Kathy Flanders - Academia.edu (original) (raw)

Papers by Kathy Flanders

Research paper thumbnail of Inducible Nitric Oxide Production and Expression of Transforming Growth Factor-β1 in Serum and CSF after Cerebral Ischaemic Stroke in Man

Nitric Oxide-biology and Chemistry, 1998

A residual blood supply to the ischaemic brain is a crucial determinant for tissue survival. Earl... more A residual blood supply to the ischaemic brain is a crucial determinant for tissue survival. Early changes in the vascular network and subsequent angiogenesis may be mediated by short-lived molecules like nitric oxide (NO) or growth factors such as transforming growth factor-β1 (TGF-β1). Although TGF-β1 can inhibit NO production, this interaction has not been studied after ischaemia in humans. Serum

Research paper thumbnail of Smad3 Signaling Is Required for Epithelial-Mesenchymal Transition of Lens Epithelium after Injury

American Journal of Pathology, 2004

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract... more Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-␤ and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-␤2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-␤ expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, ␣-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-␤ in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery . Certain cells have an inherent plasticity such that their morphology and phenotype can be modulated by various growth factors and extracellular stimuli. As an example,

Research paper thumbnail of Imatinib mesylate for the treatment of steroid-refractory sclerotic-type cutaneous chronic graft-versus-host disease

Biology of Blood and Marrow Transplantation, 2015

Sclerotic skin manifestations of chronic graft-versus-host disease (ScGVHD) lead to significant m... more Sclerotic skin manifestations of chronic graft-versus-host disease (ScGVHD) lead to significant morbidity, including functional disability from joint range of motion (ROM) restriction. No superior second-line therapy has been established for steroid-refractory disease. Imatinib mesylate is a multi-kinase inhibitor of several signaling pathways implicated in skin fibrosis with in vitro antifibrotic activity. We performed an open label pilot Phase 2 trial of imatinib in children and adults with corticosteroid refractory ScGVHD. Twenty patients were enrolled in a 6 month trial. Eight received a standard dose (adult: 400 mg daily; children: 260 mg/m(2) daily). Due to poor tolerability, 12 additional patients underwent a dose escalation regimen (adult: 100 mg daily initial dose up to 200 mg daily maximum; children initial dose 65 mg/m(2) daily up to 130 mg/m(2) daily). Fourteen patients were evaluable for primary response, improvement in joint range of motion (ROM) deficit, at 6 months. Primary outcome criteria for partial response (PR) was met in 5/14 (36%), stable disease (SD) in 7/14 (50%), and progressive disease (PD) in 2/14 (14%) patients. Eleven (79%) patients, including 5 PR and 6 with SD, demonstrated a positive gain in ROM (range 3-94% improvement in deficit). Of 13 patients with measurable changes at 6 months, the average improvement in ROM deficit was 24.2% (IQR: 15.5% to 30.5%; p=0.011). This trial is registered at http://clinicaltrials.gov as NCT007020689.

Research paper thumbnail of Transient adenoviral gene transfer of Smad7 prevents injury-induced epithelial–mesenchymal transition of lens epithelium in mice

Laboratory Investigation, 2004

We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad i... more We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFb/ activin signaling, on injury-induced epithelial-mesenchymal transition (EMT) of lens epithelium in mice. A volume of 3 ll of adenoviral solution was injected into the right lens of adult male C57BL/6 mice (n ¼ 56) at the time of capsular injury made using a hypodermic needle under general anesthesia. A mixture of recombinant adenovirus carrying CAG promoter-driven Cre (Cre adv) and mouse Smad7 complementary DNA (Smad7 adv) was administered to induce Smad7 expression, while control lenses were treated with Cre adv alone. After healing intervals of 2, 3, 5, and 10 days, animals were killed 2 h after labeling with bromodeoxyuridine (BrdU) and eyes were processed for histology. During healing, marked expression of Smad7 was observed in lens epithelial cells in the Smad7 adv group with loss of nuclear translocation of Smads2/3, while little Smad7 and abundant nuclear Smads2/3 were seen in cells in the Cre adv group. Lens epithelial cells in the Cre adv control group exhibited a fibroblastic appearance at days 5 and 10 and the capsular break was sealed with fibrous tissue, while Smad7 adv-treated cells around the capsular break retained their epithelial morphology and the break was not sealed. Expression of snail mRNA, and a-smooth muscle actin, lumican, and collagen VI proteins, markers of EMT, was observed in control-treated eyes, but not in cells of the Smad7 adv group at day 5 with minimal expression at day 10. Additionally, cell proliferation increased in epithelium infected with Smad7 adv consistent with suppression of injury-induced upregulation of TGFb1 in epithelium. We conclude that gene transfer of Smad7 in mice prevents injury-induced EMT of lens epithelial cells and sealing of the capsular break with fibrous tissue.

Research paper thumbnail of Conditional overexpression of TGF-β1 disrupts mouse salivary gland development and function

Laboratory Investigation, 2010

Transforming growth factor-β (TGF-β) signaling is known to affect salivary gland physiology by in... more Transforming growth factor-β (TGF-β) signaling is known to affect salivary gland physiology by influencing branching morphogenesis, regulating ECM deposition, and controlling immune homeostasis. To study the role of TGF-β1 in the salivary gland, we created a transgenic mouse (β1 glo ) that conditionally over-expresses the active TGF-β1 upon genomic recombination by the Cre recombinase. The β1 glo mice were bred with a MMTV (mouse mammary tumor virus)-Cre (MC) transgenic line that expresses the Cre recombinase predominantly in the secretory cells of both the mammary and salivary glands. Although most of the double positive (β1 glo /MC) pups die either in utero or just after birth, clear defects in salivary gland morphogenesis could be seen such as reduced branching and increased mesenchyme. The β1 glo /MC mice that survived into adulthood, however, had hyposalivation due to salivary gland fibrosis and acinar atrophy. Increased TGF-β signaling was observed in the salivary gland with elevated phosphorylation of Smad2 and a concomitant increase in ECM deposition. In particular, aberrant TGF-β1 overexpression caused salivary gland hypofunction in this mouse model because of the replacement of normal glandular parenchyma with interstitial fibrous tissue. These results further implicate TGF-β in pathological cases of salivary gland inflammation and fibrosis that occur with chronic infections in the glands or with the autoimmune disease, Sjögren's syndrome or with the radiation therapy given to head-and-neck cancer patients.

