Rare B(A)02/O01 was found in Sichuan again: a case report

B(A) is a rare ABO blood subgroup. Here we reported a B(A)02/O01 case. One 25-year-old female patient showed inconsistent forward and reverse blood grouping results based on micro-column gel agglutination assay. PCR-SSP and PCR-SBT based genotyping indicated that the patient was B(A)02/O01 heterozygous.     

Serological?identification and molecular?characterization?of?B?(A)?02?subtype?in?patients?and blood donors from?Eastern?China

This study was designed to identify the rare type ABO blood groups, B(A) 02, from Eastern China. Three samples with discordant serological results during routine blood type identification and four samples from one sample’ family were selected. All of them were detected by serological method. The exon 6 and 7 of the ABO genes were amplified by PCR and sequenced. They were typed as AsubB by serology and as BO by genotype. In AsubB samples, nt 700C>G mutation was detected in B gene, which was previously defined as B(A)02 alleles. In these seven samples, six showed B(A)02/O01 and one showed B(A)02/O02.B(A)02 allele was found to be more common in this study than B(A)04 which is considered to be more frequent than B(A)02. The careful identification of rare blood types is important for the safety of clinical blood transfusion.     

ABO blood group system

The accuracy of regular serum methods to detect ABO blood groups can be negatively affected by some factors, such as irregular antibodies, autoantibodies or effects of diseases leading to false or weak agglutination. This study aimed to accurately identify ambiguous ABO blood groups by serological and gene detection methods. The samples were collected in the First Affiliated Hospital of Nanjing Medical University from December 2018 to December 2019. ABO genotyping was performed by polymerase chain reaction-sequence specific primer (PCR-SSP) method in 20 samples, and ABO exons 6 and 7 or FUT1 and FUT2 genes were sequenced in 5 samples. The genes detected in the 21 specimens included 4 cases of A/B, 2 cases of A205/O01, 3 cases of A/O01, 3 cases of A/O02, 1 case of O01/O01, 1 case of O01/O02, 1 case of B/O01, 1 case of B/O02, 1 case of Bel/O01, 1 case of Cisab01/O01, 1 case of rare B/O04, 1 case of Bombay-like Bmh, 1 case of new gene showing c.261del G of exon 6, c.579 T > C of exon 7 and B new/O01. This study suggests that ABO blood group genotyping technology combined with serological typing can be used for accurately typing ambiguous blood groups.     

Serological and genetic analysis of ABO ambiguous blood group samples

The accuracy of regular serum methods to detect ABO blood groups can be negatively affected by some factors, such as irregular antibodies, autoantibodies or effects of diseases leading to false or weak agglutination. This study aimed to accurately identify ambiguous ABO blood groups by serological and gene detection methods. The samples were collected in the First Affiliated Hospital of Nanjing Medical University from December 2018 to December 2019. ABO genotyping was performed by polymerase chain reaction-sequence specific primer (PCR-SSP) method in 20 samples, and ABO exons 6 and 7 or FUT1 and FUT2 genes were sequenced in 5 samples. The genes detected in the 21 specimens included 4 cases of A/B, 2 cases of A205/O01, 3 cases of A/O01, 3 cases of A/O02, 1 case of O01/O01, 1 case of O01/O02, 1 case of B/O01, 1 case of B/O02, 1 case of Bel/O01, 1 case of Cisab01/O01, 1 case of rare B/O04, 1 case of Bombay-like Bmh, 1 case of new gene showing c.261del G of exon 6, c.579 T>C of exon 7 and B new/O01. This study suggests that ABO blood group genotyping technology combined with serological typing can be used for accurately typing ambiguous blood groups.     

