Joel R. Ross

Evidence that the fate of class II-disparate corneal grafts is determined by the timing of class II expression.

While the immune privilege nature of the eye affords significant protection to corneal grafts, immunologic rejection is the leading cause of graft failure. Class II antigens are normally absent or expressed at very low levels in normal cornea. The role that class II antigens play in causing graft rejection is particularly interesting. Class II molecules are present on many cells of the immune system, and therefore are important in modulating and mediating immune reactions. The absence of class II bearing cells in the cornea leads to the interesting issue of whether or not those antigens can induce immune rejection of a corneal graft. In the current study we have made use of a pair of congenic rat strains that differ only at class II loci to determine the impact of these antigens on corneal graft survival. Central corneal grafts were not rejected. Recipients preimmunized with skin grafts rejected 100% of the class II disparate corneas with a median survival time (MST) of 15.5 days. When class II disparate Langerhans-cell-containing (LC+) corneas were grafted, the corneas were not rejected, but they immunized the recipients as evidenced by the rejection of a second corneal graft on the contralateral eye. Immunofluorescent stains demonstrated transient expression of class II antigens on graft epithelium after transplantation. This temporary appearance of class II provides a target for rejection in the preimmunized animals but is of insufficient duration to both prime naive animals and provide a target for antigraft effectors.

The differential effects of donor versus host Langerhans cells in the rejection of MHC-matched corneal allografts

The fate of MHC-identical, multiple minor H—disparate corneal grafts was examined in the rat. Although skin grafts exchanged between LEW and F344 rats were invariably rejected, only 26% of the corresponding corneal grafts underwent rejection. The immunologic privilege of the minor H-disparate corneal grafts was due, at least in part, to the absence of donor-derived Langerhans cells. Corneal grafts were normally devoid of donor-derived Langerhans cells; however, grafts pretreated with latex beads became infiltrated with donor-derived Langerhans cells and were rejected by 59% of the naive minor H—compatible recipients. By contrast, untreated LEW corneal grafts underwent rejection in 26% of the naive F344 hosts even though the grafts became heavily infiltrated with host-derived Langerhans cells. The immunologic privilege of minor H—disparate corneal grafts was not the result of efferent blockade or suppression of the immune response. F344 hosts bearing long-term surviving LEW corneal allografts were challenged with LEW skin grafts. In all cases, orthotopic skin grafts were rejected acutely. Moreover, all previously clear corneal grafts underwent rejection following skin graft rejection. Thus, the unique absence of donor-derived Ia+ passenger cells and the avascular graft bed conspire to provide the primary minor H-disparate corneal graft with an immunologic privilege not shared by other organ grafts.

Prevention of the induction of allospecific cytotoxic T lymphocyte and delayed-type hypersensitivity responses by ultraviolet irradiation of corneal allografts.

The effect of ultraviolet radiation (UVR) on the immunogenicity of corneal allografts was examined in a mouse model. Corneal allografts differing from the host at the entire MHC and multiple minor H loci were subjected to 200 mJ/cm2 of UVB irradiation immediately prior to heterotropic transplantation. Analysis of cytotoxic T lymphocyte and delayed-type hypersensitivity responses revealed that UVR treated corneal grafts failed to induce either CTL or DTH responses in C57BL/6 recipients. UVB treatment abolished the immunogenicity of highly immunogenic corneal grafts containing either resident or infiltrating donor-specific Langerhans cells. Sequential grafting experiments demonstrated that UVB-treated grafts rendered the hosts anergic to subsequent immunization with highly immunogenic corneal limbus grafts that contained dense concentrations of Ia+ Langerhans cells of donor origin. The results indicate that UV treatment not only reduces the immunogenicity of the corneal allograft but may also render it tolerogenic.

A translatable predictor of human radiation exposure.

