Characterization of a reduced peptide bond analogue of a promiscuous CD4 T cell epitope derived from the Plasmodium falciparum malaria vaccine candidate merozoite surface protein 1

Use of synthetic peptides as vaccine components is hampered by their susceptibility to enzymatic degradation and rapid clearance from biological fluids. Introduction of non-natural structural modifications can render peptides more resistant to enzymatic degradation, encouraging attempts to profile such non-natural ligands as components of synthetic sub-unit vaccines. We have compared the antigenic and immunogenic properties of a series of non-natural peptide analogues derived from a promiscuous T cell epitope of the major Plasmodium falciparum malaria vaccine candidate merozoite surface protein 1 (MSP-1). A series of HLA class II restricted MSP-1(38-58)-specific TCC established from three volunteers were characterized for their minimal epitope and fine specificity. T cell stimulatory activities of a series of pseudo-peptide analogues with single reduced peptide bond Psi-[CH2-NH] modifications were compared with those of single d-amino acid replacement analogues. Compared to reduced peptide bond analogues the single d-amino acid replacement analogues turned out to be less suitable for stimulation of TCC. In particular, the reduced peptide analogue carrying a Psi-[CH2-NH] backbone modification between positions V52 and L53 of MSP-1(38-58) demonstrated properties that would make it a more suitable vaccine component than the unmodified parent peptide. First, the pseudo-peptide stimulated a number of TCC restricted by a range of HLA class II alleles. Second, trypsin treatment in combination with T cell stimulation assays provided evidence for increased resistance to proteolytic digestion. Third, the parasite-binding anti-MSP-1 mAb 7.27 recognized best this particular pseudo-peptide in competition ELISA experiments and its immunogenicity in out-bred Aotus monkeys was superior to that of the parent peptide eliciting antibodies cross-reactive with native MSP-1.     

Cell adhesion-related proteins as specific markers of sponge cell types involved in allogeneic recognition

Sponge immunocyte identification is of interest to comparative immunologists since characterizing these cells will allow investigations into the mechanisms of non-self recognition in the oldest animal phylum. Here, we report that polyclonal antibodies raised against the core protein of a proteoglycan involved in cell adhesion in the marine sponge Microciona prolifera are specific markers for archaeocytes, the totipotent sponge cells. Archaeocytes are mobilized upon allogeneic contact and they accumulate in the contact zone. A second type of cell, the gray cells, are specifically recognized by monoclonal antibodies raised against CD44, a hyaluronan receptor. Gray cells do also accumulate in the contact area. Specific staining of a third sponge cell type, the rhabdiferous cells, shows that these do not accumulate upon allografting. These specific cell markers allow tracking of archaeocytes and gray cells, and show that they play an active role in sponge allogeneic reactions.     

Clinical Experience with Tilavist: An Overview of Efficacy and Safety

A programme of clinical studies was carried out to determine the basic efficacy and safety of 2% nedocromil sodium eye drops (Tilavist) in treating allergic conjunctivitis, in 2,905 patients from 3–76 years of age. Results of all the double-masked placebo comparative studies completed to date - five in vernal keratoconjunctivitis (VKC), five in perennial (PAC) and 16 in seasonal allergic conjunctivitis (SAC) - have been assessed in a statistical overview analysis. Nedocromil sodium, administered four times daily to 153 patients with VKC, was significantly more effective than placebo (155 patients) and in the clinicians' opinion gave good control in 76% of cases, compared with 46% for placebo (p < 0.001). Patients with chronic symptoms of PAC also responded better to nedocromil sodium given four times daily (n = 146) rather than twice daily (n = 86), and significantly more patients (p < 0.001) were effectively controlled by four times daily treatment with nedocromil sodium (72%) than with placebo (47%; n= 156). Twice-daily dosage with nedocromil sodium (n = 677) was adequate for SAC, however, and the treatment was statistically better than placebo (p < 0.01-p < 0.001) whether dosed twice or four times daily. Speed of action was assessed in seven SAC studies in which 79% of all patients (n = 295) using nedocromil sodium had experienced relief of symptoms when questioned, half of them within 15 minutes and 74% during the first hour after dosing. Test treatments were well-accepted by both adults and children, and there were no major adverse events. Minor irritations reported more frequently with nedocromil sodium than placebo were stinging or burning of the eyes on application of the drops and a distinctive taste, noted by 5% of the active treatment group (n = 1,552).     

Recurrent and novel mutations of GCDH gene in Chinese glutaric acidemia type I families

Glutaric acidemia type I is caused by mutations of the glutaryl-CoA dehydrogenase (GCDH) gene resulting in loss of GCDH enzyme activity. Patients present with progressive dystonia and lesions in basal ganglia. Dietary treatment, when instituted from the early neonatal period, markedly reduces dystonia and morbidity. Early diagnosis and prenatal diagnosis will be facilitated by knowledge of locally prevalent GCDH mutations. Several common GCDH mutations have been found in different ethnic groups. GCDH mutations were studied in 5 Chinese glutaric acidemia type I families. We detected two novel recurrent mutations (A219T and IVS10-2A>C) which were found in two unrelated families. An asymptomatic carrier of IVS10-2A>C was also found on screening of 120 individuals. Other mutations were identified, including two other novel (R386G & IVS3+1G>A) and two known mutations (G178R & R355H). Fibroblasts from patients carrying the novel mutations were confirmed to be deficient for GCDH activity. This is the first report of GCDH mutations describing recurrent mutations in Chinese patients. The carrier rate of IVS10-2A>C may be particularly high in Chinese.     

Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C

Chromatin assembly and DNA replication are temporally coupled, and DNA replication in the absence of histone synthesis causes inviability. Here we demonstrate that chromatin assembly factor Asf1 also affects DNA replication. In budding yeast cells lacking Asf1, the amounts of several DNA replication proteins, including replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase epsilon (Pol epsilon), are reduced at stalled replication forks. In contrast, DNA polymerase alpha (Pol alpha) accumulates to higher than normal levels at stalled forks in asf1Delta cells. Using purified, recombinant proteins, we demonstrate that RFC directly binds Asf1 and can recruit Asf1 to DNA molecules in vitro. We conclude that histone chaperone protein Asf1 maintains a subset of replication elongation factors at stalled replication forks and directly interacts with the replication machinery.     

Specific biochemical markers of bone metabolism and cytokine study confirm the diagnosis of malignant infantile osteopetrosis at birth using cord blood sample

AIMS: To investigate the serum creatine kinase isoenzyme pattern, specific biochemical markers of bone metabolism, and cytokines in a Chinese family with osteopetrosis, and correlate abnormalities with the pathophysiology of this condition. METHODS: A Chinese female baby was diagnosed with malignant infantile osteopetrosis at the age of 3 weeks by clinical history and biochemical investigations. We studied the laboratory and radiological manifestations of this index case and her family members. RESULTS: Serum CK-BB fraction of our index patient was elevated to 18.0% (normal 1.6-7.6%). Her biochemical markers of bone resorption including serum C-terminal telopeptide concentration and urine N-terminal telopeptide to creatinine ratio were decreased to 0.54 microg/L (normal 0.72-1.56 microg/L) and 159 x 10(-6) (normal 372-900 x 10(-6)), respectively. Serum cytokines including soluble receptor activator of nuclear factor kappa-B ligand (sRANKL) concentration was suppressed to 0.11 pmol/L (normal 0.23-0.82 pmol/L) and osteoprotegerin (OPG) concentration was 4.9 pmol/L (normal 2.8-4.9 pmol/L), resulting in an elevated OPG to sRANKL ratio of 44.5 (normal 3.8-19.4) in favour of bone formation. CONCLUSIONS: If left untreated, this condition is usually fatal within the first year of life. With early diagnosis, management including bone marrow transplantation can be planned ahead and will result in a better survival.     

Predicting the trajectory of will to live in terminally ill patients

Will to live has been shown to vary considerably during the final course of a terminal illness. The goal of this study was to identify illness-related and demographic variables predicting will to live among dying patients. Subjects were 168 patients with cancer who were admitted for palliative care. Will to live was measured twice daily for the duration of hospitalization by using a self-report 100-mm visual analogue scale. Will-to-live data for each patient were summarized into two statistics, intercept and slope, by using simple linear regression analyses. Intercept-slope pairs for all patients were classified into the following five clusters by using spatial and conceptual criteria: patients with sustained high will to live (58%), patients with sustained moderate will to live (11%), patients with sustained low will to live (3%), will-to-live relinquishers (18%), and will-to-live acquirers (10%). Discriminant analyses revealed seven variables that accounted for 69% of the variance in cluster membership: anxiety, shortness of breath, nausea, length of survival from time of admission, having a diagnosis of colon cancer, having no religion, and living with a spouse.     

Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia

Thirteen enterovirus 71 (EV71) isolates were obtained from both fatal and non-fatal infections of patients seen in Peninsula Malaysia and in Sarawak during an outbreak of hand, foot and mouth disease (HFMD) in Malaysia in 1997, with incidences of fatal brainstem encephalomyelitis. The isolates were identified using immunofluorescence staining, neutralization assays, and partial sequencing of the 5' untranslated regions (UTR). Assessment of the potential genetic relationships of the isolates using the partial 5'UTR sequences suggested clustering of the isolates into at least two main clusters. Isolates from Peninsula Malaysia were found in both clusters whereas Sarawak-derived isolates clustered only in cluster II. Isolates derived from fatal infections, however, occurred in both clusters and no distinctive nucleotide sequences could be attributed to the fatal isolates. Examination of the nucleotide sequences revealed at least 13 nucleotide positions in all the isolates which differ completely from the previously reported EV71 5'UTR sequences. In addition, at least 11 nucleotide position differences within the 5'UTR were noted which differentiated cluster I from cluster II. Predicted secondary RNA structures drawn using the nucleotide sequences also suggested differences between isolates from the two clusters. These findings suggest the presence of at least two potentially virulent EV71 co-circulating in Malaysia during the 1997 HFMD outbreak.