Saliva contains a large number of proteins that participate in the protection of oral tissue. Exosomes are small vesicles (30-100 nm in diameter) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We have recently demonstrated that exosomes are present in human whole saliva. In this study, we found that whole saliva contained at least two types of exosomes (exosome I and exosome II) that are different in size and protein composition. Proteomic analysis revealed that both types of exosomes contained Alix, Tsg101 and Hsp70, all exosomal markers, immunoglobulin A and polymeric immunoglobulin receptor, whereas they had different protein compositions. Most of dipeptidyl peptidase IV known as CD26 in whole saliva, was present on the exosome II and metabolically active in cleaving chemokines (CXCL11 and CXCL12). Human whole saliva exosomes might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.

Altered gut microbiome is associated with systemic inflammation and cirrhosis decompensation. However, the correlation of the oral microbiome with inflammation in cirrhosis is unclear. Our aim was to evaluate the oral microbiome in cirrhosis and compare with stool microbiome. Outpatients with cirrhosis (with/without hepatic encephalopathy [HE]) and controls underwent stool/saliva microbiome analysis (for composition and function) and also systemic inflammatory evaluation. Ninety-day liver-related hospitalizations were recorded. Salivary inflammation was studied using T helper 1 cytokines/secretory immunoglobulin A (IgA), histatins and lysozyme in a subsequent group. A total of 102 patients with cirrhosis (43 previous HE) and 32 age-matched controls were included. On principal component analysis (PCA), stool and saliva microbiome clustered far apart, showing differences between sites as a whole. In salivary microbiome, with previous HE, relative abundance of autochthonous families decreased whereas potentially pathogenic ones (Enterobacteriaceae, Enterococcaceae) increased in saliva. Endotoxin-related predicted functions were significantly higher in cirrhotic saliva. In stool microbiome, relative autochthonous taxa abundance reduced in previous HE, along with increased Enterobacteriaceae and Enterococcaceae. Cirrhotic stool microbiota demonstrated a significantly higher correlation with systemic inflammation, compared to saliva microbiota, on correlation networks. Thirty-eight patients were hospitalized within 90 days. Their salivary dysbiosis was significantly worse and predicted this outcome independent of cirrhosis severity. Salivary inflammation was studied in an additional 86 age-matched subjects (43 controls/43 patients with cirrhosis); significantly higher interleukin (IL)-6/IL-1β, secretory IgA, and lower lysozyme, and histatins 1 and 5 were found in patients with cirrhosis, compared to controls.

CONCLUSIONS

Dysbiosis, represented by reduction in autochthonous bacteria, is present in both saliva and stool in patients with cirrhosis, compared to controls. Patients with cirrhosis have impaired salivary defenses and worse inflammation. Salivary dysbiosis was greater in patients with cirrhosis who developed 90-day hospitalizations. These findings could represent a global mucosal-immune interface change in cirrhosis.

Various bacterial strains were tested for their ability to stimulate immunoglobulin A (IgA) plasmocytes to populate the duodenal lamina propria in axenic mice. The mice were associated with the strains for at least 4 weeks. The strains inhabiting the conventional mouse intestine and belonging to the genera Lactobacillus, Streptococcus, Eubacterium, Actinobacillus, Micrococcus, Corynebacterium, and Clostridium (including the extremely oxygen-sensitive ones) are only slightly or nonimmunogenic, whereas the strains belonging to the genera Bacteroides and Escherichia have an immunogenic effect. The same result was obtained with Bacteroides and Escherichia strains isolated from the digestive tract of other animal species. The kinetics of appearance of intestinal IgA plasmocytes are similar in axenic mice monoassociated with a stimulatory strain and in conventional mice. The association of two or more strains with axenic mice leads either to the same or a greater number of duodenal IgA plasmocytes as that obtained with the most stimulatory strain monoassociated with axenic mice. The maximum stimulation recorded in all of these trials represents about two-thirds of that observed in conventional mice and was obtained in the duodenum of gnotoxenic mice harboring four bacterial strains isolated from the conventional mouse microflora. The orally administered killed cells of two immunogenic strains, E. coli and Bacteroides sp., are as immunogenic as the living cells, provided that their concentration in the digestive tract is sufficient.

