Antibodies to HLA-E may account for the non-donor-specific anti-HLA class-Ia antibodies in renal and liver transplant recipients.

The non-donor-specific anti-HLA-Ia antibodies correlate significantly with lower graft survival in organ transplant patients. Based on our earlier findings that anti-HLA-E murine monoclonal antibodies (MEM-E/02 and 3D12) reacted with different HLA-Ia alleles and the peptides shared by HLA-E and HLA class, Ia alleles inhibited the HLA-Ia reactivity of the anti-HLA-E antibodies in normal non-alloimmunized males, the possibility of that anti-HLA-E IgG may account for the non-donor-specific anti-HLA-Ia antibodies in the allograft recipients was examined by multiplex-Luminex®-immunoassay. About 73% of renal and 53% of liver transplant patients' sera with high level of anti-HLA-E IgG showed reactivity to different non-donor HLA-Ia alleles. About 50% renal and 52% liver allograft recipients' sera with low level of anti-HLA-E IgG had no reactivity to any HLA-Ia alleles; however, the IgG isolated from the same sera with protein-G columns showed the presence of anti-HLA-E IgG with HLA-Ia reactivity. Furthermore, both recombinant HLA-E and the IgG-free serum containing soluble HLA-E (sHLA-E) inhibited HLA-Ia reactivity of anti-HLA-E murine monoclonal IgG significantly. The data suggest that the HLA-Ia reactivity of the anti-HLA-E antibody accounts for the non-donor-specific anti-HLA-Ia antibodies. It is proposed that the sHLA-E heavy chain, shed in circulation after organ transplantation, may expose cryptic epitopes of HLA-E to elicit anti-HLA-E IgG antibodies, which may cross react with HLA-Ia alleles due to the peptide sequences shared between them. This study provides a new explanation for the presence of non-donor-specific antibodies for non-existing HLA-Ia alleles, frequently observed and correlated with survival in organ transplant recipients.

Human leukocyte antigen class II DQ alpha and beta epitopes identified from sera of kidney allograft recipients.

BACKGROUND: Epitopes are the sites to which antibodies bind. Both alpha and beta peptide chains of the human leukocyte antigen-DQ heterodimers (DQA1 and DQB1, respectively) contain polymorphic regions. We can identify DQA1 and DQB1 epitopes by DQ single antigen beads assay of the antibodies, correlating the beads' reaction patterns with either DQA1 or DQB1 alleles. METHODS: Sera from 74 transplant patients and 35 mouse DQB1 monoclonal antibodies were tested with DQ single antigen beads for their DQ allelic and serological specificities. Epitopes were defined by amino acids shared by the positive antigens of the antibodies. Unique amino acids were identified as potential epitope sites by comparing the peptide sequences of all human leukocyte antigen class II alleles. For the absorption or elution, patient's serum sample was absorbed by a homozygous B-lymphoblast cell line of specific DQ typing, the eluted antibody then tested with single antigen beads to demonstrate that the antibody reacted to a single epitope shared by multiple DQ antigens. RESULTS: Three DQA1 and 15 DQB1 epitopes were identified. We found that 21 patients produced antibodies against one of the DQA1 epitopes; 27 patients produced antibodies against one of the DQB1 epitopes. CONCLUSION: The DQA1 and DQB1 epitopes identified here seem to be immunogenic and to elicit DQ antibodies. For the DQB1 epitopes, multiple DQ serological specificities that were detected in the serum of a transplant patient could be explained as a single donor-specific DQ antibody reacting to a mismatched DQ epitope of the donor. Ten examples are shown here.

A colorimetric assay for studying effector secretion through the bacterial type III secretion system.

We have devised a colorimetric method that monitors secretion of effector proteins into host cytoplasm through the bacterial type III secretion machinery. Here we used constructs of effectors fused with Bordetella adenylate cyclase as a reporter, but evaluated the effector translocation by quantifying cell viability, rather than by measuring the intracellular cAMP concentration. This is based on our findings that cells infected by a secretion-competent bacterium expressing the fusion protein lost their viability under our experimental conditions. Cell death was quantified using commercially available reagents and basic research equipment. An observation that cell death was potentiated when the infected cells were treated with 2-deoxyglucose and sodium azide suggests that the depletion of intracellular ATP is partly involved in the process. Using enteropathogenic Escherichia coli, we demonstrated that the method was applicable to at least three effectors of bacteria, Tir, EspF, and Map, and was useful for studying a secretion signal sequence for Tir. This technically simple and inexpensive method is a good alternative to the existing procedure for studying the mechanism by which effectors are secreted through the type III secretion system in a high-throughput format.

