IFN-gamma mRNA Research Articles

Foxp3 Expression Is Induced in Peripheral Blood Mononuclear Cells from Patients with CTCL and GVHD after Extracorporeal Photopheresis

Anti-tumor immunity mediated by cytotoxic CD8+ T lymphocytes (CTL) is thought to underlie the efficacy of extracorporeal photopheresis (ECP) for cutaneous T cell lymphoma (CTCL). In contrast, induction of immune tolerance mediated by regulatory T cells (Treg) is hypothesized to underlie the effect of ECP for graft-versus-host disease (GVHD). In this translational research study, peripheral blood mononuclear cells (PBMC) from patients (Pt) with Sézary Syndrome (SS), a leukemic form of CTCL, and GVHD pre- and post-ECP were studied for expression of the CTL cytokine IFN-gamma and of the nuclear transcription factor forkhead box P3 (Foxp3), a specific marker of Treg. Total RNA from PBMCs at pre-ECP, 2 day, 1 month, 3 months, and 6 months post- ECP were quantitated by real-time quantitative reverse transcript-PCR (RT-Q-PCR) for Foxp3 and IFN-gamma mRNA expression levels, normalized to endogenous control gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Foxp3 levels at baseline varied but increased at 2 day and 1 month post-ECP in GVHD by 9.5-fold and 27.6-fold, respectively (Fig.1). Similarly,foxp3 also increased in 3 of 4 CTCL patients (Pt#1, #2 and #4). Foxp3 levels rose steadily in clinical responders, Pt#1 and Pt#2, during the treatment course, and highest levels were detected at 6 months post-ECP with 13.1-fold and 17.4-fold increase, respectively. Pt#1 and #2 experienced partial clinical response and decreased circulating tumor cells with ECP. The levels of interferon-gamma had only < 2-fold up or down over the treatment course from baseline among CTCL post-ECP samples. While the level of IFN-gamma was decreased by half at 2 day and 1 month post-ECP in GVHD compared to that in pre-ECP (Fig.2). These results suggest that Tregs (detected by Foxp3) but not IFN-gamma are induced during ECP in responding CTCL patients as in GVHD. ECP might thus have common mechanisms in these two T cell mediated diseases. The study will continue to enroll patients to further study the effects of ECP. [Display omitted] [Display omitted]

Analyses of Vaccination Protocols for Leptospira interrogans Serovar Autumnalis in Hamsters

Leptospirosis, caused by Leptospira spp., is a zoonotic disease found worldwide. Killed whole cell leptospiral vaccines have been used as effective vaccines to elicit specific antibodies for protection. However, the involvement of cytokine responses after vaccination is not well characterized. Hamsters were immunized with killed L. interrogans serovar Autumnalis before challenge to study cytokine mRNA expression levels (interferon [IFN]-gamma, tumor necrosis factor [TNF]-alpha, interleukin [IL]-10, and IL-4). Vaccinated groups showed 92-100% survival rates, whereas control hamsters died within 6-10 days. However, live organisms were detected in vaccinated groups, and mild to moderate pathology was observed early in infection. IFN-gamma and TNF-alpha mRNA expression levels correlated with the severity of infection and lung pathology, whereas IL-4 and IL-10 expression levels were significantly higher in vaccinated groups. In summary, commonly used vaccines changed the cytokine profiles and protected hamsters from death but failed to stimulate sterile immunity and were unable to prevent the occurrence of pathology.

