Dental visiting behaviours among primary schoolchildren: Application of the health belief model.

OBJECTIVES: This study aimed to develop and validate a new instrument based on the health belief model and to use the instrument to investigate the determinants of regular dental attendance among primary schoolchildren. METHODS: A cross-sectional study was conducted using a newly developed measurement scale based on the HBM, 4 health-promoting schools participated in the study and 958 students studying in grades 4-6 completed the questionnaire. The psychometric properties of the instrument were analysed, and a path analysis model was used to identify the determinants of regular dental attendance. RESULTS: The instrument had good internal consistency (Cronbach's α = 0.826-0.925) and a factor structure identical to HBM. Overall, the schoolchildren's health beliefs on caries treatment were positive. The determinants of regular dental visit were school location (ß = -0.13), mother's education level (ß = 0.15), susceptibility (ß = -0.18) and barriers (ß = -0.11). CONCLUSION: This study provided evidence that HBM is applicable to children's dental visiting behaviour and their health beliefs towards adherence to caries treatment. Although children had a positive attitude towards dental visits, environmental obstacles would interfere with dental visits. The newly developed instrument could be used to identify high-risk children and help design oral health interventions for these children. Moreover, policy makers should increase the accessibility of dental resources to enhance the utilization of dental care among schoolchildren.

How does graphene grow on complex 3D morphologies?

The growth of two-dimensional materials into three-dimensional geometries holds the promise for high performance hybrid materials and novel architectures. The synthesis of such structures, however, proceeds in fundamentally different flow regimes compared to conventional CVD where pressure differences and wall collisions are neglected. We here demonstrate the remarkable stability of graphene growth under varying fluid dynamic flow regimes. We investigate the growth process across different flow conditions using confined growth in refractory pores. Analysis of the growth rate reveals a transport-limited process which allows experimental determination of the gas diffusion coefficient. The diffusion coefficient was found to be constant for large pore dimension but scales with pore dimension as the pore size decreases below the mean free path providing clear evidence for previously predicted Knudsen molecular-flow conditions for atomic confinement. Surprisingly, changes to the flow conditions by two orders of magnitude do not cause qualitative changes of the graphene growth process. This unique behavior was attributed to rarefied flow conditions by scaling analysis and an analytical relation between growth rate and constriction could be extracted that proves accurate throughout the investigated conditions. Our results demonstrate a fundamentally different growth process compared to traditional CVD processes that is akin to atomic layer deposition and highlight the feasibility of high-quality 2D-material growth on 3D morphologies with ultra-high aspect ratios.

Oral health disparities of children among Southeast Asian immigrant women in arranged transnational marriages in Taiwan.

This study assessed the oral health disparities and oral health care needs of children whose parents are Southeast Asian immigrant women in arranged transnational marriages. We used the baseline data of the Lay Health Advisor Approach to Promote Oral Health Program (LHA-POHP) to explore the disparities in oral health between immigrant and native children, and the factors associated with their oral health. A cross-sectional community-based study was conducted to collect data from mothers and their preschool children in Southern Taiwan in 2011. A total of 590 (440 natives, 150 immigrants) children aged 4-6 years and their mothers completed the questionnaire and oral examination. Multiple regression models were used to analyze the association between children's oral health and their related factors. The caries index was 6.05 in immigrant children and 3.88 in native children (p < 0.001). The caries prevalence of maxillary anterior teeth in the labial surfaces was higher among immigrants, ranging from 14.7 to 22%. The factor associated with children's caries index was maternal tooth brushing frequency (adjusted odds ratio [aOR] = 8.95, 95% confidence interval [CI] 1.95-41.05). When the mothers did not direct children to brush teeth after eating sweets, their children were more likely to have decayed teeth (aOR = 3.54, 95% CI 1.04-12.03). Children's filled teeth were related to their dental regular check-ups (aOR = 2.28, 95% CI 1.26-4.10). Disparities in oral health among immigrant and native children were observed. The findings suggest that culturally adequate oral health promotion intervention programs should be implemented for immigrants.

