Trial of a Soviet diagnostic kit for determining class M immunoglobulins to the hepatitis A virus by immunoenzyme analysis (an anti-HAV IgM test system)

The authors have studied the effectiveness of the first Soviet test system for the diagnosis of hepatitis A by means of the enzyme immunoassay (Diagn-A-Hep), developed at the Institute of Poliomyelitis and Viral Encephalitides, Moscow, under the conditions of different epidemic situations. In the process of this trial the high specificity and sensitivity of this test system, established earlier in the certification and commission trials, have been confirmed. Diagn-A-Hep has proved to be highly effective in the diagnosis of acute forms of hepatitis A and permitted its detection in patients during the incubation period, as well as in patients with anicteric and asymptomatic subclinical forms. Besides clinical diagnosis, the kit Diagn-A-Hep may be used in large-scale seroepidemiological surveys of the immune structure of the population, as well as in detection of HAV in different material under test.     

Fabrication of a three-dimensional nanostructured biomaterial for tissue engineering of bone

A plasma process for the surface modification of HA powders has been developed. Acrylic acid and acrylic acid/octadiene plasma deposited films onto HA particles have demonstrated to interact with SBF allowing the calcium dissolution-precipitation mechanism. Therefore, a nanostructured composite between HA and a self-assembling peptide scaffold (RAD16-I) has been developed. The differentiation of mESC in this scaffold has been studied, in order to test the osteogenic capacity of the new composite material. We have observed that the mESC can be induced to produce Ca salts (mineralization) in a 3D-microenvironment and moreover, this activity can be enhanced by the presence of HA particles into the nanofiber scaffold.     

A microtiter solid-phase radioimmunoassay for hepatitis A antigen and antibody.

A microtiter solid phase radioimmunoassy for hepatitis A antigen (HA Ag) and antibody (anti-HA) was developed. The test was more sensitive than immune adherence hemagglutination for detecting HA Ag and almost as sensitive for detecting anti-HA. The specificity and sensitivity of reagents were examined and optimum conditions for the test were determined. Radioimmunoassay, immune adherence hemagglutination, and immune electron microscopy were compared for detecting anti-HA. A serologic response to HA Ag was detected in paired sera from patients with type A hepatitis but not from patients with type B or non-A, non-B hepatitis by all three techniques.     

Decrease in Plasma Levels of α-Synuclein Is Evident in Patients with Parkinson’s Disease after Elimination of Heterophilic Antibody Interference

There is substantial biochemical, pathological, and genetic evidence that α-synuclein (A-syn) is a principal molecule in the pathogenesis of Parkinson disease (PD). We previously reported that total A-syn levels in cerebrospinal fluid (CSF), measured with the specific enzyme-linked immunosorbent assay (ELISA) developed by ourselves, were decreased in patients with PD, and suggested the usefulness of A-syn in CSF and plasma as a biomarker for the diagnosis of PD. After our report, a considerable number of studies have investigated the levels A-syn in CSF and in blood, but have reported inconclusive results. Such discrepancies have often been attributed not only to the use of different antibodies in the ELISAs but also to interference from hemolysis. In this study we measured the levels of A-syn in CSF and plasma by using our own sandwich ELISA with or without heterophilic antibody (HA) inhibitor in 30 patients with PD and 58 age-matched controls. We thereby revealed that HA interfered with ELISA measurements of A-syn and are accordingly considered to be an important confounder in A-syn ELISAs. HA produced falsely exaggerated signals in A-syn ELISAs more prominently in plasma samples than in CSF samples. After elimination of HA interference, it was found that hemolysis did not have a significant effect on the signals obtained using our A-syn ELISA. Furthermore, plasma levels of A-syn were significantly lower in the PD group compared with the control group following elimination of HA interference with an HA inhibitor. Our results demonstrate that HA was a major confounder that should be controlled in A-syn ELISAs, and that plasma A-syn could be a useful biomarker for the diagnosis of PD if adequately quantified following elimination of HA interference.     

