Viral interleukin-10 gene therapy to induce tolerance to solid organ transplants in mice.

In this study, a novel gene therapy approach to prolong allograft survival was designed. Autologous (syngeneic) hematopoietic stem cell-enriched bone marrow cells (HSC; lin(-)) engineered with the vIL-10 gene (vIL-10-HSC) were injected (4 to 6 x 10(6) cells, i.v.) into lethally (9.5 Gy) or sublethally (4 Gy) irradiated CBA/J mice 6 weeks prior to allogeneic heart (C57BL/6) transplantation (Tx). Cardiac allograft survival was significantly (P <.004) prolonged in lethally (71 +/- 40 days) and sublethally (114 +/- 15 days) irradiated mice that received vIL-10-HSC compared to controls that received no HSC (11 +/- 1 days), unengineered HSC, or vector-DNA-engineered HSC (12 to 16 days). Tolerant graft histopathology demonstrated mild arteritis/venulitis (grade 0.7) and rejection (grade 1.0). Intragraft expression of costimulatory molecules (B7.1, B7.2), cytokines (IL-2, IL-4, mIL-10, IFN-gamma), and iNOS molecules were markedly lower in tolerant grafts that survived for >100 days; recipient T lymphocytes demonstrated hyporeactivity to donor and third-party antigens in mixed lymphocyte cultures. These findings have important implications and potential therapeutic applications in transplantation and autoimmune diseases.

Biological effects of genes in the Grc and EC region of the rat major histocompatibility complex.

PROBLEM: To study the mechanism of action of major histocompatibility complex (MHC)-linked genes affecting reproduction, growth, and susceptibility to chemical carcinogens. METHOD OF STUDY: Tumors derived from rat embryonic fibroblasts were transfected with cosmids from the Grc and its linked regions, the unrelated A region, and a nonMHC region, or with genes from the Grc, Grc-linked, and nonMHC regions, to determine whether they could suppress tumor growth as determined by in vitro (soft agar) and in vivo assays. RESULTS: Tumor fibroblasts transfected with cosmids from the Grc or from the EC region decreased tumor growth in both the in vitro and in vivo assays. Transfection with individual genes from the Grc had no effect on tumor growth in either assay. CONCLUSIONS: The effects of the Grc on reproduction, growth, and tumorigenesis are mediated by extended genetic effects, i.e., by the conformation of the DNA in this region. Similar effects were seen following transfection with cosmids from the Grc-linked EC region, and this finding strengthens the hypothesis that the conformation of the DNA in this general region is critical for its function. A similar effect has been described for the locus control region (LCR) in the beta-globin gene family in the human.

Infusion of donor leukocytes to induce tolerance in organ allograft recipients.

To further enhance chimerism, 229 primary allograft recipients have received perioperative intravenous infusion of a single dose of 3 to 6 X 10(8) unmodified donor bone marrow (BM) cells/kg body weight. In addition, 42 patients have been accrued in a concurrent protocol involving multiple (up to three) sequential perioperative infusions of 2 x 10(8) BM cells/kg/day from day 0-2 posttransplantation (PTx). Organ recipients (n = 133) for whom BM was not available were monitored as controls. The infusion of BM was safe and except for 50 (18%), all study patients have optimal graft function. Of the control patients, allografts in 30 (23%) have been lost during the course of follow-up. The cumulative risk of acute cellular rejection (ACR) was statistically lower in the study patients compared with that of controls. It is interesting that, 62% of BM-augmented heart recipients were free of ACR (Grade > or = 3A) in the first 6 months PTx compared to controls. The incidence of obliterative bronchiolitis was also statistically lower in study lung recipients (3.8%) compared with the contemporaneously acquired controls (31%). The levels of donor cell chimerism were at least a log higher in the peripheral blood of majority of the study patients compared with that of controls. The incidence of donor-specific hyporeactivity, as determined by one-way mixed leukocyte reaction, was also higher in those BM-augmented liver, kidney, and lung recipients that could be evaluated compared to controls.

Immunogenetic and oncogenic properties of rat trophoblast cell lines.

PROBLEM: The nature of major histocompatibility complex (MHC) antigen expression on rat placentas, trophoblast cell lines, and tumors derived from trophoblast cells was explored. METHOD OF STUDY: Cytohistochemical and flow cytometric analysis and molecular techniques. RESULTS: MHC antigen expression and genomic imprinting on the placenta and on trophoblast cells varies with the time of gestation and with the type of MHC antigen. CONCLUSIONS: There is no correlation in trophoblast cells between class I expression and cell ploidy, on the one hand, and malignant potential, on the other hand. Genomic imprinting of class I antigens in the rat placenta is a quantitative phenomenon.

Recent studies on the structure of the grc region in the rat.