Terrorism using radiological dirty bombs or improvised nuclear devices is recognized as a major threat to both public health and national security. In the event of a radiological or nuclear disaster, rapid and accurate biodosimetry of thousands of potentially affected individuals will be essential for effective medical management to occur. Currently, health care providers lack an accurate, high-throughput biodosimetric assay which is suitable for the triage of large numbers of radiation injury victims. Here, we describe the development of a biodosimetric assay based on the analysis of irradiated mice, ex vivo-irradiated human peripheral blood (PB) and humans treated with total body irradiation (TBI). Interestingly, a gene expression profile developed via analysis of murine PB radiation response alone was inaccurate in predicting human radiation injury. In contrast, generation of a gene expression profile which incorporated data from ex vivo irradiated human PB and human TBI patients yielded an 18-gene radiation classifier which was highly accurate at predicting human radiation status and discriminating medically relevant radiation dose levels in human samples. Although the patient population was relatively small, the accuracy of this classifier in discriminating radiation dose levels in human TBI patients was not substantially confounded by gender, diagnosis or prior exposure to chemotherapy. We have further incorporated genes from this human radiation signature into a rapid and high-throughput chemical ligation-dependent probe amplification assay (CLPA) which was able to discriminate radiation dose levels in a pilot study of ex vivo irradiated human blood and samples from human TBI patients. Our results illustrate the potential for translation of a human genetic signature for the diagnosis of human radiation exposure and suggest the basis for further testing of CLPA as a candidate biodosimetric assay.

Class I disparate corneal grafts enjoy afferent but not efferent blockade of the immune response

Class I antigens are normally expressed on cells in all three layers of the cornea. In congenic rats that differ only at the single Class I locus RT1 A, central orthotopic corneal grafts were rejected 18% of the time with a mean survival time (MST) of 11.5 days. Pre-immunized recipients always rejected Class I disparate corneal grafts (100%, MST = 13.3 days). Surprisingly, the presence of donor Langerhans cells in the cornea at the time of grafting did not increase the rejection of grafts (20%, MST = 14.0 days). To determine if long term surviving grafts enjoyed immune priviledged in the form of efferent blockade, the recipients were challenged with skin grafts 4 to 6 weeks following corneal transplantation. All of the corneal grafts underwent rejection (100%, MST = 14.7 days). A number of important conclusions may be drawn from these studies. A single Class I mismatch is a weak barrier to successful engraftment of corneal grafts. However if the recipient has previously been exposed to donor antigens, a single Class I disparity is sufficient to provoke rejection of all subsequent corneal grafts. The susceptibility of long term surviving grafts to rejection induced by skin grafts indicates the orthotopic corneal grafts are antigenic but not immunogenic.

A clinical model of dermal wound angiogenesis

Full‐thickness dermal biopsies were performed in healthy volunteers to establish the range of angiogenic responses in wound healing in a normal population. Four‐millimeter punch biopsies were made in the forearms of 15 healthy volunteers. Each wound was evaluated microscopically 4–5 times per week for 2 weeks. A semiquantitative wound scoring system to evaluate the neovasculature at the wound periphery was investigated. A vascular score was calculated for each wound at each observation. Two independent observers analyzed the microscopic wound images using the scoring system. At the end of the 14‐day period, repeat biopsies were performed on some of the volunteers, and the granulation tissue was stained with anti‐CD31. The Kaplan‐Meier method was used to estimate the distribution of the time to reach predetermined target average vascular scores. A mixed‐effects regression model indicated that time, age, and observer were predictors for the average vascular score outcome. The pattern and time course for wound neovascularization was highly reproducible in this group of healthy volunteers, and the assay was feasible and well tolerated. This wound angiogenesis model may be useful for monitoring the effects of antiangiogenic agents on normal wound neovascularization. (WOUND REP REG 2003;11:306–313)

Radiation Injury Treatment Network (RITN): Healthcare professionals preparing for a mass casualty radiological or nuclear incident

