H. Hugh Fudenberg

Thymus-Derived Rosette-Forming Cells in Various Human Disease States: Cancer, Lymphoma, Bacterial and Viral Infections, and Other Diseases

Lymphocytes that bind in vitro to sheep erythrocytes in a rosette formation are thymus-derived. A modified technique that does not detect the total number of rosette-forming cells (RFC) was used to study normal subjects and various disease states. Of 100 healthy subjects, 95 had more than 15% RFC (mean 28.4+/-6.5%). We studied 104 patients with solid tumors, who were classified according to clinical status and stage of therapy. Of 19 newly diagnosed patients, 13 had less than 15% RFC. Of 44 untreated patients undergoing relapse, 32 had less than 15% RFC. In both categories, patients with metastases had fewer RFC than patients with localized disease. 11 patients were studied 2 wk after cessation of therapy; four of them showed less than 15% RFC. Only one of 30 patients in remission had less than 15% RFC. In seven patients followed for various periods of time, the numbers of RFC correlated generally with clinical status. 11 patients with chronic lymphatic leukemia had very low percentages of RFC. 21 of 21 patients with symptoms of viral upper respiratory diseases had less than 15% RFC. RFC returned to normal values between 5 days and 7 wk after disappearance of clinical symptoms. 20 patients with bacterial infections had normal numbers of RFC. Of 25 patients with miscellaneous nonimmunologically related diseases, two had low numbers of RFC. It appears that the percentage of RFC may be valuable in evaluating not only immunological defenses but also the status of patients with solid tumors, lymphomas, viral diseases and, perhaps, bacterial infections.

The Human Rosette-Forming Cell as a Marker of a Population of Thymus-Derived Cells

Sheep red blood cells can surround, in vitro, some human peripheral blood lymphocytes in a formation called a rosette. The number of rosetteforming cells (RFC) in 50 normal persons had a wide range (4-40%). The organs of 13 human fetuses (11-19 wk conceptional age) were examined for the presence of RFC. The thymus possessed the highest percentage of RFC, the maximum being 65% of total thymocytes in two 15-16 wk fetal specimens. Blood RFC were always present and their number slightly increased in the oldest fetuses. The bone-marrow showed 0-8% in the six fetuses studied. RFC were found in the spleen around the 13th wk and in the liver around the 17th wk of gestation. These observations lead to the hypothesis that human blood RFC may be chiefly thymic derived. Studies of patients with immunological disorders support this hypothesis: one patient with Nezelof syndrome had no blood RFC and four patients with Wiskott-Aldrich syndrome had a low number of blood RFC (1 and 1.5%). Patients with acquired hypogammaglobulinemia showed a normal percentage of RFC. With the fetal thymocytes, the percentage of inhibition with anti-mu serum increased with the fetal age to become complete in the oldest fetuses studied. Incubation of the oldest fetal thymocytes or the blood lymphocytes with anti-gamma serum of anti-mu serum completely inhibited the rosette formation. These results suggest that mu-chain determinants are present on human fetal thymocytes and blood RFC. The significance of the presence of gamma-chain determinants on these cells is unclear.

Rosette-Forming Cells, Immunologic Deficiency Diseases and Transfer Factor

Abstract Lymphocytes from peripheral blood of normal subjects and of patients with various immunologic deficiency diseases were studied to determine the percentage able to bind with sheep erythrocytes in a formation called a rosette. These rosette-forming cells represent thymus-derived cells. Patients with Nezelof syndrome, Wiskott-Aldrich syndrome, and chronic mucocutaneous candidiasis, all diseases with defects in cellular immunity, had low percentages of these cells. Six patients with acquired hypogammaglobulinemia, a defect in humoral immunity, had normal numbers. Three patients with Wiskott-Aldrich syndrome responded clinically and immunologically to administration of transfer factor, and these patients showed significant increases in rosette-forming cells. One patient with Wiskott-Aldrich syndrome and those with chronic mucocutaneous candidiasis who did not respond to transfer factor did not show increases in rosette-forming cells.

