«Self-Reactive» T Cells. IV. «Self-Reactive» T Cells Induce Polyclonal Differentiation of IgM-Producing B Cells in vivo and in vitro

Intravenous injection of lymphoblasts (generated in vitro by different T- or B-specific mitogens) induced a polyclonal activation of IgM-producing B cells in vivo in the spleens of syngeneic recipient mice. This polyclonal differentiation of host-derived B cells to IgM-producing plaque-forming cells was stimulated by host-derived «self-reactive» T cells activated in the splenic lymphoid cell population in response to the syngeneic lymphoblast graft. We found a stable factor in the supernatants of cultures of proliferating «self-reactive» T cells that induced (antigen-independent) polyclonal maturation, but not proliferation of IgM-producing B cells.     

Similarity between in vitro and in vivo cellular aging

In vivo and in vitro cellular aging were compared by determining the division capacity of individual, cloned cells of (1) the primary stromal population of human bone marrow, obtained from donors of various ages, and (2) the population at various passage levels of an in vitro subcultivated culture of the same origin. We find a strong similarity between the two series of data. This observation provides a further argument that cellular aging in vitro represents a biologically relevant phenomenon.     

Relationship between in vivo mitomycin c exposure,sister chromatid exchange induction and in vitro mitogenic proliferation. I. Development of murine splenic cell system

In vivo administration of mitomycin C (MMC) to C57BL/6J mice induced a rapid, initial suppression (24 hours post injection) of in vitro mitogenesis. This was followed by of the mitogen concentration–response curves were found for two of the three mitogens, phytohemagglutinin and concanavalin A, but not for lipopolysaccharide. By 144 hours post injection, the dose-response curves and magnitude of the responses had returned to approximately control (untreated) levels. This approach provides a model system for the functional assessment of in vivo cellular damage by MMC.     

Macrophages influence Salmonella host-specificity in vivo

The generalist Salmonella enterica serovar Typhimurium causes disease in many animal species, but the closely related host-specific serovar Typhi only causes disease in humans. Typhi and Typhimurium share major virulence loci; hence it is not known exactly why Typhi does not cause disease in mice. We tested the hypothesis that macrophages contribute to Salmonella host-specificity in mice. No significant difference in survival of the two serovars was observed in vitro in mouse macrophage cell lines and primary murine peritoneal and bone marrow-derived macrophages after 24 h. In contrast, differential survival was observed following infection in vivo. When BALB/c mice were infected intraperitoneally (i.p.), both Typhi and Typhimurium induced neutrophil influx into the peritoneum and macrophages were the major cell type containing internalized bacteria at 0.5 and 4 h post-infection for both serovars. The number of Typhimurium in macrophages remained high at 4 h post-infection, but the number of Typhi in macrophages decreased substantially within 4 h after i.p. infection. These results indicate that macrophages are able to distinguish Typhi from Typhimurium when infected in vivo but no significant differences were observed after 24 h in vitro, suggesting that the differential killing of the two serovars by macrophages requires additional factors within the host.     

α-Bungarotoxin receptors in the chick ciliary ganglion: behaviour in vivo and in vitro

Cholinergic nicotinic receptors have been investigated in the ciliary ganglion of adult chick by means of [¹²⁵I]α-bungarotoxin binding to whole and homogenized ganglia. The behaviour of receptors has been compared in vivo (after simultaneous pre- and postganglionic axotomy) and in vitro (ganglia in organ culture). A decrease in receptor number was found both in vivo and in vitro, although the results differed quantitatively in the two cases. The loss of receptors was more evident in whole than in homogenized ganglia, suggesting that mechanisms of receptor degradation and masking may occur simultaneously. Additional experiments in vitro indicate that the behaviour of the receptors is influenced by inhibitors of energy metabolism (dinitrophenol plus iodoacetic acid, sodium cyanide) but not by an inhibitor of protein synthesis (cycloheximide). The receptor modifications in vitro are accompanied by a decrease of other cholinergic markers, such as acetylcholinesterase and cholinacetyltransferase activities. Morphological investigations of organ-cultured ganglia indicate that the biochemical modifications described above are not due to significant degenerative changes of ganglionic neurons.The results indicate that the α-bungarotoxin receptors behave rather similarly in vivo and, under our conditions, in vitro and suggest a dependence of these receptors on peripheral signals. These signals would appear to be mediated by energy-dependent processes without the synthesis of proteins being required.     

