Control of IBMIR in neonatal porcine islet xenotransplantation in baboons.

The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.

Phase 1 trial of dichloroacetate (DCA) in adults with recurrent malignant brain tumors.

BACKGROUND: Recurrent malignant brain tumors (RMBTs) carry a poor prognosis. Dichloroacetate (DCA) activates mitochondrial oxidative metabolism and has shown activity against several human cancers. DESIGN: We conducted an open-label study of oral DCA in 15 adults with recurrent WHO grade III - IV gliomas or metastases from a primary cancer outside the central nervous system. The primary objective was detection of a dose limiting toxicity for RMBTs at 4 weeks of treatment, defined as any grade 4 or 5 toxicity, or grade 3 toxicity directly attributable to DCA, based on the National Cancer Institute's Common Toxicity Criteria for Adverse Events, version 4.0. Secondary objectives involved safety, tolerability and hypothesis-generating data on disease status. Dosing was based on haplotype variation in glutathione transferase zeta 1/maleylacetoacetate isomerase (GSTZ1/MAAI), which participates in DCA and tyrosine catabolism. RESULTS: Eight patients completed at least 1 four week cycle. During this time, no dose-limiting toxicities occurred. No patient withdrew because of lack of tolerance to DCA, although 2 subjects experienced grade 0-1 distal parasthesias that led to elective withdrawal and/or dose-adjustment. All subjects completing at least 1 four week cycle remained clinically stable during this time and remained on DCA for an average of 75.5 days (range 26-312). CONCLUSIONS: Chronic, oral DCA is feasible and well-tolerated in patients with recurrent malignant gliomas and other tumors metastatic to the brain using the dose range established for metabolic diseases. The importance of genetic-based dosing is confirmed and should be incorporated into future trials of chronic DCA administration.

Shutdown of immunological priming and presentation after in vivo administration of adenovirus.

Adenoviral (Adv) vectors are widely used in both experimental and clinical trials for vaccination and gene therapy. Recombinant Adv can evoke potent innate immune responses and adaptive immune responses to encoded antigens. However, how Adv infection affects the response to subsequently encountered antigens is poorly understood. We show that intravenously administered replication defective (E1 and E3 deleted) Adv educes functional changes in dendritic cells (DC) resulting in impaired priming of cytotoxic T lymphocytes (CTL) more than 7 days after Adv treatment. Generalized DC activation was indicated by transient upregulation of CD86 and reduced endocytosis of fluorescent beads. It is known that CD8+ DC are predominantly responsible for uptake and presentation (cross-presentation) of exogenous antigens to CD8+ CTL. Hence, impaired endocytosis in CD8+, but not CD8-, DC at 7 days after Adv administration provided an explanation for the impaired CTL response to antigen at this time. Shutdown of cross-presentation was confirmed using cytochrome c (cytc), an agent that selectively depletes cross-presenting DC. Adv-infection rendered CD8+ DC resistant to depletion by cytc. As the cross-presentation pathway underlies CD8 T-cell responses to many cancers and to vaccines or viruses that do not directly infect DC, systemic Adv administration may impair these responses.

Local effector failure in mesothelioma is not mediated by CD4+ CD25+ T-regulator cells.

The aim of the present study was to define the point at which mesothelioma T-cell responses fail in order to design better immunotherapies. A murine model of mesothelioma was used which was established with asbestos. Inoculation of tumour cells into syngeneic mice results in progressing tumours with similar histopathology to human mesothelioma. The tumour cells secrete a marker tumour antigen similar to secreted tumour-associated products, such as mesothelin. The mesothelioma microenvironment contains stromal elements including dendritic cells, effector CD8(+) and CD4(+) T-cells, and CD4(+) T-regulatory (Tregs) cells, all of which are activated in situ, implying chronic inflammation. Tumour antigens are rapidly transported to draining lymph nodes wherein tumour-specific T-cell responses are generated. Despite the generation of potent CD8(+) cytotoxic lymphocyte in lymphoid organs, those that infiltrate tumours cannot restrain tumour growth suggesting local suppression. Splenic Tregs did not suppress protective responses in adoptive transfer experiments suggesting that systemic Tregs play little role in regulating anti-mesothelioma immune responses. Finally, removal of CD25(+) Tregs from the tumour site and lymphoid organs did not alter tumour growth with or without interleukin (IL)-2 or IL-21 immunotherapy. Tregs are not potent regulators of anti-mesothelioma immunity and targeting these cells may not improve results.

