Increased circulating interleukin-12 (IL-12) p40 in pulmonary sarcoidosis

In sarcoidosis, a T helper 1 (Th1) response is an essential event and the up-regulation of interleukin-12 (IL-12) has been detected in affected disease sites. In order to investigate the clinical usefulness of circulating IL-12, we measured the serum concentrations of IL-12 by ELISA and performed immunohistochemistry using specific MoAbs for IL-12 in the lungs and scalene lymph nodes of patients with sarcoidosis. The serum concentration of IL-12 p40 was detectable in all 45 patients with pulmonary sarcoidosis and 18 normal controls, whereas that of IL-12 p70 was undetectable. The serum concentrations of IL-12 p40 in pulmonary sarcoidosis were significantly higher than those of the normal controls, especially in cases with abnormal intrathoracic findings detected by chest roentogenogram. The serum concentrations of interferon-gamma (IFN-gamma) also increased compared with those of normal controls and there was a significant positive correlation between the serum concentrations of IL-12 p40 and IFN-gamma. Furthermore, serum angiotensin-converting enzyme (ACE) and lysozyme, which are known to be useful markers for disease activity in sarcoidosis, correlated well with the serum concentrations of IL-12 p40. The positive 67Ga scan group (for lung field) had significantly elevated serum IL-12 p40 levels compared with those of the negative group. No bioactivity of IL-12 p70 was detected in three sarcoid cases sera by using the IL-12 responsive cell line. Finally, the immunohistochemical approach revealed that IL-12 p40 was expressed in the epithelioid cells and macrophages of sarcoid lungs and lymph nodes. We concluded that the production of IL-12 p40 was far greater in the sera and we have demonstrated this to be a useful clinical marker for disease activity and the Th1 response in pulmonary sarcoidosis.     

Indirect enzyme‐linked Immunosorbent assay for detection of cow's milk in goat's milk

An indirect enzyme‐linked immunosorbent assay (ELISA) has been developed for the specific detection of cow's milk (1–25%) in goat's milk. The test uses polyclonal antibodies raised in rabbits against bovine whey proteins (BWP). The anti‐BWP antibodies were recovered from the crude antiserum by immunoadsorption and elution from a column containing immobilized BWP. The anti‐BWP antibodies were biotinylated and rendered cow's milk specific by mixing them with lyophilized ovine and caprine whey proteins. Streptavidin‐peroxidase was used to detect the biotinylated anti‐BWP antibodies bound to bovine milk proteins immobilized on 96‐well plates. The colour developed by the subsequent enzymic conversion of the substrate gave clear absorbance differences when assaying mixtures of goat's milk containing variable amounts of cow's milk.     

Molecular and biological analysis of echovirus 9 strain isolated from a diabetic child

The full-length infectious cDNA clone was constructed and sequenced from the strain DM of echovirus 9, which was recently isolated from a 6-week-old child at the clinical onset of type 1 diabetes. Parallel with the isolate DM, the full-length infectious cDNA clone of the prototype strain echovirus 9 Barty (Barty-INF), was constructed and sequenced. Genetic relationships of the sequenced echo 9 viruses to the other members of the human enterovirus type B species were studied by phylogenetic analyses. Comparison of capsid protein sequences showed that the isolate DM was closely related to both prototype strains: Hill and Barty-INF. The only exception was the inner capsid protein VP4 where serotype specificity was not evident and the isolate DM clustered with the strain Hill and the strain Barty-INF with echovirus 30 Bastianni. Likewise, the nonstructural protein coding region, P2P3, of isolate DM was more similar to strain Hill than to strain Barty-INF. However, like echovirus 9 Barty, the isolate DM contained the RGD-motif in the carboxy terminus of capsid protein VP1. By blocking experiments using an RGD-containing peptide and a polyclonal rabbit antiserum to the alpha(v)beta(3)-integrin, it was shown that this molecule works as a cellular receptor for isolate DM. By using primary human islets, it was shown that the isolate DM is capable of infecting insulin-producing beta-cells like the corresponding prototype strains did. However, only isolate DM was clearly cytolytic for beta-cells. The infectious clones that were made allow further investigations of the molecular features responsible for the diabetogenicity of the isolate DM.     

