The effects of alphaGalCer-induced TCRValpha24 Vbeta11(+) natural killer T cells on NK cell cytotoxicity in umbilical cord blood

Purpose The first objective of this study was to investigate in vitro effects of -galactosylceramide (GalCer) on the proliferation of umbilical cord blood (UCB) natural killer T (NKT) cells and enhancement of their cytotoxicity. The second one is to examine whether purified NKT cells could affect the cytotoxicity of UCB-NK cells either in the presence or absence of dendritic cells (DCs).Methods Mononuclear cells (MNCs) from UCB were cultured for 2 weeks in the presence of IL-2 (100 U/ml), with or without GalCer. The effect of neutralizing monoclonal antibodies (MoAb) against TCRV24 and CD1d was also examined. TCRV24 V11 double positive NKT cells were purified by FACS sorter and then cocultured with syngeneic isolated UCB^–CD56⁺NK cells in either the presence or absence of DCs. The cytotoxicity against various malignant cell targets and cytokine production was determined.Results The addition of GalCer induced human NKT cells to proliferate in UCB-MNCs to a greater extent than in adult PB-MNCs. However, it suppressed the cytotoxic activity against malignant cell targets. Anti-TCRV24 and CD1d MoAb recovered the cytotoxicity by inhibiting the proliferation of UCB-NKT cells. NKT cells cocultured with auto-DCs significantly increased NK cell cytotoxicity against K562, and Raji cells and produced IFN- at much higher levels than UCB-NKT cells alone.Conclusion In UCB samples, GalCer–pulsed DCs and NKT cells acted together to enhance NK cytotoxicity in vitro.     

Relationship between the tumor necrosis factor alpha polymorphism and the serum C-reactive protein levels in inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, including ulcerative colitis (UC) and Crohn's disease (CD). The aim of the study was to determine the prevalence of the tumor necrosis factor alpha (TNF-) promoter polymorphisms at positions –238 and –308, and to measure the serum CRP levels in CD and UC patients and in a healthy population. The TNF- gene polymorphisms were determined by the PCR-RFLP method. Samples of 74 CD and 50 UC patients and 138 healthy Hungarian volunteers were examined. The GA substitution at position –308 (designated the TNF2 allele) was significantly less frequent among IBD patients than in the control group (P=0.0009); 15% of the CD patients and 18% of the UC patients carried the mentioned allele, which was significantly less frequent compared with the healthy population (33%, P=0.0035 and P=0.036, respectively). No difference in the GA substitution at position –238 was observed. We found the median CRP levels to be significantly higher in the active phase of the disease than in the inactive phase among the –308A allele carriers (P=0.002), while this difference was not significant when the CRP levels in the active and inactive phases were compared among the –308GG homozygous patients (P=0.084). The decreased frequency of the TNF2 allele (known to be associated with elevated TNF- production) in IBD may determine the severity of IBD through its interaction with plasma CRP levels, and may modify the pathogenesis of this chronic inflammatory disease.     

The sensitivity of head and neck squamous cell carcinomas to tumor necrosis factor-α

Sensitivities to recombinant human tumor necrosis factor- (TNF-) and chemotherapeutic agents (cisplatin, peplomycin, methotrexate) were evaluated in 20 tumor cells of head and neck squamous cell carcinomas, using a dye uptake method. Also, numbers of TNF receptors of these tumor cells were measured by Scatchard plot analysis. There was no relationship between the number of TNF- receptors and the sensitivity to TNF-. Furthermore, there was no correlation between the sensitivity to TNF- and that to chemotherapeutic drugs, nor between the sensitivity to TNF- and the clinical response to chemotherapy including of cisplatin and peplomycin. The sensitivity to TNF- was higher in poorly differentiated carcinomas than in well differentiated ones.Abbreviations BSA Bovine serum albumin - CDDP Cisplatin - 5-Fu 5-fluorouracil - IC50 Inhibition concentration 50 - MTX Methotrexate - PLM Peplomycin - TNF- Tumor necrosis factor-     

Tumor necrosis factor alpha (TNF-α) gene polymorphism in alopecia areata

Tumor necrosis factor alpha (TNF-) phenotypes of two polymorphic systems were determined in 50 patients with alopecia areata, a common inflammatory disease of the skin. The distribution of TNF- T1, T2 phenotypes differed between patients with the patchy form of disease and patients with totalis/universalis disease. There was no significant difference in the distribution of TNF- G,A phenotypes between patient groups. The results of this study provide evidence of genetic heterogeneity between the two forms of alopecia areata, and suggest that the TNF- gene or a closely linked locus within the major histocompatibility complex may play a role in the pathogenesis of the patchy form of disease.     

