Predicting the periodic risk of anthrax in livestock in Victoria, Australia, using meteorological data.

Cases of anthrax in livestock are infrequently and irregularly reported in the state of Victoria, Australia; however, their impact on individual livestock, farming communities and the government agencies tasked with containing these outbreaks is high. This infrequency has been anecdotally associated with differences in annual and local weather patterns. In this study, we used historical anthrax cases and meteorological data from weather stations throughout Victoria to train a generalized linear mixed effects model to predict the daily odds of a case of anthrax occurring in each shire in the coming 30 days. Meteorological variables were transformed to deviations from the mean values for temperature or cumulative values for rainfall in the shire across all years. Shire was incorporated as a random effect to account for meteorological variation between shires. The model incorporated a post hoc weighting for the frequency of historic cases within each shire and the spatial contribution of each shire to the recently redefined Australian Anthrax Belt. Our model reveals that anthrax cases were associated with drier summer conditions (OR 0.96 (95% CI 0.95-0.97) and OR 0.98 (95% CI 0.97-0.99) for every mm increase in rainfall during September and December, respectively) and cooler than average spring (OR 0.20 (95% CI 0.11-0.52) for every °C increase in minimum daily temperature during November and warmer than average summer temperatures (OR 1.45 (95% CI 1.29-1.61) for every °C increase in maximum daily temperature during January. Cases were also preceded by a 40-day period of cooler, drier temperatures (OR 0.5 (95% CI 0.27-0.74) for every °C increase in maximum daily temperature and OR 0.96 (95% CI 0.95-0.97) for every mm increase in rainfall followed by a warmer than average minimum (or nightly) temperature 10 days immediately before the case (OR 1.46 (95% CI 1.35-1.58) for every °C increase in maximum daily temperature). These coefficients of this training model were then applied daily to meteorological data for each shire, and output of these models was presented as a choropleth and timeline plot in a Shiny web application. The application builds on previous spatial modelling and provides Victorian agencies with a tool to engage at-risk farmers and guide discussions towards anthrax control. This application can contribute to the wider rejuvenation of anthrax knowledge and control in Victoria and corroborates the anecdote that increased odds of disease can be linked to meteorological events.

Hepatopathy in Victorian dogs consuming pet meat contaminated with indospicine.

BACKGROUND: Indospicine is an arginine analogue and a natural toxin occurring only in Indigofera plant species, including Australian native species. It accumulates in the tissues of grazing animals, persisting for several months after ingestion. Dogs are particularly sensitive to indospicine toxicity and can suffer fatal liver disease after eating indospicine-contaminated pet meat. METHOD: A disease outbreak investigation was launched following notification to Agriculture Victoria of a cluster of 18 dogs displaying acute, severe, hepatopathy in the East Gippsland Shire in June 2021. RESULTS: Between June and September 2021, 24 pet dogs died, and 40 others experienced liver disease after eating commercially prepared pet meat found to contain indospicine. The investigation identified the toxin in serum and liver samples from affected dogs and at high levels in some samples of pet meat eaten by the dogs. Twenty-six horses that were moved from the Northern Territory and processed at a Pet Meat Processing facility (knackery) in eastern Victoria over a period of 14 days in late May-early June 2021 were identified as the likely source of the indospicine toxin in the pet meat. Pet meat produced by the knackery and on-sold by several retailers was determined to be the cause of the illness and death in the dogs. CONCLUSION: This is the first report of severe and frequently fatal hepatopathy in dogs in Victoria relating to consumption of pet meat contaminated with indospicine.

High speed scintillation autoradiography.

Impregnation of nuclear track emulsion with liquid scintillator and exposure at -85 degrees C allows rapid autoradiographic labeling. With tritiated thymidine of high specific activity (40 to 60 curies per millimole), exposure time can be shortened to 20 to 60 minutes, allowing complete sample processing within 4 hours. In experiments requiring isotopes with low incorporation rates or low specific activity, exposure time can be shortened from months to several days.

Primary bioassay of human tumor stem cells.

