Effect of streptavidin on cardiac allograft prolongation is due to host T-Cell suppression.

AIM: The aim of this study was to evaluate the effectiveness of streptavidin immunomodulation in the high-responder WF-to-Lewis combination. METHODS/RESULTS: We examined the effects of streptavidin on the proliferative response of T cells in coculture studies. Two to 200 microg/mL streptavidin significantly (P < .001) suppressed the proliferation of Lewis T cells to WF by 76%-83% compared with untreated responders. Next, we studied the survival of WF cardiac allografts in Lewis recipients pretreated with streptavidin. A 5-day course of peritransplantation recipient treatment with streptavidin doses of 8, 12, 20, 40, and 60 mg/kg combined with single dose of 0.5 mL antilymphocyte serum (ALS) significantly (P < .001) prolonged cardiac allograft survival from MST of 7 +/- 0.5 and 8 +/- 0.5 days in naive and ALS-treated controls to 15 +/- 1, 20 +/- 3, 16 +/- 3, 17 +/- 3, and 23 +/- 2 days, respectively. In contrast, posttransplantation administration of 80 mg/kg streptavidin resulted in animal death, suggesting toxicity of this dose. Additionally, 10 mg/kg or 20 mg/kg streptavidin administration for 10 consecutive days resulted in significant graft prolongation (MST of 18 +/- 1 and 21 +/- 1 days, respectively; P < .001). CONCLUSION: Although peritransplantation streptavidin treatment is effective in prolonging rat cardiac allografts in the high-responder WF-to-Lewis combination, it does not induce permanent graft survival as observed in the low-responder combination of Lewis-to-ACI. Our finding of in vitro immunomodulatory effect of streptavidin on T-cell proliferation suggests that its in vivo effect is partly due to prevention of T-cell activation following antigen exposure.

Single photon emission CT and positron emission tomography in the evaluation of neurologic disease.

Widely available SPECT allows imaging of certain critical components of neurotransmission, providing clinically and experimentally significant information. Future efforts may be directed toward developing innovative techniques to delineate dynamic neurochemical changes in vivo.

The role of nuclear medicine in the evaluation of pancreatic disease.

F-18 FDG PET in patients with nonendocrine pancreatic cancer and somatostatin receptor imaging in patients with endocrine pancreatic cancer have an important role in detecting or confirming the presence of a mass in the pancreas--a crucial step in the management of these patients. Both agents also have an important role in staging and in defining which patients are good candidates for resection surgery. Somatostatin receptor imaging also has a crucial role in selecting from the various systemic therapeutic options available. I-131 MIBG therapy can be of value therapeutically, mostly palliative, in patients who demonstrate a markedly elevated concentration of tumor radioactivity.

Cerebral SPECT imaging: effect on clinical management.

This study, performed in 94 consecutive patients referred for evaluation, demonstrates the clinical utility of cerebral SPECT imaging. In a significant percentage of patients (47%), the additional information provided by SPECT resulted in an alteration in clinical management. Long-term follow-up will be necessary to determine the effect of these management decisions on patient outcome.

Induction of specific unresponsiveness to rat islet allografts by intrathymic UVB donor spleen cells.

