Molecular chaperones facilitate soluble expression of recombinant non-toxic mutant CRM197 of diphtheria toxin in Escherichia coli / 生物工程学报

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.

Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model

Foxp3 is a master regulator of CD4⁺CD25⁺ regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4⁺ or CD8⁺ cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3DTR mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma.

Construção, clonagem e expressão do fragmento B da toxina diftérica de Corynebacterium diphtheriae (cepa PW- 8) em Mycobacterium bovis BCG sub-cepa Moreau / Construction, cloning and expression of the fragment B of diphtheria toxin from Corynebacterium diphtheriae (strain PW-8) in Mycobacterium bovis BCG Moreau sub-strain

A vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado...

The diphtheria vaccine currently used in Brazil (DTP), despite its history of high efficacy in the prevention of diphtheria, is associated with episodes of toxicity and vaccine reactogenicity in the vaccinee, resulting from the presence in the vaccine of residual proteins derived from the production process or detoxification. Strategies for the development of new vaccines more effective and economically viable against diphtheria have been the subject of intense investigation. The alternative proposed by our group is the use of the vaccine against tuberculosis (Mycobacterium bovis BCG Moreau sub strain) as a vector for the gene that encodes the 58.3 kDa fragment B of the diphtheria toxin (DTB). In our project the dtb gene was cloned into the bifunctional vector pUS977 for cytoplasmic expression and recombinant BCG (rBCG) clones, selected after transformation of BCG, were tested for expression of the DTB polypeptide and antigenicity against polyclonal antibodies anti- diphtheria toxoid by immunoblotting. The integrity and identity of the DNA sequence encoding the dtb gene carried by the plasmid construct pUS977dtbPW8 was confirmed by DNA Sequencing and Analysis of Similarity. The immunogenicity of the rBCG expressing the DTB was investigated in BALB/c mice and the results revealed a specific seroconversion (IgG)...

Evaluation of Immune Response for Vi-CRM(197) Conjugated Vaccine against Salmonella enterica serovar Typhi in Mice

Typhoid fever, a serious systemic infection caused by Salmonella enterica serovar Typhi, breaks out in developing countries. However, existing vaccines only induce relatively low protective effects with humoral responses and do not stimulate secondary immune response, especially to young people. The objective of this study is to evaluate the immunogenicity of the vaccine containing virulence capsular polysaccharide (Vi) conjugated with the optimal ratios of non-toxic variant of diphtheria toxin (CRM(197)) in mice. Six-week-old BALB/c female mice were injected intraperitoneally three times at intervals of 14 days and sera were collected on days 0, 14, 28, 42 and 56 post-injection. The efficacy of the vaccine was evaluated by comparing between negative control group injected with PBS and vaccine groups injected with Vi or Vi-CRM(197) conjugate of different ratio. Vi and CRM(197)-specific antibody responses were evaluated using enzyme-linked immunosorbent assay. The result showed that Vi-CRM(197)-1 group revealed the highest and significant Vi-specific IgG immune responses among the other groups and Vi group (p < 0.01). In conclusion, Vi-CRM(197)-1 conjugate vaccine induced the highest humoral immune response in mice and may be used as an effective vaccine to replace the existing typhoid vaccine for infants under 2 years old.

