Donor cell replacement in mice transplanted in utero is limited by immune-independent mechanisms.

In utero transplantation (IUTx) therapy with allogeneic cells results in negligible peripheral blood (PBL) chimerism in nonablated humans with progressive diseases. IUTx has been successful only in immunocompromised fetuses. Because early treatment has great potential for halting disease progression, mechanisms preventing cell expansion must be identified and corrected. The hypothesis that factors in addition to allogenicity are responsible for low-level expansion is tested here by transplanting congenic cells into nonablated normal and mucopolycaccharidosis type VII (MPSVII) murine fetuses. MPSVII mice lack the enzyme beta-glucuronidase (GUSB-), accumulate glycosaminoglycans, and progressively develop severe storage disease. Low levels of enzyme can reverse storage. Evidence presented elsewhere showed that allogeneic donor cells overexpressing GUSB are negligible and their corrective effects are lost post-IUTx in MPSVII mice. We find that (1) congenic donor PBL cells, like allogeneic cells, are negligible in PBL of normal GUSB+ and MPSVII GUSB- hosts post-IUTx; (2) congenic, unlike allogeneic cells, are retained long term in both GUSB+ and GUSB- recipients; and (3) sufficient GUSB is produced to alleviate storage for up to 11.5 months in multiple tissues of GUSB- hosts. GUSB+ and GUSB- animals repopulate to a similar extent, indicating that donor GUSB enzyme does not initiate an immune response in the MPSVII null recipients. We conclude that the initial expansion of congenic and allogeneic cells is limited post-IUTx by non-immune-related mechanisms and the level of PBL cells is not necessarily indicative of graft failure following congenic IUTx. The mechanism limiting initial expansion may differ from that supporting long-term cell retention.

Nonablative neonatal marrow transplantation attenuates functional and physical defects of beta-glucuronidase deficiency.

The toxicity of preparative regimens render neonatal bone marrow transplantation (BMT) for progressive childhood diseases a controversial treatment. Ablative BMT in neonatal mice with or without the lysosomal storage disease mucopolysaccharidosis type VII (MPS VII) show high morbidity and developmental disruption of both brain and bone structure. In this investigation, BMT was performed with a high dose of congenic, normal bone marrow into nonablated newborn mice. Recipients had lifelong, multilineage, peripheral blood chimerism with the donor beta-glucuronidase-positive (GUS(+)) cells that was both well tolerated and therapeutic. Three daily injections of normal adult marrow increased the average life span by at least 6 months and corrected the functional breeding deficits typical of the MPS VII mice. Twelve months after injection, several structural features of femurs were more like that of normal mice than of untreated MPS VII mice. Periosteal circumference and bone cortical thickness were significantly improved in males and cortical density did not differ significantly from values in normal females. Significant reduction of lysosomal glycosaminoglycan storage corresponded directly with GUS enzyme activity and percentage of histochemically GUS(+) cells in visceral organs and hematopoietic tissues such as thymus, spleen, peripheral blood, and bone marrow. By all criteria tested, BMT into neonatal MPS VII mice in the absence of any preparative regimen is a successful therapy.

A genetically myeloablated MPS VII model detects the expansion and curative properties of as few as 100 enriched murine stem cells.

Causes of transplantation failures are often difficult to assess due to our inability to monitor hematopoietic stem cell (HSC) homing, distribution, and amplification in situ. We have developed a mouse model that permits histochemical localization of 1000-fold enriched HSC and quantification of their long-term expanded progeny in situ. The mice are genetically myeloablated (c-kit receptor mutated, W41/W41) and are beta-glucuronidase null (GUSB ; gus(mps)/gus(mps)). The GUSB- mice with mucopolysaccharidosis type VII (MPS VII), like a large number of human patients with similar diseases, have systemic lysosomal storage disease that leads to premature death. Congenic GUSB+, Lineage(lo), Sca-1(hi), c-Kit(hi), Hoechst(lo) HSC, at doses of 30, 100, 250, and 425 cells, implanted and amplified in adult W41/W41, gus(mps)/gus(mps) recipients in a dose-dependent manner. At autopsy, primary recipients of 100 and 425 donor cells had histologically identifiable donor GUSB+ cells in multiple sites and showed both myeloid and lymphoid expansion in bone marrow. Donor cells were rare in the liver and spleen of 100-cell recipients, but lysosomal storage was significantly reduced. The life span was significantly extended in engrafted recipients of 250 (36.7 +/- 3.84 weeks,p = 0.0316) and 425 (40.7 +/-1.53 weeks,p = 0.0033) cells compared to untreated mice (26.4 +/- 1.53 weeks). Secondary hosts of marrow from the recipients of 425 cells demonstrated continued expansion of the GUSB+ cells. Results indicate the genetically myeloablated MPS VII mice can be used to trace and enumerate donor cells long-term and to follow early engraftment events in situ.

