Expansion and cell surface Ig isotype switching in the antigen-binding cell population of nu/nu mice and nu/+ littermates

Swiss-Webster nu/nu splenocytes placed in modified Mishell-Dutton culture containing sheep red blood cells (SRC) generated increased numbers of antigen-binding cells (ABC) compared with antigen-free cultures. In contrast Balb/c nu/nu cultures did not expand their ABC population in response to SRC, suggesting that strain background may influence the effect of the nu/nu gene on T-dependent immune responses. Cell surface Ig isotype analysis indicated that the SRC-induced expanded ABC population exhibited a significant decrease in cell surface IgD and a significant increase in ABC bearing both IgM and IgG. The Swiss-Webster nu/+ littermate cell surface Ig isotype patterns were generally similar to the nu/nu ABC patterns, but with different kinetics.     

Studies on defense mechanisms against Candida albicans infection in congenitally athymicnude (nu/nu) mice

The defense mechanisms against Candida albicans infection were studied by using a mouse thigh lesion model in congenitally athymic nude (nu/nu) mice and their normal littermates (nu/+). Nu/nu mice were more resistant to C. albicans infection than nu/+ mice judging from the course of the thigh lesion, the results of CFUs (colony-forming units) of C. albicans in the lesion, and histopathological observations. Histopathological and serological studies revealed that granulocytic cellular infiltration was predominant, and there were few indications of development of cell-mediated immunity to protect Candida infection in Candida-infected nu/nu and nu/+ mice. These results confirmed that lower susceptibility of nu/nu mice to C. albicans infection as compared with nu/ + mice was due to accelerated non-specific defense mechanisms in nu/nu mice, and that cell-mediated or humoral immunity played a minor role in the defense against Candida infection in this experimental model.Furthermore, treatment with high titer of rabbit anti-C. albicans serum was effective to control the number of Candida cells in thigh lesions of BALB/c mice.Above experimental results seem to clearly indicate the great variability of defense manifestation according to the experimental model exployed.     

Conformational states of chromatin nu bodies induced by urea.

Monomer chromatin nu bodies (nu1) from chicken erythrocyte nuclei were exposed to 0-10 M urea plus 0.2 mM EDTA (PH 7). Alterations in nu1 conformation were examined using hydrodynamic methods (i.e., S, eta, and (formula: see text)), thermal denaturation, circular dichroism, reactivity of histone thiol groups to N-ethyl maleimide, and electron microscopy. The two domains of a nu body (i.e., the DNA-rich shell and the protein-rich core) aeared to respond differently to the destabilizing effects of increasing urea; DNA conformation and stability exhibited noncooperative changes; the core protein structure revealed cooperative destabilization between 4 and 7 M urea. Companion studies on the conformation of the inner histone "heterotypic tetramer" also revealed cooperative destabilization with increasing urea concentration.     

Regional variations in the distributions of small intestinal intraepithelial lymphocytes (IELs) in BALB/c +/+, nu/+, and nu/nu mice

Regional variations in intraepithelial lymphocytes (IELs) in the small intestine were examined in BALB/c +/+, nu/+, and nu/nu mice. The small intestine was obtained from 11- to 12-week-old mice and divided equally into three (proximal, middle, and distal) parts. The IELs were isolated from each part of the intestine, and the total numbers of IELs in nu/+ and nu/nu mice were about a fifth of those in +/+ mice. Regional variations in the distribution of the IEL alphabeta, but not the gammadelta T-cell subset were found by use of flow cytometry in +/+ and nu/+ mice. On the other hand, such differences were not found in nu/nu mice, suggesting that thymus-independent development of T cells is not different among regions. Different local expansion of thymus-dependent alphabeta T cells may cause the regional variations seen in the distribution of alphabeta T cell IELs in +/+ and nu/+ mice.     

