Natural pet food: a review of natural diets and their impact on canine and feline physiology.

The purpose of this review is to clarify the definition of "natural" as it pertains to commercial pet food and to summarize the scientific findings related to natural ingredients in pet foods and natural diets on the impact of pet health and physiology. The term "natural," when used to market commercial pet foods or pet food ingredients in the United States, has been defined by the Association of American Feed Control Officials and requires, at minimum, that the pet food be preserved with natural preservatives. However, pet owners may consider natural as something different than the regulatory definition. The natural pet food trend has focused on the inclusion of whole ingredients, including meats, fruits, and vegetables; avoiding ingredients perceived as heavily processed, including refined grains, fiber sources, and byproducts; and feeding according to ancestral or instinctual nutritional philosophies. Current scientific evidence supporting nutritional benefits of natural pet food products is limited to evaluations of dietary macronutrient profiles, fractionation of ingredients, and the processing of ingredients and final product. Domestic cats select a macronutrient profile (52% of ME from protein) similar to the diet of wild cats. Dogs have evolved much differently in their ability to metabolize carbohydrates and select a diet lower in protein (30% of ME from protein) than the diet of wild wolves. The inclusion of whole food ingredients in natural pet foods as opposed to fractionated ingredients may result in higher nutrient concentrations, including phytonutrients. Additionally, the processing of commercial pet food can impact digestibility, nutrient bioavailability, and safety, which are particularly important considerations with new product formats in the natural pet food category. Future opportunities exist to better understand the effect of natural diets on health and nutrition outcomes and to better integrate sustainable practices in the production of natural pet foods.

Human leukemias with mutated FLT3 kinase are synergistically sensitive to FLT3 and Hsp90 inhibitors: the key role of the STAT5 signal transduction pathway.

17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of the molecular chaperone heat shock protein 90, results in cell type-specific inhibition of proliferation of leukemic cells. GTP14564 is a tyrosine kinase inhibitor actively against FLT3. The current study evaluated the single and combined effects of 17-AAG and GTP14564, and the role of FLT3 in their inhibitory effects. The importance of FLT3 mutations was demonstrated using small interfering RNA (siRNA) targeted to FLT3. Similar to FLT3 siRNA, GTP14564 inhibited FLT3 internal tandem duplication (ITD) cells (MV4;11) and FLT3 amplified wild-type cells (SEMK2-M1), but not wild-type FLT3 cells (RS4;11). However, when RS4;11 cells were stimulated with FLT3-ligand, phosphorylation of STAT5 and GTP14564 inhibition were observed. Responses to GTP14564 in all cell types were directly related to the level of STAT5 phosphorylation in the cells. We observed synergistic effects of combined 17-AAG and GTP14564 in cell lines with FLT3-ITD and amplified wild-type FLT3. Combined treatment with 17-AAG and GTP14564 reduced the levels of p-FLT3 and p-STAT5, enhanced G0/G1 arrest and apoptosis in FLT3-ITD and amplified wild-type FLT3. The combination of 17-AAG with FLT3 kinase inhibitors can enhance targeted therapy in leukemias with FLT3 mutations, such as MLL fusion gene leukemias.

Preclinical studies targeting normal and leukemic hematopoietic cells with Yttrium-90-labeled anti-CD45 antibody in vitro and in vivo in nude mice.

A study was undertaken to investigate the suitability of using a high affinity (Kd = 1.1 nM) anti-CD45 monoclonal antibody for delivering the high energy beta-particle emitting isotope (90)Y to lymphohematopoietic target cells in vivo. The antibody, AHN-12, recognized the tyrosine phosphatase CD45 expressed on the surface of normal and malignant hematopoietic cells and studies showed that it reacted with both CD45-expressing normal peripheral blood cells and leukemia cells from patients. The antibody was readily labeled with (90)Y using the highly stable chelate 1B4M-DTPA and the radioimmunoconjugate was designated (90)Y-anti-CD45. The agent selectively bound to CD45(+) B cell line Daudi, but not CD45(-) control cells and significantly (p = 0.007) more bound to Daudi tumors growing in athymic nude mice than did a control non-reactive antibody. Moreover, biodistribution data correlated well to an anti-Daudi effect observed against established tumors in nude mice. The effect was dose dependent and irreversible with the best results in mice receiving a single dose of 137 microCi (90)Y-anti-CD45. These mice displayed a significantly (p < 0.0095) better anti-tumor effect than a control (90)Y-labeled antibody and survived over 135 days with no evidence of tumor. Histology studies showed no significant injury to kidney, liver, or small intestine even at 254 microCi, the highest dose tested. Because radiolabeled anti-CD45 antibody can be used to deliver radiation selectively to lymphohematopoietic tissue, these data indicate that this agent may be used to improve treatment of hematopoietic malignancies, particularly leukemia and lymphoma, when combined with hematopoietic stem cell transplantation in a future clinical trial.

