Grey Collie Research Articles

Cyclic neutropenia in a basset hound

A six-month-old male basset hound was referred for intermittent malaise and pyrexia that repeated every 10–11 days. Sequential blood counts and bone marrow aspirates demonstrated severe neutropenia and increased myeloid...

Repeated Lentivirus-Mediated Granulocyte Colony-Stimulating Factor Administration to Treat Canine Cyclic Neutropenia

Cyclic neutropenia occurs in humans and gray collie dogs, is characterized by recurrent neutropenia, and is treated by repeated injections of recombinant granulocyte colony-stimulating factor (rG-CSF). As dose escalation of lentivirus may be clinically necessary, we monitored the outcome of four sequential intramuscular injections of G-CSF-lentivirus (3 × 10(7) IU/kg body weight) to a normal dog and a gray collie. In the normal dog absolute neutrophil counts were significantly increased after each dose of virus, with mean levels of 27.75 ± 3.00, 31.50 ± 1.40, 35.05 ± 1.68, and 43.88 ± 2.94 × 10(3) cells/μl, respectively (p<0.001), and elevated neutrophil counts of 31.18 ± 7.81 × 10(3) cells/μl were maintained for more than 6 years with no adverse effects. A gray collie dog with a mean count of 1.94 ± 1.48 × 10(3) cells/μl received G-CSF-lentivirus and we observed sustained elevations in neutrophil levels for more than 5 months with a mean of 26.00 ± 11.00 × 10(3) cells/μl, significantly increased over the pretreatment level (p<0.001). After the second and third virus administrations mean neutrophil counts of 15.80 ± 6.14 and 11.52 ± 4.90 × 10(3) cells/μl were significantly reduced compared with cell counts after the first virus administration (p<0.001). However, after the fourth virus administration mean neutrophil counts of 15.21 ± 4.50 × 10(3) cells/μl were significantly increased compared with the previous administration (p<0.05). Throughout the nearly 3 years of virus administrations the dog gained weight, was healthy, and showed neutrophil counts significantly higher than pretreatment levels (p<0.001). These studies suggest that patients with cyclic and other neutropenias may be treated with escalating doses of G-CSF-lentivirus to obtain a desired therapeutic neutrophil count.

A Parameter Estimation Model of G-CSF: Mathematical Model of Cyclical Neutropenia

We investigate the FFT (Fast Fourier Transform) model and G-CSF (granulocyte colony-stimulating factor) treatment of CN (Cyclical Neutropenia). We collect grey collies and normal dog’s data from CN and analyze the G-CSF treatment. The model develops the dynamics of circulating blood cells before and after the G-CSF treatment. This is quite natural and useful for the collection of laboratory data for investigation. The proposed interventions are practical. This reduces the quantity of G-CSF required for potential maintenance. This model gives us good result in treatment. The changes would be practical and reduce the risk side as well as the cost of treatment in G-CSF.

Cyclic neutropenia in animals

To the editor: Pacheco et al. [1] recently published in these pages an allometric scaling analysis of cyclic neutropenia (CN) in mammals, using as reference points the hereditary disorders in humans and in gray collie dogs [2]. They make a central assumption ‘‘that the biological defect in CN is the same in dogs and in humans.’’ However, virtually all human cases derive from mutations in the neutrophil elastase (ELA2) gene [3], whereas the disorder in dogs represents a similar hematological phenotype, in association with partial albinism, because of the defects in the adaptor protein complex 3 (AP3) b-subunit, directing trans-Golgi export of transmembrane cargo proteins to lysosomes [4]. Thus, the canine disorder better models Hermansky–Pudlak syndrome Type 2, [5] than human CN. Although the genetic defects in man and dog may share common downstream mechanisms involving elastase trafficking or elicitation of the unfolded protein response, the resultant kinetics of apoptosis induction and recovery in myeloid precursors may not be the same, and thus the assumption may not be correct.

Cyclic neutropenia in mammals

Cyclic neutropenia (CN) has been well documented in humans and the gray collie. A recent model of the architecture and dynamics of hematopoiesis has been used to provide insights into the mechanism of cycling of this disorder. It provides a link between the cycling period and the cells where the mutated ELA2 is expressed. Assuming that the biologic defect in CN is the same in dogs, and the observation that the structure of hematopoiesis is invariant across mammals, we use allometric scaling techniques to correctly predict the period of cycling in the gray collie and extend it to other mammals from mice to elephants. This work provides additional support for the relevance of animal models to understand disease but cautions that disease dynamics in model animals are different and this has to be taken into consideration when planning experiments.

