B-cell function in canine X-linked severe combined immunodeficiency.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gammac) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors and has a similar clinical phenotype to human XSCID. We have previously shown that the block in T-cell development is more profound in XSCID dogs than in genetically engineered gamma c-deficient mice. In this study we evaluated the B-cell function in XSCID dogs. In contrast to the marked decrease in peripheral B-cells in gamma c-deficient mice, XSCID dogs have increased proportions and numbers of peripheral B-cells as observed in XSCID boys. Canine XSCID B-cells do not proliferate following stimulation with the T-cell-dependent B-cell mitogen, pokeweed mitogen (PWM); however, they proliferate normally in response to the T-cell-independent B-cell mitogen, formalin-fixed, heat-killed Staphylococcus aureus. Canine XSCID B-cells are capable of producing IgM but are incapable of normal class-switching to IgG antibody production as demonstrated by in vitro stimulation with PWM and immunization with the T-cell-dependent antigen, bacteriophage PhiX174. Similar results have been reported for XSCID boys. Thus, it appears that gamma c-dependent cytokines have differing roles in human and canine B-cell development than in the mouse making the XSCID dog a valuable model for studying the role of these cytokines in B-cell development and function.

Canine X-linked severe combined immunodeficiency.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gamma c) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors. The most striking clinical feature is a failure to thrive or 'stunted' growth. Recurrent or chronic infections begin at the time of decline of maternal antibody, usually between six and eight weeks of age. Affected dogs rarely survive past three to four months of age. The major pathologic feature of canine XSCID is a small, dysplastic thymus. Grossly identifiable lymph nodes, tonsils, and Peyer's patches are absent in XSCID dogs. During the neonatal period, XSCID dogs have few, if any, peripheral T cells and increased number of peripheral B cells. Some XSCID dogs do develop phenotypically mature, nonfunctional T cells with age, however, the absolute number of peripheral T cells remain significantly decreased compared to age-matched normal dogs. An interesting finding is that as soon as T cells begin to appear in XSCID dogs they rapidly switch from a CD45RA+ (naive) phenotype to a CD45RA- (activated or memory phenotype). One of the characteristic findings in XSCID dogs is an absent or markedly depressed blastogenic response of T cells in response to stimulation through the T cell receptor and when the necessary second messengers for cellular proliferation are directly provided that by-pass signals delivered through ligand-receptor interaction. The proliferative defect is due to the inability of T cells to express a functional IL-2 receptor. Canine XSCID B cells do not proliferate following stimulation with T cell-dependent B cell mitogens, however, they proliferate normally in response to T cell-independent B cell mitogens. Canine XSCID B cells are capable of producing IgM but are incapable of class-switching to IgG antibody production following immunization with the T cell-dependent neoantigen, bacteriophage phiX174. The number of thymocytes in the XSCID thymus is approximately 0.3% of the thymocytes present in the thymus of age-matched normal dogs. The proportion of CD4-CD8- thymocytes in XSCID dogs is increased 3.5-fold and the CD4+CD8+ population is decreased 2.3-fold. These findings demonstrate that (1) a functional gamma c is required for normal B and T cell function, (2) early T cell development is highly dependent upon a functional gamma c, and (3) B cell development can occur through a gamma c-independent pathway.

Bone marrow transplantation for canine X-linked severe combined immunodeficiency.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma chain which is a subunit of the receptors of IL-2, IL-4, IL-7, IL-9 and IL-15. Bone marrow transplantation (BMT) of human XSCID patients without pretransplant conditioning (cytoablation) results in engraftment of donor T-cells and reconstitution of T-cell function but engraftment of few, if any, donor B cells with resultant poor reconstitution of humoral immune function. In this study, we show that XSCID dogs can be transplanted with allogeneic bone marrow cells resulting in engraftment of both donor B and T cells and reconstitution of full systemic immune function including normal humoral immune function without the need for cytoablation.

Canine X-linked severe combined immunodeficiency. A model for investigating the requirement for the common gamma chain (gamma c) in human lymphocyte development and function.

Our laboratory has identified and characterized an X-linked severe combined immunodeficiency (XSCID) in dogs that is due to mutations in the common gamma (gamma c) subunit of the interleukin-2 (IL2), IL4, IL7, IL9, and IL15 receptors. Canine XSCID, unlike genetically engineered gamma c-deficient mice, has a clinical and immunologic phenotype virtually identical to human XSCID. It appears that species-specific differences exist in the role of the gamma c and its associated cytokines in mice compared to their role in humans and dogs, suggesting gamma c-deficient dogs may be a more relevant model for studying the role of the gamma c in humans. We are utilizing this model for a variety of studies to address: 1. Fundamental questions concerning the role of the gamma c in cytokine regulation and lymphocyte development. 2. The pathogenesis of XSCID. 3. Strategies for improving bone marrow transplantation outcome. 4. Development and evaluation of strategies for gene therapy. 5. Human hematopoietic stem cell development.

