PVG Rats Research Articles

Host‐derived angiogenesis maintains bone blood flow after withdrawal of immunosuppression

A novel method of living bone allotransplantation combining microvascular repair of the nutrient circulation, implantation of host-derived arteriovenous (AV) bundles, and short-term immunosuppression is described. We hypothesized that neoangiogenesis from the implanted vessels would maintain graft viability and circulation after withdrawal of FK506 (Tacrolimus) immunosuppression. Vascularized femoral transplantation was performed between DA and PVG rats. In addition to microsurgical pedicle anastomoses, a saphenous AV bundle from the recipient animal was implanted in the medullary space. Ninety-seven rats were randomly allocated to groups differing in immunosuppression and AV bundle patency. Implanted vessels significantly improved capillary density and bone blood flow in nonimmunosuppressed and immmunosuppressed groups, respectively. A lower incidence of spontaneous AV bundle thrombosis was found with Tacrolimus treatment. More viable osteocytes were seen at 4 weeks when the AV bundle was patent. Further investigations may confirm host-derived neoangiogenesis as an alternative to tolerance induction or immunosuppression in bone allotransplantation.

Microvascular Thrombosis and Cardiac Allograft Vasculopathy in Rat Heart Transplantation

We studied association between microvascular thrombosis (MT) and cardiac allograft vasculopathy (CAV) in rat heart transplants (Tx). Male PVG (RT1c) rats were transplanted into ACI (RT1av1) (n=29). ACI into ACI (n=31) and sham-operated rats (n=33) served as controls. Recipient rats received cyclosporine (10 mg/kg/day) on days 0–9 post-Tx. Grafts and native hearts were harvested 10 days, 1, 2 and 3 mo. post-Tx. Hearts were processed for immunofluorescence and light microscopy. MT was defined as presence of fibrin and capillary antithrombin binding (CATB). CAV was assessed by computer-assisted image analysis. PVG-ACI donor hearts had the highest deposition of fibrin (Median: 14.0–16.1%, 10 days-3 mo. post-Tx) and CATB (Median: 12.8–18.4%, 10 days-3 mo. post-Tx). PVG-ACI native hearts also had high levels of fibrin (Median: 8.0–18.3%, 10 days-3 mo. post-Tx) and CATB (Median: 5.8–19.0%, 10 days-3 mo. post-Tx). ACI-ACI donor and native hearts had significantly lower (p<0.01) deposition of fibrin (Median: 1.0–5.6%, 10 days-3 mo. post-Tx) and CATB (Median: 2.0–7.9%, 1–3 mo. post-Tx) than donor and native hearts from PVG-ACI rats. Rats with a sham operation had negligible amounts of fibrin (Median: 0.4–1.0%, 10 days-3 mo. post-Tx) and CATB (Median: 0.8–1.8%, 10 days-3 mo. post-Tx). A strong association was found between presence of fibrin and CATB and development of CAV. These data show that MT in rat cardiac grafts is associated with CAV as found in humans. This model could be used to study pathogenesis of CAV and evaluate new therapies.

Submaximal force accurately predicts submaximal work

Several investigators have used work loops to evaluate neuromuscular function. Within a work loop, muscle must become activated, produce force while shortening, relax, and undergo passive lengthening as it would in many voluntary activities. However, to our knowledge investigators have only reported results from maximally activated work loops. Our purpose for conducting this study was to characterize submaximal work loop performance and its relation to submaximal isometric force. We isolated gastrocnemius of female PVG rats in situ with nerve and vascular supply intact. Achilles tendons were attached to a servo motor which controlled position and recorded force. We set onset and offset of the stimulation pulse train (130 Hz) to minimize negative work for the cycle. We imposed cycle frequencies of 2 and 4 Hz, which represent walking and running gait and excursion lengths of 2 and 4 mm, which are associated with gait and maximal flexion/extension. Current was adjusted to achieve tetanic forces that approximated 25%, 50%, 75% and 100% of maximum force. We used regression analyses to determine relationships of twitch and tetanic force with work. Our data indicated that work was highly related to tetanic force: R2=.98 for 2 Hz, both 2 and 4 mm excursions; R2=.90 and .93 for 6 Hz, 2 and 4 mm excursion respectively. Work was also highly related to twitch force: R2=.83 and .87 for 2 Hz, 2 and 4 mm respectively, and R2=.93 and .97 for 6 Hz, 2 and 4 mm respectively (p<.001 for all regressions). Despite many factors that influence work production such as force-length, force-velocity, activation and relaxation kinetics, and their interactions, it seems that work produced at various stimulation intensities can be predicted from tetanic force.

