American cutaneous leishmaniasis: In situ immune response of patients with recent and late lesions

TNF‐α, IFN‐γ, IL‐10, IL‐17, CD68 and CD57 were evaluated in biopsies of patients with American cutaneous leishmaniasis living in Sorocaba, Brazil. The analyses were performed considering the time of lesions from 23 patients with recent lesions (Group I) and 19 patients with late lesions (Group II). All patients were infected with Leishmania (Viannia) braziliensis. Immunostaining cells for CD68, CD57, TNF‐ α, IFN‐γ, IL‐10 and IL‐17 were performed by immunohistochemistry. Except for CD68 and IL‐17, the distribution of in situ for CD57, IL‐10, TNF‐α and IFN‐γ showed that patients with recent lesions expressed higher levels than those with late lesions. The comparison of cytokine expression/group showed that IL‐10 was significantly higher than IL‐17 and IFN‐γ (similar data were shown in IL‐17 compared with TNF‐α), suggesting an immunological balance between inflammatory‐anti‐inflammatory agents. This balance was similar for two groups of patients. In conclusion, these data suggested that (i) patients from Group I had recent lesions (in the beginning of chronic phase) compared to those from Group II and (ii) the modulation of inflammatory response in patients with recent American cutaneous leishmaniasis was correlated with IL‐10 expression in skin lesions preventing the development of mucosal forms. The parasite treatment also prevented the evolution of severe forms.     

Analysis of iron superoxide dismutase‐encoding DNA vaccine on the evolution of the Leishmania amazonensis experimental infection

The present work aimed to evaluate the immunogenicity of Leishmania amazonensis iron superoxide dismutase (SOD)‐encoding DNA experimental vaccine and the protective properties of this DNA vaccine during infection. The SOD gene was subcloned into the pVAX1 plasmid, and it was used to immunize BALB/c mice. Twenty‐one days after the last immunization, mice were sacrificed (immunogenicity studies) or subcutaneously challenged with L. amazonensis (studies of protection), and alterations in cellular and humoral immune responses were evaluated, as well as the course of infection. Mice only immunized with pVAX1‐SOD presented increased frequencies of CD4⁺IFN‐γ⁺, CD8⁺IFN‐γ⁺ and CD8⁺IL‐4⁺ lymphocytes; moreover, high levels of IgG2a were detected. After challenge, mice that were immunized with pVAX1‐SOD had increased frequencies of the CD4⁺IL‐4⁺, CD8⁺IFN‐γ⁺ and CD8⁺IL‐4⁺ T lymphocytes. In addition, the lymph node cells produced high amounts of IFN‐γ and IL‐4 cytokines. Increased IgG2a was also detected. The pattern of immunity induced by pVAX1‐SOD partially protected the BALB/c mice from a challenge with L. amazonensis, as the animals presented reduced parasitism and lesion size when compared to controls. Taken together, these results indicate that leishmanial SOD modulates the lymphocyte response, and that the elevation in IFN‐γ possibly accounted for the decreased skin parasitism observed in immunized animals.     

In situ immunopathological changes in cutaneous leishmaniasis due to Leishmania donovani

Cutaneous leishmaniasis in Sri Lanka is a newly established parasitic disease caused by the usually visceralizing Leishmania donovani. Skin lesions manifest as non‐itchy, non‐tender papules, nodules or ulcers. In situ cytokine expression provides clues for immunopathogenesis of this localized form of disease. Skin biopsies from 58 patients were analyzed for histological appearance and in situ cytokine expression of T‐helper 1 (Th1) and T‐helper 2 (Th2) cytokines, namely interferon (IFN)‐γ, interleukin (IL)‐12A, tumor necrosis factor (TNF)‐α, IL‐4 and IL‐10 by real‐time RT‐PCR. Significant up‐regulation of the Th1 cytokine IFN‐γ and down‐regulation of the Th2 cytokine IL‐4 were seen in patients compared to healthy controls. Significantly elevated tissue expression of IFN‐γ and TNF‐α was seen in lesions that presented later than 6 months from the time of onset, while IL‐4 expression was more prominent in lesions that responded poorly to antimony therapy. A prominent Th1 response appears to support resolving of lesions, whereas a Th2‐biased milieu tends to favor poor responsiveness to antimony and delayed lesion healing in L. donovani infections in Sri Lanka.     

