Heat-labile Escherichia coli toxin enhances the induction of allergen-specific IgG antibodies in epicutaneous patch vaccination.

Epicutaneous allergen-specific immunotherapy (EPIT) is proposed as an alternative route for allergen-specific immunotherapy (AIT). The induction of allergen-specific blocking IgG antibodies represents an important mechanism underlying AIT, but has not been investigated for EPIT. Here, we compared the induction of allergen-specific blocking IgG in outbred guinea pigs which had been immunized with recombinant birch pollen allergen Bet v 1 using patch delivery system (PDS) with or without heat-labile toxin (LT) from Escherichia coli or subcutaneously with aluminum hydroxide (Alum)-adsorbed rBet v 1. Only subcutaneous immunization with Alum-adsorbed rBet v 1 and epicutaneous administration of rBet v 1 with PDS in combination with LT from E. coli induced allergen-specific IgG antibodies blocking allergic patients' IgE, but not immunization with rBet v 1 via PDS alone. Our results suggest that patch vaccination with rBet v 1 in combination with LT may be a promising strategy for allergen-specific immunotherapy against birch pollen allergy.

Detection of antibody responses against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis proteins in children with community-acquired pneumonia: effects of combining pneumococcal antigens, pre-existing antibody levels, sampling interval, age, and duration of illness.

We evaluated the effects of combining different numbers of pneumococcal antigens, pre-existing antibody levels, sampling interval, age, and duration of illness on the detection of IgG responses against eight Streptococcus pneumoniae proteins, three Haemophilus influenzae proteins, and five Moraxella catarrhalis proteins in 690 children aged <5 years with pneumonia. Serological tests were performed on acute and convalescent serum samples with a multiplexed bead-based immunoassay. The median sampling interval was 19 days, the median age was 26.7 months, and the median duration of illness was 5 days. The rate of antibody responses was 15.4 % for at least one pneumococcal antigen, 5.8 % for H. influenzae, and 2.3 % for M. catarrhalis. The rate of antibody responses against each pneumococcal antigen varied from 3.5 to 7.1 %. By multivariate analysis, pre-existing antibody levels showed a negative association with the detection of antibody responses against pneumococcal and H. influenzae antigens; the sampling interval was positively associated with the detection of antibody responses against pneumococcal and H. influenzae antigens. A sampling interval of 3 weeks was the optimal cut-off for the detection of antibody responses against pneumococcal and H. influenzae proteins. Duration of illness was negatively associated with antibody responses against PspA. Age did not influence antibody responses against the investigated antigens. In conclusion, serological assays using combinations of different pneumococcal proteins detect a higher rate of antibody responses against S. pneumoniae compared to assays using a single pneumococcal protein. Pre-existing antibody levels and sampling interval influence the detection of antibody responses against pneumococcal and H. influenzae proteins. These factors should be considered when determining pneumonia etiology by serological methods in children.

[Novel vaccination concepts on the basis of modern insights into immunology]. / Neue Impfstoffkonzepte auf Basis moderner Erkenntnisse der Immunologie.

Since their introduction more than 200 years ago, vaccines have prevented millions of deaths caused by infectious diseases. This progress was possible because these vaccines protect through antibodies, which are relatively easily stimulated. In the meantime, we understand that diseases such as AIDS, tuberculosis, malaria and hepatitis C cannot be tackled by these conventional approaches. Recent insights into immunology provide the basis for the development of custom-tailored vaccines to successfully combat these threatening infections. These new generation vaccines comprise components that modulate the mediators of immunity (B cells, T cells, antigen-presenting cells and cytokines) in such a way that the best possible immune response develops. Alternative application methods offer the possibility to further improve the immune response. Thus, hope remains that the remarkable increase in knowledge in the areas of immunology and infectious disease research will help to successfully control infectious diseases.

Effects of nicotine on visuo-spatial selective attention as indexed by event-related potentials.

