WO1999013726A1

WO1999013726A1 - Neuronal cells and an immunosuppressant and anti-inflammatory factor

Info

Publication number: WO1999013726A1
Application number: PCT/US1998/019670
Authority: WO
Inventors: Paul Ronald Sanberg; Robert W. Engleman
Original assignee: University of South Florida; University of South Florida St Petersburg
Current assignee: University of South Florida; University of South Florida St Petersburg
Priority date: 1997-09-19
Filing date: 1998-09-18
Publication date: 1999-03-25
Anticipated expiration: 2000-03-19

Abstract

A method of preventing or modulating immunologic and inflammatory reactions in a host comprising administering to the host a sufficient amount of an anti-inflammatory factor or cells secreting such a factor; and monitoring the host. The treatment can be accomplished by injecting neuronal cells into a body cavity, by implanting neuronal cells with a graft, and by administering to the site of the graft an isolated immunomodulating factor obtained from the neuronal cell supernatant, all of which inhibit such rejection or inflammation.

Description

NEURONAL CELLS AND AN EMMUNOSUPPRESSANT AND ANTI-INFLAMMATORY FACTOR

BACKGROUND

Field of Use

The present invention is in the field of immunosuppression, and transplantation and more particularly in the field of modulating autoimmune disease, inflammatory disease, and preventing organ, tissue, or cell graft rejection, by administering neuronal cells or a factor isolated therefrom.

Background Information

The principal biological impediment to successful transplantation from one patient to another (allograft) is the immunologic rejection reaction that results from the genetic dissimilarity between the two individuals. It is rarely possible to perform a transplant between a genetically identical pair (monozygotic twins) (isograft).

The host (graft recipient) responds to the donor (graft source) histocompatibility antigens and may mount a full scale rejection of the graft. A single chromosomal complex of closely linked genes makes up the code for the major histocompatibility antigens. The major histocompatibility complex (MHC) in humans is called the HLA system (for human leukocyte antigens). The HLA system has at least seven separate antigens which each may have 8-50 known variations. HLA-A, HLA-B and HLA-C are MHC class I; HLA-D, HLA-DR, HLA-DQ are in MHC class II. Both Class I and II antigens can elicit an immunologic response, class I antigens are targets of cytotoxic T cells, and class II antigens stimulate the proliferative response of the mixed lymphocyte culture (MLC). Class I and Class II are distributed differently on cells within a person. Class I antigens are present on almost all nucleated cells, including T and B lymphocytes and platelets. Class II antigens are found only on B lymphocytes, monocytes, macrophages, some types of vascular endothelial and epidermal Langerhans cells and activated T lymphocytes. Lymphocytes are categorized as T or B cells. T cells mount a cellular response against mismatched HLA antigens. There are subsets of T cells: helper T cells, cytotoxic T and suppressor cells. T cells start a cascade of events that also involve B lymphocytes, antibodies, and activated macrophages.

Class II antigens activate helper T cells which release various factors, including interleukin-2 and macrophage-stimulated lymphokine. Class I antigens stimulate cytotoxic T cells to develop interleukin-2 receptors. Next stimulated are macrophages and other accessory cells to release interleukin-1, which in turn also influences the release of interleukin-2. Interleukin-2 then interacts with specific interleukin-2 receptors on activated helper and cytotoxic T cells, initiating their DNA synthesis and proliferation. In addition, interleukin-2 maintains the viability of activated T cell clones. In essence, alloantigens and interleukin-1 activate helper T cells and subsequently macrophages, cytotoxic T cells, antibody-releasing B cells.

The mixed lymphocyte culture (MLC) is used to predict cell-mediated graft rejection. A reaction is induced by culturing mononuclear leukocytes (WBC including T cells, B cells, natural killer (NK) cells, mononuclear phagocytes, and dendritic cells) from one individual with mononuclear WBC from another individual. The cells are typically isolated from peripheral blood. When the MHC types from the two individuals differ, a large proportion of the mononuclear cells proliferate in four to seven days. This proliferative response, usually measured by incorporation of ³H-thymidine into DNA during cell replication, is called an allogeneic reaction. If the cells from both sources proliferate, it is a two-way reaction. If one set of cells is inactivated to prevent their replication (e.g., gamma irradiation) or cannot replicate in the system, a one-way reaction occurs, with the irradiated cells acting as stimulators and the non-irradiated cells as responders which are activated to proliferate, produce cytokines and express cytokine receptors. The degree of T cell activation (stimulation index or SI) is calculated by taking the ratio of ³H-thymidine incorporation in stimulated and unstimulated responder T cells.

