Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/312—Phosphonates
  • C12N2310/3125—Methylphosphonates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
  • C12N2310/321—2'-O-R Modification
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/33—Chemical structure of the base
  • C12N2310/334—Modified C
  • C12N2310/3341—5-Methylcytosine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/34—Spatial arrangement of the modifications
  • C12N2310/345—Spatial arrangement of the modifications having at least two different backbone modifications
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/34—Spatial arrangement of the modifications
  • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

• the present invention refers to the use of Smad7antisense oligonucleotides (ODN) for the treatment of diseases mediated by the nuclear transcription factor NF-kB.
• ODN Smad7 antisense oligonucleotides
• the invention refers to the use of Smad7 antisense oligonucleotides (ODN) for the treatment of chronic inflammatory diseases and tumours that result to be associated with an abnormal and increased activity of the nuclear transcription factor NF-kB.
• ODN Smad7 antisense oligonucleotides
• an example is represented by Helicobacter Pylori (Hp) -associated diseases, such as gastric carcinoma.
• Hp Helicobacter Pylori
• the NF-kB family includes proteins involved in the regulation of multiple intracellular processes that are relevant either in the mechanisms of inflammation or apoptosis and cell death.
• NF-kB NF-kB proteins
• the NF-kB proteins are present in the cytoplasm of most cells in homodimeric or heterodimeric complexes.
• Each NFkB component contains an amino terminal region necessary for binding to the DNA, a dimerization domain, and a sequence necessary for the protein translocation into the nucleus (NLS).
• NF-kB is located in the cytoplasm of non-stimulated cells in an inactive form due to the interaction with IkB, a class of inhibitory proteins. Such proteins interacts with NF-kB components and mask the NLS sequence thus preventing the translocation into the nucleus.
• IkB In response to activating stimuli, such as those generated during bacterial infections, inflammatory or neoplastic processes, IkB is phosphorylated by intracellular kinases denominated IkK, and eventually degraded. This allows NF-kB to migrate into the nucleus, where it interacts with DNA targets, and regulates the transcription of multiple genes, that encode for cytokines and prolnflammatory enzymes (e.g TNF- , IL-1 , I- CAM-1 , IL-8, COX-2, iNOS) and/or other molecules involved in the inflammatory response underlying the aetiology of several pathologies. Among the genes induced by NF-kB there is IkB ⁇ .
• cytokines and prolnflammatory enzymes e.g TNF- , IL-1 , I- CAM-1 , IL-8, COX-2, iNOS
• NF-kB also results in the synthesis of IkB ⁇ , which in turn suppresses NF-kB signalling.
• NF-kB signalling a negative feed-back loop
• an excessive activity of the transcription factor has been documented in a variety of human inflammatory diseases, such as Helicobacter pylori-associated chronic gastritis, bronchial asthma, and auto-immune diseases, including rheumatoid arthritis, psoriasis and multiple sclerosis, neuro-degenerative pathologies such as the Alzheimer's and Parkinson's disease, chronic intestinal Inflammatory diseases (IBD) (Li Q.
• IBD chronic intestinal Inflammatory diseases
• NF-kB activation a mechanism underlying the persistent NF- kB activation remain unknown. It is however conceivable that the activation of the transcription factor could rely either on the sustained stimulation by inflammatory cytokines (e.g. IL-1 , TNF- ⁇ ), viral and bacterial products (e.g. lipopolysaccharides), or on reduced activity of counter-regulatory molecules, such as TGF- ⁇ 1.
• inflammatory cytokines e.g. IL-1 , TNF- ⁇
• viral and bacterial products e.g. lipopolysaccharides
• counter-regulatory molecules such as TGF- ⁇ 1.
• Hp activates NF-kB in gastric epithelial tumoural cellular lines, by involving different kinases, such as IKKs (Maeda et al., 2001) and a kinase inducing NF-kB (NIK) (Maeda et al., 2000).
• IKKs aeda et al., 2001
• NIK a kinase inducing NF-kB
• TGF- ⁇ 1 could have an essential and negative role in the control of NF-kB activity. Indeed, animals with either targeted deletion of TGF- ⁇ 1 gene or resistant to the action of this protein exhibit an increased expression of inflammatory molecules that could depend on NF-kB activity.
• TGF- ⁇ 1 knock-out mice develop lesions that are similar to those caused by Hp Infection, such as hyperplasia and gastric dysplasia.
• restoration of TGF- ⁇ 1 activity results in a decreased NF-kB activity and resolution of the lesions.
• a defective TGF- ⁇ 1 activity can contribute to a sustained activation of NF-kB.
• the author of the present invention has shown, in a previous study, that a defective activity of TGF- ⁇ 1 in Crohn's disease is also associated with high expression of Smad7 (Monteleone et al., 2001).
• Smad7 is a protein that, interacting with the type I TGF- ⁇ 1 receptor, prevents binding and phosphorylation of Smad2/3 (Hayashi et al., 1997), thereby inhibiting Smad2/3 translocation into the nulceus.
• Hp Helicobacter pylori
• Ernst et al., 2000 There is enormous heterogeneity in the consequences of infection.
• the Infection inevitably causes a chronic gastric inflammation.
• the majority of infected individuals are asymptomatic.
• a minority of patients develop clinically relevant gastritis, and some of these go on to develop gastric ulcers, gastric carcinoma or a low-grade B cell lymphoma.