Correlation Between Cyclosporine-Induced Nephrotoxicity in Reduced Nephron Mass and Expression of Kidney Injury Molecule-1 and Aquaporin-2 Gene

Sirolimus (SRL) has been shown to exacerbate cyclosporine (CsA)–induced nephrotoxicity. The expression of the kidney injury molecule-1 (KIM-1) is markedly upregulated in the postischemic rat kidney. We sought to correlate drug-induced nephrotoxicity and the expression of KIM-1 and aquaporin-2 (AQP-2) in male PVG rats with 2 kidneys (2K), 1 kidney (1K), and half a kidney (1/2K) treated with SRL alone, CsA alone, or a combination of both (SRL-CsA). After 7 days of treatment, the 2K group treated with SRL-CsA showed a significant decrease in creatinine clearance compared with the 2K SRL alone and 2K CsA alone groups (1.2 vs 2.47 vs 2.46 mL/min; P < .001). There was a trend toward deterioration of creatinine clearance in the 1K and 1/2K groups treated with SRL-CsA. The KIM-1 expression in the 2K SRL-CsA group was significantly upregulated compared with that in the 2K SRL alone and 2K CsA alone groups ( P = .02). The AQP-2 expression was comparable in the 3 groups. After 1 week of treatment washout, the 2K, 1K, and 1/2K groups treated with SRL alone demonstrated a significantly higher creatinine clearance rate than did the groups treated with SRL-CsA ( P = .04, P = .02, and P = .004). The expression of KIM-1 and AQP-2 was similar among the treatment groups. SRL-CsA–induced nephrotoxicity resulted in overexpression of KIM-1, suggesting injury to the proximal tubule. Treatment with SRL alone may enable earlier reversal of tubular injury.

A new strain of rat for functional analysis of PINA

Long Evans cinnamon (LEC) rat is an animal model for human Wilson disease (WD) due to a deletion in Atp7b, the copper transporter defective in WD patients. Previously, we have demonstrated presence of an alternative product termed PIneal Night-specific ATPase (PINA) generated by an intronic promoter in Atp7b gene. Analysis of LEC rat in this study demonstrates that PINA is absent in the LEC pineal establishing its usefulness for investigating PINA function. Studies of the LEC pineal, however, revealed an additional defect in serotonin N-acetyltransferase (NAT), the key enzyme in melatonin production. Linkage studies confirm that the NAT phenotype is entirely independent of PINA mutation in the pineal gland of LEC rats, and sequence analysis demonstrates that NAT defect is due to a point mutation in NAT coding region. In addition, we demonstrate that the cinnamon coat color of the LEC rat is unlinked to PINA and NAT deficiencies in these animals. To facilitate further functional analysis of PINA in pineal physiology, we crossed LEC rats with PVG rats that are wildtype for PINA, NAT and coat color, and obtained rats that are defective only in PINA/Atp7b locus (termed LPP rats) and normal for NAT activity and coat color. Furthermore, we have identified the deletion breakpoint of Atp7b gene in LPP rats, which allows simplified genotyping of mutant animals. The separation of PINA mutation from both NAT and coat color mutations in the new LPP rats will permit better functional studies of PINA in pineal circadian physiology.

