Erythropoietin improves the survival of fat tissue after its transplantation in nude mice

Background

Autologous transplanted fat has a high resorption rate, providing a clinical challenge for the means to reduce it. Erythropoietin (EPO) has non-hematopoietic targets, and we hypothesized that EPO may improve long-term fat graft survival because it has both pro-angiogenic and anti-apoptotic properties. We aimed to determine the effect of EPO on the survival of human fat tissue after its transplantation in nude mice.

Methodology/Principal Findings

Human fat tissue was injected subcutaneously into immunologically-compromised nude mice, and the grafts were then treated with either 20 IU or 100 IU EPO. At the end of the 15-week study period, the extent of angiogenesis, apoptosis, and histology were assessed in the fat grafts. The results were compared to vascular endothelial growth factor (VEGF)-treated and phosphate-buffered saline (PBS)-treated fat grafts. The weight and volume of the EPO-treated grafts were higher than those of the PBS-treated grafts, whose weights and volumes were not different from those of the VEGF-treated grafts. EPO treatment also increased the expression of angiogenic factors and microvascular density, and reduced inflammation and apoptosis in a dose-dependent manner in the fat grafts.

Conclusions/Significance

Our data suggest that stimulation of angiogenesis by a cluster of angiogenic factors and decreased fat cell apoptosis account for potential mechanisms that underlie the improved long-term survival of fat transplants following EPO treatment.     

Stromal vascular fraction cells plus sustained release VEGF/Ang-1-PLGA microspheres improve fat graft survival in mice

Autologous fat transplantation is increasingly applied in plastic and reconstructive surgery. Stromal vascular fraction cells (SVFs) combined with angiogenic factors, such as VEGF (vascular endothelial growth factor A) and Ang-1 (angiogenin-1), can improve angiogenesis, which is a critical factor for graft survival. However, direct transplant with such a mixture is insufficient owing to the short half-life of angiogenic factors. In this study, we evaluated whether a double sustained release system of VEGF/ANG-1-PLGA (poly (lactic-co-glycolic acid)) microspheres plus SVFs can improve angiogenesis and graft survival after autologous fat transplantation. VEGF/ANG-1-PLGA-sustained release microspheres were fabricated by a modified double emulsion–solvent evaporation technique. Human aspirated fat was mixed with SVF suspension plus VEGF/ANG-1 sustained release microspheres (Group C), SVF suspension (Group B) alone, or Dulbecco’s modified Eagle’s medium as the control (Group A). Eighteen immunocompromised nude mice were injected with these three mixtures subcutaneously at random positions. After 8 weeks, the mean volume of grafts was greater in the SVFs plus VEGF/ANG-1-PLGA group than in the control and SVFs groups (1.08 ± 0.069 ml vs. 0.62 ± 0.036 ml, and 0.83 ± 0.059 ml, respectively). Histological assessments showed that lower fibrosis, but greater microvascular density in the SVFs plus VEGF/ANG-1-PLGA group than in the other groups, though the SVFs group also had an appropriate capillary density and reduced fibrosis. Our findings indicate that SVFs plus VEGF/ANG-1-PLGA-sustained release microspheres can improve angiogenesis and graft survival after autologous fat transplantation.     

The effect of hyperbaric oxygenation on the viability of human fat injected into nude mice

Autologous free-fat injection for the correction of soft-tissue defects has become a common procedure in plastic surgery. The main shortcoming of this method for achieving permanent soft-tissue augmentation is the partial absorption of the injected fat, an occurrence that leads to the need for both overcorrection and repeated fat reinjection. Improving the oxygenation of the injected fat has been suggested as a means of helping to overcome the initial critical phase that occurs postinjection (when the fat cells are nourished by osmosis), increasing phagocyte activity, accelerating fibroblast activity and collagen formation, and enhancing angiogenesis. In addition, the hyperbaric oxygen-mediated decrement in endothelial leukocyte adhesion will decrease cytokine release, thereby reducing edema and inflammatory responses. The purpose of the present study was to examine the effect of hyperbaric oxygenation on improving the viability of injected fat. Adipose tissue obtained from human breasts by suction-assisted lipectomy was injected into the subcuticular nuchal region in nude mice. The mice were then exposed to daily hyperbaric oxygen treatments, breathing 100% oxygen at 2 atmospheres absolute (ATA) for 90 minutes. The duration of the administered hyperbaric oxygen therapy was 5, 10, or 15 days, according to the study group. Mice exposed to normobaric air alone served as the control group, and each group included 10 animals. The rats were killed 15 weeks after fat injection. The grafts were dissected out, weight and volume were measured, and histologic evaluation was performed. In all of the study groups, at least part of the injected fat survived, giving the desired clinical outcome. No significant differences could be found between the groups regarding fat weight and volume. Histopathologic examination of the dissected grafts demonstrated a significantly better integrity of the fat tissue in the group that received hyperbaric oxygen for 5 days (p = 0.047). This finding was manifested by the presence of well-organized, intact fat cells, along with a normal appearance of the fibrous septa and blood vessels. The worst results were found in animals treated by hyperbaric oxygenation for 15 consecutive days. An inverse correlation was found between an increased dose of the high-pressure oxygen and fat tissue integrity (r = -0.87, p = 0.076). The toxic effects of highly reactive oxygen species on fat cells might explain the failure of an excessively high dose of hyperbaric oxygen to provide any beneficial outcome. The clinical relevance of these results should be further investigated.     

