Passive Transfer Of Serum Research Articles

Plasmodium falciparum: Immune Pressure inSaimiri sciureusMonkeys Can Select for a Parasite Population Inducing a Protective Immunity That Is Not Controlled by Antibody

Morales-Betoulle, M. E., de la Salmonière, Y.-O. G., Zwetyenga, J., Le Scanf, C., Jouin, H., and Michel, J. C. 1998.Plasmodium falciparum: Immune pressure inSaimiri sciureusmonkeys can select for a parasite population inducing a protective immunity that is not controlled by antibody.Experimental Parasitology90, 49–57. Protective immunity against aPlasmodium falciparumblood infection can be passively transferred by antibodies in humans and in the primate experimental malaria modelSaimiri sciureus.We report here the emergence of a novel virulent parasite population after such passive transfer of hyperimmune serum in splenectomized monkeys. These FUP-2 parasites have been partially genotyped and phenotyped. Although no genotypic variation was detected for four polymorphic loci compared to the original FUP-1 parasite population, FUP-2-infected erythrocytes exhibit little or no detectable surface determinants, including those reacting with antibodies raised against FUP-1 surface antigens. In addition, FUP-2-infected erythrocytes exhibit no rosetting or autoagglutination. Interestingly, althoughSaimirimonkeys control efficiently FUP-2 parasites after repetitive infections, this protection cannot be passively transferred to naive recipients. Our results suggest that antibody-mediated and antibody-independent T-cell-mediated protective responses may cooperate in controllingP. falciparuminfection in splenectomizedSaimirimonkeys.

Resistance to IHN virus infection in rainbow trout is increased by glucan while subsequent production of serum neutralizing activity is decreased

Protection against the pathogenic effects of viral challenge can be achieved by passive transfer of sera from individuals that have survived a viral challenge (convalescent sera). However, individuals may differ in regard to the degree of benefit each derives from components of injected sera. We tested the hypothesis that glucan-induced potentiation of the innate immune system in rainbow troutOncorhynchus mykisswould enhance the ability of an individual to benefit from passively transferred convalescent sera. Fish pre-injected with glucan, then with convalescent sera from survivors of an IHNV challenge, were fully protected when exposed to the virus. Furthermore, glucan injections resulted in higher survival even without the benefit of convalescent serum injections. These effects were both time- and concentration-dependent, evident at 15 days but peaking at 3 weeks after a dose of <80μg glucan injected into rainbow trout weighing about 2·0g. In fish that survived IHNV challenge, high to moderate virus neutralization titres developed in sera only if the fish had received neither glucan nor convalescent serum. We speculate that efficient inactivation and/or clearance of virus by the innate immune system can preclude orchestration of the full potential of the acquired immune system.

Contribution of donor-specific antibodies to acute allograft rejection: evidence from B cell-deficient mice.

The role of T lymphocytes in acute allograft rejection is well established. The involvement of B lymphocytes in this process, however, is more controversial. A series of reports showed that mice without a functional B-cell compartment rejected allografts with the same kinetics as control animals. In rats, however, alloantibodies were found to play a decisive role in allograft rejection. To provide an explanation for the discrepant results, we readdressed the role of B cells and antibodies in mice with disrupted immunoglobulin mu chain genes. The use of cyclosporine (CsA), which strongly suppresses T cells, allowed us to focus specifically on the function of B cells. C57BL/6 mice rendered B cell deficient by targeted disruption of the immunoglobulin mu chain gene (referred to as microMT/microMT mice) and microMT/+ control mice with one functional mu chain were heterotopically transplanted with fully MHC-disparate BALB/c hearts. CsA was administered subcutaneously by Alzet osmotic pumps. Normal and immune serum specific for donor hearts was given to assess the role of antibodies in the rejection process. Both B cell-deficient microMT/microMT and heterozygous microMT/+ mice were found to reject transplanted hearts within a similar period of time. In contrast, when T cells were partially suppressed with CsA, graft survival was significantly prolonged in microMT/microMT mice as compared with heterozygous controls. Passive transfer of donor-specific immune serum, obtained from microMT/+ animals rejecting allogeneic hearts, to CsA-treated microMT/microMT mice significantly accelerated allograft rejection as opposed to recipients treated with normal serum. B lymphocytes and antibodies play an important role in acute allograft rejection particularly when the dominant T-cell compartment is partially suppressed.

