Studies on human peripheral blood lymphocytes in vitro. I. Biological and physicochemical properties of the pokeweed mitogen.

The saline extract from the roots of Phytolacca americana (pokeweed) possesses three biological properties; hemagglutinin, leukagglutinin, and mitogen. Fractionation and further purification on calcium phosphate column chromatography revealed that the biologically active substance was eluted in the front moving fraction with 0.05 M phosphate buffer pH 7.5. Analytical separation on polyacrylamide gels in disc electrophoresis yielded a single homogeneous band with an R(f) value of 0.43 containing all three biological activities. This fraction had an ultraviolet absorption spectrum similar to PHA, was stable to both periodate and mercaptoethanol treatment and gave a single band in double diffusion and immunoelectrophoretic analysis against the antibody prepared to the crude PWM saline extract. Absorption studies with red cells or stroma revealed that the hemagglutinin could be selectively removed without significantly altering the mitogen, whereas absorption with leukocytes resulted in loss of both the mitogenic and leukagglutinating activities.

Studies on human peripheral blood lymphocytes in vitro. II. Morphological and biochemical studies on the transformation of lymphocytes by pokeweed mitogen.

A study of the kinetics of RNA and DNA synthesis in PWM-stimulated lymphocytes revealed that RNA synthesis preceded the onset of DNA synthesis by approximately 24 hr and that DNA synthesis and transformation was maximal between 66 to 78 hr. Histochemical and radioautographic studies on PWM stimulated cultures indicated that at 72 hr 50 to 60% of the cell population had been transformed by PWM, and that a distinct cell type bearing cytologic resemblance to the early plasma cell had emerged. The RNA sedimentation profile for newly synthesized RNA in PWM-stimulated cells showed that a large peak of 45 to 50 S material was formed after 24 and 40 hr. PWM thus produces a distinctive transformation of human peripheral blood lymphocytes.

Infectious mononucleosis: immunoglobulin synthesis by cell lines.

Immunoglobulin synthesis by 16 long-term suspension cultures of mononuclear cells derived from peripheral blood of nine patients with heterophile-positive infectious mononucleosis (IM) has been demonstrated by radioimmunoelectrophoretic techniques. All cell lines synthesized molecules with IgG (gamma) heavy chain specificity. 14 cell lines produced molecules with IgM (mu) heavy chain specificity and 11 cell lines produced molecules with IgA (alpha) heavy chain specificity. No detectable synthesis of molecules with IgD (delta) heavy chain specificity was observed by these cell lines derived from peripheral blood of patients with IM. 13 cell lines produced molecules with type K (kappa) light chain specificity and 6 cell lines produced molecules with type L (lambda) light chain specificity. Of interest, 9 of 16 lines produced IgG (gamma), IgA (alpha), and IgM (mu) heavy chain molecules and 5 of these cell lines produced molecules with type K (kappa) and type L (lambda) light chain specificity as well. Further characterization by combined polyacrylamide gel filtration, immunodiffusion, and radioautography indicated the presence of newly synthesized immunoglobulin molecules with both heavy and light polypeptide chains in close association as well as free light polypeptide chain synthesis. Investigation of the localization of immunoglobulin in single cells by immunofluorescent techniques revealed that 5-22% of cells in these lines were strongly reactive with a fluorescein isothiocyanate-conjugated rabbit antisera directed against the antigenic determinants of human IgG and cross-reactive with the determinants common to IgA and IgM. No heterophile antibody, heteroagglutinin, or hemolytic antibody could be demonstrated in these cell lines derived from peripheral blood of patients with heterophile-positive infectious mononucleosis.

Etiology of granulomatous disease of the gut (Crohn's disease).

