Search

Charge-altering releasable transporters (CARTs) are a class of oligonucleotide delivery vehicles shown to be effective for delivery of messenger RNA (mRNA) both in vitro and in vivo. Here, we exploited the chemical versatility of the CART synthesis to generate CARTs containing the small-molecule drug fingolimod (FTY720) as a strategy to increase mRNA delivery and expression in lymphocytes through a specific ligand−receptor interaction. Fingolimod is an FDA-approved small-molecule drug that, upon in vivo phosphorylation, binds to the sphingosine-1-phosphate receptor 1 (S1P1), which is highly expressed on lymphocytes. Compared to its non-fingolimod-conjugated analogue, the fingolimod-conjugated CART achieved superior transfection of activated human and murine T and B lymphocytes in vitro. The higher transfection of the fingolimod-conjugated CARTs was lost when cells were exposed to a free fingolimod before transfection. In vivo, the fingolimod-conjugated CART showed increased mRNA delivery to marginal zone B cells and NK cells in the spleen, relative to CARTs lacking fingolimod. Moreover, fingolimod-CART-mediated mRNA delivery induces peripheral blood T-cell depletion similar to free fingolimod. Thus, we show that functionalization of CARTs with a pharmacologically validated small molecule can increase transfection of a cellular population of interest while conferring some of the targeting properties of the conjugated small molecule to the CARTs.

Targeting of numerous transmembrane proteins to the cell surface is thought to depend on their recognition by cargo receptors that interact with the adaptor machinery for anterograde traffic at the distal end of the Golgi complex. We report here on consortin, a novel integral membrane protein that is predicted to be intrinsically disordered, i.e. that contains large segments whose native state is unstructured. We identified consortin as a binding partner of connexins, the building blocks of gap junctions. Consortin is located at the trans-Golgi network (TGN), in tubulovesicular transport organelles, and at the plasma membrane. It directly interacts with the TGN clathrin adaptors GGA1 and GGA2, and disruption of this interaction by expression of a consortin mutant lacking the acidic cluster-dileucine (DXXLL) GGA interaction motif causes an intracellular accumulation of several connexins. RNA interference-mediated silencing of consortin expression in HeLa cells blocks the cell surface targeting of these connexins, which accumulate intracellularly, whereas partial depletion and redistribution of the consortin pool slows down the intracellular degradation of gap junction plaques. Altogether, our results show that, by studying connexin trafficking, we have identified the first TGN cargo receptor for the targeting of transmembrane proteins to the plasma membrane. The identification of consortin provides in addition a potential target for therapies aimed at diseases in which connexin traffic is altered, including cardiac ischemia, peripheral neuropathies, cataracts and hearing impairment. Sequence accession numbers. GenBank: Human CNST cDNA, NM_152609; mouse Cnst cDNA, NM_146105.

Auditory neuropathy is a particular type of hearing impairment in which neural transmission of the auditory signal is impaired, while cochlear outer hair cells remain functional. Here we report on DFNB59, a newly identified gene on chromosome 2q31.1-q31.3 mutated in four families segregating autosomal recessive auditory neuropathy. DFNB59 encodes pejvakin, a 352-residue protein. Pejvakin is a paralog of DFNA5, a protein of unknown function also involved in deafness. By immunohistofluorescence, pejvakin is detected in the cell bodies of neurons of the afferent auditory pathway. Furthermore, Dfnb59 knock-in mice, homozygous for the R183W variant identified in one DFNB59 family, show abnormal auditory brainstem responses indicative of neuronal dysfunction along the auditory pathway. Unlike previously described sensorineural deafness genes, all of which underlie cochlear cell pathologies, DFNB59 is the first human gene implicated in nonsyndromic deafness due to a neuronal defect.

Gjb2andGjb6,two contiguous genes respectively encoding the gap junction protein connexin26 (Cx26) and connexin 30 (Cx30) display overlapping expression in the inner ear. Both have been linked to the most frequent monogenic hearing impairment, the recessive isolated deafness DFNB1. Although there is robust evidence for the direct involvement of Cx26 in cochlear functions, the contribution of Cx30 is unclear since deletion ofCx30strongly downregulatesCx26both in human and in mouse. Thus, it is imperative that any role of Cx30 in audition be clearly evaluated. Here, we developed a newCx30knock-out mouse model (Cx30Δ/Δ) in which half ofCx26expression was preserved. Our results show thatCx30andCx26coordinated expression is dependent on the spacing of their surrounding chromosomic region, and thatCx30Δ/Δmutants display normal hearing. Thus, in deaf patients withGJB6deletion as well as in the previousCx30knock-out mouse model, defectiveCx26expression is the likely cause of deafness, and in contrast to current opinion, Cx30 is dispensable for cochlear functions.

Lipopolysaccharide (LPS) injected into the trachea of rats was found to induce the secretion of leukemia inhibitory factor (LIF) into bronchoalveolar lavage (BAL) fluid with a maximum expression of LIF after 2-12 h. The acute pulmonary neutrophilic inflammation caused by the intratracheal injection of bacterial endotoxin (LPS) could be inhibited by the intratracheal coinjection of recombinant LIF. Compared with intratracheal injection of LPS alone, intratracheal coinjection of LIF and LPS decreases the number of BAL neutrophils obtained 6 h later by approximately 50% (P < 0.0001). LIF decreased the amount of the proinflammatory cytokine tumor necrosis factor (TNF), but not the amount of the anti-inflammatory cytokine interleukin (IL)-6, in the BAL fluid of LPS-injected rats. Similarly, intravenous LIF was found to decrease TNF expression, but increase IL-6 expression, in the serum of rats receiving intravenous LPS. Intravenous LIF, even in the absence of LPS, was found to cause IL-6 expression. In conclusion, intratracheal LPS initiates the secretion of endogenous LIF into the alveolar space where LIF may contribute to the downregulation of LPS-initiated acute neutrophilic inflammation by downregulating expression of TNF. LIF may down-regulate LPS-initiated TNF expression at least in part indirectly by upregulating expression of IL-6, a cytokine known to downregulate LPS-initiated TNF expression.

