IFN-gamma mRNA Research Articles

Strong HLA‐DR antigen expression on cancer cells relates to better prognosis of colorectal cancer patients: Possible involvement of c‐myc suppression by interferon‐γin situ

Strong HLA-DR antigen expression on cancer cells relates to better prognosis of colorectal cancer patients, although the precise mechanism is controversial. From an immunological point of view, HLA-DR antigen, induced by interferon (IFN)-gamma, is required for tumor-associated antigen recognition by CD4(+) T cells. For instance, as reported previously, the expression of HLA-DR antigen in normal colorectal epithelium immediately adjacent to cancer coincided significantly with the existence of IFN-gamma mRNA in the tissue. From another aspect, IFN-gamma has been revealed to suppress c-myc expression in vivo through a stat1-dependent mechanism, which is important for cell growth, cell cycle and chromosome instability. In the present study, strong HLA-DR-positive expression on cancer cells was significantly related to better prognosis for colorectal cancer patients. High IFN-gamma mRNA expression in situ indicated significantly less activation of c-myc mRNA expression. Further, HLA-DR antigen expression in cancer cells, as well as Dukes stages, was an independent factor for better long-term survival by multivariate analysis. Taken together, IFN-gamma, which induces HLA-DR antigens on the cell surface, also suppresses c-myc expression in situ, and is a possible non-immunological mechanism involved in the better long-term survival of colorectal cancer patients.

Th2-dominated antitumor immunity induced by DNA immunization with the genes coding for a basal core peptide PDTRP and GM-CSF

Our previous study showed that DNA vaccination with a plasmid vector encoding a core peptide of mucin1 (PDTRP) provided modest protection against challenge with tumor cells that expressed mucin1 protein. We report here that a DNA vaccine comprising a modified PDTRP plasmid and GM-CSF coding sequence at the C-terminus induced better protection against tumor challenge. The increased protection was directly correlated with a stronger PDTRP-specific immune response induced by the GM-CSF fusion plasmid. The plasmid encoding GM-CSF and the target PDTRP antigen induced a greater PDTRP-specific Th proliferation, antibodies, and cytotoxicity. Interestingly, the modified plasmid vaccine predominantly enhanced the type 2 immune responses manifested by an increased IgG1 to IgG2a antibody ratio and a greater induction of GATA-3 and IL-4 mRNA than that of T-bet and IFN-gamma mRNA in spleen cells from vaccinated mice. In addition, protection against tumor challenge in vaccinated mice showed that there was no significant change in mice survival after in vivo CD8+CTL depletion, indicating that antitumor immunity augmented by plasmid encoding GM-CSF and target PDTRP gene vaccine was dominated by Th2 immune response.

Association Between Il-18 Gene Promoter Polymorphisms and Inflammatory Bowel Disease in a Japanese Population

Interleukin-18 (IL-18) is a pleiotropic cytokine that induces the production of interferon (IFN)-gamma and also to regulate Th2 cytokines. Recently, association studies between IL-18 gene promoter polymorphisms and several Th1- or Th2-mediated inflammatory diseases were reported. In inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), recent evidence suggests that IL-18 is involved in the pathogenesis. Using DNA direct sequencing, we investigated IL-18 gene promoter polymorphisms at -607C/A and -137G/C. Allele, genotype, and haplotype frequencies were determined in 210 Japanese patients with UC, 205 patients with CD, and 212 controls. In UC, the -137C allele frequency was significantly higher in the proctitis-type patients than in controls (Pc = 0.0068). The -137 genotype frequency was also significantly different in the proctitis-type patients than in controls (Pc = 0.032). No other allele and genotype frequencies were significantly associated with UC after Bonferroni correction. Furthermore, the frequency of haplotype 2 (-607A, -137C), which had a lower promoter activity and IFN-gamma mRNA level than the other haplotypes as previously reported, was significantly higher in the proctitis-type patients than in controls (Pc = 0.01). In CD, we could not find any significant differences. IL-18 gene promoter polymorphisms may not be associated with disease susceptibility but related to the extent of disease in UC.

Deficiency of regulatory B cells increases allergic airway inflammation.

