Fatma Vatansever

Antimicrobial strategies centered around reactive oxygen species – bactericidal antibiotics, photodynamic therapy, and beyond

Reactive oxygen species (ROS) can attack a diverse range of targets to exert antimicrobial activity, which accounts for their versatility in mediating host defense against a broad range of pathogens. Most ROS are formed by the partial reduction in molecular oxygen. Four major ROS are recognized comprising superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen ((1)O2), but they display very different kinetics and levels of activity. The effects of O2•- and H2O2 are less acute than those of •OH and (1)O2, because the former are much less reactive and can be detoxified by endogenous antioxidants (both enzymatic and nonenzymatic) that are induced by oxidative stress. In contrast, no enzyme can detoxify •OH or (1)O2, making them extremely toxic and acutely lethal. The present review will highlight the various methods of ROS formation and their mechanism of action. Antioxidant defenses against ROS in microbial cells and the use of ROS by antimicrobial host defense systems are covered. Antimicrobial approaches primarily utilizing ROS comprise both bactericidal antibiotics and nonpharmacological methods such as photodynamic therapy, titanium dioxide photocatalysis, cold plasma, and medicinal honey. A brief final section covers reactive nitrogen species and related therapeutics, such as acidified nitrite and nitric oxide-releasing nanoparticles.

Transcranial Low-Level Laser Therapy Improves Neurological Performance in Traumatic Brain Injury in Mice: Effect of Treatment Repetition Regimen

Low-level laser (light) therapy (LLLT) has been clinically applied around the world for a spectrum of disorders requiring healing, regeneration and prevention of tissue death. One area that is attracting growing interest in this scope is the use of transcranial LLLT to treat stroke and traumatic brain injury (TBI). We developed a mouse model of severe TBI induced by controlled cortical impact and explored the effect of different treatment schedules. Adult male BALB/c mice were divided into 3 broad groups (a) sham-TBI sham-treatment, (b) real-TBI sham-treatment, and (c) real-TBI active-treatment. Mice received active-treatment (transcranial LLLT by continuous wave 810 nm laser, 25 mW/cm2, 18 J/cm2, spot diameter 1 cm) while sham-treatment was immobilization only, delivered either as a single treatment at 4 hours post TBI, as 3 daily treatments commencing at 4 hours post TBI or as 14 daily treatments. Mice were sacrificed at 0, 4, 7, 14 and 28 days post-TBI for histology or histomorphometry, and injected with bromodeoxyuridine (BrdU) at days 21–27 to allow identification of proliferating cells. Mice with severe TBI treated with 1-laser Tx (and to a greater extent 3-laser Tx) had significant improvements in neurological severity score (NSS), and wire-grip and motion test (WGMT). However 14-laser Tx provided no benefit over TBI-sham control. Mice receiving 1- and 3-laser Tx had smaller lesion size at 28-days (although the size increased over 4 weeks in all TBI-groups) and less Fluoro-Jade staining for degenerating neurons (at 14 days) than in TBI control and 14-laser Tx groups. There were more BrdU-positive cells in the lesion in 1- and 3-laser groups suggesting LLLT may increase neurogenesis. Transcranial NIR laser may provide benefit in cases of acute TBI provided the optimum treatment regimen is employed.

Far infrared radiation (FIR): its biological effects and medical applications

Abstract Far infrared (FIR) radiation (λ=3–100 μm) is a subdivision of the electromagnetic spectrum that has been investigated for biological effects. The goal of this review is to cover the use of a further sub-division (3–12 μm) of this waveband, that has been observed in both in vitro and in vivo studies, to stimulate cells and tissue, and is considered a promising treatment modality for certain medical conditions. Technological advances have provided new techniques for delivering FIR radiation to the human body. Specialty lamps and saunas, delivering pure FIR radiation (eliminating completely the near and mid infrared bands), have became safe, effective, and widely used sources to generate therapeutic effects. Fibers impregnated with FIR emitting ceramic nanoparticles and woven into fabrics, are being used as garments and wraps to generate FIR radiation, and attain health benefits from its effects. Zusammenfassung Ferne Infrarotstrahlung (far infrared, FIR) (λ=3–100 μm) ist ein Unterbereich des elektromagnetischen Spektrums, der hinsichtlich seiner biologischen Effekte von wissenschaftlichem Interesse ist. Das vorliegende Review konzentriert sich auf den Spektralbereich von 3–12 μm, der sowohl in In-vitro- als auch in In-vivo-Studien mit Blick auf die Stimulation von Zellen und Gewebe untersucht wurde und der eine vielversprechende Behandlungsmodalität für verschiedene medizinische Konditionen darstellt. Dank des technischen Fortschrittes konnten verschiedene neue Techniken zur Applikation von FIR-Strahlung am menschlichen Körper entwickelt werden. Spezielle Lampen und Saunas, die reine FIR-Strahlung (ohne Anteile von Nahinfrarot- und Mittelinfrarotstrahlung) liefern, sind immer sicherer und effektiver geworden und werden verbreitet für therapeutische Zwecke genutzt. Fasern, die mit FIR-emittierenden Keramik-Nanopartikeln imprägniert und zu Stoffen weiterverarbeitet werden, finden Verwendung als Kleidung oder Verbandsstoffe, die aufgrund der generierten FIR-Strahlung gesundheitliche Vorteile bewirken können.

