Adhya, Sankar (University of Wisconsin, Madison), and Harrison Echols. Glucose effect and the galactose enzymes of Escherichia coli: correlation between glucose inhibition of induction and inducer transport. J. Bacteriol. 92:601-608. 1966.-The inhibitory effect of glucose on the induction of the enzymes required for galactose utilization ("glucose effect") was studied in Escherichia coli. Experiments on the uptake into the cell of labeled inducers (d-galactose-C(14) and d-fucose-H(3)) pointed to inhibition at the level of inducer transport as the possible primary mechanism of the glucose effect in the case of the gal enzymes. This interpretation was supported by the finding that a mutant constitutive for the lac enzymes was resistant to glucose inhibition of galactose induction of the gal enzymes; the mutant had acquired a glucose-resistant alternative transport mechanism for galactose via the constitutively synthesized galactoside permease. Further support for the transport inhibition model was provided by the finding that glucose did not substantially inhibit induction of the gal enzymes when glucose and galactose were produced intracellularly by beta-galactosidase hydrolysis of lactose, even if excess glucose was added. The inducer uptake experiments also showed that d-galactose and d-fucose probably enter the cell via different transport systems, although uptake of both compounds was inhibited by glucose.

Uninducible galactose nonfermenter mutants of Saccharomyces cerevisiae have been isolated and mapped in the gal i locus. They appear to be analogous to the i(s) mutations found in the regulatory genes of the Escherichia coli lactose and galactose systems. The susceptibility to suppression by an ochre suppressor of an i allele in an i(s)i(-) double mutant suggests that the i locus in S. cerevisiae codes for a protein.

The carbohydrate binding specificity of Datura stramonium agglutinin was studied by analyzing the behavior of a variety of complex-type oligosaccharides on a D. Stramonium agglutinin-Sepharose column. Oligosaccharides which contain Gal beta 1----4GlcNAc-beta 1----4(Gal beta 1----GlcNAc beta 1----2)Man units are retarded in the column so long as the pentasaccharide unit is not substituted by other sugars. Oligosaccharides which contain unsubstituted Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----4GlcNAc beta 1----2)Man groups and those in which there is at least one Gal beta 1----4GlcNAc repeating unit present on an outer chain bind to the column and are eluted with buffer containing N-acetylglucosamine oligomers. Binding was not affected by the inner core portion of complex oligosaccharides nor by the presence of a bisecting N-acetylglucosamine residue. With these principles in mind, the column can be used as an effective tool for the analysis of complex-type, asparagine-linked sugar chains.

Galectin-1 (Gal-1), the prototype of a family of beta-galactoside-binding proteins, has been shown to attenuate experimental acute and chronic inflammation. In view of the fact that endothelial cells (ECs), but not human polymorphonuclear leukocytes (PMNs), expressed Gal-1 we tested here the hypothesis that the protein could modulate leukocyte-EC interaction in inflammatory settings. In vitro, human recombinant (hr) Gal-1 inhibited PMN chemotaxis and trans-endothelial migration. These actions were specific as they were absent if Gal-1 was boiled or blocked by neutralizing antiserum. In vivo, hrGal-1 (optimum effect at 0.3 micro g equivalent to 20 pmol) inhibited interleukin-1beta-induced PMN recruitment into the mouse peritoneal cavity. Intravital microscopy analysis showed that leukocyte flux, but not their rolling velocity, was decreased by an anti-inflammatory dose of hrGal-1. Binding of biotinylated Gal-1 to resting and postadherent human PMNs occurred at concentrations inhibitory in the chemotaxis and transmigration assays. In addition, the pattern of Gal-1 binding was differentially modulated by PMN or EC activation. In conclusion, these data suggest the existence of a previously unrecognized function of Gal-1, that is inhibition of leukocyte rolling and extravasation in experimental inflammation. It is possible that endogenous Gal-1 may be part of a novel anti-inflammatory loop in which the endothelium is the source of the protein and the migrating PMNs the target for its anti-inflammatory action.

