Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion.

Candida albicans is both a commensal and an opportunistic fungal pathogen. Invading hyphae of C. albicans secrete candidalysin, a pore-forming peptide toxin. To prevent cell death, epithelial cells must protect themselves from direct damage induced by candidalysin and by the mechanical forces exerted by expanding hyphae. We identify two key Ca2+-dependent repair mechanisms employed by epithelial cells to withstand candidalysin-producing hyphae. Using camelid nanobodies, we demonstrate candidalysin secretion directly into the invasion pockets induced by elongating C. albicans hyphae. The toxin induces oscillatory increases in cytosolic [Ca2+], which cause hydrolysis of PtdIns(4,5)P2 and loss of cortical actin. Epithelial cells dispose of damaged membrane regions containing candidalysin by an Alg-2/Alix/ESCRT-III-dependent blebbing process. At later stages, plasmalemmal tears induced mechanically by invading hyphae are repaired by exocytic insertion of lysosomal membranes. These two repair mechanisms maintain epithelial integrity and prevent mucosal damage during both commensal growth and infection by C. albicans.

Viral control in chronic HIV-1 subtype C infection is associated with enrichment of p24 IgG1 with Fc effector activity.

OBJECTIVE: Postinfection HIV viral control and immune correlates analysis of the RV144 vaccine trial indicate a potentially critical role for Fc receptor-mediated antibody functions. However, the influence of functional antibodies in clade C infection is largely unknown. DESIGN: Plasma samples from 361 chronic subtype C-infected, antiretroviral therapy-naive participants were tested for their HIV-specific isotype and subclass distributions, along with their Fc receptor-mediated functional potential. METHOD: Total IgG, IgG subclasses and IgA binding to p24 clade B/C and gp120 consensus C proteins were assayed by multiplex. Antibody-dependent uptake of antigen-coated beads and Fc receptor-mediated natural killer cell degranulation were evaluated as surrogates for antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), respectively. RESULTS: p24 IgG1 was the only subclass associated with viral control (P = 0.01), with higher p24-specific ADCP and ADCC responses detected in individuals with high p24 IgG1. Although p24 IgG1 levels were enriched in patients with elevated Gag-specific T-cell responses, these levels remained an independent predictor of low-viral loads (P = 0.04) and high CD4+ cell counts (P = 0.004) after adjusting for Gag-specific T-cell responses and for protective HLA class I alleles. CONCLUSION: p24 IgG1 levels independently predict viral control in HIV-1 clade C infection. Whether these responses contribute to direct antiviral control via the recruited killing of infected cells via the innate immune system or simply mark a qualitatively superior immune response to HIV, is uncertain, but highlights the role of p24-specific antibodies in control of clade C HIV-1 infection.

Enhanced binding of antibodies generated during chronic HIV infection to mucus component MUC16.

Transmission of HIV across mucosal barriers accounts for the majority of HIV infections worldwide. Thus, efforts aimed at enhancing protective immunity at these sites are a top priority, including increasing virus-specific antibodies (Abs) and antiviral activity at mucosal sites. Mucin proteins, including the largest cell-associated mucin, mucin 16 (MUC16), help form mucus to provide a physical barrier to incoming pathogens. Here, we describe a natural interaction between Abs and MUC16 that is enhanced in specific disease settings such as chronic HIV infection. Binding to MUC16 was independent of IgG subclass, but strongly associated with shorter Ab glycan profiles, with agalactosylated (G0) Abs demonstrating the highest binding to MUC16. Binding of Abs to epithelial cells was diminished following MUC16 knockdown, and the MUC16 N-linked glycans were critical for binding. Further, agalactosylated VRC01 captured HIV more efficiently in MUC16. These data point to a novel opportunity to enrich Abs at mucosal sites by targeting Abs to MUC16 through changes in Fc glycosylation, potentially blocking viral movement and sequestering the virus far from the epithelial border. Thus, next-generation vaccines or monoclonal therapeutics may enhance protective immunity by tuning Ab glycosylation to promote the enrichment of Abs at mucosal barriers.

