Molecular mechanisms involved in the activation and regulation of the alpha 1-adrenergic receptor subtypes.

The adrenergic receptors (ARs) belong to the superfamily of membrane-bound G protein coupled receptors (GPCRs). Our investigation has focused on the structure-function relationship of the alpha 1b-AR subtype used as the model system for other GPCRs. Site-directed mutagenesis studies have elucidated the structural domains of the alpha 1b-AR involved in ligand binding, G protein coupling or desensitization. In addition, a combined approach using site-directed mutagenesis and molecular dynamics analysis of the alpha 1b-AR has provided information about the potential mechanisms underlying the activation process of the receptor, i.e. its transition from the 'inactive' to the 'active' conformation.

Characterization of cocoa butter and cocoa butter equivalents by bulk and molecular carbon isotope analyses: Implications for vegetable fat quantification in chocolate

The fatty acids from cocoa butters of different origins, varieties, and suppliers and a number of cocoa butter equivalents (Illexao 30-61, Illexao 30-71, Illexao 30-96, Choclin, Coberine, Chocosine-Illipe, Chocosine-Shea, Shokao, Akomax, Akonord, and Ertina) were investigated by bulk stable carbon isotope analysis and compound specific isotope analysis. The interpretation is based on principal component analysis combining the fatty acid concentrations and the bulk and molecular isotopic data. The scatterplot of the two first principal components allowed detection of the addition of vegetable fats to cocoa butters. Enrichment in heavy carbon isotope (C-13) of the bulk cocoa butter and of the individual fatty acids is related to mixing with other vegetable fats and possibly to thermally or oxidatively induced degradation during processing (e.g., drying and roasting of the cocoa beans or deodorization of the pressed fat) or storage. The feasibility of the analytical approach for authenticity assessment is discussed.

Desensitization and phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol 12-myristate 13-acetate.

Glucagon-like peptide-1 (GLP-1) stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor linked to activation of the adenylyl cyclase pathway. Here, using insulinoma cell lines, we studied homologous and heterologous desensitization of GLP-1-induced cAMP production. Preexposure of the cells to GLP-1 induced a decrease in GLP-1-mediated cAMP production, as assessed by a 3- to 5-fold rightward shift of the dose-response curve and an approximately 20 percent decrease in the maximal production of cAMP. Activation of protein kinase C by the phorbol ester phorbol 12-myristate 13-acetate (PMA) also induced desensitization of the GLP-1-mediated response, leading to a 6- to 9-fold shift in the EC50 and a 30% decrease in the maximal production of cAMP. Both forms of desensitization were additive, and the protein kinase C inhibitor RO-318220 inhibited PMA-induced desensitization, but not agonist-induced desensitization. GLP-1- and PMA-dependent desensitization correlated with receptor phosphorylation, and the levels of phosphorylation induced by the two agents were additive. Furthermore, PMA-induced, but not GLP-1-induced, phosphorylation was totally inhibited by RO-318220. Internalization of the GLP-1 receptor did not participate in the desensitization induced by PMA, as a mutant GLP-1 receptor lacking the last 20 amino acids of the cytoplasmic tail was found to be totally resistant to the internalization process, but was still desensitized after PMA preexposure. PMA and GLP-1 were not able to induce the phosphorylation of a receptor deletion mutant lacking the last 33 amino acids of the cytoplasmic tail, indicating that the phosphorylation sites were located within the deleted region. The cAMP production mediated by this deletion mutant was not desensitized by PMA and was only poorly desensitized by GLP-1. Together, our results indicate that the production of cAMP and, hence, the stimulation of insulin secretion induced by GLP-1 can be negatively modulated by homologous and heterologous desensitization, mechanisms that involve receptor phosphorylation.

Anti-CTLA-4 therapy broadens the melanoma-reactive CD8+ T cell response.

