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PubMed1984

Disruption of regulatory gene GAL80 in Saccharomyces cerevisiae: effects on carbon-controlled regulation of the galactose/melibiose pathway genes.

In Saccharomyces cerevisiae, the transcriptional expression of the galactose-melibiose catabolic pathway genes is under the control of at least three regulatory genes, GAL4, GAL80, and GAL3. We have isolated the GAL80 gene and have studied the effect of a null mutation on the carbon-controlled regulation of the MEL1 and GAL cluster genes. The null mutation was achieved in vivo by replacing the chromosomal wild-type GAL80 allele with an in vitro-created GAL80 deletion-disruption mutation. Enzyme activities and RNA levels for the GAL cluster and MEL1 genes were constitutively expressed in the null mutant strain grown on glycerol-lactate and were higher than in the isogenic wild-type yeast strain when compared after growth on galactose. Carbon catabolite repression of the GAL cluster and MEL1 genes, which occurs at the level of transcription, is retained in the null mutant. Deletion of the GAL80 gene in a gal4 cell does not restore GAL cluster and MEL1 gene expression. The data demonstrate that (i) the GAL80 protein is a purely negative regulator, (ii) the GAL80 protein does not mediate carbon catabolite repression, and (iii) the GAL4 protein is not simply an antagonizer of GAL80-mediated repression.

Chromosome Deletion↗

PubMed1992

Regulation by thyroid hormone of nuclear and mitochondrial genes encoding subunits of cytochrome-c oxidase in rat liver and skeletal muscle.

Biogenesis of mitochondria involves the expression of genes located on nuclear chromosomes as well as on mitochondrial DNA. We studied the coordination of the two genomes by measuring transcript levels for nuclear (IV, Va, and VIc) and mitochondrial (II and III) subunits of cytochrome-c oxidase after altering the mitochondrial content of rat muscle and liver by altering the thyroid state of the animals. Tissue levels of these mRNAs were generally decreased in hypothyroid animals and were up-regulated again after thyroid hormone (T3) treatment. However, significant increases in the levels of all nuclear transcripts were observed in the liver 24 h after T3 treatment, but were delayed or remained unaltered (VIc) in muscle. In contrast, levels of mitochondrial transcripts were elevated early in muscle and late in liver. The abundance of the corresponding polypeptides, which were analyzed by immunoblotting, changed in direction and magnitude according to the changes in their mRNAs, indicating pretranslational control. We conclude that the two genomes are regulated by T3 not through a common coordinating mechanism, but via two separate pathways, which respond to T3 with tissue-specific kinetics. S1-nuclease protection analysis showed that probably only one transcript for subunit VIc is present in both tissues, thus excluding the possibility that the tissue-specific response is due to the expression of two isogenes. The abundance of mitochondrial DNA was unaltered despite the observed changes in mitochondrial transcripts, indicating that mitochondrial gene expression is regulated by transcriptional mechanisms and not by gene dosage as has been postulated by others.

Animals↗

PubMed2001

Protease-active extracellular protein preparations from Porphyromonas gingivalis W83 induce N-cadherin proteolysis, loss of cell adhesion, and apoptosis in human epithelial cells.