A Drug-Free Zone--Lymph Nodes as a Safe Haven for HIV.

Understanding where and how the HIV latent reservoir persists is essential for developing rational HIV cure strategies. In a recent paper in Nature, Lorenzo-Redondo et al. (2016) demonstrate that HIV persists and actively evolves within lymph nodes due to low antiretroviral drug penetration, revealing the need to target these drug-privileged sites.

Independent evolution of Fc- and Fab-mediated HIV-1-specific antiviral antibody activity following acute infection.

Fc-related antibody activities, such as antibody-dependent cellular cytotoxicity (ADCC), or more broadly, antibody-mediated cellular viral inhibition (ADCVI), play a role in curbing early SIV viral replication, are enriched in human long-term infected nonprogressors, and could potentially contribute to protection from infection. However, little is known about the mechanism by which such humoral immune responses are naturally induced following infection. Here, we focused on the early evolution of the functional antibody response, largely driven by the Fc portion of the antibody, in the context of the evolving binding and neutralizing antibody response, which is driven mainly by the antibody-binding fragment (Fab). We show that ADCVI/ADCC-inducing responses in humans are rapidly generated following acute HIV-1 infection, peak at approximately 6 months postinfection, but decay rapidly in the setting of persistent immune activation, as Fab-related activities persistently increase. Moreover, the loss of Fc activity occurred in synchrony with a loss of HIV-specific IgG3 responses. Our data strongly suggest that Fc- and Fab-related antibody functions are modulated in a distinct manner following acute HIV infection. Vaccination strategies intended to optimally induce both sets of antiviral antibody activities may, therefore, require a fine tuning of the inflammatory response.

Lack of protection following passive transfer of polyclonal highly functional low-dose non-neutralizing antibodies.

Recent immune correlates analysis from the RV144 vaccine trial has renewed interest in the role of non-neutralizing antibodies in mediating protection from infection. While neutralizing antibodies have proven difficult to induce through vaccination, extra-neutralizing antibodies, such as those that mediate antibody-dependent cellular cytotoxicity (ADCC), are associated with long-term control of infection. However, while several non-neutralizing monoclonal antibodies have been tested for their protective efficacy in vivo, no studies to date have tested the protective activity of naturally produced polyclonal antibodies from individuals harboring potent ADCC activity. Because ADCC-inducing antibodies are highly enriched in elite controllers (EC), we passively transferred highly functional non-neutralizing polyclonal antibodies, purified from an EC, to assess the potential impact of polyclonal non-neutralizing antibodies on a stringent SHIV-SF162P3 challenge in rhesus monkeys. Passive transfer of a low-dose of ADCC inducing antibodies did not protect from infection following SHIV-SF162P3 challenge. Passively administered antibody titers and gp120-specific, but not gp41-specific, ADCC and antibody induced phagocytosis (ADCP) were detected in the majority of the monkeys, but did not correlate with post infection viral control. Thus these data raise the possibility that gp120-specific ADCC activity alone may not be sufficient to control viremia post infection but that other specificities or Fc-effector profiles, alone or in combination, may have an impact on viral control and should be tested in future passive transfer experiments.

Divergent antibody subclass and specificity profiles but not protective HLA-B alleles are associated with variable antibody effector function among HIV-1 controllers.

