bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a new HLA‐B*78 allele in a Hispanic family
Authors: Gaidulis L. , Krausa P. , Schaub B. , Senitzer D.
Published: March 2003

A new B*78 variant, B*7804, was detected in three members of a Hispanic family. The novel B*78 sequence differs from B*78021 by two substitutions: T at nucleotide 527 (all other B*78s have A) and T at nucleotide 583 (all other B*78s have a C). Both nucleotide substitutions encode amino acid changes at codons 152 and 171, respectively ( Note).
Screening for the IDDM high‐risk genotype. A rapid microtitre plate method using serum as source of DNA
Authors: Cinek O. , Wilkinson E. , Paltiel L. , Saugstad O.D. , Magnus P. , Rønningen K.S.
Published: March 2003

Norwegian babies born with the HLA‐DRB1*0401‐DQA1*03‐DQB1*0302/DRB1*03‐DQA1*05‐DQB1*0201 genotype have an estimated 17% lifetime risk of developing insulin‐dependent diabetes mellitus (IDDM). Identifying these children is important for future prevention, and for studies of the non‐genetic factors involved in IDDM. The aim of the study was to develop a rapid screening method for this high‐risk genotype. DNA was extracted from serum collected during routine newborn screening for phenylketonuria and hypothyreosis. The second exons of HLA‐DQA1 and DQB1 were co‐amplified using biotinylated primers, amplicons were hybridized to a set of seven probes immobilized on a microtitre plate using a single hybridisation temperature, and detected colorimetrically by streptavidin‐HRP conjugate and tetramethylbenzidine substrate. The DRB1*04 subtyping was performed using six different probes at identical conditions. The prevalence of the DRB1*0401‐DQA1*03‐DQB1*0302/DRB1*03‐DQA1*05‐DQB1*0201 genotype among 1,026 Norwegian babies was 2.7% (CI 95%: 1.7–3.7%). The new high‐throughput genetic screening method for IDDM risk can easily be automated and included in newborn screening programs.
HLA‐B*1559: a hybrid allele including exon 2 of HLA‐B35 and exon 3 of HLA‐B15 and serologically typed as B35
Authors: Magira E. , Beznik‐Cizman B. , Monos D.
Published: March 2003

Acknowledgments:

This work was supported in part by office of Naval Research cooperative agreement # N‐00014‐96‐2‐0016 to the National Marrow Donor Program and by funding from the E.B. Foerderer Fund of the Children’s Hospital of Philadelphia to D.M.

The new allele HLA‐B*1559 described is of Hispanic origin. This allele carries the exon 2 of HLA‐B*35 alleles, and is identical to HLA‐B*3501 and the exon 3 of HLA‐B*1530. Serologically it is typed as B35. The most likely scenario for its generation seems to be a gene conversion event that involved the, frequent in Hispanic populations, B*1522 allele and a B*1530 allele. A segment of the B*1530 allele that included at least the sequences 412–419 would be transferred on a B*1522 background ( Note).
Seven novel HLA‐A alleles carry previously observed polymorphisms
Authors: Steiner N.K. , Edson S.M. , Mitton W. , Ng J. , Hartzman R.J. , Hurley C.K.
Published: March 2003

This paper describes seven novel HLA‐A locus alleles which carry previously observed polymorphisms.
Two novel DRB alleles detected by PCR‐SSO and confirmed by sequencing: DRB1*0317 and DRB3*0210
Authors: Buhlmann A. , Albis‐Camps M. , Holtkamp B. , Blasczyk R.
Published: March 2003

We here describe two new DRB alleles (DRB1*0317 and DRB3*0210) which were detected by polymerase chain reaction using sequence‐specific oligonucleotides (PCR‐SSO) and confirmed by direct sequencing. The exon 2 sequence of DRB1*0317 is the result of an intergenic recombination event yielding a hybrid allele between 5′DRB1 and 3′ DRB3 sequences with the recombination breakpoint located between codons 39–51. The new DRB3*0210 allele is closest related to DRB3*02021 except for a single nucleotide position at codon 51. Here, the sequence AGG at codon 51 which was a group‐specific motif for all DRB3*02 alleles described previously, is replaced by the DRB consensus sequence ACG ( 1).
HLA class II associations with Type 1 diabetes mellitus: a multivariate approach
Authors: Schipper R.F. , Koeleman B.P.C. , Bruining G.J. , Schreuder G.M.tH. , Verduijn W. , De Vries R.R.P. , Roep B.O.
Published: March 2003

Abstract: The association of HLA class II phenotype with the development of insulin‐dependent (Type 1) diabetes mellitus (IDDM) is well established but the contribution of the various HLA‐DR and ‐DQ alleles and haplotypes to disease predisposition is not fully understood. We have determined haplotype and genotype odds ratios, and further employed multivariate tree analysis to explore the contribution of individual HLA‐DRDQ haplotypes to the genetic risk for developing IDDM in the Dutch population. Next to haplotype and genotype odds ratios, multivariate tree analysis techniques provide overall risk calculations for each modeled parameter, and offer insight in the interaction of the model parameters via tree‐shaped reports, in which subsequent stratifications on the data can easily be followed. We compared 206 Dutch IDDM patients with 840 serologically typed random healthy unrelated Dutch Caucasoid controls. The multivariate tree analysis showed that the HLA‐DR7DQ9 and DR15DQ6 haplotype were strongly associated with disease protection (OR=0.04, P=0.0003, and OR=0.07, P=<0.0001, respectively). The highest ORs were found for the DR4DQ8/DR8DQ4 genotype (OR=21.04, P=0.001), followed by DR4DQ8/DR17DQ2 (OR=12.45, P< 0.0001) and DR9DQ9/DR17DQ2 (OR=10.87, P=0.02). DR4DQ8 homozygous and DR17DQ2 homozygous individuals have a disease OR of 9.0 and 3.0 (P=0.01 and 0.03), respectively. In conclusion, the results from haplotype, genotype, and tree analyses provide insight into the disease associations for combinations of HLA‐DRDQ haplotypes. We confirm that the DR9DQ9/DR17DQ2 genotype is associated with susceptibility in the Dutch population, which has previously been noticed as a HLA risk genotypes in Asian populations only.
New DR5 sequences: a novelDRB1*11122 allele identified in Paiwan tribe members of Taiwan and a corrected sequence for the DRB1*1201 allele
Authors: Wu S. , Shiao Y.‐M. , Lai S.‐M. , Lai C.‐Y. , Chen S.‐P. , Chen Y.‐F. , Sidebottom D.A. , Hildebrand W.H. , Chou F.‐C. , Tsai M.‐F.
Published: March 2003