Nicotine has been shown to specifically reduce reaction times to invalidly cued targets in spatial cueing paradigms. In two experiments, we used event-related potentials to test whether the facilitative effect of nicotine upon the detection of invalidly cued targets is due to a modulation of perceptual processing, as indexed by early attention-related event-related potential components. Furthermore, we assessed whether the effect of nicotine on such unattended stimuli depends upon the use of exogenous or endogenous cues. In both experiments, the electroencephalogram was recorded while non-smokers completed discrimination tasks in Posner-type paradigms after chewing a nicotine polacrilex gum (Nicorette 2 mg) in one session and a placebo gum in another session. Nicotine reduced reaction times to invalidly cued targets when cueing was endogenous. In contrast, no differential effect of nicotine on reaction times was observed when exogenous cues were used. Electrophysiologically, we found a similar attentional modulation of the P1 and N1 components under placebo and nicotine but a differential modulation of later event-related potential components at a frontocentral site. The lack of a drug-dependent modulation of P1 and N1 in the presence of a behavioral effect suggests that the effect of nicotine in endogenous visuo-spatial cueing tasks is not due to an alteration of perceptual processes. Rather, the differential modulation of frontocentral event-related potentials suggests that nicotine acts at later stages of target processing.

Small-fragment genomic libraries for the display of putative epitopes from clinically significant pathogens.

Taking advantage of whole genome sequences of bacterial pathogens in many thriving diseases with global impact, we developed a comprehensive screening procedure for the identification of putative vaccine candidate antigens. Importantly, this procedure relies on highly representative small-fragment genomic libraries that are expressed to display frame-selected epitope-size peptides on a bacterial cell surface and to interact directly with carefully selected disease-relevant high-titer sera. Here we describe the generation of small-fragment genomic libraries of Gram-positive and Gram-negative clinically significant pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae, Enterococcus faecalis, Helicobacter pylori, Chlamydia pneumoniae, the enterotoxigenic Escherichia coli, and Campylobacter jejuni. Large-scale sequencing revealed that the libraries, which provide an average of 20-fold coverage, were random and, as demonstrated with two S. aureus libraries, highly representative. Consistent with the comprehensive nature of this approach is the identification of epitopes that reside in both annotated and putatively novel open reading frames. The use of these libraries therefore allows for the rapid and direct identification of immunogenic epitopes with no apparent bias or difficulty that often associate with conventional expression methods.

Totally extraperitoneal laparoendoscopic repair of lumbar hernia.

BACKGROUND: The repair of congenital and acquired lumbar hernias has remained a significant surgical challenge for over three centuries. Transperitoneal laparoendoscopic techniques have been reported that have achieved success in repairing these difficult hernias using a variety of synthetic mesh. Careful review of the surgical literature addressing the repair of lumbar hernia reveals that only fourteen successful cases have been reported using minimally invasive techniques. All of these cases elected a transperitonal approach to repair. Encouraged by established success in the repair of inguinal hernia using an extraperitoneal approach, the repair of a large inferior triangle lumbar hernia was attempted using overlapping synthetic mesh technique while remaining entirely in an extraperitoneal plane. METHODS: A seventy-eight-year-old patient presented for repair of a large symptomatic right lumbar hernia, one year following iliac bone harvest for lumbar laminectomy/fusion. Under general anesthesia, the patient was placed in a lateral decubitus position with lumbar roll in place. Using a muscle splitting dissection through the lateral abdominal musculature, a plane was developed bluntly between the transversalus muscle and the peritoneum. Using a three trocar technique, the plane was matured posteriorly, achieving an ample working space to identify the hernia and complete a synthetic mesh (PTFE) repair. RESULTS: A large inferior triangle lumbar hernia was successfully repaired using overlapping synthetic mesh technique while remaining entirely in an extraperitoneal plane. Eighteen month reevaluation including physical examination and computer tomographic (CT) study confirms successful repair without recurrence of symptoms. CONCLUSIONS: A totally extraperitoneal approach to the identification, mobilization, and repair of lumbar hernia can be successfully accomplished using established laparoendoscopic surgical techniques.

Laparoscopic options in the treatment of splenic artery aneurysms.

