ABSTRACT A new molecular subtyping approach was developed which is based on the amplification and sequencing of a repetitive region of the P1 gene of Mycoplasma pneumoniae. It allows the differentiation of all known subtypes and variants of M. pneumoniae as well as the identification of new subtypes directly in clinical samples to characterize endemic and epidemic M. pneumoniae infections.

The presymptomatic diagnosis of malignant hyperthermia (MH) susceptibility is based on the in vitro contracture test in Europe and the caffeine–halothane contracture test in North America. Both tests are invasive in requiring an open muscle biopsy undertaken in a specialized center. There are 23 MH investigation centers in Europe, but testing facilities are limited to only 6 centers in the United States.1,2The locus of the ryanodine receptor of skeletal muscle on chromosomes 19q 13.1 has been shown to link to MH, and several mutations have been identified on this gene.3Molecular genetic diagnosis of MH susceptibility in persons from MH families with identified mutations in the ryanodine receptor gene has recently been introduced.1,4A 29-yr-old woman contacted our MH investigation unit to be tested for MH susceptibility. Being pregnant, she was concerned about a possible cesarean delivery because she had a history of a clinical MH episode. She underwent adenotomy as a 6-yr-old child in 1982. The clinical and laboratory findings on the clinical grading scale for MH episodes ranked the episode in the highest category.5The child was discharged without permanent sequelae on the 11th postoperative day. In 1986, MH testing became available in Switzerland, and an open muscle biopsy and in vitro contracture test were performed on her parents rather than on the child, because she did not consent to being tested. Although the father was MH negative, the mother was MH susceptible. As part of our research program, the frozen muscle biopsy of the mother was subsequently investigated for MH-associated mutations in the ryanodine receptor gene. Mutation G2434R, known to be causative for MH, was identified. Therefore, the patient contacting our center was tested for this mutation and found to be carrier of G2434R. She agreed to have the umbilical blood of her baby taken at labor, to have the newborn assessed for MH susceptibility. Vaginal delivery was uneventful, with the mother receiving lumbar epidural analgesia with 0.12% ropivacaine and 2 μg/ml fentanyl. Umbilical cord blood was collected in an EDTA tube and sent to the Swiss MH investigation unit. DNA was automatically extracted using the MagNA Pure DNA isolation kit I (Roche Diagnostics, Rotkreuz, Switzerland). Exon 45 of the ryanodine receptor gene was amplified by polymerase chain reaction using the following primer set: forward: AGA ACG CCA ATG TGG TGG T; reverse: CTG CAT GAG GCG TTC AAA G. Presence of the mutation was proven by an automated sequencing technique (Applied Biosystems, Rotkreuz, Switzerland) and is shown in figure 1. The results were discussed in detail with the mother, and she was given a warning card about the MH status of her newborn son.This is the first report of MH susceptibility in a newborn by molecular genetic testing of umbilical cord blood. Although knowledge of MH status may be of lesser importance during daily life, it is valuable to confirm or exclude MH susceptibility before surgery in individuals from families with known MH susceptibility. Although elective procedures can be performed during either regional anesthesia or intravenous anesthesia without triggering agents, patients are likely to be exposed to inhalation agents or succinylcholine during emergency or obstetric interventions, and volatile anesthetics are preferred in pediatric anesthesia, because venous access can be established after induction of anesthesia.Muscle biopsy and contracture testing must be performed in specialized centers and may not be readily available. Most MH centers do not perform biopsies in infants and children because of the limited availability of skeletal muscle. Therefore, the MH status of this newborn would have remained unknown for at least the first decade of his life.This report emphasizes some significant points:* University Hospital Basel, Basel, Switzerland. thierry.girard@unibas.ch