Generally, the closer the histocompatibility match (the lower the SI), the less immunosuppression by drugs is required for graft acceptance. To prevent rejection, the best current protocol is sequential, quadruple therapy. Initially prednisone, azathioprine and anti-lymphocyte (or antithymoblast) globulin are administered. Usually, globulin is used for about the first seven days postoperatively, and cyclosporine is started at day five. If transplant function is not good at 5 days, globulin is continued longer and cyclosporine is added later. Over time, conditions permitting, prednisone and cyclosporine are tapered off. Use of these immunosuppressive drugs varies depending on the immune privileged status of the graftment, degree of mismatch and type of transplant. For example, in neurotransplant immunosuppression is limited as much as possible, as even small doses increase the risk of death and complications due to viral, fungal and bacterial infections.

The drugs have other important side effects. Azathioprine may depress bone marrow production of white cells and platelets, cause jaundice and impair kidney function. Prednisone, used in most transplant recipients, must be regulated carefully to prevent complications of infection, development of Cushinoid features, hypertension, increased bruising and severe acne. In children with transplants, growth is inhibited. Cyclosporine inhibits production and release of interleukin-2 by helper T cells and inhibits release of IL-1 by macrophages. Consequently, patients on cyclosporine must be closely monitored for hepatotoxicity, nephrotoxicity and increased incidence of cancer, including lymphomas.

Although kidney transplants are the most common transplant, totaling some 9000 in 1988, other organs are increasingly replaced, including the heart, lung, combined heart and lung, liver hepatocytes, bone marrow, whole pancreas and pancreatic islet cells.

Neural transplantation has been tried as a therapy in several animal models of Parkinson's disease and other neurodegenerative disorders (Bjorklund and Stenevi, Brain Res. 177:555-60, 1979; Sanberg et al., CELL TRANSPLANTATION FOR HUNTINGTON'S DISEASE, R.G. Landes Company, Austin TX, 1994, p 124). This experimental treatment has been applied clinically in Parkinson's disease (PD) patients with favorable results (Lindvall et al., Science 247:574-77, 1990; Kordower et al., New Engl. J. Med. 332:1118-24, 1995; Freeman et al., Ann. Neurol. 38:379-88, 1995). Recently preliminary clinical trials of neural transplantation in Huntington's disease (HD) patients also were conducted (Kurth et al., Amer. Soc. Neurol. Transplant. Abstr. 3:15, 1996). Alternative graft sources have been explored, such as encapsulated cells and genetically engineered cells (Emerich et al., 1996, ibid. ; Kawaja et al., J. Neurosci. 12:2849-64, 1992). HNT neuronal cells are neuron-like cells which differentiate from an embryonal cell line isolated from a human teratocarcinoma (Ntera2 or NT2/D1 cells) after retinoic acid treatment. RA treatment also increases the surface expression of MHC class I molecules (Mol. & Cell. Bio 13: 6157, 1993). Other neuronal cells have also been developed which may find use in the instant invention.

The HCN-1 cell line is derived from parental cell lines from the cortical tissue of a patients with unilateral megalencephaly growth (Ronnett GN. etal. Science 248:603-5, 1990). HCΝ-1 A cells have been induced to differentiate to a neuronal-like morphology and stain positively for neurofilament, neuron-specific enolase and p75ΝGFR, but not for myelin basic protein, S-100 or glial fibrillary acidic protein (GFAP). Because these cells also stain positively for γ-amino butyric acid and glutamate, they appear to become neuro-transmitting bodies. Earlier Poltorak M et al. (Cell Transplant 1(1):3-15, 1992) observed that HCN-1 cells survived in the brain parenchyma and proposed that these cells may be suitable for intracerebral transplantation in humans.

Ronnet GV et al. (Neuroscience 63(4): 1081-99, 1994) reported that HCN-1 cells grew processes resembling neurons when exposed to nerve growth factor, dibutyryl cyclic AMP and isobutylmethylxanthine.

The nerve cells also can be administered with macrophages which have been activated by exposure to peripheral nerve cells. Such activated macrophages have been shown to clean up the site of CNS trauma, for example a severed optic nerve, after which new nerve extensions started to grow across the lesion. Implanting macrophages exposed to CNS tissue (which secretes a chemical to inhibit macrophages) or nothing at all resulted in little or no regeneration (Lazarov-Spiegler et al. FASEB J. 10:1, 1996).

Fetal pig cells have been implanted into patients with neurodegenerative diseases, such as Parkinson's disease and Huntington's chorea, and intractable seizures, in whom surgical removal of the excited area would otherwise have been performed. Such cells, if properly screened for retroviruses, could also be used in the inventive method.

Neural crest cells are isolated and cultured according to Stemple and Anderson (U.S. Patent No. 5,654,183), which is incorporated herein by reference, with the modification that basic fibroblast growth factor (bFGF) is added to the medium at concentrations ranging from 5 to 100 ng/ml in 5 ng/ml increments. Neural crest cells so cultured are found to be stimulated by the presence of FGF in increasing concentrations about 1 or 5 ng/ml. Such cells differentiate into peripheral nerve cells. which can be used in the instant invention.

