Hunting for the elusive target antigen in gestational alloimmune liver disease (GALD).

The prevailing concept is that gestational alloimmune liver disease (GALD) is caused by maternal antibodies targeting a currently unknown antigen on the liver of the fetus. This leads to deposition of complement on the fetal hepatocytes and death of the fetal hepatocytes and extensive liver injury. In many cases, the newborn dies. In subsequent pregnancies early treatment of the woman with intravenous immunoglobulin can be instituted, and the prognosis for the fetus will be excellent. Without treatment the prognosis can be severe. Crucial improvements of diagnosis require identification of the target antigen. For this identification, this work was based on two hypotheses: 1. The GALD antigen is exclusively expressed in the fetal liver during normal fetal life in all pregnancies; 2. The GALD antigen is an alloantigen expressed in the fetal liver with the woman being homozygous for the minor allele and the father being, most frequently, homozygous for the major allele. We used three different experimental approaches to identify the liver target antigen of maternal antibodies from women who had given birth to a baby with the clinical GALD diagnosis: 1. Immunoprecipitation of antigens from either a human liver cell line or human fetal livers by immunoprecipitation with maternal antibodies followed by mass spectrometry analysis of captured antigens; 2. Construction of a cDNA expression library from human fetal liver mRNA and screening about 1.3 million recombinants in Escherichia coli using antibodies from mothers of babies diagnosed with GALD; 3. Exome/genome sequencing of DNA from 26 presumably unrelated women who had previously given birth to a child with GALD with husband controls and supplementary HLA typing. In conclusion, using the three experimental approaches we did not identify the GALD target antigen and the exome/genome sequencing results did not support the hypothesis that the GALD antigen is an alloantigen, but the results do not yield basis for excluding that the antigen is exclusively expressed during fetal life., which is the hypothesis we favor.

Immunomodulatory Properties of Mesenchymal Stromal Cells: Still Unresolved "Yin and Yang".

Mesenchymal stromal cells (MSC) are mesodermal elements characterized by the ability to differentiate into several types of cells present mainly in connective tissues. They play a key function in tissue homeostasis and repair. Furthermore, they exert a strong effect on both innate and adaptive immune response. The main current of thought considers MSC as strong inhibitors of the immune system. Indeed, the first description of MSC immunomodulation pointed out their inability to induce alloimmune responses and their veto effects on mixed lymphocyte reactions. This inhibition appears to be mediated both by direct MSC interaction with immune cells and by soluble factors. Unfortunately, evidence to support this notion comes almost exclusively from in vitro experiments. In complex experimental systems, it has been shown that MSC can exert immunosuppressive effects also in vivo, either in murine models or in transplanted patients to avoid the graft versus host disease. However, it is still debated how the small number of administered MSC can regulate efficiently a large number of host effector lymphocytes. In addition, some reports in the literature indicate that MSC can trigger rather than inhibit lymphocyte activation when a very low number of MSC are co-cultured with lymphocytes. This would imply that the ratio between the number of MSC and immune cells is a key point to forecast whether MSC will inhibit or activate the immune system. Herein, we discuss the conflicting results reported on the immunomodulatory effects of MSC to define which features are relevant to understand their behavior and cross-talk with immune cells.

A Functionally Different Immune Phenotype in Cattle Is Associated With Higher Mastitis Incidence.

A novel vaccine against bovine viral diarrhea (BVD) induced pathogenic antibody production in 5-10% of BVD-vaccinated cows. Transfer of these antibodies via colostrum caused Bovine neonatal pancytopenia (BNP) in calves, with a lethality rate of 90%. The exact immunological mechanisms behind the onset of BNP are not fully understood to date. To gain further insight into these mechanisms, we analyzed the immune proteome from alloreactive antibody producers (BNP cows) and non-responders. After in vitro stimulation of peripheral blood derived lymphocytes (PBL), we detected distinctly deviant expression levels of several master regulators of immune responses in BNP cells, pointing to a changed immune phenotype with severe dysregulation of immune response in BNP cows. Interestingly, we also found this response pattern in 22% of non-BVD-vaccinated cows, indicating a genetic predisposition of this immune deviant (ID) phenotype in cattle. We additionally analyzed the functional correlation of the ID phenotype with 10 health parameters and 6 diseases in a retrospective study over 38 months. The significantly increased prevalence of mastitis among ID cows emphasizes the clinical relevance of this deviant immune response and its potential impact on the ability to fight infections.

