Deletion of Cdh16 Ksp-cadherin leads to a developmental delay in the ability to maximally concentrate urine in mouse.

Ksp-cadherin (cadherin-16) is an atypical member of the cadherin superfamily of cell adhesion molecules that is ubiquitously expressed on the basolateral membrane of epithelial cells lining the nephron and the collecting system of the mammalian kidney. The principal aim of the present study was to determine if Ksp-cadherin played a critical role in the development and maintenance of the adult mammalian kidney by generating and evaluating a mouse line deficient in Ksp-cadherin. Ksp-null mutant animals were viable and fertile, and kidneys from both neonates and adults showed no evidence of structural abnormalities. Immunolocalization and Western blot analyses of Na+-K+-ATPase and E-cadherin indicated that Ksp-cadherin is not essential for either the genesis or maintenance of the polarized tubular epithelial phenotype. Moreover, E-cadherin expression was not altered to compensate for Ksp-cadherin loss. Plasma electrolytes, total CO2, blood urea nitrogen, and creatinine levels were also unaffected by Ksp-cadherin deficiency. However, a subtle but significant developmental delay in the ability to maximally concentrate urine was detected in Ksp-null mice. Expression analysis of the principal proteins involved in the generation of the corticomedullary osmotic gradient and the resultant movement of water identified misexpression of aquaporin-2 in the inner medullary collecting duct as the possible cause for the inability of young adult Ksp-cadherin-deficient animals to maximally concentrate their urine. In conclusion, Ksp-cadherin is not required for normal kidney development, but its absence leads to a developmental delay in maximal urinary concentrating ability.NEW & NOTEWORTHY Ksp-cadherin (cadherin-16) is an atypical member of the cadherin superfamily of cell adhesion molecules that is ubiquitously expressed on the basolateral membrane of epithelial cells lining the nephron and the collecting system. Using knockout mice, we found that Ksp-cadherin is in fact not required for kidney development despite its high and specific expression along the nephron. However, its absence leads to a developmental delay in maximal urinary concentrating ability.

Gut microbiota. Antimicrobial peptide resistance mediates resilience of prominent gut commensals during inflammation.

Resilience to host inflammation and other perturbations is a fundamental property of gut microbial communities, yet the underlying mechanisms are not well understood. We have found that human gut microbes from all dominant phyla are resistant to high levels of inflammation-associated antimicrobial peptides (AMPs) and have identified a mechanism for lipopolysaccharide (LPS) modification in the phylum Bacteroidetes that increases AMP resistance by four orders of magnitude. Bacteroides thetaiotaomicron mutants that fail to remove a single phosphate group from their LPS were displaced from the microbiota during inflammation triggered by pathogen infection. These findings establish a mechanism that determines the stability of prominent members of a healthy microbiota during perturbation.

Effects of etonogestrel treatment in the reproductive organs and uterine arteries of nonoophorectomized guinea pigs.

The endometria of women treated with long-term progestin-only contraceptives (LTPOCs) display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow, oxidative stress, and unpredictable focal abnormal endometrial bleeding. Because human studies on the effects of LTPOC treatment are constrained for ethical and practical reasons, we assessed the suitability of nonoophorectomized guinea pigs (GPs) to best mimic the hormonal milieu of women. The present study demonstrates that treatment of GPs parallels the morphological changes following LTPOC treatment of the human endometrium and ovaries. Specifically, treatment resulted in larger hyperemic, uteri compared with controls. Histopathologic and immunohistochemical analysis demonstrated fewer endometrial glands, decreased luminal mucus, increased numbers of blood vessels, and focal hemorrhage. While increased staining for the cell mitosis marker, Ki67, was present in the zona functionalis, no such increase occurred in the basalis. Lastly, effects on vasomotor features of uterine arteries suggest changes that favor increased resistance and reduced blood flow promoting decreased ability to withstand elevations in transmural pressure.

Effective treatment of human lung cancer by targeting tissue factor with a factor VII-targeted photodynamic therapy.

