Clinical markers of asthma and IgE assessed in parents before conception predict asthma and hayfever in the offspring.

BACKGROUND: Mice models suggest epigenetic inheritance induced by parental allergic disease activity. However, we know little of how parental disease activity before conception influences offspring's asthma and allergy in humans. OBJECTIVE: We aimed to assess the associations of parental asthma severity, bronchial hyperresponsiveness (BHR), and total and specific IgEs, measured before conception vs. after birth, with offspring asthma and hayfever. METHODS: The study included 4293 participants (mean age 34, 47% men) from the European Community Respiratory Health Survey (ECRHS) with information on asthma symptom severity, BHR, total and specific IgEs from 1991 to 1993, and data on 9100 offspring born 1972-2012. Adjusted relative risk ratios (aRRR) for associations of parental clinical outcome with offspring allergic disease were estimated with multinomial logistic regressions. RESULTS: Offspring asthma with hayfever was more strongly associated with parental BHR and specific IgE measured before conception than after birth [BHR: aRRR = 2.96 (95% CI: 1.92, 4.57) and 1.40 (1.03, 1.91), respectively; specific IgEs: 3.08 (2.13, 4.45) and 1.83 (1.45, 2.31), respectively]. This was confirmed in a sensitivity analysis of a subgroup of offspring aged 11-22 years with information on parental disease activity both before and after birth. CONCLUSION & CLINICAL RELEVANCE: Parental BHR and specific IgE were associated with offspring asthma and hayfever, with the strongest associations observed with clinical assessment before conception as compared to after birth of the child. If the hypothesis is confirmed in other studies, parental disease activity assessed before conception may prove useful for identifying children at risk for developing asthma with hayfever.

The locus C11orf30 increases susceptibility to poly-sensitization.

A number of genetic variants have been associated with allergic sensitization, but whether these are allergen specific or increase susceptibility to poly-sensitization is unknown. Using data from the large multicentre population-based European Community Respiratory Health Survey, we assessed the association between 10 loci and specific IgE and skin prick tests to individual allergens and poly-sensitization. We found that the 10 loci associate with sensitization to different allergens in a nonspecific manner and that one in particular, C11orf30-rs2155219, doubles the risk of poly-sensitization (specific IgE/4 allergens: OR = 1.81, 95% CI 0.80-4.24; skin prick test/4+ allergens: OR = 2.27, 95% CI 1.34-3.95). The association of rs2155219 with higher levels of expression of C11orf30, which may be involved in transcription repression of interferon-stimulated genes, and its association with sensitization to multiple allergens suggest that this locus is highly relevant for atopy.

The occupational contribution to severe exacerbation of asthma.

The goal of this study was to identify occupational risk factors for severe exacerbation of asthma and estimate the extent to which occupation contributes to these events. The 966 participants were working adults with current asthma who participated in the follow-up phase of the European Community Respiratory Health Survey. Severe exacerbation of asthma was defined as self-reported unplanned care for asthma in the past 12 months. Occupations held in the same period were combined with a general population job-exposure matrix to assess occupational exposures. 74 participants reported having had at least one severe exacerbation event, for a 1-yr cumulative incidence of 7.7%. From regression models that controlled for confounders, the relative risk (RR) was statistically significant for low (RR 1.7, 95% CI 1.1-2.6) and high (RR 3.6, 95% CI 2.2-5.8) biological dust exposure, high mineral dust exposure (RR 1.8, 95% CI 1.02-3.2), and high gas and fumes exposure (RR 2.5, 95% CI 1.2-5.5). The summary category of high dust, gas, or fumes exposure had RR 3.1 (95% CI 1.9-5.1). Based on this RR, the population attributable risk was 14.7% among workers with current asthma. These results suggest occupation contributes to approximately one in seven cases of severe exacerbation of asthma in a working population, and various agents play a role.

Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells.