Generic construction of single component particles that elicit humoural and cellular immune responses without the need for adjuvants.

Insoluble, pure protein particles could be advantageous as single-entity vaccines or as carriers for small peptide epitopes. Dense gas anti-solvent precipitation was employed to produce pure protein particles which were found to be insoluble in water. As particulate and multimerized antigens are more immunogenic and hence more advantageous for vaccination, particles were produced via this method using ovalbumin as a model antigen. The particles produced had a mean diameter of approximately 300nm, and remained as discrete particles at low pH. At neutral pH or in the presence of electrolyte, the particles exhibited predictable flocculation behaviour to produce aggregates 1-5microm in diameter. Immunisation of mice with these flocculates elicited specific ovalbumin antibody production, T-cell proliferation and a cytotoxic T-cell response, all in the absence of adjuvant. Thus, dense gas processing could be used as a generic method to produce pure protein particulate vaccines.

Identification of genes involved with tick infestation in Bos taurus and Bos indicus.

Tick resistant cattle could provide a potentially sustainable and environmentally sound method of controlling cattle ticks. Advances in genomics and the availability of the bovine genome sequence open up opportunities to identify useful and selectable genes controlling cattle tick resistance. Using quantitative real-time PCR and theAffymetrix bovine array platform, differences in gene expression of skin biopsies from tick resistant Bos indicus (Brahman) and tick susceptible Bos taurus (Holstein-Friesian) cattle following tick challenge were examined. We identified 138 significant differentially-expressed genes, including several immunologicallhost defence genes, extracellularmatrix proteins, and transcription factors as well as genes involved in lipid metabolism. Three key pathways, represented by genes differentially expressed in resistant Brahmans, were identified; the development of the cell-mediated immune response, structural integrity of the dermis and intracellular Ca2+ levels. Ca2+, which is implicated in host responses to microbial stimuli, may be required for the enhancement or fine-tuning of transcriptional activation of Ca2+ -dependant host defence signalling pathways.

A microstructurally informed model for the mechanical response of three-dimensional actin networks.

We propose a class of microstructurally informed models for the linear elastic mechanical behaviour of cross-linked polymer networks such as the actin cytoskeleton. Salient features of the models include the possibility to represent anisotropic mechanical behaviour resulting from anisotropic filament distributions, and a power law scaling of the mechanical properties with the filament density. Mechanical models within the class are parameterized by seven different constants. We demonstrate a procedure for determining these constants using finite element models of three-dimensional actin networks. Actin filaments and cross-links were modelled as elastic rods, and the networks were constructed at physiological volume fractions and at the scale of an image voxel. We show the performance of the model in estimating the mechanical behaviour of the networks over a wide range of filament densities and degrees of anisotropy.

Molecular cloning and characterization of pig, cow and sheep MAdCAM-1 cDNA and the demonstration of cross-reactive epitopes amongst mammalian homologues.

Full-length cDNA clones for the pig, cow and sheep mucosal addressin cellular adhesion molecule (MAdCAM)-1 homologues were isolated from Peyer's patches by a combination of reverse transcription (RT)-polymerase chain reaction and 5' and 3' RACE strategies. Degenerate primers based on conserved amino acid (aa) sequences within the N-terminal immunoglobulin (Ig)-like domains of the human and rodent MAdCAM-1 molecules were used for initial sequencing of the Ig-like domains. MAdCAM-1 transcripts of 1425 bp, 1525 bp and 1510 bp obtained for the pig, cow and sheep contained an open-reading frame for proteins of 390, 424 and 418 aa, respectively. The pig and ruminant MAdCAM-1 had two N-terminal Ig-like domains, a mucin-like region and a third Ig-like domain found in rodent but not human MAdCAM-1. Antibodies raised against bacterially expressed N-terminal Ig-like domains of pig, human and sheep MAdCAM-1 demonstrated the existence of cross-reactive epitopes, raising the possibility of producing monoclonal antibodies which can be used as multi-species MAdCAM-1-targeting reagent for the development of mucosal vaccines.

Inter- and intra-strain variation and PCR detection of the internal transcribed spacer 1 (ITS-1) sequences of Australian isolates of Eimeria species from chickens.

