WO2000032209A2 - Submucosa modulation of mammalian immune response - Google Patents

Abstract

A composition and method for locally suppressing the cell mediated immune response of a vertebrate species is described. The method comprises contacting the site in need of immune suppression with a composition comprising vertebrate submucosa.

Description

SUBMUCOSA MODULATION OF MAMMALIAN IMMUNE RESPONSE

Field of the Invention

The present invention relates to the use of vertebrate submucosa to suppress the cell mediated immune response of vertebrates.

Background of the Invention

A significant complication in conducting organ transplantation results from organ rejection by the host immune system. The ability to suppress the immune response and prevent rejection of implanted xenografts is critical for the ultimate success of the implanted graft/organ. Historically, agents which suppress portions of the immune system invariably have systemic effects which create associated problems in the recipient. A substance that modulates a local immune response is desirable. Such a material can be used to induce the acceptance of xenogeneic grafts, such as kidney, hearts or other transplanted organs.

T helper (Th) lymphocytes can be classified into two subsets (Thl and Th2) based on their cytokine secretion patterns. The differentiation of naive CD4⁺ lymphocytes to either the Thl or Th2 phenotype is influenced by the local environment during stimulation. Thl lymphocytes produce interleukin-2 (IL-2), interferon-γ (IFN- γ), and tumor necrosis factor-β (TNF-β), while Th2 lymphocytes produce IL-4, IL-5, IL-6, and IL-10.

The classification of T helper (Th) lymphocytes into two subsets Thl and Th2 has been used to analyze immune responses to xenotransplants. Thl cells initiate the cell mediated immune response through macrophage activation and stimulation of complement fixing antibody (Ab) production (IgG_2a and IgG_2b in mice). This pathway is associated with traditionally accepted criteria of inflammation and leads to the differentiation of CD8⁺ cells to a cytotoxic phenotype and, ultimately, rejection of a xenograft. Th2 cells mediate the humoral immune response, which does not involve macrophage activation and leads to production of non-complement fixing Ab (IgG, in mice). The Th2 pathway, which tends to be anti-inflammatory in its cellular and cytokine response, is not typically associated with graft rejection and may promote immune tolerance to a xenograft. Thl and Th2 pathways are mutually inhibitory due to the action of IL- 10 on Thl cells and IFN-γ on Th2 cells, so that generally one pathway is dominant. Which pathway dominates is determined by the type of antigen and by the cytokines present when lymphocytes are activated (Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 383: 787-793, 1996).

It is known that compositions comprising the tunica submucosa and the basilar portions of the tunica mucosa of the intestine of warm-blooded vertebrates can be used as tissue graft materials in sheet form. See U.S. Patent No. 4,902,508. The compositions described and claimed in that patent are characterized by excellent mechanical properties, including high compliance, a high burst pressure point, and an effective porosity index which allows such compositions to be used beneficially for vascular graft constructs. The graft materials disclosed in that patent are also useful in tendon, ligament and other connective tissue replacement applications. Furthermore, intestinal submucosa has been used as a scaffold for regenerating other tissues including urinary bladder and dura mater. When used in such applications the preferred graft constructs appear to serve as a matrix for the regrowth of the tissues replaced by the graft constructs.

Vertebrate submucosa is a plentiful by-product of commercial meat production operations and is thus a low cost tissue graft material, especially when the submucosa is used in its native sheet configuration. Furthermore, it is known that submucosa can be fluidized by comminuting and/or protease digestion, without loss of its apparent biotropic properties, for use in less invasive methods of administration (e.g., injection or topical) to host tissues in need of repair. See U.S. Patent No. 5,275,826. When used as a xenogeneic graft material, vertebrate submucosa induces specific remodeling in the organ or tissue into which it is placed. Common events to tissue remodeling include widespread and rapid neovascularization, proliferation of granulation mesenchymal cells, biodegradation of implanted submucosa, and lack of immune rejection. Intestinal submucosa has undergone extensive immunologic testing in over 600 cross-species implants and has never been shown to elucidate a rejection reaction.

One explanation for the apparent immune tolerance to xenogeneic vertebrate submucosa is that submucosa elicits an immune response which is inherently benign (i.e. a Th2 dominant immune response). The present invention is directed to the use of vertebrate submucosa to locally suppress the cell mediated immune response in a vertebrate species by suppressing the Thl immune response. The method comprises the steps of contacting a site in need of immune suppression with an effective amount of a composition comprising vertebrate submucosa.

Detailed Description of the Invention

The present invention is directed to the use of vertebrate submucosa to moderate the immune response of vertebrate species to an implanted xenograft. The immune response to a xenograft comprising porcine intestinal submucosa that is implanted in mice is predominantly Th2 mediated. Therefore the submucosa implant appears to suppress the Thl immune response. Furthermore, the immuno suppressant effect of intestinal submucosa is localized and does not significantly impair the host's ability to respond to an immunological challenge. The impact of implanted submucosa on a host's ability to respond to other antigens was investigated by implanting xenogenic intestinal submucosa (porcine) in mice and measuring the effect on the host systemic response to other antigens. Well established immunologic phenomena were evaluated to minimize ambiguity of any interaction between submucosa implantation and the expected normal course of the response. The results indicate that vertebrate submucosa elicits a Th2 dominant immune response to the submucosa xenograft but does not significantly impact the hosts' response to other antigenic challenges.

In accordance with one embodiment of the present invention, a method for locally suppressing the cell mediated immune response in a vertebrate species is provided. The method comprises the steps of contacting a site in need of immune suppression with an effective amount of a composition comprising vertebrate submucosa. Preferably, the submucosa comprises intestinal submucosa of a warmblooded vertebrate, and one particularly preferred source of the submucosa is the small intestine of warm-blooded vertebrates. Suitable submucosa comprises the tunica submucosa delammated from the tunica muscularis and at least the luminal portion of the tunica mucosa. In one preferred embodiment of the present invention the submucosa is intestinal submucosa comprising the tunica submucosa and basilar portions of the tunica mucosa including the lamina muscularis mucosa and the stratum compactum which layers are known to vary in thickness and in definition dependent on the source vertebrate species. Submucosa tissue can also be prepared from other organs of vertebrate species, for example, from the urogenital system, including the urinary bladder (see U.S. Patent Nos. 5,554,389), and other portions of the digestive tract including the stomach (see published PCT application no. WO98/25636). The disclosures of U.S. Patent Nos. 5,554,389 and published PCT application no. WO98/25636 are expressly incorporated herein.

