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PubMed1967

Virulence of Escherichia coli Serotypes for Mice.

A study was undertaken to determine whether virulence in mice could be used to assess the pathogenicity of a variety of Escherichia coli serotypes. Sixty-one E. coli strains isolated from animals, poultry, or humans were serotyped to determine their O, K, and H antigens, and were administered to mice via the intraperitoneal route with and without a mucin adjuvant. The ld(50) dose was then determined for each serotype. The results indicated that the source of the serotype may be associated with virulence for mice. Serotypes isolated from nonenteric, systemic sources showed a greater virulence for mice inoculated intraperitoneally than did the enteric and the nonenteric, nonsystemic (localized) isolates. It was observed that not all serotypes belonging to a specific serogroup were virulent for mice and that the presence or absence of a K antigen had no effect on the virulence of strains of one serotype.

Journal Article↗
PubMed2001

Isolation and characterization of a Salmonella enterica serotype Typhi variant and its clinical and public health implications.

We report the isolation and characterization of a member of the family Enterobacteriaceae isolated from the gallbladder pus of a food handler. Conventional biochemical tests suggested Salmonella enterica serotype Typhi, but the isolate agglutinated with poly(O), 2O, 9O, and Vi Salmonella antisera but not with poly(H) or any individual H Salmonella antisera. 16S rRNA gene sequencing showed that there were two base differences between the isolate and Salmonella enterica serotype Montevideo, four base differences between the isolate and serotype Typhi, five base differences between the isolate and Salmonella enterica serotype Typhimurium, and six base differences between the isolate and Salmonella enterica serotype Dublin, indicating that the isolate was a strain of S. enterica. Electron microscopy confirmed that the isolate was aflagellated. The flagellin gene sequence of the isolate was 100% identical to that of the H1-d flagellin gene of serotype Typhi. Sequencing of the rfbE gene, which encoded the CDP-tyvelose epimerase of the isolate, showed that there was a point mutation at position +694 (G-->T), leading to an amino acid substitution (Gly-->Cys). This may have resulted in a protein of reduced catalytic activity and hence the presence of both 2O and 9O antigens. We therefore concluded that the isolate was a variant of serotype Typhi. Besides antibiotic therapy and cholecystectomy, removal of all stones in the biliary tree was performed for eradication of the carrier state.

Amino Acid Sequence↗
PubMed2004

Successful gene transfer using adeno-associated virus vectors into the kidney: comparison among adeno-associated virus serotype 1-5 vectors in vitro and in vivo.

BACKGROUND/AIM: Gene transfer into the kidney has great potential as a novel therapeutic approach. However, an efficient method of gene transfer into the kidney has not been established. We explored the transduction efficiency of renal cells in vitro and in vivo using adeno-associated virus (AAV) serotype 1-5 vectors encoding the beta-galactosidase gene. METHODS: In the in vitro study, rat kidney epithelial cell line NRK52E cells were transfected with AAV serotype derived vectors. In the in vivo study, AAV serotype derived vectors were selectively injected into the kidney using a catheter-based gene delivery system in rats and mice mimicking the clinical procedure. The efficiency of gene expression was histologically evaluated on the basis of the beta-galactosidase expression. RESULTS: AAV serotype 1, 2, and 5 vectors transduced in rat kidney epithelial cell line NRK52E cells in vitro, whereas AAV serotype 3 or 4 vectors showed no transduction. In addition, the kidney-specific injection of AAV serotype 2 vectors successfully transduced in tubular epithelial cells, but not in glomerular, blood vessel, or interstitial cells in vivo, whereas the rest of the serotypes showed no transduction. CONCLUSION: Since kidney-specific gene delivery via the renal artery by catheterization is highly feasible in humans, these findings provide useful information for promising strategies in renal gene therapy.

Animals↗
PubMed2006

Serotypes, virulence genes, and PFGE profiles of Escherichia coli isolated from pigs with postweaning diarrhoea in Slovakia.