Strategies for antigen choice and priming of dendritic cells influence the polarization and efficacy of antitumor T-cell responses in dendritic cell–based cancer vaccination

Dendritic cells (DCs) primed with tumor antigens (Ags) can stimulate tumor rejection. This study was aimed at evaluating the polarization of T-cell responses using various DC Ag-priming strategies for vaccination purposes. DCs cocultured with irradiated apoptotic tumor cells, DC-tumor fusions, and DCs pulsed with freeze-thaw tumor lysate Ags served as Ag-primed DCs, with EG7 tumor cells (class II negative) expressing OVA as the model Ag. DCs loaded with class I– and class II–restricted OVA synthetic peptides served as controls. Primed DCs were assessed by the in vitro activation of B3Z OVA-specific CD8 T cells and the proliferation of OVA-specific CD8 and CD4 T cells from OT-I and OT-II TCR transgenic mice, respectively. In vivo responses were measured by tumor regression following treatment with Ag-primed DCs and by CTL assays. Quantification of IL-2, IL-4, IL-5, IFN-, and TNF- by cytometric bead array (CBA) assay determined the polarization of TH1/TH2 responses, whereas H-2 K^b /SIINFEKL tetramers monitored the expansion of OVA-specific T cells. DC-EG7 hybrids stimulated both efficient class I and class II OVA responses, showing that DC-tumor hybrids are also capable of class II cross-presentation. The hybrids also induced the most potent CTLs, offered the highest protection against established EG7 tumors and also induced the highest stimulation of IFN- and TNF- production. DCs cocultured with irradiated EG7 were also effective at inducing OVA-specific responses, however with slightly reduced potency to those evoked by the hybrids. DCs loaded with lysates Ags were much less efficient at stimulating any of the OVA-specific T-cell responses, showed very little antitumor protection, and stimulated a weak TH1 response, overbalanced by an IL-5 TH2 response. The strategy of Ag-loading clearly influences the ability of DCs to polarize T cells for a TH1/TH2 response and thus determines the outcome of the elicited immune response, during various vaccination protocols.Abbreviations DC Dendritic cell - FSC Forward scatter - SSC Side scatter - TC Tumor cells This work was supported by Grant 9853 from the Leukaemia Research Fund, UK; a JRC studentship from GKT; and the Lewis Family Research Trust     

TNF-α induced altered signaling mechanism in human neutrophil

In the present study we investigated the TNF- induced signal transduction mechanism in human neutrophil. Exogenously added TNF- affects both PKC activity and its translocation from cytosol to the membrane. Endogenous protein phosphorylation pattern is inhibited in TNF- induced neutrophil in Ca-dependent and Ca-independent manner, including a major 47 and 66 kDa cytosolic proteins, which may be implicated in superoxide anion generation. However TNF- dose dependently enhances the expression of -PKC isotype but not the -PKC. Morphology and cell cytotoxicity are studied in TNF- treated neutrophil to understand the TNF- induced cell death or apoptosis and these experiment is further confirmed by DNA fragmentation analysis. These results clearly demonstrate that TNF- induces cellular death of human neutrophil at least in part by enhanced expression of Ca-independent -PKC. These observations provide an insight towards understanding the function of -PKC in apoptotic pathway.     