Phosphorus requirements of broiler chicks six to nine weeks of age as influenced by phytase supplementation.

Two trials of similar design were conducted to determine the nonphytate phosphorus (NPP) requirements for broilers from 42 to 63 d of age in diets with or without phytase supplementation. Male broilers of a commercial strain were grown to 42 d on nutritionally complete diets with NRC (1994) recommended levels of Ca and NPP. At 42 d, the birds were placed on experimental diets and fed to 63 d. The experimental treatments consisted of a 2 x 6 factorial arrangement with two levels of phytase (0 or 800 U/kg) and six levels of NPP (0.10 to 0.35% in 0.05% increments). Body weight gain, feed conversion, and mortality were determined during the period. At 49, 56, and 63 d, excreta samples were taken, and samples of birds were killed for tibia ash determination. The lowest level of NPP, with or without phytase supplementation, was sufficient for maximum BW gain, feed conversion, and livability. Using nonlinear regression, levels of NPP needed to optimize tibia ash in the absence of phytase were 0.31 +/- 0.004%, 0.23 +/- 0.02%, and 0.22 +/- 0.029% at 49, 56, and 63 d, respectively. When diets were supplemented with 800 U/kg of phytase, the NPP requirement for optimum tibia ash was 0.15 +/- 0.049% at 49 d. At 56 and 63 d, no more than 0.10% NPP (lowest level tested) was sufficient to maximize tibia ash. Compared to current NRC (1994) recommendations, the application of these reduced dietary phosphorus levels could markedly reduce excreta excretion of phosphorus by broilers.

Phosphorus requirements of broiler chicks three to six weeks of age as influenced by phytase supplementation.

Two studies of identical design were conducted in battery brooders utilizing male chicks of a commercial strain. The birds were grown to 3 wk on diets with adequate P and from 3 to 6 wk were fed diets ranging from 0.10 to 0.45% nonphytate P (nPP) in increments of 0.05%, with or without supplementation with 800 units of phytase per kilogram of diet. Measurements included BW gain, feed conversion ratio (FCR), mortality, tibia ash, and fecal P content. Nonlinear regression was used to estimate nPP needs for optimizing BW gain, feed conversion, and tibia ash. In the absence of phytase, nPP levels of 0.33, 0.186, and 0.163% were required to optimize tibia ash, BW gain, and FCR, respectively. The estimated level for optimum tibia ash is in close agreement with current NRC (1994) recommendations. In the presence of 800 units of phytase per kilogram, nPP levels of 0.24, 0.151, and 0.109% were needed to optimize tibia ash, BW gain, and FCR, respectively. Fecal phosphorus levels were markedly reduced at the lower P levels. Further studies are needed to determine whether maximum tibia ash values are needed to sustain optimum production of market broilers.

Evaluation of normal yellow dent corn and high available phosphorus corn in combination with reduced dietary phosphorus and phytase supplementation for broilers grown to market weights in litter pens.

