Taenia crassiceps: effect of normal and immune serum on metacestodes in vitro.

The effect of normal and immune serum on Taenia crassiceps larvae in vitro was assessed by Evans blue dye uptake and electron microscopy. Normal guinea pig, rabbit, goat, and fetal calf serum did not have any significant detrimental effects upon the larvae after 7 days of culture in vitro. Culture for 7 days in normal mouse serum resulted in some loss of tegumental microtriches but the tegument itself remained intact. Culture in hyperimmune rabbit serum resulted in complete loss of the tegument and disruption of subtegumental structures within 48 hr. The effects of immune mouse serum in vitro closely paralleled those previously seen during early immune damage in vivo. Immune serum taken 2 to 4 weeks after secondary intraperitoneal infection with T. crassiceps metacestodes caused loss of the larval tegument and degeneration of the subtegumental tissues after 7 days in culture, whereas immune mouse serum taken 6 weeks after secondary infection caused only minor ultrastructural changes and appeared to be less toxic to larvae than normal mouse serum. Although complement appeared to increase the number and severity of the tegumental lesions, the presence of heat-labile components of complement was not essential for mediation of tegumental damage by immune mouse serum.     

The blocking effect of PGE2 on lymphocyte reactivity in vitro is influenced by high concentrations of human serum

PGE2 solubilized in human serum as well as dialyzed or heat inactivated human serum was tested for modulating natural cytotoxicity as compared to PGE2 solubilized in culture medium. PGE2 dissolved in human serum failed to affect cytotoxicity, while that solubilized in dialyzed or heat inactivated serum suppressed in vitro cytotoxic activity of the lymphocytes similarly to PGE2 in culture medium. It is concluded that the immunomodulatory potential of PGE2 observed in vitro is inhibited by high concentrations of human serum and that in vitro findings with this compound might not reflect the situation in vivo.     

Graft-versus-host induced immunosuppression: depressed T cell helper function in vitro.

The cause of graft-versus-host (GVH) induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) was investigated by in vitro restoration experiments employing a double compartment culture vessel. The two culture compartments were separated by a cell impermeable membrane. Restoring cells were placed in one chamber and responding GVH spleen cells plus SRBC were placed in the other chamber. It was demonstrated that thymus, lymph node, and spleen cells restored the PFC response whereas bone marrow cells did not. Treatment of the restoring cells with anti-theta serum plus complement abrogated restoration. Supernatants obtained from antigen free cell cultures restored nearly as well as whole cell suspensions. The degree of restoration was not increased by allogeneic or xenogeneic antigenic stimulation of the restoring cells. Thymus and lymphoid cells obtained from animals experiencing a GVH reaction restored as well as normal cells, however spleen cells were unable to restore by day 5 post-GVH induction. The results suggest that GVH induced immunosuppression of the PFC response is due, at least in part, to a depressed T cell factor production by splenic T cells.     

Graft-versus-host reactivity of mouse thymocytes: effect of in vitro treatment with anti-TL serum.

The lymph ducts efferent from prefemoral nodes of sheep were cannulated and the lymph flow monitored during immune responses to injected allogeneic lymphocytes or xenogeneic murine P815 mastocytoma cells. Changes in the lymph began 5–6 days after injection of allogeneic cells but at 3–4 days after injection of xenogeneic cells, in both systems the number of large cells in the lymph increased to reach peak values of up to 40% of the total. The in vitro cytotoxic activity of lymph cells, cell supernatants, or cell free lymph was determined by measuring the release of ⁵¹Cr from prelabeled target lymphocytes or P815 cells. The cytotoxic mechanisms that were detected in the allogeneic and xenogeneic systems were similar; in both cases the lymph cells were cytotoxic only during the large cell response, and when the immunoblast numbers had returned to normal levels in the lymph no further cell-mediated cytotoxic effects were detected. During the blast response lymphocytes alone caused some target cell damage but their cytotoxic effector function was greatly increased in cultures containing complement or normal blood white cells. It was concluded that the lymph immunoblasts caused some target cell damage by direct action, but the majority of their cytotoxic activity was associated with synthesis and secretion of complement-dependent antibody (C.D.A.) and leukocyte-dependent antibody (L.D.A.).     

Graft versus host-induced immunosuppression: mechanism of depressed T-cell helper function in vitro.

The cellular basis of graft versus host (GVH)-induced immunosuppression was investigated. Results showed that thymus, lymph node, and splenic T cells from normal mice and thymus and lymph node T cells from GVH mice, when cultured on one side of a cell impermeable membrane, restored the plaque-forming cell (PFC) response to sheep erythrocytes of GVH-immunosuppressed spleen cells (GVH-SC) cultured on the other side of the membrane. The restoring ability of T cells present in GVH-SC was inhibited by splenic accessory (A) cells. A direct relationship was shown between the proportion of splenic A cells and the degree of suppression of the PFC response during the first 10 days of the GVH reaction. Normal or GVH A cells reconstituted the PFC response of normal cells and GVH-SC depleted of their A-cell fraction. An optimum ratio of A: nonadherent (NA) cells (1: 10) was required for maximum reconstitution. Larger proportions of A cells inhibited the PFC response. The results suggest that GVH-induced immunosuppression is due, at least in its initial phase, to a depressed T-cell helper function caused by a marked increase of A cells in the spleen.     