Effect of thymosin alpha 1 on the antitumor activity of tumor-associated macrophage-derived dendritic cells

We have previously suggested that thymosin ₁ (thy1), an immunomodulating thymic hormone, can activate tumor-associated macrophages to a tumoricidal state in a murine model bearing a transplantable T-cell lymphoma of spontaneous origin designated as Dalton's lymphoma (DL). Since tumor-infiltrating dendritic cells (DC) also play an important role in the host's antitumor response and are as such in an immunocompromised state in a tumor-bearing host, in the present investigation we studied if thy1 is able to influence the differentiation of tumor-associated macrophages (TAM) into DC with granulocyte macrophage colony stimulating factor (GM-CSF), interleukin (IL)-4 and tumor necrosis factor (TNF) and whether these TAM-derived DC show enhanced antitumor activity. It was observed that DC generated from thy1-administered tumor-bearing mice showed augmented antitumor activity in vitro. Adoptive immunotherapy using TAM-derived DC showed a significant delay in the tumor growth and a prolongation of the survival time in tumor-bearing mice. DC obtained from TAM of thy1-administered mice also produced an enhanced amount of cytokines like IL-1 and TNF-. This is the first study of its kind regarding the effect of thy1 on the differentiation of DC from TAM and the role of TAM-derived DC in tumor progression.     

α-Tocopheryl succinate sensitizes established tumors to vaccination with nonmatured dendritic cells

Purpose: Dendritic cells (DCs) are considered potential candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC-based vaccines have exhibited minimal effectiveness against established tumors in mice and human cancer patients. The use of appropriate adjuvants can enhance the efficacy of DC-based cancer vaccines in treating established tumors. Methods: In this study we have employed -tocopheryl succinate (-TOS), a nontoxic esterified analogue of vitamin E, as an adjuvant to enhance the effectiveness of DC vaccines in treating established murine Lewis lung (3LL) carcinomas. Results: We demonstrate that locally or systemically administered -TOS in combination with nonmatured DCs injected intratumorally (i.t.) or subcutaneously (s.c.) significantly inhibits the growth of preestablished 10-day tumors (mean tumor volume of 77.5 ± 17.8 mm³ on day 30 post–tumor injection) as compared to -TOS alone (mean tumor volume of 471 ± 68 mm³ on day 30 post–tumor injection). Additionally, the adjuvant effect of -TOS was superior to that of cyclophosphamide (CTX). The mean tumor volume on day 28 post–tumor injection in mice treated with CTX+DCs was 611 ± 94 mm³ as compared to 105 ± 36 mm³ in mice treated with -TOS+DCs. Analysis of purified T lymphocytes from mice treated with -TOS+DC revealed significantly increased secretion of IFN- as compared to T cells from the various control groups. Conclusion: This study demonstrates the potential usefulness of -tocopheryl succinate, an agent nontoxic to normal cell types, as an adjuvant to augment the effectiveness of DC-based vaccines in treating established tumors.Abbreviations AO acridine orange - CTX cyclophosphamide - DC dendritic cell - dUTP deoxyuridine triphosphate - FACS fluorescence-activated cell sorter - FBS fetal bovine serum - FITC fluorescein isothiocyanate - GM-CSF granulocyte-macrophage colony-stimulating factor - IFN- interferon-gamma - IL-4 interleukin-4 - NaS sodium succinate - OCT optimal cutting temperature - PBS phosphate-buffered saline - PI propidium iodide - Tdt terminal deoxynucleotidyl transferase - TNF- tumor necrosis factor alpha - -TOS -tocopheryl succinateSupported by grants 1 RO1 CA94111-02 from the NIH and DAMD 17010126 from the DOD.     

The limited proteolysis of tumor necrosis factor-α

The limited proteolysis of human recombinant TNF- by trypsin yields two stable products resulting from cleavage after Arg6 and Arg44. In solution these two products remain associated together in a trimer with a Stokes' radius slightly greater than the radius of intact TNF- and, therefore, could not be separated from each other under nondenaturing conditions. This limited digest retains at least 20% of the activity of the original TNF- sample, and has a tertiary structure that is similar to that of the native protein by circular dichroism. On the other hand, incorrectly folded, inactive TNF- undergoes extensive digestion following similar treatment with trypsin. These results indicate that the active form of TNF- has a tight core structure which is maintained afterN-terminal cleavage and removal.     