UNLABELLED: Understanding the coordination between humoral and cellular immune responses may be the key to developing protective vaccines, and because genetic studies of long-term HIV-1 nonprogressors have associated specific HLA-B alleles with spontaneous control of viral replication, this subject group presents an opportunity to investigate relationships between arms of the adaptive immune system. Given evidence suggesting that cellular immunity may play a role in viral suppression, we sought to determine whether and how the humoral immune response might vary among controllers. Significantly, Fc-mediated antibody effector functions have likewise been associated with durable viral control. In this study, we compared the effector function and biophysical features of HIV-specific antibodies in a cohort of controllers with and without protective HLA-B alleles in order to investigate whether there was evidence for multiple paths to HIV-1 control, or whether cellular and humoral arms of immunity might exhibit coordinated profiles. However, with the exception of IgG2 antibodies to gp41, HLA status was not associated with divergent humoral responses. This finding did not result from uniform antibody responses across subjects, as controllers could be regrouped according to strong differences in their HIV-specific antibody subclass specificity profiles. These divergent antibody profiles were further associated with significant differences in nonneutralizing antibody effector function, with levels of HIV-specific IgG1 acting as the major distinguishing factor. Thus, while HLA background among controllers was associated with minimal differences in humoral function, antibody subclass and specificity profiles were associated with divergent effector function, suggesting that these features could be used to make functional predictions. Because these nonneutralizing antibody activities have been associated with spontaneous viral control, reduced viral load, and nonprogression in infected subjects and protection in vaccinated subjects, understanding the specific features of IgGs with potentiated effector function may be critical to vaccine and therapeutic antibody development. IMPORTANCE: In this study, we investigated whether the humoral and cellular arms of adaptive immunity exhibit coordinated or compensatory activity by studying the antibody response among HIV-1 controllers with different genetic backgrounds.

Enhanced phagocytic activity of HIV-specific antibodies correlates with natural production of immunoglobulins with skewed affinity for FcγR2a and FcγR2b.

While development of an HIV vaccine that can induce neutralizing antibodies remains a priority, decades of research have proven that this is a daunting task. However, accumulating evidence suggests that antibodies with the capacity to harness innate immunity may provide some protection. While significant research has focused on the cytolytic properties of antibodies in acquisition and control, less is known about the role of additional effector functions. In this study, we investigated antibody-dependent phagocytosis of HIV immune complexes, and we observed significant differences in the ability of antibodies from infected subjects to mediate this critical effector function. We observed both quantitative differences in the capacity of antibodies to drive phagocytosis and qualitative differences in their FcγR usage profile. We demonstrate that antibodies from controllers and untreated progressors exhibit increased phagocytic activity, altered Fc domain glycosylation, and skewed interactions with FcγR2a and FcγR2b in both bulk plasma and HIV-specific IgG. While increased phagocytic activity may directly influence immune activation via clearance of inflammatory immune complexes, it is also plausible that Fc receptor usage patterns may regulate the immune response by modulating downstream signals following phagocytosis--driving passive degradation of internalized virus, release of immune modulating cytokines and chemokines, or priming of a more effective adaptive immune response.

High-throughput, multiplexed IgG subclassing of antigen-specific antibodies from clinical samples.

In vivo, the activity of antibodies relies critically on properties of both the variable domain, responsible for antigen recognition, and the constant domain, responsible for innate immune recognition. Here, we describe a flexible, microsphere-based array format for capturing information about both functional ends of disease-specific antibodies from complex, polyclonal clinical serum samples. Using minimal serum, we demonstrate IgG subclass profiling of multiple antibody specificities. We further capture and determine the subclass of epitope-specific antibodies. The data generated in this array provides a profile of the humoral immune response with multi-dimensional metrics regarding properties of both variable and constant IgG domains. Significantly, these properties are assessed simultaneously, and therefore information about the relationship between variable and constant domain characteristics is captured, and can be used to predict functions such as antibody effector activity.

Determining the phagocytic activity of clinical antibody samples.

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination(1-3). Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 297(4-6). Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome. Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis(7). Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response(8). Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 µm fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple FcγRs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of FcγRs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection(9).

Evaluation of vascular endothelial growth factor addition in vitro on mouse inner ear progenitor cell cultures.

