Dry eye is matched by increased intrasubject variability in tear osmolarity as confirmed by machine learning approach. / El ojo seco está relacionado con un aumento intrasujeto de la variabilidad de osmolaridad lagrimal confirmado por tecnología de aprendizaje de máquinas.

OBJECTIVE: Because of high variability, tear film osmolarity measures have been questioned in dry eye assessment. Understanding the origin of such variability would aid data interpretation. This study aims to evaluate osmolarity variability in a clinical setting. MATERIAL AND METHODS: Twenty dry eyes and 20 control patients were evaluated. Three consecutive osmolarity measurements per eye at 5min intervals were obtained. Variability was represented by the difference between both extreme readings per eye. Machine learning techniques were used to quantify discrimination capacity of tear osmolarity for dry eye. RESULTS: Mean osmolarities in the control and dry eye groups were 295.1±7.3mOsm/L and 300.6±11.2mOsm/L, respectively (P=.004). Osmolarity variabilities were 7.5±3.6mOsm/L and 16.7±11.9mOsm/L, for the control and dry eye groups, respectively (P<.001). Based on osmolarity, a logistic classifier showed an 85% classification accuracy. CONCLUSIONS: In the clinical setting, both mean osmolarity and osmolarity variability in the dry eye group were significantly higher than in the control group. Machine learning techniques showed good classification accuracy. It is concluded that higher variability of tear osmolarity is a dry eye feature.

High prevalence of Demodex spp. infestation among patients with posterior blepharitis: correlation with age and cylindrical dandruff. / Alta prevalencia de infestación por Demodex spp. en pacientes con blefaritis posterior: correlación con edad y caspa cilíndrica.

OBJECTIVE: To determine prevalence of Demodex spp. and infestation index (II) by the parasite among patients of different ages with blepharitis and to assess association with occurrence of cylindrical dandruff (CC). MATERIALS AND METHODS: Prospective study including patients diagnosed with posterior blepharitis between 2013 and 2015, without previous acaricide treatment, was conducted by Fundación Oftalmológica Los Andes (Chile). Four eyelashes were randomly extracted from each eyelid for parasite detection. The II was calculated as the ratio between the total number of demodex specimens found in the total number of eyelashes removed. A semi-quantitative determination of the CC was performed. RESULTS: A total of 178 patients (91 men, 87 women), with a mean age of 58.49±20.66 years, were included. It was found that 83.7% of patients were infested with Demodex folliculorum with a mean II of 0.96±0.84 mites/eyelash. The II was significantly higher in patients over 50 years (p<.0001). Patient age and II showed a Pearson correlation coefficient (R2) of 0.12 (p<.0001). CC was observed in 71.4% of patients, with those over 50 years-old showing significantly higher values. A positive correlation was also observed between the amount of CC and the II (R2=.33; p<.0001). CONCLUSIONS: The prevalence of infestation by Demodex folliculorum is high in patients with posterior blepharitis. The II by the parasite is positively correlated with age and with the occurrence of CC on the eyelid border.

[Conjunctival impression cytology in patients with normal and impaired OSDI scores]. / Citología de impresión conjuntival en pacientes con valores OSDI normales y alterados.

PURPOSE: To describe goblet cell density and Nelson grading in different areas of the ocular surface using conjunctival impression cytology (CIC) among patients with normal and impaired Ocular Surface Disease Index (OSDI) scores. MATERIAL AND METHODS: Patients (n=166) under assessment for dry eye were recruited between 2011 and 2012 and classified according to the OSDI score in 4 categories (normal and impaired). Cytological study (CIC plus Papanicolaou staining) using the Nelson grading system, with modifications in staging, and goblet cell counting were performed on the nasal, temporal, inferior, and superior bulbar conjunctival surfaces. RESULTS: Nelson grading was significantly higher in patients with a severely impaired OSDI score (1.41±0.14) compared to normal patients (0.86±0.09) (P<.01). Goblet cell density was significantly reduced in patients with a severely impaired OSDI score (310.24±56.24 cells per sample) compared with normal subjects (497.31±50.07 cells per sample) (P<.001). Compared with the photoexposed bulbar conjunctiva, goblet cell density on the non-photoexposed conjunctiva was significantly higher both in patients with mild (P<.01) and moderate (P<.001) OSDI scores. CONCLUSION: Patients with severely impaired OSDI scores have less goblet cells and a higher Nelson grade. Goblet cells are more abundant on the non-photoexposed conjunctiva.

