Risk of second cancer after lymphohematopoietic neoplasm

People living with lymphohematopoietic neoplasms (LHNs) are known to have increased risks of second cancer; however, the incidence of second cancers after LHNs has not been studied extensively in Australia. The Australian Cancer Database was used to analyze site-specific risk of second primary cancer after LHNs in 127,707 patients diagnosed between 1983 and 2005. Standardized incidence ratios (SIRs) were calculated using population rates. Overall, patients with an LHN had nearly twice the risk of developing a second cancer compared to the Australian population. Among 40,321 patients with non-Hodgkin's lymphoma (NHL), there was over a fourfold significant increase in melanoma, Kaposi sarcoma, cancer of the lip, connective tissue and peripheral nerves, eye, thyroid, Hodgkin's disease (HD) and myeloid leukemia. Among 6,396 patients with HD, there was over a fourfold significant increase in melanoma, Kaposi sarcoma, cancer of the lip, oral cavity and pharynx, female breast, uterine cervix, testis, thyroid, NHL and myeloid leukemia. Among the 33,025 patients with lymphoid and myeloid leukemia, significant excess were seen for cancers of the lip, eye, connective tissue and peripheral nerves, NHL and HD. Among the 13,856 patients with plasma cell tumors, there was over fourfold significant increase for melanoma, cancer of the connective tissue and peripheral nerves and myeloid leukemia. Our findings provide evidence of an increased risk of cancer, particularly ultraviolet radiation- and immunosuppression-related cancers, after an LHN in Australia. Copyright © 2010 UICC.

Study of leukemia inhibitory factor polymorphism within an Australian multiple sclerosis population

OBJECTIVE: To examine a polymorphism within the 3' untranslated region of the leukemia inhibitory factor gene for an association with multiple sclerosis within an Australian case-control population. METHODS: A test group of 121 unrelated multiple sclerosis patients, of Caucasian origin, and 121 controls, matched for ethnicity, sex and age (+/-5 years) were included in the study. The LIF 3' UTR StuI polymorphism was genotyped by restriction fragment length polymorphism analysis. Statistical analysis of genotype and allele frequencies included Hardy-Weinberg law and conventional contingency table analysis incorporating the standard chi-squared test for independence. RESULTS: Allelic and genotype frequencies did not demonstrate a significant association between the case and control groups for the tested LIF 3' UTR StuI polymorphism. CONCLUSION: The results indicate that the LIF 3' UTR StuI polymorphism is not associated with multiple sclerosis, however we cannot exclude the hypothesis that other polymorphic alleles of LIF could be implicated in MS susceptibility.

Packed red blood cells suppress myeloid dendritic cell and monocyte inflammatory responses in a whole blood model of transfusion

Purpose/Objective: The basis for poor outcomes in some patients post transfusion remains largely unknown. Despite leukodepletion, there is still evidence of immunomodulatory effects of transfusion that require further study. In addition, there is evidence that the age of blood components transfused significantly affects patient outcomes. Myeloid dendritic cell (DC) and monocyte immune function were studied utilising an in vitro whole blood model of transfusion. Materials and methods: Freshly collected (‘recipient’) whole blood was cultured with ABO compatible leukodepleted PRBC at 25% blood replacement-volume (6hrs). PRBC were assayed at [Day (D) 2, 14, 28and 42 (date-of expiry)]. In parallel, LPS or Zymosan (Zy) were added to mimic infection. Recipients were maintained for the duration of the time course (2 recipients, 4 PRBC units, n = 8).Recipient DC and monocyte intracellular cytokines and chemokines (IL-6, IL-10, IL-12,TNF-a, IL-1a, IL-8, IP-10, MIP-1a, MIP-1b, MCP-1) were measured using flow cytometry. Changes in immune response were calculated by comparison to a parallel no transfusion control (Wilcoxin matched pairs). Influence of storage age was calculated using ANOVA. Results: Significant suppression of DC and monocyte inflammatory responses were evident. DC and monocyte production of IL-1a was reduced following exposure to PRBC regardless of storage age (P < 0.05 at all time points). Storage independent PRBC mediated suppression of DC and monocyte IL-1a was also evident in cultures costimulated with Zy. In cultures co-stimulated with either LPS or Zy, significant suppression of DC and monocyte TNF-a and IL-6 was also evident. PRBC storage attenuated monocyte TNF-a production when co-cultured with LPS (P < 0.01 ANOVA). DC and monocyte production of MIP-1a was significantly reduced following exposure to PRBC (DC: P < 0.05 at D2, 28, 42; Monocyte P < 0.05 all time points). In cultures co-stimulated with LPS and zymosan, a similar suppression of MIP-1a production was also evident, and production of both DC and monocyte MIP-1b and IP-10 were also significantly reduced. Conclusions: The complexity of the transfusion context was reflected in the whole blood approach utilised. Significant suppression of these key DC and monocyte immune responses may contribute to patient outcomes, such as increased risk of infection and longer hospital stay, following blood transfusion.

