD Fellowship Program). There are no competing interests.

Sequence Variant in the TRIM39-RPP21 Gene Readthrough is Shared Across a Cohort of Arabian Foals Diagnosed with Juvenile Idiopathic Epilepsy.

Juvenile idiopathic epilepsy (JIE) is a self-limiting neurological disorder with a suspected genetic predisposition affecting young Arabian foals of the Egyptian lineage. The condition is characterized by tonic-clonic seizures with intermittent post-ictal blindness, in which most incidents are sporadic and unrecognized. This study aimed to identify genetic components shared across a local cohort of Arabian foals diagnosed with JIE via a combined whole genome and targeted resequencing approach: Initial whole genome comparisons between a small cohort of nine diagnosed foals (cases) and 27 controls from other horse breeds identified variants uniquely shared amongst the case cohort. Further validation via targeted resequencing of these variants, that pertain to non-intergenic regions, on additional eleven case individuals revealed a single 19bp deletion coupled with a triple-C insertion (Δ19InsCCC) within the TRIM39-RPP21 gene readthrough that was uniquely shared across all case individuals, and absent from three additional Arabian controls. Furthermore, we have confirmed recent findings refuting potential linkage between JIE and other inherited diseases in the Arabian lineage, and refuted the potential linkage between JIE and genes predisposing a similar disorder in human newborns. This is the first study to report a genetic variant to be shared in a sub-population cohort of Arabian foals diagnosed with JIE. Further evaluation of the sensitivity and specificity of the Δ19InsCCC allele within additional cohorts of the Arabian horse is warranted in order to validate its credibility as a marker for JIE, and to ascertain whether it has been introduced into other horse breeds by Arabian ancestry.

High incidence of B cell lymphomas derived from thymectomized AKR mice expressing TL.4 antigen.

AKR mice, 6-12 mo after birth, display a high incidence of spontaneous T cell lymphomas that can be prevented by thymus removal at the age of 1-3 mo. We report here the presence of dormant preleukemic cells among bone marrow cells of 8-12-mo-old AKR mice that have been thymectomized when 40-60 d old. Transplantation of bone marrow cells from these thymectomized AKR donors into syngeneic or hybrid (AKR X DBA/2)F1 intact or thymectomized recipients resulted in lymphoma development of AKR origin in 80-100% of the recipients. Analysis, by flow microfluorometry, of the antigenic cell surface phenotypes of the developing lymphomas revealed that all tumors were B cell lymphomas, since the cells stained with class-specific anti-IgM reagents and other reagents specific for B cells (RA3-2C2, RA3-6B2, anti-I-A, and anti-Fc receptor), and were Thy-1-. All these B cell tumors also expressed two T cell differentiation antigens, TL.4, found exclusively on T cell lymphomas, and Lyt-1 antigen, previously shown (11) to be expressed on some B cell neoplasms. The surface markers mu, I-A, RA3-2C2, and TL.4 identified by immunofluorescence, were shown to be integral membrane components synthesized by the tumor cells, rather than passively acquired proteins.

Subsecond induction of alpha4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions.

Leukocyte recruitment to target tissue is initiated by weak rolling attachments to vessel wall ligands followed by firm integrin-dependent arrest triggered by endothelial chemokines. We show here that immobilized chemokines can augment not only arrest but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium. Furthermore, when presented in juxtaposition to vascular cell adhesion molecule 1 (VCAM-1), the endothelial ligand for the integrin very late antigen 4 (VLA-4, alpha4beta1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1. Chemokines potentiate VLA-4 tethering within <0.1 s of contact through Gi protein signaling, the fastest inside-out integrin signaling events reported to date. Although VLA-4 affinity is not altered upon chemokine signaling, subsecond VLA-4 clustering at the leukocyte-substrate contact zone results in enhanced leukocyte avidity to VCAM-1. Endothelial chemokines thus regulate all steps in adhesive cascades that control leukocyte recruitment at specific vascular beds.

Linear accelerator stereotactic radiosurgery can modulate the clinical course of Hemangioblastoma: Case series and review of the literature.

