Activation of human peripheral blood T cells does not lead to increased P-glycoprotein expression.

The expression of P-glycoprotein (Pgp) on normal human lymphocytes, and its drug exclusion capacity, implies that Pgp might be involved in cytokine secretion. We used two-color flow cytometry to detect simultaneously Pgp expression and IL-2 accumulation in resting and mitogen-activated human lymphocytes. Among resting lymphocytes from five healthy donors less than 1% were Pgp+ as determined by reactivity with the anti-Pgp monoclonal antibody (mAb) 4E3. The percentage of Pgp+ lymphocytes increased to 3% after 24 hr of mitogenic stimulation that induced maximal production of cytoplasmic IL-2. The percentage of lymphocytes that coexpressed membrane Pgp and cytoplasmic IL-2 accounted for < 10% of the total IL-2 producing lymphocytes. Finally, mitogen-induced cytoplasmic IL-2 accumulation was enhanced by stimulation in the presence of monensin but not the Pgp functional inhibitor verapamil. Because mAb 4E3 detected lower than expected numbers of Pgp+ lymphocytes, we compared the binding of mAbs MRK16 and 4E3 concomitant with doxorubicin (DOX)-uptake by K562 and R7 tumor cells and purified CD8+ lymphocytes. The MRK16 mAb was found to be sensitive but not very specific (30%). In contrast, the sensitivity of 4E3 was equivalent to MRK16 (98%) and was highly specific (98.5%). There was also a positive association between DOX efflux and the level of Pgp expression as detected by 4E3 but not MRK16. Thus, human T cells do not markedly up-regulate their expression of functional Pgp molecules as detected by mAb 4E3 following activation, suggesting that Pgp does not play a major role in IL-2 secretion by activated T cells.

Impairments in immune cell function in B cell chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (B-CLL) is a common clonal B cell leukemia that is often accompanied by a multitude of immune abnormalities. Each immune defect may be linked to several of the common complications affecting B-CLL patients. Furthermore, the combined abnormalities constitute a significant immunodeficiency for each patient. Importantly, some of the immune dysfunctions are potentially very relevant to the in vivo survival status of the leukemic B cell. The elucidation of these abnormalities in the circulating non-malignant immune cells of B-CLL patients has generated important insights into the biology of the disease. This discussion reviews the immune abnormalities of the clonal malignant B cells, the polyclonal B cells, and the immunoregulatory T cells and natural killer cells in B-CLL. In addition, we indicate the potential for immunotherapeutic protocols as innovations in treating this disease.

L-Canavanine modulates cellular growth, chemosensitivity and P-glycoprotein substrate accumulation in cultured human tumor cell lines.

L-Canavanine (L-CAV) is a naturally occurring L-arginine analog that induces the formation of non-functional proteins in a variety of organisms. Previous studies have shown that L-CAV is cytotoxic for several human tumor cell lines. In this study, we have evaluated the cytotoxicity of L-CAV for both parental and multi-drug resistant (MDR) human tumor cells. We have also determined the effect of L-CAV exposure on cellular expression and activity of the MDR P-glycoprotein (P-gp) membrane efflux pump, and the effect of L-CAV on cellular accumulation of P-gp substrates. The effect of pre-treatment with non-cytotoxic doses of L-CAV on cellular sensitivity to ten standard antineoplastic agents was also evaluated, in order to assess the chemosensitization potential of L-CAV. 3-(4,5-Dimethylthiazol-)2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assays revealed that the MDR variants of human uterine sarcoma and leukemic cells were equally sensitive to L-CAV as compared with their respective parental controls. Although the presence of free L-CAV in the uptake media did not influence cellular accumulation of P-gp substrates, cells cultured for 72 h in 250 microM L-CAV accumulated from 16 to 23% less P-gp substrate than untreated controls. Although L-CAV-cultured sarcoma cells accumulated 17% less doxorubicin (DOX) than untreated controls, they were three times more sensitive to its cytotoxic effects. L-CAV-treated cells were also significantly more sensitive to cisplatin, 5-fluorouracil, mitoxantrone and bleomycin than were untreated controls. Indirect immunofluorescence revealed that 72-h exposure to as much as 1000 microM L-CAV did not alter cellular expression of P-gp. These studies suggest that L-CAV may be equally cytotoxic for both parental and MDR tumor cells, and that L-CAV neither induces the expression of, nor is a substrate for, P-gp. The observation that L-CAV pre-treatment reduces cellular accumulation of DOX, yet sensitizes tumor cells to DOX and other DNA-targeting antineoplastic drugs, suggests a role for L-CAV as a chemosensitizer for the chemotherapy of cancer.

