Dog Characteristics and Gel Analysis.

<p>Animal number: Corresponds to the gel lanes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068114#pone-0068114-g002" target="_blank">Figure 2</a>; Result: ‘+’ denotes FMC positive; blank denotes negative; Intensity: relative intensity of Y chromosomal band to background gel; Months since parturition: duration of time passed since last litter containing male offspring to date of blood draw; Male sibling if no litters prior: presence of male sibling in birth litter if nulliparous at time of blood draw.</p

Y-chromosome DNA Is Present in the Blood of Female Dogs Suggesting the Presence of Fetal Microchimerism

<div><p>Fetal microchimerism has been suggested to play contradictory roles in women’s health, with factors including age of the recipient, time elapsed since microchimerism occurred, and microchimeric cell type modulating disease. Both beneficial and harmful effects have been identified in wound healing and tissue regeneration, immune mediated disease, and cancer. This area of research is relatively new, and hindered by the time course from occurrence of fetal microchimerism to the multi-factorial development of disease. Dogs represent an excellent model for study of fetal microchimerism, as they share our environment, have a naturally condensed lifespan, and spontaneously develop immune-mediated diseases and cancers similar to their human counterparts. However, fetal microchimerism has not been described in dogs. These experiments sought preliminary evidence that dogs develop fetal microchimerism following pregnancy. We hypothesized that Y chromosomal DNA would be detected in the peripheral blood mononuclear cells of female dogs collected within two months of parturition. We further hypothesized that Y chromosomal DNA would be detected in banked whole blood DNA samples from parous female Golden Retrievers with at least one male puppy in a prior litter. Amplification of DNA extracted from five female Golden Retrievers that had whelped within the two months prior to collection revealed strong positive bands for the Y chromosome. Of banked, parous samples, 36% yielded positive bands for the Y chromosome. This is the first report of persistent Y chromosomal DNA in post-partum female dogs and these results suggest that fetal microchimerism occurs in the canine species. Evaluation of the contributions of fetal microchimeric cells to disease processes in dogs as a model for human disease is warranted.</p></div

Nested PCR of dilutions of male: female blood, with male to female ratios of 1∶1 to 1∶90,000.

<p>Bands of Y chromosomal DNA are detected in as small as 1∶60,000-fold M:F diluted samples, but not in 1∶90,000-fold diluted samples.</p

Presence of 320 bp segments of Y-chromosomal DNA following nested PCR amplification performed on banked female Golden Retriever whole-blood DNA samples.

<p>Initial PCR was performed with 650 bp amplicon primers followed by 320 bp nested primer amplification. The PCR products were electrophoresed in a 1% agarose gel with Tri-borate containing Gel-Red and visualized with Bio-Doc-UVA Imaging System. (L) = 100 bp DNA ladder; (+) = male DNA positive control; (−) = female nulliparous DNA negative control; (W) = water template control; (black) numbers = female samples positive for the presence of 320 bp Y-chromosome DNA segments; (gray) numbers = female samples negative for the presence of 320 bp Y-chromosome DNA segments.</p

PCR reactions for positive and negative controls.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068114#pone-0068114-g001" target="_blank">Figure 1A</a> represents the native primer for dog Y-specific DNA fragment of 650 bp, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068114#pone-0068114-g001" target="_blank">Figure 1B</a> represents the nested primer or ∼320 bp within the 650 bp fragment.</p

