Search

Many innate immune receptors function collaboratively to detect and elicit immune responses to pathogens, but the physical mechanisms that govern the interaction and signaling crosstalk between the receptors are unclear. In this study, we report that the signaling crosstalk between Fc gamma receptor (FcγR) and Toll-like receptor (TLR)2/1 can be overall synergistic or inhibitory depending on the spatial proximity between the receptor pair on phagosome membranes. Using a geometric manipulation strategy, we physically altered the spatial distribution of FcγR and TLR2 on single phagosomes. We demonstrate that the signaling synergy between FcγR and TLR2/1 depends on the proximity of the receptors and decreases as spatial separation between them increases. However, the inhibitory effect from FcγRIIb on TLR2-dependent signaling is always present and independent of receptor proximity. The overall cell responses are an integration from these two mechanisms. This study presents quantitative evidence that the nanoscale proximity between FcγR and TLR2 functions as a key regulatory mechanism in their signaling crosstalk.

Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40 phox by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40 phox and p67 phox at the phagosomal membrane. Pharmacologic inhibition of PI(3)P synthesis at the phagosome decreased the ROS production both in differentiated PLB-985 cells and human neutrophils. It also releases p67 phox , the key cytosolic subunit of the oxidase, and p40 phox from the phagosome. The knockdown of the PI(3)P phosphatase MTM1 or Rubicon or both increases the level of PI(3)P at the phagosome. That increase enhances ROS production inside the phagosome and triggers an extended accumulation of p67 phox at the phagosome. Furthermore, the overexpression of MTM1 at the phagosomal membrane induces the disappearance of PI(3)P from the phagosome and prevents sustained ROS production. In conclusion, PI(3)P, indeed, regulates ROS production by maintaining p40 phox and p67 phox at the phagosomal membrane., (© Society for Leukocyte Biology.)

Cover legend: Macrophages phagocytosing RFP-labeled E.coli. GFP-APPL2 labels the phagosomal membrane. Image produced by N. Condon. See Yeo et al. Traffic 2016; 17(9):1014-1026. Read the full article on doi:10.1111/tra.12415., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Neutrophils kill microorganisms by inducing exocytosis of granules with antibacterial properties. Four isoforms of the "a" subunit of V-ATPase-a1V, a2V, a3V, and a4V-have been identified. a2V is expressed in white blood cells, that is, on the surface of monocytes or activated lymphocytes. Neutrophil associated-a2V was found on membranes of primary (azurophilic) granules and less often on secondary (specific) granules, tertiary (gelatinase granules), and secretory vesicles. However, it was not found on the surface of resting neutrophils. Following stimulation of neutrophils, primary granules containing a2V as well as CD63 translocated to the surface of the cell because of exocytosis. a2V was also found on the cell surface when the neutrophils were incubated in ammonium chloride buffer (pH 7.4) a weak base. The intracellular pH (cytosol) became alkaline within 5 min after stimulation, and the pH increased from 7.2 to 7.8; this pH change correlated with intragranular acidification of the neutrophil granules. Upon translocation and exocytosis, a2V on the membrane of primary granules remained on the cell surface, but myeloperoxidase was secreted. V-ATPase may have a role in the fusion of the granule membrane with the cell surface membrane before exocytosis. These findings suggest that the granule-associated a2V isoform has a role in maintaining a pH gradient within the cell between the cytosol and granules in neutrophils and also in fusion between the surface and the granules before exocytosis. Because a2V is not found on the surface of resting neutrophils, surface a2V may be useful as a biomarker for activated neutrophils., (© Society for Leukocyte Biology.)

The dengue virus (DENV) circulates between humans and mosquitoes and requires no other mammals or birds for its maintenance in nature. The virus is well-adapted to humans, as reflected by high-level viraemia in patients. To investigate its high adaptability, the DENV induction of host type-I interferon (IFN) was assessed in vitro in human-derived HeLa cells and compared with that induced by the Japanese encephalitis virus (JEV), a closely related arbovirus that generally exhibits low viraemia in humans. A sustained viral spread with a poor IFN induction was observed in the DENV-infected cells, whereas the JEV infection resulted in a self-limiting and abortive infection with a high IFN induction. There was no difference between DENV and JEV double-stranded RNA (dsRNA) as IFN inducers. Instead, the dsRNA was poorly exposed in the cytosol as late as 48 h post-infection (p.i.), despite the high level of DENV replication in the infected cells. In contrast, the JEV-derived dsRNA appeared in the cytosol as early as 24 h p.i. Our results provided evidence for the first time in DENV, that concealing dsRNA in the intracellular membrane diminishes the effect of the host defence mechanism, a strategy that differs from an active suppression of IFN activity.

The IRG system of IFNγ-inducible GTPases constitutes a powerful resistance mechanism in mice against Toxoplasma gondii and two Chlamydia strains but not against many other bacteria and protozoa. Why only T. gondii and Chlamydia? We hypothesized that unusual features of the entry mechanisms and intracellular replicative niches of these two organisms, neither of which resembles a phagosome, might hint at a common principle. We examined another unicellular parasitic organism of mammals, member of an early-diverging group of Fungi, that bypasses the phagocytic mechanism when it enters the host cell: the microsporidian Encephalitozoon cuniculi. Consistent with the known susceptibility of IFNγ-deficient mice to E. cuniculi infection, we found that IFNγ treatment suppresses meront development and spore formation in mouse fibroblasts in vitro, and that this effect is mediated by IRG proteins. The process resembles that previously described in T. gondii and Chlamydia resistance. Effector (GKS subfamily) IRG proteins accumulate at the parasitophorous vacuole of E. cuniculi and the meronts are eliminated. The suppression of E. cuniculi growth by IFNγ is completely reversed in cells lacking regulatory (GMS subfamily) IRG proteins, cells that effectively lack all IRG function. In addition IFNγ-induced cells infected with E. cuniculi die by necrosis as previously shown for IFNγ-induced cells resisting T. gondii infection. Thus the IRG resistance system provides cell-autonomous immunity to specific parasites from three kingdoms of life: protozoa, bacteria and fungi. The phylogenetic divergence of the three organisms whose vacuoles are now known to be involved in IRG-mediated immunity and the non-phagosomal character of the vacuoles themselves strongly suggests that the IRG system is triggered not by the presence of specific parasite components but rather by absence of specific host components on the vacuolar membrane.

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.

