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Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient's tumour possesses a unique set of mutations ('the mutanome') that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient's individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4(+) T cells. Vaccination with such CD4(+) immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4(+) T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient's tumour with vaccines produced 'just in time'.

Developments in sequencing technologies have not only led to a rapid generation of genomic and transcriptional data from cancer patients, but also revealed the vast diversity of cancer-specific changes in patient tumors. Among these, mutation changes in the protein sequence can result in novel epitopes recognized by the immune system and, therefore, can be employed in the development of personalized vaccines. Thanks to its easy design and scalable GMP production, vaccines based on mRNAs coding for mutated epitopes have emerged as a reliable strategy for the exploitation of the potential of patient-specific genomic data. In this review, we provide an overview of recent developments in actively personalized vaccinations, with a special focus on the promise of mRNA vaccines., (© 2015 S. Karger AG, Basel.)

The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-β chains directly from single antigen-specific CD8(+) or CD4(+) T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro-transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8(+) and CD4(+) T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes., (©2014 American Association for Cancer Research.)

The transcription of tumor mutations from DNA into RNA has implications for biology, epigenetics and clinical practice. It is not clear if mutations are in general transcribed and, if so, at what proportion to the wild-type allele. Here, we examined the correlation between DNA mutation allele frequency and RNA mutation allele frequency. We sequenced the exome and transcriptome of tumor cell lines with large copy number variations, identified heterozygous single nucleotide mutations and absolute DNA copy number, and determined the corresponding DNA and RNA mutation allele fraction. We found that 99% of the DNA mutations in expressed genes are expressed as RNA. Moreover, we found a high correlation between the DNA and RNA mutation allele frequency. Exceptions are mutations that cause premature termination codons and therefore activate nonsense-mediated decay. Beyond this, we did not find evidence of any wide-scale mechanism, such as allele-specific epigenetic silencing, preferentially promoting mutated or wild-type alleles. In conclusion, our data strongly suggest that genes are equally transcribed from all alleles, mutated and wild-type, and thus transcribed in proportion to their DNA allele frequency.

Studying distribution is necessary to understand and manage the dynamics of species with spatially structured populations. Here we studied the distribution in Tetranychus evansi and T. urticae, two mite pests of tomato, in the scope of evaluating factors that can influence the effectiveness of Integrated Pest Management strategies. We found greater positive density-dependent distribution with T. evansi than T. urticae when assayed on single, detached tomato leaves. Indeed, T. evansi distribution among leaflets increased with initial population density while it was high even at low T. urticae densities. Intensity and rate of damage to whole plants was higher with T. evansi than T. urticae. We further studied the circadian migration of T. evansi within plant. When T. evansi density was high the distribution behavior peaked between 8 am and 3 pm and between 8 pm and 3 am local time of Kenya. Over 24 h the total number of mites ascending and descending was always similar and close to the total population size. The gregarious behavior of T. evansi combined with its rapid population growth rate, may explain why few tomato plants can be severely damaged by T. evansi and how suddenly all the crop can be highly infested. However the localisation and elimination of the first infested plants damaged by T. evansi could reduce the risk of outbreaks in the entire crop. These findings suggest also that an acaricide treated net placed on the first infested plants could be very effective to control T. evansi. Moreover circadian migration would therefore accentuate the efficiency of an acaricide treated net covering the infested plants.

Background: Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. Here, we present an integrated map of the genome, transcriptome and immunome of an epithelial mouse tumor, the CT26 colon carcinoma cell line., Results: We found that Kras is homozygously mutated at p.G12D, Apc and Tp53 are not mutated, and Cdkn2a is homozygously deleted. Proliferation and stem-cell markers, including Top2a, Birc5 (Survivin), Cldn6 and Mki67, are highly expressed while differentiation and top-crypt markers Muc2, Ms4a8a (MS4A8B) and Epcam are not. Myc, Trp53 (tp53), Mdm2, Hif1a, and Nras are highly expressed while Egfr and Flt1 are not. MHC class I but not MHC class II is expressed. Several known cancer-testis antigens are expressed, including Atad2, Cep55, and Pbk. The highest expressed gene is a mutated form of the mouse tumor antigen gp70. Of the 1,688 non-synonymous point variations, 154 are both in expressed genes and in peptides predicted to bind MHC and thus potential targets for immunotherapy development. Based on its molecular signature, we predicted that CT26 is refractory to anti-EGFR mAbs and sensitive to MEK and MET inhibitors, as have been previously reported., Conclusions: CT26 cells share molecular features with aggressive, undifferentiated, refractory human colorectal carcinoma cells. As CT26 is one of the most extensively used syngeneic mouse tumor models, our data provide a map for the rationale design of mode-of-action studies for pre-clinical evaluation of targeted- and immunotherapies.

Species of the family Phytoseiidae are known as predatory mites, some of them being used in crops to control mite pests, all around the world. Neoseiulus (=Cydnodromus) californicus is among the most commonly used Phytoseiidae species in biological control programs, especially in vineyards, orchards and vegetable fields. This species is distributed world-wide but has never been reported from Australia. On the other hand, specimens morphologically close to N. californicus have been assigned to a species called Neoseiulus wearnei, only reported from Australia. Investigations based on morphological and molecular comparisons were carried out to investigate whether these two taxa are conspecific. Morphological analyses showed no significant difference between specimens identified as N. wearnei and N. californicus. Similarly, genetic distances between these taxa were null, showing that all these specimens belong to the same species. Although it is not yet possible to conclude that all the specimens identified as N. wearnei are N. californicus, we can conclude that N. californicus is present in Australia. The information about the biology of N. californicus can thus now be applied to the Australian population of this species for biological control purposes.

