Differential characteristics of pediatric sinusitis in patients who underwent endoscopic sinus surgery: children vs. adolescents.

OBJECTIVES: We hypothesised that paediatric chronic sinusitis patients might have various clinical characteristics, depending on age, compared symptoms, physical findings and clinical features in younger children and older adolescent patients who underwent endoscopic sinus surgery. DESIGN: A retrospective review of medical records. SETTING: A total of 195 paediatric patients who underwent Endoscopic sinus surgery were enrolled and medical records were reviewed. PARTICIPANTS: The subjects were divided into children (age < 12 years, n = 70, mean age = 9.6 years) and adolescents (age ≥ 12 years, n = 125, mean age = 14.7 years). MAIN OUTCOME MEASURES: Subjective symptoms, physical findings, CT images and clinical features were compared in children and adolescent groups. RESULTS: Cough and nasal obstruction were more common in adolescents, and sleep disturbance was more common in children. Nasal mucosal injection was more common in adolescents, whereas tonsils were larger in children. Septal deviation was a more common finding in adolescents, and total CT score and serum total IgE levels were higher in children. There was no statistical difference in rate of recurrence after endoscopic sinus surgery. CONCLUSION: The clinical features of paediatric chronic sinusitis differed between the younger and older groups. Symptomatic, anatomical and clinical differences between these two groups suggest that further study of paediatric chronic sinusitis should stratify patients by age to better understand the effects of treatment on each age group.

The Tat-conjugated N-terminal region of mucin antigen 1 (MUC1) induces protective immunity against MUC1-expressing tumours.

Mucin antigen 1 (MUC1) is overexpressed on various human adenocarcinomas and haematological malignancies and has long been used as a target antigen for cancer immunotherapy. Most of the preclinical and clinical studies using MUC1 have used the tandem repeat region of MUC1, which could be presented by only a limited set of major histocompatibility complex haplotypes. Here, we evaluated N-terminal region (2-147 amino acids) of MUC1 (MUC1-N) for dendritic cell (DC)-based cancer immunotherapy. We used Esherichia coli-derived MUC1-N that was fused to the protein transduction domain of human immunodeficiency virus Tat protein for three reasons. First, mature DCs do not phagocytose soluble protein antigens. Secondly, tumour cells express underglycosylated MUC1, which can generate epitopes repertoire that differs from normal cells, which express hyperglycosylated MUC1. Finally, aberrantly glycosylated MUC1 has been known to impair DC function. In our study, Tat-MUC1-N-loaded DCs induced type 1 T cell responses as well as cytotoxic T lymphocytes efficiently. Furthermore, they could break tolerance in the transgenic breast tumour mouse model, where MUC1-positive breast cancers grow spontaneously. Compared with DCs pulsed with unconjugated MUC1-N, DCs loaded with Tat-conjugated MUC1-N could delay tumour growth more effectively in the transgenic tumour model as well as in the tumour injection model. These results suggest that the recombinant N-terminal part of MUC1, which may provide a diverse epitope repertoire, could be utilized as an effective tumour antigen for DC-based cancer immunotherapy.

Protective anti-tumour immune responses by murine dendritic cells pulsed with recombinant Tat-carcinoembryonic antigen derived from Escherichia coli.

