Targeting of a cholecystokinin-DNA complex to pancreatic cells in vitro and in vivo

The carboxy terminal octapeptide of cholecystokinin (CCK8) is a hormone that binds high affinity receptors in a number of tissues including pancreas and pancreatic tumours. As part of our studies to develop effective gene therapy for the treatment of pancreatic cancers, we have investigated various gene delivery systems that depend on CCK8 receptor targeting. In this paper,we describe the synthesis of a CCK8-DNA complex designed to deliver foreign DNA to cholecystokinin receptor-positive cells. CCK8 was ligated to avidin and then complexed to linearis biotinylated DNA (pSV-CAT). The uptake of P-32-labelled CCK8-DNA complex by rat pancreatic acini was linear with time over 4 h with 65-70% of uptake inhibited by 100 nM CCK8. The complex appeared to be internalised since it could not be removed by acid wash. When administered intra-arterially, the complex was rapidly removed from the circulation with no evidence of targeted delivery to the pancreas, However, following a single intraperitoneal dose, the pancreas accumulated-5- 8% of the total administered complex by 24 h. These results suggest that peptide-dependent gene delivery to CCK receptor positive cells in vivo is feasible but, when administered directly into the circulation, diffusional barriers across the endothelium may limit distribution to peripheral tissues. Intraperitoneal administration therefore may be a useful alternative for targeting the pancreas.

Distribution of interleukin-2,-4,-10, tumour necrosis factor-alpha and transforming growth factor-beta mRNAs in oral lichen planus

In the present study. MRNA for the cytokines interleukin-2 (IL-2), IL-4, IL-10 tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor beta-1 (TGF-beta-1) were investigated in oral lichen planus (OLP) lesions using in situ hybridization with S-35-labelled oligonucleotide probes on frozen tissue sections. In addition, the expression of interferon-gamma (IFN-gamma), IL-10 and IL-4 mRNAs was analysed in cultured lesional T lymphocytes from oral lichen planus by polymerase chain reaction. Cells expressing mRNA for IL-2, IL-4, IL-10, TNF-alpha and TGF-beta(1) were found in all the biopsies studied. Approximately 1-2% of the total number of infiltrating cells in the lesions were positive for each of the different cytokine mRNAs. Most biopsies contained basement membrane-oriented, mRNA-positive cells. In the cultured T-cell lines, message for IFN-gamma was detected in all the patients, IL-10 in all but one, and IL-4 in just one of the seven patients investigated. The results suggest that mRNA for both pro- and anti-inflammatory cytokines, i.e., mixed T-helper 1 (T(H)1) and T(H)2 cytokine profiles, are generated simultaneously by a limited number of cells in chronic lesions of OLP. (C) 1999 Elsevier Science Ltd. All rights reserved.

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by Smac/DIABLO release from mitochondria

In previous studies we have shown that the sensitivity of melanoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)induced apoptosis was determined largely by the level of expression of death receptor TRAIL receptor 2 on the cells. However, approximately one-third of melanoma cell lines were resistant to TRAIL, despite expression of high levels of TRAIL receptor 2. The present studies show that these cell lines had similar levels of TRAIL-induced activated caspase-3 as the TRAIL-sensitive lines, but the activated caspase-3 did not degrade substrates downstream of caspase-3 [inhibitor of caspase-activated DNase and poly(ADP-ribose) polymerase]. This appeared to be due to inhibition of caspase-3 by X-linked inhibitor of apoptosis (XIAP) because XIAP was bound to activated caspase-3, and transfection of XIAP into TRAIL-sensitive cell lines resulted in similar inhibition of TRAIL-induced apoptosis. Conversely, reduction of XIAP levels by overexpression of Smac/ DIABLO in the TRAIL-resistant melanoma cells was associated with the appearance of catalytic activity by caspase-3 and increased TRAIL-induced apoptosis. TRAIL was shown to cause release of Smac/DIABLO from mitochondria, but this release was greater in TRAIL-sensitive cell lines than in TRAIL-resistant cell lines and was associated with downregulation of XIAP levels. Furthermore, inhibition of Smac/DIABLO release by overexpression of Bcl-2 inhibited down-regulation of XIAP levels. These results suggest that Smac/DIABLO release from mitochondria and its binding to XIAP are an alternative pathway by which TRAIL induces apoptosis of melanoma, and this pathway is dependent on the release of activated caspase-3 from inhibition by XIAP and possibly other inhibitor of apoptosis family members.

