Claudia R. Libertin

Effects of gamma rays, ultraviolet radiation, sunlight, microwaves and electromagnetic fields on gene expression mediated by human immunodeficiency virus promoter.

Previous work by our group and others has shown the modulation of human immunodeficiency virus (HIV) promoter or long terminal repeat (LTR) after exposure to neutrons and ultraviolet radiations. Using HeLa cells stably transfected with a construct containing the chloramphenicol acetyl transferase (CAT) gene, the transcription of which is mediated by the HIV-LTR, we designed experiments to examine the effects of exposure to different types of radiation (such as gamma rays, ultraviolet and sunlight irradiations, electromagnetic fields and microwaves) on HIV-LTR-driven expression of CAT. These results demonstrated ultraviolet-light-induced transcription from the HIV promoter, as has been shown by others. Exposure to other DNA-damaging agents such as gamma rays and sunlight (with limited exposures) had no significant effect on transcription mediated by HIV-LTR, suggesting that induction of HIV is not mediated by just any type of DNA damage but rather may require specific types of DNA damage. Microwaves did not cause cell killing when cells in culture were exposed in high volumes of medium, and the same cells showed no changes in expression. When microwave exposure was carried out in low volumes of medium (so that excessive heat was generated) induction of HIV-LTR transcription (as assayed by CAT activity) was evident. Electromagnetic field exposures had no effect on expression of HIV-LTR. These results demonstrate that not all types of radiation and not all DNA-damaging agents are capable of inducing HIV. We hypothesize that induction of HIV transcription may be mediated by several different signals after exposure to radiation.

Osteomyelitis Caused by Rhodococcus equi in a Renal Transplant Recipient

SummaryWe report the first case of osteomyelitis due toRhodococcus equi, which occurred in a renal transplant patient. Infection with this organism is rare and usually causes a distinct clinical syndrome resembling pulmonary tuberculosis. We investigated by time-kill curve analysis various antimicrobial combinations forin vitro efficacy. The literature is briefly reviewed, and aspects of diagnosis and therapy are discussed.ZusammenfassungWir berichten über den ersten Fall vonRhodococcus equi-Osteomyelitis, der bei einem Nierentransplantatempfänger auftrat. Infektionen mit diesem Erreger sind selten, meistens verursacht er ein klinisches Syndrom, das der Lungentuberkulose ähnelt. DieIn-vitro-Wirksamkeit verschiedener Kombinationen antimikrobieller Substanzen wurde durch Analyse von Abtötungskurven bestimmt. Es wird eine kurze Literaturübersicht gegeben und diagnostische und therapeutische Fragen werden diskutiert.

The hemolysin/bacteriocin produced by enterococci is a marker of pathogenicity

The hemolysin/bacteriocin produced by some strains of Enterococcus faecalis is active in the lysis of human, rabbit, and horse erythrocytes, but not those from sheep. In this study, we determined that 20% of clinical enterococcal isolates tested in the clinical microbiology laboratory produced hemolysin and that pathogenic human E. faecalis were more likely to be hemolysin-producing isolates. Among the organisms isolated from different anatomic sites, variability in the degree of hemolysin production existed. We used an isogenic pair of E. faecalis organisms to demonstrate that hemolysin production was due to a hemolysin/bacteriocin determinant transmissible by a plasmid and was not strain dependent. This determinant may be linked to antibiotic resistance genes in some instances. Also, the erythrocyte lysis occurred only when hemolysin was in the presence of E. faecalis organisms, suggesting a bacterial cell dependency for activity of the hemolysin.

Low doses of neutrons induce changes in gene expression.

Studies were designed to identify genes induced in fibroblasts after exposure to low-dose neutron radiation but not after gamma rays. Our past work had shown similar modulation of transcripts for alpha-tubulin, beta- and gamma-actins, ornithine decarboxylase and interleukin 1 after exposure to either neutrons or gamma rays. However, differences in the expression of beta-protein kinase C and c-fos genes were observed, with both being induced after exposure to gamma rays but not neutrons. Recently we have identified two genes that are induced after exposure to neutrons but not gamma rays: Rp-8 (a gene associated with apoptosis) and the long terminal repeat (LTR) of the human immunodeficiency virus (HIV). Induction of Rp-8 mRNA was demonstrated in Syrian hamster embryo (SHE) fibroblasts and was found to be induced in cells exposed to neutrons administered at low (0.005 Gy/min) and high dose rate (0.12 Gy/min). No induction of other genes associated with apoptosis such as Rp-2, bcl-2 and Tcl-30 was observed. The induction of transcription from the LTR of HIV was demonstrated in HeLa cells bearing a transfected construct of the chloramphenicol acetyl transferase (CAT) gene driven by the HIV-LTR promoter. Measurements of CAT activity and CAT transcripts after irradiation demonstrated an unresponsiveness to gamma rays over a broad range of doses (0.1-3 Gy). Twofold induction of the HIV-LTR was detected after exposure to neutrons (0.48 Gy) administered at low (0.05 Gy/min) but not high (0.12 Gy/min) dose rates. Ultraviolet-mediated HIV-LTR induction, however, was inhibited by exposure to low-dose-rate neutron irradiation. These results are interesting in light of reports that Rp-8 is induced during apoptosis and that HIV causes apoptosis.

