Vesna Jovanovic

Corneal collagen cross-linking

Corneal collagen cross-linking (CXL) with riboflavin and ultraviolet-A (UVA) is a new technique of corneal tissue strengthening by using riboflavin as a photosensitizer and UVA to increase the formation of intra and interfibrillar covalent bonds by photosensitized oxidation. Keratocyte apoptosis in the anterior segment of the corneal stroma all the way down to a depth of about 300 microns has been described and a demarcation line between the treated and untreated cornea has been clearly shown. It is important to ensure that the cytotoxic threshold for the endothelium has not been exceeded by strictly respecting the minimal corneal thickness. Confocal microscopy studies show that repopulation of keratocytes is already visible 1 month after the treatment, reaching its pre-operative quantity and quality in terms of functional morphology within 6 months after the treatment. The major indication for the use of CXL is to inhibit the progression of corneal ectasias, such as keratoconus and pellucid marginal degeneration. CXL may also be effective in the treatment and prophylaxis of iatrogenic keratectasia, resulting from excessively aggressive photoablation. This treatment has also been used to treat infectious corneal ulcers with apparent favorable results. Combination with other treatments, such as intracorneal ring segment implantation, limited topography-guided photoablation and conductive keratoplasty have been used with different levels of success.

Influence of the microenvironment of thiol groups in low molecular mass thiols and serum albumin on the reaction with methylglyoxal.

Methylglyoxal (MG), a reactive alpha-oxoaldehyde that is produced in higher quantities in diabetes, uremia, oxidative stress, aging and inflammation, reacts with the thiol groups (in addition to the amino and guanidino groups) of proteins. This causes protein modification, formation of advanced glycated end products (AGEs) and cross-linking. Low molecular mass thiols can be used as competitive targets for MG, preventing the reactions mentioned above. Therefore, this paper investigated how the microenvironment of the thiol group in low molecular mass thiols (cysteine, N-acetylcysteine (NAcCys), carboxymethylcysteine (CMC) and glutathione (GSH)) and human serum albumin (HSA) affected the thiol reaction with MG. The SH group reaction course was monitored by (1)H-NMR spectroscopy and spectrophotometric quantification. Changes in the HSA molecules were monitored by SDS-PAGE. The microenvironment of the SH group had a major effect on its reactivity and on the product yield. The reactivity of SH groups decreased in the order Cys>GSH>NAcCys. CMC did not react. The percentages of the reacted SH groups in the equilibrium state were almost equal, regardless of the ratio of thiol compound/MG (1:1, 1:2, 1:5): 38.1 + or - 0.9%; 38.2 + or - 0.7% and 39.0 + or - 0.8% for Cys; 26.5 + or - 0.6%; 26.6 + or - 2.6% and 27.4 + or - 2.5% for GSH; 10.8 + or - 0.9%; and 11.2 + or - 0.7% and 12.2 + or - 0.9% for NAcCys, respectively. Our results explain why substances containing alpha-amino-beta-mercapto-ethane as a pharmacophore are successful scavengers of MG. In equilibrium, HSA SH reacted in high percentages both with an insufficient amount and with an excess of MG (55% and 65%, respectively). An analysis of the hydrophobicity of the microenvironment of the SH group on the HSA surface showed that it could contribute to high levels of SH modification, leading to an increase in the scavenging activity of the albumin thiol.

Corneal collagen cross-linking outcomes: review.

Keratoconus is a condition characterized by biomechanical instability of the cornea, presenting in a progressive, asymmetric and bilateral way. Corneal collagen cross-linking with riboflavin and UVA (CXL) is a new technique of corneal tissue strengthening that combines the use of riboflavin as a photo sensitizer and UVA irradiation. The studies showed that CXL was effective in halting the progression of keratoconus over a period of up to four years. The published studies also revealed a reduction of max K readings by more than 2 D, while the postoperative SEQ was reduced by an average of more than 1 D, and refractive cylinder decreased by about 1 D. No eyes lost any line of BCDVA. Moreover, there was no significant decrease in endothelial cell density. It was also found that CXL treatment was effective with reducing corneal and total wavefront aberrations. Corneal cross-linking has also led to an arrest and/or even a partial reversal of keratectasia in the treatment of iatrogenic ectasia after excimer laser ablation. A primary intervention such as CXL should be considered to potentially increase the biomechanical stability of the corneal tissue and postpone the need of lamellar or penetrating keratoplasty.

