We assembled the genome of Leptospermum scoparium 'Crimson Glory' using a combination of Illumina paired-end sequencing, high-throughput chromosome conformation capture (Hi-C) and high density ...genetic mapping. As 'Crimson Glory' is a variety of mānuka, this is the first genome assembly for a plant species culturally recognised as a treasure (taonga) by the indigenous Māori of Aotearoa New Zealand. The mānuka genome spans a total of 297 Mbp organised in 11 pseudo-chromosomes that are syntenic with the Eucalyptus genome. A large proportion of the genome assembly corresponds to fungal and bacterial sequences, indicating the presence of an associated microbiome. A total of 31,220 protein-coding gene models were detected throughout the genome, including genes involved in biosynthesis of biologically active phenylpropanoids, triketones and terpenes, as well as genes involved in biotic resistance. The mānuka genome sequence will help shed new light on the genetic control of unique characters such as nectar and foliage biochemical composition, flowering time and disease resistance.
Abstract
CD4 T cells and antibody are required for optimal acquired immunity to C. muridarum genital tract infection, and T cell-mediated IFNγ production is necessary to clear infection in the ...absence of humoral immunity. However, the role of protective T cell-independent immune responses against primary infection remain unclear. We addressed this problem by inoculating wild-type and immune-deficient mice with a plaque-purified strain (CM001) isolated from C. muridarum Nigg stock capable of enhanced extragenital replication. Genital CM001 inoculation resulted in transient dissemination to the lungs and spleen in wild-type mice prior to clearance. However, CM001 genital infection induced lethality in STAT1−/− and IFNG−/− mice, where IFNγ signaling is absent, and in Rag1−/− mice that lack T and B cells but are competent for innate IFNγ signaling. In contrast, muMT, nude, and Tcra−/− mice survived. These data collectively indicate that IFNγ prevents lethal CM001 infection in the absence of T cells and suggest a B cell co-requirement for protection. Adoptive transfer of convalescent immune sera, but not naïve IgM antibody, to Rag1−/− mice significantly increased survival time and transfer of naïve B cells completely rescued Rag1−/− mice against CM001 lethality. Protection was associated with a significant reduction in the lung chlamydial burden of genitally infected mice. These data reveal an important synergism between T-independent B cell responses and innate IFNγ in chlamydial host defense, and suggest cooperative interactions between T-independent IgG and IFNγ are essential for limiting extragenital dissemination.
Background: Previous studies suggest that antibody engagement of red blood cells (RBCs) can result in the actual loss of detectable target antigen in the setting of autoimmune hemolytic anemia. ...However, many of these situations appear to represent complement mediated-masking of the target antigen. Recent studies suggest that complement-independent antigen-loss can occur in a variety of murine models. However, whether a similar form of RBC antigen loss can occur independent of complement in humans remains unknown. As previous studies suggest that anti-RhD fails to induce significant complement activation following engagement of RhD-positive RBCs, we evaluated the potential impact of anti-RhD antibody injection on RhD antigen levels in a RhD-positive patient being treated for idiopathic thrombocytopenic purpura.
Methods: Pre- and post-anti-RhD infusion samples were obtained from the hospital blood bank. Direct antiglobulin tests (DATs) were performed and assessed via flow cytometry on pre- and post-infusion samples. RBCs from pre- and post-infusion were also treated with Warm Autoantibody Removal Medium (WARM; ImmucorGamma, Norcross, GA), a sulphydryl/enzyme reagent based on the ZZAP method, in order to remove bound antibody. Post-WARM treated cells from pre- and post-anti-RhD infusion samples were then used to perform DATs. The presence of RhD antigen was assessed in pre- and post-infusion samples with in vitro anti-RhD, from the same lot number used to treat the patient, followed by anti-IgG and anti-complement as the secondary antibody. To account for the potential impact of bound antibody on antigen detection, antigen levels were also assessed after anti-RhD removal with the WARM method. Finally, in order to determine whether antigen loss was RhD specific, cellano (k) antigen detection was tested using anti-k antibody both pre- and post-WARM treatment.
Results: As predicted, the pre-anti-RhD infusion DAT was negative, while the post-anti-RhD injection DAT was positive (MFI 25). Post-WARM treatment, pre-and post-RhD infusion DATs showed minimal reactivity (MFI 3 and 5, respectively with background MFI of 2.7). Reduced RhD antigen levels were observed in post-anti-RhD infusion samples when compared to pre-infusion samples, while no complement could be detected in either pre- or post-anti-RhD infusion samples. The detection of antigen loss post-anti-RhD infusion was even more pronounced after RBCs were treated with WARM to remove previously bound anti-RhD antibody administered in vivo. In contrast, no difference in k antigen level could be detected pre- or post-anti-RhD infusion. As expected, post-WARM treatment, k antigen was no longer detectable pre- or post-anti-RhD infusion samples.
