Differences in normal values for murine white blood cell counts and other hematological parameters based on sampling site.

OBJECTIVE AND DESIGN: The effect of blood sampling site on the hemogram and neutrophil adhesion molecules was examined in BALB/c mice. MATERIALS AND METHODS: Blood samples were drawn from the tail, eye, and heart during anesthesia with ketamine and xylazine. Cell numbers were quantified with an automated counter and flow cytometry was used to quantify CD11b and CD18. RESULTS: Total white blood cell (WBC) counts were highest from tail, lower from eye, and significantly lower from heart blood. In general, differences between tail and heart counts reflected changes in all cell types. RBCs, platelets and hematocrits were significantly increased in tail compared to heart blood. Although CD18 levels were not different, CD11b was significantly higher on neutrophils from tail compared to heart blood. CONCLUSIONS: In anesthetized BALB/c mice, sampling site readily influences blood counts and neutrophil CD11b. The findings underscore the need to standardize sampling site when measuring these parameters.

Eotaxin represents the principal eosinophil chemoattractant in a novel murine asthma model induced by house dust containing cockroach allergens.

Asthma represents a serious health problem particularly for inner city children, and recent studies have identified that cockroach allergens trigger many of these asthmatic attacks. This study tested the concept that asthma-like pulmonary inflammation may be induced by house dust containing cockroach allergens. An aqueous extract was prepared from a house dust sample containing endotoxin and high levels of cockroach allergens. BALB/c mice were immunized with the house dust extract (HDE) and received two additional pulmonary challenges. Bronchoalveolar lavage (BAL) eosinophil counts and eotaxin levels were significantly increased in immunized mice exposed to the HDE, whereas neutrophils were the predominant BAL inflammatory cell in the unimmunized mice. Kinetics studies in immunized mice demonstrated a peak pulmonary inflammatory response 48 h after the last challenge. The allergic response in this model was further confirmed by histological and physiological studies demonstrating a significant influx of eosinophils and lymphocytes in the peribronchial area, and severe airway hyperreactivity through whole-body plethysmography. The specificity of the response was established by immunizing with HDE and challenging with purified cockroach allergen, which induced pulmonary eosinophilia and airway hyperreactivity. Ab inhibition of eotaxin significantly inhibited the number of BAL eosinophils. These data describe a novel murine model of asthma-like pulmonary inflammation induced by house dust containing endotoxin and cockroach allergens and further demonstrate that eotaxin represents the principal chemoattractant for the recruitment of the pulmonary eosinophils.

Development and optimization of cytokine ELISAs using commercial antibody pairs.

The measurement of cytokines in plasma and other fluids often requires the use of an enzyme-linked immunosorbant assay (ELISA). In the research environment, a valuable assay is one that yields reliable results in the shortest amount of time for the least cost. To achieve this goal, a protocol has been outlined to develop sandwich ELISAs for cytokines using commercial antibodies. These guidelines for ELISA development include selecting antibody concentrations, choosing an appropriate buffer, reducing plasma interference and evaluating the optimal length for incubation periods. In addition, the protocol for a rapid IL-6 ELISA is presented. This ELISA allows measurement of IL-6 in a reduced amount of time by raising the concentration of antibodies used and increasing the temperature for incubation. By following the guidelines presented, cost-effective, cytokine ELISAs can be developed that yield low background, detect a wide range of concentrations, and are suitable for use in the research setting.

Combination immunotherapy with soluble tumor necrosis factor receptors plus interleukin 1 receptor antagonist decreases sepsis mortality.