Research paper thumbnail of Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in mice

Laboratory Investigation, 2004

Retinal pigment epithelial (RPE) cells dedifferentiate and undergo epithelial-mesenchymal transit... more Retinal pigment epithelial (RPE) cells dedifferentiate and undergo epithelial-mesenchymal transition (EMT) following retinal detachment, playing a central role in formation of fibrous tissue on the detached retina and vitreous retraction (proliferative vitreoretinopathy (PVR)). We have developed a mouse model of subretinal fibrosis with implications for PVR in which retinal detachment is induced without direct damage to the RPE cells. Transforming growth factor-b (TGF-b) has long been implicated both in EMT of RPEs and the development of PVR. Using mice null for Smad3, a key signaling intermediate downstream of TGF-b and activin receptors, we show that Smad3 is essential for EMT of RPE cells induced by retinal detachment. De novo accumulation of fibrous tissue derived from multilayered RPE cells was seen following experimental retinal detachment in eyes of wild type, but not Smad3-null mice. Expression of a-smooth muscle actin, a hallmark of EMT in this cell type, and extracellular matrix components, lumican and collagen VI, were also not observed in eyes of Smad3-null mice. Our data show that induction of PDGF-BB by Smad3-dependent TGF-b signaling is likely an important secondary proliferative component of the disease process. The results suggest that blocking the Smad3 pathway might be beneficial in prevention/treatment of PVR.

Research paper thumbnail of TGF-β Rescues Target-deprived Preganglionic Sympathetic Neurons in the Spinal Cord

European Journal of Neuroscience, 1996

Transforming growth factors P (TGF-P), a family of pleiotropic cytokines, are widely distributed ... more Transforming growth factors P (TGF-P), a family of pleiotropic cytokines, are widely distributed in the developing and adult nervous system. In order to further determine the neural functions of TGF-P, we have localized the TGF-P isoforms 1, 2 and 3 in the adult rat adrenal medulla and studied the neuroprotective capacity of one representative family member, TGF-P2, for those spinal cord neurons which innervate adrenal chromaffin cells and which die after destruction of the adrenal medulla. Unilateral electrothermal destruction of the adrenal medulla led to the disappearance of 25% of sympathetic preganglionic neurons, which are located in the intermediolateral (IML) column of thoracic spinal cord segments 7-1 0 and can be selectively marked by NADPHdiaphorase. The neurons which disappeared following adrenomedullectomy constitute the full set of neurons that innervate the adrenal medulla. Implantation of gelfoam soaked with 0.5 pg TGF-P2 into the adrenal wound cavity rescued all spinal cord neurons in the IML ipsilaterally to the lesioned side. Cytochrome c was not effective. Injections of ['251]TGF-P2 into the adrenal medulla did not result in retrograde transport and subsequent labelling of spinal cord neurons, suggesting that TGF-P may exert its neuroprotective actions by indirect mechanisms. TGF-P applied to cultured adrenocortical cells did not overtly increase the amount of mRNA for fibroblast growth factor-2, an established trophic molecule for sympathetic preganglionic spinal cord neurons. The mechanisms by which TGF-P exerts its neurotrophic effect are therefore unclear. Even so, our data provide the first evidence that TGF-P may play an important role in vivo in the control of maintenance of a population of spinal cord neurons.

Research paper thumbnail of Expression of Smad7 in Mouse Eyes Accelerates Healing of Corneal Tissue after Exposure to Alkali

The American Journal of Pathology, 2005

Damage to the cornea from chemical burns is a serious clinical problem that often leads to perman... more Damage to the cornea from chemical burns is a serious clinical problem that often leads to permanent visual impairment. Because transforming growth factor (TGF)-beta has been implicated in the response to corneal injury, we evaluated the effects of altered TGF-beta signaling in a corneal alkali burn model using mice treated topically with an adenovirus (Ad) expressing inhibitory Smad7 and mice with a targeted deletion of the TGF-beta/activin signaling mediator Smad3. Expression of exogenous Smad7 in burned corneal tissue resulted in reduced activation of Smad signaling and nuclear factor-kappaB signaling via RelA/p65. Resurfacing of the burned cornea by conjunctival epithelium and its differentiation to cornea-like epithelium were both accelerated in Smad7-Ad-treated corneas with suppressed stromal ulceration, opacification, and neovascularization 20 days after injury. Introduction of the Smad7 gene suppressed invasion of monocytes/macrophages and expression of monocyte/macrophage chemotactic protein-1, TGF-beta1, TGF-beta2, vascular endothelial growth factor, matrix metalloproteinase-9, and tissue inhibitors of metalloproteinase-2 and abolished the generation of myofibroblasts. Although acceleration of healing of the burned cornea was also observed in mice lacking Smad3, the effects on epithelial and stromal healing were less pronounced than those in corneas treated with Smad7. Together these data suggest that overexpression of Smad7 may have effects beyond those of simply blocking Smad3/TGF-beta signaling and may represent an effective new strategy for treatment of ocular burns.

