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M Filipec

Publications and source records attributed to M Filipec.

At least 19 recordsLinked to original sources
PubMed2006

Morphological and immunocytochemical characterization of snake-like chromatin cells.

Snake-like chromatin (SLC) is a nuclear alteration occurring under various pathological conditions and in different tissues. The aim of this study was the morphological and immunocytochemical characterization of SLC-positive conjunctival epithelial cells from keratoconjunctivitis sicca (KCS) patients. Impression cytology specimens from the upper bulbar conjunctiva of 10 controls and 10 KCS patients with a high incidence of SLC cells were assessed, the morphology of SLC nuclei evaluated by light microscopy, and proliferation markers, nucleolar proteins, lamins and cytokeratin filaments detected immunocytochemically. In KCS patients, SLC cells with a normal nuclear shape, with nuclear membrane notching (2.3% of cells) and with binuclear dumb-bell structures (4.4% of cells) were observed. The most striking features of SLC cells were the absence of an A/C lamin signal, the redistribution of fibrillarin into two spots adjacent to SLC structures and cytokeratin 14 positivity in the strangulation belt of the dumb-bell structures. The deficiency of lamin A/C is the probable reason for the disintegration of chromatin from the nuclear lamina in SLC cells. The occurrence of SLC-positive cells, SLC-positive dumb-bell shaped nuclei and SLC-positive binucleated cells, together with the absence of mitotic markers, leads to the conclusion that the SLC phenomenon might be a form of nuclear segregation.

Adult↗
PubMed2004

Corneal stromal cells selectively inhibit production of anti-inflammatory cytokines by activated T cells.

The eye has been described as an immunologically privileged site where immunity is purely expressed. It has been demonstrated that administration of antigen into the eye induces only a weak immune response. However, the anterior part of the eye represents an important protective barrier against pathogens and other harmful invaders from the outer environment. Therefore, effective immune mechanisms, which operate locally, need to be present there. Because the cornea has been shown to be a potent producer of various cytokines and other molecules with immunomodulatory properties, we investigated a possible regulatory role for the individual corneal cell types on cytokine production by activated T cells. Mouse spleen cells were stimulated with the T cell mitogen concanavalin A in the presence of either corneal explants or cells of corneal epithelial or endothelial cell lines and the production of T helper 1 (Th1) or Th2 cytokines was determined by enzyme-linked immunosorbent assay (ELISA) or reverse transcription-polymerase chain reaction (RT-PCR). We found that the cornea possesses the ability to inhibit, in a dose-dependent manner, production of the inhibitory and anti-inflammatory cytokines interleukin (IL)-4 and IL-10 by activated T cells. The production of cytokines associated with protective immunity [IL-2, IL-1beta, interferon (IFN)-gamma ] was not inhibited under the same conditions. Corneal explants deprived of epithelial and endothelial cells retained the ability to suppress production of anti-inflammatory cytokines. This suppression was mediated by a factor produced by corneal stromal cells and occurred at the level of cytokine gene expression. We suggest that by this mechanism the cornea can potentiate a local expression of protective immune reactions in the anterior segment of the eye.

Animals↗
PubMed2004

Quantitative evaluation of the corneal endothelium in the mouse after grafting.

BACKGROUND/AIM: Corneal graft survival depends critically on the quality of the endothelium. In this study the authors aimed to evaluate corneal endothelium in mice at different times after transplantation and to correlate endothelial integrity with corneal graft survival. METHODS: Syngeneic and allogeneic corneal grafts at various times (days 0-60) after engraftment were examined in flat mount preparation by confocal microscopy, by evaluating the hexagonal pattern of the endothelial monolayer using actin staining of the cell cortex. Corneas from untreated mice and from mice, who were grafted after removal of draining lymph nodes served as controls. RESULTS: In control corneas, more than 90% of the posterior surface was covered by endothelium. Syngeneic grafts were always covered by 54-99% of endothelium. In contrast, the posterior surface of corneal allografts showed great variation in the degree of endothelial cell coverage (0-98%). In addition, clinical opacity grading measure was not a reliable predictor of endothelial coverage. CONCLUSION: In corneal allografts there is progressive loss of endothelium over time, unlike with syngeneic grafts. However, in the early stages of allograft rejection, the grade of graft opacity does not accurately reflect the degree of endothelial cell coverage. Although corneal opacity grade is considered the main determinant of graft rejection, the data suggest that both the grade of corneal opacity plus a sufficient post-graft time duration (>8 weeks in the mouse) are required for the diagnosis of irreversible graft rejection.

