Doxorubicin Delivered Using Nanoparticles Camouflaged with Mesenchymal Stem Cell Membranes to Treat Colon Cancer.

PURPOSE: The primary goal of the present study was to design doxorubicin (DOX)-loaded superparamagnetic iron oxide (SPIO) nanoparticles (NPs) coated with mesenchymal stem cell (MSC) membranes and explore their effect on colon cancer in vitro and in vivo. METHODS: DOX-SPIO NPs were coated with MSC membranes using an extruder, and the morphological characteristics of MSC membrane-camouflaged nanodrug (DOX-SPIO@MSCs) evaluated by transmission electron microscopy (TEM) and NP-tracking analysis. Drug loading and pH response were assessed by UV spectrophotometry. Intracellular colocalization was analyzed using NP-treated MC38 cells stained with 3,3'-dioctadecyloxacarbocyanine perchlorate and Hoechst 33342. Cellular uptake was analyzed using an inverted fluorescence microscope and flow cytometry and cytotoxicity evaluated by cell counting kit-8 assay. Biological compatibility was assessed by hemolysis analysis, immunoactivation test and leukocyte uptake experiments. Furthermore, intravenous injection of chemotherapy drugs into MC38 tumor-bearing C57BL/6 mice was used to study anti-tumor effects. RESULTS: Typical core-shell NP structures were observed by TEM. Particle size remained stable in fetal bovine serum and phosphate-buffered saline (PBS). Compared with DOX-SPIO, DOX-SPIO@MSCs improved cellular uptake efficiency, enhanced anti-tumor effects, and reduced the immune system response. Animal experiments demonstrated that DOX-SPIO@MSCs enhanced tumor treatment efficacy while reducing systemic side effects. CONCLUSION: Our experimental results demonstrate that DOX-SPIO@MSCs are a promising targeted nanocarrier for application in treatment of colon cancer.

A novel GABAergic dysfunction in human Dravet syndrome.

OBJECTIVE: Dravet syndrome is a rare neurodevelopmental disease, characterized by general cognitive impairment and severe refractory seizures. The majority of patients carry the gene mutation SCN1A, leading to a defective sodium channel that contributes to pathogenic brain excitability. A γ-aminobutyric acid (GABAergic) impairment, as in other neurodevelopmental diseases, has been proposed as an additional mechanism, suggesting that seizures could be alleviated by GABAergic therapies. However, up to now the physiological mechanisms underlying the GABAergic dysfunction in Dravet syndrome are still unknown due to the scarce availability of this brain tissue. Here we studied, for the first time, human GABAA -evoked currents using cortical brain tissue from Dravet syndrome patients. METHODS: We transplanted in Xenopus oocytes cell membranes obtained from brain tissues of autopsies of Dravet syndrome patients, tuberous sclerosis complex patients as a pathological comparison, and age-matched controls. Additionally, experiments were performed on oocytes expressing human α1ß2γ2 and α1ß2 GABAA receptors. GABAA currents were recorded using the two-microelectrodes voltage-clamp technique. Quantitative real-time polymerase chain reaction, immunohistochemistry, and double-labeling techniques were carried out on the same tissue samples. RESULTS: We found (1) a decrease in GABA sensitivity in Dravet syndrome compared to controls, which was related to an increase in α4- relative to α1-containing GABAA receptors; (2) a shift of the GABA reversal potential toward more depolarizing values in Dravet syndrome, and a parallel increase of the chloride transporters NKCC1/KCC2 expression ratio; (3) an increase of GABAA currents induced by low doses of cannabidiol both in Dravet syndrome and tuberous sclerosis complex comparable to that induced by a classical benzodiazepine, flunitrazepam, that still persists in γ-less GABAA receptors. SIGNIFICANCE: Our study indicates that a dysfunction of the GABAergic system, considered as a feature of brain immaturity, together with defective sodium channels, can contribute to a general reduction of inhibitory efficacy in Dravet brain, suggesting that GABAA receptors could be a target for new therapies.

Neutrophil membrane-coated nanoparticles inhibit synovial inflammation and alleviate joint damage in inflammatory arthritis.

