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1.  Schistosoma mansoni-Mediated Suppression of Allergic Airway Inflammation Requires Patency and Foxp3+ Treg Cells 
The continual rise of asthma in industrialised countries stands in strong contrast to the situation in developing lands. According to the modified Hygiene Hypothesis, helminths play a major role in suppressing bystander immune responses to allergens, and both epidemiological and experimental studies suggest that the tropical parasitic trematode Schistosoma mansoni elicits such effects. The focus of this study was to investigate which developmental stages of schistosome infection confer suppression of allergic airway inflammation (AAI) using ovalbumin (OVA) as a model allergen. Moreover, we assessed the functional role and localization of infection-induced CD4+Foxp3+ regulatory T cells (Treg) in mediating such suppressive effects. Therefore, AAI was elicited using OVA/adjuvant sensitizations with subsequent OVA aerosolic challenge and was induced during various stages of infection, as well as after successful anti-helminthic treatment with praziquantel. The role of Treg was determined by specifically depleting Treg in a genetically modified mouse model (DEREG) during schistosome infection. Alterations in AAI were determined by cell infiltration levels into the bronchial system, OVA-specific IgE and Th2 type responses, airway hyper-sensitivity and lung pathology. Our results demonstrate that schistosome infection leads to a suppression of OVA-induced AAI when mice are challenged during the patent phase of infection: production of eggs by fecund female worms. Moreover, this ameliorating effect does not persist after anti-helminthic treatment, and depletion of Treg reverts suppression, resulting in aggravated AAI responses. This is most likely due to a delayed reconstitution of Treg in infected-depleted animals which have strong ongoing immune responses. In summary, we conclude that schistosome-mediated suppression of AAI requires the presence of viable eggs and infection-driven Treg cells. These data provide evidence that helminth derived products could be incorporated into treatment strategies that specifically target suppression of immune responses in AAI by inducing Treg cells.
Author Summary
Infections with schistosomes, such as S. mansoni, S. japonicum and S. haematobium, are considered a major public health concern. Morbidity arises through granulomatous responses to eggs that become trapped in infected tissues. Interestingly, schistosomes belong to the group of helminths that have been shown to reduce allergy or autoimmunity. Indeed, the evidence provided by epidemiological surveys and experimental animal models has been so overwhelming that such helminths are now included in the Hygiene Hypothesis. However, since helminths provoke immunological responses that are similar to those seen in allergy (increased eosinophilia and IgE) it is suggested that additional mechanisms dampen such allergic responses. Helminth-induced regulatory T cells (Treg) are considered a component of these modulatory networks. Using an allergic airway inflammation model, we have elucidated that schistosome-mediated protection requires patency, that is, active egg production from fecund female worms. In addition, protection was shown to be mediated by infection-induced Treg. Interestingly, in endemic countries it is usually individuals with strong patent infections that show reduced allergic prevalence. Thus, further research into the immunomodulatory capacity of schistosome-egg derived factors may elucidate novel drug candidates or enhance treatment strategies to reduce allergic responses on the cellular level.
doi:10.1371/journal.pntd.0002379
PMCID: PMC3744427  PMID: 23967364
2.  Ly6Chi Monocyte Recruitment Is Responsible for Th2 Associated Host-Protective Macrophage Accumulation in Liver Inflammation due to Schistosomiasis 
PLoS Pathogens  2014;10(8):e1004282.
Accumulation of M2 macrophages in the liver, within the context of a strong Th2 response, is a hallmark of infection with the parasitic helminth, Schistosoma mansoni, but the origin of these cells is unclear. To explore this, we examined the relatedness of macrophages to monocytes in this setting. Our data show that both monocyte-derived and resident macrophages are engaged in the response to infection. Infection caused CCR2-dependent increases in numbers of Ly6Chi monocytes in blood and liver and of CX3CR1+ macrophages in diseased liver. Ly6Chi monocytes recovered from liver had the potential to differentiate into macrophages when cultured with M-CSF. Using pulse chase BrdU labeling, we found that most hepatic macrophages in infected mice arose from monocytes. Consistent with this, deletion of monocytes led to the loss of a subpopulation of hepatic CD11chi macrophages that was present in infected but not naïve mice. This was accompanied by a reduction in the size of egg-associated granulomas and significantly exacerbated disease. In addition to the involvement of monocytes and monocyte-derived macrophages in hepatic inflammation due to infection, we observed increased incorporation of BrdU and expression of Ki67 and MHC II in resident macrophages, indicating that these cells are participating in the response. Expression of both M2 and M1 marker genes was increased in liver from infected vs. naive mice. The M2 fingerprint in the liver was not accounted for by a single cell type, but rather reflected expression of M2 genes by various cells including macrophages, neutrophils, eosinophils and monocytes. Our data point to monocyte recruitment as the dominant process for increasing macrophage cell numbers in the liver during schistosomiasis.
Author Summary
Schistosomiasis is an important neglected tropical disease caused by parasitic worms of the genus Schistosoma. During infection with S. mansoni, parasite eggs become trapped in the liver and elicit granulomatous inflammation characterized by accumulations of immune cells intermixed with liver cells around the eggs. This inflammation is responsible for disease symptoms, but also plays an important role in protecting the host against liver damage that can be caused by egg products. Granulomas, by definition, contain a significant number of macrophages (phagocytic cells of the immune system). Recent work has emphasized that macrophage numbers in inflammation can increase due either to recruitment of precursor cells (called monocytes) from the blood, or as a result of proliferation of tissue-resident macrophages. Local proliferation has been noted in other worm infections, during which the immune response is Th2-like and IL-4 produced by Th2 cells promotes macrophages to become “alternatively (or M2) activated”. We examined the origin of the increased numbers of macrophages in liver inflammation due to schistosomiasis, in which there is also a prominent Th2 response. We found that the cells mostly originated from monocytes recruited into the tissue from the blood. This response was critical for host survival during infection.
doi:10.1371/journal.ppat.1004282
PMCID: PMC4140849  PMID: 25144366
3.  IL-10R Blockade during Chronic Schistosomiasis Mansoni Results in the Loss of B Cells from the Liver and the Development of Severe Pulmonary Disease 
PLoS Pathogens  2012;8(1):e1002490.
In schistosomiasis patients, parasite eggs trapped in hepatic sinusoids become foci for CD4+ T cell-orchestrated granulomatous cellular infiltrates. Since the immune response is unable to clear the infection, the liver is subjected to ongoing cycles of focal inflammation and healing that lead to vascular obstruction and tissue fibrosis. This is mitigated by regulatory mechanisms that develop over time and which minimize the inflammatory response to newly deposited eggs. Exploring changes in the hepatic inflammatory infiltrate over time in infected mice, we found an accumulation of schistosome egg antigen-specific IgG1-secreting plasma cells during chronic infection. This population was significantly diminished by blockade of the receptor for IL-10, a cytokine implicated in plasma cell development. Strikingly, IL-10R blockade precipitated the development of portal hypertension and the accumulation of parasite eggs in the lungs and heart. This did not reflect more aggressive Th2 cell responsiveness, increased hepatic fibrosis, or the emergence of Th1 or Th17 responses. Rather, a role for antibody in the prevention of severe disease was suggested by the finding that pulmonary involvement was also apparent in mice unable to secrete class switched antibody. A major effect of anti-IL-10R treatment was the loss of a myeloid population that stained positively for surface IgG1, and which exhibited characteristics of regulatory/anti-inflammatory macrophages. This finding suggests that antibody may promote protective effects within the liver through local interactions with macrophages. In summary, our data describe a role for IL-10-dependent B cell responses in the regulation of tissue damage during a chronic helminth infection.
Author Summary
Schistosomiasis is a chronic disease that affects approximately 200 million people. Immune modulation is a hallmark of chronic disease and serves to protect the host from severe pathology. A significant percentage of people infected with schistosomiasis fail to undergo this protective modulation and can develop portal hypertension with resulting pulmonary complications. Here we show that schistosome-specific antibody-secreting B cells accumulate in the liver as the infection progresses to the chronic state and that this accumulation is dependent on the cytokine Interleukin-10. Blocking the IL-10R results in not only the loss of B cells from the liver, but also the development of severe pulmonary pathology. We found similar changes in disease progression in mice genetically unable to mount normal antibody responses. We believe that antibody is important for triggering the production of anti-inflammatory factors, including IL-10 itself, by other immune cells called macrophages. Our data suggest that during chronic schistosomiasis IL-10 promotes the development of a population of B cells within the liver that is responsible for minimizing inflammation and preventing the development of disease in the lungs. Our findings provide a mouse model that may be of use for studying the development of pulmonary complications due to chronic schistosomiasis.
