Sexual and aggressive interactions in a visible burrow system with provisioned burrows

The visible burrow system (VBS) is a habitat providing burrows and an open area for mixed-set rat colonies. Provisioning of food and water in the burrows makes it unnecessary for potentially defensive animals to leave the burrows to eat/drink on the surface, and enables evaluation of new types of agonistic interactions that may emerge when this necessity is removed. In such colonies, subordinate males showed high magnitude tunnel guarding behavior, occupying a tunnel opening onto the surface and confronting the dominant. Dominants, in response, made lunges into the tunnels, but quickly retreated without gaining entry, apparently stopped by contact with the defender's vibrissae. Dominants also made and continued to make lateral attacks to the wall adjacent to the tunnels guarded by subordinates, although these were useless in terms of affording contact with the subordinate. Dominant-female agonistic interactions were more frequent than those of dominants and subordinates. These were largely initiated by the male, and involved female defensive behavior. Nonetheless, females, unlike subordinates, failed to show tunnel guarding and continued to utilize the surface freely. They also spent more time in the vicinity of the dominant over days of colony formation. This apparent paradox may reflect that females were seldom wounded, and that the initial site of male contact with females was the female's anogenital area, findings suggesting that interactions of males and females often reflect male sexual advances, countered by female defenses that effectively protect nonestrus females from mounting and copulation.     

Lack of correlation between expression of Epstein-Barr virus (EBV) latent membrane protein and bcl-2 oncoprotein in vivo.

AIMS--To evaluate whether there is any correlation between the expression of Epstein-Barr virus (EBV) latent membrane protein (LMP) and oncoprotein bcl-2 in the lymph node biopsy specimens of a Chinese patient with EBV-related reactive lymphoproliferation who later developed T cell lymphoma after a short period of time. METHODS--Immunohistochemistry, with a standard alkaline phosphatase antialkaline phosphatase (APAAP) method and New Fuchsin as a chromogen, was used for single staining of bcl-2 or LMP. Double immunostaining combining APAAP and indirect immunofluorescence was performed for dual labelling of LMP and bcl-2. RESULTS--bcl-2 was expressed in 10-30% of cells in the first lymph node biopsy specimen (EBV-associated lymphoproliferative disorder) and 30-50% of cells in the second lymph node biopsy specimen (T cell lymphoma). LMP was expressed in the first biopsy specimen but not in the second. Double immunostaining results showed that around 78% of LMP positive cells were bcl-2 negative and 94% bcl-2 positive cells were LMP negative. Among the very small fraction of LMP and bcl-2 double positive cells, the intensity of bcl-2 staining was heterogeneous and was not always stronger than that observed in LMP negative bcl-2 positive cells. CONCLUSIONS--The expression of bcl-2 protein is independent of LMP protein status in vivo. Several mechanisms may be involved in EBV associated lymphomagenesis, and bcl-2 induction may occur independently of LMP expression.     

84例周围血管病的足甲襞微循环观察

对84例周围血管病(脉管炎68例,静脉炎16例)和25例正常健康人进行了足甲襞微循环的对比观察。结果:患病组足甲襞微循环的血管形态、流态、管周状态均较正常健康组有明显差异(P<0.01)。提示:更接近病变部位的局部微循环观察更能代表病变的程度,对临床医生判断病情、选择用药和疗效观察都有很大帮助和参考价值。     

Contrasting Roles of Mannan-Specific Monoclonal Immunoglobulin M Antibodies in the Activation of Classical and Alternative Pathways by Candida albicans

