Natural killer activity in the rat. III. Characterization of transplantable large granular lymphocyte (LGL) leukemias in the F344 rat

Six transplantable large granular lymphocyte (LGL) tumor lines in F344 rats were examined for natural killer (NK) and antibody-dependent cell-mediated cytotoxicity. Tumor cells from all six lines were highly cytotoxic, even at low effector to target ratios, when tested against NK-susceptible targets, but were unreactive against an NK-resistant target (C58NT)D) and a macrophage-susceptible target (P815). Three lines showed significant levels of lysis against antibody-coated tumor cells. After in vivo transplantation, the levels of cytotoxicity steadily increased in three lines and decreased in one. The cytotoxic activity of one line (RNK-16) remained high through 12 transplant generations. Tumor cells injected i.p. spread via the lymphatics to regional lymph nodes, mediastinal nodes, blood, and eventually the bone marrow. Leukemia occurred concurrently with organ enlargement and increased levels of NK. Studies in (F344 X W/Fu)F1 rats clearly demonstrated that the cytotoxic cells from leukemic animals were the transplanted tumor cells themselves and not merely the activation of normal host LGL. These results demonstrate that naturally occurring, transplantable LGL leukemias are an easily obtainable and excellent source of materials for those studies requiring a large number of functionally active LGL.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Modulation of lymphocyte function with inhibitory CD2: loss of NK and NKT cells

Analysis of the NK cell developmental pathway suggests that CD2 expression may be important in regulating NK maturation. To test this hypothesis, we developed mice containing only an inhibitory CD2 molecule by linking the extracellular domain of CD2 to an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) motif. Mice containing the CD2 Tg(ITIM) transgene, introduced into a CD2 KO background, have no morphologically detectable lymph nodes, although development of the thymus appears normal. In addition, these mice had major loss of both NK and NKT subsets in peripheral organs, while T and B cell frequencies were intact. Expression of CD2 was low on T cells and lacking on B cells and functional defects were observed in these populations. NKT cells expressing CD4 were absent, while the CD8⁺ and double negative NKT cells were retained. Small subsets of NK cells were detected but expression of CD2 on these cells was very low or absent, and their maturation was impaired. Based on the phenotype described here, we believe that these mice represent a unique model to study lymphoid organ and lymphocyte development.     

A nationwide school fruit and vegetable policy and childhood and adolescent overweight: A quasi-natural experimental study

BackgroundSchool free fruit and vegetable (FFV) policies are used to promote healthy dietary habits and tackle obesity; however, our understanding of their effects on weight outcomes is limited. We assess the effect of a nationwide FFV policy on childhood and adolescent weight status and explore heterogeneity by sex and socioeconomic position.Methods and findingsThis study used a quasi-natural experimental design. Between 2007 and 2014, Norwegian combined schools (grades 1–10, age 6 to 16 years) were obligated to provide FFVs while elementary schools (grades 1–7) were not. We used 4 nationwide studies (n = 11,215 children) from the Norwegian Growth Cohort with longitudinal or cross-sectional anthropometric data up to age 8.5 and 13 years to capture variation in FFV exposure. Outcomes were body mass index standard deviation score (BMI_SDS), overweight and obesity (OW/OB), waist circumference (WC), and weight to height ratio (WtHR) at age 8.5 years, and BMI_SDS and OW/OB at age 13 years. Analyses included longitudinal models of the pre- and post-exposure trajectories to estimate the policy effect. The participation rate in each cohort was >80%, and in most analyses <4% were excluded due to missing data. Estimates were adjusted for region, population density, and parental education. In pooled models additionally adjusted for pre-exposure BMI_SDS, there was little evidence of any benefit or unintended consequence from 1–2.5 years of exposure to the FFV policy on BMI_SDS, OW/OB, WC, or WtHR in either sex. For example, boys exposed to the FFV policy had a 0.05 higher BMI_SDS (95% CI: −0.04, 0.14), a 1.20-fold higher odds of OW/OB (95% CI: 0.86, 1.66) and a 0.3 cm bigger WC (95% CI: −0.3, 0.8); while exposed girls had a 0.04 higher BMI_SDS (95% CI: −0.04, 0.13), a 1.03 fold higher odds of OW/OB (95% CI: 0.75, 1.39), and a 0-cm difference in WC (95% CI: −0.6, 0.6). There was evidence of heterogeneity in the policy effect estimates at 8.5 years across cohorts and socioeconomic position; however, these results were inconsistent with other comparisons. Analysis at age 13 years, after 4 years of policy exposure, also showed little evidence of an effect on BMI_SDS or OW/OB. The main limitations of this study are the potential for residual confounding and exposure misclassification, despite efforts to minimize their impact on conclusions.ConclusionsIn this study we observed little evidence that the Norwegian nationwide FFV policy had any notable beneficial effect or unintended consequence on weight status among Norwegian children and adolescents.

