Immunohistological study of senile brains by using a monoclonal antibody recognizing β amyloid precursor protein: significance of granular deposits in relation with senile plaques

Summary Immunochemical analyses revealed that a monclonal antibody Am-3 recognized amyloid precursor protein (APP) in senile plaques extracted from Alzheimer's brain, but did not recognize amyloid protein. Immunohistochemically, however, the staining pattern of Am-3 in frozen section of Alzheimer's brain was almost the same with that of rabbit polyclonal antibody to amyloid peptide which could recognize both amyloid protein and APP. In other words, APP was present in senile plaques of various types, cerebrovascular amyloid and granular deposits. The granular deposits were 5–10 m in size and laminarily distributed in the 1st, 3rd and 4th layers of cerebral cortex. They were especially abundant in 1st and 4th layers where senile plaques were usually fewer in number. Although the distribution in the cerebral cortex was different between the senile plaques and the granular deposits, the number of the granular deposits was well correlated with that of senile plaques. The granular deposits were negative in Congo-red birefringence, but contained amyloid protein as well as APP fragment judging from positive staining by both Am-3 and polyclonal antibody to synthetic amyloid peptide. Thus, they could be regarded as pre-amyloid.     

Immunocytochemical distribution of S-100 protein in patients with Down's syndrome

Summary The immunohistochemical distribution of S-100 protein in patients with Down's syndrome was investigated as part of a study aimed at ascertaining a possible involvement in the syndrome (trisomy 21) of this protein, which has recently been shown to be coded in chromosome 21. No appreciable differences in the cell distribution of the antigen could be observed between patients with Down's syndrome and normal subjects. The possibility of an overexpression or abnormal expression of the molecule as a consequence of chromosome 21 triplication, not detectable by immunocytochemical methods alone, remains to be investigated.Supported in part by grants from Consiglio Nazionale delle Ricerche and Ministero della Pubblica Istruzione to F. M. and L. L.     

Generalized pharmacokinetic modeling for drugs with nonlinear binding: I. Theoretical framework

The following integrodifferential equation is proposed as the basis for a generalized treatment of pharmacokinetic systems in which nonlinear binding occurs $$\phi '(c_u )c'_u = - q(c_u ) + g*c_u + f$$ where c_u≡unbound plasma drug concentration, f≡drug input rate,'indicates the derivative of a function, and * indicates the convolution operation: (g* c_u)(t)=∫ ₀ ^t g(t?u)c_udu.Possible physical interpretations of the functions q, g and f are: q (c_u)≡ rate at which drug leaves the sampling compartment, g * c_u ≡ rate at which drug returns to the sampling compartment from the peripheral system (tissues that are kinetically distinct from the sampling compartment), and φ(c_u) ≡ amount of drug in the sampling compartment. The approach assumes that drug binding is sufficiently rapid that it may be treated as an equilibrium process. It may be applied to systems in which nonlinear binding occurs within the sampling compartment, i.e., in the systemic circulation or in tissues to which drug is rapidly distributed. The proposed relationship is a generalization of most existing models for drugs with nonlinear binding. It can serve as a general theoretical framework for such models or as the basis for “model-independent” methods for analyzing the pharmacokinetics of drugs with nonlinear binding. Computer programs for the numerical solution of the integrodifferential equation are presented. Methods for pharmacokinetic system characterization, prediction and bioavailability are presented and demonstrated.     