Recently we transplanted LBS neuronal cells into the brains of immunosuppressed rodents (Kleppner et al., J. Comp. Neurol. 357:618-32, 1995; Miyazono et al., Brain Pathol. 4:575, 1994; Trojanowski et al., Exp. Neurol. 122:283-94, 1993). In vivo studies indicate that transplanted LBS neuronal cells can survive, mature and integrate into host brain (Kleppner et al., 1995, ibid. ; Mantione et al., 1995, ibid. ; Trojanowski et al., 1993, ibid.).

SUMMARY OF DISCLOSURE It is an object of this invention to prevent graft rejection and modulate autoimmune diseases. This can be performed by the administration of secretory cells which secret a factor capable of preventing graft rejection and modulating autoimmune diseases. The secretory cells are relatively long-lived and obviate the need for frequent parenteral administration of the factor.

A method of preventing or modulating an immunologic or inflammatory reaction in a host comprises administering to the host a sufficient amount of effective secretory or neuronal cells, their supernatant or a factor isolated therefrom; and monitoring the host. The neuronal cells, supernatant and isolated factor can be administered by injection or other routes.

In another embodiment, the secretory cells are encapsulated before administration. The secretory cells can be administered with a cell, tissue or organ transplant, such as pancreatic islet cells. These secretory cells can be administered with solid organ transplants.

In another embodiment, the secretory cells are implanted into a body cavity containing or adjacent to inflammation. Examples of a suitable body cavity include a ventricle of a brain receiving a transplant or affected by Alzheimer's and other autoimmune diseases, abdominal cavity, or inflamed joint. Also disclosed is a method of preventing or modulating an immunologic or inflammatory reaction in a host comprising administering to the host an immunomodulating factor which is secreted by neuronal cells; not present in supernatant from THP and PBL cultures; present in neuronal culture medium dialyzed against a 2 kilodalton membrane; less active after dialysis of neuronal culture medium against an 8 kilodalton membrane; diluted and consequently exerts less inhibition of proliferation induced by pokewood mitogen or phytohemaglutinin; inactivated by peptidases; and capable of preventing inflammation.

Such a factor can be administered systemically by subcutaneous, intradermal. intramuscular or intravenous routes.

In another embodiment, there is disclosed a cellular transplant comprising cells secreting an anti-inflammatory protein, which has been identified in the supernatant of neuronal cells but not in the supernatant of THP or PBL cultures and at least one other cell type capable of secreting at least one protein. The co- transplant may also be encapsulated with a liquid permeable, degradation-resistant material, such as an alginate.

An immunomodulating factor which is secreted by neuronal cells; not present in a supernatant from THP and PBL cultures; present in neuronal culture medium dialyzed against a 2 kilodalton membrane; less active after dialysis of neuronal culture medium against an 8 kilodalton membrane; diluted and exerts less inhibition of proliferation induced by pokewood mitogen or phytohemaglutinin; inactivated by peptidases; and capable of preventing inflammation. In particular, this factor is produced by NT2 and LBS-Neurons human neuronal cells. Also disclosed are cells which are recombinantly engineered to produce the factor.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a bar graph displaying stimulation indices for hNT cells and supernatant, indicating that both strongly inhibit stimulation in the MLC assay.

Figure 2 is a bar graph illustrating the effect of different processing of hNT culture media on the media's ability to inhibit mitogen-induced proliferation. Figure 3 is a bar graph showing the dosage-related effect of different dilutions of culture medium. Figure 4 is a bar graph showing that cultured medium causes immunosuppression without cytotoxicity.

Figure 5 is a bar graph illustrating the lack of immunosuppression by non- neuronal cell cultures. Figure 6 shows a loss of immunosuppressive activity after various treatments which inactivate proteins.

DETAILED DESCRIPTION Research was undertaken to investigate whether donor cells in general can be rendered less immunogenic but retain their therapeutic potential. Grafts composed of cells that possess imuno-modulating or immuno-suppressive characteristics would require less stringent matches across MHC barriers and less toxic drug therapy.

Study of neuronal cells in this context began with an evaluation of whether the retinoic acid (RA) treatment of NT2 precursor cells induces sufficient expression of MHC molecules on neuronal cells to make them immunogemc in a mixed lymphocyte culture (MLC) in which neuronal cells are combined with responder T cells to see if T cells proliferate. Because of a previous report that RA increased MHC I surface expression, we assumed that there would be a brisk response by responder T cells. However, in numerous experiments disclosed herein, neuronal cells and its supernatant (and a factor therein) effectively suppressed T cell proliferation without altering responder T cell viability, indicating that Neuronal cells and/or a factor(s) from neuronal cells suppressed MHC-induced T cell proliferation. Thus, neuronal cells and/or a factor from neuronal cells may suppress rejection of neuronal cells alone or as co-grafts with other transplanted cells, tissues and organs. As the data below show, not only do the neuronal cells produce marked suppression, but their supernatant also suppresses the MLC, and thus neuronal cells constitutively express a factor(s) which can be preserved, modulate auto immunities and abrogate GVHO disease. Definitions: "Beneficial effect" is an observable improvement over the baseline clinically observable signs and symptoms. For example, a beneficial effect could include improvements in graft survival, decreased inflammation or improvements in the disease treated (see below for changes in Huntington's disease, etc.).