Modification of Maternal CMV by Pre-infection α-gB Antibody Transfer in Guinea Pigs † 821

Inbred JY-9 strain guinea pigs (gp) were infected with gpCMV in early pregnancy. 7 pregnant dams were treated with pooled hyperimmune serum from non-infected animals previously immunized with glycoprotein B (gB), on days 0, 2 and 5, day 1 being the day of infection. 6 control pregnant dams received pooled non-immune(NI) serum as above. NI dams appeared sicker and had 83% pregnancy loss by in utero resorption and abortion compared to 14% in passively immunized (PI) dams(p<0.01). Mean placental weight was 4.3g in NI dams and 5.6g in PI dams(p=0.02). Mean pup weight was 9.8g in NI dams and 31.5g in PI dams(p<0.02). Mean pup length was 3.7cm in NI dams vs. 6.6cm in PI dams(p<0.02). All maternal blood cultures were positive on day 7 post-infection. On day 12-14, 83% of NI dams vs. 28% PI dams had positive blood cultures(p<0.05). Placental tissue cultures were positive in 80% of NI dams compared to 33% of PI dams(p<0.05). Pup tissue cultures were positive in 50% of NI dams compared to 25% of PI dams(NS). All placentae showed features of immaturity consistent with mid-term gestation, on H&E stains. NI placentae had marked lobular congestion with focal mononuclear cell infiltrates in the lobules and foci of necrosis with neutrophilic infiltrates in the trophoblastic septae. Less severe mononuclear cell infiltrates and no necrosis or neutrophilic infiltrates were seen in PI placentae. Frozen sections of PI placentae showed less disorganization of structure with decreased number of infected cells by immunoperoxidase stains. Passive transfer of anti-gB serum initiated 24 hours pre-infection does not prevent, but significantly modifies the effects of primary maternal CMV.

Contributions of Antibody and T Cell Subsets to Protection Elicited by Immunization with a Replication-Defective Mutant of Herpes Simplex Virus Type 1

Replication-defective mutants of herpes simplex virus 1 (HSV-1) elicit immune responses in mice that reduce acute and latent infection after corneal challenge and are protective against development of disease. To understand the basis for the protective immunity induced by this new form of immunization, we investigated the contribution of various components of the immune response to protection against corneal infection and disease. Passive transfer of sera from mice immunized with the replication-defective mutant virus,d301, its parental HSV-1 strain, or uninfected cell lysate was used to examine the role of antibody. Despite posttransfer neutralizing antibody titers equivalent to those in control mice directly immunized with mutant virus, recipients of immune serum showed no reductions in primary replication in the eye, keratitis, or latent infection of the nervous system. However, immune serum protected mice from encephalitis and death. To examine the contribution of T cell subsets to protection, mice were immunized once with mutant virus and then were depletedin vivoof CD4+or CD8+T cells prior to corneal challenge. CD4 depletion resulted in higher titers of challenge virus in the eye at 3 to 4 days after challenge compared to control mice. Latent infection of the nervous system was increased by depletion of CD4+T cells but not by depletion of CD8+T cells. Keratitis developed only in a portion of the CD8+T cell-depleted mice, suggesting that an immunopathologic potential of CD4+T cells is held in check when immune CD8+T cells are also present. Taken together, these data support a role for antibody induced by immunization with a replication-defective virus principally in protecting the central nervous system from disease, roles for CD4+T cells in reducing primary replication in the eye and protecting against latent infection of the nervous system, and a role for CD8+T cells in regulating the immunopathologic activity of CD4+T cells.

Activity of sera from patients with Lyme disease against Borrelia burgdorferi.

