Time and Temperature Stability of TGF-ß1, EGF and IGF-1 in 20% and 100% Human Serum.

Autologous serum eye drops (ASEDs) are used as a treatment for severe dry eye disease. The concentration and stability of various growth factors in ASEDs is determinative for their efficiency. We therefore assessed the concentrations of transforming growth factor beta 1 (TGF-ß1), epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1) in ASEDs following storage at 4-8, -20, -80 and -156 °C. Twenty % and 100% sera from eight healthy volunteers were analysed by the sandwich enzyme immunoassay at different time intervals up to seven months. The mean levels of TGF-ß1 and EGF in undiluted and 20% serum did not differ significantly from the baseline levels in fresh serum for any storage conditions after 7 days at 4-8 °C, as well as after 4- and 7-month preservation at sub-zero temperatures. In 20% serum, no IGF-1 concentration decrease was found following 7 days of preservation at 4-8 °C. However, a decrease to 78 % and 81 % (P < 0.01) of baseline values was found in 20% serum after 4-month storage at -20 °C and 7-month storage at -156 °C, respectively. A more pronounced decrease in IGF-1 was observed in undiluted serum. All assessed growth factors present in 20% frozen serum remained stable for up to 7 months. The highest stability was achieved at -80 °C. At -20 and -156 °C, some decrease in IGF-1 occurred. Our results indicate that 20% ASEDs can be stored frozen up to 7 months under proper conditions.

Oncology wounds - recommendations for care based on multidisciplinary cooperation.

BACKGROUND: Oncology wounds and wounds of other etiology are rare but serious complications, which significantly impair patients quality of life. Preventive and curative interventions and education of healthcare personnel and patients reduce the risk of either their occurrence or their impact and consequences. A working group of authors from professional groups (the Supportive Care Group of the Czech Society for Oncology, the Czech Society for Wound Healing, the Society for Radiation Oncology, Biology and Physics, and the Czech Nurses Association) prepared recommendations for care. A comprehensive approach to the treatment of oncological wounds, including symptomatic treatment of associated healing complications, prevention, early detection, interdisciplinary cooperation and education are essential to deal with wounds related to chemotherapy administration, radiotherapy and oncological treatment in general. The proper choice of local care products and the eventuality of active oncological treatment are important elements of care in ulcerating tumors. PURPOSE: A basic summary of recommended interventions to prevent and treat oncology wounds in daily practice, defined based on expert societies guidelines, trials and literature data, proven practice and on the consensus opinions of the authors group members. The recommended procedures contribute to the reduction of the development, severity and consequences of oncological wounds and wounds of other etiology in oncological  patients.

Recommendation for preventive and therapeutic skin care of patients undergoing radiotherapy.

BACKGROUND: Radiation dermatitis is a very common reaction to radiotherapy, affecting approx. 95% of patients with varying intensity. It is crucial to minimize its side effects. The working group that prepared this document includes physicians, nurses, representatives of the Society for Radiation Oncology, Biology and Physics of the Czech Medical Association of J. E. Purkyně, the Supportive Treatment and Care Section of the Czech Society for Oncology of the Czech Medical Association of J. E. Purkyně, the Czech Wound Management Association, the Oncological Section of Czech Association of Nurses, and dermatologists. The document has been approved by the committees of these associations. PURPOSE: Recommendation for preventive and therapeutic skin care of patients undergoing radiotherapy in the Czech Republic.

Oral cavity complications in oncological and hemato-oncological patients.

BACKGROUND: Oral cavity injuries are very significant complications in the treatment of oncological and hemato-oncological patients. Preventive and curative interventions and patient education reduce the risk of complications and their consequences. A working group of authors from professional groups prepared recommendations for care. PURPOSE: A basic summary of recommended interventions to prevent and treat oral cavity injuries in daily practice, defined on the basis of expert societies guidelines, trials, literature data and proven practice and on the consensus opinions of the authors group members. RESULTS: Preventive measures and patient education are essential in the approach to dealing with oral injuries in chemotherapy, radiotherapy, risky targeted treatment and osteonecrosis of the jaw. Local care products are an important element of care, in case of infections, their antimicrobial action is essential, in case of graft-versus-host disease or in connection with targeted oncological therapy, corticoids are used. CONCLUSION: The recommended procedures contribute to the reduction of the development, severity and consequences of oral complications in oncological and hemato-oncological patients.

[Tear Osmolarity in Patients with Severe Dry Eye Syndrome Before and After Autologous Serum Treatment: a Comparison with Tear Osmolarity in Healthy Volunteers]. / Osmolarita slz u pacientu s tezkým syndromem suchého oka pred a po aplikaci autologního séra. Porovnání s hodnotami zdravých dobrovolníku.

