Proximity-dependent mapping of the HCMV US28 interactome identifies RhoGEF signaling as a requirement for efficient viral reactivation.

Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.

Non-replicating adenovirus based Mayaro virus vaccine elicits protective immune responses and cross protects against other alphaviruses.

Mayaro virus (MAYV) is an alphavirus endemic to South and Central America associated with sporadic outbreaks in humans. MAYV infection causes severe joint and muscle pain that can persist for weeks to months. Currently, there are no approved vaccines or therapeutics to prevent MAYV infection or treat the debilitating musculoskeletal inflammatory disease. In the current study, a prophylactic MAYV vaccine expressing the complete viral structural polyprotein was developed based on a non-replicating human adenovirus V (AdV) platform. Vaccination with AdV-MAYV elicited potent neutralizing antibodies that protected WT mice against MAYV challenge by preventing viremia, reducing viral dissemination to tissues and mitigating viral disease. The vaccine also prevented viral-mediated demise in IFN⍺R1-/- mice. Passive transfer of immune serum from vaccinated animals similarly prevented infection and disease in WT mice as well as virus-induced demise of IFN⍺R1-/- mice, indicating that antiviral antibodies are protective. Immunization with AdV-MAYV also generated cross-neutralizing antibodies against two related arthritogenic alphaviruses-chikungunya and Una viruses. These cross-neutralizing antibodies were protective against lethal infection in IFN⍺R1-/- mice following challenge with these heterotypic alphaviruses. These results indicate AdV-MAYV elicits protective immune responses with substantial cross-reactivity and protective efficacy against other arthritogenic alphaviruses. Our findings also highlight the potential for development of a multi-virus targeting vaccine against alphaviruses with endemic and epidemic potential in the Americas.

Rat and human cytomegalovirus ORF116 encodes a virion envelope glycoprotein required for infectivity.

Herpesviruses encode multiple glycoproteins required for different stages of viral attachment, fusion, and envelopment. The protein encoded by the human cytomegalovirus (HCMV) open reading frame UL116 forms a stable complex with glycoprotein H that is incorporated into virions. However, the function of this complex remains unknown. Herein, we characterize R116, the rat CMV (RCMV) putative homolog of UL116. Two R116 transcripts were identified in fibroblasts with three proteins expressed with molecular weights of 42, 58, and 82 kDa. R116 is N-glycosylated, expressed with late viral gene kinetics, and is incorporated into the virion envelope. RCMV lacking R116 failed to result in productive infection of fibroblasts and siRNA knockdown of R116 substantially reduced RCMV infectivity. Complementation in trans of an R116-deficient virus restored ability of the virus to infect fibroblasts. Finally, UL116 knockdown also decreased HCMV infectivity indicating that R116 and UL116 both contribute to viral infectivity.

Wavelength- and irradiance-dependent changes in intracellular nitric oxide level.

SIGNIFICANCE: Photobiomodulation (PBM) refers to the beneficial effects of low-energy light absorption. Although there is a large body of literature describing downstream physiological benefits of PBM, there is a limited understanding of the molecular mechanisms underlying these effects. At present, the most popular hypothesis is that light absorption induces release of nitric oxide (NO) from the active site of cytochrome c oxidase (COX), allowing it to bind O2 instead. This is believed to increase mitochondrial respiration, and result in greater overall health of the cell due to increased adenosine triphosphate production. AIM: Although NO itself is a powerful signaling molecule involved in a host of biological responses, less attention has been devoted to NO mechanisms in the context of PBM. The purpose of our work is to investigate wavelength-specific effects on intracellular NO release in living cells. APPROACH: We have conducted in-depth dosimetry analyses of NO production and function in an in vitro retinal model in response to low-energy exposure to one or more wavelengths of laser light. RESULTS: We found statistically significant wavelength-dependent elevations (10% to 30%) in intracellular NO levels following laser exposures at 447, 532, 635, or 808 nm. Sequential or simultaneous exposures to light at two different wavelengths enhanced the NO modulation up to 50% of unexposed controls. Additionally, the immediate increases in cellular NO levels were independent of the function of NO synthase, depended greatly on the substrate source of electrons entering the electron transport chain, and did not result in increased levels of cyclic guanosine monophosphate. CONCLUSIONS: Our study concludes the simple model of light-mediated release of NO from COX is unlikely to explain the wide variety of PBM effects reported in the literature. Our multiwavelength method provides a novel tool for studying immediate and early mechanisms of PBM as well as exploring intracellular NO signaling networks.

