Core Protein Antibody Research Articles

İmmunsupresif Tedavi Alan Hastalarda HBV Reaktivasyonu ve Antiviral Profilaksinin Değerlendirilmesi

Aim: Patients with chronic hepatitis B and people with a history of hepatitis B (HBV) infection are at risk of HBV reactivation (HBVr) when they receive immunosuppressive therapy. In this study, we aimed to evaluate the hepatitis B serology, risk groups and antiviral prophylaxis of patients receiving various immunosuppressive therapies due to rheumatological diseases.
 Material and Method: The study included 375 patients over 18 years of age who received tumor necrosis factor-α (TNF-α) inhibitor, tyrosine kinase inhibitor, steroids, methotrexate or anti-CD20 antibodies due to rheumatic diseases in a training and research hospital between May 2022 and May 2023. Hepatitis B surface antigen (HbsAg), hepatitis B surface antibody (anti-Hbs), hepatitis B core protein antibody (anti-Hbc IgG) serologies, immunosuppressive therapies and oral antivirals were retrospectively analyzed.
 Results: The average age of the 375 patients included in the study was 43.77±13.07 years. 193 (51.5%) of the patients were male. 11 patients were HbsAg positive, 150 patients were anti-Hbs positive, 19 patients were isolated anti-Hbc IgG positive, and 79 patients were both anti-Hbs and anti-Hbc IgG positive. According to serological findings, 109 (29%) patients had HBV exposure. All three test results of 194 (51.7%) patients were negative. A total of 85 (22.7%) patients received oral antiviral prophylaxis due to the use of immunosuppressive agents. In terms of HBVr, 16.5% were evaluated as high risk, 75.3% as moderate risk, and 8.2% as low risk. Out of 85 patients 79 received entecavir, 5 reveived tenofovir disoproxil fumarate (TDF) and 1 received tenofovir alafenamide fumarate (TAF). The mean duration for the immunosuppressive therapy was 6.41±4.20 years. The mean duration of oral antiviral prophylaxis among patients was 1.02±1.72 years. HBVr was not observed in any of our patients.
 Conclusion: Before patients receive immunosuppressive therapy, hepatitis B serologies and prophylaxis indication should be evaluated firstly. In addition, as a preventive medicine activity, hepatitis B vaccinations of unvaccinated patients should be completed as quickly as possible.

Abstract 13141: Accumulation of Aggrecan at Sites of Increased Pulmonary Arterial Pressure in Idiopathic Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a lethal condition lacking curative pharmacotherapy. Expansion of the extracellular matrix occurs in early stages of pulmonary angiopathy, but the presence of individual matrix components warrants further investigation. Accumulation of the osmotically active matrix proteoglycan aggrecan has been associated with swelling and disruption of vessel wall integrity in systemic arteries. Whether aggrecan is present to any significant extent in PAH tissue, and what potential role it may have, is not known. Methods: Paraffin-embedded lung tissue from 11 patients with idiopathic PAH was imaged using synchrotron-based phase contrast micro-CT at the TOMCAT beamline, Swiss Light Source. Image analysis was performed in Fiji and Amira. Imaged blocks were subsequently sectioned for histology, immunohistochemistry with an aggrecan core protein antibody and RNAscope in situ hybridization. qPCR was performed to investigate gene expression. Failed donor lungs were used as controls. Results: Aggrecan core protein was identified in vascular lesions of all 11 patients with idiopathic PAH, localized to cellular rather than fibrotic or collagenous lesions. RNAscope in situ hybridization confirmed local production of ACAN mRNA in diseased vessels. Quantification of repeated immunohistochemistry demonstrated significantly increased accumulation of aggrecan in patients with idiopathic PAH compared to failed donor lung controls. ACAN and ADAMTS15 mRNA were also found to be up-regulated in pulmonary arteries from patients with IPAH, indicating ongoing proteolytic turnover. Image analysis and three-dimensional renderings of pulmonary arteries identified aggrecan in lumen-reducing lesions containing cellular connective tissue, at sites of intrapulmonary bronchopulmonary shunting and at sites of elevated pulmonary blood pressure. Conclusions: Our findings indicate local production and accumulation of aggrecan in pressure-related lesions of idiopathic PAH. This work strengthens the hypothesis that aggrecan plays a role in arterial adaptations to altered hemodynamics and is the first to suggest a role for aggrecan in pulmonary arterial homeostasis and idiopathic PAH.