Recent studies have shown that galectin-3 (Gal-3; also known as LGALS3), a beta-galactoside-binding lectin, promotes cell migration during re-epithelialization of corneal wounds. The goal of this study was to characterize the molecular mechanism by which Gal-3 stimulates cell migration. We demonstrate here that exogenous Gal-3, but not Gal-1 or Gal-8, promotes cell scattering and formation of lamellipodia in human corneal epithelial cells in a beta-lactose-inhibitable manner. alpha3beta1 integrin was identified as the major Gal-3-binding protein in corneal epithelial cells by affinity chromatography of cell lysates on a Gal-3-Sepharose column. Preincubation of cells with anti-alpha3 integrin function-blocking antibody significantly inhibited the induction of lamellipodia by Gal-3. Furthermore, exogenous Gal-3 activated both focal adhesion kinase, a key regulator of integrin-dependent intracellular signaling, and Rac1 GTPase, a member of the family of Rho GTPases, well known for its role in the reorganization of the actin cytoskeleton and formation of lamellipodial extensions. Experiments involving knockdown of beta-1,6-N-acetylglucosaminytransferase V, an enzyme that synthesizes high-affinity glycan ligands for Gal-3, revealed that carbohydrate-mediated interaction between Gal-3 and complex N-glycans on alpha3beta1 integrin plays a key role in Gal-3-induced lamellipodia formation. We propose that Gal-3 promotes epithelial cell migration by cross-linking MGAT5-modified complex N-glycans on alpha3beta1 integrin and subsequently activating alpha3beta1-integrin-Rac1 signaling to promote lamellipodia formation.

G(M1)-gangliosidosis is an autosomal recessive lysosomal lipid storage disorder, caused by mutations of the lysosomal beta-galactosidase (beta-gal) and results in the accumulation of G(M1). The underlying mechanisms of neurodegeneration are poorly understood. Here we demonstrate increased autophagy in beta-gal-deficient (beta-gal(-/-)) mouse brains as evidenced by elevation of LC3-II and beclin-1 levels. Activation of autophagy in the beta-gal(-/-) brain was found to be accompanied with enhanced Akt-mTOR and Erk signaling. In addition, the mitochondrial cytochrome c oxidase activity was significantly decreased in brains and cultured astrocytes from beta-gal(-/-) mouse. Mitochondria isolated from beta-gal(-/-) astrocytes were morphologically abnormal and had a decreased membrane potential. These cells were more sensitive to oxidative stress than wild type cells and this sensitivity was suppressed by ATP, an autophagy inhibitor 3-methyladenine and a pan-caspase inhibitor z-VAD-fmk. These results suggest activation of autophagy leading to mitochondrial dysfunction in the brain of G(M1)-gangliosidosis.

A virulent strain of Mycoplasma pneumoniae was metabolically labeled with [3H]palmitate and studied for binding to glycolipids and to WiDr human colon adenocarcinoma cells. The organism binds strongly to sulfatide and other sulfated glycolipids, such as seminolipid and lactosylsulfatide which all contain terminal Gal(3SO4) beta 1-residues and weakly to some neolactoseries neutral glycolipids. M. pneumoniae do not bind gangliosides including the sialylneolacto-series and other neutral glycolipids that were tested. Only metabolically active M. pneumoniae cells bind to sulfatide, as binding is maximal in RPMI medium at 37 degrees C and almost completely abolished in nutrient-deficient medium or by keeping the cells at 4 degrees C. Dextran sulfate but not other sulfated or anionic polysaccharides at 10 micrograms/ml completely inhibits binding of M. pneumoniae to purified sulfatide. Dextran sulfate does not inhibit binding to the neolacto-series neutral glycolipids. Dextran sulfate partially inhibits adhesion of M. pneumoniae to cultured human colon adenocarcinoma cells (WiDr). The biological relevance of these data is suggested by our finding that sulfatide occurs in large amounts in human trachea, lung, and WiDr cells. Thus, there are at least two distinct receptors that mediate binding of M. pneumoniae to cells: glycolipids containing terminal Gal(3SO4) beta 1-residues as reported here, and glycoproteins containing terminal NeuAc alpha 2-3Gal beta 1-4GlcNAc sequences (Roberts, D. D., Olson, L. D., Barile, M. F., Ginsburg, V., and Krivan, H. C. (1989) J. Biol. Chem. 264, 9289-9293).