Vaccine-Induced Skewing of T Cell Responses Protects Against Chikungunya Virus Disease.

Chikungunya virus (CHIKV) infections can cause severe and debilitating joint and muscular pain that can be long lasting. Current CHIKV vaccines under development rely on the generation of neutralizing antibodies for protection; however, the role of T cells in controlling CHIKV infection and disease is still unclear. Using an overlapping peptide library, we identified the CHIKV-specific T cell receptor epitopes recognized in C57BL/6 infected mice at 7 and 14 days post-infection. A fusion protein containing peptides 451, 416, a small region of nsP4, peptide 47, and an HA tag (CHKVf5) was expressed using adenovirus and cytomegalovirus-vectored vaccines. Mice vaccinated with CHKVf5 elicited robust T cell responses to higher levels than normally observed following CHIKV infection, but the vaccine vectors did not elicit neutralizing antibodies. CHKVf5-vaccinated mice had significantly reduced infectious viral load when challenged by intramuscular CHIKV injection. Depletion of both CD4+ and CD8+ T cells in vaccinated mice rendered them fully susceptible to intramuscular CHIKV challenge. Depletion of CD8+ T cells alone reduced vaccine efficacy, albeit to a lesser extent, but depletion of only CD4+ T cells did not reverse the protective phenotype. These data demonstrated a protective role for CD8+ T cells in CHIKV infection. However, CHKVf5-vaccinated mice that were challenged by footpad inoculation demonstrated equal viral loads and increased footpad swelling at 3 dpi, which we attributed to the presence of CD4 T cell receptor epitopes present in the vaccine. Indeed, vaccination of mice with vectors expressing only CHIKV-specific CD8+ T cell epitopes followed by CHIKV challenge in the footpad prevented footpad swelling and reduced proinflammatory cytokine and chemokines associated with disease, indicating that CHIKV-specific CD8+ T cells prevent CHIKV disease. These results also indicate that a T cell-biased prophylactic vaccination approach is effective against CHIKV challenge and reduces CHIKV-induced disease in mice.

Effect of ambient temperature and intracellular pigmentation on photothermal damage rate kinetics.

Computational models predicting cell damage responses to transient temperature rises generated by exposure to lasers have implemented the damage integral (Ω), which time integrates the chemical reaction rate constant described by Arrhenius. However, few published reports of empirical temperature histories (thermal profiles) correlated with damage outcomes at the cellular level are available to validate the breadth of applicability of the damage integral. In our study, an analysis of photothermal damage rate processes in cultured retinal pigment epithelium cells indicated good agreement between temperature rise, exposure duration (τ), and threshold cellular damage. Full-frame thermograms recorded at high magnification during laser exposures were overlaid with fluorescence damage images taken 1 h postexposure. From the image overlays, pixels of the thermogram correlated with the boundary of cell death were used to extract threshold thermal profiles. Assessing photothermal responses at these boundaries standardized all data points, irrespective of laser irradiance, damage size, or optical and thermal properties of the cells. These results support the hypothesis that data from boundaries of cell death were equivalent to a minimum visible lesion, where the damage integral approached unity (Ω = 1) at the end of the exposure duration. Empirically resolved Arrhenius coefficients for use in the damage integral determined from exposures at wavelengths of 2 µm and 532 nm and durations of 0.05-20 s were consistent with literature values. Varying ambient temperature (Tamb) between 20°C and 40°C during laser exposure did not change the τ-dependent threshold peak temperature (Tp). We also show that, although threshold laser irradiance varied due to pigmentation differences, threshold temperatures were irradiance independent.

Real-time optoacoustic temperature determination on cell cultures during heat exposure: a feasibility study.

Objective/Purpose: In order to study the effects of hyperthermia and other temperature-related effects on cells and tissues, determining the precise time/temperature course is crucial. Here we present a non-contact optoacoustic technique, which provides temperatures during heating of cultured cells with scalable temporal and spatial resolution. METHODS: A thulium laser (1.94 µm) with a maximum power of 15 W quickly and efficiently heats cells in a culture dish because of low penetration depth (1/e penetration depths of 78 µm) of the radiation in water. A repetitively Q-switched holmium laser (2.1 µm) is used simultaneously to probe temperatures at different locations in the dish by using the photoacoustic effect. Due to thermoelastic expansion of water, pressure waves are emitted and measured with an ultrasonic hydrophone at the side of the dish. The amplitudes of the waves are temperature dependent and can be used to calculate the temperature/time course at any location of probing. RESULTS: We measured temperatures of up to 55 °C with a heating power of 6 W after 10 s, and subsequent lateral temperature profiles over time. Within this profile, temperature fluctuations were found, likely owing to thermal convection and water circulation. By using cultured retinal pigment epithelial cells, it is shown that the probe laser pulses alone cause no biological damage, while immediate cell damage occurs when heating for 10 s at temperatures exceeding 45 °C. CONCLUSIONS: This method shows great potential not only as a noninvasive, non-contact method to determine temperature/time responses of cells in culture, but also for complex tissue and other materials.