Sera from patients with Lyme disease were evaluated for their ability to kill Borrelia burgdorferi in vivo and in vitro. Separate groups of C3H mice received sera from seropositive humans with early- or late-stage Lyme disease or from seronegative controls. Eighteen to 24 hours after passive transfer of sera, the mice were challenged with 100,000 low-passage B. burgdorferi strain B31 or CA287 organisms. Sera from subjects with late-stage Lyme disease protected the mice against infection after challenge with B. burgdorferi, but sera from subjects with early-stage Lyme disease were not protective. Late-stage sera also inhibited the growth of B. burgdorferi in microcultures on Barbour-Stoenner-Kelly media better than early-stage sera. Immunoblot analysis revealed that the protective properties of late-stage sera were associated with a response of antibodies to multiple proteins. This response included strong reactivity with the outer-surface proteins A and B, which was lacking in early-stage sera.

Passive Transfer of Immune Serum Reduces the L1/Adult Ratio of Worms Recovered from the Intestines of Strongyloides stercoralis-Infected Gerbils

In Strongyloides stercoralis-infected gerbils it had been observed previously that the ratio of first-stage larvae to adult worms decreases after 3 wk of infection. Serum obtained from gerbils after this decrease in fecundity, but before worms were expelled, was transferred passively to other infected gerbils during the peak of worm fecundity. The immune serum caused a significant decrease in the L1/adult ratio but had no effect on the number of uterine eggs, the length, or the intestinal and ovarian ultrastructure of adult worms. Apparently a factor(s) in the immune serum either killed eggs, after oviposition, or the hatched larvae, without damaging the adult worms.

Antibody alone does not prevent experimental cytomegalovirus retinitis in mice with retrovirus-induced immunodeficiency (MAIDS).

Passive-transfer studies were performed to assess the ability of antibody alone to reduce the frequency and/or severity of necrotizing retinitis caused by murine cytomegalovirus (MCMV) in C57BL/6 mice with retrovirus-induced immunodeficiency syndrome (MAIDS). Initial experiments showed a gradual decline in the ability of mice to initiate humoral immunity during the evolution of MAIDS so that neither MCMV-specific IgM nor IgG could be detected during late-stage MAIDS. Passively administered hyperimmune MCMV immunoglobulin, however, could be detected within the serum of mice with MAIDS for at least 9 days after intraperitoneal injection and protected these animals in preliminary experiments from systemic MCMV disease and death when administered 24 h prior to intraperitoneal challenge with a lethal dose of virus. Nonetheless, passive transfer of hyperimmune MCMV serum to mice with MAIDS failed to reduce intraocular MCMV titers, frequency of retinitis, or severity of retinitis when administered 24 h prior to subretinal MCMV inoculation. Whereas whole eyes of MAIDS animals that received normal mouse serum and were injected subretinally with MCMV had an ocular MCMV titer of 4.3 log10 and a frequency of retinitis of 89% (severity score = 55%), whole eyes of antibody-treated mice with MAIDS had an ocular MCMV titer of 4.3 log10 and a frequency of retinitis of 87% (severity score = 57 %). Passive transfer of a neutralizing MCMV-specific monoclonal antibody also failed to reduce the frequency or severity of MCMV retinitis when administered to mice with MAIDS prior to subretinal MCMV inoculation. Our findings suggest that antibody immunotherapy alone will not be effective therapeutically for cytomegalovirus retinitis in patients with AIDS.

Protective immunization with plasmid DNA containing the outer surface lipoprotein A gene of Borrelia burgdorferi is independent of an eukaryotic promoter.

Plasmid DNA encoding the outer surface lipoprotein A (OspA) of Borrelia burgdorferi under the control of either strong eukaryotic/viral or its own bacterial promoter was injected intramuscularly (m. tibialis anterior) or intradermally into BALB/c and AKR/N mice. OspA-specific antibodies and OspA-reactive T helper 1 cells (Th1) were induced only with those plasmids containing the ospA structural gene including its own regulatory control region immediately upstream. In the absence of the ospA promoter, no or only marginal immune responses to OspA were obtained, even when strong eukaryotic promoter/enhancer elements were present. Together with the finding that the ospA promoter is active in a mouse B-lymphoma line, the data suggest that spirochetes are able to express at least part of their genes in the mammalian environment. Mice previously vaccinated with the relevant ospA plasmid DNA were protected against subsequent experimental challenge with a virulent strain of B. burgdorferi, as measured by the appearance of antibodies to a prominent protective epitope (LA-2) and the failure to re-isolate spirochetes from ear biopsies. In addition, C.B-17 severe-combined immunodeficient mice could be protected against infection by passive transfer of immune sera from ospA plasmid DNA-inoculated normal mice. Protective LA-2-related antibody titers obtained after repeated immunization persisted for 200 days and longer. This simple procedure of immunization using plasmid DNA consisting of a prokaryotic gene under the control of its own promoter holds great promise for the development of alternative subunit vaccines against bacterial infections, including Lyme disease. In addition, the availability of this novel prokaryotic promoter element now allows the study of the basis for the differential expression of bacterial genes in prokaryotic and eukaryotic environments.

