Genome instability in the F1-progeny of mice irradiated by ionizing radiation as determined by micronucleus assay

The F1-progeny of BALB/c male mice chronically exposed to low-dose gamma-radiation (0.1; 0.25 and 0.5 Gy; dose rate 0.01 Gy/day) as well as the F1-progeny of females exposed to acute X-radiation (0.5; 1.0 and 2.0 Gy; dose rate 0.1 Gy/min) shown the significant elevated micronuclei frequencies in bone marrow erythrocytes, as compared to the F1-progeny of unirradiated males and females. The increase in the micronuclei frequency in the F1-progeny was determined by the dose of irradiation of parents. The values of elevated micronuclei frequency in the F1-progeny of chronically irradiated males and acutely irradiated females for a dose of 0.5 Gy were comparable. The micronuclei frequencies in the F1-progeny of irradiated females and males for this dose were in 1.5 and in 1.6 times higher than ones in the F1-progeny of unirradiated mice correspondingly. The results suggest the possibility of transfer of genome instability from irradiated parents to the somatic cells of the F1-progeny via non-lethally damaged germ cells of parents.     

Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting

Sera from 28 patients with bullous pemphigoid (BP), four patients with cicatricial pemphigoid (CP), and 24 controls (normal volunteers and patients with pemphigus, systemic lupus erythematosus, or other skin diseases) were tested against extracts of human epidermis by immunoblotting techniques. The extraction buffer included 1% SDS, 5% beta-mercaptoethanol, and six protease inhibitors with various specificities. BP sera from individual patients showed different patterns of reactivity with the same epidermal extract, and each pattern consisted of one or more bands. A total of five bands of 240 kD, 200 kD, 180 kD, 97 kD, and 77 kD reacted with BP sera; the 240-kD band reacted with one CP sera, and none of these bands was detected by the control sera. The 240-kD and 180-kD bands reacted very strongly with some sera and were most frequently observed (43% and 29%, respectively). The 200-kD, 97-kD, and 77-kD bands were less frequently observed (25%, 7%, and 7%, respectively), but when present, their reactions were usually strong. Eleven percent of the BP sera did not react with any bands. Contrary to previous reports, this study shows that BP autoantibodies react with several protein bands, as detected by immunoblotting. We have recently shown by immunoelectron microscopy that BP autoantibodies bind to the basal cell hemidesmosomes. It remains to be determined which of these protein bands represent specific hemidesmosomal proteins and which antibody-antigen interactions are relevant to the pathogenesis of this disease.     

Modulation of F1 cytotoxic potentials by graft-vs-host reaction. Cooperative non-H-2- and H-2D region-gene control of F1 natural resistance to graft-vs-host reaction-associated immunosuppression

Our previous study revealed that in F1 mice raised by crossing C3H/He or AKR/J mice with various H-2-congenic B10-series strains, parental H-2k spleen cells (SC) could not induce the graft-vs-host reaction (GvHR)-associated immunosuppression (GAIS). We also elucidated that a limited number of non-H-2 genes of parental C3H/He or AKR/J mice that had been incorporated into the F1 hybrids determined the F1 resistance to the GAIS, and the present study was done to explore the mechanism implicated in this type of F1 resistance to GAIS. SC from B10.AL mice carrying an rH-2 (K:k I:k S:k D:d) haplotype but not SC from H-2K B10.BR (k k k k) mice induced GAIS of in vitro CTL responses to third-party alloantigens in H-2k/d (C3H/He x B10.D2)F1 recipients mice. Further, SC from H-2k/a (C3H/He x B10.A)F1 mice carrying heterozygous C3H/B10 non-H-2 background but not SC from the same H-2k/a (B10.BR x B10.A)F1 mice but carrying homozygous B10/B10 background induced GAIS in H-2k/d (C3H/He x B10.D2)F1 recipients. Although C3H/He-, B10.BR-, and C3H.OH (d d d k)-SC were incapable of inducing GAIS in (C3H/He x B10.D2)F1 (k/d k/d k/d k/d) recipients, they were all good inducers of GAIS in (C3H.OH x B10.BR)F1 (d/k d/k d/k k/k) recipients. Exactly the same pattern of co-operative non-H-2 AKR and H-2D region-gene control of GAIS was observed on GvHR induced in H-2k/d (AKR/J x B10.D2)F1 recipients. These results suggest that the non-H-2 genes of C3H/He or AKR/J strain inhibit the functional expression of certain antigenic determinant(s) when it is encoded by heterozygous but not homozygous gene(s) linked tightly to H-2D region of k haplotype. Thus, the F1 resistance to GAIS is mediated by immune response of F1 recipients who miss the antigenic determinant(s) against that expressed on cell surface of GvHR-inducing T lymphocytes.     