Frequency of MIC antibody in rejected renal transplant patients without HLA antibody.

Antibodies to MICA and MICB antigens were sought in the sera of 139 kidney transplant recipients. MICA*001, *002, *007, *008, and MICB*002 antigens were produced in Escherichia coli and then tested using enzyme-linked immunosorbent assay plates. Among 35 normal sera, 6% had MIC antibodies, and among 14 sera from pregnant women, 21% had MIC antibodies. Among 34 patients with functioning transplants with human leukocyte antigen (HLA) antibodies, 24% had MIC antibodies, and 19% of 32 patients without HLA antibodies had MIC antibodies. Among 46 patients who lost grafts with HLA antibodies, 26% had MIC antibodies, and among 27 failed patients without HLA antibodies, 37% had MIC antibodies. We conclude that antibodies to MIC are produced in the course of immunization by pregnancies and kidney transplants. They also occurred more frequently in rejected patients (30%) than in those with functioning grafts (21%).

Association of kidney transplant failure and antibodies against MICA.

Despite the progress in renal transplantation, acute rejection and graft failure still occur and chronic rejection continues to be the main problem in long-term allograft survival. Although kidney transplant rejection has been linked to anti-HLA antibodies, not all patients with failed kidney transplants have anti-HLA antibodies, indicating that other loci may be involved. Sera of 63 patients who experienced kidney rejection were compared against sera of 82 patients with functioning transplants. Sera were examined for IgG and IgM anti-HLA Class I and II antibodies. They were also tested by cytotoxicity against panels of 26 endothelial cell lines, 8 MHC class I chain-related gene A (MICA) recombinant cell lines, and 28 B lymphoblast cell lines. Among patients whose transplants failed, 65% had anti-HLA antibodies compared with 45% of those with functioning kidneys (p < 0.05). Similarly, among those whose transplants failed, 41% had anti-endothelial cell antibodies in contrast to 22% in functioning patients (p < 0.05). Among patients whose grafts failed, 52% had anti-MICA antibodies versus 21% of those with functioning grafts (p < 0.001). Eleven patients with failed grafts and 32 with functioning grafts were negative for all of the above. However, 6 of the former and 7 of the latter showed positive cytotoxicity against B lymphoblasts (p < 0.05). Taking all antibodies together, 92% of patients with graft failure had antibodies as opposed to 70% of patients with functioning grafts (p < 0.001). We postulate that antibodies against HLA, MICA, endothelial cells, and B lymphoblasts could be independently involved in the slow process of chronic graft failure.

Predictive value of HLA antibodies and serum creatinine in chronic rejection: results of a 2-year prospective trial.

In this large collaborative study, 2,231 transplanted patients with functioning kidneys were tested for HLA antibodies, then examined 2 years later for graft survival. Among 478 patients with antibodies, 15.1% failed in 2 years, compared to 6.8% failure in 1,753 patients without antibodies (P=0.00000002). Cytotoxicity testing correlated better with outcome than flow cytometry or ELISA testing on HLA coated beads, possibly because it detected non-HLA antigens. When the patients were further broken down into those with serum creatinine at the time of testing of 0.5-1.9, 4.4% of antibody patients failed at 2 years, compared to 4.3% of patients without antibodies. This 0.1% difference increased among patients with serum creatinine values of 2.0-2.9 to 17.9%, and among those with 3.0-3.9 to 16.3%. We conclude that HLA antibodies posttransplantation is predictive of subsequent graft failure, and its predictive value can be enhanced among patients with higher serum creatinine values.

Predicting operational tolerance in pediatric living-donor liver transplantation by absence of HLA antibodies.

