Heterozygous ATM mutations do not contribute to early onset of breast cancer.

Ataxia telangiectasia (AT) is a recessive syndrome, including cerebellar degeneration, immunologic defects and cancer predisposition, attributed to mutations in the recently isolated ATM (ataxia telangiectasia, mutated) gene. AT is diagnosed in 1/40,000 to 1/100,000 live births, with carriers calculated to comprise approximately 1% of the population. Studies of AT families have suggested that female relatives presumed to be carriers have a 5 to 8-fold increased risk for developing breast cancer, raising the possibility that germline ATM mutations may account for approximately 5% of all breast cancer cases. The increased risk for breast cancer reported for AT family members has been most evident among younger women, leading to an age-specific relative risk model predicting that 8% of breast cancer in women under age 40 arises in AT carriers, compared with 2% of cases between 40-59 years. To test this hypothesis, we undertook a germ-line mutational analysis of the ATM gene in a population of women with early onset of breast cancer, using a protein truncation (PTT) assay to detect chain-terminating mutations, which account for 90% of mutations identified in children with AT. We detected a heterozygous ATM mutation in 2/202 (1%) controls, consistent with the frequency of AT carriers predicted from epidemiologic studies. ATM mutations were present in only 2/401 (0.5%) women with early onset of breast cancer (P = 0.6). We conclude that heterozygous ATM mutations do not confer genetic predisposition to early onset of breast cancer.

Systemic cellular hypersensitivity induced by an intestinally absorbed antigen.

Neonatal mice given intact living Schistosoma mansoni eggs or soluble schistosome egg antigens intragastrically developed systemic cellular hypersensitivity as shown by markedly accelerated, augmented granulomatous inflammation around S. mansoni eggs subsequently injected intravenously into the pulmonary microvasculature. To achieve partial sensitization in adult mice schistosome eggs had to be administered intragastrically with bicarbonate; full sensitization occurred when the eggs were injected intraduodenally. These data indicate that under appropriate conditions intestinal administration of antigen can result in systemic cellular immune sensitization.

Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria.

Rat small intestinal epithelial cell lines have been established in vitro and subcultured serially for periods up to 6 mo. These cells have an epithelioid morphology, grow as monolayers of closely opposed polygonal cells, and during the logarithmic phase of growth have a population doubling time of 19--22 h. Ultrastructural studies revealed the presence of microvilli, tight junctions, an extensive Golgi complex, and the presence of extracellular amorphous material similar in appearance to isolated basement membrane. These cells exhibit a number of features characteristic of normal cells in culture; namely, a normal rat diploid karyotype, strong density inhibition of growth, lack of growth in soft agar, and a low plating efficiency when seeded at low density. They did not produce tumors when injected in syngeneic animals. Immunochemical studies were performed to determine their origin using antisera prepared against rat small intestinal crypt cell plasma membrane, brush border membrane of villus cells and isolated sucrase-isomaltase complex. Antigenic determinants specific for small intestinal epithelial (crypt and villus) cells were demonstrated on the surface of the epithelioid cells, but they lacked immunological determinants specific for differentiated villus cells. An antiserum specifically staining extracellular material surrounding the cells cultured in vitro demonstrated cross-reactivity to basement membrane in rat intestinal frozen sections. It is concluded that the cultured epithelioid cells have features of undifferentiated small intestinal crypt cells.

Macromolecular absorption. Mechanism of horseradish peroxidase uptake and transport in adult and neonatal rat intestine.

The immature small intestine of neonatal mammals is permeable to gamma globulins as a source of passive immunity. Allegedly, macromolecular absorption ceases when the epithelial cell membrane matures. However, some evidence exists that adult animals retain a limited capacity to transport antigenic and biologically active quantities of large molecules. In this study, the mechanism of absorption of the tracer protein, horseradish peroxidase (HRP), was tested in neonatal and adult rat gut sacs. Transport into serosal fluid was quantitated by enzymatic assay and monitored morphologically by histochemical techniques. A greater transport of HRP was noted in the adult jejunum compared to adult ileum and neonatal intestine. Morphologically, the uptake mechanism in adult intestine was similar to the endocytosis previously reported in neonatal animals Like other endocytotic processes, HRP uptake in adult rats is an energy-dependent process as determined by metabolic inhibitors and temperature-controlled studies. An understanding of the mechanism whereby macromolecules are bound to intestinal membranes and engulfed by them is necessary before the action of physiologic macromolecules such as enterotoxins can be appreciated.

