Drosophila is a component of an expandable extrachromosomal domain of the nucleus. To better understand its function, we studied the dynamics and localization of GFP-tagged EAST. In live larval salivary glands, EAST-GFP is highly mobile and localizes to the extrachromosomal nucleoplasm. When these cells are permeabilized, EAST-GFP rapidly associated with polytene chromosomes. The affinity to chromatin increases and mobility decreases with decreasing salt concentration. Deleting the C-terminal residues 1535 to 2301 of EAST strongly reduces the affinity to polytene chromosomes. The bulk of EAST-GFP co-localizes with heterochromatin and is absent from transcriptionally active chromosomal regions. The predominantly chromosomal localization of EAST-GFP can be detected in non-detergent treated salivary glands of pupae as they undergo apoptosis, however not in earlier stages of development. Consistent with this chromosomal pattern of localization, genetic evidence indicates a role for EAST in the repression of gene expression, since a lethal east mutation is allelic to the viable mutation suppressor of white-spotted. We propose that EAST acts as an ion sensor that modulates gene expression in response to changing intracellular ion concentrations.The EAST protein of  Chro, Skeletor and east has been shown to lead to defects in mitosis and meiosis Chro affect the structure and organization of larval polytene chromosomes The nuclear matrix as an internal non-chromosomal scaffold of the cell nucleus remains a controversial concept in eukaryotic cell biology In this report, we describe that the predominantly extrachromosomal EAST protein has the ability to associate with chromosomes and that this behavior is linked to a negative role in gene regulation.rd instar larval salivary glands and performed fluorescence recovery after photo bleaching (FRAP). An extremely fast recovery demonstrated that EAST-GFP is highly mobile, suggesting that EAST does not associate with a static internal nucleoskeleton  skeleton . For simTo evaluate if EAST might associate with an ‘elusive’ structure termed the nuclear matrix, salivary glands expressing EAST-GFP were subjected to the first step of the Nuclear Matrix extraction protocol whereby cells are treated with detergent buffer. The permeabilization using saponin or Triton-X-100 results in a rapid re-localization of EAST-GFP onto polytene chromosomes . An incueasthop7 mutants .To investigate the relationship between transcription and the binding of EAST to chromosomes, we performed a run-on transcription assay by including BrUTP into detergent buffer. Transcriptionally active regions that incorporate high levels of BrUTP contain low levels of EAST-GFP , while D mutants . Loss of+ or K+ ions) in the detergent buffer. At 100 mM or lower concentrations, the bulk of EAST-GFP associates with polytene chromosomes  and C-terminally deleted EASTΔC-GFP (residues 1-1520) at 50 mM salt. The truncated version of EAST was able to associate with polytene chromosomes, yet the banding patterns was less pronounced . Moreoveeast was identical to that of a gene called suppressor of white-spotted or su(wsp) . The su(wsp) mutation suppresses the reduction of eye pigmentation caused by 4 spotted alleles of the white gene (wsp1-4), which are characterized by lesions in the upstream region of weast suppresses the wsp phenotype. Like the viable mutation su(wsp)1, the pupal lethal allele easthop7 restores eye pigmentation to almost wildtype levels (easthop7 and su(wsp)1 is indistinguishable from homozygous su(wsp)1 in terms of eye pigmentation (sp)su are allelic. To understand the molecular nature of the su(wsp)1 mutation, we sequenced the coding region of the genomic DNA of the east locus. At codon 1956, we found a point mutation that introduces a premature stop codon, reducing the length of EAST protein from 2301 to 1955 amino acids. In summary, as has already been inferred from the genetic interactions between spw and su(wsp), the product of the east/su(wsp) gene might act as a negative regulator of gene expression that binds to the upstream regulatory region of the w gene.We learned that the sequence of e levels . The traentation . Heterozsmid-GAL4 UAS-eastFL-GFP pupae contained two types of cells: small cells lacking EAST-GFP with fragmented and condensed chromosomes and larger cells maintaining high levels of EAST-GFP expression, in which polytene chromosomes appeared to have escaped from apoptotic fragmentation. As has been previously reported, cell death in salivary glands is preceded by changes in the levels and localization of filamentous actin and nuclear lamin The intra-nuclear localization of EAST-GFP depends on the developmental stage. The fusion protein shows a mainly extrachromosomal pattern throughout larval and prepupal development . During su(wsp)/east would inhibit white by interacting with its upstream regulatory sequences In this study we show that the nuclear EAST protein has the intrinsic ability to associate with chromosomes and that this behavior is linked to an involvement in gene regulation. A previous study postulated that the gene product of Because the characterization of EAST protein was carried out in permeabilized cells under non-physiological conditions it might be argued that the association with polytene chromosomes is an experimental artifact. However, we were able to observe co-localization with DNA in untreated cells while they undergo apoptosis. Moreover, despite permeabilization, cells were still able to perform transcription and replication, showing that chromatin binding of EAST did not occur under conditions that caused a general block of nuclear functions.The affinity to polytene chromosomes is sensitive to the salt concentration of the detergent buffer. We observed co-localization of the bulk of EAST-GFP with polytene chromosomes in permeabilized cells at concentrations at and below 100 mM salt, which is lower than the estimated physiological concentration of 150 mM. The release of EAST-GFP from chromosomes with increased salt concentrations (150 mM and above) is consistent with the observation that the fusion protein does not display a discernible chromosomal localization pattern in intact cells.su(wsp)1 allele, suggesting that this region is required to mediate the suppression of pigmentation seen in the wsp alleles. The C-terminus therefore might promote binding to chromatin or recruit other factors involved in repression. Although the N-terminal residues 1-1955 seem to be deficient in gene silencing, they are sufficient for viability.The C-terminal residues 1520 to 2301 promote affinity to chromatin but are dispensable for nuclear import. This domain is also involved in tethering EAST to a nuclear remnant during mitosis white transcripts do not differ between wildtype and the allele wsp despite the lesions in the promoter regions white transcription between su(wsp)1wsp1 and wsp1 adults, confirming the conclusion of the two earlier studies that altered white transcription does not appear to be responsible for the reduction in eye pigmentation . To test if sequence variations in white transcripts might explain the difference in pigmentation we sequenced cDNAs corresponding to white transcripts derived from wsp1 and su(wsp) wsp1 adults. However, we were unable to detect any evidence for genomic mutations or RNA editing.On the molecular level, the exact role of EAST in gene regulation remains a mystery. Two previous studies showed that the expression levels of east mutants Taken together, our results suggest that EAST could act as an ion sensor that fine-tunes the expression of target genes in response to fluctuating intra-cellular salt concentrations . Assumineast function leads to an acceleration of cell death in muscles, while overexpression of EAST-GFP in salivary glands attenuates apoptosis . These two opposite phenotypes could be explained by EAST counteracting the loss of K+ ions during apoptosis.In a previous study we proposed that the increase in nuclear volume in response to EAST overexpression was caused by an expansion of a non-chromosomal nucleoskeleton east alleles used have been previously described UAS-eastFL-GFP and UAS-eastΔC-GFP have been described ftz-GAL4 and smid-GAL4wsp1 and y2sc1su(wsp)1wsp1 were obtained from the Bloomington Drosophila stock center.The 2, 1 mM EGTA, 2 mM DTT, 50 µg/ml saponin, 20 µ/ml of 1 Roche EDTA free protease inhibitor cocktail dissolved in 1 ml water) that was based on the cytoskeleton extraction buffer Permeabilization of larval salivary glands was performed using detergent buffer  rd instar larval salivary glands was carried out as previously described Drosophila lamin Dm0 . Images of fluorescently labeled samples were acquired by laser scanning confocal miscroscopy using the Zeiss LSM 510 Meta or Zeiss LSM 510 upright microscopes. Image processing was carried out using Adobe Photoshop, Zeiss LSM 5 image browser and Metamorph (Molecular Devices).Antibody staining of whole-mount 3


In a recent study, the majority of three genomes of previously unknown positive-sense single-stranded (ss) RNA viruses were assembled from reverse-transcribed whole-genome shotgun libraries. The present contribution comparatively analyzes these genomes with respect to representative viruses from established viral taxa.RNA viruses have been isolated that infect marine organisms ranging from bacteria to whales, but little is known about the composition and population structure of the Picornavirales that fall into a well-supported clade of marine picorna-like viruses, the characterized members of which all infect marine protists. A temporal and geographic survey indicates that the JP genomes are persistent and widespread in British Columbia waters. The third genome, SOG, encodes a putative RNA-dependent RNA polymerase (RdRp) that is related to the RdRp of viruses in the family Tombusviridae, but the remaining SOG sequence has no significant similarity to any sequences in the NCBI database.Two of the genomes (JP-A and JP-B), appear to be polycistronic viruses in the proposed order Tombusviridae, SOG lacks a capsid and movement protein conserved within this family and SOG is thus likely more distantly related to the Tombusivridae than the RdRp phylogeney indicates.The complete genomes of these viruses permitted analyses that resulted in a more comprehensive comparison of these pathogens with established taxa. For example, in concordance with phylogenies based on the RdRp, our results support a close homology between JP-A and JP-B and RsRNAV. In contrast, although classification of the SOG genome based on the RdRp places SOG within the  RNA viruses of every classification have been isolated from the ocean; nevertheless, the marine RNA virus community remains largely uncharacterized. Although there are several examples of RNA viruses that infect marine animals  these orPicorna-like viruses are a "superfamily" of positive-sense single-stranded RNA (ssRNA) viruses that have similar genome features and several conserved protein domains . PreviouTombusviridae and the genus Umbravirus. Moreover, in both libraries, a high percentage of sequence fragments were part of only a few contiguous segments of sequence (contigs). Specifically, in the SOG sample 59% of the sequence fragments formed a single contig. Similarly, 66% of JP sequence fragments contributed to only four contigs that represented two viral genomes. Using a RT-PCR-based approach to increase the amount of sequence for each dominant contig resulted in the assembly of three complete viral genomes. This contribution analyzes these genomes from three previously unknown marine RNA viruses and investigates their similarities and differences with respect to representative genotypes from established viral taxa.In a recent study, reverse-transcribed whole-genome shotgun