Cell lines established from the lepidopteran insect Spodoptera frugiperda (fall armyworm; Sf9) are used routinely as hosts for the expression of foreign proteins by recombinant baculovirus vectors. We have examined the pathway of protein glycosylation and secretion in these cells, using human tissue plasminogen activator (t-PA) as a model. t-PA expressed in Sf9 cells was both N glycosylated and secreted. At least a subset of the N-linked oligosaccharides in extracellular t-PA was resistant to endo-beta-N-acetyl-D-glucosaminidase H, which removes immature, high-mannose-type oligosaccharides. This refutes the general conclusion from previous studies that Sf9 cells cannot process immature N-linked oligosaccharides to an endo-beta-N-acetyl-D-glucosaminidase H-resistant form. A nonglycosylated t-PA precursor was not detected in Sf9 cells, even with very short pulse-labeling times. This suggests that the mammalian signal sequence of t-PA is efficiently recognized in Sf9 cells and that it can mediate rapid translocation across the membrane of the rough endoplasmic reticulum, where cotranslational N glycosylation takes place. However, t-PA was secreted rather slowly, with a half-time of about 1.6 h. Thus, a rate-limiting step(s) in secretion occurs subsequent to translocation and N glycosylation of the t-PA polypeptide. Treatment of Sf9 cells with tunicamycin, but not with inhibitors of oligosaccharide processing, prevented the appearance of t-PA in the extracellular medium. This suggests that N glycosylation per se, but not processing of the N-linked oligosaccharides, is required directly or indirectly in baculovirus-infected Sf9 cells for the secretion of t-PA. Finally, the relative efficiency of secretion decreased dramatically with time of infection, suggesting that the Sf9 host cell secretory pathway is compromised during the later stages of baculovirus infection.

Absence of a structural basis for intracellular recognition and differential localization of nuclear and plasma membrane-associated forms of simian virus 40 large tumor antigen.

The simian virus 40 large tumor antigen (T-ag) is found in both the nuclei (nT-ag) and plasma membranes (mT-ag) of simian virus 40-infected or -transformed cells. It is not known how newly synthesized T-ag molecules are recognized, sorted, and transported to their ultimate subcellular destinations. One possibility is that these events depend upon structural differences between nT-ag and mT-ag. To test this possibility, we compared the structures of nT-ag and mT-ag from simian virus 40-infected cells. No differences between the two forms of T-ag were detected by migration in polyacrylamide gels, by Staphylococcus aureus V8 partial proteolytic mapping of methionine- or proline-containing peptides, or by two-dimensional tryptic peptide mapping of methionine-containing peptides. The carboxy-terminal, methionine-containing tryptic peptide was identified in the two-dimensional maps and was shown to be identical in nT-ag and mT-ag. Thus, a structural basis for the recognition and differential localization of T-ags could not be demonstrated. The carboxy terminus of the T-ag encoded by mutant dlA2413 is derived from the alternate open reading frame of the simian virus 40 early region, in analogy with the theoretical early gene product, T*-ag. We used this mutant to identify peptides unique to T*-ag. None of these peptides were detected in maps of mT-ag; only wild-type T-ag-specific peptides were found. These findings suggest that T*-ag does not represent the membrane-associated form of T-ag, but that mT-ag is encoded within the same reading frame used for nT-ag.

Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors.

The baculovirus-insect cell expression system is well-suited for recombinant glycoprotein production because baculovirus vectors can provide high levels of expression and insect cells can modify newly synthesized proteins in eucaryotic fashion. However, the N-glycosylation pathway of baculovirus-infected insect cells differs from the pathway found in higher eucaryotes, as indicated by the fact that glycoproteins produced in the baculovirus system typically lack complex biantennary N-linked oligosaccharide side chains containing penultimate galactose and terminal sialic acid residues. We recently developed a new type of baculovirus vector that can express foreign genes immediately after infection under the control of the viral ie1 promoter. These immediate early baculovirus expression vectors can be used to modify the insect cell N-glycosylation pathway and produce a foreign glycoprotein with more extensively processed N-linked oligosaccharides. These vectors can also be used to study the influence of the late steps in N-linked oligosaccharide processing on glycoprotein function. Further development could lead to baculovirus-insect cell expression systems that can produce recombinant glycoproteins with complex biantennary N-linked oligosaccharides structurally identical to those produced by higher eucaryotes.

Engineering N-glycosylation pathways in the baculovirus-insect cell system.

The inability to produce eukaryotic glycoproteins with complex N-linked glycans is a major limitation of the baculovirus-insect cell expression system. Recent studies have demonstrated that metabolic engineering can be used to extend the glycoprotein processing capabilities of lepidopteran insect cells. This approach is being used to develop new baculovirus-insect cell expression systems that can produce more authentic recombinant glycoproteins and obtain new information on insect N-glycosylation pathways.

Long-term stability of baculoviruses stored under various conditions.