PROBLEM: To explore the nature of the genes in the grc. METHOD: Chromosome walking of the grc and sequencing new genes. RESULTS: The RT1.O,N genes have been aligned, and two new types of genes, RT1.S1,2 and Rprl,2,3, have been discovered and mapped. An extensive physical map of the grc region is presented. CONCLUSION: The disease associations in both the rat and the human appear to cluster in two regions that map in approximately the same place in both species, given the translocation that occurred in the major histocompatibility complex (MHC) of the rat relative to the MHC of the human.

Physical mapping and structural analysis of new gene families RT1.S and Rps2r in the grc region of the rat major histocompatibility complex.

Five new genes were identified in the growth and reproduction complex (grc) region: RT1.S1, RT1.S2, Rps2r1, Rps2r2, and Rps2r3. The class Ib RT1.S1 and RT1.S2 genes have five distinct exons (1, 4, 5, 6, 7) similar to other class I major histocompatibility complex genes but the conventional exons 2 and 3 are absent. The genes are 97% similar, have CAAT and TATA boxes much upstream of the conventional position, obey the GT/AG rule in their exon-intron boundaries, and are transcribed at a low level in thymus and testis but not in the liver and spleen. The region between exon 1 and exon 4 was analyzed by obtaining transcripts by reverse transcription-polymerase chain reaction (RT-PCR) amplification which revealed the presence of four alternatively spliced mRNA transcripts of RT1.S1: 1) S1-1 (clones 14 and 16) has no exon between exons 1 and 4; 2) S1-2 (clones 7 and 8) has an exon of 45 nucleotides that can translate into 15 amino acids; 3) S1-3 (clone 5) has an exon of 42 nucleotides with a stop codon; and 4) S1-4 (clone 10) has two exons of 42 and 38 nucleotides, respectively, with stop codons. Only one RT1.S2 mRNA transcript was obtained, and it has an exon of 45 nucleotides between exon 1 and exon 4 which can form a peptide identical to the S1-2 isoform for that region. The 45 nucleotide exon between exon 1 and exon 4 was unique for RT1.S1 and RT1.S2 and only matched a sequence in the RT1.O intron region (nucleotides 2905 - 2949). The three ribosomal-protein-S2-related (Rps2r) genes are 94% - 98% similar; they are related to the genes encoding ribosomal protein S2 of the black rat and the LLRep3 genes of the mouse and the human and to the genes encoding Saccharomyces cerevisiae S4, Escherichia coli S5, and other members of prokaryote S5 family. The Rps2r1 gene is located just outside of the grc region. The Rps2r2 and Rps2r3 genes are in the grc and have multiple stop codons in their genomic sequences. The Rps2r1 mRNA transcript was identified by RT-PCR in the thymus and testis but not in the liver and spleen.

Physical mapping of the E/C and grc regions of the rat major histocompatibility complex.

Alignment of class I-hybridizing cosmids from an R21 (AlBlDlEugrc+) genomic DNA library gave two contigs: one [150 kilobases (kb)] encompassed the E/C region, or a large part thereof, and the other (110 kb) contained the grc region which has genes influencing resistance to chemical carcinogens (rcc), fertility (ft), and growth (dw-3). Amplification of gene sequences in the four cosmids in the E/C region using Eu-specific and LW2 (RT1.C)-specific primers showed that each cosmid contained both Eu-like and C-like genes. They are clearly different but closely associated, and they show some variation from the prototypic E (Eu) and C (LW2) genes, respectively. Comparison of DNA from grc+ and grc- strains of rats showed that the deletion in the grc- strains was approximately 50 kb, and that it was located on two of the three cosmids in the grc-region contig. The use of specific class I probes showed that the grc region contained tandemly duplicated RT1.O-RT1.N genes and that the RT.BM1 loci lay outside of the grc region. Neither contig reacted with probes specific for class II, TNFA, Hsp70, or RT1.M genes. The data presented here and the previous data in the literature (summarized in Gill et al. 1995) suggest that the gene order in the major histocompatibility complex (MHC) and MHC-linked region of the rat is: A-E/C-grc-M.

Expression of the rat MHC class I gene in the human B lymphoblastoid cell line Hmy 2 CIR.

Rat MHC class I cDNA (e.g. RT1.A1) can be expressed optimally as a heterodimer consisting of the rat heavy chain and the human beta2-microglobulin in the human B lymphoblastoid cell line Hmy 2 CIR, which offers several advantages. Gene expression was detected by flow cytometry using anti-human beta2-microglobulin and anti-rat heavy chain antibodies.

Nucleotide sequence and structural analysis of the rat RT1.Eu and RT1.Aw3l genes, and of genes related to RT1.O and RT1.C.