Indirect examination of exocrine pancreatic function in vivo by determination of chymotrypsin activity in animal intestine using a synthetic substrate.

Using the substrate N-acetyl-L-tyrosyl-p-aminobenzoic acid, we determined chymotrypsin activity in the small intestine of calf, pig, and poultry. Orally administered N-acetyl-L-tyrosyl-p-aminobenzoic acid is enzymatically cleaved in vivo, and the released p-aminobenzoic acid is determined by HPLC. We found that the p-aminobenzoic acid concentration in plasma and urine was significantly influenced by the feeding of soya flour. After soybean flour feeding, the p-aminobenzoic acid concentration significantly increased in the plasma of calves and hens, in contrast to pigs, where the p-aminobenzoic acid concentration significantly decreased. This shows that the oral administration of N-acetyl-L-tyrosyl-p-aminobenzoic acid with subsequent determination of p-aminobenzoic acid is suitable for the estimation of exocrine pancreatic function and for determination of changes in intestinal proteolytic activity caused by antinutritive substances.

cAMP accumulation in T-cells inhibits anti-CD3 monoclonal antibody-induced actin polymerization.

The results presented in this report offer a novel explanation for how stimulation of the beta-adrenergic receptor (beta AR) inhibits the ability of T cells to proliferate after interaction with immobilized anti-CD3 monoclonal antibody (mAb). Accordingly, T cells binding to immobilized anti-CD3 mAb but not anti-CD4 mAb undergo time-dependent F-actin assembly with concomitant formation of pseudopodia. This process is completely inhibited in the presence of isoproterenol (ISO) indicating that stimulation of the beta AR on T cells interferes with the biochemical processes responsible for the assembly of actin. To confirm these observations, we quantitated the formation of F-actin in T cells stimulated with immobilized anti-CD3 mAb in the presence of cAMP elevating agents. The results show that stimulation of the beta AR on T-cells, as well as the addition of forskolin or dibutyryl cAMP, abrogates the formation of F-actin.

Costimulatory signals modulate the antiproliferative effects of agents that elevate cAMP in T cells.

Stimulation of highly purified human T cells with immobilized anti-CD3 monoclonal antibody (mAb) in the presence of cAMP-inducing agents results in inhibition of proliferation by these T cells. In the present study, experiments were performed to determine how costimulatory signals modulate the inhibitory effects of cAMP-elevating agents on proliferation and interleukin 2 (IL-2) secretion by anti-CD3 mAb-stimulated T cells. Accordingly, the level of anti-CD3 mAb-induced T cell proliferation was determined in the presence or absence of accessory cells, anti-CD28 mAb, or phorbol myristic acid (PMA) in the presence of the adenylyl cyclase (AC)-linked receptor agonists prostaglandin E2 (PGE2), or isoproterenol (ISO) as well as the AC activator forskolin (FSK) or the cAMP analog dibutyryl-cAMP (dB-cAMP). While all three costimulators enhanced the level of anti-CD3 mAb-induced T cell proliferation and IL-2 secretion, they were variable in their ability to overcome the immunosuppressive effects of the cAMP elevating agents. The order of potency of the costimulatory signals in reversing the inhibitory effects of cAMP-elevating agents on anti-CD3 mAb-induced T cell proliferation and IL-2 secretion was PMA > accessory cells > anti-CD28 mAb. Differences were noted in the ability of the costimulatory signals to overcome the immunosuppressive effects of the various cAMP-inducing agents. Thus, the effects of PGE2 or ISO on T cell proliferation or IL-2 secretion were more readily overcome by costimulatory signals than those elicited by FSK or dB-cAMP. Experiments designed to investigate the mechanisms involved in these effects showed that neither accessory cells nor anti-CD28 mAb altered the level of cAMP accumulation or protein kinase A (PKA) activity in T cells stimulated with cAMP-elevating agents. However, PMA was found to decrease both cAMP accumulation and PKA activity in T cells stimulated with PGE2 or ISO but not FSK. These results suggest that the overall immunosuppressive effects of naturally occurring substances such as PGE2 or catecholamines may be altered by costimulatory signals when antigen-specific T cells interact with antigen-presenting cells.

