Repeated Injections Of Low Doses Research Articles

IRF2 maintains the stemness of colonic stem cells by limiting physiological stress from interferon

The physiological stresses that diminish tissue stem-cell characteristics remain largely unknown. We previously reported that type I interferon (IFN), which is essential for host antiviral responses, is a physiological stressor for hematopoietic stem cells (HSCs) and small intestinal stem cells (ISCs) and that interferon regulatory factor-2 (IRF2), which attenuates IFN signaling, maintains their stemness. Here, using a dextran sodium sulfate (DSS)-induced colitis model, we explore the role of IRF2 in maintaining colonic epithelial stem cells (CoSCs). In mice with a conditional Irf2 deletion in the intestinal epithelium (hereafter Irf2ΔIEC mice), both the number and the organoid-forming potential of CoSCs were markedly reduced. Consistent with this finding, the ability of Irf2ΔIEC mice to regenerate colon epithelium after inducing colitis was severely impaired, independently of microbial dysbiosis. Mechanistically, CoSCs differentiated prematurely into transit-amplifying (TA) cells in Irf2ΔIEC mice, which might explain their low CoSC counts. A similar phenotype was induced in wild-type mice by repeated injections of low doses of poly(I:C), which induces type I IFN. Collectively, we demonstrated that chronic IFN signaling physiologically stresses CoSCs. This study provides new insight into the development of colitis and molecular mechanisms that maintain functional CoSCs throughout life.

Effects of Testosterone on Erythropoiesis in a Female Mouse Model of Anemia of Inflammation.

The anemia of inflammation is a common problem in inflammatory and autoimmune diseases. We characterized a mouse model of anemia of chronic inflammation induced by repeated injections of low doses of heat-killed Brucella abortus (HKBA), and determined the effects of T administration on erythropoiesis in this model. Female C57BL/6NCrl mice were injected weekly with HKBA for 10 wk. Weekly injections of T or vehicle oil were started 4 wk later. Control mice were injected with saline and vehicle oil in parallel. HKBA-injected mice had significantly lower hemoglobin, hematocrit, mean corpuscular volume, reticulocyte hemoglobin, transferrin saturation (TSAT), and tissue nonheme iron in liver and spleen, enlarged spleen, and up-regulated hepatic expression of inflammatory markers, serum amyloid A1, and TNFα, but down-regulated IL-6, bone morphogenic protein 6, and hepcidin compared with saline controls. HKBA also reduced serum hepcidin and increased serum erythropoietin. Bone marrow erythroid precursors were substantially reduced in HKBA-injected mice. Cotreatment with T increased the percentage of late-stage erythroid precursors in the bone marrow relative to HKBA-injected and saline controls and reversed HKBA-induced suppression of hemoglobin and hematocrit. T also normalized serum erythropoietin, TSAT, and reticulocyte hemoglobin without correcting the expression of the hepatic inflammation markers. Conclusions are that low-dose HKBA induces moderate anemia characterized by chronic inflammation, decreased iron stores, and suppression of erythroid precursors in the bone marrow. T administration reverses HKBA-induced anemia by stimulating erythropoiesis, which is associated with a shift toward accelerated maturation of erythroid precursors in the bone marrow.

Neuronal loss in the caudal intralaminar thalamic nuclei in a primate model of Parkinson’s disease

In light of postmortem human studies showing extensive degeneration of the center median (CM) and parafascicular (Pf) thalamic nuclei in Parkinson's disease patients, the present study assessed the extent of neuronal loss in CM/Pf of non-human primates that were rendered parkinsonian by repeated injections of low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In order to determine the course of CM/Pf degeneration during the MPTP intoxication, motor-asymptomatic animals with partial striatal dopamine denervation were also used. The Cavalieri's principle for volume estimation and the unbiased stereological cell count method with the optical dissector technique were used to estimate the total number of neurons in the CM/Pf. We found substantial neurons loss in the CM/Pf in both, motor-symptomatic MPTP-treated monkeys in which the striatal dopamine innervation was reduced by more than 80%, and in motor-asymptomatic MPTP-treated animals with 40-50% striatal dopamine loss. In MPTP-treated parkinsonian monkeys, 60 and 62% neurons loss was found in CM and Pf, respectively, while partially dopamine-depleted asymptomatic animals displayed 59 and 52% neurons loss in the CM and Pf, respectively. Thus, our study demonstrates that the CM/Pf neurons loss is an early phenomenon that occurs prior to the development of parkinsonian motor symptoms in these animals. In contrast, the neighboring mediodorsal nucleus of the thalamus was only mildly affected (18% neurons loss) in the parkinsonian monkeys. Together with recent findings about the possible role of the CM/Pf-striatal system in cognition, our findings suggest that the pathology of the thalamostriatal system may precede the development of motor symptoms in PD, and may account for some of the cognitive deficits in attentional set-shifting often seen in these patients. Future studies in this animal model, and in monkeys with selective lesion of CM or Pf, are needed to further elucidate the role of the CM/Pf-striatal system in normal and parkinsonian conditions.

