Identification and characterization of two new soluble nuclear antigens reactive with sera of patients with connective tissue diseases

Two antigens, temporarily referred to as MU and TM antigens after the original serum, that are closely related to the nucleolus have been identified in the nuclear soluble extract. Both antigens reacted with the sera of a small number of patients with various connective tissue diseases, and both were different from antigens described previously in these disorders. MU antigen was sensitive to the digestion with RNase and trypsin, and was shown to be equivalent to the nuclear ribosomal components in the nuclear extract. TM antigen was resistant to the digestion with RNase and trypsin, and was localized in the nucleolus and extranucleolar portion of nuclei, but the precise nature of TM has not been established.     

Widespread occurrence of "87 kDa," a major specific substrate for protein kinase C

An 87-kDa phosphoprotein, identified previously as a major, specific substrate for Ca2+/phospholipid/diacylglycerol-dependent protein kinase (protein kinase C) in broken cell preparations from rat brain, has been characterized with respect to its species, tissue, and subcellular distribution. A similar protein was present in monkey, human, mouse, and bovine brain and in Torpedo californica electric organ. The protein was also identified in a variety of nonneuronal rat and bovine tissues. The rat protein had an apparent molecular mass 4-7 kDa lower, and was slightly more acidic, than the protein in bovine tissues. The 87-kDa proteins from various bovine tissues were identical by the following criteria: each was phosphorylated by exogenous protein kinase C, was of comparable molecular mass, generated multiple spots within the pH range of 4.4-4.9 upon isoelectric focusing, yielded identical patterns upon digestion with Staphylococcus aureus V8 protease, and was recognized by a specific 87-kDa antiserum. The relative concentrations of the 87-kDa protein in bovine tissues were highest in brain, spleen, and lung, moderate in testis, pancreas, adrenal, kidney, and liver, and lowest in heart and skeletal muscle. In the brain, the 87-kDa protein was concentrated in the synaptosomal membrane and in the cytosol. The membrane-bound protein was extractable with nonionic detergents but not with NaCl. This species, tissue, and subcellular distribution of the 87-kDa protein is similar to that of protein kinase C.     

Characterization of platelet-specific alloantigens by immimoblotting: localization of Zw and Bak antigens

The glycoprotein localization of the platelet-specific antigens Zwa, Zwb and Baka and their presence on tryptic fragments of glycoproteins was studied by immunoblotting. Human platelets were solubilized and pre-cleared from platelet-associated IgG. The glycoproteins were separated on SDS polyacrylamide gels, transferred to nitrocellulose and incubated with platelet antibodies, followed by 125I-radiolabelled anti-human Ig antibodies. Glycoprotein IIb/IIIa were isolated from platelet lysates by immuno-affinity chromatography. These proteins were subjected to trypsin digestion, and then used for the immunoblot procedure with platelet antibodies. A glycoprotein specifically reacting with either anti-Zwa or anti-Zwb was found, with an apparent molecular weight of 88 kDa. This protein co-migrated, and was probably identical with, glycoprotein IIIa. After trypsin digestion the smallest fragment, reactive with IgG anti-Zwa or IgM anti-Zwb, had a molecular weight of approximately 23 kDa. IgG anti-Baka and anti-Leka antibodies reacted with a protein of 130 kDa from platelets of Bak(a+) donors. This protein was identified as glycoprotein IIb.     

Respiratory viral antigens in autopsy lung tissue specimens from patients with cancer or myocardial infarction.

Using immunoenzyme histochemical analysis, we retrospectively examined lung tissue specimens obtained at autopsy from 118 patients with cancer who had received chemotherapy and 20 patients who had died after myocardial infarction. Respiratory viral antigens were demonstrated in lung tissue specimens from eight of 118 cancer patients and two of 20 myocardial infarction patients. Most of the patients with demonstrable viral antigens were febrile and had signs of pulmonary infection, but in no case was pulmonary viral infection considered clinically. The following viral antigens were demonstrated: influenza A virus (6 patients), respiratory syncytial virus (2), influenza B virus (1), and parainfluenza virus type 1 (1).     