Vaccination with in vitro transcribed RNA encoding tumor antigens is an emerging approach in cancer immunotherapy. Attempting to further improve RNA vaccine efficacy, we have explored combining RNA with immunomodulators such as rapamycin. Rapamycin, the inhibitor of mTOR, was used originally for immunosuppression. Recent reports in mouse systems, however, suggest that mTOR inhibition may enhance the formation and differentiation of the memory CD8(+) T-cell pool. Because memory T-cell formation is critical to the outcome of vaccination approaches, we studied the impact of rapamycin on the in vivo primed RNA vaccine-induced immune response using the chicken ovalbumin-expressing B16 melanoma model in C57BL/6 mice. Our data show that treatment with rapamycin at the effector-to-memory transition phase skews the vaccine-induced immune response toward the formation of a quantitatively and qualitatively superior memory pool and results in a better recall response. Tumor-infiltrating immune cells from these mice display a favorable ratio of effector versus suppressor cell populations. Survival of mice treated with the combined regimen of RNA vaccination with rapamycin is significantly longer (91.5 days) than that in the control groups receiving only one of these compounds (32 and 46 days, respectively). Our findings indicate that rapamycin enhances therapeutic efficacy of antigen-specific CD8(+) T cells induced by RNA vaccination, and we propose further clinical exploration of rapamycin as a component of immunotherapeutic regimens., (©2013 AACR.)

The family Phytoseiidae includes many species of predators involved in the control of mite pests of crops all over the world. In European vineyards, these natural enemies play a key role in plant protection as their presence usually makes the use of acaricides unnecessary. Each species has its specific biological features. It is thus of great interest to identify the species reported on grape, Vitis vinifera. The present paper, based on a world database of the family Phytoseiidae and on an analysis of more than 250 publications, presents the 54 species of Phytoseiidae belonging to 15 genera reported on V. vinifera in Europe, and identification keys to species. Online versions of the key (dichotomous and polytomous) with illustrations are available at http://wwwl.montpellier.inra.fr/CBGP/phytoseiidae/sitewebvineyards2/index.htm. An analysis of the biogeographic distribution of these species and their prey has also been carried out. Most species reported on V. vinifera in Europe are rare; only five species are frequently observed: Kampimodromus aberrans, Typhlodromus (Typhlodromus) pyri, Typhlodromus (Typhlodromus) exhilaratus, Euseius finlandicus and Phytoseius finitimus. The 12 countries where Phytoseiidae have been reported from grapes have been unevenly surveyed, the most well-known faunas being from Italy, Greece and France. These species are reported to prey upon the main species of mite pests of grapes.

Synthetic antigen-encoding mRNA is increasingly exploited as a tool for delivery of genetic information of complete antigens into professional antigen presenting dendritic cells for HLA haplotype-independent antigen-specific vaccination against cancer. Two strategies for mRNA-based antitumor vaccination have emerged into the clinical setting. One is transfection of autologous dendritic cells with synthetic mRNA for adoptive transfer into the patient. The other is direct injection of naked synthetic mRNA. Both methods have proven to be feasible and safe and to elicit antigen-specific immune responses. The design of novel synthetic vaccines employing synthetic mRNA requires further in-depth investigation of its bioavailability and immune pharmacology. This chapter summarizes the state-of-art in this field and describes methods elementary for preclinical studies of mRNA-based antitumor vaccine protocols.

Next generation sequencing enables identification of immunogenic tumor mutations targetable by individualized vaccines. In the B16F10 melanoma system as pre-clinical proof-of-concept model, we found a total of 563 non-synonymous expressed somatic mutations. Of the mutations we tested, one third were immunogenic. Immunization conferred in vivo tumor control, qualifying mutated epitopes as source for effective vaccines.

Multiple genetic events and subsequent clonal evolution drive carcinogenesis, making disease elimination with single-targeted drugs difficult. The multiplicity of gene mutations derived from clonal heterogeneity therefore represents an ideal setting for multiepitope tumor vaccination. Here, we used next generation sequencing exome resequencing to identify 962 nonsynonymous somatic point mutations in B16F10 murine melanoma cells, with 563 of those mutations in expressed genes. Potential driver mutations occurred in classical tumor suppressor genes and genes involved in proto-oncogenic signaling pathways that control cell proliferation, adhesion, migration, and apoptosis. Aim1 and Trrap mutations known to be altered in human melanoma were included among those found. The immunogenicity and specificity of 50 validated mutations was determined by immunizing mice with long peptides encoding the mutated epitopes. One-third of these peptides were found to be immunogenic, with 60% in this group eliciting immune responses directed preferentially against the mutated sequence as compared with the wild-type sequence. In tumor transplant models, peptide immunization conferred in vivo tumor control in protective and therapeutic settings, thereby qualifying mutated epitopes that include single amino acid substitutions as effective vaccines. Together, our findings provide a comprehensive picture of the mutanome of B16F10 melanoma which is used widely in immunotherapy studies. In addition, they offer insight into the extent of the immunogenicity of nonsynonymous base substitution mutations. Lastly, they argue that the use of deep sequencing to systematically analyze immunogenicity mutations may pave the way for individualized immunotherapy of cancer patients.

Next generation sequencing (NGS) has enabled high throughput discovery of somatic mutations. Detection depends on experimental design, lab platforms, parameters and analysis algorithms. However, NGS-based somatic mutation detection is prone to erroneous calls, with reported validation rates near 54% and congruence between algorithms less than 50%. Here, we developed an algorithm to assign a single statistic, a false discovery rate (FDR), to each somatic mutation identified by NGS. This FDR confidence value accurately discriminates true mutations from erroneous calls. Using sequencing data generated from triplicate exome profiling of C57BL/6 mice and B16-F10 melanoma cells, we used the existing algorithms GATK, SAMtools and SomaticSNiPer to identify somatic mutations. For each identified mutation, our algorithm assigned an FDR. We selected 139 mutations for validation, including 50 somatic mutations assigned a low FDR (high confidence) and 44 mutations assigned a high FDR (low confidence). All of the high confidence somatic mutations validated (50 of 50), none of the 44 low confidence somatic mutations validated, and 15 of 45 mutations with an intermediate FDR validated. Furthermore, the assignment of a single FDR to individual mutations enables statistical comparisons of lab and computation methodologies, including ROC curves and AUC metrics. Using the HiSeq 2000, single end 50 nt reads from replicates generate the highest confidence somatic mutation call set.

