Modulation of interferon-γ receptor during human T lymphocyte alloactivation

Previous work has shown that neutralization of physiologically secreted interferon(IFN)-γ or blockade of its receptor during T lymphocyte activation inhibits both proliferation and cytotoxic T lymphocyte generation, suggesting that IFN-γ plays a crucial role in T lymphocyte induction and differentiation. In this study, the kinetics of the surface expression of the 90-kDa IFN-γ receptor (IFN-γR) was followed during human mixed lymphocyte reaction (MLR) to alloantigens. IFN-γR mRNA is constitutively expressed on resting peripheral blood lymphocytes emerging from nylon wood column (NW-PBL) and its expression increases two- to threefold on alloactivated NW-PBL. IFN-γR protein is poorly expressed on the membrane of resting CD3⁺ cells, but up-modulates after 3-day MLR and sharply down-modulates at day 6. Both the p55 and the p75 chains of interleukin-2 receptor (IL-2R) were shown to up-modulate in parallel with IFN-γR, whereas they were still highly expressed at day 6. After alloactivation, IFN-γ and IL-2 secretion starts at 24 h, peaks at day 3 and decreases just when IFN-γR and IL-2R begin to up-modulate. Proliferation peaks at day 6. Lastly, stimulation with distinct cell populations showed that the intensity of lymphocyte proliferation, IFN-γR membrane up-modulation, and IFN-γ and IL-2 secretion are regulated in a parallel manner, thus suggesting that they are interrelated. Taken as whole these results demonstrate that increased expression of IFN-γR on T lymphocytes can be a critical event during their activation, and strongly support the hypothesis that IFN-γ/IFN-γR interaction provides a signal for its progression.     

Effect of chronic elevation of plasma calcium concentration by PTH or vitamin D3 on blood pressure and hypotensive activity of nifedipine in rats

The influence of a chronically elevated total plasma calcium concentration on blood pressure and heart rate was investigated in conscious normotensive rats. The plasma calcium concentration was elevated by continuous subcutaneous infusion with parathormone (PTH) after parathyreoidectomy, and by oral treatment with vitamin D₃. In both groups an elevated blood pressure was observed at the 1st day of treatment only. Blood pressure was returned to pretreatment values from the second day of treatments onaards. No significant changes in heart rate, due to hypercalcaemia, were observed. In hypercalcaemic rats, the calcium entry inhibitor nifedipine proved more potent in reducing blood pressure than in normocalcaemic control animals. Hydralazine was equipotent in reducing blood pressure in both groups. It is suggested, that the vasoconstrictor effects of a chronically elevated total plasma calcium concentration in conscious rats does not obviously result in a chronically elevated blood pressure, but rather in an enhanced sensitivity of the blood pressure towards calcium entry blockade.     

Purification of bovine conglutinin using pepsin digestion

This paper describes a new method for the purification of bovine conglutinin based on the relative resistance of this protein to pepsin digestion. First, conglutinin is purified by absorption on yeast, then the preparation is treated with 2% pepsin (w/w) at 4°C for 18 hr, and finally gel filtrated on agarose A5m. The yield is 60–75% and conglutinin thus prepared appears physically, immunochemically and functionally intact. This procedure allows for a rapid production of sufficient amounts of conglutinin for immune complex detection or purification methods.     

hCGβ-C3d3基因免疫BALB/c小鼠选择性促进B细胞克隆扩增

评价分子佐剂C3d在基因免疫中选择性促进抗原特异性B细胞增殖作用。分别用质粒pCMV4-hCG-βC3d3和pCMV4-hCGβ免疫BALB/c小鼠,免疫剂量为50 pmol。间隔3周相同剂量加强免疫1次。加强免疫后第1周及第2周,MTT法分析各组小鼠脾脏B细胞和T细胞增殖;ELISPOT法分析两免疫组小鼠脾脏hCGβ抗原特异性IgG分泌细胞。结果显示在加强免疫后第1周及第2周,hCG-βC3d3免疫组B细胞增殖能力显著高于hCGβ免疫组及正常对照组;而各组间T细胞增殖状况未见明显差异。hCG-βC3d3免疫组较hCGβ免疫组小鼠脾细胞分泌IgG的量和分泌hCGβ抗体的细胞数量均明显增加。结果表明分子佐剂C3d对于B细胞具有选择性克隆扩增作用。     

Differential distribution of growth associated protein (GAP-43) in the motor nuclei of the adult rat conus medullaris

