Decline in age-dependent, MK801-induced injury coincides with developmental switch in parvalbumin expression: somatosensory and motor cortex.

MK801-induced activation of caspase-3 is developmentally regulated, peaking at postnatal day (P) 7 and decreasing with increasing postnatal age thereafter. Further, at P7, cells displaying activation of caspase-3 lack expression of calcium binding proteins (CaBPs). To further explore this relationship, we investigated postnatal expression of calbindin (CB), calretinin (CR) and parvalbumin (PV) in two brain regions susceptible to MK801-induced injury, the somatosensory cortex (S1) and layer II/III of motor cortex (M1/M2). Expression of CB and especially PV was low to absent prior to P7 but substantially increased from P7 through to P21 and adulthood. In contrast, CR expression was more variable at early developmental ages, stabilized to lower levels after P7 and showed a marked decline by P21. The results suggest that not only does calcium buffering capacity increase developmentally but also acquisition of enhanced buffering may be one mechanism by which neurons survive agent-induced alterations in calcium homeostasis.

Late onset of development of natural anti-nonGal antibodies in infant humans and baboons: implications for xenotransplantation in infants.

If an ABO-incompatible heart is transplanted into an infant before natural antibodies have developed to the specific donor carbohydrate A/B antigen(s), then B-cell tolerance to the donor A/B antigen is achieved, and these antibodies never develop. Anti-carbohydrate antibodies play a role in the rejection of wild type (WT) and alpha1,3-galactosyltransferase gene-knockout (GT-KO) pig xenografts. We investigated development of these antibodies in infant baboons and humans. Serum samples from infant baboons (n = 42) and humans (n = 42) were tested by flow cytometry for immunoglobulin M and immunoglobulin G binding to peripheral blood mononuclear cells from WT and GT-KO pigs, and for complement-dependent cytotoxicity. The presence of anti-blood group antibodies was tested in baboon serum. In infant baboons and humans, cytotoxic anti-Galalpha1,3Gal antibodies develop during the first 3 months, and steadily increase with age, whereas cytotoxic anti-nonGal antibodies are either absent or minimal in the majority of cases throughout the first year of life. Anti-blood group antibodies were not detected before 16 weeks of age. Our data suggest GT-KO pig organ/cell transplants could be carried out in early infancy in the absence of preformed cytotoxic anti-nonGalalpha1,3Gal antibodies.

Decline in age-dependent, MK801-induced injury coincides with developmental switch in parvalbumin expression: cingulate and retrosplenial cortex.

Age-dependent, MK801-induced, activated caspase-3 expression in the postnatal brain is generally not observed in neurons expressing calcium-binding proteins (CaBPs), suggesting that apoptosis and calcium buffering are inversely related. In regions such as the cingulate and retrosplenial cortex, injury peaks at postnatal Day 7 (P7) and rapidly diminishes thereafter, whereas expression of calbindin (CB) and calretinin (CR) was relatively low from P0 to P7 and steadily increased from P7 to P14. At ages thereafter, CB and CR expression either remained stable then declined or rapidly declined. Parvalbumin (PV) was generally low-absent prior to P7 but expression dramatically increased from P10 onwards, peaking at P21. These studies suggest calcium entry (through N-methyl-D-aspartate receptor (NMDARs)) and buffering (by CaBPs) are integral to normal CNS maturation. Because schizophrenia is associated with glutamate hypo-function, developmental injury, and aberrant CaBP expression, our data indicate that this postnatal brain injury model may offer important insights into the nature of this disorder.

Selected physiologic compatibilities and incompatibilities between human and porcine organ systems.

The shortage of donor organs is a major barrier to clinical organ transplantation. Although xenotransplantation is considered one of the alternatives to human organ transplantation, there are immunologic and physiologic incompatibilities between humans and pigs. With the exception of coagulation, the major potential physiologic incompatibilities relating to function of the kidney, heart, liver, lungs, pancreatic islets, and hormones are reviewed. Some of these physiologic differences can be overcome by producing genetically altered pigs to improve compatibility with humans. The possibility of producing such pigs for organ transplantation is considered.

Induction of diabetes in cynomolgus monkeys with high-dose streptozotocin: adverse effects and early responses.

OBJECTIVES: Streptozotocin (STZ) has been widely used to induce diabetes in nonhuman primates, although it has been found difficult to achieve complete diabetes without serious adverse effects. We have investigated different types and dosages of STZ to find a way to safely induce complete diabetes in cynomolgus monkeys. METHODS: After adequate hydration, 10 monkeys received STZ. Five monkeys received conventional STZ (Sigma) at a dosage of 1250 mg/m ("high dose"; n = 4) or 60 mg/kg ("low dose"; n = 1; Group 1). Five monkeys received Zanosar STZ (Sicor Pharmaceuticals, Irvine, CA) at 150 mg/kg (high dose; n = 5; Group 2). RESULTS: High-dose Group 1 monkeys became completely diabetic (n = 4), but a protein-losing nephropathy was observed in 3 of the 4 monkeys. The monkey that received 60 mg/kg STZ failed to become fully diabetic (C-peptide, > 1.86 ng/mL). Group 2 (high-dose Zanosar-treated) monkeys became completely diabetic but with no apparent adverse effects. A triphasic blood glucose response to STZ was documented in all the high-dose STZ-treated monkeys. Low-dose STZ failed to result in a triphasic response. CONCLUSIONS: (1) High-dose Zanosar STZ induced diabetes safely in cynomolgus monkeys without adverse effects. (2) A triphasic blood glucose response suggested the complete induction of diabetes.

Antibodies directed to pig non-Gal antigens in naïve and sensitized baboons.

