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T Bock

Publications and source records attributed to T Bock.

At least 19 recordsLinked to original sources
PubMed2001

Linomide does not prevent spontaneous autoimmune thyroiditis in NOD mice.

Linomide is a potent immunomodulator and has been reported to prevent type 1 diabetes mellitus in non-obese diabetic (NOD) mice and to reduce the incidence of other autoimmune diseases in animal models. The mechanisms of action seem to involve antigen expression by down regulation of macrophage activity and to antagonise the activation of Th1 cells during the cellular immune response. With the purpose to investigate the effect of Linomide on the incidence of spontaneous autoimmune thyroiditis (AIT) in female NOD mice we administered Linomide in drinking water (100 mg/kg/day) to NOD mice from 5th to 19th week of age. The mice were sacrificed at the end of week 19. None of the mice developed diabetes during the study period. The incidence of thyroiditis was evaluated on paraffin HE-stained sections and graduated on a scale from 0 to 4. Thirty-two percent of 37 mice treated with Linomide developed thyroiditis compared to 45% of 22 controls (p=0.31, chi2 =1.00). Among the mice who developed thyroiditis no difference in the degree of thyroiditis was found. Therefore no beneficial effect of Linomide on the incidence of spontaneous AIT in NOD mice could be demonstrated.

Adjuvants, Immunologic↗
PubMed2000

Sustained expression of exendin-4 does not perturb glucose homeostasis, beta-cell mass, or food intake in metallothionein-preproexendin transgenic mice.

Activation of glucagon-like peptide (GLP)-1 receptor signaling promotes glucose lowering via multiple mechanisms, including regulation of food intake, glucose-dependent insulin secretion, and stimulation of beta-cell mass. As GLP-1 exhibits a short t(12) in vivo, the biological consequences of prolonged GLP-1 receptor signaling remains unclear. To address this question, we have now generated metallothionein promoter-preproexendin (MT-Ex) transgenic mice. MT-Ex mice process preproexendin correctly, as is made evident by detection of circulating plasma exendin-4 immunoreactivity using high pressure liquid chromatography and an exendin-4-specific radioimmunoassay. Despite elevated levels of exendin-4, fasting plasma glucose and glucose clearance following oral and intraperitoneal glucose tolerance tests are normal in MT-Ex mice. Induction of transgene expression significantly reduced glycemic excursion during both oral and intraperitoneal glucose tolerance tests (p < 0.05) and increased levels of glucose-stimulated insulin following oral glucose administration (p < 0.05). Despite evidence that exendin-4 may induce beta-cell proliferation, beta-cell mass and islet histology were normal in MT-Ex mice. MT-Ex mice exhibited no differences in basal food intake or body weight; however, induction of exendin-4 expression was associated with reduced short term food ingestion (p < 0.05). In contrast, short term water intake was significantly reduced in the absence of zinc in fluid-restricted MT-Ex mice (p < 0.05). These findings illustrate that sustained elevation of circulating exendin-4 is not invariably associated with changes in glucose homeostasis, increased beta-cell mass, or reduction in food intake in mice in vivo.

Animals↗
PubMed2000

Effective ex vivo generation of granulopoietic postprogenitor cells from mobilized peripheral blood CD34(+) cells.

OBJECTIVE: Neutropenia following high-dose chemotherapy and peripheral blood progenitor cell (PBPC) transplantation might be abrogated by an additional transplantation of ex vivo generated granulopoietic postprogenitor cells (GPPC). Therefore, the ex vivo expansion of CD34(+) PBPC was systematically studied aiming for optimum GPPC production. MATERIALS AND METHODS: CD34(+) PBPC were cultured in serum-free medium comparing different (n = 32) combinations of stem cell factor (S), interleukin 1 (1), interleukin 3 (IL-3) (3), interleukin-6 (6), erythropoietin (E), granulocyte colony-stimulating factor (G), granulate-macrophage colony-stimulating factor (GM), daniplestim (D, a novel IL-3 receptor agonist), and Flt3 ligand (FL) under various culture conditions. Ex vivo generated cells were assessed by flow cytometry, morphology, and progenitor cell assays. RESULTS: Addition of G +/- GM but not GM alone to cultures stimulated with S163E effectively induced the generation of GPPC. GPPC production was maximum after 12 to 14 days. Best expansion rates were observed when cells were cultured at 1.5x10(4)/mL in 21% O(2). Modifications of culture conditions were either less or equally effective (i.e., modification of starting cell concentrations, low oxygen, addition of serum albumin or autologous plasma, repetitive feeding). Comparison of different cytokine combinations revealed that the optimum GPPC expansion cocktail consisted of S6GD+FL (day 12: 130-fold cellular expansion, 32% myeloblasts/promyelocytes, 49.4% myelocytes/metamyelocytes, 12.4% bands/segmented), which furthermore expanded CD34(+) cells (3.4-fold) and clonogenic progenitors (13.4-fold). CONCLUSION: Using the S6DG+FL expansion cocktail, GPPC could be effectively produced ex vivo starting from positively selected CD34 PBPC, possibly enabling amelioration or even abrogation of posttransplant neutropenia.