Phagocytes such as dendritic cells (DC) and macrophages employ phagocytosis to take up pathogenic bacteria into phagosomes, digest the bacteria and present the bacteria-derived peptide antigens to the adaptive immunity. Hence, efficient antigen presentation depends greatly on a well-regulated phagocytosis process. Lipids, particularly phosphoinositides, are critical components of the phagosomes. Phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P3 ] is formed at the phagocytic cup, and as the phagosome seals off from the plasma membrane, rapid disappearance of PI(3,4,5)P3 is accompanied by high levels of phosphatidylinositol-3-phosphate (PI3P) formation. The sorting nexin (SNX) family consists of a diverse group of Phox-homology (PX) domain-containing cytoplasmic and membrane-associated proteins that are potential effectors of phosphoinositides. We hypothesized that SNX3, a small sorting nexin that contains a single PI3P lipid-binding PX domain as its only protein domain, localizes to phagosomes and regulates phagocytosis in DC. Our results show that SNX3 recruits to nascent phagosomes and silencing of SNX3 enhances phagocytic uptake of bacteria by DC. Furthermore, SNX3 competes with PI3P lipid-binding protein, early endosome antigen-1 (EEA1) recruiting to membranes. Our results indicate that SNX3 negatively regulates phagocytosis in DC possibly by modulating recruitment of essential PI3P lipid-binding proteins of the phagocytic pathways, such as EEA1, to phagosomal membranes., (© 2012 Blackwell Publishing Ltd.)

Neutrophils are pivotal to host defence during infectious diseases. However, activated neutrophils may also cause undesired tissue damage. Ample examples include small-vessel inflammatory diseases (vasculitis) that are associated with anti-neutrophil cytoplasmic autoantibodies (ANCA) residing in the patients' plasma. In addition to being an important diagnostic tool, convincing evidence shows that ANCA are pathogenic. ANCA-neutrophil interactions induce important cellular responses that result in highly inflammatory necrotizing vascular damage. The interaction begins with ANCA binding to their target antigens on primed neutrophils, proceeds by recruiting transmembrane molecules to initiate intracellular signal transduction and culminates in activation of effector functions that ultimately mediate the tissue damage., (© 2012 The Author. Clinical and Experimental Immunology © 2012 British Society for Immunology.)

Dengue virus causes ∼50-100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.

Production of ROS by the leukocyte NADPH oxidase is essential for the destruction of pathogenic bacteria inside phagosomes. The enzyme is a complex of cytosolic and membranous subunits that need to assemble upon activation. Biochemical data suggest that the complex is renewed continuously during activity. Furthermore, it is generally assumed that complex assembly and activity occur in parallel. However, information about the oxidase assembly in individual phagosomes in live cells is scarce. We studied the dynamic behavior of the crucial cytosolic NADPH oxidase component p67(phox) during phagocytosis by videomicroscopy. p67(phox) is involved in the regulation of electron flow from NADPH to oxygen, leading to superoxide radical formation inside the phagosome. p67(phox)-citrine, expressed in myeloid PLB-985 cells, accumulated at the phagosomal membrane during phagocytosis of yeast particles. Using photobleaching techniques (FRAP, FLIP), we demonstrated that p67(phox)-citrine diffused freely in this phagosomal membrane, but the phagosomal pool of p67(phox)-citrine did not exchange with the cytosolic pool. This result suggests that once assembled in the NADPH oxidase complex, p67(phox) is stable in this complex. Furthermore, the time of the presence of p67(phox)-citrine at the phagosome increased substantially in the presence of complement in the opsonizing serum compared with decomplemented serum. PI(3)P also accumulated around phagosomes for twice as long in the presence of complement. The presence of p67(phox)-citrine was correlated with the duration of phagosomal ROS production in different opsonization conditions. These data support the critical role of p67(phox) for ROS production on the level of individual phagosomes.

Cytotoxic T lymphocytes eliminate infected cells upon surface display of antigenic peptides on major histocompatibility complex I molecules. To promote immune evasion, UL49.5 of several varicelloviruses interferes with the pathway of major histocompatibility complex I antigen processing. However, the inhibition mechanism has not been elucidated yet. Within the macromolecular peptide-loading complex we identified the transporter associated with antigen processing (TAP1 and TAP2) as the prime target of UL49.5. Moreover, we determined the active oligomeric state and crucial elements of the viral factor. Remarkably, the last two residues of the cytosolic tail of UL49.5 are essential for endoplasmic reticulum (ER)-associated proteasomal degradation of TAP. However, this process strictly requires additional signaling of an upstream regulatory element in the ER lumenal domain of UL49.5. Within this new immune evasion mechanism, we show for the first time that additive elements of a small viral factor and their signaling across the ER membrane are essential for targeted degradation of a multi-subunit membrane complex.

Pemphigoid gestationis (PG) is an autoimmune sub-epidermal bullous dermatosis of pregnancy associated with circulating autoantibodies targeting the extracellular non-collagenous (NC) 16A domain of bullous pemphigoid (BP) 180 antigen. In order to determine whether BP180 regions other than NC16A are recognized by PG autoantibodies, we have analyzed the reactivity of 15 PG patient sera against several BP180 antigenic sites by sensitive methods such as immunological screening and ELISA. Most PG sera tested (13 of 15) reacted with an epitope (amino acid 508-541) mapped in the NC16A domain. Of note, nine of 15 PG patient sera reacted with at least one additional antigenic site other than NC16A. Specifically, two epitopes in the BP180 extracellular domain and five epitopes in the intracellular one were recognized by three and seven PG sera, respectively. In addition, a representative intracellular epitope was recognized by PG autoantibodies as a portion of BP180 antigen both in denaturating and native conditions. Finally, reactivity against epitopes additional to NC16A was also detected at an early stage of the disease. The identification and characterization of hitherto unrecognized epitopes targeted by PG patient autoantibodies provide novel insights into the pathophysiology of humoral immune response to BP180 in PG.

Responses of circulating hemocytes were studied in Lymnaea stagnalis exposed to 10, 30, 90, and 270 microg/L fomesafen for 24 and 504 h. Flow cytometry was used to quantify fomesafen-induced production of reactive oxygen species (ROS), phagocytic activity on Escherichia coli, and oxidative burst when hemocytes were challenged by E. coli or phorbol 12-myristate-13-acetate (PMA). Lysosomal membrane damage was assessed, using the neutral-red retention time (NRRT) assay. Exposure to fomesafen for 24 h resulted in increase in ROS levels and decreases in phagocytosis and the oxidative burst in PMA-stimulated hemocytes. After 504 h, intracellular levels of ROS returned to normal, but phagocytosis of E. coli was still inhibited and the associated oxidative burst significantly reduced. After both durations of exposure, decreases of NRRT indicated that lysosome membrane fragility increased with fomesafen concentration. Potential implications for the health and survival of the snails and consequences on populations are discussed.