Abstract: We report herein the identification of a new DRB1 allele using sequence‐based typing (SBT). This novel allele, HLA‐DRB1*11122, was found in an aboriginal individual (SWP71) from the Paiwan tribe in the southern part of Taiwan. This individual was typed by SBT method as having an HLA genotype of HLA‐A*24021/24021, HLA‐B*4001/4002, HLA‐DRB1*11122/15011, HLA‐DRB3*0202, and HLA‐DRB5*01011. This new allele differs from DRB1*1112 in the polymorphic exon 2 only at codon 34 (CAA→CAG; both specify glutamine) and from DRB1*1110 in the exon 2 sequence only at codon 32 (CAT→TAT; H32T). The most likely candidate allele which is found in the aboriginal populations of Taiwan and which may mutate into this new allele is DRB1*11011. DRB1*11122 allele differs from DRB1*11011 allele in the polymorphic exon 2 at both codon 34 (CAA→CAG) and codon 37 (TAC→TTC; T37F). This novel HLA‐DRB1*11122 allele was also found in another aboriginal individual (SWP90) from the same Paiwan tribe. This SWP90 individual was typed by SBT method as having an HLA genotype of HLA‐A*24021/24021, HLA‐B*4002/5502, HLA‐DRB1*11122/1201, and HLA‐DRB3*01011/0202. However, the original DRB1*1201 sequence from HERLUFF was found to be erroneously reported and the corrected sequence from SWP90 is now presented herein.
Polymorphism in RANTES chemokine promoter affects extent of sarcoidosis in a Japanese population
Authors: Takada T. , Suzuki E. , Ishida T. , Moriyama H. , Ooi H. , Hasegawa T. , Tsukuda H. , Gejyo F.
Published: March 2003

Abstract: RANTES, a member of C‐C chemokine, is known to be produced at sites of granulomatous reactions in the lung of sarcoidosis. RANTES is a potent eosinophil and lymphocyte attractant with particular preference for CD45RO+ T cells and eosinophils. Polymorphism of the RANTES promoter has recently been shown to be related to allergic and infectious diseases; atopic dermatitis, asthma, and polymyalgia rheumatica. Considering that this might affect sarcoidosis, we studied polymorphism of the RANTES gene in 114 patients with sarcoidosis and 136 healthy control subjects. Their genotypes were determined using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Although no difference in the genotype distribution between healthy control subjects and sarcoidosis patients was identified, the difference in the frequencies of the patients with three or more organ involvement was significant (P<0.01) with the frequency of those in AA genotype being elevated (P<0.05). BAL findings in 48 out of 114 patients who underwent bronchoscopy were reviewed. The CD4/8 ratio of lymphocytes in bronchoalveolar lavage fluid in the patients with AA genotype was significantly increased (P<0.05). From the results, we suggest that in RANTES gene polymorphism the homozygous A allele might be a genetic risk factor for extent disease of sarcoidosis.
The nucleotide diversity of MICA and MICB suggests the effect of overdominant selection
Authors: Elsner H.‐A. , Schroeder M. , Blasczyk R.
Published: March 2003

Abstract: The major histocompatibility complex (MHC) class I‐related molecules A and B (MICA and MICB) are stress‐inducible cell surface antigens that are recognized by immunocytes bearing the receptor NKG2D. In our study we estimated the average number of synonymous (πs) and nonsynonymous nucleotide substitutions (πa) per site in exons 2–4 of MICA and MICB. In exons 2 and 3 of MICB only nonsynonymous substitutions were found, and in exon 3 of MICA πa clearly exceeded πs. This finding is in contrast to the evolutionary parameters found in most other genes, and is reminiscent of the elevated πa values caused by overdominant selection in the peptide‐binding region of conventional MHC class I molecules. It may be explained by the hypothetical interaction with nonpeptide antigens, or by resistance against pathogens.
CD antigens 2001
Authors: Mason D.Y. , André P. , Bensussan A. , Buckley C. , Civin C. , Clark E. , De Haas M. , Goyert S. , Hadam M. , Hart D. , Hořejší V. , Meuer S. , Morissey J. , Schwartz‐Albiez R. , Shaw S. , Simmons D. , Uguccioni M. , Van Der Schoot E. , Viver E. , Zola H.
Published: March 2003

Abstract: The most recent Human Leucocyte Differentiation Antigen Workshop (“HLDA7”) took place in 2000 in Harrogate, UK and the proceedings are about to be published (Leucocyte Typing VII). New Sections were introduced in this Workship (Dendritic cells, Stem/progenitor cells, Erythroid cells and Carbohydrate Structures) and monoclonal antibodies were selected for which at least some molecular data were already available (to avoid “blind” screening of reagents against known specificities). A total of more than 80 new CD specificities were established (previously the average was less than 30 new CD specificities per Workshop) and these are listed in this article. There is already evidence for the existence of many new leucocyte surface molecules for study at the next HLDA Workshop (in Adelaide in 2004), and we have listed in this article a number of such potential CD candidates (identified following the production of monoclonal antibodies or via gene cloning). There are also today an increasing number of lineage‐ and/or stage‐restricted leucocyte‐associated molecules localised within the cell cytoplasm (or nucleus): they will certainly prove of intense in the future for many laboratories studying human haematopoietic cells (regardless of whether a new “intracellular CD” categorisation scheme is devised for such molecules).
EFFECT OF CHRONIC HEPATITIS C INFECTION ON LYMPHOCYTE SUBSETS IN RENAL TRANSPLANT PATIENTS
Authors: Akman B , Özdemir FN , Agca E , Emiroglu R , Turan M , Mehmet H
Published: March 2003

