pyrophosphate and Disease-Models--Animal

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitously expressed enzyme that is best known for its role during mineralization processes in bones and skeleton. The enzyme metabolizes phosphate compounds like inorganic pyrophosphate and pyridoxal-5'-phosphate to provide, among others, inorganic phosphate for the mineralization and transportable vitamin B6 molecules. Patients with inherited loss of function mutations in the

Topics: Alkaline Phosphatase; Animals; Anxiety; Bone and Bones; Calcification, Physiologic; Depression; Diphosphates; Disease Models, Animal; Gene Expression; Humans; Hypophosphatasia; Mutation; Seizures; Severity of Illness Index; Tooth; Vitamin B 6

Pseudoxanthoma elasticum is a prototype of heritable ectopic mineralization disorders, with phenotypic overlap with generalized arterial calcification of infancy and arterial calcification due to CD73 deficiency. Recent observations have suggested that the reduced inorganic pyrophosphate/phosphate ratio is the cause of soft connective tissue mineralization in these disorders. PXE International, a patient advocacy organization, supports research in part by sponsoring biennial research symposia on these disorders; the latest meeting was held in September 2016 at Thomas Jefferson University, Philadelphia. This report summarizes the progress in pseudoxanthoma elasticum and other ectopic mineralization disorders, as presented in the symposium, with focus on translational aspects of precision medicine toward improved diagnostics and treatment development for these currently intractable disorders.

Topics: 5'-Nucleotidase; Alkaline Phosphatase; Animals; Biopsy, Needle; Clinical Trials as Topic; Congresses as Topic; Diphosphates; Disease Models, Animal; Etidronic Acid; Genetic Predisposition to Disease; GPI-Linked Proteins; Humans; Immunohistochemistry; Internationality; Mice; Mutation; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Rare Diseases; Vascular Calcification

As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth - where calcium phosphate crystals are deposited and grow within an extracellular matrix - is essential for dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition in which teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibers (Sharpey's fibers) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth-suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here, with clinical examples given, namely tissue-nonspecific alkaline phosphatase and phosphate-regulating gene with homologies to endopeptidases on the X chromosome. Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia and X-linked hypophosphatemia, respectively, where the levels of local and systemic circulating mineralization determinants are perturbed. In X-linked hypophosphatemia, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating small integrin-binding ligand N-linked glycoproteins, such as matrix extracellular phosphoglycoprotein and osteopontin, and the phosphorylated peptides proteolytically released from them, such as the acidic serine- and aspartate-rich-motif peptide, may accumulate locally to impair mineralization in this disease.

Topics: Alkaline Phosphatase; Alveolar Process; Animals; Calcification, Physiologic; Calcium Phosphates; Dental Enamel Proteins; Diphosphates; Disease Models, Animal; Endopeptidases; Extracellular Matrix; Familial Hypophosphatemic Rickets; Humans; Hypophosphatasia; Periodontal Ligament

ttw (tiptoe walking), a known model mouse of OPLL (ossification of the posterior longitudinal ligament of the spine) is reviewed. ttw is a natural mutant which shows generalized ectopic calcification followed by ossification in the Achiles tendon, ear and spinal ligaments, etc. The trait is caused by a homozygous nonsense mutation in the gene for NPPS (nucleotide pyrophosphatase) , a cell-membrane enzyme that produces pyrophosphate. Its pathomechanism is the decrease of extracellular pyrophosphate due to insufficiency of NPPS. ttw is a excellent model for ectopic calcification and ossification. Several interesting genes related to ectopic calicification have been identified by studies using this mouse.

Topics: Animals; Calcinosis; Codon, Nonsense; Diphosphates; Disease Models, Animal; Genes, Recessive; Mice; Mice, Mutant Strains; Ossification of Posterior Longitudinal Ligament; Pyrophosphatases

Patients with chronic kidney disease have increased cardiovascular mortality from a combination of increased atherosclerotic disease, left ventricular hypertrophy and increased prevalence of vascular calcification (VC). Previously VC was thought to be a passive process which involved the deposition of calcium and phosphate into the vessel wall. However, recent studies have shown that VC is a highly regulated, cell-mediated process similar to bone formation, in that it is associated with expression of bone-related proteins, such as type I collagen and alkaline phosphatase. Animal and in vitro models of VC have shown that a multitude of factors including phosphate, matrix gla protein (MGP) and fetuin are involved in regulating VC. Certain factors induce calcification whereas others inhibit the process. Despite these insights, it is still not fully known how VC is regulated and a treatment for VC remains elusive. Ongoing research will hopefully elucidate these mechanisms and thereby produce targets for future therapeutic intervention. This review will highlight some of the scientific models of VC and how they have increased the understanding of this complex process.

Topics: Alkaline Phosphatase; alpha-Fetoproteins; Animals; Apoptosis; Atherosclerosis; Calcinosis; Calcium-Binding Proteins; Cardiovascular Diseases; Collagen Type I; Diphosphates; Disease Models, Animal; Extracellular Matrix Proteins; Humans; Hypertrophy, Left Ventricular; Inflammation; Kidney Failure, Chronic; Matrix Gla Protein; Mice; Osteopontin; Phosphorus; Prevalence; Risk Factors; Vascular Diseases; Vitamin D; Vitamins

Recent progress in genetics and mouse genomics enables researchers to unveil the molecular basis for mouse phenotypes that express pathologic calcification in soft tissue and/or articular tissues. A newly identified multipass transmembrane protein, ANK, appears to function as an inorganic pyrophosphate (PPi) transporter or regulator of PPi transport. Abnormal extracellular PPi (ePPi) metabolism has been implicated in abnormal calcification, decreased concentrations predisposing to basic calcium phosphate (BCP) deposition, and increased concentrations promoting calcium pyrophosphate dihydrate (CPPD) crystal deposition in articular tissues. The chromosomal location of human ANK overlaps the locus identified in several kindreds affected with familial chondrocalcinosis. Deficient generation of ePPi by the ectoenzyme nucleoside triphosphate pyrophosphohydrolase also results in excessive ossification and ectopic deposition of BCP crystals in tiptoe-walking mice and PC-1 null mice. Recent studies reinforce the important regulatory role of ePPi in pathologic and physiologic calcification.

Topics: Animals; Calcinosis; Diphosphates; Disease Models, Animal; Homeostasis; Humans; Membrane Proteins; Mice; Mice, Knockout; Mice, Transgenic; Phosphate Transport Proteins; Pyrophosphatases; Transforming Growth Factor beta

Topics: Animals; Arthropathy, Neurogenic; Calcium Pyrophosphate; Cartilage, Articular; Cholesterol; Chondrocalcinosis; Crystallization; Diphosphates; Disease Models, Animal; Durapatite; Hand; Hemochromatosis; Humans; Hydroxyapatites; Knee Joint; Menisci, Tibial; Ochronosis; Osteoarthritis; Postoperative Complications; Radiography; Shoulder Joint; Uric Acid

Pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder, is caused by inactivating mutations in the ABCC6 gene. The encoded protein, ABCC6, a transmembrane transporter, has a specialized efflux function in hepatocytes by contributing to plasma levels of inorganic pyrophosphate, a potent inhibitor of mineralization in soft connective tissues. Reduced plasma inorganic pyrophosphate levels underlie the ectopic mineralization in pseudoxanthoma elasticum. In this study, we characterized the pathogenicity of three human ABCC6 missense variants using an adenovirus-mediated liver-specific ABCC6 transgene expression system in an Abcc6

Topics: Adenoviridae; Animals; Calcinosis; Diphosphates; Disease Models, Animal; Humans; Mice; Multidrug Resistance-Associated Proteins; Mutation, Missense; Pseudoxanthoma Elasticum; Skin