Galanin is a neuropeptide with multiple inhibitory actions on neurotransmission and memory. In Alzheimer's disease (AD), increased galanin-containing fibers hyperinnervate cholinergic neurons within the basal forebrain in association with a decline in cognition. We generated transgenic mice (GAL-tg) that overexpress galanin under the control of the dopamine beta-hydroxylase promoter to study the neurochemical and behavioral sequelae of a mouse model of galanin overexpression in AD. Overexpression of galanin was associated with a reduction in the number of identifiable neurons producing acetylcholine in the horizontal limb of the diagonal band. Behavioral phenotyping indicated that GAL-tgs displayed normal general health and sensory and motor abilities; however, GAL-tg mice showed selective performance deficits on the Morris spatial navigational task and the social transmission of food preference olfactory memory test. These results suggest that elevated expression of galanin contributes to the neurochemical and cognitive impairments characteristic of AD.

Previous studies have shown that two subpopulations of cells with astrocytic properties coexist in the mouse hippocampus, which display distinct morphological and functional characteristics, specifically a nonoverlapping expression of either AMPA-type glutamate receptors (GluR cells) or glutamate transporters (GluT cells). Use of transgenic mice with hGFAP promoter-controlled EGFP expression and patch-clamp recordings allow reliable identification of the two cell types in hippocampal slices. Extending functional characterization, we report here the complete lack of gap junctional tracer coupling in GluR cells, while GluT cells are shown to be extensively coupled. This distinction is valid in immature as well as adult animals. Analysis of transgenic mice expressing beta-Gal under regulatory elements of the Cx43 promoter revealed the absence of Cx43 in GluR cells. Experiments using gap junction blockers demonstrated that passive currents, displayed primarily by GluT cells, do not reflect intercellular coupling but are attributable to intrinsic membrane properties of individual cells. This study supports the notion that the two subpopulations of hGFAP-EGFP-positive cells represent distinct cell types with contrasting physiological properties. Since GluR cells do not participate in the astrocytic gap junctional network, their functional role must be different from spatial buffering of ions or signaling molecules, i.e., properties generally assigned to astrocytes.

beta-Galactosidase (beta-gal) has been widely used as a transgene reporter enzyme, and several substrates are available for its in vitro detection. The ability to image beta-gal expression in living animals would further extend the use of this reporter. Here we show that DDAOG, a conjugate of beta-galactoside and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO), is not only a chromogenic beta-gal substrate but that the cleavage product has far-red fluorescence properties detectable by imaging. Importantly, the cleavage substrate shows a 50-nm red shift, enabling its specific detection in a background of intact probe, a highly desirable feature for in vivo imaging. Specifically, we show that beta-gal-expressing 9L gliomas are readily detectable by red fluorescence imaging in comparison with the native 9L gliomas. We furthermore show that herpes simplex virus amplicon-mediated LacZ gene transfer into tumors can be transiently and thus serially visualized over time. The results indicated that in vivo real-time detection of beta-gal activity is possible by fluorescence imaging technology.

There are no data currently available on gender and racial variation in smallpox vaccine immune responses. We recruited 1076 healthy adults 18-40 years old who received one dose of the US-licensed smallpox vaccine (Dryvax). Vaccinia neutralizing antibody titers in each subject's serum were determined using a high throughput neutralization assay based on a recombinant, beta-gal expressing vaccinia virus. Results are reported as the serum dilution inhibiting 50% of virus activity (ID(50)). The median ID(50) for all subjects was 132.2 (inter-quartile range (IQR)=78.8, 205.6). While no significant differences were observed with race and ethnicity, females had significantly higher neutralizing antibody titers than males (158.5 [93.2, 255.8] vs. 124.1 [75.2, 185.9]; p<0.0001). As expected, time since vaccination was also associated with variations in neutralizing antibody titers in our subjects. These data indicate that neutralizing antibody titers following primary smallpox vaccination vary by gender.

OBJECTIVE

Cellular senescence is a unique cellular response pathway thought to be closely associated with the aging process. The senescent phenotype is characterized by the loss of a cell's ability to respond to proliferative and apoptotic stimuli even while normal metabolic activity and vitality is maintained. Recently, a novel biomarker, senescent-associated beta-galactosidase (SA-beta-gal), was found to identify cells with the senescent phenotype. In the present study, we examined whether human prostatic epithelial cells adopt a senescence-associated phenotype after prolonged culture and analyzed a series of human benign prostatic hyperplasia (BPH) specimens to determine whether the cellular senescence process might be a factor in the development of BPH.