Protocatechuic acid inhibits neurotoxicity induced by MPTP in vivo

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in substantia nigra (SN) with the presence of α-synuclein inclusions termed Lewy bodies. The neuroprotective effects of protocatechuic acid (PAc) both in vitro and in vivo have been reported. However, little is known about the effects of PAc on neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in vivo. In this study, we demonstrated that PAc inhibited the reduction of the latent periods in a rotarod test, and the contents of dopamine (DA) and its metabolites in striatum, and furthermore, it ameliorated the pathology in SN and the decreases in the expression of tyrosine hydroxylase (TH) in SN of C57BL/6J mice induced by MPTP. Taken together, our results indicate for the first time that PAc has neuroprotective effects on MPTP treated C57BL/6J mice and may be useful in clinical treatment of PD.     

Disruption of Sertoli cell vimentin filaments in prepubertal rats: An acute effect of butylparaben in vivo and in vitro

Parabens are p-hydroxybenzoic acid ester compounds widely used as preservatives in foods, cosmetics, toiletries and pharmaceuticals. We have recently shown that butylparaben induces spermatogenic cell apoptosis in prepubertal rats. We have conducted the present study for further information. Three-week-old male Sprague–Dawley rats (n = 8) were given a single oral dose of 1000 mg/kg butylparaben. The rats were sacrificed under anesthesia at 3, 6 and 24 h after administration and their testes were collected for histopathological and immunohistochemical examination. Results showed a gradual collapse of Sertoli cell vimentin filaments and decreased actin staining intensity without accompanying changes in the pattern of tubulin expression, while spermatogenic cells became separated from the basement membrane and sloughed into the lumen in the butylparaben-treated rats, compared to the controls. To determine the direct effects of butylparaben on Sertoli cells, primary Sertoli cell cultures with and without butylparaben treatment were examined. Toluidine blue staining in butylparaben treated-cultured Sertoli cells showed an increased number and size of vacuoles in their cytoplasm. In agreement with the in vivo experiment, the in vitro study also clearly demonstrated disruption of vimentin filaments in Sertoli cells after butylparaben treatment. Considering both our present and previous reports, we can speculate that butylparaben-induced disruption of Sertoli cell vimentin filaments may lead to precocious release of spermatogenic cells from underlying Sertoli cells, and the released cells may undergo apoptosis owing to loss of support provided by the Sertoli cells.     

Nitrous oxide and xenon increase noradrenaline release in the cerebral cortex in vivo and in vitro

Noradrenaline in the central nervous system plays an important role in regulating physiological functions, and is a key mechanistic component of general anesthesia. The purpose of this present study was to determine if nitrous oxide and xenon modulate noradrenaline release in the cerebral cortex. We performed a series of in vivo and in vitro experiments in rats. For the in vivo experiments, noradrenaline release was measured by microdialysis in the prefrontal cortex with exposure to 0, 30 or 60% nitrous oxide. For the in vitro experiments, noradrenaline release was measured from cerebrocortical slices before and after incubation with 0, 15, 30, or 60% nitrous oxide in Ca²⁺-containing buffer, Ca²⁺-free buffer, or in Ca²⁺-containing buffer with 10⁻⁶ M tetrodotoxin (TTX). For the in vivo and in vitro experiments 60% xenon was also used. In the in vivo experiment, following exposure to nitrous oxide, noradrenaline release concentration-dependently increased. In the in vitro experiment, under Ca²⁺-containing conditions, noradrenaline release from cerebrocortical slices increased significantly during exposure to nitrous oxide in a concentration-dependent manner. Under Ca²⁺-free conditions, 60% nitrous oxide produced a significant release of noradrenaline. There were no significant differences in nitrous oxide-increased noradrenaline release between with and without TTX. Xenon also significantly increased noradrenaline release in the prefrontal cortex and from the cerebrocortical slices. The nitrous oxide-induced increase in noradrenaline release may be due to both excitation of the locus coeruleus-noradrenergic neuron and direct stimulation of its axon terminals.     

