Point-of-care oral-based diagnostics.

Many of the target molecules that reside in blood are also present in oral fluids, albeit at lower concentrations. Oral fluids are, however, relatively easy and safe to collect without the need for specialized equipment and training. Thus, oral fluids provide convenient samples for medical diagnostics. Recent advances in lab-on-a-chip technologies have made minute, fully integrated diagnostic systems practical for an assortment of point-of-care tests. Such systems can perform either immunoassays or molecular diagnostics outside centralized laboratories within time periods ranging from minutes to an hour. The article briefly reviews recent advances in devices for point-of-care testing with a focus on work that has been carried out by the authors as part of a NIH program.

Antiviral activities in human saliva.

In this review, the authors survey the large number of antibacterial and antiviral proteins present in human saliva. Of interest, most of these antibacterial proteins display antiviral activity, typically against specific viral pathogens. The review focuses on one protein that interacts with both bacteria and viruses-gp340, originally referred to as salivary agglutinin. In the oral cavity, soluble gp340 binds to and aggregates a variety of bacteria, and this is thought to increase bacterial clearance from the mouth. However, when bound to the tooth surface, gp340 promotes bacterial adherence. In the oral cavity, most gp340 is found soluble in saliva and can function as a specific inhibitor of infectivity of HIV-1 and influenza A. In contrast, in the female reproductive track, most gp340 is bound to the cell surface, where it can promote HIV-1 infection.

Structural and Functional Characteristics of the Microbiome in Deep-Dentin Caries.

Dental caries is a cariogenic bacteria-mediated, fermentable carbohydrate-driven dynamic disease. The new ecological hypothesis for dentin caries suggests that an alteration in the microbial community and the presence of specific metabolic pathway genes contribute to the initiation and progression of caries. This study aimed to determine the structural and functional characteristics of a microbial community of human deep-dentin carious lesions. Sixteen deep-dentin carious lesions were obtained from the first permanent molars of 8 patients aged 9 to 18 y. Shotgun metagenomic sequencing was used to measure the microbial composition and abundance at the phylum, class, order, family, genus, and species levels. Functional analysis of the DNA sequencing data set was also performed and compared among different layers of the lesions using DIAMOND software against the Kyoto Encyclopedia of Genes and Genomes database. This study found that in the deep-dentin carious lesions, Actinobacteria (35.8%) and Firmicutes (31.2%) were the most prevalent phyla, followed by Bacteroidetes (13.6%), Proteobacteria (3.6%), and Fusobacteria (2.5%). The microbial composition varied among the individuals, but there were no significant differences in the distribution of the relative microbial abundance between the superficial layers and the deep layers. Although 14.5% of the top 10 taxa were identified as Lactobacillus at the genus level, only 25% of the deep-dentin carious samples showed Lactobacillus as the most abundant genus. Other abundant taxa included Actinomyces (10.5%), Olsenella (9.4%), Prevotella (8.8%), Propionibacterium (7.2%), Streptococcus (3.9%), Selenomonas (3.7%), Corynebacterium (1.9%), Leptotrichia (1.4%), and Parascardovia (1.1%). The most abundant pathway identified in the KEGG database was the metabolic pathway containing 101,427 annotated genes, which consisted of 51.4% of all annotated genes. The carbohydrate metabolism pathway, amino acid metabolism, and membrane transport were the functional traits of the level 2 pathways. These findings suggest that the potent interaction within the microbial communities in deep-dentin carious lesions may play a fundamental role in caries etiology.

Transcription factor ERG variants and functional diversification of chondrocytes during limb long bone development.

During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27-amino acid segment located approximately 80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes.

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

A model of decreased functional alpha-1-proteinase inhibitor. Pulmonary pathology of dogs exposed to chloramine T.

The objective of this study was to develop an animal model representative of chronic human alpha-1-proteinase inhibitor deficiency. Eight dogs were treated with a mild oxidizing agent, chloramine T, with varying regimens for 3--27 wk. The capacity of the serum to inhibit both trypsin and elastase was examined and found to respond differently. Although immunologically determined levels of protease inhibitor did not change, the ability of serum to inhibit elastase in an in vitro assay decreased in direct response to chloramine T treatment. The trypsin inhibitory capacity was less affected. Emphysemalike alterations in lung morphology were observable when histologic sections were evaluated both subjectively and objectively by mean linear intercept measurements. The data suggest that this model parallels the emphysema associated with the genetic alpha-1-proteinase inhibitor deficiency in man.

