Polymorphisms Related to Iron Homeostasis Associate with Liver Disease in Chronic Hepatitis C.

Dysregulation of iron metabolism in chronic hepatitis C (CHC) is a significant risk factor for hepatic cirrhosis and cancer. We studied if known genetic variants related to iron homeostasis associate with liver disease progression in CHC. Retrospective analysis included 249 CHC patients qualified for antiviral therapy between 2004 and 2014. For all patients, nine SNPs within HFE, TFR2, HDAC2, HDAC3, HDAC5, TMPRSS6, and CYBRD1 genes were genotyped. Expression of selected iron-related genes, was determined with qRT-PCR in 124 liver biopsies, and mRNA expression of co-inhibitory receptors (PD-1, Tim3, CTLA4) was measured in 79 liver samples. CYBRD1 rs884409, HDAC5 rs368328, TFR2 rs7385804, and TMPRSS6 rs855791 associated with histopathological changes in liver tissue at baseline. The combination of minor allele in HDAC3 rs976552 and CYBRD1 rs884409 linked with higher prevalence of hepatocellular carcinoma (HCC) during follow up (OR 8.1 CI 2.2-29.2; p = 0.001). Minor allele in HDAC3 rs976552 associated with lower hepatic expression of CTLA4. Tested polymorphisms related to iron homeostasis associate with histopathological changes in the liver. The presence of both HDAC3 rs976552 G and CYBRD1 rs884409 G alleles correlates with HCC occurrence, especially in the group of patients with elevated AST (>129 IU/L). rs976552 in HDAC3 could impact immunological processes associated with carcinogenesis in CHC.

Development of Staphylococcus aureus tolerance to antimicrobial photodynamic inactivation and antimicrobial blue light upon sub-lethal treatment.

Antimicrobial photodynamic inactivation (aPDI) and antimicrobial blue light (aBL) are considered low-risk treatments for the development of bacterial resistance and/or tolerance due to their multitargeted modes of action. In this study, we assessed the development of Staphylococcus aureus tolerance to these phototreatments. Reference S. aureus USA300 JE2 was subjected to 15 cycles of both sub-lethal aPDI (employing an exogenously administered photosensitizer (PS), i.e., rose Bengal (RB)) and sub-lethal aBL (employing endogenously produced photosensitizing compounds, i.e., porphyrins). We demonstrate substantial aPDI/aBL tolerance development and tolerance stability after 5 cycles of subculturing without aPDI/aBL exposure (the development of aPDI/aBL tolerance was also confirmed with the employment of clinical MRSA and MSSA strain as well as other representatives of Gram-positive microbes, i.e. Enterococcus faecium and Streptococcus agalactiae). In addition, a rifampicin-resistant (RIFR) mutant selection assay showed an increased mutation rate in S. aureus upon sub-lethal phototreatments, indicating that the increased aPDI/aBL tolerance may result from accumulated mutations. Moreover, qRT-PCR analysis following sub-lethal phototreatments demonstrated increased expression of umuC, which encodes stress-responsive error-prone DNA polymerase V, an enzyme that increases the rate of mutation. Employment of recA and umuC transposon S. aureus mutants confirmed SOS-induction dependence of the tolerance development. Interestingly, aPDI/aBL-tolerant S. aureus exhibited increased susceptibility to gentamicin (GEN) and doxycycline (DOX), supporting the hypothesis of genetic alterations induced by sub-lethal phototreatments. The obtained results indicate that S. aureus may develop stable tolerance to studied phototreatments upon sub-lethal aPDI/aBL exposure; thus, the risk of tolerance development should be considered significant when designing aPDI/aBL protocols for infection treatments in vitro and in clinical settings.

Neutrocyte-to-lymphocyte ratio predicts the presence of a replicative hepatitis C virus strand after therapy with direct-acting antivirals.