Rheumatoid arthritis is a common chronic inflammatory disorder and a major cause of disability. Despite the progress made with recent clinical use of anti-cytokine biologics, the response rate of rheumatoid arthritis treatment remains unsatisfactory, owing largely to the complexity of cytokine interactions and the multiplicity of cytokine targets. Here, we show a nanoparticle-based broad-spectrum anti-inflammatory strategy for rheumatoid arthritis management. By fusing neutrophil membrane onto polymeric cores, we prepare neutrophil membrane-coated nanoparticles that inherit the antigenic exterior and associated membrane functions of the source cells, which makes them ideal decoys of neutrophil-targeted biological molecules. It is shown that these nanoparticles can neutralize proinflammatory cytokines, suppress synovial inflammation, target deep into the cartilage matrix, and provide strong chondroprotection against joint damage. In a mouse model of collagen-induced arthritis and a human transgenic mouse model of arthritis, the neutrophil membrane-coated nanoparticles show significant therapeutic efficacy by ameliorating joint damage and suppressing overall arthritis severity.

Therapeutic microparticles functionalized with biomimetic cardiac stem cell membranes and secretome.

Stem cell therapy represents a promising strategy in regenerative medicine. However, cells need to be carefully preserved and processed before usage. In addition, cell transplantation carries immunogenicity and/or tumourigenicity risks. Mounting lines of evidence indicate that stem cells exert their beneficial effects mainly through secretion (of regenerative factors) and membrane-based cell-cell interaction with the injured cells. Here, we fabricate a synthetic cell-mimicking microparticle (CMMP) that recapitulates stem cell functions in tissue repair. CMMPs carry similar secreted proteins and membranes as genuine cardiac stem cells do. In a mouse model of myocardial infarction, injection of CMMPs leads to the preservation of viable myocardium and augmentation of cardiac functions similar to cardiac stem cell therapy. CMMPs (derived from human cells) do not stimulate T-cell infiltration in immuno-competent mice. In conclusion, CMMPs act as 'synthetic stem cells' which mimic the paracrine and biointerfacing activities of natural stem cells in therapeutic cardiac regeneration.

Acetylcholine receptors from human muscle as pharmacological targets for ALS therapy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons that leads to progressive paralysis of skeletal muscle. Studies of ALS have revealed defects in expression of acetylcholine receptors (AChRs) in skeletal muscle that occur even in the absence of motor neuron anomalies. The endocannabinoid palmitoylethanolamide (PEA) modified the clinical conditions in one ALS patient, improving muscle force and respiratory efficacy. By microtransplanting muscle membranes from selected ALS patients into Xenopus oocytes, we show that PEA reduces the desensitization of acetylcholine-evoked currents after repetitive neurotransmitter application (i.e., rundown). The same effect was observed using muscle samples from denervated (non-ALS) control patients. The expression of human recombinant α1ß1γδ (γ-AChRs) and α1ß1εδ AChRs (ε-AChRs) in Xenopus oocytes revealed that PEA selectively affected the rundown of ACh currents in ε-AChRs. A clear up-regulation of the α1 subunit in muscle from ALS patients compared with that from non-ALS patients was found by quantitative PCR, but no differential expression was found for other subunits. Clinically, ALS patients treated with PEA showed a lower decrease in their forced vital capacity (FVC) over time as compared with untreated ALS patients, suggesting that PEA can enhance pulmonary function in ALS. In the present work, data were collected from a cohort of 76 ALS patients and 17 denervated patients. Our results strengthen the evidence for the role of skeletal muscle in ALS pathogenesis and pave the way for the development of new drugs to hamper the clinical effects of the disease.

Enxerto autólogo de células-tronco derivadas do tecido adiposo: uma nova visão de sua aplicação no tratamento de queimados e na cirurgia plástica reparadora / Adipose tissue-derived stem cell autologous grafts: a new approach to application in the treatment of burn victims and reconstructive plastic surgery