INTRODUCTION: Dry eye syndrome (DES) is a multifactorial disease of the tears and ocular surface. Recently, treatment with autologous serum eye drops (AS-ed) has been frequently used in these patients. Significant improvement correlates well with clinical, laboratory and subjective findings. It is assumed that one of the key factors in the development of the disease is increased tear osmolarity. The aims of our study were to test tear osmolarity measurements in clinical practice, to examine if osmolarity values differ before and after a 3-month application of 20% AS-ed, and to determine if the values differ between patients with severe DES and healthy individuals. METHODS: The study included 35 patients with severe DES (Schirmer test<5 mm/5 min) and 23 healthy volunteers. Tear osmolarity values (TearLab Osmolarity System), the Schirmer test (ST1), vital ocular surface staining and subjective feelings (the OSDI questionnaire) were assessed in patients with DES before and after treatment with 20% AS-ed. Further, the tear osmolarity values were compared between healthy subjects and patients with DES before and after treatment with AS-ed. RESULTS: The values of OSDI and vital staining significantly decreased in patients with DES after the treatment (p<0.0001). ST1 and tear osmolarity did not change significantly after the treatment. ST1 values in healthy individuals were significantly higher (p<0.0001) and the OSDI values significantly lower (p<0.0001) than the results obtained in patients before and after treatment. Tear osmolarity was not statistically different between healthy subjects (306 mosmol/l) and patients with DES (302 and 301 mosmol/l before and after treatment respectively). CONCLUSION: We demonstrated a positive effect of AS treatment on the ocular surface in patients with DES. However, the osmolarity values did not differ before and after treatment with AS, and they also did not differ significantly between DES patients and healthy individuals. In accordance with other recent studies, our results raise questions concerning the value of the TearLab Osmolarity System for evaluating therapeutic effect and also as a tool for DES diagnosis.

Is copper imbalance an environmental factor influencing keratoconus development?

Keratoconus is a bilateral disease characterized by progressive corneal thinning leading to irregular astigmatism that results in significant visual impairment. Despite extensive research, the exact etiopathogenesis of keratoconus remains unknown. Many copper-dependent enzymes such as superoxide dismutases, cytochrome c oxidase and lysyl oxidase have been shown to be altered in keratoconic corneas, and a decrease of copper levels in the diseased tissue has been reported as well. We propose a hypothesis linking all the putative pathways of keratoconus development and suggest that copper imbalance in corneal tissue may be an independent risk factor for the disease. The assessment of copper levels and its distribution in keratoconic corneas warrants further investigation.

Macular corneal dystrophy and associated corneal thinning.

PURPOSE: To identify the molecular genetic cause of macular corneal dystrophy (MCD) in four probands, and characterize phenotypic similarities between MCD and keratoconus. METHODS: We performed ophthalmological examination, Scheimpflug imaging (Pentacam, Oculus Inc.), histopathological examination of excised corneal buttons, and direct sequencing of the CHST6 coding region. RESULTS: Pentacam measurements were taken in six eyes of three probands. All showed diffuse corneal thinning with paracentral steepening of the anterior corneal surface that was graded as keratoconus by the integrated software, but without associated ectasia of the posterior corneal surface or regional thinning. Homozygous or compound heterozygous CHST6 mutations were identified in all cases, including two novel mutations, c.13C>T; p.(Arg5Cys) and c.289C>T; p.(Arg97Cys). DISCUSSION: Localized elevation of the anterior corneal curvature can occur in MCD in the absence of other features of keratoconus. The identification of a further two Czech probands with the compound allele c.[484C>G; 599T>G] supports the enrichment of this allele in the study population.

Ocular surface injuries in autoimmune dry eye. The severity of microscopical disturbances goes parallel with the severity of symptoms of dryness.

Autoimmune dry eye (Sjögren's syndrome, SS) is a chronic systemic disease characterized by salivary and lacrimal gland inflammation and tissue damage leading to keratoconjunctivitis sicca and xerostomia. In this review attention has been devoted to the cause of the development of oxidative injuries of the ocular surface of patients suffering from SS. It was shown that lacrimal glands and diseased conjunctival epithelium reveal increased expression of pro-inflammatory cytokines which are released into the tear fluid. A high amount of pro-inflammatory cytokines highly induce the elevated expression and activity of enzymatic systems that generate reactive oxygen and nitrogen species. An abundant amount of these toxic products leads to a decrease in antioxidants and to the formation of cytotoxic related oxidants, such as peroxynitrite. All these factors, together with reactive oxygen species from polymorphonuclear leukocytes, contribute to the development of oxidative injuries at the ocular surface. From the clinical point of view it is important that the level of severity of the above described microscopical disturbances found in conjunctival epithelial cells goes parallel with the level of severity of dry eye symptoms.