Recently, we showed that intrathymic (i.t.) injection of UVB-irradiated (600 J/m2) spleen cells induces donor-specific unresponsiveness to cardiac allografts in the sublethally irradiated (200 rads, TBI) recipients in the Lewis-to-ACI rat combination. This study examined if i.t. injection of UVB donor SC could induce specific unresponsiveness to neovascularized (islet) allografts in the same rat combination of Lewis-to-ACI. Streptozotocin-induced diabetic ACI rats pretreated with sublethal TBI (200 rads) 7 days prior to intraportal transplantation of freshly isolated Lewis islets reject their grafts in 10.2 +/- 2.9 days compared with rejection of islets in 8.5 +/- 2.6 days in unmodified controls. Recipient pretreatment with i.t. injection of UVB donor SC combined with sublethal TBI (200 rads) 7 days prior to islet transplantation induced indefinite graft survival (> 200 days) in 3 of 7 animals. Similar treatment failed to prevent acute rejection of third-party (WF) islets, thus demonstrating donor-specificity. Treatment with sublethal TBI (200 rads) on the day of islet transplantation led to permanent graft survival in 2 of 5 animals that received i.t. inoculation of UVB donor SC 7 days prior to islet transplantation. Conditioning of the recipients with a sublethal TBI dose of 300 rads on the day of islet transplantation, which alone delayed graft rejection for only 19 days, led to indefinite graft survival (> 150 days) in all animals pretreated with i.t. injection of UVB donor SC. Extrathymic inoculation of donor UVB SC via subcutaneous, intraperitoneal, intratesticular, and intravenous routes, respectively in similarly prepared animals failed to prolong islet survival, thus confirming the privileged position of the thymus in the induction of tolerance. Our findings suggest that this new strategy of immunomodulation with donor UVB SC is potentially useful for induction of donor-specific unresponsiveness.

Induction of donor-specific unresponsiveness to rat cardiac allografts by intrathymic injection of UV-B-irradiated donor spleen cells.

This study examined the role of intrathymic injection of allogeneic spleen cells in induction of donor-specific unresponsiveness to heart allografts in the Lewis-to-ACI rat combination. Intrathymic injection of naive Lewis SC led to rejection in naive or sublethally irradiated (200 rads TBI) ACI recipients at times equivalent to those obtained in control animals. Intrathymic injection of UV-B-irradiated Lewis SC, on the other hand, led to indefinite cardiac allograft survival (> 300 days) in sublethally irradiated ACI recipients; similar treatment failed to prevent rejection of third-party (Wistar Furth) cardiac allografts, which demonstrates the specificity of the immunologic unresponsiveness thus induced. The finding that intrathymic injection of untreated allogeneic SC does not prevent rejection of subsequently transplanted allograft suggests that modulation of major histocompatibility complex class II molecule by methods such as UVB may be critical to induction of unresponsiveness. Inoculation of UV-B donor SC in extrathymic sites (subcutaneous, intraperitoneal and intratesticular) did not significantly prolong graft survival in similarly prepared animals, thus confirming the privileged position of the thymus in the induction of tolerance. When the unresponsive recipients of cardiac allografts were made diabetic at 100 days and rechallenged with a second-set donor-type neovascularized pancreatic islet grafts, three of four animals accepted permanently (> 100 days) the islet grafts, thus indicating tolerance to donor alloantigens. To define the underlying mechanisms of specific tolerance in this model, in vitro MLR and in vivo adoptive transfer studies failed to demonstrate suppressor activity in the long-term cardiac allograft recipients. In contrast CML assays using 51Cr-release showed that T cells obtained from the unresponsive animals had no detectable cytotoxic activity to Con A-stimulated donor blast targets. The latter finding suggests clonal anergy or deletion of cytotoxic T cells to donor alloantigens. Our results confirm the role of the thymus as a privileged site for the induction and maintenance of specific immunologic unresponsiveness to organ allografts and suggest that this approach may be potentially useful in clinical transplantation of immediately vascularized allografts and neovascularized grafts.

Induction of donor-specific unresponsiveness to rat cardiac allografts by pretreatment with intrathymic donor MHC class I antigens.