Vaccination generates serum‐mediated protection against Onchocerca lienalis microfilariae in the mouse

Mice vaccinated with an aqueous extract of Onchocerca lienalis microfilariae (mf) plus adjuvant exhibited 51% protection against challenge with live mf. Protection was ablated by prior treatment of the extract with heat or proteinase K, indicating the involvement of proteinaceous epitope(s). A 54% level of protection was conferred on naive mice by passive transfer of serum from vaccinated donors, suggesting that host resistance was principally mediated by humoral components of the immune response. In contrast, the protection induced by sensitization of mice with living mf is transferable with cells, but not serum. Western blot data reveal different antigen recognition profiles for serum antibodies from these two groups of mice. These results indicate that vaccination and infection activate distinct protective mechanisms against Onchocerca mf in the mouse.

Isotype-specific antibody responses to the surface-exposed antigens of adult and larval stages of Dictyocaulus viviparus in infected and vaccinated calves

The antibody responses to the surface-exposed antigens of living larval and adult Dictyocaulus viviparus were measured by quantitative immunofluorescence using sera from calves infected with, or vaccinated against, the parasite. In infected animals, the surface of the sheath of the third-stage larvae (L 3) (retained cuticle of second-stage larvae (L 2)) proved highly immunogenic despite the fact that it is thought to be shed prior to parasite penetration of the host intestine. When responses to the surface of exsheathed larvae (L 3 cuticle) were measured, a high level of heterophile IgM antibody was detected in the serum of animals that had not been previously exposed to the parasite and, following infection, a specific IgG response was detected against the exsheathed L 3 surface. The antibody response, however, was less marked than that observed against the intact L 3 sheath. Responses of patently infected animals to the adult surface showed an initial IgM response that was superseded with time by IgG 1 and IgG 2 responses. Vaccinated animals showed only low level responses to the surfaces of the L 3 sheath, L 3 cuticle and adult stages following immunisation with two doses of irradiated larvae. The immunised animals produced a strong antibody response to the larval surface antigens following challenge with infective larvae but they failed to produce antibody to the surface of adult parasites. These results show that the surfaces of all the stages of D. viviparus examined are immunogenic in infected calves and, depending on the developmental stage, infection regime, or time of infection, high levels of parasite-specific IgG 1 or IgM are stimulated. It has previously been shown that significant levels of protective immunity can be obtained in naive animals following passive transfer of serum from infected calves. Thus, the antibody responses detected in the work reported here may be of relevance in protective immunity against dictyocaulosis.

Protection of mice against Schistosoma mansoni infection by passive transfer of sera from infected rabbits.

Sera from rabbits infected with unattenuated Schistosoma mansoni cercariae conferred significant levels of protection against S. mansoni challenge (P < 0.001) after passive transfer to mice. Infected rabbit sera were only effective in conferring protection when transferred during the first week of infection, and were not effective when administered against liver-stage worms. Immunoglobulins isolated from the infected rabbit sera with Protein A-Sepharose were shown to be responsible for the transfer of protection to mice. Immunofluorescence studies demonstrated that the sera were more reactive against the surface of three hour-old mechanically transformed schistosomula than against the surfaces of lung-stage schistosomula. The sera from infected rabbits reacted polyspecifically against antigens in cercaria, schistosomula, and the worm and egg stages of the S. mansoni life-cycle. The host parasite relationship of S. mansoni in the rabbit is discussed.

Neuroinvasive properties of herpes simplex virus type 1 glycoprotein variants are controlled by the immune response.

