Establishment of AIDS-related primary-effusion lymphoma (PEL) cell lines that proliferate continuously without serum.

The gammaherpesvirus dually-infected (HHV8/EBV) PEL cell line, BC-1, was weaned gradually from fetal bovine serum (FBS) during successive feedings with RPMI 1640 medium containing human transferrin and selenium dioxide as the only additives. A serum-free cell line (sfBC-1) emerged that was 100% major histocompatibility complex (MHC) class II negative, compared with 10% MHC class II-negative cells before serum removal. In contrast, MHC class I expression by sfBC-1 cells slightly exceeded that of BC-1 cells. BC-1 and sfBC-1 cells were indistinguishable in six polymorphic genetic loci, confirming their relatedness and sfBC-1 cells contained HHV8 and EBV. These findings were not attributable to dual infection because the PEL cell line, BCBL-1, which is infected with HHV8 but not EBV, also contained MHC class II positive (45%) and class II negative (55%) cells. Moreover, a serum-free BCBL-1 (sfBCBL-1) cell line was established and the sfBCBL-1 cells were MHC class I up modulated and 100% MHC class II negative. The serum-free cell lines established in this study may be useful for exploring PEL-cell autocrine-growth pathways and for assessing MHC class II-negative PEL cells for tumorigenesis in animal model systems.

Arsenic can mediate skin neoplasia by chronic stimulation of keratinocyte-derived growth factors.

Although numerous epidemiological studies have shown that inorganic arsenicals are human skin carcinogens, there is currently no accepted mechanism for its action or an established animal model for its study. We observed increased mRNA transcripts and secretion of keratinocyte growth factors, including granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-alpha (TGF-alpha) and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in primary human epidermal keratinocytes cultured in the presence of low micromolar concentrations of sodium arsenite. Total cell numbers, as well as c-myc expression and incorporation of [3H]thymidine, both indicators of cell proliferation, were also elevated in keratinocyte cultures treated with sodium arsenite. As an in vivo model, the influence of arsenic on mouse skin tumor development was studied in transgenic TG.AC mice which carry the v-Ha-ras oncogene, and can serve as a genetically initiated model for skin carcinogenesis. Following low-dose application of 12-O-tetradecanoyl phorbol-13-acetate (TPA), a marked increase in the number of skin papillomas occurred in transgenic mice receiving arsenic in the drinking water as compared to control drinking water. Papillomas did not develop in arsenic-treated transgenic mice that had not received TPA or arsenic-treated wild-type FVB/N mice, suggesting that arsenic is neither a tumor initiator or promoter but rather an enhancer. Injection of anti-GM-CSF antibodies following application of TPA in transgenic mice reduced the number of papillomas. Consistent with that observed in human keratinocyte cultures, increases in GM-CSF and TGF-alpha mRNA transcripts were found within the epidermis of arsenic-treated mice when compared to controls within 6 weeks of treatment. These results suggest that arsenic enhances papilloma development via the chronic stimulation of keratinocyte-derived growth factors and represents the first example of a chemical carcinogen that acts in this manner. These studies suggest that in vitro studies with human keratinocyte cultures examined in conjunction with TG.AC transgenic mice can provide a useful model for examining the tumor enhancing properties of environmental chemicals.

Incidence and reactivity patterns of skeletal and heart (SH) reactive autoantibodies in the sera of patients with myasthenia gravis.

Serum from patients with myasthenia gravis (MG) contain antibodies to numerous skeletal muscle components in addition to the acetylcholine receptor (AChR). Certain non-AChR skeletal muscle autoantibodies have been shown by absorption to cross-react with cardiac muscle, leading to the designation of 'skeletal and heart' or SH antibodies. This study describes a new procedure for the extraction of human cardiac muscle which allows direct determination of SH antibody reactivity. Serologic evaluation of 17 patients with MG revealed 9/17 (53%) were seropositive for SH antibody to cardiac muscle. Absorption of selected MG serum samples with cardiac muscle extracts, reduced or eliminated reactivity to skeletal muscle in all cases, confirming the presence of cross-reactive antibodies. Immunoblot analysis of cardiac muscle extracts demonstrated several distinct antigenic components, which were unrelated to the acetylcholine receptor or to previously identified striational muscle proteins. Serum samples from individual MG patients displayed different immunoblot reactivity patterns ot the antigens in cardiac muscle extracts, providing the first evidence of multiple heart-reactive SH antibodies in MG.

Immunological identity of a 60 kd oncofetal protein induced in rats by chemical carcinogens and released by transformed cells.