• IL-8 interleukin-8
• the host also mounts an adaptive immune response. There is local production of anti-Hp IgA and IgG and importantly, there is a local Th1 cell response with increased synthesis of interferon (IFN)-y, TNF- ⁇ and IL-12 (Ernst et al., 2000, Israel et al., 2001 , D'Elios et al., 1997).
• IFN interferon
• Th1 response contributes to tissue injury in patients
• animal models of Hp infection clearly show that Th1 cells are essential in the development and maintenance of lesions associated with HP-infection (Smythies et al., 2000, Eaton et al., 2001 ).
• the mechanism by which Th1 cell inflammatory response is generated and maintained in the Hp-infected gastric mucosa remains however unclear. It is however conceivable that changes in the mechanisms involved in the control of the gastro-intestinal homeostasis can have a decisive role in the pathogenesis of Hp infection -associated lesions.
• TGF- ⁇ 1 a molecule that normally suppresses both Th1 -cell-mediated immune responses and NF- kB activity
• TGF- ⁇ 1 -mediated signal transduction can influence the onset of additional pathologies correlated to excessive activation of NF-klB (Hahm et al., 2002).
• the therapeutic intervention in diseases characterised by increased activity of NF-kB is mostly symptomatic, and in most cases not curative.
• the antisense oligonucleotides are short oligonucleotidic sequences complementary to the messenger RNA (mRNA) which encodes for the protein target (it is Smad7 in the present case). Such sequences, coupling with the m-RNA, form a hybrid double strand chain, which causes the activation of ubiquitous catalytic enzymes, such as RNases H, that eventually degrade the hybrid DNA/RNA chains thereby preventing the protein translation.
• mRNA messenger RNA
• RNases H ubiquitous catalytic enzymes
• NF-kB antagonises the biological action of TGF- ⁇ 1 by enhancing Smad7 itself. This would trigger a positive feed-back loop which eventually amplified NF-kB signalling.
• the experiments performed by the author of the present invention show a marked reduction of active Smad3 levels in gastric biopsies taken from patients with Hp Infection, despite the normal production of TGF- ⁇ 1 and increased expression of TGF- ⁇ 1 receptors.
• the reduction of phosphorylated Smad3 is associated with a marked expression of Smad7 both in epithelial and in the limba lamina mononuclear cells (LPMC).
• the in vitro treatment of the gastric biopsies with the Smad7 antisense oligonucleotides reduces the levels of Smad7 protein, enhances Smad3 activation and leads to a reduction in the expression of Inflammatory Th1 type molecules such as T-bet and IFN- ⁇ . It is also shown that IFN- ⁇ induces Smad7 in gastric biopsies of normal subjects, whereas neutralisation of IFN ⁇ y activity reduces Smad7 expression in the gastric mucosa of Hp-infected patients.
• TGF- ⁇ 1 causes a marked suppression of the translocation and accumulation of NFkB/p65 in the nucleus, the binding of NF-kB to DNA target and activation of NF-kB- dependent genes induced by stimulation with TNF- .
• TGF : ⁇ 1 does not reduce the activation of NF-kB induced by TNF- ⁇ .
• antisense phosphorothioate oligonucleotides for Smad7 up to 21 nucleotides in length comprising a portion of at least 10 nucleotides of the following sequence: 5'-GTXYCCCCTTCTCCCXYCAGC-3 ' (SEQ ID No 1) wherein X is a nucleotide comprising a nitrogen base selected from the group consisting of cytosine, 5-methylcytosine and 2'-O- methylcytosine and wherein Y is a nucleotide comprising a nitrogen base selected from the group consisting of guanine, 5-methylguanine and 2'-O- methylguanine, with the provision that at least one of nucleotides X or Y comprises a methylated nitrogen base or its complementary sequence, for the preparation of a drug useful for the treatment of diseases mediated by an altered activation of the nuclear transcription factor NF-kB.
• oligonucleotidic sequences of the several stereoisomers of antisense oligonucleotides according to the present invention such as diastereoisomers and enantiomers, with respect to the phosphorus atoms of the internucleosidic bonds present in the sequence.
• the antisense oligonucleotides used according to the present invention can have at least one nucleotide of the sequence methylphosphonated, for instance, at only one end 5' or 3' or at both ends 5' and 3' or along the oligonucleotidic sequence.
• methylphosphonated nucleotide can be X or Y, so that the methylphosphonated internucleosidic bond is the bond between the indicated nucleotides.
• the antisense oligonucleotides used according to the present invention can further have at least one nucleotide of the sequence that is a
• a preferred form of the present invention is represented by the use of an antisense oligonucleotide having the sequence: 5'-GTXGCCCCTTCTCCCXGCAGC-3 ' (SEQ ID No 3) wherein X is 5-methyl 2'-deoxicytidine 5'-monophosphate.
• An additional preferred form is the use of an antisense oligonucleotide having the sequence: 5'-ZTXGCCCCTTCTCCCXGCAZ-3 ' (SEQ ID No 2) wherein X is 5 methyl 2'-deoxicytidine 5'-monophosphate and Z is 2'- deoxiguanosine methylphosphone.
• the invention is referred to the use of the aforesaid antisense oligonucleotidic sequences for the preparation of a drug for the treatment of Inflammatory and tumoral pathologies mediated by altered activation of the nuclear transcription factor NF-kB.
