Effect of gloved hand disinfection on hand hygiene before infection-prone procedures on a stem cell ward.

BACKGROUND: Hand hygiene compliance even before infection-prone procedures (indication 2, 'before aseptic tasks', according to the World Health Organization (WHO)) remains disappointing. AIM: To improve hand hygiene compliance by implementing gloved hand disinfection as a resource-neutral process optimization strategy. METHODS: We performed a three-phase intervention study on a stem cell transplant ward. After baseline evaluation of hand hygiene compliance (phase 1) gloved hand disinfection was allowed (phase 2) and restricted (phase 3) to evaluate and differentiate intervention derived from learning and time effects. The incidence of severe infections as well as of hospital-acquired multidrug-resistant bacteria was recorded by active surveillance. FINDINGS: Hand hygiene compliance improved significantly from 50% to 76% (P < 0.001) when gloved hand disinfection was allowed. The biggest increase was for infection-prone procedures (WHO 2) from 31% to 65%; P < 0.001. Severe infections decreased by trend (from 6.0 to 2.5 per 1000 patient-days) whereas transmission of multidrug-resistant organisms was not affected. CONCLUSION: Gloved hand disinfection significantly improved compliance with the hand hygiene, especially in activities relevant to infections and infection prevention. Thus, this process optimization may be an additional, easy implementable, resource-neutral tool for a highly vulnerable patient cohort.

Expression of alpha and beta parvalbumin is differentially regulated in the rat organ of corti during development.

The expression of two calcium-binding proteins of the parvalbumin (PV) family, the alpha isoform (alphaPV) and the beta isoform known as oncomodulin (OM), was investigated in the rat cochlea during postnatal development and related to cholinergic efferent innervation. Using RT-PCR analysis, we found that OM expression begins between postnatal day 2 (P2) and P4, and peaks as early as P10, while alphaPV mRNA begins expression before birth and remains highly expressed into the adult period. Both in situ hybridization and immunoreactivity confirm that OM is uniquely expressed by the outer hair cells (OHCs) in the rat cochlea and occurs after efferent innervation along the cochlear spiral between P2 and P4. In contrast to OM expression, alphaPV immunoreactivity is expressed in both inner hair cells (IHCs) and OHCs at birth. Following olivocochlear efferent innervation, OHCs demonstrate weak OM immunoreactivity beginning at P5 and diminished alphaPV immunoreactivity after P10. In organ cultures isolated prior to the efferent innervation of OHCs, OM immunoreactivity failed to develop in OHCs, but alphaPV immunoreactivity remained present in both IHCs and OHCs. In contrast, organ cultures isolated after efferent innervation of OHCs show OHCs with low levels of OM immunoreactivity and high levels of alphaPV immunoreactivity. This study suggests that OM and alphaPV are differentially regulated in OHCs during cochlear development. Our findings further raise the possibility that the expression of PV proteins in OHCs may be influenced by efferent innervation.

Development and maintenance of otoconia: biochemical considerations.

The first part of this review deals with recent advances in the understanding of biochemical mechanisms of otoconial morphogenesis. Most important in this regard is the molecular characterization of otoconin 90, the principal matrix protein of mammalian calcitic otoconia, which was found to be a homologue of the phospholytic enzyme PLA2. The unique and unexpected expression pattern of this protein required radical rethinking of traditional concepts. The new data, when integrated with existing information, provide a rational basis for an explanation of the mechanisms leading to crystal nucleation and growth. Based on this information, a hypothetical model is presented that posits interaction of otoconin 90 with microvesicles derived from the supporting cells as a key event in the formation of otoconia. The second part of the review is directed at the controversial subject of maintenance of mature otoconia and systematically analyzes the available indirect information on this topic. A synthesis of these theoretical considerations is viewed in relation to the pathogenesis of the important otoneurologic entities of BPPN and senile otoconial degeneration. The last part of the review deals with several animal models that promise to help elucidate normal and abnormal mechanisms of otoconial morphogenesis, including mineral deficiencies, mutations with selective otoconial agenesis, as well as targeted disruption of essential genes.