To investigate the effect of the X-linked immunodeficiency (Xid) B cell defect on the response to the cockroach allergen in mice. Two cockroach allergen immunization and challenge protocols were employed to sensitize CBA/J wild-type and CBA/CaHN-btk(-/-)xid/J (Xid) mice. Blood and tissue samples were collected 24 and 48 hrs after the last intratracheal antigen challenge and were analyzed for several parameters of allergic inflammation. Nearly equivalent amounts of serum IgE were detected in Xid and CBA/J mice after short-term antigen challenge despite the B cell deficiency in Xid mice. A decreased concentration of IgE was detected in CBA/J mice after repeated allergen challenges but not in the Xid mice. Correlating with the discrepancy in serum IgE levels, higher levels of IL-13, IL-5, IL-10 and CCL5 were measured in whole lung homogenates from allergen-challenged Xid mice compared to CBA/J mice. In addition, draining lymph node cells from Xid mice expressed elevated levels of IL-4, IL-5, IL-10 and IFNgamma mRNA compared to cells from CBA/J mice after in vitro culture with cockroach antigen. An increase in lung inflammation, interstitial eosinophilia and mucus production was also observed in allergen-challenged Xid mice. CD95L expression increased on B-1a cells following allergen challenge, which was accompanied by an increase in lung CD4(+) Th cell apoptosis in wild-type CBA/J mice. In contrast, Xid mice did not have an increase in CD4(+) T cell apoptosis following allergen challenge. These data suggest a regulatory role for B-1a cells in reducing cytokine production, pulmonary inflammation, and CD4(+) T cell survival during cockroach allergen-induced airway inflammation.

A canine model of cutaneous late-phase reactions: prednisolone inhibition of cellular and cytokine responses.

Immunoglobulin E (IgE)-mediated late-phase reactions can be induced in atopic humans by intradermal injection of relevant allergens or anti-IgE antibodies. The histology of these reactions resembles that of naturally occurring atopic dermatitis. Strikingly similar responses can be induced in dogs, suggesting that a canine model could prove valuable for preclinical investigation of drugs targeting late-phase reactions. This study was designed to characterize the cellular, cytokine and chemokine responses after intradermal anti-IgE injection in untreated and prednisolone-treated dogs. Normal beagles were untreated or treated with prednisolone before intradermal injection of polyclonal rabbit anti-canine IgE or normal rabbit IgG. Biopsies were taken before injection and 6, 24 and 48 hr after injection. Samples were evaluated by histological and immunohistochemical staining, as well as by real-time quantitative polymerase chain reaction analysis. Dermal eosinophil and neutrophil numbers increased dramatically within 6 hr after injection of rabbit anti-canine IgE, and remained moderately elevated at 48 hr. The numbers of CD1c(+) and CD3(+) mononuclear cells were also increased at 6 hr. The real-time quantitative polymerase chain reaction demonstrated marked increases in mRNA expression for interleukin-13 (IL-13), CCL2, CCL5 and CCL17. Levels of mRNA for IL-2, IL-4, IL-6 and IFN-gamma did not change within the limits of detection. Prednisolone administration suppressed the influx of neutrophils, eosinophils, CD1c(+) and CD3(+) cells, as well as expression of IL-13, CCL2, CCL5 and CCL17. These data document the cytokine and chemokine responses to anti-IgE injection in canine skin, and they demonstrate the ability of the model to characterize the anti-inflammatory effects of a known therapeutic agent.

Is Survivin a Possible Target Antigen for Cellular Immunotherapy in Multiple Myeloma?.

Survivin is a member of the inhibitors of apoptosis family and is expressed in different types of malignancies including multiple myeloma. Cytotoxic T cells recognizing survivin epitopes can be elicited in vitro and by vaccination in human individuals. We performed this study to investigate whether survivin peptide specific CD8 T cells occur in patients with multiple myeloma (MM) and in healthy individuals. To detect antigen specific T-cells, we incubated PBMC from HLA-A2.1 positive patients and healthy individuals with antigen-presenting-cells (C1R-A2) pulsed or not pulsed with a recently described, HLA-A2.1 binding survivin derived peptide (Survpep). We used quantitative real time PCR to measure IFN-gamma mRNA expression for the detection of peptide specific CTL in 23 patients and 21 healthy individuals. A peptide stimulation index (SI, test peptide reactivity / no peptide reactivity) of 2.0 or greater was considered a positive response. CD8 T cells recognizing Survpep could be detected in vitro in 9/23 (39%) patients with MM (SI ranging from 2.9 to 83) and 1/21 (5%) healthy individuals (SI 7). Positive responses were confirmed by 4-colour flow cytometry for intracellular IFN-gamma production. T cells producing IFN-gamma were further analyzed for expression of CD45RA and CCR7 to determine phenotypic characteristics in two patients (SI 83 and 63) identifying them as terminally differentiated effector T cells (CD8+, CD45RA+, CCR7−). Additionally, positive expression of survivin antigen in tumor cells was confirmed by imminohistochemical analysis of patients bone marrow specimen. In conclusion we provide for the first time evidence for T cell reactivity against Survivin antigen in patients with MM. Our data suggest (1) the immunogenicity of survivin antigen in multiple myeloma and (2) that survivin may be a useful candidate antigen for cellular immunotherapy of multiple myeloma. [Display omitted]

