WO2009105629A2 - Méthodes de traitement et prévention de l'entérocolite nécrosante - Google Patents

Méthodes de traitement et prévention de l'entérocolite nécrosante Download PDF

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WO2009105629A2
WO2009105629A2 PCT/US2009/034660 US2009034660W WO2009105629A2 WO 2009105629 A2 WO2009105629 A2 WO 2009105629A2 US 2009034660 W US2009034660 W US 2009034660W WO 2009105629 A2 WO2009105629 A2 WO 2009105629A2
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tgf
derivative
intestinal
ccl
bacterial
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WO2009105629A3 (fr
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Akhil Maheshawari
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UAB Research Foundation
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UAB Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present disclosure relates to the treatment and prevention of necrotizing enterocolitis (NEC) and related disease states and conditions.
  • the disclosure also relates to the treatment and prevention of neonatal NEC and related disease states and conditions.
  • the disclosure further relates to pharmaceutical compositions and medicaments comprising Transforming Growth Factor (TGF)-/3 for use in the methods of treatment and prevention disclosed herein.
  • TGF Transforming Growth Factor
  • NEC BACKGROUND NEC is an acquired gastrointestinal disease affecting 5-15% of neonates born weighing less than 1500 grams and is a leading cause of morbidity and mortality in these patients. Histopathologically, NEC is characterized by a severe inflammatory response, ischemic changes, and necrosis. Although the etiology of NEC is not fully established, the disease is associated in premature infants with intestinal ischemia, formula feeding, and abnormal bacterial colonization.
  • NEC is seen almost exclusively in premature infants born in the late 2 nd /early 3 rd trimester of pregnancy, NEC may occur in the premature neonate as the normal mucosal mechanisms of tolerance to bacterial products are developmentally regulated and deficient in the preterm intestine.
  • the art is currently lacking method for the treatment and prevention of NEC and related disease states and conditions. Due to the severity of NEC and related disease states and conditions and the mortality and morbidity associated with the foregoing, especially in neonates, new methods of treatment and prevention are needed.
  • the present disclosure provides methods for the treatment and prevention of NEC and related disease states and conditions as well as pharmaceutical compositions and medicaments for use in such methods.
  • FIG. IA demonstrates the severe inflammatory response, ischemic changes, and necrosis often associated with NEC.
  • FIG. IA (upper panel) shows intra-operative gross morphological appearance of NEC showing an area of infarction.
  • FIG. IA shows an eosin-hematoxylin photomicrograph showing inflammation (thin arrows). Thick arrows indicate intramural air, which represents trapped gas bubbles from bacterial fermentation and is a pathognomonic finding of NEC.
  • FIG. 1 B shows PCR microarray indicating upregulation of pro-inflammatory cytokines and transcriptional regulators in NEC.
  • FIG. 1C is data from last 10 years in a neonatal intensive care unit showing that NEC affects extremely preterm infants born during the late 2 nd trimester and the early 3 rd trimester of pregnancy
  • FIG. 2A shows immunohistochemical data demonstrating that fetal intestinal macrophages express TNF- ⁇ and CD 14, unlike macrophages in the adult intestine:
  • Photomicrographs of small intestinal tissue sections (magnification 10Ox) from (i) 20- wk human fetus (ii) 29-wk premature neonate (iii) full-term neonate and (iv) adult show DAB (brown) staining for HAM56, a pan-macrophage marker.
  • insets on the right show higher- magnification (100Ox) immunofluorescence photomicrographs show immunoreactivity for HAM56 (green) and TNF- ⁇ (red).
  • FIG. 2B shows that murine fetal intestinal macrophages respond to LPS to produce TNF- ⁇ in vitro.
  • Intestinal macrophages from the El 5 and El 8 murine fetus unlike those from the term neonate (Dl postnatal) and adult mouse, respond to LPS in vitro and produce TNF-o; (means ⁇ SEM).
  • FIG. 3 A shows fetal tissue-conditioned media (T-CM) suppression of macrophage cytokine production improves with gestational maturation, but remains significantly lower than adult.
  • FIG. 3B T-CM suppression of neutrophil chemotactic activity in LPS-stimulated macrophage cultures improves with maturation. Neutrophil chemotactic activity of supernatants from macrophage cultures was measured using a standard fluorescence-based microchemotaxis assay; data are depicted as means ⁇ SEM of the number of neutrophils migrating towards test samples through a polycarbonate filter.
  • T-CMs derived from 3-5 fetuses in each fetal group and 3 adult tissue samples T-CM suppression of LPS-induced NF- ⁇ B activation in macrophages improves with maturation. Unlike T-CMs prepared from adult intestinal tissue, fetal T-CMs did not block LPS-induced NF-/cB activation in macrophages.
  • the bar diagram (means ⁇ SEM) shows the ratio of phosphorylated: total NF-/cB p65 (rel A).
  • Data are representative of 3 independent experiments, each performed with T-CMs derived from 3-5 fetuses in each fetal group and 3 adult tissue samples
  • FIG. 4A shows the effect of LPS stimulation on monocyte derived macrophage expression of 23 pro-inflammatory cytokines and chemokines.
  • Bar diagram shows data from a quantitative PCR microarray. Data were normalized against GAPDH and depicted as fold-change above unstimulated macrophages. Data are representative of 3 independent experiments, each performed with T-CMs derved from 3-5 fetuses in each fetal group.
  • FIG. 4B shows the effect of LPS stimulation on monocyte derived macrophage expression of 23 pro-inflammatory cytokines and chemokines after pre-treatment with TCM derived from 10-14 week fetal intestine.
  • Bar diagram shows data from a quantitative PCR microarray. Data were normalized against GAPDH and depicted as fold- change above unstimulated macrophages. Data are representative of 3 independent experiments, each performed with T- CMs derved from 3-5 fetuses in each fetal group.
  • FIG. 4C shows the effect of LPS stimulation on monocyte derived macrophage expression of 23 pro-inflammatory cytokines and chemokines after pre-treatment with TCM derived from 20-24 week fetal intestine.
  • Bar diagram shows data from a quantitative PCR microarray. Data were normalized against GAPDH and depicted as fold-change above unstimulated macrophages. Data are representative of 3 independent experiments, each performed with T- CMs derved from 3-5 fetuses in each fetal group.
  • FIG. 4D shows the effect of LPS stimulation on monocyte derived macrophage expression of 23 pro-inflammatory cytokines and chemokines after pre-treatment with TCM derived from adult intestine. Bar diagram shows data from a quantitative PCR microarray. Data were normalized against GAPDH and depicted as fold-change above unstimulated macrophages. Data are representative of 3 independent experiments, each performed with T-CMs derved from 3-5 fetuses in each fetal
  • FIG. 5(A) shows TGF- ⁇ bioactivity increases with maturation.
  • TGF-/3 bioactivity was quantified using a luciferase assay to measure TGF- ⁇ -mediated activation of the platelet activator inhibitor- 1 promoter in mink lung epithelial cells.
  • Data are representative of 3 independent experiments, each performed with T-CMs derived from 3 fetuses in each fetal group and 3 adult tissue samples.
  • FIG. 5(B) shows T-CM activation of smad signaling in macrophages increases with maturation.
  • Treatment of monocyte-derived macrophages with fetal and adult T-CMs induced the phosphorylation of smad2, a key mediator of the TGF-j3-activated signaling pathway.
  • Bar diagram shows densitometric analysis of the blots (means ⁇ SEM). Data are representative of 3 independent experiments, each performed with a distinct set of T-CMs and utilized three different monocyte donors.
  • FIG. 5(C) shows T-CM suppression of LPS-induced cytokine production in macrophages was reversed upon neutralization of TGF-/3 in the conditioned media.
  • Bar diagram shows that T- CMs prepared from human fetal intestinal tissue of different gestational ages suppressed LPS- induced TNF- ⁇ (means ⁇ SEM) production in macrophages in a maturation dependent manner. In some wells, excess neutralizing anti-TGF-/3 (or isotype control) antibody was added. The gestational age-related suppression of cytokine production (grey bars) was reversed by anti- TGF-(S antibody (hatched bars). Data are representative of 3 independent experiments, each performed with a distinct set of T-CMs.
  • FIG. 5(D) shows the amino acid sequence of human TGF-/S 2 , isoform 1 (SEQ ID NO: 1) and isoform 2 (SEQ ID NO: 2).
