Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.

Local chickens in East African region: their production and potential.

Indigenous chickens (IC) contribute socioeconomically to household food security in the region of East Africa. However, their potential and improvement are not well documented. This review is aimed at exploring the production and potential of indigenous chickens in East Africa. The various tools for literature search such as google search and Google scholars, agricultural journals, animal sciences and health journals, poultry related journals, and country online databases were used to gather information. IC were primarily reared by women and were kept predominantly under scavenging systems where the conditions of management (feeding, housing, and health care) are poor. They presented a high variation in their reproduction and production characteristics. The products (meat and eggs) were of good quality and preferred by the local consumers. Despite the variation and potential of IC, improvements in the village system were constrained by diseases and loss due to Newcastle, Gumboro, and Ecto-endo parasites and predators. Farmers primarily used traditional methods to control the diseases, and some used conventional medications and vaccines. Due to the potential of IC, the exploration of various strategies for improvement supported by the details of their genetic variability and adaptation as well as different management conditions was a goal of this review.

Quantitative measurement of alterations in DNA damage repair (DDR) pathways using single cell network profiling (SCNP).

BACKGROUND: Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity. METHODS: Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates. RESULTS: Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples. CONCLUSIONS: SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.

Voreloxin is an anticancer quinolone derivative that intercalates DNA and poisons topoisomerase II.

BACKGROUND: Topoisomerase II is critical for DNA replication, transcription and chromosome segregation and is a well validated target of anti-neoplastic drugs including the anthracyclines and epipodophyllotoxins. However, these drugs are limited by common tumor resistance mechanisms and side-effect profiles. Novel topoisomerase II-targeting agents may benefit patients who prove resistant to currently available topoisomerase II-targeting drugs or encounter unacceptable toxicities. Voreloxin is an anticancer quinolone derivative, a chemical scaffold not used previously for cancer treatment. Voreloxin is completing Phase 2 clinical trials in acute myeloid leukemia and platinum-resistant ovarian cancer. This study defined voreloxin's anticancer mechanism of action as a critical component of rational clinical development informed by translational research. METHODS/PRINCIPAL FINDINGS: Biochemical and cell-based studies established that voreloxin intercalates DNA and poisons topoisomerase II, causing DNA double-strand breaks, G2 arrest, and apoptosis. Voreloxin is differentiated both structurally and mechanistically from other topoisomerase II poisons currently in use as chemotherapeutics. In cell-based studies, voreloxin poisoned topoisomerase II and caused dose-dependent, site-selective DNA fragmentation analogous to that of quinolone antibacterials in prokaryotes; in contrast etoposide, the nonintercalating epipodophyllotoxin topoisomerase II poison, caused extensive DNA fragmentation. Etoposide's activity was highly dependent on topoisomerase II while voreloxin and the intercalating anthracycline topoisomerase II poison, doxorubicin, had comparable dependence on this enzyme for inducing G2 arrest. Mechanistic interrogation with voreloxin analogs revealed that intercalation is required for voreloxin's activity; a nonintercalating analog did not inhibit proliferation or induce G2 arrest, while an analog with enhanced intercalation was 9.5-fold more potent. CONCLUSIONS/SIGNIFICANCE: As a first-in-class anticancer quinolone derivative, voreloxin is a toposiomerase II-targeting agent with a unique mechanistic signature. A detailed understanding of voreloxin's molecular mechanism, in combination with its evolving clinical profile, may advance our understanding of structure-activity relationships to develop safer and more effective topoisomerase II-targeted therapies for the treatment of cancer.

The economic burden to the public health system of treating non-viral injecting-related injury and disease in Australia (a cost of illness analysis).

OBJECTIVE: We estimated the cost to the public health system of treating Injecting-Related Injuries and Diseases (IRIDs) in the three most populous states in Australia in the 12 months over 2005/06. METHODS: We conducted a cost of illness analysis from the perspective of the public health system. Costs of treating IRIDs in the community were estimated from health service utilisation surveys of injecting drug users and physicians (yielding data on Government subsidised physician visits, medicines prescribed and emergency department presentations). Data on admitted hospitalisations in public hospitals due to IRIDs were extracted from State Government databases. Appropriate costs were attached to all Government-borne services and prescriptions to estimate the total cost to the public health system of treating IRIDs in 2005/06 in Queensland, NSW and Victoria. RESULTS: Our estimate of the cost to the public health system of treating IRIDs in Queensland, NSW and Victoria in 2005/06 was $20 million. CONCLUSION: IRIDs are an under-recognised harm resulting from injecting drug use, but the economic burden of IRIDs in Australia are non-negligible. Research is needed to identify cost effective programs to reduce the clinical and economic burden caused by IRIDs, particularly to reduce hospitalisations due to IRIDs. IMPLICATIONS: General practitioners, clinicians and other health workers need to be alert to IRIDs in their injecting drug user clients to prevent progression to more serious disease and consequent elevation of the associated economic costs.