The objective of this study was to confirm the presence of seven species of Eimeria involved in chicken coccidiosis in Australia by comparing internal transcribed spacer 1 (ITS-1) sequences, ITS-1 polymerase chain reaction (PCR) methods and to apply phylogenetic analysis to assess evolutionary relationships of Australian isolates. Twenty-two distinct ITS-1 regions of 15 Australian Eimeria isolates were sequenced, and analysed using maximum parsimony, distance and maximum likelihood methods. Poor bootstrap support, resulting from high ITS-1 sequence heterogeneity between all species groups, resulted in polychotomy of the Eimeria species in all three trees generated by these analyses. Percentage identity analyses revealed two distant ITS-1 lineages in both E. mitis and E. maxima at the same levels that separate the two species E. tenella and E. necatrix. One E. maxima lineage consisted of Australian isolates, the other American isolates, with one European sequence (originating from the same isolate) in each lineage. One Australian E. praecox sequence was only distantly related (33% variation) to three E. praecox sequences from Australian and European isolates. Short and long ITS-1 variants were isolated from both E. tenella (cloned line) and E. necatrix isolates with deletions (106 and 73 bp, respectively) in the short variants within the 3' region of the ITS-1 sequence. ITS-1 sequences of strains of both E. brunetti and E. acervulina species varied the least. Apart from E. maxima, all of the ITS-1 sequences of the six remaining individual species clustered to the exclusion of other species in all phylogenetic trees. Published ITS-1 tests for E. necatrix, E. acervulina, E. brunetti and E. tenella, combined with three new tests for E. mitis, E. praecox and Australian E. maxima amplified all respective Australian isolates specifically in a nested format using conserved ITS-1 PCR products as template to improve the sensitivity. All PCR tests were confirmed against a collection of 24 Australian chicken Eimeria isolates and contaminating species were detected in some instances. In conclusion, once the genetic variation between species and strains is determined, the ITS-1 is a good target for the development of species-specific assays, but the ITS-1 sequences alone do not seem suitable for the confirmation of phylogenetic inferences for these species. This study reports the first attempt at the analysis of the phylogeny and sequence comparison of the Eimeria species involved in chicken coccidiosis in Australia.

Myosins of Babesia bovis: molecular characterisation, erythrocyte invasion, and phylogeny.

Using degenerate primers, three putative myosin sequences were amplified from Australian isolates of Babesa bovis and confirmed as myosins (termed Bbmyo-A, Bbmyo-B, and Bbmyo-C) from in vitro cultures of the W strain of B. bovis. Comprehensive analysis of 15 apicomplexan myosins suggests that members of Class XIV be defined as those with greater than 35% myosin head sequence identity and that these be further subclassed into groups bearing above 50-60% identity. Bbmyo-A protein bears a strong similarity with other apicomplexan myosin-A type proteins (subclass XIVa), the Bbmyo-B myosin head protein sequence exhibits low identity (35-39%) with all members of Class XIV, and 5'-sequence of Bbmyo-C shows strong identity (60%) with P. falciparum myosin-C protein. Domain analysis revealed five divergent IQ domains within the neck of Pfmyo-C, and a myosin-N terminal domain as well as a classical IQ sequence unusually located within the head converter domain of Bbmyo-B. A cross-reacting antibody directed against P. falciparum myosin-A (Pfmyo-A) revealed a zone of approximately 85 kDa in immunoblots prepared with B. bovis total protein, and immunofluorescence inferred stage-specific myosin-A expression since only 25% of infected erythrocytes with mostly paired B. bovis were immuno-positive. Multiplication of B. bovis in in vitro culture was inhibited by myosin- and actin-binding drugs at concentrations lower than those that inhibit P. falciparum. This study identifies and classifies three myosin genes and an actin gene in B. bovis, and provides the first evidence for the participation of an actomyosin-based motor in erythrocyte invasion in this species of apicomplexan parasite.

A msp1alpha polymerase chain reaction assay for specific detection and differentiation of Anaplasma marginale isolates.

