Experimental Biology and Medicine

Protein S Structure and Function. Discovery of Protein S Function. The unraveling of the process of blood coagulation has slowly occurred throughout the 20th century. During the 1970s, the focus slowly began to change to questions dealing with regulatory mechanisms that could be important in thrombotic disease. A search for other inhibitors and/or regulatory factors began concurrent with studies on antithrombin III and its interaction with heparin. With the advent of improved chromatographic tools, it became possible to purify trace proteins and analyze their structures. In the early 1970s, Stenflo purified a new vitamin K-dependent protein that was designated protein C (1). It was observed that proteases like trypsin or thrombin could convert it to a serine protease (2). Kisiel and coworkers (3), closely followed by Walker and co-workers (4), recognized that this protease was unique in that it inhibited blood coagulation and that the mechanism appeared to be through the proteolysis of factor Va. Subsequently, it was observed that factor VIII was also a substrate (5, 6). Interestingly, it was found that the active form of factor Va was a better substrate than the circulating form of factor V (4), and the same was found to be true of factors VIII and Villa (6). Proteolysis of factors Va (4) and Villa (7) was observed to be dependent upon the presence of calcium ions and phospholipid vesicles. In 1980, Walker (8) proposed that activated protein C needed a cofactor protein for the expression of its anticoagulant activity and suggested that this protein was protein S. This discovery evolved from the earlier observations that since proteases derived from vitamin K-dependent proteins, factors Xa, IXa, and Vila, all required cofactor proteins for the maximum expression of their activity, it was possible that activated protein C might also need a cofactor protein.

The immune response is characterized by the recognition of self versus nonself, specificity, in-ducibility, and memory. These hallmarks are manifested by the activity of lymphocytes, their differentiated progeny, and some of their soluble products. Specificity of the immune response is ultimately due to cell membrane receptors of T and B lymphocyte clones which bind specifically to antigen. This interaction results in a complex series of events leading to expansion and differentiation of the responding clones, and immunologic memory of the previous exposure to the antigenic stimulus. B lymphocytes extend their specific recognition of antigen by the production of soluble, antigen-specific immunoglobulins with combining sites identical (or very similar) to the heavy and light chain V regions of the B cell membrane immunoglobulin receptor (1) for antigen. In contrast to B lymphocytes, most antigen-specific T lymphocyte activity is mediated by cell membrane-associated receptors which recognize fragments of antigen processed by antigen-presenting cells and associated with class I or II major histocompatibility gene complex (MHC) products (2). The T cell receptor (TCR) for antigen is a heterodimeric molecule consisting of α- and β- or γ- and δ-chains linked by disulfide bonds (2–4), and the organization and sequence of T cell receptor genes demonstrate a significant similarity to immunoglobulin V region genes (3, 4). In addition, murine and human TCR β-chains and immunoglobulin light chains bear cross-reactive epitopes detected by antibodies to synthetic peptides corresponding to the TCR Vβ region (5). However, only low affinity binding of nominal antigen by T cell receptor α-chains has been demonstrated (6). This may be due to conformational changes in the molecule in an aqueous environment and the nature and amount of antigen used. In addition, the affinity of T cell receptors for nominal (non-MHC associated) or MHC-associated antigen may be too low to demonstrate specific binding by conventional methods.

Vitamin A (retinol and its derivatives) is essential for a variety of biologic processes, many of which are related to growth, cellular differentiation, and cell-cell or cell-substrate interactions. Within the last 5 years, there has been a renaissance of interest in vitamin A and immunity that is based largely on three important advances. First, a number of controlled, field-based or hospital-based, studies have demonstrated that vitamin A supplementation can decrease mortality in preschool children in populations at high risk for vitamin A deficiency. Although a mechanistic link between child survival and improved immunity has yet to be established, it seems quite probable that at least some of the benefit of vitamin A is due to restoration of epithelial barriers and improved resistance to respiratory and gastrointestinal infections. Second, vitamin A, either in the form of retinol or its metabolite, retinoic acid, has been shown to stimulate the rejection of certain immunogenic tumors; this effect may be due to enhanced immunologic surveillance. Third, there has been a swell of interest in vitamin A's mechanism of action in nearly all organ systems as the result of the identification of nuclear receptors for retinoic acid and, consequently, advances in understanding the role of retinoic acid in gene regulation.

