Use of MALDI-TOF for identification and surveillance of gramnegative bacteria in captive wild psittacines / Uso do MALDI-TOF para identificação e monitoramento de bactérias Gram negativas em psitacídeos cativos

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines

Abstract Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Resumo Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines / Uso do MALDI-TOF para identificação e monitoramento de bactérias Gram-negativas em psitacídeos cativos

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines.

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Utilização do yacon ( Smallanthus sonchifolius ) na proteção contra colonização intestinal de frangos de corte infectados por Salmonella Enteritidis / Use of yacon (Smallanthus sonchifolius) protect against intestinal colonization of broilers infectes by Salmonella Enteritidis]

Os frutanos do tipo inulina são oligossacarídeos que favorecem a multiplicação de determinados gêneros bacterianos no intestino, promovendo um efeito prebiótico. Este trabalho avaliou o efeito da inulina extraída de raízes de yacon (Smallanthus sonchifolius) sobre a colonização intestinal de frangos de corte experimentalmente infectados por Salmonella Enteritidis. Sessenta frangos de corte com um dia de idade foram divididos em três grupos de tratamento, com duas repetições, criados até 21 dias. As aves do grupo yacon receberam 100mg de inulina/dia, via oral, por três dias consecutivos. No sétimo dia de vida, as aves tratadas e o controle positivo foram desafiados pela via oral com uma cultura de S. Enteritidis. Não foram observadas diferenças de desempenho zootécnico entre os grupos. O índice de infectividade das aves suplementadas com yacon foi menor até o sexto dia após o desafio, mas, ao término do experimento, foi superior ao controle positivo. Os dados deste trabalho demonstram que o uso da inulina nos três primeiros dias de vida promoveu uma redução da colonização intestinal dos frangos por Salmonella Enteritidis na primeira semana após o desafio. Novos estudos são necessários para determinar a dose e o tempo de tratamento ideal para um efeito protetor de maior duração.(AU)

The fructan inulin-type oligosaccharides favor the multiplication of some bacterial genera in the intestine, promoting a prebiotic effect. This study evaluated the effect of inulin extracted from yacon roots (Smallanthus sonchifolius) on intestinal colonization of broilers experimentally infected with Salmonella Enteritidis. Sixty-one day old chicks were grouped into three treatments, with two replicates, and reared until 21 days. Birds in the yacon group received 100mg of inulin/day orally for three consecutive days. On the seventh day of life the treated birds and the positive control were challenged orally with a culture of S. Enteritidis. There were no differences between groups in live performance. The infectivity index of the chicks supplemented with yacon was lower until the sixth day after the challenge, but at the end of the experiment it was higher than the positive control. Data from this study show that the use of inulin during the first 3 days of life caused a reduction of intestinal colonization of chickens by Salmonella Enteritidis in the first week after challenge. Further studies are needed to determine the dose and the ideal time of treatment necessary for a longer protective effect.(AU)

Virulence and antimicrobial resistance of Klebsiella pneumoniae isolated from passerine and psittacine birds.

Klebsiella pneumoniae is considered one of the most important Gram-negative opportunistic pathogens. The contact between humans and birds poses health risks to both. The aim of this study was to investigate the resistance and virulence of K. pneumoniae isolates from psittacines and passerines, seized from illegal trade in Brazil. We analysed 32 strains isolated from birds of the orders Psittaciformes and Passeriformes by polymerase chain reaction (PCR) for virulence factor genes. Antibiotic resistance was assessed by disk diffusion assay and PCR. The results indicated that fimH (100%), uge (96.8%), kfu (81.2%) and irp-2 (68.7%) were the most common virulence genes, followed by kpn (46.8%), K2 (43.7%), mrkD (34.3%) and iroN (15.6%). The combination of virulence genes resulted in a great diversity of genotypes and the heterogeneity of the strains is also confirmed in the analysis by amplified fragment length polymorphism. The susceptibility profiles of the K. pneumoniae showed 25% of multiple antibiotic resistance strains. We identified seven strains that presented non-extended spectrum beta lactamase blaSHV variants SHV-1 and SHV-11 and one strain positive to the blaTEM-1 gene. Plasmid-mediated quinolone resistance was present in 10 strains (10/32). The data obtained in this study reveal the pathogenic potential of this pathogen and highlight the need for surveillance and monitoring.