Intranodal immunization with antigen-encoding naked RNA may offer a simple and safe approach to induce antitumor immunity. RNA taken up by nodal dendritic cells (DC) coactivates toll-like receptor (TLR) signaling that will prime and expand antigen-specific T cells. In this study, we show that RNA vaccination can be optimized by coadministration of the DC-activating Fms-like tyrosine kinase 3 (FLT3) ligand as an effective adjuvant. Systemic administration of FLT3 ligand prior to immunization enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodal RNA. Unexpectedly, plasmacytoid DCs (pDC) were found to be essential for the adjuvant effect of FLT3 ligand and they were systemically expanded together with conventional DCs after treatment. In response to FLT3 ligand, pDCs maintained an immature phenotype, internalized RNA, and presented the RNA-encoded antigen for efficient induction of antigen-specific CD8(+) T-cell responses. Coadministration of FLT3 ligand with RNA vaccination achieved remarkable cure rates and survival of mice with advanced melanoma. Our findings show how to improve the simple and safe strategy offered by RNA vaccines for cancer immunotherapy.

This study deals with the long-term effect of agroforestry management (trees within vine crops) on communities of phytoseiid mites. Several plots were considered: vineyards co-planted with Sorbus domestica or Pinus pinea, monocultures of vines and monocultures of S. domestica or P. pinea. All vine plots included two vine cultivars, Syrah and Grenache. Phytoseiid mites have been surveyed in these plots during several years within the previous 10 years. In 2010, samplings were again carried out in these same plots, from May to September, twice a month. Significantly higher densities of Phytoseiidae were observed on the cultivar Syrah (0.85 phytoseiids per leaf) than on Grenache (0.26 phytoseiids per leaf). Furthermore, significantly higher phytoseiid mite densities were observed in the monocultural grapevine plot than in the two co-planted ones. The main species found was Typhlodromus (Typhlodromus) exhilaratus in all vine plots considered. However, Kampimodromus aberrans was observed in the grapevine plots co-planted with the two trees, but never in the monocultural vine plot. Surprisingly, this phytoseiid species was not found on the co-planted trees, nor in the neighbouring uncultivated vegetation. Several hypotheses are discussed to explain such an unexpected distribution. Furthermore, contrary to what has been observed previously, agroforestry management did not seem to favour phytoseiid mite development, especially on the Grenache cultivar. Again, some hypotheses are developed to explain such observations and density modifications.

While the endeavor to vaccinate against cancer has been pursued for over 20 years, only recently was the first tumor vaccine approved. Among the different antigen formats assessed for vaccination, coding messenger RNA (mRNA) is emerging as a particularly attractive option. It can code for all types of transcript based proteins, is easy and cost efficient to produce, has a favorable safety profile and enables induction of combined immune responses. Within the last few years major developments have been achieved in this field. Clinical approaches use mRNA either for direct administration or for engineering of adoptively transferred dendritic cells. However, there are still challenges to be overcome for successful clinical application of mRNA-based immunotherapies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Strategies for antibody-mediated cancer immunotherapy, such as active immunization with virus-like particle (VLP)-based vaccines, are gaining increasing attention. We developed chimeric hepatitis B virus core antigen (HBcAg)-VLPs that display a surface epitope of the highly selective tumor-associated cell lineage marker claudin-18 isoform 2 (CLDN18.2) flanked by a mobility-increasing linker. Auto-antibodies elicited by immunization with these chimeric HBcAg-VLPs in 2 relevant species (mouse and rabbit) bind with high precision to native CLDN18.2 at physiologic densities on the surface of living cells but not to the corresponding epitope of the CLDN18.1 splice variant that differs by merely one amino acid. The induced auto-antibodies are capable of efficiently killing CLDN18.2 expressing cells in vitro by complement-dependent and antibody-dependent cell-mediated cytotoxicity. Moreover, they provide partial protective immunity against the challenge of mice with syngeneic tumor cells stably expressing CLDN18.2. Our study provides a first proof-of-concept that immunization combining VLPs as antigen carriers with specific conformational epitopes of a highly selective differentiation antigen may elicit auto-antibodies with high cytocidal and tumoricidal potential., (© 2011 AACR.)

The date palm mite, Oligonychus afrasiaticus (McGregor) (Acari: Tetranychidae), is a serious pest of palm date fruits. Life cycle, fecundity, and longevity of this mite were studied on fruits of four date palms, Phoenix dactylifera L. (Arecales: Arecaceae)(varieties: Deglet Noor, Alig, Kentichi, and Besser), under laboratory conditions at 27 = 1 °C, 60 ± 10% RH. Total development time of immature female was shorter on Deglet Noor fruits than on the other cultivars. O. afrasiaticus on Deglet Noor had the highest total fecundity per female, while low fecundity values occurred on Besser. The comparison of intrinsic rates of natural increase (r(m)), net reproductive rates (R(o)), and the survival rates of immature stage of O. afrasiaticus on the host plants suggests that O. afrasiaticus performs better on Deglet Noor fruits. The mite feeding on Alig showed the lowest intrinsic rate of natural population increase (r(m) = 0.103 day (-1)). The estimation of difference in susceptibility of cultivars to O. afrasiaticus is crucial for developing efficient pest control programs. Indeed, less susceptible cultivars can either be left unsprayed or sprayed at low threshold.

RNAs with optimized properties are increasingly investigated as a tool to deliver the genetic information of complete antigens into professional antigen-presenting dendritic cells for HLA haplotype-independent antigen-specific vaccination against cancer. As the dose of the antigen and duration of its presentation are critical factors for generating strong and sustained antigen-specific immune responses, improvement of the immunobioavailability of RNA-based vaccines has been a recurrent subject of research. Substantial increase of the amount of antigen produced from RNA can be achieved by optimizing RNA stability and translational efficiency. Both features are determined by cis-acting elements in the RNA, namely the 5' cap, the poly(A) tail, and the sequence of the coding and non-coding regions, which interact with corresponding trans-acting factors. This article summarizes recent developments in identifying optimized RNA for expression of foreign proteins in dendritic cells, as well as their implications for immunotherapy based on antigen-encoding RNA.

