Teratogenic bioactivation of cyclophosphamide in vitro.

Day 10 rat embryos grown as cultured explants $\text{in vitro}$ developed characteristic defects when culture media contained cyclophosphamide (6.25 micrograms per milliliter or more), an hepatic microsomal fraction, and cofactors for a monooxygenase system. Cyclophosphamide concentrations as high as 250 micrograms per milliliter were innocuous when either the microsomal material or cofactors were omitted from the medium. These experiments represent the first direct demonstration of bioactivation of a proteratogen.     

Production and distribution of entactin and GP-2 in M1536-B3 cells

The endodermally differentiated mouse embryonal carcinoma cell line M1536-B3 produces the basement membrane-associated glycoproteins entactin and GP-2 in cell culture. Immunological techniques coupled with light and electron microscopy were used to study the intracellular distribution and fate of these molecules. The two proteins were localized in cisternae of rough endoplasmic reticulum and in discrete membrane-bound vesicles. There was no evidence for their presence in the Golgi apparatus. The membrane-bound vesicles appeared to fuse with the plasma membrane and thereby transfer their contents to discrete foci on the cell surface. These foci became apparent as cell-to-cell contact was made and were prominent at cell-cell contact sites. They eventually coalesced into continuous densely stained extracellular bands of amorphous material. These bands formed a honeycomb of spaces lined by cells. It appeared that production of the extracellular matrix was probably stimulated by cell-cell contact, and that cell density rather than age of the cell cultures dictated the pattern of extracellular distribution. These results support the hypothesis that these molecules are involved in cell adhesion and multicellular organization.     

Suppression of in vitro antibody responses by Listeria primed spleen cells.

Spleen cells from mice primed with virulent Listeria monocytogenes do not develop an anti-SRBC plaque forming cell response to SRBC in culture. Furthermore, when Listeria primed spleen cells are co-cultured with normal spleen cells and SRBC, the anti-SRBC response of the normal cells is suppressed. Listeria primed spleen cells from T cell depleted donors are equally effective at immunosuppression. The immunosuppressive effect does not appear to be due to the presence of the bacterium or its products per se in the cultures. Furthermore, the effect cannot be transferred across a 0.45 μm pore membrane. Kinetic studies show that the immunosuppressive effect develops by 2 days post-Listeria inoculation and peaks by Day 6. Low doses of Listeria are not immunosuppressive and produce some enhancement effect. From these results, it is suggested that a population of non-T cell dependent cells develop in Listeria primed hosts that nonspecifically suppress the response of B cells to an unrelated antigen in culture.     

Increased amphetamine stereotypy and longer haloperidol catalepsy in spontaneously hypertensive rats

Spontaneously hypertensive rats (SHR) of both sexes and from two sources exhibited enhanced stereotyped behavior following amphetamine (AM) administration. Both the intensity and the duration of sniffing and licking were higher in SHR than in Wistar or Sprague Dawley controls. SHR exhibited longer catalepsy after haloperidol (HALO) than did controls. Rats made hypertensive by the Goldblatt one kidney method showed neither increased AM stereotypy nor longer HALO catalepsy. The increased effect of AM in SHR was not due to greater drug accumulation in the brain. These findings are discussed in terms of altered brain catecholamine metabolism in SHR, and are related to experiments of chronic stress and the sensitization of AM's effects.     

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured human fetal aortic smooth muscle cells.

Cultured human fetal aortic smooth muscle cells derived from the abdominal aorta converted benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) $\text{via}$ cytochrome P-450-dependent monooxygenation to metabolites detectable by both a highly sensitive radiometric assay and high pressure liquid chromatography (HPLC). Cells incubated with ³H-BaP transformed this substrate primarily to phenols. ¹⁴C-DMBA was converted to metabolites that cochromatographed with 12-hydroxymethyl-7-methylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz-[a]anthracene, 7,12-dihydroxymethylbenz[a]anthracene, and trans-8,9-dihydrodiol-7,12-DMBA. Exposure of cells in culture to 13 μM 1,2-benz[a]anthracene resulted in increased oxidative metabolism of both BaP and DMBA. In the case of BaP, total phenol formation was increased, while with DMBA all metabilities detected by HPLC were increased. Support for the potential role of metabolism of polycyclic aromatic hydrocarbons by aortic smooth muscle cells in the etiology of atherosclerosis was obtained.     

