BACKGROUND

We have studied the cytokine production patterns in gastric mucosal biopsy specimens with and without the Helicobacter pylori infection, using a reverse transcription-polymerase chain reaction (RT-PCR) method capable of detecting low levels of specific mRNA.

METHODS

Total RNA was prepared from biopsy specimens with the acid guanidinium thiocyanate-phenol-chloroform method. cDNA was synthesized by M-MLV RTase and amplified using the oligonucleotide primers specific for interleukin-1 beta (IL-1beta), IL-2, IL-<em>3</em>, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>), IFN-beta, and IFN-gamma by PCR methods.

RESULTS

Although IL-1 beta and IFN-gamma mRNA were detected in most specimens, IL-2, IL-<em>3</em>, IL-4, IL-5, IL-9, TNF-<em>alpha</em>, and IFN-beta mRNA were not detected at all. The expressions of IL-7 and IL-8 mRNA were significantly higher in H. pylori-positive gastritis than in H. pylori-negative normal controls. There was a significant correlation between the expression of IL-8 mRNA and the severity of gastritis both in the antrum and in the corpus. On the other hand, there was a significant correlation between the expression of IL-7 mRNA and the severity of gastritis only in the corpus.

CONCLUSIONS

These findings suggest that some cytokines, especially IL-7 and IL-8, play some roles in H. pylori-associated gastritis.

OBJECTIVE

We investigated the antiproliferative efficacy of the addition of alpha-interferon to the somatostatin analogue octreotide in patients with metastasized gastroenteropancreatic tumors unresponsive to octreotide monotherapy.

METHODS

In an open prospective trial, 21 patients with metastasized neuroendocrine gastroenteropancreatic tumors (nine patients with carcinoid syndrome, eight with nonfunctioning tumors, four with gastrinoma) were treated with 5 x 10(6) IU alpha-interferon tiw in addition to 200 microg of octreotide tid. All patients, including 16 patients with preceding monotherapy with 200 microg of octreotide tid, had tumor progression documented by computed tomography before entering the study. Growth response (computed tomography documented) and biochemical response were assessed at 3-month intervals.

RESULTS

Inhibition of tumor growth was observed in 14 patients (67%), 11 of whom had preceding octreotide monotherapy; complete regression was observed in one patient lasting for 49 months and stable disease (stand-still) in 13 patients lasting for 3 to 52 months (median, 12 months). Seven patients failing this combination therapy exhibited a significantly shorter overall survival (median, 23 months; range, 5 to 42 months) than the 14 patients responding to this regimen (median, 68 months; range, 12 to 112 months; p = 0.007). Two patients are still alive. Biochemical response was achieved in 69% of patients with functioning tumors: in three of four patients with gastrinoma and in six of nine patients with carcinoid syndrome.

CONCLUSIONS

These data suggest that the addition of alpha-interferon to octreotide has antiproliferative efficacy in a subgroup of patients with advanced metastatic disease unresponsive to octreotide monotherapy. Prolonged survival was seen in the responder group.

Lymphocytes that inhibit hematopoiesis may have a pathogenic role in some forms of bone marrow failure, and lymphocyte-mediated suppression may also be important in the normal regulation of bone marrow function. We have investigated the mechanism of in vitro suppression of hematopoiesis by T cells by using the methylcellulose colony culture system. Total peripheral blood T cells and separated subpopulations of helper (OKT4+) and suppressor (OKT8+) cells that have been stimulated by exposure to lectin suppress autologous colony formation by bone marrow myeloid (CFU-C) and erythroid (BFU-E) progenitor cells. Medium conditioned by these cells is also inhibitory, indicating that the suppressor activity is a soluble factor. A strong correlation existed for the concentration of <em>interferon</em> and the degree of hematopoietic suppressor activity in these supernatants; both activities peaked at days <em>3</em> to 5 of incubation and had sharply declined by day 7. <em>Interferon</em> production was enhanced by exposure of lymphocytes to sheep red blood cells during the rosetting procedure. Specific antiserum and a monoclonal antibody directed against gamma-(immune) <em>interferon</em> abrogated the inhibitory activity for hematopoiesis produced by lectin-stimulated T cells; an antiserum to <em>alpha</em>-<em>interferon</em> was generally much less effective in neutralizing activity. We infer from these results that gamma-<em>interferon</em> is the mediator of hematopoietic suppression generated by lectin-treated T-cells.

The NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1 proteins) have only approximately 20% amino acid sequence identity. Nevertheless, these proteins show several functional similarities, such as their ability to bind to the same RNA targets and to inhibit the activation of protein kinase R in vitro. A critical function of the A/NS1 protein is the inhibition of synthesis of <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) during viral infection. Recently, it was also found that the B/NS1 protein inhibits IFN-<em>alpha</em>/beta synthesis in virus-infected cells. We have now found that the expression of the B/NS1 protein complements the growth of an influenza A virus with A/NS1 deleted. Expression of the full-length B/NS1 protein (281 amino acids), as well as either its N-terminal RNA-binding domain (amino acids 1 to 9<em>3</em>) or C-terminal domain (amino acids 94 to 281), in the absence of any other influenza B virus proteins resulted in the inhibition of IRF-<em>3</em> nuclear translocation and IFN-beta promoter activation. A mutational analysis of the truncated B/NS1(1-9<em>3</em>) protein showed that RNA-binding activity correlated with IFN-beta promoter inhibition. In addition, a recombinant influenza B virus with NS1 deleted induces higher levels of IRF-<em>3</em> activation, as determined by its nuclear translocation, and of IFN-<em>alpha</em>/beta synthesis than wild-type influenza B virus. Our results support the hypothesis that the NS1 protein of influenza B virus plays an important role in antagonizing the IRF-<em>3</em>- and IFN-induced antiviral host responses to virus infection.

We demonstrate here that Sendai virus (SeV) blocks <em>alpha</em> <em>interferon</em> (IFN-<em>alpha</em>) signaling to signal transducers and activators of transcription (STATs) in HeLa cells. IFN-<em>alpha</em>-stimulated tyrosine phosphorylation of STATs and subsequent formation of the IFN-stimulated gene factor <em>3</em> transcription complex were inhibited in SeV-infected cells, resulting in inefficient induction of IFN-stimulated gene products. None of the components of the signaling pathway-type I IFN receptor subunits Jak1, Tyk2, Stat1, Stat2, and p48-was degraded. Moreover, tyrosine phosphorylation of Jak1 in response to IFN-<em>alpha</em> was unaffected at the early phase of infection, suggesting that oligomerization of the receptor subunits proceeded normally. In contrast to Jak1, IFN-<em>alpha</em>-stimulated tyrosine phosphorylation of Tyk2 was partially inhibited. Therefore, this partial inhibition of activation of Tyk2 probably contributes to the subsequent failure in the activation of STATs.

Apoptosis has been identified as a mechanism of pancreatic islet beta-cell death in autoimmune diabetes. Proinflammatory cytokines are candidate mediators of beta-cell death in autoimmune diabetes, and these cytokines can induce beta-cell death by apoptosis. In the present study, we examined whether transfection of human islet beta-cells with an anti-apoptotic gene, bcl-2, can prevent cytokine-induced beta-cell destruction. Human islet beta-cells were transfected by a replication-defective herpes simplex virus (HSV) amplicon vector that expressed the bcl-2 gene (HSVbcl-2) and, as a control, the same HSV vector that expressed a beta-galactosidase reporter gene (HSVlac). Two-color immunohistochemical staining revealed that 95+/-<em>3</em>% of beta-cells transfected with HSVbcl-2 expressed Bcl-2 protein compared with 14+/-<em>3</em>% of beta-cells transfected with HSVlac and 19+/-4% of nontransfected beta-cells. The bcl-2-transfected beta-cells were fully protected from impaired insulin secretion and destruction resulting from incubation for 5 days with the cytokine combination of interleukin (IL)-1beta, tumor necrosis factor (TNF)-<em>alpha</em>, and <em>interferon</em> (IFN)-gamma. In addition, the bcl-2-transfected islet cells were significantly protected from cytokine-induced lipid peroxidation and DNA fragmentation. These results demonstrate that cytokine-induced beta-cell dysfunction and death involve mechanisms subject to regulation by an anti-apoptotic protein, Bcl-2. Therefore, bcl-2 gene therapy has the potential to protect human beta-cells in pancreatic islets, or islet grafts, from immune-mediated damage in type 1 diabetes.

