Myelostimulatory activity of recombinant human interleukin-2 in mice

In a series of studies designed to extend our understanding of interleukin-2 (IL-2) and to study the effect of biologic response modifiers on bone marrow, we observed that administering recombinant human (rH) IL-2 to normal mice resulted in an increase in the frequency of colony-forming units-culture (CFU-C) in bone marrow. In addition, rH IL-2 was able to accelerate host recovery from cyclophosphamide (CTX)- or radiation-induced bone marrow depression and peripheral blood leukopenia. Not only can rH IL-2 accelerate, in a dose-dependent manner, the return of bone marrow, peripheral blood cellularity, and CFU-C frequency to normal levels following cytoreduction by CTX or irradiation, but it also significantly increases CFU-C frequency to greater than normal levels. Furthermore, rH IL-2 can significantly prolong survival of animals receiving a lethal dose of irradiation or CTX. Thus, multiple mechanisms are responsible for the synergistic therapeutic activity associated with rH IL-2 and CTX. rH IL-2 does not act only as an immunomodulatory agent in the presence or absence of suppressor T cells, but also accelerates host recovery from cytoreductive agents, resulting in decreased leukopenia and perhaps resistances to secondary infection. Thus, rH IL-2 plus chemotherapy may increase therapeutic activity against neoplastic disease, not only by adding immune stimulation to the direct antitumor effect of the drug but also by allowing delivery of higher, more effective doses of chemotherapy.     

Ectopic expression of rhombotin-2 causes selective expansion of CD4-CD8- lymphocytes in the thymus and T-cell tumors in transgenic mice

Although the proto-oncogene rhombotin-2 (RBTN-2) is widely expressed in most tissues, it is not expressed in T cells. We investigated the potential for overexpression of RBTN-2 to cause tumors in T cells and other tissues by constructing transgenic mice that expressed RBTN-2 under control of the metallothionein-1 promoter. Despite overexpression of RBTN-2 in all tissues, transgenic mice developed T-cell tumors only, thus indicating that tumorigenesis caused by RBTN-2 is T-cell-specific. Thymic tumors were found between 37 and 71 weeks and were invariably associated with metastasis to nonlymphoid organs. Thymuses from apparently healthy transgenic mice were also examined. In some mice there was an 10-fold increase in the CD4-CD8- thymocyte subset, yet the total number of thymocytes was the same as that in wild-type mice. Thymic homeostasis was maintained by a compensatory reduction in the CD4+CD8+ subset. The expansion of CD4-CD8- thymocytes was associated with increased expression of RBTN-2 and with increased cell proliferation. No differences were found in the proportion of thymocytes undergoing apoptosis in transgenic mice. Furthermore, RBTN-2- induced expansion of CD4-CD8- cells did not block differentiation of these cells. Thymuses with 30% CD4-CD8- cells were essentially monoclonal, indicating that all thymic immunophenotypes were derived from a single clone. Overall, our data are consistent with the following scenario: (1) RBTN-2 expression in T cells causes selective and polyclonal proliferation of CD4-CD8- thymocytes accompanied by a compensatory decrease in other thymocyte subsets; (2) a clone with growth advantage and differentiation potential is selected and populates the thymus; and (3) this clone eventually breaches homeostasis of the thymus, accompanied or followed by metastasis to other organs.     

Modulation of megakaryocytopoiesis by thrombopoietin: the c-Mpl ligand

We have further characterized the biological activities, mechanism of action, and target cell populations of recombinant human and murine thrombopoietin (rhTPO and rmTPO) in in vitro human and murine model systems. Alone, hTPO or mTPO stimulated the maturation of immature murine megakaryoblasts as measured in a single cell assay. The combination of hTPO or mTPO and interleukin-6 (IL-6) resulted in a further increase in megakaryocyte differentiation in this system. Murine TPO stimulated mouse megakaryocyte progenitor development. Human megakaryocyte progenitor development was potentiated by hTPO alone and further augmented in the presence of the early-acting cytokines (IL-3) or kit ligand/stem cell factor (KL/SCF). To further define the mechanism of action of TPO, neutralization studies were performed with antisera to IL-3, granulocyte-macrophage colony-stimulating factor (GM- CSF), IL-1 beta, and IL-11. No diminution in TPO activity was observed in the presence of these antisera. Moreover, because adhesive interactions are known to modulate hematopoiesis, we studied whether hTPO might alter such interactions between human bone marrow (BM) megakaryocytes and human BM stromal fibroblasts. No changes were observed in either megakaryocyte expression of the surface molecules lymphocyte function-associated antigen-1, very late activation antigen- 4, or intercellular adhesion molecule-1 or the adhesion of megakaryocytes to stromal fibroblasts after treatment with the growth factor. Furthermore, no induction of secretion of the cytokines IL-1 alpha, IL-1 beta, GM-CSF, IL-6, granulocyte-CSF, tumor necrosis factor- alpha, transforming growth factor-beta 1, or transforming growth factor- beta 2 by primary human BM megakaryocytes was noted after treatment of the cells with hTPO. To address whether TPO affects very primitive hematopoietic progenitors, we studied the residual cells from the BMs of mice treated with high doses of 5-fluorouracil. Although no effect of mTPO alone was noted on the viability or replication of such primitive murine progenitor populations, the triple combination of IL-3 + KL/SCF + TPO stimulated growth of megakaryocyte progenitors. These results indicate that TPO is a highly lineage-specific growth factor whose primary biological effects are likely to be direct modulation of the growth and maturation of committed megakaryocyte precursors and immature megakaryoblasts.     

