Repeatability of subjective evaluation of lameness in horses.

REASONS FOR PERFORMING STUDY: Previous studies have suggested that agreement between equine veterinarians subjectively evaluating lameness in horses is low. These studies were limited to small numbers of horses, evaluating movement on the treadmill or to evaluating previously-recorded videotape. OBJECTIVES: To estimate agreement between equine practitioners performing lameness evaluations in horses in the live, over ground setting. METHODS: 131 mature horses were evaluated for lameness by 2-5 clinicians (mean 3.2) with a weighted-average of 18.7 years of experience. Clinicians graded each limb using the AAEP lameness scale by first watching the horse trot in a straight line only and then after full lameness evaluation. Agreement was estimated by calculation of Fleiss' (kappa). Evaluators agreed if they picked the same limb as lame or not lame regardless of the severity of perceived lameness. RESULTS: After only evaluating the horse trot in a straight line clinicians agreed whether a limb was lame or not 76.6% of the time (kappa= 0.44). After full lameness evaluation clinicians agreed whether a limb was lame or not 72.9% of the time (kappa= 0.45). Agreement on forelimb lameness was slightly higher than on hindlimb lameness. When the mean AAEP lameness score was >1.5 clinicians agreed whether or not a limb was lame 93.1% of the time (kappa= 0.86), but when the mean score was < or = 1.5 they agreed 61.9% (kappa= 0.23) of the time. When given the task of picking whether or not the horse was lame and picking the worst limb after full lameness evaluation, clinicians agreed 51.6% (kappa= 0.37) of the time. CONCLUSIONS: For horses with mild lameness subjective evaluation of lameness is not very reliable. POTENTIAL RELEVANCE: A search for and the development of more objective and reliable methods of lameness evaluation is justified and should be encouraged and supported.

Characterization of human tumor-infiltrating lymphocytes expanded in hollow-fiber bioreactors for immunotherapy of cancer.

We attempted to grow tumor-infiltrating lymphocytes (TIL) from 34 fresh tumors of eight different histologies using flasks for the initiation phase and hollow fiber bioreactors to expand TIL to therapeutic numbers. Overall success rate was 76% (26/34) including melanoma (9/14, 64%) and renal cell carcinoma (11/11, 100%). The mean number of days required to reach successful initiation (1 x 10(9) TIL) for all tumor types was 29 +/- 16 days (mean +/- S.D.). Therapeutic doses of TIL required an average of 88 +/- 23 days (initiation plus expansion) with an average TIL number of 3.2 x 10(10) +/- 2.8 x 10(10). TIL phenotype was predominantly CD4+ in 53% (16/30) and CD8+ in 47% (14/30), renal cell carcinoma samples accounted for 12/14 of the predominantly CD8+ TIL. Cells bearing the natural killer (NK) phenotype represented only 0-7% of TIL while LAK phenotype represented 0-68% (mean 11 +/- 15%); LAK was the predominant phenotype in one patient with kidney cancer. Cytotoxicity tests showed consistent NK and LAK activity in addition to cytolysis of autologous tumor. Autologous tumor cell restricted cytolysis was noted for three TIL cultures. The overall success rate and characteristics of TIL were similar to our results with TIL expanded in semi-permeable plastic bags. This work confirms that hollow-fiber bioreactors are a suitable alternative to semi-permeable bags and roller bottle systems for the expansion of human TIL for therapeutic use in cancer patients.

Combination interferon-alpha2a and 13-cis-retinoic acid enhances radiosensitization of human malignant glioma cells in vitro.