Adjuvants, Immunologic↗
PubMed1999

KDR receptor: a key marker defining hematopoietic stem cells.

Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34(+) cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+ cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR- subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+ fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors.

Animals↗
PubMed1999

Mutational pattern of hepatitis B virus on sequential therapy with famciclovir and lamivudine in patients with hepatitis B virus reinfection occurring under HBIg immunoglobulin after liver transplantation.

Famciclovir (FCV) and lamivudine (LAM) reduce viral replication in patients with recurrent hepatitis B virus (HBV) infection after orthotopic liver transplantation (OLT). Eighteen of 20 patients with insufficient response to FCV were treated with 100 mg LAM daily after OLT. These patients had shown nonresponse (n = 5), partial response (n = 7), or breakthrough (n = 6) during FCV therapy. Despite passive immunoprophylaxis with hepatitis B immunoglobulin after liver transplantation, HBV reinfection had occurred in 14 of 15 transplanted patients. HBV-DNA levels and the regions A to E of the HBV-DNA polymerase gene were analyzed before and after treatment failure to either therapy. Within 4 weeks on LAM, all but 1 patient showed a 95% average reduction of the HBV-DNA level. As with FCV, we did not observe any severe side-effects attributable to LAM. However, 7 patients developed a breakthrough within 12, 29 (n = 2), 32, 37, 54, and 145 weeks under treatment with LAM associated with the methionine-to-valine signature mutation (M552V) in the YMDD motif in all. With FCV, no unique, but a dominant, resistance pattern with the L528M mutation was identified for patients with breakthrough under FCV. In contrast, nonresponders or patients with partial response to FCV did not exhibit such mutations. Our results indicate that the L528M mutation is a risk factor for LAM breakthrough, because breakthrough during LAM occurred earlier in patients with this mutation (50 +/- 10 weeks vs. 120 +/- 21 weeks). Because breakthrough on either treatment is frequent for this specific group of patients, the use of combination therapy should be explored.

2-Aminopurine↗
PubMed1999

Inhibition of insulin-specific autoreactive T-cells by sulphatide which is variably expressed in beta cells.

AIMS/HYPOTHESIS: Sulphatide and insulin are present in the secretory granules and at the surface of beta cells in islets of Langerhans. Insulin autoantibodies and T-cell reactivity against insulin exist during the development of Type I (insulin-dependent) diabetes during which active beta cells may be more vulnerable than passive. Our aims were to examine the presence of sulphatide in active and passive beta cells and to clarify whether sulphatide influences the direct autoimmunity against insulin. METHODS: We incubated rat islets in 2.8, 11.0 or 20.0 mmol/l glucose for 24 h and did an electron microscopic evaluation after labelling with a specific anti-sulphatide monoclonal antibody. The reactivity of an insulin-specific T-cell clone isolated from a patient with Type I diabetes, was examined using insulin or insulin B-chain (B11-27) peptide incubated together with sulphatide. RESULTS: We detected lower amounts of sulphatide per insulin secretory granule in active compared with passive beta cells (p = 0.003). The presence of sulphatide in vitro at doses of 43-8.3 micromol/l resulted in greatly reduced proliferation (median 3.4 % of control value, p = 0. 0004) of the insulin-specific T-cell clone. No inhibition was found using the precursor of sulphatide, galactosylceramide, or GM1. Sulphatide did not reduce non-aspecific proliferation (induced by phorbol myristate acetate plus anti-CD3) or specific proliferation induced by insulin peptide. CONCLUSION/INTERPRETATION: These results imply that sulphatide possibly affect processing of the insulin molecule. Sulphatide which has been reported to interfere with phagosome-lysosome fusion, conceivably interacts with insulin. We hypothesize a (patho)physiological role of sulphatide, variably expressed in beta cells, by reducing the antigenicity of insulin. [Diabetologia (1999) 42: 1212-1218]