Residual HCV-RNA can persist in liver tissue and peripheral blood mononuclear cells (PBMCs) long after antiviral therapy of chronic hepatitis C in patients repeatedly negative for viral RNA in serum. This occult infection associates with impaired immune response and the risk of lymphoproliferative disorders or progressive liver disease. There are currently no monitoring strategies for patients after treatment. We investigated if serum inflammation markers and interferon lambda (IFNL) genotype can be predictors of the presence of HCV-RNA and the replicative HCV-RNA (-) strand in patients who reached sustained virological response after interferon-free therapy. Forty-two consecutive patients who remained HCV-RNA negative in serum 24 weeks after the end of treatment (EOT) and during the follow-up were enrolled. Total HCV-RNA and HCV-RNA (-) strand were detected using ultrasensitive RT-PCR in PBMCs collected 12-15 months after EOT. Polymorphisms within IFNL3-IFNL4 region (rs12979860 and ss469415590) were genotyped with allele-specific PCR. Viral RNA was found in PBMCs from 31 (74%) patients, and of those 29 (69%) were also positive for HCV-RNA (-). Neither normalization of alanine aminotransferase nor IFNL genotype predicted the presence of residual HCV-RNA. A significantly higher neutrocyte-to-lymphocyte ratio (NLR) 24 weeks after the start of treatment predicted elimination of replicative HCV-RNA strand (OR 0.23; 95% CI 0.10-0.86; P = 0.019). Patients with no HCV-RNA (-) in PBMCs showed a greater increase in neutrocyte count between EOT and baseline (P = 0.028). Lack of significant elevation of NLR after therapy with direct-acting antivirals could predict the presence of residual replicative HCV-RNA strand in PBMCs.

Structural Transformation to Attain Responsible BIOSciences (STARBIOS2): Protocol for a Horizon 2020 Funded European Multicenter Project to Promote Responsible Research and Innovation.

BACKGROUND: Promoting Responsible Research and Innovation (RRI) is a major strategy of the "Science with and for Society" work program of the European Union's Horizon 2020 Framework Programme for Research and Innovation. RRI aims to achieve a better alignment of research and innovation with the values, needs, and expectations of society. The RRI strategy includes the "keys" of public engagement, open access, gender, ethics, and science education. The Structural Transformation to Attain Responsible BIOSciences (STARBIOS2) project promotes RRI in 6 European research institutions and universities from Bulgaria, Germany, Italy, Slovenia, Poland, and the United Kingdom, in partnership with a further 6 institutions from Brazil, Denmark, Italy, South Africa, Sweden, and the United States. OBJECTIVE: The project aims to attain RRI structural change in 6 European institutions by implementing action plans (APs) and developing APs for 3 non-European institutions active in the field of biosciences; use the implementation of APs as a learning process with a view to developing a set of guidelines on the implementation of RRI; and develop a sustainable model for RRI in biosciences. METHODS: The project comprises interrelated research and implementation designed to achieve the aforementioned specific objectives. The project is organized into 6 core work packages and 5 supporting work packages. The core work packages deal with the implementation of institutional APs in 6 European institutions based on the structural change activation model. The supporting work packages include technical assistance, learning process on RRI-oriented structural change, monitoring and assessment, communication and dissemination, and project management. RESULTS: The project is funded by Horizon 2020 and will run for 4 years (May 2016-April 2020). As of June 2018, the initial phase has been completed. The participating institutions have developed and approved APs and commenced their implementation. An observation tool has been launched by the Technical Assistance Team to collect information from the implementation of APs; the Evaluation & Assessment team has started monitoring the advancement of the project. As part of the communication and dissemination strategy, a project website, a Facebook page, and a Twitter account have been launched and are updated periodically. The International Scientific Advisory Committee has been formed to advise on the reporting and dissemination of the project's results. CONCLUSIONS: In the short term, we anticipate that the project will have a considerable impact on the organizational processes and structures, improving the RRI uptake in the participating institutions. In the medium term, we expect to make RRI-oriented organizational change scalable across Europe by developing guidelines on RRI implementation and an RRI model in biosciences. In the long term, we expect that the project would help increase the ability of research institutions to make discoveries and innovations in better alignment with societal needs and values. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/11745.

Interferon lambda polymorphisms associate with body iron indices and hepatic expression of interferon-responsive long non-coding RNA in chronic hepatitis C.

