Solid phase indirect radioimmunoassays for the rapid diagnosis of Sindbis virus antigen.

Indirect radioimmunoassays have been developed for the rapid detection of Sindbis virus. Dilutions of Sindbis virus from tissue culture fluids have been immobilized and allowed to react with rabbit anti-Sindbis virus antibodies. The bound antibodies were assayed either by 125I-labelled anti-rabbit IgG-antibodies or alternatively by addition of human complement and 125I-labelled anti-human C1q antibodies or 125I-labelled protein A.     

T-helper-2 cytokine responses to Sj97 predict resistance to reinfection with Schistosoma japonicum

Although schistosomiasis is effectively treated with Praziquantel, rapid reinfection with rebound morbidity precludes effective control based on chemotherapy alone and justifies current efforts to develop vaccines for these parasites. Using a longitudinal treatment-reinfection study design with 616 participants 7 to 30 years of age, we evaluated the relationship between cytokine responses to Schistosoma japonicum soluble adult worm extract (SWAP), Sj97, Sj22.6, and Sj67, measured 4 weeks after treatment with Praziquantel, and resistance to reinfection in a population from Leyte, The Philippines, where S. japonicum is endemic. S. japonicum transmission was high: 54.8% and 91.1% were reinfected within 6 and 18 months, respectively. A Th2 bias in the following cytokine ratios, interleukin-4 (IL-4)/IL-12, IL-5/IL-12, IL-13/IL-12, IL-4/gamma-IFN (IFN-gamma), IL-5/IFN-gamma, and IL-13/IFN-gamma, in response to SWAP predicted a 1.4- to 2.9-month longer time to reinfection (P < 0.05) and a 27 to 55% lower intensity of reinfection (P < 0.05). Similarly, a Th2 bias in response to Sj97 predicted a 1.6- to 2.2-month longer time to reinfection (P < 0.05) and a 30 to 41% lower intensity of reinfection (P < 0.05). Only a high IL-5/IL-10 ratio in response to Sj22.6 predicted a 3.0-month-longer time to reinfection (P = 0.03). Cytokine responses to Sj67 were not associated with protection. In a large population-based treatment-reinfection study we found that Th2 responses to SWAP and Sj97 consistently predicted resistance to reinfection. These findings underscore Th2-type immune responses as central in human resistance to S. japonicum and support Sj97 as a leading vaccine candidate for this parasite.     

Influence of etoposide on the retention of radiolabeled low-density lipoprotein in the arterial wall.

BACKGROUND: Cardiovascular toxicity of chemotherapy for testicular cancer is a matter of discussion, since highly efficient agents can achieve cure of the disease, even in the metastasized setting. Acute ischemic events during the treatment period and a persistently elevated serum cholesterol thereafter are observations of particular concern in these patients, and the underlying basic mechanisms are unknown to date. OBJECTIVE: To evaluate etoposide, which is part of the standard treatment for testicular cancer, as a potential cause of atherogenesis in an experimental model. SETTING: Aortic 125I-labeled low-density lipoprotein retention was studied in 72 cholesterol-fed rabbits under etoposide treatment and was quantified according to the morphologically assessed type of surface lining. Aortic cholesterol content was determined both by Sudan III staining and quantitatively by a biochemical assay. RESULTS: A reduced uptake of 125I-labeled low-density lipoprotein in the arterial wall was observed in the etoposide-treated animals, which resulted in a size reduction of sudanophilic areas and cholesterol content. Whereas the breakdown of 125I-labeled low-density lipoprotein in the liver was not significantly enhanced, the plasma decay of 125I-labeled low-density lipoprotein was faster and serum cholesterol was lower in the etoposide group than in controls. CONCLUSION: Unexpectedly, we found an improvement of arterial wall lipid metabolism under etoposide treatment and can thus exclude this substance as a promoter of atherogenesis in this model.     