Chronic hepatitis C (HCV) infection is prevalent in renal allograft recipients. Consequently, this group is more likely to develop liver complications and have poorer graft outcome. The interaction between viral and host factors under immunosuppressive therapy is important in highlighting the response to HCV infection after renal transplantation. The aim of this study was to assess changes that occur in the lymphocyte subsets of HCV‐infected kidney recipients, and to determine how these relate to allograft rejection. We investigated 50 patients (33 males and 17 females; mean age 32.0±9.9years; mean time since transplantation: 27.6±26.5months) who were receiving standard immunosuppressive therapy. Data concerning acute or chronic allograft rejection were retrieved from the patients' records. Lymphocyte subsets (CD4, CD8, CD3, CD19, CD16–56), serum albumin levels, and anti‐HCV positivity were evaluated after renal transplantation, and findings in the HCV‐positive and ‐negative groups were compared. Seven of the 50 patients had HCV infection. Anti‐HCV positivity was detected before transplantation in three cases, and these patients received interferon therapy before transplantation. Four patients were found to be HCV positive after transplantation. The anti‐HCV (+) patients had significantly lower serum albumin (P<0.02), lower CD4/CD8 (P<0.02) ratios, and a lower percentage of CD 19 (P<0.001) and a higher percentage of CD8 (P<0.01) cells. Chronic allograft rejection was observed in 71.4% of the HCV‐positive patients (P<0.04), whereas no acute rejection was detected in this group. The findings indicate that HCV infection may alter the immune response in renal transplant recipients, and that the immunogenic potential of the virus may be higher in this patient group because of low serum albumin levels. It appears that this immunomodulatory action and low albumin levels may increase the risk of chronic rejection for HCV‐infected kidney recipients.
NEW ADVANCES IN THE MOLECULAR BIOLOGY OF ISCHEMIA/REPERFUSION: MAPK PATHWAYS
Authors: Toledo‐Pereyra LH , Lopez‐Neblina* F
Published: March 2003

Ischemia/reperfusion (I/R) is an important pathological condition frequently associated with the organ/cell transplantation response. The molecular mechanisms involved in the development and characterization of the I/R lesion are represented, in some way, by the changes occurring in the intracytoplasmic phosphorylated proteins (MAPK) which transmit the I/R signal downstream to the nucleus. The MAPKinases are at the center of these developments. As an example, one of the MAPK signaling pathways is represented by the activation of NF‐kB. In this pathway, the MAPK phosphorilates the IkB fraction of the NF‐kB complex, allowing this fraction to be degraded by ubiquitination and proteolytic degradation by the ubiquitin‐proteome system. Free NF‐kB is translocated into the nucleus, with its subsequent effect on the activation and transcription of DNA with production of RNA, which would then copy the template of certain inflammatory cytokines (TNF‐α, IL1‐α, IL6), adhesion molecules (P‐selectin, E‐selectin, ICAM‐1, VCAM‐1) and chemokines (IL8) for future expression. At the same time, NF‐kB can be activated by TNF‐α and IL‐1, which would then reach TNFR1, TRADD, TRAF2, MEK, JNK and c‐Jun activation or return to the previous IkB route.

MAPK pathways appear to be important in the regulation of the inflammatory response after I/R. p38, ERK and JNK are the best‐known MAPK systems, and their modification has direct implications for the I/R syndrome. Inhibition of p38 MAPK has been demonstrated to have a beneficial effect after various types of ischemia. ERK inhibition might play a role in the increased production of TNF‐α. The role of JNK is not as clear as the other two proteins. A better understanding of the MAPK regulatory mechanisms intervening in I/R would allow us to develop an improved and effective anti‐inflammatory therapy for this pathological condition.
JOINT PAIN AND ARTHRITIS IN RENAL TRANSPLANT RECIPIENTS, AND CORRELATION WITH CYCLOSPORINE THERAPY
Authors: Kart‐Köseoglu H , Yücel AE , Isýklar I , Türker I , Akçalý Z , Haberal M
Published: March 2003

To determine the prevalence of joint pain and arthritis in renal transplant recipients, and to investigate relationships with various laboratory and clinical parameters.

Eighty‐two patients who underwent renal transplantation (RT) and had a joint examination reported on their level of joint pain and arthritis by questionnaire. The patients were then followed for a year, with repeated joint examinations and laboratory tests every 3 months. Thirty‐one of 82 patients (37.8%) complained of joint pain before the RT, and seven of these 31 individuals reported continued pain after the operation. Seventeen of the 82 (20.7%) began to suffer joint pain after the RT. Six (7.3%) and three (3.7%) of the 82 patients developed arthritis before and after the transplantation, respectively. We found no statistical association between the joint pain and sex, age, cause of renal failure, dialysis duration, time since transplantation, bone mineral density, human leukocyte antigen type, serum levels of urate, parathormone, calcium, alkaline phosphatase, phosphorus, C‐reactive protein, erythrocyte sedimentation rate, donor source (living‐related or cadaveric), or presence of HCV infection. However, levels of serum cyclosporine higher than 200 ng/ml were significantly correlated with the joint pain. This study shows that joint pain is common before and after RT. In renal transplant recipients, joint pain is significantly correlated with serum cyclosporine levels.
HISTOCHEMICAL DETECTION OF PANCREATIC TISSUE IN VITAMIN C‐ADMINISTERED GUINEA PIGS
Authors: Kaplan B , Gonul B , Erdogan D , Elbeg S , Akyol G
Published: March 2003