Induction of major histocompatibility complex antigens within the myocardium of patients with active myocarditis: a nonhistologic marker of myocarditis

The histologic diagnosis of active myocarditis is frequently difficult to establish. A nonhistologic marker of immune activation would be clinically useful in identifying cases of immune-mediated myocarditis. A viral etiology with subsequent autoimmunity to cardiac antigens has been implicated in human myocarditis. Because autoimmunity and viral disease are commonly associated with increased expression of major histocompatibility complex (MHC) antigens on targeted tissue, we examined endomyocardial biopsy samples from patients with active myocarditis for abnormal levels of MHC antigen expression. Thirteen patients with active myocarditis and eight control patients with other well-defined cardiac diagnoses (coronary disease, amyloidosis or neoplasm) were studied. A sensitive radioimmunoassay was developed that utilized monoclonal antibodies to human MHC class I and class II antigens in order to quantitate the expression of both of these antigens within each biopsy. Abnormal MHC class I and class II antigen expression was present in 11 of 13 myocarditis specimens and 1 of 8 control samples (specificity 88%, sensitivity 84.6%). Active myocarditis samples had approximately a 10-fold increase in MHC class I and class II expression. Immunoperoxidase staining localized abnormal MHC expression primarily within microvascular endothelium and along myocyte surfaces (11 of 13). This study is the first to demonstrate a marked increase in major histocompatibility complex antigen expression within the myocardium of patients with active myocarditis. The identification of abnormal histocompatibility antigen expression within an endomyocardial biopsy may prove a useful adjunct to the histologic diagnosis of myocarditis.     

Differential association of endogenous proenkephalin-derived peptides with membranes of microsomes from rat striatum, adrenal medulla and heart ventricle

Proenkephalin-derived peptides, in common with other prohormones, are associated with membranes of microsomes and secretory granules in the bovine adrenal medulla. Post-translational processing of the precursor molecule varies depending upon the tissue. The relationship between post-translational events in different tissues was examined by studying the membrane association of endogenous proenkephalin-derived peptides in the crude microsomal fraction of rat adrenal medulla, brain striatum and heart ventricle. [Met]-Enkephalin and synenkephalin (proenkephalin(1-70)) immunoreactivities were quantified by radioimmunoassay after sequential enzymatic digestion with trypsin and carboxypeptidase B. Between 60 and 75% of total immunoreactive peptides present in intact microsomes of the three tissues were associated with membranes and specifically released with 2 M KSCN (pH 7.4). Analysis of the chromatographic profile of materials present in the soluble and associated fractions produced the following results. In the three tissues the materials associated with microsomal membranes corresponded to peptides larger than 3-5 kDa and displayed synenkephalin and [Met]-enkephalin immunoreactivity. Adrenal and heart microsomes showed a continuous pattern of membrane-associated proenkephalin-derived peptides of high, intermediate and low molecular weights containing the synenkephalin and [Met]-enkephalin sequences. These tissues, however, presented quantitative differences, as the highest concentrations belonged to materials larger and smaller than 12.5 kDa in adrenal and heart microsomes respectively. On the other hand, brain striatal microsomes displayed a discontinuous pattern of associated materials, with the absence of some products of high and intermediate molecular weight. Only in the soluble fraction of striatal microsomes were peptides detected of high and intermediate molecular weight containing the [Met]-enkephalin but not the synenkephalin sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Type C Viral Antigens in Man. I. Antigens Related to Endogenous Primate Virus in Human Tumors

Partially purified extracts from 33 human tumors of several histologic types were used as competing antigens in a radioimmunoassay for the p30 protein of an endogenous primate type C virus (M7). Antigens immunologically related to the p30 protein of the M7 virus were detected in two tumors. Like viral p30 antigens previously identified in tissues of several other primates, the antigens found in human tumors crossreact with the p30 protein of the feline RD-114 virus but are unrelated by similar immunologic criteria to the p30 proteins of several other mammalian type C viruses. Gel filtration shows that most of the antigenic activity co-chromatographs with authentic p30 protein. These results, along with those showing nucleic acid sequences related to those of an endogenous primate type C virus in the DNA of human cells, make it clear that humans, like other primates, have type C viral sequences in their genome and can, in some circumstances, express at least one type C viral protein.     