Apoptosis and production of TNF-α by tumor-associated inflammatory cells in histological grade III breast cancer

Tumor necrosis factor alpha (TNF-) is a cytokine that acts as an important mediator of the apoptotic process that also demonstrates selective citotoxicity against malignant breast tumor cells. In the present study, the presence of apoptotic tumor cells and the synthesis of TNF- by inflammatory cells were investigated in tissue samples from grade III invasive breast cancer with long-term follow-up. In situ detection of tumor apoptotic cells was investigated by direct immuno-peroxidase of digoxigenin-labeled genomic DNA. The production of TNF- and tumor cell proliferation were investigated by immunohistochemical procedures. Our data demonstrated that patients with a clinical history of cancer recurrence and metastasis presented a lower number of cancerous apoptotic cells, higher tumor proliferation rates, and lower TNF- expression rates by inflammatory cells than what is observed among patients diagnosed with the same histopathological breast cancer type but in the absence of tumor recurrence and metastasis.     

Combined lack of estrogen receptors α and β affects vascular iNOS protein expression

Endothelial and vascular smooth muscle cells express both estrogen receptor (ER) and . Recent findings indicate that vascular ER and ER may substitute for one another. Here, we investigate vascular morphology, contractility and protein expression in intact aorta from adult (4 months old) female mice lacking both ER and ER (DERKO). The body weights were 17% higher (P<0.01) in DERKO than in wild-type mice. Vascular morphology, investigated in paraffin sections from aorta stained with hematoxylin-eosin or van Gieson, was identical in DERKO and wild-type mice. Endothelial cells were clearly visible in aorta of both DERKO and wild-type animals. Morphometric analysis of media thickness and wall to lumen ratio using a computerized image analyzing system demonstrated no differences between the two groups of mice. The vascular expression of endothelial nitric oxide synthase (eNOS, NOS III) and inducible nitric oxide synthase (iNOS, NOS II) was investigated using Western blotting. Aorta from both DERKO and wild-type mice expressed iNOS protein, but the iNOS expression was 3 times lower (P<0.05) in DERKO compared to wild-type mice. No difference in eNOS protein level between the two groups of animals was observed. Force responses to noradrenaline, determined either in the absence or in the presence of the nitric oxide synthase inhibitor l-NAME and the cyclo-oxygenase inhibitor indomethacin, were unaffected by the lack of functional ER/ER. In summary, combined lack of functional ER and ER lowers the vascular expression of iNOS but has no effects on morphology, eNOS expression, and noradrenaline sensitivity in the intact aorta.This study was supported by the Swedish Medical Research Council (grant no. K99–0X-13017-01A), the Swedish Heart and Lung Foundation, KaroBio AB and the Åke Wiberg, Magnus Bergvall and Crafoord Foundations.     

Interleukin-6 Induced Suppression of Bovine Parathyroid Hormone Secretion

Calcium disturbances in the critically ill coincide with elevations of proinflammatory cytokines. The effects of tumor necrosis factor- (TNF-) and interleukin-6 (IL-6) on parathyroid hormone (PTH) secretion were investigated. IL-6 and TNF- had no acute effect on PTH secretion in extracellular Ca²⁺ concentrations of 0.5, 1.25 and 3.0 mM. In contrast to TNF-, cultures for 24 h in the presence of l0 ng/mL of IL-6 showed decreased PTH secretion by 51% and 29% in 0.5 mM and 1.25 mMCa²⁺ respectively. Neither IL-6 nor TNF-, affected cytoplasmic Ca²⁺ of the cells. We conclude that PTH secretion in vitro can be suppressed by IL-6 at clinically relevant concentrations. This suppression may aggravate hypocalcemia of the critically ill and attenuate the conventionally strong stimulation of the PTH release by reduction in serum calcium.     

