Contaminated Stream Water as Source for Escherichia coli O157 Illness in Children.

In May 2016, an outbreak of Shiga toxin-producing Escherichia coli O157 infections occurred among children who had played in a stream flowing through a park. Analysis of E. coli isolates from the patients, stream water, and deer and coyote scat showed that feces from deer were the most likely source of contamination.

Radiation and primary response to lipopolysaccharide: bone marrow-derived cells and susceptible organs.

BACKGROUND: The major goal of this study was to determine whether radiation significantly alters bone marrow-derived cell distribution and mass of sensitive organs after challenge with lipopolysaccharide (LPS). MATERIALS AND METHODS: C57BL/6 mice were exposed whole-body to 0 or 3 gray (Gy) gamma-radiation (60Co) and injected intraperitoneally with 0.1 ml saline or 1 mg/kg LPS (E. coli serotype 0111:B4) 10 days later. Subsets from each group were euthanized at 60 min and 1, 7 and 14 days post-injection for analyses. RESULTS: Body mass was low 1 day after LPS, especially in irradiated animals. LPS-induced splenomegaly and hepatomegaly were attenuated by radiation, whereas thymic atrophy was enhanced. However, radiation had no effect on LPS-induced changes in oxygen radical production by liver phagocytes. The numbers of all major leukocyte populations (lymphocytes, monocyte-macrophages, granulocytes) were altered by both radiation and LPS at virtually all time points of testing. In general, the LPS-induced changes in leukocytes were diminished by radiation. Significant radiation x LPS interactions were especially prominent at day 1 after LPS administration. In contrast, mice receiving both radiation and LPS had lower red blood cell (RBC) and platelet counts than those receiving either agent alone. CONCLUSION: The data show that radiation had a highly significant influence on LPS-induced changes in mass of several body organs, leukocytes, RBC, and platelets, and thus may increase severity of infection due to Gram-negative bacteria.

Radiation and primary immune response to lipopolysaccharide: lymphocyte distribution and function.

INTRODUCTION: Lipopolysaccharide (LPS) is a major cause of septic shock and death due to infection with Gram-negative bacteria. The purpose of this study was to quantify the effects of whole-body irradiation on lymphocyte populations during response to challenge with LPS. MATERIALS AND METHODS: C57BL/6 mice (n = 10/group) were irradiated whole-body with 3 gray (Gy) gamma-rays in a single fraction at 0.8 Gy/min. LPS (E. coli serotype 0111:B4) at 1 mg/kg was injected intraperitoneally 10 days later and mice were euthanized at 60 min and days 1, 7, and 14 post-inoculation for analyses. RESULTS: Significant interactions between radiation and LPS were noted in circulating and splenic lymphocyte subpopulations, including T-, B-, and NK-cells, particularly at the early time points. There were significant interactions on circulating, but not splenic, CD62L+ T-cell populations. However, there were no interactions on CD62L+ B-cells. Finally, there were significant interactions in both early and late blastogenic responses. CONCLUSION: The data support that response to infection with Gram-negative bacteria may be significantly compromised by exposure to ionizing radiation.

Proton radiation and TNF-alpha/Bax gene therapy for orthotopic C6 brain tumor in Wistar rats.

