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PubMed1980

Isolation and characterization of ovine prolactin and growth-hormone messenger RNAs.

Methods are described in this paper for obtaining and characterizing highly enriched preparations of ovine prolactin (PRL) and growth-hormone (GH) messenger RNAs. Purification steps include phenol extraction, oligo-(dT)-cellulose chromatography, sucrose-gradient ultracentrifugation, and polyacrylamide-gel electrophoresis. This sequence of procedures results in messenger preparations that are about 92% pure for prolactin mRNA and 78% pure for the growth-hormone mRNA species. Thus, these two closely related mRNAs can be isolated from the same tissue source at a purity adequate for cloning and nucleic acid hybridization experiments. Translation experiments with the cap analogue 7-methylguanosine, and end-labeling of the nucleic acid before and after beta-oxidation indicate that both messages possess blocked 5'-termini, and that these are part of previously described cap structures. Polyadenosine tracts of 30-160 residues were found at the 3'-ends of both purified species. Finally, sizing experiments suggest both mRNAs contain approx. 30% more bases than accounted for by the coding regions and the poly(A)-tracts. Their physical characteristics thus agree with those of most eucaryotic messages to date.

Animals↗
PubMed2006

Immunological parameters in the sera of patients with atopic dermatitis and airborne allergy treated with allergy vaccines.

Patients with atopic disorders present an increased production of IgE, which is usually limited to specific antibodies against various environmental allergens. It has also been suggested that the production of these antibodies may be influenced by effective specific immunotherapy (SIT). Of course, a decline of serum antigen specific IgE in the course of such a treatment cannot explain the clinical efficacy of SIT and is probably not a key mechanism. However, SIT may at least participate in the final clinical result. In this study, 37 patients with atopic dermatitis were treated with allergy vaccines (Novo-Helisen Depot) for a time period of 48 months. The control group consisted of 29 patients with atopic dermatitis who were treated with classical methods. The clinical score (W-AZS), total IgE and antigen specific IgE (asIgE) in the sera of patients were assessed before treatment and after 24 and 48 months of therapy (FEIA CAP System, Pharmacia). There was a significant difference between the two investigated groups from both the clinical and immunological standpoints after 2 and 4 years of observation. There was a significant decrease of serum total IgE and asIgE (directed against airborne allergens) in the course of specific immunotherapy. In the control group, the total IgE level tended to increase, and this tendency was also recorded in case of asIgE measurements. We also evaluated the influence of specific immunotherapy on the serum level of IFN-G, sIL-2R, IL-4, IL-5 and IL-10 before treatment and after 4 years of therapy with the quantitative 2-step colorimetric sandwich ELISA method (R and D Systems). In the group of patients treated with allergy vaccines, a significant decrease of the serum sIL-2R level was observed after 48 months of therapy (p<0.01). In the control group, a significant increase of serum IL-4 (p<0.01) as well as IL-5 (p<0.05) was registered at the end of the observation period. There was no significant correlation between the clinical score and serum level of any of the investigated cytokines in either group of patients before the treatment or after 48 months of therapy.

Adolescent↗
PubMed1978

[Evaluation of the antigenicity of heart valve leaflets using inbred rat strain combinations (author's transl)].

Experiments with large, non-inbred animals yield no reliable evidence in the antigenicity of heart valves. With inbred strains of rats, however, it is possible to obtain evidence on the immunological reactions of heart valve leaflet transplants. Our experiments were undertaken on the inbred rat strains (CDR, LEW, CAP and BD5, using combinations as follows: syngeneic strain combination (CDF leads to CDF), weakly allogenic (RtH-1-identical) strain cimbination (LEW leads to CDF), strongly allogeneic (RtH-1-incompatible) strain combination (CAP leads to CDF). Strain BD5 was used for the control skin grafts. Either one or two heart valve leaflets were heterotopically intra-aortally transplanted. Sensitization was verified humorally by transplantation antibody titers and cellularly by subsequent donorspecific skin transplants. The following results were achieved: Allogeneic heart valve leaflets are antigenic. Intravascular grafting of one heart valve leaflet induces the same degree of sensitization as the grafting of two leaflets. In the RtH-1-identical system, sensitization is revealed only by subsequent skin grafting, whereas in the RtH-1-incompatible system, sensitization is humorally demonstrated as well. This indicates that in heart valve leaflet transplantation cellmediated reactivity is a more sensitive indicator than the humoral one. The greater the immunological difference is, the clearer the sensitization will be. In the strongly allogeneic system, the sensitization is even demonstrated humorally; skin grafts are rejected as "white grafts". Improved long-term results may be expected in allogeneic vital heart valve transplants if consideration is given to the HLA compatibility between donor and recipient; the appearance of delayed insufficiency could be greatly lessened or even deferred.