Single nucleotide polymorphisms (SNPs) within DNA region containing interferon lambda 3 (IFNL3) and IFNL4 genes are prognostic factors of treatment response in chronic hepatitis C (CHC). Iron overload, frequently diagnosed in CHC, is associated with unfavorable disease course and a risk of carcinogenesis. Its etiology and relationship with the immune response in CHC are not fully explained. Our aim was to determine whether IFNL polymorphisms in CHC patients associate with body iron indices, and whether they are linked with hepatic expression of genes involved in iron homeostasis and IFN signaling. For 192 CHC patients, four SNPs within IFNL3-IFNL4 region (rs12979860, rs368234815, rs8099917, rs12980275) were genotyped. In 185 liver biopsies, histopathological analyses were performed. Expression of five mRNAs and three long non-coding RNAs (lncRNAs) was determined with qRT-PCR in 105 liver samples. Rs12979860 TT or rs8099917 GG genotypes as well as markers of serum and hepatocyte iron overload associated with higher activity of gamma-glutamyl transpeptidase and liver steatosis. The presence of two minor alleles in any of the tested SNPs predisposed to abnormally high serum iron concentration and correlated with higher hepatic expression of lncRNA NRIR. On the other hand, homozygosity in any major allele associated with higher viral load. Patients bearing rs12979860 CC genotype had lower hepatic expression of hepcidin (HAMP; P = 0.03). HAMP mRNA level positively correlated with serum iron indices and degree of hepatocyte iron deposits. IFNL polymorphisms influence regulatory pathways of cellular response to IFN and affect body iron balance in chronic hepatitis C virus infection.

Analysis of polymorphism and hepatic expression of duodenal cytochrome b in chronic hepatitis C.

BACKGROUND AND AIM: Pathological iron overload is commonly found in chronic hepatitis C (CHC) patients and considered as a negative prognostic factor of the disease. A single nucleotide polymorphism (SNP) rs884409 in duodenal cytochrome b gene (CYBRD1) is implicated in the pathogenesis of hemochromatosis. In our study we investigated the impact of the CYBRD1 genotype and expression on iron overload in CHC patients. METHODS: Liver biopsy specimens and whole blood samples from 243 patients with CHC were included in the study. Iron deposits in hepatocytes, serum markers of iron overload, and expression profile of gene-regulators of iron homeostasis were analyzed. Genotyping and analysis of gene expression of the CYBRD1 were performed. The frequency of SNP and the expression levels of CYBRD1 were compared between the groups of patients with and without markers of iron overload. RESULTS: The single nucleotide variant rs884409 G was associated with elevated serum iron levels, increased markers of liver inflammation, and oxidative stress. Hepatic expression of CYBRD1 was associated with the expression of Tfr2, Id1, and HO-1 genes, serum ferritin levels, and with increased iron accumulation in liver. CONCLUSION: These results implicate CYBRD1 involvement in iron homeostasis in CHC.

Factors Determining Staphylococcus aureus Susceptibility to Photoantimicrobial Chemotherapy: RsbU Activity, Staphyloxanthin Level, and Membrane Fluidity.

Photoantimicrobial chemotherapy (PACT) constitutes a particular type of stress condition, in which bacterial cells induce a pleiotropic and as yet unexplored effect. In light of this, the key master regulators are of putative significance to the overall phototoxic outcome. In Staphylococcus aureus, the alternative sigma factor σ(B) controls the expression of genes involved in the response to environmental stress. We show that aberration of any sigB operon genes in S. aureus USA300 isogenic mutants causes a pronounced sensitization (>5 log10 reduction in CFU drop) to PACT with selected photosensitizers, namely protoporphyrin diarginate, zinc phthalocyanine and rose bengal. This effect is partly due to aberration-coupled staphyloxanthin synthesis inhibition. We identified frequent mutations in RsbU, a σ(B) activator, in PACT-vulnerable clinical isolates of S. aureus, resulting in σ(B) activity impairment. Locations of significant changes in protein structure (IS256 insertion, early STOP codon occurrence, substitutions A230T and A276D) were shown in a theoretical model of S. aureus RsbU. As a phenotypic hallmark of PACT-vulnerable S. aureus strains, we observed an increased fluidity of bacterial cell membrane, which is a result of staphyloxanthin content and other yet unidentified factors. Our research indicates σ(B) as a promising target of adjunctive antimicrobial therapy and suggests that enhanced cell membrane fluidity may be an adjuvant strategy in PACT.

Murine Model Imitating Chronic Wound Infections for Evaluation of Antimicrobial Photodynamic Therapy Efficacy.

