The effects of diode laser on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilm and <Emphasis Type="Italic">Escherichia coli</Emphasis> lipopolysaccharide adherent to titanium oxide surface of dental implants. An in vitro study

Effective decontamination of biofilm and bacterial toxins from the surface of dental implants is a yet unresolved issue. This in vitro study aims at providing the experimental basis for possible use of diode laser (λ 808 nm) in the treatment of peri-implantitis. Staphylococcus aureus biofilm was grown for 48 h on titanium discs with porous surface corresponding to the bone-implant interface and then irradiated with a diode laser (λ 808 nm) in noncontact mode with airflow cooling for 1 min using a Ø 600-μm fiber. Setting parameters were 2 W (400 J/cm²) for continuous wave mode; 22 μJ, 20 kHz, 7 μs (88 J/cm²) for pulsed wave mode. Bactericidal effect was evaluated using fluorescence microscopy and counting the residual colony-forming units. Biofilm and titanium surface morphology were analyzed by scanning electron microscopy (SEM). In parallel experiments, the titanium discs were coated with Escherichia coli lipopolysaccharide (LPS), laser-irradiated and seeded with RAW 264.7 macrophages to quantify LPS-driven inflammatory cell activation by measuring the enhanced generation of nitric oxide (NO). Diode laser irradiation in both continuous and pulsed modes induced a statistically significant reduction of viable bacteria and nitrite levels. These results indicate that in addition to its bactericidal effect laser irradiation can also inhibit LPS-induced macrophage activation and thus blunt the inflammatory response. The λ 808-nm diode laser emerges as a valuable tool for decontamination/detoxification of the titanium implant surface and may be used in the treatment of peri-implantitis.     

Action of antimicrobial photodynamic therapy on heterotypic biofilm: <Emphasis Type="Italic">Candida albicans</Emphasis> and <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis>

The increase in survival and resistance of microorganisms organized in biofilms demonstrates the need for new studies to develop therapies able to break this barrier, such as photodynamic therapy, which is characterized as an alternative, effective, and non-invasive treatment. The objective was to evaluate in vitro the effect of antimicrobial photodynamic therapy on heterotypic biofilms of Candida albicans and Bacillus atrophaeus using rose bengal (12.5 μM) and light-emitting diode (LED) (532 nm and 16.2 J). We used standard strains of B. atrophaeus (ATCC 9372) and C. albicans (ATCC 18804). The biofilm was formed in the bottom of the plate for 48 h. For the photodynamic therapy (PDT) experimental groups, we added 100 μL of rose bengal with LED (P+L+), 100 μL of rose bengal without LED (P+L?), 100 μL of NaCl 0.9 % solution with LED (P?L+), and a control group without photosensitizer or LED (P?L?). The plates remained in agitation for 5 min (pre-irradiation) and were irradiated with LED for 3 min, and the biofilm was detached using an ultrasonic homogenizer for 30 s. Serial dilutions were plated in BHI agar and HiChrom agar and incubated at 37 °C/48 h. There was a reduction of 33.92 and 29.31 % of colony-forming units per milliliter (CFU/mL) for C. albicans and B. atrophaeus, respectively, from the control group to the group subjected to PDT. However, statistically significant differences were not observed among the P+L+, P+L?, P?L+, and P?L? groups. These results suggest that antimicrobial photodynamic therapy using rose bengal (12.5 μM) with a pre-irradiation period of 5 min and LED for 3 min was not enough to cause a significant reduction in the heterotypic biofilms of C. albicans and B. atrophaeus.     

