The expression of interleukin-1 alpha, TNF and VEGF in corneal cells of patients with bullous keratopathy

Bullous keratopathy (BK) is a chronic corneal edema with or without subepithelial bullae as a result of a loss of the endothelial cells. 15 patients with BK after cataract surgery with intraocular lens implantation, due to Fuchs dystrophy (n = 3) or corneal endothelial trauma (n = 12) were included in the study. All patients were treated by amniotic membrane transplantation (AMT). Corneal epithelial cells in patients suffering from BK secreted 3.91 +/- 3.09 pg/mL of IL-1 alpha, 4446 +/- 16.8 pg/mL of TNF and 81.43 +/- 37.81 pg/mL of VEGF-I. Levels of all 3 investigated cytokines were significantly higher as compared to controls (p < 0.005). Amniotic membranes that were used to treat investigated patients contained 638.98 +/- 613.98 pg/mL of IL-1ra, 0.026 +/- 0.009 pg/mL of sTNF and 81.39 +/- 21.01 pg/mL of VEGF-R. Beneficial clinical effect of the AMT in treating BK could be explained by its natural production of pro-inflammatory cytokine antagonists such as IL-ra, sTNF antagonist and VEGF-R.     

The mechanisms of action of phototherapy in the treatment of the most common dermatoses

Phototherapy denotes the use of ultraviolet (UV) light in the management of several dermatoses. Most phototherapy regimens utilize ultraviolet radiation of different wavelenghts. Currently, irradiations with broadband UVB (290-320 nm), narrowband UVB (311-313 nm), 308 nm excimer laser, UVA 1 (340-400 nm), UVA with psoralen (PUVA), and extracorporeal photochemotherapy (photopheresis) are being used. The interplay of the various photobiologic pathways is far from being completely understood. Disordes that may benefit from such approach are numerous, with psoriasis, atopic dermatitis, cutaneous T-cell lymphomas, morphea, and vitiligo as main indications. The immunomodulatory effects of UVB radiation primarily affect the epidermis and superficial dermis, while UVA radiation affects mid and deep dermal components, especially blood vessels. UVB radiation is absorbed by endogenous chromophores, such as nuclear DNA, which initiates a cascade of events. Absorption of UV light by nucleotides causes the formation of DNA photoproducts and suppresses DNA synthesis. In addition UV light stimulates synthesis of prostaglandins and cytokines that play important roles in immune suppression. It may reduce the number of Langerhans cells, cutaneous T lymphocytes and mast cells in the dermis. UV radiation can also affect extranuclear molecular targets located in the cytoplasm and cell membrane. Immune suppression, alteration in cytokine expression, and cell cycle arrest may all contribute to the suppression of disease activity. PUVA is a form of chemophototherapy which uses UVA light to activate chemicals known as psoralens, hence psoralen ultraviolet A. The conjunction of psoralens with epidermal DNA inhibits DNA replication and causes cell cycle arrest. Psoralen photosensitization also causes an alteration in the expression of cytokines and cytokine receptors. Psoralens interact with RNA, proteins and other cellular components and indirectly modify proteins and lipids via singlet oxygen-mediated reactions or by generating of free radicals. Infiltrating lymphocytes are strongly suppressed by PUVA, with variable effects on different T-cell subsets. Psoralens and UV radiation also stimulate melanogenesis. Extracorporeal photopheresis is technique used in treatment of erythrodermic cutaneous lymphomas. It is very potent in induction of lymphocyte apoptosis. Despite the introduction of numerous effective systemic medications and biologic agents in dermatology, phototherapy remains a reliable, and often preferred option for several dermatoses.     

Transplantation of amniotic membrane in corneal ulcers and persistant epithelial defects

Amniotic membrane transplantation (AMT) leads to reduction of inflammatory symptoms and causes faster epithelisation in corneal ulcers and persistant epithelial defect. 21 patients with corneal ulcer (n = 18) or non-healing epithelial defect (n = 3) unresponsive to conventional treatment were included in the study. All patients were treated by AMT. Corneal epithelial cells in patients suffering from corneal ulcer secreted 3.51 +/- 1.79 of IL-1alpha, 64.27 +/- 31.53 pg/mL of TNFalpha and 209.07 +/- 201.82 pg/mL of VEGF. Levels of all 3 investigated cytokines were significantly higher as compared to controls (p < 0.005). Amniotic membranes that were used contained 775.69 +/- 613.98 pg/mL of IL-1alpha, 0.036 +/- 0.033 pg/mL of sTNF and 175.01 +/- 166.63 pg/mL of VEGF-R. Supporting effect of the AMT could be explained by the fact that AM secretes its natural antinflammatory antagonists IL-1ra, sTNF and VEGF-R.     