The preparation of submucosa for use in accordance with this invention is described in U.S. Patent Nos. 4,902,508 and 5,554,389. To summarize, submucosa is prepared from vertebrate intestine (or other organ source), preferably harvested from porcine, ovine or bovine species, but not excluding other species, by subjecting the intestinal tissue to abrasion using a longitudinal wiping motion to remove the outer layers, comprising smooth muscle tissues, and the innermost layer, i.e., at least the luminal portion of the tunica mucosa. The submucosa is rinsed with saline and optionally sterilized; it can be stored in a hydrated or dehydrated state. Lyophilized or air dried submucosa can be rehydrated and used in accordance with this invention without significant loss of its cell proliferative activity. Native submucosa as a starting material is a relatively acellular collagenous matrix and the process of preparing intestinal submucosa for use as the collagenous matrix component of the present invention produces a collagenous matrix devoid of intact cells. Accordingly the submucosa collagenous matrix prepared in accordance with the present invention is acellular.

In accordance with one embodiment multiple sheets of submucosa can be overlapped with each other to form a multi-layered construct. The individual layers can be fix to one another using standard techniques know to those skilled in the art, including the use of sutures, staples and biocompatible adhesives such as collagen binder pastes. In one embodiment the layers are fused together by compressing the overlapped regions under dehydrating conditions, optionally with the addition of heat as described in US Patent No. 5,711,969, the disclosure of which is expressly incorporated herein. In one embodiment the multi-layered submucosa constructs are perforated to allow fluids to readily pass through the graft construct and prevent pockets of fluids from accumulating between the layers. The formation of perforated multilayered constructs is described in US Patent No, 5,755,791, the disclosure of which is expressly incorporated herein.

The vertebrate submucosa of the present invention can be conditioned to alter the viscoelastic properties of the submucosa by stretching the material in a longitudinal or lateral direction as described in U.S. Patent No. 5,275,826, the disclosure of which is expressly incorporated herein by reference. In accordance with one embodiment submucosa delaminated from the tunica muscularis and luminal portion of the tunica mucosa is conditioned to have a strain of no more than 20%. The submucosa is conditioned by stretching, chemically treating, enzymatically treating or exposing the tissue to other environmental factors. In one embodiment submucosa is conditioned by stretching in a longitudinal or lateral direction so that the submucosa tissue has a strain of no more than 20%.

The vertebrate submucosa can be sterilized using conventional sterilization techniques including glutaraldehyde tanning, formaldehyde tanning at acidic pH, ethylene oxide treatment, propylene oxide treatment, gas plasma sterilization, gamma radiation, electron beam and peracetic acid sterilization. Sterilization techniques which do not adversely affect the mechanical strength, structure, and biotropic properties of the submucosa are preferred. For instance, strong gamma radiation may cause loss of strength of the sheets of submucosa. Preferred sterilization techniques include exposing the graft to peracetic acid, 1-4 Mrads gamma irradiation (more preferably 1-2.5 Mrads of gamma irradiation) or gas plasma sterilization; peracetic acid sterilization is the most preferred sterilization method. Typically, the submucosa is subjected to two or more sterilization processes. After the submucosa is sterilized, for example by chemical treatment, the tissue may be wrapped in a plastic or foil wrap and sterilized again using electron beam or gamma irradiation sterilization techniques.

It is also known that vertebrate submucosa can be fluidized by comminuting and/or enzymatic digestion, without loss of its apparent biotropic properties, for use in less invasive methods of administration (e.g., by injection or topical application) to host tissues in need of repair. See U.S. Patent No. 5,275,826, the disclosure of which is expressly incorporated herein by reference. More particularly, the native or fluidized submucosa formulation can be treated with an enzyme for a period of time sufficient to solubilize all or a major portion of the submucosa components. Preferably the submucosa is digested with an enzyme that hydrolyzes the structural components of the submucosa to produce a suspension or homogenous solution of submucosa components. Submucosa can be enzymatically treated with proteases (for example, a collagenase or trypsin or pepsin), glycosaminoglycanases or a combination of proteases and glycosaminoglycanases. Optionally, other appropriate enzymes (i.e. those that hydrolyze the structural components of the submucosa without substantially adversely impacting the biotropic properties of the material) can be used alone or in combination with proteases and glycosaminoglycanases. The tissue digest can be optionally filtered to provide a homogenous solution of partially solubilized submucosa.

The viscosity of fluidized submucosa for use in accordance with this invention can be manipulated by controlling the concentration of the submucosa component and the degree of hydration. The viscosity can be adjusted to a range of about 2 to about 300,000 cps at 25°C. Higher viscosity formulations, for example, gels, can be prepared from the submucosa digest solutions by dialyzing the digested material and then adjusting the pH of such solutions to about 6.0 to about 7.0.

The present invention also contemplates the use of powder forms of submucosa. In one embodiment a powder form of submucosa is prepared by pulverizing submucosa under liquid nitrogen to produce particles ranging in size from 0.1 to 1 mm². The particulate composition is then lyophilized overnight and sterilized to form a solid substantially anhydrous particulate composite. Alternatively, a powder form of submucosa can be formed from fluidized submucosa by drying the suspensions or solutions of comminuted submucosa. The results shown in Examples 1 and 2 suggest a Th2 dominant host response to the submucosa implant and such a response is compatible with graft acceptance. Although Th2 dominance does not guarantee graft acceptance, it appears that Thl dominance is not compatible with acceptance of cellular xenogeneic grafts. Thl mediated rejection of acellular xenografts has not been described, although destruction of glutaraldehyde treated bovine pericardial heart valve grafts by an apparent (uncharacterized) immune process has been reported.

It is possible that the Th2 dominant response is beneficial in several ways, including prevention of Thl mediated rejection of the submucosa graft. Organ graft rejection is based on killing graft cells and interrupting its blood supply which result in a non-functional organ. The acellular nature of submucosa makes this mechanism of rejection unlikely, since the cells which populate an submucosa graft and its blood supply are host-derived and thus non-immunogenic. The fact that submucosa is gradually resorbed also likely plays some role in the positive host response, as this decreases the xenoantigen load over time and leaves only host tissue remaining. The immune response to submucosa is not associated with clinical or histologic evidence of rejection. The immunohistochemical and histopathologic analyses are consistent with Th2 dominant immune response. In addition, assays of both the local intragraft and systemic immune activities indicate Th2 dominance.

Whether a Th2 dominant response to submucosa predisposes a host animal to infection by inhibiting Thl responses systemically (IL-10 activity) has been investigated. It seems clear that submucosa has marginal or no effect on the normal immune response of BALB/c or C57BL/6 mice to various anti genie challenges, including DNP-Ovalbumin Challenge, Contact Dermatitis Assay, Inactivated Influenza Virus Challenge and Xenogeneic Skin Graft Rejection Assay (see Example 1). In summary, the results of these assays suggest that exposure to submucosa does not cause predisposition to infection or other immuno insufficiency due to the Th2 dominant response to submucosa. The collected observations of this study show that any influence of submucosa in these assays was only in the magnitude and timing, but not in fundamental character, of the immune response. Specifically, the anti-DNP Ab response was slightly altered in the relative amounts of Thl and Th2 associated Ab types produced, but not leading to a complete polarization to only one or the other type. Similarly, the contact dermatitis model showed suppression of the intensity of ear swelling response, but the expected normal response was present and significantly greater than non-treated controls.