Immunolocalization of elastase in human emphysematous lungs.

The current working hypothesis concerning the pathogenesis of human pulmonary emphysema proposes that neutrophils migrate through the alveolar interstitium and degranulate, releasing proteolytic enzymes into the interstitium. These enzymes, in particular elastase, can bind to and degrade interstitial elastin. This report describes an immunohistochemical, ultrastructural technique that utilizes polyclonal antibodies to localize neutrophil elastase in human lungs. Using both the immunoperoxidase and the immunogold methods on thin, embedded sections of surgically resected human emphysematous lung tissue, elastase was localized in neutrophils in the lung interstitium and extracellularly in association with interstitial elastic fibers in human lungs that showed local emphysema of varying severity. Quantitative morphometric data were obtained from the lungs of eight patients undergoing lobectomy for removal of pulmonary carcinomas. Patients had preoperative forced expiratory volume (FEV1)% levels ranging from 55 to 77. There was a correlation between a quantitative measure of the local distribution of neutrophil elastase in contact with alveolar interstitial elastin and the local presence of emphysematous change as determined by mean linear intercept of the various histologic sections. These data support the validity of the "protease-protease inhibitor balance hypothesis" as an explanation of the pathogenesis of human pulmonary emphysema.

Role of protein kinase C-delta in the regulation of collagen gene expression in scleroderma fibroblasts.

Working with cultured dermal fibroblasts derived from control individuals and patients with systemic sclerosis (SSc), we have examined the effects of protein kinase C-delta (PKC-delta) on type I collagen biosynthesis and steady-state levels of COL1A1 and COL3A1 mRNAs. Rottlerin, a specific inhibitor of PKC-delta, exerted a powerful, dose-dependent inhibition of type I and type III collagen gene expression in normal and SSc cells. Optimal rottlerin concentrations caused a 70-90% inhibition of type I collagen production, a >80% reduction in COL1A1 mRNA, and a >70% reduction in COL3A1 mRNA in both cell types. In vitro nuclear transcription assays and transient transfections with COL1A1 promoter deletion constructs demonstrated that rottlerin profoundly reduced COL1A1 transcription and that this effect required a 129-bp promoter region encompassing nucleotides -804 to -675. This COL1A1 segment imparted rottlerin sensitivity to a heterologous promoter. Cotransfections of COL1A1 promoter constructs with a dominant-negative PKC-delta expression plasmid showed that suppression of this kinase silenced COL1A1 promoter activity. The results indicate that PKC-delta participates in the upregulation of collagen gene transcription in SSc and suggest that treatment with PKC-delta inhibitors could suppress fibrosis in this disease.

Restricted VH gene usage by murine hybridomas directed against the human N, but not M, blood group antigen.

The M and N human blood group antigens are complex glycopeptide determinants at the amino terminus of the red blood cell membrane glycoprotein, glycophorin A. The heavy and light chain variable region cDNA sequences were determined for seven murine monoclonal antibodies recognizing glycophorin A. Three of the antibodies were anti-M and four were anti-N. Each of the anti-M antibodies was composed of VH and VL regions derived from distinct germline gene families (VH1 (J558), VH4 (X24), VH5 (7183), VK5, VK8, and VK19). In contrast, all four anti-N heavy chains were composed of VH regions derived from the VH2 (Q52) germline gene family and all used the same J4 gene segment. In addition, two of the anti-N light chains were composed of VK regions from the VK8 germline gene family and used the J1 gene segment. Since each anti-N hybridoma was derived from different mice immunized by different protocols, these results suggest that the murine immune response to the N, but not the M, human blood group antigen is restricted.

Analysis of the human gene encoding latent transforming growth factor-beta-binding protein-2.

