Evaluation of RIDA®GENE norovirus GI/GII real time RT-PCR using stool specimens collected from children and adults with acute gastroenteritis.

BACKGROUND: Norovirus is the leading cause of epidemic and sporadic acute gastroenteritis (AGE) in the United States. Widespread prevalence necessitates implementation of accurate norovirus detection assays in clinical diagnostic laboratories. OBJECTIVE: To evaluate RIDA®GENE norovirus GI/GII real-time RT-PCR assay (RGN RT-PCR) using stool samples from patients with sporadic AGE. STUDY DESIGN: Patients between 14 days to 101 years of age with symptoms of AGE were enrolled prospectively at four sites across the United States during 2014-2015. Stool specimens were screened for the presence of norovirus RNA by the RGN RT-PCR assay. Results were compared with a reference method that included conventional RT-PCR and sequencing of a partial region of the 5'end of the norovirus ORF2 gene. RESULTS: A total of 259 (36.0%) of 719 specimens tested positive for norovirus by the reference method. The RGN RT-PCR assay detected norovirus in 244 (94%) of these 259 norovirus positive specimens. The sensitivity and specificity (95% confidence interval) of the RGN RT-PCR assay for detecting norovirus genogroup (G) I was 82.8% (63.5-93.5) and 99.1% (98.0-99.6) and for GII was 94.8% (90.8-97.2) and 98.6% (96.9-99.4), respectively. Seven specimens tested positive by the RGN-RT PCR that were negative by the reference method. The fifteen false negative samples were typed as GII.4 Sydney, GII.13, GI.3, GI.5, GI.2, GII.1, and GII.3 in the reference method. CONCLUSIONS: The RGN RT-PCR assay had a high sensitivity and specificity for the detection of norovirus in stool specimens from patients with sporadic AGE.

Purification and properties of an extracellular pectin lyase produced by the strain of Penicillium oxalicum in solid-state fermentation.

A pectin lyase (PNL, EC 4.2.2.10) produced extracellularly by the strain of Penicillium oxalicum in solid-state fermentation medium containing deoiled mandarin orange peel meal was purified to apparent homogeneity by a protocol that included ammonium sulfate precipitation, DEAE-Sephadex A-50 and Sephadex G-100 chromatography. The enzyme had molecular mass of 50 kD, as determined by SDS- PAGE and showed optimum pH and temperature at 8.0 and 50 degrees C respectively. It had an isoelectric point (pI) of 5.0 and showed a K(m) of 1.1 mg/ml of citrus pectin. The enzyme was strongly inhibited by Mo4+, Ag+ and Pb2+ and also by polyphenolic compounds, in particular tannic acid.

Cumulative risk and trends in prostate cancer incidence in Mumbai, India.

BACKGROUND: Information relating to cancer incidence trends in a community forms the scientific basis for the planning and organization of prevention, diagnosis and treatment of cancer. We here estimated the cumulative risk and trends in incidence of prostate cancer in Mumbai, India, using data collected by the Bombay Population-based Cancer Registry from the year 1986 to 2000. METHODS: During the 15 year period, a total of 2864 prostate cancer cases (4.7% of all male cancers and 2.4% of all cancers) were registered by the Bombay Population-based Cancer Registry. For evaluation of the trend, we applied a linear regression model based on the logarithm of the observed incidence rates. The annual percentage changes were also computed for the evaluation. Cumulative incidence rates percentages were calculated by adding up the age specific incidence rates at single ages and then expressed as a percentage. RESULTS: Analysis of the trends in age-adjusted incidence rates of prostate cancer during the period 1986 to 2000 showed no statistically significant increase or decrease and the rates proved stable across the various age groups (00-49, 50-69 and 70+) also. The probability estimates indicated that one out of every 59 men will contract a prostate cancer at some time in his whole life and 99% of the chance is after he reaches the age of 50. CONCLUSION: The stability in age adjusted-incidence rates indicates that there are no changes in the etiological factors for prostate cancer in Mumbai, India. These findings may be of general interest because changes in diagnostic practices are confounded in the time trends of prostate cancer change in many western countries preventing inferences on the changes in risk.

MHC class I molecules can direct proteolytic cleavage of antigenic precursors in the endoplasmic reticulum.

