Inactivation of multidrug-resistant bacteria and bacterial spores and generation of high-potency bacterial vaccines using ultrashort pulsed lasers.

Multidrug-resistant organisms (MDROs) represent a continuing healthcare crisis with no definitive solution to date. An alternative to antibiotics is the development of therapies and vaccines using biocompatible physical methods such as ultrashort pulsed (USP) lasers, which have previously been shown to inactivate pathogens while minimizing collateral damage to human cells, blood proteins, and vaccine antigens. Here we demonstrate that visible USP laser treatment results in bactericidal effect (≥3-log load reduction) against clinically significant MDROs, including methicillin-resistant Staphylococcus aureus and extended spectrum beta-lactamase-producing Escherichia coli. Bacillus cereus endospores, which are highly resistant to conventional chemical and physical treatments, were also shown to be effectively inactivated by USP laser treatment, resulting in sporicidal (≥3-log load reduction) activity. Furthermore, we demonstrate that administration of USP laser-inactivated E. coli whole-cell vaccines at dosages as low as 105 cfu equivalents without adjuvant was able to protect 100% of mice against subsequent lethal challenge. Our findings open the possibility for application of USP lasers in disinfection of hospital environments, therapy of drug-resistant bacterial infections in skin or bloodstream via pheresis modalities, and in the production of potent bacterial vaccines.

Non-invasive monitoring of arthritis treatment response via targeting of tyrosine-phosphorylated annexin A2 in chondrocytes.

BACKGROUND: The development and optimization of therapies for rheumatoid arthritis (RA) is currently hindered by a lack of methods for early non-invasive monitoring of treatment response. Annexin A2, an inflammation-associated protein whose presence and phosphorylation levels are upregulated in RA, represents a potential molecular target for tracking RA treatment response. METHODS: LS301, a near-infrared dye-peptide conjugate that selectively targets tyrosine 23-phosphorylated annexin A2 (pANXA2), was evaluated for its utility in monitoring disease progression, remission, and early response to drug treatment in mouse models of RA by fluorescence imaging. The intraarticular distribution and localization of LS301 relative to pANXA2 was determined by histological and immunohistochemical methods. RESULTS: In mouse models of spontaneous and serum transfer-induced inflammatory arthritis, intravenously administered LS301 showed selective accumulation in regions of joint pathology including paws, ankles, and knees with positive correlation between fluorescent signal and disease severity by clinical scoring. Whole-body near-infrared imaging with LS301 allowed tracking of spontaneous disease remission and the therapeutic response after dexamethasone treatment. Histological analysis showed preferential accumulation of LS301 within the chondrocytes and articular cartilage in arthritic mice, and colocalization was observed between LS301 and pANXA2 in the joint tissue. CONCLUSIONS: We demonstrate that fluorescence imaging with LS301 can be used to monitor the progression, remission, and early response to drug treatment in mouse models of RA. Given the ease of detecting LS301 with portable optical imaging devices, the agent may become a useful early treatment response reporter for arthritis diagnosis and drug evaluation.

Selective imaging of solid tumours via the calcium-dependent high-affinity binding of a cyclic octapeptide to phosphorylated Annexin A2.

The heterogeneity and continuous genetic adaptation of tumours complicate their detection and treatment via the targeting of genetic mutations. However, hallmarks of cancer such as aberrant protein phosphorylation and calcium-mediated cell signalling provide broadly conserved molecular targets. Here, we show that, for a range of solid tumours, a cyclic octapeptide labelled with a near-infrared dye selectively binds to phosphorylated Annexin A2 (pANXA2), with high affinity at high levels of calcium. Because of cancer-cell-induced pANXA2 expression in tumour-associated stromal cells, the octapeptide preferentially binds to the invasive edges of tumours and then traffics within macrophages to the tumour's necrotic core. As proof-of-concept applications, we used the octapeptide to detect tumour xenografts and metastatic lesions, and to perform fluorescence-guided surgical tumour resection, in mice. Our findings suggest that high levels of pANXA2 in association with elevated calcium are present in the microenvironment of most solid cancers. The octapeptide might be broadly useful for selective tumour imaging and for delivering drugs to the edges and to the core of solid tumours.

Florid cemento-osseous dysplasia: review of an uncommon fibro-osseous lesion of the jaw with important clinical implications.

