The Impact of Colleague Peer Review on the Radiotherapy Treatment Planning Process in the Radical Treatment of Lung Cancer.

AIMS: Modern radiotherapy uses techniques to reliably identify tumour and reduce target volume margins. However, this can potentially lead to an increased risk of geographic miss. One source of error is the accuracy of target volume delineation (TVD). Colleague peer review (CPR) of all curative-intent lung cancer plans has been mandatory in our institution since May 2013. At least two clinical oncologists review plans, checking treatment paradigm, TVD, prescription dose tumour and critical organ tolerances. We report the impact of CPR in our institution. MATERIALS AND METHODS: Radiotherapy treatment plans of all patients receiving radical radiotherapy were presented at weekly CPR meetings after their target volumes were reviewed and signed off by the treating consultant. All cases and any resultant change to TVD (including organs at risk) or treatment intent were recorded in our prospective CPR database. The impact of CPR over a 13 month period from May 2013 to June 2014 is reported. RESULTS: One hundred and twenty-two patients (63% non-small cell lung carcinoma, 17% small cell lung carcinoma and 20% 'clinical diagnosis') were analysed. On average, 3.2 cases were discussed per meeting (range 1-8). CPR resulted in a change in treatment paradigm in 3% (one patient proceeded to induction chemotherapy, two patients had high-dose palliative radiotherapy). Twenty-one (17%) had a change in TVD and one (1%) patient had a change in dose prescription. In total, 6% of patients had plan adjustment after review of dose volume histogram. CONCLUSION: The introduction of CPR in our centre has resulted in a change in a component of the treatment plan for 27% of patients receiving curative-intent lung radiotherapy. We recommend CPR as a mandatory quality assurance step in the planning process of all radical lung plans.

Effects of rapid antigen degradation and VEE glycoprotein specificity on immune responses induced by a VEE replicon vaccine.

Genetic vaccines are engineered to produce immunogens de novo in the cells of the host for stimulation of a protective immune response. In some of these systems, antigens engineered for rapid degradation have produced an enhanced cellular immune response by more efficient entry into pathways for processing and presentation of MHC class I peptides. VEE replicon particles (VRP), single cycle vaccine vectors derived from Venezuelan equine encephalitis virus (VEE), are examined here for the effect of an increased rate of immunogen degradation on VRP vaccine efficacy. VRP expressing the matrix capsid (MA/CA) portion of SIV Gag were altered to promote rapid degradation of MA/CA by various linkages to co-translated ubiquitin or by destabilizing mutations and were used to immunize BALB/c mice for quantitation of anti-MA/CA cellular and humoral immune responses. Rapid degradation by the N-end rule correlated with a dampened immune response relative to unmodified MA/CA when the VRP carried a glycoprotein spike from an attenuated strain of VEE. In contrast, statistically equivalent numbers of IFNgamma(+)T-cells resulted when VRP expressing unstable MA/CA were packaged with the wild-type VEE glycoproteins. These results suggest that the cell types targeted in vivo by VRP carrying mutant or wild type glycoprotein spikes are functionally different, and are consistent with previous findings suggesting that wild-type VEE glycoproteins preferentially target professional antigen presenting cells that use peptides generated from the degraded antigen for direct presentation on MHC.

Flow cytometry-based assay for titrating dengue virus.

Plaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.

Future vaccines against emerging encephalitides.

The vaccine strategies available for control of emerging encephalitides range in a continuum from traditional approaches to those utilizing new technologies. In this report, we explore the use of live attenuated vaccines where the attenuating mutations have been selected in a rational way to improve attenuation without sacrificing effectiveness. A strategy for paired lethal and resuscitating mutations is presented that will greatly reduce the possibility of reversion to virulence. Finally, we describe an example of a vaccine vector system that could be rapidly adapted for use against these virus diseases as they emerge.

Diffraction enhanced imaging contrast mechanisms in breast cancer specimens.

