Corticosteroids inhibit the generation of lymphokine-activated killer activity in vitro

Summary In phase-I clinical trials of adoptive immunotherapy using lymphokine-activated killer (LAK) cells plus recombinant interleukin-2 (rIL-2) (Cetus) for the treatment of malignant glioma, we observed that blood mononuclear cells (MNC) from patients dependent on dexamethasone for management of cerebral edema produced substantially less LAK activity as compared to MNC of normal blood donors or glioma patients not receiving steroid therapy. Therefore, we examined the in vitro effects, brought about by therapeutically attainable concentrations of various corticosteroids, on the proliferative response, production of interferon (IFN-), and induction of LAK activity from blood MNC of normal donors. Incubation in media containing rIL-2 (1000 U/ml) with either dexamethasone, hydrocortisone, methylprednisolone, or prednisolone profoundly affected all of these parameters. First, while 0.01 g/ml of either dexamethasone or hydrocortisone caused a slight enhancement of the mitogenic response of lymphocytes to phytohemagglutinin, a dose-dependent decline occurred as concentrations increased to 10 g/ml. The addition of prednisolone and methylprednisolone elicited a dose-dependent inhibition of lymphocyte proliferation over the entire concentration range tested. At 0.1 g/ml or higher, dexamethasone, hydrocortisone, methylprednisolone and prednisolone significantly (P<0.02) inhibited the production of IFN-: respectively 18.9%, 4.4%, 2.2%, and 12.3% of the IFN- produced by MNC in the absence of steroids. All four corticosteroids inhibited the induction of LAK activity. Compared to MNC that had been incubated with 1000 U/ml rIL-2 alone, MNC cultured with rIL-2 and 10 g/ml either dexamethasone or prednisolone demonstrated significantly lower cytotoxicity (P<0.05) for the natural-killer-cell-resistant cell line, Daudi. Culturing MNC with hydrocortisone had a more dramatic result, causing a significant decline (P<0.01) in lytic activity at both 1.0 g/ml and 10 g/ml, while incubation with methylprednisolone produced a significant drop (P<0.02) in LAK-mediated cytotoxicity at 0.1 g/ml as well as 1.0 g/ml and 10 g/ml. When cytotoxicity was expressed as lytic units per million effectors, a dose-response decline in lytic activity was once again apparent, with hydrocortisone, methylprednisolone and prednisolone showing significant inhibition (P<0.05) at both 1.0 g/ml and 10 g/ml and dexamethasone at 10 g/ml (P<0.01). These results indicate that corticosteroids commonly used in the management of cerebral tumors and other malignancies inhibit induction of LAK activity in vitro, and this may explain why it is often difficult to generate LAK activity from blood MNC of patients who are receiving chronic steroid therapy.     

Tumor-infiltrating lymphocyte secretion of IL-6 antagonizes tumor-derived TGF-beta 1 and restores the lymphokine-activated killing activity

IL-6 is a multifunctional cytokine that regulates cell growth, differentiation, and cell survival. Many tumor cells produce TGF-beta1, which allows them to evade CTL-mediated immune responses. IL-6 antagonizes TGF-beta1 inhibition of CD3 cell activation. However, whether IL-6 restores NK activity, which also is suppressed by TGF-beta1, is not known. We used canine transmissible venereal tumor (CTVT), which produces TGF-beta1, as a model to determine whether IL-6 restores lymphokine-activated killer (LAK) activity. During the progression phase, CTVT cells stop expressing MHC molecules. During the regression phase, the number of surface MHC molecules increases dramatically on about one-third of tumor cells. Tumor cells that stop expressing MHC should be targeted by NK cells. In this study, we found that TGF-beta1 secreted by CTVT cells suppressed LAK cytotoxicity. Interestingly, tumor-infiltrating lymphocytes (TIL) isolated from regressing CTVT secrete high concentrations of IL-6 and antagonize the anti-LAK activity of tumor cell TGF-beta1. TIL also produce IL-6 during progression phase, but the concentration is too low to block the anti-LAK activity of TGF-beta1. There is probably a threshold concentration of IL-6 needed to reverse TGF-beta1-inhibited LAK activity. In addition, in the absence of TGF-beta1, IL-6 derived from TIL does not promote the activity of LAK. This new mechanism, in which TIL manufacture high concentrations of IL-6 to block tumor TGF-beta1 anti-LAK activity, has potential applications in cancer immunotherapy and tumor prognosis.     