The long-term stability of baculoviruses under various storage conditions was investigated. The results showed that the titers of baculovirus working stocks were significantly reduced after the viruses had been stored in polypropylene tubes for several months or a year at 4 degrees C. The virus was not lost by sticking to the plastic tubes, because the same results were obtained when the virus was stored in glass tubes. The major factor contributing to the loss of viral infectivity was exposure to light. Virus stocks stored at temperatures ranging from -85 degrees to 37 degrees C were quite stable, as long as they were protected from light. Finally, viral infectivity was not significantly reduced by multiple freeze-thaw cycles. These results demonstrated that baculovirus working stocks may be stored frozen or refrigerated with little long-term loss of infectivity, as long as they are protected from light.

Biosynthesis and subcellular localization of a lepidopteran insect alpha 1,2-mannosidase.

Like lower and higher eucaryotes, insects have alpha 1,2-mannosidases which function in the processing of N-glycans. We previously cloned and characterized an insect alpha 1,2-mannosidase cDNA and demonstrated that it encodes a member of a family of N-glycan processing alpha 1,2-mannosidases (Kawar, Z., Herscovics, A., Jarvis, D.L., 1997. Isolation and characterisation of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9. Glycobiology 7, 433-443). These enzymes have similar protein sequences, require calcium for their activities, and are sensitive to 1-deoxymannojirimycin, but can have different substrate specificities and intracellular distributions. We recently determined the substrate specificity of the insect alpha 1,2-mannosidase, SfManI (Kawar, Z., Romero, P., Herscovics, A., Jarvis, D.L., 2000. N-glycan processing by a lepidopteran insect and 1,2-mannosidase. Glycobiology 10, 347-355). Now, we have examined the biosynthesis and subcellular localization of SfManI. We found that SfManI is partially N-glycosylated and that N-glycosylation is dramatically enhanced if the wild type sequon is changed to one that is highly utilized in a mammalian system. We also found that an SfManI-GFP fusion protein had a punctate cytoplasmic distribution in insect cells. Colocalization studies indicated that this fusion protein is localized in the Golgi apparatus, not in the endoplasmic reticulum or lysosomes. Finally, N-glycosylation had no influence over the substrate specificity or subcellular localization of SfManI.

SV40 T-antigen as a dual oncogene: structure and function of the plasma membrane-associated population.

SV40 T-antigen (T-ag) is localized in both the nucleus (nT-ag) and plasma membrane (pmT-ag) of cells and provides multiple functions necessary for cell transformation. The pmT-ag population is structurally very similar to the nT-ag. Transport to the cell surface is by an unknown mechanism that does not involve the secretory pathway. The disposition of T-ag in the membrane exposes both the amino and the carboxyl terminus on the exterior of the cell. Nuclear-transport-defective mutants of T-ag can transform established cells in culture, but not primary cells, suggesting that non-nuclear forms of T-ag may mediate some transformation-related process(es). A non-cytolytic protein extraction technique utilizing 1-butanol solubilized from SV40-transformed cells a multimeric complex composed of pmT-ag and at least five cellular proteins ranging in size from 35,000 (35K) to 60K M. Both amino- and carboxylterminal T-ag-specific monoclonal antibodies co-precipitated T-ag and the 35-60K Mr proteins, but antibodies against the internal portion of T-ag precipitated only uncomplexed T-ag. The growth state of the cells markedly influenced the expression of the T-ag-containing surface complexes; more complexes were recovered from actively dividing cells than from confluent cell cultures, and suspension cells yielded more complexes than cells on a substratum. The complex exhibited a highly dynamic association with the cell membrane, as demonstrated by pulse-chase analysis. The characteristics of growth-dependent expression and rapid turnover rate suggest a functional role for the membrane complex. The identities of the cellular proteins in the complex with pmT-ag are unknown, although one member (56K) is recognized by p53-specific monoclonal antibodies.

Insect cell hosts for baculovirus expression vectors contain endogenous exoglycosidase activity.

Four different insect cell lines that can be used as hosts for baculovirus infection were assayed for the presence of endogenous exoglycosidases. All four cell lines, derived from Spodoptera frugiperda, Trichoplusia ni, Bombyx mori, or Malacosoma disstria, contained N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, beta-galactosidase, and sialidase activities. Exoglycosidase activities were found in cell lysates as well as cell-free supernatants from uninfected and wild-type baculovirus infected cells. Oligosaccharide analysis of cellular glycoproteins using lectins recognizing Gal beta 1, 3GalNAc, Gal beta 1, 4GlcNAc, and NeuAc alpha 2,6Gal demonstrated that only Gal beta 1,3GalNAc was present. The demonstration that these cells contain exoglycosidases raises the possibility that the oligosaccharides of baculovirus-expressed glycoproteins are subject to enzymatic degradation.