• Inflammatory pathologies are selected from the group consisting of Helicobacter pylori Infection-associated diseases, bronchial asthma, rheumatoid arthritis, psoriasis, multiple sclerosis,
• Alzheimer's disease, Parkinson's disease, intestinal chronic Inflammatory diseases (IBD) and neoplastic pathologies With regard to the neoplastic pathologies in which the treatment with the indicated Smad7 antisense oligonucleotides is aimed at regulating NF-kB activation and expression of NF-kB-dependent genes, there is a preferential choice for carcinoma and lymphoma, particularly those associated with Helicobacter pylori Infection.
• figure 1 shows the ELISA assay of active TGF- ⁇ 1 at level of the gastric mucosa colonised from Helicobacter pylori performed on total protein extracted from gastric biopsies of 8 Hp-positive and 8 Hp-negative patients.
• FIG. 1 Each point represents the value of active TGF- ⁇ 1 in mucosal samples collected from a single individual and the horizontal bars indicate the median value;
• figure 2 shows immunohistochemical staining for the type I and type II TGF- ⁇ 1 receptors in paraffin sections of gastric tissue from Hp- positive and Hp-negative patients, in both right panels, it is shown the immunohistochemical staining with a control rabbit isotype IgG corresponding to the gastric sections of Hp-positive patients shown in the left panels (X 40 magnification).
• the example is representative of 4 separate experiments, analysing in total gastric biopsies from 8 Hp infected and 7 Hp-negative patients;
• figure 3 shows reduced phosphorylation of Smad3 in Hp- colonised gastric mucosa.
• Panel A Representative expression of both phosphorylated (p-
• Panel B Quantitative analysis of the active/inactive Smad3 ratio in gastric biopsies from the 13 Hp-positive and 13 Hp-negative patients, measured through densitometric analysis of the Western Blots. The values are expressed in arbitrary units (u.a.) and each point represents the value (u.a.) of the active/inactive Smad3 ratio in the biopsy taken from on a single individual, while the horizontal lines indicate the mean value. Some samples have very similar levels of u.a. so that only 8 points for every group are evident in figure.
• Panel C Representative expression of p-Smad3 and total
• Smad3 proteins in lamina limbal mononuclear cells isolated from the gastric mucosa of 3 Hp-positive and 3 Hp-negative patients.
• the example is representative of two separate experiments analysing LPMC samples from 5 Hp-positive and 5 Hp-negative patients; figure 4 shows the increase of Smad7 expression in gastric biopsies of patients infected with Hp Infection.
• Panel A Representative expression of Smad7 and ⁇ -actin proteins in gastric biopsies taken from 3 Hp-positive and 3 Hp-negative patients.
• the example is representative of four separate experiments analysing in total biopsies from 13 Hp-positive and 13 Hp-negative patients.
• Panel B is representative of four separate experiments analysing in total biopsies from 13 Hp-positive and 13 Hp-negative patients.
• FIG. 1 The example is representative of two separate experiments analysing samples from 5 patients; figure 5 shows the induction of Smad7 by IFN- ⁇ in gastric mucosa.
• Panel A Representative expression of Smad7 (upper blot) and ⁇ -actin (lower blot) proteins in gastric mucosal samples collected from Hp- negative patients, and cultured for two hours in medium alone (UNS) or in the presence of filtrates of cultural broth of Hp or Brucella (control), or 100 ng/ml recombinant IFN- ⁇ (rh-IFN- ⁇ ).
• the example is representative of three separate experiments analysing gastric mucosal samples from 4 Hp- negative patients.
• Panel B Panel B.
• RT-PCR gel showing IL-8 transcripts in AGS cells cultured for one hour in medium alone (UNS), or in the presence of filtrates of cultural broth of Hp or Brucella (control), or 100 ng/ml recombinant IFN- ⁇ (rh-IFN- ⁇ ).
• Panel C Neutralisation of endogenous IFN- ⁇ inhibits Smad7 in Hp-infected gastric mucosal samples.
• Western Blot showing Smad7 protein in gastric mucosal samples collected from Hp-positive patients, and subsequently cultured for 24 hours in medium alone (UNS) or in presence of an IFN- ⁇ neutralising or control antibody, respectively.
• the example is representative of three separate experiments analysing mucosal samples from 4 Hp-positive patients.
• FIG. 6 shows the interaction between IFN- ⁇ and STAT1 protein.
• Panel A IFN ⁇ y enhances STAT1 activation in normal gastric mucosa. Normal gastric biopsies were pre-incubated with Tyrphostin B42 (TB42), an inhibitor of JAK2-STAT1 , or DMSO for 30 minutes before the stimulation with IFN- ⁇ (100 ng/ml) for 1 hour.
• UNS biopsies cultured in medium alone. Total proteins were analysed for the content of p-Tyr- STAT1 (upper blot) and of Total STAT1 (lower blot). The example is " representative of three separate experiments analysing gastric mucosal samples from 4 normal control patients.
• Panel B IFN ⁇ y enhances STAT1 activation in normal gastric mucosa. Normal gastric biopsies were pre-incubated with Tyrphostin B42 (TB42), an inhibitor of JAK2-STAT1 , or DMSO for 30 minutes before the stimulation with IFN- ⁇ (
• Panel B Gastric biopsies collected from a Hp-positive patient cultured in medium alone (M), in presence of a control sense oligonucleotide (S), or a specific Smad7 antisense oligonucleotide (AS), for 24 hours.
• M medium alone
• S control sense oligonucleotide
• AS specific Smad7 antisense oligonucleotide
• FIG. 8 Inhibition of IFN ⁇ y secretion in supernatants of organ cultures of biopsies from Hp-infected patients by Smad7 antisense oligonucleotide (AS); figure 8, panel A shows the TGF- ⁇ 1 -mediated inhibition of nuclear translocation of NF-kB/p65 induced by TNF- ⁇ in normal LPMC.