G-CSF treatment of canine cyclical neutropenia: A comprehensive mathematical model

To study the effects of different G-CSF temporal treatment schemes using a comprehensive mathematical model of the mammalian hematopoietic system that couples the pharmacokinetics of granulocyte colony-stimulating factor (G-CSF) to the hematopoietic stem cell, neutrophil, platelet, and erythrocyte dynamics. Data from cyclical neutropenic (CN) grey collies are used to build an extended model that reproduces the dynamics of circulating blood cells found in laboratory data from the dogs with and without daily G-CSF therapy. The effects of varying the treatment initiation time, and whether injections are given daily, every other day, or every three days, are examined. The mathematical model is able to reproduce the large variation in data that occurs from one dog to another. Different drug delivery times, with no other changes in the model parameters, can have significant long-term effects on neutrophil numbers. The frequency of drug delivery also has long-term effects on the oscillations. Using a realistic representation of the effects of G-CSF on the tissue-level hematopoietic system, the model matches a wide range of laboratory data. This implies that it would be possible to generate individualized predictions for specific dogs if data were available in real time. The proposed interventions are practical and may reduce the amount of G-CSF required while potentially maintaining or even improving the treatment effects.

Erratum

Animal GeneticsVolume 38, Issue 2 p. 191-191 Free Access Erratum This article corrects the following: BRIEF NOTES: Elimination of neutrophil elastase and adaptor protein complex 3 subunit genes as the cause of trapped neutrophil syndrome in Border collies J. R. Shearman, A. N. Wilton, Volume 38Issue 2Animal Genetics pages: 188-189 First Published online: February 15, 2007 First published: 30 March 2007 https://doi.org/10.1111/j.1365-2052.2007.01600.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Shearman & Wilton (2007), the following errors were published: The title should be: Elimination of neutrophil elastase and the genes for adaptor protein complex 3 subunits as the cause of trapped neutrophil syndrome in Border collies The second sentence of the third paragraph should be: Canine cyclic neutropenia, called Gray collie syndrome,5 is caused by mutations in the adaptor-related protein complex 3, beta 1 subunit gene (AP3B1). The last sentence of the third paragraph should be: ELA2 and four AP3 subunit genes (AP3B1, AP3M1, AP3D1 and AP3S1) are good candidates for TNS. We apologize for these errors. Reference Shearman J. R. & Wilton A. N. (2007) Elimination of neutrophil elastase and adaptor protein complex 3subunit genes as the cause of trapped neutrophil syndrome in Border collies. Animal Genetics 38, x– x. Wiley Online LibraryCASWeb of Science®Google Scholar Volume38, Issue2April 2007Pages 191-191 ReferencesRelatedInformation

High frequency spikes in long period blood cell oscillations

Several hematological diseases are characterised by oscillations of various blood cell populations. Two of these are a variant of chronic myelogenous leukemia (CML) and cyclical neutropenia (CN). These oscillations typically have long periods ranging from 20 to 60 days, despite the fact that the stem cell cycling time is thought to be of the order of 2-3 days. Clinical data from humans and laboratory data from the grey collie animal model of CN is suggestive of the idea that these long period oscillations may also contain higher frequency spiky oscillations. We show how such oscillations can be understood in the context of slow periodic stem cell oscillations, by analysing a two component differential-delay equation model of stem cell and neutrophil populations.

An Adult Dog with Cyclic Neutropenia Treated by Lentivirus- Mediated Delivery of Granulocyte Colony-Stimulating Factor

