Th17 cells in inflammation and autoimmunity

T helper 17 (Th17), a distinct subset of CD4⁺ T cells with IL-17 as their major cytokine, orchestrate the pathogenesis of inflammatory and autoimmune diseases. Dysregulated Th17 cells contribute to inflammatory and autoimmune diseases. Candidate biologics are in development for targeting IL-17, IL-17 receptors or IL-17 pathways. Several drugs that impact the IL-17 pathway are already in clinical trials for the treatment of autoimmune diseases. In this review we provide evidence for the role of Th17 cells in immune-mediated diseases. An understanding of the role of Th17 in these conditions will provide important insights and unravel novel targets for therapeutic intervention.     

Memory T-Cell Responses to Vibrio cholerae O1 Infection

Vibrio cholerae O1 can cause diarrheal disease that may be life-threatening without treatment. Natural infection results in long-lasting protective immunity, but the role of T cells in this immune response has not been well characterized. In contrast, robust B-cell responses to V. cholerae infection have been observed. In particular, memory B-cell responses to T-cell-dependent antigens persist for at least 1 year, whereas responses to lipopolysaccharide, a T-cell-independent antigen, wane more rapidly after infection. We hypothesize that protective immunity is mediated by anamnestic responses of memory B cells in the gut-associated lymphoid tissue, and T-cell responses may be required to generate and maintain durable memory B-cell responses. In this study, we examined B- and T-cell responses in patients with severe V. cholerae infection. Using the flow cytometric assay of the specific cell-mediated immune response in activated whole blood, we measured antigen-specific T-cell responses using V. cholerae antigens, including the toxin-coregulated pilus (TcpA), a V. cholerae membrane preparation, and the V. cholerae cytolysin/hemolysin (VCC) protein. Our results show that memory T-cell responses develop by day 7 after infection, a time prior to and concurrent with the development of B-cell responses. This suggests that T-cell responses to V. cholerae antigens may be important for the generation and stability of memory B-cell responses. The T-cell proliferative response to VCC was of a higher magnitude than responses observed to other V. cholerae antigens.Vibrio cholerae is a gram-negative bacterium that can cause a severe, acute secretory diarrhea. Serological differentiation of V. cholerae strains is based on the O-side chain of the lipopolysaccharide (LPS) component of the outer membrane. Of the more than 200 serogroups of V. cholerae identified, only the O1 and O139 serogroups can cause epidemic cholera (44). These pathogens are noninvasive and colonize the mucosal surface of the small intestine (44).Natural infection with V. cholerae is known to provide protection against subsequent disease, but the mechanism of this protective immunity is not fully characterized. The vibriocidal antibody is a complement-dependent bactericidal antibody that is associated with protection from infection. However, no known threshold level of the vibriocidal antibody confers complete protection from V. cholerae infection, and some individuals with low serum vibriocidal antibody titers are still protected. This suggests that the vibriocidal titer may be a surrogate marker (16, 45). Elevated serum immunoglobulin A (IgA) antibody levels specific for the B subunit of cholera toxin (CTB), the major structural subunit of a type IV pilus (TcpA), and LPS are also associated with protective immunity in areas where cholera is endemic (19). However, after natural infection, the serum levels of these antibodies wane more rapidly than protective immunity (19). Patients with cholera develop memory B-cell responses of both the IgG and the IgA isotype to at least two V. cholerae protein antigens, CTB and TcpA. These responses are detectable for at least 1 year after infection and persist even after V. cholerae antigen-specific antibody-secreting cells and serum antibody titers have returned to baseline (18). B-cell memory responses also develop for the T-cell independent antigen LPS, but these responses wane more rapidly than memory B-cell responses to protein antigens, suggesting that durable memory B-cell responses to some V. cholerae antigens may be T-cell dependent (18).We have recently demonstrated that cholera patients mount a primed T-cell response in the mucosa after V. cholerae O1 infection (6). We hypothesize that protection from cholera may be mediated by memory B cells capable of an anamnestic response in the gut mucosa and that these memory B cells may depend on stimulation provided by memory T cells for their development and maintenance. T cells may contribute to the activation of B cells during V. cholerae infection by secreting stimulatory cytokines and direct contact with B cells in lymph nodes. Therefore, T cells may have an important role in protective immunity to V. cholerae infection.We characterized the memory T-cell responses to V. cholerae antigens following natural V. cholerae infection and compared these with serological responses to the same antigens. Previously, our group has studied various V. cholerae antigens, including mannose-sensitive hemagglutinin, TcpA, CTB, and LPS (22, 33, 37). We also included in the present study responses to a novel antigen, V. cholerae cytolysin/hemolysin (VCC) (31, 32). The hly gene that encodes the VCC protein is widespread across both pathogenic and environmental strains of V. cholerae, suggesting that VCC may impart an advantage to the organism (42). Although the precise role of VCC in V. cholerae infection is unknown, VCC is the primary virulence factor in V. cholerae infection with non-O1, non-O139 strains that do not produce cholera toxin (12, 46). The immune response to VCC is not well understood; however, recent studies suggest that VCC may promote a Th2 response in V. cholerae infection (2). In addition, the cytolytic activity of VCC may generate epithelial destruction that allows other V. cholerae antigens to penetrate the mucosa and promote the inflammatory response observed in V. cholerae infection (35, 39).     

