Effect of repeated exposure to AQUI-S® on the viability and growth of Neoparamoeba perurans.

There have been recent efforts amongst immunologists to develop approaches for following individual fish during challenges with viral and bacterial pathogens. This study contributes to assessing the feasibility of using such approaches to study amoebic gill disease (AGD). Neoparamoeba perurans, agent of AGD, has been responsible for widespread economic and fish loss in salmonid aquaculture. With the emergence of AGD in Europe, research into infection dynamics and host response has increased. This study investigated the effect of repeat exposure to anaesthesia, a necessary requirement when following disease progression in individual fish, on N. perurans. In vitro cultures of N. perurans were exposed every 4 days over a 28-day period to AQUI-S® (isoeugenol), a popular anaesthetic choice for AGD challenges, at a concentration and duration required to sedate post-smolt salmonids. Population growth was measured by sequential counts of amoeba over the period, while viability of non-attached amoeba in the culture was assessed with a vital stain. AQUI-S® was found to be a suitable choice for in vivo ectoparasitic challenges with N. perurans during which repetitive anaesthesia is required for analysis of disease progression.

Identification and characterisation of TLR18-21 genes in Atlantic salmon (Salmo salar).

Teleost fish possess many types of toll-like receptor (TLR) some of which exist in other vertebrate groups and some that do not (ie so-called "fish-specific" TLRs). In this study, we identified in Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs seven TLRs that are not found in mammals, including six types of fish-specific TLRs (one TLR18, one TLR19, and four TLR20 members (two of which are putative soluble forms (s)) and one TLR21. Phylogenetic analysis revealed that teleost TLR19-21 are closely related with murine TLR11-TLR13, whilst teleost TLR18 groups with mammalian TLR1, 2, 6 and 10. A typical TLR protein domain structure was found in all these TLRs with the exception of TLR20b(s) and TLR20c(s). TLR-GFP expression plasmids transfected into SHK-1 cells showed that salmon TLR19, TLR20a and TLR20d were preferentially localised to the intracellular compartment. Real time PCR analysis suggested that salmon TLR19-TLR21 are mainly expressed in immune related organs, such as spleen, head kidney and gills, while TLR18 transcripts are more abundant in muscle. In vitro stimulation of primary head kidney cells with type I IFN, IFNγ and IL-1ß had no impact on TLR expression. Infectious salmon anaemia virus (ISAV) infection, in vivo, down-regulated TLR20a, TLR20b(s), TLR20d and TLR21 in infected salmon kidney tissue. In contrast, up-regulation of TLR19 and TLR20a expression was found in posterior kidney in rainbow trout with clinical proliferative kidney disease (PKD).

Magnetosphere-Ionosphere-Thermosphere Coupling Study at Jupiter Based on Juno's First 30 Orbits and Modeling Tools.

The dynamics of the Jovian magnetosphere is controlled by the interplay of the planet's fast rotation, its solar-wind interaction and its main plasma source at the Io torus, mediated by coupling processes involving its magnetosphere, ionosphere, and thermosphere. At the ionospheric level, these processes can be characterized by a set of parameters including conductances, field-aligned currents, horizontal currents, electric fields, transport of charged particles along field lines including the fluxes of electrons precipitating into the upper atmosphere which trigger auroral emissions, and the particle and Joule heating power dissipation rates into the upper atmosphere. Determination of these key parameters makes it possible to estimate the net transfer of momentum and energy between Jovian upper atmosphere and equatorial magnetosphere. A method based on a combined use of Juno multi-instrument data and three modeling tools was developed by Wang et al. (2021, https://doi.org/10.1029/2021ja029469) and applied to an analysis of the first nine orbits to retrieve these parameters along Juno's magnetic footprint. We extend this method to the first 30 Juno science orbits and to both hemispheres. Our results reveal a large variability of these parameters from orbit to orbit and between the two hemispheres. They also show dominant trends. Southern current systems are consistent with the generation of a region of sub-corotating ionospheric plasma flows, while both super-corotating and sub-corotating plasma flows are found in the north. These results are discussed in light of the previous space and ground-based observations and currently available models of plasma convection and current systems, and their implications are assessed.

Interferon response following infection with genetically similar isolates of viral haemorrhagic septicaemia virus (VHSV) exhibiting contrasting virulence in rainbow trout.

