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Background: Alcohol exposure induces TGFβ1 and renders the lung susceptible to injury and disrepair. We determined that TGFβ1 regulates myofibroblast differentiation through the loss of Thy-1 expression and consequent induction of α-SMA. TGFβ1 is important for T helper 17 (Th17) differentiation and IL-17 secretion, which in turn participates in tissue repair. We hypothesized that alcohol induces Th17 differentiation via TGFβ1 and that IL-17 produced by these cells contributes to the development of profibrotic lung myofibroblasts., Methods: Primary lung fibroblasts (PLFs) were treated with alcohol, TGFβ1, and IL-17 and then analyzed for Thy-1 expression and cell morphology. Naïve and Th17-polarized CD4 + T cells were exposed to alcohol and assessed for IL-17 expression. CD4 + T cells from alcohol-fed mice were analyzed for Th17 and IL-17 expression. Lungs of control-fed, bleomycin-treated and alcohol-fed, bleomycin-treated mice were analyzed for IL-17 protein expression., Results: Alcohol-treated PLFs expressed lower levels of Thy-1 than untreated cells. TGFβ1 or IL-17 exposure suppressed PLF Thy-1 expression. When administered together, TGFβ1 and IL-17 additively down-regulated Thy-1 expression. Exposure of naïve and Th17-polarized CD4 + T cells to alcohol induced the Th17 phenotype and augmented their production of IL-17. CD4 + Th17 + levels are elevated in the peripheral compartment but not in the lungs of alcohol-fed animals. Treatment of the PLFs with IL-17 and alcohol induced α-SMA expression. Induction of α-SMA and myofibroblast morphology by IL-17 occurred selectively in a Thy-1 - fibroblast subpopulation. Chronic alcohol ingestion augmented lung-specific IL-17 expression following bleomycin-induced lung injury., Conclusions: Alcohol exposure skews T cells toward a Th17 immune response that in turn primes the lung for fibroproliferative disrepair through loss of Thy-1 expression and induction of myofibroblast differentiation. These effects suggest that IL-17 and TGFβ1 contribute to fibroproliferative disrepair in the lung and targeting these proteins could limit morbidity and mortality following lung injury in alcoholic individuals., (© 2019 by the Research Society on Alcoholism.)

Background and Objective: Dendritic cells are able to prime and polarize naïve T cells towards either a T helper 1 or a T helper 2 response, depending on the antigen type and concentration, the costimulatory signals and the local cytokine millieu. In this investigation, we analyzed the response of human dendritic cells to stimulation with different concentrations of Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans., Material and Methods: Using different concentrations of P. gingivalis ATCC33277 and A. actinomycetemcomitans ATCC 33384, we determined the expression of the maturation markers CD80 and CD86 from purified human dendritic cells by flow cytometry. We also evaluated the mRNA expression levels for the cytokines interleukin-1beta, interleukin-2, interleukin-5, interleukin-6, interleukin-10, interleukin-12, interleukin-13, interferon-gamma, tumor necrosis factor-alpha and tumor necrosis factor-beta by quantitative real-time reverse transcription-polymerase chain reaction., Results: Both P. gingivalis and A. actinomycetemcomitans led to dendritic cell maturation, but the expression of CD80 was higher when the dendritic cells were stimulated with A. actinomycetemcomitans. Although both pathogens induced a T helper 1 pattern of cytokine expression, A. actinomycetemcomitans-stimulated dendritic cells expressed interleukin-1beta, interleukin-12, interferon-gamma, tumor necrosis factor-alpha and tumor necrosis factor-beta at lower bacterial concentrations than P. gingivalis. While 10(6) bacteria/mL of P. gingivalis or 10(4) bacteria/mL of A. actinomycetemcomitans induced expression of interleukin-12p40, the expression of other cytokines required 10 to 100-fold higher concentrations of bacteria., Conclusion: These results demonstrate that A. actinomycetemcomitans is a more potent immunogen than P. gingivalis because, at least in vitro, it induces stronger differentiation and activation of dendritic cells. In addition, our data also show that for a given strain, the bacterial load determines the pattern of cytokines that are expressed.

