Salivary (SD-type) cystatins: over one billion years in the making--but to what purpose?

Human saliva contains relatively abundant proteins that are related ancestrally in sequence to the cystatin superfamily. Most, although not all, members of this superfamily are potent inhibitors of cysteine peptidases. Four related genes have been identified, CST1, 2, 4 and 5, encoding cystatins SN, SA, S, and D, respectively. CST1, 4, and probably CST5 are now known to be expressed in a limited number of other tissues in the body, primarily in exocrine epithelia, and the term SD-type cystatin is more appropriate than 'salivary cystatin'. These genes are co-ordinately regulated in the submandibular gland during post-natal development. The organization of these tissue-specifically-expressed genes in the genome, and their phylogeny, indicate that they evolved from an ancestral housekeeping gene encoding the ubiquitously expressed cystatin C, and are members of a larger protein family. Their relationship to rat cystatin S, a developmentally regulated rodent submandibular gland protein, remains to be established. In this review, the evolution of the SD-type cystatins in the cystatin superfamily, their genomics, expression, and structure-function relationships are examined and compared with known cystatin functions, with the goal of providing clues to their biological roles.

Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease.

Cysteine peptidases (CPs) are phylogenetically ubiquitous enzymes that can be classified into clans of evolutionarily independent proteins based on the structural organization of the active site. In mammals, two of the major clans represented in the genome are: the CA clan, whose members share a structure and evolutionary history with papain; and the CD clan, which includes the legumains and caspases. This review focuses on the properties of these enzymes, with an emphasis on their potential roles in the oral cavity. The human genome encodes at least (but possibly no more than) 11 distinct enzymes, called cathepsins, that are members of the papain family C1A. Ten of these are present in rodents, which also carry additional genes encoding other cathepsins and cathepsin-like proteins. Human cathepsins are best known from the ubiquitously expressed lysosomal cathepsins B, H, and L, and dipeptidyl peptidase I (DPP I), which until recently were considered to mediate primarily "housekeeping" functions in the cell. However, mutations in DPP I have now been shown to underlie Papillon-Lefevre syndrome and pre-pubertal periodontitis. Other cathepsins are involved in tissue-specific functions such as bone remodeling, but relatively little is known about the functions of several recently discovered enzymes. Collectively, CPs participate in multiple host systems that are active in health and in disease. They are involved in tissue remodeling and turnover of the extracellular matrix, immune system function, and modulation and alteration of cell function. Intracellularly, CPs function in diverse processes including normal protein turnover, antigen and proprotein processing, and apoptosis. Extracellularly, they can contribute directly to the degradation of foreign proteins and the extracellular matrix. However, CPs can also participate in proteolytic cascades that amplify the degradative capacity, potentially leading to pathological damage, and facilitating the penetration of tissues by cancer cells. We know relatively little regarding the role of human CPs in the oral cavity in health or disease. Most studies to date have focused on the potential use of the lysosomal enzymes as markers for periodontal disease activity. Human saliva contains high levels of cystatins, which are potent CP inhibitors. Although these proteins are presumed to serve a protective function, their in vivo targets are unknown, and it remains to be discovered whether they serve to control any human CP activity.

Transforming growth factor beta 1 dysregulation in a human oral carcinoma tumour progression model.

A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.

Expression of type 2 cystatin genes CST1-CST5 in adult human tissues and the developing submandibular gland.

