Whole-exome analysis reveals novel somatic genomic alterations associated with outcome in immunochemotherapy-treated diffuse large B-cell lymphoma.

Lack of remission or early relapse remains a major clinical issue in diffuse large B-cell lymphoma (DLBCL), with 30% of patients failing standard of care. Although clinical factors and molecular signatures can partially predict DLBCL outcome, additional information is needed to identify high-risk patients, particularly biologic factors that might ultimately be amenable to intervention. Using whole-exome sequencing data from 51 newly diagnosed and immunochemotherapy-treated DLBCL patients, we evaluated the association of somatic genomic alterations with patient outcome, defined as failure to achieve event-free survival at 24 months after diagnosis (EFS24). We identified 16 genes with mutations, 374 with copy number gains and 151 with copy number losses that were associated with failure to achieve EFS24 (P<0.05). Except for FOXO1 and CIITA, known driver mutations did not correlate with EFS24. Gene losses were localized to 6q21-6q24.2, and gains to 3q13.12-3q29, 11q23.1-11q23.3 and 19q13.12-19q13.43. Globally, the number of gains was highly associated with poor outcome (P=7.4 × 10(-12)) and when combined with FOXO1 mutations identified 77% of cases that failed to achieve EFS24. One gene (SLC22A16) at 6q21, a doxorubicin transporter, was lost in 54% of EFS24 failures and our findings suggest it functions as a doxorubicin transporter in DLBCL cells.

Activation of TAK1 by MYD88 L265P drives malignant B-cell Growth in non-Hodgkin lymphoma.

Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHLs) including the rare lymphoplasmacytic lymphoma, Waldenström's macroglobulinemia (WM). Using whole-exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B-cell survival, therapeutic targeting of MYD88 signaling pathways may be clinically useful. However, we are lacking a thorough characterization of the role of intermediary signaling proteins on the biology of MYD88L265P-expressing B cells. We report here that MYD88L265P signaling is constitutively active in both WM and diffuse large B-cell lymphoma cells leading to heightened MYD88L265P, IRAK and TRAF6 oligomerization and NF-κB activation. Furthermore, we have identified the signaling protein, TAK1, to be an essential mediator of MYD88L265P-driven signaling, cellular proliferation and cytokine secretion in malignant B cells. Our studies highlight the biological significance of MYD88L265P in NHL and reveal TAK1 inhibition to be a potential therapeutic strategy for the treatment of WM and other diseases characterized by MYD88L265P.

TGF-ß upregulates CD70 expression and induces exhaustion of effector memory T cells in B-cell non-Hodgkin's lymphoma.

Transforming growth factor beta (TGF-ß) has an important role in mediating T-cell suppression in B-cell non-Hodgkin lymphoma (NHL). However, the underlying mechanism responsible for TGF-ß-mediated inhibition of effector memory T (Tm) cells is largely unknown. As reported here, we show that exhaustion is a major mechanism by which TGF-ß inhibits Tm cells, and TGF-ß mediated exhaustion is associated with upregulation of CD70. We found that TGF-ß upregulates CD70 expression on effector Tm cells while it preferentially induces Foxp3 expression in naive T cells. CD70 induction by TGF-ß is Smad3-dependent and involves IL-2/Stat5 signaling. CD70+ T cells account for TGF-ß-induced exhaustion of effector Tm cells. Both TGF-ß-induced and preexisting intratumoral CD70+ effector Tm cells from B-cell NHL have an exhausted phenotype and express higher levels of PD-1 and TIM-3 compared with CD70- T cells. Signaling transduction, proliferation and cytokine production are profoundly decreased in these cells, and they are highly susceptible to apoptosis. Clinically, intratumoral CD70-expressing T cells are prevalent in follicular B-cell lymphoma (FL) biopsy specimens, and increased numbers of intratumoral CD70+ T cells correlate with an inferior patient outcome. These findings confirm TGF-ß-mediated effector Tm cell exhaustion as an important mechanism of immune suppression in B-cell NHL.

Effect of its deaminated metabolite, 2',2'-difluorodeoxyuridine, on the transport and toxicity of gemcitabine in HeLa cells.