"Mammal" includes humans and other mammals who would reasonably benefit from treatment of immune and inflammatory disorders, including pets like dogs, cats and horses.

"NT2/D1 precursor cells" as used herein refers to a special cell line available from Layton Bioscience, Inc. (Gilroy, CA). This cell line has been developed from a previously described human teratocarcinoma cell line (termed Ntera2/clone DI or NT2 cells) (Andrews et al. LAB. INVEST. 50:147-162, 1981). These cells are precursors for LBS-Neurons™ human neuronal cells. NT2/D1 cells are unique among other teratocarcinoma cell lines because these cells act like progenitor cells whose progeny are restricted to the neuronal lineage (Andrews, ibid.)

"LBS-Neuron human neuronal cells" as used herein refers to the special neuronal cell line disclosed in U.S. Patent No. 5,175,103 to Lee et al. Briefly,

NT2/D1 precursor cells are induced to differentiate into neurons by administration of 10 μM retinoic acid which is replenished three times weekly for 5 weeks, after which the cells are replated with special manipulations to become more than 99 % pure neuronal cells. These are the cells which are used in the subsequent experiments. Alternately, for human use, there is a cell line manufacmred without antibiotics (used in the research grade neuronal cells) and under good manufacturing practices (GMP) which is termed LBS NEURONS™ human neuronal cells (Layton Bioscience, Inc.).

"Immunosuppressant" as used herein is a substance which prevents or attenuates immunologic phenomena. For example, such immunologic phenomena include inflammation, autoimmunity, GVHD and graft rejection. Examples of current immunosuppressants include but are not limited to cyclosporine A, cyclophosphamide, prednisone and tacrolimus (FK506).

"Body cavity" can be an existing body cavity, like a sinus, ventricle of the brain, abdominal cavity, joint space, or potential space which becomes a space upon injection of the neuronal cells (e.g., the fat pad surrounding the kidney). A "liquid permeable, degradation resistant material" as used herein can be any material meeting these standards. It must be resistant to biological processes and preferable not elicit them (non-inflammatory) and may be any biologically compatible material such as a polymer polyimide, poly amide, Teflon, Tyvek, polyester and naturally derived polymeric materials such as alginates. The material must be so formulated as to permit the therapeutic agent to leave the implant.

"Therapeutic agent" as used herein means the co-transplanted neuronal cells themselves or chemical entities secreted by these cells. Examples of chemical entities secreted by the cells include but are not limited to proteins and hormones. Specific replacement proteins include, but are not limited to cytokines, insulin, growth hormone, Factor VIII, and Factor IX. Examples of hormones include, but are not limited to thyroid hormone, estradiol and acetylcholine.

The factor(s) disclosed herein appears to be a protein because its activity is decreased by high heat, low pH and the proteases trypsin and carboxypeptidase. The protein has a mass between 2 and 10 kd. Preliminary evidence suggests a mass of about 3-6 kd, or approximately 5 kd. Additional tests indicate that the factor is not TGF-β or IL-10.

EXAMPLES Preparation of Neuronal Cells

Human neuronal cells (fresh or cryopreserved) or medium as described elsewhere in detail were used (Borlongan et al., 1995a, ibid. ; Kleppner et al.. 1995, ibid. ; Mantione et al., 1995; Trojanowski et al., 1993, ibid.). Fresh neuronal cells were prepared as follows: First, growth medium containing DMEM (high glucose), 10% (v/v) FBS and 2 mM glutamine was prepared. In a sterile area (e.g., a Class II biological safety cabinet), the surface of the T-25 tissue culture flask containing the neuronal cells was carefully wiped with 70% alcohol. The cap of the neuronal cells cell shipping container was removed. With a sterile Pasteur pipette, any remaining medium from the inside of the cap and the neck of the tissue culture flask was removed by aspiration. All of the shipping medium from the tissue culture flask was removed, and the culture was fed with 5 ml of neuronal cells inhibitor medium. The flask was capped loosely. The cells were placed at 37°C under 6% CO₂ in a humidified incubator. The medium was replaced with fresh 5 ml of neuronal cells inhibitor medium (Layton Bioscience, Inc., Gilroy, CA) at three days after receipt. On day 6 after receipt, the neuronal cells inhibitor medium was replaced with neuronal cell conditioned medium (Layton Bioscience, Inc.). On day 8 after receipt, the experiment was carried out.

On the day of experiment, the neuronal cells were prepared as follows:

1. The neuronal cells cell supernatant was aliquoted and stored for future analysis or used in future experiments requiring neuronal cells conditioned medium.