A study was conducted to determine the extent fecal P levels could be reduced while maintaining performance. Various strategies were employed including the use of a high available phosphorus hybrid of corn (HAPC), supplementation with phytase enzyme, and reduced dietary P levels. The use of HAPC resulted in a 50% reduction in phytate-bound dietary P as compared with a normal yellow dent corn (YDC) diet. Dietary nonphytate P was maintained at either NRC (1994) recommendations for appropriate age periods or reduced by 0.075 or 0.15%. Portions of the diets were supplemented with 1,000 units of phytase/kg. Male chicks of a commercial strain were grown to 56 d on the test diets. Broilers fed diets with HAPC had BW, feed conversion, livability, and tibia ash that were equal to or superior to those fed diets with YDC with considerably reduced fecal P content at any dietary level of nonphytate P. Phytase supplementation enabled birds to maintain live performance at lower levels of nonphytate P, further reducing the fecal P output. One of the greatest contributions of phytase was a reduction in mortality at the lower levels of nonphytate P. Dietary P levels could be reduced by 0.075% under NRC (1994) recommendations without adversely affecting live performance; a reduction of 0.15% in conjunction with phytase supplementation maintained BW, feed conversion, and livability but reduced tibia ash. The extent to which dietary P levels can be reduced over the entire feeding program is subject to further research.

Nonphytate phosphorus requirement and phosphorus excretion of broiler chicks fed diets composed of normal or high available phosphate corn with and without microbial phytase.

A study was conducted to evaluate the ability of the young (0 to 3 wk) broiler chicken to utilize the P provided by a high available P corn [HAPC; 0.27% total P and 0.17% nonphytate P] in comparison with yellow dent corn (YDC; 0.23% total P and 0.03% nonphytate P), and to determine the extent to which supplementation with exogenous phytase enzyme could reduce the demands for dietary P and subsequently reduce P excretion. Diets prepared using the two types of corn differed in the amount of phytate-bound P, with the HAPC diets containing approximately 50% less phytate-bound P. Treatment diets were prepared by varying the amount of dicalcium phosphate, and ranged from 0.10 to 0.50% nonphytate P for YDC diets, and from 0.18 to 0.50% nonphytate P for HAPC diets. Sublots of each diet were supplemented with 800 units/kg phytase. Each diet was fed to six pens of five male chicks of a commercial broiler strain from 1 to 21 d of age. Regression analysis was used to estimate nonphytate P requirements for each corn type with and without phytase supplementation. The greatest need for nonphytate P was for maximum tibia ash, with requirements of 0.39, 0.29, 0.37, and 0.32% in diets with YDC, YDC plus phytase, HAPC, and HAPC plus phytase, respectively. Addition of phytase liberated approximately 50% of the phytate-bound P from each diet. These levels were sufficient to support body weight, feed conversion, and livability. Fecal P content of broilers fed diets with YDC at the NRC (1994) recommended level of 0.45% nonphytate P was 1.21%, whereas at the respective requirement points indicated above, the P content was 1.09, 0.87, 0.78, and 0.64% in feces from broilers fed diets with YDC, YDC plus phytase, HAPC, and HAPC plus phytase, respectively. Thus, fecal P output could be reduced while maintaining optimum performance by the use of reduced dietary nonphytate P, introduction of HAPC, and phytase supplementation. One of the greatest benefits of phytase supplementation appeared to be maintaining livability at lower dietary levels of nonphytate P.

Early testicular biopsy in males with acute lymphoblastic leukemia: lack of impact on subsequent event-free survival.

PURPOSE: Children with acute lymphoblastic leukemia (ALL) who had bulky disease (lymphomatous features) at diagnosis had the highest rate of testicular relapse (20%) of any ALL subgroup on previous Children's Cancer Group (CCG) studies in the late 1980s. To limit curative, but sterilizing, testicular irradiation to those with testicular disease, testicular biopsies were performed to detect occult testicular disease within the first 6 months of treatment. Testicular irradiation then was provided to those with occult disease to increase disease-free survival. Identification of those with occult disease was believed to be a factor that would influence ultimate survival in such patients in that era. PATIENTS AND METHODS: One hundred ninety-nine patients had bilateral testicular wedge biopsies performed during the first maintenance therapy phase of the four different chemotherapy regimens. Patients with positive biopsy results were treated with testicular irradiation and continued on therapy. RESULTS: Eleven of 199 biopsy results (5.5%) were judged positive. Patients with positive biopsy results given testicular radiation had a 45% subsequent adverse event rate, compared with 36% for those with a negative biopsy results (P = 0.4). The survival rates for the two groups were similar. The low rate of positive biopsy specimens resulted in discontinuation of the procedure before closure of the study. CONCLUSION: Positive testicular biopsy results early in remission identified patients at a slightly higher risk of subsequent adverse events but did not influence survival. However, because negative biopsy results (94.5%) did not alter the prescribed treatment, the small number of positive biopsy results did not warrant undertaking the procedure in most male patients with ALL, and this procedure was abandoned.