OCP1, an F-box protein, co-localizes with OCP2/SKP1 in the cochlear epithelial gap junction region.

Immunohistochemical data indicate that OCP1 co-localizes exactly with OCP2 in the epithelial gap junction region of the guinea pig organ of Corti (OC). Despite the abundance of OCP1 in the OC, gaining access to its coding sequence -- and, in particular, the 5' end of the coding sequence -- proved unexpectedly challenging. The putative full-length OCP1 cDNA -- 1180 nucleotides in length -- includes a 67 nucleotide 5' leader sequence, 300 codons (including initiation and termination signals), and a 216 nucleotide 3' untranslated region. The cDNA encodes a protein having a predicted molecular weight of 33,700. The inferred amino acid sequence harbors an F-box motif spanning residues 52--91, consistent with a role for OCP1 and OCP2 in the proteasome-mediated degradation of select OC proteins. Although OCP1 displays extensive homology to an F-box protein recently cloned from rat brain (NFB42), clustered sequence non-identities indicate that the two proteins are transcribed from distinct genes. The presumptive human OCP1 gene was identified in the human genome databank. Located on chromosome 1p35, the inferred translation product exhibits 94% identity with the guinea pig OCP1 coding sequence.

Proteomics and the inner ear.

The inner ear, one of the most complex organs, contains within its bony shell three sensory systems, the evolutionary oldest gravity receptor system, the three semicircular canals for the detection of angular acceleration, and the auditory system--unrivaled in sensitivity and frequency discrimination. All three systems are susceptible to a host of afflictions affecting the quality of life for all of us. In the first part of this review we present an introduction to the milestones of inner ear research to pave the way for understanding the complexities of a proteomics approach to the ear. Minute sensory structures, surrounded by large fluid spaces and a hard bony shell, pose extreme challenges to the ear researcher. In spite of these obstacles, a powerful preparatory technique was developed, whereby precisely defined microscopic tissue elements can be isolated and analyzed, while maintaining the biochemical state representative of the in vivo conditions. The second part consists of a discussion of proteomics as a tool in the elucidation of basic and pathologic mechanisms, diagnosis of disease, as well as treatment. Examples are the organ of Corti proteins OCP1 and OCP2, oncomodulin, a highly specific calcium-binding protein, and several disease entities, Meniere's disease, benign paroxysmal positional vertigo, and perilymphatic fistula.

The otoconia of the guinea pig utricle: internal structure, surface exposure, and interactions with the filament matrix.

A unique feature of the vertebrate gravity receptor organs, the saccule and utricle, is the mass of biomineral structures, the otoconia, overlying a gelatinous matrix also called "otoconial membrane" on the surface of the sensory epithelium. In mammals, otoconia are deposits of calcium carbonate in the form of composite calcite crystals. We used quick-freezing, deep etching to examine the otoconial mass of the guinea pig utricle. The deep-etching step exposed large expanses of intact and fractured otoconia, showing the fine structure and relationship between their internal crystal structure, their surface components, and the filament matrix in which they are embedded. Each otoconium has a compact central core meshwork of filaments and a composite outer shell of ordered crystallites and macromolecular aggregates. A distinct network of 20-nm beaded filaments covers the surface of the otoconia. The otoconia are interconnected and secured to the gelatinous matrix by surface adhesion and by confinement within a loose interotoconial filament matrix. The gelatinous matrix is a dense network made of yet another type of filament, 22 nm in diameter, which are cross-linked by shorter filaments, characteristically 11 nm in diameter. Our freeze-etching data provide a structural framework for considering the molecular nature of the components of the otoconial complex, their mechanical properties, and the degree of biological versus chemical control of otoconia biosynthesis.