The aim of this study was to investigate the effects of fasting (F) periods and vitamin C supplementation on the serum C‐peptide levels and endocrine pancreas of guinea pigs. Vitamin C supplementation (a single intraperitoneal dose of 500 mg/kg−1) was given at the beginning of the fasting period. The animals were divided into seven groups (n=6 each) as follows: Group I (untreated group) was given food and water ad libitum; Group II, III and IV were acute (24 h, 48 h) and chronic F (120 h) respectively; while Groups V, VI and VII were acute and chronic F with supplementary vitamin C (VitC + F24 h, VitC + F48 h and VitC + F120 h, respectively). Serum C‐peptide levels were measured and pancreatic tissues were collected for immunohistochemical evaluation. Serum C‐peptide levels in the VitC + F24 and VitC + F48 groups were higher than in the F24 and F48 groups (P<0.05). Immunohistochemical examination of the endocrine pancreas confirmed that vitamin C administration in fasting may indirectly increase glucagon and insulin secretions.
RELATIONSHIPS BETWEEN HLA‐A, ‐B, ‐DQ AND ‐DR ANTIGENS AND INTERSTITIAL FIBROSIS IN RENAL ALLOGRAFTS
Authors: Özdemir BH , Çolak T , Turan M , Demirhan B , Haberal M
Published: March 2003

Although renal transplant recipients tend to exhibit similar clinical and immunological changes over time, some allograft biopsies show early interstitial fibrosis (IF). To evaluate the relationship between HLA‐antigens and IF in renal allografts, we reviewed the HLA‐A, ‐B, ‐DQ, and ‐DR antigens in 88 renal transplant recipients.

For each antigen type, we determined the number of patients who did and did not possess the antigen. We then determined the mean time to onset of IF for each of these two groups, and statistically compared the means. In the second part of the analysis, we divided the 88 patients into those who did and did not show IF at 6 months post‐transplantation, and then calculated the prevalence of each antigen type in the IF(+) and IF(–) groups. This same procedure was repeated with the patients grouped according to the presence of IF at 12 months post‐transplantation. The groups of patients with HLA‐B8, ‐B27, ‐DQ2, ‐DQ5, ‐DQ6, ‐DQ7, ‐DR4, ‐DR13, and ‐DR15, had significantly shorter times to IF onset after transplantation than the corresponding groups without these antigens (P<0.05). The groups that were IF(+) at 6 and 12 months post‐transplantation had significantly higher frequencies of HLA‐B8, ‐B27, ‐DQ2, and ‐DR4 than the corresponding IF(–) groups at these two timepoints (P<0.05).

The results indicate that any kidney recipient with these antigens is predisposed to developing diffuse IF in their graft relatively soon after transplantation. We conclude that although there is multiple etiological agents in the pathogenesis of IF one can not exclude a HLA association in such cases.
KIDNEY TRANSPLANTATION AT ONE CENTER IN 2001
Authors: Basaran O , Emiroğlu R , Tezel E , Moray G , Bilgin N , Haberal M
Published: March 2003

The number of patients with end‐stage renal disease increases, with kidney transplantation remaining the treatment of choice for this dismal disease. From January 1 to December 31, 2001, 48 kidney transplantations were performed at Baskent University Faculty of Medicine. Thirty‐nine of the recipients were male and nine were female, and the average age of the patients was 31.8. Thirty‐seven of these transplantations were performed from living donors, and 11 were from cadaver organs. Of these 48 patients, one died during the post‐operative period as a result of septicemia. Graft nephrectomy was performed in one patient because of massive bleeding. There were 16 acute‐rejection episodes in 12 patients during follow up, and all patients recovered. At the end of the year, the overall patient and graft survival rates were 98% and 95.8%, respectively. Despite these good results, there is an increasing number of patients on the waiting list and we are not able to use the full capacity of our center because of continued organ shortages.
RENAL TRANSPLANTATION OFFERS BETTER SURVIVAL IN HCV‐INFECTED END‐STAGE RENAL DISEASE PATIENTS
Authors: Siren S , Nurhan ÖF , Münire T , Sedat B , Zübeyde A , Mehmet H
Published: March 2003

Hepatitis C virus (HCV) infection is known to increase morbidity and mortality in the dialysis population. Renal transplantation is an offered treatment option after a careful pretransplant evaluation. This study assessed the impact of HCV infection on patient and allograft survival rates in a selected group of dialysis patients and kidney transplant recipients.

The study included 252 end‐stage renal disease patients who were receiving hemodialysis (HD) treatment or who received renal transplantation at our centre in 1995–96. Of the total, 116 [94 HCV (–) and 22 HCV (+)] underwent transplantation and 134 [106 HCV (–) and 30 HCV (+)] remained on HD. We retrospectively investigated 5 years of follow‐up findings in the records of these patients. All 22 HCV (+) individuals underwent liver biopsy to ensure there was no advanced liver disease before transplantation. None of the recipients or HD patients showed decompensation related to liver disease during follow up.

