Bovine monocyte TLR2 receptors differentially regulate the intracellular fate of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium.

Pathogenic mycobacterial organisms have the capacity to inhibit macrophage activation and phagosome maturation. Although the mechanism is complex, several studies have incriminated signaling through TLR2 receptors with subsequent activation of the MAPK pathway p38 (MAPKp38) and overproduction of IL-10 in the survival of pathogenic mycobacterial organisms. In the present study, we compared the response of bovine monocytes with infection by Mycobacterium avium subspecies paratuberculosis (MAP), the cause of paratuberculosis in ruminants, with the closely related organism M. avium subspecies avium (Maa), which usually does not cause disease in ruminants. Both MAP and Maa induced phosphorylation of MAPKp38 by bovine monocytes; however, addition of a blocking anti-TLR2 antibody partially prevented MAPKp38 phosphorylation of MAP-infected monocytes but not Maa-infected monocytes. Addition of anti-TLR2 antibody enhanced phagosome acidification and phagosome-lysosome fusion in MAP-containing phagosomes and enabled monocytes to kill MAP organisms. These changes were not observed in Maa-infected monocytes. The effect on phagosome maturation appears to occur independently from the previously described inhibitory effects of IL-10 on phagosome acidification and organism killing, as IL-10 production was not affected by addition of anti-TLR2 antibody to monocyte cultures. Therefore, signaling through the TLR2 receptor appears to play a role in phagosome trafficking and antimicrobial responses in MAP-infected bovine mononuclear phagocytes.

Effects of nuclear factor-kappaB on regulation of cytokine expression and apoptosis in bovine monocytes exposed to Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To evaluate the role of the nuclear factor-kappaB (NF-kappaB) in the response of bovine monocytes to exposure to Mycobacterium avium subsp paratuberculosis (MAP). SAMPLE POPULATION: Monocytes from healthy adult Holstein cows that were known to be negative for MAP infection. PROCEDURES: Monocytes were incubated with MAP organisms with or without a specific inhibitor of the NF-kappaB pathway (pyrrolidine dithiocarbamate), and activation of the NF-kappaB pathway was detected by use of an electrophorectic mobility shift assay. The capacities of monocytes to express tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, and IL-12; to acidify phagosomes; to phagocytize and kill MAP organisms; and to undergo apoptosis were evaluated. RESULTS: Addition of MAP organisms to monocytes activated the NF-kappaB pathway as indicated by increased NF-kappaB-DNA binding. Addition of pyrrolidine dithiocarbamate prevented nuclear translocation of NF-kappaB, decreased expression of TNF-alpha and IL-10, and increased IL-12 expression. Treatment of MAP-exposed monocytes with pyrrolidine dithiocarbamate increased the rate of apoptosis but failed to alter phagosome acidification, organism uptake, or organism killing by those cells. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that NF-kappaB rapidly translocated to the nucleus after exposure of bovine monocytes to MAP organisms. These data suggest that NF-kappaB is involved in initiation of inflammatory cytokine transcription and inhibition of apoptosis but that it is not directly involved in phagosome acidification or organism killing.

Role of the mitogen-activated protein kinase pathway in the differential response of bovine monocytes to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium.

We compared the kinetics of activation and antimicrobial activities of MAPK-p38 and MAPK-ERK in bovine monocytes infected with Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium avium subsp. avium (Maa). Monocytes were incubated with MAP or Maa organisms with or without a specific inhibitor of the MAPK-p38 pathway (SB203580), and MAPK phosphorylation and antimicrobial functions of monocytes were evaluated. At early time points MAPK-p38 phosphorylation was greater in MAP-infected bovine monocytes than in Maa-infected monocytes. At later time points MAPK-p38 phosphorylation by both organisms was similar. MAPKp38 phosphorylation in MAP-infected monocytes was similar to negative control cells, whereas in Maa-infected this activation remained greater than negative control cells. Increase phosphorylation MAPK-ERK was similar at all time points for both organisms. Bovine monocytes had minimal capacity to kill MAP organisms, to acidify MAP-containing phagosomes, or to form phagolysosome. Alternatively, bovine monocytes were able to kill Maa organisms. Addition of SB203580 to monocyte cultures increased phagosome acidification, phagolysosome formation, and killing of MAP and Maa organisms. Taken together these data indicate that early transient activation of MAPK-p38 in bovine mononuclear phagocytes by MAP organisms may be a key mechanism involved in the capacity of MAP to survive in bovine monocytes.