Type 2 cystatins comprise a class of cysteine peptidase inhibitor presumed to mediate protective functions at various locations, including the oral cavity. Seven cystatin genes are clustered within a 300-kb region of human 20p11.2. "Salivary" cystatins, encoded by CST1, 2, 4, and 5, are present in saliva at significant levels but have also been reported in other secretions, such as tears, suggesting that during their evolution, these genes have acquired mechanisms directing differential tissue-specific expression. However, their patterns of expression, which might also provide additional clues to their individual functions, have not been determined. Gene-specific RNase protection assays were used to examine the qualitative and quantitative distribution of expression of these seven genes within a collection of 23 adult human tissues. The CST3 gene, encoding cystatin C, was expressed at modest levels in all tissues examined. The presumptive pseudogenes CSTP1 and CSTP2 were not expressed at detectable levels in any tissue. The CST1, 2, 4, and 5 genes were expressed in differential, tissue-specific patterns. Expression of CST2 and CST5 was restricted to the submandibular and parotid glands, while CST1 and CST4 were expressed in these tissues and in the lacrimal gland. Immunohistochemistry studies localized expression to the serous-type secretory end pieces. Coexpression of CST1 and CST4 was also observed in the epithelial lining of the gallbladder and seminal vesicle. The CST1 product was detected in the tracheal glands and CST4 in the kidney and prostate. Despite their different adult patterns of expression, analysis of CST1, 2, 4, and 5 mRNA levels in infant submandibular glands demonstrated a coordinate upregulation of expression of between 3.5 and 9 months of age. The patterns of cystatin gene expression are consistent with several proposed oral functions of the salivary cystatins but also suggest they are important in other locations and that, despite their close sequence similarity, they are individually specialized.

Isolation of Cladonema Pax-B genes and studies of the DNA-binding properties of cnidarian Pax paired domains.

Pax genes encode nuclear transcription factors that are involved in developmental control. They contain a conserved DNA-binding domain, the paired domain. The DNA-binding specificity of paired domains is directly related to the gene regulation function of Pax proteins. Pax genes were previously divided into five groups on the basis of a phylogenetic analysis of paired domains. In this study, two highly similar cnidarian Pax-B genes from Cladonema californicum, a jellyfish with eyes, were found and sequenced. In an effort to understand the function of the cnidarian Pax genes isolated in this and a previous study, we characterized the consensus DNA sequences bound by the cnidarian paired domains using a PCR-based method and electrophoretic mobility shift assays. The consensus DNA sequences obtained are very similar to those bound by mammalian Pax proteins. Comparison of known consensus sequences indicates that they are all partially palindromic, but this characteristic is most prominent in cnidarians, which suggests that the DNA sequences bound by the ancestral paired domain could have been palindromic. Also, cnidarian paired domains, like those of Pax-2/5/8, possess a broader binding specificity than other paired domains, which implies that the common ancestor of Pax-2/5/8 and Pax-4/6 paired domains could also have had a similar broad DNA-binding specificity. Thus far, a definitive Pax-6 gene has not been found in several cnidarian species examined, which is consistent with a later origin of the Pax-6 gene and raises two possibilities: the Pax genes of cnidarians are multifunctional and control two or more developmental pathways, including eye development, or they use a Pax-independent pathway for eye development. Whether this pathway does exist and is unique to cnidarians or it whether it represents a true master control under which Pax-6 was later included remains to be determined.

Chemopreventive effects of green tea polyphenols correlate with reversible induction of p57 expression.

Green tea polyphenols are known to induce apoptosis in certain types of tumor cells. However, the mechanism(s) that enables normal cells to evade the apoptotic effect is still not understood. In this study, Western blot analysis combined with cycloheximide treatment was used to examine the effects of green tea polyphenols on the expression levels of p57, a cyclin-dependent kinase and apoptosis inhibitor, in normal human keratinocytes and in the oral carcinoma cell lines SCC25 and OSC2. The results showed that the most potent green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), induced p57 in normal keratinocytes in a dosage- and time-dependent manner, while the levels of p57 protein in oral carcinoma cells were unaltered. The differential response in p57 induction was consistent with the apoptosis status detected by annexin V assay. The data suggest that the chemopreventive effects of green tea polyphenols may involve p57-mediated cell cycle regulation in normal epithelial cells.

Evolution of paired domains: isolation and sequencing of jellyfish and hydra Pax genes related to Pax-5 and Pax-6.