The overall 5‐year patient survival rates for the kidney recipient and HD groups were 85.2% and 74.5%, respectively. Comparison of outcomes for the HCV (+) recipients had a significantly higher 5‐year survival rate than the HCV (+) HD patients (P<0.04). The 3‐year graft survival rates for the HCV (+) and HCV (–) transplant recipients were comparable, but the risks of chronic rejection and graft loss at 5 years were higher in the HCV (+) group (P<0.02, P<0.006, respectively). In conclusion, renal transplantation should be the preferred therapy in HCV‐infected dialysis patients because it improves the survival rates. HCV infection is associated with increased rates of chronic rejection and graft loss at 5 years post‐transplantation.
PANEL‐REACTIVE ANTIBODY POSITIVITY AND ASSOCIATED HLA‐ANTIBODIES IN TURKISH END‐STAGE RENAL DISEASE PATIENTS
Authors: Özdemir FN , Turan M , Sezer S , Arat Z , Gülmüş A , Külah E , Haberal M
Published: March 2003

Pre and postrenal transplantation panel reactive antibody (PRA) screening is associated with increased rates of hyperacute or acute‐graft rejection and graft loss. It has been suggested that phenotypic HLA‐antigens are involved in PRA sensitization. This study investigated the relationships between HLA‐specific antibody frequencies and PRA sensitization in Turkish patients with end‐stage renal disease (ESRD). Three hundred and forty patients on the renal transplantation waiting list participated. We determined the level of PRA sensitization and the candidates' class I and II HLA‐antibody profile. Panel reactive antibody levels greater than 30% were accepted as positive. The frequencies of the different antibodies in the sensitized group were calculated. Twenty‐four (7%) of the 340 patients showed PRA‐ABC positivity and 34 (10%) showed DR positivity. Thirty‐nine (11.5%) of the candidates were PRA‐positive. The most frequent class I HLA‐antibodies in this group were A2, A34, and B56, and the most frequent class II antibodies were DR1, DR7, DR10, DR11, DR14, DR52, DQ1, DQ4, DQ6, and DQ7. Analysis showed that the presence of each of these HLA class II antibodies was significantly correlated with PRA positivity. Apart from the presence of HLA‐A2, A24, and DR11 antibodies, the HLA profile for the PRA‐positive candidates differed from that of the general Turkish population. In conclusion, identification of the associated HLA‐specific antibodies and correlation with the Turkish population HLA‐antigen distribution will identify high‐risk patients as candidates for transplantation.
Two new HLA class I alleles recognised by PCR sequence‐specific primer and sequencing based typing: B*3805 and Cw*0408
Authors: García‐Sánchez F. , Fernández‐de‐Velasco J. , Lillo R. , Álvarez‐Doval A. , Rodríguez M.A. , Aviles M.J. , Balas A. , Vicario J.L.
Published: March 2003

Abstract: Two new HLA class I alleles have been recognised by molecular‐based typing. B*3805 was initially identified by polymerase chain reaction using sequence‐specific primers (PCR‐SSP) and afterwards confirmed by sequencing based typing (SBT) studies in a Spanish Caucasian blood cord unit. A unique nucleotide change throughout exons 2, 3 and 4, leading to the amino acid replacement Ser11Ala, differentiates B*3801 and *3805. This position behaves as a dimorphic residue in HLA‐B and ‐C loci, and seems to be structurally unrelated to peptide and TcR recognition. Cw*0408 was first detected by SBT in two African American bone marrow donors in combination with its most structurally related allele, Cw*04011. The single amino acid change found between Cw*04011 and Cw*0408 was Thr163Leu, a residue involved in pocket A of the peptide‐binding cleft. This new allele could be the result of a gene conversion event between Cw*04011 and any of the Cw*03 alleles.
Complete cDNA sequences of the HLA‐DRB1*14011, *1402, *1403 and *1404 alleles
Authors: Corell A. , Cox S.T. , Soteriou B. , Ramon D. , Madrigal J.A. , Marsh S.G.E.
Published: March 2003

Abstract: Sequencing studies of HLA class II molecules have been focused almost exclusively on the highly polymorphic exon 2. In this study the complete cDNA sequence of four alleles of the DR14 lineage (DR52 group) are reported for the first time. The HLA‐DRB1*1402 and *1403 sequences were shown to be identical to the previously determined DRB1*13011 sequence, also of the DR52 group, in exons 1, 3, 4, 5 and 6. HLA‐DRB1*14011 and *1404 were identical to DRB1*13011 in exons 1, 4, 5 and 6 sequences while they showed specific features within their exon 3 sequence. Both alleles showed a synonymous substitution at the third base of codon 114. However, DRB1*14011 also has a non‐synonymous substitution at the first base of codon 112 which results in a histidine to tyrosine substitution. This is a novel substitution as Histidine 112 is conserved in all known HLA class II B genes.
HLA Class I typing of volunteers for a bone marrow registry: QC analysis by DNA‐based methodology identifies serological typing discrepancies in the assignment of HLA‐A and B antigens
Authors: Gourley I. S. , Kearns J. , McKeen M. , Birkos S. , Brown M. , Kamoun M.
Published: March 2003

Abstract: Until recently, the majority of newly recruited volunteer donors were typed for HLA‐A and ‐B by serology onto the National Marrow Donor Program Registry. Quality control of this serological typing performed by contracted laboratories was carried out by retesting approximately 1% of each laboratory's test volume utilizing DNA‐based techniques (SSOP). The criteria used for selection included samples presumed to be homozygotes, samples with split antigen specificities and samples with antigens considered to be difficult to define. Out of 1983 samples analyzed, 156 HLA‐A (3.9%) and 265 HLA‐B (6.7%) locus discrepancies were identified. Review of these discrepancies by both the serological and QC laboratory revealed that the majority of discrepancies were due to errors in serological typing. Serological discrepancies were categorized as follows: blank antigens identified (36.8%) and misassignments (63.2%). Misassignments were defined as either the incorrect assignment of antigens within a group (“wrong split”), or a complete misassignment. Antigens reported as blanks most frequently belonged to the A19 and A28 groups and to the B70, 46 and 40 groups. The most frequent misassignments within groups were the A19 and A10 groups, and the B40 and B15 groups. Other HLA‐A misassignments included A2 vs A28 or A2 vs A69, while other HLA‐B misassignments included B35 and B70. This QC analysis showed that serological typing of class I antigens for the purposes of NMDP registry typing is prone to a significant error rate. Careful evaluation and selection of contracted laboratories by the NMDP suggests methodological limitations rather than poor performance as the main cause of these observations.
Characterization of a new HLA‐A allele, A*0256, identified in a Caucasian individual
Authors: Czachurski D. , Rausch M. , Scherer S. , Opelz G. , Mytilineos J.
Published: March 2003