Influence of sucrose on growth and sensitivity of <Emphasis Type="Italic">Candida albicans</Emphasis> alone and in combination with <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> and <Emphasis Type="Italic">Streptococcus mutans</Emphasis> to photodynamic therapy

This study has evaluated the effects of photodynamic inactivation (PDI) using erythrosine as photosensitizer and green light-emitting diode (LED) on biofilms of Candida albicans alone and in combination with Enterococcus faecalis and Streptococcus mutans. We have also evaluated the effect of sucrose on biofilm formation and bacterial growth and sensitivity to PDI. Biofilms were formed in suspension of 10⁶ cells/ml on plates before being grown in broth culture with and without sucrose and incubated for 48 h. Next, the treatment was applied using erythrosine at a concentration of 400 μM for 5 min and green LED (532 ± 10 nm) for 3 min on biofilms alone and in combination. The plates were washed and sonicated to disperse the biofilms, and serial dilutions were carried and aliquots seeded in Sabouraud agar before incubation for 48 h. Next, the colony-forming units per milliliter (CFU/ml; log₁₀) were counted and analyzed statistically (ANOVA, Tukey test, P ≤ 0.05). Results show that S. mutans favors the growth of C. albicans in biofilms with sucrose, with treatment not being effective. However, when the biofilm was grown without sucrose, we found a reduction in biofilm formation and a significant decrease in the PDI treatment (P < 0.0001). In conclusion, both growth and sensitivity to PDI in biofilms of C. albicans are strongly influenced by bacterial combination, and the presence of sucrose affected directly the growth and sensitivity of the biofilm to PDI as sucrose is the substrate for construction of the exopolysaccharide matrix.     

Repeated applications of photodynamic therapy on <Emphasis Type="Italic">Candida glabrata</Emphasis> biofilms formed in acrylic resin polymerized

Previous studies have been suggested that photodynamic therapy (PDT) can be used as an adjuvant treatment for denture stomatitis. In this study, we evaluated the effects of multiple sessions of PDT on Candida glabrata biofilms in specimens of polymerized acrylic resin formed after 5 days. Subsequently, four applications of PDT were performed on biofilms in 24-h intervals (days 6–9). Also, we evaluated two types of PDT, including application of laser and methylene blue or light-emitting diode (LED) and erythrosine. The control groups were treated with physiological solution. The effects of PDT on biofilm were evaluated after the first and fourth application of PDT. The biofilm analysis was performed by counting the colony-forming units. The results showed that between the days 6 and 9, the biofilms not treated by PDT had an increase of 5.53 to 6.05 log (p?=?0.0271). Regarding the treatments, after one application of PDT, the biofilms decreased from 5.53 to 0.89 log. When it was done four applications, the microbial reduction ranged from 6.05 log to 0.11 log. We observed that one application of PDT with laser or LED caused a reduction of 3.36 and 4.64 compared to the control groups, respectively (p?=?0.1708). When it was done four applications of PDT, the reductions achieved were 1.57 for laser and 5.94 for LED (p?=?0.0001). It was concluded that repeated applications of PDT on C. glabrata biofilms showed higher antimicrobial activity compared to single application. PDT mediated by LED and erythrosine was more efficient than the PDT mediated by laser and methylene blue.     

Sustained effects of blue light on <Emphasis Type="Italic">Streptococcus mutans</Emphasis> in regrown biofilm

In prior studies, exposure of Streptococcus mutans in biofilm to blue light using high fluences of up to 680 J/cm² did not interfere with bacterial capability to reform an initial biofilm; however, a delayed antibacterial effect was observed. Our aim was to determine the sustained effecttts of blue light-emitting diode (LED) curing light on the pathogenicity of the newly formed biofilm. S. mutans were grown to form biofilm that was exposed to blue light (wavelengths, 460–480 nm) for 1, 3, and 7 min (equivalent to 37, 112, and 262 J/cm², respectively). Then, bacteria were suspended and allowed to regrow into new biofilms. The regrown biofilms were assessed for bacterial quantification by optical density (OD) measurement and quantitative polymerase chain reaction (qPCR), bacterial viability and extracellular polysaccharide production by fluorescent staining using confocal scanning laser microscopy, acid production by bacteria (acidogenicity), and bacterial survival at low pH (aciduricity) using qPCR. Bacterial growth in the regrown biofilms was increased when samples were previously exposed to light; however, under the confocal microscopy, a higher proportion of dead bacteria and a reduction in polysaccharide production were observed. The acidogenicity from the regrown biofilm was lowered as fluences of light increased. The aciduricity of the regrown biofilm was decreased, meaning less growth of bacteria into biofilm in low pH with increasing fluences. Blue light has sustained effects on S. mutans bacteria grown into new biofilm. Although bacterial growth in biofilm increased, bacterial viability and virulence characteristics were impaired. The cariogenic potential over time of S. mutans previously exposed to blue light when grown on tooth surfaces is yet to be determined.     