Single nucleotide polymorphisms (SNPs) of inflammatory cytokine genes were examined in 84 adult Japanese patients with chronic immune thrombocytopenic purpura (ITP) and 56 race-matched healthy controls. The SNPs examined were within the genes encoding tumour necrosis factor (TNF)-alpha (-238 G/A and -308 G/A), TNF-beta (+252 G/A), and interleukin (IL)-1beta (-511 C/T and +3953 T/C). Of these SNPs, the frequency of the TNF-beta (+252) G/G phenotype was significantly higher in ITP patients than in healthy controls (21% vs. 7%, P = 0.04, odds ratio = 3.6, 95% confidence interval 1.1-11.1), while no significant association was detected for the other SNPs. The distribution of the TNF-beta (+252) phenotype was not associated with human leucocyte antigen class II alleles or the therapeutic response in ITP patients. The frequency of circulating anti-glycoprotein IIb/IIIa antibody-producing B cells was significantly higher in ITP patients with the TNF-beta (+252) G/G phenotype than in those with the G/A or A/A phenotype (11.9 +/- 4.9 vs. 6.8 +/- 4.9 and 3.7 +/- 2.8 per 10(5) peripheral blood mononuclear cells; P = 0.02 and P < 0.001, respectively). These findings suggest that the SNP located at TNF-beta (+252) contributes to susceptibility to chronic ITP by controlling the autoreactive B-cell responses to platelet membrane glycoproteins.

Theiler's murine encephalomyelitis virus (TMEV) belongs to the Picornaviridae genus. DA subgroup strains of this virus induce early, non-fatal polioencephalomyelitis followed by demyelination in the spinal cord, with virus persistence. We investigated the use of DA strain as a vector for the introduction of a foreign gene into the central nervous system. Human lymphotoxin (LT) gene was inserted in the L region, the most upstream part of the polyprotein coding region of DA genome. Expression of LT was demonstrated by an immunoblot and an enzyme-linked immunosorbent assay on BHK-21 cells that were infected with the recombinant virus. In addition, the expressed LT showed cytotoxicity against L-929 cells.

The effect of cultivation conditions on the biosynthesis of human lymphotoxin in recombinant Escherichia coli SG20050/pLT21 strain was studied. Cells of the producing strain were grown in Luria broth containing chloramphenicol. The highest biomass yield of the recombinant strain and plasmid DNA stability were observed under these conditions. To enhance the level of lymphotoxin production an inoculate containing freshly obtained or frozen with glycerol transformants of the producing strain were used, and the cultivation process was performed at 32 degrees C. As a result, lymphotoxin was synthesized in a soluble form without the formation of inclusion bodies. A study of the protein synthesis dynamics during the cultivation of E. coli at 32 degrees C showed that the highest lymphotoxin activity was observed during the exponential growth phase, being maximal at the end of the exponential phase and at the beginning of the stationary phase. The set of indicated methods allowed us to maximize and stabilize the production of lymphotoxin in a biologically active form with a final yield of 18-20% from cell protein.

CD30 expression was evaluated by immunohistochemistry in lesional skin biopsies of eight patients with active atopic dermatitis (AD) and three patients with allergic contact (nickel-induced) dermatitis (ACD). CD30 expression was also assessed in a large panel of CD4+ and CD8+ T-cell clones generated from the skin biopsies of four patients with AD. Finally, the levels of soluble CD30 (sCD30) were measured in the serum of 41 patients with AD, 19 patients with ACD, and 60 healthy controls. In all specimens of lesional AD skin, where the great majority of infiltrating cells were CD4+ T cells, remarkable numbers of cells were CD30+, whereas virtually no CD30+ cells were found in the skin of patients with ACD. In CD4+ T-cell clones generated from the lesional AD skin, most of which produced both interleukin (IL)-4 and interferon-gamma (IFN-gamma) (Th0-like cells) or IL-4 and IL-5, but not IFN-gamma (Th2-like cells), CD30 expression directly correlated with the ability to produce IL-4 and IL-5, but was inversely related to IFN-gamma production. High levels of sCD30 (correlated with disease activity: r = 0.618) were detected in the serum of most AD patients, whereas there was no increase of sCD30 levels in the serum of patients with ACD. These data support the view that Th0/Th2-type responses predominate in the skin of patients with AD and suggest that the presence of CD30+ T cells in tissues and/or increased levels of sCD30 in biologic fluids are indicative of Th2-dominated responses.

