Search

Background Ulcerative colitis (UC) is a chronic inflammatory condition affecting the colonic epithelium, with potential roles for the inflammasome, complement activation, T cells, and the microbiome in pathogenesis. We applied an established method of microarray-based gene expression analysis to a set of 128 UC biopsies (from 113 patients), to elucidate the molecular changes associated with active UC. Aims Our aim was to describe the molecules most associated with UC disease activity (the endoscopic Mayo score) and to annotate these molecules into biological processes. Methods 128 UC colonic biopsies were collected at the University of Alberta Hospital (Edmonton, Alberta) and Cedars-Sinai Hospital (Los Angeles, California) during standard of care colonoscopy. Biopsies were processed using Affymetrix microarrays. Gene expression data from the population was visualized using volcano plots (showing fold change and association between genes and endoscopic Mayo score), and heatmaps (showing expression of the top 30 genes in a previously established cell panel). Overexpression of top genes was analyzed using Gene Ontology and KEGG pathways. Results The volcano plot (Figure 1A) showed strong associations between the endoscopic Mayo score and components of innate immunity, e.g. complement factor B (CFB), C1-inhibitor (also known as SERPING1), chitinase 3-like 1 (CHI3L1), and inflammasome genes (ZBP1 and PIM2). Moderate associations with calprotectin (S100A8 and S100A9), other inflammasome components (CASP1 and NLRP3), and T cell transcripts (i.e. CTLA4, PDL1) were observed. Targets of biologic therapy (TNFA, ITGA4/B7, IL12B) were weakly associated with the endoscopic Mayo score. Expression of the top genes in a cell panel (Figure 1B) showed primary expression in monocytes, macrophages, dendritic cells, and polymorphonucleocytes, with some expression in colon epithelial and endothelial cells. Minimal expression was found in CD4/CD8 T cells or NK cells. Pathway analysis represented extracellular matrix remodeling, complement regulation, and TNFA signaling, but revealed no pathways associated with adaptive immunity (Table 1). Conclusions UC disease activity, as assessed by the endoscopic Mayo score, was strongly associated with tissue remodeling and molecules of innate immunity that were largely found in myeloid cells, colon epithelium, and endothelium. Cognate T cells were not dominant features of UC disease activity. These data suggest that the driver of ongoing UC activity is independent of the cognate T cell response. Funding Agencies None

Background Eosinophilic esophagitis (EoE) is a complex, chronic, allergic disease that commonly presents as dysphagia in young adults. Co-occurring allergic or atopic disease, including food allergies and asthma are seen in approximately 70% of cases. Allergy to house dust mite (HDM) tropomyosin, Der p 10, and cross-reactivity to shrimp has been well described. We discuss a presentation of EoE in the setting of positive HDM skin prick testing that subsequently improved with dietary elimination of shrimp, that has never been described in the literature. Purpose To highlight an underlying mechanism in EoE and the importance of a multidisciplinary approach to this disease. Method A 45-year-old male with a medical history significant for ulcerative colitis (diagnosed two years prior and maintained in deep endoscopic remission with adalimumab) and asthma (with seasonal, rare usage of budesonide and formoterol inhaler) was seen by his outpatient gastroenterologist following three weeks of new-onset dysphagia and subjective intermittent food bolus impaction. The patient was pre-emptively started on daily proton pump inhibitor prior to endoscopy, but discontinued this within less than two weeks as he felt like it induced loose bowel movements. Esophagogastroduodenoscopy (EGD) was performed and revealed proximal esophageal furrows. Histology of esophageal biopsies was compatible with EoE: distal esophagus >100 eosinophils/hpf with eosinophil degranulation, mid and proximal esophagus >25 eosinophils/hpf with eosinophil degranulation. The patient self-identified that his episodes of dysphagia and food bolus impaction were associated with shrimp ingestion, thus he eliminated shrimp from his diet. Result(s) Two-month clinical follow-up demonstrated symptomatic remission despite no other active treatment or additional food elimination. Consultation with an allergist demonstrated the patient had positive skin prick testing to HDM (Dermatophagoides pteronyssinus and farina) but negative testing to shrimp, despite clinical improvement with dietary shrimp elimination. Thus his EoE was attributed to HDM cross-reactivity, specifically to the Der p 10 antigen. Follow-up EGD and biopsy to confirm endoscopic and histologic remission with shrimp elimination is scheduled in two months. Conclusion(s) This is the first case description of a patient with new onset EoE attributed to Der p 10, in the setting of positive skin prick to HDM and negative skin prick to shrimp, with subsequent symptomatic resolution of EoE following dietary shrimp elimination. Shrimp and HDM share a protein, Der p 10. Der p 10 is commonly the culprit antigen in patients that have allergic reactions to HDM and (or) shrimp, but often only demonstrate positive skin prick testing to HDM (with negative testing to shrimp). This case demonstrates a role for involvement of an allergist and skin prick testing in select cases of EoE, as this may result in a relatively simple food elimination and avoidance of further therapeutic interventions. Please acknowledge all funding agencies by checking the applicable boxes below None Disclosure of Interest None Declared