Since intrathymic injection of UV-B-irradiated spleen cells induces donor-specific unresponsiveness in the sublethally irradiated (200 rads TBI) recipients, while intrathymic injection of naive SC leads to acute graft rejection, we hypothesized that presentation of MHC class I rather than MHC class II antigens to immature T cells in the thymus may convey a tolerogenic signal to the recipient. The present study was designed to examine if intrathymic injection of naive MHC class I-positive resting T lymphocytes can induce antigen-specific unresponsiveness to cardiac allografts in the Lewis-to-ACI rat combination. The results showed that intrathymic injection of resting Lewis T cells consistently induced indefinite graft survival (> 300 days) in sublethally irradiated (200 rads TBI) ACI recipients while similar treatment failed to prevent the rejection of third-party (Wistar-Furth) cardiac allografts, thus demonstrating the specificity of the immunologic unresponsiveness to donor alloantigens. Examination of the timing of intrathymic antigen presentation relative to cardiac transplantation that would achieve 100% permanent graft survival in the Lewis-to-ACI rat combination showed that the optimal time was 7 days before allografting, while peritransplant and immediate post-transplant intrathymic inoculation of donor T cells was relatively ineffective in the induction of unresponsiveness to donor grafts. We also showed that removal of the antigen-containing thymus in the sublethally irradiated recipients with functioning cardiac allografts consistently caused graft rejection if performed earlier than 21 days after heart transplantation; thymectomy after 21 days of organ transplantation did not affect indefinite survival of the grafts. Thus, it appears that the maintenance of peripheral tolerance to the grafts after 21 days of transplantation may be dependent on the presence of a new clone of antigen-specific tolerant host T cells. These results confirm the immunologic privileged position of the thymus in the induction of central and peripheral tolerance, and suggest that pretreatment with intrathymic MHC class I alloantigens is potentially useful in the induction of unresponsiveness to donor vascularized allografts in adult animals and in man.

Amino-dextran-deferoxamine: a potential polymeric heterobifunctional agent for high-level 111In-labeling of anti-melanoma monoclonal antibody TP41.2.

Amino-dextran-10 (ADX-10) was partially oxidized to polyaldehyde-ADX which was then reacted with deferoxamine (DFO) to form a Schiff's base and converted into a secondary amine, ADX-DFO (I) with ten moles of DFO per mole of ADX. ADX-DFO was chelated with Indium or 111In to yield ten moles of In or 111In per mole of ADX-DFO. A selective maleimide derivatization of (I) with sulfosuccinimidyl-4-(p-maleimidophenyl) butyrate yielded (II), which contained 3 moles of maleimide groups per mole of (II). The sulfhydryl-amidinium derivatization of the monoclonal antibody (MoAb) TP41.2 with 2-IT produced (III). Compounds (II) and (III) were combined to form the thioether space-arm linkage of (IV), which was subsequently radiolabeled with 111In to yield (V). MoAb-DFO-111In, (VI), was also prepared for a control study. Direct cell binding revealed the immunoreactivity of (V) to be 79.7% and that of (VI) to be 60.3%. The in vitro stability of (V) at 4, 24, and 48 hours resulted in 1.7%, 7.0% and 16.0% hydrolysis respectively, as compared with 2.1%, 8.7% and 18.5% hydrolysis of the control (VI), at the same time intervals. In a biodistribution study in non-tumor rats at 4, 24, and 48 hours post-injection, the liver concentration at 48 hours was 2.97% (ID/g) for (V) and 4.84% (ID/g) for (VI). This novel technique for radiolabeling antibodies allows for a high level of radiometallic labeling, preservation of immunoreactivity, and reduction of uptake by the liver.

Induction of donor-specific tolerance to rat cardiac and small bowel allografts by intrathymic inoculation of donor T-cells.

We have shown that donor-specific unresponsiveness to cardiac and islet allografts can be induced by intrathymic (IT) inoculation of uv-B-irradiated spleen cells or resting T-cells in the Lewis-to-ACI rat combination. To examine if tolerance to cardiac and small intestinal allografts can be induced in adult animals, we employed intrathymic inoculation of resting donor T-cells combined with sublethal total body irradiation of 200 rads on Day -7 relative to organ transplant and consistently induced permanent donor-specific cardiac allograft survival (> 300 days) and small bowel transplant (SBT) survival (> 100 days) in the Lewis-to-ACI rat combination. Similarly, IT inoculation of T-cells on Day -7 combined with 1 ml ALS on Days -7 and 0 relative to SBT resulted in indefinite donor-specific graft survival (> 100 days) in all recipients without any evidence of graft-versus-host disease (GVHD) in the high responder of Wistar-Furth (WF)-to-Lewis rat combination. In contrast, WF SBT was promptly rejected in recipients pretreated with intravenous administration of donor T-cells and ALS, thus confirming the privileged position of the thymus in the induction of tolerance. The long-term unresponsive ACI recipients challenged 150 days after cardiac transplant with a second-set graft specifically and permanently (> 120 days) accepted the second-set SBT without rejecting the primary cardiac grafts and without developing GVHD. Third-party grafts were rejected in a normal fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoreactive 111In-cyclodextrin inclusion complex: a new heterobifunctional reagent for antibody labeling.

