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Supernatants from four murine B and one T-cell lymphoma cultures were tested for their suppressive effect on allogeneic response in vitro (MLR), on their effect on mitogenic response to ConA and LPS and on their effect in vivo on allogeneic stimulation of BALB/c mice by C57BL/6J spleen cells. Two out of four B-cell supernatants (WEHI-231 and 24-666) were also tested for their effect on induction of IL-2 like activity by ConA in BALB/c spleen cells and on the effect of their injection on subsequent allogeneic response of spleen cells from BALB/c mice immunized against C57BL/6J spleen cells. The supernatants from all lymphoma lines inhibited MLR but only supernatants from the B-cell lymphoma lines also inhibited mitogenic stimulation in vitro and allogeneic immunization. The two B-cell supernatants tested also inhibited induction of IL-2 like activity and decreased the ability of spleen cells from the injected mice to respond to allogeneic stimulation. Partial characterization of supernatants from WEHI-231 and 24-666 lymphoma cultures revealed the non-protein nature of the suppressor factor(s), indicated a molecular weight of less than 5 Kd and suggested a ganglioside or a sialyated glycoproteinic nature.

The direct linkage of the B cell maturation process and infiltration of the thymus with mature B cells was studied in SJL mice. Phenotypically and functionally, B cells in the thymus of old SJL mice are mature B cells; IgM+, IgD+, Ly-1-, and evince a high proliferative response to lipopolysaccharide and a low one to dextran sulphate. Memory B cells can be found in the thymus of mice immunized with T-dependent or T-independent antigens. Chronic depletion and B cell maturation arrest induced by fractionated total lymphoid irradiation or by neonatal splenectomy eliminate B cells from the thymus and block their migration from the periphery to the thymus. When examined in adoptive transfer experiments, thymus B cells were found to possess a normal migration pattern and homing receptors; their migration pattern did not differ from that of lymph node or splenic B cells. It is evident, therefore, that the large number of normal functioning B cells in the thymus of SJL mice reflects a massive infiltration of the thymus by mature B cells from the periphery due to thymus dysfunction rather than to an abnormal in situ differentiation of intrathymic B cell precursors.

Plasma membranes (PM) isolated from mouse splenic lymphocytes were successfully fused to rat basophilic leukemia (RBL) cells using intact Sendai virus virions (SV). A two-step procedure was used in which SV were first fused with the PM to create PM + SV vesicles; the vesicles formed were then incubated with the RBL cells. Insertion of lymphocyte PM into the RBL cell's membrane endows a high rate of serotonin secretion upon stimulation of the implanted RBL cells with antimouse Ig antibodies or with concanavalin-A. The results of the present work clearly suggest that activation signals can be delivered via implantation of foreign membrane preparation containing specific receptors, thus rendering the target cells susceptible to stimulation by specific reagents.

Long-term cultures of highly purified B chronic lymphatic leukemia cells (B-CLL), were established. The B-CLL lymphocytes acquired the CD8 T cell marker in long-term cultures while still retaining their surface immunoglobulins. In confirmation of previous results, the cultured B-CLL lymphocytes released factor(s) into the culture medium that suppressed the allogeneic mixed-lymphocyte reaction. No correlation was found between the appearance of the CD8 T cell surface marker and onset of suppressor activity.

Previously reported studies revealed that extensive immunization with the Methanol Extraction Residue (MER) of BCG tubercle bacillus in Incomplete Freund's Adjuvant (IFA) induced marked suppression of T and B cell functions in vivo and in vitro. The purpose of the present work was to determine whether immunosuppression induced by hyperimmunization with MER is correlated with changes in morphological characteristics and in phenotype pattern of spleen and peritoneal lymphoid cells. Hyperimmunization with MER resulted in a marked increase in the number of spleen cells and in enlargement and granulation of spleen and peritoneal cells. Similar changes in size and granulation were also observed in isolated fractions of splenic T and B cells. Extensive immunization with MER also induced marked decrease in the total number of T cells (Thy 1,2 positive). The decrease in T cells was observed in all three T-cell subsets investigated: Lyt-1, Lyt-2 and L3T4 positive. Although the number of splenic B cells was decreased in samples (10,000 cells), taken from MER hyperimmunized mice, this decrease was compensated by overall increase in the number of spleen cells. The marked decrease in the percentage of splenic T cells was counterbalanced by marked increase in the splenic macrophage population: increase in MAC-1, MAC-2 and MAC-3 positive cells. It is concluded that extensive immunization with MER induces morphological changes in spleen and peritoneal cells, marked decrease in the number of splenic T cells and marked increase of the splenic macrophagic population. It is postulated that these changes are correlated with induction of immunosuppression by a similar procedure of extensive immunization with the agent.

