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.

Osteogenic sarcoma. Immunologic parameters before and during immunotherapy with tumor-specific transfer factor.

18 patients with osteogenic sarcoma were followed by serial measurements in vitro of tumor-specific cell-mediated cytotoxicity and of "active" and total rosette-forming T-cells. 13 of these patients have had or are currently receiving injections of osteogenic sarcoma-specific dialyzable transfer factor derived from healthy donors. In three patients with very small lesions, cytotoxicity was high before amputation and decreased within 2 mo after removal of tumor. Cytotoxicity was low at time of diagnosis in all patients with large tumor masses. The cytotoxicity of the patients' lymphocytes increased after administration of tumor-specific transfer factor in all patients so treated. Patients receiving nonspecific transfer factor showed evidence of declining cell-mediated cytotoxicity. Tumor-specific transfer factor may produce an increase in cell-mediated cytotoxicity to the tumor in patients with osteogenic sarcoma. This possibility is suggested by the pain and edema that occurred in the area of the tumor in patients who had metastatic disease when therapy was started and by lymphocytic infiltrates in the tumor, as well as by the increase in cell-mediated cytotoxicity and the increase in percentage of active rosette-forming cells from subnormal to normal. Serial measurements of cell-mediated cytotoxicity are helpful in monitoring the efficacy of transfer factor and other modes of therapy in these patients, and these measurements are the best available criteria for selection of donors of tumor-specific transfer factor.

Delay of tumor growth in hamsters treated with lynestrenol and effect of Staphylococcus aureus Cowan A.

The action of lynestrenol, a synthetic progesterone-like substance, was studied in Syrian golden hamsters inoculated with cells transformed by herpes simplex virus type I. Daily ip injections of 1 mg lynesterol/kg delayed tumor growth. Lynestrenol alone only slightly increased the survival of the animals. This effect was more marked when the animals were also given injections of Staphylococcus aureus Cowan A. Survivors showed an increased resistance to challenge with infected cells. Hamsters previously given injections of killed transformed cells and then challenged with living cells showed a facilitation of tumor growth. This facilitation was inhibited by lynestrenol alone or combined with S. aureus Cowan A.

Angioimmunoblastic lymphadenopathy with paraproteinemia. Analysis of the clinical and biological characteristics and their prognostic significance.

We report a case of a double M component appearing in the course of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD). Lymphocyte function, especially the decrease in T-suppressor cells, could play an essential role in the mechanisms of the disease. In the presence of an M component, the prognosis for AILD seems to be more pessimistic. Arguments for considering AILD as a premalignant disease are reviewed.

Human autologous and allogeneic rosettes.

Human red blood cells can bind to human peripheral blood lymphocytes. The percentage of these rosette-forming cells (TEh) depends on various conditions including temperature and the use of selected foetal calf serum. It is improved by toluidine blue. Prior incubation at 37 degrees C is not necessary. ABO and rhesus antigen D have no influence on rosette formation. Analysis of human rosettes in T and B cell rich populations, in patients with chronic lymphatic leukaemia or T cell lymphoma provides further evidence that human autologous and allogeneic rosette-forming cells belong to a T cell subpopulation.

Immunogold staining: an alternative method for lymphocyte subset enumeration. Comparison with immunofluorescence microscopy and flow cytometry.

An immunogold staining procedure for light microscopic enumeration of peripheral blood lymphocyte subsets defined by monoclonal antibodies (OKT3, OKT4, OKT8, OKIa1, Leu 1, Leu 4, Leu 2a, Leu 3a, Leu 10, Leu 12, B1) is described. It uses colloidal gold-labeled goat anti-mouse Ig (GAM G40 and GAM G30) as second layer and a methyl-green pyronin counterstain. Performed on small volumes of blood without sophisticated laboratory equipment, this method allows accurate cell type recognition and permanent records, essential for longitudinal observations. By enumerating the gold particles on positively labeled cells, it was shown that the staining reactivity depended on the monoclonal antibody used. Lymphocytes reacting with OKT8 or OKIa1 or B1 exhibited the strongest labeling whereas OKT4+ cells were weakly labeled. When compared with flow cytometry analysis in healthy subjects, the accuracy, precision and sensitivity of both methods were very similar. Similarly, a close correlation (97%) was found between immunogold staining and immunofluorescence microscopy in 35 patients with various diseases suggesting that immunogold staining may be useful in a clinical context.

T antigen expression by human lymphocytes in relation to E rosette affinity.

