Gamma irradiation of animal sera for inactivation of viruses and mollicutes--a review.

Animal-derived materials such as animal sera represent a low, but finite, risk for introduction of an adventitious agent (virus or mollicute) into a biological bulk harvest during upstream manufacturing processes involving mammalian cell substrates. Viral and mollicute (Mycoplasma sp. and Acholeplasma sp.) contamination events have been relatively rare, but many of those that have been reported have been attributed to use of infected animal sera in growth media during cell expansion. The risk of introduction of viruses and mollicutes may be mitigated by elimination of the use of animal sera and implementation instead of chemically defined or serum- and animal-derived material-free cell culture media. When use of animal sera is unavoidable, however, mitigation of the risk of introducing an adventitious contaminant may involve treatment of the sera to inactivate potential contaminants. Gamma irradiation is one of the most widely employed methods for viral and mollicute inactivation in animal sera. In this article, we review the inactivation results reported for viral and mollicute inactivation in frozen serum. Studies performed to assess the impact of gamma irradiation on serum quality and performance are also discussed. The available data indicate that inactivation of mollicutes in serum is essentially complete at the gamma radiation doses normally employed (25-40 kGy), while the efficacy and kinetics for viral inactivation in serum by gamma irradiation appear to be dependent in part upon the size of the target virus.

Effect of exogenous frequency exposure on human cells.

Effects of exogenous bioresonance oscillations on biological and morphological characteristics of continuous human lung and liver cells were studied. Proliferative activities and viability of cells decreased after exposure to frequencies of 8 and 78.5 Hz. This was paralleled by cytopathic disorders in lung and liver cells. The frequency of 72 Hz exhibited a less intense effect on both cell types. Cell contamination with mycoplasmas was provisionally suppressed after cell exposure according to F44 and F45 programs and to 97.5 and 69.5 Hz frequencies.

Depleted uranium: all the questions about DU and Gulf War syndrome are not yet answered.

For 15 years, the debate about depleted uranium (DU) and its detrimental effects on the health of veterans of the Gulf War of 1991, on the Iraqi people and military (and subsequently on the people of Kosovo, Afghanistan, and Iraq during the second war) has remained unresolved. Meanwhile, the number of Gulf War veterans who have developed the so-called Gulf War syndrome has risen to about one-third of the 800,000 U.S. forces deployed, and unknown proportions of those involved in the subsequent wars. Uncounted civilians and personnel of other nations that fought in Iraq and other wars since 1991 have also been afflicted. The veterans have suffered from multiple serious physiological disorders and have received little or no official recognition, medical relief, or compensation. We need to take another look at this issue, using a holistic and interactive model for the toxic matrix of exposures, identifying the major roadblocks to resolving the scientific questions, and finding appropriate medical and political responses. This commentary is such an attempt.

Efficient inactivation of viruses and mycoplasma in animal sera using UVC irradiation.

Transmission of viruses by animal sera represents a considerable risk for humans and animals particularly when the serum is used for the production of pharmaceutical products such as vaccines. Procedures applicable for inactivating large numbers of different viruses, both enveloped and non-enveloped, are therefore mandatory. For this purpose we have developed and validated UVC irradiation as the virus-inactivation procedure of choice for serum to be used in an industrial setting. Spiking experiments in foetal calf serum (FCS) were performed by independent contract laboratories and revealed constantly high clearance rates for various viruses such as bovine parvovirus, parainfluenza type III virus, bovine diarrhoea virus, foot-and-mouth disease virus and different forms of mycoplasmas. UVC-treated sera maintained their growth-promoting activities for various cell types (MRC-5, Vero, CHO). Conventional growth curves generated in the presence of 10% and 1% UVC-treated FCS differed only slightly from controls, indicating the lack of significant damage during UVC exposure. Experiments using a sensitive photometric-based acid phosphatase assay (APA), which correlates well with the more tedious cell counting procedure, confirmed these findings even in the presence of minimal serum requirements. UVC treatment of animal sera appears advantageous compared to currently recommended inactivation procedures, such as Gamma irradiation, for at least three reasons: (i) it possesses a high inactivation capacity for parvoviruses, a pathogen that cannot be destroyed easily by conventional methods; (ii) it causes no noticeable impairment in cell growth and (iii) it can be performed in a controlled manner at the production site.

Genetic exchange of transposon and integrative plasmid markers in Mycoplasma pulmonis.

