An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss,<Emphasis Type="Italic"> Physcomitrella patens</Emphasis>

The moss Physcomitrella patens is the only land plant known to date with highly efficient homologous recombination in its nuclear DNA, making it a unique model for plant functional genomics approaches. For high-throughput production of knockout plants, a robust transformation system based on polyethylene glycol-mediated transfection of protoplasts was developed and optimised. Both the DNA conformation and pre-culture of plants used for protoplast isolation significantly affected transformation efficiencies. Employing a newly developed PCR high-throughput method, the gene-targeting efficiency in more than 1,000 plants transformed with different cDNA-based knockout constructs was determined and analysed with regard to the length and intron/exon structure of the homologous gene locus. Different targeting constructs, each containing an identical selectable marker gene, were applied as batch DNA in a single transformation experiment and resulted in double-knockout plants. Thus, the fast and efficient generation of multiple targeted gene-knockouts is now feasible in Physcomitrella.Communicated by U. Kück     

Brain oscillatory responses to an auditory-verbal working memory task in mild cognitive impairment and Alzheimer's disease.

We report preliminary findings on EEG oscillatory correlates of working memory in mild cognitive impairment (MCI) and Alzheimer's disease (AD). Event-related desynchronization (ERD) and synchronization (ERS) of the 1-20 Hz EEG frequencies were studied using wavelet transforms in elderly controls, MCI patients and mild probable AD patients performing an auditory-verbal Sternberg memory task. Behaviourally, the AD patients made more errors than the controls and the MCI group. Statistically significant differences during the encoding of the memory set were found between the controls and the MCI group, such that the latter group showed ERD in the approximately 10-20 Hz frequencies. The findings may reflect different, compensatory encoding strategies in MCI. During retrieval, the most obvious differences were observed between the controls and the AD group: the ERD in the approximately 7-17 Hz frequencies was absent in the AD group particularly in anterior and left temporal electrode locations. This finding might indicate that AD is associated with deficient lexical-semantic processing during the retrieval phase in working memory tasks. Future studies with larger patient groups are needed to establish the diagnostic value of ERD/ERS patterns in MCI and AD.     

Comparison of real-time PCR and culture for detection of Porphyromonas gingivalis in subgingival plaque samples

Porphyromonas gingivalis is a major pathogen in destructive periodontal disease in humans. Detection and quantification of this microorganism are relevant for diagnosis and treatment planning. The prevalence and quantity of P. gingivalis in subgingival plaque samples of periodontitis patients were determined by anaerobic culture and real-time PCR amplification of the 16S small-subunit rRNA gene. The PCR was performed with primers and a fluorescently labeled probe specific for the P. gingivalis 16S rRNA gene. By the real-time PCR assay, as few as 1 CFU of P. gingivalis could be detected. Subgingival plaque samples from 259 adult patients with severe periodontitis were analyzed. P. gingivalis was detected in 111 (43%) of the 259 subgingival plaque samples by culture and in 138 (53%) samples by PCR. The sensitivity, specificity, and positive and negative predictive values of the real-time PCR were 100, 94, 94, and 100%, respectively. We conclude that real-time PCR confirms the results of quantitative culture of P. gingivalis and offers significant advantages with respect to the rapidity and sensitivity of detection of P. gingivalis in subgingival plaque samples.     

Regulatory role of interleukin-10 in experimental group B streptococcal arthritis

Intravenous inoculation of CD-1 mice with 10(7) CFU of type IV group B Streptococcus (GBS) results in a high incidence of diffuse septic arthritis, associated with high levels of systemic and local production of interleukin-1beta (IL-1beta) and IL-6. In this study, the role of the anti-inflammatory cytokine IL-10 in the evolution of GBS systemic infection and arthritis was evaluated. IL-10 production was evident in sera and joints of GBS-infected mice. Neutralization of endogenous IL-10 by administration of anti-IL-10 antibodies (1 mg/mouse) at the time of infection resulted in worsening of articular lesions and 60% mortality associated with early sustained production of IL-6, IL-1beta, and tumor necrosis factor alpha (TNF-alpha). The effect of IL-10 supplementation was assessed by administering IL-10 (100, 200, or 400 ng/mouse) once a day for 5 days, starting 1 h after infection. Treatment with IL-10 had a beneficial effect on GBS arthritis, and there was a clear-cut dose dependence. The decrease in pathology was associated with a significant reduction in IL-6, IL-1beta, and TNF-alpha production. Histological findings showed limited periarticular inflammation and a few-cell influx in the articular cavity of IL-10-treated mice, confirming clinical observations. In conclusion, this study provides further information concerning the role of IL-10 in regulating the immune response and inflammation and calls attention to the potential therapeutic use of IL-10 in GBS arthritis.     

