<\/p>\n
Original Article<\/p>\n
Medical Student: Andres X. Crowley<\/p>\n
Mentor: Kenneth A. Andreoni MD<\/p>\n
Specific Aim: There is a growing observation in the renal transplant literature that the<\/p>\n
appearance of anti-HLA (Human Leukocyte Antigen) antibodies, especially donor<\/p>\n
specific anti-HLA antibodies (DSAs), after solid organ transplantation, correlates with<\/p>\n
rapid loss of renal graft function. We and others hypothesize that upon kidney<\/p>\n
transplantation, some patients generate donor-specific anti-HLA antibodies (DSAs).<\/p>\n
These antibodies may bind to donor tissue and create organ dysfunction through<\/p>\n
activation of complement. The complement system may either act alone to damage tissue<\/p>\n
directly or recruit neutrophils that release proteolytic enzymes causing allograft injury.<\/p>\n
To test this hypothesis, this study will 1) investigate the isotypes and subclasses of human<\/p>\n
immunoglobulin (DSA) found in the serum of patients with clinically and pathologically<\/p>\n
defined acute humoral rejections; 2) assess DSA activation of complement pathways<\/p>\n
(both classical and alternative) and neutrophil activation (note: part 2 of the original<\/p>\n
proposal was not completed due to time restraints when this draft of the results was<\/p>\n
submitted)<\/p>\n
Background and Significance: Both Acute and Chronic Humoral Rejection are now<\/p>\n
accepted as major contributors to renal and extra-renal allograft loss. Graft injury<\/p>\n
mediated by antibodies is clinically important during the early as well as late course post-<\/p>\n
transplantation. The improving ability of immunosuppressive medications to decrease<\/p>\n
the incidence and severity of acute cellular rejection has direct correlation to short-term<\/p>\n
success in organ transplantation. Improvements in long term graft survival have been<\/p>\n
more difficult to achieve1. “Chronic rejection” and “chronic renal allograft nephropathy”<\/p>\n
are terms for renal graft long-term injury leading to fibrosis and graft dysfunction from a<\/p>\n
combination of immunological and non-immunological causes. Acute and chronic,<\/p>\n
cellular and humoral injuries are all well described in renal allografts. It has been<\/p>\n
observed for many years that the combination of acute cellular and humoral rejection has<\/p>\n
dismal renal graft outcome. <\/p>\n
In a recent review, Dr. Terasaki cites over 33 publications that associate the presence of<\/p>\n
HLA antibodies with acute and chronic rejection in kidney, heart, lung, pancreas, and<\/p>\n
liver grafts. The majority of studies have been in kidney graft recipients with a<\/p>\n
significant interest in highly sensitized cardiac recipients2. HLA antibodies of Class I and<\/p>\n
Class II origin that can be defined against the specific allograft of interest are termed<\/p>\n
“Donor Specific Antibodies”. Detailed investigation of the actual DSA induced injury in<\/p>\n
the renal tissue has not been thoroughly explored. Whether cellular injury results in a<\/p>\n
byproduct of alloantibody production, or whether HLA antibody production causes direct<\/p>\n
renal injury, such as by endothelial activation of other mechanisms, needs to be defined3-<\/p>\n
7. <\/p>\n
Since acute and chronic humoral rejections are associated with the presence of HLA<\/p>\n
antibodies in the serum, we will seek to determine whether theses DSAs cause injury to<\/p>\n
the allograft. We have obtained serum from renal transplant recipients after a clinically<\/p>\n
and pathologically documented acute humoral rejection episode. We will define the<\/p>\n
subclass and subtypes of these donor-specific HLA antibodies (DSAs), attempt to<\/p>\n
demonstrate binding of these antibodies to donor tissue, and create an in-vitro model to<\/p>\n
investigate whether these antibodies activate complement and lead to neutrophil<\/p>\n
infiltration and subsequent tissue injury. Results obtained from the proposed studies<\/p>\n
should advance our understanding of humoral rejection and may help us develop novel<\/p>\n
treatments for renal graft recipients undergoing antibody-mediated graft injury.<\/p>\n
Materials and Methods:<\/p>\n
Sources of patient sera: Serial recipient serum samples and protocol renal graft biopsies<\/p>\n
have been collected from patients with and without intravenous immunoglobulin (IVIG)<\/p>\n
treatment at UNC under IRB approved patient consent. In this set of initial experiments,<\/p>\n
the serum samples of 4 patients with clinically diagnosed acute humoral rejection were<\/p>\n
examined. Positive and negative controls were provided by One Lambda (Canoga Park,<\/p>\n
CA). <\/p>\n