doi:10.1371/journal.ppat.1002490
PMCID: PMC3266936  PMID: 22291593
4.  TWO CASES OF SARCOMATOSIS WITH PURPURA HÆMORRHAGICA 
The purpura accompanying the two foregoing cases of sarcoimatosis would seem to find its explanation in the coexistence of several factors, the main feature being an involvement of the vascular system by the sarcomatous elements. There existed in Case I a direct lesion of the vessel wall whereby the sarcoma cells invaded directly the various coats, and were found mainly between the intima and the adventitia, dissecting their way, as it were, along these tracts in the vessel walls. There was further an extensive involvement of the perivascular lymphatics, from which point, indeed, it would seem that the sarcoma cells had invaded the walls of the vessels themselves. In Case II, moreover, not only was there a definite invasion of the lymph spaces near the vessels, but, furthermore, there was undoubted evidence of the existence of emboli of sarcoma cells in the lumina of the blood vessels; and in the immediate vicinity of such conditions hæmorrhages were invariably found. While some vessels, and indeed a great many, were quite free from such emboli, in others the lumina were completely occluded by spindle cells, so as to preclude the possibility that these were merely a collection of desquamated endothelial cells, such as is frequently found as the result of post-mortem changes. That such an embolic condition can exist is by no means an unreasonable supposition, and, while it is generally recognised that multiple sarcomata are usually made up of small round cells, in this case we have an undoubted example of sarcomatosis of the spindle-celled variety. There are numerous instances of this " embolic purpura," as it may be called, especially in French and German literature, the condition being associated with rheumatism, valvular lesions of the heart, and other diseases which induce directly or indirectly the formation of emboli. Krauss, Gimard, Leloir, and others have insisted with considerable emphasis on the embolic origin of many purpuric conditions, and in some instances they have verified their observations by histological examination. Leloir assumes that, in addition to the presence of the ordinary emboli and the changes in the vessel walls with desquamative endarteritis, the blood itself may be much altered chemically, and that in the cachectic conditions clots may be thrown down from the circulating blood and be carried onward to form capillary emboli, with resulting hæmorrhagic infarctions. Krogerer, some ten years ago, in examining the skin removed from patients with symptomatic purpura, found definite thromboses in the smaller veins, and even in the arteries. According to his view, the alterations in the vessel walls gave rise to slowed circulation and tendency to thrombosis, bringing about a liability to hæmorrhages. His plates bear out his theories regarding the thrombi, many of which show considerable organization. But a careful examination of the purpuric areas shows further that a mere invasion of the vascular system by sarcoma cells can not explain all the various blood effusions present. On examining the skin, for instance, in those areas where large irregular hæmorrhages had occurred, there was but little evidence of vascular invasion, while the emboli, on the other hand, seemed to exist mainly in the localized smaller and more circumscribed patches. One must therefore conclude that in such instances a combination of factors will alone afford a rational explanation of the purpura, and that in the general condition of the patient we shall find another cause for the enormous effusions of blood. In both of our cases there were high fever, cachexia, and a rapid progressive asthenia, all being the results of a sarcomatosis, and implying also grave alterations in the composition of the blood. From this we may infer an altered condition of the vessel walls, and hence probably a combination of circumstances sufficient to explain the incidence of hæmorrhage. The raised cutaneous nodules in our second case, some of which were hæmorrhagic, can not be regarded as pure sarcomatous metastases, for on microscopic examination they merely revealed hæmorrhage or necrosis, or both, and sometimes plugging of the vessels. There was nowhere in these nodules evidence of new growths. Such elevations, then, must have been produced rather by a temporary serous or cellular exudation coincident with or following upon the hæmorrhage—a probability which is emphasized by the fact that during the last days of the patient's illness many of the nodules diminished in size. Whether the œdema and infiltration were secondary to the embolic process in the subcutaneous vessels or whether they were merely coincident with the hæmorrhage would be difficult to decide. The ringlike spots, however, are of special interest, inasmuch as it has been shown that they have been present in more than one case of sarcoma. It is not impossible that such spots may be definitely related either to the embolic processes or to a direct invasion of the cutaneous vessels, though, so far as we know, there do not exist any experimental proofs to bear out such a theory. From what has been said, however, it is evident that the cutaneous vessels were plugged during the last few days of the illness, at a time when the walls of the smaller vessels and capillaries were already greatly enfeebled. The result of the embolic formation may therefore mean a decided deficiency in the supply of nutriment to the involved area, the collateral circulation naturally being poor under the circumstances. As soon, then, as the vessels had become plugged, the surrounding blood supply would be poured in to a limited extent, and, on meeting the enfeebled vessels, might possibly break through their thin walls, thus producing a zone of hæmorrhage around the area deprived of its normal nutrition. In other words, the condition may be regarded as in many respects analogous to that presented in embolic infarcts in regions with end arteries, central necrosis with peripheral congestion and hæmorrhage being induced, the latter being chiefly limited to the outer zone of the necrotic area. The cutaneous vessels under such circumstances may be regarded as end arteries in a functional sense, since the collateral circulation would be so diminished under the altered conditions that no complete nourishment could be afforded to the area supplied normally by the plugged vessel. Von Recklinghausen has directed especial attention to the occurrence of cutaneous hæmorrhages following embolic or thrombotic occlusion of peripheral arteries. The possibility of some toxic condition as a factor in the production of the purpura in our cases may also be suggested; but while we would not exclude this possibility, we are unable to find any positive evidence in its favour. Focal necroses, which are often associated with toxic and infectious processes, were present only in direct association with the hæmorrhages, and were not distributed in the liver, spleen, and kidneys in the manner characteristic of toxic infections. Nevertheless the absence of these necroses does not exclude the possibility of the existence of some form of toxæmia. Infection demonstrable by bacteriological examination was absent, and there is no reason to regard our cases as allied to the infectious purpuras. The thermic theory suggested by Fagge at all events finds no place in the production of the multiple tumours in our cases, inasmuch as in each instance extensive visceral growths had given rise to the metastases.
PMCID: PMC2117935  PMID: 19866815
5.  Aberrant Receptor-Mediated Endocytosis of Schistosoma mansoni Glycoproteins on Host Lipoproteins 
PLoS Medicine  2006;3(8):e253.
Background
Bilharzia is one of the major parasitic infections affecting the public health and socioeconomic circumstances in (sub) tropical areas. Its causative agents are schistosomes. Since these worms remain in their host for decades, they have developed mechanisms to evade or resist the immune system. Like several other parasites, their surface membranes are coated with a protective layer of glycoproteins that are anchored by a lipid modification.
Methods and Findings
We studied the release of glycosyl-phosphatidylinositol (GPI)-anchored proteins of S. mansoni and found them in the circulation associated with host lipoprotein particles. Host cells endocytosed schistosomal GPI-anchored proteins via their lipoprotein receptor pathway, resulting in disturbed lysosome morphology. In patients suffering from chronic schistosomiasis, antibodies attacked the parasite GPI-anchored glycoproteins that were associated with the patients' own lipoprotein particles. These immunocomplexes were endocytosed by cells carrying an immunoglobulin-Fc receptor, leading to clearance of lipoproteins by the immune system. As a consequence, neutral lipids accumulated in neutrophils of infected hamsters and in human neutrophils incubated with patient serum, and this accumulation was associated with apoptosis and reduced neutrophil viability. Also, Trypanosoma brucei, the parasite that causes sleeping sickness, released its major GPI-anchored glycoprotein VSG221 on lipoprotein particles, demonstrating that this process is generalizable to other pathogens/parasites.
Conclusions
Transfer of parasite antigens to host cells via host lipoproteins disrupts lipid homeostasis in immune cells, promotes neutrophil apoptosis, may result in aberrant antigen presentation in host cells, and thus cause an inefficient immune response against the pathogen.
The finding that GPI-anchored schistosome proteins are transferred from the parasite surface to human lipoproteins may explain how the parasites interfere with an effective immune response.
Editors' Summary
Background.
More than 200 million people live in a close but uneasy alliance with schistosomes, a type of parasitic worm. Like many parasites, schistosomes have a complicated life cycle. They start life by reproducing in fresh-water snails. The snails release free-swimming, infectious parasites, which burrow into the skin of people who swim in the water. The parasites then migrate to the veins draining the gut and mature into 10–20 mm-long adult worms. The worms mate and lay eggs, some of which pass into the feces and so back into water where they hatch and infect fresh snails. Schistosomiasis does not kill many people but it does cause serious health problems. Most of these are caused by the human immune system responding to eggs that get trapped in the veins of the liver, spleen, and gut. Immune cells recognize proteins on the eggs as foreign and organize a hard shell of immune cells and tough fibres around the egg. Eventually, these fibres block the blood vessels in the liver, spleen, and gut, causing locally raised blood pressure, organ damage, and potentially fatal bleeding.
Why Was This Study Done?
Although the immune system mounts a vigorous attack against schistosome eggs, the parasites themselves somehow evade the immune response—adult worms pull off this feat of “invisibility” for years. The researchers who did this study wanted to find out whether the release of glycoproteins (proteins decorated with sugars) from the surface of the schistosome worms is involved in this immune evasion in some way. These glycoproteins (which are anchored to the parasite's surface by a structure called a GPI-anchor; GPI stands for glycosyl-phosphatidylinositol, a sort of fat or lipid) are the major antigens of schistosomes—the molecules that the immune system normally recognizes on foreign intruders.
What Did the Researchers Do and Find?
The researchers first showed that GPI-anchored schistosomal glycoproteins are released into the circulation of patients and there become attached to human lip oproteinparticles (water-soluble carrier molecules that take fats around the body). Then, using cells grown in the laboratory, the researchers discovered that lipoprotein particles loaded with parasite glycoproteins could enter mammalian cells through an interaction with a protein called the low-density lipoprotein receptor, which normally helps cells absorb the lipids needed to make membranes. Once in the cell, the parasite glycoproteins travelled to cellular regions called lysosomes, which they seemed to disrupt. In addition, the researchers found that the parasite glycoproteins could enter mammalian cells by a second route: This involved the glycoproteins being taken up by neutrophils (a type of immune cells). Many of these neutrophils then died, possibly because of the large amount of lipid they accumulated.
