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Internal medicine (IM) residency programs select applicants based on several metrics. Factors predicting success during residency are unclear across studies. To identify whether specific applicant or resident factors are associated with IM resident performance using ACGME milestones. We tested for associations between applicant factors available prior to the start of IM residency and resident factors measured during IM residency training, and resident performance on ACGME milestones across three consecutive years of IM training between 2015-2020. Univariable and multivariable linear regression modeling was used to test associations. Eighty-nine categorical IM residents that completed 3 consecutive years of training were included. Median age was 28 years (IQR 27-29) and 59.6% were male. Mean ACGME milestone scores increased with each post-graduate year (PGY) from 3.36 (SD 0.19) for PGY-1, to 3.80 (SD 0.15) for PGY-2, to 4.14 (SD 0.15) for PGY-3. Univariable modeling suggested referral to the clinical competency committee (CCC) for professionalism concerns was negatively associated with resident performance during each PGY. No applicant or resident factors included in the final multivariable regression models (age at starting residency, USMLE Step scores, interview score, rank list position, ITE scores) were associated with ACGME milestone scores for PGY-1 and PGY-2. Referral to the CCC for professionalism was negatively associated with resident performance during PGY-3. Residency selection factors did not predict resident milestone evaluation scores. Referral to the CCC was associated with significantly worse resident evaluation scores, suggesting professionalism may correlate with clinical performance.

Creating phase change lines and their corresponding labels in Microsoft Excel® remains a difficulty for many behavior analysts who want these display features to be integrated into the graph itself. Previous methods designed to address this issue have had limited utility across the types of data sets commonly analyzed by behavior analysts. The purpose of this article is to provide a fully functional method for integrating phase change lines and labels into Microsoft Excel® line graphs. This method is a combination of previous recommendations and allows for easy integration of new data and exportation of graphical displays to other software programs (e.g., Microsoft Word® and PowerPoint®)., Competing Interests: Compliance with Ethical StandardsThe authors declare that they have no conflict of interest.

Public Law 99-457 extended the landmark Public Law 94-142 legislation to include early intervention for infants and toddlers with or at-risk for development of developmental disabilities. Currently over 300,000 infants and toddlers and their families in the United States receive services through Part C of the Individuals with Disabilities Education legislation. The law fostered interagency collaborations and included the child's parent or caregiver as an integral part of the intervention team. This article reviews the 26 years of legislation associated with educating young children with disabilities and the resulting early intervention service delivery system. Analyses and review of studies of Part C services are offered to inform policies that enhance early identification, family engagement, and intervention delivery., (Copyright © 2012 Wiley Periodicals, Inc.)

Objective: Allosensitization of left ventricular assist device recipients has been associated with perioperative transfusion of cellular blood products. The relative sensitizing contribution of leukofiltered cellular blood products, however, remains unclear. We investigated the pattern of sensitization in left ventricular assist device recipients in relation to cellular blood product transfusions received., Methods: Seventy-one consecutive nonsensitized recipients of the HeartMate left ventricular assist device (Thoratec Corporation, Pleasanton, Calif) as a bridge to transplantation were reviewed. Panel-reactive HLA antibody levels at consecutive times after device implantation were correlated with perioperative cellular blood product transfusions., Results: Fifty-four patients received leukofiltered cellular blood products (transfused), whereas 17 patients received only fresh-frozen plasma (nontransfused). Among nontransfused patients, 58.8% (10/17) became sensitized during mechanical support, versus 35.2% of transfused patients (19/54, P = .15). There was a trend toward more sensitization during the 12 weeks after device placement in nontransfused patients. Kaplan-Meier analysis revealed significantly more sensitization in nontransfused patients than in transfused patients, despite equal rates of transplantation (P = .05). A dose-response analysis revealed significant trends toward less sensitization and lower peak panel-reactive antibody level with more cellular blood product transfusions (P = .04). Multivariate Cox regression revealed only increasing transfusions to be associated with a reduced risk of sensitization (hazard ratio 0.18, P = .01)., Conclusions: Sensitization becomes more prevalent with increasing length of support. Avoidance of perioperative leukocyte-filtered cellular blood product transfusions does not decrease the incidence or degree of HLA sensitization. Conversely, cellular blood product transfusions may be associated with lessened alloimmunization and may mitigate the sensitization seen in recipients of the HeartMate left ventricular assist device as a bridge to transplantation.

The National Marrow Donor Program maintains a registry of volunteer donors for patients in need of a hematopoietic stem cell transplantation. Strategies for selecting a partially HLA-mismatched donor vary when a full match cannot be identified. Some transplantation centers limit the selection of mismatched donors to those sharing mismatched antigens within HLA-A and HLA-B cross-reactive groups (CREGs). To assess whether an HLA mismatch within a CREG group ("minor") may result in better outcome than a mismatch outside CREG groups ("major"), we analyzed validated outcomes data from 2709 bone marrow and peripheral blood stem cell transplantations. Three-hundred and ninety-six pairs (15%) were HLA-DRB1 allele matched but had an antigen-level mismatch at HLA-A or HLA-B. Univariate and multivariate analyses of engraftment, graft-versus-host disease, and survival showed that outcome is not significantly different between minor and major mismatches (P = .47, from the log-rank test for Kaplan-Meier survival). However, HLA-A, HLA-B, and HLA-DRB1 allele-matched cases had significantly better outcome than mismatched cases (P < .001). For patients without an HLA match, the selection of a CREG-compatible donor as tested does not improve outcome.

