Enzyme-lined Immunosorbent Assay Research Articles

WNT6 is an effective marker for osteosarcoma diagnosis and prognosis.

Wingless-Type MMTV Integration Site Family, Member 6 (WNT6) is a member of the Wnt family and its expression is abnormal in different human cancer cell lines. The purpose of this study was to investigate the clinical significance of WNT6 in osteosarcoma.The levels of WNT6 mRNA and protein in tissue and serum were detected through quantitative real-time polymorperase chain reaction (qRT-PCR) and Enzyme Lined Immunosorbent Assay (ELISA), respectively. Chi-square test was performed to estimate the association of WNT6 expression with clinical parameters among osteosarcoma patients. Receiver operation characteristic (ROC) curve was plotted to determine diagnostic performance of serum WNT6 in osteosarcoma. Survival analysis was performed using Kaplan–Meier method. Cox regression analysis was adopted to evaluate prognostic significance of WNT6 expression among osteosarcoma patients.Compared with the controls, WNT6 mRNA and protein levels were significantly elevated in patients with osteosarcoma (P > .05 for all). Furthermore, WNT6 upregulation showed positive correlation with patients’ age (P < .001), tumor grade (P < .001) and distant metastasis (P = .001). WNT6 might be a diagnostic marker for osteosarcoma with an AUC of 0.854 combining a specificity of 88.4% and a sensitivity of 77.8%. Survival analysis result indicated that high WNT6 expression predicted poor survival (log rank test, P = .001). WNT6 might be a potential prognostic biomarker for osteosarcoma (HR = 2.227, 95%CI = 1.061–10.842, P = .027).WNT6 may be a diagnostic and prognostic marker in osteosarcoma.

Human DKK1 and human HSP70 fusion DNA vaccine induces an effective anti-tumor efficacy in murine multiple myeloma.

Dickkopf-1 (DKK1) is an ideal target for the immunotherapy of multiple myeloma. Heat Shock protein70 (HSP70) is a class of important molecular chaperone to promote antigen presentation. Homologous xenogeneic antigens can enhance immunogenicity and induce stronger anti-tumor immune response than that of allogeneic ones. Therefore, we constructed human DKK1 and human HSP70 DNA fusion vaccine (hDKK1-hHSP70), and then determined its anti-tumor immuno- genicity and anti-tumor effects on immunizing BALB/c mice already inoculated with NS-1 murine multiple myeloma cells in prophylactic and therapeutic models using cytotoxic T lymphocytes, enzyme-lined immunosorbent assay, flow cytometry, immunohistochemistry and Hochest staining. The side effects of vaccines were also monitored. We found that hDKK1-hHSP70 fusion vaccine could significantly inhibit tumor growth and prolonged the survival of the mice, whether prophylactic or therapeutic immunotherapy in vivo, by eliciting both humoral and cellular tumor-specific immune responses. A significant decrease of proliferation and increase of apoptosis were also observed in the tumor tissues injected with hDKK1-hHSP70 vaccine. These findings showed the xenogeneic homologous vaccination had stronger immunogenicity and minimal toxicity. Our study may provide an effective and safety immonutheraphy strategy for multiple myeloma.

Seroprevalence of Foot and Mouth Disease Virus (FMDV) and associated risk factors in unvaccinated sheep and goats in Pyawbwe and Meikhtila townships of Myanmar

