Natalizumab Exerts Direct Signaling Capacity and Supports a Pro-Inflammatory Phenotype in Some Patients with Multiple Sclerosis

Natalizumab is a recombinant monoclonal antibody raised against integrin alpha-4 (CD49d). It is approved for the treatment of patients with multiple sclerosis (MS), a chronic inflammatory autoimmune disease of the CNS. While having shown high therapeutic efficacy, treatment by natalizumab has been linked to progressive multifocal leukoencephalopathy (PML) as a serious adverse effect. Furthermore, drug cessation sometimes induces rebound disease activity of unknown etiology. Here we investigated whether binding of this adhesion-blocking antibody to T lymphocytes could modulate their phenotype by direct induction of intracellular signaling events. Primary CD4⁺ T lymphocytes either from healthy donors and treated with natalizumab in vitro or from MS patients receiving their very first dose of natalizumab were analyzed. Natalizumab induced a mild upregulation of IL-2, IFN-γ and IL-17 expression in activated primary human CD4⁺ T cells propagated ex vivo from healthy donors, consistent with a pro-inflammatory costimulatory effect on lymphokine expression. Along with this, natalizumab binding triggered rapid MAPK/ERK phosphorylation. Furthermore, it decreased CD49d surface expression on effector cells within a few hours. Sustained CD49d downregulation could be attributed to integrin internalization and degradation. Importantly, also CD4⁺ T cells from some MS patients receiving their very first dose of natalizumab produced more IL-2, IFN-γ and IL-17 already 24 h after infusion. Together these data indicate that in addition to its adhesion-blocking mode of action natalizumab possesses mild direct signaling capacities, which can support a pro-inflammatory phenotype of peripheral blood T lymphocytes. This might explain why a rebound of disease activity or IRIS is observed in some MS patients after natalizumab cessation.     

Anti-idiotypic protein domains selected from protein A-based affibody libraries

Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.     

Climate‐driven build‐up of temporal isolation within a recently formed avian hybrid zone

Divergence in the onset of reproduction can act as an important source of reproductive isolation (i.e., allochronic isolation) between co‐occurring young species, but evidence for the evolutionary processes leading to such divergence is often indirect. While advancing spring seasons strongly affect the onset of reproduction in many taxa, it remains largely unexplored whether contemporary spring advancement directly affects allochronic isolation between young species. We examined how increasing spring temperatures affected onset of reproduction and thereby hybridization between pied and collared flycatchers (Ficedula spp.) across habitat types in a young secondary contact zone. We found that both species have advanced their timing of breeding in 14 years. However, selection on pied flycatchers to breed earlier was weaker, resulting in a slower response to advancing springs compared to collared flycatchers and thereby build‐up of allochronic isolation between the species. We argue that a preadaptation to a broader niche use (diet) of pied flycatchers explains the slower response to raising spring temperature, but that reduced risk to hybridize may contribute to further divergence in the onset of breeding in the future. Our results show that minor differences in the response to environmental change of co‐occurring closely related species can quickly cause allochronic isolation.     

A sparse neural code for some speech sounds but not for others

The precise neural mechanisms underlying speech sound representations are still a matter of debate. Proponents of 'sparse representations' assume that on the level of speech sounds, only contrastive or otherwise not predictable information is stored in long-term memory. Here, in a passive oddball paradigm, we challenge the neural foundations of such a 'sparse' representation; we use words that differ only in their penultimate consonant ("coronal" [t] vs. "dorsal" [k] place of articulation) and for example distinguish between the German nouns Latz ([lats]; bib) and Lachs ([laks]; salmon). Changes from standard [t] to deviant [k] and vice versa elicited a discernible Mismatch Negativity (MMN) response. Crucially, however, the MMN for the deviant [lats] was stronger than the MMN for the deviant [laks]. Source localization showed this difference to be due to enhanced brain activity in right superior temporal cortex. These findings reflect a difference in phonological 'sparsity': Coronal [t] segments, but not dorsal [k] segments, are based on more sparse representations and elicit less specific neural predictions; sensory deviations from this prediction are more readily 'tolerated' and accordingly trigger weaker MMNs. The results foster the neurocomputational reality of 'representationally sparse' models of speech perception that are compatible with more general predictive mechanisms in auditory perception.     