Conclusion: These results provide an example of antigen loss in the setting of anti-RhD administration. Moreover, the anti-RhD effect on the RhD-antigen appears to be antigen specific, as the RhD immune globulin did not modulate the k antigen on the same cell. Taken together, these results suggest that anti-RhD can induce the loss of detectable antigen independent of complement and may therefore influence the rate and magnitude of RBC clearance in settings of anti-RhD infusion, incompatible RBC transfusion and autoimmune hemolytic anemia.
No relevant conflicts of interest to declare.
Introduction: Polyclonal anti-D preparations are among the most successful prophylactic immunotherapies in existence today. The mechanism(s) of action of polyclonal anti-D are not well understood, ...and no monoclonal therapy developed to date has been as effective as polyclonal preparations at preventing pregnancy associated RhD sensitization. Furthermore, no prophylactic therapies exist to prevent alloimmunization to non-RhD antigens. We have recently developed a transgenic mouse model of RBC alloimmunization to KEL, with sensitization to KEL occurring through pregnancy as well as through transfusion. We hypothesized that polyclonal anti-KEL (“KELGAM”) would prevent KEL RBC alloimmunization, and herein test this hypothesis in a controlled transfusion setting.
Materials and Methods: Polyclonal anti-KEL (KELGAM) was generated in IgHa congenic mice following repeat transfusions of transgenic KEL RBCs. This polyclonal anti-KEL was then passively infused into naïve C57BL/6 (IgHb) mice, at a dose that led to maximal clearance of KEL RBCs. 50 microliters of lipophilically labeled KEL RBCs were transfused into these passively immunized mice, with evaluation by flow cytometry of anti-KEL RBC binding, complement positivity, transfused RBC clearance, and active recipient anti-KEL responses. Controls included animals transfused in the absence of passively infused anti-KEL, animals given a 3rd party polyclonal antibody prior to KEL RBC transfusion, and animals given anti-KEL but transfused with RBCs expressing a 3rdparty RBC antigen. 16 weeks post-transfusion, animals initially treated with or without KELGAM prior to KEL RBCs were transfused again with KEL RBCs, with responses compared to that of naïve animals.
Results: In 3 of 4 experiments (n=3-5 mice/group/experiment), mice treated with IgHa KELGAM prior to KEL RBC transfusion generated no detectable anti-KEL glycoprotein IgHb alloantibodies by 4 months post-transfusion. In contrast, 100% of control mice transfused with KEL RBCs in the absence of KELGAM or transfused with KEL RBCs in the presence of a 3rdparty antibody generated anti-KEL glycoprotein IgHb alloantibodies (adjusted MFI >100 using sera at a 1:2 dilution). Approximately 50% of DiI labeled KEL RBCs cleared within 24 hours in the KELGAM group, whereas KEL RBCs didn’t begin clearing in the control groups until 5-7 days post-transfusion at which time the recipients began to generate anti-KEL. The DiI labeled KEL RBCs had surface bound IgG and C3b until 24 hours post-transfusion, after which time neither bound antibody, C3b, nor KEL antigen could be detected. In contrast, DiI labeled KEL RBCs in the control groups had detectable KEL antigen on their surface until 5 days post-transfusion, at which time the recipients began to generate anti-KEL. Animals initially treated with KELGAM prior to transfusion were able to generate anti-KEL upon a second exposure to KEL RBCs 4 months later, with similar responses to naïve animals seeing KEL RBCs for the first time.
Conclusions: Polyclonal anti-KEL (KELGAM) effectively eliminates primary anti-KEL glycoprotein alloantibody responses to transfused KEL RBCs. However, the mechanism of action of KELGAM may be different from that of polyclonal anti-D. The dose of KELGAM used for these studies saturates all detectable KEL antigen sites on the transfused RBCs. Distinct from what has been described with polyclonal anti-D, the transfused RBCs remaining in circulation from 24 hours after KELGAM treatment until day 28 post-transfusion modulate KEL antigen expression such that they no longer bind to KELGAM during in vitro testing. Furthermore, the addition of an excess of KELGAM does not lead to KEL RBC clearance rates beyond 50%. KELGAM transfusion recipients are not tolerized to the KEL antigen, as re-challenge with KEL RBCs months later leads to robust anti-KEL responses. Ongoing studies are further investigating whether KELGAM recipients may experience antibody mediated immune deviation, whether KELGAM is capable of suppressing responses to other antigens co-expressed on the same RBC, and whether KELGAM is effective at eliminating pregnancy induced anti-KEL alloimmunization. A goal of these studies includes the eventual translation of this knowledge to the setting of hemolytic disease of the fetus and newborn in humans.
