DNA shuffling and vaccines.

One challenge of biotechnology is to find ways to optimize enzymes, cytokines, vaccines or transgenes in new contexts that are typically not found in nature. The approach of DNA shuffling is a test-tube process that takes advantage of recombination to generate libraries of chimeric genes, which can then be screened to identify the encoded proteins improved in one or more functions. DNA shuffling of two or more genes that are structurally similar and homologous in function is particularly efficacious in generating large libraries of functionally novel molecules. Other directed evolution methods, such as those involving directed or random mutagenesis, have several limitations compared to the DNA shuffling recombination process. A wide variety of genes have been submitted to DNA shuffling, and significant improvements in various functional parameters have been obtained. Several examples of the application of DNA shuffling to vaccine development, therapeutics and gene therapy are discussed here.

Monocyte-derived CD1a+ and CD1a- dendritic cell subsets differ in their cytokine production profiles, susceptibilities to transfection, and capacities to direct Th cell differentiation.

We describe a phenotypically and functionally novel monocyte-derived dendritic cell (DC) subset, designated mDC2, that lacks IL-12 synthesis, produces high levels of IL-10, and directs differentiation of Th0/Th2 cells. Like conventional monocyte-derived DC, designated mDC1, mDC2 expressed high levels of CD11c, CD40, CD80, CD86, and MHC class II molecules. However, in contrast to mDC1, mDC2 lacked expression of CD1a, suggesting an association between cytokine production profile and CD1a expression in DC. mDC2 could be matured into CD83+ DC cells in the presence of anti-CD40 mAbs and LPS plus IFN-gamma, but they remained CD1a- and lacked IL-12 production even upon maturation. The lack of IL-12 and CD1a expression by mDC2 did not affect their APC capacity, because mDC2 stimulated MLR to a similar degree as mDC1. However, while mDC1 strongly favored Th1 differentiation, mDC2 directed differentiation of Th0/Th2 cells when cocultured with purified human peripheral blood T cells, further indicating functional differences between mDC1 and mDC2. Interestingly, the transfection efficiency of mDC2 with plasmid DNA vectors was significantly higher than that of mDC1, and therefore mDC2 may provide improved means to manipulate Ag-specific T cell responses after transfection ex vivo. Taken together, these data indicate that peripheral blood monocytes have the capacity to differentiate into DC subsets with different cytokine production profiles, which is associated with altered capacity to direct Th cell differentiation.

Interleukin-15 up-regulates the expression of CD154 on synovial fluid T cells.

To investigate the role of the CD40-CD154 interaction in rheumatoid arthritis (RA), we analysed the expression of CD154 on CD3+ and CD4+ T cells in synovial fluid (SF) from patients with RA and in peripheral blood (PB) from patients and normal controls. As interleukin (IL)-15 is a potent activator of synovial T cells we wanted to study whether IL-15 also regulated the expression of CD154 on these T cells. Freshly isolated synovial T cells did not express significant levels of CD154, as evaluated using flow cytometry, whereas the expression of CD86 and human leucocyte antigen (HLA)-DR was significantly elevated on SF T cells when compared with PB T cells from patients or controls. Synovial T cells could up-regulate their CD154 expression following activation with phorbol 12-myristate 13-acetate (PMA) + ionomycin or anti-CD3 + anti-CD28 monoclonal antibodies (mAbs), but the maximal level of expression remained lower than in control T cells. IL-15 significantly increased the expression of CD154 on SF and PB T cells from patients, whereas IL-2 had minimal effects. Furthermore, IL-15 induced extensive proliferation in SF T cells. Our results show that SF T cells up-regulate the expression of CD154 in the presence of IL-15, a cytokine present in the synovium of patients with RA. These results further emphasize the role of IL-15 in the pathogenesis of RA.

Regulation of UT-OC-3 ovarian carcinoma cells by cytokines: inhibitory effects on cell proliferation and activation of transcription factors AP-1 and NF-kappaB.