Cytokine profile of Plasmodium falciparum-specific T cells in non-immune malaria patients

CD3⁺ T cells are important sources of both pro‐ and anti‐inflammatory cytokines during Plasmodium falciparum malaria. We studied the frequency of interleukin‐2 (IL‐2), gamma interferon (IFN‐γ), tumour necrosis factor‐alpha (TNF‐α) and IL‐10 expressing CD3⁺ cells in 10 non‐immune malaria patients with uncomplicated malaria and in one patient with cerebral malaria after P. falciparum‐specific and non‐specific mitogenic stimulation. Analysis by fluorescence‐activated cell sorting was performed after drug‐induced clearance of parasites to allow previously sequestered T cells to be detected in peripheral blood. CD3⁺ cells of patients responded to P. falciparum infected erythrocytes with significant increases in the percentage of IL‐2, IFN‐γ, and TNF‐α, but also IL‐10, positive cells. CD3⁺ cells from malaria‐naïve donors were also responsive to specific stimulation albeit to a much lesser extent. Mitogenic stimulation of PBMC revealed no significant differences between cells of patients and controls. CD3⁺ cells of the patient with cerebral malaria were hyporesponsive both to the infecting parasite isolate as well as to our laboratory‐adapted P. falciparum isolate, whereas two patients with uncomplicated disease were more responsive to their infecting parasites than to the laboratory‐adapted isolate. The results indicate that the increased responsiveness of in vivo primed compared to malaria‐naïve CD3⁺ cells is Plasmodium‐specific and biased towards production of IFN‐γ and TNF‐α.     

Tumor necrosis factor‐α and interferon‐γ directly impair epithelial barrier function in cultured mouse cholangiocytes

Abstract: Background/Aims: In primary biliary cirrhosis (PBC), cytokines from CD4 ⁺ T lymphocytes were suggested to contribute to the intralobular bile duct damage together with cellular immunity by CD8 ⁺ T lymphocytes. Recently, we reported that immunolocalization of 7H6 – a tight junction (TJ)‐associated protein – was significantly diminished in cholangiocytes in the PBC liver. In this study, we examined the direct effects of several cytokines – tumor necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), interleukin‐2 and 4 (IL‐2 and 4) – on TJ in immortalized mouse cholangiocytes. Moreover, we examined the inhibitory effect of ursodeoxycholic acid (UDCA) on cytokine‐induced changes in paracellular permeability. Methods: Barrier function of TJ was evaluated by measuring transepithelial electrical resistance (TER) and ³H‐inulin flux. We also performed immunostaining and immunoblotting for TJ‐associated proteins – claudin‐1 and ‐3, occludin, zonula occluden‐1 (ZO‐1) and 7H6. Results: TNF‐α and IFN‐γ, but neither IL‐2 nor IL‐4, significantly decreased TER (P < 0.005). ³H‐inulin flux studies confirmed IFN‐α‐induced increases in paracellular permeability of cholangiocytes (P < 0.001). In immunostaining and immunoblotting studies, TJ‐associated proteins were well preserved in TNF‐α‐ or IFN‐γ‐treated cells. Ursodeoxycholic acidhas been found to have no inhibitory effect on increased paracellular permeability induced by TNF‐α or IFN‐γ. Conclusion: These findings show that TNF‐α and IFN‐γ disrupt barrier function of TJ in cholangiocytes without major structural changes to TJ and suggest that disruption of TJ function and subsequent leakage of the bile constituents may influence the aggravation of cholestasis in PBC.     