Lung cancer is the leading cause of cancer death. Conventional photodynamic therapy (PDT) using a nontargeted photosensitizer (ntPDT) is a treatment option for central- and peripheral-type early-stage lung cancer. However, ntPDT can cause severe side effects for normal tissue due to its non-selective distribution. To improve the selectivity and effectiveness of ntPDT for human lung cancer, we hypothesized that tissue factor would be a common yet specific biomarker and a potential therapeutic target for both lung cancer cells and lung tumor vascular endothelial cells in factor VII-targeted PDT (fVII-tPDT), which uses the fVII-Sn(IV) chlorin e6 conjugate for the treatment of human lung cancer. We first identified that tissue factor is indeed expressed on the human non-small cell lung cancer (NSCLC) lines A549 and H460 as well as on tumor vascular endothelial cells of A549 tumor xenografts from nude mice, but it is not expressed by vascular endothelial cells in healthy mouse organs including the lungs. We then demonstrated that fVII-targeting in fVII-tPDT significantly enhanced (up to 25-fold) the in vitro effect of ntPDT on the destruction of A549 and H460 lung cancer cells via the rapid induction of apoptosis and necrosis. We further demonstrated that in vivo administration of fVIItPDT significantly inhibited or eliminated subcutaneous A549 and H460 tumor xenografts in an athymic nude (ATN) mouse model without any obvious side effects. We conclude that fVII-tPDT is effective and safe for the treatment of human lung cancer in preclinical studies and that this methodology holds therapeutic potential for lung cancer patients.

Effective treatment of chemoresistant breast cancer in vitro and in vivo by a factor VII-targeted photodynamic therapy.

BACKGROUND: The purpose of this study was to test a novel, dual tumour vascular endothelial cell (VEC)- and tumour cell-targeting factor VII-targeted Sn(IV) chlorin e6 photodynamic therapy (fVII-tPDT) by targeting a receptor tissue factor (TF) as an alternative treatment for chemoresistant breast cancer using a multidrug resistant (MDR) breast cancer line MCF-7/MDR. METHODS: The TF expression by the MCF-7/MDR breast cancer cells and tumour VECs in MCF-7/MDR tumours from mice was determined separately by flow cytometry and immunohistochemistry using anti-human or anti-murine TF antibodies. The efficacy of fVII-tPDT was tested in vitro and in vivo and was compared with non-targeted PDT for treatment of chemoresistant breast cancer. The in vitro efficacy was determined by a non-clonogenic assay using crystal violet staining for monolayers, and apoptosis and necrosis were assayed to elucidate the underlying mechanisms. The in vivo efficacy of fVII-tPDT was determined in a nude mouse model of subcutaneous MCF-7/MDR tumour xenograft by measuring tumour volume. RESULTS: To our knowledge, this is the first presentation showing that TF was expressed on tumour VECs in chemoresistant breast tumours from mice. The in vitro efficacy of fVII-tPDT was 12-fold stronger than that of ntPDT for MCF-7/MDR cancer cells, and the mechanism of action involved induction of apoptosis and necrosis. Moreover, fVII-tPDT was effective and safe for the treatment of chemoresistant breast tumours in the nude mouse model. CONCLUSIONS: We conclude that fVII-tPDT is effective and safe for the treatment of chemoresistant breast cancer, presumably by simultaneously targeting both the tumour neovasculature and chemoresistant cancer cells. Thus, this dual-targeting fVII-tPDT could also have therapeutic potential for the treatment of other chemoresistant cancers.

WAG-F8(m1Ycb) rats harboring a factor VIII gene mutation provide a new animal model for hemophilia A.

BACKGROUND: We recently described an inherited coagulopathy arising in an inbred colony of WAG/RijYcb rats. The bleeding phenotype, demonstrated by both male and female rats, included periarticular hemorrhage, spontaneous bruising, prolonged bleeding from minor wounds and maternal peripartum deaths. Coagulation testing of affected rats revealed normal prothrombin time but prolongation of activated partial thromboplastin time to twice that of controls. OBJECTIVE: To determine the specific coagulation factor and the underlying genetic defect responsible for the inherited coagulopathy in the WAG/RijYcb rats. RESULTS: Evaluation of individual clotting factor activities revealed that the affected animals had a specific deficiency of factor (F) VIII (FVIII). The FVIII gene (F8) has an autosomal location on chromosome 18 in rats, in contrast to its location on the X chromosome in mice and humans. Sequencing of F8 cDNA led to the identification of a point mutation resulting in a substitution, Leu176Pro, in the A1 domain, that is predicted to disrupt the tertiary structure of the FVIII molecule. Administration of human plasma or human recombinant FVIII corrects the coagulation abnormality in the affected animals. CONCLUSIONS: We have now identified the genetic basis of the hemostatic defect in the WAG/RijYcb rat colony. The larger size of rats relative to mice and the presence of this coagulation defect in both sexes provide a unique model, well-suited to the development of novel therapies for acquired and hereditary FVIII deficiencies.