It is generally acknowledged that the age of antibiotics could come to an end, due to their widespread, and inappropriate use. Particularly for chronic wounds alternatives are being thought. Antimicrobial Photodynamic Therapy (APDT) is a potential candidate, and while approved for some indications, such as periodontitis, chronic sinusitis and other niche indications, its use in chronic wounds is not established. To further facilitate the development of APDT in chronic wounds we present an easy to use animal model exhibiting the key hallmarks of chronic wounds, based on full-thickness skin wounds paired with an optically transparent cover. The moisture-retaining wound exhibited rapid expansion of pathogen colonies up to 8 days while not jeopardizing the host survival. Use of two bioluminescent pathogens; methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa permits real time monitoring of the pathogens. The murine model was employed to evaluate the performance of four different photosensitizers as mediators in Photodynamic Therapy. While all four photosensitizers, Rose Bengal, porphyrin TMPyP, New Methylene Blue, and TLD1411 demonstrated good to excellent antimicrobial efficacy in planktonic solutions at 1 to 50 µM concentrations, whereas in in vivo the growth delay was limited with 24-48 h delay in pathogen expansion for MRSA, and we noticed longer growth suppression of P. aeruginosa with TLD1411 mediated Photodynamic Therapy. The murine model will enable developing new strategies for enhancement of APDT for chronic wound infections.

Fine-tuning recA expression in Staphylococcus aureus for antimicrobial photoinactivation: importance of photo-induced DNA damage in the photoinactivation mechanism.

Bacterial cell envelope is generally accepted as the primary target for a photo-induced oxidative stress. It is plausible that DNA damage occurs during the antimicrobial photoinactivation. Here we investigate the correlation between DNA damage and photoinactivation by evaluating the level of RecA-based DNA repair system in Staphylococcus aureus. By using exogenous photosensitizers (new methylene blue (NMB), toluidine blue O (TBO), 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), zinc phthalocyanine (ZnPc), Rose Bengal (RB)) and ALA-induced endogenous porphyrin-dependent blue light (405 nm), several outcomes were observed: (i) an increase of DNA damage (from gel electrophoresis in DNA damage assay), (ii) an increase of recA expression (luminescence assay in recA-lux strain), and (iii) an increase of RecA protein level (Western blotting). When recA expression was repressed by novobiocin, or abolished by deleting the gene, S. aureus susceptibility towards photoinactivation was increased at approximately a hundred-fold. The absence of RecA increases DNA damage to yield bactericidal effect. In novobiocin-resistant mutant (gyrB), as opposed to wild type, neither RecA protein level nor cell's susceptibility was affected by photoinactivation (when novobiocin is present). This is to suggest that GyrB-dependent inhibition mediated recA repression. Therefore, we have established the role of RecA in DNA damage during photoinactivation. With the use of rifampicin mutation frequency and Ames tests, we demonstrated that photoinactivation did not increase S. aureus mutagenesis and potentially is not mutagenic toward eukaryotic cells. The results suggest that the treatment is considered safe. In conclusion, we provide an evidence that recA inhibitor may serve as therapeutic adjuvant for antimicrobial photoinactivation. Clinical relevance of our findings warrants further investigations.

Photodynamic Inactivation of Candida albicans with Imidazoacridinones: Influence of Irradiance, Photosensitizer Uptake and Reactive Oxygen Species Generation.