Vitamin A deficiency, even in relatively early stages, is associated with impairment of linear growth, cartilage, and bone development and changes in epithelial cell differentiation and function (1). If vitamin A deficiency is allowed to persist, animals either succumb or, if they survive, develop progressive xerophthalmia leading to blindness. In parts of the developing world, particularly Southeast Asia and sub-Saharan Africa, xerophthalmia remains a significant, and preventable, public health problem (2).

Both clinical and experimental evidence has shown that vitamin A deficiency is associated with decreased resistance to infection. Vitamin A deficiency could affect immunity through a number of mechanisms including (i) changes in lymphopoiesis and lymphocyte maturation, (ii) abnormal cytokine production, (iii) altered membrane structure affecting receptors for antigens, accessory molecules, or cytokines, (iv) increased penetration of bacteria, vires, and parasites through epithelial barriers, and (v) impaired clearance of pathogens by cytotoxic and phagocytic mechanisms.

The copper content of dog serum and its distribution to copper binding proteins was compared with that of rat and mouse. Total serum Cu concentrations of dogs and mice were one third those of the rat. Plasma ceruloplasmin, determined by azide-inhibitable oxidase activity with two substrates, was 8-fold less in the dog and 9- to 20-fold less in the mouse than in the rat, and, in both dogs and mice, there was 70–75% less ceruloplasmin Cu, determined by atomic absorption after gel filtration. In the dog, the largest proportion of total and exchangeable serum Cu was with the transcuprein fraction. Only one third as much Cu was with albumin in the dog (and mouse) versus the rat, and this was released much more readily through dialysis. In dogs and mice, the exchangeable (nonceruloplasmin) serum copper pool was half the size of that in rats and humans. Especially in the mouse (but also in rats and dogs), a small proportion of the exchangeable pool appeared bound to ferroxidase II. We conclude that the dog may rely more on transcuprein and low molecular weight complexes and less on albumin and ceruloplasmin for transport of copper to cells.

T150R1 is a synthetic copolymer with Na⁺ ionophore activity. We demonstrated previously that T150R1, when injected into mice, produces rapid thymic involution with depletion of cortical thymocytes. Elevated serum ACTH and corticosterone levels, as well as abrogation of the effects of T150R1 on the thymus by adrenalectomy and hypophysectomy, suggested a pituitary-mediated mechanism. In this work, we investigated the ability of T150R1, and of the related ionophore copolymer T130R2, to stimulate ACTH in vitro from the mouse anterior pituitary cell line AtT-20. Copolymer-induced ACTH release was dose-, time-, and temperature-dependent. Hormone induction peaked at 30°C for T150R1 and 37°C for T130R2. The temperature dependence of ACTH release paralleled that of ionophore activity measured in red blood cells, providing evidence that the ability to induce ACTH is related to the ionophore property of the copolymers. Peak ionophore activity and hormonal release occurred at the temperatures when the copolymers form partially soluble complexes which interact optimally with cell membranes. Cotreatment with exogenous phospholipase C inhibited the effects of T150R1, which suggests that the enzyme either blocks the insertion of T150R1 into the cell membrane or that the phospholipase C-induced increase in intracellular calcium inhibits the ionophore activity of T150R1. These data support an ionophore mechanism for copolymer-induced ACTH release in which changes in the physicochemical structure of the copolymers may affect their interaction with cell membranes. The data also suggest that direct stimulation of pituitary ACTH accounts for at least some of the in vivo immunomodulatory effects of T150R1.

