Miriam Segall

HLA alloimmunization in patients requiring Ventricular assist device support

BACKGROUND Ventricular assist devices (VADs) are often necessary to maintain circulation in patients with heart failure prior to cardiac transplantation. However, the use of such devices has been reported to be associated with a high incidence of development of human leukocyte antigen (HLA) antibodies, due perhaps, according to some investigators, to immune-activating properties of the VAD itself. We looked at HLA antibody formation in our patients during VAD support to determine the rate and potential causes of antibody formation. METHODS Between 1995 and 2000, 54 patients were placed on a VAD at our institution. We reviewed clinical and blood transfusion history and HLA antibody testing of the 29 patients without HLA antibodies prior to implantation. HLA antibody testing was performed by an anti-globulin-augmented cytotoxicity method or by a commercial enzyme-linked immunoassay (ELISA) kit. RESULTS Eight of 29 patients (28%) developed HLA antibodies. Patients who developed HLA antibodies after VAD implantation received significantly more total peri- and post-operative transfusions than did those who remained negative (99 transfusions vs 34 transfusions, p = 0.0014). Within this small study group, gender, age, etiology of heart failure, previous cardiac surgery and duration of VAD support showed no statistically significant correlation with formation of HLA antibodies. CONCLUSIONS Our data suggest that HLA alloimmunization during VAD support may be due to extensive blood transfusion. The rate of HLA alloimmunization does not appear to be greater than that reported in other populations of multi-transfused patients. Leukoreduction of cellular components, as well as plasma, or other initiatives is needed to reduce the rate of alloimmunization and, potentially, the wait to transplantation.

Insulin-dependent diabetes—Associated HLA-D region encoded determinants☆☆☆

We have studied the relative frequency of Dw specificities (defined with homozygous typing cells or primed LD (lymphocyte defined) typing reagents) associated with DR4 and DR2 in the normal and insulin-dependent diabetic population. Our findings demonstrate that there is a highly significantly increased frequency of Dw4 in DR4 positive diabetics as compared with normals and a significantly decreased frequency of Dw2 and Dw12 in the few DR2 positive insulin-dependent diabetics that we have found. In addition, we have used PLT reagents to define a new LD specificity, LD-MN2, that is associated with DR2 and is found significantly more frequently in DR2+ IDD patients than in DR2+ normals. These results suggest that determinants of import in the association between HLA-D and IDD may be more closely related to Dw than to DR.

Cloned Cytotoxic and Non‐Cytotoxic Lymphocytes in Mouse and Man: Their Reactivities and a Large Cell Surface Membrane Protein (LMP) Differentiation Marker System

The recognition that supernatants of mitogen activated (Morgan et al. 1976, Gillis & Smith 1977) or mixed leukocyte (Ryser et al. 1978) cultures can facilitate the propagation of T lymphocytes either isolated from normal peripheral blood or obtained following activation in vitro has allowed both the expansion of T lymphocyte populations containing different types of cells and the cloning of cells belonging to functionally disparate subpopulations. The ability to clone T lymphocytes, both cytotoxic and non-cytotoxic, with maintenance of function and antigen specificity must be regarded as a major advance in cellular immunology. Cellular interactions within the immune system, which consists of an enormously complex series of positive and negative regulatory networks, can now be studied in a reductionist view involving the various contributing cell types, provided each of them can be cloned. It thus becomes conceptually possible to disassemble the immune system into its component parts and reconstruct it, step by step,

Graft-vs-Host Disease After Solid Organ Transplant

We identified 10 solid organ transplant recipients with a histologic diagnosis of graft-vs-host disease (GVHD). Histologic slides were reviewed, and information on the transplant, HLA match, and blood product transfusion history was obtained. Molecular testing to evaluate the presence of donor lymphocytes (chimerism) was done in 1 case. All patients underwent at least 1 gastrointestinal biopsy; 1 patient also underwent a skin biopsy and 1 patient a liver biopsy; all specimens showed grade I to IV acute GVHD. Six patients had a diagnosis of GVHD within 3 months of blood product transfusion and/or solid organ transplantation, which is the time frame in which GVHD reportedly occurs after solid organ transplantation; 4 patients had a distant history of blood product transfusion or solid organ transplantation. In 1 patient, a molecular technique using the polymorphic marker DIS80 documented donor lymphocytes in the colonic tissue and blood (chimerism). Although histologic findings of GVHD are quite specific, they are not pathognomonic. A GVHD-like histologic pattern can be seen in other conditions such as drug reactions and viral infections. Demonstration of donor lymphocytes in the involved organ helps support the diagnosis of GVHD in questionable cases.