Background Ulcerative colitis (UC) is a chronic inflammatory condition affecting the colonic epithelium. We used an established microarray-based system to analyze a set of 128 UC biopsies (113 patients), assessing gene expression associated with the colon’s response to injury in UC. Aims Our aim was to describe the burden of injury in UC biopsies and to explore molecular heterogeneity across disease activity, as assessed by the endoscopic Mayo score. Methods 128 UC colon biopsies were collected at the University of Alberta Hospital (Edmonton, AB) and Cedars-Sinai Hospital (Los Angeles, CA) during standard of care colonoscopy and processed using Affymetrix microarrays. Principal component analysis (PCA) and archetypal analysis (AA) visualized relationships between biopsies and previously annotated injury-associated transcript sets. AA assigned each biopsy to one of three groups, and scores to each biopsy relating it to all three groups. Results Spearman correlations (Table 1A) were highest between the endoscopic Mayo score and the injury-repair-associated transcripts (IRRAT, 0.64, P=4.7x10-16), immunoglobulin transcripts highly associated with chronic injury and fibrosis (IGT, 0.63, P=3.0x10-15), endothelial transcripts (ENDAT, 0.61, P=1.8x10-14), and parenchymal dedifferentiation i.e. epithelial solute carrier loss (CT2, -0.60, P=6.5x10-14). PCA separated injury from no injury in PC1 (Figure 1A). T cell transcripts (QCATs), interferon-gamma inducible transcripts (GRITs) and targets of biologics (IL12, TNFA, ITGA4/B7) separated from injury transcripts in PC2. We assigned three AA groups and visualized biopsies in PCA (Figure 1B, colored by AA membership). Group 1 (grey, N=44) biopsies had little parenchymal dedifferentiation and low expression of injury-associated transcripts. Groups 2 (red, N=44) and 3 (blue, N=40) had increased expression of injury-associated transcript sets and dedifferentiation compared to Group 1 (Table 1). Although Group 3 was endoscopically similar to Group 1 (P>0.05), Group 3 showed elevated injury-associated transcript set expression (e.g. IRRAT) and increased parenchymal dedifferentiation (CT2). Conclusions Assessment of UC biopsies using AA and previously annotated injury-associated gene sets reveals two groups of biopsies that are endoscopically similar though one group has increased molecular abnormalities, thus revealing heterogeneity unrelated to the Mayo score. A molecular system based around PCA and AA could enhance and refine UC disease assessment by allowing for quantitation and qualification of injury in biopsies obtained at endoscopy i.e. a level of resolution beyond conventional endoscopic scoring. Funding Agencies None