Attenuation of signaling pathways stimulated by pathologically activated FGF-receptor 2 mutants prevents craniosynostosis.

Craniosynostosis, the fusion of one or more of the sutures of the skull vault before the brain completes its growth, is a common (1 in 2,500 births) craniofacial abnormality, approximately 20% of which occurrences are caused by gain-of-function mutations in FGF receptors (FGFRs). We describe a genetic and pharmacological approach for the treatment of a murine model system of Crouzon-like craniosynostosis induced by a dominant mutation in Fgfr2c. Using genetically modified mice, we demonstrate that premature fusion of sutures mediated by Crouzon-like activated Fgfr2c mutant is prevented by attenuation of signaling pathways by selective uncoupling between the docking protein Frs2alpha and activated Fgfr2c, resulting in normal skull development. We also demonstrate that attenuation of Fgfr signaling in a calvaria organ culture with an Fgfr inhibitor prevents premature fusion of sutures without adversely affecting calvaria development. These experiments show that attenuation of FGFR signaling by pharmacological intervention could be applied for the treatment of craniosynostosis or other severe bone disorders caused by mutations in FGFRs that currently have no treatment.

Effect of lameness on culling in dairy cows.

The purpose of this study was to assess the effect of lameness on dairy cow survival. Cox's proportional hazards regression models were fitted to single-lactation data from 2520 cows in 2 New York State dairy herds. Models were controlled for the time-independent effects of parity, projected milk yield, and calving season, and for the time-dependent effects of lameness and culling. Other common diseases were found to be nonconfounding and so were not included in any of the final models. Survival was measured as the time from calving until death or sale. Cows were censored if they reached the start of the next lactation or end of the study, whichever occurred first. All models were stratified by herd. For all lameness diagnoses combined, survival in the herd decreased for those cows becoming lame during the first half of lactation, with a hazard ratio of up to 2 times that of a nonlame cow. Foot rot diagnosed during the second or third months of lactation decreased survival during the same time period (hazard ratio=5.1; 95% confidence interval=1.6 to 16.2). Sole ulcers diagnosed in the first 4 mo of lactation decreased survival in several subsequent periods in which the strongest association was between diagnosis in the third and fourth months of lactation and exit from the herd during that same period (hazard ratio=2.7; 95% confidence interval=1.3 to 6.0). Foot warts were not associated with decreased survival in this analysis. Lameness was never associated with increased survival in any of the models.

A receptor-like inositol lipid phosphatase is required for the maturation of developing cochlear hair bundles.

A screen for protein tyrosine phosphatases (PTPs) expressed in the chick inner ear yielded a high proportion of clones encoding an avian ortholog of protein tyrosine phosphatase receptor Q (Ptprq), a receptor-like PTP. Ptprq was first identified as a transcript upregulated in rat kidney in response to glomerular nephritis and has recently been shown to be active against inositol phospholipids. An antibody to the intracellular domain of Ptprq, anti-Ptprq, stains hair bundles in mice and chicks. In the chick ear, the distribution of Ptprq is almost identical to that of the 275 kDa hair-cell antigen (HCA), a component of hair-bundle shaft connectors recognized by a monoclonal antibody (mAb) that stains inner-ear hair bundles and kidney glomeruli. Furthermore, anti-Ptprq immunoblots a 275 kDa polypeptide immunoprecipitated by the anti-HCA mAb from the avian inner ear, indicating that the HCA and Ptprq are likely to be the same molecule. In two transgenic mouse strains with different mutations in Ptprq, anti-Ptprq immunoreactivity cannot be detected in the ear. Shaft connectors are absent from mutant vestibular hair bundles, but the stereocilia forming the hair bundle are not splayed, indicating that shaft connectors are not necessary to hold the stereocilia together; however, the mice show rapid postnatal deterioration in cochlear hair-bundle structure, associated with smaller than normal transducer currents with otherwise normal adaptation properties, a progressive loss of basal-coil cochlear hair cells, and deafness. These results reveal that Ptprq is required for formation of the shaft connectors of the hair bundle, the normal maturation of cochlear hair bundles, and the long-term survival of high-frequency auditory hair cells.