Polyclonal antimannan immunoglobulin G (IgG) activates the classical complement pathway and accelerates initiation of the alternative pathway by Canidida albicans. This dual role was assessed for two antimannan IgM monoclonal antibodies (MAbs). MAb B6.1 is specific for an epitope on the acid-labile portion of C. albicans phosphomannan; MAb B6 is specific for an epitope on the acid-stable region. Both MAbs were potent activators of the classical pathway but poor facilitators of alternative pathway initiation.Candida albicans activates the human complement system via both the classical and the alternative pathways, leading to deposition of opsonic complement fragments on the yeast cell surface (8, 10, 18). In previous studies, we described a critical role for naturally occurring antimannan immunoglobulin G (IgG) in complement activation by C. albicans. Those studies used a kinetic assay for C3 deposition on the yeast and immunofluorescence evaluation of the sites of C3 binding (10, 17, 18). Deposition of C3 onto C. albicans cells incubated in normal human serum (NHS) occurs rapidly via the classical pathway and can be detected within the first 2 min of incubation. If the classical pathway is blocked by chelation of Ca²⁺ with EGTA, C3 deposition occurs via the alternative pathway, but C3 deposition is delayed and a 6-min incubation is required before bound C3 is readily detectable on the yeast surface. Removal of naturally occurring antimannan IgG from the serum by mannan absorption profoundly delays accumulation of C3 on the yeast cell surface, with 12 min or more of incubation being required before appreciable amounts of bound C3 are detected. However, this 12-min delay can be overcome by supplementation of the mannan-absorbed serum with affinity-purified human antimannan IgG in the absence of EGTA to mediate classical pathway initiation or shortened to 6 min in the presence of EGTA to allow antibody-facilitated activation of the alternative pathway. These observations demonstrate a dual role for antimannan IgG in serum from healthy adults in complement activation by C. albicans. Antimannan IgG mediates activation of the classical pathway and facilitates initiation of the alternative pathway (17, 18).In studies described above, we used polyclonal antimannan IgG purified from pooled human plasma. Since C. albicans cells express a number of immunodominant mannan components recognized by rabbits (15, 16), the human polyclonal antimannan IgG likely contains a range of specificities for distinct mannan determinants. It has been shown that rabbit antibodies that are reactive with three different cell wall determinants of group A streptococci display differential abilities to activate the classical or alternative pathway (2). Although the antibodies specific for three different cell wall epitopes all activated the classical pathway, only antibody specific for the N-acetyl-d-glucosamine epitope activated the alternative pathway (2). In a separate study, capsular as well as noncapsular antibodies were found to direct classical-pathway-mediated killing of Haemophilus influenzae type b, whereas only the capsular antibodies promoted killing by the alternative pathway (12). These studies provide evidence that epitope specificity may influence the ability of an antibody to activate the alternative pathway and prompted us to examine whether antibodies that recognize different mannan determinants are able to mediate activation of the classical and alternative pathways by C. albicans.Two IgM monoclonal antibodies (MAbs) that recognize distinct mannan determinants were compared for their abilities to activate the classical or alternative pathway. MAb B6.1 is specific for an acid-labile component of the Candida phosphomannan complex, and MAb B6 is specific for an acid-stable component (5). The MAbs were produced commercially (Montana ImmunoTech, Inc., Bozeman, Mont.).C. albicans CA-1 was grown as yeast forms to stationary phase in glucose (2%)-yeast extract (0.3%)-peptone (1%) broth for 24 h at 37°C as described elsewhere (4, 6, 10). The mannan of CA-1 yeast was purified as described previously (7, 18) and coupled to CNBr-Sepahrose 4B (Pharmacia Biotech, Uppsala, Sweden) (18).Pooled NHS was prepared from peripheral blood from at least 10 healthy adult donors and stored at −80°C. C3 was isolated from frozen human plasma (9, 13) and stored at −80°C until used. C3 was labeled with ¹²⁵I as described previously (3) by use of IODO-GEN reagent (Pierce, Rockford, Ill.). NHS was absorbed with mannan-Sepharose 4B to remove antimannan antibodies (18).Kinetics of C3 binding were assayed by the method of Kozel et al. (10). To determine whether MAb B6 or B6.1 activates the classical pathway, 2 × 10⁶ yeast cells were incubated at 37°C in 1 ml of a complement binding medium that contained (i) 40% NHS, mannan-absorbed serum, or mannan-absorbed serum supplemented with MAb B6 or B6.1, (ii) sodium Veronal (5 mM)-buffered saline (142 mM, pH 7.3) containing 0.1% gelatin, 1.5 mM CaCl₂, and 1 mM MgCl₂, and (iii) ¹²⁵I-labeled C3. To study whether MAb B6 or B6.1 plays a role in alternative pathway initiation, yeast cells were incubated in the manner described above except that the binding medium was not supplemented with Ca²⁺ and contained 5 mM EGTA and 5 mM MgCl₂. At various time intervals from 2 to 16 min, 50-μl samples were withdrawn in duplicate and added to 200 μl of phosphate-buffered saline–0.1% sodium dodecyl sulfate–20 mM EDTA in Millipore MABX-N12 filter plates fitted with BV 1.2-μm-pore-size filter membranes (Millipore, Bedford, Mass.). The cells were washed with phosphate-buffered saline–0.1% sodium dodecyl sulfate, and filter-bound radioactivity was determined with a gamma counter. Nonspecific binding was estimated from cells incubated in NHS containing EDTA and was subtracted from the total counts.Mannan absorption of serum profoundly delayed C3 accumulation on yeast from 2 min to approximately 10 min (Fig. ​(Fig.11 and ​and2).2). However, addition of either MAb B6 or MAb B6.1 at 50 μg per ml of reaction mixture to the absorbed serum generated rapid activation kinetics characteristic of C3 deposition via the classical pathway (Fig. ​(Fig.1)1) (10, 17, 18). This observation was not unexpected, as polyvalent IgM is known to be a potent activator of the classical pathway. Open in a separate windowFIG. 