libraries were used to characterize two marine RNA virus communities . PositivThe two assembled genomes (JP-A and JP-B) from the Jericho Pier sampling site Figure  are singThe JP-A genome is positive-sense, 9212 nt in length with a 632 nt 5' untranslated region (UTR) followed by 2 predicted open reading frames (ORFs) of 5067 nt  and 3044 nt  separated by an intergenic region (IGR) of 149 nt Figure . ORF 2 iE value < 0.001) to nonstructural protein motifs of several viruses, including members of the families Dicistroviridae (Drosophila C virus), Marnaviridae (HaRNAV), Comoviridae (Cowpea mosaic virus) and the unassigned genus Iflavirus (Kakugo virus). The top matches for ORF 1 were to RsRNAV , a newly sequenced, unclassified positive-sense ssRNA virus that infects the widely distributed diatom Rhizosolenia setigera  and Drosophila C virus , a positive-sense ssRNA virus that infects fruit flies. Comparison of the protein sequence predicted to be encoded by ORF 2 of JP-A to known viral sequences shows that it has significant similarities to the structural proteins of viruses from the families Dicistroviridae (Drosophila C virus), Marnaviridae (HaRNAV), and the genus Iflavirus (Varroa destructor virus 1). The sequences that are most similar to ORF 2 of JP-A were the structural protein regions of RsRNAV , HaRNAV  and SssRNAV .Comparison to known viral sequences shows that the protein sequence predicted to be encoded by ORF 1 of JP-A contains conserved sequence motifs characteristic of a type III viral Helicase (aa residues 430 to 545), a 3C-like cysteine protease (aa residues 1077 to 1103) and a type I RdRp (aa residues 1350 to 1591)  searchesThe JP-B RNA genome is also likely from a positive-sense ssRNA virus. The 8839 nt genome consists of a 5' UTR of 774 nt followed by two predicted ORFs of 4842 nt  and 2589 nt  separated by an IGR of 298 nt (nt position 5617 to 5913) Figure . The 3' E value < 0.001) to nonstructural genes from positive-sense ssRNA viruses from a variety of families, including the Comoviridae (Peach rosette mosaic virus), Dicistroviridae (Taura syndrome virus), Marnaviridae (HaRNAV), Sequiviridae  and Picornaviridae . The top scoring sequences  were to a RdRp sequence from RsRNAV and a partial picorna-like virus RdRp from an unidentified virus  amplified from the same JP station during an earlier study  and HaRNAV  and SssRNAV [E value = 1 × 10-40. identities = 156/588 (26%)].The position of core sequence motifs conserved among positive-sense ssRNA viruses and BLAST searches of the NCBI database with the translated JP-B genome suggest that nonstructural proteins are encoded by ORF1, and the structural proteins are encoded by ORF2. We identified conserved sequence motifs in ORF 1 characteristic of a type III viral Helicase (aa residues 328 to 441), a 3C-like cysteine protease (aa residues 882 to 909) and a type I RdRp (aa residues 1143 to 1408)  searchesDicistroviridae. Several of these viruses contain internal ribosome entry sites (IRES) . This virus belongs to the genus Necrovirus in the family Tombusviridae that has a host range restricted to higher plants , a member of the unclassified genus Umbravirus whose known members infect only flowering plants [We identified the eight conserved motifs of the RdRp  in the Sr plants . SOG is g plants .Tombusviridae and genus Umbravirus, the remaining SOG sequence has no detectable similarity to any other known sequence. A Bayesian maximum likelihood tree based on alignments of the SOG RdRp with the available Umbravirus sequences and representative members of the Tombusviridae indicates that the SOG genome forms a well supported clade  with the single member of the genus Avenavirus, OCSV . The novelty of JP-A, JP-B and SOG, as revealed by sequence analyses and genome characterization, suggests that most of the diversity in the marine RNA virus community remains uncharacterized. Furthermore, these results raise the hypothesis that the genomes of these marine RNA viruses that we propose to infect single-celled eukaryotes may be more similar to the ancestral RNA viruses that gave rise to those that infect higher organisms.The shotgun libraries were constructed from seawater samples collected from two stations, JP (Jericho Pier), a site in English Bay adjacent to the city of Vancouver, British Columbia and SOG (Strait of Georgia), located in the central Strait of Georgia next to Powell River, B.C. Figure .The locations of the stations where one or both of the JP genomes were detected are shown in Figure Concentrated virus communities were produced as described by Suttle et al. . Twenty E values were < 0.001. The details for viruses used in phylogenetic analyses are listed in additional file A detailed description of the whole genome shotgun library construction protocol can be found in Culley et al. . BrieflyThe 5' and 3' ends of the environmental viral genomes were cloned using the 5' and 3' RACE systems (Invitrogen) according to manufacturer's instructions. The 3' RACE with the SOG genome required the addition of a poly (A) tract with poly (A) polymerase (Invitrogen) according to manufacturer directions before cDNA synthesis. cDNA was synthesized directly from extracted viral RNA from the appropriate library. Three clones of each 5' and 3' end were sequenced.Taq DNA polymerase (Invitrogen) in 1× Platinum Taq buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, and 0.2 μM of each primer  with primers JP-A 5 and 6 and JP-B 6 and 7  declare that they have no competing interests.AC contributed to the design of the study, performed the lab work, analyzed the data and drafted the manuscript. AL contributed to the design of the study, analyzed the data and helped prepare the manuscript. CS was involved in the conceptualization and design of the research and in manuscript preparation. AC, AL and CS have read and approved this manuscript.PCR primers used to complete the three genome sequences. The table provides detailed information about the primers used to complete the three viral genome sequences.Click here for fileVirus sequence details. Organized by taxonomic group, the table provides the full name, acronym and NCBI accession number for the viruses used in phylogenetic analyses.Click here for file


MPM) has been tested for its anti-inflammatory activity. Preliminary in vitro results have already indicated that MPM could exert such an anti-inflammatory activity.Over the last 10 years, whey proteins have received considerable attention in the area of functional foods and nutraceuticals. In this paper, a novel fermented whey protein-based product described as a gel-like Malleable Protein Matrix (The systemic anti-inflammatory activity of the MPM was explored using the oxazolone-induced atopic contact dermatitis mouse model (ACD). Parameters including ear thickness, side effects as well as neutrophil extravasation were monitored.In the ACD model, the MPM exhibited an anti-inflammatory effect comparable to that of hydrocortisone (positive control). Mice fed with MPM showed strong reduction of the ear inflammation while no side effects, as compared to hydrocortisone, were observed. The MPM seemed to reduce neutrophil extravasation in tissue as evidenced by blood polymorphonuclear cells and ear myeloperoxidase content.The anti-inflammatory activity demonstrated in the ACD model suggests that the mechanism of action of the MPM is different than that of hydrocortisone and could become a relevant product for people suffering from dermatological manifestations associated with immune dysfunctions such as allergies, eczema, dermatitis, and autoimmune diseases. Modern life-styles which leads to obesity, stress and inactivity, is a major cause of immunological diseases, particularly those associated with chronic inflammation which are on the upswing during the last decade -3. Many Lactoferrin (LF), a minor whey protein, has been extensively studied. LF assists the phagocytosis process in neutrophils, increases production of interleukin-8 (IL-8)  and stimLactobacillus commonly used in many fermented dairy products [lactobacilli act on Peyer's patches to stimulate IgA production, phagocytosis process and possess anti-inflammatory and anti-allergic activities by reducing the production of cytokines and immunoglobulin E (IgE) [lactobacilli are able to increase Th1 profile while others increase Th2 profile [lactobacilli could act both as immunostimulating and anti-inflammatory agents. Some studies also indicate that the effects of probiotics acting in synergy with food ingredients can be more intense than the probiotics alone [In addition, some Lactic Acid Bacteria (LAB) have shown immunomodulatory and anti-inflammatory activities. The genus products  is the mproducts . The eff E (IgE) -27. Cyto profile . These rcs alone . Moreovecs alone -32.lactobacilli, a novel fermented whey protein-based ingredient, called Malleable Protein Matrix (MPM) [in vitro that MPM reduces pro-inflammatory cytokines and inhibits the cytokines production following LPS stimulation on CaCo2 cells [Considering the positive effects on the immune system of both whey proteins and probiotic ix (MPM) , was tesix (MPM) . It was ix (MPM) ,34. On tix (MPM) . Moreoveo2 cells . These rLactobacillus casei [The objective of this present study was to evaluate the systemic anti-inflammatory potential of MPM and to determine how its complex composition may lead to synergistic effects. For this purpose, the oxazolone-induced atopic contact dermatitis mouse model (ACD) was used. This ACD mouse model requires two distinct phases . First, us casei .. Briefly, the MPM is obtained by a protein specific recuperation procedure following the fermentation of sweet whey by a proprietary Lactobacillus kefiranofaciens strain (R2C2) isolated from kefir grains and adapted to grow in whey [ and was diluted in deionized double-distilled water to a final concentration of 10 mg/mL. For the mouse ACD model, the 4-ethoxy-methylene-2-phenyloxazol-5-one (oxazolone) (Sigma-Aldrich Canada) was required at a concentration of 5% (w/v) in acetone to cause inflammation.The Malleable Protein Matrix (MPM) was obtained from Technologie Biolactis inc.  in whey . The com and were used at 20 days of age for studies in the mouse ACD model. The animals were housed in filter top isolator cages in a room kept at 20–23°C with humidity maintained between 35–45% with a 12-hour light-dark cycle and free access to a standard laboratory pelleted Rodent Lab Diet 5001 . The experimental protocols used were approved by the Animal Care Committee of the INRS- Institut Armand-Frappier (Comité Institutionnel des Soins aux Animaux et de leur Utilisation (CISAU)) and were performed in accordance with the recommendations of the Canadian Council on Animal Care as specified in the Guide to the Care and Use of Experimental Animals (CISAU # 0306-01 and # 0410-01).CD-1 female mice were obtained from Charles River Laboratories al. [.After a week adaptation in the animal facility, the mice were separated in groups of 10 animals. The grouping was randomized according to the weight of the rodents. The murine model of ACD was based on those firstly described by Garrigue et al.  and modiThe dose-response curve has been done in the prophylactic anti-inflammatory mouse ACD model. Groups of 10 CD-1 mice received each day by gavages (per os (p.o)), 100 μL of reconstituted lyophilized MPM at three doses 20% (w/v), 10% (w/v) and 5% (w/v), 100 μL of water or 100 μL of water-soluble hydrocortisone (10 mg/mL). The mouse ACD was performed as described previously and ear thickness was measured every day.The prophylactic anti-inflammatory potential of MPM was evaluated by the administration of MPM seven days prior to sensitization. Groups of 10 CD-1 mice received each day by gavages (per os (p.o)), 100 μL of reconstituted lyophilized MPM, 100 μL of water or 100 