Inherited frontotemporal dementia in nine British families associated with intronic mutations in the tau gene.

Genetic screening of 171 patients with frontotemporal lobar degeneration disclosed 14 patients, across nine pedigrees, with mutations in the intron to exon 10 in the tau gene, a region regulating the splicing of exon 10 via a stem loop mechanism. Thirteen of these patients had the +16 splice site mutation and one had the +13 splice site mutation. Affected members of all nine families presented with changes in behaviour and social conduct that were prototypical of frontotemporal dementia (FTD). In all patients with the +16 splice site mutation, the behavioural profile was characterized by disinhibition, restless overactivity, a fatuous affect, puerile behaviour and verbal and motor stereotypies. The single patient with the +13 mutation presented a contrasting picture of apathy and inertia. In addition, all patients had evidence of semantic loss. Pathologically, five of the six patients so far autopsied shared frontotemporal atrophy with involvement of the substantia nigra. The underlying histology was that of microvacuolar-type cortical degeneration with a few swollen cells. Tau pathology was widespread throughout the brain and present in neurones and glial cells, mostly in the frontal and temporal cortical regions. This was in the form of neurofibrillary tangles and amorphous tau deposits (pre-tangles); Pick bodies were not observed. Ultrastructurally, the tau filaments had a twisted, ribbon-like morphology distinct from the paired helical filaments of Alzheimer's disease. One patient died from an unrelated illness whilst in the early clinical stages of FTD. In this patient, cortical microvacuolar and astrocytic changes were absent, though there were scattered neurones and glial cells, immunoreactive to tau, throughout the cortical and subcortical regions. The disease process underlying the neurodegeneration within these inherited forms of FTD may therefore stem directly from early, primary alterations in the function of tau. All eight families with the +16 mutation seem to be part of a common extended pedigree, possibly originating from a founder member residing within the North Wales region of Great Britain.

Modulation of beta-amyloid production and fibrillization.

Alzheimer's disease (AD) is the most common cause of dementia in old age and presently affects an estimated 4 million people in the U.S.A. and 0.75 million people in the U.K. It is a relentless, degenerative brain disease, characterized by progressive cognitive impairment. In the final stages of the disease, patients are often bedridden, doubly incontinent and unable to speak or to recognize close relatives. Pathological changes of Alzheimer's disease include extensive neuronal loss and the presence of numerous neurofibrillary tangles and senile plaques in the brain. The senile plaques contain amyloid fibrils derived from a 39-43-amino-acid peptide referred to as beta-amyloid or A beta. The basic theory of the so-called 'amyloid hypothesis' is that the deposition of aggregated forms of A beta in the brain parenchyma triggers a pathological cascade of events that leads to neurofibrillary tangle formation, neuronal loss and the associated dementia [1]. Here we discuss progress towards the identification of inhibitors of A beta production and fibrillization.

Fluorescence anisotropy: a method for early detection of Alzheimer beta-peptide (Abeta) aggregation.

Time-resolved anisotropy measurements (TRAMS) have been used to study the aggregation of the beta-amyloid (Abeta) peptide which is suspected of playing a central role in the pathogenesis of Alzheimer's Disease (AD). The experiments, which employ small quantities of fluorescently-labelled Abeta, in addition to the untagged peptide, have shown that the sensitive TRAMS technique detects the presence of preformed "seed" particles in freshly prepared solutions of Abeta. More importantly, as 100 microM solutions of Abeta containing tagged Abeta at a concentration level of either 0.5 or 1 microM are incubated, the TRAMS prove capable of detection of the peptide aggregation process through the appearance of a continuously increasing "residual anisotropy" within the time-resolved fluorescence data. The method detects Abeta aggregation in its earliest stages, well before complexation becomes apparent in more conventional methods such as the thioflavin T fluorescence assay. The TRAMS approach promises to provide a most attractive route for establishment of a high-throughput procedure for the early detection of the presence of amyloid aggregates in the screening of biological samples.

alpha-Synuclein implicated in Parkinson's disease catalyses the formation of hydrogen peroxide in vitro.

