VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity

Cytotoxic T lymphocytes (CTLs) eliminate infected and neoplastic cells through directed release of cytotoxic granule contents. Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic. VAMP8 was posited to represent the cytotoxic granule vesicular SNARE protein mediating exocytosis in mice. In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes. Upon stimulation, these endosomes rapidly trafficked to and fused with the plasma membrane, preceding fusion of cytotoxic granules. Knockdown of VAMP8 blocked both recycling endosome and cytotoxic granule fusion at immune synapses, without affecting activating signaling. Mechanistically, VAMP8-dependent recycling endosomes deposited syntaxin-11 at immune synapses, facilitating assembly of plasma membrane SNARE complexes for cytotoxic granule fusion. Hence, cytotoxic granule exocytosis is a sequential, multivesicle fusion process requiring VAMP8-mediated recycling endosome fusion before cytotoxic granule fusion. Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated.     

Human SERPINB12 Is an Abundant Intracellular Serpin Expressed in Most Surface and Glandular Epithelia

The intracellular serine protease inhibitors (serpins) are an important family of proteins that protect cells form proteinase-mediated injury. Understanding the tissue and cellular expression pattern of this protein family can provide important insights into their physiologic roles. For example, high expression in epithelial tissues, such as lung, may suggest a biologic function in cellular defense, secretion, or selective absorption. Although the expression pattern of many of the intracellular serpins has been well described, one member of this class, SERPINB12, has not been carefully examined. We generated a mouse monoclonal antibody directed against human SERPINB12 and delineated its specificity and tissue and cell type distribution pattern through immunoblotting and immunohistochemistry, respectively. This monoclonal antibody was human specific and did not cross-react with other human intracellular serpins or mouse Serpinb12. SERPINB12 was found in nearly all the tissues investigated. In addition, this serpin was found in multiple cell types within individual tissues but primarily the epithelium. These data suggest that SERPINB12, like some other intracellular serpins, may play a vital role in barrier function by providing protection of epithelial cells.     

DEG/ENaC but not TRP channels are the major mechanoelectrical transduction channels in a C. elegans nociceptor

Many nociceptors detect mechanical cues, but the ion channels responsible for mechanotransduction in these sensory neurons remain obscure. Using in?vivo recordings and genetic dissection, we identified the DEG/ENaC protein, DEG-1, as the major mechanotransduction channel in ASH, a polymodal nociceptor in Caenorhabditis elegans. But DEG-1 is not the only mechanotransduction channel in ASH: loss of deg-1 revealed a minor current whose properties differ from those expected of DEG/ENaC channels. This current was independent of two TRPV channels expressed in ASH. Although loss of these TRPV channels inhibits behavioral responses to noxious stimuli, we found that both mechanoreceptor currents and potentials were essentially wild-type in TRPV mutants. We propose that ASH nociceptors rely on two genetically distinct mechanotransduction channels and that TRPV channels contribute to encoding and transmitting information. Because mammalian and insect nociceptors also coexpress DEG/ENaCs and TRPVs, the cellular functions elaborated here for these ion channels may be conserved.     

Neuroprotective effects of genistein in VSC4.1 motoneurons exposed to activated microglial cytokines

Pro-inflammatory cytokines released from activated microglia may be responsible for neuronal damage and resulting motor deficits associated with CNS disorders such as spinal cord injury, Parkinson’s disease, and multiple sclerosis. Estrogen (17β-estradiol) is capable of ameliorating motoneuron death following spinal cord injury, but has a number of deleterious side effects. Genistein (GEN), an estrogen receptor beta agonist and potent antioxidant, may represent an alternative to estrogen in treating neurodegenerative disorders. However, little is known about the neuroprotective effects of GEN. We therefore tested whether GEN would prevent apoptosis in cultured motoneurons following exposure to pro-inflammatory cytokines released from IFN-γ activated microglia. Exposure of ventral spinal cord 4.1 motoneurons to microglial cytokine supernatant in vitro caused significant apoptosis and reduced mitochondrial membrane potential. An increase in reactive oxygen species, intracellular Ca²⁺, calpain, caspases, cytochrome c, and the bax:bcl-2 ratio were also noted. GEN treatment reversed apoptotic death and cellular changes following cytokine exposure and was associated with increased expression of estrogen receptor β suggesting that GEN may promote neuroprotection via receptor-mediated pathways. The addition of ICI 182, 780, an estrogen receptor antagonist following GEN treatment attenuated neuroprotection, suggesting that GEN may act mainly via estrogen receptor β to protect VSC4.1 motoneurons. We conclude that GEN protects cultured ventral spinal cord 4.1 cells from inflammatory insult and thus may represent a potential beneficial therapy in the treatment of neurodegenerative disorders.     