A characteristic feature of neoplastic transformation is the loss of external control by cytokines and extracellular matrix of cellular differentiation, migration, and mitogenesis. Because suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine-induced signaling, it has been hypothesized that an aberrant SOCS expression plays a role in neoplastic transformation. This study reports on a constitutive SOCS-<em>3</em> expression in cutaneous T-cell lymphoma (CTCL) cell lines. SOCS-<em>3</em> protein is constitutively expressed in tumor cell lines (but not in nonmalignant T cells) obtained from affected skin from a patient with mycosis fungoides (MF) and from peripheral blood from a patient with Sezary syndrome (SS). In contrast, constitutive SOCS-<em>3</em> expression is not found in the leukemic Jurkat T-cell line, the MOLT-4 acute lymphoblastic leukemia cell line, and the monocytic leukemic cell line U9<em>3</em>7. Expression of SOCS-<em>3</em> coincides with a constitutive activation of STAT<em>3</em> in CTCL tumor cells, and stable transfection of CTCL tumor cells with a dominant negative STAT<em>3</em> strongly inhibits SOCS-<em>3</em> expression, whereas transfection with wild-type STAT<em>3</em> does not. Moreover, the reduced SOCS-<em>3</em> expression in cells transfected with the dominant negative STAT<em>3</em> is associated with an increased sensitivity to <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>). In conclusion, evidence is provided for a constitutive SOCS-<em>3</em> expression in cancer cells obtained from patients with CTCL. Moreover, the findings indicate that the aberrant expression of SOCS-<em>3</em> is mediated by a constitutive activation of STAT<em>3</em> in CTCL cells and affects the IFN-<em>alpha</em> sensitivity of these cells. (Blood. 2001;97:1056-1062)

A gene encoding chicken <em>interferon</em> (ChIFN) was cloned from a cDNA library made from primary chick embryo cells that had been "aged" in vitro so as to produce copious amounts of IFN upon induction. The coding region is predicted to produce a signal peptide of <em>3</em>1 amino acids and a mature protein of 162 amino acids with a molecular weight of 18,957. There are four potential N-glycosylation sites and six cysteine residues. Three disulfide bonds are possible, with two being common to most mammalian type I IFNs. A motif of 10 amino acids surrounding Cys-1<em>3</em>7 is highly conserved: It shows 80% homology with mammalian type I IFNs, but only <em>3</em>0% with a reported fish IFN. The T-rich <em>3</em>' UTR displays the canonical element AATAAA required for polyadenylation, and contains six repeats of the octamer CTATTTAT that may be involved in down-regulating translation. Northern blots demonstrate that the accumulation of ChIFN mRNA correlates with induction of ChIFN determined by bioassay. Biologically active protein was synthesized in transfected mouse L cells using mRNA prepared in vitro from the cloned sequence. This activity was neutralized by a monoclonal antibody prepared against purified ChIFN. The ChIFN gene shows sequence identity at the amino acid/nucleotide level with consensus mammalian IFNs as follows: <em>alpha</em> (24/2<em>3</em>%), beta (20/24%), omega (2<em>3</em>/4<em>3</em>%), tau (20/4<em>3</em>%), gamma (<em>3</em>/<em>3</em>1%), and with flatfish IFN (16/<em>3</em>5%). The conserved features of the predicted ChIFN protein and the general similarity of predicted secondary structure suggest a molecule that fits the five <em>alpha</em>-helix three-dimensional topology reported for type I mammalian IFNs.(ABSTRACT TRUNCATED AT 250 WORDS)

We conducted a prospective cohort study in Leyte, the Philippines, among 611 Schistosoma japonicum-infected participants 7-<em>3</em>0 years old, all of whom were treated with praziquantel at baseline. To detect hepatic fibrosis, abdominal ultrasound was performed at baseline and 12 months after treatment. Stool for assessment of S. japonicum infection was collected at baseline and at <em>3</em>, 6, 9, and 12 months after treatment. Cytokines (interleukin [IL]-4, IL-5, IL-10, IL-1<em>3</em>, tumor necrosis factor- <em>alpha</em> , and <em>interferon</em>- gamma ) produced by peripheral-blood mononuclear cells in response to soluble worm antigen preparation (SWAP), soluble egg antigen (SEA), and control medium were measured once 4 weeks after treatment. IL-4 to SWAP and IL-10 to both SWAP and SEA were associated with the presence of baseline fibrosis after adjustment for potential confounding variables (P<.0<em>3</em>, for all). In participants with fibrosis at baseline, IL-4 to SWAP and IL-5 and IL-1<em>3</em> to both SWAP and SEA were associated with persistent fibrosis at 12 months after treatment (P<.05, for all). Males showed consistently stronger T helper 2 (Th2) cytokine responses to both SWAP and SEA than did females (P<.02, for all). These results suggest an independent role for Th2-biased cytokine responses to S. japonicum antigens in persistent hepatic fibrosis and indicate that Th2 cytokines may contribute to the male-biased prevalence of fibrosis.