BACKGROUND: The assessment of patients with ulcerative colitis (UC) remains a challenge despite rapidly evolving medical therapy. Current assessment with endoscopic scoring and histopathology lacks the granularity to correlate strongly with response to therapy. AIMS: Utilizing a microarray based system for colonic epithelial assessment we looked for stratification of endoscopically similar UC biopsies using molecularly heterogeneous archetypes. METHODS: Molecular data from 71 UC biopsies (from 61 patients) was obtained from microarray analysis. The top 300 genes correlating with the endoscopic Mayo score (2/3 vs 0/1) were used for an unsupervised analytical method called archetypal analysis, and grouped the biopsies into three distinct clusters. Logistic regression modeling was used to compare archetype scores or cluster membership to endoscopic Mayo score and PC1 in predicting mucosal healing. A contingency table was generated to show evidence of mucosal healing within each of the archetype clusters. We then assessed a subset of biopsies from the original set of 71 (selected due to the availability of biopsies before and after therapy, all on TNF therapy) plus one IBDU case. Patients were classified as ‘responders’ (Mayo 2/3 score to Mayo 0/1 score by last obtained biopsy or scope) or ‘non-responders’ (Mayo 2/3 score that did not decrease to 0/1 by last obtained biopsy or scope). RESULTS: We found three unique groups of biopsies using archetypal analysis (A1: lack of inflammation, A2: inflammation and response to wounding, A3: inflammation). Logistic regression showed that the only models with statistically significant predictive value (p-value < 0.05) were those that contained archetype scores or cluster membership. Response rates differed between archetype clusters with statistical significance (Table 1), while the mayo score distribution within these clusters was not statistically different. In our subset of serial biopsies, the majority of initial biopsies were found to have an A2 archetype, moving to an A1 archetype in follow-up that mirrored response to treatment (Figure 1). CONCLUSIONS: This archetypal analysis suggests there is potentially important heterogeneity in UC biopsies that is not accessible by endoscopic Mayo score. Serial biopsies showed dynamic shifts in the archetype composition between biopsies. This may be a useful tool for both initailly prognosticating patients and assessing response to treatment over time with increased granularity and reliablity. FUNDING AGENCIES: None

BACKGROUND: Ulcerative colitis (UC) is a chronic, idiopathic inflammatory condition affecting the colonic epithelium, with the inflammasome, T cells, complement activation, and microbiome dysbiosis contributing to pathogenesis. AIMS: We applied a previously established method of microarray molecular analysis to a set of 71 UC biopsies (from 61 patients), to elucidate the molecular changes associated with active UC, specifically comparing UC to T-cell mediated rejection (TCMR), as a prototype of a sterile, T-cell mediated disease. METHODS: 71 for-cause UC colonic biopsies were collected at the U of A Hospital in Edmonton, AB and Cedars-Sinai Hospital in LA, California. These biopsies were processed using Affymetrix GeneChip microarrays and the data analyzed in R programming language. Gene expression data was displayed using volcano plots (showing the fold change and association between the genes and endoscopic Mayo score) and heatmaps (showing expression of the top 30 genes in a cell panel). We then analyzed overexpression of the top genes using the DAVID tool(1), and compared the results to similar results for top genes overexpressed in TCMR. RESULTS: The volcano plot (Figure 1) showed strong associations between the endoscopic Mayo score and decay accelerating factor (CD55), and moderate associations with calprotectin genes (S100A8/S100A9), with lesser associations for effector T cell transcripts (i.e. CTLA4, interferon gamma (IFNG), and chemokine ligand (CXCL13)), many IFNG inducible transcripts, inflammasome transcripts (CASP1), and toll-like receptors (TLR5). NLRP3 (incriminated in mouse UC models) did not pass the IQR filter and was not significant. Expression of the top genes in a cell panel showed primary expression in monocytes, macrophages, and dendritic cells, with some expression in epithelial and endothelial cells, while minimal expression was found in CD4/CD8 T cells, or in NK cells. Pathway analysis showed major differences between pathway terms. CONCLUSIONS: Our findings show that while transcripts associated with cognate T cell inflammatory processes (TCMR) are expressed in UC, there are substantial differences between the pathogenesis of a pure T cell-mediated disease and UC. In our cohort, the top genes associated with the endoscopic Mayo score in UC were not those associated with effector T cells: T cell-associated transcripts were only moderately associated with the Mayo score, and pathway analysis showed significant differences between TCMR and UC, which is much more an inflammatory environment with strong associations to CD55. This suggests that cognate T cell recognition is present in UC but supplemented by a second source of inflammation. REFERENCES: 1. Huang et al. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols (2009). FUNDING AGENCIES: None