Research paper thumbnail of Therapeutic Effect of Topical Administration of SN50, an Inhibitor of Nuclear Factor-κB, in Treatment of Corneal Alkali Burns in Mice

The American Journal of Pathology, 2005

We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear ... more We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear factor-B, in a corneal alkali burn model in mice. An alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. SN50 (10 g/ l) or vehicle was topically administered daily for up to 12 days. The eyes were processed for histological or immunohistochemical examination after bromodeoxyuridine labeling or for semiquantification of cytokine mRNA. Topical SN50 suppressed nuclear factor-B activation in local cells and reduced the incidence of epithelial defects/ulceration in healing corneas. Myofibroblast generation, macrophage invasion, activity of matrix metalloproteinases, basement membrane destruction, and expression of cytokines were all decreased in treated corneas compared with controls. To elucidate the role of tumor necrosis factor (TNF)-␣ in epithelial cell proliferation, we performed organ culture of mouse eyes with TNF-␣, SN50, or an inhibitor of c-Jun N-terminal kinase (JNK) and examined cell proliferation in healing corneal epithelium in TNF-␣ ؊/؊ mice treated with SN50. An acceleration of epithelial cell proliferation by SN50 treatment was found to depend on TNF-␣/JNK signaling. In conclusion, topical application of SN50 is effective in treating corneal alkali burns in mice.

Research paper thumbnail of Smad3 Signaling Is Required for Epithelial-Mesenchymal Transition of Lens Epithelium after Injury

The American Journal of Pathology, 2004

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract... more Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-␤ and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-␤2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-␤ expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, ␣-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-␤ in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery . Certain cells have an inherent plasticity such that their morphology and phenotype can be modulated by various growth factors and extracellular stimuli. As an example,

Research paper thumbnail of Murine pregnancy decidua produces a unique immunosupressive molecule related to transforming growth factor β-2

The Journal of Immunology

Non-T small lymphocytic suppressor cells in murine allopregnancy release a potent immunosuppressi... more Non-T small lymphocytic suppressor cells in murine allopregnancy release a potent immunosuppressive factor in vitro that is neutralized by rabbit anti-transforming growth factor (TGF)-beta. Previous studies have suggested that the decidual suppressor factor (DSF) is smaller than TGF-beta 1, and in this paper, we show that DSF on HPLC-sieving columns also elutes later than TGF-beta 2. Nevertheless, DSF has the ability to promote anchorage-independent growth of NRK fibroblasts similar to TGF-beta s. Using turkey antibodies specific for TGF-beta 1 or beta 2, we show that DSF is related to TGF-beta 2 rather than TGF-beta 1, and this relationship was confirmed by using a panel of murine mAb to TGF-subtypes. PAGE and Western blotting showed that the TGF-beta 2-reactive molecules in HPLC-purified DSF was slightly smaller than TGF-beta 2 and approximately 20 to 23 kDa. The DSF molecule is therefore closely related to TGF-beta 2 but as released from decidua, differs in size. The TGF-beta 2-r...

Research paper thumbnail of Transforming growth factor-beta s in mammary tumorigenesis: promoters or antipromoters?

Progress in clinical and biological research

Research paper thumbnail of Complex regulation of TGF beta expression by retinoic acid in the vitamin A-deficient rat

Development (Cambridge, England), 1991

We report the results of a histochemical study, using polyclonal antipeptide antibodies to the di... more We report the results of a histochemical study, using polyclonal antipeptide antibodies to the different TGF beta isoforms, which demonstrates that retinoic acid regulates the expression of TGF beta 2 in the vitamin A-deficient rat. Basal expression of TGF beta 2 diminished under conditions of vitamin A deficiency. Treatment with retinoic acid caused a rapid and transient induction of TGF beta 2 and TGF beta 3 in the epidermis, tracheobronchial and alveolar epithelium, and intestinal mucosa. Induction of TGF beta 1 expression was also observed in the epidermis. In contrast to these epithelia, expression of the three TGF beta isoforms increased in vaginal epithelium during vitamin A deficiency, and decreased following systemic administration of retinoic acid. Our results show for the first time the widespread regulation of TGF beta expression by retinoic acid in vivo, and suggest a possible mechanism by which retinoics regulate the functions of both normal and pre-neoplastic epithelia.

Research paper thumbnail of TRPA1 is required for TGF-β signaling and its loss blocks inflammatory fibrosis in mouse corneal stroma

Laboratory investigation; a journal of technical methods and pathology, 2014

We examined whether the loss of transient receptor potential ankyrin 1 (TRPA1), an irritant-sensi... more We examined whether the loss of transient receptor potential ankyrin 1 (TRPA1), an irritant-sensing ion channel, or TRPA1 antagonist treatment affects the severity inflammation and scarring during tissue wound healing in a mouse cornea injury model. In addition, the effects of the absence of TRPA1 on transforming growth factor β1 (TGF-β1)-signaling activation were studied in cell culture. The lack of TRPA1 in cultured ocular fibroblasts attenuated expression of TGF-β1, interleukin-6, and α-smooth muscle actin, a myofibroblast the marker, but suppressed the activation of Smad3, p38 MAPK, ERK, and JNK. Stroma of the healing corneas of TRPA1(-/-) knockout (KO) mice appeared more transparent compared with those of wild-type mice post-alkali burn. Eye globe diameters were measured from photographs. An examination of the corneal surface and eye globes suggested the loss of TRPA1 suppressed post-alkali burn inflammation and fibrosis/scarring, which was confirmed by histology, immunohistoch...

Research paper thumbnail of Epithelial-mesenchymal transition as a therapeutic target for prevention of ocular tissue fibrosis

Endocrine, metabolic & immune disorders drug targets, 2008

Fibrotic diseases are characterized by the appearance of myofibroblasts, the key cell type involv... more Fibrotic diseases are characterized by the appearance of myofibroblasts, the key cell type involved in the fibrogenic reaction, and by excess accumulation of extracellular matrix with resultant tissue contraction and impaired function. Myofiborblasts are generated by fibroblast-myofibrobalst conversion, and in certain tissues through epithelial-mesenchymal transition (EMT), a process through which an epithelial cell changes its phenotype to become more like a mesenchymal cell. Although inflammatory/fibrogenic growth factors/cytokines produced by injured tissues orchestrate the process of EMT, transforming growth factor beta (TGFbeta) is believed to play a central role in the process. Unlike fibrotic lesions in kidney or other tissues where myofibroblasts are generated from both fibroblasts and epithelial cells, fibrotic lesions in the eye crystalline lens are derived only from lens epithelial cells without contamination of fibroblast-derived myofibroblasts. Thus, this tissue is suit...