[Immunohistochemical characterization of collagen IV in control corneas and in corneas obtained from patients suffering from posterior polymorphous corneal dystrophy]. / Imunohistochemická charakterizácia kolagénu IV v zdravých rohovkách a rohovkách pacientov so zadnou polymorfnou dystrofiou.

The expression of all six chains of collagen IV was studied using the indirect fluorescent immunohistochemistry in seven control corneas and seven corneas obtained from patients suffering from the posterior polymorphous corneal dystrophy. Heterogeneous staining, especially in the epithelial basement membrane and Descemet's membrane, was observed in the control corneas. An increase of the staining intensity for the alpha1 and alpha2 chains was observed, especially in the Descemet's membrane and the corneal stroma in samples obtained from the patients compared to the control tissues.

[The application of the autologous serum eye drops results in significant improvement of the conjunctival status in patients with the dry eye syndrome]. / Aplikace autologního séra-ocních kapek vede ke statisticky významnému zlepsení stavu spojivky pacientu se syndromem suchého oka--pilotní studie.

AIM: To detect the changes on the conjunctiva surface before and after the application of the autologous serum (AS) eye drops in patients with dry eye syndrome, using both clinical and laboratory approaches, supplemented with subjective assessing the discomfort status. MATERIALS AND METHODS: The AS eye drops were applied during the period of 3 months in 8 patients with dry eye syndrome (Schirmer test < 5 mm and break-up time < 5 seconds), with the highest (maximum) frequency 8 times a day. The clinical (Schirmer test, break-up time, rose Bengal staining, examination of the tear meniscus, detritus and superficial punctate keratitis) and laboratory examinations (morphological assessment of the conjunctiva, detection of apoptotic cells) were performed at the start and at the end of the 3 months treatment period. Each day, patients reported their ocular status (dryness, discomfort, foreign body sensation, light sensitivity). RESULTS: The AS eye drops application improved significantly the values of the Schirmer test, detritus and superficial punctate keratitis as well. The goblet cells density on the conjunctival surface increased and the number of apoptotic cells decreased. The intensity of unpleasant feelings reported by the patients decreased significantly in all of the assessed categories. CONCLUSION: Because the application of AS eye drops caused the improvement of conjunctival status as well as the decrease of the severity of difficulties reported by the patients, the AS eye drops application should become common therapeutic practice in patients with dry eye syndrome.

Sequencing of the CHST6 gene in Czech macular corneal dystrophy patients supports the evidence of a founder mutation.

AIMS: To characterise the role of the carbohydrate sulfotransferase gene (CHST6) in macular corneal dystrophy (MCD) in Czech patients. METHODS: The coding region of the CHST6 gene was directly sequenced in 10 affected and five unaffected members from eight apparently unrelated MCD families. The type of MCD was determined by enzyme-linked immunosorbent assay of antigenic keratan sulfate (KS) in serum and by immunohistochemical staining of corneas with monoclonal anti-KS antibody. RESULTS: The following changes in the coding sequence of the CHST6 gene were observed; homozygous mutation of c.1A>T (p.M1?); homozygous mutation c.599T>G (p.L200R); compound heterozygosity for c.599T>G and c.614G>A (p.R205Q); compound heterozygosity for c.494G>A (p.C165Y) and c.599T>G; heterozygous c.599T>G mutation and no other change in the coding sequence. One proband exhibited no changes. The pathogenic mutation c.599T>G (p.L200R) was in allelic association with the c.484C>G (p.R162G) polymorphism. Nine patients from seven families were of MCD type I including the subtype IA. CONCLUSION: Four different CHST6 missense mutations, of which p.C165Y is novel, were identified. Allelic association of the c.[484C>G; 599T>G] in six probands out of eight, as well as occurrence of this particular allele in a heterozygous state in one healthy control individual, supports a common founder effect for MCD in the Czech Republic.

Nitric oxide synthase induction and cytotoxic nitrogen-related oxidant formation in conjunctival epithelium of dry eye (Sjögren's syndrome).