Until now it was assumed that the murine poliovirus (PV) receptor homolog gene (MPH) had been identified. Alternative splicing of MPH transcripts generates two glycoproteins named MPH alpha and MPH beta which share an identical N-terminal region composed of three immunoglobulin (Ig)-like domains and different C-terminal regions. Using a degenerate PCR strategy, we describe the identification of a second human PVR-related gene (PRR2), which encodes two glycoproteins, PRR2 alpha (short form) and PRR2 delta (long form). They present 69 and 73% identity with MPH alpha and MPH beta, respectively. In contrast, the human PVR protein exhibits 51% identity which is moreover restricted to the three Ig domains of the murine protein. We therefore propose that PRR2, and not PVR, is the true human homolog of MPH. In addition, Northern blot analysis showed that two mRNA isoforms of 3.0 kb (PRR2 alpha) and 4.4 kb (PRR2 delta) are ubiquitously found in various normal human tissues. In situ hybridization allowed us to map PRR2 to the 19q13.2-q13.4 bands of the human genome, in the same chromosomal region as PVR.

To understand the genetic predisposition to selective immunoglobulin A deficiency (IgAD), we performed a genome-wide association study in 430 affected individuals (cases) from Sweden and Iceland and 1,090 ethnically matched controls, and we performed replication studies in two independent European cohorts. In addition to the known association of HLA with IgAD, we identified association with a nonsynonymous variant in IFIH1 (rs1990760G>A, P = 7.3 x 10(-10)) which was previously associated with type 1 diabetes and systemic lupus erythematosus. Variants in CLEC16A, another known autoimmunity locus, showed suggestive evidence for association (rs6498142C>G, P = 1.8 x 10(-7)), and 29 additional loci were identified with P < 5 x 10(-5). A survey in IgAD of 118 validated non-HLA autoimmunity loci indicated a significant enrichment for association with autoimmunity loci as compared to non-autoimmunity loci (P = 9.0 x 10(-4)) or random SNPs across the genome (P < 0.0001). These findings support the hypothesis that autoimmune mechanisms may contribute to the pathogenesis of IgAD.

The immune system responds vigorously to microbial infection while permitting lifelong colonization by the microbiome. Mechanisms that facilitate the establishment and stability of the gut microbiota remain poorly described. We found that a regulatory system in the prominent human commensal Bacteroides fragilis modulates its surface architecture to invite binding of immunoglobulin A (IgA) in mice. Specific immune recognition facilitated bacterial adherence to cultured intestinal epithelial cells and intimate association with the gut mucosal surface in vivo. The IgA response was required for B. fragilis (and other commensal species) to occupy a defined mucosal niche that mediates stable colonization of the gut through exclusion of exogenous competitors. Therefore, in addition to its role in pathogen clearance, we propose that IgA responses can be co-opted by the microbiome to engender robust host-microbial symbiosis.

An inversion of chromosome 14, inv(14)(q11,q32), is frequently observed in human T cell tumors; the cytogenetic breakpoints are of interest because the T cell receptor alpha-chain and immunoglobulin heavy chain genes reside on chromosome bands 14q11 and 14q32, respectively. We have investigated the structure of the alpha-chain genes in a T cell line harboring the chromosome 14 inversion. On the normal chromosome 14, a V alpha segment has rearranged nonproductively with a J alpha segment. In contrast, the inverted chromosome features an unprecedented rearrangement in which an immunoglobulin heavy chain variable gene segment (VH) on chromosome band 14q32 has joined with a J alpha segment from band 14q11. The VH-J alpha C alpha rearrangement is productive at the genomic level and therefore may encode a hybrid immunoglobulin/T cell receptor polypeptide.