Research paper thumbnail of Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death

Proceedings of the National Academy of Sciences of the United States of America, Jan 15, 1993

To delineate specific developmental roles of transforming growth factor beta 1 (TGF-beta 1) we ha... more To delineate specific developmental roles of transforming growth factor beta 1 (TGF-beta 1) we have disrupted its cognate gene in mouse embryonic stem cells by homologous recombination to generate TGF-beta 1 null mice. These mice do not produce detectable amounts of either TGF-beta 1 RNA or protein. After normal growth for the first 2 weeks they develop a rapid wasting syndrome and die by 3-4 weeks of age. Pathological examination revealed an excessive inflammatory response with massive infiltration of lymphocytes and macrophages in many organs, but primarily in heart and lungs. Many lesions resembled those found in autoimmune disorders, graft-vs.-host disease, or certain viral diseases. This phenotype suggests a prominent role for TGF-beta 1 in homeostatic regulation of immune cell proliferation and extravasation into tissues.

Research paper thumbnail of Induction of transforming growth factor beta in hormonally treated human prostate cancer

British journal of cancer, 1994

Transforming growth factor beta-1 (TGF-beta 1) has been proposed as a mediator of tumour growth i... more Transforming growth factor beta-1 (TGF-beta 1) has been proposed as a mediator of tumour growth in a number of tumours and cell lines including prostate, and in a recent study was shown to be up-regulated in the stroma of breast cancer tissue following treatment with the anti-oestrogen tamoxifen. Immunolocalisation of the intracellular form of TGF-beta 1 confirmed that the source of the stromal TGF-beta 1 was the peritumoral fibroblasts. We present here the results of a study in which five patients with hormonally unresponsive prostatic carcinoma and seven patients responding to a luteinising hormone-releasing hormone analogue had prostate biopsies taken before and during treatment. These were stained for TGF-beta expression prior to treatment and at either relapse or 3 months later respectively. Six of seven clinically responding tumours and three of five relapsed tumours showed up-regulation of extracellular TGF-beta 1, again primarily in the stroma, with no apparent up-regulation...

Research paper thumbnail of Imatinib mesylate for the treatment of steroid-refractory sclerotic-type cutaneous chronic graft-versus-host disease

Biology of Blood and Marrow Transplantation, 2015

Sclerotic skin manifestations of chronic graft-versus-host disease (ScGVHD) lead to significant m... more Sclerotic skin manifestations of chronic graft-versus-host disease (ScGVHD) lead to significant morbidity, including functional disability from joint range of motion (ROM) restriction. No superior second-line therapy has been established for steroid-refractory disease. Imatinib mesylate is a multi-kinase inhibitor of several signaling pathways implicated in skin fibrosis with in vitro antifibrotic activity. We performed an open label pilot Phase 2 trial of imatinib in children and adults with corticosteroid refractory ScGVHD. Twenty patients were enrolled in a 6 month trial. Eight received a standard dose (adult: 400 mg daily; children: 260 mg/m(2) daily). Due to poor tolerability, 12 additional patients underwent a dose escalation regimen (adult: 100 mg daily initial dose up to 200 mg daily maximum; children initial dose 65 mg/m(2) daily up to 130 mg/m(2) daily). Fourteen patients were evaluable for primary response, improvement in joint range of motion (ROM) deficit, at 6 months. Primary outcome criteria for partial response (PR) was met in 5/14 (36%), stable disease (SD) in 7/14 (50%), and progressive disease (PD) in 2/14 (14%) patients. Eleven (79%) patients, including 5 PR and 6 with SD, demonstrated a positive gain in ROM (range 3-94% improvement in deficit). Of 13 patients with measurable changes at 6 months, the average improvement in ROM deficit was 24.2% (IQR: 15.5% to 30.5%; p=0.011). This trial is registered at http://clinicaltrials.gov as NCT007020689.

Research paper thumbnail of Semisynthetic derivatives of glucagon: (des-His1)N epsilon-acetimidoglucagon and N alpha-Biotinyl-N epsilon-acetimidoglucagon

Biochemistry

N epsilon-Acetimidoglucagon to be used for semisynthesis was prepared by reacting glucagon with m... more N epsilon-Acetimidoglucagon to be used for semisynthesis was prepared by reacting glucagon with methyl acetimidate hydrochloride at pH 10.2, favoring acetimidation of the sole epsilon-amino group. N epsilon-Acetimidoglucagon was isolated from the crude acetimidoglucagon mixture by anion-exchange chromatography at pH 9.4, producing a derivative which was identical with native glucagon on isoelectric focusing and which by amino acid analysis had greater than 98% of the lysine blocked. The yield was greater than that obtained when tetrahydrophthalic anhydride was used as a chromatographic handle to remove peptides with unreacted amino groups. N epsilon-Acetimidoglucagon closely resembled native glucagon in its biological activity and binding affinity, eliminating the need for deprotection. Semisynthetic N alpha-biotinyl-N epsilon-acetimidoglucagon, prepared by reacting (N-hydroxysuccinimido)biotin with N epsilon-acetimidoglucagon and purified by cation-exchange chromatography, was homo...

Research paper thumbnail of Definition of smad3 phosphorylation events that affect malignant and metastatic behaviors in breast cancer cells

Cancer research, 2014

Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negativ... more Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFβ, the TGFβ receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFβ-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition, and invasive activity. Moreover, all TGFβ responses were completely lost on mutation of the...