Diagnostic value of non-invasive bio-markers for stage-specific diagnosis of hepatic fibrosis in patients with advanced schistosomiasis japonica

Hepatic fibrosis caused by schistosome infection can be fatal. Its management depends on the degree of fibrosis present. To assess the diagnostic value of bio-markers in patients with advanced schistosomiasis japonica at different stages of disease progression, 84 advanced schistosomiasis japonica patients and nine controls were recruited in The People's Republic of China. Fibrosis was histologically assessed in wedge liver biopsies using the Chinese criteria for fibrosis (F) Stages. Seven selected hepatic fibrosis bio-markers were assessed and compared between the groups. The method of area under receiver operating characteristic curves (AUROCs) was used as a measurement of diagnostic efficacy. Our results showed that routine laboratory test results were normal for the controls but were significantly elevated or decreased in patients with fibrosis. While serum hyaluronic acid (HA) and matrix metalloproteinase (TIMP)-1 levels were shown to be elevated in patient groups compared with controls, the levels of platelet derived growth factor (PDGF)-BB were markedly lower. To distinguish F≥2 from no fibrosis or mild fibrosis, HA gave a high AUROC of 0.938 (95% confidence interval (CI), 0.886-0.990). Combining the aspartate aminotransferase (AST) to platelet ratio index (APRI) and HA/100 showed an AUROC of 0.958 (95% CI, 0.914-1.000). APRI in combination with TIMP-1/100 provided an AUROC of 0.873 (95% CI, 0.805-0.942) for the diagnosis of fibrosis stages greater than 2. We conclude that AST and APRI levels were reliable and sensitive markers for differentiating significant hepatic fibrosis in patients with advanced schistosomiasis japonica. HA and TIMP-1 show potential as additional markers for the diagnosis of fibrosis and cirrhosis in advanced schistosomiasis patients.     

Identification of a highly conserved H1 subtype-specific epitope with diagnostic potential in the hemagglutinin protein of influenza A virus

Subtype specificity of influenza A virus (IAV) is determined by its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). For HA, 16 distinct subtypes (H1–H16) exist, while nine exist for NA. The epidemic strains of H1N1 IAV change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. The recent introduction of pandemic A/H1N1 IAV (H1N1pdm virus) into humans re-emphasizes the public health concern about H1N1 IAV. Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1 IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from H1N1pdm patients. One epitope, P5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1 HA [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. This previously unidentified linear epitope is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (χ² = 51.81, P<0.01, Pearson correlation coefficient R = 0.741) with a hemagglutination inhibition test. The highly conserved H1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1 IAVs.     

血清铜蓝蛋白联合透明质酸检测在肝纤维化患者诊断中的应用

目的 探讨血清铜蓝蛋白(CP)联合透明质酸(HA)检测在肝纤维化患者诊断中的应用前景.方法 选取2015年1月至2018年12月本院收治的疑似肝纤维化患者226例为研究对象,对患者的CP、HA水平进行联合检测,根据各项指标检测水平对患者做出诊断,并与金标准诊断方法肝穿刺活检结果进行对比.对CP、HA单独检测和联合检测的...     

Allergy to insect stings. IV. Diagnosis by radioallergosorbent test (R.A.S.T.)

Radioallergosorbent tests (RAST(s)) have been developed and assessed for the diagnosis of insect hypersensitivity by using a purified allergen from honeybee venom, phospholipase A, and crude yellow jacket venom. Sera from 193 patients positive both by history and skin test to one of these insects were compared with various groups of control sera. Eighty percent of sera from skin test-positive patients were RAST positive; positive RAST were found in 16% of sera tested from skin test-negative patients. A highly positive RAST correlates well with a positive skin test and clinical sensitivity, but serum IgE is not measurable in many patients with mast cell or basophil bound antibody. Since biologically important reactions of antigen with IgE require that the antibody be cell bound, skin testing would be preferred to RAST if one were limited to a single test for the diagnosis of insect allergy.     

