Inflammatory cytokine production in whole blood modified by liposome-encapsulated hemoglobin

The effect of Neo Red Cells (NRC), liposome-encapsulated hemoglobin, on production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) were studied in whole blood preparations ex vivo. Venous blood was collected with heparin and incubated in a CO2 incubator. Treatment of blood samples with NRC reduced the constitutive levels of TNF-alpha and IL-6. Lipopolysaccharide (LPS) treatment for 24 h increased production of TNF-alpha and IL-6 in a dose-dependent manner. Pretreatment with NRC (5%) for 24 h markedly potentiated the LPS-induced TNF-alpha production and, that of IL-6 to a lesser extent. Northern blotting analysis of total RNA in whole blood showed that pretreatment with NRC caused a marked increase in TNF-alpha mRNA expression in response to LPS. It is concluded that NRC potentiates LPS-induced TNF-alpha and IL-6 production in whole blood ex vivo, and that the potentiating effect of NRC on LPS-induced TNF-alpha production can be attributed, at least in part, to an increase in its mRNA expression.     

Effect of macrolides on cytokine mRNA expression in human whole blood model

Recently, low dose and long term use of Macrolides (Mls) has been reported to be effective in treatment of chronic lower respiratory tract infections, however its mechanism is still obscure. We evaluated the effect of Mls (EM, AZM, RKM) on cytokine mRNA expressions. We preincubated the whole blood with several concentrations of Mls and removed the Mls and then stimulated human whole blood with LPS as an experimental vivo model. In order to examine cytokine mRNA expressions, we used the RT-PCR method. Cytokine mRNA expressions were suppressed significantly (p < 0.05) by pretreatment with EM, AZM; moreover, the suppression was peaked at low concentrations (0.04 approximately 0.2 microgram/ml). Although, Cytokine mRNA expressions were not suppressed by pretreatment with RKM. These results suggest that EM, AZM have suppression on Cytokine mRNA expressions, and consequently, this suppression has a reasonable effect for DPB patients.     

Effect of intravenous anesthetics on spontaneous and endotoxin-stimulated cytokine response in cultured human whole blood

BACKGROUND: Various anesthetics have been suggested to interfere with the immune system. The ability of leukocytes to express surface receptors and mediators is fundamental to a successful host defense. Therefore, the effects of intravenous anesthetics on cytokine release by leukocytes and expression of surface molecules known to modulate this response were determined. METHODS: Concentration-dependent effects of thiopentone, etomidate, propofol, ketamine, midazolam, and fentanyl on spontaneous and endotoxin (lipopolysaccharide; 1 microg/ml)-stimulated cytokine release were studied in whole blood from volunteers (n = 6) cultured for 25 h. In addition, expression of the lipopolysaccharide-recognition molecule CD14 and the major histocompatibility complex class II molecule human leukocyte locus A system-DR (HLA-DR) on monocytes were assessed using flow cytometry. RESULTS: All anesthetics studied elicited only minor effects on spontaneous cytokine release even at pharmacologic concentrations. However, expression density of CD14 was reduced in the presence of thiopentone, etomidate, and propofol, whereas HLA-DR was unaffected. Lipopolysaccharide-stimulated tumor necrosis factor response was inhibited by thiopentone (12.8% [median]; 7.6-18.8 [25-75 percentile]) of control, and ketamine (46.4% [median]; 44.4-56.4 [25-75 percentile]), at pharmacologic concentrations, whereas it was augmented even in the presence of low concentrations of propofol (172.3% [median]; 120.5-200.7 [25-75 percentile]). Ketamine additionally decreased the concentration of interleukin (IL)-1beta (14.8% [median]; 12.0-18.0 [25-75 percentile]). Release of IL-1 receptor antagonist (IL-1ra) was inhibited by thiopentone, etomidate, and propofol, whereas the same anesthetics increased IL-10 concentration simultaneously. Midazolam and fentanyl did not alter the concentrations of any cytokine. CONCLUSIONS: These results suggest a complex modulation of the cytokine response by the studied anesthetics in cultured whole blood. Although effects on spontaneous cytokine release by leukocytes were negligible, some anesthetics affected their ability to respond to lipopolysaccharide.     

Interferon gamma production in whole peripheral blood culture in acute hepatitis B

Database federation enables biological researchers to utilize resources more effectively, creating an environment in which the researcher can query multiple data sources without spending time learning new query mechanisms or issuing redundant queries which need to be integrated. Several mechanisms exist to federate databases. The ENQUire system is a network database federation system which uses a World-Wide-Web (WWW) interface to connect the users to various databases. Generic queries entered via a query generator form are sent in parallel to multiple databases, and the results are presented to the user in a unified format. All forms building, query generation, and results translation is done on the fly, and individual database translation modules can be added dynamically. ENQUire is a flexible answer to the problems of database federation on the WWW.     