Notch signaling mediated by Delta-like ligands 1 and 4 controls the pathogenesis of chronic GVHD in mice.

Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic cell transplantation (allo-HCT) and remains an area of unmet clinical need with few treatment options available. Notch blockade prevents acute GVHD in multiple mouse models, but the impact of Notch signaling on cGVHD remains unknown. Using genetic and antibody-mediated strategies of Notch inhibition, we investigated the role of Notch signaling in complementary mouse cGVHD models that mimic several aspects of human cGVHD in search of candidate therapeutics. In the B10.D2→BALB/c model of sclerodermatous cGVHD, Delta-like ligand 4 (Dll4)-driven Notch signaling was essential for disease development. Antibody-mediated Dll4 inhibition conferred maximum benefits when pursued early in a preventative fashion, with anti-Dll1 enhancing early protection. Notch-deficient alloantigen-specific T cells showed no early defects in proliferation or helper polarization in vivo but subsequently exhibited markedly decreased cytokine secretion and enhanced accumulation of FoxP3+ regulatory T cells. In the B6→B10.BR major histocompatibility complex-mismatched model with multi-organ system cGVHD and prominent bronchiolitis obliterans (BO), but not skin manifestations, absence of Notch signaling in T cells provided long-lasting disease protection that was replicated by systemic targeting of Dll1, Dll4, or both Notch ligands, even during established disease. Notch inhibition decreased target organ damage and germinal center formation. Moreover, decreased BO-cGVHD was observed upon inactivation of Notch1 and/or Notch2 in T cells. Systemic targeting of Notch2 alone was safe and conferred therapeutic benefits. Altogether, Notch ligands and receptors regulate key pathogenic steps in cGVHD and emerge as novel druggable targets to prevent or treat different forms of cGVHD.

Screening RhoA/ROCK inhibitors for the ability to prevent chronic rejection of mouse cardiac allografts.

BACKGROUND: Chronic rejection of transplanted organs is a major obstacle in organ transplantation. The main symptoms of chronic rejection are vessel occlusion and tissue fibrosis. Macrophages play a crucial role in chronic rejection. We showed previously that RhoA deletion or RhoA/Rock inhibition using Y27632 inhibitor reorganizes macrophage actin cytoskeleton, prevents macrophage movement to the cardiac allografts, and abrogates chronic rejection in rodent models. Although besides Y27632 there are other RhoA/ROCK inhibitors available commercially, their efficacy in inhibition of chronic rejection remains unknown. METHODS: We screened four RhoA/Rock inhibitors for their ability to inhibit chronic rejection of BALB/c [H-2d] mouse cardiac allografts heterotopically transplanted into C57BL/6 [H-2b] recipients. We also tested the effect of inhibitors on macrophages in vitro. RESULTS: We found that out of four tested compounds, the Fasudil and Azaindole, inhibited vessel occlusion, tissue fibrosis, decreased M2 macrophage infiltration and abrogated chronic rejection of mouse cardiac allografts. The remaining inhibitors, SAR-407899 and SLX-2119, decreased only tissue fibrosis, and were ineffective or only slightly effective in inhibiting vessel occlusion. We also found that Azaindole and Fasudil affected actin cytoskeleton and protein expression in mouse peritoneal macrophages CONCLUSION: Results of these studies might help in development of anti-chronic rejection therapy for clinical use.

Jk3 alloantibodies during pregnancy-blood bank management and hemolytic disease of the fetus and newborn risk.

