Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the ³H-labeled natural isomers of nicotine or cotinine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 10⁸ M⁻¹ were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly- -lysine werecoated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5–10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and ³H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.     

Evidence for persistence of adenovirus in the tear film a decade following conjunctivitis

Chronic papillary conjunctivitis has been described following adenoviral conjunctivitis. It is unknown however, how long adenovirus is able to persist in the tear film and conjunctiva. To determine if adenovirus persists in the ocular surface following adenoviral conjunctivitis, 304 patients with a history of adenovirus conjunctivitis from whom an adenovirus had been isolated 10 years previously were sent a questionnaire regarding persistent or recurrent symptoms and were invited to attend. Patients were examined and samples of tears and conjunctival cells were collected from both eyes using tear film washes, filter paper, and swabs, the latter for virus isolation. Extracted DNA from the ocular samples was amplified using primers for herpes simplex virus (thymidine kinase) and adenovirus (hexon) genes. Adenovirus amplicons were sequenced and compared to original serotype. Thirty patients attended, 19 of whom had persistent papillary conjunctivitis. Evidence of adenovirus DNA was detected in 17 of 30 patients, 15 of whom also had evidence of a chronic papillary conjunctivitis. Adenovirus DNA was significantly associated with papillary conjunctivitis (P = 0.03). Adenovirus amplicons were successfully sequenced from six patients. Four patients harbored type 3 adenovirus, the same serotype with which they were infected originally 10 years previously. Two patients were infected originally with adenovirus serotype 3 but the current serotype was type 4. Infection of the ocular surface with adenovirus may predispose to the development of a persistent or recurrent conjunctivitis, the presence of which, appears to be associated with evidence of long term persistence of adenovirus DNA.     

Hemolytic uremic syndrome after bone marrow transplantation: clinical characteristics and outcome in children.

Hemolytic uremic syndrome (HUS) is an uncommon but potentially life-threatening complication of hematopoietic stem cell transplantation. We retrospectively studied the medical records of 293 children who underwent allogeneic bone marrow transplantation at St. Jude Children's Research Hospital between 1992 and 1999 to describe the clinical course of and to identify risk factors for transplant-associated HUS. Conditioning regimens included cyclophosphamide, cytarabine, and total body irradiation for patients with hematologic malignancies (n = 244); patients with nonmalignant diseases (n = 49) received disease-specific regimens. Grafts from unrelated or mismatched related donors were depleted of T lymphocytes, whereas matched sibling grafts were unmanipulated. All patients received cyclosporine as prophylaxis for graft-versus-host disease. Recipients of grafts from matched siblings also received pentoxifylline or short-course methotrexate. HUS developed in 28 (9.6%) patients at a median of 171 days after transplantation. We identified older donor age (P = .029), use of antithymocyte globulin in the conditioning regimen (P = .008), and recipient CMV seronegativity (P = .011) as being associated with an increased risk of HUS. With a multiple regression analysis, the use of antithymocyte globulin (beta = .86; P = .04) and recipient cytomegalovirus seronegativity (beta = .93; P = .035) remained significant risk factors for the development of HUS.     

Temporal Variation of the Merozoite Surface Protein-2 Gene of Plasmodium falciparum