Receptor tyrosine kinases and their activation in melanoma

Receptor tyrosine kinases (RTKs) and their downstream signalling pathways have long been hypothesized to play key roles in melanoma development. A decade ago, evidence was derived largely from animal models, RTK expression studies and detection of activated RAS isoforms in a small fraction of melanomas. Predictions that overexpression of specific RTKs implied increased kinase activity and that some RTKs would show activating mutations in melanoma were largely untested. However, technological advances including rapid gene sequencing, siRNA methods and phospho-RTK arrays now give a more complete picture. Mutated forms of RTK genes including KIT, ERBB4, the EPH and FGFR families and others are known in melanoma. Additional over- or underexpressed RTKs and also protein tyrosine phosphatases (PTPs) have been reported, and activities measured. Complex interactions between RTKs and PTPs are implicated in the abnormal signalling driving aberrant growth and survival in malignant melanocytes, and indeed in normal melanocytic signalling including the response to ultraviolet radiation. Kinases are considered druggable targets, so characterization of global RTK activity in melanoma should assist the rational development of tyrosine kinase inhibitors for clinical use. © 2011 John Wiley & Sons A/S.

RanGTPase : a candidate for myc-mediated cancer progression

Background Ras-related nuclear protein (Ran) is required for cancer cell survival in vitro and human cancer progression, but the molecular mechanisms are largely unknown. Methods We investigated the effect of the v-myc myelocytomatosis viral oncogene homolog (Myc) on Ran expression by Western blot, chromatin immunoprecipitation, and luciferase reporter assays and the effects of Myc and Ran expression in cancer cells by soft-agar, cell adhesion, and invasion assays. The correlation between Myc and Ran and the association with patient survival were investigated in 14 independent patient cohorts (n = 2430) and analyzed with Spearman's rank correlation and Kaplan-Meier plots coupled with Wilcoxon-Gehan tests, respectively. All statistical tests were two-sided. Results Myc binds to the upstream sequence of Ran and transactivates Ran promoter activity. Overexpression of Myc upregulates Ran expression, whereas knockdown of Myc downregulates Ran expression. Myc or Ran overexpression in breast cancer cells is associated with cancer progression and metastasis. Knockdown of Ran reverses the effect induced by Myc overexpression in breast cancer cells. In clinical data, a positive association between Myc and Ran expression was revealed in 288 breast cancer and 102 lung cancer specimens. Moreover, Ran expression levels differentiate better or poorer survival in Myc overexpressing breast (χ2 = 24.1; relative risk [RR] = 9.1, 95% confidence interval [CI] = 3.3 to 24.7, P <. 001) and lung (χ2 = 6.04; RR = 2.8, 95% CI = 1.2 to 6.3; P =. 01) cancer cohorts. Conclusions Our results suggest that Ran is required for and is a potential therapeutic target of Myc-driven cancer progression in both breast and lung cancers. © 2013 The Author.