BACKGROUND: The Kidd-null phenotype, Jk(a-b-), occurs in individuals who do not express the JK glycoprotein. Jk(a-b-) individuals can make an antibody against the Jk3 antigen, a high-incidence antigen present in more than 99.9% of most populations. This presents many challenges to the blood bank including identification of the antibody, masking of other antibodies, and how to provide transfusion support given the rarity of Jk3-negative blood products. Kidd antibodies may cause acute and delayed hemolytic reactions as well as hemolytic disease of the fetus and newborn (HDFN). In this article, we present a series of four practical cases of pregnant women with the anti-Jk3 alloantibody that demonstrate a range of clinical presentations of Kidd-related HDFN. STUDY DESIGN AND METHODS: We retrospectively reviewed the clinical and blood bank records for four patients and their newborns encountered at institutions in Tennessee, Missouri, Hawaii, and Guam with an anti-Jk3 identified during pregnancy. RESULTS: Two cases showed no significant evidence for HDFN, while two cases were of mild-to-moderate severity requiring early delivery due to elevated middle cerebral artery (MCA) flow velocities but requiring only phototherapy for hyperbilirubinemia. No intrauterine or neonatal transfusions were necessary. Anti-Jk3 alloantibody titers ranged from 2 to 128. CONCLUSION: Clinical manifestations of anti-Jk3 HDFN are generally mild to moderate. Anti-Jk3 titers were not found to correlate directly with HDFN severity. We suggest a titer of 16 to 32 as a cutoff for implementing enhanced monitoring of fetal MCA flow velocities, as such titers may be indicative of elevated HDFN risk.

Immunocontraceptive target repertoire defined by systematic identification of sperm membrane alloantigens in a single species.

Sperm competence in animal fertilization requires the collective activities of numerous sperm-specific proteins that are typically alloimmunogenic in females. Consequently, sperm membrane alloantigens are potential targets for contraceptives that act by blocking the proteins' functions in gamete interactions. Here we used a targeted proteomics approach to identify the major alloantigens in swine sperm membranes and lipid rafts, and thereby systematically defined the repertoire of these sperm-specific proteins in a single species. Gilts with high alloantibody reactivity to proteins in sperm membranes or lipid rafts produced fewer offspring (73% decrease) than adjuvant-only or nonimmune control animals. Alloantisera recognized more than 20 potentially unique sperm membrane proteins and five sperm lipid raft proteins resolved on two-dimensional immunoblots with or without prior enrichment by anion exchange chromatography. Dominant sperm membrane alloantigens identified by mass spectrometry included the ADAMs fertilin α, fertilin ß, and cyritestin. Less abundant alloantigens included ATP synthase F1 ß subunit, myo-inositol monophosphatase-1, and zymogen granule membrane glycoprotein-2. Immunodominant sperm lipid raft alloantigens included SAMP14, lymphocyte antigen 6K, and the epididymal sperm protein E12. Of the fifteen unique membrane alloantigens identified, eleven were known sperm-specific proteins with uncertain functions in fertilization, and four were not previously suspected to exist as sperm-specific isoforms. De novo sequences of tryptic peptides from sperm membrane alloantigen "M6" displayed no evident homology to known proteins, so is a newly discovered sperm-specific gene product in swine. We conclude that alloimmunizing gilts with sperm membranes or lipid rafts evokes formation of antibodies to a relatively small number of dominant alloantigens that include known and novel sperm-specific proteins with possible functions in fertilization and potential utility as targets for immunocontraception.

Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion.

Most humans harbor both CD177neg and CD177pos neutrophils but 1-10% of people are CD177null, placing them at risk for formation of anti-neutrophil antibodies that can cause transfusion-related acute lung injury and neonatal alloimmune neutropenia. By deep sequencing the CD177 locus, we catalogued CD177 single nucleotide variants and identified a novel stop codon in CD177null individuals arising from a single base substitution in exon 7. This is not a mutation in CD177 itself, rather the CD177null phenotype arises when exon 7 of CD177 is supplied entirely by the CD177 pseudogene (CD177P1), which appears to have resulted from allelic gene conversion. In CD177 expressing individuals the CD177 locus contains both CD177P1 and CD177 sequences. The proportion of CD177hi neutrophils in the blood is a heritable trait. Abundance of CD177hi neutrophils correlates with homozygosity for CD177 reference allele, while heterozygosity for ectopic CD177P1 gene conversion correlates with increased CD177neg neutrophils, in which both CD177P1 partially incorporated allele and paired intact CD177 allele are transcribed. Human neutrophil heterogeneity for CD177 expression arises by ectopic allelic conversion. Resolution of the genetic basis of CD177null phenotype identifies a method for screening for individuals at risk of CD177 isoimmunisation.

The 25th anniversary of the Eurotransplant Acceptable Mismatch program for highly sensitized patients.