Nitric oxide synthase and cytokines gene expression analyses in Leishmania-infected RAW 264.7 cells treated with an extract of Pelargonium sidoides (Eps® 7630)

A modern aqueous-ethanolic formulation of the roots of Pelargonium sidoides (Eps 7630), elaborated from the traditional herbal medicine used in areas of southern Africa, is effectively employed for the treatment of ENT and respiratory tract infections in modern phytotherapy. Previous studies have demonstrated antibacterial and immunomodulatory activities. To gain insight into the mode of action at the molecular level, gene expression analyses for the inducible nitric oxide synthase and the cytokines interleukin (IL)-1, IL-12, IL-18, tumour necrosis factor (TNF)-alpha, interferon (IFN)-alpha, and IFN-gamma, were performed using reverse transcription-polymerase chain reaction (RT-PCR). The experiments were carried out in parallel in non-infected and in Leishmania major-infected RAW 264.7 cells and the expression profiles were compared with those mediated by IFN-gamma+LPS. Eps 7630 induced low mRNA levels in non-infected cells, and it considerably up-regulated the transcript expressions in parasitised cells. Interestingly, and in contrast to activation by IFN-gamma+LPS, Eps 7630 also stimulated infected cells to produce IFN-gamma mRNA. A similar expression profile was observed for the methanol-insoluble fraction (MIF) of Eps 7630 and gallic acid, a trace constituent of the extract, while the methanol-soluble fraction and umckalin, an exclusive and representative member of the occurring coumarins, proved to be devoid of any remarkable gene-inducing capabilities. The present results provide not only convincing support for the improvement of immune functions as previously demonstrated in functional bioassays, but also evidence for activation at the transcriptional level and suggest that the underlying inducing principle is located in the MIF.

Effects of specific interleukin-1beta-converting enzyme inhibitor on ischemic acute renal failure in murine models1

To study the effect of selective interleukin-1beta-converting enzyme (ICE, caspase-1) inhibitor on ischemic acute renal failure (ARF). Mouse models of ischemic ARF were treated with the specific ICE inhibitor AC-YVAD-CMK. A renal function assay and renal morphological studies were employed to estimate the renal protective effect of AC-YVAD-CMK. The survival rate of mouse models was also analyzed by a time series test. Furthermore, renal ICE activity, mature interleukin-18 (IL-18) protein expression and interferon-gamma (IFN-gamma) mRNA expression were also detected by fluorescent enzyme-linked immunosorbent assay (ELISA), ELISA, and semi-quantitative reverse transcription-polymerase chain reaction, respectively. The levels of blood urea nitrogen (BUN) and serum creatinine (Scr) increased remarkably in the model controls compared with the sham-operated groups (P<0.01). Typical renal tubular necrosis was found in the model controls. Renal ICE activity, mature IL-18 protein expression, and IFN-gamma mRNA expression were also increased significantly in the model controls compared with the sham-operated groups. The levels of BUN and Scr in the AC-YVAD-CMK therapy group were decreased significantly compared with the untreated model controls (P<0.01). Renal tubulointerstitial lesion was also attenuated significantly (P<0.05). AC-YVAD-CMK therapy alleviated the clinical features of ARF, and increased the survival rate (P<0.01). Furthermore, AC-YVAD-CMK therapy also decreased ICE activity, mature IL-18 protein expression, and IFN-gamma mRNA expression in renal tissue (P<0.05). The selective ICE inhibitor AC-YVAD-CMK can effectively protect the kidney from acute ischemic lesions. This protective effect is associated with decreased renal ICE activity and suppressed IL-18 maturation and IFN-gamma mRNA transcription.

