Role of the endogenous nitric oxide inhibitor asymmetric dimethylarginine (ADMA) and brain-derived neurotrophic factor (BDNF) in depression and behavioural changes: clinical and preclinical data in chronic kidney disease.

BACKGROUND: Patients with chronic kidney disease (CKD) exhibit a high prevalence of neuropsychiatric alterations, including depression and behavioural changes. CKD is also associated with decreased physical activity not fully explained by co-morbidities. In patients without CKD, the brain-derived neurotropic factor (BDNF) as well as the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) had been suspected to be involved in major depression. The aim of our study was to examine the role of ADMA and BDNF in the behaviour of haemodialysis patients (CKD5D) as well as in a rat model of 5/6 nephrectomy and chronic ADMA infusion alone. METHODS: Eleven (5F/6M) CKD5D patients underwent Beck Depression Inventory (BDI) testing along with analysis of ADMA and BDNF. Male Sprague-Dawley rats were randomly assigned to four groups: (i) saline infusion; (ii) ADMA (250 µg/kg/day) infusion via osmotic mini pumps; (iii) 5/6 nephrectomy; (iv) untreated controls. After 28 days, the animals underwent behavioural tests measuring anxiety, locomotion and investigative behaviour. Animals were sacrificed, blood samples were drawn and analysed and hippocampal immunohistology for BDNF was performed. RESULTS: In CKD5D patients, decreased BDNF levels correlated with higher scores of depression (Pearson r = -0.8156, P = 0.002). ADMA infusion led to a significant decrease of BDNF while the decrease of BDNF in 5/6 nephrectomy was not significant. However, an attenuated hippocampal BDNF expression could be detected in 5/6 nephrecomized animals. Decreased spontaneous locomotor activity was shown in ADMA-infused rats [15.9 (13.5-26.1) lines crossed/min] and 5/6 nephrectomy [14.6 (6.1-20.2) lines crossed/min] when compared with controls [32.5 (15.3-42.4) lines crossed/min]. Anxiety-like behaviour tested by hole investigation time was significantly more pronounced in 5/6 nephrectomy [24 (6-44) s] when compared with ADMA infusion [64 (28-93) s] and controls [33 (26-65) s]. CONCLUSIONS: Progressive renal failure in rats is accompanied by a marked increase of ADMA and a decrease in BDNF. 5/6 nephrectomy leads to significantly decreased exploratory behaviour and locomotion. Both behaviours could be reproduced by ADMA infusion alone. Indicators of anxiety were more pronounced in ADMA-infused animals when compared with 5/6 nephrectomized rats. Furthermore, an inverse relationship of BDNF and BDI in 11 CKD5D patients was shown.

Effect of a single dialysis session on cognitive function in CKD5D patients: a prospective clinical study.

BACKGROUND: Cognitive function declines in parallel to the decrease in glomerular filtration rate, best epitomized by the markedly reduced cerebral performance in patients undergoing maintenance haemodialysis [chronic kidney disease stage 5 dialysis (CKD5D)]. Aside from structural permanent damage, there seems to be a reversible part of low cognitive performance. The potential effect of a single dialysis session on cognitive function remains still elusive. The aim of the study was to assess cognitive function using a widespread test battery and avoiding excluding effects of circadian variations. METHODS: Twenty-eight medically stable CKD5D patients (age: 54.9 ± 13.2 years, dialysis vintage: 46.2 ± 51.0 month) at two tertiary care centres with outpatient dialysis units were enrolled. Cognitive testing was always performed twice within 24 h, 1 h prior to haemodialysis (T1pre-dialysis) as well as 19 h after the end of dialysis (T2post-dialysis) including assessment of memory, attention and concentration, executive functioning, word fluency and psychomotor speed by using a well-validated neuropsychological test battery. Patients were randomized into two groups. One group was examined before (T1pre-dialysis) and after (T2post-dialysis) Dialysis Session 1. The other group was first examined the day after Dialysis Session 1 (T2post-dialysis) and then before Dialysis Session 2 (T1pre-dialysis) in order to exclude potential learning effects. Twenty age-matched subjects with normal excretory renal function were used for comparison. RESULTS: Neuropsychological testing found that the CKD5D performed significantly worse on measures of alertness, attention, working memory, logical and visual memory, word fluency and executive functions compared with non-CKD subjects. No differences in short-term memory, selective attention, as well as problem-solving and planning were found between CKD5D patients and non-CKD subjects. A single haemodialysis session led to a significant improvement in logical (Rivermead Behaviour Memory Test story: P < 0.001) and visual memories [Rey-Osterrieth Complex Figure Test (RCFT) memory quotient: P < 0.001], psychomotor speed [Trail Making Test (TMT) B: P = 0.020], activity planning (executive functions) (RCFT copy/points deduction: P < 0.001) and concentration (TMT A: P < 0.001). CONCLUSION: Our data demonstrate improvements in memory functions, executive functions and psychomotor abilities after a single dialysis session, pointing to a reversible component of low cognitive performance in CKD5D.