Alterations in the expression of Ia antigens in F1 hybrid mice

The Ia.8 and 9 specificities detected either by conventional or monoclonal antisera (Ia.m3, 4) are present in strains bearing the b H-2 haplotype, but absent from those with the k haplotype. It would be expected that the (b x k)F₁ hybrids would have approximately half the amount of these specificities found on the b parent, but the Ia.8 and 9 specificities are absent or reduced in this F₁ hybrid, though not on F₁ LPS blasts. Examination of appropriate H-2 congenic strains demonstrated that only the k haplotype confers the absence of these specificities on H-2 ^b — it was not observed with b, d, q, r or s haplotypes. In the k haplotype the gene(s) responsible for this effect is mapped to the I-A ^k subregion. The reason for this low expression effect is not clear but the observation has important implications for the relationship of Ia specificities and Ir genes and may serve to explain the low responder status of certain F₁ hybrids, e. g., to TNP-mouse serum albumin, as observed elsewhere.     

Differential effects of alloimmunization on T cells mediating graft-vs-host splenomegaly or the allogeneic effect in F1 mice.

Spleen cells from normal BALB/c mice or mice immunized 10 or 30 days previously with C57BL/6 spleen cells were tested for a) their capacity to produce graft-vs-host (GVH) reactions in newborn F1 mice and b) their capacity to produce an allogeneic effect in adult F1 mice immunized with Type III pneumococcal polysaccharide. GVH reactivity of alloimmune spleen cells obtained 10 or 30 days after immunization was significantly increased as compared to the reactivity of normal spleen cells in that a) at comparable cell doses, higher spleen indices were obtained with alloimmune cells than normal cells, and b) alloimmune cells produced severe runting at lower cell doses than normal cells. By comparison, the capacity of alloimmune spleen cells to produce an allogeneic effect was reduced 50% on a per cell basis as compared to normal spleen cells at both 10 and 30 days after immunization. These results give further evidence that T cells producing the allogeneic effect are distinct from the T cell populations which interact to produce GVH splenomegaly.     

Genetic and cellular requirements for primary antibody immune response to Thy-1 antigens in mice.

An effective anti-Thy-1 response requires at least three conditions to be simultaneously fulfilled--1) The responder has to share an H-2 haplotype, or at least K and D alleles, with the donor of the Thy-1 disparate thymocytes, 2) The responder has to differ from the donor by some non-H-2 cell-surface antigens, and 3) The responder has to possess functionally capable T cells. All these requirements are consistent with the hypothesis that Thy-1 antigens and the non-H-2 antigen(s), the latter serving as carrier, form a complex that is viewed as a variant of self and then recognized in the context of the H-2 molecules by the responder's T cells. An attractive feature of the proposed hypothesis is that it could be applicable to a variety of cell-surface and also exogenous antigens.     

Valency of antibody binding to enveloped virus particles as determined by surface plasmon resonance

A simple method is described for determining the valency of binding of immunoglobulin G to immobilized influenza A virus. Where there is a free Fab arm (monovalent binding), a second virus particle is captured. This is detected by surface plasmon resonance. The methodology should be applicable to all enveloped and nonenveloped viruses.     

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/10J) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.     

Cell-mediated immunity to non-HLA antigens of the host by donor lymphocytes in patients with chronic graft-vs-host disease

Forty-four patients with aplastic anemia or leukemia were given marrow grafts from siblings selected on the basis of HLA-A and -B identity and mutual nonreactivity of their lymphocytes in mixed leukocyte culture (MLC). Twenty-two to 1089 days after grafting, their lymphocytes (of donor origin) were tested for reactivity in MLC to lymphocytes from the host (cryopreserved before grafting), the marrow donor, and unrelated individuals. Lymphocytes from 14 of 22 long-term survivors with chronic graft-vs-host disease (GVHD) showed unidirectional reactivity in response to host lymphocytes manifested as high stimulation indices (SI) and high relative responses (RR). Lymphocytes from only 1 of 12 long-term survivors without chronic GVHD showed unidirectional reactivity to host lymphocytes. Statistical analysis showed that lymphocytes from patients with chronic GVHD displayed anti-host responses that were significantly higher than those of lymphocytes from either marrow donors (p < 0.001) or patients without GVHD (p = 0.03). Lymphocytes from 5 patients with and 5 without acute GVHD, tested shortly after marrow grafting, failed to show responses to host cells. The results are consistent with a participation of cell-mediated immunity of graft against host in chronic GVHD.     

