Frequency analysis of immunoglobulin V-gene expression and functional reactivities in bone marrow B cells

Frequency of immunocompetent B cells in bone marrow has been determined in vitro under culture conditions that allow the development in vitro under culture conditions that allow the development of every growth-inducible B cell into a clone of IgM-secreting PFC. Three limiting dilution culture systems were employed: a specific helper assay with SRBC as antigen and using activated T helper cells, a nonspecific helper assay using Con A-induced factors as a source of help, and polyclonal activation with LPS. From unseparated, normal C57BL/6J bone marrow 1 in 2200 to 1 in 2820 B cells were induced to form a clone of PFC to SRBC in each of the 3 systems. This corresponds to a frequency of 1 SRBC-specific clone in every 900 IgM-secreting LPS-reactive clones. The frequencies of specific plaque-forming B cell clones in terms of LPS-reactive B cells was 1 in 36 for NNP1-SRBC, 1 in 58 for TNP30-SRBC, 1 in 75 for NIP1-SRBC, and 1 in 230 for TNP3-SRBC. These frequencies of v-gene expression in bone marrow B cells are of the same magnitude as the corresponding frequencies for splenic B cells. Bone marrow B cells are also fully susceptible to stimulation by antigen in combination with either specific or nonspecific T cell help, as well as polyclonal activation by LPS, since every 3rd Ig-positive cells in marrow could be induced to form a clone of IgM-secreting cells. There is thus no difference in immunocompetence between surface Ig-bearing B cells from bone marrow and spleen.     

Frequency analysis of the antibody specificity repertoire of mitogen-reactive B cells and "spontaneously" occurring "background" plaque-forming cells in nude mice

The antibody specificity repertoire of lipopolysaccharide (LPS)-reactive B cells has been determined in the spleens and bone marrow (BM) of C57BL/Ka athymic nude mice using a limiting dilution culture system that allows the growth and development of every LPS-reactive B cell into a clone of IgM-secreting cells. In addition, the numbers of "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells as well as the "background" IgM antibody specificity repertoire has been assessed in spleens and BM. The frequencies of antigen-specific LPS-reactive B cells of C57BL/Ka nude and thymus-bearing mice showed a great similarity and ranged from 1 in 1000 to 1 in 2500 for sheep red blood cells (SRBC), horse red blood cells (HRBC), and goat red blood cells (GRBC), from 1 in 10 to 1 in 25 for 5-iodo-3-nitrophenyl-coupled (SRBC), from 1 in 15 to 1 in 150 for 4-hydroxy-3,5-dinitrophenyl-coupled SRBC, and from 1 in 70 to 1 in 140 for 2,4,6-trinitrophenyl-coupled SRBC. The specificity repertoire of the "background" IgM-secreting cells differed from that of age-matched thymus-bearing controls and was different in young and old C57BL/Ka nude mice. Within the limitations of having assessed only a minor fraction of the total B-cell antibody specificity repertoire and supposing that nude mice are largely devoid of functional T cells, the data presented suggest that the generation of the specificity repertoire of newly-formed B cells is hardly or not affected by T cells. On the other hand, T cells do affect the expression of the established repertoire, represented by "background" immunoglobulin-secreting cells.     

Early development of Ig-secreting cells in young of germ-free BALB/c mice fed a chemically defined ultrafiltered diet

The influence of antigenic stimulation on the early development of the "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells has been studied in mice. To evaluate the effect of such exogenous stimulation by an evolving microbial microflora, the young of BALB/c mice that were kept under germ-free conditions and fed a low molecular weight chemically defined synthetic diet (GF-CD) were compared with the young of conventional BALB/c mice fed natural ingredients (CV-NI). The young were first suckling maternal milk and between Days 15 and 18 changed to the same diet as their parents. Background Ig-secreting cells in the spleen were enumerated in the protein A plaque assay. The specificity repertoire of the IgM-secreting cells was determined with plaque assays specific for sheep red blood cells (SRBC) that were haptenized with different concentrations of nitroiodophenyl (NIP), 4-hydroxy-3.5-dinitrophenyl (NNP), and 2,4,6-trinitrophenyl (TNP). The results show that during the first few weeks of life the numbers of background IgM-, IgG-, and IgA-secreting cells in the spleen develop faster in CV-NI mice than in GF-CD mice. At 4 weeks of age equal numbers of IgM- and IgG-secreting cells were found in both groups of mice, but the number of IgA-secreting cells remained reduced in GF-CD mice during the whole period of observation. The frequencies of IgM-secreting cells specific for the differently haptenized SRBC were the same in both groups of mice during the observation period of 10 weeks. This suggests that the ontogenetic appearance of IgM-, IgG-, and IgA-secreting cells in the spleen, and the specificity repertoire of the IgM-secreting cells, as far as was tested in our panel, is independent of exogenous antigenic and/or mitogenic stimulation. However, during neonatal development the rate of development of the background Ig synthesis is enhanced by environmental antigenic stimulation.     