Research paper thumbnail of Inducible Nitric Oxide Production and Expression of Transforming Growth Factor-β1 in Serum and CSF after Cerebral Ischaemic Stroke in Man

Nitric Oxide-biology and Chemistry, 1998

A residual blood supply to the ischaemic brain is a crucial determinant for tissue survival. Earl... more A residual blood supply to the ischaemic brain is a crucial determinant for tissue survival. Early changes in the vascular network and subsequent angiogenesis may be mediated by short-lived molecules like nitric oxide (NO) or growth factors such as transforming growth factor-β1 (TGF-β1). Although TGF-β1 can inhibit NO production, this interaction has not been studied after ischaemia in humans. Serum

Research paper thumbnail of Smad3 Signaling Is Required for Epithelial-Mesenchymal Transition of Lens Epithelium after Injury

American Journal of Pathology, 2004

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract... more Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-␤ and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-␤2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-␤ expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, ␣-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-␤ in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery . Certain cells have an inherent plasticity such that their morphology and phenotype can be modulated by various growth factors and extracellular stimuli. As an example,

Research paper thumbnail of Imatinib mesylate for the treatment of steroid-refractory sclerotic-type cutaneous chronic graft-versus-host disease

Biology of Blood and Marrow Transplantation, 2015

Sclerotic skin manifestations of chronic graft-versus-host disease (ScGVHD) lead to significant m... more Sclerotic skin manifestations of chronic graft-versus-host disease (ScGVHD) lead to significant morbidity, including functional disability from joint range of motion (ROM) restriction. No superior second-line therapy has been established for steroid-refractory disease. Imatinib mesylate is a multi-kinase inhibitor of several signaling pathways implicated in skin fibrosis with in vitro antifibrotic activity. We performed an open label pilot Phase 2 trial of imatinib in children and adults with corticosteroid refractory ScGVHD. Twenty patients were enrolled in a 6 month trial. Eight received a standard dose (adult: 400 mg daily; children: 260 mg/m(2) daily). Due to poor tolerability, 12 additional patients underwent a dose escalation regimen (adult: 100 mg daily initial dose up to 200 mg daily maximum; children initial dose 65 mg/m(2) daily up to 130 mg/m(2) daily). Fourteen patients were evaluable for primary response, improvement in joint range of motion (ROM) deficit, at 6 months. Primary outcome criteria for partial response (PR) was met in 5/14 (36%), stable disease (SD) in 7/14 (50%), and progressive disease (PD) in 2/14 (14%) patients. Eleven (79%) patients, including 5 PR and 6 with SD, demonstrated a positive gain in ROM (range 3-94% improvement in deficit). Of 13 patients with measurable changes at 6 months, the average improvement in ROM deficit was 24.2% (IQR: 15.5% to 30.5%; p=0.011). This trial is registered at http://clinicaltrials.gov as NCT007020689.

Research paper thumbnail of Transient adenoviral gene transfer of Smad7 prevents injury-induced epithelial–mesenchymal transition of lens epithelium in mice

Laboratory Investigation, 2004

We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad i... more We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFb/ activin signaling, on injury-induced epithelial-mesenchymal transition (EMT) of lens epithelium in mice. A volume of 3 ll of adenoviral solution was injected into the right lens of adult male C57BL/6 mice (n ¼ 56) at the time of capsular injury made using a hypodermic needle under general anesthesia. A mixture of recombinant adenovirus carrying CAG promoter-driven Cre (Cre adv) and mouse Smad7 complementary DNA (Smad7 adv) was administered to induce Smad7 expression, while control lenses were treated with Cre adv alone. After healing intervals of 2, 3, 5, and 10 days, animals were killed 2 h after labeling with bromodeoxyuridine (BrdU) and eyes were processed for histology. During healing, marked expression of Smad7 was observed in lens epithelial cells in the Smad7 adv group with loss of nuclear translocation of Smads2/3, while little Smad7 and abundant nuclear Smads2/3 were seen in cells in the Cre adv group. Lens epithelial cells in the Cre adv control group exhibited a fibroblastic appearance at days 5 and 10 and the capsular break was sealed with fibrous tissue, while Smad7 adv-treated cells around the capsular break retained their epithelial morphology and the break was not sealed. Expression of snail mRNA, and a-smooth muscle actin, lumican, and collagen VI proteins, markers of EMT, was observed in control-treated eyes, but not in cells of the Smad7 adv group at day 5 with minimal expression at day 10. Additionally, cell proliferation increased in epithelium infected with Smad7 adv consistent with suppression of injury-induced upregulation of TGFb1 in epithelium. We conclude that gene transfer of Smad7 in mice prevents injury-induced EMT of lens epithelial cells and sealing of the capsular break with fibrous tissue.

Research paper thumbnail of Conditional overexpression of TGF-β1 disrupts mouse salivary gland development and function

Laboratory Investigation, 2010

Transforming growth factor-β (TGF-β) signaling is known to affect salivary gland physiology by in... more Transforming growth factor-β (TGF-β) signaling is known to affect salivary gland physiology by influencing branching morphogenesis, regulating ECM deposition, and controlling immune homeostasis. To study the role of TGF-β1 in the salivary gland, we created a transgenic mouse (β1 glo ) that conditionally over-expresses the active TGF-β1 upon genomic recombination by the Cre recombinase. The β1 glo mice were bred with a MMTV (mouse mammary tumor virus)-Cre (MC) transgenic line that expresses the Cre recombinase predominantly in the secretory cells of both the mammary and salivary glands. Although most of the double positive (β1 glo /MC) pups die either in utero or just after birth, clear defects in salivary gland morphogenesis could be seen such as reduced branching and increased mesenchyme. The β1 glo /MC mice that survived into adulthood, however, had hyposalivation due to salivary gland fibrosis and acinar atrophy. Increased TGF-β signaling was observed in the salivary gland with elevated phosphorylation of Smad2 and a concomitant increase in ECM deposition. In particular, aberrant TGF-β1 overexpression caused salivary gland hypofunction in this mouse model because of the replacement of normal glandular parenchyma with interstitial fibrous tissue. These results further implicate TGF-β in pathological cases of salivary gland inflammation and fibrosis that occur with chronic infections in the glands or with the autoimmune disease, Sjögren's syndrome or with the radiation therapy given to head-and-neck cancer patients.