Intratumoral rhIL‐12 administration in head and neck squamous cell carcinoma patients induces B cell activation

The objectives of this study were to investigate the effects of intratumorally (i.t.) administered recombinant human interleukin-12 (rhIL-12) on the distribution and function of B cells in the primary tumors, the locoregional lymph nodes and peripheral blood of head and neck squamous cell carcinoma (HNSCC) patients. The initial characterization of the patients participating in the phase Ib and phase II studies has previously been reported. After rhIL-12 treatment, fewer secondary follicles with a broader outer region of the mantle zones and an increase in interfollicular B-blasts were seen in the enlarged lymph nodes compared with control HNSCC patients. The size of the germinal center (GC) was diminished, partly due to a decrease in the number of CD57+ GC cells that have been associated with immune suppression. These changes did not correlate with signs of apoptosis or CXCR5 expression by B cells. Strikingly, in 3 out of 4 IL-12 treated patients, increased IFN-gamma mRNA expression by B cells was detected. In addition, a highly significant IgG subclass switch was seen in the plasma with more IgG1, less IgG2 and more IgG4, indicating a switch to T helper 1 phenotype. Finally, peritumoral B cell infiltration was a positive prognostic sign for overall survival in the 30 HNSCC patients investigated, irrespective of IL-12 treatment. In conclusion, these data indicate that after i.t. IL-12 treatment in HNSCC, significant activation of the B cell and the B cell compartment occurred and that the presence of tumor infiltrating B cells correlated with overall survival of HNSCC patients.

Hyperbaric Oxygen Prevents Early Death Caused by Experimental Cerebral Malaria

BackgroundCerebral malaria (CM) is a syndrome characterized by neurological signs, seizures and coma. Despite the fact that CM presents similarities with cerebral stroke, few studies have focused on new supportive therapies for the disease. Hyperbaric oxygen (HBO) therapy has been successfully used in patients with numerous brain disorders such as stroke, migraine and atherosclerosis.Methodology/Principal FindingsC57BL/6 mice infected with Plasmodium berghei ANKA (PbA) were exposed to daily doses of HBO (100% O2, 3.0 ATA, 1–2 h per day) in conditions well-tolerated by humans and animals, before or after parasite establishment. Cumulative survival analyses demonstrated that HBO therapy protected 50% of PbA-infected mice and delayed CM-specific neurological signs when administrated after patent parasitemia. Pressurized oxygen therapy reduced peripheral parasitemia, expression of TNF-α, IFN-γ and IL-10 mRNA levels and percentage of γδ and αβ CD4+ and CD8+ T lymphocytes sequestered in mice brains, thus resulting in a reduction of blood-brain barrier (BBB) dysfunction and hypothermia.Conclusions/SignificanceThe data presented here is the first indication that HBO treatment could be used as supportive therapy, perhaps in association with neuroprotective drugs, to prevent CM clinical outcomes, including death.

TH-17 cells in rheumatoid arthritis

IntroductionThe aim of this study was to quantify the number of T-helper (TH)-17 cells present in rheumatoid arthritis (RA) synovial fluid (SF) and to determine the level of interleukin (IL)-17 cytokine in RA, osteoarthritis (OA) and normal synovial tissue, as well as to examine SF macrophages for the presence of IL-23, IL-27 and interferon (IFN)-γ.MethodsPeripheral blood (PB) mononuclear cells from normal and RA donors and mononuclear cells from RA SF were examined either without stimulation or after pretreatment with IL-23 followed by stimulation with phorbol myristate acetate (PMA) plus ionomycin (P/I). The abundance of TH-17 cells in RA SF was determined by flow cytometry. IL-17 levels were quantified in synovial tissue from RA, OA and normal individuals by ELISA and IL-23 was identified in SFs by ELISA. RA SF and control in vitro differentiated macrophages were either untreated or treated with the toll-like receptor (TLR) 2 ligand peptidoglycan, and then IL-23, IL-27 and IFN-γ mRNA levels were quantified by real-time polymerase chain reaction (RT-PCR).ResultsTreatment with P/I alone or combined with IL-23 significantly increased the number of TH-17 cells in normal, RA PB and RA SF. With or without P/I plus IL-23, the percentage of TH-17 cells was higher in RA SF compared with normal and RA PB. IL-17 levels were comparable in OA and normal synovial tissues, and these values were significantly increased in RA synovial tissue. Although IL-17 was readily detected in RA SFs, IL-23 was rarely identified in RA SF. However, IL-23 mRNA was significantly increased in RA SF macrophages compared with control macrophages, with or without TLR2 ligation. IL-27 mRNA was also significantly higher in RA SF compared with control macrophages, but there was no difference in IL-27 levels between RA and control macrophages after TLR2 ligation. IFN-γ mRNA was also detectable in RA SF macrophages but not control macrophages and the increase of IFN-γ mRNA following TLR2 ligation was greater in RA SF macrophages compared with control macrophages.ConclusionThese observations support a role for TH-17 cells in RA. Our observations do not strongly support a role for IL-23 in the generation of TH-17 cells in the RA joint, however, they suggest strategies that enhance IL-27 or IFN-γ might modulate the presence of TH-17 cells in RA.