We have investigated the contrast mechanisms of the refraction angle, and the apparent absorption images obtained from the diffraction enhanced imaging technique (DEI) and have correlated them with the absorption contrast of conventional radiography. The contrast of both the DEI refraction angle image and the radiograph have the same dependence on density differences of the tissues in the visualization of cancer; in radiography these differences directly relate to the contrast while in the DEI refraction angle image it is the density difference and thickness gradient that gives the refraction angle. We show that the density difference of fibrils in breast cancer as measured by absorption images correlate well with the density difference derived from refraction angle images of DEI. In addition we find that the DEI apparent absorption image and the image obtained with the DEI system at the top of the reflectivity curve have much greater contrast than that of the normal radiograph (x8 to 33-fold higher). This is due to the rejection of small angle scattering (extinction) from the fibrils enhancing the contrast.

Thalidomide in low doses is effective for the treatment of resistant or relapsed multiple myeloma and for plasma cell leukaemia.

Thalidomide is an effective treatment for relapsed multiple myeloma (MM), but is associated with a significant side effect profile at higher doses. In a recent study, only half of the enrolled patients were able to tolerate the maximum dose of 800 mg/day [Singhal, S., et al. (1999) "Antitumor activity of thalidomide in refractory multiple myeloma", New Engl. J. Med. 341, 1565-1571]. Moreover, the dose-response relationship has not been defined. We report our use of low dose thalidomide in a small cohort of 12 patients-eight with relapsed or refractory MM and four with plasma cell leukaemia (PCL). Five of the 12 (42%) patients had a partial response, showing a median fall in their PP/BJP of 80% (63-90%) at a median dose of 175 mg (100-300 mg) with negligible side effects. Three of four patients with PCL showed an impressive response to treatment with thalidomide as a single agent. No patient who failed to show any evidence of response at low dose (<150 mg/day) responded to higher doses. In this study, thalidomide induces a similar rate of response at a lower and better tolerated dose than previously reported and produced "best ever" responses in patients with resistant PCL.

Improving the detection of simulated masses in mammograms through two different image-processing techniques.

RATIONALE AND OBJECTIVES: The purpose of this study was to determine whether contrast-limited adaptive histogram equalization (CLAHE) or histogram-based intensity windowing (HIW) improves the detection of simulated masses in dense mammograms. MATERIALS AND METHODS: Simulated masses were embedded in portions of mammograms of patients with dense breasts; the mammograms were digitized at 50 microm per pixel, 12 bits deep. In two different experiments, images were printed both with no processing applied and with related parameter settings of two image-processing methods. A simulated mass was embedded in a realistic background of dense breast tissue, with its position varied. The key variables in each trial included the position of the mass, the contrast levels of the mass relative to the background, and the selected parameter settings for the image-processing method. RESULTS: The success in detecting simulated masses on mammograms with dense backgrounds depended on the parameter settings of the algorithms used. The best HIW setting performed better than the best fixed-intensity window setting and better than no processing. Performance with the best CLAHE settings was no different from that with no processing. In the HIW experiment, there were no significant differences in observer performance between processing conditions for radiologists and nonradiologists. CONCLUSION: HIW should be tested in clinical images to determine whether the detection of masses by radiologists can be improved. CLAHE processing will probably not improve the detection of masses on clinical mammograms.

How golden hamsters (Mesocricetus auratus) discriminate top from bottom flank scents in over-marks.

Using a habituation-discrimination paradigm, the authors investigated what cues male golden hamsters (Mesocricetus auratus) use to determine the top and bottom positions in flank gland over-marks. A difference in the ages of 2 hamsters' marks did not, by itself, produce differential memory or evaluation of the 2 scents. A spatial configuration of marks suggestive of an overlap was sufficient for the apparently overlapping scent to be remembered or valued more than the apparently underlying scent. Cues from the overlap of 2 hamsters' marks were also sufficient. These results, consistent with those previously found for responses to hamster vaginal scent over-marks, suggest that hamsters use similar cues to analyze scent over-marks that are different in chemical composition and in social functions.

PE2 cleavage mutants of Sindbis virus: correlation between viral infectivity and pH-dependent membrane fusion activation of the spike heterodimer.

