Treatment of polycystic kidney disease with a novel tyrosine kinase inhibitor

Treatment of polycystic kidney disease with a novel tyrosine kinase inhibitor. BACKGROUND: We have previously demonstrated an essential role for increased epidermal growth factor receptor (EGFR) activity in mediating renal cyst formation and biliary epithelial hyperplasia in murine models of autosomal recessive polycystic kidney disease (ARPKD). This study was designed to determine whether or not treatment with a newly developed inhibitor of EGFR tyrosine kinase activity (EKI-785) would reduce renal and biliary abnormalities in murine ARPKD. METHODS: Balb/c-bpk/bpk (BPK) litters were treated with EKI-785, an EGFR-specific tyrosine kinase inhibitor. Animals were treated by intraperitoneal injection beginning at postnatal day 7 and were treated until postnatal day 24 or 48. EKI-785's effectiveness was measured by a reduction in the renal cystic index, an increased life span, and maintenance of normal renal function. RESULTS: Treatment of BPK mice with EKI-785 resulted in a marked reduction of collecting tubule (CT) cystic lesions, improved renal function, decreased biliary epithelial abnormalities, and an increased life span. Untreated cystic animals died of renal failure at postnatal day 24 (P-24) with a CT cystic index of 4.8, a maximal urine osmolarity of 361 mOsm, and moderate to severe biliary abnormalities. Cystic animals treated with EKI-785 to postnatal day 48 (P-48) were alive and well with normal renal function, a reduced CT cystic index of 2.0 (P < 0.02), a threefold increased in maximum urinary concentrating ability (P < 0.01), and a significant decrease in biliary epithelial proliferation/fibrosis (P < 0.01). CONCLUSION: This study demonstrates that EKI-785 has therapeutic effectiveness in improving histopathologic abnormalities and decreasing mortality in murine ARPKD.     

Capecitabine induces rapid, sustained response in two patients with extensive oral verrucous carcinoma.

PURPOSE: Oral verrucous carcinoma (VC) has been traditionally treated with surgery or radiation with frequent recurrences and significant morbidity. We describe rapid and dramatic response to oral capecitabine in two patients with advanced refractory VC. EXPERIMENTAL DESIGN: VC is a rare tumor of the oral cavity. It does not metastasize but over time causes morbidity and mortality through local invasion. Radiation and surgery have been the main treatment modalities but are plagued by mutilating effects, recurrences, and possibly malignant degeneration in some cases. To date, no effective chemotherapy regimens have been described in the international literature. The clinical records of two elderly females with extensive oral VC are described. Both patients were prescribed one cycle of capecitabine, 1,000 mg bid for 14 days. Response was documented by photography in one patient. Immunohistochemical evaluation of three 5-fluorouracil metabolizing enzymes on pretreatment biopsies from both patients was also performed. A review of the literature with emphasis on treatment of oral VC is presented in view of our findings. RESULTS: Examination of the oral cavity before treatment revealed extensive involvement with an evenly spreading, exophytic, warty, whitish lesion in both patients. Microscopic examination of H&E-stained slides from biopsies of these lesions confirmed the clinical suspicion of VC. Both patients underwent treatment with oral capecitabine for one cycle (2 weeks on/1 week off) at a reduced dose of 1000 mg, p.o., bid. Both had a dramatic response with near complete resolution of their lesions within 3 weeks of initiating therapy. A durable partial response was documented at 1 year in the first patient and 6 months in the second. Immunohistochemical evaluation of pretreatment biopsies from both patients revealed a high level of expression of thymidine phosphorylase, a key enzyme in the metabolism of capecitabine. CONCLUSIONS: Oral VC is a rare entity with a progressive course over years and limited options in terms of treatment. Preliminary observations in two elderly patients demonstrate that capecitabine, an oral fluoropyrimidine, is well tolerated and may induce rapid, clinically significant response. Although not curative, it may provide a cost-effective alternative for elderly patients with a significant improvement in their quality of life.     

The Utah paradigm of skeletal physiology: an overview of its insights for bone, cartilage and collagenous tissue organs

In a 1960 paradigm of skeletal physiology, effector cells (chondroblasts, fibroblasts, osteoblasts, osteoclasts, etc.) regulated by nonmechanical agents wholly determined the architecture, strength, and health of bones, joints, fascia, ligaments, and tendons. Biomechanical and tissue-level phenomena had no roles in that paradigm. Subsequent studies and evidence slowly revealed skeletal tissue-level mechanisms and their functions, including biomechanical ones, as well as "game rules" that seem to govern them. That slow discovery process found that effector cells are only parts of tissue-level mechanisms, as kidney cells are only parts of nephrons and wheels are only parts of cars. Normally all those things help to determine skeletal architecture, strength, and health, and adding them to the 1960 paradigm led to the still-evolving Utah paradigm of skeletal physiology that concerns, in part, how load-bearing skeletal organs adapt to the voluntary mechanical loads on them. That caused controversies this article does not try to resolve; instead, it describes some issues they concern. In that regard, controversy can depend on how one assesses the relevance of facts to a problem more than on their accuracy. If a paradigm added new facts to a former one and the new one's advocates viewed all those facts as relevant, but the former's advocates questioned the relevance of some of the new facts, their views about a problem could differ even though each view depended on accurate facts. Readers would make their own judgments about the bearing of those ideas on this article's content. Received: October 5, 1999 / Accepted: January 13, 2000     