Carcinoembryonic antigen (CEA) is over-expressed on various human cancer cells and has been the target of immunotherapies using dendritic cells (DCs) pulsed with CEA-specific RNA or peptides, or transduced by CEA-expressing adenovirus or vaccinia virus. Because activated DCs do not phagocytose soluble protein antigens efficiently and pure immature DCs are not obtained easily ex vivo, an efficacious whole CEA protein-loaded DC vaccine has not been reported. To improve the antigen delivery into DCs, we utilized CEA conjugated to a protein-transduction domain, human immunodeficiency virus transactivating Tat. Furthermore, we purified the truncated non-glycosylated CEA from Escherichia coli to overcome the safety concerns and immunosuppressive functions associated with the native CEA protein. Using confocal microscopy and fluorescence activating cell sorter analysis, we demonstrated that the Tat-CEA protein entered the cytoplasm of DCs efficiently within 10 min of co-culture, compared with the negligible amount of CEA into DCs 30 min later. CEA-specific T cell proliferation and cytotoxic T cell responses were enhanced significantly in mice immunized with Tat-CEA-pulsed DCs [DC (Tat-CEA)] compared with those immunized with CEA-pulsed DCs [DC (CEA)]. T helper type 1 responses were more prominent in the DC (Tat-CEA) immunized mice whose splenocytes secreted more interferon-gamma and less interleukin-4 than those from DC (CEA) immunized mice. In vivo, the DC (Tat-CEA) vaccine delayed tumour growth significantly and prolonged survival of tumour-bearing mice. These results suggest that protective epitopes are well preserved on bacteria-derived recombinant Tat-CEA. This strategy may provide a basic platform for DC-based anti-CEA vaccines that could be utilized in combination with advanced immune-enhancing therapeutics.

Expression of chemokine genes in human dermal microvascular endothelial cell lines infected with Orientia tsutsugamushi.

Scrub typhus, caused by Orientia tsutsugamushi, is characterized by local as well as systemic inflammatory manifestations. The main pathologic change is focal or disseminated multiorgan vasculitis, which is caused by the destruction of endothelial cells and perivascular infiltration of leukocytes. We investigated the regulation of chemokine induction in transformed human dermal microvascular endothelial cells (HMEC-1) in response to O. tsutsugamushi infection. The monocyte chemoattractant protein-1 (MCP-1) and interleukin 8 (IL-8) mRNAs were induced, and their levels showed a transitory peak at 3 and 6 h, respectively. The RANTES transcript was detected at 6 h after infection, with increased levels evident by 48 h. The induction of the MCP-1 and IL-8 genes was not blocked by cycloheximide, suggesting that de novo protein synthesis of host cell proteins is not required for their transcriptional activation. Heat- or UV-inactivated O. tsutsugamushi induced a similar extent of MCP-1 and IL-8 responses. The induction of MCP-1 and IL-8 transcripts in the endothelial cells by O. tsutsugamushi was not blocked by the inhibitors of NF-kappaB. Furthermore, the activation of NF-kappaB was not detected in HMEC-1 stimulated with O. tsutsugamushi. These results demonstrate that heat-stable molecules of O. tsutsugamushi induce the MCP-1 and IL-8 genes and the induction of the chemokine genes may be mediated by an NF-kappaB independent mechanism. We also showed that another major transcription factor, activator protein-1 (AP-1), was up-regulated in HMEC-1 after O. tsutsugamushi infection. This suggests the possible involvement of AP-1 in the chemokine gene expression.

Orientia tsutsugamushi infection: overview and immune responses.

Orientia tsutsugamushi, an obligate intracellular bacterium, was isolated for the first time in 1930. Infections by virulent strains are characterized by fever, rash, eschar, pneumonia, myocarditis, and disseminated intravascular coagulation. Here we review the general aspects of O. tsutsugamushi and immune responses in terms of inflammation, protective immune mechanisms, and immunogenic antigens.

Microtubule- and dynein-mediated movement of Orientia tsutsugamushi to the microtubule organizing center.

The host cell microfilaments and microtubules (MTs) are known to play a critical role in the life cycles of several pathogenic intracellular microbes by providing for successful invasion and promoting movement of the pathogen once inside the host cell cytoplasm. Orientia tsutsugamushi, an obligate intracellular bacterium, enters host cells by induced phagocytosis, escapes to the cytosol, and then replicates in the cytosol. ECV304 cells infected with O. tsutsugamushi revealed the colocalization of the MT organizing center (MTOC) and cytosolic orientiae by indirect immunofluorescence assay. Using immunofluorescence microscopy in the presence and absence of MT-depolymerizing agents (colchicine and nocodazole), it was shown that the cytosolic oriential movement was mediated by MTs. By transfection study (overexpression of dynamitin [also called p50], which is known to associate with dynein-dependent movement), the movement of O. tsutsugamushi to the MTOC was also mediated by dynein, the minus-end-directed MT-related motor. Although the significance of this movement in the life cycle of O. tsutsugamushi was not proven, we propose that the cytosolic O. tsutsugamushi bacteria use MTs and dyneins to propel themselves from the cell periphery to the MTOC.