Evaluation of antineutrophil IgG antibodies in persistently neutropenic dogs.

BACKGROUND: Immune-mediated neutropenia (IMN) is one of several causes of persistent neutropenia in dogs. A test to detect IMN in dogs is not available. HYPOTHESIS: A flow cytometric immunofluorescence assay will provide a sensitive method for detection of antineutrophil antibodies in dogs. ANIMALS: The study included 12 neutropenic dogs and 20 healthy dogs. METHODS: An indirect immunofluorescence assay was used to detect immunoglobulin G (IgG) binding to dog neutrophils. Leukoagglutination was evaluated by light microscopy. Neutrophil distribution in scatter plots, neutrophil fluorescence intensity, and the percentage of neutrophils with increased fluorescence intensity was evaluated by use of flow cytometry. RESULTS: Antineutrophil antibodies were detected in the serum of 5 of 6 dogs with a clinical diagnosis of IMN. Leukoagglutination was present in 3 dogs. Four dogs had altered neutrophil distribution in forward-angle versus side-angle light scatter plots. Five of 6 dogs had increased neutrophil fluorescence intensity and 4 of 6 dogs had an increased percentage of neutrophils with increased fluorescence intensity. CONCLUSIONS AND CLINICAL IMPORTANCE: The flow cytometric test for antineutrophil antibodies detects dogs with a clinical diagnosis of IMN. Testing for antineutrophil antibodies should include observation for leukoagglutination, observation of scatter plots for altered distribution of the neutrophil population, observation of the shape of the fluorescence histogram, determination of neutrophil fluorescence intensity, and determination of the percentage of neutrophils with increased fluorescence intensity.

An indirect flow cytometric test for detection of anti-neutrophil antibodies in dogs.

OBJECTIVE: To develop a clinically applicable assay for detection of serum anti-neutrophil antibodies in dogs. SAMPLE POPULATION: Serum samples of 20 healthy dogs and 20 sick dogs. PROCEDURES: An indirect immunofluorescence assay was developed in which canine serum was incubated with paraformaldehyde-fixed neutrophils and subsequently incubated with fluorescein-conjugated rabbit anti-dog IgG. Neutrophil median fluorescence intensity and the percentage of neutrophils with an increase in fluorescence intensity were determined by use of a flow cytometer. RESULTS: Neutrophils incubated with serum from healthy and sick dogs had a normally distributed curve when displayed as a histogram. Alloantibodies or immune complexes that significantly affected test results were not detected. Hyperglobulinemia did not appear to affect test results. The neutrophil donor did not significantly affect test results. With 1 exception, results for the sick dogs did not differ appreciably from those for healthy dogs. Serum from a dog with steroid-responsive neutropenia had a greater neutrophil fluorescence value and percentage of neutrophils with an increase in fluorescence intensity, compared with either healthy or sick dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The indirect immunofluorescence test gave consistent results for healthy and sick dogs and detected anti-neutrophil antibodies in a dog with steroid-responsive neutropenia. Definitive evaluation of the test will be dependent on evaluation of persistently neutropenic dogs and correlation of test results with a response to immunosuppressive therapy.

Role of the MAPK(ERK) pathway in regulation of cytokine expression by Mycobacterium avium subsp paratuberculosis-exposed bovine monocytes.

OBJECTIVE: To evaluate the role of the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK(ERK)) pathway in the interaction between Mycobacterium avium subsp paratuberculosis (MAP) organisms and bovine monocytes. SAMPLE POPULATION: Monocytes obtained from healthy adult Holstein dairy cows that were not infected with MAP organisms. PROCEDURES: Monocytes and MAP organisms were incubated together with or without a specific inhibitor of the MAPK(ERK) pathway (PD98059), and the capacity of monocytes to express tumor necrosis factor alpha (TNF)-alpha and interleukin (IL)-10 and -12, produce nitric oxide, acidify phagosomes, kill MAP organisms, and undergo apoptosis was evaluated. RESULTS: The MAPK(ERK) pathway was activated within 10 minutes after addition of MAP organisms to monocytes. Addition of PD98059 to monocyte-MAP mixtures decreased monocyte TNF-alpha and IL-12 mRNA expression but had no effect on IL-10 mRNA expression. Treatment with PD98059 failed to induce significant alterations in phagosome acidification, organism killing, nitric oxide production, or apoptosis of MAP-exposed monocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the MAPK(ERK) pathway was activated during the interaction of MAP organisms with monocytes, which initiated TNF-alpha and IL-12 mRNA expression but failed to initiate antimicrobial activity. The MAPK(ERK) pathway may be involved in initiating proinflammatory and proimmune responses in MAP infection in cattle.