This report is based on a review of the various investigations attempting to determine the etiology of Crohn's disease. It has paid special attention to three main possible etiologic mechanisms, reported in the literature up to the time of this review; namely, immunologic, unknown transmissible agents or known such as L-forms, protoplasts, or spheroplasts, viral or infectious RNA and/or DNA, and a combination of the preceding causative possibilities. It has discussed the unrewarding immunological mechanisms, the failure to confirm the various investigations suggesting a transmissible agent and/or the speculations concerning the possible combination of factors. It also has included the remarks of Sacchar and Auslander, who stated that four important pieces were missing to allow acceptance of transmissible agent as the cause of Crohn's disease. This review has demonstrated that up to the present time the etiology of Crohn's disease has remained a puzzle and concluded that further investigations are necessary to resolve the etiology of Crohn's disease with the suggestion that evidence for an infectious agent will require experiments that satisfy Koch's postulates.

An epidemiologic study of inflammatory bowel disease in Rochester, New York. Hospital incidence.

The epidemiological understanding of inflammatory bowel disease has been limited by the referral bases of most inflammatory bowel disease studies. The Colitis-Ileitis Study Group of Rochester, N.Y., developed a community-wide, computerized cumulative registry of all inflammatory bowel disease patients hospitalized at the 8 community hospitals for 1973-86. Clinical data were abstracted from each of the 1651 identified hospital charts. All of these patients resided in Monroe County (city and suburbs) and the 5 contiguous counties (Genesee/Finger Lakes Region, population 1,030,640). Of the 1651 hospital patients identified in the study, 1358 resided in Monroe County (Rochester and its immediate suburbs, population 702,238). Incidence, defined as time of onset of symptoms of inflammatory bowel disease, rose from baseline rates in the 1930s to peak in 1980 (Crohn's disease = 50.29/10(5) per decade, ulcerative colitis = 35.12/10(5) per decade) and declined through 1986. For Crohn's disease, the age-specific incidence rates peaked in the 20-29-yr-old group in each of the 5 decades studied. Ulcerative colitis seems to occur at all ages and may have a bimodal distribution. There was a period effect, with the 1970s having the highest incidence of Crohn's disease and ulcerative colitis for each age group. However, the age-specific incidence rate for Crohn's disease showed a 40% decrease in the 1980s compared with the 1970s in the 10-39-yr-old group (p less than 0.001). The age-specific incidence rate for ulcerative colitis showed a 50% decrease in the 1980s compared with the 1970s in the 10-49-yr-old group (p less than 0.001).

The cause of death in inflammatory bowel disease: a comparison of death certificates and hospital charts in Rochester, New York.

OBJECTIVE: This study examines the causes of death from Crohn's disease and ulcerative colitis by comparing death certificates with hospital charts as part of an ongoing, community-based analysis in Rochester, NY. METHODS: A registry of 1358 inflammatory bowel disease patients followed from January 1973 to December 1989 was analyzed for the cause of death by a study of death certificates as well as by a study of hospital records, including surgical pathology and autopsy records. A panel of physicians defined specific criteria for diagnosis, cause of death, and relation of death to inflammatory bowel disease. RESULTS: One hundred and thirty patients (59 with ulcerative colitis and 71 with Crohn's disease) from the registry were found to have death certificates recorded by Monroe County during this period. There was an 80% concordance of the death certificate to the hospital record for the cause of death and its relationship to inflammatory bowel disease. Discordance was noted in cases of colon cancer and surgical complications. CONCLUSIONS: Sixty-eight percent of Crohn's disease and 78% of ulcerative colitis patients died from causes unrelated to their inflammatory bowel disease. Deaths caused by Crohn's disease decreased from 44% in the 1973-1980 period to 6% in the 1981-1989 period. Crohn's disease was a direct cause of death in 25% of the female patients, whereas only 6% of male patients died directly of Crohn's disease. Colorectal cancer caused 14% of the deaths in ulcerative colitis patients, three times more often than in Crohn's disease patients. Excluding cancer, there were only two deaths directly due to ulcerative colitis, both in the first 2 yr after diagnosis.