Neuroinvasiveness is a property central to the pathogenesis of herpes simplex virus (HSV) and most isolates demonstrate this property. Exceptions are HSV strains KOS and ANG, for which we have previously shown that the non-neuroinvasive phenotype is referable to single amino acid changes in glycoprotein D for ANG or glycoprotein B for KOS. Because glycoproteins B and D are immunologically significant, the possibility that the phenotype has an immunologic basis was examined. Nonimmunosuppressed mice could not be killed with any dose of these non-neuroinvasive viruses after footpad inoculation, but in cyclophosphamide-suppressed animals, the ratios of plaque-forming units to LD50 decreased by at least four orders of magnitude to levels comparable with that of ANG-path, a neuroinvasive derivative of ANG. KOS and ANG induced a more rapid circulating neutralizing Ab response than did ANG-path, and mice were protected when these agents were co-infected with the neuroinvasive strain. The noninvasive viruses engendered an enhanced mononuclear cell infiltrate in infected spinal ganglia which consisted of increased numbers of CD4+ and CD8+ T cells and an increased production and secretion of IgG. HSV-specific Ab-secreting cells were also observed. In addition, passive transfer of anti-HSV mouse serum protected immunosuppressed mice from lethal HSV challenge. Selective in vivo depletion of T lymphocytes increased the detectable levels of both KOS and ANG viruses in the spinal ganglia at 6 days postinfection, but it did not alter the ratios of plaque-forming units to LD50 or affect the HSV-induced increase in ganglionic IgG. Taken together, these data indicate that in these systems there is an immunologic basis for the control of HSV-1 neuroinvasiveness and that humoral, rather than cell-mediated immunity, is playing the major role.

Attempts to protect severe combined immunodeficient (scid) mice with antibody enriched for reactivity to Cryptosporidium parvum surface antigen-1

Cryptosporidium paruum is a protozoal pathogen which infects the gastrointestinal epithelium of mammals causing diarrhoea, the duration and severity of which is determined by the immunocompetency of the host. Currently, there is no effective treatment or prevention. We evaluated the ability of surface antigen-1 (SA-1), defined as those antigens recognized by neutralizing mAb 17.41, to elicit a protective antibody response when used as an immunogen. A SA-1 enriched fraction was obtained by immunoaffinity chromatography and was used to immunize a naive Holstein calf. SA-1 immune serum from this calf detected C. parvum epitopes to a 1:10 000 dilution in a dot blot assay, and sporozoite surface epitopes at a 1:10 000 dilution in a live immunofluorescence assay. Western blot analysis showed that SA-1 immune bovine serum recognized a similar pattern of C. parvum antigens as the defining mAb 17.41. Oral passive transfer of SA-1 immune bovine serum did not protect severe combined immunodeficient (scid) mice or suckling BALB /c mice from initial infection with C. paruum, or terminate a persistent infection in scid mice.

Immunization with viruslike particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection

We tested the ability of vaccination with virus-like particles (VLPs) to protect domestic rabbits against papillomas induced by the cottontail rabbit papillomavirus (CRPV). A recombinant baculovirus system that expressed only the L1 major papillomavirus structural protein or L1 plus the minor L2 protein was used in insect cells as the source of VLPs. Groups of 10 rabbits were immunized with native or denatured VLPs from CRPV or type 1 bovine papillomavirus by using Freund's adjuvant. Alum was used as the adjuvant for an additional group immunized with CRPV L1-L2 VLPs. Animals were challenged with 5 x 10(10) and 2 x 10(11) particles on opposing flanks. No protection was seen in rabbits immunized with native or denatured bovine papillomavirus L1-L2 or with denatured CRPV L1-L2. In these groups, the lower and higher challenge doses resulted in 27 of 30 animals with extensive papillomas, with each of the remaining animals having a smaller number of persistent papillomas. Progression to carcinoma developed in 20 rabbits. Animals inoculated with native CRPV VLPs composed of L1 alone or L1-L2 developed many fewer lesions; the lower and higher challenge doses resulted in 17 of 29 and 5 of 29 rabbits, respectively, with no lesions, and the remainder developed only one to eight papillomas, which all regressed except for those on 1 rabbit. None developed cancer within 1 year of infection. Rabbits vaccinated with native CRPV VLPs developed high-titer antibodies in an enzyme-linked immunosorbent assay based on native VLPs, and passive transfer of serum or immunoglobulin G from rabbits immunized with CRPV VLPs protected against CRPV challenge. We conclude that native VLPs can induce antibody-mediated, type-specific protection against experimental papillomavirus infection.

Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus.

The simian immunodeficiency virus (SIV) macaque model of AIDS has provided a valuable system with which to investigate vaccine approaches for protection against human immunodeficiency virus type 1 (HIV-1) infection. In particular, the ability of macaques persistently infected with attenuated infectious molecular clones of SIV to resist challenge with the pathogenic parental swarm has conclusively demonstrated that protective immunity can be achieved by immunization prior to exposure. The breadth of these protective responses and the immunological correlates of protection, however, have not been identified. In addition, vaccine studies have mainly employed lymphocyte-tropic strains of HIV-1 and SIV. Recent studies have implicated macrophage-tropic strains in the transmission of HIV-1 and have suggested that these virus strains should be examined in vaccine strategies. Macrophage-tropic viruses may confer additional advantages in the induction of protective immunity by replication in antigen-presenting cells. In this study, the immune response of rhesus macaques inoculated with an attenuated macrophage-tropic recombinant of SIVmac239 (SIV/17E-Cl) was evaluated with respect to protective immunity by heterologous challenge at various times after infection. Vigorous type-specific neutralizing-antibody responses restricted to SIV/17E-Cl were evident by 2 weeks postinfection. By 7 months, however, cross-reactive neutralizing antibodies emerged which neutralized not only SIV/17E-Cl but also the heterologous primary isolate SIV/DeltaB670. Challenge of SIV/17E-Cl-infected monkeys with SIV/DeltaB670 at various times postinfection demonstrated that protective responses were associated with the appearance of cross-reactive neutralizing antibodies. Furthermore, passive transfer of sera from SIV/17E-Cl-infected animals passively protected two of four naive recipients.

Autoantibodies to desmoplakin I and II in patients with erythema multiforme.

Erythema multiforme (EM) represents a syndrome of chronic recurrent inflammatory skin disease. Depending on the severity and extent of skin and mucosal involvement, it is defined either as EM minor or EM major. In this study we demonstrate the presence of autoantibodies (aAbs) against desmoplakin I and II, two major proteins of the desmosomal plaque, in six of six patients with the severe variant of EM, EM major. Light microscopic studies of lesional skin and mucous membranes localized in vivo bound immunoglobulin G (IgG) in a dotted desmosomal pattern along the cytoplasmic membranes of keratinocytes. By immunoelectronmicroscopy, in vivo bound IgG was confined to the desmosomal plaques. These findings were confirmed by indirect immunolocalization studies that demonstrated the presence of IgG aAbs in the serum of patients during active disease. These aAbs did not only bind to desmosomal plaques of epithelial cells where they colocalized with defined murine monoclonal antibodies directed against desmoplakin I and II, but also labeled the intercalated discs of myocardial cells. Biochemical characterization of circulating IgG aAbs revealed desmoplakin I and II as actual target autoantigens. By passive transfer of serum into newborn mice, in vivo binding of serum aAbs to keratinocytes was shown. The findings presented in this study imply a humoral immune response in certain patients with EM major and indicate a potential pathogenetic role of aAbs against desmoplakin I and II in this disease.

Resistance against Taenia hydatigena in sheep after passive transfer of serum or colostrum.

The role of antibody in the resistance of sheep to infection with Taenia hydatigena metacestodes was examined using passive transfer of immunoglobulin. The immunoglobulin either was experimentally transferred in serum, or was transferred from immune ewes to their new-born lambs in colostrum. Pooled serum from donor lambs which had received one, light, oral infection did not protect recipients although the donors themselves were immune. However, transfer of pooled serum from donors which had either received three oral infections, or three immunizations with solubilized T. hydatigena oncospheres in a water-in-oil adjuvant, resulted in 70-80% fewer cysts in the recipients. Colostrum from ewes infected with three high or low doses of T. hydatigena eggs was transferred to their lambs. A short acting protection (one to three weeks) was observed in the lambs. Comparisons by ELISA and Western blot, of the anti-T. hydatigena oncosphere antibody content of the donor sera, the sera of the recipients collected 24 h and seven days after transfer, the sera of the lambs and ewes, and the colostrum of the ewes, indicated that resistance to the challenge infection depends upon a critical level of antibody.