Cell-enlargement-related polypeptides are induced via beta(1)-adrenoceptors in mouse parotids.

Induction of cell and gland enlargement (growth-in-size) and induction of a group of secretory polypeptides (polypeptides C-G) seem to occur in close relationship in mouse parotid glands stimulated chronically by the nonselective beta-adrenergic agonist isoproterenol. To determine whether beta(1), beta(2), or both subtypes of beta-adrenergic receptors are involved in those responses, dose-dependency studies were carried out during a 7-day period of daily stimulations to assess the relative abilities of the selective beta-adrenergic agonists dobutamine (beta(1)) and salbutamol (beta(2)) to induce polypeptides C-G and growth-in-size. The relative abilities of the selective beta-adrenoceptor antagonists atenolol (beta(1)) and I.C.I. 118.551 (beta(2)) to interfere with the induction of both responses by chronic treatment with the various beta-adrenergic agonists were also studied. Parotid growth-in-size was assessed by evaluating wet weight, whole protein content, and light microscopy histology. The presence of polypeptides C-G was evaluated after SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Under these experimental conditions, dobutamine was found to be at least one order of magnitude more potent than salbutamol at inducing growth-in-size. Dobutamine was also found to be clearly stronger than salbutamol as an inducer of polypeptides C-G. On the other hand, atenolol was more effective than I.C.I. 118.551 at preventing the induction of polypeptides C-G and growth-in-size by isoproterenol, dobutamine, or salbutamol. Taken together, these results suggest that in mouse parotid glands, polypeptides C-G and growth-in-size are induced preferentially via adrenergic receptors of the beta(1)-subtype.

Plasma membrane desialylation and DNA synthesis are induced via adrenergic receptors of the beta 1-subtype in mouse parotid glands.

Induction of DNA synthesis and plasma membrane desialylation in the early prereplicative period in isoproterenol-stimulated mouse parotid glands seem to occur in a close relationship. beta 1, beta 2 or both subtypes of adrenergic receptors could be involved in those responses. To discriminate between those alternatives, dose-dependency studies were addressed to assess the abilities of the selective beta-adrenergic agonists dobutamine (beta 1) and salbutamol (beta 2) to induce plasma membrane desialylation and DNA synthesis. Likewise, the abilities of the selective beta-adrenoceptor antagonists atenolol (beta 1) and ICI 118,551 (beta 2) to interfere with the induction of both responses were also studied. Dobutamine was found to be at least 10-fold more potent than salbutamol in inducing DNA synthesis whereas ICI 118,551 was much weaker than atenolol, as inhibitor of the agonist-induced DNA synthesis. On the other hand, dobutamine was also more potent than salbutamol in provoking plasma membrane desialylation. However, whenever an increase in the rate of DNA synthesis was induced by either agonist, a constant 50% reduction in the levels of sialic acid in the plasma membranes was observed. In the same direction, atenolol, at variance to ICI 118,551, was able to produce a full suppression of the plasma membrane desialylation induced by either selective agonist. Taken together, these results suggest that in mouse parotid glands, both DNA synthesis and plasma membrane desialylation during the early prereplicative period are induced preferentially via adrenergic receptors of the beta 1-subtype.

Transcriptional and posttranscriptional control in synthesis of growth marker polypeptides in mouse parotids.

The chronic daily administration of isoproterenol provokes in mouse parotid glands the induction and progressive accumulation of a family of secretory polypeptides named polypeptides C, D, E, F, and G (polypeptides C-G). These polypeptides, which seem to be part of the family of proline-rich proteins, have been considered as molecular markers of the growth-in-size response in the mouse parotid acinar cells. In the present study, two pharmacological approaches were used to determine whether the induction and the postsecretory reappearance of polypeptides C-G may be distinguished from each other. First, actinomycin D, a transcriptional inhibitor, was found to interfere with the induction by isoproterenol but not with the postsecretory reappearance. Second, pilocarpine, a secretagogue that was found to be a very weak inducer of polypeptides C-G, was able to provoke secretion and then reappearance of the whole group of isoproterenol-induced polypeptides. Accordingly, these data suggest that the induction of polypeptides C-G is dependent on transcriptional activity and that it is unrelated to secretion stimulation. By contrast, the postsecretory reappearance of polypeptides C-G may occur even when transcriptional activity is inhibited and it would be related to the secretory activity.