• Western blot shows NF-kB/p65 in nuclear extracts from normal LPMC (inset). Quantitative analysis of the NFkBp65/histone-1 ratio.
• FIG. 11 shows the TGF- ⁇ 1 -mediated induction of lkB ⁇ transcription in normal LPMC.
• Agarose gel shows both IkB ⁇ and ⁇ -actin transcripts in normal LPMC;
• figure 12 shows the TGF- ⁇ 1 effect on the activity of the IkB ⁇ promoter in primary fibroblasts isolated from human foetal gut. Luciferase activity was analysed and normalised to the activity of pRLTK. It was then expressed as entity of the induction in comparison to the untreated control sample.
• FIG. 13 panel A shows the Western blot of NF-kB/p65 in nuclear extracts from LPMC isolated from the colon of 5 IBD patients and and 5 normals.
• FIG. 13 panel B shows the Western blot of IkB- ⁇ in cytosolic extracts from LPMC isolated from the colon of 5 IBD patients (3 CD and 2 UC) and 5 normals.
• FIG. 13 panel B shows the Western blot of IkB- ⁇ in cytosolic extracts from LPMC isolated from the colon of 5 IBD patients (3 CD and 2 UC) and 5 normals.
• One of two representative experiments analysing cells from 5 CD, 3 UC and 8 samples is shown.
• the blot shows IkB ⁇ in the same LPMC samples analysed for nuclear NF-kB (panel A).
• Panel A Western blots showing NF-kB/p65 in nuclear extracts from IBD LPMC.
• Panel B shows the two representative EMSA blots showing the bonding activity of NF-kB to DNA in IBD LPMC; figure 15, panel A, shows the effect of treatment of IBD LPMC with an antisense oligonucleotide for Smad7 or control sense oligonucleotide. The antisense but not the control DNA inhibits SmadJ expression.
• the arrows indicates Smad7 protein as revealed by a specific polyclonal antibody.
• figure 15, panel B shows that the inhibition of Smad7 increases IkB ⁇ expression in IBD LPMC.
• Figure 15, panel C shows that the inhibition of Smad7 is accompanied by accumulation of NF-kB/p65 at the nuclear level in IBD LPMC.
• Inset quantitative analysis of p65/histone- 1 ratio in LPMC from 3 CD and 1 UC.
• FIG. 16 shows the inhibition of TNF-a-induced NF-kB activation by Smad7 antisense oligonucleotides and its dependence on TGF- ⁇ 1 activity in IBD LPMC.
• the basal binding activity of NF-kB is reduced in comparison with cells pretreated with medium or sense oligonucleotide for Smad7;
• figure 17 shows PCR products for Smad7 in gastric (AGS and
• EXAMPLE 1 Induction and regulation of Smad7 in the gastric mucosa of Hp-infected patients.
• MATERIALS AND METHODS 44 patients (20 men and 24 women, median age 42, range from 21 to 74 years) who underwent esophagogastroduodenoscopy for dyspeptic symptoms were studied. Among these individuals, 22 patients were Hp positive as confirmed by urease quick test and histology.
• gastric biopsy specimens were taken: one from the antrum for urease quick test, four from the antrum and corpus for the histological and immunohistochemical test and the residual samples from the antrum for the measurement of TGF- ⁇ 1 and of Smad3 and Smad7 expression. Additional biopsy specimens were taken from the gastric antrum of 8 Hp-positive and 4 Hp-negative patients and used for the preparation of the organ cultures. None of the enrolled patients had received antibiotics within the 2 months preceding the study. The informed consent was obtained from ail patients and the protocol was approved by the local ethical committee. Organ cultures and gastric LPMCs isolation Mucosal biopsies were cultured as described in Fais et al., 1992.
• biopsies collected from the gastric antrum of 4 Hp- negative patients were placed on iron grids with the mucosa layer oriented upwards in a Petri dish and immersed in a serum free medium containing RPMI 1640 (Sigma, Milan) supplemented with 10% HL-1 (BioWhittaker, Verviers, Belgium), penicillin (100U/ml) and streptomycin (100 ⁇ g/ml) (LifeTechnologies-GibcoBRL, Milan).
• Cultures were performed with or without the addition of a broth culture filtrate derived from Hp 60190 strain (wild type) or a Brucella control strain (final dilution 1 :3) (Ricci et al., 1996) or the addition of recombinant IFN- ⁇ (rh-IFN- ⁇ , final concentration 100 ng/ml) (Peprotech EC LTD, London) for 1-4 hours. Further, the cultures were stimulated with rh-IFN- ⁇ in the presence or absence of a Jak2/STAT1 inhibitor such as Tyrphostin B42 (TB42) (100 ⁇ M, Inalco S.p.A., Milan) or DMSO (Sigma) for a period varying from 30 up to 120 minutes.
• a Jak2/STAT1 inhibitor such as Tyrphostin B42 (TB42) (100 ⁇ M, Inalco S.p.A., Milan) or DMSO (Sigma) for a period varying from 30 up to 120 minutes.
• TB42 and DMSO were preincubated for 30 minutes before the addition of rh-IFN- ⁇ .
• gastric biopsies collected from 4 Hp-positive patients were cultured in the presence or absence of a control or IFN- ⁇ neutralising antibodies (5 ⁇ g/ml, R&D Systems, Abingdon, UK) for 24 hours and examined for the content of Smad7.