Administration of Ag85B showed therapeutic effects to Th2-type cytokine-mediated acute phase atopic dermatitis by inducing regulatory T cells

Increase in the number of patients with atopic dermatitis (AD) has been recently reported. T helper (Th) cells that infiltrate AD skin lesions are Th2-type dominant; reduced exposure to environmental Th1-cytokine-inducing microbes is believed to contribute to the increased number of AD patients. Regulatory type immune responses have been also associated with the occurrence of AD. It has been reported that antigen 85B (Ag85B) purified from mycobacteria is a potent inducer of Th1-type immune response in mice as well as in humans. In this study, we have examined the effect of plasmid DNA encoding Ag85B derived from Mycobacterium kansasii on AD skin lesions induced by oxazolone (OX) application. Th2-cytokine mediated mouse AD model with immediate type response followed by a late phase reaction was developed by repeated applications of low-dose OX to sensitized mice. Mice were immunized with plasmid DNA encoding cDNA of Ag85B before OX sensitization or during repeated elicitation phase. Both therapies were associated with significant suppression of immediate type response, clinical appearance, dermal cell infiltration, reduced IL-4 production, and augmented IFN-gamma mRNA expression compared to placebo-treated mice. Additionally, increased number of Foxp3(+) regulatory T cells were observed in the skin sections in Ag85B treated mice. The results of this study suggest that Ag85B DNA vaccine is a potential therapy for Th2 type dermatitis.

Adenosine A2Areceptor activation inhibits T helper 1 and T helper 2 cell development and effector function

Adenosine is an immunosuppressive nucleoside, and adenosine A(2A) receptors inhibit T-cell activation. We investigated the role of A(2A) receptors in regulating T helper (Th)1- and Th2-cell development and effector function. A(2A)-receptor stimulation suppressed the development of T-cell receptor (TCR) -stimulated naive T cells into both Th1 and Th2 cells, as indicated by decreased IFN-gamma production by cells developed under Th1-skewing conditions and decreased interleukin (IL) -4, IL-5, and IL-10 production by cells developed under Th2-skewing conditions. Using A(2A) receptor-deficient mice, we demonstrate that A(2A) receptor activation inhibits Th1- and Th2-cell development by decreasing the proliferation and IL-2 production of naive T cells, irrespective of whether the cells are expanded under Th1- or Th2-skewing environment. Using in vivo established Th1 and Th2 cells, we further demonstrate the nonselective nature of A(2A) receptor-mediated immunosuppressive effects, because A(2A) receptor activation decreased IFN-gamma and IL-4 secretion and mRNA level of TCR-stimulated effector Th1 and Th2 cells, respectively. A(2A) receptor mRNA expression in both Th1 and Th2 effector cells increased following TCR stimulation. In summary, these data demonstrate that A(2A) receptor activation has strong inhibitory actions during early developmental, as well as late effector, stages of Th1- and Th2-cell responses.

Murine metal-induced systemic autoimmunity: baseline and stimulated cytokine mRNA expression in genetically susceptible and resistant strains.