Anaplasma marginale is the causative agent of bovine anaplasmosis, a disease which can be protected by vaccination with the less pathogenic Anaplasma species, A. centrale. Currently, there is no polymerase chain reaction (PCR) assay available which differentiates between different species of Anaplasma or which can differentiate isolates of A. marginale within outbreaks and between different countries. A molecular test specific for A. marginale would be ideal for the identification of Anaplasma species in wild ruminants, as possible reservoirs of anaplasmosis, and to differentiate between A. marginale from A. centrale. A PCR assay was designed to amplify the major surface protein 1alpha gene of the rickettsial bovine pathogen, A. marginale both as an inter- and intra-specific test. The test did not amplify A. centrale or A. ovis, and discriminated A. marginale by amplifying repeat regions within the msp1alpha gene which vary in number between many isolates. The nested A. marginale amplicons varied in size from 630 to 1190bp representing one to eight internal repeats. All 22 Australian isolates tested amplified a 630bp product (one repeat) in contrast to all 19 non-Australian isolates tested. Eight sequences from Australian isolates from different geographical regions confirmed the conserved nature of the Australian A. marginale msp1alpha genes. The Australian 'repeat unit' MSP1a deduced amino acid sequence has been designated as Australian type 1. The msp1alpha PCR method developed here enabled the amplification and comparison of A. marginale isolates originating from North and South America, Africa, Israel and Australia. The method is sensitive and specific for A. marginale. Although additional msp1alpha products were amplified from at least two Australian isolates, the results suggest limited introduction of A. marginale into Australia.

Without CD4 help, CD8 rejection of pig xenografts requires CD28 costimulation but not perforin killing.

Although CD4 cells are major mediators in cellular rejection of fetal pig pancreas (FPP) in the mouse, rejection still occurs in the absence of CD4 cells, albeit with delayed kinetics. CD4 cell-independent mechanisms of cellular rejection are poorly understood. To investigate the involvement of CD8 T cells in FPP rejection and their activation requirements, we used mice transgenic for anti-CD4 Ab; this is the most complete model of CD4 cell deficiency. We showed that in such mice FPP was infiltrated with CD8 cells starting from 2 wk posttransplantation and FPP was eventually rejected 8 wk posttransplantation. Ab depletion of CD8 cells greatly improved the survival of FPP and reduced cell infiltration at the graft site. This suggests that CD8 cells can mediate the rejection of porcine xenografts in the absence of CD4 cells. This CD8-mediated rejection of FPP is independent of their perforin-mediated lytic function, as graft survival was not affected in mice deficient in perforin. The production of IFN-gamma and IL-5 by the graft infiltrates indicates that CD8 cells may act through cytokine-mediated mechanisms. Remarkably, in the absence of CD4 cells, lymphocyte infiltration at the graft site was absent in mice transgenic for CTLA4Ig such that the islet grafts flourished beyond 24 wk. In contrast, rejection was little affected by CD40 ligand deficiency. Therefore, we show that CD8 cells are activated to mediate FPP rejection independent of perforin and that this CD4-independent activation of CD8 cells critically depends on B7/CD28 costimulation.

Molecular cloning of a C-type lectin superfamily protein differentially expressed by CD8alpha(-) splenic dendritic cells.

Dendritic cells (DC) are potent antigen presenting cells that activate naive T cells. It is becoming increasingly clear that DC are not a homogeneous cell population, but comprise different subpopulations that differ in ontogeny and function. To further the molecular characterisation of DC, we screened for genes that were differentially expressed amongst DC subsets and could therefore give insight into their varying biological functions. Using Representational Difference Analysis (RDA) we identified a gene (CIRE) that is expressed at higher levels in the myeloid-related CD8alpha(-) DC than in the lymphoid-related CD8alpha(+) DC. CIRE is a 238 amino acid type II membrane protein, of approximately 33 kDa in size, whose extracellular region contains a C-type lectin domain. Northern blot analysis revealed that CIRE is almost exclusively expressed in DC and was not detected in organs such as heart, brain, kidney, liver, and thymus. T cells failed to express message for CIRE, whilst B cells expressed very low levels. These data here further substantiated by Northern blot analysis of 18 cell lines of various origins (myeloid, macrophage, B and T cell) where only one cell line, which was of myeloid origin and could give rise to DC, expressed mRNA for CIRE. Semi-quantitative RT-PCR suggested that CIRE is down-regulated upon activation. CIRE shares 57% identity with human DC-SIGN, a molecule that has been shown to be the ligand of ICAM-3 and that is also a receptor that binds HIV and facilitates trans-infection of T cells.