Treatment of Streptozotocin-Induced Diabetes Mellitus in Rats by Transplantation of Islet Cells from Two Major Histocompatibility Complex Disparate Rats in Combination with Intra Bone Marrow Injection of Allogeneic Bone Marrow Cells

We have established a new method for the transplantation of allogeneic pancreatic islets obtained from two different rat strains in combination with a newly developed bone marrow transplantation (BMT) method in which bone marrow cells (BMCs) are directly injected into the bone marrow cavity (intra bone marrow BMT [IBM-BMT]). Streptozotocin-induced diabetic Brown Norway (BN: RT1A(n)) rats were injected with fludarabine, irradiated with 5.0 Gy x 2, and BMCs from two allogeneic rat strains, Fischer 344 (F344: RT1A(1)) and PVG (PVG: RT1A(c)), were then directly injected into the bone marrow cavity (IBM-BMT). Simultaneously, approximately 600 pancreatic islets (PIs) from F344 and PVG rats were mixed and transplanted into the liver by way of the portal vein. All the recipients thus treated showed normoglycemia 30 days after the treatment. Hematolymphoid cells were completely reconstituted with the two donor-type cells, and immunologic tolerance to F344 and PVG major histocompatibility complex (MHC) determinants were induced. The transplantation of PIs from two MHC-disparate donors was completely achieved in combination with IBM-BMT, resulting in the improvement of blood glucose levels and the amelioration of diabetes mellitus.

Nitric Oxide Contributes to Resistance of the Brown Norway Rat to Experimental Autoimmune Encephalomyelitis

The Brown Norway (BN) rat is reported to be resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and a number of mechanisms have been suggested to explain this resistance. In work reported here we provide evidence that such resistance in the BN rat can be accounted for, at least in part, by their ability to produce higher levels of nitric oxide (NO) than susceptible strains of rats. Spleen cells from the BN rat make significantly more NO following in vitro stimulation than do cells from the Lewis or PVG rat and following in vivo immunization using complete Freund's adjuvant (CFA) the BN rat makes substantially more NO than either susceptible strain. If carbonyl iron is used as adjuvant in vivo there is no increase in NO levels in the BN rat and they are rendered highly susceptible to EAE. Immunizing with CFA simultaneously with neuroantigen and carbonyl iron drives up NO levels and the resistance is restored. EAE produced using carbonyl iron is characterized by extensive macrophage/microglia presence in the central nervous system lesions of the BN rat yet the cytokine profile in the lymph nodes does not differ from that in the EAE Lewis rats.

Short photoperiod inhibition of growth in body mass and reproduction in ACI, BUF, and PVG inbred rats

Laboratory rats have been generally considered non-photoresponsive, but strains of laboratory rats have been found to be variable for this trait. Young males of both the Fischer (F344) and Brown Norway strains (BN) suppress reproductive development, food intake and body mass in short winter photoperiod (short days (SD); 8 h light:16 h darkness), and food restriction interacts with SD to enhance the effect of SD alone. Conversely, young male Harlan Sprague Dawley outbred rats, along with other outbred laboratory rats tested, have little or no response to SD except when unmasked by food restriction or other treatments, and have generally been considered nonphotoperiodic. In order to assess how widespread this trait might be among rat strains, and to test for uncoupling of reproductive and nonreproductive responses, we tested 3 additional inbred strains, including ACI, PVG and BUF rats, for photoresponsiveness and for unmasking of photoperiodic responses by food restriction. Young males of all three inbred strains exhibited photoresponsiveness in testis mass (5-20% lower in SD), seminal vesicle mass (20-50% lower in SD), and body mass (5-10% lower in SD). Food restriction also suppressed reproduction, but there was little or no interaction with the effects of photoperiod. The results are consistent with the hypothesis that laboratory rats are genetically variable for photoperiodism, and that photoresponsiveness may be widespread among inbred rat strains, as all five inbred strains tested have shown photoperiodic responses. The results are particularly important because standard research protocols may unknowingly manipulate this pathway in rats, causing unsuspected variability among or within studies.

