The bispecific antibody 500A2 x 96.5 targets T-lymphocytes activated in vivo with staphylococcal enterotoxin B (SEB) against CL62 melanoma cells in vitro.

Bispecific antibodies (BAb) direct T-lymphocytes to lyse selected tumour targets, both in vitro and in vivo. Significant tumour cell lysis with BAb requires pre-expansion of T-lymphocytes, which may be achieved in vitro by the addition of anti-CD3 monoclonal antibody plus interleukin-2 (IL-2), but anti-CD3 may cause immunosuppression. We investigated an alternative agent for in vivo immunostimulation, staphyloccal enterotoxin B (SEB), which selectively activates certain T-cell subsets and may result in less immunosuppression than with anti-CD3. We activated T-lymphocytes in vivo with SEB, expanded them in vitro with IL-2, and directed them against a tumour target with the BAb 500A2 x 96.5, specific for the murine CD3 antigen and the melanoma p97 antigen expressed by the CL62 tumour. C3H mice received SEB 50 micrograms intraperitoneally (i.p.). After 18 h mice were sacrificed and splenocytes extracted and either passed over a nylon wool column to isolate T-lymphocytes, or cultured in vitro for 3 to 7 days with 100 U ml-1 of IL-2. A 4-h chromium-release assay was used to assess the ability of T-lymphocytes to lyse the tumour target CL 62 in the presence or absence of the bispecific antibody 500A2 x 96.5. The addition of BAb significantly enhanced tumour lysis by SEB activated cells after a period of in vitro culture with IL-2. In vivo SEB results in the activation of T-lymphocytes which may be directed by bispecific antibodies to increase the lysis of selected tumour targets in vitro.

Expression of epidermal growth factor receptor and proliferating cell nuclear antigen predicts response of esophageal squamous cell carcinoma to chemoradiotherapy.

BACKGROUND: Multimodality therapy with chemotherapy and radiotherapy followed by surgery may improve survival in patients with esophageal squamous cell carcinoma compared with each of the individual treatment options. Histologic assessment of resected tumors after chemoradiotherapy shows that some patients have a complete response with no residual tumor, whereas other patients derive no benefit. The ability to predict response to chemoradiotherapy would allow treatment to be planned accordingly. METHODS: Expression of the tumor growth and proliferation proteins epidermal growth factor receptor (EGFR) and proliferating cell nuclear antigen (PCNA) was determined using immunohistochemical staining of pretreatment endoscopic biopsies from patients with esophageal squamous cell carcinoma who were randomized to chemoradiotherapy before surgery. Response to chemoradiotherapy was assessed by histologic examination of the resected specimens. Response to chemoradiotherapy and survival were correlated with EGFR and PCNA expression individually and with both markers combined as EGFR/PCNA: RESULTS: Of 14 patients available for study, 6 had a complete histologic response (CR) to chemoradiotherapy with no residual tumor in the resected specimen, 3 had a partial response (PR) to chemoradiotherapy, and the remaining 5 had minimal response (MR). Of the nine patients with a CR or PR, tumors of eight patients were negative for one or both markers. Of the five patients with an MR, four tumors were positive for both EGFR and PCNA (P < 0.05, Fisher's exact test). Comparison of survival from the date of randomization shows that patients with tumors negative for one or both markers had a significant survival advantage (P = 0.0003, log-rank test). CONCLUSIONS: Evaluation of PCNA and EGFR status of pretreatment biopsies may identify a group of patients likely to derive the greatest benefit from chemoradiotherapy before surgery in terms of histologic response and long term survival.

The biological significance of locoregional recurrence following breast conserving therapy.

Although locoregional recurrence of breast cancer following mastectomy is usually a harbinger of systemic failure, patients experiencing locoregional disease recurrence after breast-conservation therapy enjoy a better prognosis. Tumor recrudescences after a breast-preserving surgical procedure represent both residual tumor and new primary breast carcinomas. Patient prognosis is dependent on the biological characteristics of both the original and recurrent neoplasms. At present, the best predictor of patient outcome is the extent of disease at the times of treatment. While many studies report no statistically significant adverse impact of locoregional recurrence on survival, small sample sizes may obscure such an effect.

Plasma lipoproteins and fatty liver in dairy cows.