Wilson's disease is a rare autosomal recessively inherited disorder of copper metabolism resulting in accumulation of copper in many organs. The Kayser-Fleischer corneal ring represents a typical symptom in Wilson patients. ATP7B, the gene product of the Wilson's disease gene, is expressed in hepatocytes and plays a critical role in copper transport and secretion. Mutations encompassing this gene leading to amino acid substitutions reduce the affinity of the ATPase enzyme to copper and prevent the transport of copper through the hepatocytes into the bile duct. More than 200 mutations in the 21 coding exons of the Wilson's disease gene ATP7B have been described, the most common European one being Hisl069Gln (30-60% in European patients). Wilson's disease may present with various clinical symptoms, commonly as liver and neuropsychiatric disease. The diagnosis of Wilson's disease (prevalence 1:30000) is based on a combination of clinical and neurological symptoms (Kayser-Fleischer corneal ring, MRI), a multi-parametric clinical chemistry panel (ceruloplasmin, 24 h-copper excretion in urine, serum copper) and molecular genetic analysis of ATP7B gene mutations. Symptomatic patients are treated with the chelating agents D-penicillamine or trientine, and in a second phase often with zinc. Lifelong therapy is required and provides life expectancy near to normal. Here, we report on the long-term follow-up of a 30-year-old Wilson patient displaying a compound heterozygosity of His1069Gln -Asnl270Ser mutations in the ATP7B gene as diagnosed by LightCycler real time polymerase chain reaction and DNA sequencing of exon 18. Molecular family-pedigree analysis over four generations revealed seven additional heterozygous mutation carriers. Treatment of the index patient with high doses of chelating agent D-penicillamine (1500 mg/d) in combination with zinc (150 mg/d) could reverse copper encephalopathy to normal and markedly reduced neurological symptoms. Kayser-Fleischer corneal ring was persistent. Wilson's disease should be taken into consideration in case of any liver disease of unknown origin or neuropsychiatric symptoms. Current diagnostic panels (international scoring system) encompassing clinical chemistry and molecular genetics for screening and confirmation of Wilson's disease are discussed.

BACKGROUND:Treatment of HIV-1-infected individuals with antiretrovirals can result in sustained suppression of plasma viral RNA at concentrations below the detection limit of available assays. However, continuing virus replication has been detected in patients with viral RNA in plasma suppressed for months to years, and many cell types are known to act as reservoirs or carriers for the virus. In vitro, erythrocytes bind HIV-1 immune complexes, so we tested for a circulating pool of HIV-1 associated with erythrocytes in people with HIV-1 infection.METHODS:We investigated 82 chronically HIV-1-infected individuals. Plasma, white cells, and erythrocytes were tested for HIV-1 RNA by RT-PCR.FINDINGS:Erythrocyte-associated HIV-1 RNA was detected in 80 of 82 individuals. In 23, plasma HIV-1 RNA had been undetectable (<20 copies/mL) for up to 32 months; in corresponding erythrocyte samples, there were up to 82878 HIV-1 RNA copies per mL whole blood. HIV-1 associated with erythrocytes in vivo was shown to be infectious. Within the subgroup of patients with undetectable plasma viral load, higher numbers of HIV-1 associated with erythrocytes were correlated with a history of advanced clinical stages of HIV-1 infection (p=0.014).INTERPRETATION:A pool of HIV-1 is associated with erythrocytes even after long-term suppression of viral RNA in plasma. This finding is direct evidence for continuing virus replication or release in these individuals. Quantification of this viral pool may help to judge suppression of HIV-1 replication in individuals with undetectable plasma HIV-1 RNA.

