Expression of sialyl-Lewis X, an E-selectin ligand, in inflammation, immune processes, and lymphoid tissues.

The carbohydrate structure sialyl-Lewis X (SLex) can function as a ligand for E-selectin, formerly known as endothelial leukocyte adhesion molecule-1 (ELAM-1). This study was performed to analyze the expression of SLex by leukocytes and other cell types in the context of inflammatory and immune processes. Human peripheral blood cells were examined by flow cytometry using monoclonal antibody CSLEX1 directed against SLex. Cell surface SLex was found in abundance on nearly all isolated polymorphonuclear leukocytes (PMN) and monocytes, and at low levels on a substantial portion (up to 40%) of natural killer cells. This moiety was expressed also on approximately 10% of peripheral blood T cells. Immunohistochemistry was performed on various human tissues involved in inflammatory or immune processes and on secondary lymphoid tissues. In acute appendicitis, endothelial cells of postcapillary venules expressed E-selectin, and most PMN, both within vessels and extravasated, expressed SLex. A substantial number of monocytes/macrophages in inflamed appendiceal, synovial, and dermal tissues also reacted with antibody CSLEX1; however, only rare tissue macrophages in uninflamed nonlymphoid sites showed expression of SLex. These observations are consistent with the concept that SLex on circulating PMN and monocytes functions as a ligand for endothelial E-selectin in the development of inflammatory reactions. SLex-positive lymphocytes also were seen, notably, T lymphocytes in inflamed skin. An unexpected finding was that the CSLEX1 antibody also reacted with venular endothelium in certain lymphoid tissues and in inflamed appendix, but not with endothelium in normal appendix. Whether the SLex antigen identified on endothelium represents de novo expression or passive adsorption remains to be determined.     

Markers of thrombin and plasmin generation in patients with inherited thrombophilia.

AIM--To determine the prevalence of a biochemically detectable hypercoagulable state, defined in terms of increased thrombin or plasmin generation, in patients with phenotypically characterised thrombophilia. METHODS--Plasma concentrations of the prothrombin activation peptide F1.2 and fibrin degradation (FbDP) and fibrinogen degradation products (FgDP) were measured by enzyme immunoassay in 104 patients deficient in natural anticoagulants, and 35 unaffected relatives. RESULTS--Increased concentrations of F1.2, FbDP, and FgDP were present in 18, 25, and 19 of 104 patients, respectively. There were no correlations between F1.2, FbDP, and FgDP concentrations, or between these parameters and concentrations of natural anticoagulants except for a negative correlation between protein C concentrations and FgDP (rho = -0.46, p = 0.009). CONCLUSION--A biochemically detectable hypercoagulable state is present in some patients with asymptomatic thrombophilia. Markers of plasmin generation may be increased more frequently in thrombophilia than markers of thrombin generation. This finding should prompt the inclusion of markers of plasmin generation in prospective longitudinal cohort studies to determine the predictive value of a hypercoagulable state, defined by either excessive thrombin or plasmin generation, for the development of venous thromboembolism.     

Cell responses in dorsal layers of macaque lateral geniculate nucleus as a function of intensity and wavelength

We studied the relationship between light intensity and cell response to various wavelengths and wavelength combinations in the dorsal, parvocellular layers of the macaque lateral geniculate nucleus. When response is plotted as a function of the logarithm of stimulus intensity, the slope and shape of curves depends on wavelength. For wavelengths near the crossover point between excitatory and suppressive responses, nonmonotonic curves are common. Consequently, the form of spectral-response functions depends on stimulus intensity. Responses to combined stimuli made up of wavelengths close together near one spectral extreme are approximately additive. If one wavelength is near the crossover point, responses are nonadditive so that a midspectral wavelength, only producing a weak excitatory response, is able to occlude more vigorous responses to wavelengths near the spectral ends. Responses of parvocellular layer cells are consistent with their being a result of linear interaction of opponent cone mechanisms, the response of each of which follows a modified hyperbolic tangent function (22). Responses to all wavelength combinations, even those showing strikingly nonadditive effects, could be predicted from the additive opponent model described above.     

Novel OCTN2 mutations: No genotype–phenotype correlations: Early carnitine therapy prevents cardiomyopathy

Primary systemic carnitine deficiency or carnitine uptake defect (OMIM 212140) is a potentially lethal, autosomal recessive disorder characterized by progressive infantile‐onset cardiomyopathy, weakness, and recurrent hypoglycemic hypoketotic encephalopathy, which is highly responsive to L ‐carnitine therapy. Molecular analysis of the SLC22A5 (OCTN2) gene, encoding the high‐affinity carnitine transporter, was done in 11 affected individuals by direct nucleotide sequencing of polymerase chain reaction products from all 10 exons. Carnitine uptake (at Km of 5 μM) in cultured skin fibroblasts ranged from 1% to 20% of normal controls. Eleven mutations (delF23, N32S, and one 11‐bp duplication in exon 1; R169W in exon 3; a donor splice mutation [IVS3+1 G > A] in intron 3; frameshift mutations in exons 5 and 6; Y401X in exon 7; T440M, T468R and S470F in exon 8) are described. There was no correlation between residual uptake and severity of clinical presentation, suggesting that the wide phenotypic variability is likely related to exogenous stressors exacerbating carnitine deficiency. Most importantly, strict compliance with carnitine from birth appears to prevent the phenotype. © 2002 Wiley‐Liss, Inc.     