The epigenetic impact of DNA methylation in chronic myelogenous leukemia (CML) is not completely understood. To elucidate its role we analyzed 120 patients with CML for methylation of promoter-associated CpG islands of 10 genes. Five genes were identified by DNA methylation screening in the K562 cell line and <em>3</em> genes in patients with myeloproliferative neoplasms. The CDKN2B gene was selected for its frequent methylation in myeloid malignancies and ABL1 as the target of BCR-ABL translocation. Thirty patients were imatinib-naïve (mostly treated by <em>interferon</em>-<em>alpha</em> before the imatinib era), <em>3</em>0 were imatinib-responsive, 50 were imatinib-resistant, and 10 were imatinib-intolerant. We quantified DNA methylation by bisulfite pyrosequencing. The average number of methylated genes was 4.5 per patient in the chronic phase, increasing significantly to 6.2 in the accelerated and 6.4 in the blastic phase. Higher numbers of methylated genes were also observed in patients resistant or intolerant to imatinib. These patients also showed almost exclusive methylation of a putative transporter OSCP1. Abnormal methylation of a Src suppressor gene PDLIM4 was associated with shortened survival independently of CML stage and imatinib responsiveness. We conclude that aberrant DNA methylation is associated with CML progression and that DNA methylation could be a marker associated with imatinib resistance. Finally, DNA methylation of PDLIM4 may help identify a subset of CML patients that would benefit from treatment with Src/Abl inhibitors.

<em>Interferon</em> regulatory factors (IRFs) are a family of transcription factors involved in the regulation of the <em>interferons</em> (IFNs) and other genes that may have an essential role in antiviral defense in the central nervous system, although this is currently not well defined. Therefore, we examined the regulation of IRF gene expression in the brain during viral infection. Several IRF genes (IRF-2, -<em>3</em>, -5, -7, and -9) were expressed at low levels in the brain of uninfected mice. Following intracranial infection with lymphocytic choriomeningitis virus (LCMV), expression of the IRF-7 and IRF-9 genes increased significantly by day 2. IRF-7 and IRF-9 gene expression in the brain was widespread at sites of LCMV infection, with the highest levels in infiltrating mononuclear cells, microglia/macrophages, and neurons. IRF-7 and IRF-9 gene expression was increased in LCMV-infected brain from IFN-gamma knockout (KO) but not IFN-<em>alpha</em>/betaR KO animals. In the brain, spleen, and liver or cultured glial and spleen cells, IRF-7 but not IRF-9 gene expression increased with delayed kinetics in the absence of STAT1 but not STAT2 following LCMV infection or IFN-<em>alpha</em> treatment, respectively. The stimulation of IRF-7 gene expression by IFN-<em>alpha</em> in glial cell culture was prevented by cycloheximide. Thus, (i) many of the IRF genes were expressed constitutively in the mouse brain; (ii) the IRF-7 and IRF-9 genes were upregulated during viral infection, a process dependent on IFN-<em>alpha</em>/beta but not IFN-gamma; and (iii) IRF-7 but not IRF-9 gene expression can be stimulated in a STAT1-independent but STAT2-dependent fashion via unidentified indirect pathways coupled to the activation of the IFN-<em>alpha</em>/beta receptor.

Toll-like receptors (TLRs) constitute a family of innate receptors that recognize and respond to a wide spectrum of microorganisms, including fungi, bacteria, viruses, and protozoa. Previous studies have demonstrated that ligands for TLR<em>3</em> and TLR9 induce potent innate antiviral responses against herpes simplex virus type 2 (HSV-2). However, the factor(s) involved in this innate protection is not well-defined. Here we report that production of beta <em>interferon</em> (IFN-beta) but not production of IFN-<em>alpha</em>, IFN-gamma, or tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) strongly correlates with innate protection against HSV-2. Local delivery of poly(I:C) and CpG oligodeoxynucleotides induced significant production of IFN-beta in the genital tract and provided complete protection against intravaginal (IVAG) HSV-2 challenge. There was no detectable IFN-beta in mice treated with ligands for TLR4 or TLR2, and these mice were not protected against subsequent IVAG HSV-2 challenge. There was no correlation between levels of TNF-<em>alpha</em> or IFN-gamma in the genital tract and protection against IVAG HSV-2 challenge following TLR ligand delivery. Both TNF-<em>alpha</em>(-/-) and IFN-gamma(-/-) mice were protected against IVAG HSV-2 challenge following local delivery of poly(I:C). To confirm that type I <em>interferon</em>, particularly IFN-beta, mediates innate protection, mice unresponsive to type I <em>interferons</em> (IFN-<em>alpha</em>/betaR(-/-) mice) and mice lacking IFN regulatory factor-<em>3</em> (IRF-<em>3</em>(-/-) mice) were treated with poly(I:C) and then challenged with IVAG HSV-2. There was no protection against HSV-2 infection following poly(I:C) treatment of IFN-<em>alpha</em>/betaR(-/-) or IRF-<em>3</em>(-/-) mice. Local delivery of murine recombinant IFN-beta protected C57BL/6 and IRF-<em>3</em>(-/-) mice against IVAG HSV-2 challenge. Results from these in vivo studies clearly suggest a strong correlation between IFN-beta production and innate antiviral immunity against HSV-2.