Transforming growth factor-beta inhibits the in vitro generation of lymphokine-activated killer cells and cytotoxic T cells

Summary The effect(s) of purified transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) on the induction and function of lymphokine-activated killer (LAK) cells and cytotoxic T lymphocytes (CTL) was examined. The addition of TGF-beta, but not PDGF, to cultures containing fresh C57BL/6 mouse splenocytes or human peripheral blood lymphocytes plus recombinant interleukin-2 markedly inhibited the development of mouse and human LAK cell activity (measured after 3 days for cytotoxicity against cultured or fresh tumor targets in 4-h ⁵¹Cr release assays). The addition of TGF-beta, but not PDGF, to a one-way, C57BL/6 anti-DBA/2, mixed lymphocyte reaction effectively blocked the generation of allospecific CTL as well. However, TGF-beta did not inhibit the effector function of LAK cells or of allospecific CTL when added directly to the short-term cytolytic assay. A second form of homodimeric TGF-beta, type 2, was also found to be suppressive on the development of murine LAK cells and allospecific CTL. Collectively, these data demonstrate that the peptide TGF-beta is a potent inhibitor of LAK cell and CTL generation in vitro.     

Role of CD44 in lymphokine-activated killer cell-mediated killing of melanoma

In the current study, we examined the potential significance of CD44 expression on lymphokine-activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells, and NKT cells. Treatment of melanoma-bearing CD44 WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma-bearing CD44 KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2-treated CD44 KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2-treated CD44 WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred CD44 WT LAK cells were significantly more effective than CD44 KO LAK cells at controlling tumor growth in vivo. Next, the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44^lo) or elevated (CD44^hi) expression of CD44 are generated and that the CD44^hi cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44 KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.     

IL-4 regulates IL-2 induction of lymphokine-activated killer activity from human lymphocytes

IL-4 is a pluripotent lymphokine acting on various cell types. We investigated the role of human IL-4 on the generation of lymphokine-activated killer (LAK) activity. Human IL-4 alone did not induce LAK activity and inhibited IL-2 induction of LAK activity from unstimulated PBMC, peripheral blood null cells, spleen cells, and lymph node cells in a dose-dependent manner. IL-4 also inhibited several phenomena induced by IL-2 such as cell proliferation, augmentation of NK activity, increase of Leu-19+ cells, and expression of IL-2R(p55) on either CD3+ or Leu-19+ cells. IL-4, however, augmented cell proliferation with other T cell mitogens including PHA, Con A, PMA, or allo-MHC Ag with or without IL-2. In contrast to unstimulated cells, IL-4 alone induced marked cell proliferation and LAK activity as well as Leu-19+ cells from in vitro IL-2 preactivated PBMC or null cells, and did not inhibit IL-2 induced cell proliferation, LAK activity, Leu-19+ cells and IL-2R(p55) expression, but rather augmented them with low doses of IL-2. Although IL-4 alone induced LAK activity from peripheral blood of some patients previously given IL-2, IL-4 inhibited in vitro LAK generation with IL-2 from these cells in most cases. Therefore, IL-4 appears to directly inhibit the IL-2 activation pathway via IL-2R(p70) and prevent resting LAK precursors from proliferating and differentiating into final effector cells. However, once cells were sufficiently preactivated by IL-2, IL-4 induced LAK activity and did not inhibit IL-2 activation of these cells. These data suggest an immunoregulatory role of IL-4 on human null cells and T cells.     

Suppression of lymphokine-activated killer induction by neutrophils

Peripheral blood polymorphonuclear neutrophils (PMN) suppressed the induction of PBL lymphokine-activated killer (LAK) function by rIL-2 in vitro. The suppression depended on the concentration of PMN in the IL-2 culture, and required intact PMN. However, PMN did not require treatment with immunoregulators such as IL-2, LPS, or TNF to express the suppressive activity, and no direct contact with PBL was needed for the suppression. Addition of anti-TNF antibodies had no effect on the suppression, suggesting that no endogenous TNF in the culture was involved in the suppression. PMN did not inhibit LAK function by preventing utilization of IL-2 by PBL or by selective depletion of NKH-1+ cells which constitute the majority of LAK precursors in PBL. The suppression was reversed by superoxide dismutase but not by catalase, suggesting that superoxide anion, not hydrogen peroxide, was involved in the suppression. No other suppressive factor was detectable in PMN culture supernates. Our results of PMN regulating LAK induction in vitro suggest that PMN may have a role in determining the outcome of immunotherapy with IL-2 in vivo.     