Distribution and activity of amphotericin B in humans

Concentrations of amphotericin B (AmB) in tissues obtained at autopsy from eight patients were measured by high-performance liquid chromatography (HPLC). The patients had received doses of 101-2,688 mg of antibiotic. Highest concentrations of the drug were found in the liver; in one patient the amount of AmB in the liver was 41% of the total dose. No evidence of metabolism of the drug was observed, and bioassay of ethanol extracts of tissue showed that the drug retained activity. Three of the patients had histologic evidence of aspergillar or candidal infection in tissues with concentrations of AmB ranging from 2.5 to 166 micrograms/g. With two patients, the concentrations of the drug in the tissues exceeded by greater than or equal to 10-fold the minimum inhibitory concentrations of the drug in isolates that had been obtained from the same tissues. Unknown factors present in tissues appear to limit the in vivo activity of AmB.     

Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping.

Sequences of antigenic determinants were identified by limited proteolysis of peptide antigens bound to an immobilized monoclonal antibody and direct molecular weight determination of the monoclonal antibody-bound peptide fragments by 252Cf plasma desorption mass spectrometry. The epitope peptides to the monoclonal antibody h453 [Burger, R., Zilow, G., Bader, A., Friedlein, A. & Naser, W. (1988) J. Immunol. 141, 553-558] were isolated from immobilized antigen-antibody complexes by partial trypsin digestion. A synthetic eicosapeptide comprised of the C-terminal sequence of the human complement component polypeptide des-Arg77-C3a as well as guinea pig des-Arg78-C3a was used as an antigen. Conditions were developed under which trypsin specifically degraded the antigens without inactivation of the immobilized antibody. After proteolysis, epitope peptides were dissociated from the antibody with 4 M MgCl2. The antigenic peptides were purified by HPLC and identified by 252Cf plasma desorption mass spectrometry. The epitope recognized by h453 resides on the C-terminal tryptic peptides of human (residues 70-76) and guinea pig (residues 70-77) C3a. As an estimation of accuracy this method is able to provide, trypsin digestion of immune complexes caused cleavage of the antigen within a distance of two amino acid residues upstream from the epitope.     

Localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization

Dengue viral antigens have been demonstrated in several types of naturally infected human tissues, but little is known of whether these same tissues have detectable viral RNA. We studied tissue specimens from patients with serologically or virologically confirmed dengue infections by immunohistochemistry (IHC) and in situ hybridization (ISH), to localize viral antigen and RNA, respectively. IHC was performed on specimens obtained from 5 autopsies and 24 biopsies and on 20 blood-clot samples. For ISH, antisense riboprobes to the dengue E gene were applied to tissue specimens in which IHC was positive. Viral antigens were demonstrated in Kupffer and sinusoidal endothelial cells of the liver; macrophages, multinucleated cells, and reactive lymphoid cells in the spleen; macrophages and vascular endothelium in the lung; kidney tubules; and monocytes and lymphocytes in blood-clot samples. Positive-strand viral RNA was detected in the same IHC-positive cells found in the spleen and blood-clot samples. The strong, positive ISH signal in these cells indicated a high copy number of viral RNA, suggesting replication.     

风湿性心脏病患者心房成纤维细胞的原代培养

目的建立一种人心房成纤维细胞原代培养简单有效的方法。方法新鲜右心耳组织取自心外科风湿性心脏病手术患者,采用0.25%胰蛋白酶和0.2%Ⅱ型胶原酶消化法分离细胞,用含2O%胎牛血清的DMEM培养液进行培养并传代,显微镜下观察心房成纤维细胞生长状况。波形蛋白(Vimentin)、α平滑肌肌动蛋白(α-SMA)、Ⅷ因子(VWF)抗体对细胞进行免疫荧光鉴定。结果获取大量细胞,形态典型;Vimentin、α-SMA表达阳性,VWF阴性,鉴定为心房肌成纤维细胞。结论胰蛋白酶和Ⅱ型胶原酶消化法是一种简单有效的人心房成纤维细胞原代培养方法。     

Viral etiology of juvenile- and adult-onset squamous papilloma of the larynx.