  • FIG. 6(A) shows mRNA expression of TGF-(S 2 , but not of TGF-(S 1 or TGF-/3 3 , increases with intestinal maturation. Data are normalized against GAPDH and depicted as fold-change above 10-14 wk fetal intestine (means ⁇ SEM). Data are representative of 3 independent experiments, each performed with T-CMs derived from 3 subjects in each group.
  • FIG. 6(B) shows TGF-(S 2 immunoreactivity increases with intestinal maturation.
  • TGF-(S 2 immunoreactivity green; open arrow) becomes progressively more prominent in the intestinal epithelium with maturation.
  • Insets with the 12-wk intestine show high-magnification photomicrographs (100Ox) of the villus and inter- villus area (V & IVA) and the muscularis externa (ME).
  • the insets top to bottom
  • Data are representative of 3 different fetuses or adults in each group.
  • the bottom right panel shows that TGF-/? 2 immunoreactivity in lamina limba cells co-localizes with ⁇ -smooth muscle actin ( ⁇ -SMA; arrows), indicating that cells expressing TGF-(S 2 are of the myofibroblast lineage.
  • FIG. 6(C) shows concentrations of total and active fractions of TGF- ⁇ 2 increased with maturation. All measurements were performed by ELISA and are shown as means ⁇ SEM. Data are representative of 3 independent experiments, each performed with T-CMs derived from 3-5 fetuses in each fetal group and 3 adult tissue samples.
  • FIG. 6(D) shows TGF-(S 2 is the most important of the three TGF-(S isoforms in T-CM downregulation of LPS-induced macrophage cytokine production: Two of the three TGF-(S isoforms were immunoprecipitated in separate T-CM aliquots to obtain T-CM derivatives containing only one of the three TGF - ⁇ isoforms.
  • T-CM derivatives containing TGF-(S 2 were most effective in suppressing TNF- ⁇ production.
  • Data are representative of 3 independent experiments, each performed with T-CMs derived from 3 subjects in each group.
  • the inset shows recombinant TGF-(S 2 is the most potent of the three isoforms in suppressing LPS-induced TNF-o; production in macrophages.
  • Monocyte-derived macrophages were treated with 0-2000 pg/mL of the three TGF-(S isoforms 2 hrs prior to LPS stimulation. Data are representative of 3 independent experiments.
  • FIG. 7(A) shows NEC is associated with decreased TGF-/3 2 expression.
  • TGF-/3 2 concentrations in intestinal tissue resected surgically for NEC are lower than the mid-gestation fetal and preterm neonatal intestine.
  • Data show measurements by ELISA (means ⁇ SEM).
  • TGF-/3 2 mRNA expression (top left) and TGF-/3 bioactivity (top right) in the normal fetal, preterm neonatal intestine and NEC-affected intestinal tissue are shown.
  • Data are representative of 3 independent experiments, each performed with T-CMs derived from 5 tissue samples of NEC (each from a different patient) and 5 fetal intestinal tissue samples (different fetuses).
  • FIG. 8(A) shows induction of NEC-like intestinal injury by intraperitoneal administration of PAF and LPS in 12-day-old wild type and transgenic DNIIR mice that express a defective, dominant negative TGF-/3 RII when supplemented with zinc.
  • Three days of zinc supplementation resulted in about 50% expression of the transgene and a partial inhibition of TGF-/3 signaling, whereas 7 days of zinc supplementation completely abrogated TGF-/3 signaling.
  • Mice were sacrificed 2 hrs after PAF and LPS administration and mucosal injury was graded on a 4-point scale.
  • Bar diagram (means ⁇ SEM) shows the severity of mucosal injury in wild type controls, wild type mice after PAF-LPS administration, DNIIR mice after 3 days of zinc supplementation and PAF-LPS, DNIIR mice after 7 days of zinc supplementation and PAF-LPS, and finally, DNIIR mice which received 3 days of zinc supplementation and then 100 ng TGF-(S 2 by gavage 2 hrs prior to PAF-LPS.
  • PAF-LPS- induced mucosal injury was more severe in the DNIIR mice, whereas enteral TGF-(S 2 protected DNIIR mice against NEC-like mucosal injury.
  • FIG. 8B shows TGF-(S 2 is expressed in human milk in biologically relevant concentrations but was not detected in infant formula.
  • High-magnification photomicrographs (100Ox) show immunoreactivity on the epithelium (solid arrow) and cells in the lamina intestinal (open arrow).
  • FIG. 9 shows deficient TGF-
  • epithelial and stromal cell-derived TGF-(S downregulates the inflammatory responses of intestinal macrophages to low levels.
  • the present disclosure shows that the down regulation of inflammatory responses in the intestinal mucosa is deficient in the fetal intestinal cells because of a developmental deficiency of TGF-/3 2 .
  • the present disclosure also shows that TGF-/3 2 expression was reduced in tissue samples of NEC to levels lower than expected for the gestational age.
  • transgenic mice deficient in TGF-/3 2 signaling showed significantly more severe mucosal injury after administration of PAF and LPS than mice with normal TGF-/3 2 signaling.
  • enteral administration of recombinant TGF - ⁇ 2 prior to the administration of PAF and LPS protected against mucosal injury in this mouse model.
  • the present disclosure shows that down regulation of inflammatory responses in intestinal cells, such as but not limited to, macrophages, is an effect of TGF-/3 2 , and that enteral administration of TGF-/3 2 and/or TGF- ⁇ 2 derivatives treats and/or prevents NEC-like mucosal injury by correcting the developmental deficiency of gut mucosal tolerance to bacteria and/or bacterial antigens and/or products. Therefore, in one embodiment the present disclosure relates to methods for treating and/or preventing NEC in a subject in need of such treatment/prevention.
  • the present disclosure relates to methods for treating and/or preventing a disease state or condition characterized by, at least in part, un-regulated or improperly regulated intestinal cytokine release in a subject in need of such treatment/prevention.
  • the disease states and conditions include, but are not limited to, sepsis and chronic infections of the bowel.
  • the present disclosure relates to methods for treating a subject having intestinal mucosal damage or preventing such intestinal mucosal damage in a subject in need of such treatment/prevention.
  • the intestinal mucosal damage is caused by, at least in part, NEC or a disease state or condition characterized by, at least in part, un-regulated or improperly regulated intestinal cytokine release.
  • the disease states and conditions include, but are not limited to, sepsis and chronic infections of the bowel.
  • the present disclosure relates to methods for alteration of the cytokine response of immature intestinal cells, such, as but not limited to, immature intestinal epithelial cells, to mucosal injury, bacterial and/or bacterial antigens and/or products so that the cytokine response is matured.
  • a matured cytokine response include a decrease in at least one of the following factors: chemokines having both the C-C and CXC motifs (such as but not limited to CXCL-I, CXCL-5, CXCL-8, CCL-2, CCL-3, CCL-4 and CCL-5), interleukins (such as but not limited to IL-104 IL-I 1 S, IL-1F5, IL-1F7, IL-
  • IL-1F9 IL-6, IL-8, IL-12o
  • IL-12/3 IL-17 ⁇ , IL-17/3, IL-18 ⁇ , IL-18/3 and IL-23 ⁇
  • other cytokines such as but not limited to GM-CSF and TNF ⁇ .
  • compositions and medicaments containing TGF-/3 2 and/or TGF-/3 2 derivatives for use in the disclosed methods.
  • prevent refers to administering a compound prior to the onset of clinical symptoms of a disease state/condition so as to prevent any symptom, aspect or characteristic of the disease state/condition. Such preventing and suppressing need not be absolute to be useful.
  • Te terms "treat”, “treating” and “treatment” as used herein refers to administering a compound after the onset of clinical symptoms of a disease state/condition so as to reduce or eliminate any symptom, aspect or characteristic of the disease state/condition. Such treating need not be absolute to be useful.
  • in need of treatment refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, and may include the knowledge that the patient is ill as the result of a disease state/condition that is treatable by a compound, pharmaceutical composition or medicament of the disclosure.
  • in need of prevention refers to a judgment made by a caregiver that a patient requires or will benefit from prevention. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, and may include the knowledge that the patient may become ill as the result of a disease state/condition that is treatable by a compound, pharmaceutical composition or medicament of the disclosure.
  • the term "individual”, “subject” or “patient” as used herein refers to any animal, including mammals, such as, but not limited to, mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, or humans.
  • mammals such as, but not limited to, mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, or humans.
  • the term may specify male or female or both, or exclude male or female.
  • terapéuticaally effective amount in reference to the treating, preventing or suppressing of a disease state/condition, refers to an amount of a compound either alone or as contained in a pharmaceutical composition or medicament that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of the disease state/condition. Such effect need not be absolute to be beneficial.
