Selective recruitment of Th2-type cells and evasion from a cytotoxic immune response mediated by viral macrophage inhibitory protein-II

The viral CC chemokine macrophage inhibitory protein-II (vMIP-II) encoded by human herpes virus 8 (HHV-8) binds to multiple chemokine receptors, however, its ability to control the initial recruitment of specific leukocyte subtypes from the peripheral circulation has not been fully clarified. Here we show that vMIP-II blocks the firm arrest and transmigration of monocytes or Th1-like T lymphocytes triggered by RANTES immobilized on activated human microvascular endothelium (HMVEC) under flow conditions. The internalization of the receptors CCR1 and CCR5 that mediate arrest and transmigration of these cells in response to RANTES was prevented by vMIP-II, supporting its role as an antagonist of CCR1 and CCR5. In contrast, vMIP-II triggered the firm arrest of eosinophils and Th2-like T cells by engaging CCR3, as confirmed by its down-regulation. Immunohistochemical analysis of HHV-8-associated Kaposi's sarcoma lesions marked by vMIP-II expression and mononuclear cell infiltration revealed a predominance of Th2-type CCR3(+) lymphocytes over Th1-type CXCR3(+)/CCR5(+) leukocytes, indicating that as a CCR3 agonist vMIP-II can drive a Th2-type immune response in vivo. Thus, our data provide evidence for a immunomodulatory role of vMIP-II in directing inflammatory cell recruitment away from a Th1-type towards a Th2-type response and thereby facilitating evasion from cytotoxic reactions.     

Tetrodotoxin block of A-fibre afferents from skin and muscle - a tool to study pure C-fibre effects in the spinal cord

The properties of tetrodotoxin (TTX)-resistant C-fibre afferents of the dorsal roots were tested in Sprague-Dawley rats. Dorsal roots (L4-L6) were blocked with TTX (0.5-1 micro M) and the amplitude of the first response of the dorsal horn superficial interneurones (cord dorsum potential, CDP) to electrical stimulation of peripheral C-fibres in combination with natural noxious stimulation was taken as measure for intact conductivity of different kinds of noxious input by means of the C-fibre refractory period. After blockade of dorsal roots with TTX, formerly masked CDPs from muscle C-fibre afferents were uncovered. Noxious pressure to the gastrocnemius soleus muscle belly and noxious pinch to the calcanean tendon proved to be TTX resistant and therefore was propagated centrally. For cutaneous heat nociceptors it could also be shown that conductivity was intact after blockade of the dorsal roots with TTX. However, we could not exclude the TTX resistance of non-nociceptive receptors of muscle or skin. Nevertheless, blockade of afferents with TTX together with suitable stimulation techniques proves to be a reliable method to investigate central effects from C-fibre afferents without contaminating effects from A-fibres in the rat.     

Der Stoffwechsel bei vollständigem Fasten. Unterschiedliches Verhalten bei Männern und Frauen sowie bei Normalpersonen und Adipösen

Zusammenfassung Bei 24 idealgewichtigen und 24 adipösen Personen beiderlei Geschlechts wurden zirkulierende Substrate (Blutzucker, freie Fettsäuren, Ketonkörper) und Hormone (Insulin, Wachstumshormon, Pankreasglukagon) während sechstägigem Fasten bestimmt. Der Blutzucker sank bei Normalpersonen unter Nulldiät auf tiefere Werte als bei Adipösen. Der Konzentrationsanstieg der freien Fettsäuren und Ketonkörper erfolgte bei Normalgewichtigen rascher als bei Fettsüchtigen und bei Frauen rascher als bei Männern. Das Plasmainsulin fiel bei Adipösen stärker ab als bei Normalpersonen. Alle untersuchten Gruppen wiesen nach 1–3 Fastentagen einen signifikanten Anstieg des Pankreasglukagons auf, dieser erfolgte jedoch bei normalgewichtigen Frauen rascher als bei Männern. Wachstumshormon (GH) stieg bei normalgewichtigen Männern unter Nulldiät signifikant an, bei adipösen Männern jedoch nicht. Bei z.T. auffallend hohen Nüchternwerten zeigten normalgewichtige Frauen keinen signifikanten GH-Anstieg, jedoch signifikant höhere GH-Konzentrationen als adipöse Frauen nach 1–6 Fastentagen. Ausgehend von den allen untersuchten Gruppen gemeinsamen Stoffwechselveränderungen bei Nulldiät werden die diesbezüglichen Unterschiede zwischen männlichen und weiblichen sowie zwischen normalgesichtigen und adipösen Personen diskutiert.     

