Development of nalidixic acid amphotericin B vancomycin (NAV) medium for the isolation of Campylobacter ureolyticus from the stools of patients presenting with acute gastroenteritis.

Recently, Campylobacter ureolyticus has been detected for the first time in the faeces of patients with acute gastroenteritis using polymerase chain reaction (PCR) techniques. Cultural isolation of C. ureolyticusis is not possible using the established selective methods for the isolation of thermophilic Campylobacter spp. from faeces. The aim of the current study is to develop a new selective medium capable of isolating C. ureolyticus from faecal samples. The newly-developed medium consists of Anaerobe Basal Agar with 10 g/L additional agar, 2 g/L sodium formate and 3 g/L sodium fumarate dibasic, to which 10 mg/L nalidixic acid, 10 mg/L amphotericin B and 20 mg/L vancomycin (NAV) are added as selective agents. Validation studies have shown that this experimental selective medium completely inhibits growth of Candida spp. and of Enterococcus spp. and permits reduced growth of selected coliforms and Proteus spp. Growth of Campylobacter ureolyticus on NAV medium is optimal in anaerobic and enriched hydrogen atmospheres. Additionally, an overnight enrichment step using Bolton broth to which 2 g/L sodium formate, 3 g/L sodium fumarate dibasic and the NAV supplement are added, in place of the commercial Bolton broth supplement, allows improved recovery of C. ureolyticus from patients' faeces.

Ex vivo analysis of resident hepatic pro-inflammatory CD1d-reactive T cells and hepatocyte surface CD1d expression in hepatitis C.

Hepatic CD1d-restricted and natural killer T-cell populations are heterogeneous. Classical 'type 1' α-galactosylceramide-reactive CD1d-restricted T cells express 'invariant' TCRα ('iNKT'). iNKT dominating rodent liver are implicated in inflammation, including in hepatitis models. Low levels of iNKT are detected in human liver, decreased in subjects with chronic hepatitis C (CHC). However, high levels of human hepatic CD161(±) CD56(±) noninvariant pro-inflammatory CD1d-restricted 'type 2' T cells have been identified in vitro. Unlike rodents, healthy human hepatocytes only express trace and intracellular CD1d. Total hepatic CD1d appears to be increased in CHC and primary biliary cirrhosis. Direct ex vivo analysis of human intrahepatic lymphocytes (IHL), including matched ex vivo versus in vitro expanded IHL, demonstrated detectable noninvariant CD1d reactivity in substantial proportions of HCV-positive livers and significant fractions of HCV-negative livers. However, α-galactosylceramide-reactive iNKT were detected only relatively rarely. Liver CD1d-restricted IHL produced IFNγ, variable levels of IL-10 and modest levels of Th2 cytokines IL-4 and IL-13 ex vivo. In a novel FACS assay, a major fraction (10-20%) of hepatic T cells rapidly produced IFNγ and up-regulated activation marker CD69 in response to CD1d. As previously only shown with murine iNKT, noninvariant human CD1d-specific responses were also augmented by IL-12. Interestingly, CD1d was found selectively expressed on the surface of hepatocytes in CHC, but not those CHC subjects with history of alcohol usage or resolved CHC. In contrast to hepatic iNKT, noninvariant IFNγ-producing type 2 CD1d-reactive NKT cells are commonly detected in CHC, together with cognate ligand CD1d, implicating them in CHC liver damage.

Antigen-specific T lymphocyte proliferation decreases over time in advanced chronic hepatitis C.

To evaluate T cell immunity in advanced liver disease, antigen-specific lymphoproliferative (LP) responses were prospectively studied in the context of the Hepatitis C Antiviral Long-term Treatment against Cirrhosis trial. Peripheral blood responses to hepatitis C virus (HCV), tetanus and Candida protein antigens were measured at baseline, month 12 (M12), M24, M36 and M48 in 186 patients randomized to either low-dose peginterferon-alfa-2a (PEG-IFN) only or observation. Liver histology was evaluated at baseline, M24 and M48. Patients with cirrhosis (Ishak 5-6) were less likely to have positive LP responses to HCV at baseline than patients with fibrosis (15%vs 29%, P = 0.03) and had lower levels of HCV c100 responses at baseline, M24 and M48 (P = 0.11, P = 0.05, P = 0.02, respectively). For 97 patients with complete longitudinal data, the frequency of positive LP responses to HCV, tetanus and Candida antigens declined over time (P < 0.003), and the slope of this decline was greater in the PEG-IFN treatment group than the observation group (P < 0.02). Lower levels of tetanus LP responses were associated with fibrosis progression and clinical outcomes (P = 0.009). Poorer CD4+ T cell proliferative function was associated with more advanced liver disease in chronic hepatitis C and may be further affected by long-term PEG-IFN treatment.

