Sunny with a chance of gastroenteritis: predicting swimmer risk at California beaches.

Traditional beach management that uses concentrations of cultivatable fecal indicator bacteria (FIB) may lead to delayed notification of unsafe swimming conditions. Predictive, nowcast models of beach water quality may help reduce beach management errors and enhance protection of public health. This study compares performances of five different types of statistical, data-driven predictive models: multiple linear regression model, binary logistic regression model, partial least-squares regression model, artificial neural network, and classification tree, in predicting advisories due to FIB contamination at 25 beaches along the California coastline. Classification tree and the binary logistic regression model with threshold tuning are consistently the best performing model types for California beaches. Beaches with good performing models usually have a rainfall/flow related dominating factor affecting beach water quality, while beaches having a deteriorating water quality trend or low FIB exceedance rates are less likely to have a good performing model. This study identifies circumstances when predictive models are the most effective, and suggests that using predictive models for public notification of unsafe swimming conditions may improve public health protection at California beaches relative to current practices.

Predicting water quality at Santa Monica Beach: evaluation of five different models for public notification of unsafe swimming conditions.

Bathing beaches are monitored for fecal indicator bacteria (FIB) to protect swimmers from unsafe conditions. However, FIB assays take ∼24 h and water quality conditions can change dramatically in that time, so unsafe conditions cannot presently be identified in a timely manner. Statistical, data-driven predictive models use information on environmental conditions (i.e., rainfall, turbidity) to provide nowcasts of FIB concentrations. Their ability to predict real time FIB concentrations can make them more accurate at identifying unsafe conditions than the current method of using day or older FIB measurements. Predictive models are used in the Great Lakes, Hong Kong, and Scotland for beach management, but they are presently not used in California - the location of some of the world's most popular beaches. California beaches are unique as point source pollution has generally been mitigated, the summer bathing season receives little to no rainfall, and in situ measurements of turbidity and salinity are not readily available. These characteristics may make modeling FIB difficult, as many current FIB models rely heavily on rainfall or salinity. The current study investigates the potential for FIB models to predict water quality at a quintessential California Beach: Santa Monica Beach. This study compares the performance of five predictive models, multiple linear regression model, binary logistic regression model, partial least square regression model, artificial neural network, and classification tree, to predict concentrations of summertime fecal coliform and enterococci concentrations. Past measurements of bacterial concentration, storm drain condition, and tide level are found to be critical factors in the predictive models. The models perform better than the current beach management method. The classification tree models perform the best; for example they correctly predict 42% of beach postings due to fecal coliform exceedances during model validation, as compared to 28% by the current method. Artificial neural network is the second best model which minimizes the number of incorrect beach postings. The binary logistic regression model also gives promising results, comparable to classification tree, by adjusting the posting decision thresholds to maximize correct beach postings. This study indicates that predictive models hold promise as a beach management tool at Santa Monica Beach. However, there are opportunities to further refine predictive models.

Antitumor effects of Newcastle Disease Virus in vivo: local versus systemic effects.

Newcastle Disease Virus (NDV) has interesting anti-neoplastic and pleiotropic immune stimulatory properties. The virus preferentially replicates in and kills tumor cells and appears to be safe and to varying degrees effective in phase II-clinical studies in the US and in Europe. Here we have compared various lytic and non-lytic strains of NDV with regard to their antitumor effects after local or systemic application. As tumor models we used human metastatic melanoma xenotransplants in nude mice and murine metastatic colon carcinoma (CT26), renal carcinoma (Renca) and lymphoma (ESb) cell lines. Intra or peri-tumoral application of NDV or NDV infected tumor cells showed more pronounced antitumor activity than systemic application even when in the latter case much higher dose ranges were used. In the CT26 colon carcinoma model the non-lytic strain Ulster showed stronger antitumor activity than the lytic strain 73T. In the human MeWo melanoma xentransplant model strong antitumor bystander effects were observed by 20% admixture of melanoma cells pre-infected in vitro with NDV (either strain Ulster or Italien). Virus therapy of pre-established human melanomas by intra-tumoral injection of NDV was effective with the lytic strain Italien but not with the non-lytic strain Ulster. Systemic anti-metastatic effects were never observed with NDV alone in contrast to previous results obtained with NDV modified tumor vaccines.

Tumor stimulator cell modification by infection with Newcastle Disease Virus: analysis of effects and mechanism in MLTC-CML cultures.