Objective: In this study, a serological survey was conducted in unvaccinated sheep and goat populations at Pyawbwe and Meikhtila townships of Mandalay region in Myanmar to determine the seroprevalence and associated risk factors of foot and mouth disease (FMD). Materials and methods: A total of 110 sheep and 107 goat sera samples were randomly collected from Pyawbwe. Similarly, 108 sheep and 109 goat sera were collected from Meikhtila. All samples were tested for the presence of non-structural protein (NSP) specific antibodies to FMD virus (FMDV) by Ceditest FMDV-NSP Enzyme-lined Immunosorbent Assay (ELISA), and were confirmed by Liquid Phase Blocking ELISA (LPB ELISA) . Results: Overall seroprevalence was 42.4%(n=184/434) by Ceditest-NSP ELISA, and 46.8%(n=203/434) by LPB ELISA against FMDV serotype O. The presence of antibodies against FMDV serotype O was higher (P<0.01) as compared to those of serotype A and Asia-1. The seroprevalence in Meikhtila (49.77%) was higher (P<0.01) than that of Pyawbwe (35.2%). The seropositivity in sheep and goats that were in-contact (77.19%) with infected cattle and pigs was higher (P<0.01) as compared to those in-contact with non-infected animals (37.14%). Similarly, the seropositivity in sheep and goats from high animal trade areas (49.4%) was higher (P<0.05) than that of those from low animal trade areas (37.97%). Conclusion: Rearing of sheep and goats in-contact with FMDV-infected cattle and pigs, and high animal trading areas are the major associated risk factors for FMDV infection for sheep and goats in the study areas in Myanmar. http://doi.org/10.5455/javar.2017.d204 XML PubReader

EXPRESSION OF NUCLEAR FACTOR OF ACTIVATED T CELLS (NFAT) AND DOWNSTREAM TARGETS IN GROUND SQUIRREL CARDIAC MUSCLE

13-lined ground squirrels undergo remarkable adaptive changes during hibernation where the animals enter deep torpor and allow body temperature (Tb) to fall as low as 0-5°C. While hibernating, reversible cardiac hypertrophy occurs to increase contractility of the heart; this helps the heart to pump colder and more viscous blood, while avoiding heart failure as a result of sustained or chronic hypertrophy. We were interested in studying the role of the nuclear factor of activated T-cell (NFAT) family of transcription factors and their recently discovered sarcolemmal targets, myoferlin and myomaker, with the adaptations taking place in cardiac muscle during hibernation. Whole heart samples were collected at 6 different time points over the torpor-arousal cycle including euthermic control (EC), entrance into torpor (EN), early torpor (ET), late torpor (LT), early arousal (EA), and interbout arousal (IA) back to euthermia; each stage differs in squirrel Tb and respiration rates. Total protein extracts were prepared and immunoblotting was performed using antibodies specific for mammalian NFATc1, c2, c3, c4, myoferlin, and myomaker. Bioinformatic analysis was conducted on the 1500 bp region upstream of the myomaker transcription start site to identify novel NFAT transcription factor binding sites. DNA-binding enzyme-lined immunosorbent assay (ELISA) was performed over the torpor-arousal timecourse to characterize NFAT-binding to the NFAT-binding sequence identified. In cardiac muscle, NFATc1, c2, and c3 increased during torpor whereas NFATc4 protein levels were elevated throughout the torpor-arousal cycle. A spike in myoferlin protein levels was observed during ET (2.68 fold increase compared to control) with a smaller increase during EA (1.69 fold increase). Myomaker also increased significantly in cardiac muscle during ET (1.88 fold increase) and to a much lesser extent at EA. Bioinformatics analysis and cross-referencing using the NFAT consensus binding sequence resulted in the identification of a putative novel NFAT binding site and DNA-binding ELISAs showed that NFATc1 and c3 were able to bind to this site. This study is the first to observe myomaker expression in cardiac tissue and to identify a possible NFAT-binding region within its promoter. Elevated protein levels of NFATc1-4, myoferlin, and myomaker at selected stages of the torpor-arousal cycle appear to be a regulatory mechanism that contributes to reversible cardiac hypertrophy in response to the changing physiological and environmental stresses encountered during hibernation. The identification of novel targets involved in cardiac hypertrophy provides insight into how heart failure resulting from sustained cardiac overload might be prevented.