C-Fos Expression in the Rat Cerebral Cortex during Systemic GvH Reaction

Objective: It is becoming clear that the CNS receives signals from the peripheral immune system. In order to identify the areas of the brain that receive information about a specific immune response to allogeneic antigens, we studied the expression of c-Fos, a neural activation marker, in the cerebral cortex following the induction of a graft-vs.-host reaction (GvHR) in rats. Methods: C-Fos expression in the brain was studied by immunohistochemistry. GvHR was induced in (WKY × PVG)F₁ rats by injecting 5 × 10⁸ spleen cells from PVG rats. Control rats received syngeneic cells. Results: No c-Fos immunoreactivity (IR) was observed in animals undergoing GvHR in the nucleus tractus solitarii (NTS), the locus coeruleus (LC), the organum vasculosum of lamina terminalis (OVLT), the paraventricular nucleus (PVN) or the central amygdaloid nucleus (Ce). In contrast, 3 days after GvH induction c-Fos IR was observed in the piriform cortex and several other olfactory-related regions indicating the stimulation of the olfactory pathway during GvHR. Strong c-Fos IR was also observed in the occipital visual cortex of animals undergoing a GvHR, suggesting that GvHR can affect visual functions. In addition, GvHR induced c-Fos IR in the prefrontal cortex (Cg3, orbital cortex), a region that has interconnections with most sensory modalities. Double-staining studies indicate that the cells that express the c-Fos signal are neurons. Conclusion: We have defined the distribution of brain neurons that are affected during the induction phase of GvHR. Our results also indicate that the integration and processing of information from the immune system at CNS levels involve different areas during different types of immune responses.

Molecular basis for complement component 6 (C6) deficiency in rats and mice

Complement component C6 is a part of the lytic membrane attack complex formed during complement activation. Animal modeling to define the role of C5a vs. C5b-9 in human disease has used rodents deficient in C6, yet the molecular basis for the deficiencies has not been ascertained. Oligonucleotides derived from a 493 bp EST sequence of the rat C6 gene were used to isolate full-length transcripts of rat C6 mRNA. Sequence analysis confirmed that the derived amino acid sequence for rat C6 is highly homologous to human and mouse. We identified a 31 bp deletion in exon 10 of the C6 gene that leads to C6 deficiency in a strain of PVG rats (PVG/c-) and developed a PCR-based genotyping test. In addition, we identified four point mutations in the mouse C6 gene that may result in C6 deficiency observed in the Peru-Coppock mouse strain. A serendipitous finding from this study was a coagulation defect in the C6 deficient mice and rats. C6 deficient mice or rats demonstrated prolonged tail bleeding times that was reversed by treatment with purified rat C6 protein. Further, adenosine diphosphate induced platelet aggregation were markedly reduced in C6 deficient rats. The molecular basis for these coagulations defects is unknown at present.

Phenotype and natural killer cell sensitivity of a radiation-induced acute T-cell leukaemia (Roser leukaemia) in PVG rats.

A radiation-induced T-cell leukaemia [Roser leukaemia (RL)] in the rat was conditioned for growth in vitro by repeated in vivo-in vitro passages. This in vitro cell line, termed RL-T, maintained its leukaemia-inducing property when transferred to syngeneic PVG rats. It expresses several T-cell markers and the T-cell alpha/beta receptor-CD3 complex. RL-T, furthermore, expresses major histocompatibility complex (MHC) I antigens, both classical (RT1.A) and nonclassical (RT1.C), which makes it susceptible to killing by alloreactive natural killer cells in vitro.

