Immunological studies of coagulation factor XIII.

Human fibrin-stabilizing factor (Factor XIII) has been studied immunologically by the preparation of specific anti-Factor XIII antiserum in rabbits. On immunodiffusion it was found that normal plasma produced two precipitin lines. One of the precipitin lines was identical with that present in soluble platelet extract (the alpha-component), the other with that present in normal serum (beta-component). Plasma and serum of patients with congenital Factor XIII deficiency contained only the beta-component. By adsorption it was possible to prepare a second antiserum with solely anti-alpha-activity that did not react with the serum or plasma of XIII-deficient patients. Both antisera neutralized the clot-stabilizing activity of normal plasma. The action of thrombin on fibrinogen-free plasma or platelet extract abolished the immunoprecipitin alpha-line but did not reduce the capacity to neutralize antibody as measured by clot stabilization. It is concluded that the plasma Factor XIII molecule consists of two immunologically identifiable components, alpha and beta. The clot-stabilizing activity and thrombin-reactive site are situated on the alpha-component. Patients with congenital Factor XIII deficiency are devoid of immunologically identifiable or functional alpha-component but retain immunologically identifiable beta-component. It is this beta-component that accounts for the observed immunologically detectable Factor XIII in those patients devoid of clot-stabilizing activity.

Evidence for a second cell cycle block at G2/M by p53.

Wild type p53 can induce cell cycle arrest at specific points in the cell cycle, in particular G1/S, an ability lost by most p53 mutants. We have previously reported that p53 mutant genes can rescue REF52 cells from ras-induced growth arrest and that over expression of wild type p53 inhibits cell growth in these cells. In this paper we examined whether p53 can also induce cell cycle arrest at the G2/M boundary of the cell cycle. To accomplish this we used the REF52 cell line and the temperature sensitive p53val135 mutant allele. Cells were enriched in the late G1 and early S phases before the temperature shift. REF52 cells expressing mutant-p53val135 alone with an activated H-ras gene arrest primarily at the G1/S and G2/M parts of the cell cycle at the restrictive temperature, as determined by flow cytometry analysis. These results suggest that the anti-proliferative activity of p53 may be involved in regulation of the cell cycle at the G2/M restriction point as well as transit through G1/S and initiation of DNA synthesis.

Pathways of T cell suppression.

The methods of detection of suppressor cells which are generated during an immune response are reviewed. The requirements and the mechanisms for their induction as well as their relationship to other functionally important cell subpopulations (e.g., helper T cells, cytotoxic T cells) are examined. Their nature and the mechanism of their action are reviewed. The control of the generation of suppressor cells by well-defined subregions of the I region of the MHC is one of the most important recent developments. Suppressor cells have been shown to play an important role in a variety of systems such as allotypic suppression, the poor response of some strains of mice to certain polypeptides, the progress of tumor growth, etc. However, their role in other phenomena such as tolerance to self antigens and thus their involvement in the development of autoimmunity is not yet clear. In the NZB/W murine lupus model loss of suppressor cells has been implicated in the progress of autoimmune disease. Soluble mediators with suppressive activity probably represent the effector molecules derived from suppressor cells. Some of them have now been shown to contain antigens coded by the I-J subregion of the MHC. The mechanism of their action and the target cell varies from system to system and is not yet well understood.

Insidious rifampin-associated renal failure with light-chain proteinuria.

A patient who was receiving rifampin treatment for tuberculosis developed heterogenous light-chain proteinuria and insidious renal failure after a period of fluid restriction. The renal damage was characterized pathologically by an interstitial nephritis with invasive tubular casts and an associated renal vein thrombosis. The possible role of the light-chain proteinuria in the pathogenesis of the renal failure is discussed.

Development of anti-hLH antibodies after therapy with posterior pituitary extract.

This report describes the appearance of high affinity antibodies to human LH in a girl who had been treated for diabetes insipidus with injections of pitressin tannate, plus occasional nasal insufflations of posterior pituitary powder. Immunological studies indicated that the antibody was a 7S IgG directed against the beta subunit of LH, which is not species-specific. The demonstration of immunoassayable LH in a commercially available pitressin preparation strongly suggests that this patient was immunized by bovine or porcine LH. Although studies of her urinary LH excretion and serum LH (by an interstitial cell bioassay system) suggest that at least some of her endogenous LH is not bound by the antibody, the possibility remains that this type of immunization may have important implications for the development and maintenance of normal adult pituitary-ovarian relationships.

The double gammopathies. Clinical and immunological studies.