A cDNA clone encoding the human endoplasmic reticulum (ER) resident protein IP90 was isolated and sequenced. It predicts a transmembrane protein with a large ER luminal region showing sequence similarity to calreticulin and a short cytoplasmic domain containing a COOH-terminal RKPRRE sequence that may be relevant to its retention in the ER. It is 95% homologous to the canine ER membrane phosphorprotein called pp90 or calnexin (Wada, I., Rindress, D., Cameron, P. H., Ou, W.-J., Doherty, J. J., II, Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). Previously, in lymphocytes, we have characterized IP90 as a protein associated with partially assembled multichain proteins including the T cell receptor, the membrane immunoglobulin, and the heavy chain of the major histocompatibility complex class I molecules (Hochstenbach, F., David, V., Watkins, S., and Brenner, M. B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4734-4738). Here, we show that within a short metabolic labeling period, IP90 associates transiently with many different newly synthesized proteins. However, in a T cell line that cannot assemble a complete T cell receptor because it lacks the alpha subunit, the unassembled T cell receptor beta chains, which are retained in the ER, remain associated with IP90 throughout a prolonged chase time period. Together, these data offer further evidence suggesting that IP90 may act in assisting protein assembly and/or in the retention within the ER of unassembled protein subunits.

BACKGROUND

The mechanisms of sublingual immunotherapy (SLIT) are less well understood than those of subcutaneous immunotherapy (SCIT).

OBJECTIVE

To determine the effects of grass-pollen SLIT on oral mucosal immune cells, local regulatory cytokines, serum allergen-specific antibody subclasses and B cell IgE-facilitated allergen binding (IgE-FAB).

METHODS

Biopsies from the sublingual mucosa of up to 14 SLIT-treated atopics, nine placebo-treated atopics and eight normal controls were examined for myeloid dendritic cells (mDCs) (CD1c), plasmacytoid dendritic cells (CD303), mast cells (AA1), T cells (CD3) and Foxp3 using immunofluorescence microscopy. IL-10 and TGF-beta mRNA expression were identified by in situ hybridization. Allergen-specific IgG and IgA subclasses and serum inhibitory activity for binding of allergen-IgE complexes to B cells (IgE-FAB) were measured before, during and on the completion of SLIT.

RESULTS

Foxp3(+) cells were increased in the oral epithelium of SLIT- vs. placebo-treated atopics (P=0.04). Greater numbers of subepithelial mDCs were present in placebo-treated, but not in SLIT-treated, atopics compared with normal controls (P=0.05). There were fewer subepithelial mast cells and greater epithelial T cells in SLIT- compared with placebo-treated atopics (P=0.1 for both). IgG(1) and IgG(4) were increased following SLIT (P<0.001). Peak seasonal IgA(1) and IgA(2) were increased during SLIT (P<0.05). There was a time-dependent increase in serum inhibitory activity for IgE-FAB in SLIT-treated atopics.

CONCLUSIONS

SLIT with grass pollen extract is associated with increased Foxp3(+) cells in the sublingual epithelium and systemic humoral changes as observed previously for SCIT.

The T cell receptor alpha/beta (TCR-alpha/beta) is encoded by variable (V), diversity (D), joining (J), and constant (C) segments assembled by recombination during thymocyte maturation to produce a heterodimer that imparts antigenic specificity to the T cell. Unlike immunoglobulins (Igs), which bind free antigen, the ligands of TCR-alpha/beta are cell surface complexes of intracellularly degraded antigens (i.e., peptides) bound to and presented by polymorphic products of the major histocompatibility complex (MHC). Therefore, antigen recognition by T cells is defined as MHC restricted. A model has been formulated based upon the similarity between TCR-alpha/beta V region and Ig Fab amino acid sequences, and the crystal structure of the MHC class I and Ig molecules. This model predicts that the complementarity determining regions (CDR) 1 and 2, composed of TCR V alpha and V beta segments, primarily contact residues of the MHC alpha helices, whereas V/J alpha and V/D/J beta junctional regions (the CDR3 equivalent) contact the peptide in the MHC binding groove. Because polymorphism in MHC proteins is limited relative to the enormous diversity of antigenic peptides, the TCR may have evolved to position the highly diverse junctional residues (CDR3), where they have maximal contact with antigen bound in the MHC peptide groove. Here, we demonstrate a definitive association between CDR3 sequences in both TCR alpha and beta chains, and differences in recognition of antigen fine specificity using a panel of I-Ed-restricted, myoglobin-reactive T cell clones. Acquisition of these data relied in part upon a modification of the polymerase chain reaction that uses a degenerate, consensus primer to amplify TCR alpha chains without foreknowledge of the V alpha segments they utilize.