In accordance with the present invention a submucosa implant is used to elicit a localized Th2 immune response in a particular region of the host's body. Thus if the submucosa is placed in proximity to a xenograft or other source of nonendogenous biomaterial that would normally elicit a Thl immune response, the submucosa provides a protective effect and prevents the rejection of the xenograft. In accordance with one embodiment, the submucosa is intestinal submucosa comprising the tunica submucosa delaminated from the tunica muscularis and at least the luminal portion of the tunica mucosa of vertebrate intestine.

The vertebrate submucosa can be implanted into the host site of the xenograft implant either before implantation of the xenograft or simultaneously with the implantation of the xenograft. In accordance with one embodiment the immunogenic biomaterial is wrapped or encapsulated with solid sheets of submucosa. Alternatively, the immunogenic biomaterial may be contacted with fluidized submucosa. In particular, fluidized submucosa can be injected into the proposed site of implantation to prepare the site for implantation of the xenograft. Additional injections of fluidized submucosa can optionally be administered after implantation of the xenograft. The fluidized submucosa can be comminuted and/or digested with an enzyme for a period of time sufficient to solubilize the submucosa.

In accordance with one embodiment, vertebrate submucosa is utilized as a carrier for immunogenic material. In this embodiment, the method of protecting immunogenic biomaterial from the immune system of a host organism comprises the step of encapsulating the biomaterial within a construct comprising vertebrate submucosa and implanting the composition into the host organism. In accordance with one embodiment the immunogenic material comprises a population of non-autologous cells that are embedded in the submucosa matrix or attached to the matrix surface. In one embodiment the cells are cultured on the submucosa in vitro before implantation into the host. Alternatively, for larger immunogenic biomaterial, one or more sheets of submucosa can be shaped into a tube or sphere that encapsulates the biomaterial.

Furthermore it is anticipated that compositions comprising fluidized vertebrate submucosa or submucosa components will be capable of modulating an immune response. Accordingly, the present invention is also directed to a method of treating an autoimmune disorder, said method comprising the step of administering a pharmaceutical composition comprising vertebrate submucosa to a host afflicted with an autoimmune disorder. Vertebrate submucosa prepared from non-human and non-Old World

Apes can be subject to further modification as an added precaution of avoiding an immune response. Galactosyl- (l,3)galactose (referred to as the Gal epitope) is a glycosyl modification of cell surface components and some serum proteins in all mammals, except humans and Old World apes. The epitope comprises a terminal galactose moiety linked to another galactose moiety through an 1-3 linkage. It has been shown that human serum contains naturally occurring IgG and IgM antibodies directed against this epitope. It is estimated that 1% of all circulating IgG in humans is anti-Gal. This high level of anti- Gal epitope antibodies is thought to be produced in response to endogenous bacteria in the gastrointestinal system; the lipopolysaccharides of those bacteria contain the Gal epitope. Xenogeneic transplantation of organ tissue into a human host results in IgG and IgM antibodies binding to the Gal epitope (especially for those epitopes located on endothelial cells), the initiation of an inflammatory reaction, and vascular thrombosis and hyperacute xenograft rejection of the transplant. Accordingly, a major obstacle to successful xenotransplantation of porcine and other non-Old World ape vertebrate species organs into humans is the presence of Gal epitopes on the tissues of those organs. The Gal epitope has also been found in porcine submucosa prepared in accordance with the procedures disclosed in U.S. Patent Nos. 4,902,508 and 5,281,422. It is not known whether the Gal epitope exists as a naturally occurring component of the submucosa or whether the epitope is a remnant of cell lysis, and remains attached to the submucosa during processing of the submucosa. There have been no reports of vertebrate submucosa graft constructs inducing an immune response after implantation in the animal systems in which it has been tested, including rats, mice, dogs, cats, rabbits, sheep and monkeys. However, due to the association of the Gal epitope with hyperacute xenograft whole organ transplant rejection in humans, the preferred submucosa material used as described in the present invention would comprise submucosa substantially free of the Gal epitope. Thus in one embodiment of the present invention, isolated vertebrate submucosa is treated with galactosidase to be substantially free of the Gal epitope prior to use of the submucosa in accordance with the present invention. The term "Gal free submucosa" refers to submucosa that is substantially free of all detectable amounts of the Gal epitope as determined by antibody and lectin assays.

It is anticipated that each of the various forms of vertebrate submucosa (native, fluidized, protease or GAGase treated and powder forms) has the Gal epitope associated with the material. In accordance with the present invention, those various forms of submucosa can be further modified to reduce the amount of Gal epitope present in the submucosa. Alternatively, the native submucosa can be first enzymatically treated to reduce the amount of Gal epitope present in the tissue before the tissue is fluidized, enzymatically treated, or formed into powder form. In one embodiment, native submucosa is treated with α-galactosidase to produce a Gal epitope depleted submucosa. In a preferred embodiment the submucosa is hydrolyzed with α-galactosidase until it is substantially free of detectable amounts of the Gal epitope. The Gal free tissue can then optionally be further manipulated to produce the described fluidized, protease/GAGase treated, and powder forms of submucosa.

Gal free submucosa can be prepared in accordance with the present invention by contacting the submucosa with an enzymatic solution wherein the enzyme destroys or separates the Gal epitope from the submucosa. Preferably the enzyme is α-galactosidase. The submucosa is contacted with the enzyme under conditions (including temperature, pH, salt concentration, etc) suitable for enzymatic activity. The digestion is conducted for a time sufficient to reduce the Gal epitope content of the submucosa. Preferably, the Gal epitope concentration associated with the submucosa is reduced by greater than 50%, more preferably Gal epitope concentration is reduced by greater than 90% and in accordance with one embodiment vertebrate submucosa is enzymatically treated to be substantially free of detectable amounts of the Gal epitope. After the submucosa has been enzymatically digested to deplete the Gal epitope content, the tissue is repeatedly washed in saline or a suitable buffered solution to remove the cleaved epitope and the enzyme. Alternatively, after enzymatic digestion to deplete Gal epitope content, the tissue can be dialyzed against a buffered solution to remove the cleaved epitope and enzyme.

In accordance with one embodiment the submucosa is treated with α- galactosidase at a concentration ranging from about 5 to about 100 units/ml, and more preferably about 10 to about 50 units/ml for 6-12 hours. Each digestion reaction typically comprises approximately about 10 to about 100 mg of submucosa, and more preferably about 40 to about 60 mg of submucosa. Accordingly, about 0.2 to about 5 units of enzyme are added per 1 mg of submucosa, and more preferably about 0.25 to about 2 units of enzyme are added per 1 mg of submucosa and the tissue is incubated at 37°C for 6-12 hours.

