Evaluation of Hemagglutinin Protein-Specific Immunoglobulin M for Diagnosis of Measles by an Enzyme-Linked Immunosorbent Assay Based on Recombinant Protein Produced in a High-Efficiency Mammalian Expression System

Recombinant hemagglutinin (H) of the measles virus (MV) expressed in a mammalian high-expression system based on the Semliki Forest virus replicon was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulin M (IgM) and IgG in patients with acute-phase measles. One hundred twelve serum specimens from 70 patients with measles were analyzed. Case definition was based on a commercial IgM ELISA that utilizes MV-infected cells (MV-ELISA) (Enzygnost; Behring Diagnostics); the clinical criteria of the Centers for Disease Control and Prevention (Atlanta, Ga.); and/or the increase in hemagglutinin test titers, neutralization test titers, and levels of MV-specific IgG whenever paired sera were available. The initial time courses of the IgM signal after the onset of rash are similar in the H- and MV-ELISAs. On days 0 to 19, both ELISAs detected IgM in 67 of 68 (98.5%) sera. Average maximal levels of IgM seem to persist, however, about 10 days longer in the MV-ELISA (up to day 25) than in the H-ELISA (day 15). From days 20 to 29 and 30 to 59, the H-ELISA detected only 64.3 (9 of 14) and 19.2% (5 of 26), respectively, of sera that were IgM positive by MV-ELISA. At least up to day 30, the performance of the H-ELISA seemed to be similar to that reported for commercial ELISAs based on whole MV. Our results demonstrate that MV H-specific IgM can be used to diagnose most measles cases from a single serum specimen collected within 19 days after the onset of rash and that the recombinant protein used in this study is suitable for this purpose.     

Inhibition of vascular endothelial growth factor (VEGF) signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts

Angiogenesis inhibitors are receiving increased attention as cancer therapeutics, but little is known of the cellular effects of these inhibitors on tumor vessels. We sought to determine whether two agents, AG013736 and VEGF-Trap, that inhibit vascular endothelial growth factor (VEGF) signaling, merely stop angiogenesis or cause regression of existing tumor vessels. Here, we report that treatment with these inhibitors caused robust and early changes in endothelial cells, pericytes, and basement membrane of vessels in spontaneous islet-cell tumors of RIP-Tag2 transgenic mice and in subcutaneously implanted Lewis lung carcinomas. Strikingly, within 24 hours, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and patency and blood flow ceased in some vessels. By 7 days, vascular density decreased more than 70%, and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells. Vessels in Lewis lung tumors, which lacked endothelial fenestrations, showed less regression. In both tumors, pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels acquired a more normal phenotype. Vascular basement membrane persisted after endothelial cells degenerated, providing a ghost-like record of pretreatment vessel number and location and a potential scaffold for vessel regrowth. The potent anti-vascular action observed is evidence that VEGF signaling inhibitors do more than stop angiogenesis. Early loss of endothelial fenestrations in RIP-Tag2 tumors is a clue that vessel phenotype may be predictive of exceptional sensitivity to these inhibitors.     

Human immunodeficiency virus 1 favors the persistence of infection by activating macrophages through TNF

Macrophages play a major role in HIV-1 persistence. In the present paper, we demonstrate that the absence of apoptosis in HIV-1-infected primary human monocyte-differentiated macrophages (MDM) correlates with an increase in anti-apoptotic (Bcl-2 and Bcl-x(L)) and a decrease in pro-apoptotic (Bax and Bad) proteins. This is associated with macrophage activation as shown by tumor necrosis factor (TNF) production and NF-kappaB activation upon infection. TNF production was shown to be involved in the upregulation of Bcl-2 and Bcl-x(L) because this increase was abolished by an anti-TNF anti-serum or an inhibitor of TNF synthesis. In parallel, inhibition of TNF production induced an increase in the number of apoptotic cells. Furthermore, using an inhibitor of NF-kappaB activation, we demonstrated that TNF-induced upregulation of Bcl-x(L) and Bcl-2 occurs, respectively, through a NF-kappaB-dependent and an NF-kappaB-independent pathway.     

IgE-mediated anaphylaxis caused by bites of the pigeon tick Argas reflexus: cloning and expression of the major allergen Arg r 1

BACKGROUND: Anaphylactic reactions caused by bites of the European pigeon tick Argas reflexus are repeatedly reported. This soft-backed tick is a parasite of wild pigeons colonizing urban buildings and houses. Occasionally the ticks can bite human beings, inducing anaphylactic reactions in sensitized patients. OBJECTIVE: Our aim was to characterize the major allergen implicated in a series of anaphylactic reactions caused by Argas bites and to produce the allergen as recombinant protein for diagnostic purposes. METHODS: Protein extracts were prepared from whole A reflexus bodies, and IgE immunoblots were performed with sera from 13 patients who had an anaphylactic reaction with pigeon tick bites. A cDNA expression library was constructed from whole ticks and screened with a polyclonal rabbit antiserum raised against the major allergen. RESULTS: The cDNA coding for the dominant allergen Arg r 1 could be isolated. It encodes a protein belonging to the lipocalin family. Allergenicity of the recombinant Arg r 1 was confirmed by immunoblot, ELISA, and intradermal skin tests. CONCLUSION: The dominant allergen of A reflexus has been isolated and the corresponding cDNA cloned. The recombinant protein, a lipocalin, was expressed in Escherichia coli and was shown to be immunoreactive in vitro and in vivo. Recombinant Arg r 1 was used as a diagnostic tool in a series of anaphylactic reactions caused by pigeon tick bites.     