Antimicrobial photodynamic therapy using a diode laser with a potential new photosensitizer, indocyanine green-loaded nanospheres, may be effective for the clearance of Porphyromonas gingivalis.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) is a new treatment method for the removal of infectious pathogens using a photosensitizer and light of a specific wavelength, e.g., toluidine blue with a wavelength of about 600 nm. We explored a new photosensitizer and focused on indocyanine green (ICG), which has high absorption at a wavelength of 800-805 nm. We investigated the bactericidal effect of PDT on Porphyromonas gingivalis using a new photosensitizer, ICG-loaded nanospheres with an 805 nm wavelength low-level diode laser irradiation. METHODS: We designed ICG-loaded nanospheres coated with chitosan (ICG-Nano/c) as a photosensitizer. A solution containing Porphyromonas gingivalis (10(8)  CFU/mL) with or without ICG-Nano/c (or ICG) was prepared and irradiated with a diode laser or without laser irradiation as a negative control. The irradiation settings were 0.5 W with a duty ratio of 10%, for 3-100 ms in repeated pulse (RPT) or continuous wave mode. CFU were counted after 7 d of anaerobic culture. RESULTS: We observed that ICG-Nano/c could adhere to the surface of P. gingivalis. When ICG-Nano/c was used for aPDT, irradiation with RPT 100 ms mode gave the lowest increase in temperature. Laser irradiation with ICG-Nano/c significantly reduced the number of P. gingivalis (i.e., approximately 2-log10 bacterial killing). The greatest bactericidal effect was found in the RPT 100 ms group. However, laser irradiation (RPT 100 ms) with ICG, as well as without photosensitizer, had no effect on the number of bacteria. CONCLUSIONS: Within the limits of this study, ICG-Nano/c with low-level diode laser (0.5 W; 805 nm) irradiation showed an aPDT-like effect, which might be useful for a potential photodynamic periodontal therapy.

Minimally invasive approaches to treating chemosis of the eyes from unusual dural arteriovenous fistulae.

Chemosis of the eyes is usually attributed to carotid cavernous sinus dural arteriovenous fistulae. Herein, we reviewed unusual cases in which chemosis of the eyes originated from dural ateriovenous fistulae (dAVFs) that were distinctly different from carotid cavernous sinus fistulae. Cases in which ocular symptoms were related to increased intracranial pressure either due to sinus thrombosis or cortical venous drainage without involvement of superior or inferior ophthalmic veins were excluded in this review. Several different types of dural AVFs were associated with chemosis, and these included dAVFs harboring a feeding artery from branches of the external carotid artery directly draining to the superior ophthalmic vein or cavernous sinus via the superior petrous sinus, posterior fossa dAVFs draining via the inferior petrous sinus and cavernous sinus to the ophthalmic vein, a fistula between the ophthalmic artery or branches of the internal carotid artery and inferior ophthalmic vein, or tentorial fistula with a drainage vein to the cavernous sinus via the vein of Galen. This study reviews the symptomatology, treatment options, and cerebrovascular abnormalities observed for these unusual dAVF's with chemosis.

Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity.

To increase the antitumor effects of cytosine deaminase (AdCD) gene therapy and induce more potent antitumor immunity, Th1 cytokine interleukin-18 encoded adenovirus (AdIL18) was combined with adenovirus encoding CD (AdCD) for the therapy of established murine B16 melanoma. Combination therapy of the tumor-bearing mice with AdIL 18 and AdCD/5FC inhibited the growth of the subcutaneous B16 tumors more significantly, compared with AdIL 18 or AdCD/5FC alone. In vivo depletion analysis with anti-CD4, anti-CD8 or anti-NK 1.1 McAb illustrated that both CD8+ T cells and CD4+ T cells played key roles in the augmented antitumor response of the combined therapy. Peptide/MHC tetramer represents a powerful and general tool for rapid, highly sensitive, and direct analysis of antigen-specific T cells. In this study, we prepared H-2Kb/TRP-2180-188 tetramer, which was demonstrated to bind H-2Kb-restricted, B16 melanoma-specific CD8+ T cells. B16 specific H-2Kb/TRP2180-188 tetramer was used to stain the tumor-specific CD8+ T cells and the results showed that CD8+ tetramer+ T cells were about 3-5% of the splenic CD8+ T cells derived from tumor-bearing mice after combined therapy. The CTL cytotoxicity was markedly induced in mice after combined therapy, suggesting efficient induction of tumor-specific CD8+ T cells after combined gene therapy with AdCD/5FC/AdIL18. IL-18 gene transfer could significantly augment the cytotoxicity of NK cells and macrophages, and increase the production of interleukin-2 and interferon-gamma, as compared with treatments with AdCD/5FC, AdlacZ/5FC or PBS. These data suggested that in vivo IL-18 gene transfer could augment the antitumor effects of CD suicide gene therapy through efficient induction of antitumor immunity.