Addition of interleukin-2in vitro augments detection of lymphokine-activated killer activity generatedin vivo

Summary Thein vivo administration of repetitive weekly cycles of interleukin-2 (IL-2) to patients with cancer enhances the ability of freshly obtained peripheral blood lymphocytes (PBL) to lyse both the natural-killer(NK)-susceptible K562 and the NK-resistant Daudi targets. Lysis of both targets is significantly augmented by inclusion of IL-2 in the medium during the cytotoxicity assay. This boost is much greater for cells obtained following thein vivo IL-2 therapy than for cells obtained prior to the initiation of therapy or for cells from healthy control donors. In addition to direct lytic activity, the PBL obtained followingin vivo IL-2 show a rapid increase in lymphokine-activated killer (LAK) activity with more prolongedin vitro IL-2 exposure, indicating that LAK effectors primedin vivo respond with secondary-like kinetics to subsequent IL-2in vitro. Lymphocytes from healthy control individuals, cultured in IL-2 under conditions attempting to simulate thein vivo IL-2 exposure, function similarly to PBL obtained from patients following IL-2, in that low-level LAK activity was significantly boosted by inclusion of IL-2 during the cytotoxic assay and the cells also responded with secondary-like kinetics to subsequent IL-2in vitro. The augmentation of the LAK effect was also dependent on the dose of IL-2 added during the 4-h⁵¹Cr-release cytotoxicity assay, with higher doses of IL-2 having a more pronounced effect. While continuous infusion of IL-2 induces a greater cytotoxic potential per milliliter of blood obtained from patients, the peak serum IL-2 levels attained are greater with bolus IL-2 infusions. These pharmacokinetic results, together with the IL-2 dose dependence of LAK activity generatedin vivo shown in this report, suggest that a combination of treatment with bolus IL-2 infusions superimposed on continuous IL-2 infusion may transiently expose IL-2 dependent LAK cells, activatedin vivo, to higher concentrations of IL-2, facilitating theirin vivo cytotoxic potential.This work was supported by NIH contract NO1 CM-47669-02, NIH grants CA-32685, RR-031086, NO1 CM-47669-03, and American Cancer Society grant CH-237     

Additive effects of antitumor drugs and lymphokine-activated killer cell cytotoxic activity in tumor cell killing determined by lactate-dehydrogenase-release assay

Summary The effect of pretreatment with antitumor drugs on lymphokine-activated killer (LAK) cell cytotoxic activity, determined by lactate-dehydrogenase(LDH)-release assay, was investigated. LAK cells were induced by incubating peripheral blood lymphocytes of healthy donors in medium containing interleukin-2 (IL-2) and monoclonal anti-CD3 antibody for 6–7 days. A human lung squamous carcinoma cell line, SQ-5, was used as an adherent target. After 24 h exposure of the target cells to cisplatin, doxorubicin, or mitomycin C, the drugs were washed off and LAK cells were added at an E/T ratio of 5. During further incubation for 48 h, LDH release from cisplatin- or doxorubicin-pretreated target cells was markedly higher than that from non-pretreated target cells. The combination of cisplatin and LAK cells has an additive cytotoxic effect and that of mitomycin C and LAK cells does not; there may also be an additive effect late in the toxicity mechanism between doxorubicin and LAK cells.     

Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils

Neutrophils play a central role in the innate immune response and a critical role in bacterial killing. Most studies of neutrophil function have been conducted under conditions of ambient oxygen, but inflamed sites where neutrophils operate may be extremely hypoxic. Previous studies indicate that neutrophils sense and respond to hypoxia via the ubiquitous prolyl hydroxylase/hypoxia-inducible factor pathway and that this can signal for enhanced survival. In the current study, human neutrophils were shown to upregulate hypoxia-inducible factor (HIF)-1α-dependent gene expression under hypoxic incubation conditions (3 kPa), with a consequent substantial delay in the onset of apoptosis. Despite this, polarization and chemotactic responsiveness to IL-8 and fMLP were entirely unaffected by hypoxia. Similarly, hypoxia did not diminish the ability of neutrophils to phagocytose serum-opsonized heat-killed streptococci. Of the secretory functions examined, IL-8 generation was preserved and elastase release was enhanced by hypoxia. Hypoxia did, however, cause a major reduction in respiratory burst activity induced both by the soluble agonist fMLP and by ingestion of opsonized zymosan, without affecting expression of the NADPH oxidase subunits. Critically, this reduction in respiratory burst activity under hypoxia was associated with a significant defect in the killing of Staphylococcus aureus. In contrast, killing of Escherichia coli, which is predominantly oxidase independent, was fully preserved under hypoxia. In conclusion, these studies suggest that although the NADPH oxidase-dependent bacterial killing mechanism may be compromised by hypoxia, neutrophils overall appear extremely well adapted to operate successfully under severely hypoxic conditions.     