Patterns of infection and day care utilization and risk of childhood acute lymphoblastic leukaemia.

To investigate if decreased exposure to common childhood infections is associated with risk of childhood acute lymphoblastic leukaemia (ALL) we conducted a case-control study of 1842 newly diagnosed and immunophenotypically defined cases of ALL under age 15, and 1986 matched controls in the US. Data regarding day care, sibship size and common childhood infections were obtained through parental interviews. Data were analysed stratified by leukaemia lineage and separately for 'common' childhood ALL (age 2-5 years, CD19, CD10-positive). Neither attendance at day care nor time at day care was associated with risk of ALL overall or 'common' ALL. Ear infections during infancy were less common among cases, with odds ratios of 0.86, 0.83, 0.71 and 0.69 for 1, 2-4, 5+ episodes, and continuous infections respectively (trend P = 0.026). No effect of sibship size or birth interval was seen. With one exception (ear infections), these data do not support the hypothesis that a decrease in the occurrence of common childhood infection increases risk of ALL.

Xenotransplantation of human lymphoid malignancies is optimized in mice with multiple immunologic defects.

While it is known that mice with genetic immune defects are useful for establishing durable engraftment of human tumor xenografts, the relative role of components of host innate and adoptive immunity in engraftment has not been determined. We directly compared the ability of four strains of genetically immunodeficient mice (NOD/SCID, SCID, Nude and Rag-1-deficient) to successfully engraft and support the human cell lines Daudi, Raji, Namalwa and Molt-4 as subcutaneous tumors. We additionally examined the effect of further immunosuppression of the mice by whole body irradiation at a dose of 600 cGy for Nude and Rag-1 and 300 cGy for SCID mice and by administration of anti-natural killer (asialo-GM1) antibody on tumor growth. Mice with each of the defects supported xenografts to varying degrees. We found differences in growth characteristics in the cell lines tested, with Namalwa consistently producing the largest tumors. With all cell lines studied, optimal growth was achieved using NOD/SCID mice. Overall, tumor growth was somewhat enhanced by pretreatment with radiation with little additional benefit from the addition of anti-asialo-GM1 antibody. The importance of multiple components of the innate and adoptive immune system in xenotransplantation were best demonstrated when results in untreated NOD/SCID mice were compared to SCID, nude and RAG-1-deficient mice. The NOD/SCID mouse with or without additional immunosuppression provides the optimal model for the study of the biology and treatment of human leukemias and lymphomas.

AF4 encodes a ubiquitous protein that in both native and MLL-AF4 fusion types localizes to subnuclear compartments.

Acute leukemia with t(4;11)(q21,q23) translocation results from the in-frame fusion of the MLL to the AF4/FEL gene. In previous studies, we and others demonstrated that AF4 transcripts are present in a variety of hematopoietic and nonhematopoietic human cells. To further study the wild-type and leukemia fusion AF4, we used glutathione S-transferase (GST)-fusion proteins as immunogens to produce rabbit polyclonal antibodies that were specific for normal and chimeric AF4 proteins. Using Western blotting analysis, we demonstrated that the AF4 gene encodes proteins with apparent molecular weight of 125 and 145 kD. A 45-kD protein coprecipitated with AF4 protein in immunoprecipitation. Also, the anticipated MLL-AF4-encoded 240-kD protein was detected in all cell lines with t(4;11) translocations; fusion proteins were present in lesser quantity than the wild-type AF4. The proteins recognized by the antibodies are of the predicted sizes of the AF4 and MLL-AF4-encoded proteins based on previous DNA sequencing analysis. The MLL-AF4 fusion protein had a similar subcellular distribution as AF4. Both t(4;11) and non-t(4;11) leukemic cells showed a similar pattern of punctate nuclear staining in all cell lines tested using confocal immunofluorescence microscopy. AF4 antibodies should be useful for further elucidation of the function of AF4 in normal cellular physiology, as well as the function of MLL-AF4 in leukemogenesis. The antibodies should also be helpful for the diagnosis of the MLL-AF4 fusion proteins in t(4;11) leukemias.