日本血吸虫大陆株26kDa谷胱甘肽S┐转移酶编码区基因的克隆及序列测定

从日本血吸虫大陆株成虫分离总ＲＮＡ，逆转录合成ｃＤＮＡ第一链，根据菲律宾株２６ｋＤａ谷胱甘肽Ｓ－转移酶（ＧＳＴ）的ｃＤＮＡ序列，设计并合成引物，扩增出２６ｋＤａ的编码区基因，并克隆到ｐＢｌｕｅｓｃｒｉｐｔ质粒。初步酶切鉴定后，从两端对插入片段进行核苷酸序列测定。结果与菲律宾株比较，核苷酸同源性为９９．８％，仅第５８２位碱基不同，菲律宾株为Ａ，而大陆株为Ｇ。比较从ｃＤＮＡ推导出的氨基酸序列，两者１００％相同。测序结果也与曼氏血吸虫进行了比较。     

125I-氯丙嗪-酪胺测定大鼠纹状体多巴胺D2受体的初探

探讨碘标记小分子物质氯丙嗪测定大鼠纹状体神经细胞多巴胺D2受体数目、亲和力的影响。采用Iodogen法标记氯丙嗪与酪胺的合成物作为放射性配基,以不同浓度的125I氯丙嗪酪胺合成物与未标记的氯丙嗪竞争结合D2受体。结果表明125I氯丙嗪酪胺配基与正常大鼠纹状体多巴胺D2受体结合,有较好的亲和力,且数据较稳定,提示以125I氯丙嗪酪胺为放射性配基可作为深入研究多巴胺D2受体的较理想指标。     

Cellular co-operation by mouse lymphocytes to chemically defined antigens.

When populations of mouse spleen cells were treated with a highly radioactive 125I-labelled haptenic conjugate (125I-labelled PDG-N) and subsequently injected into X-irradiated, immunized (N-10-C) syngeneic BALB/c mice, it was found that after autoradiography with 125I-labelled N and C haptenic conjugates, the numbers of ABC to both the N and C haptens decreased. The number of ABC found were significantly lower than those found in mice that had been reconstituted with cells "suicided" with another 125I-labelled antigen (125I-labelled BSA) and subsequently immunized with N-10-C.     

Comparison of the cloned genes of the 26- and 28-kilodalton glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni.

Both Schistosoma japonicum and S. mansoni contain 28- and 26-kDa glutathione S-transferases (GSTs). Despite their immunological cross-reactivity using rabbit antisera, the S. japonicum 28-kDa GST (Sj28) is weakly immunogenic relative to the S. mansoni protein (Sm28) in mouse immunization experiments using GSTs purified from adult worms. The difference in immunogenicity is also observed during schistosome infection in mice. Using surface-labeled living S. japonicum worms, evidence was obtained for a surface location of Sj28 comparable to that reported for the S. mansoni molecule. The nucleotide and deduced amino acid sequences of cDNA clones corresponding to Sj28 and Sm28 were compared. Despite obvious homology (77% identity), differences were found in regions known to contain T epitopes in the S. mansoni protein which may be an explanation for the striking differences in immunogenicity in regard to antibody production in mice. The 26-kDa GSTs of these two parasites (Sj26 and Sm26) are also closely related on the basis of nucleotide and deduced amino acid sequences, there being 82% identity in the putative coding regions. When the amino acid sequences of Sj28 and Sm28 were compared with those of Sj26 and Sm26, the overall sequence identity was approximately 20%. However, a relatively conserved region was identified in otherwise structurally different molecules which may participate in common properties of these enzymes.     

A Modified Crossed Radioimmunoelectrophoretic Method for Determination of Allergen-Specific IgG Antibodies

A crossed radioimmunoelectrophoretic (CRIE) method for detection of specific IgG antibodies in patients' sera against horse hair and dander was developed. The unacceptably high non-specific binding encountered when substituting 125I-labelled antihuman IgG for 125I-labelled antihuman IgE in an ordinary CRIE was eliminated by the combined use of 125I-labelled Protein A as detector, and F(ab')2-fragments of the allergen-specific rabbit antibodies. The low background binding thus obtained makes the method useful for detection of specific IgG in sera where the ratio between specific and non-specific IgG is low. Therefore the method should also be applicable to other antigen/allergen systems.     

Interaction between herpes simplex type 1-induced Fc receptor and human and rabbit immunoglobulin G (IgG) domains.