Example 1

The Effect of Vertebrate Submucosa on a Host's Immune Response to Antigens The effect of submucosa on antibodies produced response to dinitrophenol-ovalbumin conjugate (an injected soluble antigen), the cell mediated response to dmitrofluorobenzene (a topically-applied skin sensitizing agent), and Ab response to influenza virus (injected antigen) were measured. The effect of submucosa implantation on rejection of a concomitant xenogeneic skin graft was also examined. Two strains of mice were used to determine whether or not the observed effects were strain-dependent.

MATERIALS AND METHODS

Surgery Female BALB/c and C57BL/6 mice (approximately 25 g) were anaesthetized with methoxyflurane, and the abdomen clipped and disinfected. An incision was made along the ventral midhne and a subcutaneous pocket prepared. A 1 cm x 1cm square graft of porcine submucosa was placed m the pocket. The skin was closed with a continuous suture of 6-0 polypropylene. Control animals were subjected to surgery and closed without implantation of tissue All procedures were performed using aseptic technique. All mice were treated prophylactically with oral enrofloxacm (5 mg/kg) for 7

until sacπfice.

DNP-Ovalbumm Challenge The experimental groups for this model are described in Table 1 On the specified day follow mg submucosa implantation, the animals were injected mtrapentoneally with 200 μl of 1 mg/ l dinitrophenol-ovalbumin conjugate (DNP- ovalbumm, Solid Phase Sciences, San Rafael, CA) emulsified in complete Freund's adjuvant (Sigma Chemical Company, St. Louis, MO). Blood was drawn 3 weeks following challenge and clotted at 37 °C The serum fraction was stored frozen at

20 °C until analyzed by sandwich ELLS A for antibodies (Ab) against DNP-ovalbumin Table 1. Experimental Groups for DNP-Ovalbumin Challenge

Challenge Bleed

Group Strain n Treatment Day Day

A BALB/c 5 Sham 1 22

B BALB/c 5 SIS SQ 1 22

C BALB/c 5 SIS SQ 7 28

D BALB/c 5 SIS SQ 14 35

E BALB/c 5 SIS SQ 28 49

F C57BL/6 5 Sham 1 22

G C57BL/6 5 SIS SQ 1 22

Anti-DNP Ab were assayed using a sandwich ELISA method. Briefly, wells of microliter plates (Nalge Nunc International, Rochester, NY) which were previously coated with DNP bovine serum albumin conjugate. The plates were washed with PBS containing 0.1% (w/v) gelatin and 0.05% (v/v) Tween 20. After washing, serial dilutions of serum were added and the plates incubated for 2 hours at RT. The plates were again washed and incubated with goat anti-mouse IgG,, IgG_2a, or IgG_2b Ab conjugated to alkaline phosphatase (Southern Biotechnology Associates,

Birmingham, AL). Total Ig levels were detected with alkaline phosphatase conjugated to goat anti-mouse Ig (H+L) specific Ab. After incubation for 1 hour, the plates were washed and p-nitrophenyl phosphatase substrate was added to obtain an optimal color development. Plates were read at 405 nm on an ELISA reader (Bio-Tek Instruments, Winooski, VT). Optimal working dilutions of the Ab conjugates and specificity for the target isotype were established using plates coated with purified mouse myeloma proteins (Sigma).

Contact Dermatitis Assay The experimental groups for this model are described in the Table 2. On the specified day following submucosa implantation, the animals were sensitized by application of 20 μl of 0.5% dinitrofluorobenzene (DNFB; Sigma) in a mixture of acetone and olive oil (3:1) to the shaved abdomen. Five days after sensitization with DNFB, the thickness of each ear of each animal were measured using a spring-loaded caliper (Mitutoyo). One ear was then challenged with 20 μl of 0.2% DNFB while the other ear was left untreated. The thickness of each ear was measured again after 24 hours. The ratio of post-challenge to pre-challenge thickness was calculated for each ear.

Table 2. Experimental Groups for Contact Dermatitis Assay

Sensitize Challenge

Group Strain n Treatment Day Day

H BALB/c 5 Sham 1 6

I BALB/c 5 SIS SQ 1 6

J BALB/c 5 SIS SQ 7 12

K C57BIJ6 5 Sham 1 6

L C57BL/6 5 SIS SQ 1 6

M C57BL 6 5 SIS SQ 7 12

Inactivated Influenza Virus Challenge

The experimental groups for this model are described in Table 3. On the specified day following submucosa implantation, the animals were injected subcutaneously with 0.44 ml of 114 μg/ml inactivated human influenza virus (strain HlNl; provided by Dr. C.C. Wu, Animal Disease Diagnostic Laboratory, Purdue University) without adjuvant. Blood was drawn at 3 weeks following challenge and clotted at 37 °C. The serum fraction was stored frozen at -20 °C until analyzed for Ab against the influenza virus.

Anti-virus Ab was assayed by a sandwich ELISA using plates coated with 200 HAU/well of inactivated influenza virus. Otherwise, the assay was performed exactly as described above. Table 3. Experimental Groups for Virus Challenge

Challenge Bleed

Group Strain n Treatment Day Day

N BALB/c 5 Sham 14 35

O BALB/c 5 SIS SQ 14 35

P BALB/c 5 SIS SQ 21 42

Q C57BL/6 5 Sham 14 35

R C57BL/6 5 SIS SQ 14 35

S C57BL 6 5 SIS SQ 21 42

Xenogeneic Skin Graft Rejection Assay The experimental groups for this model are described in Table 4. On the specified day following submucosa implantation, the mice were again anesthetized, as above, and implanted with a 1 cm x 1 cm graft of rat skin in place of a surgically- prepared full thickness defect on the back. The graft was sutured in place with 6-0 polypropylene. The fresh skin graft was harvested from a Sprague-Dawley rat under anesthesia immediately prior to implantation. The mice were recovered and monitored daily for graft rejection. Rejection was defined as 80% necrosis of the graft at visual observation. Photographs were taken to document graft status.

Table 4 Expeπmental Groups for Skin Xenograft Assay

Xenograft

Group Strain n Treatment Implant Day

T BALB/c 5 Sham 7

U BALB/c 5 SIS SQ 7

V BALB/c 5 SIS SQ 14 w C57BL/6 5 Sham 7

X C57BL/6 5 SIS SQ 7

Y C57BL/6 5 SIS SQ 14

RESULTS

DNP-Ovalbumin Challenge The antι~DNP Ab levels were determined 3 weeks after DNP-OVA challenge for each group of BALB/c mice.

When challenged 1 day after surgery, the level of anti-DNP Ab was suppressed m animals implanted with submucosa. When challenged 14 days following surgery, submucosa implantation appeared to increase anti-DNP Ab levels slightly There was no difference between submucosa treated and control animals when challenged at day 7 or 28 following surgery. Submucosa had no effect on IgG, levels for animals challenged at day 1 or 28, but may have increased IgG_j, slightly for the day 7 and 14 animals IgG_2a levels were suppressed in submucosa treated animals when challenged at day 1 , but were unaffected by submucosa at all other challenge times The submucosa implanted group showed lower levels of anti-DNP

IgG_2a but equivalent IgG,, when challenged at 1 day, resulting in lower total Ab In contrast, the submucosa implanted group challenged at day 14 showed greater total Ab and IgG, levels, but equivalent IgG_2a, compared to control.

