A novel SERPINA12 variant and first European patients with diffuse palmoplantar keratoderma.

BACKGROUND: Hereditary palmoplantar keratodermas (hPPKs) comprise a heterogeneous group of skin disorders characterized by persistent palmoplantar hyperkeratosis. Loss-of-function variants in a serine peptidase inhibitor, SERPINA12, have recently been implicated in autosomal recessive diffuse hPPK. The disorder appears to share similarities with another hPPK associated with protease overactivity, namely Nagashima-type PPK (NPPK) caused by biallelic variants in SERPINB7. OBJECTIVES: The aim of this study was to enhance the understanding of the clinical and genetic characteristics of serine protease-related hPPKs caused by variants in SERPINA12 and SERPINB7. METHODS: Whole-exome sequencing (WES) was performed for hPPK patients. Haplotype analysis was completed for the patients with identified recessive SERPINA12 variants and their available family members. In addition, the current literature of SERPINA12- and SERPINB7-related hPPKs was summarized. RESULTS: The phenotype of SERPINA12-related hPPK was confirmed by reporting three new SERPINA12 patients, the first of European origin. A novel SERPINA12 c.1100G>A p.(Gly367Glu) missense variant was identified confirming that the variant spectrum of SERPINA12 include both truncating and missense variants. The previously reported SERPINA12 c.631C>T p.(Arg211*) was indicated enriched in the Finnish population due to a plausible founder effect. In addition, SERPINA12 hPPK patients were shown to share a similar phenotype to patients with recessive variants in SERPINB7. The shared phenotype included diffuse transgradient PPK since birth or early childhood and frequent palmoplantar hyperhidrosis, aquagenic whitening and additional hyperkeratotic lesions in non-palmoplantar areas. SERPINA12 and SERPINB7 hPPK patients cannot be distinguished without genetic analysis. CONCLUSIONS: Recessive variants in SERPINA12 and SERPINB7 leading to protease overactivity and hPPK produce a similar phenotype, indistinguishable without genetic analysis. SERPINA12 variants should be assessed also in non-Asian patients with diffuse transgradient PPK. Understanding the role of serine protease inhibitors will provide insights into the complex proteolytic network in epidermal homeostasis.

Uncovering the genetic architecture of broad antisocial behavior through a genome-wide association study meta-analysis.

Despite the substantial heritability of antisocial behavior (ASB), specific genetic variants robustly associated with the trait have not been identified. The present study by the Broad Antisocial Behavior Consortium (BroadABC) meta-analyzed data from 28 discovery samples (N = 85,359) and five independent replication samples (N = 8058) with genotypic data and broad measures of ASB. We identified the first significant genetic associations with broad ASB, involving common intronic variants in the forkhead box protein P2 (FOXP2) gene (lead SNP rs12536335, p = 6.32 × 10-10). Furthermore, we observed intronic variation in Foxp2 and one of its targets (Cntnap2) distinguishing a mouse model of pathological aggression (BALB/cJ strain) from controls (BALB/cByJ strain). Polygenic risk score (PRS) analyses in independent samples revealed that the genetic risk for ASB was associated with several antisocial outcomes across the lifespan, including diagnosis of conduct disorder, official criminal convictions, and trajectories of antisocial development. We found substantial genetic correlations of ASB with mental health (depression rg = 0.63, insomnia rg = 0.47), physical health (overweight rg = 0.19, waist-to-hip ratio rg = 0.32), smoking (rg = 0.54), cognitive ability (intelligence rg = -0.40), educational attainment (years of schooling rg = -0.46) and reproductive traits (age at first birth rg = -0.58, father's age at death rg = -0.54). Our findings provide a starting point toward identifying critical biosocial risk mechanisms for the development of ASB.

The Golgi associated ERI3 is a Flavivirus host factor.