Cells infected with herpes simplex virus type 1 (HSV-1) express a cell surface receptor able to bind to the Fc region of immunoglobulin G (IgG). The ability of HSV-1-infected cells to bind 125I-labelled human and rabbit IgG and IgG fragments was studied to localize the site of interaction to the C gamma 2 or C gamma 3 domains of IgG. 125I-labelled IgG and IgG Fc fragments consisting of C gamma 2 and C gamma 3 domains bound strongly to HSV-infected cells and did not bind to uninfected cells. In contrast, 125I-labelled F(ab')2, Facb [consisting of F(ab')2 and C gamma 2 domains] and pFc' (consisting of C gamma 3 domains) fragments did not bind to any of these cells. Unlabelled IgG and IgG Fc fragments inhibited the interaction between 125I-labelled rabbit IgG Fc and the HSV Fc receptor, whereas F(ab')2, Facb and pFc' fragments failed to inhibit this interaction. These data indicate that the HSV Fc receptor requires both the C gamma 2 and C gamma 3 domains for interaction with the IgG molecule analogous to the known interaction of protein A of Staphylococcus aureus, the Fc binding proteins of Group A, C and G streptococci, and certain human rheumatoid factors.     

Protein-binding of secretin in human plasma

Protein-binding of endogenous plasma secretin and of 125I-labelled secretin incubated with charcoal-treated plasma examined by gel filtration on a Sephacryl S-200 Superfine column (16 X 980 mm) showed that secretin in plasma appears both to be bound to at least two different plasma proteins where albumin appears to be the major binding protein, and also to occur as a free molecular form. In addition, protein-binding studied by incubating 125I-labelled secretin with charcoal-treated plasma under various conditions followed by charcoal separation of bound from free label indicated the presence of more specific secretin-binding sites on the plasma proteins with an avidity comparable to that otherwise reported for albumin as a binding protein. The protein-binding of 125I-labelled secretin was optimal or reached equilibrium after 2 days incubation at 20 degrees C and first after 8 days incubation at 4 degrees C. Also, the protein-binding of 125I-labelled secretin was higher at an incubation temperature of 20 than of 4 degrees C; was optimal at pH 7.4; increased with increasing amounts of charcoal-treated plasma up to an amount of 800 microliters in our assay system before levelling off; and increased in a constant and predictable manner with increasing amounts of 125I-labelled secretin at least with the amounts of labelled secretin examined here.     

Molecular interactions between human IgG, IgM rheumatoid factor and streptococcal IgG Fc receptors

Group A streptococci type M15 were previously shown to bind both human IgG via the Fc component and a purified monoclonal IgM kappa rheumatoid factor (IgM RF). Using 125I-labelled IgG and 125I-labelled IgM RF, the present study gave association constants of 2.2 x 10(7) and 2.9 x 10(8) M-1, respectively. The binding of 125I-IgG to the streptococci was inhibited by unlabelled IgG, IgG Fc and fragment D of staphylococcal protein A but not by the IgM RF or F(ab')2 of anti-idiotype antibodies to RF (anti-Id RF). Inversely, unlabelled IgM RF and anti-Id RF inhibited the binding of 125I-IgM RF markedly and unlabelled human IgG and IgG Fc only slightly or moderately, respectively. Thus, group A streptococci type M15 showed different binding sites for IgG Fc and the antibody combining sites of a human monoclonal RF. The findings were still more complex on a background of previous reports showing that streptococcal IgG Fc receptors and RFs bind to the same amino acids on the Fc molecule. This complex pattern may play a role in the pathogenesis of rheumatoid arthritis.     

Further characterisation of the Schistosoma japonicum protein Sj23, a target antigen of an immunodiagnostic monoclonal antibody.

Sj23, the 23-kDa target antigen in Schistosoma japonicum adult worms of the hybridoma monoclonal antibody (mAb) I-134, has been identified and cloned from cDNA libraries, mAb I-134 has been successfully used in immunodiagnostic assays to detect S. japonicum infection in Philippine patients. Sequence analysis has shown that Sj23 is the homologue, with 84% amino acid identity, of Sm23, a 23-kDa molecule from S. mansoni worms previously described from our laboratory. The domain structures of Sj23 and Sm23 are strikingly similar to the human membrane proteins ME491, CD37, CD53 and TAPA-1, which may suggest a functional role for the schistosome molecules in cellular proliferation.     