The anti-DNP Ab levels were also determined in C57BL 6 mice The data for identically treated BALB/c mice are repeated for reference. All of the curves overlap for submucosa and control animals in the assays for total Ab, IgG,, and IgG_2a There was no difference in anti-DNP Ab levels between submucosa implanted and control for C57BL/6 mice.

Influenza Virus Challenge

Anti-influenza Ab levels were determined 3 weeks after challenge, for animals challenged 14 and 21 days after surgery. The Ab levels for submucosa and control animals overlap for all total Ab and both isotypes. Submucosa implanted animals showed no significant difference from the control groups in anti-influenza Ab production in either strain of mice at either challenge time.

Contact Dermatitis Assay

The contact dermatitis data are summarized in Table 5. Analysis of variance (ANOVA) of control ears showed that there was no significant difference (p=0.47) between groups, as expected. ANOVA between treated and control ears showed a that the treated ears had a significantly (p=0.0001) greater pos pre- challenge thickness ratio. ANOVA of treated ears showed that there was a significant difference between treatment groups, with p=0.02.

Table 5. Contact Dermatitis Assay Data

Mean Std. Dev. Mean Std.Dev.

Group Strain Treated Treated Control Control

H BALB/c 1.62 0.36 0.90 0.23

I BALB/c 1.95 0.20 1.06 0.07

J BALB/c 1.54 0.18 0.95 0.09

K C57BIJ6 1.82 0.15 0.98 0.12

L C57BL/6 2.13 0.39 1.03 0.12

M C57BIJ6 1.64 0.27 0.96 0.08

F tests for equal variances for treated ears between sham (H and K), 1 day (I and L), and 7 day (J and M) pairs showed that there were no significant differences in variance between these groups, with p values of 0.06, 0.25, and 0.47, respectively. Significance was defined as p<0.05. Student's t-tests between these groups (assuming equal variances) showed that none of the means were significantly different, with 2 tailed p values of 0.28, 0.39, and 0.52, respectively. Identical analysis of data for control ears also showed no significant differences. The data for these pairs of groups were thus considered to be from the same populations and were pooled, as described in Table 6.

Table 6. Pooled Data from Contact Dermatitis Assay

Mean Std.Dev. Mean Std.Dev.

Treatment Groups Treated Treated Control Control

Sham 1 day H+K 2.04 0.31 1.05 0.18

SIS 1 day I+L 1.59 0.28 0.94 0.10

SIS 7 day J+M 1.72 0.22 0.96 0.08

Student's t-test analysis of pooled data for treated ears showed that there was no significant difference between Sham and submucosa 7 day groups (p=0.29). Student's t-test also showed that the submucosa 1 day group had a significantly (p=0.025) reduced inflammatory response to DNFB challenge relative to control.

Skin Xenograft Assay

The skin xenograft rejection data are summarized in Table 7. ANOVA by treatment group showed that there was a significant difference between the groups (p=0.03). F tests for equal variance between mouse strains with identical treatment showed that the variances were not significantly different for the sham (N and Q), submucosa 7 day (O and R), and submucosa 14 day (P and S) pairs, with p values of 0.30, 0.12, and 0.83, respectively. Student's t-tests of the same pairs showed that the sham treatments were significantly different (p=0.03), while the submucosa 7 day and submucosa 14 day were not significantly different (p=0.06 and ρ=1.0, respectively). Since all pairs could not be considered to come from the same populations, data were not pooled and the two strains of mice were considered separately. ANOVA by treatment group showed that submucosa had no influence on the time required for rat skin graft rejection in either the BALB/c (p=0.10) or C57BL/6 (p=0.88) mice.

Table 7. Skin Xenograft Rejection Data.

Mean Days to

Group Strain Rejection Std.Dev.

N BALB/c 12.2 2.4

O BALB/c 11.2 2.6

P BALB/c 9.0 1.4

Q C57BL/6 8.6 1.8

R C57BL6 8.4 1.3

S C57BL/6 9.0 2.4

Discussion

The immune response to submucosa implantation may have a minimal and very transient effect on the simultaneous response to other antigens. Only the Ab response at 1 day but not thereafter, to injected DNP-ovalbumin with adjuvant and the contact dermatitis model, were affected. The response to injection of inactivated influenza virus without adjuvant and xenogeneic skin graft were not affected. There were also differences related to the mouse strain used.

The Ab response to DNP-ovalbumin given 1 day after submucosa implantation with powerful adjuvant shows a relatively minor (approximately 5 fold) suppression of IgG_2a production, but no effect on IgG,. This suggests that the Thl response may have been suppressed slightly due to the Th2 mediated immune response to submucosa. No effect was noted when the animals were challenged on day 7. A different effect was observed when challenge was performed on day 14. In these animals, the IgG_2a was not affected, but IgG, was enhanced. No effect of submucosa was observed when animals were challenged on day 28.

The contact dermatitis model showed a decrease in swelling response to DNFB challenge relative to control animals when sensitized 1 day after submucosa implantation, but not when sensitized after 7 days.

Genetic predisposition of the host animal was noted in the DNP- ovalbumin Ab response and the skin xenograft response. In the C57BL/6 mice, there was no difference between submucosa implanted and control animals on Ab production when challenged after 1 day, while differences were noted in the BALB/c mice. The C57BL/6 mice also rejected the skin xenografts more rapidly than BALB/c mice, although there was no effect of submucosa in either strain of mice. The strain specific responses do not appear to have confounded the analysis of the effect of submucosa implantation. In summary, the exposure to submucosa does not cause predisposition to infection or other immune insufficiency due to the Th2 dominant response to submucosa. The collected observations show that any influence of submucosa in these assays was only in the magnitude and timing, but not in fundamental character, of the immune response. Specifically, the anti-DNP Ab response was slightly altered in the relative amounts of Thl and Th2 associated Ab types produced, but not leading to a complete polarization to only one or the other type. Similarly, the contact dermatitis model showed suppression of the intensity of ear swelling response, but the expected normal response was present and significantly greater than non-treated controls.

Example 2

Characterization of the Immune Response to Porcine Submucosa

The nature and kinetics of the host response to subcutaneously implanted porcine submucosa in a BALB/c mouse model was investigated. Both the local and systemic response to porcine submucosa were probed. Serum Ab directed against submucosa were typed and quantified by ELISA. Host cells invading the graft were analyzed by histopathologic and immunohistochemical staining techniques. Finally, cytokine expression by cells invading the graft and by splenic lymphocytes was determined by RT-PCR and ELISA, respectively.