A novel megakaryocyte growth and development factor (MGDF) has been identified in aplastic canine plasma, and its cDNAs have been cloned from canine, murine, and human sources. Purified canine MGDF isolated by procedures involving MpI receptor affinity chromatography exists in at least two forms, with apparent molecular masses of 25 kDa and 31 kDa, that share the N-terminal amino acid sequence APP-ACDPRLLNKMLRDSHVLH. Human, dog, and mouse cDNAs for MGDF are highly conserved and encode open reading frames for proteins of 353, 352, and 356 amino acids, respectively, including predicted signal peptides. Canine MGDF and recombinant human MGDF support the development of megakaryocytes from human CD34+ progenitor cell populations in liquid culture and promote the survival of a factor-dependent murine cell line (32D) engineered to express MpI. These biological activities are blocked by the soluble extracellular domain of MpI. These data demonstrate that MGDF is a novel cytokine that regulates megakaryocyte development and is a ligand for the MPI receptor.

To ascertain whether a spirometer can measure tidal volume (TV) during high frequency oscillatory ventilation (HFOV) and high frequency jet ventilation (HFJV), and to analyse the effect of changes in ventilator settings.The study was performed with paediatric porcine lung models submitted to HFOV with a Sensormedics 3100 ventilator and HFJV with a Paravent Pat(e)(R) ventilator connected to a D-Fend spirometer. Programmed frequency, amplitude, and mean airway pressure (MAP) were changed in the ventilator, and TV and pressures were recorded using the spirometer.The spirometer measured TV in the paediatric lung models and piglets, but could not measure TV less than 8 ml, when the pressure amplitude was higher than 55 cmH(2)O or the MAP was higher than 30 cmH(2)O. With HFOV there was a correlation between amplitude and tidal volume, and a positive correlation between pressure and TV with HFJV. With both respirators there was a negative correlation between frequency and TV.The D-Fend spirometer can measure tidal volume and pressure during HFOV and HFJV. However, it does not work with volumes lower than 8 ml, and high amplitude or mean airway pressure.

1. The articular capsule or ligament, of the primary spines of the sea-urchin Eucidaris tribuloides behaves as a classical excitable tissue, responding with a shortening or contracture to a variety of stimuli including the cholinergic agonists acetylcholine (ACh), methacholine (MeACh), carbamylcholine (CCh) and nicotine. 2. d-Tubocurarine failed to decrease the amplitude of the acetylcholine-induced contractures, while the contractures elicited by methacholine were blocked by atropine. Neostigmine, but not eserine, increased the amplitude of ACh-induced contractures, suggesting the presence of AChE in the preparation. 3. The cholinergic agonists induced contracture of the ligament, but had quite different kinetics, the rate of rise of the contracture being fastest for ACh and decreasing in the following order: CCh, MeACh and nicotine. 4. Tyramine and octopamine exert an inhibitory action on "catch" and a relaxing effect on the contracting ligament. 5. The time courses of the contractures elicited in the same preparation were virtually identical to those of "catch". Therefore, we propose that "catch" and contracture are only two different aspects of the same phenomenon; namely the contracture of the muscle fibers present in the ligament.

1. This paper describes the effects of several cholinergic agonists and antagonists, and of β-phenylethylamine (PEA) and some of its derivatives, on the articular capsule, or ligament, of the primary spines of Eucidaris tribuloides. 2. Carbamylcholine (CCh), methacholine (MeACh), nicotine, and muscarine exert a stiffening effect similar to that of acetylcholine (ACh), although the time course of their actions varies widely. 3. Atropine induced stiffening and blocked and responses to muscarine and MeACh. The responses to MeACh were blocked also by 4-diphenylacetoxy-N-methylpiperidine, suggesting the presence in the ligament of type M3 muscarinic receptors, in addition to nicotinic ones. d-Tubocurarine induced stiffness of the ligament and failed to block the responses to ACh and nicotine. 4. While ACh induced only a slight desensitization, CCh caused a long-lasting blockade of the stiffening effects of the cholinergic agonists. This shows that the receptors for ACh have a site or sites that recognize the ester moieties of these molecules. 5. Eserine and neostigmine potentiate the responses to acetylcholine, indicating the presence of aeetyl-cholinesterase in the ligament. 6. β-Phenylethy lamine, epinephrine, norepinephrine, and dopamine induce diphasic responses; usually a brief softening followed by a slow and irreversible stiffening of the ligament. 7. In contrast to the above, tyramine and octopamine elicit a simple softening of ligaments which are stiff as a result of handling or by exposure to cholinergic agonists. However, tyramine and octopamine do not soften ligaments which become stiff as a result of exposure to adrenergic agonists.

The similar proximal and distal attachments to the stereom of primary spine ligament in the echinoid Eucidaris tribuloides are described, from thin sections and SEM studies on frozen and fractured spine articulations and ligaments from decalcified material. The orthogonal structure of the general stereom is modified on the attachment zones where bundles of collagen cylinders enter approximately hexagonally arranged channels. Straps of collagen extend in parallel series between adjacent bundles via regularly placed ports and collagen loops rather than non-striated 'tendons' pass over skeletal trabeculae. The regular pattern of collagen straps is most evident on the proximal and distal attachment zones. Mechanical features of the non-adhesive mode of attachment are considered, together with similarities and differences between insertion of muscle cells and mutable collagenous tissue (ligament) in echinoderms.

The mCAT-1 gene encodes a basic amino acid transporter that also acts as the receptor for murine ecotropic leukemia viruses. Targeted mutagenesis in embryonic stem cells has been used to introduce a germ-line null mutation into this gene. This mutation removes a domain critical for virus binding and inactivates amino acid transport activity. Homozygous mutant pups generated from these cells were approximately 25% smaller than normal littermates, very anemic, and died on the day of birth. Peripheral blood from homozygotes contained 50% fewer red blood cells, reduced hemoglobin levels, and showed a pronounced normoblastosis. Histological analyses of bone marrow, spleen, and liver showed a decrease in both erythroid progenitors and mature red blood cells. Mutant fetal liver cells behaved normally in in vitro hematopoietic colony-forming assays but generated an anemia when transplanted into irradiated C.B.-17 SCID mice. Furthermore, reconstitution of the white cell compartment of SCID mice by mutant fetal liver cells was less complete than that observed with a mixed population of wild-type and heterozygous fetal liver cells. Primary embryo fibroblasts from mutant mice were completely resistant to ecotropic retrovirus infection. Thus, mCAT-1 not only appears to be the sole receptor for a group of murine ecotropic retroviruses associated with hematological disease but also plays a critical role in both hematopoiesis and growth control during mouse development.