Enzyme replacement therapy improves reproductive performance in mucopolysaccharidosis type VII mice but does not prevent postnatal losses.

Mice with mucopolysaccharidosis type VII (MPS VII) are devoid of beta-glucuronidase and accumulate glycosaminoglycans in lysosomes resulting in bone dysplasia, learning disabilities, and decreased mobility. MPS VII males do not breed and, while MPS VII females occasionally mate with heterozygous males, they do not maintain their young postnatally. Heterozygous matings produce less than 25% MPS VII offspring, but until now it was unclear whether this results from prenatal or postnatal losses. The administration of recombinant beta-glucuronidase from birth significantly reduces glycosaminoglycan storage in most tissues, increases life span, and improves the animal's cognitive ability and mobility. To determine whether reproductive failure is corrected by such therapy, male and female MPS VII mice were injected with enzyme at weekly intervals from birth to 5 wk of age (6xinj). Enzyme-replaced MPS VII mice bred when mated together. The 6xinj MPS VII males mated repeatedly until they were killed 135 d postinjection. All mated 6xinj MPS VII females gave birth to two litters, but maintained few of their young. Selective loss of MPS VII offspring was observed in matings between heterozygotes. Analysis of 379 preterm fetuses from heterozygous matings showed a frequency of 24.6% MPS VII pups, indicating that the decreased number of MPS VII pups produced by mating heterozygotes results from postnatal losses. The ovaries of young adult MPS VII mice have follicles and corpora lutea, and the testes generate sperm. Results suggest that the reproductive failure in MPS VII mice is related to impaired mobility and/or impaired cognitive function, and enzyme replacement restores mating capacity.

Characterization of additional host restriction-modification systems in the unicellular cyanobacterium Cyanothece sp.

In order to develop a gene transfer system for the unicellular diazotrophic cyanobacterium Cyanothece sp. strain BH68K, this organism has been further investigated for the presence of additional host restriction-modification enzymes other than Csp68KI, previously reported for Cyanothece sp. Analysis of cell extracts by phosphocellulose and Mono Q fast protein liquid chromatography (FPLC) has led to the identification of three new restriction endonucleases. These enzymes have been designated Csp68KII, Csp68KIII, and Csp68KVI. Csp68KII is an isoschizomer of AsuII and restricts DNA at the recognition sequence 5'-TT/CGAA-3'. Cleavage occurred between thymine and cytosine producing 2 bp 5' overhang ends. The third restriction endonuclease, Csp68KIII, is an isoschizomer of AvaIII and restricts DNA at the recognition sequence 5'-ATGCA/T-3'. Cleavage occurred between the 3' adenosine and thymine nucleotides producing 4 bp 3' overhang ends. The fourth enzyme identified, Csp68KVI, recognizes CGCG and cleaves this sequence between the internal guanine and cytosine nucleotides producing blunt ends.

Phenotypic variation in exopolysaccharide production in the marine, aerobic nitrogen-fixing unicellular cyanobacterium Cyanothece sp.

The aerobic nitrogen-fixing cyanobacterium, Cyanothece sp. BH68K produces non-mucoid variants defective in exopolysaccharide (EPS) production at a high frequency. The EPS-producing wild-type colonies (EPS(+)) have a characteristic smooth and shiny appearance which allows them to be easily distinguished from the EPS(-) variants. When grown on agar plates lacking a source of combined nitrogen, the EPS(-) variants exhibited a yellow phenotype typical of nitrogen starvation. These EPS(-) variants showed varying degrees of reversion back to the EPS(+) phenotype. After reversion, they exhibited normal diazotrophic growth on agar plates. Alcian blue and ruthenium red staining indicated that the EPS is an acidic polysaccharide, which is present as a loose network around the cell, and which can be completely removed by low speed centrifugation. The accumulation of EPS takes place mainly during the stationary phase. All EPS(-) variants failed to produce any EPS. Analysis of growth of wild-type and EPS(-) variants revealed that EPS production is beneficial for diazotrophic growth on solid medium, but not in liquid medium. In addition, EPS phenotypic alteration may have some advantage in the dispersal of cells from one place to another in the natural environment.

Identification of a nuclease and host restriction-modification in the unicellular, aerobic nitrogen-fixing cyanobacterium Cyanothece sp.