Cell membrane receptors on bovine mononuclear phagocytes involved in phagocytosis of Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To determine cell membrane receptors involved in phagocytosis of Mycobacterium avium subsp paratuberculosis (MAP) organisms. SAMPLE POPULATION: Monocytes were obtained from healthy adult Holstein dairy cows that were test negative for MAP infection on the basis of bacteriologic culture of feces and serologic test results. PROCEDURES: Monocytes or bovine macrophage cell line (BoMac) cells were incubated with MAP organisms for 30, 60, or 120 minutes with or without inhibitors of integrins, CD14, or mannose receptors. Phagocytosis was evaluated by light microscopy or by flow cytometry. CD11a/CD18, CD11b, and CD14 expression on monocytes and BoMac cells was evaluated by use of flow cytometry. RESULTS: Monocytes and BoMac cells rapidly phagocytized MAP organisms. However, compared with BoMac cells, monocytes had a greater total capacity to phagocytize MAP organisms. Addition of neutralizing anti-integrin antibodies (anti-CD11a/CD18 and anti-CD11b) substantially inhibited phagocytosis by monocytes during the first 60 minutes of incubation with MAP organisms, but were less effective at 120 minutes of incubation. Anti-CD11a/CD18 and anti-CD11b antibodies were less effective in inhibiting phagocytosis by BoMac cells. Addition of inhibitors of CD14 or mannose receptors also inhibited phagocytosis of MAP by monocytes. Addition of a combination of integrin and mannose inhibitors had an additive effect in reducing phagocytosis, but addition of integrin and CD14 inhibitors did not have an additive effect. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple receptors are involved in phagocytosis of MAP organisms. Although CD11/CD18 receptors appear to be the major receptors used by MAP at early time points, mannose receptors and CD14 also contribute substantially to phagocytosis.

Hemophagocytic syndrome in dogs: 24 cases (1996-2005).

OBJECTIVE: To determine the frequency, potential causes, and clinical and clinicopathologic features of hemophagocytic syndrome in dogs. DESIGN: Retrospective study. ANIMALS: 24 client-owned dogs. PROCEDURES: Records for dogs in which diagnostic bone marrow specimens (including an aspiration smear and core biopsy material) were obtained from 1996 to 2005 were reviewed. Inclusion criteria were presence of bicytopenia or pancytopenia in the blood and > 2% hemophagocytic macrophages in the bone marrow aspirate. RESULTS: Of 617 bone marrow specimens evaluated, evidence of hemophagocytic syndrome was detected in 24 (3.9%). The Tibetan Terrier breed was overrepresented among dogs with hemophagocytic syndrome. Clinical signs associated with hemophagocytic syndrome included fever, icterus, splenomegaly, hepatomegaly, and diarrhea. Hemophagocytic syndrome was associated with immune-mediated, infectious, and neoplastic-myelodysplastic conditions and also occurred as an idiopathic condition. Overall, dogs with infection-associated hemophagocytic syndrome had better 1-month survival rates than dogs with immune-associated and idiopathic hemophagocytic syndrome. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that hemophagocytic syndrome may occur more frequently in dogs than has previously been suspected on the basis of the paucity of reported cases. Although most dogs had definable underlying disease conditions, idiopathic hemophagocytic syndrome was also identified. Hemophagocytic syndrome of any cause is potentially life-threatening; however, the prognosis should be adjusted on the basis of the associated disease process and potential for successful treatment.

Pure white cell aplasia in a dog.