Possible sources and functions of L-homoarginine in the brain: review of the literature and own findings.

L-Homoarginine is a cationic amino acid derivative, which is structurally related to L-arginine and lysine. Several lines of evidence point to nervous tissue as an important target of homoarginine action. In the mammalian brain homoarginine can be detected in noticeable quantities, but its origin is currently poorly explored. In part I of this review we try to show that both uptake and transport into brain (carried out by cationic amino acid transporters) and local synthesis in the brain (carried out by the homoarginine-synthesizing enzymes L-arginine:glycine amidinotransferase and ornithine transcarbamylse) might contribute to homoarginine brain content. We then give a brief overview about the multiple effects of homoarginine on the healthy brain and show that both homoarginine excess and deficiency are potentially harmful to the central nervous system. In part II, we shortly report about own experiments with regard to the cellular localization of cationic amino acid transporters, as well the enzymes L-arginine:glycine amidinotransferase and ornithine transcarbamylse, in human and rat brains.

Enzymes of urea synthesis are expressed at the ocular surface, and decreased urea in the tear fluid is associated with dry-eye syndrome.

PURPOSE: The present study aims at determining whether enzymes of urea synthesis are expressed in the human lacrimal gland and in tissues of ocular surface (conjunctiva, cornea), to give evidence for the hypothesis that urea can be locally formed from ocular tissues and is important for the composition of the tear fluid. METHODS: The presences of enzymes (arginase 1, 2 and agmatinase) that directly contribute to the formation of urea were investigated in the lacrimal gland and tissues of ocular surface by RT-PCR and immunohistochemistry. We collected tear fluid, aqueous humour, and blood samples from a total of 38 subjects, and tear fluid samples from a total of 78 subjects, with and without dry-eye syndrome (DES, keratoconjunctivitis sicca), and determined the urea concentration. RESULTS: The enzymes arginase 1, 2 and agmatinase were expressed in all tissues examined except for arginase 1, which was not expressed in the cornea. There was no correlation of urea concentration in tear fluid with aqueous humour and blood plasma (r = 0.13, p = 0.58 and r = 0.45, p = 0.05 respectively). However, correlation of urea concentration between aqueous humour and blood plasma was highly significant (r = 0.7, p = 0.0001). The concentration of urea in the tear fluid of patients with DES compared to healthy control group was significantly reduced (p < 0.0001). CONCLUSION: Enzymes that are directly involved in the formation of urea are expressed in ocular tissues. This may imply that in the ocular surface is a well-coordinated system of enzymes that can produce urea which might be independent of external urea supply.

Agmatinase, an inactivator of the putative endogenous antidepressant agmatine, is strongly upregulated in hippocampal interneurons of subjects with mood disorders.

