Role of Glutamine-Glutamate/GABA cycle and potential target GLUD2 in alleviation of rheumatoid arthritis by Tripterygium hypoglaucum (levl.) Hutch based on metabolomics and molecular pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium hypoglaucum (levl.) Hutch (Celastraceae) (THH), as a traditional Chinese medicine, was clinically exploited to treat rheumatoid arthritis (RA), yet the underlying mechanism for this effect remains largely unclear. AIM OF THE STUDY: This study aimed to examine the beneficial effects of THH extract (THHE) against rheumatoid arthritis and its regulating role in differential metabolic pathways and potential targets. MATERIALS AND METHODS: In the present study, the Lewis rat model with rheumatoid arthritis induced by adjuvant was established and administrated THHE for 14 days. Untargeted/targeted metabolomics analysis were used for determining the changes of differential metabolites, and molecular docking method was further developed to verify predicted targets and investigate the therapeutic mechanism of THH extract on RA. RESULTS: The results showed that THH extract could obviously improve body weight, significantly decrease the joint index and swelling degree of the RA model rats to reduce damage in the joint. Meanwhile, THHE could significantly suppress the releases of IL-1α, IL-1ß and MMP3, but also the expression levels of IL-4 and IL-10 and percentage of Treg cells were significantly improved, a result consistent with inhibitory effects on multiplication of macrophages, inflammatory cell infiltration and fibro genesis in the synovial tissues. Furthermore, 516 differential metabolites were identified by serum metabolic profiles analysis, including vitamin, organic acids and derivatives, lipids and lipid-like molecule, hormone, amino acids and derivatives, and other compounds, which targeted 47 metabolic pathways highly correlated with immunosuppression, such as citrate cycle (TCA cycle), sphingolipid metabolism, urea cycle, arachidonic acid metabolism and amino acid metabolism (such as Glutamine-Glutamate metabolism). Targeted metabolomics was used to verify that L-Glutamate and Glutamine changed significantly after THHE administration for 14 days, and many active ingredients of THHE could be successfully docked with glutamate dehydrogenase 2 (GLUD2). CONCLUSION: This study indicated that the Glutamine-Glutamate/GABA cycle played essential regulation roles in protective effect of THHE on rat RA following adjuvant-induced damage, and GLUD2 as an attractive target also provides great potential for development of therapy agents for rheumatoid arthritis and autoimmune diseases with less unfavorable tolerability profile.

The CD4-CD8- MAIT cell subpopulation is a functionally distinct subset developmentally related to the main CD8+ MAIT cell pool.

Mucosa-associated invariant T (MAIT) cells are unconventional innate-like T cells that recognize microbial riboflavin metabolites presented by the MHC class I-like protein MR1. Human MAIT cells predominantly express the CD8α coreceptor (CD8+), with a smaller subset lacking both CD4 and CD8 (double-negative, DN). However, it is unclear if these two MAIT cell subpopulations distinguished by CD8α represent functionally distinct subsets. Here, we show that the two MAIT cell subsets express divergent transcriptional programs and distinct patterns of classic T cell transcription factors. Furthermore, CD8+ MAIT cells have higher levels of receptors for IL-12 and IL-18, as well as of the activating receptors CD2, CD9, and NKG2D, and display superior functionality following stimulation with riboflavin-autotrophic as well as riboflavin-auxotrophic bacterial strains. DN MAIT cells display higher RORγt/T-bet ratio, and express less IFN-γ and more IL-17. Furthermore, the DN subset displays enrichment of an apoptosis gene signature and higher propensity for activation-induced apoptosis. During development in human fetal tissues, DN MAIT cells are more mature and accumulate over gestational time with reciprocal contraction of the CD8+ subset. Analysis of the T cell receptor repertoire reveals higher diversity in CD8+ MAIT cells than in DN MAIT cells. Finally, chronic T cell receptor stimulation of CD8+ MAIT cells in an in vitro culture system supports the accumulation and maintenance of the DN subpopulation. These findings define human CD8+ and DN MAIT cells as functionally distinct subsets and indicate a derivative developmental relationship.

[Efficacy of acupoint catgut embedding combined with ginger-partitioned moxibustion on chronic fatigue syndrome of spleen-kidney yang deficiency syndrome and its effects on T lymphocyte subsets and activity of NK cell].