Nicotine induction of theta frequency oscillations in rodent hippocampus in vitro

The hippocampus is an area important for learning and memory and exhibits prominent and behaviourally relevant theta (4–12 Hz) and gamma (30–100 Hz) frequency oscillations in vivo. Hippocampal slices produce similar types of oscillatory activity in response to bath-application of neurotransmitter receptor agonists. The medial septum diagonal band area (MS/DB) provides both a cholinergic and GABAergic projection to the hippocampus, and although it plays a major role in the generation and maintenance of the hippocampal theta rhythm in vivo, there is evidence for intrinsic theta generation mechanisms in the hippocampus, especially in area CA3. The aim of this study was to examine the role of the nicotinic receptor (nAChR) in the induction of oscillatory field activity in the in vitro preparation of the rat hippocampus. Bath-application of a low concentration of nicotine (1 μM) to transversely-cut hippocampal slices produced persistent theta-frequency oscillations in area CA3 of the hippocampus. These oscillations were reduced by both GABA_A receptor antagonists and ionotropic glutamate receptor antagonists, indicating the involvement of local GABAergic and glutamatergic neurons in the production of the rhythmic theta activity. The nicotine-induced theta activity was inhibited by non-selective nAChR antagonists and partially by an α7* nAChR antagonist. The induction of theta frequency oscillations in CA3 by nicotine was mimicked α7* nAChR agonists but not by non-α7* nAChR agonists. In conclusion, theta activity in the hippocampus may be promoted by tonic stimulation of α7* nAChRs, possibly via selective stimulation of theta-preferring interneurons in the hippocampus that express post-synaptic α7* nAChRs.     

The in vitro and in vivo toxicity of graphene quantum dots

Graphene quantum dots (GQD) generate intrinsic fluorescence, and improves aqueous stability of graphene oxide (GO) while maintaining wide chemical adaptability and high adsorption capacity. Despite GO's remarkable advantages in bio-imaging, bio-sensing and other biomedical applications, its biosafety issues are still unclear. Here we report a detailed and systematic study on the in vitro and in vivo toxicity of GQD. The GQD sample was prepared through a facile oxidation approach and fully characterized by means of AFM, TEM, FTIR, XPS and elemental analysis. In vitro experiments showed that GQD exhibits very low cytotoxicity owing to its ultra-small size and high oxygen content. Then, the in vivo biodistribution experiment of GQD revealed no material accumulation in main organs of mice and fast clearance of GQD through kidney. In order to mimic clinic drug administration, mice were injected with GQD and GO (as comparison) multiple times for in vivo toxicity tests. We found that GQD showed no obvious influence on mice owing to its small size, while GO appeared toxic, even caused death to mice due to GO aggregation inside mice. In brief, GQD possesses no obvious in vitro and in vivo toxicity, even under multi-dosing situation.     

The repulsive guidance molecule,RGMa, promotes retinal ganglion cell survival in vitro and in vivo

The repulsive guidance molecule, RGMa, and its receptor Neogenin, regulate neuronal cell death during development, but little is known about their expression and roles in the adult CNS. Here, we show that Neogenin is expressed in the adult rodent retina, particularly on retinal ganglion cells. To determine whether the Neogenin/RGMa pathway is important in the fully developed retina, we examined its contribution to damage-induced neurodegeneration. The effects of RGMa on survival of retinal ganglion cells (RGCs) were examined in vitro and in vivo. Using cultured whole-mount retinal explants, we showed that the addition of RGMa increased RGC survival and that this effect was mediated by the Neogenin receptor. Immunohistochemical analysis indicated that the inhibition of cell death by RGMa resulted from reduced caspase-3 activation. Then, using an in vivo model of RGC apoptosis after optic nerve transection, we demonstrated that intraocular injection of RGMa at 3 and 7 days after axotomy greatly reduced RGC death 14 days postaxotomy. This study provides the first evidence that RGMa is a molecular target for neuroprotection in retinal pathologies, and suggests that targeting “dependence receptors” such as Neogenin has therapeutic potential for the treatment of neuropathologies in the adult CNS.     