HLA genetic profile of Mapuche (Araucanian) Amerindians from Chile

Amerindian Mapuche (Araucanians) are now living in Chile and Argentina at both sides of Andean Mountains. They are anthropologically and genetically different from southernmost South America Patagonian Amerindians. Most of the HLA alleles found in our Mapuche sample are frequent or very frequent in North and South America Amerindians: (1) Class I: A*02:01, A*03:01, A*68:01, B*39:09, B*51:01, (2) Class II: DRB1*03:01, DRB1*04:03, DRB1*07:01, DRB1*08:02, DRB1*14:02, DRB1*16:02. One of the nine most frequent extended haplotypes seems to be from European origin, suggesting the existence of a degree of admixture with Europeans in our Mapuche sample. It has been calculated of about 11 % admixture. Three of the extended haplotypes are also found in other Amerindians and five of them are newly found in Mapuche Amerindians: A*68:01-B*39:09-DRB1*08:02-DQB1*04:02; A*68:01-B*51:01-DRB1*04:03-DQB1*03:02; A*29:01-B*08:01-DRB1*03:01-DQB1*02:01; A*02:01-B*15:01-DRB1*04:03-DQB1*03:02; A*33:01-B*14:02-DRB1*07:01-DQB1*03:03. The medical importance of calculating HLA profile is discussed on the diagnostic (HLA and disease) and therapeutical bases of HLA pharmacogenomics and on the construction of a virtual transplantation HLA list profile. Also, anthropological conclusions are drawn.     

The functional A allele was resurrected via recombination in the human ABO blood group gene

Functional A and B alleles are distinguished at two critical sites in exon 7 of the human ABO blood group gene. The most frequent nonfunctional O alleles have one-base deletion in exon 6 producing a frameshift, and it has the A type signature in two critical sites in exon 7. Previous studies indicated that B and O alleles were derived from A allele in human lineage. In this study, we conducted a phylogenetic network analysis using six representative haplotypes: A101, A201, B101, O01, O02, and O09. The result indicated that the A allele, possibly once extinct in the human lineage a long time ago, was resurrected by a recombination between B and O alleles less than 300,000 years ago.     

Induction of Plasmodium falciparum-specific CD4+ T cells and memory B cells in Gabonese children vaccinated with RTS,S/AS01(E) and RTS,S/AS02(D)

The recombinant circumsporozoite protein (CS) based vaccine, RTS,S, confers protection against Plasmodium falciparum infection in controlled challenge trials and in field studies. The RTS,S recombinant antigen has been formulated with two adjuvant systems, AS01 and AS02, which have both been shown to induce strong specific antibody responses and CD4 T cell responses in adults. As infants and young children are particularly susceptible to malaria infection and constitute the main target population for a malaria vaccine, we have evaluated the induction of adaptive immune responses in young children living in malaria endemic regions following vaccination with RTS,S/AS01(E) and RTS,S/AS02(D). Our data show that a CS-specific memory B cell response is induced one month after the second and third vaccine dose and that CS-specific antibodies and memory B cells persist up to 12 months after the last vaccine injection. Both formulations also induced low but significant amounts of CS-specific IL-2(+) CD4(+) T cells one month after the second and third vaccine dose, upon short-term in vitro stimulation of whole blood cells with peptides covering the entire CS derived sequence in RTS,S. These results provide evidence that both RTS,S/AS01(E) and RTS,S/AS02(D) induced adaptive immune responses including antibodies, circulating memory B cells and CD4(+) T cells directed against P. falciparum CS protein. TRIAL REGISTRATION: ClinicalTrials.gov NCT00307021.     

ABO genotyping by PCR-RFLP and cloning and sequencing

A refined PCR-RFLP based method was established to genotype ABO blood groups. The main objective of this study was to make the techniques also suitable for working with degraded DNA. Specific primer design was carried out to choose fragments shorter than 200 bp as necessary in forensic and archaeological applications. Four fragments of exon 6 and 7 of the ABO gene were amplified and digested by in total 7 restriction endonucleases. Particular attention was paid to the base changes at nucleotide positions 261(delG), 297, 526, 703, 721, 771, 796 and 1060(delC) in order to distinguish the six common alleles A101, A201, B, O01, O02 and O03. Furthermore, this method also enables determination of seven of the less frequent alleles: A104, A204, Ax02, Ax03, O05, O06 and O07. The method was applied successfully to a series of blood samples with known phenotypes and genotypes as well as DNA extracted from a thirty year old blood stain and an ancient tooth sample. However, working with ancient DNA requires additional cloning and sequencing of the RFLP-typing results due to DNA post mortem damages such as deaminations, which could lead to false typing results.     