BACKGROUND: The role of anti-human leukocyte antigen (HLA) antibodies in operational tolerance (OT) after pediatric living-donor liver transplantation (LDLT) remains inconclusive. We investigated whether the presence of HLA antibodies impeded the development of OT. METHODS: We retrospectively examined the prevalence of anti-HLA antibodies in pediatric LDLT recipients before transplantation and at 3 weeks after transplantation and analyzed the significance of those antibodies in relation to later OT. Forty pediatric LDLTs were performed between April 1996 and December 2000 and followed up through July 2011, with sera available for measurement of HLA antibodies. Seventeen patients achieved OT (mean follow-up, 4571.9±544.7 days) and 23 patients did not achieve OT (mean follow-up, 4532.0±425.4 days). Protocol liver biopsy was done for 14 OT patients and 16 non-OT patients. Their sera were tested for anti-HLA class I and II antibodies using the LABScreen single antigen beads test, in which a 1000 mean fluorescence value was considered positive. RESULTS: The prevalence of antibodies after transplantation in non-OT patients was higher than in OT patients (95.2% vs. 73.3%; P<0.001). The highest mean fluorescence intensity of antibodies was significantly higher in non-OT patients than in OT patients. The prevalence of HLA-B, HLA-C, HLA-DQ, and HLA-DR antibodies was significantly higher in non-OT patients than in OT patients. The highest mean fluorescence intensity of HLA-A, HLA-B, and HLA-DQ observed in non-OT patients was significantly higher than those in OT patients. CONCLUSIONS: In our study, posttransplantation HLA antibodies were associated with the future absence of OT. A prospective study with more patients is necessary to confirm the predictive value of HLA antibodies for OT.

Incidence and impact of de novo donor-specific alloantibody in primary renal allografts.

BACKGROUND: To date, limited information is available describing the incidence and impact of de novo donor-specific anti-human leukocyte antigen (HLA) antibodies (dnDSA) in the primary renal transplant patient. This report details the dnDSA incidence and actual 3-year post-dnDSA graft outcomes. METHODS: The study includes 189 consecutive nonsensitized, non-HLA-identical patients who received a primary kidney transplant between March 1999 and March 2006. Protocol testing for DSA via LABScreen single antigen beads (One Lambda) was done before transplantation and at 1, 3, 6, 9, and 12 months after transplantation then annually and when clinically indicated. RESULTS: Of 189 patients, 47 (25%) developed dnDSA within 10 years. The 5-year posttransplantation cumulative incidence was 20%, with the largest proportion of patients developing dnDSA in the first posttransplantation year (11%). Young patients (18-35 years old at transplantation), deceased-donor transplant recipients, pretransplantation HLA (non-DSA)-positive patients, and patients with a DQ mismatch were the most likely to develop dnDSA. From DSA appearance, 9% of patients lost their graft at 1 year. Actual 3-year death-censored post-dnDSA graft loss was 24%. CONCLUSION: We conclude that 11% of the patients without detectable DSA at transplantation will have detectable DSA at 1 year, and over the next 4 years, the incidence of dnDSA will increase to 20%. After dnDSA development, 24% of the patients will fail within 3 years. Given these findings, future trials are warranted to determine if treatment of dnDSA-positive patients can prevent allograft failure.

The role of immunoglobulin-G subclasses and C1q in de novo HLA-DQ donor-specific antibody kidney transplantation outcomes.

BACKGROUND: Anti-HLA-DQ antibodies are the predominant HLA class II donor-specific antibodies (DSAs) after transplantation. Recently, de novo DQ DSA has been associated with worse allograft outcomes. The aim of this study was to determine the further complement-binding characteristics of the most harmful DQ DSA. METHODS: Single-antigen bead technology was used to screen 284 primary kidney transplant recipients for the presence of posttransplantation DQ DSA. Peak DSA sera of 34 recipients with only de novo DQ DSA and of 20 recipients with de novo DQ plus other DSAs were further analyzed by a modified single-antigen bead assay using immunoglobulin (Ig)-G subclass-specific reporter antibodies and a C1q-binding assay. RESULTS: Compared with recipients who did not have DSA, those with de novo persistent DQ-only DSA and with de novo DQ plus other DSAs had more acute rejection (AR) episodes (22%, P=0.005; and 36%, P=0.0009), increased risk of allograft loss (hazards ratio, 3.7, P=0.03; and hazards ratio, 11.4, P=0.001), and a lower 5-year allograft survival. De novo DQ-only recipients with AR had more IgG1/IgG3 combination and C1q-binding antibodies (51%, P=0.01; and 63%, P=0.001) than patients with no AR. Furthermore, the presence of C1q-binding de novo DQ DSA was associated with a 30% lower 5-year allograft survival (P=0.003). CONCLUSIONS: The presence of de novo persistent, complement-binding DQ DSA negatively impacts kidney allograft outcomes. Therefore, early posttransplantation detection, monitoring, and removal of complement-binding DQ might be crucial for improving long-term kidney transplantation outcomes.