Time-dependent antimicrobial effect of photodynamic therapy with TONS 504 on <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa (P. aeruginosa) is a major cause of infectious keratitis, which itself is a major cause of blindness worldwide. We have now evaluated the time-dependent effectiveness of photodynamic antimicrobial chemotherapy (PACT) with the chlorin derivative TONS 504 and a light-emitting diode (LED) on P. aeruginosa in vitro. PACT with TONS 504 (10 mg/L) and irradiation (30 J/m²) by an LED device that delivers light centered on a wavelength of 660 nm was applied to 1?×?10⁶ colony-forming units of P. aeruginosa in liquid medium. The bacteria were then cultured at 37 °C for various times before assay of viability by determination of colony formation on agar plates. The effect of a second irradiation at 3 h after the initial LED exposure was also examined. Bacterial growth was markedly inhibited between 3 and 9 h after PACT with TONS 504, with the maximal effect being apparent at 3 h. Furthermore, a second exposure to LED irradiation at 3 h after the first treatment enhanced the inhibitory effect on bacterial growth. PACT with TONS 504 thus inhibited the growth of P. aeruginosa in a time-dependent manner, and an additional irradiation exposure applied 3 h after the first LED treatment greatly increased the effectiveness of PACT. This antibacterial system thus warrants further evaluation with regard to its potential effectiveness for the treatment of infectious keratitis.     

Curcumin-mediated anti-microbial photodynamic therapy against <Emphasis Type="Italic">Candida dubliniensis</Emphasis> biofilms

The purpose of this study was to evaluate the effectiveness of anti-microbial photodynamic therapy (aPDT) mediated by curcumin (Cur) associated with LED light against biofilms of Candida dubliniensis, and further, investigate cellular uptake and drug penetration through the biofilms under confocal laser scanning microscopy (CLSM). Four C. dubliniensis strains were tested: three clinical isolates from HIV-positive patients and one reference strain (CBS 7987). Biofilms were treated with three Cur concentrations (20.0, 30.0, and 40.0 μM). All samples were incubated in the dark for 20 min and exposed to a 5.28 J/cm² of LED light fluence. Additional samples of each strain were treated either with Cur or LED light only. Control samples had neither Cur nor light. After aPDT, results were read using the XTT salt reduction method. The data were statistically analyzed by two-way ANOVA followed by Games-Howell post-hoc test (α?=?0.05). Confocal laser scanning microscopy was used to verify both the uptake of Cur by yeast cells and its penetration through the biofilm. The results showed that aPDT promoted significant reduction on the metabolism of the biofilm-organized cells of C. dubliniensis. Further, while Cur was rapidly taken up by C. dubliniensis cells, a longer time interval was required to allow Cur penetration into biofilm cells. Based on these results, aPDT associating LED and Cur presents promising potential on fungal control of biofilms of C. dubliniensis.     

Photodynamic therapy controls of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> intradermal infection in mice