We investigated the individual and combined effects of cis-retinoic acid (CRA) and/or IFN-alpha (IFN) and/or radiation therapy (RT) against a human glioma cell line (American Type Culture Collection; U373MG) to evaluate the possible radiosensitization properties of these agents in vitro. Glioma cells were incubated for 24 h in 96-well plates (2 x 10(2) cells/well) in standard culture medium. Sets of U373 (n = 12) were exposed to CRA (3 x 10(6) microM), IFN (25 units/ml), CRA plus IFN, or standard culture medium. After an additional 24 h of incubation, the U373 cells were subjected to increasing radiation doses (up to 16 Gy). Glioma cells were harvested 92 h after irradiation, and cell survival curves were determined from [3H]thymidine incorporation data (over the last 24 h). The experiment was repeated for both the untreated control group and the combined CRA/IFN group. To verify the [3H]thymidine assays, a clonogenic assay was also performed. Single cell suspensions of U373 cells were plated out in six-well plates (n = 3). After chemical and RT treatment, colonies of 50 cells or more were counted, and cell survival curves were generated as fractions of nonirradiated controls. The amount of RT (in Gy) that would cause a 50% survival fraction (lethal dose 50 or LD50) was calculated from the survival curves by regression analysis. The following LD50s were obtained: [table: see text] The results showed that for both the [3H]thymidine incorporation assay and the clonogenic assay, the combination of IFN/CRA rendered U373 cells more susceptible to ionizing radiation than the untreated control or either single agent alone.

Modulation of renal carcinoma cells in vitro: comparison after transduction with retroviral vector containing a human IFN-gamma gene versus incubation with soluble IFN-gamma.

Transduction of renal cell carcinoma (RCC) cells in vitro with a gene for human interferon-gamma (IFN-gamma) results in enhanced expression of key molecules needed for immune recognition. In this study, we investigated the ability of soluble IFN-gamma protein to enhance expression of these key molecules. RCC cells were incubated with and without IFN-gamma, then analyzed by flow cytometry for the percent positive and mean intensity fluorescence (MIF), a measurement of mean antigen density, of HLA-I, HLA-II, ICAM-1, and tumor antigens (URO-2, URO-3, and URO-4). Results were compared to those seen for RCC cells transduced by IFN-gamma. The ability of cytotoxic T lymphocytes (CTL) to lyse RCC targets was measured using a standard [51Cr]lytic assay. Control cells were 99% (MIF 751) and 98% (MIF 315) positive for HLA-I and ICAM-1, respectively, with 2% (MIF 8) positive for HLA-II. Incubation with IFN-gamma protein resulted in 98% positive HLA-I (MIF 2288), 98% positive ICAM-1 (MIF 1132), and 95% positive HLA-II (MIF 287). The results for the cells incubated with IFN-gamma protein were similar to those for the transduced line. Importantly, the enhanced expression was maintained for several days after irradiation and cryopreservation. Expression of the URO tumor markers was not affected. Protein-treated RCC targets showed superior CTL lysis compared with untreated cells. Our results show that IFN-gamma protein incubation in vitro enhances expression of important immune recognition molecules to levels expressed by transduced cells. Increased expression may enhance tumor recognition by the host's immune system, as in the case of tumor cell vaccines. There may be no advantage to IFN-gamma transduction over in vitro incubation with IFN-gamma protein.

Inhibition of lysophosphatidic acid-induced neurite retraction and cell rounding by SR 57746A.

Rapid neurite retraction and transient rounding of serum-starved NG108-15 and PC12 cells by lysophosphatidic acid (LPA) is retarded and reduced by pre-incubation of the cells with the small non-peptidic molecule, SR 57746A, which exhibits neurotrophic properties. The compound also antagonizes the redistribution of filamentous actin by LPA in both cell types. We hypothesize that the SR 57746A attenuation of LPA-induced effects may account for at least some of the neuroprotective properties of this molecule.

Characterization of tumor-infiltrating lymphocytes derived from human tumors for use as adoptive immunotherapy of cancer.