Abstract: HLA‐A typing by the PCR‐SSP method in a male 21‐year‐old Caucasian individual revealed a very rare allele combination corresponding to a unique reaction pattern. Therefore, the result was examined using sequence‐based typing. Sequencing of exons 2 and 3 of the HLA‐A locus after allelic separation with specific primers revealed the sequence of a new allele, similar to A*0245. Sequencing of exons 1 and 4 resulted in no additional inconclusive positions. The sequence pattern of the new allele HLA‐A*0256 might have been generated as a result of a double crossing over recombination of an A*0201 and either an A*03 or an A*11.
Characterization of a novel HLA‐C allele, Cw*0315
Authors: Chapple S.D.J. , Agudelo J.D. , Barnardo M.C.N.M. , Vargas‐Cuero A.L. , Fuggle S.V.
Published: March 2003

Abstract: The presence of an unusual HLA class I reactivity pattern was detected in a Caucasoid‐Asian individual by PCR‐sequence specific primer (PCR‐SSP) typing. Exons 2 and 3 were characterized using PCR‐sequence‐based typing (PCR‐SBT) and were found to contain a novel Cw*03 sequence, Cw*0315. In the region studied, Cw*0315 was comprised mainly of the Cw*0302 sequence, but at four positions it contained nucleotides normally only found in other HLA Cw locus alleles. These positions each resulted in an amino acid substitution.
Major histocompatibility complex class II DOA sequences from three Antarctic seal species verify stabilizing selection on the DO locus
Authors: Decker D.J. , Stewart B.S. , Lehman N.
Published: March 2003

Abstract: To provide additional support for the sequence conservation and hence the regulatory role of the MHC class II DOA locus, we obtained the nucleotide sequences of exon 2 and exon 3, along with the intervening intron, of the Ross seal, and sequences from the exon 2 region from the Weddell and leopard seals. These are the first reports of the sequences of this locus from a carnivore species. The results demonstrate strong conservation among mammals for the exon sequence and produce a gene genealogy that is consistent in topology with a species tree.
An additional susceptibility gene for juvenile idiopathic arthritis in the HLA class I region on several DR‐DQ haplotypes
Authors: Smerdel A. , Lie B.A. , Finholt C. , Ploski R. , Førre Ø. , Undlien D.E. , Thorsby E.
Published: March 2003

Abstract: Juvenile idiopathic arthritis (JIA) is an HLA‐associated rheumatic disease with onset in childhood. We recently reported that allele 5 at microsatellite D6S265 in the HLA class I region is associated with JIA, independent of linkage disequilibrium with the high risk DR8‐DQ4 haplotype. In the present study, we investigated whether alleles at D6S265, or other markers in this region, also modify the risk for JIA on other haplotypes, i.e., DRB1*1301‐DQB1*0603 or DRB1*1101/4‐DQB1*0301. We observed a significant association with allele 6 at D6S265 on the DRB1*1301‐DQB1*0603 haplotype. We also noted an association with allele 3 at D6S265, when carried on the DRB1*1101/4‐DQB1*0301 haplotype. Our results further support an additional JIA susceptibility gene in the HLA class I region in linkage disequilibrium with alleles at D6S265.
Study on the synthesis and characterization of peptides containing phosphorylated tyrosine
Authors: Bonewald L.F. , Weintraub S.T. , Bibbs L. , Kates S.A. , Khatri A. , Medzihradszky K.F.
Published: March 2003

Phosphorylation and dephosphorylation are key events in receptor‐mediated and post‐receptor‐mediated signal transduction. Synthetic phosphopeptides have been shown to have dramatic agonist or antagonist effects in several of these signaling pathways. For its 1997 study, the Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group assessed the ability of member laboratories to synthesize phosphotyrosine peptides. Participating laboratories were requested to synthesize and submit the following crude peptide, H–Glu–Asp–Tyr–Glu–Tyr(PO3H2)–Thr–Ala–Arg–Phe–NH2, for evaluation by amino acid analysis, sequence analysis, RP‐HPLC, MALDI‐TOF and ESI mass spectrometry. Prior to analysis of submitted peptides from ABRF members, the Peptide Synthesis Research Group synthesized and characterized the nonphosphorylated form of the peptide, the doubly phosphorylated form and the peptides singly phosphorylated on either the first or the second tyrosine. These peptide standards were separated easily by HPLC and capillary electrophoresis and the phosphotyrosine was detected readily by Edman degradation sequence analysis. No differences were seen by amino acid analysis and the expected masses were observed by mass spectrometry. The two singly phosphorylated peptides were easily distinguished by MALDI‐PSD. Analysis of the peptides submitted from member facilities revealed that all but four of the 33 samples contained the correct product as determined by HPLC and mass spectrometry. HPLC analysis indicated that 20 of the 33 submitted samples contained greater than 75% correct product, five contained less than 50% correct product and four did not contain any correct product. By ESI/MS, an additional singly charged ion at m/z 535.5 was detected in five of the 33 submitted samples; this ion was subsequently shown to represent Ac‐TARF‐NH2. No correlation was found to exist between coupling time and percentage correct product; however, a correlation may exist between a greater percentage of correct product and the use of non‐protected phosphotyrosine.
Kinetic and thermodynamic control of the relative yield of the deamidation of asparagine and isomerization of aspartic acid residues
Authors: Capasso S. , Di Cerbo P.
Published: March 2003