A silkworm larvae plasma test for detecting peptidoglycan in cerebrospinal fluid is useful for the diagnosis of bacterial meningitis

The silkworm larvae plasma (SLP) test has been established based on a cascade reaction triggered by either peptidoglycan or (1, 3)-beta-D-glucan to produce melanin. We applied this test to the diagnosis of bacterial meningitis. Cerebrospinal fluid (CSF) obtained from patients with bacterial meningitis due to gram-positive bacteria, gram-negative bacteria, or fungi, showed positive reactions to the test. In contrast, CSF from patients with viral meningitis or noninfectious illnesses gave negative reactions. Therefore, this test seems to be useful for diagnosis of bacterial and fungal meningitis. When this test was used together with two types of limulus tests, an endotoxin-specific test, and a conventional test, meningitis was further characterized as gram-positive, gram-negative or fungal meningitis. The SLP test requires a computerized instrument for quantitative colorimetric measurement. A qualitative alternative of this test also can be accomplished by visually observing the darkening color. Thus, this method can be applied for simple and rapid diagnosis of meningitis.     

Practical determination of hyaluronan by a new noncompetitive fluorescence-based assay on serum of normal and cirrhotic patients

A practical fluorescence-based assay method for determination of hyaluronan (HA) was developed. Plates were coated with hyaluronan-binding proteins (HABP) obtained from bovine cartilage and successively incubated with samples containing standard solutions of hyaluronan or serum from normal and cyrrhotic patients, biotin-conjugated HABP, and europium-labeled streptavidin. After release of europium from streptavidin with enhancement solution the final fluorescence is measured in a fluorometer. The method is specific for HA even in the presence of substantial amounts of other glycosaminoglycans (chondroitin, dermatan sulfate, and heparan sulfate, and heparin) or proteins. It is possible to quantify HA between 0.2 and 500.0 microg/L. Analyses of HA concentration in 545 normal subjects and 40 cirrhotic patients gave average values of 14.5 and 542.0 microg/L, respectively. It was also shown that older subjects (> or =51 years old) have more HA (28.0 microg/L) than younger subjects (12.0 to 14.0 microg/L). This new sandwich technique has shown high precision and sensitivity similar to those of a recently described fluorescence-based assay method, being able to measure HA in amounts as small as 0.2 microg/L. In addition, this noncompetitive assay avoids preincubation, consumes less time (<5 h) than the previous competitive fluorescence-based assay (>72 h), and avoids the use of radioactive materials.     

Limitations of immunofluorescence tests in the diagnosis of infectious mononucleosis

The relative value of heterophil agglutinins (HA) and of specific EBV antibodies in the diagnosis of infectious mononucleosis (IM) was assessed in 108 cases of the disease and in 280 controls. Among the 108 cases 93 were HA-positive by sheep cells in at least one of their sera, while 15 were HA-negative by the same test. Among the 280 controls false-positive HA tests were not encountered except in eight cases with the horse cell microtitre tests. With one of the two slide tests at least two false-positive tests and 12 false-negative tests were also found but these sera had low titres in microtitre tests. The HA life-span was found to be unexpectedly long in a few cases, sheep cell HA lasting up to 8 to 10 months and horse cell HA up to 21 to 23 months.Many false-positive tests may therefore not be true false-positives and may result from the persistence of HA following unrecognized mononucleosis months before. Virtually all cases of IM had (or developed) antibodies to Epstein-Barr virus, viral capsid antigen (EBV-VCA), whereas only half of the controls were EBV-VCA-positive. The comparative analysis of nonspecific and specific test results in mononucleosis allows the following conclusions: (1) horse cell microtitre tests and the monospot test are more sensitive than sheep cell microtitre tests and the monotest; (2) false-negative results are occasionally seen with the latter tests but not with the former; (3) more false-positive results, however, are probably seen with the former tests; and (4) specific EBV-IgM and EBV-EA antibody tests are useful in the diagnosis of selected borderline cases of mononucleosis.     