Relationship of interleukin-6 and tumor necrosis factor-alpha with muscle mass and muscle strength in elderly men and women: The health ABC study

Background. A decline in muscle mass and muscle strength characterizes normal aging. As clinical and animal studies show it relationship between higher cytokine levels and low muscle mass, the aim of this study was to investigate whether markers, of inflammation are associated with muscle mass and strength in well-functioning elderly persons. Methods. We Used baseline data (1997-1998) of the Health, Aging, and Body Composition (Health ABC) Study on 3075 black and white men and women aged 70-79 years. Midthigh muscle cross-sectional area (computed tomography), appendicular muscle mass (dual-energy x-ray ab absorptiometry). isokinetic knee extensor strength (KinCom). and isometric inip strength were measured. plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were assessed by enzyme-linked immunosorbent assay (ELISA). Results. Higher cytokine levels were generally associated with lower muscle mass and lower muscle strength. The most consistent relationship across the gender and race groups was observed for IL-6 and grip strength: per SD increase in IL-6, grip strength was 1.1 to 2.4 kg lower (p < .05) after adjustment for age, clinic Site. health status, medications, physical activity. smoking. height. and body fat. Ail overall measure of elevated cytokine level was created by combining the levels of IL-6 and TNF-alpha. With the exception of white men, elderly persons having high levels of IL-6 (> 1.80 pg/ml) as well as high levels of TNF-alpha (> 3.20 pg/ml) had a smaller muscle area, less appendicular mass. a lower knee extensor strength. and a lower grip strength compared to those with low levels of both cytokines. Conclusions. Higher plasma concentrations of IL-6 and TNF-alpha are associated with lower muscle mass and lower muscle strength in well-functioning older men and women. Higher cytokine levels. as often observed in healthy older persons. may contribute to the loss Of muscle mass and strength that accompanies aging.

Association of tumor necrosis factor-alpha polymorphisms and ozone-induced change in lung function

Ozone is a major air pollutant with adverse health effects which exhibit marked inter-individual variability. In mice, regions of genetic linkage with ozone-induced lung injury include the tumor necrosis factor-alpha (TNF), lymphotoxin-alpha (LTA), Toll-like receptor 4 (TLR4), superoxide dismutase (SOD2), and glutathione peroxidase (GPX1) genes. We genotyped polymorphisms in these genes in 51 individuals who had undergone ozone challenge. Mean change in FEV1 with ozone challenge, as a percentage of baseline, was -3% in TNF -308G/A or A/A individuals, compared with -9% in G/G individuals (p = 0.024). When considering TNF haplotypes, the smallest change in FEV1 with ozone exposure was associated with the TNF haplotype comprising LTA +252G/TNF -1031T/TNF -308A/TNF -238G. This association remained statistically significant after correction for age, sex, disease, and ozone concentration (p = 0.047). SOD2 or GPX1 genotypes were not associated with lung function, and the TLR4 polymorphism was too infrequent to analyze. The results of this study support TNF as a genetic factor for susceptibility to ozone-induced changes in lung function in humans, and has potential implications for stratifying health risks of air pollution.

Molecular adjuvant effects of a conformationally biased agonist elf human C5a anaphylatoxin

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSFKPMPLaR) was used as a molecular adjuvant in stimulating Ab responses against peptide epitopes derived from human MUC1 glycoprotein and the human mu and kappa opioid receptors. C57BL6 mice were immunized with the MUC1 epitope (YKQGGFLGL); the C5a agonist (YSFKPMPLaR); YSFKPMPLaR and YKQGGFLGL together, but unconjugated; a C5a-active, MUC1 epitope construct (YKQGGFLGLYSFKPMPLaR); and a C5a-inactive, reversed moiety construct (YSFKPMPLaRYKQGGFLGL). High Ab titers specific for the MUC1 epitope were observed Only in mice immunized with the C5a-active epitope construct. Similar results were obtained in BALB/c mice immunized with the C5a-active, MUC1 epitope construct, Abs from the sera of the C57BL6 mice were predominately of the IgG2a, IgC2b, and IgM isotypes and were reactive against human recombinant MUC1 and MUC1 expressed by the Panc-1 M1F.15 pancreatic cell line, When compared with the corresponding KLH-epitope conjugates in C57BL6 mice, the epitope-C5a agonist constructs produced titers of specific IgG Abs of isotypes distinct from those generated by the keyhole limpet hemocyanin-epitope conjugates, Rabbits immunized with a mu opioid receptor epitope-C5a agonist construct (GDLSDPCGNRTNLGGRDSLYSFKPMPLaR) or a kappa opioid receptor epitope-C5a agonist construct (FPGWAEPDSNGSEDAQLYSFKPMPLaR) generated high titer, epitope-specific Ab responses, Ab titers generated in response to the opioid epitope-C5a agonist constructs were comparable to those generated by the opioid KLH-epitope conjugates, The results of this study are discussed in terms of possible mechanisms by which the conformationally biased C5a agonist serves as a molecular adjuvant.