What Does This Mean?
These results provide some tantalising clues to how schistosomes might evade the immune response. First, just binding to lipoprotein particles might change how they are seen by the immune system (possible they are not as clearly recognized as foreign substances) and weaken the immune response against them. On the other hand, the damage done to neutrophils by lipid accumulation might also contribute to how schistosomes hide in the human hosts. Neutrophils are an important type of immune cell, and their destruction could compromise the immune system's response to schistosomes. Furthermore, although the researchers do not investigate this possibility, other cells of the immune system that have might also take up these lipids and be damaged. Finally, even if immune cells are not killed outright by lipid accumulation, disruption of their lysosomes might also affect how well the immune system recognizes schistosomes as foreign. The full details of the complex interplay between schistosomes and their hosts remain a mystery, but these results provide intriguing new avenues to explore that might eventually suggest new treatments for schistosomiasis.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030253.
• World Health Organization information on schistosomiasis
• US Centers for Disease Control and Prevention information for the public and for professionals on schistosomiasis
• MedlinePlus encyclopedia entry on schistosomiasis
• Wikipedia page on schistosomiasis (note: Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030253
PMCID: PMC1502155  PMID: 16942390
6.  Optimization of Mucosal Responses after Intramuscular Immunization with Integrase Defective Lentiviral Vector 
PLoS ONE  2014;9(9):e107377.
Many infectious agents infiltrate the host at the mucosal surfaces and then spread systemically. This implies that an ideal vaccine should induce protective immune responses both at systemic and mucosal sites to counteract invasive mucosal pathogens. We evaluated the in vivo systemic and mucosal antigen-specific immune response induced in mice by intramuscular administration of an integrase defective lentiviral vector (IDLV) carrying the ovalbumin (OVA) transgene as a model antigen (IDLV-OVA), either alone or in combination with sublingual adjuvanted OVA protein. Mice immunized intramuscularly with OVA and adjuvant were compared with IDLV-OVA immunization. Mice sublingually immunized only with OVA and adjuvant were used as a positive control of mucosal responses. A single intramuscular dose of IDLV-OVA induced functional antigen-specific CD8+ T cell responses in spleen, draining and distal lymph nodes and, importantly, in the lamina propria of the large intestine. These results were similar to those obtained in a prime-boost regimen including one IDLV immunization and two mucosal boosts with adjuvanted OVA or vice versa. Remarkably, only in groups vaccinated with IDLV-OVA, either alone or in prime-boost regimens, the mucosal CD8+ T cell response persisted up to several months from immunization. Importantly, following IDLV-OVA immunization, the mucosal boost with protein greatly increased the plasma IgG response and induced mucosal antigen-specific IgA in saliva and vaginal washes. Overall, intramuscular administration of IDLV followed by protein boosts using the sublingual route induced strong, persistent and complementary systemic and mucosal immune responses, and represents an appealing prime-boost strategy for immunization including IDLV as a delivery system.
doi:10.1371/journal.pone.0107377
PMCID: PMC4161417  PMID: 25210766
7.  THE CORRELATION BETWEEN THE HISTOLOGICAL CHANGES AND THE FATE OF LIVING TUBERCLE BACILLI IN THE ORGANS OF TUBERCULOUS RABBITS 
It has been found that although there is some parallelism between the quantity of tubercle bacilli demonstrable histologically and the number of colonies that can be isolated from a given tissue, the culture method is far the more efficient in indicating quantitative relations. Tubercle bacilli were not perceived in the organs of rabbits 1 day after infection with the modified BCG although as many as 1,500 colonies were isolated from one of them. This may be solely because it is difficult to see widely dispersed single minute acid-fast rods in the diffuse infiltrations of mononuclears with their hyperchromatic nuclei and sparse cytoplasm. Later, with the formation of tubercle, the parallelism is much closer. The culture method gives evidence concerning the number of living tubercle bacilli in the tissue. The significance of the accumulation of acid-fast particles in the tissues has been discussed. It has been seen that from the beginning this accumulation is greater in the Kupffer cells of the liver, in the macrophages of the spleen and in the reticular cells of the bone marrow than within the mononuclears of the lung, the organ where the bacilli grow with the greatest rapidity and are destroyed with the greatest difficulty. Acid-fast particles are more prominent with the bovine than with the human bacillus or the BCG, the microorganism that is destroyed with the greatest difficulty thus leaving more incompletely digested bacillary debris at a given time within the cells. Thus it seems permissible to conclude from the presence of acid-fast material that some tubercle bacilli are undergoing destruction even 24 hours after infection. The initial accumulation of polynuclear leucocytes corresponds with the subsequent severity of the infection. Despite the greater primary localization of bacilli in the liver, this initial inflammatory reaction with all three infections is much greater in the lung than in the liver. In each organ it is more intense with the bovine than with the less virulent strains. The multiplication of the bacillus and its accumulation within large mononuclear and young epithelioid cells is accompanied by an intense formation of new mononuclears by mitosis. The more rapid the growth of the bacillus, the more conspicuous the regeneration of these cells. Thus with all strains mitosis is more intense in the more susceptible organ, as in the lung compared with the liver; with the most virulent strain the most extensive and diffuse accumulation of these new cells corresponds with the greater rise in the numbers of bovine bacilli after the lag of the 1st week. With the maturation of the epithelioid cells and the formation of tubercles the bacilli have already been greatly reduced numerically and the speed of this process diminishes with the virulence of the three strains used. The faster the development of tubercle the faster the destruction of the bacillus and the earlier the resorption of the tubercle. Tubercle bacilli never accumulate in such large numbers in the mononuclears of the liver as they do in the lung. Though at first the tubercles in the liver may be more numerous than those in the lung they never attain the same size. The formation of new mononuclears by mitosis is restricted and Langhans' giant cells appear very early (1st and 2nd weeks). In the lung, giant cells are not found until much later with the BCG and the human bacillus (4th week); they were not noted in the interstitial tubercles with the bovine type, but the extension of these tubercles was accompanied by an unabated mitosis of mononuclears until the death of the animal. The liver tubercles are resorbed early even with the bovine infection. Associated with these histological differences are the slow initial growth and the early and complete destruction of the tubercle bacilli even of bovine type in the liver, and the more rapid initial growth in the lung, with the later destruction of the BCG and the human bacillus and the unabated growth of the bovine bacillus. Similar differences were observed between the splenic pulp and corpuscle. In the former the accumulation of acid-fast particles was much greater and the tubercles developed earlier. Mitosis of mononuclears was less frequent and giant cells appeared earlier. Tubercle bacilli, always intracellular, disappeared from the tubercles in the pulp sooner than from those in the corpuscle, and the tubercles themselves first disappeared from the pulp. Consequently with the persistence of bacilli mitosis continued in the tubercles of the corpuscle and these attained a much larger size. Moreover individual resistance is linked with the ability to form mature tubercles early. In two animals simultaneously infected with the same strain and killed at the same time, the destruction or retardation of the bacillus is greater in that rabbit in which maturation of the tubercle and of epithelioid cells has proceeded further (Figs. 15 and 16). These observations indicate that the mononuclears of different organs or even of the same organ, as in the different parts of the spleen, have a different capacity to destroy the tubercle bacillus, and that the transformation of the mononuclear into the mature epithelioid cell follows its destruction of the tubercle bacilli. In the lung the more virulent types of bacillus are destroyed within the epithelioid cells of interstitial tubercles but persist in foci of tuberculous pneumonia. In this organ in rabbits infected with the human strain and to a lesser degree in rabbits infected with the bovine strain, the parasite largely disappears from the epithelioid cells of interstitial tubercles. But with both strains tubercle bacilli in large numbers may accumulate within epithelioid cells lying free in the alveoli. With the human type they are numerous within the cells and free in caseous material in the localized foci of caseous pneumonia. With the bovine infection, this caseous pneumonia is more often widespread and in the areas of caseous pneumonia the greater part of the vast accumulation of bovine bacilli in the lungs is found; as many as 200,000 colonies have been isolated from 10 mg. of tissue (Fig. 11). Flooding of the respiratory passages by the caseation of tuberculous lesions into the bronchi plays an important rôle in dissemination of tubercle bacilli through the lung. The process on the contrary is predominantly interstitial when the bovine bacillus is held in check (Fig. 12). Thus there is apparently some factor acting in the alveoli that favors the growth of the parasite. The accumulation of tubercle bacilli is seen especially in the peripheral epithelioid cells in immediate contact with the alveolar space. In the same lung the bacilli are much fewer in the interstitial tubercles. The accumulation in human tuberculosis of large numbers of tubercle bacilli in the tissues lining cavities is well known. Novy and Soule (20) have shown that within certain limits the growth of the bacillus in vitro is proportional to the oxygen tension of its environment. Corper, Lurie and Uyei (21) have confirmed these observations and have noted further that a difference in the gaseous environment of the bacilli equal to the difference between the conditions existing in the alveolar air and the venous blood is sufficient to cause a considerable increase in the growth of the microorganism in vitro. Loebel, Shorr and Richardson (22) by the use of Warburg's manometer have found that the oxygen consumption of tuberculous tissue is such that a tubercle 0.5 mm. thick would completely exhaust the oxygen of the air before it reached the center. These observations suggest