Animals↗
PubMed2000

ATP synthases in the year 2000: evolving views about the structures of these remarkable enzyme complexes.

This introductory article briefly summarizes how our views about the structural features of ATP synthases (F0F1) have evolved over the past 30 years and also reviews some of our current views in the year 2000 about the structures of these remarkably unique enzyme complexes. Suffice it to say that as we approach the end of the first year of this new millinium, we can be conservatively confident that we have a reasonably good grasp of the overall "low-resolution" structural features of ATP synthases. Electron microscopy techniques, combined with the tools of biochemistry, molecular biology, and immunology, have played the leading role here by identifying the headpiece, basepiece, central stalk, side stalk, cap, and in the mitochondrial enzyme, the collar around the central stalk. We can be reasonably confident also that we have a fairly good grasp of much of the "high-resolution" structural features of both the F1 moiety comprised of fives subunit types (alpha, beta, gamma, delta, and epsilon) and parts of the F0 moiety comprised of either three (E. coli) or at least ten (mitochondria) subunit types. This information acquired in several different laboratories, either by X-ray crystallography or NMR spectroscopy, includes details about the active site and subunit relationships. Moreover, it is consistent with recently reported data that the F1 moiety may be an ATP driven motor, which, during ATP synthesis, is driven in reverse by the electrochemical proton gradient generated by the electron transport chain. The real structural challenges of the future are to acquire at high resolution "complete" ATP synthase complexes representative of different stages of the catalytic cycle during ATP synthesis and representative also of key regulatory states.

Crystallography, X-Ray↗
PubMed2002

Phthalic anhydride allergy: development and characterization of optimized hapten-carrier conjugates for improved diagnosis.

BACKGROUND: At present the diagnosis of IgE-mediated hypersensitivity to phthalic anhydride (PA) is based on conjugates that are not characterized or standardized. The aim of this study was to develop optimized and molecularly characterized PA conjugates that can be used to improve the diagnosis of PA-allergy. METHODS: The PA conjugates were synthesized and the number of haptens bound on a carrier protein was estimated by matrix-assisted laser desorption/ionization time of light (MALDI-TOF) mass spectrometry. The ability of conjugates to bind IgE and IgG antibodies was measured by enzyme-linked immunosorbent assay (ELISA). Reactivity of the conjugates in vivo was evaluated by skin prick testing. RESULTS: The most active IgE-binding conjugates had a PA : HSA molar ratio of 80 : 1. In the optimal conjugates the average numbers of PA haptens per carrier molecule of human serum albumin (HSA) were 14-16. In ELISA, all 13 patients and none of the 20 controls had IgE antibodies to optimized PA conjugate. The sensitivity and specificity of the ELISA was comparable to commercial CAP RAST. PA conjugates elicited positive test results in skin prick testing showing that conjugates are immunologically active also in vivo. CONCLUSIONS: These results indicate that optimized and molecularly characterized PA-HSA conjugates can be used both in vitro and in vivo assays to improve the diagnosis of PA allergy.

Adult↗
PubMed2006

RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates.