BACKGROUND: Postweaning diarrhoea (PWD) in pigs is usually the main infectious problem of large-scale farms and is responsible for significant losses worldwide. The disease is caused mainly by enterotoxigenic E. coli (ETEC) and Shiga-toxin producing E. coli (STEC). In this study a total of 101 E. coli isolated from pigs with PWD in Slovakia were characterized using phenotypic and genotypic methods. RESULTS: These 101 isolates belonged to 40 O:H serotypes. However, 57% of the isolates belonged to only six serotypes (O9:H51, O147:H-, O149:H10, O163:H-, ONT:H-, and ONT:H4), including two new serotypes (O163:H- and ONT:H4) not previously found among porcine ETEC and STEC isolated in other countries. Genes for EAST1, STb, STa, LT and Stx2e toxins were identified in 64%, 46%, 26%, 20%, and 5% of isolates, respectively. PCR showed that 35% of isolates carried genes for F18 colonization factor, and further analyzed by restriction endonuclease revealed that all of them were F18ac. Genes for F4 (K88), F6 (P987), F17, F5 (K99), F41, and intimin (eae gene) adhesins were detected in 19 %, 5%, 3%, 0.9%, 0.9%, and 0.9% of the isolates, respectively. The study of genetic diversity, carried out by PFGE of 46 representative ETEC and STEC isolates, revealed 36 distinct restriction profiles clustered in eight groups. Isolates of the same serotype were placed together in the dendrogram, but high degree of polymorphism among certain serotypes was detected. CONCLUSION: Seropathotype O149:H10 LT/STb/EAST1/F4 (14 isolates) was the most commonly detected followed by O163:H- EAST1/F18 (six isolates), and ONT:H4 STa/STb/Stx2e/F18 (five isolates). Interestingly, this study shows that two new serotypes (O163:H- and ONT:H4) have emerged as pig pathogens in Slovakia. Furthermore, our results show that there is a high genetic variation mainly among ETEC of O149:H10 serotype.

Journal Article↗
PubMed1999

Bacterial adhesion of Actinobacillus actinomycetemcomitans serotypes to titanium implants: SEM evaluation. A preliminary report.

BACKGROUND: In this study, the adherence ability of Actinobacillus actinomycetemcomitans serotypes to titanium implant surfaces was evaluated to demonstrate if any selective adherence occurs according to the serotypes of the microorganism. METHODS: The study material included 3 reference strains of A. actinomycetemcomitans serotypes a, b, and c (ATCC 29523, ATCC 43718, ATCC 33384) and 2 clinical isolates of A. actinomycetemcomitans serotypes d and e (IDH 781, IDH 1705), together with commercially available titanium blade implants. For each strain, bacterial suspensions with identical concentrations (5 x 10(7) cells/ml) were prepared and 0.5 ml of each was added on to the implant surfaces, which had been precoated with glycine-bovine serum albumin (BSA). After incubation at 37 degrees C for 60 minutes in 5% CO2 in air, the implants with attached bacteria were prepared for scanning electron microscopic (SEM) observations. Bacterial adhesion was quantified on the textured body surfaces of the implants, and results were statistically analyzed with analysis of variance followed by Duncan's test. The surface ultrastructure of the bacterial cells was also evaluated descriptively. RESULTS: The tested strains adhered to implant surfaces in different quantities. Serotype a (ATCC 29523) showed the highest adherence affinity (statistically significant, P <0.01). When compared with each other, serotypes b, c, and d (ATCC 43718, ATCC 33384, and IDH 781) attached equally well, whereas serotype e (IDH 1705) had a statistically significant low adherence capability. CONCLUSIONS: It is suggested that in vitro A. actinomycetemcomitans adhesion to implant surfaces is strain dependent.

Aggregatibacter actinomycetemcomitans↗
PubMed1987

Gastroenteritis caused by human rotaviruses (serotype three) in a suckling mouse model.