Frequency analysis of functional immunoglobulin C and V gene expression in murine B cells at various ages

The frequencies of lipopolysaccharide- (LPS) reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of mice at different ages. A limiting dilution culture system was employed that allows the growth and development of every LPS-reactive B cell into a clone an IgM-secreting cells that are capable of switching to other Ig heavy chain isotypes (C gene expression). The secretion of IgM and IgG1 was assessed in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V gene expression) were detected with the use of plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The frequencies of LPS-reactive B cells in the spleen and BM of C3H/Tif, C57BL/Ka, BALB/c, and CBA/Rij mice appeared to be similar in 6- to 12- and 100-wk-old animals, as was the switch frequency to IgG1 secretion in three strains tested. Moreover, no age-related changes were observed in the frequencies of antigen-specific B cells within the pool of LPS-reactive B cells in the spleen and BM of C57BL/Ka mice. The frequencies ranged from 1 in 10 to 1 in 20 for NIP4- and NNP2-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 4000 for SRBC, HRBC, and GRBC. The specificity repertoire of the "spontaneously" occurring ("background") IgM-secreting cells in the spleen and BM, on the other hand, did differ between young and old C57BL/Ka mice. During aging the frequencies of the tested specificities decreased in the spleen but increased in the BM. Our data indicate that in unintentionally immunized mice the clonal selection of B lineage cells by antigen takes place at the level of the mature, antigen-reactive B cell.     

Vitamin C enhancement of brood rearing by caged honeybees fed a chemically defined diet

Pollen collected by bees was sampled during a 3-h period once a week from April to October 1983 and analyzed for vitamin C (L-ascorbic acid and dehydroascorbic acid). The levels were highly variable and ranged from a low of 136 μg/g pollen (April) to a high of 1943 μg/g pollen (May). Overall, caged honeybees fed diets containing 1,000 and 2,000 μg/g L-ascorbic acid reared significantly more bees to the sealed stage than bees fed diets with 500 μg/g ascorbic acid or control bees. The levels of vitamin C in prepupae reared by bees ranged from 64.5 to 103.5 μg/g body mass. Vitamin C is either synthesized from simple precursors or from symbiotic microorganisms in the gut since honeybees fed the ascorbic acid-free control had equivalent levels of ascorbic acid to those fed the enriched diets. The total diet consumption by bees during the 10-week study showed that the four diets were equally attractive.     

Pyrroloquinoline quinone improves growth and reproductive performance in mice fed chemically defined diets

Growth, reproductive performance, and indices of collagen maturation and expression were investigated in Balb/c mice fed chemically defined, amino acid-based diets with or without the addition 6 micro Mpyrroloquinoline quinone (PQQ)/kg diet. The diets were fed to virgin mice for 8 weeks before breeding. At weaning, the pups from successful pregnancies were fed the same diet as their respective dams. Reproductive performance was compromised in mice fed diets devoid of PQQ, and their offspring grew at slower rates than offspring from mice fed diets supplemented with PQQ. Successful mating (confirmed vaginal plugs) was not affected by the presence or absence of PQQ; however, pup viability (number of pups at parturition/number of pups at Day 4 of lactation) was decreased in PQQ-deprived mice. Conception (percentage of females giving live births) and fertility (percentage of births) were also decreased in PQQ-deprived mice. The slower rates of growth in offspring from PQQ-deprived mice were associated with decreased steady-state mRNA levels for Type I procollagen alpha(1)-chains in skin and lungs from neonatal mice. Values for lysyl oxidase accumulation as protein in PQQ-deficient mice also tended to be lower than corresponding values from PQQ-supplemented or -replete mice. Skin collagen solubility was increased in PQQ-deprived mice. These results indicate that PQQ supplementation can improve reproductive performance, growth, and may modulate indices of neonatal extracellular matrix production and maturation in mice fed chemically defined, but otherwise nutritionally complete diets.     

