Effect of atmospheric carbon dioxide concentration on the cultivation of bovine Mycoplasma species.

Recommendations for bovine mycoplasma culture CO2 concentrations are varied and were not empirically derived. The objective of this study was to determine whether the growth measures of bovine mycoplasma isolates differed when incubated in CO2 concentrations of 10 or 5% or in candle jars (2.7 ± 0.2% CO2). Growth of Mycoplasma bovis (n = 22), Mycoplasma californicum (n = 18), and other Mycoplasma spp. (n = 10) laboratory isolates was evaluated. Isolate suspensions were standardized to approximately 108 cfu/mL and serially diluted in pasteurized whole milk to achieve test suspensions of 102 and 106 cfu/mL. One hundred microliters of each test dilution was spread in duplicate onto the surface of a modified Hayflick's agar plate. Colony growth was enumerated on d 3, 5, and 7 of incubation. A mixed linear model included the fixed effects of CO2 treatment (2.7, 5, or 10%), species, day (3, 5, or 7), and their interactions, with total colony counts as the dependent variable. Carbon dioxide concentration did not significantly affect overall mycoplasma growth differences, but differences between species and day were present. Colony counts (log10 cfu/mL) of M. bovis were 2.6- and 1.6-fold greater than M. californicum and other Mycoplasma spp., respectively. Growth at 7 d of incubation was greater than d 3 and 5 for all species. These findings were confirmed using field isolates (n = 98) from a commercial veterinary diagnostic laboratory. Binary growth responses (yes/no) of the field isolates were not different between CO2 treatments but did differ between species and day of incubation. On average, 57% of all field isolates were detected by 3 d of incubation compared with 93% on d 7. These results suggest that the range of suitable CO2 culture conditions and incubation times for the common mastitis-causing Mycoplasma spp. may be broader than currently recommended.

Supplementation of culture medium with L-carnitine improves the development and cryotolerance of in vitro-produced porcine embryos.

Porcine oocytes and embryos contain substantial amounts of lipid, with little known regarding its metabolic role during development. This study investigated the role of lipid metabolism and the interaction between carbohydrate and lipid substrates in porcine embryos. Following in vitro fertilisation, presumptive zygotes were transferred to culture medium supplemented with L-carnitine, a co-factor required for the metabolism of fatty acids. In porcine zygote medium-3 (PZM-3), which contains pyruvate and lactate, 3mM L-carnitine was the only dose that improved cleavage rates compared with the control. In the absence of carbohydrates, all doses of L-carnitine from 1.5 to 12mM increased cleavage rates compared with the control. Culture in a PZM-3-based sequential media system (Days 0-3: pyruvate and lactate; Days 4-7: glucose) significantly increased blastocyst cell numbers compared with culture in standard PZM-3. Supplementing PZM-3 with 3mM L-carnitine produced blastocysts with cell numbers equivalent to those obtained in the sequential media system. After vitrification, the post-warming survival rates of blastocysts obtained in media supplemented with 3mM L-carnitine were significantly greater than those of blastocysts obtained in standard PZM-3. In conclusion, L-carnitine supplementation improved embryo development when the medium contained pyruvate and lactate or was lacking carbohydrates completely, indicating a role for fatty-acid metabolism when the embryo's requirements for carbohydrates are not adequately met.

Efficiency of induction of immunoglobulin synthesis by autologous human T cells and T cell clones: relation to surface isotype of the B cell.

Efficient immunoglobulin (Ig) production was induced when human B cells were cultured with autologous T cells activated by immobilized CD3 mAb in cultures supplemented with IL-2. Negatively purified B cells or B cells positively selected with mAb to CD19, CD21 or CD72 surface antigens produced IgM, IgG and IgA, whereas B cells selected for surface IgD or IgM produced predominantly IgM indicating that little or no isotype switching was occurring. Results are compared with reports describing high levels of mu to gamma and mu to alpha switching in single B cell systems. The limited proliferation of B cells in our culture system may account for the difference. When untreated T and B cells were cultured together in the presence of immobilized CD3 mAb, B cell numbers peaked at 6-10 days whereas T cells continued to proliferate maximally. All 52 T cell clones tested induced the production of IgM, IgG and IgA from unselected or CD19 selected B cells, but efficiency of production of Ig overall and of the different isotypes varied with different T clones. All T clones which induced high IgM, IgG and IgA production induced IgE production too, but some less active T clones also induced IgE production under non-switching conditions indicating that direct contact with activated T clone cells efficiently induces IgE as well as IgG and IgA production from B cells already expressing these isotypes. Less Ig was produced with optimal numbers of untreated T clone cells than with X-irradiated cells, confirming that proliferating T cells can inhibit as well as activate Ig production from B cells.

Is the activated T helper stimulus able to induce Ig isotype switching in cultured human B cells?

It is confirmed that large amounts of IgM, IgG, and IgA are produced when human B cells are cultured with T cells activated by immobilized CD3 antibody (CD3 system). IL-2 was essential; lower levels of Ig production with different isotype ratios were obtained if IL-4 or IL-6 replaced IL-2. Depletion of sIgG+ or sIgA+ cells from the B population to be cultured markedly reduced production of IgG or IgA. Cultures of B cells selected with the pan-B markers CD19, CD72, or CD21 contained similar levels of Ig of all three isotypes, whereas B cells selected for sIgM or sIgD expression produced IgM but very little IgG or IgA indicating that little isotype switching was occurring. Production of IgG or IgA from cells expressing these isotypes was more efficient than production of IgM from IgM+IgD+ cells. These results are considered in the light of the demonstration by others of the production of multiple isotypes from single sIgM+-selected B cells. Cloned human T cells from a single donor induced production of all three isotypes, but the proportions varied indicating that the potent T-B cell interactions inducing B cell activation may override and conceal the operation of isotype specific cell interactions. Some T clones used at an optimal dose were as effective untreated as X-irradiated, whereas with other clones maximum Ig production was not achieved without irradiation.

