Successful induction of clinically competent dendritic cells from granulocyte colony-stimulating factor-mobilized monocytes for cancer vaccine therapy

Recent studies have suggested that dendritic cell (DC)-based immunotherapy is one promising approach for the treatment of cancer. We previously studied the clinical toxicity, feasibility, and efficacy of cancer vaccine therapy with peptide-pulsed DCs. In that study, we used granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood monocytes as a cell source of DCs. However, previous investigations have suggested that G-CSF-mobilized peripheral blood monocytes produce reduced levels of proinflammatory cytokines such as interleukin (IL)-12 and tumor necrosis factor (TNF)-α. These T helper (Th)-1-type cytokines are thought to promote antitumor immune response. In this study, we assessed the functional abilities of DCs generated from G-CSF-mobilized monocytes obtained from 13 patients with CEA-positive advanced solid cancers. Peripheral blood mononuclear cells were obtained from leukapheresis products collected before and after systemic administration of G-CSF (subcutaneous administration of high-dose [5–10 μg/kg] human recombinant G-CSF for five consecutive days). In vitro cytokine production profiles after stimulation with lipopolysaccharide (LPS) were compared between monocytes with and without G-CSF mobilization. DCs generated from monocytes were also examined with respect to cytokine production and the capacity to induce peptide-specific T cell responses. Administration of G-CSF was found to efficiently mobilize peripheral blood monocytes. Although G-CSF-mobilized monocytes (G/Mo) less effectively produced Th-1-type cytokines than control monocytes (C/Mo), DCs generated from G/Mo restored the same level of IL-12 production as that seen in DCs generated from C/Mo. T cell induction assay using recall antigen peptide and phenotypic analyses also demonstrated that DCs generated from G/Mo retained characteristics identical to those generated from C/Mo. Our results suggest that G-CSF mobilization can be used to collect monocytes as a cell source for the generation of DCs for cancer immunotherapy. DCs generated in this fashion were pulsed with HLA-A24-restricted CEA epitope peptide and administered to patients safely; immunological responses were induced in some patients.     

Amino-terminal amino acid sequence and chemical and functional properties of a membrane attack complex-inhibitory factor from human erythrocyte membranes

The protein corresponding to P-18 (Sugita et al. (1988) J. Biochem, 104, 633-637) was isolated from native human erythrocyte, and newly designated membrane attack complex-inhibitory factor (MACIF). The amino-terminal sequence of this protein was determined to be Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr. Endoglycosidase F digestion of MACIF decreased its molecular weight by about 6K on SDS-PAGE. On the other hand, endoglycosidase H, neuraminidase, or endo-alpha-N-acetylgalactosaminidase treatment had no effect on the molecular weight, indicating that MACIF has complex-type N-linked oligosaccharide chains, but no O-linked chain. MACIF was highly resistant against trypsin digestion and heat treatment. The inhibitory activity of MACIF on the hemolysis of EC5-8 cells was comparable to that on EC5-7 cells, indicating that MACIF inhibited the binding of C9 to the intermediate cells, or the subsequent C9 polymerization.     

Progesterone attenuates cocaine-induced responses

In this review, we summarize literature focused on how progesterone alters cocaine-induced psychomotor, reinforcement, and physiological responses. Clinical studies suggest that progesterone attenuates the subjective effects of cocaine. Similarly, preclinical studies have demonstrated that cocaine-induced reward and psychomotor responses are attenuated after progesterone administration. In rats progesterone also reduces the reinforcement effects of cocaine attenuates acquisition, escalation, reinstatement of cocaine self-administration, and cocaine-seeking behaviors. Progesterone also counteracts the facilitatory effects of estrogen on cocaine self-administration and psychomotor activation. These findings suggest that progesterone has a potential in clinical applications as a treatment for cocaine addiction. Constantly changing progesterone serum levels in female humans and rats affect the female's reinforcement responses to cocaine and may in part contribute to the known sex differences in cocaine responses.     

Behavior of the groundwater in a riverine-type mangrove forest

The behavior of groundwater and physical properties of bottom sediment in a riverine-type mangrove forest which is composed of a tidal creek and fringing mangrove swamps were investigated through field observations at Iriomote Island, Japan. After the tidal water ebbed from the swamp surface to the creek, groundwater levels at swamp sites near the creek fell by up to 15 cm by the next flood tide, although the fall was negligible at sites far from the creek and at the open coast outside the mangrove forest. The amount of groundwater discharged to the creek from the swamp depended strongly both on the tidal range and the presence of the steep bank which separates the tidal creek from the fringing mangrove swamp. Based on the fall of groundwater level, the bulk hydraulic conductivity of the swamp was estimated to be 1.5×10⁻² cm/s. This value is two to three orders of magnitude larger than that measured in a laboratory using small scale sediment core samples collected in the swamp. These results suggest that the presence of crowded, intricate and large animal burrows as well as sediment layers rich in mangrove humus increases permeability in the mangrove swamp. Further, it is suggested that the mangrove topography with the steep bank of the tidal creek plays an important role which enhances material exchanges through groundwater between the mangrove swamp and the adjacent offshore waters.     