Characterization of occult hepatitis B in high-risk populations in Kenya

Occult hepatitis B infection (OBI) is defined as the presence of hepatitis B virus (HBV) DNA in the liver or serum in the absence of detectable HBV surface antigen (HBsAg). OBI poses a risk for the development of cirrhosis and hepatocellular carcinoma. The prevalence of OBI in Kenya is unknown, thus a study was undertaken to determine the prevalence and molecular characterization of OBI in Kenyan populations at high risk of HBV infection. Sera from two Nairobi cohorts, 99 male sex workers, primarily having sex with men (MSM-SW), and 13 non-MSM men having HIV-positive partners, as well as 65 HBsAg-negative patients presenting with jaundice at Kenyan medical facilities, were tested for HBV serological markers, including HBV DNA by real-time PCR. Positive DNA samples were sequenced and MSM-SW patients were further tested for hepatitis C virus (HCV) infection. Of the 166 HBsAg-negative samples tested, 31 (18.7%; 95% confidence interval [CI] 13.5-25.3) were HBV DNA positive (i.e., occult), the majority (20/31; 64.5%) of which were HBV core protein antibody positive. HCV infection was not observed in the MSM-SW participants, although the prevalence of HBsAg positivity was 10.1% (10/99; 95% CI 5.6-17.6). HBV genotype A was predominant among study cases, including both HBsAg-positive and OBI participants, although the data suggests a non-African network transmission source among MSM-SW. The high prevalence of HBV infection among MSM-SW in Kenya suggests that screening programmes be instituted among high-risk cohorts to facilitate preventative measures, such as vaccination, and establish entry to treatment and linkage to care.

Hepatitis Serology in Patients Receiving Immunosuppressive Treatment

Amaç: İmmünsupresif ilaç kullananlarda, hepatit B reaktivasyon riskine yönelik tarama yapılması önerilmektedir. Bu çalışmada immünsüpresif tedavi öncesi, Enfeksiyon hastalıkları kliniğine yönlendirilen hastalarda hepatit B virüsü (HBV), hepatit C virüsü (HCV) ve hepatit A virüsü (HAV) sonuçları retrospektif olarak irdelenmesi amaçlandı.Materyal ve Metod: Çalışmamızda Ocak 2018 - Aralık 2018 tarihleri arasında immünsüpresif tedavi öncesi, Enfeksiyon hastalıkları kliniğine yönlendirilen 148 hastanın hepatit B yüzey antijeni (HBsAg), hepatit B yüzey antikoru (Anti-HBs), hepatit B core protein antikoru (Anti-HBc IgG), anti hepatit C virüs antikoru (Anti-HCV) ve anti hepatit A virüs antikoru (Anti-HAV) hasta dosyalarından ve hastane otomasyon sisteminden retrospektif olarak incelendi.Bulgular: HBsAg pozitiflik oranı %4.1, Anti-HBs pozitiflik oranı %31.8, İzole Anti-HBc IgG pozitiflik oranı %1.4 ve iki hastada ise Anti-HBs bakılmamıştı. Anti-HCV pozitifliği %1.4 tespit edildi. Hastaların %79.7 Anti-HAV bakılmamış, Anti-HAV pozitifliği %12.2 ve Anti-HAV negatifliği %8.1 olarak saptandı. Sonuç: İmmünsüpresif tedavi öncesi tarama yapılmasına rağmen bazı taramaların eksik yapıldığı tespit edildi. Bu nedenle bu hastaları takip eden hekimlere eğitim verilmesi ve özellikle HBV enfeksiyonu konusunda farkındalık oluşturulmasını önermekteyiz.