HPV late gene expression is initiated as an infected basal cell migrates through the differentiating layers of the epidermis, resulting in the onset of vegetative viral DNA replication and the expression of viral late proteins. We have used a large synthetic immunoglobulin library displayed on phage (diversity 6.5 x 10(10) phage) to isolate three Fabs (TVG405, 406, and 407) which recognize distinct epitopes on the E4 late protein of HPV16. A C-terminal monoclonal (TVG404) was generated by hybridoma technology, and N-terminal polyclonal antiserum was prepared by peptide immunization (alpha N-term). The most potent antibody (TVG405) had an affinity for E4 of approximately 1.0 nM. All antibodies recognized the protein in paraffin-embedded archival material, allowing us to map events in the late stages of virus infection. Expression of E4 in vivo does not coincide with synthesis of the major virus coat protein L1, but precedes it by 1 or 2 cell layers in premalignant lesions caused by HPV16 and by up to 20 cell layers in HPV63-induced warts. In higher grade lesions associated with HPV16, E4 is produced in the absence of L1. By contrast, vegetative viral DNA replication and E4 expression correlate exactly and in some lesions begin as the infected epithelial cell leaves the basal layer. Differentiation markers such as filaggrin, loricrin, and certain keratins are not detectable in E4-positive cells, and nuclear degeneration is delayed. HPV16 E4 has a filamentous distribution in the lower epithelial layers, but associates with solitary perinuclear structures in more differentiated cells. Antibodies to the N-terminus of the protein stained these structures poorly. Our findings are compatible with a role for the HPV16 E4 protein in vegetative DNA replication or in modifying the phenotype of the infected cell to favor virus synthesis or virus release. The Fabs will be of value in the evaluation of model systems for mimicking HPV infection in vitro.

We have used the gel retardation assay to investigate the binding of nuclear proteins to the domain B1 of the SV40 enhancer, which contains the GT-II motif. Four proteins (GT-IIA, GT-IIB alpha, GT-IIB beta and GT-IIC) were detected, three of which were present in nuclear extracts from several cell lines. The fourth protein (GT-IIC) showed a clear cell-specificity, being absent from the lymphoid cell extracts tested. The results of methylation interference assays and of the binding of the proteins to mutated templates indicate that the domain B1 contains three distinct, but overlapping, protein-binding motifs (GT-IIA, B and C). The cell-specific binding of protein GT-IIC in vitro correlates with the in vivo enhancer activity of its cognate motif, strongly suggesting that this protein acts as a positive trans-acting enhancer factor. Two of the proteins also recognize other enhancer motifs; protein GT-IIB alpha binds to the microE3 motif present in the immunoglobulin heavy chain enhancer; protein GT-IIC binds to an enhancer motif of the polyomavirus mutant PyEC9.1 adapted to growth in F9 embryonal carcinoma cells, but not to the corresponding wild-type sequence.