Neutralization epitopes on the antigenic domain II of the Orientia tsutsugamushi 56-kDa protein revealed by monoclonal antibodies.

Monoclonal antibodies (MoAbs) reactive with the authentic Orientia tsutsugamushi 56-kDa protein were generated. MoAb FS10 and FS15 showed in vitro, as well as, in vivo neutralizing activity upon O. tsutsugamushi infection. Deletion mutants of the gene for 56-kDa protein of O. tsutsugamushi Boryong were expressed to map the binding region. FS10 and FS15 are bound to amino acids (aa) located in an antigenic domain II, at residues 140-160 and 187-214, respectively. Computer modeling indicated that aa 146-153 were important for antigenicity against FS10. A sequence for aa 142-150 was highly homologous between oriential strains. These results suggest that the antigenic determinant for neutralizing MoAbs is an epitope within aa 140-160. Furthermore, this region may be important for the adhesion/invasion or intracellular survival of O. tsutsugamushi within host cells.

Cellular invasion of Orientia tsutsugamushi requires initial interaction with cell surface heparan sulfate.

Role of transmembrane heparan sulfate proteoglycans on invasion of Orientia tsutsugamushi into host cells was investigated. Pretreatment with heparan sulfate and heparin inhibited the infection of O. tsutsugamushi for L cell, mouse fibroblast, whereas other glycosaminoglycans had little effect. These same treatments were also shown to reduce the infection in a dose-dependent manner, and enzymatic treatment of cells with heparitinase, but not chondroitinase ABC, inhibited the infection. In addition, mutant cell lines of Chinese hamster ovarian cell defective in heparan sulfate synthesis but not chondrotin sulfate synthesis and defective in all glycosaminoglycan synthesis showed marked reduction in susceptibility to infection by O. tsutsugamushi. Also mutant cell lines, which express heparan sulfate proteoglycans at low level, showed intermediate level of infectivity. Finally O. tsutsugamushi bind to(35)S-labelled heparin. Collectively, these findings provide strong evidence that heparan sulfate proteoglycans contribute to the attachment of O. tsutsugamushi to the cells.

Expression of chemokine genes in murine macrophages infected with Orientia tsutsugamushi.

Scrub typhus, caused by Orientia tsutsugamushi infection, is characterized by local as well as systemic inflammatory manifestations. Inflammation is initiated by O. tsutsugamushi-infected macrophages and endothelial cells in the dermis. We investigated the regulation of chemokine induction in macrophage cell line J774A.1 in response to O. tsutsugamushi infection. The mRNAs for macrophage inflammatory proteins 1alpha/beta (MIP-1alpha/beta), MIP-2, and macrophage chemoattractant protein 1 were induced within 30 min, and their levels showed a transitory peak for 3 to 12 h. However, the lymphotactin, eotaxin, gamma interferon-inducible protein 10, and T-cell activation gene 3 mRNAs were not detected by RNase protection assays. Heat-killed O. tsutsugamushi induced a similar extent of chemokine responses. Induction of the chemokine genes was not blocked by the eukaryotic protein synthesis inhibitor cycloheximide, suggesting that de novo synthesis of host cell protein is not required for these transcriptional responses. The induction of chemokine mRNAs by O. tsutsugamushi was blocked by the inhibitors of NF-kappaB activation. Furthermore, O. tsutsugamushi induced the nuclear translocation and activation of NF-kappaB. These results demonstrate that heat-stable molecules of O. tsutsugamushi induce a subset of chemokine genes and that induction involves activation of the transcription factor NF-kappaB.

Homotypic and heterotypic antibody responses to a 56-kilodalton protein of Orientia tsutsugamushi.