Double-stranded RNA (dsRNA) produced during viral replication is believed to be the critical trigger for activation of antiviral immunity mediated by the RNA helicase enzymes retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). We showed that influenza A virus infection does not generate dsRNA and that RIG-I is activated by viral genomic single-stranded RNA (ssRNA) bearing 5'-phosphates. This is blocked by the influenza protein nonstructured protein 1 (NS1), which is found in a complex with RIG-I in infected cells. These results identify RIG-I as a ssRNA sensor and potential target of viral immune evasion and suggest that its ability to sense 5'-phosphorylated RNA evolved in the innate immune system as a means of discriminating between self and nonself.

Animals↗
PubMed1995

Clinical symptoms and immunologic reactivity to bee and wasp stings in beekeepers.

We compared the history of sting reactions with venom skin prick test (SPT) and CAP RAST reactions in beekeepers in order to assess the value of structured questions and symptom backgrounds. The study population consisted of 102 beekeepers, who were 25-75 years of age. Bee and wasp venom SPT was performed with concentrations of 10, 100, and 300 micrograms/ml. The CAP Phadiatop test was used in the screening of IgE antibodies against common inhalant allergens. Forty-two beekeepers had never experienced large local or systemic reactions after a bee sting. Of the 31 subjects with a history of systemic reactions, 13 had experienced these during the previous year. A significant difference (P < 0.01) between systemic reactors and nonreactors was found in bee venom CAP and SPT (300 micrograms/ml). However, due to considerable overlap, these tests are unable to discriminate between allergic and nonallergic beekeepers. Both bee venom SPT (300 micrograms/ml) and CAP tests were positive in 65% of systemic reactors and in 34% of nonreactors (P = 0.008). Venom SPT (300 micrograms/ml) correlated significantly with CAP for both venoms. No correlation was observed between venom allergy and atopy. Clinically, the most practical concentration for evaluating bee and wasp venom sensitization by SPT proved to be 300 micrograms/ml.

Adult↗
PubMed2006

A cryopyrin-associated periodic syndrome with joint destruction.

OBJECTIVE: Describe four generations (11 members) of a family with a cryopyrin-associated periodic syndrome (CAPS), including joint destruction, associated with a CIAS1-gene mutation and good responses to anakinra. METHODS: In addition to detailed questioning and physical examination, six family members underwent haematological, immunological and biochemical testing. Exon 3 of the CIAS1 gene was sequenced in search of a mutation in the 1q44 region. RESULTS: During childhood or adolescence, four family members developed different combinations of the following CAPS manifestations: deafness (3/4); arthritis (4/4) with joint destruction for two of them; nervous (cerebral demyelinization, 2/4), cutaneous (livedo and/or urticaria, 3/4) and eye lesions (episcleritis and/or papilloedema, 4/4); IgA hypergammaglobulinaemia (4/4) and inflammatory syndrome (3/4). Sequencing of six family members' CIAS1-gene exon 3 identified a heterozygous mutation, c.1043C > T. Pertinently, this CAPS is distinct from chronic infantile neurological cutaneous and arthritis syndrome/neonatal onset multisystemic inflammatory disease syndrome and Muckle-Wells syndrome (MWS), which also result from exon 3 mutations in this gene. Moreover, this family did not have the usual neurological manifestations, typical morphological features and frequent amyloidosis of MWS. CONCLUSIONS: We describe a previously unreported form of CAPS with atypical neurological signs, joint destruction and livedo. This observation extends the clinical spectrum associated with CIAS1 mutations. Anakinra, an interleukin-1-receptor antagonist, prescribed to two family members, was highly effective.

Adolescent↗
PubMed1993

Eubacterial components similar to small nuclear ribonucleoproteins: identification of immunoprecipitable proteins and capped RNAs in a cyanobacterium and a gram-positive eubacterium.