A simple method has been developed to support human tumor stem cell colony growth in soft agar. The technique appears suitable for culture of a variety of neoplasms of differing histopathology. Tumor stem cell colonies arising from different types of cancer have differing growth characteristics and colony morphology. This bioassay should be suitable for clinical studies of effects of anticancer drugs or irradiation on human tumor stem cells.

Lymphocyte stimulation: selective destruction of cells during blastogenic response to transplantation antigens.

The blastogenic response of human lymphoid cells toward any individual's transplantation antigens can be deleted by the addition of tritiated thymidine of high specific activity during the incubation of the lymphoid cells in mixed leukocyte culture. After the immunocompetent clones which responded to histocompatibility antigens had been destroyed, the remaining population still retained its capacity to respond to unrelated antigens, including other transplantation antigens.

Classical swine fever in Victorian domestic pigs: evidence of disease freedom.

OBJECTIVE: Australia is currently regarded as free of classical swine fever (CSF), a highly contagious disease of pigs caused by a pestivirus. This study aimed to provide additional evidence that the Victorian domestic pig population is free of CSF. DESIGN: A structured representative sero-prevalence survey of Victorian domestic pigs at slaughter. METHOD: Three-hundred and ninety-one pigs from 23 holdings were sampled at the time of slaughter between March 2016 and October 2017. RESULTS: All samples were negative for CSF virus Ab on ELISA. Because of uncertainty in the sensitivity of the CSF Ab ELISA, estimates of the true prevalence of CSF were calculated using Bayesian methods. The median and upper bound of the 95% credible intervals for the true prevalence of CSF was zero when the diagnostic sensitivity of the CSF Ab ELISA was assumed to range from 0.75 to 0.95. CONCLUSION: These results provide evidence that the population of domestic pigs in Victoria in 2016-2017 was free of CSF.

Immunoglobulin synthesis and total body tumor cell number in IgG multiple myeloma.

Studies of synthesis of IgG paraproteins were performed in 10 patients who had IgG myeloma in order to quantitate cellular immunosynthetic functions and derive estimates of the number of tumor cells present in such patients. Serial in vitro studies demonstrated constancy in the cellular rate of IgG paraprotein secretion for up to 8 months. Average molecular synthesis rates in different patients ranged from 12,500 to 85,000 molecules of IgG per minute per myeloma cell. Estimated total body tumor cell number ranged from 0.5 x 10(12) to 3.1 x 10(12) myeloma cells, and could be correlated with the degree of skeletal damage observed on roentgenograms (P = <0.01). Serial measurements of tumor cell number may prove useful in characterizing the growth rate and natural history of multiple myeloma. Myeloma is the first metastatic human malignancy in which quantitative measurements of the body's burden of malignant cells have been obtained.

Immunology of the lower respiratory tract. Functional properties of bronchoalveolar lymphocytes obtained from the normal canine lung.

While the alveolar macrophage has been studied extensively, little attention has been directed toward the immune functions of the bronchoalveolar lymphocyte. These cells, obtained by bronchopulmonary lavage of the normal canine lung, are derived from the air side of the alveolar-capillary membrane of the lower respiratory tract. The distribution of lymphocyte types within the bronchoalveolar cell population was determined and compared with that of leukocytes from blood and spleen.IgG synthesis in vitro was used as a measure of bone marrow-derived lymphocyte (B cell) function, and the blastogenic response of cell cultures to phytohemagglutinin was used as a measure of the presence or absence of thymus-dependent lymphocytes (T cells). De novo synthesis of IgG by bronchoalveolar cells was demonstrated consistently by two independent radio-immunoassays. Therefore, B cells are present in the air spaces of normal canine lungs. T cells were generally not detectable in aliquots of the same cell populations but could be recruited into alveolar spaces after local irritation. The distribution of lymphocyte types within the bronchoalveolar cell population is unique and distinctly different from that of blood and spleen. The spleen is rich in both T cells and B cells, blood is rich in T cells but poor in B cells, whereas the lung lacks T cells and contains substantial numbers of immunoglobulin-producing B cells. The findings indicate that bronchoalveolar lymphocytes do not reflect simply the lymphoid composition of peripheral blood.

Primary bioassay of human myeloma stem cells.