Full immunologic reconstitution following nonconditioned bone marrow transplantation for canine X-linked severe combined immunodeficiency.

Bone marrow transplantation in human X-linked severe combined immunodeficiency (XSCID) without pretransplant conditioning results in engraftment of donor T cells and reconstitution of T-cell function but engraftment of few, if any, donor B cells and poor reconstitution of humoral immune function. Since bone marrow transplantation remains the most effective treatment of XSCID patients, better strategies are necessary to achieve optimum long-term results. Canine XSCID, like human XSCID, is due to mutations in the common gamma chain (gamma c) gene and has clinical and immunologic features identical to those of human XSCID, making it a true homolog of the human disease. We have successfully performed bone marrow transplantation in three XSCID dogs without pretransplant conditioning, using untreated bone marrow cells from mixed lymphocyte culture-nonreactive normal littermates. Unlike the experience in human XSCID patients, all three dogs engrafted both donor B and T cells and attained full reconstitution of immunologic function. Normal percentages of T cells and T-cell mitogenic responses were attained by 3 months posttransplant. CD3+ T cells after transplantation expressed the CD45RA isoform indicating that the cells were recent thymic emigrants derived from immature progenitors. Serum IgG levels were within normal range by 5 months posttransplant. Immunization with the T-dependent antigen, bacteriophage phiX174, demonstrated normal antibody titers, immunologic memory, and class-switching. Polymerase chain reaction (PCR) analysis of the gamma c locus showed that 100% of circulating T cells and 30% to 50% of circulating B cells were donor-derived. None of the dogs developed clinically evident graft-versus-host disease (GVHD). Thus, canine XSCID provides a model to determine the optimal conditions for bone marrow transplantation in human patients, and to develop and test strategies for somatic gene therapy.

Postnatal development of T cells in dogs with X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency disease (XSCID) in both humans and dogs results from mutations in the common gamma-chain, gamma c, which is a common component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. Although human and canine XSCID share similar features, such as a failure to thrive, hypogammaglobulinemia, an absent T cell mitogenic response, and thymic dysplasia, near normal percentages of T cells are observed in some affected dogs, whereas XSCID boys have few, if any, circulating T cells. In this study, PBL were analyzed by flow cytometry beginning shortly after birth until 9 wk of age. XSCID dogs < 3 wk of age had an elevated number of B cells and were nearly devoid of T cells, phenotypically resembling most human XSCID patients. At 5 wk of age, however, T cells appeared in approximately one-half of the XSCID dogs, although the absolute number of T cells was one-third of normal in these dogs. While the percentage of CD45RA+ T cells in normal dogs gradually decreased with age from > 90% in neonates to < 40% by 3 to 5 yr of age, in XSCID dogs a rapid decline in the percentage of CD45RA+ T cells was observed, resulting in < 10% CD45RA+ T cells by 7 to 9 wk of age. Maternal engraftment was not detected in any of the XSCID dogs by using a sensitive PCR assay. The appearance of nonmaternally derived T cells in XSCID dogs that undergo a rapid switch from CD45RA+ to CD45RA- suggests that limited thymic emigration and peripheral expansion of T cells can occur in the absence of a functional gamma c.

The use of fibreoptic bronchoscopy with sterile catheter in the diagnosis of pneumonia.

The use of fibreoptic bronchoscopy with sterile catheter sampling of pulmonary secretions was evaluated in 70 patients with a provisional diagnosis of pneumonia. In 37 patients quantitative analysis of the sterile catheter isolates was performed (colony forming units (CFU) per ml). Potential bacterial pathogens were isolated in 37 patients and in the quantitative analysis, 14 of 22 isolates were grown in counts greater than or equal to 10(3) CFU/ml. Sterile catheter increased the bacterial isolation rate as in only 19 patients blood (2) or sputum (18) cultures yielded the same organisms. Sputum cultures showed a 25% false-positive rate in patients with no growth from sterile catheter. Quantitative analysis did not yield any further information in patients receiving antibiotics. Atypical or fungal pneumonia was diagnosed in 22 patients, while ten patients had other pathology simulating pneumonia. Sterile catheter sampling of pulmonary secretions at fibreoptic bronchoscopy proved to be a valuable tool in the diagnosis of bacterial pneumonia.

Tracheobronchopathia osteochondroplastica.

The clinical, pathological and physiological features of two patients suffering from tracheobronchopathia osteochondroplastica (TO) are described. Unequivocal evidence of extrapulmonary airways obstruction was not able to be obtained by lung function testing, despite extensive central airway involvement in both patients. TO is a rare condition of which there is only one other clinical report from this country. As the bronchoscopic appearance may closely resemble that of endobronchial neoplasms, TO should be remembered in the differential diagnosis of patients with haemoptysis.