High-grade tumors of the brain remain virtually incurable with current therapeutic regimens, new approaches to augment existing therapies need to be explored. The major goal of this pilot study was to evaluate the feasibility of gene therapy using plasmid DNA encoding tumor necrosis factor-alpha and bax together with proton radiation in an immunocompetent animal model with orthotopic brain tumor. C6 glioma cells were stereotactically implanted into the left hemibrain of Wistar rats (day 0). On day 5, the appropriate groups received intratumoral pGL1-TNF-a and pGL1-Bax (10 microg each), parental plasmid pWS4 (20 microg), or PBS. Hemibrain proton irradiation (10 Gy, 90 MeV, single fraction) was delivered 18-20 hr later. Rats were euthanized when signs of illness appeared. In addition, a subset of animals from each group was euthanized on day 9 for immune and other assays. By day 9, 25%, 20%, and 10% of rats treated with PBS, pWS4, or pGL1-TNF-alpha/pGL1-Bax, respectively, had been euthanized due to weight loss or other signs of illness, whereas all rats treated with pGL1-TNF-alpha/pGL1-Bax + radiation or radiation alone were healthy (P<0.05). At this same time, the pGL1-TNF-alpha/pGL1-Bax + radiation group had significantly elevated lymphocyte percentages (P<0.005 or less) and a relatively high level of lymphocytic infiltrate within tumors. Although the rats treated with pGL1-TNF-alpha/pGL1-Bax had the highest levels of activated T helper (CD4+/CD71+) and T cytotoxic (CD8+/CD71+) cells, the values were not significantly different compared to the pWS4-injected control group. Splenocytes in all tumor cell-injected groups had higher mean values for DNA and protein synthesis compared to the non-tumor cell injected control group, whereas oxygen radical production by phagocytes was consistently higher in groups injected with plasmid or treated with radiation. Body, hemibrain, and spleen masses, white blood cell, red blood cell and platelet counts, hemoglobin, hematocrit, and transforming growth factor-beta1 levels in plasma were similar among groups. The results demonstrate that treatment with pGL1-TNF-alpha/pGL1-Bax combined with proton hemibrain irradiation is safe under the conditions used. Overall, these data support further investigation of this unique combination therapy.

Hypergravity-induced immunomodulation in a rodent model: hematological and lymphocyte function analyses.

The major purpose of this study was to quantify hypergravity-induced changes in erythrocyte and thrombocyte characteristics, spontaneous and mitogen-induced lymphoblastogenesis, and capacity of splenocytes to secrete immunoregulatory cytokines. C57BL/6 mice were subjected to chronic 1, 2, and 3 G; subsets were euthanized after 1, 4, 7, 10, and 21 days of centrifugation. Erythrocyte counts, hematocrit, and hemoglobin were significantly reduced by day 21 in both centrifuged groups. Hemoglobin concentration and volume per red blood cell were generally low, but an early, transient spike above normal was noted in thrombocyte counts in the 3-G group. Fluctuations above and below normal in blood and spleen cell spontaneous blastogenesis were dependent on the length of centrifugation time and not on the level of gravity. Depression in splenocyte responses to phytohemagglutinin and lipopolysaccharide due to gravity were noted when the data were expressed as stimulation indexes. Cytokine production by spleen cells was primarily affected during the first week of centrifugation: IL-2, IL-4, and tumor necrosis factor-alpha increased, whereas interferon-gamma decreased. These findings, although not identical to those reported for spaceflight, indicate that altered gravity can influence both hematological and functional variables that may translate into serious health consequences during extended missions.

Changes in the activation and reconstitution of lymphocytes resulting from total-body irradiation correlate with slowed tumor growth.

Alterations in cytokine secretion, activation marker expression, and immune cell concentrations were investigated at sequential time points following delivery of total-body irradiation (TBI) to C57BL/6 mice (n = 64) in the Lewis lung tumor model. Significantly slower tumor growth was observed when a 3-Gy dose of TBI was administered 2 h prior to tumor implantation (p < 0.05). The antitumor effect was correlated with an increased CD4:CD8 T cell ratio and heightened leukocyte blastogenesis. TBI was also found to induce an expansion of natural killer (NK) cells in the blood and spleen of tumor-bearing animals 10 days after irradiation (2.8 x 10(6) NK cells/spleen in test mice compared to 8.9 x 10(5) NK cells/spleen in normal control animals). However, no significant differences were found in NK cell levels within the tumor tissue. Enhanced production of interleukin (IL)-12 and IL-18 from spleen supernatants was consistent with an augmentation of the NK cell response. Significant reductions in transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor, both of which are associated with immune suppression, were also noted. Furthermore, TBI induced changes in expression of CD25 and CD71 activation markers, suggesting that radiation may alter tumor surveillance. Taken together, the relative percentages and activation status of immune cell compartments support the conclusion that these TBI-induced changes function to slow tumor progression.