Characterization in vitro and in vivo of a novel porcine parainfluenza virus 5 isolate in Korea

A novel porcine parainfluenza 5 (pPIV5), KNU-11, in the genus Rubulavirus of the subfamily Paramyxovirinae, was isolated from the lung of a piglet in Korea in 2011. To understand the importance of this virus as an infectious agent, in vitro and in vivo characteristics of KNU-11 virus was investigated. KNU-11 was remarkably cytopathogenic, showing distinct cell rounding and clumping evident in porcine alveolar macrophage (PAM), porcine kidney (PK-15), and swine testicle (ST) cells within 12 h postinfection and capable of hemagglutinating guinea pig red blood cells. Interestingly, this cytopathology was found to be absent in cell lines from other mammalian species. To evaluate the in vitro immunity of the pPIV5 isolate, we sought to explore alteration of inflammatory cytokine and chemokine expression in PAM cells infected with KNU-11 by using quantitative real-time RT-PCR. Most cytokine and chemokine genes including type 1 interferons (IFN-α/β) and IFN-related antiviral genes were found to be significantly elevated in KNU-11 virus-infected PAM cells. A serum neutralization test-based serosurvey demonstrated that neutralizing antibodies against KNU-11 are readily detected in domestic swine populations, suggesting high prevalence of pPIV5 in Korean pig farms. Animal studies showed that KNU-11 fails to establish an acute respiratory illness, indicating that pPIV5 is non- or very mildly pathogenic to pigs.     

Neural heterogeneities and stimulus properties affect burst coding in vivo

Many neurons tend to fire clusters of action potentials called bursts followed by quiescence in response to sensory input. While the mechanisms that underlie burst firing are generally well understood in vitro, the functional role of these bursts in generating behavioral responses to sensory input in vivo are less clear. Pyramidal cells within the electrosensory lateral line lobe (ELL) of weakly electric fish offer an attractive model system for studying the coding properties of burst firing, because the anatomy and physiology of the electrosensory circuitry are well understood, and the burst mechanism of ELL pyramidal cells has been thoroughly characterized in vitro. We investigated the coding properties of bursts generated by these cells in vivo in response to mimics of behaviorally relevant sensory input. We found that heterogeneities within the pyramidal cell population had quantitative but not qualitative effects on burst coding for the low frequency components of broadband time varying input. Moreover, spatially localized stimuli mimicking, for example, prey tended to elicit more bursts than spatially global stimuli mimicking conspecific-related stimuli. We also found small but significant correlations between burst attributes such as the number of spikes per burst or the interspike interval during the burst and stimulus attributes such as stimulus amplitude or slope. These correlations were much weaker in magnitude than those observed in vitro. More surprisingly, our results show that correlations between burst and stimulus attributes actually decreased in magnitude when we used low frequency stimuli that are expected to promote burst firing. We propose that this discrepancy is attributable to differences between ELL pyramidal cell burst firing under in vivo and in vitro conditions.     

Mechanisms of human smooth muscle cell proliferation and transplant vasculopathy induced by HLA class I antibodies: In vitro and in vivo studies

