An application of multivariate ranks to assess effects from combining factors: metal exposures and semen analysis outcomes.

In studies of environmental effects on human health outcomes, it is often difficult to assess the effects of a group of exposure variables when the individual exposures do not appear to have statistically significant effects. To address this situation, we propose a method of U-scores applied to subsets of multivariate data. We illustrate the usefulness of this approach by applying it to data collected as part of a study on the effects of metal exposure on human semen parameters. In this analysis, profiles (pairs) of metals containing copper and/or manganese were negatively correlated with total motile sperm and profiles containing copper were negatively correlated with sperm morphology; profiles containing selenium and chromium were positively correlated with total motile sperm.

Systemic polyomavirus genome increase and dissemination of capsid-defective genomes in mammary gland tumor-bearing mice.

BALB/c mice that developed tumors 7 to 8 months following neonatal infection by polyomavirus (PYV) wild-type strain A2 were characterized with respect to the abundance and integrity of the viral genome in the tumors and in 12 nontumorous organs. These patterns were compared to those found in tumor-free mice infected in parallel. Six mice were analyzed in detail including four sibling females with mammary gland tumors. In four of five mammary gland tumors, the viral genome had undergone a unique deletion and/or rearrangement. Three tumor-resident genomes with an apparently intact large T coding region were present in abundant levels in an unintegrated state. Two of these had undergone deletions and rearrangements involving the capsid genes and therefore lacked the capacity to produce live virus. In the comparative organ survey, the tumors harboring replication-competent genomes contained by far the highest levels of genomes of any tissue. However, the levels of PYV genomes in other organs were elevated by up to 1 to 2 orders of magnitude compared to those detected in the same organs of tumor-free mice. The genomes found in the nontumorous organs had the same rearrangements as the genomes residing in the tumors. The original wild-type genome was detected at low levels in a few organs, particularly in the kidneys. The data indicate that a systemic increase in the level of viral genomes occurred in conjunction with the induction of tumors by PYV. The results suggest two novel hypotheses: (i) that genomes may spread from the tumors to the usual PYV target tissues and (ii) that this dissemination may take place in the absence of capsids, providing an important path for a virus to escape from the immune response. This situation may offer a useful model for the spread of HPV accompanying HPV-induced oncogenesis.

Oncogenesis of mammary glands, skin, and bones by polyomavirus correlates with viral persistence and prolonged genome replication potential.

A correlation between polyomavirus-induced oncogenesis and viral persistence on the one hand and/or prolonged genome replication potential on the other was established with respect to their respective organ distributions. Prolonged replication potential is defined as the capacity of a genome to replicate in a given organ from the time of infection up to the onset of oncogenesis. This conclusion was derived following intraperitoneal infection of BALB/c mice with wild-type strain A2. Viral genomes were used as parameters of persistence and replication and were detected by Southern blotting and PCR analysis. The major tumor target organs (mammary gland, skin, and bone), which have not been previously analyzed for persistence, were compared with other, non-tumor-prone organs (kidney, liver, lung, spleen, and salivary gland). A progressive loss of viral genomes was observed in all tissues as a function of time postinfection; however, genomes were shown to persist through 20 weeks postinfection in the mammary glands, skin, and bones to an extent similar to that in the previously described kidneys (D. J. McCance, J. Virol. 39:958-962, 1981; W. P. Rowe, J. W. Hartley, J. D. Estes, and R. J. Huebner, Natl. Cancer Inst. Monogr. 4:189-209, 1960). Thus, tumors arise among organs that sustain a persistent infection, but not all such organs develop tumors (e.g., the kidney). The capacity of organs to support de novo replication at various ages, including the age reached when the first tumors are detected, was also determined using a 3-day infection period for ages between 0 and 7 weeks. For all organs tested, a higher level of genomes was observed in organs of mice infected as neonates than in those infected after the age of 3 weeks. However, marked organ-specific differences were seen in the degree and timing of loss of replication. In particular, viral genome replication, although reduced, was maintained in the mammary glands, skin, and bones of adult animals, in contrast to the kidneys. We conclude that organ-specific oncogenesis correlates with two organ-specific parameters: persistence of viral genomes and prolonged viral genome replication potential. This may reflect a requirement for continued viral genome replication and/or gene expression for tumorigenesis. In turn, these parameters may be linked to the tissue-specific continued capacity for cellular division.

Characterization of the mammary hyperplasia, dysplasia and neoplasia induced in athymic female adult mice by polyomavirus.