Use of cue configuration geometry for spatial orientation in human infants (Homo sapiens).

Research with both rats and human infants has found that after inertial disorientation, the geometry of an enclosed environment is used in preference over distinctive featural information during goal localization. Infants (Homo sapiens, 18-24 months) were presented with a toy search task involving inertial disorientation in 1 of 2 conditions. In the identical condition, 4 identical hiding boxes in a rectangular formation were set within a circular enclosure. In the distinctive condition, 4 distinctive hiding boxes were used. Infants searched the goal box and its rotational equivalent significantly more than would be expected by chance in the identical condition, showing that they were sensitive to the geometric configuration of the array of boxes. Unlike the results of studies using a rectangular enclosure, however, in the distinctive condition, infants searched at the correct location significantly more than at other locations.

Molecular cloning of F4/80-like-receptor, a seven-span membrane protein expressed differentially by dendritic cell and monocyte-macrophage subpopulations.

A novel dendritic cell (DC) surface molecule termed F4/80-like-receptor (FIRE) has been selected based on its differential expression between DC subsets. The gene encoding FIRE has been cloned and sequenced, and mAbs specific for FIRE have been produced. FIRE is a seven-transmembrane-spanning molecule with two epidermal growth factor-like domains in the extracellular region. It is a novel member of the epidermal growth factor/transmembrane-7 protein subfamily and shows similarity to the macrophage marker F4/80. FIRE is expressed by CD8- DC, but not by CD8+ DC, and it is down-regulated on DC activation. It is expressed by blood monocytes and by some tissue macrophages, but not by most macrophage cell lines or by lymphoid cells. FIRE is a useful marker of myeloid cells with a DC developmental potential.

The comparative efficacy of CTLA-4 and L-selectin targeted DNA vaccines in mice and sheep.

The access of antigens to antigen presenting cells (APCs) appears to be a rate-limiting step in the generation of immune responses to DNA vaccines. The cytotoxic T lymphocyte antigen 4 (CTLA-4) and L-selectin represent attractive ligands for use in the targeting of antigen to APCs and lymph nodes. CTLA-4 binds with high affinity to the B7 membrane antigen on APCs, while L-selectin functions as a lymphocyte homing marker and binds to CD34 on the surface of high endothelial venule cells. DNA vaccines encoding human immunoglobulin (HIg), fused to either CTLA-4 or L-selectin, have been shown to generate up to 10,000-fold higher anti-HIg antibody responses than DNA vaccines encoding HIg alone. In this study, the ability of CTLA-4 or L-selectin mediated targeting to enhance the humoral immune response to an alternate vaccine antigen was investigated. DNA vaccines encoding CTLA-4-HIg and L-selectin-HIg fused to the host-protective 45W antigen from Taenia ovis were constructed. In BALB/c mice, the L-selectin targeted vaccine did not improve either the magnitude or speed of antibody responses of vaccinated mice. In contrast, the CTLA-4 targeted DNA vaccine generated 45W-specific antibody responses which were up to 30-fold higher than those achieved with non-targeted DNA vaccination. The kinetic of the antibody response generated following CTLA-4 targeted DNA vaccination was also significantly faster than that achieved with non-targeted DNA vaccination, or with adjuvanted protein vaccination. Vaccination of outbred sheep with DNA vaccines expressing either murine or ovine CTLA-4 targeted antigen failed to enhance immune responses. These findings indicate that CTLA-4 targeting may find application in the improvement of DNA vaccines, but requires further development for applications in large animal species.

The human IgG3 hinge mediates the formation of antigen dimers that enhance humoral immune responses to DNA immunisation.