Effects of artesunate on parasite recrudescence and dormancy in the rodent malaria model Plasmodium vinckei

Artemisinin (ART) is the recommended first line therapy for treating uncomplicated and drug-resistant Plasmodium falciparum, the most pathogenic form of malaria. However, treatment failure following ART monotherapy is not uncommon and resistance to this rapidly acting drug has been reported in the Thai-Cambodian border. Recent in vitro studies have shown that following treatment with dihydroartemisinin (DHA), the development of ring-stage parasites is arrested for up to 20 days. These arrested (i.e. dormant) rings could be responsible for the recrudescence of infection that is observed following ART monotherapy. To develop a better understanding of the stage-specific effects of ART and determine if dormancy occurs in vivo, the ART derivative artesunate (AS) was used to treat mice infected with the synchronous rodent malaria parasites P. vinckei petteri (non-lethal) and P. v. vinckei (lethal). Results show that in both the non-lethal and lethal strains, ring-stage parasites are the least susceptible to treatment with AS and that the day of treatment has more of an impact on recrudescence than the total dose administered. Additionally, 24 hrs post-treatment with AS, dormant forms similar in morphology to those seen in vitro were observed. Finally, rate of recrudescence studies suggest that there is a positive correlation between the number of dormant parasites present and when recrudescence occurs in the vertebrate host. Collectively, these data suggest that dormancy occurs in vivo and contributes to recrudescence that is observed following AS treatment. It is possible that this may represent a novel mechanism of parasite survival following treatment with AS.     

Recovery Efficiency and Limit of Detection of Aerosolized Bacillus anthracis Sterne from Environmental Surface Samples