The present study was designed to investigate the growth regulatory effects of cytokines in UT-OC-3 ovarian cystadenocarcinoma cells in vitro. The effects of interleukin-6 (IL-6), interferons alpha (IFN-alpha) and gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor alpha (TNF-alpha), and transforming growth factor beta1 (TGF-beta1) were investigated by (125)I-deoxyuridine ((125)IUdR) incorporation assay. In order to understand better the molecular mechanisms of the observed effects, the activation of DNA-binding proteins was studied by electrophoretic mobility shift assay. In addition, cellular DNA was tested by fragmentation analysis to determine if the most growth inhibitory cytokines are able to induce programmed cell death (apoptosis). After 48h in culture, TGF-beta1, TNF-alpha, IFN-alpha and IL-6 showed a clear inhibitory effect on (125)IUdR incorporation (P<0.005), and IFN-gamma and GM-CSF caused even more significant inhibition (P<0.001). IFN-alpha and IFN-gamma were both growth inhibitory after 72h in culture (P<0.005). Similarly, GM-CSF induced a slight inhibition (P<0.05), whereas TGF-beta1 and TNF-alpha almost blocked DNA synthesis (P<0.001) after 72h. IL-6 had no statistically significant effect on cell proliferation after 72h. Transcription factors AP-1 and NF-kappaB were both constitutively expressed in UT-OC-3 cells. The binding activity of AP-1 was found to be stimulated by the growth inhibitory cytokines, TGF-beta1 and TNF-alpha, and the binding of NF-kappaB was stimulated by TNF-alpha. Apoptosis does not seem to be induced by any of these cytokines in the UT-OC-3 ovarian cancer cell model.

Molecular breeding of allergy vaccines and antiallergic cytokines.

Molecular breeding, also called DNA shuffling, is a technology that enables the generation of large libraries of novel genes and vectors, from which improved variants can be selected based on functional properties. In a common format, it involves recursive recombination and mutation, performed by random fragmentation of related DNA sequences, followed by reassembly of the fragments in a self-priming polymerase chain reaction. As in natural evolution, the technique takes advantage of crossovers, deletions, insertions, inversions and point mutations of genes to generate large pools of related sequences. Molecular breeding can be used to generate improved variants of proteins used as therapeutics, such as vaccine antigens, growth factors and immunomodulatory molecules. Moreover, the technology can be applied to evolve entire viruses or vectors, including DNA vaccines. Cytokines downregulating allergic immune responses and allergens are attractive targets for evolution by molecular breeding. This review describes approaches to generate chimeric allergens with T cell epitopes from multiple allergen homologues, while reducing the recognition by preexisting IgE. In addition, the results and applications of molecular breeding in the evolution of improved antiallergic cytokines are discussed.

Evolution of a cytokine using DNA family shuffling.

DNA shuffling of a family of over 20 human interferon-alpha (Hu-IFN-alpha) genes was used to derive variants with increased antiviral and antiproliferation activities in murine cells. A clone with 135,000-fold improved specific activity over Hu-IFN-alpha2a was obtained in the first cycle of shuffling. After a second cycle of selective shuffling, the most active clone was improved 285,000-fold relative to Hu-IFN-alpha2a and 185-fold relative to Hu-IFN-alpha1. Remarkably, the three most active clones were more active than the native murine IFN-alphas. These chimeras are derived from up to five parental genes but contained no random point mutations. These results demonstrate that diverse cytokine gene families can be used as starting material to rapidly evolve cytokines that are more active, or have superior selectivity profiles, than native cytokine genes.

Synovial fluid T cells from patients with rheumatoid arthritis are refractory to the T helper type 2 differentiation-inducing effects of interleukin-4.

