The aggregation of cytochrome C may be linked to its flexibility during refolding

Large-scale expression of biopharmaceutical proteins in cellular hosts results in production of large insoluble mass aggregates. In order to generate functional product, these aggregates require further processing through refolding with denaturant, a process in itself that can result in aggregation. Using a model folding protein, cytochrome C, we show how an increase in final denaturant concentration decreases the propensity of the protein to aggregate during refolding. Using polarised fluorescence anisotropy, we show how reduced levels of aggregation can be achieved by increasing the period of time the protein remains flexible during refolding, mediated through dilution ratios. This highlights the relationship between the flexibility of a protein and its propensity to aggregate. We attribute this behaviour to the preferential urea-residue interaction, over self-association between molecules

The role of γδ T cells in airway epithelial injury and bronchial responsiveness after chlorine gas exposure in mice

BACKGROUND: Acute exposure to chlorine (Cl(2)) gas causes epithelial injury and airway dysfunction. γδ T cells are present in the mucosal surface of the airways and may contribute to the injury/repair response of the epithelium. METHODS: C57Bl/6J (wild type) and TCR-δ(-/- )mice exposed to Cl(2 )(400 ppm) for 5 minutes underwent measurements of airway responses to i.v. methacholine (MCh) at 1, 3, and 5 days after exposure. Bronchoalveolar lavage was performed to determine epithelial and leukocyte counts, and protein content. Tissue repair was assessed by proliferating cell nuclear antigen (PCNA) immunoreactivity and by expression of keratinocyte growth factor (KGF) mRNA by real-time PCR. RESULTS: Wild type mice developed a greater degree of airway hyperresponsiveness to MCh at 1 day post exposure to Cl(2 )compared with TCR-δ(-/- )mice. Epithelial cell counts in BAL after Cl(2 )exposure were greater in TCR-δ(-/- )mice, but macrophages showed a later peak and granulocyte numbers were lower in TCR-δ(-/- )than in wild type mice. Both groups had increased levels of total protein content in BAL after Cl(2 )exposure that resolved after 3 and 5 days, respectively. Epithelial proliferating cell nuclear antigen staining was increased at 1 and 3 days post exposure and was similar in the two groups. KGF mRNA was constitutively expressed in both groups and did not increase significantly after Cl(2 )but expression was lower in TCR-δ(-/- )mice. CONCLUSION: The severity of airway epithelial injury after Cl(2 )is greater in TCR-δ(-/- )mice but the inflammatory response and the change in airway responsiveness to methacholine are reduced. The rates of epithelial regeneration are comparable in both groups

Induction of T Lymphocytes Specific for Bovine Viral Diarrhea Virus in Calves with Maternal Antibody

Passive antibody to bovine viral diarrhea virus (BVDV) acquired through colostrum intake may interfere with the development of a protective immune response by calves to this virus. The objective of this study was to determine if calves, with a high level of maternal antibody to bovine viral diarrhea virus (BVDV), develop CD4+, CD8+, or γδ T lymphocyte responses to BVDV in the absence of a measurable humoral immune response. Colostrum or milk replacer fed calves were challenged with virulent BVDV at 2-5 weeks of age and/or after maternal antibody had waned. Calves exposed to BVDV while passive antibody levels were high did not mount a measurable humoral immune response to BVDV. However, compared to nonexposed animals, these animals had CD4+, CD8+, and γδ T lymphocytes that were activated by BVDV after exposure to in vitro BVDV. The production of IFNγ by lymphocytes after in vitro BVDV exposure was also much greater in lymphocytes from calves exposed to BVDV in the presence of maternal antibody compared to the nonexposed calves. These data indicate that calves exposed to BVDV while maternal antibody levels are high can develop antigen specific CD4+, CD8+, and γδ T lymphocytes in the absence of an active antibody response. A manuscript presented separately demonstrates that the calves with T lymphocytes specific for BVDV in this study were also protected from virulent BVDV genotype 2 challenge after maternal antibody became undetectable

cDNA Sequence and Fab Crystal Structure of HL4E10, a Hamster IgG Lambda Light Chain Antibody Stimulatory for γδ T Cells