The large set of peptides presented by MHC (major histocompatibility complex) class I molecules are generated by proteolysis of diverse precursors in the cytoplasm and possibly in the endoplasmic reticulum (ER). To define the potential peptide trimming events in the ER, we analyzed proteolytic products generated in isolated microsomes. The residues flanking the N terminus of the final antigenic peptide were rapidly removed within the microsomes but only in the presence of appropriate MHC molecules. Remarkably, the precursor peptide was bound to the MHC molecules in a distinct conformation and required an aminopeptidase activity to generate the optimal peptide. The MHC molecules are therefore not only the final repositories of antigenic peptides, but they can also direct their excision from longer precursors.

Mutation of the invariant chain transmembrane region inhibits II degradation, prolongs association with MHC class II, and selectively disrupts antigen presentation.

The invariant chain (Ii) is a key player in regulating the MHC Class II antigen presentation pathway. Here we used site-directed mutagenesis to identify functionally important regions of the invariant chain in regulating antigen presentation function in transfected cells. Mutation of Ii residues 42-53 caused a defect in the presentation of the ovalbumin 247-265/A(k) epitope, but not in the inhibition of presentation of two hen egg lysozyme epitopes, HEL34-45/A(k) and HEL74-88/A(b), from endogenously expressed antigens. The mutation did not prevent ER translocation, trimerization, or association with MHC Class II molecules and had no obvious effect on endosomal targeting of Ii. It did, however, increase the half-life of the invariant chain, suggesting that sequences in this region influence the degradation of the invariant chain and as a consequence its function in antigen presentation.

ER aminopeptidases generate a unique pool of peptides for MHC class I molecules.

We define here the specificity and significance of proteases in the endoplasmic reticulum (ER) that generate peptides for presentation by major histocompatibility complex (MHC) class I molecules. We show that aminopeptidases efficiently trimmed all residues except proline that flank the NH2-termini of antigenic precursors in the ER and caused an accumulation of X-P-Xn peptides. An aminopeptidase inhibitor blocked peptide trimming in the ER and, consequently, the generation of peptide-loaded MHC molecules. Peptide trimming in the ER is therefore a key step in the MHC class I antigen-processing pathway and also explains the paradox of why many MHC class I molecules display peptides with the X-P-Xn motif despite the inability of the transporter associated with antigen processing to transport such peptides from the cytoplasm.

Introducing endogenous antigens into the major histocompatibility complex (MHC) class II presentation pathway. Both Ii mediated inhibition and enhancement of endogenous peptide/MHC class II presentation require the same Ii domains.

The invariant chain (Ii) plays a key role in regulating the antigen presentation function of major histocompatibility complex (MHC) class II molecules. Ii also influences the presentation of usually excluded endogenously synthesized proteins into the MHC class II presentation pathway. To evaluate the role of Ii in the generation of peptide-MHC class II complexes derived from endogenously synthesized proteins, we tested mutant Ii constructs in two model systems. Co-expression of wild-type Ii inhibits the presentation of hen-egg lysozyme (HEL) 35-45/Ak complex, but enhances the presentation of ovalbumin (OVA) 247-265/Ak complex from endogenously synthesized HEL or OVA precursors. The differential sensitivity of these antigens to chloroquine was consistent with their being processed in distinct compartments. Nevertheless, with a panel of Ii deletion constructs we show here that both the Ii-mediated inhibition and enhancement functions require the endosomal targeting and CLIP residues. Surprisingly, the Ii mutant lacking the endoplasmic reticulum lumenal residues 126-215, despite apparently lower expression, was at least as effective as full-length Ii in antigen presentation assays. Thus, alternative pathways exist for processing endogenously expressed antigens, and Ii-mediated inhibition and enhancement of peptide/MHC class II expression depend upon the same regions, with neither requiring the 89 C-terminal, lumenal Ii residues.

Quantitative analysis of the immune response to mouse non-MHC transplantation antigens in vivo: the H60 histocompatibility antigen dominates over all others.

Minor histocompatibility Ags (minor H Ags) are substantial impediments to MHC-matched solid tissue and bone marrow transplantation. From an antigenic standpoint, transplantation between MHC-matched individuals has the potential to be remarkably complex. To determine the extent to which the immune response is simplified by the phenomenon of immunodominance, we used peptide/MHC tetramers based on recently discovered minor H Ags (H60, H13, and HY) and monitored in vivo CD8 T cell responses of female C57BL/6 mice primed with MHC-matched, but background-disparate, male BALB.B cells. CD8 T cells against H60 overwhelmed responses to the H13 and HY throughout primary and secondary challenge. H60 immunodominance was an inherent quality, overcoming a lower memory precursor frequency compared with that of H13 and evoking a T cell response with diverse TCRV beta usage. IFN-gamma staining examining congenically defined minor H Ags extended H60 dominance over additional minor H Ags, H28, H4, and H7. These four minor H Ags accounted for up to 85% of the CD8 T cell response, but H60 stood out as the major contributor. These findings show that immunodominance applies to antigenically complex transplantation settings in vivo and that the responses to the H60 minor H Ag dominates in this model. We suggest that immunodominant minor H Ags are those that result from the absence of a self analog.