Ly-4.2: a cell membrane alloantigen of murine B lymphocytes. III. In vitro studies

The alloantigenic specificity Ly-4.2 is present on a restricted population of murine lymphocytes which have previously been shown to have some of the properties generally ascribed to B lymphocytes, both with regard to distribution and function. In the study reported herein, the effect of anti-Ly-4.2 and anti-Thy-1.2 (θ) antisera have been examined in various in vitro systems. (a) T cell-mediated lysis of ⁵¹Cr-labeled P815-X2 target cells by immune allogeneic peritoneal exudate cells is inhibited by anti-Thy-1.2, but not affected by the anti-B (Ly-4.2) reagent. (b) Antibody-dependent lymphocyte-mediated lysis of ⁵¹Cr-labeled sheep red cells was only slightly inhibited by anti-Ly-4.2 and anti-Ig antisera, and not at all by anti-Thy-1.2 antisera, indicating that this type of cell lysis is mediated by neither T (Thy-l⁺) nor B (Ly-4.2⁺,Ig⁺) cells. (c) The response of lymph node lymphocytes to various mitogens was affected thus: PHA, completely inhibited by anti-Thy-1.2 but not by anti-Ly-4.2; Con A, largely inhibited by anti-Thy-1.2, and slightly by anti-Ly-4.2; PWM (pokeweed mitogen), partially inhibited by both antisera; E. coli endotoxin lipopolysaccharide, greatly inhibited by anti-Ly-4.2 but only slightly by anti-Thy-1.2. The findings demonstrate that anti-Thy-1.2 reacts predominantly with T cells and anti-Ly-4.2 with B cells.     

Primary and secondary antibody responses of rabbits to bacteriophage T2: kinetic and quantitative analysis and suppression by antimacrophage globulin.

Antibody synthetic capacity of popliteal lymph node cells removed from rabbits at various times after immunization with bacteriophage T2 was assayed by radioimmunoassay of tissue culture fluid after incubation with ¹⁴C-leucine. Antibody synthesis began on day 2; IgM synthesis peaked on day 3; IgG synthesis peaked on day 5 and again on day 14. Reinjection of T2 one month later elicited an enhanced response which peaked sharply on day 2. The primary and secondary responses, but not priming for the secondary response, were suppressed by injection of goat antimacrophage globulin (AMG), but only when AMG was injected 1 to 3 days before T2. AMG reacted strongly with rabbit peritoneal macrophages and only slightly with rabbit lymphocytes or erythrocytes. Thus, macrophages appear to participate in the induction of antibody responses of rabbit lymph nodes to T2 and their function inhibited by AMG apparently operates only during the early phase of induction.     

Deep hypothermia and circulatory arrest: effect on cerebrospinal fluid electrolytes in newborn lambs.

Cerebrospinal fluid (CSF) Na, K, and acid-base changes were studied in 13 new-born lambs anesthetized with α-chloralose (60 mg/kg) or diethylether during 90 min of normothermic (37 °C) or hypothermic (20 °C) circulatory arrest. CSF K concentration increased linearly from 3.1 to 23.2 meq/liter during 90 min of normothermic circulatory arrest. During hypothermic circulatory arrest, animals anesthetized with α-chloralose exhibited an exponential increase in CSF K concentration from 3.1 to 13.6 meq/liter and animals anesthetized with diethylether had an exponential increase in CSF K concentration from 3.3 to 12.7 meq/liter. The rate of increase in CSF K concentration in hypothermic and normothermic animals between 60 and 90 min of circulatory arrest was the same. CSF Na concentration decreased slightly in both hypothermic and normothermic animals, with a greater decrease in the normothermic group.Although CSF pH and bicarbonate were significantly decreased during normothermia as well as hypothermia, both CSF pH and bicarbonate showed greater decreases during normothermia. Mean pH values after 90 min of circulatory arrest were 6.34, 6.87, and 6.77, respectively, in the normothermic, α-chloralose-hypothermic, and diethylether-hypothermic groups; corresponding values for bicarbonate were 7.7, 13.8, and 12.2 meq/liter.CSF pCO₂ increased linearly from 40.2 to 190.0 Torr during 90 min of normothermic circulatory arrest, from 28.6 to 92 Torr in the ether-hypothermic group, and from 28 to 81 Torr in the α-chloralose-hypothermic group.     