A 3-year-old Irish Wolfhound was evaluated because of acute onset of lethargy and fever. Severe neutropenia (0/microL; reference interval 2500-11,200/microL) was associated with granulocyte aplasia in the bone marrow (myeloid:erythroid ratio, 0.009:1). Antineutrophil antibodies were assessed by an indirect immunofluorescence assay using flow cytometry. When normal canine leukocytes were incubated with the patient's serum and anti-IgG, a marked shift was observed in the forward-angle light scatter of the neutrophil population, and the monocyte cluster disappeared, possibly the result of fragmentation or lysis. Both neutrophil fluorescence intensity (309 +/- 11 median channel units [MCU], control values 107-152 MCU) and the percentage of neutrophils with increased fluorescence intensity (61 +/- 5%, control values 3.8-13.7%) were increased in the patient's serum, consistent with the presence of antineutrophil antibodies. Repeated episodes of neutropenia occurred while treatment with steroidal and nonsteroidal immunosuppressive therapy was initiated and modified. The neutrophil count eventually stabilized in the low-normal range, and the dog was maintained for the next 15 months on prednisone (0.4 mg/kg PO q 48 h) and azathioprine (2 mg/kg daily). During this period, the dog developed immune-mediated hemolytic anemia and thrombocytopenia, decubital ulcers, nasal aspergillosis, and eventually, multi-organ septicemia, which led to euthanasia on day 784. A diagnosis of pure white cell aplasia was made in this dog, based on the many similarities to human patients with pure white cell aplasia, including severe neutropenia with selective granulocyte aplasia, serum antineutrophil antibodies, remission dependent on treatment with immunosuppressive therapy, and recurrent bacterial infections.

A retrospective study of the incidence and the classification of bone marrow disorders in the dog at a veterinary teaching hospital (1996-2004).

BACKGROUND: An 8-year retrospective study was conducted to evaluate the prevalence and the classification of canine bone marrow disorders in a clinical pathology service at a university referral hospital. ANIMALS: Dogs evaluated for bone marrow disorders at a veterinary teaching hospital. HYPOTHESIS: A better understanding of the spectrum and the prevalence of canine bone marrow disorders can be achieved with a multiyear retrospective study. METHODS: Bone marrow aspirate smears, core biopsy specimens, and case records from 717 dogs were reviewed. RESULTS: Bone marrow specimens were first categorized based on the presence or the absence of a primary bone marrow disorder. Nondysplastic and nonmalignant pathologic changes were placed into 14 subcategories. Frequently observed pathologic disorders included nonregenerative immune-mediated anemia, pure red cell aplasia, bone marrow necrosis, myelofibrosis, and hemophagocytic syndrome. Dysmyelopoiesis (n = 61) was subcategorized into myelodysplastic syndromes (n = 27), and congenital (n = 1) and secondary (n = 33) dysmyelopoiesis. One hundred twenty-six cases of neoplasia were divided into acute leukemia (n = 46), chronic leukemia (n = 7), stage 5 malignant lymphoma (n = 28), multiple myeloma (n = 25), malignant histiocytosis (n = 11), metastatic mast-cell tumor (n = 3), sarcoma (n = 5), and carcinoma (n = 1). CONCLUSIONS AND CLINICAL IMPORTANCE: This study provides a general indication of the spectrum and the prevalence of canine bone marrow disorders at a referral center in North America.

Detection of activated platelets in dogs with primary immune-mediated hemolytic anemia.

Thromboembolism is a major cause of morbidity and mortality in dogs with immune-mediated hemolytic anemia (IMHA). To the authors' knowledge, the role of platelets in thromboembolic events associated with IMHA has not been extensively investigated. In the study reported here, we evaluated cell membrane expression of P-selectin with flow cytometry to determine whether platelets circulate in an activated state in association with primary IMHA. Median P-selectin expression for 20 dogs with primary IMHA was 8.1-fold greater, compared with values for 20 healthy dogs. Fifteen of 20 dogs (75%) with IMHA had P-selectin median fluorescence intensity (MFI) values that exceeded the reference interval for healthy dogs. Additionally, P-selectin MFI after activation of platelets with phorbol myristate acetate was 2.1-fold greater for dogs with IMHA than for healthy control dogs. Despite treatment of all dogs with immunosuppressive therapy and 18 dogs with subcutaneously administered low-dose unfractionated heparin, 7 dogs developed clinical signs consistent with thromboembolism. These data provide support for the hypothesis that platelets circulate in an activated state in many dogs with IMHA.

Aplastic anemia in cats - clinicopathological features and associated disease conditions 1996-2004.