OBJECTIVE: To observe the efficacy differences between acupoint catgut embedding combined with ginger-partitioned moxibustion and regular acupuncture on chronic fatigue syndrome (CFS) of spleen-kidney yang deficiency syndrome, and to explore its effects on T lymphocyte subsets and activity of NK cell. METHODS: A total of 60 patients with CFS of spleen-kidney yang deficiency syndrome were randomly divided into a catgut embedding combined with ginger-partitioned moxibustion (CECGP) group and a regular acupuncture group, 30 cases in each one. The patients in the CECGP group were treated with acupoint catgut embedding combined with ginger-partitioned moxibustion; the acupoint catgut embedding was applied at Guanyuan (CV 4), Shenshu (BL 23), Pishu (BL 20), Zusanli (ST 36), Qihai (CV 6), once a week, while the ginger-partitioned moxibustion was applied at Guanyuan (CV 4), Qihai (CV 6) and Zusanli (ST 36), once every three days for consecutive one month. The patients in the regular acupuncture group were treated with regular acupuncture at Guanyuan (CV 4), Shenshu (BL 23), Pishu (BL 20), Zusanli (ST 36), Qihai (CV 6), once a day, 6 treatments per week (one day for rest) for consecutive one month. The clinical symptom scores, fatigue scale-14 (FS-14), fatigue assessment instrument (FAI), laboratory test results and total effective rate were compared between the two groups before and after treatment. RESULTS: (1) After treatment, the clinical symptom scores, FS-14 and FAI were reduced in the two groups (all P<0.05); after treatment, the clinical symptom scores, FS-14 and FAI in the CECGP group were significantly lower than those in the regular acupuncture group (all P<0.05). (2) After treatment, the CD4+/CD8+, natural killer cell% (NK%), CD3+%, CD% were all increased in the two groups (all +4 P<0.05); the CD4+/CD8+, CD3+%, CD% in the CECGP group were significantly higher than those in the regular acupuncture group (all P<0.05). (3) After treatment, the total effective rate was 96.7% (29/30) in the CECGP group, which was similar to 93.3% (28/30) in the regular acupuncture group (P>0.05). CONCLUSIONS: The acupoint catgut embedding combined with ginger-partitioned moxibustion, which could effectively relieve the symptoms, regulate T lymphocyte subsets and the activity of NK cell, is an effective method for CFS of spleen-kidney yang deficiency syndrome.

Epigenetic landscapes reveal transcription factors that regulate CD8+ T cell differentiation.

Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8+ T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8+ T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8+ T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8+ T cell differentiation.

Analysis of the roles of dietary protein and calcium in fluoride-induced changes in T-lymphocyte subsets in rat.

The roles of dietary protein (Pr) and calcium (Ca) levels on the changes in T-lymphocyte subsets induced by excessive fluoride (F) intake were assessed using rats that were malnourished for 120 days as a model. The CD4+ and CD8+ T-lymphocytes in the spleen tissue were determined by flow cytometry and immunofluorescence assay. The percentages of CD3+ , CD4+ , and CD8+ T-lymphocytes were reduced in the spleen of rats exposed to excessive F, and malnutrition aggravated these changes in the T-lymphocytes. In addition, the mRNA expression levels of IL-1ß, IL-2, IL-6, TNF-α, and IFN-γ in the spleen were downregulated significantly. We also reported herein the increased apoptosis ratio following caspase-9 and caspase-3 upregulation in the spleen of rats exposed to excessive amount of F. Light and transmisison electron microscopy revealed the irregularly arranged lymphocytes, few lymph nodules and the apoptotic characteristic of lymphocytes, which are caused by the increased expression of caspase. In addition, Pr and Ca supplementation reversed the morphologic and T-lymphocytic changes in spleen under malnutrition. Taken together, our results revealed an endogenous caspase-mediated mechanism of regulating the apoptosis of the T-lymphocyte subsets, as well as the immune-related cytokine secretion, which reduces the immune function in F-induced rats. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1587-1595, 2017.

Impact of prebiotic supplementation on T-cell subsets and their related cytokines, anthropometric features and blood pressure in patients with type 2 diabetes mellitus: A randomized placebo-controlled Trial.