Abstract: Selective deamidation of Asn67 of RNase A to β‐Asp67 and Asp67 residues at neutral pH initially produces greater amounts of the β‐Asp derivative. As the reaction proceeds the relative concentration of [Asp67]–RNase A increases and, at equilibrium, becomes predominant. Such a discrepancy between the kinetic and thermodynamic control on reaction products is discussed in light of information from X‐ray three‐dimensional analysis and the lower thermodynamic stability of the β‐Asp derivative relative to the parent enzyme.
Synthesis and application of peptide immunogens related to the sperm tail protein tpx‐1, a member of the CRISP superfamily of proteins
Authors: Keah H.H. , Hearn M. T. W. , O'Bryan M.K. , De Kretser D.M.
Published: March 2003

Abstract: The synthesis of peptides containing 0, 1 and 2 cysteine residues related to the human sperm tail protein, tpx‐1, is described. These synthetic peptides, following conjugation to keyhole limpet hemocyanin modified with maleimidobenzoic acid N‐hydroxysuccinimide ester, were used as immunogens to generate polyclonal antibodies in female New Zealand white rabbits. The binding characteristics of the derived antipeptide sera were evaluated using indirect and competitive ELISA procedures. Western immunoblot experiments also confirmed that these synthetic peptide immunogens are able to generate high‐titer polyclonal antibodies capable of cross‐reacting with the mature tpx‐1 protein present in crude rat sperm tail/testis preparations as well as in outer dense fiber preparations. Consequently, these synthetic peptides represent promising candidates for investigations into the role of tpx‐1 in the immunoregulation of sperm function in the rat and other mammalian models, with the derived antisera also providing an avenue to explore possible sites of expression of tpx‐1 proteins in other tissues.
A new insect neurotoxin AngP1 with analgesic effect from the scorpion Buthus martensii Karsch: purification and characterization
Authors: Guan R.‐J. , Wang M. , Wang D. , Wang D.‐C.
Published: March 2003

Abstract: An insect toxin named BmK AngP1 was purified from the venom of the scorpion Buthus martensii Karsch (BmK). It also shows an evident analgesic effect on mice, but is interestingly devoid of mammalian toxicity. Bioassay showed that the CPU value of AngP1 was 0.01 μg/body (≈ 30 mg) for the excitatory insect toxicity and 43.0% inhibition efficiency for analgesia at a dose of 5 mg/kg. However, even at the dosage of 10 mg/kg no detectable toxicity on mice could be found. The isoelectric point (pI) value for AngP1 was 4.0, and its molecular mass analyzed by MALDI‐TOF MS was 8141.0. The first 15 N‐terminal residues of AngP1 were determined by Edman degradation and showed high similarity to that of other excitatory scorpion insect toxins. The circular dichroism spectroscopy measured on a JASCO J‐720 system showed that there were 10.4% α‐helix, 46.2% β‐strand and 14.1% turn structure in this peptide. Under two conditions single crystals of AngP1 were obtained.
Evaluation of the conformational propensities of peptide isosteres as a basis for selecting bioactive pseudopeptides
Authors: Gupta S. , Payne J.W.
Published: March 2003

Abstract: Our aim was to compare the repertoires of conformers formed by the model zwitterionic peptides AA and AAA in aqueous solution with the conformational profiles of a range of their peptide isosteres, so as to facilitate selection of isosteres for synthesis and testing as biologically stable surrogates of bioactive di‐ and tripeptides. Comparisons were based upon the results of conformational analysis using a random search approach implemented within the SYBYL molecular modelling package, using zwitterionic molecules, simulated aqueous solvation using a dielectric constant of 80 and allowing all torsions to vary. For each compound, individual conformers were grouped on the basis of specific combinations of psi, phi and omega torsions and, using their energies, the aggregated percentage for each group was calculated using a Boltzmann distribution and displayed using a 3D pseudo Ramachandran plot relating percentage conformer to psi and phi torsions. Retroamide, N‐methylamide and thioamide isosteres showed the best match to natural peptides and to the molecular recognition parameters defined for substrates of peptide transporters. The results should aid rational design of therapeutic agents in various areas, e.g. oral delivery of drugs by peptide transporters and of peptidase inhibitors. This approach may usefully be applied to various biochemical and pharmaceutical topics.
Effect of viscosity on the deamidation rate of a model Asn‐hexapeptide
Authors: Li R. , Topp E.M. , Hageman M.J.
Published: March 2003

Abstract:  The effect of viscosity on the deamidation rate of a model Asn‐containing hexapeptide (l‐Val‐l‐Tyr‐Pro‐l‐Asn‐Gly‐l‐Ala) was assessed in aqueous solution and in solids containing varying amounts of poly(vinyl pyrrolidone) (PVP) and water. Stability studies were conducted at 0.1 mg/mL peptide and 0–50% PVP (w/w) in aqueous solution, and at 5% (w/w) peptide and different relative humidities (31.6, 53.1, 74.4 and 96%) in the solid state. The parent peptide and its deamidation products were analysed by reverse‐phase high‐performance liquid chromatography. Deamidation rates decreased with increasing solvent viscosity in a manner described by a semi‐empirical mathematical model developed to describe this relationship. The results suggest that the motion of the Asn side‐chain along the reaction coordinate is a function of the macroscopic solvent viscosity. However, the apparent energy barrier for the diffusive movement of the side‐chain appears to be less than the energy barrier for that associated with macroscopic viscosity. The dependence of the deamidation rate on viscosity in both viscous solution and hydrated solids further demonstrates the importance of mobility in peptide deamidation.
Practical protocols for stepwise solid‐phase synthesis of cysteine‐containing peptides
Authors: Angell Y. M. , Alsina J. , Barany G. , Albericio F.
Published: March 2003