Development of T-cell function in relation to T-cell set diversification in nu/nu mice

The differentiation pattern of splenic T-cell populations in germ- and pathogen-free nu/nu mice, as compared to nu/+ littermates, is characterized by two abnormal features: the expression of TL determinants on peripheral T cells and the delayed onset of their differentiation from the predominant Lyt-123:TL+ set into TL- cells of Lyt-1+ and Lyt-123+ phenotype, which, in these mice, does not occur until 10 weeks of age. We report here that the delayed onset of mitogen- or alloantigen-induced interleukin-2 synthesis and T-cell proliferation as well as the development of cytotoxic T-lymphocyte activity of enriched T-cell populations is strictly correlated with the time point of T-cell subset diversification in nu/nu mice and depends in particular on the presence of the Lyt-1 (TL-:Lyt-2-) T-cell set which is lacking in splenic T-cell populations of germ-free young nu/nu mice.     

Tissue-specific down-regulation of RIPK 2 in Mycobacterium leprae-infected nu/nu mice

RIPK 2 is adapter molecule in the signal pathway involved in Toll-like receptors. However, there has been no reported association between receptor-interacting serine/threonine kinase 2 (RIPK 2) expression and the infectious diseases involving mycobacterial infection. This study found that its expression was down-regulated in the footpads and skin but was up-regulated in the liver of Mycobacterium leprae-infected nu/nu mice compared with those of the M. leprae non-infected nu/nu mice. It was observed that the interlukin-12p40 and interferon-gamma genes involved in the susceptibility of M. leprae were down-regulated in the skin but were up-regulated in the liver. Overall, this suggests that regulation of RIPK 2 expression is tissue-specifically associated with M. leprae infection.     

Thymus differentiation and T-cell specificity in nu/nu +/+ mouse aggregation chimaeras

Thymus development and T cell differentiation were studied in mouse chimaeras produced by aggregating pre-implantation embryos of thymus-deficient nude BALB/c (nu/nu) and wild-type C57BL/6 (+/+) mice and vice versa. Chimaeras showed mosaic distribution of skin and coat pigmentation, of hair follicles, of glucosephosphate isomerase within all tested organs and of lymphocytes expressing the different major transplantation antigens (H-2). When tested for their capacity to generate vaccinia virus-specific and self-H-2 specific cytotoxic T cells, all chimaeras of BALB/c (nu/nu) H-2d in equilibrium C57BL/6 (+/+) H-2b type generated T cells of one or both parental origins that were specific for virus and for self-H-2 of the +/+ (H-2b) type only. In contrast, some BALB/c (+/+) H-2d in equilibrium C57BL/6 (nu/nu) H-2b chimaeras generated vaccinia virus-specific cytotoxic T cells specific for either H-2d (+/+) type or for H-2b (nu/nu) type. These asymmetrical results can be interpreted to indicate the following: (i) The +/+ thymus part alone is functional, but because of asymmetrical cross-reactivities of anti-self-H-2 specificities, the observed T cell restriction phenotypes differ. (ii) Both nu/nu and +/+ thymus parts are functional but immune response defects may be exaggerated in such chimaeras producing unexpected non-responsiveness to vaccinia virus linked to H-2d in H-2b (+/+) in equilibrium H-2d (nu/nu).     

Histochemical studies of 3 beta- and 20alpha-hydroxysteroiddehydrogenase in the adrenals and ovaries of the nu/nu mouse.

Adrenal glands and ovaries of nu/+ and nu/nu female mice were examined histochemically throughout the postnatal life for changes in distribution, intensity and appearance of activities of 3 steroid-converting enzymes. While the 3beta-OH-steroid- and pregnenolone-dehydrogenase did not reveal any difference, the 20alpha-hydroxysteroid-dehydrogenase (20alpha-OH SDH) showed a constant behaviour: the onset of the enzyme activity was detectable in the adrenals of nu/+ mice after the second week of age, in nu/nu mice 3--4 weeks later. The localization of 20alpha-OH-SDH was strictly confined to the zone of small cells, a zone lying between the x-zone and the zona fasciculata. In nu/+ mice, after reached sexual maturity at about 6--8 weeks of age, the enzyme progressively disappeared from the adrenals and simultaneously appeared in the ovaries. In nu/+nu mice, which had been kept separated from mating animals, the 20alpha-OH SDH persisted in adrenals even in adult stages. After pregnancy, both--nu/nu and nu/+, showed 20alpha-OH SDH-activity in ovaries and none in adrenals. In both, the enzyme was localized in involuting corpora lutea, whilst in nu/nu it was also present in the intersitial cells. This study indicates no disturbance in the formation of progesterone, but the process of its degradation, as indicated by 20alpha-OH SDH-activity, is definitely different in nu/+ and nu/nu.     