A retrospective study of 128 feline bone marrow reports identified 13 cases of aplastic anemia. Clinical diagnoses included chronic renal failure (n=5), feline leukemia virus infection (n=2), hyperthyroidism treated with methimazole (n=1) and idiopathic aplastic anemia (n=5). In some cats, starvation may play a role in the development of marrow aplasia. Some cats with aplastic anemia can have prolonged survival without resolution of the pancytopenia.

Regulation by Jun N-terminal kinase/stress activated protein kinase of cytokine expression in Mycobacterium avium subsp paratuberculosis-infected bovine monocytes.

OBJECTIVE: To evaluate activation of Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway in bovine monocytes after incubation with Mycobacterium avium subsp paratuberculosis (Mptb) organisms. SAMPLE POPULATION: Bovine monocytes obtained from 4 healthy adult Holstein dairy cows. PROCEDURES: Bovine monocytes were incubated with Mptb organisms with or without a specific inhibitor of the JNK/SAPK pathway (SP600125) for 2, 6, 24, or 72 hours. Expression of interleukin (IL)-1beta, IL-10, IL-12, IL-18; transforming growth factor-beta (TGF-beta); and tumor necrosis factor-alpha (TNF-alpha) and the capacity of Mptb-infected monocytes to acidify phagosomes and kill Mptb organisms were evaluated. Phosphorylation status of JNK/SAPK was evaluated at 10, 30, and 60 minutes after Mptb incubation. RESULTS: Compared with uninfected control monocytes, Mptb-infected monocytes had increased expression of IL-10 at 2 and 6 hours after incubation and had increased expression of TNF-alpha, IL-1beta, IL-18, and TGF-beta at 2, 4, and 6 hours. Additionally, Mptb-infected monocytes had increased expression of IL-12 at 6 and 24 hours. Addition of SP600125 (specific chemical inhibitor of JNK/SAPK) resulted in a decrease in TNF-alpha expression at 2, 6, and 24 hours, compared with untreated Mptb-infected cells. Addition of SP600125 resulted in a decrease in TGF-beta expression at 24 hours and an increase in IL-18 expression at 6 hours. Addition of SP600125 failed to alter phagosome acidification but did enhance the capacity of monocytes to kill Mptb organisms. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of JNK/SAPK may be an important mechanism used by Mptb to regulate cytokine expression in bovine monocytes for survival and to alter inflammatory and immune responses.

Evaluation of dysmyelopoiesis in cats: 34 cases (1996-2005).

OBJECTIVE: To further classify dysmyelopoiesis as diagnosed by use of a general classification scheme and to determine clinical features and laboratory test results that could be used to differentiate between the various forms of dysmyelopoiesis in cats. DESIGN: Retrospective case series. Sample Population-Bone marrow slides from 34 cats. PROCEDURES: Medical records of cats in which dysmyelopoiesis was diagnosed on the basis of blood and bone marrow analyses from 1996 to 2005 were reviewed. Criteria for inclusion in the study were findings of > 10% dysplastic cells in 1 or more hematologic cell lines in the bone marrow and concurrent cytopenias in the blood. Cats that met these criteria were classified into subcategories of myelodysplastic syndromes or secondary dysmyelopoiesis on the basis of reevaluation of slides. RESULTS: Of 189 bone marrow slides reviewed, 34 (14.9%) had > 10% dysplastic cells in 1 or more cell lines. Cats were subcategorized as having myelodysplastic syndrome with excessive numbers of blast cells (n = 13), myelodysplastic syndrome with refractory cytopenias (8), a variant form of myelodysplastic syndrome (1), and secondary dysmyelopoiesis (12). Findings of dysmyelopoiesis and autoagglutination in cats with myelodysplastic syndrome and in those with immune-mediated anemia complicated differentiating between the 2 conditions. CONCLUSIONS AND CLINICAL RELEVANCE: Differentiating cats with myelodysplastic syndromes from cats with immune-mediated hemolytic anemia was difficult because severe anemia and autoagglutination may be concurrent findings in both conditions. Differentiating between myelodysplastic syndrome with excessive numbers of blast cells and myelodysplastic syndrome with refractory cytopenias was useful in predicting clinical outcome.

A retrospective study of aplastic pancytopenia in the dog: 9 cases (1996-2003).