Until now, the expression and possible role of nitric oxide and nitrogen related oxidants in the human dry eye have not been investigated. Therefore, we examined immunohistochemically nitric oxide synthase isomers (NOS), enzymes generated nitric oxide, nitrotyrosine, a cytotoxic byproduct of nitric oxide and malondialdehyde, a byproduct of lipid peroxidation, in conjunctival epithelium of patients with dry eye, Sjögren's syndrome (SS). Moreover, in conjunctival epithelium of patients with dry eye (SS) the immunohistochemical staining of some pro-inflammatory cytokines was demonstrated: mature interleukin-1 beta (IL-1beta), interleukin 6 (IL-6), interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha). Conjunctival epithelial cells were obtained by the method of impression cytology. Normal eyes served as controls. In contrast to the normal eyes where endothelial nitric oxide synthase (NOS3) as well as inducible nitric oxide synthase (NOS2) were only slightly expressed in conjunctival epithelium, in dry eye both NOS (mainly NOS2) were gradually expressed along the severity of dry eye symptoms which was in accord with pro-inflammatory cytokine immunodetection (IL-1beta, IL-6, IL-8, TNF-alpha) in dry eye conjunctival cytology samples. This was in contrast to normal eyes where the staining of pro-inflammatory cytokines was weak or completely absent. Peroxynitrite formation (demonstrated by nitrotyrosine residues) and lipid peroxidation (evaluated by increased malondialdehyde staining) were also found in conjunctival epithelium of dry eye with highly pronounced symptoms of dryness. In conclusion, results point to the suggestion that reactive nitrogen species are involved in the pathogenesis or self-propagation of autoimmune dry eye (SS).

The role of conjunctival epithelial cell xanthine oxidoreductase/xanthine oxidase in oxidative reactions on the ocular surface of dry eye patients with Sjögren's syndrome.

Previous papers examined lipid peroxidase levels and myeloperoxidase activity as products of oxidative and inflammatory reactions in the tear fluid of patients suffering from dry eye. The aim of the present paper was to investigate whether the enzymes xanthine oxidoreductase/xanthine oxidase known to generate reactive oxygen species contribute to oxidative reactions on the ocular surface. Xanthine oxidoreductase/xanthine oxidase were examined immunohistochemically as well as histochemically in conjunctival epithelial cells of patients suffering from dry eye. Patients with verified autoimmune dry eye (Sjögren's syndrome) participated in our study; normal eyes served as controls. Conjunctival epithelial cells were obtained by the method of impression cytology using Millicell membranes. The results revealed a pronounced expression, as well as activity of xanthine oxidoreductase/xanthine oxidase in the conjunctival epithelium of dry eye. It is suggested that reactive oxygen species which are generated by this enzymatic system, contribute to oxidative reactions on the eye surface of patients with ocular manifestations of autoimmune disease (Sjögren's syndrome).

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.

[Dry eye syndrome in patients with conjunctival concretions]. / Syndrom suchého oka u nemocných se spojivkovými konkrementy.

PURPOSE: To establish the possible correlation between the presence of conjunctival concretions and the instability of the tear film. METHODS: The group consisted of 50 asymptomatic patients with accidentally detected conjunctival concretions, presented mostly for refractive errors at the outpatient department of the Department of Ophthalmology, School of Medicine Hospital, Prague, Czech Republic. The age of the patients ranged 24-69 years, mean 44.1 years. In 35 patients (group A) were present the conjunctival concretions only, in the remaining 15 patients (group B), together with the concretions, the Meibomian glands dysfunction was also discovered. The complete eye examination was performed, including detailed analysis of tarsal conjunctiva and fornices, Schirmer's test I, the tear film break-up time (BUT) and rose bengal staining. In five patients, the mucin ferning test (MFT) was also performed. RESULTS: Advanced to severe tear deficiency was detected in 42.8 % of patients of the group A, and in 46.6 % of patients in the group B respectively. BUT was shorter or extremely shorter (< 10 sec) in 51.4 % of patients in the group A and in 60 % of patients in the group B. The pathological staining with bengal rose was not detected even in a single patient. MFT was normal in all 5 examined patients (classification I and II). CONCLUSION: In patients with conjunctival concretions, the tear film deficiency may be present (decreased values of the Schirmer's test and BUT). The tear film break-up time (BUT) is surprisingly shorter in younger patients (younger than 45 years of age), in patients with and also without Meibomian glands dysfunction as well. In older patients (over 45 years of age), the defect of the aqueous layer is more pronounced. Patients with conjunctival concretions are potentially affected with the dry eye syndrome.

Nuclear pre-mRNA compartmentalization: trafficking of released transcripts to splicing factor reservoirs.