The ability to clone primary tumors in soft agar has proven useful in the study of the kinetics and biological properties of tumor stem cells. We report the development of an in vitro assay which permits formation of colonies of human monoclonal plasma cells in soft agar. Colony growth has been observed from bone marrow aspirates from 75% of the 70 patients with multiple myeloma or related monoclonal disorders studied. Growth was induced with either 0.02 ml of human type O erythrocytes or 0.25 ml of medium conditioned by the adherent spleen cells of mineral oil-primed BALB/c mice. 5-500 colonies appeared after 2-3 wk in culture yielding a plating efficiency of 0.001-0.1%. The number of myeloma colonies was proportional to the number of cells plated between concentrations of 10(5)-10(6) and back-extrapolated through zero, suggesting that colonies were clones derived from single myeloma stem cells. Morphological, histochemical, and functional criteria showed the colonies to consist of immature plasmablasts and mature plasma cells. 60-80% of cells picked from colonies contained intracytoplasmic monoclonal immunoglobulin. Colony growth was most easily achieved from the bone marrow cells of untreated patients or those in relapse. Only 50% of bone marrow samples from patients in remission were successfully cultured. Tritiated thymidine suicide studies provided evidence that for most myeloma patients, a very high proportion of myeloma colony-forming cells was actively in transit through the cell cycle. Velocity sedimentation at 1 g showed myeloma stem cells sedimented in a broad band with a peak at 13 mm/h. Antibody to granulocyte colony-stimulating factor did not reduce the number or size of the colonies. Increased numbers of myeloma colonies were seen when the marrow was depleted of colony-stimulating factor elaborating adherent cells before plating. This bioassay should prove useful in studying the in vitro biological behavior of certain bone marrow-derived (B)-cell neoplasia. In addition, systematic and predictive studies of anticancer drug effects on myeloma stem cells should now be feasible.

Kinetics of tumor growth and regression in IgG multiple myeloma.

Studies of immunoglobulin synthesis, total body tumor cell number, and tumor kinetics were carried out in a series of patients with IgG multiple myeloma. The changes in tumor size associated with tumor growth or with regression were underestimated when the concentration of serum M-component was used as the sole index of tumor mass. Calculation of the total body M-component synthetic rate (corrected for concentration-dependent changes in IgG metabolism) and tumor cell number gave a more accurate and predictable estimate of changes in tumor size. Tumor growth and drug-induced tumor regression were found to follow Gompertzian kinetics, with progressive retardation of the rate of change of tumor size in both of these circumstances. This retardation effect, describable with a constant alpha, may be caused by a shift in the proportion of tumor cells in the proliferative cycle. Drug sensitivity of the tumor could be described quantitatively with a calculation of B(O), the tumor's initial sensitivity to a given drug regimen. Of particular clinical significance, the magnitude of a given patient's tumor regression could be predicted from the ratio of B(O) to alpha. Mathematical proof was obtained that the retardation constant determined during tumor regression also applied to the earlier period of tumor growth, and this constant was used to reconstruct the preclinical history of disease. In the average patient, fewer than 5 yr elapse from the initial tumor cell doubling to its clinical presentation with from 10(11) to more than 10(12) myeloma cells in the body. The reduction in total body tumor mass in most patients responding to therapy ranges from less than one to almost two orders of magnitude. Application of predictive kinetic analysis to the design of sequential drug regimens may lead to further improvement in the treatment of multiple myeloma and other tumors with similar growth characteristics.

Immunology of the lower respiratory tract. II. The plaque-forming response of canine lymphoid tissues to sheep erythrocytes after intrapulmonary or intravenous immunization.