Cell-specific coupling of the cloned human 5-HT1F receptor to multiple signal transduction pathways

We recently described the cloning of a fifth member of the 5-hydroxytryptamine (5-HT)₁ (serotonin₁) receptor class that inhibits adenylyl cyclase, namely the human 5-HT_1F receptor (Adham et al. 1993 a). In the present study we have examined in greater detail the functional coupling of the 5-HT_1F receptor in two different cell lines, NIH-3T3 and LM(tk^–) fibroblasts (receptor densities of 1.7 and 4.4 pmol/mg protein, respectively). The maximal inhibitory response elicited by 5-HT was significantly greater in NIH-3T3 as compared to LM(tk^–) cells, whereas the EC₅₀ values were comparable.To investigate the relationship between receptor occupancy and inhibition of cAMP accumulation mediated by 5-HT_1F receptors in NIH-3T3 cells (and hence the degree of receptor reserve), we used the irreversible receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The half-maximal response required only about 10% receptor occupancy, consistent with a receptor reserve of 90% (88±2.1%, n = 4) for 5-HT-induced inhibition of FSCA. Despite the presence of such a high degree of receptor reserve, a range of intrinsic activities was displayed by structurally diverse classes of compounds. For example, sumatriptan and lysergol were as efficacious as 5-HT itself and thus acted as full agonists, whereas metergoline and 1-NP behaved as partial agonists and as shown previously (Adham et al. 1993a), methiothepin was a silent antagonist (K_b = 438 nM).We have also investigated activation of additional signal transduction pathways by the 5-HT_1F receptor and found that the responses differ in the two cell lines with respect to stimulation of phospholipase C. For example, in NIH-3T3 cells no elevation of inositol phosphates (IP) of [Ca²⁺]_i was observed even at very high agonist concentrations (100 M). In contrast, in LM(tk^–) cells concentrations of 5-HT as low as 10 nM induced stimulation of IP and a rapid increase of [Ca²⁺]i. The 5-HT_1F receptor failed to alter arachidonic acid release in either cell line.The maximal increase in IP accumulation in LM(tk^–) cells was modest, averaging about 100% above basal. The increases of IP and [Ca²⁺]_i required 5-HT concentrations less than one order of magnitude greater than those inhibiting FSCA (EC₅₀ = 17, 55 and 8 nM, respectively), and both responses were blocked by 100 M methiothepin. All three responses (cAMP, IP, and [Ca²⁺]_i) were sensitive to pertussis toxin pre-treatment, suggesting the involvement of G_i/G_o protein(s) in these signal transduction pathways. [Ca²⁺]_i was also elevated by sumatriptan, which may provide a mechanism by which this drug causes constriction of the vasculature. In conclusion, these data indicate that the human 5-HT_1F receptor can couple to multiple effectors, and that this coupling is cell-type dependent.Abbreviations FSCA forskolin-stimulated cAMP accumulation - [Ca²⁺] intracellular free calcium concentration - AA arachidonic acid - EEDQ N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline - CHO chinese hamster ovary cell - LM(tk^–) mouse fibroblast cell - B_max maximal binding site density - K_i apparent dissociation constant obtained from competition binding studies - G protein guanine nucleotide-binding protein - HBS HEPES-buffered saline - IP inositol phosphates - IP₃ inositol 1,4,5 trisphosphate - PLC phospholipase C - K_b antagonist dissociation constant - K_d equilibrium dissociation constant - N-1F-6 stable NIH-3T3 cells expressing the cloned 5-HT_1F receptor - L-1F-3 stable LM(tk^–) cells expressing the cloned 5-HT_1F receptor - PTX pertussis toxin - BSA bovine serum albumin - METH methiothepin - SUMA sumatriptan - 5-MeO-DMT 5-methoxy-N,N-dimethyltryptamine - 1-NP 1-(1-napthyl)piperazine - 5-CT 5-carboxyamidotryptamine Correspondence to: N. Adham at the above address     

Natural killer activity and serum immunosuppressive acidic protein levels in esophageal and gastric cancers