Dairy cows were classified on the basis of a histological study after a hepatic biopsy conducted in the second week post partum (mild fatty liver, moderate fatty liver, severe fatty liver). Plasma lipoproteins were separated into various density classes by repeated ultracentrifugation. The results indicate that the beginning of lactation is associated with a low concentration of the 1.006 to 1.063 g ml-1 lipoprotein fraction. The lowest concentrations occurred in cows with severe steatosis or during the evolution of moderate steatosis.

Influence of liver fat on experimental Escherichia coli mastitis in periparturient cows.

Eleven cows with a wide range of liver fat (5.7 to 51.4 per cent) at seven days post partum were experimentally infected in a single quarter with a capsular Escherichia coli at 10 days post partum. The results suggested that a fatty liver in itself does not influence the severity of mastitis. All animals had clinical mastitis 10 hours after infection but no animals became severely ill and no treatment was given. Four out of five animals in the group with less than 20.2 per cent liver fat had bacteria in their milk at 10 hours after infection but these bacteria were eliminated by 12 hours. The six animals in the group with more than 28.3 per cent fat in their liver retained viable bacteria in the udder for much longer; with two animals bacteria were shed and abnormal milk was secreted for up to four months despite antibiotic therapy.

Histological measurement of fat content of liver of dairy cows.

A total of 275 liver biopsies were performed on dairy cows in 6 Friesian and one Guernsey herd during the first 2 weeks after calving. Liver samples were processed by 2 independent histological procedures: (a) formalin-fixed frozen sections were stained with oil-red O (ORO), or (b) samples fixed in glutaraldehyde-osmium tetroxide were embedded in plastic and sections stained with toluidine blue (TOLB). The sections were then subjected to stereological point-counting procedures to assess the quantities of stainable fat present within the liver cells. Estimates of liver cell fat by the 2 techniques were highly correlated, although those obtained by the TOLB method were consistently lower than those obtained by the ORO method. The analytical sampling error was slightly lower in the TOLB method. The simpler ORO method should prove an acceptable alternative in the routine histology laboratory to the tedious and technically demanding TOLB method.

Haematology of subclinical fatty liver in dairy cows.

The relationship between liver fat content and haematology was investigated in 369 cows from eight herds sampled in the second week after calving. High levels of fat in the liver were associated with a depression in total white cell count and in neutrophils, eosinophils and lymphocytes. There was no correlation between liver fat content and the percentage of E rosetting lymphocytes, packed cell volume or haemoglobin concentration. The changes in peripheral white cell counts may be related to the increased incidence of post parturient disease in cows with fatty liver.

Blood composition and liver fat in post parturient dairy cows.

Cows from three different herds were used to investigate the relationship between plasma D(-)-3-hydroxybutyrate, serum free fatty acid and blood glucose concentrations and the amount of fat present in the liver in the week after calving. The study was particularly concerned with the diagnostic value of D(-)-3-hydroxybutyrate estimations. These estimations did not make a significant contribution to diagnosis of fatty liver nor did they reflect accurately the nutritional status of the cows.

Comparison of biochemical and histological methods of estimating fat content of liver of dairy cows.

The degree of fatty infiltration of the liver was estimated in two herds of dairy cows using biochemical and stereological methods. Estimation of hepatic triglyceride content biochemically or of fractional volume of fat in hepatocytes stereologically are both reliable methods of assessing fatty infiltration of the liver in the dairy cow. Estimation of hepatic total lipid content is not considered an acceptable alternative because the high basal level of non-triglyceride lipid masks the increase in hepatic triglyceride content which is characteristic of fatty liver.

A fat mobilisation syndrome in dairy cows in early lactation.

High yielding dairy cows undergo a period of energy deficit in early lactation and mobilise body reserves for milk production. In this period a high proportion of cows develop moderate or severe fatty liver. Fat is deposited in other tissues at the same time, particularly skeletal muscle. The relationship between measurements of fat mobilisation and fat deposition in liver and muscle has been investigated in a group of 19 cows in the post calving period. Cows with higher levels of indices of mobilisation, namely plasma free fatty acid and D(--) -3- hydroxybutyrate concentration and subcutaneous adipose tissue loss, had higher indices of fat deposition in liver and skeletal muscle. Statistical analysis of the data indicated that fatty liver was part of a more general fat mobilisation syndrome which was not specific to any one organ or tissue.

Incidence and severity of fatty liver in dairy cows.

