Vaccination of BALB/c mice with an avirulent Mycoplasma pneumoniae P30 mutant results in disease exacerbation upon challenge with a virulent strain.

Mycoplasma pneumoniae is a significant human respiratory pathogen that causes high morbidity worldwide. No vaccine to prevent M. pneumoniae infection currently exists, since the mechanisms of pathogenesis are poorly understood. To this end, we constructed a P30 cytadhesin mutant (P-130) with a drastically reduced capacity for binding to erythrocytes and an inability to glide on glass substrates. This mutant was determined to be avirulent and cannot survive in the lungs of BALB/c mice. We also ascertained that the previously identified P30 gliding motility mutant II-3R is avirulent and also cannot be recovered from the lungs of mice after infection. Mutant P130 was then assessed for its efficacy as a live attenuated vaccine candidate in mice after challenge with wild-type M. pneumoniae. After vaccination with the P-130 P30 mutant, mice showed evidence of exacerbated disease upon subsequent challenge with the wild-type strain PI1428, which appears to be driven by a Th17 response and corresponding eosinophilia. Our results are in accordance with other reports of vaccine-induced disease exacerbation in rodents and emphasize the need to better understand the basic mechanisms of M. pneumoniae pathogenesis.

Ultra-trace analysis of 36Cl by accelerator mass spectrometry: an interlaboratory study.

A first international (36)Cl interlaboratory comparison has been initiated. Evaluation of the final results of the eight participating accelerator mass spectrometry (AMS) laboratories on three synthetic AgCl samples with (36)Cl/Cl ratios at the 10(-11), 10(-12), and 10(-13) level shows no difference in the sense of simple statistical significance. However, more detailed statistical analyses demonstrate certain interlaboratory bias and underestimation of uncertainties by some laboratories. Following subsequent remeasurement and reanalysis of the data from some AMS facilities, the round-robin data indicate that (36)Cl/Cl data from two individual AMS laboratories can differ by up to 17%. Thus, the demand for further work on harmonising the (36)Cl-system on a worldwide scale and enlarging the improvement of measurements is obvious.

Pulmonary eosinophilia correlates with allergen deposition to the lower respiratory tract in a mouse model of asthma.

BACKGROUND: Eosinophilic infiltration into the airways is frequently associated with allergic asthma; however, the role of antigen deposition in mediating this phenomenon has not been studied in detail. OBJECTIVE: Using a murine model of ovalbumin (OVA) allergy, we examined how differential deposition of OVA during antigen challenge affects pulmonary eosinophilia, immune response and airway hyper-reactivity (AHR). METHODS: Differential allergen deposition to the upper respiratory tract (URT) alone or combined upper and lower respiratory tract (ULRT) was accomplished by administering OVA intranasally to either anaesthetized or unanaesthetized mice, respectively. BALB/c mice (6-7 weeks old) were sensitized with OVA-alum via the intraperitoneal route, and then challenged intranasally using OVA, with or without anaesthesia. AHR, enumeration of inflammatory cells and quantitative measurement of inflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BALF), lung histopathology and immune responses were subsequently assessed. RESULTS: In sensitized animals challenged via the ULRT route, a profound eosinophilia and goblet cell hyperplasia was observed in lung tissue. Conversely, sensitized mice receiving an identical challenge dose via the URT route alone exhibited only negligible levels of inflammation. Interestingly, AHR and OVA-specific IgG(1) and IgE systemic responses were comparable between the two groups. CONCLUSION: This study indicates that direct exposure of allergen in the deep lung is highly correlated with airway eosinophilia and lung inflammation, but does not correlate with AHR or immune response.

Long-term reconstitution of mice after ex vivo expansion of bone marrow cells: differential activity of cultured bone marrow and enriched stem cell populations.