Ultrastructural and gas-chromatographic analysis of the preputial glands of male nude (nu/nu) mice

Summary The preputial glands of male nude (nu/nu) mice were analyzed by a combination of electron microscopy and gas chromatography to determine whether or not they are affected, like developing hairs and nails, by the nu/nu genotype. Results of the analyses revealed no differences between the glands of nude and normal male mice in either their ultrastructural characteristics or lipid secretory products.     

Induction of T cell activity in athymic (nu/nu) mice infected with Trypanosoma rhodesiense

Infection of C57BL/10 (B10)3 nu/nu mice with Trypanosoma rhodesiense results in the development of significant T-cell reactivity in spleen and lymph nodes. The proliferative responses to mitogens, such as concanavalin A (Con A) and phytohemagglutinin (PHA), and in mixed-lymphocyte reactions (MLR) to alloantigens are enhanced compared with control uninfected nu/nu mice. These results serve to emphasize the stimulatory nature of trypanosomes on the immune system.     

Ultrastructural features of chromatin nu bodies

Spread chromatin fibers and isolated chromatin fragments prepared from chicken erythrocyte nuclei were stained with dilute aqueous uranyl acetate. High-resolution electron micrographs reveal two new morphological features exhibited by many of the chromatin nu bodies: (a) lateral association of the nu body with the connecting strand, and (b) a centrally stained spot approximately 15 A wide, possibly corresponding to a hole or crevice within the nu body.     

A cell population in nu/nu spleen can prevent generation of cytotoxic lymphocytes by normal spleen cells against self antigens of the nu/nu spleen.

Spleen cells from athymic nu/nu mice contain two kinds of physically separable active cells that can have very different effects on the generation of CL (cytotoxic lymphocytes) by normal LN cells in an in vitro response against allogeneic stimulator cells. They can provide an accessory cell required for the activation of CLP (cytotoxic lymphocyte precursor cells) which need not be H-2 identical to the CLP and will function normally even when H-2 identical to the stimulator cells. They can also provide a suppressor cell that prevents the activation of CLP that can recognize the H-2 of the nu/nu mouse. Thus, with A, B, and C to represent three H-2 differnt mouse strains, a culture containing CLP from strain A and nu/nu spleen cells from strain B or strain (A x B)F1 will produce CL against strain C or (A x C)F1 stimulator cells but not against strain B or strain (A x B)F1 stimulator cells unless the suppressor cell is first removed. It is proposed that the in vivo role of the suppressor cell in a normal mouse is to prevent the activation of CLP reactive against self.     

Increased membrane immunoglobulin capping of B cells from C57Bl/6 lpr/lpr and C57Bl/6 nu/nu mice

When the capping of membrane immunoglobulin on spleen B cells from normal C57Bl/6 mice (B6) is taken as reference, a faster capping rate is found for cells of age-matched B6 mice which are congenic at the lymphoproliferation (lpr) or nude (nu) loci. Though both congenic strains can be characterized by an abnormal T-lineage cell content, the nature of the abnormality itself is very different since B6 nudes lack thymus-processed/influenced lymphocytes whereas B6 mice with the lpr phenotype suffer from an invasion of all lymphoid organs with cells of a particular T-cell subset. Moreover, the more "normal" capping rate of B cells from the double congenic B6 mice (nu/nu, lpr/lpr) is intriguing. Since other mice homozygous at the lpr locus (MRL-1) or at the nu locus (BALB/c nude) also cap faster than their congenic controls (MRL-n and BALB/c, respectively), the observed effects do not appear to depend on a peculiarity of the B6 genetic background. If the faster capping of B cells of nu congenic and of lpr congenic mice had a common origin, it might be that T cells would control in some way the mobility of B-cell membrane immunoglobulins: both congenic mice have in their spleen a very low proportion of mature T cells together with a very high proportion of prethymic/thymic immature T-cell types, either of which might affect B-cell behavioral responses to membrane immunoglobulin clustering.     