We have characterized mammary oncogenesis induced after polyomavirus infection of adult female nude mice regarding histopathogenesis, viral replication and viral and cellular oncogene expression. A unique transient generalized epithelial hyperplasia was observed (starting at 2 weeks post infection), preceding the development of dysplasias (onset 6 weeks post infection) and multiple neoplasias (onset 6 weeks post infection) in all glands. The ductal epithelium was the target for neoplastic transformation, and the occurrence of numerous ductal dysplasias coincided with the appearance of frank tumors. Stromal abnormalities were also seen. Tumor growth was not dependent upon ovarian hormones, and new tumors continued to develop in ovariectomized mice. Viral replication, high although variable, preceded but did not correlate with oncogenesis. Most but not all tumors contained high levels of unintegrated viral DNA. Tumors produced very low levels of live virus. Viral gene expression was markedly increased in the tumors compared with the infected but morphologically normal glands. The expression of c-myc was moderately increased (fourfold); changes in c-int-2 and c-Ha-ras expression were slight and inconsistent, while expression of c-neu and c-int-1 was unchanged.

Organ- and age-specific replication of polyomavirus in mice.

A novel organ- and age-specific pattern of polyomavirus DNA replication in mice is described. Two broadly defined classes of response to polyomavirus infection were observed: class I organs (mammary gland, bone, and skin) responded with high levels of replication in neonate mice and moderate levels in adults; class II organs (kidney, liver, and lung) responded with high levels in neonates and very low levels in adults. Thus, aging affected replication in all organs, and organ specificity was superimposed on this age-related decrease. We argue that the organ- and age-specific pattern likely reflects in part the activities of a multiplicity of general or tissue-specific, age-dependent transcription factors, which modulate viral replication or viral transcription or both. Interestingly, the majority of tumors in mice infected as neonates or as immunoincompetent adults originate in class I organs, suggesting that the ability to replicate in adult tissues is an important factor controlling polyomavirus oncogenesis. From the analysis of the infection process in adult mammary glands, a novel mode of polyomavirus infection emerged which contrasts with that derived from observations of tissue culture systems. A nonproductive infection was seen, characterized by very low levels of live virus (in the range of 10(-4) PFU per cell) and maintenance of the viral genome in an unintegrated, moderately replicating state. Maintenance of the viral genome was accomplished without integration into host cell DNA in all three tumor-prone organs, both prior to as well as beyond oncogenesis.

Biochemical evidence for the presence of arginine decarboxylase activity in Trypanosoma cruzi.

Trypanosoma cruzi was found to release 14CO2 from radiolabeled arginine, and this effect was inhibited by either DL-alpha-difluoromethylarginine or monofluoromethylagmatine, both specific inhibitors of arginine decarboxylase (ADC). Furthermore, agmatine, which can be derived metabolically only by ADC-mediated arginine decarboxylation, was produced when T. cruzi was incubated with radiolabeled arginine, and agmatine production was inhibited in the presence of DL-alpha-difluoromethylarginine. These results constitute direct biochemical evidence for the presence in T. cruzi of ADC, an enzyme that does not occur in mammalian cells.

Immunological elimination of infected cells as the candidate mechanism for tumor protection in polyomavirus-infected mice.

The uniformly lethal development of mammary tumors in polyomavirus-infected adult female nude mice was prevented by adoptive cell transfer of polyomavirus-immune splenocytes or peritoneal cells. Transferred immune cells also lowered the growth rate of emerging tumors. The induction of other relatively less frequent tumors of the skin and bone was decreased as well. Using in situ hybridization of whole-body sections as well as hybridization of nucleic acids from the mammary glands, we show for the first time that transferred immune cells, but not normal cells, virtually eliminated virus signal in the whole mouse and in the mammary glands. Since infected and tumorous mammary glands produce very little infectious virus, it appears that a major mechanism mediating the prevention of polyomavirus oncogenesis involves the immunological elimination of nonproductively and persistently infected cells.

Binding of the specific ligand to Fc receptors on Trypanosoma cruzi increases the infective capacity of the parasite.