Isolates of viral haemorrhagic septicaemia virus (VHSV) were identified which are genetically similar yet, based on their isolation history were considered likely to differ in virulence in juvenile rainbow trout. An experimental infection study was performed in order to verify this hypothesis and provide an experimental infectivity model with which to investigate the basis for susceptibility of rainbow trout to this commercially important virus. Significant differences in mortality were obtained following both intraperitoneal (IP) injection and immersion challenges with an early marine (DK-M.Rhabdo) and early rainbow trout VHSV isolate (DK-F1) respectively. Expression of Type I IFN, Mx1 (an IFN-inducible protein), and viral genes (encoding nucleo-, phospho-, matrix, glyco- and non-viron proteins) was studied in sequential tissue samples using real-time quantitative PCR (QPCR). Resulting data revealed a significant increase in IFN and Mx1 expression detected in fish challenged by IP injection with both isolates. Expression levels of these genes were directly related to the degree of viral replication as measured by the expression of VHSV RNAs. In immersion-challenged fish a significant increase in Mx1 was observed only when using the virulent isolate DK-F1; however no elevated host response was detectable in fish challenged with the marine isolate DK-M.Rhabdo. Quintessentially the inability to detect any virus in trout challenged with the marine isolate via immersion suggests the virus was incapable of establishing infection. The mechanisms for this appear to be more related to initial cellular entry and replication rather than due to the overcoming of initial infection via an elevated host innate immune response.

Histology, immunocytochemistry and qRT-PCR analysis of Atlantic salmon, Salmo salar L., post-smolts following infection with infectious pancreatic necrosis virus (IPNV).

Infectious pancreatic necrosis (IPN) is a very serious viral disease in terms of its impact on production of Atlantic salmon, Salmo salar L., fry and post-smolts. Post-smolts of Atlantic salmon were injected with infectious pancreatic necrosis virus (IPNV) and cohabited with naive fish to produce natural infection. Cohabitant fish were sampled every 2 days, up to day 36 post-infection (p.i.). From 90 cohabitant fish, 11 (12.2%) were positive by immunohistochemistry (IHC). The first detection of IPNV by IHC occurred on day 16 p.i. which coincided with the onset of mortality in this group. Besides the pancreas, the liver was found to be a key target organ for IPNV. For the first time, the virus was observed in the islets of Langerhans and in the kidney corpuscles of Stannius which suggests that the virus could affect the fish's metabolism. The liver of two fish, which showed the most widespread presence of IPNV by IHC, had a pathology including focal necrosis and widespread presence of apoptotic hepatocytes, many of which did not stain for virus by IHC. Up-regulation of cytokine gene expression was found only in the IHC-positive (IHC+ve) fish and reflected the level of infection as determined by IHC positivity of the liver. In most fish, interferon (IFN), Mx, γIFN and γIP were up-regulated in liver and kidney, while only IFN and Mx were up-regulated in gill. IL1ß and TNFα were not induced in any tissue. The gill showed variable levels of constitutive expression of IL1ß and γIFN. The two fish with liver pathology had the highest level of IFN expression, especially relative to the level of Mx expression, in the liver compared with the other IHC+ve fish which did not have a liver pathology. The results suggest that following widespread infection of hepatocytes, the cells may over-produce IFN, resulting in apoptosis of neighbouring cells with subsequent death from liver failure.

The effect of sub-culturing on the basal level of type I interferon (IFN) gene expression in the Salmon Head Kidney (SHK-1) cell line.

Over sub-culturing a cell line generates a selective pressure which can result in key cellular functions being altered such as gene and protein expression. The present study set out to determine whether serial sub-culturing affects the antiviral state of the Salmon Head Kidney (SHK-1) cell line. Cells were cultured under constant conditions and real-time PCR was performed to measure the level of interferon (IFN) and Mx gene expression over different passage numbers. A significant increase in the basal level of IFN and Mx gene expression was recorded at passage number 58 (3 and 14-fold increase versus passage number 53), suggesting a sub-culturing effect on the type I IFN response in SHK-1 cells. Passage dependent variations in morphology and cell sub-populations have been previously observed in SHK-1 cells. Such variations in cell sub-types were suspected to be responsible for the fluctuations in IFN and Mx gene expression recorded in this study.