METHODS

A primary culture of epithelial cells was established from the normal tissue of the peripheral zone of a radical prostatectomy specimen and was serially passaged until senescence. Forty-three human prostate specimens were obtained subsequent to radical prostatectomy or transrectal ultrasound-guided biopsy. The cultured cells and tissue specimens were histochemically stained to reveal the expression of SA-beta-gal, the cellular senescence biomarker.

RESULTS

As has been reported for other types of cultured cells, human prostatic epithelial cells demonstrated widespread expression of the cellular senescence marker, SA-beta-gal, on prolonged culture. In our survey of hypertrophied human prostate tissues, 17 specimens (40%) of the 43 analyzed demonstrated positive staining for SA-beta-gal. In these tissues, SA-beta-gal expression was noted only in the epithelial cells. No statistical correlation (P = 0.42) between the chronologic age of the patient donor and SA-beta-gal expression was found. However, a high prostate weight (greater than 55 g) was found to correlate strongly with the expression of the SA-beta-gal biomarker (P = 0. 0001).

CONCLUSIONS

Cultured prostatic epithelial cells expressed SA-beta-gal on reaching replicative senescence in vitro. The survey of human BPH specimens for the senescent marker showed that prostatic epithelial cells in patients with BPH with more advanced enlargement of the prostate (greater than 55 g prostate weight) expressed SA-beta-gal, and the prostates from patients with BPH that weighed less than 55 g tended to lack senescent epithelial cells. On the basis of these results, we propose that the accumulation of senescent epithelial cells may play a role in the development of the prostatic enlargement associated with BPH.

BACKGROUND

Endothelial regeneration and angiogenesis in the intima of the arterial wall are key events in the pathogenesis of transplantation arteriosclerosis. The traditional hypothesis that damaged endothelial cells are replaced by remaining cells of the donor vessel has been challenged by recent observations, but the cell origins of large arteries and microvessels are still not well established.

RESULTS

Aortic segments were allografted between Balb/c and TIE2-LacZ (C57BL/6) mice expressing beta-galactosidase (gal) in endothelial cells. beta-gal+ cells in TIE2-LacZ vessels grafted to Balb/c mice completely disappeared, whereas the positive cells found in Balb/c aorta allografted into TIE2-LacZ mice 4 weeks after surgery indicated a host origin. En face analysis of allograft vessels displayed a unique distribution of beta-gal+ cells on the surface at 3 days, 1 week, and 4 weeks. Interestingly, 35+/-19% beta-gal+ cells were found in arterial segments allografted into chimeric mice with TIE2-LacZ bone marrows. Furthermore, endothelial cells of microvessels within allografts had a beta-gal+ staining in the media at 1 week and in the neointimal lesions and adventitia at 4 weeks. Allograft studies in chimeric mice demonstrated that beta-gal+ cells of microvessels in transplant arteriosclerosis were derived from bone marrow progenitors.

CONCLUSIONS

We provide strong evidence that endothelial cells of neointimal lesions in allografts are derived from circulating progenitor cells and that bone marrow-derived progenitors are responsible for angiogenesis of the allograft, that is, the formation of microvessels in transplant arteriosclerosis.

Our previous studies showed immunological and functional similarities, as well as partial sequence homology, between the enzymatically inactive alternatively spliced variant of human beta-galactosidase (S-gal) and the 67-kDa elastin/laminin-binding protein (EBP) from sheep. To define the genetic origin of the EBP further, a full-length human S-gal cDNA clone was constructed and subjected to in vitro transcription/translation. The cDNA was also transfected into COS-1 cells and into the EBP-deficient smooth muscle cells (SMC) from sheep ductus arteriosus (DA). In vitro translation yielded an unglycosylated form of the S-gal protein, which immunoreacted with anti-beta-galactosidase antibodies and bound to elastin and laminin affinity columns. S-gal cDNA transfections into COS-1 and DA SMC increased expression of a 67-kDa protein that immunolocalized intracellularly and to the cell surface and, when extracted from the cells, bound to elastin. The S-gal-transfected cells displayed increased adherence to elastin-covered dishes, consistent with the cell surface distribution of the newly produced S-gal-encoded protein. Transfection of DA SMC additionally corrected their impaired elastic fiber assembly. These results conclusively identify the 67-kDa splice variant of beta-galactosidase as EBP.