Calcifications can disrupt organ function in the cardiovascular system and the kidney, and are particularly common in patients with chronic kidney disease (CKD). Fetuin-A deficient mice maintained against the genetic background DBA/2 exhibit particularly severe soft tissue calcifications, while fetuin-A deficient C57BL/6 mice remain healthy. We employed molecular genetic analysis to identify risk factors of calcification in fetuin-A deficient mice. We sought to identify pharmaceutical therapeutic targets that could be influenced by dietary of parenteral supplementation. We studied the progeny of an intercross of fetuin-A deficient DBA/2 and C57BL/6 mice to identify candidate risk genes involved in calcification. We determined that a hypomorphic mutation of the Abcc6 gene, a liver ATP transporter supplying systemic pyrophosphate, and failure to regulate the Trpm6 magnesium transporter in kidney were associated with severity of calcification. Calcification prone fetuin-A deficient mice were alternatively treated with parenteral administration of fetuin-A dietary magnesium supplementation, phosphate restriction, or by or parenteral pyrophosphate. All treatments markedly reduced soft tissue calcification, demonstrated by computed tomography, histology and tissue calcium measurement. We show that pathological ectopic calcification in fetuin-A deficient DBA/2 mice is caused by a compound deficiency of three major extracellular and systemic inhibitors of calcification, namely fetuin-A, magnesium, and pyrophosphate. All three of these are individually known to contribute to stabilize protein-mineral complexes and thus inhibit mineral precipitation from extracellular fluid. We show for the first time a compound triple deficiency that can be treated by simple dietary or parenteral supplementation. This is of special importance in patients with advanced CKD, who commonly exhibit reduced serum fetuin-A, magnesium and pyrophosphate levels.

Topics: alpha-2-HS-Glycoprotein; alpha-Fetoproteins; Animals; Calcinosis; Diphosphates; Disease Models, Animal; Female; Kidney; Liver; Magnesium; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Microvessels; Minerals; Multidrug Resistance-Associated Proteins; Renal Insufficiency, Chronic; TRPM Cation Channels

Trauma-induced calcification is the pathological consequence of complex injuries which often affect the central nervous system and other parts of the body simultaneously. We demonstrated by an animal model recapitulating the calcification of the above condition that adrenaline transmits the stress signal of brain injury to the calcifying tissues. We have also found that although the level of plasma pyrophosphate, the endogenous inhibitor of calcification, was normal in calcifying animals, it could not counteract the acute calcification. However, externally added pyrophosphate inhibited calcification even when it was administered after the complex injuries. Our finding suggests a potentially powerful clinical intervention of calcification triggered by polytrauma injuries which has no effective treatment.

Topics: Adrenergic Antagonists; Animals; Brain Injuries, Traumatic; Cardiotoxins; Diphosphates; Disease Models, Animal; Epinephrine; Female; Gene Expression Regulation; Mice, Inbred C57BL; Muscle, Skeletal; Ossification, Heterotopic; Receptors, Adrenergic; Vascular Calcification; X-Ray Microtomography

Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson-Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP → phosphate) was greater than eNPP activity (ATP → pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate → phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Antigens, CD; Aortic Diseases; Apyrase; Calcinosis; Diphosphates; Disease Models, Animal; Gene Knock-In Techniques; Humans; Lamin Type A; Levamisole; Longevity; Male; Mice; Mice, Transgenic; Myocytes, Smooth Muscle; Phosphoric Diester Hydrolases; Progeria; Pyrophosphatases; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter ABCC6. It was recently shown that the absence of ABCC6-mediated adenosine triphosphate release from the liver and, consequently, reduced inorganic pyrophosphate levels underlie the pathogenesis of PXE. Given that tissue-nonspecific alkaline phosphatase (TNAP), encoded by ALPL, is the enzyme responsible for degrading inorganic pyrophosphate, we hypothesized that reducing TNAP levels either by genetic or pharmacological means would lead to amelioration of the ectopic mineralization phenotype in the Abcc6

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Diphosphates; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Humans; Liver; Male; Mice; Mice, Knockout; Multidrug Resistance-Associated Proteins; Mutation; Niacinamide; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Skin; Sulfonamides; Vascular Calcification

One diagnostic feature of craniosynostosis syndromes is mandibular dysgenesis. Using three mouse models of Apert, Crouzon and Pfeiffer craniosynostosis syndromes, we investigated how embryonic development of the mandible is affected by fibroblast growth factor receptor 2 (

Topics: Animals; Cell Proliferation; Chondrocytes; Craniosynostoses; Diphosphates; Disease Models, Animal; Embryo, Mammalian; Mandible; Mice; Models, Biological; Osteoblasts; Osteogenesis; Receptor, Fibroblast Growth Factor, Type 2

Mesenchymal stem cells (MSCs) can protect against cartilage breakdown in osteoarthritis (OA) via their immunomodulatory capacities. However, the optimization strategy for using MSCs remains challenging. This study's objective was to identify the in vivo effects of metformin-stimulated adipose tissue-derived human MSCs (Ad-hMSCs) in OA. An animal model of OA was established by intra-articular injection of monosodium iodoacetate into rats. OA rats were divided into a control group and two therapy groups (treated with Ad-hMSCs or metformin-stimulated Ad-hMSCs). Limb nociception was assessed by measuring the paw withdrawal latency and threshold. Our data show that metformin increased IL-10 and IDO expression in Ad-hMSCs and decreased high-mobility group box 1 protein, IL-1β, and IL-6 expression. Metformin increased the migration capacity of Ad-hMSCs with upregulation of chemokine expression. In cocultures, metformin-stimulated Ad-hMSCs inhibited the mRNA expression of RUNX2, COL X, VEGF, MMP1, MMP3, and MMP13 in IL-1β-stimulated OA chondrocytes and increased the expression of TIMP1 and TIMP3. The antinociceptive activity and chondroprotective effects were greater in OA rats treated with metformin-stimulated Ad-hMSCs than in those treated with unstimulated Ad-hMSCs. TGF-β expression in subchondral bone of OA joints was attenuated more in OA rats treated with metformin-stimulated Ad-hMSCs. Our findings suggest that metformin offers a promising option for the clinical application of Ad-hMSCs as a cell therapy for OA.

Topics: Adipose Tissue; Animals; Anti-Inflammatory Agents; Cell Movement; Cells, Cultured; Chondrocytes; Cytoprotection; Diphosphates; Disease Models, Animal; Humans; Imidazoles; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interleukin-10; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Metformin; Nociception; Osteoarthritis; Rats; Rats, Wistar

Generalized arterial calcification of infancy (GACI) is a rare, life-threatening disorder caused by loss-of-function mutations in the gene encoding ectonucleotide pyrophosphatase phosphodiesterase 1 (

Topics: Animals; Blood Pressure; Cardiovascular System; Diphosphates; Disease Models, Animal; Enzyme Replacement Therapy; Humans; Mice; Mice, Inbred BALB C; Organ Specificity; Phosphoric Diester Hydrolases; Pyrophosphatases; Vascular Calcification

Sucrosomial

Topics: Anemia, Iron-Deficiency; Animals; Diphosphates; Disease Models, Animal; Female; Ferric Compounds; Ferrous Compounds; Hep G2 Cells; Hepcidins; Humans; Inflammation; Intestinal Absorption; Intestines; Iron; Iron Deficiencies; Mice, Inbred BALB C

Understanding the uptake of a drug by diseased tissue, and the drug's subsequent spatiotemporal distribution, are central factors in the development of effective targeted therapies. However, the interaction between the pathophysiology of diseased tissue and individual therapeutic agents can be complex, and can vary across tissue types and across subjects. Here, we show that the combination of mathematical modelling, high-resolution optical imaging of intact and optically cleared tumour tissue from animal models, and in vivo imaging of vascular perfusion predicts the heterogeneous uptake, by large tissue samples, of specific therapeutic agents, as well as their spatiotemporal distribution. In particular, by using murine models of colorectal cancer and glioma, we report and validate predictions of steady-state blood flow and intravascular and interstitial fluid pressure in tumours, of the spatially heterogeneous uptake of chelated gadolinium by tumours, and of the effect of a vascular disrupting agent on tumour vasculature.