Method for monitoring of the protein amino group changes during carbonylation

OBJECTIVES Carbonylation of the protein amino, guanidino and thiol groups is one of the important causes of vascular complications in diabetes. We developed a simple spectrophotometric method for monitoring of the changes in the protein amino group contents during carbonylation. DESIGN AND METHODS The method is based on the reaction of amino group with p-benzoquinone in the slightly alkaline media. It was applied during carbonylation in vitro with methylglyoxal and in vivo in 13 patients with type 2 diabetes and 20 healthy persons. RESULTS The method is simple, fast, precise (RSD in the range of 1.2-1.8%) and accurate (recovery about 100%). The content of amino groups in human serum albumin isolated from diabetics was significantly lower (p<0.01) in comparison with a control group. CONCLUSION The method developed is suitable for quantification of protein amino groups during in vitro carbonylation as well as for clinical practice.

Endothelial Keratoplasty without Descemet’s Membrane Stripping: Histologic and Ultrastructural Findings

Aim: To report histologic and ultrastructural findings of endothelial keratoplasty (EK) performed without Descemet’s membrane stripping. Methods: Clinical techniques, histology, and transmission electron microscopy. Results: A 55-year-old woman was referred to us, after 2 unsuccessful penetrating keratoplasties (PKs), for pseudophakic bullous keratopathy. An 8.0-mm EK without Descemet’s membrane stripping was performed, and clarity was restored to the failed penetrating regraft. A year later, the lamellar graft failed, and a third PK was performed for intractable corneal edema. Light microscopy of a semi-thin section of the trephined corneal button showed both the recipient’s and the donor’s Descemet’s membrane, the well-preserved structure of the full-thickness graft, and marked edema of the adherent stromal carrier of the endothelial transplant. The host endothelium was absent at the interface, and the donor endothelium was atrophic. Electron microscopy revealed regularity and even spacing of collagen fibrils as well as quiet keratocytes on both sides of Descemet’s membrane at PK-EK interface. Conclusion: These findings suggest a lack of proliferation and hypercellular scarring, and offer further support to the already proven merits of EK.

The influence of fatty acids on determination of human serum albumin thiol group.

During investigation of the changes of the Cys34 thiol group of human serum albumin (HSA) (isolated by affinity chromatography with Cibacron Blue (CB)) in diabetes, we found that the HSA-SH content was higher (11-33%) than the total serum thiol content. The influence of fatty acids (FA) binding to HSA on this discrepancy was investigated in vitro (using fluorescence and CD spectroscopy and GC) and with HSA samples from diabetic (n=20) and control groups (n=17). HSA-bound FA determine the selection of HSA molecules by CB and enhance reactivity and/or accessibility of the SH group. A high content of polyunsaturated FA (35.6%) leads to weaker binding of HSA molecules to CB. Rate constants of DTNB reaction with the SH group of HSA applied to a CB column, bound-HSA and unbound-HSA fractions, were 4.8×10(-3), 21.6×10(-3), and 11.2×10(-3) s(-1), respectively. The HSA-SH group of diabetics is more reactive compared with control individuals (rate constants 20.9×10(-3)±4.4×10(-3) vs 12.9×10(-3)±2.6×10(-3) s(-1), P<0.05). Recovery values of the SH group obtained after chromatography of HSA with bound stearic acid ranged from 110 to 140%, while those for defatted HSA were from 98.5 to 101.7%. Thus, HSA-bound FA leads to an increase of HSA-SH content and a contribution to total serum thiols, which make the determination of the thiol group unreliable.

[Acquired amegakaryocytic thrombocytopenia: three case reports and a literature review].

