Development of a transformation system for Crinipellis perniciosa,the causal agent of witches' broom in cocoa plants

Protoplasts of the pathogenic plant fungus, Crinipellis perniciosa, were transformed to hygromycin B resistance using the pAN7-1 plasmid, which contains the Escherichia coli hph gene under the control of Aspergillus nidulans regulatory sequences. The pAN7-1 plasmid was introduced by PEG/CaCl(2) treatment. Transformation frequencies of 1.6-2.5 transformants/microg of DNA were achieved. About 54% of the transformants were abortive and 40 analyzed transformants were mitotically stable and showed different hygromycin B resistance levels. The presence of the hph gene was checked by PCR in five transformants and the integration of multiple plasmid copies into different genome sites was observed by Southern analysis. This is the first report of a C. perniciosa transformation system and represents an important step for further research into genetic manipulation of this fungal plant pathogen.     

Sputum induction as a research tool for the study of human respiratory mucus

The study objectives were to compare in vitro transportability and physical properties of respiratory mucus, obtained invasively by direct collection (DC) right after endotracheal intubation and non-invasively by sputum induction with 3% hypertonic saline solution inhalation (SI) 24 h before the anesthesia. Twenty-two patients with no pulmonary disease scheduled for elective abdominal surgical procedures were studied. The parameters analyzed and the main results are as follows. (1) Transportability by cilia (MCT), SI was higher than DC (0.94+/-0.25 and 0.62+/-0.25; P<0.001). There was a significant correlation between the two methods and DC could be estimated by: DC=0.21+(0.44 SI) (r=0.44; P<0.001). (2) Transportability by cough (CC), SI was higher than DC (68.23+/-32.1 and 33.58+/-19.04 mm; P=0.002). (3) Contact angle (CA), SI was lower than DC (10+/-3 degrees and 22+/-14 degrees ; P=0.025). (4) Rheological properties (no significant difference obtained between SI and DC). These results indicated that SI changes mucus physical properties and transportability in non-expectorators.     

Development of CD4+ T cell lines that suppress an antigen-specific immune response in vivo

It has been suggested for many years that the regulation of the immune system for the maintenance of peripheral tolerance may involve regulatory/suppressor T cells. In the past few years, several investigators have demonstrated that these cells can be generated in vitro. It has also been shown that they can inhibit the progression of various autoimmune disease models when infused into susceptible mice. We have generated two murine T cell lines in the presence of KLH-specific T cell clones from BALB/c or DBA2 mice. The lines are characterized by a low proliferative response to mitogens, the capacity to secrete high amounts of IL-10 and TGF-beta, and small amounts of IFN-gamma. Interestingly, these cells are unable to produce IL-2, IL-4 or IL-5. The study of the surface phenotype of both lines revealed CD4+, CD25high, CD44low and CTLA-4- cells. When injected intravenously in (CBy.D2) F1 mice, these cells were able to inhibit 50-100% of the TNP-specific antibody production, when the hapten was coupled to KLH. In the present study we offer another evidence for the existence of regulatory T cells in the T lymphocyte repertoire, suggesting that they can also regulate immune responses to foreign antigens. Furthermore, we demonstrate an alternative pathway to generate these cells different from approaches used thus far.     

Clinical evaluation of eosinophils in the sputum.

The sputum differential eosinophil/neutrophil count was done in 384 patients using Leishman staining. The patients were distributed in four groups: bronchial asthma (197 patients); chronic bronchitis with wheezing (45 patients); chronic bronchitis and/or emphysema without wheezing (73 patients); other pulmonary diseases (64 patients). Eosinophils were present in patients from all groups but more frequently (P less than 0.001) in asthma: 142 (72%) of 197 patients. In bronchial asthma and chronic bronchitis with wheezing the percentages of eosinophils were more frequently (P less than 0.001) above 80%: 57% and 58% of the patients respectively. The other two groups had more cases with 19% or less eosinophils. There is no percentage level specific for asthma but levels above 80% of eosinophils are strongly suggestive of asthma or of chronic bronchitis with wheezing.     

Acquired immune deficiency in Haitians: opportunistic infections in previously healthy Haitian immigrants

We describe acquired immune deficiency manifested by opportunistic infections in 10 previously healthy heterosexual Haitian men. The opportunistic pathogens included Toxoplasma gondii (in four patients), Cryptococcus neoformans (in one), Pneumocystis carinii (in four patients), and Candida albicans (in three). Six of the patients also had Mycobacterium tuberculosis. Immunologic studies of three patients showed a decrease in the numbers and activity of helper T cells, with normal or increased populations of suppressor T cells. Serologic markers for previous infections from hepatitis A, cytomegalovirus, and herpes simplex virus were detected in several patients. Six of the patients died despite specific antimicrobial therapy. The clinical and immunologic findings in these 10 Haitians are similar to those reported in drug addicts and homosexuals with the acquired immune-deficiency syndrome.     

The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation

In a human in-vitro fertilization (IVF) programme, the effect of co- culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.      

Interleukin-12 mediates resistance to Trypanosoma cruzi in mice and is produced by murine macrophages in response to live trypomastigotes.

Host resistance to infection by Trypanosoma cruzi is dependent on both natural and acquired immune responses. During the first week of infection in mice, NK cell-derived gamma interferon (IFN-gamma) is involved in controlling intracellular parasite replication, mainly through the induction of NO biosynthesis by activated macrophages. Interleukin-12 (IL-12) has been shown to be a powerful cytokine in inducing IFN-gamma synthesis by NK cells, as well as in mediating resistance to different intracellular protozoa. We have therefore studied the ability of T. cruzi to elicit IL-12 synthesis by macrophages and the role of this cytokine in controlling parasite replication during acute infection in mice. Our results show that macrophages cultured in the presence of live trypomastigote forms (but not epimastigotes) release IL-12 that can induce IFN-gamma production by normal spleen cells. IL-12 was detected in as little as 12 h after the addition of the trypomastigotes, and the level of IL-12 peaked at 48 h after the initial macrophage-parasite incubation. The addition of anti-IL-12 monoclonal antibody to macrophage-trypomastigote supernatants dose-dependently inhibited IFN-gamma production by naive splenocytes. Finally, the in vivo role of IL-12 in resistance to infection by T. cruzi was analyzed. Mice treated with anti-IL-12 monoclonal antibody had significantly increased parasitemia and mortality in comparison with those of control infected mice treated with control antibody. Together, these results suggest that macrophage-derived IL-12 plays a major role in controlling the parasitemia in T. cruzi-infected mice and that the animal's resistance during the acute phase of infection may, at least in part, be a consequence of postinfection levels of IL-12.