that a factor responsible for the greater multiplication of the bacillus in the cells of the alveoli may be the greater oxygen tension of the alveolar air. In the liver, spleen and bone marrow even with the bovine infection many instances were found of the effective destruction of the parasite synchronously with the maturation of epithelioid cells and the formation of tubercle. On the other hand, in the spleen and bone marrow of some rabbits, living bacilli persisted within the epithelioid cells of isolated tubercles even 2 months after infection, a condition never found with the human type or BCG infection. Thus the epithelioid cell is the means of defense for the rabbit against the bovine type bacillus, and as such it is usually adequate in the liver, spleen and bone marrow though ineffective in the lung and kidney. In the latter, descending infection, and the occasional colony-like multiplication of bacilli in unorganized material, tubular casts, determine the long persistence of large numbers of bacilli in this organ. In differentiating the mononuclear phagocyte of the connective tissues into the monocyte and clasmatocyte Sabin and her coworkers (23) have maintained that the clasmatocyte can efficiently destroy the tubercle bacillus but that the monocyte and its derivatives, the epithelioid and Langhans' giant cells, cannot. With the progress of the disease they have noted that the monocytes accumulate in great numbers in the foci of infection and overflow into general circulation (4). White (24) and Sabin and her coworkers have concluded that tuberculosis is specifically a disease of the monocyte, and that this cell and its derivatives act as incubators for the tubercle bacillus. Doan and Sabin (25) have therefore sought, with indecisive results, to protect the body against tuberculosis by an antimonocytic serum. However it has been shown here that although an intense multiplication of mononuclears is associated with the growth of the tubercle bacillus, their transformation into mature epithelioid cells is constantly associated with its destruction, and the rapidity of the destruction varies with the rapidity of the maturation of tubercle. Even in the bovine infection the epithelioid cells destroy the bacilli in the liver, spleen and bone marrow as a rule, and even in the lung, keep them in check in the interstitial tubercles. The appearance of giant cells is associated with cessation or diminution of mononuclear regeneration by mitosis, and is coincident with cessation of multiplication or marked reduction in the number of living bacilli. They therefore appear earlier and in larger numbers in these organs or parts of organs that first destroy the bacillus (Figs. 16 and 17). They were not observed even 2 months after the bovine infection in the interstitial tubercles in the lung. Their absence and the continued mitosis of mononuclears, which accounts for the massive pneumonic and interstitial consolidation of the lung with this infection, were associated with the failure of the lung to destroy effectively the bovine parasite. The formation of giant cells in the pneumonic foci in the bovine infection would seem to be an exception to this rule. The Langhans giant cells have often been considered an indication of the chronicity of the pathological process. It would appear that they are formed from existing epithelioid cells when the multiplication of the bacillus has ceased and the stimulus for the formation of new cells has decreased or stopped. Giant cells were most conspicuous in the liver and splenic pulp where, with the BCG infection, no caseation ever developed, and in the liver before caseation was seen anywhere in the body. In the human and bovine infections, giant cells formed in the liver before caseation appeared. Hence caseation is not a necessary requirement for giant cell formation, as maintained by Medlar (26), though these cells frequently form about caseous material. Lymphocytes and granulation tissue do not cause the destruction of tubercle bacilli, these being destroyed in their absence. They usually appear about tubercles due to all strains and in all organs, after the greater part of the microorganisms have been destroyed (Fig. 18). The bacilli are not destroyed in the lung with bovine infection where the tubercles are usually little permeated by lymphocytes and granulation tissue. There is however, no constant relation between granulation tissue and destruction of tubercle bacilli, for in the lung after the human infection and even in other organs after the bovine infection isolated tubercles may be surrounded and penetrated by lymphocytes and granulation tissue at a time when considerable numbers of living bacilli are still histologically demonstrable within the epithelioid cells. Caseation is usually not caused by the local accumulation of tubercle bacilli. At first, when the BCG (after 1 week) and the human microorganism (after 2 weeks) are present in the cells in very large numbers as demonstrated both histologically and by culture (Figs. 4 and 13) there is no necrosis of these cells. An exception to this rule found in the lung with the bovine infection is considered below. Later, after the bacilli have been destroyed to a great extent and even though the number of bacilli is small, caseation appears (Fig. 14). After this preliminary destruction the extent of caseation apparently varies with the number of residual bacilli. With the least virulent microorganism, the BCG, few bacilli remained in the liver in the 4th week and no caseation was seen. In the tubercles of the splenic corpuscle at the same time bacilli were somewhat more numerous and there was scant caseation. On the other hand with the human bacillus after 4 weeks more bacilli survived and caseation was more extensive in both organs; with the bovine microorganism tubercle bacilli were much more numerous and caseation was far advanced. In the lung, however, caseation appeared with the first considerable accumulation of the bovine bacilli present 2 weeks after inoculation. That the bovine bacillus is primarily more injurious to the lung of rabbits than the BCG or the human bacillus is suggested by the greater intensity of the initial inflammation and by the more conspicuous accumulation of cells in the alveoli evident from the very beginning of infection. Maximow (27) showed that bovine bacilli even in small numbers cause the death of cells in tissue cultures of rabbit lymph nodes whereas the BCG or the human bacillus may accumulate within the cells in tremendous numbers without injuring them. Nevertheless in the liver, spleen and bone marrow of the living animal, caseation does not appear at the time when bovine bacilli are most abundant, but after they have been greatly reduced in numbers. Large numbers of the less virulent types of tubercle bacilli accumulated in different organs a short time after infection do not cause caseation, and with the bovine infection caseation under the same conditions occurs only in the lung. Later when the animal is sensitized caseation occurs in various organs in the presence of the small numbers of tubercle bacilli that remain in the tissues after most of them have been destroyed, and the extent of this caseation varies with the numbers of residual bacilli. These observations suggest that a large number of bacilli fail to cause necrosis soon after infection whereas a few bacilli produce caseation in the animal that is sensitized. Many investigators have held that caseation is due to sensitization. Krause (28), Huebschman (29) and Pagel (30) think that caseation is caused by the action of tuberculin-like substances on the sensitized tissues of the allergic animal. Rich and McCordock (31) view the process in essentially the same light. Recently Schleussing (32) has suggested that caseation is a coagulation necrosis in Weigert's sense of an allergically inflamed tissue, and is similar to the necrosis of the Arthus phenomenon.
PMCID: PMC2132067  PMID: 19869977
8.  Identification of Targets of CD8+ T Cell Responses to Malaria Liver Stages by Genome-wide Epitope Profiling 
PLoS Pathogens  2013;9(5):e1003303.
CD8+ T cells mediate immunity against Plasmodium liver stages. However, the paucity of parasite-specific epitopes of CD8+ T cells has limited our current understanding of the mechanisms influencing the generation, maintenance and efficiency of these responses. To identify antigenic epitopes in a stringent murine malaria immunisation model, we performed a systematic profiling of H2b-restricted peptides predicted from genome-wide analysis. We describe the identification of Plasmodium berghei (Pb) sporozoite-specific gene 20 (S20)- and thrombospondin-related adhesive protein (TRAP)-derived peptides, termed PbS20318 and PbTRAP130 respectively, as targets of CD8+ T cells from C57BL/6 mice vaccinated by whole parasite strategies known to protect against sporozoite challenge. While both PbS20318 and PbTRAP130 elicit effector and effector memory phenotypes in both the spleens and livers of immunised mice, only PbTRAP130-specific CD8+ T cells exhibit in vivo cytotoxicity. Moreover, PbTRAP130-specific, but not PbS20318-specific, CD8+ T cells significantly contribute to inhibition of parasite development. Prime/boost vaccination with PbTRAP demonstrates CD8+ T cell-dependent efficacy against sporozoite challenge. We conclude that PbTRAP is an immunodominant antigen during liver-stage infection. Together, our results underscore the presence of CD8+ T cells with divergent potencies against distinct Plasmodium liver-stage epitopes. Our identification of antigen-specific CD8+ T cells will allow interrogation of the development of immune responses against malaria liver stages.
Author Summary
Vaccination against malaria is feasible, as demonstrated with radiation-attenuated sporozoite vaccine, which protects experimental animals and humans by targeting the clinically silent liver stages. Potent protection largely depends on CD8+ T cells, a type of white blood cell that is tailor-made to kill obligate intracellular pathogens. Malaria-infected cells display fragments of parasite proteins, which are then recognised and targeted by CD8+ T cells. How CD8+ T cells are activated following immunisation and how they execute protective functions are key considerations for vaccination. However, characterisation of CD8+ T cells is hampered by the lack of identified malaria protein targets. Of concern, the circumsporozoite protein, which is the basis of the most advanced malaria vaccine candidate (RTS,S), is not an essential target of CD8+ T cells induced by attenuated sporozoites in several mouse strains. In this study, we have made considerable advances by identifying for the first time, fragments of malaria proteins that are targeted by CD8+ T cells generated by vaccination in a relevant mouse strain, C57BL/6. Notably, CD8+ T cells against one of the target proteins elicit partial protection against infection. Our study exemplifies how immunisation by complex pathogens can be dissected to identify distinct antigens for subunit vaccine development.
doi:10.1371/journal.ppat.1003303
PMCID: PMC3649980  PMID: 23675294
9.  Antigen-sensitized CD4+CD62Llow memory/effector T helper 2 cells can induce airway hyperresponsiveness in an antigen free setting 
Respiratory Research  2005;6(1):46.