Cytokines play an important and complex role in the pathogenesis of systemic autoimmune diseases. In susceptible H-2s mice, inorganic mercury (Hg) induces lymphoproliferation, antinucleolar antibodies against the 34-kDa-protein fibrillarin, and systemic immune-complex (IC) deposits. Here, we report extensive analysis of cytokine mRNA levels in susceptible A.SW (H-2s) and resistant A.TL (H-2tl) mice under unstimulated conditions and during oral treatment with Hg and/or silver nitrate (Ag). Cytokine mRNA expression in lymphoid tissues was assessed using the ribonuclease protection assay and phosphorimaging. Baseline expression of IL-2 and IFN-gamma mRNA was higher in A.SW than in A.TL mice. In A.SW mice, Hg treatment caused early up-regulation of IL-2 and IFN-gamma levels, followed by substantial expression of IL-4 mRNA, which was significant compared to control A.SW and Hg-treated A.TL mice. Hg-exposed A.TL mice exhibited unchanged IFN-gamma, reduced IL-2 and greatly increased IL-10 mRNA expression. Ag-treated A.SW mice, which develop antifibrillarin antibodies (AFA) but exhibit minimal immune activation and no IC deposits, showed an early increase in IL-2 and IFN-gamma mRNA, but only a small and delayed rise in IL-4 mRNA. In conclusion, H-2-linked resistance to Hg-induced AFA is characterized by low constitutive expression of IL-2 and IFN-gamma mRNA, which is not increased by Hg, and a marked increase in IL-10 expression. Conversely, the key features of H-2-linked susceptibility to Hg- and Ag-induced AFA are up-regulation of IL-2, IFN-gamma and IL-4 mRNA expression, and down-regulation of IL-10 expression.

Increased secretion of IL-18 in vitro by peripheral blood mononuclear cells of patients with bronchial asthma and atopic dermatitis.

This study was performed to determine whether or not IL-18, formerly called IFN-gamma-inducing factor, is involved in the pathogeneses of allergic disorders. Peripheral blood mononuclear cells (PBMC) were obtained from patients with allergic bronchial asthma (BA), patients with atopic dermatitis (AD) and controls who did not have any allergic disease, and then cultured with lipopolysaccharide (LPS) or phytohaemagglutinin (PHA). The concentrations of IL-18, IFN-gamma and IL-13 in supernatant fluids were determined by enzymatic immunoassaying, and the expression of IFN-gamma messenger (m) RNA in the cells was measured by colorimetric microplate assaying. IL-18 secretion in the BA patients (geometric mean (gm) = 189 pg/ml) and AD patients (gm = 172 pg/ml) was significantly higher than that in non-allergic controls (gm = 118 pg/ml). In contrast, IFN-gamma secretion in the BA patients (gm = 7.3 IU/ml) and AD patients (gm = 6.8 IU/ml) was significantly lower than that in non-allergic controls (gm = 20.7 IU/ml). The amounts of IL-13 in supernatant fluids and IFN-gamma mRNA in cells were not statistically different among the BA patients, AD patients and non-allergic controls. The possible involvement of IL-18 in allergic disorders is discussed.

The expression of IL-2, IL-4 and interferon-gamma (IFN-gamma) mRNA using liver biopsies at different phases of acute exacerbation of chronic hepatitis B.

To investigate the hypothesis that Th1 phenotype cytokines are associated with the increasing activity of hepatitis and Th2 phenotype cytokines with decreasing activity in the liver of chronic viral hepatitis, expressions of the mRNA of the cytokines IL-2, IFN-gamma and IL-4 in the liver of 23 patients with chronic hepatitis B were investigated by reverse transcription polymerase chain reaction. Patients were divided into three groups according to the phase of acute exacerbation of hepatitis as increasing (n = 9), decreasing (n = 8), and stable phase (n = 6). Both IL-2 and IFN-gamma mRNA were preferentially expressed in increasing phase than in decreasing phase (P < 0.01, P < 0.05, respectively) and associated with the high serum alanine aminotransferase (ALT) level. On the other hand, IL-4 mRNA was detected in decreasing phase with significant frequency compared with increasing phase (P < 0.05). However, expression of IL-4 mRNA was not associated with serum ALT level. Our results suggest that Th1 phenotype cytokines up-regulate and Th2 phenotype cytokines down-regulate the liver inflammation of chronic viral hepatitis.