Effects of tumor stimulator cell modification by infection with Newcastle Disease Virus (NDV) are described as analysed in vitro in mixed lymphocyte tumor cell cultures (MLTC). Direct antitumor effects were seen with human melanoma or colon-carcinoma cells in a dose- and time-dependent manner when using live but not UV inactivated virus. When T cell stimulation was measured by [3H]-thymidine uptake, NDV infected tumor stimulator cells did not show an augmentation but rather an inhibitory effect in comparison to non-infected stimulator cells. Virus infected tumor stimulator cells were, however, capable of augmenting the induction of tumor specific cytotoxic T cells in MLTC-CML assays when using murine ESb lymphoma immune cells and syngeneic NDV modified ESb cells as stimulators. A CML stimulatory effect was also shown for NDV modified third party cells and thereof derived conditioned medium. These effects are most likely explained by interferon- which is induced in tumor cells by NDV infection and by interferon-á which is induced in responder cells when stimulated with NDV infected stimulator cells.

In vivo 1H-NMR microimaging with respiratory triggering for monitoring adoptive immunotherapy of metastatic mouse lymphoma.

The metastatic ESb-MP murine lymphoma in DBA/2 mice has been used as a model for investigating metastatic disease and its cure by adoptive immunotherapy (ADI) as monitored by in vivo multislice spin-echo 1H NMR microimaging at 7 T. isoflurane inhalation anesthesia facilitated long measurement sessions, and respiratory gating with a fiber-optic sensor greatly reduced motional artifacts. With T2 weighting (TR = 2 s, TE = 30 ms) mean signal-to-noise ratios of 30 and 15 for kidney and liver, respectively, were achieved in 20 min (100-micron pixels, 1-mm slices, 25-mm field of view). Without the use of contrast agents, metastases with diameters > or = 0.3 mm in the imaged plane could be detected as hyperintense lesions in kidney (contrast ratio ca. 1.4) and liver (contrast ratio ca. 2) with a confidence level of > 98%. For the first time the complete eradication of late-stage macroscopic metastases by ADI could be demonstrated noninvasively by MRI.

Intra-pinna induction of specific antitumor immune T cell functions.

Upon inoculation of highly metastatic ESb lymphoma cells into the ear pinna of syngeneic mice a potent specific antitumor immune response is induced which prevents the outgrowth of the tumor cells at this particular site. When the tumor cell inoculated pinna was resected at different times after antigen application, systemic protective antitumor immunity, tested by s.c. tumor challenge 7 days later, could still be induced. This was true even for a situation in which the pinna was resected as early as two hours after tumor cell inoculation. Protective antitumor immunity was found to be even augmented when the pinna was removed three days after tumor cell inoculation as compared to the non-resected situation. Pinna resection had no effect on the induction of a tumor specific cytotoxic T lymphocyte response while resection after 48 h had an augmenting effect on the delayed type hypersensitivity (DTH) response. An explanation for some of these findings was found when lacZ-labeled lymphoma cells were used for single cell detection in tissue sections. As early as 15 min after intra-pinna inoculation disseminated single tumor cells were detected in the local draining lymph node as well as in the spleen. Whether these tumor cells can prime T cells for protective immunity directly or only after processing and presentation by specialized host cells remains to be elucidated.

Characterization of a murine lymphoma cell line by 31P-NMR spectroscopy: in vivo monitoring of the local anti-tumor effects of systemic immune cell transfer.

The intradermal ESb-MP murine T-cell lymphoma in syngeneic DBA/2 mice has been used as a model for adoptive immunotherapy (ADI). Cultured ESb-MP cells were characterized in suspension by 31P-NMR spectroscopy (MRS) at 11.7 T, and solid primary tumors were examined by 31P-MRS in vivo at 7.0 Tesla using surface-coil techniques. Growing tumors contained relatively high levels of phosphomonoesters (PME, predominantly phosphoethanolamine), nucleotides (NTP) and Pi, low levels of phosphodiesters (PDE) and no phosphocreatine. Mean tissue pH was found to be 6.7-6.9. The spectra of ESb-MP cells cultured in RPMI medium (containing choline but no ethanolamine) also showed low PDE and no phosphocreatine at an intracellular pH of 7.4; however, only a trace amount of phosphoethanolamine was detected and significant levels of nucleoside mono- and diphosphates were observed. The complete ADI treatment protocol involved low-dose irradiation (5 Gy) followed by i.v. transfer of immune spleen cells from allogeneic B10.D2 donors and resulted in 100% remission (responders); no treatment or incomplete ADI (irradiation or immune cell transfer alone) resulted in no remissions (nonresponders). In vivo MRS could best discriminate between responders and non-responders on the basis of tissue pH, which increased in responders to 7.0 by day 5-6 after complete ADI. Following therapy, the sum of PME + Pi (both absolute and as a percent of total phosphates) decreased significantly only for responders but only after a visible decrease in tumor volume was apparent.