Silencing Toll-like Receptor-9 inPseudomonas aeruginosaKeratitis

To determine the effects of silencing Toll-like receptor (TLR) 9 signaling in Pseudomonas aeruginosa keratitis. Corneal TLR9 mRNA levels were tested by RT-PCR in C57BL/6 (B6, susceptible) and BALB/c (resistant) mice and compared. The response of B6 mice to CpG DNA, which binds TLR9, was tested after subconjunctival injection of mice with control or CpG DNA; TLR9, IL-1beta, macrophage inflammatory protein (MIP)-2, IL-4, IL-10, IL-12, IL-18, and IFN-gamma levels were measured by RT-PCR. Langerhans cells (LCs) were stimulated with CpG DNA and treated with TLR9 or control siRNA, and mRNA levels of TLR9, IL-1beta, and MIP-2 were detected by RT-PCR. In addition, IL-1beta levels were tested by ELISA. Then B6 mice were injected subconjunctivally with control or TLR9 siRNA before infection and treated topically afterward. Slit lamp, clinical score, RT-PCR, ELISA, myeloperoxidase assay, and plate counts were performed. TLR9 mRNA levels were sixfold higher in B6 than in BALB/c corneas the day after injection. B6 mice injected with CpG DNA exhibited an increase in corneal mRNA for TLR9, IL-1beta, MIP-2, IL-12, and IFN-gamma over controls. LCs stimulated with CpG DNA and treated with TLR9 siRNA exhibited reduced TLR9, IL-1beta, and MIP-2 levels compared with controls. Finally, B6 mice treated with TLR9 siRNA showed decreases in corneal opacity, polymorphonuclear leukocyte number, IL-12 and IFN-gamma mRNA, IL-1beta, and MIP-2 protein compared with those treated with control siRNA. Fewer corneas perforated in these mice, but bacterial loads were higher than in controls. Signaling through TLR9 appears important in P. aeruginosa keratitis, and silencing TLR9 signaling reduces inflammation but likely contributes to decreased bacterial killing in the cornea.

Gene expression analysis of the CD4+ T‐cell clones derived from gingival tissues of periodontitis patients

The function of T cells infiltrating periodontitis lesions is complex and has not been fully elucidated. Here, we established T-cell clones from the gingival tissues of periodontitis patients and examined their gene expression. A total of 57 and 101 T-cell clones were established by means of immobilized anti-CD3 antibody and IL-2 from gingival tissues and peripheral blood, respectively. The gingival T-cell clones were derived from three patients, and the peripheral blood T-cell clones from two of these patients and a further patient whose gingival T-cell clones were not established. Gingival tissues were also obtained from a further 19 periodontitis patients. The expression of cytokines and molecules related to both regulatory function and tissue destruction were examined by means of reverse-transcription polymerase chain reaction. All the gingival T-cell clones expressed mRNA for TGF-beta1, CTLA-4, and CD25, and all the T-cell clones from peripheral blood expressed IFN-gamma and TGF-beta1 mRNAs. Most but not all the T-cell clones from gingival tissues and peripheral blood expressed mRNA for IFN-gamma and, CD25 and CTLA-4, respectively. The frequency of T-cell clones and gingival tissues expressing FOXP3, a possible master gene for mouse CD4(+)CD25(+) regulatory T cells, was very high (97%, 93%, and 100% for gingival T-cell clones, peripheral blood T-cell clones, and gingival tissues, respectively). Whereas the frequency of IL-4-expressing T-cell clones was lower for gingival T-cell clones (70% vs. 87%), the frequency of the gingival T-cell clones expressing IL-10 and IL-17 was higher than peripheral blood T-cell clones (75% vs. 62% for IL-10, 51% vs. 11% for IL-17). A similar expression profile was observed for gingival T-cell clones compared with gingival tissue samples with the exception of IL-4 expression, where the frequency of positive samples was lower in the gingival tissues (70% vs. 11%). These results suggest that the individual T cells infiltrating gingival lesions can express mRNA for both Th1 and Th2 cytokines as well as regulatory cytokines simultaneously.