A survey of the incidence and severity of post-parturient fatty liver in high yielding dairy cows was carried out on 151 cows and 46 heifers from four Friesian herds and one Guernsey herd. In Friesian and Guernsey cows from the second lactation onwards fat occupied a significant proportion (15 to 32 per cent) of the hepatic parenchyma at one week after calving but decreased to low values at eight weeks after calving. Fatty liver was insignificant in heifers of either breed. When the fatty liver was classified according to the amount of fat at one week after calving, 66 per cent of Friesian cows and 33 per cent of Guernsey cows had a moderate or severe fatty liver. The severity of the fatty liver was related to the loss of condition score after calving but not the condition score at calving.

Morphometric methods in veterinary pathology: a review.

The stereological procedures available for estimating volume, surface area and number of the structural components of cells and tissue are reviewed. The applications of stereology to the study of liver, mammary gland, lung and placenta are discussed with particular emphasis on the aspects of interest to veterinary pathologists. Stereology provides the cell biologist and the pathologist with a powerful tool for describing biological structure in quantitative terms at various levels of organization from the organ to the organelle.

The pathology of post-parturient fatty liver in high-yielding dairy cows.

The pathology of post-parturient fatty liver has been studied in a group of 20 high-yielding dairy cows; 10 had less than 20 per cent fat in the liver cell and were classified as having a mild fatty liver and 10 had more than 30 per cent fat in the liver cell and were classified as having severe fatty liver. Severe fatty liver was accompanied by a number of morphological changes including the occurrence of lipogranulomas, increased liver cell volume, decreased volume of rough endoplasmic reticulum per liver cell and evidence of mitochondrial damage. The latter two changes were reflected in reduced albumin levels and increased activities of mitochondrial enzymes observed in the blood of cows with severe fatty liver. Reduced fertility may be one of the consequences of severe fatty liver in the dairy cow.

A correlated biochemical and stereological study of periparturient fatty liver in the dairy cow.

Liver lipids were estimated in cows eight weeks before and one and eight weeks after calving by two independent procedures. Liver lipid was estimated stereologically in Araldite-embedded sections and using thin layer chromatography and densitometry of the separated lipids. The results obtained by these methods were compared. There were significant increases in the hepatic content (g/kg liver wet weight) of total lipid, triacylglycerol and non-esterified fatty acids one week after calving, with no significant changes in the phospholipid and cholesterol content around calving. The liver lipid composition (g lipid/100 g total lipid) changed one week after calving, with an increased proprotion of triacylglycerol and a decreased proportion of phospholipids and cholesterol. There was no change in the proportion of non-esterified fatty acids. The volume fracton of the hepatocyte occupied by fat droplets increased one week after calving. There was a high correlation between the triacylglycerol content of the liver determined biochemically and the volume fraction of fat droplets lying within the hepatocyte cytoplasm determined stereologically.

Effects of food deprivation on ketonaemia, ketogenesis and hepatic intermediary metabolism in the non-lactating dairy cow.

1. The aim of this work was to investigate why non-lactating dairy cows are less susceptible to the development of ketonaemia during food deprivation than are dairy cows in early lactation. 2. The first experiment (Expt. A) consisted of determining the effect of 6 days of food deprivation on the concentrations of ketone bodies, and of metabolites related to the regulation of ketogenesis, in jugular blood and liver of non-lactating cows. 3. During the food deprivation, blood ketone-body concentrations rose significantly, but to a value that was only 16% of that achieved in lactating cows deprived of food for 6 days [Baird, Heitzman & Hibbitt (1972) Biochem. J. 128, 1311--1318]. 4. In the liver, food deprivation caused: a rise in ketone-body concentrations; a fall in the concentration of glycogen and of various intermediates of the Embden-Meyerhof pathway and the tricarboxylic acid cycle; an increase in cytoplasmic reduction; a decrease in the [total NAD+]/[total NADH] ratio; a decrease in energy charge. These changes were all qualitatively similar to those previously observed in the livers of the food-deprived lactating cows. 5. There appeared therefore to be a discrepancy in the food-deprived non-lactating cows between the absence of marked ketonaemia and the occurrence of metabolic changes within the liver suggesting increased hepatic ketogenesis. This discrepancy was partially resolved in Expt. B by the observation in two catheterized non-lactating cows that, although there was a 2-fold increase in hepatic ketogenesis during 6 days of food deprivation, ketogenesis from the splanchnic bed as a whole (i.e. gut and liver combined) declined slightly owing to cessation of gut ketogenesis.