Animals↗
PubMed1999

Topical budesonide treatment reduces endothelial expression of intercellular adhesion molecules (vascular cell adhesion molecule-1 and P-selectin) and eosinophil infiltration in nasal polyps.

Infiltration of eosinophil granulocytes and endothelial expression of vascular cell adhesion molecule-1 (VCAM-1) and P-selectin was investigated in biopsies of polyps and inferior turbinates from 16 patients with nasal polyps, by the use of immunohistochemical staining and stereological quantification before, during and after topical treatment with budesonide (Rhinocort Turbuhaler). Before glucocorticoid treatment a higher density of eosinophil profiles (p < 0.005), making up a larger proportion of the total cellular infiltrate (p < 0.0005), was found in the polyps compared with the inferior turbinates. Endothelial VCAM-1 expression was higher in polyps than in inferior turbinates (p < 0.005), in contrast with the expression of P-selectin, which was more frequently expressed in the inferior turbinates (p < 0.05). Topical glucocorticoid treatment reduced the density of eosinophil profiles (p < 0.05) and the endothelial expression of VCAM-1 (p < 0.007) and P-selectin (p < 0.02) in polyps. Eosinophil counts and VCAM-1 expression returned to pre-treatment levels 8 weeks after discontinuation of budesonide treatment. The observed reduction in endothelial expression of cellular adhesion molecules may interfere with cellular recruitment in nasal polyps and thus contribute to the known anti-inflammatory effect of glucocorticoid in nasal polyposis.

Administration, Topical↗
PubMed1999

Unbiased estimation of total beta-cell number and mean beta-cell volume in rodent pancreas.

We describe a method for unbiased assumption-free estimation of the total number of beta-cells and the mean beta-cell volume in mouse and rat pancreas based on light microscopy. Such a method, which takes advantage of one of the most recent developments in stereology, the fractionator, has not previously been described. It relies on repeated fractionation of the tissue using systematic uniform random sampling combined with an unbiased counting principle. The method was applied to eight BALB/cBom male mice (56 days) and six Lewis/MOL male rats (47 days). In mice, the total number of beta-cells was 1.06 +/- 0.07 x 10(6) (mean +/- SEM) per pancreas with a mean beta-cell volume of 1280 +/- 17 microm3, while in rats the total beta-cell number was 2.76 +/- 0.42 x 10(6) per pancreas with a mean beta-cell volume of 1170 +/- 65 microm3. Furthermore, the results showed that in both species the biological variability in the total beta-cell volume is due to differences in the number of beta-cells rather than variability of the mean beta-cell volume. The method can be used to give a precise description of number and volume of beta-cells at different ages, and will make it possible to estimate the contributions of hyper/hypotrophia and hyper/hypoplasia to a given induced or spontaneous change in the total beta-cell mass.

Animals↗
PubMed1999

Cytokine production in NOD mice on prophylactic insulin therapy.