Therapeutic efficacies of chitosan against Pneumocystis pneumonia of immunosuppressed rat

This study was designed to investigate the therapeutic efficacy of chitosan on Pneumocystis pneumonia (PCP) in immunosuppressed rats. The PCP rat model was established using intramuscular injections of dexamethasone sodium phosphate. To estimate treatment effects of chitosan on rat PCP, weight gain, lung weight, lung weight/body weight (LW/BW) ratio and per cent survival were measured and the HSP70 mRNA expression of Pneumocystis carinii was detected using real‐time PCR analysis. Rat lung tissues were stained with HE, and their pathological changes, inflammatory cells and alveolar macrophages were observed by light microscopy. Rat lymphocyte numbers and the concentrations of IL‐10, IFN‐γ and TNF‐α were measured by flow cytometry and ELISA analysis. Additionally, the ultrastructure of P. carinii was examined by electron microscopy to evaluate the effects of chitosan on the protist. Our results demonstrated that chitosan has some apparent treatment effects on rat PCP by reducing HSP70 mRNA expression and lung inflammation, increasing the concentrations of IL‐10 and IFN‐γ as well as CD4⁺ T‐lymphocyte numbers, reducing the CD8⁺ T‐lymphocyte numbers and the concentration of TNF‐α and inducing significant ultrastructural damage to P. carinii. Although its precise therapeutic mechanism has yet to be determined, these results lay a theoretical foundation for PCP chitosan therapy.     

Vaccine potential of HLA‐A2 epitopes from Leishmania Cysteine Protease Type III (CPC)

Although the precise host‐defence mechanisms are not completely understood, T‐cell‐mediated immune responses are believed to play a pivotal role in controlling parasite infection. In this study, the potential HLA*A2 restricted peptides were predicted and the ability of peptides to bind HLA‐A*02 was confirmed by a MHC stabilization assay. Two of the peptides tested stabilized HLA‐A*02: (a) LLATTVSGL (P1) and (b) LMTNGPLEV (P3). The potential of the peptides to generate protective immune response was evaluated in patients with treated visceral leishmaniasis as well as in healthy control subjects. Our data suggest that CD8⁺ T‐cell proliferation against the selected peptide was significantly higher compared to unstimulated culture conditions. The stimulation of peripheral blood mononuclear cells with epitopes individually or as a cocktail upregulated IFN‐γ production, which indicates its pivotal role in protective immune response. The IFN‐γ production was mainly in a CD8⁺ T‐cells‐dependent manner, which suggested that these epitopes had an immunoprophylactic potential in a MHC class I‐dependent manner. Moreover, no role of the CD3⁺ T cell was observed in the IL‐10 production against the selected peptides, and no role was found in disease pathogenesis. Further studies on the role of these synthetic peptides may contribute significantly to developing a polytope vaccine idea towards leishmaniasis.     

Differential in vitro CD4+/CD8+ T‐cell response to live vs. killed Leishmania major

Clinical trials of killed Leishmania vaccines showed a limited efficacy compared with leishmanization (LZ). The reason for this difference in protection against cutaneous leishmaniasis (CL) is not known and in vivo studies on T‐cell function may provide valuable information. Nevertheless, there are limited studies on the nature of the stimulatory effects of live vs. killed parasites on human T cells in vitro. A total of nine Leishmanin Skin Test+ volunteers with a history of self‐healing CL (HCL) and seven healthy volunteers were included in this study. 5,6‐carboxyfluroescein diacetate succinimidyl ester‐labelled CD4⁺/CD8⁺ lymphocytes were cultured with killed Leishmania Lysate (Killed LL) or live Leishmania major (Live LM) and analysed for proliferation using flow cytometry. Culture supernatants were used for cytokine titration. In HCL volunteers, upon stimulation with killed LL, the number of proliferated CD4⁺/CD8⁺ cells was significantly more than that of unstimulated (P < 0·001) or live LM stimulated (P < 0·05) cells, or cells from controls (CD4⁺/CD8⁺: P < 0·05/P < 0·001). Stimulation of CD4⁺ cells with Live LM (P < 0·001) or Killed LL (P < 0·05) induced a significantly higher IFN‐γ production compared with that of controls, but Live LM induced significantly (P < 0·05) more IFN‐γ than Killed LL. A significantly (P < 0·05) higher IFN‐γ production was observed when CD8⁺ cells were stimulated with Live LM. Cells from HCL volunteers showed significantly more IL‐10 production to Live LM stimulation compared with that of controls (CD4⁺: P < 0·05 /CD8⁺: P < 0·001) or cells stimulated with Killed LL (CD4⁺/CD8⁺: P < 0·001/P < 0·0005). Whereas Killed LL induced more proliferation response in purified T cells, Live LM induced cytokine production without significant induction of proliferation. The results from healed CL volunteers in this study could be implicated in further studies on T‐cell response in vaccinated individuals.     