Effect of isoproterenol analogs on the polypeptide composition of mouse parotid glands: relationship to enhanced growth.

The chronic administration of isoproterenol ((+-)-1-(3,4)-dihydroxyphenyl)-2-isopropylaminoethanol) induces both the accumulation of a family of secretory polypeptides (polypeptides C, D, E, F and G) and growth in size in mouse parotid glands. Eleven isoproterenol analogs including minor structural modifications either at the aromatic ring, at the ethanol-derived residue or at the end group bonded to the amino of the side chain, were analysed regarding their ability to produce those two responses. Analogs were distributed into two groups, namely inducers and noninducers. Inducer isoproterenol analogs provoked a massive accumulation of polypeptides C, D, E, F and G and were active in producing parotid gland enlargement. Noninducer isoproterenol analogs produced neither changes in the polypeptide composition nor growth response in these glands. Thus, a correlation between accumulation of polypeptides C, D, E, F and G and the growth in size response in parotid glands was more firmly established. In considering the chemical structure of the isoproterenol analogs with regard to their inducer or noninducer character, the three main domains taken into account appeared to participate in the inductive process. However, while an intact ethanol-derived domain was found to be absolutely necessary for the inductive ability of the analogs, both the aromatic ring as well as the substituent on the side-chain amino group could experience several modifications without resulting in loss of the inductive character.

Secretory character of a group of isoproterenol-induced polypeptides in mouse parotid glands.

The secretory nature of the isoproterenol-induced mouse parotid polypeptides C, D, E, F, and G (molecular weights 64,000, 61,000, 51,500, 38,000, and 37,000, respectively) is documented. Polypeptides C, D, E, F, and G, accumulated in response to successive daily stimulations with isoproterenol, were detected in a fraction enriched in hypertrophic parotid acinar cells. These cells, characterized by an increased content of cytoplasmic granules, maintain a secretory responsiveness to isoproterenol, which has been evidenced by light microscopy, enzymatic analysis, and unidimensional SDS-polyacrylamide gel electrophoresis. Thus, a parallelism in the loss and recovery of both secretory granules, alpha-amylase and polypeptides C, D, E, F, and G, was observed. Moreover, after secretion stimulation, polypeptides C, D, E, F, and G were detected in the fluid collected directly from parotid gland cannulation. Given the secretory character of polypeptides C, D, E, F, and G, mechanisms explaining both their progressive accumulation along the chronic administration of isoproterenol, as well as their progressive disappearance observed after suspending that treatment, are discussed.

Changes in the content and distribution of sialic acid on the basal surface of isoproterenol-stimulated mouse parotid acinar cells.

The presence, distribution and content of sialic acid on the cell surface in collagenase-dispersed acini obtained both from unstimulated as well as from in vivo isoproterenol-stimulated mouse parotid have been studied. To this end, sialic acid residues have been qualitatively and quantitatively analyzed by 1) cytochemical labeling by wheat germ agglutinin (WGA), 2) biochemical procedures and 3) isotopic labeling by [3H]WGA (WGA-N-[acetyl-3H]-acetylated). Electron microscopy revealed striking differences in the binding of ferritin-conjugated WGA at the basal, lateral and apical cell surface. Unstimulated acinar cells showed a heavy patch-distributed binding of ferritin-conjugate on the basal cell surface while it was homogeneous and very scarce on the lateral one and absent on the apical cell surface. During the first few hours after isoproterenol, the WGA binding sites at the basal cell surface became homogeneously distributed. This fact was coincident with a loss of about 60 to 70% both in the content of neuraminidase-releasable sialic acid and in the binding of [3H]WGA to the acinar surface. These findings suggest that the release of sialic acid as free residues, which has been involved in the isoproterenol-triggered cell proliferation-inducing mechanism in the mouse parotid, would occur at the glycocalyx corresponding to the basal plasma membrane of the acinar cells.

Changes in the polypeptide composition related to the growth response in chronically isoproterenol-stimulated mouse parotid glands.