Directly bone-anchored implants for fixation of aural epistheses

In cases of missing outer ears, generally, epistheses are attached to eye-glasses which gives insecure attachment and a fairly poor cosmetic result. A new method for stable episthesis attachment is being tested in Gothenburg, Sweden. Threaded, cylindrical titanium implants are inserted with a meticulous technique in the temporal bone of the patient. At the next stage, 3–4 months later, the implants are connected to abutments which are allowed to penetrate the skin, To these abutments a silicone episthesis is attached. Presently, 7 patients who had no outer ear because of congenital disorders, hereditary diseases or status post trauma or tumour surgery have been operated and followed for up to $\text{1}\text{2}$ have been no problems reported with the bone anchorage or the skin penetration.     

Hevein-specific recombinant IgE antibodies from human single-chain antibody phage display libraries

IgE antibodies distinctively recognising allergenic epitopes would be ideal reagents in immunodiagnostics to detect and quantify allergens, as well as for the development of allergy diagnostics and therapeutics. We have isolated recombinant human IgE antibodies specific for the major latex allergen, hevein, from antibody phage display libraries using a green fluorescent protein (GFP)-hevein fusion as a selection antigen. Human IgE phage display libraries were constructed by combining the IgE heavy chain genes to kappa and lambda light-chain genes which were isolated from lymphocytes of a latex allergic patient. The screening of antibody libraries resulted in the enrichment of two hevein-binding scFvs designated as 1A4 and 1C2. Both antibodies showed specific binding to the hevein that could be inhibited by both the recombinant GFP-hevein and native hevein isolated from latex examination gloves. The scFvs were prone to aggregate and, thus, for further characterisation, they were converted to Fab fragments with human IgG1 or IgE isotype. Similar hevein-binding properties of the 1A4 and 1C2 Fab fragments and human IgE serum pool, conventionally used in the detection of latex allergens, demonstrate the potential utility of these recombinant antibodies for the analysis of latex allergen.     

Characterization of genomically amplified segments using PCR: optimizing relative-PCR for reliable and simple gene expression and gene copy analyses

Gene amplification is one of the mechanisms for oncogene activation in solid tumors. The size of the amplified regions may vary considerably among individual tumors, and more than one gene may be affected within the same amplicon. The main objective in analyzing genomic amplifications has therefore been to map the shortest region involved and to identify genes with increased expression as a result of the increased gene copy number. To facilitate such an analysis, we have developed simple polymerase chain reaction (PCR) procedures using the internal standards beta-actin (ACTB) and L1Hs for gene expression and gene copy number analyses, respectively. We used cDNA derived from pancreatic carcinoma cell lines, and genomic DNA extracted from the same cell lines, as templates in the gene expression and in the gene copy number analyses, respectively. To determine the optimal number of PCR cycles, dilution series of the templates were made. Furthermore, competing primers were used to adjust for differences in target sequence levels. We show that by these simple means it is possible to determine optimal conditions for expression analyses. In addition, the procedure was adapted for the analysis of gene copy number changes at the genomic level using L1Hs as the internal standard. This PCR method makes it possible to produce detailed gene copy number profiles of amplified genomic regions.     

Infection of human endothelial cells with Staphylococcus aureus induces transcription of genes encoding an innate immunity response

Staphylococcus aureus is a gram-positive bacterium frequently isolated from patients with bloodstream infections. Endothelial cells (EC) play an important role in host defence against bacteria, and recent reports have shown that infection of EC with S. aureus induces expression of cytokines and cell surface receptors involved in activating the innate immune response. The ability of S. aureus to invade nonphagocytic cells, including EC, has been documented. However, the knowledge of the role of EC in pathogenesis of S. aureus infection is still limited. In this study, we investigate the gene-expression program in human EC initiated by internalized S. aureus, using microarray analysis. We found 156 genes that were differentially regulated at least threefold, using arrays representing 14,239 genes. Many of the upregulated genes code for proteins involved in innate immunity, such as cytokines, chemokines and cell adhesion proteins. Other upregulated genes encode proteins involved in antigen presentation, cell signalling and metabolism. Furthermore, intracellular bacteria survived for days without inducing EC death.     

Temporal expression and cellular origin of CC chemokine receptors CCR1, CCR2 and CCR5 in the central nervous system: insight into mechanisms of MOG-induced EAE

Background  

The CC chemokine receptors CCR1, CCR2 and CCR5 are critical for the recruitment of mononuclear phagocytes to the central nervous system (CNS) in multiple sclerosis (MS) and other neuroinflammatory diseases. Mononuclear phagocytes are effector cells capable of phagocytosing myelin and damaging axons. In this study, we characterize the regional, temporal and cellular expression of CCR1, CCR2 and CCR5 mRNA in the spinal cord of rats with myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE). While resembling human MS, this animal model allows unique access to CNS-tissue from various time-points of relapsing neuroinflammation and from various lesional stages: early active, late active, and inactive completely demyelinated lesions.