BACKGROUNDS AND AIMS: Type 2 diabetic mellitus (T2DM) asone of the main causes of morbidity and mortality is associated with immune system disturbances and metabolic abnormalities. In the current study we aimed to evaluate the effects of oligofructose-enriched inulin on T-cell subsets and their related cytokines, anthropometric and metabolic parameters in patients with T2DM. METHODS: Forty-six diabetic females patients were randomly allocated into intervention (n=27) and control (n=22) groups. Subjects in the intervention group received a daily dose of 10g of oligofructose-enriched inulin and subjects in control group received a placebo for two months. Anthropometric variables, metabolic parameters including fasting serum glucose (FSG), hemoglobin A1c (HbA1c), lipid profile and blood pressure were measured at the beginning and after two months. Immune markers also included serum interleukin (IL)-4, IL-12 and interferon (IFN)-γ concentrations were assessed and CD3(+), CD4(+), CD8(+) and CD11b(+)T-cell counts were determined by flow cytometry at baseline and end of the trial. RESULTS: After two months intervention, significant improvements in anthropometric variables, blood pressure and serum lipids occurred in prebiotic-treated group (P<0.001). Serum IL-4, IL-12 and IFN-γ concentrationsalso significantly decreased in intervention group (P<0.001). No significant changes in CD3(+), CD4(+), CD8(+) and CD11b(+) T-cell counts were observed in treatment groups after intervention. CONCLUSION: The present study showed several beneficial effects of oligofructose-enriched inulin on the improvement of the glycemic status, lipid profile, and immune markers in patients with T2DM. Further studies are needed to confirming our findings and to better clarify the underlying mechanisms.

IL-1 receptor antagonist-deficient mice develop autoimmune arthritis due to intrinsic activation of IL-17-producing CCR2(+)Vγ6(+)γδ T cells.

Interleukin-17 (IL-17)-producing γδ T (γδ17) cells have been implicated in inflammatory diseases, but the underlying pathogenic mechanisms remain unclear. Here, we show that both CD4(+) and γδ17 cells are required for the development of autoimmune arthritis in IL-1 receptor antagonist (IL-1Ra)-deficient mice. Specifically, activated CD4(+) T cells direct γδ T-cell infiltration by inducing CCL2 expression in joints. Furthermore, IL-17 reporter mice reveal that the Vγ6(+) subset of CCR2(+) γδ T cells preferentially produces IL-17 in inflamed joints. Importantly, because IL-1Ra normally suppresses IL-1R expression on γδ T cells, IL-1Ra-deficient mice exhibit elevated IL-1R expression on Vγ6(+) cells, which play a critical role in inducing them to produce IL-17. Our findings demonstrate a pathogenic mechanism in which adaptive and innate immunity induce an autoimmune disease in a coordinated manner.

Inflammation, immunity, and hypertensive end-organ damage.

For >50 years, it has been recognized that immunity contributes to hypertension. Recent data have defined an important role of T cells and various T cell-derived cytokines in several models of experimental hypertension. These studies have shown that stimuli like angiotensin II, deoxycorticosterone acetate-salt, and excessive catecholamines lead to formation of effector like T cells that infiltrate the kidney and perivascular regions of both large arteries and arterioles. There is also accumulation of monocyte/macrophages in these regions. Cytokines released from these cells, including interleukin-17, interferon-γ, tumor necrosis factorα, and interleukin-6 promote both renal and vascular dysfunction and damage, leading to enhanced sodium retention and increased systemic vascular resistance. The renal effects of these cytokines remain to be fully defined, but include enhanced formation of angiotensinogen, increased sodium reabsorption, and increased renal fibrosis. Recent experiments have defined a link between oxidative stress and immune activation in hypertension. These have shown that hypertension is associated with formation of reactive oxygen species in dendritic cells that lead to formation of gamma ketoaldehydes, or isoketals. These rapidly adduct to protein lysines and are presented by dendritic cells as neoantigens that activate T cells and promote hypertension. Thus, cells of both the innate and adaptive immune system contribute to end-organ damage and dysfunction in hypertension. Therapeutic interventions to reduce activation of these cells may prove beneficial in reducing end-organ damage and preventing consequences of hypertension, including myocardial infarction, heart failure, renal failure, and stroke.

Metabolic and immune response of young turkeys originating from parent flocks fed diets with inorganic or organic selenium.