The compound of interest, N-5-azido-2-nitrobenzoylaminomethyl-111In-acetylacetone-alpha-cycl odextrin (CD) (V) was synthesized by the selective tosylation of alpha-CD to form 6-tosyl-6-deoxy-CD, which was then reacted with NaN3 to form 6-azido-6-deoxy-CD (II). This was followed by catalytic hydrogenation to yield III. Compound III and 111In-acetylacetone were mixed to form an inclusion complex, which was then reacted with N-5-azido-2-nitrobenzoyloxysuccinimide to yield compound V. Anti-melanoma MAbTP41.2 was added to compound V, followed by immediate photoreactivation labeling by u.v. light at 320 nm. The final product VI was purified from a Sephadex G-50 column. 111In-DTPA-MAbTP41.2 was also prepared as a control. Immunoreactivity via the cell-binding assay of VI was 87%, compared with 57% by the BADTPA method. Biodistribution in non-tumor rats yielded a liver concentration in %ID/g of 3.5, 1.7 and 1.0 for compound VI, compared to the 5.5, 5.2 and 3.1 for the BADTPA compound, at 4, 24 and 48 h post-injection, respectively.

Reduced hepatic accumulation of radiolabeled monoclonal antibodies with indium-111-thioether-poly-L-lysine-DTPA-monoclonal antibody-TP41.2F(ab')2.

In an attempt to improve bifunctional chelate labelling of Mab, we investigated the use of a polyamino acid backbone for multiple DTPA substitutions. Poly-L-lysine (PL) (3.8 Kd, n = 25) was partially acetylated with MADTPA to yield 11 moles of DPTA per mole of PL. The average numbers of DTPA on PL were directly quantified with MADTPA-C-14. The remaining epsilon amino groups on PL-DTPA (I) were measured with TNBS reagent. A selective maleimide derivatization of (I) with S-SMPB yielded (II), which contains 2.3 moles of maleimide groups per mole of (I). The sulfhydryl activation of Mab-TP41.2F(ab')2 with 2-Iminothiolane hydrochloride produced (III), containing 1.3 moles of sulfhydryl groups per mole of Mab. Compounds (II) and (III) were combined to form a single thioether-spaced chain linkage of Mab-PL-DTPA (IV), which was subsequently chelated with 111In to yield (V), which was the compound of interest. Indium-111-PL-DTPA (VI) and 111In-DTPA-MabTP41.2F(ab')2 (VII) also were prepared for control studies. Direct cell binding assay revealed the mean immunoreactivity of (V) to be 79.4% and that of (VII) to be 39.5%. In a biodistribution study on melanoma tumor-bearing athymic mice at 4, 24, and 48 hr postinjection, the tumor/blood and tumor/liver ratios at 48 hr were 11.6 and 1.2 for (V), compared to 3.7 and 0.13, respectively, with (VII). Thus, the PL configuration for radiolabeled antibodies seems to result in decreased hepatic accumulation and retained tumor activity. The findings suggest that further studies of this new compound are warranted.

Improvement by affinity chromatography on antiidiotypic monoclonal antibodies (MAbs) of immunoreactivity and in vivo targeting of radiolabelled anti-HMW-MAA MAb TP61.5 in nude mice bearing human melanoma lesions.