Evaluation of RTS,S/AS02A and RTS,S/AS01B in Adults in a High Malaria Transmission Area

Background

This study advances the clinical development of the RTS,S/AS01B candidate malaria vaccine to malaria endemic populations. As a primary objective it compares the safety and reactogenicity of RTS,S/AS01B to the more extensively evaluated RTS,S/AS02A vaccine.

Methodology

A Phase IIb, single centre, double-blind, controlled trial of 6 months duration with a subsequent 6 month single-blind follow-up conducted in Kisumu West District, Kenya between August 2005 and August 2006. 255 healthy adults aged 18 to 35 years were randomized (1∶1∶1) to receive 3 doses of RTS,S/AS02A, RTS,S/AS01B or rabies vaccine (Rabipur®; Chiron Behring GmbH) at months 0, 1, 2. The primary objective was the occurrence of severe (grade 3) solicited or unsolicited general (i.e. systemic) adverse events (AEs) during 7 days follow up after each vaccination.

Principal Findings

Both candidate vaccines had a good safety profile and were well tolerated. One grade 3 systemic AE occurred within 7 days of vaccination (RTS,S/AS01B group). No unsolicited AEs or SAEs were related to vaccine. A marked increase in anti-CS antibody GMTs was observed post Dose 2 of both RTS,S/AS01B (31.6 EU/mL [95% CI: 23.9 to 41.6]) and RTS,S/AS02A (16.7 EU/mL [95% CI: 12.9 to 21.7]). A further increase was observed post Dose 3 in both the RTS,S/AS01B (41.4 EU/mL [95% CI: 31.7 to 54.2]) and RTS,S/AS02A (21.4 EU/mL [95% CI: 16.0 to 28.7]) groups. Anti-CS antibody GMTs were significantly greater with RTS,S/AS01B compared to RTS,S/AS02A at all time points post Dose 2 and Dose 3. Both candidate vaccines produced strong anti-HBs responses. Vaccine efficacy in the RTS,S/AS01B group was 29.5% (95% CI: −15.4 to 56.9, p = 0.164) and in the RTS,S/AS02A group 31.7% (95% CI: −11.6 to 58.2, p = 0.128).

Conclusions

Both candidate malaria vaccines were well tolerated over a 12 month surveillance period. A more favorable immunogenicity profile was observed with RTS,S/AS01B than with RTS,S/AS02A.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00197054","term_id":"NCT00197054"}}NCT00197054     

Immunocytochemistry and heterogeneity of rat brain vimentin

In unfixed cryostat sections of the brains of early postnatal and adult rats, we screened for cells containing vimentin-positive intermediate filaments (VI+-IFs) by applying a panel of four monoclonal antibodies (Mabs VI-01, VI-02, VI-05 and VI-5B3) using indirect immunofluorescence. All of the Mabs stained VI+-IFs in the stromal part of the choroid plexus, in endothelial cells of blood vessels and in meninges in both adult and immature brains, although with varying strength (VI-5B3 and VI-01 stained more strongly than VI-05 and VI-02). In the brain parenchyma of adults, intense staining was mainly localized in ventricular ependymal cells (VI-5B3/VI-01 greater than VI-02/VI-05) and fibrous astrocyte-like cells (FAs). In the immature brain, the ependymal cells were activated in appearance, with evidence of cell enlargement, greater spreading of VI+-IFs within the cytoplasm and more pronounced VI+ cytoplasmic protrusions into the brain parenchyma. VI+-FAs were found near the ependymal and meningeal borders as well as in the white matter tracts of adult brain (VI-5B3/VI-01 greater than VI-05 greater than VI-02). In immature animals, VI+-FAs were less frequently encountered in the forebrain regions, except in and near the subepenydmal layer (in the adjacent parenchyma) as well as in submeningeal layers. Weaker staining was usually elicited by Mabs VI-02 and VI-05. In the cerebellum, Bergmann cell fibers were stained in both age groups. In adults, the most intense fluorescence usually occurred in segments close to the pia (VI-5B3/VI-01 greater than VI-05 greater than VI-02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential analysis of Bacillus anthracis after pX01 plasmid curing and comprehensive data on Bacillus anthracis infection in macrophages and glial cells