Neurotrophic effect of bone marrow mesenchymal stem cells for erectile dysfunction in diabetic rats

It has been demonstrated that intracavernous injection of bone marrow-derived mesenchymal stem cells (BM-MSCs) had beneficial effects on improving erectile function in type-1 diabetic rats. This study was designed to investigate the neurotrophic effect of BM-MSCs for type-1 diabetic rats. Streptozocin-induced type-1 diabetic rats were randomly divided into three groups: diabetic group, BM-MSCs-treated group and BM-MSCs-conditioned medium-treated group. At the 3d, 1 and 2w time points after BM-MSCs injection, three randomly selected rats in MSCs group were sacrificed and penile samples were harvested to detect BM-MSCs in penile tissue. Four weeks after intracavernous injection of BM-MSCs or BM-MSCs-conditioned medium, intracavernous pressure (ICP) was assessed to evaluate the erectile function. Immunohistochemistry was used to track labelled BM-MSCs in penile tissue and to detect neuronal nitric oxide synthase (nNOS) and neurofilament (NF) positive fibres in penile dorsal nerve. Enzyme lined immunosorbent assay (ELISA) was used to measure the concentrations of vascular endothelial growth factor (VEGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in BM-MSCs-conditioned medium. BM- MSCs secreted detectable levels of VEGF, BDNF and NGF. Intracavernous injection of BM-MSCs improved erectile function in diabetic rats. The functional improvement was accompanied by promoted nNOS and NF positive nerve fibres within penile dorsal nerve in type-1 diabetic rats. Histological data revealed a time-dependent decrease in the number of BM-MSCs in the corpus cavernosum following injection. Furthermore, the beneficial effect of BM-MSCs was partially repeated by BM-MSCs-conditioned medium. Intracavernous injection of BM-MSCs is effective in improving nerve regeneration in diabetic rats. Paracrine effects of BM-MSCs are probably involved in the improvement.

Platelet Microparticles: Making Blood a Bad Humor

Ancient physicians based their practice of medicine on the humoral theory of disease. This theory posited that substances called humors make up the body and determine health and disease. When in balance, these humors — blood, yellow bile, black bile, and phlegm — caused health; out of balance, they caused disease. This theory also related humors to the 4 elements: earth, fire, water, and air. Of the humors, blood was unique in its content of all 4 elements. Now relegated to the realm of quaint and discredited ideas of the past, the humoral theory of disease nevertheless has elements of truth. Importantly, this theory recognized the importance of blood in disease pathogenesis and the diversity of bodily constituents that can influence disease. With scientific advance, earth, fire, water, and air have been replaced by cells, cytokines, chemokines, and prostanoids, among many others. The list of these substances is ever increasing, with disciplines of genomics, proteomics, metabolomics, and nucleomics filling the blood to the brim with noxious mediators. Reading today’s literature, we can be wistful about a time when physicians could understand disease with only 4 elements, in contrast to today’s 4 thousand or even 4 million. As an article in this issue of The Journal demonstrates, the blood of patients with connective tissue disease (CTD) has novel elements that can drive disease and serve as biomarkers to elucidate pathogenesis and assess disease activity1. Thus, using an enzyme-lined immunosorbent assay (ELISA), Oyabu, et al provide evidence that the blood of patients with CTD has increased levels of platelet-derived microparticles (PDMP) and that elevations of PDMP are related to Raynaud’s phenomenon (RP). This article is important in substantiating the value of solid-phase methods to detect particles and in delineating clinical associations. As shown in … Address correspondence to Dr. D.S. Pisetsky, 151G Durham VA Medical Center, 508 Fulton Street, Durham, NC 27705, USA. E-mail address: dpiset{at}acpub.duke.edu

Novel Recombinant Engineered gp41 N-terminal Heptad Repeat Trimers and Their Potential as Anti-HIV-1 Therapeutics or Microbicides

