Avonex Combination Trial in relapsing--remitting MS: rationale, design and baseline data.

OBJECTIVE: To review the rationale, design and baseline data of the Avonex Combination Trial (ACT), an investigator-run study of intramuscular interferon beta-1a (IM IFNbeta-1a) combined with methotrexate (MTX) and/or IV methylprednisolone (IVMP) in relapsing-remitting multiple sclerosis (RRMS) patients with continued disease activity on IM IFNbeta-1a monotherapy. METHODS: Eligibility criteria included RRMS, Expanded Disability Status Scale score 0-5.5, and >or=1 relapse or gadolinium-enhancing MRI lesion in the prior year while on IM IFNbeta-1a monotherapy. Subjects continued IFNbeta-1a 30 mcg IM weekly and were randomized in a 2 x 2 factorial design to adjunctive weekly placebo or MTX 20 mg PO, with or without IVMP 1,000 mg/day for three days every other month. ACT was industry-supported, and collaboratively designed and governed by an Investigator Steering Committee with independent Advisory and Data Safety Monitoring Committees. Study operations, MRI analysis and aggregated data were managed by the Cleveland Clinic MS Academic Coordinating Center. RESULTS: In total 313 subjects were enrolled with clinical and MRI characteristics typical of RRMS. Most subjects (86.9%) qualified with a clinical relapse, with or without an enhancing MRI lesion, in the preceding year. At baseline, 21.4% had enhancing lesions, and 5.1% had anti-IFNbeta neutralizing antibodies. ACT's management and operational structures functioned well. CONCLUSION: This study provides an innovative model for academic-industry collaborative MS research and will enhance understanding of the utility of combination therapy for RRMS patients with continued disease activity on an established first-line treatment.

Single vs. dual vessel porcine extracorporeal liver perfusion.

BACKGROUND: The use of porcine extracorporeal liver perfusion (PECLP) to provide temporary hepatic support for patients in fulminant hepatic failure has been limited by the fact that individual perfusions can be sustained for only a few hours. Inadequate liver function and/or hemodynamic instability are the major contributing factors for early interruption of PECLP. Recent reports suggest that the choice of single (portal vein only) vs dual (portal vein and hepatic artery) vessel perfusion may influence the duration of perfusion. We hypothesize that PECLP with single vessel perfusion (SVP) is associated with worse liver function and greater hemodynamic instability than PECLP with dual vessel perfusion (DVP). MATERIALS AND METHODS: To eliminate the potentially confounding influences of liver failure and xenograft rejection, liver isografts procured from White-Landrace pig donors were perfused by either SVP or DVP via an extracorporeal circuit established with normal White-Landrace pig recipients. The function of perfused livers was evaluated by measuring production of bile and Factors V and VIII, clearance of ammonia and lactate, and extraction of O(2) at baseline and at 0, 1, 3, 6, 12, and 24 h after initiation of PECLP. The impact of PECLP on recipient hemodynamic status was assessed by monitoring BP, heart rate, urine output, O(2) saturation, etc. Among other parameters evaluated were serum albumin and total protein and hepatic release of IL-1beta and nitric oxide to assess their possible contributions to hemodynamic instability. RESULTS: DVP and SVP livers cleared ammonia and lactate similarly. Both approaches were associated with progressive hypoalbuminemia and hypoproteinemia. DVP livers produced more bile and Factor V and were associated with less recipient hypotension and IL-1beta and NO release than SVP livers. CONCLUSIONS: Livers with DVP function better than livers with SVP. The duration of PECLP can be limited by recipient hypotension, although this complication is less severe with DVP than with SVP.

Distribution of, and immune response to, chicken anti-alpha Gal immunoglobulin Y antibodies in wild-type and alpha Gal knockout mice.

Chicken antibodies (immunoglobulin Y; IgY) to the alpha Gal epitope (galactose alpha-1,3-galactose) bind to alpha Gal antigens of mouse and porcine tissues and endothelial cells in vitro and block human anti-alpha Gal antibody binding, complement activation and antibody-dependent cell-mediated lysis mechanisms. The activities and toxicity of anti-alpha Gal IgY have not been tested in vivo. In this study, we tested the effects of multiple injections of affinity-purified anti-alpha Gal IgY (AP-IgY) in both wild-type (WT) and alpha-1,3-galactosyltransferase knockout (Gal KO) mice. WT and Gal KO mice were injected once, twice, three, or four times intravenously (i.v.) with AP-IgY and killed at 1 hr or 24 hr. Mice displayed no toxicity to four injections of AP-IgY. Heart, lung, liver, kidney, spleen and pancreatic tissue were evaluated using immunohistochemical techniques for the presence of the alpha Gal epitope using the GSI-B4 lectin, and for bound IgY, as well as mouse IgM and IgG. The binding of AP-IgY antibodies to the endothelium of WT mouse tissues was essentially identical to the pattern of binding of the GSI-B4 lectin after injection of WT mice and death at 1 hr. WT mice killed 24 hr after i.v. injection of AP-IgY showed little remaining bound IgY in their endothelia, indicating that IgY is cleared over that time period. We also evaluated the blood drawn at the time of death for the presence of anti-alpha Gal IgY, anti-IgY IgM and anti-IgY IgG by enzyme-linked immunosorbent assay. Anti-alpha Gal IgY was almost undetectable in WT mouse sera at all injection and killing times. In contrast, Gal KO mouse sera showed increasing anti-alpha Gal IgY levels until 24 hr after the fourth injection, when anti-alpha Gal IgY levels were almost undetectable. Anti-IgY IgM and IgG levels in WT and Gal KO mouse sera showed a typical increase in anti-IgY IgM 24 hr after the second injection (3 days after the first injection) and an increase in anti-IgY IgG 24 hr after the third injection (5 days after the first injection). These results show that IgY binds to alpha Gal epitopes in the WT mice and is cleared sometime over a 24-hr time period and that IgY is an expected immunogen in mice eliciting a rather typical anti-IgY IgM and IgG response.