Correlation of antibiotic-induced endotoxin release and cytokine production in Escherichia coli-inoculated mouse whole blood ex vivo

Escherichia coli were incubated in mouse whole blood ex vivo supplemented with beta-lactam antibiotics that possessed preferential affinities for penicillin-binding proteins (PBPs). After 4 h, viable bacteria were undetectable in the presence of any of the 3 antibiotics tested, whereas significant increases in colony-forming units were detected in samples not treated with antibiotics. Differential levels of endotoxin in platelet-rich plasma were detected using the limulus amebocyte lysate assay, according to differential antibiotic affinities for the various PBPs. Levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in antibiotic-treated cultures after 8 h of incubation correlated well with the levels of endotoxin at 4 h (r = .96, P < .0001 for TNF-alpha; r = .91, P = .0002 for IL-6). These data indicate that differential affinities of beta-lactam antibiotics for PBPs affect both endotoxin and cytokine responses ex vivo in mouse blood and correlate with in vivo protective efficacy of these antibiotics in gram-negative experimental models.     

Lead differentially modifies cytokine production in vitro and in vivo

An imbalance between helper T cell type 1 (Th1) and helper T cell type 2 (Th2) activation can result in immunodysregulations leading to impaired cell-mediated immunity with an increased incidence of infectious disease or cancer and/or aberrant humoral immunity that may culminate with an autoimmune disease. Mercury, a heavy-metal toxicant, is known to induce renal autoimmunity characterized by a predominant Th2 response. Lead, another metal toxicant, causes enhanced B cell activities and impairs host resistance to several bacterial and viral infections. In addition, Pb was reported to enhance Th2 proliferation and inhibit Th1 proliferation. The differential effects of Pb on Th subset activation have been further investigated. In vitro IL-4 production by a Th2 clone was significantly increased by the addition of PbCl2, whereas IFN gamma production by a Th1 clone was decreased by the addition of PbCl2. When BALB/c mice were subcutaneously exposed to PbCl2, ex vivo Il-4 production by anti-CD3-stimulated splenic T cells was enhanced, but IFN gamma production was inhibited. Additionally, the plasma IL-4 and IgE levels of Pb-exposed mice were increased, and the plasma IFN gamma levels were significantly lowered in the absence of any additional exogenous antigen. In vitro, ex vivo, and in vivo treatment with HgCl2 produced similar findings. This study is the first report of the preferential activation of a Th2 response by Pb in vivo and suggests that PB, like Hg, may induce autoimmune responses by upsetting the balance between Th1- and Th2-like cells, which could enhance production of antibodies to self antigens.     

Endogenous and exogenous factors in tumor development and cell differentiation in gastrointestinal tumors

The full-length copy of the hemagglutinin gene RNA of the influenza virus A/Alma Ata/1417/84 (Hsw1 N1-serovariant) has been synthesized and cloned on Escherichia coli plasmid pBR327. The complete nucleotide sequence of the cloned DNA copy was determined by the Maxam-Gilbert procedure. The predicted amino acid, sequence of HA1 hemagglutinin subunit was compared with the sequences of HA1 subunits from other H1N1-subtype influenza virus strains. It has been found that the structure of the HA1-subunit of the studied strain is most similar to the structure of the identical region of the A/New Jersey/18/76 hemagglutinin.     

Natural killer cell depletion fails to influence initial CD4 T cell commitment in vivo in exogenous antigen-stimulated cytokine and antibody responses

The role played by NK- and NK1.1-expressing T cells in CD4 T cell activation and induction of immune responses in vivo is controversial. These effector cells of the innate immune response are hypothesized to play a pivotal role in shaping initial T cell activation, with some groups reporting that classical NK cells are required for optimal Th1-like T cell activation, and others supporting a role for NK1.1+ alphabeta T cells in Th2 generation. Here, we examine the impact of in vivo NK cell depletion on the development of exogenous Ag-specific cytokine and Ab responses using a murine model of human immediate hypersensitivity. OVA-specific immune responses were induced in 1) C57Bl/6 bg/bg and bg/+ mice, 2) BALB/c mice pretreated with anti-asialoGM1 or control Ab, and 3) C57Bl/6 mice depleted of NK1.1-expressing cells by in vivo administration of anti-NK1.1 mAb PK136. Depletion efficacy was assessed by functional assays and flow cytometric analysis. Each of these approaches indicated that depletion of NK cells and NK1.1+ CD4+ T cells fails to alter the Th1:Th2 balance of Ag-driven cytokine synthesis, as indicated by OVA-stimulated cytokine synthesis in primary bulk culture. Similarly, the kinetics and intensity of effector responses such as OVA-specific IgG2a and IgE synthesis were neither increased nor decreased in any of the three models examined. The results argue that NK cells and peripheral NK1.1+ T cells do not play an essential role in shaping the induction of Ag-specific immune responses to soluble exogenous Ags, the most common class of inhalant allergen.     