Gemcitabine is a pyrimidine analog effective against many solid tumors. Following intravenous administration, deaminases in the plasma rapidly convert the parent compound, gemcitabine, to its deaminated metabolite, 2',2'-difluorodeoxyuridine (dFdU), resulting in an elimination half-life for gemcitabine of 8min. The half-life of dFdU, however, is upwards of 14h, yielding plasma concentrations that are frequently 10-20-fold higher than that of gemcitabine. The uptake of gemcitabine into tumor cells is facilitated by both concentrative (hCNT) and equilibrative (hENT) nucleoside transporters. Recently, it was observed that dFdU is a substrate for hCNT as well. The purpose of this study was to investigate the effects of dFdU on gemcitabine uptake and efflux via hENT1 and hENT2 in HeLa cells. Our results suggest that dFdU is a substrate for both hENT1 and hENT2 as well as a competitive inhibitor of gemcitabine transport at concentrations >100-fold lower than those typically achieved in plasma (IC(50)=0.45 and 1.2µM for hENT1/2 and hENT2, respectively). However, inhibition of gemcitabine uptake is time-dependent, as dFdU limits gemcitabine uptake into HeLa cells by more than 80% during short (<20s) incubation periods but increases net gemcitabine retention as incubation length increases. While dFdU enhances the accumulation of gemcitabine by up to 1.5-fold following a 60 min incubation, dFdU did not enhance gemcitabine cytotoxicity. In conclusion, this is the first report of an interaction between dFdU and gemcitabine suggesting that the deaminated metabolite may play an important role in the disposition of gemcitabine in tumor cells.

MicroRNA expression in tumor cells from Waldenstrom's macroglobulinemia reflects both their normal and malignant cell counterparts.

MicroRNAs (miRNAs) are involved in the regulation of many cellular processes including hematopoiesis, with the aberrant expression of differentiation-stage specific miRNA associated with lymphomagenesis. miRNA profiling has been essential for understanding the underlying biology of many hematological malignancies; however the miRNA signature of the diverse tumor clone associated with Waldenstrom's macroglobulinemia (WM), consisting of B lymphocytes, plasmacytes and lymphoplasmacytic cells, has not been characterized. We have investigated the expression of over 13 000 known and candidate miRNAs in both CD19(+) and CD138(+) WM tumor cells, as well as in their malignant and non-malignant counterparts. Although neither CD19(+) nor CD138(+) WM cells were defined by a distinct miRNA profile, the combination of all WM cells revealed a unique miRNA transcriptome characterized by the dysregulation of many miRNAs previously identified as crucial for normal B-cell lineage differentiation. Specifically, miRNA-9(*)/152/182 were underexpressed in WM, whereas the expression of miRNA-21/125b/181a/193b/223/363 were notably increased (analysis of variance; P<0.0001). Future studies focusing on the effects of these dysregulated miRNAs will provide further insight into the mechanisms responsible for the pathogenesis of WM.

Chronic renal failure from the English-speaking Caribbean: 2007 data / Fallo renal crónico en el Caribe anglófono: datos del 2007

AIM: Development of the renal registry to include patients at different stages of chronic kidney disease (CKD). BACKGROUND: The 2007 renal registry include cases at different stages of CKD based on the current guidelines according to the National Kidney Foundation (NKF) Kidney Disease Outcome Initiative (K/DOQI) staging. There was an increase in the number of participating countries, with the addition of Antigua and Barbuda, St Lucia and Turks and Caicos. METHODS: Data were collected using a questionnaire form. Data were stored and analysed in Words Excel for Windows or SPSS 12.0. RESULTS: Data were available for Antigua and Barbuda (n = 43), British Virgin Islands (n = 69), Cayman Islands (n = 45), Trinidad and Tobago (n = 564), Jamaica (n = 920), Turks and Caicos (n = 64), St Lucia (n = 51) and Bahamas (n = 121). The registry identified hypertension, diabetes mellitus and Chronic Glomerulonephritis (CGN) as the commonest causes of chronic kidney disease (CKD) and end-stage renal disease (ESRD) in these countries. The leading cause of death reported was listed as ischaemic heart disease/heart failure, sepsis and cerebrovascular accident. CONCLUSIONS: The majority of patients with CKD and ESRD had hypertension, diabetes mellitus and CGN as the major causes. Collection of data for patients with CKD at different stages was met with some challenges, and resulted in underestimation of the true number of persons with CKD across these Caribbean countries. More emphasis will continue to be placed on improving data collection so the true incidence, prevalence and healthcare burden of CKD is known in the Caribbean. A web based programme is being developed to improve data collection.