In the process of developing a gene transfer system for the marine, unicellular, nitrogen-fixing cyanobacterium Cyanothece sp. strain BH68K, two major restriction barriers have been identified. A cell wall-associated nuclease exhibited non-site-specific degradation of covalently closed circular and linear double-stranded DNA molecules, including Cyanothece sp. strain BH68K chromosomal DNA. The nuclease is easily released from intact cells by using water or buffer containing Triton X-100. Nuclease activity was undetectable in cell extracts prepared from water-washed cells. Comparison of the restriction endonuclease susceptibility of Cyanothece sp. strain BH68K DNA to that of Anabaena sp. strain PCC 7120 revealed that these organisms have a nearly identical pattern of restriction and therefore may contain similar systems for DNA methylation. Restriction by DpnI, MboI, and Sau3AI indicated the presence of adenine methylation. Cyanothece sp. strain BH68K cell extracts contain a type II restriction endonuclease, Csp68KI. The activity of Csp68KI was easily detected in cell extracts without extensive purification. Csp68KI is an isoschizomer of AvaII and recognizes the nucleotide sequence 5'-GG(A/T)CC-3'. Cleavage occurs between the guanosine nucleotides producing 3-bp 5' overhang ends.

Transcriptional regulation of G-protein alpha i subunit genes in LLC-PK1 renal cells and characterization of the porcine G alpha 1-3 gene promoter.

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) function as signal transducers for a variety of hormone-coupled enzyme and ion transport systems in eukaryotic cells. The expression of pertussis toxin-sensitive G-proteins (Gi) which couple their cognate receptors and effectors are regulated by cell cycle-dependent events in porcine LLC-PK1 renal epithelial cells. G alpha i-2 and G alpha i-3 isoforms are detected in these cells, and like G alpha i-2 (Holtzman, E. J., Soper, B. W., Stow, L. L., Ausiello, D. A., and Ercolani, L. (1991) J. Biol. Chem. 266, 1763-1771), we now demonstrate that G alpha i-3 mRNA and protein is coordinately expressed in these cells during differentiation. To gain further insights into these events, the porcine G alpha i-3 gene minimal promoter was characterized and found 67 base pairs upstream from the major transcription start site. The 56-base pair minimal promoter lacked TATAAA and GC boxes but did contain a sequence GGAAGTG conserved in both the human and porcine gene that could potentially bind an adenovirus E4TF1 transcription factor. In cells stably transfected with G alpha i-2 or G alpha i-3 gene 5'-flanking sequences fused to firefly luciferase cDNA reporter, temporal 10-15-fold transcriptional activation of both genes occurred before cellular polarization. Utilizing mobility shift assays which compared nuclear extracts from cells before and after cell polarization, a motif in the 5' region of the gene promoter GTACTTCCGCT was identified that bound an induced nuclear protein complex during transcriptional activation. In polarized cells complemented with the human glucocorticoid receptor, dexamethasone decreased G alpha i-2 but increased G alpha i-3 basal transcription and mRNA content 3-fold. These studies demonstrate that both G alpha i genes are dynamically regulated in LLC-PK1 cells by both growth, differentiation, and hormone signals.

Regulation of the G-protein alpha i-2 subunit gene in LLC-PK1 renal cells and isolation of porcine genomic clones encoding the gene promoter.

Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme and ion transport systems in eukaryotic cells. We have studied G-protein-coupled processes that appear to be developmentally regulated in polarized pig kidney cells (LLC-PK1). Following trypsinization, LLC-PK1 cells differentiate from a rounded cell type to a fully polarized epithelium by 7 days of culture. During this differentiation, the expression of G-protein alpha i-2 subunit mRNA was not detected until day 4 of culture, it peaked at day 6, and declined thereafter. In contrast, G-protein alpha s subunit mRNA which peaked on day 4 was easily detected on all culture days. The presence of the alpha i-2 protein on epithelial cell basolateral membranes followed the same pattern of mRNA expression during culture. To understand the developmental expression of the alpha i-2 subunit in non-polarized cells and its potential regulation by hormones and second messengers in polarized cells at the transcriptional level, genomic DNA segments encoding the alpha i-2 gene promoter were isolated from an EMBL-3 porcine genomic library. S1 nuclease analysis of LLC-PK1 mRNA with cRNA probes derived from these DNA segments revealed major and a minor transcriptional start sites 131 and 171 base pairs upstream of the translation initiation site. The porcine and human alpha i-2 subunit genes shared a 78% sequence identity in their 5' flanks which suggested an evolutionary conservation of cis elements required to influence their transcription. The porcine alpha i-2 gene promoter was identified by fusing DNA segments encoding putative 5'-flanking areas of the gene to a plasmid that contained a firefly luciferase reporter gene but lacked a promoter. The minimal promoter was found between -130 and -60 base pairs from the major transcription start site. No typical "TATA-like" sequences were found. However, a "GC" box and a "TGTGG" sequence were two potential cis elements required for basal transcription of the porcine gene promoter which shared a 76% sequence identity to the promoter of another GTP-binding protein, the human c-Ha-ras proto-oncogene. Transcription of the gene was inhibited following treatment of renal cells with 10(-8) M dexamethasone.(ABSTRACT TRUNCATED AT 400 WORDS)