Increased tubulointerstitial recruitment of human CD141(hi) CLEC9A(+) and CD1c(+) myeloid dendritic cell subsets in renal fibrosis and chronic kidney disease

Dendritic cells (DCs) play critical roles in immune-mediated kidney diseases. Little is known, however, about DC subsets in human chronic kidney disease, with previous studies restricted to a limited set of pathologies and to using immunohistochemical methods. In this study, we developed novel protocols for extracting renal DC subsets from diseased human kidneys and identified, enumerated, and phenotyped them by multicolor flow cytometry. We detected significantly greater numbers of total DCs as well as CD141(hi) and CD1c(+) myeloid DC (mDCs) subsets in diseased biopsies with interstitial fibrosis than diseased biopsies without fibrosis or healthy kidney tissue. In contrast, plasmacytoid DC numbers were significantly higher in the fibrotic group compared with healthy tissue only. Numbers of all DC subsets correlated with loss of kidney function, recorded as estimated glomerular filtration rate. CD141(hi) DCs expressed C-type lectin domain family 9 member A (CLEC9A), whereas the majority of CD1c(+) DCs lacked the expression of CD1a and DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), suggesting these mDC subsets may be circulating CD141(hi) and CD1c(+) blood DCs infiltrating kidney tissue. Our analysis revealed CLEC9A(+) and CD1c(+) cells were restricted to the tubulointerstitium. Notably, DC expression of the costimulatory and maturation molecule CD86 was significantly increased in both diseased cohorts compared with healthy tissue. Transforming growth factor-β levels in dissociated tissue supernatants were significantly elevated in diseased biopsies with fibrosis compared with nonfibrotic biopsies, with mDCs identified as a major source of this profibrotic cytokine. Collectively, our data indicate that activated mDC subsets, likely recruited into the tubulointerstitium, are positioned to play a role in the development of fibrosis and, thus, progression to chronic kidney disease.

Human CD141(+) (BDCA-3)(+) dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141(+) DC subset. CD141(+) DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-beta, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c(+) DC subset. Polyinosine-polycytidylic acid (poly I:C)-activated CD141(+) DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8(+) cytotoxic T lymphocytes than poly I:C-activated CD1c(+) DCs. Importantly, CD141(+) DCs, but not CD1c(+) DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141(+) DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8 alpha(+) DC subset. The data demonstrate a role for CD141(+) DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.

Manufacturing and use of human placenta-derived mesenchymal stromal cells for phase I clinical trials: Establishment and evaluation of a protocol

Background/Aim. Mesenchymal stromal cells (MSCs) have been utilised in many clinical trials as an experimental treatment in numerous clinical settings. Bone marrow remains the traditional source tissue for MSCs but is relatively hard to access in large volumes. Alternatively, MSCs may be derived from other tissues including the placenta and adipose tissue. In an initial study no obvious differences in parameters such as cell surface phenotype, chemokine receptor display, mesodermal differentiation capacity or immunosuppressive ability, were detected when we compared human marrow derived- MSCs to human placenta-derived MSCs. The aim of this study was to establish and evaluate a protocol and related processes for preparation placenta-derived MSCs for early phase clinical trials. Methods. A full-term placenta was taken after delivery of the baby as a source of MSCs. Isolation, seeding, incubation, cryopreservation of human placentaderived MSCs and used production release criteria were in accordance with the complex regulatory requirements applicable to Code of Good Manufacturing Practice manufacturing of ex vivo expanded cells. Results. We established and evaluated instructions for MSCs preparation protocol and gave an overview of the three clinical areas application. In the first trial, MSCs were co-transplanted iv to patient receiving an allogeneic cord blood transplant as therapy for treatmentrefractory acute myeloid leukemia. In the second trial, MSCs were administered iv in the treatment of idiopathic pulmonary fibrosis and without serious adverse effects. In the third trial, MSCs were injected directly into the site of tendon damage using ultrasound guidance in the treatment of chronic refractory tendinopathy. Conclusion. Clinical trials using both allogeneic and autologous cells demonstrated MSCs to be safe. A described protocol for human placenta-derived MSCs is appropriate for use in a clinical setting, relatively inexpensive and can be relatively easily adjusted to a different set of regulatory requirements, as applicable to early phase clinical trials.