Background: The use of left ventricular assist devices is associated with human leukocyte antigen (HLA) allosensitization. We investigated whether prophylactic treatment with low-dose intravenous immunoglobulin (IVIG), analogous to the use of IgG anti-D (anti-Rh) in preventing Rh immunization, can abrogate HLA allosensitization after left ventricular assist device implantation., Methods: We retrospectively reviewed the data from 84 consecutive heart failure patients who underwent implantation of a left ventricular assist device as a bridge to transplantation. After implantation, panel reactive antibody (PRA) was measured biweekly to assess sensitization (defined by PRA > 10%). Patients who were sensitized before left ventricular assist device implantation were excluded from further analysis (n = 12). Patients who either did not require perioperatively transfusions of cellular blood products or received other immunomodifying regimens were also excluded from further analysis (n = 21). The rest of the patients were divided into two groups based on whether they received IVIG, 10 g daily for 3 days (IVIG group, n = 26; non-IVIG group, n = 25). The decision as to whether patients received IVIG was not randomized but was based on surgeon preference., Results: The sensitization rates (expressed as ratio of sensitized patients to total patients at risk) in the two groups were similar at consecutive time points (2, 4, 6, 8, 12, 20 weeks) after left ventricular assist device implantation. Also, mean PRA at the same time points did not differ between the two groups. Overall, 34.6% (9 of 26) of the IVIG group became sensitized during mechanical support, compared with 32% (8 of 25) of the non-IVIG group (p = 1.0). A PRA of 90% or greater (high-degree sensitization) occurred in 15.3% (4 of 26) of the IVIG group and 12.0% (3 of 25) of the non-IVIG group (p = 0.5)., Conclusions: The use of low-dose prophylactic IVIG after left ventricular assist device implantation affects neither the incidence nor the severity of HLA allosensitization.

Allografts used in the repair of congenital heart defects in children induce a persistent broad HLA antibody response. We have previously shown that a 3-month course of mycophenolic mofetil (MMF) significantly reduces the HLA class I antibody response to valved allograft implantation in children. The purpose of this study was to determine if this reduction in HLA antibody persists after discontinuation of MMF. We conducted follow-up (mean 2 +/- 0.5 years) of seven patients who had received allograft placement for repair of congenital heart defects. These patients received 3 months of immunosuppression with MMF following allograft implantation. When compared to historical controls, patients who received MMF following surgery showed a significantly decreased HLA class I antibody response at 2 years postimplantation. This study demonstrates the ability to persistently alter the HLA class I antibody response using 3 months of MMF following allograft implantation in children.

Sensitization to HLA antigens and the subsequent development of HLA antibodies in children under consideration for heart transplantation is a significant impediment to survival after listing. This is related both to the frequent need for prospective donor-specific crossmatching (thus limiting donor availability and increasing pretransplant morbidity and mortality), and to the increased risk of adverse outcomes after transplantation. This article will review the scope of this problem in children under consideration for heart transplantation, the different methods available for diagnosing HLA sensitization, the known causes of HLA sensitization, the consequences of these preformed antibodies on outcomes before and after heart transplantation, and the different methods of preventing and treating this sensitization that are currently available. Improved methods of diagnosing, preventing, and treating this problem can only lead to better outcomes for children who require heart transplantation.

Background: Circulating human leukocyte antigen (HLA) panel-reactive antibodies (PRA > 10%) have been independently associated with increased risk of rejection and mortality in patients who undergo cardiac transplantation. Cryopreserved allografts used to repair heart defects induce broadly reactive HLA antibodies in children that persist for an undetermined duration of time. The purpose of this study was to prospectively determine the level of HLA sensitization several years after implantation of cryopreserved allografts in children., Methods: We conducted late follow-up of 13 children previously screened for PRA before and after implantation of valved and nonvalved allografts who are alive and free from allograft replacement. Panel-reactive antibodies against HLA class I and II antigens were determined using flow cytometry and classified as high reactive (>50% PRA), low reactive (11% to 50%), or absent (0% to 10%). Follow-up PRA was compared with PRA obtained 3 months after initial allograft implantation., Results: Elevated HLA class I PRA persisted at late follow-up in 12 of 13 children, although it decreased significantly from high to low or from low to absent in 12 of 13 patients (p < 0.001). Elevated HLA class II PRA persisted at late follow-up in 6 of 13 children (46%) and had decreased significantly from prior levels (p = 0.011)., Conclusions: Circulating HLA antibodies induced by cryopreserved allograft tissue persist up to 8 years after implantation although they decrease with time. Therefore, children who have received cryopreserved allografts before cardiac transplantation may be at greater risk for transplant rejection.

A 24-year-old woman experienced severe tricuspid valve regurgitation 6 years after heart transplantation. Tricuspid valve replacement was performed using a cryopreserved mitral valve homograft. Severe tricuspid valve regurgitation recurred within 4 months, associated with an increase in the panel reactive antibody titers from zero to 72%. Tricuspid valve replacement was repeated with a porcine bioprosthesis with excellent recovery and function for >2 years. The mitral valve homograft displayed inflammatory features consistent with humoral immune-mediated destruction.

Background: Valved allografts induce a brisk, broadly reactive human leukocyte antigen (HLA) antibody response in children after implantation. Mycophenolic mofetil (MMF) is a powerful immunosuppressant that inhibits the proliferation of both T cells and B cells and has been reported to possibly reduce HLA panel reactive antibody (PRA) in sensitized transplant recipients., Methods: The purpose of this study was to determine whether MMF can blunt the HLA antibody response to valved allografts in children. Eight patients completed (of 28 approached) a pilot study to determine the effects of 3 months of twice daily MMF (600 mg/m(2)/dose) on the HLA antibody response measured before surgery, at 1 month, and at 3 months after implantation. Patients were 7.5 +/- 4 yrs old (mean +/- standard deviation [SD]), with 5 patients undergoing repair of tetralogy of Fallot, 2 Ross procedures, and 1 aortic valve replacement., Results: In contrast to historical controls with a virtual 100% HLA class I PRA response to valved allograft implantation, MMF markedly decreased the HLA class I antibody response at 1 and 3 months postimplantation. In 6 cases where the HLA type of the donor was defined, PRA specificity correlated with incompatible antigens on the allograft. One patient withdrew after 2 weeks due to a sinus infection that was successfully treated with oral antibiotics, and 3 patients had a transient adverse effect of postoperative vomiting., Conclusions: This study demonstrates the ability to pharmacologically abrogate the HLA class I antibody response to valved allograft implantation in children using MMF.