Restoration of cytotoxic T lymphocyte function in malignant pleural effusion: interleukin-15 vs. interleukin-2.

The present study attempts to define the role of interleukin-15 (IL-15), as compared with IL-2, in generating cytotoxic T lymphocytes (CTL) from the malignant effusions of cancer patients. Effusion-associated lymphocytes (EAL) from malignant effusion were incubated with IL-15 or IL-2 with or without alphaCD3. Proliferation and cytotoxicity assays were performed. IL-15 was found to have at least an equivalent, if not higher, activity to IL-2 in terms of lymphocyte proliferation and generation of CTL from EAL. The proliferative response of EAL, cocultured with IL-15, with or without alphaCD3, was partly inhibited by pretreatment with an anti-IL2 receptor beta chain monoclonal antibody (mAb). The proliferative response of EAL, cocultured with alphaCD3, IL-2, or both, was partly inhibited by pretreatment with an anti-IL-2 receptor alpha chain mAb. Overnight [5lCr] release assays against K562, Daudi, and the patients' autologous tumor cells were done to evaluate EAL's cytolytic activity. MHC class I Ab blocked the stimulated cytolytic activity of EAL against autologous tumors. An mAb depletion assay showed that the phenotype of the restored EAL was CD16-CD4-CD8+; thus, the restored activity of EAL was CTL activity. The results suggest that both IL-15 and IL-2 can restore CTL activity from EAL in the presence of T cell receptor (TCR)-CD3 engagement, but the effect of IL-15 was superior.

Depressed cytolytic activity of peripheral blood mononuclear cells in unusually high paclitaxel concentrations: reversal by IL-2 and IL-12.

BACKGROUND: Human lymphocyte function was inhibited by high concentrations of paclitaxel and the effect was reversed by interleukin (IL)-2. However, there was no parallel study determining the relationship between paclitaxel concentrations in the lymphocyte cultures and pharmacokinetic analysis in human patients, nor was there any study on the reversal by cytokines, other than IL-2, of the paclitaxel-induced suppression of lymphocyte cytotoxicity. METHODS: We tested the effect of different doses of paclitaxel with various incubation times on the cytolytic activity of peripheral blood mononuclear cells (PBMNCs) against K-562 target cells. RESULTS: Our results showed that using a schedule similar to that for treating patients with tolerable doses of paclitaxel, no inhibition of cytolytic activity of PBMNCs was seen. When the paclitaxel concentration was increased 10-fold, the cytolytic activity of PBMNCs was significantly reduced. This suppression was reversed by the simultaneous addition of a low dose (10 U/ml) of IL-2 or IL-12. Addition of granulocyte macrophage-colony stimulating factor (10 U/ml) did not affect the cytolytic activity of PBMNCs, whereas addition of IL-4 reduced it. Time kinetic studies revealed that, with the addition of IL-2 or IL-12, most of the mononuclear cellular cytolytic activity recovered within 48 to 72 hours. CONCLUSIONS: These findings suggested that, to reduce the toxicity on mononuclear cellular function when high-dose paclitaxel treatment is elected in clinical practice, paclitaxel should be infused over a longer duration of time, or the treatment should be combined with the administration of a low dose of IL-2 or IL-12.

Treatment of B-cell lymphoma with chimeric IgG and single-chain Fv antibody-interleukin-2 fusion proteins.