trans-Urocanic acid (UCA) acts as a chromophore for UV radiation in the epidermis and isomerizes to cis-UCA which then initiates some of the changes leading to UV-induced immunosuppression. The mechanism of the immunomodulation by cis-UCA is unknown at present, but one possibility is that the interaction between cis-UCA and keratinocytes causes the release of immunosuppressive cytokines locally. To test this hypothesis, PAM-212 cells, a murine keratinocyte cell line, were incubated with 0.10–100 μg/mL trans- and cis-UCA for 6 or 24 h, respectively. The expression of interleukin (IL)-10, transforming growth factor (TGF)-β and tumor necrosis factor (TNF)-α messenger RNA (mRNA) was then measured by reverse transcription-polymerase chain reaction in comparison with the mRNA for the house-keeping gene, β-actin. No change or significant induction of any of the cytokine messages occurred. However, the expression of IL-10 messenger RNA (mRNA) was induced 24 h after UVB irradiation (300 J/m2) and that of TNF-α mRNA occurred 6 h after treatment with phorbol myristate acetate. The expression of IL-10 protein was also examined by immunostaining in both PAM-212 cells and B16-F10 murine melanoma cells between 3 and 48 h after incubation with 10 and 100 μg/mL cis- and trans-UCA. No alteration was seen with either isomer at either concentration. In contrast, UVB irradiation of both cell lines resulted in a marked increase in intracellular IL-10 protein at 24 and 48 h. Therefore the upregulation of the immunosuppressive cytokines, IL-10, TNF-α and TGF-β, in keratinocytes is unlikely to be the mechanism by which cis-UCA induces immunosuppression in mice.

In our model of partially appropriable invention, if the US Plant Variety Protection Act (PVPA) of 1970 succeeded in strengthening intellectual property protection for plant breeders, it should have led to increases in investment in varietal R&D, improved varietal quality, and enhanced royalties. These hypotheses are tested using data on US wheat. We find that the PVPA may have stimulated public (but not private-sector) investment in wheat varietal improvement but that the PVPA did not cause any increase in experimental or commercial wheat yields. These findings indicate that the PVPA had little impact on excludability or appropriability in wheat varieties.

The binding of unfractionated heparin to endothelium is thought to be responsible for the rapid and saturable phase of unfractionated heparin clearance. Thrombin can induce endothelial cells to express and/or secrete a number of heparin binding proteins that have the potential to increase the binding of unfractionated heparin and to a lesser extent the binding of low molecular weight heparin. To explore this possibility, we examined the binding of unfractionated heparin and low molecular weight heparin to thrombin-activated endothelial cells. Cultured human umbilical vein endothelial cells were used to determine the binding of 125 I-labeled unfractionated heparin and low molecular weight heparin to untreated and to thrombin-activated cells. After thrombin treatment, we obtained a time-dependent increase in the binding of radiolabeled unfractionated heparin. In contrast, there was much less binding of low molecular weight heparin, and a time-dependent increase was not apparent. After 30, 45, and 60 minutes of thrombin treatment, the binding of unfractionated heparin was significantly higher than that of low molecular weight heparin. The increase in binding of unfractionated heparin to thrombin-activated cells also was demonstrated using fluorescently labeled unfractionated heparin followed by fluorescence microscopy. The average fluorescence intensity of thrombin-treated cells increased by 44% when compared with resting cells. The present results indicate that thrombin can increase the binding of unfractionated heparin to human umbilical vein endothelial cells. Thus, an activated endothelium may contribute to the variability of the anticoagulant response to unfractionated heparin. In contrast, the binding of low molecular weight heparin is much less affected, which may account for its better bioavailability and longer half-life.

SummaryPreviously, we demonstrated in a rat model of heparin-induced osteoporosis that low molecular weight heparin (LMWH) produces less bone loss than unfractionated heparin, and that only heparin increases osteoclast number and activity. In contrast, both heparin and LMWH were found to decrease osteoblast function to a similar extent, possibly because at the doses tested both agents produced maximal inhibition. To examine the relative effects of heparin and LMWH on osteoblast function more closely we used an in vitro bone nodule assay, together with measurements of alkaline phosphatase (ALP) activity. Both agents inhibited bone nodule formation and ALP activity in a concentration-dependent manner, but 6 to 8-fold higher concentrations of LMWH were required to achieve equivalent effects. The effect of heparin on osteoblast function was both chain-length and negative charge-dependent because the ability of defined heparin fragments to inhibit nodule formation correlated with their molecular weight (r = 0.98), and N-desulfated heparin was less inhibitory than heparin. In contrast, the effect of heparin on osteoblast function was pentasaccharide-independent because heparin with low affinity for antithrombin had similar activity to heparin with high antithrombin activity. These findings help to explain mounting clinical evidence that the risk of osteoporosis is lower with LMWH than with heparin.