Presence of mRNA for interferon-gamma (IFN-gamma) in blood mononuclear cells is associated with an active stage I sarcoidosis.

Peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage (BAL) cells obtained from sarcoidosis patients were investigated for the presence of mRNA for IFN-gamma. RNA was extracted from freshly obtained cells. In 12 cases PBMC were studied and in seven cases BAL cells were also available for concomitant investigation. In eight patients mRNA for IFN-gamma was present in blood and was significantly associated with stage I disease (P = 0.030). By comparison, BAL cells were less frequently positive for IFN-gamma transcripts (P = 0.039). Only one patient had simultaneous expression of IFN-gamma gene in blood and BAL cells. All other patients including four positive in blood were negative in BAL. The presence of IFN-gamma transcripts in PBMC was significantly associated with the capacity of these cells to generate IFN-gamma in unstimulated cultures. PBMC from patients showing IFN-gamma mRNA in blood were frequently high IFN-gamma producers in anti-CD3 MoAb-stimulated cultures.

Analysis of cytokine profiles in synovial T cell clones from chlamydial reactive arthritis patients: predominance of the Th1 subset.

Subpopulations of human T cells (Th0, Th1 and Th2) can be distinguished by their cytokine-secretion pattern. Evidence is increasing from other studies that the outcome of a human disease may depend on the subpopulation of T cells that predominates at the site of inflammation. Reactive arthritis serves as a useful model of chronic inflammatory diseases, because the triggering antigen can be identified. Using this triggering antigen we raised 33 T cell clones reactive with Chlamydia trachomatis and 25 T cell clones that were not reactive, all from the synovial fluid of two patients suffering from Chlamydia-induced arthritis. Their cytokine secretion patterns for interferon-gamma (IFN-gamma), IL-2 and IL-4 were analysed, as also were mRNAs for IFN-gamma and IL-10 by in situ hybridization. Out of the 33 antigen-reactive clones 23 showed a Th1 pattern with IFN-gamma but not IL-4 secretion, while the remaining 10 exhibited a Th0 pattern. The clones that did not react with Chlamydia expressed all patterns of cytokine secretion, including a Th2 pattern, thus providing a control population that excludes bias in the sampling procedure. CD4 and CD8 clones displayed a similar cytokine-secretion pattern. In addition this study demonstrates for the first time the expression of IL-10 mRNA in T cell clones derived from synovial fluid, and this was not confined to the Th2 subset. The Th1 response that Chlamydia provoke can be regarded as appropriate for such an obligate intracellular pathogen.

High numbers of autoantigen-reactive mononuclear cells expressing interferon-gamma (IFN-gamma), IL-4 and transforming growth factor-beta (TGF-beta) are present in cord blood.

Umbilical cord blood of neonates and peripheral blood of healthy adults were analysed by in situ hybridization for numbers of mononuclear cells (MNC) expressing the cytokines IFN-gamma, TGF-beta and IL-4 mRNA without culture and after culture in the presence of acetylcholine receptor (AChR), myelin basic protein (MBP) and peripheral myelin protein P2. These antigens were chosen since they represent autoantigens in putatively immune-mediated neurological diseases. The numbers of cells expressing cytokine mRNA after 72 h culture in the presence of AChR, MBP and P2 were higher in cord blood than in peripheral blood of healthy adults. IFN-gamma, TGF-beta and IL-4 were always elevated in parallel. In cord blood there was a pronounced reactivity to several of the tested antigens, while such broad reactivity was not found in peripheral blood of healthy adults. No differences in cytokine mRNA expression were found between cord blood and peripheral blood of adults when cells were analysed without culture. The results show a capacity of cord blood cells to react to several autoantigens by the up-regulation of cytokine mRNA expression.