The diamine agmatine may serve as a precursor in polyamine synthesis. In addition, agmatine may also act as a neurotransmitter, binding to imidazoline receptors. Behaviorally, agmatine exerts antidepressant-like effects. The enzyme agmatinase degrades agmatine. The gene coding for human agmatinase is located on chromosome 1p36, a gene locus which has been linked to bipolar disorder and major depression, but the enzyme has not yet been studied in the context of neuropsychiatric diseases. We analyzed agmatinase protein expression in postmortem hippocampi of individuals with affective disorders. Data from eleven patients with mood disorders (unipolar and bipolar depression) and twelve matched control cases were compared by immunocytochemical and morphometrical analysis. Agmatinase protein was detected in a subset of hippocampal interneurons. The protein was localized to perikarya, neurites and putative nerve endings contacting hippocampal pyramidal neurons and dentate gyrus granule cells. The number and the numerical density of agmatinase-immunopositive cell bodies were strongly elevated in depressive patients. In addition, a significantly increased density of agmatinase-immunoreactive punctate profiles was observed in the CA(4) region in unipolar and bipolar depression. The reported increased expression of agmatinase suggests a functional relevance of the enzyme in the pathophysiology of human affective disorders. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Expression analysis of ADAM17 during mouse eye development.

ADAM17 (a disintegrin and metallopeptidase domain 17) is crucial for eye morphogenesis. In this study we analysed the expression pattern of ADAM17 during mouse eye development. ADAM17 expression in adult retina was examined using the reverse transcription-polymerase chain reaction (RT-PCR) and verification of the RT-PCR products by DNA sequencing. Immunohistochemistry was performed to evaluate the ADAM17 expression pattern in mouse eyes at developmental stages of embryonic day (E) 12, E14, E16, E18, postnatal day (P) 0, P1, P4, P7, P14, P 30 and P175 (adult). We detected ADAM17 mRNA in adult retina tissue. ADAM17 protein was expressed in non-pigmented ciliary epithelial cells and in retinal vessels from P7 onwards during eye development. In corneal epithelial cells and endothelium, ADAM17 protein was present from P14 onwards. Although, mice in which the functional ADAM17 gene is significantly reduced develop multiple eye malformations, the expression of ADAM17 is not ubiquitous over the entire eye. Its expression pattern during development suggests that not only TNF-alpha but additional membrane-anchored substrates of ADAM17 play an important role in eye formation.

Asymmetric dimethylarginine may mediate increased heat pain threshold in experimental chronic kidney disease.

BACKGROUND: Thermal sensitivity in uraemia is decreased. Non-selective synthetic nitric oxide synthase (NOS) inhibitors significantly attenuate thermal hyperalgesia in preclinical models. The aim of our study was to evaluate the effect of experimental uraemia, which is associated with an increase of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA), on thermal sensitivity in rats. Furthermore, we intended to study the effect of chronic ADMA infusion alone on thermal sensitivity. METHODS: Male Sprague-Dawley rats (n = 54), 10 weeks old, weight 370-430 g, were randomly assigned to three groups receiving either (i) isotonic saline or (ii) ADMA via osmotic mini pumps or (iii) underwent 5/6 nephrectomy (Nx). After 14 days, 50% of all animals from all groups underwent thermal sensitivity testing and terminal blood draw. After 28 days, the remaining animals underwent the same procedures. Thermal sensitivity examination was performed by the hot-plate test, measuring time from heat exposition to first paw licking or jumping of the animal. RESULTS: While the median [interquartile range] latency time between heat exposition to first paw licking or jumping of the animal in the NaCl infusion group remained unchanged between Day 14 (8.4 [6.75-11.50] s) and Day 28 (7.35 [6.10-7.90] s) both, ADMA infusion and 5/6 nephrectomy tended to increase the thermal pain threshold at Day 14 (9.25 [6.55-12.18] s) and (9.50 [5.8 ± 11.0] s), respectively, compared to NaCl on Day 14 (8.4 [6.75-11.50] s). This difference became statistical significant at Day 28 where the median latency time in the ADMA group (13.10 [11.85-15.95] s) and in the 5/6 Nx group (13.50 [10.85-17.55] s) were significantly higher than in the NaCl group (7.35 [6.10-7.90] s). CONCLUSIONS: Induction of progressive renal failure in rats by 5/6 nephrectomy, which is accompanied by a marked increase of the serum levels of the endogenous NOS inhibitor ADMA, leads to a significantly increased heat pain threshold at 28 days. The sole infusion of ADMA into healthy rats leads to the same increase in heat pain threshold.