Rush Hymenoptera venom immunotherapy: a safe and practical protocol for high-risk patients

BACKGROUND: Hymenoptera venom immunotherapy in allergic patients is a well-established treatment modality for the prevention of systemic anaphylactic reactions caused by insect stings. A variety of therapy regimens exists, from conventional to rush and ultrarush modalities that operate on continuous or intermittent schedules. OBJECTIVE: The aim of this study was to report the 8-year experience with our rush venom immunotherapy regimen in predominantly high-risk patients and to compare data on safety and convenience with the results of 26 studies published from 1978 to 2001. METHODS: One hundred one patients allergic to bee, yellow jacket, or hornet venom were treated with rush Hymenoptera venom immunotherapy. Diagnosis and selection of patients for venom immunotherapy were carried out according to the recommendations of the European Academy of Allergology and Clinical Immunology. We used a 4-day regimen, and the incidence and nature of systemic reactions (SRs) were documented. Fifty-two patients were treated with honeybee venom, and 49 were treated with yellow jacket venom. RESULTS: One hundred (99%) patients reached the maintenance dose. We observed 8 injection-related SRs (0.47% of all injections given) in 7 (6.9%) patients. The number of SRs was higher in patients treated with bee venom extract (12%) compared with in patients receiving yellow jacket venom extract (2%). There was no significant difference in the risk of SRs between female and male patients. The incidence of SRs was considerably lower than the average of 17.8% reported in the literature. CONCLUSION: With a rush immunotherapy regimen over a time period of 8 years in predominantly high-risk patients, the incidence of SRs was low, despite the high number of patients with bee venom allergy, who are more likely to have side effects. Epinephrine as rescue medication was never necessary, and the regimen proved to be safe and convenient for both the patients and the medical staff.     

Site-independent prognostic value of chromosome 9q loss in primary gastrointestinal stromal tumours

Although the significance of tumour site for estimating malignant potential in gastrointestinal stromal tumours (GISTs) has recently been recognized, site-specific genetic patterns have not to date been defined. This study examined 52 c-kit-positive primary GISTs (with a mean follow-up of 42.3 months in 51 cases) from three different locations (35 gastric, 12 small intestinal, and five colorectal) using comparative genomic hybridization (CGH). In general, tumour site correlated with key prognostic factors, including tumour size, mitotic rate, proliferative activity, and probable malignant potential. Furthermore, several DNA copy number changes showed a site-dependent pattern. These included losses at 14q (gastric 83%, intestinal 35%; p = 0.001), losses at 22q (gastric 46%, intestinal 82%; p = 0.02), losses at 1p (gastric 23%, intestinal 88%; p = 1 x 10(-5)), losses at 15q (gastric 14%, intestinal 59%; p = 0.002), losses at 9q (gastric 14%, intestinal 53%; p = 0.006), and gains at 5p (gastric 11%, intestinal 53%; p = 0.002). These data demonstrate strong site-dependent genetic heterogeneity in GISTs that may form a basis for subclassification. Prognostic evaluation of DNA copy number changes identified losses at 9q as a site-independent prognostic marker associated with shorter disease-free survival (p = 0.03) and overall survival (p = 0.002). Furthermore, 9q loss also appeared to carry prognostic value in predicting overall survival for patients with advanced or progressive GISTs (p = 0.003).     

Risk factors for pneumonia in urban-dwelling HIV-infected women: a case-control study in Nairobi,Kenya

In sub-Saharan Africa, respiratory tract infections (RTI) are the leading cause of serious morbidity and mortality in HIV-infected persons. This study sought to investigate demographic, socioeconomic, and environmental risk factors for pneumonia in a cohort of HIV-infected women. The authors performed a nested case-control study in a cohort of HIV-1-infected adults followed in Nairobi, Kenya. Thirty-nine women who developed pneumonia during the follow-up period were selected as cases, and 66 women who did not develop pneumonia were randomly chosen to serve as control subjects. A questionnaire was administered in subjects' homes that assessed demographics, home environment, and socioeconomic status. Women were followed in the cohort for a median of 36.8 months (range, 27.3-39.3). Adjusting for length of follow-up period, factors associated with lower socioeconomic status (lower monthly spending [OR = 3.2; 95% CI, 1.2-8.4 per 10,000 Kenyan shilling decrease], having no savings [OR = 4.1; 95% CI, 1.4-11.9], less sturdy home construction material such as mud or cement walls [OR = 2.6; 95% CI, 1.1-5.9] or dirt floors [OR = 2.8; 95% CI, 1.0-7.6], and lack of a window in the home [OR = 5.5; 95% CI, 0.9-32.2]) and being widowed (OR = 4.3; 95% CI, 1.2-15.1) or single (OR = 3.3; 95% CI, 1.0-11.2) were associated with an increased risk of pneumonia. In multivariate analysis, widowed (AOR = 5.9; 95% CI, 1.3-26.3), single (AOR = 7.7; 95% CI, 1.6-36.4), and divorced (AOR = 4.5; 95% CI, 1.0-20.1) women, those without savings (AOR = 3.7; 95% CI, 1.2-11.7), and those living in more crowded and contagious conditions (AOR = 1.5; 95% CI, 1.1-2.1) remained at increased risk of pneumonia. If confirmed by prospective investigation, these findings could help identify persons and subpopulations of HIV-infected women with the greatest risk of pneumonia.     

Strain variation among Bordetella pertussis isolates from Québec and Alberta provinces of Canada from 1985 to 1994

Pulsed-field gel electrophoresis and gene typing were able to differentiate among 3,597 Bordetella pertussis isolates circulating in Alberta and Québec Provinces, Canada, from 1985 to 1994 and distinguish them from the strains used in vaccine production. This study provides a baseline for continued surveillance of prevalent and emerging strains of B. pertussis in Canada.