Interferon-gamma (IFN-gamma) production by human T lymphocytes upon Legionella pneumophila stimulation in vitro.

In vitro immune responses to Legionella pneumophila were investigated. When human peripheral blood lymphocytes (PBL) from healthy volunteers were stimulated with formalin-killed L. pneumophila for 7 days in vitro, strong proliferative responses were observed. The responding cells were shown to be a CD4 T cell subset. It was also found that the CD4 T cells secreted significant amounts of IFN-gamma into the PBL culture supernatant. The production of IFN-gamma and IL-4 by PBL was measured semiquantitatively by reverse transcriptase-assisted polymerase chain reaction (RT-PCR) methods. Formalin-killed or live L. pneumophila-stimulated PBL expressed the mRNA for IFN-gamma but not the mRNA for IL-4. The results suggest that the whole bacterium, as opposed to the supernatant, predominantly stimulates Th1 type helper T cells. The cloned T cells specific for L. pneumophila expressed the mRNA for IFN-gamma but not for IL-4. In contrast to formalin-killed or live L. pneumophila stimulation, when PBL were stimulated with the bacterial culture supernatant, the proliferating T cells produced the mRNA for IL-4 as well as for IFN-gamma. A significant correlation between the proliferative response to formalin-killed L. pneumophila and IFN-gamma release in culture was observed (r = 0.6932, P < 0.001) in PBL from 30 healthy volunteers. From these in vitro studies, it is suggested that the whole L. pneumophila bacterium and their soluble antigens stimulate T cells in a manner which results in a different pattern of cytokine production.

IL-4 mRNA expression by peritoneal cells during episodes of peritonitis in patients on continuous ambulatory peritoneal dialysis.

Peritoneal cells were isolated from dialysates drained from nine patients on continuous ambulatory peritoneal dialysis (CAPD) during episodes of peritonitis. Levels of expression of mRNA for the regulatory cytokines, interferon-gamma (IFN-gamma) and IL-4, were investigated daily, where possible, during the first 5 days of peritonitis. Cytokine mRNA levels were compared with those of peripheral blood mononuclear cells (PBMC) stimulated in vitro with phorbol 12-myristate 13-acetate (PMA) plus phytohaemagglutinin (PHA). Peritoneal cells expressed low levels of IFN-gamma mRNA; for four of nine patients, IL-4 mRNA levels greater than those expressed by stimulated PBMC were detected. There was no pattern of cytokine mRNA expression associated with the types of organisms detected in dialysates at initiation of peritonitis. However, in contrast to those patients with a transient, resolving peritonitis, significant IL-4 mRNA expression was detected in cells isolated early in the episodes of peritonitis in patients who suffered recurrent peritonitis within 30 days of the initial peritonitis episode. These results suggest an association between early IL-4 mRNA expression and susceptibility to further infections. The known anti-inflammatory effects of IL-4 may explain this association.

Altered Th1/Th2 cytokine profiles in the intestinal mucosa of patients with inflammatory bowel disease as assessed by quantitative reversed transcribed polymerase chain reaction (RT-PCR).

Cytokines serve a central function as key factors in the regulation of the intestinal immune response and mediation of tissue damage in inflammatory bowel disease (IBD). Abnormalities in the expression of immunoregulatory cytokines such as IL-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) may indicate a dysregulation of intestinal immunity probably associated with pathogenic events. Therefore, cytokine mRNA concentrations were determined in the mucosa of patients with IBD at sites of active (n = 13) and inactive (n = 12) ulcerative colitis (UC), active (n = 11) and inactive (n = 11) Crohn's disease (CD) and in control patients (n = 14) using quantitative RT-PCR. IL-10 mRNA concentrations were significantly increased in patients with both active UC (P < 0.001) and active CD (P < 0.005) compared with control patients. IFN-gamma mRNA concentrations were also significantly increased both in patients with active UC (P < 0.02) and active CD (P < 0.05) compared with control patients, whereas IL-2 mRNA levels were significantly (P < 0.02) increased only in active CD. IL-4 mRNA expression in the intestinal mucosa was frequently below the detection limit. Our results demonstrate that chronic intestinal inflammation in patients with CD is characterized by an increase of Th1-like cytokines. Furthermore, the increased IL-10 mRNA expression at sites of active IBD suggests that IL-10 is an important regulatory component involved in the control of the inflammatory response in inflammatory bowel disease.