Pax proteins are a family of transcription factors with a highly conserved paired domain; many members also contain a paired-type homeodomain and/or an octapeptide. Nine mammalian Pax genes are known and classified into four subgroups: Pax-1/9, Pax-2/5/8, Pax-3/7, and Pax-4/6. Most of these genes are involved in nervous system development. In particular, Pax-6 is a key regulator that controls eye development in vertebrates and Drosophila. Although the Pax-4/6 subgroup seems to be more closely related to Pax-2/5/8 than to Pax-3/7 or Pax-1/9, its evolutionary origin is unknown. We therefore searched for a Pax-6 homolog and related genes in Cnidaria, which is the lowest phylum of animals that possess a nervous system and eyes. A sea nettle (a jellyfish) genomic library was constructed and two pax genes (Pax-A and -B) were isolated and partially sequenced. Surprisingly, unlike most known Pax genes, the paired box in these two genes contains no intron. In addition, the complete cDNA sequences of hydra Pax-A and -B were obtained. Hydra Pax-B contains both the homeodomain and the octapeptide, whereas hydra Pax-A contains neither. DNA binding assays showed that sea nettle Pax-A and -B and hydra Pax-A paired domains bound to a Pax-5/6 site and a Pax-5 site, although hydra Pax-B paired domain bound neither. An alignment of all available paired domain sequences revealed two highly conserved regions, which cover the DNA binding contact positions. Phylogenetic analysis showed that Pax-A and especially Pax-B were more closely related to Pax-2/5/8 and Pax-4/6 than to Pax-1/9 or Pax-3/7 and that the Pax genes can be classified into two supergroups: Pax-A/Pax-B/Pax-2/5/8/4/6 and Pax-1/9/3/7. From this analysis and the gene structure, we propose that modern Pax-4/6 and Pax-2/5/8 genes evolved from an ancestral gene similar to cnidarian Pax-B, having both the homeodomain and the octapeptide.

cDNA cloning of an abundant human lacrimal gland mRNA encoding a novel tear protein.

An abundant 1.05 kb human lacrimal gland mRNA has been characterized by cDNA cloning. It encodes a predicted 180 residue, 20546 Da secreted protein, with a charge of +11 at ph 7 and 24.5% proline, designated as Basic Proline-rich Lacrimal Protein (BPLP), Southern blot analysis is consistent with a single BPLP gene. BPLP lacks any distinct repetitive structure, and is unrelated to the salivary proline-rich protein super-family. The pre-proprotein shows modest overall similarity to a superfamily comprising human PRPb, the mouse MSG proteins, and rat VCS-alpha 1, VCS-beta 1 and submandibular apomucin. BPLP also contains a domain with similarity to the Zp2 protein domain found in several otherwise unrelated proteins. Northern blot analysis indicated that the BPLP gene is also expressed at modest levels in the human submandibular gland, and in situ hybridization demonstrated expression of BPLP in the secretory endpieces of the human lacrimal gland. The BPLP cDNA clone defines a new human tear protein, and should provide a useful phenotypic marker of differentiation in in vitro studies of lacrimal gland function.

A major human lacrimal gland mRNA encodes a new proline-rich protein family member.

PURPOSE: To examine the existence of novel protein products of the human lacrimal gland. METHODS: cDNA clones corresponding to a highly abundant human lacrimal gland mRNA were isolated and sequenced. Tissue distribution of expression was studied by Northern blot analysis, RNase protection analysis, and in situ hybridization. RESULTS: A highly abundant 600-base mRNA was identified, and corresponding cDNA clones were isolated. The mRNA has a 134-residue open reading frame encoding a secreted protein of 13458 Da. This protein shows 45.5% similarity to human salivary acidic proline-rich protein PRP 1 and has a similar domain structure, but, unlike other members of the proline-rich protein family, it lacks a conserved repetitive domain. The lacrimal proline-rich protein is encoded by a single gene, designated as LPRP. Expression of LPRP was also detected in the human submandibular, von Ebners, sublingual, and parotid glands. LPRP was expressed in the acinar cells of the lacrimal gland, and in the submandibular gland expression of the LPRP and PRP 1 genes was localized to the serous acini and demilunes. CONCLUSIONS: The human lacrimal gland produces a previously unknown member of the proline-rich protein family. By analogy with other proline-rich proteins, this LPRP most likely mediates protective functions in the eye, such as modulation of the microflora. In contrast to other proline-rich protein genes, LPRP is expressed in the lacrimal acinar cells, and other anterior exocrine glands. LPRP should be a useful marker for human lacrimal gland acinar cell function in vitro.

Direct mapping of seven genes encoding human type 2 cystatins to a single site located at 20p11.2.