We analyzed homotypic and heterotypic antibody responses to a type-specific antigen (Tsa), a 56-kDa protein of Orientia tsutsugamushi, by using sera from mice immunized with strains Gilliam, Karp, Kato, and Boryong. We generated a series of deletion constructs of the tsa gene and expressed them as MalE fusion proteins. Variable domain I (VD I) showed strong responses to homotypic antibodies. Antigenic domain II (AD II) from Boryong and Karp showed cross-reactivities to each other. VD III showed no responses to any of the antibodies. Sera from Kato-immunized mice showed only homotypic responses to AD III. On the other hand, sera of the mice immunized with Gilliam, Karp, or Boryong showed homotypic as well as heterotypic responses to this region. VD IV showed the strongest heterotypic antibody responses among the fragments tested. These data suggest that VD I is important in homotypic antibody responses and that AD II, AD III, and VD IV are important in heterotypic antibody responses of the mice to Tsa.

Protective immunity of microsphere-based mucosal vaccines against lethal intranasal challenge with Streptococcus pneumoniae.

Mucosal vaccination of capsular polysaccharide (PS) of Streptococcus pneumoniae and subsequent creation of the first line of immunological defense in mucosa were examined. Mucosal as well as systemic antibody responses to PS were evoked by peroral or intranasal immunization of BALB/c mice with PS-cholera toxin B subunit (CTB) conjugates entrapped in the alginate microspheres (AM). The bacterial colonization at the lung mucosa was most profoundly inhibited (<95%) by intranasal immunization with the naked conjugate (PS-CTB). The mice vaccinated orally with encapsulated conjugate [AM(PS-CTB)] showed significant reduction on the level of pneumococcal bacteremia (<99%). Eighty percent of the mice perorally immunized with AM (PS-CTB) were protected from lethal intranasal challenge with S. pneumoniae, whereas more than 60% of the mice in the other control groups died of infection. Our novel approach may prove to be important in the development of a mucosal vaccine that will provide protection of mucosal surfaces of host.

Peroral immunization of microencapsulated human VP8 in combination with cholera toxin induces intestinal antibody responses.

To develop an orally delivered subunit vaccine for rotavirus infection, a trypsin cleavage product of VP4, recombinant VP8*, was expressed in Escherichia coli. The recombinant VP8* (rVP8*), purified by affinity chromatography, was reactive against human rotavirus positive serum in Western-blot analysis. To further evaluate the immunogenicity of the oral-delivered rVP8*, it was encapsulated with alginate-microsphere and administered in combination with cholera toxin (CT) as a mucosal adjuvant perorally into mice. The ELISPOT assay showed that the number of rVP8*-specific IgG1 antibody secreting cells increased about 3-fold and about 2-fold in spleen and Peyer's patch, respectively as compared to non-immune mice. In addition, the number of rVP8*-specific IgA antibody secreting cells increased about 2-fold in Peyer's patch. Finally, rVP8*-specific IgA antibody response was significantly enhanced in the intestinal fluids from the mice immunized perorally with encapsulated rVP8* and CT. Taken together, these results indicate that rVP8* possessed proper immunogenicity and it would be potentially useful as a subunit vaccine against rotavirus-associated disease through peroral immunization.

Novel mucosal immunization with polysaccharide-protein conjugates entrapped in alginate microspheres.

A novel mucosal immunization was examined using biocompatible and biodegradable alginate microspheres containing a conjugate of polysaccharide antigen and cholera toxin B subunit (CTB). In order to prepare the alginate microspheres with diameters of less than 5 microm, a new diffusion-controlled interfacial gelation technique was developed. Also, in order to improve the mucosal immune response, a pneumococcal capsular polysaccharide type 19 (PS19) was conjugated to the CTB (PS19-CTB). This conjugate was subsequently encapsulated into the alginate microspheres. The loading content of PS19-CTB to the alginate microspheres was 60%. An in vitro sustained release pattern was observed with the antigen-loaded microspheres, showing 80% antigen release within one day. Mucosal and systemic immunities following oral immunization with the alginate microspheres were studied. Balb/c mice were immunized perorally three times at intervals of two weeks. Peroral immunization with 25 microg of PS19-CTB entrapped in the alginate microspheres evoked both the mucosal IgA and systemic IgM responses to PS19 in small intestine and in sera, respectively. The results suggest that both the mucosal and systemic antibody responses could be induced by oral administration of the PS19-CTB antigen entrapped in alginate microspheres.