BACKGROUND: The retrogastric and often intrapancreatic position of splenic artery aneurysms (SAA) has discouraged many surgeons from attempting the laparoscopic resection of SAA. Only two reports of successful laparoscopically resected SAA have appeared in the surgical literature. METHODS/RESULTS: The successful laparoscopic resection of a large expanding SAA was accomplished using a modification of currently described techniques. CONCLUSIONS: The semilateral decubitus position affords excellent access to the lesser sac, allowing excision of SAA with good visualization of the splenic artery and splenic hilar vessels should splenic hypoperfusion demand splenic resection. Excision of SAA is preferred to ligation except when dense adhesions or intrapancreatic arterial course preclude safe dissection. Pseudoaneurysms from trauma or pancreatitis are likely best treated with intraarterial embolization but significant complications should be expected in this high-risk subset of patients.

Bacterial phage receptors, versatile tools for display of polypeptides on the cell surface.

Four outer membrane proteins of Escherichia coli were examined for their capabilities and limitations in displaying heterologous peptide inserts on the bacterial cell surface. The T7 tag or multiple copies of the myc epitope were inserted into loops 4 and 5 of the ferrichrome and phage T5 receptor FhuA. Fluorescence-activated cell sorting analysis showed that peptides of up to 250 amino acids were efficiently displayed on the surface of E. coli as inserts within FhuA. Strains expressing FhuA fusion proteins behaved similarly to those expressing wild-type FhuA, as judged by phage infection and colicin sensitivity. The vitamin B(12) and phage BF23 receptor BtuB could display peptide inserts of at least 86 amino acids containing the T7 tag. In contrast, the receptors of the phages K3 and lambda, OmpA and LamB, accepted only insertions in their respective loop 4 of up to 40 amino acids containing the T7 tag. The insertion of larger fragments resulted in inefficient transport and/or assembly of OmpA and LamB fusion proteins into the outer membrane. Cells displaying a foreign peptide fused to any one of these outer membrane proteins were almost completely recovered by magnetic cell sorting from a large pool of cells expressing the relevant wild-type platform protein only. Thus, this approach offers a fast and simple screening procedure for cells displaying heterologous polypeptides. The combination of FhuA, along with with BtuB and LamB, should provide a comprehensive tool for displaying complex peptide libraries of various insert sizes on the surface of E. coli for diverse applications.

Ligand-mediated protection against phage lysis as a positive selection strategy for the enrichment of epitopes displayed on the surface of E. coli cells.

We present a novel strategy, termed CISTEM, which allows direct in vivo screening of polypeptides displayed on the surface of E. coli cells by a combination of ligand-mediated protection and phage-mediated selection. The effectiveness of this new approach was demonstrated by displaying the T7.tag on the surface of E. coli as a fusion with the outer membrane protein A, the receptor for bacteriophage K3. A monoclonal T7.tag antibody was used as protective ligand for T7.tag-displaying cells and phage K3 for the elimination of unprotected cells. When populations of bacteria, containing between 6 to 10,000 cells displaying the T7.tag and approximately 10(8) cells displaying an unrelated OmpA fusion protein, were infected with phage K3, specific and antibody-dependent survival of T7.tag displaying cells was observed, yielding an enrichment factor of up to 10(7)-fold. The CISTEM technology was used to select sequences from a T7.tag-based, randomised library and the results were compared to those obtained from selection by MACS with the same library. Together, these results reveal a novel in vivo screening strategy in which an E. coli phage receptor is used as display plafform and selection is performed in suspension upon addition of a protective ligand and a bacteriophage. Extentions and modifications of the basic strategy should lead to novel applications for the identification of protein-ligand interactions.

Two distinct pathways remove mammalian cohesin from chromosome arms in prophase and from centromeres in anaphase.