Small nuclear ribonucleoprotein (snRNP) particles play an important role in the processing of pre-mRNA. snRNPs have been identified immunologically in a variety of cells, but none have ever been observed in prokaryotic systems. This report provides the first evidence for the presence of snRNP-like components in two types of prokaryotic cells: those of the cyanobacterium Synechococcus leopoliensis and those of the gram-positive eubacterium Bacillus subtilis. These components consist of snRNP-immunoreactive proteins and RNAs, including some with the snRNP-unique 5' m2,2,7G (m3G) cap. Immunoreactivity was determined by immunoprecipitation procedures, with either antinuclear-antibody-positive (RNP- and Sm-monospecific) patient sera or a m3G monoclonal antibody, with radiolabelled cell extracts that were preadsorbed with antinuclear-antibody-negative sera. S. leopoliensis immunoprecipitates showed the presence of high-molecular-mass proteins (14 to 70 kDa) and RNAs (138 to 243 nucleotides) that are analogous in size to proteins and RNAs found in human (HEp-2) cell immunoprecipitates but absent in Escherichia coli immunoprecipitates. Thin-layer chromatography of S. leopoliensis immunoprecipitates confirmed the presence of a capped nucleotide similar to a capped nucleotide in HEp-2 immunoprecipitates; no such nucleotide was observed in E. coli immunoprecipitates. Immunoreactive RNAs (117-170 nucleotides) were identified in a second eubacterium, B. subtilis, as well. This work suggests that snRNPs or their evolutionary predecessors predate the emergence of eukaryotic cells.

Antibodies, Antinuclear↗
PubMed2000

Usefulness of currently available methods for the diagnosis of Anisakis simplex allergy.

BACKGROUND: Serodiagnosis of anisakiosis and Anisakis allergy is difficult since many Anisakis antigens show cross-reactivity complications. In the present study, we assess the usefulness of the major immunologic methods currently available for the diagnosis of Anisakis allergy. METHODS: Four tests (skin prick test, CAP-FEIA system, Western blotting, and an antigen-capture ELISA using O-deglycosylated antigen bound by the monoclonal antibody UA3) were applied to Anisakis-free subjects and subjects with confirmed Anisakis allergy. RESULTS: The skin prick test, CAP-FEIA, and the antigen-capture ELISA identified Anisakis allergy sera with 100% sensitivity, while Western blotting showed 96%/ sensitivity. The antigen-capture ELISA also showed 100% specificity, but CAP-FEIA showed a specificity of only 50%. In Western blotting, none of the bands detected were specific for either Anisakis-free or Anisakis allergy subjects. The skin prick test was not applied to the Anisakis-free subjects, so its specificity could not be determined. CONCLUSIONS: On the basis of these results, we suggest that the most appropriate procedure for diagnosis of Anisakis allergy suspected on clinical grounds is the antigen-capture ELISA using UA3, or, alternatively, a preliminary skin prick test with a positive result subsequently confirmed by UA3-ELISA.

Adolescent↗
PubMed1998

Latex allergy in fruit-allergic patients.

The purpose of this study was to investigate the prevalence of latex allergy in fruit-allergic patients, and to assess its clinical significance. Fifty-seven fruit-allergic patients and 50 non-fruit-allergic atopic patient controls were studied. All patients were questioned about conventional immediate symptoms after contact with latex products. Patients also underwent skin prick testing and determination of specific serum IgE to latex, as well as a screening test for environmental allergens. Immunologic latex sensitization occurred in 49/57 (85.9%) fruit-allergic patients, who showed a positive STP and/or CAP to latex, but in only two controls (P < 0.001). Six out of 57 (10.5%) fruit-allergic patients suffered from clinically relevant latex allergy. Symptoms included contact urticaria, angioedema, conjunctivitis, generalized urticaria, and moderate anaphylactic reactions. No control reported symptoms with latex products (P = 0.052). In all patients, clinical symptoms to fruits preceded a history of latex allergy. The fruits most associated were melon, peach, and banana. From our data, we conclude that there is a potential for allergic reactions to latex in patients with allergy to fruit. All patients with fruit allergy should be screened for individual risk of latex allergy.

Adolescent↗
PubMed2006

Community-acquired pneumonia in Shanghai, China: microbial etiology and implications for empirical therapy in a prospective study of 389 patients.