Abstract We have previously demonstrated that the nonspecific binding of unfractionated heparin (UFH) to plasma proteins has a marked modulating effect on its anticoagulant activity. Since some heparin-binding proteins are also acute-phase-reactant proteins, we explored the possibility that the induction of the acute-phase response can increase the plasma concentrations of heparin-binding proteins. The recovery of a fixed amount of UFH or low-molecular-weight heparin (LMWH) added in vitro to rat plasma samples obtained at various time intervals after the administration of intravenous endotoxin or subcutaneous turpentine was compared with that of saline-treated control animals. The anti–factor Xa activity was measured in the plasma samples before and after the addition of a chemically modified low-affinity heparin (LAH) to displace the proportion of the added heparin that is reversibly bound to plasma proteins. Our results show that at 6 hours post–endotoxin and at 24 hours post–turpentine treatment, virtually no anti–factor Xa activity could be measured in the plasma samples, while the expected levels were obtained for control plasma. After the addition of LAH to displace protein-bound UFH, essentially the same anti–factor Xa levels were measured in the plasma from all three treatment groups. These results indicate that induction of the acute-phase reaction can dramatically increase the levels of heparin-binding proteins in rat plasma. In addition, we compared the anti–factor Xa recovery of UFH with that of an LMWH from the plasma of endotoxin- and saline-treated rats and demonstrated that LMWH binds less to plasma proteins than UFH, even in plasma in which the levels of heparin-binding proteins are markedly elevated. The recovery of a fixed amount of UFH added in vitro to human plasma from septic patients was also reduced, but not to the same extent as seen in rat plasma. Removal of candidate heparin-binding and acute-phase proteins by immunodepletion indicated that vitronectin plays an important role in the nonspecific binding of UFH in patient plasma.

Cyclosporine A (CsA) has been shown to be an effective therapeutic agent for a wide variety of cutaneous diseases yet its exact mechanism of action is still unclear, although one well-defined effect of CsA is the inhibition of T-cell-derived cytokine expression. We recently demonstrated in vitro that CsA inhibits cell proliferation and suppresses cytokine gene expression in keratinocytes. In this study, we report the in vivo effects of CsA on skin cytokine gene expression as determined by reverse-transcriptase polymerase chain reaction. C57BL6 mice (female, 8-10 weeks old) were subcutaneously injected with CsA in olive oil (0, 5 and 10 mg/kg) every other day for 3 weeks. Treatment with 5 mg/kg CsA inhibited both interleu-kin (IL)-1Α and tumor necrosis factor Α gene expression by about 70 and 90%, respectively, relative to vehicle control levels. However, IL-6 gene expression did not significantly change. Injection of 10 mg/kg CsA inhibited expression of all three genes by 80-90% relative to control levels. These data show that CsA can inhibit constitutive cytokine gene expression in mouse skin.

A major cellular protein (P2; approximately 70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors colchicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc70). The inference that P2 and hsc70 are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc70; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp70 with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp70 in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp70 antibodies in human and CHO cells; and (v) a cDNA clone for hsc70 has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc70 in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.

trans-Urocanic acid (UCA) acts as a chromophore for UV radiation in the epidermis and isomerizes to cis-UCA which then initiates some of the changes leading to UV-induced immunosuppression. The mechanism of the immunomodulation by cis-UCA is unknown at present, but one possibility is that the interaction between cis-UCA and keratinocytes causes the release of immunosuppressive cytokines locally. To test this hypothesis, PAM-212 cells, a murine keratinocyte cell line, were incubated with 0.10-100 micrograms/mL trans- and cis-UCA for 6 or 24 h, respectively. The expression of interleukin (IL)-10, transforming growth factor (TGF)-beta and tumor necrosis factor (TNF)-alpha messenger RNA (mRNA) was then measured by reverse transcription-polymerase chain reaction in comparison with the mRNA for the house-keeping gene, beta-actin. No change or significant induction of any of the cytokine messages occurred. However, the expression of IL-10 messenger RNA (mRNA) was induced 24 h after UVB irradiation (300 J/m2) and that of TNF-alpha mRNA occurred 6 h after treatment with phorbol myristate acetate. The expression of IL-10 protein was also examined by immunostaining in both PAM-212 cells and B16-F10 murine melanoma cells between 3 and 48 h after incubation with 10 and 100 micrograms/mL cis- and trans-UCA. No alteration was seen with either isomer at either concentration. In contrast, UVB irradiation of both cell lines resulted in a marked increase in intracellular IL-10 protein at 24 and 48 h. Therefore the upregulation of the immunosuppressive cytokines, IL-10, TNF-alpha and TGF-beta, in keratinocytes is unlikely to be the mechanism by which cis-UCA induces immunosuppression in mice.

