Local immune response to injection of Plasmodium sporozoites into the skin.

Malarial infection is initiated when the sporozoite form of the Plasmodium parasite is inoculated into the skin by a mosquito. Sporozoites invade hepatocytes in the liver and develop into the erythrocyte-infecting form of the parasite, the cause of clinical blood infection. Protection against parasite development in the liver can be induced by injection of live attenuated parasites that do not develop in the liver and thus do not cause blood infection. Radiation-attenuated sporozoites (RAS) and genetically attenuated parasites are now considered as lead candidates for vaccination of humans against malaria. Although the skin appears as the preferable administration route, most studies in rodents, which have served as model systems, have been performed after i.v. injection of attenuated sporozoites. In this study, we analyzed the early response to Plasmodium berghei RAS or wild-type sporozoites (WTS) injected intradermally into C57BL/6 mice. We show that RAS have a similar in vivo distribution to WTS and that both induce a similar inflammatory response consisting of a biphasic recruitment of polymorphonuclear neutrophils and inflammatory monocytes in the skin injection site and proximal draining lymph node (dLN). Both WTS and RAS associate with neutrophils and resident myeloid cells in the skin and the dLN, transform inside CD11b(+) cells, and induce a Th1 cytokine profile in the dLN. WTS and RAS are also similarly capable of priming parasite-specific CD8(+) T cells. These studies delineate the early and local response to sporozoite injection into the skin, and suggest that WTS and RAS prime the host immune system in a similar fashion.

Type I and type III interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia.

Interferons (IFNs) are a group of cytokines with a well-established antiviral function. They can be induced by viral infection, are secreted and bind to specific receptors on the same or neighbouring cells to activate the expression of hundreds of IFN stimulated genes (ISGs) with antiviral function. Type I IFN has been known for more than half a century. However, more recently, type III IFN (IFNλ, IL-28/29) was shown to play a similar role and to be particularly important at epithelial surfaces. Here we show that airway epithelia, the primary target of influenza A virus, produce both IFN I and III upon infection, and that induction of both depends on the RIG-I/MAVS pathway. While IRF3 is generally regarded as the transcription factor required for initiation of IFN transcription and the so-called "priming loop", we find that IRF3 deficiency has little impact on IFN expression. In contrast, lack of IRF7 reduced IFN production significantly, and only IRF3(-/-)IRF7(-/-) double deficiency completely abolished it. The transcriptional response to influenza infection was largely dependent on IFNs, as it was reduced to a few upregulated genes in epithelia lacking receptors for both type I and III IFN (IFNAR1(-/-)IL-28Rα(-/-)). Wild-type epithelia and epithelia deficient in either the type I IFN receptor or the type III IFN receptor exhibit similar transcriptional profiles in response to virus, indicating that none of the induced genes depends selectively on only one IFN system. In chimeric mice, the lack of both IFN I and III signalling in the stromal compartment alone significantly increased the susceptibility to influenza infection. In conclusion, virus infection of airway epithelia induces, via a RIG-I/MAVS/IRF7 dependent pathway, both type I and III IFNs which drive two completely overlapping and redundant amplification loops to upregulate ISGs and protect from influenza infection.

Dendritic cells sequester antigenic epitopes for prolonged periods in the absence of antigen-encoding genetic information.

Studies with a number of viral systems have shown, on the basis of the ability of a host to prime naïve T cells, that viral antigens persist in the infected host well beyond complete clearance of the infection and even when viral antigen is undetectable by the most sensitive methods. This has led to a reasonable assumption that the antigen persists through persistence of antigen-encoding genetic information (DNA or RNA) that resides in the host at a subdetectable level. Here, we demonstrate that epitopes, or epitope precursors, of a model antigen (ovalbumin) persist in a host for prolonged periods (weeks), well beyond the time at which the intact antigen has disappeared, and in the complete absence of genetic information encoding it. Dendritic cells are shown to be the site of this epitope sequestration in vivo, as well as in cultures in vitro. For sequestration to occur, the uptaken antigen must be significantly large, that is, the epitope and its 18-mer precursor are not sequestered. Dendritic cells are shown to create an hsp90-dependent intracellular pool of epitopes or epitope precursors that continues to release epitopes for presentation on the major histocompatibility complex I molecules for prolonged periods. Demonstration of such long-term sequestration of antigenic epitopes inside dendritic cells presents new opportunities for stimulation of immune response against cancers and viruses.

