Fractionation and characterization of the immunosuppressive substance in crude extracellular products released by Streptococcus intermedius.

The noncytotoxic immunosuppressive substance detected in crude extracellular products of Streptococcus intermedius (CEP-SI) was fractionated by two steps of preparative isoelectric focusing in sucrose gradients using ampholytes of pH range from 3.5 to 6 and 4 to 5, respectively. The in vitro and in vivo suppressor effects of the most highly purified fraction of CEP-Si, designated fraction 3' (F3'EP-Si), corresponded well with those of the original CEP-Si. F3'EP-Si was sensitive to the effects of alpha, gamma, and delta chymotrypsin, trypsin, and heating. It contained approximately 1% of the total amount of protein found in the original CEP-Si, corresponding to a single band on analytical isoelectric focusing, stainable by Coomassie Blue and of isoelectric point of 4.25. The absorption spectrum of F3'EP-Si had a maximum at 260 nm but its biological activity was resistant to deoxyribonuclease and ribonuclease A and it did not contain material stainable by methylene blue. It was also resistant to neuraminidase and did not contain material stainable by periodic acid schiff. We conclude that the substance responsible for the suppressor activity of CEP-Si is a protein of molecular weight approximately 90,000, which adheres to Sephadex of cellulose acetate and forms complexes with other, nonactive constituents of CEP-Si.

Evidence for the synthesis and release of strongly immunosuppressive, noncytotoxic substances by Streptococcus intermedius.

Products secreted by Streptococcus intermedius were studied for their effects on the immune response. Three different preparations of crude extracellular products from S. intermedius (CEP-Si) were found to have powerful suppressor activity in vitro as shown by inhibition of human lymphocyte proliferation (uptake of [3H]thymidine) and protein synthesis in response to a wide variety of stimulants, including mitogens and antigens, and suppression of plaque formation by human cells in response to sheep erythrocytes. CEP-Si was noncytotoxic, because cells incubated with high concentrations of CEP-Si and subsequently washed were viable and recovered their ability to respond to mitogens, and because leukocyte migration was not inhibited by CEP-Si, nor was the release of leukocyte migration inhibitory factor from sensitized lymphocytes. The possibility of antigen or mitogen competition was excluded. The effects of CEP-Si in vitro were time dependent and did not require the presence of monocytes. Cells pretreated with CEP-Si and then washed suppressed plaque formation by fresh autologous cells in highly stimulated cultures. CEP-Si injected into C57BL/6 mice also strongly suppressed their immune response to sheep erythrocytes, and the in vivo suppression was correlated with the effects of CEP-Si in vitro.

Correlation between B-cell mitogenicity and immunosuppressor effects of a protein released by porcine monocytes infected with African swine fever virus.

Virus-free supernatants of cultured swine monocytes infected by African swine fever virus (ASFV) suppressed in vitro proliferation of porcine and human blood mononuclear cells in response to phytohemagglutinin and the in vivo primary immune response of C57BL/6 mice against sheep RBC. The supernatants were fractionated by discontinuous ion-exchange chromatography and subfractionated by double-step preparative isoelectric focusing. The pool of the most purified active subfractions (F5'EP-ASFV) is made up of heat-unstable material, can be stained by silver nitrate, and has an isoelectric point of 3.88, a maximal optical density at 280 nm, and a mass of 36,000 daltons. In vivo kinetic studies in nonimmunized C57BL/6 mice were performed on days 1, 2, 3, 5, and 7 after injection with 50 micrograms of F5'EP-ASFV protein. Compared with the untreated mice, the treated mice had a noticeable increase in nonspecific immunoglobulin-secreting splenic plaque-forming cells (PFC) with the following isotype profile: IgG2a greater than IgG2b greater than IgG3 greater than IgG1 congruent to IgM. Three days after treatment with the active material, specific IgM PFC against sheep RBC increased up to 23-fold. In C57BL/6 mice immunized against sheep RBC 2 days after treatment with F5'EP-ASFV, the increase in nonspecific PFC was followed by a suppression of specific PFC response in the respective isotype. When C57BL/6 mice were treated after priming with sheep RBC, however, there was little or no suppression of specific PFC and the increase in nonspecific PFC was considerably lower than that in the other F5'EP-ASFV-treated mice. In this case, kinetic curves of specific vs nonspecific PFC of each isotype were mirror images. Mice treated with 200 micrograms of F5'EP-ASFV protein died with hemorrhagic diastasis.