Induction of cAMP-dependent protein kinase (PKA) activity in T cells after stimulation of the prostaglandin E2 or the beta-adrenergic receptors: relationship between PKA activity and inhibition of anti-CD3 monoclonal antibody-induced T cell proliferation.

Recently, we have shown that T cells exposed to concentrations of prostaglandin E2 (PGE2) or the beta-adrenergic receptor agonist isoproterenol (ISO) that elicit equimolar levels of cAMP accumulation do not inhibit anti-CD3 monoclonal antibody-induced T cell proliferation to the same extent. This report extends these studies by investigating the induction of cAMP-dependent protein kinase (PKA) in T cells stimulated with PGE2 or ISO. The kinetics of PKA activity induced by PGE2 or ISO in T cells are similar but PGE2 induces more PKA activity. When T cells were treated with concentrations of PGE2 or ISO that elicited similar PKA activities, PGE2 was found to be more immunosuppressive than ISO. T cells stimulated with PGE2 or ISO showed similar levels of increased PKA activity in both the cytosolic and the particulate fractions. Quantitation of the activity of PKA I and PKA II isozymes in T cells stimulated with PGE2 or ISO revealed that both types were activated; however, while PGE2 induced the utilization of an equal amount of both isozymes in T cells, ISO-treated cells utilized twice as much PKA I compared to PKA II. Overall, these results suggest that qualitative differences in the concentration of cAMP and PKA activity are important elements in modulatory T cell proliferative responses.

Modulation of T cell proliferation by stimulation of the beta-adrenergic receptor: lack of correlation between inhibition of T cell proliferation and cAMP accumulation.

The presence of beta-adrenergic receptors on T cells suggests the potential for modulating T cell function upon binding of an appropriate agonist. Utilizing highly purified human T cells we assessed the proliferative capacity of T cells upon stimulation with immobilized anti-CD3 monoclonal antibody (mAb) in the presence of the beta-adrenergic agonist isoproterenol (ISO). The proliferative response of T cells and their CD4+, CD8+, or CD45RO+ subsets to anti-CD3 mAb was inhibited in a dose-dependent manner by ISO. In parallel experiments, various concentrations of prostaglandin E2 (PGE2) were added to anti-CD3 mAb-stimulated T cells and their subsets. Similar dose-dependent effects were observed with the important exception that PGE2 was considerably more immunosuppressive than ISO. The results also showed that PGE2 was a much more effective inhibitor of anti-CD3 mAb-induced interleukin 2 synthesis by T cells than was ISO. Because both the beta-adrenergic and PGE2 receptors are linked to adenylyl cyclase, the magnitude and kinetics of cAMP accumulation in T cells and their subsets were determined after stimulation with ISO or PGE2. The results show that PGE2 induced a greater and more sustained accumulation of cAMP than ISO. Moreover, these differences could not be ascribed to differential modulation of cAMP phosphodiesterase activity. Correlation of the degree of inhibition of anti-CD3 mAb-induced T cell proliferation by PGE2 or ISO with the level of accumulation of cAMP in these stimulated T cells indicate that, on an equimolar basis, cAMP elicited by PGE2 is more immunosuppressive than that induced by ISO. These results suggest that qualitative differences in cAMP accumulation in T cells have an important role in the subsequent modulation of anti-CD3 mAb-induced T cell proliferation.