C-Rel-Dependent Priming of Naive T Cells by Inflammatory Cytokines

The intrinsic refractoriness of naive T cells for cytokine production is counteracted by cells of the innate immune system. Upon sensing danger via Toll-like receptors, these cells upregulate T cell costimulatory molecules and secrete cytokines that enhance T cell activation. We show that cytokine-mediated priming of naive T cells requires the NF-kappaB family member c-Rel. In resting naive cells c-Rel is associated primarily with IkappaBbeta, an inhibitory molecule that is not effectively degraded by TCR signals. Exposure of T cells to proinflammatory cytokines, TNF-alpha and IL-1beta, shifts c-Rel to IkappaBalpha-associated complexes that are readily targeted by the TCR. As a consequence, IL-2 and IFN-gamma mRNA are produced more quickly, and at higher levels, in cytokine-primed T cells. This mechanism does not operate in effector T cells where cytokine gene expression is c-Rel-independent. We propose that c-Rel plays a crucial role as a target of innate signals in T cells.

TGF‐β1 production by CD4+CD25+ regulatory T cells is not essential for suppression of intestinal inflammation

Naturally occurring CD4+ CD25+ regulatory T cells (Treg) are potent suppressors of CD4+ and CD8+ T cell responses in vitro and inhibit several organ-specific autoimmune diseases. While most in vitro studies suggest that CD4+ CD25+ Treg cells adopt a cytokine-independent but cell contact-dependent mode of T cell regulation, their precise mechanism of suppression in vivo remains largely unknown. Here we examine the functional contribution of Treg cell-derived TGF-beta1 and effector T cell responsiveness to TGF-beta in CD4+ CD25+ T cell-mediated suppression of inflammatory bowel disease (IBD). We show that CD4+ CD25+ Treg cells from either TGF-beta1+/+ or neonatal TGF-beta1-/- mice can suppress the incidence and severity of IBD as well as colonic IFN-gamma mRNA expression induced by WT CD4+ CD25- effector T cells. Furthermore, TGF-beta-resistant Smad3-/- CD4+ CD25+ Treg cells are equivalent to WT Treg cells in their capacity to suppress disease induced by either WT or Smad3-/- CD4+ CD25- effector T cells. Finally, anti-TGF-beta treatment exacerbates the colitogenic potential of CD4+ CD25- effector T cells in the absence of CD4+ CD25+ Treg cells. Together, these data demonstrate that in certain situations CD4+ CD25+ T cells are able to suppress intestinal inflammation by a mechanism not requiring Treg cell-derived TGF-beta1 or effector T cell/Treg cell responsiveness to TGF-beta via Smad3.

The Immunological Effect of Photopheresis in Children with Newly Diagnosed Type 1 Diabetes

Photopheresis has been claimed to have immune-modulating effects, but the mechanisms of action are unknown. This study investigated the immune effect of photopheresis in children with type 1 diabetes, with a focus on the balance of Th1- and Th2-like cytokines. Ten children with newly diagnosed type 1 diabetes (10-17 y) were treated with five double treatments of photopheresis and 10 children matched for disease, age, and gender were given placebo tablets and sham pheresis. Expression of IFN-gamma and IL-4 mRNA was determined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and secretion of IFN-gamma, IL-10, and IL-13 in cell-culture supernatants by ELISA after stimulation with glutamic acid decarboxylase (GAD65) (a.a. 247-279), the ABBOS peptide (a.a. 152-169), insulin, phytohemagglutinin (PHA), and keyhole limpet hemocyanin (KLH). Photopheresis changed antigen-stimulated immune balance in line with a Th2-like shift. Thus, the ratio of IFN-gamma/IL-4 mRNA expression after in vitro stimulation with a peptide of the autoantigen GAD65 was reduced after treatment in the photopheresis group. The IFN-gamma/IL-4 mRNA expression ratio after in vitro stimulation with insulin was also lower in children treated with photopheresis compared with the placebo group. Photopheresis has an immune-modulating effect in children with type 1 diabetes, causing a Th2-like deviation.