Herpes simplex virus type 1/adeno-associated virus hybrid vectors mediate site-specific integration at the adeno-associated virus preintegration site,AAVS1, on human chromosome 19

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 vectors is their instability of transgene expression. By contrast, vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four rep genes, rep68, -78, -40, and -52. Of those, rep68 or rep78 are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV vectors is the small transgene capacity ( approximately 4.6 kb). In this study, we constructed HSV/AAV hybrid vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV rep68, rep78, both rep68 and -78, or all four rep genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the hybrid vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the hybrid vector. All hybrid vectors could be packaged into HSV-1 virions, although those containing rep sequences had lower titers than vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in hybrid vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette, rep in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV hybrid vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.     

Phosphate dependent,ruthenium red insensitive Ca2+ uptake in mung bean mitochondria

Energy linked Ca²⁺ uptake into mung bean mitochondria has been studied. Using arsenazo III as a monitor of extramitochondrial Ca²⁺, we observe a respiration-linked uptake of Ca²⁺ which requires phosphate and is insensitive to ruthenium red. The rate of uptake is of the order of 5 nmol/mg protein/min. Acetate, sulphate and thiosulphate are unable to support Ca²⁺ uptake. The results suggest that although plant mitochondria accumulate Ca²⁺ in an energy dependent fashion, it is not via a simple electrophoretic uniport mechanism.     

Experimentally increased nest temperature affects body temperature,growth and apparent survival in blue tit nestlings

The thermal environment experienced by birds during early postembryonic development may be an important factor shaping growth and survival. However, few studies have directly manipulated nest temperature (T _n) during the nestling phase, and none have measured the consequences of experimental heat stress on nestlings’ body temperature (T _b). It is therefore not known to what extent any fitness consequences of development in a thermally challenging environment arise as a direct, or indirect, effect of heat stress. We, therefore, studied how experimentally increased T _n affected T _b in 8–12 d old blue tit Cyanistes caeruleus nestlings, to investigate if increased thermoregulatory demands to maintain normothermic T _b influenced nestling growth and apparent long‐term survival. Nestlings in heated nest‐boxes had significantly higher T _b compared to unheated nestlings during most of the experimental period. Yet, despite facing T _n  50°C (as measured in the bottom of the nest cup below the nestlings), the highest nestling T _b recorded was 43.8°C with nestlings showing evidence of controlled facultative hyperthermia without any increased nestling mortality in heated nests. However, body mass gain was lower in these nestlings compared to nestlings from control nest‐boxes. Contrary to our prediction, a larger proportion of nestlings from heated nest‐boxes were recaptured during their first winter, or subsequently recruited into the breeding population as first‐ or second‐year breeders. This result should, however, be treated with caution because of low recapture rates. This study highlights the importance of the thermal environment during nestling development, and its role in shaping both growth patterns and possibly also apparent survival.     

Oxidation of ω-hydroxylated fatty acids and steroids by SS-isoenzyme of liver alcohol dehydrogenase

The SS-isoenzyme of alcohol dehydrogenase from horse liver was found to be active towards ω1- and ω2-hydroxylated fatty acids, an ω-hydroxylated steroid, ethanol and a 3β-hydroxysteroid. The main part of all these activities disappeared after carboxymethylation of a cysteineresidue at the active site of LADH_SS. The ω-hydroxyfatty acid dehydrogenase activity of LADH_SS was of similar magnitude as that of LADH_EE whereas the ω-hydroxysteroid dehydrogenase activity of LADH_SS was considerably higher than that of LADH_EE.