The human high-molecular-weight melanoma-associated antigen (HMW-MAA) represents a useful marker for immunoscintigraphy in patients with melanoma. Since injection of a radiolabelled anti-HMW-MAA monoclonal antibody (MAb) visualizes only about 60% of melanoma lesions, approaches are being developed to increase the sensitivity of immunoscintigraphy. One of them aims at improving the immunoreactivity of radiolabelled anti-HMW-MAA MAbs, since this approach may improve the targeting of radiolabelled MAbs to melanoma lesions. We have previously shown that affinity chromatography on insolubilized anti-idiotypic MAbs is a useful method for purifying immunoreactive anti-HMW-MAA MAb TP61.5 from 125I-labelled MAb preparations and that not all the anti-idiotypic MAbs are useful for this purpose. Since the increasing number of available anti-idiotypic MAbs is likely to facilitate the application of this procedure in many antigenic systems, we have now tested criteria to select anti-idiotypic MAbs suitable for the purification procedure. Furthermore, we have investigated the effect of the increase in immunoreactivity of 125I-MAb TP61.5 on its in vivo targeting to human melanoma lesions transplanted into nude mice. Among the 3 anti-idiotypic MAbs tested, the most effective in purifying immunoreactive MAb TP61.5 molecules following radiolabelling is MAb TK7-110, with which 125I-MAb TP61.5 displays an immunoreactivity similar to that displayed with melanoma cells. This parameter may represent a useful criterion to identify anti-idiotypic MAbs suitable for the purification procedure, if the present results are confirmed with a large number of anti-idiotypic MAbs in different antigenic systems. We have also shown that an incubation time for up to 4 hr of 125I-MAb TP61.5 with insolubilized MAb TK7-110 is the most effective in increasing immunoreactivity and in recovering immunoreactive MAb applied to the affinity matrix. The increase in the immunoreactive fraction of 125I-MAb TP61.5 significantly increases its specific localization in human melanoma lesions transplanted into nude mice. These results suggest that purification of radiolabelled immunoreactive anti-HMW-MAA MAb TP61.5 by affinity chromatography using anti-idiotypic MAb TK7-110 represents a useful approach to increasing the sensitivity of immunoscintigraphy in patients with melanoma.

Migration patterns of dendritic cells in the rat: comparison of the effects of gamma and UV-B irradiation on the migration of dendritic cells and Lymphocytes.

To further define the underlying mechanisms of immune suppression induced by UV-B irradiation, we have examined the kinetics of homing patterns of in vitro UV-B-irradiated and gamma-irradiated-thoracic duct lymphocytes (TDL) compared to dendritic cells (DC). Our findings show that 111In-oxine-labeled TDL specifically home to the spleen, liver, lymph nodes, and bone marrow with subsequent recirculation of a large number of cells from the spleen to lymph nodes. In contrast, DC preferentially migrate to the spleen and liver with a relatively insignificant distribution to lymph nodes and an absence of subsequent recirculation. Splenectomy prior to cell injection significantly diverts the spleen-seeking DC to the liver but not to the lymph nodes, while the homing of TDL to lymph nodes is significantly increased. In vitro exposure of 111In-oxine labeled TDL to gamma irradiation does not significantly impair immediate homing to lymphoid tissues but inhibits cell recirculation between 3 and 24 hr. In contrast, gamma irradiation does not affect the tissue distribution of labeled DC, suggesting that DC are more radioresistant to gamma irradiation than TDL. Unlike the findings in animals injected with gamma-irradiated cells, UV-B irradiation virtually abolished the homing of TDL to lymph nodes and significantly reduced the homing of the spleen-seeking DC to the splenic compartment while a large number of cells were sequestered in the liver. The results of in vitro cell binding assay show that TDL, unlike DC, have the capacity to bind to high endothelial venules (HEV) within lymph node frozen sections while gamma and UV-B irradiation significantly inhibit the binding of TDL to lymph node HEV. These findings suggest that: (i) DC, unlike TDL, are unable to recirculate from blood to lymph nodes through HEV; (ii) although gamma irradiation impairs TDL recirculation, it does not affect DC tissue distribution; and (iii) UV-B irradiation impairs both TDL and DC migration patterns. We conclude that the lack of capacity of irradiated TDL to home to lymph nodes is due to damage to cell surface homing receptors and that the failure of DC to home to the lymph node microenvironment is related to the absence of HEV homing receptors on their cell surface.