Macrophages from A/J mice are permissive for growth of Legionella pneumophila, an intracellular opportunistic pathogen that grows preferentially in macrophages. Macrophages from other mouse strains are highly resistant to growth of Legionella. In the present study, it was found that macrophages from A/J mice are readily activated by pretreatment with lipopolysaccharide (LPS), so that the cells do not permit Legionella to replicate in vitro, as occurs when untreated macrophages from A/J mice are cultured with these organisms for 48 hr. The augmentation of Legionella growth inhibition by LPS-activated macrophages from nonpermissive BDF, mice also occurred. After in vitro infection, there was a 1000-fold increase in the number of Legionella in A/J macrophages and approximately a 10-fold increase in BDF₁ macrophages, but LPS treatment of macrophages from either strain resulted in marked growth restrictions. This suppression was both dose dependent as well as dependent upon the time of addition of the LPS to the macrophages. Furthermore, the lipid A component of LPS was found to be as effective as the intact LPS in activating macrophages to inhibit the intracellular growth of Legionella. Further studies concerning the mechanisms involved are clearly warranted and in progress.

Previously, we demonstrated that naloxone, an opiate antagonist, prolonged survival of strain 13 guinea pigs infected with Pichinde virus. Thus, endogenous opiates may be involved in the pathogenesis of this viral disease. To determine whether endogenous opiate levels were affected by Pichinde viral infection, β-endorphin concentrations in plasma and cerebrospinal fluid (CSF) of normal and infected strain 13 guinea pigs were measured by radioimmunoassay. Cerebrospinal fluid β-endorphin concentrations were 78.0 ± 13.2 pg/ml on postinoculation day (PID) 7, 59.0 ± 5.6 pg/ml on PID 12, and 58.8 ± 5.4 pg/ml on PID 14. These values were significantly higher than baseline levels of CSF β-endorphin: 30.8 ± 1.9 pg/ml. Plasma β-endorphin concentrations of infected animals increased significantly to 202.1 ± 17.9 pg/ml on PID 7 and to 154.2 ± 21.4 pg/ml on PID 12 from a mean baseline value of 84.2 ± 13.1 pg/ml. After a primer intravenous injection of β-endorphin (10, 15, or 30 μg/kg), followed by constant infusion of β-endorphin (15, 45, or 90 μg/kg-hr) to control noninfected guinea pigs, heart rate (except with the lowest dose) and mean blood pressure decreased markedly. Under these experimental conditions, concentrations of plasma and CSF β-endorphin increased simultaneously with different magnitude. Because both Pichinde viral infection and β-endorphin administration produced a similar trend of cardiovascular disturbances, leading to hypotension and bradycardia, increased concentrations of plasma and CSF β-endorphin may play a partial role in the pathophysiological mechanisms of Pichinde virus infection.

A preparation of a triacontanol-containing compound was studied for its effect on cells involved in the inflammatory response. C57BL/6 mice were injected intraperitoneally with various concentrations of this compound and investigated for total body weight, wet weight of thymus tissue, number of thymus cells and splenocytes, interleukin 1 production of spleen monocytes, and response of splenocytes to the T cell mitogen, phytohemagglutinin. Mice treated with the triacontanol preparation exhibited decreased total body weight, 24% reduction in thymus weights, 39% decrease in the number of thymus cells, and 21% depression in total splenocytes. Splenic monocytes of these animals produced a significantly reduced amount of interleukin 1 and splenocytes had a significantly depressed response to phytohemagglutinin. It is concluded that triacontanol has an inhibitory effect on at least some of the cells responsible for inflammation.

We demonstrated previously that in Escherichia coli-infected rats, the heat necessary for the febrile response is a result of thermogenesis in brown adipose tissue (BAT). To investigate whether senescent rats have an impaired febrile response to infection and whether such an impairment is a result of attenuated sympathetically activated thermogenesis in BAT, we assessed body temperature and the increase in mitochondrial guanosine 5′-diphosphate (GDP) binding sites in interscapular BAT in response to E. coli administration in young and senescent male F-344 rats. There was a significant delay of 2 hr in the onset of fever in the older animals. In addition, in senescent rats, the peak fever (1.0 ± 0.1 Δ°C vs 2.2 ± 0.1) and the cumulative fever (383 ± 43 Δ°C · min vs 775 ± 69) were significantly less than in the young rats (P < 0.005). Baseline levels of GDP binding were the same in young and old rats. In young rats, during the rising phase of the fever, E. coli infection resulted in a 50% increase in the density of GDP binding sites in BAT mitochondria. In contrast, there was no increase in GDP binding in the older rats following infection. The failure to increase GDP binding may be a result of a reduced ability to unmask reserve GDP binding sites. Alternatively, there may be fewer total GDP binding sites (masked and unmasked) in senescent rats and these sites may already be unmasked. Collectively, these data suggest that the impaired febrile response with age is due to reduced thermogenesis in BAT.

