CREB coactivators CRTC2 and CRTC3 modulate bone marrow hematopoiesis.

Populations of circulating immune cells are maintained in equilibrium through signals that enhance the retention or egress of hematopoietic stem cells (HSCs) from bone marrow (BM). Prostaglandin E2 (PGE2) stimulates HSC renewal and engraftment through, for example, induction of the cAMP pathway. Triggering of PGE2 receptors increases HSC survival in part via the PKA-mediated induction of the cAMP response element-binding protein (CREB) signaling pathway. PKA stimulates cellular gene expression by phosphorylating CREB at Ser133 and by promoting the dephosphorylation of the cAMP- responsive transcriptional coactivators (CRTCs). We show here that disruption of both CRTC2 and CRTC3 causes embryonic lethality, and that a single allele of either CRTC2 or CRTC3 is sufficient for viability. CRTC2 knockout mice that express one CRTC3 allele (CRTC2/3m mice) develop neutrophilia and splenomegaly in adulthood due to the up-regulation of granulocyte-colony stimulating factor (G-CSF); these effects are reversed following administration of neutralizing anti-G-CSF antiserum. Adoptive transfer of CRTC2/3m BM conferred the splenomegaly/neutrophilia phenotype in WT recipients. Targeted disruption of both CRTC2 and CRTC3 in stromal cells with a mesenchymal Prx1-Cre transgene also promoted this phenotype. Depletion of CRTC2/3 was found to decrease the expression of Suppressor of Cytokine Signaling 3 (SOCS3), leading to increases in STAT3 phosphorylation and to the induction of CEBPß, a key regulator of the G-CSF gene. As small molecule inhibition of JAK activity disrupted CEBPß induction and reduced G-CSF expression in CRTC2/3m stromal cells, our results demonstrate how cross-coupling between the CREB/CRTC and JAK/STAT pathways contributes to BM homeostasis.

Hypercortisolemia and Depressive-like Behaviors in a Rhesus Macaque (Macaca mulatta) Involved in Visual Research.

A 10-y-old cranially implanted rhesus macaque (Macaca mulatta) involved in visual research was presented for dull mentation and weight loss. Physical examination revealed alopecia and poor body conditioning, and bloodwork revealed marked hypercortisolemia (23 µg/dL). Differential diagnoses for hypercortisolemia, weight loss, and alopecia included Cushing and pseudo-Cushing syndromes. To further evaluate hypercortisolemia, we compared the urine cortisol:creatinine ratio (UCCR) at baseline and after low-dose dexamethasone suppression (LDDS) testing in the presenting animal and healthy naïve and implanted working monkeys. At baseline, UCCR was 10 times higher in the presenting macaque (118.1 ± 7.1) than in naïve animals (12.5 ± 12.8) and 3 times higher than in healthy implanted working macaques (44.4 ± 6.9); however, levels were suppressed similarly by dexamethasone in both the presenting animal and healthy controls. In addition, healthy implanted working macaques had significantly higher baseline UCCR levels than naïve controls, suggesting chronic stress in working animals. Abdominal ultrasonography and radiographs of the presenting animal revealed marked bilateral adrenal mineralization but no overt adrenal tumor or hyperplasia. Overall, these results excluded endogenous Cushing syndrome and prompted us to evaluate different causes of pseudo-Cushing syndrome, including depression. Using videorecordings to evaluate behavior, we used published criteria for macaque models of depression models, including huddling, to make a presumptive diagnosis of depression. The macaque was treated with fluoxetine (2 mg/kg PO daily), provided increased environmental enrichment, and followed over time by regular UCCR assessment and videorecordings. The animal improved clinically and behaviorally, and UCCR returned to levels observed in working implanted macaques (44.4) after 8 wk of treatment. This case highlights the potential effect of research-related work on stress and pathologic behaviors in macaques and demonstrates the utility of UCCR and LDDS for screening behavioral and hypothalamic-pituitary-adrenal abnormalities in these animals.

Lactate as a diagnostic test for septic peritoneal effusions in dogs and cats.

Lactate concentration in peritoneal fluid was evaluated and compared to blood lactate concentration in dogs and cats with septic and nonseptic abdominal effusions. All dogs with septic effusions had a peritoneal fluid lactate concentration >2.5 mmol/L and a peritoneal fluid lactate concentration higher than blood lactate, resulting in a negative blood to fluid lactate difference. In dogs, the diagnostic accuracy of the peritoneal fluid lactate concentration and the blood to fluid lactate difference in differentiating septic peritoneal effusion was 95% and 90%, respectively. Peritoneal fluid lactate concentration and blood to fluid lactate difference were not accurate tests for detecting septic peritoneal effusions in cats.

Detection of carcinoma micrometastases in bone marrow of dogs and cats using conventional and cell block cytology.

BACKGROUND: The presence of human breast carcinoma micrometastases in bone marrow is associated with poor overall survival, poor breast-cancer-specific survival, poor disease-free survival, and poor distant disease-free survival. In veterinary practice, the detection of micrometastases as a component of clinical staging is a routine practice for lymphomas and mast cell tumors, but not for carcinomas. OBJECTIVES: This prospective study evaluated whether the identification of micrometastases from various carcinomas in dogs and cats in bone marrow using cell block cytology is technically feasible and whether it correlates with routine cytologic examination. METHODS: Thirteen dogs and 4 cats with various types of carcinomas were available for analysis. Routine and cell block cytologic evaluation combined with immunocytochemical staining with antibodies to CKAE1/AE3 and CK7 were performed on all bone marrow samples. RESULTS: Bone marrow micrometastasis was demonstrated by both methods in 2 dogs with advanced disease. In one case cells were immunoreactive for both CKAE1/AE3 and CK7. CONCLUSIONS: This study demonstrates that cell block cytology is a practical and useful method for bone marrow evaluation and is suitable for cytokeratin immunocytochemical analysis.