Experience with a dynamic inexpensive video-conferencing system for frozen section telepathology.

AIM: To evaluate the feasibility of an inexpensive, generally applicable video-conferencing system for frozen section telepathology (TP). METHODS: A commercially widely available PC-based dynamic video-conferencing system (Picture-Tel LIVE, model PCS 100) has been evaluated, using two, four and six ISDN channels (128-384 kilobits per second (kbs)) bandwidths. 129 frozen sections have been analyzed which were classified by TP as benign, uncertain (the remark probably benign, or probably malignant was allowed), malignant, or not acceptable image quality. The TP results were compared with the original frozen section diagnosis and final paraffin diagnosis. RESULTS: Only 384 kbs (3 ISDN-2 lines) resulted in acceptable speed and quality of microscope images, and synchronous image/speech transfer. In one of the frozen section cases (0.7%), TP image quality was classified as not acceptable, leaving 128 frozen sections for the analysis. Five of these cases were uncertain by TP, and also deferred by frozen section procedure (FS). One more benign and three malignant FS cases were classified as uncertain by TP. Three additional cases were uncertain by FS, but benign according to TP (in agreement with the final diagnosis). In one case, FS diagnosis was uncertain but TP was malignant (in agreement with the final diagnosis). Thus, test efficiency (i.e., cases with complete agreement) was 120/128 (93.8%, Kappa = 0.88) between FS and TP. Sensitivity was 93.5%, specificity 98.6%, positive and negative predictive values were 97.7% and 96.0%. Between TP and final diagnosis agreement was even higher. More importantly, there was not a single discrepancy as to benign-malignant. Moreover, there was a clear learning effect: 5 of the 8 FS/TP discrepancies occurred in the first 42 cases (5/42 = 11.9%), the remaining 3 in the following 86 cases (3/86 = 3.5%). DISCUSSION: The results are encouraging. However, TP evaluation is time-consuming (5-15 min for one case instead of 2-4 min although speed went up with more experience) and is more tiring. The system has the following technical drawbacks: no possibility to point at objects or areas of interest in the life image at the other end, resolution (rarely) may become suboptimal (blocky), storage of images evaluated (which is essential for legal reasons) is not easy and no direct control of a remote motorized microscope. Yet, all users were positive about the system both for telepathology and personal contact by video-conferencing. CONCLUSION: With a relatively simple videoconferencing system, accurate dynamic telepathology frozen section diagnosis can be obtained without false positive or negative results, although a limited number of uncertain cases will have to be accepted.     

A possible mechanism for the action of some myxoviruses

Summary The redox properties of some myxoviruses [Fowl plaque virus strain Rostock (FPV), New Castle Disease virus strain Italy (NDV), B/Hong Kong, A/Port Chalmers, A/Victoria, A/Scotland, and A/Fort Dix (FD)] have been investigated by means of electron spin resonance (ESR) and electron microscopic studies as well as by the determination of the hemagglutination (HA) titer (antigen efficiency). The results have shown that viruses decrease the spin concentration of Cu²⁺ by acting as a reducing species (electron donor) which will result in the inactivation (oxidation) of the virus. Addition of an oxidizing substance, such as H₂O₂, to a virus suspension also leads to an oxidation of the viruses and, thus, to their inability to reduce Cu²⁺. This result is confirmed by the decrease of the HA titer of viruses with increasing Cu²⁺ concentrations. H₂O₂ could not be applied for the HA titer test since it interacts with the erythrocytes of the chicken blood used for this determination. Therefore, another oxidizing substance (oxidized glutathione, GSS) was selected which exhibited a slightly less pronounced effect than Cu²⁺. Since reduced glutathione (GSH) exerts a similar but less pronounced effect than GSS, it might be concluded that viruses have a redox system of their own and act as reducing or oxidizing substance depending on the biological receptor system. Electron microscopic studies confirm this hypothesis. As can be seen by the electron micrographs, increasing concentrations of either Cu²⁺, GSS, H₂O₂, KMnO₄, or GSH will, finally, result in a complete destruction of the virus. Because of structural similarities it might be assumed that other types of viruses behave very similarly.     