BACKGROUND: The protease-induced cytotoxicity of P. gingivalis may partly result from alteration of the extracellular matrix and/or surface receptors that mediate interaction between the host cells and their matrix. While P. gingivalis-induced degradation of E-cadherin has been documented, there is no information on the effects of P. gingivalis proteases on other members of this family of cell adhesion proteins. METHODS: Human epithelial KB cells were exposed to protease-active extracellular protein preparations from isogenic mutants of P. gingivalis. Quantification of apoptosis was performed by visualization of nuclei stained with 4,6'-diamidino-2-phenylindole. Alteration of cell adhesion proteins was examined by immunoblotting of cell lysates using monoclonal antibodies to those proteins. RESULTS: Treated cells exhibited loss of cell adhesion properties with apoptotic cell death subsequently observed. These effects correlated with the different levels of cysteine-dependent proteolytic activities of the isogenic mutants tested. Cleavage of N-cadherin was observed in immunoblots of lysates from detached cells. There was a direct correlation between the kinetics of N-cadherin cleavage and loss of cell adhesion properties. Loss of cell adhesion, as well as N-cadherin cleavage, could be inhibited by preincubation of P. gingivalis protease active extracellular protein preparations with the cysteine protease inhibitor TLCK. In control experiments, the cleavage of N-cadherin was detected after treatment of KB cells with trypsin but not after cell dissociation by a non-enzymatic method. CONCLUSIONS: These results suggest that extracellular proteases from P. gingivalis can induce degradation of N-cadherin, which could have implications for the pathogenicity of this bacterium.

Analysis of Variance↗

PubMed1993

Analysis of activated T cell infiltrates in rat renal allografts by gamma camera imaging after injection of 123iodine-interleukin 2.

Activated T cells bearing receptors for interleukin 2 (IL-2) play an important role in immunity and in immunopathological processes such as allograft rejection. In order to investigate the presence of activated T cells in lymphocytic infiltrates in transplanted kidneys, we investigated the uptake and retention of radioactivity after an intravenous injection of radioiodinated IL-2 in experimentally transplanted rats. IL-2 was enzyme radiolabelled with 123iodine using a glucose oxidase/lactoperoxidase method and shown to retain specific binding on an IL-2 receptor positive cell line, C58E6. To examine the kinetics of 123iodine-interleukin 2 (123I-IL-2) uptake in vivo, animals that had been transplanted five days previously with allogeneic or syngeneic grafts were injected with 123I-IL-2 and then imaged using an external gamma camera. Radioactivity was measured at time points up to 240 min after intravenous injection of 123I-IL-2. Four groups of animals were examined: allogeneic grafts (n = 7); syngeneic grafts (n = 6); ischaemic native kidneys (n = 5) all following injection with 123I-IL-2; and allogeneic transplants (n = 5) after injection of 123I-lactalbumin, an irrelevant molecule of similar molecular weight to IL-2. The peak radioactivity after injection was measured and the amount of radioactivity retained in the graft at increasing time intervals after injection was expressed as a function of initial peak radioactivity. At four hours after injection of 123I-IL-2, mean retention of activity by rejecting grafts was 77(+/- 2.68)% of peak activity, compared to 45(+/- 6.38)% in syngeneic controls (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1979

Effect of carrageenan on the non-specific resistance of mice to injected syngeneic tumour cells, alone or in mixtures.

The mechanisms of non-specific resistance to syngeneic methylcholanthrene-induced fibrosarcomas of mice were investigated. Results showed that a small tumour graft of 0.05 X 10(5) cells is greatly enhanced in growth when admixed with large numbers of cell fragments, killed cells or viable non-replicating cells. The enhancement of small tumour grafts in cell mixtures was found to be non-specific. Carrageenan, a known anti-macrophage agent, significantly increased tumour growth in normal mice. However, it did not enhance the increased tumour growth of 0.05 X 10(5) cells mixed with 10(6) viable, non-replicating mitomycin C-treated tumour cells. The latter observation indicates that carrageenan and admixed cells interfere with the same tumour-inhibitory mechanism and therefore cannot produce additive effects. The results give support to the concept of a non-specific macrophage "surveillance" system which appears crucial in controlling tumour growth, since it determines the establishment of small numbers of tumour cells while they can still be easily destroyed.

Animals↗

PubMed2006

Functional hypersplenism in mice induced by adoptive transfer of syngeneic spleen cells.