Identifying potential virulence determinants in viral haemorrhagic septicaemia virus (VHSV) for rainbow trout.

We identified viral haemorrhagic septicaemia virus (VHSV) isolates classified within Genotype Ib which are genetically similar (>99.4% glycoprotein amino acid identity) yet, based on their isolation history, were suspected to differ in virulence in juvenile rainbow trout. The virulence of an isolate recovered in 2000 from a viral haemorrhagic septicaemia disease episode in a marine rainbow trout farm in Sweden (SE-SVA-1033) was evaluated in juvenile rainbow trout via intraperitoneal injection and immersion challenge alongside 3 isolates recovered from wild-caught marine fish (DK-4p37, DK-5e59 and UKMLA98/6HE1) suspected of being of low pathogenicity to trout. Mortality data revealed that isolate SE-SVA-1033 caused VHSV-specific mortality in both intraperitoneal and immersion challenges (75.0 and 15.4%, respectively). The remaining Genotype Ib isolates caused significantly lower mortalities using the same experimental infection routes (<35.0 and <2.0%, respectively). Having identified VHSV isolates with clear differences in their pathogenicity, coding and inter-genic non-coding regions of 2 isolates (SE-SVA-1033 and DK-4p37) were determined and compared in order to identify potential markers responsible for the observed differences in virulence. Only 4 predicted amino acid substitutions were identified across the genome sequenced; these occurred in the N (R46G), G (S113G), NV (L12F) and L (S56A) proteins. These findings form the basis for further studies aimed at determining the biological significance of these mutations and suggest that small changes at the molecular level can cause significant changes in the virulence properties of VHSV isolates.

A sensitive loop-mediated isothermal amplification (LAMP) method for detection of Renibacterium salmoninarum, causative agent of bacterial kidney disease in salmonids.

Loop-mediated isothermal amplification (LAMP) is a novel technique for nucleic acid amplification with high specificity, sensitivity and rapidity and does not require expensive equipment or reagents. In the present study, we developed and evaluated a LAMP method for the rapid detection of Renibacterium salmoninarum causing the bacterial kidney disease in salmonids. This method was more sensitive than quantitative real-time polymerase chain reaction (qPCR). Using DNA template extracted from cultured R. salmoninarum, the LAMP method gave an amplification signal from template diluted to 10(-8) while the limit of detection of qPCR was10(-7). The LAMP method was also highly specific and did not amplify DNA purified from five other Gram-positive and -negative bacterial fish pathogens. The method also worked well using extracts of macrophages infected with R. salmoninarum and kidney material from rainbow trout, which were positive for R. salmoninarum by qPCR and crude R. salmoninarum culture. There was some evidence for inhibitors of the LAMP reaction in the kidney samples, which was overcome by diluting the sample.

Molecular characterization of IRF3 and IRF7 in rainbow trout, Oncorhynchus mykiss: functional analysis and transcriptional modulation.

Interferon regulatory factors (IRF) 3 and 7 in mammals are known to be crucial in regulating the type I interferon (IFN) response to viral infection as part of transcriptional complexes binding to IRF-binding elements (IRF-Es) and interferon stimulatory response elements (ISREs) within IFN and interferon-stimulated genes (ISGs). Here we report the sequencing and characterization of full-length cDNA homologues of rainbow trout (rt)IRF7 and, for the first time in fish, IRF3. RtIRF3 consists of 2127 bp with a 159 bp 5'-UTR-containing two upstream AUGs and a 573 bp 3'-UTR. RtIRF7 was found to be 2055 bp, with a 102 bp 5'-UTR and a 705 bp 3'-UTR. The open reading frames (ORFs) translate into 464 amino acid and 415 amino acid proteins, respectively, each possessing a putative DNA-binding domain (DBD) containing a tryptophan cluster, which is characteristic of all IRF family members. The presence of putative IRF association domain (IAD)s, serine-rich C terminal domains (poorly conserved in trout IRF3), and phylogenetic analysis places the two genes in the IRF3 subfamily. Both genes were found to be upregulated by poly I:C, type I recombinant rainbow trout (r) IFN (second isoform, type I rIFN), type II rIFN (rIFNgamma), LPS, and rIL-1beta in the trout macrophage cell line, RTS-11. Poly I:C and type I rIFN also induced IRF3 and IRF7 expression in a trout fibroblast cell line (RTG-2). Transient transfection of RTG-2 cells with each IRF fused to GFP revealed a predominant cytoplasmic distribution found most intensely around the nucleus and, to a lesser extent, within cell nuclei. Transient transfection of rtIRF3 in the Mx-1-luciferase reporter cell line, RTG-P1, revealed a modest increase in luciferase activity relative to the vehicle control, which was lost in cells over-expressing a DBD-truncated form of rtIRF3. Both full-length and DBD-truncated forms of rtIRF7 increased reporter activity relative to the control, although to a non-significant extent. Electromobility shift assays (EMSAs) did not reveal a specific interaction between each IRF and the ISRE element found in the Mx-1 promoter, although the Mx-1 ISRE bound specifically to endogenous transcriptional complexes. These data support the premise that rtIRF3 and rtIRF7 are important molecules in the regulation of antiviral responses in fish, with the impact of rIFNgamma on rtIRF3/7 expression implying a role for these IRFs in immune processes other than type I IFN-driven antiviral responses.