Early diagnostic esophagoscopy in the management of corrosive burns of the esophagus

Effective methods developed to review and study the care of patients in hospital have not been applicable to ambulatory care, in which definitive diagnosis is the exception rather than the rule. A reasonable alternative to using diagnosis as the basis for assessing ambulatory care is to use the problems or requests presented by the patients themselves. A system has been developed for classifying and coding this information for flexible computer retrieval. Testing indicates that the system is simple in design, easily mastered by nonphysicians and provides reliable, useful data at a low cost.     

Measuring pKa of activation and pKi of inactivation for influenza hemagglutinin from kinetics of membrane fusion of virions and of HA expressing cells

The data for the pH dependence of lipid mixing between influenza virus (A/PR/8/34 strain) and fluorescently labeled liposomes containing gangliosides has been analyzed using a comprehensive mass action kinetic model for hemaglutinin (HA)-mediated fusion. Quantitative results obtained about the architecture of HA-mediated membrane fusion site from this analysis are in agreement with the previously reported results from analyses of data for HA-expressing cells fusing with various target membranes. Of the eight or more HAs forming a fusogenic aggregate, only two have to undergo the "essential" conformational change needed to initiate fusion. The mass action kinetic model has been extended to allow the analysis of the pKa for HA activation and pKi for HA inactivation. Inactivation and activation of HA following protonation were investigated for various experimental systems involving different strains of HA (A/PR/8/34, X:31, A/Japan). We find that the pKa for the final protonation site on each monomer of the trimer molecule is 5.6 to 5.7, irrespective of the strain. We also find that the pKi for the PR/8 strain is 4.8 to 4.9. The inactivation rate constants for HA, measured from experiments done with PR/8 virions fusing with liposomes and X:31 HA-expressing cells fusing with red blood cells, were both found to be of the order of 10(-4) s(-1). This number appears to be the minimal rate for HA's essential conformational change at low HA surface density. At high HA surface densities, we find evidence for cooperativity in the conformational change, as suggested by other studies.     

Validation of DNA test for hip dysplasia failed in Danish Labrador Retrievers

Canine hip dysplasia is characterized by poor hip joint conformation and laxity. The disease is a complex trait influenced by both genetics and environment. Diagnosis and quantification of hip dysplasia are performed by radiographic examination of the hip joint and the diagnosis is used for making breeding decisions in many breeds. A prognostic genetic test (the Dysgen test) based on seven associated SNPs has been developed in a study based on Spanish Labrador Retrievers. In our study this test has been evaluated in 39 Danish Labrador Retrievers with known radiographic hip score: 14 with hip dysplasia (grade D or E) and 25 without hip dysplasia (grade A or B). There was no significant correlation between the Dysgen test results and the radiographic hip status (P = 0.3203) in these dogs, indicating that Dysgen test results obtained for Danish Labrador Retrievers have no prognostic value.     