Buoyant density analysis of myeloid colony-forming cells in germfree and conventional mice.

Granulocyte-macrophage colony-forming cells (CFUc), in the bone marrow of germfree and conventioal CBA mice, were compared quantitatively and qualitatively. Cells were separated on the basis of their buoyant density by equilibrium centrifugation in continuous albumin density gradients. CFUc in the density subpopulations were detected by culture in agar containing three different types of colony stimulating factor (CSF). The sources of the CSF were post-endotoxin mouse serum (CSFES), mouse lung conditioned medium (CSFMLCM) and human urine (CSFHU). Mice were removed from the germfree environment and the buoyant density status of their CFUc was examined 1, 4 and 8 weeks later. No difference was found between germfree and conventional mice in the number of nucleated cells per femur or in their modal density. Neither was the number of CFUc per femur different. The cell cycle status of CFUc, as determined by the thymidine suicide technique was not significantly different. Functional heterogeneity was found among the density subpopulations for both groups of mice. This depended on the type of CSF. The density distribution of CFUc was significantly different in germfree mice. There were proportionately more low density CFUc. The mean modal density of CFUc under CSFES stimulation was less by 0.0045 g/cm3 in germfree mice. The removal of mice from the germfree environment resulted in a shift of the distribution to higher densities. The trend was towards the conventional situation. The significance of the buoyant density status of CFUc is discussed.     

Morphological and functional properties of rat dorsal root ganglion cells cultured in a chemically defined medium

A culture procedure for dorsal root ganglion (DRG) cells is presented using a completely defined culture medium without antibiotics, in combination with mechanical dissociation procedures. This culture procedure allows all dorsal root ganglion cell types to be cocultured for periods of at least 106 days. Some of the dorsal root ganglion neurons, which could be identified by their neurofilaments and the presence of fluoride resistant acid phosphatase, regained their original T-cell appearance within two weeks. After one month in culture ganglion-like reaggregates appeared. Schwann cells, satellite cells and fibroblasts were identified using morphological criteria. All neurons tested maintained excitability during, at least, the first 35 days in culture, since in all cases action potentials could be evoked by current pulses. The method has proved to be useful in the study of morphological, cytochemical and electrophysiological aspects of dorsal root ganglion cell differentiation in vitro.     

Functional analysis of defined mutations in the immunoglobulin heavy-chain enhancer in transgenic mice.

We have analyzed the effect of defined mutations in the mouse immunoglobulin heavy-chain enhancer after introduction into the germline of transgenic mice. We have tested a mutation of the enhancer octamer motif, a double mutation of the octamer motif and the microB-site, and a triple mutation in the microE2, microE3 and microE4-sites. All constructs are expressed in the spleen of transgenic mice. Furthermore, expression is exclusively detectable in lymphoid organs and not in several nonlymphoid tissues. Whereas mutations in the microE-sites have a more pronounced effect on transgene activity in thymocytes as compared to bone marrow and spleen cells, the octamer/microB double mutation shows significantly reduced expression levels only in B-cells. Finally, our results demonstrate that the intronic heavy-chain enhancer element does not contribute to the increase steady state levels of heavy-chain mRNA after stimulation of spleen cells with LPS.     

Reorganization of porcine thyroid cells into functional follicles in a chemically defined, serum- and thyrotropin-free medium

In the serum-free, chemically defined medium NCTC 109, freshly isolated porcine thyroid cells aggregate and form functional follicles in culture even in the absence of thyrotropin. The follicular pattern observed under light and electron microscopy express the main morphological characteristics of in vivo thyroid cells. Follicles are large, replete with dense colloid, and the apical pole of cells is characterized by well-developed microvilli and the presence of aminopeptidase N. The index of iodide transport activity (125I-C/M ratio) decreases vs. days of culture to a resting value of about 1 or 2 at day 2. Addition of thyrotropin (200 microU/ml final concentration) at day 4 is followed by a 10-fold increase in iodide transport activity within 24 h and a 40-fold increase 4 d later. Incorporation and organification of iodide are dose dependent between 0 and 250 microU/ml thyrotropin; highest concentrations (4,000--16,000 muU/ml) are significantly inhibitory. In the absence of thyrotropin each cell synthesizes 8.2 pg thyroglobulin/d. Acute stimulation by thyrotropin at day 4 resulted in a slight decrease in the quantity of thyroglobulin present in the cell layer but in an increase in the total amount of thyroglobulin recovered in both cells and medium, reaching 34.3 pg/cell/d. The protein exported into the medium is thyroglobulin, as shown by SDS PAGE and immunological properties. Here we demonstrate that porcine thyroid cells can be maintained in culture as resting, highly differentiated, follicular-associated cells, sensitive to acute stimulation by thyrotropin.     