A series of plasmid DNA constructs containing the 45W antigen gene from Taenia ovis were used to investigate the impact of antigen dimerisation on the humoral immune response to genetic immunisation. Genes encoding dimeric 45W were generated via fusion to the hinge region of human IgG3 (hIg). This region was selected because it is compact and contains 11 inter-chain disulphide-bridges. The DNA encoding the IgG3 hinge contains four exons, with the last three exons being repeats and possibly superfluous. Plasmids containing the 45W gene linked to exons 1-2, 1-3 or 1-4 of the hIgG3 hinge, were compared to a control plasmid containing a form of the 45W gene which encodes secreted, monomeric 45W protein. Western blot analysis was used to investigate the formation of the fusion-proteins in transfected Cos-7 cells. The full-length fusion construct expressed predominantly dimeric forms of the fusion-protein, while truncation of the hinge region decreased the abundance of dimeric fusion-protein and increased the proportion monomeric fusion antigen. In immunised BALB/c mice, 45W-specific antibody titres were increased 3 to 4-fold via fusion to the full-length hinge region, whereas the truncated constructs were similar to the control. IgG subclass analysis indicated that all mice generated predominantly IgG1, IgG2a and IgG2b antibodies. Therefore, these results suggest that the efficient formation of dimeric antigen, via fusion to the full-length hinge of human IgG3, can increase the immunogenicity of expressed antigens without altering the form of the immune response elicited by DNA immunisation.

Cell-associated ovalbumin is cross-presented much more efficiently than soluble ovalbumin in vivo.

To better understand the antigenic requirements for cross-presentation, we compared the in vivo efficiency of presentation of cell-associated vs soluble OVA with the OT-I (CD8) and OT-II (CD4) TCR transgenic lines. Cross-presentation of cell-associated OVA was very efficient, requiring as little as 21 ng of OVA to activate OT-II cells and 100-fold less to activate OT-I cells. In contrast, soluble OVA was presented inefficiently, requiring at least 10,000 ng OVA for activation of either T cell subset. Thus, cell-associated OVA was presented 500-fold more efficiently than soluble OVA to CD4 T cells and 50,000-fold more efficiently to CD8 T cells. These data, which represent the first quantitative in vivo analysis of cross-presentation, show that cell-associated OVA is very efficiently presented via the class I pathway.

Enhanced survival of grafts genetically endowed with the ability to block CD2 and B7.

In a model of transplantation rejection, we have tested whether a graft manipulated to secrete immunomodulators could protect itself from immune destruction. An insulinoma cell line having the NOD genotype but also expressing the neoantigen, SV40 T antigen, was transfected with CTLA4Ig or LFA3Ig to block signals in the co-stimulatory/adhesion pathways. This neoantigen is potent at inducing graft rejection. Secretion of CTLA4Ig and LFA3Ig by transfectants promoted survival of the insulinoma graft in young NOD mice. In immunodeficient mice, cell growth was similar for all transfectants. However, in immunocompetent NOD mice the survival/growth of test grafts was significantly better than that of the controls. Graft survival was enhanced additively, when the two test transfectants were cotransplanted. Endowing the graft the ability to secrete immunomodulators that block individual co-stimulatory/adhesion signals can contribute to transplantation success. Blockade of two signals (CD2 and CD28) in these pathways enhances this success.

Protection of xenografts by a combination of immunoisolation and a single dose of anti-CD4 antibody.

Immunoisolation is the separation of transplanted cells from cells of the immune system using a semipermeable membrane. Using one such immunoisolation capsule-the TheraCyte device-we have assessed the survival of encapsulated xenogeneic tissue in vivo as well as the contribution of CD4+ve T cells to encapsulated xenograft rejection. The foreign body reaction to the TheraCyte capsule in vivo was assessed by transplanting empty capsules into normal mice. These capsules elicit a foreign body response by the host animal. Encapsulated CHO, NIT-1, and PK-15 cells were placed in culture and in immunodeficient mice to investigate their growth characteristics in the TheraCyte device. These cell lines survive both in culture and in immunodeficient SCID mice. Xenogeneic PK cells were also transplanted into normal C57BL/6 mice. These cells do not survive in normal mice despite the absence of direct contact between infiltrating and encapsulated cells. In addition, the survival of encapsulated cells in mice treated with a single dose of anti-CD4 antibody was examined. This was assessed using two systems: 1) histological analysis of capsule sections; 2) a quantitative luciferase reporter system using PK cells transfected to express luciferase. In both cases, anti-CD4 antibody contributed to prolonged encapsulated xenogeneic cell survival. Encapsulated xenogeneic cells survive in immunodeficient mice but not normal mice. Treatment of normal mice with anti-CD4 antibody results in prolonged survival of xenogeneic cells that can be measured using a luciferase reporter system. These results highlight the contribution of CD4+ve T cells to encapsulated xenograft rejection.