Infections caused by Staphylococcus aureus lead to skin infections, as well as soft tissues and bone infections. Given the communal resistance to antibiotics developed by strains of this bacterium, photodynamic therapy emerges as a promising alternative treatment to control and cure infections. Females of the Balb/C mice were infected with 10⁸ CFU of methicillin-resistant S. aureus (MRSA) and divided into four distinct groups: P?L? (negative control group), P+L? (group exposed only to curcumin), P?L+ (group exposed only to LED incidence of 450 nm, 75 mW/cm², and 54 J/cm² for 10 min), and P+L+ (group exposed to curcumin followed by 10 min of LED irradiation) (n = 24). The mice were euthanized 48 and 72 h after infection, and biologic materials were collected for analysis of the bacterial load, peripheral blood leukocyte counts, and draining lymph nodes cell counts. The normalization of data was checked and the ANOVA test was applied. The bacterial load in the draining lymph node of P+L+ group was lower when compared to the control groups 72 h post infection (p < 0.0001), indicating that the LED incidence associated with curcumin controls of the staphylococci intradermal infection. The number of the total lymph node cells shows to be lower than control groups in the two availed times (p < 0.01). The histological analysis and the counting of white blood cells did not show differences among cells in the blood and in the tissue of infection. This is the first report showing that photodynamic therapy may be effective against MRSA infection in a murine model of intradermal infection.     

Virulence factors of fluconazole-susceptible and fluconazole-resistant <Emphasis Type="Italic">Candida albicans</Emphasis> after antimicrobial photodynamic therapy

This study evaluated the effects of antimicrobial photodynamic therapy (aPDT) mediated by Photodithazine® (PDZ) and LED light on the virulence factors of fluconazole-susceptible (CaS) and fluconazole-resistant (CaR) Candida albicans. Standardized suspensions of strains were prepared (10⁷), and after 48 h of biofilm formation, these strains were incubated with PDZ (100 mg/L) for 20 min and exposed to LED light (660 nm, 37.5 J/cm²). Additional samples were treated with PDZ or light only, and the control consisted of biofilms that received no treatment. After aPDT, the cells were recovered and the virulence factors were evaluated. To analyze the capacity of adhesion, cells were recovered after aPDT and submitted to the adhesion process in the bottom of a 96-well plate. After this, metabolic activity tests (XTT assay) and cell viability (colony forming units per milliliter, CFU/mL) were applied. To evaluate the biofilm-forming ability after aPDT, the cells recovered were submitted to biofilm formation procedures, and the biofilm formed was evaluated by XTT, CFU/mL, and total biomass (crystal violet) tests. Lastly, the capacity for synthesizing protease and phospholipase enzymes after aPDT was evaluated by fluorimetric tests. Data were analyzed by two- or three-way ANOVA tests (p?≤?0.05). It was verified that aPDT reduced the viability of both strains, fluconazole-susceptible and fluconazole-resistant C. albicans. It was also observed that the CaR strain had lower susceptibility to the aPDT when compared with the CaS strain. However, regarding the virulence factors evaluated, it was demonstrated that aPDT did not alter the adherence and biofilm formation ability and enzymatic production.     

Enhancement of photodynamic inactivation of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilms by disruptive strategies

Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers and visible light. On the one hand, near-infrared treatment (NIRT) has also bactericidal and dispersal effects on biofilms. In addition, dispersal biological tools such as enzymes have also been employed in antibiotic combination treatments. The aim of this work was to use alternative approaches to increase the PDI efficacy, employing combination therapies aimed at the partial disruption of the biofilms, thus potentially increasing photosensitizer or oxygen penetration and interaction with bacteria. To that end, we applied toluidine blue (TB)-PDI treatment to Staphylococcus aureus biofilms previously treated with NIRT or enzymes and investigated the outcome of the combined therapies. TB employed at 0.5 mM induced per se 2-log drop in S. aureus RN6390 biofilm viability. Each NIRT (980-nm laser) and PDI (635-nm laser) treatment induced a further reduction of 1-log of viable counts. The combination of successive 980- and 635-nm laser treatments on TB-treated biofilms induced additive effects, leading to a 4.5-log viable count decrease. Proteinase K treatment applied to S. aureus of the Newman strain induced an additive effect on PDI mortality, leading to an overall 4-log decrease in S. aureus viability. Confocal scanning laser microscopy after biofilm staining with a fluorescent viability test and scanning electron microscopy observations were correlated with colony counts. The NIRT dose employed (227 J/cm²) led to an increase from 21 to 47 °C in the buffer temperature of the biofilm system, and this NIRT dose also induced 100% keratinocyte death. Further work is needed to establish conditions under which biofilm dispersal occurs at lower NIRT doses.     