Early IL‐10 predominant responses are associated with progression to chronic hepatitis C virus infection in injecting drug users

Summary. The critical events in clearance or persistence of hepatitis C virus (HCV) infection are unknown but likely to be determined early in acute infection. Type 1 and type 2 cytokine production was assessed by HCV peptide ELISpot and multiplex in vitro cytokine production assays in longitudinally collected samples from 20 untreated participants enrolled in the Australian Trial in Acute Hepatitis C (ATAHC); a prospective cohort of acute HCV infection (77% injecting drug users, IDU). Significantly higher interleukin‐10 (IL‐10) production (P = 0.048), in the relative absence of interferon‐gamma (IFN‐γ) and IL‐2 production, was present early in HCV infection in those who progressed to chronic infection. In contrast, viral clearance was associated with a greater magnitude and broader specificity of IFN‐γ (magnitude P < 0.001, breadth P = 0.004) and IL‐2 responses, in the relative absence of IL‐10. Early IL‐10 production was correlated with higher HCV RNA level at baseline (P = 0.046) and week 12 (P = 0.018), while IFN‐γ and IL‐2 production was inversely correlated with HCV RNA level at baseline (IFN‐γP = 0.020, IL‐2 P = 0.050) and week 48 (IFN‐γP = 0.045, IL‐2 P = 0.026). Intracellular staining (ICS) indicated the HCV‐specific IFN‐γ response was primarily from CD8⁺ T cells and NK cells, whereas IL‐10 production was predominantly from monocytes, with a subset of IL‐10 producing CD8⁺ T cells present only in those who progressed to chronic infection. IL‐10, an immunoregulatory cytokine, appears to play a key role in progression to chronic HCV infection.     

Co‐existence of Echinococcus granulosus infection and cancer metastasis in the liver correlates with reduced Th1 immune responses

A possible relationship between cancer and Echinococcus granulosus infection has been postulated. As T cells are critical players in immune responses against both infections and malignancies, in an experimental model of secondary echinococcosis and breast cancer, this study aims to observe the progression of cancer and to determine the characters of T‐cell responses. 4T1 breast tumour cells were subcutaneously injected into mammary region, whereas protoscoleces were intraperitoneally inoculated into the mice. Hydatid cysts, tumours and metastases were determined with macroscopic and histopathological evaluation. T cells found in spleen, liver and tumour were characterised by flow cytometric analysis of CD3, CD4, CD8, CD25, CCR5, CCR3, IL‐4 and IFN‐γ. In the mice inoculated both with protoscoleces and with breast tumour cells, increased frequency of cancer metastasis was observed in the liver. The amount of CD4⁺ T cells was increased in the liver and in the spleen of mice infected with E. granulosus. However, co‐existence of echinococcosis and metastatic lesions in the liver was associated with significant reduction in the IFN‐γ⁺ and CCR5⁺ Th1 cells and increase in the CD25⁺ T cells. Our results may indicate an immunological link between cystic echinococcosis and cancer that allows tumour metastasis to flourish in the liver.     

Differential IFN‐γ production by adult and neonatal blood CD56+ natural killer (NK) and NK‐like‐T cells in response to Trypanosoma cruzi and IL‐15

Early interferon‐gamma (IFN‐γ) release by innate cells is critical to direct type 1 immune response able to control intracellular pathogens like Trypanosoma cruzi. Although CD56^bright natural killer (NK) cells are reported to be potent early IFN‐γ producers, other CD56⁺ cells like CD56^dim NK cells and NK‐like T cells have recently been shown to also release IFN‐γ. We have here studied the contribution of each CD56⁺ lymphocyte populations in early IFN‐γ production in both adults and neonates. On this purpose, we analysed the kinetics of IFN‐γ production by RT‐PCR, ELISA and flow cytometry from 2 h onwards after T. cruzi and IL‐15 stimulation and sought for the responding CD56⁺ cells. CD56^bright and CD56^dimCD16^? NK cells were the more potent IFN‐γ early producers in response to IL‐15 and parasites in adults and neonates. In both age groups, the majority of IFN‐γ producing cells were NK cells. However, on the contrary to neonates, CD3⁺CD56⁺ NK‐like T cells and CD3⁺CD56^? ‘classical’ T cells also contributed to early IFN‐γ production in adults. Altogether, our results support that whereas NK cells responded almost similarly in neonates and adults, cord blood innate CD56⁺ and CD56^? T cells displayed major quantitative and qualitative defects that could contribute to the well‐known neonatal immune immaturity.     