Pharmacokinetics and biodistribution of radioimmunoconjugates of anti-CD19 antibody and single-chain Fv for treatment of human B-cell malignancy.

The comparative advantages and disadvantages of intact antibodies and single-chain Fv as immunotoxins and radioimmunoconjugates have been widely discussed but not directly compared. In this study, the in vivo properties of anti-CD19 B43 monoclonal antibody and its derived single-chain Fv (FVS191) were studied in athymic nude mice bearing CD19-positive human lymphomas. B43 mab and FVS191 were labeled with iodine-125 using iodine-beads, and immunoreactivities were determined to be 57% and 72%, respectively. Scatchard analysis showed a similar high affinity for both. The results of pharmacokinetic studies revealed that FVS191 had a rapid biphasic clearance from the circulation (T1/2alpha=2.5 min, T1/2beta=3.7 h); The T1/2alpha and T1/2beta phases of B43 mab were determined to be 0.72 h and 57 h respectively. Biodistribution studies compared the uptake of labeled antibodies by CD19-positive and by CD19-negative tumors. The peak percentages of injected dose were 5.7% at 12 h for B43 and 2.45% at 1 h for FVS191. Radiolocalization indices (RI) demonstrated tumor-specific uptake for both, but higher uptake for B43. The optimal RI was seen at 15 min for FVS191 and 6 h for B43. FVS191 was unstable in vivo, approximately 50% of the injected dose being degraded in blood in 100 min. Radioactivity detected in the urine was present mainly as the deiodinized form of FVS191. The results suggest that B43 mab is favored over FVS191 in biodistribution properties and in vivo stability. Because B43 Mab showed early tumor-specific uptake, high RI values, and favorable tissue-to-blood ratios, it is a potential candidate for radioimmunotherapy and immunotoxin therapy of B-cell leukemia and lymphoma.

Resistance of t(4;11) (MLL-AF4 fusion gene) leukemias to stress-induced cell death: possible mechanism for extensive extramedullary accumulation of cells and poor prognosis.

Acute leukemias of the t(4;11) (MLL-AF4 fusion gene) type frequently have high white blood counts and extramedullary disease in multiple organs. In the present study we evaluated the hypotheses that this extensive disease is the result of extramedullary survival of leukemia cells due to resistance to stress-induced cell death. Leukemias with t(4;11)(MLL-AF4) were found to be resistant to the cell death that results from serum deprivation in vitro when compared with B lineage acute leukemias without t(4;11)(MLL-AF4). Cells with t(4;11)(MLL-AF4) did not have increased doubling time or increased numbers of cells in cycle. These results suggest that the alteration in cellular homeostasis in these leukemias is due to abnormalities of cellular destruction rather than cellular proliferation when compared to other leukemias. Our results are consistent with the hypothesis that death of non-t(4;11) leukemias occurs in the microenvironment outside of the bone marrow as a result of deficient cellular and humoral growth factors. Resistance to death signals in t(4;11) leukemias results in extensive accumulation of leukemia cells in extramedullary sites and likely contributes to the poor prognosis of these leukemias.

Utilization of spent hen meal in diets for broiler chickens.