BACKGROUND

Mesenchymal stem cells (MSC) are pluripotent progenitors for a variety of cell types, including fibroblasts and muscle cells. Their involvement in the tissue repair of allografts during the development of chronic rejection has been hypothesized, but not yet substantiated, by experimental evidence.

METHODS

Rat MSC were isolated from circulation using an aortic pouch allograft as a trapping device. The plasticity of these cells was examined in differentiation cultures. One of the resulting MSC lines was immortalized and transduced to express a marker gene. The -labeled cells were then transferred to F344 rats bearing Lewis (LEW) cardiac allografts to measure their localization and contribution to graft tissue repair.

RESULTS

The MSC isolated from circulation exhibited multipotential for differentiation in culture, developing into various lineages including osteoblasts, lipocytes, chondrocytes, myotubes, and fibroblasts. Intravenous engraftment of the -labeled cells into recipients of heart transplant resulted in migration of the beta-gal+ cells into the lesions of chronic rejection in the cardiac grafts and homing of the cells to the bone marrow. The majority of beta-gal+ cells present in the allografts exhibited fibroblast phenotypes, and a small number of the cells expressed desmin, indicative of myocyte differentiation.

CONCLUSIONS

MSC vigorously migrated into the site of allograft rejection. This data suggests that they may be attracted to this site to actively participate in tissue repair during chronic rejection. In addition, given the robust migration, the inhibition of MSC differentiation toward fibroblast progeny and induction toward the myocyte lineage may serve as a new strategy for treatment of chronic rejection and allograft tissue repair.

BACKGROUND

IgA nephropathy (IgAN) is characterized by IgA1-containing immune complexes in mesangial deposits and in the circulation. The circulating immune complexes (CIC) are composed of galactose- (Gal) deficient IgA1 and IgG or IgA1 antibodies specific for the Gal-deficient IgA1; interactions of these CIC with mesangial cells (MC) were studied.

METHODS

Binding, internalization, and catabolic degradation of myeloma IgA1 protein as a standard control and the isolated CIC were studied using human MC, hepatoma cell line HepG2 expressing the asialoglycoprotein receptor (ASGP-R), and monocyte-like cell line U937 expressing the Fc(alpha)-R (CD89). Biochemical and molecular approaches were used to assess expression of CD89 and ASGP-R by MC.

RESULTS

At 4 degrees C, radiolabeled IgA1 bound to MC and HepG2 cells in a dose-dependent and saturable manner. The binding was inhibited by IgA-containing CIC or excess IgA1 or its Fc fragment but not by the Fab fragment of IgA1. At 37 degrees C, the cell-bound IgA1 was internalized and catabolized. In addition to IgA1, HepG2 cells also bound (in a Ca2+-dependent manner), internalized, and catabolized asialoorosomucoid (ASOR), other asialo-(AS)-glycoproteins, and secretory component (SC). The binding by MC appeared to be restricted to IgA1 or AS-IgA1 and was not Ca2+-dependent. Furthermore, MC and HepG2 cells internalized and catabolized IgA1-containing CIC. Using RT-PCR with ASGP-R- or CD89-specific primers, mRNAs of the two respective genes were not detected in MC.

CONCLUSIONS

The data showed that the ability of MC to bind IgA1 and IgA1-containing CIC in vitro was mediated by an IgA receptor that was different from CD89 or ASGP-R and had a higher affinity for IgA-CIC than for uncomplexed IgA.