Antitumor activity of cell walls from Brucella abortus.

The ip inoculation of inactivated Brucella abortus, strain B19 R, protected mice against a subsequent graft of an ascites lymphoma. The bacterial components responsible for this effect were investigated. Centrifugation supernatants of sonicated bacteria supposed to contain mainly cytoplasmic products did not offer protection against the lymphoma. Cell walls (CW's) prepared by enzyme digestion of pellets of lysed bacteria and checked for purity by electron microscopy prolonged survival of mice and induced cytotoxic macrophages in their peritoneal cavities. CW peptidoglycan (PG) did not seem to play an important part in this effect. Enzyme digestion of CW, in particular by lysozyme, was found to reduce a PG characteristic component (diaminopimelic acid) without altering CW antitumor activity. Conversely, a purified PG preparation did not influence tumor growth. Extraction of CW by an ether:water mixture did not alter its antitumor activity, while incubation in NaOH abolished its activity almost completely. All CW preparations were found to elicit hypersensitivity reactions in Brucella-infected animals.

Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology.

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus that has had a significant economic impact on Atlantic salmon farming in Europe, North America and Chile. Monoclonal antibodies (mAbs) were developed against Segment 3 (encoding the viral nucleoprotein, NP) of the virus. Six of the mAbs were shown to be specific to ISAV and recognised all isolates from Scotland, Norway and Canada. They reacted with ISAV in enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique (IFAT) and western blotting. They were also used to develop a novel detection method based on Luminex (Bio-Plex) bead-based flow cytometric technology for the detection of ISAV in the plasma of Atlantic salmon (Salmo salar L.) smolts experimentally infected with ISAV. Fish were challenged by intraperitoneal (i.p.) injection of virus at 50% Tissue Culture Infective Dose (TCID50) = 2.8 x106 per animal. Virus present in plasma of infected fish, collected at 0, 4, 8, 12, 16, 21 and 28 days post infection using a non-lethal sampling method (n = 12 at each time point), was quantified using the optimised Bio-Plex assay. The results obtained with this assay were compared with absolute quantification of the virus by RT-qPCR using SYBR Green I and TaqMan chemistries. The Bio-Plex assay developed using the NP mAbs appears to be a rapid, sensitive method for detecting and quantifying ISAV in small volumes of fish plasma and has the potential to be multiplexed for the detection of other fish pathogens (e.g. during co-infections). To our knowledge this is the first report of the use of Luminex (Bio-Plex) technology for the detection of a fish pathogen.

N-Myc gene amplification is a major prognostic factor in localized neuroblastoma: results of the French NBL 90 study. Neuroblastoma Study Group of the Société Francaise d'Oncologie Pédiatrique.