After the 2001 anthrax incidents, surface sampling techniques for biological agents were found to be inadequately validated, especially at low surface loadings. We aerosolized Bacillus anthracis Sterne spores within a chamber to achieve very low surface loading (ca. 3, 30, and 200 CFU per 100 cm²). Steel and carpet coupons seeded in the chamber were sampled with swab (103 cm²) or wipe or vacuum (929 cm²) surface sampling methods and analyzed at three laboratories. Agar settle plates (60 cm²) were the reference for determining recovery efficiency (RE). The minimum estimated surface concentrations to achieve a 95% response rate based on probit regression were 190, 15, and 44 CFU/100 cm² for sampling steel surfaces and 40, 9.2, and 28 CFU/100 cm² for sampling carpet surfaces with swab, wipe, and vacuum methods, respectively; however, these results should be cautiously interpreted because of high observed variability. Mean REs at the highest surface loading were 5.0%, 18%, and 3.7% on steel and 12%, 23%, and 4.7% on carpet for the swab, wipe, and vacuum methods, respectively. Precision (coefficient of variation) was poor at the lower surface concentrations but improved with increasing surface concentration. The best precision was obtained with wipe samples on carpet, achieving 38% at the highest surface concentration. The wipe sampling method detected B. anthracis at lower estimated surface concentrations and had higher RE and better precision than the other methods. These results may guide investigators to more meaningfully conduct environmental sampling, quantify contamination levels, and conduct risk assessment for humans.Anthrax, the spectrum of diseases caused by infection with Bacillus anthracis, is not considered a communicable disease but is generally acquired via environmental exposures. Many anthrax cases through history have been the result of agricultural or industrial exposure to B. anthracis spores (33). The disease most often presents itself as a cutaneous infection; however, there are both gastrointestinal and inhalational forms of the disease. Inhalational anthrax is typically rapidly fatal, even with treatment. In general, inhalation exposures require specific conditions, such as poor ventilation and activities that disturb dust containing B. anthracis spores (13).Because diagnosing anthrax in its early stages in human and animal hosts is difficult and B. anthracis spores are extremely stable in the environment, this microorganism has been investigated, developed, and deployed as a biological weapon throughout the 20th century. Use of this microorganism has seen varied success during World War I (9) and subsequently. It is generally accepted that there was an accidental release of B. anthracis spores from a weapons manufacturing or development facility in 1979 in Sverdlovsk, USSR (now Yekaterinaburg, Russia) (10, 26). In 1993, an attempt by a civilian group, Aum Shinrikyo, to use this microorganism to attack a civilian population in a Tokyo suburb did not result in any casualties (22, 28).In 2001, envelopes containing a powder formulation of B. anthracis were mailed in the United States to several individuals. These letters were the presumed cause of 22 cases of clinical anthrax, 11 inhalational and 11 cutaneous, with 5 fatalities, all of whom suffered from inhalational disease (34). According to congressional testimony, the powdered spore suspension was “easily dispersed into the air” (29). Of the 11 individuals with inhalational disease, 2 had no history of handling mail or having any other direct contact with these threat letters (11, 21). Of the remaining nine individuals, eight were thought to have been exposed through handling or processing mail (20) but may never have picked up or directly handled the actual threat letters. Thus, some individuals who contracted inhalational disease may have been exposed to aerosols that were generated from residual spore material deposited on contaminated surfaces. This conclusion was borne out by a study conducted on the scene of one contamination incident, which demonstrated that spores could be reaerosolized from surfaces during simulated office activities—e.g., paper handling, foot traffic, moving containers—after a period of no entry and no ventilation for several days (38). McCleery et al. (25) found that reaerosolization of spores is possible in postal facilities.In the mail-related instance of 2001, aerosol exposures occurred. Since spore-contaminated surfaces can become sources for aerosol generation, nonporous surfaces (walls, desks, lockers, etc.) were decontaminated to reduce risk while porous surfaces (draperies and sofas) were removed. To determine the efficacy of decontamination, contaminated buildings were first sampled for the presence of B. anthracis spores followed by treatment by a variety of techniques. Postdecontamination sampling was used to determine efficacy (37) and to assess the safety for reoccupancy.The Government Accountability Office (GAO) reported that additional methodological validation of sampling collection and analytical methods should be conducted to enhance the interpretation of negative sampling results because initial samples from two postal facilities were negative, but later samples were positive (17). The GAO (17) report defined validation as “… a formal and independently administered empirical process. For validation, the overall performance characteristics of a given method must be certified as meeting the specified requirements for intended use and as conforming with applicable standards.” Currently, there is no preexisting standard for a presumable safe level of surface contamination with B. anthracis spores that may be assessed through sampling and analysis.Development of independent standards for assessing the requirements for surface sampling methods requires an understanding of the rate at which spores leave surfaces to become entrained in aerosols, the potential for aerosol exposure by humans, and the infectivity of inhaled spores. Inhalation infectivity has been researched, but estimates of a lethal dose vary (14, 15). Bartrand et al. (5) conducted a risk analysis on the mortality of guinea pigs and rhesus monkeys exposed to B. anthracis spores and found a 50% lethal dose (LD₅₀; i.e., the dose at which 50% of subjects die) of about 100,000 spores inhaled for 1-μm particles. Limitations of relating exposure to inhalation infectivity include quantification of the ability of spores to move from stasis on a surface to entrainment as an aerosol, quantification of exposures to the resultant aerosol, uptake by humans, room size and ventilation characteristics, and exposure time. Despite these limitations, it is necessary to standardize the performance of surface sampling methods.Brown et al. evaluated wipe (6), swab (7), and vacuum (8) spore collection methods with B. atrophaeus. These studies have added significant information to the understanding of recovery efficiencies for these three sampling methods; however, sampling performance was not evaluated at very low spore surface loading concentrations. Sampling performance measures at very low surface loading of B. anthracis are needed to aid in the decision making for decontamination and other interventions (31, 38).The goal of this study was to evaluate the current CDC environmental surface sampling methods for B. anthracis (12) as slightly modified based on subsequent CDC research (19, 30). We estimated B. anthracis Sterne sampling limit of detection (LOD), recovery efficiency (RE), and measurement precision for three sampling methods (swab, wipe, and vacuum) and two surfaces (steel and carpet) by allowing spores to settle from an aerosol in a controlled environment. In addition, we compared sample analyses performed at three laboratories to determine the level of interlaboratory variability.     

