Opposing consequences of IL-23 signaling mediated by innate and adaptive cells in chemically induced colitis in mice.

The interleukin-23 (IL-23) pathway has emerged as a promising therapeutic target for inflammatory bowel disease. Although the pathogenic role of IL-23 receptor (IL-23R) on T lymphocytes is well established, its function on innate immune cells has not been thoroughly examined. Here we investigate the consequence of IL-23R deletion in dextran sulfate sodium (DSS)-induced colitis. In IL23R(-/-) and IL23p19(-/-) mice, we observed decreased weight loss and reduced leukocyte infiltrate following DSS exposure. Surprisingly, when the IL-23R(-/-) allele was crossed into Rag2(-/-) mice, we observed exacerbated disease, increased epithelial damage, reduced pSTAT3 in the epithelium, and delayed recovery of IL23R(-/-)Rag2(-/-) mice. This phenotype was rescued with exogenous IL22-Fc, and epithelial pSTAT3 was restored. Depletion of Thy1(+) innate lymphoid cells eliminated the majority of IL-22 production in the colon lamina propria of DSS-treated Rag2(-/-) mice, suggesting that these are the major IL-23 responsive innate cells in this context. In summary, we provide evidence for opposing consequences of IL-23R on innate and adaptive lymphoid cells in murine colitis.

Analysis of vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) after oral rabies vaccination campaigns in Germany and Austria.

To eradicate rabies in foxes, almost 97 million oral rabies vaccine baits have been distributed in Germany and Austria since 1983 and 1986, respectively. Since 2007, no terrestrial cases have been reported in either country. The most widely used oral rabies vaccine viruses in these countries were SAD (Street Alabama Dufferin) strains, e.g. SAD B19 (53.2%) and SAD P5/88 (44.5%). In this paper, we describe six possible vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) detected during post-vaccination surveillance from 2001 to 2006, involving two different vaccines and different batches. Compared to prototypic vaccine strains, full-genome sequencing revealed between 1 and 5 single nucleotide alterations in the L gene in 5 of 6 SAD isolates, resulting in up to two amino acid substitutions. However, experimental infection of juvenile foxes showed that those mutations had no influence on pathogenicity. The cases described here, coming from geographically widely separated regions, do not represent a spatial cluster. More importantly, enhanced surveillance showed that the vaccine viruses involved did not become established in the red fox population. It seems that the number of reported vaccine virus-associated rabies cases is determined predominantly by the intensity of surveillance after the oral rabies vaccination campaign and not by the selection of strains.

Evaluation of aldrithiol-2-inactivated preparations of HIV type 1 subtypes A, B, and D as reagents to monitor T cell responses.

The development of HIV vaccines is an urgent priority and there is need to generate reagents representing multiple subtypes that can be used to screen HIV-1-specific responses. We used Aldrithiol-2 (AT-2), a mild oxidizing reagent, to eliminate the infectivity of HIV while maintaining its structure and ability to be processed for presentation to T cells. Inactivated subtype A, B, and D viruses were evaluated for their ability to stimulate T cell responses in PBMC samples from 18 U.S. subjects infected with HIV-1 subtype B and 32 Ugandan subjects infected with subtypes A and D or recombinants AC and AD. Five HIV-1-negative samples were also analyzed. T cell responses to AT-2-inactivated viral isolates were monitored by interferon-gamma (IFN-gamma) intracellular cytokine secretion (ICS) analysis; matched microvesicle preparations served as negative controls. Among the 18 subtype B infected subjects, 39% had CD3(+) CD4 (+) IFN-gamma responses and 67% had CD3(+) CD8(+) IFN-gamma responses. Of the 32 Ugandan subjects, 34% demonstrated CD3(+) CD4(+) IFN-gamma responses and 78% demonstrated CD3(+) CD8(+) IFN-gamma responses. Both subtype-specific and cross-reactive responses were observed. Responses to the AT-2 viruses tended to be lower in magnitude than those detected by a set of overlapping gag peptides. Robust lymphoproliferative responses to AT-2 viruses were seen in a subset of subjects. In conclusion, AT-2-inactivated HIV-1 virions stimulated both CD4 and CD8 HIV-1-specific responses and may provide an additional reagent for screening HIV-1-specific responses in HIV seropositives and vaccinees.

Regular exposure to rabies virus and lack of symptomatic disease in Serengeti spotted hyenas.