The pathogenic potential of human rotaviruses of serotypes 1 through 4 was evaluated in suckling mice. Oral inoculation of three different human rotaviruses of serotype 3 into 5-6 day old CD-1 mice caused disease characterized by diarrhea and dehydration. The mean 50% diarrhea inducing dose (DD50) was 5 X 10(5) pfu. Histopathological examination of small intestines revealed villus epithelial cell vacuolization localized to the distal one-third of the villus. Only Serotype 3 rotaviruses exhibited a rapid phase of viral growth in the intestine between 7 and 12 hours post-inoculation. Larger inocula of rotavirus serotypes 1, 2, and 4 did not cause disease or typical histopathologic changes. However, immunoperoxidase staining for rotavirus antigen was positive in all serotypes tested indicating that infection can occur without apparent disease and is not serotype specific. This convenient in-vivo model can be used to evaluate attenuation of human origin vaccine candidates of serotype 3.

Animals↗
PubMed2000

Symptomatic human rotavirus subgroups, serotypes & electropherotypes in Hyderabad, India.

A total of 352 stool specimens obtained from children under 2 yr of age with acute diarrhoea, between January 1998 and March 1999, were screened for the presence of rotavirus by RNA-PAGE. Symptomatic human rotaviruses were detected in 57 of 352 (16.19%) specimens by RNA-PAGE. These 57 samples were tested for rotavirus double stranded RNA pattern and among these, 46 samples were tested for subgroup and serotype specificities. Among the 46 strains tested, 29 strains were found to be subgroup II and remaining 17 strains were subgroup I, indicating that subgroup II strains are more predominant than subgroup I strains. Subgroup I and II strains were circulating concurrently throughout the study period. Seventeen strains with 'short' RNA pattern and subgroup I specificity could not be assigned as serotype 2 strains as they exhibited cross-reactivity to MAbs specific for more than one serotype. Of the 29 subgroup II strains with 'long' RNA pattern, 16 (55.17%) were serotype 1, 8 (27.58%) were serotype 4. Five (17.24%) showed dual reactivity to serotypes 1 and 3. Our results indicated that serotype 1 and G2-like strains are predominant in Hyderabad. None of the virus strains showed an unusual RNA pattern.

DNA, Viral↗
PubMed2000

[Mode of serotype inheritance in exoconjugant progeny of the ciliate Dileptus anser].

Two clones of Dileptus anser, originally isolated from natural reservoirs and referred to below as B and D clones, were found to display different serotypes, when cultured under identical laboratory conditions. On being tested with two different polyclonal rabbit immune sera against each particular clone (the classic immobilization test) these clones showed no cross-reaction. At a standard dilution (1:50) and at a standard exposure time (4 h), either of the two immune sera immobilized 100% or commonly 0% of homologous and heterologous clone cells, respectively. In addition, the difference in serotypes was confirmed by the immunofluorescence analysis. By crossing (conjugation) between B (mating type I) and D (mating type III) cells, exconjugant F1 clones were obtained. Their serotypes were then tested (the same immobilization test) with antisera against both the "parental" clones: some clones were tested before their sexual maturation in ca. one month after conjugation, while others were examined in approximately 4 months after conjugation, i.e. after reaching maturity. Each of the F1 clones could react with both immune sera, which means that they possessed the intermediate, "hybrid" phenotype. Five different F1 clones were selected, and each of them was back-crossed to both "parental" clones, B and D. We succeeded in raising 25 exconjugant F2 (B1, to be more exact) clones from F1 x B crosses and 26 clones from F1 x D crosses. The conventional testing of these clones in 5-10 weeks after conjugation provided quite unexpected results, since among them no segregation for "parental" serotypes was observed. Each of the 51 tested clones demonstrated the "hybrid" serotype--seemingly the same as that of F1 clones. Such a non-Mendelian inheritance of the character is hardly to explain from the standard, canonical assumptions on the genetic control of serotype difference between original "parental" clones (different alleles in one locus? different loci?). Also it does not seem likely that the absence of segregation could result from differential survival of various phenotypes in F2 (although the total viability of exconjugant clones appeared rather low). The above data obviously need further confirmations and experimental analyses. We attempt to discuss the obtained results in terms of the epigene hypothesis (Tchuraev, 1975) and in relation to the epigenetic control of serotype expression in species of the Paramecium aurelia complex and in Tetrahymena thermophila, which are "the chosen few" subjects in ciliate genetics.