Background: Recognition of the immunogenicity of standard cryopreserved allografts has led to the development of new decellularized allografts (CryoValve SG; CryoLife, Inc, Kennesaw, Ga). This preliminary study examined the HLA antibody response to these decellularized allografts and compared it with the response to standard allograft material., Methods: We prospectively measured the frequency of panel-reactive HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DR/DQ) alloantibodies in 14 children (age 8.5 +/- 7.9 years) receiving decellularized, cryopreserved allografts, including 6 undergoing allograft patch insertion and 8 with a valved pulmonary allograft. We compared them with 20 historical control subjects (age 1.7 +/- 2.4 years) undergoing implantation of standard cryopreserved allografts, 8 with valves and 12 with allograft patch. All patients had panel-reactive antibody levels measured before and at 1, 3, and 12 months after the operation. HLA class I and class II panel-reactive antibody levels were determined with a sensitive flow cytometry technique., Results: We found panel-reactive antibody levels in decellularized allografts to be elevated slightly from preoperative levels for both class I and class II antibodies at 1, 3, and 12 months (P >.05). The panel-reactive antibody level for both class I and class II antibodies were significantly lower for decellularized allografts as compared to standard allografts. Functionally, the allografts were similar with decellularized valved grafts showing a peak echo-determined systolic gradient of 13 +/- 15 mm Hg at 8 +/- 2.6 months postoperatively as compared to a gradient of 24 +/- 18 mm Hg measured 12 +/- 6 months postoperatively in standard allografts (P =.11)., Conclusions: Decellularized grafts elicited significantly lower levels of class I and class II HLA antibody formation at 1, 3, and 12 months after implantation than did standard cryopreserved allografts. Early hemodynamic function of decellularized grafts was similar to that of standard cryopreserved allograft valves. Further experience is necessary to determine whether the reduced immunogenicity of decellularized allografts will truly allow tissue ingrowth and improved long-term durability in patients.

We have developed a high throughput HLA typing methodology that is a modification of the standard sequence-specific primer method. This approach is distinct from other methods using an automated DNA analyzer, as more than one gene is typed in a single lane. We have optimized the method for use on an ABI 373 automated genotyping machine. Primers were designed to preferentially amplify DNA fragments of the generic allelic groups of the DQA1 and DQB1 loci. PCR products representing alleles at the DQA1 locus were amplified using a different fluorescent dye than the PCR products from the DQB1 locus. Only three PCR reactions are required for low resolution typing of DQA1 and DQB1. Use of different labeled primers enables genotyping for both loci in a single gel lane, allowing for 64 samples to be typed at low resolution for both DQA1 and DQB1 on a single gel. Automated allele assignments were determined based on DNA migration distance through a polyacrylamide gel using a standard genotype allele-calling program. Accuracy of this method is greater than 98% for both loci. The strategy described here may be adapted to include more loci or to produce higher resolution typing of alleles encoded by these loci. It can be readily optimized for use on other slab gel or capillary electrophoresis systems.

Fifteen blood group O and B recipients have been transplanted with kidneys from subtype A2 living donors since April 1992. ABO red cell grouping was performed by local licensed blood banks with A2 subtype determined using an anti-A1 lectin and, retrospectively, by a polymerase chain reaction (PCR)-based molecular method. All grafts functioned immediately and no patient has required dialysis. Three patients each experienced one reversible rejection episode. With the exception of one cardiac death at 9months and one patient with profound toxicity to calcineurin inhibitors, all allografts continue to function normally. One donor, mistyped as a group A2 using lectin, was by PCR typing an A1O1 nonsecretor; the graft continues to function normally at 30 months. Transplantation of living donor A2 renal allografts into non-A recipients produces excellent long-term allograft survival and expands the potential living donor pool for nonblood group A recipients.

Background: The purpose of this study was to prospectively assess the effects of azathioprine on the humoral immune response to HLA alloantigens and allograft function in children receiving cryopreserved valved allografts., Methods: We randomized 13 children to receive azathioprine or not to receive azathioprine (controls) after receiving a cryopreserved valved allograft. Azathioprine patients received intraoperatively 4 mg/kg of azathioprine and 2.0 +/- 0.5 mg/kg once daily for 3 months after operation. Panel reactive antibodies against HLA class I and class II alloantigens were measured before, 1 month, and 3 months after operation., Results: Panel reactive antibodies were not significantly different between the azathioprine and control groups before (0.0% +/- 0% versus 1.6% +/- 1%), 1 month (59% +/- 17% versus 71% +/- 12%), or 3 months (84% +/- 15% versus 96% +/- 1.3%) after operation. There were no differences in degree of allograft valve stenosis between azathioprine (31.5 +/- 26 mm Hg, 13.4 +/- 7 months postoperatively) and control groups (25.4 +/- 11 mm Hg, 17.2 +/- 10 months postoperatively) or allograft valve insufficiency., Conclusions: Azathioprine does not significantly decrease the immune response to HLA alloantigens or affect the function of cryopreserved valved allografts used in children to repair congenital heart defects.

Background: The purpose of this study was to prospectively determine the immunogenicity of nonvalved allograft tissue used to repair congenital heart defects., Methods and Results: We prospectively analyzed the immune response of 11 children, 1.4 months to 10 years of age, who required nonvalved allografts to alleviate stenosis during repair of congenital heart defects. In 7 patients, pulmonary arterial grafts were used; in 3 patients, monocusp pulmonary artery grafts were used; and in 1 patient, a section of glutaraldehyde-preserved allograft pericardium was used. We measured the level of HLA panel-reactive antibody (PRA) before surgery, 1 week after, 1 month after, and 3 months after surgery. PRA was determined by the antiglobulin technique and flow cytometry. HLA class I and class II antibodies measured by either technique were negligible before and 1 week after surgery. Nine of 11 patients (82%) exhibited a significant immune response at 1 month after surgery that further increased at 3 months. The measured PRA for class I antibodies with the antiglobulin technique increased to 43+/-36% at 1 month and to 69+/-38% at 3 months after surgery. Flow cytometry class I PRA measurements were similar. Class II PRA increased to 26+/-34% at 1 month and to 41+/-36% at 3 months. Age negatively correlated with the degree of elevation of PRA, but neither allograft area nor the area indexed to patient body surface area correlated with PRA., Conclusions: Cryopreserved nonvalved allografts induce a strong HLA antibody response in the majority of children.