The natural killer (NK) activity of peripheral blood mononuclear cells and serum immunosuppressive acidic protein (IAP) levels were examined in patients with esophageal or gastric cancer, before and after surgery. Patients with stage IV esophageal or stage IV gastric cancer had significantly lower NK activity (39.5±14.8% and 37±11.6%, respectively), and also higher serum IAP levels (778±264 g/mL and 633±156 g/mL, respectively), than the corresponding control values (50±5.6% and 375±26 g/mL, respectively). Patients with esophageal or gastric cancer who underwent curative resection had high NK activity (54.8±11.6% and 54.8±8.0%, respectively), and low IAP levels (471±116 g/mL and 490±42 g/mL, respectively), compared with those who underwent non-curative resection. Patients who underwent non-curative resection had lower NK activity and higher serum IAP levels than those who underwent curative resection, even 1 month after surgery. Mononuclear cells in the regional lymph nodes and tumor specimens showed significantly lower NK activity than those in the peripheral blood and spleen. Thus, NK activity and the IAP level reflected the immunocompetence, clinical course, and surgical curability of those patients. NK cells appeared not to have any significant antitumor activity in the regional lymph nodes or in the tumor itself, although they were still active in the peripheral blood.     

Nonlinear Binding of Valproic Acid (VPA) and E-Δ2-Valproic Acid to Rat Plasma Proteins

The binding characteristics of valproic acid (VPA) and its pharmacologically active monounsaturated metabolite, E-²-VPA, to rat plasma proteins were compared. The plasma free fraction was determined by a rapid equilibrium procedure, which minimizes the interfering effects of nonesterified fatty acids liberated by in vitro lipolysis. Nonlinear binding behavior was observed with both compounds over their respective pharmacologic concentration range. Multiple binding-site models were invoked to explain the binding isotherm. The 2-unsaturated compound has a much higher affinity for the rat plasma proteins (mainly albumin) than its saturated precursor. The equilibrium association constants for the high- and intermediate-affinity sites were more than an order of magnitude higher with E-²-VPA than with VPA (10⁴–10⁶ versus 10³ M ^–1). This difference in binding affinity was also reflected by a lower plasma free fraction for E-²-VPA compared with VPA (<<10 versus >20% at total concentrations of less than 100 µg/ml). A more pronounced dose- and concentration-dependent variation in the distribution and clearance kinetics is predicted for the 2-unsaturated analogue compared to VPA. Also, the structural dependency in plasma protein binding observed with these branched-chain fatty acids may provide insights into the mechanism of interaction between fatty acyl molecules and albumin.     

EB病毒LMP1在鼻咽癌细胞中通过IκBα的降解活化NF-κB

目的　阐明鼻咽癌细胞中EB病毒编码的潜伏膜蛋白 1(LMP1)活化核转录因子NF κB的机制。方法　利用强力霉素Dox诱导表达LMP1的鼻咽癌细胞株Tet on LMP1 HNE2为实验模型 ,首先应用免疫印迹方法测定Dox诱导后不同时相LMP1的表达动力学以及IκBs蛋白量及功能的改变。进而用间接免疫荧光法检测NF κB的亚细胞定位。最后采用瞬间共转染及报道基因活性分析分析NF κB的活性。结果　在鼻咽癌细胞Tet on LMP1 HNE2中 ,Dox处理 15分钟后LMP1的表达迅速升高并维持与较高水平直至 12 0分钟。LMP1的诱导性表达导致IκBα的磷酸化并降解 ,但IκBα蛋白总量无改变。继IκBα的磷酸化并降解 ,NF κB(P6 5 )自胞浆易位至胞核且活性升高。IκBα的显性负性突变子抑制NF κB(P6 5 )的核易位及报道活性。LMP1的诱导性表达并未引起IκBβ蛋白水平变化。结论　在鼻咽癌细胞Tet on LMP1 HNE2中 ,EB病毒LMP1通过IκBα的磷酸化并降解激活核转录因子NF κB的活性 ,并且 ,LMP1诱导的NF κB活性能被IκBα的显性负性突变子完全抑制。IκBβ在此信号传导途径中无改变。LMP1表达前后IκBα蛋白总量维持恒定可能是由于NF κB的活化迅速启动了IκBα的重头合成这一自身调节环路所致。     