OBJETIVO: Desarrollar el registro renal incluyendo pacientes en diferentes etapas de la enfermedad crónica del riñón (ECR). ANTECEDENTES: El registro renal 2007 incluye casos en diferentes etapas de la ECR, sobre la base de los lineamientos actuales de la estadificación según la iniciativa para los resultados de la enfermedad crónica renal (K/DOQI) propuesta por la Fundación Nacional del Riñón (NKF). Hubo un aumento en el número de países participantes, al añadirse Antigua y Barbuda, Santa Lucia e Islas Turcas y Caicos. MÉTODOS: Los datos fueron recogidos utilizando un cuestionario. Luego fueron almacenados y analizados usando Excel para Windows, o mediante SPSS 12.0. RESULTADOS: Hubo a disposición datos para Antigua y Barbuda (n = 43), Islas Vírgenes Británicas (n = 69), Islas Cayman (n = 45), Trinidad y Tobago (n = 564), Jamaica (n = 920), Islas Turcas y Caicos (n = 64), Santa Lucia (n = 51) y Bahamas (n = 121). El registro identificó la hipertensión, la diabetes mellitus y la glomerulonefritis crónica (GNC) como las causas más comunes de la enfermedad crónica del riñón (ECR) y la enfermedad renal terminal (ERT) en estos países. La principal causa de muerte según los reportes, fueron la cardiopatía isquémica/fallo cardíaco, la sepsis y el accidente cardiovascular. CONCLUSIONES: La mayoría de los pacientes con ECR y ERT sufrían de hipertensión, diabetes mellitus y GNC como causas mayores. La recogida de datos para los pacientes con ECR tuvo algunas dificultades, por lo que se subestimó el número real de personas con ECR en todos estos países caribeños. Se seguiría haciendo un mayor énfasis en mejorar la recogida de datos, de modo que la verdadera incidencia, prevalencia y carga de atención a la salud de la ECR sea conocida en el Caribe. Se halla en curso el desarrollo de un programa en la red de Internet, a fin de mejorar la recogida de datos.

Prevalence of zoonotic agents in dogs visiting hospitalized people in Ontario: implications for infection control.

Visitation of hospitalized people by dogs is becoming commonplace, but little is known about the potential health risks of introducing dogs to healthcare settings. This cross-sectional study evaluated the prevalence of zoonotic agents in a group of 102 visitation dogs from a variety of sources across Ontario. Between May and July 2004, owners were interviewed by a standardized questionnaire while dogs underwent a standardized physical examination. One specimen of faeces, hair-coat brushings and one rectal, aural, nasal, oral and pharyngeal swab were collected from each dog and tested for 18 specific pathogens. All dogs were judged to be in good health. Zoonotic agents were isolated from 80 out of 102 (80%) dogs. The primary pathogen was Clostridium difficile, which was isolated from 58 (58%) faecal specimens. Seventy-one percent (41/58) of these isolates were toxigenic. Extended-spectrum beta-lactamase Escherichia coli was isolated from one (1%) dog, extended-spectrum cephalosporinase E. coli was isolated from three (3%) dogs, and organisms of the genus Salmonella were isolated from three (3%) dogs. Pasteurella multocida or Pasteurella canis was isolated from 29 (29%) oral swabs, and Malassezia pachydermatis was isolated from eight (8%) aural swabs. Giardia antigen was present in the faeces of seven (7%) dogs, while Toxocara canis and Ancylostoma caninum were detected in two (2%) dogs and one (1%) dog, respectively. Methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Campylobacter spp., Microsporum canis, group A streptococci, Pseudomonas aeruginosa and Cryptosporidium spp. were not detected. Further information is needed before the full implications of these findings for infection control can be assessed properly.

The pulmonary environment promotes Th2 cell responses after nasal-pulmonary immunization with antigen alone, but Th1 responses are induced during instances of intense immune stimulation.