This study examined multifiber baroreceptor nerve activity (BNA) as a function of carotid sinus wall distension in 19 rabbits. Analysis estimated mechanical or viscoelastic properties of the sinus wall and their influence on BNA. In six sinuses, properties were altered by treatment with the enzyme protease to remove the endothelium and with nifedipine to passively relax smooth muscle. Properties were estimated from dynamic and steady state wall response to a 45 mm Hg step increase and decrease in intrasinus pressure (ISP) of 20 min. Control wall response had fast and slow (creep) portions with a viscosity increase from 1,370 N(s)/m to 17,864 N(s)/m during step-up in ISP. Wall elasticity averaged 77 N/m; which estimated the relationship of force and change in steady state response. Control BNA response also had fast and slow (resetting) portions. A BNA and wall response relationship (BNA/m) was defined as transduction-gain (T-G) with proportional and dynamic components. In the subgroup, wall creep and baroreceptor resetting were abolished by protease treatment, suggesting an endothelial mediator which influenced sinus smooth muscle. Histology data indicated enzyme damage was limited to tunica intima tissues, and nifedipine did not block Ca²⁺ channels on neural structures. By comparison of responses before and after treatments the proportional component of T-G was equated to an elastic influence (1/E), with E = 7.5 × 10⁻⁶ m/BNA, while the dynamic component was equated to a viscous influence (1/V), with V = 1.53 × 10⁻⁴ m(s)/BNA. A simple but fundamental relationship for baroreceptor-tissue linkages was estimated by BNA/m = 1/(Vs + E), a first-order transfer function.

The validity of hemodynamic measurements by the reference sample method with microspheres injection into the aorta, via a carotid artery catheter, was evaluated in rats and compared with the results obtained after left ventricle injection. In the aorta injection group, a good mix of microspheres was observed in 83% of the animals. Moreover, a symmetrical distribution of microspheres was observed in 10 out of 12 rats (83%). An excellent correlation between right and left kidney-testes blood flows was observed (r = 0.93 and 0.96, respectively; P < 0.01). Mean arterial pressure was not modified during microspheres injection into the aorta. Cardiac output (104 ± 26 vs 101 ± 23 ml/min, NS) and portal blood flow (14.2 ± 3.3 vs 13.5 ± 2.2 ml/min, NS) were similar after aorta and left ventricle injections series, respectively. Our results indicate that the injection of microspheres into the aorta is an adequate and easy approach to systemic and splanchnic hemodynamic measurements. This approach could be a good alternative to left ventricle injection of microspheres in experimental studies in rats.

We evaluated the effects of volume expansion with saline (0.5 ml kg^-1 min^-1, n = 13) and with 10% mannitol in saline (0.5 ml kg^-1 min^-1, n = 13) on the cardiorenal actions of endothelin-1 (ET) in rats anesthetized with sodium pentobarbital. We also evaluated to what extent the calcium channel antagonist, verapamil (0.02 mg kg^-1 min^-1), altered the cardiorenal actions of endothelin in volume-expanded rats (n = 10 with saline and n = 10 with mannitol). In five rats from each group, renal blood flow was measured with an electromagnetic flow probe. Sixty minutes after surgery, control clearances were collected, ET (110 ng kg^-1 min^-1) was then infused for 30 min, and recovery clearances were collected for 60 min. ET caused a similar increase in mean arterial blood pressure and decrease in renal blood flow and the glomerular filtration rate in the saline and mannitol groups. Verapamil significantly attenuated but did not abolish the ET-induced increase in mean arterial blood pressure in both saline- and mannitol-treated rats. By contrast, the calcium channel antagonist had no effect on the ET-induced decrease in either the glomerular filtration rate or renal blood flow in saline-treated rats, but significantly attenuated these responses to ET in mannitol-expanded animals. These data demonstrate that (i) the systemic and renal responses to ET are not affected by expansion with saline or mannitol and (ii) the renal vasoconstriction prompted by endothelin is not affected by verapamil in saline-expanded rats, but is attenuated by the Ca²⁺ channel antagonist during expansion with mannitol. These data suggest that during volume expansion with mannitol, but not with saline, the ET-induced renal vasoconstriction occurs primarily at intrarenal resistance sites that are dependent upon extracellular Ca²⁺.