The human type 2 cystatins are encoded by a multigene family of eight to nine members. Five functional genes (CST1-CST5) and two pseudogenes (CSTP1 and CSTP2) have previously been cloned. We have developed a panel of short genomic fragments that serve as gene-specific probes for these seven genes under hybridization conditions used for Southern blots or fluorescence in situ hybridization (FISH). Analysis of BssHII-digested genomic DNA using these probes was consistent with association of all seven genes with a 905-kb fragment, suggesting physical clustering at a single site. The cluster was assigned to chromosome 20 by screening a somatic cell hybrid panel with an STS for CST2. This assignment was confirmed for each gene by FISH using repeat-free gene-specific genomic probes ranging in size from 2.3 to 0.8 kb. All the loci mapped to 20p11.2.

The human type 2 cystatin gene family consists of eight to nine members, with at least seven genes clustered at a single locus on human chromosome 20.

The family of type 2 cystatin proteins are a class of cysteine proteinase inhibitors found in a variety of human fluids and secretions, where they appear to provide protective functions. To establish the size of the human gene family encoding these proteins, we isolated cosmid and lambda genomic clones. Restriction mapping, partial sequence analysis, and hybridization studies identified a total of seven distinct genes, six of which correspond to known genes and proteins. Sequence analysis showed that the seventh gene, CSTP2, is an apparent pseudogene carrying a nonsense mutation in exon 1 distinct from that in CSTP1. Southern blots of genomic DNA probed with gene-specific probes accounted for all but one or two sets of fragments containing exon 1, and one or two sets of fragments containing exon 3, indicating that the human type 2 cystatin gene family consists of eight or nine members. Southern blot analysis of large restriction fragments using these gene-specific probes indicates that all seven of the cloned type 2 cystatin genes are clustered at a single locus on human chromosome 20. This locus is no larger than about 910 kb, and possibly as small as 365 kb. We designate this as the CST locus and suggest a numbering system for the cystatin genes.

Proteins with molecular masses of 50 and 80 kilodaltons encoded by genes downstream from the fimbrilin gene (fimA) are components associated with fimbriae in the oral anaerobe Porphyromonas gingivalis.

Flanking DNA regions of the fimbrilin gene (designated fimA), which encodes the major subunit protein of Porphyromonas (Bacteroides) gingivalis fimbriae, were cloned in several manners from the P. gingivalis chromosome into Escherichia coli by screening with probes derived from a 2.5-kb SacI DNA fragment previously cloned. A total of 10.4 kb of DNA fragments from the P. gingivalis genome was cloned in the pUC plasmid. Expression of the fimA gene and possible flanking genes in the fragments cloned was examined in a pUC plasmid vector system and in a bacteriophage T7 RNA polymerase-promoter expression vector system. The results show that in the pUC plasmid system, a 45-kDa protein, a product of fimA, was only poorly expressed as a precursor of the fimbrilin protein (FimA) and could be detected from cell extracts in Western blotting (immunoblotting) analysis as a sharp band but not in colony immunoblotting analysis. On the other hand, in the T7 RNA polymerase-promoter system, the product of fimA and products of the possible flanking genes responsible for fimbriation were overproduced as thick bands of the 45-kDa protein and as 63-, 50-, and 80-kDa proteins, respectively, in stained electrophoresis gels. All of the recombinant proteins were insoluble and seemed to be expressed as precursors with leader peptides. The 63-kDa, 45-k*Da (a truncated protein of the 50-kDa protein), and 80-kDa proteins were purified after solubilization with sodium dodecyl sulfate. N-terminal amino acid sequences of the 45-k*Da and 80-kDa proteins were analyzed up to the first 35 residues with a gas-phase sequencer. Monospecific antibodies directed to the recombinant proteins, i.e., the 63-kDa, 45-k*Da, and 80-kDa proteins, were raised in rabbits. By using the antibodies, localization of their matured proteins in P. gingivalis was investigated by Western blotting analysis. Immunoblotting analysis suggests that at least the 50- and 80-kDa proteins, encoded by genes downstream from the fimA gene, are minor components associated with fimbriae.

Genomic cloning, physical mapping, and expression of human type 2 cystatin genes.