Long lasting complex nocturnal hallucinations during Osmotic Release Oral System (OROS) methylphenidate treatment in a 7-year old girl

We report a case of a girl with Attention deficit hyperactivity disorder (ADHD) and Oppositional defiant disorder (ODD) who experienced a 3-hour episode of nocturnal complex bizarre visual hallucinations when treated with 18 mg Osmotic Release Oral System (OROS) methylphenidate (MPH). Nocturnal polysomnography performed two weeks later revealed REM sleep reduction (17%) and fragmentation . Two episodes of confusional arousals were recorded. This finding is typical of parasomnia associated with NREM sleep - disorder of arousal. We hypothesize that this preexisting sleep impairment represents a factor of vulnerability to MPH sleep side effects. In our search of literature, we found no report of nocturnal hallucination alone during treatment with stimulants.     

Peptidoglycan Hydrolases as Species-Specific Markers to Differentiate Lactobacillus helveticus from Lactobacillus gallinarum and Other Closely Related Homofermentative Lactobacilli

We propose a new method that allows accurate discrimination of Lactobacillus helveticus from other closely related homofermentative lactobacilli, especially Lactobacillus gallinarum. This method is based on the amplification by PCR of two peptidoglycan hydrolytic genes, Lhv_0190 and Lhv_0191. These genes are ubiquitous and show high homology at the intra-species level. The PCR method gave two specific PCR products, of 542 and 747 bp, for 25 L. helveticus strains coming from various sources. For L. gallinarum, two amplicons were obtained, the specific 542 bp amplicon and another one with a size greater than 1,500 bp. No specific PCR products were obtained for 12 other closely related species of lactobacilli, including the L. acidophilus complex, L. delbrueckii, and L. ultunensis. The developed PCR method provided rapid, precise, and easy identification of L. helveticus. Moreover, it enabled differentiation between the two closely phylogenetically related species L. helveticus and L. gallinarum.     

The Tracing of Mycobacteria in Drinking Water Supply Systems by Culture, Conventional, and Real Time PCRs

Mycobacteria are widely present in diverse aquatic habitats, where they can survive for months or years while some species can even proliferate. The resistance of different mycobacterial species to disinfection methods like chlorination or ozonation could result in their presence in the final tap water of consumers. In this study, the culture method, Mycobacterium tuberculosis complex conventional duplex PCR for detection of non-tuberculous mycobacteria (NTM) and quantitative real-time PCR (qPCR) to detect three subspecies of M. avium species (M. a. avium, M. a. hominissuis, and M. a. paratuberculosis) were used to trace their possible path of transmission from the watershed through the reservoir and drinking water plant to raw drinking water and finally to households. A total of 124 samples from four drinking water supply systems in the Czech Republic, 52 dam sediments, 34 water treatment plant sludge samples, and 38 tap water household sediments, were analyzed. NTM of 11 different species were isolated by culture from 42 (33.9 %) samples; the most prevalent were M. gordonae (16.7 %), M. triplex (14.3 %), M. lentiflavum (9.5 %), M. a. avium (7.1 %), M. montefiorenase (7.1 %), and M. nonchromogenicum (7.1 %). NTM DNA was detected in 92 (76.7 %) samples. By qPCR analysis a statistically significant decrease (P < 0.01) was observed along the route from the reservoir (dam sediments), through water treatment sludge and finally to household sediments. The concentrations ranged from 10⁰ to 10⁴ DNA cells/g. It was confirmed that drinking water supply systems (watershed–reservoir–drinking water treatment plant–household) might be a potential transmission route for mycobacteria.     

Reference Genes for Real-Time PCR Quantification of Messenger RNAs and MicroRNAs in Mouse Model of Obesity

Obesity and metabolic syndrome is increasing health problem worldwide. Among other ways, nutritional intervention using phytochemicals is important method for treatment and prevention of this disease. Recent studies have shown that certain phytochemicals could alter the expression of specific genes and microRNAs (miRNAs) that play a fundamental role in the pathogenesis of obesity. For study of the obesity and its treatment, monosodium glutamate (MSG)-injected mice with developed central obesity, insulin resistance and liver lipid accumulation are frequently used animal models. To understand the mechanism of phytochemicals action in obese animals, the study of selected genes expression together with miRNA quantification is extremely important. For this purpose, real-time quantitative PCR is a sensitive and reproducible method, but it depends on proper normalization entirely. The aim of present study was to identify the appropriate reference genes for mRNA and miRNA quantification in MSG mice treated with green tea catechins, potential anti-obesity phytochemicals. Two sets of reference genes were tested: first set contained seven commonly used genes for normalization of messenger RNA, the second set of candidate reference genes included ten small RNAs for normalization of miRNA. The expression stability of these reference genes were tested upon treatment of mice with catechins using geNorm, NormFinder and BestKeeper algorithms. Selected normalizers for mRNA quantification were tested and validated on expression of NAD(P)H:quinone oxidoreductase, biotransformation enzyme known to be modified by catechins. The effect of selected normalizers for miRNA quantification was tested on two obesity- and diabetes- related miRNAs, miR-221 and miR-29b, respectively. Finally, the combinations of B2M/18S/HPRT1 and miR-16/sno234 were validated as optimal reference genes for mRNA and miRNA quantification in liver and 18S/RPlP0/HPRT1 and sno234/miR-186 in small intestine of MSG mice. These reference genes will be used for mRNA and miRNA normalization in further study of green tea catechins action in obese mice.