Effect of radiation therapy on survival in surgically resected retroperitoneal sarcoma: a propensity score-adjusted SEER analysis.

BACKGROUND: Currently no prospective randomized trial has measured the efficacy of radiation therapy for resected retroperitoneal sarcomas (RPS). Our objective was to determine the effect of radiation therapy on disease-specific and overall survival between propensity score-matched surgically resected RPS patients using the Surveillance, Epidemiology, and End Results (SEER) database. PATIENTS AND METHODS: The study population consisted of patients with histologically confirmed RPS who underwent surgical resection between 1988 and 2006. Exclusion criteria included multiple malignancies, distant metastasis, and unknown grade or stage. Cox modeling was used to determine covariate associations with disease-specific survival. Propensity score methods were used to perform survival analysis in patients who received radiation matched with patients who underwent surgery alone. RESULTS: Prior to matching, there were 762 patients (558 surgery only, 204 surgery with radiation). Factors independently associated with radiation therapy were age (P = 0.037), geographic region (P = 0.041), grade (P = 0.047), stage (P = 0.003), and surgery type (P = 0.01). Cox modeling demonstrated that age, sex, grade, and stage were independently associated with survival. Propensity scoring (309 matched pairs) and survival analysis using Kaplan-Meier methods demonstrated no difference between propensity score-matched patients receiving radiation therapy and those who did not (P = 0.35). CONCLUSION: At present, SEER patients with surgically resected RPS who received radiation therapy did not demonstrate survival benefit.

Point-of-care detection of lactate in cerebrospinal fluid.

PURPOSE: Measurements of cerebrospinal fluid (CSF) lactate can aid in detecting infections of the central nervous system and surrounding structures. Neurosurgical patients with temporary lumbar or ventricular CSF drainage harbor an increased risk for developing infections of the central nervous system, which require immediate therapeutic responses. Since blood gas analyzers enable rapid blood-lactate measurements, we were interested in finding out if we can reliably measure CSF-lactate by this point-of-care technique. METHODS: Neurosurgical patients on our intensive care unit (ICU) with either lumbar or external ventricular drainage due to a variety of reasons were included in this prospective observational study. Standard of care included measurements of leucocyte counts, total protein and lactate measurements in CSF by the neurochemical laboratory of our University Medical Center twice a week. With respect to this study, we additionally performed nearly daily measurements of cerebrospinal fluid by blood gas analyzers to determine the reliability of CSF-lactate measured by blood gas analyzers as compared to the standard measurements with a certified device. RESULTS: 62 patients were included in this study. We performed 514 CSF-lactate measurements with blood gas analyzers and compared 180 of these to the in-house standard CSF-lactate measurements. Both techniques correlated highly significantly (Pearson correlation index 0.94) even though lacking full concordance in a Bland-Altman plotting. Of particular importance, regular measurements enabled immediate detection of central infection in three patients who had developed meningitis during the course of their treatment. CONCLUSION: Blood gas analyzers measure CSF-lactate with sufficient reliability and can help in the timely detection of a developing meningitis. In addition to and triggering established CSF diagnostics, CSF-lactate measurements by blood gas analyzers may improve surveillance of patients with CSF drainage. This study was retrospectively registered on April 20th 2020 in the German trial register. The trial registration number is DRKS00021466.

Filopodia are enriched in a cell cohesion molecule of Mr 80,000 and participate in cell-cell contact formation in Dictyostelium discoideum.

During the early phase of Dictyostelium discoideum development, cells undergo chemotactic migration to form tight aggregates. A developmentally regulated surface glycoprotein of Mr 80,000 (gp80) has been implicated in mediating the EDTA-resistant type of cell cohesion at this stage. We have used a monoclonal antibody directed against gp80 to study the topographical distribution of gp80 on the cell surface. Indirect immunofluorescence studies showed that gp80 was primarily localized on the cell surface, with a higher concentration at contact areas. Immunoelectron microscopy was carried out by indirect labeling using protein A-gold, and a nonrandom distribution of gp80 was revealed. In addition to contact regions, gold particles were found preferentially localized on filopodia. Quantitative analysis using transmission electron microscopy (TEM) showed that approximately 60% more gold particles were localized in contact regions in comparison with the noncontact regions, and the filopodial surfaces had a twofold higher gold density. Both TEM and scanning electron microscopy showed that contact areas were enriched in filopodial structures. Filopodia often appeared to adhere to either smooth surfaces or similar filopodial structures of an adjacent cell. These observations suggest that the formation of stable cell-cell contacts involves at least four sequential steps in which filopodia and gp80 probably play an important role in the initial stages of recognition and cohesion among cells.

