Derivation of transplantable 7,12-dimethylbenz[a]anthracene-induced chicken fibrosarcoma lines: differences in metastasizing properties and organ specificity.

Several transplantable 7,12-dimethylbenz[a]anthracene-induced SC chicken fibrosarcoma (CHCT-NYU) lines were studied for their ability to grow in internal organs after iv injection (artificial metastases) into 1- to 3-week-old chickens. Some tumor lines were recently derived, whereas others were studied after many serial subcutaneous transplantations. STriking similarities as well as differences were found between tumor lines' ability to grow in various organs. Artificial metastases were seen primarily in the stomach, pancreas, lungs, heart, and muscle, and occasionally also in the kidneys and in the liver. Agammaglobulinemic recipients showed more extensive organ involvement than normal recipients of the same age. Whole-body gamma-irradation also enhanced the incidence of artificial metastases, particularly in lungs. Antibody from the serum of a primary tumor-bearing host reduced the growth of the corresponding tumor in many organs. The metastatic pattern of line CHCT-NYU4 was a relatively stable property, since there was little difference in the internal localization of these tumor cells studied before and after 15 serial subcutaneous transplants. However, intravenous transplantation of tumor cells from line CHCT-NYU4 taken from the liver, lungs, and pancreas of a single recipient established sublines with changes in organ specificity. After a few such serial transplants of liver-derived tumor, a line was derived that grew virtually in the liver alone. A subline with preference for growth in lungs was also obtained, but its ability to grow in the pancreas persisted. A pancreas-derived tumor line also grew in the liver and lungs. Subcutaneous transplants of tissue fragments of the lung-derived tumor line caused the appearance of spontaneous metastases in lungs. The incidence of spontaneous metastases with the lung-derived line was much greater than that with the liver-derived line or with the original CHCT-NYU4 line.

Chemical carcinogen-induced transplantable fibrosarcomas in histocompatible chickens. II. Effect of age and thymectomy on tumor incidence.

The incidence of primary fibrosarcoma 2-4 months after im injection of 7,12-dimethylbenz[a]anthracene (DMBA) was studied in normal, neonatally thymectomized and bursectomized SC (B2/B2) chickens. The tumor incidence was not significantly increased in thymectomized chickens inoculated at 1 week of age, but thymectomized animals inoculated at 4 weeks of age developed a higher incidence of tumors than did controls. Bursectomy did not affect tumor induction. Whereas thymectomy and bursectomy clearly reduced T- and B-cell immune responses, respectively, neither the carcinogen nor the presence of tumor had a detectable effect on the immune response. The effect of varying the age of chickens at the time of carcinogen injection was also studied. DMBA injected into chickens at 8-12 weeks of age produced a significantly lower incidence of tumors than did a similar dose of DMBA injected into chickens at 1-4 weeks of age. Thus DMBA-induced tumors in the chicken may present an interesting model for studies on immune surveillance.

Detection of monoclonal antibodies for tumor diagnosis with the use of inhibition of micro-enzyme-linked immunosorbent assay.

A micro-enzyme-linked immunosorbent assay (ELISA) test and an ELISA inhibition test were developed and used to detect 4 monoclonal antibodies potentially useful for serodiagnosis of cancer. The 4 antibodies used in conjunction detected 73% of 71 sera from cancer patients and 8% of 42 sera from normal persons. Separately, the 4 antibodies reacted to tumors from various sites such as lung, breast, colon, stomach, and ovary. The ELISA inhibition assay may be useful for detecting culture supernatants reactive against tumor-associated serum antigens. Eventually, a panel of monoclonal antibodies detecting various tumors may be obtainable.

Use of monoclonal antibodies to sialylated Lewisx and sialylated Lewisa for serological tests of cancer.

A new monoclonal antibody, CSLEX1, directed against sialylated Lewisx was tested in parallel with a monoclonal antibody, CSLEA1, directed against sialylated Lewisa antigen. In tests with a solid-phase radioimmune sandwich assay, the sialylated Lewisx monoclonal antibody detected sera from certain cancer patients that were negative with the sialylated Lewisa monoclonal antibody. Some sera from cancer patients showed the reverse reaction. We conclude that the combined use of these two monoclonal antibodies detects a wider range of sera from cancer patients than the use of a single antibody alone. It should be possible in the future to use multiple monoclonal antibodies to increase detection.

Detection of tumor-associated antigens in the sera of lung cancer patients by three monoclonal antibodies.