In this report, we evaluated the short-term expansion of murine bone marrow mononuclear cells (BMMNC) and enriched stem cell populations to determine the capacity of these cells for long-term rescue and engraftment to lethally irradiated recipients. In our study, nonadherent bone marrow mononuclear cell (NBM-MNC) and Thy1+Lin- stem cells populations were cultured with interleukin-3 (IL-3) or IL-3 plus stem cell factor (SCF) for periods up to 6 days. By day 6 of culture, the mononuclear cells (MNC) decreased to 6% of input cell number, whereas Thy1+Lin- cells increased by 2310%. Doses of 95,000; 100,000; 50,000; and 250,000 NBM-MNCs at 0, 1, 2, and 6 days of culture, respectively, rescued 50% of lethally irradiated mice. When 250,000 MNCs were cultured for 0, 1, 2, and 6 days, 71, 61, 100, and 50% of the animals survived lethal irradiation for greater than 24 weeks. In contrast, doses of 8,000 and 21,000 Thy1+Lin- cells cultured 0 and 1 day, respectively, yielded 50% survival rates. These same cells cultured for 6 days failed to rescue recipients even at high doses. Twenty thousand Thy1+Lin- cells cultured for 0, 1, 2, and 6 days, even in the presence of SCF, produced decreasing survival rates of 86, 43, 26, and 0%, respectively. The proliferative responses of these different populations in combination with their long-term rescue abilities indicated that the absolute number of long-term rescue units (LD50, 24 weeks) in the cultured Thy1+Lin- population decreased faster than in similarly cultured NBM-MNCs. Studies evaluating donor cell engraftment demonstrated that animals rescued with cultured Thy1+Lin- and NBM-MNCs maintained high levels of donor reconstitution [7]. The percent donor T cell engraftment did not significantly change between 2 and 17 months post-bone marrow transplantation (post-BMT). Therefore, those animals who received sufficient cells to survive lethal irradiation generally established and maintained high levels of donor engraftment. The data suggest a role for accessory cells and/or factors in the preservation of stem cell activity.

Selection of murine lymphoid and hematopoietic cells using polystyrene tissue culture devices containing covalently immobilized antibody.

We have established rapid procedures that negatively deplete and positively select for specific murine cell populations. By using polystyrene tissue culture flasks containing a covalently bound mouse anti-rat antibody and specific anti-mouse, cell-surface antigen antibodies, we easily and efficiently depleted greater than 90% of the mature lineage cells from murine bone marrow. This selection procedure resulted in an enrichment of progenitor colonies (CFU-Cs) in murine bone marrow. Using the same polystyrene tissue culture devices, we can directly isolate CD117+ (c-kit+) murine hematopoietic cells. As few as 2000 of these CD117+ cells rescued and reconstituted lethally irradiated recipients in a murine bone marrow transplant model.

Immune response to a mucosally administered aflatoxin B1 vaccine.

In the present study, a mucosal vaccine was used in an effort to elicit serum IgG and intestinal secretory IgA against the mycotoxin aflatoxin B1 (AFB) in chickens. AFB was coupled to carrier proteins (BSA and porcine thyroglobulin) for use as a vaccine and ELISA coating antigen, respectively. Seven-day-old broiler chicks were divided into groups of 10 and immunized with one of four vaccine preparations: 1) AFB-BSA conjugate alone, 2) AFB-BSA linked to the B subunit of the recombinant heat-labile enterotoxin of Escherichia coli (rLT-B), 3) AFB-BSA admixed with rLT-B, or 4) AFB-BSA mixed with cholera toxin (CT). Each vaccine preparation was administered perorally, intrarectally, or intraperitoneally, with a booster immunization given 2 wk later. Sera and feces were collected weekly and assayed using isotype specific ELISA. All three routes of immunization elicited significant serum IgG responses; however, the intraperitoneal route was strongest for all vaccine preparations tested. The serum IgG immune response to the AFB-BSA conjugate was enhanced by co-administration of rLT-B but not by covalent coupling to rLT-B or coadministration with CT. Secretory IgA anti-CT and anti-rLT-B antibodies were detected in fecal supernatants, but no anti-AFB responses could be detected. As all 12 treatment groups produced significant levels of serum IgG anti-AFB, any of these approaches, including oral administration without adjuvant, may afford the chicken some level of protection through simple immuno-interception of free AFB.

Rapid thinning of Pine Island Glacier in the early Holocene.