Dominant exudative vitreoretinopathy: a sporadic case with a normal platelet aggregation study.

Case report of a woman with the characteristics of dominant exudative vitreoretinopathy. These anomalies are however unilateral and the family study is not very contributive. A platelet aggregation study carried out on the patient and 3 family members is normal.

Enhanced expansion of the thymic CD8+ cell subset as a potential mechanism for the generation of enhanced antitumor cytotoxicity by thymocytes from low-dose melphalan-treated MOPC-315 tumor bearers.

We have previously shown that thymocytes from low-dose melphalan (L-phenylalanine mustard)-treated MOPC-315-tumor-bearing mice (melphalan TuB) are able to generate an enhanced level of anti-MOPC-315 cytotoxicity, as compared to thymocytes from untreated MOPC-315-tumor-bearing mice or thymocytes from untreated or low-dose melphalan-treated normal mice, upon in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of recombinant interleukin-2 (rIL-2). Here we show that the generation of enhanced anti-MOPC-315 cytotoxicity by melphalan TuB thymocytes depends on the ability of the thymocytes to proliferate. In addition, the ability of melphalan TuB thymocytes to generate an enhanced level of anti-MOPC-315 cytotoxicity correlated with their ability to proliferate more readily than thymocytes from untreated tumor-bearing mice and thymocytes from untreated or melphalan-treated normal mice in response to stimulation with MOPC-315 tumor cells plus a low concentration of rIL-2. Moreover, although fresh melphalan TuB thymocytes do not contain a higher percentage of phenotypically mature cells (i.e., CD4-/CD8+ or CD4+/CD8-) than do thymocytes from normal mice or untreated tumor-bearing mice, after a 5-day culture with both MOPC-315 tumor cells and a low concentration of rIL-2, cultures of thymocytes from melphalan TuB contained a much higher percentage of CD4-/CD8+ (but not CD4+/CD8-) cells than did cultures of thymocytes from the other two sources. Since CD4-/CD8+ cells were previously shown to be responsible for the exertion of antitumor cytotoxicity by thymocytes stimulated with MOPC-315 in vitro, our results indicate that the enhanced antitumor cytotoxicity exerted by melphalan TuB thymocytes following in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of rIL-2 is due, at least in part, to an expansion of the pool of CD4-/CD8+ effector cells.

Presence of an enlarged pool of MOPC-315-specific cytotoxic T lymphocyte precursors in the thymuses of mice that eradicated a large MOPC-315 tumor as a consequence of low-dose melphalan therapy.

We have previously shown that, as a consequence of low-dose melphalan (L-phenylalanine mustard) treatment, thymocytes from mice bearing a large, day-10 MOPC-315 tumor, but not thymocytes from normal mice, acquire the ability to generate an enhanced level of antitumor cytotoxicity upon in vitro stimulation with MOPC-315 tumor cells plus low concentrations (9.0-90 IU/ml) of exogenous interleukin-2 (IL-2). Here we show that the time interval between tumor inoculation and low-dose melphalan therapy as well as the magnitude of tumor burden at the time of the chemotherapy are important for the ability of the drug to render thymocytes more responsive to in vitro stimulation with MOPC-315 tumor cells plus low concentrations of recombinant IL-2 (rIL-2). Specifically, the chemotherapy was found to be effective in enhancing the thymic antitumor reactivity only if the mice bore a large, late-stage tumor. Comparison of thymocytes from untreated mice bearing a large, late-stage tumor to thymocytes from normal mice revealed that tumor-bearer thymocytes contained approximately a three-fold higher frequency of cytotoxic T lymphocyte precursors (CTLp) for MOPC-315-associated antigens. Following curative low-dose melphalan therapy of mice bearing a large, late-stage MOPC-315 tumor, the frequency of CTLp for MOPC-315-associated antigens increased further, reaching a level approximately tenfold higher than that found among thymocytes of normal mice. At the same time, the frequency of CTLp for an antigenically unrelated allogeneic tumor (EL4) as well as the overall percentage of mature cells was not increased. The cells responsible for the exertion of the enhanced antitumor cytotoxicity following in vitro stimulation of thymocytes from mice treated with low-dose melphalan when they have a large, late-stage MOPC-315 tumor are of the CD8+/CD4- phenotype. Thus, the enhanced level of antitumor cytotoxicity generated by thymocytes from mice that are treated with low-dose melphalan when they have a large, late-stage MOPC-315 tumor is due, at least in part, to the presence of an enlarged pool of CTLp specific for MOPC-315-associated antigens, which mature into CD8+/CD4- effector cells upon stimulation with MOPC-315 tumor cells plus low concentrations of rIL-2.