CD5⁺ B cells have been shown to be disproportionately associated with autoimmune diseases and transformation. In many cases, their apparent ability to bypass self-tolerance is manifested by the production of autoantibodies. These observations, plus the hypothesis that CD5⁺ B cells represent a distinct B cell lineage, encourage studies into the conditions and factors that influence their development. In the present study, we employed a well-established assay for murine CD5⁺ B cell function, i.e., their ability to augment the responses of lgH^b-linked idiotypic determinants on anti-(4-hydroxy-3-nitrophenyl) acetyl (NP) antibody (Nb^b) idiotype-bearing CD5^- B cells to a T-independent antigen, together with multiple methods of cell surface phenotyping, to evaluate the potential for interleukin (IL) 4 to effect maturation of CD5⁺ B cells, CD5⁺, IgM⁺, Thy-1^-, and NP^b idiotype-specific cells panned on antibody-coated petri dishes or sorted by flow cytometry from spleen were capable of augmenting NP^b idiotypic responses of NP-KLH-primed responder cells to NP-Ficoll. Splenic B cell populations depleted of CD5⁺ B cells failed to affect idiotype expression even after 2 days in culture, a time when a small percentage of CD5⁺ B cells appeared to be regenerated. However, idiotype-specific regulatory activity could be restored in CD5^- splenic B cell populations by culture for 2 days with recombinant IL-4. Cells responsible for idiotype-specific regulatory activity after culture with IL-4 were in fact CD5⁺, lgM⁺, and Thy-1.2^- B cells, demonstrating that IL-4 can drive the functional, if not the phenotypic, maturation of splenic B cells associated with the CD5⁺ B cell lineage. The results illustrate one possible mechanism by which T cells could control the maturation of cells belonging to the CD5⁺ B cell lineage.

Specific receptors for bombesin/gastrin-releasing peptide, somatostatin, and EGF were investigated in 15 human colon cancer specimens. Eight of 15 clinical specimens (15%) of colon cancer showed the presence of somatostatin receptors. Octapeptide somatostatin analogs, RC-160 and RC-121, showed 10 times higher binding affinity for somatostatin receptors on colon cancer membranes than somatostatin. Analysis of ¹²⁵I-Tyr⁴-bombesin binding data revealed the presence of specific binding sites in six (40%) specimens of human colon cancer. Scatchard analysis of ¹²⁵I-labeled bombesin indicated a single class of receptors in three specimens with an apparent K_d value of 2.5 nM and two classes of receptors with high (K_d = 0.4 ± 0.2 nM) and low affinity (K_d = 1.6 ± 0.4 μM) in three other specimens. The ¹²⁵I-Tyr⁴-bombesin binding capacities in the colon cancers for high affinity binding sites were from 6 to 228 fmol/mg protein and for low affinity binding sites 76 ± 15 pmol/mg protein. None of the membrane preparations made from normal colonic mucosa specimens showed specific binding for ¹²⁵I-Tyr⁴-bombesin. Five pseudononapeptide (ψ^13–14) bombesin (6–14) antagonists, with different modifications at Positions 6 and 14, synthesized in our laboratory, inhibited the binding of ¹²⁵I-Tyr⁴-bombesin in nanomolar concentrations. No correlation was found between the degree of differentiation and the presence of binding sites for somatostatin or bombesin. Specific binding of EGF was detected in 80% of colon cancer specimens. EGF binding capacity in colon cancer membranes was on average twice as high as in normal colon mucosa (50 ± 21 vs 28 ± 14 fmol/mg protein, respectively). Specific binding sites for somatostatin and EGF, but not bombesin, were also demonstrated in human colon cancer cell line HT-29. In HCT-116 colon cancer line only EGF receptors were found. These receptor findings and our in vivo studies on inhibition of colon cancer growth support the merit of continued evaluation of somatostatin analogs and bombesin/gastrin-releasing peptide antagonists in the management of colonic carcinoma.