Phase I trial of interleukin-2 after unmodified HLA-matched sibling bone marrow transplantation for children with acute leukemia

Allogeneic bone marrow transplantation (BMT) for advanced acute leukemia is associated with a high risk of relapse. It is postulated that interleukin-2 (IL-2) administered after BMT might induce or amplify a graft-versus-leukemia effect and thereby reduce the relapse rate. To identify an IL-2 regimen for testing this hypothesis, a phase I trial of IL-2 (Roche) was performed in children in complete remission (CR) without active graft-versus-host disease (GVHD) off immunosuppressive agents after unmodified allogeneic matched-sibling BMT for acute leukemia beyond first remission. Beginning a median of 68 days after BMT, 17 patients received escalating doses of induction IL-2 (0.9, 3.0, or 6.0 x 10(6) IU/m2/d representing levels I, II, and III) for 5 days by continuous intravenous infusion (CIV). After 6 days of rest, they received maintenance IL-2 (0.9 x 10(6) IU/m2/d) for 10 days by CIV infusion. Levels I and II were well-tolerated, but, of 6 patients at level III, 1 developed pulmonary infiltrates, 1 developed hypotension (both resolved), and 1 died of bacterial sepsis and acute respiratory distress syndrome. Grade II acute GVHD developed in 1 patient at level I and 1 at level III. The maximum tolerated dose of induction IL-2 was level II. IL-2 induced lymphocytosis, with an increase in CD56+ and CD8+ cells. Ten patients remain in CR at 5+ to 67+ months. Thus, a regimen of IL-2 has been identified that did not induce a high incidence of acute GVHD when administered to children after unmodified allogeneic BMT. Its clinical activity will be assessed in a phase II trial.     

The Interaction Between an Individual’s Acculturation and Community Factors on Physical Inactivity and Obesity: A Multilevel Analysis

Objectives. We examined whether the interactions between primarily speaking English at home and community-level measures (median household income and immigrant composition) are associated with physical inactivity and obesity.Methods. We pooled the 2005 and 2007 Los Angeles County Health Survey data to construct a multilevel data set, with community-level median household income and immigrant density as predictors at the community level. After controlling for individual-level demographic variables, we included the respondent’s perceived community safety as a covariate to test the hypothesis that perceived public safety mediates the association between acculturation and health outcomes.Results. The interaction between community median household income and primarily speaking English at home was associated with lower likelihoods of physical inactivity (odds ratio [OR] = 0.644; 95% confidence interval [CI] = 0.502, 0.825) and obesity (OR = 0.674; 95% CI = 0.514, 0.882). These odds remained significant after we controlled for perceived community safety.Conclusions. Resources in higher-income areas may be beneficial only to residents fully integrated into the community. Future research could focus on understanding how linguistic isolation affects community-level social learning and access to resources and whether this differs by family-level acculturation.Immigrants face the challenge of assimilating into their host country while maintaining values, beliefs, and behaviors from their homelands. Both acculturation and ethnic identity can influence health, and the construct of acculturation has been included in more and more health studies.1,2 Despite long-standing sociocultural theories of behavior that suggest that one’s behavior is the result of a dynamic interplay between internal, individual-level factors and social-cultural context,3–6 few public health studies have explored this interaction.7 A critical review noted that studies of acculturation tend to

separate culture from the larger social structure and the dynamic social processes in which behavior and beliefs are generated, and to relegate consideration of the socio-economic challenges associated with immigration, poor English language skills, and poverty, to their effects as separate or confounding variables.8^(p981)

For an immigrant, the interaction between acculturation status and the larger social structure in the host society could be important for health, as an inadequate level of acculturation in some contexts might result in reduced access to resources. In particular, undocumented immigrants have no federal coverage of health care under the Affordable Care Act. Therefore, access to resources may differ by legal status. However, in some settings, ethnic identity may buffer and even be protective against public health challenges in the United States (e.g., immigrants may maintain their dietary customs, which often include more whole foods, despite the excessive availability of processed foods in the United States).9 From a methodological perspective, multilevel models can provide a better understanding of this kind of interaction, whereby community-level factors, individual-level acculturation, and the cross-level interaction effects between the two can all be included as regressors of the outcome variable. However, very few public health studies have considered the cross-level interaction between acculturation and community-level factors on health behaviors and health outcomes. In an attempt to fill this research gap, we used population-based survey data to explore the cross-level interaction between community-level factors (median household income and immigrant composition) and individual-level linguistic acculturation (language preference at home).This study includes 2 independent variables that have been infrequently considered in previous studies of immigrant health: community immigrant composition and perceived community safety. Among various community-level factors that could influence residents’ health outcomes, community immigrant composition has begun to receive academic attention.10 Aside from individual-level acculturation indicators such as language preference and place of birth, living in a community with a high proportion of immigrants may be an independent predictor of one’s level of acculturation since people who are less acculturated may choose to live in ethnic enclaves.11 Perceived community safety has been shown to be a strong predictor of individual-level health outcomes such as having a mental health disorder or being overweight.12–15 The causal pathways between an unsafe community and negative health outcomes such as obesity could operate through reduced physical activity16–20 or through stress, which can disrupt energy metabolism and food intake regulation.21–24Because acculturation has been shown to be associated with one’s perception of community safety25 and predicts many different health behaviors and health outcomes,1,2 it is likely that an individual’s level of acculturation could modify the impact of community-level factors on health outcomes. Because understanding of these causal mechanisms is still far from conclusive, a study of the interaction between individuals’ level of acculturation and community-level factors could help reveal the complex pattern of acculturation and health. From the perspective of public health interventions, a good understanding of acculturation, perceived safety, and health could inform intersectoral collaboration between public safety, K-12 (kindergarten through 12th grade) and adult education, immigrant services, and public health agencies.     

