Extensive multiple test centre evaluation of the VecTest malaria antigen panel assay

To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest Malaria) is a rapid, one-step procedure using a 'dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The 'VecTest Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for > 15 weeks in dry conditions up to 45 degrees C and in humid conditions up to 37 degrees C. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.     

Ultrasonographic assessment of bone maturity in newborns

BACKGROUND: Traditionally, the bone maturity at birth has been estimated from the radiological presence and size of the ossified distal femoral epiphysis. This study was conducted in a search for a sonographic tool for the evaluation of neonatal bone maturity. METHODS: We examined sonographically 256 neonates within 24 h of birth. Gestational ages ranged from 36 to 42 weeks (mean: 39.4; median: 40). Birth weights ranged from 1,945 to 5,000 g (mean: 3,175; median: 3,180). The distal femoral epiphysis was imaged on the coronal plane sonogram of the distal femur with the knee at 90 degrees flexion and the distal femoral epiphysis maximal height was recorded. The acetabulum was imaged using Graf's method in the coronal plane image and the acetabular diameter recorded. RESULTS: It was found that plotting the distal femoral epiphysis against neonatal birth weight and gestational age provided a simple method for assessing the bone maturity. According to our study, a neonate can be regarded as bone maturity percentile X when plotting distal femoral epiphysis height or acetabulum diameter against birth weight and gestational age or when averaging the four readings. CONCLUSIONS: We suggest performing sonography of the distal femoral epiphysis as a bedside tool for the assessment of skeletal maturity in newborns.     

Invasion of human epithelial cells by Campylobacter upsaliensis

Few data exist on the interaction of Campylobacter upsaliensis with host cells, and the potential for this emerging enteropathogen to invade epithelial cells has not been explored. We have characterized the ability of C. upsaliensis to invade both cultured epithelial cell lines and primary human small intestinal cells. Epithelial cell lines of intestinal origin appeared to be more susceptible to invasion than non-intestinal-derived cells. Of three bacterial isolates studied, a human clinical isolate, CU1887, entered cells most efficiently. Although there was a trend towards more efficient invasion of Caco-2 cells by C. upsaliensis CU1887 at lower initial inocula, actual numbers of intracellular organisms increased with increasing multiplicity of infection and with prolonged incubation period. Confocal microscopy revealed C. upsaliensis within primary human small intestinal cells. Both Caco-2 and primary cells in non-confluent areas of the infected monolayers were substantially more susceptible to infection than confluent cells. The specific cytoskeletal inhibitors cytochalasin B, cytochalasin D and vinblastine attenuated invasion of Caco-2 cells in a concentration-dependent manner, providing evidence for both microtubule- and microfilament-dependent uptake of C. upsaliensis. Electron microscopy revealed the presence of organisms within Caco-2 cell cytoplasmic vacuoles. C. upsaliensis is capable of invading epithelial cells and appears to interact with host cell cytoskeletal structures in order to gain entry to the intracellular environment. Entry into cultured primary intestinal cells ex vivo provides strong support for the role of host cell invasion during human enteric C. upsaliensis infection.     

COX-2: Where are we in 2003? - Be strong and resolute: continue to use COX-2 selective inhibitors at recommended dosages in appropriate patients

Cyclooxygenase (COX)-2 selective inhibitors have been shown to have comparable efficacy to nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) in the treatment of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Large outcome studies have shown that patients with OA and RA not taking low-dose aspirin have fewer symptomatic and complicated upper GI events when treated with COX-2 selective inhibitors than with nonselective NSAIDs. When used in recommended dosages, there is no convincing evidence that patients treated with COX-2 selective inhibitors have an increased incidence of cardiovascular thrombotic events, including non-fatal myocardial infarction, than patients treated with either placebo or nonselective NSAIDs other than naproxen. Co-therapy with low-dose aspirin is recommended in patients with OA and RA at increased risk for cardiovascular events; the need for gastroprotective therapy in such patients is controversial.     

Genomic imprinting controls matrix attachment regions in the Igf2 gene

Genomic imprinting at the Igf2/H19 locus originates from allele-specific DNA methylation, which modifies the affinity of some proteins for their target sequences. Here, we show that AT-rich DNA sequences located in the vicinity of previously characterized differentially methylated regions (DMRs) of the imprinted Igf2 gene are conserved between mouse and human. These sequences have all the characteristics of matrix attachment regions (MARs), which are known as versatile regulatory elements involved in chromatin structure and gene expression. Combining allele-specific nuclear matrix binding assays and real-time PCR quantification, we show that retention of two of these Igf2 MARs (MAR0 and MAR2) in the nuclear matrix fraction depends on the tissue and is specific to the paternal allele. Furthermore, on this allele, the Igf2 MAR2 is functionally linked to the neighboring DMR2 while, on the maternal allele, it is controlled by the imprinting-control region. Our work clearly demonstrates that genomic imprinting controls matrix attachment regions in the Igf2 gene.     

The non-coding RNAs as riboregulators

The non-coding RNAs database (http://biobases.ibch.poznan.pl/ncRNA/) contains currently available data on RNAs, which do not have long open reading frames and act as riboregulators. Non-coding RNAs are involved in the specific recognition of cellular nucleic acid targets through complementary base pairing to control cell growth and differentiation. Some of them are connected with several well known developmental and neuro-behavioral disorders. We have divided them into four groups. This paper is a short introduction to the database and presents its latest, updated edition.     

Complement depletion facilitates the infection of multiple brain tumors by an intravascular, replication-conditional herpes simplex virus mutant

Intravascular routes of administration can provide a means to target gene- and virus-based therapies to multiple tumor foci located within an organ, such as the brain. However, we demonstrate here that rodent plasma inhibits cell transduction by replication-conditional (oncolytic) herpes simplex viruses (HSV), replication-defective HSV, and adenovirus vectors. In vitro depletion of complement with mild heat treatment or in vivo depletion by treatment of athymic rats with cobra venom factor (CVF) partially reverses this effect. Without CVF, inhibition of cell infection by HSV is observed at plasma dilution as high as 1:32, while plasma from CVF-treated animals displays anti-HSV activity at lower dilutions (1:8). When applied to the therapy of intracerebral brain tumors, in vivo complement depletion facilitates the initial infection (assayed at the 2-day time point) by an intra-arterial replication-conditional HSV of tumor cells, located within three separate and distinct human glioma masses. However, at the 4-day time point, no propagation of HSV from initially infected tumor cells could be observed. Previously, we have shown that the immunosuppressive agent, cyclophosphamide (CPA), facilitates the in vivo propagation of an oncolytic HSV, delivered intravascularly, within infected multiple intracerebral masses, by inhibition of both innate and elicited anti-HSV neutralizing antibody response (K. Ikeda et al., Nat. Med. 5:881-889, 1999). In this study, we thus show that the addition of CPA to the CVF treatment results in a significant increase in viral propagation within infected tumors, measured at the 4-day time period. The concerted action of CVF and CPA significantly increases the life span of athymic rodents harboring three separate and large glioma xenografts after treatment with intravascular, oncolytic HSV. Southern analysis of viral genomes analyzed by PCR reveals the presence of the oncolytic virus in the brains, livers, spleens, kidneys, and intestine of treated animals, although none of these tissues displays evidence of HSV-mediated gene expression. In light of clinical trials of oncolytic HSV for malignant brain tumors, these findings suggest that antitumor efficacy may be limited by the host innate and elicited humoral responses.