Symbolic logic analysis of cause of dealth in humans: Application to 108 patients after coronary artery bypass graft surgery

Over the past two decades there has been increasing interest in the development of an objective, or formalized “medical logic”, and many authors have employed classical symbolic logic as a part of their approach. On the other hand, it has become clear that certain patterns of reasoning which are commonplace in evaluating patients clinicopathologically are awkward to handle in classical symbolic logic. The present paper proposes an extension of classical symbolic logic which addresses three problems in medical reasoning: (i) the problem of provisional diagnosis, (ii) the problem of inaccessible data, and (iii) the problem of the adequate discharge summary. It is proved mathematically that with a suitably constructed logic, the system “complains” until all questions involving threats to the patient's health are either answered or shown to be unanswerable because of inaccessibility of data. To illustrate this method, the cause of death was studied in 108 patients who had been autopsied at The Johns Hopkins Hospital after coronary artery bypass surgery. The analysis disclosed that 46% of patients suffered a fatal complication which could be attributed to events in the perioperative period; in 15% of patients the cause of death was unexplained by the analysis. Computerized symbolic logic analysis is a useful supplement to intuitive reasoning in assigning cause of death to patients with complex medical histories.     

Effects of 2-formylpyridine monothiosemicarbazonato copper II on red cell components

2-Formylpyridine monothiosemicarbazonato copper II (CuL⁺) is readily taken up by red cells and is initially bound to glutathione and hemoglobin. Glutathione was depleted within 5 hr of incubation, presumably by oxidation mediated by CuL⁺ and O₂ with concomittant generation of toxic oxygen species. Cupric ion was slowly transferred from CuL⁺ to hemoglobin within about 7 hr, and hemoglobin was oxidized until the major form prevailing after 10 hr was α₂β₂⁺. Little increase in hemolysis due to addition of CuL⁺ dissolved in the radical scavenger dimethyl sulfoxide was observed with prolonged incubation. Strong inhibition of red cell hexokinase by CuL⁺ was observed when the enzymes in red cell lysates and hemoglobin-free red cell lysates were examined. CuL⁺ was also an effective inhibitor of yeast hexokinase. However, the inhibitory effect of CuL ⁺ within the red cells was less pronounced. It is suggested that even though intracellular accumulation of CuL ⁺ creates an oxidizing environment and is potentially capable of inhibiting thiol enzymes such as hexokinase, protective effects are exerted in the red cell by the presence of hemoglobin, of radical scavengers, and of high levels of enzymes that detoxify toxic oxygen species. Address reprint requests to Dr. W.E. Antholine, Department of Radiology, or Dr. F. Taketa, Department of Bio     

Cell attachment to a substratum and cell surface proteases.

The polytripeptide (Tyr-Ala-Glu)_n, n～-175, has been reported to undergo an α-helix-disordered chain transition in aqueous medium (Ramachandran, J., et al. (1971) Biopolymers, 10, 1829–1851). We find from circular dichroism and infrared spectroscopy that, upon transferring (Tyr-Ala-Glu)₉ from aqueous buffer at neutral pH to dioxane-containing media at acidic pH, and in certain other circumstances, a transition from the disordered state to the antiparallel β structure occurs. Molecular weight studies and the independence of the transition from concentration suggest that the β structure is intramolecular. (Tyr-Ala-Glu)₄ shows no evidence for the occurrence of any conformational change under similar conditions.     

Immunologic properties of mouse thymus cells: membrane antigen patterns associated with various cell subpopulations

The membrane antigen components of mouse thymus cells and fractions derived from BSA density gradient centrifugation were assayed by quantitative cytotoxicity tests. Two subpopulations were identified on the basis of average density and antigen patterns. The major subpopulation consisted of small lymphoid cells and comprised 80%–90% of all cells, was of high relative density and rich in θ, TL, G_IX, Ly-A, Ly-B, and Ly-C, but contained little or no H-2. The minor subpopulation was chiefly large lymphoid cells, comprised 10%–15% of cells, was of low relative density, was relatively rich in H-2 but low in θ and Ly antigens, and contained no detectable TL or G_IX. This minor subpopulation was identical in density and antigen patterns to those cells remaining in the thymus after short-term cortisone treatment or whole-body irradiation. It could also be reproduced by treating whole thymus with anti-TL or anti-θ sera. The antigenic attributes of this minor subpopulation differed from those of spleen lymphocytes only with respect to average density.     