PEG-rHuMGDF injected daily in normal mice causes a rapid dose-dependent increase in megakaryocytes and platelets. At the same time that platelet numbers are increased, the mean platelet volume (MPV) and platelet distribution width (PDW) can be either decreased, normal, or increased depending on the dose and time after administration. Thus, PEG-rHuMGDF at a low dose causes decreases in MPV and PDW, MGDF at an intermediate dose causes an initial increase followed by a decrease in MPV and PDW, and PEG-rHuMGDF at higher doses causes an increase in MPV and PDW followed by a gradual normalization of these platelet indices. In addition to the expected thrombocytosis after 7 to 10 days of daily injection of high doses of PEG-rHuMGDF, a transient decrease in peripheral red blood cell numbers and hemoglobin is noted accompanied in the bone marrow by megakaryocytic hyperplasia, myeloid hyperplasia, erythroid and lymphoid hypoplasia, and deposition of a fine network of reticulin fibers. Splenomegaly, an increase in splenic megakaryocytes, and extramedullary hematopoiesis accompany the hematologic changes in the peripheral blood and marrow to complete a spectrum of pathologic features similar to those reported in patients with myelofibrosis and megakaryocyte hyperplasia. However, all the PEG-rHuMGDF-initiated hematopathology including the increase in marrow reticulin is completely and rapidly reversible upon the cessation of administration of PEG-rHuMGDF. Thus, transient hyperplastic proliferation of megakaryocytes does not cause irreversible tissue injury. Furthermore, PEG-rHuMGDF completely ameliorates carboplatin-induced thrombocytopenia at a low-dose that does not cause the hematopathology associated with myelofibrosis.

Megakaryocyte growth and development factor (MGDF) administered intraperitoneally (IP) to mice causes a dose-dependent thrombocytosis accompanied by a decrease in mean platelet volume. MGDF increases the number of megakaryocytes in the bone marrow and spleen. MGDF does not affect the circulating number of leukocytes. Carboplatin, a chemotherapeutic agent that causes thrombocytopenia in humans, administered to mice as a single IP injection at a nonlethal dose causes a significant, but reversible thrombocytopenia. The carboplatin-induced thrombocytopenia is accompanied by an increase in circulating endogenous MGDF that precedes the return of circulating platelets to a normal level. MGDF mRNA is constitutively present in the liver. After carboplatin treatment, hepatic MGDF mRNA does not increase in concordance with circulating MGDF. Circulating soluble MGDF receptor levels (c-mpl) do not change significantly during the course of carboplatin-induced thrombocytopenia. MGDF injected IP once daily beginning 1 day after injection of carboplatin reverses carboplatin-induced thrombocytopenia in a dose-dependent fashion. The normalization of circulating platelet numbers in carboplatin plus MGDF-treated mice is accompanied by a normalization of megakaryocyte numbers in the bone marrow. In conclusion, MGDF, by increasing the number of marrow megakaryocytes and circulating platelets is an effective therapy for carboplatin-induced thrombocytopenia in mice.

Stem cell factor (SCF) administered as daily bolus injections in dose-response experiments in mice causes a progressive and dramatic dose-dependent panleukocytosis characterized by neutrophilia, eosinophilia, monocytosis, and lymphocytosis. SCF causes circulating platelet numbers to be dose-dependently increased after 2 weeks of daily injections. Leukemia inhibitory factor (LIF) administered as daily bolus injections in mice causes a peripheral leukopenia that is largely due to peripheral lymphopenia. LIF causes thrombocytosis peaking after approximately 1 w. Coinjection of SCF and LIF for 1 to 2 wk in mice does not cause a much greater thrombocytosis than the maximum thrombocytosis achievable with SCF or LIF alone. On the other hand, daily injection of SCF for 5 days followed by daily injection of LIF for 5 to 6 d in mice causes a very substantial increase in platelets that was lineage-specific in terms of not being accompanied by a generalized leukocytosis. In contrast, only a very modest thrombocytosis was noted in SCF-primed LIF-treated rats. LIF causes a large increase in the cytoplasmic volume of splenic megakaryocytes in mice, but not in rats. In conclusion, SCF-induced priming followed by LIF-induced maturation of megakaryocytes causes a substantial selective increase in the numbers of circulating platelets in mice.

Maternally administered recombinant human granulocyte colony- stimulating factor (rhG-CSF) has been shown to cross the placenta and induce a peripheral neutrophilia and increases in the marrow and spleen neutrophil storage pools in fetal and newborn rats. In the present study, we have used this model system to investigate the efficacy of prenatally administered rhG-CSF on neonatal defense to a lethal challenge with Group B-beta hemolytic Streptococcus (GBS). Pregnant rats were injected with rhG-CSF twice daily beginning 6 days before parturition. At birth, all pups were infected with a dose of GBS that is lethal for 90% of infected pups (LD90). Survival was monitored daily for 5 days. Survival of infected pups from saline-treated mothers beyond 60 hours after infection was 10%. No difference in survival was observed among pups from mothers treated 2 and 4 days before parturition. In contrast, we determined that survival was 82.5% among infected pups from mothers treated for 6 days before parturition with rhG-CSF. Our results demonstrate that maternal administration of rhG- CSF augments neonatal defenses against a lethal bacterial challenge.

We studied the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration to pregnant rats upon fetal and neonatal myelopoiesis. Pregnant rats were treated with rhG-CSF twice daily for 2, 4, and 6 days before parturition. rhG-CSF crossed the placenta and reached peak fetal serum concentrations 4 hours after administration. Peak fetal serum levels were 1,000-fold lower than levels detected in the dam. Hematopoietic effects of rhG-CSF were assessed by cytologic analysis of the newborn blood, spleen, bone marrow, thymus, and liver. White blood cell counts were increased twofold to fourfold in newborns. This increase was due to circulating numbers of polymorphonuclear cells (PMN). rhG-CSF induced a myeloid hyperplasia in the newborn marrow consisting of immature and mature myeloid cells in the day-2 and day-4 treated pups. Bone marrow of pups treated for 6 days contained mostly hyper-segmented PMN with little or no increase in myeloid precursors. An increase in the number of postmitotic (PMN, bands, and metamyelocytes) and mitotic (promyeloblasts, myeloblasts, and metamyeloblasts) myeloid cells in the spleen of neonates was observed. No change was detected in splenic lymphocytes or monocytes. No effect of rhG-CSF was noted in the newborn liver or thymus. These results demonstrate that maternally administered rhG-CSF crosses the placenta and specifically induces bone marrow and spleen myelopoiesis in the fetus and neonate. The significant myelopoietic effects of rhG-CSF at low concentrations in the fetus suggest an exquisite degree of developmental sensitivity to this cytokine and may provide enhanced defense mechanisms to the neonate.