A 60,000 dalton (60 kd) oncofetal protein was previously shown to be produced by tumors in tumor-bearing rats and by target tissues within 3 weeks of carcinogen treatment. The factor is released to and accumulates in the blood in vivo and in the conditioned medium of cultured transformed cells in vitro. A polyclonal antibody produced against the 60 kd factor purified from the plasma of a rat carrying the N-2-fluorenylphthalamic acid-induced transplantable Hepatoma 7777, was tested against the 60 kd factor from various sources. Based on the results of immunoprecipitation of biochemical activity associated with the 60 kd factor, it was determined that these anti-60 kd antibodies cross-reacted with the factor released by a dimethylbenzanthracene-induced rat mammary carcinoma, with the factor in rat tumor cytosol and with rat spontaneous lymphoma cells, but not with a 60 kd factor isolated from pooled cancer patient plasma. Furthermore, these antibodies cross-reacted with the 60 kd factor induced within 21 days of treatment of the rats with a range of carcinogens from 8 chemical structural groups. The anti-60 kd factor antibodies did not cross-react with a 35 kd factor having similar biochemical activity found in normal adult cells.

Myasthenia gravis. Identification of skeletal and heart muscle antigens not related to the acetylcholine receptor.

Myasthenia gravis (MG) is a neuromuscular disease thought to have an autoimmune etiology. The acetylcholine receptor has been considered the primary site of antibody binding; however, other muscle components may be involved in the pathogenesis of myasthenia gravis. This study describes a hypertonic sucrose extract of skeletal muscle (Muscle-HSE) that reacts with antibodies in myasthenic sera. The active component in Muscle-HSE is not the acetylcholine receptor as demonstrated by the inability of this extract to bind [125I]alpha-bungarotoxin. Muscle-HSE does, however, contain two distinct antigenic components reactive with MG sera. One antigen reacted with 70% (14/20) of myasthenic sera in the passive hemagglutination assay. This antigen was detected in the HSE of both skeletal muscle and heart, and was unaffected by treatment with Triton X-100. The second antigen reacted with 10% (2/20) of MG sera in the complement fixation assay, was unique to skeletal muscle, and was inactivated by Triton X-100.

Characterization of a 60,000-dalton oncofetal protein from the plasma of tumor-bearing rats.

A tumor cell-associated protein, previously shown to be present in the circulation of carcinogen-treated and tumor-bearing animals and cancer patients, has now been identified in the cytosol of embryonic tissue. This oncofetal protein, which is absent from the plasma of normal animals, has been purified from the plasma of tumor-bearing rats by a series of steps including ammonium sulfate fractionation and chromatography on Sepharose CL-6B and on CM Affi-Gel Blue. The tumor and fetal-associated 60-kd rat factors appear to be identical based on their reactivity to polyclonal antibody produced against the tumor factor. The factor, assayed by its ability to induce the transport of RNA from isolated nuclei, is a phosphoprotein with a minimum molecular weight of 60,000, as determined by polyacrylamide gel electrophoresis. In its purified form it is phosphorylated in the presence of the catalytic subunit of heart muscle protein kinase and ATP but does not exhibit auto-phosphorylating activity. 32P-orthophosphate is also incorporated into the phosphoprotein in vivo.

Detection of polysaccharide cell wall antigen of Neisseria gonorrhoeae in a rabbit model by counterimmunoelectrophoresis.

A rabbit chamber model was developed and inoculated with 10(9) colony-forming units (cfu) of viable Neisseria gonorrhoeae to determine whether the lipopolysaccharide-derived Gc2 polysaccharide cell wall antigens could be detected by counterimmunoelectrophoresis (CIE). Four hours after inoculation, a polymorphonuclear leukocyte response was noted in the chambers; this response was followed by progressive phagocytosis of the organisms and a fall in number of cfu/ml. All visible bacteria were intracellular, and chamber fluids were sterile 6 hr after inoculation. Use of sero specific antisera permitted detection by CIE of the Gc2 polysaccharide antigen in sera of all rabbits within 48 hr after inoculation of the chambers, whereas blood cultures remained sterile throughout the experiment. At 2-6 hr after inoculation, the Gc2 polysaccharide antigen was also detected as a single precipitin band in the chamber fluid of inoculated rabbits. At 24 hr the precipitin band was not observed; rather, a halo above the antigen well was noted. The halo was found to be a nonspecific complex containing the Gc2 polysaccharide antigen and no antibody. In the rabbit model studied, CIE was sufficiently sensitive to detect concentrations of the Gc2 polysaccharide antigen of greater than or equal to 0.97 microgram/ml in serum and chamber fluid.