The effect of interleukin-8 on the viability of injected adipose tissue in nude mice

Adipose tissue injection as a free graft for the correction of soft-tissue defects is a widespread procedure in plastic surgery. The main problem in achieving long-term soft-tissue augmentation is partial absorption of the injected fat and hence the need for overcorrection and re-injection. The purpose of this study was to improve the viability of the injected fat by the use of interleukin-8. The rationale for the use of interleukin-8 was its abilities to accelerate angiogenesis and attract inflammatory cells and fibroblasts, providing the injected adipocytes more feeding vessels and a well-established graft bed to enhance their viability. Human adipose tissue, obtained by suction-assisted lipectomy, was re-injected into the subcutis in the scalp of nude mice. Interleukin-8 (0.25 ng) was injected subcutaneously to the scalp as a preparation of the recipient site 24 hours before the fat injection and was added to the fat graft itself (25 ng per 1 cc of injected fat). In the control group, pure fat without interleukin-8 was injected and no interleukin-8 was added for the preparation of the recipient site. One cubic centimeter of fat was injected in each animal in both the study and control groups. There were 10 animals in each group. The animals were euthanized 15 weeks after the procedure. Graft weight and volume were measured and histologic evaluation was performed. In addition, triglyceride content and adipose cell sizes were measured as parameters for fat cells viability. Histologic analysis demonstrated significantly less cyst formation in the group treated with interleukin-8. No significant differences were found between the groups with regard to graft weight and volume or the other histologic parameters investigated. No significant differences were demonstrated in adipose cell sizes and their triglyceride content. In conclusion, less cyst formation, indicating improved quality of the injected fat, can be obtained by the addition of interleukin-8. Further studies of various dosages of interleukin-8 and their long-term effect are required before these encouraging results could be applied clinically.     

Revascularization determines volume retention and gene expression by fat grafts in mice

Autologous fat transplantation is a popular and useful technique in plastic and reconstructive surgery. The efficiency and survival of such grafts is predictable in many cases, but there are still issues to be resolved, such as how to improve graft volume retention. To address the issue of volume retention, we studied the effect of revascularization from the recipient on the size and function of adipocytes in fat grafts. Treatment of mice with TNP-470, an angiogenesis inhibitor, reduced blood flow from the recipient into the graft after subcutaneous transplantation of epididymal fat. The weight of transplanted tissues and the size of adipocytes in the grafts were significantly lower in mice treated with TNP-470 (TNP mice) than in control mice. Expression of genes for enzymes related to lipid accumulation was decreased in the grafts of TNP mice compared with control mice. Moreover, the expression of adipocyte-derived angiogenic peptides, VEGF and leptin, was significantly lower in the grafts of TNP mice than in grafts from control animals. The expression of VEGF and leptin by cultured human adipocytes was increased in the presence of conditioned medium from cultured vascular endothelial cells. These results show that the inhibition of the revascularization of fat grafts after transplantation reduces graft volume retention and cellular function. Early and adequate revascularization may be important for both the supply of nutrients and vasoactive interactions between vascular endothelial cells and adipocytes in graft.     