OBJECTIVE: Inhibition of tumor necrosis factor (TNF) or interleukin 1 (IL-1) alone has not improved sepsis survival in human clinical trials; therefore, it has been suggested that blockade of both may be successful. We tested whether combination immunotherapy would improve survival in mice subjected to a lethal lipopolysaccharide (LPS) challenge or the sepsis model of cecal ligation and puncture. DESIGN: Mice were treated with the combination immunotherapy and challenged with either a lethal dose of lipopolysaccharide or a septic challenge induced by cecal ligation and puncture. SETTING: University research laboratory. SUBJECTS: Adult, female Balb/c mice. INTERVENTIONS: Mice were treated with the combination of the IL-1 receptor antagonist plus a polyethylene glycol-linked dimer of the TNF soluble receptor. MEASUREMENTS AND MAIN RESULTS: LPS lethality was reduced in the treated mice with a decrease in biologically active TNF in the plasma and peritoneal fluid. In the cecal ligation and puncture (CLP) model of sepsis, this combination immunotherapy for 1 day decreased plasma and peritoneal levels of IL-6 and the murine chemokines KC and MIP-2. However, treatment did not result in a reduction in the hypothermia or peripheral blood alterations that occur after CLP, and the 1-day therapy did not result in an improvement in survival. In contrast, when combination immunotherapy was extended to 3 days there was a significant improvement in survival. CONCLUSIONS: These data demonstrate that inhibition of both TNF and IL-1 will decrease the lethality of sepsis initiated by CLP if the combination immunotherapy is provided for a sufficient amount of time.

Differential local and systemic regulation of the murine chemokines KC and MIP2.

We characterized the relative biological activity and expression of two murine chemokines that may serve as functional homologues for human IL-8, KC, and macrophage inflammatory protein 2 (MIP2). Recombinant chemokines were produced in bacterial expression systems and antibodies specific for KC or MIP2 were raised. In vitro assays showed that KC elicited 4-fold greater neutrophil chemotaxis compared with MIP2, while MIP2 elicited significantly greater release of elastase. Lipopolysaccharide- (LPS) stimulated macrophages (8 h) secreted more MIP2 (approximately 10 ng/mL) compared with KC (approximately 4 ng/ml) and expression of either murine chemokine was independent of TNFalpha or IL-1beta production. Thioglycollate (thio) and glycogen (gly) induced peritonitis produced more KC (thio = 7.1 and gly = 2.5 ng/mL) in the peritoneum compared with MIP2 (thio = 4.5 and gly = 0.3 ng/mL). Plasma KC levels were very high after either challenge (approximately 24 ng/mL), which was >50-fold more than the systemic increase in MIP2 (approximately 0.3 ng/mL). Our data demonstrate that while KC and MIP2 have similar in vitro production characteristics, KC appears to be a more potent and systemically distributed chemokine during acute in vivo inflammation, while MIP2 expression appears limited to localized expression.

Critical role of CD14 for production of proinflammatory cytokines and cytokine inhibitors during sepsis with failure to alter morbidity or mortality.

We investigated the immunopathophysiologic responses during sepsis induced by cecal ligation and puncture (CLP) in CD4-deficient (CD14 knockout [CD14KO]) mice. Our studies were designed to specifically test the role of CD14 in the inflammatory response to sepsis and to ascertain if alterations would improve morbidity or mortality. Sepsis was induced using the CLP model with appropriate antibiotic treatment. The severity of sepsis increased in the CD14KO mice with increasing puncture size (18 gauge [18G], 21G, and 25G). Following CLP, body temperature (at 12 h) and gross motor activity levels of the sham and 25G CLP groups recovered to normal, while the 21G and 18G CLP groups exhibited severe hypothermia coupled with decreased gross motor activity and body weight. There were no significant differences in survival, temperature, body weight, or activity levels between CD14KO and control mice after 21G CLP. However, CD14KO mice expressed two- to fourfold less pro-inflammatory (interleukin-1beta [IL-1beta], tumor necrosis factor [TNF], and IL-6) and anti-inflammatory (IL-10, IL-1 receptor antagonist, and TNF receptors I and II) cytokines in the blood after 21G CLP. Plasma levels of the chemokines macrophage inflammatory protein 2alpha and KC were similarly reduced in CD14KO mice. A similar trend of decreased cytokine and cytokine inhibitor levels was observed in the peritoneal cavity of CD14KO mice. Our results indicate that the CD14 pathway of activation plays a critical role in the production of both pro-inflammatory cytokines and cytokine inhibitors but has minimal impact on the morbidity or mortality induced by the CLP model of sepsis.