The contact site A glycoprotein mediates cell-cell adhesion by homophilic binding in Dictyostelium discoideum.

Dictyostelium discoideum expresses a developmentally regulated cell surface glycoprotein of Mr 80,000 (gp80), which has been implicated in the formation of the EDTA-resistant contact sites A at the cell aggregation stage. To determine whether gp80 participates directly in cell binding and, if so, its mode of action, we conjugated purified gp80 to Covaspheres (Covalent Technology Corp., Ann Arbor, MI) and investigated their ability to bind to cells. The binding of gp80-Covaspheres was dependent on the developmental stage of the cells, with maximal interaction at the late aggregation stage. Scanning electron microscopic studies revealed the clustering of gp80-Covaspheres at the polar ends of these cells, similar to the pattern of gp80 distribution on the cell surface as reported earlier (Choi, A. H. C., and Siu, C.-H., 1987, J. Cell Biol., 104:1375-1387). Precoating cells with an adhesion-blocking anti-gp80 monoclonal antibody inhibited the binding of gp80-Covaspheres, suggesting that Covasphere-associated gp80 might undergo homophilic interaction with gp80 on the cell surface. Quantitative binding of 125I-labeled gp80 to intact cells gave an estimate of 1.5 X 10(5) binding sites per cell at the aggregation stage. Binding of soluble gp80 to cells was blocked by precoating cells with the anti-gp80 monoclonal antibody. The ability of gp80 to undergo homophilic interaction was further tested in a filter-binding assay, which showed that 125I-labeled gp80 was able to interact with gp80 bound on nitrocellulose in a dosage-dependent manner. In addition, reassociation of cells was significantly inhibited in the presence of soluble gp80, suggesting that gp80 has a single cell-binding site. These results are consistent with the notion that gp80 mediates cell-cell binding at the aggregation stage of development via homophilic interaction.

Genome-wide DNA methylation profiling reveals cancer-associated changes within early colonic neoplasia.

Colorectal cancer (CRC) is characterized by genome-wide alterations to DNA methylation that influence gene expression and genomic stability. Less is known about the extent to which methylation is disrupted in the earliest stages of CRC development. In this study, we have combined laser-capture microdissection with reduced representation bisulfite sequencing to identify cancer-associated DNA methylation changes in human aberrant crypt foci (ACF), the earliest putative precursor to CRC. Using this approach, methylation profiles have been generated for 10 KRAS-mutant ACF and 10 CRCs harboring a KRAS mutation, as well as matched samples of normal mucosa. Of 811 differentially methylated regions (DMRs) identified in ACF, 537 (66%) were hypermethylated and 274 (34%) were hypomethylated. DMRs located within intergenic regions were heavily enriched for AP-1 transcription factor binding sites and were frequently hypomethylated. Furthermore, gene ontology analysis demonstrated that DMRs associated with promoters were enriched for genes involved in intestinal development, including homeobox genes and targets of the Polycomb repressive complex 2. Consistent with their role in the earliest stages of colonic neoplasia, 75% of the loci harboring methylation changes in ACF were also altered in CRC samples, though the magnitude of change at these sites was lesser in ACF. Although aberrant promoter methylation was associated with altered gene expression in CRC, this was not the case in ACF, suggesting the insufficiency of methylation changes to modulate gene expression in early colonic neoplasia. Altogether, these data demonstrate that DNA methylation changes, including significant hypermethylation, occur more frequently in early colonic neoplasia than previously believed, and identify epigenomic features of ACF that may provide new targets for cancer chemoprevention or lead to the development of new biomarkers for CRC risk.

Extra-nodal extension of sentinel lymph node metastasis is a marker of poor prognosis in breast cancer patients: A systematic review and an exploratory meta-analysis.