In this study, a computer-assisted reverse immunology approach was utilized in order to identify potentially antigenic peptides derived from the differentiation antigen TRP-2, a melanosomal protein frequently expressed in melanoma. Among the seven peptides complying with HLA-A2.1-binding motifs, two induced specific CD8(+) cytotoxic T lymphocytes. HLA-A2.1(+) melanoma cells expressing TRP-2 were lysed by clones specific for TRP-2(360-368) (TLDSQVMSL) peptide, thus identifying it as a naturally processed epitope. Other T-cell clones directed against TRP-2(476-484) (VMGTLVALV) were unable to lyse HLA-matched TRP-2(+) cell lines. The role of intracellular proteolytic processing in the generation of this epitope was investigated by transfecting mini-genes encoding the TRP-2(476-484) peptide alone or carrying N- or C-terminal extensions. Specific T-cell clones recognized target cells expressing the cytotoxic T-lymphocyte (CTL)-defined epitope or its C-terminally extended precursor, but failed to recognize cells expressing the N-terminally extended TRP-2(476-484) peptide, suggesting the presence of a negative processing signal (NPS). Regarding C-terminus-flanking regions, mutational analysis indicates that the GLY485 residue plays a key role in the processing of the TRP-2(476-484) epitope. Interestingly, proteasome inhibitors preventing the generation of the MART-1/Melan-A(27-35) immunodominant melanoma tumor-associated antigen (TAA) promoted detectable presentation of TRP-2(476-484) epitope in HLA-A2.1(+) and TRP-2(+) tumor lines, as witnessed by cytokine release by specific T-cell clones.

C-type lectin-like inhibitory receptors are heterodimers consisting of CD94 and NKG2-A-B molecules expressed on NK cells and on a subset of activated T lymphocytes. Their inhibitory effects on NK cytotoxicity and on the NK-like activity of T cell clones have been demonstrated, but no data are currently available on antigen-specific class I-restricted cytotoxic T lymphocytes (CTL). We have generated a panel of HLA-A2.1-restricted CTL clones directed against a nonapeptide derived from a melanoma-associated antigen, dopachrome tautomerase (TRP-2). All clones were CD8+ and TCR alphabeta+. About half of them expressed a CD94bright phenotype, whereas the remaining were CD94dim. Only the CD94bright CTL expressed the NKG2-A-B gene, consistent with the expression of a C-type, lectin-like, inhibitory CD94/NKG2-A-B heterodimer. Both CD94bright and CD94dim clones appeared to require similar amounts of synthetic epitope sensitizing target cells. Addition of anti-CD94 mAb resulted in a significant increase of specific killing by CD94bright, but not by CD94dim clones in the presence of suboptimal concentrations of peptide, whereas, when optimal amounts were used, the mAb did not induce a significant modulation of the cytotoxicity. Antigen-induced inward [Ca2+]i fluxes were unaffected, but an enhancement of TCR down-modulation could be observed in the presence of anti-CD94 mAb at high concentration of antigenic peptide. The analysis of the TCR-Vbeta repertoire of the CTL clones by RT-PCR and immunofluorescence revealed that all clones regardless of CD94 phenotype shared Vbeta22 expression. Most importantly, sequence analysis showed that they all expressed identical Vbeta22 TCR rearranged with Jbeta2.1 and Cbeta2. Taken together, these data indicate that different expression of functionally active lectin-like inhibitory receptors can be detected in CTL clones sharing identical TCR sequence and peptide specificity.

In this work, we addressed the possibility to enhance the "in vitro" generation of CTLs recognizing tumor-associated antigens (TAAs) by using an inactivated recombinant vaccinia virus encoding B7.1 and B7.2 costimulatory molecules (rVV-B7.1/2). Antigen presenting cells (APCs) infected by rVV-B7.1/2 and pulsed with MART-1/Melan-A27-35 HLA-A2.1-restricted peptide induced significantly higher specific cytotoxic activity than peptide-loaded APCs infected by wild-type VV, both in VV-sensitized and naive donors. When APCs were infected with a rVV encoding both MART-1/Melan-A27-35 and B7-1/2 (rVV-B7.1/2-M), a significantly more effective CTL generation was observed as compared with cultures stimulated by APCs infected with a rVV encoding the TAA epitope only (rVV-M). These enhancing effects were detectable irrespective of a previous VV-specific sensitization. Most importantly, fibroblasts, devoid of antigen-presenting capacity upon peptide pulsing or infection with rVV-M, could be turned into effective APCs after infection by rVV encoding TAA epitopes and costimulatory molecules. In these experiments, by using separate recombinant viral constructs, we observed a predominant role of B7-1 as compared with B7-2 in the induction of TAA-specific CTLs. Taken together, our data indicate that replication-incompetent rVV encoding TAA epitopes and costimulatory molecules are able to induce highly effective generation of tumor-specific CTLs. Therefore, these vectors could represent valuable clinical tools for immunotherapy of melanoma patients.