The increasing applicability of antifungal treatments, the limited range of available drug classes and the emergence of drug resistance in Candida spp. suggest the need for new treatment options. To explore the applicability of C. albicans photoinactivation, we examined nine structurally different imidazoacridinone derivatives as photosensitizing agents. The most effective derivatives showed a >10(4)-fold reduction of viable cell numbers. The fungicidal action of the three most active compounds was compared at different radiant powers (3.5 to 63 mW/cm2), and this analysis indicated that 7 mW/cm2 was the most efficient. The intracellular accumulation of these compounds in fungal cells correlated with the fungicidal activity of all 9 derivatives. The lack of effect of verapamil, an inhibitor targeting Candida ABC efflux pumps, suggests that these imidazoacridinones are not substrates for ABC transporters. Thus, unlike azoles, a major class of antifungals used against Candida, ABC transporter-mediated resistance is unlikely. Electron paramagnetic resonance (EPR)-spin trapping data suggested that the fungicidal light-induced action of these derivatives might depend on the production of superoxide anion. The highest generation rate of superoxide anion was observed for 1330H, 1610H, and 1611. Singlet oxygen production was also detected upon the irradiation of imidazoacridinone derivatives with UV laser light, with a low to moderate yield, depending on the type of compound. Thus, imidazoacridinone derivatives examined in the present study might act via mixed type I/type II photodynamic mechanism. The presented data indicate lack of direct correlation between the structures of studied imidazoacridinones, cell killing ability, and ROS production. However, we showed for the first time that for imidazoacridinones not only intracellular accumulation is necessary prerequisite of lethal photosensitization of C. albicans, but also localization within particular cellular structures. Our findings present IA derivatives as efficient antifungal photosensitizers with a potential to be used in local treatment of Candida infection.

Antimicrobial photodynamic therapy with fulleropyrrolidine: photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies.

A family of N-methylpyrrolidinium fullerene iodide salts has been intensively studied to determine their applicability in antimicrobial photodynamic therapy (APDT). This study examined in vitro the efficacy of a C60 fullerene functionalized with one methylpyrrolidinium group to kill upon irradiation with white light gram-negative and gram-positive bacteria, as well as fungal cells, and the corresponding mechanism of the fullerene bactericidal action. The in vitro studies revealed that the high antistaphylococcal efficacy of functionalized fullerene could be linked to their ability to photogenerate singlet oxygen and superoxide anion. Following Staphylococcus aureus photoinactivation, no modifications of its genomic DNA were detected. In contrast, photodamage of the cell envelope seemed to be a dominant mechanism of bactericidal action. In in vivo studies, a 2 log10 reduction in the average bioluminescent radiance between treated and non-treated mice was reached. One day post APDT treatment, moist and abundant growth of bacteria could be observed on wounds of non-fulleropyrrolidine and dark control mice. APDT-treated wounds stayed visibly clear up to the third day. Moreover, cytotoxicity test on human dermal keratinocytes revealed great safety of using the sensitizer toward eukaryotic cells. These data indicate potential application of functionalized fullerene as antistaphylococcal sensitizer for superficial infections.

mRNA heptaplex protocol for distinguishing between menstrual and peripheral blood.

The identification of menstrual blood is an important issue in forensic biology, but currently, there are no confirmatory methods for its detection. Here, we demonstrate a highly reliable simple heptaplex method that allows for the discrimination between menstrual and peripheral blood. The test has been used successfully in criminal casework, in which the origin of blood on a rape victim's underwear and trousers was questioned as being menstrual or traumatic peripheral blood. To solve this problem, transcripts of the following genes were used: mucin 4 (MUC4), human ß-defensin 1 (HBD1), two matrix metalloproteinases (MMP7, MMP11), δ-aminolevulinate synthase 2 (ALAS2), hemoglobin alpha (HBA) and glucose 6-phosphate dehydrogenase (G6PDH). The sensitivity of the test is 0.3ng of RNA. The possibility of the detection and differentiation of menstrual and peripheral blood in mixtures that contain other body fluids was investigated. Reliable detection is possible for menstrual blood stains that are up to 1-2 years old if stored at room temperature. This easy approach, thanks to the amplification of 4 vaginal and 2 blood markers, minimizes the risk of false negative results.

Multiresistant strains are as susceptible to photodynamic inactivation as their naïve counterparts: protoporphyrin IX-mediated photoinactivation reveals differences between methicillin-resistant and methicillin-sensitive Staphylococcus aureus strains.

