Identification and characterization of a conserved,stage-specific gene product of Plasmodium falciparum recognized by parasite growth inhibitory antibodies

We have identified a novel conserved protein of Plasmodium falciparum, designated D13, that is stage-specifically expressed in asexual blood stages of the parasite. The predicted open reading frame (ORF) D13 contains 863 amino acids with a calculated molecular mass of 99.7 kDa and displays a repeat region composed of pentapeptide motives. Northern blot analysis with lysates of synchronized blood stage parasites showed that D13 is highly expressed at the mRNA level during schizogony. The first N'-terminal 138 amino acids of D13 were expressed in Escherichia coli and the purified protein was used to generate anti-D13 monoclonal antibodies (MAbs). Using total lysates of blood stage parasites and Western blot analysis, these MAbs stained one single band of approximately 100 kDa, corresponding to the predicted molecular mass of ORF D13. Western blot analysis demonstrated further that D13 is expressed during schizogony, declines rapidly in early ring stages and is undetectable in trophozoites. D13 protein is localized in individual merozoites in a distinct area, as demonstrated by indirect immunofluorescence analysis. After subcellular fractionation, D13 was confined to the pelleted fraction of the parasite lysate and its extraction by alkaline carbonate buffer treatment indicated that D13 is not a membrane-integral protein. Inclusion of certain anti-D13 MAbs into in vitro cultures of blood stage parasites resulted in considerable reduction in parasite growth. The N'-terminal domain encompassing 158 amino acids is 94 and 95%, respectively, identical at the amino acid level between Plasmodium knowlesi, Plasmodium yoelii, and P. falciparum. In summary, we describe a novel stage-specifically expressed, highly conserved gene product of P. falciparum that is recognized by parasite growth inhibitory antibodies.     

Mother-child interaction in autistic and nonautistic children: characteristics of maternal approach behaviors and child social responses

The nature of mother-child interaction in autism and the maternal approach characteristics that elicit social response in children with autism were examined in two studies. Mother-child play sessions of 24 preschool children with autism and 24 typically developing preschoolers were compared in Study 1, and play sessions of 9 mothers with their autistic child and with their nonautistic child were compared in Study 2. Mother-child interactions were coded using the Approach Withdrawal Interaction Coding System to quantify maternal approach behaviors and child responses. Results of Study 1 indicate that, although the quantity of approaches did not differ between mothers with their autistic children and mothers with their nonautistic children, there were qualitative differences. Mothers used more physical contact, more high-intensity behaviors, and fewer social verbal approaches with autistic children. Results of Study 2 replicated these findings with mothers showing a similar pattern of approach toward their autistic children but not their nonautistic children. Although autistic children displayed lower contingency to maternal approaches in general, they showed greater responsiveness to approaches involving increased physical proximity and/or containing nonverbal object use. Mothers socially engaged both autistic and nonautistic children. The implications for parent training and intervention are discussed.     

Identification of genes induced in alkaloid-producing cultures of Claviceps sp.

In order to identify genes which are expressed during alkaloid synthesis in an axenic culture of Claviceps sp. (strain ATCC 26245), a cDNA library from a producing culture was differentially screened with cDNA from producing (cDNA+) and non-producing (cDNA–) cultures, respectively. Altogether, ten cDNA clones were obtained, the alkaloid-synthesis-correlated expression of which was confirmed by Northern analyses. Evaluation of their nucleotide and derived amino-acid sequences identified one gene unequivocally, coding for dimethylallyltryptophan-synthase (DMAT-S), the initial enzyme of the specific alkaloid pathway. For two other genes significant homologies to known fungal genes were detected: one clone showed homology to the Neurospora crassa ccg1 gene, coding for a clock-regulated putative general stress protein; seven cDNA clones, derived from the same gene, which is highly expressed under these conditions, contained typical hydrophobin domains and long stretches of asparagine/glycine repeats (like QID3 from Trichoderma harzianum), thus probably representing a cell-wall constituent. These data show that this is not only a successful approach to clone genes specific for the alkaloid-pathway of C. purpurea, but also of genes which might be involved in the differentiation of sclerotial hyphae, the prerequisite for alkaloid synthesis. Received: 22 November 1996     

Characterization of a monoclonal anti-Bw4 antibody (Tü109): Evidence for similar epitopes on the Bw4 and Bw6 antigens