PTPRQ is a novel phosphatidylinositol phosphatase that can be expressed as a cytoplasmic protein or as a subcellularly localized receptor-like protein.

PTPRQ (rPTP-GMC1) is a member of the type III receptor-like protein tyrosine phosphatase family. PTPRQ has very low activity against phosphotyrosine but is active against phosphatidylinositol phosphates that are involved in regulation of survival, proliferation, and subcellular architecture. Here, we report that PTPRQ can be expressed as a cytosolic or a receptor-like protein and that the form, subcellular localization, and cell types in which it is expressed are regulated by alternative promoter use and by alternative splicing. The first promoter drives expression of transcripts encoding a transmembrane protein in human podocytes and lung. PTPRQ protein is localized to the basal membrane of human podocytes, beginning when podocyte progenitors can first be identified in the embryonic kidney. A second promoter drives expression of a transcript that can encode a cytoplasmic protein containing the catalytic site. This is the major PTPRQ transcript in rat mesangial cells and human testis and is upregulated in mesangial cells in a rat model of mesangial proliferative glomerulonephritis. Differential regulation of expression of the transmembrane vs cytosolic forms, in different cell types during development or response to injury, may be a mechanism through which PTPRQ, with its activities against membrane phospholipids and against phosphotyrosine, can target specific substrates under different conditions.

Hemangiomas and hemangiosarcomas in inbred laboratory mice.

Hemangiomas and hemangiosarcomas are rare, naturally developing tumors of blood vessels. In a retrospective study covering a period of 57 contiguous months between 1986 and 1991, four cavernous hemangiomas, eight capillary hemangiomas, and three hemangiosarcomas developed spontaneously in A/J, BALB/cJ, BALB/cByJ, C57BL/6J, NU/J, and 129/SvJ inbred mouse strains. When data were correlated to actual numbers of each sex in the colony, no sexual dimorphism was determined. At the time of submission, mice ranged in age from 100 to 434 days, with a mean of 217 days. Tumors principally affected the skin but were also diagnosed in the seminal vesicles, liver, muscle tissue, and cerebellum. Immunohistochemistry, with antisera directed against human von Willebrand factor (factor VIII-related antigen) as an endothelial cell marker, labeled mouse endothelial cells inconsistently. A monoclonal antibody directed against the smooth muscle actin isoform, but not the sarcomeric muscle actin isoform, outlined vascular structures in tumors and normal vascular smooth muscle in adjacent normal tissues.

The effect of iron and ethanol on rat hepatocyte collagen synthesis.

1. Carbonyl iron (2.5% w/w) in rat chow was used to induce iron loading in rat hepatocytes. 2. Acute exposure of cultured hepatocytes from control and iron-loaded rats to ethanol (25-100 mM) resulted in a significant inhibition of protein synthesis. 3. Inhibition of protein synthesis in hepatocytes from iron-loaded rats was primarily due to impaired amino acid uptake by these cells. 4. High concentrations of ethanol stimulated the rate of protein degradation by hepatocytes from iron-loaded rats. 5. Acute administration of ethanol to hepatocytes from control animals did not stimulate the absolute rates of collagen biosynthesis nor induce Type I procollagen mRNA. 6. Acute administration of ethanol did not inhibit procollagen synthesis. 7. Iron overload induced Type I procollagen mRNA and increased the absolute rates of collagen synthesis in hepatocytes. 8. These findings may be relevant for the development of hepatic fibrosis in patients with genetic hemochromatosis who consume excess ethanol.