Hemangioblastomas (HB) are benign low grade vascular tumors most frequently occurring in the cerebellum, brain stem, and spinal cord. Often associated with Von Hippel Lindau disease (VHL), the lesions are often multifocal requiring complex resection and are difficult to control. Linear Accelerator (LINAC) Stereotactic Radiosurgery (SRS) has been demonstrated to provide additional tumor control. In this case series, we present our multi-center experience utilizing LINAC SRS in fourteen patients with 23 lesions. We observed a tumor control rate of 87% and found interval changes in the peritumoral enhancement to correlate with treatment outcome. In our study, SRS treatment was also well-tolerated in both cystic and noncystic patients with multifocal disease. Disease control was achieved in all but three patients post-resection and no longitudinal radiation-induced secondary malignancy was observed. SRS response correlated highly with lesion size and radiation dose. We conclude that LINAC SRS is safe and effective for patients with HB and should be considered in addition to surgery in asymptomatic, VHL patients, deep seated lesions and isolated lesions.

Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.

Stem cell homing and repopulation are not well understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 were found to be critical for murine bone marrow engraftment by human severe combined immunodeficient (SCID) repopulating stem cells. Treatment of human cells with antibodies to CXCR4 prevented engraftment. In vitro CXCR4-dependent migration to SDF-1 of CD34+CD38-/low cells correlated with in vivo engraftment and stem cell function. Stem cell factor and interleukin-6 induced CXCR4 expression on CD34+ cells, which potentiated migration to SDF-1 and engraftment in primary and secondary transplanted mice. Thus, up-regulation of CXCR4 expression may be useful for improving engraftment of repopulating stem cells in clinical transplantation.

The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow.

The chemokine SDF-1 plays a central role in the repopulation of the bone marrow (BM) by circulating CD34(+) progenitors, but the mechanisms of its action remain obscure. To extravasate to target tissue, a blood-borne cell must arrest firmly on vascular endothelium. Murine hematopoietic progenitors were recently shown in vivo to roll along BM microvessels that display selectins and integrins. We now show that SDF-1 is constitutively expressed by human BM endothelium. In vitro, human CD34(+) cells establish efficient rolling on P-selectin, E-selectin, and the CD44 ligand hyaluronic acid under physiological shear flow. ICAM-1 alone did not tether CD34(+) cells under flow, but, in the presence of surface-bound SDF-1, CD34(+) progenitors rolling on endothelial selectin rapidly developed firm adhesion to the endothelial surface, mediated by an interaction between ICAM-1 and its integrin ligand, which coimmobilized with SDF-1. Human CD34(+) cells accumulated efficiently on TNF-activated human umbilical cord endothelial cells in the absence of SDF-1, but they required immobilized SDF-1 to develop firm integrin-mediated adhesion and spreading. In the absence of selectins, SDF-1 also promoted VLA-4-mediated, Gi protein-dependent tethering and firm adhesion to VCAM-1 under shear flow. To our knowledge, this is the first demonstration that SDF-1 expressed on vascular endothelium is crucial for translating rolling adhesion of CD34(+) progenitors into firm adhesion by increasing the adhesiveness of the integrins VLA-4 and LFA-1 to their respective endothelial ligands, VCAM-1 and ICAM-1.

Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function.

The chemokine stromal-derived factor-1 (SDF-1) controls many aspects of stem cell function. Details of its regulation and sites of production are currently unknown. We report that in the bone marrow, SDF-1 is produced mainly by immature osteoblasts and endothelial cells. Conditioning with DNA-damaging agents (ionizing irradiation, cyclophosphamide, and 5-fluorouracil) caused an increase in SDF-1 expression and in CXCR4-dependent homing and repopulation by human stem cells transplanted into NOD/SCID mice. Our findings suggest that immature osteoblasts and endothelial cells control stem cell homing, retention, and repopulation by secreting SDF-1, which also participates in host defense responses to DNA damage.

The murine C'-terminally alternatively spliced form of p53 induces attenuated apoptosis in myeloid cells.