Objectives: Very little is known regarding the immune response to cryopreserved allograft valves and patch material used in the surgical repair of congenital heart defects., Methods: We prospectively measured the frequency of panel reactive antibodies directed against HLA class I (HLA-A, B, and C) and class II (HLA-DR/DQ) alloantigens in 24 children receiving cryopreserved allografts. We compared them with results in 11 previously reported control patients. Sixteen of the study patients underwent placement of a valved conduit (11 pulmonic, 5 aortic) between the right ventricle and pulmonary arteries, 6 underwent patch angioplasty of stenotic vessels with cryopreserved pulmonary artery, and 2 underwent placement of a pulmonary monocusp patch. Study patients had panel reactive antibodies measured before, 1 month, 3 months, and 1 year after the operation., Results: With allograft implantation, panel reactive antibodies increased from 1.9% +/- 5% before the operation to 62% +/- 33% at 31 +/- 8 days after the operation, 92% +/- 15% at 3.3 +/- 0.6 months after the operation, and 85% +/- 18% at 1.1 +/- 0.2 years after the operation. The control group showed no change in panel reactive antibodies, with a level of 1.6% +/- 1% before the operation, 3.2% +/- 1% 28 +/- 5 days after the operation, and 1.7% +/- 1% 2.7 +/- 0.3 months after the operation. Class II antibodies (anti-HLA-DR/DQ) rose to 49% +/- 35% at 30 +/- 8 days and 70% +/- 26% at 3.3 +/- 0.6 months after the operation., Conclusions: Cryopreserved allograft material induces a marked response that involves both class I and class II anti-HLA antibodies within 3 months after operation in children. This alloantibody response may represent a form of "rejection," may have implications for those who require subsequent cardiac transplantation, and may play a role in early allograft failure.

Introduction: Data from the UCLA/UNOS and Collaborative Transplant Studies Registries indicate that mismatched HLA-DR alloantigens expressed on a former donor renal allograft should not be repeated because of significantly poorer long-term survival., Methods: Retransplant candidates waiting for another renal allograft were screened for HLA class II alloantibodies (aAb) using direct complement-dependent cytotoxicity and several sensitive aAb binding assays., Results: When screened by complement-dependent cytotoxicity, 46% of the patients were aAb negative. In contrast, using aAb binding assays, 90% of the patients had HLA-DR aAb specific for previous HLA-DR allograft mismatches. Most important, no directly cytotoxic HLA-DR antibody was detected in 9 of 27 patients., Conclusion: Our studies suggest that crossing the same HLA-DR mismatch in a subsequent transplant may result in poorer survival due to underlying donor-specific HLA-DR aAb. If confirmed in a retrospective study of retransplant patients, B cell donor cross-matches using antiglobulin complement-dependent cytotoxicity or flow cytometry would appear essential if this barrier were to be crossed.

Background: The genetic basis for control of alloantibody responses against foreign HLA histocompatibility antigens has never been delineated. The most likely postulate would be that HLA class II alloantigens of the host regulate the response through their ability to present processed HLA allopeptide fragments for the cognate interaction between CD4+ T lymphocytes and B lymphocytes that leads to IgG antibody synthesis., Methods: We have analyzed our allosensitized transplant patient population with regard to humoral responsiveness to a serologically defined public HLA class I epitope, Bw4. Peptides representing the linear sequence of the Bw4 epitope (amino acids 74-86) and the alternative Bw6 epitope were synthesized and assayed for binding to a panel of HLA homozygous lymphoblastoid B cells using a quantitative fluorescence binding assay., Results: We found that 73% of patients who have produced a HLA-Bw4-specific alloantibody express either the HLA-DRB1*01 or HLA-DRB1*03 alloantigen; 19% of the remaining responders expressed HLA-DRB1*04. Analysis of the United Network for Organ Sharing Transplant Registry indicated that the survival of cadaver renal allografts mismatched for Bw4 was significantly compromised in sensitized DRB1*01+ or DRB1*03+ recipients (P<0.01). In vitro, the Bw4 peptide bound strongly to DRB1*01+ and DRB1*03+ lymphoblastoid B cells; no similar binding was observed with Bw6 peptide. These findings were confirmed using murine fibroblast lines transfected with HLA-DR alpha/beta genes and by solid-phase enzyme-linked immunosorbent assay using purified HLA-DR alloantigen., Conclusions: We conclude that there are at least two human Ir genes, HLA-DRB1*01 and HLA-DRB1*03, that confer a high risk for both humoral allosensitization and renal allograft failure in situations of HLA-Bw4 incompatibility. These findings may be of future benefit in devising new antigen matching strategies for reducing the risk of humoral HLA allosensitization and chronic allograft rejection.