OBJECTIVE: The current study was aimed at the investigation of differences in response to photoinactivation between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates. Moreover, we aimed to elucidate if the observed variation resulted from antimicrobial resistance mechanisms and strains' susceptibility to antibiotic therapy. BACKGROUND DATA: Because of the emergence of multidrug resistance, the development of alternative antimicrobial strategies seems to be required. The concept of photodynamic inactivation (PDI) involves cell exposure to appropriate wavelength light that leads to the excitation of photosensitizer molecules, resulting in the production of reactive oxygen species responsible for cell inactivation and death. Recently, we have demonstrated a strain-dependent response of S. aureus to photoinactivation, and observed elevated resistance to PDI among MRSA strains. Nevertheless, the mechanism underlying this phenomenon remains unexplained. METHODS: S. aureus response to protoporphyrin IX (PPIX)-mediated photoinactivation was studied for 424 MRSA/MSSA isolates. VITEK 2 Advanced Expert System was used to detect antimicrobial resistance mechanisms and strains' susceptibility to antibiotictherapy. RESULTS: Data obtained demonstrated that MRSA are significantly more resistant to photoinactivation than MSSA strains; however, the difference observed did not result from antimicrobial susceptibility or resistance mechanisms. Furthermore, regardless of the strains' origin, a similar effectiveness of PDI could be achieved. Moreover, it was determined that the ability to form biofilms in vitro, and the presence of mec element, does not explain the observed differences between MRSA and MSSA strains. CONCLUSIONS: PDI could be highly effective against multidrug resistant pathogens as well as their naïve counterparts. Nevertheless, regardless of the antimicrobial resistance mechanism, the difference in response to PDI between MRSA and MSSA exists.

The agr function and polymorphism: impact on Staphylococcus aureus susceptibility to photoinactivation.

Staphylococcus aureus is an important human pathogen that causes healthcare-associated and community-acquired infections. Moreover, the growing prevalence of multiresistant strains requires the development of alternative methods to antibiotic therapy. One effective therapeutic option may be antimicrobial photodynamic inactivation (aPDI). Recently, S. aureus strain-dependent response to PDI was demonstrated, although the mechanism underlying this phenomenon remains unexplained. The aim of the current study was to investigate statistically relevant correlations between the functionality and polymorphisms of agr gene determined for 750 methicillin-susceptible and methicillin-resistant S. aureus strains and their responses to photodynamic inactivation using protoporphyrin IX. An AluI and RsaI digestion of the agr gene PCR product revealed existing correlations between the determined digestion profiles (designations used for the first time) and the PDI response. Moreover, the functionality of the agr system affected S. aureus susceptibility to PDI. Based on our results, we conclude that the agr gene may be a genetic factor affecting the strain dependent response to PDI.

Discovering the mechanisms of strain-dependent response of Staphylococcus aureus to photoinactivation: oxidative stress toleration, endogenous porphyrin level and strain's virulence.

BACKGROUND: Staphylococcus aureus is generally known to be susceptible to photoinactivation. However, the phenomenon of its strain-dependent response to photodynamic treatment has been reported. Moreover, the factors determining the emerging variation among strains according to photoinactivation remain unclear. METHODS: This work aimed to investigate any relevant correlation between bacterial toleration of oxidative stress, porphyrin level, photosensitizer uptake and strain's virulence of studied methicillin-susceptible and methicillin-resistant S. aureus strains and their response to photodynamic inactivation (using protoporphyrin diarginate, toluidine blue O and 5-aminolevulinic acid). RESULTS: Obtained data let to demonstrate that studied factors have limited impact on strain response to PDI. However, we have shown that multicomponent sensitizing agent i.e. consisted of PPArg2, ALA and TBO would eliminate the S. aureus elevated resistance to photoinactivation and that both highly virulent and low virulent S. aureus strains could be easily eradicated with the use of PDI. Moreover, we have shown that photodynamic inactivation could decrease the virulence of S. aureus extracellular fraction. CONCLUSION: The mechanism underlying strain-dependent response to photoinactivation is complex and multifactorial nevertheless with the use of several sensitizing agents the elevated resistance to photodynamic treatment can be omitted.

Sub-lethal photodynamic inactivation renders Staphylococcus aureus susceptible to silver nanoparticles.

Staphylococcus aureus is a common etiological factor in infections of burns and other chronic wounds. The development of an effective and fast-acting treatment would be enormously beneficial and is highly desired. We focused on testing the bactericidal efficacy of photoinactivation using a known photosensitizer (protoporphyrin IX, PPIX) in sequential combination with silver nanoparticles against S. aureus. Using PPIX-based photoinactivation followed by silver nanoparticles we obtained a high bactericidal effect (7 log10 units reduction) with limited harmful effects on human epidermal keratinocytes. Moreover, we observed that the use of silver nanoparticles prevents bacterial re-growth 24 h post-PDI treatment. A sequential combination of photoinactivation and silver nanoparticles represents a potentially effective antibacterial approach.