Cyclic neutropenia occurs in humans and gray collie dogs, is characterized by recurrent neutropenia, and is treated by daily injections of recombinant granulocyte colony-stimulating factor (G-CSF). After showing that canine recombinant G-CSF increased neutrophil counts in an affected dog, we administered intramuscularly 2 x 10(9) infectious units (IU) of a lentiviral vector encoding canine G-CSF cDNA. Elevated, therapeutic neutrophil production was obtained for nearly 18 months. Lentiviral vector treatment provided a mean neutrophil count of 29,230 +/- 12,930 cells/microl, which was significantly increased over both the pretreatment value (5,240 +/- 4,800 cells/microl; p < 0.0001) and the neutrophil count during G-CSF administration (17,820 +/- 11,100 cells/microl; p < 0.0001). By systemic infusion of recombinant G-CSF to normal dogs we estimated that 2 x 10(9) IU of lentivirus delivered 3.5 microg of G-CSF per kilogram per day. After lentiviral vector treatment the gray collie gained weight, showed no clinical signs of infection and fever, and no longer needed housing in a pathogen-free environment. Genomic DNA harvested from muscle at the injection sites was positive for provirus, whereas gonad, lung, spleen, heart, liver, kidney, and noninjected muscle samples were negative. These studies show that an adult animal is responsive long-term to lentivirus-mediated G-CSF delivery, suggesting this approach may be applied for treatment of adult patients with cyclic and other neutropenias.

Observations on the Pathophysiology and Mechanisms for Cyclic Neutropenia

We review the basic pathology of cyclical neutropenia in both humans and the grey collie, and examine the role that mathematical modeling of hematopoietic cell production has played in our understanding of the origins of this fascinating dynamical disease.

A mathematical model of hematopoiesis: II. Cyclical neutropenia

Cyclical neutropenia is a dynamical disease of the hematopoietic system marked by an oscillation in circulating leukocyte (e.g. neutrophil) numbers to near zero levels and then back to normal. This oscillation is also mirrored in the platelets and reticulocytes which oscillate with the same period. Cyclical neutropenia has an animal counterpart in the grey collie. Using the mathematical model of the hematopoietic system of Colijn and Mackey [A mathematical model of hematopoiesis: I. Periodic chronic myelogenous leukemia. Companion paper to the present paper.] we have determined what parameters are necessary to mimic laboratory and clinical data on untreated grey collies and humans, and also what changes in these parameters are necessary to fit data during treatment with granulocyte colony stimulating factor (G-CSF). Compared to the normal steady-state values, we found that the major parameter changes that mimic untreated cyclical neutropenia correspond to a decreased amplification (increased apoptosis) within the proliferating neutrophil precursor compartment, and a decrease in the maximal rate of re-entry into the proliferative phase of the stem cell compartment. For the data obtained during G-CSF treatment, good fits were obtained only when parameters were altered that would imply that G-CSF led to higher amplification (lower rate of apoptosis) in the proliferating neutrophil precursors, and a elevated rate of differentiation into the neutrophil line.

459. Long-Term Treatment of Canine Cyclic Neutropenia by G-CSF-Lentivirus

Cyclic neutropenia (CN) occurs both in man and grey collie dogs and is characterized by recurrent periods of neutropenia leading to bacterial infections and shortened life expectancy. Daily rG-CSF provides therapy for patients and dogs. To treat CN by gene therapy we constructed a VSV-G pseudotyped lentivirus encoding canine G-CSF cDNA regulated by CMV promoter, cPPT from HIV 1 and PRE from human hepatitis B virus. At 7 weeks of age when an affected grey collie dog was weaned, serial blood counts were monitored. These data showed the dog's absolute neutrophil counts (ANC) were cycling and observations over 4 cycles established a 12 day periodicity. The ANC mean|[plusmn]|SD during nadirs and peaks were 560|[plusmn]|787 cells/|[mu]|l and 10,760|[plusmn]|1,120 cells/|[mu]|l respectively and overall counts were 5,240|[plusmn]|4,790 cells/|[mu]|l. Having established that neutrophils were cycling, recombinant canine G-CSF was administered subcutaneously at a dose of 1.5 |[mu]|g/kg per weekday for two months. Although ANC continued to cycle, we observed significant increases in neutrophils both at the nadirs and the peaks of cycles (p<0.0001) with mean values of 4,860|[plusmn]|2,670 cells/|[mu]|l and 28,870|[plusmn]|9,830 cells/|[mu]|l respectively. Over this period the mean count was 17,820|[plusmn]|11,100 cells/|[mu]|l. After demonstrating that rG-CSF elevated neutrophil production cytokine administration was stopped and we administered IM 2|[times]|109 infectious units(IU) of G-CSF-lentivirus. The dog showed no ill effects to the virus injections. Serial monitoring of blood cells showed elevated ANC for over 17 months with mean value of 29,230|[plusmn]|12,930 cells/|[mu]|l (n=217) that was significantly increased over both no treatment and rG-CSF treatment (p<0.0001). The increases in ANC from recombinant G-CSF and lentivirus administration were associated with absence of clinical signs of infection and fever.