Extensive polymorphism of key parasite antigens is likely to hamper the effectiveness of subunit vaccines against Plasmodium falciparum infection. However, little is known about the extent of the antigenic repertoire of naturally circulating strains in different areas where malaria is endemic. To address this question, we conducted a study in which blood samples were collected from parasitemic individuals living within a small hamlet in Western Irian Jaya and subjected to PCR amplification using primers that would allow amplification of the gene encoding merozoite surface protein-2 (MSP2). We determined the nucleotide sequence of the amplified product and compared the deduced amino acid sequences to sequences obtained from samples collected in the same hamlet 29 months previously. MSP2 genes belonging to both major allelic families were observed at both time points. In the case of the FC27 MSP2 family, we observed that the majority of individuals were infected by parasites expressing the same form of MSP2. Infections with parasites expressing 3D7 MSP2 family alleles were more heterogeneous. No MSP2 alleles observed at the earlier time point were detectable at the later time point, either for the population as a whole or for individuals who were assayed at both time points. We examined a subset of the infected patients by using blood samples taken between the two major surveys. In no patients could we detect reinfection by a parasite expressing a previously encountered form of MSP2. Our results are consistent with the possibility that infection induces a form of strain-specific immune response against the MSP2 antigen that biases against reinfection by parasites bearing identical forms of MSP2.The development of a host-protective immune response against Plasmodium falciparum takes several years and many episodes of infection, at least for children living in areas where malaria is endemic. One of the reasons for this is believed to be the large number of distinct parasite strains circulating within an area of endemicity and the assumption that exposure must occur to a sufficiently large sample of these before lasting immunity is induced. However, the detailed epidemiology of endemic malaria infection remains poorly understood at the molecular level, and there is surprisingly little nucleotide sequence data to support the concept of a large repertoire of antigenically distinct strains.There are at least six antigenically diverse proteins of the asexual stage that are known to be the target of potentially protective host responses. The definition of antigenically distinct strains involves identification of the allelic form expressed at all antigenically diverse loci—the extended antigenic haplotype. The loci would include merozoite surface protein-1 to -3 (3), apical membrane antigen-1 (17), S-antigen (6), and P. falciparum erythrocyte membrane protein-1 (PfEMP-1) (5). Such a complete molecular definition of infecting parasites is a highly ambitious task, particularly in the case of blood samples collected from patients harboring mixed infections. Accordingly, most studies focus on one or other of the antigenically diverse antigens. We have elected to study merozoite surface protein-2 (MSP2) (27), a 45- to 50-kDa glycoprotein anchored in the merozoite surface by a glycosylphosphatidylinositol anchor. This surface protein is a promising candidate for inclusion in a malaria subunit vaccine, as both in vitro and in vivo studies have demonstrated the ability of immune responses to MSP2 to inhibit parasite multiplication (23, 25). However, the efficacy of any subunit vaccine containing a single form of MSP2 may be limited by the presence of antigenically distinct parasite strains within an area of endemicity. We will adopt the recently proposed convention for parasite genes and gene products of denoting the gene sequence as MSP2 and the protein as MSP2.Sequence polymorphism has been described for MSP2 genes of both laboratory-maintained isolates (29) and field isolates (14, 16, 19, 30). Comparison of MSP2 gene sequences from these isolates reveals highly conserved 5′ and 3′ sequences that flank a central variable region. This central region is composed of repeats flanked by nonrepetitive sequences. The nonrepetitive sequences are one or other of two distinct forms that define two allelic families, FC27 and IC-1/3D7 (29). The central repeats are more variable and define the individual alleles of MSP2. There is a correlation between the general form of the central repeat sequence and the allelic family. For example, FC27 family members have variants of a central 96-bp pair sequence that may be present in one to four copies followed by a 21-bp partial repeat and a variably represented 36-bp sequence that may be present in one to five copies. In contrast, alleles belonging to the 3D7 family show a central repeat region made up of variable numbers of 12- to 24-bp repeats separated by repeating 6-bp sequences.Field studies aimed at defining the antigenic diversity of MSP2 have approached the problem by determining MSP2 gene structure by various forms of PCR. The rationale for this is that P. falciparum is haploid and MSP2 has been shown to be present in all laboratory and field isolates examined (8–10, 15). Most studies examining the distribution and frequency of different allelic forms of MSP2 have enumerated the presence of the allelic families (11, 12, 14). Whereas a skewed distribution of predominantly 3D7 family alleles exists among laboratory-adapted strains, in the field a more even distribution of FC27 and 3D7 alleles occurs. Often FC27 family alleles are more prevalent than 3D7 alleles, and novel FC27 and 3D7 family alleles have been found in field malaria strains (14, 16). Some field studies examining recurrent MSP2 infections have been performed, but these have classified MSP2 alleles on the basis of family and length of the central repeat region (7, 11, 14). This makes it difficult to form conclusions about the repertoire of repeat sequences in the circulating pool of parasites and to infer the possible action of immune responses to MSP2 repeats. We were interested to examine the sequences of MSP2 alleles circulating in an area of endemicity over time and to determine the persistence of various MSP2 alleles within a localized area. This study describes MSP2 genotypes from malaria-infected inhabitants of the Oksibil region of Irian Jaya and allows comparison of the variation in MSP2 sequences seen over a 2.5-year period within the region as a whole and in particular individuals.     

Which treatment for whom for ADHD? Moderators of treatment response in the MTA

Using receiver operating characteristics, the authors examined outcome predictors (variables associated with outcome regardless of treatment) and moderators (variables identifying subgroups with differential treatment effectiveness) in the Multimodal Treatment Study of Children with Attention-Deficit/Hyperactivity Disorder (ADHD; MTA). Treatment response was determined using parent- and teacher-reported ADHD and oppositional defiant symptoms, with levels near or within the normal range indicating excellent response. Among 9 baseline child and family characteristics, none predicted but 3 moderated treatment response. In medication management and combined treatments, parental depressive symptoms and severity of child ADHD were associated with decreased rates of excellent response; when these 2 characteristics were present, below-average child IQ was an additional moderator. No predictors or moderators emerged for behavioral and community comparison treatments. The authors discuss conceptual and clinical implications of research on treatment moderators.