Assessment of florfenicol as a possible treatment for chlamydiosis in koalas (Phascolarctos cinereus).

OBJECTIVE: Because of limited availability of chloramphenicol to veterinary suppliers, a preliminary study was performed to predict whether an analogue, florfenicol, is an efficacious treatment for chlamydiosis in koalas. METHODS: Florfenicol was administered to koalas with naturally occurring chlamydiosis at 20 mg/kg SC (n = 3) and at 5 mg/kg (n = 3) and 10 mg/kg (n = 3) IV. The estimated areas under the plasma concentration versus time curves (AUC) were compared with the minimum inhibitory concentration to inhibit Chlamydia pecorum. Clinical data were also examined from field trials conducted on koalas (n = 19) with naturally occurring chlamydiosis and treated with florfenicol at a range of dosages (5-20 mg/kg SC and 6-15 mg/kg IV). Florfenicol binding to proteins in plasma was also determined. RESULTS: Florfenicol was not detectable in plasma 24 h post-administration at 20 mg/kg SC. The estimated AUC0-24 h following administration at 10 mg/kg IV suggests florfenicol might be effective against Chlamydia spp. via this route. Florfenicol binding to plasma proteins was 13.0% (± 0.30 SEM). After treatment with florfenicol in field trials, 5 of 19 koalas (26%) were released without further treatment, 4 with no long-term follow-up; 6 (32%) required additional treatment with chloramphenicol to resolve chlamydiosis; 7 (36%) failed to clinically improve, of which 3 had clinical signs and/or necropsy findings suggestive of antibiotic-related gastrointestinal dysbiosis; another koala died within minutes of florfenicol administered IV at 7 mg/kg. CONCLUSION: When administered at dosages tolerable in the field, florfenicol is a problematic treatment for chlamydiosis based on equivocal outcomes and plasma concentrations below those that inhibit the pathogen.

Plasma concentrations of chloramphenicol after subcutaneous administration to koalas (Phascolarctos cinereus) with chlamydiosis.

Nine mature koalas with chlamydiosis, typically keratoconjunctivitis and/or urogenital tract infection, were treated with daily subcutaneous injections of chloramphenicol at 60 mg/kg for 45 days (five koalas), or for a shorter duration (four koalas). All koalas were initially positive for Chlamydia pecorum as determined by real-time polymerase chain reaction (qPCR). Plasma chloramphenicol concentrations were determined at t = 0, 1, 2, 4, 8, and 24 h after the day 1 injection (nine koalas) and after the day 15 injection (seven koalas). Chloramphenicol reached a median (and range) maximum plasma concentration of 3.03 (1.32-5.03 µg/mL) at 4 (1-8 h) after the day 1 injection and 4.82 (1.97-27.55 µg/mL) at 1 (1-2 h) after day 15. The median (and range) of AUC(0-24) on day 1 and day 15 were 48.14 (22.37-81.14 µg·h/mL) and 50.83 (28.43-123.99 µg·h/mL), respectively. The area under the moment curve (AUMC)(0-24) median (and range) for day 1 and day 15 were 530.03 (233.05-798.97 h) and 458.15 (291.72-1093.58 h), respectively. Swabs were positive for chlamydial DNA pretreatment, and all koalas except one, produced swabs negative for chlamydial DNA during treatment and which remained so, for 2-63 days after treatment, however whether chloramphenicol treatment prevented long-term recrudescence of infection was not established. At this dose and dosing frequency, chloramphenicol appeared to control mild chlamydial infection and prevent shedding, but severe urogenital disease did not appear to respond to chloramphenicol at this dosage regime. For koalas affected by severe chlamydiosis, antibiotics alone are not sufficient to effect a cure, possibly because of structural or metabolic changes associated with chronic disease and inflammation.

Using quantitative polymerase chain reaction to correlate Chlamydia pecorum infectious load with ocular, urinary and reproductive tract disease in the koala (Phascolarctos cinereus).

Complex interactions between Chlamydia pecorum infection, the immune response and disease exist in the koala. We used quantitative polymerase chain reaction to investigate the relationship between C. pecorum infectious load and ocular and urogenital tract disease. Chlamydia pecorum shedding was generally higher in animals with chronic, active disease than in animals with inactive disease. The absence of ocular disease was generally associated with low levels of shedding, but relatively high levels of shedding in the urogenital tract were detected in some koalas without clinical disease signs. These results suggest a complex disease pathogenesis and clinical course in C. pecorum-infected koalas.

Sapogenin levels in Narthecium ossifragum plants and Ovis aries lamb faeces during two alveld outbreaks in Møre og Romsdal, Norway, 2001.