In vitro evaluation of photodynamic therapy using curcumin on <Emphasis Type="Italic">Leishmania majo</Emphasis>r and <Emphasis Type="Italic">Leishmania braziliensis</Emphasis>

Cutaneous leishmaniasis is an infectious disease caused by the Leishmania protozoan. The conventional treatment is long-lasting and aggressive, in addition to causing harmful effect. Photodynamic therapy has emerged as a promising alternative treatment, which allows local administration with fewer side effects. This study investigated the photodynamic activity of curcumin on Leishmania major and Leishmania braziliensis promastigote. Both species were submitted to incubation with curcumin in serial dilutions from 500 μg/ml up to 7.8 μg/ml. Control groups were kept in the dark while PDT groups received a fluency of 10 J/cm² at 450 nm. Mitochondrial activity was assessed by MTT assay 18 h after light treatment, and viability was measured by Trypan blue dye exclusion test. Morphological alterations were observed by Giemsa staining. Confocal microscopy showed the uptake of curcumin by both tested Leishmania species. Mitochondrial activity was inconclusive to determine viability; however, Trypan blue test was able to show that curcumin photodynamic treatment had a significant effect on viability of parasites. The morphology of promastigotes was highly affected by the photodynamic therapy. These results indicated that curcumin may be a promising alternative photosensitizer, because it presents no toxicity in the dark; however, further tests in co-culture with macrophages and other species of Leishmania should be conducted to determine better conditions before in vivo tests are performed.     

Photodynamic inactivation of pathogenic species <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Candida albicans</Emphasis> with lutetium (III) acetate phthalocyanines and specific light irradiation

Photodynamic inactivation (PDI) is a light-associated therapeutic approach suitable for treatment of local acute infections. The method is based on specific light-activated compound which by specific irradiation and in the presence of molecular oxygen produced molecular singlet oxygen and other reactive oxygen species, all toxic for pathogenic microbial cells. The study presents photodynamic impact of two recently synthesized water-soluble cationic lutetium (III) acetate phthalocyanines (LuPc-5 and LuPc-6) towards two pathogenic strains, namely, the Gram-negative bacterium Pseudomonas aeruginosa and a fungus Candida albicans. The photodynamic effect was evaluated for the cells in suspensions and organized in 48-h developed biofilms. The relatively high levels of uptakes of LuPc-5 and LuPc-6 were determined for fungal cells compared to bacterial cells. The penetration depths and distribution of both LuPcs into microbial biofilms were investigated by means of confocal fluorescence microscopy. The photoinactivation efficiency was studied for a wide concentration range (0.85–30 μM) of LuPc-5 and LuPc-6 at a light dose of 50 J cm^?2 from red light-emitting diode (LED; 665 nm). The PDI study on microbial biofilms showed incomplete photoinactivation (<3 logs) for the used gentle drug-light protocol.     

Photoinactivation effect of eosin methylene blue and chlorophyllin sodium-copper against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>

The use of eosin methylene blue according to Giemsa as photosensitizer is presented for the first time in this paper. The present study evaluated the potential application of chlorophyllin sodium copper salt (CuChlNa) and eosin methylene blue according to Giemsa (EMB) as antimicrobial photosensitizers (aPS) for photodynamic inactivation (PDI) of Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria. The experiments were performed using S. aureus stain ATCC 25923 and E. coli ATCC 25922 in which five aPS concentrations (0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 μM for S. aureus and 0.0, 5.0, 10.0, 20.0, 40.0, and 50.0 μM for E. coli) were prepared and added in 2 mL of a saline solution containing the bacterial inoculum. After aPS incubation, the samples were divided into two groups, one kept in the dark and another submitted to the illumination. Then, the bacterial inactivation was determined 18 h after the incubation at 37 °C by counting the colony-forming units (CFU). The results revealed that both EMB and CuChlNa can be used as aPS for the photoinactivation of S. aureus, while only EMB was able to photoinactivate E. coli. Nevertheless, a more complex experimental setup was needed for photoinactivation of E. coli. The data showed that EMB and CuChlNa presented similar photoinactivation effects on S. aureus, in which bacterial growth was completely inhibited at photosensitizer (PS) concentrations over 5 μM, when samples were previously incubated for 30 min and irradiated by a light dose of 30 J cm^?2 as a result of an illumination of 1 h at 8.3 mW cm^?2 by using a red light at 625 nm with a 1 cm beam diameter and output power of 6.5 mW. In the case of E. coli, bacterial growth was completely inhibited only when combining a PS incubation period of 120 min with concentrations over 20 μM.     