Decreased anti-donor major histocompatibility complex class I and increased class II alloantibody response in allograft tolerance in adult rats.

Permanent tolerance to allografts can be induced in adult rats by donor-specific transfusions (DST) prior to transplantation. We have previously reported, in a model of heart allograft, the presence of a heavy leukocyte infiltrate, in the allograft which displayed a strong allospecific cytotoxicity when tested in vitro against donor cells, and a strong accumulation of mRNA for granzyme A and perforin in vivo. In contrast, there was a major decrease in the accumulation of mRNA for interleukin-2 and interferon-gamma. These results suggested that the DST-induced tolerance was associated with a decrease in type-1 T helper (Th1) cell function. The major role of preformed antibodies in xeno and allorejection is clearly established. Nevertheless, the consequences of alloantibody production in acute rejection and tolerance induction remains to be elucidated. We here analyze the alloantibody response in rejecting and DST-treated recipients. We show that, after transplantation, tolerant recipients, in contrast to rejecting ones, mount a low IgM alloresponse that switches to low IgG production. Detailed analysis of IgG alloantibodies in DST-treated recipients revealed that their production decrease was not equally distributed. Whereas rejecting animals mounted a strong anti-class I and II IgG alloantibody response, DST-treated recipients produced anti-class II and low titers of anti-class I IgG alloantibodies. Furthermore, among IgG subclasses, tolerant recipients predominantly produced IgG2a, a profile which, in the rat, is compatible with a Th2-controlled response. Finally, the passive transfer of immune serum from rejecting animals to DST-treated recipients could abrogate the tolerance. We suggest that the absence of anti-class I alloantibodies combined with preserved and/or increased anti-class II production plays a major role in graft tolerance in this model. These results reinforced the role of alloantibodies in rejection and in induction of tolerance.

Mechanisms of hyperacute rejection in porcine liver transplantation. Antibody-mediated endothelial injury.

Hyperacute rejection after OLT is an unusual event. We have demonstrated previously that hepatic hyperacute rejection can occur in the presence of high titers of donor-specific cytotoxic antibody. This study addresses the issues of antibody localization within the allograft and the consequences of antibody deposition and complement activation within the transplanted organ. A porcine model of liver transplantation was used and 3 experimental groups were studied. Group I recipients (n = 6) were specifically sensitized to their liver donors with skin grafts from their liver donors before hepatic grafting. Group II recipients (n = 6) underwent third-party skin graft sensitization before liver transplantation. Group III recipients (n = 6) underwent liver grafting without prior sensitization. After liver transplantation, serum complement (CH50) levels declined promptly in all groups; a statistically significant drop as compared with control animals was seen only in group I (P < 0.05). Liver biopsies from donor-specific sensitized recipients showed massive injury by light microscopy within 30 min of revascularization, demonstrating fibrinoid necrosis of vessels and neutrophil influx. On immunofluorescent examination, liver specimens from donor-specific sensitized animals showed intense IgG, IgM, and C3 deposition in the vessels of the portal triads. Antibody deposition was not seen in third-party sensitized animals or control animals. On electron microscopy, control animals and third-party sensitized animals showed minimal ultrastructural alterations. In comparison, livers from donor-specific sensitized animals showed severe microvascular injury with destruction of endothelial cells, edema, hemorrhage, and hepatocyte necrosis. In a passive serum transfer experiment carried out by infusing serum from a skin graft-sensitized pig directly into the portal vein of the skin graft donor, severe liver injury was evident, with identification of antibody deposition by light and electron microscopy and by immunofluorescence. These studies demonstrate that tissue injury in hyperacute hepatic rejection is mediated by antibody deposition within the grafted liver and is associated with systemic activation of the complement cascade.