In 2014, the Eurotransplant Acceptable Mismatch (AM) program celebrated its 25th anniversary. The AM program was initiated to enhance transplantation of highly sensitized patients awaiting a renal transplant within the Eurotransplant region. Unlike the regular renal transplant allocation, in which the histocompatibility parameters consist of the degree of compatibility with the patient's human leucocyte antigen (HLA) type and the absence of unacceptable antigens, the AM program is based on compatibility of the possible donor with the combination of the patient's HLA type and the acceptable antigens. These acceptable antigens are defined as HLA antigens to which the patient has never made antibodies. This strategy aims at the prediction of a negative cross match. Since the start of the program almost 2000 patients participated and more than 1000 patients were transplanted with excellent transplant outcome, comparable to that of non-immunized transplant recipients within Eurotransplant. Progressive insights have led to fine-tuning of the AM program through the years, as well as to novel initiatives, including a recent consortium study to determine the feasibility of a Europe-wide AM program. The current review will tell the story of the AM program in a historical perspective, but will also provide an open-minded look into the future of transplanting highly sensitized patients.

Hemolytic disease of the fetus and newborn caused by anti-Lan.

BACKGROUND: Antibodies to the high-incidence red blood cell (RBC) antigen Lan (Langereis) are typically immunoglobulin G and have been shown to fix complement and cause hemolysis of Lan antigen-positive RBCs. Only three cases of hemolytic disease of the fetus and newborn (HDFN) have been reported involving anti-Lan and all have been characterized as "mild." CASE REPORT: A 26-year-old Hispanic female presented in her fifth pregnancy for routine obstetric care. Due to progressively rising anti-Lan titers, middle cerebral artery (MCA) Dopplers were performed. At 32 weeks of gestation, the antibody titer had reached 128; the MCA Doppler indicated that fetal anemia was severe. An intrauterine transfusion with Lan antigen-negative RBCs was performed and a viable infant was delivered 25 days later. DISCUSSION: Three cases of HDFN associated with anti-Lan have been previously reported. While these cases have been associated with somewhat variable serologic findings, none have resulted in fetal demise or severe symptomatology requiring pre- or postnatal intervention other than routine phototherapy. The current report, however, suggests that in some instances anti-Lan can result in a more severe form of HDFN requiring more aggressive prenatal therapy. CONCLUSION: In spite of previous case reports suggesting that anti-Lan is associated with relatively mild HDFN, this case suggests that in some instances, this antibody can cause severe HDFN requiring prenatal intervention.

Genetic variation of the HNA-3a encoding gene.

BACKGROUND: Antibodies against the human neutrophil alloantigen-3a (HNA-3a) play an important role in transfusion-related acute lung injury. The HNA-3a and -3b alloantigens result from a single-nucleotide exchange in the choline transporter-like protein 2 gene (CTL2). We sought for additional polymorphisms that might impair antibody binding to or genotyping of the HNA-3a or -3b antigens. STUDY DESIGN AND METHODS: CTL2-specific complementary DNA (cDNA) fragments were generated from 67 unrelated blood donors followed by DNA sequencing. Polymerase chain reaction with sequence-specific primers (PCR-SSP) was used to test a higher number of donors for relevant new single-nucleotide polymorphisms (SNPs). The granulocyte agglutination test recommended for HNA-3a antibody detection was performed to check HNA-3a antibody binding to the products of the CTL-2 gene variants. RESULTS: Two new missense mutations were demonstrated in the CTL2 cDNA: a 537C>T* exchange leading to a Leu153Phe amino acid substitution and 988C>T variation predicting Thr301Met change. The inherited 537T variant is located in HNA-3a allele results impaired granulocyte agglutination by four of 14 antibodies tested while 988T remains nearly unaffected. CONCLUSIONS: The Leu153Phe exchange next to the HNA-3a/b defining amino acid position can impede the binding of HNA-3a alloantibodies. The HNA-3a genotyping by PCR-SSP might produce misleading results in HNA-3ab heterozygous individuals with the additional CTL2-537T variation of the HNA-3a antigen. These findings must account for the development of new screening assays.

Cell therapy to promote transplantation tolerance: a winning strategy?