Differentiation of acute and chronic myeloid leukemic blasts into the dendritic cell lineage: analysis of various differentiation-inducing signals

Purpose: Ex vivo differentiation of myeloid leukemic blasts into dendritic cells (DCs) holds significant promise for use as cellular vaccines, as they may present a constellation of endogenously expressed known and unknown leukemia antigens to the immune system. Although variety of stimuli can drive leukemiaDC differentiation in vitro, these blast-derived DCs typically have aberrant characteristics compared with DCs generated from normal progenitors by the same stimuli. It is not clear whether this is due to underlying leukemogenic mechanisms (e.g., specific oncogenes), genetic defects, stage of maturation arrest, defects in cytokine receptor expression or signal transduction pathways, or whether different stimuli themselves induce qualitatively dissimilar DC differentiation. Methods: To assess what factors may contribute to aberrant leukemic blastDC differentiation, we have examined how the same leukemic blasts (AML and CML) respond to different DC differentiation signals—including extracellular (the cytokine combination GM-CSF+TNF-+IL-4) and intracellular (the protein kinase C agonist PMA, the calcium ionophore A23187, and the combination of PMA plus A23187) stimuli. Results: We have found that the same leukemic blasts will develop qualitatively different sets of DC characteristics in response to differing stimuli, although no stimuli consistently induced all of the characteristic DC features. There were no clear differences in the responses relative to specific oncogene expression or stage of maturation arrest (AML vs CML). Signal transduction agonists that bypassed membrane receptors/proximal signaling (in particular, the combination of PMA and A23187) consistently induced the greatest capability to activate T cells. Interestingly, this ability did not clearly correlate with expression of MHC/costimulatory ligands. Conclusions: Our findings suggest that signal transduction may play an important role in the aberrant DC differentiation of leukemic blasts, and demonstrate that direct activation of PKC together with intracellular calcium signaling may be an effective method for generating immunostimulatory leukemia-derived DCs.This work was supported by NIH CA85208, CA95829, and ASCO Young Investigator Award (M.A.K-D)     

Contribution of cytokines on the suppression of lung metastasis

Weekly injection of a protein-bound polysaccharide PSK in mice with Lewis Lung Cancer (LLC) significantly decreased the number of lung metastatic foci concomitant with enhancement of cytostatic activity in the bronchoalveolar lavage (BAL) cells. These effects were more marked when the agent was given intratracheally, inducing a larger number of pulmonary macrophages, lymphocytes and neutrophils concomitant with increases in BAL tumor necrosis factor- (TNF-), mouse inflammatory protein- (MIP-1), mouse inflammatory protein- (MIP-1), interleukin-1 (IL-1) and interleukin-6 (IL-6), but not interleukin-2(IL-2) and interleukin-4 (IL-4). Pre-treatment with anti TNF- antibody reduced these effects. The time course and production of PSK-induced cytokines were similar between the tumor-bearing mice and control mice. BAL neutrophils in mice with LLC showed a tendency toward acceleration of O2- production compared with circulating neutrophils. Pulmonary macrophage phagocytosis was also significantly higher in the LLC mice.These results suggest that enhancement of cytostasis appears to be induced by activation and/or improvement of function in inflammatory and immune cells through cytokines under immunomodulator treatment in lung metastasis, possibly via a TNF--dependent mechanism.     

Acute Multiple Sclerosis Characterized by Extensive Mononuclear Phagocyte Infiltration

In this study, we examine the clinical, neuroradiological, and immunohistochemical findings of a 51 year old white female who died 27 months after onset of acute multiple sclerosis despite treatment with interferon-, azathioprine, corticosteroids, and cyclophosphamide. Immunohistochemical studies revealed extensive gliosis and mononuclear phagocyte infiltration with corresponding upregulation of proinflammatory cytokines (eg. IFN-, TNF-). The significance of immunohistochemical findings with respect to clinical presentation is discussed.     