Investigation of peripheral blood cell count alterations in cases with hypersplenism, and an understanding of the relationship between splenic function and hematopoietic cell production require suitable experimental animal models. Previously described methods are either traumatic or require surgical intervention. We suggest a relatively simple method for achievement of a state mimicking hypersplenism in mice by intraperitoneal inoculation of syngeneic spleen cells. Mice were inoculated intraperitoneally with 3 x 10(7) splenocytes suspended in 0.3 ml phosphate buffered saline (PBS). After 2 months, the inoculated animals showed a progressive decrease in the peripheral white blood cell (WBC) counts and hyperplastic bone marrow that persisted until the experimental end point (7 months). Five days after inoculation of splenocytes stained with carboxy-fluorescein diacetate succinimidyl ester (CFSE), the majority of the stained cells was present in the peritoneal cavity (33%) and in the liver (13%), whereas the percentage of stained cells in the peripheral blood and the spleen cell suspension was negligible. The mitogen response of the peripheral blood mononuclear cells (PBMC) from treated mice to concanavalin A (Con A) remained unaltered. Splenocyte-inoculated mice that were further splenectomized did not show leukocytosis after splenectomy, as was observed in animals in which the spleen was removed without any pretreatment. The lack of any signs of discomfort in animals from the study group, in comparison with the visibly ill appearance and even death of mice in which hypersplenism was achieved by repeated injections of methylcellulose (MC), which served as controls, favors the convenience of the method.

Adoptive Transfer↗

PubMed1991

IL-2 gene transfer in immunotherapy of cancer: local administration of IL-2-activated lymphocytes and X63-m-IL-2 cells constitutively producing IL-2 inhibits growth of plasmacytomas in syngeneic mice.

Peritumoral administration of X63-m-IL-2 cells, transformed by IL-2 cDNA and constitutively producing large quantities of IL-2, was effective in mediating regression of X63-Ag8.653 plasmacytomas transplanted in syngeneic mice. Injection of killer cells with LAK activity substantially enhanced the tumor-inhibitory effect of IL-2-producing cells. In vitro activation of murine bone marrow and spleen cells with IL-2 produced by the X63-m-IL-2 cell line resulted in generation of lymphocytes with oncolytic activity against X63-Ag8.653 plasmacytomas and a variety of other target cell lines. The cytolytic activity of adherent IL-2-activated killer cells (A-LAK) was substantially higher than that of a nonadherent subset. Bone marrow and spleen cells from both healthy control and tumor-bearing mice contained LAK precursors, but the cytolytic activity derived from tumor-bearing mice was lower than that from healthy controls.

Animals↗

PubMed1997

Control of Escherichia coli type 1 fimbrial gene expression in stationary phase: a negative role for RpoS.

Expression of type 1 fimbriae in Escherichia coli is subject to phase-dependent control, with many regulatory inputs from proteins which organise or rearrange the structure of DNA. Inversion of a DNA segment carrying the promoter for expression of fimA, the gene encoding the fimbrial subunit protein, makes a key contribution to the switching of cells between fimbriate and afimbriate states. We have discovered that transcription of fimA is repressed as cells enter stationary phase. This repression is not seen in isogenic strains deficient in rpoS, the gene coding for the stationary phase-specific sigma factor RpoS. RpoS-deficient strains are also altered in the frequency of inversion of the fimA promoter segment. In the strains used in this study, inversion is catalysed by an integrase-like site-specific recombinase encoded by the fimB gene. We report that fimB transcription is repressed strongly as wild-type cells enter stationary phase but that this repression is alleviated in cells deficient in RpoS. These data suggest that RpoS has a negative regulatory role which may be indirect, at both the fimA and the fimB promoters.

Bacterial Proteins↗

PubMed2002

Detection of the proliferated donor cells in bone grafts in rats, using a PCR for a Y-chromosome-specific gene.