Vascular smooth muscle cells (SMC) play an important role in the pathophysiology of transplant vasculopathy (TV), a major cause of late death in patients receiving an organ transplant. In this review we describe the proliferative effect in vitro and in vivo of HLA class I antibodies on human SMC. We have developed an experimental model using segments of human mesenteric arteries transplanted in the position of the infrarenal aorta in immunodeficient mice (SCID/beige). Weekly injections of transplanted mice with a monoclonal antibody towards HLA class I provoked typical lesions of TV after 6 weeks in the human graft while transplanted mice receiving an irrelevant antibody did not develop any significant lesion. In vitro, the anti-HLA antibodies were mitogenic to SMC and we showed that they activate a stress-induced signaling pathway implicating matrix metalloproteinases (MMP) and neutral sphingomyelinase 2 (nSMase-2). The proliferative effect of anti-HLA antibodies could be blocked by pharmacological inhibitors or by siRNA. Administration of pharmacological inhibitors diminished the development of TV in grafted mice injected with anti-HLA antibodies demonstrating an important role of the MMP/nSMase-2 pathway in antibody-induced TV. This observation opens new perspectives for the management of TV in clinical settings.     

Ex vivo expansion of memory CD8 T cells from lymph nodes or spleen through in vitro culture with interleukin-7

Interleukin-7 (IL-7) increases lymphocyte numbers, a critical feature of immune reconstitution, through mechanisms that are still poorly understood. Part of the problem is that IL-7 is produced in limited amounts by non-lymphoid cells, making in vivo studies of the cytokine's activity a challenge. To overcome this, we developed an in vitro system by which lymphocytes from secondary immune organs could be cultured to produce IL-7 responsive cells. Using this method, we showed that CD8^hiCD44^hi T cells accumulate in culture with IL-7 from a population of lymph node or splenic cells. These results were validated when a similar lymphocyte subset was found in mice expressing a constitutively active form of STAT5b, a key transducer of IL-7 signals. Interestingly, IL-7-expanded cells also up regulated the activation marker, CD69. The IL-7-derived CD44^hiCD69^hi cells were not generated from naïve cells, but expanded from an existing population, since culture in IL-7 of naïve lymphocytes from OT-1/Rag1^−/− mice did not produce CD44^hiCD69^hi cells. Using the in vitro culture system to study lymphocytes from mice deficient in the apoptotic protein, BIM, we were able to attribute the expansion of CD8^hiCD44^hiCD69^hi T cells to the proliferative and not survival activity of IL-7. The in vitro culture system provides an important new methodology to examine the activities of this essential as well as immunotherapeutic cytokine.     

An in vitro investigation of the hematopoietic micro-environment in young and aged mice

Dexter-type cultures derived from the bone marrow of young and aged mice were established as in vitro correlates to the hematopoietic microenvironment and inoculated two weeks later with fresh bone marrow-derived stem cells (colony-forming units, CFUs) from young, syngeneic mice. Such cultures allowed the observation, quantitation and evaluation of interactions between aged or young microenvironments and the young stem cells. The hematopoietic microenvironments derived from aged marrow were found to support a greater total nucleated cellularity and a significantly greater number of CFUs. Also, the production of CFUs on aged monolayers occurred at an elevated rate. Though cyclic variations in total cellularity were noted in all cultures, the granulocyte-macrophage lineage always predominated. Lymphocyte populations in all cultures were seen to decline rapidly with time as other cell types became more abundant. The number of megakaryocytes in the aged marrow-derived cultures was significantly elevated in the early time periods post-refeeding. Differences in the adherent cell population densities were noted with the aged monolayers being somewhat less dense. However, there were no differences in morphologically identifiable cell types comprising the adherent layers derived from marrow of young and old mice.From these results, we conclude that there are differences in the ability of aged versus young hematopoietic microenvironments to support normal young stem cells in vitro and that the microenvironmental influences present in the in vitro system are reflective of those seen in the in vivo marrow microenvironment.     

