Lack of effect of opioid peptides,morphine and naloxone on superoxide formation in human neutrophils and HL-60 leukemic cells

Summary There are controversial reports in the literature concerning the effects of opioids on superoxide (O ₂ ^– ) formation in phagocytes, these agents being either inhibitory or stimulatory. We re-examined this issue and compared the effects of the Chemotactic peptide, N-formyl-l,-methionyl-l-leucyl-l-phenylalanine (fMet-Leu-Phe), phorbol myristate acetate (PMA), ATP, platelet activating factor (PAF), cytochalasin B (CB) and prostaglandin E₁ (PGE₁) with those of various opioids on O ₂ ^– formation in human neutrophils and HL-60 leukemic cells under defined experimental conditions. In the presence of CB, fMet-Leu-Phe and PAF concentration-dependently activated O ₂ ^– formation in neutrophils with EC₅₀ values of 20 nM and 100 nM, respectively. In the absence of CB, fMet-Leu-Phe and PAF were much less effective. PAF synergistically enhanced O ₂ ^– formation induced by fMet-Leu-Phe. ATP at a concentration of 100 M and the opioids, methionine enkephalin, -endorphin, dynorphin, [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin, [d-Ala²-d-Leu⁵]-enkephalin and morphine at concentrations between 10 pM to 1 M did not activate O ₂ ^– formation. ATP but not \-endorphin potentiated fMet-Leu-Phe-induced O ₂ ^– formation. O ₂ ^– formation induced by a maximally stimulatory concentration of PMA (100 ng/ml) was enhanced by fMet-Leu-Phe but was unaffected by methionine enkephalin or PGE₁. PMA at a non-stimulatory concentration (2 ng/ml) potentiated the effect of fMet-Leu-Phe but did not induce responsiveness to PAF, ATP or -endorphin. PGE₁ strongly inhibited fMet-Leu-Phe-induced O ₂ ^– formation, whereas morphine, methionine enkephalin and the opioid antagonist, naloxone, were without effect. In HL-60 cells differentiated with dibutyryl cAMP, fMet-Leu-Phe, PAF and ATP but not -endorphin activated O ₂ ^– formation. Our results show that O ₂ ^– formation is differentially regulated by various classes of intercellular signal molecules and that opioids do not play a role in the regulation of O ₂ ^– formation. The precise definition of the experimental conditions and control experiments with established modulators of O ₂ ^– formation are essential to evaluate the role of opioids in the regulation of this effector system.Send offprint requests to R. Seifert at the above address     

Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable,epigenetic gene silencing: a mammalian cell culture model of gene variegation

Heterochromatin protein 1 (HP1) is a key component of constitutive heterochromatin in Drosophila and is required for stable epigenetic gene silencing classically observed as position effect variegation. Less is known of the family of mammalian HP1 proteins, which may be euchromatic, targeted to expressed loci by repressor-corepressor complexes, and retained there by Lys 9-methylated histone H3 (H3-MeK9). To characterize the physical properties of euchromatic loci bound by HP1, we developed a strategy for regulated recruitment of HP1 to an expressed transgene in mammalian cells by using a synthetic, hormone-regulated KRAB repression domain. We show that its obligate corepressor, KAP1, can coordinate all the machinery required for stable gene silencing. In the presence of hormone, the transgene is rapidly silenced, spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with KAP1, HP1, and the H3 Lys 9-specific methyltransferase, SETDB1, over a highly localized region centered around the promoter. Remarkably, silencing established by a short pulse of hormone is stably maintained for >50 population doublings in the absence of hormone in clonal-cell populations, and the silent transgenes in these clones show promoter hypermethylation. Thus, like variegation in Drosophila, recruitment of mammalian HP1 to a euchromatic promoter can establish a silenced state that is epigenetically heritable.     

Transfer factor (TF) treatment of patients with HBs-Ag-positive chronic active hepatitis

Summary It is a clinically and experimentally well supported working hypothesis that infection with hepatitis B virus may result in chronic active hepatitis in patients with suspected immune deficiencies. On this basis, a pilot study was performed in order to evaluate the effect of specific transfer factor (TF) in the treatment of HB_s-Ag-positive chronic active hepatitis. From the leukocytes of 500 ml venous blood each of 40 volunteers that had completely recovered from acute virus hepatitis B within the last 6 months, a unique TF pool (40 units of TF) was prepared according to the method of Lawrence. Preexaminations indicated that this preparation was able to enhance cellular immune reactions in vitro. Thirteen patients with HB_s-antigenemia and chronic active hepatitis (i.e., two liver biopsies within the last 6 or more months with the histological criteria of chronic aggressive hepatitis according to de Groote, elevated serum levels of bilirubin, alkaline phosphatase, transaminase activities, and/or -globulines) were randomized: Seven received s.c. injections of two units of TF each on days 1 and 15, the other six saline. Conversion of skin reactions to some ubiquitous antigens occurred in the TF group, but no significant and constant drop of HB_s-Ag serum titers was observed. Although some of the biochemical parameters seemed to ameliorate in the TF group, the differences versus the control group did not prove to be significant within the limited number of patients under observation. The in vitro reactivity of patients' lymphocytes to HB_s-Ag, tested by means of the³H-thymidine uptake, was never found enhanced after TF application. In the used doses, specific TF was not effective in the treatment of HB_s-Ag-positive chronic active hepatitis; unfavorable side-effects were not observed.     