No Brasil, 1 milhão de acidentes com queimaduras acontecem por ano e as infecções são responsáveis por 75% dos óbitos nestes pacientes, além de deixar lesões que ocasionam deformidades nas áreas atingidas. Sendo assim, o objetivo deste trabalho é fornecer uma visão atual sobre células-tronco mesenquimais (MSCs), com ênfase nas células-tronco derivadas do tecido adiposo (ADSCs), associadas a gel de plasma, gel de fibrina e membranas (scaffold). O uso de géis e membranas tendem a auxiliar o crescimento celular visando sua possível aplicação na Cirurgia Plástica Reparadora para o tratamento pacientes queimados ou que necessitam de enxerto de pele. O presente trabalho abordou de forma exploratória e narrativa o tema células-tronco mesenquimais, células-tronco mesenquimais derivadas do tecido adiposo, gel de fibrina, gel de plasma e scaffold. O tipo de pesquisa empregada foi conduzido com coleta de informações utilizando-se a Biblioteca Virtual em Saúde (BVS) e PubMed. O número absoluto de artigos publicados relacionados ao tratamento de queimaduras é considerável. Até o momento, a quantidade de pesquisas relacionadas à terapia com células-tronco derivadas do tecido adiposo, gel de fibrina, gel de plasma e scaffold para o tratamento de queimaduras apresenta-se escassa. O autoenxerto de ADSCs associado a biocurativos torna-se uma perspectiva promissora na Cirurgia Plástica Reparadora para o tratamento e recuperação de pacientes que sofreram queimaduras ou outros acidentes que necessitam de enxerto de pele. Estes recursos podem reduzir a dor e prover a dessecação da lesão, promovendo neovascularização e a reepitelização da ferida.

In Brazil, 1 million burn accidents occur annually, and subsequent wound infections account for 75% cases of deaths among these patients, in addition to inducing deformities in the affected areas. Therefore, the aim of this study was to discuss the current status of mesenchymal stem cells, with an emphasis on adipose-derived stem cells (ADSCs), in combination with plasma gel, glue fibrin, and membranes (scaffold). The use of gels and membranes supports cell growth, and aims at potential application in reconstructive plastic surgery for the treatment of burn patients or individuals requiring skin grafts. This study explores and discusses the role of mesenchymal stem cells, adipose-derived mesenchymal stem cells, glue fibrin, plasma gel, and the scaffold. This research collected information from the Virtual Health Library (VHL) and PubMed. A considerable number of articles have been published on burn treatment. However, there is little research on burn treatment with ADSCs, glue fibrin, plasma gel, and scaffold. An ADSC autograft combined with a biological dressing is promising in reconstructive plastic surgery for the treatment and recovery of burn patients or individuals with other injuries that require skin grafts. These features can reduce pain and aid in drying of the lesion, thus promoting neovascularization and wound reepithelialization.

Fibroblasts Rendered Antifibrotic, Antiapoptotic, and Angiogenic by Priming With Cardiosphere-Derived Extracellular Membrane Vesicles.

BACKGROUND: Cardiosphere-derived cells mediate therapeutic regeneration in patients after myocardial infarction and are undergoing further clinical testing for cardiomyopathy. The beneficial effects of cardiosphere-derived cells are mediated by the secretion of exosomes and possibly other extracellular membrane vesicles (EMVs). OBJECTIVES: This study sought to investigate the effect of cardiosphere-derived EMVs (CSp-EMVs) on fibroblasts in vitro and tested whether priming with CSp-EMVs could confer salutary properties on fibroblasts in vivo. METHODS: CSp-EMVs were isolated from serum-free media conditioned for 3 days by cardiospheres. Dermal fibroblasts were primed with CSp-EMVs for 24 h followed by exosomal micro-ribonucleic acid profiling. In vivo, we injected CSp-EMV-primed or -unprimed dermal fibroblasts (or CSp-EMVs) in a chronic rat model of myocardial infarction and defined the functional and structural consequences. RESULTS: CSp-EMVs amplified their own biological signals: exposure of "inert" fibroblasts to CSp-EMVs rendered the fibroblasts therapeutic. Intramyocardially injected CSp-EMV-primed (but not unprimed) fibroblasts increased global pump function and vessel density while reducing scar mass. CSp-EMV priming caused fibroblasts to secrete much higher levels of stromal-cell-derived factor 1 and vascular endothelial growth factor and dramatically changed the micro-ribonucleic acid profile of fibroblast-secreted EMVs in vitro. The priming was followed by significant angiogenic and cardioprotective effects. CONCLUSIONS: CSp-EMVs alter fibroblast phenotype and secretome in a salutary positive-feedback loop. The phenotypic conversion of inert cells to therapeutically active cells reveals a novel mechanism for amplification of exosome bioactivity.

Profiling neurotransmitter receptor expression in the Ambystoma mexicanum brain.