Mouse Models for the Evaluation of Osteocyte Functions

Osteocytes establish an extensive intracellular and extracellular communication system via gap junction-coupled cell processes and canaliculi, through which cell processes pass throughout bone, and the communication system is extended to osteoblasts on the bone surface. To examine the osteocyte function, several mouse models were established. To ablate osteocytes, osteocytes death was induced by diphtheria toxin. However, any types of osteocyte death result in necrosis, because dying osteocytes are not phagocytosed by scavengers. After the rupture of cytoplasmic membrane, immunostimulatory molecules are released from lacunae to bone surface through canaliculi, and stimulate macrophages. The stimulated macrophages produce interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha), which are the most important proinflammatory cytokines triggering inflammatory bone loss. Therefore, the osteocyte ablation results in necrosis-induced severe osteoporosis. In conditional knockout mice of gap junction protein alpha-1 (GJA1), which encodes connexin 43 in Gap junction, using dentin matrix protein 1 (DMP1) Cre transgenic mice, osteocyte apoptosis and enhanced bone resorption occur, because extracellular communication is intact. Overexpression of Bcl-2 in osteoblasts using 2.3 kb collagen type I alpha1 (COL1A1) promoter causes osteocyte apoptosis due to the severe reduction in the number of osteocyte processes, resulting in the disruption of both intracellular and extracellular communication systems. This mouse model unraveled osteocyte functions. Osteocytes negatively regulate bone mass by stimulating osteoclastogenesis and inhibiting osteoblast function in physiological condition. Osteocytes are responsible for bone loss in unloaded condition, and osteocytes augment their functions by further stimulating osteoclastogenesis and further inhibiting osteoblast function, at least partly, through the upregulation of receptor activator of nuclear factor-kappa B ligand (RANKL) in osteoblasts and Sost in osteocytes in unloaded condition.

Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.

Diphtheria Antibodies and T lymphocyte Counts in Patients Infected with HIV-1

We assessed the IgG levels anti-diphtheria (D-Ab) and T cell counts (CD4+ and CD8+) in HIV-1 infected subjects undergoing or not highly active antiretroviral therapy (HAART). Approximately 70% of all HIV-1 patients were unprotected against diphtheria. There were no differences in D-Ab according to CD4 counts. Untreated patients had higher D-Ab (geometric mean of 0.62 IU/ml) than HAART-patients (geometric mean of 0.39 IU/ml). The data indicated the necessity of keeping all HIV-1 patients up-to-date with their vaccination.

Protective antitumor immunity induced by tumor cell lysates conjugated with diphtheria toxin and adjuvant epitope in mouse breast tumor models / 癌症

Cancer cell vaccine-based immunotherapy has received increasing interest in many clinical trials involving patients with breast cancer. Combining with appropriate adjuvants can enhance the weak immunogenic properties of tumor cell lysates (TCL). In this study, diphtheria toxin (DT) and two tandem repeats of mycobacterial heat shock protein 70 (mHSP70) fragment 407-426 (M2) were conjugated to TCL with glutaraldehyde, and the constructed cancer cell vaccine was named DT-TCL-M2. Subcutaneous injection of DT-TCL-M2 in mice effectively elicited tumor-specific polyclonal immune responses, including humoral and cellular immune responses. High levels of antibodies against TCL were detected in the serum of immunized mice with ELISA and verified with Western blot analyses. The splenocytes from immunized mice showed potent cytotoxicity on Ehrlich ascites carcinoma cells. Moreover, the protective antitumor immunity induced by DT-TCL-M2 inhibited tumor growth in a mouse breast tumor model. DT-TCL-M2 also attenuated tumor-induced angiogenesis and slowed tumor growth in a mouse intradermal tumor model. These findings demonstrate that TCL conjugated with appropriate adjuvants induced effective antitumor immunity in vivo. Improvements in potency could further make cancer cell vaccines a useful and safe method for preventing cancer recurrence after resection.

Dimensionamiento de los sistemas de microfiltración y ultrafiltración para la producción de la Toxina Diftérica elaborada en el Instituto Nacional de Higiene “Rafael Rangel” / Microfiltration and ultrafiltration sistems dimension for using in Difteria Toxin production in the Instituto Nacional de Higiene "Rafael Rangel"