GAP-43 is normally produced by neurons during developmental growth and axonal regeneration, but it is also expressed in specific regions of the normal adult nervous system. We studied the protein expression of GAP-43 within the conus medullaris portion of the spinal cord in adult male rats. Immunohistochemistry for choline acetyltransferase (ChAT) was first performed to identify specific efferent autonomic and motor nuclei in lumbosacral segments of the spinal cord. Adjacent sections were then processed for GAP-43 immunoreactivity (IR). We show GAP-43 IR in the superficial portion of the dorsal horn, the intermediolateral nucleus, and the dorsal commissural tract. We also demonstrate a differential distribution of GAP-43 IR between different motor nuclei of the conus medullaris. Using densitometry, the most prominent GAP-43 IR was detected in the dorsolateral and dorsomedial motor nuclei, which represent the human Onufs nucleus homologue. Confocal microscopy of double immunofluorescent labeling for ChAT and GAP-43 demonstrate GAP-43 IR in the neuropil of the autonomic and motor nuclei, and many of the GAP-43 IR arbors are in close apposition with the efferent cholinergic neurons. We note that the efferent neurons of both the autonomic and somatic nuclei, which are ultimately responsible for the integrated normal control of the lower urinary tract, bowel and sexual functions, are heavily innervated by GAP-43 enriched projections. We speculate that these functionally related neurons retain a physiological GAP-43-associated synaptic plasticity throughout adult life.     

alpha(v)beta(3) Integrin in central nervous system tumors

alpha(v)beta(3) Is an integrin specifically expressed in endothelial cells of newly forming blood vessels. Integrin-mediated angiogenesis is hypothesized to play a central role in the development and the progression of central nervous system neoplasms. Accordingly, it is considered a potential target for antiangiogenic therapy. In the current study, we compare the expression of alpha(v)beta(3) in ependymomas, oligodendrogliomas, pilocytic astrocytomas, medulloblastomas, and vestibular schwannomas (acoustic neuromas). Samples of 5 tumors of each of the 5 tumor types were harvested surgically and frozen. After the pathological diagnosis was confirmed, immunohistochemistry was performed using an anti- alpha(v)beta(3) monoclonal antibody (LM609). The expression of alpha(v)beta(3) was assessed using a 4-tiered (0-3) grading scheme reflecting the percentage of positively staining vessels. All vestibular schwannomas demonstrated strong (grade 3) alpha(v)beta(3) expression. The expression was uniformly prominent in Antoni B regions of the tumors. Of 5 ependymomas, 4 demonstrated uniformly strong alpha(v)beta(3). Oligodendrogliomas, medulloblastomas, and pilocytic astrocytomas demonstrated more variable alpha(v)beta(3). alpha(v)beta(3) may contribute significantly to angiogenesis in vestibular schwannomas and ependymomas. Despite the high vascular density of oligodendrogliomas, pilocytic astrocytomas, and medulloblastomas, these tumors had variable moderate alpha(v)beta(3) expression. This discrepancy suggests temporal and/or regional variability in the angiogenesis in these types of tumor. This study provides the first demonstration of alpha(v)beta(3) expression in vestibular schwannomas, medulloblastomas, and pilocytic astrocytomas.     

Gastrointestinal stromal tumours (GISTs) negative for KIT (CD117 antigen) immunoreactivity

Gastrointestinal stromal tumours (GISTs) are currently defined as mesenchymal tumours of the gastrointestinal tract that express KIT receptor tyrosine kinase. However, a small subgroup of tumours that fulfil the clinical and morphological criteria for GISTs lack KIT expression. So far, the biological features of these tumours have rarely been addressed. The present study describes seven gastrointestinal stromal neoplasms that presented clinicopathological features typical of GISTs but showed absence of CD117 expression as detected by immunohistochemistry. The tumours originated from the stomach (n = 5), duodenum (n = 1), and colon (n = 1), showing histologically either predominantly epithelioid (n = 3), mixed spindled and epithelioid (n = 2), or anaplastic/spindle cell (n = 2) type features. CD34 and alpha-smooth muscle actin (alpha-SMA) positivity was present in four and three tumours, respectively. Chromosomal analysis was performed in two cases, both showing losses of chromosomes 14, 22, and 1p, which is the characteristic feature of GISTs. Dual-colour interphase fluorescence in situ hybridization (FISH) analysis, utilizing chromosome 1p-, 14-, and 22-specific probes, revealed a similar cytogenetic profile in the remaining five tumour specimens. Mutational analysis of exons 9, 11, 13, and 17 of KIT, and exons 12 and 18 of PDGFRA was performed in all cases by denaturing high-pressure liquid chromatography (DHPLC) pre-screening, followed by direct sequencing. None of the tumours showed KIT mutant isoforms. Three tumours harboured PDGFRA exon 18 activating mutations; two were Asp --> Val(842) missense substitutions and one was a DIM842-844 amino acid deletion. KIT and PKC theta (protein activated in interstitial cells of Cajal and GISTs) expression was determined by western immunoblotting of the total cell lysates from three tumour biopsies. None of these three tumours expressed KIT, while all specimens showed expression of PKC theta protein. These findings indicate that there is a subgroup of KIT-negative GISTs that exhibit the same morphological, cytogenetic, and molecular features as KIT-positive tumours. While intragenic PDGFRA activating mutations are present in some of these tumours, the oncogenic events underlying the pathogenesis of the others remain unknown.     