Transforming growth factor (TGF)-beta is secreted as an inactive complex, which frequently contains a large molecular weight binding protein designated latent TGF-beta-binding protein (LTBP). Recently, the LTBPs have been shown to be a gene family that contains three known members and exhibits a multidomain structure containing cysteine-rich motifs that are also found in the fibrillin gene family. The present work seeks to characterize the gene encoding LTBP-2 and to compare its features to that of the other LTBPs and to the fibrillins. Human fibroblast libraries were used to isolate cDNA encoding LTBP-2 which was then used to identify LTBP-2 transcripts and to isolate the corresponding LTBP-2 gene. The cloned cDNA encodes a 195 kDa protein containing 20 epidermal growth factor (EGF)-like repeats, three repeats containing eight cysteines, and one segment that appears to be a hybrid of the two. Single exons encode EGF repeats while the eight-cysteine repeats are encoded in two exons. Northern analysis identified two transcripts of 7.5 and 9.0 kb, with the presently analyzed cDNA probably corresponding to the 7.5 transcript. Phylogenetic sequence comparisons demonstrated that LTBP-3 is more similar to LTBP-1 than LTBP-2, while LTBP-2 shows the most similarity to the fibrillins. These analyses suggest that LTBP-1 diverged from LTBP-3, and that LTBP-2 diverged from LTBP-1. Within the fibrillin family, fibrillin-1 is nearest to the LTBPs. While the domain structure of LTBP-2 is similar to that of the other LTBPs, LTBP-2 possesses unique regions that make it the largest member of the LTBP family. LTBP-2 may have dual functions as a member of the TGF-beta latent complex and as a structural component of microfibrils.

Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.

Comparison of upstream regions of X- and Y-chromosomal amelogenin genes.

The amelogenin genes encode abundant enamel proteins that are required for the development of normal tooth enamel. These genes are active only in enamel-forming ameloblasts within the dental organ of the developing tooth, and are part of a small group of genes that are active on both sex chromosomes. The upstream regions of the bovine X- and Y-chromosomal and the sole murine X-chromosomal amelogenin genes have been cloned and sequenced, and conservation at nearly 60% is found in the 300 bp upstream of exon 1 for the 3 genes. A region of the bovine X-chromosomal gene that has inhibitory activity when assayed by gene transfer into heterologous cells includes motifs that have a silencing activity in other genes, and may be important to the mechanism that represses amelogenin expression in non-ameloblast cells in vivo. A comparison of sequences from three genes has led to the identification of several regions with conserved motifs that are strong candidates for having positive or negative regulatory functions, and these regions can now be tested further for interaction with nuclear proteins, and for their ability to regulate expression in vivo.

Tropoelastin binding to fibulins, nidogen-2 and other extracellular matrix proteins.

Elastic fibers in vessel walls and other tissues consist of cross-linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (K(d) about 1 nM) of tropoelastin for microfibrillar fibulin-2 and the recently described nidogen-2 isoform. More moderate affinities were observed for fibulin-1, laminin-1 and perlecan, while several other ligands such as collagens, nidogen-1, fibronectin and BM-40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin-2 and nidogen-2, while the reaction with fibulin-1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions.

Amino acid derived latent isocyanates: irreversible inactivation of porcine pancreatic elastase and human leukocyte elastase.

Several amino acid derived azolides (I) have been synthesized and investigated for their inhibitory activity toward human leukocyte elastase and porcine pancreatic elastase. The inhibitory activity was found to be dependent on the nature of the precursor amino acid ester. Thus, compounds derived from L-valine methyl ester 3, L-norvaline methyl ester 5, DL-norleucine methyl ester 9, and L-methionine methyl ester 10 were found to inhibit irreversibly both enzymes. Compound 10 was found to be a specific and selective inhibitor of human leukocyte elastase. In contrast to these, inhibitors derived from glycine methyl ester 1, D-valine methyl ester 4, and D-norvaline methyl ester 6 were found to be inactive. The results of the present study show that latent isocyanates derived from appropriate amino acids can serve as selective inhibitors of serine proteases and are of potential pharmacological value.

A flow cytometric assay of neutrophil degranulation.