Se evaluó el uso de la tecnología de Flujo de Filtración Tangencial (FFT), para obtener la toxina diftérica a partir de cultivos de la bacteria Corynebacterium diphtheriae, usando el proceso de Microfiltración (MF), para eliminar el paquete celular y posteriormente, a partir del filtrado obtenido, concentrar y diafiltrar la toxina diftérica usando el proceso de Ultrafiltración (UF). Se determinaron características de los filtros, condiciones de trabajo y dimensionamiento de los equipos a adquirir para la producción industrial de Toxina Diftérica. Se evaluaron el flujo, tiempo, rendimiento del proceso y las características del producto obtenido, utilizando cultivos con Toxina Diftérica en un equipo de filtración de laboratorio, diseñado para producir el efecto de FFT. Seseleccionó las membranas tipo cassettes, formato Médium Screen, porosidad 0,2 μm, como las adecuadas para el proceso de MF, ya que mostraron 100% de transmisión de la Toxina Diftérica, ausencia de restos celulares y flujo promedio de filtrado de 9.16 L/m2h. Así mismo, se seleccionaron las membranas tipo cassettes, formato Omega, porosidad 10 y 30 kDa, como las adecuadas para el proceso de UF, ya que mostraron 100% de recuperación de la toxina, ausencia de toxina en el filtrado y adecuados flujos de filtrado (97,5 y 125,9 L/m2h, respectivamente), Estos resultaron permitieron dimensionar, considerando las variables a utilizar en la producción industrial (Volumen 650 a 950 Litros, Tiempo de Procesos, 3 a 5 horas), el área de filtración de los equipos de MF y UF a adquirir, estimados en 20m2 y 5m2, respectivamente.

Tangential Flow Filtration (TFF) technology was evaluated to process diphtheria toxin which is produced by Cory ne - bacterium diphtheriae bacterium. Microfiltration (MF) is used to retain cells while allowing passage of the toxin to the filtrate stream. The filtrate is collected and further pro - cessed by Ultrafiltration (UF) to concentrate the toxin and to maximize the wash of small species by a Diafiltration step. Both, MF and UF processes were evaluated to specify the filters and corresponding critical process parameters to scale-up the application. As part of the evaluation, flow rate, processing time, yield and product attributes were characterized. The cell harvest containing the diphtheria toxin was processed using a laboratory scale TFF system designed to product the TFF effect. The evaluation demonstrated that a cassette in medium screen format and membrane with 0.2 μm pore is the right selection for the MF step. It showed 100% of toxin transmission without the presence of cellular debris and average process flux of 9.16 L/m2h. The UF step was conducted using the same laboratory equipment with cassettes in medium screen format with pores of 10 and 30 kD. It showed 100% retention of the toxin with a process flux of 97,5 and 125,9 L/m2h, respectively. These results were used to scale-up the application to process the industrial volume of 650 a 950 liters between 3 to 5 hours of processing time. Membrane area sizing of MF and UF to be acquired is estimated in 20 m2 and 5m2, respectively.

Effect of recombinant adenovirus Ad-DT-A in targeted therapy for malignant cancer cell lines with loss of IGF2 imprinting / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the feasibility of IGF2 imprinting system in target gene therapy for tumors.</p><p><b>METHODS</b>The mouse H19 enhancer, DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312. The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid, and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5, namely, Ad-EGFP and Ad-DT-A. Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infections. The effect of the IGF2 imprinting system was tested by fluorescence microscopy. RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MOI), respectively. The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8 (LOI). Human breast cancer cell line MCF-7 (MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro. The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors.</p><p><b>RESULTS</b>The expression of EGFP protein, DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line. Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ± 6.4)% compared with that in the control group, and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9)%. The anti-tumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice, and the results showed that the Ad-DT-A inhibited the tumor growth, with on inhibition rate of 36.4%.</p><p><b>CONCLUSIONS</b>The recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed, while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting. It might play an important role in future target gene therapy against malignant tumors based on loss of IGF2 imprinting.</p>

Fibrinogen binds to nontoxigenic and toxigenic Corynebacterium diphtheriae strains

The production of fibrinous exudates may play an important role in determining the outcome of bacterial infection. Although pseudomembrane formation is a characteristic feature of diphtheria, little is known about the fibrinogen (Fbn)-binding properties of Corynebacterium diphtheriae strains and the influence of the gene that codes for diphtheria toxin (tox gene) in this process. In this study we demonstrated the ability of C. diphtheriae strains to bind to Fbn and to convert Fbn to fibrin. Bacterial interaction with rabbit plasma was evaluated by both slide and tube tests. Interaction of microorganisms with human Fbn was evaluated by both enzyme linked immunosorbent assay (ELISA) and fluorescein isothiocyanate-conjugated (FITC) Fbn binding assays. Nontoxigenic and toxigenic strains formed bacterial aggregates in the presence of plasma in the slide tests. The ability to convert Fbn to a loose web of fibrin in the plasma solution in the tube tests appeared to be a common characteristic of the species, including strains that do not carry the tox gene. Fbn binding to C. diphtheriae strains occurred at varying intensities, as demonstrated by the FITC-Fbn and ELISA binding assays. Our data suggest that the capacity to bind to Fbn and to convert Fbn to fibrin may play a role in pseudomembrane formation and act as virulence determinants of both nontoxigenic and toxigenic strains.