In view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Oncolytic vesicular stomatitis virus (VSV) is a promising novel therapeutic agent for the treatment of cancer. The aim of this study was to evaluate the potential of recombinant VSV containing the M51R mutation in the matrix (M) protein gene administered intravenously as an effective and safe therapeutic agent for treating mice with experimental breast cancer metastases. Recombinant VSV(M51R)-LacZ was generated and characterized in vitro on human and murine breast cancer cells. Breast cancer metastases were established in immune-competent Balb/c mice by intravenous injection of syngeneic 4T1 cells. The vector was infused into the tumor-bearing animals via the tail vein, and productive infection of pulmonary breast cancer lesions was assessed by X-gal stainings of frozen lung sections. To evaluate potential systemic toxicity, histology of major organs and serum chemistries were analyzed. To assess effectiveness, buffer- or vector-treated tumor-bearing mice were followed for survival and the results were analyzed by the Kaplan-Meier method and the log-rank test. We found that VSV(M51R)-LacZ efficiently replicated and lysed human breast cancer cells but was partially attenuated in 4T1 cells in vitro. We also demonstrated that its maximum tolerated dose after intravenous infusion in normal Balb/c mice was elevated by at least 100-fold over that of the parental VSV vector containing the wild-type M gene. When VSV(M51R)-LacZ was repeatedly injected intravenously into mice bearing syngeneic 4T1 tumors, the virus was able to infect multiple breast cancer lesions in the lungs without apparent toxicities, which led to significant prolongation of animal survival (P=.003). In conclusion, systemic administration of M mutant VSV is both effective and safe in the treatment of experimental breast cancer metastases in immune-competent mice, suggesting that further development of this approach may have potential for clinical application in patients.

BACKGROUND

The population of senescent disc cells has been shown to increase in degenerated or herniated discs. However, the mechanism and signaling pathway involved in the senescence of nucleus pulposus (NP) chondrocytes are unknown.

OBJECTIVE

To demonstrate the mechanisms involved in the senescence of NP chondrocytes.

METHODS

Senescence-related markers were assessed in the surgically obtained human NP specimens.

METHODS

NP specimens remaining in the central region of the intervertebral disc were obtained from 25 patients (mean: 49 years, range: 20-75 years) undergoing discectomy. Based on the preoperative magnetic resonance images, there were 3 patients with Grade II degeneration, 17 patients with Grade III degeneration, and 5 patients with Grade IV degeneration.

METHODS

We examined cell senescence markers (senescence-associated beta-galactosidase [SA-beta-gal], telomere length, telomerase activity, p53, p21, pRB, and p16) and the hydrogen peroxide (H(2)O(2)) content as a marker for an oxidative stress in the human NP specimens.

METHODS

SA-beta-gal expression, telomere length, telomerase activity, and H(2)O(2) content as well as their relationships with age and degeneration grades were analyzed. For the mechanism involved in the senescence of NP chondrocytes, expressions of p53, p21, pRB, and p16 in these cells were assessed with immunohistochemistry and Western blotting.

RESULTS

The percentages of SA-beta-gal-positive NP chondrocytes increased with age (r=.82, p<.001), whereas the telomere length and telomerase activity declined (r=-.41, p=.045; r=-.52, p=.008, respectively) However, there was no significant correlation between age and H(2)O(2) contents (p=.18). The NP specimens with Grade III or Grade IV degeneration showed significantly higher percentages of SA-beta-gal-positive NP chondrocytes than those with Grade II degeneration (p=.01 and p=.025, respectively). Immunohistochemistry showed that the senescent NP chondrocytes in all the specimens expressed p53, p21, and pRB, but a few NP chondrocytes in only two specimens expressed p16. Western blotting showed that the expressions of p53, p21, and pRB displayed a corresponding pattern, that is, a strong p53 expression led to strong p21 and pRB expressions and vice versa.

CONCLUSIONS

Our in vivo study demonstrated that senescent NP chondrocytes increased or accumulated in the NP with increasing age and advancing disc degeneration. The NP chondrocytes in the aging discs exhibited characteristic senescent features such as an increased SA-beta-gal expression, shortened telomeres, and decreased telomerase activity. We further demonstrated that the telomere-based p53-p21-pRB pathway, rather than the stress-based p16-pRB pathway, plays a more important role in the senescence of NP chondrocytes in an in vivo condition. Our results suggest that prevention or reversal of the senescence of NP chondrocytes can be a novel therapeutic target for human disc degeneration.