The spike glycoprotein E2 of Sindbis virus (SIN) is synthesized in the infected cell as a PE2 precursor protein, which matures through cleavage by a cellular furin-like protease. Previous work has shown that SIN mutants impaired in PE2 cleavage are noninfectious on BHK-21 cells, the block in infection being localized at a step after virus-receptor interaction but prior to RNA replication. Here, we studied the membrane fusion properties of SIN PE2 cleavage mutants and observed that these viruses are impaired in their ability to form an E1 homotrimer and to fuse with liposomes at a mildly acidic pH. The block in spike rearrangement and fusion could be overridden by exposure of the mutant viruses to very low pH (<4.5). Cleavage mutants with second-site resuscitating mutations in PE2 were highly infectious for BHK-21 cells. The ability of these viruses to form E1 homotrimers and to fuse at a mildly acidic pH was completely restored despite a sustained lack of PE2 cleavage.

Immunopathogenesis and immune modulation of Venezuelan equine encephalitis virus-induced disease in the mouse.

The course of Venezuelan equine encephalitis (VEE) disease in immunodeficient and immunologically normal mice was compared to define the role of the immune system in this disease process. Immunocompetent mice infected with VEE exhibited a biphasic illness characterized by an early self-limiting lymphoid phase and a fatal CNS phase. The lymphoid phase of the illness was characterized by extensive viral replication within spleen, thymus, Peyer's patches, and lymph nodes, was accompanied by a high-titered serum viremia, and resolved with the production of VEE-specific IgM class antibody at 72 h postinfection (p.i.). Immunocompetent animals survived an average of 6.8 +/- 1.2 days before succumbing to fulminant encephalitis. In contrast, SCID mice infected with VEE showed a persistent replication of virus throughout all organs tested beginning at 24 h p.i. VEE-infected SCID mice exhibited a severe spongiform encephalopathy with 100% mortality and an average survival time of 8.9 +/- 0.9 days. These studies indicated that the characteristic organ tropism of VEE in the mouse is due in large part to an early anti-viral state, the establishment of which is dependent upon the presence of an intact immune system. Finally, the CNS pathology in a VEE-infected mouse had a significant immunologic component. However, in contrast to other neurovirulent alphaviruses, VEE was directly cytopathic for the cells of the CNS, even in the absence of an immune response.

Trophic factor secreting kidney cell lines: in vitro characterization and functional effects following transplantation in ischemic rats.

Several kidney cell lines were investigated for their ability to produce glial cell line-derived neurotrophic factor (GDNF). Cell line-conditioned medium was analyzed using ELISA and two cell lines were identified which produce GDNF in physiologically active concentrations. ELISA analyses revealed that conditioned medium from these two cell lines also contained PDGF, bFGF, TGFbeta1 and TGFbeta2. Both of these cell lines were then transplanted into the striatal penumbra of rats, 1 h following middle cerebral artery occlusion. Behavioral testing revealed that both cell lines reduced the deficit associated with cerebral ischemia and reduced the infarct volume relative to controls. Reduction of infarct volume was likely achieved by the action of GDNF and/or other growth factors produced by the cells.

Role of alpha/beta interferon in Venezuelan equine encephalitis virus pathogenesis: effect of an attenuating mutation in the 5' untranslated region.

Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas. In the adult mouse model, cDNA-derived, virulent V3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotide 3 (nt 3) of the 5' untranslated region (UTR) of the V3000 genome resulted in a virus (V3043) that was avirulent in mice. The mechanism of attenuation by the V3043 mutation was studied in vivo and in vitro. Kinetic studies of virus spread in adult mice following s.c. inoculation showed that V3043 replication was reduced in peripheral organs compared to that of V3000, titers in serum also were lower, and V3043 was cleared more rapidly from the periphery than V3000. Because clearance of V3043 from serum began 1 to 2 days prior to clearance of V3000, we examined the involvement of alpha/beta interferon (IFN-alpha/beta) activity in VEE pathogenesis. In IFN-alpha/betaR(-/-) mice, the course of the wild-type disease was extremely rapid, with all animals dying within 48 h (average survival time of 30 h compared to 7.7 days in the wild-type mice). The mutant V3043 was as virulent as the wild type (100% mortality, average survival time of 30 h). Virus titers in serum, peripheral organs, and the brain were similar in V3000- and V3043-infected IFN-alpha/betaR(-/-) mice at all time points up until the death of the animals. Consistent with the in vivo data, the mutant virus exhibited reduced growth in vitro in several cell types except in cells that lacked a functional IFN-alpha/beta pathway. In cells derived from IFN-alpha/betaR(-/-) mice, the mutant virus showed no growth disadvantage compared to the wild-type virus, suggesting that IFN-alpha/beta plays a major role in the attenuation of V3043 compared to V3000. There were no differences in the induction of IFN-alpha/beta between V3000 and V3043, but the mutant virus was more sensitive than V3000 to the antiviral actions of IFN-alpha/beta in two separate in vitro assays, suggesting that the increased sensitivity to IFN-alpha/beta plays a major role in the in vivo attenuation of V3043.