Pine Island Glacier, a major outlet of the West Antarctic Ice Sheet, has been undergoing rapid thinning and retreat for the past two decades. We demonstrate, using glacial-geological and geochronological data, that Pine Island Glacier (PIG) also experienced rapid thinning during the early Holocene, around 8000 years ago. Cosmogenic (10)Be concentrations in glacially transported rocks show that this thinning was sustained for decades to centuries at an average rate of more than 100 centimeters per year, which is comparable with contemporary thinning rates. The most likely mechanism was a reduction in ice shelf buttressing. Our findings reveal that PIG has experienced rapid thinning at least once in the past and that, once set in motion, rapid ice sheet changes in this region can persist for centuries.

The pharmacokinetics and pharmacodynamics of d- and dl-verapamil in rabbits.

The pharmacokinetics and pharmacodynamics of d- and dl-verapamil were studied in conscious rabbits in randomized cross-over fashion. Following a single intravenous dose, there was a biexponential decline in plasma concentration with time. No differences were observed in the pharmacokinetic properties of the compounds. The mean (+/- SD) clearances of d- and dl-verapamil were 0.13 +/- 0.03 and 0.12 +/- 0.05 L/min/kg, respectively. The mean (+/- SD) steady-state volume of distribution was 9.7 +/- 5.2 L/kg for d-verapamil and 8.1 +/- 4.1 L/kg for dl-verapamil. No difference was observed between the compounds in their binding to plasma proteins. The mean (+/- SD) half-life in plasma was 98.7 +/- 63.8 min for d-verapamil and 96.3 +/- 38.0 min for dl-verapamil. In contrast to the lack of stereoselective differences in the pharmacokinetic properties of verapamil, there were marked differences in the pharmacodynamics of d- and dl-verapamil. dl-Verapamil appeared to prolong the PR interval to a greater degree than did d-verapamil, consistent with the more potent calcium channel effects of the l-enantiomer. Similarly, dl-verapamil had more potent hypotensive effects compared with the d-enantiomer, which produced no effects on systemic arterial pressure. Chronotropic effects, judged to be caused by autonomic reflexes in response to the hypotensive effects of the compound, were also statistically greater for dl-verapamil than for d-verapamil. These results demonstrate stereo-selective pharmacodynamic effects in vivo of verapamil.

Intranasal immunization of guinea pigs with an immunodominant foot-and-mouth disease virus peptide conjugate induces mucosal and humoral antibodies and protection against challenge.

Guinea pigs immunized intranasally with a keyhole limpet hemocyanin-linked peptide, corresponding to the prominent G-H loop of the VP1 protein of foot-and-mouth disease virus, raised substantial levels of antipeptide and virus-neutralizing antibodies in sera and of peptide-specific secretory immunoglobulin A in nasal secretions. In groups of animals immunized intranasally without adjuvant, 86 percent were fully protected upon challenge with homotypic virus. Surprisingly, animals given the peptide conjugates plus the mucosal adjuvant cholera toxin were afforded only partial protection in that primary lesions were observed in most animals, although spread to other feet was prevented. These results indicate that intranasal inoculation with the peptide offers a potential route of vaccination against foot-and-mouth disease and may be useful for eliciting protection in the upper respiratory tracts of susceptible animals.

A modified live Mycoplasma gallisepticum vaccine to protect chickens from respiratory disease.

The aim of this study was to assess the efficacy of a modified live Mycoplasma gallisepticum vaccine (GT5) for the protection of chickens against infection and respiratory disease. GT5 was constructed by the reconstitution of the avirulent high passage R (R(high)) strain with the gene encoding the major cytadhesin GapA. GT5 expressed GapA on its surface yet retained the phenotypic characteristics of the parental R(high) strain. Birds vaccinated with GT5 were protected upon challenge with the virulent low passage R (R(low)) strain as evidenced by a complete absence of tracheal lesions 2 and 4 weeks post-challenge, in contrast to sham immunized/challenged control birds. Modest amounts of IgG, and little, if any secretory IgA or IgM anti-M. gallisepticum were found in tracheal washings following vaccination. However, copious amounts of specific IgA were found following challenge, especially in sham immunized birds. This suggests that the tracheal IgG elicited by GT5 vaccination may have been responsible for blocking the initial colonization of R(low), thereby resulting in protection.