Novel derivatives of spirohydantoin induce growth inhibition followed by apoptosis in leukemia cells

Hydantoin derivatives possess a variety of biochemical and pharmacological properties and consequently are used to treat many human diseases. However, there are only few studies focusing on their potential as cancer therapeutic agents. In the present study, we have examined anticancer properties of two novel spirohydantoin compounds, 8-(3,4-difluorobenzyl)-1'-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1] octane-3,4'-imidazolidine]-2',5'-dione (DFH) and 8-(3,4-dichlorobenzyl)-1'-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1]octane-3,4'-imidazolidine]-2',5'-dione (DCH). Both the compounds exhibited dose- and time-dependent cytotoxic effect on human leukemic cell lines, K562, Reh, CEM and 8ES. Incorporation of tritiated thymidine ([H-3) thymidine) in conjunction with cell cycle analysis suggested that DFH and DCH inhibited the growth of leukemic cells. Downregulation of PCNA and p-histone H3 further confirm that the growth inhibition could be at the level of DNA replication. Flow cytometric analysis indicated the accumulation of cells at subG1 phase suggesting induction of apoptosis, which was further confirmed and quantified both by fluorescence-activated cell sorting (FACS) and confocal microscopy following annexin V-FITC/propidium iodide (PI) staining. Mechanistically, our data support the induction of apoptosis by activation of the mitochondrial pathway. Results supporting such a model include, elevated levels of p53, and BAD, decreased level of BCL2, activation and cleavage of caspase 9, activation of procaspase 3, poly (ADP-ribosyl) polymerase (PARP) cleavage, downregulation of Ku70, Ku80 and DNA fragmentation. Based on these results we discuss the mechanism of apoptosis induced by DFH and its implications in leukemia therapy. (C) 2008 Elsevier Inc. All rights reserved.

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H2O2, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl(+) cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl(+) CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl(+) cells. NAC enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

Synthesis of 2-(5-((5-(4-chlorophenyl)furan 2-yl)methylene)-4-oxo-2-thioxo-thiazolidi n-3-yl)acetic acid derivatives and evaluation of their cytotoxicity and induction of apoptosis in human leukemia cells

In order to explore the anticancer effect associated with the thiazolidinone framework, several 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid derivatives 5(a-1) were synthesized. Variation in the functional group at C-terminal of the thiazolidinone led to set of compounds bearing amide moiety. Their chemical structures were confirmed by H-1 NMR, IR and Mass Spectra analysis. These thiazolidinone compounds containing furan moiety exhibits moderate to strong antiproliferative activity in a cell cycle stage-dependent and dose dependent manner in two different human leukemia cell lines. The importance of the electron donating groups on thiazolidinone moiety was confirmed by MTT and Trypan blue assays and it was concluded that the 4th position of the substituted aryl ring plays a dominant role for its anticancer property. Among the synthesized compounds, 5e and 5f have shown potent anticancer activity on both the cell lines tested. To rationalize the role of electron donating group in the induction of cytotoxicity we have chosen two molecules (5e and 5k) having different electron donating group at different positions. LDH assay, Flow cytometric analysis and DNA fragmentation suggest that 5e is more cytotoxic and able to induce the apoptosis. (C) 2009 Elsevier Ltd. All rights reserved.

Synthesis and in vitro cytotoxic evaluation of novel diazaspiro bicyclo hydantoin derivatives in human leukemia cells: A SAR study

To study the structure activity relationship (SAR) on the cytotoxic activity and probe the structural requirement for the potent antitumor activity, a series of novel diazaspiro bicyclo hydantoin derivatives were designed and synthesized. Their structures were confirmed by H-1 NMR, LCMS and IR analyses. The antiproliferative effect of these compounds were determined against human leukemia, K562 (chronic myelogenous leukemia) and CEM (T-cell leukemia) cells using trypan blue and MTT assay, and the SAR associated with the position of N-terminal substituents in diazaspiro bicyclo hydantoin have also been discussed. It has been observed that these compounds displayed strong, moderate and weak cytotoxic activities. Interestingly, compounds having electron withdrawing groups at third and fourth position of the phenyl ring displayed selectively cytotoxic activities to both the cell lines tested with IC50 value lower than 50 mu M. In addition, the cytotoxic activities of the compounds 7(a-o) bearing the substituents at N-3 position of diazaspiro bicyclo hydantoin increases in the order alkene > ester > ether and plays an important role in determining their antitumor activities. The position and number of substituents in benzyl group attached to N-8 of diazaspiro bicyclo hydantoin nucleus interacted selectively with specific targets leading to the difference of biochemical and pharmacological effects.