Background
Airway hyperresponsiveness (AHR) is one of the most prominent features of asthma, however, precise mechanisms for its induction have not been fully elucidated. We previously reported that systemic antigen sensitization alone directly induces AHR before development of eosinophilic airway inflammation in a mouse model of allergic airway inflammation, which suggests a critical role of antigen-specific systemic immune response itself in the induction of AHR. In the present study, we examined this possibility by cell transfer experiment, and then analyzed which cell source was essential for this process.
Methods
BALB/c mice were immunized with ovalbumin (OVA) twice. Spleen cells were obtained from the mice and were transferred in naive mice. Four days later, AHR was assessed. We carried out bronchoalveolar lavage (BAL) to analyze inflammation and cytokine production in the lung. Fluorescence and immunohistochemical studies were performed to identify T cells recruiting and proliferating in the lung or in the gut of the recipient. To determine the essential phenotype, spleen cells were column purified by antibody-coated microbeads with negative or positive selection, and transferred. Then, AHR was assessed.
Results
Transfer of spleen cells obtained from OVA-sensitized mice induced a moderate, but significant, AHR without airway antigen challenge in naive mice without airway eosinophilia. Immunization with T helper (Th) 1 elicited antigen (OVA with complete Freund's adjuvant) did not induce the AHR. Transferred cells distributed among organs, and the cells proliferated in an antigen free setting for at least three days in the lung. This transfer-induced AHR persisted for one week. Interleukin-4 and 5 in the BAL fluid increased in the transferred mice. Immunoglobulin E was not involved in this transfer-induced AHR. Transfer of in vitro polarized CD4+ Th2 cells, but not Th1 cells, induced AHR. We finally clarified that CD4+CD62Llow memory/effector T cells recruited in the lung and proliferated, thus induced AHR.
Conclusion
These results suggest that antigen-sensitized memory/effector Th2 cells themselves play an important role for induction of basal AHR in an antigen free, eosinophil-independent setting. Therefore, regulation of CD4+ T cell-mediated immune response itself could be a critical therapeutic target for allergic asthma.
doi:10.1186/1465-9921-6-46
PMCID: PMC1180472  PMID: 15921525
10.  An Inducible Transgenic Mouse Model for Immune Mediated Hepatitis Showing Clearance of Antigen Expressing Hepatocytes by CD8+ T Cells 
PLoS ONE  2013;8(7):e68720.
The liver has the ability to prime immune responses against neo antigens provided upon infections. However, T cell immunity in liver is uniquely modulated by the complex tolerogenic property of this organ that has to also cope with foreign agents such as endotoxins or food antigens. In this respect, the nature of intrahepatic T cell responses remains to be fully characterized. To gain deeper insight into the mechanisms that regulate the CD8+ T cell responses in the liver, we established a novel OVA_X_CreERT2 mouse model. Upon tamoxifen administration OVA antigen expression is observed in a fraction of hepatocytes, resulting in a mosaic expression pattern. To elucidate the cross-talk of CD8+ T cells with antigen-expressing hepatocytes, we adoptively transferred Kb/OVA257-264-specific OT-I T cells to OVA_X_CreERT2 mice or generated triple transgenic OVA_X CreERT2_X_OT-I mice.
OT-I T cells become activated in OVA_X_CreERT2 mice and induce an acute and transient hepatitis accompanied by liver damage. In OVA_X_CreERT2_X_OT-I mice, OVA induction triggers an OT-I T cell mediated, fulminant hepatitis resulting in 50% mortality. Surviving mice manifest a long lasting hepatitis, and recover after 9 weeks. In these experimental settings, recovery from hepatitis correlates with a complete loss of OVA expression indicating efficient clearance of the antigen-expressing hepatocytes. Moreover, a relapse of hepatitis can be induced upon re-induction of cured OVA_X_CreERT2_X_OT-I mice indicating absence of tolerogenic mechanisms.
This pathogen-free, conditional mouse model has the advantage of tamoxifen inducible tissue specific antigen expression that reflects the heterogeneity of viral antigen expression and enables the study of intrahepatic immune responses to both de novo and persistent antigen. It allows following the course of intrahepatic immune responses: initiation, the acute phase and antigen clearance.
doi:10.1371/journal.pone.0068720
PMCID: PMC3711822  PMID: 23869228
11.  Robust Stimulation of Humoral and Cellular Immune Responses following Vaccination with Antigen-Loaded β-Glucan Particles 
mBio  2010;1(3):e00164-10.
β-Glucan particles (GPs) are purified Saccharomyces cerevisiae cell walls treated so that they are primarily β1,3-d-glucans and free of mannans and proteins. GPs are phagocytosed by dendritic cells (DCs) via the Dectin-1 receptor, and this interaction stimulates proinflammatory cytokine secretion by DCs. As the hollow, porous GP structure allows for high antigen loading, we hypothesized that antigen-loaded GPs could be exploited as a receptor-targeted vaccine delivery system. Ovalbumin (OVA) was electrostatically complexed inside the hollow GP shells (GP-OVA). Incubation of C57BL/6J mouse bone marrow-derived DCs with GP-OVA resulted in phagocytosis, upregulation of maturation markers, and rapid proteolysis of OVA. Compared with free OVA, GP-OVA was >100-fold more potent at stimulating the proliferation of OVA-reactive transgenic CD8+ OT-I and CD4+ OT-II T cells, as measured by in vitro [3H]thymidine incorporation using DCs as antigen-presenting cells. Next, immune responses in C57BL/6J mice following subcutaneous immunizations with GP-OVA were compared with those in C57BL/6J mice following subcutaneous immunizations with OVA absorbed onto the adjuvant alum (Alum/OVA). Vaccination with GP-OVA stimulated substantially higher antigen-specific CD4+ T-cell lymphoproliferative and enzyme-linked immunospot (ELISPOT) responses than that with Alum/OVA. Moreover, the T-cell responses induced by GP-OVA were Th1 biased (determined by gamma interferon [IFN-γ] ELISPOT assay) and Th17 biased (determined by interleukin-17a [IL-17a] ELISPOT assay). Finally, both the GP-OVA and Alum/OVA formulations induced strong secretions of IgG1 subclass anti-OVA antibodies, although only GP-OVA induced secretion of Th1-associated IgG2c antibodies. Thus, the GP-based vaccine platform combines adjuvanticity and antigen delivery to induce strong humoral and Th1- and Th17-biased CD4+ T-cell responses.
IMPORTANCE
Most licensed vaccines work by promoting protective antibody responses. However, for many infectious diseases, antibody-mediated protection appears to play a relatively minor role, and vaccination has met with limited success. While live-attenuated organisms generally elicit T-cell responses, their use in vaccines is limited by the potential for causing disease. Thus, there is an urgent need for new vaccine platforms that deliver antigens in such a manner as to promote strong T-cell-mediated responses. Here we designed a novel vaccine platform consisting of yeast-derived β-glucan particles (GPs) that combines antigen delivery and adjuvant activity. GPs loaded with the model antigen ovalbumin (OVA) stimulated robust humoral and T-cell responses in mice. In addition, the cellular response was Th1 and Th17 biased. This work has implications for the design of vaccines that stimulate biased T-cell responses as well as for understanding how immunity to fungal pathogens develops.
doi:10.1128/mBio.00164-10
PMCID: PMC2925077  PMID: 20802824
12.  Oral antigen exposure in newborn piglets circumvents induction of oral tolerance in response to intraperitoneal vaccination in later life 
Background
We previously determined that newborn piglets orally gavaged with Ovalbumin (OVA) responded to systemic OVA re-exposure with tolerance; if adjuvants were included in oral vaccine, piglets responded with antibody-mediated immunity (Vet Immunol Immunopathol 161(3–4):211–21, 2014). Here, we will investigate whether newborn piglets gavaged with a vaccine comprised of OVA plus unmethylated CpG oligodeoxynucleotides (CpG; soluble component; OVA/CpG) combined with OVA plus CpG encapsulated within polyphosphazene microparticles (MP; particulate component) responded with systemic and mucosal immunity. To monitor the response to systemic antigen re-exposure, piglets were i.p.-immunized with OVA plus Incomplete Freund’s Adjuvant (IFA) one month later.
Results
Newborn piglets (n = 5/group) were gavaged with a combined soluble and particulate vaccine consisting of OVA (0.5-0.05 mg) plus 50 μg CpG and 0.5 mg OVA plus 50 μg CpG encapsulated within a polyphosphazene MP (0.5 mg) referred to as OVA/CpG + MP. Control piglets were gavaged with saline alone. Piglets were i.p. immunized with 10 mg OVA (or saline) in IFA at four weeks of age and then euthanized at eight weeks of age. We observed significantly higher titres of serum anti-OVA immunoglobulin (Ig) IgM, IgA, IgG, IgG1, IgG2 and IgG in piglets immunized with 0.05 mg OVA/CpG + MP relative to saline control animals. Thus, a single oral exposure at birth to a combined soluble and particulate OVA vaccine including adjuvants can circumvent induction of oral tolerance which impacts response to i.p. vaccination in later life. Further, piglets gavaged with 0.05 mg OVA/CpG + MP generated significant anti-OVA IgG and IgG1 titres in lung compared to saline control piglets but results were comparable to titres measured in parenteral control piglets. Peripheral blood mononuclear cells (PBMCs) ex vivo-stimulated with OVA showed markedly decreased production of IL-10 cytokine after 72 hours relative to animal-matched cells incubated with media alone. No production of IFN-γ was observed from any groups.