Animals↗
PubMed2004

Prevention of corneal allograft rejection in a mouse model of high risk recipients.

AIM: To determine the effectiveness of treatment with immunosuppressive drugs and monoclonal antibodies (mAb) after penetrating keratoplasty in two different models of high risk mouse recipients. METHODS: Corneas were grafted orthotopically in mouse models of high risk recipients with either neovascularisation of the graft bed or presensitisation to graft donor antigens. Recipients were treated with mAb against CD4(+) or CD8(+) cells or against T cells, or were treated with cyclosporin A (CsA) or mycophenolate mofetil (MMF), or a combination of both drugs. RESULTS: Control untreated recipients with neovascularised graft bed or presensitised to the graft donor antigens rejected corneal allografts in 12.5 (SD 2.3) and 9.9 (1.6) days, respectively. Treatment of graft recipients with a neovascularised graft bed with mAb anti-CD4 or anti-T cells, but not with mAb anti-CD8 or with immunosuppressive drugs, resulted in a significant prolongation of graft survival; 75% and 28.5%, respectively, of grafts survived for more than 45 days after grafting. However, none of the treatments were successful in presensitised recipients. CONCLUSIONS: Treatment of high risk recipients with mAb anti-CD4 is more effective in preventing corneal allograft rejection than the treatment with mAb anti-CD8 or the immunosuppressive drugs MMF and CsA. However, the effectiveness of the treatment depends on the recipients' pretransplantation risk type.

Animals↗
PubMed2004

[Thygeson's keratitis--clinical characteristics and therapy].

PURPOSE: The evaluation of clinical manifestations and therapeutical modalities Thygeson's keratitis (Thygeson's superficial punctate keratitis-TSPK) in a group of patients with long follow-up in the Cornea and Immunology Clinic of the Department of Ophthalmology, General Teching Hospital, Charles University in Prague. PATIENTS AND METHODS: The group of 7 patients (13 eyes) at the mean age of 20.7 years (9-39) with clinical diagnosis of TSPK was evaluated retrospectively. The course of the disease, symptoms and signs of the disease, efficacy of the therapy and primary established diagnosis were evaluated. RESULTS: The average onset of the disease was 12.5 years (6-27) and the average duration was 6 years (2-10). TSPK was bilateral in six patients, while unilateral the disease was only in one patient. The clinical picture was characterized by recurrent episodes of photophobia, tearing and burning and foreign body sensation in the eyes. The examination revealed whitish fine granular asterisk-form or dendriform intraepithelial opacities, sometimes slightly above the niveau of the surrounding epithelium. In the acute phase the corneal epithelium above the lesions was disrupted. Subjective symptoms and sometime also the objective findings diminished after local corticosteroids administration. The most common primary diagnoses the TSPK patients were treated for herpetic keratitis. CONCLUSION: TSPK is a rare, relapsing corneal disease with the onset mostly in the first and third decade of life. TSPK is mostly bilateral, but may be also unilateral and findings are asymmetrical in almost all cases. Relapses frequently occur in connection with physical or psychological stress. Concerning the permanent damage to the cornea and potential to decrease visual acuity TSPK can be considered as a benign and during several years self-limited disease. Subjective symptoms however may significantly deteriorate patient's quality of life. Local treatment with corticosteroids diminishes subjective symptoms, number and duration of relapses but does not cure the disease. The disease is often misdiagnosed and treated incorrectly.

Adolescent↗
PubMed2003

FK 506 and aminoguanidine suppress iNOS induction in orthotopic corneal allografts and prolong graft survival in mice.