The purpose of this study was to determine the nature of the CD4(+) Th cell responses induced after nasal-pulmonary immunization, especially those coinciding with previously described pulmonary inflammation associated with the use of the mucosal adjuvant, cholera toxin (CT). The major T cell population in the lungs of naive mice was CD4(+), and these cells were shown to be predominantly of Th2 type as in vitro polyclonal stimulation resulted in IL-4, but not IFN-gamma, production. After nasal immunization with influenza Ag alone, Th2 cytokine mRNA (IL-4 and IL-5) levels were increased, whereas there was no change in Th1 cytokine (IL-2 and IFN-gamma) mRNA expression. The use of the mucosal adjuvant, CT, markedly enhanced pulmonary Th2-type responses; however, there was also a Th1 component to the T cell response. Using in vitro Ag stimulation of pulmonary lymphocytes, influenza virus-specific cytokine production correlated with the mRNA cytokine results. Furthermore, there was a large increase in CD4(+) Th cell numbers in lungs after nasal immunization using CT, correlating with the pulmonary inflammatory infiltrate previously described. Coincidentally, both macrophage-inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta mRNA expression increased in the lungs after immunization with Ag plus CT, while only MIP-1beta expression increased when mice were given influenza Ag alone. Our study suggests a mechanism to foster Th1 cell recruitment into the lung, which may impact on pulmonary immune responses. Thus, while Th2 cell responses may be prevalent in modulating mucosal immunity in the lungs, Th1 cell responses contribute to pulmonary defenses during instances of intense immune stimulation.

Eosinophilia, interleukin-5, and tumour necrosis factor-alpha in asthmatic children.

BACKGROUND: There are few paediatric studies of the interrelationships between inflammatory markers and asthma severity. We therefore assessed the relationships between eosinophil-associated markers, cytokines, and asthma severity in asthmatic children aged 8-12 years. METHODS: Forty-five children were tested twice, 2 weeks apart. Asthma severity was measured in terms of symptoms, lung function, medication needs, and histamine responsiveness. Peripheral inflammatory markers measured included eosinophil numbers, serum ECP, IL-5, and TNF-alpha and mononuclear cell IL-5, and TNF-alpha production. RESULTS: Histamine responsiveness was correlated with circulating eosinophils (r = 0.56, P = 0.0001) and serum ECP (r = 0.54, P = 0.003). Eosinophilia was increased in children with severe as opposed to mild airway hyperresponsiveness (P = 0.02) and those who lost days at school as opposed to those who did not (P = 0.01). There were no other associations between markers of asthma severity and inflammation. Children taking inhaled corticosteroids had lower serum IL-5 levels than those on beta-agonists +/- cromolyn (mean and 95% CI: 20.5 [11.7-35.7] pg/ml vs 64.3 [26.6-155.4] pg/ml; P = 0.04). Cellular IL-5 production correlated with serum TNF-alpha (r = 0.63, P = 0.0062) and IL-5 (r = -0.59, P = 0.005). CONCLUSION: Serum levels of TNF-alpha and IL-5 were not related to peripheral eosinophilia and asthma severity in these children but were related to their own cellular production ex vivo. This study confirms that eosinophilia is the index of inflammation that is most closely related to the clinical severity of childhood asthma.

Immunoglobulin A (IgA) responses and IgE-associated inflammation along the respiratory tract after mucosal but not systemic immunization.

The purpose of the present study was to determine the extent of immunologic responses, particularly immunopathologic responses, within the upper and lower respiratory tracts after intranasal immunization using the mucosal adjuvant cholera toxin (CT). BALB/c mice were nasally immunized with influenza virus vaccine combined with CT. The inclusion of the mucosal adjuvant CT clearly enhanced generation of antibody responses in both the nasal passages and lungs. After nasal immunization, antigen-specific immunoglobulin A (IgA) antibody-forming cells dominated antibody responses throughout the respiratory tract. However, IgG responses were significant in lungs but not in nasal passages. Furthermore, parenteral immunization did not enhance humoral immunity in the upper respiratory tract even after a nasal challenge, whereas extrapulmonary lymphoid responses enhanced responses in the lung. After nasal immunization, inflammatory reactions, characterized by mononuclear cell infiltration, developed within the lungs of mice but not in nasal passages. Lowering dosages of CT reduced, but did not eliminate, these adverse reactions without compromising adjuvancy. Serum IgE responses were also enhanced in a dose-dependent manner by inclusion of CT. In summary, there are differences in the generation of humoral immunity between the upper respiratory tract and the lung. As the upper respiratory tract is in a separate compartment of the immune system from that stimulated by parenteral immunization, nasal immunization is an optimal approach to generate immunity throughout the respiratory tract. Despite the promise of nasal immunization, there is also the potential to develop adverse immunopathologic reactions characterized by pulmonary airway inflammation and IgE production.

Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord.

Recent advances in the isolation and characterization of neural precursor cells suggest that they have properties that would make them useful transplants for the treatment of central nervous system disorders. We demonstrate here that spinal cord cells isolated from embryonic day 14 Sprague-Dawley and Fischer 344 rats possess characteristics of precursor cells. They proliferate as undifferentiated neurospheres in the presence of EGF and bFGF and can be maintained in vitro or frozen, expanded and induced to differentiate into both neurons and glia. Exposure of these cells to serum in the absence of EGF and bFGF promotes differentiation into astrocytes; treatment with retinoic acid promotes differentiation into neurons. Spinal cord cells labeled with a nuclear dye or a recombinant adenovirus vector carrying the lacZ gene survive grafting into the injured spinal cord of immunosuppressed Sprague-Dawley rats and non-immunosuppressed Fischer 344 rats for up to 4 months following transplantation. In the presence of exogenously supplied BDNF, the grafted cells differentiate into both neurons and glia. These spinal cord cell grafts are permissive for growth by several populations of host axons, especially when combined with exogenous BDNF administration, as demonstrated by penetration into the graft of axons immunopositive for 5-HT and CGRP. Thus, precursor cells isolated from the embryonic spinal cord of rats, expanded in culture and genetically modified, are a promising type of transplant for repair of the injured spinal cord.

Calreticulin binding and other biological activities of survival peptide Y-P30 including effects of systemic treatment of rats.

Neuron survival-promoting peptide Y-P30, purified from oxidatively stressed neural cell lines, inhibits the appearance of microglia and rescues neurons 1 week after direct application to lesions of the rat cerebral cortex (7). Y-P30 affinity matrices treated with solubilized membranes from a variety of cell lines including human neuroblastoma SY5Y, mouse hippocampal cells HN 33.1, and human promonocytes HL-60, as well as with cerebral cortex tissue from both humans and rats, showed highly specific binding to calreticulin, a ubiquitous calcium binding protein that may be critical for integrin function. Treatment of cultures with 0.1 nM Y-P30 stabilized all these cell types whether differentiated or not, while 1 microM peptide also inhibited the morphological differentiation of the HL-60 cells into macrophages. Western analysis of the medium of SY5Y cell cultures suggested Y-P30-stimulated release of calreticulin, a result consistent with its other biological activities. Likewise, single dose systemic application of Y-P30 in unoperated rats and in rats with cerebral cortex lesions produced significant reductions in cerebral cortex membrane-associated calreticulin. Both direct and intravenous treatment with peptide also reduced cortical neuron atrophy 4 days after these lesions but only direct application consistently inhibited the appearance of ED-1(+) monocyte derivatives. We suggest that in vitro and in vivo mechanisms of Y-P30 effects are similar and involve the targeting of calreticulin. The results also suggest that some of these activities are apparent in the cerebral cortex after systemic application of this peptide.

Identification of a survival-promoting peptide in medium conditioned by oxidatively stressed cell lines of nervous system origin.