Transcranial low-level laser therapy enhances learning, memory, and neuroprogenitor cells after traumatic brain injury in mice

Abstract. The use of transcranial low-level laser (light) therapy (tLLLT) to treat stroke and traumatic brain injury (TBI) is attracting increasing attention. We previously showed that LLLT using an 810-nm laser 4 h after controlled cortical impact (CCI)-TBI in mice could significantly improve the neurological severity score, decrease lesion volume, and reduce Fluoro-Jade staining for degenerating neurons. We obtained some evidence for neurogenesis in the region of the lesion. We now tested the hypothesis that tLLLT can improve performance on the Morris water maze (MWM, learning, and memory) and increase neurogenesis in the hippocampus and subventricular zone (SVZ) after CCI-TBI in mice. One and (to a greater extent) three daily laser treatments commencing 4-h post-TBI improved neurological performance as measured by wire grip and motion test especially at 3 and 4 weeks post-TBI. Improvements in visible and hidden platform latency and probe tests in MWM were seen at 4 weeks. Caspase-3 expression was lower in the lesion region at 4 days post-TBI. Double-stained BrdU-NeuN (neuroprogenitor cells) was increased in the dentate gyrus and SVZ. Increases in double-cortin (DCX) and TUJ-1 were also seen. Our study results suggest that tLLLT may improve TBI both by reducing cell death in the lesion and by stimulating neurogenesis.

Clostridium difficile infection: molecular pathogenesis and novel therapeutics

The Gram-positive anaerobic bacterium Clostridium difficile produces toxins A and B, which can cause a spectrum of diseases from pseudomembranous colitis to C. difficile-associated diarrhea. A limited number of C. difficile strains also produce a binary toxin that exhibits ADP ribosyltransferase activity. Here, the structure and the mechanism of action of these toxins as well as their role in disease are reviewed. Nosocomial C. difficile infection is often contracted in hospital when patients treated with antibiotics suffer a disturbance in normal gut microflora. C. difficile spores can persist on dry, inanimate surface for months. Metronidazole and oral vancomycin are clinically used for treatment of C. difficile infection but clinical failure and concern about promotion of resistance are motivating the search for novel non-antibiotic therapeutics. Methods for controlling both toxins and spores, replacing gut microflora by probiotics or fecal transplant, and killing bacteria in the anaerobic gut by photodynamic therapy are discussed.

Photodynamic Therapy of Murine Mastocytoma Induces Specific Immune Responses against the Cancer/Testis Antigen P1A

Photodynamic therapy (PDT) involves the intravenous administration of photosensitizers followed by illumination of the tumor with visible light, leading to local production of reactive oxygen species that cause vascular shutdown and tumor cell death. Antitumor immunity is stimulated after PDT because of the acute inflammatory response that involves activation of the innate immune system, leading to stimulation of adaptive immunity. We carried out PDT using benzoporphyrin derivative and 690-nm light after 15 minutes, in DBA/2 mice bearing either the mastocytoma, P815, which expresses the naturally occurring cancer/testis antigen P1A, or the corresponding tumor P1.204 that lacks P1A expression. Tumor cures, significantly higher survival, and rejection of tumor rechallenge were obtained with P815, which were not seen with P1.204 or seen with P815 growing in nude mice. Both CD4 and CD8 T cells had higher levels of intracellular cytokines when isolated from mice receiving PDT of P815 tumors than P1.204 tumors and CD8 T cells from P815-cured mice recognized the peptide epitope of the P1A antigen (LPYLGWLVF) using pentamer staining. Taken together, these findings show that PDT can induce a potent antigen- and epitope-specific immune response against a naturally occurring mouse tumor antigen.