Immunomodulatory effect of β‐carotene on T lymphocyte subsets in patients with resected colonic polyps and cancer

Results from a number of studies suggest that beta-carotene-containing foods prevent the initiation or progression of various cancers. One possible mechanism for this effect could be enhancement of the immune response. The aim of this study was to determine whether beta-carotene modulates T lymphocyte subsets in patients affected with colonic polyps or cancerous lesions. Patients with previous adenomatous colonic polyps (n = 18) or colon cancers (n = 19) were randomized to receive placebo or beta-carotene (30 mg/day) for three months. Percentages of T lymphocyte subsets were determined using flow cytometry in blood samples collected before randomization and at three months. T lymphocyte subsets of 14 normal control subjects were also determined for comparison. Initially, there was no difference in total leukocyte counts, percentage of lymphocytes, and various subsets of lymphocytes among the three groups, although in cancer patients there was a lower percentage of CD4 and interleukin-2 (IL-2) receptor-positive (IL-2R+) cells than in patients with polyps and in controls. After supplementation with beta-carotene, a significant increase in IL-2R+ T lymphocytes (from 12.7 +/- 3.0% to 26.0 +/- 1.9%) and CD4+ lymphocytes (from 40.9 +/- 3.1% to 45.6 +/- 3.2%) was seen only in the cancer patients. These percentages remained unchanged in patients with adenomatous polyps receiving placebo or beta-carotene. We concluded that beta-carotene increased the number of IL-2R+ T lymphocytes and CD4+ lymphocytes, which in turn may produce IL-2 only in patients with cancer who may already have some deficiency in their immune system. This increase in activated T lymphocytes may mediate cytotoxic reactions to cancer cells via cytokine production.

Candida lusitaniae—An opportunistic pathogen

Candida lusitaniae is a newly recognized opportunistic pathogen amongst immunocompromised patients. The epidemiology and pathology remain largely unknown. This case report describes a 27-year-old female with vasculitis who acquired a fatal C. lusitaniae fungemia.

THE EFFECTS OF MULTIPLE UV EXPOSURES ON HIV‐LTR EXPRESSION

Abstract— Previous studies have shown that cellular stress agents such as UV radiation induce transcription from the long terminal repeat (LTR) of the human immunodeficiency virus (HIV). Using HeLa cells stably transfected with the HIV‐LTR sequence, which transcriptionally drives the chloramphenicol acetyl transferase (CAT) reporter gene, we examined the effects of multiple exposures to UVC (254 nm) on HIV‐LTR‐CAT expression. Low doses (± 5 J m‐2) had no effect on CAT expression, but up to 29‐fold induction was observed with 10 J m‐2 when cells were harvested 48 h after completion of the exposure. Little difference was noted in induction levels when cells were exposed to one 25 J m‐2 dose, viable cells were harvested at 24 h, 48 h or 72 h, and cell lysates were assayed for CAT expression. Two sequential 12.5 J m‐2 exposures, given 24 h apart, resulted in an additive effect on CAT expression; these two exposures produced CAT activity equivalent to that induced following a single 25 J m‐2 dose. This additive effect was not evident at the lower doses (≤5 J m‐2) or at the higher doses. Maximal induction was observed using doses from 25 to 37.5 J m‐2. Multiple exposures with either the low (≤5 J m ‐2) or high doses (>25 J m ‐2) did not result in an additive effect. Our data suggest that HIV‐LTR requires a specific threshold UV dose in order to elicit induction; a maximal induction dose is also evident; exposures higher than this maximal dose contribute no more to HIV‐LTR induction in viable cells.

