Porcine spermadhesin PSP-I/PSP-II stimulates macrophages to release a neutrophil chemotactic substance: modulation by mast cells

The complex of porcine seminal plasma heterodimers I and II (PSP-I/PSP-II), which are heterodimers of glycosylated spermadhesins, is the major component of porcine seminal fluid. The proinflammatory and immunostimulatory activities of this spermadhesin complex suggest its participation in modulation of the uterine immune activity that may ensure reproductive success. Spermadhesin PSP-I/PSP-II induced the migration of neutrophils into the peritoneal cavity of rats via activation of resident cells. In the present study, we have investigated the involvement of macrophages and mast cells in the neutrophil chemotactic activity of PSP-I/PSP-II and the underlying mechanism. Macrophages and mast cells were isolated, cultured, and stimulated with purified PSP-I/PSP-II. Pharmacological modulation was performed using the glucocorticoid dexamethasone, indomethacin (cyclooxygenase inhibitor), MK886 (leukotriene inhibitor), and the supernatant of spermadhesin-stimulated mast cells. Macrophages stimulated with PSP-I/PSP-II released into the culture supernatant a neutrophil chemotactic substance. This activity was partly inhibited by both dexamethasone (85%) and the supernatant of spermadhesin-stimulated mast cells (74%) but not by indomethacin and MK886. An anti-tumor necrosis factor (TNF) alpha antibody neutralized (by 68%) the neutrophil chemotactic activity of PSP-I/PSP-II-stimulated macrophages. An anti-interleukin (IL)-4 antibody blocked the inhibitory activity of spermadhesin-stimulated mast cells on release of a neutrophil chemotactic substance by PSP-I/PSP-II-stimulated macrophages. As a whole, these data indicate that the neutrophil migration-inducing ability of spermadhesin PSP-I/PSP-II involves the release of the inflammatory cytokine TNFalpha by stimulated macrophages and that this activity is modulated by the lymphokine IL-4 liberated by mast cells. The balance between these two cytokines may control onset of the local inflammatory reaction, avoiding excessive neutrophil recruitment that would lead to tissue damage.     

Contribution of Multiparameter Flow Cytometry Immunophenotyping to the Diagnostic Screening and Classification of Pediatric Cancer

Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 35), with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56^hi/GD2⁺/CD81^hi), primitive neuroectodermal tumors (CD271^hi/CD99⁺), Wilms tumors (>1 cell population), rhabdomyosarcoma (_nuMYOD1⁺/_numyogenin⁺), carcinomas (CD45⁻/EpCAM⁺), germ cell tumors (CD56⁺/CD45⁻/NG2⁺/CD10⁺) and eventually also hemangiopericytomas (CD45⁻/CD34⁺). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.     

Evolution of cytochrome P-450 proteins [published erratum appears in Mol Biol Evol 1988 Mar;5(2):199]

Thirty-four cytochrome P-450 sequences from one bacterial and six vertebrate species have been aligned with the aid of a computer alignment algorithm. Phylogenetic trees were constructed using the unweighted-pair-group and neighbor-joining methods. The two trees differed at only a single branch point near the base of the tree. The cytochrome P-450 superfamily of proteins clustered into eight families and contained 16 gene-duplication events. The first gene duplication occurred approximately 1,360 Myr before the present (Mybp) and gave rise to cytochrome P-450s found in two different cellular organelles, the mitochondria and the endoplasmic reticulum. Both groups utilize cholesterol or its metabolites as substrates, implying that cholesterol existed greater than 1,360 Mybp. The fourth gene duplication (approximately 900 Mybp) gave rise to the drug-metabolizing P-450s. These proteins aid in the detoxification of foreign chemicals, as opposed to the metabolism of endogenous compounds. The importance of the capacity to metabolize drugs is reflected in 11 further gene duplications occurring in this lineage. The first occurred approximately 800 Mybp and gave rise to the two major P-450 families, the phenobarbital and 3-methylcholanthrene families. An apparent increase in the rate of cytochrome P-450 evolution is noted between the bird-mammal divergence (300 Mybp) and the mammalian radiation (75 Mybp).