Performance evaluation of an UASB reactor used for combined treatment of domestic sewage and excess aerobic sludge from a trickling filter.

This work aimed at evaluating the influence of the excess sludge produced in a trickling filter (TF) on the performance of a UASB reactor used for the combined treatment of domestic sewage and aerobic sludge. During phase 1 of the research, the UASB reactor/TF system was fed with domestic sewage pumped directly from the sewer collector of Arrudas stream, in Belo Horizonte-Brazil. During phase 2, besides feeding the reactor with domestic sewage, the UASB reactor was also fed with the aerobic sludge from the trickling filter. The UASB reactor, with a volume of 420 litres, was operated at a mean hydraulic detention time of 5.6 hours in both operational phases. After 133 days of continuous monitoring, no detrimental effect was noticed on the performance of the UASB reactor regarding the return of the aerobic sludge produced in the TF. On the contrary, the COD results indicated a higher percentage of compliance with the discharge standards set forth by the Brazilian environmental legislation. During phase 2 of the research, when the UASB reactor was used for combined treatment of domestic sewage and excess aerobic sludge from the TF, the anaerobic effluent presented mean concentrations of 108 mgCOD x L(-1), 57 mgBOD x L(-1) and 18 mgTSS x L(-1).

Semi-purification of an immunosuppressor substance secreted by Streptococcus mutans that plays a role in the protection of the bacteria in the host.

Crude extracellular products of Streptococcus mutans (CEP-Sm) suppress the proliferative response to phytohaemagglutinin of human peripheral blood mononuclear cells and the primary immune response of C57BL/6 mice to sheep erythrocytes. This immunosuppressive effect favours the survival of the microorganism, and the bacteria lose the ability to secrete immunosuppressor substance if previously subcultured several times. Cells incubated with CEP-Sm and subsequently washed recover the ability to proliferate. Traces of CEP-Sm or to short a time of contact between CEP-Sm and the target immune system induced higher proliferative ratios or higher in vivo immune responses than controls, respectively. The proliferative values of cultures supplemented with CEP-Sm were parallel to the control values up to a certain time, after which they dropped abruptly. This drop is followed by a proliferation, and the higher the amount of CEP-Sm added to the cultures, the shorter the time until the proliferation. CEP-Sm was fractionated by means of ion exchange chromatography followed by double preparative isoelectrofocusing, ending in a subfraction of isoelectric point between 3.9 and 4.2, containing a heat-unstable material stainable by Coomassie blue but unstainable by periodic acid Schiff or methylene blue, and having a maximum optical density of 280 nm.

Role of adherent T cells and of B cells in the immune response to purified protein derivative of tuberculin.

The role of monocytes, B cells, and adherent and nonadherent T cells in the response of purified protein derivative of tuberculin (PPD) as measured by [3H]thymidine uptake in vitro has been explored. The response to PPD was found to be highly dependent on monocytes, to approximately the same extent as previously found for the responses to concanavalin A (Con A) and phytohemagglutinin. The response to PPD was also found to be highly dependent on a population of adherent T cells different from the adherent T cell population involved in the response to Con A. In the case of PPD, the effect was additive, as opposed to the potentiating effect seen for Con A. Further, the adherent T cells involved in the response to PPD were much more sensitive to hypotonic shock than those in the response to Con A. B cells were also found to be important in the response to PPD, although only to a slight extent.

Role of B and T lymphocytes in the specific immunosuppression induced by a protein released by porcine monocytes infected with African swine fever virus.