Shoulder periprosthetic joint infection caused by <Emphasis Type="Italic">Propionibacterium acnes</Emphasis>

Background

Propionibacterium acnes is increasingly recognized as an important cause of shoulder periprosthetic joint infection (PJI). We performed a retrospective cohort study of P. acnes shoulder PJI to analyze the clinical, diagnostic, and treatment characteristics.

Methods

Patients diagnosed with shoulder PJI caused by P. acnes were retrospectively analyzed in two university hospitals. Patient data were retrieved through chart review. The outcome was evaluated at patient follow-up visits.

Results

The study included 20 patients with shoulder PJI (median age, 65 years; range, 54–77 years); 75 % were males. The median time from prosthesis implantation to diagnosis of PJI was 34 months (range, 2–60 months). Most PJI (55?%) were diagnosed >24 months after arthroplasty, followed by delayed manifestation 3–24 months after arthroplasty in 30?%. The diagnosis of PJI was preoperatively confirmed in 50?% of patients and suspected in 20 %. Persistent pain was present in 90?%, local signs of inflammation in 60?%, and radiological signs of loosening in 70 % of patients. P. acnes was cultured in joint aspirate in 33?%, periprosthetic tissue in 60?%, and sonication fluid in 89?% of patients. In four patients, coinfection with coagulase-negative staphylococci was found. One-stage prosthesis exchange was performed in four patients (20?%) and two-stage exchange in 15 patients (75?%); in one patient the prosthesis was not re-implanted. After a median follow-up of 26 months (range, 12–47 months), 18 patients (90?%) showed no signs or symptoms of infection.

Conclusion

P. acnes PJI typically manifested several years after implantation. In 30?% of patients, PJI was not suspected before surgery. In patients with persistent pain or prosthesis loosening, low-grade PJI should be excluded, including infection caused by P. acnes.

     

Enhancement of photo-bactericidal effect of tetrasulfonated hydroxyaluminum phthalocyanine on <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

At the present time, photodynamic inactivation (PDI) is receiving considerable interest for its potential as an antimicrobial therapy. The results of our study indicate that enhancement of the phototoxic effect on Pseudomonas aeruginosa can be achieved by combination of tetrasulfonated hydroxyaluminum phthalocyanine (AlPcS₄) and bimetallic gold/silver nanoparticles (Au/Ag-NPs) synthesized by the cell-free filtrate of Aureobasidium pullulans. The bimetallic nanoparticles were characterized by a number of techniques including UV-vis, XPS, TEM, and SEM-EDS to be 14 ± 3 nm spherical particles coated with proteins. The effect of diode lasers with the peak-power wavelength ? = 650 nm (output power of 10 and 40 mW; radiation intensity of 26 and 105 mW/cm²) in combination with the AlPcS₄ and the bimetallic nanoparticles mixture on the viability of P. aeruginosa rods was shown. Particularly high efficiency of killing bacterial cells was obtained for the light intensity of 105 mW/cm², after 20, 30, and 40 min of irradiation corresponding to 126, 189^, and 252 J/cm² energy fluences. For AlPcS₄+Au/Ag-NPs treatment, the viable count reduction were equal to 99.90, 99.96, and 99.975%, respectively. These results were significantly better than those accomplished for irradiated separated assays of AlPcS₄ and Au/Ag-NPs.     