Heterologous protein expression affects the death kinetics of baculovirus-infected insect cell cultures: a quantitative study by use of n-target theory.

The death of cultured insect cells after baculovirus infection is a time-dependent event. Without a quantitative model, it is difficult to characterize its kinetics. Our group has shown that the cell survival rate can be characterized by use of the n-target theory, which involves only two parameters: the number of hypothetical inactivation targets (n) and the first-order death rate (k). In this study, we used different recombinant viruses to examine the effect of heterologous protein expression on the cell survival rate. The proteins expressed were beta-galactosidase, human T-cell leukemia virus type I p40x, human interleukin-2, and human tissue plasminogen activator (tPA). The survival rate was affected by protein expression, but the n value remained constant if the protein expression level was high (above 30 mg/L). Low-level expression of secreted, glycosylated tPA resulted in a reduced n value, which was restored to the normal value when the tPA signal peptide and prosequence were deleted. In addition, if the n value was normal (10-11), the level of protein expression correlated negatively with the death rate. However, if the n value was reduced by unfavorable culture conditions or foreign protein expression, the expression level correlated positively with the death rate. A dimensionless plot with kt as the dimensionless time shows that alteration of the k value while retaining constant n is equivalent to a rescaling of time. Therefore, the survival curves with constant n reduce to a single curve on the dimensionless plot.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells.

Baculoviruses are currently used as vectors for the transient high-level expression of foreign gene products in insect cells. In this study, we demonstrate that baculoviruses can also be made to continuously express a foreign gene product by using the promoter from IE1, an immediate early viral gene, to produce stably-transformed insect cells. This approach gave levels of foreign gene expression lower than those usually obtained with the lytic baculovirus expression vector system. Expression, however, was continuous and stable, and a complex human glycoprotein (tissue plasminogen activator) was processed more efficiently. We conclude that stable transformation is a feasible approach for baculovirus-mediated foreign gene expression in lepidopteran cells, particularly for products that are relatively poorly-expressed and/or processed in lytically infected cells.

Region-specific deletions of RIM1 reproduce a subset of global RIM1α(-/-) phenotypes.

The presynaptic protein RIM1α mediates multiple forms of presynaptic plasticity at both excitatory and inhibitory synapses. Previous studies of mice lacking RIM1α (RIM1α(-/-) throughout the brain showed that deletion of RIM1α results in multiple behavioral abnormalities. In an effort to begin to delineate the brain regions in which RIM1 deletion mediates these abnormal behaviors, we used conditional (floxed) RIM1 knockout mice (fRIM1). By crossing these fRIM1 mice to previously characterized transgenic cre lines, we aimed to delete RIM1 selectively in the dentate gyrus (DG), using a specific preproopiomelanocortin promoter driving cre recombinase (POMC-cre) line , and in pyramidal neurons of the CA3 region of hippocampus, using the kainate receptor subunit 1 promoter driving cre recombinase (KA-cre). Neither of these cre driver lines was uniquely selective to the targeted regions. In spite of this, we were able to reproduce a subset of the global RIM1α(-/-) behavioral abnormalities, thereby narrowing the brain regions in which loss of RIM1 is sufficient to produce these behavioral differences. Most interestingly, hypersensitivity to the pyschotomimetic MK-801 was shown in mice lacking RIM1 selectively in the DG, arcuate nucleus of the hypothalamus and select cerebellar neurons, implicating novel brain regions and neuronal subtypes in this behavior.

Indoor nitrous acid and respiratory symptoms and lung function in adults.

BACKGROUND: Nitrogen dioxide (NO2) is an important pollutant of indoor and outdoor air, but epidemiological studies show inconsistent health effects. These inconsistencies may be due to failure to account for the health effects of nitrous acid (HONO) which is generated directly from gas combustion and indirectly from NO2. METHODS: Two hundred and seventy six adults provided information on respiratory symptoms and lung function and had home levels of NO2 and HONO measured as well as outdoor levels of NO2. The association of indoor HONO levels with symptoms and lung function was examined. RESULTS: The median indoor HONO level was 3.10 ppb (IQR 2.05-5.09), with higher levels in homes with gas hobs, gas ovens, and in those measured during the winter months. Non-significant increases in respiratory symptoms were observed in those living in homes with higher HONO levels. An increase of 1 ppb in indoor HONO was associated with a decrease in forced expiratory volume in 1 second (FEV1) percentage predicted (-0.96%; 95% CI -0.09 to -1.82) and a decrease in percentage FEV1/forced vital capacity (FVC) (-0.45%; 95% CI -0.06 to -0.83) after adjustment for relevant confounders. Measures of indoor NO2 were correlated with HONO (r = 0.77), but no significant association of indoor NO2 with symptoms or lung function was observed. After adjustment for NO2 measures, the association of HONO with low lung function persisted. CONCLUSION: Indoor HONO levels are associated with decrements in lung function and possibly with more respiratory symptoms. Inconsistencies between studies examining health effects of NO2 and use of gas appliances may be related to failure to account for this association.