Cytokine mRNA levels in human fat tissue after injection lipolysis with phosphatidylcholine and deoxycholate

Injections with Lipostabil, a phosphatidylcholine and deoxycholate containing substance, have become a popular technique to treat localized fat accumulation and lipomas. It is believed that the injected substance leads to fat cell destruction with subsequent acute panniculitis followed by a repair process of the treated fat tissue. We sought to investigate the mRNA expression of cytokines within the acute stage of panniculitis following Lipostabil injections. Tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-8, and IL-10 mRNA expression were determined using quantitative real-time reverse transcriptase polymerase chain reaction in treated and adjacent non-treated fat tissue of lipomas 48 h after injection in seven patients. Following injection lipolysis with Lipostabil, TNF-alpha, IL-6, IL-8, and IL-10 mRNA levels were significantly elevated in treated fat tissue compared to non-treated fat. No significant differences were found for IL-4. Both in treated and non-treated fat tissue, mRNA of IFN-gamma, IL-2, and IL-5 was not expressed. Injection lipolysis induces acute panniculitis within the treated fat tissue, associated with changes of the cytokine profile. However, their pathogenetic relevance with respect to clinical dissolving of fat needs further investigation.

Effects of Xiehuo Bushen Decoction on survival and differentiation of transplanted neural stem cells in brains of rats with intracerebral hemorrhage

To investigate the effects of Xiehuo Bushen Decoction (XHBSD), a compound Chinese herbal medicine, on the survival and differentiation of transplanted neural stem cells (NSCs) in brains of rats with intracerebral hemorrhage, and to explore the mechanism of Xiehuo Bushen formula in promoting the survival of transplanted NSCs. NSCs separated from hippocampuses of neonatal SD rats were cultured. Sixty-five panel reactive antibody (PRA) positive SD rats were selected by lymphocytotoxicity methods. The PRA positive rats were made into intracerebral hemorrhagic model and divided into three groups: cerebral hemorrhage group (n=15), NSCs transplanted group (n=25) and XHBSD group (n=25). XHBSD was orally administered after 5-bromodeoxyuridine (BrdU)-marked NSCs were transplanted in brains of rats with intracerebral hemorrhage in the XHBSD group. Rats in the other two groups were administered distilled water. The expressions of interferon gamma (IFN-gamma) and interleukin-4 (IL-4) mRNAs were measured by reverse transcription polymerase chain reaction (RT-PCR); the numbers of BrdU and 200 kD neurofilament (NF200) positive cells were detected by double-labeling immunofluorescence method. The expression of IFN-gamma mRNA was down-regulated significantly in the XHBSD group, but the expression of IL-4 mRNA was up-regulated significantly (P<0.05). The numbers of BrdU and NF200 positive cells were also increased remarkably in the XHBSD group. XHBSD can promote the survival and differentiation of transplanted NSCs, which may be related to inducing the expression of IL-4 mRNA and inhibiting the expression of IFN-gamma mRNA.