We sought to determine whether transplanted allogeneic bone mesenchymal stem cells can survive and increase the amount of proteoglycans in intervertebral discs. We used the rabbit intervertebral disc as a model, creating three groups: an uninjected control group, a group injected with saline, and a group injected with 1 x 10(5) of bone mesenchymal stem cells containing trace marker gene LacZ from young rabbits. At 1, 3, and 6 months, X-gal staining and DNA-polymerase chain reaction of the neomycin-resistant gene were used to ascertain cell location. Reverse transcription-polymerase chain reaction and enzyme-linked immunosorbant assay analysis were done to evaluate the effect on the disc matrix. Transplanted mesenchymal stem cells were located and identified in the group injected with mesenchymal stem cells, whereas we detected none in the saline and control groups. The amount of mRNA and protein of proteoglycan and collagen Type II in the mesenchymal stem cells group was increased, whereas the amount of collagen Type I did not change. We found no changes in the saline group. Our data suggest transplanted allogenic bone mesenchymal stem cells can survive and increase proteoglycan amount, supporting its potential use as a treatment of intervertebral disc degeneration.

OBJECTIVE

Gene therapy offers a promising alternative for the treatment of ocular diseases. However, the implementation of this type of therapy is actually hampered by the lack of an efficient ocular gene delivery carrier. The main objective of the present work was to assess the effectiveness and investigate the mechanism of action of a new type of nanoparticle made of two bioadhesive polysaccharides, hyaluronic acid (HA) and chitosan (CS), intended for the delivery of genes to the cornea and conjunctiva.

METHODS

The nanoparticles were obtained by a very mild ionotropic gelation technique. They were loaded with either the model plasmid pEGFP or pbeta-gal. Transfection and toxicological studies were conducted in human corneal epithelial (HCE) and normal human conjunctival (IOBA-NHC) cell lines. The mechanism of internalization of the nanoparticles by the corneal and conjunctival cells was investigated by using fluorescence confocal microscopy.

RESULTS

The nanoparticles had a size in the range of 100 to 235 nm and a zeta-potential of -30 to +28 mV. The results of the transfection studies showed that HA-CS nanoparticles were able to provide high transfection levels (up to 15% of cells transfected), without affecting cell viability. The confocal images indicated that HA-CS nanoparticles were internalized by fluid endocytosis and that this endocytic process was mediated by the hyaluronan receptor CD44.

CONCLUSIONS

The results give evidence of the potential of HA-CS nanoparticles for the targeting and further transfer of genes to the ocular surface.

OBJECTIVE

Transplantation is limited by a lack of human organ donors. Organs derived from animals, most likely the pig, represent a potential solution to this problem. For the heart, 90-day median graft survival of life-supporting pig hearts transplanted to nonhuman primates has been considered a reasonable standard for entry into the clinical arena. Overcoming the immune barrier to successful cardiac xenotransplantation is most appropriately first explored with the non-life-supporting heterotopic model.

METHODS

We performed a series of 7 heterotopic heart transplantations from CD46 transgenic pigs to baboons using a combination of therapeutic agents largely targeted at controlling the synthesis of anti-pig antibodies. Rituximab (anti-CD20) and Thymoglobulin (rabbit antithymocyte globulin [ATG]; SangStat Medical Corp, Fremont, Calif) were used as induction therapy. Baseline immunosuppression consisted of splenectomy, tacrolimus, sirolimus, steroids, and TPC (an anti-Gal antibody therapeutic). Rejection events were not treated.

RESULTS

By using Kaplan-Meier analysis, median graft survival was 96 days (range, 15-137 days; 95% confidence interval, 38-99 days). Only 2 grafts were lost as a result of rejection, as defined by cessation of graft palpation. There was no evidence of a consumptive coagulopathy, infectious complications were treatable, and no posttransplantation lymphoproliferative disorders occurred. No cellular infiltration was observed.

CONCLUSIONS

This study reports the longest median survival to date (96 days) of pig hearts transplanted heterotopically into baboons. Duplication of these results in the orthotopic life-supporting position could bring cardiac xenotransplantation to the threshold of clinical application.

BACKGROUND

Recently, it has been shown that stem/progenitor cells may repair damaged/lost endothelial cells in vein grafts and wire-injured arteries. In the present study, we investigated endothelial cell turnover and regeneration in apolipoprotein E (apoE)(-/-)/transgenic mice carrying LacZ genes driven by an endothelial TIE2 promoter.