SNS-032 is a potent and selective CDK 2, 7 and 9 inhibitor that drives target modulation in patient samples.

PURPOSE: SNS-032 (formerly BMS-387032) is a potent, selective inhibitor of cyclin-dependent kinases (CDK) 2, 7 and 9, currently in phase 1 clinical trial for chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). We used the MM cell line RPMI-8226 to evaluate the relationship between duration of SNS-032 exposure, target modulation of CDKs 2, 7 and 9, and induction of apoptosis. We also assessed target modulation in patient peripheral blood mononuclear cells (PBMCs) from phase 1 solid tumor patients treated with SNS-032. METHODS: Proliferation and colony forming assays were used to evaluate cytotoxicity, Western blot analyses to evaluate target modulation, FACS analysis to assess cell cycle distribution, RT-PCR to evaluate transcriptional inhibition. RESULTS: SNS-032 blocks the cell cycle via inhibition of CDKs 2 and 7, and transcription via inhibition of CDKs 7 and 9. Treatment of RPMI-8226 MM cells at 300 nM (IC(90)) for 6 h was sufficient for commitment to apoptosis. This correlated with inhibition of CDKs 2, 7 and 9, as reflected in substrate signaling molecules. SNS-032 activity was unaffected by human serum. Target modulation was observed in PBMC from treated patients. CONCLUSIONS: These results demonstrate SNS-032 target modulation of CDKs 2, 7 and 9, and establish 6 h exposure as sufficient to commit RPMI-8226 MM cells to apoptosis. Combined with the demonstration of target modulation in PBMC from phase 1 solid tumor patients treated with SNS-032, these data support the ongoing clinical study of SNS-032 in MM and CLL.

Prevalence and predictors of injecting-related injury and disease among clients of Australia's needle and syringe programs.

OBJECTIVE: To identify lifetime prevalence and predictors of self-reported injecting-related injuries and diseases (IRID) and/or injecting-related problems (IRP) among a national cross-sectional sample of injecting drug users. METHODS: 1,961 clients of 45 needle and syringe programs (NSPs) who participated in the 2006 Australian NSP Survey self-completed an item regarding lifetime experience of eight separate IRIDs and IRPs. RESULTS: Sixty-nine per cent of participants reported a history of IRID/IRP, with a mean of 1.9 injuries/problems (range 0-8). Lifetime prevalence of specific injuries/problems ranged from problems finding a vein (43%) to endocarditis (4%). Factors independently associated with IRID/IRP included bisexual identity; daily or more frequent injecting; injection of pharmaceutical preparations; female gender; longer injecting history; and hepatitis C antibody-positive serostatus. CONCLUSIONS: Consistent with existing literature, results suggest that vascular injury and localised infections are common among IDUs; and that treatment-seeking is often delayed until serious complications arise. IMPLICATIONS: Findings support the imperative for co-ordinated and timely treatment and prevention activities to reduce the severity and burden of these prevalent injecting outcomes.

A novel zinc finger transcription factor with two isoforms that are differentially repressed by estrogen receptor-alpha.

Estrogen receptor-alpha (ERalpha) can induce the expression of genes in response to estrogen by binding to estrogen response elements in the promoters of target genes. There is growing evidence that ERalpha can alter patterns of gene expression in response to ligand by regulating the activity of other factors through a direct protein-protein interaction. To identify other factors that are regulated by ERalpha, a yeast two-hybrid screen was performed that identified a novel Cys(2)His(2) zinc finger protein named ZER6. The ZER6 protein contains a Kruppel-associated box domain and six Cys(2)His(2) zinc fingers. Transcripts from the ZER6 gene can have alternate 5' exons and encode either a p71 or p52 isoform. The p52-ZER6 protein interacts strongly with ERalpha in the presence of 17beta-estradiol, whereas the p71-ZER6 isoform has a HUB-1 amino-terminal domain that inhibits the interaction with ERalpha. A consensus ZER6 binding element was defined using PCR-assisted binding site selection. In COS-1 cells, both the p52 and p71 isoforms can activate transcription through the ZER6 binding element; however, in the presence of ERalpha, transactivation by the p52 isoform is specifically repressed. Overexpression of the p52 isoform was able to abrogate activation by p71-ZER6. Expression of ZER6 was largely restricted to the mammary gland with a lower level of expression in the kidney. We conclude that ZER6 is a novel zinc finger transcription factor in which regulation of transcription in hormone-responsive cells can be controlled by the relative level of expression of two distinct isoforms.