The infective capacity of Trypanosoma cruzi was significantly increased after treatment with monoclonal IgG1 antibodies, whether or not specific for the parasite; minimal or no change in infectivity was seen after treatment with IgG2a, IgG2b or IgG3 monoclonal antibodies. The stimulatory effect was evidenced by elevated numbers of trypanosomes invading mammalian host cells in vitro compared to parasites treated with medium alone. Greater infectivity was also induced by pure human Fc, suggesting a role for Fc receptors on the organism. This inference received support in the fact that protein A inhibited the stimulatory effect of Fc. In addition, Fc-treated parasites incubated with fluorescein-labelled F(ab')2 from goat anti-human IgG exhibited fluorescence detectable by both ultraviolet microscopy and flow cytometry. 125I-Fc binding to T. cruzi was found to be saturable at 0 degrees and was inhibited by cold Fc but not by bovine serum albumin (BSA) or orosomucoid. Interestingly, 125I-Fc binding was greater at 37 degrees and it was not saturable with the concentrations that did saturate at 0 degrees. Possibly, Fc might up-regulate expression of its own receptor and greater endocytosis could take place at 37 degrees. Significant increases in infectivity were detectable after a 40 min pretreatment with Fc--hinting that Fc could trigger a chain of biochemical events underlying the phenomenon--and were reversible, becoming undetectable 2 hr after Fc removal. The average number of Fc receptors per parasite, determined at 0 degrees (at which binding saturation was possible), was estimated as 5 x 10(5), the dissociation constant was of the order of 10(-6)-10(7)M. The present results define an important biological role for an Fc-binding T. cruzi surface component and expose the capacity of this organism to exploit even elements of the immune system in its quest to attain intracellular localization, required for multiplication.

Possible roles for zinc in destruction of Trypanosoma cruzi by toxic oxygen metabolites produced by mononuclear phagocytes.

The effects of a single nutrient deficiency on immune function is now most extensively characterized using the dietary zinc deficient murine model. Deficiencies in zinc have rapid adverse effects on host defenses of humans and rodents. This impaired defense seems to be, in part, the result of a reduction in number of lymphocytes available for surveillance since residual lymphocytes are able to carry out many normal functions. In vitro, the lymphocytes were able to proliferate at a normal rate as well as produce antibodies or interleukin 2 in response to mitogens or antigens even when cultured in autologous serum to reduce the possibility of restoration of zinc deficient functions. Conversely, mononuclear phagocytes (MNP) from deficient mice had a significantly reduced capacity to associate with and kill the parasite Trypanosoma cruzi (T. cruzi) which causes Chaga's disease. Moreover, indicating the specificity of the deficient function, a short incubation of ZnCl2 but not other metals completely restored the capacity of MNP from deficient mice to take up and kill T. cruzi. Dependency on H2O2 production by the MNP's oxygen burst for killing of T. cruzi.suggested that MNP from zinc deficient mice might produce smaller amounts of H2O2. The possibility that zinc might play an integral role in the oxygen burst seemed evident from the ability of zinc to quickly restore the killing capacity of MNP from the zinc deficient mice. Further, the renewed interest in the role of metals in the production of highly reactive oxidants in biological systems prompted a literature search to identify enzymes and/or reactions known to be involved in the generation of oxygen radicals or toxic oxygen metabolites that might be zinc dependent. The literature review provided herein indicates many possible roles for zinc in the generation of toxic oxygen species. The data indicated that normal levels of H2O2 are produced by MNP from zinc deficient mice. The amount of H2O2/mg macrophage protein is normal in response to phorbol or opsonized zymosan but reduced in response to direct stimulation by T. cruzi. However, the reduced H2O2 production by T. cruzi-stimulated zinc deficient MNP was due to reduced stimulation as a result of fewer T. cruzi associated with the MNP. Thus, H2O2 levels/parasite were the same as zinc adequate controls. Yet, this does not preclude the possibility that reduced killing of T. cruzi by MNP from zinc deficient mice may be due to a function for zinc in the actual killing process or in the production of some other agent important in the killing of T. cruzi.

Zinc requirement for macrophage function: effect of zinc deficiency on uptake and killing of a protozoan parasite.

The effects of suboptimal levels of zinc, an essential trace element, on the ability of murine macrophages to associate with and destroy a pathogenic parasite, Trypanosoma cruzi, were evaluated. Young adult A/J mice were fed zinc-deficient, zinc-adequate or restricted amounts of a zinc-adequate diet for 28 days. On the basis of weight loss and parakeratosis, the zinc-deficient mice were further divided into moderately and severely zinc-deficient on Day 28. Both the percentage of mouse peritoneal macrophages (MPM) with associated parasites and the number of parasites per 100 macrophages were significantly lower for macrophages from moderately and severely deficient mice compared to MPM from mice fed restricted or zinc-adequate diet. Furthermore, MPM from both zinc-deficient groups of mice killed fewer internalized parasites than did MPM from restricted or zinc-adequate mice. Pretreatment of MPM from zinc-deficient mice with 5 micrograms zinc/ml for 30 min completely restored both their capacity to take up and kill the parasites. Other trace metals tested, including copper, manganese and nickel, failed to reverse the effects of zinc deficiency. These results point to an important role for zinc in the biochemical events associated with macrophage uptake and killing of the parasite.

