Frequencies of background immunoglobulin-secreting cells in mice as a function if organ, age, and immune status

The influence of hereditary absence of thymus and spleen upon the numbers, organ, and class distribution of background immunoglobulin Ig-secreting cells was studied in mice by means of the protein-A plaque assay. In young adult BALB/c mice the spleen contained the largest number of Ig-secreting cells (about 0.5% ). The absolute number of Ig-secreting cells in the spleen was larger than the estimate for all lymph nodes together. Between 8 and 40 weeks of age, the number of Ig-secreting cells in spleen and lymph nodes increased by a factor of 3, maximally. In the same period, the number of Ig-secreting cells in the bone marrow, however, increased by a factor of 20, so that it became the major site of Ig synthesis. Hereditary absence of the spleen did hardly or not at all affect the number of Ig-secreting cells in the other lymphoid organs. However, the athymic state did affect the organ distribution. The most consistent finding was the decreased number of Ig-secreting cells in the Peyer's patches.The class distribution of Ig-secreting cells was found to be independent of the presence of the spleen, but did depend on the presence of the thymus. Athymic mice had a higher percentage of IgM-secreting cells and a lower percentage of IgA-secreting cells. The percentage of IgG₁- and IgG₂-secreting cells did not differ clearly between normal and athymic mice. Percent-wise, most IgM-secreting cells occurred in the spleen, whereas most IgG₁-, IgG₂-, and IgA-secreting cells occurred in the bone marrow, lymph nodes, and Peyer's patches.The specificity repertoire of the background Ig-secreting cells was tested by determining the frequencies of IgM-producing cells with specificity for a panel of six different antigens. These frequencies ranged from 1 in 85 for nitroiodophenyl(NIP)-conjugated sheep erythrocytes (SRBC) till 1 in 1500 for unconjugated SRBC and were found to be the same for the spleen of germ-free and specific pathogen-free (SPF) C3H mice, and for spleen, bone marrow, and thymus of SPF C3H mice.     

The distribution of cytoplasmic immunoglobulin containing cells over various lymphoid organs of congenitally athymic (nude) mice as a function of age.

The distribution of cells containing cytoplasmic immunoglobulin (C-Ig cells) over various lymphoid organs was studied in congenitally athymic (nude) mice as a function of age. The C-IgM, C-IgG and C-IgA cells were enumerated in spleen, bone marrow, mesenteric lymph node and Peyer''s patches of nude mice and their heterozygous littermates of 6, 40 and 100 weeks of age. In the nude as well as in the heterozygous mice an age-related shift was observed in the localization of the C-Ig cells. In young mice of both groups the majority of these cells resided in the spleen, whereas in adult and old mice the bone marrow was found to be the major C-Ig cell organ, indicating that this shift is not dependent on the presence of the thymus. In young and adult nude and heterozygous mice C-Ig cell numbers in the spleen were comparable, whereas C-Ig cell numbers in the other lymphoid organs were higher in the heterozygous mice than in the nude mice. The total C-Ig cell number in young and adult nude mice was lower than in heterozygous mice of the same age, whereas in old nude mice they were as high as in heterozygous mice of the same age, indicating a retarded development of the immunological activity in nude mice. C-Ig cells in nude mice were almost exclusively of the IgM class, although in the bone marrow of the oldest animals also a substantial number of C-IgG and C-IgA cells was observed. Our finding that nude mice can live up to at least two years of age indicates that the age-related deterioration of the thymus-dependent limb of the immune system is not the cause of ageing, but rather a consequence of it.     