Imidazoacridinone derivatives as efficient sensitizers in photoantimicrobial chemotherapy.

The objective of this study was to investigate a new potential photosensitizer (PS) in the photodynamic inactivation (PDI) of microorganisms in vitro (11 reference strains and 13 clinical isolates, representing common Gram-positive and Gram-negative human pathogens), with special emphasis on Candida albicans. We studied the light-induced cytotoxicity of the imidazoacridinone derivative C1330 toward fungal cells grown in planktonic form. We examined the influence of various parameters (time of incubation, PDI quencher effect, and C1330 accumulation in C. albicans cells) on the efficacy of light-dependent cytotoxicity. Additionally, we checked for the potential cyto- and phototoxic activity of C1330 against human dermal keratinocytes. In our research, we used a broadband incoherent blue light source (380 to 470 nm) with an output power of 100 mW/cm(2). In vitro studies showed that the C1330 action against C. albicans was a light-dependent process. C1330 was an efficient photosensitizer in the photodynamic inactivation of C. albicans, which reduced the growth of planktonic cells by 6.1 log10 units. Efficient accumulation of PS in the nucleus and vacuoles was observed after 30 min of incubation, which correlated with the highest photokilling efficacy. Significant changes in intracellular structure were observed upon illumination of C1330-incubated C. albicans cells. In the case of the human HaCaT cell line, approximately 40% of cells survived the treatment, which indicates the potential benefit of further study of the application of C1330 in photoantimicrobial chemotherapy. These data suggest that PDI may be a viable approach for the treatment of localized C. albicans infections.

Diagnosis and treatment difficulties in 18-year-old male patient with hereditary hemochromatosis, chronic hepatitis B, Gilbert syndrome and ulcerative colitis.

Among possible causes of chronic hepatitis in adolescents most common are infections, autoimmune disorders and metabolic diseases. Thus, diagnostic procedures should be multidirectional. This study reports diagnosis and treatment difficulties in an 18-year-old male patient with hereditary hemochromatosis (HH), ulcerative colitis (UC), chronic hepatitis B (CHB) and Gilbert syndrome. The presented case illustrates problems in diagnostics related to the presence of numerous disease conditions in one patient. It should be taken into consideration that these diseases coexisting in one patient can mutually affect their symptoms creating specific diagnostic difficulties.

The connection between agr and SCCmec elements of Staphylococcus aureus strains and their response to photodynamic inactivation.

OBJECTIVE: The aim of this work was to analyze the presence of specific types of agr and SCCmec in Staphylococcus aureus strains and to determine the correlation between these types of genes and the response of S. aureus strains to photodynamic inactivation. BACKGROUND: S. aureus is an important human pathogen that is still one of the most common etiological factors of nosocomial infections. The genetic factor connected with high pathogenicity of S. aureus strains is the agr locus, which encodes a molecule responsible for activation of virulence genes. The characteristic feature of strains resistant to methicillin (MRSA) is the presence of the gene determining the resistance to ß-lactam antibiotics. This gene is a part of a mobile genetic element known as Staphylococcal Chromosome Cassette mec (SCCmec). Polymorphic differences in the agr locus and SCCmec cassette enable classification of strains into different groups. MATERIALS AND METHODS: We cultured and incubated each strain with defined dose of photosensitizer (protoporphyrin diarginate). Next, strains were irradiated with a red light at a dose of 12 J/cm(2). After an 18-h incubation, the Colony Forming Units were counted and the results were analyzed statistically. Furthermore, the genetic profile of the studied strains was determined with the use of the Multiplex PCR reaction both for agr and SCCmec elements. RESULTS: The results agreed with previous data, confirming that the response to photodynamic inactivation varies among different S. aureus strains. We also found a connection between some of the agr and SCCmec groups and the response of analyzed S. aureus strains to photoinactivation. CONCLUSION: Unfortunately, those relations are not specific enough to determine a diagnostically important pattern, which could enable predictions of strain response to PDI. Nevertheless, we can conclude that the connection between the response of S. aureus strains to photoinactivation and the strain specific agr/SCCmec pattern could be observed.