Effects of IL-4 on macrophage functions: increased uptake and killing of a protozoan parasite (Trypanosoma cruzi).

We studied whether interleukin-4 (IL-4) could modulate two macrophage functions relevant to their microbicidal activity (uptake and killing), using non-invasive [amastigote (AMA)] forms of the protozoan parasite Trypanosoma cruzi. Treatment of cultures of mouse resident peritoneal macrophages (MPM) with the supernatant of cultures of cells transfected with IL-4 cDNA increased both the capacity of the MPM to take up the organisms and the rate of intracellular killing with respect to MPM mock-treated with medium alone. The presence in the medium of a monoclonal antibody specific for IL-4 during MPM treatment inhibited both effects, pointing to recombinant IL-4 (rIL-4) as the active principle in the supernatant. Kinetic studies revealed that at least a 24-hr pretreatment of the MPM with the rIL-4-containing supernatant was required for these effects to be produced. The rate of intracellular parasite killing was also significantly increased when the rIL-4 treatment was applied after AMA ingestion by MPM. This result confirmed that MPM could be activated by rIL-4 for greater intracellular killing and showed that this enhancement was not necessarily dependent on the initial rIL-4-mediated increase in parasite load. The use of scavengers of reactive oxygen reduction intermediates indicated that hydrogen peroxide, superoxide anion and singlet oxygen, but apparently not hydroxyl radicals, were involved in parasite killing modulated by rIL-4. These results document for the first time the capacity of IL-4 to enhance the microbicidal activity of macrophages and suggest that this lymphokine might play a role in host defence against T. cruzi infection.

Recombinant tumor necrosis factor enhances macrophage destruction of Trypanosoma cruzi in the presence of bacterial endotoxin.

We examined in this work whether rTNF inhibits the capacity of Trypanosoma cruzi to multiply within murine macrophages or enhances the ability of the phagocytic host cells to destroy internalized parasites. We found that rTNF would not alter the fate of the trypanosomes within macrophages over a 48-h incubation period unless the latter cells were also treated with 1 ng/ml bacterial endotoxin (LPS). Treatment of macrophages with rTNF plus LPS, but not separate treatment with either rTNF or LPS, resulted in a significant decrease in the number of organisms per 100 macrophages with respect to values obtained with mock-treated macrophages. In addition, there was a significant reduction in the proportion of infected macrophages over the 48-h incubation period, indicating parasite clearance by the host cells. The combined effects of rTNF and LPS were seen when macrophages from CBA/J were used but not with LPS-insensitive macrophages from C3H/HeJ mice. Increased trypanosome killing by CBA/J macrophages treated with rTNF plus LPS was not seen when catalase was present in the culture medium, indicating a role for hydrogen peroxide in the cytotoxic effect. These results show that rTNF can affect the fate of T. cruzi within macrophages if LPS is present and point to destruction of internalized organisms rather than inhibition of parasite multiplication as the most likely explanation.

Impairment of macrophage function by inhibitors of ornithine decarboxylase activity.

The effects of irreversible inhibition of ornithine decarboxylase on the capacity of murine macrophages to take up a protozoan organism (Trypanosoma cruzi) or inert particles were investigated. Incubation of macrophage cultures with four different ornithine decarboxylase inhibitors, namely, DL-alpha-difluoromethylornithine (DFMO, 0.5 to 20 mM), delta-methyl-acetylenic putrescine (1 to 5 mM), monofluoromethyldehydroornithine ethyl ester (1 to 5 mM), and monofluoromethyldehydroornithine methyl ester (1 to 5 mM), before the addition of the parasites significantly reduced the percentage of macrophages with parasites, indicating that some of the host cells were no longer capable of binding or ingesting the parasite. The average number of trypanosomes per 100 macrophages was also diminished, denoting a lesser phagocytic capacity as a consequence of the treatments. These effects were reversible within 2 h after removal of excess DFMO. No alteration in parasite-macrophage interaction was seen when the trypanosomes were treated with DFMO. That the effects of DFMO on the macrophages probably resulted from a reduction in polyamine levels caused by inhibition of ornithine decarboxylase was indicated by the fact that these effects were not seen when the macrophages were incubated with DFMO in the presence of putrescine, the product of ornithine decarboxylation by ornithine decarboxylase. DFMO treatment of macrophages also inhibited the capacity of these cells to ingest killed parasites or latex beads and thus appeared to generally affect phagocytosis. An effect of DFMO on the susceptibility of macrophages to penetration by the parasites seemed less likely because no significant alteration in cell-parasite association occurred when myoblasts--which, not being phagocytic, can be infected only by membrane penetration--were treated with DFMO. Taken together, these results emphasize a role of ornithine decarboxylase activity and polyamine biosynthesis in macrophage function.