No relevant conflicts of interest to declare.
Resolution of
Chlamydia
genital tract infection is delayed in the absence of MyD88. In these studies, we first used bone marrow chimeras to demonstrate a requirement for MyD88 expression by ...hematopoietic cells in the presence of a wild-type epithelium. Using mixed bone marrow chimeras we then determined that MyD88 expression was specifically required in the adaptive immune compartment. Furthermore, adoptive transfer experiments revealed that CD4+ T cell expression of MyD88 was necessary for normal resolution of genital tract infection. This requirement was associated with a reduced ability of MyD88
−/−
CD4+ T cells to accumulate in the draining lymph nodes and genital tract when exposed to the same inflammatory milieu as wild-type CD4+ T cells. We also demonstrated that the impaired infection control we observed in the absence of MyD88 could not be recapitulated by deficiencies in TLR or IL-1R signaling. In vitro, we detected an increased frequency of apoptotic MyD88
−/−
CD4+ T cells upon activation in the absence of exogenous ligands for receptors upstream of MyD88. These data reveal an intrinsic requirement for MyD88 in CD4+ T cells during
Chlamydia
infection and indicate that the importance of MyD88 extends beyond innate immune responses by directly influencing adaptive immunity.
Objective
Chlamydia trachomatis
infections are a significant cause of reproductive tract pathology. Protective and pathological immune mediators must be differentiated to design a safe and effective ...vaccine.
Methods
Wild‐type mice and mice deficient in
IL
‐22 and
IL
‐23 were infected intravaginally with
C
hlamydia muridarum,
and their course of infection and oviduct pathology were compared. Local genital tract and draining lymph node immune responses were also examined in
IL
‐23‐deficient mice.
Results
IL
‐22‐ and
IL
‐23‐deficient mice exhibited normal susceptibility to infection and oviduct pathology.
IL
‐23 was required for the development of a
C
hlamydia
‐specific Th17 response in the lymph nodes and for production of
IL
‐22 and
IL
‐17 in the genital tract. However, influx of
T
h1 and innate immune cells was not compromised in the absence of
IL
‐23.
Conclusion
IL
‐22 and
IL
‐23 play either redundant or minimal roles in the pathogenesis of
C
hlamydia
infection in the mouse model. Induction of
T
h17‐associated cytokines by a
C
hlamydia
vaccine should be avoided as these responses are not central to resolution of infection and have pathologic potential.
To analyze results of 127 patients undergoing myeloablative therapy followed by marrow transplantation for relapsed or refractory Hodgkin's disease.
Twenty-three patients had primary refractory ...disease, 34 were in early first relapse or second complete remission (CR), and 70 had refractory first relapse or disease beyond second CR. Preparative regimens included total-body irradiation (TBI) and chemotherapy (n = 61) or chemotherapy only (n = 66). Sixty-eight patients received autologous marrow, six syngeneic marrow, and 53 allogeneic marrow.
The 5-year actuarial probabilities of survival, event-free survival (EFS), relapse, and nonrelapse mortality for the entire group were 21%, 18%, 65%, and 49%, respectively. HLA-identical allogeneic marrow recipients had a statistically lower relapse rate compared with recipients of autologous marrow, but survival, EFS, and nonrelapse mortality rates were not significantly different. In the multivariate analysis, higher performance status and absence of bulky disease predicted for improved EFS and lower relapse rates, while fewer prior treatment regimens predicted for improved EFS and lower nonrelapse mortality rates. Additionally, the univariate analysis showed that patients who underwent transplantation with disease refractory to chemotherapy or beyond second CR had a worse outcome compared with those who had less advanced disease.
Outcome with transplantation for patients with Hodgkin's disease is improved if transplantation is performed early after relapse when disease burden is less, tumor chemosensitivity is greater, and the patient is likely to have a better performance status. The use of HLA-matched sibling marrow results in a lower relapse rate and, thus, for some individuals, may be preferable to the use of autologous marrow.
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