Studies were carried out to test whether tolerance to alloantigens and to heterologous proteins could be induced in (NZB X NZW)F1 (B/W) female mice, compared with females of various other mouse strains, including BALB/c, C3H/eb, C57Bl/Ka and (BALB/c X C57Bl/6)F1. Untreated BALB/c and B/W mice were resistant to tolerance induction by deaggregated BSA, while all other strains were susceptible, as indicated by their lack of response to antigen challenge. Tolerance induction to BSA was further potentiated in all mouse strains including BALB/c with the exception of B/W, following prior conditioning of the mice with total lymphoid irradiation (TLI). Similarly, specific and permanent tolerance to H-2 incompatible alloantigens was successfully induced in TLI conditioned BALB/c, C3H/eb, (BALB/c X C57Bl/6)F1 injected with bone marrow cells, however, B/W mice were resistant. Stable chimeras could be established in TLI treated B/W mice only across a semi-allogeneic combination (BALB/c--greater than B/W). No graft vs host disease (GVHD) was observed in any of the chimeras including B/W mice. We conclude that B/W mice are resistant to tolerance induction to heterologous proteins and alloantigens, even after TLI conditioning. We postulate that this phenomenon is a function of both the intrinsic properties of the haemopoietic stem cells, including their differentiated progeny, as well as characteristics of their cellular microenvironment.

(1) Evidence is provided that tolerance induced in mice to HSA depends on a suppressive mechanism which inhibits the responsiveness of potentially competent cells. (2) the suppression is apparently the function of suppressor T cells which are comparatively radiosensitive and are resistant to cortisone. (3) The cortisone resistant suppressor cells are apparently the progeny of cortisone-sensitive precursors, the differentiation of which into mature suppressors seems to be signalled by antigen. (4) The precursor lymphocytes are comparatively short functioning cells (about 14 days), while the mature suppressor T cells function for at least two months.

In order to label dopamine D2 receptors selectively we tritiated the potent benzamide neuroleptic, YM-09151-2 (26.7 Ci/mmol). The binding of [3H]-YM-09151-2 to canine striatal membranes was saturable and specific with a KD of 57 pmol/l and Bmax of 36 pmol/g tissue as determined by Scatchard analysis. The KD, but not the Bmax, of [3H]-YM-09151-2 increased 6-fold in the absence of sodium chloride. [3H]-YM-09151-2 labeled 40% more sites than [3H]-spiperone in the same tissue homogenate. [3H]-YM-09151-2 binding was inhibited by dopaminergic drugs in a concentration and stereoselective manner with the appropriate dopamine D2 receptor profile. Thus, dopamine agonists inhibited [3H]-YM-09151-2 binding to canine striatal membranes with the following rank order of potency: (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than (-)-noradrenaline greater than serotonin greater than (-)-isoprenaline. Dopaminergic antagonists competed for [3H]-YM-09151-2 binding with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than SCH-23390 greater than (-)-butaclamol. Furthermore, dopamine agonists recognized 2 states of the receptor labeled by [3H]-YM-09151-2, Dhigh2 and Dlow2. The Dhigh2 state of the receptor could be converted to Dlow2 by guanine nucleotides and sodium ions as is the case for [3H]-spiperone binding to D2 receptors. [3H]-YM-09151-2 appears to be a more selective ligand for dopamine D2 receptors than [3H]-spiperone, since YM-09151-2 displays approximately 9-fold lower affinity than spiperone for cortical serotonergic (S2) receptors. [3H]-YM-09151-2 may become a useful tool for the selective characterization of dopamine D2 receptors.

Total lymphoid irradiation (TLI) results in long-lasting changes in the characteristics of both T and B cells suggestive of arrested maturation. A characteristic feature of immature B cells is their high susceptibility to tolerance induction. This study examines the susceptibility to tolerance to bovine serum albumin (BSA) of TLI-treated mice. Two experimental protocols were designed. In the first, tolerance to BSA was induced in TLI-treated adult (BALB/c X C57BL/6)F1 mice, and the ability of B cells of those mice to respond to BSA was assessed in an adoptive transfer system. In the second experimental protocol, tolerance was induced in adoptive hosts reconstituted with purified B cells originating from TLI-treated mice and with splenic T cells of normal, untreated mice. Results obtained in these two systems clearly demonstrated that splenic B cells of TLI-treated mice are highly susceptible to tolerance induction. This high susceptibility of B cells is linked neither with the elevation of immature T cells nor with induced T suppressor cells which arise due to the long-term malfunction of the thymus. Tolerance could be induced in TLI cells even 4 months after termination of the treatment. Thus, maturation processes of B cells in TLI-treated mice are arrested for long periods of time.