Peptides derived from N-terminal heptad repeat (NHR) of the HIV-1 gp41 are generally poor inhibitors of HIV-1 entry, because they tend to aggregate and do not form a trimeric coiled-coil. In this study, we have fused portions of gp41 NHR, e.g. N36 or N28, to the T4 fibritin trimerization domain, Foldon (Fd), thus constructing novel NHR trimers, designated N36Fd or N28Fd, which could be expressed in Escherichia coli cells. The purified N36Fd and N28Fd exhibited SDS-resistant trimeric coiled-coil conformation with improved alpha-helicity compared with the corresponding N-peptides. They could interact with a C-peptide (e.g. C34) to form stable six-helix bundle and possessed potent anti-HIV-1 activity against a broad spectrum of HIV-1 strains. N28Fd was effective against T20-resistant HIV-1 variants and more resistant to proteinase K compared with T20 (enfuvirtide), a C-peptide-based HIV fusion inhibitor. Therefore, N28Fd trimer has great potentials for further development as an affordable therapeutic or microbicide for treatment and prevention of HIV-1 infection.

Targeting of PED/PEA-15 Molecular Interaction with Phospholipase D1 Enhances Insulin Sensitivity in Skeletal Muscle Cells

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) is overexpressed in several tissues of individuals affected by type 2 diabetes. In intact cells and in transgenic animal models, PED/PEA-15 overexpression impairs insulin regulation of glucose transport, and this is mediated by its interaction with the C-terminal D4 domain of phospholipase D1 (PLD1) and the consequent increase of protein kinase C-alpha activity. Here we show that interfering with the interaction of PED/PEA-15 with PLD1 in L6 skeletal muscle cells overexpressing PED/PEA-15 (L6(PED/PEA-15)) restores insulin sensitivity. Surface plasmon resonance and ELISA-like assays show that PED/PEA-15 binds in vitro the D4 domain with high affinity (K(D) = 0.37 +/- 0.13 mum), and a PED/PEA-15 peptide, spanning residues 1-24, PED-(1-24), is able to compete with the PED/PEA-15-D4 recognition. When loaded into L6(PED/PEA-15) cells and in myocytes derived from PED/PEA-15-overexpressing transgenic mice, PED-(1-24) abrogates the PED/PEA-15-PLD1 interaction and reduces protein kinase C-alpha activity to levels similar to controls. Importantly, the peptide restores insulin-stimulated glucose uptake by approximately 70%. Similar results are obtained by expression of D4 in L6(PED/PEA-15). All these findings suggest that disruption of the PED/PEA-15-PLD1 molecular interaction enhances insulin sensitivity in skeletal muscle cells and indicate that PED/PEA-15 as an important target for type 2 diabetes.

Antiprion Prophylaxis by Gene Transfer of a Soluble Prion Antagonist

Prion diseases are untreatable neurodegenerative disorders characterized by accumulation of PrP(Sc), an aggregated isoform of the normal prion protein PrP(C). Here, we delivered the soluble prion antagonist PrP-Fc(2) to the brains of mice by lentiviral gene transfer. Although naïve mice developed scrapie at 175 +/- 5 days postintracerebral prion inoculation (dpi), gene transfer before inoculation delayed disease onset by 72 +/- 4 days. At 170 days postintracerebral prion inoculation, PrP(Sc) accumulation and prion infectivity in PrPFc-treated brains were reduced by 3.6 and 4.2 logs, respectively. When PrP-Fc(2) was delivered 30 days after prion inoculation, survival of the treated animals was extended by 25 days. We then used tissue-specific recombination to express PrP-Fc(2) in the entire central nervous system, in only astrocytes, or in only oligodendrocytes. Oligodendrocyte-restricted PrP-Fc(2) expression impaired PrP(Sc) deposition and delayed disease even though oligodendrocytes are completely resistant to prion infection, suggesting that PrP-Fc(2) affords protection via noncell autonomous mechanisms. These results suggest that somatic gene transfer of prion antagonists may be effective for postexposure prophylaxis of prion diseases.