Treatment of canine cyclic neutropenia by lentivirus-mediated G-CSF delivery

AbstractCyclic neutropenia is a rare disease that occurs both in humans and gray collie dogs and is characterized by recurrent severe neutropenia leading to bacterial infections and shortened life expectancy. Daily injections of recombinant granulocyte colony-stimulating factor (rG-CSF) are effective in shortening the period of severe neutropenia and reducing infections. After demonstrating that rG-CSF induced elevated neutrophil production in an affected dog, cytokine administration was stopped and 109 infectious units (IUs) of a lentivirus pseudotyped with vesicular stomatitis virus G protein (VSV-G) encoding canine G-CSF cDNA was administered intramuscularly. Serial blood cell counts showed elevated neutrophil production for longer than 17 months. Although neutrophil counts continued to cycle, the range at nadirs was from 3710 to 5300 cells/μL, well above the nadirs before lentivirus administration. After the injection of lentivirus, mean neutrophil counts ± SD were 12 460 ± 4240 cells/μL, significantly increased over both pretreatment values of 3040 ± 2540 cells/μL (P < .0001) and neutrophil counts during G-CSF administration of 10 290 ± 4860 cells/μL(P < .007). The changes in blood counts from lentivirus injection were associated with absence of clinical signs of infection and fever. The gray collie continued to gain weight and was no longer housed in a pathogen-free environment. Genomic DNA from muscle at injection sites was positive for provirus, whereas gonad, lung, spleen, heart, liver, kidney, leukocytes, and noninjected muscle samples were all negative for provirus. Thus, intramuscular administration of lentivirus encoding G-CSF provided sustained therapeutic levels of neutrophils, suggesting this approach may be applied for long-term treatment of patients with cyclic and other neutropenias.

Oscillations in cyclical neutropenia: new evidence based on mathematical modeling

We present a dynamical model of the production and regulation of circulating blood neutrophil number. This model is derived from physiologically relevant features of the hematopoietic system, and is analysed using both analytic and numerical methods. Supercritical Hopf bifurcations and saddle-node bifurcations of limit cycles are shown to exist. We make the estimation of kinetic parameters for dogs and then apply the model to cyclical neutropenia (CN) in the grey collie, a rare disorder in which oscillations in all blood cell counts are found. We conclude that the major cause of the oscillations in CN is an increased rate of apoptosis of neutrophil precursors which leads to a destabilization of the hematopoietic stem cell compartment.

Cloning and sequencing of the canine neutrophil elastase cDNA.

Human cyclic neutropenia, also referred to as cyclic hematopoiesis, is a rare disease characterized by periodic fluctuations in blood cell production by the bone marrow and a corresponding recurrent severe neutropenia every 19-21 days. This results in bacterial infections and shortened life expectancy. Platelets, monocytes, lymphocytes, and reticulocytes cycle with the same periodicity. It has been determined that the neutrophil elastase (NE) gene is mutated in all cases of human cyclic hematopoiesis. Currently, the only animal model for this disease is the grey collie dog, in which there is a strikingly similar periodic neutropenia every 12-14 days. Towards the validation of this animal model, we have cloned and sequenced the canine NE cDNA from a normal dog.

Modeling Complex Neutrophil Dynamics in the Grey Collie

We have developed a mathematical model for the peripheral regulation of neutrophil production mediated by granulocyte colony-stimulating factor. We have used that model to show that the pattern of neutrophil oscillations in nine grey collies is consistent with the hypothesis that cyclical neutropenia is due to an oscillatory stem cell input to the neutrophil regulatory system, and not due to autonomous oscillations in the peripheral neutrophil regulatory system. In the process of interfacing our model with the laboratory data, we have estimated parameters for the peripheral neutrophil control system consistent with higher than normal apoptotic cell loss within the recognizable neutrophil precursors. This is in agreement with other experimental data. Our estimated model parameters also predict that the peripheral neutrophil production system is globally stable in the grey collies we studied. This further supports our hypothesis that the origin of the oscillatory behavior in cyclical neutropenia is in the stem cell population, consistent with other clinical and experimental evidence.