Soil properties and productivity in two long-term crop rotations differing with respect to organic matter management on an Albic Luvisol.

This study was based on a three-factor experiment carried out since 1980 on a loamy sand (Albic Luvisol) in which arable crops were grown in two 4-years rotations: RotA (grain maize, winter wheat, spring barley and silage maize) and RotB [grain maize, winter wheat plus mustard green manure (GM), spring barley and grass-clover ley (GCL)]. The soil in RotB with an increased input of OM (GM and 1-year GCL) accumulated significantly larger amounts of soil organic carbon and soil microbial biomass C, had higher activities of dehydrogenase and acid phosphatase enzymes and gave significantly higher winter wheat grain yields compared to the soil in RotA. However, in the absence of liming, the soil in RotB, contrary to that in RotA, became more acidic, had reduced activity of alkaline phosphatase and lower contents of Ca and Mg, and contained a diminished proportion of the >0.5 mm macroaggregates fraction. These soil deteriorative effects of crop rotations delivering larger amounts of OM have not been reported so far. In both rotations FYM applied once per 4-year rotation at 40 Mg ha-1 improved all the tested soil properties and had mitigating effects on the negative changes found in the soil of RotB.

HLA class I-restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterization of patterns of cytokine release.

Cytotoxic T lymphocytes (CTL) are important to the control of viral replication and their presence may be important to disease outcome. An understanding of the spectrum of proteins recognized by hepatitis C virus (HCV)-specific CTL and the functional properties of these cells is an important step in understanding the disease process and the mechanisms of persistent infection, which occurs in the majority of HCV-infected individuals. In this report we identify HCV-specific CTL responses restricted by the HLA class I molecules A2, A3, A11, A23, B7, B8, and B53. The epitopes recognized by these intrahepatic CTL conform to published motifs for binding to HLA class I molecules, although in some cases we have identified CTL epitopes for which no published motif exists. The use of vectors expressing two different strains of HCV, HCV-1 and HCV-H, revealed both strain-specific and cross-reactive CTL. These HCV-specific CTL were shown to produce cytokines including IFN-gamma, TNF-alpha, GM-CSF, IL-8, and IL-10 in an antigen- and HLA class I-specific manner. These studies indicate that the CTL response to HCV is broadly directed and that as many as five different epitopes may be targeted in a single individual. The identification of minimal epitopes may facilitate peptide-specific immunization strategies. In addition, the release of proinflammatory cytokines by these cells may contribute to the pathogenesis of HCV-induced liver damage.

Transgenic plants: an emerging approach to pest control.

Insect pests are a major cause of damage to the world's commercially important agricultural crops. Current strategies aimed at reducing crop losses rely primarily on chemical pesticides. Alternatively transgenic crops with intrinsic pest resistance offer a promising alternative and continue to be developed. The first generation of insect-resistant transgenic plants are based on insecticidal proteins from Bacillus thuringiensis (Bt). A second generation of insect-resistant plants under development include both Bt and non-Bt proteins with novel modes of action and different spectra of activity against insect pests.

Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms.

Field tests of corn co-expressing two new delta-endotoxins from Bacillus thuringiensis (Bt) have demonstrated protection from root damage by western corn rootworm (Diabrotica virgifera virgifera LeConte). The level of protection exceeds that provided by chemical insecticides. In the bacterium, these proteins form crystals during the sporulation phase of the growth cycle, are encoded by a single operon, and have molecular masses of 14 kDa and 44 kDa. Corn rootworm larvae fed on corn roots expressing the proteins showed histopathological symptoms in the midgut epithelium.

The immunopathogenesis of HBV infection.

Clinical manifestations of hepatitis B virus (HBV) infection are a balance between viral and host factors. The immune response against any virus consists of a coordinated defence of innate immunity and acquired, virus-specific immunity. In acute HBV, immune responses associated with recovery include vigorous, polyclonal CD4 T cells directed against multiple epitopes within HBV; antibodies directed against surface envelope proteins (anti-HBs), the development of which requires the presence of a CD4 response; and HBV-specific cytotoxic T lymphocytes (CTLs). HBV-specific CTLs can induce death of infected hepatocytes as well as produce cytokines. Most individuals with acute HBV recover without evidence of massive liver destruction; this, plus evidence from transgenic animal models, suggests that these cytokines produced by T cells play an important role in controlling HBV replication. Individuals who fail to mount a vigorous response in acute HBV develop chronic infection. In these cases, the persisting ineffective immune response appears to be responsible for liver damage and is likely to initiate the process of hepatic fibrosis. Based on our current understanding of the immune response in acute and chronic HBV, several groups are investigating the prospect of manipulating the immune response in chronic HBV.