Total body x-irradiation has been utilized in the treatment of several human diseases, including leukemia, where it is followed by bone marrow transplantation, and in some autoimmune disorders. Recently, it was reported that total body irradiation appeared useful in the treatment of Friend leukemia virus infection in mice. In this report, the effect of x-irradiation on the replication of human immunodeficiency virus (HIV) In vitro in CD4⁺ cells was examined. MT-4 cells and HIV strain human T cell lymphotropic virus Type Ill_B were used to conduct this study. Infected MT-4 cells were irradiated at the time of infection or following infection with x-ray doses of 25–300 cGy. Doses of 50, 150, and 300 cGy enhanced HIV replication by 1.6-, 2-, and 4.8-fold, respectively. Irradiating the cells prior to infection also resulted in similar enhancement of HIV replication. This phenomenon was also observed with wild-type HIV isolates grown in peripheral blood mononuclear and in HIV chronically infected cells. In addition, the enhancement was associated with a radiation-induced increase in intracellular levels of cAMP. The use of the cAMP-dependent protein kinase A inhibitor, H-8, inhibited HIV replication by 65%. These data suggest that in vitro exposure to low doses of x-ray enhances HIV replication partially via a cAMP-dependent pathway.

The aim of this study was to determine whether the circadian changes in ornithine decarboxylase (ODC) activity of different segments of the small intestine were governed by factors other than food intake. First, the effects of fasting on mucosal ODC activity were examined. The results indicate that mucosal ODC activity in 24 hr and 48 hr fasted rats decreased significantly compared with ad libitum-fed rats. Second, the circadian rhythm of mucosal ODC activity was characterized by measuring mucosal ODC activity in fasted rats at four time points (09:00, 15:00, 21:00, and 03:00 hr; light period: 06:00–18:00 hr). The results from this study indicate that there is a detectable baseline ODC activity in different segments of fasting intestine. In duodenum, mucosal ODC activity was highest at 15:00 hr (light period), a time at which the rat was normally not eating. In jejunum and ileum, mucosal ODC activity increased between 21:00 and 03:00 hr (dark period). The observation that small intestine exhibits a distinct circadian rhythm of ODC activity in fasted rats suggests that not only food but also intrinsic factors can modulate physiologic oscillations in mucosal ODC activity.

The scarce bioavailability of β-interferon (IFN-β) after intramuscular administration is probably due either to the binding of IFN-β to interstitial matrix, or to lymphatic absorption and/or to local breakdown by lysosomal proteinases from muscle. In this work, we first showed that after intramuscular injection, the apparent bioavailability of natural human IFN-β is about 10% of that of recombinant IFN-α₂ and then we evaluated the effects of proteinase inhibitors and albumin on IFN-β incubated at 37°C with muscle homogenate. IFN biological activity decreased spontaneously by about 20% after incubation for 6 hr at 37°C in Hanks' solution, but it was almost completely lost after incubation with muscle homogenate. Proteinase inhibitors (α₁-antitrypsin, α₂-macroglobulin, aprotinin, soybean trypsin inhibitor, leupeptin, EP-459, and EP-475) failed to block the inactivation of IFN-β by muscle proteinases, whereas albumin exerted a partial but consistent protection.

Pregnant mice congenic with C57BL/10 (B10.A, B10.BR, B10.D2, B10.A(2R), B10.A(5R), B10.A(15R), B10.A(1R), B10.A(18R), and B10.OL) were fed Purina Mouse Chow or the same diet plus 200 IU of vitamin A daily. The pregnant dams were sacrificed on the eighteenth day of gestation, and the fetuses were sexed and examined for defects in mandibular development.