The aim of this study was to verify the hypothesis that the health and growth of turkey poults may be improved by supplementing diets fed to parent flocks with available selenium. Experimental poults originated from parent flocks fed with diets containing 0.3 mg/kg inorganic selenium (control group Se(M)) and organic selenium (experimental group Se(O)). Egg yolk selenium content was comparable in both flocks (0.72 and 0.70 mg/kg d.m., respectively). Eggs from the Se(O) flock had a significantly lower content of thiobarbituric acid reactive substances - TBARS (31.13 vs. 53.10 nmol/g, p > 0.001). Se(O) group poults were characterized by higher activity of glutathione peroxidase (7.54 vs. 5.92 U/mL, P = 0.001) and superoxide dismutase (89.30 vs. 79.23 U/mL, P = 0.026). The thigh muscles of Se(O) group birds had significantly higher selenium concentrations (0.74 vs. 0.57, p = 0.045) and a significantly lower TBARS content (38.42 vs. 65.01, p = 0.001). No differences were found between the groups with respect to the content of total protein, albumins and uric acid, and the activites of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (DLH) in day-old poults. On day 28, groups Se(O) and Se(M) differed in the activity of ALT (20.50 vs. 26.33, p = 0.05) and SOD (87.29 vs. 100.02 U/mL, p = 0.035). There were no differences between the groups regarding the percentages of T lymphocyte subpopulations CD4+, CD8+, CD4+CD8+ and B lymphocyte subpopulations (IgM+) at 1 and 28 days of age. Over the experimental period, mortality rates were similar in both groups (7.32 and 8.87%), and so were the final body weights of birds (1108 vs. 1135 g). The results of the study show that the dietary supplementation of organic selenium in turkey parent flocks reduces the rate of oxidation processes in the egg and in the tissues of newly-hatched poults, yet it has no effect on the analyzed parameters of cell-mediated immunity and the growth performance of birds during the first five weeks of their life.

Stepwise development of MAIT cells in mouse and human.

Mucosal-associated invariant T (MAIT) cells display two evolutionarily conserved features: an invariant T cell receptor (TCR)alpha (iTCRalpha) chain and restriction by the nonpolymorphic class Ib major histocompatibility complex (MHC) molecule, MHC-related molecule 1 (MR1). MR1 expression on thymus epithelial cells is not necessary for MAIT cell development but their accumulation in the gut requires MR1 expressing B cells and commensal flora. MAIT cell development is poorly known, as these cells have not been found in the thymus so far. Herein, complementary human and mouse experiments using an anti-humanValpha7.2 antibody and MAIT cell-specific iTCRalpha and TCRbeta transgenic mice in different genetic backgrounds show that MAIT cell development is a stepwise process, with an intra-thymic selection followed by peripheral expansion. Mouse MAIT cells are selected in an MR1-dependent manner both in fetal thymic organ culture and in double iTCRalpha and TCRbeta transgenic RAG knockout mice. In the latter mice, MAIT cells do not expand in the periphery unless B cells are added back by adoptive transfer, showing that B cells are not required for the initial thymic selection step but for the peripheral accumulation. In humans, contrary to natural killer T (NKT) cells, MAIT cells display a naïve phenotype in the thymus as well as in cord blood where they are in low numbers. After birth, MAIT cells acquire a memory phenotype and expand dramatically, up to 1%-4% of blood T cells. Finally, in contrast with NKT cells, human MAIT cell development is independent of the molecular adaptor SAP. Interestingly, mouse MAIT cells display a naïve phenotype and do not express the ZBTB16 transcription factor, which, in contrast, is expressed by NKT cells and the memory human MAIT cells found in the periphery after birth. In conclusion, MAIT cells are selected by MR1 in the thymus on a non-B non-T hematopoietic cell, and acquire a memory phenotype and expand in the periphery in a process dependent both upon B cells and the bacterial flora. Thus, their development follows a unique pattern at the crossroad of NKT and gammadelta T cells.

Clinical holistic medicine: a pilot study on HIV and quality of life and a suggested cure for HIV and AIDS.

This study was undertaken to examine the association between the immunological impact of HIV (measured by CD4 count) and global self-assessed quality of life (QOL) (measured with QOL1) for people suffering from HIV, to see if the connection was large and statistically strong enough to support our hypothesis of a strong QOL-immunological connection through the nonspecific, nonreceptor-mediated immune system, and thus to give a rationale for a holistic cure for HIV. This cross-sectional population study in Uganda included 20 HIV infected persons with no symptoms of AIDS and a CD4 count above 200 mill./liter. The main outcome measures were CD4 count, global QOL measured with the validated questionnaire QOL1, translated to Luganda and translated back to English. We found a large, clinically significant correlation between the number of T-helper cells (CD4) and global self-assessed quality of life (QOL1) (r = 0.57, p = 0.021), when controlled for age, gender, and years of infection. Together with other studies and holistic medicine theory, the results have given rationale for a holistic cure for HIV. We suggest, based on our findings and theoretical considerations, that HIV patients who improve their global QOL, also will improve their CD4 counts. Using the technique of holistic medicine based on the life mission theory and the holistic process theory of healing, we hypothesize that the improvement of QOL can have sufficient biological effect on the CD4, which could avoid or postpone the development of AIDS. A holistic HIV/AIDS cure improving the QOL draws on hidden resources in the person and is thus affordable for everybody. Improving global QOL also means a higher consciousness and a more ethical attitude, making it more difficult for the HIV-infected person to pass on the infection.