Antibodies to saline extractable tissue antigen in sera of patients with renal diseases

Multiple serum samples originating from 110 renal allograft recipients were examined against saline extract of normal human kidney by means of double diffusion gel precipitation. Eleven recipients were found to be positive; 99 of 106 sera from these patients were positive. Pretransplantation sera were available from 7 of these recipients and 6 patients were found "positive." The precipitation reaction was composed of one line. Identity reactions were formed between the lines produced by sera from all patients except 1. Sera of patients from end-stage renal disease produced similar reaction; however, only 3 of 234 sera from patients with nonrenal diseases precipitated the kidney extract. None of 154 normal sera were positive. Several positive sera also were positive in complement fixation tests with human kidney extract. Evidence was presented that the antibodies under study combined with a nonorgan-specific but species-restricted tissue antigen. The hypothesis was advanced that these antibodies are autoantibodies formed in response to a sequestered antigen released as a result of tissue damage. Apparently, the antigen is released frequently in immunogenic form from injury to kidney but infrequently from injury to other organs.     

鼠疫疫苗Ⅱa期临床血清对小鼠的被动保护水平及其与抗体效价的相关性分析

目的根据"动物法则"验证鼠疫疫苗临床有效性免疫学指标为F1抗体效价和V抗体效价,为疫苗的临床有效性评价提供依据。方法将BALB/c小鼠随机分为6组,每组10只。其中5组用含有不同效价的F1抗体和V抗体的鼠疫疫苗IIa期临床人血清腹腔被动免疫小鼠,另外1组为对照;3 h后各实验组分别以6 MLD鼠疫耶尔森菌标准强毒菌株141株(简称鼠疫强毒菌)攻击,对照组以2MLD鼠疫强毒菌攻击,观察期14 d,详细记录14 d内的动物死亡时间,并对攻击小鼠的存活数据进行Kaplan-Meier生存分析和Spearman相关性分析。结果鼠疫疫苗IIa期临床人血清被动保护小鼠结果显示,对照组小鼠在2 MLD强毒菌攻击下的存活率为0.0%,平均存活时间为6.5 d;实验组临床人血清被动免疫小鼠在6 MLD强毒菌攻击下的存活率分别为10.0%、0.0%、40.0%、40.0%、90.0%,小鼠平均存活时间分别提高到9.1 d、9.9 d、11.2 d、11.4 d、13.9 d。F1抗体效价与小鼠平均存活时间具有显著相关性(P=0.000、r=1.000);V抗体效价与小鼠平均存活时间具有显著相关性(P=0.014、r=0.949)。F1抗体效价与小鼠存活率具有显著相关性(P=0.000、r=1.000);V抗体效价与小鼠存活率具有显著相关性(P=0.014、r=0.949)。结论鼠疫疫苗IIa期临床人血清可对小鼠提供被动保护,且保护力水平与F1抗体效价和V抗体效价具有显著相关性。     

Mobility of elastin chains as determined by 13C nuclear magnetic resonance

Relaxation times and integrated intensities of ¹³C have been obtained from nuclear magnetic resonance spectra of elastin in unstretched calf ligamentum nuchae and indicate that about 80% of the backbone carbonyl carbons have short rotational correlation times, τ_R ～ 40 nanoseconds. τ_R is reduced by only a factor of two when the ligament is in contact with 2 m-KCNS, a strong denaturant. By contrast, the highly ordered chains of collagen in insoluble calf achilles tendon give no spectrum until denatured in 2 m-KCNS, when t_R decreases by many orders of magnitude. These results show that elastin is composed largely of highly mobile chains under physiological conditions, suggesting that configurational entropy has an important role in its elastic properties.     

Reversal of advanced murine lupus in NZB/NZW F1 mice by treatment with monoclonal antibody to L3T4

Murine lupus in NZB/NZW F1 (B/W) mice can be prevented by weekly treatment with monoclonal antibodies (MAb) to L3T4 (on "helper/inducer" T cells) if treatment is begun prior to the onset of clinical illness. To determine whether anti-L3T4 could reverse as well as prevent murine lupus, we monitored a cohort of 30 B/W females until age 7 mo, when severe autoimmune disease was established, and then we examined the effects of weekly treatment with MAb to L3T4. The rate of target cell clearance by MAb was considerably slower in old B/W mice than it was in young B/W mice or in normal (BALB/c and C57BL/6) mice. Nonetheless, treatment with anti-L3T4 depleted 90% of circulating L3T4+ cells over 3 mo. In treated mice, the concentration of anti-DNA antibodies fell by 80%, renal insufficiency was reversed, and 1 yr survival was 75% compared to 17% in controls. These findings indicate that L3T4+ cells play an important role in perpetuating murine lupus in B/W mice even after severe disease is present. Because the L3T4 antigen in mice is homologous to the Leu-3/T4 (CD4) antigen in humans, these findings suggest that treatment with CD4 MAb may be effective in people with systemic lupus erythematosus.     