A retrospective review of 1135 patients with paraproteinemias recorded 28 (2.5%) as having two M components. This group included 11 patients with myeloma, 6 with lymphoproliferative disease, 5 with a nonlymphoproliferative malignancy, and 6 with a double gammopathy of undetermined significance. In 13 cases in which the M components were measured over a period of time, three distinct patterns were observed, which may reflect the cellular and subcellular origin of the two proteins: 1) In 2 cases the minor component remained relatively stable while the dominant protein changed with time and treatment, suggesting the origin to be two cell lines--the minor arising from a quiescent clone of the monoclonal gammopathy of undetermined significance, and the major M component arising from a more rapidly proliferating plasma cell line; 2) a discordant pattern was seen in 4 patients, suggesting that the two M components arose from two separate plasma cell clones; 3) seven cases in which the proteins behaved in a concordant manner probably arose from a single plasma cell clone with incomplete class switching, producing two M components with different heavy chains.

Macrophage T cell interaction. Induction in vitro of a mediator with potent antisuppressor activity.

A simple in vitro method is described for the induction of a potent mediator that interferes with suppressor cell function. The mediator consists of three easily identifiable components, Ig, class II determinants and antigen, that form a unique complex similar to, or identical with, the complexes detected in vivo within 3-6 h after immunization. The formation of the antisuppressor mediator in vitro takes place in two steps: the first involves a macrophage-T cell interaction which generates an 'intermediate complex' containing antigen and class II determinants. In the second step the addition of immunochemically purified IgG from normal mouse serum to the macrophage-T cell supernate generates potent antisuppressor activity, which is assayed by the conversion of suppression to immunity. It is suggested that the IgG interacts with the 'intermediate complex' giving rise to the final complex identical to that found in vivo 6 h after immunization. No activity is detected when IgG is added to a supernate of antigen-fed macrophages in the absence of T cells. Furthermore, the T cell plays an additional important role in the formation of the final complexes since it restricts the source of the IgG that will generate the antisuppressor activity. In other words the antisuppressor function is detected only if the IgG matches the donor of the T cell in the Igh locus. The T cell involved in the formation of the complex is the Ly1+ subpopulation. This method should allow elucidation of the genetic, cellular and molecular mechanisms in the activation of this important T cell pathway.

Liposomes as immune adjuvants: T cell dependence.

The T cell dependence of the immune adjuvant action of liposomes containing the soluble antigens bovine serum albumin (BSA) and chicken immunoglobulin (CIgG) was studied with use of a quantitative enzyme-linked immunosorbent assay to measure serum antibody levels. Normal BALB/c mice, adult thymectomized mice, and congenitally athymic (nu+/nu+) mice were intravenously inoculated with liposomes containing BSA (Lip-BSA). The high levels of serum anti-BSA antibody that were seen in the normal group were decreased in the adult thymectomized group and were almost completely abrogated in the nu+/nu+ group. Reconstitution of nu+/nu+ mice with normal thymocytes and cortisone-resistant thymocytes led to a partial restoration of the anti-BSA antibody production after Lip-BSA immunization. Examination of the class of immunoglobulin produced in normal mice, immunized with Lip-BSA, showed an early low IgM response and a sustained higher IgG response that was primarily due to the IgG1 subclass. Trypsin removal of BSA exposed on the liposome surface decreased the resulting serum anti-BSA antibody level by 30% to 50%. Animals could be primed equally with a very low dose (0.2 micrograms) of Lip-BSA or with peritoneal macrophages that had phagocytosed the same dose of Lip-BSA. The adjuvant effect of liposomes containing CIgG on the number and type of specific anti-CIgG antibody-producing cells in the spleen was an early increase in IgM-producing cells followed by a substantially higher increase in IgG-producing cells. These observations suggest that liposome encapsulation of a soluble T-dependent antigen stimulates the helper T cell, not the suppressor T cell population, and that this stimulation involves uptake by macrophages.

Cytodiagnosis of lymphoid proliferations by fine needle aspiration biopsy. Adjunctive value of flow cytometry.

OBJECTIVE: To determine the accuracy of fine needle aspiration biopsy (FNAB) complemented by flow cytometry (FC) for the diagnosis of reactive and neoplastic lymphoid proliferations and subclassification of malignant lymphomas. STUDY DESIGN: Forty-one FNABs of lymphoid lesions on which FC had been performed were evaluated retrospectively. All cases were correlated with histology or clinical follow-up. RESULTS: Twelve FNABs were diagnosed as reactive. Eleven of the 12 were confirmed as reactive on follow-up. One was a case of posttransplant lymphoproliferative disorder. Twenty-five FNABs diagnosed as lymphoma were confirmed by histology. In 22 of these 25 cases, there was 100% correlation between the subclassification given on FNAB with FC and that given on histology. Two of the remaining cases, which were correctly called follicular center cell lymphoma, showed discrepancies in grading. One case called Hodgkin's disease on FNAB was T-cell lymphoma on histology. Of four FNABs given an inconclusive diagnosis, two were lymphoma on follow-up, and two were reactive. CONCLUSION: FNAB examination, when it includes immunophenotyping by FC, is a useful technique for distinguishing reactive lymphoid proliferations from malignant lymphomas and for the subclassification of lymphomas.