Serotonergic mechanisms are necessary for central respiratory chemoresponsiveness in situ

Evidence from in vivo and in vitro experiments conclude that serotonin (5-HT) neurons are involved in and play an important role in central respiratory CO₂/H⁺ chemosensitivity. This study was designed to assess the importance of 5-HT neurons and 5-HT receptor activation in the frequency and amplitude components of the hypercapnic response of the respiratory network in the unanesthetized perfused in situ juvenile rat brainstem preparation that exhibits patterns of phrenic nerve discharge similar to breathing in vivo. Exposure to a hypercapnic perfusate increased phrenic burst frequency and/or amplitude, the neural correlates of breathing frequency and tidal volume in vivo. Hypercapnic responses were also assessed during exposure to ketanserin (5-HT₂ receptor antagonist), and 8-OH-DPAT (inhibiting 5-HT neurons via 5-HT_1A autoreceptors). Neither of these drugs substantially altered baseline activity, however, both abolished hypercapnic responses of the respiratory network. These data illustrate that 5-HT neurons and 5-HT receptor activation are not required for respiratory rhythm generation per se, but are critical for CO₂ responses in situ, supporting the hypothesis that 5-HT neurons play an important role in central ventilatory chemosensitivity in vivo.     

Effects of cerebrolysin on rat Schwann cells in vitro

Although the peripheral nervous system (PNS) is capable of regeneration, these processes are limited. As a potential means to augment PNS regeneration, the effects of cerebrolysin (CL), a proteolytic peptide fraction, were tested in vitro on Schwann cell (SC) proliferation, stress resistance, phagocytic and cluster-forming capacity. Primary SC/fibrocyte co-cultures were prepared from dorsal root ganglia of 5–7-day-old rats. SCs were subjected to mechanical stress by media change and metabolic stress by serum glucose deprivation (SGD). Cell survival was assessed using MTT test. SC proliferation was determined by counting BrdU-labeled cells. SC clustering was studied by ImageJ analysis of S100 immunostaining. Wallerian degeneration (WD) was evaluated by measuring acetylcholine-esterase staining within sciatic nerves in vitro. It was found that CL caused no effect on MTT turnover in the tested doses. CL inhibited SC proliferation in a dose-dependent manner. Media change and additional SGD stress inhibited SC clustering. CL enhanced the reorganization of SC clusters and was able to counteract SGD-induced cluster defects. Moreover, CL accelerated WD in vitro. CL was able to enhance the functions of SCs that are relevant to nerve regeneration. Thus, our findings suggest that CL may be suitable for therapeutic usage to enhance PNS regeneration/reconstruction.     

Vitamin C treatment of mouse bone marrow-derived dendritic cells enhanced CD8 memory T cell production capacity of these cells in vivo

Vitamin C has been found to stimulate dendritic cells (DCs) to secrete more IL-12 and thereby drive naïve CD4⁺ T cells to differentiate into Th1 cells. In the present study, we evaluated the effect of these vitamin C-treated DCs on CD8⁺ T cell differentiation both in vitro and in vivo. Mouse bone marrow-derived DCs were prepared in the presence of GM-CSF and IL-15. With vitamin C treatment, these DCs, when LPS-stimulated, secreted more IL-12p70 and IL-15 than did untreated DCs. And when co-cultured with T cells, they yielded a higher frequency of IFN-γ⁺ CD8⁺ T cells. Moreover, we found that administering vitamin C-treated and tumor lysate-loaded DCs into mice yielded a higher frequency of CD44^high CD62L^low CD8⁺ effector and effector memory T cells, which showed an increased ex vivo killing effect of the tumor cells. These DCs also elicited enhanced protective effects against inoculated tumor cells, most probably by way of the increased cytotoxic T cells, as was revealed by the decreased growth of the inoculated tumor cells in these mice. This ex vivo vitamin C treatment effect on DCs can be considered as a strategy for boosting DC vaccination potency against tumors.     

Morphologic revertants of murine sarcoma virus transformed nonproducer BALB/3T3: Selective techniques for isolation and biologic properties in vitro and in vivo

Revertant clones of murine sarcoma virus transformed nonproducer BALB/3T3 cells have been isolated by selection of iododeoxyuridine-resistant colonies in methylcellulose. Each revertant contained a rescuable sarcoma virus but demonstrated both in vitro and in vivo biologic properties of nontransformed BALB/3T3 cells. Further, each clone was highly susceptible to retransformation by added sarcoma virus. The evidence indicates that these lines comprise a new class of morphologic revertants of murine sarcoma virus transformed cells.