Anti-idiotype (Id) antibodies (Abs) have been shown to be effective in treatment of B-cell lymphoma in animal models and in clinical trials. The combination of interleukin-2 (IL-2) can augment the therapeutic effect of anti-Id Abs. To further improve the power of the combined therapy, a monoclonal anti-Id Ab, S5A8, specifically recognizing a murine B-cell lymphoma 38C13, was genetically modified to contain the IL-2 domain and thus use the unique targeting ability of Abs to direct IL-2 to the tumor site. Two forms of the anti-Id-IL-2 fusion proteins were constructed: one configuration consisting of mouse-human chimeric IgG (chS5A8-IL-2) and the other containing only the variable light (VL) and variable heavy (VH) Ab domains covalently connected by a peptide linker (scFvS5A8-IL-2). Both forms of the anti-Id-IL-2 fusion proteins retained IL-2 biological activities and were equivalent in potentiating tumor cell lysis in vitro. In contrast, the antigen-binding ability of scFvS5A8-IL-2 was 30- to 40-fold lower than that of the bivalent chS5A8-IL-2. Pharmacokinetic analysis showed that scFvS5A8-IL-2 was eliminated about 20 times faster than chS5A8-IL-2. Finally, it was shown that chS5A8-IL-2 was very proficient in inhibiting 38C13 tumor growth in vivo, more effectively than a combined therapy with anti-Id Abs and IL-2, whereas scFvS5A8-IL-2 did not show any therapeutic effect. These results demonstrate that the anti-Id-IL-2 fusion protein represents a potent reagent for treatment for B-cell lymphoma and that the intact IgG fusion protein is far more effective than its single-chain counterpart.

Paradoxical effect of GM-CSF gene transfer on the tumorigenicity and immunogenicity of murine tumors.

The effect of granulocyte/macrophage colony-stimulating factor (GM-CSF) gene transfer on the tumorigenicity and immunogenicity of 2 different murine tumor lines was determined. Transduction of B16 melanoma cells with the GM-CSF gene rendered the cells more immunogenic. In contrast, transduction of NG4TL4 fibrosarcoma in FVB/N mice (NG) with the GM-CSF gene showed increased tumorigenicity in a high producer line (NG-MGh). The parent NG or NG-MG cells induced the same level of cytotoxic T-lymphocyte (CTL) response and the same magnitude of tumor transplantation immunity. However, the proliferation of the NG-MGh cells was increased 2- to 10-fold. There was no increase in apoptosis in the NG cells and there was no increase of NG-MGh cells in S-phase, hence the increase of the proliferative activity appeared to be indeed inherent to the cells. Mixing the splenocytes from the NG-MGh tumor bearers with the NG tumor cells did not increase tumorigenicity but totally inhibited the growth of the NG tumor, indicating that suppressor cells were not present. Mixing 10,000 rad X-irradiated NG-MGh cells with viable NG tumor cells resulted in 3- to 10-fold increased NG tumor growth rate. The in vitro proliferation of NG cells was increased by adding both GM-CSFs and macrophages and not by either one alone, suggesting that interaction between macrophages and GM-CSFs resulted in the production of tumor growth enhancing factor(s). Our findings suggest that transduction of NG tumor cells with the GM-CSF gene increases tumorigenicity, which is attributed both to an increased inherent proliferative ability of the tumor cells and to the in vivo production of a tumor growth enhancing factor(s) at the tumor site.

Cross regulation by IL-10 and IL-2/IL-12 of the helper T cells and the cytolytic activity of lymphocytes from malignant effusions of lung cancer patients.