Factors affecting outcomes of liver transplantation: an analysis of OPTN/UNOS database.

This was a historic cohort analysis based on 110,521 patients who underwent liver transplant between 1987 and July 2011 in the United States and were reported to the UNOS registry. In addition to univariate Kaplan-Meier survival analyses, we used cox proportional hazard analysis and multiple logistic regression analysis to evaluate hazard ratios adjusted for clinical factors. The overall 5- and 10-year patient survival rates were 81% and 72%, respectively, for 4,412 recipients of living donor livers and 73% and 59%, respectively, for 106,109 recipients of deceased donor livers. Multivariate analyses suggest that these differences are due to demographics, including patient age rather than differences due to the donor organs. Recipients of zero HLA-mismatched livers had significantly worst graft survival (HR 1.29, p = 0.02) compared with those given an HLA mismatched graft. This appears to be due in part to graft versus host disease. Among recipients who experienced GVHD, multivariate analysis revealed that zero mismatch of HLA-A (HR 2.75), zero mismatch of HLA-B (HR 4.79), recipient age > 65 (HR 2.57) and Asian recipient (HR 2.70) were significant risk factors for GVHD respectively.

A report of the epidemiology of de novo donor-specific anti-HLA antibodies (DSA) in "low-risk" renal transplant recipients.

The donor specific anti-HLA antibody (DSA) has been increasingly recognized as the major cause of allograft loss. Despite this, no published reports exist describing the true epidemiology of de novo DSA.Here we describe the epidemiology of DSA based on the results of one of the longest running antibody study in consecutive renal transplant recipients. The study includes 224 non-sensitized, non-HLA-identical patients who received a primary kidney transplant between 3/1999-3/2006. Protocol testing for DSA was done pre-transplant, at 1, 3, 6, 9, and 12 months, and then annually. DSA was tested using single antigen beads. Data from the East Carolina University transplant cohort indicate that the prevalence of DSA in the first year post-transplant is 12.1 cases per 100. The average annual incidence of DSA is 4.7 per 100 cases, per year. The highest incidence of DSA was in the first year post transplant. Although deceased donors and African-Americans have a higher incidence rate of DSA than the comparator living donors and non-African American groups, respectively, these factors were not associated with DSA onset. The one factor found to be predictive of DSA was DQ mismatch (p = 0.036). Based on these epidemiologic findings in combination with previous reports showing DSA is a cause of allograft failure, it seems reasonable that at least annual testing should be done even in "low-risk" transplant patients, because every year a new 5% of patients will develop DSA.

HLA-E monoclonal antibodies recognize shared peptide sequences on classical HLA class Ia: relevance to human natural HLA antibodies.

The non-classical HLA-Ib molecule, HLA-E share several peptide sequence similarities with the heavy chains of classical HLA class Ia (-B and -C) molecules. Therefore, the antibodies to HLA-E, that recognize shared sequences, may bind to HLA-Ia alleles. This hypothesis is tested by examining the affinity of HLA-E monoclonal antibodies (HLA-E-MAbs) to HLA-Ia molecules and by inhibiting the antibody binding to both HLA-E and HLA-Ia with the shared peptide sequence(s). Single recombinant HLA molecule-coated beads are used for antibody binding. The antibody binding is evaluated by measuring mean fluorescence index [MFI] with Luminex multiplex flow-cytometric technology. The peptide-inhibition experiments are carried out with synthetic shared peptides, most prevalent to HLA-E and HLA-Ia alleles. The number of HLA-Ia alleles recognized by the HLA-E-MAbs varies with the density of the antigen (quantity of antigen-coated beads) and dilution of MAb. Binding of HLA-E-MAbs to beta2 microglobulin (beta(2)m)-free HLA-Ia antigens confirms the location of the epitopes on the heavy chain (HC) of the antigens. Strikingly, the nature of alleles of HLA-Ia recognized by different HLA-E-MAbs is identical. The binding of HLA-E-MAbs to the HLA-Ia is inhibited dosimetrically by the adjacent peptides, (115)QFAYDGKDY(123) and (137)DTAAQI(142), but not by (126)LNEDLRSWTA(135), another closer shared peptide sequence. The inhibitory peptide sequences in HLA-E are at the alpha2-helix terminal facing beta(2)m. The HLA-Ia alleles recognized by HLA-E-MAb (e.g., MEM-E/02) are similar to those recognized by the natural anti-HLA antibodies found in the sera of healthy non-alloimmunized males. This study postulates that some, if not all, of the natural HLA-Ia antibodies seen in healthy males could be anti-HLA-E antibodies cross-reacting with HLA-Ia alleles.