A survival-promoting peptide has been purified from medium conditioned by Y79 human retinoblastoma cells and a mouse hippocampal cell line (HN 33.1) exposed to H2O2. A 30 residue synthetic peptide was made on the basis of N-terminal sequences obtained during purification, and it was found to exhibit gel mobility and staining properties similar to the purified molecules. The peptide maintains cells and their processes in vitro for the HN 33.1 cell line treated with H2O2, and in vivo for cortical neurons after lesions of the cerebral cortex. It has weak homology with a fragment of a putative bacterial antigen and, like that molecule, binds IgG. The peptide also contains a motif reminiscent of a critical sequence in the catalytic region of calcineurin-type phosphatases; surprisingly, like several members of this family, the peptide catalyzes the hydrolysis of para-nitrophenylphosphate in the presence of Mn2+. Application of the peptide to one side of bilateral cerebral cortex lesions centered on area 2 in rats results in an increase in IgG immunoreactivity in the vicinity of the lesions 7 d after surgery. Microglia immunopositive for IgG and ED-1 are, however, dramatically reduced around the lesions in the treated hemisphere. Furthermore, pyramidal neurons that would normally shrink, die, or disintegrate were maintained, as determined by MAP2 immunocytochemistry and Nissl staining. These survival effects were often found in both hemispheres. The results suggest that this peptide operates by diffusion to regulate the immune response and thereby rescue neurons that would usually degenerate after cortical lesions. The phosphatase activity of this molecule also suggests the potential for direct neuron survival-promoting effects.

Effect of dietary intake of omega-3 and omega-6 fatty acids on severity of asthma in children.

We assessed the clinical and biochemical effects in asthmatic children of fish oil supplementation and a diet that increases omega-3 and reduces omega-6 fatty acids. Thirty nine asthmatic children aged 8-12 yrs participated in a double-blind, randomized, controlled trial for 6 months during which they received fish oil capsules plus canola oil and margarine (omega-3 group) or safflower oil capsules plus sunflower oil and margarine (omega-6 group). Plasma fatty acids, stimulated tumour necrosis factor alpha (TNFalpha) production, circulating eosinophil numbers and lung function were measured at baseline and after 3 and 6 months of dietary modification. Day and night symptoms, peak flow rates and medication use were recorded for 1 week prior to laboratory visits. Plasma phospholipid omega-3 fatty acids were significantly greater in the omega-3 group at 3 and 6 months compared to the omega-6 group (p<0.001). In the omega-3 group TNFalpha production fell significantly compared with baseline (p=0.026), but the magnitude of change between groups did not reach significance (p=0.075). There were no significant changes in clinical outcome measures. Dietary enrichment of omega-3 fatty acids over 6 months increased plasma levels of these fatty acids, reduced stimulated tumour necrosis factor alpha production, but had no effect on the clinical severity of asthma in these children.

Serological evidence of increased Coccidioides immitis infections in western Canada in 1996.

OBJECTIVE: To investigate the epidemiology of Coccidioides immitis infection in persons returning to western Canada from C immitis endemic zones in southwestern United States. DESIGN: Review of C immitis serology requests from 1996. METHODS: Data were based on review of enzyme immunoassay and immunodiffusion results from 1993 to 1996 inclusive. Detailed information on clinical presentation, treatment and outcome of disease process was obtained through questionnaires and interviews with physicians who submitted Coccidioides serology requests in 1996. RESULTS: Positive serology for C immitis increased from 4.7% to 5.2% (between 1993 and 1995 inclusive) to 10.7% in 1996. Enzyme immunoassay for immunoglobulin G and/or immunoglobulin M or immunodiffusion was positive in 25 patients in 1996. The mean age of these patients was 62 years, and the predominant clinical presentation was pulmonary infiltrate with fever. All patients with positive serology were known to have travelled to central or southwestern Arizona or southern California. CONCLUSIONS: Travel to a defined coccidioidomycosis endemic zone presents a risk for the older traveller. Serology for C immitis supported the clinical, histological and microbiological diagnoses in patients who had travelled to this defined endemic zone.

Assessment of food chemical intolerance in adult asthmatic subjects.

BACKGROUND: Identification of food chemical intolerance in asthmatic subjects can be reliably assessed by changes in the forced expiratory volume in one second (FEV1) in response to double blind, placebo controlled challenges on a strict elimination diet. However, this method is cumbersome and time consuming. A study was undertaken to determine whether changes in bronchial responsiveness to histamine following food chemical challenge without an elimination diet might be a faster, more convenient method. METHODS: Eleven adult asthmatic subjects were challenged twice with metabisulphite, aspirin, monosodium glutamate, artificial food colours, sodium nitrite/ nitrate, 0.5% citric acid solution (placebo), and sucrose (placebo) on separate days. During the first set of challenges subjects consumed a normal diet. Bronchial responsiveness to histamine was assessed 90 minutes after each challenge. A greater than twofold increase in bronchial responsiveness was considered positive. For one month prior to and during the second set of challenges subjects followed a strict elimination diet and FEV1 was monitored during and for two hours after each challenge. A fall in FEV1 of 20% or more was considered positive. RESULTS: Of the 77 food chemical challenges performed on an unmodified diet, 20 were positive (six placebo responses). In two subjects it was not possible to perform a histamine test after one of the chemical challenges because of poor spirometric function. Of the 77 food chemical challenges performed on an elimination diet, 11 were positive (no placebo responses). Excluding the two challenges in which there were no corresponding histamine tests, only on two occasions did the positive responses in both methods coincide, giving the unmodified diet method a sensitivity of 22%. CONCLUSIONS: Strict dietary elimination and measurement of FEV1 after double blind food chemical challenge remains the most reliable method for the detection of food chemical intolerance in asthmatic subjects.