Release and reconstitution of Fc receptors from normal human mononuclear cells.

Nylon-fibre non-adherent cells isolated from human peripheral blood, release their Fc receptors for IgG upon culture in vitro in medium containing 0-2% foetal calf serum. The majority of Fc+ nylon-fibre non-adherent cells was reconstituted using supernates from different cell populations of human peripheral blood. The best reconstituting activity was detected in the supernates from glass-adherent cells, or unfractionated Lymphoprep cells, while those obtained from nylon-fibre non-adherent cells gave variable results sometimes containing no activity at all. It was shown that reconstitution was achieved by the uptake of Fc receptors present in the supernate which inhibited Fc rosette formation. Inhibition was also achieved with supernates from peripheral blood cells of a patient with monocytic leukaemia. Only autologous but not allogeneic cell supernates could reconstitute Fc receptors. This suggests that the reconstitution of Fc receptors on peripheral blood Fc-IgG lymphocytes by monocytic cells is under genetic control.

Soluble factors in immune sera of mice. II. Reversible loss of surface immunoglobulin induced in vitro.

Serum collected from BALB/c mice at different time intervals during primary immunization induces in vitro a decrease of Ig+ cells in short-term cultures of normal spleen cells. The decrease was shown to be caused by loss of surface immunoglobulin (Ig) for the following reasons: no cell loss was detected which coulc account for it; the number of Ig+ cells returned to normal levels when the serum was removed and the cells were cultured further in fresh medium. The serum activity was recovered in a fraction with molecular weight less than 10,000 Daltons. The factor was active at 4 degrees C and on cells treated with high doses of Con A. Only a portion (25-30%) of the Ig+ cells are affected by this factor since after the initial decrease no further changes were observed during the 7 hr culture period. No changes in the distrubution of surface Ig was detected by fluorescent techniques on the remaining Ig+ cells. This factor was detected as early as 6 hr after immunization but its concentration was found 4-fold higher on the 7th day.

Cancer and depression: cancer presenting with depressive illness: an autoimmune disease?

It is proposed that some cases of depressive illness in cancer patients may be caused by immunological interference with the activity of serotinin, one of the neurotransmitters thought to be implicated in depression. This interference could be mediated in two ways. Antibody induced against a protein released from cancer cells could, on the basis of cross-reactivity with CNS tissue, bind to receptors for serotonin and block them. Such primary antibodies could stimulate the production of anti-idiotypic antibodies, which would act as an alternative receptor for serotonin and reduce its synaptic availability.

Soluble factors in immune sera of mice. I. Specific and non-specific suppressive activity.

Serum collected from mice 7 days following immunization with heterologous erythrocytes (SRBC, HoRBC) contained potent and specific immunosuppressive activity. When the serum was filtered through a Diaflo UM-10 membrane the activity was recovered in the filtrate, indicating that a factor less than 10,000 Daltons is responsible for it. Filtrates obtained from animals immunized with soluble antigens (BSA, POL) in FCA suppressed the 7S response to SRBC but had no effect on the 19S response. This non-specific suppressive effect on the7S response was also shown by filtrates obtained from sera of animals injected with FCA or other adjuvants (LPS, poly A:U). The 19S response to SRBC was either not affectd (LPS, poly A:U) or actually enhanced. (FCA). The SRBC-induced filtrates markedly suppressed the espression of DH to SRBC.

Inhibition of antisuppression by the acute murine graft-versus-host reaction.

Profound suppression of both humoral and cell-mediated immunity is a significant systemic effect of graft-versus-host reactions. Although no complete explanation has been advanced for this immunosuppression suppressor cells have been implicated. The data presented in this paper indicate that acute GVH reactions in (C57BL/6J X A/J) F1-hybrid mice induced by the injection of A/J cells severely disrupts the function of the antisuppressor T-cell pathway at both its induction and effector stages. Results show that within 3 weeks of induction of the reaction, Ly1+-T antisuppressor inducer cells lose their ability to generate the serum factor that mediates antisuppression. This factor is normally taken up by and activates Ly2+ T cells which then inhibit suppressor T-cell function. The data also reveal that Ly2+ T cells collected 2 weeks after induction lose their ability to be activated by the antisuppressor factor produced in normal mice. These cells are thus unable to function as antisuppressor effector cells. The uptake of the antisuppressor factor by Ly2+ T cells depends on the expression of Ia antigens on the surface of these cells. Experiments have shown that these antigens are absent from the surface of T cells derived from mice with GVH reactions. This finding may provide an explanation for the inability of these cells to function as antisuppressor effectors. Antisuppression is an important T-cell pathway that is intimately associated with the regulation of immune function. It is possible that the immunosuppression arising in mice with GVH reactions may stem, in part, from unopposed suppressor T-cell activity that results from widespread interference by the reaction with a pathway that normally inhibits suppressor cell activity.