Differential protein expression in tears of patients with primary open angle and pseudoexfoliative glaucoma

Primary open angle (POAG) and pseudoexfoliative glaucoma (PXG) are the most common primary and secondary forms of glaucoma, respectively. Even though the patho-physiology, aqueous humor composition, risk factors, clinical features, therapy and drug induced ocular surface changes in POAG and PXG have been widely studied, to date information concerning tear protein characterization is lacking. Tears are a source of nourishment for ocular surface tissues and a vehicle to remove local waste products, metabolized drugs and inflammatory mediators produced in several ophthalmic diseases. In glaucoma, the proteomic definition of tears may provide insights concerning patho-physiology of the disease and ocular surface modifications induced by topical therapy. Our study aimed at characterizing protein patterns in tears of patients with medically controlled POAG and PXG. A comparative tears proteomic analysis by label-free LC-MS(E) highlighted differences in the expression of several proteins in the two glaucoma sub-types and control subjects, highlighting inflammation pathways expressed in both diseases. Results were independently reconfirmed by SDS-PAGE and linear MALDI-TOF MS, validating altered levels of Lysozyme C, Lipocalin-1, Protein S100, Immunoglobulins and Prolactin Inducible Protein. Moreover, we found a differential pattern of phosphorylated Cystatin-S that distinguishes the two pathologies. The most relevant results suggest that in both pathologies there may be active inflammation pathways related to the disease and/or induced by therapy. We show, for the first time, tear protein patterns expressed under controlled intraocular pressure conditions in POAG and PXG subjects. These findings could help in the understanding of molecular machinery underlying these ophthalmologic diseases, resulting in early diagnosis and more specific therapy.     

Pharmacokinetic analysis of topical tobramycin in equine tears by automated immunoassay

ABSTRACT: BACKGROUND: Ophthalmic antibiotic therapy in large animals is often used empirically because of the lack of pharmacokinetics studies. The purpose of the study was to determine the pharmacokinetics of topical tobramycin 0.3% ophthalmic solution in the tears of normal horses using an automated immunoassay analysis. RESULTS: The mean tobramycin concentrations in the tears at 5, 10, 15, 30 minutes and 1, 2, 4, 6 hours after administration were 759 ([PLUS-MINUS SIGN]414), 489 ([PLUS-MINUS SIGN]237), 346 ([PLUS-MINUS SIGN]227), 147 ([PLUS-MINUS SIGN]264), 27.6 ([PLUS-MINUS SIGN]28.4), 14.8 ([PLUS-MINUS SIGN]66.6), 6.7 ([PLUS-MINUS SIGN]18.6), and 23.4 ([PLUS-MINUS SIGN]73.4) mg/L. Mean tobramycin concentration was maintained above the MIC90 for commonly isolated bacteria for 68.5 min. CONCLUSION: A single dose of topical tobramycin resulted in therapeutic concentrations of tobramycin in the tears for 1 h after administration. Therapeutic levels of tobramycin remained in equine tears 6 times longer than was reported in rabbit tears.     

Collagen type IX and HNK-1 epitope in tears of patients with pseudoexfoliation syndrome

Pseudoexfoliation syndrome (PEX) is an age-related condition, which may cause open-angle glaucoma and has increasing interest since it seems to affect additional human tissues, i.e., cardiovascular tissue, skin, and still lacks elucidated pathogenesis. Collagen type IX and HNK-1 epitope have been considered characteristic constituents of the aqueous humour of PEX patients, since their amounts were increased in PEX aqueous humour compared to normal eyes. Since it has been proposed that the initial manifestations of PEX syndrome occur in conjunctiva, the present study was undertaken to investigate the presence of the same antigens in tears of PEX patients and their possible use as the biochemical markers for early diagnosis. Tears of PEX patients and healthy individuals were subjected to western blotting analysis for various basement membrane components identified in aqueous humour. It was found that collagen type IX and HNK-1 epitope were present in tears, the amount of the former being increased 2.7 times compared to normal (P<0.05), surprisingly high as compared with total protein or lysozyme activity in tears, which were found to be increased in PEX patients about 25% with no statistical differences (P approximately 0.4). The results suggest the possible use of tears' collagen type IX for the diagnosis of PEX syndrome.     