Anti-CTLA-4 treatment improves the survival of patients with advanced-stage melanoma. However, although the anti-CTLA-4 antibody ipilimumab is now an approved treatment for patients with metastatic disease, it remains unknown by which mechanism it boosts tumor-specific T cell activity. In particular, it is unclear whether treatment amplifies previously induced T cell responses or whether it induces new tumor-specific T cell reactivities. Using a combination ultraviolet (UV)-induced peptide exchange and peptide-major histocompatibility complex (pMHC) combinatorial coding, we monitored immune reactivity against a panel of 145 melanoma-associated epitopes in a cohort of patients receiving anti-CTLA-4 treatment. Comparison of pre- and posttreatment T cell reactivities in peripheral blood mononuclear cell samples of 40 melanoma patients demonstrated that anti-CTLA-4 treatment induces a significant increase in the number of detectable melanoma-specific CD8 T cell responses (P = 0.0009). In striking contrast, the magnitude of both virus-specific and melanoma-specific T cell responses that were already detected before start of therapy remained unaltered by treatment (P = 0.74). The observation that anti-CTLA-4 treatment induces a significant number of newly detected T cell responses-but only infrequently boosts preexisting immune responses-provides strong evidence for anti-CTLA-4 therapy-enhanced T cell priming as a component of the clinical mode of action.

RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium and biocontrol model organism Pseudomonas fluorescens CHA0, the GacS/GacA two-component system upregulates the production of biocontrol factors, i.e. antifungal secondary metabolites and extracellular enzymes, under conditions of slow, non-exponential growth. When activated, the GacS/GacA system promotes the transcription of a small regulatory RNA (RsmZ), which sequesters the small RNA-binding protein RsmA, a translational regulator of genes involved in biocontrol. The gene for a second GacA-regulated small RNA (RsmY) was detected in silico in various pseudomonads, and was cloned from strain CHA0. RsmY, like RsmZ, contains several characteristic GGA motifs. The rsmY gene was expressed in strain CHA0 as a 118 nt transcript which was most abundant in stationary phase, as revealed by Northern blot and transcriptional fusion analysis. Transcription of rsmY was enhanced by the addition of the strain's own supernatant extract containing a quorum-sensing signal and was abolished in gacS or gacA mutants. An rsmA mutation led to reduced rsmY expression, via a gacA-independent mechanism. Overexpression of rsmY restored the expression of target genes (hcnA, aprA) to gacS or gacA mutants. Whereas mutants deleted for either the rsmY or the rsmZ structural gene were not significantly altered in the synthesis of extracellular products (hydrogen cyanide, 2,4-diacetylphloroglucinol, exoprotease), an rsmY rsmZ double mutant was strongly impaired in this production and in its biocontrol properties in a cucumber-Pythium ultimum microcosm. Mobility shift assays demonstrated that multiple molecules of RsmA bound specifically to RsmY and RsmZ RNAs. In conclusion, two small, untranslated RNAs, RsmY and RsmZ, are key factors that relieve RsmA-mediated regulation of secondary metabolism and biocontrol traits in the GacS/GacA cascade of strain CHA0.

Characterization of the Morphological and Molecular Changes Associated with Diabetic Peripheral Neuropathy in Type 2 Diabetes

More than 246 million individuals worldwide are affected by diabetes mellitus (DM) and this number is rapidly increasing (http://www.eatlas. idf.org). 90% of all diabetic patients have type 2 DM, which is characterized by insulin resistance and b-cell dysfunction. Even though diabetic peripheral neuropathy (DPN) is the major chronic complication of DM its underlying pathophysiological mechanisms still remain unknown. To get more insight into the DPN associated with type 2 DM, we characterized the rodent model of this form of diabetes, the db/db mice. The progression of pathological changes in db/db mice mimics the ones observed in humans: increase of the body weight, insulin insensitivity, elevated blood glucose level and reduction in nerve conduction velocity (NCV). Decreased NCV, present in many peripheral neuropathies, is usually associated with demyelination of peripheral nerves. However, our detailed analysis of the sciatic nerves of db/db mice exposed for 4 months to hyperglycemia, failed to reveal any signs of demyelination in spite of significantly reduced NCV in these animals. We therefore currently focus our analysis on the structure of Nodes of Ranvier, regions of intense axo-glial interactions, which also play a crucial role in rapid saltatory impulse conduction. In addition we are also evaluating molecular changes in somas of sensory neurons projecting through sciatic nerve, which are localized in the dorsal root ganglia. We hope that the combination of these approaches will shed light on molecular alterations leading to DPN as a consequence of type 2 DM.