Arginine decarboxylase inhibitors reduce the capacity of Trypanosoma cruzi to infect and multiply in mammalian host cells.

The capacity of blood (trypomastigote) forms of Trypanosoma cruzi to infect mouse peritoneal macrophages or rat heart myoblasts in vitro was inhibited by treatment of the trypomastigotes with DL-alpha-difluoromethylarginine (F2Me Arg), monofluoromethylagmatine, or (E)-alpha-monofluoromethyl-3-4-dehydroarginine--all irreversible inhibitors of arginine decarboxylase. Similar results were obtained when F2MeArg-treated parasites were incubated with rat heart myoblasts. The inhibitory effects were characterized by marked reductions in both the proportion of infected cells and the number of parasites per 100 host cells. The concentrations of the arginine decarboxylase inhibitors that affected infectivity had no detectable effect on either the concentration or motility of the parasite and, therefore, could not have affected the collision frequency. F2MeArg appeared to inhibit the ability of T. cruzi to penetrate the host cells since the drug had no significant effect on the extent of parasite binding to the surface of the host cells. The inhibitory effect of F2MeArg was markedly reduced or abrogated in the presence of either agmatine or putrescine, as would have been expected if F2MeArg acted by inhibiting arginine decarboxylase. Addition of F2MeArg to macrophage or myoblast cultures immediately after infection or at a time when virtually all of the intracellular parasites had transformed into the multiplicative amastigote form, resulted in a markedly reduced parasite growth rate. This effect was also prevented by exogenous agmatine. These results indicate the importance of polyamines and polyamine biosynthesis in the following two important functions of T. cruzi: invasion of host cells and intracellular multiplication. Furthermore, concentrations of the inhibitors tested that affected the parasite did not alter the viability of the host cells, the cellular density of the cultures, or the ability of uninfected myoblasts to grow. Thus, arginine decarboxylase inhibitors may have a potential application in chemotherapy against T. cruzi infection.

Stimulatory effects of retinoic acid on macrophage interaction with blood forms of Trypanosoma cruzi: involvement of transglutaminase activity.

The effects of retinoic acid (RA; vitamin A acid) on macrophage function were investigated by measuring the capacities of mouse peritoneal macrophages to associate with (i.e., bind and internalize) and kill the unicellular parasite Trypanosoma cruzi. The presence of 10(-8) to 10(-6) M RA in co-cultures of macrophages and blood forms of the parasite markedly increased their interaction as evidenced by significant increases in both the percentage of phagocytes associating with parasites and the average number of parasites per 100 cells. A similar effect was produced when either the macrophages or the trypanosomes were pretreated with RA, suggesting that both cell types could contribute to the noted effect. Although RA might have enhanced parasite-macrophage association by binding to both, its ability to stimulate phagocytosis was independently evidenced by a significant increase in the uptake of latex particles. RA-treated macrophages also took up larger numbers of dead T cruzi, denoting that parasite viability (i.e., infectivity) was not necessary for the production of the RA effect. The minimum pretreatment time for RA to significantly stimulate macrophage association with T. cruzi was 30 min, although a 45-min pretreatment was necessary for a maximal effect to be seen under our experimental conditions. The RA effect was reversible because, once optimally induced, it remained demonstrable for only 30 to 60 min after removal of the reagent; however, the effect persisted for at least 3 hr if RA was not removed. Transglutaminase activity appeared to be involved in the RA effect, because the latter was abrogated when the macrophages were treated with RA in the presence of cystamine, methylamine, or monodansylcadaverine, all of which inhibit transglutaminase activity by different mechanisms. RA also increased the capacity of macrophages to kill parasites internalized before the treatment. This cytotoxic capacity was inhibited by catalase, indicating that H2O2 played a role in the killing mechanism. RA treatment significantly increased the proportion of macrophages capable of reducing nitroblue tetrazolium. The present results indicated that RA was capable of activating macrophages, leading to greater uptake and killing of a protozoan parasite.