Interleukin-6 enhances the induction of human lymphokine-activated killer cells

Summary Human peripheral blood mononuclear cells develop a powerful lytic capacity when cultured in vitro with interleukin-2 (IL-2), becoming lymphokine-activated killer cells (LAK cells). As part of an investigation into means of influencing this process, the effect of other cytokines has been examined. In this study we describe the ability of interleukin-6 (IL-6) to regulate the induction and function of human LAK cells. The results show that substitution of IL-6 for IL-2 did not lead to the development of functional LAK cells, nor was IL-6 able to alter the lytic capacity of established LAK cells. However, when IL-6 was included with IL-2 during the induction phase of the LAK cells, the resulting cells displayed considerably greater lytic activity than those prepared with IL-2 alone. This effect was IL-6 dose-related. These results indicate that LAK cell development may be positively regulated in vitro; the implications of this observation for the clinical usage of LAK cells are discussed.     

Bilirubin inhibits the chemotactic activity of human polymorphonuclear leukocytesin vitro

Using the migration of human polymorphonuclear leukocytes in agarosein vitro, it was established that bilirubin inhibits migration (chemotaxis) of these cells upon stimulation with both complement-derived (zymosan-activated serum) and bacteria-derived (abacterial filtrate ofE. coli) chemotactic agents.     

Ethidium bromide inhibits rat brain acetylcholinesterase activity in vitro

Ethidium bromide (EtBr), a fluorescent dark red compound and stain for double-stranded DNA and RNA was used to study acetylcholinesterase (AChE) activity in vitro together with kinetic parameters of this enzyme in the striatum (ST), hippocampus (HP), cerebral cortex (CC) and cerebellum (CB) of adult rats. AChE activity in vitro in the ST, HP, CC and CB was significantly reduced (p<0.05) in the presence of EtBr at concentrations of 0.00625, 0.0125, 0.025, 0.05 and 0.1 mM. For the analysis of the kinetic three concentrations of EtBr were tested (0.00625, 0.025 and 0.1 mM). An uncompetitive inhibition type was observed in the ST, HP and CC, whereas in the CB the inhibition type was mixed. These data indicate that EtBr should be considered a strong inhibitor of AChE activity demonstrating that there is an interaction between this compound and the cholinergic system.     

Prerequisite for the induction of lymphokine-activated killer cells from T lymphocytes

Human blood mononuclear cells were separated into Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells by means of a combination of the Percoll gradient method and C-mediated cytolysis using mAb. When purified Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells were cultured with rIL 2 (500 U/ml) for 6 days in a medium supplemented with 10% FCS, Leu-11+7-NK cells responded at the maximum level and Leu-11-7+ cells responded moderately as shown by both cell-proliferation response and cytotoxic activity generated. On the other hand, Leu-11-7-T cells did not respond at all to rIL-2. However, when Leu-11-7-T cells were cultured with rIL-2 in a medium supplemented with 10% autologous serum, they showed considerable responsiveness to rIL-2. In addition, much greater response to Leu-11-7-T cells were produced by the addition of monocytes. Monocyte cytokines, neither IL 1, IFN-gamma, TNF, nor their combination were able to substitute for monocytes in the induction culture. In contrast, the response level of Leu-11+7- NK cells remained unchanged irrespective of supplementation with autologous serum to medium or the addition of monocytes to the culture. These results indicated that culture conditions in the experiments significantly affected the results as to determination of lymphokine-activated killer cell precursors, especially the result pertaining to the conversion of T lymphocytes to lymphokine-activated killer cells. Under appropriate conditions, not only NK cells but also T cells are important precursors of lymphokine-activated killer cells.     