Analysis of lipocyte viability after liposuction

Free fat grafts from liposuction aspirate can be used as donor material for soft-tissue augmentation. The purpose of this study was to attempt to identify a subpopulation of adipose cells within liposuction aspirate with the greatest viability and, it is hoped, a greater chance for increased survival after transplantation. Liposuction samples were obtained from 20 individuals (16 women, four men; age range, 27 to 49 years). These samples were then centrifuged at 50 g. At 2-minute intervals, specimens from three different areas (superficial, middle, deep) were obtained from each specimen. After collagenase degradation, the specimens were stained with trypan blue, and the number of viable cells were counted. The bottom (deepest) layer consistently contained the highest number of viable cells after centrifugation: 250 percent more viable cells when compared with the top layer (p < 0.0001) and 140 percent more viable cells when compared with the middle layer (p < 0.0002). Centrifugation beyond 2 minutes did not increase the number or proportion of viable adipocytes. When using aspirated fat from liposuction for soft-tissue augmentation, centrifugation for 2 minutes at 50 g will stratify the adipocytes, with more viable cells being found at the deepest layer. Using only this bottom portion of the fat layer for transplantation will yield a fat graft with a greater number of viable adipocytes, potentially improving fat graft survival and decreased fat graft resorption.     

Cellular survival in rat vein-to-artery grafts

Microsurgical models of vein-to-artery graft surgery have been developed in rats as a means of assessing vein graft adaptation and neo-intimal hyperplasia. Neo-intimal hyperplasia in these grafts is often attributed, at least in part, to an adaptive response by venous smooth muscle cells to the increased intraluminal pressure of the arterial pressure. However, considerable evidence suggests complete or near-complete cellular replacement in these grafts. A series of experiments were undertaken in which male vein or artery grafts were placed into either allogeneic female nude rat hosts or into syngeneic WKy female hosts as a means of determining donor cell survival. Grafts were removed at postsurgery week 2 or week 6 and the fate of the donor male cells assessed by PCR amplification of the testis-determining gene Sry. The Sry gene was undetectable in 2-week male to female vein grafts. When left for 6 weeks, donor cells were detectable in vein grafts only after multiple 50-cycle PCR analyses. Minimal donor cell survival was not due to an allograft response, as donor male cells were readily detectable in WKy male to female nude rat artery-to-artery grafts. These data were not nude rat specific, as poor donor cell survival was also evidenced in syngeneic male to female vein-to-artery grafts. In conclusion, we demonstrate only marginal survival of donor cells in rat vein-to-artery grafts. Neo-intimal hyperplasia in these grafts was not a consequence of donor venous smooth muscle cell proliferation.     

Long-term survival of corneal allografts is dependent on intact CD1d-reactive NKT cells

BALB/c mice that tolerate the allogeneic grafts develop allogeneic-specific anterior chamber-associated immune deviation. Because CD1d-reactive NKT cells are essential for anterior chamber-associated immune deviation, we postulated that the survival of C57BL/6 (B6) cornea graft in BALB/c mice was also dependent on CD1d-reactive NKT cells. The B6 corneal graft rejection rate in BALB/c vs Jalpha281 knockout (KO) mice, which lack NKT cells, was measured. While there were no difference in the early phase of rejection, the survival rates at 12 wk after grafting for BALB/c and Jalpha281 KO mice were 50 and 0%, respectively. Because anti-CD1d mAb abrogated the corneal graft survival in the wild-type mice we concluded that CD1d-reactive NKT cells were essential for graft survival. Moreover, allospecific T regulatory (Tr) cells correlated with acceptance of B6 grafts in BALB/c mice, and the adoptive transfer of these allospecific Tr cells to Jalpha281 KO mice allowed a 50% survival rate of B6 cornea grafts. In conclusion, CD1d-reactive NKT cells are required for induction of allospecific Tr cells and are essential for survival of corneal allografts. Mechanisms that contribute to cornea graft acceptance may lead to new therapies for improvement in graft survival in high-risk corneas and other transplanted tissues and grafts.     

Announcements

The presence of cytotoxic T-lymphocyte precursor cells (CTLp) within BALB/c nude mice was analyzed. Allogeneic T lymphocytes were injected intravenously into BALB/c nude mice. The resulting graft versus host reaction circumvented the thymus defect of the nude mice and induced the differentiation of CTLp into anti-H-2 cytotoxic T lymphocytes.     