Preparation,characterization and storage of human ferritin for use as a standard for the assay of serum ferritin

Summary. This paper describes studies on the source, preparation, characterization and storage of human ferritin for use as a standard for the immunoassay of serum ferritin. Ferritin was prepared from the liver or spleen by methods including either ultracentrifugation or cadmium sulphate crystallization. Preparations were characterized by polyacrylamide gel electrophoresis in dissociating and non-dissociating buffers, iso-electric focusing, analysis of amino acid composition and measurement of protein content. The protein content of solutions of liver or spleen ferritin may be determined by the method of Lowry with bovine serum albumin as standard. Lyophilization under carefully controlled conditions in buffer containing high concentrations of albumin provides a stable preparation of ferritin. Accelerated degradation and collaborative immunological studies of two lyophilized preparations of ferritin, one from liver and one from spleen, indicate that either is an acceptable reference material.     

Production of Spherocytosis and Increased Osmotic Fragility in Normal Erythrocytes by Sodium Stress

Red cells from normal human subjects were exposed to hypertonic sodium chloride solutions for varying periods of time. The cell changes commonly observed in hereditary spherocytosis were induced, including microspherocyte formation with corresponding increase in osmotic fragility. Solutions of magnesium sulphate of equal osmolarity did not produce these changes. These observations lend support to the theory that excessive sodium permeability is concerned in the pathogenesis of hereditary spherocytosis. Increased influx of sodium and water into erythrocytes leads to spherocytosis, but only increased efflux of sodium leads to irreversible microspherocytosis.     

Interleukin‐6 limits influenza‐induced inflammation and protects against fatal lung pathology

Balancing the generation of immune responses capable of controlling virus replication with those causing immunopathology is critical for the survival of the host and resolution of influenza‐induced inflammation. Based on the capacity of interleukin‐6 (IL‐6) to govern both optimal T‐cell responses and inflammatory resolution, we hypothesised that IL‐6 plays an important role in maintaining this balance. Comparison of innate and adaptive immune responses in influenza‐infected wild‐type control and IL‐6‐deficient mice revealed striking differences in virus clearance, lung immunopathology and generation of heterosubtypic immunity. Mice lacking IL?6 displayed a profound defect in their ability to mount an anti‐viral T‐cell response. Failure to adequately control virus was further associated with an enhanced infiltration of inflammatory monocytes into the lung and an elevated production of the pro‐inflammatory cytokines, IFN‐α and TNF‐α. These events were associated with severe lung damage, characterised by profound vascular leakage and death. Our data highlight an essential role for IL‐6 in orchestrating anti‐viral immunity through an ability to limit inflammation, promote protective adaptive immune responses and prevent fatal immunopathology.     

Corticobasal and ataxia syndromes widen the spectrum of C9ORF72 hexanucleotide expansion disease

Recently, a hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 was reported as the cause of chromosome 9p21‐linked frontotemporal dementia‐amyotrophic lateral sclerosis (FTD‐ALS). We here report the prevalence of the expansion in a hospital‐based cohort and associated clinical features indicating a wider clinical spectrum of C9ORF72 disease than previously described. We studied 280 patients previously screened for mutations in genes involved in early onset autosomal dominant inherited dementia disorders. A repeat‐primed polymerase chain reaction amplification assay was used to identify pathogenic GGGGCC expansions. As a potential modifier, confirmed cases were further investigated for abnormal CAG expansions in ATXN2. A pathogenic GGGGCC expansion was identified in a total of 14 probands. Three of these presented with atypical clinical features and were previously diagnosed with clinical olivopontocerebellar degeneration (OPCD), atypical Parkinsonian syndrome (APS) and a corticobasal syndrome (CBS). Further, the pathogenic expansion was identified in six FTD patients, four patients with FTD‐ALS and one ALS patient. All confirmed cases had normal ATXN2 repeat sizes. Our study widens the clinical spectrum of C9ORF72related disease and confirms the hexanucleotide expansion as a prevalent cause of FTD‐ALS disorders. There was no indication of a modifying effect of the ATXN2 gene.