The role of hyaluronic acid as a mediator and regulator of cervical ripening

During pregnancy, hyaluronic acid (HA) concentration in the human cervix is very low, but increases rapidly at the onset of labour. HA has a high affinity for water molecules and hence can maintain tissue hydration. HA can stimulate collagenase production in rabbit cervix, and also stimulates migration and function of polymorphonuclear leukocytes in the tissues. It is an endogenous regulator of interleukin- 1 (IL-1). We hypothesized that HA plays an essential role during cervical ripening. The effect of exogenous application of HA (20 mg) on non-pregnant and pregnant (day 23) rabbit cervices was compared with controls. HA induced cervical ripening in both pregnant and non- pregnant animals, and cervical water content was significantly increased. Tissue collagen was markedly decreased. The localization and distribution of HA and HA receptor CD44 was determined in non-pregnant and pregnant human cervical connective tissue using biotinylated HA binding protein and CD44 monoclonal antibodies. Both were widely distributed in the connective tissues, especially around the blood vessels and cervical glands. The effect of IL-8 (50, 100, 150 and 200 ng/ml) on HA production and hyaluronidase (HAase) activity was investigated in cultures of lower uterine segment collected during elective Caesarean sections. HA production was stimulated in a dose- dependent manner; there was no effect on hyaluronidase activity. HA administration (0.5, 1 and 2 mg/ml) stimulated the activities of collagenase and gelatinase together with IL-8 production in the culture supernatants. Thus HA may play an important role in cervical ripening, being involved in the regulation of cervical tissue water content, collagenolytic enzymes and cytokines.      

Sodium 2-propenyl thiosulfate derived from garlic induces phase II detoxification enzymes in rat hepatoma H4IIE cells

There is evidence that onions and garlic protect against cancer in humans. It has been suggested that this effect is partly due to the organosulfur compounds in Allium vegetables and that these substances act through induction of phase II detoxification enzymes. Here, we hypothesized that alk(en)yl thiosulfates, sodium n-propyl thiosulfate (NPTS), and sodium 2-propenyl thiosulfate (2PTS), which were identified in onions and garlic, respectively, may induce phase II enzymes. Therefore, rat hepatoma cells (H4IIE) were cultured with 1 to 100 μmol/L of NPTS or 2PTS for 48 hours at 37°C; and the activities and messenger RNA (mRNA) expression levels of phase II enzymes in H4IIE cells were investigated. The effects of diallyl trisulfide and tert-butylhydroquinone, known as phase II inducers, were also examined as positive controls and compared with the responses of NPTS and 2PTS. Quinone reductase (QR) activity and mRNA expression levels of QR and epoxide hydrolase 1 were significantly increased by 2PTS (P < .05-.005). In particular, QR activity was increased at a relatively low concentration of 2PTS (10 μmol/L). However, glutathione S-transferase activity and mRNA expression levels of glutathione S-transferase A5 and uridine diphosphate glucuronosyl transferase 1A1 were not changed by 2PTS. In contrast, NPTS did not affect the activities and mRNA expression levels of these phase II enzymes. These results show that 2PTS can induce phase II enzymes, and its inductive effect is comparable or superior to that of diallyl trisulfide and tert-butylhydroquinone.     

Fetal calf serum inhibits virus genome expression in Madin-Darby canine kidney cells persistently infected with influenza A virus

A cell line of Madin-Darby canine kidney (MDCK) cells persistently infected with human influenza A virus has been established and designated as MDCK-IVpi cells. Production of progeny virus in MDCK-IVpi cells was suppressed when the cells were incubated in the presence of 10% fetal calf serum (FCS). FCS impaired virus mRNA synthesis in MDCK-IVpi cells, which resulted in a scarcity of virus proteins for virion formation. However, MDCK-IVpi cells well supported the growth of superinfecting heterologous influenza viruses, even in the presence of FCS. A certain fetuin-like substance in FCS might be responsible for the observed inhibition of virus replication.     