The administration of isoproterenol induces DNA-synthesis mitosis and growth (increase in size) responses in mouse parotid glands. Both responses were uncoupled by means of daily stimulations with isoproterenol in such a way that the DNA-synthesis mitosis response was observed during the first 4 days only, whereas the growth response was continuous since the first stimulation until about day 12. In parallel to the chronic stimulation by isoproterenol, drastic changes in the polypeptide composition of parotid glands were observed. These modifications, consisting basically of the reduction in content of a couple of major poly peptides (polypeptides A and B) together with the reciprocal massive accumulation of five new polypeptides (polypeptides C, D, E, F and G), were also progressive and continuous along the chronic stimulation by isoproterenol, even after the disappearance of the DNA-synthesis mitosis response. Thus, a relationship between specific changes in the mouse parotid content of polypeptides A, B, C, D, E, F and G and the isoproterenol-induced growth response, rather than with the DNA-synthesis mitosis response, is suggested. The correlation is firmly supported by the progressive recovery of the normal polypeptide composition upon suspending isoproterenol treatment, which allows parotid glands to return to normal size parameters.

Changes in the polypeptide composition of mouse parotid glands after stimulation of secretion and DNA synthesis by isoproterenol.

The polypeptide composition of mouse parotid glands has been analysed by unidimensional SDS-polyacrylamide slab gel electrophoresis and Coomassie blue staining after isoproterenol stimulation of secretion and DNA synthesis. Two polypeptides (polypeptides A and B) are lost within 2 h and their restoration in the glands occurs according to a chronology which is identical to that of the alpha-amylase activity. On the other hand, five clearly defined new bands appear consistently during the late prereplicative period of isoproterenol-stimulated mouse parotid acinar cells (polypeptides C, D, E, F and G). These new polypeptides are induced by doses of isoproterenol which provoke secretion and DNA synthesis, but not by doses which provoke only secretion. Although no function has been assigned to any of the above-described polypeptides, a relation between polypeptides A and B and secretion and between polypeptides C, D, E, F and G and the proliferative response is suggested.

Purification of plasma membranes from mouse parotid gland and membrane reorganization in response to isoproterenol.

Two highly purified plasma membrane fractions have been obtained from mouse parotid glands by a combination of differential centrifugation and isopycnic centrifugation in discontinuous sucrose gradients. The membranes were characterized by enzymic, chemical and morphological criteria. The effect of isoproterenol, which induces parotid acinar cells to proliferate, upon sialic acid and five different enzyme activities located in the plasma membrane phosphodiesterase (EC 3.1.4.1), Mg2+-ATPase (EC 3.6.1.4), leucine aminopeptidase (EC 3.4.1.1), protein kinase (EC 2.7.1.37) and sialyltransferase (EC 2.4.99.1), were quantified along the cell cycle. Plasma membrane sialic acid content falls 30% within 30 min and remains depressed for at least 6 h with the major restoration towards normal levels occurring between 12 and 16 h later. In contrast multiple daily isoproterenol injections lead to a more than 2-fold elevation of sialic acid content. Sialyltransferase activity rises 2-fold by 12 h after isoproterenol treatment and then rapidly falls. This enzyme has a pH optimum of 6.5, requires a divalent cation for activity and is inhibited by Triton X-100. Other enzyme activities showed markedly different changes after isoproterenol stimulation, either increasing, decreasing or remaining unaltered. These continuous functional modifications suggest an active role of the plasma membrane in the control of the proliferative cycle.

The application of a simple, sensitive disc assay for sialyltransferase activity to particulate and soluble enzyme preparations and its use in measuring enzymatic activity in human plasma samples.

A simple assay for the determination of sialyltransferase activity is described. The method involves the isolation of the radioactive reaction product on cellulose paper discs washed with trichloroacetic acid, thereby greatly facilitating the handling of large numbers of assays. This procedure is both more accurate and more sensitive than that involving precipitation with protein denaturants and separation by centrifugation, and is applicable to both soluble and particulate enzyme preparations. The application of the method to the determination of sialytransferase in human plasma is detailed. Enzyme activity is elevated two-fold in both cancer patients and patients with non-malignant disease. This suggests that the diagnostic use of determinations of sialyltransferase activity may be limited by its non-specificity.