Ability to regenerate limbs and central nervous system (CNS) is unique to few vertebrates, most notably the axolotl (Ambystoma sp.). However, despite the fact the neurotransmitter receptors are involved in axonal regeneration, little is known regarding its expression profile. In this project, RT-PCR and qPCR were performed to gain insight into the neurotransmitter receptors present in Ambystoma. Its functional ability was studied by expressing axolotl receptors in Xenopus laevis oocytes by either injection of mRNA or by direct microtransplantation of brain membranes. Oocytes injected with axolotl mRNA expressed ionotropic receptors activated by GABA, aspartate+glycine and kainate, as well as metabotropic receptors activated by acetylcholine and glutamate. Interestingly, we did not see responses following the application of serotonin. Membranes from the axolotl brain were efficiently microtransplanted into Xenopus oocytes and two types of native GABA receptors that differed in the temporal course of their responses and affinities to GABA were observed. Results of this study are necessary for further characterization of axolotl neurotransmitter receptors and may be useful for guiding experiments aimed at understanding activity-dependant limb and CNS regeneration.

Isolated Bowman layer transplantation to manage persistent subepithelial haze after excimer laser surface ablation.

In a 21-year-old patient with persistent dense subepithelial haze after photorefractive keratectomy, unresponsive to retreatment, a stromal flap, 9.0 mm in diameter and approximately 60 microm in thickness, was excised and an unsutured 9.0 mm donor Bowman layer was transplanted onto the stromal bed. The corrected distance visual acuity improved from 20/40 (0.5) before surgery to 20/18 (1.2) with a scleral-supported contact lens 2 months after transplantation. With optical coherence tomography, the transplanted Bowman layer was seen as a fine white line bordering the anterior host stroma. No recurrence of stromal haze was seen throughout the 6-month follow-up. Isolated Bowman layer transplantation may be a new technique for the management of anterior stromal opacities or complicated epithelial wound healing such as persistent corneal haze after excimer laser surface ablation.

The antiepileptic drug levetiracetam stabilizes the human epileptic GABAA receptors upon repetitive activation.

PURPOSE: GABAA receptors from the brain of patients afflicted with mesial temporal lobe epilepsy (MTLE) become less efficient (run-down) when repetitively activated by GABA. Experiments were designed to investigate whether the antiepileptic drug, levetiracetam (LEV), which is used as an adjunctive treatment for medically intractable MTLE, counteracts the GABAA receptor run-down. METHODS: GABAA receptors were microtransplanted from the brains of patients afflicted with MTLE into Xenopus oocytes. The GABA-current run-down, caused by repetitive applications of GABA, was investigated using the standard two-microelectrode voltage-clamp technique. Additionally, the GABA-current run-down was investigated directly on pyramidal neurons in human MTLE cortical slices. RESULTS: It was found that, in oocytes injected with membranes isolated from the MTLE neocortex, the GABA-current run-down was inhibited by a 3-h pretreatment with 0.5-100 microM LEV. Moreover, the GABAA receptors of pyramidal neurons in human neocortical slices exhibited a current run-down that was significantly reduced by 1 microM LEV. Interestingly, the run-down in oocytes injected with membranes isolated from the MTLE hippocampal subiculum was not affected by LEV. CONCLUSIONS: We report that the antiepileptic LEV strengthens GABA inhibition of neuronal circuits by blocking the receptor run-down in the cortex whilst leaving the run-down of GABAA receptors in the hippocampal subiculum unaltered. These findings point to the GABAA receptor run-down as an important event in epileptogenesis and as a possible target for testing and screening antiepileptic drugs.

Microtransplantation of neurotransmitter receptors from cells to Xenopus oocyte membranes: new procedure for ion channel studies.

The Xenopus oocyte is largely used as a cell expression system for studying both structure and function of transmitter receptors and ion channels. Messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional ion channels. A new method was developed further to transplant neurotransmitter receptors from human brain or cultured cell lines to the membrane of Xenopus oocytes. This method represents a modification of the method used many years ago of injecting into oocytes membrane vesicles from Torpedo electroplaques, yielding the expression of functional Torpedo acetylcholine receptors. We describe this approach by extracting membrane vesicles from human hippocampus or temporal neocortex and from mammalian cell lines stably expressing glutamate or neuronal nicotinic receptors. Because the human neurotransmitter receptors are "microtransplanted" with their native cell membranes, this method extends the usefulness of Xenopus oocytes as an expression system for addressing issues in many fields, including channelopathies.

Passive transfer of experimental autoimmune labyrinthitis.