Florid cemento-osseous dysplasia (FCOD) is a rare, benign, multifocal fibro-osseous dysplastic process affecting tooth-bearing areas of the jaw, characterized by replacement of normal trabecular bone with osseous tissue and dense acellular cementum in a fibrous stroma. It is one clinicopathologic variant in a spectrum of related non-neoplastic fibro-osseous lesions known as cemento-osseous dysplasias (CODs), thought to arise from elements of the periodontal ligament. Diagnosis primarily relies upon radiographic and clinical findings; unnecessary biopsy should be avoided, as inoculation with oral pathogens may precipitate chronic infection in these hypovascular lesions. Appropriate management of uncomplicated FCOD consists of periodic radiographic follow-up. Accordingly, it is important that both radiologists and clinicians performing endodontic interventions possess familiarity with this entity in order to prevent misdiagnosis and inappropriate intervention, which may result in a protracted clinical course. Lesions are usually asymptomatic in the absence of infection, typically discovered on routine dental radiographs or imaging performed for unrelated indications. Radiographically, the condition typically manifests as widespread non-expansile intraosseous masses of varying internal lucency and sclerosis that surround the root apices of vital teeth or edentulous areas in the posterior jaw. While all CODs share similar microscopic features, FCOD is distinguished by its multifocal distribution, involving two or more quadrants of the maxilla and mandible, often in a bilateral symmetric fashion. The vast majority of cases are sporadic, though few exhibit an autosomal dominant familial inheritance pattern. In this pictorial review, we discuss the radiologic characteristics of this entity, pertinent clinical and histologic features, differential diagnoses, and management options.

Selective photonic disinfection of cell culture using a visible ultrashort pulsed laser.

Microbial contamination of cell culture is a major problem encountered both in academic labs and in the biotechnology/pharmaceutical industries. A broad spectrum of microbes including mycoplasma, bacteria, fungi, and viruses are the causative agents of cell culture contamination. Unfortunately, the existing disinfection techniques lack selectivity and/or lead to the development of drug-resistance, and more importantly there is no universal method to address all microbes. Here, we report a novel, chemical-free visible ultrashort pulsed laser method for cell culture disinfection. The ultrashort pulsed laser technology inactivates pathogens with mechanical means, a paradigm shift from the traditional pharmaceutical and chemical approaches. We demonstrate that ultrashort pulsed laser treatment can efficiently inactivate mycoplasma, bacteria, yeast, and viruses with good preservation of mammalian cell viability. Our results indicate that this ultrashort pulsed laser technology has the potential to serve as a universal method for the disinfection of cell culture.

Chemical-free inactivated whole influenza virus vaccine prepared by ultrashort pulsed laser treatment.

There is an urgent need for rapid methods to develop vaccines in response to emerging viral pathogens. Whole inactivated virus (WIV) vaccines represent an ideal strategy for this purpose; however, a universal method for producing safe and immunogenic inactivated vaccines is lacking. Conventional pathogen inactivation methods such as formalin, heat, ultraviolet light, and gamma rays cause structural alterations in vaccines that lead to reduced neutralizing antibody specificity, and in some cases, disastrous T helper type 2-mediated immune pathology. We have evaluated the potential of a visible ultrashort pulsed (USP) laser method to generate safe and immunogenic WIV vaccines without adjuvants. Specifically, we demonstrate that vaccination of mice with laser-inactivated H1N1 influenza virus at about a 10-fold lower dose than that required using conventional formalin-inactivated influenza vaccines results in protection against lethal H1N1 challenge in mice. The virus, inactivated by the USP laser irradiation, has been shown to retain its surface protein structure through hemagglutination assay. Unlike conventional inactivation methods, laser treatment did not generate carbonyl groups in protein, thereby reducing the risk of adverse vaccine-elicited T helper type 2 responses. Therefore, USP laser treatment is an attractive potential strategy to generate WIV vaccines with greater potency and safety than vaccines produced by current inactivation techniques.

Pathogen reduction in human plasma using an ultrashort pulsed laser.

Pathogen reduction is a viable approach to ensure the continued safety of the blood supply against emerging pathogens. However, the currently licensed pathogen reduction techniques are ineffective against non-enveloped viruses such as hepatitis A virus, and they introduce chemicals with concerns of side effects which prevent their widespread use. In this report, we demonstrate the inactivation of both enveloped and non-enveloped viruses in human plasma using a novel chemical-free method, a visible ultrashort pulsed laser. We found that laser treatment resulted in 2-log, 1-log, and 3-log reductions in human immunodeficiency virus, hepatitis A virus, and murine cytomegalovirus in human plasma, respectively. Laser-treated plasma showed ≥70% retention for most coagulation factors tested. Furthermore, laser treatment did not alter the structure of a model coagulation factor, fibrinogen. Ultrashort pulsed lasers are a promising new method for chemical-free, broad-spectrum pathogen reduction in human plasma.