Some rare inherited forms of Parkinson's disease (PD) are due to mutations in the gene encoding a 140-amino acid presynaptic protein called alpha-synuclein. In PD, and some other related disorders such as dementia with Lewy bodies, alpha-synuclein accumulates in the brain in the form of fibrillar aggregates, which are found inside the neuronal cytoplasmic inclusions known as Lewy bodies. By means of an electron spin resonance (ESR) spin trapping method, we show here that solutions of full-length alpha-synuclein, and a synthetic peptide fragment of alpha-synuclein corresponding to residues 61-95 (the so-called non-Abeta component or NAC), both liberate hydroxyl radicals upon incubation in vitro followed by the addition of Fe(II). We did not observe this property for the related beta- and gamma-synucleins, which are not found in Lewy bodies, and are not linked genetically to any neurodegenerative disorder. There is abundant evidence for the involvement of free radicals and oxidative stress in the pathogenesis of nigral damage in PD. Our new data suggest that the fundamental molecular mechanism underlying this pathological process could be the production of hydrogen peroxide by alpha-synuclein.

Pick's disease is associated with mutations in the tau gene.

Recently, mutations within the tau gene have been associated with some familial forms of frontotemporal dementia. To investigate whether tau gene mutations are also associated with Pick's disease, we analyzed the tau gene in 30 cases of pathologically confirmed Pick's disease. Two coding mutations were identified in separate cases of Pick's disease. A glycine-to-arginine mutation at codon 389 was detected in 1 case and a lysine-to-threonine mutation at codon 257 was identified in another. Analysis of dephosphorylated tau from the brain of the patient with the codon 389 mutation revealed a prominent band representing tau, with four microtubule-binding domains and no amino terminal inserts. This is in contrast to Pick's disease without any tau gene mutations, which consist of tau with mainly three microtubule-binding domains and only a trace of tau, with four microtubule-binding domains. Functional analysis of tau with these two mutations demonstrated a reduced ability of tau to promote microtubule assembly. Surprisingly, these mutations increased tau's susceptibility to calpain I digestion, suggesting that this feature may be related to the formation of a Pick type of histology. Moreover, these data suggest that Pick's disease is not a separate entity but part of the frontotemporal dementia disease spectrum.

Ultrastructural localization of sulfated and unsulfated keratan sulfate in normal and macular corneal dystrophy type I.

Keratan sulfate (KS) proteoglycans are of importance for the maintenance of corneal transparency as evidenced in the condition macular corneal dystrophy type I (MCD I), a disorder involving the absence of KS sulfation, in which the cornea becomes opaque. In this transmission electron microscope study quantitative immuno- and histochemical methods have been used to examine a normal and MCD I cornea. The monoclonal antibody, 5-D-4, has been used to localize sulfated KS and the lectin Erythrina cristagalli agglutinin (ECA) to localize poly N -acetyllactosamine (unsulfated KS). In normal cornea high levels of sulfated KS were detected in the stroma, Bowman's layer, and Descemet's membrane and low levels in the keratocytes, epithelium and endothelium. Furthermore, in normal cornea, negligible levels of labeling were found for N -acetyllactosamine (unsulfated KS). In the MCD I cornea sulfated KS was not detected anywhere, but a specific distribution of N -acetyllactosamine (unsulfated KS) was evident: deposits found in the stroma, keratocytes, and endothelium labeled heavily as did the disrupted posterior region of Descemet's membrane. However, the actual cytoplasm of cells and the undisrupted regions of stroma revealed low levels of labeling. In conclusion, little or no unsulfated KS is present in normal cornea, but in MCD I cornea the abnormal unsulfated KS was localized in deposits and did not associate with the collagen fibrils of the corneal stroma. This study has also shown that ECA is an effective probe for unsulfated KS.

Proteoglycans on normal and migrating human corneal endothelium.