  • salts are meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
  • inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phospho
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S. M., et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19).
  • Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
  • the term "prodrug” is meant to include functional derivatives of the compounds disclosed which are readily convertible in vivo into the required compound.
  • administering shall encompass the treatment of the various disease states/conditions described with the compound specifically disclosed or with a prodrug which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient.
  • Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985. Methods of Treatment and Prevention
  • the present disclosure describes the use of TGF-/3 2 and/or TGF-(S 2 derivatives to prevent or treat NEC and/or a disease state or condition characterized by, at least in part, unregulated or improperly regulated intestinal cytokine release in a subject in need of such treatment/prevention.
  • the disease states and conditions include, but are not limited to, sepsis and chronic infections of the bowel.
  • the teachings of the present disclosure provides for treating and/or preventing NEC in a subject in need of such treatment/prevention.
  • the method of treatment/prevention comprises the steps of identifying a subject in need of such treatment/prevention and administering to said subject TGF-(S 2 and/or a TGF-(S 2 derivative.
  • the TGF-(S 2 and/or a TGF-/3 2 derivative is administered in a therapeutically effective amount.
  • the TGF-/3 2 and/or a TGF-(S 2 derivative may be administered by itself or as a part of a pharmaceutical composition or medicament.
  • 3 2 and/or a TGF-(S 2 derivative inhibits, at least in part, a cytokine response in the intestine to mucosal injury, bacterial invasion, bacterial products, bacterial antigens or combinations of the foregoing.
  • administration of TGF- j8 2 and/or a TGF-/3 2 derivative alters the cytokine response of immature intestinal cells, such, as but not limited to, immature intestinal epithelial cells and/or macrophages, to mucosal injury, bacterial and/or bacterial antigens and/or products so that the cytokine response is matured.
  • a matured cytokine response include a decrease in at least one of the following factors: chemokines having both the C-C and CXC motifs (such as but not limited to CXCL-I, CXCL-5, CXCL-8, CCL-2, CCL-3, CCL-4 and CCL-5), interleukins (such as but not limited to IL-Io; IL- 1/3, IL-1F5, IL-1F7, IL-1F8, IL-1F9, IL-6, IL-8, IL-12o ⁇ IL- 120, IL- 17a; IL- 17ft IL-18a, IL- 18(8 and IL-23 ⁇ ), and other cytokines (such as but not limited to GM- CSF and TNFo;).
  • the subject is a premature infant.
  • the subject is a premature infant having a gestational age of 32 weeks or less.
  • the administration is enteral administration.
  • the teachings of the present disclosure provides for treating and/or preventing a disease state or condition characterized by, at least in part, un-regulated or improperly regulated intestinal cytokine release in a subject in need of such treatment/prevention.
  • the disease states and conditions include, but are not limited to, sepsis and chronic infections of the bowel.
  • the method of treatment/prevention comprises the steps of identifying a subject in need of such treatment/prevention and administering to said subject TGF-/3 2 and/or a TGF-(S 2 derivative.
  • the TGF-(S 2 and/or a TGF-/3 2 derivative is administered in a therapeutically effective amount.
  • the TGF-(S 2 and/or a TGF-j8 2 derivative may be administered by itself or as a part of a pharmaceutical composition or medicament.
  • the administration of TGF-(S 2 and/or a TGF-(S 2 derivative inhibits, at least in part, a cytokine response in the intestine to mucosal injury, bacterial invasion, bacterial products, bacterial antigens or combinations of the foregoing.
  • administration of TGF-(S 2 and/or a TGF -/3 2 derivative alters the cytokine response of immature intestinal cells, such, as but not limited to, immature intestinal epithelial cells and macrophages, to mucosal injury, bacterial and/or bacterial antigens and/or products so that the cytokine response is matured.
  • immature intestinal cells such as but not limited to, immature intestinal epithelial cells and macrophages
  • a matured cytokine response include a decrease in at least one of the following factors: chemokines having both the C-C and CXC motifs (such as but not limited to CXCL-I, CXCL-5, CXCL-8, CCL-2, CCL-3, CCL-4 and CCL-5), interleukins (such as but not limited to IL-l ⁇ , IL- lft IL-1F5, IL-1F7, IL- 1F8, IL-1F9, IL-6, IL-8, IL-12a, IL-12ft IL-17 ⁇ , IL-H 1 S, IL-18a, IL-18/3 and IL-23 ⁇ ), and other cytokines (such as but not limited to GM-CSF and TNFo;).
  • chemokines having both the C-C and CXC motifs such as but not limited to CXCL-I, CXCL-5, CXCL-8, CCL-2, CCL-3, CCL-4 and CCL-5
  • the subject is a premature infant. In still a further embodiment, the subject is a premature infant having a gestational age of 32 weeks or less.
  • the administration is enteral administration.
  • the teachings of the present disclosure provides for treating a subject having intestinal mucosal damage or preventing such intestinal mucosal damage in a subject in need of such treatment/prevention.
  • the intestinal mucosal damage is caused by, at least in part, NEC or a disease state or condition characterized by, at least in part, un-regulated or improperly regulated intestinal cytokine release.
  • the disease states and conditions include, but are not limited to, sepsis and chronic infections of the bowel.
  • the method of treatment/prevention comprises the steps of identifying a subject in need of such treatment/prevention and administering to said subject TGF-(S 2 and/or a TGF -/3 2 derivative.
  • the TGF-(S 2 and/or a TGF-(S 2 derivative is administered in a therapeutically effective amount.
  • the TGF -/3 2 and/or a TGF-/3 2 derivative may be administered by itself or as a part of a pharmaceutical composition or medicament.
  • the administration of TGF-(S 2 and/or a TGF-(S 2 derivative inhibits, at least in part, a cytokine response in the intestine to mucosal injury, bacterial and/or bacterial antigens.
  • administration of TGF-/3 2 and/or a TGF-(S 2 derivative alters the cytokine response of immature intestinal cells, such, as but not limited to, immature intestinal epithelial cells, to mucosal injury, bacterial invasion, bacterial products, bacterial antigens or combinations of the foregoing so that the cytokine response is matured.
  • a matured cytokine response include a decrease in at least one of the following factors: chemokines having both the C-C and CXC motifs (such as but not limited to CXCL-I, CXCL-5, CXCL-8, CCL-2, CCL-3, CCL-4 and CCL-5), interleukins (such as but not limited to IL- lot, IL- 1/3, IL- 1F5, IL-1F7, IL-1F8, IL-1F9, IL-6, IL-8, IL-12 ⁇ , IL-12/3, IL-17 ⁇ , IL-17/3, IL-18 ⁇ , IL-18/3 and IL-23 ⁇ ), and other cytokines (such as but not limited to GM-CSF and TNF ⁇ ).
  • the subject is a premature infant.
  • the subject is a premature infant having a gestational age of 32 weeks or less.
  • the administration is enteral administration.
  • teachings of the present disclosure provide for alteration of the cytokine response of immature intestinal cells, such, as but not limited to, immature intestinal epithelial cells, to mucosal injury, bacterial invasion, bacterial products, bacterial antigens or combinations of the foregoing so that the cytokine response is matured.
  • a matured cytokine response include a decrease in at least one of the following factors: chemokines having both the C-C and CXC motifs (such as but not limited to CXCL-I, CXCL-5, CXCL-8, CCL-2, CCL-3, CCL-4 and CCL-5), interleukins (such as but not limited to IL-Io; IL-1/3, IL-1F5, IL-1F7, IL-1F8, IL-1F9, IL-6, IL-8, IL-12 ⁇ , IL-12/3, IL- 17a, IL- 17/3, IL-18a, IL-18/3 and IL-23 ⁇ ), and other cytokines (such as but not limited to GM- CSF and TNF ⁇ ).
  • chemokines having both the C-C and CXC motifs such as but not limited to CXCL-I, CXCL-5, CXCL-8, CCL-2, CCL-3, CCL-4 and CCL-5
  • interleukins such as but
  • the method comprises the steps of identifying a subject in need of such alteration and administering to said subject TGF-/3 2 and/or a TGF-/3 2 derivative.
  • the TGF-(S 2 and/or a TGF-(S 2 derivative is administered in a therapeutically effective amount.
  • the TGF-(S 2 and/or a TGF-(S 2 derivative may be administered by itself or as a part of a pharmaceutical composition or medicament.