Invasive breast cancer is the most common malignancy in women. Its most common site of metastasis is represented by the lymph nodes of axilla, and the sentinel lymph node (SLN) is the first station of nodal metastasis. Axillary SLN biopsy accurately predicts axillary lymph node status and has been accepted as standard of care for nodal staging in breast cancer. To date, the morphologic aspects of SLN metastasis have not been considered by the oncologic staging system. Extranodal extension (ENE) of nodal metastasis, defined as extension of neoplastic cells through the nodal capsule into the peri-nodal adipose tissue, has recently emerged as an important prognostic factor in several types of malignancies. It has also been considered as a possible predictor of non-sentinel node tumor burden in SLN-positive breast cancer patients. We sought out to clarify the prognostic role of ENE in SLN-positive breast cancer patients in terms of overall and disease-free survival by conducting a systematic review and meta-analysis. Among 172 screened articles, 5 were eligible for the meta-analysis; they globally include 624 patients (163 ENE+ and 461 ENE-) with a median follow-up of 58 months. ENE was associated with a higher risk of both mortality (RR = 2.51; 95% CI: 1.66-3.79, p < 0.0001, I(2) = 0%) and recurrence of disease (RR = 2.07, 95% CI: 1.38-3.10, p < 0.0001, I(2) = 0%). These findings recommend the consideration of ENE from the gross sampling to the histopathological evaluation, in perspectives to be validated and included in the oncologic staging.

Three-dimensional modelling and finite element analysis of the human mandible during clenching.

BACKGROUND: Until recently, very few papers have been published concerning the development, analysis and experimental verification of three-dimensional, finite element modelling of the human adult edentulous mandible. The purpose of this study was to improve the method of modelling by using computer-aided engineering (CAE) and computer-aided design (CAD) methods and to utilize the model in analyzing maxillofacial problems. METHODS: The model geometry was derived from position measurements taken from 28 diamond blade cut cross-sections of an average size human adult edentulous mandible and generated using a special sequencing method. Data on anatomical, structural, functional aspects and material properties were obtained from measurements and published data. The materials were idealized as transversely isotropic. The complete model consisted of 258 solid elements and 1635 nodes. RESULTS: The model was solved for displacements and stresses during clenching. In general, the observed displacement and stresses (tensile and compressive) were highest around the condylar region. Compressive stress was also observed around the premolar and molar bite points. CONCLUSION: This investigation has shown that the use of computer-aided modelling in conjunction with the finite element analysis could be effectively utilized in biomechanical analysis of the mandible. It could help to investigate many functional problems and could reduce the time of extensive experimentations.

Current perspectives: calcium phosphate nanocoatings and nanocomposite coatings in dentistry.

The purpose of coatings on implants is to achieve some or all of the improvements in biocompatibility, bioactivity, and increased protection from the release of harmful or unnecessary metal ions. During the last decade, there has been substantially increased interest in nanomaterials in biomedical science and dentistry. Nanocomposites can be described as a combination of two or more nanomaterials. By this approach, it is possible to manipulate mechanical properties, such as strength and modulus of the composites, to become closer to those of natural bone. This is feasible with the help of secondary substitution phases. Currently, the most common composite materials used for clinical applications are those selected from a handful of available and well-characterized biocompatible ceramics and natural and synthetic polymers. This approach is currently being explored in the development of a new generation of nanocomposite coatings with a wider range of oral and dental applications to promote osseointegration. The aim of this review is to give a brief introduction into the new advances in calcium phosphate nanocoatings and their composites, with a range of materials such as bioglass, carbon nanotubes, silica, ceramic oxide, and other nanoparticles being investigated or used in dentistry.

Internalization of virus binding proteins during entry of reovirus into K562 erythroleukemia cells.

Virus overlay protein blot assays of cell membranes of mouse L929 fibroblasts have revealed multiple reovirus binding proteins with molecular masses ranging from approximately 26 to 200 kDa. To determine whether this observation is unique to L cells, membranes of human K562 erythroleukemia cells and human A431 epidermoid cells were subjected to virus overlay protein blot assays. The profiles of reovirus binding proteins of these cells are similar to that of L cells and reovirus is capable of binding to at least 30 membrane proteins. To determine the fate of reovirus binding proteins during viral entry into K562 cells which are infectible by reovirus, cell surface proteins were derivatized with biotin. During viral entry, biotinylated cell surface proteins with molecular masses of 55, 74, 78, 80, 90, 94, 98, and 115 kDa became internalized. The 90- and 115-kDa proteins also bound reovirus, indicating that they are likely to be reovirus receptors. Thus many virus binding proteins are present at the surface of host cells but very few are internalized during entry of reovirus. K562 cells also express glycophorin A which is the putative reovirus receptor on erythrocytes. However, during entry of reovirus into K562 cells, glycophorin A did not appear to become internalized. Reovirus could be shown to attach to erythrocytes but viral entry into these cells could not be demonstrated.