Ultrashort pulsed laser treatment inactivates viruses by inhibiting viral replication and transcription in the host nucleus.

Ultrashort pulsed laser irradiation is a new method for virus reduction in pharmaceuticals and blood products. Current evidence suggests that ultrashort pulsed laser irradiation inactivates viruses through an impulsive stimulated Raman scattering process, resulting in aggregation of viral capsid proteins. However, the specific functional defect(s) in viruses inactivated in this manner have not been demonstrated. This information is critical for the optimization and the extension of this treatment platform to other applications. Toward this goal, we investigated whether viral internalization, replication, or gene expression in cells were altered by ultrashort pulsed laser irradiation. Murine Cytomegalovirus (MCMV), an enveloped DNA virus, was used as a model virus. Using electron and fluorescence microscopy, we found that laser-treated MCMV virions successfully internalized in cells, as evidenced by the detection of intracellular virions, which was confirmed by the detection of intracellular viral DNA via PCR. Although the viral DNA itself remained polymerase-amplifiable after laser treatment, no viral replication or gene expression was observed in cells infected with laser-treated virus. These results, along with evidence from previous studies, support a model whereby the laser treatment stabilizes the capsid, which inhibits capsid uncoating within cells. By targeting the mechanical properties of viral capsids, ultrashort pulsed laser treatment represents a unique potential strategy to overcome viral mutational escape, with implications for combatting emerging or drug-resistant pathogens.

Food safety and bioavailability evaluations of four vegetables grown in the highly arsenic-contaminated soils on the Guandu Plain of northern Taiwan.

Arsenic contamination in a large area of agricultural fields on the Guandu Plain of northern Taiwan was confirmed in a survey conducted in 2006, but research concerning the relationship between bioavailable As concentrations in contaminated soils and crop production in Taiwan is not available. Pot experiments were conducted to examine the growth and accumulation of As in four vegetable crops grown in As-contaminated soils and to assess As intake through consumption. The phytotoxic effects of As in soils were not shown in the pot experiments in which vegetable crops were grown in soils contaminated with different As levels in situ collected from Guandu Plain (120-460 mg/kg) or artificially spiked As-contaminated soils (50-170 mg/kg). Experimental results showed that the bioavailable As extracted with 0.5M NaHCO3 from soils can be used to estimate As concentrations in vegetables. The As concentrations in the vegetables were compared with data shown in the literature and As limits calculated from drinking water standards and the provisional tolerance weekly intake (PTWI) of inorganic As established by the Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO). Although the As levels in the vegetables were not high and the bioavailability of As in the soils was quite low, long-term consumption may result in higher As intake in the human body.

Studies of inactivation mechanism of non-enveloped icosahedral virus by a visible ultrashort pulsed laser.

BACKGROUND: Low-power ultrashort pulsed (USP) lasers operating at wavelengths of 425 nm and near infrared region have been shown to effectively inactivate viruses such as human immunodeficiency virus (HIV), M13 bacteriophage, and murine cytomegalovirus (MCMV). It was shown previously that non-enveloped, helical viruses such as M13 bacteriophage, were inactivated by a USP laser through an impulsive stimulated Raman scattering (ISRS) process. Recently, enveloped virus like MCMV has been shown to be inactivated by a USP laser via protein aggregation induced by an ISRS process. However, the inactivation mechanism for a clinically important class of viruses--non-enveloped, icosahedral viruses remains unknown. RESULTS AND DISCUSSIONS: We have ruled out the following four possible inactivation mechanisms for non-enveloped, icosahedral viruses, namely, (1) inactivation due to ultraviolet C (UVC) photons produced by non-linear optical process of the intense, fundamental laser beam at 425 nm; (2) inactivation caused by thermal heating generated by the direct laser absorption/heating of the virion; (3) inactivation resulting from a one-photon absorption process via chromophores such as porphyrin molecules, or indicator dyes, potentially producing reactive oxygen or other species; (4) inactivation by the USP lasers in which the extremely intense laser pulse produces shock wave-like vibrations upon impact with the viral particle. We present data which support that the inactivation mechanism for non-enveloped, icosahedral viruses is the impulsive stimulated Raman scattering process. Real-time PCR experiments show that, within the amplicon size of 273 bp tested, there is no damage on the genome of MNV-1 caused by the USP laser irradiation. CONCLUSION: We conclude that our model non-enveloped virus, MNV-1, is inactivated by the ISRS process. These studies provide fundamental knowledge on photon-virus interactions on femtosecond time scales. From the analysis of the transmission electron microscope (TEM) images of viral particles before and after USP laser irradiation, the locations of weak structural links on the capsid of MNV-1 were revealed. This important information will greatly aid our understanding of the structure of non-enveloped, icosahedral viruses. We envision that this non-invasive, efficient viral eradication method will find applications in the disinfection of pharmaceuticals, biologicals and blood products in the near future.