Interleukin-12 and -18 Levels in Peritoneal Dialysate Effluent Correlate With the Outcome of Peritonitis in Patients Undergoing Peritoneal Dialysis: Implications for the Type I/Type II T-Cell Immune Response

We previously showed that a positive impact of peritoneal defense response on the outcome of peritoneal dialysis (PD)-related peritonitis is characterized by an increased pattern of peritoneal CD4/CD8 T-cell ratio with a predominant CD4(+)-T helper subtype 1 phenotype. To further explore longitudinal changes in peritoneal immunity during PD-related peritonitis, we examined the production of interleukin 12 (IL-12), IL-18, and interferon gamma (IFN-gamma) in peritoneal dialysate effluent (PDE) and kinetic expression of the transcription factors T box expressed in T cells (T-bet) and guanine adenine thymine adenine (GATA) binding protein 3 (GATA-3) in peritoneal T cells during peritonitis. Correlations between these observations and responses to antibiotics were analyzed. IL-12, IL-18, and IFN-gamma protein and IFN-gamma, T-bet, and GATA-3 messenger RNA (mRNA) were measured in PDE during various phases of peritonitis in 40 patients undergoing PD. Patients were divided into 2 groups according to whether they had a rapid versus delayed response to antibiotic treatment. In the early phase of peritonitis, IL-12, IL-18, and IFN-gamma levels in PDE were significantly greater in the rapid-response group (P < 0.05). Changes in peritoneal IL-12 and IL-18 levels preceded changes in IFN-gamma levels. The kinetics of IFN-gamma, T-bet, and GATA-3 mRNA expression in peritoneal T cells, measured by means of real-time polymerase chain reaction, differed between the 2 groups. In the rapid-response group, IFN-gamma and T-bet mRNA expression increased, whereas that of GATA-3 decreased over time. Results were opposite in the delayed-response group, with IFN-gamma and T-bet levels decreasing and GATA-3 levels increasing over time. These data suggest that local IL-12 and IL-18 production is part of a protective early immune response to PD-related peritonitis. High IL-12 and IL-18 levels in PDE during the early phase of peritonitis correlated with a predominant type 1 immune response and favorable outcome.

Milk Casein‐Based Diet Containing TGF‐β Controls the Inflammatory Reaction in the HLA‐B27 Transgenic Rat Model

A casein-based formula containing TGF-beta has been successfully used in adolescents during acute episodes of Crohn's disease. The role played by this molecule requires confirmation. We have examined the capacity of a TGF-beta containing diet to control the intestinal inflammation in HLA-B27 transgenic rats, and compared its effects with a similar diet devoid of TGF-beta. Three groups of rats were studied. HLA-B27/hbeta2M transgenic rats were fed with a casein-based rat-adapted diet containing TGF-beta or a control casein-based diet without TGF-beta. Fischer control animals were fed the latter. Body weight, dietary intake, tissue weights, fecal samples, leukocyte counts, and acute phase response were analyzed. Intestinal inflammation was assessed by histology, myeloperoxidase, and mRNA expression of cytokines. MUC2 protein expression was assessed by immunohistochemistry. Breakdown of muscle protein was examined. The test diet improved diarrhea increasing the fecal dry matter and the colonic inflammation as shown by a lower inflammatory score (2.43 +/- 1.13 vs 4.42 +/- 0.53, p < .05), lower mucosal thickness (431.25 +/- 72.29 vs 508.57 +/- 81.32 microm, p = .08) and decreased IFNgamma mRNA expression. MUC2 protein expression was increased in HLA rats fed the TGF-beta diet compared with HLA rats fed the control diet, but restitution to normal pattern was not observed. The test diet also decreased leukocytosis and the acute phase response and improved the muscle catabolic response. The TGF-beta containing diet has a beneficial effect in an animal model of intestinal inflammation. Our observations support a potential role for dietary TGF-beta in the restoration of immune homeostasis.

Characterization of the B‐cell immune response elicited in BALB/c mice challenged with Neospora caninum tachyzoites