Humans carry one gene encoding cystatin C and six to eight genes with homology to an S-like cystatin hybridization probe. However, the precise composition and organization of the cystatin gene family remains to be established. Further, the pattern of tissue-specific expression has not been fully defined. We have previously shown that the type 2 cystatin genes are clustered together in a ca. 270 kb region (the CST locus). To determine the structure of this region, we have sought to clone the entire CST locus. Our approach has been to isolate cosmid and lambda genomic clones carrying cystatin genes and then to use "walk" probes derived from the end regions of these clones to identify other clones, which extend them. To date, we have obtained over 320 kb of distinct sequences. Based on restriction maps, sequencing, and hybridization analyses, we have identified eight apparently nonallelic copies of cystatin genes. These include one gene for cystatin C, four closely related genes encoding S-like cystatins, and three genes encoding relatively divergent sequences. Complete assembly of these clones into an unambiguous contiguous sequence is hampered by the presence of flanking locus-specific repetitive-like sequences. RNase protection assays used to characterize the tissue-specific patterns of expression showed that cystatin C is expressed at modest, comparable levels in all tissues examined, whereas expression of the CST 1 gene, encoding cystatin SA-I, was found to be restricted to a small subset of tissues, with the highest level in the submandibular gland.(ABSTRACT TRUNCATED AT 250 WORDS)

High-yield recovery of recombinant DNA from poorly growing cosmid and lambda genomic clones.

Certain genomic sequences cannot be recovered efficiently in cosmid or lambda bacteriophage clones, presenting a barrier to efforts to construct a contiguous cloned library of a genome. We have encountered such sequences during our efforts to isolate cosmid and bacteriophage lambda clones carrying members of the human type 2 cystatin gene family. Several cosmid clones constructed in the pWE 15 vector did not survive purification, and using standard techniques, we were unable to obtain significant amounts of cosmid DNA from those clones we could purify. Similarly, several lambda bacteriophage clones constructed in the lambda DASH II vector could not be purified, and those lambda clones we were able to isolate gave low titers in liquid lysates. In this paper, we describe generally applicable methods for preparing high yields of recombinant DNA from such recalcitrant cosmid and lambda clones constructed in these vectors.

A statistical approach to the ecology of Porphyromonas gingivalis.

Previous studies have suggested that infections with Porphyromonas gingivalis, associated with periodontal disease, may consist of one clonal type. It has also been shown that each individual patient carries a unique clonal type of P. gingivalis, as assessed by DNA fingerprinting. This issue was further examined by random collection of multiple isolates of P. gingivalis from multiple sites in several patients, and characterization of these isolates by DNA fingerprinting. Although most patients appeared to be infected exclusively by one clonal type of P. gingivalis, at least one patient was found to harbor two distinct clonal types. This indicates that the simultaneous presence of different clonal types of P. gingivalis can occur. A statistical method was developed for retrospective analysis of these data for estimation of whether the remainder of these patients were actually infected with single or multiple clonal types of P. gingivalis. With this statistical method, a confidence interval was calculated for estimation of the true proportion of a single observed clonal type in the potential population of P. gingivalis that might be recovered from an infected patient. Statistically, the sampling of small numbers of sites per patient or isolates per site leads to a wide confidence interval for the estimated true proportion of the observed clonal type in the infecting P. gingivalis population. For example, when five sites in an oral cavity yield indistinguishable P. gingivalis isolates, then the true proportion of this clonal type in the total P. gingivalis population in the infected oral cavity is estimated to be in the interval between 55% and 100% (at a 95% confidence level).(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic heterogeneity of Porphyromonas (Bacteroides) gingivalis by genomic DNA fingerprinting.