Immunolocalization of Keratan Sulfate in Rat Spinal Tissues Using the Keratanase Generated BKS-1(+) Neoepitope: Correlation of Expression Patterns with the Class II SLRPs, Lumican and Keratocan.

This study has identified keratan sulfate in fetal and adult rat spinal cord and vertebral connective tissues using the antibody BKS-1(+) which recognizes a reducing terminal N-acetyl glucosamine-6-sulfate neo-epitope exposed by keratanase-I digestion. Labeling patterns were correlated with those of lumican and keratocan using core protein antibodies to these small leucine rich proteoglycan species. BKS-1(+) was not immunolocalized in fetal spinal cord but was apparent in adult cord and was also prominently immunolocalized to the nucleus pulposus and inner annulus fibrosus of the intervertebral disc. Interestingly, BKS-1(+) was also strongly associated with vertebral body ossification centers of the fetal spine. Immunolocalization of lumican and keratocan was faint within the vertebral body rudiments of the fetus and did not correlate with the BKS-1(+) localization indicating that this reactivity was due to another KS-proteoglycan, possibly osteoadherin (osteomodulin) which has known roles in endochondral ossification. Western blotting of adult rat spinal cord and intervertebral discs to identify proteoglycan core protein species decorated with the BKS-1(+) motif confirmed the identity of 37 and 51 kDa BKS-1(+) positive core protein species. Lumican and keratocan contain low sulfation KS-I glycoforms which have neuroregulatory and matrix organizational properties through their growth factor and morphogen interactive profiles and ability to influence neural cell migration. Furthermore, KS has interactive capability with a diverse range of neuroregulatory proteins that promote neural proliferation and direct neural pathway development, illustrating key roles for keratocan and lumican in spinal cord development.

High prevalence of occult hepatitis C infection in predialysis patients.

BACKGROUNDOccult hepatitis C virus (HCV) infection (OCI) may be associated with extrahepatic diseases and it is known that the patients with chronic kidney disease (CKD) who are on hemodialysis (HD) present a higher prevalence of this type of infection than the general population, with a worse clinical outcome. However, there are no data in the literature to assess the presence of OCI in patients prior to the initiation of renal replacement therapy (RRT). Therefore, this study aimed to evaluate the occurrence and epidemiological aspects of OCI in patients with Predialysis CKD. We hypothesize that this infection could occur before RRT initiation.AIMTo research the status in predialysis patients when HD patients have high prevalence of OCI.METHODSA cross-sectional study was conducted between 2015 and 2017. Adults with creatinine clearance < 60 mL/min·1.73 m2 (predialysis patients) were recruited to the study. Pregnant and postpartum women, patients with glomerulopathies, and patients showing positivity for serological markers of hepatitis B virus (HBV), HCV or human immunodeficiency virus infection were excluded. Patients were diagnosed with OCI according to test results of anti-HCV antibody negativity and HCV RNA positivity in either ultracentrifuged serum or, if serum-negative, in peripheral blood mononuclear cells.RESULTSAmong the 91 total patients included in the study, the prevalence of OCI was 16.5%. Among these 15 total OCI patients, 1 was diagnosed by 14 ultracentrifuged serum results and 14 were diagnosed by peripheral blood mononuclear cell results. Compared to the non-OCI group, the OCI patients presented higher frequency of older age (P = 0.002), patients with CKD of mixed etiology (P = 0.019), and patients with markers of previous HBV infection (i.e., combined positivity for anti-hepatitis B core protein antibody and anti-hepatitis B surface protein antibody) (P = 0.001).CONCLUSIONAmong predialysis patients, OCI involved the elderly, patients with CKD of mixed etiology, and patients with previous HBV infection.

High prevalence of hepatitis markers in immigrant populations: a prospective screening approach in a real-world setting.