In vitro evidence of involvement of the epithelial y+ transporter in ß-defensin production on the ocular surface.

To analyse the hypothesis as to whether there is a functional relationship between human cationic amino acid transporters (hCATs, y(+) transporter, the main transporter of L-arginine and L-lysine) and human ß-defensin (important components of immune function) production on the ocular surface, arginase and nitrate monoxide synthase (NOS), enzymes that compete for L-arginine, were inhibited by norNOHA (N(omega)-hydroxy-nor-L-arginine) and/or L-NAME (NG-nitro-L-arginine methyl ester) in cultured human corneal epithelial cells. In addition, the transport activity of hCAT proteins was inhibited or activated through α-tocopherol or PMA (phorbol myristate acetate), respectively. Concentrations of the human inducible ß-defensins (hBD) 2 and 3 were determined by ELISA experiments. The basic expression of hBD3 in non-stimulated HCE cells significantly exceeded that of hBD2. Both ß-defensins also differed as to how readily their excretion could be stimulated. HBD2 excretion rate was 3.5 time more by L-NAME, whereas norNOHA had no effect. In contrast, hBD3 excretion was increased by norNOHA by a factor of 1.5 but L-NAME alone had no effect. The excretion of both ß-defensins was increased 3- and 6-fold by combined administration of L-NAME, norNOHA and interleukin (IL)-1ß. Administration of α-tocopherol increased hBD2 excretion twofold. No effect was observed for hBD3. With PMA, on the other hand, a reduction in secretion for both ß-defensins was observed. These in vitro findings provide evidence of a functional association between CAT proteins and ß-defensins 2 and 3 opening up a new field of research with pharmacological perspectives for treatment of inflammatory diseases such as keratitis or dry eye disease.

Roles of human beta-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells.

Human beta-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. In this study, the expression and inducibility of beta-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human beta-defensins (hBD) was determined by RT-PCR and immunohistochemistry in tissues of the ocular surface and lacrimal apparatus. Cultured corneal and conjunctival epithelial cells were stimulated with proinflammatory cytokines and supernatants of different ocular pathogens. Real-time PCR and ELISA experiments were performed to study the effect on the inducibility of hBD2 and 3. Expression and inducibility of mouse beta-defensins-2, -3 and -4 (mBD2-4) were tested in a mouse ocular surface scratch model with and without treatment of supernatants of a clinical Staphylococcus aureus (SA) isolate by means of immunohistochemistry. Here we show that hBD1, -2, -3 and -4 are constitutively expressed in conjunctival epithelial cells and also partly in cornea. Healthy tissues of the ocular surface, lacrimal apparatus and human tears contain measurable amounts of hBD2 and -3, with highest concentrations in cornea and much lower concentrations in all other tissues, especially tears, suggesting intraepithelial storage of beta-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1beta is a very strong inductor of hBD2 and Staphylococcus aureus increases both hBD2 and hBD3 production in corneal and conjunctival epithelial cells. A murine corneal scratch model demonstrated that beta-defensins are only induced if microbial products within the tear film come into contact with a defective epithelium. Our finding suggests that the tear film per se contains so much antimicrobial substances that epithelial induction of beta-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of beta-defensins at the ocular surface and lacrimal apparatus and show how beta-defensins are regulated specifically.

Cationic amino acid transporters and beta-defensins in dry eye syndrome.