Molecular and serologic survey of Orientia tsutsugamushi infection among field rodents in southern Cholla Province, Korea.

Field rodents were collected from six areas in southern Cholla Province, Korea from October to December 1993. Twenty-eight (24%) of the 119 Apodemus agrarius were seropositive (> 1:10) for Orientia tsutsugamushi by the passive hemagglutination assay (PHA). Of the seropositive cases, 11 specimens had antibody titers greater than 1:80. No seropositive specimens were found among the eight Crocidura lasiura collected. On the other hand, the polymerase chain reaction (PCR) amplified about 520 basepairs of a gene encoding the 56-kD protein from the genomic DNA of 12 strains of O. tsutsugamushi tested. This target DNA sequence was amplified from the 11 (8.7%) blood specimens of A. agrarius, and one of the eight C. lasiura also showed evidence of O. tsutsugamushi infection by PCR. Only one of the PCR-positive samples was also PHA-positive. These results suggest that the PCR combined with a serologic assay more accurately detects the degree of infection of rodents with rickettsiae-causing scrub typhus in epidemiologic surveys.

Mapping of antigenic determinant regions of the Bor56 protein of Orientia tsutsugamushi.

The 56-kDa protein (Bor56) of Orientia tsutsugamushi is an immunoprotective antigen and is the target molecule of neutralizing antibodies. This antigen is recognized by almost all of the serum antibodies produced by patients in the convalescence phase of scrub typhus. We expressed the Bor56 open reading frame in Escherichia coli and generated from it a series of deletion constructs as MalE fusion proteins. Antibody-binding domains were characterized by using patient sera, mouse monoclonal antibodies (MAbs), and Bor56-immunized-mouse sera. None of the antibodies bound to a fusion protein containing the carboxy-terminal 140 amino acids (aa) of the Bor56 protein, suggesting that the carboxy-terminal domain of Bor56 is not exposed on the surface of the molecule. Human immunoglobulin M (IgM) antibodies predominantly bound to antigenic domain I (AD I; amino acids [aa] 19 to 113) and AD III (aa 243 to 328). Human IgG antibodies also showed preferential binding to AD I. The epitope recognized by strain-specific MAb (KI4) or group-specific MAb (KI57) was mapped to AD II (aa 142 to 203). Mouse serum antibodies, elicited by immunization with deletion mutants, consistently bound to AD III. Moreover, the carboxy-terminal 140 aa of the Bor56 protein did not elicit an antibody response in C3H/HeDub mice. A model of the antigenic structure of Bor56 is presented and discussed. These results suggest that antigenic fragments from AD I and AD III are useful in the induction of humoral immunity against O. tsutsugamushi. These antigenic analyses provide an important foundation for further analyses of the neutralizing-antibody responses generated during rickettsial infections. They also provide potential peptide substrates for diagnostic assays and vaccine strategies.

Induction of neutralizing antibody in mice by immunization with recombinant 56 kDa protein of Orientia tsutsugamushi.

Anti-oriential antibody inhibits Orientia tsutsugamushi attachment to, and penetration of, host cells. However, O. tsutsugamushi antigens that induce the production of a neutralizing antibody have not been identified. The authors immunized mice and rabbits with the recombinant 56 kDa protein of O. tsutsugamushi fused to the maltose binding protein of Escherichia coli (MBP-Bor56) and analysed their effect on O. tsutsugamushi attachment to or penetration of L929 cells. O. tsutsugamushi attachment and penetration were measured by using an indirect immunofluorescent antibody assay (IFA). O. tsutsugamushi growth in L929 cells was determined by [3H]thymidine uptake assay. By IFA, we observed a 96% reduction of attachment or penetration of O. tsutsugamushi treated with rabbit anti-MBP-Bor56 sera. [3H]thymidine uptake showed that mouse anti-MBP-Bor56 sera caused a 91% reduction in O. tsutsugamushi growth, when compared to mouse anti-MBP sera. These results suggest that the 56 kDa protein of O. tsutsugamushi plays an important role in O. tsutsugamushi attachment to or penetration of cells.