  • the administration of TGF-/3 2 and/or a TGF-/3 2 derivative inhibits, at least in part, a cytokine response in the intestine to mucosal injury, bacterial and/or bacterial antigens.
  • the patient in need of such alternation is a subject suffering from at risk for NEC a disease state or condition characterized by, at least in part, un-regulated or improperly regulated intestinal cytokine release, such as but not limited to, sepsis and chronic infections of the bowel.
  • the subject is a premature infant.
  • the subject is a premature infant having a gestational age of 32 weeks or less.
  • the administration is enteral administration. Creation and Selection of TGF-fo and TGF-fo Derivatives The peptide structures of the three members of the TGF-/3 family are highly similar.
  • TGF-/31 contains 390 amino acids and TGF- /32 and TGF-/33 each contain 412 amino acids, although a second isoform of TGF -/32 exists comprising 442 amino acids.
  • TGF-/32 contains 412 amino acids, although a second isoform of TGF -/32 exists comprising 442 amino acids.
  • They each have an N-terminal signal peptide of 19-30 amino acids that they require for secretion from a cell, a propeptide region (called latency associated peptide or LAP), and a 112-114 amino acid C-terminal region that becomes the mature TGF-/3 molecule following its release from the pro-region by proteolytic cleavage.
  • LAP latency associated peptide
  • the mature TGF-/3 protein dimerizes to produce a 25 KDa active molecule with many conserved structural motifs.
  • TGF-/3 has nine cysteine residues that are conserved among its family; eight form disulfide bonds within the molecule to create a cysteine knot structure characteristic of the TGF-/3 superfamily while the ninth cysteine forms a bond with the ninth cysteine of another TGF-/3 molecule to produce the dimer.
  • Other conserved residues in TGF-/3 are thought to form secondary structure through hydrophobic interactions.
  • the region between the fifth and sixth conserved cysteines houses the most divergent area of TGF-/3 molecules that is exposed at the surface of the molecule and is implicated in receptor binding and specificity of TGF -/3.
  • the present disclosure contemplates the use of TGF-/32 and TGF-/32 derivatives in the methods of treatment and prevention disclosed herein.
  • TGF-/3 2 includes isoforms 1 (SEQ ID NO: 1) and isoform 2 (SEQ ID NO: 2), as well as naturally occurring variants of the foregoing; furthermore, the term also includes the full length polypeptide, the polypeptide with the signal sequence (amino acids 1-19 for isoform 1) removed and the polypeptide with the signal sequence and pro-peptide (amino acids 1-302 for isoform 1) removed.
  • the TGF-/3 2 may be provided as a homodimer or as a heterodimer with other TGF-/3 family members (such as TGF-jSi or TGF-/3 3 ). It is noted that the carboxy terminal regions of isoform 1 and 2 of TGF-/32 are identical in amino acid content.
  • TGF-j3 2 derivative refers to a TGF-/32 polypeptide having one or more insertions, deletions or substitutions.
  • an active fragment of TGF-/32 is used; in a specific embodiment, the active fragment of TGF-/32 contains all 9 conserved cysteine residues.
  • one or more or all of the 9 conserved cysteine residues are maintained; in an alternate embodiment, amino acids 72, 140 and 241 are maintained.
  • the TGF-/32 derivative may have an activity that is comparable to or increased (in one embodiment, 50% or more) as compared to the wild-type TGF-/32 polypeptide activity and as such may be used to increase a TGF-/32 activity; alternatively, the TGF-/32 derivative may have an activity that is decreased (in one embodiment, less than 50%) as compared to the wild-type TGF-/32 activity and as such may be used to decrease a TGF-/32 activity.
  • deletions, additions and substitutions can be selected to generate a desired TGF-/32 derivative. For example, it is not expected that deletions, additions and substitutions in the non-conserved areas of TGF-/32 would alter TGF-/32 activity. Likewise conservative substitutions or substitutions of amino acids with similar properties is expected to be tolerated.
  • TGF-/32 derivatives having functional and chemical characteristics similar to those of naturally occurring TGF-/32.
  • a "conservative amino acid substitution” may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
  • any native residue in the polypeptide may also be substituted with alanine.
  • amino acid substitutions also encompass non-naturally occurring amino acid residues which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties. It will be appreciated by those of skill in the art that nucleic acid and polypeptide molecules described herein may be chemically synthesized as well as produced by recombinant means.
  • Naturally occurring residues may be divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, VaI, Leu, He; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, GIn; 3) acidic: Asp, GIu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: GIy, Pro; and 6) aromatic: Trp, Tyr, Phe.
  • non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.
  • Such substituted residues may be introduced into regions of the TGF-/32 derivatives that are homologous with non-human TGF- ⁇ 2 orthologs, or into the non-homologous regions of the molecule.
  • the hydropathic index of amino acids may be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte et al., J. MoI. Biol., 157:105-131, 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within +/- 2 may be used; in an alternate embodiment, the hydropathic indices are with +/- 1; in yet another alternate embodiment, the hydropathic indices are within +/- 0.5.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-
  • hydrophilicity values are within +/- 2
  • the hydrophilicity values are with +/- 1
  • the hydrophilicity values are within +/- 0.5.
  • Desired amino acid substitutions can be determined by those skilled in the art at the time such substitutions are desired.
  • amino acid substitutions can be used to identify important residues of TGF-/32, or to increase or decrease the affinity of TGF-/32 with a particular binding target in order to increase or decrease TGF-/32 activity.
  • a skilled artisan will be able to determine suitable derivaitves of the polypeptide as set forth in any of SEQ ID NOS: 1-2, including combinations thereof, using well known techniques. For identifying suitable areas of the molecule that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of TGF -/32 to such similar polypeptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides.
  • One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of that information, one skilled in the art may predict the alignment of amino acid residues of TGF-/32 with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test TGF-/32 derivatives containing a single amino acid substitution at each desired amino acid residue. The derivatives can then be screened using activity assays know to those skilled in the art and as disclosed herein. Such derivatives could be used to gather information about suitable substitution.
  • PDB protein structural data base
  • Additional methods of predicting secondary structure include “threading” (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87, 1997; Suppl et al., Structure, 4(l):15-9, 1996), “profile analysis” (Bowie et al., Science, 253:164-170, 1991; Gribskov et al., Meth. Enzym., 183:146- 159, 1990; and Gribskov et al., Proc. Nat. Acad. ScL, 84(13): 4355 Pharmaceutical Compositions and Modes of Administration
  • the present disclosure provides for a pharmaceutical composition and medicaments comprising, consisting of or consisting essentially of TGF-/3 2 and/or a TGF-/3 2 derivative for use in the methods described herein.
  • the TGF-/? 2 and/or a TGF- ⁇ 2 derivative is present in a therapeutically effective amount.
  • Such pharmaceutical compositions may be used in the manufacture of a medicament for use in the methods of treatment and prevention described herein.
  • pharmaceutical compositions may be used in the manufacture of a medicament for treating and/or preventing NEC or a disease or condition characterized, at least in part, by an un-regulated or improperly regulated cytokine response to mucosal injury, bacterial and/or bacterial antigens and/or products.
  • unregulated or improperly regulated cytokine response such as, but not limited to, the inappropriate cytokine response of immature intestinal cells, such, as but not limited to, immature intestinal epithelial cells, to mucosal injury, bacteria and/or bacterial antigens and/or products.
  • This inappropriate cytokine response may be caused by the lack of normal developmental cues that would normally regulate such cytokine response in mature intestinal epithelial cells.
  • the disease or condition is NEC.
  • the disclosed pharmaceutical compositions and medicaments may also comprise a pharmaceutically acceptable carrier.
  • the compounds of the disclosure are useful in both free form and in the form of pharmaceutically acceptable salts.
  • compositions described herein including, but not limited to, vehicles, adjuvants, excipients, or diluents, are well-known to those who are skilled in the art.
  • the pharmaceutically acceptable carrier is chemically inert to the active compounds and has no detrimental side effects or toxicity under the conditions of use.
  • the pharmaceutically acceptable carriers can include polymers and polymer matrices.
  • the compounds described in the instant disclosure can be administered by any conventional method available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in combination with additional therapeutic agents.
  • the compounds described are administered in therapeutically effective amount.
  • the therapeutically effective amount of the compound and the dosage of the pharmaceutical composition administered will, of course, vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration, the age, health and weight of the recipient; the severity and stage of the disease state or condition; the kind of concurrent treatment; the frequency of treatment; and the effect desired.
