Presence Of Normal Mouse Serum Research Articles

TAxI-peptide targeted Cas12a ribonuclease protein nanoformulations increase genome editing in hippocampal neurons.

Gene therapy approaches that utilize Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleases have tremendous potential to treat human disease. However, CRISPR therapies delivered by integrating viral vectors are limited by potential off-target genome editing caused by constitutive activation of ribonuclease functions. Thus, biomaterial formulations are being used for the delivery of purified CRISPR components to increase the efficiency and safety of genome editing approaches. We previously demonstrated that a novel peptide identified by phage display, TAxI-peptide, mediates delivery of recombinant proteins into neurons. In this report we utilized NeutrAvidin protein to formulate neuron-targeted genome-editing nanoparticles. Cas12a ribonucleases was loaded with biotinylated guide RNA and biotinylated TAxI-peptide onto NeutrAvidin protein to coordinate the formation a targeted ribonuclease protein (RNP) complex. TAxI-RNP complexes are polydisperse with a 14.3nm radius. The nanoparticles are stable after formulation and show good stability in the presence of normal mouse serum. TAxI-RNP nanoparticles increased neuronal delivery of Cas12a in reporter mice, resulting in induced tdTomato expression after direct injection into the dentate gyrus of the hippocampus. TAxI-RNP nanoparticles also increased genome editing efficacy in hippocampal neurons versus glia. These studies demonstrate the ability to assemble RNP nanoformulations with NeutrAvidin by binding biotinylated peptides and gRNA-loaded Cas12a ribonucleases into protein nanoparticles that target CRISPR delivery to specific cell-types in vivo. The potential to deliver CRISPR nanoparticles to specific cell-types and control off-target delivery to further reduce deleterious genome editing is essential for the creation of viable therapies to treat nervous system disease.

Reaction of complement factors varies with prion strains in vitro and in vivo

Roles of complement factors in prion infection of the central nervous system remain unclear. In this study, we assessed the strain-dependent reactivity of complement factors in prion infections of Neuro2a (N2a) cells and mouse brains. N2a cells persistently infected with either Chandler or 22L scrapie strains were cultured in the presence of normal mouse serum (NMS), followed by staining with phosphatidylserine binding protein and early apoptosis marker Annexin V. The proportion of Annexin V positive cells was increased both in Chandler- and 22L-infected cells. Preincubation of NMS with anti-C1q, C3 and/or C9 antibodies reduced Annexin V positive cells in Chandler-infected cells, while only anti-C3 antibodies were effective on 22L-infected cells. The immunohistochemistry showed that deposition of C1q and C3 was different between Chandler- and 22L-infected mouse brains. These results indicate that the reactivity of complement factors differs between prion strains both in vitro and in vivo.

Functional activity of individual abscess neutrophils from mice

In the absence of antibiotic therapy, viable bacteria can persist within intra-abdominal abscesses in mice for at least 10 weeks. The mechanisms contributing to this survival are unknown, but abscess-derived neutrophils have impaired abilities to kill, in vitro, organisms engulfed in vivo. In order to determine whether subpopulations of abscess neutrophils might be discernible on the basis of phenotypic or functional criteria, cells from murine intra-abdominal abscesses were examined for phagocytic activity, CR3 expression, and H2O2 production in response to soluble and particulate stimuli. With respect to phagocytosis of Proteus mirabilis, abscess cells were no less efficient than peritoneal exudate neutrophils; no significant subpopulation of cells was incapable of phagocytosis in the presence of normal mouse serum. Using flow cytometry to examine abscess neutrophils for CR3 expression, we found that no subpopulations of cells were observed with unstimulated cells or with cells incubated with either phorbol 12-myristate 13-acetate or bacteria and serum. Intracellular H2O2 levels were measured by using the probe 2',7'-dichlorofluorescin diacetate. In general, incubation with phorbol 12-myristate 13-acetate resulted in similar increases in H2O2 production in all cells of the population. However, stimulation with bacteria and serum revealed a variable but consistent, poorly responsive subpopulation of neutrophils in abscess cell populations. Cell-sorting experiments showed that cells from the poorly responsive section of the FACS profile contained significantly higher numbers of abscess-derived bacteria, suggesting the presence of a subpopulation of viable abscess neutrophils harboring persisting viable bacteria.