Genetic separation of GvL and GvH reactivity in new recombinant-inbred tumor-resistant mouse strains.

From a cross between a tumor-susceptible syngeneic mouse strain (DBA/2) and a tumor resistant MHC congenic strain (B10.D2) new recombinant inbred mouse strains were established over many generations of inbreeding and tumor resistance selection. The resistance selected against one DBA/2 derived malignant tumor (ESb) extended to other DBA/2 malignant tumors (SL 2, MDAY-D2) and was thus of more general significance. Since tumor resistance had an immunological basis and since the two parental strains differed in multiple minor histocompatibility antigens (H) as well as in viral superantigens (vSAGs) we determined specificities of cytotoxic T lymphocyte (CTL) responses in vitro. All CTL responses from tumor resistant strains showed not only antitumor reactivity but also rather strong anti-minor H reactivity. There was no relationship between cytolysis and the DBA/2 type vSAG-7 (Mls(a)) expression. We also tested the capacity of immune cells from 7 resistant lines to transfer graft versus leukemia and graft versus host reactivity to ESb tumor bearing DBA/2 mice. Immune cells from one subline were capable of transfering complete protection without development of chronic GVH over a period of 4 months. The resistant parental line B10.D2 and most of the other sublines also were able to transfer GvL reactivity but this was usually associated later with chronic GVH disease caused mortality. These results demonstrate the potential of this genetic approach to separate GvL from GvH reactivity.

Effective immune rejection of advanced metastasized cancer.

A cellular cancer therapy is described with unique efficiency even in late-stage disease. in situ activated tumor-immune T cells, induced in allogeneic, tumor-resistant, MHC identical but superantigen different donor mice (B10.D2) could transfer strong graft-versus-leukemia (GvL) effects accompanied by only mild graft-versus-host (GvH) reactivity. Systemic immune cell transfer into 5 Gy irradiated DBA/2 mice bearing up to 4 week established syngeneic tumors and macrometastases led to massive infiltration of tumor tissues by CD4 and CD8 donor T lymphocytes. Upon interaction of immune CD4 donor T cells with host antigen presenting cells in synergy with immune CD8 donor T cells attacking the tumor cells directly, primary tumors (1.5 cm diameter) were encapsulated and rejected from the skin and liver metastases eradicated. For the first time, such adoptive cellular immunotherapy (ADI) was followed in individual live animals by P-31-NMR spectroscopy of primary tumors. An approximately 25,000 fold excess of metastatic tumor cells could be rejected as revealed quantitatively by FACScan analysis of lacZ gene transfected tumor cells.

Persistence of dormant tumor cells in the bone marrow of tumor cell-vaccinated mice correlates with long-term immunological protection.

Live proliferation-competent and irradiated proliferation-incompetent L5178 murine lymphoma cells (Eb cell line) were compared for their potency to induce systemic anti-tumor immunity in syngeneic DBA/2 mice. The tumorigenic potential in vivo of live Eb cells was suppressed through local secretion of interleukin 4 (IL4) or alternatively by injection of parental cells at a site refractory to tumor growth. Inoculation of nontumorigenic doses of live Eb or Eb-IL4 cells led to long-lasting specific and systemic T-cell-mediated antitumor response requiring both CD4+ and CD8+ T lymphocytes. Irradiated cells offered only limited short-term protection, which could be marginally improved by IL4. The more effective protection offered by vaccination with live tumor cells correlated with rapid migration and persistence of tumor cells in the bone marrow of host animals after tumor cell inoculation. In contrast, irradiated Eb-lacZ cells had a short persistence. Tumor cells recovered from the bone marrow of host animals injected with live Eb-IL4 cells still expressed IL4. These observations indicate that in the course of vaccination with live Eb or Eb-IL4 cells, a fraction of these cells escaped destruction by host mechanisms and persisted in a dormant state in the bone marrow for long periods of time. Persistence of dormant tumor in the bone marrow correlated with the duration of anti-tumor immunity.

Adoptive transfer of protective immunity against a high metastatic tumor-cell variant by small numbers of tumor-specific in-situ activated peritoneal effector T-cells.