Isovaleric and -methylbutyric acidemias induced by hypoglycin A: mechanism of Jamaican vomiting sickness.

Hypoglycin A, the causative agent of the Jamaican vomiting sickness, produced a marked increase in concentration of isovaleric acid in the plasma of rats, when administered in a single dose. alpha-Methylbutyric acid, a position isomer, also accumulated. The use of hypoglycin A reproduced some features of human isovaleric acidemia. Accumulation of these branched pentanoic acids may be another factor contributing to the pathogenesis of the Jamaican vomiting sickness.

Very-low-density lipoprotein in intestinal lymph: evidence for presence of the A protein.

Rat mesenteric lymph very-low-density lipoproteins of intestinal origin contain components which are antigenically identical to plasma high-density lipoprotein. Because the antigenicity of the latter most likely resides in its apoprotein (the A protein), it is concluded that intestinal very-low-density lipoproteins contain the A protein. This and other evidence supports the concept that the intestine is a source of plasma very-low-density lipoprotein.

Intestinal uptake of macromolecules: effect of oral immunization.

Animals were orally immunized with horseradish peroxidase and bovine serum albumin, and absorption of these antigens was studied. In comparison with controls, a consistent and significant decrease in peroxidase uptake was noted in both germ-free and conventional rats immunized with peroxidase; a similar decrease in serum albumin uptake was also noted in animals immunized with serum albumin. There was no difference in the uptake of an unrelated macromolecule. These observations suggest that local immunization interferes specifically with the intestinal uptake of macromolecular antigens.

Ethanol stimulates triglyceride synthesis by the intestine.

In vivo ethanol given acutely or chronically by two dietary means resulted in significant increases in [1-(14)C]palmitate incorporation into triglyceride by intestinal slices or microsomes derived from intestinal slices. In vitro, 2.6 percent ethanol, an amount comparable to that found in t..e intestinal lumen of social drinkers, also resulted in significant increases in [1-(14)C]palmitate incorporation into triglyceride. Pyrazole, an inhibitor of alcohol dehydrogenase, diminished the stimulatory effect of ethanol both in vivo and in vitro. These data may provide a new insight into the effects of alcohol, and specifically on the possible contribution of intestinal triglyceride synthesis to alcoholic hyperlipemia and the alcohol-induced fatty liver.

Reversal of oncogenesis by the expression of a major histocompatibility complex class I gene.

The classical transplantation antigens (the major histocompatibility complex class I antigens) play a key role in host defense against cells expressing foreign antigens. Several naturally occurring tumors and virally transformed cells show an overall suppression of these surface antigens. Since the class I molecules are required in the presentation of neoantigens on tumor cells to the cytotoxic T lymphocytes, their absence from the cell surface may lead to the escape of these tumors from immunosurveillance. To test this possibility, a functional class I gene was transfected into human adenovirus 12-transformed mouse cells that do not express detectable levels of class I antigens; the transformants were tested for expression of the transfected gene and for changes in oncogenicity. The expression of a single class I gene, introduced by DNA-mediated gene transfer into highly tumorigenic adenovirus 12-transformed cells, was sufficient to abrogate the oncogenicity of these cells. This finding has important implications for the regulation of the malignant phenotype in certain tumors and for the potential modulation of oncogenicity through derepression of the endogenous class I genes.

Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome.

The hCHK2 gene encodes the human homolog of the yeast Cds1 and Rad53 G2 checkpoint kinases, whose activation in response to DNA damage prevents cellular entry into mitosis. Here, it is shown that heterozygous germ line mutations in hCHK2 occur in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in the TP53 gene. These observations suggest that hCHK2 is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors, and they also provide a link between the central role of p53 inactivation in human cancer and the well-defined G2 checkpoint in yeast.