Three dimensional reconstruction of quadrivalents and mapping of translocation breakpoints of the mouse translocations T(2;8) 26H and T(9;17)138Ca.

Three dimensional reconstruction from electron micrographs of serial sections reveals 18 synaptonemal complexes and a cross-shaped quadrivalent in the mouse pachytene oocytes of the heterozygous reciprocal translocations T(2;8)26H and T(9;17)138Ca. The unambiguous identification of translocation breakpoints on the quadrivalents has allowed the mapping of the translocation breakpoints on the chromosomes. The translocation breakpoints of T(2;8)26H are mapped at 73% and 45% from the telocentric centromeres of chromosomes 2 and 8, while those of T(9;17)138Ca are mapped at 41% and 45% from the telocentric centromeres of chromosomes 9 and 17 respectively. This report represents the first study of serial section reconstruction of a mammalian oocyte.

Does the beta-adrenergic receptor function as a reovirus receptor?

A reovirus (type 3) receptor has previously been identified on the mouse thymoma R1.1 cell line and shown to be structurally similar to the mammalian beta-adrenergic receptor [M. S. Co, G. N. Gaulton, A. Tominaga, C.J. Homcy, B.N. Fields, and M.I. Greene (1985). Proc. Natl. Acad. Sci. USA 82, 5315-5318]. To determine whether beta-adrenergic receptors are universal recognition signals for reovirus binding, we studied the human epidermoid carcinoma cell line A431 which is known to possess large numbers of functional beta-adrenergic receptors and was found in the present study to be susceptible to reovirus infection. It was observed that unlike beta-adrenergic agonists, reovirus binding and internalization did not result in the triggering of cellular adenylate cyclase activity. The presence of reovirus also had no effect on cellular response to the agonist (-)-isoproterenol, nor on the binding of the hydrophilic antagonist [3H]CGP-12,177 to intact A431 cells. Furthermore, sequestration of beta-adrenergic receptors from the cell surface by (-)-isoproterenol had no effect on reovirus binding. Conversely, the binding of [3H]CGP-12,177 to cells with internalized reovirus receptors was found to be normal. These data strongly suggest that reovirus and beta-adrenergic receptors on A431 cells are distinct from each other. Similar observations were also made with the mouse L fibroblasts, with the exception that these cells appear to possess few functional beta-adrenergic receptors.

Calcofluor staining of cellulose during microcyst differentiation in wild-type and mutant strains of Polysphondylium pallidum.

Calcofluor White ST was used to monitor the morphological events in the biogenesis of cellulose in the microcyst wall of the wild-type strain (WS-320) and two developmental mutants (mic-1 and mic-2) of Polysphondylium pallidum. During encystment, the cell surface acquires a Calcofluor-specific material which appears to be cellulose because of its sensitivity to purified cellulase. Cellulose-containing vesicles appear distributed throughout the cytoplasm of encysting cells of the three strains. Later, the cellulose-rich vesicles appear near the cell surface. Subsequently, the cell surface stains with Calcofluor, and the vesicles are no longer detectable. Intracellular vesicles resembling the cellulose-rich vesicles in size, in the timing of appearance, and in cellular location are also seen in thin sections. These vesicles are surrounded by a single unit membrane, and their amorphous matrix, which contains a dense irregular core, further implicates them as the basis for the bilayered microcyst wall.

Inhibition of cell-cell binding at the aggregation stage of Dictyostelium discoideum development by monoclonal antibodies directed against an 80,000-dalton surface glycoprotein.

Monoclonal antibodies were prepared against a putative cell-cell adhesion molecule, a surface glycoprotein with an apparent Mr of 80,000 (gp80), from Dictyostelium discoideum. Seven monoclonal antibodies directed against gp80 were characterized and found to fall into three distinct classes. Class I consisted of one monoclonal antibody, is monospecific for gp80, and probably recognizes the peptide portion of the molecule. This class was capable of blocking the EDTA-resistant contact sites effectively. Class II recognized the carbohydrate moiety of gp80 and cross-reacted with a large number of glycoproteins. These monoclonal antibodies partially inhibited cell reassociation. Class III recognized gp80 and one other glycoprotein of Mr 95,000. This class had no effect on cell-cell binding. The class I monoclonal antibody was most potent in inhibiting cell reassociation at the aggregation stage of development. Its effect decreased drastically as development progressed and became negligible by the culmination stage. These observations are consistent with a direct role of gp80 in cell-cell binding and suggest a transient function for gp80 at the aggregation stage.