Enzymatically quiescent heparanase augments T cell interactions with VCAM-1 and extracellular matrix components under versatile dynamic contexts.

During their migration into inflammatory sites, immune cells, such as T cells, secrete extracellular matrix (ECM)-degrading enzymes, such as heparanase, which, under mildly acidic conditions, degrade heparan sulfate proteoglycans (HSPG). We have previously shown that at pH 7.2, human placental heparanase loses its enzymatic activity, while retaining its ability to bind HSPG and promote T cell adhesion to unfractionated ECM. We now demonstrate that the 65-kDa recombinant human heparanase, which is devoid of enzymatic activity, but can still bind HSPG, captures T cells under shear flow conditions and mediates their rolling and arrest, in the absence or presence of stromal cell-derived factor 1 alpha (SDF-1 alpha; CXCL12), in an alpha(4)beta(1)-VCAM-1-dependent manner. Furthermore, heparanase binds to and induces T cell adhesion to key ECM components, like fibronectin and hyaluronic acid, in beta(1) integrin- and CD44-specific manners, respectively, via the activation of the protein kinase C and phosphatidylinositol 3-kinase intracellular signaling machineries. Although the nature of the putative T cell heparanase-binding moiety is unknown, it appears that heparanase exerts its proadhesive activity by interacting with the T cells' surface HSPG, because pretreatment of the cells with heparinase abolished their subsequent response to heparanase. Also, heparanase augmented the SDF-1 alpha-triggered phosphorylation of Pyk-2 and extracellular signal-regulated kinase-2 implicated in integrin functioning. Moreover, heparanase, which had no chemotactic effect on T cells on its own, augmented the SDF-1 alpha-induced T cell chemotaxis across fibronectin. These findings add another dimension to the known versatility of heparanase as a key regulator of T cell activities during inflammation, both in the context of the vasculature and at extravascular sites.

In vitro induction of anti-type 2 T cells by glycyrrhizin.

Glycyrrhizin (GR), an active component of licorice roots, has been previously shown to induce the generation of anti-type 2 T cells (anti-BI2T cells), which are able to counteract the activity of burn-induced CD8+ type 2 T cells (BI2T cells), in thermally injured and normal mice. In the present study, anti-BI2T cells were generated in vitro in cultures of spleen cells stimulated with GR. Anti-BI2T cells were induced in vitro when splenic mononuclear cells (SMNC) were stimulated in vitro for 24 to 72 h with 0.1-10 micrograms/ml of GR. Anti-BI2T cell activity was detected when suppressor macrophages (M phi) were depleted from SMNC after stimulation with GR. However, the GR-stimulated generation of anti-BI2T cells required the participation of M phi or their sonicated fractions. Anti-BI2T cells induced in vitro by GR were identified as Vicia villosa lectin adherent IFN7 producing-CD4+ T cells lacking the ability to produce IL-2, IL-4 or IL-10. These results indicate that anti-BI2T cells are generated by GR in vitro. M phi are not only necessary for the generation of anti-BI2T cells induced by GR but also inhibit their activity.

Iron chelation suppresses mononuclear cell activation, modifies lymphocyte migration patterns, and prolongs rat cardiac allograft survival in rats.

Iron influences host immunity, in part by affecting the function and migration patterns of T cell subpopulations. Removal of iron stores from the body by chelation decreases proliferation and differentiation of T cells, as shown in models of autoimmunity and pancreatic islet transplantation. We have examined the influence of iron chelation on rejection of vascularized heart allografts in rats. Two protocols were investigated: "treated" recipients received desferrithiocin (30 mg/kg/day) orally for 10 days beginning the day of transplantation; "pretreated" hosts received a similar dose of the drug for 10 days before engraftment. Graft survival increased from about 7 days in untreated animals to 14-16 days in both treatment groups (P < 0.001). Histological and immunoperoxidase studies of allografts at day 7 showed that iron chelation resulted in only a mild reduction in cell infiltration, but in a marked decrease in graft edema and interstitial hemorrhage and essentially complete suppression of mononuclear cell activation and cytokine production. Chelation therapy was also found to inhibit profoundly cytokine (TNF-alpha) production in rats treated with LPS, consistent with the effects observed in situ in the allografts. In vitro studies showed that pretreatment significantly inhibited the mixed lymphocyte reaction. Chelation also influenced migration of T lymphocyte subsets: treatment stimulated migration of CD4+ lymphocytes to peripheral lymph nodes; pretreatment strikingly and selectively increased CD8+ cell migration into parathymic lymph nodes draining the graft, with the opposite effect on nondraining node groups. We conclude that treatment with iron-chelating agents has several effects on host alloresponsiveness in a rat heart graft model secondary to inhibition of immune activation; these include prolongation of graft survival, inhibition of the mixed lymphocyte reaction (pretreatment), marked depression of cytokine production, and alteration in recirculation patterns of lymphocyte subpopulations.