AMPK as a metabolic switch in rat muscle,liver and adipose tissue after exercise

An increasing body of evidence has revealed that activation of adenosine monophosphate (AMP)‐activated protein kinase (AMPK)‐activated protein kinase increases fatty acid oxidation by lowering the concentration of malonyl coenzyme A (CoA), an inhibitor of carnitine palmitoyl transferase 1. Studies carried out primarily in skeletal muscle suggest that AMPK modulates the concentration of malonyl CoA by concurrently phosphorylating and inhibiting acetyl CoA carboxylase (ACC), the rate limiting enzyme in malonyl CoA synthesis, and phosphorylating and activating malonyl CoA decarboxylase (MCD), an enzyme involved in its degradation. We have recently observed that AMPK and MCD activities are increased and ACC activity diminished in skeletal muscle, liver and, surprisingly, in adipose tissue 30 min following exercise (treadmill run) in normal rats. In liver and adipose tissue these changes were associated with a decrease in the activity of glycerol‐3‐phosphate acyltransferase (GPAT), which catalyses the first committed reaction in glycerolipid synthesis and, which like ACC, is phosphorylated and inhibited by AMPK. Similar changes in ACC, MCD and GPAT were observed following the administration of 5‐aminoimidazole 4‐carboxamide‐riboside (AICAR), further indicating that the exercise‐induced alterations in these enzymes were AMPK‐mediated. Conclusions: (1) AMPK plays a major role in regulating lipid metabolism in multiple tissues following exercise. (2) The net effect of its activation is to increase fatty acid oxidation and diminish glycerolipid synthesis. (3) The relevance of these findings to the regulation of muscle glycogen repletion in the post‐exercise state and to the demonstrated ability of AMPK activation to decrease adiposity and increase insulin sensitivity in rodents remains to be determined.     

gp49B1 suppresses stem cell factor-induced mast cell activation-secretion and attendant inflammation in vivo

We report that gp49B1, a mast cell membrane receptor with two immunoreceptor tyrosine-based inhibitory motifs (ITIM), constitutively inhibits mast cell activation-secretion induced by stem cell factor (SCF), a tissue-derived cytokine that also regulates mast cell development. The intradermal injection of SCF into the ears of gp49B1 null (gp49B(-/-)) mice elicited approximately 4- and 2.5-fold more degranulating mast cells and tissue swelling caused by edema, respectively, than in gp49B(+/+) mice. SCF did not induce tissue swelling in mast cell-deficient mice, and the responsiveness of gp49B(-/-) mice to mast cell-associated amine and lipid mediators was unaltered. When gp49B(+/+) and gp49B(-/-) mice were pretreated with antagonists of the amines, SCF-induced tissue swelling was reduced by >90% and 60%, respectively, and it was reduced by >90% in both genotypes when a cysteinyl leukotriene receptor antagonist was also provided. Hence, the dominant contribution of secretory granule amines to SCF-induced tissue swelling is the result of gp49B1-mediated inhibition of the production of cysteinyl leukotrienes by mast cells. Our findings also provide the first example of an ITIM-bearing receptor that constitutively suppresses inflammation generated in vivo independently of the adaptive immune response by a receptor that signals through intrinsic tyrosine kinase activity rather than immunoreceptor tyrosine-based activation motifs.     

Inhalation anesthetic-induced neuroinvasion by an attenuated strain of West Nile virus in mice

There are contradictory reports regarding the effects of inhalation anesthetics on the immune system. Measurable immune responses have been studied in vitro, but little is known about the in vivo effects in the intact organism. We used an attenuated, non-neuroinvasive, nonlethal strain of the encephalitic West Nile virus, termed WN-25, which can become lethal in combination with environmental stressors, to study possible modulatory immune effects of inhalation anesthetics in mice. Both single short-term exposure and repeated exposure to halothane and nitrous oxide were studied. Exposure to 30% CO2 served as a positive control. Mortality, brain invasion, spleen weight, and antiviral antibodies served as the experimental endpoints. Halothane and nitrous oxide led to viral brain invasion, increased mortality, and suppressed immune response in a concentration- and time-dependent manner. Repeated exposures had a cumulative effect. Assessment of the stability of the viral attenuation did not demonstrate any alteration in the character of the virus, suggesting an increased access to the brain by inhalation anesthetics that led to the fatal encephalitis. These findings may be of special concern to populations at risk, such as operating room staff and patients undergoing general anesthesia in endemic areas of encephalitic virus species, in which subclinical infection may develop into an overt disease.     