The Wiskott-Aldrich Syndrome: RESULTS OF TRANSFER FACTOR THERAPY

12 patients with Wiskott-Aldrich syndrome were treated with therapeutic doses of transfer factor in an attempt to induce cellular immunity. Clinical improvement was noted after transfer factor therapy in 7 of the 12 patients treated. Because this disease has a variable course and temporary spontaneous improvement can occur, the observed improvement cannot necessarily be attributed to the transfer factor. However, in two patients repeated remissions consistently followed transfer factor administration on repeated occasions. This included freedom from infections, regression of splenomegaly, and clearing of eczema. An unexpected finding was a decrease in bleeding in 3 of the 10 patients who had bleeding. Conversion of skin reactivity was obtained in all seven patients who clinically seemed to respond to transfer factor. In vitro studies performed after the administration of transfer factor demonstrated that the lymphocytes of the patients now produced migration inhibitory factor in response to appropriate test antigens, but did not undergo increased radioactive thymidine incorporation in response to the same antigens. A defect in the monocyte IgG receptors has been found in certain patients with the disease, and the current study shows that all patients with defective monocyte IgG receptors responded to transfer factor, whereas only one patient with normal receptors showed any response. This test may thus prove to be useful in predicting the results of transfer factor therapy in patients with Wiskott-Aldrich syndrome, although evaluation of a larger series of patients will be necessary to confirm this point. We conclude that cellular immunity can be induced, that there appears to be clinical benefit in certain patients with Wiskott-Aldrich syndrome by the use of transfer factor, and that this mode of therapy warrents trial in these patients and others with defects of cellular immunity.

Genetically determined immune deficiency as the predisposing cause of “autoimmunity” and lymphoid neoplasia

Burnet has postulated that autoimmunity arises from emergence of “forbidden clones” which make auto-antibody. Our postulate, in contradistinction, is that auto-antibody-producing cells are always present, producing enough autoantibody to remove damaged or aged tissues. In normals, the number of such cells is kept in check by normal T-cells. When T-cell function is defective, the “governing system” controlling auto-antibody producing clones fails, and autoimmune disease, mediated in most diseases by cellular immunity, emerges. The governing mechanism in normals may be one analogous to that which limits the amount of antibody produced to a given antigen, and keeps the antibody (and Ig levels) from continuously rising in normal subjects. Experimental data for this concept are provided by several lines of evidence: 1) Normals have low levels of rheumatoid factor and other “auto-antibodies” in their serum.

ASSOCIATION BETWEEN IMMUNOGLOBULIN ALLOTYPES AND IMMUNE RESPONSES TO HÆMOPHILUS INFLUENZÆ AND MENINGOCOCCUS POLYSACCHARIDES

Serum samples were collected from 20 healthy White and 33 Black infants before and after immunisation with three doses of diphtheria-pertussis-tetanus vaccine and with one dose of Haemophilus influenzae type b polyribose phosphate vaccine and meningococcal group A and group C polysaccharide vaccines. Antibodies to these immunogens were measured and sera were allotyped for several Gm, A2m, and Km antigens. A highly significant association was found between the Km(1) allotype and the immune responses (difference between post-immunisation and pre-immunisation antibody levels) to H. influenzae and meningococcus C polysaccharides in the White children.

Binding of [125I]corticotropin releasing factor to blood immunocytes and its reduction in Alzheimer's disease

The binding of [125I]iodine-labelled corticotropin releasing factor (CRF) was studied using peripheral blood lymphocytes from normal donors and Alzheimers disease (AD) patients. The high affinity binding of [125I]CRF was found in the membranes of various immunocytes. Monocytes and T cells displayed binding which was several times greater than the binding of brain (cortical) cells. The immunocyte CRF binding was significantly (P less than 0.001) lower in 14 out of 18 (78%) AD patients relative to non-AD controls, suggesting the association of CRF in the pathology of AD. Our data demonstrate that blood immunocytes can be used to analyze deficiency of neurohormone sites in neuropsychiatric diseases, e.g., AD.