Coffee and alcohol consumption and the risk of pancreatic cancer in two prospective United States cohorts

Although most prospective cohort studies do not support an association between coffee consumption and pancreatic cancer, the findings for alcohol are inconsistent. Recently, a large prospective cohort study of women reported statistically significant elevations in risk of pancreatic cancer for both coffee and alcoholic beverage consumption. We obtained data on coffee, alcohol, and other dietary factors using semiquantitative food frequency questionnaires administered at baseline (1986 in the Health Professionals Follow-Up Study and 1980 in the Nurses’ Health Study) and in subsequent follow-up questionnaires. Data on other risk factors for pancreatic cancer, including cigarette smoking, were also available. Individuals with a history of cancer at study initiation were excluded from all of the analyses. During the 1,907,222 person-years of follow-up, 288 incident cases of pancreatic cancer were diagnosed. The data were analyzed separately for each cohort, and results were pooled to compute overall relative risks (RR). Neither coffee nor alcohol intakes were associated with an increased risk of pancreatic cancer in either cohort or after pooling the results (pooled RR, 0.62; 95% confidence interval, 0.27–1.43, for >3 cups of coffee/day versus none; and pooled RR, 1.00; 95% confidence interval, 0.57–1.76, for >=30 grams of alcohol/day versus none). The associations did not change with analyses examining different latency periods for coffee and alcohol. Similarly, no statistically significant associations were observed for intakes of tea, decaffeinated coffee, total caffeine, or alcoholic beverages. Data from these two large cohorts do not support any overall association between coffee intake or alcohol intake and risk of pancreatic cancer.

Early Australian experience with infliximab, a chimeric antibody against tumour necrosis factor-alpha, in the treatment of Crohn's disease: is its efficacy augmented by steroid-sparing immunosuppressive therapy?

Background: Tumour necrosis factor-alpha (TNF-alpha) plays an important role in the pathology of Crohn's disease. Infliximab, a chimeric antibody against TNF-alpha, has been shown in controlled clinical trials to be effective in two-thirds of patients with refractory or fistulating Crohn's disease. The factors that determine a clinical response in some patients but not others are unknown. Aims: To document the early Australian experience with infliximab treatment for Crohn's disease and to identify factors that may determine a beneficial clinical response. Methods: Gastroenterologists known to have used infliximab for Crohn's disease according to a compassionate use protocol were asked to complete a spreadsheet that included demographic information, Crohn's disease site, severity, other medical or surgical treatments and a global clinical assessment of Crohn's disease outcome, judged by participating physicians as complete and sustained (remission for the duration of the study), complete but unsustained (remission at 4 weeks but not for the whole study) or partial clinical improvement (sustained or unsustained). Results: Fifty-seven patients were able to be evaluated, with a median follow-up time of 16.4 (4-70) weeks, including 23 patients with fistulae. There were 21 adverse events, including four serious events. Fifty-one patients (89%) had a positive clinical response for a median duration (range) of 11 (2-70) weeks. Thirty patients (52%) had a remission at 4 weeks, 10 of whom had remission for longer than 12 weeks. Forty-two per cent of fistulae closed. Sustained remission (P = 0.065), remission at 4 weeks (P = 0.033) and a positive clinical response of any sort (P = 0.004) were more likely in patients on immunosuppressive therapy, despite there being more smelters in this group. Conclusion: This review of the first Australian experience with infliximab corroborates the reported speed and efficacy of this treatment for Crohn's disease. The excellent response appears enhanced by the concomitant use of conventional steroid-sparing immunosuppressive therapy.

Development of disabled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle

Jembrana disease virus (JDV) is a newly isolated and characterised bovine lentivirus. It causes an acute disease in Ball cattle (Bos javanicus). which can be readily transmitted to susceptible cattle with 17% mortality. There is as yet no treatment or preventive vaccine. We have developed a gene transfer vector system based on JDV that has three components. The first of the components is a bicistronic transfer vector plasmid that was constructed to contain cis-sequences from the JDV genome, including 5 '- and 3 ' -long terminal repeats (LTRs), 0.4 kb of truncated gag and 1.1 kb of 3 ' -env, a multiple cloning site to accommodate the gene(s) of interest for transfer, and an internal ribosome entry site plus the neomycin phosphotransferase (Neo) gene cassette for antibiotic selection. The second element is a packaging plasmid that contains trans-sequences. including gag, pol. vif, tar and rev: but without the env and packaging signals. The third is a plasmid encoding the G glycoprotein of vesicular stomatitis virus (VSV-G) to supply the vector an envelope for pseudotyping. Cotransfection of 293T cells with these three plasmid components produced VSV-G pseudotyped. disabled, replication defective, bicistronic JDV vectors encoding the green fluorescent protein (EGFP) and the Neo resistance selection maker simultaneously with a titre range of (0.4-1.2) x 10(6) CFU/ml. Transduction of several replicating primary and transformed cells from cattle, primate and human sources and importantly growth-arrested cells with the JDV vectors showed high efficiency of EGFP gene transfer at 35-75%, which was stable and the expression of EGFP was long term. Furthermore, these JDV vectors were designed to suit the inclusion and expression of genes corresponding to JDV specific proteins, such as gag or env, for the development of vaccines for Jembrana disease. This strategy should also be applicable to other bovine diseases as wall. The design and construction of the JDV vector system should facilitate the study of the lentivirology and pathogenesis of the diseases associated with JDV or other bovine virus infections. To our knowledge, this is the first such vector system developed from a cattle virus. (C) 2001 Elsevier Science B.V. All rights reserved.