Bacillus anthracis is a gram-positive bacterial organism responsible for anthrax. This organism has two pathogenic plasmids: pX01 and pX02. The genetic function of pX01, which comprises about 198 kb, is not known, except for a region called the pathogenic island, which contains three genes-pag, lef, and cya-that code for three toxic proteins. A 2-D difference gel electrophoresis (2-D DIGE) system was used to verify the existence of proteins controlled by the pX01 plasmid, and protein regulation data were obtained using DeCyder software. A total of 1728 proteins were identified in the wild-type strain of this organism and 1684 in the pX01 plasmid. Twenty-seven of these proteins disappeared and eight appeared when the pX01 plasmid was removed. An additional 52 proteins were downregulated and 15 were upregulated when this plasmid was removed. A total of 102 proteins have been identified using the MALDI-TOF method of analysis, including 49 whose functions are unknown. Among these, 31 participate in metabolic processes, two in cellular processes, 15 in the processing of genetic information, and five in the processing of extracellular information. Another seven proteins participate in bacterial virulence and pathogenesis. We investigated the functions of these proteins in other bacteria, particularly the B. anthracis derivative H9041. Bacterial growth differed between pX01+/pX02+ B. anthracis and its pX01-/pX02+ derivative as did the cytotoxicity of macrophages infected by pX01+/pX02+ B. anthracis and the pX01-pX02+ derivative. We also found that S100B protein levels increased in the host infected with pX01+/pX02+ B. anthracis or its pX01-/pX02+ derivative. These data suggest that the pX01 plasmid plays a key role in the regulation of protein functions in B. anthracis.     

Allele and haplotype frequencies of human leukocyte antigen-A, -B, -C,-DRB1, and -DQB1 in Chinese patients with hematological diseases

The human leukocyte antigen (HLA) system plays a central role in the immune response to pathogens, as well as in organ and allogenic hematopoietic stem cell transplantation (HSCT). Finding a five-locus (i.e., HLA-A, -B, -C, -DRB1, and -DQB1) matched unrelated donor for a patient awaiting HSCT is a major clinical challenge, due to the lack of HLA-identical sibling donors and the high polymorphism of HLA. To date, most studies providing HLA allele frequencies (AF) and haplotype frequencies (HF) in Chinese populations have focused on donors instead of the recipients and have provided data for three loci (HLA-A, -B, and -DR); however, data from five-locus HLA typing in a large sample of patients, especially those with hematological diseases, remains unavailable. Therefore, this study was designed to determine HLA AF and two-, three-, four- and five-locus HF in a large cohort of Chinese Han patients with hematological diseases. The AF and the HF were determined using high-resolution HLA typing data from 2,878 patients. The total number of HLA-A, -B, -C, -DRB1, and -DQB1 alleles was determined to be 48, 92, 49, 52, and 24, respectively. Hardy-Weinberg equilibrium (HWE) analyses indicated significant deviations from HWE for HLA-A, -C, -DRB1, and -DQB1 AF, but not for HLA-B locus. The three most common alleles at each locus were A*11:01, A*24:02, A*02:01; B*46:01, B*40:01, B*13:02; C*01:02, C*07:02, C*06:02; DRB1*09:01, DRB1*15:01, DRB1*07:01; DQB1*03:01, DQB1*03:03, and DQB1*06:01. Our data may help to determine whether the current bone marrow registry contains sufficient diversity to meet the demand.     