Assessment of tissue heating under tunable near-infrared radiation.

The time-temperature effects of laser radiation exposure are investigated as a function of wavelength. Here, we report the thermal response of bulk tissue as a function of wavelength from 700 to 1064 nm. Additionally, Monte Carlo simulations were used to verify the thermal response measured and predict damage thresholds based on the response.

Maxwell's equations-based dynamic laser-tissue interaction model.

Since its invention in the early 1960s, the laser has been used as a tool for surgical, therapeutic, and diagnostic purposes. To achieve maximum effectiveness with the greatest margin of safety it is important to understand the mechanisms of light propagation through tissue and how that light affects living cells. Lasers with novel output characteristics for medical and military applications are too often implemented prior to proper evaluation with respect to tissue optical properties and human safety. Therefore, advances in computational models that describe light propagation and the cellular responses to laser exposure, without the use of animal models, are of considerable interest. Here, a physics-based laser-tissue interaction model was developed to predict the dynamic changes in the spatial and temporal temperature rise during laser exposure to biological tissues. Unlike conventional models, the new approach is grounded on the rigorous electromagnetic theory that accounts for wave interference, polarization, and nonlinearity in propagation using a Maxwell's equations-based technique.

Hyperthermia sensitizes pigmented cells to laser damage without changing threshold damage temperature.

We studied the efficacy of mild hyperthermia as a protective measure against subsequent laser-induced thermal damage. Using a well established in vitro retinal model for laser bioeffects, consisting of an artificially pigmented human retinal pigment epithelial (RPE) cell culture (hTERT-RPE1), we found both protection and sensitization to laser damage that depended upon the location of pigment granules during the hyperthermia preconditioning (PC). Photothermal challenge of cell monolayers consisted of 16 independent replicate exposures of 65 W/cm2 at 514 nm and post laser damage was assessed using fluorescence indicator dyes. Untreated cells had 44% damage, but when melanosome particles (MPs) were intracellular or extracellular during the hyperthermia treatment, laser-induced cell damage occurred 94% or 25% of the time, respectively. Using a recently published method called microthermography, we found that the hyperthermia pretreatment did not alter the threshold temperature for cell death, indicating an alteration in absorption or localization of heat as the mechanism for sensitization and protection. Raman microspectroscopy revealed significant chemical changes in MPs when they were preconditioned within the cytoplasm of cells. Our results suggest intracellular pigment granules undergo chemical modifications during mild hyperthermia that can profoundly affect absorption or heat dissipation.

Predictors of creatinine rise post-endovascular abdominal aortic aneurysm repair.

BACKGROUND: Endovascular abdominal aortic aneurysm repair involves manipulation of the aorta around the renal arteries. Fenestrated grafts involve the direct cannulation, stenting and injecting of contrast into the renal arteries. These procedures may be associated with an acute post-operative creatinine rise. METHODS: We retrospectively examined data from all endovascular aortic repairs at our institution from 2005 to 2009, where contrast dosage had been recorded. Renal impairment was defined as a 25% increase in creatinine during the 5-day postoperative period. Univariable analysis was undertaken for a number of likely predictors, including: age, contrast dosage, preoperative creatinine, graft type (fenestrated or standard), diabetes mellitus, hypertension, hypercholesterolaemia, ischaemic heart disease, aspirin therapy, statins therapy, non-steroidal anti-inflammatory drug use, preoperative N-acetyl-cysteine and intravenous pre-hydration. Multivariable analysis was then applied to variables with a univariable P-value of < 0.05. RESULTS: We identified 106 consecutive cases, with complete data for 102. Twenty per cent of patients developed renal impairment (22/102). Contrast dose (P = 0.043) and fenestrated grafts (P = 0.006) were identified as significant risk factors for post-operative creatinine increase (P = 0.043). Multivariable analysis demonstrated that fenestrated grafts were a risk factor independent of contrast dosage (P < 0.05). CONCLUSIONS: Patients who received a fenestration graft (P < 0.01) and increased contrast dose (P < 0.05) were at a significant increased risk of a 25% post-operative creatinine rise. The risk of fenestration grafts persisted when multivariable regression was performed to control for contrast dosage (P < 0.05). Other variables investigated were not found to be significant in this study.