BACKGROUND: As pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GT-KO) are available, primate antibodies to pig non-Gal antigens can be studied. METHODS: Sera from 56 baboons were tested for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from both wild-type (WT) and GT-KO pigs by flow cytometry. Complement-dependent cytotoxicity was measured in 39 sera. Antibody and cytotoxicity responses were measured in two baboons exposed to a GT-KO pig heart, one not immunosuppressed and one that received only cobra venom factor. RESULTS: IgM and IgG bound to 95% and 79% of WT PBMC, and 32% and 9% GT-KO PBMC, respectively (WT vs. GT-KO, P<0.01). Whereas 97% of sera were cytotoxic to WT PBMC, only 64% were cytotoxic to GT-KO PBMC, and the level of cytotoxicity was less (mean 60% vs. 25% lysis, P<0.05). In the two baboons exposed to GT-KO hearts, anti-non-Gal antibodies increased markedly, peaking after 2 (IgM) and 3 (IgG) weeks, associated with an increase in lysis of GT-KO PBMC. CONCLUSIONS: Two-thirds of baboon sera demonstrated cytotoxicity to GT-KO PBMC. After GT-KO organ transplantation, if an elicited antibody response develops, it is likely to cause rapid graft rejection.

Allosensitized humans are at no greater risk of humoral rejection of GT-KO pig organs than other humans.

BACKGROUND: The availability of pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GT-KO) has enabled study of the incidence and cytotoxicity of primate antibodies directed to antigens other than Galalpha1,3Gal (Gal), termed non-Gal antigens. METHODS: Sera from 27 healthy humans and 31 patients awaiting renal allotransplantation, who were either unsensitized [panel reactive antibodies (PRA) < 10%] or allosensitized (PRA > 70%), were tested by flow cytometry for binding of immunoglobulin M (IgM) and IgG to peripheral blood mononuclear cells (PBMC) from both wild-type (WT) and GT-KO pigs. Complement-dependent cytotoxicity to WT and GT-KO PBMC was also measured. RESULTS: IgM and IgG from all 27 (100%) healthy human sera bound to WT PBMC, while 78% and 63% of these sera had IgM and IgG that bound to GT-KO PBMC, respectively. Mean binding to WT PBMC was significantly greater than GT-KO PBMC. Whereas 100% of sera were cytotoxic to WT PBMC, only 61% were cytotoxic to GT-KO PBMC, and the extent of lysis was significantly less. Neither mean binding of IgM and IgG nor cytotoxicity of unsensitized and allosensitized sera to WT and GT-KO PBMC was significantly different to that of healthy sera. CONCLUSIONS: More than half of the healthy humans tested had cytotoxic antibodies to GT-KO PBMC, but allosensitized patients will be at no greater risk of rejecting a pig xenograft by a humoral mechanism.

Buccal mucosal cell immunohistochemistry: a simple method of determining the ABH phenotype of baboons, monkeys, and pigs.

BACKGROUND: Baboons and monkeys fail to express ABH antigens on red blood cells (RBCs), and the A or H antigens are expressed only weakly on the surface of pig RBCs. Baboons and monkeys have been previously blood typed by detection of ABH antigens in the saliva after administration of pilocarpine. A reliable method to ABH type pigs is by immunohistochemical staining of renal distal tubules in kidney biopsies. We describe a simple and efficient method to blood type baboons, monkeys, and pigs. METHODS: Baboons (n = 14) and cynomolgus monkeys (n = 8) were blood typed by staining of buccal mucosal smears and by determining the presence of serum anti-A or B antibodies following human type O adsorption. Pigs (n = 11) were tested for ABH type by immunohistochemistry for the presence of A, B, and H antigens using monoclonal antibodies on (i) renal biopsies, (ii) RBCs, and (iii) buccal mucosal smears, without pilocarpine administration, in addition, (iv) after adsorption on human type O RBCs to remove anti-human antibodies, the pig sera were typed by hemagglutination assay for the presence of anti-A or B antibodies using human A and B RBCs. RESULTS AND CONCLUSIONS: There was complete consistency among the results obtained using all of the above methods, except that no determination could be made from staining of RBCs in one pig. Staining of buccal mucosal cells proved to be the preferred method in all three species because: (i) expression of A or H antigen is weak on pig RBCs, making an accurate blood type determination difficult, and A, B, and H expression is non-existent on baboon and monkey RBCs, (ii) neither venepuncture nor organ biopsy is necessary, (iii) time-consuming adsorption of anti-human antibodies from the sera of the test animal is not required, and (iv) it proved a quick method of evaluation.

Incidence and cytotoxicity of antibodies in cynomolgus monkeys directed to nonGal antigens, and their relevance for experimental models.

The recent availability of pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GT-KO) has enabled the study of incidence and cytotoxicity of antibodies of cynomolgus monkeys directed to antigens other than Galalpha1,3Gal (Gal), termed nonGal antigens. To this aim, sera from 21 cynomolgus monkeys were tested by flow cytometry for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from wild-type (WT) and GT-KO pigs. The sera were also tested for complement-dependent cytotoxicity to WT and GT-KO PBMC. Anti-WT IgM and IgG were found in 100% and 95%, respectively, and anti-GT-KO IgM and IgG in 76% and 66%, respectively, in the sera of the monkeys tested (P < 0.01). Whereas 100% of sera were cytotoxic to WT PBMC, only 76% were cytotoxic to GT-KO PBMC, and the level of cytotoxicity was significantly less (P < 0.01). Although the incidence and cytotoxicity of antibodies in monkey sera to GT-KO pig PBMC are significantly less than to WT PBMC, approximately three-quarters of the monkeys tested had cytotoxic antibodies to GT-KO PBMC. This incidence of cytotoxicity is significantly higher than that found in baboons and humans, suggesting the baboon may be an easier and possibly more suitable model to study antibody-mediated rejection of transplanted GT-KO pig organs and cells.