Exosomes are MHC-bearing vesicles secreted by a wide array of cells. We have previously shown that donor-haplotype exosomes from bone marrow dendritic cells (DCs) injected before transplantation significantly prolong heart allograft survival in congenic and fully MHC-mismatched Lewis rats. Here we show that donor exosomes administered after transplantation are similarly able to prolong allograft survival, however, without inducing tolerance. We therefore tested the effect of exosomes combined with short-term LF 15-0195 (LF) treatment, which blocks the maturation of DCs, so that donor-MHC antigens from exosomes could be presented in a more tolerogenic environment. LF treatment does not preclude the development of a strong antidonor cellular response, and while LF, but not exosome, treatment inhibits the antidonor humoral response and decreases leukocyte graft infiltration, allografts from LF-treated recipients were either acutely or strongly chronically rejected. Interestingly, when combined with LF treatment, exosomes induced a donor-specific allograft tolerance characterized by a strong inhibition of the antidonor proliferative response. This donor-specific tolerance was transferable to naïve allograft recipients. Moreover, exosomes/LF treatment prevented or considerably delayed the appearance of chronic rejection. These results suggest that under LF treatment, presentation of donor-MHC antigens (from exosomes) can induce regulatory responses that are able to modulate allograft rejection and to induce donor-specific allograft tolerance.

Major Histocompatibility Complex (MHC) class II molecules, including Human Leukocyte Antigen (HLA)-DR, present peptide fragments from proteins degraded in the endocytic pathway. HLA-DR is targeted to late-endocytic structures named MHC class II-containing Compartments (MIIC), where it interacts with HLA-DM. This chaperone stabilizes HLA-DR during peptide exchange and is critical for successful peptide loading. To follow this process in living cells, we have generated cells containing HLA-DR3/Cyan Fluorescent Protein (CFP), HLA-DM/Yellow Fluorescent Protein (YFP), and invariant chain. HLA-DR/DM interactions were observed by Fluorescence Resonance Energy Transfer (FRET). These interactions were pH insensitive, yet occurred only in internal structures and not at the limiting membrane of MIIC. In a cellular model of infection, phagosomes formed a limiting membrane surrounding internalized Salmonella. HLA-DR and HLA-DM did not interact in Salmonella-induced vacuoles, and HLA-DR was not loaded with antigens. The absence of HLA-DR/DM interactions at the limiting membrane prevents local loading of MHC class II molecules in phagosomes. This may allow these bacteria to successfully evade the immune system.

During phagocytosis and phagosome maturation, complex membrane traffic events must be coordinated. We have observed, using fluorescent fluid-phase and membrane markers, that in the human neutrophil, internalization of nonopsonized, Gram-positive bacteria, but not of latex beads, is accompanied by a rapid and localized formation of pinosomal structures. This pinocytic response is calcium-dependent but insensitive to actin cytoskeleton disruption and wortmannin treatment. Contrary to what we observe, endosomal structures usually are considered to participate in phagosome formation by providing necessary membrane to forming phagosomes. Instead, our results show a coupling between neutrophil secretory and membrane-retrieval processes during phagosome maturation, and we suggest that the observed, localized pinocytic response is linked to the secretion of azurophilic granules toward nascent phagosomes. Accordingly, M and M-like protein-expressing Streptococcus pyogenes bacteria, which are able to survive inside neutrophil phagosomes, inhibit both the secretion of azurophilic granules to phagosomes and pinosome formation.

In late endosomes and lysosomes of antigen presenting cells major histocompatibility complex class II (MHC II) molecules bind peptides from degraded internalized pathogens. These compartments are called MHC class II compartments (MIICs), and from here peptide-loaded MHC II is transported to the cell surface for presentation to helper T-lymphocytes to generate an immune response. Recent studies from our group in mouse dendritic cells indicate that the MHC class II on internal vesicles of multivesicular late endosomes or multivesicular bodies is the main source of MHC II at the plasma membrane. We showed that dendritic cell activation triggers a back fusion mechanism whereby MHC II from the inner membranes is delivered to the multivesicular bodies' outer membrane. Another type of MIIC in B-lymphocytes and dendritic cells is more related to lysosomes and often appears as a multilaminar organelle with abundant MHC II-enriched internal membrane sheets. These multilaminar lysosomes have a functioning peptide-loading machinery, but to date it is not clear whether peptide-loaded MHC II molecules from the internal membranes can make their way to the cell surface and contribute to T cell activation. To obtain detailed information on the membrane organization of multilaminar lysosomes and investigate possible escape routes from the lumen of this organelle, we performed electron tomography on cryo-immobilized B-lymphocytes and dendritic cells. Our high-resolution 3-D reconstructions of multilaminar lysosomes indicate that their membranes are organized in such a way that MHC class II may be trapped on the inner membranes, without the possibility to escape to the cell surface.

B cell antigen receptors (BCRs) are multimeric transmembrane protein complexes comprising membrane-bound immunoglobulins (mIgs) and Ig-alpha/Ig-beta heterodimers. In most cases, transport of mIgs from the endoplasmic reticulum (ER) to the cell surface requires assembly with the Ig-alpha/Ig-beta subunits. In addition to Ig-alpha/Ig-beta, mIg molecules also bind two ER-resident membrane proteins, BAP29 and BAP31, and the chaperone heavy chain binding protein (BiP). In this article, we show that neither Ig-alpha/Ig-beta nor BAP29/BAP31 nor BiP bind simultaneously to the same mIgD molecule. Blue native PAGE revealed that only a minor fraction of intracellular mIgD is associated with high-molecular-weight BAP29/BAP31 complexes. BAP-binding to mIgs was found to correlate with ER retention of chimeric mIgD molecules. On high-level expression in Drosophila melanogaster S2 cells, mIgD molecules were detected on the cell surface in the absence of Ig-alpha/Ig-beta. This aberrant transport was prevented by coexpression of BAP29 and BAP31. Thus, BAP complexes contribute to ER retention of mIg complexes that are not bound to Ig-alpha/Ig-beta. Furthermore, the mechanism of ER retention of both BAP31 and mIgD is not through retrieval from a post-ER compartment, but true ER retention. In conclusion, BAP29 and BAP31 might be the long sought after retention proteins and/or chaperones that act on transmembrane regions of various proteins.

Bullous pemphigoid (BP) and gestational pemphigoid (PG) are subepidermal blistering disorders associated with autoantibodies directed against two components of hemidesmosomes: the BP antigen 180 (BP180) and the BP antigen 230 (BP230). Autoantibodies against the extracellular domain (ECD) of BP180 are thought to play an initiatory role in subepidermal blister formation. To characterize the targeted antigenic sites on BP180, we have assessed the reactivity of sera from BP and PG patients against eukaryotic recombinant proteins encompassing various portions of the ECD and the intracellular domain (ICD) of BP180. Twenty-two of 22 (100%) BP sera that immunoblotted BP180 in keratinocyte extracts, bound a mutant form consisting of the entire ECD of BP180, whereas only three of these 22 sera (14%) reacted against the ECD of BP180 lacking the NC16A membrane proximal region. Thirteen out of the 22 (59%) BP sera recognized the ICD of BP180. Circulating IgG from a representative BP patient that was affinity purified against the ECD of BP180 did not bind the ICD when reblotted, indicating that there was no antigenic cross-reactivity between the ECD and the ICD of BP180. Reactivity against the ICD of BP180 was further ascertained by immunofluorescence microscopy studies showing that nine of the 22 (41%) BP sera stained COS-7 cells expressing the ICD of BP180. Using deletion mutants of the ICD of BP180, the majority of the sera was found to recognize the central region of the ICD of BP180. Specifically, an immunodominant region was localized to an 87-amino acid segment located towards the NH2-terminus of BP180. In contrast to BP sera, five of six (83%) PG sera contained IgG that recognized exclusively the NC16A region, whereas none bound to the ICD of BP180. Together, the results indicate that in BP, autoantibody reactivity to BP180 is not exclusively restricted to the NC16A region, but that additional antigenic determinants exist on the ICD of BP180. The observed heterogeneous immune response against BP180 might reflect intramolecular epitope spreading. Because the ICD ofBP180 harbors functionally important regions, it is possible that autoantibodies against the ICD of BP180 have pathogenic significance for the progression of the disease.

Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis, angiogenesis, and vascular permeability. In contrast to its transient expression during the formation of new blood vessels, VEGF and its receptors are continuously and highly expressed in some adult tissues, such as the kidney glomerulus and choroid plexus. This suggests that VEGF produced by the epithelial cells of these tissues might be involved in the induction or maintenance of fenestrations in adjacent endothelial cells expressing the VEGF receptors. Here we describe a defined in vitro culture system where fenestrae formation was induced in adrenal cortex capillary endothelial cells by VEGF, but not by fibroblast growth factor. A strong induction of endothelial fenestrations was observed in cocultures of endothelial cells with choroid plexus epithelial cells, or mammary epithelial cells stably transfected with cDNAs for VEGF 120 or 164, but not with untransfected cells. These results demonstrate that, in these cocultures, VEGF is sufficient to induce fenestrations in vitro. Identical results were achieved when the epithelial cells were replaced by an epithelial-derived basal lamina-type extracellular matrix, but not with collagen alone. In this defined system, VEGF-mediated induction of fenestrae was always accompanied by an increase in the number of fused diaphragmed caveolae-like vesicles. Caveolae, but not fenestrae, were labeled with a caveolin-1-specific antibody both in vivo and in vitro. VEGF stimulation led to VEGF receptor tyrosine phosphorylation, but no change in the distribution, phosphorylation, or protein level of caveolin-1 was observed. We conclude that VEGF in the presence of a basal lamina-type extracellular matrix specifically induces fenestrations in endothelial cells. This defined in vitro system will allow further study of the signaling mechanisms involved in fenestrae formation, modification of caveolae, and vascular permeability.

Maturation of phagosomes is characterized by changes in their polypeptides, phosphorylated proteins and phospholipid composition. Kinetic analyses have shown that a variety of proteins associate and dissociate from latex-containing phagosomes at precise intervals during phagolysosome biogenesis. In an attempt to link these temporal biochemical modifications to functional changes, we have examined the in vivo fusion properties of aging endosomes and phagosomes. Using an in vivo fusion assay at the electron microscope, we measured the rate of exchange of bovine serum albumin-gold (5 and 16 nm particles) between endosomes and latex-bead-containing phagosomes. The results obtained indicate that the maturation of phagosomes is accompanied by changes of their fusion properties. Early phagosomes were shown to fuse preferentially with early endocytic organelles and to gradually acquire the ability to fuse with late endocytic organelles. Furthermore, the transfer of bovine serum albumin-gold from endosomes to phagosomes is size-dependent, a process also modulated by the maturation of these organelles, in agreement with the concept that transient fusion events occur between endosomes and phagosomes. Biochemical analysis showed variations in the levels of rab proteins associated with phagosomes during maturation while other 'fusion' proteins, including synaptobrevin1 and synaptobrevin2, remained constant.

The study was set up to determine the relationship between the human sperm acrosome reaction and fertilization in couples undergoing routine in-vitro fertilization (IVF) treatment. Prospective data analysis was carried out on all IVF patients during a 6 month period. Exceptions were those patients having insufficient sperm concentration to allow both acrosome reaction determination and insemination. The main outcome measures were the prediction of fertilization in IVF patients using flow cytometric analysis of the spontaneous and ionophore-induced acrosome reaction [giving the acrosomal response to ionophore challenge (ARIC) score] in the male partner's spermatozoa versus standard analytical methods of sperm motion parameters and morphology. Stepwise logistic regression indicated only two independent factors predictive of fertilization: ARIC score (chi 2 = 109.6, P < 0.0001) and post-Percoll % motility (chi 2 = 8.8, P < 0.003). Of patients with an ARIC score of > 10, 92% had > 30% of oocytes fertilized; 100% of patients with an ARIC score of < 10 had < 30% fertilization of oocytes. The sensitivity and specificity of the assay system were 1.00 and 0.82 respectively. The results would indicate that the ARIC test as measured by flow cytometric analysis of CD46 binding is a sensitive and specific assay for use in the prediction of fertilization in IVF patients, thus enabling direct channelling of those patients with ARIC scores of < 10 into the more invasive micro-assisted fertilization schemes.

Polyclonal antibodies were generated against a 9-amino acid, synthetic peptide corresponding to the selectivity filter in the pore region of K(+)-channel proteins. The sequence of amino acids in the ion-conducting pore region of K+ channels is the only highly conserved region of members of this protein family. The objectives of the present work were (i) to determine whether the anti-channel pore peptide antibody was immunoreactive with known K(+)-channel proteins and (ii) to demonstrate the usefulness of the antibody by employing it to identify a newly discovered K(+)-channel protein. Anti-channel pore peptide was immunoreactive with various K(+)-channel subtypes native to a number of different species. Immunoblot analysis demonstrated affinity of the antibody for the drk1, maxi-K, and KAT1 K(+)-channel proteins. Studies also suggested that the anti-channel pore peptide antibody did not immunoreact with membrane proteins other than K+ channels. The anti-channel pore peptide antibody was used to establish the identity of a 62-kDa chloroplast inner envelope polypeptide as a putative component of a K(+)-channel protein. It was concluded that an antibody generated against the conserved pore region/selectivity filter of K+ channels has broad but selective affinity for this class of proteins. This K(+)-channel probe may be a useful tool for identification of K(+)-channel proteins in native membranes.

The content of alkaline phosphatase (ALP) was determined in neutrophils isolated from patients with acute bacterial infections by a standard enzyme assay. Compared with control cells, patient cells exhibited about a fivefold increase in ALP activity. There was no difference between the ALP Km values of control and patient cells, which indicates that the elevated activity in patient cells was due to the presence of increased amounts of the enzyme. The ALP isozyme in both cell types was determined to be the tissue-unspecific ALP. The fact that much of the ALP activity was measurable only in the presence of detergent suggested that the enzyme was localized in the secretory vesicles, a putative reservoir of plasma membrane components. The amount and subcellular distribution of two other secretory vesicle membrane proteins, i.e., cytochrome b and complement receptor 3, were not altered; hence, we conclude that there was no general increase in amounts of secretory vesicle membrane constituents in the patient cells.