Estradiol and progesterone differentially regulate formalin-induced nociception in ovariectomized female rats

Clinical and preclinical studies have found sex-specific differences in the discrimination and perception of inflammatory stimuli. The emerging picture suggests that the biological basis of these differences resides in the regulatory activity of gonadal hormones in the central nervous system. This study describes the effects of ovarian hormones in inflammatory pain processes. Ovariectomized rats received estradiol and/or progesterone, and the number of paw flinches was measured after 1, 2.5 or 5% formalin administration. Both estradiol and progesterone altered the number of flinches only after 1% formalin administration. Estradiol significantly reduced the overall number of flinches during Phase II of the formalin nociceptive response while progesterone attenuated Phase I of the response. After co-administration of estradiol and progesterone, progesterone reversed estradiol's analgesic effect in Phase II, however, estradiol did not reverse progesterone's analgesic activity in Phase I. To determine if estradiol effects are receptor-mediated, tamoxifen (selective estrogen receptor mediator, 15 mg/kg) or alpha-estradiol (an inactive isomer of estradiol, 20 microg) were utilized. Tamoxifen decreased the number of formalin-induced flinches during Phase II while alpha-estradiol did not affect any formalin-induced responses. When co-administered with estradiol, tamoxifen failed to reverse estradiol's effect, suggesting both tamoxifen and estradiol activate similar intracellular mechanisms. Although Western blot analysis detected the presence of estradiol alpha and beta and progesterone B receptors in the spinal cord, hormone replacement treatments had no effects on the levels of these receptors. We postulate that the mechanisms by which estradiol and progesterone induce analgesia occur through the activation of their receptor at the spinal cord level.     

Modes of overinitiation, dnaA gene expression, and inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia coli

The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.     

High pressure treatment under subfreezing temperature results in drastic inactivation of enveloped and non-enveloped viruses

Some viruses are sensitive to high pressure. The freeze-pressure generation method (FPGM) applies pressure as high as 250 MPa on a substance, simply by freezing a pressure-resistant reservoir in which the substance is immersed in water. Here we examined whether the FPGM successfully inactivates herpes simplex virus type 1 (HSV-1), an enveloped DNA virus belonging to the human Herpesviridae, and encephalomyocarditis virus (EMCV), an envelope-free RNA virus belonging to the Picornaviridae. After the treatment, HSV-1 drastically reduced the ability to form plaque in Vero cells in vitro as well as to kill mice in vivo. EMCV that had been pressurized failed to proliferate in HeLa cells and induce interferon response. The results suggest that the FPGM provides a feasible procedure to inactivate a broad spectrum of viruses.     

Investigating sources of variability in metabolomic data in the EPIC study: the Principal Component Partial R-square (PC-PR2) method

The key goal of metabolomic studies is to identify relevant individual biomarkers or composite metabolic patterns associated with particular disease status or patho-physiological conditions. There are currently very few approaches to evaluate the variability of metabolomic data in terms of characteristics of individuals or aspects pertaining to technical processing. To address this issue, a method was developed to identify and quantify the contribution of relevant sources of variation in metabolomic data prior to investigation of etiological hypotheses. The Principal Component Partial R-square (PC-PR2) method combines features of principal component and of multivariable linear regression analyses. Within the European Prospective Investigation into Cancer and nutrition (EPIC), metabolic profiles were determined by ¹H NMR analysis on 807 serum samples originating from a nested liver cancer case–control study. PC-PR2 was used to quantify the variability of metabolomic profiles in terms of study subjects age, sex, body mass index, country of origin, smoking status, diabetes and fasting status, as well as factors related to sample processing. PC-PR2 enables the evaluation of important sources of variations in metabolomic studies within large-scale epidemiological investigations.     

Temporal effects of estrogen and progesterone on behavioral and endocrinological responses to acute cocaine administration.

Estrogen and progesterone have been postulated to play a key role modulating cocaine-induced behavioral and neurochemical activation in female rats. This study investigated the temporal relationship between estrogen and progesterone in the modulation of cocaine-induced behavioral alterations. Ovariectomized Fischer rats received s.c. injections of estradiol benzoate 48 hr prior to cocaine or saline treatment and one s.c. injection of progesterone concurrently or 1, 4, 20, 24, 30, 44 or 48 hr after estrogen treatment. Forty-eight hours after estrogen treatment rats received either a single i.p. injection of 15 mg/kg of cocaine or 0.9% saline. Overall, cocaine induced increases in locomotor behaviors (ambulatory and rearing activity). A bimodal interaction between estrogen and progesterone was observed in the modulation of all locomotor activities. A gradual increase in behaviors, which peaked when progesterone was administered 24 hr after estrogen was followed by an inhibition of both ambulatory and rearing activity when progesterone was administered for a shorter period of time. This estrogen and progesterone interaction was not observed in the modulation of cocaine-induced stereotypic activity. However, shorter administration of progesterone in relation to estrogen administration resulted in lowered benzoylecgonine plasma levels when compared to longer progesterone administration times. On the other hand, longer administration of progesterone (48 hr of estrogen and progesterone) caused increases in corticosterone levels in cocaine-treated rats. Thus, the temporal interaction between estrogen and progesterone in the regulation of cocaine metabolism and hypothalamic-pituitary-axis (HPA) activation do not completely correlate with that observed for locomotor behavioral activation. Taken together, these results suggest that temporal interactions between estrogen and progesterone may underlie some of the previously reported estrous cycle and sex effects on cocaine-induced behavioral and endocrinological alteration.