This study describes the use of total genomic DNA fingerprinting with the use of restriction endonucleases to characterize clinical isolates of Porphyromonas gingivalis (Bacteroides gingivalis) obtained from patients with periodontitis or with root-canal infections. The majority of independent isolates had a unique DNA fingerprint, indicating extensive genetic heterogeneity within this species. Twenty-nine distinct DNA fingerprints were found among the 33 isolates investigated. This is in contrast to biotyping and serotyping, where only one type and three types, respectively, have been reported. The observed heterogeneity indicates that DNA fingerprinting is a sensitive measure of genetic dissimilarity between P. gingivalis isolates and is able to characterize individual isolates. These results have ecological implications, indicating that there is considerable natural diversity in the global population of P. gingivalis, and that there are likely to be relatively large numbers of genetically distinct clonal lines. Furthermore, DNA fingerprinting is a sensitive and powerful tool for longitudinal and cross-sectional epidemiological studies. This technique provides far greater discrimination between isolates than either biotyping or serotyping, and will be most helpful in, for example, the analysis of distribution of clonal lines within one periodontal patient, or the analysis of the transmission to and turnover of strain populations within a patient population, since the probability of two strains with the same DNA fingerprint being found by chance is small.

Expression of glutamine/glutamic acid-rich proteins in rat submandibular glands.

The expression of GRP transcripts was found to be highly specific to the rat submandibular gland. GRP cross-reactive species were detected in the saliva of both inbred and outbred rat strains. There was no evidence of GRP transcripts in RNA prepared from bovine, ovine, porcine or murine submandibular glands. Thus the GRPs differ from the family of PRPs that are expressed in several species. The restriction of GRP expression to the rat suggests a relatively recent origin for a functional GRP gene, presumably after rat-mouse divergence. The ontogeny of the relative steady-state levels of GRP transcripts was assessed by dot blot analysis. Maximal levels of RNA-encoding GRP were detected at 6 months; there was a significant age-related decline at both 12 and 18 months. There was, however, no significant age-related alteration in the size of transcripts which encode this protein family.

Genetic and tissue-specific variation in the expression of a closely linked murine multigene family on chromosome 15 that encodes salivary and lacrimal proteins.

The murine submandibular gland (SMG) produces a novel class of highly acidic salivary proteins encoded by one or more highly abundant mRNA transcripts. In inbred mice, these transcripts are encoded by members of a multigene family comprising approximately 8-12 homologues. Most, and probably all, of these homologues are clustered at a new locus near belted (bt) on chromosome 15, which we designate Spt (salivary protein). Although physically closely linked, Spt genes differ in their patterns of expression both in strains of mice and in their tissues. One gene, Spt-1, is expressed at high levels in the SMG of all inbred strains examined. This gene is also expressed at significant levels in the lacrimal gland. A second gene, Spt-2, appears to be present as a single copy in some strains and as two copies in others. This gene is expressed at high levels only in the SMG of those strains carrying two copies, and Spt-2 mRNA is not detectable in the SMG of strains carrying only one copy. In contrast to Spt-1, the Spt-2 gene is not expressed at detectable levels in the lacrimal gland.

Determination of the epidemiology and transmission of nontypable Haemophilus influenzae in children with otitis media by comparison of total genomic DNA restriction fingerprints.

It is assumed that the causative bacteria in children suffering from otitis media reach the middle ear via the eustachian tube. The purpose of this investigation was to use endonuclease restriction of bacterial chromosomal DNA to compare isolates of nontypable (NT) Haemophilus influenzae obtained from the nasopharynx and from middle ear (ME) effusions of patients with otitis media. Strains of NT H. influenzae were isolated from the nasopharynx (NP) and affected ME from a group of 13 unrelated children with otitis media with effusion (OME). For 12 of these children, identical strains were isolated from the NP and ME in a first episode of OME. Each of these 12 sets differed from the other 11. Six of these children suffered from a second episode of OME with NT H. influenzae. Five of these children with recurrence again had identical NP and ME strains. These results suggest that at the time of an episode of OME, there is one predominant strain of NT H. influenzae that colonizes both the NP and ME. The strains of NT H. influenzae isolated from all six of the second episodes were different from strains from the first episode, indicating turnover of the predominant strain in the NT H. influenzae population between episodes. When we investigated three siblings with concurrent episodes of OME, we found that they shared several similar strains of NT H. influenzae, thereby demonstrating that within a family, transmission of NT H. influenzae from child to child is possible. These results from DNA fingerprinting were essentially identical when compared with results from outer membrane protein subtyping performed on the same set of strains. The analysis of endonuclease restriction patterns of total genomic DNA provides a sensitive measure of genetic dissimilarity between strains and represents an easily applicable method for epidemiological and transmission studies of bacterial infections associated with NT H. influenzae.