Purpose: To describe the history, composition, and activities of the Radiation Injury Treatment Network (RITN). The Radiation Injury Treatment Network® is a cooperative effort of the National Marrow Donor Program and the American Society for Blood and Marrow Transplantation. The goals of RITN are to educate hematologists, oncologists, and stem cell transplant practitioners about their potential involvement in the response to a radiation incident and provide treatment expertise. Injuries to the marrow system readily occur when a victim is exposed to ionising radiation. This focus therefore leverages the expertise of these specialists who are accustomed to providing the intensive supportive care required by patients with a suppressed marrow function. Following a radiological incident, RITN centres may be asked to: Accept patient transfers to their institutions; provide treatment expertise to practitioners caring for victims at other centres; travel to other centres to provide medical expertise; or provide data on victims treated at their centres. Moving forward, it is crucial that we develop a coordinated interdisciplinary approach in planning for and responding to radiological and nuclear incidents. The ongoing efforts of radiation biologists, radiation oncologists, and health physicists can and should complement the efforts of RITN and government agencies. Conclusion: RITN serves as a vital partner in preparedness and response efforts for potential radiological and nuclear incidents.

Hematopoietic cell infusion for the treatment of nuclear disaster victims: New data from the Chernobyl accident

Purpose: To present previously unavailable data on the use of stem cell administration to aid recovery of victims of the Chernobyl disaster. On 26 April 1986, an accident at Unit 4 of the Chernobyl Nuclear Power Plant took place during the planned test of one of the safety systems. The diagnosis of acute radiation syndrome (ARS) was confirmed in 134 individuals exposed to high levels of radiation. There were nine patients heretofore unreported in the scientific literature who underwent intraosseous injections of allogeneic bone marrow cells in Kyiv. Conclusions: Transplantation was associated with significantly shortened time to recovery of granulocyte and platelet counts in these patients. While current guidelines would certainly include the use of cytokines, these data provide an indication of the effectiveness of stem cell transplant to treat victims of radiation exposure.

Histopathologic features of rejecting orthotopic corneal xenografts.

Discordant xenogeneic Hartley guinea pig corneal buttons were transplanted orthotopically to either naive or pre-immune Lewis rats. Recipients were sacrificed serially and grafts were immediately frozen and subsequently examined using standard immunohistologic techniques. Corneal xenografts remained clear in naive recipients for 7 days, at which point they rapidly became opaque and edematous. In pre-immunized recipients, corneal xenografts were rejected much more quickly, becoming opaque and edematous by day 3 post-transplantation. Histologic examination of grafts revealed severe stromal edema and diffuse inflammatory cell infiltrates composed of mononuclear cells and neutrophils. Infiltrates were present as early as day 2 in xenografts from both presensitized and naive recipients. The infiltrates were densest in the posterior half of the grafts with fewer cells penetrating into the epithelium. Immunoperoxidase staining confirmed the presence of OX-19+ T cells as well as a substantial infiltrate of OX-42+ neutrophils/macrophages. Additionally, IgG was deposited throughout the grafts in a diffuse manner. Deposition of IgG was accelerated in presensitized recipients, with intense staining of the entire graft detected by day 2. Examination of the rejected grafts suggests that rejection occurs via mechanisms similar to those seen in corneal allografts. This, in turn, implies corneal xenografts may be amenable to standard immunosuppressive regimens.

Acceptance of H-Y-disparate corneal grafts despite concomitant immunization of the recipient

Male-specific, H-Y antigen is a widely utilized antigen system for analyzing the role of non-MHC transplantation antigens in graft rejection. In this study, we examined the role of H-Y antigen in corneal graft rejection. Orthotopic corneal grafts from male LBNF1 rats were transplanted to syngeneic female LBNF1 recipients. The male corneal grafts survived beyond 100 days on naive female recipients. In other experiments, hosts bearing clear male corneal grafts and systemically immunized with subcutaneous inoculations of male splenocytes followed by full-thickness male skin grafts failed to reject their corneal grafts, even though the male skin grafts were swiftly rejected. The inability of female hosts to reject existing male corneal grafts suggested that the cornea failed to express H-Y transplantation antigen. Further experiments, however, revealed that male-specific antigen was expressed on corneal grafts. Hosts bearing clear male grafts in the left eye rejected subsequent male skin grafts and promptly rejected male corneal grafts transplanted to the contralateral eye. Interestingly, the original male corneal grafts remained clear during the rejection of both the skin graft and the second corneal graft. The results indicate that corneal grafts representing minor histocompatibility disparities enjoy immunologic privilege in the naive host, even if the host is subsequently immunized systemically.