STUDY OBJECTIVE: Our previous report demonstrated that there was impairment of local cellular immunity with elevated interleukin-10 (IL-10) and undetectable IL-12 in neoplastic pleural effusion. These findings suggest that the local immune reactions favor the T-helper type 2 (Th2) pathway instead of Th1 pathway. The present study was designed to examine whether local cellular immunity could be manipulated by IL-2 and/or IL-12 treatment, and to determine their effect on the helper T-cell pathways and the cytolytic activity of the effusion-associated lymphocytes (EALs). DESIGN: Using malignant pleural effusions obtained from four patients suffering from adenocarcinoma of lung, we separated the tumor cells from the EALs with Ficol-Hypaque centrifugation, followed by Percoll density centrifugation. To test whether the cytolytic function of lymphocytes could be enhanced by culturing with IL-2 and/or IL-12, lymphocytes were incubated with recombinant IL-2 with/without IL-12 for 6 days. Following this, the tumoricidal activity was assessed in an overnight 5'chromium-release assay. Autologous tumor cells for measuring specific antitumor activity, Daudi cells susceptible to lymphokine-activated killer cells, and NK-susceptible K562 cells were used as target cells. MEASUREMENTS AND RESULTS: After treatment in vitro with IL-2, IL-12, or IL-2 plus IL-12, the Th pathway shifted from Th2 to Th1 type (increased gamma-interferon production). To further study the effect of cytokine treatment on the cytolytic activity of EALs, it was found that after 6-day culturing, the EALs failed to kill any of the three tumor targets, whereas the 6-day cultured peripheral blood lymphocytes (PBLs) gave low level of cytotoxicity against all three tumor targets. Stimulation with IL-2 alone partially restored the immunocompetence of EALs to kill the tumor targets. Stimulation with IL-12 alone showed no significant effect on their cytolytic activity. However, IL-12 synergized with IL-2 to increase the cytolytic activity of EALs and PBLs against autologous tumor targets. This synergistic effect was not found for Daudi cells and K562 cells. CONCLUSIONS: These results suggest that EALs activated with IL-12 in the presence of a low concentration of IL-2, which converted the EALs from Th2 pathway to Th1 pathway, could be an alternative source of antitumor effectors for adoptive immunotherapy of cancer.

Restoration of the immunocompetence by IL-2 activation and TCR-CD3 engagement of the in vivo anergized tumor-specific CTL from lung cancer patients.

The present study investigates the nature of the immunosuppressed state of the lymphocytes obtained from the malignant pleural effusion (effusion associated lymphocytes, EAL) of lung cancer patients. The immunocompetence of EAL from 13 patients was assessed by determining their T-helper cell phenotype, proliferative response to alpha CD3-activation, and their cytolytic activity against three tumor targets: the autologous tumor, Daudi, and K562. Flow cytometry analysis showed that the lymphocytes in EAL were predominantly T cells with < 1% natural killer cells. The T-helper cell phenotype was found to be predominantly of Th2 type, but could be readily converted to Th1 type by culturing the EAL in vitro, and this conversion was augmented by interleukin-2 (IL-2) or IL-2 plus alpha CD3. To test the cytolytic activity of EAL, it was found that after 6-day culturing, the EAL remained in an immunosuppressed state so that they failed to kill any of the three tumor targets. Stimulation with IL-2 partially restored the immunocompetence of EAL. Further engagement of TCR-CD3 by alpha CD3 fully restored the cytolytic activity of the EAL to kill the autologous tumor target but not Daudi or K562 tumor cells, and thus seemed to be tumor specific. The specificity was further confirmed by testing the activated EAL and normal donor peripheral blood lymphocytes against a variety of tumor targets and control targets. Furthermore, the killing by EAL against the autologous tumor target seemed to be major histocompatibility complex-restricted and was inhibited by anti-human leukocyte antigen class I antibody. The EAL from lung cancer patients also showed much reduced responsiveness to the alpha CD3 stimulation to induce proliferation, and addition of IL-2 restored the responsiveness. These results suggest that, through close contact with tumor cells, anergy of cytotoxic T lymphocytes (CTLs) was induced in vivo at a localized site. IL-2 stimulation and TCR-CD3 engagement could reverse the anergic state and restored the full competence of CTLs in EAL to mediate the specific anti-tumor killing against the autologous tumor. Proper manipulation of EAL may prove useful as a source of anti-tumor effectors for cancer adoptive immunotherapy.

Interleukin-2 and interleukin-7 augment the cytolytic activity and expand the antitumor killing spectrum of alpha CD3-induced activated killer cells: potential use in the immunotherapy of non-immunogenic tumors.