IL-4 regulation of murine lymphokine-activated killer activity in vitro. Effects on the IL-2-induced expansion, cytotoxicity, and phenotype of lymphokine-activated killer effectors

The in vitro incubation of B6 splenocytes with purified, mouse rIL-4 for 4 to 5 days was sufficient to generate lymphokine-activated killer (LAK) activity. In addition, rIL-4 augmented LAK cytotoxic activity when combined with rIL-2, as measured in a 4 h 51Cr-release assay against fresh, syngeneic MCA-sarcoma (MCA-102 and MCA-105) cells. Interestingly, this augmentation was not observed against the cultured YAC-1 target. LAK generation and augmentation of cytotoxicity by rIL-4 was species-specific, because human rIL-4 (up to 20,000 U/ml) failed to elicit these effects in the mouse splenocyte cultures. When 5-day B6 LAK cells (splenocytes incubated in rIL-2 at 1000 U/ml for 5 days) were split and recultured in the combination of rIL-2 plus rIL-4 for 4 additional days at least a twofold greater expansion in cell number resulted compared to similar cells cultured in either rIL-2 or rIL-4 alone. Moreover, LAK cells expanded in rIL-2 plus rIL-4 exhibited substantial increases in in vitro cytolytic activity (on a per cell basis) against MCA-102 and MCA-105 sarcoma cells, but not against YAC-1 targets. FACS analysis or negative selection using Lyt-2 or NK-1.1 mAb plus C revealed no differences in effector phenotype(s) of LAK cells expanded in rIL-2 alone compared to rIL-2 plus rIL-4 to account for the differences observed in both expansion and cytolytic activity by rIL-4. The majority of cells was Thy-1+, Lyt-2+, T3+, and ASGM-1+. However, a marked increase in the granule-associated serine esterase, BLT-E, was found only in LAK cells expanded in the combination of both lymphokines. Collectively, these studies show that rIL-4 has potent regulatory activities on splenic LAK generation, expansion, and cytotoxic function in the mouse.     

IL-4 inhibits macrophage-mediated killing of Plasmodium falciparum in vitro. A possible parasite-immune evasion mechanism.

Although a number of mechanisms have been put forward for immunity to malaria, their importance remains to be clarified. One of the important findings is that nonactivated monocytes and macrophages showed marked antiplasmodial activity in vitro. Recently we postulated that parasites may induce host factors that may depress the natural antiplasmodial activity of monocytes. In this investigation we identify IL-4 as a lymphokine that could function in this capacity. Human monocytes and macrophages in the absence of antiplasmodial antibody showed substantial killing of the asexual erythrocytic forms of Plasmodium falciparum as determined by a radiometric assay. Suppression of this killing was seen if the mononuclear phagocytes were pretreated with human rIL-4 at concentrations of 10 to 250 U with optimum activity between 100 and 250 U/2 x 10(5) cells. Cells from some individuals were rendered completely inactive by the IL-4 treatment. In contrast, IL-4 did not affect the neutrophil-mediated anti-P. falciparum activity. Our work identifies a potentially important parasite immune evasion mechanism involving IL-4 suppression of macrophage antiparasite activity.     

Ovine myometrial cytosol inhibits phospholipase A activity, in vitro

Myometrium obtained from pregnant ewes (30-80 days gestation) contains a factor which inhibits phospholipase A2 (PLA2) activity. The activity of this moiety was assessed using an in vitro porcine pancreatic PLA2 assay system. Inhibitory activity was associated with a 35-45000 dalton molecular weight fraction, heat-labile, sensitive to protease degradation and did not partition into organic solvents. These data are indicative that PLA2-inhibitory activity resides in a protein moiety. Dixon-plot analysis of myometrial-inhibitory activity was indicative that the inhibition of PLA2 activity was of a non-competitive nature (Ki = 4.1 +/- 0.7 micrograms/ml, ca 118 nmol/l). Myometrial phospholipase-inhibitory protein(s) may be involved in the suppression of eicosanoid biosynthesis by the uterine tissues throughout gestation thus inhibiting uterine contractile activity.     

Ovine myometrial cytosol inhibits phospholipase A activity, in vitro

Myometrium obtained from pregnant ewes (30–80 days gestation) contains a factor which inhibits phospholipase A₂ (PLA₂) activity. The activity of this moiety was assessed using an in vitro porcine pancreatic PLA₂ assay system. Inhibitory activity was associated with a 35–45000 dalton molecular weight fraction, heat-labile, sensitive to protease degradation and did not partition into organic solvents. These data are indicative that PLA₂-inhibitory activity resides in a protein moiety. Dixon-plot analysis of myometrial-inhibitory activity was indicative that the inhibition of PLA₂ activity was of a non-competitive nature (K_i = 4.1 ± 0.7μg/ml, ca 118 nmol/1). Myometrial phospholipase-inhibitory protein(s) may be involved in the suppression of eicosanoid biosynthesis by the uterine tissues throughout gestation thus inhibiting uterine contractile activity.     

