Motoko Nishimura

HLA-A null allele with a stop codon, HLA-A*0215N, identified in a homozygous state in a healthy adult.

A healthy adult having no serologically detectable HLA class I A locus antigens was identified. The parents of the individual are consanguineous. Results of a family study indicated that the individual is homozygous for the B46-Cw1-DR8.1 haplotype, which was shown to be positively associated with A*0207 in our previous study. The HLA-A null individual is healthy and exhibits no apparent immunological abnormality. Total RNAs extracted from peripheral blood were converted to cDNAs. The reverse transcriptase-polymerase chain reaction (PCR) product, which is of the same size as the normally expressed gene, was easily obtained from the cDNAs with HLA-A locus-specific primers. The nucleotide sequence of this null allele (A*0215N) was the same as that of A*0207 except for a single nucleotide substitution which resulted in a stop codon in exon 4. From its nucleotide sequence, a truncated molecule was expected to be produced; however, the immunoprecipitation study failed to detect the predicted product. Genomic DNAs from 29 unrelated individuals who expressed only one HLA-A antigen with HLA-B46, were analyzed by a PCR-sequence-specific oligonucleotide method. None of the samples possessed this stop codon. Therefore, A*0215N is likely to be a rare allele generated by a single point mutation from A*0207.

Enhancement of the Expression of Progesterone Receptor on Progesterone‐Treated Lymphocytes After Immunotherapy in Unexplained Recurrent Spontaneous Abortion

PROBLEM: The immunological mechanism of an effective immunotherapy with paternal lymphocytes for unexplained recurrent spontaneous abortion (RSA) is not yet clear. Previous studies revealed that progesterone plays an important role in maintaining normal pregnancy and lower expression of progesterone receptor (PGR) on lymphocytes was found in RSA. Therefore, it was of interest to investigate whether immunotherapy for RSA would be able to enhance the expression of PGR on lymphocytes of RSA.

Effects of the Immunosuppressant FK506 on a Penetrating Keratoplasty Rejection Model in the Rat

PURPOSE The immunosuppressive effects of FK506 on allogeneic corneal transplantation were tested in a rat model. METHODS Inbred-strain Lewis rats were used as recipients, and Fisher rats were used as donors. Intraperitoneal injection of FK506 (0.3, 1.0, and 3.0 mg/kg per day) was administered for 2 weeks, and the grafts were inspected by clinical evaluation. Mixed lymphocyte culture assay, using lymphocytes from recipients of penetrating keratoplasty as responder cells and irradiated splenocytes from naive Fisher or Brown Norway as stimulator cells, was used to identify allogeneic stimulation. The rejection process was studied by histology and immunohistochemistry. RESULTS The rat strain combination developed 100% graft rejection in about 2 weeks after the penetrating keratoplasty. FK506 prolonged the graft survival in a dose-dependent manner, as observed by clinical evaluation. In mixed lymphocyte culture assay, Lewis rats that had been primed to allogeneic stimulation at the time of cornea transplantation presented significant proliferation to Fisher stimulator splenocytes. FK506 suppressed this primed lymphocyte proliferation. Immunohistochemical and histologic studies confirmed the clinical evaluations. Untreated rat corneas, at the second postoperative week, presented a large number of helper/inducer T cells, macrophages, IL-2 receptor-expressing cells, and Ia-antigen-expressing cells. In the same period, FK506-treated rats appeared normal and had no cellular infiltration. Corneas rejected after FK506 cessation had less intense cell infiltration than the control corneas. CONCLUSIONS These data indicate that FK506 prolonged the corneal graft survival and can be a potentially useful drug in the immunotherapeutic arsenal to suppress corneal graft rejection.