Active specific immunotherapy targeting tumor-associated antigens (TAA) requires reagents of high immunogenicity and safety. To address this issue, we constructed a recombinant vaccinia virus carrying a minigene insert encoding the HLA-A2.1-restricted MART-1/Melan-A27-35 melanoma TAA (rVV-M). To facilitate the entry of the antigenic epitope into the endoplasmic reticulum, a sequence coding for adenovirus E3/19K leader peptide was added. This rVV-M was made replication-incompetent by treatment with psoralen and UV light. Infection with rVV-M rendered HLA-A2.1 EBV-transformed lymphoblastoid cells sensitive to the cytotoxic effects of HLA-class-I-restricted, MART-1/Melan-A27-35-specific cytotoxic T lymphocytes (CTL). The capacity of rVV-M to generate HLA-A2.1-restricted MART-1/Melan A-specific CTL was demonstrated from tumor-infiltrating-lymphocyte (TIL) cultures and from healthy donors' peripheral-blood mononuclear cells (PBMC). MART-1/Melan-A27-35-specific CTL were generated from TIL after 2 weekly stimulation courses. Infection with rVV-M elicited a higher CTL response than addition of exogenous peptide, whereas, when a similar protocol was used to stimulate PBMC of healthy donors, significant and specific cytotoxic activity could be observed only upon rVV-M infection but not upon exogenous peptide addition. All CTL generated upon rVV-M stimulation were also able to efficiently kill melanoma cell lines expressing both MART-1/Melan-A and HLA-A2.1. In addition, TNF-α production could be induced in rVV-M-stimulated CTL upon co-culture with COS-7 cells transiently transfected with MART-1/Melan-A and HLA-A2.1 genes. This safe and highly immunogenic reagent could be of use in TAA-targeted clinical immunotherapy. Int. J. Cancer 71:491-496, 1997. © 1997 Wiley-Liss Inc.

International Journal of CancerVolume 71, Issue 6 p. 1119-1121 Letter to the EditorFree Access High expression of MAGE-3 protein in squamous-cell lung carcinoma Claude Fischer, Claude Fischer Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorFred Gudat, Fred Gudat Department of Pathology, University of Basel, Basel, SwitzerlandSearch for more papers by this authorPeter Stulz, Peter Stulz Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorChristoph Noppen, Christoph Noppen Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorChristoph Schaefer, Christoph Schaefer Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorPaul Zajac, Paul Zajac Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorMarkus Trutmann, Markus Trutmann Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorThomas Kocher, Thomas Kocher Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorMarkus Zuber, Markus Zuber Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorFelix Harder, Felix Harder Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorMichael Heberer, Michael Heberer Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorGiulio C. Spagnoli, Corresponding Author Giulio C. Spagnoli Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSurgical Research Laboratory, Z.L.F., 20, Hebelstrasse, 4031, Basel, Switzerland. Fax:—41 61 265 3990.Search for more papers by this author Claude Fischer, Claude Fischer Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorFred Gudat, Fred Gudat Department of Pathology, University of Basel, Basel, SwitzerlandSearch for more papers by this authorPeter Stulz, Peter Stulz Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorChristoph Noppen, Christoph Noppen Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorChristoph Schaefer, Christoph Schaefer Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorPaul Zajac, Paul Zajac Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorMarkus Trutmann, Markus Trutmann Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorThomas Kocher, Thomas Kocher Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorMarkus Zuber, Markus Zuber Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorFelix Harder, Felix Harder Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorMichael Heberer, Michael Heberer Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSearch for more papers by this authorGiulio C. Spagnoli, Corresponding Author Giulio C. Spagnoli Departments of Surgery and Research, University of Basel, Basel, SwitzerlandSurgical Research Laboratory, Z.L.F., 20, Hebelstrasse, 4031, Basel, Switzerland. Fax:—41 61 265 3990.Search for more papers by this author First published: 06 December 1998 https://doi.org/10.1002/(SICI)1097-0215(19970611)71:6<1119::AID-IJC34>3.0.CO;2-5Citations: 26AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume71, Issue611 June 1997Pages 1119-1121 ReferencesRelatedInformation

