Phenotypic and functional activation of hyporesponsive KIRnegNKG2Aneg human NK-cell precursors requires IL12p70 provided by Poly(I:C)-matured monocyte-derived dendritic cells.

A functionally responsive natural killer (NK)-cell repertoire requires the acquisition of inhibitory NKG2A and killer immunoglobulin-like receptors (KIR) through pathways that remain undefined. Functional donor NK cells expressing KIRs for non-self class I MHC ligands contribute to a positive outcome after allogeneic hematopoietic stem cell transplantation (alloHSCT) by targeting HLA-matched recipient leukemic cells. Insofar as circulating donor conventional dendritic cells (DC) reconstitute with comparable kinetics with donor NK cells after alloHSCT, we used hyporesponsive KIRnegNKG2Aneg precursor cells to evaluate how specific DC subtypes generate a functionally active NK-cell repertoire. Both monocyte-derived DCs (moDC) and Langerhans-type DCs (LC) induce KIRnegNKG2Aneg precursor cells to express the inhibitory receptors NKG2A and KIR, without requiring cell proliferation. Poly(I:C)-matured moDCs significantly augmented the expression of NKG2A, but not KIR, in an IL12p70-dependent manner. Although all DC-stimulated KIRnegNKG2Aneg cells were able to acquire cytolytic activity against class I MHC-negative targets, the ability to secrete IFNγ was restricted to cells that were stimulated by IL12p70-producing, poly(I:C)-matured moDCs. This critical ability of poly(I:C)-matured moDCs to provide IL12p70 to developing KIRnegNKG2Aneg precursors results in a dom4inant, multifunctional, NKG2Apos NK-cell population that is capable of both cytolysis and IFNγ production. Poly(I:C)-matured moDCs are, therefore, the most effective conventional DC subtype for generating a functionally competent NK-cell repertoire by an IL12p70-dependent mechanism.

Combined analysis of modeled and monitored SO2 concentrations at a complex smelting facility.

UNLABELLED: Vale Canada Limited owns and operates a large nickel smelting facility located in Sudbury, Ontario. This is a complex facility with many sources of SO2 emissions, including a mix of source types ranging from passive building roof vents to North America's tallest stack. In addition, as this facility performs batch operations, there is significant variability in the emission rates depending on the operations that are occurring. Although SO2 emission rates for many of the sources have been measured by source testing, the reliability of these emission rates has not been tested from a dispersion modeling perspective. This facility is a significant source of SO2 in the local region, making it critical that when modeling the emissions from this facility for regulatory or other purposes, that the resulting concentrations are representative of what would actually be measured or otherwise observed. To assess the accuracy of the modeling, a detailed analysis of modeled and monitored data for SO2 at the facility was performed. A mobile SO2 monitor sampled at five locations downwind of different source groups for different wind directions resulting in a total of 168 hr of valid data that could be used for the modeled to monitored results comparison. The facility was modeled in AERMOD (American Meteorological Society/U.S. Environmental Protection Agency Regulatory Model) using site-specific meteorological data such that the modeled periods coincided with the same times as the monitored events. In addition, great effort was invested into estimating the actual SO2 emission rates that would likely be occurring during each of the monitoring events. SO2 concentrations were modeled for receptors around each monitoring location so that the modeled data could be directly compared with the monitored data. The modeled and monitored concentrations were compared and showed that there were no systematic biases in the modeled concentrations. IMPLICATIONS: This paper is a case study of a Combined Analysis of Modelled and Monitored Data (CAMM), which is an approach promulgated within air quality regulations in the Province of Ontario, Canada. Although combining dispersion models and monitoring data to estimate or refine estimates of source emission rates is not a new technique, this study shows how, with a high degree of rigor in the design of the monitoring and filtering of the data, it can be applied to a large industrial facility, with a variety of emission sources. The comparison of modeled and monitored SO2 concentrations in this case study also provides an illustration of the AERMOD model performance for a large industrial complex with many sources, at short time scales in comparison with monitored data. Overall, this analysis demonstrated that the AERMOD model performed well.

Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies.

BACKGROUND: Based on its selective cell surface expression in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), receptor tyrosine kinase ROR1 has recently emerged as a promising target for therapeutic monoclonal antibodies (mAbs). To further assess the suitability of ROR1 for targeted therapy of CLL and MCL, a panel of mAbs was generated and its therapeutic utility was investigated. METHODOLOGY AND PRINCIPAL FINDINGS: A chimeric rabbit/human Fab library was generated from immunized rabbits and selected by phage display. Chimeric rabbit/human Fab and IgG1 were investigated for their capability to bind to human and mouse ROR1, to mediate antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and internalization, and to agonize or antagonize apoptosis using primary CLL cells from untreated patients as well as MCL cell lines. A panel of mAbs demonstrated high affinity and specificity for a diverse set of epitopes that involve all three extracellular domains of ROR1, are accessible on the cell surface, and mediate internalization. The mAb with the highest affinity and slowest rate of internalization was found to be the only mAb that mediated significant, albeit weak, ADCC. None of the mAbs mediated CDC. Alone, they did not enhance or inhibit apoptosis. CONCLUSIONS AND SIGNIFICANCE: Owing to its relatively low cell surface density, ROR1 may be a preferred target for armed rather than naked mAbs. Provided is a panel of fully sequenced and thoroughly characterized anti-ROR1 mAbs suitable for conversion to antibody-drug conjugates, immunotoxins, chimeric antigen receptors, and other armed mAb entities for preclinical and clinical studies.

Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia.