From 1991 to 1995, we initiated cultures of 94 fresh tumor samples of various histologies in an effort to grow tumor-infiltrating lymphocytes (TIL) using flasks and subsequent expansion in semipermeable bags. The five most prevalent tumor types from which TIL were successfully initiated were melanoma (25 successful initiates in 34 tumor samples, 74% success rate), colorectal cancer (12 of 18, 67%), renal cell carcinoma (9 of 12, 75%), breast (4 of 5, 80%), and sarcoma (5 of 7, 71%). The overall success rate for all tumors was 67 of 94 (71%). There were no instances of contamination from the time of culture initiation through harvesting of the final cell product for clinical use. The mean number of days to reach successful initiation (> 5 x 10(8) cells) was 35 +/- 24 days (mean +/- SD). TIL were then expanded from these successful initiates for either a repeated low-dose therapy (TIL reinfusion numbers of 5 x 10(8)-5 x 10(9) or for a repeated high-dose therapy (> 5 x 10(9)-5 x 10(10). The mean number of days to expand a TIL culture from the time of initiation to treatment for a first low-dose TIL was 59 days (range, 27-94 days) compared with 80 days (range, 33-209 days) for high-dose TIL. For patients who received a second or third high-dose TIL treatment, the average number of days needed to expand TIL was 39 days (n = 10) if there was no intervening cryopreservation of TIL, compared with 49 days (n = 10) if the culture had to be reestablished from cryopreserved TIL. For patients who received a second or third low-dose TIL, the mean number of days needed to expand TIL was 23 days (n = 3) if there was no intervening cryopreservation compared with 42 days (n = 17) if cultures had to be reestablished after cryopreservation of TIL. Low-dose TIL displayed predominantly CD4+ phenotype in 76% of 42 cultures, whereas high-dose TIL displayed predominantly CD8+ phenotype in 84% of 44 cultures. Cells bearing the natural killer (NK) phenotype (CD3-, CD56+) and the lymphokine activated killer (LAK) phenotype (CD3+, CD56+) were present in both low- and high-dose TIL cultures, but these phenotypes were never predominant. Cytotoxicity testing consistently demonstrated the persistence of NK and LAK activity in addition to the killing of allogeneic and autologous melanoma tumor targets. This work confirms that TIL cultures from most tumor types can be successfully established and expanded for therapeutic use, and repeated expansion from continuous TIL culture or cryopreserved TIL for repeated treatments is feasible. Such cultures are predominantly T lymphocytes that are phenotypically heterogeneous, and these phenotypes do not remain constant during prolonged time in culture.

Tumor localization by tumor infiltrating lymphocytes labeled with indium-111 in patients with metastatic renal cell carcinoma, melanoma, and colorectal cancer.

BACKGROUND: Adoptive immunotherapy with autologous tumor infiltrating lymphocytes (TIL) is a promising approach for cancer bio-therapy. One issue, however, is whether such cells actually migrate to sites of tumor after intravenous infusion. There have been several reports of tumor uptake of radiolabeled TIL in patients with metastatic melanoma, but efforts to visualize tumor with radiolabeled TIL in other tumor types reportedly have been unsuccessful. METHODS: Eight patients with metastatic cancer (5 renal, 2 melanoma, 1 colon) received an intravenous infusion of 2 to 100 billion autologous TIL, including 50 million TIL which had been conjugated to 500 microCi Indium-111, co-administered with interleukin-2 (IL-2). One patient received 1 gm/m2 of cyclophosphamide one day prior to TIL; seven patients received interferon alpha 2b for 4 days prior to receiving TIL. Total body gamma camera imaging, including single photon emission computerized tomography (SPECT), was performed at 24 and 48 hours. RESULTS: All eight patients had demonstrable uptake of 111-Indium-labeled TIL into one or more known sites of tumor. There were no known sites of tumor which were not imaged. Metastatic sites imaged included bone, brain, mediastinal and perihilar lymph nodes, lung and liver parenchyma, abdominal periaortic nodes, and a pelvic mass. One patient served as a negative control in that the TIL scan was negative at a time when she had no evident disease, but a few weeks later had a positive TIL scan which lead to a diagnosis of axillary recurrence. CONCLUSION: Uptake of radiolabeled TIL, whether CD8+ or CD4+, by metastatic renal cell carcinoma and other carcinomas was similar to that previously reported in melanoma. Pretreatment with cyclophosphamide was not a prerequisite for imaging, and TIL uptake did not predict tumor response.

Evidence for nerve growth factor-potentiating activities of the nonpeptidic compound SR 57746A in PC12 cells.