Absence of cytokine receptor-dependent specificity in red blood cell differentiation in vivo

Erythropoietin (EPO) is required for red blood cell development, but whether EPO-specific signals directly instruct erythroid differentiation is unknown. We used a dominant system in which constitutively active variants of the EPO receptor were introduced into erythroid progenitors in mice. Chimeric receptors were constructed by replacing the cytoplasmic tail of constitutively active variants of the EPO receptor with tails of diverse cytokine receptors. Receptors linked to granulocyte or platelet production supported complete erythroid development in vitro and in vivo, as did the growth hormone receptor, a nonhematopoietic receptor. Therefore, EPOR-specific signals are not required for terminal differentiation of erythrocytes. Furthermore, we found that cellular context can influence cytokine receptor signaling.     

New etiopathogenic, clinical and therapeutic findings in multiple endocrine neoplasia type 1

Multiple Endocrine Neoplasia type 1 (MEN 1) syndrome comprises tumors or hyperplasia of different glands, including parathyroid, pituitary, adrenal cortex and the gastroenteropancreatic system. The vast majority of MEN 1 are found in familial clusters, although a few cases are sporadic. Hypercalcemia and/or nephrocalcinosis are the first and most common clinical manifestation in familial MEN 1 syndrome, followed by islet cell tumors (especially those secreting gastrin or insulin) and pituitary dysfunction due to either functioning or non-functioning microadenomas. Genetic studies indicate that familial MEN 1 syndrome is inherited through a dominant gene with incomplete penetrance and variable expression. The diagnosis of MEN 1 syndrome is mainly based on the careful assessment of the clinical history, symptoms physical evaluation along with the assay of serum electrolytes (i.e., calcium, phosphorus, etc.) and hormonal substances (i.e., gastrin, insulin, pancreatic polypeptide, prolactin, adrenocorticotropic hormone, etc.). In addition, several provocative tests have been used to identify endocrine tumors (particularly those of the gastroenteropancreatic system) and imaging techniques play a crucial role for the diagnostic approach in MEN 1 syndrome. Even though in the long term, the prognosis of MEN 1 syndrome is unfavourable. Recently, however, many therapeutic strategies, including both surgical and pharmacological options, have been developed to reduce the size of the neoplasm and control symptoms associated with hormone oversecretion.     

Lipopolysaccharide induction of MARCKS-related protein and cytokine secretion are differentially impaired in microglia from LPS-nonresponsive (C3H/HeJ) mice

Many events involved in activation of microglia and leukocytes by lipopolysaccharide (LPS) are mediated by protein kinase C (PKC), and we have recently demonstrated that a major PKC substrate, MARCKS-related protein (MRP), is selectively induced by LPS in murine microglia. In microglia from LPS-nonresponsive (C3H/HeJ) mice, induction of MRP and secretion of CSF-1 required much higher LPS concentrations (> or = 100 ng/ml) than in normal (C3H/OuJ) microglia (< or = 10 ng/ml). By contrast, TNF alpha production was not significantly increased in C3H/HeJ microglia even at 1 microgram LPS/ml. Microglia expressed PKC isoforms alpha, beta, delta, and zeta (but not gamma and epsilon); PKC isoform levels were similar in both normal and C3H/HeJ microglia and no significant change in response to LPS was noted. Our results indicate that LPS alters PKC substrate (rather than kinase) expression, and that the Lpsd mutation in C3H/HeJ mice differentially affects regulation of several gene products implicated in microglial function.     