• biopsies collected from the antrum of 4 Hp- positive patients were cultured in the presence or absence of Smad7 sense and antisense oligonucleotides (10 ⁇ g/ml). Both Smad7 sense and antisense oligonucleotide were combined with 2000 Reagent lipofectamine (Invitrogen Italy, St.
• oligonucleotides 20 minutes before using, according to the instructions of the manufacturer.
• the characteristics of both oligonucleotides have been described in previous studies (Monteleone et al., 2001). Briefly, the phosphorothioate single strand oligonucleotides match the region 107-128 (5'- GCTGCGGGGAGMGGGGCGAC-3') of the human Smad7 complementary DNA sequence and were synthesized in the sense and antisense orientation.
• the Petri dishes with the organ cultures were placed in a hermetic container with 95% O 2 / 5%CO 2 at 37°C at 1 bar. After 24 hours, the total proteins were extracted from tissue and analysed for the expression of cytokines by Western Blotting.
• the supernatants of the organ cultures were collected and used to measure IFN- ⁇ by ELISA (Peprotech).
• Gastric LPMCs were isolated by standard methods including the sequential use of DTT, EDTA and collagenase as previously described in detail in previous studies (Monteleone et al., 1997). The percentage of epithelial cells contaminating LPMC preparations was ⁇ 5% in any case. The isolated cells were counted and checked for viability, using
• 0.1 % trypan blue solution (this was between 89 and 93%).
• RNA extraction Total RNA, cDNA preparation and RT-PCR were carried out as previously described (Monteleone et al., 1997).
• PCR primers were as follow: IL-8, FWD: 5'-TGCAGCTCTGTGTGAAGG-3 ' (SEQ ID No 4); REV: 5 ' - ATTGCATCTGGCMCCCTAC-3 ' (SEQ ID No 5); ⁇ -actin, FWD: 5'-GGCACCACACCTTCTACA-3 ' (SEQ ID No 8); REV: 5'-CAGGTCTTTGCGGATGTC-3 ' (SEQ ID No 9).
• TGF- ⁇ 1 ELISA Frozen biopsies were homogenised in liquid nitrogen and the total protein extract were collected in buffer containing 10 mM Hepes (pH).
• phosphorylated Smad3 For the detection of phosphorylated Smad3 (p-Smad3), 200 ⁇ g/sample of total proteins were separated on a 10% SDS-PAGE gel. Phosphorylated Smad3 was detected using a rabbit anti-human pSmad2/3 (final dilution 1 :500) (Saint-Cruz biotechnology) and followed by a HRP- peroxidase conjugated goat anti rabbit IgG (final dilution 1 :10000). The reaction was detected with a chemiluminescence kit (West Dura, Pierce, Rockford, IL, USA).
• blots were stripped and incubated with a rabbit anti human Smad3 antibodies (2 ⁇ g/ml, Upstate, Lake Placid, NY, USA) and subsequently with goat anti-rabbit antibody conjugated to HRP-peroxidase (dilution 1 :10000).
• Smad7 was analyzed using a specific rabbit anti-human Smad7 antibody (H-79; final dilution 1 :500, SantaCruz Biotechnology).
• Goat anti rabbit antibodies conjugated to HRP-peroxidase (dilution 1 :50000) were used to detect the antigen-antibody binding and the immunoreactivity was visualized as above described.
• IFN- ⁇ and T-bet were analyzed using specific goat anti-human IFN- ⁇ and anti-T-bet antibodies (dilution final 1:500, Saint Cruz Biotechnology).
• Rabbit anti-goat antibodies conjugated to HRP peroxidase (dilution 1:20000) were used to detect primary antibodies binding and the immunoreactivity was visualised as above described.
• the stain was performed with ponceau S (Sigma).
• the blots were stripped and analyzed for the content of ⁇ -actin, as internal control, using specific mouse anti human ⁇ - actine antibodies (dilution 1 :5000, Sigma), followed by goat anti-mouse antibodies conjugated to HRP peroxidase (dilution 1 :30000).
• blots were stripped and incubated with rabbit anti human polyclonal antibodies that recognise the total STAT1 forms (dilution final 1 :2000, Saint Cruz Biotechnology) followed by a goat anti-mouse antibodies conjugated to HRP-peroxidase (dilution 1 :20000, Dako). The intensity of bands was analyzed by densitometry using a computer assisted system (Total lab, AB.EL Sience-Ware Sri, Rome). Statistic analysis Differences between groups were compared using either the Mann-Whitney U test for not normally distributed data, or the Student test if the observations were consistent with a sample deriving from a normally distributed population.
• TGF- ⁇ 1 negatively regulates mucosal immune response and a defective production of TGF- ⁇ 1 has been associated with the development of gastritis in experimental models (Letterio et al., 1998, Yang et al., 1999). The authors have analysed if Hp infection-related gastritis is associated with changes in the production of active TGF- ⁇ 1. Since severed gastric mucosal cell types are able to synthesize TGF- ⁇ 1 , the cytokine was measured in total protein extracts from whole gastric mucosa rather than in single purified cell types.
• Smad7 protein expression was increased in all patients with Hp infection- associated gastritis compared with Hp-negative patients (figure 4, panel A).
• Hp-positive patients have a mean value of Smad7/ ⁇ - actin ratio of 0.652 densitometry arbitrary units (range 0.4-0.98) that is significantly higher than that found in Hp-negative patients (mean 0.125; range 0.001-0.3) (P ⁇ 0.001), (figure 4, panel B).