We investigated whether cytokines produced primarily by monocytes/macrophages (IL-1alpha), Th1-lymphocytes (IFNgamma), or Th2-lymphocytes (IL-4) are modulated in diabetes-prone NOD mice by insulin treatment as used in prophylaxis studies. The cytokines were measured by ELISA in plasma and in supernatants of spleen cells activated ex vivo by lipopolysaccharide plus phytohemagglutinin. Insulin, 0.25-0.50 IU/day, was given subcutaneously for 8 weeks starting in 4-week-old female mice. The insulin-treated and control NOD mice showed similar weight gains and, by the end of the study, both groups exhibited cell infiltration in about 25% of their islets. IL-1alpha, IFNgamma and IL-4 were generally below detection in plasma of prediabetic animals and controls. Diabetic NOD mice, aged 28-45 weeks, had significantly elevated plasma IL-1alpha: 154+/-39 pg/ml (mean+/-SEM, p<0.0001). While ex vivo activated NOD splenocytes released similar amounts of IL-1alpha, insulin therapy increased the levels from 99+/-17 to 155+/-19 pg/10(6) cells (p<0.05). Supernatants of activated splenocytes from prediabetic NOD mice had lower levels of IL-4 (<15 pg/10(6) cells) compared with those from BALB/c mice (88+/-22 pg/10(6) cells; p<0.01), and this deficiency was partially compensated for when the NOD mice were given insulin (27+/-8; p<0.01). The levels of IFNgamma were comparable and largely unaffected by insulin treatment. Hence, insulin therapy appears to partially normalize an otherwise deficient Th2 response in NOD mice.

Animals↗
PubMed1999

Previous maternal abortion, longer gestation, and younger maternal age decrease the risk of type 1 diabetes among male offspring.

OBJECTIVE: To identify possible influences and interactions of perinatal determinants in the subsequent development of type 1 diabetes. RESEARCH DESIGN AND METHODS: The data were obtained from children born in Denmark during the periods 1978-1982 and 1984-1986 and admitted to a Danish hospital with newly diagnosed type 1 diabetes between 1978 and 1995; 857 patients fulfilled the criteria. The study was conducted by combining and analyzing two national registries: the National Patient Registry and the Medical Birth Registry. For each diabetic child, two control children were randomly selected, matched by sex, time, and district of delivery. RESULTS: By multivariate logistic regression analysis, the following significant determinants were identified. Male offspring showed decreased risk when born of mothers who had had one or more abortions (odds ratio [OR] 0.66 [95% CI 0.48-0.92]) and with long duration of gestation (linearly with OR 0.91 per week [0.85-0.99]), while increased risk was found for high maternal age (linearly with OR 1.03 per year [1.00-1.06]). Female offspring showed no such association. No significant differences between diabetic patients and control subjects were found with respect to paternal age, maternal parity, placental weight or any of the birth size parameters, or interventions and complications during delivery. CONCLUSIONS: The findings show that perinatal determinants may influence the risk of subsequent development of type 1 diabetes in a sex-specific manner.

Abortion, Spontaneous↗
PubMed1999

Intermittent feeding and fasting reduces diabetes incidence in BB rats.

Food intake may be one of several factors which influence the risk of development of insulin dependent diabetes mellitus, but the influence of the pattern of food supply has not been studied previously. The aim of the present study was to investigate the effect of intermittent feeding and fasting upon diabetes in BB rats. This study included three groups. Group 1 served as control and included 77 animals, 79% became diabetic. In groups 2 and 3, after weaning, food but not water was withdrawn from the animals: 24 h twice a week in group 2; 24 h every second day in group 3. Group 2 included 40 BB rats, 50% (p < 0.004) became diabetic. Group 3 included 44 BB rats, 52% (p < 0.01) became diabetic. No differences were seen between sexes. Degree of insulitis was not influenced by changed food supply. Regarding blood glucose, no influence was seen among diabetic animals, among non-diabetic animals changed food supply reduced blood glucose values obtained at the end of the study. Intermittent feeding and fasting tended to reduce mean age at the time of diagnosis of diabetes, significance was reached only in female animals from group 3 compared to group 1. Body weight was obtained weekly. Intermittent feeding and fasting caused a reduced weight gain in group 2 as well as in group 3 compared to control animals; however, most pronounced in group 3 and also more pronounced among males compared to females. For pre-diabetic and non-diabetic animals comparable influence on body weight was seen. The main conclusion in the study is that intermittent feeding and fasting reduced diabetes incidence in BB rats.

Age Factors↗
PubMed1998

Short-term effects of early-acting and multilineage hematopoietic growth factors on the repair and proliferation of irradiated pure cord blood (CB) CD34+ hematopoietic progenitor cells.