Hematopoietic dynamics in grey collies

Using Lomb periodogram analysis we have quantified variations in the peripheral neutrophil and platelet counts of the cyclical neutropenia animal model–the grey collie. We found that the amplitudes of the oscillations in these two cell lineages vary concomitantly. Further, the power spectrum and the shape of the oscillations in the absolute neutrophil counts vary together with the amplitude of the oscillations. As the amplitude of the oscillations increases, the height of the second subharmonic increases, giving rise to a distorted oscillation with two peaks per cycle. The particular dynamics of the absolute neutrophil counts can be reproduced by a combination of a delayed peripheral feedback, representing the peripheral control of granulopoiesis through granulocyte colony stimulating factor, together with a sinusoidal input representing an oscillatory input from the pluripotential stem cells to the granulocytic lineage. The same pluripotential stem cell input is probably responsible for the sinusoidal oscillations observed in the other cell lineages.

Cyclical Neutropenia and the Peripheral Control of White Blood Cell Production

Cyclical neutropenia (CN) is an interesting dynamic hematological disease in which the neutrophils spontaneously oscillate from approximately normal levels to near zero with a period between 19 and 21 days. In the only known animal model for this disorder, the grey collie, the disease's single apparent difference from human CN is the smaller period of 11–15 days. CN can be treated using the cytokine G-CSF which decreases the period (to about 14 days in humans), increases the mean value, and elevates the amplitude of the oscillations. After reviewing the clinical and laboratory data on this disease, we examine the proposition that CN is due to a loss of stability in the peripheral negative feedback control of neutrophil production. This is accomplished by the development of a physiologically realist mathematical model for the system. We conclude that there is no consistent way in which such a destabilization can give rise to either the clinical or laboratory characteristics of CN. Rather it seems more likely that the oscillations of CN are generated within the pluripotential stem cell population.

Cloning and expression of the cDNA encoding rat granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) acts on precursor hematopoietic cells to control the production and maintenance of neutrophils. Recombinant G-CSF (re-G-CSF)is used clinically to treat patients with neutropenia and has greatly reduced the infection risk associated with bone marrow transplantation. Cyclic hematopoiesis, a stem cell defect characterized by severe recurrent neutropenia, occurs in man and grey collie dogs, and can be treated by administration of re-G-CSF. Availability of the rat G-CSF cDNA would benefit the use of rats as models of gene therapy for the treatment of cyclic hematopoiesis. In preliminary rat experiments, retroviral-mediated expression of canine G-CSF caused neutralizing antibody formation which precluded long-term increases in neutrophil counts. To overcome this problem we cloned the rat G-CSF cDNA from RNA isolated from skin fibroblasts. The rat G-CSF sequence shared a high degree of identity in both the coding and non-coding regions with both the murine G-CSF (85%) and human G-CSF (74%). The signal peptides of murine and human G-CSF both contained 30 amino acids (aa), whereas the deduced signal sequence for rat G-CSF possessed 21 aa. A retrovirus encoding the rat G-CSF cDNA synthesized bioactive G-CSF from transduced vascular smooth muscle cells.

Granulocyte colony-stimulating factor expression from transduced vascular smooth muscle cells provides sustained neutrophil increases in rats.

Granulocyte colony-stimulating factor (G-CSF) regulates granulocyte precursor cell proliferation, neutrophil survival, and activation. Cyclic hematopoiesis, a disease that occurs both in humans and grey collie dogs is characterized by cyclical variations in blood neutrophils. Although the underlying molecular defect is not known, long-term daily administration of recombinant G-CSF eliminates the severe recurrent neutropenia, indicating that expression of G-CSF by gene therapy would be beneficial. As a prelude to preclinical studies in affected collie dogs, we monitored hematopoiesis in rats receiving vascular smooth muscle cells transduced to express G-CSF. Cells transduced with LrGSN, a retrovirus expressing rat G-CSF, were implanted in the carotid artery and control animals received cells transduced with LASN, a retrovirus expressing human adenosine deaminase (ADA). Test animals showed significant increases in neutrophil counts for at least 7 weeks, with mean values of 3,670 +/- 740 cells/microliter in comparison to 1,870 +/- 460 cells/microliter in controls (p < 0.001). Thus, in rats G-CSF gene transfer targeted at vascular smooth muscle cells initiated sustained production of 1,800 neutrophils/microliter, a cell number that would provide clinical benefit to patients. Lymphocytes, red cells and platelets were not different between control and test animals (p > 0.05). These studies indicate that retrovirally transduced vascular smooth muscle cells can provide sustained clinically useful levels of neutrophils in vivo.