BACKGROUND: Aplastic pancytopenia is defined by the presence of pancytopenia in blood and a hypocellular bone marrow with the hematopoietic space replaced by adipose tissue. Several causes of acquired aplastic pancytopenia are known; however, in some cases, an underlying cause is never determined. OBJECTIVE: The objective of this retrospective study was to identify the incidence, potential causes, and outcome of aplastic pancytopenia in dogs. METHODS: Bone marrow cytologic and core biopsy reports were reviewed to identify dogs diagnosed with aplastic pancytopenia between July 1, 1996 and June 30, 2003. Four-hundred eighty-six bone marrow reports that included aspirate and core biopsy evaluations were reviewed. Signalment, treatment given, previous and current disease conditions, clinical signs of disease, clinical laboratory data, therapy, response to therapy, and survival time were recorded. RESULTS: Nine dogs (1.85% of bone marrow samples reviewed) met the criteria for inclusion. Two dogs (22%) had associated diseases that included monocytic ehrlichiosis and Sertoli cell tumor. In 7 dogs (78%), the cause of aplastic pancytopenia could not be definitively determined, although an idiosyncratic drug reaction to griseofulvin was suspected in 1 of the dogs. The median age of dogs diagnosed with aplastic pancytopenia was 3.2 years, and apparent breed or sex predilection was not identified. Median HCT, total WBC count, and platelet count on the day of presentation were 21.8%, 1.0 x 10(3)/microL, and 2.0 x 10(3)/microL, respectively. Six of 9 dogs diagnosed with aplastic pancytopenia died or were euthanized within 21 days. Two dogs had complete hematologic recovery. One dog was living 3 years after diagnosis, but hematologic recovery was never documented. CONCLUSIONS: Aplastic pancytopenia is diagnosed infrequently and idiopathic aplastic pancytopenia may account for up to 67% or more of canine cases. Although the prognosis is guarded, some dogs with aplastic pancytopenia recover.

Sideroblastic anemia in 7 dogs (1996-2002).

Sideroblastic anemia is an anemic condition characterized by chronic hypochromic anemia and the presence of large iron deposits in erythroid cells. Seven dogs with sideroblastic anemia were evaluated retrospectively. Historical, clinical, and clinicopathologic findings were reviewed to determine whether the condition was idiopathic or associated with disease conditions or drug or toxin exposure. Associated diseases were identified in 6 affected dogs and included acute hepatitis, pancreatitis, acute hepatitis and pancreatitis, inflammatory disease, glomerulonephritis, and myelofibrosis. None of the dogs had a history of recent exposure to drugs or toxins. One dog had no evidence of associated disease. Regardless of the associated disease condition, sideroblastic anemia was characterized by moderate to severe nonregenerative and frequently hypochromic anemia with prominent dysplastic features in bone marrow that were most prominent in the erythroid series. Survival varied from days to years. Identification of large numbers of siderocytes or sideroblasts in blood or bone marrow is inconsistent with a diagnosis of iron deficiency and should prompt a search for inflammatory disease conditions, including hepatitis, pancreatitis, and glomerulonephritis.

Recognition and classification of dysmyelopoiesis in the dog: a review.

Dysmyelopoiesis is defined as a hematologic disorder characterized by the presence of cytopenias in the blood and dysplastic cells in one or more hematologic cell lines in the blood or bone marrow. The causes of dysmyelopoiesis include acquired mutations in hematopoietic stem cells (i.e., myelodysplastic syndromes [MDSs]), congenital defects in hematopoiesis, and dysmyelopoietic conditions associated with various disease processes, drug treatments, or toxin exposure. Two major subtypes of MDSs (i.e., MDS with refractory cytopenias and MDS with excess myeloblasts) have been described that differ in clinical presentation, response to treatment, and survival time. The most frequently occurring causes of secondary dysmyelopoiesis include immune-mediated hematologic diseases, lymphoid malignancies, and exposure to chemotherapeutic drugs. Differentiation of the various causes of dysmyelopoiesis is essential for establishing an appropriate therapeutic plan and for determining prognosis.

Differentiating benign and malignant causes of lymphocytosis in feline bone marrow.