PURPOSE: To assess the relevance of N-Myc gene amplification (NMA) as a prognostic factor in localized neuroblastoma (NB) and to evaluate whether less intensive adjuvant treatment is advisable in infants without NMA. PATIENTS AND METHODS: Assessment of NBs included clinical and imaging data to allow tumor-node-metastasis (TNM) staging, biologic determinations (N-Myc gene analysis), and standard histology and work-up to eliminate metastatic spread (metaiodobenzylguanidine [MIBG] scintigraphy and extensive bone marrow staging). Resectability was defined according to imaging findings. Chemotherapy was indicated in children older than 1 year at diagnosis who had postoperative residual disease or lymph node (LN) involvement, in infants with NMA, or as primary treatment in children with an unresectable NB, including dumbbell tumors. Radiotherapy was recommended in children older than 1 who presented with persistent gross residual disease at the end of therapy. RESULTS: Between 1990 and 1994, 316 consecutive children who presented with a localized NB were registered in the NBL 90 study. The median age was 12 months, and 42 patients had dumbbell tumors (13%). NMA was found in 22 of 225 assessable children (10%) and correlated with adverse prognostic indicators such as age older than 1 year, an abdominal primary tumor, a large tumor (T3), and unresectability. Among 186 children who had primary excision, five died of surgery-related complications. Primary chemotherapy was given to 130 patients, which allowed removal of the tumor in all but four. The 5-year overall survival (OS) and event-free survival (EFS) rates were, respectively, 91% and 84% with a median follow-up time of 36 months. The outcome of infants and older children was similar (P = .2). EFS of patients with resectable tumors was slightly better than with unresectable primary tumors (EFS, 89% v 78%; P = .02). In dumbbell NBs, neurologic recovery was achieved in 74% of cases that presented with symptoms, and initial laminectomy was avoided in 75% of children. In a univariate analysis, large tumors, high neuron-specific enolase (NSE) and lactate dehydrogenase (LDH) levels, positive LNs, macroscopic residue, and NMA adversely influenced outcome. In the multivariate analysis, NMA was the most powerful unfavorable predictive indicator: OS and EFS rates for these children were 36% and 32%, compared with 98% and 90% in nonamplified tumors (P < .001). CONCLUSION: Our data confirm the overall good prognosis of localized NBs, even when unresectable. NMA is the most relevant adverse prognostic factor in localized NBs, and more intensive treatment should be investigated in these patients. Prospective studies of other biologic factors are warranted to tailor therapy more accurately. The EFS of children who underwent primary surgery was excellent, and further justifies elimination of adjuvant treatment provided they have no NMA. Despite the elimination of postoperative therapy, infants with non-NMA tumors have an excellent outcome, which suggests that initial chemotherapy can be further reduced in case of unresectable NBs.

Diminution of circulating polymorphonuclear leucocytes by administration of a monoclonal antibody reacting with granulocytes and monocytes.

The monoclonal antibody 3A35 which binds mouse polymorphonuclear leucocytes (PMN) and monocytes was injected intravenously (i.v.) into normal mice. A great diminution of circulating PMN was observed. The percentage of PMN passed through a minimum (2.5%) 20 min after antibody injection and returned to normal value (18.3%) within 24 h. After repeated daily injections, the ability of the antibody to induce granulopenia attenuated. Moreover, mice bearing the 3A35-producing hybridoma as an ascitic tumor had a normal percentage of blood PMN and a normal granulopoiesis as judged from bone marrow cytological examination. Thus, the monoclonal antibody produced a transitory diminution of PMN but could not induce a lasting granulopenia.

Serum anti-p53 antibodies in patients with squamous cell carcinoma of the esophagus.

Immunoassay of serum anti-p53 antibodies was performed in a series of 63 patients with squamous cell carcinoma of the esophagus. p53 alterations were also analyzed with DGGE to detect gene mutations (n=53) and by immunohistochemistry to assess overexpression of p53 (n=43). An immune response was observed in 16 sera (25%). The corresponding biopsies all had a p53 gene mutation or overexpression of protein p53. We were unable to demonstrate any significant relationship between habitual tumor parameters (localization, cell differentiation, TNM stage) and development of p53 alterations. However, none of the patients with a localized tumor developed an immune response, while some of them had a muted gene or overexpressed p53.

Puerperal presentation of a living abdominal pregnancy.

BACKGROUND: Abdominal and heterotopic pregnancies appear to be increasing in incidence. CASE: We report a case of puerperal presentation of a living heterotopic pregnancy in an African woman. The patient presented 6 days postpartum with fever and abdominal pain. The correct diagnosis of heterotopic pregnancy was not considered, and for 9 days she was treated for presumed puerperal sepsis. It was only upon abdominal x-ray that the diagnosis was made. The patient underwent laparotomy with delivery of a living male neonate weighing 2000 g. He subsequently died of respiratory failure on day 3 of life. CONCLUSION: Although still rare, the increasing incidence of abdominal pregnancies in both developed and developing countries mandates awareness of this diagnosis, particularly in pregnant or postpartum women presenting with abdominal pain.