Topics: Animals; Antineoplastic Agents; Blood Vessels; Cell Line, Tumor; Colorectal Neoplasms; Contrast Media; Diphosphates; Disease Models, Animal; Female; Gadolinium; Glioma; Humans; Hydrodynamics; Image Processing, Computer-Assisted; Mice; Mice, Inbred C57BL; Mice, Nude; Models, Theoretical; Regional Blood Flow; Stilbenes; Transplantation, Heterologous

Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6

Topics: Acute Disease; Animals; ATP-Binding Cassette Transporters; Calcinosis; Chronic Disease; Diphosphates; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Etidronic Acid; Female; Liver; Male; Mice, Inbred C57BL; Mice, Knockout; Multidrug Resistance-Associated Proteins; Phenotype; Pseudoxanthoma Elasticum; Transgenes

Various disorders including pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI), which are caused by inactivating mutations in

Topics: Administration, Oral; Adult; Aged; Animals; ATP-Binding Cassette Transporters; Calcium; Connective Tissue; Diphosphates; Disease Models, Animal; Female; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Multidrug Resistance-Associated Proteins; Myocardium; Phosphoric Diester Hydrolases; Pregnancy; Pseudoxanthoma Elasticum; Pyrophosphatases; Vascular Calcification; Young Adult

Mutations in the proximal tubular sodium-dependent phosphate co-transporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis, however the relative contribution of genotype, dietary calcium and phosphate, and modifiers of mineralization such as pyrophosphate (PPi) to the formation of renal mineral deposits is unclear. In the present study, we used Npt2a-/- mice to model the renal calcifications observed in these disorders. We observed elevated urinary excretion of PPi in Npt2a-/- mice when compared to WT mice. Presence of two hypomorphic Extracellular nucleotide pyrophosphatase phosphodiesterase 1 (Enpp1asj/asj) alleles decreased urine PPi and worsened renal calcifications in Npt2a-/- mice. These studies suggest that PPi is a thus far unrecognized factor protecting Npt2a-/- mice from the development of renal mineral deposits. Consistent with this conclusion, we next showed that renal calcifications in these mice can be reduced by intraperitoneal administration of sodium pyrophosphate. If confirmed in humans, urine PPi could therefore be of interest for developing new strategies to prevent the nephrocalcinosis and nephrolithiasis seen in phosphaturic disorders.

Topics: Animals; Diphosphates; Disease Models, Animal; Female; Humans; Injections, Intraperitoneal; Kidney Calculi; Male; Mice; Mice, Knockout; Mutation; Phosphoric Diester Hydrolases; Pyrophosphatases; Sodium-Phosphate Cotransporter Proteins, Type IIa; Treatment Outcome

Mammalian tissues calcify with age and injury. Analogous to bone formation, osteogenic cells are thought to be recruited to the affected tissue and induce mineralization. In the heart, calcification of cardiac muscle leads to conduction system disturbances and is one of the most common pathologies underlying heart blocks. However the cell identity and mechanisms contributing to pathological heart muscle calcification remain unknown. Using lineage tracing, murine models of heart calcification and in vivo transplantation assays, we show that cardiac fibroblasts (CFs) adopt an osteoblast cell-like fate and contribute directly to heart muscle calcification. Small-molecule inhibition of ENPP1, an enzyme that is induced upon injury and regulates bone mineralization, significantly attenuated cardiac calcification. Inhibitors of bone mineralization completely prevented ectopic cardiac calcification and improved post injury heart function. Taken together, these findings highlight the plasticity of fibroblasts in contributing to ectopic calcification and identify pharmacological targets for therapeutic development.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Calcification, Physiologic; Calcinosis; Cardiomyopathies; Cell Differentiation; Cell Lineage; Cell Separation; Diphosphates; Disease Models, Animal; Female; Fibroblasts; Humans; Male; Mice, Inbred C57BL; Myocardial Infarction; Myocardium; Osteogenesis; Phosphates; Phosphoric Diester Hydrolases; Pyrophosphatases

Topics: Anemia, Iron-Deficiency; Animals; Diphosphates; Disease Models, Animal; Injections, Intraperitoneal; Iron; Peritoneal Dialysis; Pilot Projects; Rabbits; Random Allocation; Reference Values; Risk Factors; Treatment Outcome

Inositol pyrophosphates (PP-IPs) comprising inositol, phosphate, and pyrophosphate (PP) are essential for multiple functions in eukaryotes. Their role in fungal pathogens has never been addressed. Cryptococcus neoformans is a model pathogenic fungus causing life-threatening meningoencephalitis. We investigate the cryptococcal kinases responsible for the production of PP-IPs (IP7/IP8) and the hierarchy of PP-IP importance in pathogenicity. Using gene deletion and inositol polyphosphate profiling, we identified Kcs1 as the major IP6 kinase (producing IP7) and Asp1 as an IP7 kinase (producing IP8). We show that Kcs1-derived IP7 is the most crucial PP-IP for cryptococcal drug susceptibility and the production of virulence determinants. In particular, Kcs1 kinase activity is essential for cryptococcal infection of mouse lungs, as reduced fungal burdens were observed in the absence of Kcs1 or when Kcs1 was catalytically inactive. Transcriptome and carbon source utilization analysis suggested that compromised growth of the KCS1 deletion strain (Δkcs1 mutant) in the low-glucose environment of the host lung is due to its inability to utilize alternative carbon sources. Despite this metabolic defect, the Δkcs1 mutant established persistent, low-level asymptomatic pulmonary infection but failed to elicit a strong immune response in vivo and in vitro and was not readily phagocytosed by primary or immortalized monocytes. Reduced recognition of the Δkcs1 cells by monocytes correlated with reduced exposure of mannoproteins on the Δkcs1 mutant cell surface. We conclude that IP7 is essential for fungal metabolic adaptation to the host environment, immune recognition, and pathogenicity.. Cryptococcus neoformans is responsible for 1 million cases of AIDS-associated meningitis and ~600,000 deaths annually. Understanding cellular pathways responsible for pathogenicity might have an impact on new drug development. We characterized the inositol polyphosphate kinases Kcs1 and Asp1, which are predicted to catalyze the production of inositol pyrophosphates containing one or two diphosphate moieties (PP-IPs). Using gene deletion analysis and inositol polyphosphate profiling, we confirmed that Kcs1 and Asp1 are major IP6 and IP7 kinases, respectively. Kcs1-derived IP7, but not Asp1-derived IP8, is crucial for pathogenicity. Global expression profiling and carbon source utilization testing suggest that IP7-deficient cryptococci cannot adapt their metabolism to allow growth in the glucose-poor environment of the host lung, and consequently, fungal burdens are significantly reduced. Persistent asymptomatic Δkcs1 mutant infection correlated with decreased mannoprotein exposure on the Δkcs1 mutant surface and reduced phagocytosis. We conclude that IP7 is crucial for the metabolic adaptation of C. neoformans to the host environment and for pathogenicity.

Topics: Adaptation, Physiological; Animals; Cryptococcosis; Cryptococcus neoformans; Diphosphates; Disease Models, Animal; Gene Deletion; Inositol Phosphates; Lung; Mice; Phosphotransferases (Phosphate Group Acceptor); Virulence; Virulence Factors

Chronic kidney disease (CKD) is generally associated with disturbances of mineral and bone metabolism. They contribute to the development of vascular calcification (VC), a strong, independent predictor of cardiovascular risk. Pyrophosphate (PPi), an endogenous inhibitor of hydroxyapatite formation, has been shown to slow the progression of VC in uremic animals. Since in patients with CKD treatment is usually initiated for already existing calcifications, we aimed to compare the efficacy of PPi therapy with that of the phosphate binder sevelamer, using a uremic apolipoprotein-E knockout mouse model with advanced VCs. After CKD creation or sham surgery, 12-week-old female mice were randomized to one sham group and four CKD groups (n = 18-19/group). Treatment was initiated 8 weeks after left nephrectomy allowing prior VC development. Uremic groups received either intraperitoneal PPi (high dose, 1.65 mg/kg or low dose, 0.33 mg/kg per day), oral sevelamer (3 % in diet), or placebo treatment for 8 weeks. Both intima and media calcifications worsened with time in placebo-treated CKD mice, based on both quantitative image analysis and biochemical measurements. Progression of calcification between 8 and 16 weeks was entirely halted by PPi treatment, as it was by sevelamer treatment. PPi did not induce consistent bone histomorphometry changes. Finally, the beneficial vascular action of PPi probably involved mechanisms different from that of sevelamer. Further studies are needed to gain more precise insight into underlying mechanisms and to see whether PPi administration may also be useful in patients with CKD and VC.

