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.

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.

Modulation of bovine neutrophil functions by monoclonal antibodies.

The main objective of the study reported here was to generate a panel of monoclonal antibodies (MAB) to bovine neutrophil surface antigens, and to identify MAB that modulate neutrophil chemotaxis, respiratory burst, and phagocytosis. A further objective was to study MAB reactivity with resting and activated neutrophils, to identify activation antigens and adhesion molecules. A panel of 14 MAB was generated by producing murine hybridomas. Neutrophils incubated with MAB at 4 C for 2 hours were used in chemotaxis, respiratory burst, and phagocytosis assays. Chemotaxis was evaluated in Boyden chambers, using Escherichia coli endotoxin-activated fetal bovine serum as the chemoattractant. Respiratory burst was determined by measuring chemoluminescence of neutrophils incubated with 5-amino-2,3-dihydro-1,4-phthalazinedione, and serum opsonized zymosan. Phagocytosis was determined by flow cytometry, using fluorescein-labeled Staphylococcus aureus. The MAB S7G8, S5F8G10, S7E10, and S5F8B8 enhanced chemotaxis (to > 125% of control). The MAB S7E10 and S8D9 enhanced respiratory burst activity (to > 125% of control), whereas MAB S2G8, S4G10, S8G10, and S5F8B8 caused inhibition (to < 75% of control). The MAB S2G8, S4G10, S8G10, and S5F8G10 enhanced phagocytosis (to > 125% of control). Chemotaxis, respiratory burst, and phagocytosis values of neutrophils not bound with MAB served as controls for comparison. The MAB binding for nonactivated neutrophils (at 4 C) ranged from 9 to 100%, and for activated neutrophils (at 37 C; at 37 C with phorbol myristate acetate) from 90 to 100%. Binding of MAB S4F5, S5F8B8, S6C6, S7E10, S8D9, and S5F8G10 increased when neutrophils had been incubated at 37 C.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophil surface antigens, and monoclonal antibody production and characterization.

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophils (surface antigen-specific) were optimized. Sensitivity of the 2 methods was compared. A panel of 14 murine monoclonal antibodies (MAB) to surface antigens of bovine polymorphonuclear neutrophilic leukocytes (neutrophils) was produced by hybridoma technology, and their isotypes were determined by whole-cell ELISA. Monoclonal antibody reactivity with neutrophils, eosinophils, and lymphocytes isolated on phosphate-buffered saline solution and on Ficoll-sodium diatrizoate were compared. Biochemical characterization of antigens recognized by MAB was performed by immunoblot analysis. Neutrophil plasma membranes were isolated on sucrose gradients (20, 32, and 50%) and purified for polypeptide characterization. Neutrophil surface proteins were characterized by external labeling with 125I. The flow cytometric method was proven to be more sensitive and rapid than ELISA to screen hybridoma supernatants. This method allowed light-scatter gating of live neutrophil populations for analysis, which eliminated nonspecific binding of antibodies to contaminating cells and dead neutrophils. The optimal conditions for flow cytometric analyses were 5 x 10(5) neutrophils and 1 micrograms of fluorescein-labeled F(ab')2/assay as the second antibody. The optimal conditions for hybridoma screening by ELISA were neutrophil concentration of 2.5 x 10(5)/well, using a 96-well polystyrene microtitration plate as solid support, and 2,2'-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 as the chromatogenic substrate. Tissue culture plates as solid support and 3,3', 5,5'-tetramethyl benzidine, with H2O2 as the chromogenic substrate, were equally as sensitive. Panel MAB reacted differently with neutrophils, eosinophils, and lymphocytes. Isolation of these cells from blood on Ficoll-sodium diatrizoate generally did not alter MAB reactivity. Coomassie blue-stained gels of neutrophil plasma membrane proteins contained about 25 polypeptide bands, 13 of which were major bands. Autoradiography revealed about 11 surface proteins, 5 of which were heavily labeled with 125I. Monoclonal antibody S7G8 identified a 65-kd protein and MAB S8G10 identified 65- and 70-kd proteins. On the basis of molecular weight, MAB S7G8 and S8G10 are comparable to human CD15, CD16, and CD64 molecules. The MAB generated in this study are potential candidates to discern bovine neutrophil function and heterogeneity.

Flow cytometric study of oxidative burst activity in bovine neutrophils.

A flow cytometric procedure was evaluated to measure the oxidative burst activity (hydrogen peroxide formation) of bovine neutrophils. The method involves measuring the oxidation of intracellular dichlorofluorescein to fluorescent dichlorofluorescein (DCF). Phorbol myristate acetate (PMA) was used to perturb the neutrophil plasma membrane. The sources of variation introduced into the DCF assay were also examined. The sources of variation were attributable to the isolation of neutrophils from blood, variation between duplicate assays and duplicate flow cytometric determinations of oxidative product formation, variation in neutrophil oxidative product formation among cows, and the variation (over repeated daily and weekly neutrophil isolations) in neutrophil oxidative product formation. A final objective was to determine effects of dexamethasone on oxidative product formation, and whether differences existed between blood and mammary neutrophils in oxidative product formation. There was an increasing trend in the formation of DCF with increasing time of incubation and with increasing PMA concentration. Increasing the concentration of PMA decreased lag time and increased the rate of oxidative product formation. The increase in DCF formation was statistically significant up to a PMA concentration of 10 ng/ml. This concentration was considered optimal for bovine neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)