Zinc inhibits calcineurin activity in vitro by competing with nickel

Calcineurin (CN) is a Ca(2+)/calmodulin (CaM)-dependent protein serine/threonine phosphatase that contains Zn(2+) in its catalytic domain and can be stimulated by divalent ions such as Mn(2+) and Ni(2+). In this study, the role of exogenous Zn(2+) in the regulation of CN activity and its relevance to the role of Ni(2+) was investigated. Zn(2+) at a concentration range of 10nM-10 micro M inhibited Ni(2+)-stimulated CN-activity in vitro in a dose-dependent manner and approximately 50% inhibition was attained with 0.25 micro M Zn(2+). Kinetic analysis showed that Zn(2+) inhibited the activity of CN by competing with Ni(2+). Interaction of CN and CaM was not inhibited with Zn(2+) at 10 micro M. Zn(2+) never affected the activity of cAMP phosphodiesterase 1 or myosin light-chain kinase (CaM-dependent enzymes) and rather activated alkaline phosphatase. The present results indicate that Zn(2+) should be a potent inhibitor for CN activity although this ion is essential for CN.     

Fungicidal activity of Candida albicans-induced murine lymphokine-activated killer cells against C. albicans hyphae in vitro.

Multiple intraperitoneal injections of inactivated Candida albicans cells resulted in the generation of cytotoxic peritoneal cells with phenotypical and functional properties similar to in vitro-generated lymphokine-activated killer (LAK) cells. Using an in vitro [3H]glucose uptake assay, C. albicans-induced LAK-like (CA-LAK) cells exhibited high levels of anti-hyphal activity, the effects being effector to target cell (E:T) ratio- and time-dependent. Maximal levels of anti-C. albicans activity (approximately 60%) were observed after 4 h and at E:T greater than or equal to 300:1. Similar patterns of anti-C. albicans activity were exerted by in vivo-activated natural killer (NK) cells, in vitro interleukin-2- (IL-2) generated LAK cells and polymorphonuclear cells. The anti-hyphal activity of CA-LAK cells was enriched by separation on a Percoll gradient, F2 and F3 fractions retaining most of the activity. Experiments using immunodepressed animals demonstrated that the in vivo lethality of the C. albicans hyphal form is significantly affected by in vitro pre-exposure to CA-LAK cells. While control mice receiving C. albicans alone had a median survival time of 2 d, mice receiving C. albicans pre-exposed to CA-LAK cells (E:T = 300:1) had a median survival time of 15 d. Overall, the susceptibility of the C. albicans hyphal form to CA-LAK cells suggests that C. albicans-induced effectors might play a significant role as a second-line defence mechanism against the C. albicans hyphal form.     

Withaferin a inhibits the proteasome activity in mesothelioma in vitro and in vivo

The medicinal plant Withania somnifera has been used for over centuries in Indian Ayurvedic Medicine to treat a wide spectrum of disorders. Withaferin A (WA), a bioactive compound that is isolated from this plant, has anti-inflammatory, immuno-modulatory, anti-angiogenic, and anti-cancer properties. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of WA and the molecular mechanisms involved. WA inhibited growth of the murine as well as patient-derived MPM cells in part by decreasing the chymotryptic activity of the proteasome that resulted in increased levels of ubiquitinated proteins and pro-apoptotic proteasome target proteins (p21, Bax, IκBα). WA suppression of MPM growth also involved elevated apoptosis as evidenced by activation of pro-apoptotic p38 stress activated protein kinase (SAPK) and caspase-3, elevated levels of pro-apoptotic Bax protein and cleavage of poly-(ADP-ribose)-polymerase (PARP). Our studies including gene-array based analyses further revealed that WA suppressed a number of cell growth and metastasis-promoting genes including c-myc. WA treatments also stimulated expression of the cell cycle and apoptosis regulatory protein (CARP)-1/CCAR1, a novel transducer of cell growth signaling. Knock-down of CARP-1, on the other hand, interfered with MPM growth inhibitory effects of WA. Intra-peritoneal administration of 5 mg/kg WA daily inhibited growth of murine MPM cell-derived tumors in vivo in part by inhibiting proteasome activity and stimulating apoptosis. Together our in vitro and in vivo studies suggest that WA suppresses MPM growth by targeting multiple pathways that include blockage of proteasome activity and stimulation of apoptosis, and thus holds promise as an anti-MPM agent.