One hundred sixty-one sera from lung cancer patients, including 46 samples from patients who had not yet received treatment were screened for tumor-associated antigens with 3 monoclonal antibodies, CSLEX1, CSLEA1, and CLEX5, by a new cell binding inhibition assay. We had previously determined that the antigens recognized by CSLEX1 and CSLEA1 are sialosylated Lewisx and sialosylated Lewisa, respectively. Either of these two antibodies alone reacted with about 65% of the 46 untreated patients' sera. Eighty-seven % of the 46 showed positive results with at least one of the two antibodies. The CLEX5 monoclonal antibody is presented here as recognizing a potential tumor-associated antigen. CLEX5 reacted with 54% of the 46 sera from nontreated lung cancer patients. When the results for all three antibodies were combined, the percentage of positive sera was 89% (of 46). Some interesting patterns in the serum levels of the antigens detected by these antibodies were observed. Levels of sialosylated Lewisx were significantly higher in sera from nontreated advanced stage (III and IV) patients (P less than 0.0003). In addition, levels of the antigens detected by CSLEX1 and CSLEA1 were dependent on whether or not the patient had been receiving treatment. These observations suggest potential applications of monoclonal antibodies to diagnosis and monitoring of therapies.

An immunofluorescent technique for the detection of lymphocyte alloantigens.

A triple-layer immunofluorescent technique for the detection of cell surface alloantigens has been developed. Chicken lymphoid cells were treated in sequence with: 1) chicken alloantibody, 2) rabbit anti-chicken Fc or anti-gamma chain serum and 3) FITC-labelled sheep anti-rabbit globulin. The second layer was diluted to that point where normal serum treated lymphocytes were no longer stained but without diminishing the degree of specific staining. This method enables the study of alloantigens expressed on B cells, avoiding the problem of staining of surface Ig-bearing lymphocytes.

Detection of bursa and thymus-specific alloantigens in the chicken.

CH strain chickens selected from the F2 progeny of a cross between CH (B10/10) and B14B (B14/14) strains carried bursa (BA) and thymus (TA)-specific alloantigens of B14B parental origin. The BA marker was present on 95% of bursal cells but only on 10% of splenic and blood B lymphocytes. The TA marker was expressed by 80% of thymus cells and weakly by 45% of splenic and 70% of blood T lymphocytes. The adopted immunological and genetic protocol offers a feasible approach towards detection of differentiation antigens in major histocompatibility complex-homozygous but 'minor' alloantigen-segregating populations.

Immunofluorescent detection of differentiation alloantigens (CA1) in the chicken.

Lymphoid cell surface alloantigens were detected by a triple-layer immunofluorescence technique. An antiserum raised against B locus-incompatible lymphoid cells also reacted with previously undefined differentiation antigens (CA1) which segregated between B locus homozygous (B14/B14) chickens of the same strain as the antiserum donor. CA1-positive chickens reacted with the antiserum by agglutination of red cells and staining of all peripheral lymphocytes and thymocytes but not bursal cells. Cross-absorption experiments have demonstrated that antigens expressed by red cells, T cells (both thymic and peripheral) and B cells (peripheral only) respectively, were of distinct specificity.

Committed precursors of B and T lymphocytes in chick embryo bursa of Fabricius, thymus, and bone marrow.

Lymphocyte precursors in bursa of Fabricius, thymus and bone marrow (BM) of chick embryos were studied at different stages of incubation over 12-21 days, and for their state of commitment to B or T cell lines of development. Cell suspensions were fractionated on albumin gradients to remove nonlymphoid cells and incubated in vitro with bursopoietin, a specific inducer of B cells, or crude chicken thymus extract, a specific inducer of T cells, or ubiquitin, a nonspecific inducer. Precursors were identified by increases in numbers of cells bearing surface alloantigens as determined by immunofluorescence, either Bu-1 (specific to B cells) or Th-1 (specific to T cells). Precursors inducible to Bu-1+ cells were found in bursal cells and BM cells from all age groups but not in thymic cells. Precursors inducible to Th-1+ cells were found in thymic preparations and BM cells at all ages, but in significant numbers in bursa on day 12 only. Because B and T precursors were never found together in bursa or thymus, or only in very unequal amounts, it was concluded that precursors in these organs were not multipotential but were separately committed to one or other line of development. This argument did not apply to BM cells, for which other evidence was obtained. Bu-1+ cells were specifically induced in BM cells with bursopoietin and then removed by complement-dependent cytolysis wih anti-Bu-1 antiserum. When the remaining cells were incubated with ubiquitin, only Th-1+ cells were induced, showing that Bu-1 and Th-1 precursors were separately committed. Surface IgM was never induced on either bursal or BM lymphocytes. The Ia (or B-L) antigen was inducible on 12- to 21-day bursal cells, but could not be generated on BM cells until day 14 onwards. The pattern of occurrence of committed lymphocyte precursors in the developing chick embryo suggests that these cells are released into the circulation from both central lymphoid organs at their respective times of high lymphopoietic activity, and accumulate in the BM at least up to the time of hatching. Moreover, the presence of committed B precursors in bursa and committed T precursors in thymus at times and in quantities appropriate to the known features of avian lymphopoiesis leads us to conclude that in vitro induction is analogous to a true stage of in vivo B and T cell differentiation.

Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. II. Effect of prior immunization with transplantable tumors on primary tumor incidence.