Effects of baculovirus infection on IE1-mediated foreign gene expression in stably transformed insect cells.

Previously, we produced transformed insect cell lines that can express a selected foreign protein constitutively, in the absence of baculovirus infection (D. L. Jarvis, J. G. W. Fleming, G. R. Kovacs, M. D. Summers, and L. A. Guarino, Bio/Technology 8:950-955, 1990). These cells contain stably integrated copies of chimeric genes consisting of the promoter from an immediate-early baculovirus gene, IE1, and the sequences encoding either human tissue plasminogen activator or Escherichia coli beta-galactosidase. Transcription of the integrated genes in these cells is specifically controlled by the IE1 promoter. The purpose of this study was to determine how baculovirus infection influences IE1-mediated foreign protein production by these stably transformed insect cell lines. The results showed that viral infection transiently stimulated and then strongly inhibited the production of both tissue plasminogen activator, a secreted protein, and beta-galactosidase, an intracellular protein. These effects reflected virus-induced changes in the steady-state levels of RNA produced by the integrated genes. Transient assays showed that expression of the viral IEN gene alone could account for the increased levels of RNA observed early in infection. The precise mechanism accounting for the decreased levels of RNA observed later in infection was not determined. However, we obtained evidence that the native IE1 promoter remains active throughout infection, which suggested indirectly that the integrated IE1 promoter is transcriptionally inactivated at late times of baculovirus infection. Thus, the same promoter behaved quite differently late in infection, depending on its local environment. Neither methylation nor degradation appeared to be responsible for inactivating IE1-mediated expression of the integrated genes. The significance of these results with respect to the baculovirus-host interaction and the practical applications of stably transformed insect cell lines are discussed.

Biosynthesis and processing of the Autographa californica nuclear polyhedrosis virus gp64 protein.

gp64 is a major virion envelope glycoprotein of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). gp64 plays an important role in AcMNPV infection, probably mediating penetration of one form of the virus into host cells through the endocytic pathway. gp64 also represents an excellent probe for studying the membrane glycoprotein processing capabilities of baculovirus-infected insect cells, which are used widely as a eucaryotic expression system. The goals of this study were to characterize gp64 biosynthesis and processing and determine how N-glycosylation and N-linked oligosaccharide processing influence the fate and function of gp64 in AcMNPV-infected insect cells. We found that gp64 was synthesized in a biphasic fashion, with peaks at 8 and 24 hr postinfection in both the intracellular and extracellular fractions. Interestingly, the first peak preceded detectable budded virus (BV) production, suggesting that gp64 is shed from infected cells early in infection. Transcriptional regulation accounted for the biphasic mode of gp64 protein synthesis, as transcription initiated at a consensus early motif during early times of infection, at a late motif during late times of infection, and there was a lag between the peak of early and the onset of late transcription. In vitro transcription-translation assays showed that the second ATG in the AcMNPV gp64 long open reading frame is used as the translational initiation codon and that downstream sequences encode a functional signal peptide. Pulse-chase analyses, endoglycosidases, and various inhibitors were used to show that some N-linked oligosaccharides on gp64 are processed by glucosidases and alpha-mannosidases in AcMNPV-infected insect cells. These experiments also revealed that at least two differentially processed gp64 glycoforms are produced in these cells and that both can reach the cell surface and assemble into progeny BV. However, N-linked oligosaccharide processing was not required for gp64 cell surface expression, its assembly into infectious BV, or its fusogenic activity. This suggested that any gp64 glycoform produced during infection, regardless of its N-linked carbohydrate structure, can have essentially normal biological properties. By contrast, transport of gp64 to the cell surface, production of infectious BV, and fusogenic activity were reduced in the absence of N-glycosylation, indicating that this modification is necessary for optimal gp64 function.