Genes of the MAGE family encode tumor-specific antigens recognized by cytotoxic T-lymphocytes in a variety of neoplasms. We investigated the protein expression of these antigens as related to the gene expression, in esophageal squamous cell carcinoma by using monoclonal antibodies recognizing MAGE gene products. Esophageal squamous cell carcinomas were found to express both MAGE-1 (4 out of 15 samples) and MAGE-3 (7 out of 15 samples) genes, by RT-PCR. Immunoblotting revealed MAGE-1 and MAGE-3 gene products in 2 and 6 out of 15 samples, respectively. Immunohistochemistry performed on 12 samples showed MAGE-1 protein expression, limited to single tumor cells, in 2 cases. MAGE-3 gene product was detectable in 7 cases: in 5 of them over 50% of neoplastic cells were positive. Considering the high percentages of tumor cells expressing MAGE-3 antigen, the use of epitope-based vaccines could be envisaged in patients displaying appropriate HLA-class I phenotype.

OBJECTIVE:The authors' objective is to identify MAGE-1 tumor antigen in clinical melanoma specimens and to verify the extent of its expression in tumors where evidence of specific gene transcripts can be obtained.BACKGROUND DATA:The MAGE-1 gene encodes a tumor-associated antigen that can be recognized by specific cytotoxic T lymphocytes. Transcription of the MAGE-1 gene has previously been demonstrated in various malignancies, but the production of the specific gene product and its distribution in neoplastic tissues have not yet been addressed.METHODS:Total cellular mRNA was extracted from six melanoma biopsies, reverse-transcribed and tested in 25-45 cycles of reverse polymerase chain reaction (rtPCR) in the presence of primers' pairs specific for the beta-actin-positive control gene and for the MAGE-1-encoding gene. Concurrently, portions of these specimens were lysed and probed for MAGE-1 protein by immunoblotting. Additional material from the same biopsies was analyzed following immunohistological staining with MAGE-1-specific monoclonal antibodies.RESULTS:MAGE-1 gene transcription could be demonstrated following 25 cycles of rtPCR in one out of six biopsies and in three more following 35 cycles of rtPCR. 2/6 samples were negative even after 45 cycles of rtPCR. MAGE-1 protein production could be detected by immunoblotting in the lysates from biopsies showing evidence of specific gene transcription. Cells positive for MAGE-1 protein expression could be identified by immunohistochemistry on snap-frozen sections in three of the four tumors displaying specific transcripts. Distribution of positivity ranged between focal cellular areas and single positive cells in the different tumors.CONCLUSIONS:The MAGE-1 tumor antigen can be detected by specific monoclonal antibodies in clinical tumor specimens. The pattern of positivity observed in samples showing evidence of MAGE-1 gene expression suggests a relevant heterogeneity regarding MAGE-1 antigen production within individual tumor specimens.

The MAGE-1 gene encodes a protein encompassing a HLA-A1-restricted target epitope for cytolytic T lymphocytes. Monoclonal antibodies directed against the MAGE-1 protein were tested for usage in immunohistology of routine pathology material. Seven formalinfixed, paraffin-embedded malignant melanomas were studied by the Avidin-Biotin complex (ABC) method with or without different antigen retrieval methods. Native, frozen tissues from the same tumours were used to validate the results by immunohistochemistry on frozen sections, by PCR for mRNA and by protein demonstration in tissue extracts using western blotting. Of 4 monoclonal antibodies tested, mAB 34B and mAB 77B were highly efficient in detecting MAGE-1 protein in deparaffinised sections with the regular ABC method after microwave pretreatment. In a series of an additional 28 patients 75% expressed MAGE-1, 50% in a substantial proportion. Follow-up studies in 6 patients indicate that the expression pattern remains stable but may change substantially within a short range. Immunohistology is thus a rapid and well-established method that might be used to select and monitor HLA-A1 positive patients with malignant melanoma and other candidate tumours for MAGE-1-directed immuno-therapy.