2. The cells were washed twice with PBS + Ca²⁺ + Mg²⁺. 3. A solution consisting of 0.5 ml of a 0.025% (v/v) trypsin and 0.01 % (w/v) EDTA solution in PBS (without the calcium and magnesium supplements) / 25 cc was added. The trypsin and EDTA solution was distributed evenly over the cell monolayer by swirling the flask. The flask was incubated for two minutes at room temperature. At this time, the neurons lifted off the monolayer. 4. The trypsinization process was stopped by adding 5 ml of neuronal cells conditioned medium containing 10% (v/v) FBS.

5. Pasteur pipette with pipette aid were used to draw up cells and medium 2-3 times.

6. The cell solution was spun at 300 X g for 10 minutes at room temperature. 7. The supernatant was removed with Pasteur pipettes, leaving the cell pellet at the bottom of the tube.

8. With a pipette aid. 0.1 ml of fresh neuronal cells conditioned medium was added.

9. A 10 μl sample was removed for viability cell count by trypan blue exclusion.

10. The remaining sample was transferred into a sterile microvial via sterile pipette. This cell solution was used for future tests.

Immediately before and after neuronal cells cell experiments, viability cell counts were performed by the trypan blue exclusion method and revealed at least 95% survival rate for neuronal cells. MHC Typing of NT2 and LBS Neuronal cells

Using Terasaki and Lambda serological typing trays (One Lambda, Inc., Canoga Park, CA) and purified antibodies with broad MHC class I or class II specificity (Becton Dickinson, Sparks, MD) in flow cytometric analysis, we analyzed the NT2 and hNT neuronal cell surface expression of MHC antigens. Cells were incubated at room temperature for 30 min, followed by the addition of rabbit complement, ethidium bromide and acridine orange for 60 min. Cell viability was scored.

In addition, MHC genotyping was performed. DNA was extracted from NT2 and hNT neuronal cells and analyzed with sequence-specific primers in the polymerase chain reactions (SSP-PCR) (Class I, ABC, Unitray" , Pel-freez Clinical Systems, Inc., Brown Deer, WI and "SSP-DR Typing", Biosynthesis, Inc., Louiseville, TX.). The kit provides specific primers for each HLAall of interest. The amplified DNA was loaded onto an agarose gel and the electrophoretic patterns analyzed (data not shown ). The immature NT2 cells had detectable MHC class I and II surface protein expression as determined by flow cytometric analysis, but did not have a consistent MHC pattern which could be determined with Terasaki typing. By genotyping, NT2 cells have an HLA type of Al B8 DR3. In contrast, hNT neuronal cells did consistently express MHC class I and II markers in a discernible pattern. hNT neuronal cells were found to have an Al B8 Bw6 Cw7 DR3 DR52 genotype, a common Caucasian set of antigens.

We hypothesized that, because almost all people are exposed to Type Al (and should be immunologically reactive to cells of this type), this MHC class I and II expression by differentiated neuronal cells should stimulate T cell activation and proliferation in a modified mixed lymphocyte culture (MLC) where stimulator T cells were substituted by irradiated LBS- Neurons cells. LBS Neuronal Cells and Supernatant Suppress T Lymphocyte Activation The MLC were set up with total volumes per well of 200 μl, consisting of 100 μl of responder cells (e.g., A) and 100 μl of inducer cells

(e.g., B or neuronal cells or medium conditioned by neuronal cells only). In each well, the final cell concentration was 1 X 10⁶. The modified MLC

(m-MLC) consisted of 100 μl each of 10⁶ stimulator cells and responder

PBL. Blood was collected in heparinized containers from normal donors.

Peripheral blood leukocytes (PBL) were isolated using Accu-Prep (Accurate

Chemical Corp., Westbury NY) and rinsed three times with HBSS (Gibco- BRL, Gaithersburg, MD). Viability and concentrations were established before use in assays.

The MLC were incubated 96 hours at 37°C with 5 % carbon dioxide.

Each culture received a pulse of ³H-thymidine (0.5 μCi/well) and was further incubated 18-22 hours at 37° with 5% carbon dioxide. Plates were refrigerated and harvested. The incorporation of ³H-thymidine is an indication of induced proliferation and was determined by gamma counting on an Inotech reader.

In some experiments, stimulator PBL or hNT cells (hNTx cells) were irradiated at 2,500 rads for 3 min. MLC were set up using either a supplemented DMEM, a supplemented RPMI, hNT supernatant (hNT-sup) or the supernatant from irradiated hNT cedlls (hNTx-sup).

Stimulation index (SI) is a ratio of the measured induced proliferation of responder cells in the presence of irradiated stimulator cells, divided by the induced proliferation of responder cells in thepresence of irradiated responder cells, which is summarized below.

