Detection of human antibodies against Plasmodium falciparum sporozoites using synthetic peptides.

A large peptide consisting of about 40 (Asn-Ala-Asn-Pro) repeats of Plasmodium falciparum circumsporozoite protein, (NANP)40, was synthesized. It was recognized specifically by monoclonal antibodies produced against P. falciparum sporozoites. Moreover, this peptide strongly inhibited the binding of such monoclonal antibodies to antigens present in a sporozoite extract. The (NANP)40 peptide was employed without any carrier to develop an enzyme-linked immunosorbent assay to detect sporozoite-specific serum antibodies arising after natural malaria infections. Antibodies were detected in a high percentage (43.1%) of European patients suffering from acute P. falciparum malaria and in Africans living in an area of Gabon endemic for malaria. In the latter group, the frequency of antisporozoite antibodies increased with age, reaching 65.9% in individuals more than 40 years old. There was a significant correlation between the results obtained with an immunofluorescence assay with glutaraldehyde-fixed sporozoites and those obtained by enzyme-linked immunosorbent assay with (NANP)40. Therefore, such synthetic peptides representing the repetitive epitope of P. falciparum circumsporozoite protein can be used for the detection of antisporozoite antibodies and for the epidemiological studies required to obtain base-line data concerning the immune status of individuals before their participation in a sporozoite vaccine trial.     

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions cytocauds. The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.     

The interaction of Tamm-Horsfall protein with the extracellular matrix.

Tamm-Horsfall protein (THP) is the major glycoprotein component of urine, yet its biological role remains obscure. Recent reports have suggested that a concanavalin A (Con A)-binding fraction of THP from pregnancy urine can bind the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1). In order to investigate this claim in relation to THP from normal adult urine we raised monoclonal antibodies to THP and sought THP/TNF-alpha interactions in three separate assay systems. We found no evidence that THP binds to TNF-alpha under physiological conditions, but we observed that it exerts a weak, probably not physiologically relevant, but reproducible inhibitory effect on the toxicity of TNF-alpha for monolayers of L929 cells, even when the cells are pretreated with the THP, and washed before addition of the cytokine. Since our preparations of THP do not interact directly with TNF-alpha we postulated an interaction with the cells themselves, or with their extracellular matrix. The THP was found by ELISA, immunoblotting and immunohistology, to bind to as yet unidentified components of the extracellular matrix in a manner dependent on cations, pH and carbohydrates. These data, considered in the light of the published amino acid sequence and biochemical properties, suggest that THP is a member of a structural glycoprotein family known to modulate cell adhesion.     

Performance characteristics of HIV-1 culture and HIV-1 DNA and RNA amplification assays for early diagnosis of perinatal HIV-1 infection

A plasma HIV-1 RNA amplification assay (RNA assay), a quantitative peripheral blood mononuclear cell (PBMC) microculture (culture), and a PBMC HIV-1 DNA amplification assay (DNA assay) were compared for diagnosis of HIV-1 infection in infants receiving zidovudine in Pediatric AIDS Clinical Trials Group protocol 185; assays were performed for all 24 infected and 100 uninfected infants. HIV-1 infection was defined as >or=2 positive cultures or positive antibody to HIV-1 at >or=18 months. Cultures were performed at birth and 6 and 24 weeks of age; DNA and RNA assays were performed on cryopreserved specimens. The sensitivity of culture and DNA and RNA assays at birth was 20.8%, 10.5%, and 26.7%, respectively. At older ages, sensitivity typically exceeded 80%, remaining highest for the RNA assay (>85%). Assay specificity was >99%. Positive predictive values exceeded 93% for each assay at each age; negative predictive values were highest (>90%) for the RNA assay. At birth (P < 0.005) and age 6 weeks (P < 0.001), a significantly larger proportion of infected infants were identified by means of the RNA assay than by the other assays. The diagnostic performance of the RNA assay matched or exceeded that of culture and the DNA assay. Given that RNA assays require less blood volume and yield rapid results, our study adds to existing data suggesting that RNA assays may be used for early diagnosis of HIV-1 infection in infants.     

Immune complexes and complement hypercatabolism in patients with leprosy.

The occurrence of immune complexes in the serum and the level of the C3 breakdown product C3d in the plasma from patients with leprosy were studied by quantitative methods and the results were compared in various forms of the disease. These studies were performed on sixty-two samples from twenty-six patients. The serum 125I-C1q binding activity was found to be increased by more than 2 s.d., as compared to the normal values, in most of the sera from patients with erythema nodosum leprosum (ENL) (80%) and uncomplicated lepromatous leprosy (82%), but also in the sera from patients with tuberculoid leprosy (58%). In vitro studies suggested that immune complexes involving mycobacterial antigens were present in leprosy sera. An increased C3d level (greater than 2s.d.) was also found in most of the plasma from patients with ENL (70%), but rarely in the plasma from patients with uncomplicated lepromatous leprosy (18%) and never in tuberculoid leprosy patients' plasma. The absence of a significant correlation between the 125I-C1q binding activity and the C3d level in leprosy patients may suggest that extravascular immune complexes are involved in the complement activation occurring in ENL. The quantitation of C3d in plasma may be of some practical interest in the early diagnosis of ENL complications of leprosy.     

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.      

Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test. A comparative study with 125I-labelled C1q binding and Raji-cell RIA tests

Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample is incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes are detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody.

The conglutinin-binding (KgB) test does not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG is 3 μg/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 μg/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the ¹²⁵I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, ¹²⁵I-labelled Clq BA and Raji-cell radioimmunoassay (RIA).

Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application.