Therapy of Anemia in Kidney Failure, Using Plasmid Encoding Erythropoietin

Numerous studies using erythropoietin (EPO) gene delivery vectors, either viral or nonviral, have shown uncontrolled EPO expression leading to transient or sustained erythrocytosis and, more recently, severe autoimmune anemia. Therefore, there is a need to develop other EPO gene delivery systems that allow sustained and adjustable expression of EPO. We have examined a new approach of delivering plasmid encoding mouse EPO cDNA into mouse skeletal muscle, using an amphiphilic block copolymer. Repeated injections of low doses of block copolymer-EPOcDNA formulations increased hematocrit in a dose-dependent manner for more than 9 months, without any initial overshoot. Low doses of block copolymer-EPOcDNA formulations prevented autoimmune anemia in immunocompetent Swiss mice and prevented or reversed chronic anemia in an acquired mouse model of renal failure. We conclude that repeated injections of low doses of block copolymer-DNA formulations that do not induce (1) inflammation at the injection site, (2) overexpression of EPO, or (3) the production of anti-EPO neutralizing auto-antibodies hold promise for in vivo expression of therapeutic proteins, in particular for systemic delivery.

Identification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice

AbstractThe pathogenic effects of antiplatelet antibodies were investigated in mice. Monoclonal antibodies (mAbs) of different immunoglobulin G subclass directed against mouse GPIIbIIIa, GPIIIa, GPIbα, GPIb-IX, GPV, and CD31 were generated and characterized biochemically. MAbs against GPIb-IX, GPV, CD31, and linear epitopes on GPIIIa had mild and transient effects on platelet counts and induced no spontaneous bleeding. Anti-GPIbα mAbs induced profound irreversible thrombocytopenia (< 3% of normal) by Fc-independent mechanisms but only had minor effects on hematocrits. In contrast, injection of intact mAbs, but not F(ab)2 fragments, against conformational epitopes on GPIIbIIIa, induced irreversible thrombocytopenia, acute systemic reactions, hypothermia, decreased hematocrits, and a paradoxical loss of surface GPIIbIIIa on platelets in vivo, the latter suggesting the formation of platelet-derived microparticles. Blockage of platelet-activating factor receptors inhibited the acute reactions, but not thrombocytopenia, loss of GPIIbIIIa, and decreases in hematocrits. Repeated injections of low doses of anti-GPIIbIIIa antibodies resulted in profound thrombocytopenia and bleeding, whereas no acute systemic reactions were observed. These data strongly suggest that the identity of the target antigen recognized by antiplatelet antibodies determines the mechanisms of platelet destruction and the severity of bleeding in mice, the latter depending on previously unrecognized anti-GPIIbIIIa-specific inflammatory mechanisms.

Identification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice

The pathogenic effects of antiplatelet antibodies were investigated in mice. Monoclonal antibodies (mAbs) of different immunoglobulin G subclass directed against mouse GPIIbIIIa, GPIIIa, GPIbα, GPIb-IX, GPV, and CD31 were generated and characterized biochemically. MAbs against GPIb-IX, GPV, CD31, and linear epitopes on GPIIIa had mild and transient effects on platelet counts and induced no spontaneous bleeding. Anti-GPIbα mAbs induced profound irreversible thrombocytopenia (&lt; 3% of normal) by Fc-independent mechanisms but only had minor effects on hematocrits. In contrast, injection of intact mAbs, but not F(ab)2 fragments, against conformational epitopes on GPIIbIIIa, induced irreversible thrombocytopenia, acute systemic reactions, hypothermia, decreased hematocrits, and a paradoxical loss of surface GPIIbIIIa on platelets in vivo, the latter suggesting the formation of platelet-derived microparticles. Blockage of platelet-activating factor receptors inhibited the acute reactions, but not thrombocytopenia, loss of GPIIbIIIa, and decreases in hematocrits. Repeated injections of low doses of anti-GPIIbIIIa antibodies resulted in profound thrombocytopenia and bleeding, whereas no acute systemic reactions were observed. These data strongly suggest that the identity of the target antigen recognized by antiplatelet antibodies determines the mechanisms of platelet destruction and the severity of bleeding in mice, the latter depending on previously unrecognized anti-GPIIbIIIa-specific inflammatory mechanisms.

The Preferential Induction of a Th1 Immune Response by DNA-Based Immunization Is Mediated by the Immunostimulatory Effect of Plasmid DNA

In the present study, we have investigated the T cell response to the HBsAg, normally secreted as multivalent particles, and to β-galactosidase, a cytoplasmic antigen, delivered as plasmid DNAs. We found that cytokines characteristic of a Th1 phenotype are produced in mice immunized by these plasmid DNAs. Using repeated injections of low doses of purified antigen, we demonstrated that neither prolonged presence of the antigen nor site of immunization resulted in an immune response with characteristics resembling those obtained with DNA-mediated immunization. Analysis of immune responses induced in mice by coinjection of plasmid DNA and β-galactosidase or HBsAg demonstrated that the coinjected DNA stimulated a Th1 response against the injected antigen. These data therefore strongly suggest that the strong immune response obtained after intramuscular DNA immunization was due to the adjuvant effect of the plasmid DNA which is also responsible for the selective activation of CD4+T cells with a Th1 phenotype.