Reflection-based near-field ellipsometry for thin film characterization.

This paper presents a near-field ellipsometry method for nano-scale thin film characterization. The method is based on a reflection configuration of near-field optical detection. In the method, a new set of ellipsometry equations is established by taking into consideration the near-field sample-probe interaction and the topography of the thin film. Experimental and simulation results have shown that the proposed near-field ellipsometry is able to attain precise thin film characterization with nano-scale lateral resolution.

Inactivation of enveloped virus by laser-driven protein aggregation.

Ultrafast lasers in the visible and near-infrared range have emerged as a potential new method for pathogen reduction of blood products and pharmaceuticals. However, the mechanism of enveloped virus inactivation by this method is unknown. We report the inactivation as well as the molecular and structural effects caused by visible (425 nm) femtosecond laser irradiation on murine cytomegalovirus (MCMV), an enveloped, double-stranded DNA virus. Our results show that laser irradiation (1) caused a 5-log reduction in MCMV titer, (2) did not cause significant changes to the global structure of MCMV virions including membrane and capsid, as assessed by electron microscopy, (3) produced no evidence of double-strand breaks or crosslinking in MCMV genomic DNA, and (4) caused selective aggregation of viral capsid and tegument proteins. We propose a model in which ultrafast laser irradiation induces partial unfolding of viral proteins by disrupting hydrogen bonds and/or hydrophobic interactions, leading to aggregation of closely associated viral proteins and inactivation of the virus. These results provide new insight into the inactivation of enveloped viruses by visible femtosecond lasers at the molecular level, and help pave the way for the development of a new ultrafast laser technology for pathogen reduction.

Prospects for a novel ultrashort pulsed laser technology for pathogen inactivation.

The threat of emerging pathogens and microbial drug resistance has spurred tremendous efforts to develop new and more effective antimicrobial strategies. Recently, a novel ultrashort pulsed (USP) laser technology has been developed that enables efficient and chemical-free inactivation of a wide spectrum of viral and bacterial pathogens. Such a technology circumvents the need to introduce potentially toxic chemicals and could permit safe and environmentally friendly pathogen reduction, with a multitude of possible applications including the sterilization of pharmaceuticals and blood products, and the generation of attenuated or inactivated vaccines.

Studies of inactivation of encephalomyocarditis virus, M13 bacteriophage, and Salmonella typhimurium by using a visible femtosecond laser: insight into the possible inactivation mechanisms.

We report experimental results on the inactivation of encephalomyocarditis virus, M13 bacteriophage, and Salmonella typhimurium by a visible femtosecond laser. Our results suggest that inactivation of virus and bacterium by a visible femtosecond laser involves completely different mechanisms. Inactivation of viruses by a visible femtosecond laser involves the breaking of hydrogen∕hydrophobic bonds or the separation of the weak protein links in the protein shell of a viral particle. In contrast, inactivation of bacteria is related to the damage of their DNAs due to irradiation of a visible femtosecond laser. Possible mechanisms for the inactivation of viruses and bacteria are discussed.

TLR4 through IFN-ß promotes low molecular mass hyaluronan-induced neutrophil apoptosis.

Intratracheal administration of low molecular mass (LMM) hyaluronan (200 kDa) results in greater neutrophil infiltration in the lungs of TLR4(-/-) mice compared with that in wild-type mice. In general, enhanced neutrophil infiltration in tissue is due to cell influx; however, neutrophil apoptosis also plays an important role. We have assessed the effects of TLR4 in the regulation of neutrophil apoptosis in response to administration of LMM hyaluronan. We found that apoptosis of inflammatory neutrophils is impaired in TLR4(-/-) mice, an effect that depends upon the IFN-ß-mediated TRAIL/TRAILR system. IFN-ß levels were decreased in LMM hyaluronan-treated TLR4-deficient neutrophils. The treatment of inflammatory neutrophils with IFN-ß enhanced the levels of TRAIL and TRAILR 2. LMM hyaluronan-induced inflammatory neutrophil apoptosis was substantially prevented by anti-TRAIL neutralizing mAb. We conclude that decreased IFN-ß levels decrease the activity of the TRAIL/TRAILR system in TLR4-deficient neutrophils, leading to impaired apoptosis of neutrophils and resulting in abnormal accumulation of neutrophils in the lungs of LMM hyaluronan-treated mice. Thus, TLR4 plays a novel homeostatic role in noninfectious lung inflammation by accelerating the elimination of inflammatory neutrophils.