μL of water-soluble hydrocortisone (10 mg/mL). The mouse ACD was performed as described previously and ear thickness was measured every day. The mice's weight was measured twice a week. The spleen's weight was measured at the end of the protocol and was normalized in accordance to each mouse's weight.The therapeutic anti-inflammatory potential of MPM was evaluated by the administration of MPM, soluble hydrocortisone or water as in the prophylactic protocol, but only after the first challenge. The other parameters were followed as described. in accordance with manufacturer's instructions. The cell counts were obtained by passage of 20 μL of preparation in a Flow Cytometry Epics XL cytometer . The lymphocytes, monocytes and polymorphonuclears (PMN) were separated in accordance with cell size and cell granulometry.At the end of the prophylactic protocol of mouse ACD, the blood of each mouse was taken and white blood cell counts evaluated by flow cytometry. Briefly, the red blood cells were lysed with Optilyse C Sigma-Aldrich, Oakville, On) were mixed with 2.9 mL of 50 mM phosphate potassium buffer containing 0.117 mg/mL of o-dianisidine  and 0.0005% hydrogen peroxide. The oxydation of o-dianisidine kinetic was followed at 460 nm with a spectrophotometer Varian Cary 300  during 5 min at 25°C.The method for the evaluation of MPO content was adapted from those developed by Bradley et al.  and Xia The inflammatory mouse ACD experiments were performed with groups counting 10 mice/group and two independent experiments. The statistical analysis of data was performed by the biostatistical service of INRS-Institut Armand-Frappier. Statistical analysis used was a repeated measure one-way ANOVA test that permits the comparison between groups during the entire experiment independently of each day. When the ANOVA test was not possible because of interactions between groups, a Student test was run for comparison of groups at each day.MPM is a whey-fermented product, which by its composition, has a high potential as an anti-inflammatory agent. The oxazolone-induced atopic contact dermatitis (ACD) model was used for the demonstration of MPM's effect on inflammatory diseases. Figure The consumption of hydrocortisone is associated with a negative effect on mice growth which is clearly demonstrated by the cessation of growth in the mice who received hydrocortisone Figure . The MPMThe polymorphonuclear (PMN) cell counts were higher in MPM and hydrocortisone fed groups compared to the water control group Figure . The bloin vitro on CaCo2 cells stimulated with LPS [MPM contains a variety of ingredients including whey proteins and peptides, LAB and their related exopolysaccharides, group B vitamins and calcium Table . All thewith LPS  suggestiThe anti-inflammatory potential of MPM has been confirmed in these studies with the murine ACD model. This model of inflammation has proven to be a sensitive and useful tool to determine efficacy and potency of several anti-inflammatory and immunosuppressive drugs used in dermatological disorders such as dermatitis and psoriasis. Glucocorticoids, such as hydrocortisone, are commonly used to relieve skin and joint inflammation and have been used as a positive control group in these experiments . This mop.o. either in a prophylactic  in circulation as well as a reduced MPO content in ear Figures  and 5. RIt is previously demonstrated that MPM enhances some cytokines, blood PMN cells and glutathione production by leukocytes  indicatiMPM possesses a strong anti-inflammatory effect comparable to hydrocortisone when examined in the ACD model. The anti-inflammatory effects of consumption of MPM occur without the undesirable side effects normally associated with hydrocortisone. Therefore, MPM would be an alternative of choice for children and young adult suffering from chronic inflammatory of various diseases such as ACD. The consumption of the MPM could act as a preventive or a therapeutic nutraceutical in the case of inflammatory diseases like atopic dermatitis or related diseases such as, psoriasis. Psoriasis is a chronic inflammatory disease with similar effects on the immune system to that observed for ACD.Technologie Biolactis (TB) was the industrial sponsor of a Natural Science and Engineering Research Council of Canada (NSERC) grant obtained by INRS (CD). Collaborative research conventions and agreements intervened between TB, INRS and NSERC. INRS is a minor shareholder of TB (less than 1%) and does not have any vote. The findings of the present study are covered by a patent application (PCT CA2002/001899). JB was an on-site scholar of Fond de Recherche en Santé du Québec (FRSQ) and part of the scholarship was covered by TB.JB design the animal studies, carried out the animal and other experiments, perform the statistical analysis and drafted the manuscript. CD participated in the design of animal studies, data interpretation and the statistical analysis. CD revised the manuscript for the intellectual content and language. PL participated in the design of animal studies, data interpretation and revised the manuscript for the intellectual content and language. All authors read and approved the final manuscript.


Although the human β-defensins hBDs today seem to have diverse functional activities in innate antimicrobial immunity, a few reports also indicated an altered expression of these antimicrobial peptides (AMPs) in tissues of cancers such as oral squamous cell carcinoma. The present work was aimed on the study of hBD gene expression in basal cell carcinoma (BCC) which is the most common cancer in humans.Twenty-two non-ulcerated BCCs  have been analysed for the presence of hBD (1–3) mRNA by quantitative real-time RT-PCR. As controls, non-lesional skin specimens of BCC patients as well as samples of healthy subjects were assessed by RT-PCR.hBD-1 levels in healthy controls and non-lesional skin of BCC patients were significantly (P < 0.05) higher than the levels observed in tumour tissue. Moreover, BCCs showed significantly (P < 0.05) increased mRNA expression of hBD-2 as compared to controls. There was no significant (P > 0.05) difference between lesional mRNA levels for hBD-3 and those levels observed in controls. The mRNA expression of hBDs (1–3) found in nodular and superficial BCCs did not significantly (P > 0.05) differ.The gene expression patterns of hBD-1 and hBD-2 are for the first time shown to be significantly altered in non-ulcerated BCCs as compared to intra-individual and inter-individual controls, respectively. The present findings may indicate that beside the antimicrobial activity of AMPs, hBDs may also play a role in the pathogenesis of BCC. However, functional and immunohistological studies investigating hBDs in patients with BCC are needed to confirm our data. Basal cell carcinoma (BCC) is the most common cancer in humans. It is classified, together with squamous cell carcinoma, as non-melanoma skin cancer. The incidence of BCC is increasing worldwide by up to 10% a year. Although mortality is low as BCC rarely metastasises, this malignancy causes considerable morbidity and places a huge burden on healthcare services worldwide. Three main types of BCCs are generally distinguished with regard to the histopathological growth pattern: nodular, superficial, and morphoeic. BCCs are believed to derive from the epidermis, specifically the basal cell layer and the outer root sheath of the hair follicle. The development of BCC is clearly associated with mutated p53 tumour-suppressor gene and constitutive activation of sonic hedgehog signalling molecules that regulate cell proliferation and cell fate determination. Risk factors for the development of BCC include advanced age, fair skin colour, chronic ultraviolet exposure, and immunosuppression .BCCs are 10 times more common in persons who have undergone a solid organ transplant and herpes-virus like DNA sequences have been found in these tumours. The possibility that certain types of human papilloma virus, either alone or in conjunction with ultraviolet radiation, may play a role in the pathogenesis of particular cancers is suggested by several lines of evidence . The humIn this prospective pilot study, 28 patients , who were suspected of having non-ulcerated BCC on the basis of clinical and dermoscopic features, underwent complete tumour excision in our dermatologic surgery. Three millimetre punch biopsies were harvested from the centre of the tumour  as well as an adjacent healthy skin site approximately 1 cm in distance to the tumour border . In addition to the BCC patient group, we included 27 subjects  who underwent cosmetic surgery . Punch biopsies were taken from the centre of excised skin tissue that was otherwise rejected. We have not controlled age and gender for the healthy subjects, since these parameters do not significantly influence AMP expression. However, we sought to obtain skin specimens from facial as well as body skin in controls to exclude any bias that may arise from anatomic variations of AMP expression [® Lipid Tissue Kit  following the manufacturer's protocol. Prior to cDNA synthesis RNA was digested with RNase-free DNase I . cDNA was synthesized by reverse transcription from DNase I treated RNA using MultiScribe™ reverse transcriptase enzyme and random hexamers primers . Real-time PCR was performed using a Taqman SYBR Green PCR Master Mix and GeneAmp® Sequence Detection System (Applied Biosystems). PCR Primers for AMPs and the Housekeeping Gene (GAPDH) were designed using the computer program Primer Express (PE Biosystems) and produced by the custom oligonucleotide synthesis service TIB Molbiol (Germany). The primers for hBDs (1–3) and GAPDH are given in Table t method was used as previously suggested by Livak and Schmittgen [t values (logarithms) were re-transformed.Quantitative analysis of real-time RT-PCR was performed as previously suggested : total cfor hBDs –3 and GAfor hBDs –3 and GAhmittgen . A standData analysis was performed using the statistical package MedCalc Software . Non-normal distribution of data was confirmed by the D'Agostino-Pearson test. Hence, data were expressed in medians and lowest and highest values (range). The results were analysed using paired or independent non-parametric tests including the Wilcoxon-rank test and the Mann-Whitney test. We constrained experiment-wise error rates due to multiple comparisons to the standard alpha (P) level of < 0.05 by the Bonferroni method.Fifteen of the BCCs investigated were localised on the face and 13 on the trunk and lower extremities. Histological examination revealed 18 nodular BCCs and 10 superficial BCCs. Six of the nodular BCCs on the face showed ulceration on histology and were therefore excluded from further evaluation. In the control group, we harvested 11 biopsies from the face and 16 on the trunk and lower extremities. The medians and range of mRNA expression of AMPs in BCC patients n = 22) and healthy controls (n = 27) are shown in Fig. 2 and heaCurrent information on AMP structure, expression, and biologic activity has established a firm foundation for further study of the impact that these peptides might have in human disease. For example, human α-defensin (hAD) expression has previously been linked to different types of tumours and cell lines. hAD-1 has been detected in lung tumours and in the submandibular glands of patients with oral carcinomas. By RT-PCR, mass spectrometry and flow cytometric analysis, hADs (1–3) have been shown to be expressed by cell lines deriving from renal cell carcinomas and the expression of a specific hAD precursor peptide has been shown to be upregulated in human leukemic cells. It has been suggested that hADs could belong to tumour substrates that modulate malignant cell growth and enhance immune escape in tumours -14. MoreWe have shown that mRNA expression of hBDs