α2-Adrenergic receptor subtype alterations in the brainstem in the sudden infant death syndrome

Background: The sudden infant death syndrome (SIDS) is still the main cause of postneonatal infant death. However, the causes and mechanisms of SIDS have never been completely elucidated. Catecholamines, via α2-adrenergic receptor (α2-AR) interactions, are known to influence brainstem autonomic and respiratory activity. Aims: To examine the catecholaminergic system abnormalities in SIDS victims, we investigated the alterations of α2-AR subtypes. Subjects and methods: We examined the developmental changes of α2-AR subtypes in the brainstem, especially in cardiorespiratory nuclei, in 21 SIDS victims and 17 age-matched controls by means of immunohistochemical methods. For statistical analysis, the χ²-test or Fisher’s exact probability test was performed. Results: There was a significant decrease in α2A-AR immunoreactivity in the solitary nucleus and ventrolateral medulla (VLM) in the medulla oblongata in SIDS victims compared with in control cases, but there were no significant differences of the α2B and α2C-AR immunoreactivity in the brainstem between SIDS victims and controls. Conclusion: α2A-AR immunoreactivity was selectively decreased in the solitary nucleus and VLM in the medulla oblongata in SIDS victims, so there was no possibility that it was secondary to chronic hypoxia or repeated ischemia. It may be related to some impairment of the cardiorespiratory neuronal system. Therefore, SIDS victims may be vulnerable to asphyxia, hypoxia, and/or hypercapnia, and fail to exhibit brainstem responses.     

小鼠胃癌MAGE3抗原H-2K~k限制性抗原肽的筛选

本研究对小鼠胃癌MAGE3抗原H 2Kk 限制性的抗原肽进行了筛选。用CTL表位预测的基序法对易与H 2Kk 结合的多肽序列进行了预测并人工合成了 4个多肽。在体外检测了各多肽刺激H 2Kk 型小鼠T细胞增殖和分泌IFN γ的能力 ,同时测定了各多肽诱导出的CTL对小鼠前胃癌细胞株MFC细胞的杀伤活性。结果显示 ,抗原肽MAGE31 3 0 1 3 7可有效地刺激特异性T细胞的增殖和分泌细胞因子IFN γ ,其诱导出的CTL细胞对MFC细胞也有较高的杀伤活性。这些实验结果证实抗原肽MAGE31 3 0 1 3 7可以作为多肽疫苗来对表达MAGE3抗原的H 2Kk 型小鼠胃癌进行免疫治疗。     

C3 binds with similar efficiency to Fab and Fc regions of IgG immune aggregates

The covalent binding reaction of the third component of complement (C3) with rabbit IgG immune aggregates has been studied by enzymic digestion of C3b-IgG adducts. In these adducts C3b was radioactively labeled in the free thiol group generated during activation of the internal thioester of C3. Trypsin digestion of ¹⁴C-labeled C3b-IgG adducts degrades C3b to a small antibody-bound ¹⁴C-labeled C3 fragment (¹⁴C-C3frg), whereas the antibody remains unaltered. Papain digestion of trypsin-treated ¹⁴C-C3frg-IgG complexes generated Fc and Fab fragments bearing equivalent amounts of covalently bound ¹⁴C-C3frg (43% and 40%, of the total C3 present in the aggregates, respectively). Hydroxylamine treatment of the ¹⁴C-C3frg-Fab and ¹⁴C-C3frg-Fc complexes released a ¹⁴C-C3frg of similar size (about 3–4 kDa) in which the N-terminal residue was the radiolabeled Cys¹⁰¹⁰. A fragment with the same radioactive N terminus and characteristics was obtained by sequential trypsin and papain digestion of purified C3 labeled with iodo–[¹⁴C] acetamide. Affinity-purified ¹⁴C-C3frg-Fc complexes digested with pepsin generated a mixture of radioactive peptides, most probably complexes formed by ¹⁴C-C3frg and Cγ2 or the hinge digestion products, and ¹⁴C-C3frg-pFc' complexes. The latter was also immunoprecipitated with anti-Fc-Sepharose from the pepsin digestion supernatants of ¹⁴C-labeled-C3b-IgG complexes. Taken together these data indicate that, during complement activation through the alternative pathway by IgG immune aggregates, C3 is not bound to a single site on the antibody molecule. Both Fab and Fc regions of IgG are equally efficient targets for C3 anchorage. In addition, the data confirm the pFc' as a region of C3 attachment within the Fc portion, and strongly suggest that C3b is bound either to the Cγ2 domain or the hinge or both.