The aim of this study was to compare the effectiveness of immunosuppressant FK 506 and the specific inhibitor of inducible nitric oxide synthase (iNOS) aminoguanidine (AG) in prevention of corneal graft rejection and to investigate the iNOS expression in the rejection process. Orthotopic corneal allografting in mice was performed (C57BL/10; H-2(b) to BALB/c; H-2(d)). FK 506 (0.3 mg/kg per day) or AG (100 mg/kg per day) was injected intraperitoneally for 4 weeks. Grafted mice without therapy served as controls. Immunohistological evaluation of iNOS-positive cells and macrophage infiltration in grafts 27th day after grafting was performed. Within 4 weeks FK 506 prevented graft rejection in 71% and AG in 57% of animals compared to 29% of clear grafts in controls. A significant proportion of iNOS-positive cells was detected in the rejected grafts of the control and AG-treated groups. The treatment with FK 506 resulted in the inhibition of iNOS expression to a high degree in the rejected corneas. Non-rejected corneas of all groups and non-transplanted corneas exhibited no iNOS-positive cells. A massive infiltration of macrophages was detected in the rejected grafts, whereas non-rejected grafts exhibited only slight infiltration of macrophages. The presented data suggest that overexpression of iNOS and/or activation of iNOS is one of the several influential factors that contribute to the rejection process and that iNOS suppression delays corneal allograft rejection. FK 506 and AG are effective drugs in preventing corneal allograft rejection. Higher beneficial effect of FK 506 on graft survival could be explained by its well-known selective T-cell immunosuppression.

Animals↗
PubMed2002

Evaluation of corneal graft rejection in a mouse model.

Corneal graft rejection presents clinically and in experimental models as opacification and is considered to be the result of endothelial cell dysfunction or loss. However, recovery from opacification can occur suggesting either (a) that new endothelial cells can regenerate if the original cells were lost, or (b) that sufficient numbers of original cells can regain function if the opacification was due to temporary dysfunction. In this perspective, previous experimental studies of allograft rejection plus some new data are reviewed to support the latter mechanism.

Animals↗
PubMed2002

Xanthine oxidoreductase and xanthine oxidase in human cornea.

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.

Adult↗
PubMed2001

Kinetics of leukocyte and myeloid cell traffic in the murine corneal allograft response.

BACKGROUND: Little information exists on the trafficking of myeloid and lymphoid cells between the transplanted cornea and the secondary lymphoid tissue. This study reports on changes in the cornea and the draining lymph node (DLN) from the time of graft emplacement. METHODS: Using a mouse corneal graft model (C57BL/10Sn to BALB/c), eyes and submandibular DLN were examined by immunohistochemistry and three-color flow cytometry for evidence of T cell activation and dendritic cell (DC) conditioning (up-regulation of costimulatory molecules) at various times (15 min to 24 days; n=4 for each time). RESULTS: In the DLN, early (2 hr) DC conditioning was sustained throughout allograft rejection whereas a remarkable drop in percentage of activated CD4+ and CD8+ T cells (P <0.001) was followed by a biphasic rise in activated CD4+ and, to a lesser extent, CD8+ T cells (24 hr, P <0.001 and 6 days, P <0.01). CD11b+ and MOMA-2+ macrophages, MHC Class II+ cells, CD86+ DC, and neutrophils were the earliest cells infiltrating the cornea (at 24 hr), whereas T cells appeared after 2 days, with CD4+ T cells being confined largely to the graft recipient border. CONCLUSIONS: Immediate and rapid changes in T cell and DC populations in the DLN correlate with the type of cellular infiltration in the corneal graft. The data are consistent with a model in which CD4+ T cell help for CD8+ cytotoxic T cells could be provided by sequential two-way activation of T cells and DC in the DLN. The majority of cells infiltrating the graft were macrophages and neutrophils, with fewer DC and T cells.

Animals↗
PubMed2001

Different mechanisms in inhibition of rat macrophage nitric oxide synthase expression by FK 506 and cyclosporin A.