To study the effect of hydroxychloroquine on cytokine production by monocytes and T cells, cells were pretreated with varying concentrations of hydroxychloroquine and stimulated with lipopolysaccharide (monocytes), phytohemagglutinin or anti-CD-<em>3</em> monoclonal antibodies (T cells). Interleukin 1 <em>alpha</em> (IL-1-<em>alpha</em>), IL-6 and tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) production were measured from the stimulated monocytes and IL-2, IL-4 and gamma <em>interferon</em> (IFN-gamma) were measured from the stimulated T cells. Hydroxychloroquine inhibited production of IL-1-<em>alpha</em> (monocytes) and IL-6 (T cells and monocytes). In contrast IL-2, IL-4, TNF-<em>alpha</em> and IFN-gamma production were not affected. Preferential inhibition of IL-1-<em>alpha</em> production by monocytes and IL-6 production by T cells and monocytes may contribute to its antiinflammatory effect in autoimmune diseases.

The induction of indoleamine 2,<em>3</em>-dioxygenase (IDO) activity has been implicated in the antiproliferative action of <em>interferon</em> (IFN)-gamma on tumor cells and the inhibition of intracellular pathogens. Earlier studies have demonstrated that the expression of the IDO gene is induced strongly by IFN-gamma, but very poorly by IFN-<em>alpha</em> despite the presence of a sequence highly homologous to the IFN-<em>alpha</em>-responsive sequence element (<em>interferon</em>-stimulated response element (ISRE)) in its IFN-gamma-responsive control region. In addition, a sequence with a partial homology to the IFN-gamma-responsive sequence (GAS) identified by Lew et al. (Lew, D.J., Decker, T., Strehlow, I., and Darnell, J.E., Jr. (1991) Mol. Cell. Biol. 11, 182-191) in a human gene for a guanylate-binding protein and to the X box sequence found in all major histocompatibility complex class II genes was found. Deletion experiments have indicated that the ISRE homolog (but not the GAS-related or the X box-related sequence) was essential for an inducibility by IFN-gamma. To investigate the lack of inducibility by IFN-<em>alpha</em> despite the presence of an ISRE homolog, the binding of this ISRE homolog to the IFN-<em>alpha</em>-stimulated gene factor <em>3</em> (ISGF<em>3</em>) was examined. Gel mobility shift experiments and competition experiments indicated that this ISRE homolog did not form a stable complex with ISGF<em>3</em>. This may account for a poor inducibility by IFN-<em>alpha</em>. This inability to bind ISGF<em>3</em> appears to be (at least in part) due to minor differences between the nucleotide sequence of the ISRE homolog present in the IDO gene promoter and the ISRE consensus sequence found in IFN-<em>alpha</em>-inducible genes. An IFN-gamma-inducible DNA-binding factor was identified with characteristics different from ISGF<em>3</em>: (i) the IFN-gamma-inducible factor was detected in the nuclear extracts, but not in the cytoplasmic extracts; and (ii) the appearance of this DNA-binding factor required new protein synthesis, which could explain the dependence on new protein synthesis for the induction of IDO by IFN-gamma.

Recognition of viral RNA by cytoplasmic retinoic acid inducible gene I (RIG-I)-like receptors initiates signals leading to the induction of type I <em>interferon</em> (IFN) transcription via transcription factors such as <em>interferon</em> regulatory factor <em>3</em> (IRF<em>3</em>) and nuclear factor κB (NF-κB). Here, we describe a new signalling pathway that involves protein kinase C <em>alpha</em> (PKCα), histone deacetylase 6 (HDAC6) and beta-catenin (β-catenin), which is essential for IFN gene induction following virus infection. Knockdown of PKCα in various human cells, including primary cells, inhibited Sendai virus (SeV)-mediated IFN induction and enhanced virus replication. In the absence of this pathway IRF<em>3</em> becomes activated, but does not bind to its promoter and is thus unable to support transcription. Mechanistically, SeV infection induced the activation of PKCα, which promoted its interaction with HDAC6 and enhanced its deacetylation activity in a phosphorylation-dependent manner. Further downstream, HDAC6 caused deacetylation of β-catenin and enhanced its nuclear translocation and promoter binding. In the nucleus, β-catenin acted as a co-activator for IRF<em>3</em>-mediated transcription. Our findings suggest an important role of a novel signalling pathway mediated by PKCα-HDAC6-β-catenin in controlling IRF<em>3</em>-mediated transcription.