Synthetic peptides which inhibit the interaction between C1q and immunoglobulin and prolong xenograft survival.

BACKGROUND: Acute vascular xenograft rejection (AVXR), also termed delayed xenograft rejection (DXR), occurs when hyperacute rejection (HAR) is prevented by strategies directed at xenoreactive natural antibodies and/or complement activation. We have hypothesized that AVXR/DXR is initiated in part by early components of the complement cascade, notably C1q. We have developed synthetic peptides (termed CBP2 and WY) that interfere with the interaction between C1q and antibody. METHODS: CBP2 and the WY-conjugates were used as inhibitors of immunoglobulin aggregate binding to solid phase C1q. Inhibition of complement activation by the peptides of the classical system was determined using lysis assays with sensitized sheep red blood cells or porcine aortic endothelial cells as targets and of the alternate complement pathway using guinea pig red blood cells as targets. Two transplant models were used to study the effects of administering peptides to recipients: rat heart transplant to presensitized mouse, and guinea heart transplant to PVG C6-deficient rats. RESULTS: CBP2 and WY-conjugates inhibited immunoglobulin aggregate binding to C1q. The peptides also inhibited human complement-mediated lysis of sensitized sheep red blood cells and porcine aortic endothelial cells in a dose-dependent manner and the WY-conjugates prevented activation of the alternate complement pathway as shown by inhibition of guinea pig red blood cells lysis with human serum. In addition, the use of the peptides and conjugates resulted in significant prolongation of xenograft survival. CONCLUSIONS: The CBP2 and WY peptides exhibit the functional activity of inhibition of complement activation. These peptides also prolong xenograft survival and thus provide reagents for the study of the importance of C1q and other complement components in transplant rejection mechanisms.

IgY antiporcine endothelial cell antibodies effectively block human antiporcine xenoantibody binding.

Avian IgY antibodies are structurally different from mammalian IgGs and do not fix mammalian complement components or bind human Fc receptors. As these antibody-mediated interactions are believed to play significant roles in both hyperacute rejection (HAR) and acute vascular xenograft rejection (AVXR), IgY antibodies to xenoantigen target epitopes may inhibit these rejection processes. In this report, we show that chicken IgY antibodies to alpha-Gal antigen epitopes and to other porcine aortic endothelial cell (PAEC) antigens block human xenoreactive natural antibody binding to both porcine and rat cardiac tissues and porcine kidney tissues. Chicken IgY antibodies blocked complement-mediated lysis of PAECs by human serum, and inhibited antibody-dependent cell-mediated lysis of PAECs by heat-inactivated human serum plus peripheral blood leukocytes. Binding of IgY to porcine endothelial cells did not affect cell morphology nor expression of E-selectin. These results suggest that avian IgYs could be of potential use in inhibiting pig-to-human xenograft rejection.

Early histopathology of small intestinal discordant xenografts.

A descriptive study of a new model enabling serial biopsies of ongoing hyperacute rejection of small intestinal discordant xenografts is presented. In a series of guinea-pig-to-Lewis rat small bowel xenotransplants (n=7), aboral free ends of Thierry-Vella loops constructed from the graft were sequentially biopsied at one-minute intervals up to ten minutes post-reperfusion and less frequently thereafter. In a guinea pig-to-guinea pig (n=6) isograft series, biopsy controls for preservation/ischemia-reperfusion injury were obtained. Xenoantibody sequestration in this model was evaluated in a separate series of transplants, utilizing an ELISA assay for rat anti-guinea pig natural antibodies. Pathologic evaluation revealed a unique series of events characterized with microcirculatory failure and thrombosis progressing from the submucosal vasculature to the lumen. Within the system's detection limits, complement deposition and P-selectin expression occurred as early as one minute post-reperfusion, preceding the staining for IgM and IgG. Using rat serum ELISAs, no significant difference in xenoantibody sequestration was detected between the xenograft and isograft groups. The guinea pig-to-rat discordant small bowel xenotransplantation is an efficient small animal model to dissect the very early pathophysiologic events during hyperacute rejection.