• Smad7 was also observed in the LPMC samples collected from patients with Hp Infection, as shown in figure 4, panel C.
• the Smad7 expression was analysed in gastric biopsies of 5 Hp-positive patients before and after a successful eradicating therapy. Hp eradication results in a reduction of
• gastric biopsies taken from Hp-positive patients were cultured in the presence of neutralising IFN- ⁇ antibodies and then analysed for Smad7 content.
• panel C the neutralising IFN- ⁇ but not the control antibody reduced significantly Smad7 expression.
• IFN- ⁇ acts by activating intracellular signals linked to the STAT1 pathway (Horvath et al., 1997) and several previous studies have shown that IFN- ⁇ -induced STAT1 activation can be associated with S ad7 induction in monocyte cell lines (Ulla et al., 1999).
• Hp colonised biopsies release IFN- ⁇ in the culture supernatants and the concentration levels of the protein were not affected by the addition of Smad7 sense oligonucleotide, as shown in figure 7, panel C.
• Smad7 inhibition on the contrary caused a marked reduction of levels of IFN- ⁇ secreted in the culture supernatant.
• the results reported in the previous example show that the colonization of the stomach by Helicobacter pylori involves a drastic reduction in Smad3 phosphorylation, a crucial step in the TGF- ⁇ 1- mediated signal transduction mechanism, despite the elevated expression of TGF- ⁇ 1 receptors and synthesis of active TGF- ⁇ 1.
• TGF- ⁇ 1 factor signal pathway The failure in TGF- ⁇ 1 factor signal pathway is associated with increased expression of Smad7, an inhibitory protein of the Smad3 phosphorylation process mediated by TGF- ⁇
• Smad7 an inhibitory protein of the Smad3 phosphorylation process mediated by TGF- ⁇
• Smad7 antisense oligonucleotide causes a marked reduction in the synthesis of inflammatory molecules secreted by Th1 cells, which seem to have a key role in the activation of other Inflammatory pathways such as the NF-kB.
• EXAMPLE 2 Role of Smad ⁇ in sustaining the increased activity of NF-kB in chronic intestinal Inflammatory diseases (IBD).
• mucosal samples were taken from 11 CD patients with moderate to severe activity. The primary site of the lesions was the colon in 9 patients, while in 2 patients both the colon and terminal ileum were involved. Eight patients were treated with mesalazine and/or with antibiotics and the remaining 3 with corticosteroids. The indication for surgery was the unresponsiveness to pharmacological treatment. In addition, mucosal samples were taken from 3 patients with UC undergoing colectomy, due to unresponsiveness to medical therapy. As controls, 14 patients undergoing colectomy for colonic cancer were recruited. In each of these 14 patients the mucosal samples were collected from macroscopically and histologically unaffected areas. The approval was obtained by the local ethical committee.
• IBD LPMC were resuspended in RPMI 1640 supplemented with HL-1 and cultured in the presence or absence of a Smad7 antisense oligonucleotide or control (sense) oligonucleotide (both used at a final concentration of 2 ⁇ g/ml) for 24 hours in absence of lipofectamine.
• Smad7 anti-sense and sense oligonucleotide sequences have previously been described (Monteleone et al., 2001).
• phosphorothioate single strand oligonucleotides matching the region 107- 128 (5'-GCTGCGGGGAGAAGGG GCGAC-3 ') of the human Smad7 coding sequence were synthesised in sense and antisense orientation. Since the phosphorylated anti-sense oligonucleotide contains two CpG motifs that could have immunostimulatory activity, the cytosine in dinucleotides was replaced with a 5-methylcytosine. In addition phosphodiester bonds were replaced with methylphosphonates at the ends of the oligonucleotide to increase the stability of molecule.
• IBD LPMC were treated with the antisense oligonucleotide in the presence or absence of a neutralising TGF- ⁇ 1 antibody (5 ⁇ g/ml; R&D Systems, Abingdon, UK). After 24 hours, an aliquot of LPMC was used to extract the proteins for Smad7 analysis. To assess the effect of IkB ⁇ , the cells were washed and resuspended in RPMI 1640 plus HL-1 and cultured in the presence or absence of TGF ⁇ 1 (5 ng/ml) for 7-14 hours and then stimulated with TNF- ⁇ .
• TGF- ⁇ 1 antibody 5 ⁇ g/ml
• TGF ⁇ 1 5 ng/ml
• RNA extraction, preparation of complementary DNA (cDNA) and RT-PCR Total RNA, preparation of cDNA and RT-PCR were carried out as previously described (Monteleone et al., 1997).
• PCR primers were the followings: IL-8, FWD, 5'-TGCAGCTCTGTGTGAAGG-3' (SEQ ID No 4); REV, 5'-ATTGCATCTGGCAACCCTAC-3' (SEQ ID No 5); IkB ⁇ , FWD: 5'-ATCACCMCCAGCCAGAMT-3' (SEQ ID No 6); REV: 5'-GCACCCMGGACACCMAAG-3' (SEQ ID No 7); ⁇ -actin, FWD: 5'-GGC ACC ACA CCT TCT ACA-3' (SEQ ID No 8); REV: 5'-CAG GTC TTT GCG GAT GTC-3' (SEQ IDs No 9).
• RT-PCR products were electrophoresed on 1 % agarose gel containing 0.3 ⁇ g/ml of ethidium bromide.