1Effect of MAb B6 or B6.1 on the kinetics of C3 deposition on C. albicans cells via the classical pathway. Yeast cells were incubated in a C3 binding medium containing (i) 40% NHS (•), (ii) 40% mannan-absorbed NHS (○), (iii) 40% mannan-absorbed NHS supplemented with MAb B6 (▴), or (iv) 40% mannan-absorbed NHS supplemented with MAb B6.1 (▿) at 50 μg per ml of reaction mixture. C3 deposition patterns from three independent assays were similar; results from one representative assay are shown.Open in a separate windowFIG. 2Effect of MAb B6 or B6.1 on the kinetics of C3 deposition on C. albicans cells via the alternative pathway. Yeast cells were incubated in a C3 binding medium containing (i) 40% NHS (•), (ii) 40% NHS–EGTA (■), (iii) 40% mannan-absorbed NHS containing EGTA (○), (vi) 40% mannan-absorbed NHS containing EGTA supplemented with MAb B6 (▴), or (iv) 40% mannan-absorbed NHS supplemented with MAb B6.1 (▿) at 50 μg per ml of reaction mixture. C3 deposition patterns from four independent assays were similar; results from one representative assay are shown.The effects of MAbs B6 and B6.1 on activation of the alternative pathway were assessed by addition of the antibodies to mannan-absorbed serum in the presence of EGTA. The results (Fig. ​(Fig.2)2) showed that neither MAb B6 nor MAb B6.1 at 50 μg per ml of reaction mixture altered the alternative pathway activity of the mannan-absorbed serum. To determine whether the inability of MAb B6 or B6.1 to facilitate initiation of the alternative pathway was influenced by antibody concentration, the experiment represented in Fig. ​Fig.22 was repeated with mannan-absorbed serum that was supplemented with 10 to 160 μg of MAb B6 or B6.1 per ml. These antibody concentrations were chosen because in our previous studies we found that affinity-purified human antimannan IgG activates both the classical and alternative pathways (17). However, at 10, 40, or 160 μg per ml of reaction mixture, both antibodies failed to enhance alternative pathway activity of mannan-absorbed serum but promoted classical pathway activity (data not shown).The observation that both MAbs were unable to enhance alternative pathway activity was unexpected. Our previous studies showed that addition of polyclonal antimannan IgG to mannan-absorbed NHS containing EGTA produced C3 binding kinetics that were indistinguishable from the kinetics observed with nonabsorbed NHS containing EGTA (17). We further demonstrated IgG-dependent initiation of the alternative pathway by C. albicans using the six purified alternative pathway proteins (17).There are at least three possible explanations for the failure of MAbs B6 and B6.1 to facilitate activation of the alternative pathway. First, it is possible that antimannan antibodies of the IgM class are unable to enhance C3 deposition via the alternative pathway. However, there is evidence that polyclonal IgM is able to enhance alternative pathway-mediated lysis of rabbit erythrocytes by NHS (11, 14). Second, the ability of an antibody to facilitate deposition of C3 via the alternative pathway could be epitope specific; MAbs B6 and B6.1 could have the wrong epitope specificity. As noted above, Eisenberg and Schwab (2) found that polyclonal antibodies specific for one antigen found on group A streptococcal cell walls were able to facilitate initiation of the alternative pathway, whereas antibodies specific for two other antigens were not. If antibody-facilitated activation of the alternative pathway is dependent on epitope specificity, such a finding might influence strategies for induction of protective immunity to Candida. Optimal immunization may require an immunogen that induces antibodies with epitope specificities needed to facilitate activation of the alternative pathway. Finally, we cannot exclude the possibility that human antimannan antibodies are able to facilitate activation of the alternative pathway, whereas mouse antibodies lack this capability.In studies involving a murine model of disseminated candidiasis, MAb B6.1 was shown to be protective, whereas MAb B6 was not (4). However, the protection mechanisms remain to be elucidated. In an in vitro assay, MAb B6.1 but not MAb B6 was found to enhance candidacidal activity of polymorphonuclear leukocytes in the presence of fresh mouse serum, suggesting the involvement of mouse complement in the killing (1). Although assessing the role of complement in MAb B6.1-mediated protection was beyond the scope of this study, our observation that the two antibodies mediate similar kinetics of C3 deposition for C. albicans does not preclude the possibility that the composition and/or accessibility of opsonic complement fragments bound to the yeast cells might differ following complement activation by these two antibodies. Alternatively, the concerted action of several protective functions, including activation of the complement system, may be required for MAb B6.1-mediated protection.     