Possible mechanisms underlying development of transfusion‐related acute lung injury: roles of anti‐major histocompatibility complex class II DR antibody

Summary.  Anti‐major histocompatibility complex (anti‐MHC) antibodies (Abs) and antipolymorphonuclear neutrophil (anti‐PMN) Abs are generally considered as the main causes of the development of transfusion‐related acute lung injury (TRALI), which is one of the most severe and sometimes lethal side effects of transfusion. These Abs are postulated to activate recipients leucocytes, resulting in the release of soluble factors such as reactive oxygen species and detrimental cytokines and chemokines. The harmful effects on the lung tissues and resident leucocytes of these malignant factors are suspected to be profoundly involved in TRALI reactions. Several reports have indicated the principle effect of biologically active lipids on the pathogenesis of TRALI. However, the precise mechanisms of TRALI development remain unclear. To resolve this issue, we have been investigating cytokines that induce continuous inflammation of the lungs, specifically focusing on the cytokines derived from activated PMNs. We observed that the granulocyte–macrophage colony‐stimulating factor (GM‐CSF) markedly enhances the expression of MHC class II DR in PMNs. Moreover, MHC class II DR‐expressing PMNs were also proved to express a high‐affinity receptor for immunoglobulin E (IgE) (FcεRI) and to produce tumour necrosis factor‐α, interferon‐γ and interleukin‐18 following a challenge with an anti‐MHC class II DR monoclonal Ab (MoAb) or anti‐DR antiserum. It is strongly suggested that amongst various inflammatory mediators, at least these three cytokines may contribute to the duration of inflammatory reactions in the lungs. Furthermore, FcεRI expression, in GM‐CSF‐treated PMNs, suggests the involvement of PMNs in IgE‐mediated immune reactions.

Role of anti-Naka antibody, monocytes and platelets in the development of transfusion-related acute lung injury

Background and Objectives  Transfusion‐related acute lung injury (TRALI) is one of the most serious side‐effects of transfusion. We report here the first two cases of TRALI caused by anti‐Naka (anti‐CD36) antibody from a single blood donor. The aim of this study was to clarify the role of the anti‐Naka antibody in TRALI development.

Interference with TRALI-causing anti-HLA DR alloantibody induction of human pulmonary microvascular endothelial cell injury by purified soluble HLA DR

Background and Objective  Antibodies to human leucocyte antigens (HLA) and human polymorphonuclear neutrophil (PMN) antigens are considered etiologic agents of transfusion‐related acute lung injury (TRALI). The aim of this study was to clarify the role of anti‐HLA DR antibodies in the pathophysiology of TRALI and the ability of purified soluble HLA DR (psHLA DR) to inhibit the release of cytokines in an in vitro model.

Immunosuppressive effects of chloroquine: potential effectiveness for treatment of post‐transfusion graft‐versus‐host disease

Post‐transfusion graft‐versus‐host disease (PT‐GVHD) is a fatal adverse effect of blood transfusion. In spite of its severity, there is no effective treatment at present for PT‐GVHD. Previously, we reported that chloroquine (CH) inhibited the cytotoxicity of cytotoxic T‐cell (CTL) clones and tumour necrosis factor β (TNF β) production by TNF β‐producing clones in vitro, both the clones being derived from peripheral blood lymphocytes (PBMCs) of PT‐GVHD patients. To explore the possibility of utilizing CH for the treatment of PT‐GVHD, we extended our investigation of the immunosuppressive effects of CH in vitro to PBMCs derived from healthy donors. Our results show that CH inhibits the mixed lymphocyte reaction (MLR) between allogeneic PBMCs, production of inflammatory cytokines such as TNF α, interleukin‐1β (IL‐1β) and interferon γ (IFN γ) in mixed lymphocyte culture and natural killer cell activity, and, further, reduces the number of alloreactive CTL precursors.