THE ROLES OF DONOR LEUKOCYTES AND SOLUBLE MHC CLASS I IN TRANSPLANTATION TOLERANCE

A275 Aims: Spontaneous induction of tolerance occurs in the DA (RT1a) to PVG (RT1c) rat model following orthotopic liver transplantation (OLT), while heart grafts, unless accompanied by a liver transplant, are acutely rejected in 8 days. The liver, unlike the heart allograft, is a source of both secreted soluble MHC molecules and resident leukocytes which may be responsible for spontaneous tolerance. In the present study, we measured levels of circulating soluble MHC class I molecules (sMHC) following OLT and tested whether sMHC and leukocytes could substitute for a liver transplant in protecting DA cardiac allografts from acute rejection in PVG recipients. Methods: PVG rats received either a heterotopic cardiac allograft or an OLT from DA donors. MHC class I heavy chains (RT1Aa, RT1A1c or RT1Au) and β2microglobulin were produced using a prokaryotic expression system, refolded around appropriate single peptides and purified by gel filtration using FPLC. Recombinant sMHC were continuously administered intravenously to graft recipients using osmotic mini infusion pumps. Allotype specific ELISA were developed and used to quantify levels of circulating MHC levels in the recipient sera. Results: Naïve DA rats had circulating levels of sRT1Aa of ∼350ng/ml while RT1A1c was <50ng/ml in naïve PVG rats. Following OLT, donor sMHC (RT1Aa) rise rapidly, peaking around 400ng/ml before returning to a plateau of ∼200ng/ml, 2 weeks after transplantation. Recipient RT1A1c levels also increased briefly to ∼100ng/ml following OLT. Infusion, at the time of transplant, of sRT1Aa alone failed to prolong cardiac allograft survival (median survival time, mst = 8d) despite serum levels of 2000ng /ml. However, combined administration of 10-150 million donor lymph node or spleen cells together with continuous sRT1Aa (serum levels >1000ng/ml) significantly prolonged allograft survival (mst = 12d versus 6.5d for cells alone). Similarly, pre-treatment with sRT1Aa alone also prevented rapid rejection (mst = 12d). Third party sMHC or cells were ineffective. Conclusions: These findings support the concept that soluble MHC class I may play a role in spontaneous liver tolerance. It is noteworthy that the sRT1Aa used in this study was of prokaryotic (not eukaryotic) origin and therefore lacked glycosylation and presented only a single peptide rather than a range of naturally selected peptides.

Macrophages play a role in the early phase of corneal allograft rejection in rats.

Rat corneal allograft rejection is delayed by repeated local injection of liposomes filled with clodronate (dichloromethylene diphosphonate), which selectively deplete macrophages. Various administration schedules of liposomes were tested to determine the optimum schedule for prevention of graft rejection. Cell subpopulations in the anterior segment of the eye were studied at different time points after transplantation to assess the kinetics of the immune response. AO rats were grafted orthotopically with corneal buttons from PVG rats. Postoperatively, rats remained untreated or received clodronate liposomes subconjunctivally. Clodronate liposomes were injected five times on postoperative days (PODs) 0, 2, 4, 6, and 8; or once, on POD 0 or 6; or twice on PODs 0 and 2 or PODs 0 and 6. Grafts were examined for signs of rejection clinically and immunohistologically. All untreated rats rejected their grafts as did all five rats that received clodronate liposomes once on POD 6. In all the other administration schedules tested, graft survival was prolonged compared with the untreated control group (P <0.01). Injections of clodronate liposomes on PODs 0 and 2 proved to be the most effective treatment. Histologically reduced influx of virtually all cell types tested was found in this group. To prevent or delay graft rejection, it is necessary to administer clodronate liposomes in the early phase after corneal transplantation. These results suggest a role for macrophages in the afferent phase of corneal graft rejection.