Immune response to C. novyi-NT immunotherapy

Abstract Clostridium novyi-NT (CVN-NT) spores germinate in hypoxic regions of tumors and have successfully cured induced neoplasia in mouse models and resulted in objective tumor responses in naturally developing neoplasia in the dog. The objective of this pilot, descriptive, prospective, clinical investigation, was to evaluate and describe the immune response to CNV-NT spores to better understand which immune pathways might play a role in the response to this bacteriolytic immunotherapy. Intratumoral injection of CNV-NT spores result in increased phagocytosis and NK cell-like function after treatment. Intravenous injection of CNV-NT spores resulted in increased LPS-induced TNF-α production, LTA-induced IL-10 production and NK cell-like function post-treatment. Increased NK cell-like function was sustained to 28 (intratumoral) or 56 (intravenous) days post-treatment, and increased phagocytic function was sustained to 28 days post-treatment suggesting that CNV-NT spores induce longer-term immune cell function changes. Future investigations evaluating long-term immune system changes and associations between immune function and tumor remission rates should include evaluation of these pathways

Identification of immunologic and clinical characteristics that predict inflammatory response to C. Novyi-NT bacteriolytic immunotherapy

Abstract Background Clostridium novyi-NT (CNV-NT), has shown promise as a bacterolytic therapy for solid tumors in mouse models and in dogs with naturally developing neoplasia. Factors that impact the immunologic response to therapy are largely unknown. The goal of this pilot study was to determine if plasma immune biomarkers, immune cell function, peripheral blood cytological composition and tumor characteristics including evaluation of a PET imaging surrogate of tumor tissue hypoxia could predict which dogs with naturally developing naïve neoplasia would develop an inflammatory response to CNV-NT. Results Dogs that developed an inflammatory response to CNV-NT had a higher heart rate, larger gross tumor volume, greater tumor [64Cu]ATSM SUVMax, increased constitutive leukocyte IL-10 production, more robust NK cell-like function and greater peripheral blood lymphocyte counts compared to dogs that did not develop an inflammatory response to CNV-NT. Of these, unstimulated leukocyte IL-10 production, heart rate, and gross tumor volume appeared to be the best predictors of which dogs will develop an inflammatory response to CNV-NT. Conclusions Development of inflammation in response to CNV-NT is best predicted by pretreatment unstimulated leukocyte IL-10 production, heart rate, and gross tumor volume

Pharmacokinetics and safety of TCMCB07, a melanocortin‐4 antagonist peptide in dogs

Abstract The melanocortin‐4 receptor (MC4R) antagonistic peptide TCMCB07 was developed for the treatment of cachexia. The objectives of this study were to examine pharmacokinetics and safety of TCMCB07 administered subcutaneously to healthy dogs. Dogs were treated with high‐ (2.25 mg kg−1) (n = 5) and low‐dose TCMCB07 (0.75 mg kg−1) (n = 5) once daily for 28 days with a 14‐day washout period between groups. Histamine levels, complete blood count, chemistry panel, blood pressure, 24‐hour Holter recording, and pharmacokinetic parameters were monitored in the high‐dose group. Physical examination changes were limited to weight gain and darkening of the coat color. There was no elevation of plasma histamine within 24 hours of injection but there was a significant elevation of plasma histamine across time. An approximately doubled eosinophil count and an approximately 25% increase, and then 25% decrease back to pre‐treatment plasma phosphorous were also found, although both remained within the reference interval. Serial blood pressure and 24‐hour Holter monitors revealed no clinically relevant changes. A difference was found in the AUC between dosing groups and a significant effect of dose, time, and interaction was noted for Vd. Low‐dose TCMCB07 had a Cmax of 2.1 ug ml−1 at day 28, compared to high‐dose TCMCB07 which had a Cmax 3.6 ug ml−1 at day 28. Once‐daily subcutaneous administration of TCMCB07 was well‐tolerated for up to 28 days in dogs when administered at doses one and three times (0.75 mg kg−1 and 2.25 mg kg−1) the predicted therapeutic dose and pharmacokinetic parameters are described. Significance Statement Melanocortin‐4 receptor (MC4R) antagonistic peptide TCMCB07 is safe at both low and high doses in dogs. Therapy was tolerated well as determined by physical examination, clinical pathology, and cardiovascular parameters; darkening of the coat was noted with treatment and resolved with discontinuation. Pharmacokinetics are described and further study in the naturally occurring canine model is warranted