Inhibition of humoral immunity in vivo by monoclonal antibody to L3T4: studies with soluble antigens in intact mice

Monoclonal antibody (MAb) to the mouse "helper" T cell antigen L3T4 inhibits the T cell response to class II major histocompatibility antigens on antigen-presenting cells in vitro and in thymectomized mice. To examine the effect of MAb to L3T4 on humoral immunity in euthymic mice, we treated BALB/c mice with 1 mg of anti-L3T4 i.p. at the time of immunization with either bovine serum albumin (BSA) or chicken egg ovalbumin (OA) in complete Freund's adjuvant. Administration of MAb to L3T4 selectively depleted greater than 90% of L3T4+ cells from the blood, spleen, and lymph nodes, but it had little effect on thymocytes. Mice treated with anti-L3T4 were unable to generate an IgG response to either BSA or OA. Treatment with anti-L3T4 also prevented the antigen-specific IgM response to these antigens, although it did not prevent nonspecific stimulation of IgM anti-BSA and anti-OA antibodies induced by adjuvant in the absence of antigen. Humoral immunity was inhibited even when treatment was delayed until 48 hr after immunization. These findings indicate that T cell help for humoral immunity can be abrogated in intact mice by MAb to L3T4.     

Graft-vs-host reactions in F1 mice induced by parental lymphoid cells: nature of recruited F1 cells.

Graft versus host (GVH) reactivity of parental lymph node (LN) cells was assayed by measurements of 3H-thymidine incorporation in vivo. Mitomycin (Mit.) treatment of parental cells abolished their proliferative activity but the combination of such Mit.-treated parental cells with F1 LN cells resulted in much higher proliferation than either one population alone. This recruitment into proliferation of F1 cells was prominent on days 3 and 4 after cell injection and amounted to 35 to 51% of the total activity seen after injection of untreated parental cells alone. The F1 cell sensitive to recruitment was resistant to anti-Thy 1.2 treatment, was not removed by carbonyl iron-magnet separation; and was not present in thymus. The parental cell inducing recruitment was, however, sensitive to anti-Thy 1.2. When spleen cells from hapten immune F1 donors were injected together with Mit.-treated parental LN cells and boosted with hapten on another carrier, a typical "allogeneic effect" was observed in the anti-hapten immune response. It was concluded that Mit.-treated parental T cells exerted a mitogenic effect on F1 B cells resulting in extensive recruitment similar to that seen in murine mixed lymphocyte reactions.     

Androgen receptor ligand-binding domain interaction and nuclear receptor specificity of FXXLF and LXXLL motifs as determined by L/F swapping

The androgen receptor (AR) ligand-binding domain (LBD) binds FXXLF motifs, present in the AR N-terminal domain and AR-specific cofactors, and some LXXLL motifs of nuclear receptor coactivators. We demonstrated that in the context of the AR FXXLF motif many different amino acid residues at positions +2 and +3 are compatible with strong AR LBD interaction, although a preference for E at +2 and K or R at +3 was found. Pairwise systematic analysis of F/L swaps at +1 and +5 in FXXLF and LXXLL motifs showed: 1) F to L substitutions in natural FXXLF motifs abolished AR LBD interaction; 2) binding of interacting LXXLL motifs was unchanged or increased upon L to F substitutions; 3) certain noninteracting LXXLL motifs became strongly AR-interacting FXXLF motifs; whereas 4) other nonbinders remained unaffected by L to F substitutions. All FXXLF motifs, but not the corresponding LXXLL motifs, displayed a strong preference for AR LBD. Progesterone receptor LBD interacted with some FXXLF motifs, albeit always less efficiently than corresponding LXXLL motifs. AR LBD interaction of most FXXLF and LXXLL peptides depended on classical charge clamp residue K720, whereas E897 was less important. Other charged residues lining the AR coactivator-binding groove, K717 and R726, modulated optimal peptide binding. Interestingly, these four charged residues affected binding of individual peptides independent of an F or L at +1 and +5 in swap experiments. In conclusion, F residues determine strong and selective peptide interactions with AR. Sequences flanking the core motif determine the specific mode of FXXLF and LXXLL interactions.     

Confocal microscopy of giant vesicles supports the absence of HIV-1 neutralizing 2F5 antibody reactivity to plasma membrane phospholipids

The broadly neutralizing anti-HIV-1 2F5 monoclonal antibody recognizes a gp41 epitope proximal to the viral membrane. Potential phospholipid autoreactivity at cell surfaces has raised concerns about the use of this antibody for development of vaccines or immunotherapy. In this study, confocal microscopy of giant unilamellar vesicles (GUVs) was used to assess 2F5 reactivity with phospholipids assembled into bilayers with surface charge and curvature stress approximating those of the eukaryotic plasma membranes. Antibody partitioning into lipid bilayers required the specific recognition of membrane-inserted epitope, indicating that 2F5 was unable to directly react with GUV phospholipids, even under fluid phase segregation conditions. Our results thus support the feasibility of raising 2F5-like neutralizing responses through vaccination, and the medical safety of mAb infusions.