Proteoglycans are of fundamental importance to the normal functioning of the cornea. They consist of a core protein to which one or more glycosaminoglycan chains are attached. Cell surface proteoglycans are known to mediate many aspects of cell behaviour including cell adhesion, control of extracellular matrix deposition, cell proliferation, cell migration, leukocyte adhesion and modulation of growth factor activity. This paper describes the first investigation into the distribution and function of the three main classes of proteoglycans on human corneal endothelium. Immuno-gold labelling techniques were used at the light, scanning and transmission electron microscope level to localise heparan sulphate, chondroitin sulphate and keratan sulphate proteoglycans on human corneal endothelium. Human corneas were freeze-wounded and kept in organ culture for 3 days in order to study the distribution of proteoglycans on migrating corneal endothelium. An Optimas image analysis system was used to quantify the change in proteoglycan labelling during cell migration. Labelling for chondroitin sulphate and heparan sulphate was at very low levels on normal corneal endothelium while keratan sulphate labelling was at high levels. The wound healing experiments showed that migrating cells had increased labelling for heparan sulphate and chondroitin sulphate with greatly decreased labelling for keratan sulphate. Statistical analysis showed these changes were highly significant (P<0.001). Transmission electron microscopy revealed that chondroitin sulphate and keratan sulphate were present throughout Descemet's membrane while heparan sulphate was concentrated at the interface of Descemet's membrane and the migrating corneal endothelial cells. The pattern of occurrence of chondroitin sulphate, heparan sulphate and keratan sulphate on the human endothelium in normal and wounded cornea suggests that these proteoglycans are linked to the process of cell migration.

Keratan sulphate in the trabecular meshwork and cornea.

PURPOSE: A study was made of the distribution of keratan sulphate in the human anterior chamber. METHODS: The monoclonal antibody, 5-D-4, was used in immuno-electron microscopy to visualise keratan sulphate distribution in the anterior chamber of 16 normal eyes, 7 Fuchs' dystrophy corneas, and a macular dystrophy cornea. RESULTS: Keratan sulphate was detected in normal human aqueous humour and also on the surface of trabecular cells in the uveal meshwork. Normal corneal stroma showed an increase in keratan sulphate labelling from anterior to posterior, with marked labelling in the posterior region of Descemet's membrane. The apical surface of the corneal endothelium labelled positively, but showed considerable variation in the level of labelling from cell to cell. The macular dystrophy cornea had the classic histopathological features of a type I case, including a highly abnormal Descemet's membrane. No keratan sulphate was detected in the macular dystrophy patient's corneal stroma or serum. The Fuchs' endothelial dystrophy corneas showed a normal distribution of keratan sulphate labelling in the stroma. The Fuchs' endothelial cells labelled for keratan sulphate but were highly abnormal in appearance, often exhibiting long filopodia and appearing to be actively migrating. CONCLUSIONS: This work has shown that keratan sulphate has a much wider distribution than was previously believed. The detection of keratan sulphate on the trabecular and endothelial cell surfaces also suggests a possible role for this molecule in cell adhesion and/or migration.

Cell surface-associated keratan sulfate on normal and migrating corneal endothelium.

PURPOSE: To investigate cell surface-associated keratan sulfate on the corneal endothelium. METHODS: Immunolabeling techniques were used at the light, scanning, and transmission electron microscopic level to localize keratan sulfate on the corneal endothelium. The investigation included human, bovine, and rabbit corneal endothelia. A quantitative study of the relationship between cell size and keratan sulfate levels was conducted on normal bovine corneal endothelium. Changes in the distribution of keratan sulfate and chondroitin sulfate on endothelial cell surfaces were investigated on organ cultured bovine corneas during endothelial wound healing. Changes in the levels of keratan sulfate during endothelial wound healing were investigated in organ cultured human corneas and in vivo in rabbit corneas. Inhibition-enzyme-linked immunosorbent assay also was used to detect keratan sulfate in the aqueous humor. RESULTS: A variegated distribution of keratan sulfate was revealed on normal human, bovine, and rabbit corneal endothelia. Some cells had high levels of keratan sulfate on their surfaces whereas others, sometimes immediately adjacent, had little or none. Wound healing experiments resulted in changes of keratan sulfate levels on the migrating endothelial cells in bovine, human, and rabbit. In wounded organ cultured bovine corneas, there was a decrease in keratan sulfate levels and an increase in chondroitin sulfate levels on migrating endothelial cells. Keratan sulfate was detected in bovine aqueous humor. CONCLUSIONS: The pattern of occurrence of keratan sulfate and chondroitin sulfate on the corneal endothelial cells in normal and wounded cornea suggests that these glycosaminoglycans have differing roles in endothelial adhesion and migration.