In yeast, anaphase depends on cohesin cleavage. How anaphase is controlled in vertebrates is unknown because their cohesins dissociate from chromosomes before anaphase. We show that residual amounts of the cohesin SCC1 remain associated with human centromeres until the onset of anaphase when a similarly small amount of SCC1 is cleaved. In Xenopus extracts, SCC1 cleavage depends on the anaphase-promoting complex and separin. Separin immunoprecipitates are sufficient to cleave SCC1, indicating that separin is associated with a protease activity. Separin activation coincides with securin destruction and partial separin cleavage, suggesting that several mechanisms regulate separin activity. We propose that in vertebrates, a cleavage-independent pathway removes cohesin from chromosome arms during prophase, whereas a separin-dependent pathway cleaves centromeric cohesin at the metaphase-anaphase transition.

Jak2-Stat5 interactions analyzed in yeast.

Many cytokine receptors employ Janus protein tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats) for nuclear signaling. Here, we have established yeast strains in which an autoactivated Jak2 kinase induces tyrosine phosphorylation, dimerization, nuclear translocation, and DNA binding of a concomitantly expressed Stat5 protein. Transcriptional activity of Stat5 on a stably integrated, Stat-dependent reporter gene required the C-terminal fusion of the VP16 transactivation domain. In such yeast strains, the interaction between Jak2 and Stat5 was analyzed without interference by other mammalian proteins involved in regulating Jak-Stat signaling, and mutant versions of both proteins were analyzed for their ability to productively interact. Complexes between Jak2 and Stat5 were found to be stable under stringent co-immunoprecipitation conditions. Deletion of the Jak homology regions 2-7 (JH2-JH7) of Jak2, leaving only the kinase domain (JH1) intact, reduced the ability of the kinase to phosphorylate Stat5, whereas deletion of the JH2 domain caused an increased enzymatic activity. A site-directed R618K mutation in the Stat5 SH2 domain abolished the phosphorylation by Jak2, while deletion of the C terminus led to Stat5 hyperphosphorylation. A single phosphotyrosine-SH2 domain interaction was sufficient for the dimerization of Stat5, but such dimers bound to DNA very inefficiently. Together, our data show that yeast cells are appropriate tools for studying Jak-Stat or Stat-Stat interactions. Our mutational analysis suggests that the Stat5 SH2 domain is essential for the interaction with Jak2 and that the kinase domain of Jak2 is sufficient for Jak2-Stat5 interaction. Therefore, the Jak kinase domain may be all that is needed to cause Stat phosphorylation in situations where receptor docking is dispensable.

STAT5 involvement in the differentiation response of primary chicken myeloid progenitor cells to chicken myelomonocytic growth factor.

We have investigated the activation of the STAT5 protein during the differentiation of myelomonocytic cells. In the human U937 promonocytic cell line, STAT5 activation occurred in response to several inducers of monocytic differentiation (phorbol ester, 1alpha,25-dihydroxyvitamin D3, and retinoic acid). In the promyelocytic HL60 cell line, STAT5 was activated in the course of either phorbol ester-induced monocytic differentiation or DMSO-induced granulocytic differentiation. To test for involvement of STAT5 in the differentiation of primary nonimmortalized cells, chicken myeloid progenitor cells transfected with the ts21-E26 avian retrovirus were studied. At 37 degrees C the temperature-sensitive oncoprotein of the ts21-E26 virus (p135(gag-myb-ets)) installs a differentiation block that is released by a shift to the nonpermissive temperature (42 degrees C). Both proliferation and differentiation of ts21-E26-transfected myeloblasts require the continuous presence of chicken myelomonocytic growth factor (cMGF). Addition of cMGF to growth factor-starved cells rapidly caused STAT5 tyrosine phosphorylation, DNA binding, and transcriptional activity at both permissive and nonpermissive temperatures. Moreover, the temperature shift-induced onset of myelomonocytic differentiation strictly correlated with the appearance of activated STAT5 in ts21-E26-infected myeloblasts, but not in cells infected with wtE26 retrovirus. These data position STAT5 in a nuclear signaling cascade induced by the cMGF receptor and suggest a contribution of STAT5 to the process of myelomonocytic differentiation or to the functional changes that accompany the maturation of myeloid progenitor cells to a terminally differentiated stage.

GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression.