Proteolytic 'defences' and the accumulation of oxidized polypeptides in cataractogenesis and atherogenesis

Over the last few years, it has been clearly established that normal plasma contains low levels of oxidized polypeptides, and that these accumulate in tissues during several age-related pathologies. In contrast, normal mammalian aging, contrary to conventional dogma, is not clearly associated with enhanced levels of oxidized proteins, except in extracellular connective tissues, whose proteins can, for example, be oxidized by the neutrophil oxidative burst. Since mildly oxidized proteins are susceptible to accelerated degradation in most experimental systems, the question arises as to how the accumulation of oxidized proteins can take place. Such accumulation requires an excess of production (or deposition) over removal, which might reflect alterations in capacity or rate of production or removal. This chapter discusses our presently limited knowledge of rates and control of proteolysis of oxidized proteins in two pathologies, cataractogenesis and atherogenesis. It commences with a brief summary of current understanding of the mechanisms of protein oxidation, and of the observed accumulation of oxidized proteins in several pathologies.     

薏苡种质资源萌发期抗旱性鉴定及抗旱指标筛选

干旱是影响薏苡生产的主要因素之一,鉴定薏苡种质资源的抗旱性,筛选抗旱指标,培育抗旱品种,对薏苡产业的发展具有重要意义。本研究首先以抗旱性不同的6份薏苡种质为材料,通过调查5个不同浓度的PEG-6000水溶液模拟干旱胁迫下的发芽率,确定了薏苡萌发期模拟干旱胁迫的最适PEG-6000水溶液渗透势为-0.1 MPa。然后以-0.1 MPa的PEG-6000水溶液模拟干旱胁迫,探讨50份薏苡种质发芽势、发芽率、萌发指数、芽长、芽粗、芽鲜重、芽干重、根长、根粗、根鲜重和根干重的变化,利用综合评价法对50份薏苡种质进行萌发期抗旱性鉴定及抗旱指标筛选。结果表明,干旱胁迫对薏苡萌发期各指标均有极显著影响。筛选出萌发期抗旱性强的薏苡种质分别为yy18-1、yy14-3和yy13-1,可为薏苡抗旱育种、抗旱机理及干旱调控缓解机制的研究提供基础材料。芽长、芽干重、根长、根鲜重和根干重可作为薏苡种质资源萌发期简单、直观的抗旱性评价指标。     

Effect of tissue-type plasminogen activator and its inhibitor in haemostasis system function in norm and pathology

The paper is dedicated to the study of structure and physiological functions of tissue type plasminogen activator and its inhibitor PAI-1, changes of these parametres in normal and pathology conditions. The interrelation of the coagulation and the fibrinolytic system during the range of pathologies was studied. It was demonstrated that simultaneous analysis of the coagulation and fibrinolytic systems analysis allow to diagnose the thrombotic complications.     

Detection of anti-HIV IgA in tears of children born to seropositive mothers is highly specific

In order to improve the diagnosis of HIV infection in children born to seropositive mothers, 86 children were previously tested by Western blotting for anti-HIV IgA in tears and IgG in serum, at a median age of 9.2 months. To determine the exact value of the assay, 68/86 children of the same cohort were retested 9 months later.Nine children (13.4 %) were seropositive and all had anti-HIV IgA in tears. Eight of them had possessed lachrymal antibodies 9 months earlier. The ninth child was seronegative when 9 months old and then seroconverted. Four children (6 %), known to be seronegative, had an indeterminate Western blot pattern and no HIV IgA in tears. Fifty four (80.6 %) were seronegative at 18 months; none of them had ever had anti-HIV IgA in tears. This highlights the fact that only the children without lachrymal HIV IgA at the age of 9 months became seronegative at the age of 18 months.Our results clearly show that the detection of anti-HIV IgA in tears is a highly specific and reliable diagnostic test in children aged less than 15 months, born to seropositive mothers.     

Factor structure of regional cerebral blood flow and glucose metabolism rate as a tool to study the default mode of the brain

The functional connectivity of anatomical and functional brain structures in the state of operational rest was assessed on the basis of positron emission tomography (PET) data to study the so-called default mode of the brain, i.e., the brain’s spontaneous activity at rest. It is concluded that the possibility of identifying neuroanatomical systems of the default mode (default mode network) in routine clinical PET studies of the cerebral blood flow and glucose metabolism is important for studying the functional organization of the brain in the normal state and its rearrangements in pathologies.     