insignificantly differ between non-lesional skin of BCC patients and healthy controls indicating that there exists no constitutional difference in hBD gene expression. Nevertheless, we observed significantly altered expression of hBD-1 and hBD-2 in BCCs as compared to controls. The alterations observed in BCCs were however independent from tumour subtypes. The hBD gene expression patterns observed in BCCs do not appear to be substantially different from those seen in inflammatory conditions such as atopic dermatitis and psoriasis ,15,16. HThe virtual absence of hBD-2 in controls observed in the present study, gives support to previous reports that this AMP is not normally produced but are upregulated under pathological conditions such as inflammation  and malignancies . We have demonstrated that hBD-2 levels are significantly upregulated in BCC. Although we sought to include into the study only BCC without evidence of ulceration, we cannot fully exclude that impairment of skin barrier functions associated with an increased invasion of microbes, contributed to the upregulation of hBD-2 in tumour tissue investigated. Whereas impairment of skin barrier functions are common in BCC, it has been demonstrated that germ colonization of BCC lesions is usually indifferent from normal skin ,19. YoshThe gene expression patterns of hBD-1 and hBD-2 are for the first time shown to be significantly altered in non-ulcerated BCCs as compared to intra-individual and inter-individual controls, respectively. The heterogeneity of hBD expression in BCC indicates that beside the antimicrobial activity of AMPs, hBDs have other unique functions that may also play a role in the pathogenesis of BCC. The altered expression of hBDs by transformed keratinocytes may impair host immune recognition and control of cellular proliferation and/or differentiation ,21. The BCC: basal cell carcinoma; AMP: antimicrobial peptide; hBD: human β-defensin; hAD: human α-defensin.The author(s) declare that they have no competing interests.TG drafted the manuscript, participated in the design of the study, and performed the statistical analysis and interpretation of data. MS carried out the molecular genetic studies and participated in the sequence alignment. JH, FGB and MS obtained the biological samples. AK conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


High-degree microsatellite instability (MSI-H) is a feature of approximately 15% of sporadic colorectal cancers. Patients with MSI-H cancers have been reported to have a better prognosis than those with non-MSI-H cancers. The MSI-H subset is also characterised by a dense infiltrate of intra-epithelial lymphocytes and the hypothesis that the latter represents an efficacious immune response contributing to improved outcome is very attractive.Data for this review were identified by searches of MEDLINE, PubMed, and cross references from relevant articles using the search terms 'microsatellite instability', 'colorectal cancer' and 'immunology', 'immune response' or 'immunogenicity'.A total of 38 articles were identified by the search criteria and a further 95 articles by cross-referencing. The relevance of the articles to be interviewed was established by hand searching. Out of a total of 133 articles identified, 47 articles were rejected due to lack of relevance. A total of 86 articles were included in the review, pertaining to microsatellite instability in colorectal cancer, and immune mechanisms in colorectal cancer.It is suggested that this distinct group of colorectal cancers may have inherent immunogenic properties and that further elucidation of these may be invaluable to the development of successful immunotherapy. Instead epigenetic modulation of MMR genes, particularly hMLH1, such as hypermethylation of the promoter region leads to silencing and MMR deficiency ,18. HypeAll colorectal cancers display some degree of microsatellite instability if enough markers are studied . In 1997p53 mutation. A number of human genes implicated in pathways regulating growth control and apoptosis, for example IGF-IIR, TGFβ-RII and BAX, contain microsatellite repeats within the coding regions of their transcribed sequences. More recently the BRAF gene has been noted to be commonly mutated in sporadic MSI-H, as well as in serrated polyps, suggesting that serrated polyps are likely precursors of these cancers . Succesin vitro conditions. The same group have also identified MHC class-II restricted epitopes by induction of helper (CD4+) T cells from the peripheral blood mononuclear cells of normal volunteers and MSI-H colorectal cancer patients [Studies on abnormal peptides characteristic of MSI-H have demonstrated their potential for immune recognition. A peptide generated by frameshift mutations in TGFβRII, characteristic of the mutator phenotype, has been used to pulse dendritic cells, which in turn induced activated cytotoxic (CD8+) T cells . One cytpatients . They alpatients . They alin vivo due to the truncation of β2-microglobulin that is characteristic of these tumours [2-microglobulin protein is an essential co-factor for MHC Class I and its defective function would render effective presentation of antigens impossible. However, whilst this defect negates antigen presentation by the tumour itself there is now considerable evidence that the direct presentation of antigen by a tumour may be relatively unimportant. Instead, the concept of "cross-priming" in which APC's such as dendritic cells, and to a lesser extent macrophages, pick up antigens released by dead tumour cells and subsequently present them to T cells may be more important in vivo [2-microglobulin mutations may be of relatively little importance. Instead a pool of abnormal peptides generated by the mutator phenotype may be recognised and harvested as TSA's by immature dendritic cells. On acquisition of non-self antigens these dendritic cells mature into professional APC's, with a natural propensity to appropriately present antigen and co-stimulate T lymphocytes [Tumour specific antigens must be appropriately presented to the host immune system if a specific immune response is to occur. This normally requires the presentation of antigen in conjunction with the appropriate Major Histocompatibility Complex (MHC) molecules that interact with the T cell receptor (TCR): Class I MHC for presentation to cytotoxic (CD8+) T cells and Class II MHC for presentation to helper (CD4+) T cells . It has  tumours . The β2- in vivo ,50. Thusin vitro studies of tumour infiltrating lymphocytes in MSI-H colorectal cancer, mentioned earlier, that demonstrated HLA Class II restricted T helper cell activity and the presence of dendritic cells [Antigen presenting cells may obviate the need for direct presentation by tumour cells and cross-priming may involve the use of HLA Class II machinery. Immunohistochemical studies have demonstrated that HLA Class II expression is increased in MSI-H colorectal cancer compared to MSS cancers, although this was not associated with improved survival . Additioic cells ,45. EithIt has recently become evident that appropriate co-stimulation is pivotal to effective antigen presentation . Co-stimFor many years the presence of a pronounced infiltrate of lymphocytes in colorectal cancer has been associated with improved prognosis ,56 but cIn a study specific to MSI-H colorectal cancer, Dolcetti and colleagues demonstrated increased immunostaining for IEL's when compared to MSS cancers . These lEβ7+) lymphocytes in MSI-H colorectal cancer compared to MSS cancers [One immunohistochemical study, involving relatively small numbers and two groups that differed considerably in size (n = 17 v n = 7), has noted the increased presence of CD8+ CD103+  T lymphocyte subsets and local cytokine profiles. Immune responses are promoted by the Th1 subset of CD4+ lymphocytes and their associated cytokines [Interferon (IFN)-γ, Tumour necrosis factor (TNF)-α, Interleukin (IL)-2 and IL-12). By contrast the Th2 subset of CD4+ lymphocytes and cytokines such as IL-10 promote tolerance to unrecognised antigens and prevent successful antigen-specific responses. A microarray analysis performed in our laboratory has demonstrated the upregulation of several pro-inflammatory cytokines in MSI-H colorectal cancer . As wellThis microarray analysis also demonstrated that the heat shock protein family is up-regulated in MSI-H colorectal cancer . This isOur microarray findings have been validated using quantitative real-time RT-PCR across several genes of interest and demonstrate clear differences in the tumour biology of MSI-H and MSS cancers at the mRNA level.To counteract immune surveillance solid cancers may employ mechanisms to evade the immune system. Several possible methods of immune escape have been identified in colorectal cancer, such as the down-regulation of MHC proteins and co-stimulatory molecules ,71. The in vitro studies of lymphocytes infiltrating colorectal cancer have failed to demonstrate specific anti-tumour activity even though analysis of their receptor usage strongly suggested antigen-specificity [Alternative mechanisms of immune escape identified in colorectal cancer include the release of mediators that suppress the function of tumour infiltrating lymphocytes: so-called "tumour counter-attack". In the past cificity ,73. Thescificity . Fas-L rcificity . The autDolcetti and colleagues showed that MSI-H colorectal cancers stained significantly more heavily for markers of apoptotic cell death . SubsequAn alternative explanation for the increased apoptosis observed in MSI-H colorectal cancer has been the theory of "effete malignancy". This concept proposes that the mutator phenotype affects peptides or pathways involved in cell processes fundamental to life. Thus, the accumulation of mutations adversely influences the tumour cells' viability and hence, leads to apoptotic cell death . The antThe apoptosis observed in these cancers, whether induced by effete malignancy or an immune response, is of significance for another reason. The death of tumour cells releases intra-cellular proteins into the tumour micro-environment. This would be a vital step in the promotion of TSA release, recognition and presentation to the host T lymphocytes. It might also release heat shock proteins, upregulated in MSI-H colorectal canecr, which may promote both innate and antigen-specific arms of the immune response. The importance of dendritic cells in immune responses has already been mentioned  and immuClearly, the mutator phenotype may influence the function of several cellular processes and these may include those that are designed to nullify the host response. Equally it has been suggested that it is the effect of microsatellite instability on other cellular processes, unrelated to tumour immunology, which leads to the survival benefit observed in MSI-H colorectal cancer. Effete malignancy may, for instance, limit a tumour's ability grow and survive . AlternaThere is now an accumulation of evidence that promotes the argument that MSI-H colorectal cancer is subject to a marked, possibly antigen-specific, immune response. We acknowledge that this may not be the sole determinant of improved prognosis although it seems likely to be a major influence. We believe that MSI-H may be a natural paradigm of host-tumour interactions in immunogenic colorectal cancers and as such further studies are required to clarify the nature of immune responses in these tumours. The use of heat shock proteins and abnormal peptides, derived from frameshift mutations in coding microsatellites, already hold promise in advancing the field of immunotherapy ,86. UltiThe author(s) declare that they have no competing interests.AB searched and evaluated the literature and drafted the manuscript.SAB and SD revised the manuscript.All authors read and approved the final manuscript.