  • a daily dosage of active ingredient can be expected to be about 0.1 to 15 micrograms ( ⁇ g) per kilogram (kg) of body weight. In one embodiment, the total amount is between about
  • 0.1 ⁇ g/kg and about 1 ⁇ g/kg of body weight in an alternate embodiment between about 1 ⁇ g/kg and about 5 ⁇ g/kg of body weight; in yet another alternate embodiment between 5 ⁇ g/kg and about 15 ⁇ g/kg of body weight.
  • the above described amounts may be administered as a series of smaller doses over a period of time if desired. As would be obvious, the dosage of active ingredient may be given other than daily if desired.
  • the total amount of the compound administered will also be determined by the route, timing and frequency of administration as well as the existence, nature, and extent of any adverse side effects that might accompany the administration of the compound and the desired physiological effect. It will be appreciated by one skilled in the art that various conditions or disease states, in particular chronic conditions or disease states, may require prolonged treatment involving multiple administrations.
  • Dosage forms of the pharmaceutical compositions described herein contain from about 0.1 ⁇ g to about 15 ⁇ g of active ingredient (i.e. the compounds disclosed) per unit.
  • the active ingredient will ordinarily be present in an amount of about 0.5-95% weight based on the total weight of the composition.
  • Multiple dosage forms may be administered as part of a single treatment.
  • the active ingredient can be administered enterally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as milk, elixirs, syrups and suspensions. It can also be administered parenterally, in sterile liquid dosage forms.
  • the active ingredient can also be administered intranasally (nose drops) or by inhalation via the pulmonary system, such as by propellant based metered dose inhalers or dry powders inhalation devices.
  • Other dosage forms are potentially possible such as administration transdermally, via patch mechanism or ointment.
  • formulations suitable for enteral or oral administration may be liquid solutions, such as a therapeutically effective amount of the compound dissolved in diluents, such as milk, water, saline, buffered solutions, infant formula, and expressed breast milk, other suitable carriers, or combinations thereof.
  • diluents such as milk, water, saline, buffered solutions, infant formula, and expressed breast milk, other suitable carriers, or combinations thereof.
  • the active ingredients can then be mixed to the diluent just prior to administration.
  • formulations suitable for enteral or oral administration may be capsules, sachets, tablets, lozenges, and troches.
  • the formulation may contain a predetermined therapeutically effective amount of the active ingredient, as solids or granules, powders, suspensions and suitable emulsions.
  • Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, propylene glycol, glycerin, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
  • diluents such as water and alcohols, for example, ethanol, benzyl alcohol, propylene glycol, glycerin, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
  • Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.
  • Tablet forms can include one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the patient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the compound can be administered in a physiologically acceptable diluent in a pharmaceutically acceptable carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl- l,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and
  • Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyldialkylammonium halides, and alkylpyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl .beta.-aminopropionates, and 2-alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.
  • the parenteral formulations typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • HLB hydrophile-lipophile balance
  • compositions of the present invention are also well-known to those who are skilled in the art. The choice of excipient will be determined in part by the particular compound, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following methods and excipients are merely exemplary and are in no way limiting.
  • the pharmaceutically acceptable excipients preferably do not interfere with the action of the active ingredients and do not cause adverse side-effects.
  • Suitable carriers and excipients include solvents such as water, alcohol, and propylene glycol, solid absorbants and diluents, surface active agents, suspending agent, tableting binders, lubricants, flavors, and coloring agents.
  • the compounds of the present disclosure can be made into aerosol formulations to be administered via inhalation.
  • aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen.
  • Such aerosol formulations may be administered by metered dose inhalers. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • sterile liquid excipient for example, water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
  • the requirements for effective pharmaceutically acceptable carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J.B.
  • Formulations suitable for topical administration include pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, as well as creams, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.
  • an inert base such as gelatin and glycerin, or sucrose and acacia
  • formulations suitable for rectal administration may be presented as suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • suitable methods of administering a compound of the present invention to an patient are available, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective reaction than another route. Results
  • FIG. IA shows intra-operative gross morphological appearance of NEC showing an area of infarction.
  • FIG. IA shows an eosin-hematoxylin photomicrograph showing inflammation (thin arrows). Thick arrows indicate intramural air, which represents trapped gas bubbles from bacterial fermentation and is a pathognomonic finding of NEC.
  • FIG. IB shows PCR microarray indicating upregulation of pro-inflammatory cytokines and transcriptional regulators in NEC (expressed as fold-change over normal fetal intestine).
  • FIG. 1C is data from last 10 years in a neonatal intensive care unit showing that NEC affects preterm infants born during the late 2 nd trimester and the early 3 rd trimester of pregnancy
  • Fetal intestinal macrophages unlike intestinal macrophages from the adult, respond to LPS and other bacterial products and produce inflammatory mediators, including TNF- ⁇ .
  • resident macrophages are the first phagocytic cells of the innate immune system to encounter bacteria and bacterial products that breach the epithelium. Located in the subepithelial lamina basement, these cells play a role in protecting against pathogens and in regulating inflammatory responses to commensal bacteria. Therefore, to investigate the development of mucosal tolerance to bacterial products, human fetal macrophages and adult intestinal macrophages were compared. Consistent with earlier reports, macrophages were identifiable in the human fetal intestine at 10 weeks gestation.
  • macrophages in the full term neonate (iii) and adult (iv) intestine macrophages in the fetus (i) and premature (ii) intestine expressed CD 14, a key component of the LPS receptor complex, and tumor necrosis factor (TNF)-G! (FIG. 2A).
  • murine intestinal macrophages were studied in vitro. Human fetal intestinal macrophages were not available for these studies because of technical difficulties in the standard isolation protocols and immunoselection (lack of specific markers for human intestinal macrophages). Therefore, murine intestinal macrophages, which can be purified by immunoselection for CDl Ib, were used as a model system. Data from the mouse were consistent with immunohistochemical analysis on human tissue; unlike macrophages isolated from the neonatal (Dl postnatal) adult mouse intestine (Adult), El 5 and El 8 fetal intestinal macrophages produced TNF- ⁇ upon exposure to LPS in vitro (FIG. 2B). These finding shows that macrophages in the developing intestine, in contrast to those in the mature intestine, can produce an inflammatory response upon stimulation with bacterial products
  • macrophages are derived from blood monocytes, which differentiate under the influence of various epithelial/stromal cell-derived factors present in the ECM. In response to these factors, the macrophages become downregulated for their inflammatory response to bacterial products and their surface expression of innate immune receptors such as CD 14.
  • the biochemical microenvironment seen by monocytes newly recruited to the intestinal mucosa can be simulated in vitro by preparing conditioned media from explanted intestinal tissue or from cell-free intestinal stroma. Treatment of monocytes with these intestinal T-CMs in vitro induces functional and phenotypic characteristics of intestinal macrophages such as tolerance to bacterial products such as LPS and downregulation of CD 14 and other innate immune receptors.
  • T-CMs murine fetal intestinal macrophages (El 5) were treated with T-CMs derived from the adult mouse jejunum. As shown in FIG. 2B (inset), T-CMs from adult intestine downregulated the LPS-induced TNF- ⁇ response of El 5 fetal macrophages, indicating differential expression of one or more ECM factors in the adult versus fetal intestine that suppress LPS-induced macrophage cytokine production.
  • peripheral blood monocytes from healthy adult volunteers were treated in vitro with T-CMs prepared from 10-14, 15-19, 20-24 wks gestation human fetal intestinal tissue and from adult jejunum using: (1) ex planted intestinal tissue after removing the epithelial layer to expose the lamina intestinal; (2) intact ex planted intestinal tissue without removing the intestinal epithelium; and (3) primary epithelial cells isolated from fetal intestinal tissue.
  • Blood monocytes from healthy adult volunteers were treated with fetal/adult T-CMs in vitro prior to LPS stimulation.
  • T-CMs suppressed LPS-induced cytokine production (TNF- ⁇ , IL-6, IL- 1/3, and IL-8) in the monocyte-derived macrophages in a dose-dependent fashion (individual doses not depicted in figure), and this suppression increased with maturation (FIG. 3A).
  • fetal T-CMs showed increasing suppressive effects on LPS-induced cytokine production with maturation, fetal T-CMs were not as effective as adult T-CM in suppressing LPS-induced cytokine production.
  • the suppressive effect of these conditioned media on macrophage cytokine production was not related to the experimental protocol used for preparing the T-CMs.
  • T-CMs did not affect total protein synthesis or the synthesis of anti-inflammatory cytokines such as TGF-/3 and IL-10 by the monocyte-derived macrophages (data not depicted).