SR 57746A (1-[2-(naphth-2-yl)ethyl]-4-(3-trifluoromethylphenyl)-1,2,5,6- tetrahydropyridine hydrochloride) exhibits neurotrophic activities in vivo and in vitro. We used the rat pheochromocytoma PC12 cell line to investigate in vitro cellular changes induced by SR 57746A. A significant increase in the percentage of cells bearing neurite-like processes was obtained in cells treated by SR 57746A and nerve growth factor (NGF) compared with NGF treatment alone. SR 57746A added alone, however, had no effect on morphogenesis or on survival of cells in serum-free medium. In contrast, SR 57746A induced a "priming" effect on PC12 cells for neurite outgrowth within 6 h of addition of the protein tyrosine kinase inhibitor genistein. An increase in alpha-actinin content resulted from treatment with SR 57746A. Expression of NGF-mediated acetylcholinesterase and choline acetyltransferase was enhanced within 5 days by SR 57746A. The molecule also induced rapid F-actin redistribution. Within 2 min of incubation, outgrowth of F-actin-containing filopodia was clearly visible at the cell periphery, as previously shown with NGF. It is interesting that this effect of SR 57746A could be mimicked by protein tyrosine kinase inhibitors and abolished by preincubation with sodium orthovanadate, a protein tyrosine phosphatase inhibitor.

Migration of activated lymphocytes when adoptively transferred into cannulated rat brain.

Migration of fluorescent DNA-labeled or 111Indium-labeled activated lymphocytes was studied in normal rat brain bearing surgically implanted cannulas. The migration of allogeneic cytotoxic T lymphocytes (CTL), derived from the DA rat (DA anti Fischer CTL), and of syngeneic concanavalin A (ConA)-activated lymphocytes (Fischer Con A blasts), was determined in Fischer rats between 2 h and 7 days post instillation into parietal brain. Whole body nuclear imaging indicated that the majority of the radiolabeled lymphocytes, either syngeneic or allogeneic, were present in the brain at 2 and 18 h. Autoradiography of brain slices demonstrated that label was located throughout the brain and in both hemispheres at all time points. By direct tissue radioassay, approximately 60% of the injected dose was present between 2 and 18 h; this decreased to 18% by day 7. By fluorescence microscopy, large numbers of lymphocytes were visible up to 3-4 days. The lymphocytes traveled from the instillation site into both cerebral hemispheres primarily following white matter tracts. Preferential localization of fluorescently labeled lymphocytes was seen in the corpus callosum, internal and external capsules, anterior commissures, lateral olfactory tracts, white matter connections in the caudate and putamen, mammillothalamic and optic tracts. Overall, gray matter contained fewer cells although perivascular spaces within it had high concentrations of cells, indicating these spaces may act as points of egress.

Increased ICAM-1 expression in the early stages of murine chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is considered to be a model for scleroderma, and vascular changes are considered to be important in that disease. We have examined the expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function associated antigen-1 (LFA-1) using monoclonal antibodies and immunohistochemistry in very early murine cGVHD. ICAM-1 is found on a number of cell types including endothelial cells. It is the natural ligand for LFA-1 found on leukocytes. Adherence of leukocytes to ICAM-1 positive cells is mediated by LFA-1 and this binding is thought to play an important role in a number of cell adhesion events in immune reactions. Experimental cGVHD across minor histocompatibility barriers is established by the iv inoculum of B10.D2 spleen cells into a sublethally irradiated BALB/c host. Ear and skin biopsies were taken at Days 0-5 and from Days 14 to 120 postinoculum. In comparison to the control group (BALB/c spleen cells given to a sublethally irradiated BALB/c host), the cGVHD mice show increased ICAM-1 expression by Day 3 on endothelial cells and mononuclear cells (MNC) and on fibroblast-like cells by Day 4. By Day 14, there are increasing numbers of ICAM-1-expressing cells and increased epidermal reactivity for ICAM-1; and finally, an increased number of LFA-1 positive infiltrating MNC. These changes wane by Day 28 and are gone by Day 120. These results support the concept that ICAM-1/LFA-1 interactions play a role in the immune regulation of cGVHD. The very early upregulation of ICAM-1 on the endothelium indicates that this cell type may be playing a primary role in cGVHD, and this model should provide a simple system in which to test regulation of endothelial activation in vivo.