Topics: Animals; Apolipoproteins E; Diphosphates; Disease Models, Animal; Disease Progression; Infusions, Parenteral; Mice; Mice, Knockout; Renal Insufficiency, Chronic; Uremia; Vascular Calcification

Plasma levels of pyrophosphate, an endogenous inhibitor of vascular calcification, are reduced in end-stage renal disease and correlate inversely with arterial calcification. However, it is not known whether the low plasma levels are directly pathogenic or are merely a marker of reduced tissue levels. This was tested in an animal model in which aortas were transplanted between normal mice and Enpp1(-/-) mice lacking ectonucleotide pyrophosphatase phosphodiesterase, the enzyme that synthesizes extracellular pyrophosphate. Enpp1(-/-) mice had very low plasma pyrophosphate and developed aortic calcification by 2 months that was greatly accelerated with a high-phosphate diet. Aortas of Enpp1(-/-) mice showed no further calcification after transplantation into wild-type mice fed a high-phosphate diet. Aorta allografts of wild-type mice calcified in Enpp1(-/-) mice but less so than the adjacent recipient Enpp1(-/-) aorta. Donor and recipient aortic calcium contents did not differ in transplants between wild-type and Enpp1(-/-) mice, demonstrating that transplantation per se did not affect calcification. Histology revealed medial calcification with no signs of rejection. Thus, normal levels of extracellular pyrophosphate are sufficient to prevent vascular calcification, and systemic Enpp1 deficiency is sufficient to produce vascular calcification despite normal vascular extracellular pyrophosphate production. This establishes an important role for circulating extracellular pyrophosphate in preventing vascular calcification.

Topics: Animals; Aorta; Aortic Diseases; Calcium; Diphosphates; Disease Models, Animal; Disease Progression; Mice, Inbred C57BL; Mice, Knockout; Phosphoric Diester Hydrolases; Phosphorus, Dietary; Pyrophosphatases; Time Factors; Vascular Calcification

Individuals with neurofibromatosis type-1 (NF1) can manifest focal skeletal dysplasias that remain extremely difficult to treat. NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase-activating protein neurofibromin. We report here that ablation of Nf1 in bone-forming cells leads to supraphysiologic accumulation of pyrophosphate (PPi), a strong inhibitor of hydroxyapatite formation, and that a chronic extracellular signal-regulated kinase (ERK)-dependent increase in expression of genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank, causes this phenotype. Nf1 ablation also prevents bone morphogenic protein-2-induced osteoprogenitor differentiation and, consequently, expression of alkaline phosphatase and PPi breakdown, further contributing to PPi accumulation. The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization. They also suggest that altered PPi homeostasis contributes to the skeletal dysplasias associated with NF1 and that some of the NF1 skeletal conditions could be prevented pharmacologically.

Topics: Adolescent; Alkaline Phosphatase; Animals; Bone Development; Bone Diseases, Developmental; Bone Morphogenetic Protein 2; Calcification, Physiologic; Cells, Cultured; Child; Child, Preschool; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type II; Diphosphates; Disease Models, Animal; Durapatite; Humans; Immunoglobulin G; Infant; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinases; Neurofibromatosis 1; Neurofibromin 1; Osteoblasts; Osteogenesis; Phosphate Transport Proteins; Phosphoric Diester Hydrolases; Pyrophosphatases; Recombinant Fusion Proteins; Sp7 Transcription Factor; Transcription Factors

The ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival.

Topics: Animals; Bacterial Proteins; Biological Transport; Deferoxamine; Diphosphates; Disease Models, Animal; Humans; Iron; Membrane Proteins; Meningitis, Meningococcal; Mice; Microbial Viability; Neisseria meningitidis; Transferrin

Progerin is a mutant form of lamin A responsible for Hutchinson-Gilford progeria syndrome (HGPS), a premature aging disorder characterized by excessive atherosclerosis and vascular calcification that leads to premature death, predominantly of myocardial infarction or stroke. The goal of this study was to investigate mechanisms that cause excessive vascular calcification in HGPS.. We performed expression and functional studies in wild-type mice and knock-in Lmna(G609G/+) mice expressing progerin, which mimic the main clinical manifestations of HGPS. Lmna(G609G/+) mice showed excessive aortic calcification, and primary aortic vascular smooth muscle cells from these progeroid animals had an impaired capacity to inhibit vascular calcification. This defect in progerin-expressing vascular smooth muscle cells is associated with increased expression and activity of tissue-nonspecific alkaline phosphatase and mitochondrial dysfunction, which leads to reduced ATP synthesis. Accordingly, Lmna(G609G/+) vascular smooth muscle cells are defective for the production and extracellular accumulation of pyrophosphate, a major inhibitor of vascular calcification. We also found increased alkaline phosphatase activity and reduced ATP and pyrophosphate levels in plasma of Lmna(G609G/+) mice without changes in phosphorus and calcium. Treatment with pyrophosphate inhibited vascular calcification in progeroid mice.. Excessive vascular calcification in Lmna(G609G) mice is caused by reduced extracellular accumulation of pyrophosphate that results from increased tissue-nonspecific alkaline phosphatase activity and diminished ATP availability caused by mitochondrial dysfunction in vascular smooth muscle cells. Excessive calcification is ameliorated on pyrophosphate treatment. These findings reveal a previously undefined pathogenic process in HGPS that may also contribute to vascular calcification in normal aging, because progerin progressively accumulates in the vascular tissue of individuals without HGPS.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Animals; Aorta; Cells, Cultured; Diphosphates; Disease Models, Animal; Lamin Type A; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mitochondria, Muscle; Muscle, Smooth, Vascular; Progeria; Treatment Outcome; Vascular Calcification

Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder, is characterized by early mineralization of blood vessels, often diagnosed by prenatal ultrasound and usually resulting in demise during the first year of life. It is caused in most cases by mutations in the ENPP1 gene, encoding an enzyme that hydrolyzes ATP to AMP and inorganic pyrophosphate, the latter being a powerful anti-mineralization factor. Recently, a novel mouse phenotype was recognized as a result of ENU mutagenesis - those mice developed stiffening of the joints, hence the mutant mouse was named 'ages with stiffened joints' (asj). These mice harbor a missense mutation, p.V246D, in the Enpp1 gene. Here we demonstrate that the mutant ENPP1 protein is largely absent in the liver of asj mice, and the lack of enzymatic activity results in reduced inorganic pyrophosphate (PPi) levels in the plasma, accompanied by extensive mineralization of a number of tissues, including arterial blood vessels. The progress of mineralization is highly dependent on the mineral composition of the diet, with significant shortening of the lifespan on a diet enriched in phosphorus and low in magnesium. These results suggest that the asj mouse can serve as an animal model for GACI.

Topics: Animals; Base Sequence; Calcification, Physiologic; Calcium; Diet; Diphosphates; Disease Models, Animal; DNA Mutational Analysis; Genotyping Techniques; Kaplan-Meier Estimate; Kidney; Mice; Mice, Mutant Strains; Molecular Sequence Data; Phenotype; Phosphoric Diester Hydrolases; Phosphorus; Pyrophosphatases; RNA, Messenger; Spectrometry, X-Ray Emission; Tomography, X-Ray Computed; Vascular Calcification; Vibrissae

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PPi ) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF-hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PPi ) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP-OE) resulted in a significant decrease in cytosolic PPi , and short and long-chain polyP levels. Additionally, the TcVSP-OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PPi and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.