BALB/c mice given total lymphoid irradiations (TLI) were injected i.p. with bovine serum albumin (BSA) in saline, and challenged with DNP-BSA in complete Freund's adjuvant 6 weeks later. The latter animals made no anti-DNP antibody response as measured by a modified Farr assay, but made a normal anti-DNP response after challenge with DNP-BGG in adjuvant. Normal mice or mice given whole body irradiation were not tolerized by the i.p. injection of BSA in saline. Spleen cells from unresponsive mice (TLI + BSA in saline) suppressed the adoptive secondary anti-DNP response of sublethally irradiated syngeneic hosts given BSA-primed T cells, DNP-BSA-primed B cells, and DNP-BSA in saline. The suppressor cells were antigen specific, and were inactivated by in vitro treatment with anti-Thy 1.2 antiserum and complement. The findings suggest that soluble antigens administered to mice after TLI evoke a state of tolerance that is maintained by antigen-specific suppressor T cells. A similar mechanism may be involved in the maintenance of tolerance to allografts. These findings may have important clinical implications for patients treated with TLI for lymphoid malignancies.

The effect of fractionated total lymphoid irradiation (TLI) on the induction of cells blocking in vivo B cell maturation was examined. (BALB/c x C57BL/6)F1 mice received 200 rds lymphoid irradiation daily for 8 days. One month after termination of the irradiation splenic T cells of TLI-treated mice were transferred, together with normal bone marrow cells, to lethally irradiated hosts. In addition, splenic cells or splenic B cells of TLI mice were transferred to irradiated hosts. Two months later, we checked the reconstituted mice for their ability to produce in vivo anti-trinitrophenyl (TNP)-Ficoll antibodies and their capacity to respond in vitro to dextran sulfate and lipopolysaccharide. In parallel, stained spleen cells were analyzed on the fluorescence-activated cells sorter using monoclonal anti-mu, anti-delta and anti-B220 surface marker antibodies. The results indicate that splenic T cells originating from TLI-treated mice cause maturation arrest of normal and TLI-treated B cells, since normal bone marrow cells injected together with those cells have immature phenotypic and functional features. In addition, neither splenic cells nor splenic B cells of TLI-treated mice can mature upon transfer to normal irradiated hosts. The B cells are IgM+, IgD-; they respond with a high proliferation rate to dextran sulfate and low proliferation rate to lipopolysaccharide, and poorly to TNP-Ficoll challenge.

The enzyme adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at lambda exmax = 350 nm and lambda emmax = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence (lambda exmax = 305 nm, lambda emmax = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.

Ultrasonically guided oocyte collection gradually replaces laparoscopy in many in vitro fertilization (IVF) centers. In the present study we compare the efficacy of both methods in our IVF program. One hundred twenty patients were prospectively randomized into two groups. Sixty women underwent oocyte collection during laparoscopy under general anesthesia, and 60 women under ultrasound guidance with local anesthesia. Superovulation was induced with human menopausal gonadotropin/human chorionic gonadotropin. For the oocyte collection a cannula with a trocar was used for single percutaneous puncture, through which both ovaries could be reached by the aspiration needle. The number of aspirated oocytes and transferred embryos was higher in the laparoscopy group as compared with the ultrasound group (5.3 versus 4.0 per woman and 3.0 versus 2.3 per woman, respectively); but the clinical pregnancy rate per cycle was similar in both groups (12.5% versus 14.5%). Ultrasound aspiration seems to be as effective as laparoscopy in terms of oocyte retrieval and conception rates. Because the procedure is simple and inexpensive, we believe that it may replace laparoscopy as a method for oocyte collection in most patients who undergo IVF.

The effect of fractionated total lymphoid irradiation (TLI) on the maturation of B cells was examined in mice. At various times after irradiation of the mice, their spleen cells were tested for the mitogenic responses to dextran sulfate and lipopolysaccharide. In parallel the cells were stained with fluorescent anti-Thy-1 and various anti-Ig antisera, and were analyzed on a fluorescence-activated cell sorter (FACS). By comparison with normal spleen cells the cells of TLI-treated mice gave a high response to dextran sulfate and a low response to lipopolysaccharide. FACS analysis revealed that TLI-treated spleen contains elevated numbers of B cells bearing high IgM and low IgD on the surface. The B cells of TLI-treated mice retained these immature characteristics even two to four months after the last irradiation. These findings indicate that TLI causes a longlasting blockage in B cell maturation processes.