Gamma interferon activation site (GAS) elements are short stretches of DNA, originally defined as a requirement for the rapid transcriptional induction of genes in response to interferon-gamma (IFN-gamma). The protein complex binding to GAS sequences in IFN-gamma-treated cells, the gamma interferon activation factor (GAF), is a dimer of Stat1, the prototype of a family of cytokine-responsive transcription factors, the signal transducers and activators of transcription. To date, seven different Stats are known (excluding alternatively spliced or processed forms), six of which recognize the same small palindromic consensus sequence TTCN2-4 GAA that defines a GAS element. Because one or several Stats take part in nuclear signaling in response to most cytokines or growth factors, the GAS sequence has changed from being viewed as a specific site for IFN-activated GAF to becoming the general nuclear end of the Jak-Stat signaling pathways. This review focuses on the identification and definition of GAS elements, their interaction with Stat transcription factors, and their contribution to the specificity of cytokine-induced gene expression.

Jaks, Stats and the immune system.

Changes in gene expression are necessary for an adaptive response of cells to immunological stimuli and thus for their proper function in the context of the immune system. Regulatory inputs usually originate from cell surface receptors and in many cases affect the transcription rates of specific genes by modulating the activity of transcription factors. The Jak-Stat signalling paradigm has received large attention by molecular immunologists because it applies to nuclear signalling by all cytokine receptors. In its simplest form it requires only two protein components downstream of the receptor: Janus family protein tyrosine kinases (Jaks) which are usually receptor-associated, and signal transducer and activator of transcription (Stat) family transcription factors which carry the receptor-generated signal to the nucleus and stimulate gene expression. Here we give a brief overview of both recent progress and open questions concerning the Jak and Stat molecules, their regulation, and the biological implications of their activity.

Activation of different Stat5 isoforms contributes to cell-type-restricted signaling in response to interferons.

Tyrosine phosphorylation and activation of the transcription factor Stat5 occur in response to stimuli like granulocyte-macrophage colony-stimulating factor, interleukin-3, or erythropoietin that stimulate both proliferation and differentiation of hematopoietic cells. It is unclear whether Stat5 is part of a proliferative response or part of the events leading to cellular differentiation. Here we report that agents promoting differentiation but not proliferation of hematopoietic cells, like phorbol ester or both types of interferons (IFNs), activate Stat5 in promonocytic U937 cells. Both IFN types caused tyrosine phosphorylation and DNA binding of predominantly one Stat5 isoform (Stat5a) despite expression of both Stat5a and Stat5b proteins. Monocytic differentiation of U937 cells led to a strong decrease in IFN-gamma-mediated activation of Stat5 but not of Stat1. Transactivation of Stat5-target genes occurred in response to IFN-gamma, which activates both Stat5 and Stat1, but not in response to granulocyte-macrophage colony-stimulating factor, which activates only Stat5. Tyrosine phosphorylation of Stat5 is not generally part of the IFN response. IFN-gamma did not cause Stat5 activation in HeLa cells, despite the expression of both Stat5 isoforms at similar levels. By contrast, IFN-alpha caused tyrosine phosphorylation and DNA binding of exclusively the b isoform of Stat5, and activated Stat5b formed a DNA binding activity previously found in HeLa cells and designated IFN-alpha activation factor 2. Taken together, our results demonstrate that ligand binding of IFN receptors leads to an isoform-specific activation of Stat5 in a restricted number of cell lineages. Moreover, they suggest that Stat5 might be part of the differentiation response of myeloid cells.

Activation of Stat 5b in erythroid progenitors correlates with the ability of ErbB to induce sustained cell proliferation.

Self renewal of normal erythroid progenitors is induced by the receptor tyrosine kinase c-ErbB, whereas other receptors (c-Kit/Epo-R) regulate erythroid differentiation. To address possible mechanisms that could explain this selective activity of c-ErbB, we analyzed the ability of these receptors to activate the different members of the Stat transcription factor family. Ligand activation of c-ErbB induced the tyrosine phosphorylation, DNA-binding, and reporter gene transcription of Stat 5b in erythroblasts. In contrast, ligand activation of c-Kit was unable to induce any of these effects in the same cells. Activation of the erythropoietin receptor caused specific DNA-binding of Stat 5b, but failed to induce reporter gene transcription. These biochemical findings correlate perfectly with the selective ability of c-ErbB to cause sustained self renewal in erythroid progenitors.