Topics: Animals; Cells, Cultured; Chagas Disease; Chlorocebus aethiops; Cloning, Molecular; Diphosphates; Disease Models, Animal; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Polyphosphates; Protozoan Proteins; Pyrophosphatases; Trypanosoma cruzi; Vacuoles; Vero Cells; Virulence Factors

Vascular calcification is a major risk factor of cardiovascular mortality, particularly for patients with end-stage renal disease and diabetes. Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood. To clarify this, we studied how nuclear factor-kappa B (NF-κB) induction, a mediator of inflammation, might promote vascular calcification. Activation of NF-κB by tumor necrosis factor (TNF) promoted inorganic phosphate-induced calcification in human aortic smooth muscle cells. Pyrophosphate (an inhibitor of calcification) efflux to the extracellular matrix was suppressed along with the decreased expression of ankylosis protein homolog (ANKH), a transmembrane protein that controls pyrophosphate efflux of cells. The restoration of ANKH expression in these cells overcame the decreased pyrophosphate efflux and calcification. Tristetraprolin, a downstream product of NF-κB activation, may mediate destabilization of ANKH mRNA as its knockdown by shRNA increased ANKH expression and decreased calcification. Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels. In contrast, the inhibition of NF-κB maintained ANKH expression and attenuated vascular calcification both in vivo and in vitro. Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression. Thus, TNF-activated NF-κB promotes inflammation-accelerated vascular calcification by inhibiting ankylosis protein homolog expression and consequent pyrophosphate secretion.

Topics: Animals; Atherosclerosis; Diphosphates; Disease Models, Animal; Disease Progression; Down-Regulation; Genes, Reporter; HEK293 Cells; Humans; I-kappa B Proteins; Inflammation Mediators; Kidney Failure, Chronic; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; NF-KappaB Inhibitor alpha; Osteogenesis; Phosphate Transport Proteins; Promoter Regions, Genetic; Rats; Rats, Wistar; RNA Interference; RNA Stability; Signal Transduction; Time Factors; Transfection; Tristetraprolin; Tumor Necrosis Factor-alpha; Vascular Calcification

The effects of the tubulin-binding vascular-disrupting agents (VDAs), combretastatin A4 phosphate (CA4P), OXi4503/CA1P and OXi8007, in subcutaneous mouse models of the Ewing's sarcoma family of tumours (ESFTs) have been investigated alone and in combination with doxorubicin. Delay in subcutaneous tumour growth was observed following treatment of mice with multiple doses of OXi4503/CA1P but not with CA4P or OXi8007. A single dose of OXi4503/CA1P caused complete shutdown of vasculature by 24h and extensive haemorrhagic necrosis by 48h. However, a viable rim of proliferating cells remained, which repopulated the tumour within 10 days following the withdrawal of treatment. Combined treatment with doxorubicin 1h prior to administration of OXi4503/CA1P enhanced the effects of OXi4503/CA1P causing a synergistic delay in tumour growth (p<0.001). This study demonstrates that OXi4503/CA1P is a potent VDA in ESFT and in combination with conventional cytotoxic agents represents a promising treatment strategy for this tumour group.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bibenzyls; Bone Neoplasms; Cell Proliferation; Diphosphates; Disease Models, Animal; Doxorubicin; Drug Evaluation, Preclinical; Mice; Mice, Nude; Necrosis; Neoplasm Transplantation; Neovascularization, Pathologic; Sarcoma, Ewing; Stilbenes

As first-generation small-molecule vascular disrupting agents (VDA) have begun to enter clinical trials, second-generation agents are under active development. One such agent is the combretastatin A4 disodium phosphate (CA4P) analogue OXi4503 (CA1P).. C3H/HeJ mice bearing KHT sarcomas were treated with CA4P and OXi4503 and the effect on tumor vasculature was determined by evaluating the extent of vascular shutdown (Hoechst-33342 vessel staining) and tumor perfusion inhibition (dynamic contrast-enhanced magnetic resonance imaging). Dynamic contrast-enhanced magnetic resonance imaging and tumor necrosis end points also were used to examine the pathophysiologic tumor effects following repeated exposures to these agents.. Single doses of either agent (CA4P, 100 mg/kg; OXi4503, 25 mg/kg) resulted in an 80% to 90% reduction in tumor perfusion 4 hours after treatment. Whereas recovery in tumor perfusion was observed 48 hours posttreatment, this recovery was significantly slower in mice treated with OXi4503. Tumors re-treated with either VDA 72 hours after the first drug exposure showed a similar reduction and recovery in tumor perfusion. Histologic evidence showed the presence of a smaller viable rim after exposure to OXi4503 than that observed after CA4P treatment. Furthermore, the extent of recovery of tumor necrosis 72 hours after drug treatment was less for OXi4053.. The present studies show that the second-generation VDA OXi4503 possesses significant antivascular effects in solid tumors. Importantly, the vasculature of tumors of mice that had received an initial dose this agent was as responsive to a subsequent treatment.

Topics: Animals; Cell Survival; Diphosphates; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Injections, Intraperitoneal; Magnetic Resonance Imaging; Mice; Mice, Inbred C3H; Neovascularization, Pathologic; Radiography; Sarcoma, Experimental; Stilbenes; Time Factors; Transplantation, Heterologous; Xenograft Model Antitumor Assays

Paracoccidioidomycosis (PCM) is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. Diagnosis of PCM is sometimes difficult outside the endemic countries, thus a rapid and conclusive method for diagnosis of PCM has been anticipated. We compared the sensitivities of a nested PCR method for detecting the gp43 gene and a commercial kit for detecting (1-3)-beta-D-glucan in the blood of experimentally infected mice. Blood samples were collected from mice at 0 (soon after inoculation), 6, 12, 24, 48, and 72 hours and 5, 7, 10, 14, 17, 21, 24, 28 and 56 days after the intravenous inoculation of 10(6) yeast cells of P. brasiliensis, and were separated into clots and plasma. The (1-3)-beta-D-glucan detection kit in the plasma showed positive reactions in some samples within 7 days and 28 and 56 days after infections. In contrast, the PCR method was more sensitive than the (1-3)-beta-D-glucan detection kit throughout the observation period. The clot samples yielded more sensitive PCR-results than did the plasma samples. Although 24 hours is required for the PCR detection, it was confirmed to provide an accurate diagnosis of PCM.

Topics: Adult; Aged; Animals; Antigens, Fungal; beta-Glucans; Biomarkers; Child; Diphosphates; Disease Models, Animal; Drug Combinations; Female; Fungal Proteins; Genes, Fungal; Glucans; Glycoproteins; Humans; Male; Mice; Mice, Inbred Strains; Middle Aged; Nitrates; Paracoccidioides; Paracoccidioidomycosis; Polyethylenes; Polymerase Chain Reaction; Sodium Fluoride

The cortical thick ascending limb (CTAL) absorbs Cl- via a Na+-K+-Cl- cotransport at the apical membrane and several Cl- channels at the basolateral membrane, including a 9-pS channel having several properties of the cystic fibrosis transmembrane conductance regulator (CFTR). Having checked that CFTR mRNA is present in the mouse CTAL, we investigated whether this channel is a CFTR molecule by applying the patch-clamp technique to CTALs microdissected from CFTR knockout mice (cftrm1Unc). The 9-pS channel was active in cell-attached patches from tubules of mice homozygous for the disrupted cftr gene [CFTR (-/-)] at the same frequency and with the same activity (NPo) as in normal [CFTR (+/+)] or heterozygous [CFTR (+/-)] mice. The conductive properties of the channel, studied on inside-out patches, were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) tubules, as were the sensitivities to internal pH and internal ATP, two typical features of this channel. In addition, the Cl- absorption in isolated, microperfused CTALs and the Na+-K+-Cl- cotransport activity were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) mice. These results show that the 9-pS Cl- channel is distinct from CFTR, and that the CFTR protein has no influence on the Cl- absorption in this part of the renal tubule.