Some immunobiological aspects of host responses to an immunosuppressive protein (p36) released by porcine monocytes upon infection with African swine fever virus were analysed in a murine system. Treatment of normal, adult C57BL/6 mice with p36 (i) significantly delayed allogenic skin graft rejection; (ii) suppressed the specific plaque-forming cell response to immunization with heterologous erythrocytes; but (iii) induced marked increases in the numbers of 'background' splenic Ig-secreting plaque-forming cells. Cytofluorometric analysis of spleen cells revealed that a considerable fraction of all B cells, as well as CD4 and CD8 T lymphocytes, undergo blast transformation after p36 treatment. The immunosuppressive effects do not seem to result from 'antigenic competition', for they cannot be induced by even higher doses of pig albumin or by culture products of non-infected pig monocytes. Suppression of specific antibody responses and stimulation of 'background' plaque-forming cells are both T cell-dependent, since they are markedly reduced in thymectomized mice and in animals treated with anti-CD4 or anti-CD8 antibodies. This suggests the relationship between non-specific stimulation and specific suppression of 'unrelated' immune responses and reinforces the notion that viral-associated immunosuppression may be due to overstimulation. The present murine experimental model may prove valuable in the study of immunosuppression associated with infection, even for microorganisms which do not infect mice.

Study of the immunopotentiator effects of dialyzable leukocyte extracts obtained out of leukocytes previously used for interferon production.

The existence of immunopotentiator factors able to induce blastogenesis, chemotaxis and able of inhibiting migration of leukocytes was investigated in alcohol precipitates of different batches of dialyzed leukocyte extracts (DLE). These were prepared either out of fresh leukocytes from a large pool of buffy coats obtained from all blood collected from healthy donors with irrelevant antigen sensitiveness (DLE-NS) or from similar leukocytes which were incubated during 19 h for interferon production (DLE-NS-I). Alternatively, the same factors were investigated on a batch of DLE obtained from a donor exquisitely sensitive to Candida antigen (C-DLE-CS). It was observed that all the three batches contained equal amounts of either specific or nonspecific immunological enhancing factors. The similarity of the intensity of specific factors between the so-called nonspecific batches (DLE-NS and DLE-NS-I) and the specific batch (C-DLE-CS) was interpreted to be the result of the random selection of the donors for the preparation of the nonspecific batches, which consequently contain randomized relevant specificities. It is postulated, therefore, that batches of DLE which represent a large waste of leukocytes used for interferon production can be a useful tool for double-blind DLE therapy purposes.

Effects of thymosin and evidence of monocyte suppression of both T- and B-cell functions in two cases of 'common variable immunodeficiency'.

We have studied two patients with common variable immunodeficiency (CVID), impaired cell-mediated immunity, and high percentages of monocytes in their peripheral blood. Removal of monocytes from cultures of peripheral blood mononuclear cells from both patients increased the in vitro responses to phytohaemagglutinin (PHA) and concanavalin A (Con A) but not to purified protein derivative (PPD), as measured by [3H]thymidine uptake. Similarly, supernatants of monocyte cultures from both patients, unlike supernatants of normal monocytes, suppressed the in vitro responses to PHA and Con A but enhanced the response to PPD by cultured mononuclear cells from the patients and from normal donors. Addition of unfractionated mononuclear cells from both patients to normal mononuclear cells suppressed both pokeweed mitogen (PWM) stimulation and IgG production; this effect was abrogated by removal of monocytes from the patients' mononuclear cell populations. This effect of thymosin on both patients' mononuclear cells was assayed in vitro. Thymosin was ineffective in vitro with cells from the first patient; for the other patient, [3H]thymidine uptake by mononuclear cells stimulated with PPD increased, whereas uptake by Con A-stimulated cells decreased, as did the percentage of E rosette-forming cells, providing further evidence of heterogeneity of the CVID syndrome. The effects of thymosin were also dependent on monocytes.

Erythema multiforme associated with autoreactivity to 17 alpha-hydroxyprogesterone.

We report a case of a 23-year-old woman who was afflicted with disseminated skin erythema multiforme-like eruptions that started at the menarche, relapsed at the premenstrual periods, dramatically spread during two pregnancies and cleared after abortion; the skin lesions responded dramatically to thalidomide treatment. A high-affinity binding factor to 17 alpha-hydroxyprogesterone (17-OHP) was found in the serum of this patient. Her lymphocytes did not proliferate in vitro after exposure to exogenous 17-OHP but showed significant chromatin activation. There was a decreased expression of HLA antigens at the surface of the patient's blood lymphocytes. This is a unique well-documented case of erythema multiforme most possibly due to autoreactivity to 17-OHP; the precise mechanism(s) of this autoreactivity has not been established.