Dengue virus (DENV) is a mosquito-borne Flavivirus classified into four serotypes (DENV-1-4) that causes Dengue fever (DF), Dengue hemorrhagic Fever (DHF) or Dengue shock syndrome (DSS). An estimated 390 million people are at risk for infection with DENV and there are no effective vaccines or therapeutics. We utilized RNA chromatography coupled with quantitative mass spectrometry (qMS) to identify host RNA binding proteins (RBPs) that interact with DENV-2 RNA. We identified ERI3 (also PRNPIP and PINT1), a putative 3'-5' RNA exonuclease, which preferentially associates with DENV-2 genomic RNA via interactions with dumbbell structures in the 3' UTR. ERI3 is required for accumulation of DENV-2 genomic RNA and production of infectious particles. Furthermore, the mosquito homologue of ERI3 is required for DENV-2 replication in adult Aedes aegypti mosquitos implying that the requirement for ERI3 is conserved in both DENV hosts. In human cells ERI3 localizes to the Golgi in uninfected cells, but relocalizes near sites of DENV-2 replication in infected cells. ERI3 is not required for maintaining DENV-2 RNA stability or translation of the viral polyprotein, but is required for viral RNA synthesis. Our results define a specific role for ERI3 and highlight the importance of Golgi proteins in DENV-2 replication.

Recurrence rate after absorbable tack fixation of mesh in laparoscopic incisional hernia repair.

BACKGROUND: The mesh fixation technique in laparoscopic incisional hernia repair may influence the rates of hernia recurrence and chronic pain. This study investigated the long-term risk of recurrence and chronic pain in patients undergoing laparoscopic incisional hernia repair with either absorbable or non-absorbable tacks for mesh fixation. METHODS: This was a nationwide consecutive cohort study based on data collected prospectively concerning perioperative information and clinical follow-up. Patients undergoing primary, elective, laparoscopic incisional hernia repair with absorbable or non-absorbable tack fixation during a 4-year interval were included. Follow-up was by a structured questionnaire regarding recurrence and chronic pain, supplemented by clinical examination, and CT when indicated. Recurrence was defined as either reoperation for recurrence or clinical/radiological recurrence. RESULTS: Of 1037 eligible patients, 84·9 per cent responded to the questionnaire, and 816 were included for analysis. The median observation time for the cohort was 40 (range 0-72) months. The cumulative recurrence-free survival rate was 71·5 and 82·0 per cent after absorbable and non-absorbable tack fixation respectively (P = 0·007). In multivariable analysis, the use of absorbable tacks was an independent risk factor for recurrence (hazard ratio 1·53, 95 per cent c.i. 1·11 to 2·09; P = 0·008). The rate of moderate or severe chronic pain was 15·3 and 16·1 per cent after absorbable and non-absorbable tack fixation respectively (P = 0·765). CONCLUSION: Absorbable tack fixation of the mesh was associated with a higher risk of recurrence than non-absorbable tacks for laparoscopic mesh repair of incisional hernia, but did not influence chronic pain.

Diesel exhaust particle exposure increases severity of allergic asthma in young mice.

BACKGROUND: Epidemiologic studies have reported an association between diesel exhaust particle (DEP) exposure, allergic sensitization, and childhood wheezing, although the mechanisms remain unclear. While DEP is known to augment allergic responses in adult animal models, its effects on sensitization and asthma severity in young animals is unknown. OBJECTIVE: To examine the impact of different doses of DEP and allergen co-exposure on allergic sensitization and asthma characteristics in young mice, and whether Th17 as well as Th2 responses are induced. METHODS: Lungs of 3-week-old wild-type Balb/c mice were exposed by pharyngeal aspiration nine times over 3 weeks to DEP at 1.2 or 6.0 mg/kg body weight, house dust mite (HDM) at 0.8, 1.2 or 6.0 mg/kg of DEP in combination with HDM, or the same volume (50 µL) of 0.9% sterile saline. RESULTS: In young mice, exposure to 1.2 mg/kg of DEP caused no detectable lung inflammation, but 6.0 mg/kg of DEP induced neutrophilic influx. Compared to HDM or DEP alone, mice exposed to either dose of DEP together with HDM demonstrated increased allergen-specific IgE, lung inflammation, airway hyperreactivity, goblet cell metaplasia, Th2/Th17 cytokines, dendritic cells, activated T cells, effector T cells, and IL-17(pos) and IL-13(pos) /IL-17A(pos) T effector cells. CONCLUSIONS AND CLINICAL RELEVANCE: In young mice, co-exposure to DEP and HDM together exacerbated allergic sensitization and induced key characteristics of more severe asthma, including IL-17A, IL-17(pos) and IL-13(pos) /IL-17A(pos) T effector cells. While exposure to 1.2 mg/kg DEP alone caused no detectable changes, it did exacerbate allergic sensitization and asthma characteristics to a similar degree as a five-fold higher dose of DEP. This study demonstrates that exposure to DEP, even at a dose that alone causes no inflammation, exacerbates allergic asthma in young animals and suggests the importance of preventive measures to reduce the exposure of children to traffic related air pollution.