Materials and Methods Surgery

Female BALB/c mice (approximately 25 g) were anaesthetized with methoxyflurane, and the abdomen clipped and disinfected. An incision was made along the ventral midline and a subcutaneous pocket prepared using aseptic technique. A 1cm x 1 cm piece of porcine submucosa (SIS; Cook Biotech, Inc., West Lafayette, IN), BALB/c mouse abdominal muscle, or rat abdominal muscle was placed in the pocket and secured at the comers to the underside of the skin with 6-0 Prolene®. The skin was closed with a continuous suture of 6-0 Prolene®. Control animals were subjected to surgery and closed without implantation of tissue. All mice were treated prophylactically with oral enrofloxacin (5 mg/kg) for 7 days or until sacrifice. Animals were sacrificed after 1, 2, 3, 5, 7, 14, or 35 days, with n=6 per treatment group, and n- -1 for sham operated controls. The treatment groups and time points are summarized in Table 8.

Table 8. Summary of study design. Number of animals (n) per timepoint, by treatment group.

Evaluation Treatment Timepoint Rat abdominal Mouse abdominal (Days) Sham muscle muscle SIS

1 1 6 6 6

2 1 6 6 6

3 1 6 6 6

5 1 6 6 6

7 1 6 6 6

14 1 6 6 6

35 1 6 6 6

On the designated sacrifice date, blood was drawn from methoxyflurane anaesthetized animals by intracardiac puncture. Animals were then euthanized by intracardiac injection of saturated potassium chloride solution to effect asystole. The blood was clotted in an incubator at 37 °C for 90 minutes, and the serum collected and frozen at -20°C. The graft site, including implanted tissue, underlying abdominal wall, and overlying skin, was excised. The explant was subdivided and either frozen in liquid nitrogen and stored at -80 °C for cytokine RNA analysis, fixed in 10% neutral buffered formalin for histopathologic evaluation, or placed in O.C.T. Compound® (Miles; Elkhart, IN), frozen m liquid nitrogen, and stored at -80 °C for immunohistochemical staining. The spleen was halved and frozen in O.C.T. Compound® or placed in ice cold Hank's Balanced Salt Solution (Sigma Chemical Company, St. Louis, MO) for lymphocyte culture (submucosa-implanted and control animals only).

Anti-Submucosa Ab Analysis

Serum from submucosa-implanted and control animals were assayed for anti-submucosa Ab by an ELISA method for mouse IgG,, IgG_2a, IgG_2b, and IgG₃. Microtiter plates (Nalge Nunc International, Rochester, NY) were coated overnight with 10 μg/ml of a Tπs HC1 extract of submucosa. The plates were washed with PBS containing 0.1% (w/v) gelatin and 0.05% (v/v) Tween 20. After washing, seπal dilutions of serum were added and the plates incubated for 2 hours at RT. The plates were again washed and incubated with goat anti-mouse IgG,, IgG_2a, IgG_2b, or IgG₃ antibody conjugated to alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL). After incubation for 1 hour, the plates were washed and p- nitrophenyl phosphatase substrate was added to obtain an optimal color development Plates were read at 405 nm on an ELISA reader (Bio-Tek Instruments, Winooski, VT). Optimal working dilutions of the antibody conjugates and specificity for the target isotype was established using plates coated with purified mouse myeloma proteins (Sigma).

Cytokine RNA Analysis

Total RNA isolation from snap frozen implant tissue was performed with Tπzol reagent (Life Technologies, Rockville, MD) Bπefly, the frozen tissues were homogenized with a mortar and pestle and immediately transferred to tubes containing 2.0 ml of Tπzol reagent. The homogenized samples were centπfuged at 12,000 x G and the supernatant was added to chloroform. The samples were incubated for 15 minutes on ice and centπfuged The RNA was precipitated with isopropanol, washed twice with 75% ethanol, and solubilized in diethylpyrocarbonate (DEPC; nuclease inhibitor) treated water. The concentration of total RNA was determined by spectrophotometπc analysis at 260 nm. Three μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit (Life Technologies) and oligo (dT),₆.,_g primers. The resulting cDNA was amplified using specific primers for mouse IFN-γ and IL-10, with hypoxanthine phosphoribosyl transferase (HPRT) primers used as a control. PCR- was carried out using a PCR optimizer kit (Invitrogen Corporation, San Diego, CA) to determine optimal reaction conditions for specific primers. The sense and anti-sense primers had the following sequences:

IFN-γ: 5'-TGAACGCTACACACTGCATGG and 5'-CGACTCCTTTTCCGCTTCCTGAG; IL-10: 5'-ATGCAGGACTTTAAGGGTTACTTGGTT and

5'-ATTTCGGAGAGAGGTACAAACGAGGTTT HPRT: 5'-GTTGGATACAGGCCAGACTTTGT and 5'-GATTCAACTTGCGCTCATCTTAGGC; IL4: 5'-GTTGTCATCCTGCTCTTCTTT and 5'-CTCTCTGTGGTGTTCTTCGTT.

The reaction mixture consisted of 2 μl cDNA, 10 μl of 300 mM Tris HC1 (pH 8.5), 75 mM (NH₄)₂SO₄, 2.0 mM MgCl₂, 5 μl 2.5 mM dNTPs (Invitrogen Corporation), 0.5 μl Taq DNA polymerase (2.5 U; Life Technologies), 2 μl 20 μM primer, and 31.3 μl DEPC water to make a final volume of 50 μl. The mixture was incubated at 95 °C for 5 min, and then subjected to the amplification profile of 1 min at 95 °C, 1 min at 56 °C. and 1 min at 72 °C for a duration of 35 cycles. This was followed by a final extension for 10 minutes at 72 °C. The PCR products were separated on a 2.5% agarose gel and stained with ethidium bromide and the bands were visualized and photographed by UV transillumination.

Splenic Lymphocyte Culture

All manipulations were performed using aseptic technique. Spleens were minced with forceps in HBSS and centrifuged at 100 xG for 5 minutes. The pellet was resuspended in 0.75% NH₄C1 in 5 mM Tris HC1, pH 7.6 to lyse red blood cells. The suspension was kept on ice with agitation every two minutes. After 10 minutes, 5 ml of cell culture medium containing RPMI-1640 supplemented with 1% L- glutamine, 50 μM 2-mercaptoethanol, penicillin 100 U/ml, streptomycin 100 μg/ml (all purchased from Sigma), and 2.5% fetal clone serum (Hyclone Laboratories, Logan, UT) was added. The suspension was centrifuged at 100 xG for 5 minutes and the supernatant discarded. The pellet was washed with 5 ml of medium and centrifuged, then resuspended in 3 ml of medium and left undisturbed on ice for 5 minutes. The cells were collected with a pipet, leaving behind the tissue debris. The viable cell concentration was determined with a hemacytometer, based on Trypan Blue (Sigma) dye exclusion. The cell concentration was adjusted to 2 million./ml and cells pipetted into wells of a 96 well culture plate, at 200,000 cells/well. To the cells was added either guanidine HC1 extract of submucosa or Concanavalin A (ConA; Sigma) at a final concentration of 100 μg/ml or 5 μg/ml , respectively. The cells were kept at 37 °C in a 5% C0₂ incubator for 48 hours. The supernatants were collected and stored at 20 °C until use. IL-10 and IFN-γ in lymphocyte supernatants were quantified using sandwich ELISAs. Capture and detection Ab and standard cytokines were purchased from Pharmingen. High protein binding microliter plates (Corning Glass Works, Coming, NY) were coated with capture Ab at 2 μg/ml at 4°C overnight followed by incubation with samples or standards for 2 hours, biotinylated detection Ab for 1 hour, and avidin-peroxidase conjugate (ExtrAviding®, Sigma) for 1 hour, all at RT. The sample wells were rinsed 3 times with 0.05% Tween 20 in PBS between incubations. ABTS®& (Kirkegaard & Perry Laboratories, Gaithersburg, MD) peroxidase substrate was added and the reaction stopped with 1% sodium dodecylsulfate after color development for approximately 2 hours. The plates were read at 405 nm. Standard IL- 10 and IFN-γ were assayed as 8 two-fold serial dilutions to allow quantitation of samples. All samples were assayed in triplicate and repeated twice, except when there was insufficient sample.