Frequencies of background cytoplasmic Ig-containing cells in various lymphoid organs of athymic and euthymic mice as a function of age and immune status

The distribution of background Ig-secreting cells, measured as cells containing cytoplasmic immunoglobulin (C-Ig cells), over spleen, bone marrow, lymph nodes and Peyer's patches was studied in congenitally athymic (nude) mice and heterozygous euthymic mice as a function of age and immune status (germ-free (GF) vs specific pathogen-free (SPF]. In young athymic as well as in young euthymic mice, the spleen was found to contain the great part of all C-Ig cells, irrespective of whether the mice were GF or SPF. The number of C-Ig cells in the spleen was found to be rather constant over the life span, while the number of C-Ig cells in the bone marrow of all groups of mice greatly increased with age. This indicates that the relative shift of C-Ig cells to the bone marrow is neither dependent on the presence of the thymus, nor on the microbiological status of the mice. However, at young and intermediate age the microbiological status of the mice did affect the total number of C-Ig cells per mouse. This was mainly due to the effect upon the bone marrow, mesenteric lymph nodes and Peyer's patches. At these ages the background Ig synthesis in these organs appeared to be mainly dependent on external antigenic stimulation, in contrast to the spleen, where the Ig synthesis appeared to be mainly due to endogenous stimulation. The Ig (sub)class distribution of the C-Ig cells was different for all different organs tested. Hardly or no difference in percentage distribution was found between the GF nude and GF heterozygous mice. Most C-Ig cells in spleen, bone marrow and lymph nodes of the GF mice were of the IgM isotype. C-IgG and C-IgA cells occurred in substantial percentages only in bone marrow and lymph nodes. In the lymph nodes of GF nude mice a remarkably high percentage of C-IgA cells was found.     

Lipopolysaccharide-induced suppression of graft-versus-host reactivity in mice

Reconstitution of lethally irradiated mice with spleen cells from donors that had been treated with lipopolysaccharide (LPS) intravenously and allogeneic spleen cells subcutaneously leads to a suppressed anti-host delayed-type hypersensitivity (DTH). Either donor injection alone proved to be ineffective. The state of suppression appeared to be antigen-specific, but, depending on the experimental conditions, also anti-host DTH to third-party alloantigens could be suppressed. The suppression was mediated by a population of Thy-1- suppressor cells that could also be induced in athymic nude mice. The suppressor cells specifically adhered to anti-kappa-coated plastic plates, but were not adsorbed by passage through a Sephadex G-10 column. Thus, it appears that the combined donor treatment with LPS and allogeneic spleen cells induces a population of B cells that can suppress anti-host immune reactivity.     

Secondary delayed type hypersensitivity to H-2 subregion-coded alloantigens

Secondary type delayed type hypersensitivity (DTH) in mice against class I alloantigens or non-H-2 alloantigens is characterized by an earlier appearance of DTH reactivity after booster immunization compared with the development of DTH reactivity after primary immunization. In contrast to the primary and secondary DTH against class I or non-H-2 alloantigens, the development of secondary DTH against class II alloantigens or a set of alloantigens that includes class II alloantigens is not faster than the development of primary DTH. Thus, primary and secondary immunization with class II alloantigens prevents secondary type DTH reactivity to simultaneously administered class I alloantigens and non-H-2 alloantigens.     

Genomic aberrations and survival in chronic lymphocytic leukemia

BACKGROUND: Fluorescence in situ hybridization has improved the detection of genomic aberrations in chronic lymphocytic leukemia. We used this method to identify chromosomal abnormalities in patients with chronic lymphocytic leukemia and assessed their prognostic implications. METHODS: Mononuclear cells from the blood of 325 patients with chronic lymphocytic leukemia were analyzed by fluorescence in situ hybridization for deletions in chromosome bands 6q21, 11q22-23, 13q14, and 17p13; trisomy of bands 3q26, 8q24, and 12q13; and translocations involving band 14q32. Molecular cytogenetic data were correlated with clinical findings. RESULTS: Chromosomal aberrations were detected in 268 of 325 cases (82 percent). The most frequent changes were a deletion in 13q (55 percent), a deletion in 11q (18 percent), trisomy of 12q (16 percent), a deletion in 17p (7 percent), and a deletion in 6q (7 percent). Five categories were defined with a statistical model: 17p deletion, 11q deletion, 12q trisomy, normal karyotype, and 13q deletion as the sole abnormality; the median survival times for patients in these groups were 32, 79, 114, 111, and 133 months, respectively. Patients in the 17p- and 11q-deletion groups had more advanced disease than those in the other three groups. Patients with 17p deletions had the shortest median treatment-free interval (9 months), and those with 13q deletions had the longest (92 months). In multivariate analysis, the presence or absence of a 17p deletion, the presence or absence of an 11q deletion, age, Binet stage, the serum lactate dehydrogenase level, and the white-cell count gave significant prognostic information. CONCLUSIONS: Genomic aberrations in chronic lymphocytic leukemia are important independent predictors of disease progression and survival. These findings have implications for the design of risk-adapted treatment strategies.     