Organ transplantation is currently the only effective treatment for end-stage organ failure. However, success is limited by the immune response of the recipient to allogeneic tissues (recognized by the direct and indirect alloresponses) and by the morbidity and mortality associated with the immunosuppressive drugs that are used to control alloimmunity. One solution to these problems is the induction of immunological tolerance. In our laboratory, we have selected two strategies to achieve this goal. The first is to expand and/or generate Tregs directly in vivo using infusions of 'tolerogenic' DCs into patients; the second is to purify Tregs from the blood of patients on the waiting list for a transplant, enrich and expand these cells in vitro and then inject back in vivo after transplantation. Here, we have summarized our results both in the murine and human systems on the use of Treg-based strategies to induce tolerance to the transplanted organs.

Manipulating the bioenergetics of alloreactive T cells causes their selective apoptosis and arrests graft-versus-host disease.

Cells generate adenosine triphosphate (ATP) by glycolysis and by oxidative phosphorylation (OXPHOS). Despite the importance of having sufficient ATP available for the energy-dependent processes involved in immune activation, little is known about the metabolic adaptations that occur in vivo to meet the increased demand for ATP in activated and proliferating lymphocytes. We found that bone marrow (BM) cells proliferating after BM transplantation (BMT) increased aerobic glycolysis but not OXPHOS, whereas T cells proliferating in response to alloantigens during graft-versus-host disease (GVHD) increased both aerobic glycolysis and OXPHOS. Metabolomic analysis of alloreactive T cells showed an accumulation of acylcarnitines consistent with changes in fatty acid oxidation. Alloreactive T cells also exhibited a hyperpolarized mitochondrial membrane potential (ΔΨm), increased superoxide production, and decreased amounts of antioxidants, whereas proliferating BM cells did not. Bz-423, a small-molecule inhibitor of the mitochondrial F(1)F(0) adenosine triphosphate synthase (F(1)F(0)-ATPase), selectively increased superoxide and induced the apoptosis of alloreactive T cells, which arrested established GVHD in several BMT models without affecting hematopoietic engraftment or lymphocyte reconstitution. These findings challenge the current paradigm that activated T cells meet their increased demands for ATP through aerobic glycolysis, and identify the possibility that bioenergetic and redox characteristics can be selectively exploited as a therapeutic strategy for immune disorders.

Hemolytic disease of the newborn associated with anti-Jra alloimmunization in a twin pregnancy: the first case report in Korea.

Jr(a) is a high-frequency antigen found in all ethnic groups. However, the clinical significance of the anti-Jr(a) antibody has remained controversial. Most studies have reported mild hemolytic disease of the newborn and fetus (HDNF) in Jr(a)-positive patients. Recently, fatal cases of HDNF have also been reported. We report the first case of HDNF caused by anti-Jr(a) alloimmunization in twins in Korea. A 33-yr-old nulliparous woman with no history of transfusion or amniocentesis was admitted at the 32nd week of gestation because of vaginal bleeding caused by placenta previa. Anti-Jr(a) antibodies were detected in a routine laboratory examination. An emergency cesarean section was performed at the 34th week of gestation, and 2 premature infant twins were delivered. Laboratory examination showed positive direct antiglobulin test and Jr(a+) phenotype in the red blood cells and the presence of anti-Jr(a) antibodies in the serum in both neonates. The infants underwent phototherapy for neonatal jaundice; this was followed by conservative management. They showed no further complications and were discharged on the 19th postpartum day. Preparative management to ensure the availability of Jr(a-) blood, via autologous donation, and close fetal monitoring must be performed even in cases of first pregnancy in Jr(a-) women.

Two different regulatory T cell populations that promote corneal allograft survival.