Identification and separation of pre T-cells from nu/nu mice: differentiation by preculture with thymic reticuloepithelial cells.

Spleens and lymph nodes of nu/nu, congenitally athymic, mice have 16–32% and 72–76%, respectively, of large, brain-associated-T (BAT) antigen positive cells. These BAT positive large cells were isolated from nu/nu spleens with a fluorescence-activated cell sorter (FACS-II) and then cultured on thymic reticuloepithelial cell (TRC) monolayers. Both concanavalin A and mixed lymphocyte reactivity was induced during this culture period. We concluded that these BAT positive cells are pre-T cells which can he induced to at least some T cell functions by an inductive factor produced by TRC.     

A unique error signature for human DNA polymerase nu

Human DNA polymerase nu (pol nu) is one of three A family polymerases conserved in vertebrates. Although its biological functions are unknown, pol nu has been implicated in DNA repair and in translesion DNA synthesis (TLS). Pol nu lacks intrinsic exonucleolytic proofreading activity and discriminates poorly against misinsertion of dNTP opposite template thymine or guanine, implying that it should copy DNA with low base substitution fidelity. To test this prediction and to comprehensively examine pol nu DNA synthesis fidelity as a clue to its function, here we describe human pol nu error rates for all 12 single base-base mismatches and for insertion and deletion errors during synthesis to copy the lacZ alpha-complementation sequence in M13mp2 DNA. Pol nu copies this DNA with average single-base insertion and deletion error rates of 7 x 10(-5) and 17 x 10(-5), respectively. This accuracy is comparable to that of replicative polymerases in the B family, lower than that of its A family homolog, human pol gamma, and much higher than that of Y family TLS polymerases. In contrast, the average single-base substitution error rate of human pol nu is 3.5 x 10(-3), which is inaccurate compared to the replicative polymerases and comparable to Y family polymerases. Interestingly, the vast majority of errors made by pol nu reflect stable misincorporation of dTMP opposite template G, at average rates that are much higher than for homologous A family members. This pol nu error is especially prevalent in sequence contexts wherein the template G is preceded by a C-G or G-C base pair, where error rates can exceed 10%. Amino acid sequence alignments based on the structures of more accurate A family polymerases suggest substantial differences in the O-helix of pol nu that could contribute to this unique error signature.     

Demyelination and wasting associated with polyomavirus infection in nude (nu/nu) mice

Nude (nu/nu) mice bearing human tumour heterografts were affected with posterior paralysis and wasting. There was demyelination and infection of the oligodendrocytes of the spinal cord with a papovavirus. Similar virus particles and inclusion bodies were found in the bronchial epithelium, which showed histopathological changes. Similar changes were shown by the epithelia of the renal pelvis, ureter and choroid plexus. The virus was found in a transplantable human tumour, and evidence of spread by contact was also obtained. Intracerebral injection of spinal cord suspension from infected mice resulted in virus infected cutaneous carcinomata, demyelination with virus particles in the oligodendrocytes and posterior paralysis with wasting in adult nude mice. The suspension injected intraperitoneally into newborn Syrian hamsters produced tumours similar to those produced by murine polyoma. No evidence of infection was found in mice from the colony of origin. The virus was identified as murine polyoma Wild Type A2.     

Unfolded protein response is required in nu/nu mice microvasculature for treating breast tumor with tunicamycin