Interference in thyrotropin (TSH) determination: falsely elevated TSH values in a transplanted patient

We report a falsely elevated TSH value as determined by an immunoradiometric assay. The patient, a thyroidectomized woman on substitutive therapy, underwent a kidney transplantation. She was treated for transplant rejection with OKT 3 (Orthoclone), a mouse monoclonal antibody. The discrepancy between the TSH value (TSH-Riabead II, Abbott) and the thyroid hormones level led us to suspect the presence of antimouse antibodies in the serum of this patient. Indeed, human immunoglobulins directed against mouse IgG2a and IgG2b were detected. We repeated-the TSH determination in the presence of normal mouse serum (NMS) and obtained a true TSH value. Upon fractionation of the serum with protein-A chromatography, the falsely elevated TSH immunoreactivity was detected in the IgG fraction.

Effects of Trembler mouse serum and laminin on oligodendrocyte proliferation and differentiation in culture

Oligodendrocytes in primary cultures derived from rat embryo spinal cord were examined in control medium and in Trembler mouse serum (TMS)-supplemented medium. The Oligodendrocytes were identified on the basis of the synthesis and surface expression of galactocerebrosides revealed by a monoclonal antibody directed against this component. We noticed two effects of TMS compared to control mouse serum. First, our results revealed that in TMS medium there is a mitogenic response of galactocerebroside (GalC)-positive cells. Second, in the presence of TMS, Oligodendrocytes do not develop processes as they do in the presence of normal mouse serum. When laminin, a basal lamina component was added to TMS medium, GalC+ Oligodendrocytes decreased in number and differentiation was normal. Possible explanations of the effects of TMS and laminin on oligodendrocyte proliferation and differentiation are discussed.

Lymphokine-activated killer cell and interleukin-2 inhibitors: Their role in adoptive immunotherapy

Interleukin-2 (IL-2) and the adoptive transfer of lymphokine-activated killer (LAK) cells have been used to reduce tumor mass in intraperitoneal, pulmonary, and hepatic models. Unfortunately, repeated cycles of IL-2 plus LAK cells have not been demonstrated to exert additive antitumor effects. We investigated the responses that repeated challenge with LAK cells and IL-2 provoked in normal BL/6 mice. Six immunizations of LAK cells generated in media containing fetal calf serum abrogated the ability of mice to respond to IL-2 plus LAK immunotherapy in a peritoneal carcinomatosis model. Serum from these treated mice would cause lysis of LAK cells or cultured lymphocytes in the presence of complement. LAK cells generated in the presence of normal mouse serum were not lysed. Similarly, the antitumor effects of IL-2 plus LAK immunotherapy were abrogated in mice pretreated with six courses of IL-2. We found that LAK cells cytolysis could not be generated if serum from IL-2-pretreated mice was present in vitro with splenocytes and IL-2. These data suggest that antibody against absorbed antigens on LAK cells and serum inhibitors of IL-2 are generated following repeated challenge. These observations may be important in interpreting IL-2 plus LAK immunotherapy studies in murine models and may modify our attempts to treat cancer patients.

Functional neutrophils from long-term murine bone marrow cell cultures

Murine bone marrow cell cultures that had been established for up to 26 weeks were harvested each week and found to provide functional neutrophils. Leukocytes harvested from the cultures were enriched for neutrophils using discontinuous Percoll density gradients. These cells mounted a chemiluminescence response to Proteus mirabilis in the presence of normal mouse serum (NMS). They killed several NMS-opsonised bacterial species, an activity that was blocked by a monoclonal antibody to the C3 receptor of mouse neutrophils. Cultured bone marrow neutrophils expressed both Fc and C3 receptors. C3 receptor expression could be augmented by exposure to the chemotactic peptide f-Met-Leu-Phe. We conclude that murine bone marrow cell cultures provide a useful source of functional neutrophils, and that their productivity can be sustained in long-term culture. As their receptor expression can be augmented from the resting state by exogenous stimuli, they represent a useful cell source in studies of neutrophil activation.

Macrophage type 3 complement receptors mediate serum-independent binding of Leishmania donovani. Detection of macrophage-derived complement on the parasite surface by immunoelectron microscopy.