Intracranial administrations of single or multiple source allogeneic cytotoxic T lymphocytes: chronic therapy for primary brain tumors.

Previous investigations by our group demonstrated the efficacy of single source allogeneic cytotoxic T lymphocytes (CTLs) given multiple times in reducing or curing tumor burden in the rat 9L gliosarcoma model. In this study, the lack of toxicity to normal brain when single source allogeneic CTLs were intracranially administered multiple times is documented. Additionally, the efficacy and lack of toxicity of allogeneic CTLs from multiple sources, each given once is documented. CTLs sensitized to Fischer antigen were prepared from major histocompatibility complex incompatible DA, PVG, Sprague-Dawley and Wistar-Furth rat lymphocytes. CTLs from multiple donors were administered one time each to Fischer rats bearing established 9L tumor at staggered intervals over a two week period and survival was monitored in relation to a sham treated group. Additional groups of nontumor-bearing rats received either multiple source allogeneic CTLs or single source DA anti Fischer CTLs in the same treatment regimen. Histological evaluation of the nontumor-bearing brains receiving either single or multiple source allogeneic CTL infusions showed minimal localized brain damage confined to the cannulation tract. No neuronal loss or inflammatory reaction was seen either adjacent to or remote from the administration site. Brains from the long-term survivors of the tumor-bearing animals showed no residual neoplasm; the instillation site had focal sterile abscesses; gliosis and neuronal loss did not extend into adjacent brain. The safety and potential of chronic, local allogeneic CTL administration, derived from multiple donors, as adjuvant local therapy for brain tumors was demonstrated.

A rat glioma model, CNS-1, with invasive characteristics similar to those of human gliomas: a comparison to 9L gliosarcoma.

A glioma cell line, CNS-1, was developed in the inbred Lewis rat to obtain a histocompatible astrocytoma cell line with infiltrative and growth patterns that more closely simulate those observed in human gliomas. Rats were given weekly intravenous injections for a six month period with N-nitroso-N-methylurea to produce neoplasm in the central nervous system. Intracranial tumor was isolated, enzymatically and mechanically digested, and placed into culture. The tumor cell line injected subcutaneously on the flanks of Lewis rats grew extensively in situ as cohesive tumor masses but did not metastasize. Intracranially, CNS-1 demonstrated single cell infiltration of paranchyma and leptomeningeal, perivascular, and periventricular spread with expansion of the tumor within choroid plexus stroma. CNS-1 cells titrated in right frontal brain of Lewis rats at 10(5), 5 x 10(5), 10(5), 5 x 10(4) cells per group had mean survival times ranging from 20.5 to 30.2 days. CNS-1 was immunoreactive for glial fibrillary acidic protein, S100 protein, vimentin, neural cell adhesion molecule, retinoic acid receptor alpha, intercellular adhesion molecule, and neuron specific enolase. The CNS-1 cells commonly had one or more trisomies of chromosomes 11, 13 or 18; losses, possibly random, of chromosomes (3, 5, 19, 30, X or Y) were noticed, and a marker chromosome made up of approximately 3 chromosomes was usual. Comparisons of CNS-1 to 9L gliosarcoma tumor were made. The glial CNS-1 tumor model provides an excellent system in which to investigate a variety of immunological therapeutic modalities. It spreads within brain in a less cohesive mass than 9L and is accepted without rejection in non-central nervous system sites by Lewis rats.

Mast cell immunohistochemistry: non-immunological immunostaining mediated by non-specific F(ab')2-mast cell secretory granule interaction.

During investigations of murine and human mast cell immunoreactivity with potential anti-interleukin-4 antibodies, non-specific, non-immunological labelling of mouse and human mast cells became apparent. Non-specific, non-immunological labelling was identified by (i) immunolabelling of mast cells when using control isotype primary antibodies, (ii) ability of conjugated secondary antibodies to label mast cells without prior mast cell exposure to a primary antibody, (iii) extinction of the non-specific labelling and retention of specific labelling when the pH of the diluting and washing buffers is shifted from pH 7.2 to pH 6.0, and (iv) reduction/extinction of the labelling when the antibodies are pre-incubated with soluble heparin prior to immunostaining. The site of the reactivity on the electron microscope level was shown to be confined to the mast cell secretory granules. The results of this study support the hypothesis that non-specific labelling of mast cells results from an ionic interaction between the F(ab')2 segments of antibodies and the heparin constituent of the mast cell secretory granules. This study points out the necessity of stringent controls when using immunohistochemistry to determine mast cell reactivity to various antibodies.

