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.

Pathogenesis of Morquio A syndrome: an autopsied case reveals systemic storage disorder.

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase, which results in systemic accumulation of glycosaminoglycans (GAGs), keratan sulfate and chondroitin-6-sulfate. Accumulation of these GAGs causes characteristic features as disproportionate dwarfism associated with skeletal deformities, genu valgum, pigeon chest, joint laxity, and kyphoscoliosis. However, the pathological mechanism of systemic skeletal dysplasia and involvement of other tissues remain unanswered in the paucity of availability of an autopsied case and successive systemic analyses of multiple tissues. We report here a 20-year-old male autopsied case with MPS IVA, who developed characteristic skeletal features by the age of 1.5 years and died of acute respiratory distress syndrome five days later after occipito-C1-C2 cervical fusion. We pathohistologically analyzed postmortem tissues including trachea, lung, thyroid, humerus, aorta, heart, liver, spleen, kidney, testes, bone marrow, and lumbar vertebrae. The postmortem tissues relevant with clinical findings demonstrated 1) systemic storage materials in multiple tissues beyond cartilage, 2) severely vacuolated and ballooned chondrocytes in trachea, humerus, vertebrae, and thyroid cartilage with disorganized extracellular matrix and poor ossification, 3) appearance of foam cells and macrophages in lung, aorta, heart valves, heart muscle, trachea, visceral organs, and bone marrow, and 4) storage of chondrotin-6-sulfate in aorta. This is the first autopsied case with MPS IVA whose multiple tissues have been analyzed pathohistologically and these pathological findings should provide a new insight into pathogenesis of MPS IVA.

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.

Isolation of peptide transport system-6 from brain endothelial cells: therapeutic effects with antisense inhibition in Alzheimer and stroke models.

By isolating for the first time ever a peptide transporter from the blood-brain barrier (BBB) and developing an antisense that selectively targets the brain-to-blood efflux component, we were able to deliver a therapeutic concentration of the neurotrophic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) 27 to brain in animal models of Alzheimer's and stroke. Efflux pumps at the BBB are major causes of BBB impermeability to peptides. PACAP is neuroprotective in vitro in femtomole amounts, but brain uptake of PACAP27 is limited by an efflux component of peptide transport system-6 (PTS-6). Here, we characterized, isolated, and sequenced this component of PTS-6, identifying it as beta-F1 ATPase, and colocalized it with PACAP27 on BBB endothelial cells. Antisenses targeting the BBB inhibited PACAP27 efflux, thus increasing brain uptake of PACAP27. Treatment with antisense+PACAP27 improved cognition in a mouse model of Alzheimer's disease and reduced infarct size after cerebral ischemia. This represents the first isolation from BBB tissue of a peptide transporter and shows that inhibition of peptide efflux pumps is a potential strategy for drug delivery to brain.

PKC1 is essential for protection against both oxidative and nitrosative stresses, cell integrity, and normal manifestation of virulence factors in the pathogenic fungus Cryptococcus neoformans.

Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a "top-down" approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1Delta strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism.

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.

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

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 approximately 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 approximately 4.5 x 10(4) to 1 x 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.

Neutrophil adherence to bladder microvascular endothelial cells following platelet-activating factor acetylhydrolase inhibition.

Interstitial cystitis (IC) is an inflammatory bladder condition of unknown etiology. Tryptase released from elevated numbers of activated mast cells is a proposed mediator of the inflammatory process in IC. We have previously shown that tryptase increases human bladder microvascular endothelial cell (HBMEC) calcium-independent phospholipase A(2) (iPLA(2)) activity, resulting in the production of multiple biologically active phospholipid metabolites, including platelet-activating factor (PAF), that can mediate inflammation. Because the design of selective PLA(2) inhibitors may provide a useful therapeutic strategy to reduce the inflammatory process in IC, we tested several frequently used PLA(2) inhibitors on PAF production in tryptase-stimulated HBMEC. Among the inhibitors tested, methyl arachidonyl fluorophosphonate (MAFP) was found to be a potent inhibitor of PAF-acetylhydrolase activity. Pretreatment of HBMEC with MAFP significantly increased PAF production in both unstimulated and tryptase-stimulated cells. In addition, MAFP pretreatment of tryptase-stimulated HBMEC increased both surface expression of P-selectin and polymorphonuclear leukocyte adherence to the HBMEC monolayer. These effects suggest that MAFP has a proinflammatory effect, irrespective of its ability to inhibit PLA(2).

Human immunodeficiency virus type 1 transport across the in vitro mouse brain endothelial cell monolayer.

