Antigen selection in the IgE response of allergic and nonallergic individuals

BACKGROUND: Affinity maturation within germinal centers should usually lead to an accumulation of replacement mutations in complementarity-determining regions (CDRs) of Ig genes as a result of antigen selection. A number of studies have suggested, but not statistically demonstrated, that antigen selection might not guide such an accumulation of replacement mutations in allergic IgE sequences. This has been suggested to reflect the nature of allergens themselves or of the allergic response. OBJECTIVE: We sought to investigate the role of antigen selection in the evolution of the IgE response by mean of analysis of Ig sequences derived from both allergic and nonallergic individuals. METHODS: IgE sequences were amplified from peripheral blood of allergic and nonallergic individuals by using seminested RT-PCR. Additional IgE and IgG sequences were obtained from public databases. Analysis considered replacement mutations in the CDRs as a proportion of total mutations and compared IgE sequences with IgG sequences. RESULTS: The nonallergic IgE sequences were significantly less mutated than both the allergic IgE (P < .001) and IgG (P < .01) sequences. There was a low proportion of replacement mutations in the CDRs of both nonallergic and allergic IgE sequences and a significantly increased proportion of such mutations in IgG sequences (P < .001). CONCLUSIONS: IgE antibodies in both nonallergic and allergic individuals appear to accumulate few somatic point mutations as a consequence of antigen selection. CLINICAL IMPLICATIONS: Allergic and nonallergic IgE responses might often develop along a common pathway that is distinct from the conventional germinal center reaction through which the IgG response develops.     

A common toxin fold mediates microbial attack and plant defense

Many plant pathogens secrete toxins that enhance microbial virulence by killing host cells. Usually, these toxins are produced by particular microbial taxa, such as bacteria or fungi. In contrast, many bacterial, fungal and oomycete species produce necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) that trigger leaf necrosis and immunity-associated responses in various plants. We have determined the crystal structure of an NLP from the phytopathogenic oomycete Pythium aphanidermatum to 1.35Å resolution. The protein fold exhibits structural similarities to cytolytic toxins produced by marine organisms (actinoporins). Computational modeling of the 3-dimensional structure of NLPs from another oomycete, Phytophthora parasitica, and from the phytopathogenic bacterium, Pectobacterium carotovorum, revealed a high extent of fold conservation. Expression of the 2 oomycete NLPs in an nlp-deficient P. carotovorum strain restored bacterial virulence, suggesting that NLPs of prokaryotic and eukaryotic origins are orthologous proteins. NLP mutant protein analyses revealed that identical structural properties were required to cause plasma membrane permeabilization and cytolysis in plant cells, as well as to restore bacterial virulence. In sum, NLPs are conserved virulence factors whose taxonomic distribution is exceptional for microbial phytotoxins, and that contribute to host infection by plasma membrane destruction and cytolysis. We further show that NLP-mediated phytotoxicity and plant defense gene expression share identical fold requirements, suggesting that toxin-mediated interference with host integrity triggers plant immunity-associated responses. Phytotoxin-induced cellular damage-associated activation of plant defenses is reminiscent of microbial toxin-induced inflammasome activation in vertebrates and may thus constitute another conserved element in animal and plant innate immunity.     

Chlorella viruses evoke a rapid release of K+ from host cells during the early phase of infection

Infection of Chlorella NC64A cells by PBCV-1 produces a rapid depolarization of the host probably by incorporation of a viral-encoded K(+) channel (Kcv) into the host membrane. To examine the effect of an elevated conductance, we monitored the virus-stimulated efflux of K(+) from the chlorella cells. The results indicate that all 8 chlorella viruses tested evoked a host specific K(+) efflux with a concomitant decrease in the intracellular K(+). This K(+) efflux is partially reduced by blockers of the Kcv channel. Qualitatively these results support the hypothesis that depolarization and K(+) efflux are at least partially mediated by Kcv. The virus-triggered K(+) efflux occurs in the same time frame as host cell wall degradation and ejection of viral DNA. Therefore, it is reasonable to postulate that loss of K(+) and associated water fluxes from the host lower the pressure barrier to aid ejection of DNA from the virus particles into the host.     

A shift from membranous and stromal syndecan‐1 (CD138) expression to cytoplasmic CD138 expression is associated with poor prognosis in breast cancer

Syndecan‐1 (CD138) is a transmembrane proteoglycan expressed in normal and malignant tissues. It is of interest because of a possible prognostic effect in tumors and as a target for Indatuximab, a monoclonal antibody coupled to a cytotoxic agent. To assess the prognostic role of CD138 expression in breast cancer (BCa), a tissue microarray containing 1535 BCa specimens was analyzed by immunohistochemistry. Cytoplasmic, membranous, and stromal CD138 staining was separately analyzed. In normal breast tissue, CD138 staining was limited to epithelial cell membranes. In cancers, membranous staining tended to become weaker or even disappeared (38.3% of cancers with absence of membranous staining) but cytoplasmic and stromal staining newly appeared in 29.7% and 58.1% of cancers. Loss of membranous epithelial CD138 staining as well as presence of cytoplasmic and stromal CD138 positivity were—to a variable degree—associated with high pT, high grade, nodal metastasis, estrogen receptor‐negative, progesterone receptor‐negative, human epidermal growth factor receptor 2+, and poor overall patient survival. A combined analysis of epithelial and stromal CD138 expression revealed a link to overall patient survival (P < .0001) with best prognosis for patients with stromal positivity and absence of cytoplasmic staining, the worst prognosis for cancers with cytoplasmic staining and stromal negativity and intermediate prognosis for patients having either cytoplasmic staining or stromal negativity. In multivariate analyses, CD138 was not independent of established prognostic features. In summary, these data reveal a compartment depending prognostic effect of CD138 expression in BCa with cytoplasmic positivity being linked to aggressive cancer and stromal CD138 being linked to a more favorable prognosis.     