Effects of [3H]-BIDN,a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates

1. The radiolabelled bicyclic dinitrile, [³H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([³H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of γ-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC₅₀, 35±3 nM) and dieldrin (IC₅₀, 93±7 nM), displaced [³H]-BIDN from rootworm membranes. When tested at 100 μM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane-1-thione (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [³H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands.
2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and α and β endosulphan displace bound [³H]-BIDN from rootworm membranes by a competitive mechanism.
3. Rat brain membranes were also shown to possess a population of saturable, specific [³H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [³H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm.
4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA_B receptors were unaffected.
5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl⁻ channels to which a number of insecticidally-active molecules bind.

     

Centrosome aberrations as a possible mechanism for chromosomal instability in non-Hodgkin's lymphoma.

Recently, centrosome aberrations have been described as a possible cause of aneuploidy in many solid tumors. To investigate whether centrosome aberrations occur in non-Hodgkin's lymphoma (NHL) and correlate with histologic subtype, karyotype, and other biological disease features, we examined 24 follicular lymphomas (FL), 18 diffuse large-B-cell lymphomas (DLCL), 33 mantle cell lymphomas (MCL), and 17 extranodal marginal zone B-cell lymphomas (MZBCL), using antibodies to centrosomal proteins. All 92 NHL displayed numerical and structural centrosome aberrations as compared to nonmalignant lymphoid tissue. Centrosome abnormalities were detectable in 32.3% of the cells in NHL, but in only 5.5% of lymphoid cells from 30 control individuals (P<0.0001). Indolent FL and MZBCL contained only 25.8 and 28.8% cells with abnormal centrosomes. In contrast, aggressive DLCL and MCL harbored centrosome aberrations in 41.8 and 35.0% of the cells, respectively (P<0.0001). Centrosomal aberrations correlated to lymphoma grade, mitotic, and proliferation indices, but not to the p53 labeling index. Importantly, diploid MCL contained 31.2% cells with abnormal centrosomes, while tetraploid samples harbored centrosome aberrations in 55.6% of the cells (P<0.0001). These results indicate that centrosome defects are common in NHL and suggest that they may contribute to the acquisition of chromosomal instability typically seen in NHL.     

Cerebral MR perfusion imaging: first clinical application of a 1 M gadolinium chelate (Gadovist 1.0) in a double-blinded randomized dose-finding study

The purpose of this study was to evaluate efficacy and safety of the 1 M gadolinium chelate Gadovist 1.0 for assessment of cerebral hemodynamics with dynamic susceptibility contrast-enhanced magnetic resonance (MR) imaging. Eighty-nine patients with carotid artery stenosis or cerebral infarcts were included in this multicenter, double-blinded study using five dose groups from 0.1 to 0.5 mmol/kg. Imaging was performed with 1-T scanners using a T2*-weighted fast low-angle shot (FLASH) sequence. Dose-dependent changes in quantitative and qualitative parameters describing signal-time curves and relative regional cerebral blood volume maps were investigated. For safety evaluation, vital signs, clinical and laboratory tests, and adverse events were assessed. The quantitative measurements revealed an optimal dose of 0.4 mmol/kg. The qualitative evaluation revealed that the required qualitative assessment for clinical purposes was already reached at a dose of 0. 3 mmol/kg. No significant changes in vital signs and laboratory tests were found. No serious adverse events were observed. The combined results revealed the dose of 0.3 mmol/kg as the diagnostically adequate dose given the gradient-echo sequence and field strength used. Gadovist 1.0 has been shown to be a safe and well-tolerated contrast agent. J. Magn. Reson. Imaging 2000;12:371-380.