We describe the generation and immunotherapeutic properties of syngeneic immune T cells activated in situ against the lymphoma variant ESb which metastasizes to multiple organs (liver, lung, spleen, bone) and can develop sophisticated immune escape mechanisms. Peritoneal effector cells (PEC) generated from intra-pinna tumor immunized and i.p. restimulated mice were able to transfer protective immunity in the absence of exogenous cytokines into normal or 5Gy irradiated or SCID mice which had been injected either s.c. or i.v. with 5x10(3) to 10(5) metastatic ESb tumor cells. Protective immunity was specific for ESb cells and could be followed by growth retardation of s.c. tumors as well as by prevention or retardation of death from metastases. Expression of protective immunity was independent of host T cells since it was similarly expressed in SCID-mice and sublethally irradiated mice. Preincubation in vitro for 24 h of the PEC before adoptive transfer led to pronounced decrease of protective immunity while the tumor specific cytotoxic T lymphocyte (CTL) activity was maintained or even enhanced. These results demonstrate i) that protective immunity in vivo requires more than tumor specific cytotoxic activity and ii) that in vitro culture of immune cells can change their in vivo functional properties.

In situ activation of syngeneic tumour-specific cytotoxic T lymphocytes: intra-pinna immunization followed by restimulation in the peritoneal cavity.

Tumour-specific cytotoxic T lymphocytes (CTL) are usually obtained after immunization in vivo and restimulation of immune cells in vitro. We here describe the generation of syngeneic tumour-specific CTL within no more than 9 days by priming and restimulation in vivo. This is achieved only if the correct sites are used both for primary immunization (ear pinna) and for restimulation (peritoneal cavity). The kinetics of immune T cell induction and of the secondary response in vivo will be reported. While a secondary CTL response could be generated in the peritoneal cavity, this was not possible in the spleen, no matter which routes of antigen restimulation were used. Upon transfer of immune spleen cells into the peritoneal cavity but not into the spleen, a secondary response could be generated upon in situ restimulation, indicating the importance of the correct microenvironment for this type of response. The peritoneal effector cells were true T cells and recognized a tumour-associated antigen in association with the Kd major histocompatibility (MHC class I) antigen. Finally the activated tumour-specific peritoneal exudate cells were able to transfer protective immunity without exogenous interleukin-2 into normal syngeneic mice.

Specific eradication of micrometastases by transfer of tumour-immune T cells from major-histocompatibility-complex congenic mice.

DBA/2 (H-2d) mice bearing a transplanted highly metastatic lymphoma (ESb) in a state of widely disseminated disease could be successfully treated by a combination of surgery (removal of the local tumour), irradiation (5 Gy) and adoptive immunotherapy. The immunotherapy was achieved by transfer of anti-ESb-immune spleen cells from B10.D2 mice, which express the same major histocompatibility complex (MHC) molecules as DBA/2. In contrast, anti-ESb-immune cells from MHC-disparate C57BL/6 mice did not confer protective immunity. The B10.D2 anti-ESb-immune T cells contain two types of cytolytic specificity as detected by limiting-dilution analysis: (1) clones with specificity for the ESb-tumour-associated transplantation antigen (TATA) (at low frequency), and (b) clones with specificity for minor DBA/2 histocompatibility (H) antigens (at high frequency). Immune B10.D2 cells raised against different tumour lines or against TATA-ESb tumour variants did not confer the 100% protection seen with immune cells against ESb TATA+ cells. Finally we demonstrate that the allogeneic immune cells are more potent in terms of protective immunity than corresponding syngeneic immune cells. The data suggest that the strong graft-versus-leukemia effect with immune T cells from allogeneic MHC-identical but not from MHC-disparate mice was due to T cells with MHC-restricted specificity for an ESb-associated TATA. A graft-versus-host reactivity that developed much later and could not be prevented was most likely due to T cells sensitized against normal minor H antigens of the host. Our results are of potential relevance for allogeneic bone marrow transplantation and adoptive immunotherapy protocols.

Prevention of metastatic spread by postoperative immunotherapy with virally modified autologous tumor cells. III. Postoperative activation of tumor-specific CTLP from mice with metastases requires stimulation with the specific antigen plus additional signals.

DBA/2 mice transplanted with the high metastatic syngeneic lymphoma variant ESb can be successfully treated by a combination of surgery and active-specific immunotherapy (ASI). The ASI procedure was applied postoperatively and involved vaccination either (1) with inactivated ESb tumor cells which had been modified by incubation with a low dose of Newcastle Disease Virus (NDV) or (2) with mutagenesis-derived immunogenic tumor variants. The analysis of the immune status of spleens from tumor-bearing or tumor-immune mice revealed important differences. The activation of tumor-specific cytotoxic T-lymphocyte precursors (CTLP) from mice with metastasis required stimulation with the specific antigen plus additional helper factors (IL-2 or NDV). The importance of second signal immune stimulation for the activation of sensitized tumor-specific CTLP in tumor-bearing mice is underlined.