Matings of genetically marked derivatives of Mycoplasma pulmonis resulted in the exchange of chromosomal DNA and the appearance of doubly marked transconjugants. Transposons Tn916 and Tn4001, and a series of integrative plasmids derived from their cloned antibiotic resistance genes, were used to construct antibiotic-resistant mycoplasmal derivatives to examine this phenomenon at the molecular level. Genetic exchange occurred on agar surfaces at frequencies ranging from 3.3 X 10(-4) to 6.4 X 10(-8) transconjugants per CFU. Examination of chromosomal DNA from transconjugants by hybridization revealed that the transposons or integrated plasmids were in the same chromosomal locations as in the parental strains, indicating that exchange involved the transfer of chromosomal DNA and homologous recombination. Transfer was not affected by DNase, polyethylene glycol, EDTA, or calcium chloride but was affected by treatment of either parent with trypsin. Mixing of mating strains before plating had no effect on mating frequencies, but mating did occur in liquid media. The ability to exchange chromosomal markers was limited to selected strains of M. pulmonis; mating did not occur with Acholeplasma laidlawii or M. gallisepticum. Heat and UV inactivation studies revealed that nonviable cells could act as donors in matings. The evidence presented supports a conjugationlike mechanism involving specific trypsin-sensitive membrane components.

[The inactivation of mycoplasmas and bacteria in calf serum by 60Co gamma rays]. / Versuche zur Inaktivierung von Mykoplasmen und Bakterien in Kälberseren durch 60Co-gamma-Strahlen.

Reported in this paper is the use of 60Co gamma radiation to inactivate mycoplasmas in calf serum, newborn calf serum, and fetal calf serum. A dose of 3 kGy, independent of dose rate, was found to be sufficient for inactivation in the above sera of several mycoplasmas, including Acholeplasma laidlawii, Mycoplasma (M.) orale, M. arginini, M. hyorhinis, and M. bovis. The critical dose proved to be at 2 kGy. No difference was found to exist between the above species in susceptibility to irradiation in diluted sera (50%) and 10% in Eagle MEM). Sensibility of wild mycoplasma strains was found to be identical with that of laboratory strains. Hence, 60Co gamma irradiation of sera appears to be a safe method by which to make sera mycoplasma-free. Bacillus subtilis in calf serum was inactivated by doses above 18 kGy, with the critical dose being 15 kGy.

[Decontamination of continual cell lines spontaneously infected with mycoplasmas]. / Dekontaminace kontinuálních bunecných linií spontánne infikovaných mykoplazmaty.

The continual cell lines of bovine kidneys MDBK and AUBEK, and porcine kidneys RPD and IBRS, spontaneously infected with Mycoplasma arginini and Acholeplasma laidlawii, were decontaminated by the method of selective elimination. Two elimination procedures were modified to be used for the decontamination: one based on the reduction of infection by the light treatment of the cultures, the other based on the selection of mycoplasma-free cell population through cell clonation. On the basis of a long-continued control of the cell clones a methodical procedure of the preparation of mycoplasma-free cell lines was worked out.

The photosensitizing activity of haematoporphyrin on mollicutes.

The photosensitizing activity of haematoporphyrin (HP) on Mycoplasma hominis and Acholeplasma laidlawii was studied as a function of the phase of growth and the amount of sterols in the cell membrane. Less HP was bound to cells when the membrane had a high sterol content. Both strains in the exponential but not in the stationary phase of growth were sensitive to HP treatment (above 1 microgram ml-1) in the dark. Visible light irradiation of HP-loaded cells caused in all cases a decrease of cell survival, with concomitant changes in the pattern of membrane proteins that suggested protein-protein cross-linking, and the appearance of ultrastructural alterations (rounded and lysed cells); the photosensitivity was indirectly related to the sterol content of the cell membrane. On the whole, our findings suggest that the cell membrane is a major target for HP photosensitization of mycoplasma cells.

Eliminating mycoplasmas from contaminated cell cultures.