Composition of human colonic mucin. Selective alteration in inflammatory bowel disease.

Human colonic mucin has been isolated from mucosal scrapings of fresh surgical specimens of normal controls as well as patients with Crohn's colitis and ulcerative colitis. Following sonication and ultracentrifugation, mucin fractions were separated from other soluble colonic glycoproteins by Sepharose 4B chromatography. After nuclease digestion, cesium chloride gradient centrifugation of the excluded material yielded colonic mucin with an average buoyant density of 1.52 g/ml. Subsequent chromatography of the apparently homogeneous colonic mucin on DEAE-cellulose revealed the presence of at least six distinct mucin species (mucin I-VI). Each mucin species was found to have a distinctive hexose, hexosamine, sialic acid, and sulfate content as well as blood group substance activities. Mucin from five patients with Crohn's colitis was found to represent a mixture of at least six discrete species comparable to those isolated from normal colonic specimens. However, in mucin from eight patients with ulcerative colitis there was a marked and selective reduction of one component mucin subclass, designated species IV. Normal mucin and mucin from patients with Crohn's disease contained 48 +/- 17 and 42 +/- 12 mg of species IV/g, while mucin from patients with ulcerative colitis had 5 +/- 3 mg/g solubilized glycoprotein. The selective absence of species IV was found in preparations from both sigmoid (n = 7) and ascending (n = 4) colon and could not be accounted for by an overall decrease in total mucin content. The selective reduction of species IV was also found in mucin isolated from relatively noninflamed colonic mucosa of patients with ulcerative colitis. The carbohydrate composition and blood group activities of the remaining five mucin species were similar to their normal counterparts. Based on the results to date, there appears to be an underlying selective decrease of one colonic mucin subclass in ulcerative colitis.

Effect of mixed micellar lipid on the absorption of cholesterol and vitamin D3 into lymph.

The absorption of endogenous cholesterol, labeled with tracer doses of cholesterol (14)C or cholesterol-(3)H and of near physiological doses of vitamin D(3)-(3)H was studied in rats with cannulated intestinal lymphatics. The effects of administering mixed micellar solutions of fatty acid, monoglyceride, and bile salt on the absorption of these labeled sterols was determined. It was observed that the specific activity of free cholesterol and the amounts of vitamin D(3) appearing in lymph were significantly increased during the intraduodenal administration of mixed micellar solutions of either linoleic or palmitic acid, in contrast to control rats receiving a micellar solution of taurocholate. These increases were related linearly to the lymph triglyceride level. In addition it was observed that when the linoleic acid solution was administered there was a more marked increase in the ratio of the specific activities of free and esterified cholesterol in lymph than with either the palmitic acid or taurocholate solutions. Additional studies in rats with intact lymphatics showed that the uptake of labeled cholesterol and vitamin D(3) from the intestinal lumen into the wall was similar whether the sterols were administered in taurocholate or in mixed micellar solution. These findings suggest that mixed micellar lipid increased the rate of appearance of labeled free cholesterol and vitamin D(3) in lymph by enhancing their transport out of the intestinal mucosa, rather than by an effect on uptake.

Very low density lipoproteins in intestinal lymph: origin, composition, and role in lipid transport in the fasting state.

