Viruses are obligate parasites and cannot replicate without the help of host cells. Thus they are... more Viruses are obligate parasites and cannot replicate without the help of host cells. Thus they are, by nature, adapted to efficiently transmit genetic information to cells. Among the viruses many different strategies have evolved that allow them to enter cells, and the form of their genetic information to some extent determines the later mechanisms used for replication and production of new virus particles or virions. Retroviruses are particularly of interest in this respect, because although they carry an RNA genome, there is a requirement that this genome be transcribed into a double-stranded DNA form in the infected cell, which can then be efficiently integrated into the host cell DNA. Once integrated, the virus DNA, known as a provirus, is transcribed like any other cellular gene and the virus-specific RNA is used to produce virus proteins and new genomic RNA, both of which are assembled into new virions. The integrated provirus is a stable part of the cell genome for the life of...
A candidate hand, foot, and mouth disease vaccine comprising of human enterovirus A71 (EV-A71) vi... more A candidate hand, foot, and mouth disease vaccine comprising of human enterovirus A71 (EV-A71) viruslike particles (VLPs) was tested in rabbits to evaluate the potential local and systemic effects of this vaccine. The rabbits received more than double the full human dose and one additional dose according to the n + 1 recommended scheme. The three doses were given mixed with Alhydrogel adjuvant as intramuscular (IM) injections. Vaccinations were well-tolerated, with no indication of overt toxicity in any parameter observed. An EV-A71 specific immune response in the form of antibodies that specifically reacted with the virus capsid proteins VP1 and VP0, the complete VLP, and EV-A71 viruses of different subgenotypes to that of the vaccine could be demonstrated. A boosting effect in the form of higher EV-A71 specific antibody titers was observed after the subsequent doses, and these enhanced titers were shown to be statistically significant in one-way ANOVA analyses. Fortnightly intramuscular administration of EV-A71 VLP vaccine did not result in any test article-related changes in immunotoxicity as defined by increased serum IL-6, and in general IL-6 concentrations remained below the lower limit of quantitation for the majority of animals throughout the study. Although increased indicators of inflammation at the injection site were observed in animals sacrificed immediately after the last vaccination, these largely reversed at the end of the recovery phase. No findings suggestive of systemic or delayed toxicity were recorded in this independently conducted study. In conclusion, repeated IM administration of the EV-A71 VLP vaccine were locally and systemically well-tolerated in rabbits and immunogenic, supporting the clinical development of the vaccine.
SIGLEAvailable from British Library Document Supply Centre- DSC:D51675/84 / BLDSC - British Libra... more SIGLEAvailable from British Library Document Supply Centre- DSC:D51675/84 / BLDSC - British Library Document Supply CentreGBUnited Kingdo
There are a number of publications suggesting that a virus highly related to mouse mammary tumor ... more There are a number of publications suggesting that a virus highly related to mouse mammary tumor virus (MMTV) is involved in human breast cancer but this is not universally accepted. A recent paper concludes that MMTV is probably not involved in human breast cancer, based primarily on PCR data, because less than one copy of MMTV proviral DNA per cell can be found in human breast cancer samples. However, this conclusion assumes the tumor arose from a single cisactivation event due to insertional mutagenesis. Here we outline other mechanisms by which MMTV and other related retroviruses cause tumorigenesis. Since insertional mutagenesis is not the only mechanism by which retroviruses can induce tumorigenesis, we suggest that it is not yet possible to rule out a role for a virus highly related to mouse mammary tumor virus (MMTV) in human breast cancer.
Pronuclear microinjection was used to produce transgenic mice harboring gene constructs, in which... more Pronuclear microinjection was used to produce transgenic mice harboring gene constructs, in which 110 base pairs (WAP1) or 2.4 kilobases (WAP2) of the 5' flanking sequences of the whey acidic protein (WAP) gene were fused to human growth hormone (hGH)-coding sequences. Female WAP-hGH transgenic mice expressed the transgenes in the mammary gland, the expression of the WAP2-hGH transgene mirroring that of the endogenous WAP gene. When other organs were examined, high level expression of hGH was observed in the brains of WAP2-hGH transgenic mice. Using in situ hybridization and immunohistochemistry, hGH expression from the transgene was seen to occur specifically in Bergmann glia cells. While normally neither WAP nor hGH is expressed in this type of cell, it appears that a combination of the regulatory region of the WAP gene and the hGH structural gene results in a novel tissue specificity in Bergmann glia cells.
Long-term benefits of coronary angioplasty remain limited by the treatment-induced renarrowing of... more Long-term benefits of coronary angioplasty remain limited by the treatment-induced renarrowing of arteries, termed restenosis. One of the mechanisms leading to restenosis is the proliferation of smooth muscle cells. Therefore, proliferating cells of the injured arterial wall, which can be selectively transduced by retroviruses, are potential targets for gene therapy strategies. A direct single-dose therapeutic application of retroviral vectors for inhibition of cell proliferation is normally limited by too low transduction efficiencies. Encapsulated retrovirus-producing cells release viral vectors from microcapsules, and may enhance the transduction efficiency by prolonged infection. Primary and immortal murine and porcine cells and murine retrovirus-producing cells were encapsulated in cellulose sulphate. Cell viability was monitored by analysing cell metabolism. Safety, stability, transfer efficiency and extent of restenosis using capsules were determined in a porcine restenosis model for local gene therapy using morphometry, histology, in situ beta-galactosidase assay and PCR. Encapsulation of cells did not impair cell viability. Capsules containing retrovirus-producing cells expressing the beta-galactosidase reporter gene were implanted into periarterial tissue or a pig model of restenosis. Three weeks following implantation, beta-galactosidase activity was detected in the pericapsular tissue with a transduction efficiency of approximately 1 in 500 cells. Adventitial implantation of vector-producing encapsulated cells for gene therapy may, therefore, facilitate successful targeting of proliferating vascular smooth muscle cells, and allow stable integration of therapeutic genes into surrounding cells. The encapsulation of vector-producing cells could represent a novel and feasible way to optimize local retroviral gene therapy.
International Journal of Pharmaceutics, Sep 1, 2018
The size and speed of release of proteins of different sizes from standard cellulose sulphate cap... more The size and speed of release of proteins of different sizes from standard cellulose sulphate capsules (Cell-in-a-Box®) was investigated. Proteins with molecular weights of up to around 70kD can be released. The conformation, charge and concentration of the protein being released play a role in the release kinetics. Small proteins such as cytokines can be easily released. The ability to produce cytokines at a sustained and predefined level from encapsulated cells genetically engineered to overexpress such cytokines and implanted into patients may aid immunotherapies of cancer as well as infectious and other diseases. It will also allow allogeneic rather than autologous cells to be used. We show that cells encapsulated in polymers of cellulose sulphate are able to release cytokines such as interleukin-2 (IL-2) in a stimulated fashion e.g. using phorbol 12-myristate 13-acetate (PMA) plus ionomycin. Given the excellent documented safety record of cellulose sulphate in patients, these data suggest that clinical usage of the technology may be warranted for cancer treatment and other diseases.
Background: Gut microbiota in humans and animals play an important role in health, aiding in dige... more Background: Gut microbiota in humans and animals play an important role in health, aiding in digestion, regulation of the immune system and protection against pathogens. Changes or imbalances in the gut microbiota (dysbiosis) have been linked to a variety of local and systemic diseases, and there is growing evidence that restoring the balance of the microbiota by delivery of probiotic microorganisms can improve health. However, orally delivered probiotic microorganisms must survive transit through lethal highly acid conditions of the stomach and bile salts in the small intestine. Current methods to protect probiotic microorganisms are still not effective enough. Results: We have developed a cell encapsulation technology based on the natural polymer, cellulose sulphate (CS), that protects members of the microbiota from stomach acid and bile. Here we show that six commonly used probiotic strains (5 bacteria and 1 yeast) can be encapsulated within CS microspheres. These encapsulated strains survive low pH in vitro for at least 4 h without appreciable loss in viability as compared to their respective non-encapsulated counterparts. They also survive subsequent exposure to bile. The CS microspheres can be digested by cellulase at concentrations found in the human intestine, indicating one mechanism of release. Studies in mice that were fed CS encapsulated autofluorescing, commensal E. coli demonstrated release and colonization of the intestinal tract. Conclusion: Taken together, the data suggests that CS microencapsulation can protect bacteria and yeasts from viability losses due to stomach acid, allowing the use of lower oral doses of probiotics and microbiota, whilst ensuring good intestinal delivery and release.
Background: The role of mouse mammary tumor virus (MMTV) as a causative agent in human breast car... more Background: The role of mouse mammary tumor virus (MMTV) as a causative agent in human breast carcinogenesis has recently been the subject of renewed interest. The proposed model is based on the detection of MMTV sequences in human breast cancer but not in healthy breast tissue. One of the main drawbacks to this model, however, was that until now human cells had not been demonstrated to sustain productive MMTV infection. Results: Here, we show for the first time the rapid spread of mouse mammary tumor virus, MMTV(GR), in cultured human mammary cells (Hs578T), ultimately leading to the infection of every cell in culture. The replication of the virus was monitored by quantitative PCR, quantitative RT-PCR and immunofluorescence imaging. The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody. Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine. The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences. Conclusion: Taken together, our results show that human cells can support replication of mouse mammary tumor virus.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
While striving to serve the needs of the international biomedical community of today, we would al... more While striving to serve the needs of the international biomedical community of today, we would also like to remember the Journal of Molecular Medicine (JMM)'s long past as a forum for medical research. JMM is the successor to the Berliner Klinische Wochenschrift (Berlin Clinical Weekly), established in 1864. Below are excerpts from the Berliner Klinische Wochenschrift's January issue from 110 years ago, which give hints about the challenges and solutions being addressed by medical research at that time (Fig.
Viruses are obligate parasites and cannot replicate without the help of host cells. Thus they are... more Viruses are obligate parasites and cannot replicate without the help of host cells. Thus they are, by nature, adapted to efficiently transmit genetic information to cells. Among the viruses many different strategies have evolved that allow them to enter cells, and the form of their genetic information to some extent determines the later mechanisms used for replication and production of new virus particles or virions. Retroviruses are particularly of interest in this respect, because although they carry an RNA genome, there is a requirement that this genome be transcribed into a double-stranded DNA form in the infected cell, which can then be efficiently integrated into the host cell DNA. Once integrated, the virus DNA, known as a provirus, is transcribed like any other cellular gene and the virus-specific RNA is used to produce virus proteins and new genomic RNA, both of which are assembled into new virions. The integrated provirus is a stable part of the cell genome for the life of...
A candidate hand, foot, and mouth disease vaccine comprising of human enterovirus A71 (EV-A71) vi... more A candidate hand, foot, and mouth disease vaccine comprising of human enterovirus A71 (EV-A71) viruslike particles (VLPs) was tested in rabbits to evaluate the potential local and systemic effects of this vaccine. The rabbits received more than double the full human dose and one additional dose according to the n + 1 recommended scheme. The three doses were given mixed with Alhydrogel adjuvant as intramuscular (IM) injections. Vaccinations were well-tolerated, with no indication of overt toxicity in any parameter observed. An EV-A71 specific immune response in the form of antibodies that specifically reacted with the virus capsid proteins VP1 and VP0, the complete VLP, and EV-A71 viruses of different subgenotypes to that of the vaccine could be demonstrated. A boosting effect in the form of higher EV-A71 specific antibody titers was observed after the subsequent doses, and these enhanced titers were shown to be statistically significant in one-way ANOVA analyses. Fortnightly intramuscular administration of EV-A71 VLP vaccine did not result in any test article-related changes in immunotoxicity as defined by increased serum IL-6, and in general IL-6 concentrations remained below the lower limit of quantitation for the majority of animals throughout the study. Although increased indicators of inflammation at the injection site were observed in animals sacrificed immediately after the last vaccination, these largely reversed at the end of the recovery phase. No findings suggestive of systemic or delayed toxicity were recorded in this independently conducted study. In conclusion, repeated IM administration of the EV-A71 VLP vaccine were locally and systemically well-tolerated in rabbits and immunogenic, supporting the clinical development of the vaccine.
SIGLEAvailable from British Library Document Supply Centre- DSC:D51675/84 / BLDSC - British Libra... more SIGLEAvailable from British Library Document Supply Centre- DSC:D51675/84 / BLDSC - British Library Document Supply CentreGBUnited Kingdo
There are a number of publications suggesting that a virus highly related to mouse mammary tumor ... more There are a number of publications suggesting that a virus highly related to mouse mammary tumor virus (MMTV) is involved in human breast cancer but this is not universally accepted. A recent paper concludes that MMTV is probably not involved in human breast cancer, based primarily on PCR data, because less than one copy of MMTV proviral DNA per cell can be found in human breast cancer samples. However, this conclusion assumes the tumor arose from a single cisactivation event due to insertional mutagenesis. Here we outline other mechanisms by which MMTV and other related retroviruses cause tumorigenesis. Since insertional mutagenesis is not the only mechanism by which retroviruses can induce tumorigenesis, we suggest that it is not yet possible to rule out a role for a virus highly related to mouse mammary tumor virus (MMTV) in human breast cancer.
Pronuclear microinjection was used to produce transgenic mice harboring gene constructs, in which... more Pronuclear microinjection was used to produce transgenic mice harboring gene constructs, in which 110 base pairs (WAP1) or 2.4 kilobases (WAP2) of the 5' flanking sequences of the whey acidic protein (WAP) gene were fused to human growth hormone (hGH)-coding sequences. Female WAP-hGH transgenic mice expressed the transgenes in the mammary gland, the expression of the WAP2-hGH transgene mirroring that of the endogenous WAP gene. When other organs were examined, high level expression of hGH was observed in the brains of WAP2-hGH transgenic mice. Using in situ hybridization and immunohistochemistry, hGH expression from the transgene was seen to occur specifically in Bergmann glia cells. While normally neither WAP nor hGH is expressed in this type of cell, it appears that a combination of the regulatory region of the WAP gene and the hGH structural gene results in a novel tissue specificity in Bergmann glia cells.
Long-term benefits of coronary angioplasty remain limited by the treatment-induced renarrowing of... more Long-term benefits of coronary angioplasty remain limited by the treatment-induced renarrowing of arteries, termed restenosis. One of the mechanisms leading to restenosis is the proliferation of smooth muscle cells. Therefore, proliferating cells of the injured arterial wall, which can be selectively transduced by retroviruses, are potential targets for gene therapy strategies. A direct single-dose therapeutic application of retroviral vectors for inhibition of cell proliferation is normally limited by too low transduction efficiencies. Encapsulated retrovirus-producing cells release viral vectors from microcapsules, and may enhance the transduction efficiency by prolonged infection. Primary and immortal murine and porcine cells and murine retrovirus-producing cells were encapsulated in cellulose sulphate. Cell viability was monitored by analysing cell metabolism. Safety, stability, transfer efficiency and extent of restenosis using capsules were determined in a porcine restenosis model for local gene therapy using morphometry, histology, in situ beta-galactosidase assay and PCR. Encapsulation of cells did not impair cell viability. Capsules containing retrovirus-producing cells expressing the beta-galactosidase reporter gene were implanted into periarterial tissue or a pig model of restenosis. Three weeks following implantation, beta-galactosidase activity was detected in the pericapsular tissue with a transduction efficiency of approximately 1 in 500 cells. Adventitial implantation of vector-producing encapsulated cells for gene therapy may, therefore, facilitate successful targeting of proliferating vascular smooth muscle cells, and allow stable integration of therapeutic genes into surrounding cells. The encapsulation of vector-producing cells could represent a novel and feasible way to optimize local retroviral gene therapy.
International Journal of Pharmaceutics, Sep 1, 2018
The size and speed of release of proteins of different sizes from standard cellulose sulphate cap... more The size and speed of release of proteins of different sizes from standard cellulose sulphate capsules (Cell-in-a-Box®) was investigated. Proteins with molecular weights of up to around 70kD can be released. The conformation, charge and concentration of the protein being released play a role in the release kinetics. Small proteins such as cytokines can be easily released. The ability to produce cytokines at a sustained and predefined level from encapsulated cells genetically engineered to overexpress such cytokines and implanted into patients may aid immunotherapies of cancer as well as infectious and other diseases. It will also allow allogeneic rather than autologous cells to be used. We show that cells encapsulated in polymers of cellulose sulphate are able to release cytokines such as interleukin-2 (IL-2) in a stimulated fashion e.g. using phorbol 12-myristate 13-acetate (PMA) plus ionomycin. Given the excellent documented safety record of cellulose sulphate in patients, these data suggest that clinical usage of the technology may be warranted for cancer treatment and other diseases.
Background: Gut microbiota in humans and animals play an important role in health, aiding in dige... more Background: Gut microbiota in humans and animals play an important role in health, aiding in digestion, regulation of the immune system and protection against pathogens. Changes or imbalances in the gut microbiota (dysbiosis) have been linked to a variety of local and systemic diseases, and there is growing evidence that restoring the balance of the microbiota by delivery of probiotic microorganisms can improve health. However, orally delivered probiotic microorganisms must survive transit through lethal highly acid conditions of the stomach and bile salts in the small intestine. Current methods to protect probiotic microorganisms are still not effective enough. Results: We have developed a cell encapsulation technology based on the natural polymer, cellulose sulphate (CS), that protects members of the microbiota from stomach acid and bile. Here we show that six commonly used probiotic strains (5 bacteria and 1 yeast) can be encapsulated within CS microspheres. These encapsulated strains survive low pH in vitro for at least 4 h without appreciable loss in viability as compared to their respective non-encapsulated counterparts. They also survive subsequent exposure to bile. The CS microspheres can be digested by cellulase at concentrations found in the human intestine, indicating one mechanism of release. Studies in mice that were fed CS encapsulated autofluorescing, commensal E. coli demonstrated release and colonization of the intestinal tract. Conclusion: Taken together, the data suggests that CS microencapsulation can protect bacteria and yeasts from viability losses due to stomach acid, allowing the use of lower oral doses of probiotics and microbiota, whilst ensuring good intestinal delivery and release.
Background: The role of mouse mammary tumor virus (MMTV) as a causative agent in human breast car... more Background: The role of mouse mammary tumor virus (MMTV) as a causative agent in human breast carcinogenesis has recently been the subject of renewed interest. The proposed model is based on the detection of MMTV sequences in human breast cancer but not in healthy breast tissue. One of the main drawbacks to this model, however, was that until now human cells had not been demonstrated to sustain productive MMTV infection. Results: Here, we show for the first time the rapid spread of mouse mammary tumor virus, MMTV(GR), in cultured human mammary cells (Hs578T), ultimately leading to the infection of every cell in culture. The replication of the virus was monitored by quantitative PCR, quantitative RT-PCR and immunofluorescence imaging. The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody. Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine. The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences. Conclusion: Taken together, our results show that human cells can support replication of mouse mammary tumor virus.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
While striving to serve the needs of the international biomedical community of today, we would al... more While striving to serve the needs of the international biomedical community of today, we would also like to remember the Journal of Molecular Medicine (JMM)'s long past as a forum for medical research. JMM is the successor to the Berliner Klinische Wochenschrift (Berlin Clinical Weekly), established in 1864. Below are excerpts from the Berliner Klinische Wochenschrift's January issue from 110 years ago, which give hints about the challenges and solutions being addressed by medical research at that time (Fig.
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