The balance between T helper type 1 (Th1) and Th2 cytokines is thought to be important in the initiation and outcome of autoimmune diseases. The goal of the present study was to compare the production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) by synovial fluid (SF) and peripheral blood (PB) CD4+ and CD8+ cells from patients with rheumatoid arthritis (RA) using three-colour immunofluorescence staining and flow cytometry, and to investigate the capacity of IL-4, IL-10 and IL-12 to modify the cytokine production profile of SF T cells. The frequency of IFN-gamma-producing CD4+ and CD8+ cells was significantly increased in SF when compared with PB. In contrast to IFN-gamma, the expression of IL-4 in SF and PB T cells was comparable. The majority of IL-4-producing cells in SF belonged to Th0/T cytotoxic (Tc) type 0 phenotype, whereas there were significantly more Th2/Tc2 cells in PB than in SF. Interestingly, IL-4 was unable to induce differentiation of non-adherent SF mononuclear cells (SFMC) into Th2 cells, whereas PB mononuclear cells (PBMC) under similar culture conditions differentiated into cells producing high levels of IL-4, IL-10 and IL-13. In contrast, there were no major differences in the effects of IL-10 and IL-12 on the cytokine production profile of SFMC when compared with PBMC. Taken together, the present results suggest that SF T cells from patients with RA are terminally differentiated into Th1/Tc1-like phenotype, and Th2/Tc2 differentiation-inducing agents, such as IL-4, may not be able to reverse the inflammatory process occurring in the joints.

Expression of soluble human signaling lymphocytic activation molecule in vivo.

BACKGROUND: Signaling lymphocytic activation molecule (SLAM) is a novel glycoprotein expressed on activated T and B cells. Ligation of cell surface SLAM, either by anti-SLAM mAbs or the recombinant soluble form of SLAM (sSLAM), enhanced the proliferation of T and B cells in vitro. In addition, the engagement of SLAM on T cells preferentially induced IFN-gamma production even by allergen-specific TH2 clones. OBJECTIVE: In this study we investigated the expression of sSLAM in vivo in healthy individuals and in disease conditions that are associated with increased TH1 - or TH2 -cell responses. METHODS: The expression of mRNA encoding sSLAM in peripheral blood and synovial fluid (SF) lymphocytes was studied by using reverse transcriptase-PCR, and the presence of sSLAM protein in serum and SF samples was investigated by using a specific ELISA. RESULTS: Lymphocytes from patients with rheumatoid arthritis (RA) and healthy individuals consistently expressed mRNA encoding sSLAM. In addition, sSLAM protein was present in 38% of serum and 54% of SF samples from patients with RA and in 47% of serum samples from healthy individuals. The levels of sSLAM in positive serum and SF samples from patients with RA and in positive serum samples from healthy individuals were not significantly different. In contrast, the levels of sSLAM were significantly lower in patients with reactive arthritis or in patients with elevated IgE levels than in patients with RA. Similarly, the frequency of positive SF samples was significantly lower in reactive arthritis (28%) than in RA (54%). CONCLUSION: These results indicate that sSLAM is present in serum and SF, further suggesting that sSLAM regulates T- and B-cell function in vivo. Moreover, these data suggest an association between low sSLAM production and the occurrence of TH2 responses in vivo.

Interleukin-10 inhibits the capacity of synovial macrophages to function as antigen-presenting cells.

OBJECTIVE: We have investigated the effects of interleukin (IL)-10, IL-4 + granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor alpha (TNF-alpha) on the phenotype and antigen-presenting capacity of synovial fluid (SF) macrophages from patients with rheumatoid arthritis. METHODS: The effects of IL-4, IL-10, GM-CSF and TNF-alpha on the expression of surface antigens on SF macrophages were studied using flow cytometry. The effects of these cytokines on the capacity of SF macrophages to activate T cells was investigated using the allogeneic mixed lymphocyte reaction (MLR). RESULTS: IL-10 reduced the expression of CD40, CD86 and HLA-DR, and increased the expression of CD14, on SF macrophages. IL-10 had no effect on the expression of CD80. Importantly, these effects of IL-10 on the phenotype of SF macrophages appear to have functional consequences, because cells incubated with IL-10 had a significantly reduced capacity to activate T cells in MLR. The effects of IL-4, GM-CSF and TNF-alpha were generally opposite to those observed in response to IL-10. IL-4 + GM-CSF, a combination of cytokines known to induce differentiation of dendritic cells, increased the expression of CD40, CD80 and CD86, and decreased the expression of CD14 on SF macrophages. Accordingly, IL-4 + GM-CSF increased the capacity of SF macrophages to activate T cells in MLR. IL-10 inhibited the effects of IL-4 + GM-CSF on SF macrophages. CONCLUSIONS: IL-10 inhibits the antigen-presenting capacity of SF macrophages, which further emphasizes the anti-inflammatory potential of IL-10 in RA. Importantly, IL-10 is able to downregulate the APC function of SF macrophages even when they are efficiently activated.