Postmetaphase nuclear formation: loss of a chromosomal epitope coincident with apparent chromatid coalescence.

Previously, we have conceptualized mitotic nuclear formation following metaphase as a morphogenic process and have suggested that sets of chromatids, after separation from a metaphase plate, can be thought of as prenuclei. Such structures can be grouped temporally as either early or late prenuclei based on morphologic, morphometric and density characteristics. Sequential ordering of early prenuclei is of particular interest because it reveals that condensed chromatids coalesce with the resulting formation of a unique chambered structure. In this paper we describe data obtained with a newly raised monoclonal antibody (mAb-2) that initially recognizes an epitope(s) on metaphase chromosomes. Light and confocal fluorescent microscopy of early prenuclei reveal that the chromosomal epitope can no longer be detected about chromatids after their apparent coalescence. Immunoblot analysis of dispersed polypeptides of metaphase plates and early prenuclei indicates that the major protein antigens recognized by mAb-2 have apparent molecular masses of approximately 106000 and 80500 and that each is likely composed of multiple charge isomers. A dual fluorescent analysis using mAb-2 and high-titer anti-lamin B serum provides additional evidence that chromatid coalescence is a separate, early event that precedes nuclear lamina formation.

Consumption of oily fish and childhood asthma risk.

OBJECTIVE: To investigate the association between diet and airway disease in children in the light of epidemiological studies suggesting that consumption of fish more than once a week reduces the risk of developing airway hyperresponsiveness (AHR). DESIGN: Diet was assessed by a detailed food frequency questionnaire and airway disease by respiratory symptoms or airway responsiveness to exercise. METHODS: A questionnaire, containing questions about the frequency of eating more than 200 foods, was sent to the parents of 574 children in whom we had measured recent wheeze (by questionnaire), AHR (by exercise) and atopy (by skin prick tests) six months before this study. We defined current asthma as the presence of both recent wheeze and AHR. RESULTS: Response rate to the questionnaire was 81.5% (n=468.) After adjusting for confounders such as sex, ethnicity, country of birth, atopy, respiratory infection in the first two years of life and a parental history of asthma or smoking, children who ate fresh, oily fish (>2% fat) had a significantly reduced risk of current asthma (odds ratio, 0.26; 95% confidence interval, 0.09-0.72; P<0.01). No other food groups or nutrients were significantly associated with either an increased or reduced risk of current asthma. CONCLUSION: These data suggest that consumption of oily fish may protect against asthma in childhood.

Computer graphics of SEM images facilitate recognition of chromosome position in isolated human metaphase plates.

There is general agreement that at the time of mitosis chromosomes occupy precise positions and that these positions likely affect subsequent nuclear function in interphase. However, before such ideas can be investigated in human cells, it is necessary to determine first the precise position of each chromosome with regard to its neighbors. It has occurred to us that stereo images, produced by scanning electron microscopy, of isolated metaphase plates could form the basis whereby these positions could be ascertained. In this paper we describe a computer graphic technique that permits us to keep track of individual chromosomes in a metaphase plate and to compare chromosome positions in different metaphase plates. Moreover, the computer graphics provide permanent, easily manipulated, rapid recall of stored chromosome profiles. These advantages are demonstrated by a comparison of the relative position of group A-specific and groups D- and G-specific chromosomes to the full complement of chromosomes in metaphase plates isolated from a nearly triploid human-derived cell (HeLa S3) to a hypo-diploid human fetal lung cell.