Research paper thumbnail of Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in mice

Laboratory Investigation, 2004

Retinal pigment epithelial (RPE) cells dedifferentiate and undergo epithelial-mesenchymal transit... more Retinal pigment epithelial (RPE) cells dedifferentiate and undergo epithelial-mesenchymal transition (EMT) following retinal detachment, playing a central role in formation of fibrous tissue on the detached retina and vitreous retraction (proliferative vitreoretinopathy (PVR)). We have developed a mouse model of subretinal fibrosis with implications for PVR in which retinal detachment is induced without direct damage to the RPE cells. Transforming growth factor-b (TGF-b) has long been implicated both in EMT of RPEs and the development of PVR. Using mice null for Smad3, a key signaling intermediate downstream of TGF-b and activin receptors, we show that Smad3 is essential for EMT of RPE cells induced by retinal detachment. De novo accumulation of fibrous tissue derived from multilayered RPE cells was seen following experimental retinal detachment in eyes of wild type, but not Smad3-null mice. Expression of a-smooth muscle actin, a hallmark of EMT in this cell type, and extracellular matrix components, lumican and collagen VI, were also not observed in eyes of Smad3-null mice. Our data show that induction of PDGF-BB by Smad3-dependent TGF-b signaling is likely an important secondary proliferative component of the disease process. The results suggest that blocking the Smad3 pathway might be beneficial in prevention/treatment of PVR.

Research paper thumbnail of TGF-β Rescues Target-deprived Preganglionic Sympathetic Neurons in the Spinal Cord

European Journal of Neuroscience, 1996

Transforming growth factors P (TGF-P), a family of pleiotropic cytokines, are widely distributed ... more Transforming growth factors P (TGF-P), a family of pleiotropic cytokines, are widely distributed in the developing and adult nervous system. In order to further determine the neural functions of TGF-P, we have localized the TGF-P isoforms 1, 2 and 3 in the adult rat adrenal medulla and studied the neuroprotective capacity of one representative family member, TGF-P2, for those spinal cord neurons which innervate adrenal chromaffin cells and which die after destruction of the adrenal medulla. Unilateral electrothermal destruction of the adrenal medulla led to the disappearance of 25% of sympathetic preganglionic neurons, which are located in the intermediolateral (IML) column of thoracic spinal cord segments 7-1 0 and can be selectively marked by NADPHdiaphorase. The neurons which disappeared following adrenomedullectomy constitute the full set of neurons that innervate the adrenal medulla. Implantation of gelfoam soaked with 0.5 pg TGF-P2 into the adrenal wound cavity rescued all spinal cord neurons in the IML ipsilaterally to the lesioned side. Cytochrome c was not effective. Injections of ['251]TGF-P2 into the adrenal medulla did not result in retrograde transport and subsequent labelling of spinal cord neurons, suggesting that TGF-P may exert its neuroprotective actions by indirect mechanisms. TGF-P applied to cultured adrenocortical cells did not overtly increase the amount of mRNA for fibroblast growth factor-2, an established trophic molecule for sympathetic preganglionic spinal cord neurons. The mechanisms by which TGF-P exerts its neurotrophic effect are therefore unclear. Even so, our data provide the first evidence that TGF-P may play an important role in vivo in the control of maintenance of a population of spinal cord neurons.

Research paper thumbnail of Expression of Smad7 in Mouse Eyes Accelerates Healing of Corneal Tissue after Exposure to Alkali

The American Journal of Pathology, 2005

Damage to the cornea from chemical burns is a serious clinical problem that often leads to perman... more Damage to the cornea from chemical burns is a serious clinical problem that often leads to permanent visual impairment. Because transforming growth factor (TGF)-beta has been implicated in the response to corneal injury, we evaluated the effects of altered TGF-beta signaling in a corneal alkali burn model using mice treated topically with an adenovirus (Ad) expressing inhibitory Smad7 and mice with a targeted deletion of the TGF-beta/activin signaling mediator Smad3. Expression of exogenous Smad7 in burned corneal tissue resulted in reduced activation of Smad signaling and nuclear factor-kappaB signaling via RelA/p65. Resurfacing of the burned cornea by conjunctival epithelium and its differentiation to cornea-like epithelium were both accelerated in Smad7-Ad-treated corneas with suppressed stromal ulceration, opacification, and neovascularization 20 days after injury. Introduction of the Smad7 gene suppressed invasion of monocytes/macrophages and expression of monocyte/macrophage chemotactic protein-1, TGF-beta1, TGF-beta2, vascular endothelial growth factor, matrix metalloproteinase-9, and tissue inhibitors of metalloproteinase-2 and abolished the generation of myofibroblasts. Although acceleration of healing of the burned cornea was also observed in mice lacking Smad3, the effects on epithelial and stromal healing were less pronounced than those in corneas treated with Smad7. Together these data suggest that overexpression of Smad7 may have effects beyond those of simply blocking Smad3/TGF-beta signaling and may represent an effective new strategy for treatment of ocular burns.