WNT10B variants in split hand/foot malformation: Report of three novel families and review of the literature

Split‐hand/foot malformation (SHFM) is a genetically heterogeneous congenital limb malformation typically limited to a defect of the central rays of the autopod, presenting as a median cleft of hands and feet. It can be associated with long bone deficiency or included in more complex syndromes. Among the numerous genetic causes, WNT10B homozygous variants have been recently identified in consanguineous families, but remain still rarely described (SHFM6; MIM225300). We report on three novel SHFM families harboring WNT10B variants and review the literature, allowing us to highlight some clinical findings. The feet are more severely affected than the hands and there is a frequent asymmetry without obvious side‐bias. Syndactyly of third–fourth fingers was a frequent finding (62%). Polydactyly, which was classically described in SHFM6, was only present in 27% of patients. No genotype–phenotype correlation is delineated but heterozygous individuals might have mild features of SHFM, suggesting a dose‐effect of the WNT10B loss‐of‐function.     

Factors affecting the determination of threshold doses for allergenic foods: how much is too much?

BACKGROUND: Ingestion of small amounts of an offending food can elicit adverse reactions in individuals with IgE-mediated food allergies. The threshold dose for provocation of such reactions is often considered to be zero. However, because of various practical limitations in food production and processing, foods may occasionally contain trace residues of the offending food. Are these very low, residual quantities hazardous to allergic consumers? How much of the offending food is too much? Very little quantitative information exists to allow any risk assessments to be conducted by the food industry. OBJECTIVE: We sought to determine whether the quality and quantity of existing clinical data on threshold doses for commonly allergenic foods were sufficient to allow consensus to be reached on establishment of threshold doses for specific foods. METHODS: In September 1999, 12 clinical allergists and other interested parties were invited to participate in a roundtable conference to share existing data on threshold doses and to discuss clinical approaches that would allow the acquisition of that information. RESULTS: Considerable data were identified in clinical files relating to the threshold doses for peanut, cows' milk, and egg; limited data were available for other foods, such as fish and mustard. CONCLUSIONS: Because these data were often obtained by means of different protocols, the estimation of a threshold dose was very difficult. Development of a standardized protocol for clinical experiments to allow determination of the threshold dose is needed.     

A model for antigen-induced T cell unresponsiveness based on autophosphorylative protein tyrosine kinase activity

Helper T cell signaling is initiated by the aggregation of TCRwith the induction of tyrosine kinase activity as one of theearliest consequences. Here, a theoretical model for antigen-inducedunresponsiveness is presented that relies on a cascade of tyrosinephosphorylation- dephoshorylation cycles. A mechanism is describedfor both desensitization in the presence of antigen and persistentlowering of cell responsiveness after stimulus removal. An importantcomponent of the model, leading to bistability, is the presenceof autophosphorylating protein tyrosine kinases in the earlysteps of TCR signaling. One of its predictions is that, followingstimulation, the net phosphorylative activity of these receptor-associatedtyrosine kinases will remain above background level after removalof the antigen. It is proposed that this residual tyrosine kinaseactivity is linked to a deficient signal transduction capacityof the TCR system that leads to a state of prolonged unresponsiveness.In addition, the present analysis defines the notion of a signalingthreshold for hyporesponsiveness induction, associated witha durable switch and amplification of the net tyrosine kinaseactivity. This approach emphasizes the role of tyrosine kinasesin the down-regulation of cellular competence.     

Human immunodeficiency virus gp120 and derived peptides activate protein tyrosine kinase p56Ick in human CD4 T lymphocytes

Human immunodeficiency virus binds to CD4 T lymphocytes by interaction between its envelope glycoprotein gpl20 and the CD4 molecule. The latter is non-covalently associated with a src-related tyrosine kinase, p56^lck. CD4 cross-linking increases the activity of p56^lck, leading to phosphorylation of several cellular substrates. We report here that gpl60/120 increases both the autophos-phorylation of p56^lck and its enzymatic activity (reflected by phosphorylation of an exogeneous substrate) in normal T cells and the HUT78 CD4⁺ T cell line. This effect was detectable 5 min after activation and persisted for 40 min in normal T cells. It did not require gpl20 cross-linking and was associated with phosphorylation of tyrosine residue on several proteins, as shown by phosphotyrosine Western blot analysis. The pattern of proteins phosphorylated on tyrosine residues in response to gpl20 activation was distinct from that induced by anti-CD4 antibodies. p56^lck activation required its association with CD4, since p56^lck activity was not modified in HUT78 T cell lines expressing a truncated or mutated form of CD4 unable to associate with p56^lck. Peptides mimicking residues 418 to 434 and 449 to 464 of HIV-1 Bru gpl20, regions known to participate in gpl20 binding to CD4, also increased p56^lck activity and triggered phosphorylation of similar substrates. Taken together, these results show that gpl60/120 and derived peptides can transiently increase p56^lck activity without the need for CD4 cross-linking. This activation led to a specific pattern of tyrosine phosphorylation on cellular proteins that may be of significance in the biological effects of the gpl20/CD4 interaction, e.g. syncytium formation and inhibition of T cell activation.