No occurrence of de novo HLA antibodies in patients with early corticosteroid withdrawal in a 5-year prospective randomized study.

The purpose of this study was to determine the effect of early corticosteroid cessation on the occurrence of de novo human leukocyte antigen (HLA) antibody posttransplant. Renal transplant recipients (n=37) were randomized to early corticosteroid withdrawal at day 7 posttransplant (n=21 patients), or to chronic steroids (n=16), all in combination with thymoglobulin as induction agent, tacrolimus and mycophenolic acid as maintenance therapy. To establish the time course of HLA antibody appearance, sera collected pretransplant and for up to 5 years posttransplant were screened for the appearance of HLA antibodies. In this 5-year longitudinal study, only one patient in the control group developed a de novo donor-specific HLA antibody. We conclude that renal transplant recipients on steroid withdrawal by the end of week 1 are not at higher risk for developing HLA antibodies compared with a standard steroid regimen up to 5 years posttransplant.

Reappraisal of HLA antibody analysis and crossmatching in kidney transplantation.

It has been established that preformed IgG antibodies specific for donor HLA antigens may accelerate graft failure. An increasing number of studies have demonstrated adverse graft survival in patients who have anti-HLA antibodies, whether preformed or developed post-transplant. More recently, ELISA and flow cytometric techniques were introduced to overcome the limited sensitivity and specificity of the CDC assay. These emerging approaches can be reliably used to predict crossmatches in highly sensitized patients and also to monitor the development of clinically relevant anti-HLA antibody after transplantation. This retrospective study used LAT-M screening and Luminex HLA class I and II specificity assay to re-examine: (a), the impact of pre-transplant HLA antibody on long term graft survival; (b), the accuracy with which detection of HLA antibody and specificity by ELISA predicts pretransplant CDC crossmatch; (c), a comparison of Luminex and ELISA methods in detecting HLA antibodies. In this study, pre-transplant sera from 288 renal patients followed up at NCKUH were tested by the ELISA method, LAT-M. The tests showed that 19% had HLA antibodies before transplantation. Among the 234 of the patients who did not have pre-transplant antibodies, 85% enjoyed 5-year functional graft survival, 76% 10-year, and 56% 15-year functional graft survival. The corresponding functional graft survival for the 54 patients who tested HLA antibody-positive was 65%, 53% and 28% (P=0.0021). Sera from 481 patients awaiting kidney transplantation at NCKUH were tested by the ELISA method LAT-M and by CDC to determine how well HLA antibodies detected by ELISA predict the crossmatches shown by CDC. HLA antibodies found by ELISA ranged from 24% weak reactivity (OD "2") to 17% strongly reactive (OD "8"). The positive predictive value (PPV) of ELISA-detected antibodies for positive CDC crossmatch at the time of transplant was found to be 43-54%. The negative predictive value (NPV)-ELISA found no antibodies, CDC no crossmatches- was 88%. The PPV was 55% for sera with HLA class I DSA and 67% with HLA class II sera. On the other hand, NPV was 84% with sera negative for HLA class I DSA and 86% with sera negative for HLA class II DSA. Pretransplant sera from 48 patients with followup data at NCKUH were retested by both ELISA LAT-M and Luminex in order to compare the efficacy of those two methods. ELISA found pre-transplant HLA antibodies in 8 of the 48 (17%). Luminex found HLA antibodies in 27 (56%). Functional graft survival at 5, 10 and 15 years was not significantly different between the 27 patients whom Luminex identified as having pre-transplant HLA antibodies and the 21 patients Luminex found to be free of those antibodies (P=0.7197). For patients shown by Luminex to have pre-transplant class II DSA (N=8), functional graft survival was significantly lower than for those Luminex showed negative for HLA antibodies (P=0.0036). The concept of virtual XM relies on accurate HLA typing and thorough evaluation of HLA antibodies by solid-phase assays. While a negative virtual XM proved to be very reliable to rule out the presence of donor-specific HLA antibodies, it becomes more a concern whether all HLA antibodies detected by flow-beads are in fact clinically relevant. The virtual XM approach-in which antibodies are characterized by solid-phase assays prior to crossmatching-was reported to predict a negative flow XM in greater than 90% of cases. The predictive value for a correct CDC XM, however, was only 75%. A potential disadvantage of the virtual XM approach is that transplants may be excluded based on antibody results with unknown clinical relevance. Based on our results, we believe HLA antibody identification using ELISA still has a role in predicting long term graft survival and negative predictive value for CDC crossmatch before transplantation. Further analysis of HLA antibody, using Luminex, will be done to compare with present data.