The differentiation of bone marrow cells to functional T lymphocytes following implantation of thymus grafts and thymic stroma in nude and AT×BM mice

Cardiac allografts were used to compare the immunologic capacity of nude mice and adult, thymectomized, lethally irradiated, bone marrow-reconstituted (AT × BM) mice. Neither nude nor AT × BM mice were able to reject cardiac allografts of any party. However, both rejected grafts of any party following implantation of neonatal thymus or thymus from 3-week-old syngeneic mice. Irradiated syngeneic thymus grafts (800 R) were equally effective in restoring host responsiveness against allografts. In contrast, allogeneic thymus grafts restored the capacity to reject second-party heart grafts only in AT × BM mice. Second-party grafts persisted indefinitely when placed on nude mice implanted with an allogeneic, unirradiated thymus graft. Third-party grafts transplanted 17 weeks after reconstitution, however, were rejected. Irradiated nude mice given normal littermate bone marrow and simultaneously grafted with second-party thymus and heart allografts also failed to reject their second-party heart grafts. The difference in ultimate capacity to respond between AT × BM and nude mice suggests that a maturational defect exists in the nude mouse enviroment which impedes development of precursor T lymphocytes.     

FK 506 and aminoguanidine suppress iNOS induction in orthotopic corneal allografts and prolong graft survival in mice.

The aim of this study was to compare the effectiveness of immunosuppressant FK 506 and the specific inhibitor of inducible nitric oxide synthase (iNOS) aminoguanidine (AG) in prevention of corneal graft rejection and to investigate the iNOS expression in the rejection process. Orthotopic corneal allografting in mice was performed (C57BL/10; H-2(b) to BALB/c; H-2(d)). FK 506 (0.3 mg/kg per day) or AG (100 mg/kg per day) was injected intraperitoneally for 4 weeks. Grafted mice without therapy served as controls. Immunohistological evaluation of iNOS-positive cells and macrophage infiltration in grafts 27th day after grafting was performed. Within 4 weeks FK 506 prevented graft rejection in 71% and AG in 57% of animals compared to 29% of clear grafts in controls. A significant proportion of iNOS-positive cells was detected in the rejected grafts of the control and AG-treated groups. The treatment with FK 506 resulted in the inhibition of iNOS expression to a high degree in the rejected corneas. Non-rejected corneas of all groups and non-transplanted corneas exhibited no iNOS-positive cells. A massive infiltration of macrophages was detected in the rejected grafts, whereas non-rejected grafts exhibited only slight infiltration of macrophages. The presented data suggest that overexpression of iNOS and/or activation of iNOS is one of the several influential factors that contribute to the rejection process and that iNOS suppression delays corneal allograft rejection. FK 506 and AG are effective drugs in preventing corneal allograft rejection. Higher beneficial effect of FK 506 on graft survival could be explained by its well-known selective T-cell immunosuppression.     

Regulation of IFN-γ and IL-10 synthesis in vivo, as well as continuous antigen exposure, is associated with tolerance to murine skin allografts

C3H/HEJ mice are rendered hyporesponsive to multiple minor incompatible (B10.BR) skin allografts by pretreatment with irradiated B10.BR lymphoid cells injected via the portal vein, but not the lateral tail vein. As assessed by PCR with lymphocytes taken from grafted mice, or by measuring cytokines in vitro from antigen-restimulated cells, this hyporesponsiveness is associated with decreased mRNA for IFN-γ and IL-2 production, but enhanced mRNA for IL-4 and IL-10 production. In mice given B10.BR cells via the tail vein, but in addition injected every second day with anti-IFN-γ antibody, similar enhanced graft survival (with diminished IFN-γ/IL-2 and enhanced IL-4/IL-10 production) was seen. In a separate study spleen cells from pretreated mice were “parked” in lethally irradiated syngeneic mice for 21 days, along with B10.BR skin grafts to some of the recipients. Only when recipients received this reexposure to B10.BR antigen did adoptively transferred spleen cells show “persistence” of the ability to produce delayed graft rejection and preferential IL-4 production in vitro.     

Human urine-derived stem cells play a novel role in the treatment of STZ-induced diabetic mice

Human urine-derived stem cells (hUSCs) are a potential stem cell source for cell therapy. However, the effect of hUSCs on glucose metabolism regulation in type 1 diabetes was not clear. Therefore, the aim of the study was to evaluate whether hUSCs have protective effect on streptozotocin (STZ)-induced diabetes. hUSCs were extracted and cultivated with a special culture medium. Flow cytometry analysis was applied to detect cell surface markers. BALB/c male nude mice were either injected with high-dose STZ (HD-STZ) or multiple low-dose STZ (MLD-STZ). Serum and pancreatic insulin were measured, islet morphology and its vascularization were investigated. hUSCs highly expressed CD29, CD73, CD90 and CD146, and could differentiate into, at least, bone and fat in vitro. Transplantation of hUSCs into HD-STZ treated mice prolonged the median survival time and improved their blood glucose, and into those with MLD-STZ improved the glucose tolerance, islet morphology and increased the serum and pancreas insulin content. Furthermore, CD31 expression increased significantly in islets of BALB/c nude mice treated with hUSCs compared to those of un-transplanted MLD-STZ mice. hUSCs could improve the median survival time and glucose homeostasis in STZ-treated mice through promoting islet vascular regeneration and pancreatic beta-cell survival.     