We analyzed progenitor cell cultures of inner ear tissue from newborn mice, and found proliferating cells, morphologically differentiating cells and subpopulations of cells expressing either neuronal or glial markers. In addition, we observed the expression of fetal liver kinase-1, a receptor for the vascular endothelial growth factor in a subpopulation of the cultured cells. Consistent with the expression of fetal liver kinase-1, addition of vascular endothelial growth factor at a dose of 10 ng/ml increased the expansion rate of inner ear-derived progenitor cells. Together with other published data, these results suggest that the vascular endothelial growth factor might be involved in inducing or supporting cochlear repair processes.

The tobacco alkaloid nicotine demonstrates genotoxicity in human tonsillar tissue and lymphocytes.

Recent studies suggest a direct contribution of nicotine, the addictive component of tobacco and tobacco smoke, to human carcinogenesis. To assess the genotoxicity of nicotine, the DNA-damaging effect on human lymphocytes and target cells from lymphatic tissue of the palatine tonsils from 10 healthy patients was tested with the alkaline single-cell microgel electrophoresis (Comet) assay. The degree of DNA migration, a measure of possible DNA single strand breaks, alkali labile sites, and incomplete excision repair sites, was expressed as the Olive tail moment, the percentage of DNA in the tail, and the tail length. One hour exposure to nicotine at 0.125, 0.25, 0.5, 1, 2, and 4 mM induced a statistically significant dose-dependent increase of DNA migration up to 3.8-fold and 3.2-fold in tonsillar cells and lymphocytes, respectively. The lowest concentration eliciting significant DNA damage was 0.5 mM nicotine. The genotoxic effect was confirmed in a second series of experiments using nicotine of high purity from two different suppliers. There were no significant differences between the two series, excluding artifacts from the source of nicotine. Finally, DNA damage by nicotine was compared in cells incubated in medium strictly adjusted to neutral pH, with non-adjusted medium becoming alkaline with increasing nicotine concentrations. Again no differences in DNA migration were observed. The data indicate that nicotine expresses significant direct genotoxic effects in human target cells in vitro. However, no differences in DNA damage were observed in cells from smokers and nonsmokers incubated without nicotine. The lack of higher DNA damage in smokers compared to nonsmokers could be a question of nicotine dose, rapid DNA repair, or interactions with other smoke constituents. These results require further investigations on the contribution of nicotine to tobacco carcinogenesis.

Vertigo and hearing disturbance as the first sign of a glioblastoma (World Health Organization grade IV).

OBJECTIVE: To describe vertigo and hearing disturbance as a first sign of glioblastoma. STUDY DESIGN: Case report. SETTING: Ear, Nose, and Throat Department of the University of Regensberg, Germany. Primary Care Center. PATIENTS: A patient with a left temporal glioblastoma. RESULTS: A 67-year-old man presented with a 2-month history of vertigo and hearing disturbance. Radiological imaging revealed a left temporal tumor with dural inflation and erosion of the petrous bone and superior semicircular duct. The surgery involved total resection of the tumor and resurfacing of the gap in the superior canal. The histopathological examination revealed World Health Trade Organization IV glioblastoma. Postoperatively, the debilitating symptoms were relieved and the patient received radiation therapy. Tumor progression indicated a recraniotomy and a mastoidectomy. The tumor was only partially resected, and required chemotherapy. It subsequently developed otoliquorrhea and required a remastoidectomy. Histopathology of a pathological fracture of the X thoracic vertebra revealed a metastasis of the known glioblastoma. The patient died from respiratory distress syndrome. CONCLUSION: To the best of our knowledge, we are presenting the first case with transdural infiltration of bony structures by a glioblastoma at the moment of diagnosis. The transdural spread could be via the sinus petrosus and along the nervous petrosus major in the petrosal bone. Superior canal dehiscence syndrome should be considered in the differential diagnosis of vertigo and hearing disturbance. Two different processes for the etiology of the superior canal dehiscence syndrome are discussed previously in the literature; however, we present a new entity with a tumor-cause dehiscence of the bone overlying the superior canal.