Conventional corticocancellous bone grafts are known to revascularize gradually and to become repopulated with osteocytes and other types of cells. This study was conducted to study the origin of the cells found within conventional bone grafts as a function of time, using a new method which accurately identifies donor cells transplanted with the graft. The technique requires the transplantation of a 25-mm tibial diaphyseal bone graft from a male rat to a female isogenous recipient, placed anatomically in the tibia. The presence and relative proportion of cells containing the Y-chromosome (i.e., originating from the bone graft) was determined 4, 8, and 12 weeks after transplantation. At these times, genomic DNA was extracted from the grafted bones and a polymerase chain reaction (PCR) specific for the sex-determining region of the Y-chromosome (Sry) was performed, with appropriate controls. Sry-specific bands were evident in none out of three grafts at 4 weeks, two out of three at 8 weeks, and all grafts at 12 weeks. These data demonstrate that not all cells within a conventional graft result from "creeping substitution" of necrotic bone. Instead, a small number of intrinsic cells from a nonvascularized corticocancellous structural graft survive and proliferate over time.

Animals↗

PubMed2001

Chemotaxis in the human gastric pathogen Helicobacter pylori: different roles for CheW and the three CheV paralogues, and evidence for CheV2 phosphorylation.

The complete genome sequence of Helicobacter pylori has revealed the presence of a novel set of chemotaxis genes including three cheV paralogues. CheV is a bi-functional protein, the N-terminal domain being homologous to the signalling-complex linker protein CheW, while the C-terminal domain is homologous to the response-regulator CheY, but its precise function in chemotaxis is unknown. In this study, each of the three cheV paralogues were insertionally inactivated in strain 26695 to determine their importance in the chemotactic signal-transduction pathway of H. pylori. Mutation of HP0019 (cheV1) had a severe inhibitory effect on chemotaxis, as determined by a swarm-plate assay. In contrast, strains carrying single mutations in either cheV2 (HP0616) or cheV3 (HP0393) displayed wild-type swarming behaviour, as did a cheV2/cheV3 double mutant. However, expression of the cheV2 or cheV3 genes in Escherichia coli resulted in an inhibition of chemotaxis in a wild-type strain, indicating their role in chemotaxis, although these genes were unable to complement isogenic E. coli cheW or cheY mutants. The product of cheV2/HP0616 was overexpressed in E. coli and purified to homogeneity. Protein fluorescence quenching experiments showed that CheV2 was capable of binding acetyl phosphate, a small-molecule phosphodonor. The measured K(m) for acetyl phosphate was 21 mM. It is concluded that in the absence of a cheZ gene, the CheV proteins could act as phosphate sinks to control the cellular level of phospho-CheY in H. pylori. However, only CheV1 was critical for chemotaxis, indicating a specific role distinct from the other paralogues in the signal-transduction pathway. Significantly, none of the CheV proteins could substitute for the loss of CheW, as an H. pylori cheW null mutant was non-chemotactic.

Amino Acid Sequence↗

PubMed2005

Genetic transformation: a powerful tool for dissection of adaptive traits in trees.

Plant transformation and regeneration systems have become indispensable parts of gene discovery and functional characterization over the last two decades. Adoption of transformation methods in studies of plant adaptation to natural environments has been slow. This is a result of poor genomic knowledge and inefficient transformation systems for species dominating terrestrial ecosystems, and logistical difficulties in conducting field tests of genetically engineered organisms. In trees, where long generation cycles, high background polymorphism, large sizes and outcrossing systems of mating make production of near-isogenic lines and large experiments difficult, transformation is an attractive alternative for establishing direct linkages between genes and adaptively significant phenotypes. Here, we outline the capabilities, challenges, and prospects for transformation to become a significant tool for studying the ecophysiological adaptation of trees to the environment. Focusing on poplars (genus Populus) as model system, we describe how transformation-based approaches can provide insights into the genes that control adaptive traits. The availability of the poplar genome sequence, along with its large expressed sequences tag (EST) databanks, facile transformation and rapid growth, enable reverse genetic approaches to be used to test virtually any hypothesis of gene function.

Adaptation, Physiological↗

PubMed1989

Axon regeneration through blood vessel allografts after cyclosporine treatment.