On average, micrognathia occurred five times more frequently in female (1.5%) than male (0.3%) fetuses. The addition of vitamin A to the diet affected only females, reducing the frequency of this defect to that observed in males from dams fed the control diet. Micrognathia was strongly associated with micro- or anophthalmia, but not with defects of the palate. C57BL/10 fetuses had the highest frequency of micrognathia (3.2%) and B10.D2 and B10.A(5R) fetuses had the lowest (0.1%). The results suggest that a locus distal to C4 and perhaps proximal to Qa-1 may exert a moderate influence on mandibular development and a second locus proximal to E_β may have a weak effect.

The purpose of this study was to examine the effect of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the major psychoactive component of marijuana, on T lymphocyte functional competence against herpes simplex virus Type 1 (HSV1) infection. Spleen cells from C3H/HeJ (H-2^k) mice primed with HSV1 and exposed to Δ⁹-THC were examined for anti- HSV1 cytolytic T lymphocyte (CTL) activity. Flow cytometry was used to determine whether Δ⁹-THC altered T cytotoxic (Lyt-2⁺) and T helper (L3T4⁺) lymphocyte numbers or cell ratios. Nomarski optics microscopy was used to determine whether effector lymphocytes from drug-treated mice were able to bind to virally infected L929 (H-2^k) target cells. Cytotoxicity assays demonstrated that CTL from mice exposed to Δ⁹-THC were deficient in anti-HSV1 cytolytic activity. Δ⁹-THC in vivo treatment had little effect on the number of T lymphocytes expressing the Lyt-2 or L3T4 antigens. Nomarski optics microscopy revealed that the CTL from the drug-treated mice were able to bind specifically to the HSV1-infected targets. However, Δ⁹-THC In vivo exposure affected CTL cytoplasmic polarization toward the virus-infected target cell. CTL granule reorientation toward the effector cell-target cell interface following cell conjugation occurred at a lower frequency in co-cultures containing CTL from drug-treated mice. These results suggest that Δ⁹-THC elicits dysfunction in CTL by altering effector cell-target cell postconjugation events.

Rat nylon wool nonadherent bone marrow cells were propagated for up to 75 days in co-culture with stromal cells derived from either spleen or bone marrow. Interleukin (IL) 1 enhanced the ability of spleen stroma to support the long-term culture of natural killer (NK) cells, ostensibly by inducing these support cells to synthesize other cytokines. Flow cytometry studies indicated that the nylon wool separation procedure enriched the concentrations of mature NK cells from 7.9% to 38.1% for splenocytes and from 3.8% to 19.5% for bone marrow cells. Analyses of the adherent zones of suspended nylon screen NK cell cultures revealed substantial numbers of large granular lymphocytes that expressed NK 323⁺/MOM/3F12/F2-phenotypes. The presence of both mature and immature cells of the NK lineage in this matrix was inferred by the presence of both IL-2 receptor (IL-2R) positive and IL-2R negative, and OX-8⁺ and OX-8^- NK 323⁺ cells over the >4-month experimental period. Suspended nylon screen cultures displayed a greater potential for producing cytolytic cells than either co-cultures of bone marrow nonadherent cells on stromal monolayers or suspension cultures. The large granular lymphocytes produced in suspended nylon screen cultures could be transformed into active killers of YAC-1 targets by IL-2. In contrast to bone marrow nonadherent cells, more splenic nylon-wool-passed cells displayed a mature NK phenotype, but their proliferative potential and ability to be transformed into cytolytic cells by IL-2 decreased rapidly in culture. In the suspended nylon screen culture system, NK cells migrate from the underlying stroma in stages as they mature, retain their cytolytic potential, and manifest a capacity for self-renewal. Cultured cells were routinely dissociated into single cell suspensions via enzyme treatment and were reinoculated onto “fresh” nylon screen/stromal cell templates after passage through nylon wool columns. These co-cultures continued to generate cytolytic cells in numbers greater than those of the initial inoculum.