Development of Immunoglobulin M Memory to Both a T-Cell-Independent and a T-Cell-Dependent Antigen following Infection with Vibrio cholerae O1 in Bangladesh

Vibrio cholerae O1 can cause severe watery diarrhea that can be life-threatening without treatment. Infection results in long-lasting protection against subsequent disease. Development of memory B cells of the immunoglobulin G (IgG) and IgA isotypes to V. cholerae O1 antigens, including serotype-specific lipopolysaccharide (LPS) and the B subunit of cholera toxin (CTB), after cholera infection has been demonstrated. Memory B cells of the IgM isotype may play a role in long-term protection, particularly against T-cell-independent antigens, but IgM memory has not been studied in V. cholerae O1 infection. Therefore, we assayed acute- and convalescent-phase blood samples from cholera patients for the presence of memory B cells that produce cholera antigen-specific IgM antibody upon polyclonal stimulation in in vitro culture. We also examined the development of serological and antibody-secreting cell responses following infection. Subjects developed significant IgM memory responses by day 30 after infection, both to the T-cell-independent antigen LPS and to the T-cell-dependent antigen CTB. No significant corresponding elevations in plasma IgM antibodies or circulating IgM antibody-secreting cells to CTB were detected. In 17 subjects followed to day 90 after infection, significant persistence of elevated IgM memory responses was not observed. The IgM memory response to CTB was negatively correlated with the IgG plasma antibody response to CTB, and there was a trend toward negative correlation between the IgM memory and IgA plasma antibody responses to LPS. We did not observe an association between the IgM memory response to LPS and the vibriocidal titer.Vibrio cholerae continues to be a significant global health burden as a cause of severe secretory diarrhea, resulting in an estimated three to five million annual cases, with more than 100,000 deaths from rapid dehydration (47); cholera has recently become endemic in new regions (44, 45). V. cholerae is a noninvasive pathogen that colonizes the mucosal surface of the small intestine. Strains can be distinguished serologically by the O antigen of the lipopolysaccharide (LPS); V. cholerae O1 is the most common cause of cholera in South Asia as well as globally. The O1 serogroup has two major biotypes, El Tor and classical, and two major serotypes, Inaba and Ogawa (35). Natural infection with V. cholerae O1 El Tor induces protective immunity that lasts for at least 3 to 10 years in both areas where cholera is not endemic and areas where it is endemic (21). It remains unknown, however, what aspects of the adaptive immune response to cholera confer this long-term protection.V. cholerae-infected patients mount immunologic responses to both protein and polysaccharide antigens, including rises in both serum immunoglobulin G (IgG) and IgA antibodies (14). A number of these serological responses have been shown to correlate with protection against reinfection; these include the complement-dependent serum vibriocidal antibody (14) and IgA (but not IgG) responses to LPS, cholera toxin B subunit (CTB), and toxin coregulated pilus A (TcpA) (17). These serological responses, however, are short-lived (4, 32), and the association of the vibriocidal titer with protection is not absolute (36), suggesting that these responses may reflect protection from more recent exposure but that other immunologic mechanisms mediate longer-term protection. In addition to serological responses, development of mucosal immune responses to intestinal antigens can be detected in the blood, when B cells activated by antigen in the gut-associated lymphoid tissues circulate transiently in the blood as antibody-secreting cells (ASCs), before homing back to intestinal mucosal surfaces (11, 26). Circulation of ASCs specific to both LPS and CTB is seen after cholera infection, peaking around the seventh day after infection and declining by day 11 (32).Responses of the IgM isotype to cholera antigens have been less thoroughly investigated than the IgG and IgA responses. However, IgM defenses may be an important component of the overall immunologic response to cholera, since vibriocidal antibodies are principally of the IgM isotype (22) and IgM levels of pooled convalescent-phase serum samples correspond closely with vibriocidal activity (24), which in turn correlates with immunity (14). The pentameric structure of IgM facilitates strong cross-linking of antigens and activation of complement in the defense against other gram-negative enteric bacteria (2).We have recently shown development of memory B cells of both the IgG and IgA isotypes to LPS, CTB, and TcpA; these cells persisted in the circulation beyond 1 year for the protein antigens CTB and TcpA, but were not measurably above baseline levels by 9 to 12 months after infection for the polysaccharide-containing antigen LPS (16, 18). These circulating memory B cells can be detected by ex vivo polyclonal stimulation of peripheral blood mononuclear cells (PBMCs); stimulated memory B cells mature into ASCs detectable by enzyme-linked immunospot (ELISPOT) assay. Alternatively, memory B-cell responses can be detected by measuring antigen-specific antibodies secreted by maturing ASCs during the ex vivo stimulation of PBMCs in the memory B-cell assay (18).Memory B cells relevant for cholera immunity may include IgM⁺ as well as switched-memory (IgA⁺ and IgG⁺) populations. The majority of circulating IgM⁺ cells are naïve B cells, but some IgM⁺ cells bear the memory cell marker CD27⁺, and recent evidence suggests that these IgM⁺CD27⁺ cells are true memory B cells whose immunoglobulin variable region genes have undergone somatic hypermutation in response to antigen in early-stage germinal centers (39). IgM⁺ memory cells can undergo isotype switching to produce IgG, IgA, or IgE antibody, but they also have a role in producing rapid, high-affinity IgM antibody responses to acute infection (19, 37, 46). In this study, we have measured the development of memory B-cell responses of the IgA, IgG, and IgM isotypes to both a protein (CTB) and a nonprotein (LPS) antigen, and we compared these memory responses with other immunologic responses in patients after V. cholerae infection in Bangladesh.     