BioTechniquesVol. 21, No. 3 BenchmarksOpen AccessIsolation of Multiple mRNAs from a Few Eukaryotic Cells: A Fast Method to Obtain Templates for RT-PCRChristoph Noppen, Giulio C. Spagnoli & Christoph SchaeferChristoph NoppenUniversity of Basel, Basel, Switzerland, Giulio C. SpagnoliUniversity of Basel, Basel, Switzerland & Christoph Schaefer*Address correspondence to Christoph Schaefer, Departments of Surgery and Research, University of Basel, Hebelstr. 20, CH-4031 Basel, Switzerland.University of Basel, Basel, SwitzerlandPublished Online:2 Aug 2018https://doi.org/10.2144/96213bm11AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByOptimal Detection of Sentinel Lymph Node Metastases by Intraoperative Radioactive Threshold and Molecular Analysis in Patients with Melanoma16 October 2008 | Journal of Nuclear Medicine, Vol. 49, No. 11Molecular staging of the sentinel lymph node in melanoma patients: correlation with clinical outcomeAnnals of Oncology, Vol. 16, No. 11Modulating T-cell immunity to tumours: new strategies for monitoring T-cell responsesNature Reviews Cancer, Vol. 2, No. 6Affinity chromatography8 December 2009Naturally processed and concealed HLA-A2.1-restricted epitopes from tumor-associated antigen tyrosinase-related protein-21 January 2000 | International Journal of Cancer, Vol. 87, No. 2Phase I study in melanoma patients of a vaccine with peptide-pulsed dendritic cells generatedin vitro from CD34+ hematopoietic progenitor cellsInternational Journal of Cancer, Vol. 86, No. 3C-type lectin-like receptors in peptide-specific HLA class I-restricted cytotoxic T lymphocytes: differential expression and modulation of effector functions in clones sharing identical TCR structure and epitope specificityEuropean Journal of Immunology, Vol. 28, No. 4Effects of Dopamine Receptor Antisense RNA Expression Vectors in The Nervous SystemReverse transcription–polymerase chain reaction–based methodology to quantify differential gene expression directly from microdissected regions of frozen tissue sectionsMolecular Carcinogenesis, Vol. 20, No. 3 Vol. 21, No. 3 Follow us on social media for the latest updates Metrics Downloaded 30 times History Published online 2 August 2018 Published in print September 1996 Information© 2018 Author(s)PDF download

Several novel myelin-associated/oligodendrocytic basic protein (MOBP) isoforms were identified in this study by cDNA cloning. They are small, highly basic polypeptides comprising 69, 81, and 99 amino acids (8.2, 9.7, and 11.7 kDa, respectively) and show no significant homology with described proteins or domain structures. All (as yet) identified MOBP isoforms are identical in amino acids 1-68 but differ in the length and polarity of the C-terminal region. One isoform, designated MOBP81, was shown to be expressed abundantly during development. Interestingly, MOBP81 has a significant clustering of positively charged residues at positions 69-81, a feature that also has been observed for myelin basic protein (MBP) and Po. As demonstrated by in situ hybridization, MOBP gene expression occurs during development of the rat optic nerve later than that of MBP and proteolipid protein and coincides exactly with the beginning of myelin compaction. The 2.6 kb MOBP81-A transcript is localized in the processes of oligodendrocytes, whereas the 3.8 kb MOBP81-B transcript is restricted to the perinuclear region. Therefore, MOBP81-A and related mRNAs seem to be transported to the periphery of the oligodendrocytes, as is known for the transcripts of the MBP gene. The late developmental expression of the MOBP gene suggests that the MOBP proteins act at the late steps of myelin formation, possibly in myelin compaction and in the maintenance of the myelin sheath.