Analysis of donor specific antibody (DSA) levels and IgG subclasses in the recipient<\/p>\n
sera: In order to determine the IgG subclass of the Donor Specific Antibodies present in<\/p>\n
each patient’s sera, the technique of flow cytometry was utilized. Sera samples from both<\/p>\n
pre- and post- allograft transplantation were initially analyzed using pooled FlowPRA<\/p>\n
HLA Class I and Class II Screening tests (One Lambda, Canoga Park, CA). Furthermore,<\/p>\n
mouse FITC-conjugated antibody fragments specific for human IgG total and IgG<\/p>\n
subclasses 1 through 4 (SouthernBiotech, Birmingham AL) were used instead of the<\/p>\n
FITC-conjugated anti-IgG-total provided by One Lambda. This initial set of assays<\/p>\n
allowed for a cost-effective approach to screen for the presence of IgG subclasses without<\/p>\n
having to determine the HLA specificity of each antibody. <\/p>\n
Once this set of screening assays was completed, it was possible to eliminate certain IgG<\/p>\n
subclasses from further analysis, and thus conserve serum and other key reagents. The<\/p>\n
post-transplant sera samples were then analyzed using FlowPRA Single Antigen Bead<\/p>\n
HLA Antibody Detection Tests for Class I and Class II. The appropriate FITC<\/p>\n
conjugated anti-IgG antibody fragments mentioned above were used again to determine<\/p>\n
the subclass of the Donor Specific Antibodies and the HLA-specific (non-Donor)<\/p>\n
antibodies in each serum sample. <\/p>\n
(The following descriptions of additional methods, which were listed in the original<\/p>\n
proposal, were not completed during this phase of the project due to time restraints but<\/p>\n
are currently being pursued in the laboratory of Dr. Zhi Lui at UNC-Chapel Hill:<\/p>\n
1) Detection and quantification of activated complement components in patients’ sera, 2)<\/p>\n
In vitro determination of complement and neutrophil activation using Donor Specific<\/p>\n
Antibodies from patient sera, 3) Immunohistological staining of kidney graft tissue<\/p>\n
sections.)<\/p>\n
Results and Discussion:<\/p>\n
Patient 1 Summary (M.H.): IgG-1 positive for anti-DR 1, 103, 4, 7, 9, 15, 51, 53 ( ? –<\/p>\n
HLA class II) and IgG-3 positive for anti DR 1, 4, 7, 9, 53 ( ? -HLA class II) in post-op<\/p>\n
serum sample dated 7\/1\/2004 (with thymoglobin removed). The presence of ? -HLA class<\/p>\n
I specific antibodies of subclasses IgG-1 and IgG-3 were also detected during the initial<\/p>\n
screening assays, but the specificities of these antibodies were not determined due to<\/p>\n
limited sample aliquots. We focused only on defining the ? -HLA class II antibody<\/p>\n
specificities and subclasses for this patient’s sera.<\/p>\n
Patient 2 Summary (J.W.): IgG-1 positive for anti-DQ 5,6,8,9 ( ? -HLA class II) in the<\/p>\n
post-op serum sample dated 10\/4\/2004. No IgG-3 antibodies were detected. No ? -HLA<\/p>\n
class I specific antibodies were detected in the initial screening assays.<\/p>\n
Patient 3 Summary (L.R.): IgG-1 positive for anti-DR 1, 103, 4, 9, 7, 10, 51, 53 ( ? –<\/p>\n
HLA class II) and IgG-3 positive for anti DR 1, 103, 7, 53 and DQ 5 ( ? -HLA class II) in<\/p>\n
post-op serum sample dated 3\/22\/2005. In the initial screening assays, there was a<\/p>\n
significant presence of ? -HLA class I specific antibody in both samples, but only of the<\/p>\n
subclass IgG-1. Therefore, the specificities of these antibodies were not analyzed since<\/p>\n
the subclass, which was the variable in question, was already determined.<\/p>\n
Patient 4 Summary (J.K.): IgG-1 positive for anti-A 3, 8(BW6) ( ? -HLA class I) and<\/p>\n
IgG-3 positive for anti A 3, 8(BW6) ( ? -HLA class I) in post-op serum sample dated<\/p>\n
9\/13\/2002. In the initial screening assays, we detected ? -HLA class II specific antibody<\/p>\n
in both pre-op and post-op serum samples, but only of subclass IgG-1. Therefore, the<\/p>\n
specificities of these antibodies were not analyzed since the subclass, which was the<\/p>\n
variable in question, was already determined.<\/p>\n
The results obtained from this series of experiments confirm what has been reported in a<\/p>\n
related study performed by Kushihata,et al.8. We determined that the predominant IgG<\/p>\n
subclass that is produced during acute humoral rejection of a renal allograft is IgG-1. <\/p>\n
The subclass IgG-3 is also present to a lesser degree in some of the samples, and there is<\/p>\n
no detectable trace of IgG-2 or IgG-4 specific for any HLA type in the samples<\/p>\n
examined. This study is unique compared to the experiments of Kushihata, et al. in that<\/p>\n
we determined the IgG subclass of DSA detected in the sera of patients clinically<\/p>\n
diagnosed with acute humoral rejection, as opposed to those of patients who had high<\/p>\n