Immigrant populations are believed to be more frequently infected with hepatitis viruses. However, limited unbiased data are available on immigrants outside of academic centres. Therefore, the aim of this study was to perform large-scale screening for hepatitis markers in primary care centres treating mainly individuals with a migrational background in Germany. Between November 2010 and January 2012, we prospectively screened 1313 individuals treated by general practitioners at eight primary care centres in North-western Germany. Patients were eligible if they or their parents were not born in Germany. Serological screening for hepatitis B core protein antibodies, hepatitis B surface antigens (HBsAgs), and anti-hepatitis C virus antibodies was performed in each individual. HBsAg-positive and anti-hepatitis C virus-positive patients were further tested for molecular markers of viral replication. The mean age was 49.1±15.8 years. Of the patients, 45.7% were male; 87.3% had migrated to Germany from the Eastern Mediterranean area and 12.0% from Eastern Europe. Of the patients, 32.5% tested positive for hepatitis B core protein antibodies. HBsAgs were found in 3.6% of patients. Overall, hepatitis B virus DNA was detected in 2.2% of patients. Markers for hepatitis C virus infection were found in an almost similar high frequency (1.9%). Individuals with migrational background showed significant deficits in knowledge on general routes of transmission. Hepatitis virus infections are indeed significantly more prevalent in immigrant populations as compared with the general German population. These data underline the importance of introducing screening programs in this particular risk group.

Efficacy and effectiveness of infant vaccination against chronic hepatitis B in the Gambia Hepatitis Intervention Study (1986–90) and in the nationwide immunisation program

BackgroundGambian infants were not routinely vaccinated against hepatitis B virus (HBV) before 1986. During 1986–90 the Gambia Hepatitis Intervention Study (GHIS) allocated 125,000 infants, by area, to vaccination or not and thereafter all infants were offered the vaccine through the nationwide immunisation programme. We report HBV serology from samples of GHIS vaccinees and unvaccinated controls, and from children born later.MethodsDuring 2007–08, 2670 young adults born during the GHIS (1986-90) were recruited from 80 randomly selected villages and four townships. Only 28% (753/2670) could be definitively linked to their infant HBV vaccination records (255 fully vaccinated, 23 partially vaccinated [1–2 doses], 475 not vaccinated). All were tested for current HBV infection (HBV surface antigen [HBsAg]) and, if HBsAg-negative, evidence of past infection (HBV core-protein antibody [anti-HBc]). HBsAg-positive samples (each with two age- and sex-matched HBsAg-negative samples) underwent liver function tests. In addition, 4613 children born since nationwide vaccination (in 1990-2007) were tested for HBsAg. Statistical analyses ignore clustering.ResultsComparing fully vaccinated vs unvaccinated GHIS participants, current HBV infection was 0.8% (2/255) vs 12.4% (59/475), p < 0.0001, suggesting 94% (95% CI 77-99%) vaccine efficacy. Among unvaccinated individuals, the prevalence was higher in males (p = 0.015) and in rural areas (p = 0.009), but adjustment for this did not affect estimated vaccine efficacy. Comparing fully vaccinated vs unvaccinated participants, anti-HBc was 27.4% (70/255) vs 56.0% (267/475), p < 0.00001. Chronic active hepatitis was not common: the proportion of HBsAg-positive subjects with abnormal liver function tests (ALT > 2 ULN) was 4.1%, compared with 0.2% in those HBsAg-negative. The prevalence of antibodies to hepatitis C virus was low (0.5%, 13/2592). In children born after the end of GHIS, HBsAg prevalence has remained low; 1.4% (15/1103) in those born between 1990–97, and 0.3% (9/35150) in those born between 1998–2007.ConclusionsInfant HBV vaccination achieves substantial protection against chronic carriage in early adulthood, even though approximately a quarter of vaccinated young adults have been infected. This protection persists past the potential onset of sexual activity, reinforcing previous GHIS findings of protection during childhood and suggesting no need for a booster dose. Nationwide infant HBV vaccination is controlling chronic infection remarkably effectively.

Effects of Ultraviolet-A and Riboflavin on the Interaction of Collagen and Proteoglycans during Corneal Cross-linking

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and β chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.