The active ion transport is related to some vital functions for the normal eukaryotic cell metabolism. The study of transport mechanism and its regulation is a fundamental problem in cellular biology. The production of monoclonal antibodies (AcM) let us to have a probe, with large specificity, for the study of the localization, distribution and function of carrier proteins in the epithelium, as well as the study of its molecular structure, synthesis and assembling in the cell. The quality of a monoclonal antibody depends of the proper selection of each step to follow in the course of its production. In this article, we examine the strategy more currently used in the production of monoclonal antibodies and its direct use in the ionics pump's morphological, biochemical and physiological study.

The intravenous injection of rrSCF causes neutrophilia and lymphocytosis as well as the appearance of immature myeloid cells and occasional blast cells in the circulation. The marrow shows a left-shifted myeloid and erythroid hyperplasia as evidenced by increases in numbers of morphologically recognizable early myeloid and erythroid precursors. A decrease in the number of mature marrow neutrophils is noted, suggesting that the release of marrow neutrophils contributes to the peripheral neutrophilia. After 2 weeks of daily injections of rrSCF, the marrow demonstrates a remarkable mast cell hyperplasia accompanied by erythroid and lymphoid hypoplasia. rrSCF causes mast cells to appear in the circulation and causes a systemic increase in embryonic connective tissue-type mast cells. In vitro long-term culture of mouse marrow cells with rrSCF results in an outgrowth of mast cells. The coinjection of rrSCF and G-CSF for 1 week causes a synergistic increase in mature marrow neutrophils accompanied by a striking decrease in erythroid and lymphoid marrow elements. Spleens demonstrate increased granulopoiesis as well as erythropoiesis as compared to the spleens of rats treated with single growth factors. Splenic extramedullary erythropoiesis may act to compensate for the decrease in marrow erythropoiesis. The coinjection of rrSCF and G-CSF causes an increase in marrow mast cells at the end of 1 week, but the increase is much less than in rats treated with rrSCF alone. The combination of rrSCF and G-CSF causes a synergistic peripheral neutrophilia. In vivo daily administration of SCF plus GM-CSF results in a synergistic increase in marrow neutrophils, but not the striking synergistic increase in circulating neutrophils that is observed after SCF plus G-CSF. Colony-forming assays reveal a synergistic increase in CFU-GMs in the marrow, but not in peripheral blood, after coincubation with SCF plus GM-CSF as opposed to GM-CSF alone, demonstrating anatomic compartmentalization between a more primitive marrow CFU-GM subset and a more mature peripheral blood CFU-GM subset.

Recombinant rat stem cell factor (rrSCF) and recombinant human granulocyte colony-stimulating factor (G-CSF) coinjected for 1 week in rats cause a synergistic increase in mature marrow neutrophils accompanied by a striking decrease in erythroid and lymphoid marrow elements. The spleens of the same rats show increased granulopoiesis as well as increased erythropoiesis as compared with the spleens of rats treated with either growth factor alone. Splenic extramedullary erythropoiesis may act to compensate for the decrease in marrow erythropoiesis. The coinjection of rrSCF and G-CSF causes an increase in marrow mast cells at the end of 1 week, but the increase is much less than in rrSCF-alone-treated rats. The combination of rrSCF and G- CSF increases the rate of release of marrow neutrophils into the circulation and causes a dramatic synergistic peripheral neutrophilia, beginning especially after 4 days of treatment. Colony-forming assays of all experimental groups showed a synergistic increase in colony- forming unit granulocyte-macrophage (CFU-GM) in the marrow, but not in peripheral blood, after coincubation with SCF plus granulocyte- macrophage CSF (GM-CSF) as opposed to GM-CSF alone, showing anatomic compartmentalization between a more primitive marrow CFU-GM subset and a more mature peripheral blood CFU-GM subset. In vivo daily administration of SCF plus GM-CSF results in a synergistic increase in marrow neutrophils, but not the striking synergistic increase in circulating neutrophils that is observed with SCF plus G-CSF.

Although the morphology of the hematopoietic and circulating blood cells of the rat differs slightly from that of human blood cells, the basophil is the only human blood cell for which the rat does not have a readily recognizable counterpart. The morphological classification of the rat's marrow and peripheral blood cells as based on our experience is described with reference to the differing observations of previous investigators and with comparison to the human hematopoietic system. Examination of the bone marrow and peripheral blood demonstrated that control rats injected i.v. with 1% normal rat serum in sterile saline exhibit a moderate transient lymphopenia as the only significant hematologic fluctuation induced by the stresses of injection, general anesthesia, and tail bleeding. Morphologic quantitation of marrow and peripheral blood cells complements the insights obtained by the culture of marrow cells. An appreciation of the morphology of the rat's blood cells and of the reproducible and quantitative nature of marrow and peripheral blood smear examinations may lead to more in vivo investigations of hematopoiesis and leukocyte trafficking.

Recombinant rat stem cell factor (rrSCF) administered to rats as a single intravenous injection causes a dose-dependent neutrophilia and lymphocytosis as well as the appearance of immature myeloid cells and occasional blast cells in the circulation. Neutrophilia begins at 2 hours, peaks at 4 to 6 hours, and subsides between 12 and 24 hours. Lymphocytosis occurs at 0.5 hours and has subsided by 2 hours. rrSCF-induced neutrophilia and lymphocytosis are abrogated by boiling, demonstrating that endotoxin-contamination of the rrSCF preparation is not responsible for the observed hematologic effects. The bone marrow at 6 hours after injection of rrSCF shows a left-shifted myeloid and erythroid hyperplasia as evidenced by significant increases in the absolute numbers of morphologically recognizable early myeloid and erythroid precursors. A concurrent decrease in the absolute numbers of mature marrow neutrophils is noted, suggesting that the release of marrow neutrophils contributes to the peripheral neutrophilia. After 2 weeks of daily injections of rrSCF, bone marrow smears demonstrate a remarkable mast cell hyperplasia accompanied by a decrease in total marrow cellularity and by a striking erythroid and lymphoid hypoplasia. rrSCF also causes mast cells to appear in the circulation and causes a systemic increase in embryonic connective tissue-type, but not mucosal-type, mast cells. In vitro long-term culture of lineage-depleted mouse bone marrow cells with rrSCF results in an almost pure outgrowth of mast cells.