The central issue for Genetic Analysis Workshop 14 (GAW14) is the question, which is the better strategy for linkage analysis, the use of single-nucleotide polymorphisms (SNPs) or microsatellite markers? To answer this question we analyzed the simulated data using Duffy's SIB-PAIR program, which can incorporate parental genotypes, and our identity-by-state – identity-by-descent (IBS-IBD) transformation method of affected sib-pair linkage analysis which uses the matrix transformation between IBS and IBD. The advantages of our method are as follows: the assumption of Hardy-Weinberg equilibrium is not necessary; the parental genotype information maybe all unknown; both IBS and its related IBD transformation can be used in the linkage analysis; the determinant of the IBS-IBD transformation matrix provides a quantitative measure of the quality of the marker in linkage analysis. With the originally distributed simulated data, we found that 1) for microsatellite markers there are virtually no differences in types I and II error rates when parental genotypes were or were not used; 2) on average, a microsatellite marker has more power than a SNP marker does in linkage detection; 3) if parental genotype information is used, SNP markers show lower type I error rates than microsatellite markers; and 4) if parental genotypes are not available, SNP markers show considerable variation in type I error rates for different methods. A key issue in nonparametric linkage analysis is the accuracy in the estimation of the relative pair identity-by-descent (IBD) distributions. The Genetic Analysis Workshop 14 (GAW14) simulated data provide an opportunity to evaluate new or existing methods for linkage analysis since the "answers" were known to the designers of the simulated data. We applied two types of methods to find the locations of linkage and determine the power and type I errors for single-nucleotide polymorphism (SNP) and microsatellite markers according to whether or not parental genotypes are available. The first method is the affected pedigree member (APM) method implemented in Duffy's SIB-PAIR program . The secWe applied two types of methods to determine the performance (power and type I errors) for SNP and microsatellite markers with different data assumptions based on the availability of parental genotypes. The first method is the APM method implemented in Duffy's SIB-PAIR program, which was fully documented in . The secPij (M) = Pji(M) = 1/2 of the sum of frequencies for the genotypes ai/aj and aj/ai in the parental generation with ai being the alleles over the marker. Then in a full sib pair population without gender differences, the IBS and IBD probabilities are related byAssume that 1) parental mating is random; 2) in the parental population, for any genotype the two possible phase known genotypes have the same probability; 3) for each mating type that produces a sib-pair with IBD = 0, the two possible sib pairs have an equal probability to come; if the IBD = 1, the shared IBD allele has an equal probability to come from each one of the two parents. Let T = [Tij] with Tij = p(IBS = i|IBD = j) 0 ≤ j ≤ i ≤ 2 is given bywhere the transformation matrix T11 = Het(M)T21 = Hom(M)Hom(M) and Hom(M2) are the sums of all diagonal elements for the matrix [Pij(M)] and [Pij(M)]2, respectively, Het(M) and Het(M2) are the sums of all off-diagonal elements for the matrix [Pij(M)] and [Pij(M)]2, respectively, and Pi(M) is the frequency for the ith allele ai, . The above formula reduces into Lange's [Pij(M) = Pi(M)Pj(M). Our formula can transform the IBD distribution to that of IBS by the transformation matrix T or vice-versa through the inverse transformation matrix T-1. With the estimates for IBD or the IBS probabilities, the statistics for nonparametric linkage analysis can be calculated and tested in the usual manner. and HomM are the We performed all analyses without knowledge of the "answers." We still do not have the "answers," except those results appearing in the meeting abstracts.Figure To see the type I error rate, we listed the median of the numbers of significant replicates over all the markers, which could be viewed as an average type I error rate and graphically could be interpreted as the "noise level." Table Based on our single-point linkage analysis, we observed the following results with respect to the comparison of SNP vs. Microsatellite markers (one SNP marker vs. one Microsatellite marker around the same location) and the effects of the parental genotype information in the comparison on the two types of markers.1) On average, a microsatellite marker showed higher rates of significant replications than a SNP marker over the linkage locations.2) Some SNP markers provided almost equally strong linkage evidence, for example, SNP C01R0052 in the Danacaa sample  For the microsatellites, our affected sib-pair only methods showed a modest "noise" level (3%–6%) while the affected pedigree method (Duffy's APM) has a stable "noise" level of 3%. Both methods have almost the same "power." Since our method just used one affected sib-pair , it seems therefore that parental genotyping may not be very critical in linkage analysis for microssatellite markers.4) For SNP data, our affected sib-pair only IBS method has a "noise" level (4%–6%), IBS-IBD method has a high "noise" level (15%–21%) while affected pedigree method (Duffy's APM) has a stable "noise" level of 0%. The high "noise" level for IBS-IBD method reflects the fact that the IBS-IBD matrix for a SNP marker is close to singular. Thus, we conclude that for SNP data with parental genotypes, the false positive rate is very low in linkage analysis, and without parental genotype information the false positive rate can be relatively high.The different sites vary with respect to the power to detect linkage. Since the linkage evidence over a marker for a disease is inversely proportional to the number of markers which interact in determining the phenotype, our results may reflect some characteristics of the four population groups. For example, the relatively weak linkage over the four locations in the Aipotu group  for microsatellite markers there are virtually no differences in type I or type II error rates whether one uses or excludes parental genotypes. 2) On average, a microsatellite marker provides more power than a SNP marker does in linkage analysis. 3) If parental genotype information is used, SNPs show lower type I error rates than that of microsatellite markers. 4) If parental genotypes are not available, SNPs show variable type I error rates over different methods.In summary, other things being equal in the simulated sample analyzed, microsatellites are better than SNPs, although if parents are typed, SNPs can have slightly better type I error rates.APM: Affected pedigree memberGAW: Genetic Analysis WorkshopIBD: Identity-by-descentIBS: Identity-by-stateKPD: Kofendrerd Personality DisorderSNP: Single-nucleotide polymorphismQY developed the methodology, implemented the computer program, participated in the simulations and drafted the manuscript. VA participated in the simulations and GEB participated in analysis and coordination and helped to draft the manuscript.


E-cadherin, a cell surface protein involved in cell adhesion, is present in normal breast epithelium, benign breast lesions, and in breast carcinoma. Alterations in the gene CDH1 on chromosome 16q22 are associated with changes in E-cadherin protein expression and function. Inactivation of E-cadherin in lobular carcinomas and certain diffuse gastric carcinomas may play a role in the dispersed, discohesive "single cell" growth patterns seen in these tumors. The molecular "signature" of mammary lobular carcinomas is the loss of E-cadherin protein expression as evidenced by immunohistochemistry, whereas ductal carcinomas are typically E-cadherin positive.We report on E-cadherin immunostaining patterns in five cases of invasive mammary carcinomaThese were five exceptional instances in which the E-cadherin immunophenotype did not correspond to the apparent histologic classification of the lesion. These cases which are exceedingly rare in our experience are the subject of this report.Findings such as those illustrated in this study occur in virtually all biologic phenomena and they do not invalidate the very high degree of correlation between the expression of E-cadherin and the classification of breast carcinomas as ductal or lobular type on the basis of conventional histologic criteria. The utility of the E-cadherin immunohistochemical stain to distinguish between lobular and ductal carcinomas that are difficult to classify by morphologic features alone has been well-documented in recent years -8. HowevFour cases were obtained from the Pathology department of the New York Presbyterian Hospital-Weill Medical College of Cornell University and a fifth case from Memorial Sloan Kettering Cancer Center, between Jan 1999 and July 2004. All specimens were available as formalin-fixed paraffin embedded tissue blocks and hematoxylin and eosin stained slides.For E-cadherin immunostaining, four micron-thick sections were prepared from paraffin blocks containing lesional tissue. The slides were subsequently deparaffinized in three 5-minute changes of xylene and rehydrated through graded alcohols to distilled water. Heat induced epitope retrieval was performed on paraffin sections by pretreatment in a pressure