Ovarian serous cystadenoma with mural nodules of genital rhabdomyoma

We present an extremely rare case of ovarian serous cystadenoma with mural nodules of rhabdomyoma. The patient, a 48-year-old woman, was admitted to our hospital with left lower abdominal pain and vaginal bleeding. A unilocular cystic tumor, measuring 13 x 10 x 10 cm, was found in her left ovary and was removed. The tumor contained clear serous fluid, approximately 600 mL, and 2 mural nodules, up to 7.5 x 5.5 x 4.5 cm. The internal cystic wall was thin for the most part and lined with ciliated cuboidal epithelium without any malignant feature. The mural part was composed of mainly more mature muscle fibers with easily discernible cross-striations, set in abundant myxoid to fibromyxoid stroma, similar to clinical and microscopic manifestations of genital rhabdomyomas reported in other sites. Because extracardiac rhabdomyoma has never been described occurring in the ovary, especially arising in serous cystadenoma, to our knowledge, this is the first case reported in the English literature.     

Characterization of cortical neuron outgrowth in two- and three-dimensional culture systems

To improve the ability of regeneration by grafting living cells or by adding growth factor to a lesion site, it is important to find good biomaterials for neuron survival and regeneration. This study focused on two- and three-dimensional cultures in a matrix using biomaterials such as agarose, collagen, fibrin, and their mixtures, because these are considered to be suitable biomaterials for neuron outgrowth. Cortical neurons were dissected from E17 rat embryos and cultured in agarose gel, collagen gel, fibrin glue, and mixtures of collagen and fibrin. Results showed that neurons cultured in collagen gel and fibrin glue had longer periods of survival (more than 3 weeks) and better neurite extension than those observed in agarose gels. As to the survival rate according to the MTT and lactate dehydrogenase assays, fibrin glue was the most suitable biomaterial for neuron survival among the biomaterials examined. With two-dimensional fibrin plating, neuron cells exhibited cell aggregation and stress fibers, but the same results were not observed with collagen gel. There were no differences in neurite extension and survival in the mixtures of collagen and fibrin. The results suggest that collagen and fibrin can provide a suitable substrate for a three-dimensional culture matrix for neuronal survival and differentiation.     

介绍一种制备特异性TF和特异性iRNA的方法

以乙肝疫苗、人喉癌细胞膜抗原为抗原，猪脾细胞为效应细胞，经体外免疫后收集应答细胞，制备ＰＳＨＢＶ－ＴＦ ＰＳＡＣ－ｉＲＮＡ。通过抗原特异性细胞免疫功能试验证实，ＰＳＨＢＶ－ＴＦ和ＰＳＡＣ－ｉＲＮＡ都能转移特异性细胞免疫功能。采用体外免疫法制备ＰＳＨＢＶ－ＴＦ和ＰＳＡＣ－ｉＲＮＡ是可行的，并且具有诸多优点。