The organization of mouse immunoglobulin heavy chain genes has been investigated by hybridization with cloned mu and alpha cDNA probes. Restriction endonuclease fragments bearing mu and alpha constant region genes and two types of variable region (VH) genes were compared in BALB/c embryos, liver and nine plasmacytomas synthesizing IgM, IgA, IgG1, IgG2a, IgG2b and IgG3. Embryo DNA was found to contain a single copy of the C mu gene per haploid genome. In contrast, one VH probe (HPC 76) detected at least six related VH genes, while the other (S107) detected a separate set of at least four genes, indicating that the germline contains distinct sets of multiple related VH genes. Most VH genes within the two subsets remained in germline context in different plasmacytomas, providing no evidence for somatic reassortment of VH genes. One plasmacytoma was devoid of specific VH genes, including some related to the expressed VH sequence. This may mean that the translocation event creating an active heavy chain gene involves deletion of the DNA between the expressed VH and CH sequences. The context of C mu sequences in DNA from a plasmacytoma secreting IgM differed from that in embryo DNA, as did C alpha sequences in two IgA- and several IgG-secreting plasmacytomas. Unlike heavy chain expression, rearrangement was not confined to one allele and often took different forms within a single cell line, presumably varying on different homologous chromosomes. Each rearrangement, whether resulting in an active C gene or not, appeared to change sequences upstream but not downstream from the CH gene. Significantly, the eight IgG and IgA plasmacytomas examined had undergone deletions of at least half and often all C mu sequences while retaining the embryo level of C alpha sequences. Hence a deletion mechanism may be responsible for the switch in expression from one CH gene to another which occurs during differentiation of a lymphocyte clone.

BACKGROUND

Liver biopsy is currently the gold standard for assessing liver fibrosis, and no reliable noninvasive diagnostic approach is available. Therefore a suitable serologic biomarker of liver fibrosis is urgently needed.

METHODS

We used a proteomics method based on 2-dimensional gel electrophoresis to identify potential fibrosis biomarkers. Serum samples from patients with varying degrees of hepatic scarring induced by infection with the hepatitis C virus (HCV) were analyzed and compared with serum from healthy controls.

RESULTS

We observed the most prominent differences when we compared serum samples from cirrhotic patients with healthy control serum. Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) fragments, alphaalphaalphaimmunoglobulin components increased with hepatic fibrosis, whereas haptoglobin and complement components (C3, C4, and factor H-related protein 1) decreased. Novel proteins associated with HCV-induced fibrosis included ITIH4 fragments, complement factor H-related protein 1, CD5L, Apo L1, beta2GPI, and thioester-cleaved products of a2M.

CONCLUSIONS

Assessment of hepatic scarring may be performed with a combination of these novel fibrosis biomarkers, thus eliminating the need for liver biopsy. Further evaluation of these candidate markers needs to be performed in larger patient populations. Diagnosis of fibrosis during early stages will allow early treatment, thereby preventing fibrosis progression.

OBJECTIVE

Activation-induced cytidine deaminase (AID) was originally identified as an inducer of somatic hypermutations in the immunoglobulin gene. We recently revealed that ectopic AID expression serves as a link between the cellular editing machinery and high mutation frequencies, leading to human cancer development. In the current study, we investigated whether AID might contribute to the development of colitis-associated colorectal cancers.

METHODS

The expression and regulation of AID in association with proinflammatory cytokine stimulation were investigated in cultured colonic cells. Genotoxic activity of AID in colonic cells was analyzed using retroviral system. Immunohistochemistry for AID was carried out on various human colonic tissues specimens.

RESULTS

Tumor necrosis factor-alpha induced aberrant AID expression via IkappaB kinase-dependent nuclear factor (NF)-kappaB-signaling pathways in human colonic epithelial cells. Moreover, AID expression was also induced in response to the T helper cell 2-driven cytokines interleukin-4 and interleukin-13, which are activated in human inflammatory bowel disease. Aberrant activation of AID in colonic cells preferentially induced genetic mutations in the TP53 gene, whereas there were no nucleotide alterations of the APC gene. Immunohistochemistry revealed enhanced expression of endogenous AID protein not only in the inflamed colonic mucosa of ulcerative colitis patients but also in tumor lesions of colitis-associated colorectal cancers.

CONCLUSIONS

Our findings indicate that proinflammatory cytokine-mediated aberrant expression of AID in colonic epithelial cells is a genotoxic factor linking inflammation, somatic mutations, and colorectal cancer development.