[Association of the CHARGE syndrome and imperforation of the lacrymal ducts]. / Association de CHARGE et imperforation des voies lacrymales.

Clinical description of a 12-year-old girl with a number of congenital abnormalities including retarded growth and an asymmetric facies. All the clinical features observed are compatible with the CHARGE association. Ocular anomalies are important bilateral colobomata associated with nystagmus and strabismus. Three among four of the lacrymal canaliculi are missing. This anomaly has not yet been described in the CHARGE syndrome. The discussion refers to all the clinical observations especially the ophthalmological features.

Interleukin 2 requirement for the in vitro generation of antitumor cytotoxicity by thymocytes from melphalan-cured MOPC-315 tumor bearers.

We have previously shown that enhanced antitumor cytotoxicity is generated when thymocytes from melphalan (L-phenylalanine mustard; L-PAM)-treated MOPC-315 tumor bearers, but not thymocytes from normal mice, are added to the immunization culture of syngeneic normal spleen cells and MOPC-315 tumor cells (Bartik et al., Cancer Res., 47: 4848-4855, 1987). Here we show that normal spleen cells produce, upon stimulation with MOPC-315 tumor cells, helper-like factors which are sufficient for thymocytes from L-PAM-treated MOPC-315 tumor bearers, but not for thymocytes from normal mice, to develop antitumor cytotoxicity in response to stimulation with MOPC-315 tumor cells. Since one of the helper-like factors produced by in vitro-immunized spleen cells is interleukin 2 (IL-2), we assessed the exogenous IL-2 requirements for the development of anti-MOPC-315 cytotoxicity in thymocytes from L-PAM-treated MOPC-315 tumor bearers, relative to thymocytes from normal mice. Thymocytes from L-PAM-treated MOPC-315 tumor bearers were found to require a 10-fold lower concentration of recombinant IL-2 (rIL-2) than thymocytes from normal mice in order to develop antitumor cytotoxicity in response to stimulation with MOPC-315 tumor cells. The concentration of rIL-2 required for the development of anti-MOPC-315 cytotoxicity by thymocytes from L-PAM-treated MOPC-315 tumor bearers was also 10-fold lower than the concentration of rIL-2 required by thymocytes from untreated MOPC-315 tumor bearers or thymocytes from L-PAM-treated normal mice. In addition, at any concentration of rIL-2 employed, thymocytes from L-PAM-treated MOPC-315 tumor bearers developed a higher level of anti-MOPC-315 cytotoxicity than did thymocytes from normal mice, L-PAM-treated normal mice, or untreated MOPC-315 tumor bearers. The enhanced antitumor cytotoxicity exhibited by thymocytes from L-PAM-treated MOPC-315 tumor bearers, following in vitro stimulation with MOPC-315 tumor cells plus rIL-2, was evident not only against MOPC-315 tumor cells but also against other syngeneic plasmacytomas but not an allogeneic thymoma. In addition, thymocytes from L-PAM-treated MOPC-315 tumor bearers required less rIL-2 than thymocytes from normal mice to develop antitumor cytotoxicity in response to stimulation with MOPC-315-associated antigens but not in response to stimulation with an allogeneic antigenically unrelated thymoma (EL4).(ABSTRACT TRUNCATED AT 400 WORDS)

Melphalan-induced enhancement of antitumor immune reactivity in thymocytes of adult BALB/c mice bearing a large MOPC-315 tumor.