Guidelines for patient selection and performance of carotid artery stenting.

BACKGROUND: The endovascular treatment of carotid atherosclerosis with carotid artery stenting (CAS) remains controversial. Carotid endarterectomy remains the benchmark in terms of procedural mortality and morbidity. At present, there are no consensus Australasian guidelines for the safe performance of CAS. METHODS: We applied a modified Delphi consensus method of iterative consultation between the College representatives on the Carotid Stenting Guidelines Committee (CSGC). RESULTS: Selection of patients suitable for CAS needs careful consideration of clinical and patho-anatomical criteria and cannot be directly extrapolated from clinical indicators for carotid endarterectomy (CEA). Randomized controlled trials (including pooled analyses of results) comparing CAS with CEA for treatment of symptomatic stenosis have demonstrated that CAS is more hazardous than CEA. On current evidence, the CGSC therefore recommends that CAS should not be performed in the majority of patients requiring carotid revascularisation. The evidence for CAS in patients with symptomatic severe carotid stenosis who are considered medically high risk is weak, and there is currently no evidence to support CAS as a treatment for asymptomatic carotid stenosis. The use of distal protection devices during CAS remains controversial with increased risk of clinically silent stroke. The knowledge requirements for the safe performance of CAS include an understanding of the evidence base from randomized controlled trials, carotid and aortic arch anatomy and pathology, clinical stroke syndromes, the differing treatment options for stroke and carotid atherosclerosis, and recognition and management of periprocedural complications. It is critical that all patients being considered for a carotid intervention have adequate pre-procedural neuro-imaging and an independent, standardized neurological assessment before and after the procedure. Maintenance of proficiency in CAS requires active involvement in surgical/endovascular audit and continuing medical education programs. These standards should apply in the public and private health care settings. CONCLUSION: These guidelines represent the consensus of an inter-collegiate committee in order to direct appropriate patient selection and the range of cognitive and technical requirements to perform CAS. Advances in endovascular technologies and the results of randomized controlled trials will guide future revisions of these guidelines.

Adenovirus vectors expressing hantavirus proteins protect hamsters against lethal challenge with andes virus.

Hantaviruses infect humans following aerosolization from rodent feces and urine, producing hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Due to the high rates of mortality and lack of therapies, vaccines are urgently needed. Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN) or glycoproteins (AdG(N) and AdG(C)) were constructed. Ad vectors were tested for their ability to protect Syrian hamsters from a lethal ANDV infection that mimics the pulmonary disease seen in humans. When administered once, all three Ad vectors, individually or in combination, elicited a robust immune response that protected hamsters. No vaccinated animal died, and there were no obvious clinical signs of disease. Further, hantavirus RNA was not detected by sensitive reverse transcription-PCR in tissues and blood of hamsters immunized with both AdG(N) and AdG(C). Cellular immunity appeared to be important for protection because the AdN vector completely protected animals. All three Ad vectors produced strong cytotoxic T-lymphocyte responses directed to hantavirus proteins in mice. Moreover, hamsters vaccinated with AdN, AdG(N), or AdG(C) produced no detectable neutralizing antibodies yet were protected. These Ad vectors represent the first vaccines that prevent lethal hantavirus disease and, in some instances (AdG(N) and AdG(C)), provide sterile immunity. These observations set the stage for a more detailed characterization of the types of immunity required to protect humans from hantavirus infections.

The iliac bifurcation device for endovascular iliac aneurysm repair: indications, deployment options and results at 1-year follow-up of 25 cases.