We report a previously unrecognized complexity to the ecology of rabies in wildlife. Rabies-specific virus-neutralizing antibodies in spotted hyenas, the most numerous large carnivore in the Serengeti ecosystem (Tanzania, East Africa), revealed a high frequency of exposure of 37.0% to rabies virus, and reverse transcriptase (RT) PCR demonstrated rabies RNA in 13.0% of hyenas. Despite this high frequency, exposure neither caused symptomatic rabies nor decreased survival among members of hyena social groups monitored for 9 to 13 years. Repeated, intermittent presence of virus in saliva of 45.5% of seropositive hyenas indicated a "carrier" state. Rabies isolates from Serengeti hyenas differed significantly (8.5% sequence divergence) from those isolated from other Serengeti carnivores, suggesting that at least two separate strains circulate within the Serengeti carnivore community. This finding is consistent with the fact that exposure in hyenas increased with age and social status, following a pattern predicted by intraspecific age and social-status-dependent oral and bite contact rates. High seroprevalence of rabies, low basic reproductive rate of the virus (R(0)) of 1.9, a carrier state, and the absence of symptomatic rabies in a carnivore in an ecosystem with multihost and multistrain maintenance has not been previously demonstrated for rabies. Because of the substantial differences between the hyena viral isolates and those from canids and viverrids in the Serengeti, it is unlikely that spotted hyenas were the source of rabies virus that killed several African wild dog packs in the Serengeti ecosystem in the 1990s.

Selective increases in HIV-specific neutralizing antibody and partial reconstitution of cellular immune responses during prolonged, successful drug therapy of HIV infection.

Because the immune response to HIV depends on viral gene expression, we examined the HIV-specific immune responses in persons whose viral load after highly active antiretroviral therapy (HAART) was <400 on at least 3 occasions over a 12-month interval. Eleven patients were identified. While there was little change in mean HIV-binding antibody (Ab) titers in this group, two persons mounted increases in HIV envelope-specific binding antibody. Neutralizing antibody (NAb) titers against a panel of HIV-1 primary isolates (BZ167, US1, and CM237) increased post-HAART (80% neutralization titer against US1, p = 0.06; against CM237, p = 0.04). The two persons with large increases in binding antibody also had increases in primary isolate NAb. Roughly half of HAART recipients had significant increases in neutralizing antibody to the primary isolates US1 and CM237. Compared with CD4-matched, non-HAART controls, there were significant increases in NAb against the subtype B primary isolate US1 (p < 0.0009); no increases were seen against more easily neutralized primary isolate BZ167. There were no differences after HAART in antibody-directed cellular cytotoxicity (ADCC). HAART resulted in a partial restoration of lymphoproliferative responses to recall antigens (tetanus and diphtheria). New responses developed to HIV Gag p24. No patient responded to HIV Env gp160 or gp120 either before or after HAART. The data underscore the lack of functional reconstitution of HIV-specific, CD4-mediated responses despite durable suppression of viral replication. In the setting of stable anti-HIV Ab levels, the development of increased NAb in certain individuals suggests that control of the virus by HAART may assist in immune control of HIV.

A novel approach to antigen-specific deletion of CTL with minimal cellular activation using alpha3 domain mutants of MHC class I/peptide complex.

In this study, we have compared the effector functions and fate of a number of human CTL clones in vitro or ex vivo following contact with variant peptides presented either on the cell surface or in a soluble multimeric format. In the presence of CD8 coreceptor binding, there is a good correlation between TCR signaling, killing of the targets, and FasL-mediated CTL apoptosis. Blocking CD8 binding using alpha3 domain mutants of MHC class I results in much reduced signaling and reduced killing of the targets. Surprisingly, however, FasL expression is induced to a similar degree on these CTLs, and apoptosis of CTL is unaffected. The ability to divorce these events may allow the deletion of antigen-specific and pathological CTL populations without the deleterious effects induced by full CTL activation.

Evaluation of natural killer cell activity.

Natural killer (NK) cells are being appreciated not only for their ability to recognize and lyse tumor cells and virus-infected cells but also for their immunoregulatory properties. NK cells provide a first line of defense against invading pathogens with a two pronged attack, lysis of infected cells and secretion of cytokines and chemokines with potent antipathogen effects. This article describes the standard chromium release assay, which measures the ability of NK cells derived from the peripheral blood to lyse appropriate target cells.