Studies were conducted to evaluate spent hen meal (SHM) produced in commercial rendering plants as a nutrient source in diets for broiler chickens. Utilizing previously determined nutrient composition values, including digestible amino acid and TMEn content, diets were formulated to include 0, 5, 10, and 15% of SHM from three different locations. In the first experiment, conducted in battery pens from 1 to 21 d posthatch, diets were formulated with digestible amino acid requirements set at 90, 95, or 100% of NRC (1994) total amino acid requirements. In the second experiment, conducted in floor pens from 1 to 49 d posthatch, diets were formulated with digestible amino acid requirements set at 95% of NRC (1994) total amino acid requirements. Samples of birds from the second experiment were processed to determine the possible influence of SHM inclusion on carcass yield. Results of the present studies indicate that SHM from commercial rendering facilities can be utilized in diets for growing broiler chickens provided adjustments are made in nutrient content and digestibility. When formulated on the basis of digestible amino acid content, levels of SHM up to 10% appear acceptable based upon body weight, feed conversion, bone ash, and carcass yield. Higher inclusion rates generally reduced performance. It is apparent that differences in nutritional quality may exist among products produced by different rendering facilities; however, evaluation of products to assess nutrient quality may be difficult under commercial conditions. As more information is generated regarding typical amino acid content and digestibility of rendered SHM, the product may be used with greater confidence in commercial diets.

Treatment of patients with acute lymphoblastic leukemia with bulky extramedullary disease and T-cell phenotype or other poor prognostic features: randomized controlled trial from the Children's Cancer Group.

BACKGROUND: Children with acute lymphoblastic leukemia with multiple poor prognostic factors and who have a lymphomatous mass at diagnosis, whether of T- or non-T-immunophenotype, are at increased risk of short term remission and extramedullary recurrence, and are in need of better therapies. METHODS: Six hundred and ninety-four eligible patients ranging in age from 1-20 years were entered on the study. Sixty-five percent of the patients had T-cell immunophenotype. Of these, 678 were randomized to one of four regimens: Regimen A: Berlin-Frankfurt-Munster (BFM) 76/79; Regimen B: LSA2-L2 with cranial irradiation; Regimen C: LSA2-L2 without cranial irradiation; and Regimen D: the New York (NY) regimen. RESULTS: Complete remission was induced in 97% of patients. The overall event free survival (EFS) +/- the standard deviation was 60 +/- 4% 6 years after diagnosis, in contrast to 36 +/- 6% in a comparable historic group. The EFS of the 371 T-cell patients was 62 +/- 7%. EFS was best on the NY (67 +/- 7%) and the BFM (67 +/- 6%) arms. These were significantly better than the EFS on the 2 LSA-L2 regimens, with an EFS of 53 +/- 8% (Regimen B) and 42 +/- 11% (Regimen C) (P = 0.03 and 0.0003 for NY vs. Regimen B and NY vs. Regimen C; P = 0.01 and 0.0001 for BFM vs. Regimen B and BFM vs. Regimen C). Regimen C had a 3-fold greater central nervous system (CNS) recurrence rate than the identical chemotherapy Regimen B (16 +/- 5% vs. 6 +/- 4%; P = 0.02), although the difference in overall EFS did not reach the required level for significance. Testicular recurrence varied from 2-8% in comparison with 20% in the historic group. EFS was not influenced by age, gender, CNS disease at diagnosis, morphology, or immunophenotype. In addition to treatment regimen and early response rate, initial leukocyte count, hemoglobin level, liver, spleen, and lymph node enlargement, and the presence of a mediastinal mass had univariate prognostic influence on EFS. In multivariate analysis, only the kinetics of response, leukocyte count (unfavorably, P < 0.0001), and mediastinal mass status (favorably, P = 0.01) were prognostic. CONCLUSIONS: The adverse prognostic implications of lymphomatous ALL can be minimized by the NY and BFM regimens. Cranial irradiation resulted in better CNS disease control when added to the LSA2-L2 regimen, but did not improve the overall disease free survival. With improved systemic chemotherapy, there was no excess of lymph node, testicular, or other local recurrence without prophylactic irradiation to sites of initial bulk disease or to the testes.

Generation and characterization of an anti-CD19 single-chain Fv immunotoxin composed of C-terminal disulfide-linked dgRTA.