Active and late rosette-forming cells, separated on the basis of their different affinities for SRBC, were tested for their ability to react with monoclonal antibodies of the OKT series. No significant preferential distribution of T subpopulations defined by these reagents was found in the high affinity E rosette fraction, while in the low affinity T cell subset the major finding was a high number of cells lacking both OKT4 and OKT8 determinants. This seems to be related to methodology, as indicated by experiments in which sequential cycles of rosetting procedures were found to induce loss of reactivity with OKT monoclonal antibodies. The implications of these methodological observations are further discussed.

Influence of in vivo immunosuppressive drugs on production of lymphokines.

The influence in vivo of immunosuppressive drugs (cyclosporine, azathioprine, and corticosteroids) on the production of various lymphokines (alpha and gamma interferon, interleukin 2), both in organ transplant recipients and in normal volunteers taking 100 mg hydrocortisone orally has been studied. To avoid interference with the rejection process or viral infection, patients were studied in a steady state with low maintenance immunosuppression consisting of prednisolone combined with azathioprine or with cyclosporine. In patients treated with both drug regimens, significant depression of production of the three lymphokines was found. Normal volunteers challenged with 100 mg hydrocortisone showed inhibition of production of interleukin 2 and alpha and gamma interferon in 4 hr, a time corresponding to the nadir of T cell lymphopenia, affecting the OKT4 subset preferentially. The percentage of OKT8 cells remained unchanged. Percentages of large granular lymphocytes increased, but their absolute number was not significantly modified. Changes in lymphocyte markers were fully reversible after 24 hr, but interleukin 2 production remained markedly depressed, showing that the redistribution patterns induced by corticosteroids on lymphocyte subsets may be dissociated from functional consequences.

Release of tumor necrosis factor, interleukin-2, and gamma-interferon in serum after injection of OKT3 monoclonal antibody in kidney transplant recipients.

High levels of tumor necrosis factor-alpha, interleukin-2, and gamma-interferon appeared in the circulation of kidney transplant recipients after the first injection of the monoclonal antibody OKT3. This initial injection was systematically followed by fever. The three cytokines were released in all patients (n = 9), with peak serum levels of tumor necrosis factor occurring 1 hr after OKT3 injection and those of interleukin-2 and gamma-interferon after 2 hr. Cytokines were not released after the second and third OKT3 injections, when CD3+ cells had disappeared from blood. These findings suggest that circulating cytokines are released by T cells after activation by OKT3. These cytokines are probably involved in the systematic reactions observed after injection of OKT3.

An immunogold silver staining method for the light microscopic analysis of blood lymphocyte subsets with monoclonal antibodies.

This paper introduces an immunogold silver staining procedure for the identification of human lymphocyte subpopulations in optical microscopy. The described procedure is performed on cells in suspension (peripheral mononuclear cells or blood buffy-coat cells) and uses conventional antilymphocytic monoclonal antibodies (OKT3, OKT4, OKT8, OKIa1) followed by gold-labeled secondary antibodies (GAM G30 or G40) and silver sensitization. It ends with the preparation of permanent records (smears or cytocentrifuge preparations) which are counterstained with standard panoptic Wright or May-Grünwald Giemsa stain. In all cases, the surface antigens appear as numerous black dots on the lymphocytes, with a strong labeling reaction that allows one to clearly distinguish between negative and positive cells. The comparison with immunofluorescence microscopy in normal individuals and in patients indicates that this new immunostaining technic is specific and more sensitive. It has the advantages of using small amounts of blood (1 mL per test), of being rapid (three hours), and of allowing the simultaneous immunophenotypic and morphologic evaluation on smears. This makes it suitable for the analysis of other biologic specimens not already accessible with immunofluorescence. The labeled preparations apparently can be stored indefinitely, and are thus useful for longitudinal studies in the same patient. Finally, the method does not require sophisticated equipment and may be subjected to automated analysis.

Ultrastructural demonstration of retrovirus antigens with immuno-gold staining in prodromal acquired immune deficiency syndrome.

The peripheral lymphocytes of a patient with prodromal acquired immune deficiency syndrome contained giant multivesicular bodies. These were specifically stained by immuno-gold labelled polyclonal antibodies against the major core protein p24 of bovine leukaemia virus and human T cell leukaemia virus I. Moreover, the patient's serum was positive for bovine leukaemia virus by the ELISA method.

Enkephalins and endorphins: activation molecules for the immune system and natural killer activity?