A comparative study of the impact of HIV infection on natural killer cell number and function in Thais and North Americans.

Innate immunity may play a role in preventing HIV infection and progression to AIDS. Most studies of natural killer (NK) cell function have been conducted in populations with different HLA allele frequencies and HIV subtypes than those found in Southeast Asia. NK cell number and function, defined as CD3- cells expressing CD16+/CD56+ and the ability to lyse K562 cells, were enumerated in 42 HIV-seronegative Thais and 20 HIV-seronegative North Americans. The number and percentage of NK cells were similar for both groups, but cytotoxicity function expressed as lytic units (LU20) of NK cells was significantly greater in the Thai subjects compared with the North American subjects (p = 0.004). Comparisons were also conducted between the HIV-seronegative groups and HIV-infected subjects from both Thailand and North America. NK cell number and function were not significantly different between the Thai HIV-seronegative and -seropositive groups. However, the comparison between the North American HIV-seronegative and -seropositive subjects demonstrated profound impairment of NK cell number, percentage, and function (p < 0.001). Matching the Thai and North American HIV-infected subjects on CD4+ cell count revealed higher NK number and function in the Thai subjects (p < 0.001). The study indicates that NK function in both HIV-seronegative and -seropositive Thais is elevated relative to similar groups in North America.

Effect of short-term exercise training on leptin and insulin action.

The purpose of the study was to determine the effect of short-term exercise training (7 consecutive days for 60 min/d at 75% maximal oxygen consumption [VO2 max]), which did not change body mass on fasting plasma leptin concentration and insulin action. Young, lean subjects (n = 16; age, 21.9 +/- 0.6 years; body fat, 17.5% +/- 1.5%) and older subjects with relatively more adipose tissue (n = 14; age, 58.6 +/- 1.4 years; body fat, 28.3% +/- 1.3%) were studied (mean +/- SE). Fasting plasma leptin was significantly (P < .05) related to adiposity (fat mass, r = .58; % body fat, r = .76) in this population. Body mass did not change (P < .05) in any of the groups with training (71.8 +/- 2.5 v 71.9 +/- 2.5 kg). The insulin sensitivity index (SI determined from an intravenous glucose tolerance test (IVGTT) improved significantly (P < .05) in both the young group (4.8 +/- 0.6 v6.9 +/- 0.8 x 10(-4)/ min (microU/mL) and the older group (3.2 +/- 0.6 v 5.9 +/- 1.0 x 10(-4)/min (microU/mL)). Fasting leptin did not change with training in either group (10.4 +/- 1.6 v 9.2 +/- 1.0 ng/mL). These findings suggest that exercise does not independently affect the fasting plasma leptin concentration and the improvement in insulin action with exercise is not associated with an alteration in fasting leptin in healthy sedentary lean and relatively lean subjects.

Differential transcription attenuation of rabies virus genes by intergenic regions: generation of recombinant viruses overexpressing the polymerase gene.

Gene expression of nonsegmented negative-sense RNA viruses involves sequential synthesis of monocistronic mRNAs and transcriptional attenuation at gene borders resulting in a transcript gradient. To address the role of the heterogeneous rabies virus (RV) intergenic regions (IGRs) in transcription attenuation, we constructed bicistronic model RNAs in which two reporter genes are separated by the RV N/P gene border. Replacement of the 2-nucleotide (nt) N/P IGR with the 5-nt IGRs from the P/M or M/G border resulted in attenuation of downstream gene transcription to 78 or 81%, respectively. A severe attenuation to 11% was observed for the 24-nt G/L border. This indicated that attenuation in RV is correlated with the length of the IGR, and, in particular, severe downregulation of the L (polymerase) gene by the 24 nt IGR. By reverse genetics, we recovered viable RVs in which the strongly attenuating G/L gene border of wild-type (wt) RV (SAD L16) was replaced with N/P-derived gene borders (SAD T and SAD T2). In these viruses, transcription of L mRNA was enhanced by factors of 1.8 and 5.1, respectively, resulting in exaggerated general gene expression, faster growth, higher virus titers, and induction of cytopathic effects in cell culture. The major role of the IGR in attenuation was further confirmed by reintroduction of the wt 24-nt IGR into SAD T, resulting in a ninefold drop of L mRNA. The ability to modulate RV gene expression by altering transcriptional attenuation is an advantage in the study of virus protein functions and in the development of gene delivery vectors.