Auxiliary liver-heart transplantation in the rat: a new microsurgical model inducing tolerance

Objective To establish a model of immunological tolerance induced by simultaneous auxiliary liver transplantation and heterotopic heart transplantations using PVG and DA rats as donors and recipients respectively, to provide a new method for the research of liver transplantation. Methods The recipient's whole portal vein blood was transfered to the auxiliary liver allograft. The donor's heart was transplanted to the recipient's abdominal cavity by end-to-side anastomosis of the donor aorta and pulmonary artery to the recipient aorta and vena cava, respectively. Results In the syngeneic control group and the test group the recipient rats and their transplanted hearts both survived more than 100 days, while the hearts of alloheart control group only survived 6 days. The weight of the liver graft increased slowly. Conclusions In the presence of recipient liver, the auxiliary liver graft functioned well to induce tolerance. At 100 days, the auxiliary liver graft had increased in volume and weight with maintenance of its tolerance-inducing effects.

The rat RT1-A1c MHC molecule is a xenogeneic ligand recognized by the mouse activating Ly-49W and inhibitory Ly-49G receptors.

Mouse Ly-49 receptors are known to recognize xenogeneic ligands from hamster and rat. However, until now, there has been no description of a specific rat xenogeneic ligand for any mouse Ly-49 receptor. In this report, we identify RT1-A1c, a rat classical class I MHC molecule, as a ligand for the Ly-49G(BALB/c) inhibitory receptor and the closely related activating receptor, Ly-49W. Xenogeneic class I recognition of targets from PVG but not DA strain rats was mapped to the classical region of the RT1c haplotype by using Con A blasts from RT1c/RT1av1 intra-MHC recombinant rats as targets for RNK-16 cells expressing either Ly-49W or Ly-49G(BALB/c) receptors. Individual expression of class I molecules from PVG and DA rat strains in YB2/0 target cells demonstrate the xenogeneic recognition to be allele specific, because other class I molecules of the RT1c haplotype, RT1-A2c and RT1-U2c, and a classical class I molecule encoded by the RT1av1 haplotype, RT1-Aa, are not recognized by Ly-49W and -G(BALB/c). Furthermore, specificity for RT1-Ac can be transferred from Ly-49W to Ly-49P, which is normally unable to recognize RT1-Ac, by substitution of three residues shared by Ly-49W and -G(BALB/c) but not Ly-49P. These residues are located in the Ly-49 beta4-beta5 loop, which can determine class I allele specificity in mouse Ly-49 receptor interactions with mouse class I ligands, suggesting that mouse Ly-49 recognition of rat class I molecules follows similar principles of interaction. These findings have implications for xenotransplantation studies and for discerning Ly-49 recognition motifs present in MHC molecules.

Break of tolerance via donor-specific blood transfusion by high doses of steroids: a differential effect after intestinal transplantation and heart transplantation

Tolerance requires active mechanisms. How immunosuppressors affects tolerance is poorly understood. Methods RA (RT1 p)/PVG (RT1 c) rats were used as donor/recipient. Intestinal and heart transplant model were selected as highly and poorly immunogenic organs. Studied groups were 1, rejecting control; 2, received peritransplant steroids; 3, donor-specific blood transfusion (DSBT); 4, DSBT plus peritransplant steroids; and 5, DSBT+periDSBT Ste. Results Intestinal transplant recipients in group 1 died on posttransplant day (d) 18. In group 2, steroids did not change survival (17 days, P > .05 versus group 1). With DSBT (group 3), all rats survived >75 days, whereas with steroids those in group 4 survived 59 days ( P > .05 vs group 3) and group 5 survived 51 days ( P < .05 versus group 3). Survivors in group 2 were tolerant as evidenced by acceptance of secondary donor-specific (not third-party) graft. However, 100% and 33% of donor-specific secondary grafts were rejected in groups 4 and 5 ( P < .05 and P > .05 versus group 3). In heart transplants, steroid treatment had no effect on graft survival (group 1 9 days; group 2 9 days; P > .05). DSBT (group 3) induced 100% tolerance (primary: >100 days, secondary: 100%). Unlike in intestinal transplantation, adjunction peritransplant steroids (group 4) allowed 100% of primary and 83% of secondary graft acceptance ( P > .05 versus group 3). In group 5, (DSBT+periDSBT steroids) acceptance of primary and secondary grafts tended to be reduced (primary: 77 days; P > .05 versus group 3; secondary: 67%, P > .05 versus group 3). Conclusion Steroid induction did not prolong graft survival after either intestinal or heart transplant. Adjunction of steroids to a DSBT tolerogenic regimen caused rejection of primary and secondary grafts, particularly after intestinal transplantation. Routine use of steroids in the clinics must be reconsidered, particularly when immunogenic organs are transplanted and when immunomodulation is applied.