Quantitation of the blocking effect of tween 20 and bovine serum albumin in ELISA microwells

ELISA provides a highly sensitive procedure for quantitating antigens and antibodies. In that assay, microwells are coated initially with a specific ligand and then saturated with inert molecules to minimize nonspecific background. Coating can be improved by pretreating the microwells with poly-l-lysine (PLL). Proteins and Tween 20 are most often used to block vacant binding sites in enzyme-linked immunosorbent assay (ELISA). In the present study the blocking effects of Tween 20 and bovine serum albumin (BSA) were estimated using an original novel approach. In the assay the magnitude of saturation of the microwells was quantitated by measuring the enzymatic activity of alkaline phosphatase adsorbed to residual vacant sites in the microwell. Tween 20 completely saturated ELISA microwells at concentrations higher than 2 microg/ml. If the microwells were pretreated with PLL, even high concentrations of the detergent did not completely saturate the wells. In contrast, BSA completely saturated both PLL-treated and nontreated microwells at 5 microg/ml. Complementation of Tween 20-induced saturation of PLL-treated microwells was achieved only by addition of BSA at concentration required for BSA alone to reach complete saturation. This approach is applicable for assessing binding to ELISA microwells of any reagent of choice either as a ligand or as a blocking reagent.     

Delipidated serum abolishes the inhibitory effect of serum on in vitro liposome-mediated transfection

All the standard in vitro lipofection has been routinely performed in serum-free medium as the transfection activity of liposome/DNA complexes is sensitive to the presence of serum. In this study, we have demonstrated that lipid-rich serum lipoprotein included in the transfection medium strongly inhibited the transfection activity of DC-chol liposome/DNA complexes in five different cell types (CHO, 293, A2780CP, A431 and SKBR3). The levels of inhibition by serum lipoprotein were rather greater than those by serum and varied with cell types. However, this inhibition was completely abolished by delipidation of serum. Thus, delipidated serum can be included in the transfection medium. The complexes formed in the presence of serum (zeta=-18.2+/-1.07 mV), delipidated serum (zeta=-19.6+/-0.54 mV), IgG (zeta=-21.6+/-1.92 mV) or serum lipoprotein (zeta=-10.5+/-2.33 mV) were as much negatively charged as those in serum-free medium (zeta=-21.3+/-1.60 mV). The results suggest that the inhibition of liposome-mediated transfection by serum was not associated with charges of serum proteins but with lipids or lipid-associated proteins present in serum.     

Reye's syndrome: the effect of patient serum on mitochondrial respiration in vitro

Serum from patients with Reye's Syndrome stimulated state 4 respiration in isolated rat liver mitochondria. Inhibitors of specific mitochondrial functions were tested as potential antagonists of the stimulatory effect of RS serum. Oligomycin, ruthenium red, rotenone and antimycin A were all ineffective in preventing the increase in state 4 respiration, but KCN completely abolished all respiratory activity in the presence of RS serum. We conclude that the putative serum factor stimulates respiration by directly or indirectly interacting with the electron transport chain at a point beyond site II.     

Cellular immunity and blocking serum activity in chimeric mice

Mice (9 CBA × T6, 6 T6, and 7 CBA) were irradiated and repopulated with foreign (BALB/c) bone marrow. Lymph node cells from 16 of 18 repopulated mice not showing signs of graft versus host disease (GVH), were cytotoxic to host type fibroblasts but not to BALB/c fibroblasts, and sera from the same mice could block lymphocyte mediated cytotoxicity. No blocking was seen with sera from three mice which had signs of GVH. LNC from the latter three mice were cytotoxic to recipient fibroblasts.It is suggested that the blocking effect detected in vitro may protect against GVH in vivo, but the relative importance of the blocking phenomenon as compared to other mechanisms is not yet settled.     

The effect of immunosuppression on secondary Echinococcus multilocularis infections in mice.

Baron R. W. and Tanner C. E. 1976. The effect of immunosuppression on secondary Echinococcus multilocularis infections in mice. International Journal for Parasitology6: 37–42. The growth of cysts of Echinococcus multilocularis in T-cell depleted A/J mice was studied. Adult thymectomy enhances the metastasis of hydatid cysts but does not significantly affect the total cyst weight. Combined thymectomy and antithymocyte serum (ATS) treatment also increases the metastatic dissemination of the parasite and also significantly increases cyst loads. It is suggested that cell-mediated immunity controls the early phase of Echinococcus infection.