Endocytosis of uncleaved tumor necrosis factor-alpha in macrophages

Activated monocytes and macrophages secrete the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) TNF-alpha is produced as a 26 kd transmembrane protein that is cleaved to release a 17 kd soluble protein. TNF-alpha in both forms is biologically active. The intracellular trafficking of membrane-associated TNF-alpha in lipopolysaccharide-activated mouse macrophages was assessed after treatment with the metalloprotease inhibitor BB-3103, which prevents the cleavage of pro-TNF-alpha. Immunoprecipitation and immunofluorescence studies showed sustained expression of cell-associated TNF-alpha in the presence of the inhibitor. Cell immunoreactivity and surface biotinylation revealed that uncleaved TNF-alpha accumulated on the cell surface and was endocytosed, appearing in intracellular vesicles. Perturbation of post-Golgi traffic blocked the surface expression of 26 kd TNF-alpha. Tracking a bolus of TNF-alpha over time in cycloheximide-treated cells confirmed that uncleaved TNF-alpha is first transported to the cell surface and subsequently endocytosed. Vesicular structures immunoreactive for TNF-alpha were identified as endosomes by double labeling. The secretory and membrane-associated endocytic trafficking of TNF-alpha provides a mechanism for modulating the quantity of biologically active 26 kd TNF-alpha expressed on macrophages, allowing regulation of paracrine and autocrine responses.

Human trypsinogen in colorectal cancer

Trypsinogen (TRY), the precursor to the serine protease trypsin, is found in the pancreas and mediates digestive proteolysis in the small intestine. Differential display of cDNAs expressed by human colorectal tumor tissues compared with adjacent normal colonic mucosa identified an isoform of TRY (TRY2) up-regulated in colorectal cancers. Northern blot analysis of RNA isolated from a series of 28 malignant colon tumors and corresponding normal mucosa showed that TRY transcripts were up-regulated 2- to 33-fold in 29% of tumors. Further, TRY mRNA was expressed in 6 colorectal cancer cell lines, with highest levels detected in the metastatic tumor lines SW620 and HT29. Immunostaining for TRY protein expression showed intense immunoreactivity in the supranuclear cytoplasm of colon tumors in 16% of tissue specimens. To evaluate the relative contributions of 2 isoforms of TRY, TRY1 and TRY2, to total TRY mRNA expression, a semiquantitative multiplex RT-PCR assay was developed. TRY2 mRNA was detected in all 6 colorectal tumor cell lines, whereas TRY1 mRNA was expressed only in the metastatic tumor lines, showing that the high levels of TRY expression in the metastatic tumor lines are likely due to up-regulation of TRY1. Evaluation of TRY1 and TRY2 mRNA expression by multiplex RT-PCR in a series of 20 colon tumor tissues representative of the range of tumor progression showed that TRY2 mRNA was expressed much more commonly than TRY1 mRNA in normal mucosa (26% vs. 6%) as well as in primary tumor tissues (65% vs. 15%). These data demonstrate that TRY2 is the dominant TRY in colon tissue and suggest that up-regulation of TRY1 expression in colon tumors may be associated with a metastatic phenotype. (C) 2001 Wiley-Liss, Inc.

Characterisation of New Zealand isolates of infectious bursal disease virus

Isolates of infectious bursal disease virus (IBDV) were obtained from domestic poultry in New Zealand in 1997 and 1998. An in-vivo pathogenicity study carried out in specific pathogen free (SPF) chickens demonstrated the low virulence of one of the virus isolates. The nucleotide sequences of the hypervariable region of the VP2 gene of two isolates were determined and compared with published sequences of strains from other countries. The deduced amino acid sequence of the two New Zealand IBDV isolates showed 100% identity with each other, suggesting that little genetic drift had occurred. Phylogenetic analysis showed that the New Zealand isolates were more closely related to two attenuated IBDV strains (Cu1 and PBG98) than to classical (STC and 52/70), very virulent (DV86), variant (variant E) or Australian (002-73) strains. The results support the hypothesis that an attenuated strain of the virus was inadvertently introduced into the NZ poultry population in 1993.

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice

Type I diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta -cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta -cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in A-PNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta -cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(-/-) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.