BACKGROUND

Aging is associated with increased inflammation and risk of community-acquired pneumonia. Streptococcus pneumoniae co-opts the nuclear factor kappa B (NFkB)-regulated proteins polymeric immunoglobulin receptor (pIgR) and platelet-activating factor receptor (PAFr) to attach and invade cells. We sought to determine whether aging and chronic inflammation were associated with increased pIgR and PAFr levels in the lungs and increased susceptibility to S. pneumoniae infection.

METHODS

Lung protein and messenger RNA levels were quantitated using Western blot and quantitative polymerase chain reaction. NFkB activation was measured by electrophoretic mobility shift assay. Cytokine levels were measured by cytometric bead analysis. To model chronic inflammation, mice were implanted with osmotic pumps that delivered tumor necrosis factor-alpha.

RESULTS

Aged mice and those infused with tumor necrosis factor-alpha had increased levels of pIgR and PAFr in their lungs and were more susceptible to S. pneumoniae infection. During pneumonia, aged mice had reduced levels of pIgR and PAFr and less NFkB activation, despite greater bacterial burden. We determined that aged mice had decreased amounts of lung Toll-like receptors 1, 2, and 4 and reduced capacity to respond to S. pneumoniae with proinflammatory cytokine production.

CONCLUSIONS

Aged mice and, potentially, elderly humans are more susceptible to pneumonia because of a priming effect of chronic inflammation and Toll-like receptor dysfunction.

Cancer metastasis poses the greatest challenge to the eradication of malignancy. The majority of clinical and experimental evidence indicates that metastasis is a non-random, organ-specific process. Tumor cell interaction with endothelium and subendothelial matrix constitutes the most crucial factor in determining the organ preference of metastasis. A plethora of cell surface adhesion molecules, which encompass four major families (i.e., integrins, cadherins, immunoglobulins and selectins) and many other unclassified molecules, mediate tumor-host interactions. Adhesion molecules and adhesion processes are involved in most, if not all, of the intermediate steps of the metastatic cascade. Decreased E-cadherin expression and increased CD44 expression are clearly correlated with the acquisition of the invasive capacity of primary tumor cells. Similarly, altered expression pattern of many other adhesion molecules such as upregulated expression of the laminin receptors and depressed expression of fibronectin receptors (alpha 5 beta 1) appears to be involved in tumor cell invasion into the subendothelial matrix. Tumor cell-endothelium interactions involve several well-defined sequential steps that can be analyzed by the 'Docking and Locking' hypothesis at the molecular level. Tumor cell-matrix interactions are determined by the repertoire of adhesion receptors of tumor cells and the unique composition of organ-specific matrices. Our experimental data, together with others', suggest that the integrin alpha IIb beta 3 is one of the major players in these tumor-host interactions. Tumor-host interaction is a dynamic process which is constantly modulated by a host of factors including various cytokines, growth factors and arachidonate metabolites such as 12(S)-HETE. Delineation of the molecular mechanisms of tumor-host interactions may provide additional means to intervene in the metastatic process.

The potential involvement of reactive oxygen species in the expression of genes involved in immune response was examined in mesangial cells. Tumor necrosis factor (TNF-alpha) and aggregated (aggr.) IgG increased mRNA levels for the monocyte chemoattractant protein, JE/MCP-1, and the colony-stimulating factor, CSF-1. Scavengers for free radicals such as di- and tetra-methylthiourea (DMTU and TMTU) attenuated the increase in mRNA levels in response to TNF-alpha and aggr. IgG. Generation of superoxide anion by xanthine oxidase and hypoxanthine increased mRNA levels of these genes, but exogenous H2O2 did not. Addition of NADPH to activate a membrane-bound NADPH-oxidase generated superoxide and caused a dose-dependent increase in mRNA levels and further enhanced the stimulation by TNF-alpha or aggr. IgG. An inhibitor of NADPH-dependent oxidase 4'-hydroxy-3'-methoxy-acetophenone attenuated the rise in mRNA levels in response to TNF-alpha and aggr. IgG. By nuclear run-on experiments TNF-alpha, aggr. IgG and NADPH increased the transcription rates for JE/MCP-1 and CSF-1, effects inhibited by TMTU. We conclude that generation of reactive oxygen species, possibly by NADPH-dependent oxidase, are involved in the induction of the JE/MCP-1 and CSF-1 genes by TNF-alpha and IgG complexes. The concerted expression of leukocyte-directed cytokines represents a general response to tissue injury.