At no stage of tumor growth are thymocytes from MOPC-315 tumor bearers capable of bringing about the generation of enhanced antitumor cytotoxicity when added to immunization cultures of syngeneic normal spleen cells and "autochthonous" tumor cells. However, by Day 7 after low-dose melphalan [L-PAM (L-phenylalanine mustard)] therapy of mice bearing a large (greater than or equal to 20 mm) s.c. MOPC-315 tumor, their thymocytes exhibit such activity and it persists for at least 17 additional days. The ability of thymocytes from L-PAM-treated MOPC-315 tumor bearers to bring about the generation of enhanced antitumor cytotoxicity when added to immunization cultures of normal spleen cells and MOPC-315 tumor cells is evident over a 10-fold range of responder/stimulator cell ratios, and requires the presence of the thymocytes within the first day after initiation of the 5-day immunization cultures. In addition, immunization cultures containing normal spleen cells and thymocytes from L-PAM-treated MOPC-315 tumor bearers exhibit enhanced antitumor cytotoxicity by Day 4 after culture initiation that persists for at least 3 additional days. Thymocytes from L-PAM-treated MOPC-315 tumor bearers are able to bring about the generation of enhanced antitumor cytotoxicity only in response to stimulation with autochthonous tumor cells but not in response to stimulation with unrelated allogeneic EL4 tumor cells. The apparent specificity of the enhanced antitumor immune reactivity of thymocytes from L-PAM-treated MOPC-315 tumor bearers is not the result of extensive metastasis of tumor cells to the thymus. In fact, no tumor cells were found in the thymuses of MOPC-315 tumor bearers with methods that can detect 1 X 10(3) tumor cells, indicating that if MOPC-315 tumor cells metastasize at all into the thymus, the thymuses of mice bearing a large MOPC-315 tumor contain fewer than 1 X 10(3) tumor cells. Thus, thymocytes from mice which are engaged in the eradication of a large MOPC-315 tumor display enhanced antitumor immunity in response to stimulation with the autochthonous tumor cells. Such thymocytes may prove important to the outcome of low-dose L-PAM therapy for mice bearing a large MOPC-315 tumor, since the low-dose chemotherapy requires the contribution of T-cell-dependent antitumor immunity for its therapeutic effectiveness.

Porcine aortic versus bovine pericardial valves: a comparative study of unimplanted and from patient explanted bioprostheses.

This extensive morphological study (macroscopy, x-ray, histology, histochemistry and electron microscopy) compares two types of bioprosthetic valves, porcine aortic (PAV) and bovine pericardial (BPV) of various models, both unimplanted (five) and explanted (229). There were 197 PAV and 32 BPV explanted from the mitral, aortic and tricuspid positions, with a mean duration of implantation of 70.3 and 13.5 months, respectively. Within that material, a smaller, rather homogeneous, series of 11 Carpentier-Edwards PAV (CE) and 11 Ionescu-Shiley BPV (IS) explants (mean implantation period 53 and 49, mean patient-age 45 and 47 years) made the comparison of clinical and macroscopic features more valid. In the total series, the leading causes of failure were cuspal tear/perforation with calcification in the PAV group (64 per cent); non-calcified leaflet rupture (27 per cent) and infective endocarditis (27 per cent) in the BPV group. In the small series of CE PAV and IS PAV, the characteristic modes of failure were calcified juxta-commissural cusp rupture for CE and non-calcified leaflet rupture at the suture for IS. The most characteristic x-ray features were calcification of fibrous cords irradiating from the commissures and calcific nodules in the centre in PAV and large plaques extending from the commissures and leaflet base in all directions in BPV. The main microscopic features of leaflet degradation were: the soaked sponge phenomenon (loosening and plasma and fat insudation) and the nodular, protein-rich calcification, both centred in the spongiosa, in PAV explants; important macrophagic activity and destruction of collagenous structures at the outflow layer and along the suture of the leaflets, with preservation of the middle layer, and the intrinsic calcification of the deep collagenous bundles, in BPV explants. Those alterations and other features are discussed with reference to leaflet structure and design, haemodynamics and possible causal mechanisms.