The transport of endogenous lipids in the lipoproteins of mesenteric lymph was studied in fasting rats with mesenteric lymph fistulas. The lymph was found to contain, in addition to chylomicrons (S(f) >400), a significant amount of another, more dense, triglyceride-rich fraction, the very low density lipoproteins (VLDL), which showed a peak S(f) of 102. The VLDL differed from chylomicrons not only in flotation, but also in per cent lipid composition and electrophoretic mobility in agarose gel. The VLDL fraction was found to contain 47% of the triglyceride and 54% of the cholesterol of fasting lymph and, in the fasting state, was the major lipoprotein species present. When cholestyramine resin was administered intraduodenally, or bile flow was acutely diverted from the intestine, it was demonstrated that the lipids in lymph VLDL, like those in chylomicrons, were derived from the intestine and bile. These data indicate that the VLDL in intestinal lymph are not derived from the plasma but are of intestinal origin. Because certain properties of lymph VLDL were similar to those reported for plasma VLDL (per cent lipid composition, flotation coefficient, and continuing entry into plasma in the fasting state), additional comparisons between these fractions were made. Although lymph VLDL moved to the alpha(2) region in agarose gel, when they were mixed with VLDL-free serum immediately before electrophoresis they showed the alpha(2) mobility of rat serum VLDL. Furthermore, immunoelectrophoretic comparison of partially delipidated lymph and serum VLDL revealed that these fractions shared in common their major apoprotein, and possibly others as well. The fatty acid composition of lymph and serum triglycerides, as determined by gas-liquid chromatography, revealed that although they were generally similar, differences existed which most likely reflected the presence in serum of triglycerides of hepatic origin. These experiments demonstrate the importance of intestinal VLDL in the transport of endogenous lipids in mesenteric lymph in the fasting state. The similarities between intestinal lymph VLDL and plasma VLDL suggest that the latter may be derived in part from the former.

Fat absorption during inhibition of protein synthesis: studies of lymph chylomicrons.

The effect of protein synthesis inhibition on the absorption of oleic acid from micellar solution was studied in mesenteric lymph fistula rats. A micellar solution of oleic acid labeled with tracer doses of oleic acid-(14)C was administered by intraduodenal infusion to rats with indwelling mesenteric lymph cannulas. Protein synthesis was inhibited by intraperitoneal acetoxycycloheximide (ACH), 0.25 mg/kg, 1 hr before lipid infusion. Lymph chylomicrons labeled with oleic acid-(14)C were collected from control and protein inhibited animals at various times after lipid infusion and subjected to sucrose density gradient centrifugation to determine changes in size. In control animals there was a transient increase in chylomicron size during maximal triglyceride absorption; however, in protein-inhibited animals there was a marked and sustained increase in chylomicron size as late as 4 hr after lipid infusion. Triglyceride and phospholipid determinations on washed chylomicrons from both groups indicated a greater triglyceride/phospholipid ratio after protein synthesis inhibition supporting a greater chylomicron size. Electron microscopy of lymph from both groups further confirmed a markedly increased chylomicron size after protein synthesis inhibition. It is proposed that an increase in size conserves chylomicron surface components, i.e. apoprotein, during conditions of inhibition of protein synthesis. These studies clearly demonstrate that the intestinal inhibition of protein synthesis is associated with an increase in the size of intestinal lymph chylomicrons and support the concept that protein synthesis is important in the formation and transport of chylomicrons from the mucosal cell into the lymph.

Very low density lipoproteins in intestinal lymph: role in triglyceride and cholesterol transport during fat absorption.

The role of nonchylomicron very low density lipoproteins (VLDL, S(f) 20-400) in the transport of triglyceride and cholesterol was studied during lipid absorption. Various long chain fatty acids were infused intraduodenally in the form of mixed fatty acid-mono-olein-taurocholate micelles; control animals received saline or taurocholate. As compared with controls, all fatty acids (palmitic, oleic, linoleic) resulted in significant increases in chylomicron (S(f) > 400) triglyceride. In addition, palmitic acid resulted in a twofold increase in VLDL triglyceride, whereas with the absorption of oleic or linoleic acid VLDL triglyceride did not change significantly. Differences in triglyceride fatty acid composition between chylomicrons and VLDL were observed during lipid absorption. Although the absolute amount of endogenous cholesterol in intestinal lymph was not significantly affected by lipid absorption under these conditions, its lipoprotein distribution differed substantially among the lipid-infused groups. During palmitate absorption, VLDL cholesterol was similar to that in the taurocholate-infused controls, and was equal to chylomicron cholesterol. In contrast, during oleate and linoleate absorption the VLDL cholesterol fell markedly, and was less than half of the chylomicron cholesterol in these groups. The half-time of plasma survival of VLDL cholesterol-(14)C was found to be twice that of chylomicron cholesterol-(14)C. These studies demonstrate that dietary long chain fatty acids differ significantly in their effects upon the transport of triglyceride and cholesterol by lipoproteins of rat intestinal lymph. These findings, together with the observed differences in rates of removal of chylomicrons and VLDL from plasma, suggest that variations in lipoprotein production at the intestinal level may be reflected in differences in the subsequent metabolism of absorbed dietary and endogenous lipids.