Cytokine production profiles in the peritoneal fluids of patients with malignant or benign gynecologic tumors.

BACKGROUND: Cytokines play a key role in the regulation of cells of the immune system and also have been implicated in the pathogenesis of malignant diseases. Some cytokines have been shown to have potential in the diagnosis of cancer. METHODS: A total of 111 patients with ovarian, cervical, or endometrial carcinomas or benign ovarian or uterine tumors were enrolled on the study, and the levels of interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-gamma, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF), and tumor necrosis factor (TNF)-alpha were measured by cytokine specific, enzyme-linked immunoadsorbent assays. In addition, ratios of IL-2, IL-4, and IFN-gamma production were studied to characterize the type of T-cell response that occurred in the peritoneal cavities of the patients. RESULTS: High levels of M-CSF (mean for all patients, 26,050 pg/mL) and G-CSF (mean for all patients, 20,267 pg/mL) were observed in virtually all patients, but no significant differences between the study groups were observed. Similarly, no differences in the levels of IL-2, IL-4, IL-10, IFN-gamma, GM-CSF, or TNF-alpha were found. However, IL-6 levels were significantly higher in patients with ovarian carcinoma (mean +/- standard error of the mean [SEM]: 5572 +/- 1266) or benign tumors (mean +/- SEM: 4474 +/- 2008) than in those with cervical (mean +/- SEM: 1222 +/- 546) or endometrial carcinoma (mean +/- SEM: 1977 +/- 616). A predominantly Th1 type cytokine profile, irrespective of the diagnosis, was observed in patients with gynecologic tumors. CONCLUSIONS: With the exception of IL-6, the cytokine synthesis profiles in the peritoneal fluids of patients with benign and malignant gynecologic tumors were found to be similar. These results suggest that cytokine production in these patients is a result of nonspecific inflammation rather than a specific response against the tumor cells, and that skewing of cytokine synthesis toward either the Th1 or the Th2 phenotype is not the underlying mechanism resulting in the malignant process in women with gynecologic tumors.

Inhibitory effects of cytokines on ovarian and endometrial carcinoma cells in vitro with special reference to induction of specific transcriptional regulators.

In the present study, we have investigated the effects of interferons-alpha (IFN-alpha) and -gamma (IFN-gamma), interleukin-10 (IL-10) and -13 (IL-13), transforming growth factor-beta1 (TGF-beta1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha) on cell proliferation and induction of transcription factors AP-1 and NF-kappaB in UM-EC-3 human endometrial adenocarcinoma cells and UT-OC-5 ovarian carcinoma cells in vitro. In addition, cellular DNA was extracted to study if any of these factors is able to induce apoptosis. In UM-EC-3 cell line DNA synthesis was inhibited by GM-CSF, IL-10, IL-13, TGF-beta1, IFN-alpha, and IFN-gamma after 48 and 72 h in culture, whereas TNF-alpha had no significant effect on cell proliferation in any of the experiments. The inhibition of DNA synthesis was similarly observed in UT-OC-5 ovarian carcinoma cells by IL-10, TNF-alpha, and IFN-gamma after 48 and 72 h, whereas IFN-alpha had no statistically significant effect. An inhibitory effect of GM-CSF was observed only after 48 h and TGF-beta after 72 h in culture, respectively. Transcription factors AP-1 and NF-kappaB were both constitutively active in UM-EC-3 and UT-OC-5 cells. The binding activity of AP-1 was found to be stimulated by all growth-inhibitory cytokines studied in both cell lines, whereas the specific binding activity of NF-kappaB was affected moderately only by TNF-alpha in UT-OC-5 ovarian carcinoma cells. No signs of DNA fragmentation typical of apoptosis were observed in any of these studies.