Differentiation of benign and malignant causes of lymphocytosis in blood or bone marrow can be problematic. In the present study, reports of examinations of bone marrow from cats, submitted over an 8-year period, were reviewed to identify cats with increased numbers of small lymphocytes. Of 203 reports reviewed, 12 (5.9%) indicated increased numbers of small lymphocytes. Diagnoses for these cats included chronic lymphocytic leukemia (CLL; n = 2), pure red cell aplasia (PRCA; n = 4), immune-mediated hemolytic anemia (IMHA; n = 3), thymoma (n = 1), cholangiohepatitis (n = 1), and fever of unknown origin (n = 1). Several factors were identified that could be used to differentiate reactive lymphocytosis from CLL. Cats with CLL tended to be older, and lymphocytes were slightly larger and had cleaved or lobulated nuclei. Reactive lymphocytosis was associated with immune-mediated anemias and inflammatory diseases. In reactive lymphocytosis, the proliferating lymphocytes were organized into lymphoid aggregates in bone marrow and were predominately B cells. Alternatively, in CLL and thymoma, the proliferating lymphocytes were diffusely distributed and were predominately T cells. Therefore, differentiation of the causes of lymphocytosis should include evaluation of signalment, concurrent disease conditions, lymphocyte morphology, lymphocyte distribution in bone marrow, and immunophenotype. Cat age, presence of severe anemia, and evidence of inflammatory disease also should be considered.

Evaluation of flow cytometric and automated methods for detection of activated platelets in dogs with inflammatory disease.

OBJECTIVE: To evaluate platelet surface-associated P-selectin, mean platelet component concentration (MPC), mean platelet component distribution width (MPCDW), mean platelet volume (MPV), and platelet distribution width (PDW) for detection of activated platelets in dogs with septic and nonseptic inflammatory disease. ANIMALS: 20 healthy dogs and 20 dogs with septic and nonseptic inflammatory disease. Procedures-Platelet surface-associated P-selectin (expressed as the median fluorescence intensity [MFI] of the platelet population), MPC, MPCDW, MPV, and PDW were determined in 20 healthy adult dogs, and reference ranges were calculated. These parameters were also determined in 11 dogs with nonseptic and 9 dogs with septic inflammatory disease and evaluated to determine which parameters were useful for detection of activated platelets. Results-12 dogs with inflammatory disease had P-selectin greater than the upper limit of the reference range, whereas 16 dogs with inflammatory disease had MPC lower than the lower limit of the reference range. All dogs in which P-selectin was greater than the upper limit of the reference range had MPC lower than the lower limit of the reference range. The correlation coefficient for P-selectin and MPC was 0.62. Differences in the MPCDW, MPV, and PDW in most dogs with inflammatory disease (compared with healthy dogs) were found; however, the correlation coefficients for P-selectin and MPCDW, MPV, and PDW were low. CONCLUSIONS AND CLINICAL RELEVANCE: Platelet surface-associated P-selectin and MPC appeared to be useful to detect activated platelets in most dogs with septic and nonseptic inflammatory disease.

Expression of interleukin-10 and suppressor of cytokine signaling-3 associated with susceptibility of cattle to infection with Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To determine functional characteristics of monocytes obtained from cows with subclinical infection with Mycobacterium avium subsp paratuberculosis (MAP) that may have predisposed those cows to becoming infected with MAP SAMPLE POPULATION: Monocytes obtained from 5 uninfected cows and 5 cows subclinically infected with MAP in a herd with a high prevalence of paratuberculosis (ie, Johne's disease). PROCEDURES: Monocytes from uninfected and subclinically infected cows were incubated with MAP for 2, 6, 24, 72, or 96 hours. Variables measured included expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-10, IL-12, transforming growth factor-beta, and suppressor of cytokine signaling-3 (SOCS-3); apoptosis of monocytes; acidification of phagosomes; and killing of MAP. RESULTS: Monocytes from infected cows had greater expression of IL-10 and SOCS-3 at 2 hours of coincubation with MAP and lower expression of TNF-alpha and IL-12 when results for all incubation times were combined. Monocytes from infected cows had a greater capacity to acidify phagosomes. No differences were observed in the rate of apoptosis or capacity of monocytes to kill MAP organisms. CONCLUSIONS AND CLINICAL RELEVANCE: Monocytes obtained from cows with subclinical infection with MAP had upregulated expression of IL-10 and SOCS-3 within the first 2 hours after exposure to MAP organisms. Although this did not inhibit acidification of phagosomes, apoptosis of monocytes, or attenuation of the capacity to kill MAP organisms, it may have attenuated the capacity of mononuclear phagocytes to initiate inflammatory and adaptive immune responses.