Abstract: This study details a series of conditions that may be applied to ensure ‘safe’ incorporation of cysteine with minimal racemization during automated or manual solid‐phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62, 4307–4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N‐[(dimethylamino)‐1H‐1,2,3‐triazolo[4,5‐b]pyridin‐1‐ylmethylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HATU), N‐[1H‐benzotriazol‐1‐yl)‐(dimethylamino)methylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HBTU), and (benzotriazol‐1‐yl‐N‐oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N‐methylmorpholine (NMM) or N,N‐diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5–33%) of cysteine racemization. As demonstrated on the tripeptide model H‐Gly‐Cys‐Phe‐NH2, and on the nonapeptide dihydrooxytocin, the following methods are recommended: O‐pentafluorophenyl (O‐Pfp) ester in DMF; O‐Pfp ester/1‐hydroxybenzotriazole (HOBt) in DMF; N,N′‐diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6‐trimethylpyridine (TMP) in DMF (preactivation time 3.5–7.0 min in all of these cases); and HBTU/HOBt/TMP in CH2Cl2/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58‐residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6‐dimethylpyridine (lutidine), 2,3,5,6‐tetramethylpyridine (TEMP), octahydroacridine (OHA), and 2,6‐di‐tert‐butyl‐4‐(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.
β‐turn formation by a six‐residue linear peptide in solution
Authors: Gao F , Li Y , Sha Y , Lai L , Wang Y , Wu H , Qiu Y
Published: March 2003

Abstract:A model peptide AAGDYY‐NH2(B1), which is found to adopt a β‐turn conformation in the TEM‐1 β‐lactamase inhibitor protein (BLIP) in the TEM‐1/BLIP co‐crystal, was synthesized to elucidate the mechanism of its β‐turn formation and stability. Its structural preferences in solution were comprehensively characterized using CD, FT‐IR and1H NMR spectroscopy, respectively. The set of observed diagnostic NOEs, the restrained molecular dynamics simulation, CD and FT‐IR spectroscopy confirmed the formation of a β‐turn in solution by the model peptide. The dihedral angles [(φ3, ϕ3) (φ4, ϕ4)] of [(−52°, −32°) (−38°, −44°)] of Gly‐Asp fragment in the model peptide are consistent with those of a type III β‐turn. In a conclusion, the conformational preference of the linear hexapeptide B1 in solution was determined, and it would provide a simple template to study the mechanism of β‐turn formation and stability.
Structure−affinity relationships in the gp41 ELDKWA epitope for the HIV‐1 neutralizing monoclonal antibody 2F5: effects of side‐chain and backbone modifications and conformational constraints
Authors: Tian Y. , Ramesh C.V. , Ma X. , Naqvi S. , Patel T. , Cenizal T. , Tiscione M. , Diaz K. , Crea T. , Arnold E. , Arnold G.F. , Taylor J.W.
Published: March 2003

Abstract: The human monoclonal antibody, mAb 2F5, has broad HIV‐1 neutralizing activity and binds a conserved linear epitope within the envelope glycoprotein gp41 having a core recognition sequence ELDKWA. In this study, the structural requirements of this epitope for high‐affinity binding to mAb 2F5 were explored using peptide synthesis and competitive enzyme‐linked immunosorbant assay (ELISA). Expansion of the minimal epitope to an end‐capped, linear nonapeptide, Ac‐LELDKWASL‐amide, was sufficient to attain maximal affinity within the set of native gp41‐sequence peptides assayed. Scanning single‐residue alanine and d‐residue substitutions then confirmed the essential recognition requirements of 2F5 for the central DKW sequence, and also established the importance of the terminal leucine residues in determining high‐affinity binding of the linear nonapeptide. Further studies of side‐chain and backbone‐modified analogs revealed a high degree of structural specificity for the DK sequence in particular, and delineated the steric requirements of the Leu3 and Trp6 residues. The nine‐residue 2F5 epitope, flanked by pairs of serine residues, retained a high affinity for 2F5 when it was conformationally constrained as a 15‐residue, disulfide‐bridged loop. However, analogs with smaller or larger loop sizes resulted in lower 2F5 affinities. The conformational effects of the gp41 C‐peptide helix immediately adjacent to the N‐terminal end of the ELDKWA epitope were examined through the synthesis of helix‐initiated analogs. Circular dichroism (CD) studies indicated that the α‐helical conformation was propagated efficiently into the LELDKWASL epitope, but without any significant effect on its affinity for 2F5. This study should guide the design of a second generation of conformationally constrained ELDKWA analogs that might elicit an immune response that mimics the HIV‐neutralizing actions of 2F5.
Transient vesicle leakage initiated by a synthetic apoptotic peptide derived from the death domain of neurotrophin receptor, p75NTR
Authors: Medina M.L. , Bolender J.P. , Plesniak L.A. , Chapman B.S.
Published: March 2003

Abstract:  Peptides that induce apoptosis have potential as anticancer therapeutics. The design of safe, effective cancer therapeutic peptides requires characterization of the physical and chemical properties that influence activation of cell death in neoplastic cells. NTR365 is a synthetic pro‐apoptotic peptide with an amino acid sequence derived from the death domain of p75NTR. These studies were initiated to identify a potential mechanism for the apoptotic activity of NTR365 identified by Rabizadeh et al. We examined the interactions of this synthetic pro‐apoptotic peptide with phospholipid vesicles. Fluorescence experiments demonstrate that the peptide induces leakage from large unilamellar vesicles. Leakage activity is transient and dependent on the presence of anionic lipid in the vesicles. Circular dichroism studies show that the NTR365 adopts a different conformation and may have altered vesicle affinity under conditions conducive to leakage. The active conformation of NTR365 differs from that of the NMR derived conformation. A related peptide with a single substitution is not apoptotically active, does not form a helical structure in the presence of vesicles and does not induce appreciable vesicle leakage under the same conditions as NTR365. These studies suggest that the demonstrated apoptotic activity of a closely related NTR364 peptide is linked to disruption of a membrane barrier and to the ability of the peptide to form a helical structure.