Reversal of human allergic T helper 2 responses by engagement of signaling lymphocytic activation molecule.

Allergen-specific Th2 cells accumulate at high frequencies in the skin of patients with atopic dermatitis (AD), where they contribute to the induction and maintenance of the lesions that are characteristic for the disease. Attenuation of these lesions in response to successful therapy is associated with a reduction in IL-4-producing Th2 cells and the appearance of IFN-gamma-producing Th cells. In this study, we demonstrate that engagement of the signaling lymphocytic activation molecule (SLAM) by an agonistic mAb, during allergen-specific expansion of highly polarized Th2 cell populations derived from skin biopsies of AD patients, results in the generation of stable populations of IFN-gamma-producing cells. SLAM-mediated reversal of Th cell phenotype has important biologic consequences, because supernatants of these activated, allergen-specific Th cells fail to induce IgE synthesis by purified B cells costimulated by anti-CD40 mAbs. Thus, highly polarized, allergen-specific Th2 cell populations derived from the skin of AD patients can be reversed into Th cell populations that contain IFN-gamma-producing cells and that do not support IgE synthesis. These results define a new mechanism to promote Th0/Th1 differentiation and suggest a potential role for anti-SLAM mAbs in the treatment of Th2-mediated allergic diseases.

Increased expression of signaling lymphocytic activation molecule in patients with rheumatoid arthritis and its role in the regulation of cytokine production in rheumatoid synovium.

In the present study the expression and function of signaling lymphocytic activation molecule (SLAM) in lymphocytes from patients with rheumatoid arthritis (RA) were investigated. The expression levels of SLAM were significantly up-regulated on synovial fluid and synovial tissue T cells from patients with RA compared with peripheral blood T cells from the same patients or from healthy volunteers. In addition, the expression of SLAM on peripheral blood B cells from patients with RA was elevated compared with that in healthy volunteers. SLAM+ T cells in synovial fluid coexpressed CD45RO and demonstrated decreased expression of CD27, indicative of a primed phenotype. In addition, the activation state of SLAM+ T cells was enhanced, as judged by increased expression of CD25, CD28, CD69, and CD95 on these cells. Interestingly, SLAM expression on activated CD4+ and CD8+ T cells from both patients and healthy individuals could be down-regulated by IL-10, which has been previously shown to function as an anti-inflammatory molecule in rheumatoid synovium. Furthermore, anti-SLAM mAbs increased the production of IL-10, IFN-gamma, and TNF-alpha by in vitro activated synovial fluid mononuclear cells, supporting the idea that signaling through SLAM may play a role in the regulation of synovial inflammation in patients with RA. Given the fact that SLAM was recently shown to be a high affinity self ligand, our data suggest that synovial T cells may stimulate their own cytokine production through homophilic SLAM-SLAM interactions.

SLAM and its role in T cell activation and Th cell responses.

Following the initial events of T cell activation, triggered by binding of specific peptide-MHC complex to the TCR for antigen and engagement of costimulatory molecules, a number of activation molecules are expressed on the cell surface. Many of these molecules regulate T cell function, T-T cell interactions and the interaction of T cells with other cells. One such molecule is SLAM, a multifunctional 70 kDa glycoprotein member of the Ig superfamily with multiple isoforms. SLAM is rapidly induced on naive T cells and B cells following activation. Engagement of SLAM by a specific antibody (mAb A12) results in IL-2-independent T cell expansion and induction/up-regulation of IFN-gamma by activated T cells, including Th2 cells. SLAM was found to be a high-affinity self-ligand mediating molecular and cellular homophilic interactions. In this review we discuss SLAM as a receptor involved in T cell expansion and in directing immune responses to a Th0-Th1 pathway.

Soluble and membrane-bound forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B lymphocytes.