A cDNA library was constructed using mRNA isolated from the R21 strain of rats which have the major histocompatibility complex (MHC) haplotype RT1.AlBlDlEu and the growth and reproduction complex (grc) genotype grc+. The cDNA clones that hybridized with the class I probes pAG64c and pARI.5 and were 1.3-1.7 kilobases were selected. Full-length clones were identified by sequencing partially the 5' and 3' ends of each clone, by the presence of a start codon at the 5' end, and by a polyadenylation sequence at the 3' end. The full-length cDNA clones were examined for in vitro transcription by transfection into human CIR cells using electroporation, and expression was detected by flow cytometry using monoclonal antibodies specific to the heavy chains and polyclonal antibody to beta 2-microglobulin. The RT1.Eu gene was transcribed and expressed optimally, and its nucleotide and deduced amino acid sequences differed significantly from the RT1.Aa, RT1.A(l), RT.Au, LW2, and 11/3R genes but only slightly from the RT1.K gene. The high level of sequence similarity between RT1.Eu and RT1.K suggests that the two genes may have originated from a common ancestral gene. In addition, three new genes (RT1.Aw3l, RT1.C-type, and RT1.O-type) were identified. The RT1.Aw3l gene is almost identical to RT1.A(l) with the exception of an in frame deletion of 21 nucleotides in exon 2 leading to a 7 amino acid deletion in the alpha 1 domain of the deduced amino acid sequence and 11 nucleotide substitutions and insertions in the rest of the sequence. It transcribed optimally, but no significant expression was detected. The RT1.C-type gene 119 is very similar (97%) to the LW2 gene in the 3' untranslated region, which suggests that it is in the RT1.C region. It transcribed optimally, but no significant expression was detected. The RT1.O-type gene 149 has all the features of a class Ib gene, but a premature stop codon in the alpha 1 domain causes incomplete translation. Its in vitro transcription was very low, and no expression was detected. These studies, combined with previous work, indicate that in the MHC of the R21 strain three class Ia genes (Eu, A(l), Aw3l) and three class Ib genes (C-type, O-type, N) are transcribed but only two class Ia genes (Eu, A(l)) are expressed.

Clusterin expression in differentiating smooth muscle cells.

Clusterin is a heterodimeric glycoprotein, expressed by various cell types and shown to have activity in cell-cell adhesion. Cultured porcine smooth muscle cells (SMC) undergo morphological and phenotypic modulation associated with a change from a substrate-attached monolayer culture to a nodular culture in which most of the cells are present in multicellular aggregations (nodules). During that transition from monolayer to nodular cell culture (> 8 days) the expression of an mRNA and protein with significant homology to rat and human clusterin is increased. Clusterin expression continues in the nodular cell cultures and it is secreted at 0.3 micrograms/ml/24 h as a protein with an apparent molecular mass = 80 kDa. In the presence of beta-mercaptoethanol the molecular mass is approximately 40 kDa. SMC clusterin expression is regulated by culture conditions that also affect culture morphology. SMC cultures seeded on a preformed extracellular matrix composed of Matrigel form nodules within 24 h and cultures seeded on a collagen gel form nodules in 48-72 h. We establish here that Matrigel contains clusterin and propose that endogenous clusterin supports the rapid formation of nodules. The collagen gel does not contain clusterin but facilitates clusterin expression by smooth muscle cells. Nodule formation in SMC cultures growing on collagen gel is inhibited by the addition of anticlusterin antibody to SMC growing on collagen gels and the antibody effect is eliminated by preincubation with purified plasma clusterin. These results demonstrate differential expression of SMC clusterin and suggest that clusterin has a functional role in SMC modulation.

Isolation and partial polypeptide characterization of bovine neutrophil plasma membranes.

The neutrophil plasma membrane has a major role in migration, phagocytosis, and destruction of microorganisms. Neutrophils isolated from blood and mammary secretions were homogenized, and the plasma membrane fraction was isolated on discontinuous sucrose gradient (20, 32, and 50%). Purity of plasma membrane preparation was determined by use of marker enzyme analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the membrane proteins was performed under reducing conditions for polypeptide characterization. The membrane proteins were also labeled with 125I externally, using 1,3,4,6-tetrachloro-3 alpha-6 alpha-diphenylglycouril, and proteins were separated by SDS-PAGE and autoradiographed. Compared with whole cell homogenate, the plasma membrane fraction obtained at the 20/32% interface was enriched for the marker enzymes, 5'-nucleotidase (16-fold), alkaline phosphatase (5.5-fold), and total phosphatase (26-fold). The membrane fraction had minimal specific activity for beta-glucuronidase (0.4-fold), compared with whole cell homogenate. Plasma membrane protein yield was about 500 micrograms/10(9) bovine blood neutrophils. The SDS-PAGE of plasma membrane proteins revealed 25 protein bands, of which 13 were major bands. There were 3 distinct bands (18, 36, and 65 kd) in the plasma membrane-enriched fraction (20/32 interface) that were not seen in other fractions (30/50% and pellet). Further, 125I-labeling identified 5 distinct protein bands (205, 140, 65, 35, and 30 kd). Blood and mammary neutrophils had similar polypeptide patterns, except that 36- and 65-kd bands were more prominent for blood neutrophils than for mammary neutrophils.