Although naked antigen-encoding RNA has entered clinical testing, basic knowledge on how to apply this promising novel vaccine format is still pending. By comparing different administration routes, we observed surprisingly potent antigen-specific T-cell immunity upon intranodal injection of naked antigen-encoding RNA. RNA was selectively uptaken by resident dendritic cells, propagated a T-cell attracting and stimulatory intralymphatic milieu, and led to efficient expansion of antigen-specific CD8+ as well as CD4+ T cells. By intranodal treatment of mice with repeated cycles of RNA, we achieved de novo priming of naïve T cells, which became potent cytolytic effectors capable of homing to primary and secondary lymphatic tissues as well as memory T cells. In tumor-bearing mice intralymphatic RNA vaccination elicited protective and therapeutic antitumor immune responses, resulting in a remarkable survival benefit as compared with other treatment regimens. This is the first report of strong systemic antigen-specific Th1-type immunity and cancer cure achieved with naked antigen-encoding RNA in preclinical animal models., (Copyright © 2010 AACR.)

The abundance and diversity of phytoseiid mites in the vegetal ground cover of a citrus orchard were surveyed by monthly samplings from October 2008 to July 2009. Six different vegetal cover management methods were studied: herbicide application without mowing (Gly), mowing without herbicide application (PV), mowing with herbicide application (AV), late mowing without herbicide application (LMV), cover crop (Neonotonia wightii, Fabaceae) without herbicide application (PNeo) and cover crop with herbicide application (ANeo). Eleven species were present in the ground cover, with Phytoseius rex and Proprioseiopsis mexicanus as major species. Species richness and densities (1.5 ± 4.5) in the Gly treatment were very low, except for one sample 4 months after herbicide treatment. The AV and PV treatments showed poor diversity and very low mite densities (1.2 ± 2.6 and 1.4 ± 1.5, respectively). The LMV treatment showed the highest diversity and high density of phytoseiid mites (6.9 ± 8.8). The ANeo and PNeo treatments also showed generally high diversity, but with variations in time and exhibited the highest density of phytoseiid mites (13.5 ± 12.7 and 13.4 ± 9.1, respectively). Neonotonia wightii as the cover crop seems to act as a reservoir of phytoseiid mites, sustaining abundant and diverse populations all year round. Some naturally occurring plant species such as Achyranthes aspera, Amaranthus dubius and Eleutheranthera ruderalis could also constitute favourable host plants for Phytoseiidae. Results are discussed in relation to the potential of collected phytoseiid species as candidates for biological control of phytophagous mites on Guadeloupean citrus orchards.

This study focuses on the diagnostics of two natural enemy species, belonging to the genus Phytoseiulus in the family Phytoseiidae (sub-family Amblyseiinae): P. macropilis and P. persimilis. These two species are of primary importance in biological control all over the world. However, they are morphologically very similar and specific diagnostics is difficult. This study utilizes mitochondrial molecular markers (12S rRNA and Cytb mtDNA) to differentiate these two species. Morphological analyses showed significant differences between P. persimilis and P. macropilis for 17 morphological characters of the 32 considered. However, despite these significant differences, the ranges of all characters overlap. Only the serration of the macroseta on the basitarsus (StIV) allows the differentiation between P. persimilis and P. macropilis. Despite these small morphological differences, molecular results, for both mitochondrial DNA fragments considered (rRNA and Cytb mtDNA), showed a clear delineation between the specimens of P. macropilis and P. persimilis. This study emphasizes (i) that only one morphological character (serration of the seta StIV) clearly separates these two species, and (ii) the usefulness of an automatical molecular and simple diagnostic tool for accurate differentiation of the two species and ensure the morphological diagnostics. Further studies are proposed, including more DNA sequences especially for P. macropilis.

This paper focuses on the differentiation of specimens, identified as Phytoseiulus longipes, collected in four countries: Argentina, Brazil, Chile and South Africa. Two of these populations are known to feed and develop on Tetranychus evansi, whereas the two others do not. As morphologically similar specimens can sometimes belong to different species and because differences in predatory behaviours exist among the four populations considered, we tested for the presence of cryptic species. Morphological and molecular experiments (12S rDNA) were carried out. The four studied populations of P. longipes could be morphologically differentiated thanks to a combination of characters. However, these morphological differences are very small. The two populations that feed and develop on T. evansi (from Argentina and Brazil) are morphologically closer to each other than to the two other populations. Genetic distances among the four populations of P. longipes were very low, suggesting that despite their different feeding habits, all specimens belong to the same species. However, the populations associated with T. evansi showed some genetic differentiation from those that do not use this pest. This is the first time that this type of differentiation has been reported for the family Phytoseiidae. These results are of primary importance to ensure the success of biological control programs and to develop strains adapted to both crops and prey species.

Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5' mRNA cap analogs on the kinetics of the encoded protein, we found that m(2)(7,2'-O)Gpp(S)pG (beta-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and translational efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as beta-S-ARCA(D1)-capped RNA species is superior for protein expression and for efficient priming and expansion of naïve antigen-specific T cells in mice. Our findings establish 5' mRNA cap analogs as yet another module for tuning immunopharmacological properties of recombinant antigen-encoding RNA for vaccination purposes.

Morphological measurements and a mitochondrial molecular marker (COI) were used to identity specimens reported as Kampimodromus aberrans on hazelnut in the USA. Several species and populations of this genus were studied to assist with identification. Both data types showed that specimens from the USA differed from K. aberrans from other regions. USA specimens seem to belong to the same species as Kampimodromus specimens from France on hazelnut. These mites were morphologically similar to Kampimodromus coryli and K. corylosus, which according to the original descriptions, are distinguished by the presence or absence of a tooth on the movable digit of the chelicera, with K. coryli having one tooth and K. corylosus none. As chelicerae of Kampimodromus from hazelnut in the USA and France are toothless, they are assigned to the species K. corylosus. Studies showed that morphological characters traditionally used to identify Kampimodromus species, such as setal length, are of less value than other characters that are difficult to observe, such as the numbers of solenostomes and the presence of teeth on the movable digit of the chelicerae. Some synonyms are discussed.