Source and role of endolymph macromolecules.

Evaluation of some 200 endolymph proteins indicates that they are predominantly derived from plasma. However, the profile of endolymph proteins is remarkably similar to that of perilymph and entirely different from that of plasma. This supports the current consensus that perilymph rather than plasma is the (direct) source of endolymph. Although the levels of total protein of endolymph is extremely low, a few plasma-derived proteins, such as apolipoproteins J and D, are selectively enriched, conceivably for protection of cell membranes bounding the endolymphatic space. A small number of endolymph proteins, mostly glycosylated ones, are continually secreted into the endolymph by specialized epithelial cells, primarily for the maintenance of the structural and functional integrity of the extracellular superstructures comprising tectorial membrane, otoconial complex (membrane) and cupula. These complex macromolecules cannot be eliminated in the periphery of the compartment, but are transported to the endolymphatic sac for elimination. Impaired clearance of these negatively charged macromolecules by a dysfunctional endolymphatic sac will contribute to the chemical imbalance of endolymph which accompanies long-standing endolymphatic hydrops, and may be one of the reasons for the observed loss of function.

Otoconin-90, the mammalian otoconial matrix protein, contains two domains of homology to secretory phospholipase A2.

The ability to sense orientation relative to gravity requires dense particles, called otoconia, which are localized in the vestibular macular organs. In mammals, otoconia are composed of proteins (otoconins) and calcium carbonate crystals in a calcite lattice. Little is known about the mechanisms that regulate otoconial biosynthesis. To begin to elucidate these mechanisms, we have partially sequenced and cloned the major protein component of murine otoconia, otoconin-90 (OC90). The amino acid sequence identified an orphan chimeric human cDNA. Because of its similarity to secretory phospholipase A2 (sPLA2), this gene was referred to as PLA2-like (PLA2L) and enabled the identification of human Oc90. Partial murine cDNA and genomic clones were isolated and shown to be specifically expressed in the developing mouse otocyst. The mature mouse OC90 is composed of 453 residues and contains two domains homologous to sPLA2. The cloning of Oc90 will allow an examination of the role of this protein in otoconial biosynthesis and in diseases that affect the vestibular system.

Otoconial agenesis in tilted mutant mice.

The sense of balance is one of the phylogenetically oldest sensory systems. The vestibular organs, consisting of sensory hair cells and an overlying extracellular membrane, have been conserved throughout vertebrate evolution. To better understand mechanisms regulating vestibular development and mechanisms of vestibular pathophysiology, we have analyzed the mouse mutant, tilted (tlt), which has dysfunction of the gravity receptors. The tilted mouse arose spontaneously and has not been previously analyzed for a developmental or physiological deficit. Here we demonstrate that the tilted mouse, like the head tilt (het) mouse, specifically lacks otoconia and consequently does not sense spatial orientation relative to the force of gravity. Unlike other mouse mutations affecting the vestibular system (such as pallid, mocha and tilted head), the defect in the tilted mouse is highly penetrant, results in the nearly complete absence of otoconia, exhibits no degeneration of the sensory epithelium and has no apparent abnormal phenotype in other organ systems. We further demonstrate that protein expression in the macular sensory epithelium is qualitatively unaltered in tilted mutant mice.

OCP2 exists as a dimer in the organ of Corti.

OCP2 is one of the most abundant proteins in the organ of Corti (OC), comprising approximately 5% of the total protein in the supporting cell population. Although the very close homolog, Skp1p, has been implicated in regulating cell-cycle progression, the function of OCP2 in the terminally differentiated cochlea is presently unknown. We have purified recombinant OCP2 from Escherichia coli and examined the protein by analytical ultracentrifugation. Interestingly, sedimentation equilibrium data collected at 20 degrees C unequivocally indicate that, at the concentrations present in the OC, free OCP2 would exist as a dimeric species. The apparent sedimentation coefficient is independent of concentration at loading concentrations between 10 and 100 microM, indicating the absence of a significant monomer-dimer equilibrium in this concentration range. The functional significance of this finding is discussed.