Type I IFNs promote the early IFN‐γ response and the IL‐10 response in Leishmania mexicana infection

The protozoan parasite Leishmania mexicana causes chronic cutaneous disease in humans and most mouse strains. We previously showed that STAT4‐deficient mice, but not IL‐12p40‐deficient mice, have more parasites and progressively growing lesions unlike those of wild‐type mice, the lesions and parasite burdens of which plateau by 10–12 weeks post‐infection. This demonstrates a STAT4‐dependent, IL‐12/IL‐23‐independent pathway of parasite control. Type I IFNs are important in viral and other infections and can activate STAT4. We found that IFN‐α/βR‐deficient mice have a nonpersistent, early IFN‐γ defect, and a persistent, early IL‐10 defect, without changes in serum IL‐12 or LN‐derived nitric oxide. We found less IL‐10 per cell in CD25⁺CD4⁺ T cells and possibly fewer IL‐10‐producing cells in the draining LN of IFN‐α/βR‐deficient vs. wild‐type mice. IFN‐α/βR‐deficient mice have chronic, nonprogressive disease, like wild‐type mice, suggesting that IL‐10 and IFN‐γ defects may balance each other. Our data indicate that although type I IFNs help promote early Th1 responses, they are not the missing activators of STAT4 responsible for partial control of L. mexicana. Also, the lack of lesion resolution in IFN‐α/βR‐deficient mice despite lower IL‐10 levels indicates that other pathways independent of T cell IL‐10 help prevent an IL‐12‐driven clearance of parasites.     

Giardia lamblia binding immunoglobulin protein triggers maturation of dendritic cells via activation of TLR4‐MyD88‐p38 and ERK1/2 MAPKs

Much remains unknown about the mammalian immune response to Giardia lamblia, a protozoan pathogen that causes diarrhoeal outbreaks. We fractionated protein extracts of G. lamblia trophozoites by Viva‐spin centrifugation, DEAE ion exchange and gel filtration chromatography. Resultant fractions were screened for antigenic molecules by western blots analysis using anti‐G. lamblia antibodies (Abs), resulting in identification of G. lamblia binding immunoglobulin protein (GlBiP). Maturation of mouse dendritic cells (DCs) in response to recombinant GlBiP (rGlBiP) was detected by increased expression of surface molecules such as CD80, CD86 and MHC class II; these mature DCs, produced pro‐inflammatory cytokines (TNF‐α, IL‐12 and IL‐6). Especially, the truncated rGlBiP containing the heat‐shock protein 70 domain‐induced cytokine production from mouse DCs. rGlBiP‐induced DC activation was initiated by TLR4 in a MyD88‐dependent way and occurred through activation of p38 and ERK1/2 MAPKs as well as increased activity of NF‐κB and AP‐1. Moreover, CD4⁺ T cells stimulated with rGlBiP‐treated DCs produced high levels of IL‐2 and IFN‐γ. Together, our results suggest that GlBiP contributes to maturation of DCs via activation of TLR4‐MyD88‐p38, ERK1/2 MAPK, NF‐κB and AP‐1.     