Topics: Adenosine Triphosphate; Animals; Arginine Vasopressin; Carrier Proteins; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diphosphates; Disease Models, Animal; Hydrogen-Ion Concentration; Ion Transport; Kidney; Loop of Henle; Mice; Mice, Knockout; Patch-Clamp Techniques; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium-Potassium-Chloride Symporters

Due to the fact that there were difficulties in interpreting the cardiac scintigrams after 99mTC pyrophosphate had been given to patients with coronary heart disease without acute myocardial infarction, an experimental study was undertaken. The scintigraphic characteristics were examined in 10 cats following ligation of the interventricular artery at its middle third for more than 20 min, followed by myocardial reperfusion, histochemical and electron microscopic studies. Cat interventricular artery occlusion for a more than 20 min was found to be followed by specific ischemic changes in ECG and myocardial accumulation of 99mTc pyrophosphate. The histochemical and electron microscopic studies indicated that there were both reversible and irreversible cardiomyocyte lesions. Reversible myocardial changes were detected not only in the ischemic area, but in the other myocardial regions away from the basin of the ligated artery. If occlusion was short, the rate of myocardial tracer accumulation rapidly became lower; with long-term occlusion or profound myocardial damage caused by reperfusion, tracer accumulation became higher. There is experimental evidence for applying 99mTc-pyrophosphate scintigraphy in the clinical setting to reveal reversible myocardial changes that are most common in chronic coronary heart disease.

Topics: Animals; Cats; Coronary Disease; Diphosphates; Disease Models, Animal; Heart; Microscopy, Electron; Myocardium; Radionuclide Imaging; Technetium; Technetium Tc 99m Pyrophosphate

Work performed by King et al. in the 1940's and 1950's, as well as recent studies by our group, have shown that the domestic ferret is a suitable model for the study of calculus formation, offering several advantages over the rodent and dog models in current use. We have demonstrated that mineral supplementation of a moist diet accelerates calculus accumulation, and that twice-daily application of a regular dentifrice slows the initial rate of calculus formation, but permits significant accumulation by the eighth week. The present study compared calculus accumulation in female ferrets receiving mineral-supplemented cat food and a twice-daily application of either Regular Crest toothpaste (Crest), Anti-tartar Crest (Crest-AT), or Anti-tartar Colgate Gold toothpaste (Colg-AT). Animals received an ultrasonic prophylaxis, then were fed once daily for eight weeks with moist canned cat food supplemented with sucrose and mineral salts, and were scored for area and extent of calculus accumulation at four and eight weeks after prophylaxis. The data show that the groups treated with the anti-calculus dentifrices produced significantly less calculus than the group treated with regular dentifrice; the Colg-AT group also exhibited lower scores than did the Crest-AT group, especially at four weeks. These results, similar to those seen in human studies, demonstrate that the ferret is a suitable model for the study of anti-calculus dentifrices.

Topics: Analysis of Variance; Animals; Carnivora; Dental Calculus; Dentifrices; Diet, Cariogenic; Diphosphates; Disease Models, Animal; Female; Ferrets; Minerals; Sucrose; Toothpastes

Limiting the rate of reperfusion blood flow has been shown to be beneficial locally in models of ischemia-reperfusion injury. We investigated the effects of this on eicosanoids (thromboxane B2, 6-keto-PGF1 alpha, and leukotriene B4), white blood cell activation, and skeletal muscle injury as quantitated by triphenyltetrazolium chloride and technetium-99m pyrophosphate after ischemia-reperfusion injury in an isolated gracilis muscle model in 16 anesthetized dogs. One gracilis muscle in each dog was subjected to 6 hours of ischemia followed by 1 hour of limited reperfusion and then by a second hour of normal reperfusion. The other muscle was subjected to 6 hours of ischemia followed by 2 hours of normal reperfusion. Six dogs each were used as normal reperfusion controls (NR) and limited reperfusion controls (LR), with 5 dogs being treated with a thromboxane synthetase inhibitor (LR/TSI) and another five with a leukotriene inhibitor (LR/LI). LR in all three groups (LR, LR/TSI, and LR/LI) showed a benefit in skeletal muscle injury as measured by triphenyltetrazolim chloride and technetium-99m pyrophosphate when compared with NR. However, there was no significant difference between the groups with LR regarding eicosanoid levels and white blood cell activation when compared with NR. These results demonstrate that LR produces benefits by mechanisms other than those dependent upon thromboxane A2, prostacyclin, or white blood cell activation.

Topics: Animals; Blood Flow Velocity; Diphosphates; Disease Models, Animal; Dogs; Eicosanoids; Leukocytes; Muscles; Reperfusion Injury; Technetium; Technetium Tc 99m Pyrophosphate; Tetrazolium Salts; Time Factors

The susceptibility of the cardiovascular system to exposure to a high-boiling coal liquid (heavy distillate, HD) was studied in the rat using an isoproterenol (ISO) myocardial infarction model. Male Fischer rats were exposed to HD by inhalation (0.7 mg/l), 6 h/day, 5 days/week, for 6 weeks. After a 10-day recovery period, sham-exposed and HD-exposed rats were injected subcutaneously with 0, 20, 40 or 60 mg ISO/kg body weight. Blood pressure, heart rate, electrocardiogram and 99mTc uptake by the heart were measured 1 day later. A dose-related increase was observed in the uptake of 99mTc by the hearts of both sham-exposed and HD-exposed animals after ISO injection; however, uptake by the sham-exposed group was significantly greater than that of exposed groups. The most striking observation was a 20% elevation in arterial blood pressure of HD-exposed rats over that of sham-exposed animals when no ISO was injected. These results suggest that the cardiovascular system could be detrimentally affected by exposure to coal-derived complex mixtures and, possibly, to other complex organic mixtures.

Topics: Administration, Inhalation; Animals; Blood Pressure; Coal Tar; Diphosphates; Disease Models, Animal; Dose-Response Relationship, Drug; Electrocardiography; Heart Rate; Isoproterenol; Male; Myocardial Infarction; Rats; Rats, Inbred F344; Technetium; Technetium Tc 99m Pyrophosphate; Time Factors

Monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals initiated acute inflammatory reactions characterized by increased plasma extravasation and polymorphonuclear leukocyte (PMNL) accumulation in the rat subcutaneous air-pouch. Pretreatment of rats with colchicine (1 mg kg-1, s.c.) inhibited PMNL accumulation induced by either crystal type but had a greater inhibitory effect on MSU-induced plasma extravasation compared with that induced by CPPD crystals. Colchicine (1 mg kg-1, s.c.) did not reduce histamine-induced plasma extravasation in the air-pouch. The lipoxygenase product of arachidonic acid metabolism, leukotriene B4 (LTB4), was detected in MSU-induced exudates but not in CPPD-induced exudates. Pretreatment of rats with colchicine (1 mg kg-1, s.c.) inhibited LTB4 production in MSU-induced exudates.

Topics: Animals; Anti-Inflammatory Agents; Calcium Pyrophosphate; Colchicine; Diphosphates; Disease Models, Animal; Inflammation; Leukotriene B4; Male; Neutrophils; Rats; Rats, Inbred Strains; Uric Acid

Abnormal loculated or diffuse blood pools adjacent to the heart have been observed in patients with pericardial bleeding who have been imaged by gated equilibrium radionuclide ventriculography (RNV). To study the scintigraphic appearance of fresh pericardial blood, we performed equilibrium RNV in six dogs with measured volumes (10, 30, or 50 ml) of intrapericardial blood. Loculated and diffuse pericardial blood was simulated by injecting the blood either into an intrapericardial balloon, or freely into the pericardial space. Ability to detect pericardial blood was determined by blinded review, and blood volume analysis was attempted by measuring its scintigraphic thickness, brightness (relative to the left ventricle), extent, and background-subtracted count rate and a peak count index. Detection rates for 10, 30, and 50 ml were all 100% for loculated pericardial blood, and 67%, 100% and 100% for free pericardial blood, with the use of three scintigraphic views. Visually determined "extent" of the abnormal blood pool was the most reliable indicator of pericardial blood volume. When the volume was 30 ml or more, at least 40% of the heart was surrounded in 26 of 27 cases (96%); the specificity of this finding was 90%. We conclude from this animal study that RNV should be a sensitive method for detecting pericardial bleeding; visual appearance permits qualitative assessment of the volume of accumulated labeled blood.