Multiple cytokines induce a number of alterations in lipid metabolism which can produce hyperlipidemia. Recent studies have demonstrated that tumor necrosis factor (TNF) increases lipolysis, resulting in an increase in circulating FFA levels, which stimulates hepatic triglyceride production, thereby contributing to the hyperlipidemia induced by TNF. In the present investigation we have determined the effects of a variety of cytokines on lipolysis in cultured <em>3</em>T<em>3</em>-F442A adipocytes. TNF increased lipolysis approximately <em>3</em>-fold with a maximal effect at 100 ng/ml and a half-maximal increase at 5-10 ng/ml. This increase was first observed 8 h after incubation with TNF. Interleukin-1 (IL-1) and <em>interferon</em>-<em>alpha</em> (IFN), -beta, and -gamma also stimulated lipolysis in cultured adipocytes. The half-maximal increase in lipolysis occurred at approximately 10 ng/ml IL-1, 5 ng/ml IFN <em>alpha</em>, 10 ng/ml IFN beta, and 8 ng/ml of IFN gamma. Maximal lipolysis was observed at approximately 100 ng/ml for each of these cytokines, with the exception of IFN beta, for which maximal stimulation was observed at 1000 ng/ml. Neither platelet-activating factor nor IL-6 stimulated lipolysis; therefore, it is unlikely that these compounds mediate the increase in lipolysis induced by cytokines. However, indomethacin, a well known inhibitor of prostaglandin synthesis, prevented the increase in lipolysis induced by TNF, IL-1, IFN <em>alpha</em>, IFN beta, or IFN gamma. Indomethacin did not affect basal lipolysis or the acute stimulation of lipolysis induced by epinephrine. These results demonstrate that multiple cytokines can increase lipolysis and that this increase is mediated by cytokine-induced stimulation of prostaglandin synthesis.

The 'promoter' fragment from the yeast phosphoglycerate kinase (PGK) gene has been used to direct the expression of human <em>interferon</em>-<em>alpha</em>-2 (IFN <em>alpha</em> 2) on a high-copy-number plasmid in yeast. The yields of IFN <em>alpha</em> 2 are only 1-<em>3</em>% of yeast total protein, whereas the maximum yield of PGK produced by the PGK gene on a high-copy-number plasmid is at least 50%. IFN <em>alpha</em> 2 is turned over more rapidly than PGK but in addition a major reason for the relatively low level of IFN <em>alpha</em> 2 is that IFN-specific RNA levels are much lower. This does not reflect differences in plasmid copy number or integrity, or differences in the 5' and <em>3</em>' untranslated regions of the transcripts or DNA flanking regions. It appears that the presence of heterologous coding sequences, or the absence of specific yeast sequences causes a reduction in heterologous RNA levels in yeast.

The immunostimulatory capacity of dendritic cells is improved by co-electroporation with mRNA encoding CD40 ligand, constitutively active toll-like receptor 4, and CD70 (TriMix-DC). This pilot clinical trial evaluated the feasibility, safety, and immunogenicity of a therapeutic vaccination containing autologous TriMix-DC co-electroporated with mRNA encoding a human leukocyte antigen class II-targeting signal linked to 1 of 4 melanoma-associated antigens (MAGE-A<em>3</em>, MAGE-C2, tyrosinase, and gp100) in patients with advanced melanoma. Thirty-five American Joint Committee on Cancer stage III/IV melanoma patients received autologous TriMix-DC (4 administrations 2 weeks apart). Immune monitoring was performed by evaluating skin biopsies of delayed type IV hypersensitivity (DTH) reactions for presence of vaccinal antigen-specific DTH-infiltrating lymphocytes (DIL). Thereafter, patients could receive <em>interferon</em>-<em>alpha</em>-2b (IFN-α-2b) 5 MU subcutaneously <em>3</em> times weekly and additional TriMix-DC every 8 weeks. TriMix-DC-related adverse events comprised grade 2 local injection site reactions (all patients), and grade 2 fever and lethargy (2 patients). Vaccinal antigen-specific DIL were found in 0/6 patients tested at vaccine initiation and in 12/21 (57.1%) assessed after the fourth vaccine. A positive postvaccination DTH test correlated with IL-12p70 secretion capacity of TriMix-DC. No objective responses to TriMix-DC alone were seen according to RECIST. Twenty-nine patients received IFN-α-2b after the fourth vaccine without unexpected adverse events. During TriMix-DC/IFN-α-2b combination therapy, 1 partial response and 5 stable disease (disease control of >6 months with regression of metastases) were observed in 17 patients with evaluable disease at baseline. In conclusion, this study demonstrated that therapeutic vaccination with autologous TriMix-DC is feasible, safe, and immunogenic and can be combined with sequential IFN-α-2b.

