DNA sequencing-based typing of HPA-1 to HPA-17w systems.

Although several DNA-based human platelet antigens (HPA) typing techniques, such as PCR-SSP and PCR-SSO, have been established, the typing errors and the lack of interlaboratory reproducibility are still the issues of concerns. In the present study, polymerase chain reaction primers were designed for identification of all the phenotypically different HPA-1 to HPA-17w types by sequencing-based typing (SBT) method using genomic DNA samples. No discrepancies were observed between PCR-SSP typing and SBT typing in typing a panel of HPA-typed platelet donors that included all common HPA types and the rare HPA-1b, 2b, 3b, and 6bw homozygous donors.

HLA-A, -B, -DR haplotype frequencies from DNA typing data of 26,266 Chinese bone marrow donors.

HLA phenotypes of 26,266 Chinese individuals who were recruited as potential hematopoietic stem cell donors by the Shanghai Red Cross Marrow Donor Registry, part of the China Marrow Donor Program, were determined for HLA-A, -B, and -DRB1 alleles at low to intermediate resolution using DNA-based typing methods. The large sample size of the study allowed accurate calculation of the Chinese HLA haplotype frequencies. The observed alleles correspond to 19 HLA-A, 44 -B, and 13 -DR split antigens. The serologic equivalents of HLA-A36, -A80, -B78, and -DR18 alleles were not observed. A total of 2,241 distinct HLA-A, -B, -DRB1 haplotypes were identified. Three-locus haplotype frequency was estimated using the maximum likelihood method. The lowest haplotype frequency that can be reliably estimated at a 95% confidence level was 0.000057. Using this cutoff value, 1,220 haplotypes (54%) were statistically reliable and their cumulative haplotype frequency was 0.9730. The cumulative haplotype frequency of the remaining 1,021 haplotypes (46%) was 0.0270. A regression equation of p = 0.192 log N - 0.576 was derived to estimate the probability (p) of finding an HLA-A, -B, -DR split antigens-matched donor in a pool of N Chinese donors.

[Case report of a rare platelet-specific antigen HPA-10bw allele found in Chinese mainland].

A total of 1,000 Chinese blood donors were typed for human platelet antigens (HPA) using a sequence specific primers -polymerase chain reaction (PCR-SSP) based HPA genotyping method. An individual with a rare HPA-10w(a+b+) genotype was found. In order to confirm the typing results, a fragment of HPA-10 gene was amplified by PCR and then sequenced. Sequencing data showed that a single G to A substitution at nucleotide 263 occurred, resulting in amino acid change from Arg(CGA) to Gln(CAA) at position 62 of GPa protein. The substitution generated antigenic specificity HPA-10bw. The detection of an HPA-10bw allele in the Chinese population suggests that this rare allele should be considered in platelet alloimmunization, such as neonatal alloimmune thrombocytopenia (NAIT), post-transfusion thrombocytopenic purpura (PTP) and post-transfusion refractoriness to platelets (PTR).

Quantification of HTLV-I proviral load in experimentally infected rabbits.

BACKGROUND: Levels of proviral load in HTLV-1 infected patients correlate with clinical outcome and are reasonably prognostic. Adaptation of proviral load measurement techniques is examined here for use in an experimental rabbit model of HTLV-1 infection. Initial efforts sought to correlate proviral load with route and dose of inoculation and with clinical outcome in this model. These methods contribute to our continuing goal of using the model to test treatments that alleviate virus infection. RESULTS: A real-time PCR assay was used to measure proviral load in blood and tissue samples from a series of rabbits infected using HTLV-1 inocula prepared as either cell-free virus particles, infected cells or blood, or by naked DNA injection. Proviral loads from asymptomatically infected rabbits showed levels corresponding to those reported for human patients with clinically silent HTLV-1 infections. Proviral load was comparably increased in 50% of experimentally infected rabbits that developed either spontaneous benign or malignant tumors while infected. Similarly elevated provirus was found in organs of rabbits with experimentally induced acute leukemia/lymphoma-like disease. Levels of provirus in organs taken at necropsy varied widely suggesting that reservoirs of infections exist in non-lymphoid organs not traditionally thought to be targets for HTLV-1. CONCLUSION: Proviral load measurement is a valuable enhancement to the rabbit model for HTLV-1 infection providing a metric to monitor clinical status of the infected animals as well as a means for the testing of treatment to combat infection. In some cases proviral load in blood did not reflect organ proviral levels, revealing a limitation of this method for monitoring health status of HTLV-1 infected individuals.