Morphologic features of spontaneous annular tears and disc degeneration in the aging sand rat (Psammomys obesus obesus)

The sand rat, a member of the gerbil family, is a valuable small animal model in which intervertebral disc degeneration occurs spontaneously as the animal ages. Radiographic features of cervical and lumbar degeneration resemble those in human spines. We conducted a retrospective analysis of spines of 140 animals 3?41 months old focusing specifically on the presence of annular tears that are not visible by radiography and have not been described previously in the sand rat disc. During degeneration of the nucleus pulposus, notochordal cell death occurs and granular material, which stains with Alcian blue for proteoglycans, accumulates. Lamellar architecture also deteriorates and annular tears occur that are morphologically similar to the concentric, radiating and transdiscal annular tears in human discs. These tears contain granular material that provides a “marker” that can be used to distinguish the annular tears from artefactual separations during sectioning. We observed lamellar degeneration and separation in the annulus fibrosus at 4 months with associated tears that contained granular material in the nucleus. Tears that contained granular material and displacement of the degenerating nucleus were common in cervical and lumbar discs of animals older than 9 months; some specimens showed tears at 4 and 5 months. With advanced degeneration, granular globules were displaced dorsally adjacent to and into the spinal cord area and also ventrally into regions where osteophytes formed. We present morphologic data that expand the utility of this rodent model of spontaneous age-related disc degeneration and provide novel information on annular tears and disc degeneration.     

Proteomic analysis of secretion from human transplanted submandibular gland replacing lacrimal gland with severe keratoconjunctivitis sicca

Purpose: Proteomic analysis of secretions from transplanted or non-transplanted submandibular glands in patients with severe keratoconjunctivitis sicca and tears from normal eyes. Experimental design: Secretions from submandibular glands transplanted to replace lacrimal glands and non-transplanted submandibular glands were collected at 1year from 5 patients with severe keratoconjunctivitis sicca undergoing transplantation, and tears were collected from 3 normal subjects. 2-D electrophoresis (2-DE), then mass spectrometry was used to identify proteins. Western blot analysis was used to confirm protein expression. Results: We identified 34 and 11 distinct proteins in the saliva from transplanted submandibular glands and tears, respectively. The saliva from transplanted submandibular glands contained almost all the proteins abundant in tear fluid. The functions of identified proteins in the saliva from transplanted submandibular gland were mainly immune response and anti-bacterial. In total, 7 proteins showed differential expression between the saliva of transplanted and non-transplanted submandibular glands. The upregulation of short palate, lung and nasal epithelium carcinoma-associated protein 2 and carbonic anhydrase VI was confirmed by Western blot analysis. Conclusions: Identified proteins in saliva from transplanted submandibular glands may protect ocular structures. These findings can help in understanding the functional status of transplanted submandibular glands.     

Supraspinatus tendon organizational and mechanical properties in a chronic rotator cuff tear animal model

Rotator cuff tears of the shoulder are a common cause of pain and disability. The successful repair of rotator cuff tendon tears depends on the time from onset of injury to the time of surgical repair. However, the effect of time from injury to repair remains poorly understood. A rat model was used to investigate the supraspinatus tendon organizational and mechanical property changes that occur with time post-injury to understand the natural injury response in the absence of repair. It was hypothesized that increased time post-injury would result in increased detrimental changes to tendon organizational and mechanical properties. Tendons were detached at the insertion on the humerus without repair and the quantitative organizational and mechanical properties were analyzed at 1, 2, 4, 8, and 16 weeks post-detachment. Tendon detachment resulted in a dramatic decrease in mechanical properties initially followed by a progressive increase with time. The quantitative collagen fiber orientation results provided corroborating support to the mechanical property data. Based on similarities in histology and mechanical properties to rotator cuff tears in humans, the animal model presented here is promising for future investigations of the tendon's natural injury response in the absence of repair.     

Use of high resolution algorithm in computerized tomography of disseminated lung diseases]

Lobule of the lung is a principal anatomical structure of the respiratory zone in the lungs. Secondary lobule of the lung consists of about fifty primary lobules and is delineated by fibrous interlobular septa. Each lobule is delineated by interlobular septa and blood is supplied by small arterioles of pulmonary artery system. Its shape is conical and size ranges from 10 to 22 mm. The author used algorithm of high resolution in HR CT to present normal distal generations of respiratory area of the lungs and the same in 32 patients with disseminated lesions to the lungs. It is known, that lobule of the lung may be involved in very important pathologies. The author shown that significant architectural rearrangement takes place in pulmonary lobules and adjacent areas in the course of disseminated pulmonary diseases. The process include thickening of interlobular septa, consolidation of lobular area, changes in the shape of lobuli, and appearance of small cysts. The use of HR CT enables imaging in such pathologies which normally are inaccessible to conventional radiologic examinations.     