Primary carcinogen-induced (7,12-dimethyl-benz[a]anthracene; DMBA) tumor-bearing SC chickens (B2/B2) frequently showed antibodies in their sera which reacted with cells from their autochthonous tumors, chicken embryo fibroblasts (CEF), tumor cells from some transplantable tumor lines, and from approximately 10% of other primary tumors. Similar results were obtained by ELISA on glutaraldehyde-fixed cells and by immunofluorescence on viable cells. The serum antibody reactivity could be removed by absorption with CEF but not with non-cross-reacting primary tumor cells or a variety of normal tissues. Although sera from normal chickens never showed significant reactivity, a high percentage of sera from chickens that had been injected with DMBA but failed to develop detectable tumors showed antibody activity to a transplantable DMBA-induced tumor and to CEF. On the basis of previously established cross-reactivity patterns in protective immunity to transplantable carcinogen-induced fibrosarcomas, attempts were made to protect against chemical carcinogenesis by prior immunization with selected DMBA-induced transplantable tumors. Tumor-immune chickens showed a significant decrease in the development of tumors during the first 3 mo after injection of DMBA (p = 0.001) or methylcholanthrene (p = 0.033) when compared to controls. This resistance to tumor induction in immune chickens was correlated to the degree of tumor immunity to the immunizing tumor present 1 mo after carcinogen injection (p = 0.046). There was, however, no detectable difference in the incidence of tumors arising later than 3 mo after carcinogen injection. The reduction in tumor incidence in immune as compared to control chickens at 5 mo was therefore less striking than the reduction seen at 3 mo. Immunization with CEF and adjuvants or with adjuvants alone afforded no protection to tumor induction.

gamma-Irradiation-induced mortality: protective effect of protease inhibitors in chickens and mice.

Chickens (Gallus domesticus) were protected from the acute gamma-irradiation-induced mortality (within 24 hours) by the proteolytic enzyme inhibitors, soy-bean trypsin inhibitor (SBTI), lima bean inhibitor (LBTI), antipain, alpha-N-benzoyl-L-arginine ethyl ester HCl (BAEE), trasylol, and leupeptin. Several other enzyme inhibitors, p-tosyl-L-arginine methyl ester HCl (TAME), alpha-tosyl-lysyl-chloromethyl ketone HCl (TLCK) and epsilon-amino caproic acid (EACA), did not protect. EACA even increased the mortality caused by gamma-irradiation. The pattern of protective enzyme inhibitors suggests involvement of a kallikrein-like enzyme. SBTI and antipain also protected against low range lethal gamma-irradiation exposures, 690 R in BALB/c and 880 R in SJL/J mice. It is suggested that enhanced vascular permeability, which in chickens is known to be the cause of the irradiation mortality during the first 24 hours, may also contribute to the mortality in mice during the first week after irradiation.

Tissue distribution of the Pk antigen as determined by a monoclonal antibody.

Using an anti-Pk monoclonal antibody (mAb) designated CPK-1, the expression of the Pk antigen was assessed on normal human tissue from non-Pk individuals. Although the Pk antigen was detected on fibroblasts and blood vessels as previously reported, it was also found on smooth muscle cells of the digestive tract and the urogenital system. Pk was also found on glandular cells of the stomach, oesophagus and prostate. Additionally, CPK-1 reacted weakly with oesophagus squamous cells, and a small number of glomeruli and tubules in the kidney. The mechanism of expression of the Pk determinant in non-Pk individuals is discussed.

Heterogeneity of Lewis antibodies. A comparison of the reaction of human and animal reagents with synthetic oligosaccharides.

Human as well as animal anti-Lewis reagents were shown to have different binding patterns to synthetic structures chemically related to the Lewis epitopes. Two main types of cross-reactions were found: (1) Cross-reactions among type 1 Lewis epitopes (Lea, Leb and Lewis disaccharide). This type of cross-reaction among different type 1 structures was predominant in anti-Lea reagents (16 out of 18), although it was also present in some anti-Leb reagents (4 out of 14). (2) Cross-reactions of Lea and Leb with their type 2 isomers X and Y. The Leb-Y cross-reaction was more frequent (7 out of 14) than the Lea-X cross-reaction (2 out of 18). The serological property of some anti-Lewis reagents reacting with cord cells ('Lex') is also shown to be heterogeneous although probably related to common features of the type 1 Lea and Leb epitopes and independent of the type 2 X and Y epitopes.

Defective bursa regeneration after irradiation of young thymectomized chickens.

The ability of the bursa of Fabricius to regenerate after gamma-irradiation and bone marrow reconstitution was examined in chickens thymectomized (TX) immediately after hatching. Irradiation (2 X 500 R) 3 weeks after hatching was followed by impaired bursa regeneration, as judged both by bursa/body weight ratios and by bursa follicle development 3-6 weeks later in TX as compared to control birds. Germinal center formation in the spleen was deficient, and immune responses to sheep erythrocytes (SRBC) and B. abortus (BA) were moderately reduced in the TX as compared to control birds irradiated at 3 weeks but not in TX birds irradiated at 5 weeks of age.