Phylogenetic relationships among four species of the guarani group of Drosophila (Diptera, Drosophilidae) as inferred by molecular and morphological analyses

Traditionally, the Drosophila guarani species group has been divided into two subgroups: the guarani and the guaramunu subgroups. Two, out of the four species included in this research, are members of the guarani subgroup (D. ornatifrons Duda, 1927 and D. subbadia Paterson & Mainland, 1943) and two are included in the guaramunu subgroup (D. maculifrons Duda, 1927 and D. griseolineata Duda, 1927). However, some authors have suggested that D. maculifrons and D. griseolineata are much closer to some species of the Drosophila tripunctata group than to some of the species of the guarani group. To add new data to the matter under dispute, Polyacrylamide Gel Eletrophoresis (PAGE-SDS) was used for the analysis and comparison of protein composition and Random Amplified Polymorphic DNA (RAPD) analysis to find differences in genomic DNA, in addition to the analysis of quantitative morphological characters previously described. Analysis of PAGE-SDS results in a dendrogram that pointed out D. subbadia as being the most distant within the Drosophila guarani group. However, these results were not supported either by RAPD analysis or by the analysis of continuous morphological characters, which supplied the clustering of D. subbadia with D. ornatifrons. Although our data give strong support to the clustering of D. subbadia and D. ornatifrons, none of the dendrograms provided a clade comprising D. maculifrons and D. griseolineata. Thus, this research does not support the traditional subdivision of the D. guarani group into those two subgroups.

Phylogeographical structure, postglacial recolonization and barriers to gene flow in the distinctive Valais chromosome race of the common shrew (Sorex araneus).

Using one male-inherited and eight biparentally inherited microsatellite markers, we investigate the population genetic structure of the Valais chromosome race of the common shrew (Sorex araneus) in the Central Alps of Europe. Unexpectedly, the Y-chromosome microsatellite suggests nearly complete absence of male gene flow among populations from the St-Bernard and Simplon regions (Switzerland). Autosomal markers also show significant genetic structuring among these two geographical areas. Isolation by distance is significant and possible barriers to gene flow exist in the study area. Two different approaches are used to better understand the geographical patterns and the causes of this structuring. Using a principal component analysis for which testing procedure exists, and partial Mantel tests, we show that the St-Bernard pass does not represent a significant barrier to gene flow although it culminates at 2469 m, close to the highest altitudinal record for this species. Similar results are found for the Simplon pass, indicating that both passes represented potential postglacial recolonization routes into Switzerland from Italian refugia after the last Pleistocene glaciations. In contrast with the weak effect of these mountain passes, the Rhône valley lowlands significantly reduce gene flow in this species. Natural obstacles (the large Rhône river) and unsuitable habitats (dry slopes) are both present in the valley. Moreover, anthropogenic changes to landscape structures are likely to have strongly reduced available habitats for this shrew in the lowlands, thereby promoting genetic differentiation of populations found on opposite sides of the Rhône valley.

