C. López-Larrea

Molecular typing of HLA-B27 alleles

HLA-B27 represents a family of closely related antigens. Six alleles which differ in a limited number of nucleotide substitutions have been described (B*2701—B*2706). These changes are clustered in α1 and α2 domains. Polymerase chain reaction strategies were designed to amplify specific regions of class I exons 2 and 3. Amplified sequences were tested with eight sequence-specific oligonucleotides to distinguish all B27 subtypes. We also subtyped B27 in 50 healthy Spanish individuals using this procedure. The B*2705 subtype is over-represented in our population (96%). The remaining 4% carried the B*2702 allele. This finding is in agreement with the frequencies described by other techniques (cytotoxic T lymphocytes and isoeletric-focusing) for Caucasian populations. Class I oligotyping is a poorly developed field with significant potential applications. This procedure of genotyping B27 alleles is a reliable method which can be used in transplantation and B27-associated disease studies.

Molecular evolution of the N-formyl peptide and C5a receptors in non-human primates

N-formyl peptides (FMLP) and complement fragment C5a are neutrophil chemoattractants. In humans, a single-copy gene was identified for the C5a receptor, and the receptor for FMLP (FPR1) is encoded by a single gene that shows 53% amino acid similarity to the C5aR. Two other humanFPR1 homologues,FPR-like 1 (FPR2/FPRL1) andFPR-like 2 (FPRL2) have been cloned. The human C5aR, FPR1, FPRL1, and FPRL2 are physically linked. By direct sequencing or by sequencing plasmid clones we studied theC5aR andFPR genes from four non-human primates (chimpanzee, gorilla, orangutan, and macaque). The sequences showed 95%–99% similarity to the human homologues, with the major divergences observed in macaque. In these genes, the transmembrane and the cytoplasmic domains are highly conserved, while the highest divergence corresponded to the extracellular loops involved in ligand binding. Additionally, we constructed a physical map of these genes in non-human primates. In all species the four genes were physically linked and we defined the relative orientation of the four genes in primates:C5aR>FPR1>FPR2 (FPRL1)>FPRL2.

Endoplasmic Reticulum Stress Signals in Defined Human Embryonic Stem Cell Lines and Culture Conditions

Human embryonic stem cells (hESCs) are especially resistant to several cellular stresses, but the existence and induction of Endoplasmic Reticulum (ER) stress by culture conditions are unknown. Using qPCR, here, we investigated the behavior of the principal sensors of ER stress and their relation with the feeder layer, the type of conditioned media used in feeder free systems and the upregulation of several differentiation markers. We observed the preservation of pluripotency, and detected differential expression of differentiation markers in HS181 and SHEF1 hESCs growing on Adipose-derived mesenchymal stem cells (ASCs) and feeder-free system with different conditioned media (HEF-CM and ASC-CM). Taken together, these results demonstrate evidence of ER stress events that cells must resolve to survive and maintenance of markers of pluripotency. The early differentiation status defined could progress into a more differentiated state, and may be influenced by culture conditions.

Modulation of Autoimmune T-Cell Memory by Stem Cell Educator Therapy: Phase 1/2 Clinical Trial.

Background Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that causes a deficit of pancreatic islet β cells. The complexities of overcoming autoimmunity in T1D have contributed to the challenges the research community faces when devising successful treatments with conventional immune therapies. Overcoming autoimmune T cell memory represents one of the key hurdles. Methods In this open-label, phase 1/phase 2 study, Caucasian T1D patients (N = 15) received two treatments with the Stem Cell Educator (SCE) therapy, an approach that uses human multipotent cord blood-derived multipotent stem cells (CB-SCs). SCE therapy involves a closed-loop system that briefly treats the patients lymphocytes with CB-SCs in vitro and returns the “educated” lymphocytes (but not the CB-SCs) into the patients blood circulation. This study is registered with ClinicalTrials.gov, NCT01350219. Findings Clinical data demonstrated that SCE therapy was well tolerated in all subjects. The percentage of naïve CD4+ T cells was significantly increased at 26 weeks and maintained through the final follow-up at 56 weeks. The percentage of CD4+ central memory T cells (TCM) was markedly and constantly increased at 18 weeks. Both CD4+ effector memory T cells (TEM) and CD8+ TEM cells were considerably decreased at 18 weeks and 26 weeks respectively. Additional clinical data demonstrated the modulation of C–C chemokine receptor 7 (CCR7) expressions on naïve T, TCM, and TEM cells. Following two treatments with SCE therapy, islet β-cell function was improved and maintained in individuals with residual β-cell function, but not in those without residual β-cell function. Interpretation Current clinical data demonstrated the safety and efficacy of SCE therapy in immune modulation. SCE therapy provides lasting reversal of autoimmune memory that could improve islet β-cell function in Caucasian subjects. Funding Obra Social “La Caixa”, Instituto de Salud Carlos III, Red de Investigación Renal, European Union FEDER Funds, Principado de Asturias, FICYT, and Hackensack University Medical Center Foundation.