Stimulation Index (SI) = (responder + irradiated stimulator)

(responder + irradiated responder)

Figure 1 shows the SI for a variety of MLC and m-MLC. All MLC with hNT supernatant and cells showed marked depression. Moreover, even when irradiated neuronal cells replaced allogeneic T cells as the stimulator cells, Sis were always less than 0.1, and as low as 0.0006, with a mean of 0.04. Irradiated LBS-Neurons cells reduced the SI by 95-99% . This was completely unexpected, as the neuronal cells were serologically and genetically tested and had a consistent MHC pattern which generally excites an allogeneic response. A SI index comparable to the allogeneic MLC (the left-most bar) was expected, but not seen.

Lymphocyte Proliferation Assay (LPA)

PBL were suspended at 10⁶ cells/ml and plated 100 μl/well in 96- well plates. 100 μl/well of control or mitogen-containing medium was added, and cells were incubated at 37°C in 5% CO₂ for 48 hours. PBL were labeled with 3H-thymidine at 0.5 μCi/well and incubated another 18- 22 hrs. PBL were refrigerated overnight, harvested, and read on an Inotech reader. Mitogens used in LPA included a 1:250 dilution or phytohemagglutinin A (PHA), and a 1:50 dilution of pokeweed mitogen (PWM). Figure 2 shows the results of those tests. The results indicate that the conditioned medium (without or without fetal bovine serum) effectively inhibited mitogen-induced proliferation. When dialyzed against the 2 kd membrane, there was still highly effective inhibition, indicating the factor is larger than 2kd. The loss of activity when the medium was dialyzed against the 8 kd membrane indicates that the factor may be smaller than 8kd.

Figure 3 shows that dilution of the culture medium by 1:5, 1 : 10 and 1:20 produces a stepwise decrease in inhibition, which indicates a dose- related effect.

TGF-B. IL-10 and Nitric Oxide (NO) Assays

Supernatants from MLC and LPA assays collected on Day 3 were used in ELISAs for TGF-β and interleukin (IL-10) (Genzyme, Cambridge MA), or in the Greiss reagent system to detect NO (Promega Corp., Madison WI). No TGF-β, IL-10 or NO was detectable. Effect of HNT-sup on PBL Viability

Whether hNT-sup (CM) was cytotoxic to PBL was determined by detection of LDH in LPA culture supernatant (Cyto Tox 96 Assay, Promega Corp.) PBL viability in m-MLC and LPA was determined by trypan blue exclusion. To test further whether CM affected PBL viability in LPA, CM was replaced with supplemented DMEM on day 2, or vice versa, and the induced proliferation assessed. Controls were DMEM replaced with DMEM (DMEM/DMEM) and CM replaced with CM (CM/CM. Figure 4 shows that proliferation was partially suppressed after replacing DMEM- containing serum with CM (DMEM/CM). Proliferation was partially restored when replacing CM with DMEM (CM/DMEM). Hence the immunosuppression is reversible and not likely to be caused by cytotoxicity.

Specificity of the Factor for Neuronal Cells Media used in LPA included CM from mixed cultures of 10-15% hNT neurons and approximately 85 % accessory cells prior to their inhibition, or serum-free CM (without FBS - CM-SF), or CM-SF dialyzed to either a 2kd or 8 kd membrane (CM-SF-2kd and CM-SF-8kd, respectively), or supernatants from cultures of THP- 1 (THP-sup), a monocytic leukemia derived cell line (ATCC #TIB 202) of PBL (PBL-sup) cultured from a normal donor. Figure 5 shows that supernatants from non- neuronal cultures have essentially no effect in LPA, compared to hNT culture medium and NT-2 neuronal supernatant, both of which exerted considerable inhibition. Similar tests are readily performed with other neuronal cells, such as fetal nerve cells and neural crest nerve cells, and cells engineered to produce the inventive factor recombinantly.

Characteristics of the Immunosuppressive Factor of CM

CM was treated with one of the following: heat (56°C for 30 min), low pH (IN HC1 for 30 min at room temperature), trypsin attached to agarose beads (Sigma) and incubated at 37°C for 1 hr, and carboxypeptidease A on agarose beads for 1 hr. Following treatments, CM was evaluated for the retention or loss of the immunosuppressive effect. As shown in Figure 6, treatments that typically cause a protein to lose some or all of its activity (high heat, low pH, and trypsin and carboxypeptidase enzymes) resulted in increased proliferation, or loss of factor activity.

Further Tests to Identify the Immuno-modulating Factor(s)

The protein sequence(s) of unknown f actor (s) is determined. The immunosuppressive and in vivo anti-inflammatory effects of neuronal cells and the active factor(s) are determined in animal models. For example, nude, diabetic, arthritic and normal inbred mice receive skin allografts and neuronal cells or medium conditioned by neuronal cells for eight days, along with appropriate controls. Neuronal cells and the active factor(s) are observed for immunosuppressive effects in vivo, including but not limited to prolonging skin graft retention, modulating and preventing diabetes, arthritis, systemic and organ-specific autoimmunities, and permitting engraftment of allogeneic skin, tissues, organs, stem cells, bone marrow cells (with or without T cell depletion of the marrow graft).