The proposal that saponins produced by the lily bog asphodel (Narthecium ossifragum) may be the direct cause of the hepatogenous photosensitization disease alveld seen in Norwegian lambs was investigated by comparing sapogenin levels in two control and two toxic pastures, and in faeces from lambs grazing the four pastures in the Halsa and Surnadal municipalities, Møre og Romsdal county, Norway. Generally similar levels of sapogenins, determined after hydrolysis of parent plant saponins, were found in Narthecium leaves collected in June/July 2001 from the two alveld outbreak areas and two nearby control areas. Differences in the median sapogenin levels determined for leaf samples in outbreak and control areas were not statistically significant. The total level of free and conjugated sapogenins in faeces recovered from the rectums of lambs grazing the outbreak and control pastures areas varied greatly. The results obtained do not support the hypothesis that a dose-response relationship exists between Narthecium saponin levels and the occurrence of alveld outbreaks.

CD70 (TNFSF7) is expressed at high prevalence in renal cell carcinomas and is rapidly internalised on antibody binding.

In order to identify potential markers of renal cancer, the plasma membrane protein content of renal cell carcinoma (RCC)-derived cell lines was annotated using a proteomics process. One unusual protein identified at high levels in A498 and 786-O cells was CD70 (TNFSF7), a type II transmembrane receptor normally expressed on a subset of B, T and NK cells, where it plays a costimulatory role in immune cell activation. Immunohistochemical analysis of CD70 expression in multiple carcinoma types demonstrated strong CD70 staining in RCC tissues. Metastatic tissues from eight of 11 patients with clear cell RCC were positive for CD70 expression. Immunocytochemical analysis demonstrated that binding of an anti-CD70 antibody to CD70 endogenously expressed on the surface of A498 and 786-O cell lines resulted in the rapid internalisation of the antibody-receptor complex. Coincubation of the internalising anti-CD70 antibody with a saporin-conjugated secondary antibody before addition to A498 cells resulted in 50% cell killing. These data indicate that CD70 represents a potential target antigen for toxin-conjugated therapeutic antibody treatment of RCC.

Proteomic analysis of the cell-surface membrane in chronic lymphocytic leukemia: identification of two novel proteins, BCNP1 and MIG2B.

B-cell-specific plasma-membrane proteins are potential targets for either small molecule or antibody-based therapies. We have sought to annotate proteins expressed at the cell surface membrane in patients with chronic lymphocytic leukemia (CLL) using plasma-membrane-based proteomic analysis to identify previously uncharacterized and potentially B-cell-specific proteins. Proteins from plasma-membrane fractions were separated on one-dimensional gels and trypsinized fractions subjected to high-throughput MALDI-TOF mass spectrometry. Using this method, many known B-cell surface antigens were detected, but also known proteins not previously described in this disease or in this cellular compartment, including cell surface receptors, membrane-associated enzymes and secreted proteins, and completely unknown proteins. To validate the method, we show that BLK, a B-cell-specific kinase, is located in the CLL-plasma-membrane fraction. We also describe two novel proteins (MIG2B and B-cell novel protein #1, BCNP1), which are expressed preferentially in B cells. MIG2B is in a highly conserved and defined gene family containing two plasma-membrane-binding ezrin/radixin/moesin domains and a pleckstrin homology domain; the Caenorhabditis elegans homolog (UNC-112) is a membrane-associated protein that colocalizes with integrin at cell-matrix adhesion complexes. BCNP1 is a completely unknown protein with three predicted transmembrane domains, with three alternatively spliced final exons. Proteomic analysis may thus define new potential therapeutic targets.

hAG-2 and hAG-3, human homologues of genes involved in differentiation, are associated with oestrogen receptor-positive breast tumours and interact with metastasis gene C4.4a and dystroglycan.

hAG-2 and hAG-3 are recently discovered human homologues of the secreted Xenopus laevis proteins XAG-1/2 (AGR-1/2) that are expressed in the cement gland, an ectodermal organ in the head associated with anteroposterior fate determination during early development. Although the roles of hAG-2 and hAG-3 in mammalian cells are unknown, both proteins share a high degree of protein sequence homology and lie adjacent to one another on chromosome 7p21. hAG-2 mRNA expression has previously been demonstrated in oestrogen receptor (ER)-positive cell lines. In this study, we have used real-time quantitative RT - PCR analysis and immunohistochemistry on tissue microarrays to demonstrate concordant expression of hAG-2 and hAG-3 mRNA and protein in breast tumour tissues. Tumour expression of both genes correlated with OR (hAG2, P=0.0002; hAG-3, P=0.0012), and inversely correlated with epidermal growth factor receptor (EGFR) (P=0.003). Yeast two-hybrid cloning identified metastasis-associated GPI-anchored C4.4a protein and extracellular alpha-dystroglycan (DAG-1) as binding partners for both hAG-2 and hAG-3, which if replicated in clinical oncology would demonstrate a potential role in tumour metastasis through the regulation of receptor adhesion and functioning. hAG-2 and hAG-3 may therefore serve as useful molecular markers and/or potential therapeutic targets for hormone-responsive breast tumours.