The human SHIP gene is differentially expressed in cell lineages of the bone marrow and blood

The macrophage colony-stimulating factor receptor and several other hematopoietic growth factor receptors induce the tyrosine phosphorylation of a 145- to 150-kD protein in murine cells. We have previously cloned a cDNA for the murine 150-kD protein, SHIP, and found that it encodes a unique signaling intermediate that binds the SHC PTB domain through at least one tyrosine phosphorylated (NPXY) site in the carboxyl-terminal region. SHIP also contains several potential SH3 domain-binding sites, an SH2 domain for binding other tyrosine phosphorylated proteins, and an enzymatic activity that removes the phosphate from the 5 position of phosphatidylinositol 3,4,5-phosphate or from inositol 1,3,4,5-phosphate. SHIP has a negative effect on cell growth and therefore loss or modification may have profound effects on hematopoietic cell development. In this study, we have cloned a cDNA for human SHIP and examined mRNA and protein expression of SHIP and related species in bone marrow and blood cells. Flow cytometry indicates that at least 74% of immature CD34+ cells express SHIP cross-reacting protein species, whereas within the more mature population of CD33+ cells, only 10% of cells have similar expression. The majority of T cells react positively with the anti-SHIP antibodies, but significantly fewer B cells are positive. Immunoblotting detects up to seven different cross-reacting SHIP species, with peripheral blood mononuclear cells exhibiting primarily a 100-kD protein and a CD34+ acute myeloblastic leukemia expressing mainly 130-kD and 145-kD forms of SHIP. Overall, these results indicate that there is an enormous diversity in the size of SHIP or SHIP-related mRNA and protein species. Furthermore, the expression of these protein species changes according to both the developmental stage and differentiated lineage of the mature blood cell.     

Cortisol secretion rate (CSR) in hypertension and endocrine patients

With the method of isotope dilution the circadian cortisol secretion was determined in 22 patients with hypertonic disease--28.35 mg/24 h, elevated versus CSR in healthy subjects-16.63 mg/24 h. CSR is elevated also in 5 patients with bilateral adenocortical hyperplasia and with suprarenal cancer. The patients with diencephalic syndrome, progressing according to the type of Cushing disease have elevated absolute values of CSR, but recalculated per square meter of body surface and per gram creatineine excreted in 24 h-CSR proved to be normal-about 12 mg/24 h.     

Cytokine receptors, growth factors and cell cycle in human bone marrow and peripheral blood hematopoietic progenitors

BACKGROUND AND OBJECTIVE: An increasing number of growth factors have been shown to be responsible for the proliferation, survival and enhanced function of many cell types within the hemopoietic system. The action of these hemopoietic growth factors in stimulating cell growth and survival applies both to cells within the progenitor compartment and mature cells. Whether a specific cytokine influences in vivo hematopoietic progenitor cell proliferation or survival depends on cytokine-mediated modulation or target cell cytokine receptors, cell proliferation, and cell death regulator genes and other pathways. To address these issues, particularly in view of the current and future clinical use of hemopoietic growth factors, the Italian Society of Experimental Hematology organized a Meeting in Florence on July 4th, 1996. INFORMATION SOURCES: The material examined in the present review includes full papers and abstracts published in journals covered by the Science Citation Index and Medline. All the participants to the Meeting in Florence have been actively working in the field of biology and clinical application of hemopoietic growth factors. Summaries of their oral presentations at the Florence Meeting are reported in the Appendix to this article. STATE OF ART AND PERSPECTIVES: Myelopoietic growth factors particularly granulocyte (G-) colony-stimulating factor (CSF) and granulocyte-macrophage (GM)-CSF, have been available for clinical use for only a few years but they have already markedly changed the management of chemotherapy-induced neutropenia, the use of dose-intensive chemotherapy regimens and the practice and safety of autologous stem cell transplantation. While these growth factors have been rapidly introduced as routine agents in the management of cancer patients, they have continued to generate a considerable amount of fundamental research into the biology of hematopoiesis as well as the growth regulation of normal and cancer cells. For instance, one goal of cancer treatment is to protect hematopoietic stem and progenitor cells from the damaging effects of chemotherapy, while maintaining their anticancer action. Any means of preferentially and reversibly suppressing the proliferation of normal hematopoietic stem and progenitor cells while leaving the proliferation of tumor cells and their susceptibility to chemotherapy unmodified, could potentially optimize treatment efficacy. In this field, the possibility of using colony-stimulating factors as myeloprotective agents in dose-intensive chemotherapy to enhance anticancer activity could be an attractive goal of current anti-cancer treatment modalities.     

Comparison of 5-fluorouracil pharmacokinetics in whole blood, plasma, and red blood cells in patients with colorectal cancer