The aim of this study was to examine tissue from patients with breast carcinoma or benign breast disease for the presence of monoclonal-antibody-defined antigens, including the MUC1 mucin and carcinoembryonic antigen CEA. The tests were performed by sodium dodecyl sulphate/polyacrylamide gel electrophoretic separation of proteins, electrophoretic transfer to nitrocellulose membranes and immunostaining with the monoclonal antibodies. Some of the antigens identified are known to circulate at high levels in some but not necessarily all, breast carcinoma patients. Serum from a panel of ten breast cancer patients was subjected to a fractionation procedure designed to release antigen from immune complexes, and again these samples were analysed for the presence of monoclonal-antibody-defined antigens. A high frequency of positive reactions was detected by the anti-MUC1 monoclonal antibody C595 with both breast carcinoma subcellular membrane fractions as well as antigen fractions eluted from circulating immune complexes. No reactions were observed with equivalent materials from benign breast disease samples. The findings illustrate the variability in antigen expression between breast tumours. The data also indicate that a proportion of patients respond to their tumour by the production of antibodies that recognise the MUC1 antigen in their circulation.

CD9 (p24) and PECAM1 (CD31) antigens are well-defined components of the platelet plasma membrane. Both are integral glycoproteins (GPs) implicated in the adhesive and aggregative properties of human platelets. In the present report, we have investigated their subcellular localization using immunoelectron microscopy. The monospecificity of the two polyclonal antibodies used was confirmed by immunoblotting. On normal resting platelets, immunolabeling for CD9 and PECAM1 was found lining the plasma membrane and the luminal face of the open canalicular system. Some labeling was also consistently found on the alpha-granule limiting membrane. This was confirmed by double labeling experiments in which fibrinogen and von Willebrand factor (vWF) were used as alpha-granule markers. CD9 and PECAM-1 were found lining the membrane of the same granules that contained fibrinogen and vWF in their matrix. CD9 and PECAM-1 thus appear to have an intracellular distribution identical to GPIIb-IIIa, a major aggregation platelet receptor. To rule out a cross-reactivity of the two polyclonal antibodies with GPIIb/IIIa, we studied PECAM1 and CD9 expression on the platelets from a patient with type I Glanzmann's thrombasthenia whose platelets are devoid of GPIIb/IIIa. The same pattern of labeling was observed for both antigens as for normal platelets. Normal platelets were further observed after stimulation by agonists that either fail to induce (ADP) or induce granule secretion (thrombin). After treatment with ADP, platelets changed shape and centralized their granules; the plasma membrane immunolabeling remained unchanged; and gold particles were still found decorating the periphery of the centralized alpha-granules. After thrombin treatment, alpha-granules fused with the platelet membrane and secretion occurred. A significant increase of labeling was then observed on the platelet surface. From these results we conclude that the alpha-granule membrane contains two additional receptors in common with the plasma membrane. This suggests that alpha-granule membrane receptors may originate from a dual mechanism: direct targeting from the Golgi complex in megakaryocytes (for alpha-granule-specific receptors such as P-selectin) or by endocytosis from the plasma membrane (for proteins distributed in the two compartments).

TGN38/41 is an integral membrane protein predominantly located in the trans Golgi network (TGN) of rat (NRK) cells. We have used a cDNA expression system to map the epitopes recognised by a panel of antibodies raised to TGN38/41 as a preliminary step in the accurate identification of the region(s) of the molecule responsible for its correct intracellular location. These studies have confirmed the predicted topology of the molecule, and have identified a region in the cytoplasmic domain which is immunologically (and hence potentially functionally) conserved between species.

We have generated a monoclonal antibody, designated mAb 3G2, which reacts with a subsarcolemmal component of the neuromuscular junction in adult rats. mAb 3G2 immunoreactivity lies beneath and between the ACh receptor-rich synaptic gutters, around the sole plate nuclei, and at/near sarcomeric Z-disks in the vicinity of the synapse. Localization of mAb 3G2 immunoreactivity to neuromuscular junctions begins postnatally and gradually increases to adult levels. The establishment of this synaptic localization is neurally regulated, as neonatal denervation prevents its occurrence. In adults, denervation results in a loss of synaptic immunoreactivity that returns upon reinnervation. The antigen is also found at the myotendinous junction; its localization here is innervation independent. mAb 3G2 recognizes a 41 kDa protein on immunoblots of extracts of newborn muscle. Based on its distribution within muscle fibers, its developmental and neural regulation, and its molecular weight, the protein recognized by mAb 3G2 can be distinguished from other known postsynaptic proteins. Its neural dependence and developmental regulation suggest that it may participate in synaptic stabilization, perhaps as the intracellular component in a chain of proteins that serve to tether the nerve terminal to the perijunctional region of the muscle fiber.

A membrane fraction from rat distal colon contained both ouabain-sensitive and -insensitive K(+)-ATPase activities, which were measured under Na(+)-free conditions. About 38% of the ouabain-insensitive K(+)-ATPase activity was inhibited by vanadate. It was determined whether the ouabain-insensitive, vanadate-sensitive K(+)-ATPase in the colon is similar or identical to gastric H+,K(+)-ATPase. This colonic K(+)-ATPase activity was inhibited completely by monoclonal antibody HK4001, which inhibits the hog gastric H+,K(+)-ATPase activity but not Na+,K(+)-ATPase or Ca(2+)-ATPase. The colonic ATPase activity was inhibited partly by SCH 28080, which is a specific reversible inhibitor of gastric H+,K(+)-ATPase. The colonic ATPase activity was stimulated by low concentrations of K+ (its half-maximal stimulating concentration was 1 mM) and inhibited by high concentrations of K+ (its half-maximal inhibiting concentration was 10 mM), indicating that high and low K+ affinity sites are present in the colonic enzyme as in gastric H+,K(+)-ATPase and that this enzyme is not fully operative under normal physiological conditions. Two other monoclonal antibodies, which inhibit the gastric H+,K(+)-ATPase activity, did not inhibit the colonic K(+)-ATPase activity. The present results suggest that the colonic ouabain-insensitive K(+)-ATPase is partly similar but not identical to the gastric H+,K(+)-ATPase.

Binding of anti-phospholipid antibodies to circulating platelets and its consequences on platelet activation and aggregation was investigated in 11 patients with anti-phospholipid antibodies. Seven patients had mild thrombocytopenia. Nine healthy donors served as controls. Binding to platelets was investigated by performing enzyme-linked immunosorbent assays (ELISAs) with phospholipids as antigen on platelet eluates. Platelet activation was measured by flow cytofluorometry using monoclonal antibodies to an activation-specific lysosomal membrane protein. Findings in ELISA were compared with results of a conventional immunofluorescence method to detect platelet autoantibodies. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. In all thrombocytopenic patients and controls the platelets were not activated and aggregation was not impaired. There was a positive concordance of 50% between the results of immunofluorescence and ELISA. No apparent relation was found between the results of ELISA or immunofluorescence and platelet counts. It is concluded that anti-phospholipid antibodies can bind to circulating platelets. This binding is not associated with measurable aggregation abnormalities nor with platelet activation characterized by exposure of lysosomal membrane proteins. More studies are necessary to determine the exact role of anti-phospholipid antibodies in the pathogenesis of thrombocytopenia and thrombosis.