Importance of human leukocyte antigen (HLA) class I and II alleles on the risk of multiple sclerosis

Multiple sclerosis (MS) is a complex disease of the central nervous system of unknown etiology. The human leukocyte antigen (HLA) locus on chromosome 6 confers a considerable part of the susceptibility to MS, and the most important factor is the class II allele HLA-DRB1*15:01. In addition, we and others have previously established a protective effect of HLA-A*02. Here, we genotyped 1,784 patients and 1,660 healthy controls from Scandinavia for the HLA-A, HLA-B, HLA-C and HLA-DRB1 genes and investigated their effects on MS risk by logistic regression. Several allele groups were found to exert effects independently of DRB1*15 and A*02, in particular DRB1*01 (OR = 0.82, p = 0.034) and B*12 (including B*44/45, OR = 0.76, p = 0.0028), confirming previous reports. Furthermore, we observed interaction between allele groups: DRB1*15 and DRB1*01 (multiplicative: OR = 0.54, p = 0.0041; additive: AP = 0.47, p = 4 × 10(-06)), DRB1*15 and C*12 (multiplicative: OR = 0.37, p = 0.00035; additive: AP = 0.58, p = 2.6 × 10(-05)), indicating that the effect size of these allele groups varies when taking DRB1*15 into account. Analysis of inferred haplotypes showed that almost all DRB1*15 bearing haplotypes were risk haplotypes, and that all A*02 bearing haplotypes were protective as long as they did not carry DRB1*15. In contrast, we found one class I haplotype, carrying A*02-C*05-B*12, which abolished the risk of DRB1*15. In conclusion, these results confirms a complex role of HLA class I and II genes that goes beyond DRB1*15 and A*02, in particular by including all three classical HLA class I genes as well as functional interactions between DRB1*15 and several alleles of DRB1 and class I genes.     

HLA class I,KIR, and genome-wide SNP diversity in the RV144 Thai phase 3 HIV vaccine clinical trial

RV144 is the first phase 3 HIV vaccine clinical trial to demonstrate efficacy. This study consisted of more than 8,000 individuals in each arm of the trial, representing the four major regions of Thailand. Human leukocyte antigen (HLA) class I and killer cell immunoglobulin-like receptor (KIR) genes, as well as 96 genome-wide ancestry informative markers (AIMs) were genotyped in 450 placebo HIV-1-uninfected individuals to identify the immunogenetic diversity and population structure of this cohort. High-resolution genotyping identified the common HLA alleles as A*02:03, A*02:07, A*11:01, A*24:02, A*24:07, A*33:03, B*13:01, B*15:02, B*18:01, B*40:01, B*44:03, B*46:01, B*58:01, C*01:02, C*03:02, C*03:04, C*07:01, C*07:02, C*07:04, and C*08:01. The most frequent three-loci haplotype was B*46:01-C*01:02-A*02:07. Framework genes KIR2DL4, 3DL2, and 3DL3 were present in all samples, and KIR2DL1, 2DL3, 3DL1, 2DS4, and 2DP1 occurred at frequencies greater than 90 %. The combined HLA and KIR profile suggests admixture with neighboring Asian populations. Principal component and correspondence analyses comparing the RV144 samples to the phase 3 International HapMap Project (HapMap3) populations using AIMs corroborated these findings. Structure analyses identified a distinct profile in the Thai population that did not match the Asian or other HapMap3 samples. This shows genetic variability unique to Thais in RV144, making it essential to take into account population stratification while performing genetic association studies. The overall analyses from all three genetic markers indicate that the RV144 samples are representative of the Thai population. This will inform subsequent host genetic analyses in the RV144 cohort and provide insight for future genetic association studies in the Thai population.     

Expression analysis of the thyrotropin-releasing hormone receptor (TRHR) in the immune system using agonist anti-TRHR monoclonal antibodies.