Juvenile- and adult-onset laryngeal papillomas were examined for the presence of a human papillomavirus (HPV) genome and capsid antigens. DNA was isolated from a portion of tissue removed for therapeutic purposes, and the presence of a papillomavirus genome was detected by Southern transfer analysis. The viral DNA found in the 12 juvenile-onset and the 8 adult-onset laryngeal papillomas examined was identified as HPV-6 on the basis of size, restriction endonuclease digestion patterns, and homology detected under stringent conditions. Restriction endonuclease analysis of the viral genomes revealed at least four different subtypes, designated HPV-6c through HPV-6f. The most common subtype, HPV-6c, was detected in over half of the papillomas studied, including both juvenile and adult types. The remaining tissue was fixed and processed for immunocytochemistry. The immunoperoxidase technique was used with an antiserum that reacts with capsid antigen(s) common to all HPV serotypes. HPV antigen was found in two of the juvenile-onset papillomas and two of the adult-onset papillomas. The antigen was localized to the nucleus and was distributed in the superficial layers of the epithelium. HPV capsid antigen had not previously been detected in cases of adult-onset papilloma, and the HPV genome in both juvenile- and adult-onset laryngeal papillomas had not been characterized. Despite the absence of detectable viral antigen in most of the specimens examined, the presence of the HPV genome provides strong evidence for the papillomavirus etiology of these tumors.     

组织内弓形虫病原体的分离纯化

采用0.01％(W/V)浓度的植物血凝素P(PHAP)亲和分离法自RH株弓形虫感染小鼠腹腔液内分离到纯净的速殖子,纯度达99.5％,收获为73.2％。本法克服了胰酶消化法对虫体的化学损伤。采用Ficoll-Paque密度梯度离心法从感染鼠脑组织中分离到纯净的包囊。     

Differentiation and characterization of autoantibodies and their antigens in Sj?gren's syndrome.

Autoantibodies in Sj?gren's syndrome (SS) were identified by their precipitin reactions with tissue antigens in immunodiffusion. The source of antigens was a sonicated extract of human lymphoid cells (WiI2) grown in tissue culture. Three distinct precipitin systems were identified and referred to as precipitins SS-A and SS-B, and rheumatoid arthritis precipitin. These studies show that precipitins SS-A and SS-B are present in high frequency in sera of patients with SS without associated rheuamtoid arthritis (RA) and are absent or present in low frequency in many other connective tissue diseases. On the other hand patients with SS who also had clinical features compatible with RA (SS-RA) did not have precipitins SS-A and SS-B but had rheumatoid arthritis precipitin (RAP). The latter precipitin was also present in RA patients in a similar order of frequency as in SS-RA. All three precipitin systems were shown to be reactions betweeen immunoglobulins and cellular antigens. The origin of SS-A and SS-B antigens was demonstrated by immunofluorescence to be primarily from the nucleus but the origin of RAP in the cell has not been established.     

Hepatitis B virus replication in damaged endothelial tissues of patients with extrahepatic disease

Hepatitis B virus (HBV) infection may be complicated by extrahepatic manifestations such as polyarteritis nodosa (PAN), glomerulonephritis, polymyositis, and dermatitis, but the etiology of these processes is not yet clear. HBV replication has been demonstrated in a variety of extrahepatic tissues and cell types, but the possible pathogenetic role of extrahepatic HBV replication has not been fully explored in patients with extrahepatic manifestations of HBV infection. In this case series, immunohistochemistry and in situ hybridization studies were performed on extrahepatic tissues from one HBsAg-positive patient with PAN and another HBsAg-positive patient with polymyositis, using HBsAg-seronegative control subjects with the same vasculitic disorders as controls. Tissue samples from the two study patients had detectable HBV RNA, replicative intermediates of HBV DNA, as well as HBsAg and HBcAg localized to vascular endothelium. In contrast, HBsAg-negative control patients had no tissue reactivity. Our results suggest that patients with HBV-related extrahepatic disease have evidence of viral replication in damaged extrahepatic endothelial tissues. While further studies would be required to support a hypothesis of causality, these findings suggest a role for both immune complex deposition and viral replication within diseased endothelial tissue in the pathogenesis of these poorly understood extrahepatic disorders.     

Herpes simplex type 1 DNA in human brain tissue.

Herpes simplex virus type 1 (HSV-1) is known to reside latently in the trigeminal ganglia of man. Reactivation of this virus causes skin lesions and may occasionally infect other tissues, including the brain. To determine whether the brain tissue of humans free of clinical signs of HSV-1 infection contains any trace of HSV-1, we examined the DNA from brain tissue by endonuclease digestion, separation of the fragments by gel electrophoresis, and hybridization with labeled HSV-1 DNA probes. Hybrid bands were detected autoradiographically in experiments using cloned and virion-purified fragments of the HSV-1 genome. HSV-1 DNA sequences were found in 6 of 11 human brain DNA samples tested. In some cases, these bands corresponded to the bands expected for the complete viral genome, whereas others contained bands representing only a part of the genome. In some cases, the terminal fragments could be found, suggesting that the DNA was in a linear, nonintegrated form.     