In this study, direct visual evidence for local opsonization of L. donovani by macrophage (M phi)-derived complement components was obtained using immunoelectron microscopy. C3 deposition was detected on the surface of both promastigotes and amastigotes after 20 min serum-free incubation with murine resident peritoneal M phi (RPM), followed by fixation and incubation first with specific antibody directed against C3 and then with gold-labelled protein A. Gold deposition was not observed around either form of the parasite if the anti-C3 antibody was omitted. For promastigotes, the degree of C3 deposition under serum-free conditions was comparable with that observed in the presence of an exogenous (serum) source of C3, but did not result in the same severe damage to the parasite as did the latter. Addition of sodium salicyl hydroxamate, which prevents covalent binding of C3 to activator surfaces, abrogated promastigote binding. Hence, although the anti-C3 antibody did not distinguish between native C3 and its breakdown product iC3b, these data support our earlier conclusion that promastigote binding to the CR3 of murine RPM is complement dependent. For amastigotes, gold deposition and binding to murine RPM were not eliminated by sodium salicyl hydroxamate. The presence of normal mouse serum resulted in increased gold deposition, but did not mediate either enhanced binding to M phi or damage to the amastigote. These data suggest that a proportion of C3 binding to the amastigote surface may be via noncovalent linkages, and that the C3 bound may not be in the correct form to mediate binding to CR3.

Characterization of opsonins for Bacteroides fragilis in immune sera collected from experimentally infected mice.

Serum collected from mice experimentally infected with Bacteroides fragilis 23745 (immune serum) was analyzed for its ability to opsonize the in vitro ingestion of this organism by mouse peritoneal macrophages. B. fragilis was shown to be phagocytized most efficiently in the presence of immune serum although normal mouse serum demonstrated reduced, but significant, opsonic activity. Phagocytosis was greater in the presence of serum collected from animals inoculated twice with the organism than in the presence of serum from once-inoculated animals. The increased opsonic activity of serum from twice-inoculated animals compared to singly inoculated animals was associated with increases in immunoglobulin G1(IgG1), IgG2a, and IgG2b but not IgM. Adsorption analysis of immune serum with homologous or heterologous bacterial antigens indicated that both antibody and complement act synergistically in opsonizing B. fragilis, although either alone may effectively opsonize this organism. Further evaluation of antibody-mediated opsonization revealed that prior treatment of heat-inactivated immune serum with the reducing agent 2-mercaptoethanol caused a slight, but significant, decrease in opsonic activity, thus indicating that IgM is a minor opsonizing antibody for B. fragilis. When ingestion of a B. fragilis stock strain (23745) was compared to a recent clinical isolate (C-1), it was observed that the stock strain was more easily phagocytized in the presence of normal mouse serum, thus suggesting a possible anti-opsonic-phagocytic property of the clinical strain. In addition, the clinical isolate was phagocytized to a significantly greater degree in an aerobic than an anaerobic environment. Subsequent analysis of in vitro killing of B. fragilis 23745 by peritoneal macrophages reflected the previous results in that optimal killing occurred in the presence of immune serum, although normal serum promoted phagocytic killing to an intermediate degree. Thus, these studies implicate both antibody and complement, either alone or in combination, in the opsonization of B. fragilis. Moreover, the virulence of clinical B. fragilis strains may relate to their refractoriness to opsonization and phagocytosis.

Inhibition of chondrogenesis by normal mouse serum in cultured chick limb cells

Chick limb-bud mesoderm cells from embryonic stages 22–25 were cultured at high cell densities in media known to support chondrogenesis. Under these conditions the continuous presence of normal mouse serum, at a concentration of 10%, inhibits the ability of the cells to produce toluidine blue-stainable cartilage matrix materials. In contrast, mesodermal cells treated with comparable concentrations of other heterologous sera continue to differentiate much like the control cultures while growing in the presence of the test sera. The inhibitory effect of the serum was shown not to be the result of a general cytotoxic effect on protein synthesis or the inability of the cells to incorporate [ 3H] d-glucosamine. There was a significant difference however, in the distribution of the incorporated glucosamine. Less label was associated with the cell layer of the treated series, while a greater amount of the incorporated material was found to be secreted into the medium when compared with the control cultures. Studies have shown also that the serum inhibitory response is dose dependent, while the factor(s) itself is non-dialysable, stable to heat and repeated freezing and is not a conventional serum lipoprotein. Following the addition of whole or delipidated mouse serum, a significant increase in lipid droplets appears in the cytoplasm of the cells. Biochemical analyses of mouse serum-treated cells indicate that there is a marked increase in their triglyceride content as compared to the control cells. While the nature of the serum inhibitory factor remains to be determined, the accumulation of triglyceride following mouse serum treatment suggests that this may play a role in modulating the expression of the chondrogenic phenotype.