Mechanisms of cholinergic dysfunction in rabbits following recurrent aspiration of cow's milk.

Recurrent aspiration of cow's milk has been shown to alter neural control of airways in young rabbits (Gelfand et al., 1997). The purpose of this study was to define the mechanisms responsible for in vitro cholinergic hyperresponsiveness in this model. Beginning at 1 week of age, rabbits received either 0.5 mL/kg whole cow's milk or sterile saline intranasally while under light anesthesia. This was repeated each weekday for 2 weeks. At 8 weeks of age, rabbits were sacrificed. Portions of lungs underwent lavage with sterile saline. Tracheal smooth muscle (TSM) segments were also removed. Segments were assessed for acetylcholine (ACh) release by high-performance liquid chromatography ( HPLC) with electrochemical detection or acetylcholinesterase (AChE) kinetic activity by spectrophotometry. Substance P (SP), a neuropeptide that can increase ACh release from nerves, was also assessed using an enzyme immunoassay to define the content in lavage and TSM segments. Immunohistochemistry for SP within airways was also assessed. We found that recurrent aspiration of milk led to statistically significant alterations in many parameters. Acetylcholine release was significantly greater in segments of airways from rabbits that had aspirated cow's milk (27.5 +/- 1.7 vs. 20.1 +/- 1.6 pmol/min/g tissue) than saline. At the same time, AChE activity was less in the group that aspirated milk (8.7 +/- 0.4 vs. 10.2 +/- 0.5 nmol/min/mg protein) compared to saline. The amount of SP within both lavage as well as tissue homogenates was greater in the group that had aspirated the foreign protein (159.1 +/- 28.9 vs. 41.9 +/- 5.2 pmol/mg protein in lavage; 158.7 +/- 31.9 vs. 80.5 +/- 7.8 pmol/mg protein in tissues) than saline controls. While total cholinergic nerve density as assessed by choline acetyltransferase was not significantly different between groups, SP-positive immunoreactive nerves were easily identified in the group that aspirated cow's milk. This study suggests that cholinergic hyperresponsiveness caused by repeated aspiration of milk is due to several abnormalities, including prejunctional (increase in ACh release) as well as junctional (decrease in AChE) mechanisms within the airways. In addition, an upregulation of SP within airways is part of this process.

Mice that overexpress Cu/Zn superoxide dismutase are resistant to allergen-induced changes in airway control.

Within the respiratory epithelium of asthmatic patients, copper/zinc-containing superoxide dismutase (Cu/Zn SOD) is decreased. To address the hypothesis that lung Cu/Zn SOD protects against allergen-induced injury, wild-type and transgenic mice that overexpress human Cu/Zn SOD were either passively sensitized to ovalbumin (OVA) or actively sensitized by repeated airway exposure to OVA. Controls included nonsensitized wild-type and transgenic mice given intravenous saline or airway exposure to saline. After aerosol challenge to saline or OVA, segments of tracheal smooth muscle were obtained for in vitro analysis of neural control. In response to electrical field stimulation, wild-type sensitized mice challenged with OVA had significant increases in cholinergic reactivity. Conversely, sensitized transgenic mice challenged with OVA were resistant to changes in neural control. Stimulation of tracheal smooth muscle to elicit acetylcholine release showed that passively sensitized wild-type but not transgenic mice released more acetylcholine after OVA challenge. Function of the M(2) muscarinic autoreceptor was preserved in transgenic mice. These results demonstrate that murine airways with elevated Cu/Zn SOD were resistant to allergen-induced changes in neural control.

IL-10 is necessary for the expression of airway hyperresponsiveness but not pulmonary inflammation after allergic sensitization.

Cytokines play an important role in modulating inflammatory responses and, as a result, airway tone. IL-10 is a regulatory cytokine that has been suggested for treatment of asthma because of its immunosuppressive and anti-inflammatory properties. In contrast to these suggestions, we demonstrate in a model of allergic sensitization that mice deficient in IL-10 (IL-10-/-) develop a pulmonary inflammatory response but fail to exhibit airway hyperresponsiveness in both in vitro and in vivo assessments of lung function. Reconstitution of these deficient mice with the IL-10 gene fully restores development of airway hyperresponsiveness comparable to control mice. These results identify an important role of IL-10, downstream of the inflammatory cascade, in regulating the tone of the airways after allergic sensitization and challenge.

CD23 exhibits negative regulatory effects on allergic sensitization and airway hyperresponsiveness.