Diabetes In diabetic rodent model(s), neuronal cells are co-administered with pancreatic β-islet cells. The β-islet cells replace the missing insulin, and the neuronal cells and factors provide protection of the engrafted islet cells, and modulate any anti-β-islet cell inflammation associates with the pathogenesis of the diabetes. The current methods of implanting and maintaining islet cells are employed and are discussed, for example, in U.S. Patent Nos.

5,429,821; 5,5121,079; and 5,578,314. These patents disclose methods for encapsulating cells in alginate and hardening the alginate coating to prevent its degradation in the body and prevent the encapsulated cells from contacting T cells and cell-mediated reactions and death of the implant. Nevertheless, after a few weeks to months, the cells stop secreting insulin, probably due to host rejection. A combination of islet cells and anti-inflammatory neuronal cells are preferably encapsulated in a biocompatible polymer such as polyethylene, Teflon^®, polyamine or alginate as described above. The encapsulated combination is administered in an effort to provide a longer-lived implant than islet cells alone.

Other Substance Replacement Methods

Neuronal cells can be combined with other cells or tissues which secrete needed proteins or other substances in order to provide protection from immune response and thus increasing the time the implant remains active. The secretory cells may be natural producers of the desired substance, or the cells may have been genetically engineered to produce the substance. Examples of desirable categories of proteins are cytokines, hormones and growth factors. Specific replacement proteins include, but are not limited to, human growth hormone, Factor VIII, Factor IX, thyroid hormone and estradiol. Examples of suitable patients include those with inherited metabolic deficiencies and other genetic disorders (e.g., cystic fibrosis, hemophilia and multiple sclerosis) and those who acquire the deficiencies through accident, injury or other occurrence (hypothyroidism). The dose and frequency of administration depends on a variety of factors, including but not limited to patient weight, age, sex, anticipated output of the protein, mode of administration, normal rate at which the protein- secreting and/or neuronal cells stop functioning.

The dose of neuronal cells in humans varies from about 2 X 10⁵ to about 2 X 10⁸ cells.

Parkinson's Disease Co-Graft

Neural transplantation has been applied clinically in Parkinson's Disease (PD) patients with favorable results (e.g., Freeman et al. ANN. NEUROL. 38: 379- 88, 1995). Currently most successful transplantation studies are performed on immunosuppressed patients, taking drugs such as cyclosporine, which impairs the immune system and has other serious side effects. Freeman et al., 1995, describe the clinical protocol for implanting 6 Vi to 9 week old fetal cells bilaterally into the postcommissural putamen, using transplant deposits from 3-4 fetal donors on each side. The transplant deposits were separated by no more than 5 mm in three dimensions. Instead of cyclosporine, the fetal transplants are accompanied by small aliquots of immunosuppressive neuronal cells at each deposit location. That way the immunosuppressive effect is immediately adjacent the fetal cells, thereby exerting a localized protective effect.

Huntington's Chorea

Huntington's Disease (HD) is a degenerative disorder of the nervous system characterized by choreoathetosis, caudate atrophy, dementia and personality changes. Patents experience decreasing mobility, nutritional deficiencies from swallowing difficulties, slowing of cognition, memory disturbances, impairment in visuospatial ability, and deficits in a number of executive functions including word generation, mental flexibility, problem- solving and abstraction of concepts.

Pharmacological treatments have had little effect in controlling the movement and neuropsychological decline in HD patients. Recently a preliminary clinical trial of neural transplantation in HD patients was conducted (Philpott et al., CELL TRANSPLANT. 6:203-12, 1997). Three patients were given a battery of pre- and 4-6-month post-transplantation neuropsychological tests; they improved in skills most affected by basal ganglia degeneration, which is impaired in HD. Philpott et al., 1997 describe in detail the procedures of obtaining 8-10-week-old fetal lateral ganglionic eminence tissue and its implantation in the caudate and putamen.

In a clinical trial of immunosuppressive neuronal cells to be implanted with fetal lateral ganglionic eminence tissue, patients receive pre- and post-operative testing of motor and cognitive skills most affected by basal ganglia degeneration. Neuronal cells are implanted with fetal cells at 5 locations bilaterally in the caudate and putamen as described in Philpott et al., 1997. Alzheimer's Disease

At least part of the disease process in Alzheimer's Disease (AD) is alleged to be auto-inflammatory. AD is characterized by extensive convolution of the brain, including the frontal, parietal and medial temporal regions with a corresponding enlargement of the ventricular system. To arrest the inflammatory process, anti- inflammatory neuronal cells and/or the anti-inflammatory factor can be injected into the brain parenchyma or ventricles, either as individual cells or as coated pellets (see above). In addition, biochemical studies of AD have shown that choline acetyltransferase, the enzyme required for acetylcholine (ACh) synthesis is decreased in the cerebral cortex. Because the major source of neocortical ACh is a group of neurons in the basal part of the forebrain just beneath the corpus striatum - the nucleus basalis of Meynert, verified as a site of major neuronal loss, this is another target for neuronal cells co-transplant. In this site, fetal cholinergic cells can be co-transplanted with anti-inflammatory neuronal cells to provide long-functioning replacement for nucleus basalis cells.