Research paper thumbnail of Therapeutic Effect of Topical Administration of SN50, an Inhibitor of Nuclear Factor-κB, in Treatment of Corneal Alkali Burns in Mice

The American Journal of Pathology, 2005

We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear ... more We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear factor-B, in a corneal alkali burn model in mice. An alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. SN50 (10 g/ l) or vehicle was topically administered daily for up to 12 days. The eyes were processed for histological or immunohistochemical examination after bromodeoxyuridine labeling or for semiquantification of cytokine mRNA. Topical SN50 suppressed nuclear factor-B activation in local cells and reduced the incidence of epithelial defects/ulceration in healing corneas. Myofibroblast generation, macrophage invasion, activity of matrix metalloproteinases, basement membrane destruction, and expression of cytokines were all decreased in treated corneas compared with controls. To elucidate the role of tumor necrosis factor (TNF)-␣ in epithelial cell proliferation, we performed organ culture of mouse eyes with TNF-␣, SN50, or an inhibitor of c-Jun N-terminal kinase (JNK) and examined cell proliferation in healing corneal epithelium in TNF-␣ ؊/؊ mice treated with SN50. An acceleration of epithelial cell proliferation by SN50 treatment was found to depend on TNF-␣/JNK signaling. In conclusion, topical application of SN50 is effective in treating corneal alkali burns in mice.

Research paper thumbnail of Smad3 Signaling Is Required for Epithelial-Mesenchymal Transition of Lens Epithelium after Injury

The American Journal of Pathology, 2004

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract... more Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-␤ and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-␤2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-␤ expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, ␣-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-␤ in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery . Certain cells have an inherent plasticity such that their morphology and phenotype can be modulated by various growth factors and extracellular stimuli. As an example,

Research paper thumbnail of Murine pregnancy decidua produces a unique immunosupressive molecule related to transforming growth factor β-2

The Journal of Immunology

Non-T small lymphocytic suppressor cells in murine allopregnancy release a potent immunosuppressi... more Non-T small lymphocytic suppressor cells in murine allopregnancy release a potent immunosuppressive factor in vitro that is neutralized by rabbit anti-transforming growth factor (TGF)-beta. Previous studies have suggested that the decidual suppressor factor (DSF) is smaller than TGF-beta 1, and in this paper, we show that DSF on HPLC-sieving columns also elutes later than TGF-beta 2. Nevertheless, DSF has the ability to promote anchorage-independent growth of NRK fibroblasts similar to TGF-beta s. Using turkey antibodies specific for TGF-beta 1 or beta 2, we show that DSF is related to TGF-beta 2 rather than TGF-beta 1, and this relationship was confirmed by using a panel of murine mAb to TGF-subtypes. PAGE and Western blotting showed that the TGF-beta 2-reactive molecules in HPLC-purified DSF was slightly smaller than TGF-beta 2 and approximately 20 to 23 kDa. The DSF molecule is therefore closely related to TGF-beta 2 but as released from decidua, differs in size. The TGF-beta 2-r...

Research paper thumbnail of Transforming growth factor-beta s in mammary tumorigenesis: promoters or antipromoters?

Progress in clinical and biological research

Research paper thumbnail of Complex regulation of TGF beta expression by retinoic acid in the vitamin A-deficient rat

Development (Cambridge, England), 1991

We report the results of a histochemical study, using polyclonal antipeptide antibodies to the di... more We report the results of a histochemical study, using polyclonal antipeptide antibodies to the different TGF beta isoforms, which demonstrates that retinoic acid regulates the expression of TGF beta 2 in the vitamin A-deficient rat. Basal expression of TGF beta 2 diminished under conditions of vitamin A deficiency. Treatment with retinoic acid caused a rapid and transient induction of TGF beta 2 and TGF beta 3 in the epidermis, tracheobronchial and alveolar epithelium, and intestinal mucosa. Induction of TGF beta 1 expression was also observed in the epidermis. In contrast to these epithelia, expression of the three TGF beta isoforms increased in vaginal epithelium during vitamin A deficiency, and decreased following systemic administration of retinoic acid. Our results show for the first time the widespread regulation of TGF beta expression by retinoic acid in vivo, and suggest a possible mechanism by which retinoics regulate the functions of both normal and pre-neoplastic epithelia.

Research paper thumbnail of TRPA1 is required for TGF-β signaling and its loss blocks inflammatory fibrosis in mouse corneal stroma

Laboratory investigation; a journal of technical methods and pathology, 2014

We examined whether the loss of transient receptor potential ankyrin 1 (TRPA1), an irritant-sensi... more We examined whether the loss of transient receptor potential ankyrin 1 (TRPA1), an irritant-sensing ion channel, or TRPA1 antagonist treatment affects the severity inflammation and scarring during tissue wound healing in a mouse cornea injury model. In addition, the effects of the absence of TRPA1 on transforming growth factor β1 (TGF-β1)-signaling activation were studied in cell culture. The lack of TRPA1 in cultured ocular fibroblasts attenuated expression of TGF-β1, interleukin-6, and α-smooth muscle actin, a myofibroblast the marker, but suppressed the activation of Smad3, p38 MAPK, ERK, and JNK. Stroma of the healing corneas of TRPA1(-/-) knockout (KO) mice appeared more transparent compared with those of wild-type mice post-alkali burn. Eye globe diameters were measured from photographs. An examination of the corneal surface and eye globes suggested the loss of TRPA1 suppressed post-alkali burn inflammation and fibrosis/scarring, which was confirmed by histology, immunohistoch...

Research paper thumbnail of Epithelial-mesenchymal transition as a therapeutic target for prevention of ocular tissue fibrosis

Endocrine, metabolic & immune disorders drug targets, 2008

Fibrotic diseases are characterized by the appearance of myofibroblasts, the key cell type involv... more Fibrotic diseases are characterized by the appearance of myofibroblasts, the key cell type involved in the fibrogenic reaction, and by excess accumulation of extracellular matrix with resultant tissue contraction and impaired function. Myofiborblasts are generated by fibroblast-myofibrobalst conversion, and in certain tissues through epithelial-mesenchymal transition (EMT), a process through which an epithelial cell changes its phenotype to become more like a mesenchymal cell. Although inflammatory/fibrogenic growth factors/cytokines produced by injured tissues orchestrate the process of EMT, transforming growth factor beta (TGFbeta) is believed to play a central role in the process. Unlike fibrotic lesions in kidney or other tissues where myofibroblasts are generated from both fibroblasts and epithelial cells, fibrotic lesions in the eye crystalline lens are derived only from lens epithelial cells without contamination of fibroblast-derived myofibroblasts. Thus, this tissue is suit...