General epidemiological parameters of viral hepatitis A, B, C, and E in six regions of China: a cross-sectional study in 2007.

BackgroundViral hepatitis is a serious health burden worldwide. To date, few reports have addressed the prevalence of hepatitis A, B, C, and E in China. Therefore, the general epidemiological parameters of viral hepatitis remain unknown.Principal FindingsIn this cross-sectional study, we performed a serological prevalence analysis of viral hepatitis A, B, C, and E in 8,762 randomly selected Chinese subjects, which represented six areas of China. The overall prevalence of anti-Hepatitis C virus antibody (anti-HCV) was 0.58%, which was much lower than was estimated by WHO. The prevalences of Hepatitis B virus surface antigen (HBsAg), anti-Hepatitis B virus surface protein antibody (HBsAb), and anti-Hepatitis B virus core protein antibody (HBcAb) were 5.84%, 41.31%, and 35.92%, respectively, whereas in the group of subjects less than 5 years old, these prevalences were 1.16%, 46.77%, and 8.69% respectively, which suggests that the Hepatitis B virus (HBV)-carrier population is decreasing, and the nationwide HBV vaccine program has contributed to the lowered HBV prevalence in the younger generation in China. Meanwhile, a large deficit remains in coverage provided by the national HBV immune program. In addition, our data suggested the possibility that HBsAb may not last long enough to protect people from HBV infection throughout life. The overall prevalence of anti-Hepatitis A virus antibody (anti-HAV) and anti-Hepatitis E virus antibody (anti-HEV) were as high as 72.87% and 17.66%, respectively. The indices increased with age, which suggests that a large proportion of Chinese adults are protected by latent infection. Furthermore, the pattern of HEV infection was significantly different among ethnic groups in China.ConclusionsOur study provided much important information concerning hepatitis A, B, C, and E prevalence in China and will contribute to worldwide oversight of viral hepatitis.

ScFv Antibody-induced Translocation of Cell-surface Heparan Sulfate Proteoglycan to Endocytic Vesicles

Cellular uptake of several viruses and polybasic macromolecules requires the expression of cell-surface heparan sulfate proteoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (alphaHS) clones, only one, AO4B08, efficiently translocated macromolecular cargo to intracellular vesicles through induction of HSPG endocytosis. Interestingly, AO4B08-induced PG internalization was strictly dependent on HS 2-O-sulfation and appeared independent of intact N-sulfation. AO4B08 and human immunodeficiency virus (HIV)-Tat, i.e. a well known cell-penetrating peptide, were shown to compete for the internalizing PG population. To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamagnetic nanoparticles. [(35)S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phosphatidyl-inositol-anchored glypican in purified vesicles. Importantly, internalized syndecan and glypican were found to co-localize in AO4B08-containing vesicles. Our data establish HSPGs as true internalizing receptors of macromolecular cargo and indicate that the sorting of cell-surface HSPG to endocytic vesicles is determined by a specific HS epitope that can be carried by both syndecan and glypican core protein.

Potential of mean force of the hepatitis C virus core protein–monoclonal 19D9D6 antibody interaction

Antigen–antibody interactions are critical for understanding antigen–antibody associations in immunology. To shed further light on this question, we studied a dissociation of the 19D9D6-HCV core protein antibody complex structure. However, forced separations in single molecule experiments are difficult, and therefore molecular simulation techniques were applied in our study. The stretching, that is, the distance between the center of mass of the HCV core protein and the 19D9D6 antibody, has been studied using the potential of mean force calculations based on molecular dynamics and the explicit water model. Our simulations indicate that the 7 residues Gly70, Gly72, Gly134, Gly158, Glu219, Gln221 and Tyr314, the interaction region (antibody), and the 14 interprotein molecular hydrogen bonds might play important roles in the antigen–antibody interaction, and this finding may be useful for protein engineering of this antigen–antibody structure. In addition, the 3 residues Gly134, Gly158 and Tyr314 might be more important in the development of bioactive antibody analogs.