• Electrophoretic mobility shift assay (EMS A) Nuclear protein-DNA birding studies were carried out for 20 minutes at room temperature in a 20 ml reaction volume containing 10 mM
• Tris-HCI 50 M KCI, 1 mM DTT, 2.5% glycerol, 5mM MgCI 2 , 1 ⁇ g Poly
• dl-dC (dl-dC), (Sigma) 50 fmol biotin labelled oligonucleotides containing probe and 10 ⁇ g of nuclear proteins.
• the DNA probes were prepared by annealing the two consensus oligonucleotides (FWD, 5': -AGTTGAGGGGAGTTTCCCAGG- 3' SEQ ID No 10, REV, 5 , :-CGGACCCTTTCAGGGGAGTTGA-3 , I SEQ ID No 11), which were labelled at 3' end with biotin using a commercially available kit (Pierce, Rockford, IL, U.S.A.).
• a monoclonal antihuman NF-kB/p65 or p50 monoclonal (Saint Cruz Biotechnology, Saint Cruz, CA, USA) or a control anti-isotype IgG antibody (Dako Ltd) (both used at the concentration of 2.5 ⁇ g) were incubated with nuclear proteins for 45 minutes prior to adding the probes.
• a 6% non-denaturing poiyacrylamide gel was used for the electrophoretic separation.
• the labelled oiligonucleotides were detected with a chemiluminescence EMSA kit (Pierce).
• Protein extraction and Western Blot analysis LPMC were homogenised and cytosolic extracts collected in buffer A containing 10 mM Hepes (pH 7,9), 10 mM KCI, 0,1 mM EDTA and 0,2 mM EGTA.
• Nuclear extracts were prepared by dissolution of the remaining nuclei in buffer C containing 20 mM Hepes (pH 7,9), 0,4 M NaCI, 1 mM EDTA, 1 mM EGTA and 10% glycerol.
• Both buffers were supplemented with 1 mM dithiothreitol (DTT), 10 ⁇ g/ml aprotinin, 10 ⁇ g/ml leupeptin and 1 mM phenilmethansulphonyl fluoride (Sigma).
• DTT dithiothreitol
• 10 ⁇ g/ml aprotinin 10 ⁇ g/ml leupeptin
• 1 mM phenilmethansulphonyl fluoride Sigma.
• 10 ⁇ g/sample of nuclear or cytosolic proteins were separated on 10% SDS- PAGE gel.
• p65 was detected by using a rabbit anti human NF-kB/p65 antibody (final dilution 1:500) (Saint Cruz Biotechnology) and subsequently a goat antibody IgG, conjugated to horseradish-peroxidase (DAKO, Cambridgeshire, U.K.) (final dilution 1 :2500).
• the reaction was performed using an ECL kit (Amersham Pharmaceuticals, Amersham, U.K.). In order to ascertain equivalent loading and transfer of proteins, with ponceau 5 (Sigma) staining was performed .
• blots was stripped and analysed for the content of histonel using a mouse anti human-histonel monoclonal antibody (final dilution 1 :300, Saint Cruz Biotechnology) and subsequently a goat anti-mouse antibody goat antibody conjugated to horseradish-peroxidase (dilution 1 :1500, Dako Ltd). IkB ⁇ expression was detected by Western blotting using a rabbit anti human IkB ⁇ antibody (final dilution 1 :500, Saint Cruz Biotechnology). Blots were stripped and reincubated with the antibody for ⁇ -actin as previously specified (Monteleone et al., 2001).
• Smad7 was analysed using a specific rabbit anti human Smad7 antibody (final dilution 1 :400, Saint Cruz Biotechnology, Inc.). Goat anti- rabbit antibody conjugated to horseradish peroxidase (dilution 1:20,000, Dako Ltd) was used for the detection of primary antibody binding and immunoreactivity was visualised with a chemiluminescence kit (Pierce). Plasmid DNA All the plasmid DNA were prepared using a commercial extraction and isolation kit (Maxiprep, Qiagen, UK).. A luciferase reporter vector containing the nucleotides from -332 to +35 of the human IkB- ⁇ gene promoter was provided by Professor Ron Hay (St Andrews, UK).
• the control Renila luciferase vector pRLTK was purchased from Promega (Southampton, UK). Transfection analysis and reporter gene assay Primary foetal gut fibroblasts (Bajaj-Eliot et al., 1998) were transfected using the non-liposomal Effectene protocol (Qiagen) according to the manufacturer's instructions. Luciferase assays were performed using a dual luciferase kit
• firefly luciferase plkB ⁇ -Luc
• IkB ⁇ promoter The expression of firefly luciferase (plkB ⁇ -Luc) under the control of the IkB ⁇ promoter was normalised for differences in expression efficiency, by measuring the activity of a co-transfected Renilla luciferase vector (pRLTK).
• LPMC Stimulation of normal LPMCs with TNF- ⁇ alone results in a marked nuclear translocation of p65 (figure 8, panel A). Preincubation of LPMC with TGF- ⁇ 1 prevents the TNF- ⁇ -induced p65 nuclear translocation, with no change in the content of cytosolic NF-kB (figure 8, panel B). Stimulation of normal LPMC with TNF- ⁇ also increases the NF- kB binding activity, as shown in figure 9.