The inhibitory actions of progesterone: effects on male and female sexual behavior of the hamster

A series of three experiments compared the inhibitory effects of progesterone on estrogen- or androgen-induced sexual behavior in male and female hamsters. In the first experiment chronic progesterone treatment was found to have no effect on male copulatory behavior maintained after castration with testosterone propionate or estradiol benzoate. However, testosterone propionate was more effective at maintaining male behavior than estradiol benzoate. In the second experiment progesterone was found to have a slight inhibitory effect on the rate of the restoration of the intromission response after androgen treatment in males which had been castrated for 8 weeks. In the final experiment, chronic progesterone treatment markedly inhibited sexual receptivity in male and female hamsters which had been given 4 weeks of androgen or estrogen treatment and a single pretest injection of progesterone. Thus, progesterone was shown to be a potent inhibitor of androgen- or estrogen-induced estrus in both male and female hamsters. Due to the large difference in effectiveness on these two behavioral systems, we suggest that progesterone affects steroid-induced male copulatory behavior and female receptivity by different mechanisms of action.     

Differential effects of ouabain on human cell-mediated cytotoxicity. I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets.

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed ⁵¹Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10^?4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.     

Suppressor T-cell activity induced as a result of thermal injury.

Many thermally injured patients survive their initial trauma only to succumb to infection at 2 to 4 weeks after the burn. Both clinical and experimental data have suggested that acute thermal insult compromises immune function. In this report we have sequentially examined the ability of thermally injured mice to generate a specific in vitro primary antibody-forming cell (AFC) response to sheep red blood cells (SRBC) at various times after thermal injury. Thermally injured mice appear to lose the ability to generate de novo antibody-forming cells in vitro after thermal injury. The defect was dissected as to the involvement of macrophage (φ), thymus-derived cell (T-cell), or bursal equivalent (B-cell) defects. Murine B cells from burned animals exhibited normal immunological function in the in vitro AFC system. T cells from burned mice were demonstrated as not only dysfunctional in the generation of immune AFC, but also as able to suppress generation of an AFC response by syngeneic normal cells.     

Hypothesis test for causal explanations in human pathology: evaluation of pulmonary edema in 181 autopsied patients with leukemia

Symbolic logic, as used in the formal theory of scientific explanation proposed by Hempel and Oppenheim, has been suggested as the basis for automated medical diagnosis. In human autopsy pathology the determination of cause-and-effect relationships is a major area subject to logical analysis. We propose a modification of the Hempel-Oppenheim schema in which the logical relationships must only be satisfied “much” of the time, as determined by binomial significance tests. The analysis employs “certainty levels” logic with a more limited consistency requirement than classical logic. The analysis is applied to a series of 181 autopsied patients with leukemia in an attempt to determine a possible role of chemotherapeutic agents in the etiology of pulmonary edema. Among 51 patients who had received cytosine arabinoside (Ara-C) within 30 days of death, there was significantly more unexplained moderate or massive pulmonary edema than among patients with no or remote therapy (p<0.001). The results suggest that a symbolic logical analysis combined with a binomial significance test can elucidate cause-and-effect relationships observed at autopsy, especially when there are multiple possible explanations for the same effect.     

Myogenesis in vitro. Enhancement by dibutyryl cAMP

Cholera enterotoxin (CT) increased the concentration of adenosine 3′-5′-cyclic monophosphate (cAMP) in monolayer cultures of adrenal tumor cells after a 60 min lag phase in contrast to the rapid effect of adrenocorticotropin (ACTH). The change in intracellular cAMP was accompanied by the release of steroids into the culture medium and a reversible alteration of monolayer morphology.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. II. Antigen-dependent enhancement by exogenous prostaglandins of the E series.