The modulatory effect of FK 506 and cyclosporin A (CsA) on the expression of inducible nitric oxide synthase (iNOS) in macrophages and mechanisms of their action were analysed. Isolated rat peritoneal macrophages were cultured for 12 or 24 h with or without lipopolysaccharide (LPS) (5 microg/ml) and in the absence or presence of FK 506 or CsA (0.1 and 1 microg/ml). Total RNA from macrophages was isolated and the expression of the gene for iNOS was assessed by using RT-PCR. The concentration of NO2- in culture supernatants was taken as a measure of nitric oxide (NO) production. FK 506 (0.1 and 1 microg/ml) reduced the LPS-induced increase of NO2- levels by 68% and 81%, respectively. CsA (0.1 and 1 microg/ml) decreased levels of nitrites by 39% and 69%, respectively. The results obtained suggest that both immunosuppressive drugs exhibit dose-dependent inhibitory effect on NO production and that FK 506 is more potent agent than CsA, in this respect. FK 506 exhibits its inhibitory effect on a phosphatase at the transcriptional level in macrophages. iNOS expression down-regulation by CsA is occurred post-transcriptionally.

Animals↗
PubMed2001

Detection of galectin-3 in tear fluid at disease states and immunohistochemical and lectin histochemical analysis in human corneal and conjunctival epithelium.

BACKGROUND/AIM: Components of the tear fluid contribute to the biochemical defence system of the eye. To reveal whether the immune mediator and lipopolysaccharide binding galectin-3 is present in tears, tear samples were collected from eyes in healthy and pathological states. Investigation of expression of galectin-3 and galectin-3 reactive glycoligands in normal human conjunctival and corneal epithelia was also initiated as a step to understand the role of galectin-3 in ocular surface pathology. METHODS: Immunoblot analysis using either a rabbit polyclonal or a mouse monoclonal antibody against galectin-3 was employed to detect galectin-3 in tear fluid. Galectin-3 expression in tissue specimens was detected by immunocytochemistry employing A1D6 mouse monoclonal antibody, and galectin-3 reactive glycoligands were visualised by lectin histochemistry using labelled galectin-3. RESULTS: Galectin-3 was found only in tears from patients with ocular surface disorders. It was expressed in normal corneal and conjunctival epithelia but not in lacrimal glands. Inflammatory leucocytes and goblet cells found in galectin-3 containing tear fluid also expressed galectin-3. Galectin-3 binding sites were detected on the surface of conjunctival and corneal epithelial cells co-localising with desmoglein. CONCLUSIONS: This study revealed expression of galectin-3 in tear fluid obtained from patients with eye diseases. The role of this endogenous lectin (produced by inflammatory as well as epithelial cells) in antimicrobial action and inflammation modulation could be expected.

Antigens, Differentiation↗
PubMed2000

Link between a novel human gammaD-crystallin allele and a unique cataract phenotype explained by protein crystallography.

We describe a 5-year-old boy with a unique congenital cataract caused by deposition of numerous birefringent, pleiochroic and macroscopically prismatic crystals. Crystal analysis with subsequent automatic Edman degradation and matrix-associated laser desorption ionization time-of-flight mass spectrometry have identified the crystal-forming protein as gammaD-crystallin (CRYGD) lacking the N-terminal methionine. Sequencing of the CRYGD gene has shown a heterozygous C-->A transversion in position 109 of the inferred cDNA (36R-->S transversion of the processed, N-terminal methionine-lacking CRYGD). The lens protein crystals were X-ray diffracting, and our crystal structure solution at 2.25 A suggests that mutant R36S CRYGD has an unaltered protein fold. In contrast, the observed crystal packing is possible only with the mutant protein molecules that lack the bulky Arg36 side chain. This is the first described case of human cataract caused by crystallization of a protein in the lens. It involves the third known mutation in the CRYGD gene but offers, for the first time, a causative explanation of the phenotype.

Alleles↗
PubMed2000

Induction of specific transplantation immunity by oral immunization with allogeneic cells.