Monoclonal anti-rat thyrotropin-releasing hormone (TRH) receptor (TRHR)-specific antibodies (mAb) were generated by immunization with synthetic peptides of rat TRHR partial amino acid sequences; one (TRHR01) was directed against a sequence (84-98) in the extracellular portion of the rat TRHR reported to be constant among different species, including man, and the second (TRHR02) recognizes the C-terminal region sequence 399-412. In lysates from GH4C1 cells, a clonal rat pituitary cell line, both mAb recognize the TRHR in Western blot analysis, and TRHR02 immunoprecipitates the TRHR. Incubation of GH4C1 cells with the mAb causes a fluorescence shift in fluorescence-activated cell sorting analysis. The cells were stained specifically by both mAb using immunocytochemical techniques. Furthermore, TRHR01 is agonistic in its ability to trigger Ca2+ flux, and desensitizes the TRH receptor. We tested for TRHR in several rat organs and found expression in lymphoid tissues. TRHR01 recognizes the human TRHR, and analysis of human peripheral blood lymphocyte and tonsil-derived leukocyte populations showed receptor expression in non-activated and phytohemagglutinin-activated T and B cells.     

The native virulence plasmid combination affects the segregational stability of a theta-replicating shuttle vector in Bacillus anthracis var. New Hampshire

The segregational stability of a small, theta-replicating, non-mobilizable shuttle plasmid (pAEX-5E) was determined in fully virulent (pX01+/pX02+), partially cured (pX01+/pX02- and pX01-/pX02+) and fully cured (pX01-/pX02-) derivatives of Bacillus anthracis var. New Hampshire. Under the growth conditions used (L-broth, 37 degrees C, aerobic, batch culture), pAEX-5E remained segregationally stable in the pX01-/pX02+ and pX01-/pX02- derivatives for in excess of 100 culture generations, but was expelled from the pX01+/pX02+ and pX01+/pX02- derivatives (100% loss occurred after 101+/-3.8 and 54+/-6.0 culture generations, respectively). In the presence of antibiotic selection pressure to maintain pAEX-5E (5 microg erythromycin ml-1) no comparable loss of pX01 or pX02 was observed over 100 generations of growth in any of the derivatives of B. anthracis. Under these conditions the pX01+/pX02- derivative had an extended culture doubling time (td+/-S. E. of the mean) of 75.3 +/- 1.4 min compared with 47.3 +/- 1.1, 46.2 +/- 0.86 and 43.2 +/- 1.2 min for the pX01+/pX02+, pX01-/pX02+ and pX01-/pX02- derivatives, respectively. That antibiotic resistance was pAEX-5E-mediated was confirmed using a second antibiotic marker (kanamycin). After100 generations of growth in the presence of erythromycin, colonies were shown to have retained kanamycin resistance. Southern blot analysis, in conjunction with plasmid rescue to Escherichia coli confirmed that, after 100 culture generations in the presence of antibiotic selection pressure, pAEX-5E had remained structurally stable and had not integrated into the B. anthracis genome.     

Influence of HLA Class I Haplotypes on HIV-1 Seroconversion and Disease Progression in Pumwani Sex Worker Cohort