The present study describes the usefulness of blood vessel allografts to repair gaps in rat peripheral nerve after immunosuppression with cyclosporine. Isogeneic strains of rats with known histoincompatibility were used for this study. A 10-mm gap was created in the peroneal nerve of host Fischer rats. The gap was bridged by a 12-mm section of internal carotid artery removed from a Buffalo strain of rat. The host rats were divided into two groups. One group received no immunosuppression, whereas the other group was treated with cyclosporine. Untreated control rats immunologically rejected the allografted vessels and were unable to support host axonal regeneration through them. On the other hand, in cyclosporine-treated rats the allografted vessels survived. The regenerating host axons reorganized to form a functional nerve within the vessel conduit. The regenerated axons persisted even after rejection of the allografted vessel caused by cessation of immunotherapy. These results show that blood vessel allografts can serve as an effective conduit for reorganization of regenerated nerves and can bridge gaps in peripheral nerves.

Animals↗

PubMed2001

Comparative antimicrobial activities of antiseptic mouthrinses against isogenic planktonic and biofilm forms of Actinobacillus actinomycetemcomitans.

BACKGROUND: Bacteria contained in biofilms have been shown to have a decreased susceptibility to antimicrobial agents compared to those in planktonic form. Thus, in vitro biofilm models have been developed for screening oral antimicrobial formulations in an effort to produce findings more predictive of clinical activity. This study compared the antimicrobial activity of three mouthrinse formulations when tested against isogenic strains of Actinobacillus actinomycetemcomitans (Aa), one of which was a clinical isolate which forms tenacious biofilms in vitro and the other of which was a spontaneous variant which always grows planktonically. METHOD: Biofilm-forming Aa strains CU1000 and NJ4300, obtained as clinical isolates, and their respective spontaneous planktonic variants, CU1060 and NJ4350, were grown under standard laboratory conditions and exposed for 15 s to either a negative control (phosphate buffered saline [PBS]), an essential-oil containing mouthrinse (Listerine Antiseptic [LA]), an amine fluoride/stannous fluoride-containing mouthrinse (Meridol [M]), or a triclosan and PVM/MA copolymer-containing mouthrinse (Plax [P]). The cells were then washed, serially diluted, plated, and incubated for enumeration of viable bacteria. Colony-forming units (CFU)/ml were log10 transformed and the mouthrinse groups were compared to the PBS group using analysis of variance. RESULTS: All 3 mouthrinses produced statisically significant 99.99% reductions (p< or =0.0001) in both planktonic strains compared to the PBS control. Effects on the biofilm forms of the organisms were more variable. Exposure to LA produced statistically significant (p< or =0.0001) reductions in strains CU1000 and NJ4300 of 98.20% and 96.47%, respectively, compared to PBS. M and P produced much smaller reductions which were not statistically significant. CONCLUSIONS: The results of this study, in which antimicrobial mouthrinses were tested against biofilm-forming and planktonic strains of the same organism, provide a clear demonstration of the resistance to antimicrobial agents conferred by biofilm formation and provide additional support for employing tests using biofilms to more accurately assess the relative activities of antiplaque agents in vitro.

Aggregatibacter actinomycetemcomitans↗

PubMed1997

Failure to express the P-selectin gene or P-selectin blockade confers early pulmonary protection after lung ischemia or transplantation.