PURPOSE: Hematopoietic growth factor(s) (GF) may exert positive effects in vitro or in vivo on the survival of hematopoietic stem and progenitor cells after accidental or therapeutic total body irradiation. METHODS AND MATERIALS: We studied the clonogenic survival and DNA repair of irradiated (0.36, 0.73, and 1.46 Gy) CD34+ cord blood (CB) cells after short-term incubation (24 h) with GFs. CD34+ cells were stimulated with basic fibroblast growth factor (bFGF), stem cell factor/c-kit ligand (SCF), interleukin-3 (IL-3), IL-6, leukemia inhibitory factor (LIF), and granulocyte-monocyte colony stimulating factor (GM-CSF) alone or in combination in short-term serum-free liquid suspension cultures (LSC) immediately after irradiation and then assayed for clonogenic progenitors. DNA repair was evaluated by analysis of DNA strand breaks using the comet assay. Survival of CFU-GM, BFU-E, and CFU-Mix was determined and dose-response curves were fitted to the data. RESULTS: The radiobiological parameters (D[0] and n) showed significant GF(s) effects. Combination of IL-3 with IL-6, SCF or GM-CSF resulted in best survival for CFU-GM BFU-E and CFU-Mix, respectively. Combinations of three or more GFs did not increase the survival of clonogenic CD34+ cells compared to optimal two-factor combinations. The D[0] values for CFU-GM, BFU-E, and CFU-Mix ranged between 0.56-1.15, 0.41-2.24, and 0.56-1.29 Gy, respectively. As for controls, the curves remained strictly exponential, i.e., all survival curves were strictly exponential without any shoulder (extrapolation numbers n=1 for all tested GF(s). DNA repair capacity of CD34+ cells determined by comet assay, was measured before, immediately after irradiation, as well as 30 and 120 min after irradiation at 1 Gy. Notably, after irradiation the 2-h repair of cytokine-stimulated and unstimulated CD34+ cells was similar. CONCLUSION: Our data indicate that increased survival of irradiated CB CD34+ cells after short-term GF treatment is mediated through proliferative GF effects on the surviving fraction but not through improved DNA repair capacity.

Cell Survival↗
PubMed1998

Expression of intercellular adhesion molecule-1 on the vascular endothelium in nasal polyps before, during and after topical glucocorticoid treatment.

Nasal polyposis is a result of a chronic inflammatory disorder in the upper airways. In vitro studies have revealed that extravasation of leucocytes requires interactions between several sets of adhesion molecules expressed on the circulating leucocytes and the vascular endothelium. Therefore, the endothelial expression of intercellular adhesion molecule-1 (ICAM-1) in biopsies from polyps and inferior turbinates was investigated by the use of immunohistochemical staining. Biopsies were obtained from 11 patients suffering from nasal polyposis before, during and after treatment with 100 microg budesonide (Rhinocort Turbuhaler) in each nostril twice daily. Before, during and after treatment, ICAM-1 was expressed in the majority of vessels in polyps and mucosa of inferior turbinates. The intensity of endothelial ICAM-1 expression in polyps was significantly reduced during topical glucocorticoid treatment compared with the pretreatment and posttreatment levels (p < 0.005). In biopsies from the inferior turbinates, the intensity of the endothelial ICAM-1 expression was lower during treatment than after discontinuation of medical treatment (p < 0.005). In conclusion, topical budesonide treatment seems to downregulate ICAM-1 expression on the vascular endothelium in nasal polyps. Such an effect may interfere with leucocyte extravasation and partially account for the anti-inflammatory effect of local glucocorticoid treatment in human nasal polyposis.

Administration, Topical↗
PubMed1997

Purging of peripheral blood progenitor cell autografts and treatment of minimal residual disease.

Clinical success of autologous peripheral blood progenitor cell (PBPC) transplantation is challenged by relapse of malignant disease which might at least in part be mediated by graft-contaminating tumor cells. Although the clinical efficacy of tumor cell depletion still remains to be demonstrated, multiple purging strategies are currently pursued in the context of autologous stem cell transplantation. This report discusses ex vivo manipulations of PBPC transplants with respect to purging of tumor cells, including positive selection of CD34+ cells with or without negative depletion of tumor cells as well as ex vivo expansion techniques. Moreover, strategies with an adoptive immunotherapy using ex vivo-generated autologous dendritic cells for the treatment of minimal residual disease after stem cell transplantation will be discussed here.