BACKGROUND: The iliac bifurcation device (William A Cook Australia, Brisbane, QLD, Australia) is a new endovascular device for iliac aneurysm repair. We review the indications for use, device characteristics, deployment options and the results of our case series. METHODS: The most common indication for deployment is endovascular aortic aneurysm repair (EVAR) with common iliac aneurysm repair. The standard deployment sequence can be adapted to increase the utility of the device. Data were collected prospectively. Follow-up was performed with plain X-ray, ultrasound and computed tomography (CT) scan. RESULTS: Between 2004 and 2007, 25 patients had their common iliac artery aneurysm repaired using the iliac bifurcation device. There were 23 male and 2 female patients. Median age was 75 years (range 60-85). The median follow-up was 12 months (range 1-38). Twenty-one procedures were combined with EVAR. The median abdominal aortic aneurysm diameter was 60 mm (range 31-97), and the median common iliac artery aneurysm diameter was 37 mm (range 24-71). Technical success was achieved in 100% of cases. There were no acute branch vessel occlusions. There was one early type I endoleak (4%). There was one death (4%) in the 30-day period post-procedure. There was one late type I endoleak (4%). CONCLUSIONS: The iliac bifurcation device achieves endovascular common iliac artery aneurysm repair with preservation of internal iliac artery flow. There are multiple different applications of the device and complementary deployment techniques. High rates of technical success and low rates of branch vessel occlusion are possible.

In vitro model that approximates retinal damage threshold trends.

Without effective in vitro damage models, advances in our understanding of the physics and biology of laser-tissue interaction would be hampered due to cost and ethical limitations placed on the use of nonhuman primates. We extend our characterization of laser-induced cell death in an existing in vitro retinal model to include damage thresholds at 514 and 413 nm. The new data, when combined with data previously reported for 532 and 458 nm exposures, provide a sufficiently broad range of wavelengths and exposure durations (0.1 to 100 s) to make comparisons with minimum visible lesion (in vivo) data in the literature. Based on similarities between in vivo and in vitro action spectra and temporal action profiles, the cell culture model is found to respond to laser irradiation in a fundamentally similar fashion as the retina of the rhesus animal model. We further show that this response depends on the amount of intracellular melanin pigmentation.

A Phase II study of external-beam radiotherapy and endovascular brachytherapy with PTA and stenting for femoropopliteal artery restenosis.

PURPOSE: To assess the safety and seek evidence of efficacy of combined external-beam radiotherapy (EBRT) and endovascular brachytherapy in the treatment of stenotic vascular lesions. METHODS AND MATERIALS: Seventeen patients with high risk for restenosis of femoropopliteal arteries were enrolled in this study from February 2000 to August 2002. The external beam radiotherapy regimen consisted of 10 Gy in 5 fractions of 2 Gy, starting on Day 0. This was followed on Day 6 by angiography, stent placement, and intraluminal brachytherapy to a dose of 10 Gy at 1.2 mm from stent surface. The EBRT was continued from the same day to another 10 Gy in 2 Gy daily fractions for 5 days. RESULTS: The follow up ranged from 33 months to 60 months. At the time of analysis 15 of 17 patients were alive with patent stents. Of these, 10 were symptom-free. Two patients died of unrelated causes. CONCLUSIONS: The combination of EBRT and endovascular brachytherapy provided adequate dose distribution without any geographical miss or "candy wrapper" restenosis. No incidence of aneurysmal dilation of radiated vascular segment was observed. The treatment was feasible, well tolerated, and achieved 88% stenosis free survival.

Damage Thresholds for Exposure to NIR and Blue Lasers in an In Vitro RPE Cell System.

PURPOSE: Until reliable nonanimal systems of analysis are available, animal models will be necessary for ocular laser hazard analysis and for evaluating clinical applications. The purpose of this work was to demonstrate the utility of an in vitro system for laser bioeffects by identifying photothermal and photochemical cytotoxicity thresholds for continuous-wave (cw) and mode-locked (ml) laser exposures. METHODS: Exogenous melanosomes were added to hTERT-RPE1 cells in exposure wells 1 day before laser exposure. Thermal or photochemical laser exposures were delivered to artificially pigmented retinal pigment epithelial (RPE) cultures, with subsequent assay for viability 1 hour after exposure. Beam delivery for the 1-hour photochemical exposures was via a modified culture incubator. The cytoprotective effect of pretreatment with two antioxidants was investigated. RESULTS: Phagocytosis of melanosomes by the RPE cells was efficient, yielding cultures of uniform pigmentation. The damage threshold for the thermal exposure was consistent with published in vivo results. Thresholds for both blue exposures (cw and ml) were identical. Overnight treatment of cells with ascorbic acid (AA) minimized cell death from both cw and ml blue laser exposure, whereas similar treatment with N-acetyl-L-cysteine (NAC) was less effective. CONCLUSIONS: The in vitro system described is suitable for measuring meaningful thermal and photochemical laser damage thresholds. The system is also useful in comparative laser bioeffects studies, such as comparisons between cw and ml laser exposures, cells with various degrees of pigmentation, and studies determining the efficacy and mechanisms of treatments altering the response of cells to lasers.