<Emphasis Type="Italic">KRAS</Emphasis> and <Emphasis Type="Italic">BRAF</Emphasis> Mutations in 203 Esophageal Squamous Cell Carcinomas: Pyrosequencing Technology and Literature Review

Background

Epidermal growth factor receptor (EGFR) signaling is one of the most promising targets for molecular-targeted therapies in esophageal squamous cell carcinoma (ESCC). Thus, the molecular diagnosis of KRAS and BRAF mutations is clinically important in therapeutic decision making. However, the frequency of KRAS and BRAF mutations in ESCCs remains inconclusive because of the limited sample sizes of previous studies (all N ≤ 80). Pyrosequencing is a nonelectrophoretic nucleotide extension sequencing technology that can be used for mutation testing.

Methods

The frequency of KRAS and BRAF mutations was examined using a nonbiased database of 203 resected ESCCs and a high-throughput pyrosequencing assay.

Results

The validity of the KRAS pyrosequencing method was initially demonstrated by detection of all 4 types of KRAS mutations [c.35G>T (codon 12 GGT>GTT), c.35G>A (codon 12 GGT>GAT), c.34G>T (codon 12 GGT>TGT), c.38G>A mutation (codon 13 GGC>GAC)], which had been previously diagnosed using Scorpion-ARMS technology, in 9 colon cancer tissues (9 of 9; 100 %). Similar results were demonstrated for BRAF mutational status in 3 colon cancer cell lines (HCT116, Colo201, and HT29), which were validated by Sanger dideoxy sequencing. Subsequently, the KRAS mutation was found to be extremely rare (1 of 203; 0.5 %), and the BRAF mutation was absent (0 of 203; 0 %), in the dataset of 203 ESCCs.

Conclusions

These results suggest that KRAS and BRAF mutations play a limited role in the development of ESCC and that mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy in ESCC.

     

Impact of <Emphasis Type="Italic">KRAS</Emphasis> and <Emphasis Type="Italic">EGFR</Emphasis> Gene Mutations on Recurrence and Survival in Patients with Surgically Resected Lung Adenocarcinomas

Background

Oncogenic gene mutations observed in lung adenocarcinomas, such as epidermal growth factor receptor (EGFR) and KRAS, have some predictive value for chemotherapeutic drugs or EGFR–tyrosine kinase inhibitors. However, the influence of these gene alterations on patients’ prognosis remains controversial.

Methods

We retrospectively analyzed the tumors of 180 patients with completely resected pathological stage I–III lung adenocarcinoma which harbored either KRAS codon 12 mutation or EGFR gene mutations within exons 18–21 to investigate the impact of these gene mutations on the patients’ survival. Gene mutations were detected by established methods.

Results

Of 180 patients, 32 had KRAS codon 12 mutations (KRAS group), 148 had EGFR mutations within exon 18–21 (EGFR group). Pathological stage and operation mode were independent factors for disease-free survival. However, the EGFR group had better overall survival than the KRAS group (P = 0.0271). Cox proportional hazard model revealed pathological stage (P = 0.0001) and presence of EGFR gene mutations (P = 0.0408) were independent factors for overall survival. In survival after tumor recurrence, the EGFR group had a better median survival time (46.7 months) after recurrence than the KRAS group (26.0 months).

Conclusions

In patients with completely resected lung adenocarcinomas, KRAS and EGFR gene mutation status of tumors was not associated with disease-free survival. However, the presence of an EGFR gene mutation boded well for the patient’s overall survival, and thus patients with EGFR mutations have a better prognosis than those with KRAS mutations.