The vomeronasal organ is involved in discrimination of individual odors by males but not by females in golden hamsters.

The vomeronasal organ (VNO) has a wide variety of functions in terrestrial vertebrates, some of which involve responses to classical pheromones whereas others do not. We examined the role of the VNO in discrimination of individual differences in odors of male and female golden hamsters using a habituation paradigm. Removal of the VNO resulted in elimination of the ability of male hamsters to discriminate between some individually distinctive odors (e.g., flank gland secretion), but not others (e.g., urine). In females, such lesions had no effect. The type of test trial also influenced the results; in test trials employing a single, novel odor, removal of the VNO in males did have an effect but in test trials in which both the novel and the familiar odor were presented, VNO removal had no effect. It is concluded that (a) there is a sex difference in the role of the VNO in the discrimination of individual odors, (b) the role of the VNO in discrimination of individual odors varies from odor to odor, and (c) deficits due to VNO removal are more readily observed in more difficult tasks.

Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles.

RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). Open reading frame 5 (ORF5) encodes the G(L) protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114-128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in trans in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G(L), pVR21-M, and pVR100-G(L)/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G(L)/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G(L) protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies.

Mutations in the E2 glycoprotein of Venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbidity, and rapid clearance from blood of mice.

The arbovirus, Venezuelan equine encephalitis virus (VEE), causes disease in humans and equines during periodic outbreaks. A murine model, which closely mimics the encephalitic form of the disease, was used to study mechanisms of attenuation. Molecularly cloned VEE viruses were used: a virulent, epizootic, parental virus and eight site-specific glycoprotein mutants derived from the parental virus. Four of these mutants were selected in vitro for rapid binding and penetration, resulting in positive charge changes in the E2 glycoprotein from glutamic acid or threonine to lysine (N. L. Davis, N. Powell, G. F. Greenwald, L. V. Willis, B. J. Johnson, J. F. Smith, and R. E. Johnston, Virology 183, 20-31, 1991). Tissue culture adaptation also selected for the ability to bind heparan sulfate as evidenced by inhibition of plaque formation by heparin, decreased infectivity for CHO cells deficient for heparan sulfate, and tight binding to heparin-agarose beads. In contrast, the parental virus and three other mutants did not use heparan sulfate as a receptor. All eight mutants were partially or completely attenuated with respect to mortality in adult mice after a subcutaneous inoculation, and the five mutants that interacted with heparan sulfate in vitro had low morbidity (0-50%). These same five mutants were cleared rapidly from the blood after an intravenous inoculation. In contrast, the parental virus and the other three mutants were cleared very slowly. In summary, the five VEE viruses that contain tissue-culture-selected mutations interacted with cell surface heparan sulfate, and this interaction correlated with low morbidity and rapid clearance from the blood. We propose that one mechanism of attenuation is rapid viral clearance in vivo due to binding of the virus to ubiquitous heparan sulfate.

Image processing algorithms for digital mammography: a pictorial essay.