The production and serologic, as well as immunochemical properties of a cytotoxic murine IgG monoclonal antibody (Tü109) that precipitates HLA-class I molecules, are described. In the microcytotoxicity assay Tü109 supernatant was demonstrated on a panel of 424 HLA-ABC, -DR, -DQ, -MT typed normal Caucasian blood donors to define an epitope on HLA-B locus molecules in great association with the supertypic specificity Bw4. Reactivity of supernatant showed MHC linked inheritance of the Tü109 determinant and discriminated the HLA-Bw4/Bw6 associated HLA-B locus split antigens. Weak or lack of binding on lymphocytes from some HLA-Bw4 heterozygous individuals, particularly typing for HLA-Bw44, appeared to be due to qualitative and/or quantitative variations of HLA-B locus molecules on the cell surface. With Tü109 ascites fluid, however, extra-reactivity on all HLA-Bw6⁺ cells was demonstrated. Preferential binding of supernatant to HLA-Bw4, but reactivity of ascites fluid with HLA-Bw6⁺ molecules in addition, was furthermore confirmed by IEF analysis of antigens immunoprecipitated with Tü109 from cell lysates. Thus the antibody may help to analyze the evolutionary relationship of the diallelic specificities Bw4 and Bw6.     

3D facial morphometry in Italian patients affected by Aicardi syndrome

Aicardi syndrome (AIC) is a rare congenital neurodevelopmental disorder of unknown etiology, that affects almost exclusively females, originally characterized by corpus callosum agenesis, chorioretinal lacunae, and infantile spasms. The current diagnostic criteria also include qualitative facial features (prominent premaxilla, upturned nasal tip, decreased nasal bridge angle, sparse lateral eyebrows, and microphthalmia) that still need quantification. A three‐dimensional (3D) photogrammetric assessment of 11 Italian females, age 7–32 years, who satisfied AIC criteria, was performed. Linear distances and angles were computed from soft‐tissue facial landmarks coordinates. The z‐score values were calculated using data of 850 healthy reference females matched for age and compared by Mann–Whitney test (p < .01). Patients showed a shorter philtrum and right side orbital height (mean z‐scores: ?1.7, ?0.9), shorter superior, middle, and inferior facial depths (mean z‐scores: ?1.3, ?2.2, ?2.3), and a smaller length of mandibular ramus (mean z‐score: ?2.1); conversely, they showed larger nasal and lower facial widths, and lower facial convexity (mean z‐scores: 1.7, 1.4, 2.4). The inclinations of the orbit versus the true horizontal were increased bilaterally (mean z‐scores: 1.8, 1.1). Some common facial abnormalities were quantified in AIC patients using a noninvasive instrument. They may help clinicians in performing a definite AIC diagnosis in atypical or doubt cases.     

Prevalences, genotypes, and risk factors for HIV transmission in South America

HIV cross-sectional studies were conducted among high-risk populations in 9 countries of South America. Enzyme-linked immunosorbent assay screening and Western blot confirmatory testing were performed, and env heteroduplex mobility assay genotyping and DNA sequencing were performed on a subset of HIV-positive subjects. HIV prevalences were highest among men who have sex with men (MSM; 2.0%-27.8%) and were found to be associated with multiple partners, noninjection drug use (non-IDU), and sexually transmitted infections (STIs). By comparison, much lower prevalences were noted among female commercial sex workers (FCSWs; 0%-6.3%) and were associated mainly with a prior IDU and STI history. Env subtype B predominated among MSM throughout the region (more than 90% of strains), whereas env subtype F predominated among FCSWs in Argentina and male commercial sex workers in Uruguay (more than 50% of strains). A renewed effort in controlling STIs, especially among MSM groups, could significantly lessen the impact of the HIV epidemic in South America.     

Toxoplasma gondii prevents neuron degeneration by interferon-gamma-activated microglia in a mechanism involving inhibition of inducible nitric oxide synthase and transforming growth factor-beta1 production by infected microglia