Reovirus binds to multiple plasma membrane proteins of mouse L fibroblasts.

Plasma membranes from mouse L fibroblasts were isolated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were electroblotted to nitrocellulose paper and probed with 125I-labeled type 3 (T3) reovirus. Multiple protein bands with molecular weights ranging from 26 to 200 kDa were consistently recognized by the virus. Such binding was specific since it was blocked in the presence of unlabeled virus. That these proteins were exposed on the cell surface was confirmed by their susceptibility to sulfo-NHS-LC-biotin labeling of intact cells prior to membrane purification. Blots probed with wheat germ agglutinin (WGA)-gold showed a similar pattern of protein bands. These findings are consistent with the ability of WGA to block reovirus binding to L cells, and with our recent demonstration that the alpha-anomeric form of sialic acid is the minimal receptor determinant recognized by reovirus (R. W. Paul, A. H. C. Choi, and P. W. K. Lee, Virology 172, 382-385, 1989). Both type 1 and type 3 reoviruses were found to recognize the same set of multiple proteins on the blot, which is again consistent with the previous observation that the two serotypes compete with each other for binding to intact L cells.

The alpha-anomeric form of sialic acid is the minimal receptor determinant recognized by reovirus.

A series of synthetic sialosides were evaluated for their ability to interact with reovirus serotype 3. It was found that sialosides with terminal N-acetylneuraminic acid (NeuNAc) linked in either an alpha 2,3 or alpha 2,6 configuration effectively blocked the binding of reovirus to mouse L fibroblasts, in contrast to a monosaccharide mixture containing the oligosaccharide constituents. Direct binding of reovirus to the sialosides was also demonstrable using sialosides conjugated to bovine serum albumin as ligands in a solid phase binding system. Of particular significance was the finding that the conjugate containing alpha-sialic acid alone (linked to bovine serum albumin) was capable of being recognized by reovirus at a level comparable to that of the other sialoside conjugates. Virus binding was abrogated by pretreating such conjugates with neuraminidase. These results suggest that the alpha-anomeric form of sialic acid is the minimal receptor determinant for reovirus recognition.

Topography and behavior of Sertoli cells in sparse culture during the transitional remodeling phase.

We report observations on the behavior of Sertoli cells in sparse culture during the period from the time of plating to the time of initial confluence (the transitional remodeling phase). Changes in shape, structure, and polarity of cells, as well as changes in migration patterns and cell-cell association patterns, have been followed during the transitional remodeling phase with the aid of topographical markers. These markers are based upon differences between ultrastructural features of the basolateral and apicolateral surfaces. The basolateral surface is characterized by plasmalemmal blebs, whereas the apicolateral surface is characterized by filopodial extensions. Structural differences observed in situ remain evident in Sertoli cells isolated by sequential enzymatic treatments that are described. Another marker is provided by laminin-binding sites, which are detected exclusively on the blebbed, basolateral surfaces of freshly prepared Sertoli cell aggregates. The orientation described is sustained during the initial radial migration of Sertoli cells explanted on uncoated glass coverslips. Under these conditions, blebs are detected only on the dorsal surfaces, and filopodial extensions are evident only on the ventral surfaces. In contrast, Sertoli cells sparsely plated on a reconstituted basement membrane (air-dried Matrigel) migrate rapidly, display an extraordinary capacity to form elaborate cytoplasmic extensions for cell-cell and cell-substratum contacts, and readily retract blebs and filopodial extensions. These cells do not form mosaic borders, whereas cells plated on uncoated glass do form a monolayer with mosaic-like borders. Cells sparsely seeded on gelated Matrigel migrate preferentially at gaps between adjacent cell explants, and develop a compact cell-cell association pattern. These cells display few, if any, cytoplasmic extensions. We compare the behavior of Sertoli cells sparsely plated on Matrigel with the behavior of Sertoli cells in situ during different stages of development.

Particle bombardment-mediated DNA vaccination with rotavirus VP6 induces high levels of serum rotavirus IgG but fails to protect mice against challenge.