The aim of this multicenter study was to identify the causative pathogens of community-acquired pneumonia (CAP) in Shanghai, China, and to determine their susceptibility to antimicrobial agents. Pathogens obtained from 389 patients with documented CAP during 2001-2003 were identified by multiple diagnostic tools that included bacterial culture, polymerase chain reaction (PCR), and specific immunological assays. Susceptibility of the bacterial isolates was tested by the broth microdilution method. A specific pathogen was identified in 39.8% (155/389) of the patients: Haemophilus influenzae (n=80), Klebsiella spp. (n=15), Streptococcus pneumoniae (n=12), Staphylococcus aureus (n=6), Moraxella catarrhalis (n=1), other gram-negative organisms (n=9), and atypical pathogens that comprised Mycoplasma pneumoniae (n=42), Chlamydia pneumoniae (n=17), and Legionella pneumophila (n=2). Most H. influenzae isolates were susceptible to ampicillin (88.3%), and all were susceptible to macrolides. Of the S. pneumoniae isolates, 75% (9/12) were susceptible to penicillin, while 25% (3/12) were intermediately susceptible. H. influenzae and atypical pathogens are among the most important pathogens of CAP. Ampicillin, cephalosporins, and the newer fluoroquinolones can be used as empirical therapy for CAP in the Shanghai area. The efficacy of monotherapy with newer macrolides for CAP caused by S. pneumoniae requires further evaluation.

Adolescent↗
PubMed2002

The accessory molecules CD5 and CD6 associate on the membrane of lymphoid T cells.

CD5 and CD6 are closely related lymphocyte surface receptors of the scavenger receptor cysteine-rich superfamily, which show highly homologous extracellular regions but little conserved cytoplasmic tails. Both molecules are expressed on the same lymphocyte populations (thymocytes, mature T cells, and B1a cells) and share similar co-stimulatory properties on mature T cells. Although several works have been reported on the molecular associations and the signaling pathway mediated by CD5, very limited information is available for CD6 in this regard. Here we show the physical association of CD5 and CD6 at the cell membrane of lymphocytes, as well as their localization at the immunological synapse. CD5 and CD6 co-immunoprecipitate from Brij 96 but not Nonidet P-40 cell lysates, independently of both the co-expression of other lymphocyte surface receptors and the integrity of CD5 cytoplasmic region. Fluorescence resonance energy transfer analysis, co-capping, and co-modulation experiments demonstrate the physical in vivo association of CD5 and CD6. Analysis of T cell/antigen-presenting cells conjugates shows the accumulation of both molecules at the immunological synapse. These results indicate that CD5 and CD6 are structurally and physically related receptors, which may be functionally linked to provide either similar or complementary accessory signals during T cell activation and/or differentiation.

Animals↗
PubMed1988

Single RNA species injected in Xenopus oocyte directs the synthesis of active choline acetyltransferase.

In vitro synthesized complementary RNA (cRNA) transcribed from a non-full length Drosophila choline acetyltransferase (ChAT) cDNA clone will direct the synthesis of enzymatically active and immunologically recognizable protein when injected into Xenopus oocytes. The levels of ChAT activity expressed in injected oocytes are proportional, over 4 orders of magnitude difference, to the concentration of injected 'sense' orientation cRNA. GpppG capping of the in vitro synthesized cRNA is not necessary for expression of active ChAT but inclusion of the capping compound during in vitro transcription results in higher levels of enzyme expression at lower levels of cRNA injection. In addition the capped cRNA results in increasing ChAT expression by the oocytes up to 7 days after injection while uncapped cRNA results in maximum enzyme activity after a single day and decreasing levels of activity during subsequent days. A single immunologically detectable protein is produced by oocytes injected with 'sense' cRNA which has a molecular size of 75 kDa and is indistinguishable from the major form of ChAT present in Drosophila. Oocytes making enzymatically active ChAT also accumulate significant levels of acetylcholine. We conclude from these results that our our non-full length Drosophila ChAT cDNA clone contains all the necessary coding information to make a functional protein which appears to have the same size and activity as native Drosophila ChAT.