Human immunodeficiency virus type 1 (HIV-1) is associated with a neuroinflammatory dementia. Cognitive impairment remains a common complication of late-stage HIV-1 infection. Previous studies have shown that entry of HIV-1 into the central nervous system (CNS) occurs soon after infection. For these reasons, it is important to understand how HIV-1 crosses the BBB. We used primary mouse brain microvessel endothelial cell (MBEC) monolayer models to study interactions between brain endothelial cells and radioactively labeled HIV-1 CL4 (131I-HIV-1), which had been rendered noninfectious with aldithiol, and compared to radioactively labeled bovine serum albumin (131I-BSA or 125I-BSA) and detected HIV-1 on MBEC monolayer with electron microscopic analysis. The permeability of the monolayers to HIV-1 was measured by determining the percent material transported (PMT). Luminal to abluminal PMT of 131I-HIV-1 was 4.65 times greater than that of the much smaller 131I-BSA, showing that the MBEC monolayer is more permeable to HIV-1 than to BSA. Electron microscopy showed that HIV-1 was transported through a trans-cellular pathway from luminal side to basolateral space with some virus associated with the nucleus. Unlabeled HIV-1 did not affect the transport of 131I-HIV-1 or break down the MBEC monolayer. Wheatgerm agglutinin (WGA) increased 131I-HIV-1 penetration across the MBEC monolayer, consistent with absorptive endocytosis as the mechanism for HIV-1 penetration. The enhanced transport of HIV-1 was unidirectional, as the abluminal to luminal PMT of 131I-HIV-1 was not different from that of BSA nor enhanced by WGA. Characterization of the radioactivity transported from the luminal to abluminal chamber on Sepharose 4B-200 columns showed the transported radioactivity represented intact virus. MBEC monolayers preloaded from the luminal surface with 131I-HIV-1 showed most of the virus was retained by the endothelial cells, while the remainder was effluxed mainly to the luminal surface. MBEC monolayers preloaded from the abluminal surface with 131I-HIV-1 retained little virus and most of the virus was effluxed mainly to the abluminal surface. In conclusion, cell-free, intact 131I-HIV-1 crossed brain endothelial cell monolayers unidirectionally in the luminal to abluminal direction through an adsorptive endocytotic pathway. HIV-1 taken up from luminal side by monolayers of brain endothelial cells was mainly released to the luminal side. HIV-1 efflux mechanisms are different from influx mechanisms.

Relationship of chloroquine-induced redistribution of a neutral aminopeptidase to hemoglobin accumulation in malaria parasites.

To study the relationship between neutral aminopeptidase activity and hemoglobin accumulation in malaria parasites, we treated mice infected with Plasmodium berghei NYU-2 with chloroquine intraperitoneally in doses ranging from 0.3 to 3 micromol per 25 g mouse. Preparations of infected erythrocytes (normalized to represent 1000 parasites per 1000 erythrocytes) hydrolyzed 1200 nmol of leucine-p-nitroanilide per minute per milliliter of packed erythrocytes, which was 10x more than that of uninfected preparations. The activity in infected preparations was distinguished by resistance to ferriprotoporphyrin IX and puromycin and susceptibility to inhibition by ethanol and Tris. Chloroquine treatment caused the activity in unwashed membrane ghosts of infected preparations to decrease by 50% despite an increase in total activity. Concomitantly, hemoglobin in washed membrane ghosts increased. Electron microscopy revealed that the hemoglobin was retained in endocytic vesicles. Chloroquine-induced redistribution of a neutral aminopeptidase may be the cause of hemoglobin accumulation in endocytic vesicles of malaria parasites.

Mitochondrial DNA mutations activate the mitochondrial apoptotic pathway and cause dilated cardiomyopathy.

OBJECTIVE: To determine whether low frequency mitochondrial DNA (mtDNA) mutations are pathogenic. METHODS: We studied mice that express a proofreading-deficient mitochondrial DNA polymerase in the heart and develop cardiac mtDNA mutations. RESULTS: At 4 weeks of age, when point mutation levels had risen to on average two per mitochondrial genome, these mice developed severe dilated cardiomyopathy. Interstitial fibrosis first became apparent at 4 weeks of age and progressed with age. Sporadic myocytic death occurred in all regions of the heart, apparently due to apoptosis as assessed by histological analysis and TUNEL staining. The frequency of TUNEL-positive cells peaked at 4-5 weeks of age and then gradually declined. While mitochondrial respiratory function, ultrastructure, and number remained normal, cytochrome c was released from mitochondria, a known apoptotic signal. CONCLUSION: mtDNA mutations therefore are pathogenic, and seem to trigger apoptosis through the mitochondrial pathway.