INTRODUCTION Acquired amegakaryocytic thrombocytopenia (AAT) is a rare disease characterized by thrombocytopenia due to selective reduction/absence of bone marrow (BM) megakaryocytes. In the BM culture isolated reduction of colony-forming units-megakaryocyte (CFU-Mk) may occur. MATERIAL AND METHODS BM aspirates and trephine biopsies were obtained from all patients and processed by routine methods. In vitro BM culture and cytogenetic analysis was performed in one patient. RESULTS This article presents three patients with manifested signs of hemorrhagic syndrome due to severe thrombocytopenia caused by an absence/significant reduction of BM megakaryocytes. Existence of systemic or any other disease was excluded in all patients. BM culture of the second patient showed reduction of all hematopoietic progenitors. In the subsequent course of the disease in this patient, signs of dysplastic erythrocytic series and megakaryocytes were also noted, although there were no positive proofs of evolution into myelodysplastic syndrome. DISCUSSION AAT is a disease of hematopoietic stem cells manifesting in a certain period as amegakaryocytic thrombocytopenia which subsequently may progress into aplastic anemia or myelodysplastic syndrome. Patients were treated with corticosteroids, lithium carbonate, androgens, vincristine, immunoglobulins, folic acid, platelet and erythrocyte transfusions along with plasma substitution. The first patient reacted positively to the therapy. In two other patients a minimal, short-term therapeutic effect was achieved, followed by improvement of hemorrhagic syndrome and an insignificant increase in platelet count. In one patient the treatment was stopped after 4 months and the other died of bleeding after 4 months. CONCLUSION AAT is a rare disease with unpredictable course. This is a case report of three patients with AAT and different therapeutic effects.

The Effect of Topical Doxycycline on Corneal Neovascularization

Abstract Purpose: To examine the effect of topical doxycycline on human corneal neovascularization (CONV). Materials and methods: Six eyes of six patients with CONV received topical 1% doxycycline four times per day for three weeks and monitored for a total of one year. Ophthalmic evaluations included visual acuity, tonometry, slit-lamp examination, conjunctival swabs, quantification of CONV and photography. Results: CONV either disappeared or was attenuated, shortened and less dense in five of six patients. There were no adverse effects. Conclusion: Topical doxycycline was effective in reducing CONV and healing of the ocular surface.

Monitoring of the human serum albumin carbonylation level through determination of guanidino group content

Carbonylation of the protein amino, guanidine, and thiol groups with α-oxoaldehydes (which are produced in higher quantities in diabetes, uremia, oxidative stress, aging, and inflammation) is one of the important causes of vascular complications. For monitoring of the human serum albumin (HSA) carbonylation level, a spectrophotometric method based on the formation of colored adduct between guanidine group and thymol-sodium hypobromite reagent in the alkaline medium was investigated. Beers law is obeyed in the concentration range of Arg and protein guanidine groups from 1 to 40 mM. Precision of the method (relative standard deviation) was in the range of 0.9 to 2%. Accuracy was examined by the standard addition method (recovery ~100%). The method was applied for monitoring of the carbonylation level of HSA with methylglyoxal in vitro and of HSA isolated (using affinity chromatography) from sera of 21 patients with type 2 diabetes and 12 healthy persons. The content of guanidine groups in HSA isolated from diabetics (19.64 ± 1.07 mM/mM albumin) was significantly lower (P < 0.001) in comparison with a control group (21.87 ± 1.02 mM/mM albumin). The method is simple and fast, has good accuracy and precision, and is suitable for clinical practice as well for in vitro protein carbonylation experiments.

One cornea for two patients: case report.

Case reporting the use of one donor cornea for two transplantation procedures: deep anterior lamellar keratoplasty (DALK) in a case of an imminent corneal perforation caused by herpetic stromal necrosis, and Descemet stripping with endothelial keratoplasty (DSEK) in an eye with pseudophakic bullous keratopathy (PBK). Descemets membrane (DM), denuded by stromal necrosis, served as the starting point for dissection plane and creation of the recipient bed for DALK. The next steps were excision of the diseased stroma along the edge of trephination, and transplantation of a 400-450 microm thick, manually dissected lamellar graft. The remaining posterior layers of the donor cornea, 100-150 microm thick, were used as a graft in the DSEK procedure for PBK. The integrity of the globe was saved, and best-corrected visual acuity (BCVA) of 20/40 was reached after DALK in the eye with an imminent corneal perforation. A subnormal central corneal thickness (CCT) of 430 microm did not interfere with corneal shape (43.50 x 45.50 D) and function. The graft remained attached and clear after DSEK in the eye with PBK, with BCVA of 20/30 and a CCT of 653 microm. One donor cornea can be used for two lamellar keratoplasties, DALK and DSEK. Although the described obstacles may prevent this approach from becoming widely used, it may prove useful when one is confronted with a need for an urgent anterior lamellar keratoplasty, a long list of cases for DSEK, and a shortage of donor corneas.