Protective immunity to leishmaniasis has been demonstrated in murine models to be mediated by T cells and the cytokines they produce. We have previously shown that resistance to experimental Leishmania infantum infection in the dog, a natural host and reservoir of the parasite, is associated with the proliferation of peripheral blood mononuclear cells (PBMC) to parasite antigen and to the production of interleukin-2 and tumour necrosis factor. In this study we show that PBMC from asymptomatic experimentally infected dogs produce interferon-gamma upon parasite antigen-specific stimulation, whereas lymphocytes from symptomatic dogs do not. In addition, we report for the first time the lysis of L. infantum-infected macrophages by PBMC from asymptomatic dogs and by parasite-specific T cell lines derived from these animals. These T cell lines were generated by restimulation in vitro with parasite soluble antigen and irradiated autologous PBMC as antigen-presenting cells. We show that lysis of infected macrophages by T cell lines is major histocompatibility complex restricted. Characterization of parasite-specific cytotoxic T cell lines revealed that the responding cells are CD8+. However, for some animals, CD4+ T cells that lyse infected macrophages were also found. In contrast to asymptomatic dogs, lymphocytes from symptomatic dogs failed to proliferate and produce interferon-gamma after Leishmania antigen stimulation in vitro and were not capable of lysing infected macrophages. These results suggest that both the production of interferon-gamma and the destruction of the parasitized host cells by Leishmania-specific T cells play an important role in resistance to visceral leishmaniasis.

Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and ␤-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and ␤-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin-and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Lipopolysaccharide (LPS), a major component of the Gram-negative bacterial outer membrane, is sensed by mammalian cells through Toll-like receptor 4 (TLR4), resulting in activation of proinflammatory cytokine pathways. TLR4 recognizes the lipid A moiety of the LPS molecule, and the chemical composition of the lipid A determines how well it is recognized by TLR4. N. meningitidis has been reported to produce lipid A with six acyl chains, the optimal number for TLR4 recognition. Indeed, meningococcal sepsis is generally seen as the prototypical endotoxin-mediated disease. In the present study, we screened meningococcal disease isolates from 464 patients for their ability to induce cytokine production in vitro. We found that around 9% of them were dramatically less potent than wild-type strains. Analysis of the lipid A of several of the low-activity strains by mass spectrometry revealed they were penta-acylated, suggesting a mutation in the lpxL1 or lpxL2 genes required for addition of secondary acyl chains. Sequencing of these genes showed that all the low activity strains had mutations that inactivated the lpxL1 gene. In order to see whether lpxL1 mutants might give a different clinical picture, we investigated the clinical correlate of these mutations in a prospective nationwide observational cohort study of adults with meningococcal meningitis. Patients infected with an lpxL1 mutant presented significantly less frequently with rash and had higher thrombocyte counts, consistent with reduced cytokine induction and less activation of tissue-factor mediated coagulopathy. In conclusion, here we report for the first time that a surprisingly large fraction of meningococcal clinical isolates have LPS with underacylated lipid A due to mutations in the lpxL1 gene. The resulting low-activity LPS may have an important role in virulence by aiding the bacteria to evade the innate immune system. Our results provide the first example of a specific mutation in N. meningitidis that can be correlated with the clinical course of meningococcal disease.

Influenza infections are responsible for significant morbidity and mortality each year, with the highest infection rates found in the elderly population. The main strategy to reduce the impact of influenza infections in the elderly population is vaccination. However, the efficacy of influenza vaccines that are licensed for use in the elderly is relatively low (17-53%). The complex age-related changes that occur in both innate and adaptive immunity are thought to hamper the immune response to influenza immunization and to reduce protection against infection in the elderly. For the development of improved vaccines that overcome the limitations of an aged immune system, it is crucial to understand the mechanisms that lead to immune dysfunction. Here, we review the recent progress in unravelling the mechanisms behind the age-related immune dysfunction in elderly, as well as the recent developments in improving influenza vaccines and identification of new correlates of protection.

NK cells play an important role in the early defense against invading pathogens. Although it is well-established that infection leads to a substantial, local increase in NK cell numbers, little is known about the mechanisms that trigger their proliferation and migration. We here investigated the dynamics of NK cell responses following intranasal respiratory virus infection. We show that NK cell numbers increased in the airways following influenza virus infection, but find no evidence of proliferation either at the site of infection or in the draining lymph nodes. Instead, we find that the bone marrow (BM) is the primary site of proliferation of both immature and mature NK cells during infection. Using an adoptive transfer model, we demonstrate that peripheral, long-lived and phenotypically mature NK cells migrate back to the BM and proliferate there, both homeostatically and in response to infection. Thus, the BM is not only a site of NK cell development, but also an important site for proliferation of long-lived mature NK cells.