Animals↗
PubMed2006

Emergence of Vibrio cholerae O1 biotype El Tor serotype Inaba causing outbreaks of cholera in Orissa, India.

A total of 431 rectal swabs, collected from acute diarrheal cases at a surveillance site and at different diarrheal outbreak areas of Orissa from May to October 2005, were bacteriologically analyzed. Out of 265 culture-positive samples, Vibrio cholerae O1 was isolated in 56 samples (20.8%), of which 37 were the Inaba serotype and 19 were the Ogawa. The antibiogram profile revealed that all the V. cholerae O1 Ogawa and Inaba serotypes were uniformly sensitive to ampicillin, chloramphenicol, gentamicin, ciprofloxacin, norfloxacin and tetracycline. The V. cholerae O1 Inaba serotypes were resistant to furazolidone and nalidixic acid, while the Ogawa strains were resistant to furazolidone, nalidixic acid and neomycin. The multiplex polymerase chain reaction (PCR) assay on some selected strains of both serotypes revealed that all the strains were positive for ctxA and tcpA genes showing biotype El Tor. The present study revealed the emergence of V. cholerae O1 biotype El Tor serotype Inaba, which caused sporadic outbreaks of cholera in 2005. The outbreaks of diarrheal disorders in one geographical area of the state (in the Pattamundai area, Kendrapara district) in 2005 were due to V. cholerae O1 Ogawa, whereas the other outbreaks in other areas (Puri, Khurda and Dhenkanal districts) from August to October 2005 were due to V. cholerae O1 serotype Inaba. This is the first report that an emergence of V. cholerae O1 serotype Inaba caused sporadic outbreaks of cholera in different parts of Orissa. Switching over of V. cholerae O1 Ogawa strains to Inaba, causing diarrheal outbreaks in Orissa, needs close monitoring.

Cholera↗
PubMed1990

[The antigenic relationship between Brucella abortus, Brucella melitensis and Yersinia enterocolitica serotype 0:3 and 0:9].

Antigenic interrelationship between B. abortus, B. melitensis, Y. enterocolitica serotype 0:3 and serotype 0:9 were investigated in hyperimmün sera obtained from rabbits, by using agglutination and agglutinin absorbtion tests. Agglutination was determined at 1/2560 titer of B. abortus and B. melitensis hyperimmün sera when their own antigens were used. On the other hand, these sera agglutinated Y. enterocolitica serotype 0:9 antigen at 1/1280 and 1/320 titer respectively. As it was seen that high level antigenic relation was found between B. abortus and Y. enterocolitica serotype 0:9 while antigenic resemblance was found at lower level between B. melitensis and Y. enterocolitica serotype 0:9. There wasn't any significant interrelationship between B. abortus, B. melitensis and Y. enterocolitica serotype 0:3. In conclusion it is though that it is impossible to distinguish B. abortus and Y. enterocolitica serotype 0:9 infections from each other by using agglutination tests when 0 antigens are used.

Agglutination Tests↗
PubMed1979

[Serological reactivity of healthy and ill people in relation to antigens from Bacteroides-serotypes (author's transl)].