Antigens, CD34↗
PubMed1997

Physicochemical properties and in vitro toxicity of cationic liposome cDNA complexes.

The purpose of this study was to elucidate the interaction of cationic liposomes and plasmid cDNA by examining their ultrastructure, zeta potential, stability in aqueous media and protection from DNaseI digestion; their potential for hemolysis and platelet aggregation was evaluated as it may serve as an in vitro toxicity screen. Liposomes consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and dioleylphosphatidylethanolamine (DOPE) were complexed with plasmid constructs of ovine prostaglandin G/H synthase (pCMV4-PGH) or human alpha 1-antitrypsin (pCMV4-AAT) at lipid:plasmid (L/P) ratios of 3:1-8:1 (w/w). The electron micrographs showed bead-like attachment of liposomes to cDNA and coating of plasmid strands. The zeta potential showed isoelectric points at L/P ratios of 3.5-4 (DOTMA/DOPE) and 5.5-6.5, corresponding to a pKa of 6.45 (DC-Chol/DOPE). Liposome cDNA complexes were stable in water, saline and 5% dextrose for 48 h, but precipitated instantaneously in PBS. An increase in the L/P ratio corresponded with increased protection from DNaseI digestion. DOTMA/DOPE liposomes alone were highly hemolytic and DC-Chol/DOPE liposomes moderately hemolytic; hemolysis was abolished by cDNA complexation, with the exception of very high (> or = 7:1) L/P ratios. Both liposomes alone and cDNA complexes caused transient serum turbidity, while none caused platelet aggregation. It was concluded that current cationic lipid cDNA formulations are metastable and appear to have very little if any toxicity with respect to hemolytic potential and untoward interaction with other blood components.

DNA, Complementary↗
PubMed1997

Total number and size distribution of motor neurons in the spinal cord of normal and EMC-virus infected mice--a stereological study.

The encephalomyocarditis virus of the diabetogenic M-strain (EMC-M) is known to cause diabetes in mice. The EMC-M virus has also been shown to cause paresis in some of the infected animals. The clinical features include an acute ascending predominantly motor paralysis, developing within days. This resembles acute idiopathic polyneuritis. The alpha motor neurons would be a possible target for the virus, so two parameters, the total number and the size distribution of motor neurons, were therefore selected for further investigation in 6 mice with neurological involvement and compared with 6 control mice. The optical fractionator method was applied for estimating the total number of motor neurons and the 3D size distribution was estimated using the rotator method in a vertical design. No difference was found in the total number of motor neurons and the size distributions were similar in the 2 groups. This design can be used as a model for the estimation of the total number of motor neurons and their size distribution in other experimental animal models.

Animals↗
PubMed1997

Assembly of adeno-associated virus type 2 capsids in vitro.

Capsid proteins VP1, VP2 and VP3 of adeno-associated virus type 2 (AAV-2) were separately expressed by recombinant baculoviruses, purified under denaturing conditions and renatured in the presence of 0.5 M arginine, followed by dialysis against buffers of physiological ionic strength. At a protein concentration of 0.05 mg/ml, the three capsid proteins predominantly formed monomers and, to a lesser extent, oligomers, as determined by sedimentation analysis. Oligomerization increased at higher protein concentrations. The capsid protein oligomers consisted of globular, non-capsid-like structures, as detected by electron microscopy. Addition of a HeLa cell extract significantly stimulated oligomerization of the capsid proteins, probably due to interactions with HeLa cell proteins. Characterization of structures sedimenting around 60S by immunoprecipitation and electron microscopy showed that, in addition to other aggregates, empty capsid-like structures were formed in vitro. The identity of these structures as empty AAV capsids was verified by immunoelectron microscopy. Analysis of capsid formation in HeLa cells by transfection of VP expression constructs allowing separate expression of VP1, VP2 and VP3 showed that they were able to form capsids, although with a reduced efficiency as compared to VP proteins expressed from the wt cap gene. This finding suggests that the mutations introduced to allow separate capsid protein expression reduced the efficiency of capsid assembly in vivo and might also explain the reduced recovery of empty capsids employing the in vitro assembly procedure.

Animals↗