Histopathologic evaluation Paraffin-embedded tissue was sectioned to a thickness of 7 μm and stained with hematoxylin and eosin (H&E) using an automated slide stainer (Zeiss), according to standard operating procedures in the laboratory. Immunohistochemical staining

Frozen tissue was sectioned to 5 mm thickness and mounted on poly-L- lysine coated slides. The tissues were fixed in acetone at 4 °C for 5 minutes and air dried. The slides were hydrated in PBS for 5 minutes and endogenous peroxidase inhibited by incubation in PBS containing 1 mM NaN₃, 1 unit ml glucose oxidase (Sigma) and 1 mM glucose for 30 minutes at 37°C. After rinsing in PBS, the tissues were incubated with hybridoma supernatant containing monoclonal antibody against mouse CD4 (clone GK1.5), CD8 (clone 53-6.72), B220 (clone RA3-3A1/6.1), Mac-1 (clone M1/70.15.11.5.HL), I-A (clone M5/114.15.2) markers, or purified monoclonal antiMOMA2 (Serotec, Raleigh, NC) for 1 hour at RT. All hybridomas were obtained from American Type Culture Collection (Rockville, MD). After rinsing with PBS, tissues were incubated with sheep anti-rat IgG peroxidase conjugate (Amersham, Arlington Heights, PL) for 1 hour at RT. Antibodies were diluted in 1% bovine serum albumin in PBS. The tissues were rinsed and incubated with DAB peroxidase substrate (Vector Laboratories, Burlingame, CA) for 10 minutes at RT. The tissues were rinsed, counterstained with hematoxylin, dehydrated in a graded ethanol series, and cover slipped.

Results Histopathology

Sham-operated controls: There was minimal inflammation at the surgical site. A small number of mononuclear inflammatory cells with minimal amounts of hemorrhage were present at all time points. There was a small amount of disorganized fibrous connective tissue (i.e., scar tissue) present at this site. By 35 days, there was still a small number of widely scattered mononuclear inflammatory cells present. Rat abdominal muscle implanted animals: Histologic examination of these tissues showed an intense inflammatory reaction beginning as early as one day with maximum numbers of lymphocytes, plasma cells, and mononuclear macrophages present between days 2 and 14. However, there were still a large number of inflammatory cells present at day 35 with some evidence of lymphoid follicle formation in the surrounding tissues. In addition, there was necrosis of the implant with areas of hemorrhage and eosinophilic debris scattered throughout the implant site. No infectious agents were identified. These findings were consistent with cytotoxic rejection of the implant.

Mouse abdominal muscle implanted animals: Histologic examination of tissues removed from mice implanted with isogeneic tissue showed an acute inflammatory response during the first 5 days that was virtually identical to that seen in the abdominal wall of mice implanted with submucosa (described below). There was an accumulation of mononuclear inflammatory cells, a small number of lymphocytes, and occasional neutrophiles scattered throughout the tissues and surrounding the implant. By 14 days the mononuclear inflammatory response had diminished moderately. By 35 days there was a virtual absence of any inflammatory response with the exception of a few mononuclear cells in the dermis. Minimal fibrous tissue was present in the area of the implant. No lymphocytic reaction could be found by day 35. There was no evidence for necrosis or immunologic rejection at any time during the 35 day period in this study group. Submucosa implanted animals: Histologic examination of the submucosa- implanted animals showed a gradual increase in the amount of inflammatory cells which appeared to be maximal at days 7 and 14. Many of these cells were mononuclear in nature and consisted of lymphocytes and mononuclear macrophages. No infectious agents were identified. At the earliest time points (i.e., days 1-3) there was also a small population of neutrophils noted. By day 35, there was organization of minimal amounts of eosinophilic staining collagenous connective tissue. The number of inflammatory cells by day 35 had subsided to a number which was only slightly greater than the sham-operated control. These cells were exclusively mononuclear in nature. There was an intense vascularity to the submucosa-implanted site which was most notable between days 3 and 14. The submucosa tissue itself became impossible to identify by as early as 7 days.

Immunohistochemistrv

No differences between groups at any time point for CD4, CD8, B220, MHC-LΪ, or MOMA-2 Ab. Few lymphocytes were noted at any time point in any tissue. MHC-II and MOMA-2 responses were. Mac-1 showed distinct differences between rat, mouse and submucosa, and sham groups. The response of Mac-1⁺ cells was more intense and more prolonged in the rat tissue grafts than in the submucosa and mouse tissue graft groups. Sham operated mice showed only moderate levels of the Mac-1⁺ cells, consistent with a limited inflammatory response following surgery.

Lymphocyte culture

The secretion of IL-10 and IFN-γ by cultured splenic lymphocytes from submucosa implanted (SIS) and sham control (sham) mice, was determined expressed as the ratio of submucosa-challenged to Con A challenged cells for each animal. The ratio was used to account for variation between animals to allow comparisons. In 4 cases (#2, 8, and 13 for IL- 10 and #16 for IFN-γ) neither submucosa nor ConA treated cells produced measurable cytokines, and the value of the ratio was set to zero. In a single case (#15) submucosa treated cells produced a low level of DFN-γ, but the same cells treated with ConA did not produce measurable cytokine, and therefore the ratio was set to 1. This sample was clearly an outlier. For the first three days following implantation, splenic lymphocytes produced very little or no detectable EL- 10 or EFN- γ in response to stimulation with submucosa. Con A was effective in stimulating the same cells to produce IL- 10 at approximately 0.1 - 1.8 ng/ml and IFN-γ at approximately 0.4 - 46.2 ng/ml. Lymphocytes from sham operated mice similarly produced very little or no EL-10 or IFN-γ in response to submucosa stimulation. After 5 - 35 days implantation, submucosa stimulated greater IL-10 than EFN-γ production when compared for each submucosa implanted animal. Sham control mice showed some minor stimulation by submucosa in IFN-γ production at 5 days and IL-10 at 7 and 35 days. At 14 days, EL-10 production by sham control was approximately 80% of ConA stimulated cells.