Activation of B cells occurring in hosts infected with protozoan parasites has been implicated either in protective or parasite-evasion immune-mediated mechanisms. Intraperitoneal inoculation of Neospora caninum tachyzoites into BALB/c mice induces an acute response characterized by a rapid increase in the numbers of CD69-expressing peritoneal and splenic B cells. This early B-cell stimulatory effect preceded an increase in the numbers of total and immunoglobulin-secreting splenic B cells and a rise in serum levels of N. caninum-specific immunoglobulins, predominantly of the immunoglobulin G2a (IgG2a) and IgM isotypes. Increased numbers of B cells expressing the costimulatory molecules CD80 and CD86 were also observed in the N. caninum-infected mice. The B-cell stimulatory effect observed in mice challenged with N. caninum tachyzoites was reduced in mice challenged with gamma-irradiated parasites. Contrasting with the peripheral B-cell expansion, a depletion of B-lineage cells was observed in the bone-marrow of the N. caninum-infected mice. Intradermal immunization of BALB/c mice with diverse N. caninum antigenic preparations although inducing the production of parasite-specific antibodies nevertheless impaired interferon-gamma (IFN-gamma) mRNA expression and caused lethal susceptibility to infection in mice inoculated with a non-lethal parasitic inoculum. This increased susceptibility to N. caninum was not observed in naïve mice passively transferred with anti-N. caninum antibodies. Taken together, these results show that N. caninum induces in BALB/c mice a parasite-specific, non-polyclonal, B-cell response, reinforce previous observations made by others showing that immunization with N. caninum whole structural antigens increases susceptibility to murine neosporosis and further stress the role of IFN-gamma in the host protective immune mechanisms against this parasite.

Sodium Methyldithiocarbamate Inhibits MAP Kinase Activation through Toll-like Receptor 4, Alters Cytokine Production by Mouse Peritoneal Macrophages, and Suppresses Innate Immunity

Sodium methyldithiocarbamate (SMD; trade name, Metam Sodium) is an abundantly used soil fumigant that can cause adverse health effects in humans, including some immunological manifestations. The mechanisms by which SMD acts, and its targets within the immune system are not fully understood. Initial experiments demonstrated that SMD administered by oral gavage substantially decreased IL-12 production and increased IL-10 production induced by lipopolysaccharide in mice. The present study was conducted to further characterize these effects and to evaluate our working hypothesis that the mechanism for these effects involves alteration in signaling through toll-like receptor 4 and that this would suppress innate immunity to infection. SMD decreased the activation of MAP kinases and AP-1 but not NF-kappaB in peritoneal macrophages. The expression of mRNA for IL-1alpha, IL-1beta, IL-18, IFN-gamma, IL-12 p35, IL-12 p40, and macrophage migration inhibitory factor (MIF) was inhibited by SMD, whereas mRNA for IL-10 was increased. SMD increased the IL-10 concentration in the peritoneal cavity and serum and decreased the concentration of IL-12 p40 in the serum, peritoneal cavity, and intracellularly in peritoneal cells (which are >80% macrophages). Similar effects on LPS-induced cytokine production were observed following dermal administration of SMD. The major breakdown product of SMD, methylisothiocyanate (MITC), caused similar effects on cytokine production at dosages as low as 17 mg/kg, a dosage relevant to human exposure levels associated with agricultural use of SMD. Treatment of mice with SMD decreased survival following challenge with non-pathogenic Escherichia coli within 24-48 h, demonstrating suppression of innate immunity.

Compartmental responses after thoracic irradiation of mice: Strain differences

To examine and compare the molecular and cellular processes leading to radiation fibrosis and pneumonitis in C57BL/6J and C3H/HeN mice. At indicated times after various doses of thoracic irradiation, the cell populations obtained by bronchoalveolar lavage of C57BL/6J mice were differentially analyzed by cytology and assessed by RNase protection (RPA) assay for levels of cytokines and related genes. The molecular responses in bronchial alveolar lavage (BAL) populations were compared with those in whole lung of C57BL/6J mice and with those of C3H/HeN mice. The former strain develops late radiation fibrosis, whereas the latter develop subacute radiation pneumonitis. In C57BL/6J mice, a decrease in the total number of BAL cells was found 1 week after 6, 12, or 20 Gy thoracic irradiation with a subsequent dose-dependent increase up to 6 months. After 12 and 20 Gy, large, foamy macrophages and multinucleated cells became evident in BAL at 3 weeks, only to disappear at 4 months and reappear at 6 months. This biphasic response was mirrored by changes expression of mRNA for proinflammatory cytokines and the Mac-1 macrophage-associated antigen. As with BAL, whole lung tissue also showed biphasic cytokine and Mac-1 mRNA responses, but there were striking temporal differences between the two compartments, with changes in whole lung tissue correlating better than BAL with the onset of fibrosis in this strain. The radiation-induced proinflammatory mRNA responses had strain-dependent and strain-independent components. Thoracic irradiation of C3H/HeN induced similar increases in tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha/beta, and interferon (IFN)-gamma mRNA expression in lung as it did in C57BL/6J mice during the "presymptom" phase at 1-2 months. However, immediately preceding and during the pneumonitic time period at 3-4 months, TNF-alpha and IL-1alpha/beta mRNAs were highly upregulated in C3H/HeN mice, which develop pneumonitis, but not in C57BL/6J mice, which do not. At the onset of radiation fibrosis in C57BL/6J mice (5-6 months), irradiated lungs had increased levels of IL-1alpha/beta and IFN-gamma mRNA expression, but the TNF-alpha response was, notably, still muted. The major molecular and cellular events in lungs of C57BL/6J and C3H/HeN mice, which develop late fibrosis and subacute pneumonitis after thoracic irradiation respectively, take place within the interstitium and are not reflected within BAL populations. The initial proinflammatory responses are similar in the two strains, but later responses reflect the latent time to lesion development. TNF-alpha expression at 3-4 months may be important in radiation-induced pneumonitis, and its downregulation is important in avoiding this radiation-induced complication.