We investigated sera of healthy and ill people with different diagnoses to their reactivity against antigens isolated from six serotypes A--E2 (subspecies) of Bacteroides fragilis in indirect hemagglutination tests. These investigations were conducted partly in Poland and partly in the Netherlands. - In many cases antibodies towards several serotypes of Bacteroides fragilis were present in the healthy. In the Netherlands we found antibodies towards serotypes C and E2; in Poland serotypes D and E1 seem to be predominant in the healthy, whereas some reactivity towards other serotypes also could be observed (see table 1). - Hemagglutinationtiters in sera of the ill were frequently increased. In these sera we found also reactivity towards serotypes which are absent or rare in the healthy (see tables 2, 4 and 5). Sera of patients with carcinoma or sarcoma displayed high titers and especially reactivity towards those serotypes which are absent or rare in the healthy (see tables 3, 4 and 5).

Abortion, Septic↗
PubMed1994

Characterization of unique human TCR V beta specificities for a family of streptococcal superantigens represented by rheumatogenic serotypes of M protein.

The M protein of Streptococcus pyogenes plays a major role in the virulence of these bacteria. Members of the M protein superfamily are characterized by the presence of tandem segments of repeated amino acid sequences. The NH2-terminal end of the M proteins is a hypervariable region that harbors the type-specific epitopes of the molecule. Pepsin cleaves the molecule into a highly conserved carboxyl terminal half and a variable amino terminal portion referred to as pep M. In some individuals, infection with certain serotypes of group A streptococci is followed by autoimmune disorders such as rheumatic fever and acute glomerulonephritis. The serotypes of M protein that show a high degree of association with acute rheumatic fever are referred to as rheumatogenic serotypes. We have reported that one such serotype, type 5, is a superantigen to human T cells, specifically stimulating T cells bearing V beta 2, V beta 4, and V beta 8 elements. Here we extend our studies by examining other rheumatogenic serotypes for superantigenic properties. Studies with types 6, 18, 19, and 24 M proteins revealed that they are all superantigens to human T cells. The specificity to V beta 4 was shared by the rheumatogenic M proteins tested; however, each pep M serotype has its unique characteristic set of V beta specificity and these are distinct from those reported for the streptococcal pyrogenic exotoxins. The non-rheumatogenic serotype, pep M2, only stimulated V beta 2-bearing T cells. This study establishes that the structurally related M proteins represent a family of streptococcal superantigens analogous to the structurally related family of staphylococcal enterotoxin superantigens.

Amino Acid Sequence↗
PubMed1993

Pathogenicity and immunity studies of Haemophilus parasuis serotypes.

After intranasal inoculation of SPF pigs with the 7 reference strains of Haemophilus parasuis only the reference strains of serotypes 1 and 5 proved to be pathogenic and were able to produce the lesions characteristic of Glässer's disease. Even though the reference strains of serotypes 2, 3, 4, 6 and 7 proved to be apathogenic, intranasal inoculation resulted in the development of circulating antibodies as demonstrated by the complement fixation test. This serological response suggests an antigenic relationship between serotypes 1 and 3 different from the relationship seen among serotypes 2, 4, 5, 6 and 7. Pigs given an aerosol with the apathogenic serotypes 2, 3, 4 and 7, respectively, resisted challenge with a virulent Danish strain of serotype 5. These results indicate that the 7 serotypes also have antigenic determinants in common and that these determinants elicit cross-protecting immunity on the respiratory mucosa.

Animals↗
PubMed1997

Serodiagnosis of swine pleuropneumonia due to Actinobacillus pleuropneumoniae serotypes 7 and 4 using long-chain lipopolysaccharides.

A saline boiled extract (SBE), capsular polysaccharides (CPS) and long-chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 7 have been evaluated in ELISA for the serodiagnosis of swine pleuropneumonia caused by this serotype. Mean optical densities (ODs) obtained with the 3 antigens using sera from negative herds as well as from animals experimentally and naturally exposed to A. pleuropneumoniae serotypes 7 or 4 were not statistically different. The positive ELISA reaction with anti-serotype 4 sera was unexpected with the CPS, which are supposed to be serotype-specific; LPS traces present in the CPS appeared to be responsible for this reaction. In addition, sera from animals exposed to A. pleuropneumoniae serotypes 5 or 10 presented cross-reactions with the SBE and the CPS, but not with the LC-LPS. Cross-reactions were mainly due to rough LPS, as shown by immunoblotting. The LC-LPS is easily obtainable and can be used for the detection of antibodies in animals infected with A. pleuropneumoniae serotypes 7 and 4.