Rapid identification of low level hepatitis B-related viral genome in serum.

A sensitive and specific method has been developed to detect hepatitis B virus (HBV) in serum. The method involves two steps: the capture of viral genome from serum using a high affinity IgM monoclonal antibody directed against a common a domain epitope found on the envelope, and the amplification of viral DNA by the polymerase chain reaction (PCR). The amplification is initiated using "generic" primers derived from the core and pre-core sequences which are highly conserved amongst the hepadnaviruses. This rapid technique detects less than 10 infectious virions and may be useful in the study of individuals with acute and chronic liver disease of unknown etiology.

The pathogenesis of arthritis associated with acute hepatitis-B surface antigen-positive hepatitis. Complement activation and characterization of circulating immune complexes.

Circulating immune complexes were identified in cryoproteins isolated from serial samples of serum from six patients with acute viral hepatitis with and without arthritic symptoms. Cryoprecipitates were analyzed for the presence of hepatitis-B surface antigen (HBsAg) and hepatitis-B surface antibody (anti-HBs) by hemagglutination inhibition and hemagglutination. Complement components were detected by counter electrophoresis, and immunoglobulins were detected by gel diffusion. HBsAg, IgG, and IgM were identified in cryoprecipitates from all hepatitis patients, but were higher in concentration in patients with arthritis. Only cryoprecipitates from hepatitis patients with arthritis contained IgA and complement components C3, C4, and C5 as well as IgG and IgM, which disappear with resolution of the arthritis. The subtypes of IgG in these cryoprecipitates were predominantly the complement-fixing IgG1 and IgG3, HBsAg and anti-HBs were concentrated several-fold in the cryoprecipitates when compared to the serum concentration. Sequential studies in two patients demonstrated that the initial appearance of anti-HBs in the cryoprotein complex was associated with the detection in the complex of IgM suggesting a primary immune response to HBsAg. The C3 activator fragment (C3A) of the properdin complex was found in fresh serum obtained from three hepatitis patients with arthritis and not in uncomplicated hepatitis. The cryoprecipitable immune complexes from patients with arthritis converted C3PA in fresh normal sera to C3A in vitro whereas cryoprotein isolated from patients with uncomplicated hepatitis had no such effect. Thus, the transient appearance of circulating complement-fixing immune complexes in patients with the arthritis of acute hepatitis is associated with activation of both classical and alternate complement pathways and suggests that they play an important role in the pathogenesis of these serum sickness-like extrahepatic symptoms.

Cell-mediated immunity in acute and chronic hepatitis.

Peripheral lymphocytes from patients with hepatitis-B surface antigen (HBsAg)-positive and -negative acute hepatitis (AH), chronic active hepatitis (CAH), chronic persistent hepatitis (CPH), and normal controls were tested for in vitro cytotoxicity and blast transformation. Cytotoxicity was measured by chrominum (21Cr) release into the medium from 51Cr-labeled Chang liver cells after incubation for 6 h with peripheral lymphocytes at a lymphocyte target cell ratio of 200:1. Concomitant 72-h incubation studies were performed to assess thymus cell-dependent (T) lymphocyte function as measured by conccanavalin A (Con A)- stimulated incorporation of tritiated thymidine (blast transformation) and by cytotoxicity. It was found that (a) lymphocytes from patients with AH are cytotoxic to Chang liver cells compared to controls (P less than 0.001); (b) lymphocytes from patients with acute and chronic hepatitis are less cytotoxic when incubated with autologous and homologous HB2Ag-positive and -negative AH, CAH, and CPH are as cytotoxic as normal controls when stimulated with a nonspecific mitogen such as Con A; and (d) lymphocytes from patients with CAH while on prednisone therapy showed marked depression of cytotoxicity when stimulated with Con A. Thus these studies show that patients with AH have circulating T lymphocytes which are capable of causing the destruction of Chang liver cells. There is no defect in T-cell function as measured by Con A-stimulated cytotoxicity. There is a serum factor (s) in patients with acute and chronic hepatitis which inhibits spontaneous and induced lymphocyte cytotoxicity and blast transformation. Finally, prednisone treatment appears to inhibit lymphocyte cytotoxicity in patients with CAH.