Running sutures anchored with square knots are unreliable.

BACKGROUND: A previous study showed that running polypropylene sutures anchored with square knots retain only 75% of their strength compared with half hitches. The aim of this study was to investigate whether anchor knot geometry similarly affects the tensile strength of other types of sutures used in continuous closures. METHODS: Monofilament and multifilament sutures (all 3-0) were anchored with either square knots or half hitches to 1 tensionometer post, and the running ends were secured to the other. The force required to break the running suture and the site of suture failure were recorded. RESULTS: The running sutures anchored with square knots retained only 50% to 84% of the strength of the identical sutures secured with half hitches (P < .001). CONCLUSIONS: A running suture anchored with half hitches is stronger and safer in comparison with the same suture anchored with square knots. This study provokes a fundamental reconsideration of the use of square knots to anchor running sutures.

Human cell-conditioned media produced under embryonic-like conditions result in improved healing time after laser resurfacing.

BACKGROUND: Laser resurfacing procedures are continuing to grow in popularity as patients select less invasive procedures for rejuvenation of photo-damaged and aging skin. However, although physicians have begun exploring options to aid in postlaser healing, currently available treatments have little clinical evidence to support their use for wounded skin. METHODS: When grown under conditions of very low oxygen and suspension, a simulation of the embryonic environment, neonatal cells have been found to produce proteins and growth factors in types and quantities similar to those of fetal cells. The human cell-conditioned media (hCCM) produced by the cells was extracted and formulated into a gel to evaluate its efficacy in the healing of postlaser wounds. RESULTS: A split-face clinical evaluation of the material was performed, with 42 subjects undergoing combination ablative and nonablative laser procedures. Three concentrations of the hCCM were tested (× 0.1, × 1.0, × 10.0), and a dose-response trend was seen in the blinded physician evaluation, particularly in the assessment of crusting. In addition, transepidermal water loss readings showed a significant difference (p ≤ 0.05), indicating a more rapid return to normal skin barrier function with the active treatment. Histopathologic evaluation of subject biopsies showed reduced inflammation and a more normal epidermal appearance in the active treatment sites. CONCLUSIONS: The results of this clinical evaluation support the use of the soluble hCCM produced under embryonic-like conditions to accelerate wound healing after laser resurfacing procedures. The utility of the × 10 concentration appears to promote more rapid, scarless wound healing after resurfacing procedures and more normal skin recovery.

Mitigation of patient harm from testing errors in family medicine offices: a report from the American Academy of Family Physicians National Research Network.

OBJECTIVES: Little research has focused on preventing harm from errors that occur in primary care. We studied mitigation of patient harm by analysing error reports from family physicians' offices. METHODS: The data for this analysis come from reports of testing process errors identified by family physicians and their office staff in eight practices in the American Academy of Family Physicians National Research Network. We determined how often reported error events were mitigated, described factors related to mitigation and assessed the effect of mitigation on the outcome of error events. RESULTS: We identified mitigation in 123 (21%) of 597 testing process event reports. Of the identified mitigators, 79% were persons from inside the practice, and 7% were patients or patient's family. Older age was the only patient demographic attribute associated with increased likelihood of mitigation occurring (unadjusted OR 18-44 years compared with 65 years of age or older = 0.27; p = 0.007). Events that included testing implementation errors (11% of the events) had lower odds of mitigation (unadjusted OR = 0.40; p = 0.001), and events containing reporting errors (26% of the events) had higher odds of mitigation (unadjusted OR = 1.63; p = 0.021). As the number of errors reported in an event increased, the odds of that event being mitigated decreased (unadjusted OR = 0.58; p = 0.001). Multivariate logistic regression showed that an event had higher odds of being mitigated if it included an ordering error or if the patient was 65 years of age or older, and lower odds of being mitigated if the patient was between age 18 and 44, or if the event included an implementation error or involved more than one error. Mitigated events had lower odds of patient harm (unadjusted OR = 0.16; p<0.0001) and negative consequences (unadjusted OR = 0.28; p<0.0001). Mitigated events resulted in less severe and fewer detrimental outcomes compared with non-mitigated events. CONCLUSION: Nearly a quarter of testing process errors reported by family physicians and their staff had evidence of mitigation, and mitigated errors resulted in less frequent and less serious harm to patients. Vigilance throughout the testing process is likely to detect and correct errors, thereby preventing or reducing harm.