Microsatellite analysis of synchronous and metachronous tumors: a tool for double primary tumor and metastasis assessment.

Despite well-established histopathological features and the development of immunostaining of human neoplasms, there are a number of cases in which surgical pathologists cannot assure the origin of synchronous and metachronous tumors. In many cases, the classification of these lesions as either two separate primary tumors or as a single primary tumor with a metastasis has significant implications with respect to patient prognosis and recommendations for therapy. To establish the origin of tumors, we assessed tumor cell clonality using PCR-based microsatellite analysis on microdissected archival tissues for loss of heterozygosity (LOH) and microsatellite instability (MSI) in a series of 19 paired synchronous and metachronous tumors from several organs. As a control group, 15 autopsy cases with an unequivocally recognizable primary tumor and associated metastases were also examined. Based on LOH and MSI findings, and using a panel of 4 to 12 (median 7) microsatellite markers, we were able to establish the clonal pattern of microsatellite changes in 17 out of 19 (89%) biopsy cases and thus determine if they were either double primary tumors (41%) or metastases (59%). Of interest, identical or similar pattern of microsatellite abnormalities were detected in 15 primary tumors and corresponding metastasis from autopsies. Our results indicate that microsatellite analysis for LOH and MSI, as an expression of clonality, provides a useful tool to distinguish double primary neoplasms and metastases in synchronous and metachronous tumors.     

Survival of satellite cells in whole muscle transplants

The ability of satellite cells to survive the ischemic conditions at the core of orthotopically free grafted rat extensor digitorum longus muscles was examined. Cell cultures of isolated core and peripheral regions of whole muscle grafts maintained in vivo for more than 24 hours indicated that no viable cells were present in the core, whereas the number of cells from the peripheral region was greatly increased. Muscles were examined with the electron microscope to determine the fate of satellite cells of the core at various times after transplantation. The population of satellite cells in the core was reduced beginning at 18 hours and had virtually disappeared by 24-28 hours. This reduction did not appear to be the result of satellite cell death. Although there was abundant morphological evidence that myonuclei as well as myofiber cytoplasmic organelles were degenerating, there was little indication of satellite cell death in situ any time period studied. These studies suggest that satellite cells cannot survive, but migrate from the ischemic core to more peripheral regions of whole muscle transplants. In addition, they suggest that migration is an important aspect of the regeneration response in the free graft system and permits the myogenic population to contribute en masse to the centripetal wave of regeneration from the time it is initiated at the muscle periphery.     

A cytogenetic study of 53 human gliomas

Cytogenetic studies were performed on human glioma samples obtained by stereotactic biopsy, stereotactic craniotomy, or routine craniotomy. Using in situ culture and robotic harvesting techniques, we obtained suitable metaphases in 50 (94%) of 53 tumors, including 28 diffuse astrocytomas, four juvenile pilocytic astrocytomas, two gliosarcomas, three other miscellaneous astrocytomas, eight oligodendrogliomas, four mixed oligodendroglioma-astrocytomas, and four ependymomas. Cytogenetic studies were performed only on primary cultures; the mean culture time was 9.6 days (range 1-31 days). One or more chromosomally abnormal clones were observed in 35 (66%) tumors. Eleven (21%) other specimens had random nonclonal chromosome abnormalities. In four (8%) specimens, no chromosome abnormalities were noted. The results of this study suggest that grade 3 and 4 tumors are more likely to contain an abnormal clone than tumors of grade 1 or 2 (p less than 0.01). The most common numeric chromosome abnormalities were -6, +7, -10, -13, -14, -15, -18, and -Y. The most common structural abnormalities involved 1p, 6q, 7q, 8p, 9p, 11p, 11q, 13q, and 19q. Four tumors had two or more independent clones and ten contained subclones demonstrating karyotype evolution. With in situ culture and robotic harvesting techniques, cytogenetic studies can be successful on nearly all human gliomas, including those derived from small stereotactic biopsies.