Epidemiologia Molecular, Evolução Viral e Perfil de Resistência aos Antirretrovirais em uma População de Pacientes Infectados por Hiv-1 e/ou Hiv-2 Residentes em Diferentes Ilhas do Arquipélago de Cabo Verde

O diagnóstico sorológico da infecção pelo HIV-1 e HIV-2 teve início em Cabo Verde em 1987, mas pouco se sabe a respeito da diversidade genética desses vírus nessas ilhas, localizadas na costa Ocidental Africana. Neste estudo, caracterizamos a epidemiologia molecular do HIV-1 e HIV-2 em Cabo Verde, analisamos a origem dos principais clados de HIV introduzidos no país e descrevemos a ocorrência de mutações de resistência aos antirretrovirais (DRM) em indivíduos virgens de tratamento (ARTn) e pacientes em tratamento (ARTexp) oriundos das diferentes ilhas. Amostras de sangue, dados sociodemográfico e clínico-laboratoriais foram obtidos de 221 indivíduos HIV positivos entre 2010-2011. As amostras foram sequenciadas na região da polimerase (1300 pares de bases) e análises filogenéticas e de bootscan foram realizadas para a subtipagem viral. Os algoritmos disponibilizados nos sites Stanford HIV Database e HIV-GRADE e.V. Algorithm Homepage foram utilizados para avaliar a existência de DRM em pacientes positivos para HIV-1 e HIV-2, respectivamente. Os estudos evolutivos e filogeográficos foram realizados através do programa BEAST. Entre os 221 pacientes analisados, sendo 169 (76,5%) HIV-1, 43 (19,5%) HIV-2 e 9 de (4,1%) co-infectados pelo HIV-1 e pelo HIV-2, 67% eram do sexo feminino. As medianas de idade foram de 34 (IQR = 1-75) e 47 (IQR = 12-84) para o HIV-1 e HIV-2, respectivamente. A infecção pelo HIV-1 é causada pelo subtipo G (36,6%), CRF02_AG (30,6%), subtipo F1, (9,7%), URFs (10,4%), subtipo B (5,2%), CRF05_DF (3,0%), subtipo C (2,2%), CRF06_cpx (0,7%), CRF25_cpx (0,7%) e CRF49_cpx (0,7%), e todas as infecções por HIV-2 pertencem ao grupo A. De acordo com as análises filogeográficas e de origem do HIV, estima-se que o HIV-2 foi o primeiro tipo viral introduzido em Cabo Verde e possui relações filogenéticas com sequências referências de Portugal. O HIV-1 entrou no país mais tarde, primeiramente pelo subtipo G, evidenciando relações com sequências da África Central e de Portugal. Transmissão de DRM (TDRM) foi observada em 3,4% (2/58) de pacientes HIV-1 ARTn (1,7% NRTI, NNRTI 1,7%), mas não entre os infectados com HIV-2. Entre os pacientes ARTexp, DRM foi observada em 47,8% (33/69) dos infectados pelo HIV-1 (37,7% NRTI, NNRTI 37,7%, 7,4% de PI, 33,3% para duas classes) e 17,6% (3/17) nos infectados pelo HIV-2 (17,6%, 11,8% NRTI PI, 11,8% para ambas as classes). Este estudo indica que Cabo Verde tem um cenário epidemiológico molecular complexo e único dominado pelo HIV-1 subtipo G, CRF02_AG e F1 e HIV-2 grupo A, sendo esse o primeiro tipo viral introduzido em Cabo Verde. A ocorrência de TDRM e o nível relativamente elevado de DRM entre os pacientes tratados constituem uma preocupação, pelo que o monitoramento contínuo dos pacientes em ARTexp, incluindo genotipagem são políticas públicas a serem implementadas.