Immunology of viral hepatitis.

So far, five major forms of viral hepatitis, hepatitis A, B, C, D, and E, have been identified. There appears to be at least one other form of enterically transmitted and one other parenterally transmitted hepatotropic virus, but characterization of these viruses is still preliminary. The five hepatotropic viruses have unique structures, yet all the share the property of inducing hepatocellular damage, whether through direct cytotoxicity or through induction of immune mechanisms that lead to hepatocellular necrosis. Advances in molecular biology in the past decade have enabled researchers to understand much about the structure, mechanisms of replication, and viral life cycle of each of these viruses, and successful vaccines have been developed for hepatitis A and B. However, many problems remain unsolved, including which immune system factors are important defenses against these viral infections, which components of the immune system are necessary for a successful vaccine, and what allows some viruses, such as hepatitis B and hepatitis C virus, to become persistent and lead to chronic liver disease.

Hepatitis C virus and organ transplantation.

Hepatitis C virus (HCV) is both the leading cause of cirrhosis and hepatic failure leading to liver transplantation and a cause of chronic hepatitis in approximately 10% of all transplant recipients. Beginning 5-10 years or more posttransplant, HCV causes progressive liver disease in a significant fraction of infected individuals and contributes to an increased incidence of opportunistic infection and hepatocellular carcinoma. The existence of multiple genotypes of HCV with differing biologic behaviors and the generation of antigenic diversity of the virus (quasispecies) during the course of infection, limit the capacity of the immune system to generate protective immunity. Antiviral therapy with interferon-alpha is effective in only a minority of transplant patients, and since allografts from HCV infected donors are quite efficient in transmitting the virus, great attention is paid to the appropriate use of organs from HCV-positive donors. At present, these organs should be particularly targeted for patients in emergent need of lifesaving heart, liver, or lung transplants. Issues requiring further investigation include the impact of viral superinfection on HCV-infected recipients of organs from HCV-infected donors and the use of such organs in seronegative patients who are older, diabetic, or highly sensitized, for whom quality of life issues may outweigh the long-term impact of HCV infection.

Pulmonary complications of diabetes mellitus. Pneumonia.

Diabetes mellitus is often identified as an independent risk factor for developing lower respiratory tract infections. Pulmonary infections, such as those caused by Mycobacterum tuberculosis, mucor, Staphylococcus aureus, and gram-negative bacteria may occur with an increased frequency whereas infections due to Streptococcus pneumoniae, Legionella, and influenza may be associated with increased morbidity and mortality. The predisposition to lower respiratory tract infections may represent alterations in pulmonary host defenses at several levels. The purpose of this article is to review the spectrum of pulmonary infections encountered in the diabetic patient, focusing on predisposing defects in pulmonary host defense, highlighting characteristic clinical features, and discussing diagnostic approaches, therapeutic interventions, and prophylaxis in this patient population.

Characterization of HCV-Specific Cytotoxic T-Lymphocytes from Liver Tissue.

Cellular immune responses, especially those mediated by cytotoxic T-lymphocytes (CTLs), are an important component of the host immune response in many viral infections. For many years, it has been observed that CD8(+) cells were present in large numbers in the liver of patients with chronic HCV (1), but it was unknown whether these cells represented virus-specific immune responses. In order to understand the potential role of these CD8(+) lymphocytes in the disease course, it is first necessary to define the functional characteristics of the cells, including a precise definition of the epitopes, which are recognized by these CD8(+) lymphocytes.

Lower peripheral blood CD14+ monocyte frequency and higher CD34+ progenitor cell frequency are associated with HBV vaccine induced response in HIV infected individuals.

We evaluated immunologic predictors of response to HBV vaccine administered in the presence or absence of GM-CSF in HIV infected individuals. We measured peripheral blood hematopoietic progenitor, monocyte and myeloid-derived suppressor cell (MDSC) frequencies, and expression of GMCSF receptor on monocytes and MDSCs, at baseline and 4weeks after immunization in relation to antibody response. We observed higher baseline progenitor and lower monocyte frequencies among week 16 antibody responders. Week 4 decline in MDSC frequency was associated with week 16 antibody response, while administration of GM-CSF was associated with preservation of these cells. No significant differences in GM-CSF receptor expression were observed in the presence vs. absence of GM-CSF. These findings are consistent with a positive role of progenitor cells and a potential negative role of monocytes in vaccine response. Additionally, GM-CSF augmented the preservation of peripheral blood MDSC, which may contribute to the lack of improved vaccine responses.