Three isolation techniques for primary culture of human osteoblast-like cells: A comparison

The culture of osteoblast-like cells of human origin has become an important experimental model in bone biology. We report here a comparison and evaluation of three of the most widely used systems available today: bone marrow stroma cell cultures (BMSC), human osteoblast explant cultures (hOB) and osteoblast explant cultures from collagenase-treated bone (hOB^col). Cultures from 16 bone specimens obtained from various donors were established and their expression of the osteoblast phenotype were then compared in secondary cultures by use of biochemical markers. BMSC had the highest basal and 1,25-dihy-droxyvitaminD₃ (1,25(OH)₂D.))-induced alkaline phosphatase activities in all cell isolations, with levels approximately twice those in explant cultures. Basal osteocalcin secretion was low-to-undetectable in all cell cultures but was detected in 1,25(OH)₂D₃-stimulated cultures. BMSC produced half of the amount of osteocalcin synthesized in explant cultures. The BMSC cultures also synthesized the lowest amounts of type I collagen, whereas collagen type III synthesis did not differ significantly among the various cultures. When secondary cultures were treated with 100 nM dexam-ethasone in the presence of ascorbic acid (50 u.g/mL) and B-glycerophosphate (10 mM), cultures deposited calcium mineral into the cell layer within 2-4 weeks. PTH-induced cAMP formation was detected in only 5 of 15 isolations and no consistent isolation-dependent response pattern was seen. We conclude that BMSC cultures differ significantly from explant cultures obtained from the same bone specimen. However, all cultures represent cells which can differentiate further and induce mineralization of the extracellular matrix in response to osteoinductive drugs.     

Myelination and behaviour of tenascin-C null transgenic mice

The extracellular matrix glycoprotein tenascin-C is widely expressed during development and repair, making it surprising that few abnormalities have been found in transgenic mice lacking this molecule. We have therefore re-examined the transgenic mice described by Saga et al. [Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T. & Aizawa, S. (1992) Genes Dev., 6 1821-1831] in which tenascin-C was knocked-out by homologous recombination, focusing on two aspects of the nervous system likely to reveal any abnormalities that might follow the loss of tenascin-C. First, we have determined the pattern of myelin and distribution of oligodendrocyte precursor cells in those areas, such as the optic nerve and retina where local concentrations of tenascin-C have been proposed to act as barriers to oligodendrocyte precursor migration and so prevent inappropriate myelination. Secondly, we have examined the behaviour of the mice in a number of well-characterized tests, e.g. beam-walking, passive avoidance and the Morris water maze. We find no abnormalities of myelination or oligodendrocyte precursor distribution in adult mice, showing that local concentrations of tenascin-C are not the sole mechanism responsible for the pattern of myelination in these regions of CNS. However, we do find a number of behavioural abnormalities in these mice and show that hyperlocomotion and deficits in coordination during beam walking can be ascribed to tenascin-C deficiency. The effects on coordination are, however, not seen on a 129 genetic background. Taken together, these results significantly extend the phenotype associated with tenascin-C deficiency but argue against a role in myelination.     

Linkage disequilibrium mapping of the Nova Scotia variant of Niemann–Pick disease

Niemann-Pick type D (NPD) disease is a severe degenerative disorder of the nervous system characterized by the accumulation of tissue cholesterol and sphingomyelin. Because of a founder effect, it is unusually common in southwestern Nova Scotia, Canada. We have confirmed that almost all patients from 20 affected sibships descended on both sides from a small group of Acadians who settled in this region in about the year 1767. Previously using classic linkage analysis of this large kindred, we defined the critical gene region to a 13-cM chromosome segment between D18S869 and D18S66. Seven ESTs have been positioned within this interval. Carstea et al. (Niemann Pick C disease gene: homology to mediators of cholesterol homeostasis. Science 1997: 277: 232-235) recently demonstrated that one of these ESTs is the Niemann-Pick type C (NPCI) gene, the gene disrupted in most patients with NPC disease, and we have shown that a G3097-->T mutation in the NPC1 gene is also responsible for NPD. Here we report the development of five new polymorphic microsatellite markers and the testing for complete linkage disequilibrium in our single large NPD kindred that allowed us to reduce the NPD critical region to a 1-cM (1.3-1.6 Mb) interval between D18S1398 and D18S1108. In contrast, Carstea et al., using classic linkage analysis, required more than 18 unrelated NPC families to reduce the NPC1 critical region to a 5-cM interval. Our work supports the finding that NPD is an allelic variant of NPC1, and illustrates the power of large kindreds, which are common in Atlantic Canada and other relatively isolated areas, for gene mapping and identification.     