PURPOSE: Gene expression profiling identified receptor tyrosine kinase ROR1, an embryonic protein involved in organogenesis, as a signature gene in B-cell chronic lymphocytic leukemia (B-CLL). To assess the suitability of ROR1 as a cell surface antigen for targeted therapy of B-CLL, we carried out a comprehensive analysis of ROR1 protein expression. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells, sera, and other adult tissues from B-CLL patients and healthy donors were analyzed qualitatively and quantitatively for ROR1 protein expression by flow cytometry, cell surface biotinylation, Western blotting, and ELISA. RESULTS: ROR1 protein is selectively expressed on the surface of B-CLL cells, whereas normal B cells, other normal blood cells, and normal adult tissues do not express cell surface ROR1. Moreover, cell surface expression of ROR1 is uniform and constitutive, i.e., independent of anatomic niches, independent of biological and clinical heterogeneity of B-CLL, independent of B-cell activation, and found at similar levels in all B-CLL samples tested. The antibody binding capacity of B-CLL cell surface ROR1 was determined to be in the range of 10(3) to 10(4) molecules per cell. A portion of B-CLL cell surface ROR1 was actively internalized upon antibody binding. Soluble ROR1 protein was detectable in sera of <25% of B-CLL patients and a similar fraction of healthy donors at concentrations below 200 ng/mL. CONCLUSIONS: The restricted, uniform, and constitutive cell surface expression of ROR1 protein in B-CLL provides a strong incentive for the development of targeted therapeutics such as monoclonal antibodies.

Chimeric rabbit/human Fab and IgG specific for members of the Nogo-66 receptor family selected for species cross-reactivity with an improved phage display vector.

NgR1, NgR2, and NgR3 which constitute the Nogo-66 receptor family are primarily expressed by neurons in the central nervous system (CNS) and believed to limit axonal growth and sprouting following CNS injury. In an attempt to define the expression and decipher the function of individual members of the Nogo-66 receptor family, we previously reported the generation of selective rabbit polyclonal antibodies. Here we exploit the same immune repertoires by phage display technology to generate rabbit monoclonal antibodies (mAbs) with nanomolar affinity to epitopes that are specific for NgR1 and NgR2, respectively, but at the same time conserved between mouse, rat, and human orthologs. Employing phage display vector pC3C, a newly designed phagemid optimized for the generation and selection of Fab libraries with human constant domains, rabbit mAbs were selected from chimeric rabbit/human Fab libraries, characterized in terms of specificity, affinity, and amino acid sequence, and finally converted to chimeric rabbit/human IgG. Using immunofluorescence microscopy and immunoprecipitation, we demonstrate strong and specific recognition of cell surface bound Nogo-66 receptor family members by chimeric rabbit/human IgG. The rabbit mAbs reported here together with their amino acid sequences constitute a defined panel of species cross-reactive reagents in infinite supply which will aid investigations toward a functional role of the Nogo-66 receptor family in and beyond the CNS.

Relational experiences of partnered Japanese immigrant women with affect disorders.

AIM: This paper reports the findings of a study that explored characteristics of relationships of Japanese immigrant women partnered both intraculturally and interculturally, and analysed the role of Japanese culture in these relationships. BACKGROUND: Immigration can cause shifts in interpersonal structures with partners. When there are large discrepancies in gender roles and communication styles between the original and host cultures, the psychological impact on both partners may be significant. However, currently no empirical data are available to support this assumption. METHODS: Ten cases selected from the 68 medical records of Japanese-speaking women seen at a mental health clinic from September 2001 to September 2004 were analysed. All of the 10 women met DSM IV-TR criteria of major depressive disorder and were taking antidepressants. Half of the 10 women were in intimate intercultural partnered relationships and the remainder of the matched cases were in intracultural relationships at the time of treatment. The two cohorts were matched in age (36.2 and 43.2 years), length of stay in the United States of America (12 and 16.2 years), and length of treatment (1.2 and 1 years). The length of time of the sample in individual psychodynamic psychotherapy ranged from 20 to 317 hours, depending on the intensity of therapy. FINDINGS: Inductive data analysis revealed two themes: (1) Lack of awareness of differences in culturally bound communication by Japanese women in intercultural partnerships; (2) Lack of individuality in Japanese women in intracultural partnerships. Neither group appeared to consider relational aspects of partnership, or to make efforts to improve direct communication with their partners. CONCLUSION: The influence of Japanese culture on gender role and communication styles functions contrary to the mainstream Western culture of the United States of America. In the future, interpersonal elements of cultural differences between host and original cultures in immigration should be considered in research related to immigrants' mental health. This approach should lead to effective interventions to facilitate mental health among immigrants, and regardless of purposes of immigration or length of stay in a psychosocially foreign environment.

Cultural incommensurability in psychodynamic psychotherapy in Western and Japanese traditions.

PURPOSE: To examine cultural incommensurability between clients of Japanese ancestry and Western therapists. DESIGN: Aggregated case-study method was used to analyze psychotherapy cases of Japanese individuals seen by Western therapists. Eight cases were selected for this study from a private practice client pool. FINDINGS: Themes were: (a) only observable data were valued for diagnosis and treatment; (b) cultural stereotypes hampered treatment; (c) individuation and separation occurred within a social web of norms in Japanese culture; (d) key concepts in mental health such as "death and dying" and "rape" were interpreted by clients within a Japanese cultural framework, resulting in unique psychological reactions and behaviors, which had not been recognized by Western therapists; (e) psychological effects of immigration were minimized or ignored by Western therapists; (f) culturally unfamiliar behaviors were "pathologized" by Western therapists; and (g) Western therapists showed ethnocentric biases regarding the effects of immigration. CONCLUSIONS: Various forms of misunderstanding and culturally ignorant practices were found in the treatment of eight Japanese clients by these Western therapists. These ineffective and often harmful practices were unrecognized by the Western therapists.