Testing process errors and their harms and consequences reported from family medicine practices: a study of the American Academy of Family Physicians National Research Network.

CONTEXT: Little is known about the types and outcomes of testing process errors that occur in primary care. OBJECTIVE: To describe types, predictors and outcomes of testing errors reported by family physicians and office staff. DESIGN: Events were reported anonymously. Each office completed a survey describing their testing processes prior to event reporting. SETTING AND PARTICIPANTS: 243 clinicians and office staff of eight family medicine offices. MAIN OUTCOME MEASURES: Distribution of error types, associations with potential predictors; predictors of harm and consequences of the errors. RESULTS: Participants submitted 590 event reports with 966 testing process errors. Errors occurred in ordering tests (12.9%), implementing tests (17.9%), reporting results to clinicians (24.6%), clinicians responding to results (6.6%), notifying patient of results (6.8%), general administration (17.6%), communication (5.7%) and other categories (7.8%). Charting or filing errors accounted for 14.5% of errors. Significant associations (p<0.05) existed between error types and type of reporter (clinician or staff), number of labs used by the practice, absence of a results follow-up system and patients' race/ethnicity. Adverse consequences included time lost and financial consequences (22%), delays in care (24%), pain/suffering (11%) and adverse clinical consequence (2%). Patients were unharmed in 54% of events; 18% resulted in some harm, and harm status was unknown for 28%. Using multilevel logistic regression analyses, adverse consequences or harm were more common in events that were clinician-reported, involved patients aged 45-64 years and involved test implementation errors. Minority patients were more likely than white, non-Hispanic patients to suffer adverse consequences or harm. CONCLUSIONS: Errors occur throughout the testing process, most commonly involving test implementation and reporting results to clinicians. While significant physical harm was rare, adverse consequences for patients were common. The higher prevalence of harm and adverse consequences for minority patients is a troubling disparity needing further investigation.

Collimonas arenae sp. nov. and Collimonas pratensis sp. nov., isolated from (semi-)natural grassland soils.

A polyphasic taxonomic study was performed to compare 26 novel bacterial isolates obtained from (semi-)natural grassland soils and a heathland soil in the Netherlands with 16 strains that had previously been assigned to the genus Collimonas. Genomic fingerprinting (BOX-PCR), whole-cell protein electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry of intact cells and physiological characterization (Biolog) of the isolates confirmed the existence of different strain clusters (A-D) within the genus Collimonas. Until now, only cluster C strains have been formally classified, as Collimonas fungivorans. In this study, DNA-DNA hybridizations were performed with a selection of strains representing the four clusters. The results showed that cluster B strains also belong to C. fungivorans and that strains of clusters A and D represent two novel species within the genus Collimonas. The latter novel species could be differentiated by means of phenotypic and genotypic characteristics and are classified as Collimonas arenae sp. nov. (cluster A; type strain Ter10(T) =LMG 23964(T) =CCUG 54727(T)) and Collimonas pratensis sp. nov. (cluster D; type strain Ter91(T) =LMG 23965(T) =CCUG 54728(T)).

Antimicrobial susceptibility of Bifidobacterium strains from humans, animals and probiotic products.