The effect of preoperative antimicrobial administration on culture results in dogs undergoing cystotomy.

OBJECTIVE: To evaluate the effect of preoperative antimicrobial administration on culture results in dogs undergoing cystotomy as a treatment for urolithiasis. DESIGN: Prospective controlled study. Animals-41 dogs undergoing cystotomy for cystic calculi removal. PROCEDURES: Urine samples were collected at time of anesthetic induction and during surgery prior to cystotomy, and a mucosal biopsy and culturette swab was collected during surgery from a control group, which received antimicrobials only after surgical culture sample collection, and from an experimental group, which received antimicrobials at the time of anesthetic induction. RESULTS: 17 of 41 patients had positive culture results at anesthetic induction. Twenty of 41 patients had positive results of cultures for the surgical sample. No dogs that had positive results before antimicrobial administration had negative results after antimicrobial administration. There were no significant changes to urinalysis results regardless of group. Calcium monohydrate uroliths were the most common stone removed (24/41), followed by magnesium ammonium phosphate uroliths (11/41). CONCLUSIONS AND CLINICAL RELEVANCE: There was no difference in culture results (positivity and bacterial type) when antimicrobials were given at anesthetic induction versus after surgical culture sample collection for dogs undergoing cystotomy for cystic calculi removal.

Euthanasia by CO2 inhalation affects potassium levels in mice.

We and others frequently have noted serum potassium levels of 8.0 +/- 0.85 mEq/L or greater in laboratory mice; this concentration has even been published as the upper limit of a 'normal' reference range. However, if bone fide, this potassium concentration would be incompatible with life in all species. We investigated conditions frequently encountered in the research setting to distinguish artifactual from true hyperkalemia. Variables evaluated included site of collection, time allowed for clot formation before serum separation, time elapsed between collection and analysis of samples collected in a serum separator tube, precollection method of anesthesia, and euthanasia technique. Serum potassium was measured from 75 C57BL/6NTac 10-wk-old female mice and divided into at least 5 mice per variable. Animals were euthanized by exsanguination immediately after terminal CO2 or ketamine-xylazine (KX) administration. Mice euthanized with CO2 had higher mean serum potassium (7.0 +/- 0.5 mEq/L) and range serum potassium (6.0 to 8.1 mEq/L) than did KX-treated mice. CO2 inhalation resulted in significantly lower blood pH (6.9 +/- 0.1), higher pCO2 (153.3 +/- 38.8 mm Hg), and higher lactate levels (3.9 +/- 0.9 mmol/L) than did KX anesthesia followed by exsanguination. These results suggest that antemortem respiratory acidosis from CO2 administration causes artifactual hyperkalemia in mice. Therefore, blood collection under KX anesthesia is preferable over CO2 inhalation to obtain accurate potassium values from mice.

Comparison of peritoneal fluid and peripheral blood pH, bicarbonate, glucose, and lactate concentration as a diagnostic tool for septic peritonitis in dogs and cats.

OBJECTIVE: To establish a reliable diagnostic tool for septic peritonitis in dogs and cats using pH, bicarbonate, lactate, and glucose concentrations in peritoneal fluid and venous blood. STUDY DESIGN: Prospective clinical study. ANIMALS: Eighteen dogs and 12 cats with peritoneal effusion. METHODS: pH, bicarbonate, electrolyte, lactate, and glucose concentrations were measured on 1- to 2-mL samples of venous blood and peritoneal fluid collected at admission. The concentration difference between blood and peritoneal fluid for pH, bicarbonate, glucose, and lactate concentrations were calculated by subtracting the peritoneal fluid concentration from the blood concentration. Peritoneal fluid was submitted for cytologic examination and bacterial culture. Peritonitis was classified as septic or nonseptic based on cytology and bacterial culture results. RESULTS: In dogs, with septic effusion, peritoneal fluid glucose concentration was always lower than the blood glucose concentration. A blood-to-fluid glucose (BFG) difference > 20 mg/dL was 100% sensitive and 100% specific for the diagnosis of septic peritoneal effusion in dogs. In 7 dogs in which it was evaluated, a blood-to-fluid lactate (BFL) difference < -2.0 mmol/L was also 100% sensitive and specific for a diagnosis of septic peritoneal effusion. In cats, the BFG difference was 86% sensitive and 100% specific for a diagnosis of septic peritonitis. In dogs and cats, the BFG difference was more accurate for a diagnosis of septic peritonitis than peritoneal fluid glucose concentration alone. CONCLUSIONS: A concentration difference > 20 mg/dL between blood and peritoneal fluid glucose concentration provides a rapid and reliable means to differentiate a septic peritoneal effusion from a nonseptic peritoneal effusion in dogs and cats. CLINICAL RELEVANCE: The difference between blood and peritoneal fluid glucose concentrations should be used as a more reliable diagnostic indicator of septic peritoneal effusion than peritoneal fluid glucose concentration alone.