Walking through the forest of transgenic models of human disease

Summary: In the investigation of human disease, molecular biology has provided Immunologists with several enormously powerful tools. Trans-genic and knockout mice provide animal models to investigate mechanisms, as well as aid in the design of therapies for these diseases. These mice have been useful in several different ways. First, as direct models of disease they provide direct tools for the study of the disease. Second, expression of individual molecules can be altered in the context of established disease models. We describe here some of the models in use as well as the limitations and promise of this research.     

Thermal preference behavior following clonidine, norepinephrine, isoproterenol, and ephedrine.

A thermal gradient (temperature range 7-45 degrees C) was used to assess ambient temperature (Ta) preferences of rats following treatment with clonidine (25 microg/kg), norepinephrine (NE, 250 microg/kg), isoproterenol (ISO, 50 microg/kg), and ephedrine (EPH, 10 mg/kg). Clonidine produced a preference for a temperature (31.5 degrees C) slightly warmer than that preferred after saline (28.3 degrees C), but this resulted in no significant change in posttest colonic temperature (Tc). NE, ISO and EPH produced a preference for a colder region of the gradient (20-22 degrees C) compared to saline (24.5-28.9 degrees C). Posttest Tc was reduced significantly from 37.7-37.9 degrees C after saline to 37.2 degrees C (NE), 37.3 degrees C (ISO), and 36.8 degrees C (EPH). Thus, given the opportunity to select an environmental temperature, the animals selected a Ta that resulted in significantly lower body temperatures after NE, ISO, and EPH. This suggests that paradoxical thermoregulatory effects of these thermogenic adrenergic agonists are due, at least in part, to a preference for a lower body temperature.     

Autoradiographic and SPECT imaging of cerebral opioid receptors with an iodine-123 labeled analogue of diprenorphine

The feasibility of imaging cerebral opioid receptors by single photon emission computed tomography (SPECT) has been established in baboon using a novel analog of diprenorphine (DPN) radiolabeled with iodine-123. The radioligand, [¹²³I]-O-IA-DPN (C6-O-[¹²³I]iodoallyl-DPN), was prepared in good yield (80%) with high radiochemical purity (>97%) and high specific radioactivity (>2,400 mCi/μmol). In ex vivo autoradiographic studies, with and without naltrexone blockade, [¹²³I]-O-IA-DPN specifically labeled opioid receptors throughout the mouse brain. Nonmetabolized radioligand accounted for >90% of the signal observed in extracts of whole mouse brain. SPECT imaging trials showed that [¹²³I]-O-IA-DPN selectively localized in regions of baboon brain known to have high densities of opioid receptors, such as striatum, thalamus, and temporal cortex. A much lower level of radioligand uptake and retention was noted for cerebellum, a region with few opioid binding sites. Pretreatment with naltrexone (6.5 μmol/kg) blocked [¹²³I]-O-IA-DPN binding in all brain regions. Using naltrexone blockade to define the nonspecific component for a given region of interest, total to nonspecific binding ratios increased linearly (r ≥ 0.98) over the SPECT study with maximal values for striatum (9.8), thalamus (7.1), and temporal cortex (6.9) reached at the last time point investigated (3.5 h). Specific binding for these regions, assessed as the difference between regional SPECT activity for the control and blocked states, proved irreversible over the observation period. By the end of the time course, specific [¹²³I]-O-IA-DPN binding was >85% of total radioactivity in regions rich in opioid receptors and 62% of total radioactivity in cerebellum. The aggregate data are consistent with visualization of multiple opioid receptor types. Thus, [¹²³I]-O-IA-DPN should prove useful for SPECT studies within the constraints imposed by a lack of innate selectivity for a single type of brain opioid receptor. Synapse 29:172–182, 1998. © 1998 Wiley-Liss, Inc.     

Perspectives: A proposed general model of the “mechanostat” (suggestions from a new skeletal-biologic paradigm)

This provisional general model for the skeleton's mechanostat spans the biologic “distance” between the organ and macromolecule. It could apply to bone, cartilage and fibrous tissue, and to bones, joints, ligaments and other organs made wholly or in part from the basic tissues. It suggests where small things such as a cytokine effect on some cell should fit in the overall scheme of skeletal physiology. It proposes that interlocking negative feedback loops provide mechanical-usage-dedicated message traffic routes on which nonmechanical agents could act to optimize or impair postnatal skeletal adaptations to varied mechanical and nonmechanical challenges, and treatments of disease too. It suggests that future research must try to understand the mechanostat's cell- and molecular-biologic roots. © 1996 Wiley-Liss, Inc.