Several diseases concomitant with L-arginine deficiency (diabetes, chronic kidney failure, psoriasis) are significantly associated with dry eye syndrome. One important factor that has so far been neglected is the y(+) transporter. In humans, y(+) accounts for nearly 80% of arginine transport, exclusively carrying the cationic amino acids L-arginine, L-lysine and L-ornithine. y(+) is represented by CAT(cationic amino acid transporter) proteins. L-arginine is a precursor of the moisturizer urea, which has been used in the treatment of dry skin diseases. Although urea has also been shown to be part of the tear film, little attention has been paid to it in this role. Moreover, L-arginine and L-lysine are major components contributing to synthesis of the antimicrobially active beta-defensins induced under dry eye conditions. The first results have demonstrated that transport of L-arginine and L-lysine into epithelial cells is limited by the y(+) transporter at the ocular surface.

Expression profile of aquaporins in human nasolacrimal duct epithelium.

PURPOSE: To determine whether the epithelium of the human nasolacrimal ducts contains aquaporins (AQPs), a family of membrane proteins that function as selective pores and are able to transport water, glycerol, and other small solutes across the cell plasma membrane. METHODS: Expression of AQPs 0 and 1-10 in human nasolacrimal duct tissue was determined by reverse transcription polymerase chain reaction (RT-PCR). Positive PCR amplification products were verified by direct cDNA sequencing. Western blot analysis was used to detect AQPs 3-5. Antisera specific for AQPs were used in immunohistochemical analysis to determine the presence and distribution of ten AQPs (AQP 0 and 1-9) in epithelia and subepithelial glands of the nasolacrimal ducts. All samples investigated originated from human postmortem tissue. RESULTS: In human nasolacrimal duct samples, AQPs 1, 3, 4, 5, 7, 8, 9, and 10 were identified by RT-PCR. No RT-PCR products were detected for AQPs 0, 2, and 6. All AQPs, which were detected by RT-PCR, were also confirmed by direct sequencing of the cDNA. Immunohistochemical analyses revealed AQPs 1, 3, 5, 7, 8, and 9 in human nasolacrimal duct epithelium and were detected in different cells. Expression of AQP 4 could not be detected immunohistochemically but by Western blot analysis. Protein detection of AQP 10 could not be performed due to the unavailability of an appropriate antibody. CONCLUSIONS: The results suggest specific roles for AQPs in water transport through the epithelium of the nasolacrimal ducts and underline the presumption that tear fluid components are selectively absorbed in the nasolacrimal passage.

Functional relationship between cationic amino acid transporters and beta-defensins: implications for dry skin diseases and the dry eye.

The ocular surface, constantly exposed to environmental pathogens, is particularly vulnerable to infection. Hence an advanced immune defence system is essential to protect the eye from microbial attack. Antimicrobial peptides, such as beta-defensins, are essential components of the innate immune system and are the first line of defence against invaders of the eye. High concentrations of L-arginine and L-lysine are necessary for the expression of beta-defensins. These are supplied by epithelial cells in inflammatory processes. The limiting factor for initiation of beta-defensin production is the transport of L-arginine and L-lysine into the cell. This transport is performed to 80% by only one transporter system in the human, the y(+)-transporter. This group of proteins exclusively transports the cationic amino acids L-arginine, L-lysine and L-ornithine and is also known under the term cationic amino acid transporter proteins (CAT-proteins). Various infections associated with L-arginine deficiency (for example psoriasis, keratoconjuctivitis sicca) are also associated with an increase in beta-defensin production. For the first time, preliminary work has shown the expression of human CATs in ocular surface epithelia and tissues of the lacrimal apparatus indicating their relevance for diseases of the ocular surface. In this review, we summarize current knowledge on the human CATs that appear to be integrated in causal regulation cascades of beta-defensins, thereby offering novel concepts for therapeutic perspectives.

Human parotid and submandibular glands express and secrete surfactant proteins A, B, C and D.