OBJECTIVES: The aim of this study was to assess the antimicrobial susceptibility of a taxonomically diverse set of Bifidobacterium strains to different classes of antimicrobial agents using a recently described medium. METHODS: The susceptibility of 100 strains encompassing 11 bifidobacterial species originating from humans, animals and probiotic products to 12 antimicrobial agents was tested by agar overlay disc diffusion. Based on these results, one or two strains per species were selected for susceptibility testing to nine antibiotics by broth microdilution using the Lactic acid bacteria Susceptibility test Medium (LSM) supplemented with cysteine. The genotypic basis of atypical tetracycline resistance was further characterized using PCR, Southern blotting and partial sequencing. RESULTS: Based on the distribution of inhibition zone diameters and MIC values, all strains tested were susceptible to amoxicillin, chloramphenicol, erythromycin, quinupristin/dalfopristin, rifampicin and vancomycin. Our data also reinforce earlier observations indicating that bifidobacteria are intrinsically resistant to gentamicin, sulfamethoxazole and polymyxin B. Susceptibility to trimethoprim, trimethoprim/sulfamethoxazole, ciprofloxacin, clindamycin, tetracycline and minocycline was variable. The tet(W) gene was responsible for tetracycline resistance in 15 strains including 7 probiotic isolates belonging to the taxa Bifidobacterium animalis subsp. lactis and Bifidobacterium bifidum. This gene was present in a single copy on the chromosome and did not appear to be associated with the conjugative transposon TnB1230 previously found in tet(W)-containing Butyrivibrio fibrisolvens. CONCLUSIONS: The use of the LSM + cysteine medium allowed us to discriminate between intrinsic and atypical resistance properties of bifidobacteria and sets the scene for future definition of epidemiological cut-off values for all important Bifidobacterium species. The presence of an acquired tet(W) gene in several probiotic product isolates stresses the need for a minimal safety evaluation during the selection of Bifidobacterium strains for probiotic use.

Polyphenols from Honeybush tea (Cyclopia intermedia).

The fermented leaves and stems of Cyclopia intermedia are used to brew Honeybush tea, a herbal tea indigenous to South Africa. The plant is also used to manufacture a sweet herbal infusion used for restorative properties such as soothing coughs and alleviating bronchial complaints including tuberculosis, pneumonia, and catarrh. It is claimed to have a low tannin content and no caffeine and contains various antioxidants. Continued investigations into the phenolic content of the leaves and stems of C. intermedia yielded tyrosol and a methoxy analogue, 2-[4-[O-alpha-apiofuranosyl-(1' '-->6')-beta-d-glucopyranosyloxy]phenyl]ethanol, 4-[O-alpha-apiofuranosyl-(1' '-->2')-beta-d-glucopyranosyloxy]benzaldehyde, five glycosylated flavonols, two isoflavones, four flavanones, two isoflavones, and two flavones. Structure elucidation was done by NMR, CD, and MS methods. Because flavonoids are presumed to contribute significantly toward the scavenging effects of active oxygen species, our results indicate that the tentative claimed health-promoting properties may be attributed to the presence of these and other phenolics in C. intermedia.

Antibody levels to the class I and II epitopes of the M protein and myosin are related to group A streptococcal exposure in endemic populations.

Rheumatic fever (RF)/rheumatic heart disease (RHD) and post-streptococcal glomerulonephritis are thought to be autoimmune diseases, and follow group A streptococcal (GAS) infection. Different GAS M types have been associated with rheumatogenicity or nephritogenicity and categorized into either of two distinct classes (I or II) based on amino acid sequences present within the repeat region ('C' repeats) of the M protein. Sera from ARF patients have previously been shown to contain elevated levels of antibodies to the class I-specific epitope and myosin with the class I-specific antibodies also being cross-reactive to myosin, suggesting a disease association. This study shows that immunoreactivity of the class I-specific peptide and myosin does not differ between controls and acute RF (ARF)/RHD in populations that are highly endemic for GAS, raising the possibility that the association is related to GAS exposure, not the presence of ARF/RHD. Peptide inhibition studies suggest that the class I epitope may be conformational and residue 10 of the peptide is critical for antibody binding. We demonstrate that correlation of antibody levels between the class I and II epitope is due to class II-specific antibodies recognizing a common epitope with class I which is contained within the sequence RDL-ASRE. Our results suggest that antibody prevalence to class I and II epitopes and myosin is associated with GAS exposure, and that antibodies to these epitopes are not an indicator of disease nor a pathogenic factor in endemic populations.

Gastrointestinal eosinophils.