RESULTS

To assess cell proliferation on the surface of aortas in apoE(-/-) mice and wild-type controls, BrdU was injected into the tail vein and labeled on en face preparation. BrdU-positive cells on the aortas were observed occasionally in wild-type mice and frequently at sites prone to lesion development in apoE(-/-) mice (0.18+/-0.1% versus 1.12+/-0.2%; P<0.001). Endothelial integrity tests demonstrated that the areas with high rate of cell turnover displayed Evans blue leakage, low levels of VE-cadherin expression, and increased cell attachment, as evidenced by Evans blue dye injection, immunostaining, and scanning electron microscopy, respectively. Furthermore, immunostaining for CD34, Sca-1, Flk-1, and CD133 indicated that approximately 3% to 5% of total cells on the aorta were positive in apoE(-/-) mice. En face double labeling using Ki-67 and progenitor markers revealed that 30% to 50% of progenitor(+) cells expressed Ki-67, indicating a state of proliferation. To clarify the origin of endothelial progenitor cells participating in endothelial repair in apoE(-/-) mice, a chimeric mouse model was created by bone marrow transplantation between apoE(-/-) and LacZ(+/+)/apoE(-/-) mice. Ten months after bone marrow transplantation, approximately 3% to 4% of total cells in the lesion-prone areas were beta-gal positive in apoE(-/-) with apoE(-/-)/TIE2-LacZ bone marrow mice. When cells of aortas from chimeric mice were cultivated on Matrigel-coated plates, a capillary-like structure was found, which showed beta-gal/CD31 or beta-gal/von Willebrand factor double positivity. By a combined analysis of laser dissection microscopy and nest reverse transcription polymerase chain reaction, it was found that beta-gal(+) cells were mainly expressing CD31 and CD144.

CONCLUSIONS

Our findings provide the first quantitative data on endothelial turnover and repair by progenitor cells that are, at least in part, derived from bone marrow during development of atherosclerosis in apoE(-/-) mice.

Vascular development in the embryo requires coordinated signaling through several endothelial cell-specific receptors; however, it is not known whether this is also required later during retinal vascular development or as part of retinal neovascularization in adults. The Tie2 receptor has been implicated in stabilization and maturation of vessels through action of an agonist ligand, angiopoietin 1 (Ang1) and an antagonistic ligand, Ang2. In this study, we have demonstrated that ang2 mRNA levels are increased in the retina during development of the deep retinal capillaries by angiogenesis and during pathologic angiogenesis in a model of ischemic retinopathy. Mice with hemizygous disruption of the ang2 gene by insertion of a promoterless beta-galactosidase (beta gal) gene behind the ang2 promoter, show constitutive beta gal staining primarily in cells along the outer border of the inner nuclear layer identified as horizontal cells by colocalization of calbindin. During development of the deep capillary bed or retinal neovascularization, other cells in the inner nuclear layer and ganglion cell layer, in regions of neovascularization, stain for beta gal. Thus, there is temporal and spatial correlation of Ang2 expression with developmental and pathologic angiogenesis in the retina, suggesting that it may play a role.

1. Recombinant human erythropoietin has N-linked sugar [Tsuda et al., (1988) Biochemistry 27, 5646-5654]. Here we have demonstrated the presence of O-linked sugar (0.85 mol/mol erythropoietin) composed of sialic acid and Gal beta(1-3)GalNAc. 2. To investigate the role of these sugars, erythropoietins deglycosylated to different extents were prepared using specific glycosidases. Sugars are not essential for in vitro biological activity of erythropoietin, because the fully deglycosylated erythropoietin had the full activity when assayed with in vitro bioassay methods. Asialylation yielded erythropoietin with higher affinity to the receptor than the undigested hormone and therefore an increased in vitro activity. Although erythropoietin from which N-linked or total sugars were removed also had higher affinity for the receptor, their in vitro activity remained unchanged compared with that of the undigested erythropoietin for unknown reasons. On the other hand, removal of sialic acids or N-linked sugar abolished the in vivo biological activity completely, indicating that the presence of N-linked sugar with terminal sialic acids is required for the hormone to reach target sites; full deglycosylation resulted in total loss of the in vivo biological activity of erythropoietin. 3. Incubation of asialo-erythropoietin and fully deglycosylated recombinant human erythropoietin at 70 degrees C for 15 min decreased the biological activity to 35% and 11% of the initial activity, respectively, while the undigested erythropoietin lost no activity. Thus resistance of erythropoietin to thermal inactivation is largely due to the presence of sugars, and terminal sialic acids greatly contribute to the stability.