OBJECTIVE

To determine the effects of dietary consumption of Bifidobacterium lactis (strain HN019, DR10TM) on natural immunity.

METHODS

A randomized, double blind, placebo-controlled clinical trial.

METHODS

Janeway Medical Centre, Memorial University, St Johns, Newfoundland.

METHODS

Twenty-five healthy elderly volunteers (median age 69 y; range 60-8<em>3</em> y).

METHODS

Twelve control subjects consumed 180 ml low-fat/low-lactose milk twice daily for a period of 6 weeks; 1<em>3</em> test subjects consumed milk supplemented with 1.5x1011 colony-forming units of B. lactis twice daily. Indices of natural immunity, including <em>interferon</em> production, phagocytic capacity and phagocyte-mediated bactericidal activity, were determined via peripheral blood at 0, <em>3</em>, 6 and 12 weeks post-trial commencement.

RESULTS

Subjects who consumed milk containing B. lactis for 6 weeks produced significantly enhanced levels of interferon-alpha, upon stimulation of their peripheral blood mononuclear cells in culture, in comparison to the placebo control group who received milk alone. There were also significant increases in polymorphonuclear cell phagocytic capacity among test group subjects, following consumption of milk supplemented with B. lactis, while individuals who consumed B. lactis-supplemented milk or milk alone showed enhanced phagocyte-mediated bactericidal activity.

CONCLUSIONS

The results demonstrate that dietary consumption of B. lactis HN019 can enhance natural immunity in healthy elderly subjects, and that a relatively short-term dietary regime (6 weeks) is sufficient to impart measurable improvements in immunity that may offer significant health benefits to consumers.

BACKGROUND

Financial support for this project was provided by the New Zealand Dairy Board.

In contrast to the United States, Japanese patients with chronic hepatitis C currently treated with <em>interferon</em> are generally 10 to 15 years older. Older patients, however, tend to experience more frequent adverse events. This study was conducted to clarify the effect of patient age on the efficacy and safety of combination therapy. We consecutively enrolled 208 patients with naïve chronic hepatitis C. Patients were classified into three groups according to age: younger than 50 years of age (n = 52); 50 to 59 years old (n = 8<em>3</em>); and 60 years of age or older (n = 7<em>3</em>). <em>Interferon</em> <em>alpha</em>-2b therapy was administered daily for 2 weeks, followed by <em>3</em> times per week for 22 weeks, while ribavirin was administered daily. Of the 208 study patients, discontinuation of therapy or dose reduction was required in 116 (56%) and was more frequent in older patient groups: <em>3</em>8%, 48%, and 77% for the < 50, 50-59, and>> or = 60-year-old patient groups, respectively (P < .001). Multivariate analysis showed patient age to be independently associated with adherence to therapy. A sustained virological response was achieved in 77 (<em>3</em>7%) patients, with genotype, viral load, and adherence to therapy associated with this achievement. A tendency toward a lower sustained virological response rate was seen in the older patients. In conclusion, patient age is an important factor contributing to the safety of combination therapy. Thus, treatment schedule should be modified, or other therapeutic modalities should be considered for older patients with chronic hepatitis C.

OBJECTIVE

Oxidative stress mediates activation and stimulates collagen production of cultured hepatic stellate (Ito) cells. The aim of this study was to assess whether oxidative stress contributes to hepatic fibrogenesis in chronic hepatitis C.

METHODS

In liver biopsy specimens of patients with chronic hepatitis C, the following fibrogenesis cascade was analyzed: (1) oxidative stress, determined by the presence of malondialdehyde protein adducts; (2) activation of stellate cells as indicated by their expression of <em>alpha</em>-smooth muscle actin; (<em>3</em>) stimulation of c-myb expression in stellate cells, a critical step in the activation of these cells; and (4) induction of collagen gene expression as detected by in situ hybridization.