This study investigates the effect of different cytokines on the growth and antitumor activity of the alpha CD3-induced killer cells CD3-AK, and the potential of the use of CD3-AK cells in cancer immunotherapy. Eight cytokines were tested. Only three (interleukin-2, -4 and -7) were able to support the growth of CD3-AK cells, which selectively killed different tumor targets of diversified origin. Culturing in interleukin-4 (IL-4) or IL-7 alone could maintain the growth of CD3-AK cells for 6-8 days. Only IL-2 could maintain long-term growth, but further addition of IL-4 exerted an inhibitory effect, which terminated the cell growth in 2 weeks. In contrast, despite the fact that IL-7 inhibited the proliferation of CD3-AK cells cultured in IL-2, as determined by [3H]thymidine uptake, the recovery of viable cells was not reduced. In 10 days, CD3-AK cells cultured in IL-2 alone or IL-2 plus IL-7 increased 160- or 176-fold respectively. There is an inverse relationship between the in vitro growth ability and Fas expression on the CD3-AK cells. Further, IL-7 increased the cytolytic activity of the CD3-AK cells two- to threefold. CD3-AK cells could be maintained in IL-2 or IL-2 plus IL-7 for 60-240 days or more. The long-term-cultured CD3-AK cells not only possessed a high level of cytolytic activity, but also showed a wide spectrum of killing with different tumor targets; the normally "resistant" targets, such as EL-4 lymphoma, fibrosarcoma, or melanoma, became susceptible. When the in vivo antitumor activity of the CD3-AK cells against a non-immunogenic tumor. EL-4, was tested by tumor-neutralization experiments, we found that only the long-term-cultured cells gave significant protection, with those maintained in both IL-2 and IL-7 giving the highest degree of protection. Thus, these long-term-cultured CD3-AK cells may have the potential to be used for immunotherapy of a variety of tumors whatever their immunogenicity.

IL-2 and IL-4 mediate through two distinct kinase pathways for the activation of alphaCD3-induced activated killer cells.

The present study has examined the role of IL-2 and IL-4 in the regulation of different kinase pathways for the generation of alphaCD3-induced activated killer cells, CD3-AK. It has previously been shown that the IL-2 promoted CD3-AK cell response is mediated through a PKC (protein kinase C)-dependent pathway, which is susceptible to PKC inhibitors and resistant to inhibitors of PTK (protein tyrosine kinase), and that IL-4 synergized with IL-2 to induce CD3-AK cells. However, the IL-4-promoted CD3-AK cell response was PKC-independent as assessed by its resistance to PKC inhibitors. These findings suggest a dichotomy in the pathways leading to CD3-AK cell generation. To further determine whether IL-4 mediated a different kinase pathway to activate the T cells, we studied its effect on protein tyrosine phosphorylation. IL-4 up-regulated protein tyrosine phosphorylation in CD3-AK cells in a dose-dependent fashion, and resulted in increased levels of a number of phosphorylated proteins. Of particular note was the increase of tyrosine phosphorylated p56(lck) and p59(fyn) in CD3-AK cells. The changes in global protein tyrosine phosphorylation were correlated with the up-regulation by IL-4 of CD3-AK cell cytolytic activity, and the production of granzyme A. alphaIL-4 specifically blocked all the effects which were induced by IL-4. The PTK inhibitor genistein inhibited the IL-4-augmented cytolytic activity of CD3-AK cells as well as the IL-4-induced augmentation of protein tyrosine phosphorylation to the basal level of CD3-AK cells cultured in IL-2 alone. Consistent with a dichotomy in pathways for IL-2- and IL-4-mediated CD3-AK generation, genistein had no effect on the generation of CD3-AK cells cultured in IL-2 alone. Thus while PKC is primarily involved in the generation of IL-2-promoted CD3-AK cells, PTK appears to be required for the regulation of IL-4-promoted CD3-AK response.

Reversal of multiple-site tumor cell-induced immunosuppression by specific cytokines and pharmacological agents.