Conclusion
Newborn piglets gavaged with low dose soluble and particulate OVA plus CpG ODN and polyphosphazene adjuvants produced antigen-specific antibodies in serum and lung after systemic re-exposure in later life. These data indicate circumvention of oral tolerance but not induction of oral immunity.
doi:10.1186/s12917-015-0350-8
PMCID: PMC4357157  PMID: 25889479
Piglets; Neonate; Ovalbumin; Oral; Tolerance
13.  Concerted Activity of IgG1 Antibodies and IL-4/IL-25-Dependent Effector Cells Trap Helminth Larvae in the Tissues following Vaccination with Defined Secreted Antigens, Providing Sterile Immunity to Challenge Infection 
PLoS Pathogens  2015;11(3):e1004676.
Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.
Author Summary
Despite the high prevalence of gastrointestinal helminth parasites in human and animal populations throughout the world, no vaccines are yet available and we lack understanding of how anti-parasite protective immunity may operate effectively. We have used a model system with a natural mouse nematode parasite, Heligmosomoides polygyrus, which establishes long-term chronic infection in laboratory mice through the secretion of immunosuppressive molecules. Immunization of mice with as few as 3 secreted proteins, collected from parasites in vitro, confers complete immunity to challenge infection. We show here that immunity requires specific IgG1 antibodies directed to the secreted products, acting together with innate myeloid cells that require activation through the canonical Type 2 cytokine receptor, IL-4Rα, as well as through a pathway not previously known to be involved in effector mechanisms, IL-25. These myeloid cells act to trap and envelop helminth larvae while in the submucosal tissues of the small intestine, massing in large numbers and preventing their maturation and exit into the gut lumen. Thus the combined effects of specific antibodies from the adaptive immune system, and Type 2 cytokine activation of the innate immune system, co-operate to ensure elimination of the helminth parasite.
doi:10.1371/journal.ppat.1004676
PMCID: PMC4376884  PMID: 25816012
14.  Nippocystatin, a Cysteine Protease Inhibitor from Nippostrongylus brasiliensis, Inhibits Antigen Processing and Modulates Antigen-Specific Immune Response 
Infection and Immunity  2001;69(12):7380-7386.
During infection, parasites evade the host immune system by modulating or exploiting the immune system; e.g., they suppress expression of major histocompatibility complex class II molecules or secrete cytokine-like molecules. However, it is not clear whether helminths disturb the immune responses of their hosts by controlling the antigen-processing pathways of the hosts. In this study, we identified a new cysteine protease inhibitor, nippocystatin, derived from excretory-secretory (ES) products of an intestinal nematode, Nippostrongylus brasiliensis. Nippocystatin, which belongs to cystatin family 2, consists of 144 amino acids and is secreted as a 14-kDa mature form. In vivo treatment of ovalbumin (OVA)-immunized mice with recombinant nippocystatin (rNbCys) profoundly suppressed OVA-specific proliferation of splenocytes but not non-antigen-specific proliferation of splenocytes. OVA-specific cytokine production was also greatly suppressed in rNbCys-treated mice. Although the serum levels of both OVA-specific immunoglobulin G1 (IgG1) and IgG2a were not affected by rNbCys treatment, OVA-specific IgE was preferentially downregulated in rNbCys-treated mice. In vitro rNbCys inhibited processing of OVA by lysosomal cysteine proteases from the spleens of mice. Mice with anti-nippocystatin antibodies became partially resistant to infection with N. brasiliensis. Based on these findings, N. brasiliensis appears to skillfully evade host immune systems by secreting nippocystatin, which modulates antigen processing in antigen-presenting cells of hosts.
doi:10.1128/IAI.69.12.7380-7386.2001
PMCID: PMC98825  PMID: 11705911
15.  Synthesized OVA323-339MAP octamers mitigate OVA-induced airway inflammation by regulating Foxp3 T regulatory cells 
BMC Immunology  2012;13:34.
Background
Antigen-specific immunotherapy (SIT) has been widely practiced in treating allergic diseases such as asthma. However, this therapy may induce a series of allergic adverse events during treatment. Peptide immunotherapy (PIT) was explored to overcome these disadvantages. We confirmed that multiple antigen peptides (MAPs) do not cause autoimmune responses, which led to the presumption that MAPs intervention could alleviate allergic airway inflammation without inducing adverse effects.
Results
In this study, synthesized OVA323-339MAP octamers were subcutaneously injected into ovalbumin (OVA)-sensitized and -challenged Balb/c mice to observe its effect on allergic airway inflammation, Th2 immune response, and immune regulating function. It was confirmed that OVA sensitization and challenge led to significant peritracheal inflammatory, cell infiltration, and intensive Th2 response. Treatment of OVA323-339MAP octomers in the airway inflammation mice model increased CD4+CD25+Foxp3+ T regulatory (Treg) cells and their regulatory function in peripheral blood, mediastinal draining lymph nodes, and the spleen. Furthermore, OVA323-339MAP increased IL-10 levels in bronchial alveolar lavage fluid (BALF); up-regulated the expression of IL-10, membrane-bound TGF-β1, as well as Foxp3 in lung tissues; and up-regulated programmed death-1 (PD-1) and cytotoxic T lymphocyte associated antigen 4 (CTLA-4) on the surface of Treg cells. These results were further correlated with the decreased OVA specific immunoglobulin E (sIgE) level and the infiltration of inflammatory cells such as eosinophils and lymphocytes in BALF. However, OVA323-339 peptide monomers did not show any of the mentioned effects in the same animal model.
Conclusions
Our study indicates that OVA323-339MAP had significant therapeutic effects on mice allergic airway inflammation by regulating the balance of Th1/Th2 response through Treg cells in vivo.
doi:10.1186/1471-2172-13-34
PMCID: PMC3472185  PMID: 22769043
Allergic airway inflammation; Specific immunotherapy; Multiple antigen peptide
16.  Notch Ligand Delta-Like 4-Pretreated Dendritic Cells Alleviate Allergic Airway Responses by Enhancing IL-10 Production 
PLoS ONE  2013;8(5):e63613.
The Notch pathway plays a role in the processes of cell proliferation, differentiation, and apoptosis, which affect the development and function of various organs. Dendritic cells (DCs), as professional antigen-presenting cells (APCs), induce T cell activation and promote T cell differentiation by antigen stimulation. Research has shown that Notch ligand delta-like 4 (Dll4) in APCs is associated with stimulation of a Th1-type response. However, the regulatory roles of Dll4 in the activation and function of DCs have yet to be clearly elucidated. In this study, we demonstrated that activation of Dll4-pretreated bone marrow-derived DCs by performing ovalbumin (OVA) stimulation expressed a high level of interleukin (IL)-10 without diminishing IL-12 production. By contrast, the proinflammatory cytokines, IL-1β, IL-6, and tumor necrosis factor (TNF)-α, decreased in Dll4-pretreated DCs by performing either lipopolysaccharide (LPS) or OVA stimulation. Compared to fully mature DCs, lower levels of MHC class II CD40 and higher levels of CD80 and CD86 molecules were expressed in these semi-mature like DCs. Dll4 Notch signaling also enhanced Notch ligand mRNA expression of Dll1, Dll4, and Jagged1 in DCs. Dll4-modified DCs exhibited a reduced capacity to stimulate the proliferation of OVA-specific CD4+ T cells, but actively promoted large amounts of IL-10 production in these activated T cells. Furthermore, immunomodulatory effects of Dll4-modified DCs were examined in an established asthmatic animal model. After adoptive transfer of OVA-pulsed plus Dll4-pretreated DCs in OVA-immunized mice, OVA challenge induced lower OVA-specific immunoglobulin E (IgE) and higher IgG2a antibody production, lower eotaxin, keratinocyte-derived chemokine (KC), IL-5, and IL-13 release in bronchial alveolar lavage fluid, attenuated airway hyper-responsiveness, and promoted higher IL-10 and interferon (IFN)-γ production in the spleen. In summary, our findings elucidate the new role of Dll4 in the phenotype and function of DCs and provide a novel approach for manipulating T cell-driven deleterious immune diseases.
doi:10.1371/journal.pone.0063613
PMCID: PMC3656003  PMID: 23696838
17.  Immune Evasion by Yersinia enterocolitica: Differential Targeting of Dendritic Cell Subpopulations In Vivo 
PLoS Pathogens  2010;6(11):e1001212.
CD4+ T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4+ T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4+ T cells was markedly reduced when cultured with splenic CD8α+ DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4+ or CD4−CD8α− DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α+ DCs, but not in CD4+ and CD4−CD8α− DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α+ DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α+ DCs. Three days post infection with Ye the number of splenic CD8α+ and CD4+ DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4+ and CD8α+ DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye.