Pythiosis with bone lesions in a pregnant mare.

A 9-year-old pregnant mare was referred for evaluation of a nonhealing wound of 8 weeks' duration on the lateral aspect of the left forelimb. A soft tissue mass encircled the proximal two thirds of the metacarpus; radiography revealed a moderate periosteal reaction affecting metacarpal bone i.v. Histologic and immunohistochemical examinations revealed eosinophilic granulomatous inflammation and Pythium sp in the soft tissues. The mare was treated for 12 days with antimicrobials, medicated wound dressings, debridement, and i.v. administration of sodium iodide; radiography revealed progression of the bone lesions. The mare was treated by regional arterial perfusion with miconazole and excision of affected soft tissues and the distal two thirds of metacarpal bone i.v. The mare recovered without complications and gave birth to a healthy foal. Regional perfusion of antifungal agents provides high concentrations in soft and osseous tissues and permits use of low dosages of agents administered by other routes, which reduces cost, adverse effects, and teratogenic effects.

Preliminary evaluation of human immunodeficiency virus type 1 (HIV-1) immunogen in children with HIV-1 infection.

The safety and preliminary activity of human immunodeficiency virus type 1 (HIV-1) immunogen were evaluated in 10 HIV-1-infected children with disease stage N1,2 or A1,2. Multiple inoculations of 2. 5 or 10 units (U) of HIV-1 immunogen were safe and well tolerated without an acceleration of disease progression. When antiretroviral agents were coadministered, the 10 U dose appeared to be associated with more sustained reduction in plasma HIV-1 RNA than the 2.5 U dose (median log10 HIV-1 RNA at month 18, 3.07 vs. 4.01 copies/mL in 10 U [n=4] vs. 2.5 U [n=3], respectively; P=.034). Levels of regulated-on-activation, normal T cell-expressed and -secreted chemokine produced from HIV-1 immunogen-stimulated lymphocytes in vitro were increased in the children who had HIV-1 immunogen-specific antibody responses (P<.02) and appeared to be inversely correlated with levels of plasma HIV-1 RNA (P<.01). These preliminary data warrant larger studies to determine the effectiveness of adjunctive therapy with HIV-1 immunogen in children with HIV-1 infection.

Antibody-dependent cellular cytotoxicity in HIV type 1-infected patients receiving VaxSyn, a recombinant gp160 envelope vaccine.

Antibody-dependent cellular cytotoxicity (ADCC) activity was measured in 60 human immunodeficiency virus (HIV-1)-infected patients receiving a recombinant gp160 (rgp160) envelope protein of HIV-1(NL4-3) in alum and 64 receiving placebo over a 5-year study period. There was no difference in the percentage of ADCC responders when comparing rgp160-immunized patients (mean, 78.4%) with those receiving placebo alone (mean, 81.5%) at any time point examined. Patients were further divided into progression groups regardless of their vaccine status. ADCC activity was somewhat higher in rapid than in slow-progressing groups, although the number that had detectable ADCC activity was equivalent in each group. ADCC activity of sera from rapid- and slow-progressing groups against primary or laboratory isolate envelopes was similar. This study showed that transcription with rgp160 did not appear to enhance HIV-specific ADCC activity. ADCC activity did not appear to correlate with protection against AIDS in this cohort of HIV-1-infected people.

Effect of aging on response to exercise training in humans: skeletal muscle GLUT-4 and insulin sensitivity.

The purpose of this study was to compare the effects of short-term exercise training on insulin-responsive glucose transporter (GLUT-4) concentration and insulin sensitivity in young and older individuals. Young and older women [22.4 +/- 0.8 (SE) yr, n = 9; and 60.9 +/- 1. 0 yr, n = 10] and men (20.9 +/- 0.9, n = 9; 56.5 +/- 1.9 yr, n = 8), respectively, were studied before and after 7 consecutive days of exercise training (1 h/day, approximately 75% maximal oxygen uptake). The older groups had more adipose tissue, increased central adiposity, and a lower maximal oxygen uptake. Despite these differences, increases in whole body insulin action (insulin sensitivity index, determined with an intravenous glucose tolerance test and minimal-model analysis) with training were similar regardless of age, in both the women and men (mean increase of 2.2 +/- 0.3-fold). This was accompanied by similar relative increases in muscle (vastus lateralis) GLUT-4 protein concentration, irrespective of age (mean increase of 3.1 +/- 0.7-fold). Body mass did not change with training in any of the groups. These data suggest that older human skeletal muscle retains the ability to rapidly increase muscle GLUT-4 and improve insulin action with endurance training.