Molecular determinants of vesicular storage and release of the gliotransmitter D-serine from cortical astrocytes

Neuron-astrocyte reciprocal communication at synapses has emerged as a novel signalling pathway in brain function. Astrocytes sense the level of synaptic activity and, in turn, influence its efficacy through the regulated release of 'gliotransmitters' such as glutamate, ATP or D-serine. A calcium-dependent exocytosis is proposed to drive the release of gliotransmitters but its existence is still debated. Over the last years, we have been studying the molecular determinants governing D-serine release from glia using different approaches. Using a novel bioassay for D-serine, we have been able to show that D-serine release occurs mainly through a calcium- and SNARE proteindependent mechanism just supporting the idea that this amino acid is released by exocytosis from glia. We next have pursued our exploration by confocal imaging and tracking of the exocytotic routes for Dserine- mediated gliotransmission and have shown that D-serine releasable pools are confined to synaptobrevin2/cellubrevin-bearing vesicles. To shed light onto the mechanisms controlling the storage and the release of gliotransmitters and namely D-serine, we have developed a new method for the immunoisolation of synaptobrevin 2- positive vesicles from rat cortical astrocytes in culture while preserving their content in gliotransmitters. The purified organelles are clear round shape vesicles of excellent purity with homogeneous size (40 nm) as judged by electron microscopy. Immunoblotting analysis revealed that isolated vesicles contain most of the major proteins already described for neuron-derived vesicles like synaptic vesicle protein 2 (SV2) and the proton pump H?-ATPase. In addition, we have analyzed the content for various amino acids of these vesicles by means of chiral capillary electrophoresis coupled to laser-induced fluorescence detection. The purified vesicles contain large amount of D-serine. We also detect peaks corresponding to unidentified compounds that may correspond to others amino acids. Postembedding immunogold labelling of the rat neocortex further revealed the expression of D-serine in astrocytes processes contacting excitatory synapses. Finally, we have examined the uptake properties for Dserine and glutamate inside the isolated glial vesicles. Our results provide significant support for the existence of an uptake system for D-serine in secretory glial vesicles and for the storage of chemical substances like D-serine and glutamate. 11th International Congress on Amino Acids, Peptides and Proteins 763 123

Molecular epidemiology and evolution of HIV-1 in Portugal and portuguese speaking african countries

The aims of this thesis were to better characterize HIV-1 diversity in Portugal, Angola, Mozambique and Cape Verde and to investigate the origin and epidemiological history of HIV-1 in these countries. The impact of these issues in diagnosis, disease progression and susceptibility to ARV therapy was also investigated. Finally, the nature, dynamics and prevalence of transmitted drug resistance (TDR) was determined in untreated HIV-1 infected patients. In Angola, practically all HIV-1 genetic forms were found, including almost all subtypes, untypable (U) strains, CRFs and URFs. Recombinants (first and second generation) were present in 47.1% of the patients. HIV/AIDS epidemic in Angola probably started in 1961, the major cause being the independence war, subsequently spreading to Portugal. In Maputo, 81% of the patients were infected with subtype C viruses. Subtype G, U and recombinants such as CRF37_cpx, were also present. The results suggest that HIV-1 epidemic in Mozambique is evolving rapidly in genetic complexity. In Cape Verde, where HIV-1 and HIV-2 co-circulate, subtype G is the prevailed subtype. Subtypes B, C, F1, U, CRF02_AG and other recombinant strains were also found. HIV-2 isolates belonged to group A, some being closely related to the original ROD isolate. In all three countries numerous new polymorphisms were identified in the RT and PR of HIV-1 viruses. Mutations conferring resistance to the NRTIs or NNRTIs were found in isolates from 2 (2%) patients from Angola, 4 (6%) from Mozambique and 3 (12%) from Cape Verde. None of the isolates containing TDR mutations would be fully sensitive to the standard first-line therapeutic regimens used in these countries. Close surveillance in treated and untreated populations will be crucial to prevent further transmission of drug resistant strains and maximize the efficacy of ARV therapy. In Portugal, investigation of a seronegative case infection with rapid progression to AIDS and death revealed that the patient was infected with a CRF14_BG-like R5-tropic strain selectively transmitted by his seropositive sexual partner. The results suggest a massive infection with a highly aggressive CRF14_BG like strain and/or the presence of an unidentified immunological problem that prevented the formation of HIV-1-specific antibodies. Near full-length genomic sequences obtained from three unrelated patients enabled the first molecular and phylogenomic characterization of CRF14_BG from Portugal; all sequences were strongly related with CRF14_BG Spanish isolates. The mean date of origin of CRF14_BG was estimated to be 1992. We propose that CRF14_BG emerged in Portugal in the early 1990s, spread to Spain in late 1990s as a consequence of IDUs migration and then to the rest of Europe. Most CRF14_BG strains were predicted to use CXCR4 and were associated with rapid CD4 depletion and disease progression. Finally, we provide evidence suggesting that the X4 tropism of CRF14_BG may have resulted from convergent evolution of the V3 loop possibly driven by an effective escape from neutralizing antibody response.