Up-regulation of the dolichol pathway, a "hallmark" of asparagine-linked protein glycosylation, enhances angiogenesis in vitro. The dynamic relationship between these two processes is now evaluated with tunicamycin. Capillary endothelial cells treated with tunicamycin were growth inhibited and could not be reversed with exogenous VEGF(165). Inhibition of angiogenesis is supported by down-regulation of (i) phosphorylated VEGFR1 and VEGFR2 receptors; (ii) VEGF(165)-specific phosphotyrosine kinase activity; and (iii) Matrigel(TM) invasion and chemotaxis. In vivo, tunicamycin prevented the vessel development in Matrigel(TM) implants in athymic Balb/c (nu/nu) mice. Immunohistochemical analysis of CD34 (p < 0.001) and CD144 (p < 0.001) exhibited reduced vascularization. A 3.8-fold increased expression of TSP-1, an endogenous angiogenesis inhibitor in Matrigel(TM) implants correlated with that in tunicamycin (32 h)-treated capillary endothelial cells. Intravenous injection of tunicamycin (0.5 mg/kg to 1.0 mg/kg) per week slowed down a double negative (MDA-MB-435) grade III breast adenocarcinoma growth by ～50-60% in 3 weeks. Histopathological analysis of the paraffin sections indicated significant reduction in vessel size, the microvascular density and tumor mitotic index. Ki-67 and VEGF expression in tumor tissue were also reduced. A significant reduction of N-glycan expression in tumor microvessel was also observed. High expression of GRP-78 in CD144-positive cells supported unfolded protein response-mediated ER stress in tumor microvasculature. ～65% reduction of a triple negative (MDA-MB-231) breast tumor xenograft in 1 week with tunicamycin (0.25 mg/kg) given orally and the absence of systemic and/or organ failure strongly supported tunicamycin's potential for a powerful glycotherapeutic treatment of breast cancer in the clinic.     

Changes in proliferation activity and relative distributions of lymphoid cell subpopulations in congenitally athymic nu/nu mice and lurcher mice with spontaneous olivopontocerebellar degeneration

Changes observed in mice with congenital damage of some part of the CNS-neuroendocrine-immune regulatory system are described. nu/nu mice with congenital absence of thymus and Lurcher mice with spontaneous olivopontocerebellar degeneration displayed changes in the histoarchitecture of adrenal gland, immune organs (thymus, spleen, axillar lymph nodes) and intestine. Changes were also observed in IgM+, IgG+, CD4+ and CD8+ lymphoid cell subpopulations in the main lymphoid organs--the spleen and axillar lymph nodes and in the proliferative ability of whole lymphoid cell populations. The extreme decrease of lymphoid T-cell subpopulations in athymic nu/nu mice is the consequence of the absence of thymus, the organ of their maturation. On the other hand, a relative increase of B-cell subpopulations was found in this mouse strain. A relative decrease of CD4+ lymphocytes and a different influence of immunization on B-cell subpopulations were found in the spleen in neurodeficient Lurcher mice. The high percentage of apoptotic cells, cells in the S-phase of cell cycle and increased proliferation index in nu/nu mice suggest that the turnover and renewal of lymphoid cells in the spleen in nu/nu mice is more rapid than in control immunocompetent BALB/c mice.     

Interleukin 2 production by lymphoid cells from congenitally athymic (nu/nu) mice

The ability of lymphoid cells from congenitally athymic (nu/nu) mice to produce interleukin 2 (IL 2) was investigated. Spleen or lymph node cells (superficial or mesenteric) from nude mice on an N:NIH(S)II or BALB/c genetic background were stimulated with concanavalin A (Con A) or with irradiated allogeneic (DBA/2) spleen cells that had been depleted of T cells by treatment with monoclonal anti-Thy-1.2 antibody plus complement. After 24 hr, supernatants were harvested and assayed for their ability to support the proliferation of a cloned IL 2-dependent cytolytic T cell line. With this quantitative microassay, IL 2 production was not detectable in spleen and lymph nodes of 6-wk-old N:NIH(S)II nude mice; however, by 12 mo of age, IL 2 production increased more than 100-fold to reach levels comparable to control (nu/+) animals. Con A was more potent than alloantigen in the induction of IL 2 in either nude or control (nu/+) animals. Furthermore, differences in the genetic background of nude mice resulted in corresponding differences in both numbers of T cells (defined by monoclonal anti-Thy-1 antibody) and IL 2 production. By using negative selection with monoclonal antibodies plus complement, IL 2 production in aged nude mice was shown to depend upon a subpopulation of cells that expressed Thy-1 but not Lyt-2. These data thus demonstrate that a subpopulation of IL 2-producing cells with a Thy-1+ Lyt-2- surface phenotype can develop in the apparent absence of thymic influence.