Cografting of hamster (Phodopus sungorus) and marmoset (Callithrix jacchus) testicular tissues into nude mice does not overcome blockade of early spermatogenic differentiation in primate grafts

The ectopic xenotransplantation of testicular tissues into nude mice is a tool to generate sperm from immature testes. Immunodeficient mice as recipients of xenografts offered an appropriate microenvironment for differentiation of testicular tissue from hamsters, goats, pigs, and macaques. One exception is the neotropical primate Callithrix jacchus. Spermatogenesis in testicular grafts from marmosets does not proceed beyond the spermatogonial stage. The most likely cause for the poor graft development of marmosets is a deletion of exon 10 in the luteinizing hormone-receptor (LHR) gene, which renders this species insensitive to LH but responsive to chorionic gonadotropin (CG). We investigated whether cografting of testicular tissue from Djungarian hamsters would overcome the blockade in marmoset graft development. We also tested if exogenous administration of human CG (hCG) to the recipient would stimulate development of the marmoset tissue. No difference in graft survival was noted between hamster and monkey tissue. Seminiferous lumina were present in marmoset and hamster grafts but were significantly larger in hamster grafts. In the hamster grafts, a high proportion of the tubules contained meiotic and postmeiotic germ cells. In contrast, the marmoset tubules were populated with gonocytes and premeiotic spermatogonia. These results indicate that neither normal serum androgen levels nor the high local testosterone levels were sufficient to initiate marmoset spermatogenesis, nor was administration of hCG successful in overcoming the developmental blockade in marmoset tissue. Our results indicate that the conditions needed for initiation of spermatogenesis in the marmoset are remarkably different from those present in most other mammals.     

Histologic study of the outcome of components of nerves of human corpses grafted in nude mice (a model of chimeric intercellular communication)

Human corpse nerves are microsurgically grafted in nude mouse and biopsied from 1 to 152 days after graft. Schwann cells ever survived and probably produce angiogenic factors stimulating mice vessels. Others nerves are grafted end to end with mouse sciatic nerve. Mouse's neurites invading grafts are surrounded by human Schwann cells and set up normal chimeric nerve.     

Surgical model for in vivo evaluation of cultured epidermal sheets in mice.

The objective of this study was to establish a novel surgical model for the grafting of cultured epidermal sheets in mice in order to optimize studies on the behavior of these grafts. Graft-related skin immunology and wound-healing studies also would benefit from this adapted surgical approach. Adapted tie-over surgical procedures were established for mice and promptly applied. Early-stage observation of grafts was made possible by replacing the cotton dressing with a saddle-like plastic tube dressing with a screw cap. We grafted normal Balb/c mice and athymic nude (nu/nu) mice with cultured human epidermis. Evaluation of graft rejection was carried out with the first group, whereas the second provided information on epidermal stratification and terminal differentiation. This innovation permitted direct evaluation of the grafted tissue at any time. Advances in applied transplantation research will certainly provide additional tools for human applications.     

Effect of bursectomy and thymectomy on cellular immunological reactions in chickens

The effect of thymectomy + irradiation and of bursectomy + irradiation on the rejection of skin grafts and injected homologous spleen cells was studied. Prolonged skin graft survival was found in thymectomized and irradiated chickens. Bursectomy together with irradiation and isolated irradiation did not affect the rejection of these grafts. Surgical operations together with irradiation had no effect on the rejection of an allogenein cellular graft. In one-day-old recipients, survival of the cells was prolonged in all the birds, while in 12-day-old recipients the cells were rejected at the normal time by both bursectomized and thymectomized birds. The possible explanations of these results are discussed. Dedicated to Academician Ivan Málek on the occasion of his 60th birthday     