The gut-associated lymphoid tissue (GALT) is composed of lymphocytes residing in Peyer's patches, lamina propria, and intraepithelial compartments. In addition to these features which distinguish GALT from other peripheral sites of the immune system, the gastrointestinal immune system is also composed of resident eosinophils. Eosinophils are generally considered to be peripheral blood leukocytes that have an important pro-inflammatory role in various immune disorders. Although most research concerning this cell has focused on understanding its trafficking and function in the blood and lung, recent studies have also started to elucidate its regulation and function in the gastrointestinal tract. Interestingly, eosinophil numbers in the gastrointestinal tract are substantially higher than in other tissues. At baseline (healthy conditions), most eosinophils reside in the lamina propria in the stomach and intestine. Eosinophil homing to these sites occurs during embryonic development and their levels in perinatal mice are comparable to those in adults, indicating that their homing is not dependent upon the presence of intestinal flora. Furthermore, eosinophil localization to the lamina propria at baseline is critically regulated by eotaxin, a chemokine constitutively expressed throughout the gastrointestinal tract. Although eotaxin is required for eosinophil homing, its expression in the esophagus is not sufficient for eosinophil accumulation, since this organ is devoid of eosinophils at baseline. During Th2-associated inflammatory conditions (e.g. interleukin (IL)-5 overexpression or oral allergen challenge), marked increases of eosinophils occur not only in the lamina propria but also in Peyer's patches. The accumulation of Peyer's patch eosinophils, which mainly occurs in the outer cortex and interfollicular regions, is critically regulated by IL-5 and less significantly by eotaxin, suggesting the involvement of other eosinophil chemokines in this lymphoid compartment. Preliminary investigations have shown that gastrointestinal eosinophils express the alpha4beta7 integrin and that this molecule is responsible, in part, for eosinophil homing. In summary, eosinophils are resident cells of the gastrointestinal immune system whose levels can be induced by antigen exposure under Th2 conditions, in a manner that is critically regulated by eotaxin and IL-5. We propose that eosinophils are integral members of the gastrointestinal immune system and are likely to be important in innate, regulatory and inflammatory immune responses.

A pathological function for eotaxin and eosinophils in eosinophilic gastrointestinal inflammation.

Although eosinophils have been implicated in the pathogenesis of gastrointestinal disorders, their function has not been established. Using a murine model of oral antigen-induced eosinophil-associated gastrointestinal disease, we report the pathological consequences of eosinophilic inflammation and the involvement of eotaxin and eosinophils. Exposure of mice to enteric-coated antigen promotes an extensive T helper 2-associated eosinophilic inflammatory response involving the esophagus, stomach, small intestine and Peyer's patches as well as the development of gastric dysmotility, gastromegaly and cachexia. Electron microscopy shows eosinophils in proximity to damaged axons, which indicated that eosinophils were mediating a pathologic response. In addition, mice deficient in eotaxin have impaired eosinophil recruitment and are protected from gastromegaly and cachexia. These results establish a critical pathological function for eotaxin and eosinophils in gastrointestinal allergic hypersensitivity.

Enhanced production of IL-8 in chronic but not in early ileal lesions of Crohn's disease (CD).

Distinct Th1/Th2 patterns have been observed during the evolution of CD. The aim of this study was to compare neutrophil involvement and IL-8 mRNA and protein expression during early recurrent lesions and chronic phases of CD. Twenty-nine patients with CD having ileocolonic resection with anastomosis were studied. Biopsies were obtained during surgery from the non-inflamed ileal mucosa and from chronic ileal lesions. Endoscopic ileal biopsies were also taken from early recurrent ileal lesions occurring 3 months after surgery. Neutrophil counts were performed and mucosal IL-8 levels were evaluated by competitive reverse transcriptase-polymerase chain reaction and immunohistochemistry. Early recurrent ileal lesions were characterized by low neutrophil counts and IL-8 production at the mRNA and protein levels compared with the ileal chronic lesions. The main cellular sources of IL-8 in the early recurrent lesions were neutrophils, while in chronic lesions the majority of IL-8-stained cells were CD3+ T cells and macrophages. These results confirmed that the nature of the inflammatory infiltrate and the expression of cytokine profiles may differ between the acute and chronic phases of CD.

Down-regulation of neutrophil functions by the ELR(+) CXC chemokine platelet basic protein.

