Proficiency monitoring of monoclonal antibody cocktail-based enzyme-linked immunosorbent assay for detection of allergen-specific immunoglobulin E in dogs.

The purpose of our study was to document the continued comparative proficiency of different laboratories that perform a monoclonal antibody-based enzyme-linked immunosorbent assay (macELISA) for detection of allergen-specific immunoglobulin (Ig)E in dogs. Replicate samples of 18 different sera pools were independently evaluated in a single blinded fashion by each of 16 different operators functioning in 10 different laboratories. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.0% (range: 2.7-16.1%), while the average intralaboratory interassay variance was 7.5% (range: 3.9-10.9%). The overall interassay interlaboratory variance was consistent among laboratories and averaged 11.4% (range: 8.5-12.5%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose response profiles observed in each of the laboratories were indistinguishable. Considering the positive or negative results, interassay interlaboratory concordance of results exceeded 90%. Correlation of optical density values between and among all laboratories was strong (r > 0.9, P < 0.001). Collectively, the results demonstrated that the macELISA for measuring allergen-specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory by differing operators but also among laboratories using the same monoclonal-based ELISA.

The prevalence of Bartonella, hemoplasma, and Rickettsia felis infections in domestic cats and in cat fleas in Ontario.

The prevalence of persistent bacteremic Bartonella spp. and hemoplasma infections was determined in healthy pet cats in Ontario. Blood samples from healthy cats sent to a diagnostic laboratory for routine health assessment over the course of 1 y were tested for Bartonella spp. using both polymerase chain reaction (PCR) and blood culture, and for the presence of hemoplasma by PCR. The overall prevalence of Bartonella spp. by PCR and by culture combined was 4.3% (28/646) [3.7% (24/646) Bartonella henselae, 0.6% (4/646) Bartonella clarridgeiae]. The novel B. henselae PCR developed for this study demonstrated nearly twice the sensitivity of bacterial isolation. The overall prevalence of hemoplasma was 4% (30/742) [3.3% (25/742) Candidatus Mycoplasma haemominutum, 0.7% (5/742) Mycoplasma haemofelis]. There was no significant difference between the prevalence of infection by season or by age (< or = 2 y, > 2 y). Candidatus Mycoplasma turicensis was identified, for the first time in Canada, in 1 cat. The prevalence of Bartonella (58%) and hemoplasma (47% M. haemofelis, 13% M. haemominutum) in blood from a small sampling (n = 45) of stray cats was considerably higher than that found in healthy pet cats. The prevalence of Rickettsia felis in cat fleas was also assessed. A pool of fleas from each of 50 flea-infested cats was analyzed for the presence of R. felis by PCR. Rickettsia felis was confirmed, for the first time in Canada, in 9 of the 50 samples. Therefore, the prevalence of Bartonella and hemoplasma infection in healthy pet cats is relatively low. Further, the control of cat fleas is important because of the public health significance of Bartonella and R. felis infection.

Heterogeneity of Tie2 expression in tumor microcirculation: influence of cancer type, implantation site, and response to therapy.

To evaluate the expression of the Tie2/Tek tyrosine kinase receptor in tumor blood vessels, we examined Tie2lacZ(+)/RAG1(-) mice. There was considerable heterogeneity (Tie2-negative, Tie2-positive, or Tie2-composite blood vessels) in subcutaneous xenografts of human colorectal carcinoma (HCT116; 97.5% Tie2-positive vessels) versus human melanoma (WM115; 75.9% Tie2-positive vessels). Similar patterns of Tie2 expression occurred in abdominal metastases derived from the same cell lines. Immunostaining for endothelial markers and Tie2 revealed that endogenous protein levels corresponded with transgene activity. Endothelial cells were confirmed to be of mouse origin through triple immunofluorescence staining with mouse antiserum to human nuclei, isolectin GS-IB(4), and anti-Tie2. Similar Tie2 heterogeneity was observed in clinical specimens from a variety of human cancers, including malignant melanoma and colorectal carcinoma. We also examined the effect of Tek-Delta Fc anti-angiogenic therapy on tumor growth and Tie2 expression patterns in HCT116 and WM115 subcutaneous xenografts. Tek-Delta induced extensive tumor regression in HCT116 tumors and concomitant reductions in Tie2-expressing blood vessels. However, no significant responses were seen in Tek-Delta-treated WM115 tumors. Thus, vascular heterogeneity of Tie2 expression is cancer-type specific, suggesting that the tumor microenvironment and/or direct cancer cell interactions influence Tie2 endothelial expression.

Chimerism of murine fetal bone marrow by maternal cells occurs in late gestation and persists into adulthood.

Studies of murine severe combined immune-deficient (scid/scid) fetuses gestating in transgene-tagged immune competent dams have established high frequencies of transplacental trafficking of nucleated maternal cells. Maternal cells first appeared in thymus at gestation day (gd) 12.5 and were present in more than 90% of late gestation fetuses. Morphologically heterogeneous maternal cells were located predominantly in bone marrow and thymus and also occasionally in liver, spleen, and nonlymphoid organs. We have now evaluated maternal cell chimerism in offspring with normal lymphoid development. Genetically normal blastocysts from random-bred CD1 mice were transferred to C57BL/6J- lacZ transgene-tagged ROSA26 females. Serial sectioning of fetuses followed by histochemistry for lacZ-expressing cells was used to comprehensively define organs containing maternal cells. Fetuses, sectioned in their entirety, had no detectable maternal cells before gd 16.5. Morphologically homogenous, nucleated maternal cells were first present in fetal bone marrow cavities at gd 16.5 and were evident in all offspring in later gestation. Postnatally, maternal cells were also present in bone marrow cavities into adulthood, as determined by lacZ histochemistry and PCR amplification of the maternal transgene. The frequency of maternally derived cells in postnatal bone marrow was increased compared with late gestation, and occasionally, maternal cells were detected in postnatal spleen. The normalcy of maternal cell transfer to genetically immune competent progeny and their long-term engraftment is suggestive of a functional role for maternal cells in offspring.