Cutting edge: independent roles for IRF-3 and IRF-7 in hematopoietic and nonhematopoietic cells during host response to Chikungunya infection.

The host response to Chikungunya virus is dependent on the direct action of type I IFN on infected nonhematopoietic cells. Prior studies have demonstrated that multiple host sensors coordinate an antiviral response; however, the tissue source(s) and signaling pathways for IFN production remain unknown. In this study, we demonstrate that IRF-3 and IRF-7 are functionally redundant, but lack of both factors results in lethal infection in adult mice. Reciprocal bone marrow chimeras indicated that IRF-3 or IRF-7 expression in either hematopoietic or nonhemotopoietic cell compartments was capable of inducing an antiviral response. Interestingly, redundancy of IRF-3 and IRF-7 was age dependent, as neonatal animals lacking either factor succumbed to infection. We further demonstrate that IPS-1 is essential in nonhematopoietic cells and preferentially required during early life. These results highlight the interplay between nonimmune and immune cells during Chikungunya virus infection and suggest an important role for nonhematopoietic cells as a critical source of IFN-α/ß.

Mechanism of dichotomy between CD8+ responses elicited by apoptotic and necrotic cells.

Apoptotic cells are significantly more immunogenic than necrotic cells, even though both forms are identical in antigenic content. When a combination of apoptotic and necrotic cells are used to immunize, the phenotype conferred by apoptotic cells, i.e., high immunogenicity, is dominant. However, necrotic cells are not immunosuppressive or tolerogenic. Apoptotic and necrotic cells are taken up by antigen-presenting cells in an equivalent manner. The priming of naïve T cell response is also equivalent. However, the CD8+ T cells elicited by apoptotic cells expand, accumulate, and express effector function, while those primed by the necrotic cells do not. This dichotomy does not extend to CD4+ cells. Apoptotic and necrotic cells elicit equivalent CD4+ T cell priming, accumulation, and function. The deficit in CD8+ T cell function elicited by necrotic cells can be overcome to varying degrees by anti-CD40 antibody and ligands for TLR4 and TLR9; conversely, the immunogenicity of apoptotic cells can be abrogated by blocking anti-CD154 antibody. Our results indicate that immunization with apoptotic cells leads to engagement of CD40 on antigen-presenting cells; this is essential for their ability to elicit mature functional CD8+ cells. The necrotic cells fail to engage CD40, and this failure is the basis of their lack of immunogenicity. These differences have consequences for the understanding of mechanisms of cross-presentation and for efforts toward immunotherapy of cancers and autoimmune pathologies.

Litchi-associated acute encephalitis in children, Northern Vietnam, 2004-2009.

Since the end of the 1990s, unexplained outbreaks of acute encephalitis in children coinciding with litchi harvesting (May-July) have been documented in the Bac Giang Province in northern Vietnam. A retrospective ecologic analysis of data for 2004-2009 involving environmental, agronomic, and climatic factors was conducted to investigate the suspected association between the outbreaks and litchi harvesting. The clinical, biological, and immunologic characteristics of the patients suggested a viral etiology. The ecologic study revealed an independent association between litchi plantation surface proportion and acute encephalitis incidence: Incidence rate ratios were 1.52 (95% CI 0.90-2.57), 2.94 (95% CI 1.88-4.60), and 2.76 (95% CI 1.76-4.32) for second, third, and fourth quartiles, respectively, compared with the lowest quartile. This ecologic study confirmed the suspected association between incidence of acute encephalitis and litchi plantations and should be followed by other studies to identify the causative agent for this syndrome.

Identification of a novel neuropathogenic Theiler's murine encephalomyelitis virus.