The platelet-derived neutrophil-activating peptide 2 (NAP-2, 70 amino acids) belongs to the ELR(+) CXC subfamily of chemokines. Similar to other members of this group, such as IL-8, NAP-2 activates chemotaxis and degranulation in neutrophils (polymorphonuclear [PMN]) through chemokine receptors CXCR-1 and CXCR-2. However, platelets do not secrete NAP-2 as an active chemokine but as the C-terminal part of several precursors that lack PMN-stimulating capacity. As we have previously shown, PMN themselves may liberate NAP-2 from the precursor connective tissue-activating peptide III (CTAP-III, 85 amino acids) by proteolysis. Instead of inducing cell activation, continuous accumulation of the chemokine in the surroundings of the processing cells results in the down-regulation of specific surface-expressed NAP-2 binding sites and in the desensitization of chemokine-induced PMN degranulation. Thus, NAP-2 precursors may be regarded as indirect mediators of functional desensitization in neutrophils. In the current study we investigated the biologic impact of another major NAP-2 precursor, the platelet basic protein (PBP, 94 amino acids). We show that PBP is considerably more potent than CTAP-III to desensitize degranulation and chemotaxis in neutrophils. We present data suggesting that the high desensitizing capacity of PBP is based on its enhanced proteolytic cleavage into NAP-2 by neutrophil-expressed cathepsin G and that it involves efficient down-regulation of surface-expressed CXCR-2 while CXCR-1 is hardly affected. Correspondingly, we found PBP and, less potently, CTAP-III to inhibit CXCR-2- but not CXCR-1- dependent chemotaxis of neutrophils toward NAP-2. Altogether our findings demonstrate that the anti-inflammatory capacity of NAP-2 is governed by the species of its precursors.

Stimulation of cellular sphingomyelin import by the chemokine connective tissue-activating peptide III.

The selective import of phospholipids into cells could be mediated by proteins secreted from the cells into the extracellular compartment. We observed that the supernatants obtained from suspensions of thrombin-activated platelets stimulated the exchange of pyrene (py)-labeled sphingomyelin between lipid vesicles in vitro. The proteins with sphingomyelin transfer activity were purified and identified as the chemokine connective tissue-activating peptide III (CTAP-III) and platelet basic protein. Isolated CTAP-III stimulated the exchange of py-sphingomyelin between lipid vesicles but did not affect the translocations of py-labeled phosphatidylcholine and phosphatidylethanolamine. CTAP-III rapidly increased the transfer of py-sphingomyelin from low density lipoproteins into peripheral blood lymphocytes, other immune cells, and fibroblasts. In the presence of heparin, CTAP-III was unable to insert sphingomyelin into the peripheral blood lymphocytes. The activation energy of the py-sphingomyelin transfer suggested that the translocation proceeded entirely in a hydrophobic environment. [(3)H]Sphingomyelin transferred to the cells by CTAP-III was hydrolyzed to [(3)H]ceramide and [(3)H]sphingosine after activation with tumor necrosis factor alpha. The generation of the [(3)H]sphingolipid messengers was catalyzed by acid sphingomyelinase. Our results identify CTAP-III as the first mediator of the selective (endocytosis-independent) cellular import of sphingomyelin allowing the paracrine modulation of the sphingolipid signaling.

Polyphenols, condensed tannins, and other natural products in Onobrychis viciifolia (Sainfoin).

An acetone/water extract of the fodder legume Onobrychis viciifolia afforded arbutin, kaempferol, quercetin, rutin, afzelin, the branched quercetin-3-(2(G)-rhamnosylrutinoside), the amino acid L-tryptophan, the inositol (+)-pinitol, and relatively high concentrations of sucrose (ca. 35% of extractable material). Acid-catalyzed cleavage of the condensed tannins with phloroglucinol afforded catechin, epicatechin and gallocatechin as the terminal and extender units, but epigallocatechin was only present in extender units. The condensed tannins in O. viciifolia presumably consist of hetero- and homopolymers containing both procyanidin and prodelphinidin units. Comparison of data from the present study and the literature suggests that sainfoin tannins have a highly variable composition with cis:trans ratios ranging from 47:53 to 90:10 and delphinidin:cyanidin ratios from 36:64 to 93:7. The composition of terminal and extender units in sainfoin tannins seems to be cultivar specific.