Distinct regions of periaqueductal gray (PAG) are involved in freezing behavior in hooded PVG rats on the cat-freezing test apparatus

The periaqueductal gray (PAG) is considered to be an exit relay for defensive responses. Studies have shown that the ventrolateral periaqueductal gray (vlPAG) plays a role in the expression of freezing behavior whereas dorsolateral periaqueductal gray (dlPAG) is involved on both freezing and active forms of defensive behaviors. To further elucidate this theory, lesioned vlPAG and dlPAG rats were exposed to a cat in the cat-freezing test apparatus. Subsequently, a 7-day repeated exposure to a cat was done on the vlPAG and dlPAG lesioned rats. Results showed that the vlPAG lesioned rats demonstrated significant decrease in freezing behavior and corresponding increase in locomotor activity, while the dlPAG lesioned rats failed to show any significance. Subsequent repeated exposure of the vlPAG lesioned rats to a cat for 7 days showed a gradual decrease in freezing behavior with significance shown at days 5, 6 and 7 while the dlPAG lesioned rats failed to show any changes. These results suggest that vlPAG regulates freezing behavior in hooded PVG rats.

Regulatory cell-mediated tolerance does not protect against chronic rejection.

Regulatory cells prevent graft loss to acute rejection and induce tolerance, possibly by promoting Th2 deviation. Th2 cytokines stimulate B cells, which cause alloantibody-mediated chronic rejection. We searched to determine whether regulatory cell-mediated tolerance protects or not against chronic rejection. Heart transplantation (Htx) was performed using RA (RT1P) and PVG (RT1c) rats as donor and recipients. Donor-specific blood transfusion (DSBT) was given on preTx day 12. Secondary grafts were implanted at day 100. Splenocytes were transferred from tolerant rats (and controls) into lightly irradiated (450 rad) naive PVG, which received RA Htx. Primary Htx were investigated for the development of vascular occlusion (VO), the production of Th1/Th2 intragraft cytokines, and for the nature of graft infiltrate as well as for endothelial deposition of immunoglobulin (Ig)G isotypes and complement (C3) binding. Results were compared with rejecting controls (no DSBT) and syngeneic Htx. RA Htx were rejected within 10 days (8, 9, 10x4). PreTx DSBT prolonged primary Htx survival indefinitely (>140 days) with acceptance of secondary donor-specific (but not third-party) grafts (P<0.001). Naive irradiated PVG rats given splenocytes from tolerant rats but not from controls accepted RA Htx, showing the existence of regulatory cells in allograft acceptors. Despite being tolerant, DSBT-treated rats displayed typical features of chronic rejection at day 90 (VO=77%; P<0.001 vs. VO=4% in syngeneic rats). An overt Th2 deviation, particularly intragraft production of interleukin (IL)-4, was observed at day 30. Simultaneously to this Th2 deviation, B cells emerged in the grafts and endothelial deposition of IgG1 (Th2 dependent) and C3 binding were observed. Regulatory cells that prevent graft loss to acute rejection in primary and secondary grafts do not protect against the development of chronic rejection.