Binding of HTLV-1 virions to T cells occurs by a temperature and calcium-dependent process and is blocked by certain type 2 adenosine receptor antagonists.

A flow cytometric assay that measures binding of human T-lymphotropic virus type 1 (HTLV-1) virions to target cells was used to investigate the binding process and to screen for compounds affecting viral binding. Results showed that adenosine receptor type 2 antagonists effectively inhibit viral binding at concentrations below 10 microM; no inhibition was seen when antagonist was used to pretreat cells or was added post binding, suggesting direct interference with virus attachment. Efficient HTLV-1 binding required divalent calcium ions and temperatures greater than 20 degrees C. Disruption of lipid rafts by cholesterol depletion compromised viral binding but cleavage of glycosyl-phosphatidylinositol linkages had no effect. A monoclonal antibody (mAb) that recognizes HTLV-1 envelope positions 190-209 impaired binding of virus while other anti-envelope antibodies had no effect. These findings place major constraints on the nature of the HTLV-1 cell binding process and identify a class of inhibitors that may have potential for treatment of infection.

Development of a DNA-based genotyping method for the Diego blood group system.

BACKGROUND: The paucity of appropriate reagents for serologic typing of the Diego blood group has hindered the identification of the rare Di(b-) blood donors needed to transfuse a Dib antigen-negative patient who presented with anti-Dib. Development of an alternative Di typing approach as a supplement to the current serologic typing method is an important and necessary goal. STUDY DESIGN AND METHODS: DI1 and DI2 alleles result from a single C to T substitution at nucleotide 2561 in exon 19 of the human anion exchanger gene causing a proline (DI1) to leucine (DI2) change at amino acid position 854. Allele-specific primers were designed to specifically amplify the DI1 and DI2 alleles using a PCR-based assay system. RESULTS: A PCR sequence-specific primer (SSP) method for Di genotyping was developed, and the specificity and reproducibility of the method were assessed in a blind control study using serologic tests, family segregation, and DNA sequencing analyses. A total of 1,766 DNA samples from unrelated blood donors were typed for DI1 and DI2 alleles and a single Di(b-) donor was identified. The frequency of DI1 and DI2 alleles among Chinese blood donors was 0.0357 and 0.9643, respectively. CONCLUSION: A simple, accurate, and inexpensive DNA-based PCR-SSP method was established for Di genotyping. The typing results can be visualized on a single photograph within 3 hours, making this reliable method suitable for large-scale typing of potential blood donors without serologic backup.

Monoclonally integrated HTLV type 1 in epithelial cancers from rabbits infected with an HTLV type 1 molecular clone.

In addition to T cell leukemias and lymphomas, human T cell leukemia virus type 1 (HTLV-1) infection has been associated with nonhematologic malignancies and described as the cause of one case of small-cell lung carcinoma. Infected primary epithelial cells have been isolated from sweat gland and oral mucosae of HTLV-1-infected human patients. In the present study, epithelial neoplasms developed in two rabbits experimentally infected with a molecular clone of HTLV-1 (strain K30p). Serologic detection of anti-HTLV-1 and isolation of virus from blood lymphocytes at multiple time points postinjection established a course of chronic asymptomatic infection in both. One rabbit, infected for 5.5 years after intramuscular injection of HTLV-1 DNA, developed a thymoma having features of medullary differentiation. HTLV-1 provirus was detected in both thymocytes and neoplastic epithelium isolated discretely from the thymoma by laser capture microdissection. These findings provide the first experimental evidence of HTLV-1 disease after infection by HTLV-1 DNA injection. Endometrial adenocarcinoma occurred in a second rabbit 2.5 years after its inoculation with cell-associated virus. In this second case, an epithelial cell line derived ex vivo from a metastatic lesion produced virus in culture. In tumors from each of the two rabbits, the neoplastic epithelium was infected and harbored monoclonally integrated HTLV-1 provirus. Although monoclonal provirus integration alone does not establish retroviral cause of carcinogenesis unequivocally, these and other accumulating data indicate that there may be a role for HTLV-1 in diseases associated with infection of epithelia, including some epithelial cancers.