Antigenic characterization of human hepatocellular carcinoma. Development of in vitro and in vivo immunoassays that use monoclonal antibodies.

Several libraries of monoclonal antibodies have been produced by immunization of Balb/c mice with single cell suspensions of nontrypsin-treated human hepatocellular carcinoma cell (HCC) lines in order to study the antigenic properties of transformed hepatocytes. The antibodies were characterized with regards to specificity for hepatoma-associated antigens and their capability for use as reagents in radioimmunoassays (RIAs) and tumor localization in vivo. Three such antibodies namely, P215457, PM4E9917, P232524 of the IgG2a, IgG2a, and IgG1 isotypes, respectively, not only recognized separate and distinct antigenic determinants on four human hepatoma cell lines but also reacted with epitopes present on chemically induced rat hepatoma cell lines. In contrast, only 1 of 38 other human malignant and transformed cell lines demonstrated reactivity with the three antibodies; normal human tissues were also found to be unreactive. Monoclonal antibody P215457 densely stained the plasma membrane by indirect immunofluorescence, showed rapid binding activity to HCC cells in suspension, and precipitated a 50,000-mol wt cell surface protein; antibody PM4E9917 also stained the plasma membrane and precipitated a 65,000-mol wt protein, whereas P232534 recognized cytoplasmic antigenic determinants. With these antibodies "simultaneous sandwich" RIAs were established that detect soluble hepatoma-associated antigens in culture supernatants. Finally, the Fab fragment of P215457 was found to be useful in tumor localization in vivo. This antibody fragment when labeled with 131I was shown to localize by radionuclide-imaging studies in human hepatoma grown in nude mice. Thus, these investigations demonstrate that monoclonal antibodies may be produced against epitopes that reside almost exclusively on transformed hepatocytes and such antibodies may be successfully employed in the development of in vitro and in vivo immunoassays.

Structural analysis of hepatitis B surface antigen by monoclonal antibodies.

A method has been developed for the analysis of hepatitis B surface antigen (HBsAg) antigenic structure at the molecular level that creates "fingerprints" or "signatures" of various hepatitis B viral (HBV) strains. This technique employs high affinity IgM and IgG monoclonal antibodies (anti-HBs) directed against distinct and separate determinants on HBsAg. In performing this antigenic structural analysis, separate binding curves for different monoclonal anti-HBs are generated by measuring immunoreactivity in serial dilutions of HBsAg-positive serum by radioimmunoassay. Since the HBsAg concentration in serum is unknown, the binding profiles of groups of samples are aligned by an iterative least-squares procedure to generate the numerical signature characteristic of the viral strain. The numerical signatures are then displayed on a computer-graphic plot. The signature profiles of HBsAg subtypes are a true reflection of their antigenic structure, and in vertical and horizontal transmission studies the molecular characteristics of the viral epitopes are conserved. By signature analysis we found substantial antigenic heterogeneity among the ayw3 strain both in the U.S. and France, as well as in populations of the Far East and Africa. Populations in Ethiopia, Gambia, and the Philippines were infected with two antigenically distinct HBV strains. In some newly identified HBV strains, it was found that epitopes identified by some monoclonal antibodies were absent or substantially reduced, which suggested that a genetic mutation may have occurred. Thus this study suggests that there is far more antigenic heterogeneity in HBV than previously recognized. These variants are antigenically distinct from each other at the epitope level, and were heretofore unrecognized by polyvalent anti-HBsAg antibodies.