The oligosaccharidic component of the glycoconjugates in lichen planus, granuloma annulare, seborrheic keratosis and plamoplantar keratoderma: lectin histochemical study.

It is well known that cell surface glycoconjugates play an important role in cell proliferation, adhesion and differentiation. The aim of this investigation was to define the changes of the glycoconjugate saccharidic moieties in the epidermis and derma of patients affected by several skin pathologies such as seborrheic keratosis, lichen planus, granuloma annulare and palmoplantaris keratoderma. Bioptical specimens from skin lesions as well as from normal skin were fixed in Carnoy's fluid and routinely processed. The sections were treated with HRP-lectins (PNA, DBA, SBA, WGA, ConA, LTA and UEAI). Cytochemical controls were performed for specificity of lectin-sugar reaction. Some sections were pre-treated with neuraminidase prior to staining with HRP lectins. In comparison with normal human skin, epidermal lectin binding pattern in the considered diseases showed considerable qualitative and quantitative variations. In general, in all the considered pathologies, a lack and/or a decrease in lectin binding at the epidermal layers was observed; among the various diseases, differences in cellular localisation of the sugar residues were also noted. In such respect, an exception was represented by seborrheic keratosis, where the cells of the basal layer showed PNA reactivity, which was absent in the basal layer of the normal skin. Although seborrheic keratosis and lichen planus have been studied by others authors, our findings are not in total accordance concerning lectin binding; this is probably due to the different fixatives employed. Our findings seem to reveal significant changes in keratinocyte glycoconjugate oligosaccharides in the previously mentioned diseases, providing clues to their pathogenesis.     

Estimation of C3 levels in cerebrospinal fluid by electroimmunodiffusion

C3 levels have been determined by the electroimmunodiffusion technique in the CSF of patients with a wide variety of pathologies. The patients were grouped on the basis of protein content and G/A ratio of the CSF as I) patients with normal meningeal permeability and apparent absence of local gamma-globulin synthesis; II) patients with increased meningeal permeability; III) patients with characteristics of MS, i.e. increase of IgG accompanied by a normal or slightly elevated protein content, Group III showed a lower level of C3 when expressed at % of the total protein and also as % of the total protein less gamma-globulins of the CSF. Other parameters of the CSF are also recorded. It was shown that only the expression of C3 concentration relative to the total protein content of the CSF produced meaningful analytical data.     

Partie 7. Spécificités pédiatriques

Bone scintigraphy is a nuclear imaging scan using a radiopharmaceutical composed of a bisphosphonate coupled to a radionuclide (technetium 99m). Radiopharmaceutical uptake is particularly important at the level of the bone structures having a strong osteoblastic activity. These uptakes can be due to a benign pathology (fracture, loosening of prosthesis, rheumatic pathologies, etc.) or to a malignant pathology (primary or secondary bone lesion). The high sensitivity of bone scintigraphy makes it particularly interesting at the initial stage of the pathology, especially when X-rays are normal. In addition, its specificity has clearly improved in recent years with the increasingly use of tomoscintigraphy coupled with X-ray scanning (SPECT/CT). We describe the operating principle of bone scintigraphy, normal uptakes with its variants as well as pathological uptake features in traumatic, rheumatic, prosthetic or cancerous pathologies.     

Partie 1. Généralités

Bone scintigraphy is a nuclear imaging scan using a radiopharmaceutical composed of a bisphosphonate coupled to a radionuclide (technetium 99m). Radiopharmaceutical uptake is particularly important at the level of the bone structures having a strong osteoblastic activity. These uptakes can be due to a benign pathology (fracture, loosening of prosthesis, rheumatic pathologies, etc.) or to a malignant pathology (primary or secondary bone lesion). The high sensitivity of bone scintigraphy makes it particularly interesting at the initial stage of the pathology, especially when x-rays are normal. In addition, its specificity has clearly improved in recent years with the increasingly use of tomoscintigraphy coupled with X-ray scanning (SPECT/CT). We describe the operating principle of bone scintigraphy, normal uptakes with its variants as well as pathological uptake features in traumatic, rheumatic, prosthetic or cancerous pathologies.     