Low intensity laser therapy accelerates muscle regeneration in aged rats

Abstract Background: Elderly people suffer from skeletal muscle disorders that undermine their daily activity and quality of life; some of these problems can be listed as but not limited to: sarcopenia, changes in central and peripheral nervous system, blood hypoperfusion, regenerative changes contributing to atrophy, and muscle weakness. Determination, proliferation and differentiation of satellite cells in the regenerative process are regulated by specific transcription factors, known as myogenic regulatory factors (MRFs). In the elderly, the activation of MRFs is inefficient which hampers the regenerative process. Recent studies found that low intensity laser therapy (LILT) has a stimulatory effect in the muscle regeneration process. However, the effects of this therapy when associated with aging are still unknown. Objective: This study aimed to evaluate the effects of LILT (λ=830 nm) on the tibialis anterior (TA) muscle of aged rats. Subjects and methods: The total of 56 male Wistar rats formed two population sets: old and young, with 28 animals in each set. Each of these sets were randomly divided into four groups of young rats (3 months of age) with n=7 per group and four groups of aged rats (10 months of age) with n=7 per group. These groups were submitted to cryoinjury + laser irradiation, cryoinjury only, laser irradiation only and the control group (no cryoinjury/no laser irradiation). The laser treatment was performed for 5 consecutive days. The first laser application was done 24 h after the injury (on day 2) and on the seventh day, the TA muscle was dissected and removed under anesthesia. After this the animals were euthanized. Histological analyses with toluidine blue as well as hematoxylin-eosin staining (for counting the blood capillaries) were performed for the lesion areas. In addition, MyoD and VEGF mRNA was assessed by quantitative polymerase chain reaction. Results: The results showed significant elevation (p<0.05) in MyoD and VEGF genes expression levels. Moreover, capillary blood count was more prominent in elderly rats in laser irradiated groups when compared to young animals. Conclusion: In conclusion, LILT increased the maturation of satellite cells into myoblasts and myotubes, enhancing the regenerative process of aged rats irradiated with laser. Zusammenfassung Hintergrund: Ältere Menschen leiden häufig unter Skeletterkrankungen, die ihre Tagesaktivitäten und Lebensqualität negativ beeinflussen. Häufig, aber nicht ausschließlich, lassen sich die Probleme auf Sarkopenie, Veränderungen des zentralen und peripheren Nervensystems, Durchblutungsstörungen oder regenerative Veränderungen (Muskelatrophie, Muskelschwäche) zurückführen. Determination, Ausbreitung und Differenzierung von Satellitenzellen im Regenerationsprozess werden durch spezifische Transkriptionsfaktoren, den sogenannten myogenen regulatorischen Faktoren (myogenic regulatory factors, MRFs) bestimmt. Im Alter ist die Aktivierung der MRFs ineffizient, was den Regenerationsprozess hemmt. Neuere Studien haben gezeigt, dass die Therapie mit Laserlicht niedriger Intensität (low intensity laser therapy, LILT) die Muskelregeneration stimuliert. Allerdings sind diese Effekte im Zusammenhang mit dem Altern noch wenig erforscht. Zielsetzung: Die vorliegende Studie evaluiert den Effekt der LILT (λ=830 nm) auf den Tibialis-anterior (TA)-Muskel von älteren Ratten. Material und Methoden: Es wurden an insgesamt 56 männlichen Wistar-Ratten, aufgeteilt in zwei Populations-klassen (jung, alt) mit jeweils 28 Versuchstieren, Untersuchungen durchgeführt. Jede dieser beiden Klassen wurde nach dem Zufallsprinzip in 4 Gruppen mit jeweils 7 jungen Ratten (3 Monate alt) bzw. 4 Gruppen mit jeweils 7 alten Ratten (10 Monate alt) aufgeteilt: Cryoschädigung des TA + Lasertherapie, nur Cryoschädigung, nur Lasertherapie oder weder Cryoschädigung noch Lasertherapie (Kontrollgruppe). Die Laserbehandlung erfolgte an 5 aufeinanderfolgenden Tagen. Die erste Behandlung wurde 24 Stunden nach Cryoschädigung durchgeführt (Tag 2). Am 7. Tag wurde der TA-Muskel unter Anästhesie präpariert und entnommen; anschließend wurden die Tiere eingeschläfert. Die entnommenen Gewebeproben wurden sowohl mit Toluidinblau als auch mit Hämatoxylin-Eosin eingefärbt und histologisch untersucht (Zählung der Blutgefäße). Zusätzlich wurde die Expression von MyoD and VEGF mRNA mittels quantitativer Polymerase-Kettenreaktion (quantitative polymerase chain reaction, qPCR) bestimmt. Ergebnisse: Im Ergebnis zeigte sich eine signifikante Erhöhung der MyoD- und VEGF-Expression (p<0.05). Außerdem stellten sich die kapillaren Blutgefäße bei den älteren lasertherapierten Ratten im Vergleich zu den jüngeren Ratten prominenter dar. Zusammenfassung: Die LILT verbesserte die Reifung der Satellitenzellen in Myoblasten und Myotuben, wodurch der Regenerationsprozess bei älteren, laserbehandelten Ratten verstärkt wurde.