     

<Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis> IgG seropositivities are not predictors of osteoporosis-associated bone loss: a prospective cohort study

The potential link between infection with Chlamydia pneumoniae or Helicobacter pylori and osteoporosis has not been investigated in population-based longitudinal studies. A total of 250 healthy postmenopausal women who participated in a prospective cohort study were evaluated for IgG antibodies directed against C. pneumoniae and H. p ylori, osteoprotegerin (OPG), the receptor activator of nuclear factor kappa B ligand (RANKL), CrossLaps, and osteocalcin. Bone mineral density (BMD) was measured at the femoral neck and lumbar spine at baseline and at follow-up 5.8 years later. There were no significant differences in age-adjusted bone turnover markers, OPG, RANKL, the RANKL/OPG ratio, and BMD between the C. p neumoniae and H. p ylori IgG seropositive and seronegative subjects (P > 0.05). Neither C. p neumoniae nor H. p ylori IgG seropositivity was associated with age-and body mass index-adjusted BMD at the femoral neck and lumbar spine or bone loss at the 5.8-year follow-up. In logistic regression analysis, neither C. p neumoniae nor H. p ylori IgG seropositivities predicted incident lumbar or spine osteoporosis 5.8 years later. In conclusion, neither C. p neumoniae nor H. p ylori IgG seropositivity was associated with bone turnover markers, the RANKL/OPG ratio, BMD, or bone loss in postmenopausal women. In addition, chronic infection with C. p neumoniae or H. p ylori did not predict incident osteoporosis among this group of women.     

Photodynamic antimicrobial chemotherapy (PACT) using toluidine blue inhibits both growth and biofilm formation by <Emphasis Type="Italic">Candida krusei</Emphasis>

Among non-albicans Candida species, the opportunistic pathogen Candida krusei emerges because of the high mortality related to infections produced by this yeast. The Candida krusei is an opportunistic pathogen presenting an intrinsic resistance to fluconazol. In spite of the reduced number of infections produced by C. krusei, its occurrence is increasing in some groups of patients submitted to the use of fluconazol for prophylaxis. Photodynamic antimicrobial chemotherapy (PACT) is a potential antimicrobial therapy that combines visible light and a nontoxic dye, known as a photosensitizer, producing reactive oxygen species (ROS) that can kill the treated cells. The objective of this study was to investigate the effects of PACT, using toluidine blue, as a photosensitizer on both growth and biofilm formation by Candida krusei. In this work, we studied the effect of the PACT, using TB on both cell growth and biofilm formation by C. krusei. PACT was performed using a light source with output power of 0.068 W and peak wavelength of 630 nm, resulting in a fluence of 20, 30, or 40 J/cm². In addition, ROS production was determined after PACT. The number of samples used in this study varied from 6 to 8. Statistical differences were evaluated by analysis of variance (ANOVA) and post hoc comparison with Tukey-Kramer test. PACT inhibited both growth and biofilm formation by C. krusei. It was also observed that PACT stimulated ROS production. Comparing to cells not irradiated, irradiation was able to increase ROS production in 11.43, 6.27, and 4.37 times, in the presence of TB 0.01, 0.02, and 0.05 mg/mL, respectively. These results suggest that the inhibition observed in the cell growth after PACT could be related to the ROS production, promoting cellular damage. Taken together, these results demonstrated the ability of PACT reducing both cell growth and biofilm formation by C. krusei.     

Detection of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in Nasal Polyps

To detect the presence of Helicobacter pylori in nasal polyps. A case–control study was conducted enrolling 35 patients with nasal polyps (cases) and patients undergoing septoplasty (controls). Fresh tissue samples were used for urea broth test and imprint cytology, while formalin fixed tissue sections were used for morphology, special stains and immunohistochemistry for H. pylori. Fresh stool samples from both groups were tested to correlate the gastrointestinal status. H. pylori was detected in 40.0 % (14/35) of cases and 8.5 % of controls (3/35) (p = 0.004) by immunohistochemistry. Amongst cases, eight were positive with urea broth test, six with imprint cytology (Giemsa stain), three with H & E, and nine with modified McMullen’s stain. Hyperplasia of the lining epithelium and lymphoid aggregates were significantly noticed in nasal polyps positive for H. pylori. Stool antigen test was positive in subjects who were positive for H. pylori in the nasal mucosa. There appears to be an association between H. pylori and nasal polyps. Immunohistochemistry is more sensitive and specific method to detect H. pylori. H. pylori induced inflammatory tissue reaction pattern indicates a possible causal association. Further studies are needed to prove the causal relationship between H. pylori and nasal polyps.