Quantification of Neutral Cysteine Protease Bleomycin Hydrolase and its Localization in Rat Tissues

A neutral cysteine protease, bleomycin hydrolase (BH), was found to be present in the range 3.7-131.1 ng per mg of rat tissues by enzyme-lined immunosorbent assay (ELISA). Newborn rat skin contained the highest amount of BH, and relatively high levels of BH were detected in the kidney and liver of 6-week-old male rats. The tissue distribution of BH in female rats was similar to that in male rats. Moreover, BH was detected in the extracts of erythrocytes and leukocyte-rich cells as well as in those of rat hemo-lymphocytic lineage cell lines by Western blotting. The BH level was increased at 6 weeks after birth and then slightly decreased. By immunohistochemistry, BH was localized as granular staining in the distal and proximal tubular cells of the kidney, and it was also detected in hepatocytes of the liver, in the red pulpy region of the spleen and in neurons of the brain. An immunoelectron microscopic study showed that BH-immunoreactivity was essentially located in the cytoplasm and at the outer membrane of the rough endoplasmic reticulum of epithelial cells of the kidney, as well as in that of hepatocytes of the liver. These results suggest that BH may play ubiquitous and unique roles in rat tissues.

Etiological investigation of fatal liver failure during the course of chronic hepatitis B in southeast China

The purpose of this study was to clarify the relationships between patients who had fatal liver failure during the course of chronic hepatitis B and those who were also superinfected with hepatitis A, C, D, or E virus, as well as their hepatitis B virus e system status, so that suitable measures could be adopted to decrease the mortality of patients with chronic hepatitis B. This study detected superinfections of hepatitis A, C, D, or E virus and the hepatitis B virus e system status in cases of fatal liver failure during the course of chronic hepatitis B by enzyme-lined immunosorbent assay. The frequency of superinfections of hepatitis A, C, D, and E virus was 1.4% (4/282), 6.4% (18/282), 1.8% (5/282), and 28.4% (80/282), respectively, overall, 37.9% (107/282). Hepatitis E was prominent and steady in superinfection rates during the past 12 years. In 62.1% (175/282) of patients, the causes of fatal liver failure were not clear. The serological status frequency of HBeAg(+) and anti-HBe(-), HBeAg(-) and anti-HBe(-), and HBeAg(-) and anti-HBe(+) was 20.6% (22/107), 23.4% (25/107), and 56.1% (60/107), respectively, in the group with superinfections of hepatitis A, C, D, or E virus and 31.4% (55/175), 21.1% (37/175), and 47.4% (83/175), respectively, in the group in which causes were not clear. The serological status HBeAg(+) and anti-HBe(-) was more frequent in the group in which causes were not clear than in the group with superinfections of hepatitis A, C, D, or E virus (P < 0.05). Statistically, there were no differences (P > 0.05) between the serological status HBeAg(-), anti-HBe(-) and HBeAg(-), anti-HBe(+) between the two groups. These results suggest that superinfection (107/282) is an important factor in fatal liver failure. The mortality of chronic hepatitis B can be decreased by strict food sanitation and the use of safe blood products. There were no significant relationships between hepatitis B e antigen seroconversion and fatal liver failure during the course of chronic hepatitis B.

Anticyclic citrullinated peptide antibodies in patients with mixed connective tissue disease

The clinical significance of anticyclic citrullinated peptide (CCP) antibodies in patients with mixed connective tissue disease (MCTD) was assessed. Altogether, 86 sera from MCTD patients, 96 from rheumatoid arthritis (RA) patients, 42 from systemic lupus erythematosus (SLE) patients, 23 from systemic sclerosis (SSc) patients, 21 from polymyositis/dermatomyositis (PM/DM) patients, and 17 from those with Sjögren's syndrome (SjS) were tested for anti-CCP antibodies using an enzyme-lined immunosorbent assay. Among the 96 RA patients, anti-CCP antibodies were detected in 85%, with the frequency being significantly higher than in MCTD, SLE, SSc, PM/DM, and SjS patients (9%, 14%, 13%, 14%, and 18%, respectively; P < 0.001). Among eight MCTD patients who fulfilled the diagnostic criteria for RA, only 50% had anti-CCP antibodies, and the prevalence was significantly lower than for all RA patients (p < 0.01). All eight patients who fulfilled the criteria for RA had overlap of SLE and SSc, except one patient, whereas the four anti-CCP-positive patients who did not fulfill the criteria for RA had SjS without overlapping features of SLE and SSc; moreover, most of their antibody titers were low. These results suggested that anti-CCP antibodies are associated with RA in MCTD patients, but careful diagnosis of RA is required if patients with low titers of anti-CCP antibodies lack overlapping SLE and SSc.