Endotoxin (LPS) at sublethal doses injected i.v. into rats was found to induce IL-6 mRNA expression peaking at 1 to 2 h in whole organ RNA preparations of the spleen, liver, lung, bowel, and kidney. IL-6 serum protein levels also peaked at 2 h. TNF and IL-1, generally considered to be among the most rapidly released cytokines, also induced IL-6 expression. IL-6 in turn inhibited TNF and IL-1 expression, suggesting that IL-6 may be part of a negative feedback mechanism in the cytokine cascade. Dexamethasone down-regulated and Corynebacterium parvum up-regulated IL-6 expression, although the possibility cannot be excluded that these immunomodulating factors may in part have exerted their effects indirectly via the up- and down-regulation of TNF and IL-1. IL-6 injected i.v. at a pathophysiologically relevant dose caused a peripheral neutrophilia and mild myeloproliferative effect in the bone marrow.

Endotoxin, a lipopolysaccharide (LPS) component of gram-negative bacteria, induces alveolar macrophages to express interleukin-1 (IL-1). Lipopolysaccharide and IL-1 both cause severe acute neutrophilic inflammation in the lung after intratracheal injection, suggesting that LPS-induced IL-1 expression contributes to the pathogenesis of LPS-induced acute inflammation. In the present study, the role of IL-1 in LPS-induced acute pneumonia was investigated by quantitating the acute inflammation occurring at 6 hours after the intratracheal injection of LPS as compared to the same timepoint after the intratracheal coinjection of LPS and IL-1 receptor antagonist (IL-1ra). The IL-1ra was found to inhibit LPS-induced acute inflammation (P greater than 0.0001) as measured by the number of neutrophils recovered in bronchoalveolar lavage. The LPS-induced emigration of neutrophils was inhibited by as much as 45%. Recombinant IL-1 beta-induced neutrophil emigration into the lung was inhibited by 95% when IL-1ra was coinjected intratracheally with IL-1 beta. Coinjection of recombinant IL-1 beta and LPS increased the neutrophilic exodus as compared to the intratracheal injection of either agent alone. Intratracheal injection of LPS induces a progressive increase in IL-1ra mRNA expression in whole-lung RNA preparations, suggesting that endogenous IL-1ra may play an important role as a negative feedback mechanism to downregulate LPS initiated IL-1-mediated acute inflammation. In conclusion IL-1ra inhibits both LPS- and IL-1-induced neutrophilic inflammation and may therefore prove clinically useful as an anti-inflammatory agent for the therapy of either septic or aseptic IL-1-mediated acute inflammation.

The purpose of this investigation was to compare the erythropoietic effects of recombinant interleukin 6 (IL-6) and recombinant erythropoietin (EPO) on the marrow and peripheral blood in vivo. IL-6 administered to rats as a single i.v. injection induces a selective erythroid hyperplasia of the marrow's late normoblasts at 12 and 24 h with a return to preinjection numbers of normoblasts at 48 and 72 h. The hyperplasia of late normoblasts in the marrow is accompanied by a left-shifted peripheral reticulocytosis. Daily injection of IL-6 does not induce any effects on the erythroid population of the marrow or circulation beyond those of a single injection. After daily administration of IL-6 for 4 or 7 days, the erythroid differential in the marrow and the peripheral reticulocyte count are equal to negative control values, indicating a rapid tachyphylaxis to the erythropoietic effect of IL-6. In contrast to IL-6, EPO administered as a single i.v. injection induces a panerythroid marrow hyperplasia with sequential peak increases in pronormoblasts and early normoblasts at 24 h, intermediate normoblasts at 24-48 h, and late normoblasts at 72 h. The peripheral reticulocyte count mirrors the development of erythroid precursors in the marrow by demonstrating an increasing left-shifted reticulocytosis between 24 and 72 h. Daily injection of EPO for 7 days induces a striking erythroid hyperplasia and a myeloid hypoplasia in the marrow. In summary, IL-6 in vivo is a differentiation factor that rapidly induces tolerance to its own effect, whereas EPO in vivo affects all stages of erythropoiesis and sustains erythropoiesis indefinitely. IL-6 may be one of the non-EPO factors in pokeweed mitogen spleen cell-conditioned medium that has been reported by previous investigators to enhance erythropoiesis, although many of those factors were thought to act upon an earlier stage of erythropoiesis. IL-6 is unlikely to exert an indirect erythropoietic effect in vivo via the induction of EPO because the sera of IL-6-treated rats did not contain elevated levels of EPO and because the effects of exogenously administered IL-6 and EPO are so different.

Recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was noted to support rat bone marrow colony formation in vitro. The in vivo hematologic effects of a single intravenous injection of murine GM-CSF were therefore investigated. Doses of murine GM-CSF between 0.1 and 5 micrograms/rat caused an increasing leukocytosis that did not further increase with a dose of 25 micrograms/rat. In contrast, human GM-CSF at 25 micrograms/rat did not induce any significant peripheral hematologic effects. Murine GM-CSF induced peripheral neutrophilia and monocytosis, peaking between 4 and 8 hours and subsiding to baseline by 12 hours. Neutropenia and monocytopenia, which reached a nadir at 15 minutes, preceded the leukocytosis, suggesting that GM-CSF activates these leukocytes and causes transient intravascular margination. A mild lymphopenia occurred between 2 to 8 hours. The bone marrow at 6 hours after injection of GM-CSF demonstrated a variable and slight left-shifted myeloid hyperplasia most noticeable at the level of promyelocytes and myelocytes, suggesting a myeloproliferative effect. The marrow at 6 hours also demonstrated a decrease in mature neutrophils, documenting that the marrow contributes to the increased number of circulating neutrophils. Once-daily injection of GM-CSF for 7 days induced a repetitive daily neutrophilia of the same magnitude. The marrow after 1 week of injections did not show a generalized myeloid hyperplasia, but did show an increase in eosinophils and a decrease in lymphocytes. Granulocyte-macrophage colony-stimulating factor plus granulocyte colony-stimulating factor (G-CSF) have been reported to synergize in vitro in both mouse and human bone marrow colony assays. However GM-CSF plus G-CSF in vivo, administered as either a single injection or as daily injections for 1 week, were found in the present study to induce, at most, an additive effect on circulating numbers of neutrophils. It is concluded that murine GM-CSF will be useful in the rat model to study the in vivo hematoreconstitutive effects of GM-CSF alone and in combination with other hematologic growth factors. The relatively rapid kinetics and lesser magnitude of GM-CSF-induced neutrophilia and monocytosis, as compared to G-CSF and M-CSF, respectively, and the lesser myeloproliferative effect of GM-CSF in bone marrow smears, as compared to G-CSF, might be taken to suggest that GM-CSF's natural activity is predominantly as an inflammatory rather than a myeloproliferative factor.