  • T-CMs The effect of T-CMs on the ability of monocyte-derived macrophages to recruit neutrophils upon LPS stimulation in vitro was also measured.
  • Neutrophil chemotaxis was measured by using a previously described fluorescence-based assay (Fox, et al., Cytokine 29: 135-140, 2005). Similar to the effect on macrophage cytokine production, T-CMs suppression of the chemotactic activity for neutrophils increased with maturation (FIG. 3B). Finally, the anti-inflammatory effect of T-CMs was further investigated by measuring NF-/cB activation (phosphorylation of p65/rel A) in LPS-treated monocyte-derived macrophages.
  • fetal T-CMs did not suppress NF- ⁇ B activation in these macrophages (FIG. 3C).
  • the differential effects of fetal vs. adult T-CMs in suppressing LPS-induced neutrophil chemotaxis and NF-/cB activation in macrophages further indicates the presence of one or more ECM factors that are expressed in the adult but not in the fetal intestine that promote the non-inflammatory profile of adult intestinal macrophages.
  • the efficacy of the full-term neonatal and adult intestinal tissues was not significantly different from each other.
  • TGF-/3 bioactivity in T-CMs was measured using a quantitative luciferase assay for TGF- ⁇ activation of the plasminogen activator inhibitor- 1 (PAI-I) promoter. As seen in FIG. 5 A, TGF-/3 bioactivity was low in fetal T-CMs and increased with maturation reaching a peak in the adult T-CM.
  • PAI-I plasminogen activator inhibitor- 1
  • monocyte-derived macrophages were treated with 10-14 week, 15-19 week and 20-24 week fetal T-CMs and adult T-CM and the phosphorylation of Smad2, a key transducer of TGF- ⁇ -induced signaling in monocytes/macrophages, was measured.
  • Smad2 phosphorylation correlated with the results of the PAI-I luciferase bioassay (FIG. 5B), thereby confirming that TGF-/3 activity increases in the intestinal ECM with maturation.
  • monocyte-derived macrophages were treated with 10-24 week fetal intestinal T-CMs and excess (10-50 ⁇ g/mL) neutralizing polyclonal rabbit anti-TGF- ⁇ IgG antibody (R&D) was added in some wells. This antibody neutralizes the bioactivity of all three isoforms of TGF-(S. Neutralization of TGF-(S reversed T-CM suppression of LPS-induced TNF-ce (FIG. 5C) and IL-8 production in macrophages. For IL-8, LPS increased macrophage IL-8 production from 0.72 ⁇ 0.01 ng/mL to 4.25 ⁇ 1.46 ng/mL.
  • T-CMs from 10-14, 15-19, and 20-24 wks gestation suppressed LPS-induced IL-8 production to 3.49 ⁇ 1.41 (not significant), 2.24 ⁇ 0.02 ng/mL (p ⁇ 0.05), and 1.15 ⁇ 0.22 ng/mL (p ⁇ 0.05), respectively.
  • TGF- ⁇ neutralizing antibody reversed the suppressive effect of T-CMs on macrophage IL-8 production to 3.60 ⁇ 0.18 (not significant), 3.58 ⁇ 0.23 (p ⁇ 0.05), and 2.59 ⁇ .13 ng/mL (p ⁇ 0.05) in the three gestational age-based groups, respectively.
  • TGF-(S) expression in the intestine mRNA expression of the three TGF - ⁇ isoforms, TGF- ⁇ i, TGF-(S 2 , and TGF-(S 3 was measured in the human fetal (10-24 weeks), neonatal (term newborn), and adult intestine. As shown in FIG. 6A, the expression of TGF-(S 2 , but not TGF-jSi or TGF- ⁇ 3 , increased with maturation. These data were supported by immunohistochemical analysis (FIG. 6B). In the 10-14 wk fetal intestine, minimal TGF-(S 2 expression was noted in the epithelium but was prominent in the muscularis externa.
  • TGF-(S 2 immunoreactivity was noted on epithelial cells and muscularis mucosae (MM). TGF-/3 2 immunoreactivity became stronger with maturation in the 20-24 wk fetus, term neonate, and adult. In the mature intestine, TGF-(S 2 immunoreactivity was also seen in some lamina basement cells. TGF-(S 2 co-localized in these cells with osmooth muscle actin, indicating that these TGF-(S 2 + cells were myofibroblasts (FIG. 6B). In contrast to TGF-(S 2 , immunoreactivity for TGF-JS 1 and TGF-jS 3 did not change significantly with intestinal maturation (data not shown). The effect of maturation on TGF-/S 2 expression was further confirmed by measuring total and active TGF-(S 2 in T-CMs by ELISA. As seen in FIG. 6C, both total and active TGF-/3 2 increased with maturation.
  • T-CMs containing only TGF-JS 2 were most effective in suppressing LPS-induced TNF- ⁇ production (FIG. 6D).
  • T- CMs containing only TGF-(S 1 had a smaller suppressive effect on macrophage cytokine production, whereas T-CMs containing TGF-(S 3 did not have a significant effect.
  • TGF-(Si and TGF-(S 2 ) were also measured in the same supernatants (data not shown), and the effects were similar to those on TNF- ⁇
  • IL-8 concentrations were also measured in the same supernatants (data not shown), and the effects were similar to those on TNF- ⁇
  • monocyte-derived macrophages were treated with 0-2000 pg/mL of recombinant human TGF- /Si, TGF-j8 2 , and TGF-/3 3 for 2 hrs before LPS stimulation
  • TGF-(S 2 was the most potent of the three isoforms in suppressing TNF- ⁇ (inset of FIG. 6D) and IL-8 expression (data not shown).
  • TGF-/3 intestinal ECM suppression of the inflammatory responses of macrophages is mediated through TGF-/3
  • TGF-(S 2 is the most important of the TGF-/3 isoforms for this downregulation of macrophage cytokine production.
  • TGF - ⁇ is likely to play an important role in maintaining the absence of inflammation in the normal intestinal mucosa despite exposure to bacteria and bacterial products.
  • inflammation is an important characteristic of NEC. Therefore, experiments were conducted to determine whether NEC is associated with decreased expression and/or bioactivity OfTGF-(S.
  • NEC was associated with decreased TGF-/3 2 mRNA, TGF-(S bioactivity and active TGF-(S 2 as compared to both normal intestinal tissue from premature infants as well as the fetal intestinal tissue.
  • concentrations of active TGF- ⁇ i were also lower in NEC (104 ⁇ 27 pg/mg in normal preterm intestine and 124 ⁇ 18 pg/mg in normal fetal tissue, vs. 32 ⁇ 10 pg/mg in NEC; p ⁇ 0.05).
  • the concentrations of active TGF-(S 3 were below the lower limit of detection in both normal preterm/fetal tissue and NEC.
  • the bioactivity of TGF-(S was decreased in tissue affected by NEC, further confirming the lower concentrations of TGF-(S isoforms measured by ELISA.
  • TGF-(S 2 mRNA and active TGF-/3 2 protein was measured in a previously described murine model of intestinal ischemia-reperfusion (I/R) injury, which is associated with a marked inflammatory response (Maheshwari, et al., Fetal Pediatr Pathol 23:145-157, 2004).
  • I/R intestinal ischemia-reperfusion
  • TGF-(S 2 mRNA and protein were increased in intestinal tissue following I/R injury (FIG. 7B), indicating that decreased TGF-/3 2 expression in NEC is a specific finding unrelated to possible consumption of TGF-(S 2 during mucosal inflammation.
  • TGF-(S activity such as in the premature intestine
  • TGF-(S activity can increase the severity of the mucosal inflammatory response and NEC-like injury in a murine model.
  • the three isoforms of TGF-/3 have a quantitatively different but qualitatively similar inhibitory effect on macrophage cytokine production (FIG. 6D, inset), and this redundancy renders deletion/neutralization models for individual TGF-(S isoform(s) susceptible to interference from the remaining isoforms.
  • TGF-/3 TGF-(S receptor protein II (TGF-/3 RII)
  • TGF-/3 RII TGF-(S receptor protein II
  • a transgenic DNIIR mice Serra, et al., J Cell Biol 139:541-552, 1997) was used in which a truncated, kinase-defective TGF-/3 RII transgene is controlled by a metallothionein-like promoter, MT-DNIIR.
  • mice supplementation with zinc activates the DNA regulatory element and promotes transgene expression; overexpression of the mutated TGF-/3 RII inhibits the response to all TGF-/3 species and thus creates a conditional knock-out model that avoids fetal/neonatal lethality.