Distinguishing self from nonself: immunogenicity of the murine H47 locus is determined by a single amino acid substitution in an unusual peptide.

Histocompatibility (H) Ags are responsible for chronic graft rejection and graft vs host disease in solid tissue and bone marrow transplantation among MHC-matched individuals. Here we defined the molecular basis of self-nonself discrimination for the murine chromosome 7 encoded H47 histocompatibility locus, known by its trait of graft-rejection for over 40 years. H47 encodes a novel, highly conserved cell surface protein containing the SCILLYIVI (SII9) nonapeptide in its transmembrane region. The p7 isoleucine-to-phenylalanine substitution in SII9 defined the antigenic polymorphism and T cell specificity. Despite absence of the canonical consensus motif and weak binding to D(b) MHC I, both H47 peptides were presented to CTLs. However, unlike all the other known H loci, the relative immunogenicity of both H47 alleles varied dramatically and was profoundly influenced by neighboring H loci. The results provide insights into the peptide universe that defines nonself and the basis of histoincompatibility.

Differences that matter: major cytotoxic T cell-stimulating minor histocompatibility antigens.

Despite thousands of genetic polymorphisms among MHC matched mouse strains, a few unknown histocompatibility antigens are targeted by the cytotoxic T cells specific for tissue grafts. We isolated the cDNA of a novel BALB.B antigen gene that defines the polymorphic H28 locus on chromosome 3 and yields the naturally processed ILENFPRL (IFL8) peptide for presentation by Kb MHC to C57BI/6 CTL. The CTL specific for the IFL8/Kb and our previously identified H60/Kb complexes represent a major fraction of the B6 anti-BALB.B immune response. The immunodominance of these antigens can be explained by their differential transcription in the donor versus the host strains and their expression in professional donor antigen-presenting cells.

Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages.

Natural killer (NK) cells attack tumor and infected cells, but the receptors and ligands that stimulate them are poorly understood. Here we report the expression cloning of two murine ligands for the lectin-like receptor NKG2D. The two ligands, H-60 and Rae1 beta, are distant relatives of major histocompatibility complex class I molecules. NKG2D ligands are not expressed by most normal cells but are up-regulated on numerous tumor cells. We show that mouse NKG2D is expressed by NK cells, activated CD8+ T cells and activated macrophages. Expression of either NKG2D ligand by target cells triggers NK cell cytotoxicity and interferon-gamma secretion by NK cells, as well as nitric oxide release and tumor necrosis factor alpha transcription by macrophages. Thus, through their interaction with NKG2D, H-60 and Rae1 beta are newly identified potent stimulators of innate immunity.

Bacterial proteins can be processed by macrophages in a transporter associated with antigen processing-independent, cysteine protease-dependent manner for presentation by MHC class I molecules.

MHC class I molecules present peptides derived primarily from endogenously synthesized proteins on the cell surface as ligands for CD8+ T cells. However, CD8+ T cell responses to extracellular bacteria, virus-infected, or tumor cells can also be elicited because certain professional APC can generate peptide/MHC class I (MHC-I) complexes from exogenous sources. Whether the peptide/MHC-I complexes are generated because the exogenous proteins enter the classical cytosolic, TAP-dependent MHC-I processing pathway or an alternate pathway is controversial. Here we analyze the generation of peptide/MHC-I complexes from recombinant Escherichia coli as an exogenous Ag source that could be delivered to the phagosomes or directly into the cytosol. We show that peritoneal and bone marrow macrophages generate peptide/MHC-I complexes by the classical as well as an alternate, but relatively less efficient, TAP-independent pathway. Using a novel method to detect proteolytic intermediates we show that the generation of the optimal MHC-I binding peptide in the alternate pathway requires cysteine as well as other protease(s). This alternate TAP-independent pathway also operates in vivo and provides a potential mechanism for eliciting CD8+ T cell responses to exogenous Ags.

Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library.