Anexo 4 - Matriz Tratamento Dados

A alimentação/nutrição é hoje considerada inseparável do desenvolvimento, sobretudo em países deficitários a nível da oferta face a uma população crescente como é o caso de Cabo Verde. As questões de alimentação/nutrição (Segurança Alimentar) no país têm merecido uma enorme atenção, reflectindo uma preocupação pela situação de défice alimentar, que torna o país muito dependente do exterior para satisfazer as suas necessidades alimentares. A presente dissertação tem por objecto o estudo dos Hábitos Alimentares e Comportamento dos Consumidores em Cabo Verde (caso da ilha de Santiago). Este país transitou para um segundo estado de desenvolvimento (Desenvolvimento Médio), mas continua com uma significativa percentagem da população a viver em situação de pobreza e grande vulnerabilidade, o que se reflecte nos níveis de acesso ao consumo alimentar e nutricional da população. Utiliza-se dados secundários (consultados nas diversas fontes bibliográficas), e levantamento directo de informação, com aplicação de inquéritos e entrevistas que permitiram responder às principais questões levantadas nas fases elementares e são utilizadas em processo de modelação e definição de “racionalidades”. Fez-se ainda análise mais pormenorizada de algumas situações que nos pareciam mais relevantes. Os resultados obtidos permitem evidenciar alterações nos hábitos alimentares. Através da análise antropométrica, e dos resultados obtidos, conclui-se que existem problemas e carências nutricionais. As famílias de maior dimensão são as que apresentam maior vulnerabilidade alimentar e consumos energéticos inferiores aos recomendados. Este estudo permitiu evidenciar alguns problemas nas crianças. Os problemas de obesidade afectam cerca de 11% das crianças, enquanto que a mal nutrição aguda moderada atingia cerca de 5%. Verifica-se uma tendência para a diminuição do consumo de produtos tradicionais (como é o caso do milho) e também uma maior diversificação da dieta com o aumento do rendimento, (caso do consumo do arroz) que aumenta globalmente, mas observando-se também diminuição per capita com níveis de rendimento mais elevados. Para o caso do leite há uma tendência para incremento do consumo com o rendimento. Estas alterações de consumo de leite fazem-se em duas direcções. Por um lado o aumento do rendimento induz a um maior consumo de leite, por outro a substituição do leite em pó por leite de “pacote” é uma evidência para estratos mais elevados de rendimentos. Comprova-se que a alimentação absorve uma fatia muito elevada do rendimento familiar, a elasticidade consumo alimentar ao nível do rendimento familiar é muito grande. Constata-se a partir dos modelos econométricos que o nível de educação da família é importante para a definição do padrão alimentar e que existe espaço para melhorias comportamentais da população, designadamente nas formas e escolhas de consumo alimentar