ITO pattern fabrication of glass platforms for electropolymerization of light sensitive polymer for its conjugation to bioreceptors on a micro-array

We present herein an effective and versatile method to fabricate a micro-patterned structure of conductive polymer, poly(pyrrole-benzophenone), on Indium Tin Oxide (ITO) glass chips for the subsequent photo-immobilization of various bioreceptor, antigens. Such methodologies are based on photolithography of ITO pattern fabrication on non-conductive surfaces, glass slides, and on a photo-active electrogenerated polymer films. The photo-active polymer serves as a substrate platform for the photo-immobilization of the bioreceptor reagents used for subsequent immunoreactions. We were able to show the resolution of electropolymerization on an ITO pattern as well as immobilization of more than one bioreceptor for the simultaneous detection of several analytes. The antigen micro-arrays were tested for sensitivity, specificity, and overall practicality for the simultaneous detection of analyte anti-Cholera Toxin B, anti-Hepatitis B virus surface and core protein antibodies. In addition we used our pattern ITO-poly(pyrrole-benzophenone) micro-array for the detection of serum samples of Hepatitis B virus patients previously screened by a standard hospital detection method.

Significance of Hepatitis C Virus Core Protein in the Diagnosis of Hepatitis C Virus Infection in Different Liver Diseases

The present article describes the presence of hepatitis C virus (HCV) core protein in relation to HCV antibody in different types of liver diseases caused by hepatitis viral infections. One...

Change in Decorin during Aging of Rat Placenta

By immunohistochemistry, with or without chondroitinases, decorin was found to be distributed in the extracellular matrix of chorionic villi and amnia. The strength of staining intensified with increasing gestational age. Decorin was isolated from the placenta of 13- to 20-day-old pregnant rats and identified by Western blotting, using an antidecorin core protein antibody. The molecular weight of decorin is ∼100 kDa, whereas the respective figures for the core protein treated with chondroitinase (chase) ABC and with chase B are ∼40 kDa and 43 kDa. The difference in the molecular weight between the core protein with chase ABC and B suggests that the glycosaminoglycan (GAG)- base structure on the core protein was chondroitin sulfate (CS) without dermatan sulfate (DS). The decorin content and the proportion of CS to DS in GAG increased with age. We concluded that the age-related changes in the GAG chain may be related to specific functional properties and may have a crucial role in placental tissue organization.

Development of recombinant capsid antigen/transmembrane epitope fusion proteins for serological diagnosis of animal lentivirus infections

Among animal lentiviruses, Feline immunodeficiency virus (FIV), Equine infectious anaemia virus (EIAV) and Small ruminant lentiviruses (SRLV) are important pathogens associated with a variety of clinical pictures including immunodeficiency, anaemia, arthritis, pneumonia. The detection of viral antibody response represents a practical diagnostic approach in all lentivirus infections since they remain detectable long life. Capsid antigen (CA) is the major viral core protein and specific antibodies against this antigen are usually first recognised in infected sheep, goat and horse, remaining detectable for long period. Transmembrane (TM) domain of envelope glycoprotein contains a well conserved motif known to form an immunodominant epitope in several lentiviruses. In this study a simple strategy was developed to express the entire CA and the TM epitope in a single fusion protein from equine, feline and small ruminant lentiviruses in prokaryotic system and evaluated the diagnostic utility of a purified preparation in an indirect ELISA for each of the three infections. Results demonstrate that, for FIV and SRLV infections, the combination of CA and TM fractions increases the sensitivity of diagnostic tests based only on CA. The corresponding CA/TM antigen from EIAV showed excellent agreement with Coggins test.

Characterization of low- and very-low-density hepatitis C virus RNA-containing particles.

The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.