In the present study, the spatial organization of intron-containing pre-mRNAs of Epstein-Barr virus (EBV) genes relative to location of splicing factors is investigated. The intranuclear position of transcriptionally active EBV genes, as well as of nascent transcripts, is found to be random with respect to the speckled accumulations of splicing factors (SC35 domains) in Namalwa cells, arguing against the concept of the locus-specific organization of mRNA genes with respect to the speckles. Microclusters of splicing factors are, however, frequently superimposed on nascent transcript sites. The transcript environment is a dynamic structure consisting of both nascent and released transcripts, i.e., the track-like transcript environment. Both EBV sequences of the chromosome 1 homologue are usually associated with the track, are transcriptionally active, and exhibit in most cases a polar orientation. In contrast to nascent transcripts (in the form of spots), the association of a post-transcriptional pool of viral pre-mRNA (in the form of tracks) with speckles is not random and is further enhanced in transcriptionally silent cells when splicing factors are sequestered in enlarged accumulations. The transcript environment reflects the intranuclear transport of RNA from the sites of transcription to SC35 domains, as shown by concomitant mapping of DNA, RNA, and splicing factors. No clear vectorial intranuclear trafficking of transcripts from the site of synthesis toward the nuclear envelope for export into the cytoplasm is observed. Using Namalwa and Raji cell lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has been observed. This supports a concept that the level of transcription can alter the spatial relationship among intron-containing genes, their transcripts, and speckles attributable to various levels of splicing factors recruited from splicing factor reservoirs. Electron microscopic in situ hybridization studies reveal that the released transcripts are directed toward reservoirs of splicing factors organized in clusters of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecular complexes between intron-containing genes and splicing factor reservoirs: the recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors.

Neurotoxicity of tetraphenylporphinesulfonate (TPPS4) and a hematoporphyrin derivative (Photosan) in organotypic cultures of chick embryonic dorsal root ganglia.

The neurotoxic effect of tetraphenylporphinesulfonate (TPPS4) and a hematoporphyrin derivative (HPD, Photosan) has been studied in organotypic cultures of chick dorsal root ganglia maintained in a semi-solid culture medium. The changes in two characteristics of neurite outgrowth, the mean radial length of neurites growing out from the ganglia and the area of neurite outgrowths, are used as parameters to evaluate the toxic effect. The porphyrins are tested over the concentration range 10-160 micrograms ml-1. TPPS4 is slightly more toxic than the HPD Photosan. The median inhibitory concentration (IC50) for TPPS4 is 45-50 micrograms ml-1 and for the HPD Photosan 50-60 micrograms ml-1, respectively. Nevertheless, the toxicity of the two drugs is relatively low compared to that of commonly used anticancer drugs, such as cisplatin or taxol.

Differences in the inhibition of neuritic outgrowth in organotypic cultures of rat foetal dorsal root ganglia treated with cisplatin and carboplatin: a comparative study.

Neurotoxic effect of cisplatin and carboplatin, two anticancer drugs with different neurotoxic potential, was examined in organotypic cultures of rat foetal dorsal root ganglia (DRG), maintained in a semisolid agar culture medium. Two main parameters of neurites growing out of the ganglia--the mean radial length of neurites and the area of neurite outgrowth--were used to evaluate the toxic influence of both drugs. IC50 values were calculated from the dose-response curves established at three cultivation time points (24, 48, and 72 h). Cisplatin demonstrated higher toxicity in comparison with carboplatin for all exposure times. The lowest concentration which caused neurite outgrowth alteration was 25 microM of carboplatin and 10 microM of cisplatin. IC 50 was 11.93, 8.25, 8.58 microM for cisplatin and 177.46, 47.83, 44.94 for carboplatin after 24, 48 and 72 h, respectively.

Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment.

This paper presents a new and gentle method to separate Schwann cells from fibroblasts obtained from foetal rat dorsal root ganglia (DRG). The method exploits the different growth and adhesion characteristics of fibroblasts and Schwann cells under different experimental conditions such that antiproliferative (cytotoxic) drugs or time-consuming centrifugation is not needed. Standard procedures were used to obtain mixed cultures of Schwann cells, fibroblasts and neurons. After about 5 days further purification of the cells was achieved by exploiting the different responses of Schwann cells and fibroblasts to a temperature shock. Cooling the cells with cold phosphate-buffered saline (PBS), followed by pipetting cold medium directly on top of the cells ('cold jet'), resulted in specific detachment of Schwann cells and neurons, whereas fibroblasts remained securely attached. Schwann cells attached to the surface of new, uncoated culture dishes whereas neurons did not. Two cycles of the cold jet procedure resulted in nearly pure (98-100%) cultures of Schwann cells. Besides being gentle, this method is easy and fast, and because cytotoxic drugs are not used, it does not affect cell survival negatively.