NK cell depletion results in accelerated tumor growth and attenuates the antitumor effect of total body irradiation.

Total body irradiation (TBI), given in low to moderate doses as a single modality, can enhance leukocyte populations and immune modifiers, resulting in slowed tumor progression. The aim of this study was to evaluate natural killer (NK) cell involvement in mediating the antitumor effect of TBI by depleting NK populations and monitoring tumor progression and immune status following exposure. C57BL/6 mice (n=54) were injected with anti-NK1.1, anti-asialo GM1, or rabbit serum prior to irradiation/tumor implantation. Selected animal groups were irradiated with a 3 Gy dose of gamma-rays and Lewis lung carcinoma (LLC) cells were subcutaneously implanted 2 h later. Tumor volumes, leukocyte populations, and cytokine levels in blood and spleen were measured up to 10 days post-irradiation/tumor implantation. Depletion of asialo GM1+ cells, but not NK1.1+ cells, led to significant acceleration of tumor growth (P<0.05). Challenge with exogenous antigens (rabbit antibodies or serum) when accompanied by administration of TBI resulted in: a) radioresistance of splenic lymphocytes, b) increased granulocyte and monocyte numbers, and c) enhanced production of IgG, IL-10, and IL-18 within plasma and tumor supernatants. Delivery of TBI to NK1.1+ depleted mice, did not show similar enhancement of leukocytes and/or their modulators. These data indicate that TBI, in conjunction with immune challenge, activates leukocyte parameters and redirects the immune system toward a T helper 2 (Th2) cell response. Additionally, NK cells are involved in mediating the antitumor effect of TBI, while challenge with exogenous protein attenuates the slowing of malignant growth that accompanies delivery of radiation. These findings also support the premise that radiation exposure can activate NK and some T cytotoxic lymphocytes, thereby leading to tumor suppression.

Evaluation of TNF-alpha/Bax gene therapy and radiation against C6 glioma xenografts.

Successful therapy of high-grade tumors of the brain is likely to require a combination of new therapeutic approaches. The major goal of the present study was to construct a plasmid-based bax gene vector (pGL1-Bax) and evaluate its expression in vitro and in vivo using athymic mice with subcutaneously growing C6 glioma. Preliminary experiments of efficacy and safety were also performed using pGL1-Bax alone and in combination with previously constructed pGL1-TNF-alpha, as well as with radiation. pGL1-Bax was expressed by C6 cells and was correlated with apoptosis, indicating that the construct and the bax protein were functional. Although intratumoral injections of pGL1-Bax alone, up to total doses of 450 micro g, did not significantly affect tumor growth, consistently smaller tumors were obtained when pGL1-TNF-alpha plus pGL1-Bax were injected 16-18 hr prior to tumor irradiation. Furthermore, in mice with two tumors, one treated and one untreated, progression of the untreated tumor was delayed in the animals receiving all three modalities. No prohibitive toxicities were noted, based on mouse body weights and in vitro assays of blood and spleen. Significant increases in spleen mass, total leukocyte counts, percentage of granulocytes, spontaneous blastogenesis, and CD71-expressing B cells were primarily associated with tumor presence and not treatment type. Overall, the results are promising and suggest that TNF-alpha/Bax gene therapy may be beneficial against highly malignant tumors of the brain. To our knowledge, this is the first report of bax gene therapy used together with radiation in an in vivo glioma model.

Immunohistological analysis of immune cell infiltration of a human colon tumor xenograft after treatment with Stealth liposome-encapsulated tumor necrosis factor-alpha and radiation.