PURPOSE: To compare and contrast the T regulatory cells (Tregs) induced by anterior chamber (AC) injection of antigen with those induced by orthotopic corneal allografts. METHODS: Anterior chamber-associated immune deviation (ACAID) Tregs were induced by injecting C57BL/6 spleen cells into the AC of BALB/c mice. Delayed-type hypersensitivity responses to C57BL/6 alloantigens were evaluated by a conventional ear swelling assay. Corneal allograft Tregs were induced by applying orthotopic C57BL/6 corneal allografts onto BALB/c hosts. The effects of anti-CD25, anti-CD8, anti-interferon-γ (IFN-γ), anti-IL-17A, or cyclophosphamide treatments on corneal allograft survival and ACAID were evaluated. RESULTS: Administration of either anti-CD25 or anti-IFN-γ antibodies prevented the expression of ACAID and abolished the immune privilege of corneal allografts. By contrast, in vivo treatment with anti-CD8 antibody abrogated ACAID but had no effect on corneal allograft survival. Further discordance between ACAID and corneal allograft survival emerged in experiments in which the induction of allergic conjunctivitis or the administration of anti-IL-17A abolished the immune privilege of corneal allografts but had no effect on the induction or expression of ACAID. CONCLUSIONS: Although orthotopic corneal allografts are strategically located for the induction of ACAID by the sloughing of corneal cells into the AC, the results reported here indicate that the Tregs induced by orthotopic corneal allografts are remarkably different from the Tregs that are induced by AC injection of alloantigen. Although both of these Treg populations promote corneal allograft survival, they display distinctly different phenotypes.

The use of small molecule high-throughput screening to identify inhibitors of the proteinase 3-NB1 interaction.

Anti-neutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are found in patients with small-vessel vasculitis. PR3-ANCA bind strongly to membrane PR3 (mPR3) that is presented by the NB1 receptor. We performed high-throughput screening using a small molecule library to identify compounds that inhibit PR3-NB1 binding. We established a human embryonic kidney (HEK293) cell-based system, where approximately 95 +/- 2% of the NB1-transfected cells expressed the NB1 receptor on the cell surface. Addition of 0.1 microg/ml human PR3 to 10(4) NB1-expressing HEK293 cells resulted in PR3 binding that was detected by immunofluorescence using a fluorescence plate reader assay. We identified 13 of 20 000 molecules that inhibited PR3 binding by >70%. Seven of 13 substances showed reproducible inhibition in four additional validation experiments. Two selected compounds (27519 and 27549) demonstrated a dose-dependent inhibition over a range from 6.25 to 100 microM as measured by the plate reader assay. We used flow cytometry as a second assay, and found that both compounds reproducibly inhibited PR3 binding to NB1-transfected HEK293 cells at 50 microM (inhibition to 42 +/- 4% with compound 27519 and to 47 +/- 6% with compound 27549 compared to the dimethylsulphoxide control). Furthermore, compounds 27519 and 27549 also inhibited binding of exogenous PR3 to human neutrophils. In contrast, the compounds did not decrease mPR3 expression on resting neutrophils, but reduced the tumour necrosis factor-alpha-mediated mPR3 increase on NB1(pos) neutrophils when present continuously during the assay. The findings suggest that small inhibitory compounds provide a potential therapeutic tool to reduce mPR3 by preventing its binding to NB1.

Isatis tinctoria L. combined with co-stimulatory molecules blockade prolongs survival of cardiac allografts in alloantigen-primed mice.

Memory T cells present a unique challenge in transplantation. Although memory T cells express robust immune responses to invading pathogens, they may be resistant to the effects of immunosuppressive therapies used to prolong graft survival. In previous studies, we found that compound K, the synthesized analogue of highly unsaturated fatty acids from Isatis tinctoria L., reduced acute cardiac allograft rejection in mice (Wang et al., 2009 [1]). Here, we further investigated the effect of compound K on cardiac allograft rejection in alloantigen-primed mice. We found that compound K significantly inhibited CD4(+) and CD8(+) memory T cells proliferation in a mixed lymphocyte reaction (MLR). In vivo, compound K combined with anti-CD154 and anti-LFA-1 monoclonal antibodies (mAbs) significantly extended the survival time of heart grafts in alloantigen-primed mice with no obvious toxic side effects. Furthermore, our data suggests that compound K works by reducing the expression of both IL-2 and IFN-gamma within the graft rather than enhancing expression of regulatory T cells (Tregs). Compound K can also inhibit the alloresponses of memory T cells, while increasing the proportion of CD4(+) memory T cells in the spleen of the recipients and significantly reducing the level of alloantibodies in the serum. Our study highlights the unique immune effects of compound K that may be further explored for clinical use in extending the survival of transplant grafts.