We examined the effect of HLA class I haplotypes on HIV-1 seroconversion and disease progression in the Pumwani sex worker cohort. This study included 595 HIV-1 positive patients and 176 HIV negative individuals. HLA-A, -B, and -C were typed to 4-digit resolution using sequence-based typing method. HLA class I haplotype frequencies were estimated using PyPop 32-0.6.0. The influence of haplotypes on time to seroconversion and CD4+ T cell decline to <200 cells/mm³ were analyzed by Kaplan-Meier analysis using SPSS 13.0. Before corrections for multiple comparisons, three 2-loci haplotypes were significantly associated with faster seroconversion, including A*23∶01-C*02∶02 (p = 0.014, log rank(LR) = 6.06, false-discovery rate (FDR) = 0.056), B*42∶01-C*17∶01 (p = 0.01, LR = 6.60, FDR = 0.08) and B*07∶02-C*07∶02 (p = 0.013, LR = 6.14, FDR = 0.069). Two A*74∶01 containing haplotypes, A*74∶01-B*15∶03 (p = 0.047, LR = 3.942, FDR = 0.068) and A*74∶01-B*15∶03-C*02∶02 (p = 0.045, LR = 4.01, FDR = 0.072) and B*14∶02-C*08∶02 (p = 0.021, LR = 5.36, FDR = 0.056) were associated with slower disease progression. Five haplotypes, including A*30∶02-B*45∶01 (p = 0.0008, LR = 11.183, FDR = 0.013), A*30∶02-C*16∶01 (p = 0.015, LR = 5.97, FDR = 0.048), B*53∶01-C*04∶01 (p = 0.010, LR = 6.61, FDR = 0.08), B*15∶10-C*03∶04 (p = 0.031, LR = 4.65, FDR = 0.062), and B*58∶01-C*03∶02 (p = 0.037, LR = 4.35, FDR = 0.066) were associated with faster progression to AIDS. After FDR corrections, only the associations of A*30∶02-B*45∶01 and A*30∶02-C*16∶01 with faster disease progression remained significant. Cox regression and deconstructed Kaplan-Meier survival analysis showed that the associations of haplotypes of A*23∶01-C*02∶02, B*07∶02-C*07∶02, A*74∶01-B*15∶03, A*74∶01-B*15∶03-C*02∶02, B*14∶02-C*08∶02 and B*58∶01-C*03∶02 with differential seroconversion or disease progression are due to the dominant effect of a single allele within the haplotypes. The true haplotype effect was observed with A*30∶02-B*45∶01, A*30∶02-C*16∶02, B*53∶01-C*04∶01 B*15∶10-C*03∶04, and B*42∶01-C*17∶01. In these cases, the presence of both alleles accelerated the disease progression or seroconversion than any of the single allele within the haplotypes. Our study showed that the true effects of HLA class I haplotypes on HIV seroconversion and disease progression exist and the associations of HLA class I haplotype can also be due to the dominant effect of a single allele within the haplotype.     

Structures of the asparagine-linked oligosaccharide chains of human von Willebrand factor. Occurrence of blood group A, B, and H(O) structures.

The asparagine-linked oligosaccharide chains of human von Willebrand factor (vWF) purified from pooled plasma were quantitatively liberated from the polypeptide moiety by hydrazinolysis. After N-acetylation, these were fractionated by paper electrophoresis and sequential chromatography on lectin-affinity columns of concanavalin A, Phaseolus vulgaris erythrophytohemagglutinin, Datura stramonium agglutinin, Ricinus communis agglutinin 120, and Ulex europaeus agglutinin I and on a Bio-Gel P-4 column. Their structures were investigated by sequential exoglycosidase digestion in conjunction with methylation analysis. The glycoprotein was shown to be unique in its great diversity of oligosaccharide structures. Another noteworthy finding which had not been reported previously was the occurrence of asparagine-linked oligosaccharide chains with blood group A, B, and H(O) structures. In the present study, this glycoprotein was shown to contain mono- (0.4% of the total oligosaccharides), bi-(78.2%), tri- (12.3%), and tetraantennary (2.3%) complex type oligosaccharides in addition to a series of high mannose type oligosaccharides, Man6-9GlcNAc2 (0.8%). Biantennary complex type oligosaccharide chains were those with (8.2%) and without (70.0%) a bisecting GlcNAc residue and approximately 13.2%, 2.2%, and 0.4% of these contained blood group H(O), A, and B structures, respectively. The tri- and tetraantennary complex type chains were those with and without N-acetyllactosamine repeats, and about 13.0% of the triantennary chains without the N-acetyllactosamine repeat contained the blood group H(O) structure. Occurrence of these asparagine-linked oligosaccharides with blood group A and B structures suggest that the repeated use of factor VIII/vWF pooled concentrate for the treatment of hemophiliacs could result in the production of antibodies against vWF with a different blood group from that of the patient, and this development may be pathogenic.     