Endothelial P-selectin expression contributes to the first wave of neutrophil (polymorphonuclear leukocyte: PMN) influx in several inflammatory conditions. Although remote tissue ischemia, such as a crush injury to the hindlimb, may result in P-selectin-mediated pulmonary leukosequestration, it is not known whether the lungs exhibit a similar response after hypothermic preservation or when subjected to a direct ischemic insult. To determine if P-selectin may mediate early primary graft failure, left lungs harvested from male Lewis rats were preserved for 6 hr at 4 degrees C and transplanted orthotopically into isogeneic recipients. Recipients immunodepleted of PMNs before transplantation demonstrated improved graft function; pulmonary vascular resistance was reduced approximately 6-fold, arterial oxygenation was increased approximately 3-fold, and recipient survival was increased approximately 4-fold (P < 0.05, 0.05, and 0.005, respectively). Administration of a blocking anti-P-selectin IgG 10 min before reperfusion diminished graft PMN infiltration and resulted in improved graft function and recipient survival compared with controls. To establish the role of P-selectin in normothermic pulmonary ischemia, mice were subjected to temporary left pulmonary artery ligation. After functional removal of the nonischemic right lung, mice deletionally mutant for the P-selectin gene (P-selectin-/-) exhibited reduced PMN infiltration (approximately 2-fold), improved arterial oxygenation (approximately 2-fold), and improved survival (approximately 3-fold) compared with P-selectin +/+ control mice (P < 0.05, 0.01, and 0.05, respectively). These studies isolate and identify the central role of a single gene product (P-selectin) in early PMN recruitment and tissue injury after frank pulmonary ischemia and in the setting of lung transplantation after hypothermic preservation.

Animals↗

PubMed2004

Formation of 'non-culturable' cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation.

Conditions were investigated that promote the formation of 'non-culturable' (NC) cells of Mycobacterium (Myc.) smegmatis in stationary phase. After cultivation in a rich medium, or under conditions that may be considered optimal for bacterial growth, or starvation for carbon, nitrogen or phosphorus, bacteria failed to enter a NC state. However, when grown under suboptimal conditions, resulting in a reduced growth rate or maximal cell concentration (e.g. in modified Hartman's-de Bont medium), bacteria adopted a stable NC state after 3-4 days incubation in stationary phase. Such conditions are not specific as purF and devR mutants of Myc. smegmatis also showed (transient) loss of culturability following growth to stationary phase in an optimized medium, but under oxygen-limited conditions. The behaviour of the same mutants in oxygen-sufficient but nutrient-inappropriate medium (modified Hartman's-de Bont medium) was similar to that of the wild-type (adoption of a stable NC state). It is hypothesized that adoption of a NC state may represent an adaptive response of the bacteria, grown under conditions when their metabolism is significantly compromised due to the simultaneous action of several factors, such as usage of inappropriate nutrients or low oxygen availability or impairment of a particular metabolic pathway. NC cells of wild-type Myc. smegmatis resume growth when transferred to a suitable resuscitation medium. Significantly, resuscitation was observed when either recombinant Rpf protein or supernatant derived from a growing bacterial culture was incorporated into the resuscitation medium. Moreover, co-culture with Micrococcus (Mcc.) luteus cells (producing and secreting Rpf) also permitted resuscitation. Isogenic strains of Myc. smegmatis harbouring plasmids containing the Mcc. luteus rpf gene also adopt a similar NC state after growth to stationary phase in modified Hartman's-de Bont medium. However, in contrast to the behaviour noted above, these strains resuscitated spontaneously when transferred to the resuscitation medium, presumably because they are able to resume endogenous synthesis of Mcc. luteus Rpf. Resuscitation was not observed in the control strain harbouring a plasmid lacking Mcc. luteus rpf. In contrast to wild-type, the NC cells of purF and devR mutants obtained under oxygen-limited conditions resuscitate spontaneously, presumably because the heterogeneous population contains some residual viable cells that continue to make Rpf-like proteins.

Bacterial Proteins↗

PubMed1990

Analysis of protein expression in response to osmotic stress in Escherichia coli.