Acetylcholine↗
PubMed2004

Formation of a central supramolecular activation cluster is not required for activation of naive CD8+ T cells.

Although both naive and effector T lymphocytes interact with antigen-expressing cells, the functional outcome of these interactions is distinct. Naive CD8(+) T cells are activated to proliferate and differentiate into effector cytolytic T lymphocytes (CTL), whereas CTL interact with specific targets, such as tumor cells, to induce apoptotic death. We recently observed that several molecules linked to actin cytoskeleton dynamics were up-regulated in effector vs. naive CD8(+) T cells, leading us to investigate whether T cell differentiation is accompanied by changes in actin-dependent processes. We observed that both naive and effector CD8(+) T cells underwent T cell receptor capping and formed stable conjugates with antigen-specific antigen-presenting cells. However, the characteristics of the immunological synapse were distinct. Whereas accumulation of signaling molecules at the T cell/antigen-presenting cell contact site was detectable in both naive and effector CD8(+) T cells, only effector cells developed a central supramolecular activation cluster as defined by punctate focusing of PKC theta, phospho-PKC theta, and phospho-ZAP70. Extended kinetics, CD28 costimulation, and high-affinity antigenic peptide did not promote PKC theta focusing in naive cells. Nonetheless, naive CD8(+) T cells polarized the microtubule organizing center, produced IL-2, proliferated, and differentiated into effector cells. Our results suggest that the formation of a central supramolecular activation cluster is not required for activation of naive CD8(+) T cells and support the notion that one role of an organized immune synapse is directed delivery of effector function.

Animals↗
PubMedDate not supplied

How do cell-free HIV virions avoid infecting dead-end host cells and cell fragments?

HIV faces the challenge of identifying and entering suitable host cells (i.e. activated and viable) among a wide array of receptor-positive but unsuitable targets. Lymph nodes contain resting cells, activated cells destined for apoptosis within 24 h, and cell fragments, all of which represent replicative dead ends. We postulate that 1) HIV virions have evolved the ability to probe the internal status of potential host cells from the external cell membrane by assessing the ability of cells to co-cap CD4 and chemokine receptors, and 2) the requirement for dual receptor binding in a concerted manner by three gp120 molecules is the molecular mechanism by which virions stochastically ensure high density co-capping of receptors. Cell-associated HIV accomplishes the same selective process by targeting cells capable of participating in immunological synapse formation.

Actins↗
PubMed2005

Biochemical modifications induced in rabbits by Schistosoma mansoni antigens and the beneficial effect of carnosine treatment.

This immunological study involved individual injection of the three Schistosoma mansoni antigens (Ags). soluble egg antigen (SEA), cercarial antigen preparation (CAP) or soluble worm antigen preparation (SWAP) in three rabbits groups (Ag). respectively. Three other groups each received the same specific antigen conjoined with administration of L-carnosine (Ag-C). Determination of three hepatic parameters and ten serum proteins was done. These were total protein, glycogen content and glycogen phosphorylase b activity of liver as well as serum total protein and nine protein fractions [alpha2-macrglobulin; beta-galactosidase; phosphorylase b; serum albumin; fumarase; carbonic anhydrase; beta-lactoglobulin; alpha-lactalbumin and aprotinin]. Conjoined carnosine treatment produced numerous variations. SEA-I-C group presented sex decreased parameters. In CAP-I-C animals hepatic glycogen content was increased while phosphorrylase b activity was decreased as well as seven the concentration of serum parameters; total serum protein, alpha2-macroglobulin, phosphorylase b, albumin, fumarase, carbonic anhydrase, alpha-lactalbumin and aprotinin. In SWAP-I-C group the concentration of only one fraction was decreased; carbonic anhydrase. In batch A both the Ags. of the egg and cercaria, developmental stages having transient residence in the animal host, showed more affection by the specific Ag. Although, carnosine modified the results of all the three groups in batch B yet, its effect on both the egg and cercaria Ags. was still more than that of worm.

Animals↗