Cytidine 5′-Diphosphocholine (CDP-Choline) in Stroke and Other CNS Disorders

Brain phosphatidylcholine (PC) levels are regulated by a balance between synthesis and hydrolysis. Pro-inflammatory cytokines such as tumor necrosis factor- (TNF-) and interleukin-1 (IL-1/) activate phospholipase A₂ (PLA₂) and PC-phospholipase C (PC-PLC) to hydrolyze PC. PC hydrolysis by PLA₂ releases free fatty acids including arachidonic acid, and lyso-PC, an inhibitor of CTP-phosphocholine cytidylyltransferase (CCT). Arachidonic acid metabolism by cyclooxygenases/lipoxygenases is a significant source of reactive oxygen species. CDP-choline might increase the PC levels by attenuating PLA₂ stimulation and loss of CCT activity. TNF- also stimulates proteolysis of CCT. TNF- and IL-1 are induced in brain ischemia and may disrupt PC homeostasis by increasing its hydrolysis (increase PLA₂ and PC-PLC activities) and inhibiting its synthesis (decrease CCT activity). The beneficial effects of CDP-choline may result by counteracting TNF- and/or IL-1 mediated events, integrating cytokine biology and lipid metabolism. Re-evaluation of CDP-choline phase III stroke clinical trial data is encouraging and future trails are warranted. CDP-choline is non-xenobiotic, safe, well tolerated, and can be considered as one of the agents in multi-drug treatment of stroke.     

Lectin histochemical identification of the carbohydrate moieties on N- and O-linked oligosaccharides in the duct cells of the testis of an amphibian urodele, the spanish newt (Pleurodeles waltl)

The aim of the present work was to study the carbohydrate moieties present on N- and O-linked oligosaccharides of duct cells of a urodele amphibian testis, by means of lectin histochemistry. It was found that duct cells have a carbohydrate composition that includes ensuremath (1,3)-, (1,4)- or (1,6)-linked Fuc and Man on N-linked oligosaccharides, Gal and GlcNAc on O-linked oligosaccharides, and DBA-positive GalNAc, (1,2)-linked Fuc and Neu5Ac(2,3)Gal (1,4)GlcNAc on both N- and O-linked oligosaccharides. All the duct cells showed the same lectin labelling pattern, the only exception being some sparse duct cells that showed the sequence Neu5Ac(2,6)Gal/GalNAc. The possible roles of duct cells in sperm maturation and the hypothesis for a common origin of duct and follicle (Sertoli) cells in the urodele testis are discussed.     

Interaction of alpha-lactalbumin with mini-alphaA-crystallin

A-Crystallin can function like a molecular chaperone. We have recently shown that residues 71-88 in A-crystallin represent the chaperone active site of the protein. A peptide containing the sequence of A-crystallin sequence DFVIFLDVKHFSPEDLTVK (mini A-crystallin) by itself displays the antiaggregation property of A-crystallin. We have prepared a complex of reduced -lactalbumin and mini-A-crystallin and investigated the nature, conformation, and properties of the complex by dynamic light scattering, HPLC analysis, CD spectroscopy, and fluorescence studies. Although mini-A was able to prevent the precipitation of reduced -lactalbumin, large aggregates (50-500 nm) of the complex were formed during the assay. Amino acid composition estimation revealed that -lactalbumin and mini-A-crystallin were present in 1:2 ratio in the aggregates. During our study significant red shift in the Trp fluorescence emission maximum and an increase in Bis-ANS binding to the mini A-crystallin-bound -lacatalbumin were observed. The CD spectra of the complex showed a significant loss of -helical content but the -sheet content appeared to be less affected, indicating the molten-globule state of the reduced lactalbumin in the complex. These data show that the active site of A-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form.     

Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor α

Summary Recombinant tumor necrosis factor (rTNF; optimal dose 1000 U/ml) significantly increased the density of epidermal growth factor receptor (EGF-R) in three of four glioma cell lines in culture as determined by binding analysis of anti-EGF-R monoclonal antibody (mAb) 425. Since enhancement of EGF-R expression by rTNF- was inhibited when cells were treated with the protein synthesis inhibitor cycloheximide, the effects of rTNF may be protein-synthesis-dependent. The dose of rTNF that was optimal for up-regulation of EGF-R on glioma cells did not inhibit the growth of these cells.¹²⁵I-labeled mAb 425 lysed glioma cells in culture following its internalization into the cells. After glioma cells had been treated with rTNF, the growth-inhibitory effects of the mAb were significantly enhanced, probably a reflection of the increase in EGF-R density on the tumor cell surfaces. The rTNF effects were specific to the EGF-R and did not affect unrelated glioma-associated antigens. In our previous clinical trials,¹²⁵I-labeled mAb 425 showed immunotherapeutic effects in glioma patients. The present study provides the basis for considerations of combined immunotherapy of glioma patients with¹²⁵I-labeled mAb 425 and rTNF.