An initial evaluation of subcuticular skin closure with absorbable intradermal pins.

A skin-approximating instrument has been developed to facilitate rapid subcuticular skin closure of surgical incisions using intradermally placed absorbable pins. A prospective, randomized, double-blind study was conducted comparing wound healing characteristics of incisions closed using the intradermal pins and those closed using a subcuticular closure with a synthetic braided absorbable suture. Wounds were evaluated for cosmetic effect, closure accuracy, comfort, and speed of closure by the surgeon, an independent observer, and the patient. Incisions closed using both techniques resulted in equally excellent wound healing characteristics. The instrument facilitated a significantly more rapid closure than the characteristically time-consuming technique of sewing by hand.

A role for STAT family transcription factors in myeloid differentiation.

STAT family transcription factors regulate gene expression in response to a wide variety of cytokines. A transcription factor designated differentiation-induced factor (DIF), activated by treatment of myeloid cells with the differentiating agents interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), colony-stimulating factor-1 (CSF-1) or during phorbol ester-induced differentiation, was characterized as a 112kDa protein related to, but not identical with known isoforms of STAT 5. Taken together with previously published results, our data suggest an important function for members of the STAT 5 subfamily in regulating gene expression during the process of myeloid differentiation.

Enhancement of the endo-beta-1,4-glucanase activity of an exocellobiohydrolase by deletion of a surface loop.

In the commonly accepted mechanism for enzymatic hydrolysis of cellulose, endo-beta-1,4-glucanases randomly cleave glucosidic bonds within glucan polymers, providing sites for attack by exo-cellobiohydrolases (EC 3.2.1.91). It has been proposed that hydrolysis by Trichoderma reesei cellobiohydrolase II is restricted to the ends of cellulose polymers because two surface loops cover its active site to form a tunnel. In a closely related endoglucanase, E2 from Thermomonospora fusca, access to the substrate appears to be relatively unhindered because the carboxyl-proximal loop is shortened, and the amino-proximal loop is displaced. The hypothesis was examined by deletion of a region in Cellulomonas fimi cellobiohydrolase A corresponding to part of the carboxyl-proximal loop of T. reesei cellobiohydrolase II. The mutation enhanced the endoglucanase activity of the enzyme on soluble O-(carboxymethyl)cellulose and altered its activities on 2',4'-dinitrophenyl-beta-D-cellobioside, insoluble cellulose, and cellotetraose.

Colony-stimulating factors and interferon-gamma activate a protein related to MGF-Stat 5 to cause formation of the differentiation-induced factor in myeloid cells.

The Jak-Stat pathway of intracellular signals is used by growth factor- and cytokine receptors to induce gene transcription. We have recently reported that differentiation of myeloid cells, induced by phorbol ester, interferon-gamma (IFN-gamma) or colony-stimulating factor-1 (CSF-1) is accompanied by the activation of the differentiation-induced factor (DIF). Activated DIF specifically associates with a subclass of gamma-interferon activation site (GAS)-like DNA elements. We now report that GM-CSF, which like CSF-1 promotes the generation of mature macrophages, activates DIF. No activation was observed after treatment with the granulocyte growth and differentiation factor G-CSF. Antibodies raised against a Stat family protein, designated mammary gland factor-Stat 5 (MGF-Stat 5), reacted with DIF induced by either CSF-1, GM-CSF or IFN-gamma. Antisera to other known Stats were without effect on the DIF complex in electrophoretic mobility shift assays (EMSA). A 112 kDa protein could be isolated from either GM-CSF- or IFN-gamma-treated cells by GAS oligonucleotide precipitation. This protein reacted with antibodies to both MGF-Stat 5 and phosphotyrosine. MGF-Stat 5 and closely related proteins thus define a subfamily of Stat transcription factors that are present in a variety of cell types and are required for the onset of immediate gene expression in response to differentiating stimuli.