Anticyclic citrullinated peptide antibodies in patients with mixed connective tissue disease

The clinical significance of anticyclic citrullinated peptide (CCP) antibodies in patients with mixed connective tissue disease (MCTD) was assessed. Altogether, 86 sera from MCTD patients, 96 from rheumatoid arthritis (RA) patients, 42 from systemic lupus erythematosus (SLE) patients, 23 from systemic sclerosis (SSc) patients, 21 from poymyositis/dermatomyositis (PM/DM) patients, and 17 from those with Sjögren’s syndrome (SjS) were tested for anti-CCP antibodies using an enzyme-lined immunosorbent assay. Among the 96 RA patients, anti-CCP antibodies were detected in 85%, with the frequency being significantly higher than in MCTD, SLE, SSc, PM/DM, and SjS patients (9%, 14%, 13%, 14%, and 18%, respectively; P < 0.001). Among eight MCTD patients who fulfilled the diagnostic criteria for RA, only 50% had anti-CCP antibodies, and the prevalence was significantly lower than for all RA patients (p < 0.01). All eight patients who fulfilled the criteria for RA had overlap of SLE and SSc, except one patient, whereas the four anti-CCP-positive patients who did not fulfill the criteria for RA had SjS without overlapping features of SLE and SSc; moreover, most of their antibody titers were low. These results suggested that anti-CCP antibodies are associated with RA in MCTD patients, but careful diagnosis of RA is required if patients with low titers of anti-CCP antibodies lack overlapping SLE and SSc.

Performance characteristics of a rapid new immunochromatographic test for detection of antibodies to human immunodeficiency virus.

A new immunochromatographic rapid test (Rapid Check HIV 1 and 2; Núcleo de Doenças Infecciosas) for the detection of antibodies to human immunodeficiency virus type 1 and type 2 in human samples (whole blood, serum, and plasma) was evaluated and compared to the commercially available Determine (Abbott Laboratories). When whole-blood samples were evaluated, the specificity and sensitivity of both tests were 100%. However, when plasma samples were used, sensitivity for the Rapid Check HIV 1&2 and the Determine tests were 100 and 98.58%, respectively. The observed specificity for plasma samples was 98.94% for the Rapid Check HIV 1&2 and 96.97% for the Determine test. The results presented here are encouraging and support the adoption of both tests as an alternative to enzyme-lined immunosorbent assay and/or Western blots in regions where laboratorial infrastructure is not available or for use in the management of occupational accidents for healthcare workers.

A serological survey of measles vaccine in a rural region of Eskişehir in Turkey

We designed this longitudinal study as a response to measles outbreaks which occur occasionally in Eskişehir in Turkey to investigate the incidence of primary and secondary vaccine failure. The investigation was conducted over two periods (December 1993 to October 1994 and April 1995 to October 1995). Two study groups were involved, infants aged 9–11 months and children aged 18 months to 9 y. During December 1993 to October 1994 prevaccination sera was collected from 35 infants aged 9–11 months and tested to determine if maternally derived antibodies were present. The infants were vaccinated and subsequently the 31 infants who could be traced were retested 30–40 d later to determine their response to vaccination. The following was done to determine whether seropositivity rates alter over time. The second group, a randomised sample of 117 children aged between 18 months to 9 y was chosen; all of whom had been previously vaccinated and who had no history of measles. Sera was taken and tested during December 1993 to October 1994 in order to determine whether seropositivity rates varied with time. During April 1995 to October 1995 out of all the children in both groups 123 children were retested. All sera samples were studied by an enzyme-lined immunosorbent assay (ELISA). Out of the 35 infants in group 1 only four (11.4%) had maternal antibody against measles on initial testing. Out of 31 infants followed up 30–40 d after vaccination (61.3%) were found to be seropositive for measles. In the second group, of the 117 previously vaccinated children ninety-three (71.5%) had measles IgG antibody. Seropositivity rates did not show significant difference with time after vaccination. There was no association between first and second screening seropositivity rates. We conclude that the presence of maternal antibody reduces the success of vaccination. These results suggest the vaccination policy in Turkey should be re-examined with a view to revision.