PRA values (ie high levels of HLA specific antibodies), but were not recipients of renal<\/p>\n
allografts. This distinction is important because the presence of HLA specific antibodies<\/p>\n
in the serum can be due to a variety of causes other than allograft transplantation, such as<\/p>\n
pregnancy and blood transfusion. The common finding, however, is that regardless of the<\/p>\n
source of sensitizing antigen, HLA antigen (both class I and class II) stimulates the<\/p>\n
production of IgG-1 and IgG-3 antibody, but does not stimulate the production of IgG-2<\/p>\n
and IgG-4. <\/p>\n
The significance of this finding is related to the intrinsic properties of the different IgG<\/p>\n
subclasses. IgG-1 and IgG-3 are very effective with regards to complement activation,<\/p>\n
whereas IgG-2 is not as effective, and IgG-4 does not bind complement at all9. <\/p>\n
Considering the activation of complement may lead to tissue injury by both lytic and<\/p>\n
inflammatory pathways9, 10, it is not surprising that these patients experienced symptoms<\/p>\n
of acute graft dysfunction. <\/p>\n
References<\/p>\n
1. Meier-Kriesche HU SJ, Srinivas TR, Kaplan B. Lack of improvement in renal<\/p>\n
allograft survival despite a marked decrease in acute rejection rates over the most<\/p>\n
recent era. Am J Transplant. 2004;4:378-383.<\/p>\n
2. Teraski P. Humoral theory of transplantation. Am J Transplant. 2003(3):665-673.<\/p>\n
3. Bian H HP, Mulder A, Reed EF. Anti-HLA antibody ligation to HLA class I molecules<\/p>\n
expressed by endothelial cells stimulates tyrosine phosphorylation, inositol phosphate<\/p>\n
generation, and proliferation. Hum Immunol. 1997(53):90-97.<\/p>\n
4. Harris PE BH, Reed EF. Induction of high affinity fibroblast growth factor receptor<\/p>\n
expression and proliferation in human endothelial cells by anti-HLA antibodies: a<\/p>\n
possible mechanism for transplant atherosclerosis. J Immunol. 1997(159):5697-5704.<\/p>\n
5. Liu C WK, Shi C, Heyner S, Komm B, Haddad JG. Post-transcriptional stimulation of<\/p>\n
transforming growth factor beta 1 mRNA by TGF-beta 1 treatment of transformed human<\/p>\n
osteoblasts. J Bone Miner Res. 1996(11):211-217.<\/p>\n
6. McKenna RM TS, Terasaki PI. Anti-HLA antibodies after solid organ transplantation.<\/p>\n
Transplantation. 2000(69):319-326.<\/p>\n
7. Russell PS CC, Winn HJ, Colvin RB. Coronary atherosclerosis in transplanted mouse<\/p>\n
hearts. II. Importance of humoral immunity. J Immunol. 1994(152):5135-5141.<\/p>\n
8. F. Kushihata JW, A. Mulder, F. Claas, J.C. Scornik. Human Leukocyte Antigen<\/p>\n
Antibodies and Human Complement Activation: Role of IgG Subclass,<\/p>\n
Specificity, and Cytotoxic Potential. Transplantation. October 15, 2004<\/p>\n
2004;78(7):995-1001.<\/p>\n
9. Baldwin WM 3rd PS, Brauer RB, Daha MR, Sanfilippo F. Complement in organ<\/p>\n
transplantation. Contributions to inflammation, injury, and rejection.<\/p>\n
Transplantation. March 27, 1995 1995;59(6):797-808.<\/p>\n
10. Saadi S PJ. Endothelial cell responses to complement activation In: Volanakis JE,<\/p>\n
Frank MM, eds. The human complement system in health and disease. New York,<\/p>\n
Marcel Dekker. 998 1998:335.<\/p>\n","protected":false},"excerpt":{"rendered":"
Original Article Medical Student: Andres X. Crowley Mentor: Kenneth A. Andreoni MD Specific Aim: There is a growing observation in the renal transplant literature that the appearance of anti-HLA (Human Leukocyte Antigen) antibodies, especially donor specific anti-HLA antibodies (DSAs), after solid organ transplantation, correlates with rapid loss of renal graft function. We and others hypothesize … Read more<\/a><\/p>\n","protected":false},"author":80868,"featured_media":0,"parent":2236,"menu_order":5,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-2291","page","type-page","status-publish","hentry","odd"],"acf":[],"_links_to":[],"_links_to_target":[],"_links":{"self":[{"href":"https:\/\/www.med.unc.edu\/dms\/wp-json\/wp\/v2\/pages\/2291","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.med.unc.edu\/dms\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.med.unc.edu\/dms\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/dms\/wp-json\/wp\/v2\/users\/80868"}],"replies":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/dms\/wp-json\/wp\/v2\/comments?post=2291"}],"version-history":[{"count":0,"href":"https:\/\/www.med.unc.edu\/dms\/wp-json\/wp\/v2\/pages\/2291\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/dms\/wp-json\/wp\/v2\/pages\/2236"}],"wp:attachment":[{"href":"https:\/\/www.med.unc.edu\/dms\/wp-json\/wp\/v2\/media?parent=2291"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}