In this study it is shown that both membrane-bound and soluble forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B cells. Activated B cells express the membrane-bound form of SLAM (mSLAM), the soluble (s) and the cytoplasmic (c) isoforms of SLAM, and the expression levels of mSLAM on B cells are rapidly upregulated after activation in vitro. Importantly, recombinant sSLAM and L cells transfected with mSLAM efficiently enhance B cell proliferation induced by anti-mu mAbs, anti-CD40 mAbs or Staphylococcus aureus Cowan I (SAC) in the presence or absence of IL-2, IL-4, IL-10, IL-12, or IL-15. sSLAM strongly enhances proliferation of both freshly isolated B cells and B cells derived from long-term in vitro cultures, indicating that SLAM acts not only during the initial phase of B cell activation but also during the expansion of preactivated B cells. In addition, sSLAM enhances production of IgM, IgG, and IgA by B cells activated by anti-CD40 mAbs. SLAM has recently been shown to be a high affinity self-ligand, and the present data suggest that signaling through homophilic SLAM-SLAM binding during B-B and B-T cell interactions enhances the expansion and differentiation of activated B cells.

IL-4 synergizes with IL-10 and anti-CD40 MoAbs to induce B-cell differentiation in patients with common variable immunodeficiency.

In the present study the phenotype and function of lymphocytes from patients with common variable immunodeficiency (CVI) were studied. Five out of 12 patients had abnormally low proportion of CD4+ T cells, but PBMC of these patients were capable of proliferating in response to polyclonal T-cell mitogens or PPD antigen. The phenotype of patients' B cells, as determined by expression of CD10, CD19 and CD34, was comparable to that of healthy controls. IL-4 and anti-CD40 MoAbs induced moderate B-cell differentiation in PBMC derived from patients with CVI, but the frequencies of Ig-secreting cells were generally at levels spontaneously observed in healthy controls. IL-10 was completely ineffective in inducing IgG-secreting cells in cultures of PBMC derived from patients with CVI even in the presence of anti-CD40 MoAbs, whereas high frequencies of Ig-secreting cells were induced under similar condition in cultures of PBMC derived from healthy controls. Importantly, when IL-4 was added to cultures stimulated with anti-CD40 MoAbs and IL-10, a very strong synergistic effect on the numbers of Ig-secreting cells and the levels of Ig secretion was observed in PBMC from both patients and controls. Moreover, the frequencies of Ig-secreting cells after activation with anti-CD40 MoAbs, IL-4 plus IL-10 in PBMC from some patients were comparable to those observed in PBMC from healthy controls. Taken together, these results indicate that B cells from patients with CVI have impaired capacity to differentiate into Ig-secreting cells in response to IL-10 and anti-CD40 MoAbs, and that this unresponsiveness can be restored by exogenous IL-4 in a proportion of the patients.

Regulation of proliferation of UM-SCV-1A and UM-SCV-6 vulvar carcinoma cells by cytokines.