A quantitative assay of neutrophil degranulation was developed using flow cytometry. Dog neutrophils were purified to greater than 95% purity and viability by isopyknic density centrifugation in an isosmotic medium. These cells concentrated the fluorochrome acridine orange (AO) in their azurophilic granules, but not in specific granules. Also contained in the azurophilic granules are elastase, myeloperoxidase, and approximately 50% of the lysozyme activity. The fluorochrome was released concomitantly with elastase activity, as shown by flow cytometry, fluorescence microscopy, and biochemical assay in response to the ionophore A23187. By flow cytometry, unstimulated cells are distributed in a single broad peak of high fluorescence intensity. With increasing concentrations of A23187 (0.48-4.80 microM), a greater proportion of the cells shifted to a single peak of low fluorescence intensity. Few cells with intermediate fluorescence were observed. These analyses revealed that the neutrophils degranulated in a quantal, all-or-none response.

Expression of microfibrillar proteins by bovine bladder urothelium.

OBJECTIVES: To determine the occurrence and potential function of proteins composing elastic microfibrils in the developing bovine bladder. METHODS: Monospecific antibodies, generated against two well-characterized microfibrillar proteins, microfibril-associated glycoprotein (MAGP) and fibrillin-1 (FBN1), were used in immunohistochemical analysis of full-thickness frozen sections of fetal bovine bladder. The localization of these two antibodies was compared with that of anti-type IV collagen antibody. Adjacent serial sections were stained for routine light microscopy. Cultured urothelial cells were fixed in 3.7% formaldehyde and permeabilized with 0.5% Triton X-100 before immunoanalysis. Control reactions used either preimmune serum or a monoclonal antibody to a nonmatrix protein. Poly(A+) ribonucleic acid was isolated from cultured urothelial cells and subjected to Northern analysis using specific complementary deoxyribonucleic acid probes for MAGP and FBN1. RESULTS: Both MAGP and FBN1 are expressed by the urothelium and are found in association with the underlying basement membrane, as visualized by their co-localization with type IV collagen. Furthermore, urothelial cells in culture continue to express both microfibrillar proteins. CONCLUSIONS: The developing bovine urothelium expresses major microfibrillar protein components. The role of these microfibrils in the urothelium remains to be determined, but they may have an important anchoring function.

Submandibular salivary proteases: lack of a role in anti-HIV activity.

Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity. However, less information is available regarding proteases produced by salivary glands and present in salivary secretions. In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities. These have been characterized in terms of their susceptibility to a series of protease inhibitors. The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors. We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva. This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid. Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity.

Molecular cloning and characterization of the bovine tuftelin gene.

The bovine tuftelin gene was cloned and its structure determined by DNA sequence analysis and comparison to that of bovine tuftelin cDNA. The analyses demonstrated that the cDNA contains a 1014-bp open reading frame encoding a protein of 338 residues with a calculated mol. wt of 38,630 and an isoelectric point of 5.85. These results differ from those previously published, (1991) which contained a different conceptual amino acid sequence for the carboxy terminal region and identified a different termination codon. The protein does not appear to share homology or domain motifs with any other known protein. The gene consists of 13 exons ranging in size from 66 to 1531 bp, the latter containing the encoded carboxyterminal and 3' untranslated regions. The exons are embedded in more than 28 kbp of genomic DNA. Codons are generally not divided at exon/intron borders. Several alternatively spliced transcripts were identified by DNA sequence analysis of the isolated products produced by reverse transcriptase/polymerase chain reaction.

A new frameshift mutation encoding a truncated amelogenin leads to X-linked amelogenesis imperfecta.

The amelogenin proteins are the most abundant organic components of developing dental enamel. Their importance for the proper mineralization of enamel is evident from the association between previously identified mutations in the X-chromosomal gene that encodes them and the enamel defect amelogenesis imperfecta. In this investigation, an adult male presenting with a severe hypoplastic enamel phenotype was found to have a single base deletion at the codon for amino acid 110 of the X-chromosomal 175-amino acid amelogenin protein. The proband's mother, who also has affected enamel, carries the identical deletion on one of her X-chromosomes, while the father has both normal enamel and DNA sequence. This frameshift mutation deletes part of the coding region for the repetitive portion of amelogenin as well as the hydrophilic tail, replacing them with a 47-amino acid segment containing nine cysteine residues. While greater than 60% of the protein is predicted to be intact, the severity of this phenotype illustrates the importance of the C-terminal region of the amelogenin protein for the formation of enamel with normal thickness.