Dw subtypes of DR4 in rheumatoid arthritis: Evidence for a preferential association with Dw4

We have determined the frequency of the DR4-associated Dw subtypes, defined by homozygous typing cells, in a group of rheumatoid arthritis (RA) patients on second-line drug therapy. The frequency of DR4 in these patients was 86%. Among Caucasians, the frequency of Dw4 in the DR4-positive patients was significantly increased (68%) as compared to DR4-positive normal individuals (46%; p less than 0.025). Dw4, as compared to the other DR4 subtypes tested, may also be associated with more severe disease as judged by indices of functional impairment and joint damage. In a small subgroup of non-Caucasian (black and Native American) patients, the Dw13 (DB3) subtype of DR4 was often seen, suggesting that RA may have different Dw associations in different ethnic groups.

Southern analysis of DNA polymorphism among Dw subtypes of DR4

DNA from individuals of four Dw subtypes of DR4 (Dw4, Dw10, Dw14, Dw15) were studied using Southern blotting to determine if subtype-specific DR beta or DQ beta restriction fragment polymorphism could be found. Although very little polymorphism was found among ten DR4 homozygous individuals (4 Dw4, 2 Dw10, 3 Dw14, 1 Dw15) using a Dr beta or a DQ beta probe, restriction fragment polymorphism was easily detected between different DR types (DR1-DRw8). The possible evolutionary significance of the lack of Dw-associated polymorphism relative to DR-associated polymorphism is discussed.

DNA restriction fragment length polymorphisms characteristic for Dw subtypes of DR2

DNA restriction fragment length polymorphisms (RFLP) can be easily demonstrated in DNA of cells expressing different DR specificities when class II cDNA probes are used for hybridization. Previous studies of DR4+ homozygous typing cells (HTCs) carrying different Dw subtypes, however, detected no RFLP correlating with subtypes. In contrast, we report here Southern blotting studies of DR2+ HTCs carrying different subtypes which showed RFLP patterns characteristic for each subtype, using both DR beta and DQ beta probes and several restriction enzymes. The RFLP between subtypes of DR2 was, however, appreciably lower than that found between DR specificities.

Positive remote crossmatch: impact on short-term and long-term outcome in cadaver renal transplantation.

Background. A positive crossmatch with a “current” recipient serum (drawn shortly before the proposed transplant) is a contraindication to renal transplantation because of the risk of hyperacute rejection. Conflicting data have been reported concerning the significance of a positive crossmatch with “remote” sera (obtained months or years earlier) when the current crossmatch is negative. Methods. Recipients of a first or second cadaver transplant between June 1988 and April 1994 were studied. All transplants were performed with a negative “current” crossmatch. Retrospective crossmatches using “remote” sera were performed for all sensitized recipients. Results. Recipients with a positive remote crossmatch (RXM) demonstrated a higher incidence of delayed graft function and of acute rejection and graft loss occurring in the first year posttransplant than did sensitized recipients with a negative RXM or unsensitized recipients. In multivariate analysis, only recipients with both a positive RXM and delayed graft function were at significantly higher risk for graft loss. Grafts surviving the first year demonstrated similar half-lives whether the RXM was positive or negative. Conclusions. The positive RXM, possibly in conjunction with other factors leading to very early graft damage, is a significant predictor of unfavorable transplant outcome in first and second renal transplants. This effect is seen early in the transplant course, and there seems to be no impact on outcome after the first year. Newer immunosuppressive modalities may help to reduce the early negative impact.

Pooled stimulating cells as a "standard stimulator" in mixed lymphocyte culture.

Lymphocytes from normal unrelated donors were pooled and used as stimulating cells in mixed lymphocyte culture (MLC). Different pools, each consisting of cells from 20 or 30 different donors, stimulated approximately the same amount of 3H-thymidine incorporation by a given responder, and this “plateau level” of incorporation was different for different responders. Stimulation by pools of 20 cells was highly correlated with the general “responsiveness” of responding cells as measured by their mean response to a large panel of stimulating cells. Such pools may be useful as “standard stimulators” in quantitating MLC stimulation, and perhaps also in evaluating lymphocyte responsiveness of patients.

Familial ureteral abnormalities syndrome: genomic mapping, clinical findings

Abstract. Abnormal development of the ureter during embryogenesis, when occurring in multiple family members, appears to be a genetically determined defect with autosomal dominant inheritance and high penetrance, which can lead to significant kidney damage, renal failure, and death. We have studied 48 individuals within a large kindred in which ureteral-related abnormalities (including vesicoureteral reflux, ureteropelvic junction obstruction, duplicated ureters, and medullary sponge kidney) were segregated. Family members who had not had previous diagnostic studies were evaluated for presence or absence of ureteral abnormalities and we attempted to map the locus for this familial ureteral abnormalities syndrome (FUAS). These studies identified 11 asymptomatic individuals, previously assumed to be unaffected, with minor abnormalities. When linkage analysis between the inheritance of ureteral abnormalities and six marker loci glyoxalase I (GLO-1), major histocompatibility antigens (HLA-A, B, and DR/DQ), D6S288, and factor XIII antigen (F13A1) on the short arm of chromosome 6 was performed, the lod scores significantly rejected linkage over a 77.1-cM distance. These findings are in contrast to previous data suggesting linkage between the presence of ureteral abnormalities and HLA, and indicate the possibility of genetic heterogeneity of FUAS.