Differentiation of mycobacterial species by PCR-restriction analysis of DNA (342 base pairs) of the RNA polymerase gene (rpoB)

PCR amplification-restriction analysis (PRA) of rpoB DNA (342 bp), which comprises the Rif(r) region, was used for the differential identification of 49 mycobacteria. The DNA had been used previously for the identification of mycobacterial species by comparative sequence analysis (B. J. Kim et al., J. Clin. Microbiol. 37:1714-1720, 1999). Digestion with four restriction enzymes (HaeIII, HindII, MvaI, and AccII), which were selected on the basis of rpoB DNA sequences, generated distinctive PRA patterns that allowed not only the reference strains but also the clinical isolates of mycobacteria to be distinguished. Both rapidly and slowly growing mycobacteria were distinctly differentiated by HaeIII digestion of the amplified rpoB DNA. By HindII digestion the Mycobacterium tuberculosis complex was distinguished from the other mycobacteria. Furthermore, six subspecies of Mycobacterium kansasii (subspecies I to VI) as well as the closely related Mycobacterium gastri, and other closely related species, were distinguished by simultaneous digestion of MvaI and AccII. According to the rpoB PRA scheme, 240 strains of clinical isolates could be identified. It was also possible to detect and identify M. tuberculosis directly from sputa and bronchoalveolar lavage specimens. These results suggest that PRA of rpoB DNA is a simple and feasible method not only for the differentiation of culture isolates but also for the rapid detection and identification of pathogenic mycobacteria in primary clinical specimens.     

Noninfectious Virus-Like Particle Antigen for Detection of Swine Vesicular Disease Virus Antibodies in Pigs by Enzyme-Linked Immunosorbent Assay

An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (n = 1,041). When tested using sera (n = 186) collected periodically from pigs (n = 19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed.     

Crystallization and Melting Behavior of Poly(3‐hydroxybutyrate)‐Based Blends

Summary: Blends of high molecular weight poly(R‐3‐hydroxybutyrate) (PHB) ( = 352 000 g · mol^?1), comprising of either low molecular weight poly(R‐3‐hydroxybutyrate) (D‐PHB) ( = 3 900 g · mol^?1) or poly[(R‐3‐hydroxybutyrate)‐co‐(R‐3‐hydroxyvalerate)] (PHBV) ( = 238 000 g · mol^?1) with 12 mol‐% hydroxyvalerate (HV) content as a second constituent, were investigated along with the thermal properties and morphologies. After isothermal crystallization, a lowering of the melting temperature of PHB can be observed with increasing content of the second component in the blends. This behavior points towards miscibility of the constituents both in the liquid and the solid state. Crystallization kinetics was studied under isothermal and non‐isothermal conditions. The overall kinetics of isothermal crystallization was analyzed in terms of the Avrami equation. Only one crystallization peak is observed in all cases for the PHB/D‐PHB and PHB/PHBV blends under the conditions studied. This demonstrates co‐crystallization of the constituents. The addition of D‐PHB or PHBV to PHB reduces the rate of crystallization of the blends compared to that of neat PHB. The corresponding activation energies of crystallization also decrease with an increasing concentration of the second constituent. Non‐isothermal crystallization, carried out with different cooling rates held constant, is discussed in terms of a quasi‐isothermal approach. The corresponding rate constants as functions of reciprocal undercooling show Arrhenius‐like behavior in a certain range of temperatures. At sufficiently high undercooling, the rate constants of crystallization for the isothermal process exceed those reflecting non‐isothermal conditions, whereas in the limit of low undercoolings, the rate constants become similar. Ring‐banded morphologies are observed when PHB is in excess. When the respective second component is the major component, fibrous textures of the spherulites develop.

Polarized micrograph of PHB/PHBV 90/10.     

Experimental extrinsic allergic alveolitis and pulmonary angiitis induced by intratracheal or intravenous challenge with Corynebacterium parvum in sensitized rats.

Extrinsic allergic alveolitis and pulmonary sarcoidosis are granulomatous diseases of the lung for which clinical presentation and anatomic site of granuloma formation differ. Extrinsic allergic alveolitis is caused by inhaled antigens, whereas the nature and source of the inciting antigen in sarcoidosis is unknown. To test the hypothesis that the route via which antigen is introduced to the lung contributes to the clinicopathological presentation of pulmonary granulomatous disease, rats immunized with intravenous (i.v.) Corynebacterium parvum were challenged after 2 weeks with either intratracheal (i.t.) or i.v. C. parvum. The granulomatous inflammation elicited by i.t. challenge predominantly involved alveolar spaces and histologically simulated extrinsic allergic alveolitis. In contrast, the inflammation induced by i.v. challenge was characterized by granulomatous angiitis and interstitial inflammation simulating sarcoidosis. Elevations of leukocyte counts and TNF levels in bronchoalveolar fluid, which reflect inflammation in the intra-alveolar compartment, were much more pronounced after i.t. than after i.v. challenge. Tumor necrosis factor, interleukin-6, CC chemokine, CXC chemokine, and adhesion molecule mRNA and protein expression occurred in each model. In conclusion, i.t. or i.v. challenge with C. parvum in sensitized rats caused pulmonary granulomatous inflammation that was histologically similar to human extrinsic allergic alveolitis and sarcoidosis, respectively. Although the soluble and cellular mediators of granulomatous inflammation were qualitatively similar in both disease models, the differing anatomic source of the same antigenic challenge was responsible for differing clinicopathological presentations.