Background: Laboratory results of the French ANPP/CEB guideline No. 167 and IOBC/WPRS Ring Testing Group methods for testing the side effects of pesticides on the predatory mite Typhlodromus pyri Scheuten were compared with respect to their suitability to evaluate the toxicity of three pesticides., Results: Results obtained with the ANPP/CEB guideline allow the demonstration of significant differences between two slightly toxic products, a dichlofluanid 500 g kg(-1) kWP (Euparen) 50WP) and a quinoxyfen 250 g L(-1) SCC (Legend), and a highly toxic cymoxanil 60/mancozeb 200/folpet 275 g kg(-1) WP [Remiltine F Pepite) (RFP)], on the basis of bioassays conducted in the laboratory. In contrast, results obtained with the IOBC/WPRS method classified all three as harmful., Conclusion: The evaluation of the toxicity of RFP revealed that the concentration, the quantity of the wet deposit and the food source used in the IOBC/WPRS method maximise the toxicity, in comparison with those used in the ANPP/CEB protocol. Valid criteria in controls were all respected in the ANPP/CEB tests but not in the IOBC/WPRS samples. This result revealed difficulties related to the use of the IOBC/WPRS method in laboratories.

Genetic modification of vaccines by linking the Ag to lysosomal or endosomal targeting signals has been used to route Ags into MHC class II processing compartments for improvement of CD4+ T cell responses. We report in this study that combining an N-terminal leader peptide with an MHC class I trafficking signal (MITD) attached to the C terminus of the Ag strongly improves the presentation of MHC class I and class II epitopes in human and murine dendritic cells (DCs). Such chimeric fusion proteins display a maturation state-dependent subcellular distribution pattern in immature and mature DCs, mimicking the dynamic trafficking properties of MHC molecules. T cell response analysis in vitro and in mice immunized with DCs transfected with Ag-encoding RNA showed that MITD fusion proteins have a profoundly higher stimulatory capacity than wild-type controls. This results in efficient expansion of Ag-specific CD8+ and CD4+ T cells and improved effector functions. We used CMVpp65 and NY-ESO-1 Ags to study preformed immune responses in CMV-seropositive individuals and cancer patients. We show that linking these Ags to the MITD trafficking signal allows simultaneous, polyepitopic expansion of CD8+ and CD4+ T cells, resulting in distinct CD8+ T cell specificities and a surprisingly broad and variable Ag-specific CD4+ repertoire in different individuals.

Assessment of antigen-specific T-cell responses has been greatly facilitated by development of ELISPOT and intracellular cytokine flow cytometry (CFC) assays. The use of autologous antigen presenting cells transfected with in vitro transcribed RNA as stimulators allows in principle quantification of antigen-specific T-cells independent of the knowledge of the epitopes. We describe here a cytokine secretion assay that enables simultaneous assessment of both antigen-specific CD4+ as well as CD8+ T-cells directly from clinical samples without the need for generation of dendritic cells. To this aim, bulk PBMCs were electroporated with RNA encoding the antigen fused to trafficking signal sequences derived from a MHC class I molecule and used as stimulators. With human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) as antigen we show that for measuring ex vivo T-cell responses in ELISPOT and CFC such stimulators are superior or at least equivalent to a pool of overlapping peptides representing the entire pp65 sequence as well as to untagged pp65 encoding RNA. This approach avoids the time consuming generation of dendritic cells as immune stimulators and, in particular when used in the context of the CFC, is robust, broadly applicable and fast.

Two species of Phytoseiidae are found in the same agroecosystem: Typhlodromus exhilaratus prevails in vine plots, while T. phialatus prevails in uncultivated surrounding areas. The objective of the present paper was to investigate whether the poor settlement of T. phialatus in vine plots can be explained by intraguild predation of these two species and/or cannibalism. Predatory abilities of the females on larvae and protonymphs were studied under laboratory conditions. A first experiment was conducted with only conspecific or heterospecific phytoseiid prey, in a second experiment Tetranychus urticae eggs were added to the phytoseiid prey. Oviposition, prey consumption, and escape rates of females were recorded. Oviposition and intraguild predation rates were higher for T. exhilaratus than for T. phialatus. Typhlodromus exhilaratus consumed fewer conspecifics than heterospecific phytoseiids, and oviposited when feeding on both diets. Typhlodromus phialatus consumed equal amounts of con- and heterospecifics. Although these two generalist predators belong to the type III defined by McMurtry and Croft (Annual Review of Entomology 42:291-321, 1997), our results suggest that they have different predation behaviour. However, because these results were obtained in experiments where no choice was given between the two phytoseiid species, they are difficult to link to previous studies conducted on the intraguild predation of the Phytoseiidae. The greater voracity and prolificacy of T. exhilaratus could partially explain the poor settlement of T. phialatus in vineyards and the predominance of T. exhilaratus. However, a full understanding of this phenomenon will require the study of other factors, such as susceptibility to pesticides and micro-climatic conditions, as well as the ability to cope with different food sources and host plants.

The mite Tetranychus evansi Baker & Pritchard (Tetranychidae) probably originated in South America. Because of its importance as a tomato pest in Africa, an extensive project has been conducted to detect potentially effective natural enemies in South America for the classical biological control of the pest in Africa. A search for the natural enemies of T. evansi was conducted in the Province of Tucumán, northwestern Argentina, in December 2004, and this report describes the results. One hundred predatory mites of the family Phytoseiidae referring to 11 species were collected on 11 examined species of solanaceous plants. The most abundant phytoseiid species collected were Neoseiulus californicus (McGregor) and Euseius concordis (Chant). Adults and immatures of those species, as well as of Neoseiulus idaeus Denmark & Muma, Phytoseiulus fragariae Denmark & Schicha and Proprioseiopsis cannaensis (Muma) were found in association with T. evansi, suggesting that they were developing on the pest. However, because of the possible biological differences between populations of a given species, biological studies evaluating T. evansi as a prey for those predators seem desirable.