Ratio of local to systemic chemokine concentrations regulates neutrophil recruitment.

CXC chemokines are important regulators of local neutrophil recruitment. In this study, we examined the role of the ratio of local to systemic chemokine concentrations as a significant factor determining local neutrophil recruitment. Thioglycollate was injected intraperitoneally into BALB/c mice resulting in a dose-dependent increase in neutrophil recruitment and local inflammation, as measured by peritoneal levels of interleukin 6. At the high dose of 3% thioglycollate, antibody inhibition of the murine chemokines KC and macrophage inflammatory protein-2 caused a reduction in peritoneal neutrophil recruitment by as much as 93%. A paradoxical effect was observed with a 0.3% thioglycollate intraperitoneal challenge. In this situation, inhibition of KC resulted in a significant increase in peritoneal neutrophils, and inhibition of macrophage inflammatory protein-2 also resulted in increased peritoneal neutrophils. These results were consistent with a reverse chemotactic gradient as described by the ratio of peritoneal to plasma KC levels. A higher ratio (ie, increased peritoneal chemokines compared to plasma) resulted in increased neutrophil recruitment after either the 3% or 0.3% thioglycollate challenge. Our results demonstrate that whereas sufficient local concentrations of chemokines are necessary, a critical factor dictating local neutrophil recruitment is the ratio of the local to the systemic chemokine concentrations.

Immunopathology of a two-hit murine model of acid aspiration lung injury.

In a two-hit model of acid aspiration lung injury, mice were subjected to nonlethal cecal ligation and puncture (CLP). After 48 h, intratracheal (IT) acid was administered, and mice were killed at several time points. Recruitment of neutrophils in response to acid was documented by myeloperoxidase assay and neutrophil counts in bronchoalveolar lavage (BAL) fluid and peaked at 8 h post-IT injection. Albumin in BAL fluid, an indicator of lung injury, also peaked at 8 h. When the contributions of the two hits were compared, neutrophil recruitment and lung injury occurred in response to acid but were not greatly influenced by addition of another hit. Neutrophil sequestration was preceded by elevations in KC and macrophage inflammatory protein-2alpha in plasma and BAL fluid. KC levels in BAL fluid were higher and peaked earlier than macrophage inflammatory protein-2alpha levels. When KC was blocked with specific antiserum, neutrophil recruitment was significantly reduced, whereas albumin in BAL fluid was not affected. In conclusion, murine KC mediated neutrophil recruitment but not lung injury in a two-hit model of aspiration lung injury.

Immunopathologic alterations in murine models of sepsis of increasing severity.

We investigated inflammatory and physiologic parameters in sepsis models of increasing lethality induced by cecal ligation and puncture (CLP). Mice received imipenem for antibiotic therapy, and groups were sacrificed at 2, 4, 8, 12, 16, 20, and 24 h after CLP. The severity of sepsis increased with needle puncture size (lethality with 18-gauge puncture [18G], 100%; 21G, 50%; 25G, 5%; sham treatment, 0%). While the temperature (at 12 h) and the activity and diurnal rhythm (at day 4) of the 25G-treated CLP group recovered to normal, the 21G and 18G treatment groups exhibited severe hypothermia along with decreased activities. A direct correlation was also observed between the severity of sepsis and cytokine (interleukin 1beta [IL-1beta], tumor necrosis factor [TNF], IL-6, and IL-10) concentrations in both the peritoneum and the plasma. There were substantially higher cytokine levels in the more severe CLP models than in the sham-treated one. Peritoneal and plasma TNF levels were always less than 40 pg/ml in all models. None of the cytokines in the septic mice peaked within the first hour, which is in contrast to the results of most endotoxin models. Chemokine (KC and macrophage inflammatory protein 2) profiles also correlated with the severity of sepsis. Except for the chemokines, levels of inflammatory mediators were always higher at the site of inflammation (peritoneum) than in the circulation. Our study demonstrated that sepsis of increasing severity induced increased cytokine levels both within the local environment (peritoneum) and systemically (plasma), which in turn correlated with morbidity and mortality.