The oral cavity and the salivary glands are open to the oral environment and are thus exposed to multiple microbiological, chemical and mechanical influences. The existence of an efficient defense system is essential to ensure healthy and physiological function of the oral cavity. Surfactant proteins play an important role in innate immunity and surface stability of fluids. This study aimed to evaluate the expression and presence of surfactant proteins (SP) A, B, C, and D in human salivary glands and saliva. The expression of mRNA for SP-A, -B, -C and -D was analyzed by RT-PCR in healthy parotid and submandibular glands. Deposition of all surfactant proteins was determined with monoclonal antibodies by means of Western blot analysis and immunohistochemistry in healthy tissues and saliva of volunteers. Our results show that all four surfactant proteins SP-A, SP-B, SP-C and SP-D are peptides of saliva and salivary glands. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D appear to be involved in immune defense inside the oral cavity. Furthermore, by lowering surface tension between saliva and the epithelial lining of excretory ducts, SP-B and SP-C may assist in drainage and outflow into the oral cavity. Further functions such as pellicle formation on teeth have yet to be determined.

Detection and regulation of cationic amino acid transporters in healthy and diseased ocular surface.

PURPOSE: To evaluate the presence and role of human cationic amino acid transporters (hCATs) at the ocular surface in healthy and pathologic states and under experimental inflammatory conditions. METHODS: Expression of mRNA for hCATs 1, 2, and 3 (SLC7A1, SLC7A2, and SLC7A3) was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts and in an SV40 immortalized human corneal epithelial (HCE) cell line. Localization of hCAT1 and hCAT2 was determined by immunohistochemistry in healthy tissues and in sections of different corneal abnormalities, including keratoconus, Fuchs dystrophy, and herpetic keratitis. Cultured corneal epithelial cells were stimulated with proinflammatory cytokines and supernatants of Staphylococcus aureus and Pseudomonas aeruginosa and were analyzed by real-time PCR. RESULTS: Expression of hCAT1 and hCAT2 mRNA, but not of hCAT3 mRNA, was detected in healthy conjunctiva, cornea, and nasolacrimal ducts. Human lacrimal gland revealed only hCAT2 mRNA expression. Immunohistochemistry demonstrated the presence of hCAT1 and hCAT2 in epithelial cells of cornea, conjunctiva, and nasolacrimal ducts. Goblet cells revealed no reactivity. Moreover, hCAT2 was visible in acinar cells of lacrimal gland. No changes in staining reactivity were obtained for hCAT1 in different corneal abnormalities. In contrast, hCAT2 showed increased subjective staining intensity in all corneal abnormalities. Cell culture experiments revealed that TNF-alpha and supernatant of S. aureus increased hCAT1 and hCAT2 expression significantly. Supernatant of P. aeruginosa led to an increase in hCAT2-expression only. CONCLUSIONS: These results show for the first time the distribution of hCATs in tissues of the ocular surface and lacrimal apparatus. Both transporters seem to be differently regulated under pathologic conditions of the ocular surface. Their physiological functions in amino acid transport make them potential candidates for therapeutic intervention.

Regulation of MUC16 by inflammatory mediators in ocular surface epithelial cell lines.

The aim of the present study was to evaluate the regulation of membrane-anchored mucin MUC16 by proinflammatory cytokines and bacterial components at the ocular surface. Expression and distribution of MUC16 in conjunctival (HCjE) and corneal (HCE) epithelial cell lines was monitored by RT-PCR and immunohistochemistry. To determine the regulation of MUC16, cultured HCjEs and HCEs were stimulated with different cytokines, bacterial components and bacterial supernatants, and analyzed by real-time PCR, immunodot blot and immunohistochemistry. The results indicate that MUC16 is differentially regulated between HCjEs and HCEs after challenge with inflammatory mediators and suggest shedding of MUC16 from the ocular surface epithelia into the tear film. This seems to be precisely regulated. MUC16 shedding can be differentially increased and decreased, suggesting a protective function of membrane-anchored MUC16 and supporting the hypothesis that dysregulation of membrane-anchored MUC16 at the ocular surface may be involved in dry eye pathology.

Intestinal trefoil factor/TFF3 promotes re-epithelialization of corneal wounds.