The effects of an anti-CD23 monoclonal antibody (B3B4) in CD23-deficient and CD23-overexpressing mice were compared in a murine model of allergic sensitization. After sensitization and challenge with OA, mice developed increased serum levels of OA-specific IgE and IgG(1) with airway eosinophilia and AHR when compared with nonsensitized animals. Anti-CD23 treatment was studied under two protocols: 10-d OA aerosol exposure and intraperitoneal sensitization followed by aerosol challenge. In both protocols anti-CD23 significantly reduced IgE and IgG(1) levels, abolished eosinophilia, and normalized AHR in BALB/c and wild-type CD23+/+ mice but not in CD23-/- mice. These changes were associated with increases in IFN-gamma and decreases in IL-4 production, suggesting that CD23 binding may affect not only IgE production but also the Th1/Th2 imbalance during the development of allergic AHR. Absence of CD23 in gene-deficient mice significantly enhanced OA-specific IgE and IgG(1) levels, airway eosinophilia, and AHR when compared with CD23+/+ wild-type littermates after sensitization and airway challenge. Sensitized and challenged CD23 transgenic mice also developed eosinophilic airway inflammation and methacholine hyperresponsiveness. However, the extent of AHR, BAL, and tissue eosinophilia in these animals showed a significant negative correlation with levels of CD23 expression on splenic T and B cells, demonstrating a limiting role of CD23 in the development of allergic AHR.

Anti-CD86 (B7.2) treatment abolishes allergic airway hyperresponsiveness in mice.

Allergic sensitization in asthma develops as a consequence of complex interactions between T cells and antigen-presenting cells. We have developed several in vivo models to study allergen-specific T cell and B cell function and their relevance to allergic airway hyperresponsiveness (AHR), focusing on the role of the costimulatory molecules CD80 and CD86. Treatment of mice with anti-CD86, but not anti-CD80, significantly inhibited increased serum levels of ovalbumin (OA)-specific IgE and IgG1, airway eosinophilia, and AHR both after 10 d of OA aerosol exposure (in the absence of adjuvant) and after intraperitoneal sensitization followed by repeated airway challenges. Inhibition of AHR was associated with decreased IL-4 and IL-5 levels in the BAL fluid of sensitized mice, suggesting impaired Th2 function in anti-CD86-treated animals. This effect was not seen when mice received treatment only before allergen challenge, indicating that anti-CD86 acts through inhibition of allergic sensitization and not simply by inhibiting the influx of inflammatory cells. These data suggest that the CD86 costimulatory ligand plays a major role in the development of allergic inflammation and AHR in allergen-challenged mice. Further, this study demonstrates that T-B cell interactions during allergic sensitization are amenable to therapeutic manipulation and that selective blockade of accessory signals can be an effective means for modulating distinct T cell functions.

Human respiratory syncytial virus produces prolonged alterations of neural control in airways of developing ferrets.

A dysfunction of pathways that normally cause contraction or relaxation of airways has been proposed to explain heightened levels of responsiveness produced by various insults to the airway. For example, we previously reported (4) that infection of cotton rats with the human respiratory syncytial virus (HRSV) leads to a significant decrease in an airway's nonadrenergic noncholinergic inhibitory (NANCi) response shortly after the infection. In the present study we addressed the more chronic effects of HRSV infection on airway function in young ferrets during a period of rapid somatic growth. Animals 1 wk old received HRSV or uninfected cell culture medium intranasally. In vitro studies of airway function were performed on tracheal smooth muscle (TSM) segments at 4, 8, and 24 wk of age. To evaluate neurally mediated contractile responses, frequency-response curves to electrical field stimulation (EFS) were performed with results expressed in terms of the frequency causing 50% of the maximal contractile response (ES50). In addition, contractile responses of TSM to methacholine (MCh) were also assessed with results expressed as the concentration needed to produce 50% of the maximal contractile response (EC50). To gauge NANCi responses, TSM was contracted with neurokinin A in the presence of atropine, propranolol, and indomethacin. Relaxant responses to EFS were assessed at frequencies from 5 to 30 Hz, with results expressed as mean percent relaxation. We found increased contractile responses to EFS in infected animals compared with that in the control group in both 4- and 8-wk old animals (p = 0.001 and p = 0.008, respectively). This difference had resolved by 24 wk of age. There was no difference in TSM responses to MCh between the groups at any age. Although there were no NANCi responses in 4-wk-old ferrets from either group, NANCi responses were significantly decreased in 8-wk-old ferrets previously infected with HRSV in the first week of life (p = 0.0001). A significant difference persisted (p = 0.008), albeit to a lesser degree, at 24 wk of age. These findings demonstrate that HRSV produces prolonged alterations of TSM function in ferret airways in vitro.