Expression of an enzymatically active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione S-transferase

The NH2-terminal amino acid sequence of the Mr 26 000 glutathione S-transferase (EC 2.5.1.18) of Schistosoma japonicum (Sj26) has been deduced by RNA and protein sequence analysis. Using this information, a bacterial plasmid has been constructed that directs the synthesis of the entire Sj26 molecule in Escherichia coli. Recombinant Sj26 exhibits glutathione S-transferase activity and can be readily purified from bacteria in a one-step procedure under non-denaturing conditions. The availability of recombinant Sj26 in essentially unlimited quantities will aid its assessment as a candidate vaccine molecule in schistosomiasis and could eventually lead to the rational design of a drug targetted on schistosome glutathione S-transferases.     

Radioallergosorbent test (RAST) for measurement of IgG antibodies to Aspergillus fumigatus in sera of patients with different lung diseases

A radioallergosorbent test (RAST) for measuring human anti-Aspergillus fumigatus (Af) antibodies of the IgG class is described. The use of 125I-labelled animal antibodies against human IgG is compared with the use of 125I-labelled protein A. Under optimal conditions the radioactivity binding ratio between pooled patients' serum and pooled healthy persons' serum is 8-11.5. The immunoblotting technique was used to investigate so-called non-specific binding. The results obtained show that most if not all human sera contain anti-Af antibodies of the IgG type. The difference between pathological and normal immunological response to Af antigens seems to be in the antibody titres rather than in the presence or absence of antibodies to these antigens.     

Phenotypic characterization of epidemic versus sporadic strains of methicillin-resistant Staphylococcus aureus.

Forty strains of methicillin-resistant Staphylococcus aureus (MRSA) were divided on the basis of their epidemiologic behavior into two subgroups, sporadic MRSA (SMRSA) and epidemic MRSA (EMRSA) strains. The strains were examined for binding of 125I-labelled fibronectin, vitronectin, collagen, Fc fragments of immunoglobulin G, and fibrinogen. A significant difference between EMRSA and SMRSA strains was found for binding of 125I-labelled fibrinogen and for Fc fragments of immunoglobulin G, (P < 0.05). No significant difference in the binding of 125I-labelled fibronectin and collagen was found between EMRSA and SMRSA strains. The binding of 125I-labelled vitronectin to MRSA strains was found to be aspecific. Capsular serotypes of the strains were determined with monoclonal antibodies against capsular types 5 and 8. Strains could be divided into the following four groups: types 5, 8, and 5/8 and nontypeable. More nontypeable strains were found in the EMRSA group (66.6%). Significantly more EMRSA strains (79%) than SMRSA strains (44%) produced alpha-toxin (P < 0.025). Logistic regression analysis using a combination of the parameters 125I-labelled immunoglobulin G binding, capsular type, and alpha-toxin production predicted the epidemic character with a sensitivity of 83% and a specificity of 75%.     

Modifications in the handling in vitro of 125I-labelled keyhole limpet haemocyanin by peritoneal macrophages from mice pretreated with the adjuvant Corynebacterium parvum.

Peritoneal macrophages were isolated from C. parvum-pretreated (CP) and normal CBAT6T6 mice and their in vitro handling of 125I-labelled Keyhole limpet haemocyanin (125I-labelled KLH) studied in relation to the humoral anti-KLH responses induced in corresponding animals. CP pretreatment exerted an adjuvant effect on the production of anti-KLH antibodies, both IgM and IgG, which was also demonstrable with a normally subimmunogenic dose of antigen. There was a clear difference between the handling of 125I-labelled KLH by CP and normal macrophages. The initial uptake of the antigen by CP macrophages was slower than that by normal ones. Moreover, 125I-labelled KLH was degraded to a lesser extent within CP macrophages, although the rates of antigen digestion were similar in both kinds of cells. The lower extent of 125I-labelled KLH degradation within the CP macrophages was due to a larger amount of antigen being retained on the cell membrane, where it escapes digestion. The findings suggest that intensified presentation to lymphocytes of antigen on the macrophage surface could be a causal factor in the adjuvant action of CP.     