Digital mammography systems allow manipulation of fine differences in image contrast by means of image processing algorithms. Different display algorithms have advantages and disadvantages for the specific tasks required in breast imaging-diagnosis and screening. Manual intensity windowing can produce digital mammograms very similar to standard screen-film mammograms but is limited by its operator dependence. Histogram-based intensity windowing improves the conspicuity of the lesion edge, but there is loss of detail outside the dense parts of the image. Mixture-model intensity windowing enhances the visibility of lesion borders against the fatty background, but the mixed parenchymal densities abutting the lesion may be lost. Contrast-limited adaptive histogram equalization can also provide subtle edge information but might degrade performance in the screening setting by enhancing the visibility of nuisance information. Unsharp masking enhances the sharpness of the borders of mass lesions, but this algorithm may make even an indistinct mass appear more circumscribed. Peripheral equalization displays lesion details well and preserves the peripheral information in the surrounding breast, but there may be flattening of image contrast in the nonperipheral portions of the image. Trex processing allows visualization of both lesion detail and breast edge information but reduces image contrast.

Sindbis-group alphavirus replication in periosteum and endosteum of long bones in adult mice.

Several alphaviruses, including the Sindbis-group viruses, Ross River virus, O'nyong-nyong virus, and Chikungunya virus, are associated with outbreaks of acute and persistent arthralgia and arthritis in humans. Mechanisms underlying alphavirus-induced arthralgia and arthritis are not clearly understood, though direct viral replication within or around the affected joints is thought to contribute to disease. S.A.AR86 is a Sindbis-group alphavirus closely related to the arthralgia-associated Ockelbo and Girdwood S.A viruses. Following inoculation with S.A.AR86 derived from a molecular clone, infectious virus was isolated from bone and joint tissue 1 to 6 days postinfection. Studies using either in situ hybridization or S.A.AR86-derived double promoter viruses and replicons expressing green fluorescent protein localized sites of viral replication to the periosteum, tendons, and endosteum within the epiphyses of the long bones adjacent to articular joints. These results demonstrate that alphaviruses associated with arthralgia in humans replicate within bone-associated connective tissue adjacent to articular joints in an adult mouse model.

Radiologists' preferences for digital mammographic display. The International Digital Mammography Development Group.

PURPOSE: To determine the preferences of radiologists among eight different image processing algorithms applied to digital mammograms obtained for screening and diagnostic imaging tasks. MATERIALS AND METHODS: Twenty-eight images representing histologically proved masses or calcifications were obtained by using three clinically available digital mammographic units. Images were processed and printed on film by using manual intensity windowing, histogram-based intensity windowing, mixture model intensity windowing, peripheral equalization, multiscale image contrast amplification (MUSICA), contrast-limited adaptive histogram equalization, Trex processing, and unsharp masking. Twelve radiologists compared the processed digital images with screen-film mammograms obtained in the same patient for breast cancer screening and breast lesion diagnosis. RESULTS: For the screening task, screen-film mammograms were preferred to all digital presentations, but the acceptability of images processed with Trex and MUSICA algorithms were not significantly different. All printed digital images were preferred to screen-film radiographs in the diagnosis of masses; mammograms processed with unsharp masking were significantly preferred. For the diagnosis of calcifications, no processed digital mammogram was preferred to screen-film mammograms. CONCLUSION: When digital mammograms were preferred to screen-film mammograms, radiologists selected different digital processing algorithms for each of three mammographic reading tasks and for different lesion types. Soft-copy display will eventually allow radiologists to select among these options more easily.

Improved mucosal protection against Venezuelan equine encephalitis virus is induced by the molecularly defined, live-attenuated V3526 vaccine candidate.

The genetically engineered, live-attenuated Venezuelan equine encephalitis (VEE) virus vaccine candidate, V3526, was evaluated as a replacement for the TC-83 virus vaccine. Protection from lethal subcutaneous or aerosol challenge was evaluated in vaccinated mice clinically and immunohistochemically. Subcutaneous administration of V3526 induced systemic and mucosal protection more efficiently than did the TC-83 vaccine. The bronchial IgA responses induced in mice by subcutaneous administration of vaccines significantly corresponded to the ability to survive aerosol challenge with virulent virus. Furthermore, V3526 delivered by aerosol induced more complete mucosal protection than either vaccine administered subcutaneously. The ability of V3526 to induce protection in mice warrants its consideration for further testing as a potential vaccine candidate for human use.