Bente Øvrebø and colleagues assess whether a nationwide free school fruit and vegetable policy was associated with weight outcomes in children and early adolescents in Norway.     

The restricted expression of granzyme M in human lymphocytes

We have analyzed the expression of human granzyme M (Gzm M) in various human leukocyte subsets using the specific mAb 4H10. Using FACS and Western blotting analysis we compared the expression of Gzm M with that of other granzymes (Gzm A and Gzm B) and the lytic protein perforin. Human Gzm M was constitutively highly expressed in NK cells as was perforin and Gzm A. Surprisingly, freshly isolated NK cells had very low (sometimes undetectable) levels of Gzm B. In contrast to Gzm B and perforin, Gzm M was not detected in highly purified CD4(+) and CD8(+) T cells either constitutively or after short term activation in vitro. However, low levels of Gzm M were observed in some T cell clones on prolonged passage in vitro. Gzm M was not detected in highly purified neutrophils, monocytes, or tumor cells of the myelomonocytic lineage. Examination of minor T cell subsets from human peripheral blood showed detectable Gzm M in CD3(+), CD56(+) T cells and gammadelta T cells. A histological staining procedure was developed that demonstrated a granular staining pattern for Gzm M and a cellular distribution similar to that observed by Western blotting. These data indicate that the expression of Gzm M does not always correlate with the lytic activity of cytotoxic cells. However, expression of Gzm M in NK cells, CD3(+), CD56(+) T cells, and gammadelta T cells suggests that this enzyme may play some role in innate immune responses.     

In vivo hydrodynamic delivery of cDNA encoding IL-2: rapid, sustained redistribution, activation of mouse NK cells, and therapeutic potential in the absence of NKT cells

In the present study, we have tested the ability of hydrodynamically delivered IL-2 cDNA to modulate the number and function of murine leukocyte subsets in different organs and in mice of different genetic backgrounds, and we have evaluated effects of this mode of gene delivery on established murine tumor metastases. Hydrodynamic administration of the IL-2 gene resulted in the rapid and transient production of up to 160 ng/ml IL-2 in the serum. The appearance of IL-2 was followed by transient production of IFN-gamma and a dramatic and sustained increase in NK cell numbers and NK-mediated cytolytic activity in liver and spleen leukocytes. In addition, significant increases in other lymphocyte subpopulations (e.g., NKT, T, and B cells) that are known to be responsive to IL-2 were observed following IL-2 cDNA plasmid delivery. Finally, hydrodynamic delivery of only 4 mug of the IL-2 plasmid to mice bearing established lung and liver metastases was as effective in inhibiting progression of metastases as was the administration of large amounts (100,000 IU/twice daily) of IL-2 protein. Studies performed in mice bearing metastatic renal cell tumors demonstrated that the IL-2 cDNA plasmid was an effective treatment against liver metastasis and moderately effective against lung metastasis. Collectively, these results demonstrate that hydrodynamic delivery of relatively small amounts of IL-2 cDNA provides a simple and inexpensive method to increase the numbers of NK and NKT cells, to induce the biological effects of IL-2 in vivo for use in combination with other biological agents, and for studies of its antitumor activity.     