Theiler's murine encephalitis viruses (TMEV) are divided into two subgroups based on their neurovirulence. Persistent strains resemble Theiler's original viruses (referred to as the TO subgroup), which largely induce a subclinical polioencephalomyelitis during the acute phase of the disease and can persist in the spinal cord of susceptible animals, inducing a chronic demyelinating disease. In contrast, members of the neurovirulent subgroup cause an acute encephalitis characterized by the rapid onset of paralysis and death within days following intracranial inoculation. We report herein the characterization of a novel neurovirulent strain of TMEV, identified using pyrosequencing technology and referred to as NIHE. Complete coverage of the NIHE viral genome was obtained, and it shares <90% nucleotide sequence identity to known TMEV strains irrespective of subgroup, with the greatest sequence variability being observed in genes encoding the leader and capsid proteins. The histopathological analysis of infected brain and spinal cord demonstrate inflammatory lesions and neuronal necrosis during acute infection with no evidence of viral persistence or chronic disease. Intriguingly, genetic analysis indicates the putative expression of the L protein, considered a hallmark of strains within the persistent subgroup. Thus, the identification and characterization of a novel neurovirulent TMEV strain sharing features previously associated with both subgroups will lead to a deeper understanding of the evolution of TMEV strains and new insights into the determinants of neurovirulence.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c(+) cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Tail profile: a more accurate system for analyzing DNA damage using the Comet assay.

The Comet assay (single cell gel electrophoresis assay) measures DNA strand breaks in individual cells. In the assay cells are embedded in agarose, lysed, and electrophoresed under low voltage, allowing migration of damaged DNA. The DNA is stained and subsequently viewed with an epifluorescent microscope. If DNA damage has occurred the electrophoresed DNA fragments appear as a diffuse tail behind the nucleus known as a "comet". Many computer-aided analysis systems are currently in use to quantify the amount of DNA damage that is represented by a comet image. Here, we present a novel method of analysis known as "tail profile". This method of analysis provides several advantages over currently employed methods, which rely primarily on the "tail moment" method of analysis. We compared the amount of DNA damage reported from both the tail profile and tail moment methods of analysis and observed a 26% (P<0.0001) increase in damage detected by tail profile across the 10-25 microm range of tail length, where the majority of the relevant comet data is concentrated. We further report that this increase in sensitivity is not only limited to assessing DNA damage, but also to gathering data from DNA repair assays. Furthermore, we demonstrate increased functionality and extended data analysis capabilities with the use of a compressed collection of images called a "comet chip" and through a visual representation of data called a "profile plot". Use of the custom macros enabled us to detect an unexpected characteristic of the electrophoretic profile, giving us novel insight into the nature of comet analysis. In addition to the increased analytical sensitivity proffered by this system, the tail profile macros are upgradeable and platform independent.

"It is the antigen(s), stupid" and other lessons from over a decade of vaccitherapy of human cancer.

The lessons are: (a) human cancers certainly respond to immunological manipulations. Efforts at human cancer immunotherapy are therefore worthwhile. (b) Prophylaxis is very different from therapy of pre-existing disease, and hence much enthusiasm should not be derived from successful prophylaxis studies. Even in case of infectious agents against which robust prophylaxis is routinely achieved, therapy is nearly impossible once the disease has established. (c) Studies with appropriate cancer models of mice and rats are useful. The notion that it is easy to cure cancers in mice is generally advanced the most confidently by those who have never cured a mouse of cancer by immunotherapy. (d) With a nod to James Carville, it is the antigen(s), stupid! We still do not know the identity of protective tumor antigens. If any lesson can be drawn at all, it may well be that cancer immunotherapy must move away from the one-shoe-fits-all therapeutic models of chemotherapy and must move to individualized approaches. (e) All targets are equal, but some are more equal than others. The key is specificity for cancer. That does not necessarily mean specificity for cancer cells. (f) Vaccitherapy must be attempted preferably in the minimal residual disease setting, even though this is certain to be time-taking and expensive. In the setting of bulky disease, vaccitherapy must be combined with blockade of inhibitory signals, or depletion of down-regulatory T cells. Inhibition of effector level suppression of immune response is a key. Vaccitherapy alone or immuno-modulation alone is unlikely to succeed in therapy of bulky metastatic disease.