T LYMPHOCYTES reactive to pancreatic beta-cells are thought to have a central role in the autoimmune process leading to type 1 (insulin-dependent) diabetes, but the molecular targets of these T cells have not yet been defined. As identification of such antigens may enable measures to be developed to prevent the disease, we have characterized an antigen that is recognized by insulinoma membrane-reactive T-cell clones established from a newly diagnosed type-1 diabetes patient. Subcellular fractionation studies using rat insulinoma indicate that the antigenic determinant recognized by one of these clones is an integral membrane component of the insulin secretory granule. After a 5,000-fold purification, we have defined the antigen as a monomer of relative molecular mass 38,000. As granular membrane proteins are transiently exposed on the cell surface during exocytosis, their accessibility to components of the immune system may be a function of the secretory activity of beta-cells.

Autophagic vacuoles form within 15 min of perfusing a liver with amino acid-depleted medium. These vacuoles are bound by a "smooth" double membrane and do not contain acid phosphatase activity. In an attempt to identify the membrane source of these vacuoles, I have used morphological techniques combined with immunological probes to localize specific membrane antigens to the limiting membranes of newly formed or nascent autophagic vacuoles. Antibodies to three integral membrane proteins of the plasma membrane (CE9, HA4, and epidermal growth factor receptor) and one of the Golgi apparatus (sialyltransferase) did not label these vacuoles. Internalized epidermal growth factor and its membrane receptor were not found in nascent autophagic vacuoles but were present in lysosome-like degradative autophagic vacuoles. All these results suggested that autophagic vacuoles were not formed from plasma membrane, Golgi apparatus, or endosome constituents. Antisera prepared against integral membrane proteins (14, 25, and 40 kD) of the RER was found to label the inner and outer limiting membranes of almost all nascent autophagic vacuoles. In addition, ribophorin II was identified at the limiting membranes of many nascent autophagic vacuoles. Finally, secretory proteins, rat serum albumin and alpha 2u-globulin, were localized to the lumen of the RER and to the intramembrane space between the inner and outer membranes of some of these vacuoles. The results were consistent with the formation of autophagic vacuoles from ribosome-free regions of the RER.

Many studies have established the central involvement of the Golgi apparatus in the transport and processing of plasma membrane, lysosomal, and secreted proteins. The Golgi apparatus of neurons is also involved in the axoplasmic flow of fast-moving macromolecules and in the orthograde, retrograde, and transsynaptic transport of exogenous ligands. Markers of the Golgi apparatus, based on traditional methods of enzyme cytochemistry, are not applicable to human tissues obtained at autopsy. For that reason, the Golgi apparatus of brain cells has not been examined adequately in diseases of the human nervous system. Here we report that an antiserum raised against MG-160, a 160-kDa sialoglycoprotein of medial cisternae of the Golgi apparatus of several rat cells, is a specific and easily reproducible immunocytochemical marker of the Golgi apparatus of human neurons and other cells obtained at autopsy. Application of this probe in amyotrophic lateral sclerosis has shown a fragmentation of the Golgi apparatus in motor neurons similar to that induced by depolymerization of microtubules. We suggest that the fragmentation of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis has functional implications because significant reductions of secretion of insulin and immunoglobulins have been observed in islet cells and plasma cells, respectively, treated with microtubule-disrupting agents.

Urinary mucins which express determinants for the anti-breast carcinoma monoclonal antibody, NCRC-11 (IgM), closely resemble the mammary mucins found in milk fat globules and carcinomas. An IgG3 monoclonal antibody, C595, was prepared against urinary mucins isolated on a NCRC-11 antibody affinity column, and this 'second generation' antibody was shown to have a very similar pattern of reactivity to the original NCRC-11 antibody. By immunohistology, the profile of reactivity of both antibodies with tumour and normal tissue specimens was virtually identical. Both antibodies reacted with epithelial mucins isolated from breast tumours or normal urine using an NCRC-11 antibody affinity column, although the antibodies were unreactive with other antigen preparations. Heterologous immunoradiometric assays ('sandwich' tests) confirmed that NCRC-11 and C595 epitopes were co-expressed on the same molecule. C595 antibodies inhibited the binding of radiolabelled NCRC-11 antibodies to antigen, suggesting that the two epitopes were in close topographical proximity. The protein core of the mammary mucins has recently been shown to consist predominantly of a repeated 20 amino acid sequence (Gendler et al., 1988). Peptides with this complete sequence and small fragments were synthesised, and the C595 antibody was found to recognise an epitope within this repeat. The ability to identify and synthesise monoclonal antibody-defined determinants, as well as those in the adjacent or overlapping sequences within the protein core of epithelial mucins, is viewed as a strategy for facilitating the production of antibodies of new and novel specificity to complement the panels of existing anti-breast cancer reagents.

Immunoblotting of platelets that have been subjected to SDS-PAGE has revealed that sera from normal individuals contain IgG which binds to many platelet components. This binding was seen with autologous and heterologous platelets using serum of males and of nulliparous females who had not received blood transfusions. Although binding patterns of different sera were not identical, almost all sera caused IgG binding to platelet components of 87-90 kD, 140 kD (identified as vinculin) and 220-240 kD (tentatively identified as talin and actin-binding protein). Purified IgG showed the same binding pattern as whole serum and F(ab')2 fragments retained their ability to bind to many components. The titre of IgG binding in serum was 1:50-1600 while that of alloantibodies to the PlA1 antigen was 1:3200. IgG binding components were not secreted when platelets were stimulated and were rarely associated with isolated membranes, but were located either in platelet cytoplasm or cytoskeletons. IgG binding was decreased by absorbing sera with lysed platelets or isolated cytoskeletons, but only slightly with intact platelets. Microaffinity purification of IgG which formed a major band on immunoblots showed that it was antibody with specificity for vinculin or its degradation products. These findings suggest that normal sera contain naturally occurring IgG antibodies with specificity for intracellular platelet antigens and that in some cases their titre approaches that of antibodies of pathological significance.

Recent papers have reported that there are at least two distinct binding sites of bullous pemphigoid (BP) antibodies; one is the extracellular (lamina lucida) BP antigen (E-BPA) and the other is the intracellular (basal cell cytomembrane-hemidesmosome-cytoskeleton) BP antigen (I-BPA). In attempting to characterize the properties of these two BP antigens (BPA), we have investigated the expression of E-BPA and I-BPA as well as other basement membrane zone (BMZ) components such as type IV collagen, laminin, and epidermolysis bullosa acquisita (EBA) antigen at the dermal-epibolic junction as a model system for BMZ neogenesis. I-BPA was found along the dermal-epidermal junction and throughout the whole length of the dermal-epibolic junction. However, E-BPA and other BMZ components were found along the dermal-epidermal junction and along only the most proximal portion of the dermal-epibolic junction. These results suggest 1) that emergence of E-BPA varies from that of I-BPA in such a model system of BMZ neogenesis, and 2) that the emerged E-BPA may attach to the newly formed basement membrane complex.