Plasma interference in an enzyme-linked immunosorbant assay using a commercial matched antibody pair.

In this study, severe plasma interference was repeatedly documented in an IL-1ra sandwich enzyme-linked immunosorbant assay (ELISA) using a commercial matched antibody pair. Several physical and biochemical treatments were used in an attempt to alleviate this plasma effect including the following: buffer optimization, sample dilution, increasing incubation temperature, heat treatment of plasma, increasing detergent concentrations, glutaraldehyde pretreatment of the plate and the addition of polyethylene glycol (PEG). Evaluation of several buffers demonstrated that the range of optical densities could be increased dramatically with the use of an appropriate buffer. Of the treatments examined, only the addition of polyethylene glycol (PEG) to the dilution buffer created a marked improvement in the ELISA, despite a resulting background increase. Further investigation demonstrated that 10% PEG in the dilution buffer added to biotinylated antibody and the streptavidin provided the greatest improvement to the sensitivity of the ELISA.

The effect of bone compaction on early fixation of porous-coated implants.

The effect of a bone compaction technique versus conventional drilling on the early fixation of porous-coated implants was examined in a canine model. Compaction dilation resulted in a significant increase in implant fixation stiffness (P < .01) and ultimate fixation strength (P < .01) at 0 and 3 weeks. Fixation stiffness remained significantly increased at 6 weeks (P < .01); however, the ultimate fixation strength was not statistically significant between the 2 techniques (P > .05). There was no significant difference in either fixation value at 9 weeks (P > .05). Histological examination of the bone-implant interface demonstrated an increase in the density of cancellous bone immediately adjacent to the implants placed in the compaction dilated holes. The results of this study suggest that the compaction method of host bone preparation may optimize the initial stability of the implant interface of porous-coated prostheses.

Retroviral transmission in bone allotransplantation. The effects of tissue processing.

The transmission of a retrovirus through transplantation of processed bone allografts was studied using the feline leukemia virus. The long bones of 4 previously infected donor cats were harvested and assigned to 1 of 3 treatment groups: single freeze/thaw cycle, double freeze/thaw cycle, or double freeze/thaw cycle with water flush to remove bone marrow. Cortical bone grafts and corticocancellous bone grafts from each treatment group were transplanted into individual specific-pathogen-free recipients. Samples of plasma were obtained weekly from all recipients and were tested with an enzyme-linked immunosorbent assay to detect viral antigen. For animals that tested consistently negative for viral antigen, plasma samples also were tested for antiviral antibody to feline leukemia virus measured by live cell immunofluorescence. The results of the antigen and antibody testing revealed that all of the cortical and corticocancellous bone allografts in each of the 3 treatment groups transmitted virus. The ability of the treated bone allografts to transmit a feline retrovirus suggests that routine processing and removal of bone marrow may not inhibit their ability to transmit other retroviruses, such as the human immunodeficiency virus.

Gastric dilatation-volvulus after splenic torsion in two dogs.

Two dogs developed gastric dilatation-volvulus 2 and 17 months, respectively, after splenectomy for treatment of splenic torsion. Splenic displacement and torsion may stretch the gastric ligaments, allowing increased mobility of the stomach. After splenectomy, an anatomic void may be created in the cranioventral part of the abdomen, contributing to the mobility of the stomach. Veterinarians treating dogs with isolated splenic torsion may wish to consider prophylactic gastropexy at splenectomy, to reduce the chance of future gastric dilatation-volvulus. Prophylactic gastropexy should be done only if the dog's hemodynamic status is stable enough to allow for performance of the additional surgery.

Retroviral transmission by the transplantation of connective-tissue allografts. An experimental study.