The tomato red spider mite, Tetranychus evansi, is reported as a severe pest of tomato and other solanaceous crops from Africa, from Atlantic and Mediterranean Islands, and more recently from the south of Europe (Portugal, Spain and France). A population of the predaceous mite Phytoseiulus longipes has been recently found in Brazil in association with T. evansi. The objective of this paper was to assess the development and reproduction abilities of this strain on T. evansi under laboratory conditions at four temperatures: 15, 20, 25 and 30 degrees C. The duration of the immature phase ranged from 3.1 to 15.4 days, at 30 and 15 degrees C, respectively. Global immature lower thermal threshold was 12.0 degrees C. Immature survival was high at all temperatures tested (minimum of 88% at 30 degrees C). The intrinsic rate of increase (r (m)) of P. longipes ranged from 0.091 to 0.416 female/female/day, at 15 and 30 degrees C, respectively. P. longipes would be able to develop at a wide range of temperatures feeding on T. evansi and has the potential to control T. evansi populations.

Two species of Phytoseiidae predominated in and around vineyards in an experimental site in the south of France: Typhlodromus exhilaratus is the dominant species on grapes, whereas T. phialatus occurs in the uncultivated areas surrounding these crops. To help understand their distribution, this study aims to determine their susceptibility to a fungicide (dimethomorph + mancozeb) and an insecticide (chlorpyriphos-ethyl), which have been widely used at the experimental site for at least four years. The hypothesis tested is that the effects of these two pesticides differ between T. exhilaratus and T. phialatus. Two populations of T. exhilaratus, one collected on a vine crop where the two pesticides were applied and the other in an unsprayed orchard of Sorbus domestica, and one population of T. phialatus collected on Viburnum tinus from uncultivated areas adjacent to the vine crop, were tested under laboratory conditions. The fungicide seems to affect the females of T. phialatus more than those of T. exhilaratus from vine. However, at the recommended field concentration the female mortality rates of the three strains were low and female fecundities were not significantly different. Conversely, mortality rates were very high for the three strains at the recommended field concentration of chlorpyrifos-ethyl. The strain of T. exhilaratus from vine seems to be more tolerant and 20% of females survived the recommended field rate. This suggests better survival after insecticide application than females of T. phialatus (100% mortality at a lower concentration than the recommended rate). As extrapolation of laboratory results to the field is often irrelevant, these differences could be just one of several factors affecting the spatial segregation of the two species in the agrosystem considered. Possible explanations other than pesticides for the poor settlement of T. phialatus in vine plots are discussed.

Adoptive transfer of dendritic cells (DCs) transfected with in vitro-transcribed, RNA-encoding, tumor-associated antigens has recently entered clinical testing as a promising approach for cancer immunotherapy. However, pharmacokinetic exploration of RNA as a potential drug compound and a key aspect of clinical development is still pending. While investigating the impact of different structural modifications of RNA molecules on the kinetics of the encoded protein in DCs, we identified components located 3' of the coding region that contributed to a higher transcript stability and translational efficiency. With the use of quantitative reverse transcription-polymerase chain reaction (RT-PCR) and eGFP variants to measure transcript amounts and protein yield, we showed that a poly(A) tail measuring 120 nucleotides compared with a shorter one, an unmasked poly(A) tail with a free 3' end rather than one extended with unrelated nucleotides, and 2 sequential beta-globin 3' untranslated regions cloned head to tail between the coding region and the poly(A) tail each independently enhanced RNA stability and translational efficiency. Consecutively, the density of antigen-specific peptide/MHC complexes on the transfected cells and their potency to stimulate and expand antigen-specific CD4+ and CD8+ T cells were also increased. In summary, our data provide a strategy for optimizing RNA-transfected DC vaccines and a basis for defining release criteria for such vaccine preparations.

Tetranychus evansi Baker & Pritchard is an important pest of Solanaceae in several countries. Introduced accidentally to Africa, it presently occurs in many countries of that continent. In some of them, it is considered a key pest. The suspected area of origin of this mite is South America. The objective of the present study was to identify phytoseiid mites on solanaceous plants in association with T. evansi in south and southeast Brazil for introduction in the African continent for use in a classic biological control program. Almost 1,400 predatory mites of the family Phytoseiidae were collected, on 22 solanaceous species. The Amblyseiinae were the most diverse group in this study. Twenty-three of the species found belong to this subfamily, while only three belong to the Typhlodrominae and two to the Phytoseiinae. The most abundant and most frequent phytoseiid species were Phytoseius guianensis De Leon and Galendromus annectens (De Leon) of the Phytoseiinae and Typhlodrominae, respectively. The most frequent and abundant species of Amblyseiinae was Neoseiulus tunus (De Leon). Phytoseius guianensis and N. tunus were never found in association with T. evansi and G. annectens was found only once in association with it. Two factors suggested Phytoseiulus longipes Evans as the most promising predator found in this study. It could walk very well on tomato leaves infested by T. evansi, without being hampered by the profuse webbing produced by the prey and by the trichomes. In addition, several specimens of both sexes including eggs and nymphs of the predator were found associated with T. evansi on three different plant species and in two different periods of the year, when T. evansi was the only arthropod present on the leaves.