The onset of p53-dependent apoptosis results from the accumulation of damaged DNA. Recently, it was shown that the C' terminus of the p53 protein plays a central role in sensing damaged DNA. In our present study, we examined the role of the C' terminus in the induction of apoptosis. A temperature-sensitive (ts) mutant of the alternatively spliced form of p53 (p53AS-ts) and the ts mutant of the regularly spliced form (p53RS-ts) were used to generate series of stable clones with increasing amounts of p53 protein. Apoptotic patterns induced by either the regularly spliced p53 product (p53RS) or a C'-terminally alternatively spliced p53 product (p53AS) were compared. We found that although both forms of p53 induced apoptosis following expression of the wild-type protein conformation, the kinetics were different. Apoptosis induced by the p53AS protein was attenuated compared to that induced by p53RS. The delay in the manifestation of the apoptotic features following p53AS expression was in agreement with a delay in the regulation of the expression of apoptosis-related genes. The observation that p53 with an altered C' terminus is still capable of inducing apoptosis suggests that the actual onset of the apoptotic process most probably involves structural domains other than the C' terminus of the p53 molecule. However, the fact that the apoptotic activity mediated by the p53AS product was slower than that mediated by the p53RS product suggests that the C' terminus indeed exerts a certain control on the apoptotic activity of the p53 molecule.

Hierarchy measurement for modeling network dynamics under directed attacks.

A fundamental issue in the dynamics of complex systems is the resilience of the network in response to targeted attacks. This paper explores the local dynamics of the network attack process by investigating the order of removal of the nodes that have maximal degree, and shows that this dynamic network response can be predicted from the graph's initial connectivity. We demonstrate numerically that the maximal degree M(τ) of the network at time step τ decays exponentially with τ via a topology-dependent exponent. Moreover, the order in which sites are removed can be approximated by considering the network's "hierarchy" function h, which measures for each node V_{i} how many of its initial nearest neighbors have lower degree versus those that have a higher one. Finally, we show that the exponents we identified for the attack dynamics are related to the exponential behavior of spreading activation dynamics. The results suggest that the function h, which has both local and global properties, is a novel nodal measurement for network dynamics and structure.

Analysis of the Association of Climate, Weather and Herd Immunity with the Spread of Equine Encephalosis Virus in Horses in Israel.

It is claimed that the distribution of Culicoides-borne viruses is highly influenced by climate. Equine encephalosis virus (EEV) is a Culicoides-borne orbivirus which affects horses and was recently found to be endemic in Israel. To test whether climate is a crucial factor in the geographical distribution of EEV, we collected blood samples from horses in Israel during the years 2002, 2007 and 2010 and tested them for the abundance of antibodies to EEV. Samples were also collected in 2011 from horses that were seronegative to the virus in 2010, to determine the rate of infection with EEV. It was found that seroprevalence fluctuated between the years and that in each year it was highest in a different climatic region. Interestingly, analysis of infection rate at the different farms showed a negative association with seroprevalence at prior observations. In addition, analysis of precipitation preceding the outbreak of EEV which occurred during 2008 revealed that an extremely dry period existed several months prior to the febrile outbreak with the average precipitation of spring 2008 being significantly lower than the average spring precipitation of the years 1997-2009. It is therefore conjectured that exposure to EEV is not climate specific. Rather, it is highly influenced by herd immunity and weather fluctuations which might change annually. This finding may have important implications for the prediction of the abundance of Culicoides-borne viruses in endemic regions.

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.

Effective elimination of lung metastases induced by tumor cells treated with hydrostatic pressure and N-acetyl-L-cysteine.

In previous studies, we have demonstrated that application of high hydrostatic pressure (P) to tumor cells in the presence of a slow-reacting membrane-impermeable cross-linker (CL), 2'-3'-adenosine dialdehyde, can rearrange cell surface proteins into immunogenic clusters. Here, we present evidence indicating that subsequent reduction of surface protein disulfides with N-acetyl-L-cysteine (NAC) further augments the immunogenic potential of PCL-modified tumor cells both in vitro and in vivo. Immunotherapy with PCL+NAC-modified 3LL-D122 Lewis lung carcinoma cells plus i.v. delivery of NAC in mice bearing established lung metastases provoked an antitumor response capable of eradicating the metastatic nodules as demonstrated by restoration of normal lung weight and histology. In addition, immunization with PCL+NAC-modified tumor cells gave rise to a strong delayed-type hypersensitivity recall response against parental D122 cells. We propose that this novel two-prong strategy, based on local immunization with autologous PCL+NAC-modified tumor cells and systemic boosting with NAC, could provide a practical, effective immunotherapeutic regimen for the treatment of human cancer.