Topics: Animals; Blood Volume; Diphosphates; Disease Models, Animal; Dogs; Erythrocytes; Heart Ventricles; Hemorrhage; Pericardium; Radionuclide Imaging; Technetium; Technetium Tc 99m Pyrophosphate

To determine the validity of a method for induction of experimental hemarthrosis in dogs and for the nuclear imaging of hemarthrosis, serial technetium-99m pyrophosphate [( 99mTc]PYP) flow and blood-pool scans were performed monthly in eight dogs who received bi-weekly injections of autologous blood into their femoro-tibial joints (also called stifle joint). In four control dogs, one joint was injected with saline while the other joint received only a sham injection. In addition, two dogs received intra-articular injections of autologous blood into their right stifle joint and saline into their left stifle joint. These dogs were studied with 99mTcO4 joint scintigraphy at monthly intervals. The dogs were periodically taken out of the study and explored surgically. Pathologic examination of synovial tissue was performed. Serial radiographs were also obtained and correlated with the scan and surgical findings. There was a striking abnormal increase in blood-pool activity of [99mTc]PYP in the treated stifle joints, commencing at the first examination after 1 mo of blood injections and continuing for the length of the study. All radiographs showed only minimal joint space widening and some soft-tissue swelling. On pathologic examination, both grossly and microscopically, there was profuse pannus formation, with intense inflammatory infiltrate replacing much of the subsynovial fat. The scintigraphic findings correlated well with these pathologic findings. This study not only validates this method for simulating hemophilic hemarthrosis but also suggests that [99mTc]PYP joint scintigraphy is a simple, and noninvasive method for monitoring the early changes in hemophilic arthropathy and is superior to pertechnetate imaging for this disease process. Instead of the previously recommended delayed bone images, we recommend, in addition, flow studies to assess joint hypervascularity and immediate static images to visualize the synovium and joint capsule.

Topics: Animals; Blood; Diphosphates; Disease Models, Animal; Dogs; Hemarthrosis; Hindlimb; Injections, Intra-Articular; Joints; Radionuclide Imaging; Sodium Pertechnetate Tc 99m; Technetium; Technetium Tc 99m Pyrophosphate

Topics: Animals; Calcium Pyrophosphate; Cartilage Diseases; Diphosphates; Disease Models, Animal; Humans; Macaca mulatta; Microscopy, Electron, Scanning; Monkey Diseases

Topics: Animals; Diphosphates; Disease Models, Animal; Dogs; Female; Leg Injuries; Male; Osteomyelitis; Pentetic Acid; Radionuclide Imaging; Technetium; Technetium Tc 99m Pentetate; Technetium Tc 99m Pyrophosphate

An animal model of arthritis in the rabbit was employed to assess the radioactivity contribution of joint tissues to externally monitored scintigram positivity. Bone contained the greatest total amount of radioactivity whether the imaging agent was technetium pertechnetate or pyrophosphate, although the greatest percent increase in the arthritis joints over control joints was seen in synovium. Mid-shaft bone in the same region as the arthritic joint also showed increased radioactivity compared with control.

Topics: Animals; Arthritis; Diphosphates; Disease Models, Animal; Evaluation Studies as Topic; Femur; Injections, Intra-Articular; Knee Joint; Rabbits; Radionuclide Imaging; Sodium Pertechnetate Tc 99m; Synovial Fluid; Synovial Membrane; Technetium; Technetium Tc 99m Pyrophosphate; Tissue Distribution

Extensive mucosal small-bowel infarction was produced in 8 dogs by occluding the cranial mesenteric artery. After one hour of reperfusion, 15 mCi (555 MBq) of 99mTc-pyrophosphate was intra-arterially injected into 4 dogs and venously into the other 4. Positive images were obtained in all dogs except 1 which had received an intravenous injection. Diagnostic images were obtained consistently as early as 15 minutes after injection, and the infarcted bowel could still be visualized two hours later. The average tracer content in infarcted small bowel was 0.015% I.D./g. This was about eight times the uptake found in normal dogs. The results show that experimental small-bowel infarction can be detected as early as five hours after the onset of ischemia.

Topics: Animals; Diphosphates; Disease Models, Animal; Dogs; Infarction; Intestinal Mucosa; Intestine, Small; Male; Mesenteric Arteries; Mesenteric Vascular Occlusion; Radionuclide Imaging; Technetium

An animal model has been set up to study the adherence of various radionucleotide particles to experimental carotid ulcers. 6 animals were used in all, 4 dogs and 2 sheep. Carotid angiography was performed in 2 animals and in 1 of these the conventional mode of angiography failed to detect the carotid ulcer. Intra-arterial injection of technetium labelled macroaggregates in both of these cases showed the position of the lesion in vitro. In the remaining 4 cases, intravenous radionucleotide 99m technetium labelled pyrophosphate was used and in all cases in vitro the position of the experimental carotid ulceration was revealed. Our studies show that both technetium labelled macroaggregates and technetium labelled pyrophosphate are suitable particles for use in this model. Further research is required to establish the mode of their adherence to the experimental area of ulceration. A further extension of this work would be to demonstrate the ulceration in vivo. Encouraging work in this regard has been done by Mettinger et al. (1978) in humans demonstrating, by substraction methods, increased uptake over ulcerated carotid plaques as compared to controls.

Topics: Animals; Carotid Artery Diseases; Cerebral Angiography; Diphosphates; Disease Models, Animal; Dogs; In Vitro Techniques; Radionuclide Imaging; Sheep; Technetium; Ulcer

A new rapid method for producing myocardial necrosis in rabbits was developed, using percutaneous intramyocardial injection of vasopressin in peanut oil. The 15-min procedure resulted in a mortality rate of 15% and a success rate among surviving animals of 50%. When the lesions were 24 hr old, strontium-85 and a technetium-99m-tagged agent were injected intravenously simultaneously, and the animals were killed 1,6, and 24 hr later for tissue radioassay. Strontium-85 failed to accumulate appreciably in the lesions. Three bone-seeking technetium complexes (pyrophosphate, methylene diphosphonate, and imidodiphosphonate) produced lesion-to-normal myocardial ratios of 6,5, and 14, respectively, at 1 hr, and 20,30, and 33 at 6 hr. The ratios for 99mTc-glucoheptonate were only 2 at 1 hr and 4 at 6 hr, while the ratios of 99mTc-acetylcysteine and 99mTc-citrate were even lower.

Topics: Animals; Citrates; Cysteine; Diphosphates; Diphosphonates; Disease Models, Animal; Myocardial Infarction; Rabbits; Radionuclide Imaging; Strontium Radioisotopes; Sugar Acids; Technetium; Vasopressins

The relation between the accumulation of pyrophosphate and technetium-99m in myocardium with reversible and irreversible ischmic injury was studied in dogs subjected to transitory or persistent coronary arterial occlusion. Among four dogs with coronary occlusion maintained for less than 20 minutes, none had either increased MB creatine kinase (CK) (the "myocardial" CK isoenzyme) activity serum or a positive 99mTc stannous pyrophosphate image. Seven dogs with coronary occlusion maintained for 30 or more minutes had elevated serum MB CK activity, and five of the seven had positive (abnormal) images. Thus, although false negative images may occur occasionally despite myocardial damage, both increased serum MB CK and abnormal images generally accompanied prolonged coronary occlusion. In contrast, ischemia without infarction was not associated with abnormal images. Both 99mTc and 32P labeled pyrophosphate were accumulated extensively and proportionally in myocardium from zones of infarction, and uptake of both tracers was comparable although modest in isolated mitochondria. Similar results were obtained after myocardial infarction in animals with induced profound leukopenia. Thus, phagocytosis of the radiopharmaceutical agent by leukocytes migrating into the infarct is not an essential mechanism accounting for uptake. These results indicate that abnormal images reflect uptake of pyrophosphate, associated with 99mTc, by irreversibly injured myocardium rather than leukocytic infiltration involved in the inflammatory response in the heart.