cooker using 10 mM citrate buffer pH 6.0 for two minutes. Immunohistochemical staining was performed on paraffin sections using a TechMate 500 TM automated immunostainer  according to a modified MIP protocol  using the ChemMate ABC peroxidase secondary detection system . A monoclonal antibody to E-cadherin, clone HECD-1  was used in 1:400 dilution. The peroxidase reaction was developed using DAB chromogen provided in the kit. Sections were counterstained with hematoxylin. The hematoxylin and eosin and immunohistochemical stained slides were subsequently reviewed and histopathologic features of lesional areas recorded.A summary of the morphologic and immunohistochemical features of the following five cases is presented in Table A 62 year-old woman underwent a left lumpectomy in April of 2001 which revealed invasive carcinoma, histologically classified as ductal type.The 2.3 cm invasive carcinoma was well to moderately differentiated with intermediate nuclear grade. Well-formed ducts comprised greater than 90% of the invasive carcinoma Figure . In someAn E-cadherin immunohistochemical stain was performed on representative samples of invasive, in-situ, and metastatic carcinoma. No cell membrane reactivity was demonstrated in the primary invasive carcinoma or in the nodal metastasis including areas of glandular differentiation Figure  and 1C. In this case, aberrant E-cadherin protein expression was represented by absence of cell membrane immunoreactivity in gland-forming regions of invasive as well as metastatic carcinoma which appeared to be phenotypically "ductal".A 60 year-old female presented with a left breast mass. Subsequent excisional biopsy revealed invasive duct carcinoma.The lumpectomy specimen contained an 8 mm invasive well-differentiated duct carcinoma. Although greater than 90% of the tumor exhibited glandular differentiation, very focal areas with a single cell and linear growth pattern were also present Figure . The invThe immunohistochemical stain for E-cadherin showed weak cell membrane reactivity in areas of the primary invasive carcinoma exhibiting a ductal phenotype Figure . In contFoci of intraductal carcinoma demonstrated moderate positivity for E-cadherin while those of classical LCIS were negative for this antigen.In this case, aberrant E-cadherin protein expression consisted of weak immunoreactivity in the "ductal" areas of invasive carcinoma, whereas strong positivity was appreciated in areas of invasive and metastatic carcinoma with a lobular histologic phenotype.A 62 year-old woman presented with a right breast mass. Stereotactic needle core biopsy revealed an invasive, moderately differentiated carcinoma that was classified as ductal. Subsequent wide local excision included the needle core biopsy site as well as an area of radiographic density not previously sampled.The excisional biopsy revealed two separate invasive tumors measuring 8 and 5 mm, respectively. Well-formed glands comprised 80–90% of both tumors. Adjacent classical LCIS was also present. The larger tumor was strongly positive for estrogen and progesterone receptors by immunohistochemistry. No membrane expression for HER-2/neu was appreciated. A single ipsilateral sentinel lymph node contained several cytokeratin-positive cells. Five additional axillary lymph nodes were free of metastatic disease.The immunohistochemical stain for E-cadherin of the larger tumor was weak and discontinuous Figure . There wAberrant E-cadherin protein expression in this case consisted of predominantly negative immunoreactivity in invasive tumors exhibiting a ductal histological phenotype.A 57 year-old woman presented with a mass in the left breast.In-situ carcinoma comprised the remainder of the tumor mass and had a solid growth pattern, apocrine features, and intermediate-high nuclear grade. Both invasive and in-situ carcinoma formed signet-ring cells containing bluish-tinged, intracytoplasmic mucin vacuoles and together with the nuclear atypia, had the appearance of pleomorphic lobular carcinoma. The invasive carcinoma showed nuclear reactivity for estrogen and progesterone receptors by immunohistochemistry. The subsequent left mastectomy specimen revealed residual invasive carcinoma. A single, 2.2 cm left axillary lymph node contained metastatic carcinoma with extranodal extension. Morphologically, the metastatic deposit showed a "lobular phenotype" in the form of signet-ring cells growing in a diffuse distribution and absence of glandular differentiation  and 198 in situ carcinomas were studied, all in situ and invasive ductal carcinomas showed strong membrane E-cadherin expression. Forty-one of 42 invasive and 50 of 53 in situ lobular carcinoma showed complete absence of E-cadherin expression [et al, studied 89 primary infiltrating carcinomas immunohistochemically using an antibody to E-cadherin and found that 78% of well and moderately differentiated invasive duct carcinomas (IDC) showed strong linear staining for E-cadherin. Fifty-four percent of the poorly differentiated IDC however, had reduced and heterogeneous staining. E-cadherin reactivity was absent in 86.4% of the invasive lobular carcinomas and there was weak staining in 13.6 %. Among the in situ carcinomas the majority of DCIS had strong E-cadherin expression and all cases of LCIS showed absence of staining [Several studies have demonstrated the reliability of E-cadherin as a marker for distinguishing ductal from lobular carcinoma. In a study by Acs We have separately studied a series of 132 breast carcinomas, both in situ and invasive, for E-cadherin expression . In thiset al, investigated 41 cases which were classified as such due to the presence of a "mixture in variable degrees of ductal cytology and growth pattern  and typical lobular pattern (dispersed infiltrating fashion) in the same lesion, or to occasional tubules or small nest formation in a lesion that otherwise showed a typical lobular type of infiltration and cytology" [Few studies have specifically investigated invasive carcinomas with mixed duct and lobular features (IDLC). Acs et al, investigated 80 mixed  breast carcinomas. They found that the percentage of strongly E-cadherin-reactive lobular carcinoma cells was greatest in the mixed, predominantly ductal carcinomas and the greatest numbers of strongly E-cadherin-stained lobular carcinoma cells were identified near the periphery of the mixed carcinomas, particularly along the leading edge of these tumors. In addition, they observed that the nuclear grade of the lobular carcinoma component increased as the percentage of the carcinoma that was of the ductal type increased [Goldstein ncreased . In the ncreased , four caThe cases described in the present study represent a phenomenon wherein the histological structural phenotype did not match the "genotype" as suggested by the staining pattern of the E-cadherin immunohistochemical stain. This "aberrant" staining pattern is exceedingly rare in our experience. In cases 1, 2, 3, and 5 invasive carcinomas with predominantly gland forming elements and therefore ductal phenotype lacked E-cadherin reactivity. The differential diagnostic option in cases 1–3 and 5 is tubulolobular carcinoma based on the hybrid morphology of gland (tubules) and linear (lobular) growth pattern. Tubulolobular carcinoma is a distinct subtype of mammary carcinoma, which were originally thought to represent a tubular variant of lobular carcinoma. Few studies have examined the immunoprofile of tubulolobular carcinoma with respect to the E-cadherin staining pattern. In a recent study by Wheeler et al in which 27 cases of tubulolobular carcinoma composed of intermixed, round to angulated tubules and single file cell cords with diffuse and targetoid growth pattern demonstrated strong and diffuse positivity for E-cadherin in both the cell cord and tubular components . In our et al., reported that all eight invasive lobular carcinomas showed only cytoplasmic staining for E-cadherin which was also localized to the cytoplasm in the majority of (7/9) metastatic lobular carcinomas [Cytoplasmic E-cadherin reactivity has been described in diffuse type of gastric adenocarcinomas as well as in some invasive lobular carcinomas ,21,24. AMolecular studies have shown that invasive breast cancer is a disease with multiple cytogenetic subclones which are also present in the preinvasive lesions . The twoWe speculate that E-cadherin may play a critical role in molecular classification of breast carcinoma and may further elucidate the origin of tumors with a mixed or discordant phenotype.Findings such as those illustrated in this study occur in virtually all biologic phenomena and they do not invalidate the very high degree of correlation between the expression of E-cadherin and the classification of breast carcinomas as ductal or lobular type on the basis of conventional histologic criteria. Further studies are necessary to clarify whether E-cadherin protein is non-functional or truly represents exceptional biology in breast carcinomas exhibiting aberrant staining of E-cadherin.IDLC – invasive carcinomas with mixed duct and lobular featuresLCIS – lobular carcinoma in situDCIS – ductal carcinoma in situIDC – infiltrating ductal carcinomaThe author(s) declare that they have no competing interests.MH, SS, MM, and PPR: These authors contributed equally to the preparation of this manuscript.ST and EB: Each of these authors contributed one case.