Neutrophil-specific alloantibodies and the antigens they recognize are important in clinical medicine, but little is known about the structure of these antigens. Alloimmunization to the antigen NB1 is a clinically important cause of neonatal neutropenia and febrile transfusion reactions. To study the immunochemistry of the NB1 antigen, we prepared neutrophil plasma membranes and granules by nitrogen cavitation and differential centrifugation and then analyzed them by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with alloantibodies to several neutrophil-specific antigens. Two different antisera to the neutrophil-specific antigen NB1 identified an approximately 55-Kd protein by immunoblotting on neutrophil membranes from four NB1-positive donors but not on neutrophil membranes from five NB1-negative donors. Four anti-NB1 antisera immunoprecipitated a 58- to 64-Kd protein from extracts of NB1-positive neutrophils surface-labeled with 125I using lactoperoxidase, but not from similarly treated NB1-negative neutrophils. Normal human serum did not immunoprecipitate or immunoblot any proteins from these same neutrophil preparations. The NB1 antigen was detected by immunoblotting in secondary granules but was not found in primary granules. The electrophoretic mobility of the antigen was decreased slightly by reduction, suggesting that intrachain disulfide bonds were present. After reduction, the antigen could no longer be recognized by anti-NB1 antisera, but treatment of the antigen with periodate had no effect on the ability of anti-NB1 antisera to recognize the antigen, suggesting that it is not a carbohydrate. The data suggest that the neutrophil-specific antigen NB1 is present on a 58- to 64-Kd surface glycoprotein that is also present in secondary granules, and that the NB1 epitope is not a carbohydrate but probably resides in the tertiary structure of the protein backbone.

The authors have previously shown that human neutrophils can be permeabilized with the cholesterol-complexing agent digitonin and that these cells can be induced to secrete granule contents by increasing free Ca2+ concentrations. In the studies reported here, the authors wished to determine whether secretion of granule constituents correlated with the appearance of an immunologic marker for granule membranes on the surface of the permeabilized neutrophils. For this purpose, we used flow cytometry and two fluorometrically identifiable markers, Mo1 (a granule membrane marker) and beta 2-microglobulin (beta 2m) (a plasma membrane marker). It was found that the ratio of Mo1/beta 2m increased for permeabilized neutrophils which were exposed to micromolar concentrations of free Ca2+. This increase in the detectable surface concentration of Mo1 was accompanied by the release of lysozyme, vitamin B12 binding protein, and beta-glucuronidase into the medium. Statistical analysis showed a very strong correlation between granule secretion and the Mo1/beta 2m ratio. These data thus suggest that granule membrane components were being introduced into the plasma membrane during Ca2+-induced granule discharge; this in turn suggests that secretion by permeabilized neutrophils represents true degranulation.

Glutamate dehydrogenase (GDH) was localized in rat liver by indirect electron microscopic immunogold, using different sizes of gold particles and monoclonal and polyclonal antibodies. Using the protein A-gold technique in double immunocytochemical experiments, both antibodies, at their optimal dilutions, gave similar results. A novel assessment of the distribution of GDH was made by measurements of the number of gold particles per square micrometer of cross-sectional images of individual mitochondria. The data indicate intracellular homogeneity among mitochondria in individual parenchymal cells. The enzyme is almost absent in non-parenchymal cells. Finally, GDH was found mainly in association with the mitochondrial inner membrane.

The Epstein-Barr virus (EBV)-associated membrane antigen polypeptides (350,000, 220,000, 140,000, and 85,000 daltons) are recognized by a rabbit anti-EBV serum and are present on the plasma membranes of producer cell lines, as we demonstrated previously. In this report, we show that these polypeptides are present on intact virus particles. Subcellular fractionation revealed that these antigens are distributed throughout the cell, except for the 85,000-dalton protein, which was poorly represented in the nuclear fraction. In addition, an EBV-associated protein of 160,000 daltons, which comigrates with a major component of the viral capsid, was detected in the cytoplasmic and nuclear fractions. The immunoprecipitation patterns of 13 different EBV isolates were similar, with two exceptions. First, the 350,000- and 220,000-dalton polypeptides from marmoset cell lines had slightly larger molecular sizes than the corresponding polypeptides from human cell lines. Second, B95-8 virus and B95-8-derived human and marmoset cell lines contained little of the 220,000-dalton protein; however, 883L, the human parent line of B95-8, has a normal amount of the 220,000-dalton protein. Thus, the B95-8 strain of EBV appears to be a structurally defective variant. We have not observed any variation in protein patterns associated with different EBV disease states. The 350,000-, 220,000-, and 85,000-dalton polypeptides were shown to be glycoproteins by incorporation of [3H]mannose and [3H]glucosamine and to contain N-asparagine-linked glycosyl groups by their sensitivity to tunicamycin. To simplify future work, the following nomenclature for these EBV-associated polypeptides is suggested: 350,000 (gp350), 220,000 (gp220), 160,000 (p160), 140,000 (p140), and 85,000 (gp85).

Antisera to non-enzymatically released oral epithelial cells were produced in rabbits. Reactivity with gingival sections, by immunofluorescence, was observed for intercellular (IC) and basement membrane (BM) zones. Both IC and BM reactivity could be blocked with Concanavalin A (500 and 1000 micrograms/ml respectively) and anti-human fibronectin serum. Pretreatment with pemphigus serum blocked IC reactivity only. Pemphigus antigen may reside in the material present between coherent epithelial cells and be absent from enzymatically dispersed cells.

The localization of Plasmodium brasilianum-derived antigens in short and long clefts within the cytoplasm of infected erythrocytes and in association with knobs of the host cell membrane was demonstrated by immunoelectron microscopy with monoclonal antibodies. Our results document that malaria-induced short and long clefts, previously distinguishable only by morphology, differ also in antigenic composition. Another parasite-derived antigen was found to be associated with the parasitophorous vacuole space in schizonts. In segmenters, this antigen was present in large amounts between merozoites and in the cytoplasm of infected cells. These antigens were characterized by biosynthetic labeling and gel electrophoresis.