A link between catalytic activity, IgE-independent mast cell activation, and allergenicity of bee venom phospholipase A2.

The molecular and cellular mechanisms controlling Ab isotype selection following encounter of a given Ag are still unclear, although the regulatory role of cytokines is established. In the present study we explored the possibility that the nonimmunologic interaction of an allergen with cells of the innate immune system might result in a release of mediators that promote IgE isotype selection in adaptive responses. Using the bee venom allergen phospholipase A2 (PLA2) and a mutant variant lacking enzymatic function, we show that PLA2, but not its catalytically inactive variant, is able to induce IgE-independent mediator release, including IL-4, from rodent mast cells. Assessing the in vivo relevance of these observations, we find that repeated injections of low doses of active enzyme into mice induce the synthesis of high levels of PLA2-specific IgE, while immunization with the inactive form yields no detectable IgE response. Both Ags were similarly immunogenic when high doses of Ag were used for immunization. These findings suggest that mast cells might be a source of IL-4 at the onset of specific immunity against sources of allergens such as bee venom that contain PLA2 and support the concept that the biologic action of an Ag on cells of the innate immune system can play a role in determining adaptive immune responses.

Plasma LH concentrations in the ewe in response to repeated injections of low doses of gonadotrophin releasing hormone during the follicular phase of the oestrous cycle

Oestrus was synchronized in 15 naturally cyclic ewes by the administration of a prostaglandin F2 alpha analogue. Groups of five ewes were then treated i.v. with either small doses of gonadotrophin releasing hormone (GnRH; 125 or 250 ng/injection) or saline, at 2-h intervals from day 14 of the subsequent cycle until 24 h after the onset of oestrus. Treatment with GnRH induced episodic LH release which continued until the onset of a preovulatory LH surge. Mean plasma LH concentrations over this period were significantly (P less than 0.001) higher in animals receiving 250 ng GnRH (2.44 +/- 0.11 microgram/1) than in those receiving either 125 ng GnRH (1.17 +/- 0.06 microgram/1) or saline (1.14 +/- 0.05 microgram/1). However, GnRH treatment did not influence the timing of oestrus or mean ovulation rates.

Plasma LH and FSH concentrations in prepubertal beef heifers before and in response to repeated injections of low doses of Gn-RH.

Blood samples were taken at 15-min intervals from prepubertal beef heifers, 6 aged 4 months and 6 aged 10 months, to determine plasma LH and FSH concentrations. Two animals of each age group were then subjected to 9 consecutive injections of 0.5, 2.0 or 5.0 micrograms Gn-RH at 2-h intervals. LH episodes were evident in all animals before treatment, and in 11/12 heifers occurred at a rate of between 1 and 4 per 24 h. One 10-month old heifer, later detected in oestrus, displayed 10 episodes/24 h. Mean FSH concentrations did not differ significantly between age groups. In all animals, Gn-RH injections induced an episodic pattern of LH secretion which was significantly different between Gn-RH doses, but not between age groups. A distinct episode-like response in FSH concentrations occurred to each Gn-RH injection in only one heifer. These results provide further evidence that the prepubertal period is associated with a lack of pituitary stimulation, rather than an inability to respond to Gn-RH.

ISLET IMPLANTATION NORMALISES HYPERGLYCÆEMIA CAUSED BY STREPTOZOTOCIN-INDUCED INSULITIS: Experiments in Mice

Islet-cell deterioration in juvenile diabetes mellitus may be due to an autoimmune reaction, possibly involving both circulating islet-cell antibodies and an inflammatory process in the islets of Langerhans. Replacement of deteriorated islet cells by implantation of normal ones is now under investigation in many laboratories. The present study does not support the assumption that such islet transplants should" be affected in the same way as the endogenous islets. Diabetic mice with a cell-mediated immune reaction to their pancreatic islets, induced by repeated injections of low doses of streptozotocin, were used as recipients. Iso geneic islets implanted intrasplenically in these animals were as effective in producing normoglycæmia as were those injected into animals made diabetic with a single bolus dose of streptozotocin. No inflammatory reaction was seen in the implanted islets, irrespective of the regimen of the preceding streptozotocin treatment. This finding suggests that islet-cell implantation may be attempted in insulin-requiring diabetic patients, even if the cause of the disorder is an inflammatory lesion of the patient's own islets.

Drinking induced by parenteral injections of pilocarpine

Parenteral (IP) injections of pilocarpine, in doses from 3.75–30 mg/kg, reliably produced drinking in water-satiated rats. This effect was not diminished by pretreatment with either centrally active (scopolamine, atropine) or peripherally active (methyl scopolamine, methyl atropine) cholinergic blocking agents, suggesting that pilocarpine does not induce drinking via a cholinergic mechanism. Repeated injections of low doses, but not high doses, of pilocarpine augmented drinking over trials.