Type 1 diabetes is an autoimmune disease in which the insulin-producing pancreatic beta cells are destroyed at an early age by an immune process that involves both CD4 and CD8 T lymphocytes. The identification of autoantigens in diabetes is very important for the design of antigen-specific immunotherapy. By screening a pancreatic islet cDNA library, we have identified the autoantigen recognized by highly pathogenic CD8 T cells in the non-obese diabetic mouse, one of the best animal models for human diabetes. This is the first identification, to our knowledge, of a CD8 T-cell epitope in an autoimmune disease. The peptide recognized by the cells is in the same region of the insulin B chain as the epitope recognized by previously isolated pathogenic CD4 T cells. This has very important implications for the potential use of insulin in preventative therapy.

Discrete proteolytic intermediates in the MHC class I antigen processing pathway and MHC I-dependent peptide trimming in the ER.

The antigen processing pathway generates the peptides displayed by MHC I molecules on the cell surface. Whether these peptides are generated in the cytosol or from longer intermediates transported into the ER is unclear, because peptides other than those bound to MHC I have been difficult to find. Using a novel assay, we show that N-terminally extended antigenic analogs were associated with high-molecular weight material in the cytosol and were transported by TAP. In the ER, a nonapeptide was predominant that was converted to the final octapeptide only in presence of the appropriate MHC I molecule. The existence of extended peptides and their MHC I-dependent trimming suggest a mechanism for efficiently satisfying the distinct sequence preferences of polymorphic MHC I molecules.

Presentation of out-of-frame peptide/MHC class I complexes by a novel translation initiation mechanism.

Immune surveillance by CD8 T cells requires that peptides derived from intracellular proteins be presented by MHC class I molecules on the target cell surface. Interestingly, MHC molecules can also present peptides encoded in alternate translational reading frames, some even without conventional AUG initiation codons. Using T cells to measure expression of MHC bound peptides, we identified the non-AUG translation initiation codons and established that their activity was at the level of translational rather than DNA replication or transcription mechanisms. This translation mechanism decoded the CUG initiation codon not as the canonical methionine but as the leucine residue, and its activity was independent of upstream translation initiation events. Naturally processed peptide/MHC complexes can thus arise from "noncoding" mRNAs via a novel translation initiation mechanism.

Apparent MHC-independent stimulation of CD8+ T cells in vivo during latent murine gammaherpesvirus infection.

Like EBV-infected humans with infectious mononucleosis, mice infected with the rodent gammaherpesvirus MHV-68 develop a profound increase in the number of CD8+ T cells in the circulation. In the mouse model, this lymphocytosis consists of highly activated CD8+ T cells strikingly biased toward V beta 4 TCR expression. Moreover, this expansion of V beta 4+CD8+ T cells does not depend on the MHC haplotype of the infected animal. Using a panel of lacZ-inducible T cell hybridomas, we have detected V beta 4-specific T cell stimulatory activity in the spleens of MHV-68-infected mice. We show that the appearance and quantity of this activity correlate with the establishment and magnitude of latent viral infection. Furthermore, on the basis of Ab blocking studies as well as experiments with MHC class II, beta2-microglobulin (beta2m) and TAP1 knockout mice, the V beta 4-specific T cell stimulatory activity does not appear to depend on conventional presentation by classical MHC class I or class II molecules. Taken together, the data indicate that during latent infection, MHV-68 may express a T cell ligand that differs fundamentally from both conventional peptide Ags and classical viral superantigens.

Delivery of protein to the cytosol of macrophages using Escherichia coli K-12.

Listeriolysin O (LLO) is an essential determinant of pathogenicity whose natural biological role is to mediate lysis of Listeria monocytogenes containing phagosomes. In this study, we report that Escherichia coli expressing cytoplasmic recombinant LLO can efficiently deliver co-expressed proteins to the cytosol of macrophages. We propose a model in which subsequent or concomitant to phagocytosis the E. coli are killed and degraded within phagosomes causing the release of LLO and target proteins from the bacteria. LLO acts by forming large pores in the phagosomal membrane, thus releasing the target protein into the cytosol. Delivery was shown to be rapid, within minutes after phagocytosis. Using this method, a large enzymatically active protein was delivered to the cytosol. Furthermore, we demonstrated that the E. coli/LLO system is very efficient for delivery of ovalbumin (OVA) to the major histocompatibility (MHC) class I pathway for antigen processing and presentation, greater than 4 logs compared with E. coli expressing OVA alone. Moreover, the time required for processing and presentation of an OVA-derived peptide was similar to that previously reported when purified OVA was introduced directly into the cytosol by other methods. Using this system, potentially large amounts of any protein that can be expressed in E. coli can be delivered to the cytosol without protein purification. The potential use of this system for the delivery of antigenic protein in vivo and the delivery of DNA are discussed.