Comparison of growth and exploratory behavior in mice fed an exclusively milk formula diet and mice fed a food-pellet diet post weaning

An exclusively milk formula diet stunted the growth of mice immediately following weaning. Milk-fed mice displayed a low-frequency profile of exploratory behavior, while pellet-fed mice showed high-frequency exploration. In contrast to exploratory behavior, feeding behavior did not differ significantly between milk- and pellet-fed mice. Despite showing low-frequency exploratory behavior, mice on an exclusively milk formula diet showed no difference in behavioral activities analyzed by an automatic hole-board apparatus compared to pellet-fed mice. These results suggest that the growth stunt caused by an exclusively milk formula diet retards the acquisition of active exploratory behavior without affecting the emotional state of mice.     

Amino acid and dipeptide absorption in rats fed chemically defined diets of differing fiber composition

In rats chronically fed a fiber-free diet or one of the three nutritionally equivalent fiber-containing diets, in vivo jejunal absorption of L-leucine from a free amino acid mixture of L-leucine and glycine as well as equimolar solutions of the dipeptides, L-leucyl-glycine and glycyl-L-leucine, were compared. In addition, total and brush border amino peptidase activities for the two dipeptide substrates were examined in small bowel segments. With two of three fiber diet groups, absorption of L-leucine from the free amino acid solution was reduced without a detectable change in peptide transport or aminopeptidase activities. This investigation provides evidence that peptide transport mechanisms are relatively spared with long-term feeding of fiber-containing diets similar to observations recorded in disease states associated with protein-energy malnutrition.     

Production of aflatoxins B1 and G1 in chemically defined medium

Summary Aflatoxins B₁ and G₁ were produced in a chemically defined liquid medium in stationary culture. Glucose, sucrose, and fructose were satisfactory carbon sources. Organic nitrogen compounds were essential for production of high levels of aflatoxins. Complex nitrogen sources, such as yeast extract and peptone, gave higher yields than single amino acids. Aspartate, glycine, glutamine, and glutamate were good sources of nitrogen for toxin production. Little or no aflatoxin was produced when zinc, iron, or magnesium were omitted from the medium. Manganese appeared to reduce yields of aflatoxin.     

Induction of kappa light chain synthesis in 70Z/3 B lymphoma cells by chemically defined lipid A precursors

The murine B cell lymphoma 70Z/3 is a tumor line that resembles an early B cell. It responds to lipopolysaccharide by synthesizing kappa light chains, resulting in the appearance of surface IgM. We now demonstrate that this effect is triggered by the lipid A domain of lipopolysaccharide since a defined tetraacyl disaccharide 1,4'-bisphosphate precursor of mature lipid A, designated lipid IVA (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088), is fully active in stimulating kappa chain mRNA synthesis. In contrast, the monosaccharide lipid A precursor, lipid X, shows only slight activity; but a large excess of lipid X appears to complete with lipid IVA, partially blocking its effects. Mutants of the 70Z/3 line that are unresponsive to lipopolysaccharide also fail to respond to lipid IVA. The somatic cell system described here, coupled with the use of chemically defined agonists and antagonists, offers a new approach to understanding the early events in lipopolysaccharide/animal cell interactions.     