Highly sensitive biosafety model for stem-cell-derived grafts

BACKGROUND: The recent success in the derivation of differentiated cell types from stem cells has raised prospects for the application of regenerative cell therapy. In particular, embryonic stem cells are attractive sources for cell transplantation, due to their immortality and rapid growth. These cells, however, also possess tumorigenic properties, which raises serious safety concerns and makes biosafety testing mandatory. Our goal was to establish a highly sensitive animal model for testing the proliferative potential of stem-cell grafts. METHODS: BALB/c nude mice received cell grafts of non-neoplastic MRC-5 cells containing defined numbers of mouse embryonic stem cells. We either injected 1 million viable cells into the kidney capsule, or mixed 2 million cells with Matrigel for s.c. transplantation. To analyze the possible impact of an intact immune response on tumor development, we also transplanted the cells into immunocompetent mice. Animals were sacrificed when the tumors became >1 cm and were analyzed in detail. RESULTS: The nude mouse model reproducibly allowed detection of 20 tumorigenic cells, and even as few as 2 ES cells were found to form teratoma. Interestingly, the administration of cell grafts at two different application sites resulted in different growth kinetics and tumor phenotypes. The highest level of sensitivity (100% detection of 20 tumorigenic ES cells) was achieved by s.c. injection of cells mixed with Matrigel. The influence of the immune system on tumor-cell development was demonstrated by a higher tumor rate of transplants in immunodeficient nude mice compared with immunocompetent mice. DISCUSSION: We have established a reliable animal model for routine assessment of the biosafety profile of stem-cell-derived cell transplants. This model will facilitate the generation of homogenous non-tumorigenic cell populations, and will help to integrate standardized safety systems into the application of stem-cell-derived grafts for clinical purposes.     

Thymus-dependent and thymus-independent developmental pathways for peripheral T cell receptor-gamma delta-bearing lymphocytes

To elucidate the developmental pathways of T cells that bear TCR gamma delta, we have analyzed the kinetics of expression and biochemical characteristics of gamma delta receptors in the thymus and spleen of normal and athymic (nude) mice, as well as nude mice engrafted with neonatal thymuses. TCR gamma delta-bearing thymocytes and splenocytes have a CD4-8- phenotype, and both populations express products of the C gamma 1 locus. TCR gamma delta-bearing cells develop in the thymus before their appearance in the spleen. Young nude mice have no detectable TCR gamma delta-bearing cells in their spleens. When young nude mice are given thymus grafts, TCR gamma delta-bearing cells of host origin first develop in the engrafted thymus, followed by their appearance in the spleen. In the absence of a thymus graft, the spleens of old nude mice eventually develop small numbers of TCR gamma delta + cells, as well as TCR alpha beta + cells. These results demonstrate that there is a major thymic-dependent pathway for TCR gamma delta expression, as well as a minor thymic-independent pathway seen in older nude mice. The development of TCR gamma delta + cells in the thymus before their appearance in the spleen, both in normal ontogeny as well as in the thymus-engrafted nude mouse model, suggests that thymic TCR gamma delta + cells are precursors of the thymus-dependent population of peripheral TCR gamma delta + cells.     

Effect of immune reconstitution on resistance to Brugia pahangi in congenitally athymic nude mice

The dichotomy of resistance to Brugia pahangi (Nematoda: Filarioidea) between nonsusceptible, euthymic C3H/HeN mice, heterozygotic for the "nu" gene (+/nu), and susceptible, congenitally-athymic "nude" (nu/nu) C3H/HeN mice, suggests that resistance is thymus-dependent. To test this hypothesis, the effect of syngeneic neonatal thymus grafts and neonatal thymus cell suspensions on recovery of worms at day 40 PI, and responses to Concanavalin A (Con A) were examined in reconstituted nudes. Nude recipients of a thymus graft 7 or 14 wk before subcutaneous inoculation with 50 infective larvae (L3) yielded no worms and responded strongly to Con A. Serum from these mice reacted in two lines of identity with serum from similarly-infected heterozygotes by double radial immunodiffusion against an adult worm saline extract. Nude recipients of a thymus 2 days or 3 wk before inoculation harbored an average of three or two worms, respectively. Intravenous injection of nude recipients with 10(7) or 10(8) neonatal thymus cells seven weeks before inoculation was less effective in conferring resistance to B. pahangi and responsiveness to Con A. Complete resistance to B. pahangi could be adoptively transferred to nude mice by 10(8) spleen cells obtained from infection-primed heterozygotes and injected intravenously on the day of larval inoculation. The same numbers of worms were significantly reduced. less effective when injected 3 wk before inoculation, although numbers of worms were significantly reduced. Passive transfer of primed heterozygote serum, containing high titers of antibodies to adult worm and larval antigens, failed to protect nude recipients against a larval inoculum in the absence of cellular reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)