Evaluation of bovine fibrin sealant in the dog.

Fibrin sealant (human fibrinogen-bovine thrombin) is an effective biodegradable hemostatic agent. However, there is a risk of transmission of infectious viral disease. A new bovine fibrinogen-thrombin sealant (BFTS) was tested for tissue and immune responses in intrathoracic aorta graft in dogs. Intrathoracic aorta replacement was performed in three dogs with a porous Dacron graft presealed with BFTS. Dogs were immunized preoperatively with four dermal applications of BFTS at 9-day intervals. Cellular and humoral immunity to BFTS were determined with lymphocyte blastogenesis test and enzyme-lined immunosorbent assay, respectively. Dogs were necropsied 3 weeks after aortic replacement. Histopathological examination showed that all dogs had a mild inflammatory reaction to the BFTS sealed graft, as expected in response to an inert foreign body. Assay of cellular and humoral immunity to BFTS revealed a low lymphocyte response and a moderate immunoglobulin G (Ig G) response, with no evidence of immediate Ig E (type I) or delayed (type IV) hypersensitivity reaction. We conclude that BFTS causes no adverse tissue response or immunologic reaction when used as a hemostatic agent in the dog even after multiple applications of the material.

Evaluation of an elsa for the detection of anti‐histoplasma ribosomal and antihistoplasmin antibodies in histoplasmosis

We have developed an indirect sandwich enzyme-lined immunosorbent assay (ELISA) for the detection of antibodies to Histoplasma ribosomes and histoplasmin; we used this test for demonstration of these antibodies in sera from proven cases of histoplasmosis and other infections. Serum dilutions from five negative controls used in each experiment were normalized against 50 normal sera, and a factor of the mean absorbance was used to establish a positive reaction. Antiribosomal antibodies were detected in 97% of the known histoplasmosis patients with ELISA titers ranging from 1:100 to over 1:12,800. In contrast, antibodies to histoplasmin were detected in only 75% of these sera; titers ranged from 1:100 to 1:12,800. Cross-reactions with sera from other fungal infections (blastomycosis, coccidioidomycosis, paracoccidioidomycosis, cryptococcosis, candidiasis, and aspergillosis) were seen in 46% of the cases with ribosomes and 37% with histoplasmin. Fifty percent of the sera from tuberculosis patients gave positive reactions with ribosomes and 29% with histoplasmin. These results warrant further studies on the significance of antibodies to ribosomes and histoplasmin in immunity to histoplasmosis.

Localization of protease nexin-1 on the fibroblast extracellular matrix.

Protease nexin-1 (PN-1) is a protease inhibitor that is secreted by fibroblasts and several other cultured cells. PN-1 forms complexes with certain serine proteases in the extracellular environment including thrombin, urokinase, and plasmin. The complexes then bind to the cells and are rapidly internalized and degraded. This report demonstrates that PN-1 is present on the surface of fibroblasts, bound to the extracellular matrix. Immunofluorescent studies showed that PN-1 colocalized with fibronectin on both intact cells and in preparations of extracellular matrix made from these cells. In contrast, PN-1 did not colocalize with the epidermal growth factor receptor, a plasma membrane marker. An enzyme-lined immunosorbent assay was developed which showed that the extracellular matrix contained at least 60-80% of the cellular immunoreactive PN-1. Extraction of the matrix with 2 M NaCl removed PN-1 in a form which reacted with 125I-thrombin to form complexes which were immunoprecipitated by anti-PN-1 IgG and were of identical size as complexes made from soluble PN-1 and 125I-thrombin. These data indicate that in addition to its role as a soluble protease inhibitor, PN-1 is also a component of the extracellular matrix and might control its proteolysis.