The abundance and diversity of phytoseiid mites were surveyed from April to September 2003 to 2005 in vineyards (Grenache and Syrah cultivars) co-planted with rows of Sorbus domestica or Pinus pinea and in monoculture plots of grapes in the South of France. Densities of phytoseiid mites were different on the two tree species, with P. pinea a more suitable host than S. domestica. Typhlodromus (Typhlodromus) exhilaratus was the dominant species occurring on grapes and on co-planted rows of S. domestica and P. pinea, whereas T. (T.) phialatus was the most abundant species in monoculture plots of both S. domestica and P. pinea. Factors determining the dominance of T. (T.) phialatus over T. (T.) exhilaratus in monoculture trees are discussed. In this study, agroforestry management did not affect phytoseiid diversity in vineyards, but did affect phytoseiid density, especially in 2005. The results obtained in 2003 and 2004 are not easy to discuss in this regard because of the low densities of mites observed during these 2 years (very dry climatic conditions and pesticide applications).

This study reports (1) a faunistic survey of phytoseiid mites observed inside a vine plot and in neighbouring vegetation (other vine plots and uncultivated areas) and (2) dispersal of phytoseiid mites into the plot studied. These data aim to raise some hypotheses concerning natural colonisation of a vineyard by predatory mites. The study was carried out over 3 years (1999, 2000 and 2001) in an experimental plot planted with two cultivars (Grenache and Syrah) and with Sorbus domestica in a framework of agroforestry investigations. Phytoseiid mites were collected in both cultivated and uncultivated areas surrounding the experimental plot, and their dispersal into the plot studied using "aerial" traps. Densities remained quite low compared to previous studies. The main species encountered in the uncultivated areas and in the traps was Typhlodromus phialatus. Despite the low numbers of phytoseiid mites trapped, densities of phytoseiid mites into the vine field increased during 3 years. Typhlodromus phialatus, the species mainly found in the neighbouring uncultivated areas, was rarely found in vineyards. Another morphologically close species was predominant on vines: Typhlodromus exhilaratus. However, Kampimodromus aberrans the main phytoseiid mite species in vineyards of Southern France was not found in the present survey. Hypotheses for this colonisation process are discussed.

In patients undergoing allogeneic peripheral blood stem cell (PBSC) transplantation after reduced-intensity conditioning (RIC), graft-versus-host disease (GVHD) represents a major cause of morbidity and mortality. T-cell depletion (TCD) prevents GVHD but carries potential risks of graft failure, opportunistic infections, and disease relapse. We explored ex vivo TCD of stem cell allografts that were administered after RIC treatment. Thirteen high-risk patients with hematological malignancies were treated with a fludarabine/melphalan-based RIC regimen followed by transplantation of immunomagnetically selected CD34(+) PBSC from HLA-identical sibling or matched unrelated donors. Patients were sequentially enrolled in two cohorts: group A (n=6) received antithymocyte globulin (ATG) during conditioning and 10(5) donor T cells/kg at transplantation, while group B (n=7) received 10(6) donor T cells/kg without ATG pretreatment. Primary graft failure occurred in two patients of group A and in one patient of group B. Complete donor chimerism persisting more than 1 year was achieved in two cases per cohort. Acute grade II to IV or chronic extensive GVHD were observed in a total of six patients (group A, 2; group B, 4). Procedure-related deaths were mainly due to severe pneumonia occurring in two patients of group A and in five patients of group B. These results suggest that CD34 selection of reduced-intensity PBSC allografts may cause adverse effects upon specific antimicrobial immunity which can lead to fatal infections, particularly in high-risk patients. In our study, simultaneous add-back of < or =10(6)/kg donor T cells was unable to compensate for this deficiency.

A field population of Typhlodromus pyri (Acari: Phytoseiidae) tolerant to mancozeb was selected in the laboratory. After 10 mancozeb selections the LC50 value for mancozeb was 73 times higher in the selected-10 strain compared to the standard susceptible strain. A genetic analysis using reciprocal crosses and backcrosses of female F1 progeny found no maternal effect. Resistance in the selected-10 strain was codominant in expression, dominance value was about -0.1. Backcrosses between F1 females and the susceptible strain indicate that the resistance to mancozeb could be principally conferred by a predominant gene, but additional factors would also be involved.

Interspecific predation and cannibalism are common types of interaction in phytoseiid predator guilds, but the extent and nature of these interactions have not been determined yet in phytoseiid guilds composed of African native and neotropical exotic phytoseiid predators found in cassava habitat in southern Africa. We determined in laboratory experiments the level of cannibalism and interspecific predation among the three phytoseiid mite species Euseius fustis, Iphiseius degenerans, and Typhlodromalus aripo in the absence of food and in the presence of limited or abundant quantities of two food types--Mononychellus tanajoa and maize pollen--commonly found on cassava in Africa. When confined without food, only two T. aripo females laid each two eggs within 5 days, and this species survived longer than I. degenerans and E. fustis. In the presence of con- or hetero-specific larvae or protonymphs, the three species fed more on the former than on the latter, and more on hetero-specifics than on con-specifics. Oviposition rates of the three species did not exceed 0.7 egg/female/day on con- and hetero-specific immatures. Typhlodromalus aripo and E. fustis survived longer on con-specific and hetero-specific larvae and on hetero-specific protonymphs than in the absence of any food, while T. aripo survived longer than the two other species on the same diets. Provision of limited quantity of food decreased interspecific predation rate by I. degenerans and T. aripo, but not by E. fustis, and increased oviposition rate and longevity of all three species. Provision of abundant food, however, eliminated cannibalism by all three species and further reduced interspecific predation rates, but their oviposition and longevity remained relatively unchanged compared with limited food provision. Potential consequences of cannibalism and interspecific predation among phytoseiid mites on cassava for the biological control of M. tanajoa are discussed.

The side effects of mancozeb on the predatory mite Typhlodromus pyri were studied in 4-year field trials on grapevine and in the laboratory. In the field, the effect of mancozeb varied according to previous mancozeb use. In vineyards where mancozeb had commonly been used over years, this fungicide is generally slightly toxic, in some cases moderately toxic and rarely toxic. In plots were mancozeb has never been used, its effect on T. pyri was more pronounced and varied from moderately toxic to toxic. Despite the toxicity of mancozeb, T. pyri populations have never been eradicated. Laboratory results obtained with the French CEB guideline no. 167 confirmed those of the 4-year field study: mancozeb was significantly more toxic to T. pyri populations collected in plots where it had rarely been used before the field experiment. In plots where mancozeb had been used for a long time, the susceptibility of T. pyri populations to this fungicide was reduced and female survival, fecundity but also viability of female progeny were less affected by mancozeb. Even though toxicity of mancozeb increased in controlled conditions, a significant correlation was established between field and laboratory results.