Rapid localization of indium-111-labeled inhibited recombinant tissue plasminogen activator in a rabbit thrombosis model.

The thrombus localizing properties of indium-111-recombinant tissue plasminogen activator (111In-rt-PA) have been investigated in an effort to achieve prompt and accurate detection of thrombi. Unlike previous studies with rt-PA, the active plasminogen catalytic site was permanently inhibited with peptides of chloromethyl ketone so that the radiotracer binds to fibrin without causing fibrinolysis. Thrombi were created in the external jugular vein of 14 male New Zealand white rabbits followed by injection of 111In-rt-PA. The agent cleared rapidly in vivo with a half-time of 4.6 min. The thrombus: blood ratio in nonheparinized rabbits (n = 7) was 6.39 +/- 0.86. The ratio in heparinized rabbits (n = 4) was 3.11 +/- 0.23. Thrombi were clearly visible in the planar images of both groups 1 hr postinjection. The combination of rapid thrombus localization and positive images, especially in the presence of anticoagulation, suggests that further work is warranted with rt-PA thrombus imaging.

Mn (III) hematoporphyrin. A potential MR contrast agent.

Manganese (III) hematoporphyrin (MnHP), a new and stable complex, was prepared, and its toxicity and magnetic resonance (MR) imaging properties were evaluated. In tests of acute and subacute toxicity, no deaths resulted from bolus intravenous injections of 13 or 19 mumols/kg of MnHP, but there was a 33% mortality when the dose was 38 mumols/kg. Laboratory results were normal in the surviving rats. Ultraviolet- visible spectroscopy of the urine and serum of two rats injected 24 hours previously with 38 mumols/kg MnHP revealed no free HP, suggesting in vivo stability of MnHP. Finally, using a standardized imaging protocol, there was a mean increase of 37% in the liver-to-muscle intensity ratios in four rats injected 24 hours previously with 25 mumols/kg MnHP when compared to paired controls (P less than .005). In addition, obvious visual increase in the signal intensity of the liver on T1-weighted images was seen in animals tested with 13 and 19 mumols/kg of MnHP. The results suggest that further evaluation of MnHP as an MR contrast agent for the liver is warranted.

Permanent rat cardiac allograft survival induced by ultraviolet B-irradiated donor lymphocytes and peritransplant cyclosporine.

This study examines the effect of pretreatment with 10(8) ultraviolet B-irradiated donor leukocytes (UV-DL) with or without peritransplant cyclosporine (CyA) treatment (20 mg/kg on days 0, +1, and +2 relative to transplantation) on rat cardiac allograft survival across major histocompatibility loci. A single UV-DL pretreatment on day -3 or -7 (before transplantation) significantly prolonged survival of heart allografts from Wistar-Furth rats (W/F) in Lewis recipients from 6.8 +/- 0.8 days to 18.4 +/- 2.1 and 17.6 +/- 1.5 days (p less than 0.001), respectively. Multiple UV-DL infusions on days -14 and -7 increased the mean survival time to 20.0 +/- 0.9 days (p less than 0.001). Similarly, UV-DL infusion on day -3 or -7 significantly prolonged the mean survival time of heart allografts from ACI rats in Lewis rats. A single or multiple UV-DL infusions combined with peritransplant CyA led specifically to permanent W/F cardiac allograft survival (more than 200 days) in all recipients. Similarly, UV-DL infusion combined with peritransplant CyA led to indefinite survival of ACI cardiac allografts in two thirds of Lewis recipients. Adoptive transfer of splenocytes from long-term recipients of cardiac allografts, which specifically prolonged donor test grafts in syngeneic hosts, suggests that unresponsiveness to cardiac allografts is, in part, dependent on suppressor cells. This study emphasizes the importance of UV irradiation of DLs in the modulation of alloreactivity and the induction of donor-specific unresponsiveness in adult animals.