Oral administration of antigen has been shown to be effective for both positive and negative modulation of immune responses. In the present study we characterized changes in the reactivity of the immune system after oral immunization with allogeneic spleen cells. Mice were orally immunized for 10 consecutive days with fresh allogeneic spleen cells, and the phenotype, proliferative response, cytotoxic activity and cytokine production profile of recipient spleen cells were assessed 1 or 7 days after the last immunization dose. Although no significant changes in the proportion of CD4+, CD8+ or CD25+ cells were observed in the spleen of orally immunized mice, significant activation of alloreactivity in spleen cells was found. Cells from orally immunized mice exhibited enhanced proliferation and cytotoxic activity after stimulation with specific allogeneic cells in vitro, and produced considerably higher concentrations of interferon-gamma (IFN-gamma) and significantly less interleukin (IL)-4 than did cells from control mice. The production of IL-2 was essentially unchanged and that of IL-10 was only slightly increased. The systemic allosensitization induced by oral immunization was demonstrated in vivo by increased resistance to the growth of allogeneic tumours induced by subcutaneous inoculation of high doses of tumour cells. In addition, orthotopic corneal allografts in orally immunized recipients were rejected more rapidly (in a second-set manner) than in control, untreated recipients. These data show that oral immunization with allogeneic cells modulates individual components of the immune response and that specific transplantation immunity, rather than tolerance, is induced in the treated recipients.

Administration, Oral↗
PubMed2000

Inhibitory effect of FK 506 and cyclosporin A on nitric oxide production by LPS-treated cultured rat macrophages.

We analyzed the effect of FK 506 on the production of nitric oxide by macrophages. Isolated rat peritoneal macrophages were cultured for 24 h with or without lipopolysaccharide (LPS) (5 microg/ml) and in the absence or presence of FK 506 (0.1 and 1 microg/ml). The concentration of NO2- in culture supernatants was taken as a measure of nitric oxide production. FK 506 (0.1 and 1 microg/ml) reduced the LPS-induced increase of NO2- levels by 68% and 81%, respectively. The impact of cyclosporin A (CsA) was studied in order to compare their effects. CsA (0.1 and 1 microg/ml) decreased the levels of nitrites by 39% and 69%, respectively. The results obtained suggest that both immunosuppressive drugs exhibit a dose-dependent inhibitory effect on nitric oxide production and that FK 506 is a more potent agent than CsA in this respect.

Animals↗
PubMed1999

[The Th1/Th2 paradigm. The role of the Th1 and Th2 lymphocyte subpopulations in the regulation of immune processes].

A short report on the interaction of Th1 and Th2 lymphocyte subpopulations in the regulation of immune processes is reviewed. Th1 and Th2 subsets have been characterized on the basis of cytokines they secrete and the immune functions they mediate and also on the basis of cytokine and chemokine receptor expression. The clinical and diagnostic influence of immune processes in pathological stages is also mentioned.

Cytokines↗
PubMed1999

Occurrence of cytokine receptors on different lymphoid leukaemic cells.

We have studied the expression of cytokine receptors CD25 (IL-2R alpha, 55 kD), CD116 (hGM-CSFR, 145 kD), CD117 (CSFR, 145 kD), CD120a (TNFR, 55 kD), CD120b (TNFR, 75 kD), CD121a (IL-1R, type I, 80 kD), CDw123 (IL-3R), CD124 (IL-4R, 140 kD), CD126 (IL-6R, 80 kD), CD127 (IL-7R, 75 kD), CDw128 (IL-8R), CD130 (gp130 subunit), CDw131 (common beta), CD132 (IL-2Rgamma), CD134 (OX40) and also CD95 (Fas antigen) on the lymphoid leukaemic cells. Cells from peripheral blood or bone marrow of 24 patients with disorders in lymphoid lineage mostly included acute lymphoid leukaemias (with a high leukocyte count and percentage of blasts) were analysed for the expression of surface membrane molecules by the immunofluorescence method evaluated by flow cytometry. The findings indicate that some monoclonal antibodies have a reactivity against cytokine receptors of pathological cells in individual cases, but with very variable qualitative and quantitative expression (number copies/cell). The lymphoid leukaemic cells demonstrate unique cytokine receptor profiles, which reveal the great diversity of immunophenotypes within the main functional characterisation of T and B lymphoproliferative malignancies.

Adolescent↗