Oncomodulin is expressed exclusively by outer hair cells in the organ of Corti.

Oncomodulin (OM) is a small, acidic calcium-binding protein first discovered in a rat hepatoma and later found in placental cytotrophoblasts, the pre-implantation embryo, and in a wide variety of neoplastic tissues. OM was considered to be exclusively an oncofetal protein until its recent detection in extracts of the adult guinea pig's organ of Corti. Here we report that light and electron microscopic immunostaining of gerbil, rat, and mouse inner ears with a monoclonal antibody against recombinant rat OM localizes the protein exclusively in cochlear outer hair cells (OHCs). At the ultrastructural level, high gold labeling density was seen overlying the nucleus, cytoplasm, and the cuticular plate of gerbil OHCs. Few, if any, gold particles were present over intracellular organelles and the stereocilia. Staining of a wide range of similarly processed gerbil organs failed to detect immunoreactive OM in any other adult tissues. The mammalian genome encodes one alpha- and one beta-isoform of parvalbumin (PV). The widely distributed alpha PV exhibits a very high affinity for Ca2+ and is believed to serve as a Ca2+ buffer. By contrast, OM, the mammalian beta PV, displays a highly attenuated affinity for Ca2+, consistent with a Ca2+-dependent regulatory function. The exclusive association of OM with cochlear OHCs in mature tissues is likely to have functional relevance. Teleological considerations favor its involvement in regulating some aspect of OHC electromotility. Although the fast electromotile response of OHCs does not require Ca2+, its gain and magnitude are modulated by efferent innervation. Therefore, OM may be involved in mediation of intracellular responses to cholinergic stimulation, which are known to be Ca2+ regulated. (J Histochem Cytochem 46:29-39, 1998)

Oncomodulin is abundant in the organ of Corti.

A small, acidic Ca(2+)-binding protein (CBP-15) was recently detected in extracts of the mammalian auditory receptor organ, the organ of Corti [Senarita et al. (1995) Hear. Res. 90, 169-175]. N-terminal sequence data for CBP-15 [Thalmann et al. (1995) Biochem. Biophys. Res. Commun. 215, 142-147] implied membership in the parvalbumin family and possible identity with the mammalian beta-parvalbumin oncomodulin. As shown herein, the latter conclusion is supported by strong cross-reactivity between CBP-15 and isoform-specific antibodies to oncomodulin. Moreover, we have succeeded in amplifying the guinea pig CBP-15 coding sequence from organ of Corti cDNA using degenerate oligonucleotide primers based on the rat oncomodulin sequence. The deduced amino acid sequence of guinea pig CBP-15 displays 90%, 92%, and 98% identity with mouse, rat, and human oncomodulin isoforms. Demonstration of the presence of oncomodulin in the organ of Corti is the first documentation of this substance in a postnatal mammalian tissue.

Specific proteins of the organ of Corti.

The mammalian organ of Corti has achieved a degree of perfection unequaled in other hair cell systems. Although cellular metabolism requires the coordinated action of thousands of proteins, the physical processes underlying auditory transduction in the OC are undoubtedly mediated by a much smaller subset of these. OCP1, OCP2, and CBP-15-identified by 2D-PAGE-are apparently members of this elite class. OCP1 and OCP2 are restricted to the supporting cells of the organ of Corti and adjacent epithelia. Their distribution closely parallels the boundaries of the epithelial gap junction system, implying a role in cochlear potassium and pH homeostasis. CBP-15 was recently shown to be identical to oncomodulin, the mammalian beta-parvalbumin, heretofore documented only in the placenta and neoplasms. Expression of this small calcium-binding protein in the OC is restricted to the outer hair cells, where it may function as a calcium-dependent regulatory protein.