Hepatocellular carcinoma (HCC) represents the fifth most common cancer in the world, and the third most frequent oncological cause of death. The incidence of HCC is on the increase. HCC typically develops in patients with chronic liver diseases, and cirrhosis, usually with viral etiology, is the strongest predisposing factor. Nowadays HCC diagnosis is a multistage process including clinical, laboratory, imaging and pathological examinations. The prognosis of HCC is mostly poor, because of detection at an advanced, non-resectable stage. Potentially curative treatment (surgery) is limited and really possible only for cases with small HCC malignancies. For this reason, more effective surveillance strategies should be used to screen for early occurrence of HCC targeted to the population at risk. So far, the generally accepted serological marker is alpha-fetoprotein (AFP). Its diagnostic accuracy is unsatisfactory and questionable because of low sensitivity, therefore there is a strong demand by clinicians for new HCC-specific biomarkers. In this review, we will focus on other biomarkers that seem to improve HCC diagnosis, such as AFP-L3, des-gamma-carboxyprothrombin, alpha-l-fucosidase, gamma-glutamyl transferase, glypican-3, squamous cell carcinoma antigen, a new generation of immunoglobulin M-immunocomplexes, and very promising gene-expression profiling.

OBJECTIVE

To determine the serum cytokine profiles of patients with spinal cord injury (SCI) and varying clinical presentations relative to healthy, able-bodied, age-matched control subjects.

METHODS

Cross-sectional study.

METHODS

Clinical research unit.

METHODS

People with SCI (N=56) and different clinical presentations, and healthy, able-bodied, age-matched control subjects (N=35).

METHODS

Not applicable.

METHODS

Serum levels of the proinflammatory cytokines interleukin (IL) 1beta, IL-6, tumor necrosis factor alpha (TNF-alpha), the anti-inflammatory cytokines IL-4 and IL-10, the regulatory cytokine IL-2, the IL-1 receptor antagonist (IL-1RA), and autoantibodies against myelin-associated glycoprotein and GM(1) ganglioside (anti-GM(1)) immunoglobulin (IgG and IgM), as determined by enzyme-linked immunosorbent assay. The relationship between elevated serum cytokine levels and clinical variables was also studied.

RESULTS

SCI subjects exhibited serum concentrations of IL-6, TNF-alpha, IL-1RA, and anti-GM(1) (IgG) that were greater (P<.05) than control group values. Elevated cytokine concentrations were not associated with high white blood cell counts, level of injury, or American Spinal Injury Association classification; they were evident in SCI subjects who were asymptomatic for medical complications, but were further elevated in subjects with pain, urinary tract infection (UTI), and pressure ulcers.

CONCLUSIONS

Elevated levels of circulating proinflammatory cytokines and autoantibodies are present in the serum of SCI subjects without medical complications, and are further elevated in SCI subjects with neuropathic pain, UTI, or pressure ulcers, relative to healthy, able-bodied control subjects. These findings may be indicative of a protective autoimmunity, simply a consequence of occult or evident infection, or evidence of cytokine dysregulation that may contribute to an immune-mediated impairment of axonal conduction.