  • mice develop a partial deficiency of TGF-/3 effects, whereas 7 days of zinc treatment resulted in a near complete loss of TGF- ⁇ signaling.
  • wild- type mice mice supplemented with zinc sulfate (subcutaneous, 50 ⁇ g/gm/day) for 3 days were compared with mice supplemented with zinc sulfate for 7 days.
  • NEC-like mucosal injury was induced by intraperitoneal administration of platelet-activating factor (PAF; 50 ⁇ g/kg) and LPS (1 mg/kg) in 12-14 day old pups as described previously (Sun, et al, Am J Physiol 273:G56-61, 1997; Hsueh, et al, FASEB J 1 :403-405, 1987). Appropriate wild type and DNIIR controls were maintained with and without zinc supplementation. Mice were sacrificed 2 hrs after PAF- LPS administration.
  • PAF platelet-activating factor
  • Mucosal injury was graded on a 4-point scale: grade 0: no injury; grade 1 : mild separation of lamina intestinal; grade 2: moderate separation of submucosa; grade 3: severe separation of submucosa and/or severe edema in submucosa/muscularis; grade 4: transmural injury (Musemeche, et al., J Pediatr Surg 26:1047-1049, 1999; Maheshwari, et al., Fetal Pediatr Pathol 23:145-157 2004). As seen in FIG. 8A, the deficiency of TGF-/5 signaling worsened PAF-LPS-induced mucosal injury in a dose-dependent manner.
  • TGF-ft biologically relevant concentrations of TGF-ft
  • TGF-ft both TGF-/3 RI and TGF-/3 RII components of the heteromeric TGF-/3 receptor complex are widely expressed in human fetal as well as murine intestine (FIG. 6C).
  • enteral supplementation of TGF-/3 might be effective as a therapeutic strategy to correct an endogenous deficiency state. Therefore it was determined whether enteral administration of recombinant human TGF - ⁇ 2 would prevent mucosal injury in 3 -day zinc supplemented DNIIR pups, which are partially deficient in TGF-/3 signaling.
  • the use of human TGF-/3 2 in murine pups is justified because the two peptides are nearly identical and human TGF-fe has been previously shown to be effective in mice (Penttila, et al., Pediatr Res 59:650-655, 2006).
  • TGF-(S 2 protected 3 -day zinc-supplemented DNIIR mice against mucosal injury.
  • TGF- ⁇ 2 was not protective in DNIIR mice that had received supplemental zinc for 7 days and therefore had a complete loss of TGF- ⁇ signaling (data not shown).
  • the present disclosure provides the first detailed investigation into the normal development of mucosal tolerance to bacterial products in the intestine and also provides a novel pathophysiological model for NEC and related disease states and conditions that depend, at least in part, by un-regulated or improperly regulated intestinal cytokine release that has therapeutic implications.
  • the present disclosure shows that tissue macrophages in the fetal intestine produce an inflammatory response upon stimulation with bacterial products (LPS), which contrast with the LPS-tolerance and non-inflammatory characteristics of intestinal macrophages in the adult.
  • LPS bacterial products
  • Intestinal macrophages are derived from blood monocytes that are recruited to the lamina limbal and undergo specific differentiation under the influence of various ECM components and other bioactive molecules.
  • macrophages isolated from the fetal intestine became LPS-tolerant upon treatment with conditioned media prepared from adult intestinal tissue (FIG. 9). Because macrophages in both fetal as well as the adult intestine are derived from monocytes, the present disclosure shows that that differences between fetal and adult intestinal macrophages reflects changes in the differentiating microenvironment rather than the intrinsic, programmed differences of two distinct cellular populations. Furthermore, the unique inflammatory attenuation of intestinal macrophages is acquired as a progressive maturational change from the fetal period towards adulthood, further emphasizing the importance of changes in the ECM.
  • the present disclosure shows that mucosal inflammatory responses to bacterial products are likely to be the highest in the premature intestine than at any other time in life, which explains the occurrence of NEC almost exclusively in premature infants even though altered mucosal permeability and bacterial translocation is common in critically ill patients of all ages.
  • TGF- ⁇ effects on intestinal macrophages can be traced through development and that among the three TGF-/3 isoforms, TGF-/3 2 has the most important role in intestinal macrophage differentiation in humans.
  • the effects of TGF-(S in the intestine are interesting when intestinal macrophages are compared to macrophages in other organs such as lung.
  • LPS-tolerance is also seen in alveolar macrophages, these cells contrast with intestinal macrophages as LPS-tolerance in alveolar macrophages is a postnatal phenomenon related to the exposure to bacterial flora and represents a secondary state of refractoriness.
  • TGF-/3 2 -mediated differentiation represents an endogenous mechanism to suppress mucosal inflammatory responses beginning in utero, in anticipation of postnatal bacterial colonization.
  • TGF-/3 is widely expressed in the lung, the differences between intestinal vs. alveolar macrophages can be explained on the basis of spatial differences in expression of TGF-(S 2 .
  • TGF-(S 2 is the most effective of the three TGF-(S isoforms in suppressing macrophage cytokine production in the developing intestine.
  • TGF- ⁇ the anti-inflammatory effects of TGF-/3 2 are not well-characterized because TGF-/3 2 V mouse pups die of congenital anomalies during the neonatal period.
  • the anti-inflammatory effects of TGF-(S 2 have been noted in diverse models such as the systemic inflammatory response syndrome, traumatic brain injury, and T-cell-mediated encephalomyelitis.
  • TGF-(S 2 gene polymorphisms have also been associated with an increased risk of atopy.
  • TGF-(S 2 is unique among TGF-/3 isoforms because the absence of the arg-gly-asp (RGD) integrin-binding sequence in its precursor permits activation by mechanisms other than via the ⁇ V j8 6 integrin, which is important in the mature intestine but is expressed at very low levels during the fetal period. Furthermore, phenotypic differences between transgenic mice lacking individual TGF- ⁇ isoforms and data from various in vitro model systems indicate that the three isoforms of TGF-(S may differ in their intracellular signals. TGF-(S 2 has a low intrinsic affinity for the type II TGF-(S receptor and requires betaglycan, a co-receptor, for optimal receptor activation and signaling.
  • TGF/? 2 -betaglycan dyad is believed to alter the conformation of the TGF-/3 receptor complex to transduce unique cellular signals and biological effects.
  • the present disclosure reports for the first time that TGF-/3 2 mRNA and protein expression and TGF-/3 bioactivity is decreased in NEC.
  • TGF-(S 2 expression was decreased in NEC to levels even lower than the developmentally 'low' levels of the premature/fetal intestine.
  • TGF-/5 may have a protective effect in experimental colitis.
  • the present disclosure shows that the loss of TGF-/3 signaling in mice worsened NEC-like mucosal injury, which is consistent with the data from in vitro experiments on macrophages and with assays on tissue samples from patients with NEC.
  • the gastrointestinal mucosa is known to absorb intact proteins and other macromolecules during the fetal/neonatal period, and therefore, some of the TGF-/3 2 received by gavage is likely to have been absorbed through the mucosa to reach the macrophages in the lamina limbal.
  • TGF-(S 2 ) Enteral administration of TGF-(S 2 , possibly added to premature infant formula or in human milk fortifiers, is biologically plausible because TGF-/5 2 is detectable in both amniotic fluid as well as breast milk in significant amounts, indicating that the cytokine is normally swallowed in large amounts by the fetus and the newborn infant.
  • the protective effect of enteral TGF-(S 2 against mucosal injury in mice suggests that recombinant TGF-/3 2 may have a therapeutic role in the prevention of NEC in premature infants.
  • HBSS Hanks' balanced-salt solution
  • DTT Hanks' balanced-salt solution
  • Tissues were next treated with HBSS containing 1 mM EDTA (Sigma) twice for 20 min each at 37 0 C, washed thrice, and then incubated in HBSS containing 1 mM collagenase type IV (Sigma) for 2 h at 37°C.
  • Isolated cells were suspended in 40% Percoll (Pharmacia Biotech), layered onto 75% Percoll, and centrifuged at 2000 rpm for 20 min.
  • Cells recovered from the interphase were further purified by immunoselection using CDl Ib microbeads (Miltenyi Biotec) and then allowed to adhere on polystyrene plates for 1 hr.
  • the purity of adherent macrophages was confirmed as >97% by immunostaining for F4/80, a pan-macrophage marker in mice (E- biosciences).