Topics: Animals; Chick Embryo; Coronary Disease; Creatine Kinase; Diphosphates; Disease Models, Animal; Dogs; False Negative Reactions; Leukocytes; Mitochondria; Myocardial Infarction; Myocardium; Radionuclide Imaging; Technetium; Tin Polyphosphates

The cardiac uptake of Tc-99m tagged skeletal agents was studied after myocardial injury produced by subcutaneous catecholamine injection and random foot-shock stress. Rats stressed for 2 hr developed microfocal myocardial injury, without gross change, whereas those stressed for 12 hr sustained more confluent and sometimes grossly visible damage. Tc-99m MDP and Tc-99m PPi concentrations in these hearts were significantly above control (undamaged) heart levels, producing positive gamma-camera images. Subcutaneous epinephrine injections resulted in grossly visible lesions, with tracer concentrations higher than those previously reported in vasoocclusive infarcts. We postulate that the stress-induced scattered microfocal lesions may accumulate radiopharmaceutical on a per-gram basis in the same way as the larger catecholamine-induced lesions, since tracer delivery to the injured areas in each case is probably less impeded than in frankly vasoocclusive models. Such microfoci, then, could provide an explanation for some of the "false positive" myocardial scans observed clinically.

Topics: Animals; Diphosphates; Diphosphonates; Disease Models, Animal; Epinephrine; Heart Injuries; Injections, Subcutaneous; Myocardium; Organotin Compounds; Rats; Stress, Mechanical; Technetium

Topics: Animals; Diphosphates; Disease Models, Animal; Dogs; Myocardial Infarction; Radionuclide Imaging; Technetium

Activity levels of 99TC-labeled compounds, 18F, and 85Sr were obtained at 1, 3, and 5 hr. postinjection in normal and healing fractured bone and in soft-tissue rat specimens. Serial diagnostic bone images and blood and urine kinetics were obtained in patients with each of the TC-labeled compounds. Computer-processed images were used to evaluate in vivo kinetics. 99mTC pyrophosphate provides the best overall characteristics for bone imaging. Improved quality and bioassay procedures are required, however, before any one agent can be designated the radiopharmaceutical of choice for diagnostic bone imaging.

Topics: Animals; Diphosphates; Disease Models, Animal; Fluorine; Fractures, Bone; Humans; Isotope Labeling; Kinetics; Phosphates; Radioisotopes; Radionuclide Imaging; Rats; Strontium Radioisotopes; Technetium; Tibial Fractures; Wound Healing

The pleural cavity of rats and guinea-pigs has been utilized to study the inflammatory response to immediate hypersensitivity, delayed hypersensitivity, calcium pyrophosphate and carrageenan. The mediators will be described and their possible interaction with cyclic AMP. Finally the effect of some anti-inflammatory agents on these models will be discussed.

Topics: Animals; Arthritis; Arthus Reaction; Carrageenan; Cyclic AMP; Diphosphates; Disease Models, Animal; Guinea Pigs; Humans; Hypersensitivity, Delayed; Hypersensitivity, Immediate; Inflammation; Lung; Pleura; Rats; Synovial Fluid

The quantitative relationship between abnormalities seen on technetium-99m pyrophosphate (99mTc-PYP) infarct scintigrams and the size of the myocardial infarction is unclear. We evaluated two possible determinants of 99mTc-PYP accumulation: myocardial perfusion measured with 7-10 mu microspheres and the extent of necrosis determined histologically. Hemodynamics and myocardial perfusion to small segments of the left ventricle were measured prior to, 5-10 min, and 44-48 hours following sudden occlusion of the left anterior descending coronary artery in ten awake dogs. 99mTc-PYP was injected i.v. following the third injection of microspheres and the animals were killed 2 hours later. The important findings were as follows: 1) there is a close relationship between the extent of myocardial necrosis observed and the perfusion of segments 5-10 min following coronary occlusion; and 2) that segmental myocardial perfusion is an important determinant of 99mTc-PYP accumulation by myocardial segments which contain areas of necrosis. Although the present data preclude statistical analysis of the relationship between the level of necrosis in a segment and the accumulation of 99mTc-PYP by that segment, the two do not appear to be related, a finding which would discourage use of intensity of 99mTc-PYP images for infarct size. The distribution of an abnormality on the scintigram may provide an estimate of infarct size. However, the geometry of the infarct and the resolving power of the scanning equipment will significantly limit this in many clinical situations.

Topics: Animals; Coronary Circulation; Diphosphates; Disease Models, Animal; Dogs; Female; Male; Myocardial Infarction; Myocardium; Necrosis; Perfusion; Technetium

We sought to quantitate infarct size using radioactive imaging techniques. Infarcts were created in closed chest dogs. Using a scintillation camera interfaced to a computer, infarct images were made in the anterior, left lateral, LAO, and RAO projections, 48 hours after infarction and 75 to 90 min following the intravenous injection of 15 mCi of Technetium 99m pyrophosphate (Tc-PYP). Images were computer enhanced and area was calibrated with a radioactive grid source of known dimensions. Image radioactivity was normalized for decay and dose corrected for body weight. Animals were sacrificed two hours following the injection Tc-PYP. Postmortem images were also computer enhanced and calibrated. Gross infarct area and weight were estimated and transmural biopsies were evaluated for Tc-PYP activity and analyzed for creatine phosphokinase (CPK) content. Contiguous biopsies were pathologically analyzed and graded. There was a negative correlation between tissue Tc-PYP activity and CPK content (r=0.89). Pathologic severity worsened with increased Tc-PYP activity and diminished CPK content. There was a good correlation between gross infarct area and image infarct area, both in vivo (r

Topics: Animals; Creatine Kinase; Diphosphates; Disease Models, Animal; Dogs; Evaluation Studies as Topic; Humans; Myocardial Infarction; Myocardium; Radionuclide Imaging; Technetium

The concentration of 99mTc-pyrophosphate was determined in the lower extremities of rabbits (normal, abacterial and bacterial affected soft tissues), in osteoarthritis of the hip joint (capsule and muscle) as well as in knee joint effusions. Compared with the 85Sr-concentration, reflecting the calcification capacity, concentrations of 99mTc-pyrophosphate in soft tissues were found to be lower 2 hours p.i., but were up to elevenfold higher 24 hours p.i. These findings should be due to a fixation of 99mTc-pyrophosphate in collagen containing tissues as in the soft tissue tumors (myosarcoma, synvialioma, breast cancer) presented. A mechanism of delayed equilibration could explain augmented uptake in lymph-edema, ascites and effusions in florid osteoarthritis of the knee joint. The possible dependence of 99mTc-pyrophosphate concentration in bone and soft tissue on collagenous contents is discussed.

Topics: Animals; Ascites; Bone and Bones; Breast Neoplasms; Diphosphates; Disease Models, Animal; Extremities; Freund's Adjuvant; Hip; Humans; Joint Diseases; Joint Prosthesis; Joints; Knee; Osteoarthritis; Phosphates; Rabbits; Rhabdomyosarcoma; Sarcoma, Synovial; Strontium; Strontium Radioisotopes; Technetium; Tibial Fractures; Time Factors; Tin

A model is described of acute inflammation in the pleural cavity of rats using calcium pyrophosphate as the irritant. This model would seem to simulate the pseudogout syndrome. It has been shown to be acute in onset, dominated by polymorphonuclear cells, complement independent. The advantage of the model is that volume of exudate, numbers and types of cells may be quantitated. Prostaglandins and cyclic AMP have been measured in the migrating cells. The significance of these findings has been discussed.

Topics: Animals; Calcium Phosphates; Cyclic AMP; Diphosphates; Disease Models, Animal; Indomethacin; Inflammation; Male; Pleural Effusion; Pleurisy; Prostaglandins E; Prostaglandins F; Rats; Snake Venoms

Topics: Animals; Arthritis; Arthus Reaction; Calcium; Carrageenan; Chondrocalcinosis; Diphosphates; Disease Models, Animal; Guinea Pigs; Immunity, Cellular; Kinetics; Macrophages; Phosphorus; Pleurisy; Rats

Topics: Anemia, Hypochromic; Animals; Biopharmaceutics; Bread; Diphosphates; Disease Models, Animal; Edible Grain; Flour; Food-Processing Industry; Food, Fortified; Humans; Intestinal Absorption; Iron; Iron Radioisotopes; Phosphates; Rats; Sulfates; United States; United States Food and Drug Administration