Anti‐idiotypic antibody to T‐cell receptor in multiply transfused patients may play a role in resistance to graft‐versus‐host disease

Most patients who receive multiple blood or platelet transfusions do not develop graft‐versus‐host disease (GVHD) in spite of the transfusion of donor white cells–cells that are capable of engraftment and subsequent GVHD. The object of this study was to search for the factors responsible for resistance to GVHD in such patients. Some sera from patients who have received multiple platelet transfusions inhibit the proliferation of alloreactive T‐cell clones that function as an in vitro model of donor‐derived proliferating T cells recognizing recipient alloantigens. The humoral factor in such sera was capable of binding to the T‐cell clones, but not to stimulator cells. Further analysis revealed that the humoral factor in such sera was IgG, which specifically bound to membrane molecules of the T‐cell clones. The antibody competed with WT31, a monoclonal antibody (MoAb) to T‐cell receptor (TCR), in binding to TCR of the T‐cell clones. It did not compete with CD3 or CD2 MoAb. These observations strongly favor the view that the antibody against TCR exists in the sera of multiple transfusion recipients. It is suggested that the TCR antibody binds to TCR of the T‐cell clones, thus blocking the interaction of the T‐cell clone with alloantigens of stimulator cells and resulting in inhibition of the proliferation of T‐cell clones. Furthermore, in view of T‐cell clone‐specific binding of the antibody in sera, it might be concluded that the antibody is anti‐idiotypic.

Frozen-stored granulocytes can be used for an immunofluorescence test to detect granulocyte antibodies

BACKGROUND: Donor‐ and/or recipient‐derived granulocyte antibodies are considered to be the main cause of transfusion‐related acute lung injury (TRALI), neutropenia, and febrile transfusion reactions. Several types of tests are performed to detect antibodies in donated blood and/or the serum of a transfusion recipient. Because granulocytes cannot endure the freezing‐thawing process, they cannot be stored in liquid nitrogen (LN2). Therefore, testing is time‐consuming, because freshly prepared granulocytes are needed for each testing. An attempt has been made to develop a method that uses granulocytes stored in LN2 for the granulocyte immunofluorescence test (GIFT).

Potential usefulness of protease inhibitor and chloroquine in the treatment of transfusion‐associated graft‐versus‐host disease

In a recent issue of TRANSFUSION, Burger et al.’ documented the potential for clot formation in peripheral blood progenitor cell collections. We suggest an alternative to a strict adherence to lower citrate ratios for those institutions that collect cells by using either the Spectra (COBE BCT, Lakewood, CO) or, potentially, an apheresis device from Haemonetics (Braintree, MA). The addition of heparin or ACD-A to the collection bag before initiation of the collection prevents clot formation.2 We have preferred the addition of ACD-A at 10 to 20 percent of the anticipated final volume, as heparin does not prevent platelet clumping that may affect the final mononuclear cell yield, especially if the collected component is subjected to additional processing steps3This technique with the Spectra has successfully prevented clot formation in over 2500 collections at the Fred Hutchinson Cancer Center and over 300 pediatric collections at Boston Children’s Hospital. That the addition of ACD-A before collection does not cause any loss of viability has been validated by both functional assays (colony-forming units-granulocyte-macrophage and fluorescenceactivated cell-sorting viability assay^).^ For collections performed using the same cell separator as Burger et al. (CS3000 Plus, Fenwal, Deerfield, IL) one might consider adding citrate to the bag of autologous plasma to be combined with the cells immediately upon completion. However, we have not validated this option, and because coagulation could occur in the collection chamber, one should heed the observations of Burger et al. before resorting to this option when using that particular device. Because children are particularly susceptible to citrate toxicity, as a result of the relatively higher rates at which cells are typically collected from them (1.5-2.0 mllkglmin), we routinely collect cells by using citrate ratios of 1:25 to 1:30 in combination with heparin anti~oagulation.~ This practice has allowed us to collect up to 12 blood volumes during a 4-hour period without apparent citrate toxicity.6 Jeb B. Gorlin, MD Transfusion Service, Children’s Hospital 300 LongwoodAvenue, Boston, MA 02115 Scott D. Rowley, MD Clinical Cryobiology Laboratory Fred Hutchinson Cancer Center I124 Columbia Street, Seattle, WA 981 04