Groups of dogs were immunized with sheep erythrocytes administered either directly into the lower respiratory tract (bronchoalveolar spaces) or intravenously. The hemolytic plaque-forming response (Jerne plaque assay) was studied in various canine lymphoid populations (bronchoalveolar cells, hilar lymph nodes, peripheral lymph nodes, and splenic and peripheral blood leukocytes) as a function of time after immunization and as a function of the dose of antigen administered. Serum hemagglutinating antibody titers against sheep erythrocytes were also measured. Intrapulmonary and intravenous administration of sheep erythrocytes to dogs both result in an immune response, the kinetics of which are identical to those observed in other animal species. At equivalent doses, the intravenous route is more efficient than the intrapulmonary route in generating serum hemagglutinating antibodies and antibody-forming cells. Both routes give rise transiently to circulating antibody-forming cells during the primary response; the distribution in tissues of antibody-forming cells is distinctive and unique, depending on the route of immunization. After i.v. immunization, antibody-forming cells are found predominately in spleen, blood, and bronchoalveolar spaces; after intrapulmonary immunization, they are located predominately in hilar lymph nodes, blood, and bronchoalveolar spaces. The reasons for this pattern of distribution are not known. Both routes of immunization are equally effective in populating bronchoalveolar air spaces with antibody-forming cells, which are predominately IgM-secreting and IgG-secreting cells. IgA-secreting cells were not detected.

Effects of suramin on in vitro growth of fresh human tumors.

BACKGROUND: Suramin is a polysulfonated urea recently tested in clinical trials as an anticancer agent. PURPOSE: To define tumor types for further clinical testing of suramin, we assessed the in vitro activity of suramin against fresh human tumor specimens. METHODS: Inhibition of tumor colony formation (human tumor clonogenic assay [HTCA] method) and inhibition of tritiated thymidine incorporation (TTI method) were used as indicators of drug sensitivity. RESULTS: With the use of the HTCA method, 80% or more of carcinomas of the colon, endometrium, kidney, lung (non-small-cell), and ovary as well as malignant melanoma and mesothelioma were sensitive to 200 micrograms/mL of suramin by continuous exposure. Suramin's antitumor activity was dose dependent, and it was less effective when tested at concentrations of 50 micrograms/mL or less. With the TTI method, the more slowly growing tumors (breast cancer, colon cancer, multiple myeloma, non-Hodgkin's lymphoma, prostate cancer, and sarcoma) appeared to be less sensitive to suramin. However, when the two assay methods were directly compared in melanoma and ovarian cancer specimens, individual tumors were generally more sensitive to suramin (greater inhibition relative to control) using the HTCA method, whereas the TTI method appeared to underestimate suramin's antitumor activity. There was no significant difference in the activity of suramin when tested in the presence of 10% versus 50% serum. CONCLUSION: These results provide an experimental basis for clinical evaluations of suramin therapy in patients with colon, endometrial, kidney, non-small-cell lung, and ovarian cancers as well as malignant melanoma and mesothelioma.

Antiproliferative and antitumor activity of the 2-cyanoaziridine compound imexon on tumor cell lines and fresh tumor cells in vitro.

BACKGROUND: Imexon, a 2-cyanoaziridine, is therapeutic and reverses lymphadenopathy and splenomegaly in the LP-BM5 murine retrovirus-induced immunodeficiency disease (murine AIDS). It can restore chemotherapy-induced immunosuppression. Imexon reduced the incidence of lymphoma in severe combined immune deficient mice inoculated with human lymphocytes. PURPOSE: To determine its antitumor activity, we screened imexon against fresh human tumor cells and tumor cell lines. To determine the time-concentration relationships of its cytotoxicity, we studied the effects of imexon on macromolecular synthesis and on the cell cycle. METHODS: Imexon was incubated at 1-200 micrograms/mL with various tumor cell lines, mitogen-stimulated peripheral blood lymphocytes, and fresh tumor cells. Cell survival, macromolecular synthesis, and cell cycle progression were studied. RESULTS: The concentration of imexon that caused 50% inhibition of growth was under 10 micrograms/mL for lymphocytes stimulated with mitogens. It was about 3-10 micrograms/mL for B-cell lymphomas and both multi-drug-resistant and -sensitive myeloma cell lines. Imexon inhibited four of seven fresh lymphoma and 11 of 16 fresh myeloma biopsy specimens to less than 40% of the control. A 1-hour exposure of lymphoma cells to 50-100 microgram/mL followed by removal of drug by washing the cells and continuing culture resulted in greater than 95% inhibition during the next 48-72 hours. Imexon selectively inhibited protein synthesis during the first 24-48 hours of exposure of lymphoma and myeloma cells. Cells exposed to inhibitory concentrations of imexon were blocked in cell cycle progression. CONCLUSION: Imexon may be a potentially useful agent in the treatment of malignant disease, particularly lymphoid malignancies, and should be explored further.