Recombinant human tumor necrosis factor alpha (TNF) administered i.v. to Lewis rats as a daily low dose for 1 week induces an erythroid hyperplasia of late normoblasts. Although the erythroid marrow compartment is hyperplastic, the morphology of the normoblasts is dysplastic and there is no accompanying increase in circulating red blood cells, suggesting a state of ineffective erythropoiesis. TNF administered on the same daily schedule as a high dose induces an erythroid hypoplasia of late normoblasts and a peripheral anemia with decreases in the hematocrit and hemoglobin. A tremendous myeloid hyperplasia is noted in rats treated with high-dose TNF, and the mechanism of the erythroid anemia may be in part due to the increase in neutrophils. In support of the hypothesis that the erythroid anemia may be partly myelophthisic in nature, a decrease in marrow lymphocytes was also noted. On the other hand, the dysplastic morphology of the late erythroid precursors in rats treated with low-dose TNF would also be consistent with a destructive effect of TNF on erythroid precursors as a mechanism of TNF-related anemia. In light of the in vitro inhibitory effects of TNF on erythropoiesis and myelopoiesis as reported by previous investigators, the erythroid and myeloid hyperplasia noted in vivo most likely represent indirect effects.

Macrophage colony-stimulating factor (recombinant human M-CSF) given as a single intravenous injection to Lewis rats induces a dose-dependent peripheral monocytosis, neutrophilia, and lymphopenia. The monocytosis peaks at 28 to 32 hours with a seven- to eightfold increase in the number of circulating monocytes and promonocytes. The peripheral monocytosis is accompanied by a slight increase in marrow blasts, promonocytes, and monocytes. A monocytopenia reaching a nadir at 15 minutes precedes the monocytosis, suggesting that M-CSF activates circulating monocytes and causes intravascular margination. The M-CSF-induced neutrophilia and lymphopenia are relatively mild in magnitude, are observed between 2 and 16 hours after injection, and are no longer evident at later time-points. The monocytosis was at least partially inhibited by dexamethasone. M-CSF-induced monocytosis most likely reflects a direct effect of M-CSF on marrow monocyte precursor proliferation, maturation, and release, whereas the neutrophilia and lymphopenia may reflect indirect effects mediated by the known ability of M-CSF to cause the release of other cytokines.

Multi-colony stimulating factor (Multi-CSF, interleukin-3, IL-3) and granulocyte-CSF (G-CSF) administered concurrently as an intravenous (IV) injection induce a peripheral neutrophilia that is approximately additive in comparison with the neutrophilia induced by IL-3 and G-CSF individually. The bone marrow (BM) at 12 hours is depleted of mature neutrophils and shows a left-shifted myeloid hyperplasia, consistent with the neutrophil-releasing and myeloproliferative activities of both IL-3 and G-CSF individually. The BM at 24 hours shows a replenished reserve of mature neutrophils and a synergistic left-shifted myeloid hyperplasia as compared with IL-3 and G-CSF alone. Daily IV injections of IL-3 plus G-CSF for 1 week also induce an approximately additive daily peripheral neutrophilia. The BM after a week's administration of IL-3 plus G-CSF shows a generalized myeloid hyperplasia with a synergistic increase in mature neutrophils as compared with IL-3 or G-CSF alone. Daily injection of IL-3 plus G-CSF induced a significant decrease in erythroid, lymphoid, and eosinophilic marrow precursors, possibly owing to a myelophthisic effect of the myeloid hyperplasia and despite the fact that IL-3 alone induced a significant erythroid hyperplasia.

The principle of economy that emerged from the views of William of Occam, namely that where alternative hypotheses exist, the one involving the fewest assumptions is to be preferred, is still valid in science. Indeed, it was the application of "Occam's razor" that led us to propose a new model (1) to explain that mechanism of catch contraction in the primary spines of Eucidaris tribuloides--the model that I. C. Wilkie (2) considers inadequate through its failure to invoke collagen mutability.

The electrical and mechanical responses of body, leg and jaw muscle of Peripatus to electrical excitation of their motor-nerves were examined. A small twitch was obtained from each muscle, whose strength increased stepwise with increasing stimulus strength. In jaw and body muscles as many as ten increments in height were obtained with increasing stimulus strength. Only a single twitch height was obtained from the claw retractor muscle. Tetanus:twitch ratio under supraminal stimulation was less than 2:1 for jaw muscles, about 50:1 for the claw retractor and about 6:1 for muscles moving the legs. The jaw-muscle twitch duration was 0·6 s, that for the leg muscles 1·2 s and for body muscles about 3·0 s. Large miniature junctional potentials were frequently recorded regardless of electrode location. Responses to neural stimulation consisted of small junctional potentials whose height was progressively increased with increasing stimulus strength, generally with three steps. With repetitive stimulation, facilitation of the second and third junctional potentials occurred, plus summation. A few fibres gave spikes to a single shock : most gave a few spikes sporadically, during repetitive stimulation only. No abrupt tension increments occurred in whole muscles when individual fibres spiked. We saw no evidence for peripheral inhibitory axons. The excitation of Peripatus muscle is by local graded junctional potentials at distributed nerve-on-muscle fibre synapses, together with action potentials. The latter are initiated only by larger junctional potentials compounded of multiple smaller ones summated and/ or facilitated. The details of neuromuscular physiology are not compatible with the phylogenetic status commonly proposed for Peripatus.