The biology and pathogenesis of vulvar carcinoma are poorly understood at present. In order to understand this disease better, we have used recently developed squamous cell carcinoma lines of the vulva as models. Two cell lines originating from two individuals (UM-SCV-1A and UM-SCV-6) were cultured in vitro in 10% fetal calf serum. The effects of interleukins 10 and 13, interferons alpha and gamma, granulocyte/macrophage-growth-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta (TGFbeta) on the proliferation of the cells was investigated by using radioactively labelled uridine as tracer. In addition, an investigation on the molecular structure of extracted cellular DNA was carried out to investigate whether programmed cell death (apoptosis) would be inducible by any of the factors. In UM-SCV-1A cells, interleukin-10 (IL-10) and interleukin-13 (IL-13) caused an approximately 12-fold decrease in DNA synthesis in cells cultured for 72 h (P<0.001), while GM-CSF had no significant effect. TGFbeta showed a significant inhibitory effect on deoxyuridine incorporation (P<0.001), which was 2.0- and 4.2-fold at 48 h and 72 h, respectively. TFG alpha showed a 1.2-fold inhibitory effect on DNA synthesis at 48 h (P<0.01) and a 1.5-fold inhibition at 72 h (P<0.05). Interferon gamma (IFNgamma) showed an inhibitory effect on DNA synthesis (1.3-fold; P<0.01). In UM-SCV-6 cells, both IL-10 and IL-13 showed inhibitory effects on deoxyuridine incorporation (1.3- and 1.4-fold at 48 h, respectively; P<0.001) that were even more pronounced at 72 h (2.4- and 2.5-fold respectively; P<0.001). IFNgamma caused a 3.6-fold inhibition of DNA synthesis by UM-SCV-6 cells at 72 h (P<0.001). Both TFGbeta and TNF alpha inhibited uridine incorporation (3.0- and 1.6-fold at 48 h, respectively; 2.7-fold at 72 h for both factors). GM-CSF inihibited DNA synthesis by UM-SCV-6 cells 1.3- 2.0-fold at 48 h and 72 h, respectively. In dose/response analyses, the effect of INF alpha on DNA synthesis was inhibitory in both cell lines at 48 h, while stimulatory effects were observed at 72 h. Electrophoretic analyses of DNA isolated from cells cultured in the presence or absence of different factors did not reveal DNA fragmentation. All cytokines, with the exception of IFN alpha, showed inhibitory effects on DNA synthesis by vulvar carcinoma cells. Of the factors studied, the recently described interleukins 10 and 13 showed potent inhibition of cell growth, encouraging further investigation on the molecular mechanisms of the observed inhibition. Apoptosis does not seem to be induced in the two vulvar carcinoma cell lines by any of the cytokines studied.

Pro- and anti-inflammatory cytokines in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by the accumulation of inflammatory cells into the synovium and the destruction of joints. Cytokines are important regulators of the synovial inflammation. Some cytokines, such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-1, function by promoting inflammatory responses and by inducing cartilage degradation. Other cytokines, such as IL-4, IL-10 and IL-13, function mainly as anti-inflammatory molecules. Although anti-inflammatory cytokines are present in rheumatoid joints, in progressive RA their levels obviously are too low to neutralize the deleterious effects of proinflammatory cytokines. Inhibiting the action of proinflammatory cytokines by using specific cytokine inhibitors or anti-inflammatory cytokines is the basis for new therapies currently tested in patients with RA. Promising results on the use of neutralizing anti-TNF-alpha monoclonal antibodies in the treatment of RA have been reported. The results from a trial using recombinant IL-10 in the treatment of patients with RA are available in the near future and will be important in determining the therapeutic potential of this cytokine.

The relative contribution of IL-4 and IL-13 to human IgE synthesis induced by activated CD4+ or CD8+ T cells.

The relative contribution of IL-4 and IL-13 to the regulation of IgE synthesis has remained relatively poorly characterized, partially because of lack of suitable animal models. We have studied the roles of IL-4 and IL-13 in human IgE synthesis induced by supernatants derived from activated CD4+ or CD8+ T cell clones. Neutralizing anti-IL-4 and anti-IL-13 monoclonal antibodies (mAbs) inhibited IgE synthesis induced by anti-CD40 mAbs and supernatants from CD4+ T cells by an average 61% and 42%, respectively (n = 25). Recombinant IL-13 had additive effects on IL-4-induced IgE synthesis, but only when IL-4 was present at low concentrations. Accordingly, IL-4 was the dominant IgE synthesis-inducing cytokine derived from highly polarized T helper (TH)2 cells. However, anti-IL-13 mAbs also significantly inhibited IgE synthesis induced by two of three supernatants derived from allergen-specific T(H2)-like cell lines generated from the skin of patients with atopic dermatitis. Furthermore, anti-IL-13 mAbs almost completely inhibited IgE synthesis induced by supernatants from T(H1) cells or CD8+ T cell clones. Taken together, these data indicate that IL-13, in addition to IL-4, contributes to IgE synthesis induced by all T helper cell subsets, including allergen-specific T(H2) cells. Moreover, IL-13 appears to be the major IgE synthesis-inducing cytokine derived from T(H1) cells or CD8+ T cells.