Previously, we demonstrated in a rat model of heparin-induced osteoporosis that low molecular weight heparin (LMWH) produces less bone loss than unfractionated heparin, and that only heparin increases osteoclast number and activity. In contrast, both heparin and LMWH were found to decrease osteoblast function to a similar extent, possibly because at the doses tested both agents produced maximal inhibition. To examine the relative effects of heparin and LMWH on osteoblast function more closely we used an in vitro bone nodule assay, together with measurements of alkaline phosphatase (ALP) activity. Both agents inhibited bone nodule formation and ALP activity in a concentration-dependent manner, but 6 to 8-fold higher concentrations of LMWH were required to achieve equivalent effects. The effect of heparin on osteoblast function was both chain-length and negative charge-dependent because the ability of defined heparin fragments to inhibit nodule formation correlated with their molecular weight (r = 0.98), and N-desulfated heparin was less inhibitory than heparin. In contrast, the effect of heparin on osteoblast function was pentasaccharide-independent because heparin with low affinity for antithrombin had similar activity to heparin with high antithrombin activity. These findings help to explain mounting clinical evidence that the risk of osteoporosis is lower with LMWH than with heparin.

Ultraviolet radiation can induce the transcription and release of cytokines from keratinocytes (KC's). These cytokines have the potential to modulate local and systemic immunologic responses. In this paper we report that northern blotting showed that human KC and KC lines expressed a 1.2-1.4 kb transcript for the chemokine and melanoma growth-stimulatory protein, GRO-alpha and that ultraviolet B radiation (UVB) could upregulate the expression of GRO-alpha mRNA and protein in the KC line A431. The GRO-alpha gene response to UVB was maximal at 48h post-irradiation with 70 J/m2. Reverse transcription-polymerase chain reaction (RT-PCR) revealed a 4.5-fold increase in GRO-alpha mRNA over basal levels (p < 0.001). GRO-alpha protein was measured in the culture media by enzyme-linked immunosorbent assay (ELISA). Media from unirradiated cultures contained 1166 +/- 83 pg/ml GRO-alpha protein. After UVB, a time-dependent increase in GRO-alpha protein was seen in the culture media from 6-48h. At 48h post-irradiation the GRO-alpha protein content was 27583 +/- 678 pg/ml, or 23 times the basal level. This protein release could be inhibited by 70% when the cells were pre-incubated with 10 micrograms/ml interleukin-1 receptor antagonist (IL-1RA). We also show that another potent leukocyte chemoattractant, Interleukin-8 (IL-8), was induced in A431 cells by UVB. This induction of IL-8 mRNA began as early as 3h post-irradiation, when it reached twice basal levels (p < 0.05) and reached 4.5-fold basal levels at 48h post-irradiation (p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

A protein purified from Eschericheri coli has previously been shown to have cytotoxic effects on neoplastic cells of several lineages both in vitro and in vivo. Accordingly, this protein has been named anti-neoplastic protein (ACP). Although ACP kills neoplastic cells by inducing apoptosis, it has negligible effects on various normal cells. In addition to the direct cytotoxic effects of ACP on tumour cells, previous studies have shown that in vivo ACP increases tumouricidal activity of cytotoxic lymphocytes. We investigated whether cytokines from host or tumour cells play a part in the enhanced cellular immunity seen in ACP-treated tumour-bearing mice. Growth of normal human keratinocytres (KC) was not significantly affected by subnanogram amounts of ACP, however ACP dose-dependently killed KHT cells, a murine fibrosarcoma cell line (LD50 = 8 x 10(4) ng/cell). as well as the human squamous carcinoma cell line COLO-16 (LD50 = 2.5 x 10(-4) ng/cell). Testing purified ACP on cultures of normal keratinocytes and squamous carcinoma cell lines revealed that ACP could induce both mRNA and protein for interleukin-6 (IL-6). Messenger RNA for IL-6 increased dose-dependently 4h after treatment of COLO-16 squamous carcinoma cells with 10(-4) to 10(-2) ng/cell ACP. Maximal increment was 50-fold. Interleukin-6 message remained elevated up to 24h later in both normal keratinocytes and squamous carcinoma cultures treated with ACP. Conditioned supernates from these cultures were analysed by ELISA and found to have 4-fold higher levels of IL-6 protein than untreated cells after 4h. After 24h, IL-6 did not increase above the 4h level. Boiling of the ACP preparation showed that the cytokine induction was not due to contaminating lipopolysaccharide. The cytoxic effect of ACP on tumour cells in vitro was not due to IL-6 protein induction since neither recombinant IL-6, nor the other proinflammatory cytokines, IL-1 alpha or Tumour necrosis factor-alpha (TNF-alpha) (0.1-10ng/ml) were able to kill malignant cells. We demonstrated IL-6 gene induction by ACP in the squamous carcinoma lines as well as in normal KC. This suggests that the in vivo effectiveness of ACP against tumours may be due to stimulatory effects of IL-6 on host immunity.