Actinobacillus Infections↗
PubMed1997

Serotyping and genotyping of hepatitis C virus (HCV) strains in chronic HCV infection. Commission Hepatologie du CREGG. Club de Reflexion des Cabinets de Groupes en GastroEnterologie.

Hepatitis C virus (HCV) genotypes can be established by methods based on PCR typing and serological typing. The accuracy of these methods depends on their sensitivity and specificity. These should be compared with the reference method, direct sequencing, and analysis of viral genomes. Among the serologic methods recently developed, the performance of a new serotyping assay (RIBA HCV 3.0 SIA, Chiron corporation, Emeryville) was assessed using a panel of 147 well-characterized French isolates from chronic hepatitis C patients. Definitive genotypes of the isolates were established by direct sequencing in 5' NC and in some cases in NS-5B. HCV serotypes 1, 2, and 3 were determined by measuring type specific antibodies to core and NS-4 derived peptide antigens. Of the 147 sera, serotypic-specific antibodies were detected in 136 (sensitivity, 92.5%). The specificity of the RIBA SIA HCV serotyping assay was 92.6% (including samples with mixed results); without these, the specificity was 80.1%. Analysis of the 28 discrepant samples showed that (1) a different serotype was found in 18 samples including five for genotype 1, three for genotype 2, two for genotype 3, five for genotype 4, and three for genotype 5, and that (2) ten patients showed a reactivity with mixed serotypes, one had circulating antibodies to type 1 or 2, and nine had circulating antibodies to type 1 or 3. In summary, except for genotypes 4 and 5, the results of the test were well correlated (85.7%) with those of direct sequence genotyping. The former test is rapid and does not require the strict HCV RNA storage and preservation conditions of the latter. This new method may thus be considered as an alternative for HCV typing. However, although it is convenient, its lower sensitivity compared to the molecular typing method and the discrepant results limit its routine use in a clinical context.

Antibodies, Viral↗
PubMed2005

1alpha,25-Dihydroxyvitamin D3 modulates the murine antibody response to pneumococcal capsular polysaccharide serotype 3 through IL-12.

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is a steroid hormone that regulates calcium metabolism. Besides, 1alpha,25(OH)(2)D(3 )also has pronounced immunomodulatory effects: it strongly inhibits dendritic cell (DC) maturation and impairs IL-12 production. We studied the effect of 1alpha,25(OH)(2)D(3 )on the antibody response to pneumococcal capsular polysaccharide (caps-PS) serotype 3. 1alpha,25(OH)(2)D(3) inhibited the IgG2a antibody response to caps-PS serotype 3. Besides, 1alpha,25(OH)(2)D(3) also inhibited IL-12 production and maturation of DC. Anti-IL-12 and exogenous IL-12, respectively, inhibited and stimulated the IgG2a antibody response to caps-PS serotype 3. Exogenous IL-12 abrogated the effect of 1alpha,25(OH)(2)D(3) on the IgG2a antibody response to caps-PS serotype 3, indicating that the effect of 1alpha,25(OH)(2)D(3) on the IgG2a antibody response to caps-PS serotype 3 was mediated through IL-12. In conclusion, we demonstrate that 1alpha,25(OH)(2)D(3) has an inhibitory effect on the IgG2a antibody response to caps-PS serotype 3, and that this effect was mediated trough IL-12.

Animals↗
PubMed2005

Adeno-associated virus serotypes 1 to 5 mediated tumor cell directed gene transfer and improvement of transduction efficiency.