Author Summary
Dendritic cells (DCs) are crucial in promoting immune responses against pathogens. Mouse DCs consist of different subpopulations but their role in immunity to pathogens and immune evasion is largely unclear. The enteric pathogen Yersinia enterocolitica (Ye) was shown to evade DC functions in bone marrow-derived DCs in vitro inhibiting antigen uptake and degradation, maturation and subsequently T-cell activation. However, it is controversial whether and, if so, which virulence factors of Ye (e.g. Yops) contribute to immune evasion of DCs in vivo. Using an experimental mouse infection model and a β-lactamase reporter system to track Yop injection into host cells we demonstrate here for the first time that distinct DC subpopulations are affected by Ye infection in vivo in terms of antigen uptake and degradation, cytokine production, and T-cell proliferation. Moreover, Ye infection causes the loss of 90% of CD11chiCD4+ DCs in a TLR4- and TRIF-signaling dependent manner. These data combined with results reported from infection with e.g. Mycobacterium tuberculosis, Salmonella typhimurium, or Escherichia coli suggest that the response of DCs to bacterial infections is manifold, reflecting the diversity of DC subpopulations, pathogenicity factors, and life styles of the pathogens.
doi:10.1371/journal.ppat.1001212
PMCID: PMC2991265  PMID: 21124820
18.  Macrophage-Derived Human Resistin Is Induced in Multiple Helminth Infections and Promotes Inflammatory Monocytes and Increased Parasite Burden 
PLoS Pathogens  2015;11(1):e1004579.
Parasitic helminth infections can be associated with lifelong morbidity such as immune-mediated organ failure. A better understanding of the host immune response to helminths could provide new avenues to promote parasite clearance and/or alleviate infection-associated morbidity. Murine resistin-like molecules (RELM) exhibit pleiotropic functions following helminth infection including modulating the host immune response; however, the relevance of human RELM proteins in helminth infection is unknown. To examine the function of human resistin (hResistin), we utilized transgenic mice expressing the human resistin gene (hRetnTg+). Following infection with the helminth Nippostrongylus brasiliensis (Nb), hResistin expression was significantly upregulated in infected tissue. Compared to control hRetnTg− mice, hRetnTg+ mice suffered from exacerbated Nb-induced inflammation characterized by weight loss and increased infiltration of inflammatory monocytes in the lung, along with elevated Nb egg burdens and delayed parasite expulsion. Genome-wide transcriptional profiling of the infected tissue revealed that hResistin promoted expression of proinflammatory cytokines and genes downstream of toll-like receptor signaling. Moreover, hResistin preferentially bound lung monocytes, and exogenous treatment of mice with recombinant hResistin promoted monocyte recruitment and proinflammatory cytokine expression. In human studies, increased serum resistin was associated with higher parasite load in individuals infected with soil-transmitted helminths or filarial nematode Wuchereria bancrofti, and was positively correlated with proinflammatory cytokines. Together, these studies identify human resistin as a detrimental factor induced by multiple helminth infections, where it promotes proinflammatory cytokines and impedes parasite clearance. Targeting the resistin/proinflammatory cytokine immune axis may provide new diagnostic or treatment strategies for helminth infection and associated immune-mediated pathology.
Author Summary
Parasitic helminths, which infect an estimated two billion people worldwide, represent a significant global public health problem. Infection is associated with life-long morbidity including growth retardation and organ failure. Despite these debilitating conditions, there are currently no successful vaccines against helminths. Further, great variability in the host immune response to helminths exists, with the ability of some individuals to develop immunity, while others are susceptible when re-exposed or maintain life-long chronic infections. Identifying new factors that are differentially expressed in immune versus susceptible individuals could provide new targeting strategies for diagnosis or treatment of helminth infection. Here, we identify an important immunoregulatory function for human resistin in helminth infection. Employing transgenic mice in which the human resistin gene was inserted, we show that human resistin is induced by infection with the helminth Nippostrongylus brasiliensis, where it promotes excessive inflammation and impedes parasite killing. Moreover, analysis of clinical samples from two cohorts of individuals infected with filarial nematodes or soil-transmitted helminths revealed increased resistin and serum proinflammatory cytokines compared to putatively immune individuals. Together, these studies suggest that human resistin is a detrimental cytokine that is expressed in multiple helminth infections, mediates pathogenic inflammation, and delays parasite clearance.
doi:10.1371/journal.ppat.1004579
PMCID: PMC4287580  PMID: 25568944
19.  Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis 
Neutrophils are multifaceted cells that are often the immune system’s first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system.
doi:10.1016/j.micinf.2014.02.012
PMCID: PMC4076910  PMID: 24642003
Neutrophils; Extracellular traps; NET; Mice; Human; Strongyloides stercoralis
20.  Ly6C- Monocytes Regulate Parasite-Induced Liver Inflammation by Inducing the Differentiation of Pathogenic Ly6C+ Monocytes into Macrophages 
PLoS Pathogens  2015;11(5):e1004873.
Monocytes consist of two well-defined subsets, the Ly6C+ and Ly6C– monocytes. Both CD11b+ myeloid cells populations have been proposed to infiltrate tissues during inflammation. While infiltration of Ly6C+ monocytes is an established pathogenic factor during hepatic inflammation, the role of Ly6C– monocytes remains elusive. Mice suffering experimental African trypanosome infection die from systemic inflammatory response syndrome (SIRS) that is initiated by phagocytosis of parasites by liver myeloid cells and culminates in apoptosis/necrosis of liver myeloid and parenchymal cells that reduces host survival. C57BL/6 mice are considered as trypanotolerant to Trypanosoma congolense infection. We have reported that in these animals, IL-10, produced among others by myeloid cells, limits the liver damage caused by pathogenic TNF-producing Ly6C+ monocytes, ensuring prolonged survival. Here, the heterogeneity and dynamics of liver myeloid cells in T. congolense-infected C57/BL6 mice was further dissected. Moreover, the contribution of Ly6C– monocytes to trypanotolerance was investigated. By using FACS analysis and adoptive transfer experiments, we found that the accumulation of Ly6C– monocytes and macrophages in the liver of infected mice coincided with a drop in the pool of Ly6C+ monocytes. Pathogenic TNF mainly originated from Ly6C+ monocytes while Ly6C– monocytes and macrophages were major and equipotent sources of IL-10 within myeloid cells. Moreover, Nr4a1 (Nur77) transcription factor-dependent Ly6C– monocytes exhibited IL-10-dependent and cell contact-dependent regulatory properties contributing to trypanotolerance by suppressing the production of TNF by Ly6C+ monocytes and by promoting the differentiation of the latter cells into macrophages. Thus, Ly6C– monocytes can dampen liver damage caused by an extensive Ly6C+ monocyte-associated inflammatory immune response in T. congolense trypanotolerant animals. In a more general context, Ly6C– or Ly6C+ monocyte targeting may represent a therapeutic approach in liver pathogenicity induced by chronic infection.
Author Summary
The liver is not only a central organ for efficient metabolism of nutrients and for toxin clearance, but also for immune surveillance, including elimination of intravascular infections. However, excess of nutrients like fat or of toxins like alcohol and certain medications, as well as infections can trigger overactive immune responses which destroy the liver. Such chronic inflammations are major worldwide human health problem with often lethal consequences. Thus, understanding the particular function of various liver immune cells could provide original concepts to alleviate damages in this vital organ. Here, we dissected the heterogeneity, dynamics and function of the myeloid/monocytic cell compartment in the liver of mice infected with Trypanosoma congolense parasite. We established that infiltration of Ly6C+ monocyte subset initiated liver injury in infected mice. More importantly, we revealed that another myeloid cell subset for which the role in liver injury remained elusive, the Ly6C- monocyte subset, exerted hepatoprotective function in infected mice by secreting the anti-inflammatory cytokine IL-10 and by inducing, through cell-contact, the differentiation of pathogenic Ly6C+ monocytes into macrophages expressing genes coding for anti-inflammatory molecules. Thus, augmenting Ly6C- monocyte accumulation or functionality may represent a useful intervention strategy complementing anti-infective medication in conditions of liver injury due to chronic infections.
doi:10.1371/journal.ppat.1004873
PMCID: PMC4447383  PMID: 26020782
21.  Sublingual Vaccination Induces Mucosal and Systemic Adaptive Immunity for Protection against Lung Tumor Challenge 
PLoS ONE  2014;9(3):e90001.
Sublingual route offers a safer and more practical approach for delivering vaccines relative to other systemic and mucosal immunization strategies. Here we present evidence demonstrating protection against ovalbumin expressing B16 (B16-OVA) metastatic melanoma lung tumor formation by sublingual vaccination with the model tumor antigen OVA plus synthetic glycolipid alpha-galactosylceramide (aGalCer) for harnessing the adjuvant potential of natural killer T (NKT) cells, which effectively bridge innate and adaptive arms of the immune system. The protective efficacy of immunization with OVA plus aGalCer was antigen-specific as immunized mice challenged with parental B16 tumors lacking OVA expression were not protected. Multiple sublingual immunizations in the presence, but not in the absence of aGalCer, resulted in repeated activation of NKT cells in the draining lymph nodes, spleens, and lungs of immunized animals concurrent with progressively increasing OVA-specific CD8+ T cell responses as well as serum IgG and vaginal IgA levels. Furthermore, sublingual administration of the antigen only in the presence of the aGalCer adjuvant effectively boosted the OVA-specific immune responses. These results support potential clinical utility of sublingual route of vaccination with aGalCer-for prevention of pulmonary metastases.
doi:10.1371/journal.pone.0090001
PMCID: PMC3943861  PMID: 24599269
22.  HMGB1 Mediates Endogenous TLR2 Activation and Brain Tumor Regression 
PLoS Medicine  2009;6(1):e1000010.