The glutamate transporter, GLAST, participates in a macromolecular complex that supports glutamate metabolism

GLAST is the predominant glutamate transporter in the cerebellum and contributes substantially to glutamate transport in forebrain. This astroglial glutamate transporter quickly binds and clears synaptically released glutamate and is principally responsible for ensuring that synaptic glutamate concentrations remain low. This process is associated with a significant energetic cost. Compartmentalization of GLAST with mitochondria and proteins involved in energy metabolism could provide energetic support for glutamate transport. Therefore, we performed immunoprecipitation and co-localization experiments to determine if GLAST might co-compartmentalize with proteins involved in energy metabolism. GLAST was immunoprecipitated from rat cerebellum and subunits of the Na(+)/K(+) ATPase, glycolytic enzymes, and mitochondrial proteins were detected. GLAST co-localized with mitochondria in cerebellar tissue. GLAST also co-localized with mitochondria in fine processes of astrocytes in organotypic hippocampal slice cultures. From these data, we hypothesized that mitochondria participate in a macromolecular complex with GLAST to support oxidative metabolism of transported glutamate. To determine the functional metabolic role of this complex, we measured CO(2) production from radiolabeled glutamate in cultured astrocytes and compared it to overall glutamate uptake. Within 15min, 9% of transported glutamate was converted to CO(2). This CO(2) production was blocked by inhibitors of glutamate transport and glutamate dehydrogenase, but not by an inhibitor of glutamine synthetase. Our data support a model in which GLAST exists in a macromolecular complex that allows transported glutamate to be metabolized in mitochondria to support energy production.     

Intracellular heat shock protein-70 negatively regulates TLR4 signaling in the newborn intestinal epithelium

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related mortality in premature infants, and it develops under conditions of exaggerated TLR4 signaling in the newborn intestinal epithelium. Because NEC does not develop spontaneously, despite the presence of seemingly tonic stimulation of intestinal TLR4, we hypothesized that mechanisms must exist to constrain TLR4 signaling that become diminished during NEC pathogenesis and focused on the intracellular stress response protein and chaperone heat shock protein-70 (Hsp70). We demonstrate that the induction of intracellular Hsp70 in enterocytes dramatically reduced TLR4 signaling, as assessed by LPS-induced NF-κB translocation, cytokine expression, and apoptosis. These findings were confirmed in vivo, using mice that either globally lacked Hsp70 or overexpressed Hsp70 within the intestinal epithelium. TLR4 activation itself significantly increased Hsp70 expression in enterocytes, which provided a mechanism of autoinhibition of TLR4 signaling in enterocytes. In seeking to define the mechanisms involved, intracellular Hsp70-mediated inhibition of TLR4 signaling required both its substrate-binding EEVD domain and association with the cochaperone CHIP, resulting in ubiquitination and proteasomal degradation of TLR4. The expression of Hsp70 in the intestinal epithelium was significantly decreased in murine and human NEC compared with healthy controls, suggesting that loss of Hsp70 protection from TLR4 could lead to NEC. In support of this, intestinal Hsp70 overexpression in mice and pharmacologic upregulation of Hsp70 reversed TLR4-induced cytokines and enterocyte apoptosis, as well as prevented and treated experimental NEC. Thus, a novel TLR4 regulatory pathway exists within the newborn gut involving Hsp70 that may be pharmacologically activated to limit NEC severity.