Comparison of technetium-99m-ECD to Xenon-133 SPECT in normal controls and in patients with mild to moderate regional cerebral blood flow abnormalities.

Technetium-99m-1,1-ethyl cysteinate dimer (ECD) has been proposed as a "chemical microsphere" for SPECT measurement of regional cerebral blood flow (rCBF). However, its distribution has not yet been compared in humans to an established rCBF measure. Therefore, we compared the uptake and distribution of ECD with rCBF measured by 133Xe SPECT in subjects with mild to moderate flow abnormalities and in normal volunteers. Blood and urine chemistries and vital signs were unchanged from pre-ECD values up to seven days postinjection. Profile plots demonstrated pattern agreement between rCBF ratios (133Xe) and ECD count density ratios. A significant correlation of rCBF ratios to ECD count density ratios was observed (r = 0.77), with a slope of 0.64 and intercept of 0.36. To explore whether or not the relationship between rCBF and ECD was dependent on absolute flow, ECD region of interest data were expressed in units of ml/min/100 g by equating global CBF (133Xe) and ECD global count density. A closer correlation (r = 0.88) was found for these data than for the count ratio data. The slope was closer to one (m = 0.83) and the intercept was closer to zero (b = 8.2). Also, a significant correlation was observed between ECD-derived rCBF and 133Xe rCBF in the lesion area (r = 0.92) for patients with well-demarcated rCBF lesions. The slope (0.80) suggested a slight underestimation of lesion flow by ECD. Finally, ECD clearance from cortical gray matter ROIs derived from high-resolution scans from 1 to 4 hr postinjection was slow (2.4%/hr). In summary, ECD is a safe and effective marker of regional cerebral perfusion. The distribution of ECD is linearly related to rCBF measured by 133Xe SPECT, although our data suggest a mild underestimation of flow at the high end of the normal range.

Dual-isotope brain SPECT imaging with technetium-99m and iodine-123: clinical validation using xenon-133 SPECT.

Our phantom studies indicate that the energy resolution (9.7% FWHM) of a new three-headed single-photon tomograph (PRISM-3000) separates the distribution of 99mTc from 123I for 10% asymmetric or 15% or 10% centered 99mTc windows when combined with a 10% asymmetric 123I window. This technique is now applied to the simultaneous measurement of resting rCBF and changes induced by vasodilation (1 g acetazolamide) in 10 subjects with cerebrovascular disease. Resting and vasodilated 133Xe SPECT images were obtained first. Within 48 hr, 99mTc HMPAO was given at rest, acetazolamide injected, and after 20 min either [123I] IMP or [123I] HIPDM was administered. Subjects were scanned for 99mTc and 123I simultaneously using 10% asymmetric windows. Regression analyses demonstrated a linear relationship between 133Xe SPECT and dual-isotope SPECT measurements of lesion-to-cerebellum ratios in baseline (r = 0.92), vasodilated (r = 0.86) and rest-minus-vasodilated data (r = 0.85). Technetium-99m and 123I images obtained through dual-isotope imaging are by definition in perfect anatomic registration.

Dual-isotope brain SPECT imaging with technetium-99m and iodine-123: validation by phantom studies.

Phantom studies were employed to determine whether the enhanced energy resolution (9.7% FWHM) of a new high-resolution, three-headed single-photon emission computed tomograph might permit the simultaneous acquisition of 99mTc and (123)I. Various window widths (15% and 10%) and positions (centered and asymmetric to the photopeak) were used to examine cross-contamination between these two isotopes. Brain phantom experiments using a 15% centered 99mTc window in conjunction with a 10% asymmetric (123)I window (upper half of the (123)I photopeak) demonstrated that approximately 95% of observed counts were derived from the isotope of interest. Shifting the (123)I window from asymmetric to centered resulted in a significant increase in contamination of the (123)I window. Shifting the 99mTc window from centered to asymmetric did not significantly alter image quality for 99mTc. Separate experiments employing vials with varying isotope concentrations demonstrated that quantitative recovery from mixed 99mTc and (123)I sources was equivalent to that from matched single-isotope sources (r2 > or = to 0.90).

Sodium retention and cortisol (hydrocortisone) suppression caused by dexamethasone and triamcinolone in equids.

Three ponies and 1 horse were bilaterally adrenalectomized (BADX). The initial hypoadrenal episode after BADX was reversed with 20 mg of dexamethasone (DXM) IM (n = 2) or 20 mg of triamcinolone (TMC) IM (n = 2). Nine hypoadrenal crises were reversed with 20 mg of DXM given IM (n = 4) or 20 mg of TMC given IM (n = 5). Sodium and chloride retention and potassium excretion were documented based on changes in serum electrolytes and urinary excretion. Eight intact adult horses were randomly assigned to 2 groups to study the effects of a single IM injection of DXM (0.044 mg/kg of body weight) or TMC (0.044 mg/kg). Cortisol (hydrocortisone) suppression was found to be maximal (nondetectable amounts of cortisol) by 12 hours in both groups. Cortisol was again detectable in the DXM group at 24 hours after injection and was at pretreatment values at 168 hours. Cortisol was not detectable in the TMC group for 192 hours and did not reach pretreatment values until 336 hours. The duration of the gluconeogenic effect was compared with the duration of cortisol suppression exerted by DXM and TMC in these intact animals. Assuming that the decrease in plasma glucose coincides with the decrease in glucocorticoid activity of the respective steroid, a relative hypoadrenocortical state was found in the animals treated with DXM between the 2nd and 7th day after treatment, whereas this state occurred between the 6th and 14th day after treatment with TMC.