A study of the rat passive cutaneous anaphylaxis (PCA) reaction for the assay of mouse IgE antibody

The rat PCA reaction for assay of mouse IgE antibody was investigated. PCA reactivity was examined in rats with different characteristics in terms of strain, sex, age and rearing conditions. Sprague-Dawley (S-D), Fischer and Wistar strain rats were almost equally appropriate for the assay. The male rat was recommended in spite of the comparable PCA reactivity to the female since the male showed less non-specific bluing. PCA reactivity of the germ-free rat was comparable to that of the conventional animal. PCA reactivity appeared at 3 weeks of age, increased up to 11–13 weeks and remained constant until 40 weeks of age. PCA titers assayed in the rostral, caudal, lateral or central side of the back were comparable. The PCA reaction was poor when elicited with a 15 min sensitization period, became progressively stronger to reach a plateau at 4 h and remained constant until 72 h. Heating conditions required to demonstrate thermolability of IgE antibodies were studied. Incubation of undiluted antiserum at 56°C for 2 h abolished the reaginic activity. Reaginic activity of an antiserum diluted in saline was relatively resistant to heat, but diluted reaginic antibody was equally sensitive to heat in the presence of normal mouse serum (NMS) or HGG in the dileunt. NMS must be coexistent with reaginic antibody on heating to confer thermolability since preheated NMS used for dilution had no effect on the PCA titer of an antiserum.

Fc receptor-mediated inhibition of murine B-lymphocyte activation.

Immobilized antigen-antibody complexes are able to inhibit the mitogenic response of murine spleen cells to the B-cell mitogen 8-bromo-3',5'-cyclic guanosine monophosphoric acid. This this inhibition is dependent on intact Fc fragments in the immobilized complexes. Soluble complexes do not mediate this inhibition. When lipopolysaccharide (lps) activation of B cells was studied, it was found that the mitogenic response was inhibited at all times tested between 2 and 7 days of culture. Also, the LPS-induced mitogenesis of nude spleen cells was inhibited by immobilized complexes, indicating that suppressor T cells probably play no significant role in the inhibition. Immobilized complexes inhibit polyclonal antibody responses in a serum-free system and in the presence of normal mouse serum, but are unable to inhibit in the presence of fetal calf serum (FCS). If nu/nu spleen cells are used, however, the FCS does not block the ability of the complexes to inhibit the polyclonal response. It is suggested that that antigen-antibody complexes under appropriate conditions may bind to B lymphocytes via their Fc receptors and trigger a central "off" signal which blocks proliferation and consequently antibody production.

Effect of normal mouse serum on mouse lymphocyte transformation in vitro.

AbstractMouse spleen cells were cultured in the presence or absence of one of the following mitogens: phytohemagglutinin (PHA), concanavalin A, allogeneic spleen cells or bacterial lipopolysaccharide. The addition of normal mouse serum (NMS) to the cultures usually depressed mitogen‐induced lymphocyte transformation as measured by DNA, RNA or protein synthesis, whether calculated as gross or net synthesis. The degree of depression was increased as the concentration of NMS serum increased. DNA and RNA synthesis in unstimulated cultures were also usually depressed by NMS. NMS added 24 hours or more after the start of cultures with PHA or allogeneic lymphocytes was less depressive than serum present from the start and in some cases, actually increased DNA synthesis. The presence of NMS in cultures containing supra‐optimal doses of concanavalin A could also increase DNA synthesis. Cells incubated with NMS for 24 hours survived as well as cells incubated without NMS, but subsequently responded less well to PHA. Cells passed through a column of glass wool had a lower baseline DNA synthesis, which was not inhibited by NMS; they responded less well to mitogens and this response was further depressed by NMS.It is suggested that NMS contains a factor which damps the proliferation of mouse T and B cells, without exerting an overt cytotoxic effect, and that it acts directly on lymphocytes.

The Action of Normal Mouse Serum on Influenza Virus

SUMMARY: Normal mouse serum contains a haemagglutination inhibitor and a neutralizing factor for influenza virus. The inhibitor and the neutralizing factor are heat-labile, can be destroyed by a crude filtrate of Vibrio cholerœ and are much more active towards unadapted virus than towards mouse-adapted virus. The inhibitor enters into stable combination with the virus in the presence of Ca ions. It is adsorbed by a large amount of unadapted virus, even after the haemagglutinin of the latter has been destroyed by heating at 60° for 15 min. Three strains of freshly isolated influenza B virus tested were not significantly neutralized by normal mouse serum. By growing two strains of unadapted influenza A virus in eggs in the presence of normal mouse serum, two variants which are resistant to the neutralizing action of mouse serum were produced. It appears that this type of variation also occurs when an unadapted virus is passed in mice. The theoretical implication of this type of variation in response to a normal host component and its relationship to the mouse-pathogenicity of influenza virus are discussed.