Monoclonal antibody to novel cell surface epitope on Hsc70 promotes morphogenesis of bile ducts in newborn rat liver

We previously described a cell surface reactive monoclonal antibody, MAb OC.10, which recognizes an epitope shared by rat fetal liver ductal cells, hepatic progenitor cells, mature cholangiocytes, and hepatocellular carcinomas (HCC). Here, intrasplenic injection of MAb OC.10 into newborn rats was shown by immunofluorescence microscopy to strongly label intrahepatic bile ducts. Furthermore, the in situ labeling of intrahepatic cholangiocytes by injecting MAb OC.10 increased the number of intraportal and intralobular bile ducts with well-defined lumens when compared to IgM-injected control animals. The antigen for MAb OC.10 was identified by mass spectrometry as Hsc70, a constitutively expressed heat shock protein belonging to the HSP70 family. Immunoblot analysis demonstrated that MAb OC.10 reacted with recombinant bovine Hsc70 protein, with protein immunoprecipitated from rat bile duct epithelial (BDE) cell lysates with monoclonal anti-Hsc70 antibody, and with Hsc70-FLAG protein over-expressed in human 293T cells. In addition, Hsc70-specific small interfering RNA reduced the amount of OC.10 antigen expressed in nucleofected BDE cells. Consistent with the specificity of MAb OC.10 for Hsc70, heat shock did not induce OC.10 expression in BDE cells, a characteristic of Hsp70. Immunofluorescence with BDE cells further suggested that MAb OC.10 binds a novel cell surface epitope of Hsc70. This was in contrast to a commercially available monoclonal anti-Hsc70 antibody that showed strong cytosolic reactivity. These findings demonstrate that presentation of the OC.10 epitope differs between cytosolic and surface forms of Hsc70 and may suggest distinct differences in protein conformation or epitope availability determined in part by protein–protein or protein–lipid interactions. Phage display and pepscan analysis mapped the epitope for MAb OC.10 to the N-terminal 340–384 amino acids of the ATPase domain of rat Hsc70. These findings suggest that MAb OC.10 recognizes an epitope on rat Hsc70 when presented on the cell surface that promotes morphogenic maturation of bile ducts in newborn rat liver. Furthermore, since we have shown previously that the OC.10 antigen is expressed on HCC subpopulations with oval cell characteristics, our current results indicate that Hsc70 has the potential to be expressed on the surface of certain tumor cells.     

The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells

The initiation of DNA synthesis is governed by the licensing of replication origins, which consists of assembling a pre-replication complex (pre-RC) on origins during late M- and G1-phases. In metazoans, functional replication origins do not show defined DNA consensus sequences, thus evoking the involvement of chromatin determinants in the selection of these origins. Here, we show that the onset of licensing in mammalian cells coincides with an increase in histone H4 Lys 20 monomethylation (H4K20me1) at replication origins by the methyltransferase PR-Set7 (also known as Set8 or KMT5A). Indeed, tethering PR-Set7 methylase activity to a specific genomic locus promotes the loading of pre-RC proteins on chromatin. In addition, we demonstrate that PR-Set7 undergoes a PCNA- and Cul4-Ddb1-driven degradation during S phase that contributes to the disappearance of H4K20me1 at origins and the inhibition of replication licensing. Strikingly, expression of a PR-Set7 mutant insensitive to this degradation causes the maintenance of H4K20me1 and repeated DNA replication at origins. These results elucidate a critical role for PR-Set7 and H4K20me1 in the chromatin events that regulate replication origins.     

Cholangiocyte marker-positive and -negative fetal liver cells differ significantly in their ability to regenerate the livers of adult rats exposed to retrorsine

We have used monoclonal antibodies against cell-surface developmental epitopes in combination with micromagnetic beads to isolate phenotypically defined subpopulations of cholangiocyte marker-positive fetal liver epithelial cells (CMP-FLEC). Differentiation potential was evaluated by injecting cell isolates from dipeptidyl peptidase IV (DPPIV) positive (DPPIV+) Fischer donor rats into the spleen of partially hepatectomized, DPPIV negative (DPPIV-) Fischer host rats exposed to retrorsine. At various time points, liver tissue was harvested and cells in DPPIV+ colonies were phenotyped by immunofluorescence and histochemical protocols. Functional differentiation and liver replacement were determined by comparing donor and host hepatocyte protein expression patterns and DPPIV enzyme activity in extracts from livers of host rats receiving CMP-FLEC. Our results showed that bipotentiality was retained during differentiation and maturation of CMP-FLEC, indicating that the acquisition of ductal morphology and phenotype were not indicative of lineage commitment. CMP-FLEC transplanted into the adult rat liver lost ductal and gained hepatocyte markers, and acquired protein expression patterns in 2D gels with a close similarity (>75% spot match) to host hepatocytes but differing significantly from the transplanted CMP-FLEC cell isolate (<25% spot match). The average size of donor hepatocyte colonies increased with time so that by 1 year, up to 70% of the host rat liver was replaced by CMP-FLEC derived DPPIV+ hepatocytes. Depletion of CMP-FLEC from fetal liver isolates resulted in a marked decrease in adult liver colonization, suggesting that a high percentage of the hepatocyte colonies in animals receiving total fetal liver isolates are derived from CMP-FLEC.     

The phenotype and genotype of rheumatoid arthritis in the Democratic Republic of Congo

Introduction

Little is known about rheumatoid arthritis in the black, particularly in Congolese, populations. Our objective was to describe the phenotype and genotype of rheumatoid arthritis (RA) in Congolese.