Zinc Finger Protein A20 Promotes Regeneration of Small-for-Size Liver Allograft and Suppresses Rejection and Results in a Longer Survival in Recipient Rats

Small-for-size liver allografts without immunosuppression have decreased survival compared with full-for-size grafts for the concomitant regeneration-induced accelerated rejection. This study was designed to examine the effect of zinc finger protein A20 on liver allograft regeneration and acute rejection using a high responder rat model (DA-->Lewis) of 30% partial liver transplantation. Adenovirus carrying the full length of A20 was introduced into liver grafts by ex vivo perfusion via the portal vein during preservation, physiological saline (PS), and empty Ad vector rAdEasy served as controls; then small-sized liver transplants were performed. Liver graft regeneration was assessed, as well as graft rejection, hepatocyte apoptosis, nuclear factor kappa B activation, and intercellular adhesion molecule-1 mRNA expression in liver graft sinusoidal endothelial cells (LSECs), infiltration of liver graft infiltrating mononuclear cells (LIMCs), and the subproportion of NK and NKT cells, activity of liver graft NK-like cells, interferon gamma (IFN-gamma) production, and animal survival. Ex vivo transfer of the A20 gene resulted in overexpression of A20 protein in LSECs and hepatocytes 24 h after partial liver transplantation. Regeneration of the small-sized liver allograft was augmented by A20 overexpression, the DNA synthesis of hepatocytes on d 4 post-transplant was increased in A20 group compared with PS and rAdEasy groups (P < 0.01). Hepatocyte apoptosis was inhibited by A20 (P < 0.001). On d 4 after transplantation, histological examination revealed a more exiguous cellular infiltration and mild rejection in A20 group but a more vigorous cellular infiltration in the sinusoidal area and more severe rejection in PS and rAdEasy group. Nuclear factor kappa B activation and intercellular adhesion molecule-1 mRNA expression in LSECs were suppressed by A20 overexpression. Flow cytometry analysis showed a marked down-regulation of LIMCs number by A20, including more prominent decrease in the subproportion of NK and NKT cells. Activity of liver graft NK-like cells, IFN-gamma mRNA expression in LIMCs, and serum IFN-gamma protein level were also suppressed by A20 overexpression (P < 0.05), respectively. Survival days of A20 rats were longer than those of PS rats and rAdEasy rats (P < 0.01), whereas survival days of rAdEasy rats were shorter than those of PS rats (P < 0.01). These data suggest that A20 overexpression could effectively promote small-sized liver allograft regeneration, suppress rejection, and prolong survival of recipient rat. These effects of A20 could be related to an inhibition of LSECs activation, suppression of infiltration of LIMCs, and the subpopulations such as NK and NKT cells into liver graft, and inhibition of hepatocyte apoptosis.

Early transcriptional response in the jejunum of germ-free piglets after oral infection with virulent rotavirus

Germ-free piglets were orally infected with virulent rotavirus to collect jejunal mucosal scrapings at 12 and 18 hours post infection (two piglets per time point). IFN-gamma mRNA expression was stimulated in the mucosa of all four infected piglets, indicating that they all responded to the rotavirus infection. RNA pools prepared from two infected piglets were used to compare whole mucosal gene expression at 12 and 18 hpi to expression in uninfected germ-free piglets (n = 3) using a porcine intestinal cDNA microarray. Microarray analysis identified 13 down-regulated and 17 up-regulated genes. Northern blot analysis of a selected group of genes confirmed the data of the microarray. Genes were functionally clustered in interferon-regulated genes, proliferation/differentiation genes, apoptosis genes, cytoskeleton genes, signal transduction genes, and enterocyte digestive, absorptive, and transport genes. Down-regulation of the transport gene cluster reflected in part the loss of rotavirus-infected enterocytes from the villous tips. Data mining suggested that several genes were regulated in lower- or mid-villus immature enterocytes and goblet cells, probably to support repair of the damaged epithelial cell layer at the villous tips. Furthermore, up-regulation was observed for IFN-γ induced guanylate binding protein 2, a protein that effectively inhibited VSV and EMCV replication in vitro (Arch Virol 150:1213–1220, 2005). This protein may play a role in the small intestine’s innate defense against enteric viruses like rotavirus.Electronic supplementary materialThe online version of this article (doi:10.1007/s00705-008-0118-6) contains supplementary material, which is available to authorized users.