Flt3 ligand treatment modulates parasitemia during infection with rodent malaria parasites via MyD88‐ and IFN‐γ‐dependent mechanisms

We previously showed that treatment of mice with the Flt3 ligand (Flt3L) prevents development of lethal experimental cerebral malaria and inhibits parasitemia during Plasmodium berghei ANKA (PbA) infection. In this study, we investigated the mechanisms underlying the reduction of parasitemia in Flt3L‐treated mice. Studies using gene knockout mice and antibody treatment indicated that the anti‐parasitemia effect of Flt3L was mediated by innate immune system and was dependent on MyD88, IFN‐γ, IL‐12 and natural killer (NK) cells. The number of NK cells and their ability to produce IFN‐γ was enhanced in Flt3L‐treated mice. Phagocytic activity of splenocytes was increased in Flt3L‐treated mice after PbA infection when compared with that in untreated mice, and this activity was mainly mediated by the accumulation of F4/80^midCD11b⁺ cells in the spleen. In both MyD88^?/? and IFN‐γ^?/? mice, the proportion of F4/80^midCD11b⁺ cells was not increased in the spleen of Flt3L‐treated mice after infection. These correlations suggest that NK cells produce IFN‐γ in Flt3L‐treated mice, and accumulation of F4/80^midCD11b⁺ cells in the spleen is promoted by an IFN‐γ ‐dependent manner, culminating in the inhibition of parasitemia. These findings imply that Flt3L promotes effective innate immunity against malaria infection mediated by interplay among varieties of innate immune cells.     

Induction of regulatory T cells by Opisthorchis viverrini

Opisthorchis viverrini causes public health problems in South‐East Asia. Recently, TGF‐β and IL‐10 have been reported to increase in O. viverrini‐infected hamsters but the sources of these cytokines are still unknown. In this study, the CD4⁺ T cells in infected hamsters were investigated. It was demonstrated that IL‐4⁺CD4⁺ T cells were significantly increased in hamster spleens and mesenteric lymph nodes (MLNs) during chronic infection. Interestingly, IL‐10⁺CD4⁺ T cells were also discovered at a significant level while Treg (T regulatory)‐like TGF‐ β⁺CD4⁺ T cells were in MLNs of infected hamsters. Moreover, the CD4⁺CD25⁺Foxp3⁺ Treg cell response was significantly found both in spleens and MLNs in infected hamsters. The findings were then confirmed by development of T‐cell clones against crude somatic antigens (CSAg) in immunized BALB/c mice. Five clones named TCC21, TCC23, TCC35, TCC41 and TCC108 were established. The TCC21 was found to be the TGF‐β⁺CD4⁺ while TCC35, TCC41 and TCC108 were IL‐4⁺CD4⁺ and TCC23 was IFN‐γ⁺CD4⁺. This TGF‐β⁺CD4⁺ T clone showed an inhibitory function in vitro in mononuclear cell proliferation via TGF‐β‐mediated mechanisms. This study indicated that O. viverrini‐infected hamsters could induce TGF‐β⁺ CD4⁺ Treg‐like cells. The CSAg‐specific Tregs secreted high TGF‐β, and limited immune cell proliferation.     

Prophylactic properties of a Leishmania‐specific hypothetical protein in a murine model of visceral leishmaniasis

In this work, the effect of vaccination of a newly described Leishmania infantum antigenic protein has been studied in BALB/c mice infected with this parasite species. The LiHyD protein was characterized after a proteomic screening performed with the sera from dogs suffering visceral leishmaniasis (VL). Its recombinant version was expressed, purified and administered to BALB/c mice in combination with saponin. As a result of vaccination and 10 weeks after challenge using an infective dose of L. infantum stationary promastigotes, vaccinated mice showed lower parasite burdens in different organs (liver, spleen, bone marrow and footpads’ draining lymph nodes) than mice inoculated with the adjuvant alone or the vaccine diluent. Protected mice showed anti‐Leishmania IgG2a antibodies and a predominant IL‐12‐driven IFN‐γ production (mainly produced by CD4⁺ T cells) against parasite proteins, whereas unprotected controls showed anti‐Leishmania IgG1 antibodies and parasite‐mediated IL‐4 and IL‐10 responses. Vaccinated mice showed an anti‐LiHyD IgG2a humoral response, and their spleen cells were able to secrete LiHyD‐specific IFN‐γ, IL‐12 and GM‐CSF cytokines before and after infection. The protection was correlated with the Leishmania‐specific production on nitric oxide. Altogether, the results indicate that the new LiHyD protein could be considered in vaccine formulations against VL.     