HLA class II DP epitopes.

Both alpha (DPA1) and beta (DPB1) peptide chains of class II HLA-DP heterodimers contain polymorphic regions. Five DPB1 epitopes, for which we propose numbers #4001-4005, were identified by the reaction patterns ofthe DPB1 antibodies to DPB1 alleles of DP single antigen beads. Epitopes were defined by unique amino acids shared by the positive antigens of theantibodies. The unique amino acids for #4001 are DEAV at positions 84-87; for #4002, they are DED at positions 55-57; for #4003, DEE at positions 55-57; for #4004, Q at position 33; for #4005, the unique amino acids may be GGPM at positions 84-87. An antibody to epitope #4001-shared by DPB1*01, 03, 05, 09, 10, 11, 13, 14, 17, 19- could be absorbed by the cell line of DPB1*01 typing. This eluted antibody was reactive to all the DPB1 #4001 specificities. So this test demonstrated that a single antibody was reactive to the epitope shared by multiple antigens.

Epitopes of the HLA-A, B, C, DR, DQ and MICA antigens.

The intent for this chapter was to summarize the HLA epitopes that have been defined by adsorption and elution of antibodies from single HLA antigens to date. Examples of reactions of mAb and eluted allosera with the class I, class II and MICA SA are also presented. We have identified 103 HLA class I epitopes, of which 40 were defined by mAbs and 63 by alloantibodies mostly eluted from rHLA class I single antigen cell lines. We identified 32 epitopes shared by A-locus antigens, 43 epitopes shared by B-locus antigens, 4 epitopes shared by C-locus antigens, 16 inter-locus epitopes shared by A-B loci antigens, 5 inter-locus epitopes shared by B-C loci antigens and 3 inter-locus epitopes shared by A-B-C-locus antigens. Sixty HLA-DR epitopes have been defined, most by one aa residue on the HLA DR beta chain. However, as is the case with Class I epitopes, some DR epitopes are defined by one or more alternative residue(s). Eighteen HLA-DQ epitopes have been identified on the HLA-DQB chain and on the HLA-DQA chain of HLA-DQ antigens. Most DQ epitopes were defined by one aa residue. However, almost half are characterized by several alternative combinations of aa residues. Only a few DQA epitopes have been identified. Seven MICA epitopes have been defined to date. All are defined by a single aa residue.

Enteropathogenic Escherichia coli, Shigella flexneri, and Listeria monocytogenes recruit a junctional protein, zonula occludens-1, to actin tails and pedestals.

Enteropathogenic Escherichia coli, Shigella flexneri, and Listeria monocytogenes induce localized actin polymerization at the cytoplasmic face of the plasma membrane or within the host cytoplasm, creating unique actin-rich structures termed pedestals or actin tails. The process is known to be mediated by the actin-related protein 2 and 3 (Arp2/3) complex, which in these cases acts downstream of neural Wiskott-Aldrich syndrome protein (N-WASP) or of a listerial functional homolog of WASP family proteins. Here, we show that zonula occludens-1 (ZO-1), a protein in the tight junctions of polarized epithelial cells, is recruited to actin tails and pedestals. Immunocytochemical analysis revealed that ZO-1 was stained most in the distal part of the actin-rich structures, and the incorporation was mediated by the proline-rich region of the ZO-1 molecule. The direct clustering of membrane-targeted Nck, which is known to activate the N-WASP-Arp2/3 pathway, triggered the formation of the ZO-1-associated actin tails. The results suggest that the activation of the Arp2/3 complex downstream of N-WASP or a WASP-related molecule is a key to the formation of the particular actin-rich structures that bind with ZO-1. We propose that an analysis of the recruitment on a molecular basis will lead to an understanding of how ZO-1 recognizes a distinctive actin-rich structure under pathophysiological conditions.