Immunologic competence of B cells subjected to constitutive c-myc oncogene expression in immunoglobulin heavy chain enhancer myc transgenic mice

B cell activation is associated with a marked transient rise in expression of the c-myc proto-onco-gene. A unique opportunity to examine the effects of constitutive c-myc expression upon B cell function is provided by transgenic mice in which the c-myc oncogene is regulated by the enhancer (E mu) from the immunoglobulin locus (E mu-myc mice). We have examined the immunologic competence of B cells from E mu-myc mice both in vitro and in vivo. Upon stimulation, many E mu-myc B cells can proliferate to form clones most of which contain antibody-forming cells. However, the frequency of responsive B cells from E mu-myc donors is only about 30% that of B cells from normal littermates. Thus, enforced myc expression is not sufficient to block the differentiation of all B cells, but a much larger fraction of the immunoglobulin-bearing cells from E mu-myc mice are incompetent. Upon immunization, E mu-myc mice mounted specific antibody responses, although some responses were delayed. Isotype switching can occur, since we observed hemolytic plaques of both IgM and IgG type and detected specific antibody of both classes in the serum. Moreover, the serum from nonimmunized E mu-myc mice contained normal levels of both IgM and IgG. Thus constitutive expression of the c-myc gene appears to retard B cell differentiation, but does not grossly impair immunologic function in the intact animal.     

Modulation of hepatic lipidome by rhodioloside in high-fat diet fed apolipoprotein E knockout mice

BackgroundRhodioloside is a glucoside of tyrosol isolated from Rhodiola rosea. However, its regulating effect on hepatic dyslipidemia of atherogenic mice has rarely been studied.PurposeThe specific aims of current study included to clarify lipidomic perturbation in liver tissues of apolipoprotein E deficient (apoE^−/−) mice fed with high-fat diet, and to examine the effects of rhodioloside against atherosclerosis and dyslipidemia.Study DesignThe comparisons of hepatic lipidome were executed between wide type (WT) mice fed with normal diet (NDC) and apoE^−/− mice fed with high-fat diet (Model), WT mice fed with high-fat diet (HFDC) versus the model mice, as well as the model mice versus rhodioloside-treated atherosclerotic mice.MethodsUltra high performance liquid chromatography coupled with a Q exactive hybrid quadrupole-orbitrap mass spectrometry (UPLC-MS/MS) was employed to provide an unbiased and simultaneous measurement of individual lipid species in liver tissues.ResultsMultivariate statistical analysis derived from LC-MS spectra revealed that high-fat diet and apoE deficiency caused a series of disturbances on glyerolipid metabolism, glycerophospholipid metabolism and sphingolipid metabolism. Rhodioloside administration showed atheroprotective effects on the apoE^−/− mice with regulating the levels of 1 phosphatidylcholine, 2 phosphatidylserines, 5 alkyldiacylglycerols and 3 alkenyldiacylglycerols back to normal. In particular, PC (4:0/15:0) was positively associated with high-density lipoprotein cholesterol in blood, both of which could be ameliorated by rhodioloside.ConclusionOur results identified the abnormal hepatic lipids in atherosclerosis progression that could efficiently improved by rhodioloside. These lipids contributed to biological understanding of atherogenic dyslipidemia in liver and could also served as sensitive indicators for drug target screening.     

Enhancer sequences located 3' of the mouse immunoglobulin lambda locus specify high-level expression of an immunoglobulin lambda gene in B cells of transgenic mice

In contrast to the mouse immunoglobulin heavy chain and kappa light chain genes, very little is known about the regulation of expression of the immunoglobulin lambda light chain locus. To identify elements responsible for lambda gene regulation we mapped DNaseI hypersensitive sites associated with a functionally rearranged lambda 1 gene in nuclei from the myeloma cell line J558L. Tissue-specific hypersensitive sites were identified 2.3 to 2.5 kb upstream of the CAP site of both the lambda 1 gene and the unrearranged variable (V) lambda 2 gene segments. DNA sequences flanking the lambda 1 gene were isolated and tested for their influence on expression of the lambda 1 gene after transfection into myeloma cells and after injection into fertilized mouse eggs. Two enhancer elements were identified downstream of the lambda 1 gene. A proximal element (located 4 to 10 kb 3' of the gene) enhanced expression of a lambda 1 gene in stable myeloma cell transfectants but had no effect on the expression of a heterologous reporter gene in transient assays. A second, distal element, located approximately 30 kb 3' of the gene, enhanced heterologous expression in J558L cells expressing a lambda gene but not in a non-lambda myeloma cell line (SP2/0-Ag14). Co-injection of cosmids containing the lambda 1 gene and both the proximal and distal downstream elements into fertilized mouse eggs resulted in high-level expression of the lambda 1 transgene in B cells of transgenic mice. The identification of these lambda regulatory elements, in addition to contributing to an understanding of lambda gene regulation per se, will facilitate the study of the regulation of differential expression of kappa and lambda light chain genes in the immune system.