Interferon (IFN)-gamma, the main cytokine responsible for immunological defense against Toxoplasma gondii, is essential in all infected tissues, including the central nervous system. However, IFN-gamma-activated microglia may cause tissue injury through production of toxic metabolites such as nitric oxide (NO), a potent inducer of central nervous system pathologies related to inflammatory neuronal disturbances. Despite potential NO toxicity, neurodegeneration is not commonly found during chronic T. gondii infection. In this study, we describe decreased NO production by IFN-gamma-activated microglial cells infected by T. gondii. This effect involved strong inhibition of iNOS expression in IFN-gamma-activated, infected microglia but not in uninfected neighboring cells. The inhibition of NO production and iNOS expression were parallel with recovery of neurite outgrowth when neurons were co-cultured with T. gondii-infected, IFN-gamma-activated microglia. In the presence of transforming growth factor (TGF)-beta1-neutralizing antibodies, the beneficial effect of the parasite on neurons was abrogated, and NO production reverted to levels similar to IFN-gamma-activated uninfected co-cultures. In addition, we observed Smad-2 nuclear translocation, a hallmark of TGF-beta1 downstream signaling, in infected microglial cultures, emphasizing an autocrine effect restricted to infected cells. Together, these data may explain a neuropreservation pattern observed during immunocompetent host infection that is dependent on T. gondii-triggered TGF-beta1 secretion by infected microglia.     

Epidermal growth factor receptor expression correlates with poor survival in gastric adenocarcinoma from Mexican patients: a multivariate analysis using a standardized immunohistochemical detection system.

The aim of the study was to determine epidermal growth factor receptor (EGFR) expression in gastric adenocarcinoma by standardized immunohistochemistry and to correlate EGFR expression with clinical features and patient survival. EGFR expression was investigated in paraffin sections of resection specimens of 89 gastric carcinomas from Mexican Mestizo patients using standardized immunohistochemistry with antigen retrieval (Dako EGFRpharmDx assay detection system). Membrane staining of EGFR was evaluated in the neoplastic cells and graded using a semiquantitative score (0-3+). Of the 89 carcinomas examined, staining of neoplastic cells was weak in 17 (19.1%, score 1+), moderate in 16 (18.0%, score 2+), and strong in nine cases (10.1%, score 3+). EGFR reactivity was heterogeneous, frequently showing completely negative up to 3+ positive areas within an individual tumor. EGFR reactivity score correlated with distant metastases (P=0.002) and clinical stage (P=0.033). EGFR score 0/1+ was significantly associated with an increase in patient survival when compared to score 2+/3+ (P=0.0006). In a multivariate analysis, EGFR positive cells in muscularis or subserosa (P=0.004), distant metastases (P=0.016) and residual disease (P=0.039) were significantly correlated with decreased survival. The prognosis was associated with the EGFR reactivity score (P=0.003), distant metastases (P=0.0001) and residual disease (P=0.012) in a univariate analysis. EGFR reactivity in neoplastic cells is an independent prognostic factor in gastric adenocarcinoma. The relevance of the heterogeneity in EGFR expression with regard to tumor progression, metastasis and anti-EGFR therapy needs to be studied.     

Analysis of IFN-kappa expression in pathologic skin conditions: downregulation in psoriasis and atopic dermatitis.

Interferon-kappa (IFN-kappa) is a type I IFN expressed by keratinocytes, monocytes and dendritic cells (DCs). In human keratinocytes, it is produced in response to double-stranded RNA (dsRNA) and other IFNs and protects from viral infections. In monocytes and DCs, IFN-kappa induces tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) and inhibits lipopolysaccharide (LPS)-induced IL-12. In this study, we evaluated IFN-kappa expression in skin lesions of patients with common immune-mediated inflammatory disorders using immunohistochemical techniques. IFN-kappa was not detectable in healthy skin but was strongly expressed in allergic contact dermatitis and lichen planus-affected skin. IFN-kappa was localized mainly in basal and suprabasal keratinocytes and in some leukocytes infiltrating the dermis. In contrast, IFN-kappa expression in psoriatic or atopic dermatitis (AD) pidermis was weak and detectable in only 2 of 5 patients examined. Consistently, cultured keratinocytes and monocytes obtained from psoriatic and AD patients expressed null or low levels of IFN-kappa in response to IFN-gamma, which strongly upregulates IFN-kappa in normal keratinocytes. IFN-kappa accumulated in keratinocyte cytoplasm and plasma membrane, and only limited amounts were released extracellularly. Soluble IFN-kappa did not influence keratinocyte proliferation or chemokine and membrane molecule expression, and only its membrane-associated form activated IFN-stimulated response element (ISRE) signaling. Given the difference in IFN-kappa expression levels in the skin disorders examined, IFN-kappa presence or deficiency might have different pathogenetic consequences depending also on other disease-specific intrinsic alterations.