The present study explores a model for tumor cell-induced immunosuppression and reversal of suppression by cytokines and other pharmacological agents. To simulate tumor-cell-induced suppression, a panel of suppressor agents which included CsA (cyclosporin A), SSP (staurosporine), BSO (L-buthionine-[S,R]-sulfoximine) and PMA, and a panel of anti-suppressor agents which included IL-2, IL-4, GSH (glutathione) and amiloride, were tested. These suppressor/anti-suppressor agents acted differently on four specific sites of the immune arm that affected the alpha CD3-induced T cell proliferative and cytotoxic responses. They included (1) IL-2 production, (2) PKC-regulated cytolytic granule production, (3) GSH-regulated maturation of functional granules, and (4) granule exocytosis. When a single suppressor agent was used, all the suppressor agents tested in this study inhibited the generation of alpha CD3-induced activated killer cells (CD3-AK), whereas alpha CD3-induced proliferation was inhibited by CsA, BSO, and EL-4 tumor cells. Except for EL-4, suppression induced by a single suppressor agent could be corrected by an appropriate single anti-suppressor agent. Multiple suppressor agents induced profound suppression of CD3-AK response. In most cases, multiple anti-suppressor agents were required to correct the immune defects induced by multiple suppressor agents. Finally, EL-4 tumor-cell-induced immunosuppression could not be corrected by any single anti-suppressor agent tested, but a combination of IL-4, GSH and amiloride fully restored the CD3-AK response. These results suggest that tumor cells may induce multiple immune defects that require multiple anti-suppressor agents for correcting the defects to restore the host immunocompetence.

Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and alpha CD3-induced CD3-AK cell responses.

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR-CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the alpha CD3-induced CD3-AK cell response. First, the alpha CD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-L-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR-CD3. The former pathway is primarily PTK-dependent. Activation through TCR-CD3 is a more complex event.(ABSTRACT TRUNCATED AT 250 WORDS)

IL-4 regulation of perforin gene expression and BLT-esterase production in alpha CD3-induced activated killer cells.

This present study examines Il-4 regulation of perforin gene expression and cytolytic granule production in alpha CD3-induced activated killer cells CD3-AK. After stimulation of resting T cells with alpha CD3, proliferative response could be detected at 1 day after activation. The expression of perforin mRNA and production of cytolytic granules (using BLT-E as indicator) was detected on days 2-4, and this time course correlated with the generation of lytic CD3-AK cells. These findings indicate that killer cells generation is a late event during the course of alpha CD3 activation. Generation of CD3-AK cells is primarily PKC dependent and is blocked by the depletion or inhibition of PKC by PMA or SSP. These changes are accompanied by the suppression of perforin gene expression (mRNA) and BLT-E production. However, adding IL-4 into the cultures restored the perforin mRNA expression and BLT-E production, and also the cytolytic activity of the CD3-AK cells. Furthermore, for preactivated CD3-AK cells cultured in IL-2, SSP also suppressed the perforin mRNA and BLT-E with the concomitant reduction of cytolytic activity. Similar to the resting T cells, in the SSP-maintained preactivated CD3-AK cells, switching the cytokine from IL-2 to IL-4/IL-2 restored perforin mRNA expression and BLT-E production, with concomitant restoration of the cytolytic activity. In contrast, switching from IL-4/IL-2 gave the opposite effect. These results could be reproduced by using amiloride which also inhibited PKC activity but did not affect the growth of preactivated CD3-AK cells. These findings indicate that IL-4 may play a role in the late stage of alpha CD3 activation to regulate the expression of perforin gene and probably the translation process during the generation of activated killer cells.

Regulation by glutathione of the activation and differentiation of IL-4-dependent activated killer cells.

Glutathione (GSH) was shown to regulate the generation of IL-2-dependent activated killer cells. Generation of alpha CD3-activated killer cells CD3-AK was regulated by both IL-2 and IL-4. In the present study the role of GSH in the regulation of IL-4-dependent CD3-AK cells was examined. After initial activation of mouse splenocytes by alpha CD3, subculturing the CD3-AK cells in IL-4 resulted in the production of IL-4-dependent killer cells whose proliferative and cytolytic activities were abrogated by alpha IL-4 antibody 11B11. Adding graded doses of BSO, a GSH synthetase inhibitor, into CD3-AK cells culturing in IL-4 resulted in the reduction of their proliferative and cytotoxic responses. Adding exogenous GSH reversed the inhibitory effect of BSO and restored the proliferation and cytolytic activity of IL-4-dependent CD3-AK cells. The dose requirement for BSO to affect the IL-4-dependent CD3-AK cells was similar to that for the IL-2-dependent CD3-AK cells. These findings indicate that GSH also regulates the function of IL-4 in the activation and differentiation of CD3-AK cells. To further study the mechanism for the GSH regulation of the cytolytic activity of CD3-AK cells, we found that BSO did not reduce the production of BLT-esterase which contained mostly the cytolytic granules; in fact, BLT-esterase production was often increased by BSO. Furthermore, the exocytosis and effector function of cytolytic granules were also not affected by BSO. Thus it appears that reduction of cellular GSH may result in the accumulation of defective cytolytic granules which accounts for the reduction of killer cell cytolytic activity.