Methods

All consecutive rheumatoid arthritis (RA) patients attending Kinshasa University Hospital in a three-year time period were included. Demographics, clinical features and tobacco consumption were noted. Disease Activity Score (DAS)-28 based on the erythrocyte sedimentation rate (ESR), Health Assessment Questionnaire (HAQ), anti-citrullinated peptide antibodies (CCP) antibodies and rheumatoid factor (RF) were determined. Radiographs were scored according to Sharp-van der Heijde. On a subset of patients and controls HLA-DRB1 typing was performed.

Results

A total of 114 females and 14 males aged 51.2 ± 14.9 were included. Mean duration of symptoms was four years. Moderate tobacco consumption was reported in a minority of patients. DAS-28 at first visit was >5.1 and HAQ ≥0.5 in all patients. X-rays showed joint erosions and/or joint space narrowing, mostly of a moderate grade in 55.8% of patients. Anti-CCP and/or RF were present in 48.6% of patients with available data (n = 72) and in 3.0% of controls (n = 67). Radiographic changes and nodules were more frequent in RF or anti-CCP positive patients. One copy of the shared epitope was found in 13 patients (35.1%) and 3 controls (12.5%). Two copies were found in one patient (2.7%) and in one control (4.2%).

Conclusion

Congolese patients with RA consult long after disease onset. Despite this delay, the majority presents without major damage and is RF, anti-CCP and SE negative. We put forward the hypothesis that besides different environmental factors there is probably also a particular genetic risk profile in Congolese patients, different from the HLA-DRB1 shared epitope.     

Endometrial biopsy: a valuable clinical and research tool in bovine reproduction

Studies of postpartum endometrial physiologic and immune mechanisms in cows are compromised by the difficulty in acquiring tissue of suitable quality and in sufficient quantity (Bos taurus). Endometrial biopsy sampling has attracted concern regarding potential animal ill-health and perturbed subsequent fertility. Here, we describe a method of endometrial biopsy that obtains high-quality tissue samples and does not compromise fertility. Using a Hauptner instrument, endometrial biopsies were taken at 15, 30, and 60 d postpartum from 13 mixed-breed beef cows. The effects of repeat biopsy on health (heart rate, respiration rate, color of mucous membranes, rectal temperature), onset of estrous cyclicity, and first service conception rate were monitored. Extensive daily clinical examinations revealed no signs of ill-health. All cows had resumed estrous cyclicity at 60 d postpartum. A conception rate of 77% was achieved after estrus synchronization and artificial insemination. Each biopsy yielded intact endometrial tissue and nucleic acid suitable for extensive histologic and molecular analysis, respectively. We conclude that when carried out appropriately, bovine endometrial biopsy is a safe and reliable technique for assessing postpartum uterine function or health.     

Dynamics of viral and proviral loads of feline immunodeficiency virus within the feline central nervous system during the acute phase following intravenous infection

Animal models of human immunodeficiency virus 1, such as feline immunodeficiency virus (FIV), provide the opportunities to dissect the mechanisms of early interactions of the virus with the central nervous system (CNS). The aims of the present study were to evaluate viral loads within CNS, cerebrospinal fluid (CSF), ocular fluid, and the plasma of cats in the first 23 weeks after intravenous inoculation with FIV(GL8). Proviral loads were also determined within peripheral blood mononuclear cells (PBMCs) and brain tissue. In this acute phase of infection, virus entered the brain in the majority of animals. Virus distribution was initially in a random fashion, with more diffuse brain involvement as infection progressed. Virus in the CSF was predictive of brain parenchymal infection. While the peak of virus production in blood coincided with proliferation within brain, more sustained production appeared to continue in brain tissue. In contrast, proviral loads in the brain decreased to undetectable levels in the presence of a strengthening PBMC load. A final observation in this study was that there was no direct correlation between viral loads in regions of brain or ocular tissue and the presence of histopathology.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Protection against cartilage and bone destruction by systemic interleukin-4 treatment in established murine type II collagen-induced arthritis

Destruction of cartilage and bone are hallmarks of human rheumatoid arthritis (RA), and controlling these erosive processes is the most challenging objective in the treatment of RA. Systemic interleukin-4 treatment of established murine collagen-induced arthritis suppressed disease activity and protected against cartilage and bone destruction. Reduced cartilage pathology was confirmed by both decreased serum cartilage oligomeric matrix protein (COMP) and histological examination. In addition, radiological analysis revealed that bone destruction was also partially prevented. Improved suppression of joint swelling was achieved when interleukin-4 treatment was combined with low-dose prednisolone treatment. Interestingly, synergistic reduction of both serum COMP and inflammatory parameters was noted when low-dose interleukin-4 was combined with prednisolone. Systemic treatment with interleukin-4 appeared to be a protective therapy for cartilage and bone in arthritis, and in combination with prednisolone at low dosages may offer an alternative therapy in RA.     

Graphical representation of a generalized linear model-based statistical test estimating the fit of the single-hit Poisson model to limiting dilution assays.

Standardized statistical and graphical methods for analysis of limiting dilution assays are highly desirable to enable investigators to compare and interpret results and conclusions with greater accuracy and precision. According to these requirements, we present in this work a powerful statistical slope test that estimates the fit of the single-hit Poisson model to limiting dilution experiments. This method is readily amenable to a graphical representation. This slope test is obtained by modeling limiting dilution data according to a linear log-log regression model, which is a generalized linear model specially designed for modeling binary data. The result of the statistical slope test can then be graphed to visualize whether the data are compatible or not with the single-hit Poisson model. We demonstrate this statistical test and its graphical representation by using two examples: a real limiting dilution experiment evaluating the growth frequency of IL-2-responsive tumor-infiltrating T cells in a malignant lymph node involved by a B cell non-Hodgkin's lymphoma, and a simulation of a limiting dilution assay corresponding to a theoretical non-single-hit Poisson model, suppressor two-target Poisson model.