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

Diphtheria toxin IgG levels in military and civilian blood donors in Rio de Janeiro, Brazil

Serologic data on diseases that are preventable by vaccines are necessary to evaluate the success of immunization programs and to identify susceptible subgroups. In the present study, we determined serum IgG levels against diphtheria toxin of military and civilian blood donors (N = 75; 69.3 percent males and 30.7 percent females) aged 18-64 years, from the Brazilian Army Biology Institute, Rio de Janeiro, using a commercial diphtheria kit (Diphtheria IgG ELISA; IBL, Germany). Most (63 percent) unprotected military donors were from the older age group of 41 to 64 years. In contrast, the majority (71 percent) of young military donors (18 to 30 years) were fully protected. About half of the military donors aged 31 to 40 years were protected against diphtheria. Among the civilians, about 50 percent of persons aged 18 to 30 years and 31 to 40 years had protective antibody levels against diphtheria as also did 64 percent of individuals aged 41 to 64 years. All civilians had a similar antibody response (geometric mean = 0.55 IU/mL) independent of age group. Military donors aged 18-30 years had higher IgG levels (geometric mean = 0.82 IU/mL) than military donors of 41-64 years (geometric mean = 0.51 IU/mL; P > 0.05). In conclusion, the existence of a considerable proportion of susceptible adults supports the position that reliable data on the immune status of the population should be maintained routinely and emphasizes the importance of adequate immunization during adulthood.

Murine monoclonal antibodies neutralizing the cytotoxic activity of diphtheria toxin.

In this study, murine monoclonal antibodies that specifically bound to the A and B subunits of diphtheria toxin (DT) were produced by conventional hybridoma technology using the spleens of BALB/c mice immunized with diphtheria DTP vaccine and CRM197. Monoclonal antibodies specific to the A subunit, i.e. clone AC5, as well as those specific to the B subunit, i.e. clone BB7, could neutralize the DT-mediated cytotoxicity to Vero cells in microcultures. The DT neutralizing mechanisms have yet to be determined. The MAbBB7 is hypothesized to either interfere with the DT receptor binding or with the pore forming function of the T domain of the B subunit. The MAbAC5 could neutralize the DT mediated cytotoxicity when mixed with the DT before adding to the Vero cell culture thus suggesting that the antibody interfered with the translocation of the A subunit. The A subunit-antibody complex might be too large to pass through the membrane channel formed by the T domain and thus prevent the accessibility of the A subunit to the cytosolic target. It is also possible that the MAb AC5 blocked the enzymatic active site of the enzyme catalytic subunit. While further experiments are needed to localize the epitopes of the two MAbs on the holo-DT in order to reveal the DT neutralizing mechanisms, both MAbs in their humanized forms have a high potential as human therapeutic antibodies for diphtheria.

Desenvolvimento e implementação de métodos laboratoriais aplicados ao diagnóstico fenotípico e genotípico do Corynebacterium diptheriae / Development and implementation of laboratory methods applied to phenotypic and genotypic diagnosis of Corynebacterium diphtheriae