The transmission of a retrovirus by the transplantation of allografts of connective tissues was studied in a feline model with use of the feline leukemia virus, a retrovirus with a replication cycle and pathological characteristics similar to those of the human immunodeficiency virus. The retrovirus was used to infect four specific-pathogen-free cats that were subsequently used as tissue donors. Fresh allografts of menisci, patellar ligaments, and patellar ligament and bone composites were harvested from infected donors and were transplanted into the knee joints of twelve specific-pathogen-free cats. A fresh cancellous-bone allograft was transplanted into the proximal part of the tibia of four additional specific-pathogen-free cats, which served as positive control animals. Additional grafts from infected donors were harvested and were stored at -80 degrees Celsius for ten weeks. A fresh-frozen graft was then transplanted into the knee of twelve other specific-pathogen-free cats. Samples of plasma were obtained weekly from all twenty-eight cats and were tested with both an enzyme-linked immunosorbent assay to detect the presence of viral antigen and an immunofluorescent antibody assay to determine exposure to the virus. All types of fresh and fresh-frozen connective-tissue allografts from the infected donors resulted in transmission of the retrovirus to the recipient cats. The recipients had evidence of viral antigen or rising antibody titers as early as two weeks after the transplantation. Histological examination of specimens of the allografts revealed normal incorporation of the transplanted tissues, with no sign of rejection of the graft.

Acute onset of hypokalemia and muscular weakness in four hyperthyroid cats.

Sudden onset of muscular weakness and ventroflexion of the neck were identified in 4 hyperthyroid cats. In each cat, the onset of clinical signs was associated with an acute decrease in serum potassium concentration. The cause for hypokalemia was undetermined, but could have resulted from deficits in total body potassium content or shifts of potassium from the extracellular space into the intracellular space. The 4 cats responded to administration of potassium. Hyperthyroid cats may be prone to disturbances in potassium homeostasis. Clinicians should be aware of potential changes in potassium homeostasis during the treatment of cats with hyperthyroidism.

The effect of electromagnetic pulsing on posterior lumbar spinal fusions in dogs.

This study evaluated the effect of pulsed electromagnetic fields (PEMF) on the healing of lumbar spinal fusions. Bilateral posterior facet fusions were performed at L1-2 and L4-5 in 24 adult mongrel dogs. After surgery, eight animals were stimulated with a pulse burst type signal (PEMF) for 30 minutes a day, and eight animals were stimulated with the same PEMF for 60 minutes a day. The remaining eight animals received no active PEMF stimulation and served as controls. Four animals from each group were euthanatized at 6 and 12 weeks, and the facet fusions were evaluated using high resolution radiographs and routine histology. No statistical difference in the radiographic or histologic appearance of the fusion mass could be detected between the stimulated and control groups at either 6 or 12 weeks. The results of this study suggest that PEMF stimulation had no effect on the healing of the primary posterior spinal fusions in this controlled experimental canine model.

Simple hemagglutination inhibition test for the diagnosis of toxoplasmosis.

A simple hemagglutination inhibition (HI) test for the serological diagnosis of toxoplasmosis has been developed and evaluated. A total of 84 human and 120 mouse serum samples were tested by the newly developed HI test and compared with an immunoglobulin G-indirect fluorescent antibody test. Statistical analysis of serum titers obtained by using the HI test and the immunoglobulin G-indirect fluorescent antibody test showed a correlation coefficient of 0.89. The diagnostic efficacy of HI when compared with the immunoglobulin G-indirect fluorescent antibody diagnostic test results was 96.43% for human sera and 100% for mouse sera. The unique hemagglutination antigen, derived from Toxoplasma gondii (Rh strain) exotoxin, spontaneously binds with mouse or rat erythrocytes, causing the hemagglutination reaction. In this study, 2, 4, or 8 hemagglutinating units of T. gondii exotoxin was used with Swiss/Webster mouse erythrocytes as an indicator for the HI assay. The results indicate that 8 hemagglutinating units is optimal because this concentration has the least unexplained variability. T. gondii exotoxin was stable for at least 18 months at -70 degrees C. The Toxoplasma HI test we report in this paper is shown to be a fast, easy, highly specific, and sensitive test for the diagnosis of toxoplasmosis.