Analysis of differentially expressed proteins in cancerous and normal colonic tissues

AIM: To identify and analyze the differentially expressed proteins in normal and cancerous tissues of four patients suffering from colon cancer. METHODS: Colon tissues (normal and cancerous) were homogenized and the proteins were extracted using three protein extraction buffers. The extraction buffers were used in an orderly sequence of increasing extraction strength for proteins with hydrophobic properties. The protein extracts were separated using the SDS-PAGE method and the images were captured and analyzed using Quantity One software. The target protein bands were subjected to in-gel digestion with trypsin and finally analyzed using an ESI-ion trap mass spectrometer. RESULTS: A total of 50 differentially expressed proteins in colonic cancerous and normal tissues were identified. CONCLUSION: Many of the identified proteins have been reported to be involved in the progression of similar or other types of cancers. However, some of the identified proteins have not been reported before. In addition, a number of hypothetical proteins were also identified.     

Identification of thyroglobulin in orbital tissues of patients with thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is thought to have an autoimmune pathogenesis because of its association with autoimmune thyroid disease, in particular with Graves' disease. Nevertheless, the nature of the autoimmune reaction is unclear, and a target orbital autoantigen has not been conclusively identified. A widely discussed hypothesis is that antigens constitutively shared by the thyroid and orbital tissues are targets of an autoimmune reaction. It has been also postulated that a thyroid-soluble antigen, namely thyroglobulin (Tg), is transported to orbital tissues through the lymphatics, where it accumulates and elicits autoimmune damages in susceptible individuals. Here we have investigated whether Tg is present in orbital tissues from patients with TAO. Retrobulbar tissue specimens were obtained from three patients with Graves' disease and TAO who underwent decompressive orbitotomy, and at autopsy from two patients with no thyroid or eye disease. All patients with TAO had been previously treated with radioiodine to control Graves' hyperthyroidism. Western blot analysis with a monoclonal anti-Tg antibody showed the presence of intact Tg, both in soluble and membrane-associated fractions of orbital tissue extracts from the patients with TAO, in amounts estimated to range from approximately 320 to approximately 900 pg/microg of tissue protein. In contrast, Tg was not detected in orbital tissue extracts from patients with no thyroid or eye disease. Tg was also demonstrated in orbital tissue extracts from two of three patients with TAO by enzyme-linked immunosorbent assay (ELISA), in amounts estimated to range from approximately 450 to approximately 1000 pg/microg of protein. In addition, Tg in orbital tissue extracts from patients with TAO was immunoprecipitated by a rabbit anti-Tg antibody, suggesting that it retained its native conformation. An anti-thyroxine (T4) antibody captured in solid-phase Tg from orbital tissue extracts, showing that it contained thyroid hormone residues and had therefore originated in the thyroid. Tg-anti-Tg immune complexes were not found in orbital tissues, suggesting that if an autoimmune reaction to Tg occurs in TAO, it is likely to be cell mediated.     

胃癌组织中Raf激酶抑制蛋白的表达临床意义

目的 探讨Raf激酶抑制蛋白(RKIP)在胃癌中的差异表达及临床意义.方法 采用激光捕获显微切割技术(LCM)获得12例纯化的胃腺癌细胞(GAC)及其癌旁(＞5 cm)胃黏膜上皮细胞(NGEC),应用18O/16O分别标记两种细胞样本酶切后的多肽混合物.结合纳升级液相色谱定量鉴定GAC和NGEC的差异表达蛋白质.免疫印迹法验证差异蛋白RKIP在胃癌中的表达.免疫组化检测RKIP蛋白在胃癌组织(118例)、癌旁胃黏膜组织(70例)、转移的淋巴结组织(35例)的表达.结果 共筛选出78个差异表达蛋白质,其中RKIP蛋白表达水平在GAC中较NGEC明显下调(1:4.37).免疫组化结果显示,RKIP蛋白表达与胃癌的浸润深度、TNM分期及淋巴结转移呈负相关,与分化程度呈正相关(P＜0.05).结论 胃癌组织中RKIP蛋白低表达可能影响胃癌的生物学行为.