Development of microsatellite loci and optimization of a multiplex assay for Latibulus argiolus (Hymenoptera: Ichneumonidae), the specialized parasitoid of paper wasps

Microsatellite loci are commonly used markers in population genetic studies. In this study, we present 40 novel and polymorphic microsatellite loci elaborated for the ichneumonid parasitoid Latibulus argiolus (Rossi, 1790). Reaction condition optimisation procedures allowed 14 of these loci to be co-amplified in two PCRs and loaded in two multiplex panels onto a genetic analyser. The assay was tested on 197 individuals of L. argiolus originating from ten natural populations obtained from the host nests of paper wasps. The validated loci were polymorphic with high allele numbers ranging from eight to 27 (average 17.6 alleles per locus). Both observed and expected heterozygosity values were high, ranging between 0.75 and 0.92 for HO (mean 0.83) and from 0.70 to 0.90 for HE (mean 0.85). The optimized assay showed low genotyping error rate and negligible null allele frequency. The designed multiplex panels could be successfully applied in relatedness analyses and genetic variability studies of L. argiolus populations, which would be particularly interesting considering the coevolutionary context of this species with its social host.

Characterization of SSR genomic abundance and identification of SSR markers for population genetics in Chinese jujube (Ziziphus jujubaMill.)

Chinese jujube (Ziziphus jujubaMill. [Rhamnaceae]), native to China, is a major dried fruit crop in Asia. Although many simple sequence repeat (SSR) markers are available for phylogenetic analysis of jujube cultivars, few of these are validated on the level of jujube populations. In this study, we first examined the abundance of jujube SSRs with repeated unit lengths of 1–6 base pairs, and compared their distribution with those inArabidopsis thaliana. We identified 280,596 SSRs in the assembled genome of jujube. The density of SSRs in jujube was 872.60 loci/Mb, which was much higher than inA. thaliana(221.78 loci/Mb). (A+ T)-rich repeats were dominant in the jujube genome. We then randomly selected 100 SSRs in the jujube genome with long repeats and used them to successfully design 70 primer pairs. After screening using a series of criteria, a set of 20 fluorescently labeled primer pairs was further selected and screened for polymorphisms among three jujube populations. The average number of alleles per locus was 12.8. Among the three populations, mean observed and expected heterozygosities ranged from 0.858 to 0.967 and 0.578 to 0.844, respectively. After testing in three populations, all SSRs loci were in Hardy-Weinberg equilibrium (HWE) in at least one population. Finally, removing high null allele frequency loci and linked loci, a set of 17 unlinked loci was in HWE. These markers will facilitate the study of jujube genetic structure and help elucidate the evolutionary history of this important fruit crop.

Transfer of Microsatellite Loci For The Tropical Tree Prunus africana (Hook. f.) Kalkman

Using databases of previously published primers, we optimised six nuclear microsatellite markers for Prunus africana for the purposes of studying spatial genetic structure and gene flow. To assess variability, these and three previously transferred loci were screened in populations from Kenya and South Africa. Across both populations most loci were polymorphic, with the exception of a single locus which failed to amplify in the South African samples, exhibiting between 2 and 22 alleles and levels of expected heterozygosity (He) ranged from 0.059 to 0.932. Departures from Hardy-Weinberg equilibrium were detected for all loci but not for all populations. The estimated null allele frequency was very low to moderate and no evidence for linkage disequilibrium was detected.

IgG Allotypes Reveal That Antimicrobial Humoral Immunity Persists after Reduced-Intensity Hematopoietic Cell Transplantation

Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for hematologic malignancies through graft-versus-leukemia/lymphoma (GVL) responses and replacement of the patient’s immune system. Conditioning intensity may influence hematopoietic reconstitution, persistent recipient immunity, and donor alloimmune responses. High-dose conditioning (HDC) causes rapid conversion to full donor T- and B-cell chimerism, while patients who undergo reduced-intensity conditioning (RIC) progress through a dynamic mixed chimerism extending weeks to months. We hypothesize that long-lived recipient plasma cells persist, providing beneficial antimicrobial serologic immunity, while donor B cells target allo-antigens, contributing to both GVL and GVHD. Furthermore, recipient humoral immunity persists longer following RIC than HDC, leading to fewer infections and decreased transplant-related mortality. Inherited polymorphisms in IgG heavy chain constant regions can be recognized by allotype-specific monoclonal antibodies and thus distinguish donor and recipient antibodies. We developed a quantitative ELISA by coating mouse monoclonal antibodies specific for the G1m(f) and G2m(n) allotypes. Serial dilutions of 63 patients and their donors were incubated and detected by alkaline phosphatase-conjugated anti-human IgG polyclonal antibody. Twenty-eight HDC patients were conditioned with VP-16 and total body irradiation, while 35 RIC patients received total lymphoid irradiation and anti-thymocyte globulin (TLI-ATG). One hundred fifteen of the total 126 subjects (91%) expressed G1m(f) (null allele frequency: 0.29), and 100 of 126 (79%) expressed G2m(n) (null allele frequency: 0.45). Twenty-six of the 63 pairs (41%) were informative because either the donor or recipient was homozygous null for an allotype. Following both RIC and HDC when the recipient was allotype null, the donor allotype was first detected six months post-HCT and reached 50–80% of donor levels by 12 months (n=8 pairs). Eighteen donors were homozygous allotype null, and recipient-specific antibody was prospectively determined. By 12 months after HDC, recipient antibody levels fell to <10% of pre-HCT allotype levels (n=11 pairs). In contrast, RIC patients retained the allotype at >90% of pre-HCT levels through 12 months post-HCT (n=7 pairs), demonstrating a significant difference from HDC patients in one-year median recipient allotype levels (p=0.016). We confirmed RIC humoral immune persistence by DNA chimerism detection of CD38+CD138+ plasma cells from bone marrow aspirates collected 12 months post-HCT. Analysis revealed 30–58% donor levels of plasma cells, suggesting that RIC patients maintain long-lived plasma cells. Allotype-informative patients were measured by ELISA for total IgG and allotype-specific IgG against Epstein-Barr virus (EBV), varicella zoster virus (VZV), pneumococcus, and influenza. HDC patients displayed loss of recipient antimicrobial allotype between five and eight months in conjunction with loss of bulk allotype. RIC patients maintained >90% pre-HCT allotype levels through one year correlating to levels of total IgG against all four infectious agents when they were seropositive before HCT. In addition, new donorspecific responses to influenza, pneumococcus, and VZV were detected in both sets of patients within seven to 14 months. In the allotype-informative setting, H-Y antibody analysis confirmed that donor-derived allogeneic antibodies against UTY2 and DDX3Y developed after nine and 12 months, respectively, and persisted in both HDC and RIC male patients with female donors. In summary, RIC recipients benefit from a twofold effect: persistent recipient-derived antimicrobial humoral immunity and donor-derived allogeneic B-cell responses. Our studies may explain why RIC results in decreased infectious complications in the post-HCT setting.

Allotype Reagents Distinguish Donor and Recipient Antibodies after Hematopoietic Transplantation.

Allogeneic immune responses provide beneficial graft-versus-leukemia (GVL) and detrimental graft-versus-host disease (GVHD). To characterize allogeneic B cells and their antibodies in relation to GVHD and GVL, antigen specific assays are required to distinguish donor and recipient antibodies. Inherited polymorphisms in heavy chain constant regions of immunoglobulin can be recognized by allotype specific monoclonal antibodies. We hypothesize that B cell reconstitution differs after myeloablative and nonmyeloablative (NMA) HCT with clinical implications. To test this, we developed allotype ELISAs to quantify donor and recipient antibody responses for specific infectious and allogeneic antigens. Human sera were screened by ELISA coating monoclonal antibodies specific for human allotypes (IgG1m(f), m(z), m(a), IgG2m(n), and IgG3m(g1)) at titers providing shared dynamic ranges. Pre-transplant sera from 48 patients and their donors were serially diluted, and allotype-specific immunoglobulin was detected by alkaline phosphatase-conjugated polyclonal anti-human IgG. Allotype-null sera clearly segregated from wild-type sera with 10-fold absorbency differences. Each null phenotype was confirmed by total IgG and isotype-specific quantification. Overall, IgG1m(f) was null in 8 of 96 sera (null allele frequency 29%), and IgG2m(n) was null in 23 of 96 (null allele frequency 48%). Six patients were null for both, and overall 17 of 48 donor/recipient transplant pairs were informative for either allotype. Nulls for the remaining three allotypes were infrequently recognized limiting their clinical utility. Additionally, we measured monoclonal IgG1 purified from 5 multiple myeloma patients identifying three null alleles, one wild-type, and a single intermediate polymorphism. Labeled conjugation of the wild-type monoclonal IgG1 enables competitive inhibition analysis of null allotype improving null allotype sensitivity for engraftment less than 5%. Sera were collected monthly from all HCT patients informative for allotype antibody. Three NMA HCT patients who underwent total lymphoid irradiation and anti-thymoglobulin (TLI/ATG) conditioning have donors that are null for IgG2m(n) and are being prospectively assessed for recipient antibody loss. Their recipient allotype-specific IgG persists at pretransplant recipient levels in all three patients measured six months after NMA HCT, and the lead patient expresses 100% pretransplant recipient allotype antibody ten months after HCT. Conversely, a single NMA patient null for IgG2m(n) with a wild-type donor has no detectable IgG2m(n) donor antibodies four months after HCT despite having 100% donor peripheral B cell engraftment measured 30 days after NMA HCT. In contrast, an informative patient undergoing myeloablative HCT developed 25% IgG2m(n) donor specific antibodies 3 months post-transplant, and 50% at 7 months. Others have reported donor allotype specific antibody achieves full engraftment by 6 months after myeloablative HCT (Van Tol et al. Blood 1996). Our ongoing preliminary studies suggest NMA HCT patients experience delayed donor antibody onset and prolonged recipient antibodies as compared to patients undergoing myeloablative HCT. In order to confirm this, we are measuring antigen-specific donor allotype antibody reconstitution for infectious antigens (EBV and tetanus) and allogeneic H-Y antigens.