Mycoplasmas are bothersome contaminants in cell cultures. Cells derived from various sources are subject to contamination and although many procedures have been suggested to eliminate the contaminating mycoplasmas, there is no simple, rapid and effective method for eliminating them. We have devised a method for the selective killing of mycoplasmas based on the differences between the nucleic acid metabolism of mycoplasmas and that of tissue culture cells. Whereas the nutritional requirements of mycoplasmas for nucleic acid precursors can be met by purine and pyrimidine bases, mammalian cells do not incorporate the free bases. The approach was to selectively incorporate the free base analogue 5-bromouracil (5-BrUra) into the mycoplasmas followed by photosensitization of the DNA containing 5-BrUra by low concentrations of the fluorochrome 33258-Hoechst. Such treatment renders the mycoplasma DNA very susceptible to breaks induced by visible light. The unusually high A + T content in mycoplasma DNA makes these organisms suitable candidates for the induction of breakage by the combined action of 5-BrUra, 33258-Hoechst and light because 33258-Hoechst has a high affinity for A + T base pairs and an even higher affinity for A-BrUra base pairs. We report that Mycoplasma hyorhinis and Acholeplasma laidlawii contaminating Chinese hamster cells were selectively killed by our technique, and cured clones of cells were easily obtained from the treated cell cultures. The technique proved efficient in curing BHK-21 and RAG cells from unknown contaminating mycoplasmas suggesting that this technique could be generally applied to eliminate contaminating mycoplasma strains from mammalian cell culture.

UV survival of human mycoplasmas: evidence of dark reactivation in Mycoplasma buccale.

The inactivation by ultraviolet (UV) light irradiation of mycoplasma cells of five human strains was monitored by investigating the colony-forming ability. The survival curves of five strains tested indicated that the cells of Mycoplasma buccale only are single and homogenously susceptible to UV light. The effect of the repair inhibitor, caffeine, on the colony-forming ability of UV-irradiated cells was investigated with M. buccale because of its homogenous susceptibility to UV light. The colony formation of irradiated cells was markedly depressed by post-irradiation treatment with caffeine at concentrations that had little or no effect on the colony formation of unirradiated cells. The colony-forming units (CFU) of UV-irradiated cells which were kept in broth without caffeine in the dark increased without a lag as the time in the dark increased. The colony-forming ability of the irradiated cells completely recovered after 3 hr in the dark. However, when irradiated cells were kept in the presence of caffeine, no increase in their CFU was observed. The mode of action of caffeine on UV-irradiated cells closely resembles that described for other organisms which possess dark reactivation systems for UV-induced damage in deoxyribonucleic acid (DNA). Thus, the results obtained provide evidence for the existence of a dark repair function in M. buccale.

T-mycoplasmas: Growth patterns and physical characteristics of some human strains.

Five T-mycoplasmas isolated from patients with nonspecific urethritis and five laboratory strains of T-mycoplasma were examined for differentiating biological properties. The strains differed by the presence or absence of a lag phase and the number of T-mycoplasmas constituting a colony-forming unit (cfu). Most T-mycoplasmas had no lag phase and a cfu consisting of single organisms. All had biphasic ultraviolet inactivation curves typical of suspensions containing both mononucleate and binucleate cells. Binucleate cells probably were in the process of division. They resisted sonication for 3 min. Sonication disrupted multicellular cfu in to single cells within 2 min. Stationary-phase organisms died more rapidly than exponential-phase cells. T-mycoplasmas replicated at 2 C; they grew more slowly in T-broth at 40 C and died in 2.5 min at 56 C. Inactivation curves at 45 C and 50 C differed but insufficiently to permit identification of individual strains. At pressures less than 5 psi, single-cell suspensions passed through filter membranes with 0.65-mum and 0.45-mum pores but were retained by membranes with 0.22-mum pores.

Drying and irradiation of calf and horse serum. I. Influence on the growth of cell cultures and mycoplasmas.

Gamma-irradiation of liquid and dried calf sera with 2.5 Mrads did not affect their capacity to promote the growth of chick embryo, L cell and human embryonic lung cell cultures. Drying and gamma-irradiation of horsesera did not affect their capacity to support the growth of 3 mycoplasma of the species Acholeplasma laidawii and Mycoplasma bovigenitalium.

Drying and irradiation of calf and horse serum. II. Influence on mycoplasma content of the calf sera.

Drying of calf sera produced only a negligible change in their content of Acholeplasma laidlawii organisms and reduced the content of Mycoplasma arginini organisms by 2 to 4 logs. Gamma-irradiation of liquid and dried calf sera killed M. arginini and A. laidlawii organisms at irradiation levels of 0.2-0.3 and 0.4-0.6 Mrads, respectively.