Humoral antibody and cell-mediated cytotoxicity responses of mice to rat skin xenografts.

Aspects of both the humoral and cell-mediated immune responses of mice to rat skin xenografts were studied sequentially and quantitated. Antirat lymphocytotoxic and hemagglutinating antibodies were first detectable at 7 and 6 days, respectively, after primary grafting and their appearance in serum corresponded to the time of graft rejection. Lymphocytotoxic titers were low after primary grafting but increased greater than 4-fold after secondary grafting. Cytotoxicity of mouse spleen and axillary lymph node cells for 51Cr-labeled rat lymphocyte target cells was first detectable 5 days after primary grafting but was quite low showing a peak of only 7% specific 51Cr release 6 days after grafting. In contrast, the cytotoxicity of mouse spleen and lymph node cells for allogeneic target cells after primary skin allografting was significantly greater. It is suggested that the magnitude of the cell-mediated immune response to skin xenografts is less than the response to allografts and that the quicker and more vigorous rejection of skin xenografts is due to a larger participation of humoral antibody in the rejection process.

Antimicrobial factors and bacterial correlation in chronic otitis media with effusion.

Analyses of effusions and sera from patients with otitis media with effusion demonstrated local production in the middle ear of lysozyme, IgA and IgG. The effusion IgM was markedly elevated in some patients, also indicating local production. Complement C3 with rare exception was significantly lower in effusions than sera, suggesting utilization of complement in the middle ear, perhaps in conjunction with antibodies. The presence of high levels of lysozyme and immunoglobulins in effusions correlates with the low isolation rate of microorganisms in culture and may influence survival of organisms in the middle ear.

Characterization of IgG-containing DEAE fractions of canine serum.

A high-titered canine antilymphocyte serum was chromatographed on DE-52 cellulose and the fractions were assayed for cytotoxic activity. Although cytotoxicity was associated with the first 63% of the column effluent corresponding to fractions 1 through 7, significant activity (256) was demonstrable only in fractions 2, 3, 4, and 5. An immunoelectrophoretic study of fractions 1 through 5 showed only IgGa in fraction 1, IgGa and IgGb as electrophoretically separate arcs in fraction 2, and as electrophoretically inseparable arcs in fractions 3 through 5. Cytotoxicity is postulated to be associated with the IgGb subclass, since titers correspond with the distribution of IgGb in column fractions.

A technique for separation of canine lymphocytes and their use in the lymphocytotoxic, blastogenic, and rosette assys.

When the techniques established for preparing lymphocytes from human blood were applied to canine blood, the resulting preparations were contaminated with significant numbers of other blood cells. A three-step technique is described here for the separation of canine lymphocytes from blood which eliminates most granulocytes, platelets, and erythrocytes. Defibrinated blood is incubated with iron particles to allow phagocytosis by granulocytes. When mixed with Plasmagel, erythrocytes and iron-containing granulocytes sediment rapidly, leaving a lymphocyte-rich supernate plasma which is recovered and banded by centrifugation on a Ficoll-Hypaque gradient. Any erythrocytes remaining in the lymphocyte suspension aspirated from the gradient are eliminated by hypotonic lysis and centrifugation. The resulting preparation, representing 30 per cent recovery of the circulating lymphocytes, contained 85 to 94 per cent lymphocytes with a cell viability of 99 per cent. The functional capacity of the lymphocyte preparation was tested in three assays: 1) lymphoblastic transformation using phytohemagglutinin and pokeweed mitogen, 2) E rosette formation with human erythrocytes showed a mean 5 per cent rosette forming lymphocytes, 3) antibody-mediated lymphocytotoxicity using a canine anti-lymphocyte serum showed titers reproducible within one tube dilution.

Chronic middle ear effusions. Immunochemical and bacteriological investigations.

Middle ear effusions from 100 patients (ages 6 months to 10 years) with serous otitis media were examined. The IgA, IgG, and lysozyme were demonstrated at a higher level in the effusions than the corresponding sera, indicating local production. The mucoid type contained higher level of immunoglobulins and lysozyme compared to serous type effusions. Bacteria were found in 77 percent of the effusions by means of a smear, and 52 percent yielded positive bacterial culture. The incidence of positive culture in effusions of the patients less than 6 years of age was 60 percent, while the group older than 6 years old was 32%, and the group over 8 was only 22 percent. Bacterial recovery rate was inversely related to the dramatic increase with age of IgA and IgG and lysozyme levels in effusions.