Mucin-Like Glycoprotein Associated with Photoreceptor Cells in the Postnatal Developing Rat Retina

Purpose: To examine the developmental expression of mucin-like glycoprotein associated with photoreceptor cells (MLGAPC), and to confirm that MLGAPC with a relative mass of 120 and 135 kD correspond to the cone and rod types, respectively. Methods: We isolated the cDNA of the core protein of rat MLGAPC, prepared an anti-rat MLGAPC core protein antibody and then examined the expression of the MLGAPC core protein in postnatal developing rat retinas by means of Western blotting and immunohistochemistry. Results: On postnatal early days when cone photoreceptor cells were thought to be developing, MLGAPC was detected as a single band of 120 kD and was found to be positive in the apical portion of the neuroblastic layer. After postnatal day 7, when rod photoreceptor cells were developing, MLGAPC was detected as a minor band of 120 kD and an additional major band of 135 kD and was present throughout the differentiated photoreceptor and outer plexiform layers. Conclusions: The present findings revealed the developmental expression of MLGAPC and confirmed that MLGAPC of 120 and of 135 kD correspond to the cone and rod types, respectively. This demonstration is advantageous for studying the physiological roles of MLGAPC in the photoreceptor and outer plexiform layers, since the cone- and rod-associated glycoproteins should be investigated separately.

Functional construction of the anti-mucin core protein (MUC1) antibody MUSE11 variable regions in a bacterial expression system.

A bacterial expression system for the variable region fragments (Fvs) of the anti-MUC1 tumor antigen antibody MUSE11 has been constructed. The Fv fragment showed binding specificity toward TFK-1 cells, with slightly reduced affinity compared to its parent IgG. The single-chain Fv fragment was arranged in two orders, VH-linker-VL and VL-linker-VH. However, linking the regions with a flexible peptide linker (GGGGS)(3) or with a shorter linker (GGGGS) led to a dramatic decrease in the biological activity toward the target antigen in both arrangements, suggesting that the MUSE11 antibody loses its activity when the domains are linked with polypeptide linkers. These results indicate that the variable region domains of the anti-MUC1 antibody MUSE11 have specificity only in the Fv form, and that linking the domains strongly reduces the association with its target antigen. Gel filtration analysis indicates that the scFv has a dimeric structure, suggesting that the inactivation of MUSE11 scFv is due to unfavorable intermolecular associations of the scFv chains. To our knowledge, this is the first report of a significant reduction in affinity caused by linking the variable domains in both arrangements, i.e., VH-VL and VL-VH.

Detection of circulating anti-MUC1 mucin core protein antibodies in patients with colorectal cancer.

MUC1 mucin has a unique immunogenic peptide epitope in the extracellular domain, which has been shown to induce humoral and cellular immune response. In this study, we evaluated the pathophysiological significance of circulating anti-MUC1 mucin core protein IgG antibodies (anti-MUC1 antibodies) in colorectal cancer by Western blot analysis and 51Cr release assay. Anti-MUC1 antibodies were detected in 5 of 31 (16.1%) healthy subjects and in 27 of 56 (48.2%) patients with colorectal cancer. The presence of circulating anti-MUC1 antibodies was not significantly correlated with the level of circulating antigen MUSE11 or with other clinicopathological parameters tested. The incidence of positivity for anti-MUC1 antibodies in stage I and II (staged according to the General Rules for Clinical and Pathological Studies on Cancer of the Colon and Rectum of the Japanese Research Society for Cancer of the Colon and Rectum) cancers was 45.5% and 58.8%, respectively, suggesting that positivity for these antibodies may be of use as an adjunct for the diagnosis of colorectal cancer in the early stages in the absence of serious complications such as liver diseases. Because of the epitope similarity, anti-MUC1 antibodies in the serum may function in a manner similar to that of anti-MUC1 peptide monoclonal antibodies (mAbs). We therefore observed antibody-dependent cell mediated cytotoxicity with anti-MUC1 peptide mAb using MUC1 cDNA-transfected colon cancer CHC-Y1 cells as the target. The decreased sensitivity of MUC1 transfectants to effector cells was restored to a level equivalent to that in control cells. These data suggest that the detection of circulating anti-MUC1 antibodies may be a useful adjunct for the early diagnosis and immunological analysis of colorectal cancer.