A emergência de cepas de Corynebacterium diphtheriae atoxinogênicas como agentes de endocardite e outras infecções sistêmicas aliada ao aumento do número de adultos susceptíveis à difteria enfatizam a necessidade de métodos alternativos para o diagnóstico laboratorial desta doença, especialmente para laboratórios de rotina clínica. Neste estudo avaliou-se a atividade de DNase de 91 amostras de C. diphtheriae (37 toxinogênicas e 54 atoxinogênicas) e de 564 cepas clínicas de bacilo Gram positivo não diftérico. A atividade de DNase foi detectada em todas as amostras de C. diphtheriae examinadas, previamente identificadas por métodos bioquímicos e pelo sistema API Coryne System. Diferentemente, os resultados do teste de DNase foram negativos em 93.9 porcento das cepas clínicas de bacilo Gram positivo não diftérico. Também foi documentado o valor de uma PCR espécie-específica que tem como alvo o gene dtxR como um método para diferenciação entre C. diphtheriae e colônias similares ao gênero Corynebacterium. Os resultados da PCR-dtxR foram positivos para todas as amostras de C. diphtheriae estudadas e foram concordantes com os obtidos através de metodologia bioquímica padrão. Diferentemente, os resultados da PCR-dtxR foram negativos para 100 porcento das 111 amostras de bacilos Gram positivos não diftéricos estudadas. A partir destes resultados, uma PCR multiplex utilizando três pares de oligonucleotídeos iniciadores foi desenvolvida para a detecção do C. diphtheriae e diferenciação em amostras toxinogênicas ou atoxinogênicas. Dois pares de oligonucleotídeos iniciadores têm como alvo as regiões do gene tox relativas aos domínios A e B da toxina diftérica e um terceiro par direcionado para o gene dtxR. Todas as amostras de C. diphtheriae foram identificadas pela reação de PCR multiplex em concordância com os testes bioquímicos padrão e os ensaios de citotoxicidade celular...

The emergence of non-toxigenic Corynebaterium diphtheriae strains as the causative agent of endocarditis and other systemic infections and the significant rise in the percentage of adults susceptible to diphtheria emphasize the need for new laboratory diagnostic procedures. In this study, we examine techniques as alternative procedures for differentiating C. diphtheriae from Corynebacterium-like colonies for the presumptive identification of this pathogen, especially in the diagnosis laboratory. This study evaluated the DNase activity of 91 C. diphtheriae (37 toxigenic and 54 non-toxigenic) and 564 non-diphtherial Gram-positive rod clinical strains. The DNase activity was detected in all C. diphtheriae strains examined, previously identified by both conventional biochemical methods and API Coryne System. Conversely, DNase test results were negative in 93.9 percent of the 564 non-diphtherial Gram-positive rod clinical strains. We also documented the value of a species-specific PCR assay that targets the dtxR gene as a procedure for differentiating C. diphtheriae from Corynebacterium-like colonies. The results of the PCR-dtxR were all positive for 91 C. diphtheriae strains and completely correlated with the standard biochemical methods and commercial identification system for all strains tested. In other hand, the PCR-dtxR results were negative in 100 percent of the 111 non-diphtherial Gram-positive rod strains. Considering these results, a multiplex PCR using three primers pairs was developed for detection of C. diphtheriae infection and differentiation between toxigenic and non-toxigenic strains. Two primer pairs targeted to domains A and B of tox gene and a third primer pair targeted to a region of dtxR gene. All C. diphtheriae strains were diagnosed by the multiplex PCR in agreement with standard biochemical tests and citotoxicity assay in Vero cells. Thus, these tecniques emerged as viable, cost-effective screening methods for C. diphtheriae laboratory...

Therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis (EAE).</p><p><b>METHODS</b>EAE was induced in the low-sensitive strain C57BL/6 mice with intraperitoneal injection of myelin basic protein (MBP) to simulate the human disease multiple sclerosis, followed by intramuscular injection of cationic liposome carrying the plasmid DNA SRalpha-mMIP-1alpha-DT390 in the leg muscle to elicit resistance to EAE development. The mice were then examined daily for clinical signs of EAE by an observer blind to the treatment protocol. For immunohistochemistry the mice were anesthetized and perfused with sterile PBS and paraformaldehyde, and the cerebrum, cerebellum, medulla and spinal cord were removed for preparation of serial sections. The mononuclear cells (MNCs) from the EAE mouse spleens were prepared for three-color flow cytometry analysis of the surface markers with appropriate antibodies following the BD Pharmingen cytokine staining protocol.</p><p><b>RESULTS</b>EAE model was successfully established by active MBP immunization in C57BL/6 mice. Administration of the immunotoxin mMIP-1alpha-DT390 significantly delayed the disease onset and lowered the mean clinical score for EAE as compared with the control mice. Immunohistochemistry demonstrated much less CCR5(+) infiltrating cells in the central nervous system in mMIP-1alpha-DT390-treated mice than in the control. The treatment also eliminated reactive T cells in the periphery blood without affecting the number of B cells.</p><p><b>CONCLUSION</b>The immunotoxin mMIP-1alpha-DT390 can attenuate the disease activity of EAE in mice, suggesting its potential use in the treatment of other autoimmune disorders.</p>

An expression plasmid encoding recombinant immunotoxin IP10-DT390 suppresses the experimental autoimmune encephalomyelitis / 生物医学工程学杂志

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS); it serves as a model for the human multiple sclerosis (MS). In mice, EAE is mediated by T cells specific for various myelin basic proteins which migrate from the periphery to the CNS. In search of a way to prevent the induction and progression of EAE, we observed the effects of recombinant immunotoxin IP10-DT390 on blocking or eliminating the active T cells in the EAE model. In this paper is presented an experimental gene therapy-based model in which the mice were made resistant to EAE induction by plasmid DNA encoding recombinant immunotoxin that was injected into the leg muscles of mice. The new immuno-biological construct could selectively impair autoreactive T-cell homing while the duration of clinical signs is shorter, and the new construct would not affect other components of the immune response. These data demonstrated the effectiveness of the constructs in the treatment of EAE and suggested its usefulness in the treatment of other autoimmune diseases.

Expression, purification and specific monoclonal antibodies preparation of diphtheria toxin A fragment / 生物工程学报

Diphtheria toxin A fragment (DTA) is an essential catalytic domain of diphtheria toxin (DT)-based immunotoxin. DTA protein and its antibodies play an important role in the studies on toxicology, purification and identification of DT-based immunotoxins. In this paper, DTA was expressed and purified from E. coli. After Q-Sepharose FF chromatography and (Ni+)-Sepharose affinity chromatography, 6 x His-DTA fusion protein with 90% purity was achieved. Using the purified DTA as antigen to immunize BalB/c mice, 2 hybridoma cell lines (designated as 3B6 and 3B9, respectively) secreting monoclonal antibodies (McAbs) against DTA were established. Investigations showed that both McAbs were characterized as IgG1 with titers of 1: 10(6). The binding of the McAbs to DTA was competitively inhibited by horse sera against DT. The fact that anti-DTA McAbs could be used in western blot analysis and affinity chromatography purification of DT-based immunotoxins implied that they will be useful agents in the studies on DT-based immunotoxins.

Cytotoxicity of OKT9 ScFv-Diphtheria Toxin Fusion Immunotoxin on Human Brain Tumor Cell Lines

OBJECTIVE: Immunotoxin therapy is a novel approach for the treatment of tumor, and it has been successfully used in the central nervous system. The purpose of this study is to evaluate the cytotoxicity of OKT9 ScFv-Diphtheria toxin fusion immunotoxin on various human brain tumor cell lines. METHODS: Immunotoxin which was composed of OKT9 ScFv and Diphtheria toxin was made. Its cytotoxicity on glioblastoma cell lines(U87MG, U118MG) and medulloblastoma cell line(TE671) was tested and compared with anti-cancer chemotherapeutic agents. And we also examined the relationship between its cytotoxicity and transferrin receptor expression. RESULTS: It showed most cytotoxicity on U87MG cell line and nearly no effect on U118MG cell line, moderate cytotoxicity on TE671 cell line in sixteen hours exposure experiment. In continuous exposure experiment, it showed moderate cytotoxicity on U118MG cell line, but showed strong cytotoxicity on other cell lines comparable or higher than anti-cancer chemotherapeutic agents. The relationship between its cytotoxicity and transferrin receptor expression was tested using flow cytometry, but no direct relationship could be found. CONCLUSION: Collectively, the result shows the cytotoxic effects of OKT9 ScFv-Diphtheria toxin fusion immunotoxin against various human brain tumor cell lines in continuous exposure experiment. Therefore, we suggest that this immunotoxin could be developed as a potential immunotherapeutic agent in the treatment of various human brain tumor clinically.