Alternative management of complex wounds and fistulae

The management of complex wounds and fistulae can often prove challenging to even the most skilled clinician. The incidence and complexity of fistulae vary considerably from centre to centre, however they often lead to prolonged hospital stays. Routine admissions for 4-5 days may lead to 4-5 months in the event of fistulae formation. Thus, many patients experience not only compromised physical health, but also complex psychological problems. This article provides a brief overview of the challenges and developments of managing a complex wound with multiple fistulae and a pictorial illustration of an innovative alternative wound management system.     

Molecular analysis of TGF-(beta)s and their receptors in human keratinocyte cell lines of different biological behaviour

We investigated three clonally related human keratinocyte cell lines of different biological behaviour, HaCaT (non-tumorigenic), A5 (benign, tumorigenic) and II-4RT (malignant, tumorigenic), with regard to the expression of TGF-beta-isoforms -1, -2 and -3 and that of the TGF-beta-cell-receptors TBR-I, -II and -III. In addition, we amplified and sequenced the genome of TBR-II which is known to be a target for mutations in several types of malignant tumors including squamous cell carcinomas. In all three cell lines, TGF-beta1 and -beta3 were present only in very low amounts. Western blots provided no evidence for differences in TGF-beta1 between the cell lines. However, in immunohistochemistry more cells were slightly positive for this cytokine in HaCaT than in A5 and II-4RT cells. In contrast, a significantly variable expression of TGF-beta2 was seen by both Western blot and immunohistochemistry. Thereby, the non-tumorigenic HaCaT-cells contained significantly more TGF-beta2 than the tumorigenic, benign A5 cells and the malignant II-4RT cells. TBR-I, -II and -III were present in all three cell lines. While most cells were positive for TBR-I, only part of the cells contained TBR-II and -III, however, without obvious differences between the three cell lines. The molecular analysis of all 7 exons of TBR-II by PCR amplification and direct sequencing revealed in all three cell lines correct sequences without evidence for mutations. Our study indicates differences in the expression of TGF-beta in a human model of keratinocytes of varying tumorigenicity, but presents no evidence for mutations in the functionally most important TGF-beta-receptor TBR-II. This suggests a dysregulation of cytokine control on the level of TGF-beta expression, which may be responsible for the biological behaviour.     

Evaluation of a selectively oncolytic adenovirus for local and systemic treatment of cervical cancer

Treatment options for disseminated cervical cancer remain inadequate. Here, we investigated a strategy featuring Ad5-Delta 24 RGD, an oncolytic adenovirus replication-competent selectively in cells defective in the Rb-p16 pathway, such as most cervical cancer cells. The viral fiber contains an alpha(v)beta(3) and alpha(v)beta(5) integrin-binding RGD-4C motif, allowing coxsackie-adenovirus receptor-independent infection. These integrins have been reported to be frequently upregulated in cervical cancer. Oncolysis of cervical cancer cells was similar to a wild-type control in vitro. In an animal model of cervical cancer, the therapeutic efficacy of Ad5-Delta 24 RGD could be demonstrated for both intratumoral and intravenous application routes. Biodistribution was determined following intravenous administration to mice. Further preclinical safety data were obtained by demonstrating lack of replication of the agent in human peripheral blood mononuclear cells. These results suggest that Ad5-Delta 24 RGD could be useful for local or systemic treatment of cervical cancer in patients with disease resistant to currently available modalities.     

Neural correlates of syntactic transformations

Many agrammatic aphasics have a specific syntactic comprehension deficit involving processing syntactic transformations. It has been proposed that this deficit is due to a dysfunction of Broca's area, an area that is thought to be critical for comprehension of complex transformed sentences. The goal of this study was to investigate the role of Broca's area in processing canonical and non-canonical sentences in healthy subjects. The sentences were presented auditorily and were controlled for task difficulty. Subjects were asked to judge the grammaticality of the sentences while their brain activity was monitored using event-related functional magnetic resonance imaging. Processing both kinds of sentences resulted in activation of language-related brain regions. Comparison of non-canonical and canonical sentences showed greater activation in bilateral temporal regions; a greater activation of Broca's area in processing antecedent-gap relations was not found. Moreover, the posterior part of Broca's area was conjointly activated by both sentence conditions. Broca's area is thus involved in general syntactic processing as required by grammaticality judgments and does not seem to have a specific role in processing syntactic transformations.