Under laboratory conditions, the acaricidal effect of wettable sulfur is influenced by climatic conditions and the stage of development of Tetranychus urticae. Its ovicidal effect results from the combined action of temperature and relative humidity (RH). Wettable sulfur becomes effective at 27.5 degrees C and 75% RH. Beyond this threshold, the acaricidal effect increases with rising temperature or humidity, to become complete at a temperature of 35 degrees C and 90% RH. Within the range of temperatures and humidities 20 degrees C/30% RH and 35 degrees C/90% RH the mortality of immatures (from protonymphs to teleiochrysalis) was total. Under similar experimental conditions, females usually died before the end of the experiment. Temperature and relative humidity increased the adulticidal potential of wettable sulfur. The fecundity of the sulfur-treated females and the viability of their progeny was found to depend on temperature and RH. According to the same climatic conditions, eggs were less susceptible than females, which were in turn less susceptible than juvenile stages.

This study examined young adolescents'intentions to use moralistic violence and their violence exposure, examining male-female differences. Sixth-grade students (n=702) from Georgia middle schools servicing impoverished communities participated. Data were obtained on the students' exposure to violence, family structure and education level, church attendance, gang interest, drug use, and depression status. The dependent variable, intention to use moralistic violence, was measured with an 11-item scale. Linear regression models were run separately for males and females. Males had significantly higher mean intention to use moralistic violence than females (p=0.002). Males reported being exposed to violence more than females, but exposure decreased as attendance to religious services increased. For these 11-12-year-olds, unconventional peer social norms, such as witnessing violence, increased their intention to use violence while involvement in conventional activities, such as church attendance, decreased it. The protective effect was greater for males than females., (Copyright 2001 The Association for Professionals in Services for Adolescents.)

We investigated whether a T cell-reduced allogeneic stem cell transplant (SCT) with minimal conditioning and subsequent donor lymphocyte infusions (DLI) could reduce the incidence and severity of GVHD while retaining stable engraftment. Five patients with hematological malignancies (three MM, one CLL, one Chediak-Higashi syndrome) were conditioned with TBI (200 cGy). One patient additionally received fludarabine (120 mg/m(2)). CsA and mofetyl-mycophenolate (MMF) were administered to prevent GVHD. All patients were grafted with >3 x 10(6)/kg highly purified CD34(+) cells together with 2 x 10(6)/kg CD3(+) cells (three patients) or 1 x 10(5)/kg CD3(+) cells (two patients). Quick hematopoietic recovery and initial mixed donor chimerism was observed. Treatment-related toxicity was minimal in all but one patient who died of treatment-refractory GVHD on day 112. The four other patients only achieved partial donor T cell chimerism. BM and PBMC donor chimerism was lost between day 40 and 209 despite DLI. Three patients are alive with disease and one is in CR. We conclude that T cell-reduced SCT using 200 cGy as the conditioning regimen does not result in stable hematopoietic engraftment. Predominant donor T cell chimerism is not a prerequisite for initial allogeneic hematopoietic proliferation. However for sustained long-term engraftment it is of major importance.

Graft-versus-host disease (GVHD) remains a significant complication in patients undergoing allogeneic stem cell transplantation (SCT) using a reduced intensity conditioning regimen. Although T-cell depletion (TCD) reduces the risk of GVHD after a myeloablative conditioning regimen, it is associated with an increased risk of graft failure. We have therefore examined whether TCD compromises engraftment using a fludarabine-based conditioning regimen. Fifteen patients have been transplanted using such a regimen of whom 13 underwent ex vivo TCD. All but one patient demonstrated durable engraftment and no patient receiving a TCD product developed severe GVHD. Thus, TCD may play a role in GvHD prophylaxis using such regimens.

Donor-recipient disparitiy of the minor histocompatibility antigen HA-1 is relevant for the development of graft-versus-host disease after HLA-matched sibling allogeneic bone marrow transplantation in HLA-A*0201-positive individuals. Two different alleles of HA-1 with a single amino acid polymorphism have been identified. Here we describe a time- and cost-efficient method for HA-1 typing of genomic DNA, using site-specific hybridization probes with the LightCycler. This method was compared with standard techniques as sequencing or allele-specific polymerase chain reaction (PCR) and proved to be specific, reliable and reproducible. We conclude that HA-1-subtyping using fluorescent-labeled oligonucleotides represents a attractive method for the screening of samples before allogeneic transplantation in HLA-A*0201-positive individuals.

Objective: To examine the relationships among exposure to violence; tobacco, alcohol, and other substance use; depression; church attendance; and the use of violence among very young adolescents., Methods: An 86-item confidential questionnaire was administered to 722 sixth grade students (mean age = 11.9+/-0.8 years) attending 4 middle schools serving neighborhoods in and around public housing., Results: Boys had a higher mean violence scale score than girls (P < or =.0001), and students living in public housing had higher violence scale scores than other students (P< or =.0001). Self-reported use of violence was significantly associated with exposure to violence (r =.45); age (r =.28); frequency of church attendance (r = -.14); depression (r =.28); the probability of being alive at age 25 (r = -.09); the frequency of use of cigarettes (r =.39), alcohol (r =.37), and multiple substances (r =.38); and interest in a gang (r =.37). When all of these variables were analyzed with multiple linear regression, multiple substance use, exposure to violence, interest in a gang, male gender, cigarette smoking, and depression level accounted for 49.7% of the variation in the use of violence scale., Conclusion: Recent multiple substance use and lifetime exposure to violence and victimization were the strongest correlates with the frequency that these youth reported using violence and carrying weapons.