This article summarizes some of the immunological activities of enkephalins and endorphins. Human and animal lymphocytes possess receptors for endorphins and enkephalins. These opioid peptides enhance T cell mitogen response. They decrease in vitro antibody formation. Endorphins and enkephalins will enhance human natural killer activity. This effect may be mediated by several mechanisms including the production of factor(s) enhancing natural killer activity by lymphocytes incubated in presence of opioid peptides. All these results suggest that opioid peptides may be activation molecules for the immune system and as such immunomodulatory agents.

Trisomy 11 in preleukemia.

The clinical and cytogenetic findings of a patient with a preleukemic state and trisomy 11 are reported. Trisomy 11 was present as the sole karyotypic alteration at the time of overt leukemia. Trisomy 11 presents an additional chromosomal abnormality not previously described in preleukemia.

Lynestrenol: a progesteronelike agent with immunostimulatory properties.

Lynestrenol, a synthetic progesteronelike drug, was assessed for its influence upon human leukocyte tests in vitro and in vivo. Lynestrenol markedly enhanced the lymphocyte response to phytohemagglutinin, the mixed lymphocyte culture, the active T-rosette test, the autologous red cell rosettes, and the number of nonadherent cells in the leukocyte adherence inhibition test. All these results indicate a stimulatory effect upon human T-cell function. Lynestrenol also increased monocyte phagocytosis. Lynestrenol delayed the appearance of malignant tumors in hamsters. Finally, lynestrenol, given to lung cancer patients, increased their T-cell functions as evaluated by skin tests. T-cell rosettes, and phytohemagglutinin response. Lynestrenol does possess immunostimulatory properties.

Impairment of phagocyte oxidative metabolism in hemodialyzed patients with iron overload.

In order to study the influence of iron overload on the polymorphonuclear leucocyte (PMN) metabolism of patients on chronic hemodialysis, generation of superoxide anion (O2-) by PMN in whole blood was compared in two groups of hemodialyzed patients: group A consisted of twenty-one individuals with serum ferritin levels above 1000 ng/ml and group B of nineteen individuals with serum ferritin levels below 1000 ng/ml. Whereas basal production of O2- was similar in the two groups (6.3 +/- 4.6 vs 11.5 +/- 8.3 nmoles O2- 10(6) granulocytes-1 15 min-1) (mean +/- s.e.m.), PMN response to opsonized zymosan was significantly lower in group A as compared with group B (86.5 +/- 6.3 vs 120.4 +/- 8.2 nmoles O2- 10(6) granulocytes-1 15 min-1) (p less than 0.01). Superoxide anion generation induced by the dialysis procedure was reduced in eight patients from group A (89.2 +/- 32.1) as compared with eight patients from group B (374.3 +/- 100.0 nmoles O2- 10(6) granulocytes-1 15 min-1) (p less than 0.05). These data suggest that iron overload may be involved in the impairment of neutrophil phagocytosis in patients on chronic hemodialysis.

[Immunologic and hematologic sequelae of splenectomy]. / Les séquelles immunologiques et hématologiques de la splénectomie.

This article summarizes the hematological and immunological consequences of splenectomy in children. The hematological consequences are mainly noticed in the number of white cells and platelets. Indeed, platelets can remain elevated several weeks and even several months after surgery. Rarely antiplatelets aggregant drugs will be given if the platelet count is higher than one million per cubic millimeter. The immunological consequences of splenectomy in children are mainly manifested if the surgery was performed before the age of 6 or 7. The major consequence is a rapid and often lethal infectious complication. In diseases like thalassemia major or lymphoma, there is an increased risk of infection. The immunological mechanism seems linked to a decreased antibody formation in presence of higher antibody titre needed by the reticuloendothelial system to eliminate the microorganisms responsible for the infection. Therapeutic measures can be proposed: delay the age of splenectomy, leave some splenic tissue, administration of penicillin and lastly immunization against some antigens mainly responsible of the infection.

[Preoperative autotransfusion for elective surgery]. / L'autotransfusion préopératoire pour intervention programmée.

In order to eliminate the risk of transmission of infectious diseases and of alloimmunisation, a pre-deposited autologous blood program for elective surgery has been started at the Erasmus Hospital. In 8 months, 51 patients have given 111 units of packed red cells and 111 units of fresh plasma. Two vasovagal reactions were observed. Four patients were withdrawn from the program in view of a hematocrit decreasing below 34%. Out of these 51 patients, 27 (53%) were transfused with only autologous blood. The autologous blood not distributed to these patients was given to other patients (10% of packed red cells and 24% of plasma). This pre-deposited autologous program has allowed to operate more than 50% of patients, undergoing elective surgery, only with autologous blood and to save homologous blood. The pre-deposited blood appears to be an interesting tool in transfusion safety and in the reduction of blood demand.