Assessment of combined oral theophylline and inhaled beta-adrenoceptor agonist bronchodilator therapy.

1. The bronchodilator effects of 500 microgram rimiterol by pressurized aerosol, 375 mg oral theophylline and both drugs in combination were compared in a randomized, placebo-controlled, double-blind trial in eight patients with chronic, partially reversible airways obstruction. 2. The four treatments were (i) oral theophylline, placebo aerosol (TP); (ii) oral placebo, rimiterol aerosol (PR); (iii) oral theophylline, rimiterol aerosol (TR) and; (iv) oral placebo, placebo aerosol (PP). The aerosol was administered 2 h after the oral treatment. 3. Significant bronchodilatation (% FEV1 change from control) compared to PP occurred with TP from 60 to 480 min and with TR from 60 to 300 min, whereas with PR only for 45 min (P less than 0.05). 4. The mean, peak % FEV1 increases from control were 51.8% at 125 min, 31.7% at 125 min, 26.1% at 210 min and 0.9% at 30 min for TR, PR, TP and PP respectively. 5. At 125 min (5 min after aerosol inhalation) the mean % FEV1 change from control with TR (51.8%) Was significantly greater than with PR (31.7%), TP (22.2%) (P less than 0.05) and PP (-2.4%) (P less than 0.01). 6. The mean, peak plasma theophylline levels were 10.19 microgram/ml at 120 min and 9.98 microgram/ml at 180 min with TR and TP respectively. Theophylline half-life ranged between 4.3 and 12.5 h (mean +/- s.e. mean, 8.0 +/- 0.8 h). 7. Additive bronchodilatation was produced when rimiterol was administered with theophylline at a time when therapeutic plasma theophylline levels were achieved.

Comparison of terbutaline and salbutamol aerosols.

The bronchodilator effects of terbutaline, 500 microng, and salbutamol, 200 microng, by pressurised aerosol were compared in 16 patients (eight asthma, eight chronic bronchitis with asthma). Salbutamol produced greater bronchodilatation than terbutaline for the initial hour after inhalation. Thereafter, there was no difference in action and an effective bronchodilator response was maintained by each drug for at least four hours. beta2-adrenoceptor stimulating drugs by inhalation are widely used in the treatment of asthma. The purpose of this study was to compare the bronchodilator efficacies of two of these drugs, terbutaline and salbutamol by pressurised aerosol, in patients with partially reversible airways obstruction.

Terminal exacerbation of chronic melioidosis in New South Wales.

A fatal case of melioidosis, thought to be the third recorded from New South Wales, is presented. Infection probably occurred in Queensland. The patients presented with a subcutaneous abscess complicated by pyaemia, extensive lung involvement and septicaemic shock. The diagnosis was bacteriologically confirmed shortly before death, by isolation of Pseudomonas pseudomallei from blood, pus swabs and tracheal aspirates. There is a need for greater awareness of this disease in persons who have resided in South-East Asia and in North-Eastern Australia.

Doxycycline in serum and bronchial secretions.

The concentration of doxycycline hydrochloride was measured in serum and bronchial secretions in five patients with chronic bronchitis receiving doxycycline orally in normal therapeutic dosage for seven days (200 mg day 1, 100 mg days 2 to 7). After the loading dose of 200 mg, serum concentrations ranged between 5-40 and 3-45 mug/ml (mean 4-33 mug/ml) at 3 hours, declining to between 2-28 and 1-21 mug/ml (mean 1-71 mug/ml) at 23 hours. The mean serum levels for days 2 to 7 were 2-15, 1-79, and 1-38 at 3, 8, and 23 hours respectively. There was considerable individual variability and a wide range of concentrations of doxycycline in the sputum (0-07 to 2-10 mug/ml, mean 0-34 mug/ml). During the course of treatment there was a progressive increase in sputum levels and sputum/serum concentration ratios. There was no correlation between sputum concentration and degree of purulence. The clinical efficacy of doxycycline does not appear to be related to sputum concentration, although the progressive increase in sputum doxycycline levels may be relevant in preventing recurrence of acute infection when the drug is administered as long-term prophylactic therapy.

Comparison of oral theophylline and salbutamol by inhalation in asthmatic patients.

The bronchodilator effects of 375 mg theophylline orally and 200 microg salbutamol by pressurized aerosol were compared in a randomized, cross-over, double-blind trial in fourteen asthmatic patients. The mean peak percentage FEV1 increase from control was 43.7% at 60 min after salbutamol and 30.3% at 180 min after theophylline. Salbutamol produced significantly greater bronchodilatation than theophylline for the initial 30 min (P < 0.01). Theophylline demonstrated a longer duration of action than salbutamol, with a significantly greater FEV1 response at 360 min (P < 0.02). There was no significant difference between the total effect of each drug for the 360 min period, as calculated by the areas under the respective FEV1 response curves.