Although several genes and proteins have been implicated in the development of melanomas, the molecular mechanisms involved in the development of these tumors are not well understood. To gain a better understanding of the relationship between the cell growth, tumorigenesis and differentiation, we have studied a highly malignant cat melanoma cell line that trans-differentiates into neuronal cells after exposure to a feline endogenous retrovirus RD114.To define the repertoire of proteins responsible for the phenotypic differences between melanoma and its counterpart trans-differentiated neuronal cells we have applied proteomics technology and compared protein profiles of the two cell types and identified differentially expressed proteins by 2D-gel electrophoresis, image analyses and mass spectrometry.de novo after infection of melanoma cells with RD114 virus in vitro. Expression of these proteins in trans-differentiated cells was significantly associated with concomitant down regulation of growth promoting proteins and up-regulation of neurogenic proteins (p = < 0.001). Based on their physiologic properties, >95% proteins expressed in trans-differentiated cells could be associated with the development, differentiation and regulation of nervous system cells.The melanoma and trans-differentiated neuronal cells could be distinguished by the presence of distinct sets of proteins in each. Although approximately 60–70% of the expressed proteins were shared between the two cell types, twelve proteins were induced Our results indicate that the cat melanoma cells have the ability to differentiate into distinct neuronal cell types and they express proteins that are essential for self-renewal. Since melanocytes arise from the neural crest of the embryo, we conclude that this melanoma arose from embryonic precursor stem cells. This model system provides a unique opportunity to identify domains of interactions between the expressed proteins that halt the tumorigenic potential of melanoma cells and drive them toward neurogenerative pathways involved in early neurogenesis. A better understanding of these proteins in a well-coordinated signaling network would also help in developing novel approaches for suppression of highly malignant tumors that arise from stem-like embryonic cells. Melanomas are a heterogeneous group of tumors that arise in the skin, eye, meninges and other parts of the body. While early stages of cutaneous melanomas are recognized by changes in the size, shape or color of black nevi, most cancer cells grow downward from the skin and invade neighboring tissues before they are detected as highly metastatic tumors in lymph nodes or other organs . Patientin vitro [To gain a better understanding of mechanisms involved in the growth, differentiation and transformation of cells, we have studied a fully differentiated (pigmented) highly malignant cat melanoma cell line CT1413, that trans-differentiates into neuronal cells, 48–72 hours after infection with the endogenous cat retrovirus RD114 de novo expression of pro-inflammatory cytokines, neuro-regulatory enzymes/kinases, neurotrophic factors and concomitant suppression of growth-promoting proteins. This repertoire of proteins is not only responsible for the generation of neuronal cells but it is also involved in the reversion of a highly malignant tumor into non-cancerous neuronal cells.Since RD114 virus does not replicate in the terminally trans-differentiated neuronal cells, we hypothesized that binding of virus to its receptor on the melanoma cell membrane could induce conformational changes that generate a cascade of molecular interactions through distinct signals which arrest the tumor cell growth and drive them toward neurogenetic pathways. To test this hypothesis we have analyzed comprehensive protein profiles of both the malignant melanoma and its counterpart trans-differentiated neuronal cells by 2-dimensional gel electrophoresis (2DGE) and identified differentially expressed proteins in the two cell types by Matrix-Assisted-Laser-Desorption-Ionization-Time-Of-Flight mass spectrometry (MALDI-TOF-MS). Herein we show that the trans-differentiation of melanoma into neuronal cells is directly associated with We have compared 3129 protein spots in 15 gels derived from RD114-infected and uninfected melanoma cells from two independent experiments and analyzed peptide fingerprints of 467 differentially expressed (up-regulated and downregulated) protein spots by MALDI-TOF-MS. A total of 46 proteins were confirmed unambiguously from 302 spots excised from multiple gels of both experiments  and 220 spots containing protein complexes  proteins were shared between the two cell types  expressed in neuronal cells have been shown previously to be associated with the early stages of brain development, differentiation and regulation of nervous system. Based on these biological functions, we have proposed possible roles of these 46 proteins in signaling antiviral responses, arrest in tumor cell growth, modulation of cell membranes and in altering the signal transduction pathways from malignancy to early neurogenesis and cell differentiation Figure .de novo expression of interferon gamma (ING) in the presence of tumor necrosis factor (TNF5) and interleukin-1 beta converting enzyme (ICE1) that were already present in melanoma cells , IL-2 receptor alpha chain (IL2A) and IL-6, all of which produce anti-tumorigenic and inflammatory responses. Co-expression of ING, IL-6 and other newly synthesized cytokines prompts proteases  to functionally activate these proteins by cleavage [The first line of defense against the RD114 virus infection begins with conformational changes in the melanoma cell membrane due to binding of the viral envelope glycoproteins to its receptor, the NAA transporter protein . The tollls Fig. &2C. Thiscleavage . A seriecleavage ,9. At thde novo in transdifferentiated neuronal cells after exposure of melanoma cells to the RD114 virus , the frequency of detection for this kinase was significantly higher in neuronal cells compared to melanoma Fig. . Co-exprBDNF binds to tyrosine kinase receptor with a high affinity . It can Analyses of all complexed proteins containing BDNF Fig. &4B indicProduction of BDNF stimulates expression of acetylcholinesterase (ACES), an enzyme vital for neurite formation and transmission of signals in both the sympathetic and parasympathetic arms of the nervous system . Expressde novo in trans-differentiated cells  and lipoprotein lipase (LIPL) were expressed lls Fig. . These ells Fig. . AnotherThe pyruvate kinase isoenzyme M1 (KPYM) was downregulated in trans-differentiated cells but its presence is considered important for nerve endings . LikewisOne of the most frequently detected cell membrane proteins, in both melanoma and neuronal cells was integrin VLA-4 beta I subunit (ITB/ CD29). This protein was present in both single and complex forms in both cell types Fig. . IntegriThe sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (ATA2) enzyme was detected with a high frequency in both melanoma and neuronal cells and sodium/calcium exchanger 1 (NAC1) a plasma membrane pump was expressed at a slightly higher frequency in neuronal cells than in melanoma Fig. . Both prExpression of different isomers of zona pellucida  in both melanoma and neuronal cells was totally unanticipated. Although exact function of these sperm-binding receptor proteins in melanoma or neuronal cells is not known, recent reports suggest that ZP domains particularly those of ZP2 that is upregulated in neuronal cells may transduce intracellular signals necessary for polymerization of proteins into neural filaments required for mechano-sensory dendrites ,42. It iin vitro. To our knowledge, this is the first demonstration that epigenetic stimuli from the cell surface of a naturally occurring melanoma can generate neurogenetic signals that can alter the genome-wide transcriptional and translational programs such that its oncogenic pathways are halted and pro-neuronal auto-regulatory mechanisms are prompted toward neural cell differentiation.We have shown that RD114 virus infection of cat melanoma cells induces a repertoire of novel proteins that suppress its tumorigenic potential and promote neuronal cell differentiation de novo expressed) that distinguish highly malignant melanoma cells from their counterpart trans-differentiated neuronal cells. Our proteomics studies also emphasize that although a significant number of the expressed proteins are shared between the two cell types, distinct protein-protein interactions are operative in altering signals that lead cells toward tumorigenic or neurogenic pathways.By immunohistochemistry numerous markers of neural cell neoplasms , neural specific enolase, neuro-peptide substance P, the low-affinity nerve growth factor receptor (p75NGFR), neural cell adhesion molecule (CD56/N-CAM) and growth-associated phosphoprotein-43) have been shown to enhance malignant potential of melanomas and neuronal cell-derived tumors -46. In aExpression of >69% of germ-line associated sensory or neurogenic signaling proteins, neural-specific enzymes, cytokines, neurotrophic/ growth factors, serine, threonine and tyrosine kinases in cat melanoma cells as well as in trans-differentiated counterpart cells, supports the stem cell origin of this tumor and suggests that the precursor of this melanoma is a stem-like cell rather than the primitive melanoblast committed to melanocytic differentiation only. These cells are unique in that they are fully differentiated (pigmented) and they have the ability to give rise to new cell types that express differentiation-specific proteins. In addition, these cells express many proteins involved in self-renewal of cells. These include the stem cell growth factor receptor protein (C-Kit), PDGB, BDNF, NT-3, polycomb protein 1 and others. Recently, BMI1 has been shown to be essential for efficient self-renewal of neural stem cells ,49. As cBased on our findings it can also be speculated that a high incidence of cutaneous melanomas and atypical melanocytic nevi in patients with hereditary neurofibroma, astrocytoma, glioma, meningioma, and other neuronal tumors , may alsTo gain a better insight into the complex processes of oncogenesis or neurogenesis, it would also be important to identify proteins in various natural biological settings since most substrate proteins are cleaved by proteases prior to enzyme interactions. Defining the specificity of natural targets may throw light on how biological specificities are achieved in nature. This model system offers a unique opportunity to study protein profiles in relation to specific ligand-binding interactions that are capable of inducing genetic or phenotypic changes in these cells. Characterization of protein-protein interaction domains by the use of constitutively active and dominantly negative constructs and screening of small peptides or interfering RNAs would provide new knowledge into functional significance of each of the proteins that direct cells toward distinct pathways (i.e. oncogenic versus neurogenic). These studies would also have important clinical implications for understanding neurogenerative and neuroprotective mechanisms for neurologic disorders since activated proteins may induce changes in protein subunits in a cell-type specific manner. This information would help in identifying selective inhibitors for tumors and activators/co-activators of neurogenic proteins involved in neural cell development.in vitro from a highly metastatic and melanotic tumor of the lung [2), at a density of 2 × 106 cells/ flask in medium containing 2-ug/ml polybrene [in vitro [A cat melanoma cell line CT1413) was established the lung . Cells wolybrene . This viin vitro . The RD1 was estaTo validate protein profiles two independent cell culture experiments were conducted almost 12 months apart and proteomes of both cell types were analyzed separately for differential expression profiles.7 cells from each set of experimental and control melanoma cells were removed by the use of a cell-scraper or a "rubber policeman" (i.e. not treated with trypsin to detach cells). Each cell pellet was washed again 2× with PBS and proteins were extracted sequentially by a modified two-step solubilization procedure using two reagents containing different concentrations of 5–8 M Urea, 2%–4% CHAPS, 2 M thiourea and other non-ionic and/zwitterionic detergents . The most soluble membrane proteins were removed in the first extraction and the less soluble proteins were separated in the second fraction. In order to isolate proteins as close to their natural states in cells as possible, each solubilization step was modified by rapid lysis technique (only 10–15 seconds' cell lysis with 2-seconds' sonication) followed by centrifugation for 90 minutes at 100,000 × g prior to analyzing soluble proteins present in the supernatant by isoelectric focusing (IEF).Proteins were extracted from 12 flasks each of the trans-differentiated and counterpart melanoma cells from each of the two experiments. Cells were washed 3× with phosphate buffered saline (PBS) to remove serum and other proteins, and 2 × 10All protein fractions from melanoma controls and trans-differentiated neuronal cells were analyzed separately by 2-dimensional gel electrophoresis (2DGE). In the first dimension proteins were separated by IEF on a pH gradient 3–10 and then size fractionated in the second dimension on 6–18% gradient of SDS-polyacrylamide gels. Proteins were stained with Coomassie blue and by the use of a CCD camera and an image-processing analytical program (PDQuest from BioRad) all protein spots were evaluated from each of the 15 gels (9 gels from the first experiment and 6 gels from the second experiments). This program compares both the quality and normalized quantity of each spot across 15 gels and creates a master gel-image of well-calibrated and quantifiable spots using internal reference proteins. A master gel containing 3,129 spots was used to compare each of the corresponding spots in all gels derived from melanoma controls and trans-differentiated neuronal cell. All differentially expressed (i.e. upregulated and down-regulated) proteins were identified in each gel and 467 spots including some spots common to both cell types were excised from multiple gels. Proteins were digested using ultra-pure trypsin and peptide fingerprints of each in-gel digest were analyzed by MALDI-TOF-MS. Full scan mass spectra were acquired and high mass accuracy criteria were used throughout the study with high resolution and strict trypsin specificity. Spectra were submitted to the Protein database (SWISS-PROT) for searching protein identification. In addition to using these stringent criteria, we used manual acquisitions of spectra, which yielded more reliable and reproducible results compared to automated acquisitions using PS1 software. The confidence level in our protein identification was high because almost all proteins were confirmed in corresponding spots in multiple gels and by duplicating the entire experiment for validation. In this study we included only those proteins that were most reproducible in the high stringency Feline Protein database and results were confirmed in other mammalian species (SWISS-PROT). Thus far we have identified 46 proteins among 302 spots from multiple gels tested. All spots in which proteins were not identified reproducibly by mass spectrometry from the same or different gels were NOT included in any analysis.The frequency distribution between melanoma and neuronal cells was compared using Chi-square test or 2-sided Fisher's exact test. The mean quantity for each protein was compared between melanoma and neuronal cells using the Mann-Whitney rank-sum test and the probability of a protein occurrence in the trans-differentiated neuronal versus melanoma cells was examined based on the binomial probability distribution with an expected proportion less than 0.05.The author(s) declare that they have no competing interests.


