TECHNICAL NOTE - Single embryoid body formation in a multi-well plate

Embryoid bodies (EB) formed from murine embryonic stem (ES) cells recapitulate many aspects of a developing embryo. Of specific importance, synchronous differentiation of EB recapitulates organ-specific development and is achieved in culture by formation of uniformly sized EB. The method described here demonstrates a simple and cost-effective way of generating EB from murine ES cells. Single EB are formed in a multi-well plate format and large numbers of EB are generated using a 96-well multi-well plate. Uniform single-sized EB formed in the multi-well are an ideal system for screening compounds and determining differentiation effects. Since EB contain all three germ layers, they are appropriate for studying small molecule effects on differentiation of ES such as is performed in high-throughput screening protocols

Human Immunodeficiency Virus-1 Uses the Mannose-6-Phosphate Receptor to Cross the Blood-Brain Barrier

HIV-1 circulates both as free virus and within immune cells, with the level of free virus being predictive of clinical course. Both forms of HIV-1 cross the blood-brain barrier (BBB) and much progress has been made in understanding the mechanisms by which infected immune cells cross the blood-brain barrier BBB. How HIV-1 as free virus crosses the BBB is less clear as brain endothelial cells are CD4 and galactosylceramide negative. Here, we found that HIV-1 can use the mannose-6 phosphate receptor (M6PR) to cross the BBB. Brain perfusion studies showed that HIV-1 crossed the BBB of all brain regions consistent with the uniform distribution of M6PR. Ultrastructural studies showed HIV-1 crossed by a transcytotic pathway consistent with transport by M6PR. An in vitro model of the BBB was used to show that transport of HIV-1 was inhibited by mannose, mannan, and mannose-6 phosphate and that enzymatic removal of high mannose oligosaccharide residues from HIV-1 reduced transport. Wheatgerm agglutinin and protamine sulfate, substances known to greatly increase transcytosis of HIV-1 across the BBB in vivo, were shown to be active in the in vitro model and to act through a mannose-dependent mechanism. Transport was also cAMP and calcium-dependent, the latter suggesting that the cation-dependent member of the M6PR family mediates HIV-1 transport across the BBB. We conclude that M6PR is an important receptor used by HIV-1 to cross the BBB

TECHNICAL NOTE - Single embryoid body formation in a multi-well plate

Embryoid bodies (EB) formed from murine embryonic stem (ES) cells recapitulate many aspects of a developing embryo. Of specific importance, synchronous differentiation of EB recapitulates organ-specific development and is achieved in culture by formation of uniformly sized EB. The method described here demonstrates a simple and cost-effective way of generating EB from murine ES cells. Single EB are formed in a multi-well plate format and large numbers of EB are generated using a 96-well multi-well plate. Uniform single-sized EB formed in the multi-well are an ideal system for screening compounds and determining differentiation effects. Since EB contain all three germ layers, they are appropriate for studying small molecule effects on differentiation of ES such as is performed in high-throughput screening protocols

The uptake of soluble and particulate antigens by epithelial cells in the mouse small intestine.

Intestinal epithelial cells (IECs) overlying the villi play a prominent role in absorption of digested nutrients and establish a barrier that separates the internal milieu from potentially harmful microbial antigens. Several mechanisms by which antigens of dietary and microbial origin enter the body have been identified; however whether IECs play a role in antigen uptake is not known. Using in vivo imaging of the mouse small intestine, we investigated whether epithelial cells (enterocytes) play an active role in the uptake (sampling) of lumen antigens. We found that small molecular weight antigens such as chicken ovalbumin, dextran, and bacterial LPS enter the lamina propria, the loose connective tissue which lies beneath the epithelium via goblet cell associated passageways. However, epithelial cells overlying the villi can internalize particulate antigens such as bacterial cell debris and inert nanoparticles (NPs), which are then found co-localizing with the CD11c+ dendritic cells in the lamina propria. The extent of NP uptake by IECs depends on their size: 20-40 nm NPs are taken up readily, while NPs larger than 100 nm are taken up mainly by the epithelial cells overlying Peyer's patches. Blocking NPs with small proteins or conjugating them with ovalbumin does not inhibit their uptake. However, the uptake of 40 nm NPs can be inhibited when they are administered with an endocytosis inhibitor (chlorpromazine). Delineating the mechanisms of antigen uptake in the gut is essential for understanding how tolerance and immunity to lumen antigens are generated, and for the development of mucosal vaccines and therapies

Hepatitis C Virus Genotype 1a Growth and Induction of Autophagy▿

We have previously reported that immortalized human hepatocytes (IHH) support the generation of infectious hepatitis C virus (HCV) genotype 1a (clone H77). In the present study, we have investigated the growth of HCV genotype 1a (clone H77) through serial passages and accompanying changes in IHH in response to infection. Eleven serial passages of HCV genotype 1a (clone H77) in IHH were completed. Virus replication was ascertained from the presence of HCV-specific sequences, the detection of core antigen, the virus genome copy number, and the virus titer in IHH culture fluid. Electron microscopy suggested that HCV infection induces autophagic vacuole formation in IHH. Fluorescence microscopy displayed localization of autophagic markers, microtubule-associated protein-1 light chain-3 and Apg5, on the vacuoles of HCV-infected hepatocytes. Taken together, our results suggested that HCV genotype 1a (clone H77) can be serially passaged in IHH and that HCV infection induces an autophagic response in hepatocytes

Generation of Infectious Hepatitis C Virus in Immortalized Human Hepatocytes

Progress in understanding hepatitis C virus (HCV) biology has remained a challenge due to the lack of an efficient cell culture system for virus growth. In this study, we examined HCV core protein-mediated immortalized human hepatocytes (IHH) for growth of HCV. In vitro-transcribed full-length RNA from HCV genotype 1a (clone H77) was introduced into IHH by electroporation. Reverse transcription-PCR of cellular RNA isolated from HCV genome-transfected IHH suggested that viral RNA replication occurred. IHH transfected with the full-length HCV genome also displayed viral protein expression by indirect immunofluorescence. In contrast, cells transfected with polymerase-defective HCV (H77/GND) RNA as a negative control did not exhibit expression of the viral genome. Immunogold labeling demonstrated localization of E1 protein in the rough endoplasmic reticulum of RNA-transfected IHH. Virus-like particles of ∼50 nm were observed in the cytoplasm. After being inoculated with culture media of cells transfected with the full-length HCV genome, naïve IHH displayed NS5a protein expression in a dilution-dependent manner, but expression of NS5a was inhibited by prior incubation of culture medium with HCV-infected patient sera. NS5a-positive immunofluorescence of cell culture media of IHH transfected with full-length H77 RNA yielded ∼4.5 × 10(4) to 1 × 10(5) focus-forming units/ml. A similar level of virus growth was observed upon transfection of RNA from HCV genotype 2a (JFH1) into IHH. Taken together, our results suggest that IHH support HCV genome replication and virus assembly

Panel A: Cleavage of high mannose oligosaccharides from HIV with endoglycosidase F1 (eF1) reduced the transport of I-HIV across BMEC.

<p>Panel B: The internal control of I-Albumin was unaffected. Mannose-6 phosphate, a ligand for and competitive inhibitor of the mannose-6 phosphate receptor, inhibited the uptake (panel C) and permeation (panel D) of I-HIV across BMECs.</p

Uptake (panels A and B) and transport (panels C and D) were inhibited by mannose (panels A and C) and by mannan (panels B and D).

<p>Uptake (panels A and B) and transport (panels C and D) were inhibited by mannose (panels A and C) and by mannan (panels B and D).</p