Stable prostaglandin analogs are known to induce lymphopenia and neutrophilia in a dose-dependent fashion after subcutaneous injection in rats. The purpose of the present investigation is to determine whether the prostaglandin-induced changes in circulating leukocytes might be secondary to hypotension with the ensuing release of adrenal hormones. The adrenal medullary catecholamine epinephrine was found to induce neutrophilia in both intact and adrenalectomized rats, and the glucocorticosteroid analog dexamethasone induced a profound lymphopenia in rats as reported by previous investigators. A stable analog of PGF2α (15-S-15-methyl PGF2α; M-PGF2α) at the dose of 1 mg/kg induced marked systemic hypotension 1 h after injection, with lymphopenia and neutrophilia 6 h after injection. The non-prostanoid hypotensive agent captopril, at a dose of 63 mg/kg, induced a hypotension of similar magnitude and kinetics to that induced by prostaglandin. Captopril also induced lymphopenia and neutrophilia at 6 h, although the neutrophilia was of lesser magnitude than that induced by prostaglandins. The prostaglandin-induced lymphopenia was found to be mediated, at least in part, by the hypotension-induced release of adrenal hormones, as evidenced by the abrogation of lymphopenia in prostaglandin-treated adrenalectomized rats. Captopril-treated adrenalectomized rats, however, did develop a significant lymphopenia, suggesting that hypotension can result in lymphopenia even in adrenalectomized rats. The M-PGF2α-induced neutrophilia in adrenalectomized rats, by comparison to captopril-induced neutrophilia in adrenalectomized rats, was greater than the neutrophilia expected as the result of hypotension alone. Indeed, the M-PGF2α-induced neutrophilia in adrenalectomized rats was greater than the captopril-induced neutrophilia in sham-adrenalectomized rats. Thus, a portion of the neutrophilia induced by M-PGF2α in intact rats may be mediated through adrenal-independent, hemodynamic-independent mechanisms. The possibility that M-PGF2α might be inducing neutrophilia via an endotoxin-like stress reaction was investigated by examining changes in circulating white blood cells in intact and adrenalectomized C3H/HeN (endotoxin-sensitive) and C3H/HeJ (endotoxin-resistant) mice after prostaglandin administration. No quantitative differences in the prostaglandin-induced neutrophilia were noted in C3H/HeJ mice as compared to the C3H/HeN mice. In conclusion, the effects of M-PGF2α on circulating white blood cells in the intact rat are mediated in part through hemodynamic and adrenal mechanisms, and in part through adrenal-independent, hemodynamic-independent, and endotoxin-like stress reaction-independent mechanisms. Prostaglandins may contribute directly to the genesis of neutrophilia.

This paper deals with the effects on the endplate receptors of the electro-osmotic and bath application of compounds which, although lacking pharmacological affinity, mimic the actions caused at much lower concentrations by conventional cholinergic drugs. Small cyclic molecules containing carbonyl, ether, ester, amide, and carboxyl anhydride groups were tested. Lactones, carboxyl anhydrides, cyclic amides, and ketones depolarized the endplate membrane and, in addition, most of them potentiated the depolarizing action of acetylcholine. On the contrary, cyclic ethers behaved as neuromuscular blocking agents. Dioxane, which contains two ether oxygens, caused a marked prolongation of the time course of the potentials elicited by the iontophoretic application of acetylcholine pulses. In some instances the effects of adding the compounds to the bath were different from those observed following local electro-osmotic application.

1. The changes in the consistence of the spine-test articular capsule, or ligament, of the primary spines of Eucidaris tribuloides induced by acetylcholine (ACh) have been studied. Two complementary techniques were used: (a) “forced-vibration”, which detects variations in the stiffness of the ligament along a single diametral plane; and (b) “forced-rotation” which records the spatial distribution of those changes. 2. ACh (1 μM to 1 mM) caused a rapid increase in the resistive force opposed by the ligament to passive stretching. Similar effects were elicited by several monoquaternary, N-substituted derivatives of trimethylammonium. 3. The opposite effect, i.e. softening, was induced by decamethonium, dimethylphenylpiperazine, and 2-ketoamyltrimethylammonium. 4. The involvement in these effects of ACh-binding groups with pharmacological properties similar to those of the “anionic sites” of nicotinic ACh receptors is suggested.

The apparent lifetimes of frog end-plate channels activated by several nicotinic agonists have been determined with voltage-jump and fluctuation analysis techniques. The agonists were monoquaternary, N-substituted derivatives of trimethylammonium (TMA). Methyl TMA activated channels which had apparent lifetimes about 3-4 times shorter than acetylcholine (ACh)-activated channels. This result was confirmed with single-channel recordings from embryonic chick skeletal muscle. Channel conductance and voltage dependence of channel lifetime were similar for methyl TMA- and ACh-activated channels. Methyl TMA showed no signs of blocking open end-plate channels. Ethyl TMA, acetylthiocholine, cholinethiol and carbamylcholine all activated channels similar to methyl TMA-activated channels with regard to lifetime. None of these agonists appeared to block end-plate channels in the employed concentrations. 4-ketopentyl TMA, which contains a methylene group in place of the ether oxygen of ACh, sometimes opened end-plate channels with similar apparent lifetimes as those opened by ACh. Single-channel recordings showed that bursts of current from channels activated by 4-ketopentyl TMA have similar durations as do those activated by ACh. Pentyl TMA and benzyl TMA block open end-plate channels even when delivered at doses which elicit very small currents. It is concluded that the ester moiety of ACh serves to stabilize the open conformation of the channel.

Two techniques are described that are designed to elicit long and constant postsynaptic depolarizations or agonist-induced currents by the electrophoretic application of agonists to endplate membranes. In the first technique, which can be described as a "pharmacologic voltage clamp," the current flowing through the agonist pipette is controlled by the potential across the endplate membrane. Sustained depolarizations of constant amplitude can be obtained in this manner, while the agonist current needed to produce and maintain the response is recorded. The second technique uses a signal proportional to the current flowing across a voltage-clamped endplate membrane to control the delivery of agonist. This allows control of the intensity of the agonist-induced current. The possible application of these techniques in studying receptor desensitization and obtaining samples of post-synaptic current noise for spectral analysis is discussed.