The spleen cells from CFW/D mice injected with dimethylbenzanthracene-induced leukemia virus exhibited a progressive decline in the in vitro response to heterologous erythrocyte antigens in parallel with tumor growth. Cell transfer experiments revealed that this immunodepressed state may involve a B-cell defect rather than extrinsic factors in the cellular environment since: (i) nonresponsiveness could be transferred to irradiated non-tumor-bearing mice with spleen cells, and (ii) T cells from tumorbearing mice cooperated with normal bone marrow cells, but bone marrow from tumorbearing mice did not cooperate with normal T cells. In addition, T cells from the thymic tumor could cooperate with normal bone marrow cells upon transfer to irradiated recipients. TL 485-2 cells, a T-cell line derived from the tumor, could be specifically activated with SRBC thereby indicating that the virus transformed T cells were immunocompetent. Suppressor cells, which appeared in the spleen concomitant with immunodepression and tumor development, may directly raise B-cell thresholds for T-dependent triggering signals since the antibody response of spleen cells from tumor-bearing mice could be restored by adding agents such as LPS, 2 mercaptoethanol, or T cells exogenously preactivated in normal animals. The suppressor cell could be enriched by adherence to plastic and was removed by treatment with carbonyl iron. In addition, it was unlikely that the suppressor cell was a virus-infected cell since transformed, virus-infected cells from the tumor or TL 485-2 cells were not suppressive when added to spleen cells in vitro but rather resulted in a marked, polyclonal enhancement of the PFC response. The interaction of TL 485-2 cells and normal spleen cells resulted in the release of a stimulatory factor which increased DNA synthesis in resting cells as well as increasing PFC. The role of these enhancing factors and suppressor cells in controlling tumor growth remains to be elucidated.     

Early degranulation of human neutrophils: immunocytochemical studies of surface and intracellular phagocytic events.

Degranulation of azurophil and specific granules after phagocytic challenge with E. coli for 5 sec to 10 min was investigated in the human polymorphonuclear neutrophil (PMN). PMN were stained simultaneously with fluorescein and rhodamine-labeled monospecific antisera to myeloperoxidase (MPO) and lactoferrin (LF) to identify azurophil and specific granules, respectively, within single cells. Fixation was designed to preserve or disrupt differential permeability of cell membrane to fluorescent conjugates in order to study granule translocation. Within 5 sec after phagocytic challenge, MPO and LF appeared on the cell surface coating the bacteria as granule contents leaked from the incompletely formed phagolysosomes. The phagocytic cup, shown by scanning electron microscopy as large and circular, appeared by immunofluorescent markers to be outlined by curvilinear staining for both granule markers, and was always coincident with bacterial localization. MPO and LF appeared singly or simultaneously on the cell surface, suggesting that degranulation to the surface was random. Sequential phagocytic events were demonstrated by comparing staining intensities for each granule marker on the surface and intracellularly within single cells. LF sometimes appeared on the cell surface independent of the nascent phagosome, suggesting that perturbation of the cell membrane by bacteria may cause some specific granule extrusion not limited to the phagosome. These results imply that bacteria make contact with granule-associated anti-microbial substances within 5 sec after phagocytosis is initiated and that free communication of granule constituents occurs between the newly forming phagolysosome and the extracellular space.     

H2O2− and alkyl hydroperoxide-supported para hydroxylation of aniline by alkaline hematin

Under alkaline conditions and in the presence of reductant, NADH or NADPH, and molecular oxygen, hemin catalyzes the regiospecific para-hydroxylation of aniline to form p-aminophenol [P.A. Adams, D.A. Baldwin, and M.C. Berman, J. Chem. Soc. (Lond) Chem. Commun. 856 (1979); P.A. Adams and M.C. Berman, J. Inorg. Biochem.17, 1 (1982)]. The reaction has now been studied in the presence of H₂O₂ and alkyl hydroperoxides and in the absence of oxygen and reductant. Results indicate that the H₂O₂^? and alkyl hydroperoxide-supported processes proceed via different mechanisms involving, on the one hand, the hydroperoxide anion (HO₂^?) and on the other, the undissociated alkyl hydroperoxide molecule (ROOH). The addition of superoxide dismutase to the reaction had no effect, unlike the NADH/O₂ supported reaction where the enzyme completely inhibits reaction. Similarities between the hemin-mediated peroxide-supported reactions reported here, and the cytochrome P-450-mediated peroxide-supported reactions reinforce our earlier contentions that the alkaline hemin system appears to be a good model for the in vivo activation of oxygen by hemoproteins.