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study using cornea as a model, we demonstrate the importance of trefoil factor 3 (TFF3, also known as intestinal trefoil factor) in re-epithelialization of wounds. In two different models of corneal wound healing, alkali- and laser-induced corneal wounding, we analyzed the wound healing process in in vivo as well as in combined in vivo/in vitro model in wild type (Tff3(+)(/)(+)) and Tff3-deficient (Tff3(-)(/)(-)) mice. Furthermore, we topically applied different concentrations of recombinant human TFF3 (rTFF3) peptide on the wounded cornea to determine the efficacy of rTFF3 on corneal wound healing. We found that Tff3 peptide is not expressed in intact corneal epithelium, but its expression is extensively up-regulated after epithelial injury. Re-epithelialization of corneal wounds in Tff3(-/-) mice is significantly prolonged in comparison to Tff3(+/+) mice. In addition, exogenous application of rTFF3 to the alkali-induced corneal wounds accelerates significantly in in vivo and in combined in vivo/in vitro model wound healing in Tff3(+/+) and Tff3(-/-) mice. These findings reveal a pivotal role for Tff3 in corneal wound healing mechanism and have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.

Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system.

PURPOSE: To evaluate the expression and presence of surfactant protein (SP) A and SP-D in the lacrimal apparatus, at the ocular surface, and in tears in healthy and pathologic states. METHODS: Expression of mRNA for SP-A and SP-D was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts as well as in a spontaneously immortalized conjunctival epithelial cell line (HCjE; IOBA-NHC) and a SV40-transfected cornea epithelial cell line (HCE). Deposition of SP-A and SP-D was determined by Western blot, dot blot, and immunohistochemistry in healthy tissues, in tears, aqueous humor, and in sections of different corneal abnormalities (keratoconus, herpetic keratitis, and Staphylococcus aureus-based ulceration). Cell lines were stimulated with different cytokines and bacterial components and were analyzed for the production of SP-A and SP-D by immunohistochemistry. RESULTS: The presence of SP-A and SP-D on mRNA and protein levels was evidenced in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal duct samples. Moreover, both proteins were present in tears but were absent in aqueous humor. Immunohistochemistry revealed the production of both peptides by acinar epithelial cells of the lacrimal gland and epithelial cells of the conjunctiva and nasolacrimal ducts, whereas goblet cells revealed no reactivity. Healthy cornea revealed weak reactivity on epithelial surface cells only. In contrast, SP-A and SP-D revealed strong reactivity in patients with herpetic keratitis and corneal ulceration surrounding lesions and in several immigrated defense cells. Reactivity in corneal epithelium and endothelium was also seen in patients with keratoconus. Cell culture experiments revealed that SP-A and SP-D are produced by both epithelial cell lines without and after stimulation with cytokines and bacterial components. CONCLUSIONS: These results show that SP-A, in addition to SP-D, is a peptide of the tear film. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D seem to be involved in several ocular surface diseases.

MUC16 in the lacrimal apparatus.

The aim of the present study was to determine the possible expression of the mucin MUC16 in the lacrimal apparatus. Expression and distribution of MUC16 in lacrimal gland, accessory lacrimal glands, and nasolacrimal ducts was monitored by RT-PCR and immunohistochemistry. MUC16 was expressed and detected in all tissues investigated. Comparable to conjunctiva and cornea it was membrane-anchored in accessory lacrimal glands whereas in lacrimal gland acinar cells and columnar cells of the nasolacrimal ducts it was stored in intracytoplasmic vesicles without membrane-association. Subepithelial serous glands of the nasolacrimal ducts revealed staining of the secretion product. Intracelluar production of MUC16 is present in lacrimal gland and epithelial cells of the nasolacrimal ducts but it is not clear whether this MUC16 is secreted. MUC16 seems to be shedded or secreted from the epithelial surface of subepithelial serous glands of the nasolacrimal ducts. Our results show that MUC16 is present in the whole lacrimal apparatus. Its distribution pattern suggests different physiological functions with regard to tear film physiology and tear outflow. Moreover, the results demonstrate the existence of so far not recognized qualitative differences in the secretion product of main lacrimal gland and accessory lacrimal glands (glands of Krause).