The aim of the present study was to establish an animal model of autoimmune labyrinthitis using heterologous inner ear antigen (IEAg) and to elucidate whether the experimentally induced labyrinthitis could be passively transferred. Cochlear and vestibular membranous labyrinthine tissues from bovine temporal bones were used as IEAg. Donor mice were inoculated intracutaneously at multiple sites with an emulsion consisting of equal parts of IEAg and complete Freund's adjuvant. After 10 days, mononuclear cells were collected from lymph nodes, spleen and blood of the donor mice and injected intravenously into naive recipient mice. Cellular infiltration was observed in the perilymphatic space of the cochlea of all donor and recipient mice. Endolymphatic hydrops was also observed in 63% of donor and 42% of recipient mice. These findings suggest that the experimentally induced labyrinthitis observed in this animal model was probably due to an autoimmune reaction to the IEAg and was passively transferred by a cell-mediated immune reaction.

Differential immune responses to alpha-gal epitopes on xenografts and allografts: implications for accommodation in xenotransplantation.

Xenograft recipients produce large amounts of high-affinity anti-Gal IgG in response to Galalpha1-3Galbeta1- 4GlcNAc-R (alpha-gal) epitopes on the graft. In contrast, ABO-mismatched allograft recipients undergo "accommodation," a state of very weak immune response to ABO antigens. These differences in anti-carbohydrate immune response were studied in alpha1,3galactosyltransferase knock-out mice. Pig kidney membranes administered to these mice elicited extensive production of anti-Gal IgG, whereas allogeneic kidney membranes expressing alpha-gal epitopes elicited only a weak anti-Gal IgM response. Anti-Gal IgG response to xenograft membranes depended on helper T cell activation and was inhibited by anti-CD40L antibody. These T cells were activated by xenopeptides and not by alpha-gal epitopes. Moreover, allogeneic cell membranes manipulated to express xenoproteins also induced anti-Gal IgG response. Xenoglycoproteins with alpha-gal epitopes are processed by anti-Gal B cells. Xenopeptides presented by these cells activate a large repertoire of helper T cells required for the differentiation of anti-Gal B cells into cells secreting anti-Gal IgG. Alloglycoproteins with alpha- gal epitopes have very few immunogenic peptides and fail to activate helper T cells. Similarly, ineffective helper T-cell activation prevents a strong immune response to blood group antigens in ABO-mismatched allograft recipients, thus enabling the development of accommodation.

Intrinsic defect in B cells of patients with hyper-immunoglobulin M syndrome.

We challenge the theory that the CD40-CD40 ligand is the only explanation for X-linked immunodeficiency in patients with hyper-immunoglobulin M (IgM) syndrome (HIGM1), and we demonstrate an intrinsic defect in the patients' B cells. Patients with HIGM1 have a defective CD40 ligand on their activated T-helper cells; therefore, they cannot receive signals for isotype switching when the cells are activated by T cell-dependent antigens. We activated mononuclear cells from three patients with HIGM1 and from three healthy blood donors with T cell-independent mitogens and studied their proliferative responses and Ig secretion. Normal murine plasma membrane fragments were implanted into peripheral blood mononuclear cells, and the cells were activated with Staphylococcus aureus Cowan I, pokeweed mitogen, and lipopolysaccharide. This implantation significantly augmented the proliferative responses to the mitogens in two patients. However, it augmented IGM secretion in response to B-cell mitogens in only one patient. No IgG or IgA response could be detected in the implanted mononuclear cells that originated from patients with HIGM1, unlike implanted mononuclear cells from healthy donors, which responded by IgM, IgG, and IgA antibody secretion following their stimulation with B-cell mitogens. The data suggest that the B cells of patients with HIGM1 possess an additional defect which prevents Ig isotype switching in response to T cell-independent mitogens. This defect is not located in the membrane receptors or within the membrane enzymes.

Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.

A method was developed to transplant assembled nicotinic acetylcholine receptors (AcChoRs) and Cl- channels from the electric organ of Torpedo to the membrane of Xenopus oocytes. Membrane vesicles from Torpedo electroplaques were injected into the oocytes and, within a few hours, the oocyte membrane acquired AcChoRs and Cl- channels. The mechanism of expression of these receptors and channels is very different from that which follows the injection of mRNA, since the appearance of receptors after membrane injection does not require de novo protein synthesis or N-glycosylation. This, and other controls, indicate that the foreign receptor-bearing membranes fuse with the oocyte membrane and cause the appearance of functional receptors and channels. All this makes the Xenopus oocyte an even more powerful tool for studies of the structure and function of membrane proteins.