Chlamydial inclusions were demonstrated by indirect immunofluorescence (IF) with antiserum to the chlamydial group antigen when McCoy cell monolayers infected with either Chlamydia trachomatis or Chlamydia psittaci were fixed in formaldehyde or paraformaldehyde, provided the monolayer was not allowed to dry. If these monolayers were then air dried and restained by IF with the same antiserum but with a different fluorescence conjugate, group antigen associated with inclusion-containing McCoy cells but independent of the inclusions was revealed. This antigen was not restricted to infected cells but appeared to radiate out from them, suggesting that group antigen was released from infected cells. Similar host cell-associated antigen could be shown by IF of glutaraldehyde-fixed, air-dried monolayers, but inclusions could not be stained by IF before these preparations were dried, presumably because antibody could not penetrate glutaraldehyde-fixed cells. Electron microscopic immunoperoxidase studies of paraformaldehyde-fixed, wet monolayers located group antigen within inclusions on the outer membrane of chlamydial organisms and on single-membrane vesicles. However, when dried monolayers were labeled with the same immunoperoxidase technique, no intracellular labeling occurred, but dense staining was seen at the surface of infected cells and on adjacent membranous material. These observations are compatible with the postulate that replicating chlamydiae produce outer membrane blebs containing group antigen, which are excreted by the host cells during the chlamydial developmental cycle.

Monoclonal antibodies, prepared against rat apoB, were used to examine apoB structure in serum lipoproteins and characterize the forms and localization of apoB in liver membrane fractions and cultured hepatocytes. Of the several antibodies obtained, four, having separate epitopes, were characterized. Western blot analysis showed that three (DB11, F4, and LB14) antibodies recognized both apoBL and apoBS. One antibody (HB41) recognized only apoBL. This antibody showed unusual properties. Competition ELISA assays showed that the epitope recognized by HB41 was more effectively expressed on low density lipoproteins (LDL) compared to very low density lipoproteins (VLDL). In addition, treatment of lipoproteins with detergents and sulfhydryl reducing agents also increased the expression of the HB41 epitope. Since HB41 has been found to inhibit LDL binding to hepatocyte receptors, these data indicate that the HB41 epitope is located on the carboxy-terminal side of the apoBS junction (probably within the LDL receptor binding domain). Western blotting hepatic microsomal subfractions showed that in the rough and smooth microsomes, HB41 recognized only apoBL, while in the Golgi it recognized both apoBL and a protein having a molecular weight slightly smaller. In contrast, Western blotting with a polyclonal antibody known to recognize both apoBL and apoBS showed that, in rough and smooth microsomes, proteins in addition to apoBL and apoBS having molecular weights between 120,000 and 30,000 were recognized. These proteins, likely to be proteolytic fragments of apoB, were barely detectable in the Golgi. Additional biosynthetic studies show that the [35S]methionine-labeled proteins smaller than apoB were immunoprecipitated from the rough microsome subfraction. Pulse-chase experiments show that these are produced with the same kinetics as full-size apoBL and apoBS, indicating that they are not incomplete nascent chains. Finally, immunofluorescence microscopy was used to determine the localization of monoclonal epitopes. ApoB monoclonal antibodies that recognized exclusively apoBL (HB41) and apoBL and apoBS (DB11) produced an immunofluorescence pattern characteristic of the endoplasmic reticulum, but not the Golgi. These data suggest that, in cultured rat hepatocytes, the majority of both molecular weight forms of apoB are localized in the endoplasmic reticulum, the initial site of VLDL assembly. The additional finding that proteolytic fragments of apoB are enriched in the microsomal fraction suggests that if the proteolysis occurs during subcellular fractionation, immature apoB is susceptible to proteolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Circulating antibodies reacting specifically with the adenine nucleotide translocator from liver mitochondria were detected in sera from 12 patients with proven primary biliary cirrhosis (PBC) by a solid phase double antibody immunoradiometric assay (IRMA). Furthermore these antibodies were absorbed with the isolated adenine nucleotide translocator from liver mitochondria. None of the sera from 20 normal individuals, four patients with anti-mitochondrial positive pseudolupus syndrome (PLE) sera (M-3) and three patients with syphilis (anti-M-l) had antibodies directed against this protein from inner mitochondrial membrane. The adenine nucleotide translocator as antigen in PBC could clearly be distinguished from the ATPase associated PBC specific M-2 antigen. With the present study, for the first time, a well characterized protein from inner mitochondrial membrane has been clearly defined as an autoantigen in primary biliary cirrhosis.

Evidence has been presented that the PH-20 protein functions in sperm adhesion to the egg zona pellucida (Primakoff, P., H. Hyatt, and D. G. Myles, 1985, J. Cell Biol., 101:2239-2244). The PH-20 protein migrates from its original surface domain to a new surface domain after the acrosome reaction (Myles, D. G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). The acrosome reaction is an exocytotic event that results in insertion of a region of the secretory granule membrane, the inner acrosomal membrane (IAM), into the plasma membrane. After the acrosome reaction, PH-20 protein migrates to the IAM from its initial domain on the posterior head surface. We have now found a new dynamic feature of the regulation of PH-20 protein on the sperm surface; exocytosis increases the surface expression of PH-20 protein. After the acrosome reaction there is an approximately threefold increase in the number of PH-20 antigenic sites on the sperm surface. These new antigenic sites are revealed on the surface by insertion of the IAM into the plasma membrane. Our evidence indicates that before the acrosome reaction an intracellular population of PH-20 antigen is localized to the IAM. When migration of the surface population of the PH-20 protein is prevented, PH-20 protein can still be detected on the IAM of acrosome-reacted sperm. Also, PH-20 protein can be detected on the IAM of permeabilized acrosome-intact sperm by indirect immunofluorescence. Thus, the sperm cell regulates the amount of PH-20 protein on its surface by sequestering about two-thirds of the protein on an intracellular membrane and subsequently exposing this population on the cell surface by an exocytotic event. This may be a general mechanism for regulating cell surface composition where a rapid increase in the amount of a cell surface protein is required.

Using nuclear envelopes from Chinese hamster ovary (CHO) cells as an antigen, a mouse monoclonal antibody (IgM; designated mAb CHON211) that specifically binds to components of the nuclear pore complex (nucleoporins) was isolated. Immunofluorescence localization of the antigen recognized by mAb CHON211 revealed a punctate pattern restricted to the nuclear envelope; this pattern changed dramatically during the cell cycle. When examined by electron microscopy, the antigen was largely restricted to the nucleoplasmic and cytoplasmic faces of the nuclear pore complex. Immunoblots showed that mAb CHON211 bound to a series of polypeptides enriched in the nuclear envelope fraction with apparent molecular masses ranging from 110 to 35 kDa. Using galactosyltransferase and the lectin wheat germ agglutinin as probes, we have previously shown that proteins bearing O-linked N-acetylglucosamine (GlcNAc) are restricted to the cytoplasmic and nucleoplasmic faces of the nuclear pore complex. The nuclear membrane proteins recognized by mAb CHON211 had properties very similar to those identified by galactosyltransferase and wheat germ agglutinin labeling. Removal of O-linked GlcNAc residues with beta-hexosaminidase greatly reduced the binding of mAb CHON211, strongly suggesting that O-linked GlcNAc moieties are part of the immunodeterminant. This monoclonal antibody defines a new family of antigens that are restricted to the nuclear pore and bear a common modification: O-linked GlcNAc. mAb CHON211 will be useful for defining the role of the nucleoporins in nucleo-cytoplasmic exchange.