MspI polymorphism at the human Complement component C6 gene (C6)

as •• «B2 Two RFLPs at the D12S51 locus R.Newton, D.J.Henderson, D.J.Cockburn, Y.Boyd and G.E.Moore Action Research Laboratory for the Molecular Biology of Fetal Development, Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School, Queen Charlottes and Chelsea Hospital, Goldhawk Road, London W6 0XG, 1 Genetics Laboratory, Department of Biochemistry, South Parks Road, Oxford OX1 3QU and MRC Radiobiology Unit, Chilton, Didcot, Oxon, OX11 0RD, UK

DNA analysis of HLA-DR4B1 subtypes in multiple sclerosis by specific oligonucleotide probes

Conversely to the well-established association of DR2/Dw2 with multiple sclerosis (MS) susceptibility in Caucasoids, several studies have found an association of DR4 in populations from Mediterranean origin. We have studied the distribution of the different DR4B1 subtypes in Spanish MS patients. Oligonucleotide probes were selected in order to type samples amplified by polymerase chain reaction (PCR) from Spanish DR4+ MS patients (25) and controls (28). No DR4B1 subtypes were found to be increased in MS. MS susceptibility linked to DR4 may be due to the presence of shared functional epitopes common to the different HLA-DR4B1 subtypes.

A 3’-UTR polymorphism in soluble epoxide hydrolase gene Is associated with acute rejection in renal transplant recipients

Background and Purpose Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites that play a protective role against damaging processes that may occur after re-oxygenation of the graft. We aimed to investigate whether the presence of functional polymorphisms in the gene encoding soluble epoxy hydrolase (EPHX2), which metabolizes EETs to less active compounds, may play a role in the outcome of renal transplantation. Methods In a group of 259 Caucasian renal transplant recipients and 183 deceased donors, we determined the presence of three common EPHX2 SNPs, namely rs41507953 (K55R), rs751141 (R287Q) and rs1042032 A/G. Associations with parameters of graft function and the incidence of acute rejection were retrospectively investigated throughout the first year after grafting by logistic regression adjusting for clinical and demographic variables. Results Carriers of the rs1042032 GG genotype displayed significantly lower estimated glomerular filtration rate (eGFR) (38.15 ± 15.57 vs. 45.99 ± 16.05; p = 0.04) and higher serum creatinine values (1.57 ± 0.58 vs. 1.30 ± 0.47 g/dL; p=0.02) one year after grafting, compared to patients carrying the wildtype A-allele. The same GG genotype was also associated to increased risk of acute rejection. Interestingly, this association was observed for the genotype of both recipients [OR =6.34 (1.35-29.90); p = 0.015] and donors [OR = 5.53 (1.10-27.80); p=0.042]. A statistical model including both genotypes along with other meaningful demographic and clinical variables resulted in an increased significance for the association with the recipients’ genotype [OR=8.28 (1.21-74.27); p=0.031]. Conclusions Our results suggest that genetic variability in the EETs-metabolizing gene, EPHX2, may have a significant impact on the outcome of deceased-donor renal transplantation.

Study of Genetic Polymorphism of Seventh Complement Component in Two Families with Hereditary Deficit

The results of an allelic segregation of C6 and C7 were studied in two Spanish families that have members with C7 deficiency. Absence of C7 in the affected siblings and half of the normal values in their parents were found in both families. The variant responsible for the deficiency (C7Q*0) follows a codominant autosomic inheritance pattern. Establishing allotypes of C6 and C7 by isoelectrofocusing followed by electrophoretic immunoblotting allowed haplotype assignments. In both families the haplotype form responsible for the deficiency seemed to be related to the C6B allotype.