The toxicity associated with tumor necrosis factor-alpha (TNF-alpha) has limited its usefulness as an anticancer agent. However, encapsulation of TNF-alpha in Stealth (SL) liposomes can minimize risk for toxicity and thus increase its potential as an adjuvant treatment. Our recent studies have shown that SL-TNF-alpha plus radiation is more effective at inhibiting LS174T colon tumor growth than either radiation alone or free TNF-alpha plus radiation. This increase in efficacy was coincident with a modulation of immune parameters in blood and spleen. The aim of this study was to determine if infiltration of natural killer (NK) cells, macrophages, and neutrophils into LS174T tumors was altered by SL-TNF-alpha treatment and whether any observed changes could potentially contribute to the enhanced antitumor efficacy seen with SL-TNF-alpha plus radiation treatment. Sections of excised tumors were examined histologically and quantitative analysis was performed using laser scanning cytometry. The data showed that the group receiving multiple treatments with SL-TNF-alpha plus radiation had the smallest tumors, but yet the level of necrosis was similar to that found in groups with much larger tumors. Furthermore, the necrotic areas in the SL-TNF-alpha plus radiation group had signs of recent and/or continuing cell death and the highest levels of NK cell and macrophage infiltrates. In time course experiments, a single injection of SL-TNF-alpha (but not free TNF-alpha) induced fluctuations in leukocyte infiltration into tumors that correlated inversely with our previous findings in blood and spleen. Overall, the data indicate that the mechanisms underlying the increased efficacy of SL-TNF-alpha compared to free TNF-alpha include a rapid and relatively sustained recruitment of NK cells, macrophages, and neutrophils.

Dose and timing of total-body irradiation mediate tumor progression and immunomodulation.

The major goal of this study was to examine the effects of total-body irradiation (TBI) on lung carcinoma progression and determine if changes in tumor growth could be correlated with radiation-induced alterations of immune system parameters. Lewis lung tumor cells were injected subcutaneously into syngeneic C57BL/6 mice that had been irradiated with a single 3.0 Gy dose of gamma-rays (60Co) at four time points either before or after tumor cell implantation. Subsequently, a second group of mice was irradiated 2 h prior to tumor injection with sequential doses of gamma-rays (0.46-2.66 Gy range). Assays were performed on blood and spleen from mice euthanized 16 days postimplantation. Tumor growth was consistently slower regardless of the timing of radiation exposure. However, dose of radiation influenced tumor growth delay. The preirradiated tumor-bearing mice had high CD4/CD8 T lymphocyte ratios along with increasing percentages of NKT cells in the blood supply with dose. Tumor-induced immunomodulation was also present, as evidenced by splenomegaly, low proliferative response to mitogens, and decreased spontaneous blastogenesis of leukocytes within the blood compared with normal values (P < or = 0.01). Anemia and thrombocytopenia were not observed with either tumor presence or irradiation. The present study demonstrates that a modest dose of TBI prior to tumor cell implantation resulted in a beneficial antitumor effect. A selective radiation-induced depletion of CD8+ T lymphocytes and changes in NKT cell percentages, correlated with findings from cytotoxicity assays, were indicative of a protumoricidal immune environment.

TNF-alpha gene and proton radiotherapy in an orthotopic brain tumor model.

The major goal of this study was to evaluate the safety and efficacy of TNF-alpha gene therapy (pGL1-TNF-alpha) in combination with proton radiation in an orthotopic brain tumor model. C6 glioma cells were implanted into the left hemibrain of athymic rats (day 0). On day 5, pGL1-TNF-alpha (19 microg/10 microl) was injected into the same site; appropriate control groups were included. Proton irradiation (10 Gy, single fraction) was performed 18-20 h thereafter and, on day 10, a portion of animals from each group was assayed. Nearly all tumor-bearing groups had lower body mass compared to those without tumor; brain mass was somewhat increased with plasmid (pGL1-TNF-alpha or pWS4) injection (p<0.05). Histopathological analysis of brain sections revealed that rats receiving pGL1-TNF-alpha/proton irradiation had the smallest tumors and lowest number of mitotic tumor cells, although survival time for animals kept long-term was not significantly prolonged. A decline in leukocyte populations was noted with combination treatment compared to controls (p<0.05), but no differences were found compared to groups receiving each modality alone. Based on DNA synthesis, the pGL1-TNF-alpha/proton irradiated group had the highest levels of leukocyte activation. The highest percentage of lymphocytes expressing the CD71 activation marker occurred with pGL1-TNF-alpha, whereas the proton-irradiated group had the highest percentage of activated NK cells (NK1.1+/CD71+). No significant differences were found in erythrocyte and thrombocyte numbers, hemoglobin, and hematocrit. Overall, the data indicate that pGL1-TNF-alpha/proton treatment results in a measurable antitumor effect and is safe under the conditions used.