Dissociation of NKT stimulation, cytokine induction, and NK activation in vivo by the use of distinct TCR-binding ceramides

NKT and NK cells are important immune regulatory cells. The only efficient means to selectively stimulate NKT cells in vivo is alpha-galactosylceramide (alphaGalCer). However, alphaGalCer effectively stimulates and then diminishes the number of detectable NKT cells. It also exhibits a potent, indirect ability to activate NK cells. We have now discovered another ceramide compound, beta-galactosylceramide (betaGalCer) (C12), that efficiently diminishes the number of detectable mouse NKT cells in vivo without inducing significant cytokine expression or activation of NK cells. Binding studies using CD1d tetramers loaded with betaGalCer (C12) demonstrated significant but lower intensity binding to NKT cells when compared with alphaGalCer, but both ceramides were equally efficient in reducing the number of NKT cells. However, betaGalCer (C12), in contrast to alphaGalCer, failed to increase NK cell size, number, and cytolytic activity. Also in contrast to alphaGalCer, betaGalCer (C12) is a poor inducer of IFN-gamma, TNF-alpha, GM-CSF, and IL-4 gene expression. These qualitative differences in NKT perturbation/NK activation have important implications for delineating the unique in vivo roles of NKT vs NK cells. Thus, alphaGalCer (which triggers NKT cells and activates NK cells) efficiently increases the resistance to allogeneic bone marrow transplantation while betaGalCer (C12) (which triggers NKT cells but does not activate NK cells) fails to enhance bone marrow graft rejection. Our results show betaGalCer (C12) can effectively discriminate between NKT- and NK-mediated responses in vivo. These results indicate the use of different TCR-binding ceramides can provide a unique approach for understanding the intricate immunoregulatory contributions of these two cell types.     

Use of biological monitoring tools beyond their country of origin: a case study of the South African Scoring System Version 5 (SASS5)

Biological monitoring tools are largely lacking for many countries, resulting in adoption of tools developed from other countries/regions, but in many instances, their applicability to the new system has not been explicitly evaluated. The objective of the study was to test the applicability of the South African Scoring Systems Version 5 (SASS5) to urban streams in Zimbabwe. The study evaluated the relationship between water quality variables and SASS5 indices/metrics [(SASS and average score per taxon (ASPT)] and found high degree of concordance between water chemistry parameters and SASS5 metrics, indicating that both SASS and ASPT scores are sensitive to detect environmental changes. This result can be attributed to occurrence of ubiquitous macroinvertebrate taxa sharing similar environmental tolerances with those recorded for South African systems. The applicability of SASS5 metrics need to be tested across different geographical and climatic regions in the country (taking into consideration seasonal variations that are important drivers of benthic faunal assemblages in lotic systems) and disparities among the regions compared for the adoption of the index in the entire country. The SASS5 metrics can also be further strengthened by (a) taking into account the relative abundance of taxa and (b) also improving on its ability to reflect other forms of perturbations besides eutrophication and organic pollution such heavy metal pollution.     

Energy Balance Related Behaviour: Personal,Home- and Friend-Related Factors among Schoolchildren in Europe Studied in the ENERGY-Project

Objective

To design interventions that target energy balance-related behaviours, knowledge of primary schoolchildren''s perceptions regarding soft drink intake, fruit juice intake, breakfast consumption, TV viewing and physical activity (PA) is essential. The current study describes personal beliefs and attitudes, home- and friend-related variables regarding these behaviours across Europe.

Design

Cross-sectional study in which personal, family and friend -related variables were assessed by validated questionnaires, and dichotomized as favourable versus unfavourable answers. Logistic regression analyses were conducted to estimate proportions of children giving unfavourable answers and test between-country differences.

Setting

A survey in eight European countries.

Subjects

A total of 7903 10–12 year old primary schoolchildren.

Results

A majority of the children reported unfavourable attitudes, preferences and subjective norms regarding soft drink, fruit juice intake and TV viewing accompanied with high availability and accessibility at home. Few children reported unfavourable attitudes and preferences regarding breakfast consumption and PA. Many children reported unfavourable health beliefs regarding breakfast consumption and TV viewing. Substantial differences between countries were observed, especially for variables regarding soft drink intake, breakfast consumption and TV viewing.

Conclusion

The surveyed children demonstrated favourable attitudes to some healthy behaviours (PA, breakfast intake) as well as to some unhealthy behaviours (soft drink consumption, TV viewing). Additionally, many children across Europe have personal beliefs and are exposed to social environments that are not supportive to engagement in healthy behaviours. Moreover, the large differences in personal, family and friend-related variables across Europe argue for implementing different strategies in the different European countries.