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT), one of the virulence factors produced by the bacteria, exerts its toxicity by up-regulating various signaling cascades downstream of the heterotrimeric GTPases Gq and G12/13 in an unknown fashion. Here, we present the crystal structure of the C-terminal region (residues 575-1,285) of PMT, which carries an intracellularly active moiety. The overall structure of C-terminal region of PMT displays a Trojan horse-like shape, composed of three domains with a "feet"-,"body"-, and "head"-type arrangement, which were designated C1, C2, and C3 from the N to the C terminus, respectively. The C1 domain, showing marked similarity in steric structure to the N-terminal domain of Clostridium difficile toxin B, was found to lead the toxin molecule to the plasma membrane. The C3 domain possesses the Cys-His-Asp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying Cys-His-Asp. PMT toxicities on target cells were completely abrogated when one of the amino acids constituting the triad was mutated. Our results indicate that PMT is an enzyme toxin carrying the cysteine protease-like catalytic triad dependent on the redox state and functions on the cytoplasmic face of the plasma membrane of target cells.

Retrospective antibody analysis of thirty patients with kidney graft failure.

Out of 30 patients who rejected their kidney grafts, HLA or MICA antibodies were found in 26 patients. Among the four patients in whom antibodies could not be found, two patients did not have any HLA mismatch with their mother donor, and 1 had died with a functioning graft. There was only one other patient who chronically rejected but did not have antibodies. The antibodies were shown to appear before rejection of the grafts, and before the increase in serum creatinine. In most instances, the de novo antibodies increased in strength with time. Donor specific antibodies were found in 12 patients, two had non-donor-specific antibodies. In the absence of other antibodies, six had DQ antibodies and six had MICA antibodies, and one had DP antibodies.

Clinical and anti-HLA antibody profile of nine renal transplant recipients with failed grafts: donor-specific and non-donor-specific antibody development.

This study applied the single antigen microsphere technology to the retrospective analysis of sequential post-transplant serum samples in the context of the patient's clinical course. Detailed information on nine of the study patients was presented as representative of the larger cohort and illustrative of different patterns of anti-HLA antibody development and different clinical scenarios that culminated in graft failure. Our major observations are summarized as follows: 1. These data confirm the high sensitivity of the single antigen bead method: In some patients, DSA and NDSA that were undetected by standard methods were found pre-transplant and in sequential post-transplant samples. 2. The precise role that anti-HLA antibody plays in a particular rejection are complicated in cases in which humoral rejection is not diagnosed in the biopsy: The possible involvement of ADCC and mechanisms involving an indirect role for antibody in the rejection process should be carefully investigated. 3. Although anti-HLA antibodies are associated with graft rejection, the time interval between detection and rejection can vary dramatically between patients. Both DSA and NDSA can be adsorbed by the graft and erratically detected in the circulation, in some cases remaining undetected until nephrectomy. 4. Anti-HLA antibody strengths often fluctuate widely over a patient's clinical course, with de novo DSA generally of greater strength than de novo NDSA. 5. In addition to DSA, we have observed the consistent induction of diverse, cross-reactive NDSA. This occurs not only during the post-transplant course but also after graft failure, when immunosuppression is tapered prior to nephrectomy. Our data support further studies to evaluate the value of prospective monitoring of anti-HLA antibodies to better understand the place of anti-HLA antibodies in acute rejection. This may improve our ability to reverse some acute rejection episodes. Since acute rejection has been considered a predictor of late graft loss via chronic allograft nephropathy, understanding and modifying the antibody response is critical to extending the longevity of transplanted organs. Finally, since the strong sensitization to NDSA will seriously hamper the ability to identify a compatible donor for a future transplant, these data reinforce the importance of minimizing HLA mismatches between the donor and the recipient.