Correlation Between Type of Adaptive Immune Response Against Porcine Circovirus Type 2 and Level of Virus Replication

Porcine circovirus 2 (PCV2) replication is characterized by high variation among infected pigs. This study investigated the role of immunologic responses in causing this variation. Twelve gnotobiotic pigs were inoculated with PCV2. Four of these pigs were treated with cyclosporin A (CysA) to monitor the effect of the adaptive immunity on the development of the PCV2 infection. Through lymph node biopsies at 10, 15, and 21 days postinoculation (DPI), PCV2 replication in lymphoid tissues was monitored. The production of total PCV2-specific and PCV2-neutralizing antibodies was followed, together with interferon-gamma (IFN-gamma) mRNA expression levels in peripheral blood monocytes as a marker for cellular immunity. In general, the CysA-treated pigs showed the highest PCV2 titers, indicating that the adaptive immunity is necessary to restrain PCV2 replication. Three different PCV2 replication patterns were observed in non-CysA-treated pigs. Pattern 1: In two pigs, PCV2 was not detected. They had the highest neutralizing antibody titers, appearing from 15 DPI. In these pigs a good cellular response was indicated by a peak in IFN-gamma mRNA at 15 DPI. Pattern 2: Five pigs contained low to moderate PCV2 titers at 15 DPI, remaining constant or decreasing towards 21 DPI. Lower neutralizing antibody titers were observed and no rise in IFN-gamma was detected. Pattern 3: In one pig, a low PCV2 titer at 15 DPI dramatically increased toward 21 DPI. Although an antibody response against PCV2 was mounted, no PCV2-neutralizing antibodies were detected. This pig also showed no rise in IFN-gamma. The study findings indicate that variation in the onset of the adaptive immunity may account for variation in PCV2 replication among pigs. Absence of PCV2-neutralizing antibodies may be an important factor in the development of an increased virus replication.

A detailed understanding of the enhanced hypothermic productivity of interferon‐γ by Chinese‐hamster ovary cells

Culturing CHO (Chinese-hamster ovary) cells at low temperature leads to growth arrest in the G0/G1 phase of the cell cycle and, in many cases, causes an increase in the specific productivity of recombinant protein. Controlled proliferation is often used to increase CHO specific productivity, and thus there is speculation that the enhanced productivity at low temperature is due to G0/G1-phase growth arrest. However, we show that the positive effect of low temperature on recombinant protein production is due to elevated mRNA levels and not due to growth arrest and that a cell line can still exhibit growth-associated productivity at low temperatures. Using a CHO cell producing recombinant human IFN-gamma (interferon-gamma), we show that productivity increases as the percentage of cells in the S phase of the cell cycle increases, at both 32 and 37 degrees C. The increased productivity is due to higher recombinant IFN-gamma mRNA levels. We also show that, for a given cell-cycle distribution, specific productivity increases as the temperature is lowered from 37 to 32 degrees C. Thus specific productivity is maximized when cells are actively growing (high percentage of S-phase cells) and also exposed to low temperature. These findings have important implications for cell-culture optimization and cell-line engineering, providing evidence that a CHO cell line capable of actively growing at low temperature would provide improved total protein production relative to the current growth strategies, namely 37 degrees C active growth or low-temperature growth arrest.