Hamsters are widely used to generate monoclonal antibodies against mouse, rat, and human antigens, but sequence and structural information for hamster immunoglobulins is sparse. To our knowledge, only three hamster IgG sequences have been published, all of which use kappa light chains, and no three-dimensional structure of a hamster antibody has been reported. We generated antibody HL4E10 as a probe to identify novel costimulatory molecules on the surface of γδ T cells which lack the traditional αβ T cell co-receptors CD4, CD8, and the costimulatory molecule CD28. HL4E10 binding to γδ T cell, surface-expressed, Junctional Adhesion Molecule-Like (JAML) protein leads to potent costimulation via activation of MAP kinase pathways and cytokine production, resulting in cell proliferation. The cDNA sequence of HL4E10 is the first example of a hamster lambda light chain and only the second known complete hamster heavy chain sequence. The crystal structure of the HL4E10 Fab at 2.95 Å resolution reveals a rigid combining site with pockets faceted by solvent-exposed tyrosine residues, which are structurally optimized for JAML binding. The characterization of HL4E10 thus comprises a valuable addition to the spartan database of hamster immunoglobulin genes and structures. As the HL4E10 antibody is uniquely costimulatory for γδ T cells, humanized versions thereof may be of clinical relevance in treating γδ T cell dysfunction-associated diseases, such as chronic non-healing wounds and cancer

Osteogenesi imperfetta nell\u2019interpretazione del \u201cnon accidental injury\u201d : due casi a confronto

L\u2019osteogenesi imperfetta ha da sempre posto seri problemi di diagnosi differenziale con eventuali fratture di natura non accidentale, in quanto i soggetti affetti da tale condizione patologica tendono a sviluppare fratture multiple ed in epoche diverse, paragonabili ai casi di child physical abuse. Sebbene alcune forme di osteogenesi imperfetta siano riconoscibili anche clinicamente grazie ai diversi segni secondari associati, quali le sclere blu e la lassit\ue0 legamentosa, alcune forme, spesso di gravit\ue0 inferiore, mancano di tale obiettivit\ue0, e spesso si manifestano con una aspecifica fragilit\ue0 ossea. Risulta evidente in questi casi la difficolt\ue0 nel porre una diagnosi definitiva, che pu\uf2 essere eseguita efficacemente con l\u2019analisi del profilo di sintesi del collagene da colture di fibroblasti e i test genetici. L\u2019esistenza di forme di osteogenesi imperfetta caratterizzate unicamente dalla fragilit\ue0 ossea ha spinto alcuni autori a ipotizzare l\u2019esistenza di forme nosologiche transienti ad eziopatogenesi sconosciuta, come la TBBF (Temporary Brittle Bone Disease); il rischio dell\u2019utilizzo di forme nosologiche non ancora definite ed indimostrabili sul piano obiettivo \ue8 evidente, considerando l\u2019impatto di tale argomentazione in ambito giudiziario. Il presente studio ha l\u2019obiettivo di sottolineare le difficolt\ue0 legate alla diagnosi di lesione non accidentale nel contesto di un sospetto di osteogenesi imperfetta; nel primo, l\u2019evidenza di fratture multiple in epoche diverse ha fatto insorgere il sospetto dell\u2019origine non accidentale delle lesioni: la presenza dello stesso pattern fratturativo nella sorella gemella omozigote e l\u2019esecuzione dei test genetici ha consentito di evidenziare la diagnosi corretta di osteogenesi imperfetta. Nel secondo, l\u2019osservazione di multiple fratture ossee nel contesto di un ambiente familiare considerato non sospetto per child abuse ha posto in evidenza una possibile diagnosi di osteogenesi imperfetta, in assenza di qualsiasi altro segno secondario; l\u2019esito negativo delle indagini genetiche e colturali ha consentito di sostenere come ipotesi diagnostica l\u2019origine non accidentale. Dall\u2019analisi dei casi clinici esposti risulta pertanto di notevole importanza un approccio il pi\uf9 possibile aperto ad ogni ipotesi diagnostica in tali situazioni, oltre che il ruolo decisivo delle indagini genetiche e colturali per la diagnosi delle forme prive dei segni secondari, e pertanto non obiettivabili clinicamente

Attributes of γδ intraepithelial lymphocytes as suggested by their transcriptional profile

γδ T lymphocytes in the intestinal intraepithelial layer (γδ IELs) are thought to contribute to immune competence, but their actual function remains poorly understood. Here we used DNA microarrays to study the gene expression profile of γδ IELs in a Yersinia infection system to better define their roles. To validate this approach, mesenteric lymph node CD8(+) αβ T cells were similarly analyzed. The transcription profiles show that, whereas lymph node CD8(+) αβ T cells must be activated to become cytotoxic effectors, γδ IELs are constitutively activated and appear to use different signaling cascades. Our data suggest that γδ IELs may respond efficiently to a broad range of pathological situations irrespective of their diverse T cell antigen receptor repertoire. γδ IELs may modulate local immune responses and participate in intestinal lipid metabolism, cholesterol homeostasis, and physiology. This study provides a strong basis for further investigations of the roles of these cells as well as mucosal immune defense in general

A bad salad seasoning: When aconite confused with Couscouil

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