Construction of a recombinant plasmid harbouring the rhoptry protein 1 gene of Toxoplasma gondii and preliminary observations on DNA immunity

Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A ge ne encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3.Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice wer e injected at a dosage of 100 μg recombinant plasmid DNA by intramuscular inje ction and boosted after 2 weeks. pcDNA3 and normal saline were used as control . 30, 50 and 70 days after the second immunization, NK cell activity, T lympho cyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinan t was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T ly mphocytes seen on MTT assay were higher in pcROP1 group than in the controls. T he number of CD4(+) T cells exhibited no obvious increase compared with that of the control, but CD8(+) T cells were obviously increased (P&lt;0.05). At 90 d ays after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100).Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune r esponses in immunized mice.     

Immune responses to the expressed products of the CSP antige n gene of Plasmodium falciparum southern China isolate FCC1/HN in Hela cell

Objective To construct a eukaryotic expression system with pcDNA3-PfCSP/Hela for the Circ umsporozoite protein (CSP) gene of Plasmodium falciparum (P.falciparum), t o observe the immune responses in BALB/c mice induced by the expressed proteins .Methods The recombinant plasmid pcDNA3-PfCSP was transformed into the Hela cell line. The expressed protein was isolated and analyzed by using SDS-PAGE and used for immunization of BALB/c mice by subcutaneous, intravenous, and intraperitone al adminstration.Enzyme-linked immunosorbent assay(ELISA), Dot-ELISA, Wester n blot, T lymphocyte proliferation test, natural killer cell(NKC) activity assay , and CD4(+) and CD8(+) T cell detection were used for observation of humoral an d cellular immune responses.Results Immune sera strongly reacted with the expressed protein, antibody titer was up to 1∶6400 as detected by ELISA.Western blot analysis revealed a specific b and at 38.3 Kda.When the spleen cells of normal and immunized BALB/c mice we re specifically stimulated with expressed protein, the optical densities were 0 .12±0.03 and 0.34±0.04, respectively.The latter were significantly highe r than the former (P&lt;0.01).We used the MTT colorimetric assay to measure NKC activity of mice spleen.The results showed that the NKC activity of immuni zed BALB/c mice was remarkably higher than that of the controls (P&lt;0.05). CD4(+) and CD8(+) T cells were detected by using monoclonal antibody immunofluor escence methods.The results showed that the percentage of CD4(+) and CD8(+) T cells of immunized group were significantly higher than that of control group ( P&lt;0.05).Conclusions The humoral and cell-mediated immune responses and elevated NKC activity to pr oducts made with a eukaryotic expression system could be specifically detected i n BALB/c mice.These findings indicate that the expressed protein could enhance the immune function in mice.     

Murine antibody against E2 can capture hepatitis C virus in vitro

目的　寻找可能存在的抗丙型肝炎病毒 (HCV)感染的中和抗体。方法　构建两个分别含HCVE1和E2抗原基因的真核表达载体 ,用瞬时表达法检测其在哺乳动物细胞中的表达。将构建的载体连同IL 2基因一起经皮下给BalB/c小鼠进行注射 ,然后用ELISA法检测特异性抗体产生情况。最后通过研究抗体和病毒间的相互作用分析基因免疫诱导产生的抗血清在体外与HCV的相互作用情况。结果　经过基因免疫的小鼠分别产生了E 1和E2抗体。其中 ,用含E2基因的质粒载体免疫的小鼠所产生的E2抗血清不仅可以免疫沉淀来源血清中的HCV病毒颗粒 ,而且可以和与来源株非相关的HCV病毒颗粒相互作用。结论　我们的研究表明基因免疫诱导小鼠产生的E2抗体可以和HCV病毒颗粒发生交叉反应 ,这使我们看到了诱导产生具有广泛针对性的E2抗体的希望。