Background: Transfusion of cellular blood products during left ventricular assist device (LVAD) implantation has been associated with HLA allosensitization, resulting in the need for a negative prospective cross-match and prolonged transplant waiting times. In order to prevent this risk, we developed a protocol to avoid transfusion of cellular blood products., Methods: The protocol included preoperative patient stabilization, perioperative recombinant erythropoietin and blood conservation strategies, and postoperative monitoring of mixed venous oxygen saturation (SVO2) to assure adequate peripheral oxygen delivery. Panel reactive antibody (PRA) was measured in all patients pre and post LVAD placement to assess HLA sensitization., Results: Seven consecutive patients underwent LVAD implantation without transfusion of blood or platelets, one of whom expired perioperatively. Mean hematocrit was 35.2% preoperatively, and 21.8% postoperatively, reaching a nadir of 20.2%. Postoperative SVO2 was >60% in all patients. In the six survivors, mean hematocrit reach 24.3%, 27.3%, and 33.0% by postoperative day seven, fourteen, and thirty, respectively. PRA in three patients was 0% preoperatively and remained 0% until transplantation after 33, 34, and 50 days of support. In two patients, preoperative PRA was 7% and 17%, dropped to 3% and 0% after thirty days, then progressively rose to 96% and 100% after 60 and 90 days, respectively. In one other patient, preoperative PRA was 0%, remained at 0% after thirty days, then rose to 96% by 60 days., Conclusions: Avoiding transfusion of cellular blood products in LVAD recipients is safe and well tolerated, but does not universally protect from HLA allosensitization. Other factors may also produce sensitization, such as immunogenic components of the LVAD, soluble antigen in fresh frozen plasma, or latent sensitization which is not initially evident in critically ill and possibly anergic patients.

This study shows that the broad anti-HLA antibody response against cryopreserved valved allografts used for surgical repair of congenital heart disease persists beyond 1 year after implantation. In 3 patients, there were clearly defined HLA antibody specificities consistent with the HLA phenotypes of the patients, i.e., the panel-reactive antibody was directed against major alloantigen groups that were not expressed by the antibody responders.

HLA Class I alloantigens express multiple epitopes which can be defined serologically using human HLA alloantibodies (aAb). We have shown that the vast majority of HLA antisera exhibit the CYNAP phenomenon (complement-dependent cytotoxicity (CDC) negative, adsorption positive) which can be identified by conversion to direct CDC positive reactivity with the addition of an antihuman immunoglobulin (Ig) light chain (AHG) reagent. In this study, the immunochemical mechanisms responsible for the CYNAP phenomena and how AHG overrides CYNAP have been further characterized using affinity-purified HLA aAb, class-specific anti-IgH reagents and human C1q binding assays quantified by flow cytometry. We have found that CYNAP reactions are not the result of low affinity aAb or generally caused by non-complement fixing HLA aAb. Our experiments illustrate that only anti-human IgL AHG reagents can consistently augment CDC and override CYNAP; anti-IgH have not effective. Two noncompeting HLA aAb of different epitopic specificity or one aAb in conjunction with the AHG-augmenting reagent results in striking synergy with a 200 to 400% increase in binding of C1q. We conclude from these and other experiments detailed in this article that an IgM aAb or either two adjacent, noncompeting IgG HLA aAb bound to spatially distinct epitopes on a single HLA molecule or a monospecific IgG HLA aAb in concert with the AHG binding to this HLA aAb, is required for efficient (bivalent) C1q binding and initiation of C-mediated lympholysis. In contrast, the CYNAP phenomenon usually occurs because monospecific HLA aAb directed against a single epitope cannot effect high affinity, bivalent interaction with Clq and activate complement that would ultimately lead to cytolysis.

The stability of HLA alloantibodies was studied in 128 antibody-positive, potential kidney transplant recipients over an average period of 3 years. Antibody detection was performed using an anti-human globulin-complement-dependent cytotoxicity technique. In this study, the specificity of antibodies was categorized as against either private epitopes or cross-reactive group (CREG) epitope clusters. Definable antibodies were found in 94% of patients, and 89.5% of the definable antibodies had specificity for CREG clusters. Patterns of antibody reactivity were stable in most of the patients evaluated, even though the percentage of panel-reactive antibody (PRA) often demonstrated considerable fluctuations. Of the 220 definable private-specific or CREG cluster-specific antibodies identified in the patients, nearly 80% persisted throughout the observation period. The fluctuations in % PRA were common, but usually were not due to the acquisition of new HLA antibodies. Most fluctuations were attributable to variable detection of specificities within the same CREG cluster, possibly due to technique variation or changes in antibody avidity or titer or in cell panel composition. This study demonstrates that patterns of antibody specificity are remarkably stable in this patient population, even though PRA values fluctuated. This study further suggests that HLA antibody specificity analysis is a more useful clinical parameter of lymphocytotoxicity testing than simple reporting of % PRA when identifying potential donors for individual patients.

Background: The HLA immunogenicity of cryopreserved valved allografts used in the surgical repair of congenital heart defects is unknown., Methods and Results: To determine the immunogenicity of these allografts, we measured prospectively the frequency of panel-reactive HLA class I alloantibodies (PRA) before, 1 month after, and 3 months after allograft implantation in 9 children (age, 5.4 +/- 2.1 years) and after open-heart surgery without allograft implantation in 11 age-matched control children (age, 4.0 +/- 1.5 years). PRA was determined against an HLA-select frozen T-lymphocyte panel using the antiglobulin cytotoxicity technique. After allograft implantation, PRA increased from 3.2 +/- 2.7% before surgery to 63.3 +/- 12% at 25 +/- 2 days after surgery and 99.7 +/- 0.3% at 3.4 +/- 0.3 months after surgery. The use of dithiothreitol to remove IgM alloantibodies resulted in a modest decrease in PRA at 1 month (33.2 +/- 13%) but no change at 3 months (93.0 +/- 3.4%), suggesting the initial humoral response is an IgM alloantibody that switches almost exclusively to IgG by 3 months. Control patients showed no increase in PRA over time: 1.6 +/- 1% before surgery, 3.2 +/- 1% at 28 +/- 5 days after surgery, and 1.7 +/- 1% at 2.7 +/- 0.3 months after surgery., Conclusions: Cryopreserved valved allografts in children induce a marked HLA alloantibody response that increases to broad panel reactivity within 3 months after surgery. This HLA sensitization has potential not only for causing deleterious effects on allograft function but also for limiting the future opportunity of heart transplantation in patients who receive cryopreserved valved allografts.