Analysis of the biological and molecular variability of Watermelon mosaic virus isolates from Iran

Watermelon mosaic virus (WMV) is one of the most important viruses that causes different symptoms in Cucurbitaceae. WMV is a potyvirus with a worldwide distribution, but occurs most commonly in temperate and Mediterranean regions. Cucurbit species grown in Yazd, Esfahan, West Azerbaijan, Hormozgan, and Kerman provinces were surveyed for the relative incidence of WMV in 2004–2005. A total of 757 symptomatic cucurbit and 31 weed species were collected and assayed for infection with WMV. Of 788 leaf samples from cucurbit and weed plants, 190 samples were positive by double antibody sandwich ELISA (DAS-ELISA) using specific polyclonal antibody. Among the weed species tested only colocynth (Citrullus colocynthis) was found to be infected with WMV. The coat protein (CP) gene from 18 representative isolates was PCR amplified, cloned, sequenced, and compared with the sequences available in GeneBank. Phylogenetic analysis using 778 nucleotide long sequences of the coat protein gene showed that these isolates fell into two; groups I and II. Only one isolates (KER.JI.1) was classified in the group II. This isolate had a wider host range and infected Nicotiana debneyii and Datura metel. None of the other 17 isolates could infect these two species. Members of group I were divided into three subgroups; A, B, and C. The subgroup I_B appears to be a new subgroup comprising only of the Iranian isolates. Phylogenetic analysis based on 200 nucleotides coding for the N-terminal segment of the CP showed that all Iranian isolates except KER.JI.1 clustered with the previously reported WMV strains. All Iranian isolates had a DAG amino acid triplet which is involved in aphid transmissibility. This is the first report on sequence analysis of the nearly full-length CP cDNA clones of WMV isolates from Iran.     

Evaluation of a virus derived from MDCK cells infected persistently with influenza A virus as a potential live-attenuated vaccine candidate in the mouse model

A temperature-sensitive mutant virus unable to replicate at 38 degrees C was recovered from passage 189 (IVpi-189) of Madin-Darby canine kidney cells infected persistently with influenza A. Immunofluorescent staining of the IVpi-189 virus-infected cells revealed disrupted transport of the matrix (M) 1 protein into the nucleus at non-permissive temperatures, resulting in retention of the nucleoprotein (NP) in the nucleus. Upon comparison with the parental influenza A E61-24-P15 strain used to establish persistent infection, amino acid exchanges were found in the M1 protein of IVpi-189 virus; arginine to glutamine at position 72 and threonine to alanine at position 139. When mice were inoculated intranasally with IVpi-189 virus, virus growth in the lungs was restrained and terminated rapidly. Prior intranasal inoculation with only a small dose of IVpi-189 virus induced humoral and cellular immune responses and protected mice against subsequent virulent virus challenge. These results indicate that IVpi-189 virus, an avirulent temperature-sensitive mutant, is a promising candidate for use as a live-attenuated vaccine.     

Expression of RAB27B is up-regulated in senescent human cells

Immortal SVts8 cells that express thermolabile SV40 T antigen exhibit a senescence-like phenomenon upon inactivation of the T antigen. By using a cDNA subtractive hybridization technique, RAB27B, a member of the RAB GTPase family, was found to be up-regulated in senescent SVts8 cells. The up-regulation of RAB27B depends on the p53 gene. Enhanced expression was also observed in replicative senescence in normal human fibroblasts.