Variations in HDL and VLDL levels chronic alcoholics. Influence of the degree of liver damage and of withdrawal of alcohol.

Changes in plasma HDL and VLDL levels were investigated in 284 chronic alcoholics staying in a Detoxification Centre where they initiated or continued abstinence. The data show that the variations in plasma HDL are modulated by the degree of liver injury. In severe hepatic damage HDL levels are sharply decreased. An alcohol-induced rise in HDL can occur in the only subjects with no signs of hepatic insufficiency. This elevation is rapidly reversible after withdrawal of alcohol. Such a rise might reflect enhanced synthesis and release by liver but might also be due to an accelerated turnover of the VLDL.

Fate of mouse macrophages radiolabelled with PKH-95 and injected intravenously.

Mouse macrophages purified by elutriation from thioglycollate-induced peritoneal exudate cells were labelled with indium-111-oxine and injected intravenously into mice. A substantial amount of unbound radioactivity remained in the circulation, suggesting that the radionuclide was not stably bound to the cells. Culture experiments with radiolabelled cells showed that indium-111 was released in the medium. Another cell marker, PKH-95, an iodine-125-labelled aliphatic compound insertable into the cell membrane, bound more stably than indium-111. Five minutes after injection of 125I-PKH-95-labelled macrophages, about 98% of the cells were in a non-circulating pool. It was checked that PKH-95 labelling did not compromise the viability and functions of the macrophages and that autologous erythrocytes and blood mononuclear cells labelled with PKH-95 remained in the circulation after i.v. injection. One hour after injection, 125I-PKH-95-labelled macrophages were distributed mainly in lung (36%), liver (19%) and spleen (5%). Subsequently, radioactivity decreased in the lung while increasing in liver, spleen and in an artificially induced footpad inflammation. The radioactivity accumulation in the inflammation persisted at least for 7 days. It represented a small proportion of radioactivity injected (0.2%) but was trapped very specifically in the inflammation. This raised the hypothesis that macrophages of the non-circulating pool could be released in the circulation and recruited into the inflammation with slow kinetics.

111Indium-F(ab)'2-NCA 102 monoclonal antibody: in vitro study of a specific agent for the detection of inflammatory foci.

A new antigranulocyte antibody was evaluated in vitro for the detection of inflammatory foci in man. The specificity for polymorphonuclear cells (PMN) of NCA 102, an anti-NCA 95 monoclonal IgG1, was determined with immunohistochemical and cytofluorometrical tests. Its affinity, assessed by Scatchard analysis, was 1.1 x 10(9) L/mol and the number of epitopes per granulocyte reached about 10(5). The biological properties of PMNs incubated with NCA 102 were not inhibited even when coupled with DTPA. A F(ab)'2 fragment was radiolabelled with 111Indium and incubated in the presence of whole blood. More than 65% radioactivity was selectively taken up by the PMN population. These findings indicated that NCA 102 antibody is suitable for sepsis detection.

Expression of MAGE genes in esophageal squamous-cell carcinoma.

The genes MAGE-1, -2, -3 and -4 are expressed in tumors of different histological types, but not in normal tissues, with the exception of testis and placenta. Short peptides derived from MAGE-1 and MAGE-3 gene products are recognized by cytolytic T lymphocytes when presented by HLA-class-I molecules, and represent potential targets for specific immunotherapy. We have determined whether esophageal carcinoma patients should be eligible for MAGE-peptide-based vaccine therapies. The expression of genes MAGE-1, -2, -3 and -4 in tumor samples was assessed by reverse-transcription and polymerase-chain-reaction amplification. Out of the 49 esophageal squa-mous-cell carcinomas studied, 53% expressed MAGE-1, 49% MAGE-2, 47% MAGE-3 and 71% MAGE-4. Eighty-four percent of the tumors expressed one or more of the four MAGE genes. Owing to the high incidence of MAGE gene expression in esophageal squamous-cell carcinoma, a large proportion of patients could be suitable candidates for immune therapies involving tumor-specific antigens encoded by MAGE genes.