IL-4 and IL-10 modulate autoimmune haemolytic anaemia in NZB mice

New Zealand Black (NZB) mice spontaneously develop autoimmune haemolytic anaemia (AIHA). Here the effect of injecting NZB mice with plasmids encoding IL-4 (pIL-4) or IL-10 (pIL-10) on NZB disease was tested. Both constructs delayed the development of anaemia as judged by increased haematocrit values as compared with controls, but neither altered the IgG1 to IgG2 red blood cell (RBC) bound autoantibody levels. The increased haematocrit value was associated temporally with increased RBC bound IgG in NZB mice treated with pIL-10, but not pIL-4. By contrast, up-regulation of splenic macrophage FcgammaRIIb2 mRNA was associated temporally with increased haematocrit values in NZB mice given pIL-4. However, no such increase occurred in NZB mice that inhaled a peptide containing a dominant T-cell epitope, although this treatment is known to bias the autoimmune response towards Th2 and to reduce the severity of anaemia. It is considered that IL-4 treatment, in part, ameliorates NZB anaemia by increasing the expression of the inhibitory FcgammaRIIb2 and thereby reducing the capacity of splenic macrophages to phagocytose autoantibody coated RBC, but that this mechanism does not explain the beneficial effects of the inhaled peptide.     

Morphology and Thermal Properties of MCPA6/ABS by in situ Polymerization of ε‐Caprolactam

Summary: In this work, blends of monomer casting polyamide 6 (MCPA6) and acrylonitrile‐butadiene‐styrene (ABS) were successfully prepared by in situ polymerization via the application of ε‐caprolactam as a reactive solvent. The morphology and thermal properties of MCPA6/ABS were investigated by means of wide angle X‐ray diffraction (WAXD), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM), respectively. The domain sizes of the ABS phase in MCPA6/ABS blends were much finer than those in corresponding polyamide 6 (PA6)/ABS blends prepared by simple melt blending. With an increased amount of ABS in MCPA6, the melt enthalpy (ΔH_f), the rate of crystallization (T_c) and the degree of crystallinity (X_c(DSC)) of MCPA6 in MCPA6/ABS blends were all decreased. The degree of supercooling (ΔT_d) showed a contrary trend. However, the melting temperatures of these blends were almost unchanged. All the results could be attributed to in situ polymerization and the hydrolysis reaction of ABS that occurred during the polymerization process. Furthermore, WAXD results showed that only α‐form crystals existed in the MCPA6/ABS blends, despite the ABS content and heat treatment.

SEM micrograph of the fractured surface of an MCPA6/ABS blend with an ABS content of 20 wt.‐% (×10 000).     

Multidimensional Chromatographic and Hyphenated Techniques for Hydrophilic Copolymers, 1

Summary: The chromatographic analysis of hydrophilic copolymers is complicated due to the fact that in most cases aqueous eluents must be used. In aqueous eluents different polar and ionic effects may disturb the selective interactions between the macromolecules and the stationary phase making it impossible to separate such copolymers with regard to chemical composition. Therefore, 2D chromatography combining a separation according to composition with a separation according to molar mass has been applied mostly to polymers that are soluble in organic solvents. The present contribution describes experimental approaches to analyze such hydrophilic copolymers by 2D‐chromatography. For a model polymer system resulting from the copolymerization of methacrylic acid and a poly(ethylene glycol) macromonomer, it is shown that different analytical techniques including SEC, LC‐CC, MALDI‐TOF MS and 2D chromatography can be used to analyze the different parameters of molecular heterogeneity of such copolymers.

2D separation of poly(MPEG‐MM 2), 1^st dimension: LC‐CC, 2^nd dimension: SEC.