BACKGROUND: Gene therapy is an attractive new approach for the treatment of cancer. Therefore, the development of efficient vector systems is of crucial importance in this field. Different adeno-associated virus (AAV) serotypes have been characterized so far, which show considerable differences in tissue tropism. Consequently, we aimed to characterize the most efficient serotype for this application. METHODS: To exclude all influences other than those provided by the capsid, all serotypes contained the same transgene cassette flanked by the AAV2 inverted terminal repeats. We systematically compared these vectors for efficiency in human cancer cell directed gene transfer. In order to identify limiting steps, the influence of second-strand synthesis and proteasomal degradation of AAV in a poorly transducible cell line were examined. RESULTS: AAV2 was the most efficient serotype in all solid tumor cells and primary melanoma cells with transduction rates up to 98 +/- 0.3%. Transduction above 70% could be reached with serotypes 1 (in cervical and prostate carcinoma) and 3 (in cervical, breast, prostate and colon carcinoma) using 1000 genomic particles per cell. In the colon carcinoma cell line HT-29 proteasomal degradation limited AAV1-AAV4-mediated gene transfer. Moreover, inefficient second-strand synthesis prevents AAV2-mediated transgene expression in this cell line. CONCLUSIONS: Recent advances in AAV-vector technology suggest that AAV-based vectors can be used for cancer gene therapy. Our comparative analysis revealed that, although AAV2 is the most promising candidate for such an application, serotypes 1 and 3 are valid alternatives. Furthermore, the use of self-complementary AAV vectors and proteasome inhibitors significantly improves cancer cell transduction.

Cell Line, Tumor↗
PubMed2006

Adeno-associated virus (AAV) serotypes 2, 4 and 5 display similar transduction profiles and penetrate solid tumor tissue in models of human glioma.

BACKGROUND: Adeno-associated viral (AAV) vectors are potent delivery vehicles for gene transfer strategies directed at the central nervous system (CNS), muscle and liver. However, comparatively few studies have described AAV-mediated gene transfer to tumor tissues. We have previously demonstrated that while AAV2 and Adenoviral (Ad) 5 vectors have similar broad host ranges in tumor-derived cell lines, AAV2 was able to penetrate human glioblastoma biopsy spheroids and xenografts more efficiently than Ad 5 vectors. These results suggested that AAV vectors could be suitable for therapeutic gene delivery to solid tumor tissue. In the present work, the transduction efficacy of AAV serotypes 4 and 5 were compared to AAV2, both in vitro and in intracranial GBM xenografts derived from patient biopsies implanted into nude rats. METHODS: AAV vector serotypes 2, 4, and 5 containing either the green fluorescent protein (GFP) or the bacterial beta-galactosidase (lacZ) reporter gene were added to five different human glioma cell lines, to multicellular spheroids generated from glioblastoma patient biopsies, and to spheroids xenografted intracranially in nude rats. Transduction efficiency was assessed by fluorescence imaging, histochemistry, immunohistochemistry and flow cytometry. RESULTS: While all three AAV serotypes were able to transduce the glioma cell lines when added individually or when they were administered in concert, AAV2 transduced the glioma cells most effectively compared to AAV4 or AAV5. Upon infecting glioblastoma spheroids in vitro, all three AAV serotypes efficiently transduced cells located at the surface as well as within deeper layers of the spheroids. In addition, similarly to what was observed for AAV2 16, both AAV4 and AAV5 were able to transduce human glioblastoma xenografts implanted intracranially. CONCLUSIONS: In addition to the widely used AAV2 serotype, AAV4 and AAV5 serotypes may also be used to transduce biologically diverse glioma cell lines. They also penetrate and transduce solid human tumor tissue derived from patient biopsies. Therefore, the data presented here provide a proof of principle for developing AAV4 and AAV5 as treatment vehicles for human malignant gliomas.

Animals↗