Localization of organ of Corti protein II in the adult and developing gerbil cochlea.

The distribution of organ of Corti protein II (OCP-II) was assessed in the developing and mature gerbil cochlea by light and electron microscopic immunohistochemistry. In the adult cochlea, OCP-II was expressed only in certain epithelial cells which included all supporting cells of the organ of Corti, inner and outer sulcus cells and interdental cells. Inner and outer hair cells lacked immunoreactivity. The highest gold particle labeling density was seen overlying intracellular regions devoid of organelles. In the developing inner ear, OCP-II was first detected at 2 days after birth (DAB) with the strongest staining in immature Deiters, inner phalangeal and pillar cells. Immunostaining intensity increased gradually in cells lying laterally and medially to the more centrally located supporting cells and reached adult levels in all reactive cell types around 18 DAB. The results demonstrated conclusively that OCP-II is a cytosolic protein and fail to support its role as a transcription factor postulated on the basis of its homology with p15 or a role in the control of the cycle as suggested by its near-identity with p19Skp1, a cyclin A/CDK2-associated protein. The continued high level of expression in the mature cochlea argues against OCP-II's involvement in regulating the development and differentiation of epithelial cells. The protein's unique distribution and its gradual increase in expression prior to and during the onset and maturation of hearing, however, support its potential function in the recycling of K+ effluxed from hair cells and neurons back to endolymph.

Detection of a beta-parvalbumin isoform in the mammalian inner ear.

A small, acidic calcium-binding protein (CBP-15) has been detected in the guinea pig organ of Corti, the auditory receptor organ. The apparent molecular weight (15,000) and very low isoelectric point (pI approximately 3.1) suggest that CBP-15 is a beta-parvalbumin isoform. Consistent with this hypothesis, CBP-15 exhibits extreme homology to the mammalian oncofetal parvalbumin called oncomodulin. Sequence data have now been obtained for 30 residues in the N-terminal third of CBP-15. Identity with oncomodulin is observed at all 30 positions. This finding could necessitate revision of the assumption that postnatal mammals utilize a single alpha-parvalbumin isoform in muscle and nonmuscle settings alike.

Calcium-binding proteins in organ of Corti and basilar papilla: CBP-15, an unidentified calcium-binding protein of the inner ear.

In a previous paper (Thalmann et al., 1993) we reported that the amino acid sequence of OCP2, a low molecular weight acidic protein present in extremely high concentrations in the organ of Corti and absent in the basilar papilla, exhibits a rudimentary EF-hand--a potential calcium-binding domain. The present study was undertaken to determine whether OCP2 binds 45-calcium under non-denaturing conditions following separation by isoelectric focusing and transblotting. The same criterion was used to determine whether the EF-hands of several other calcium-binding proteins (CBP) are functional in organ of Corti and basilar papilla. OCP2 exhibited no 45-calcium binding. Calmodulin, present in the organ of Corti in extremely high concentrations and lower in basilar papilla, showed strong 45-calcium binding in both structures. While calbindin represents a major protein in basilar papilla and binds 45-calcium, this protein is a minor component in the organ of Corti; whether it binds 45-calcium remains to be decided. By extending the pI range in the acidic region of isoelectric focusing, a 15 kDa, highly acidic (pI approximately 3.1) protein was revealed that constitutes a major protein in the organ of Corti; the protein was not detectable in the basilar papilla, spiral ligament/stria vascularis complex and numerous other organs tested. It remains to be resolved whether this protein represents an isoform of parvalbumin or a novel CBP. The differential make-up of CBPs between the mammalian organ of Corti and the avian basilar papilla is discussed.

cDNA cloning, tissue distribution, and chromosomal localization of Ocp2, a gene encoding a putative transcription-associated factor predominantly expressed in the auditory organs.