The rotavirus inner capsid protein VP6 contains conserved epitopes that are potential targets for eliciting protective immunity against different serotypes within the same group of rotavirus. In order to determine whether VP6 alone can induce protective immunity, an expression vector pcDNA1/EDIM6 containing gene 6 of rotavirus EDIM strain was constructed and used as a vaccine in an adult mouse model. Cloned gene 6 was determined to be 1356 nucleotides long and contained a 5' noncoding region of 23 nucleotides, a 3' noncoding region of 139 nucleotides, and a coding frame of 1194 nucleotides for a polypeptide of 397 amino acid residues. Recombinant VP6 was expressed in rabbit reticulocyte lysate and the heat-denatured recombinant VP6 migrated in SDS-gels with an apparent molecular weight of approximately 43 kDa. Five additional polypeptide bands corresponding to oligomers of recombinant VP6 were observed when the expressed product was not heat denatured. To determine the immunogenicity of recombinant VP6, female BALB/c mice were injected intramuscularly or intradermally with pcDNA1/EDIM6, or were inoculated epidermally with plasmid-coated gold beads using the Geniva Accell particle delivery device. Only intradermal injection and particle delivery elicited measurable serum anti-rotavirus IgG responses, but responses developed following particle delivery were significantly (P < 0.001) greater. However, none of the delivery methods induced serum or stool anti-rotavirus IgA responses and, when challenged with EDIM no protection against infection was observed in the immunized mice. Therefore, parenteral immunization with VP6 alone elicited large anti-rotavirus IgG responses but did not elicit protection against murine rotavirus infection in this model.

Particle-bombardment-mediated DNA vaccination with rotavirus VP4 or VP7 induces high levels of serum rotavirus IgG but fails to protect mice against challenge.

We recently reported that epidermal immunization using the PowderJet particle delivery device with plasmid vector pcDNA1/EDIM6 encoding rotavirus VP6 of murine strain EDIM induced high levels of serum rotavirus IgG but failed to protect mice against EDIM infection (Choi, A. H., Knowlton, D. R., McNeal, M. M., and Ward, R. L. (1997) Virology 232, 129-138.). This was extended to determine whether pcDNA1/EDIM4 or pcDNA1/EDIM7, which encode either rotavirus VP4 or VP7, the rotavirus neutralization proteins, could also induce rotavirus-specific antibody responses and if these responses resulted in protection. Titers of rotavirus serum IgG increased with the first dose in mice immunized with pcDNA1/EDIM7, but little or no serum rotavirus IgG was detected in mice immunized with pcDNA1/EDIM4. In vitro assays with these plasmids in rabbit reticulocyte lysates showed that VP4 was expressed but the amount was considerably lower than VP6 or VP7. To improve expression of VP4 and induction of rotavirus-specific humoral responses, the coding region of VP4 was cloned into the high-expression plasmid WRG7054 as a fusion protein containing the 22-amino-acid secretory signal peptide of tissue plasminogen activator (tPA) at its N terminus. In vitro expression of tPA::VP4 was significantly higher than unmodified VP4, and mice inoculated with WRG7054/EDIM4 generated high titers of rotavirus IgG. The coding sequence of VP7 without the first 162 nucleotides was also cloned into WRG7054, but no difference was observed between titers of serum rotavirus IgG in mice immunized with this plasmid (WRG7054/EDIM7Delta1-162) and pcDNA1/EDIM7. The rotavirus-specific IgG titers in all immune sera were predominantly IgG1 indicating induction of Th 2-type responses. None of the mice immunized with any of the VP4 or VP7 plasmids developed serum or fecal rotavirus IgA or neutralizing antibody to EDIM. When immunized mice were challenged with EDIM virus, there was no significant reduction in viral shedding relative to unimmunized controls. Therefore epidermal immunization with VP4 or VP7 alone elicited rotavirus IgG responses but did not protect against homologous rotavirus challenge.

Antibody-independent protection against rotavirus infection of mice stimulated by intranasal immunization with chimeric VP4 or VP6 protein.

This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model. BALB/c mice were intranasally or intramuscularly administered purified Escherichia coli-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP). One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured. When three 9-microg doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein. Following EDIM challenge, shedding was significantly (P = 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only. Three 9-micrograms doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated E. coli heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody. No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice. However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (P = <0.001). Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6. When MBP::VP6 was administered intranasally to B-cell-deficient microMt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls. These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody.