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Background Lentiviral vectors are being used with increasing frequency in human clinical trials. We were the first to use lentiviral vectors in clinical trials in 2003. Our lentiviral vector encoded a long RNA antisense sequence to the HIV-1 envelope and was used in an ex vivo autologous setting to provide viral load control in HIV-1 positive subjects failing anti-HIV therapy. A total of 65 subjects have been treated in Phase 1 and Phase 2 trials in six institutions. Methods Good manufacturing practices (GMP) lots of the lentiviral vector used in our clinical trials were assayed for the presence of replication competent lentivirus (RCL). RCL assays were conducted at two stages. The first testing was performed on samples collected immediately following bulk harvest of the GMP product lot and consisted of 1 × 108 cells used in production. RCL assays were also performed on aliquots of the final fill of the vector by the inoculation of at least 5% of the GMP final fill volume into C8166 cells, passaged for at least ten passages and tested for RCL by p24 enzyme-linked immunosorbent assay and vesicular stomatitis virus-G envelope DNA. Results Following 263 infusions of autologous, transduced cells, no adverse events have been detected in these subjects, with some followed for more than 8 years following infusions. More than 4.3 × 1012 VRX496 proviral copies were administered to these 65 subjects. Conclusions Data from this small population suggest that there is no apparent risk for serious adverse events with the use of lentiviral vectors. Copyright © 2013 John Wiley & Sons, Ltd.

Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy. Here we describe a novel trans-splicing strategy, which targets highly abundant pre-mRNAs, to produce therapeutic proteins in vivo. First, we used a pre-trans-splicing molecule (PTM) that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-mRNA and production of hapoA-I protein. The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice. Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo.

Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology for the repair of defective pre-messenger RNA (pre-mRNA) molecules. It is especially useful in the treatment of genetic disorders involving large genes. Although viral vectors have been used for achieving long-lasting expression of trans-splicing molecules, the immunogenicity and suboptimal safety profiles associated with viral-based components could limit the widespread application of SMaRT in the repair of genetic defects. Here, we tested whether the non-viral Sleeping Beauty (SB) transposon system could mediate stable delivery of trans-splicing molecules designed to correct the genetic defect responsible for severe combined immune deficiency (SCID). This immunological disorder is caused by a point mutation within the 12.4 kilobase (kb) gene encoding the DNA protein kinase catalytic subunit (DNA-PKcs) and is associated with aberrant DNA repair, defective T- and B-cell production, and hypersensitivity to radiation-induced injury. Using a novel SB-based trans-splicing vector, we demonstrate stable mRNA correction, proper DNA-PKcs protein production, and conference of a radiation-resistant phenotype in a T-cell thymoma cell line and SCID multipotent adult progenitor cells (MAPCs). These results suggest that SB-based trans-splicing vectors should prove useful in facilitating the correction of endogenous mutated mRNA transcripts, including the DNA-PKcs defect present in SCID cells.

Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone.

While replication-defective retroviral vectors provide excellent vehicles for the long-term expression of therapeutic genes, they also harbor the potential to induce undesired genetic changes by random insertions into the host genome. The rate of insertional mutagenesis for retroviral vectors has been determined in several different assay systems; however, the rate at which such events induce cellular transformation has not been directly determined. Such measurements are critical to determining the actual risk of carcinogenesis resulting from retroviral gene therapy. In this study, the ability of a replication-defective retroviral vector, GlnBgSvNa, to induce cellular transformation in the BALB/c-3T3 in vitro transformation assay was assessed. The transformation frequency observed in vector-transduced BALB/c-3T3 cells, which contained one to six copies of integrated provirus, was not significantly different from that of untreated control cells. The finding that GlnBgSvNa was nontransforming in this assay indicates that the rate of transformation induced by retroviral insertions is less than the spontaneous rate of cellular transformation by BALB/c-3T3 cells, or less than 1.1 x 10(-5). These results are the first to define an upper limit for the rate of transformation induced by retroviral vectors.

We used the polymerase chain reaction (PCR) to assay for the presence of retroviral vector and replication-competent retrovirus (RCR) in autopsy and biopsy specimens from patients who received inoculations of retroviral vector producer cells (VPCs) into brain tumors or apparently normal tissues surrounding resected tumors. The PCR assays were capable of detecting 1 or more proviral copies of vector or RCR in 500,000 cells. Of 113 patients treated in clinical trials between 1994 and 1997, autopsy specimens were available from 32 patients. Brain tumor biopsies were also available from 24 patients. A total of 346 specimens was analyzed. Vector DNA was detected in 55% of tumor samples and 22% of brain samples obtained from resection margins. In contrast, most of the nonbrain tissues were negative for vector DNA; only low levels (0.03%) of vector sequence were detected in 6 of 240 (2.5%) nonbrain tissues. Vector DNA was not detected in gonadal tissues from 12 men and 10 women. More importantly, RCR was not detected in any of the 134 biopsy and autopsy tissues tested, including all brain tumor, brain, and gonadal specimens. These results comprise the largest data set on molecular analysis of autopsy specimens from patients receiving retroviral gene therapy and indicate that distribution of retroviral vectors following injection of high doses of VPCs is limited to the site of inoculation.

A rapid, sensitive assay was developed that can detect the six species of Mycoplasmas that account for the vast majority of cell culture infections. This assay, a modification of the method published by Wong-Lee & Lovett [1], allows direct evaluation of culture medium by a single-step PCR method that utilizes primers complementary to conserved 16S rRNA sequences. Extensive testing of medium from uninfected cultures spiked with purified Mycoplasma DNAs showed that the method described in this report can detect the equivalent of one Mycoplasma in 15 μl culture medium; thus, evaluation of a single culture sample allows detection of Mycoplasmas in cultures infected with the equivalent of 10 or more Mycoplasmas per 15 μl (or ≥6.7×102 Mycoplasma equivalents/ml) with greater than 99.99% confidence. Comparison of results obtained with this PCR-based assay and a standard biological colony-forming assay revealed that the PCR assay is capable of detecting 0.0015-0.015 colony forming units, suggesting that the PCR assay may also be detecting nonviable Mycoplasmas. The high level of amplification achieved with this method allows direct detection of amplification products by ethidium bromide staining of agarose gels, and thus allows rapid screening of cell cultures.

Long-term marrow cultures (LTMCs) were established from 27 human marrows. Hematopoietic cells were subjected to multiple rounds of exposure to retroviral vectors during 3 weeks of culture. Seven different retroviral vectors were evaluated. LTMCs were assessed for viability, replication-competent retrovirus, progenitors capable of proliferating in immune-deficient mice, and gene transfer. The average number of adherent cells and committed granulocyte-macrophage progenitors (CFU-GM) recovered from LTMCs was 28% and 11% of the input totals, respectively. There was no evidence by marker rescue assay or polymerase chain reaction (PCR) of replication-competent virus production during LTMC. No toxicity to cellular proliferation due to the transduction procedure was observed. The adherent layers of LTMCs exposed to retroviral vectors were positive for proviral DNA by PCR and by Southern blot analysis. Fifty-three percent of 1,427 individual CFU-GM from transduced LTMC adherent layers were positive for vector-derived DNA. For neocontaining vectors, the average G418 resistance was 28% of 1,393 LTMC-derived CFU-GM. Forty percent of 187 tissues from 30 immune-deficient mice injected with human LTMC cells were positive for human DNA 4-5 weeks after adoptive transfer. These studies indicate that multiple exposures of human LTMCs to retroviral vectors result in consistent and reproducible LTMC viability and gene transfer into committed progenitors. Our results further support the use of transduced LTMC cells in clinical trials of hematopoietic stem cell gene transfer.

Lentiviral vectors are being used with increasing frequency in human clinical trials. We were the first to use lentiviral vectors in clinical trials in 2003. Our lentiviral vector encoded a long RNA antisense sequence to the HIV-1 envelope and was used in an ex vivo autologous setting to provide viral load control in HIV-1 positive subjects failing anti-HIV therapy. A total of 65 subjects have been treated in Phase 1 and Phase 2 trials in six institutions.Good manufacturing practices (GMP) lots of the lentiviral vector used in our clinical trials were assayed for the presence of replication competent lentivirus (RCL). RCL assays were conducted at two stages. The first testing was performed on samples collected immediately following bulk harvest of the GMP product lot and consisted of 1 × 10(8) cells used in production. RCL assays were also performed on aliquots of the final fill of the vector by the inoculation of at least 5% of the GMP final fill volume into C8166 cells, passaged for at least ten passages and tested for RCL by p24 enzyme-linked immunosorbent assay and vesicular stomatitis virus-G envelope DNA.Following 263 infusions of autologous, transduced cells, no adverse events have been detected in these subjects, with some followed for more than 8 years following infusions. More than 4.3 × 10(12) VRX496 proviral copies were administered to these 65 subjects.Data from this small population suggest that there is no apparent risk for serious adverse events with the use of lentiviral vectors.

The recent recommendation of the Board of Directors of the American Society of Gene and Cell Therapy that the National Institutes of Health (NIH) Recombinant DNA Advisory Committee (RAC) end its review of clinical protocols in human gene therapy and human gene marking is a welcome and necessary step in the continuing evolution of the oversight of clinical gene therapy.1 In the late 1980s and early 1990s, the RAC and the Division of Cell and Gene Therapy of the Center for Biologics Evaluation and Research at the US Food and Drug Administration (FDA) met often in informal sessions following RAC meetings to discuss and review various aspects of clinical trials in gene marking and therapy. Indeed, RAC review was a productive part of the process when these early gene transfer protocols were being considered. This pooling of expertise was a completely new approach to therapeutics that greatly aided both groups. However, owing in large part to the emergence of a strong and capable full-time group devoted to cell and gene therapy within the FDA, RAC review is no longer necessary.

We conducted a phase I hematopoietic stem cell (HSC) gene-marking trial in patients undergoing autologous blood or marrow stem cell transplant for the treatment of multiple myeloma. Between 500 and 1000 ml of bone marrow was harvested from each of 14 myeloma patients and 1 syngeneic donor. A mean of 3.3x10(9) cells per patient were plated in 20 to 50 long-term marrow culture (LTMC) flasks and maintained for 3 weeks. LTMCs were exposed on days 8 and 15 to clinical-grade neo(r)-containing retrovirus supernatant (G1Na). A mean of 8.23x10(8) day-21 LTMC cells containing 5.2x10(4) gene-marked granulocyte-macrophage progenitor cells (CFU-GM) were infused along with an unmanipulated peripheral blood stem cell graft into each patient after myeloablative therapy. Proviral DNA was detected in 71% of 68 tested blood and bone marrow samples and 150 of 2936 (5.1%) CFU-GM derived from patient bone marrow samples after transplant. The proportion of proviral DNA-positive CFU-GM declined from a mean of 9.8% at 3 months to a mean of 2.3% at 24 months postinfusion. Southern blots of 26 marrow and blood samples were negative. Semiquantitative PCR analysis indicated that gene transfer was achieved in 0.01-1% of total bone marrow and blood mononuclear cells (MNCs). Proviral DNA was also observed in EBV-transformed B lymphocytes, in CD34+ -enriched bone marrow cells, and in CFUs derived from the latter progenitors. Gene-modified cells were detected by PCR in peripheral blood and bone marrow for 24 months after infusion of LTMC cells. Sensitivity and specificity of the PCR assays were independently validated in four laboratories. Our data confirm that HSCs may be successfully transduced in stromal based culture systems. The major obstacle to therapeutic application of this approach remains the overall low level of genetically modified cells among the total hematopoietic cell pool in vivo.

Patients with recurrent malignant brain cancer, who were receiving gene therapy by intracerebral injection of murine retroviral vector producer cells (VPCs), were monitored for the presence of replication-competent retrovirus (RCR). RCR sequences were not detected by polymerase chain reaction (PCR) in any of the 608 peripheral blood leukocyte (PBL) samples analyzed. Vector DNA sequences were detected transiently in PBL samples from a subset of 34 patients. Humoral immune responses to a retroviral core protein p30 and murine VPC were detected in some patients, most frequently in patients receiving repeated administrations of VPC. RCR was not detected in biological assays of PBLs from 41 patients who had either anti-retroviral antibodies in sera and/or vector DNA in PBLs. Our data suggest that in situ generation of RCR was not detected following intracerebral inoculation of VPCs in any of the 128 patients evaluated.

The first clinical trial for gene transfer in humans was approved in the United States in 1989. In this trial, genetically marked lymphocytes that had been removed from a melanoma biopsy were transduced with a retroviral vector containing the gene conferring resistance to the neomycin analogue G-418, propagated ex vivo and returned to the patient. The purpose of this gene marking study was to determine if the G-418 resistant lymphocytes honed specifically to melanoma and to determine the lifespan of the marked lymphocytes. Results of various gene marker trials have been published (Dunbar et al. 1994; Brenner 1996). Marker trials have demonstrated some homing to tumors, and marked lymphocytes have been detectable for years past re-infusion.

The use of defined or serum-free culture conditions during retroviral transduction of hematopoietic cells would be desirable for standardization and safety reasons, as well as potentially allowing greater expansion of progenitor cells. Retroviral vector supernatants were concentrated and purified via tangential flow filtration polyethylene glycol (PEG)-precipitation, and ultracentrifugation, allowing serum-free transductions at standard multiplicities of infection (moi). Protein content of transductions using these concentrated vectors was 5-6 logs lower than in standard transductions. Transduction efficiencies of these concentrated vector preparations added back to serum-free or serum-containing media were equivalent to standard retroviral supernatant transductions of CD34-enriched progenitors. Absolute progenitor (CFU-C) numbers at the end of transduction were higher in serum-free + concentrated virus transductions, as opposed to transductions in standard vector supernatants containing fetal calf serum.

Transfer of the herpes simplex virus type-1 thymidine kinase (HSV-tk) gene into tumor cells followed by ganciclovir (GCV) administration, will provide selective tumor cell killing. We studied the effect of herpes simplex virus thymidine kinase (HSV-tk) expression level on the HSV-tk/GCV-mediated "bystander effect." Clones of HSV-tk-transduced rat glioma cells (9L) were isolated that stably expressed with different levels of HSV-tk. All clones studied had similar sensitivity to ganciclovir with IC50 values ranging from 0.45 to 1.3 microM. Within certain enzyme level thresholds, in vitro evaluation of the bystander effect has shown that clones with higher level of HSV-tk expression exhibited a better bystander effect. Interestingly, the bystander effect was observed between different cell types. Both the transduction efficiency and bystander effect are essential factors for the success of the antitumor effect by the HSV-tk/prodrug GCV suicide gene system.

A replication-competent retrovirus (RCR) was detected by S+/L- assays in three lots of retroviral vector G1Na that were harvested on consecutive days from a single culture of PA317/G1Na producer cells. Using a number of retrovirus-specific primer pairs, it was shown that this RCR was a novel recombinant created by exchanges between G1Na and helper sequence pPAM3 and was not an existing RCR introduced by cross-contamination. Sequencing of clones of DNA amplified in six independent PCR reactions confirmed that the 3' portion of this RCR was composed of retroviral envelope sequences unique to pPAM3 joined to a 3' long terminal repeat (LTR) unique to G1Na. Comparison of pPAM3 and G1Na sequences at the site corresponding to this junction revealed a short segment of patchy nucleotide identity (8 out of 10 bp), suggesting that these helper and vector sequences were joined by homologous recombination. Generation of RCR by exchanges between helper and vector sequences underscores the necessity of testing by efficient methods all retroviral vectors for the presence of RCR before their use. Production of 171 lots (855 liters) of various retroviral vectors that were free of RCR, including 42 lots of G1Na, however, indicates that the combination of exchanges required to generate an RCR are infrequent in this system.

To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable replication-competent retroviruses. The purpose of this study was to optimize methods of retroviral vector production and transduction. Studies were conducted using 22 retroviral vector producer cell lines. Inactivation of retroviral vectors was greater at 37 degrees C than at 32 degrees C. A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate. For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C. The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor. Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system. Retroviral supernatants were successfully lyophilized. The combination of glucose or sorbitol with gelatin resulted in recovery rates of 64-83%. In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses. The combination of the above methods has significantly increased the growth and transduction by this vector system.

Publisher Summary This chapter discusses the recombinant DNA advisory committee (RAC) of the National Institutes of Health (NIH) and the approval of 52 different clinical protocols in human gene transfer/gene therapy. The term gene therapy refers to the clinical trials performed solely for therapeutic objectives. The term gene transfer denotes the use of foreign genes inserted into human cells for the purposes of either therapy or marking. The most significant developments in human gene transfer in recent years have been related to vector design and the potential use of viral vectors other than retroviral as well as nonviral vectors, design of appropriate safety studies, studies in animal model systems for the treatment of cancer, use in infectious and genetic diseases, and the continued acceleration of clinical trials. Some of the human clinical trials approved in the United States are for the purposes of gene marking— that is, to determine the fate of genetically marked cells through the use of a gene conferring antibiotic resistance to the neomycin analogue G418. The majority of clinical trials continue to utilize retroviral vectors. The retroviral vectors currently in clinical trials for human gene transfer have been derived from members of the oncovirinae subfamily of the retrovidiae family. Retroviral producer cells vary in their ability to generate replication competent viruses (RCR). This can be influenced by the degree of homology between the helper and the vector genome. In some systems, the vector genome is mutated to change the gag start codon to a stop codon. Three recombinational events would be necessary to generate RCR in split packaging systems. However, the conversion of a producing cell system from a standard to a split packaging cell will not, a priori, lessen the possibility of an RCR “breakout.” The results of the breakout of RCR and the development of tumors in immunosuppressed monkeys clearly demonstrate the need for reliable and sensitive assays to detect RCR. Assays for RCR can consist of inoculation of specimens from the production lots into PG-4 cells as well as inoculation into 3T3.

Mycoplasmal infection of cell cultures remains a significant threat to diagnostic and research procedures. In certain defined situations, curing of mycoplasmal infected cultures is a reasonable exercise. Four methods of curing were compared: treatment with BM-cycline, 5 bromouracil, use of specific antisera and treatment of infected cells suspended in soft agar with antibiotics. Antisera treatments were of low efficiency of curing: 50%. None of nine infected cell lines treated with 5-bromouracil were consistently cured of mycoplasmas. The use of BM-cycline was effective for some, but not all lines and required long periods of treatment, 12-21 days. 35 naturally or deliberately infected cultures were treated in soft agar a total of 119 times. This procedure which consisted of suspending infected cultures in soft agar containing appropriate antibiotics resulted in successful mycoplasmal elimination 118/119 times. This soft agar technique took 1-3 days. In separate studies, it was shown that certain Mycoplasma fermentans strains were resisted to this and other curing methods. This may be due to their intracellular location. Such strains may be more amenable to antibiotics that penetrate mammalian cells. It is concluded that the soft agar technique is a rapid, efficient and reliable method to eliminate cell culture mycoplasmas.

Proteins resistant to proteinase K are rare because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme. Previously, only the prion proteins associated with transmissible spongiform encephalopathies, possibly related proteins in the mollicute Spiroplasma mirum, and proteinase K itself have been reported. We identified a new proteinase K-resistant protein, p40-pr, in two strains of Mycoplasma hyorhinis and in extracts of these organisms. p40-pr's are similar to prion proteins in their resistance to high doses of proteinase K and in the reversal of this resistance by strong denaturing conditions. However, p40-pr's were distinct immunologically, in relative molecular mass, and in their method of extraction. Two immunologically related forms of p40-pr were identified on sodium dodecyl sulfate (SDS) gels and Western immunoblots, a 40-kDa species in boiled samples and a 120-kDa species dissociable by boiling in SDS. Reduction with 2-mercaptoethanol did not affect the mass of p40-pr's or the 120-kDa forms. The development of proteinase K resistance of p40-pr correlated to age-dependent increases in organism protein-lipid ratios. p40-pr-like proteinase K-resistant proteins of 46 to 50 kDa were identified in four of eight additional species of the class Mollicutes but not in S. mirum. However, these mycoplasmal proteins did not react with antibody to the denatured 40-kDa form of M. hyorhinis p40-pr purified by electroelution. The chromatographically purified 46-kDa proteinase K-resistant protein of Mycoplasma orale was an arginine deiminase.

The failure of many cell culture isolates of Mycoplasma hyorhinis to grow on microbiological media has stressed the need for alternate assays to detect these organisms. The use of freshly prepared yeast extract in mycoplasmal media together with incubation in 5% CO2/air successfully detected M. hyorhinis in 12 of 12 infected cultures. These were not detected by the use of conventional mycoplasmal media using aerobic or anaerobic incubation. This assay may also be helpful in detection of other mycoplasmal species commonly isolated from cell cultures.

Low levels of high density lipoprotein (HDL) represent an important predictor of cardiovascular disease risk1,2. An increase of 1mg/dl in HDL correlates with a risk reduction of cardiovascular disease of 2|[ndash]|3%3. We have adapted spliceosome mediated RNA trans-splicing technology (SMaRT|[trade]|) to trans-splice the sequences of wild type human apolipoprotein A-I (apoA-I) into the highly abundant albumin pre-mRNA in hepatocytes with the objective of transiently increasing blood levels of HDL. ApoA-I is the major protein component of HDL. We targeted a pre-trans-splicing molecule (PTM) to bind to specific sequences in the first intron of mouse albumin pre-mRNA. The bound PTM will then trans-splice, inserting sequences for human ApoA-I into intron 1 of albumin pre-mRNA. With trans-splicing, apoA-I would be made in hepatocytes, the same cells that naturally synthesize apoA-I.

Cardiovascular disease (CVD) is the most common cause of death in Western societies, and its worldwide prevalence is increasing. A very strong predictor of CVD risk is the plasma concentration of high-density lipoprotein (HDL) or of its major protein component, ApoA-1. HDL and ApoA-1 levels exhibit an inverse relationship with the development of atherosclerosis and coronary heart disease1,2,3 and exogenous ApoA1 and HDL protect against atherosclerosis and plaque development 4,5,6,7. The present study was undertaken to determine if RNA trans-splicing could be used to increase blood levels of human ApoA-1 (hApoA1). To this end we targeted a pre-trans-splicing molecule (PTM) encoding human ApoA1 to the highly abundant albumin pre-messenger RNA (pre-mRNA). Trans-splicing between the PTM and the albumin pre-mRNA results in the production of human ApoA1 protein with two residual albumin amino acids. This fusion protein was expressed, processed and secreted as well as authentic hApoA1 and indeed the fusion protein had full activity in ATP-binding cassette transporter protein (ABC1)-mediated transfer of cellular cholesterol efflux assays.

Uridine phosphorylase activity has been used to detect mycoplasmas in cell cultures by measuring formation of14C-uracil from14C-uridine. In this report we show that all species ofMycoplasma, Acholeplasma, andUreaplasma tested exhibited uridine phorphorylase activity. Among the genusSpiroplasma, serogroups I-1, I-3, I-5, I-7, I-8, IV, XIII, and XIV lacked uridine phosphorylase activity.

A helical mycoplasma, Spiroplasma mirum strain SMCA, produced malignant transformation in mouse NIH 3T3 cells and monkey kidney CV-1 cells. The transformed cells exhibited morphological changes consistent with the transformed phenotype, grew in soft agar and produced tumors in athymic and BALB/c mice. Transmission electron microscopy revealed structures morphologically similar to mycoplasmas present in the cytoplasm of transformed but not untransformed 3T3 cells. The time of inoculation of S. mirum SMCA to 3T3 cells and the passage level of 3T3 cells affected transformation.

A radiochemical method was developed for the assay of aspartate aminotransferase and alanine aminotransferase activities in Mollicutes. Using [1-C14]α-ketoglutarate as the amino group acceptor in transamination, we found that the fermentative speciesAcholeplasma laidlawii MG of the family of Acholeplasmataceae, the fermentativeMycoplasma pneumonia FH of the family of Mycoplasmataceae, and the nonfermentativeMycoplasma salivarium VV, also of the family of Mycoplasmataceae, all had aspartate aminotransferase and alanine aminotransferase activities. The radioactive product was identified as [1-C14]l-glutamic acid.Mycoplasma pneumoniae andM. salivarium had very low activity of alanine aminotransferase. Both aminotransferases had a partial requirement for pyridoxal 5′-phosphate and were strongly inhibited by 0.1 mM aminooxyacetate.

Polyoma virus (PV) infection was transmitted through the air of an animal laboratory. Mice free of detectable antibodies to PV were exposed for 1 month to the airborne environment of laboratories housing naturally infected mice. The seroconversion rate was 75% (24/32), as measured by hemagglutination inhibition. Control mice, housed in the sterile atmosphere of a mass air flow cabinet (MAFC) in the same laboratory, had a seroconversion rate of 15.8% (3/19). Airborne transmission occurred bia PV aerosois, generated by the handling of contaminated bedding, cages, and mice during weekly housekeeping. Length of exposure to PV aerosols correlated with seroconversion. One- and 3-hour exposures resulted in seroconversion rates of 40% (6/15) and 72% (23/32), respectively. Seroconversion rates of mice continuously housed in MAFC totaled 5% (2/40). Checkerboarding mice free of detectable antibodies with mice given 10(5) mean tissue culture infective doses of PV ip resulted in an airborne infection rate of 50% (15/30) in a conventionally ventilated room during a 12-week study. The airborne transmission rate was 10% (3/30) when experiments were performed in a mass air flow room with a vertical air velocity of 30 feet/minute was used. Antibodies to PV could not be detected in any of 138 human sera, including sera from 29 animal-care technicians who handled PV-infected mice and 15 personnel who had worked with the virus.

An overview of the purpose, design, and results of the summer 1981 and winter 1982 ATEOS studies is presented. Daily sampling was conducted during 6-week periods at three urban sites and one rural site: Newark, Elizabeth, Camden, and Ringwood. Inhalable particulate matter was characterized with respect to organic and inorganic composition, the bacterial mutagenic activity of three organic fractions and fine particle mass. The study showed that the IPM fractions of the atmospheric aerosol was composed primarily of EOM (extractable organic matter) sulfate, and crustal material. There were seasonal shifts in the composition, however, with winter time space heating and local motor vehicle traffic appearing to be important factors. Each EOM fraction and the PAH associated with the nonpolar particulate matter increased by a large factor during the winter. The mutagenic activity of the EOM increased, but the types of mutagens present appear to be different in each season. The average SO/sub 4//sup -2/ levels were similar for the two seasons. There were few significant day to day excursions in concentration during the winter. The volatile organic substance measurements showed distinct local source relationships, although benzene levels were fairly constant in all urban locales. Winter time values of VOC (volatilemore » organic compounds) were generally higher than those in the summer. 7 figures, 8 tables. (DP)« less

Adenosine phosphorylase (adenosine: orthophosphate ribosyltransferase) activity is low in mammalian cells, but is abundant in certain prokaryotes. It is an appropriate target enzyme for the development of diagnostic and chemotherapeutic agents. Adenosine phosphorylase from Bacillus subtilis and mycoplasmas that commonly infect cell cultures converted the non-toxic deoxyadenosine analog, 6-methylpurine deoxyriboside (6MPDR) into the potent anti-metabolites 6-methylpurine and 6-methylpurine riboside. Consequently 6MPDR selectively killed mammalian cell cultures infected with mycoplasmas. 6MPDR was cytotoxic to 87 of 90 mycoplasma-infected cultures (96.6%). No toxicity was observed in nine different types of mycoplasma-free cell cultures. Use of a 3T6 mouse embryo fibroblast indicator cell culture improved standardization. Cytotoxicity was apparent 3–4 days after inoculation of 10 μM of 6MPDR to 3T6 cultures infected with the following mycoplasmas: M. hyorhinis, M. orale, M. arginini, M. salivarium, A, laidlawii, M. hominis, M. fermentons, M. sp . 70–159 and M. buccale . Cytotoxicity was produced in 28/28 3T6 indicator cultures inoculated with mycoplasma-infected cell cultures in the presence of 10 μM 6MPDR. The analog is apparently non-toxic to the mycoplasmas.

SUMMARYPolyoma virus was recovered from the air of an animal laboratory housing mice infected with the virus. Air samples were obtained by means of a high volume air sampler and further concentrated by high speed centrifugation. Total concentration of the air samples was 7·5 × 107. Assay for polyoma virus was by mouse antibody production tests. Airborne polyoma virus was detected in four of six samples.

A simple and rapid method of species identification of mycoplasmas by immunobinding assay is described. Small amounts of antigen of supernatant from cell cultures, broth cultures or clinical specimens were spotted onto nitrocellulose paper. This was followed by application of specific anti-mycoplasma antisera. After incubation, an enzyme-conjugated antiserum against the first antiserum was applied. A positive reaction was indicated by the development of intense blue color reaction when substrate was added. This method identified mycoplasma species with monoclonal and polyclonal antibodies. It detected 9.3 X 10(3) - 7.5 X 10(4) CFU/ml of organisms depending on mycoplasma species. For identification of mycoplasma, ureaplasma, acholeplasma and spiroplasma species, this assay is useful and rapid compared with other serological methods. In limited studies, the method correlated with microbiological assay of clinical specimens for Mycoplasma pneumoniae.

Studies were performed to compare three methods to detect mycoplasmal infection of cell cultures. The methods included microbiological assay by inoculation into broth and onto agar with anaerobic incubation, fluorescent DNA staining by Hoechst 33258, and mycoplasmal mediated cytotoxicity by 6 methylpurine deoxyriboside (6MPDR). Fluorescent DNA staining and 6MPDR assays were performed in an indicator cell culture system. A total of 2589 cell cultures were assayed. Mycoplasmas were detected in 174, an incidence of 6.7%. Species isolated were: Acholeplasma laidlawii, Mycoplasma orale, M. arginini, M. hyorhinis, M. fermentans, M. pirum, and M. pneumoniae. In separate studies, 6MPDR also detected infection with Spiroplasma mirum when this organism was deliberately inoculated into cell cultures. The efficiencies of microbiological testing, fluorescent DNA assays, and 6MPDR were 43.1, 98.8, and 97.1%, respectively.

Tests are reported on a modified laminar flow biological safety cabinet in which the return air plenum that conducts air from the work area to the high efficiency particulate air filters is under negative pressure. Freon gas released inside the cabinet could not be detected outside by a freon gas detection method capable of detecting 10 -6 cc/s. When T3 bacteriophage was aerosolized 5 cm outside the front opening in 11 tests, no phage could be detected inside the cabinet with the motor-filter unit in operation. An average of 2.8 × 10 5 plaque-forming units (PFU)/ft 3 (ca. 0.028 m 3 ) were detected with the motor-filter unit not in operation, a penetration of 0.0%. Aerosolization 5 cm inside the cabinet yielded an average of 10 PFU/ft 3 outside the cabinet with the motor-filter unit in operation and an average of 4.1 × 10 5 PFU/ft 3 with the motor-filter unit not in operation, a penetration of 0.002%. These values are the same order of effectiveness as the positive-pressure laminar flow biological safety cabinets previously tested. The advantages of the negative-pressure return plenum design include: (i) assurance that if cracks or leaks develop in the plenum it will not lead to discharge of contaminated air into the laboratory; and (ii) the price is lower due to reduced manufacturing costs.

The aliphatic diamine putrescine, a metabolic precursor of the polyamines spermidine and spermine, markedly stimulated the growth of a murine lymphoblastoid cell line (R 1.1) infected with Mycoplasma orale, under conditions of arginine limitation. The diamine acted by suppressing the growth of the mycoplasma, which use arginine as a major energy source, and thereby prevented the depletion of arginine from the medium. The antimycoplasmal effects of putrescine occurred at concentrations that were neither stimulatory nor toxic to uninfected cells.

The most frequent causes of cell culture contamination are poor techniques and housekeeping and inadequate sterility testing of supplies and culture media. The most common contaminants of cell cultures areMycoplasma, Torula sp., andPseudomonas sp. Routine procedures used in this laboratory to control or prevent accidental contamination include the use of filtered laminar air flow in transfer rooms, elimination of antibiotics wherever possible, careful execution of aseptic procedures, periodic review and discussion of techniques, sterility testing of all media components, use of approved clothing, and effective cleaning and disinfection procedures.

Evaluation of a hard-wall, dust-free hospital room for effectiveness of control of gaseous pollutants and odors was conducted in a clinical research center. The study is part of a long-range goal to improve postoperative care and burn patient care techniques by providing a known and controlled environment. Filtration through absolute filters in the air recirculation system removes particulate matter. A charged electrode system consisting of high-voltage and high-frequency units, which is claimed to neutralize “spaces charges” on particulate and gaseous material, preventing deposition on room surfaces, was evaluated. A continuous method of following in situ ventilation rates of a “slug” and “continuous” gaseous sources with N2O gas as a tracer has been developed in this work. The method appears to be an improvement over radioactive and nonradioactive tracers for the study of rooms having high ventilation rates.

Bacterial air sampling in an animal care laboratory showed that dense aerosols are generated during cage changing and cage cleaning. Reyniers and Andersen sampling showed that the airborne bacteria numbered 50 to 200 colony-forming units (CFU)/ft 3 of air. Of the viable particles collected by Andersen samplers, 78.5% were larger than 5.5 μm. A low velocity laminar air flow system composed of high-efficiency particulate air (HEPA) filters and a ceiling distribution system maintained the number of airborne viable particles at low levels, generally less than 2 CFU/ft 3 . Vertical air flow of 15 ft/min significantly reduced the rate of airborne infection by a strain of Proteus mirabilis . Other factors shown to influence airborne infection included type of cage utilized, the use of bedding, the distance between cages, and the number of animals per cage.

Publisher Summary This chapter discusses on contamination, which may originate in the tissue specimen used to initiate the cell culture, in media, especially in bovine serum, or in the general environment. The contamination may be bacterial, mycoplasmal, fungal, viral, or cellular. Detection methods described will effectively monitor microbiological contamination in cell cultures and media. This is viewed as part of an overall quality-control program. The major limitations for detection of bacterial, mycoplasmal, and fungal organisms are sample size, level of contamination, type of growth media utilized, and presence of antibiotics in the sample. Contamination in bovine sera and biologicals is more difficult to detect because of the low frequency of contamination and the low concentration of organisms in contaminated units, 1–10 organisms per milliliter or less. Antibiotics can mask contamination and yield false negatives.

Controlled, prospective studies were performed to compare detection of cell culture mycoplasmas by ratio of uptake of tritiated uridine (UdR) to tritiated uracil (U) and by microbiological culture. Culture was by standard agar and broth inoculation with aerobic and anaerobic incubation; immunofluorescent staining of indicator cell cultures was used to detect M. hyorhinis. The ratio of uptake of UdR to U ( UdR U ) and interpretation of test results were by standard published methods and performed in triplicate. 115 cell cultures were simultaneously assayed by the two techniques. 84 cultures (73.1%) yielded agreement between the 2 methods; 2 cultures (1.7%) yielded conflicting results, and 29 cultures (25.2%) yielded UdR U results in the questionable range. Conflicting results consisted of two negative UdR U tests in mouse cell cultures infected with M. orale. In separate studies, 3T-6 cultures freshly infected with M. orale yielded negative UdR U results 3 days after infection, questionable results after 10 days and a positive UdR U 17 days after infection. UdR U detected infection in fibroblast, epithelial, and lymphocyte cell cultures. Highest UdR U ratios were detected in human skin fibroblasts at early population doubling levels (PDLs), 4064 in one culture at PDL4. UdR U was determined for IMR-90, a human diploid fibroblast at 12 different PDLs using the same lot of media. UdR U gradually decreased throughout the life of the culture, from 2 125 at PDL6 to 340 at PDL36. Cultures in phase III and others exhibiting poor growth frequently yielded questionable or false-positive UdR U results and were not included in tabulations of these results. UdR U determined in endothelial cell cultures decreased as population density increased. In a representative experiment performed over a 4-day period, the UdR U values were 1 808, 955 and 356 when the number of endothelial cells in culture were 5.3 × 105, 6.6 × 105 and 1.1 × 106, respectively.