Modulation of innate immunological factors by STEALTH liposome-encapsulated tumor necrosis factor-alpha in a colon tumor xenograft model.

BACKGROUND: Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) encapsulated in sterically-stabilized PEGylated STEALTH liposomes (SL) can better and more safely augment the efficacy of other treatment modalities than free TNF-alpha. The aim of this study was to examine the effects of SL-TNF-alpha in the LS174T human colon tumor xenograft model and to correlate its administration with alterations in innate immune system parameters. MATERIALS AND METHODS: Nude mice (n = 128) were injected subcutaneously with LSI 74T cells and treated intravenously with SL-TNF-alpha SL-placebo, or free recombinant human TNF-alpha; the animals were euthanized at 6, 18, 36 and 96 hours after injection. RESULTS: Significant increases in leukocyte, granulocyte, monocyte and NK cell numbers were observed early (6 hours) in the blood from both SL-TNF-alpha and free TNF-alpha treated mice compared to the control group. In contrast, during the 18- to 36-hours interval, SL-TNF-alpha induced significantly higher (p<0.05) leukoctyte, T cell, and NK cell numbers, basal leukocyte proliferation, and CD25+ activation marker expression; the modulation occurred primarily in the spleen. CONCLUSION: These data indicate that both SL-TNF-alpha and free TNF-alpha can induce dramatic up-regulation in leukocyte populations early after injection. However, the up-regulation produced by SL-TNF-alpha was more prolonged and pronounced than that of TNF-alpha and had better correlation with cell activation. These findings suggest that sustained leukocyte recruitment and/or activation may be an important factor in the greater than additive or synergistic antitumor effects observed when SL-TNF-alpha is used in combination with other cancer therapies.

Hypergravity-induced immunomodulation in a rodent model: lymphocytes and lymphoid organs.

The major goal of this study was to quantify changes in lymphoid organs and cells over time due to centrifugation-induced hypergravity. C57BL/6 mice were exposed to 1, 2 and 3 G and the following assays were performed on days 1, 4, 7, 10, and 21: spleen, thymus, lung, and liver masses; total leukocyte, lymphocyte, monocyte/macrophage, and granulocyte counts; level of splenocyte apoptosis; enumeration of CD3+ T, CD3+/CD4+ T helper, CD3+/CD8+ T cytotoxic, B220+ B, and NK1.1+ natural killer cells; and quantification of cells expressing CD25, CD69, and CD71 activation markers. The data show that increased gravity resulted in decreased body, spleen, thymus, and liver, but not lung, mass. Significant reductions were noted in all three major leukocyte populations (lymphocytes, granulocytes, monocyte/macrophages) [correction of macrphages] with increased gravity; persistent depletion was noted in blood but not spleen. Among the various lymphocyte populations, the CD3+/CD8+ T cells and B220+ B cells were the most affected and NK1.1+ NK cells the least affected. Overall, the changes were most evident during the first week, with a greater influence noted for cells in the spleen. A linear relationship was found between some of the measurements and the level of gravity, especially on day 4. These findings indicate that hypergravity profoundly alters leukocyte number and distribution in a mammalian model and that some aberrations persisted throughout the three weeks of the study. In certain cases, the detected changes were similar to those observed after whole-body irradiation. In future investigations we hope to combine hypergravity with low-dose rate irradiation and immune challenge.