STUDY OBJECTIVE: To compare 5-fluorouracil (5-FU) pharmacokinetics in whole blood, plasma, and red blood cells in patients with colorectal cancer. DESIGN: Prospective, unblinded observational study in consecutive patients. SETTING: Large regional teaching hospital. PATIENTS: Five patients with colorectal cancer. INTERVENTIONS: Patients received folinic acid 200 mg/m2 intravenously over 2 hours, followed by 5-FU 600 mg/m2 intravenous bolus over 30 minutes, then 5-FU 600 mg/m2 intravenous infusion over 22 hours, administered on days 1 and 2. This 48-hour cycle was repeated every 14 days. MEASUREMENTS AND MAIN RESULTS: Concentrations of 5-FU in whole blood, plasma, and red blood cells were determined by high-performance liquid chromatography. ADAPT II was used for pharmacokinetic computations. The optimum model was determined for each matrix by calculating Akaike's information criteria values. Concentrations of 5-FU in whole blood were 106-115% of simultaneous plasma concentrations (median 112%), and packed red blood cell levels were 5-17% of plasma concentrations (median 11%). The drug's concentration-time profile was similar in the three matrices. The drug is reported to be unstable in whole blood, and red blood cell 5-FU concentrations were near the limit of detection (10 ng/ml), supporting plasma as the preferred matrix for therapeutic drug monitoring studies. Six pharmacokinetic models were fitted to the 5-FU individual data sets to determine the best curve fit. The optimal model for whole blood and plasma data sets was one compartment with both linear and nonlinear elimination models; a one-compartment model with nonlinear elimination provided the best curve fit for 5-FU in red blood cells. A two-compartment model with nonlinear elimination gave a similar degree of curve fit for plasma 5-FU as the one-compartment model with both linear and nonlinear elimination. CONCLUSIONS: These pharmacokinetic results provide the basis for further investigation into the ability to correlate 5-FU systemic exposure with clinical drug activity.     

Specific residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins

Two classes of homeodomain proteins, Hox and Engrailed, have been shown to act in concert with the atypical homeodomain proteins Pbx and extradenticle. We now show that specific residues located within the Pbx homeodomain are essential for cooperative DNA binding with Hox and Engrailed gene products. Within the N-terminal region of the Pbx homeodomain, we have identified a residue that is required for cooperative DNA binding with three Hox gene products but not for cooperativity with Engrailed-2 (En-2). Furthermore, there are similarities between heterodimeric interactions involving the yeast mating type proteins MATa1 and MATalpha2 and those that allow the formation of Pbx/Hox and Pbx/En-2 heterodimers. Specifically, residues located in the a1 homeodomain that were previously shown to form a hydrophobic pocket allowing the alpha2 C-terminal tail to bind, are also required for Pbx/Hox and Pbx/En-2 cooperativity. Furthermore, we show that three residues located in the turn between helix 1 and helix 2, characteristic of many atypical homeodomain proteins, are required for cooperative DNA binding involving both Hox and En-2. Replacement of the three residues located in the turn between helix 1 and helix 2 of the Pbx homeodomain with those of the atypical homeodomain proteins controlling cell fate in the basidiomycete Ustilago maydis, bE5 and bE6, allows cooperative DNA binding with three Hox members but abolishes interactions with En-2. The data suggest that the molecular mechanism of homeodomain protein interactions that control cell fate in Saccharomyces cerevisiae and in the basidiomycetes may well be conserved in part in multicellular organisms.     

Glial cell line-derived neurotrophic factor differentially stimulates ret mutants associated with the multiple endocrine neoplasia type 2 syndromes and Hirschsprung's disease

Ret is a receptor tyrosine kinase involved in several neoplastic and developmental diseases affecting the thyroid gland and tissues of neuroectodermal origin. Different ret mutations are associated with different disease phenotypes. Gain-of-function of ret is caused by gene rearrangements in thyroid papillary carcinomas and by point mutations in multiple endocrine neoplasia (MEN) type 2A syndrome (MEN2A), in familial medullary thyroid carcinoma (FMTC), and in the more severe MEN2B syndrome. Conversely, Hirschsprung's disease (HSCR) is associated with loss of function of ret. Recently, it has been shown that glial cell line-derived neurotrophic factor (GDNF), by binding to the accessory molecule GDNFR-alpha, acts as a functional ligand of Ret and stimulates its tyrosine kinase and biological activity. To ascertain whether the biological effects of ret mutations are modulated by GDNF, we have investigated the responsiveness to GDNF of ret mutants in cell lines coexpressing GDNFR-alpha and MEN2A-, MEN2B-, FMTC-, or HSCR-associated ret mutants. Here, we show that triggering of GDNF affected only ret/MEN2B, i.e. it stimulated ret/MEN2B mitogenic and kinase activities, as well as its ability to phosphorylate Shc, a bona fide Ret substrate. In contrast, ret mutants associated with MEN2A or FMTC (carrying Cys634 or Cys620 mutations) were unresponsive to GDNF. HSCR mutations, by affecting either the extracellular or the intracellular Ret domain, impaired responsiveness to GDNF. These data suggest that the phenotype of human diseases caused by ret mutations can be differentially influenced by GDNF.