T-track PCR fingerprinting for the rapid detection of genetic polymorphism.

The diversity of DNA sequences can be analyzed by comparing randomly amplified polymorphic DNA, or restriction fragment length polymorphism fragments of DNA. Such analyses are dependent on the selection of appropriate restriction enzyme(s) and/or primers. We have investigated a simpler approach to providing sensitive and specific genotyping. Cyclic extension of target sequences with dideoxythymidine generates PCR products with variable lengths. We analyzed these variable PCR products by scoring the number of variable bands and comparing the scores (numerical profiles) to establish similarities. We found that the polymorphic lengths of the PCR products were comparable among serologically defined strains. It suggests that this single PCR reaction followed by a one-step electrophoresis yields easily analyzable data that can be compared with data from other gels.

Induction of homologous immune response to Rickettsia tsutsugamushi Boryong with a partial 56-kilodalton recombinant antigen fused with the maltose-binding protein MBP-Bor56.

Although the 56-kDa protein of Rickettsia tsutsugamushi has been presumed to play important roles in generating protective immunity against scrub typhus, studies of this protein have been impeded. We used the recombinant 56-kDa protein of R. tsutsugamushi Boryong fused with the maltose-binding protein of Escherichia coli (MBP-Bor56) to analyze its ability to induce protective immunity in a C3H/HeDub murine model. Intraperitoneal immunization of mice with MBP-Bor56 resulted in an increase in the 50% minimal lethal dose of more than 160 times compared with that for the control mice. Splenic mononuclear cells from the mice immunized with MBP-Bor56 showed a dose-dependent pattern of lymphocyte proliferation response and secreted gamma interferon and interleukin-2 when stimulated with irradiated R. tsutsugamushi Boryong, which is a cytokine profile of Th1 cells. High titers of antibody to R. tsutsugamushi were also demonstrated by indirect immunofluorescent-antibody testing. These findings suggest that the 56-kDa protein of R. tsutsugamushi is one of the candidates for a vaccine against scrub typhus.

Isolation of a new Orientia tsutsugamushi serotype.

Orientia tsutsugamushi, the etiological agent of scrub typhus, is an antigenically diverse organism and many serologically distinct strains have been identified. The 56 kDa protein of O. tsutsugamushi, a major protein in the outer membrane, has been thought to be responsible for this antigenic variability. A strain of O. tsutsugamushi isolated in Korea cross-reacted with both Gilliam strain-specific and Karp strain-specific monoclonal antibodies. When its 56 kDa protein gene was cloned and analyzed, its sequence showed variation especially between 1,200 and 1,250 bp, showing that this isolate is a new O. tsutsugamushi strain.

Rapid diagnosis of scrub typhus by a passive hemagglutination assay using recombinant 56-kilodalton polypeptides.

The genes encoding the 56-kDa polypeptides were amplified by polymerase chain reaction from the genomic DNAs of three serotypes of Rickettsia tsutsugamushi, Gilliam, Karp, and Boryong. The amplified products were cloned into expression vector pIH821, and the recombinant antigens were expressed in Escherichia coli as fusion proteins with maltose-binding protein. The recombinant 56-kDa polypeptides were purified by affinity chromatography for the sensitization of sheep erythrocytes. The recombinant 56-kDa polypeptides were evaluated with 89 serum specimens from health blood donors, 94 serum specimens from scrub typhus patients, and 31 serum specimens from patients with other febrile diseases by a passive hemagglutination assay (PHA). Among the scrub typhus patients diagnosed by indirect immunofluorescent-antibody testing, the antibodies to R. tsutsugamushi were detected in 93 patients (99%). One serum specimen from a healthy person showed a false-positive reaction by this method. The recombinant PHA showed no cross-reactions with sera obtained from other febrile patients with diseases such as murine typhus, hemorrhagic fever with renal syndrome, and leptospirosis. In conclusion, this recombinant PHA could be substituted for the conventional indirect immunofluorescent-antibody test and the immunoperoxidase test.