Role of protein kinase C and cytokines on the function and production of cytolytic granules in alpha CD3-activated killer-cell-mediated killing of tumor cells.

The effects of PMA and staurosporine (PKC depletor/antagonist) and IL-2/IL4 were used to determine the role of PKC and cytokine on alpha CD3-induced activated killer cells (CD3-AK). The present study examines their effects on the production of BLT-esterase and on the effector function of CD3-AK cells as well as the cytolytic granules. The production of BLT-esterase generally correlated with the cytolytic activity of CD3-AK cells and was reduced by PKC depletor/inhibitor but increased by IL-4. In studying the effector function of CD3-AK cells, we found that adding PMA or SSP at the effector phase inhibited the PKC-dependent slow lysis. PMA, but not SSP, also reduced fast lysis, which was shown to be a PKC-independent event. Additional experiments were performed to determine the effect of PKC on the lytic granules and to ascertain whether PMA has other effects on the effector-to-target relationship unrelated to PKC. It was found that neither PMA nor SSP affects the function of cytolytic granules, as measured by hemolytic assay against anucleated target (SRBC). These findings indicate that PKC has no direct effect on the granules. During testing against the nucleated tumor target through a novel approach using non-cytolytic surrogate killers, the lytic activity of the granules was inhibited by PMA, suggesting that exocytosis or delivery of granules to nucleated target cells may require mobilization of intracellular Ca2+ in the killer cells, and this process is inhibited by PMA. Our findings indicate that PKC and cytokines regulate the production but not the lytic activity of cytolytic granules. Nonetheless, delivery of cytolytic granules from killer cells to the nucleated tumor target appears to be a Ca(2+)-dependent event unrelated to PKC.

Dichotomy of glutathione regulation of the activation of resting and preactivated lymphocytes.

The present study has examined the effect of GSH on two lines of IL-2-dependent activated killer cells, LAK cells and alpha CD3-activated killer (CD3-AK) cells. We found that GSH added during first 24 hr decreased the generation of LAK and CD3-AK cells from resting lymphocytes, whereas after 48 hr of activation, the addition of GSH increased the killer cell activity. In addition, BSO, an inhibitor of GSH biosynthesis, decreased the proliferation and cytotoxic activities of activated killer cells, and the inhibitory effect was reversed by GSH. These results indicate that GSH downregulates the generation of LAK or CD3-AK cells from resting lymphocytes, but it upregulates the further differentiation of preactivated killer cells. The effect of GSH thus varied with the state of activation of the killer cells. Culturing CD3-AK cells in GSH did not change the distribution of T cell subsets, did not affect the cells' ability to produce lymphokine (IL-2), and did not induce suppressor cells. One striking change as revealed by flow cytometry analysis was that the levels of IL-2 receptor and TCR (alpha/beta)-CD3 were reduced by 80 and 30%, respectively, after 48 hr culturing in GSH. Determination of the mRNA of IL-2 receptor suggests that a post-transcriptional block existed. It appears that the negative effect of GSH on the function of surface IL-2 receptors or T cell receptors on resting lymphocytes severely affected the signal transduction through these receptors and thus abrogated or reduced LAK or CD3-AK cell response. In contrast, for preactivated killer cells, upregulation by intracellular GSH of IL-2 utilization is a dominant effect, thus allowing further differentiation of these killer cells. Our results indicate that the balance between the activation signal (IL-2 or alpha CD3) and the immunoregulatory signal (induced by GSH) may determine the outcome of the immune response.