We report the cloning of the Ocp2 gene encoding OCP-II from a guinea pig organ-of-Corti cDNA library. The predicted open reading frame encodes a protein of 163 amino acids with an estimated molecular mass of 18.6 kDa. A homology search revealed that Ocp2 shares significant sequence similarity with p15, a subunit of transcription factor SIII that regulates the activity of the RNA polymerase II elongation complex. The Ocp2 messenger RNA is expressed abundantly in the cochlea while not significantly in any other tissues examined, including brain, eye, heart, intestine, kidney, liver, lung, thigh muscle, and testis, demonstrating that the expression of this gene may be restricted to auditory organs. A polyclonal antiserum was raised against the N-terminal region of OCP-II. Immunohistochemical staining of paraffin-embedded sections of the cochlea showed that OCP-II is localized abundantly in nonsensory cells in the organ of Corti; in addition, it was also detected, at a lower concentration, in vestibular sensory organs, as well as auditory and vestibular brain stem nuclei. The Ocp2 gene was mapped to mouse chromosome 4 as well as 11. Our results suggest that OCP-II may be involved in transcription regulation for the development or maintenance of specialized functions of the inner ear.

High resolution two-dimensional electrophoresis: technique and potential applicability to the study of inner ear disease.

Technologic progress in two-dimensional polyacrylamide gel electrophoresis (2-D Page), in combination with immunoblotting, amino acid sequencing, and computer-assisted image analysis, allowed establishment of human body fluid and tissue "reference maps," which in turn enabled meaningful comparison of data from various clinical and research centers. Altered protein profiles have been observed in plasma/serum, cerebrospinal fluid, urine, and other body fluids in numerous systemic or localized pathologic entities. Human perilymph, obtained during ear surgery or post mortem, exhibits a protein profile differing from plasma in several ways. Most interesting are the extremely high levels of high density lipoprotein-associated proteins, a group of proteins thought to play a role in atherosclerosis, nerve damage/regeneration, chronic inflammation, and Alzheimer's disease, among others. A technique is described for collection and analysis of human perilymph, using a state-of-the-art standardized 2-D Page technique. It is expected that, as in other body fluids, disease-specific protein patterns will be identified. With the possible exception of presumed perilymphatic fistula, it is not envisioned that analysis of perilymph will be used for diagnostic purposes but rather as an aid for the elucidation of the mechanisms underlying inner ear disease, whether localized or as part of systemic alterations.

Protein profile of human perilymph: in search of markers for the diagnosis of perilymph fistula and other inner ear disease.

Recent developments in high-resolution two-dimensional polyacrylamide gel electrophoresis, combined with amino acid sequencing and computer-assisted image analysis, have allowed separation of approximately 100 proteins and identification and quantitation of some 30 proteins in human perilymph. The majority of proteins were found to be present in perilymph at levels in basic agreement with the total protein gradient between perilymph and plasma (1:35). However, several striking differences were observed: (1) beta 2-transferrin, known to be absent from normal plasma but present in cerebrospinal fluid, was detected in perilymph at a concentration roughly equal to that in cerebrospinal fluid; and (2) two high-density lipoprotein-associated apolipoproteins--apo D (formerly PLS:33) and apo J or NA1 and NA2 (formerly PSL:29/30), the latter showing identity with SP40/40, or cytolysis inhibitor--were found to be present at concentrations 1 to 2 orders of magnitude higher when examined in terms of total protein and to be comparable with or higher than plasma levels when examined in terms of absolute concentrations. The functional significance of the extremely high levels of the two apolipoproteins is not known at this time. An attempt was made to use beta 2-transferrin, as well as apo D and apo J (NA1/NA2), as markers for the diagnosis of perilymph fistula, one of the most controversial and challenging problems for the otologist today. It was determined that the technique is indeed applicable when relatively pure fistula samples are analyzed. Limitations and potential improvements of the technique are discussed. In addition, the potential usefulness of two-dimensional polyacrylamide gel electrophoresis in other pathologic conditions of the inner ear is discussed briefly.