The role of BiP in endoplasmic reticulum-associated degradation of major histocompatibility complex class I heavy chain induced by cytomegalovirus proteins.

Human cytomegalovirus (HCMV1) US11 and US2 proteins cause rapid degradation of major histocompatibility complex (MHC) molecules, apparently by ligating cellular endoplasmic reticulum (ER)-associated degradation machinery. Here, we show that US11 and US2 bind the ER chaperone BiP. Four related HCMV proteins, US3, US7, US9, and US10, which do not promote degradation of MHC proteins, did not bind BiP. Silencing BiP reduced US11- and US2-mediated degradation of MHC class I heavy chain (HC) without altering the synthesis or translocation of HC into the ER or the stability of HC in the absence of US11 or US2. Induction of the unfolded protein response (UPR) did not affect US11-mediated HC degradation and could not explain the stabilization of HC when BiP was silenced. Unlike in yeast, BiP did not act by maintaining substrates in a retrotranslocation-competent form. Our studies go beyond previous observations in mammalian cells correlating BiP release with degradation, demonstrating that BiP is functionally required for US2- and US11-mediated HC degradation. Further, US2 and US11 bound BiP even when HC was absent and degradation of US2 depended on HC. These data were consistent with a model in which US2 and US11 bridge HC onto BiP promoting interactions with other ER-associated degradation proteins.

Accurate measure of laser irradiance threshold for near-infrared photo-oxidation with a modified confocal microscope.

Femtosecond mode-locked lasers are now being used routinely in multiphoton fluorescence and autofluorescence spectroscopy, are just beginning to be used in refractive surgery, and may be used in the future diagnosis of skin cancer. Pulses from these lasers induce non-linear effects in resultant tissue interactions. Using a modified confocal microscope with dispersion compensation and accurate measurements of beam diameter, a very low threshold was measured for photochemical oxidation in cultured cells. The measured threshold showed non-linear photo-oxidation at a peak irradiance and photon-flux density of 8.4x10(8) W cm-2 and 3.4x10(27) photons cm-2 s-1, respectively (90-fs pulse). The impact of these findings is significant to those using ultrashort lasers because they provide a tangible reference point (microscope-independent) for the generation of photo-oxidative stress in laser-exposed tissues, and because they highlight the importance of dispersion compensation in minimizing collateral tissue damage.

Herstatin inhibits heregulin-mediated breast cancer cell growth and overcomes tamoxifen resistance in breast cancer cells that overexpress HER-2.

Ligands of the ErbB family of receptors and estrogens control the proliferation of breast cancer cells. Overexpression of human EGF receptor HER-2 (erbB2) leads to amplified heregulin (HRG) signaling, promoting more aggressive breast cancer that is nonresponsive to estrogen and the antiestrogenic drug tamoxifen. Herstatin (Hst), a secreted HER-2 gene product, binds to the HER-2 receptor ectodomain blocking receptor activation. The aim of this study was to investigate the impact of this HER-2 inhibitor on HRG-induced signaling, proliferation, and sensitivity to tamoxifen in breast cancer cells with and without HER-2 overexpression. The expression of Hst in MCF7 cells eliminated HRG signaling through both mitogen-activated protein kinase and Akt pathways and prevented HRG-mediated proliferation. The loss in signaling corresponded to downregulation of the HRG receptors, HER-3 and HER-4, whereas HER-2 overexpression strongly stimulated the levels of both HRG receptors. Although Hst blocked HRG signaling in both parental and HER-2 transfected cells, it enhanced sensitivity to tamoxifen only in the MCF7 cells that overexpressed HER-2. To evaluate further the efficacy of Hst as an anticancer agent, His-tagged Hst was expressed in transfected insect cells, purified, and added to the breast cancer cells. As in the transfected cells, purified Hst inhibited HER-3 levels and suppressed HRG-induced proliferation of MCF7 and BT474 breast cancer cells. In contrast, the HER-2 monoclonal antibody, herceptin, downregulated HER-2, but not HER-3. These results suggest the potential use of Hst against HRG-mediated growth of breast cancers with high and low levels of HER-2 and against tamoxifen resistance in HER-2 overexpressing breast cancer.