Can biowarfare agents be defeated with light

Biological warfare and bioterrorism is an unpleasant fact of 21st century life. Highly infectious and profoundly virulent diseases may be caused in combat personnel or in civilian populations by the appropriate dissemination of viruses, bacteria, spores, fungi, or toxins. Dissemination may be airborne, waterborne, or by contamination of food or surfaces. Countermeasures may be directed toward destroying or neutralizing the agents outside the body before infection has taken place, by destroying the agents once they have entered the body before the disease has fully developed, or by immunizing susceptible populations against the effects. A range of light-based technologies may have a role to play in biodefense countermeasures. Germicidal UV (UVC) is exceptionally active in destroying a wide range of viruses and microbial cells, and recent data suggests that UVC has high selectivity over host mammalian cells and tissues. Two UVA mediated approaches may also have roles to play; one where UVA is combined with titanium dioxide nanoparticles in a process called photocatalysis, and a second where UVA is combined with psoralens (PUVA) to produce “killed but metabolically active” microbial cells that may be particularly suitable for vaccines. Many microbial cells are surprisingly sensitive to blue light alone, and blue light can effectively destroy bacteria, fungi, and Bacillus spores and can treat wound infections. The combination of photosensitizing dyes such as porphyrins or phenothiaziniums and red light is called photodynamic therapy (PDT) or photoinactivation, and this approach cannot only kill bacteria, spores, and fungi, but also inactivate viruses and toxins. Many reports have highlighted the ability of PDT to treat infections and stimulate the host immune system. Finally pulsed (femtosecond) high power lasers have been used to inactivate pathogens with some degree of selectivity. We have pointed to some of the ways light-based technology may be used to defeat biological warfare in the future.

Photodynamic Therapy and Antitumor Immune Response

Photodynamic therapy (PDT) is the combination of photosensitizers and visible light used as a treatment for cancer and infections. As a direct consequence of PDT, reactive oxygen species and other “danger signals” activate the innate immune system and by doing so generate acute inflammation. This cascade of effects is followed by priming of tumor-specific T lymphocytes that have the potential to destroy distant untreated tumor cells, in addition to developing an innate memory “shield” that can be effective in combating recurrence of the cancer. Moreover, PDT can be successfully applied for overcoming the escaping mechanisms employed by progressing tumors attempting to evade immune attack. Furthermore, PDT can assert a beneficial effect on cases of bacterial infection through attracting and accumulating neutrophils into the infected regions rather than by directly killing the bacterial cells.

Transcranial low-level light therapy produces neuroprotection, neurogenesis and BDNF after TBI in mice.

We have previously shown that transcranial low level light therapy (LLLT) can ameliorate brain damage in mice subjected to traumatic brain injury and improve neurological function. We used a 810-nm laser and delivered 18 J/cm2 at an irradiance 25 mW/cm2. LLLT was either delivered once at 4 hours after controlled cortical impact TBI, once a day for 3 days, or once a day for 14 days. One and 3 applications of LLLT had beneficial effects on the mice, with 3 being better than 1, but 14 applications had no beneficial effect. We now report immunofluorescence studies in mouse brain sections that offer some explanation for this intriguing finding. Mice were injected with BrdU for 1 week before sacrifice (a marker for proliferating cells) and antibodies to double cortin (DCX-1,a marker of migrating neurons), Tuj-1 ( a marker of neuroprogenitor cells), BDNF (brain derived neurotrophic factor) and synapsin-1 ( a marker for newly formed synaptic connections between existing neurons). We found increased BrdU incorporation indicating proliferating cells in the dentate gyrus of the hippocampus, the subventricular layer of the lateral ventricle, as well as the brain tissue surrounding the cortical lesion. Interestingly these cells were more abundant at 7 days than at 28 days post TBI. Co-labeling of BrdU with Neu-N was performed indicating that the proliferating cells were in fact neuronal in nature. Mice with 3 laser treatments had much more BrdU incorporation than mice with 14. Upregulation of BDNF was seen at 7 days, and increased expression of DCX-1 and Tuj-1 was seen at 28 days in the lesion region, indication that neuroprogenitor cells may have migrated there from sites of neurogenesis. Increased syapsin-1 was seen in the cortex at 28 days indicating that neural plasticity may be stimulated by LLLT. Taken together these data suggest that transcranial LLLT may have applications beyond TBI in areas such as neurodegenerative disease and psychiatric disorders.