  • T-CMs from murine and human intestinal tissue were prepared by using a previously reported protocol with minor modifications (Smythies, et al., In Current Protocols in Immunology. J. E. Coligan, A.M. Kruisbeek, D. H. Marguilies, E. M. Shevach, and W. Strober, editors. New York, NY: Current Protocols. 1-9, 2006).
  • the intestinal epithelium was removed by enzymatic treatment. Intestinal tissue was washed in HBSS/DTT as above and then treated twice with dispase (each time for 20 min on an agitator).
  • the remaining sub-epithelial tissue was incubated in RPMI (1 ml/gram of tissue, no serum) x 24 hrs at 37 0 C, 5% CO 2 .
  • Exfoliated epithelial cells were cultured in a similar fashion to prepare epithelial-conditioned media. These media were clarified by centrifugation and assayed for LPS (limulus lysate assay, Sigma), protease activity (Sigma), and total protein (BCA, Pierce).
  • Monocytes from healthy volunteers were isolated by Ficoll-Hypaque density centrifugation followed by positive immunoselection with CD 14 microbeads (Miltenyi) as per our previously described protocol (Maheshwari, et al., J Leukoc Biol 80:1111-1117, 2006; English, et al., J Immunol Methods 5:249-252, 1974).
  • Cells were allowed to attach (2 x 10 4 cells/well) in 96-well plates and incubated with T-CM (250, 500, and 1000 ⁇ g total protein/mL). After 2 hrs, monocyte cultures were stimulated with 500 ng/mL LPS (pre-determined optimum concentration) for 8 (for mRNA) or 18 hrs (for protein measurements).
  • TNF- ⁇ human and murine
  • IL-6 human and murine
  • IL-8/CXCL8 were measured by ELISA (R&D, Minneapolis, MN) as per the manufacturer's protocol.
  • Neutrophil chemo taxis Neutrophil chemotaxis was measured in microchemotaxis chambers using our previously described protocol (Fox, et al., Cytokine 29:135-140, 2005). Supernatants from the above macrophage cultures were used in the lower wells of the microchemotaxis chambers (NeuroProbe, Gaithersberg, MD). The number of migrating cells was read off a standard curve generated from known numbers of labeled cells.
  • NF-/cB activation Monocytes were treated with T-CMs and LPS as above.
  • NF- ⁇ B p65 phosphorylation was measured using a commercially available kit (SuperArray Biosciences).
  • TGF-/3 Excess (50 ⁇ g/mL) neutralizing polyclonal rabbit anti-human TGF-/3 antibody (R&D) was used to neutralize TGF-/3. Appropriate isotype controls were maintained. Assays for TGF-ff: TGF- J S 1 , TGF-/3 2 , and TGF-& were measured using specific ELISA kits (R&D). Total concentration of each isoform was measured by using the complete protocol including the acid-activation step to convert latent TGF- ⁇ to immunoreactive forms. For measuring the active fraction, the acid-activation step was not performed. To measure the TGF-(S 2 concentrations in the aqueous fraction of human milk samples, a variation of the standard assay was developed. Standard curves were obtained using standard solutions and milk samples 'spiked' with known amounts of recombinant TGF-/3 2 as previously described for other cytokines.
  • TGF- / 3 bioactivity was measured using a quantitative luciferase assay based on the activation of the PAI-I gene in stably transfected mink lung epithelial cells (kind gift from Dr. D. B. Rifkin, NYU Medical Center) were grown in 96-well plates. T-CMs equalized for total protein concentration were added to the reporter cells x 16 hrs. The luciferase assay is described elsewhere (Munger, et al., Kidney Int 51 :1376-1382, 1997). The signal attributable to TGF-jS was determined by comparing luciferase activity for each sample in the presence and absence of 15 ⁇ g/mL neutralizing anti-TGF-/3 antibody.
  • Smad phosphorylation was measured by western blotting. Monocytes (5 x 10 6 /well) were treated with T-CMs for 20 min (pre-determined optimum duration) and smad2 (ser423, ser425) phosphorylation was measured using polyclonal anti-phospho-smad 2 and anti-smad 2 (Santa Cruz) with appropriate secondary reagents. The relative importance of the three TGF-/3 isoforms was determined by immunoprecipitation and removal of two of the three TGF-/3 isoforms from 20-24 wk T-CMs. Specific monoclonal antibodies (R&D) were used followed by addition of sepharose- immobilized protein AJG (Pierce).
  • TGF-(S 1 , TGF-(S 2 , or TGF-(S 3 ) were obtained.
  • Monocytes were treated with T- CM or one of the three T-CM derivatives and then stimulated with LPS. IL-8 and TNF- ⁇ production was measured as above.
  • monocytes were treated with incremental 0-2000 pg/mL of recombinant human TGF-(Si, TGF-(S 2 , and TGF-(S 3 for 2 hrs before LPS stimulation and measured cytokine/chemokine production as above.
  • Mice Wild type C57B6 mice were procured from a commercial vendor (Jackson Labs,
  • DNIIR mice have been described herein.
  • Zinc sulfate was administered (50 ⁇ g/gm/day subcutaneous optimum dose) to DNIIR pups for 3 or 7 days for partial and complete inhibition of TGF- ⁇ signaling, respectively.
  • the induction of gut mucosal injury by administration of PAF and LPS has been described herein.
  • Smad phosphorylation was measured in the intestinal tissue by western blotting.
  • DNIIR mice after 0, 3, and 7 days of zinc supplementation were treated with 100 ng recombinant human TGF-(S 2 intraperitoneally and euthanized after 1 hr. Intestinal tissues were homogenized in T-PER lysis buffer (Pierce) with protease inhibitors.
  • Smad2 (ser423, ser425) phosphorylation was measured in western blots using polyclonal anti-phospho-smad 2 and anti-smad 2 (Santa Cruz) with appropriate secondary reagents.
  • Ischemia-reperfusion injury was induced in 10-12 day old wild-type mice by clamping the superior mesenteric artery for 60 and then allowing reperfusion by releasing the clamp for 90 minutes. Mice were euthanized immediately after the experiment to harvest the intestines.
  • Milk TGF-/3 2 concentrations are shown in standard box-plots and summarize the lowest concentration, lower quartile (Ql), median, upper quartile (Q3), and the highest concentration. Group comparisons were done with the Student's t test or analysis of variance for parametric and Mann-Whitney/Kruskall-Wallis with appropriate corrections for non-parametric data. A p value of 0.05 was accepted as significant.

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Abstract

L'invention porte sur l'administration de TGF-? ou d'un dérivé du TGF-? pour traiter et/ou prévenir différents états et/ou troubles pathologiques caractérisés par la réponse de la cytokine de cellules épithéliales intestinales immatures à des lésions des muqueuses, à une invasion bactérienne, à des antigènes bactériens et/ou à leurs produits, comprenant entre autres, l'entérocolite nécrosante (NEC). L'invention porte également sur une préparation pharmaceutique et sur des médicaments comprenant des doses à efficacité thérapeutique de TGF-? ou d'un dérivé du TGF-?, utilisables dans les méthodes décrites dans la description.
PCT/US2009/034660 2008-02-20 2009-02-20 Méthodes de traitement et prévention de l'entérocolite nécrosante Ceased WO2009105629A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014018621A1 (fr) * 2012-07-24 2014-01-30 The Board Of Trustees Of The University Of Illinois Méthode permettant de prédire l'apparition d'une entérocolite nécrosante
US9186379B2 (en) * 2013-02-23 2015-11-17 Stemnion, Inc. Methods for preventing or treating necrotizing enterocolitis

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CLAUD ERIKA C. ET AL.: 'Modulation of Human Intestinal Epithelial Cell IL-8 Secretion by Human Milk Factors' PEDIATRIC RESEARCH vol. 53, no. 3, 2003, pages 419 - 425 *
DATABASE NCBI 18 November 2007 Database accession no. NP003229 *
MAREK ANDRZEJ ET AL.: 'Human milk, a multipotent infant's immune system fortifier' MED SCI MONIT vol. 9, no. SUP.4, 2003, pages 75 - 81 *
URUAKPA F.O. ET AL.: 'Colostrum and its benefits: a review' NUTRITION RESEARCH vol. 22, 2002, pages 755 - 767 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014018621A1 (fr) * 2012-07-24 2014-01-30 The Board Of Trustees Of The University Of Illinois Méthode permettant de prédire l'apparition d'une entérocolite nécrosante
US9186379B2 (en) * 2013-02-23 2015-11-17 Stemnion, Inc. Methods for preventing or treating necrotizing enterocolitis

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