Functional characterization of the brain-to-blood efflux clearance of human amyloid-beta peptide (1-40) across the rat blood-brain barrier

The present study sought to characterize the brain-to-blood efflux transport of human amyloid-beta peptide (hAbeta)(1-40) across the blood-brain barrier (BBB) in rats. We determined the apparent brain-to-blood [(125)I]hAbeta(1-40) efflux clearance in rats and found it to be 11.0 microL/(ming brain). There were no significant gender differences in the apparent brain-to-blood [(125)I]hAbeta(1-40) efflux clearance. The brain-to-blood [(125)I]hAbeta(1-40) efflux transport was significantly inhibited by unlabeled hAbeta(1-40) and hAbeta(1-42) by 79.1% and 36.4%, respectively, but was not inhibited by hAbeta(1-43) and hAbeta(40-1), and was significantly facilitated by hAbeta(17-40) by 16.0%, which is one of the major proteolytic fragments of hAbeta(1-40) generated by the action of Abeta degradation enzymes, such as endothelin-converting enzyme. Pre-administration of human receptor-associated protein, a low-density lipoprotein receptor-related protein (LRP) antagonist, reduced the elimination of [(125)I]hAbeta(1-40) by 20.3%, while quinidine or verapamil, P-glycoprotein (P-gp) inhibitors, did not significantly affect the elimination. Western blot analysis suggested that LRP-1 is expressed in rat brain capillary endothelial cells. In conclusion, the partial contribution of LRP-1 and the minor contribution of P-gp suggest that the hAbeta(1-40) elimination from rat brain is mediated by as yet unidentified molecules.     

Optimizing the solid-phase immunofiltration assay. A rapid alternative to immunoassays

The technical variables of the solid-phase immunofiltration assay (SPIA) for the detection of antibodies bound to antigens on a solid-phase filter have been investigated. The binding to solid-phase filters of 125I-labelled axial filament proteins derived from Treponema phagedenis and the optimal conditions for blocking non-specific protein binding were analysed. Axial filament was applied to nitrocellulose, Hybond Nylon and Zeta Probe. After extensive rinsing, the highest amount (68%) of axial filament was observed bound to Zeta Probe. However, blocking non-specific protein binding by pre-wetting the filter with rinsing buffer containing 0.5% Tween 20, prevented the binding of protein to the filter only when nitrocellulose was used as solid phase. Tween 20 (0.5%) in the rinsing and incubation solutions was found to be necessary for the reduction of non-specific binding of contaminants in turbid sera. However, the use of such solutions resulted in a substantial leakage of antigen (47%) during rinsing procedures. Binding of antigen-specific antibody was analysed using 125I-labelled protein A. The maximal possible binding of the antibody occurred within 5 min when the antibody solution was filtered. For optimal binding of 125I-labelled protein A an incubation time of 1 h was needed. It is suggested that solid-phase immunofiltration may provide a rapid alternative for radioimmunoassays or enzyme immunoassays for the detection of specific antibodies.     

Molecular properties of T-lymphoma immunoglobulin. III. Peptide composition of the light chain.

Structural studies of T-cell immunoglobulin light chains have been carried out in order to ascertain whether they possess a unique structure or if they resemble standard kappa or lambda isotypes. T-cell immunoglobulin was isolated from 125I-labelled culture medium of monoclonal, continuously cultured T-lymphoma cells, and the purified 125I-labelled light chains were subjected to either cleavage by cyanogen bromide or digestion with trypsin. These peptides were then resolved and compared with those derived from 125I- and 131I-labelled murine kappa and lambda chains in mixed label experiments, by means of polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffers and ion exchange chromatography. The profiles obtained suggested that T-lymphoma immunoglobulin light chains are immunoglobulin polypeptides and most closely resemble kappa chains. These results support available antigenic and mRNA hybridization data, which also suggest that T cells bear kappa-like light chains.     

The use of micropeptide maps and monoclonal antibodies for antigenic variation analysis of influenza A viruses isolated in China from 1972 to 1983

125I-labelled haemagglutinin (HA) micropeptide mapping and haemagglutinin inhibition (HAI) tests with monoclonal antibodies were used in analysing antigenic variation of six strains of influenza A virus isolated from 1972 to 1983 in China. The results from micropeptide mapping generally coincided with those of sequence analysis. 125I-labelled HA micropeptide mapping is a simple, reproducible and practical method for surveillance of influenza, especially in combination with HAI test using monoclonal antibodies.