Serum and Gingival Fluid Antibodies as Adjuncts in the Diagnosis of Actinobacillus actinomycetemcomitans Associated Periodontal Disease.

Serum antibody titers to Actinobacillus actinomycetemcomitans were measured in 200 subjects by an enzyme-lined immunosorbent assay (ELISA) using whole microorganisms as antigen. Comparisons were made between titers found in periodontally normal subjects and titers in subjects with localized juvenile periodontitis (LJP), postlocalized juvenile periodontitis, generalized juvenile periodontitis or adult periodontitis. It was found that titers to all three serotypes of A. actinomycetemcomitans were elevated in LJP patients' sera, while serum antibody levels in other diseased groups were not significantly elevated to any of the serotypes. Patient sera were also examined for serum antibody to oral Haemophili previously shown to cross-react with A. actinomycetemcomitans. Similar antibody titers were found in both normal subjects and in patients with various forms of periodontal disease to Haemophilus aphrophilus, H. influenzae and H. parainfluenzae. The A. actinomycetemcomitans antibodies which were elevated in LJP patients could not be correlated with antibody titers to cross-reacting Haemophili, suggesting that these antibodies are A. actinomycetemcomitansspecific. Serum antibody responses in six of the LJP patients were assessed to autologous strains of A. actinomycetemcomitans. Each patient was found to be infected with only a single serotype of A. actinomycetemcomitans, and specific antibodies to the infecting serotype were found in the patients' sera. In families, the LJP patients had significantly elevated IgG, IgA and IgM serum antibody titers to A actinomycetemcomitans, while the IgG and IgA antibody titers in periodontally normal siblings were at levels comparable to those found in normal subjects. However, IgM serum antibodies were elevated in the periodontally normal siblings of LJP patients suggesting that the formation of IgM antibodies to A. actinomycetemcomitans may precede the clinical appearance of localized juvenile periodontitis. Gingival crevicular fluid and serum antibody levels to A. actinomycetemcomitans were compared in LJP patients. Comparable titers of IgG, IgA and IgM antibodies were found in serum and gingival fluid in most subjects; however, gingival fluid samples sometimes showed higher titers than serum, likely resulting from local antibody synthesis. The value of serum antibody determinations to A. actinomycetemcomitans in the diagnosis of Actinobacillus-associated periodontitis was also assessed. The predictive value of a positive test (significantly elevated anti-A actinomycetemcomitans IgG) was 86%, while the specificity was 89%. These results suggest that the measurement of serum or gingival crevicular fluid antibodies to A. actinomycetemcomitans may be valuable in the diagnosis of Actinobacillusassociated periodontal disease.

Antigen distribution and humoral response in the lizard, [formula omitted], after injection with [formula omitted

The response of the spiny-tailed agamid lizard, Agama caudospinosum , to administration of Leishmania agamae promastigotes was investigated. Lizards given a single injection of promastigotes showed no signs of clinical infection. Neither promastigotes nor amastigotes were found in blood and tissue impression smears, nor in blood and selected body organ cultures. However, parasite antigens were demonstrated by an immunoenzyme method only in the liver, small intestine, stomach, spleen and kidney. Non-precipitating serum antibodies with γ-electrophoretic mobility were detected by enzyme-lined immunosorbent assay 1 week post-injection and a maximum titre was reached after 6 weeks. The mean immune serum protein concentration increased significantly ( P<0.005) about two-fold over the controls after injection. Decreases occurred in the β-globulin region of anti- L.agamae sera ( P<0.001) whilst the γ-globulin fraction was increased ( P<0.005) following electrophoresis on cellulose acetate membranes. C-reactive protein was not detected in any of the sera. These data show that although A.caudospinosum failed to become infected by L.agamae promastigotes, which had been isolated from agamids, it did exhibit antigen distribution and a humoral response similar to other reptiles.