Identification of C(4)-dicarboxylate transport systems in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa utilizes preferentially C(4)-dicarboxylates such as malate, fumarate, and succinate as carbon and energy sources. We have identified and characterized two C(4)-dicarboxylate transport (Dct) systems in P. aeruginosa PAO1. Inactivation of the dctA(PA1183) gene caused a growth defect of the strain in minimal media supplemented with succinate, fumarate or malate, indicating that DctA has a major role in Dct. However, residual growth of the dctA mutant in these media suggested the presence of additional C(4)-dicarboxylate transporter(s). Tn5 insertion mutagenesis of the ΔdctA mutant led to the identification of a second Dct system, i.e., the DctPQM transporter belonging to the tripartite ATP-independent periplasmic (TRAP) family of carriers. The ΔdctA ΔdctPQM double mutant showed no growth on malate and fumarate and residual growth on succinate, suggesting that DctA and DctPQM are the only malate and fumarate transporters, whereas additional transporters for succinate are present. Using lacZ reporter fusions, we showed that the expression of the dctA gene and the dctPQM operon was enhanced in early exponential growth phase and induced by C(4)-dicarboxylates. Competition experiments demonstrated that the DctPQM carrier was more efficient than the DctA carrier for the utilization of succinate at micromolar concentrations, whereas DctA was the major transporter at millimolar concentrations. To conclude, this is the first time that the high- and low-affinity uptake systems for succinate DctA and DctPQM have been reported to function coordinately to transport C(4)-dicarboxylates and that the alternative sigma factor RpoN and a DctB/DctD two-component system regulates simultaneously the dctA gene and the dctPQM operon.

Molecular characterization of HLA-C incompatibilities in HLA-ABDR-matched unrelated bone marrow donor-recipient pairs. Sequence of two new Cw alleles (Cw*02023 and Cw*0707) and recognition by cytotoxic T lymphocytes.

While the influence of HLA-AB and -DRB1 matching on the outcome of bone marrow transplantation (BMT) with unrelated donors is clear, the evaluation of HLA-C has been hampered by its poor serological definition. Because the low resolution of standard HLA-C typing could explain the significant number of positive cytotoxic T lymphocyte precursor frequency (CTLpf) tests found among HLA-AB-subtype, DRB1/B3/B5-subtype matched patient/donor pairs, we have identified by sequencing the incompatibilities recognized by CD8+ CTL clones obtained from such positive CTLpf tests. In most cases the target molecules were HLA-C antigens that had escaped detection by serology (e.g. Cw*1601, 1502 or 0702). Direct recognition of HLA-C by a CTL clone was demonstrated by lysis of the HLA class I-negative 721.221 cell line transfected with Cw*1601 cDNA. Because of the functional importance of Cw polymorphism, a PCR-SSO oligotyping procedure was set up allowing the resolution of 29 Cw alleles. Oligotyping of a panel of 382 individuals (including 101 patients and their 272 potential unrelated donors, 5 related donors and 4 platelet donors) allowed to determine HLA-C and HLA A-B-Cw-DRB1 allelic frequencies, as well as a number of A-Cw, B-Cw, and DRB1-Cw associations. Two new HLA-Cw alleles (Cw*02023 and Cw*0707) were identified by DNA sequencing of PCR-amplified exon 2-intron 2-exon 3 amplicons. Furthermore, we determined the degree of HLA-C compatibility in 287 matched pairs that could be formed from 73 patients and their 184 potential unrelated donors compatible for HLA-AB by serology and for HLA-DRB1/ B3/B5 by oligotyping. Cw mismatches were identified in 42.1% of these pairs, and AB-subtype oligotyping showed that 30% of these Cw-incompatible pairs were also mismatched for A or B-locus subtype. The degree of HLA-C incompatibility was strongly influenced by the linkage with B alleles and by the ABDR haplotypes. Cw alleles linked with B*4403, B*5101, B18, and B62 haplotypes were frequently mismatched. Apparently high resolution DNA typing for HLA-AB does not result in full matching at locus C. Since HLA-C polymorphism is recognized by alloreactive CTLs, such incompatibilities might be as relevant as AB-subtype mismatches in clinical transplantation.