RESULTS

Treatment with d-alpha-tocopherol (1200 IU/day for 8 weeks) in 6 of these patients, who were refractory to interferon therapy, prevented the fibrogenesis cascade observed before antioxidant treatment. In addition, d-alpha-tocopherol treatment significantly decreased the carbonyl modifications of plasma proteins, a sensitive index of oxidative stress. However, 8 weeks of d-alpha-tocopherol treatment did not significantly affect serum alanine aminotransferase levels, hepatitis C virus titers, or histological degree of hepatocellular inflammation or fibrosis.

CONCLUSIONS

These data suggest that enhanced oxidative stress initiates a fibrogenesis cascade in the liver of patients with chronic hepatitis C.

T cells and other leucocytes regularly occur within and subjacent to the gut epithelium. Recent data suggest that intestinal epithelial cells may exert accessory immunological functions. Although interactions between leucocytes and accessory cells usually require expression of adhesion molecules, intestinal epithelium has generally been considered negative for intercellular adhesion molecule-1 (ICAM-1) by immunohistochemical techniques. We therefore studied the expression of ICAM-1 and lymphocyte function-associated antigen-<em>3</em> (LFA-<em>3</em>) by two colonic epithelial cell lines and found that both adhesion molecules were constitutively present at low levels. ICAM-1 protein expression could be enhanced within 4 h by cytokines, particularly after co-incubation with <em>interferon</em>-gamma (IFN) and tumour necrosis factor-<em>alpha</em> (TNF), interleukin-1 beta (IL-1), or IL-6. IFN also resulted in accumulation of ICAM-1 mRNA. Conversely, the LFA-<em>3</em> expression was virtually unaffected by cytokine stimulation. These data imply that intestinal epithelial cells under certain conditions may bear adhesion molecules required for cooperation with juxtaposed leucocytes in situ, and that the expression of some of these molecules is modulated by cytokines from activated mucosal leucocytes.

OBJECTIVE

To assess the efficacy of systemic intravenous-fluorouracil (5-FU) and subcutaneous recombinant human interferon alfa-2b (rIFN alpha-2b) in patients with measurable cancer of the biliary tree.

METHODS

Thirty-five patients (25 with cholangiocarcinoma and 10 with gallbladder carcinoma) were registered onto this phase II protocol between 1992 and 1995. Patients received a continuous infusion of 750 mg/m2/d of 5-FU on days 1 through 5 through a centrally placed venous catheter and a subcutaneous injection of 5 MU/m2 of rIFN alpha-2b on days 1, 3, and 5. Treatment cycles were repeated every 14 days; one course of therapy included four treatment cycles. Disease status was assessed every 8 weeks. Dosages were lowered for grade III mucositis. Fourteen patients had prior treatment and, before initiating this therapy, 17 patients required decompression of the biliary tree.

RESULTS

Eleven of 32 (34%) assessable patients had a partial response. The median time to disease progression was 9.5 months, and the median survival time 12 months. Grade III to IV toxic effects were granulocytopenia (14%), mucositis (20%), diarrhea (9%), and dermatitis (11%). Grade III to IV asthenia and fatigue were observed in 6% of patients.

CONCLUSIONS

Drug tolerance was better among previously untreated patients. To achieve a complete response, additional chemotherapy or radiotherapy should be considered when liver resection or transplantation is not feasible. However, if these results can be reproduced by other investigators, the regimen should be studied for adjuvant treatment of gallbladder carcinoma incidentally identified in patients undergoing cholecystectomy.

The function of the 5'-flanking region of the mouse major histocompatibility complex gene Ed <em>alpha</em> has been studied by deletion analysis with the chloramphenicol acetyltransferase gene as a transient expression marker in various cell lines. This analysis reveals the presence of several control regions on the 5' side of the gene. Sequences between base pair (bp) -87<em>3</em> and bp -<em>3</em>5<em>3</em> have a negative function in human and mouse fibroblasts but not in the mouse macrophage line WEHI-<em>3</em>. Additional positive and negative elements have been mapped between bp -<em>3</em>5<em>3</em> and bp -<em>3</em>8. A gamma-<em>interferon</em> response region has been also identified within that sequence. the 5' and <em>3</em>' boundaries of the gamma-<em>interferon</em> response region have been located between bp -164 and bp -4<em>3</em>. Inducible human cell lines showed the same gamma-<em>interferon</em> response region endpoints with the mouse cell line WEHI-<em>3</em>. A DNA fragment spanning the equivalent region of the mouse Ed beta gene confers gamma-<em>interferon</em> inducibility to the simian virus 40 and <em>alpha</em>-globin promoters in an orientation-independent manner. We further provide evidence that the conserved sequence motifs on the 5' side of all major histocompatibility complex class II genes are indispensable for gamma-<em>interferon</em> induction.