Abnormal T cell subpopulations and circulating immune complexes in the Guillain‐Barré syndrome and multiple sclerosis

Immunologic studies were performed in 21 patients with multiple sclerosis(MS) and 16 with the Guillain-Barré syndrome (GBS). Levels of thymus-derived (T) cells measured by “total” and “active” rosette formation between sheep erythrocytes and peripheral blood mononuclear cells (TEt, TEa) were within normal limits in all the patients, with the exception of four GBS patients, including one who also had received chemotherapy for lymphoma and three who were receiving steroids. When lymphocytes from the 21 patients were incubated with the bone-marrow-derived (B) lymphoblastoid cell line PGLC-33H, there were, for 12 of 18 MS patients and 11 of 16 GBS patients, significant decreases in a subpopulation of peripheral blood T lymphocytes that form “PGLC rosettes” (PGR) with the PGLC-33H cells. (Peripheral blood T cells from normal individuals formed PGR with 23.9 ± 3.8 percent of PGLC-33H cells.) Using the l25 I-Clq binding assay, immune complexes were detected in the serum of 14 of 19 MS patients and 15 of 16 GBS patients. An association between increased C1 q binding and decreased PGR values was found in 10 of 18 MS patients and 12 of 17 GBS patients. The results suggest that in both diseases the etiology may involve a decrease in the subset of T cells that bind to the IgM-producing cell line PGLC-33H, in association with the appearance of circulating immune complexes containing the infectious viral agent.

Immunofixation after electrofocusing: Improved method for specific detection of serum proteins with determination of isoelectric points I. Immunofixation print technique for detection of alpha-1-protease inhibitor

An improved method is described for direct localization of human serum proteins in polyacrylamide gel with simultaneous determination of their isoelectric points (pI). The technique employs isoelectric focusing in thin-layer polyacrylamide gels to separate the serum proteins and the pH gradient is read at 4 degrees C with a dual-membrane surface microelectrode. Subsequently, the desired proteins are localized by immunofixation in the gel or by immunofixation-printing onto cellulose acetate strips soaked in specific antiserum. No sectioning of the electrofocused gel is necessary, and the entire technique can be completed in less than 14 h. When this method is applied to the detection of the genetic variants of alpha-1-antitrypsin (alpha-1-protease inhibitor) (A1Pi system), the results indicate that it can be used to specifically localize serum proteins whose pIs differ by as little as 0.01 pH units. The resolution afforded is especially evident in the analysis of A1Pi M variants.

Cyclic variations in white cell subpopulations in the human menstrual cycle: Correlations with progesterone and estradiol

Cyclic variations in white cell subpopulations were studied in serial blood samples from 18 female volunteers (14 ovulatory and 4 nonovulatory cycles) and 2 males. Total white blood cells (WBC), lymphocytes, total and active T cells (TEt, TEa), monocytes, and granulocytes were counted, and levels of estradiol (E2), progesterone, and luteinizing hormone (LH) were measured. In the ovulatory cycles, lymphocyte counts at midcycle (Day 0) reached a minimum, coinciding with the maximum level of E2 peak (35 ng%). In daily samples, the minimum lymphocyte counts coincided with the preovulatory E2 surge (P < 0.01). Similar but less significant negative correlations (P < 0.05) were found between E2, WBC, and TEt. In contrast, TEa did not show cyclic variations. Monocyte and granulocyte counts were significantly higher in the luteal than in the follicular phase (P < 0.05); their pattern followed closely that of progesterone (P < 0.05) but not of E2. Daily evaluations of hematocrit, hemoglobin content, red blood cell count, and mean corpuscular volume failed to show cyclic variations. For the females with nonovulatory cycles and for the males studied (with sex steroid profiles corresponding to the follicular phase), no cyclic variations were found in white cell subpopulations.