HIV-1-Specific Cytotoxic T-Cell Assays.

Cytotoxic T-cells (CTL) provide the basis of protective immunity in many viral infections by appearing early in the immune response and becoming involved with the elimination of virus by lysis of infected cells. A considerable amount of evidence suggests that CTL may be an especially important component of the host defense against HIV-1 (1,2) . Strong CTL activity is seen early during the course of infection (3,4), in nonprogressor individuals, some of whom have been infected for a decade or more with HIV-1 (5,6) and also in some HIV-1 exposed sex workers (7) and babies (8,9) who remain HIV-1-negative.

HIV-1-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC).

Antibodies elicited during the course of HIV-1 infection can act as a bridge between cytolytic cells and HIV-1-infected cells or other cells that have passively absorbed appropriate HIV-1-antigens. These cytolytic cells cause lysis of the HIV-1-infected cells thereby decreasing viral load (1-5) . The effector cells for ADCC are natural killer (NK) cells, that mediate lysis in a non-MHC restricted fashion. NK cells expressing CD16(+) low affinity Fc-receptors bind to antibody which specifically binds to the antigen expressed on target cells. Two types of ADCC activity can be demonstrated in HIV-1-infected patients. The first type, the classical or indirect ADCC, is assayed using normal donor lymphocytes and HIV-1 patient sera. Lysis of HIV-1 envelope protein-coated or HIV-1-infected target cells is detected in a chromium 51 [(51)Cr] release assay. Sera from HIV-1-infected individuals appear to mediate indirect ADCC regardless of their disease status. A second type, or direct ADCC, uses NK cells freshly isolated from HIV-1-infected individuals. These NK cells are coated with anti-HIV-1 cytophilic antibodies and can readily lyse envelope-coated or HIV-1-infected target cells. The latter may be a more pertinent measure of ADCC activity since the activity of this type of NK-mediated ADCC declines as HIV-1 disease progresses (6).

Evaluation of natural killer cell activity.

The immune response to a virus infection involves both nonspecific and specific immune mechanisms. Natural killer (NK) cells are naturally-occurring cytolytic cells capable of lysing various tumor cells and virus-infected cells without previous sensitization or with a requirement for major histocompatibility (MHC) restriction. The molecular mechanisms that explain how NK cells are able to kill virus-infected cells and tumor cells while sparing self-cells have recently been elucidated (1). NK cells may play a role as a first line of defense against virus infection by mediating lysis of virus-infected cells prior to the development of specific humoral and cell-mediated defense mechanisms. Although the percentage of NK cells in HIV-1-infected patients may remain normal, the absolute numbers of some NK subsets are substantially reduced in the blood of HIV-1 patients and NK function decreases as HIV-1 infection proceeds (2-4). The interplay between NK cells and other cells of the innate and specific immune system is mediated, in part, through the release of cytokines, in particular interleukin-2 (IL-2) and γ-interferon (γ-IFN). Thus, it seems plausible that the generalized immunosuppression seen in HIV-1-infected patients may contribute to the impairment of NK activity. A dynamic balance between NK cells and cytotoxic T lymphocytes (CTL) is likely to occur (5). Therefore, any alterations in NK or CTL activity are likely to impair anti-HIV-1 cytolytic function.

Cross-clade cytotoxic T cell response to human immunodeficiency virus type 1 proteins among HLA disparate North Americans and Thais.

A globally effective vaccine will need to elicit cytotoxic T lymphocytes (CTL) capable of recognizing diverse human immunodeficiency virus type 1 (HIV-1) clades. Study of the cellular immune responses of HIV-1-infected persons may allow predictions to be made regarding useful vaccine antigen components. The frequency and magnitude of CTL responses to clade E and B Gag, Pol-RT, Env, and Nef proteins were compared in 12 HLA-characterized, clade E-infected Thais and in 10 clade B-infected North Americans using vaccinia recombinant constructs for protein expression. While responses were detected against all proteins, they were most frequent and cross-reactive to Gag in both groups. Pol-RT was recognized less frequently in Thais than North Americans. Cross-clade protein recognition was common but not uniformly present among these HLA-disparate individuals. Population-specific CTL data are needed to adequately prepare for vaccine trials outside of North America and Europe.