Magnetic hydroxyapatite bone substitutes to enhance tissue regeneration: evaluation in vitro using osteoblast-like cells and in vivo in a bone defect

In case of degenerative disease or lesion, bone tissue replacement and regeneration is an important clinical goal. In particular, nowadays, critical size defects rely on the engineering of scaffolds that are 3D structural supports, allowing cellular infiltration and subsequent integration with the native tissue. Several ceramic hydroxyapatite (HA) scaffolds with high porosity and good osteointegration have been developed in the past few decades but they have not solved completely the problems related to bone defects. In the present study we have developed a novel porous ceramic composite made of HA that incorporates magnetite at three different ratios: HA/Mgn 95/5, HA/Mgn 90/10 and HA/Mgn 50/50. The scaffolds, consolidated by sintering at high temperature in a controlled atmosphere, have been analysed in vitro using human osteoblast-like cells. Results indicate high biocompatibility, similar to a commercially available HA bone graft, with no negative effects arising from the presence of magnetite or by the use of a static magnetic field. HA/Mgn 90/10 was shown to enhance cell proliferation at the early stage. Moreover, it has been implanted in vivo in a critical size lesion of the rabbit condyle and a good level of histocompatibility was observed. Such results identify this scaffold as particularly relevant for bone tissue regeneration and open new perspectives for the application of a magnetic field in a clinical setting of bone replacement, either for magnetic scaffold fixation or magnetic drug delivery.     

Hepatitis A antigen isolated from liver and stool: immunologic comparison of antisera prepared in guinea pigs.

Morphologically similar hepatitis A antigen particles (HA Ag)3 have been detected in the stools of patients with type A hepatitis and in the livers of marmosets experimentally infected with hepatitis A virus. To investigate the humoral antibody responses to these antigens and to compare the immunologic properties of HA Ag from these two sources, we immunized guinea pigs with either marmoset liver-derived HA Ag or with human stool-derived HA Ag in complete Freund's adjuvant and measured their antibody responses by immune electron microscopy (IEM) and immune adherence hemagglutination (IAHA). Antibodies reacting with both hepatitis A antigens were elicited in both groups. As determined by IEM, no distinction was seen between the reaction of guinea pig antiserum to each HA Ag tested under code when reacted against either liver-derived or stool-derived HA Ag. Antibodies elicited to marmoset liver-derived HA Ag and human stool-derived HA Ag had similar end point dilution titers by IAHA when tested against either "light" density (1.34 g/cm3) or "heavy" density (1.40 g/cm3) stool-derived HA Ag or liver-derived HA Ag. Low levels of antibody to normal liver or stool control antigens were observed transiently but did not obscure the specific response to HA Ag. These data suggest that morphologically similar HA Ag particles from different sources and with different densities are immunologically similar and may be identical. In contrast to the heterogeneity of surface antigens of hepatitis B virus, the comparable immunogenicity and apparent antigenic homogeneity of HA Ags derived from various sources may simplify the approach to development of a vaccine against viral hepatitis, type A.     

禽流感病毒分型基因芯片的研制

[目的]禽流感病毒是一种全球重要的人和动物呼吸道病病原,快速确定其不同亚型对于全球流感监测具有重要的意义.本研究意在研制一种可同时鉴定禽流感病毒所有亚型的方法.[方法]根据GenBank上已发表的禽流感病毒不同亚型(16个HA亚型和9个NA亚型)的基因序列,设计合成了25对特异性引物和1对通用引物,然后以各亚型病毒的参考株RNA作为模板,建立扩增不同亚型的多重RT-PCR方法.参考各亚型病毒靶cDNAs区域的保守序列设计了52条亚型特异的探针,进而利用扩增的各亚型病毒的靶cDNAs对其特异性进行评价.在此基础上,将设计好的探针点制到处理好的玻片上,制备了禽流感病毒分型鉴定基因芯片,结合所建立的扩增不同亚型的多重RT-PCR方法,开发了禽流感病毒亚型鉴定基因芯片试剂.利用收集自49个地区的2653份标本对其特异性和敏感性进行了初步评价.[结果]用于评价的各亚型参考毒株均出现良好的特异性杂交信号,检测的敏感度可达2.47 PFU/mL或2.5 ng靶DNA片段,而且与禽类常见的IBV、NDV等6种病毒均无交叉反应.[结论]证明该病毒分型基因芯片具有良好的特异性、敏感性.