Our laboratory utilized two methods to produce the anti-CD19 immunotoxin containing a single-chain Fv (scFv) FVS191 and a ricin A chain (RTA). The first method produced the recombinant protein FVS191CDRTA from a fusing gene containing sequences encoding FVS191, catheptsin D proteinase digestion site (CD), and RTA. FVS191CDRTA did not show CD19 antigen binding and cytotoxic activity. The second method generated a disulfide-linked FVS191cys-dgRTA from a FVS191cys, the FVS191 with an additional C-terminal cysteine, and a deglycosylated RTA (dgRTA). The formation of FVS191cys-dgRTA is efficient; up to 70% of the proteins participating in the reaction had formed FVS191cys-dgRTA when the molar ratio of FVS191cys to dgRTA was 1:1. A competitive ELISA assay indicated that FVS191cys-dgRTA and the parental monoclonal antibody B43 possessed comparable CD19 binding abilities. The protein synthesis inhibition assay revealed that FVS191cys-dgRTA was toxic to CD19 positive cell lines, but it was less potent than the intact antibody-conjugated B43-dgRTA, which had an IC50 = 2 x 10(-11) M. 125I-Labeled FVS191 and 125I-labeled B43 were internalized by Nalm-6 cells at 37 degrees C as demonstrated by internalization studies; this result indicates that cross-linking of CD19 antigen is not required for the endocytosis of CD19 and raises the possibility that the lower cytotoxity of FVS191cys-dgRTA is not due to the monovalent binding of CD19 by FVS191cys-dgRTA. Our study with anti-CD19 scFv immunotoxin indicates that the formation of a disulfide-linked scFv immunotoxin is an alternative to the recombinant method of producing scFv immunotoxin.

An in vitro model for toxin-mediated vascular leak syndrome: ricin toxin A chain increases the permeability of human endothelial cell monolayers.

Vascular leak syndrome (VLS) is the dose-limiting toxicity observed in clinical trials of immunotoxins containing ricin toxin A chain (RTA). RTA itself is thought to cause VLS by damaging vascular endothelial cells, but the exact mechanism remains unclear. This is partially due to the paucity of appropriate models. To study VLS, we developed an in vitro model in which human umbilical vein-derived endothelial cells were first grown to confluence on microporous supports and then cultured under low pressure in the presence or absence of RTA. Endothelial cell barrier function was assessed by measuring the volume of fluid that passed through each monolayer per unit time. We found that RTA significantly increased monolayer permeability at times and concentrations consistent with the onset of VLS in patients treated with RTA-based immunotoxins. Scanning electron microscopy showed that intercellular gaps formed in endothelial monolayers exposed to RTA. Intercellular gap formation followed endothelial cell death caused by the enzymatic activity of RTA. We conclude that RTA is directly toxic to endothelial cells in vitro and speculate that this contributes to VLS in vivo.

Therapy of patients with T-cell lymphomas and leukemias using an anti-CD7 monoclonal antibody-ricin A chain immunotoxin.

Anti-CD7-dgA, DA7, consists of deglycosylated ricin A chain coupled to a mouse monoclonal anti-human CD7 antibody. This study determined the maximally tolerated dose (MTD) of this immunotoxin administered as a one hour infusion over five days to 11 patients with T-cell lymphoma (>30% CD7+ malignant cells). The MTD was 0.2 mg/kg/day or 1 mg/kg/120 hours (maximal toxicity grade 3) with vascular leak syndrome (VLS) as dose-limiting toxicity (DLT). Predictors of severe VLS included age and absence of circulating lymphoma cells. Two partial responses and one minimal response were seen. Patients with minimal lymphoma burden or T-cell large granular lymphocyte (LGL) leukemia showed the best responses. The mean maximal serum concentration of immunotoxin at the MTD was 2.5 ug/ml. The mean alpha-phase half-life was 1.5 hours and the mean beta-phase half-life was 8 hours. Repeated dosing had minimal effects on either peak serum immunotoxin concentrations or serum half-lives. While human antimouse antibodies were observed, they were low in concentration (<55 ng/ml). Human anti-ricin antibody was elevated in one patient (190 ng/ml). VLS presented with hypoalbuminemia, dyspnea, pulmonary edema, aphasia, and peripheral edema and cleared over a two week period. Serum fibronectin levels were measured in three patients and were very low in one patient who developed VLS. No specific binding of DA7 immunotoxin was seen with vascular endothelium in various human tissues.