Melphalan-induced appearance of potent antitumor immune reactivity in tumor bearer lymphocytes co-expressing the Lyt 2 and the L3T4 antigens.

We have previously shown that Sephadex G-10-adherent spleen cells from mice bearing a large MOPC-315 tumor can suppress the in vitro generation of a primary anti-MOPC-315 cytotoxic response. Here we show that following low dose melphalan (L-phenylalanine mustard; L-PAM) therapy of such tumor bearing mice their Sephadex G-10-adherent spleen cells no longer suppressed but actually brought about the generation of enhanced antitumor cytotoxicity when added to the immunization culture of normal spleen cells and MOPC-315 tumor cells. This immunopotentiating activity of the Sephadex G-10-adherent spleen cells from L-PAM treated MOPC-315 tumor bearers was attributed to T-cells which co-express the Lyt 2 and the L3T4 antigens based on results of experiments employing negative selection. Specifically, depletion of Lyt 2+ cells or of L3T4+ cells abolished the ability of the Sephadex G-10-adherent splenic cell population from L-PAM treated MOPC-315 tumor bearers to bring about the generation of enhanced antitumor cytotoxicity when added to the immunization culture of normal spleen cells. Moreover, the immunopotentiating activity was not restored when a population of Sephadex G-10-adherent spleen cells depleted of Lyt 2+ cells was admixed with a population of Sephadex G-10-adherent spleen cells depleted of L3T4+ cells. In light of the unusual phenotype of the immunopotentiating cells in the spleens of L-PAM treated MOPC-315 tumor bearing mice (i.e. Lyt 2+ L3T4+), and since the vast majority of thymocytes in normal adult BALB/c mice co-express the Lyt 2 and the L3T4 antigens, we evaluated the effect of low dose L-PAM therapy on the antitumor immune reactivity of thymocytes from MOPC-315 tumor bearing mice. A low dose of L-PAM was found to render thymocytes from MOPC-315 tumor bearers, but not from normal mice, capable of bringing about the generation of enhanced lytic activity when added to the immunization culture of normal spleen cells and MOPC-315 tumor cells. At the same time, the thymocytes from L-PAM treated MOPC-315 tumor bearers were unable to develop an antitumor cytotoxic response of their own when immunized in vitro in the absence of normal spleen cells. The possibility that the Lyt 2+ L3T4+ immunopotentiating cells in the spleens of L-PAM treated MOPC-315 tumor bearers represent immature cells that have been induced by the chemotherapy to migrate from the thymus into the spleen is discussed.

Use of Ac-L-Tyr-PAB for estimating the activity of chymotrypsin in rats.

Pancreatic secretion in rats was assessed by measuring the amounts of p-aminobenzoic acid (PABH) excreted in urine after oral administration of Ac-L-Tyr-PAB, and by determination of enzyme activity in pancreas and faeces. 3 groups of 7 rats were kept on diets with casein as the main source of nitrogen without (control K0) or with two levels of trypsin inhibitor (K1 and K2). Two other groups were fed diets with 40 and 80% of casein substituted by raw soya bean protein and having trypsin inhibitor contents equivalent to those in diets K1 and K2. Urinary excretion of PABH ranged from 99 to 105% of intake and were not different between control and experimental rats. The weight of pancreas and pancreatic activities of trypsin and chymotrypsin in all experimental rats were higher than in controls. The activity of chymotrypsin in the faeces of rats of groups K1 and K2 was greater than in K0 while in groups S1 and S2 it was about five times that in groups K1 and K2. Generally, it was greater on diets with more trypsin inhibitor. The activity of trypsin in the faeces of rats K0, K1 and S1 was several times less than in K2 and S2. It has been concluded that measurement of trypsin and chymotrypsin in faeces allows to estimate differences in the secretion of pancreatic enzymes.