Detection of chimerism following vascularized bone allotransplantation by polymerase chain reaction using a Y-chromosome specific primer

Chimerism following allogeneic organ transplantation is a phenomenon known to occur and be associated with development of immunologic tolerance in allotransplantation. However, little is known about graft cell migration following vascularized bone allografting. In this study, chimerism was assessed following vascularized tibia transplantation from male DA or PVG donors to female PVG rat recipients using a semi-quantitative polymerase chain reaction for the Y-chromosome. FK-506 (Tacrolimus) was administered after transplantation for immunosuppression. All immunosuppresssed PVG rat recipients of PVG bone grafts showed a high level of chimerism (1%) in the thymus, spleen, liver and cervical lymph nodes at 18 weeks post-transplant. Donor cells were also detected in the contralateral tibia and humerus. In non-immunosuppressed PVG rat recipients of DA bone grafts, donor cells were detected in the spleen in three of five rats within 2 weeks post-transplant. In these animals the bone grafts were severely rejected. In immunosuppressed PVG rat recipients of DA bone grafts, two of five, four of eight and eight of 10 rats showed low level chimerism (0.1%) in peripheral blood at 1, 12, and 18 weeks post-transplant. Six rats showed a high level of chimerism in the spleen and thymus. Histological studies revealed no rejection findings through 18 weeks post-transplant. Our results indicate that chimerism, or the presence of graft cells in host tissue, may occur in the face of acute rejection and be demonstrable following vascularized isograft and allograft living bone transplantation when chronic immunosuppression is maintained. Graft vascular patency during the short-term likely allows cellular migration, even in the face of acute rejection. Long-term survival and proliferation of graft marrow elements in host tissue may be possible with adequate immunosuppression.

FTY720, an immunosuppressant that alters lymphocyte trafficking, abrogates chronic rejection in combination with cyclosporine A

Chronic rejection remains the leading cause of failure after transplantation (Tx). FTY720, a new immunosuppressant altering lymphocyte trafficking, is effective against acute rejection, but its activity against chronic rejection is not known. A valid model of chronic rejection was produced. Heart transplantation (HTx) was performed using fully mismatched RA (RT1p) and PVG (RT1c) rats. Administration of donor-specific blood transfusion 12 days before HTx prolongs graft survival, but features of chronic rejection including intimal hyperplasia and vascular obliteration (VO) develop with time only in allogeneic Tx. This is therefore a valid model of chronic rejection. VO was assessed on post-Tx day 90 in six groups differing according to the maintenance immunosuppressive regimen administered. group 1, donor-specific blood transfusion only and no other treatment; group 2, FTY720 (0.3 mg/kg/day orally) for 90 days; group 3, cyclosporine A (CsA) (1 mg/kg/day orally) for 90 days; group 4, combined administration of FTY720 and CsA for 90 days; group 5, transient administration of combined FTY720 and CsA for 7 days; and group 6, syngeneic HTx (RA to RA). Graft infiltrate, endothelial immunoglobulin (Ig) G deposition, and complement binding were also examined on post-Tx day 90. In control group 1, severe VO was observed, compared with syngeneic HTx (group 6). Monotherapy with FTY720 (group 2) or with CsA (group 3) significantly but partially reduced VO. On the contrary, combined administration of FTY720 and CsA (group 4) abrogated VO. A 1-week treatment with combined FTY720 and CsA (group 5) reduced VO but only partially. In group 1, arteriosclerosis was accompanied by graft infiltrate, endothelial IgG deposition, and complement binding. In groups 2, 3, and 5, graft infiltrating scores were partially decreased compared with group 1 but remained higher than in syngeneic controls; endothelial IgG deposition and complement binding were still present. In group 4, continuous administration of combined FTY720 and CsA reduced graft infiltrate to the level of syngeneic control and abrogated both endothelial IgG deposition and complement binding. Maintenance treatment with either FTY720 or CsA monotherapy partially prevents chronic rejection; short-term treatment with combined FTY720 and CsA reduces chronic rejection only partially; and continuous treatment with combined FTY720 and CsA abrogates chronic rejection, and this is accompanied by dramatic reduction of graft infiltrating cells, endothelial IgG deposition, and complement binding. Prevention of chronic rejection by maintenance treatment with FTY720 and CsA represents indirect evidence that normal lymphocyte trafficking and function are mandatory for development of chronic rejection.