Partie 3. Infections osseuses (hors greffe sur matériel)

Bone scintigraphy is a nuclear imaging scan using a radiopharmaceutical composed of a bisphosphonate coupled to a radionuclide (technetium 99m). Radiopharmaceutical uptake is particularly important at the level of the bone structures having a strong osteoblastic activity. These uptakes can be due to a benign pathology (fracture, loosening of prosthesis, rheumatic pathologies, etc.) or to a malignant pathology (primary or secondary bone lesion). The high sensitivity of bone scintigraphy makes it particularly interesting at the initial stage of the pathology, especially when X-rays are normal. In addition, its specificity has clearly improved in recent years with the increasingly use of tomoscintigraphy coupled with X-ray scanning (SPECT/CT). We describe the operating principle of bone scintigraphy, normal uptakes with its variants as well as pathological uptake features in traumatic, rheumatic, prosthetic or cancerous pathologies.     

Partie 4. Rhumatologie

Bone scintigraphy is a nuclear imaging scan using a radiopharmaceutical composed of a bisphosphonate coupled to a radionuclide (technetium 99m). Radiopharmaceutical uptake is particularly important at the level of the bone structures having a strong osteoblastic activity. These uptakes can be due to a benign pathology (fracture, loosening of prosthesis, rheumatic pathologies, etc.) or to a malignant pathology (primary or secondary bone lesion). The high sensitivity of bone scintigraphy makes it particularly interesting at the initial stage of the pathology, especially when X-rays are normal. In addition, its specificity has clearly improved in recent years with the increasingly use of tomoscintigraphy coupled with X-ray scanning (SPECT/CT). We describe the operating principle of bone scintigraphy, normal uptakes with its variants as well as pathological uptake features in traumatic, rheumatic, prosthetic or cancerous pathologies.     

Purification, cloning and regulation of a novel acid-lipase-like protein of hamster expressed in lacrimal glands and tears during lactation

We report a novel 48-kDa tear acid-lipase-like protein (TALLP), which is markedly induced in lacrimal glands (LG) and secreted in tears of hamster dams during lactation. TALLP is undetectable in LG and tears of normal hamsters, but is also induced after gonadectomy in both sexes and this is prevented by androgen, estrogen or thyroid hormone treatment. These observations and the obliteration of TALLP upon cessation of lactation suggest that endogenous estrogens (in females) and androgens (in males) completely repress TALLP expression. Purified TALLP is monomeric, contains approximately 18% N-glycosylation and several pI isoforms. TALLP expression was tissue-specific and immunolocalized in LG acinar cells. The cDNA deduced amino-acid sequence of TALLP precursor (398 residue, containing a 19 residues signal-peptide) showed only 43-48% identity with all known mammalian acid-lipases, including even those of other rodents, suggesting that TALLP is a prototype of a new category, within the acid-lipase family. Surprisingly, although the catalytic triad residues and other sequence features important for lipolytic activity are conserved in TALLP, it has no detectable lipase activity. However, TALLP binds the polarity sensitive hydrophobic probe, 1-aminoanthracene (K(d)=12 microM). TALLP might have a unique substrate-specificity or a lipid-binding/carrier function in tears of hamster dams. This is the first report of an acid-lipase-like protein secreted in tears of any species. Since TALLP lacks the usual lipase activity, it can be an excellent model to understand better what other structural features in acid-lipases influence their catalytic activity.     

Assessment of the Genetic Diversity of Different Job's Tears (Coix lacryma-jobi L.) Accessions and the Active Composition and Anticancer Effect of Its Seed Oil

Job’s tears (Coix lachryma-jobi L.) is an important crop used as food and herbal medicine in Asian countries. A drug made of Job’s tears seed oil has been clinically applied to treat multiple cancers. In this study, the genetic diversity of Job’s tears accessions and the fatty acid composition, triglyceride composition, and anti-proliferative effect of Job’s tears seed oil were analyzed using morphological characteristics and ISSR markers, GC-MS, HPLC-ELSD, and the MTT method. ISSR analysis demonstrated low genetic diversity of Job’s tears at the species level (h = 0.21, I = 0.33) and the accession level (h = 0.07, I = 0.10), and strong genetic differentiation (G_ST = 0.6702) among all accessions. It also clustered the 11 accessions into three cultivated clades corresponding with geographical locations and two evidently divergent wild clades. The grouping patterns based on morphological characteristics and chemical profiles were in accordance with those clustered by ISSR analysis. Significant differences in morphological characteristics, fatty acid composition, triglyceride composition, and inhibition rates of seed oil were detected among different accessions, which showed a highly significant positive correlation with genetic variation. These results suggest that the seed morphological characteristics, fatty acid composition, and triglyceride composition may be mainly attributed to genetic factors. The proportion of palmitic acid and linoleic acid to oleic acid displayed a highly significant positive correlation with the inhibition rates of Job’s tears seed oil for T24 cells, and thus can be an important indicator for quality control for Job’s tears.