Immunomodulatory effects of nitrosoureas on the phenotype and functions of T cells in the thymus and periphery.

We have recently demonstrated that nitrosoureas such as 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and chlorozotocin (CLZ) can cure almost 100% of mice bearing LSA tumor, syngeneic to C57BL/6 mice. In contrast, similar or higher doses of streptozotocin (STZ) completely failed to cure LSA-bearing mice. Further studies revealed that the efficacy of nitrosoureas may depend on their immunomodulating properties. In the current study, therefore, we investigated the effect of these nitrosoureas on the immune system of normal mice. Treatment of C57BL/6 mice with 5 intraperitoneal injections of 20 mg/kg body weight of BCNU or CLZ caused an increase in the percentage of CD4(-)CD8- T cells and a decrease in the percentage of CD4(+)CD8+ T cells in the thymus. In addition, such treatment also caused an increase in the percentage of CD4+ T cells without significantly affecting the CD8+ T cells in the thymus. However, when total cellularity of the thymus was studied, BCNU and CLZ were found to decrease the total number of CD4(+)CD8+ T cells without significantly affecting the other subsets. In contrast, similar or higher (100 mg/kg body weight) doses of STZ had no significant effect on the total number and percentages of T cell subsets in the thymus. Also BCNU and CLZ but not STZ treatment caused a 50% decrease in the total number of CD4+ and CD8+ T cells in the spleen. When T cells in the spleens of nitrosourea-treated mice were functionally analysed, it was observed that BCNU and CLZ caused a dramatic decrease in the T cell responsiveness to ConA, anti-CD3 and phorbol myristate acetate plus calcium ionophore stimulation. In contrast, STZ treatment failed to significantly inhibit the T cell responsiveness to these activation signals. Using the accessory cell-dependent and -independent assays, BCNU and CLZ were found to suppress the functions of both T cells and macrophages. Also, addition of growth factors such as IL-1, IL-2, IL-4 and IL-6 failed to reconstitute the defective responsiveness of BCNU- and CLZ-treated T cells and macrophages. Together our data suggest that nitrosoureas have varying immunomodulating properties and this may in turn determine their efficacy in the treatment of cancer.

A Viscum album oligosaccharide activating human natural cytotoxicity is an interferon gamma inducer.

Commercial Viscum album extract Helixor-M contains a dialysable oligosaccharide (HM-BP) that activates natural killer (NK) cytotoxicity against K562 tumour cells when preincubated with human peripheral blood mononuclear cells (PBMC) for 72 h. The activated effector cells were exclusively found in the monocyte/macrophage subpopulation. However, when peripheral non-adherent cells (PNAC) were preincubated with HM-BP for 72 h the NK cytotoxicity of CD56+CD3- NK cells was activated. This discrepancy was found to be due to the release of prostaglandin E2 from activated monocytes/macrophages, which blocked activation of the cytotoxicity of NK cells. Analysis of the supernatant culture medium after 72 h preincubation demonstrated that HM-BP induced release of interferon gamma (IFN gamma) from T cells (preferentially from CD3+CD4+ cells) and of tumour necrosis factor alpha (TNF alpha) from monocytes/macrophages. Release of IFN gamma was the crucial step for activation of NK cytotoxicity since enhancement of NK cytotoxicity during pretreatment of PBMC or PNAC with HM-BP was completely blocked in the presence of anti-IFN gamma antibodies. Anti-interleukin-2, anti-TNF alpha or anti-IFN alpha antibodies had no effect on the HM-BP-induced enhancement of NK cytotoxicity. The activation of the NK cytotoxicity of nonadherent cells by interleukin-2 treatment was found to be synergistic to the enhancement of NK cytotoxicity by treatment with HM-BP.