A flow cytometric assay for lymphocyte HLA-B27 expression using a two-color direct immunofluorescent assay was compared to traditional microlymphocytotoxicity testing on 209 clinical samples. For the flow cytometric assay, whole blood was mixed with a monoclonal anti-B27 conjugated to fluorescein-isothiocyanate (FITC) and anti-CD3 conjugated to phycoerythrin (PE). The samples were analyzed with flow cytometry by gating on CD3 positive events and anti-B27 staining intensity was evaluated as median channel fluorescence of the histogram peak. The median channel fluorescence was least with B27 negative and B7 negative samples (84 +/- 17), intermediate with samples that were B27 negative but B7 positive (118 +/- 13), and greatest with samples that were B27 positive (155 +/- 13). In addition to cross-reactivity with the B7 antigen (n = 38), the monoclonal anti-B27 cross-reacted with HLA-B37 positive samples (n = 3) and HLA-B39 positive samples (n = 3). Using a median channel fluorescence cutoff of 136, 39 of the 40 B27 positive samples gave positive results in the flow cytometric assay for a sensitivity of 97.6%. The specificity was 95.9% with 7 false positives of 169 B27 negative samples. The flow cytometric HLA-B27 assay is a convenient, useful screening test. For greatest specificity, samples positive by flow cytometry should be confirmed by conventional microlymphocytotoxicity or by use of other monoclonal antibodies directed against B27.

The identification of serum HLA alloantibody in clinical transplantation can only be considered meaningful if the information derived from clinical tests can be used to predict crossmatch outcome. Drawing on the foundations and principles of blood transfusion medicine, this article presents a strategy for efficient, cost-effective patient HLA antibody monitoring that relies on a screening technique identical in sensitivity to the final donor crossmatch method. Application of this approach to the successful transplantation of highly immunized patients using HLA disparate cadaver renal allografts supports the contentions advanced within this article.

Monoclonal antibodies (mAb) recognizing the B7 CREG have been used to construct an epitopic map of HLA-B7. Similar studies with human HLA alloantisera have been lacking due to the polyclonal nature of the alloantibodies (aAb). Detergent-solubilized HLA Class I antigens were purified and coupled to activated CH-Sepharose 4B. Sequential affinity isolation of aAb populations using a series of HLA antigen columns enabled us to produce a battery of aAb eluates against both the private B7, B13, B27, and B omega 60 determinants and the public B7-42, B7-60, B7-60-61, B7-27-13-60, B7-42-22-27, B7-8-42-60-41, and B omega 6 epitopes. The topographic relationship of the B7 family of determinants recognized by the Ab probes was derived using crosscompetition Ab blocking assays with quantitation by indirect immunofluorescence and FACS analysis. We have found that aAb and mAb of similar specificity crossblock; Ab of different specificity give complex patterns including both overlapping blocking between the alpha domains and Ab-induced conformational change of the molecule. From these investigations, we conclude that HLA Class I alloantigens bear both multiple, topographically distinct public epitopes and separate private determinants that can be distinguished using human aAb probes. At least four discrete epitopes are expressed by each molecule of the HLA-B7 CREG and can be ascribed to unique aa substitutions on the hydrophilic beta loops of the distal heavy chain domains and also on several exposed areas of the alpha helices. These findings are extremely similar to those of the HLA-A2 CREG and suggest that possibly all Class I molecules possess a comparable, complex degree of serologic polymorphism.

The topographic architecture of the epitopes expressed on the HLA-A2 glycoprotein using murine monoclonal antibody (mAb) probes indicates at least two sterically distinct domains. Previously, we have demonstrated using human HLA alloantibodies (aAb) that multiple determinants are expressed on each HLA antigen: the highly polymorphic private epitopes and the public determinants that are shared within a family of crossreactive groups (CREG). Our objectives now focus on probing the antigenic structure of the HLA-A2-28-9-B17 CREG using highly specific aAb in conjunction with mAb that have previously been used for structural studies. Both mAb-mediated blockage of complement-dependent cytotoxic aAb and reciprocal antibody (Ab) binding inhibition assays with quantitation by fluorescence flow cytometry have been utilized. We have found that xenogeneic mAb directed against A2-69, A2-B17, and A2-28 crossblock aAb of the same serologic specificity, and vice versa, indicating that the epitopes they respectively recognize are at least in close steric proximity. However, additional HLA-A2, A28, and B17 aAb of private specificity and A2-28-9 aAb of public specificity, for which there are no known mAb counterparts, paint an additional complexity not previously known. We conclude that at least four different alloepitopes can be expressed by each serologically defined HLA antigen. Based on the primary sequence data, we have assigned the location and the amino acid substitutions which most likely account for these discrete epitopes. The unique private determinants are located on the alpha 1 domain together with the interlocus A2-B17 epitope while the public epitopes A2-69, A2-28-9 and A2-28 are located on the alpha 2 domain.

Excellent allograft survival is now routinely accomplished following renal transplantation. Changes in immunosuppression have resulted in a significant improvement in early survival for recipients of primary LRD and CD allografts. In our series, crossmatching techniques which accurately assess alloantibody reactivity and not the degree of HLA mismatch have also permitted successful transplantation of such high-risk groups as recipients of second transplants and highly sensitized recipients. However, a yearly attrition rate of allograft loss persists for all recipients. These long-term observations stress the need for newer approaches to immunosuppression in the future, which include protocols that allow for an indefinite tolerance to incompatible donor antigens.