Insulin-like growth factor binding protein-4 inhibits both basal and IGF-mediated chick pelvic cartilage growth in vitro.

This laboratory has purified a unique insulin-like growth factor binding protein (IGFBP-4) that was previously demonstrated to be inhibitory to bone cell proliferation. In this study, the hypothesis that IGFBP-4 is inhibitory to insulin-like growth factor (IGF) actions on cartilage was tested using the pelvic cartilages of 10-day-old chick embryos as an in vitro model system. Pelvic leaflets were incubated in serum-free medium for 18 h with effectors (BSA, IGF-I, IGF-II, IGFBP-4, or a combination of IGF and IGFBP-4). After the first 8 h, 1.5 microCi [3H]thymidine per well was added. Cartilage growth was assayed by TCA-insoluble [3H]thymidine incorporation into DNA. Additional experiments were conducted under similar conditions to assess the actions of the effectors on cartilage dry weight over a 72 h time period. In separate experiments, serum-free medium conditioned by chick pelvic cartilages for 72 h was assayed for IGF-II by radioreceptorassay, IGF-I by radioimmunoassay, and IGFBP by western ligand analysis. Exogenous IGF addition increased [3H]thymidine incorporation and dry weight of cartilages compared to controls. IGFBP-4 decreased both parameters in basal cartilage growth and also inhibited IGF-mediated cartilage growth. Pelvic cartilages secreted in vitro both IGF-I and IGF-II and a 32-34 kD IGFBP. In conclusion, the IGFs are stimulatory to cartilage growth in vitro and embryonic chick cartilage in vitro produces both IGF-I and II as well as an IGFBP.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta 1 mRNA in neonatal ovine molars visualized by in situ hybridization: potential role for the stratum intermedium.

Human dentine contains relatively large amounts of transforming growth factor-beta (TGF-beta), which might originate from odontoblasts. The expression of the TGF-beta 1 message in developing teeth was examined by in situ hybridization. The analysis was made on 5-microns serial sections of mandibular third molars of neonatal sheep cut from tissues that had been fixed in glutaraldehyde and paraffin-embedded. A 35S-labelled cRNA probe, complementary to TGF-beta 1 mRNA, was constructed from human TGF-beta 1 cDNA. Northern analysis of total RNA from sheep placenta and neonatal third molars demonstrated hybridization to a single 2.4 kb TGF-beta 1 transcript from both tissues, indicating cross-reactivity of the human probe in the sheep. In the neonatal molars, in situ hybridization was observed in cells of the inner enamel epithelium, mature ameloblasts and mature odontoblasts, but not within preodontoblasts before dentine matrix formation. TGF-beta 1 mRNA expression was also evident in the cells of the dental papilla but scarcely so in the stellate reticulum. The most striking feature was the appearance of hybridization signal in the cells of the stratum intermedium before hybridization was evident in the inner enamel epithelium. Control sections incubated with RNAase before incubation with probe did not show evidence of hybridization. These findings suggest that TGF-beta 1 may have an important regulatory role in the differentiation of ameloblasts and odontoblasts, perhaps by modulating matrix formation during amelogenesis or odontogenesis. They also suggest a potential novel regulatory role for the cells of the stratum intermedium.

Growth hormone stimulates cortical bone formation in immature hypophysectomized rats.

Daily subcutaneous injections of rat derived growth hormone to immature, hypophysectomized rats stimulated significant increases in body weight gain, serum osteocalcin, skeletal alkaline phosphatase and incorporation of radioactive thymidine and proline into the compact bone of femurs and tibiae. Equimolar doses of insulin-like growth factor-II did not produce similar biological effects. The data support the contention that growth hormone at equimolar concentration is a stronger osteogenic agent than is insulin-like growth factor-II in vivo.