Research paper thumbnail of Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death

Proceedings of the National Academy of Sciences of the United States of America, Jan 15, 1993

To delineate specific developmental roles of transforming growth factor beta 1 (TGF-beta 1) we ha... more To delineate specific developmental roles of transforming growth factor beta 1 (TGF-beta 1) we have disrupted its cognate gene in mouse embryonic stem cells by homologous recombination to generate TGF-beta 1 null mice. These mice do not produce detectable amounts of either TGF-beta 1 RNA or protein. After normal growth for the first 2 weeks they develop a rapid wasting syndrome and die by 3-4 weeks of age. Pathological examination revealed an excessive inflammatory response with massive infiltration of lymphocytes and macrophages in many organs, but primarily in heart and lungs. Many lesions resembled those found in autoimmune disorders, graft-vs.-host disease, or certain viral diseases. This phenotype suggests a prominent role for TGF-beta 1 in homeostatic regulation of immune cell proliferation and extravasation into tissues.

Research paper thumbnail of Induction of transforming growth factor beta in hormonally treated human prostate cancer

British journal of cancer, 1994

Transforming growth factor beta-1 (TGF-beta 1) has been proposed as a mediator of tumour growth i... more Transforming growth factor beta-1 (TGF-beta 1) has been proposed as a mediator of tumour growth in a number of tumours and cell lines including prostate, and in a recent study was shown to be up-regulated in the stroma of breast cancer tissue following treatment with the anti-oestrogen tamoxifen. Immunolocalisation of the intracellular form of TGF-beta 1 confirmed that the source of the stromal TGF-beta 1 was the peritumoral fibroblasts. We present here the results of a study in which five patients with hormonally unresponsive prostatic carcinoma and seven patients responding to a luteinising hormone-releasing hormone analogue had prostate biopsies taken before and during treatment. These were stained for TGF-beta expression prior to treatment and at either relapse or 3 months later respectively. Six of seven clinically responding tumours and three of five relapsed tumours showed up-regulation of extracellular TGF-beta 1, again primarily in the stroma, with no apparent up-regulation...

Research paper thumbnail of Imatinib mesylate for the treatment of steroid-refractory sclerotic-type cutaneous chronic graft-versus-host disease

Biology of Blood and Marrow Transplantation, 2015

Sclerotic skin manifestations of chronic graft-versus-host disease (ScGVHD) lead to significant m... more Sclerotic skin manifestations of chronic graft-versus-host disease (ScGVHD) lead to significant morbidity, including functional disability from joint range of motion (ROM) restriction. No superior second-line therapy has been established for steroid-refractory disease. Imatinib mesylate is a multi-kinase inhibitor of several signaling pathways implicated in skin fibrosis with in vitro antifibrotic activity. We performed an open label pilot Phase 2 trial of imatinib in children and adults with corticosteroid refractory ScGVHD. Twenty patients were enrolled in a 6 month trial. Eight received a standard dose (adult: 400 mg daily; children: 260 mg/m(2) daily). Due to poor tolerability, 12 additional patients underwent a dose escalation regimen (adult: 100 mg daily initial dose up to 200 mg daily maximum; children initial dose 65 mg/m(2) daily up to 130 mg/m(2) daily). Fourteen patients were evaluable for primary response, improvement in joint range of motion (ROM) deficit, at 6 months. Primary outcome criteria for partial response (PR) was met in 5/14 (36%), stable disease (SD) in 7/14 (50%), and progressive disease (PD) in 2/14 (14%) patients. Eleven (79%) patients, including 5 PR and 6 with SD, demonstrated a positive gain in ROM (range 3-94% improvement in deficit). Of 13 patients with measurable changes at 6 months, the average improvement in ROM deficit was 24.2% (IQR: 15.5% to 30.5%; p=0.011). This trial is registered at http://clinicaltrials.gov as NCT007020689.

Research paper thumbnail of Semisynthetic derivatives of glucagon: (des-His1)N epsilon-acetimidoglucagon and N alpha-Biotinyl-N epsilon-acetimidoglucagon

Biochemistry

N epsilon-Acetimidoglucagon to be used for semisynthesis was prepared by reacting glucagon with m... more N epsilon-Acetimidoglucagon to be used for semisynthesis was prepared by reacting glucagon with methyl acetimidate hydrochloride at pH 10.2, favoring acetimidation of the sole epsilon-amino group. N epsilon-Acetimidoglucagon was isolated from the crude acetimidoglucagon mixture by anion-exchange chromatography at pH 9.4, producing a derivative which was identical with native glucagon on isoelectric focusing and which by amino acid analysis had greater than 98% of the lysine blocked. The yield was greater than that obtained when tetrahydrophthalic anhydride was used as a chromatographic handle to remove peptides with unreacted amino groups. N epsilon-Acetimidoglucagon closely resembled native glucagon in its biological activity and binding affinity, eliminating the need for deprotection. Semisynthetic N alpha-biotinyl-N epsilon-acetimidoglucagon, prepared by reacting (N-hydroxysuccinimido)biotin with N epsilon-acetimidoglucagon and purified by cation-exchange chromatography, was homo...

Research paper thumbnail of Definition of smad3 phosphorylation events that affect malignant and metastatic behaviors in breast cancer cells

Cancer research, 2014

Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negativ... more Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFβ, the TGFβ receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFβ-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition, and invasive activity. Moreover, all TGFβ responses were completely lost on mutation of the...