A class I HLA molecule may bear not only a private or unique determinant, but a shared, yet discrete, public epitope. These public determinants occur with a much higher frequency in the random donor population than the associated private determinants--and thus, are encountered more often in random donor blood transfusions and in renal transplantation. Sera from highly sensitized dialysis patients have been reported to contain a restricted number of antibodies to public determinants rather than a diverse array of antibodies directed against the private HLA-AB epitopes. As detailed in this report, comprehensive serum analysis of the public antibodies in highly sensitized transplant candidates has optimized identification of potential crossmatch-compatible donors and has avoided needless crossmatches. During the past two years, the incidence of renal transplantation from cadaveric donors to highly sensitized recipients has doubled at this institution. At 10-25 months following transplantation, 70% of these allografts are functioning. Private HLA class I antigen incompatibility was not a barometer for exclusion in the final donor crossmatch of these highly sensitized recipients. Furthermore, positive donor T cell crossmatches with sera obtained more than six months prior to transplantation may not represent an impediment to successful transplantation. We conclude that the approach of detailed antibody analysis can result in an improved outlook for successful transplantation of more dialysis patients who are highly sensitized to the class I HLA alloantigens.

The human class II, HLA-linked, B cell alloantigens include the HLA-DR, MB, MT, and Te determinants. Interest in the molecular relationships of these antigens has recently intensified because of their homology to the murine Ia antigens and their possible importance in disease predisposition and transplantation. We have used alloantisera with carefully defined immunochemical as well as serologic specificity, and two immunochemical techniques, sequential immunoprecipitation with analysis by SDS-PAGE and two-dimensional gel electrophoresis, to explore the molecular relationships of the MT2, MB3, MT4, and HLA-DR5 antigenic determinants. The data presented here indicate that 1) all class II molecules that bear the DR5 antigenic determinant also bear the MT2 antigenic determinant; (2) the homozygous DR5 cell line, Swei, expresses at least two structurally distinct class II molecules, both of which bear MT2: one bears the MT2, MB3, and MT4 antigenic determinants, and the second bears the MT2, but not the MB3 or MT4 antigenic determinant; and (3) the DR5 determinant is located on at least one and possibly both of these distinct class II molecules.

Neuraminidase (Vibrio cholerae) treatment of human metastatic mammary carcinoma MDA-MB-231 cells grown in culture released 0.60-0.63 mg of N-acetylneuraminic acid from 10(9) cells. Incubation of intact cells with a modified trypsin and fractionation by gel filtration gave mainly O-glycopeptides. The presence of O-glycosyl-linked chains having one or two carbohydrate residues was confirmed by treatment of the glycopeptide fractions with galactose oxidase, followed by reduction with alkaline sodium borotritide and fractionation. The major glycopeptide fraction, which consisted of 53% carbohydrate and 47% protein, and a minor glycopeptide fraction each inhibited hemagglutination by peanut lectin. These results suggest the presence of O-beta-D-galactopyranosyl-(1 leads to 3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1 leads to 3)-L-serine (threonine) residues. The absorptive capacities for anti-HLA-A2 and anti-HLA-B8 antisera were slightly greater for intact than for lyophilized cells, which suggested that masking of these major histocompatibility antigens did not occur in intact cells.

Two well characterized Eighth International Workshop sera, 8w1090 and 8w112 , described to have anti-DR3 and anti-DR5 activity, respectively, were tested by standard and antiglobulin augmented (AHG) complement dependent cytotoxicity (CDC) assays with 21 target cells. We demonstrate that both sera, in fact, have only one antibody (most likely anti-MT2), but have different CYNAP patterns. These observations are discussed with regard to a more complete analysis of antisera that detect gene products of the HLA-DR region.

Histocompatibility (HL-A) phenotypes of 44 unrelated white patients from the greater Boston area with amyotrophic lateral sclerosis (ALS) and 200 white controls were compared. In the overall ALS group, an increased frequency of HL-A3 was noted (43% vs 25%, P less than .05). Thirty-eight patients had rapidly progressive disease; among this group the HL-A3 incidence was 50% (P less than .005). Six patients had slowly progressive disease, none had HL-A3, and five had HL-A12. The HL-A antigens may link with disease severity in ALS.

Previous studies have indicated that HLA-DR homozygous cell lines express two Ia alpha and Ia beta chains that combine to form at least two Ia molecules. This report demonstrates by two-dimensional gel electrophoresis the existence of a third structurally distinct human Ia beta chain on DR2 and DR5 cell lines. This suggests that at least five separate genes control the expression of Ia molecules on HLA-DR homozygous cell lines.

Cell mediated lympholysis (CML) has been proposed as an in vitro model of the rejection process that results from transplantation of allogeneic tissue. To date, the absolute frequencies of cytotoxic T lymphocytes (CTL) and their precursors (CTL.P) have not been directly estimated in man because of technical difficulties. Through optimizing the conditions for radiometric detection of 51Cr release and the attendant improvement in CML sensitivity, direct CTL frequency estimates have been determined in peripheral blood (PBL), spleen (SPL), and lymph nodes (LNC) after in vitro allostimulation using unrelated human cells and limiting dilution assays. The mean frequency of CTL generated from PBL is 1 in 826 cells (0.121% +/- 0.101%) which, from preliminary experiments, is significantly greater than that generated from either LNC or SPL (p less than 0.05). With restimulation of primed cells on day 10, the frequency of CTL generated from PBL was increased 400%. The CTL.P frequency (0.0064% +/- 0.0050%) was approximately 5% of the corresponding CTL frequency. The CTL.P frequencies were found to be minimal estimates as both accessory "filler" cells and T cell growth factors increased the level of detection of CTL.P an average of threefold. The limiting cell dilution assay as detailed in this report should be a powerful tool for defining the cellular requirements and related factors necessary for optimal induction of a CTL response and should provide the means for determination of the immunogenetic requirements and the allospecificity of human cytotoxic lymphocytes.

We have been using human T-lymphocyte clones specifically sensitized to detect leucocyte antigens of Type 1 (insulin-dependent) diabetic patients in the hope of detecting novel HLA antigens associated with Type 1 diabetes. We previously described two such clones which define a new class II HLA antigen, Boston-1 (BO1). BO1 is found mainly on cells of persons with particular HLA-DR antigens and, of potential significance for diabetes, BO1 identifies a distinctive subset of DR3 haplotypes. We report here that BO1+ DR3 haplotypes are overrepresented in Type 1 diabetes. That is, significantly more of the DR3-positive subjects are BO1-positive in the patient group (31%) than in the control group (8%), suggesting that a diabetes-susceptibility gene may be more common on the BO1+ than on the BO1- DR3 haplotypes. Alternative interpretations are also discussed.