Cytokine mRNA analysis

Results of RT-PCR analysis of mouse cytokine RNA in the graft explants is shown in Table 9. Sham mice exhibited no IFN-γ RNA in the surgical site at any time point. LL-10 and IL4 were detected only at days 5 and 7. Animals implanted with submucosa showed similar cytokine profiles to animals implanted with syngeneic mouse tissue. High levels of LL-10 were detected early, then tapered off to very low levels by day 35. IFN-γ was only detected at low to moderate levels at days 3 and 5 in these specimens. Animals implanted with rat xenografts also exhibited early EL-10 production, but it tapered to undetectable levels by day 14. EFN-γ production in the rat xenograft group was low or undetectable until day 3, and then continued at moderate levels through day 7. EL-4 production was similar in all samples, with low to moderate levels detected at 5, 7, and 14 days only.

Table 9. Relative band intensities of inliagraft cytokine mRNA deteπnined by RT-PCR.

SIS Rat Mouse Sham

Days IFN-γ IL-10 IL-4 IFN-γ IL-10 IL-4 IFN-γ IL-10 IL-4 IFN-γ IL-10 IL-4

1 -.- ++,++ -.- -,+/- ++,+++ _""»-. *"»" -.- -.- - - -

2 +++,++ ^~»~ ^~»~ ++,++ ++,- -r - - - I o

3 + +++ - ++ +++ - + +++ - - - - I

5 ++ +++ ++ ++ ++ + + ++ - ++ + +/-

7 - + + ++ + ++ + - + +/-

+/-

14 - + +/- - - +/- + - -

+/-

35 - +/- - - - +/- - -

Further analysis of 3 and 5 day samples shows that there is little or no EFN-γ produced in grafts on day 3, but significantly greater amounts are expressed in rat grafts at day 5 compared to all other treatments. This suggests that Thl differentiation is occurring in the rat grafts but in the not other groups at 5 days.

Anti-Submucosa Ab analysis

At day 14 and day 35, submucosa-implanted mice showed equivalent IgG, to positive control serum, but less than half as much total antibody. The positive control serum also contained significant amounts of IgG_2a and IgG_2b, which raised the total Ab level in this serum. Serum from sham operated mice and submucosa- implanted mice showed no IgG_2a or IgG_2b. No serum samples, including the positive control, showed IgG, (an IFN-γ dependent isotype) at any time point. Thus, the Ab produced in response to implantation of submucosa was exclusively Th2 associated isotype, whereas inoculation with submucosa extract in adjuvant yielded both Thl and Th2 associated Ab response.

Discussion

The data suggest a Th2 dominance in the host response to submucosa which is compatible with graft acceptance. Although Th2 dominance does not guarantee graft acceptance, it appears that Thl dominance is not compatible with acceptance of cellular xenogeneic grafts. Thl mediated rejection of acellular xenografts has not been described, although destruction of glutaraldehyde treated bovine pericardial heart valve grafts by an apparent (uncharacterized) immune process has been reported. It is anticipated that the Th2 dominant response is beneficial in several ways, including prevention of Thl mediated rejection of the submucosa graft. Organ graft rejection is based on killing graft cells and interrupting its blood supply which result in a non-functional organ. The acellular nature of submucosa makes this mechanism of rejection unlikely, since the cells which populate an submucosa graft and its blood supply are host-derived and thus non-immunogenic. The fact that submucosa is gradually resorbed also likely plays some role in the positive host response, as this decreases the xenoantigen load over time and leaves only host tissue remaining. The inflammatory cells seen at the site of submucosa implantation are almost exclusively mononuclear in nature by day 3. This mononuclear cell predominance remains through day 14 with only small numbers left by day 35. The relationship of the inflammatory cells to the subsequent deposition of extracellular matrix and organization of this matrix in the connective tissue remains unclear.

However, the Th2 pathway is also anti-inflammatory. This mechanism may keep post- implantation inflammation to a minimal level, allowing the normal remodeling response to occur. Thus, Th2 anti-inflammatory activity may be an important factor in promoting submucosa remodeling. The implications for the immunologic response to a second exposure to submucosa are not clear. Very preliminary studies with reimplantation suggested that the cellular and connective tissue responses to submucosa implantation are identical. Studies of neonatal tolerance to transplantation suggest that a Th2 dominant primary response is protective of secondary graft exposure. Whether a Th2 dominant response to submucosa predisposes a host animal to infection by inhibiting Thl responses systemically (EL-10 activity) has been investigated. It seems clear that submucosa has marginal or no effect on the normal immune response of BALB/c or C57BL/6 mice to various antigenic challenges.

It is not known whether components of submucosa direct the host cells toward Th2 differentiation (i.e. exhibit immunomodulating activity) or if the nature of the antigens in submucosa intrinsically elicit Th2 type response. In vitro studies with human cells are planned to determine whether immunomodulating activities are present within submucosa, and if present, what step of the Th stimulation pathway is affected; what cell types are affected (antigen presenting cells or lymphocytes); the mechanism of submucosa effects (e.g. modulating cytokine activity); and the components of submucosa likely responsible for immmomodulating activity.

It appears that submucosa elicits a Th2 dominant immune response in BALB/c mice. Assays of both the local (intragraft) and systemic immune activities indicate Th2 dominance. The immune response to submucosa is not associated with clinical or histologic evidence of rejection. The immunohistochemical and histopathologic analyses are consistent with Th2 dominant immune response.

Claims

CLALMS:

1. A method for locally suppressing the cell mediated immune response in a vertebrate species, said method comprising the steps of contacting a site in need of immune suppression with an effective amount of a composition comprising vertebrate submucosa.

2. The method of claim 1 wherein the submucosa is intestinal submucosa comprising the tunica submucosa delaminated from the tunica muscularis and at least the luminal portion of the tunica mucosa of vertebrate intestine.

3. The method of claim 1 wherein the submucosa tissue is treated with galactosidase to be substantially free of the Gal epitope.

4. The method of claim 1, wherein the submucosa is fluidized submucosa.

5. The method of claim 4, wherein the submucosa is digested with an enzyme for a period of time sufficient to solubilize the tissue.

6. A method of protecting immunogenic biomaterial from the immune system of a host organism said method comprising the step of encapsulating the biomaterial within a composition comprising vertebrate submucosa and implanting the composition into the host organism.

7. The method of claim 6 wherein the composition comprises intestinal submucosa delaminated from the tunica muscularis and at least the luminal portion of the tunica mucosa of vertebrate intestine.

8. The use of vertebrate submucosa in the manufacture of a pharmaceutical composition for the suppression of a cell mediated immune response in a vertebrate species.

9. The use according to claim 8 wherein the pharmaceutical composition is used to treat an autoimmune disorder.