HLA CLASS II AND SUSCEPTIBILITY AND RESISTANCE TO INSULIN-DEPENDENT DIABETES MELLITUS IN A POPULATION FROM THE NORTHWEST OF SPAIN

The role of HLA class II alleles in genetic predisposition to insulin‐dependent diabetes mellitus(IDDM) was examined using Polymerase Chain Reaction/oligonucleotide probe typing (PCR/SSOs) of eight HLA class II loci in 58 IDDM patients and 50 healthy controls from the Northwest of Spain (Asturias). We compared the distribution of HLA class II alleles, haplotypes and genotypes between IDDM patients and controls, and tested three recently proposed HLA‐IDDM susceptibility theories. By using the aetiologic fraction (δ) as an almost absolute measure of the strongest linkage of disequilibrium of a HLA marker to the putative Type I susceptibility locus, it has been found that the strength of association of the HLA markers may be quantified as follows: DQA1 *03‐DQB1 *0302 or DQA1 *0501‐DQB1 *0201 > DR3 or DR4; presence of more than one dimer DQαβ of the six proposed by Rønningen > non‐Asp57 DQβ and Arg52 DQα > Arg52 DQα > non‐Asp57 DQβ/non‐Asp57 DQβ > DRB1*0301; DQA1*0501‐DQB1*0201 > DQA1*03‐DQB1*0302; DQB1*0302. The presence of at least one Asp57 DQβ allele was the best protection HLA marker to IDDM in our population. Therefore, the above data confirm that IDDM susceptibility to HLA locus is linked more to DQ than DR.

GENETIC STRUCTURE AND ORGANIZATION OF THE MEMBRANE ATTACK COMPLEMENT COMPONENTS

Complement provides an effector defence system against infections. Complement activation, through either the classical or the alternative pathway, results in the formation of the terminal complement complex (TCC) (Miiller-Eberhard, 1986). This leads to generation of functional membrane attack complex (MAC), which damages the surface membranes of various microorganisms (Ross et al., 1984). The adaptative evolution of this effector system evolved over at least 400 million years, as complement activity related to MAC function has been described in lower vertebrates (Jensen et al., 1981). The importance of the terminal formationof MAC in host defence is demonstrated by the susceptibility of terminal complement-deficient individuals to bacterial infections (Petersen et al., 1979). MAC generation is initiated by C5b, a product formed by the action of C5 covertase. The C5b fragment acquires a metastable binding site for C6. Once C5b-6 is formed, C7, C8 (C8, C8p and C8y) and C9 bind sequentially to form a large protein complex. The complex contains one molecule each of C5b, C6, C7, C8 and around 6-18 molecules of C9 (C5b-9,) (Tschopp et a/., 1984; Kolb et al., 1972; Podack et al . , 1982). The final step is the catalytic polymerization of C9 by membrane-bound C5b-8. The transition of C 9 to poly (C9), involves major structural rearrangements of the molecule (Podack & Tschopp, 1984; DiScipio, 1987). This molecular complex is inserted into the target membrane and generates a transmembranous hydrophilic channel across the lipid bilayer (see Fig. 2b below) (Miiller-Eberhard, 1988). MAC proteins are all soluble hydrophilic molecules which can undergo hydrophilic-amphilic transition for insertion into membranes. The formation of MAC on self membranes is regulated by the S protein, also called vitronectin, which controls the activity of C5b-7 (Jenne et al., 1985). Lytic reaction by homologous complement can also be controlled by the ‘homologous restriction factors’ (HRFs) (Zalman et al., 1986), CD59 (Okada et al., 1989) and SP40,40 (Tschopp etal., 1993). The liver is an important site of the terminal plasma of the lytic complement cascade (Moms et al., 1982). The regulatory region containing the cisand trans-acting elements that control the expression of these genes has not been examined. However, evidence for production of these proteins at extrahepatic sites has also been obtained (Pettersen et al., 1987).