Prediction of doxorubicin resistance in vitro in myeloma, lymphoma, and breast cancer by P-glycoprotein staining.

Prior studies have shown that the P-glycoprotein is a cell membrane efflux pump that is quantitatively increased in expression in multidrug-resistant tumor cell lines. In this study, fresh tumor tissues from patients with multiple myeloma, malignant lymphoma, or metastatic breast cancer were studied immunohistochemically for P-glycoprotein expression and for in vitro sensitivity to doxorubicin. Twenty-six patients who were either previously untreated or in relapse after chemotherapy had tumor specimens submitted that could be evaluated in both assays. The testing was done independently and blindly in separate laboratories instead of our being provided relevant clinical data on the patients. Tumor cells from 12 of the 26 patients (46%) stained positively for P-glycoprotein. Fifteen of the 26 specimens (58%) exhibited drug resistance in vitro. Although only three (21%) of the 14 P-glycoprotein-negative tumors exhibited in vitro resistance to doxorubicin, all 12 fresh tumors that stained positively for P-glycoprotein were resistant to doxorubicin. The difference in frequency of intrinsic doxorubicin resistance between P-glycoprotein-negative and -positive tumors was highly significant (P less than .001). Similar trends were observed in each of the individual tumor categories and were statistically significant in myeloma and breast cancer. Four of the biopsy specimens that stained positively for P-glycoprotein and exhibited doxorubicin resistance were from patients who had not received prior cytotoxic chemotherapy. Similar conclusions were reached when results of drug sensitivity tests were ranked in relation to the median infective dose rather than by criteria based on correlations with clinical drug resistance. Our findings indicate that positive staining for P-glycoprotein associated with multidrug resistance predicts intrinsic cellular resistance of human cancers to doxorubicin. We anticipate that immunohistochemical staining for P-glycoprotein will prove useful in clinical oncology.

Systemic toxic effects associated with high-dose verapamil infusion and chemotherapy administration.

Aside from its more conventional uses as a cardiovascular drug, the calcium channel blocker verapamil has recently been added to chemotherapeutic regimens to reduce drug resistance in B-cell and other neoplasms that express the P-glycoprotein. We recently treated patients with continuous-infusion verapamil (0.15 mg/kg per hour to 0.60 mg/kg per hour) over a 5-day period in combination with continuous-infusion vincristine and doxorubicin plus oral dexamethasone. Seventy-one courses involving 35 hospitalized patients were prospectively studied for cardiovascular and other side effects. Cardiovascular side effects were observed most frequently and consisted of first-degree heart block, hypotension, sinus bradycardia, and junctional rhythms. We observed higher degree heart block, but the QRS interval remained narrow and the ventricular escape rate remained relatively normal. Effects on mean arterial pressure, heart rate, and PR interval were both time and dose related. Severe, symptomatic congestive heart failure was rarely observed. The most common noncardiovascular side effects were constipation, peripheral edema, and weight gain. All systemic toxic effects observed were easily treated or disappeared with either temporary or permanent discontinuation of the verapamil infusion or by a decrease in the dose of verapamil. We conclude that the cardiovascular side effects associated with continuous, high-dose intravenous verapamil therapy are significant and dose limiting but are rapidly reversible. Less cardiotoxic chemosensitizers are needed to reverse multidrug resistance in cancer.

Preparation of permanent slides of intact soft-agar colony cultures of hematopoietic and tumor stem cells.

A simple technique is described for fixing colony-containing layers of soft agar and drying them onto microscopic slides. The method is extrapolated from techniques used in immunology for permanent preservation of immunodiffusion or immunoelectrophoresis plates. Slides prepared in this fashion are eminently suitable for subsequent analysis with a variety of techniques including conventional Papanicolaou or other staining methods as well as histochemistry, immunofluorescence, and autoradiography. In addition to research applications, the technique may have diagnostic applications and should greatly enhance both qualitative and quantitative analysis of the biology of hematopoietic and tumor colony formation.

Inhibition of human melanoma colony formation by retinoids.