• IL-8 transcription is induced by NF-kB and IL-8 levels are increased in IBD (Bows et al., 1998). Since it has been established that TNF- ⁇ up regulates IL-8 transcription through a NF-kB-dependent mechanism (Baldwin et al., 1996), the authors of the present invention have determined whether TGF- ⁇ 1 inhibited IL-8 gene expression. As expected LPMCs treated with TNF- ⁇ show a marked increase in IL-8 RNA transcripts, as shown in figure 10, panel A. However the TNF- ⁇ -induced IL-8 transcripts were approximately reduced of 95% by pre-treatment with TGF ⁇ l (figure 10, pane! B).
• TGF- ⁇ 1 inhibits the NF- kB/p65 nuclear translocation and induces IkB ⁇ in normal LPMC.
• NF-kB proteins are maintained as inactive protein complexes in the cytoplasm by the inhibitory proteins lkBs, among which IkB ⁇ represents a prototype.
• Activation of NF-kB requires IkB phosphorylation which is then followed by its ubiquitination and degradation. This allows the free NF-kB dimers to translocate into the nucleus (Baldwin et al. , 1996). Therefore, one potential mechanism by which TGF- ⁇ 1 suppresses NF-kB activation could be related to its ability to prevent p65nuclear translocation.
• IkB ⁇ protein expression was analysed in cytosolic extracts isolated from LPMCs pre-incubated with TGF- ⁇ 1 for 7 hours and then treated with TNF- ⁇ for various times.
• un-treated LPMC the cytoplasmic IkB ⁇ signal disappears almost completely after 20 minutes of treatment with TNF- ⁇ and returns to the original level after 40 minutes of stimulation (figure 1 , pane! A).
• Pre-treatment of LPMC with TGF ⁇ l for 7 hours results in an increased IkB ⁇ expression, so that the following stimulation with TNF ⁇ does not decrease IkB ⁇ , (figure 11 , panel A.
• TGF- ⁇ 1 The key change in LPMCs pre-treated with TGF- ⁇ 1 (compared to cells treated with medium) prior to TNF- ⁇ stimulation was the substantial increase in IkB ⁇ / ⁇ -actin ratio at all time points after treatment (figure 11 , panel B).
• cytosolic extracts were prepared from normal LPMC treated for various time points and analysed by Western blotting.
• TGF- ⁇ 1 treatment results in an increased expression of IkB ⁇ protein for up to 28 hours, as can be observed in the panel C of figure 11.
• TGF- ⁇ 1 also increases IkB ⁇ transcripts in normal LPMCs, as shown in the panel D of figure 11.
• TGF- ⁇ 1 increases the transcriptional activity by approximately about 2,5 and 3,5-folds after 2 and 4 hours, as shown in figure 12. It has also been shown that IBD LPMC have high NF-kB/ ⁇ 65 nuclear accumulation and low IkB ⁇ cytosolic content.
• LPMC show an enhanced NF- kB/p65 activation (Neurath et al., 1998, Schreiber et al., 1998, Neurath et al., 1996)
• the nuclear proteins were isolated both from normal and IBD LPMC and analysed by Western blotting.
• a strong immunoreactivity for NF-kB/p65 was seen in nuclear extracts of IBD LPMCs in comparison to control samples (figure 13, panel A).
• the analysis of the p65/histone-1 ratio shows a mean value of 0,91 densitometric arbitrary units (range 0.54- 1.23) in patients with IBD and 0.26 in controls (range 0.08-0.4) (p ⁇ 0.02) (figure 13).
• TGF ⁇ l does not reduce the DNA binding activity in extracts of IBD LPMC, as shown in figure 14, panel B. It has been observed that Smad7 inhibition in IBD LPMC allows TGF- ⁇ 1 to down regulate NF-kB activation. In both CD and UC LPMC, there is a reduced activity of TGF ⁇ l -mediated signal cascade due to high Smad7 expression levels (Monteleone et al., 2001 ). Thus the sustained activation of NF-kB documented in IBD LPMCs could result from a defective TGF- ⁇ 1 -mediated signal transmission.
• IBD LPMCs were treated with Smad7 sense and antisense oligonucleotides and then analysed for both nuclear NF-kB/p65 and cytosolic IkB ⁇ .
• the treatment with Smad7 antisense inhibits the Smad7 expression, as shown in figure 15, panel A, clearly confirming the efficiency of oligonucleotides transfection.
• Both untreated or treated with Smad7 sense oligonucleotide LPMC show high levels of nuclear p65 and low cytosolic levels of IkB ⁇ , as shown in figure 15, panels B and C.
• IBD LPMC treated with Smad7 antisense oligonucleotide show a markedly decrease in NF-KB binding activity.
• stimulation with TNF- ⁇ results in a high NF-kB binding which was prevented by incubation with TGF- ⁇ 1.
• the authors of the present invention show that TGF- ⁇ 1 is a potent negative regulator of the transcription factor NF-kB in the human intestine.
• TGF- ⁇ 1 results in the inhibition of TNF ⁇ - induced nuclear translocation of p65 and this was associated with TGF- ⁇ 1- mediated induction of elevated IkB ⁇ levels.
• TGF ⁇ l was unable to inhibit NF-kB activation in LPMCs from patients with IBD.

Applications Claiming Priority (2)

Publications (1)

Family

ID=30131545

Family Applications (1)

Country Status (2)

Cited By (6)

Citations (1)

• 2003
  • 2003-08-11 IT IT000393A patent/ITRM20030393A1/it unknown
• 2004
  • 2004-08-06 WO PCT/IT2004/000451 patent/WO2005014011A1/en active Application Filing

Patent Citations (1)

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events