We have isolated several new genes that are specifically expressed by oligodendrocytes in the CNS. This was achieved by differential screening of a rat spinal cord cDNA library with probes derived from normal and from oligodendrocyte-free spinal cord mRNAs. Four of these genes are exclusively expressed by oligodendrocytes: Three of these are not related to known genes, whereas one encodes the myelin oligodendrocyte glycoprotein (MOG). Four other genes are expressed by oligodendrocytes as well as by Schwann cells. One gene codes for apolipoprotein D, which is thought to be involved in lipid metabolism. A second cDNA sequence codes for the recently identified galactosylceramide-synthesizing enzyme UDP-galactose:ceramide galactosyltransferase. The third gene encodes a small protein with four putative transmembrane domains that is related to a T-lymphocyte-specific membrane protein, MAL. The fourth gene encodes the rat homologue of the stearyl-CoA-desaturase 2 (SCD2) gene, which is specifically expressed in the nervous system and involved in the synthesis and regulation of long-chain unsaturated fatty acids essential for myelination. Finally, we found that a member of the beta-tubulin family is highly expressed in oligodendrocytes as well as neurons. The identification of several new proteins that may play a role in myelin synthesis and sheath formation will lead to new insight into this complex mechanism.

The human MAGE-3 gene encodes a melanoma antigenic epitope recognized by specific cytotoxic T lymphocytes, but its gene product has not been identified thus far. We produced a recombinant MAGE-3 gene product by expression cloning of the entire reading frame in the context of a fusion protein characterized by a 10-histidine tail, allowing purification by metal chelation on a nickel Sepharose column. The semipurified product was used to generate MAGE-3-specific monoclonal antibodies. One reagent could identify by immunoblotting the native MAGE-3 gene product as a M(r) 48,000 protein in lysates of cell lines showing evidence of MAGE-3 gene expression. No apparent cross-reactivity with recombinant or native MAGE-1 gene product was observed. Immunohistochemistry shows that, closely resembling the MAGE-1 gene product, MAGE-3 is a cytoplasmic protein.

Peptide specificity of cultured tumor-infiltrating lymphocytes (TIL) was systematically investigated in a group of HLA-A2.1+ metastatic melanoma patients consecutively referred to our department for surgical treatment. Seven samples from 6 patients were studied. All surgical specimens showed evidence of gp 100, MART-1/Melan-A and Tyrosinase gene expression as detectable by reverse PCR (rPCR). Cultured TIL from 2 patients displayed cytotoxic activity against autologous or HLA-matched EBV-transformed cells previously pulsed with MART-1/Melan-A27-35 peptide. In contrast, no CTL activity against gp100(280-288) or tyrosinase1-9 peptides could be observed. TIL were then repeatedly stimulated in vitro with the same peptides. After 6 restimulation courses at weekly intervals, specific recognition of gp100(280-288) and MART-1/Melan-A peptides was detectable in 3 and 5 TIL populations, respectively. In one case Tyrosinase1-9-specific CTL could be demonstrated. Two TIL populations from metastases resected from a melanoma patient at 6 months' distance showed a different peptide specificity pattern, and no specific CTL could be generated from simultaneously sampled peripheral blood mononuclear cells (PBMC). All peptide-specific CTL populations also displayed significant cytotoxic activity against HLA-A2.1 matched melanoma cell lines expressing the antigens under investigation. Our data indicate that CTL specific for MART-Melan-A27-35, gp100(280-288) or Tyrosinase1-9 peptides could be expanded with varying frequency from TIL derived from 4 out of 6 HLA-A2.1+ patients whose tumors expressed the genes encoding these tumor-associated antigens (TAA).