Artifact removal by intrinsic harmonics of tuning fork probe for scanning near-field optical microscopy.

This paper presents a new method to reduce the topographical artifacts in scanning near-field optical microscopy (SNOM) images. The method uses the harmonics caused intrinsically by the nonlinearity in the oscillation of the SNOM probe even when the probe is working in a normal condition without extra excitation. Using these intrinsic harmonics, the gradient of the received SNOM signal with respect to the probe motion is obtained. Then, taking advantage of a SNOM capable of simultaneously obtaining both the topographical and optical signals, topographical artifacts are calculated from the product of the gradient and the topographical signal, and then removed from the received SNOM signal. The effectiveness of the proposed method is demonstrated experimentally.

Near-field ellipsometry for thin film characterization.

A near-field ellipsometry method is presented for nano-scale thin film characterization. The technique fuses the topographic and ellipso-metric optical measurements that are simultaneously obtained by a scanning near-field optical microscopy (SNOM). It is shown that the proposed near-field ellipsometry is able to attain nano-scale lateral resolution and correct artifacts in characterization. The effectiveness of the proposed method is verified by simulation and experimental studies.

TLR4 is a negative regulator in noninfectious lung inflammation.

Low m.w. hyaluronan (LMW HA) has been shown to elicit the expression of proinflammatory cytokines and chemokines in various cells in vitro. However, the effects of this molecule in vivo are unknown. In this study, we report that intratracheal administration of LMW HA (200 kDa) causes inflammation in mouse lung. A lack of TLR4 is associated with even stronger inflammatory response in the lung as shown by increased neutrophil counts and elevated cytokine and chemokine concentrations. We also demonstrate that TLR4 anti-inflammatory signaling is dependent upon a MyD88-independent pathway. TLR4-mediated IL-1R antagonist production plays a negative regulatory role in LMW HA (200 kDa) induced lung inflammation. These data provide a molecular level explanation for the function of TLR4 in LMW HA (200 kDa)-induced lung inflammation, as inhibition of the beta form of pro-IL-1 promotes an anti-inflammatory response.

Therapeutic HPV DNA vaccines.

Human papillomavirus (HPV) has been associated with several human cancers, including cervical cancer, vulvar cancer, vaginal and anal cancer, and a subset of head and neck cancers. The identification of HPV as an etiological factor for HPV-associated malignancies creates the opportunity for the control of these cancers through vaccination. Currently, the preventive HPV vaccine using HPV virus-like particles has been proven to be safe and highly effective. However, this preventive vaccine does not have therapeutic effects, and a significant number of people have established HPV infection and HPV-associated lesions. Therefore, it is necessary to develop therapeutic HPV vaccines to facilitate the control of HPV-associated malignancies and their precursor lesions. Among the various forms of therapeutic HPV vaccines, DNA vaccines have emerged as a potentially promising approach for vaccine development due to their safety profile, ease of preparation and stability. However, since DNA does not have the intrinsic ability to amplify or spread in transfected cells like viral vectors, DNA vaccines can have limited immunogenicity. Therefore, it is important to develop innovative strategies to improve DNA vaccine potency. Since dendritic cells (DCs) are key players in the generation of antigen-specific immune responses, it is important to develop innovative strategies to modify the properties of the DNA-transfected DCs. These strategies include increasing the number of antigen-expressing/antigen-loaded DCs, improving antigen processing and presentation in DCs, and enhancing the interaction between DCs and T cells. Many of the studies on DNA vaccines have been performed on preclinical models. Encouraging results from impressive preclinical studies have led to several clinical trials.

Femtosecond laser treatment enhances DNA transfection efficiency in vivo.

BACKGROUND: Gene therapy with plasmid DNA is emerging as a promising strategy for the treatment of many diseases. One of the major obstacles to such therapy is the poor transfection efficiency of DNA in vivo. METHODS: In this report, we employed a very low power, near-infrared femtosecond laser technique to enhance the transfection efficiency of intradermally and intratumorally administered DNA plasmid. RESULTS: We found that femtosecond laser treatment can significantly enhance the delivery of DNA into the skin and into established tumors in mice. In addition, we found that both laser power density as well as duration of laser treatment are critical parameters for augmenting DNA transfection efficiency. The femtosecond laser technique employs a relatively unfocused laser beam that maximizes the transfected area, minimizes damage to tissue and simplifies its implementation. CONCLUSION: This femtosecond new laser technology represents a safe and innovative technology for enhancing DNA gene transfer in vivo.