Simplified procedures for determining amino acid sequences in proteins and nucleotide sequences in DNA have rapidly expanded the number of MHC molecules for which primary amino acid structure is known. These molecules will be especially valuable as tools to study the structure-function relationships of globular proteins because of the extensive polymorphism of genes coding the MHC genes products. The general three-dimensional structure of class I MHC molecules was recently deduced, but the more subtle topographical microconformations are still undefined. Definition and topographical mapping of epitopes, defined by serological or cellular immune effector products, will be critical probes for these three-dimensional studies. Comparative studies of amino acid sequences among various MHC and molecules have revealed distinct regions of hypervariability in the alpha-1 and -2 domains of class I heavy chains and the alpha-1 and beta-1 domains of most class II molecules. Mutant MHC molecules that differ from each other by no more than one to three amino acids can have structural changes which may result in a loss of the private epitopes that defined the allelic gene product. On the basis of these studies, the private epitopes are thought to be determined by one or more of the hypervariable regions. Similar studies of the relationships between specific regions of the molecule and public epitopes are not fully explored. Because public epitopes are partially conserved structures, one might expect that their structure is not principally determined by hypervariable region. In fact, however, some public epitopes, such as A2/B17 and BW4/Bw6, do map to diversity regions. Epitope mapping as a means of identifying specific topographic sites and relating these sites to specific functional regions of the molecule will be difficult unless the epitopes themselves are better defined. Thus, the capacity to distinguish spatially distinct public epitopes from cross-reactive homologous private epitopes will be important if epitope-specific immunological probes are use to map specific regions of an MHC molecule. Many investigators are interested in the possibility that some components of HLA alloimmunization are regulated through idiotypic networks. Suciu-Foca and colleagues have provided preliminary evidence that epitope-specific HLA alloantibodies bear dominant idiotypic determinants. Antibodies to these determinants appear during pregnancy, following blood tranfusion, and following renal allograft transplantation, also, the antibodies have been correlated with renal allograft survival.(ABSTRACT TRUNCATED AT 400 WORDS)

The sera of 37 renal and 12 liver allograft recipients treated with OKT3 (42), Leu2a (7), or both (2) monoclonal antibodies were serially analyzed by an enzyme-linked immunosorbent assay to determine the humoral response (IgG) to mAb. Anti-mAb IgG to the treatment mAb was detected in the serum of 23 (76%) renal and 6 (50%) liver OKT3 recipients, and all 7 Leu2a renal recipients, usually within 14 days of mAb completion, but never during the first week of Rx. Each of the 7 Leu2a recipients developed reactivity not only to Leu2a isotype (IgG1), but also to OKT3 isotype (IgG2a). In contrast, only 1 of the 42 renal and liver allograft recipients treated with OKT3 developed reactivity to the Leu2a isotype. Blocking studies indicated that the specificity of the response to the treatment mAb was directed at the idiotype--and, in some patients, to the constant domain (isotype) of the mAb administered. The antibody response to an alternate isotype (IgG2a) observed in Leu2a (IgG1)-treated patients most likely resulted from irrelevant immunoglobulin (IgG2a) in the Leu2a preparation. This reactivity appeared to be specific for the IgG2a subclass. Clinicians administering mAb therapy should be aware that various mAb preparations may contain immunoglobulin isotypes unrelated to the therapeutic mAb. Crossimmunization to the irrelevant immunoglobulins may occur, precluding subsequent use of other mAbs sharing similar isotype.

A component of the interleukin 2 receptor (IL-2R) is released in soluble form during T cell activation and can be detected in the blood during acute renal allograft rejection. This study evaluates the diagnostic utility of a sandwich enzyme immunoassay test for serum and urine IL-2R in renal allograft recipients. A rise in serum IL-2R during the week prior to the clinical diagnosis of rejection correlated better with rejection than did isolated serum IL-2R levels or urine values. For the diagnosis of acute rejection, a rise in serum IL-2R (sensitivity 73%, specificity 87%) was comparable in overall test performance to a rise in serum creatinine (sensitivity 70%, specificity 84%). Overall, the two tests had equivalent receiver operating characteristic curves. Because the etiology of false positives in creatinine and IL-2R assays differed (primarily cyclosporine toxicity and infection, respectively), the predictive value of the combined tests was superior to either alone.

The addition of an antihuman immunoglobulin (AHG) reagent to the basic complement-dependent cytotoxicity (CDC) test markedly increases the frequency of lymphocyte-reactive antibodies in many alloantisera. The extra reactivity has been previously identified as associated with alloantigens coded by the HLA complex, but definitive evidence establishing the antigenic specificity of the antibodies reactive by AHG-CDC has been lacking. We determined the nature and specificity of AHG-reactive alloantibodies through parallel testing (CDC +/- AHG) of a large battery of HLA alloantisera against panel cells composed of unrelated individuals and genotypic HLA-identical sibling pairs, and by means of differential platelet absorption and elution of alloantibody. We conclude that the AHG-CDC procedure, relative to "standard" CDC, detects subthreshold levels of alloantibody with specificity for the HLA-A, B, and C locus alloantigens. Most importantly, the AHGG-CDC technique consistently converts cytotoxicity-negative absorption-positive (CYNAP) HLA alloantibody to direct cytotoxic antibody, thus providing a more accurate assessment of the complete specificity of antibodies in complex alloantisera and patients' sera without having to resort to more cumbersome binding assays. These data should be of assistance in improving the characterization of HLA alloantisera used for serological and biochemical studies of the HLA molecules and in delineating the specificity of AHG-CDC antibody in clinical allotransplantation and single-donor platelet transfusion.