This work was undertaken for evaluation of the value of endometrial thickness as an early predictor for the success of in vitro fertilization (IVF). Endometrial changes were evaluated ultrasonographically in 47 women undergoing IVF. A high-dose gonadotropin protocol was used for induction of multiple follicular development. Thirty-seven women did not conceive following the procedure (group I), and 10 conceived (group II). Ultrasonographic endometrial measurements were performed repeatedly throughout the cycle. Serum 17 beta-estradiol and progesterone levels were evaluated concomitantly. Three consecutive growth patterns of the endometrium were observed. The first was a rapid one with a daily growth rate of about 0.5 mm from approximately 9 mm on day -3 to 12 mm on day +2 (day 0 being the day of human chorionic gonadotropin administration). The second phase, following follicular aspiration, showed a decrease in growth rate to about 0.1 mm per day until day +11, when a thickness of about 13 mm was measured. In group I growth was arrested from day +11 until menstruation, whereas in group II an accelerated growth rate of about 0.4 mm per day could be demonstrated from day +14 onward. Endometrial growth did not correlate with serum estradiol or progesterone levels. No conception occurred with an endometrial thickness below 13 mm on day +11. It is concluded that endometrial thickness follows a distinct pattern of growth in human menopausal gonadotropin-induced cycles and does not correlate with serum sex hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

The surface markers and size of suppressor cells were determined in adult (BALB/c x C57BL/Ka)F1 mice which were tolerized with a single injection of deaggregated bovine serum albumin (BSA). Suppressor cells from the spleens of tolerazided donors were assayed in a cell transfer system in which graded numbers of cells were injected into irradiated syngeneic mice along with limiting numbers of T cells primed to BSA and an excess of B cells primed to DNP-BSA. Adoptive hosts were challenged with DNP-BSA in saline, and the anti-DNP response was measured. Suppressor cells were antigen specific as shown by the inhibitory activity of BSA-tolerant spleen cells on the response to DNP-BSA, but not to DNP-BGG. Suppressor cells were eliminated by in vitro treatment with anti-Thy 1.2, anti-Ly-2.2, anti-I-J subregion antisera and C, but not with anti-Ly-1 or anti-I-A subregion antisera. Neither unprimed nor primed helper T cells were detected in the spleen of tolerized donors after in vitro treatment with anti-Ly-2.2 antisera. Both helper and suppressor T cells from the spleens of primed or tolerized donors, respectively, showed a rapid sedimentation velocity (S greater than 3.7 mm/hr).

We have developed a technique for autotransfusion of shed mediastinal blood collected in the cardiotomy reservoir. Results in the first 50 consecutive patients in whom the system was employed were studied prospectively (group A) and were compared with those of the last 50 consecutive patients operated on before application of the new method (group B). The mean preoperative hematocrit (about 42%) and the total postoperative bleeding (about 11) were similar in both groups. Only 24 patients (48%) in group A required homologous blood transfusion, versus 43 (86%) in group B (p = 0.0001). This resulted in a 57% saving of blood units in group A (mean, 1.16 +/- 1.49 units per patient versus 2.72 +/- 1.99 in group B, p less than 0.0001). Foreign blood transfusion was thus avoided in 21 of the last 25 patients (84%) of group A. 7 days after operation, the mean hematocrit in group A was 30.9 +/- 4.3, compared with 33.0 +/- 3.8 in group B (p less than 0.001). Hemolytic jaundice occurred in 7 group B patients (14%) but in none in group A (p less than 0.01). A trend towards reduction in other complications was also demonstrated. We conclude that the use of foreign blood transfusions in those undergoing open heart surgery can be reduced significantly by this method, thus avoiding immediate transfusion reactions and minimizing the risks of late complications.

The role of IgD (delta) in the induction of tolerance in murine B lymphocytes was explored in the in vivo adoptive antibody response to bovine serum albumin (BSA). Delta+ or delta- B cells purified on the fluorescence-activated cell sorter were injected, with or without purified T cells, into lethally irradiated intermediate hosts, which were rendered tolerant by an injection of deaggregated BSA one day after the cell transfer. Spleen cells from the intermediate hosts were treated with anti-Thy-1.2 antiserum and complement mixed with normal T cells and were transferred to final hosts. The ability of the B cells of the final hosts to respond to the tolerogen BSA and to dinitrophenylated human gamma globulin antigen was examined. This approach enabled examination of the function of the B cell subpopulations in an environment free of tolerogen and of induced T suppressor cells. The results revealed that in the presence of T cells the delta+ subpopulation was highly resistant to tolerance induction, whereas the delta- subpopulation was highly susceptible to tolerance induction. However, there was no difference in susceptibility to tolerance induction between the two subpopulations when tolerance was induced in the absence of T cells.