Mitozolomide and its decomposition product MCTIC were found to be more cytotoxic to BE colon carcinoma cells in vitro than to HT-29 cells, another colon carcinoma cell line. In addition mitozolomide and MCTIC induced DNA interstrand crosslinks in the BE but not the HT-29 cell line. BE cells are deficient in the repair of O6-methylguanine lesions and are designated Mer-, whereas, HT-29 cells are proficient in this repair process and are designated Mer+. Thus DNA interstrand crosslinking produced by mitozolomide and MCTIC appears to correlate with the Mer phenotype. Pretreatment of HT-29 cells (Mer+) with the DNA methylating agent MNNG allows mitozolomide or MCTIC to produce DNA interstrand crosslinks. HT-29 cells also become more sensitive to the cell killing of mitozolomide and MCTIC with MNNG pretreatment. Pretreatment of Mer- cells (BE) had little effect on either cell killing or DNA crosslinking levels induced by mitozolomide or MCTIC. DNA interstrand crosslinking induced by mitozolomide and MCTIC is probably a consequence of an initial alkylation at the O6-position of guanine followed by a delayed reaction with the opposite DNA strand.


In oromandibular dystonia (OMD) abnormal repetitive contractions of masticatory, facial, and lingual muscles as well as the presence of orobuccolingual (OBL) dyskinesias may interfere with the appropriate performance of tasks such as chewing and swallowing leading to significant dysphagia and weight loss. We present here the clinical characteristics and treatment variables of a series of patients that developed an OMD-associated eating dysfunction.We present a series of patients diagnosed and followed-up at the Movement Disorders Clinic of the Department of Neurology of University of Miami, Miller School of Medicine over a 10-year period. Patients were treated with botulinum toxin injections according to standard methods.Five out of 32 (15.6%) OMD patients experienced symptoms of eating dysfunction associated with OMD. Significant weight loss was reported in 3/5 patients (ranged for 13–15 lbs). Two patients regained the lost weight after treatment and one was lost to follow-up. Tetrabenazine in combination with other antidystonic medication and/or botulinum toxin injections provided substantial benefit to the patients with dysphagia caused by OMD.Dystonic eating dysfunction may occasionally complicate OMD leading to weight loss. Its adequate characterization at the time of history taking and clinical examination should be part of outcome measurements of the anti-dystonic treatment in clinical practice. In oromandibular dystonia (OMD) spasms of the masticatory, facial, and lingual muscles result in repetitive and sometimes sustained jaw opening, closure, deviation, or any combination of these as well as abnormal tongue movements . AlthougDue to the anatomical distribution of the muscles affected, OMD and co-existing OBL dyskinesias are associated with disturbed perioral, oral and lingual movements that may interfere with the appropriate performance of tasks such as chewing, swallowing or talking, complicated with social embarrassment  and some®, Allergan Pharmaceuticals, Irvine, CA, USA) was performed according to standard methods [® EMG needle. We recorded the muscles injected and the dose each muscle received. The mean dose of BTX-A for each patient was determined by adding the units injected per visit divided by the number of visits was used in the calculation of the mean dose (in units) of BTX-A in each group. The initial visit was not included since a lower than optimal dose of botulinum toxin was used.We performed a study of consecutive OMD patients followed-up at the Movement Disorders Clinic of the Department of Neurology of University of Miami, Miller School of Medicine over a 10-year period. We identified 53 patients with primary and secondary OMD. We excluded patients who were lost to follow up after initial evaluation or had inconclusive charts (n = 21) . Five out of 32 (15.6%) OMD patients reported symptoms of dystonic eating dysfunction. All OMD patients seen in our practice are routinely asked a set of specific questions about weight loss or eating difficulties associated with OMD . All patients were examined and followed-up by the same physician (CS). Results of treatment were analyzed using a global impression scale . The formulation and preparation of botulinum toxin type A (BTX-A) . After 9 sessions he continues experiencing the same marked benefit and no longer complains of eating difficulties.A 48-year-old female was initially evaluated for OMD of one year evolution. She complained of intermittent involuntary movements of jaw-opening accompanied by tongue thrusting. While eating her tongue would protrude causing substantial eating and swallowing difficulties that had lead to a 15 lbs weight loss (from 110 to 95 lbs). A barium swallowing test at the time revealed her swallowing function to be moderately impaired secondary to decreased bolus preparation and decreased bolus propulsion without evidence of aspiration. After the unsuccessful injections of BTX-A (4 sessions) to her lateral pterygoids (50 u/side), she was placed on a regimen that included tetrabenazine 75 mg/day, trihexyphenidyl 3 mg/day, and lorazepam 4.5 mg/day with significant improvement of her symptoms and gradual weight gain.A 32-year-old male presented with new-onset jaw-closure spasms (jaw spasms with any kind of stimulus-able to open his mouth only 1/4 inch). This process increased in severity for three weeks, after which he could not take any food in, except through a straw. For the ensuing two months there was a gradual albeit limited improvement where he was able to open his mouth 3/4 of an inch, and from then on his condition had remained stationary. He had to change to a soft, pureed diet. Chewing would result in pain, particularly on the left mandibular area. His dystonic disorder also interfered with his speech, forcing him to keep his tongue behind the teeth to prevent from biting it. The patient was successfully treated with BTX-A injections to his masseters (50 units/side). After 4 sessions he continues to experience the same marked benefit and no longer complains of eating difficulties.A 49-year-old female presented with a 2-year history of jaw opening movements, which caused substantial drinking difficulties. A year later these movements became constant and were complicated with movements of the tongue (tongue protrusion and dyskinesias) with consequent impairment of fluid and food manipulation overlapping with chewing difficulties caused by her jaw-opening OMD. The patient reported a 15 lbs weight loss due to her condition (from 130 to 115 lbs).She was subsequently placed on tetrabenazine (125 mg/day) with moderate benefit in the frequency and intensity of the jaw-opening movements. However, tongue protrusion and dyskinesias were not affected. The patient developed a hypokinetic extrapyramidal syndrome as a side effect to tetrabenazine therapy but insisted on continuing the drug (at a lowered dose of 75 mg/day) because of its beneficial effects on her symptoms. Within about 6 moths after the initiation of tetrabenazine the patient gained 10 lbs. In an effort to further control her symptoms the patients had trials with clonazepam and gabapentin. A combination of tetrabenazine (75 mg/day) and gabapentin (300 mg/day) improved her symptoms by a reported 75%. She also received BTX-A injections to lateral pterygoid muscles (25 units/side) without benefit. Ten years into her condition she still experiences substantial benefit from her treatment.A 56-year-old female presented with a chief complaint of involuntary jaw movements. The patient had a long (35-year) history of migraines for which she had received a number of treatments  with limited success. Her first trial with an atypical neuroleptic (off-label use) was 2.5 years before presentation when she had been started on ziprasidone (80 mg/day) . The patWe present here a series of 5 patients with eating dysfunction associated with OMD. The clinical and epidemiological characteristics of our OMD cohort has been described elsewhere . The preVarious studies have shown that a range of swallowing difficulties accompany focal cranio-cervical dystonias such as spasmodic dysphonia and spasmodic torticollis before treatment initiation -14. CervDysphagia has been also reported as a complication of BTX-A therapy in OMD ,17,18. HOur study has some inherent limitations commonly seen in retrospective series. Documentation of all clinical and treatment variables may not be complete. However, all study patients have been evaluated and followed by the same movement disorders specialist (CS). Hence the impact of confounding factors such as inconsistencies in the diagnosis, inaccurate history, inter-examiner differences, and under-documentation were reduced. Furthermore, there are no additional evaluations of masticatory function  other than weight loss (which may be influenced by many factors) and patient descriptions.In summary, eating dysfunction was reported in 15.6% of our OMD cases. Eating dysfunction was associated with significant weight loss in three of our patients. Interestingly, only patients with OBL dyskinesias experienced weight loss. Additional difficulties included pain during eating, social embarrassment and speech disturbance. A measure of benefit was reported in the majority of our patients with BTX-A injections and oral antidystonic medications, especially tetrabenazine. Eating dysfunction associated with OMD should be identified and adequately characterized at the time of history taking and should be part of outcome measurements of the anti-dystonic treatment. Special attention has to be paid to patients with OMD and OBL dyskinesias since in our cases they experienced significant weight loss.


CA-125 levels were assessed prior to each of the first three cycles of chemotherapy, in 81 patients with epithelial ovarian cancer receiving first-line chemotherapy. All patients have at least 1 year's follow-up. Thirty-nine patients (48%) have progressed clinically or have died within 1 year of treatment (treatment 'failures'). Three CA-125 indices previously shown to be of prognostic value are assessed for their ability to pick-out these 'failures'. When the indices examined are modified to obtain a specificity for picking out failures just exceeding 90%, the maximum sensitivity obtained was 46%. The use of CA-125 for clinical decision making in ovarian cancer requires further investigation to determine and validate a prognostic index with acceptable sensitivity and specificity, and to determine the clinical impact of treatment decisions made using such an index.


The role of the initial DNA double-strand breaks (dsb) as a determinant of cellular radiosensitivity was studied in human breast and bladder cancer cell lines. Cell survival was measured by monolayer colony-forming assay as appropriate and differences in radiosensitivity were seen . After pulsed-field gel electrophoresis (PFGE) the initial slopes of dose-response curves were biphasic with a flattening of the curves above 30 Gy. When the frequency of DNA dsb induction was assessed using a mathematical model based on the DNA fragment size distribution into the gel lane, we found a statistically significant relationship between the number of DNA dsb induced and the corresponding alpha-values and fraction surviving after 2Gy (P = 0.0049 and P = 0.0031 respectively). These results support the view that initial damage is a major determinant of cell radiosensitivity.