Expression of enhanced spontaneous and gamma-ray-induced apoptosis by lymphocytes of the wasted mouse

Mice bearing the autosomal recessive mutation wasted (wst/wst) display a disease pattern including increased sensitivity of lymphocytes to ionizing radiation, neurologic dysfunction, and immunodeficiency. Many of the features of this mouse model have suggested a premature or increased spontaneous frequency of apoptosis in thymocytes. Past work has documented an inability to establish cultured T cell lines, and abnormally high death rate of stimulated T cells in culture, and an increased sensitivity of T cells to the killing effects of ionizing radiations in the wst/wst mouse relative to controls. The experiments reported here were designed to examine splenic and thymic lymphocytes from the wasted and control mouse for signs of early apoptosis. Our results revealed enhanced expression of Rp-8 mRNA (which has been associated with apoptosis) in thymic lymphocytes and to a lesser extent in spinal cord in the wst/wst mouse relative to controls; expression of Rp-2 and Tcl-30 mRNA (also reported to be induced during apoptosis) were not detectable in spleen or thymus. Expression of Rp-2, Rp-8, and Tcl-30 mRNA in other affected tissues of the wasted mouse (brain and liver) were similar in the wasted mouse and controls. Thymus and spleen from the wasted mouse have reduced numbers of viable cells relative to controls. Higher spontaneous DNA fragmentation was observed in lymphocytes from the wasted mouse than in controls; however, gamma-ray-induced DNA fragmentation peaked at a lower dose and occurred to a greater extent in lymphocytes derived from the wasted mouse relative to controls. These results suggest that high spontaneous and gamma-ray-induced apoptosis in T cells of the wasted mouse may contribute to the mechanism underlying the observed lymphocyte and DNA repair abnormalities.

Time kill curve analysis of vancomycin and rifampin alone and in combination against nine strains of nutritionally deficient streptococci

Endocarditis caused by nutritionally deficient streptococci has a high bacteriologic and clinical failure rate, despite appropriate antimicrobial therapy. We investigated by time kill curve methodology nine clinical endocarditis isolates of nutritionally deficient streptococci to determine the in vitro efficacies of vancomycin and rifampin alone and in combination. The combination of vancomycin and rifampin demonstrated synergy and bactericidal activity in five of the strains. In one strain, this combination inhibited growth by greater than 2 log10 CFU/ml when compared to the growth control or either antibiotic alone, but it failed to be bactericidal. Indifference, defined as less than or equal to 2 log10 CFU per milliliter increase in killing of the combination compared to the next most active single agent, was demonstrated with the remaining three isolates. Changing the antibiotic concentrations in the time kill curve studies for these latter strains failed to demonstrate synergistic activity of the antibiotic combination. The vancomycin and rifampin combination may be a promising therapeutic modality for which in vivo correlation is indicated.

Cytokine and T-cell subset abnormalities in immunodeficient wasted mice

Wasted mice bear an autosomal recessive mutation (wst/wst) that manifests itself in neurologic abnormalities, immunologic deficiency, and faulty DNA repair evident by 21 days of age. The immunodeficiency is characterized by a reduction in the thymus-to-body weight ratio, low levels of IgA plasma cells at secretory sites, and increased sensitivity of T-cells to the killing effects of ionizing radiation. Experiments were designed to examine measures of T-cell activity in wasted mice. The initial experiments established that wst/wst mice have percentages of thymic and splenic Thy1+ cells equivalent to those of control littermates. Further studies of T-cell subpopulations with thymocytes revealed normal percentages of CD4+ and CD8+ cells in wst/wst mice; however, double-labeling experiments showed that CD8+ cells were predominantly CD4- in wst/wst mice, whereas in controls most CD8+ cells also expressed CD4+. Mesenteric lymph node T-cell subpopulations were similar in wasted and control mice. Because cytokines play a significant role in the regulation of the immune response and also interact with a variety of cellular systems, we examined the expression of different cytokine and related genes (IL1, IL2, IL2R, TNF, IL5, gamma-interferon, beta-TGF) in lymphoid tissues from wasted mice as well as from littermate and parental controls. Studies of RNA from lymphoid tissues of wasted mice using dot blot and Northern blot hybridizations revealed a deficiency of IL5 mRNA in thymus and spleen, decreased expression of IL2R in thymus (but not spleen), increased expression of IL1 in spleen (but not thymus), and increased expression of IL2, gamma-interferon, and beta-TGF in both spleen and thymus, relative to controls. Expression of TNF mRNA in lymphoid tissues was unaffected by the wasted mutation. These results suggest a role for cytokine imbalance in the pathogenesis of the immunodeficiency and other abnormalities of wasted mice.