We studied the effects of retinoids on the in vitro survival of melanoma colony-forming cells in biopsies obtained from ten patients with metastatic melanoma. The results indicate that specific retinoids reduce the ability of fresh human melanoma cells to form colonies in soft agar. The retinoids studied had differential effects on the survival of clonogenic melanoma cells, and these effects vary from patient to patient. The data provide support for the clinical trial of selected retinoids in micrometastatic and advanced melanoma.

Polyamines as markers of response and disease activity in cancer chemotherapy.

One hundred twenty-four patients with hematological and solid neoplasms had pretreatment urinary polyamine determinations. Putrescine, spermidine, and spermine were all significantly increased as compared to normals (p less than 0.001). Polyamine levels were directly related to disease activity and tumor burden. In patients with multiple myeloma, putrescine levels were significantly correlated with clinical disease activity as well as the in vitro labeling index of marrow plasma cells. Spermidine values reflected tumor cell burden. Serial studies in 56 patients indicated that greater than twofold rise in urinary spermidine during treatment was highly correlated with cell kill and subsequent clinical response (p less than 0.001). Serum polyamine levels in 17 patients were found to be comparable to urinary values. Our data indicate that polyamine determinations can potentially be clinically useful, i.e., baseline values as indicators of tumor cell mass and growth fraction, and increases in spermidine during treatment as an excellent marker of tumor cell kill.

Relation of estrogen receptor expression to clonal growth and antiestrogen effects on human breast cancer cells.

Expression of estrogen receptor (ER) was studied in the ER-positive human breast cancer cell line MCF-7 using immunoperoxidase staining with monoclonal antibodies to ER. Using a soft agar colony assay and liquid culture, effects of growth and the antiestrogen tamoxifen were examined. Heterogeneity in expression of ER was observed between different clones in the agar cultures as well as among cells within the same clone. Clonal expression of ER increased progressively with increasing cell number within a clone. At pharmacological doses, tamoxifen significantly reduced clonal growth but also markedly reduced the expression of ER within clones that grew despite the presence of the antiestrogen. These findings are consistent with the hypothesis that ER-positive colonies arise from ER-negative progenitors and that ER expression occurs along with differentiation of cells within clones. Furthermore, the findings are consistent with tamoxifen exerting its antineoplastic action beyond the level of the tumor stem cell. Such therapy would therefore be capable of suppression but not eradication of breast cancer clonal progenitors.

Phase I clinical investigation of 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazone] dihydrochloride with correlative in vitro human tumor clonogenic assay.

9,10-Anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazone] dihydrochloride (bisantrene) is a new anthracene bishydrazone derivative which was entered into a Phase I clinical trial (one dose weekly for 3 weeks) because it showed significant antitumor activity in a number of animal tumor models and in vitro in the human tumor stem cell assay. When possible, patients were entered into the phase I study if their tumors showed in vitro sensitivity to bisantrene and resistance to standard agents, using a human tumor stem cell assay. Thirty-one patients were treated with bisantrene over a 10-month period, starting at a dose of 70 mg/sq m/week. The appearance of leukopenia determined the dose-limiting toxicity of bisantrene. The maximally tolerated dose appeared to be 200 mg/sq m in that three of five patients tolerated these weekly-for-3-weeks doses while experiencing only mild or moderate leukopenia. In contrast, the 220-mg/sq m dose caused moderate to life-threatening leukopenia after just two weekly doses in four of five patients. Local bisantrene toxicity included mild to severe arm swelling, phlebitis, pain, urticaria, and erythema in 68% of the patients. In general, these toxicities were well tolerated and rapidly reversible, but two patients had severe local swelling for up to 6 months. In this Phase I trial, bisantrene showed clinical antitumor activity against both hematological cancer (i.e., lymphoma and myeloma) and solid tumors (i.e., bladder, lung, and renal cancer and melanoma). Of importance, four of the six responses occurred in patients whose therapy was selected on the basis of in vitro sensitivity to bisantrene using the human tumor stem cell assay. One patient with disseminated melanoma had complete disappearance of an axillary node metastasis (for more than 6 months) while developing a brain metastasis, suggesting that bisantrene does not concentrate in the central nervous system.