The cDNA sequence of the 60 kDa heat-shock protein from mouse 3T3 cells has been determined. The deduced amino acid sequence of mouse hsp60 protein differs from the corresponding proteins from Chinese hamster and human cells in 7 and 13 residues, respectively, most of which are conservative replacements.

Using antibodies to a mitochondrial molecular chaperone class of protein, which is specifically altered in mutants resistant to microtubule (MT) inhibitors, the effect of a number of MT and mitochondrial inhibitors on the cellular distribution of mitochondria and various cytoskeletal filaments was examined. Treatment of Chinese hamster ovary (CHO) or chicken embryo fibroblast (CEF) cells with the MT inhibitors podophyllotoxin, colchicine, nocodazole and vinblastine caused depolymerization of cellular MTs, but had no significant effect on the distribution patterns of mitochondria. This is attributed to the association of mitochondria with intermediate filaments (IFs) which are not destroyed under these conditions. In contrast to MT inhibitors, treatment of CEFs with the potassium ionophores nonactin and valinomycin caused aggregation of mitochondria towards the perinuclear region of the cells, without having any apparent effect on cellular MTs. This observation suggests that mitochondrial membrane potential, which is abolished by these drugs, play a role in the cellular distribution of mitochondria. In cells recovering from the effects of MT inhibitors, mitochondria have been found to surround the MT organizing complexes and upon complete recovery a realignment of MTs with mitochondria takes place. These observations suggest that MT growth in cells does not occur in a completely random manner but that mitochondria may play some role in their directional growth.

Absorption cross-sectional measurements and model predictions of 25 disks with moderate electrical conductivity were made. The disk radii span part of the resonance region, and their thicknesses are an order of magnitude less than the skin depth. A photoacoustic apparatus, with a 35-GHz microwave source, was used to measure the absorption cross sections. The disks tend to be more absorbing when they are edge-on rather than broadside to the incident microwaves. Also they absorb most efficiently near a size parameter of 1. Model predictions generally agree with the measured magnitudes and trends in the data.

In Chinese hamster ovary cells, stable mutants exhibiting specific resistance or collateral sensitivity towards the various microtubule inhibitors podophyllotoxin, colchicine, griseofulvin, taxol, nocodazole, vinblastine, and maytansine have been isolated. A number of independent mutants selected for resistance to podophyllotoxin and colchicine contain electrophoretically altered forms of two proteins, P1 and P2, of relative molecular masses of approximately 63 000 and 69 000, respectively. The proteins P1 and P2 have been shown to be microtubule related by a number of different genetic and biochemical criteria and are among the major proteins of Chinese hamster ovary cells, being present in approximately equimolar amounts with tubulin. In addition, a griseofulvin-resistant mutant contains a novel mutation (presumably nonsense) which reduces the relative molecular mass of a protein, P5 [Formula: see text], by about 20 000. Specific antibodies to protein P1 have been raised and cross-reactivity studies show that a similar protein is also present in cells from all vertebrate species examined, viz. man, monkey, mouse, Chinese hamster, Syrian hamster, and chicken. Immunofluorescence studies with anti-P1 and anti-tubulin antibodies show that, in interphase cells from various species, the P1 antibody reacts specifically with mitochondria whose overall cellular distribution is strikingly similar to the distribution of microtubules. The mitochondrial localization of the microtubule-related protein P1 provides strong suggestive evidence regarding the existence of a chemical and functional linkage between these two structures, with protein P1 playing an important role in this linkage. Some implication of these results are discussed and it is suggested that mitochondria play an important role in the dynamics of microtubules.