We studied the physiological role of complement receptor type II (CR2, C3d/EBV receptor) expressed on T cells using MT-2 cells. First, we confirmed CR2 expression on MT-2 cells by flow cytometry and found that the MW of CR2 molecules on these cells and Raji B cells were the same by SDS-PAGE analysis. When MT-2 lysates were incubated with anti-CR2 mAb HB5 and thereafter with 32P-labeled ATP, 52- and 74-kDa proteins were phosphorylated, suggesting the activation of MT-2 cells through the complex of CR2 with these proteins. In this respect, we measured lymphotoxin production by MT-2 cells when incubated with C3d or EBV. The cytotoxicity of the MT-2 supernatant against L929 cells was elevated in a dose- and time-dependent manner. Next, we confirmed EBNA expression on EBV-infected MT-2 cells and attempted to establish an EBV-positive MT-2 clone by in vitro EBV infection. However, these clones disappeared during cloning. To clarify this mechanism, we examined the EBV genome in MT-2 cells. By Southern blot analysis, BamHI digestion of DNA extracts from MT-2 cells 3 days after EBV treatment gave a 3.0-kb signal which comigrated with the EBV BamHI-W probe. The 3.0-kb signal of genomic EBV-DNA was detected at 1, 2, 3, 5, and 7 days after EBV treatment, but could not be detected at 14 days. Thus, natural ligands of CR2 stimulate CR2-positive MT-2 cells through their functionally active CR2 molecules and in vitro EBV infection of MT-2 cells might be transient.

Different activation parameters of peripheral blood mononuclear cells (PBMC) from 31 patients with oral lichen planus (OLP) were examined and compared with 23 healthy donors. Impaired spontaneous (450 +/- 241 vs 1290 +/- 480 cpm) and mitogen-induced (39580 +/- 14470 vs 67000 +/- 11810 cpm) lymphocyte blastogenesis was observed in OLP patients. Furthermore, reduced cytokine production was found after phytohemagglutinin A (PHA) stimulation for all cytokines studied-tumour necrosis factor alpha (TNF alpha, 432.2 +/- 73.4 vs 979.8 +/- 46.3 units/ml), interleukin 2 (IL-2, 156.2 +/- 14.9 vs 572.6 +/- 12.9 pg/ml), interferon gamma (IFN gamma, 48.5 +/- 11.9 vs 82.6 +/- 12.4 pg/ml) and interleukin 6 (IL-6, 253.6 +/- 57.7 vs 1,419.0 +/- 279.6 units/ml)-except for interleukin 1 beta (IL-1 beta) and lymphotoxin (LT). In contrast, unstimulated culture supernatants showed increased TNF alpha (38.2 +/- 13.1 vs 8.0 +/- 0.2 units/ml), LT (10.2 +/- 2.2 units/ml vs < 0.4) and IL-6 (18.5 +/- 5.6 units/ml vs < 0.5) activity. Similarly, elevated concentrations of TNF alpha (19.6 +/- 6.3 units/ml) and IL-6 (22.9 +/- 4.7 units/ml) were detected in the sera of OLP patients. Combination of PHA and phorbol myristate acetate (PMA) could restore OLP proliferative T cell response and cytokine production to the level of healthy donors, whereas exogenous recombinant human IL-2 (rhuIL-2) plus PMA did not seem to be an effective stimulant for OLP T cells. These results indicate an alteration in the immune condition of OLP patients and an impairment in T lymphocyte function.

Human tumour necrosis factor-beta (TNF-beta) was produced in eukaryotic cells using the insect baculovirus cloning and expression system. A novel insect signal sequence, the honey-bee (Apis mellifera) prepromelittin secretory sequence, was used to aid in the post-translational modifications, glycosylation and secretion of recombinant human TNF-beta. Human TNF-beta cDNA was cloned using the insect baculovirus vector pAcC4s. Expression of the human TNF-beta was regulated by the insect Autographa californica nuclear-polyhedrosis-virus polyhedrin promoter. The 5' end of the TNF-beta cDNA was fused to the honey-bee prepromelittin signal sequence on the baculovirus vector. Insect [Spodoptera frugiperda (Sf9)] cells infected with the recombinant baculovirus secreted high levels of recombinant human TNF-beta into the culture medium. The amount of TNF-beta secreted by the Sf9 cells was estimated to be 28 micrograms of TNF-beta/ml of culture medium at 60-72 h post infection. The secreted human TNF-beta was a 22.5 kDa polypeptide which was glycosylated. Amino acid sequencing of the N-terminus of the recombinant human TNF-beta purified from the infected Sf9-cell culture confirmed that the secreted product was indeed human TNF-beta. This demonstrates that the honey-bee prepromelittin signal sequence was efficiently recognized and accurately cleaved in the Sf9 insect cells. The insect-derived TNF-beta exhibited a high cytotoxic activity similar to that of the native human TNF-beta when assessed by cytotoxic assays using murine L929 cells. Thus the insect baculovirus expression vector can be used for the production of abundant quantities of biologically active, glycosylated human TNF-beta protein.