1. 1. Strips of dorsal longitudinal muscle of Sabellastarte magnifica exhibit apparently anomalous responses to drugs. Thus, acetylcholine causes either a contraction or a relaxation, or both. 2. 2. d -Tubocurarine fails to block the stimulating action of acetylcholine; moreover, the acetylcholine-induced contractions are larger in curarized strips. 3. 3. Nicotine exerts a combination of relaxing and stimulating effects; the former being blocked by d -tubocurarine. 4. 4. Adrenaline causes a slow and sustained contraction. 5. 5. 5-Hydroxytryptamne elicits a relaxation, but potentiates the acetylcholine-induced contractions. 6. 6. To explain these observations it is proposed that this muscle is made of different types of cells with different physiological and pharmacological properties.

1. The site and mechanism of initiation of the rhythmic action potentials controlling the somatic musculature of Ascaris have been reinvestigated. 2. Polarization of the muscle syncytium by direct current injection revealed little accommodation. Action potentials are generated continuously at this region at a frequency which depends on the membrane potential. 3. Excitatory and inhibitory nerve fibres control the membrane potential of the syncytial membrane and, therefore, the frequency of spike firing. The effects of stimulation of these fibres are described. 4. The resumption of electrical activity when cooled, quiescent preparations were warmed up was studied. The first signs of activity are slow rhythmic depolarizations on which bursts of abortive spikes are superimposed. When the amplitude of the transients in each burst increases sufficiently they unite into a large, single action potential. 5. Evidence is presented suggesting that each of the abortive spikes represents the separate, subthreshold excitation of one of the terminal branches of the muscle arm, due to a low safety margin for the conduction of impulses towards the muscle belly. 6. Small (1-2 mV.) spontaneous, apparently random, depolarizations and hyperpolarizations have been recorded with microelectrodes inserted into the syncytial region. Their possible synaptic origin is discussed.

The skin of the tropical Atlantic soapfish Rypticus saponaceus (Bloch and Schneider) releases a foamy secretion, toxic to other fishes, when the animal is disturbed. The toxicity of this exudation has been studied using an assay employing fresh water guppies. Although these survived a 24 hr exposure to pure sea water in good condition, they died within 15–30 min when immersed in a 1 : 16 solution of crude toxin; i.e. sea water (300–400 ml) in which a soapfish had been handled. The substance responsible for the effects is non-dialysable and insoluble in solutions of low ionic strength. Crude toxin solutions at neutral pH undergo rapid inactivation but they are very stable at pH 3–4. The toxic principle cannot be extracted with ethyl ether, chloroform or methyl acetate, but it is soluble in water saturated n -butanol, a property which was used to purify it. Although activity is retained after 2 hr at 65°, the results suggest that a protein or polypeptide is responsible for, or at least associated with, the toxic effects.

1. A technique has been developed for the continuous recording of the amount of blood flowing from single vessels following their transection.2. the blood is washed away by a stream of saline flowing at constant rate. the mixture of blood and saline crosses a beam of blue light directed to a phototube. the output of the associated circuit is fed into an ink writer which records the changes in the concentration of erythrocytes suspended in the saline.3. Experiments performed with this technique on the small vessels of the rabbit's ear have revealed two basic patterns of hemostasis.4. the “arterial pattern” is characterized by a sudden outburst of blood when the vessel is cut, followed within a few seconds by a marked decrease in the rate of blood outflow. Therefore, the record begins with a sharp initial peak. Rhythmic fluctuations in the rate of blood outflow which might be due to vasomotion, are often seen in this type of patterns.5. the “venous pattern” lacks the initial peak, looking roughly tr...

The effects of piperazine on Ascaris muscle cells have been investigated with electrophysiological techniques. These cells have an average resting potential of about 30 mV interrupted by rhythmic spikes of myogenic origin (1 to 7 spikes/sec). With piperazine (10-3, w/v), the average resting potential increases above 40 mV and the pacemaker activity is suppressed. These changes are similar to those temporarily produced in the same cells by the electrical stimulation of inhibitory nerve fibres. Electrophoretic application of piperazine to different areas of the muscle cells shows that this drug hyperpolarizes their membrane only when applied to the region where both excitatory and inhibitory neuromuscular synapses are located. The degree of muscle hyperpolarization induced by piperazine depends upon the extracellular chloride concentration, decreasing when a fraction of the chloride ions is replaced by the larger, supposedly nonpenetrating, sulphate anions. Piperazine may, therefore, be regarded as a pharmacological analogue of a natural inhibitory transmitter.

Electrical stimulation of the primary spines (greater than 10 cm long) of the tropical sea urchin Diadema antillarum elicits graded compound action potentials that are conducted at a constant speed of approximately equal to 27 cm/sec. Ion substitution experiments suggest that these are due to the summation of calcium spikes. Structural studies have revealed the presence of up to 21 regularly disposed nerves within the spine shaft, each nerve bundle including greater than 1000 neurites in the basal region, narrowing to slender groups of processes near the spine tip. The neurites in each nerve range in diameter from less than 0.1- approximately equal to 2 micron. Most appear to be distal processes of presumed sensory perikarya situated at the level of the tissue cone surrounding the spine base, or more proximally, although some neurites may arise from perikarya near the spine tip. These nerve tracts are thought to correspond to the nerve fibers described by Hamann [(1887) Jena Z. Naturwiss. 21, 114-176] almost a century ago in spines of Centrostephanus longispinus and, thus, to represent a long-neglected region of the echinoid nervous system.

Interleukin-6 (IL-6) administered as a single intravenous (IV) injection caused the following changes in the peripheral circulation of rats: (a) a biphasic neutrophilia with an initial peak at 1.5 hours and a second sustained wave of neutrophilia between four and 12 hours, (b) a mild lymphocytosis at 0.5 hours and a mild lymphopenia between 1.5 and four hours, and (c) a reticulocytosis between 12 and 24 hours. The bone marrow showed no significant changes at 1.5 hours, suggesting that the peripheral neutrophilia at that time is caused by demargination of intravascular neutrophils and not by release of marrow neutrophils. The bone marrow at 12 hours showed a mild left-shifted myeloid hyperplasia of myeloblasts and promyelocytes and a tremendous erythroid hyperplasia of intermediate and late normoblasts. The bone marrow at 24 hours showed a continued mild myeloid hyperplasia and striking erythroid hyperplasia. In conclusion, IL-6 in vivo acts as a stimulus for myelopoiesis and erythropoiesis and causes accompanying peripheral changes in the number of neutrophils, lymphocytes, and RBCs.