Secretory immunoglobulin A (S-IgA) was investigated in human secretions for the presence of natural antibodies (Abs) acting as the first "immune barrier" to infection before induction or boosting of specific responses. These molecules could be the secretory counterpart of the natural Abs in serum that were previously shown by our laboratory to be polyreactive to autoantigens. Significant levels of S-IgA Abs to human actin, myosin, tubulin, and spectrin were detected in 10 saliva and 8 colostrum samples from normal subjects. Computer-assisted analysis of immunoblots of extracts from human muscles showed these Abs to react with a large number of autoantigens. Their polyreactivity was confirmed by cross-inhibition and by immunoblotting studies of affinity-purified natural Abs, assayed against a large variety of surface or secreted antigens from Streptococcus pyogenes. The thiocyanate elution method showed that functional affinities of some natural Abs can be of the same order of magnitude as those of tetanus vaccine antitoxins. Moreover, nonimmune binding of these natural Abs to the gut protein Fv (Fv-fragment binding protein) can enhance their effector functions. This demonstrates that human secretions contain polyreactive auto-Abs which can also react with pathogens. These secretory Abs of "skeleton key" specificities are possibly produced by a primordial B-1-cell-associated immune system and can be involved in a plurispecific mucosal protection against pathogens, irrespective of the conventional immune response.

BACKGROUND

Immunization with cardiac myosin induces experimental autoimmune heart disease in genetically predisposed mice. These mice produce heart-specific autoantibodies, some of which are directed against the cardiac myosin isoform.

RESULTS

We have reported the presence of circulating heart-specific autoantibodies in 26% of patients with idiopathic dilated cardiomyopathy (DCM) using indirect immunofluorescence. To identify the autoantigen(s) recognized by heart-specific autoantibodies in human disease, we tested, by Western blotting, sera from 26 DCM patients, 14 of whom were cardiac antibody-positive and 12 antibody-negative, as well as sera from 12 patients with cardiac failure from ischemic or valvular heart disease and from 13 normal subjects who were cardiac antibody-negative. Crude myofibrillar proteins and myosin preparations extracted from human atrial or ventricular specimens were used as antigens. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed. The proteins were electrophoretically transferred to nitrocellulose sheets. The paper strips were incubated in sera from patients or controls at 1:100 dilution; the reaction was revealed with a peroxidase-labeled second antibody against human immunoglobulin. Twelve of the 14 DCM sera (86%) containing heart-specific antibodies reacted with both the alpha- (atrial specific) and beta- (ventricular and slow skeletal) myosin heavy chain isoforms; none of the 13 normal sera (p = 0.0001) and one of the 24 heart failure-negative control sera (4%, p = 0.0001) contained antibodies against myosin heavy chain.

CONCLUSIONS

These findings indicate that alpha- and beta-cardiac myosin heavy chain isoforms as in the murine model of autoimmune heart disease are major autoantigens in patients with idiopathic DCM.

For millennia, food has been at the center of social events, in times of joy and in times of sorrow. Protein-energy malnutrition is associated with a significant impairment of cell-mediated immunity, phagocyte function, complement system, secretory immunoglobulin A antibody concentrations, and cytokine production. Deficiency of single nutrients also results in altered immune response: this is observed even when the deficiency state is relatively mild. Of the micronutrients, zinc, selenium, iron, copper, vitamins A, C, E and B(6), and folic acid have important influences on immune responses. Overnutrition and obesity also reduce immunity. Low-birth-weight infants have a prolonged impairment of cell-mediated immunity that can be partly restored by providing extra amounts of dietary zinc. In the elderly, impaired immunity can be enhanced by modest amounts of a combination of micronutrients. These findings have considerable practical and public health significance.

This study examined the interaction of a human salivary low-molecular-weight mucin (MG2) with Staphylococcus aureus and Pseudomonas aeruginosa by using both solution-phase and solid-phase assays. In solution phase, MG2 in human submandibular-sublingual saliva (HSMSL) bound to the bacterial surface; however, the highly purified mucin isoforms (MG2a and MG2b) did not. Mucin binding appeared to be dependent on heterotypic complexing between MG2 and secretory immunoglobulin A (IgA), although other salivary molecules may also be involved. In contrast, in a solid-phase assay in which HSMSL, MG2-containing fractions with secretory IgA, and purified MG2 were immobilized onto a solid surface, there was minimal adherence of S. aureus. The collective results suggest that mucin binding to S. aureus and P. aeruginosa may be predicated on the formation of an MG2-secretory IgA complex. Such interactions may facilitate microbial clearance from the oral cavity and play an important role in preventing colonization of the oral cavity and the respiratory tract by potential pathogens.