Multiple Sclerosis and Other Autoimmune Diseases

Multiple sclerosis (MS) is a disease of chronic inflammation, demyelination and gliosis (scarring). Current therapy consists of drugs which suppress the entire immune system, particularly methylprednisone; however, there "is no evidence that their use alters the long-term course of the disease." (HARRISON'S INTERNAL MEDICINE, 13^th Ed. , 1994, McGraw- Hill, New York City, p. 2292). Also the antimetabolite azathioprine may be given orally on a chronic basis, but its use must be weighed against increased risks of hepatitis, susceptibility to infection and a possible increased cancer risk (HARRISON'S, p. 2293). A more selective, long-term therapy consists of implanting immunosuppressive neuronal cells in multiple parts of brain, particularly the ventricular system. There the cells exert their immunosuppressive effect, countering the destructive inflammation and permitting repair of glial cells, etc.

Autoimmune glomerulonephritis is another autoimmune disease that is resistant to all but the most powerful therapies. In this instance, immunosuppressive neuronal cells are preferably implanted in the abdominal cavity or in the retroperitoneal space, particularly the fat pad surrounding the affected kidney (s).

Autoimmune thyroid disease also can benefit from a local implant of neuronal cells, preferably in the neck and even inside the thyroid itself. Rheumatoid arthritis is another autoimmune disease with chronic inflammation in localized spaces, the joints. Where there is active, acute inflammation, neuronal cells are injected into the synovial cavity to provide a localized anti-inflammatory effect.

Some patients have excessive wound healing which results in abnormally large scars: The inflammatory wound healing process needs better control. In this case, neuronal cells can be given to modulate inflammation and healing.

Additionally, after abdominal surgery, intra-abdominal adhesions frequently develop, which if unchecked may interfere with organ function, such as strangulating the bowel. Neuronal cells or the isolated factor are placed in the abdomen before the surgeon closes the abdomen to limit adhesion formation.

The foregoing description and examples are intended only to illustrate, not to limit, the disclosed invention.

Claims

We claim:

1. A method of preventing or modulating an immunologic or inflammatory reaction in a host comprising: administering to the host a sufficient amount of cells secreting an anti- inflammatory factor; and monitoring the host.

2. The method of claim 1 wherein the secretory cells are administered by injection.

3. The method of claim 1 wherein the secretory cells are neuronal cells and are encapsulated before administration.

4. The method of claim 1 wherein the secretory cells are administered with a cell, tissue or organ transplant.

5. The method of claim 1 wherein the secretory cells are administered with pancreatic islet cells.

6. The method of claim 1 wherein the secretory cells are administered with solid organ transplants.

7. The method of claim 1 wherein the secretory cells are implanted into a body cavity containing or adjacent to inflammation.

8. The method of claim 7 wherein the body cavity is a ventricle of the brain, abdominal cavity, or inflamed joint.

9. A method of preventing or modulating an immunologic or inflammatory reaction in a host comprising: administering to the host an immunomodulating factor which is a) secreted by neuronal cells; b) not present in a supernatant from THP and PBL cultures; c) present in neuronal culture medium dialyzed against a 2 kilodalton membrane; d) less active after dialysis of neuronal culture medium against an 8 kilodalton membrane; e) diluted and exerts less inhibition of proliferation induced by pokewood mitogen or phytohemaglutinin; f) inactivated by peptidases; and g) capable of preventing inflammation.

10. The method of claim 9 wherein the factor is administered systemically by subcutaneous, intradermal, intramuscular or intravenous routes.

11. A cellular transplant comprising neuronal cells and at least one other cell type capable of secreting at least one therapeutic agent.

12. The cellular transplant of claim 11 wherein the therapeutic agent is a protein.

13. The cellular transplant of claim 11 further comprising a liquid permeable, degradation-resistant material which encapsulates the cells.

14. The cellular transplant of claim 13 wherein the encapsulating material is an alginate.

15. An immunomodulating factor which is: a) secreted by neuronal cells; b) not present in a supernatant from THP and PBL cultures; c) present in neuronal culture medium dialyzed against a 2 kilodalton membrane; d) less active after dialysis of neuronal culture medium against an 8 kilodalton membrane; e) diluted and exerts less inhibition of proliferation induced by pokewood mitogen or phytohemaglutinin; f) inactivated by peptidases; and g) capable of preventing inflammation.

16. The factor of claim 15, wherein the neuronal cells are selected from NT2 cells, LBS-Neurons human neuronal cells, neural crest cells, human fetal nerve cells, and pig fetal nerve cells..

17. Cells which are recombinantly engineered to produce the protein of claim 15.