Hemolytic Disease of the Newborn Associated with Anti-Jr(a) Alloimmunization in a Twin Pregnancy: The First Case Report in Korea / 대한진단검사의학회지

Jr(a) is a high-frequency antigen found in all ethnic groups. However, the clinical significance of the anti-Jr(a) antibody has remained controversial. Most studies have reported mild hemolytic disease of the newborn and fetus (HDNF) in Jr(a)-positive patients. Recently, fatal cases of HDNF have also been reported. We report the first case of HDNF caused by anti-Jr(a) alloimmunization in twins in Korea. A 33-yr-old nulliparous woman with no history of transfusion or amniocentesis was admitted at the 32nd week of gestation because of vaginal bleeding caused by placenta previa. Anti-Jr(a) antibodies were detected in a routine laboratory examination. An emergency cesarean section was performed at the 34th week of gestation, and 2 premature infant twins were delivered. Laboratory examination showed positive direct antiglobulin test and Jr(a+) phenotype in the red blood cells and the presence of anti-Jr(a) antibodies in the serum in both neonates. The infants underwent phototherapy for neonatal jaundice; this was followed by conservative management. They showed no further complications and were discharged on the 19th postpartum day. Preparative management to ensure the availability of Jr(a-) blood, via autologous donation, and close fetal monitoring must be performed even in cases of first pregnancy in Jr(a-) women.

PKCtheta is required for alloreactivity and GVHD but not for immune responses toward leukemia and infection in mice.

When used as therapy for hematopoietic malignancies, allogeneic BM transplantation (BMT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host disease (GVHD), which is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues, is a potentially lethal complication of allogeneic BMT. To enhance the therapeutic potential of BMT, we sought to find therapeutic targets that could inhibit GVHD while preserving GVL and immune responses to infectious agents. We show here that T cell responses triggered in mice by either Listeria monocytogenes or administration of antigen and adjuvant were relatively well preserved in the absence of PKC isoform theta (PKCtheta), a key regulator of TCR signaling. In contrast, PKCtheta was required for alloreactivity and GVHD induction. Furthermore, absence of PKCtheta raised the threshold for T cell activation, which selectively affected alloresponses. Most importantly, PKCtheta-deficient T cells retained the ability to respond to virus infection and to induce GVL effect after BMT. These findings suggest PKCtheta is a potentially unique therapeutic target required for GVHD induction but not for GVL or protective responses to infectious agents.

Immunomodulatory effects of blood transfusions: the synergic role of soluble HLA Class I free heavy-chain molecules detectable in blood components.

BACKGROUND: Over the past decades, the weight of the published literature demonstrates that blood transfusions can induce clinically significant immunosuppression in recipients. Several studies showed significant improved clinical outcomes in the patients receiving leukoreduced transfusions, compared with control patients who received nonleukoreduced transfusions. Moreover, the immunosuppressive potential of blood products grows with the time of their storage and becomes highest in nonleukoreduced blood products stored for a long time. STUDY DESIGN AND METHODS: The interest was previously focused on the determination of immunomodulatory soluble molecules such as soluble HLA Class I (sHLA-I) and soluble Fas ligand (sFasL) in different blood components and on the evaluation of their immunomodulatory activities. On this basis, whether soluble beta2-microglobulin free HLA Class I heavy chains (sHLA-beta2fHC) could be detected and immunochemically characterized in different blood components was evaluated. Immunomodulatory activity of detectable sHLA-beta2fHC molecules was evaluated by apoptosis inducing capacity in interleukin-2-activated antigen-specific cytotoxic T lymphocytes (CTL). RESULTS: Double-determinant immunoenzymatic assay indicates that sHLA-beta2fHC levels in red blood cells stored for up to 30 days and in random-donor platelets are significantly (p < 0.001) higher than in other blood components, and the immunochemical characterization suggests that the major source of sHLA-beta2fHC molecules might be the residual white cells that undergo membrane damage during storage. Finally, allogeneic CD8+ CTL apoptosis induction confirmed biofunctionality of sHLA-beta2fHC molecules. CONCLUSION: These data are comparable with those previously reported dealing with contaminant soluble molecules in allogeneic and autologous blood components, suggesting that sHLA-beta2fHC molecules could contribute to the immunosuppressive effects of blood transfusions.