Generation of cytotoxic T cells specific for minor histocompatibility antigens by cross challenge in vitro with H-2 disparate adherent cells.

Although it is well known that an H-2-restricted cytotoxic T cell response to minor histocompatibility antigens (MIHA) can be primed in vivo with H-2 disparate spleen cells, it has not been previously possible to induce cytotoxic T lymphocyte (CTL) precursors (CTLp) in vitro by this type of challenge. In this work, we demonstrate that the inability to cross challenge in vitro is due to the existence of inhibitory effects that can be obviated by cell fractionation, and to insufficient priming in vivo. BALB/c CTLp (H-2d) that have been repeatedly primed in vivo with B10.D2 can be challenged in vitro with C57BL10/J (H-2b) or B10.BR (H-2k)-adherent cells to generate CTL able to lyse B10.D2 (H-2d) target cells. The H-2 restriction properties of the cross-challenged CTL specific for MIHA were analyzed by using the technique of cold target competition. Within the limits of detection in bulk cultures, the entire response appeared to be H-2 unrestricted, whether the cross challenge was with intact C57BL10/J-adherent cells, or with membrane fragments of C57BL10/J presented by BALB/c adherent cells. The frequency of CTLp responsive to cross challenge was analyzed by limiting dilution, with cold target competition at each cell number to establish the restriction properties of the MIHA-specific CTL induced. We were able to detect two subsets of H-2-unrestricted CTLp responsive to intact C57BL10/J-adherent cells; one present at high frequency (1/250 T cells) and subject to suppressive effects at high cell number, and a second present at lower frequency (1/9800 T cells). There appeared to be a relatively infrequent subset of H-2-restricted CTLp as well (1/52,500 T cells). The frequency of CTLp responsive to cross challenge is of comparable magnitude to the frequency of H-2-restricted CTLp responsive to H-2-matched cells bearing MIHA. These observations are discussed in relationship to immunodominance and clonal dominance effects in the response to MIHA.

Infection of normal human thymic epithelial cells by Epstein-Barr virus (EBV) following implantation of EBV receptors.

Normal human thymic epithelial cells cannot be infected and transformed by Epstein-Barr virus (EBV). Measurements using fluorescein-labelled EBV and cytofluorograph revealed that the cells do not have receptors for the virus. Following transplantation of functional EBV receptors onto epithelial cell membranes, however, the cells were infected with EBV and expressed EBV-determined nuclear antigen (EBNA). No EBV associated antigens characteristic of the lytic cycle of the virus were detected. This method of in vitro infection by transforming viruses such as EBV may provide ways of deriving functioning thymic epithelial cell lines.

Immunotherapy of tumor-bearing mice with chemically modified tumor membrane fractions of two syngeneic tumor cell lines.

The effects of chemical modification on the immunogenicity of plasma membrane fractions of virus-induced and chemically induced tumor cell lines were tested in syngeneic tumor-bearing mice. The immunotherapeutic effects were dependent on (1) the chemical nature of the modifying reagent, which also determines the molecular site of modification in the membrane components, (2) the degree of modification, (3) the immunizing dose of modified membranes, and (4) the time schedule of immunization. Best results were obtained when the immunizing membrane samples were modified by methylation or acetylation using immunizing doses of membrane equivalents corresponding to 10(3)-10(4) cells. Up to 30% of the animals remained tumor-free and the observed survival of the animals was different from that of the control when immunotherapy was started five days after tumor transplantation. A delayed start of immunotherapy, or simultaneous immunization with tumor transplantation, or immunizations with nonmodified membrane samples led to reduced observed survival. The therapeutic effects were specific for each individual cell line. Cross-protection was not observed when mice bearing chemically induced tumors were treated with modified membrane samples derived from a syngeneic virus-induced tumor cell line.

Fusion and biochemical expression of membrane receptors in foreign living cells.

Successful transplantation of cell surface molecules from the membranes of one cell type to recipient cells of a different type is described. Plasma membranes purified from donor cells were fluoresceinated and fused to recipient cells using poly(ethylene glycol) and the fate of the transplanted membrane components was followed by fluorescence microscopy. In approximately 100 min the 'foreign' membrane components were seen to cluster and internalise. During this time, judged by the criteria of hormonal stimulation and immune cytotoxic killing, the cell surface of the recipient cell mimicked the cell surface phenotype of the donor cell.