Two strains of Escherichia coli isogenic except for the cya (adenylate cyclase) allele were grown with [35S]methionine and cysteine in minimal defined glucose medium and in this medium with 600 mM NaCl to induce osmotic stress. Cells were grown for approximately two generations. The labeled proteins were separated by 2-dimensional electrophoresis and were quantified fluorographically. Of the 263 major proteins (proteins incorporating 0.10% or more of the total radioactivity) in the cya+ control culture, radioactivity in 41 proteins was at least ten times greater in cells grown with osmotic stress. Six of these individual proteins each accounted for 1.0% or more of the total radioactive label in the cells. Conversely, radioactivity in 31 major proteins appeared to decrease at least ten times when cells grew with osmotic stress. These data indicate that the response of the bacterium to osmotic stress involves induction of some proteins and repression of others. 61% of the proteins that appear to be stimulated by salt stress were found in both strains indicating there is no obligatory requirement for cAMP.

Adenylyl Cyclases↗

PubMed2000

Early graft failure of xenogeneic islets in NOD mice is accompanied by high levels of interleukin-1 and low levels of transforming growth factor-beta mRNA in the grafts.

Early graft failure, graft rejection, and autoimmune recurrence remain unresolved issues in islet xenotransplantation in type 1 diabetes. The first aim of this study was to examine the existence of early graft failure in spontaneously diabetic autoimmune NOD mice after rat islet transplantation under technically controlled circumstances. The second aim was to examine the mediators of this early xenograft dysfunction. First, we demonstrated a higher percentage of early xenograft failure (48%) in spontaneously diabetic NOD mice as compared with chemically diabetic old NOD (13%, P < 0.05) and C57Bl/6 (7%, P < 0.01) mice. In addition, in spontaneously diabetic NOD mice, xenogeneic islets displayed early graft failure more frequently than allogeneic (23%, P < or = 0.05) or isogeneic islets (7%, P < 0.01). No early graft failure was observed in allotransplantation or isotransplantation in chemically diabetic mice. Reverse transcriptase-polymerase chain reaction analysis of cytokine mRNA in islet xenografts 8 h after transplantation showed higher levels of interleukin (IL)-1 mRNA in autoimmune diabetic mice compared with chemically diabetic old NOD mice (1.40 +/- 0.32 vs. 0.90 +/- 0.14 IL-1 copies/beta-actin copies, P < 0.05). In contrast, mRNA levels of transforming growth factor (TGF)-beta were lower in spontaneously diabetic NOD mice than in chemically diabetic old NOD mice (0.67 +/- 0.16 vs. 1.36 +/- 0.50 TGF-beta copies/beta-actin copies, P < 0.05). No differences in tumor necrosis factor-alpha, IL-6, and inducible nitric oxide synthase were seen between autoimmune and nonautoimmune diabetic mice. T-cell cytokines (IL-2, IL-4, IL-10, and gamma-interferon) were absent in all mice until 48 h after transplantation. These data suggest that early islet xenograft failure is more common in spontaneously diabetic NOD mice and could be due to a nonspecific inflammatory reaction locally in the grafts.

Animals↗

PubMed2006

[Construction of HA-1-DC nucleic-acid vaccine and induction of specific cytotoxic T lymphocytes].

The purpose of this study was to construct a HA-1-DC nucleic acid vaccine and to induce anti-leukemia effect after hematopoietic stem cell transplantation (HSCT). The dendritic cells (DCs) were generated from HSCT donors in vitro, and its immunologic activity was studied by using flow cytometry and mix lymphocyte reaction. HA-1 gene was electroporated into the cultured DCs to construct a DC nucleic acid vaccine. After transfecting for 48 hours, the expression of HA-1 protein was detected by Western blot. The DCs were cultured with isogenic lymphocytes to induce specific cytotoxic T lymphocytes (CTLs). The cytotoxicity of the CTLs was detected by LDH assay. The results showed that the DCs derived from peripheral blood monocytes (PBMCs) expressed the DC phenotype, and were effective in stimulating proliferation of the allogenic lymphocytes. After electroporating for 48 hours, HA-1 protein was detected by Western blot. The cytotoxity of inducing CTLs was higher than that in the control group. It is concluded that the minor histocompatibility antigen HA-1 can be considered as a target of immunotherapy against leukemia after HSCT.

Cancer Vaccines↗