Immune response to hepatitis B vaccine in HIV-infected subjects using granulocyte-macrophage colony-stimulating factor (GM-CSF) as a vaccine adjuvant: ACTG study 5220.

HIV-infected persons are at risk for HBV co-infection which is associated with increased morbidity and mortality. Unfortunately, protective immunity following HBV vaccination in HIV-infected persons is poor. This randomized, phase II, open-label study aimed to evaluate efficacy and safety of 40 mcg HBV vaccine with or without 250 mcg GM-CSF administered at day 0, weeks 4 and 12. HIV-infected individuals >or=18 years of age, CD4 count >or=200 cells/mm(3), seronegative for HBV and HCV, and naïve to HBV vaccination were eligible. Primary endpoints were quantitative HBsAb titers and adverse events. The study enrolled 48 subjects. Median age and baseline CD4 were 41 years and 446 cells/mm(3), 37 were on ART, and 26 subjects had undetectable VL. Vaccination was well tolerated. Seven subjects in the GM-CSF arm reported transient grade >or=2 signs/symptoms (six grade 2, one grade 3), mostly aches and nausea. GM-CSF had no significant effect on VL or CD4. Four weeks after vaccination, 26 subjects (59%) developed a protective antibody response (HBsAb >or=10 mIU/mL; 52% in the GM-CSF arm and 65% in the control arm) without improved Ab titer in the GM-CSF vs. control arm (median 11 mIU/mL vs. 92 mIU/mL, respectively). Response was more frequent in those with CD4 >or=350 cells/mm(3) (64%) than with CD4 <350 cells/mm(3) (50%), though not statistically significant. GM-CSF as an adjuvant did not improve the Ab titer or the development of protective immunity to HBV vaccination in those receiving an accelerated vaccine schedule. Given the common routes of transmission for HIV and HBV, additional HBV vaccine research is warranted.

The role of immune responses in the pathogenesis of hepatitis C virus infection.

Hepatitis C virus (HCV) is notable for the high rate of chronic infection, which occurs in nearly all individuals who become infected. Liver biopsies from individuals with chronic HCV infection are notable for the presence of numerous mononuclear cells, at least some of which are CD4+ and CD8+ T lymphocytes. The immune response to HCV is polyclonal and multispecific, both in terms of antibody and cellular immune responses. Individuals who recover from acute HCV infection appear to have quantitatively more vigorous CD4+ proliferative responses against one or more HCV proteins compared with those individuals who develop chronic disease. CD8+ responses are less well characterized, in part because of the technical difficulties involved in isolating and characterizing these cells. HCV-specific CTL can be readily isolated from the liver and PBMC of chronically infected individuals, and recognize multiple epitopes. Even individuals with the same HLA type do not consistently recognize the same epitope. Thus, there does not appear to be an immunodominant response on the CD8+ level in this infection. CD8+ cells do appear to play some role in limiting viral replication. These responses are insufficient to eradicate virus completely, however, and may cause liver injury once chronic infection is established. Cytokines produced by both CD4+ and CD8+ cells may play an important role in both inhibiting viral replication and causing liver injury. A better understanding of the role of cellular immunity in the pathogenesis of HCV infection may aid in the development of vaccines and immunotherapeutic intervention strategies.

Cytokines in viral hepatitis.

Cytokines play an important role in the defense against viral infections, both indirectly, through determination of the predominant pattern of host response, and directly, through inhibition of viral replication. However, in the context of an inflammatory response against a virus, cytokines may also lead to liver damage. The importance of this is best demonstrated in hepatitis B virus (HBV). In acute HBV infection, a vigorous polyclonal cellular immune response is critical; thus type 1 cytokine release is essential to initiating an effective immune response. The cytokines released by CD4+ and CD8+ cells also play an important role in downregulation of HBV replication, demonstrating that it is possible to control a viral infection without the death of infected cells. However, if there is a defect in the acute response, HBV becomes chronic; in that case, the presence of an ongoing suboptimal inflammatory response can activate the process of hepatic fibrosis. In hepatitis C infection, the role of cellular immune responses and cytokines is less clear. Hepatitis C may be resistant to inhibition by cytokines, so cytokines may have a more prominent role in liver damage than in controlling viral replication. Both hepatitis B and C may have specific mechanisms to inhibit cytokine production, highlighting the critical role of these molecules in recovery from infection.