The role of IL‐1β in reduced IL‐7 production by stromal and epithelial cells: a model for impaired T‐cell numbers in the gut during HIV‐1 infection

Abstract. Thang PH, Ruffin N, Brodin D, Rethi B, Cam PD, Hien NT, Lopalco L, Vivar N, Chiodi F (Karolinska Institutet, Stockholm, Sweden; National Institute of Hygiene and Epidemiology, Hanoi, Vietnam; San Raffaele Scientific Institute, Milan, Italy). The role of IL‐1β in reduced IL‐7 production by stromal and epithelial cells: a model for impaired T‐cell numbers in the gut during HIV‐1 infection. J Intern Med 2010; 268 : 181–193. Objectives. Interleukin (IL)‐7 is a key cytokine in T‐cell homeostasis. Stromal cells, intestinal epithelial cells and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL‐7 production are still unclear. We assessed whether IL‐1β and interferon (IFN)‐γ, cytokines produced during inflammatory conditions, may impact on IL‐7 production. Design. We used human intestinal epithelial cells (DLD‐1 cell line) and bone marrow stromal cells (HS27 cell line), known to produce IL‐7; IL‐7 production was evaluated at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL‐1β and/or IFN‐γ leads to changes in the gene expression of cytokines, Toll‐like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole‐genome microarray Human Gene 1.0 ST. Results. We found that IFN‐γ enhanced the expression of IL‐7 mRNA (P < 0.001) in both cell lines. IL‐1β treatment led to a significant down‐regulation (P < 0.001) of IL‐7 mRNA expression in both cell lines. The IL‐7 concentration in supernatants collected from treated DLD‐1 and HS27 cell cultures reflected the trend of IL‐7 mRNA levels. The gene profiles revealed dramatic changes in expression of cytokines and their receptors (IL‐7/IL‐7Rα; IL‐1α,IL‐1β/IL‐1R1; IFN‐γ/IFN‐γR1), of IFN regulatory factors (IRF‐1 and 2), of TLRs and of important chemo‐attractants for T cells. The microarray results were verified by additional methods. Conclusions. Our results are discussed in the setting of inflammation and T‐cell survival in the gut compartment during HIV‐1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL‐7 homeostasis and homing of T cells.     

In vivo infectivity of liver extracts after resolution of hepadnaviral infection following therapy associating DNA vaccine and cytokine genes

DNA‐based vaccination appears of promise for chronic hepatitis B immunotherapy, although there is an urgent need to increase its efficacy. In this preclinical study, we evaluated the therapeutic benefit of cytokine (IL‐2, IFN‐γ) genes co‐delivery with DNA vaccine targeting hepadnaviral proteins in the chronic duck hepatitis B virus (DHBV) infection model. Then, we investigated the persistence of replication‐competent virus in the livers of apparently resolved animals. DHBV carriers received four injections of plasmids encoding DHBV envelope and core alone or co‐delivered with duck IL‐2 (DuIL‐2) or duck IFN‐γ (DuIFN‐γ) plasmids. After long‐term (8 months) follow‐up, viral covalently closed circular (ccc) DNA was analysed in duck necropsy liver samples. Liver homogenates were also tested for in vivo infectivity in neonatal ducklings. Co‐delivery of DuIFN‐γ resulted in significantly lower mean viremia starting from week 21. Viral cccDNA was undetectable by conventional methods in the livers of 25% and 57% of animals co‐immunized with DuIL‐2 and DuIFN‐γ, respectively. Interestingly, inoculation of liver homogenates from 7 such apparently resolved animals, exhibiting cccDNA undetectable in Southern blotting and DHBV expression undetectable or restricted to few hepatocytes, revealed that three liver homogenates transmitted high‐titre viremia (3–5×10¹⁰ vge/mL) to naïve animals. In conclusion, our results indicate that IFN‐γ gene co‐delivery considerably enhances immunotherapeutic efficacy of DNA vaccine targeting hepadnaviral proteins. Importantly, we also showed that livers exhibiting only minute amounts of hepadnaviral cccDNA could induce extremely high‐titre infection, highlighting the caution that should be taken in occult hepatitis B patients to prevent HBV transmission in liver transplantation context.