Immunocytochemistry and heterogeneity of rat brain vimentin

Summary In unfixed cryostat sections of the brains of early postnatal and adult rats, we screened for cells containing vimentin-positive intermediate filaments (VI⁺-IFs) by applying a panel of four monoclonal antibodies (Mabs VI-01, VI-02, VI-05 and VI-5B3) using indirect immunofluorescence. All of the Mabs stained VI⁺-IFs in the stromal part of the choroid plexus, in endothelial cells of blood vessels and in meninges in both adult and immature brains, although with varying strength (VI-5B3 and VI-01 stained more strongly than VI-05 and VI-02). In the brain parenchyma of adults, intense staining was mainly localized in ventricular ependymal cells (VI-5B3/VI-01>VI-02/VI-05) and fibrous astrocyte-like cells (FAs). In the immature brain, the ependymall cells were activated in appearance, with evidence of cell enlargement, greater spreading of VI⁺-IFs within the cytoplasma and more pronounced VI⁺ cytoplasmic protrusions into the brain parenchyma.VI⁺-FAs were found near the ependymal and meningead borders as well as in the white matter tracts of adult brain (VI-5B3/VI-01>VI-05>VI-02). In immature animals, VI⁺-FAs were less frequently encountered in the forebrain regions, except in and near the subependymal layer (in the adjacent parenchyma) as well as in submeningeal layers. Weaker staining was usually clicited by Mabs VI-02 and VI-05. In the cerebellum, Bergmann cell fibres were stained in both age groups. In adults, the most intense fluorescence usually occurred in segments close to the pia (VI-5B3/VI-01>VI-05>VI-02). In immature animals, the Bergmann cell fibres were less straight, less smooth and thicker, and were stained along their whole length by all Mabs except VI-02. In adults, VI⁺-FAs were observed in the internal granular layer (VI-5B3 and VI-01) and, relatively more often, in the white matter (VI-05). In immature animals, a quasi-continuous mesh-work of VI⁺ cells was detected at some sites of the cerebellum, especially when VI-01 and VI-5B3 were used. With maturation, reduced staining was produced by all VI Mabs in the choroid plexus. We have thus demonstrated that VI is a common molecular denominator of cerebrospinal-fluid and/or blood-washed cells as well as of glial fibers contacting these cells. The differences in the staining of VI⁺ cells by various Mabs probably reflect an immunological heterogeneity of VI⁺-IFs based on the varying accessibility of the individual VI epitopes. This might be due to alterations in the tertiary structure of VI caused, for instance, by phosphorylation or other posttranslational processes. The actual structural state of VI may explain the variations in the immunostainability of astrocytes and also in the staining obtained using classical impregnation methods within the brain and/or its regions.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Distribution of human kappa locus IGKV2-29 and IGKV2D-29 alleles in Swedish Caucasians and Hong Kong Chinese

Polymorphism in the IGKV2-29 gene was shown to decrease the recombination frequency in B cells and to be important for immune responses to Haemophilus influenzae type b polysaccharide. By using the combination of PCR and restriction enzyme mapping, the distribution of IGKV2D-29 and IGKV2-29 gene alleles was estimated in two geographically and ethnically different groups. We found that V2D-29*01 homozygous individuals were most common in Swedish Caucasians (82%), but less common in the Chinese population of Hong Kong (28%). The homozygous V2D-29*02 genotype was found in 19% Chinese, but only in one Caucasian (1%). The frequency of the heterozygous V2D-29*01/V2D-29*02 genotype was also higher in the Chinese population (46%) compared with the Caucasians (7%). V2-29*01 homozygosity was more frequent among Caucasians (85%) than among Chinese (19%). In contrast, homozygous V2-29*02 individuals were over-represented in the Chinese population (18%), whereas only one was found among Caucasians (1%). Heterozygous V2-29*01/V2-29*02 individuals were also more common in the Chinese (63%) than the Caucasian (15%) population. Most Caucasians had the combination of V2D-29*01/V2D-29*01+V2-29*01/V2-29*01 (74%), while the most common genotype for Chinese was V2D-29*01/V2D-29*02+ V2-29*01/V2-29*02 (41%). Analysis of the association of V2D-29*02 and V2-29*02 alleles demonstrated a high degree of linkage, as for V2D-29*01 with V2-29*01. These data show a significant difference in the distribution of IGKV2D-29 and IGKV2-29 alleles among Swedish Caucasians and Hong Kong Chinese. This may help to explain differences in the occurrence of H. influenzae type b infection in the two populations. Evaluated methods for IGKV2D-29 and IGKV2-29 allele detection can be used for the screening allele polymorphisms in other particular patient groups.