Background
Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling on bone marrow-derived GBM-infiltrating DCs.
Methods and Findings
Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like tyrosine kinase 3 ligand (Flt3L) and thymidine kinase (TK) delivered into the tumor mass, we demonstrated that CD4+ and CD8+ T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs) were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV]) treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV)-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV)-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor) or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor-bearing Ad-Flt3L and Ad-TK treated mice, abolished therapeutic efficacy, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit tumor regression. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second glioma model and in an intracranial melanoma model with concomitant increases in the levels of circulating HMGB1.
Conclusions
Our data provide evidence for the molecular and cellular mechanisms that support the rationale for the clinical implementation of antibrain cancer immunotherapies in combination with tumor killing approaches in order to elicit effective antitumor immune responses, and thus, will impact clinical neuro-oncology practice.
Maria Castro and colleagues use cell line and transgenic mouse approaches to study the mechanisms underlying the immune response to glioblastoma multiforme.
Editors' Summary
Background.
Every year, more than 175,000 people develop a primary brain tumor (a cancer that starts in the brain rather than spreading in from elsewhere). Like all cancers, brain tumors develop when a cell acquires genetic changes that allow it to grow uncontrollably and that change other aspects of its behavior, including the proteins it makes. There are many different types of cells in the brain and, as a result, there are many different types of brain tumors. However, one in five primary brain tumors is glioblastoma multiforme (GBM; also known as grade 4 astrocytoma), a particularly aggressive cancer. With GBM, the average time from diagnosis to death is one year and only one person in 20 survives for five years after a diagnosis of GBM. Symptoms of GBM include headaches, seizures, and changes in memory, mood, or mental capacity. Treatments for GBM, which include surgery, radiotherapy, and chemotherapy, do not “cure” the tumor but they can ease these symptoms.
Why Was This Study Done?
Better treatments for GBM are badly needed, and one avenue that is being explored is immunotherapy—a treatment in which the immune system is used to fight the cancer. Because many tumors make unusual proteins, the immune system can sometimes be encouraged to recognize tumor cells as foreign invaders and kill them. Unfortunately, attempts to induce a clinically useful anti-GBM immune response have been unsuccessful, partly because the brain contains very few dendritic cells, a type of immune system cell that kick-starts effective immune responses by presenting foreign proteins to other immune system cells. Another barrier to immunotherapy for GBM is immune evasion by the tumor. Many tumors develop ways to avoid the immune response as they grow. For example, they sometimes reduce the expression of proteins that the immune system might recognize as foreign. In this study, the researchers test a new combined treatment strategy for GBM in which dendritic cells are encouraged to enter the brain and tumor cells are killed to release proteins capable of stimulating an effective antitumor immune response.
What Did the Researchers Do and Find?
The researchers first established brain tumors in mice. Then, they injected harmless viruses carrying the genes for Fms-like tyrosine kinase 3 ligand (Ftl3L; a protein that attracts dendritic cells) and for thymidine kinase (TK; cells expressing TK are killed by a drug called gancyclovir) into the tumor. Expression of both Flt3L and TK (but not of either protein alone) plus gancyclovir treatment shrank the tumors and greatly improved the survival of the mice. The researchers show that their strategy increased the migration of dendritic cells into the tumor provided they expressed an immune system protein called Toll-like receptor 2 (TLR2). TLR2 expression on the dendritic cells was also needed for an effective anti-tumor immune response and for tumor regression. TLR2 normally activates dendritic cells by binding to specific proteins on invading pathogens, so what was TLR2 binding to in the mouse tumors? The researchers reveal that TLR2 was responding to high-mobility-group box 1 (HMGB1), a protein released by the dying tumor cells by showing that treatment of the tumor-bearing mice with the HMGB1 inhibitor glycyrrhizin blocked the therapeutic effect of Flt3L/TK expression. Finally, the researchers report that other tumor cell types release HMGB1 when they are killed and that the Flt3L/TK expression strategy can also kill other tumors growing in mouse brains.
What Do These Findings Mean?
Results obtained in mouse models of human diseases do not always lead to effective treatments for human patients. Nevertheless, the findings of this study provide new insights into how an effective immune response against brain tumors might be brought about. Most importantly, they show that an effective strategy might need to both attract dendritic cells into the brain tumor and to kill tumor cells, so they release proteins that can activate the dendritic cells. That is, the authors suggest it's important to combine immunotherapies with tumor-killing strategies to provide effective treatments for primary and metastatic brain tumors
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000010.
The US National Cancer Institute provides information about brain tumors for patients and health professionals and about the the immune system and how it can be harnessed to fight cancer (in English and Spanish)
Cancer Research UK provides information on all aspects of brain tumors for patients and their caregivers
MedlinePlus provides links to further information about brain cancer, (including some links to information in Spanish)
The American Brain Tumor Association provides brain tumor resources and information
The National Brain Tumor Society provides educational and support services regarding brain tumors
doi:10.1371/journal.pmed.1000010
PMCID: PMC2621261  PMID: 19143470
23.  Acrolein exposure suppresses antigen-induced pulmonary inflammation 
Respiratory Research  2013;14(1):107.
Background
Adverse health effects of tobacco smoke arise partly from its influence on innate and adaptive immune responses, leading to impaired innate immunity and host defense. The impact of smoking on allergic asthma remains unclear, with various reports demonstrating that cigarette smoke enhances asthma development but can also suppress allergic airway inflammation. Based on our previous findings that immunosuppressive effects of smoking may be largely attributed to one of its main reactive electrophiles, acrolein, we explored the impact of acrolein exposure in a mouse model of ovalbumin (OVA)-induced allergic asthma.
Methods
C57BL/6 mice were sensitized to ovalbumin (OVA) by intraperitoneal injection with the adjuvant aluminum hydroxide on days 0 and 7, and challenged with aerosolized OVA on days 14–16. In some cases, mice were also exposed to 5 ppm acrolein vapor for 6 hrs/day on days 14–17. Lung tissues or brochoalveolar lavage fluids (BALF) were collected either 6 hrs after a single initial OVA challenge and/or acrolein exposure on day 14 or 48 hrs after the last OVA challenge, on day 18. Inflammatory cells and Th1/Th2 cytokine levels were measured in BALF, and lung tissue samples were collected for analysis of mucus and Th1/Th2 cytokine expression, determination of protein alkylation, cellular thiol status and transcription factor activity.
Results
Exposure to acrolein following OVA challenge of OVA-sensitized mice resulted in markedly attenuated allergic airway inflammation, demonstrated by decreased inflammatory cell infiltrates, mucus hyperplasia and Th2 cytokines. Acrolein exposure rapidly depleted lung tissue glutathione (GSH) levels, and induced activation of the Nrf2 pathway, indicated by accumulation of Nrf2, increased alkylation of Keap1, and induction of Nrf2-target genes such as HO-1. Additionally, analysis of inflammatory signaling pathways showed suppressed activation of NF-κB and marginally reduced activation of JNK in acrolein-exposed lungs, associated with increased carbonylation of RelA and JNK.
Conclusion
Acrolein inhalation suppresses Th2-driven allergic inflammation in sensitized animals, due to direct protein alkylation resulting in activation of Nrf2 and anti-inflammatory gene expression, and inhibition of NF-κB or JNK signaling. Our findings help explain the paradoxical anti-inflammatory effects of cigarette smoke exposure in allergic airways disease.
doi:10.1186/1465-9921-14-107
PMCID: PMC3852782  PMID: 24131734
Cigarette smoke; Electrophile; Inflammation; Asthma; COPD; Nrf2; NF-κB; JNK
24.  6-Thioguanine-loaded polymeric micelles deplete myeloid-derived suppressor cells and enhance the efficacy of T cell immunotherapy in tumor-bearing mice 
Cancer Immunology, Immunotherapy  2015;64(8):1033-1046.
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress effector T cell responses and can reduce the efficacy of cancer immunotherapies. We previously showed that ultra-small polymer nanoparticles efficiently drain to the lymphatics after intradermal injection and target antigen-presenting cells, including Ly6chi Ly6g− monocytic MDSCs (Mo-MDSCs), in skin-draining lymph nodes (LNs) and spleen. Here, we developed ultra-small polymer micelles loaded with 6-thioguanine (MC-TG), a cytotoxic drug used in the treatment of myelogenous leukemia, with the aim of killing Mo-MDSCs in tumor-bearing mice and thus enhancing T cell-mediated anti-tumor responses. We found that 2 days post-injection in tumor-bearing mice (B16-F10 melanoma or E.G7-OVA thymoma), MC-TG depleted Mo-MDSCs in the spleen, Ly6clo Ly6g+ granulocytic MDSCs (G-MDSCs) in the draining LNs, and Gr1int Mo-MDSCs in the tumor. In both tumor models, MC-TG decreased the numbers of circulating Mo- and G-MDSCs, as well as of Ly6chi macrophages, for up to 7 days following a single administration. MDSC depletion was dose dependent and more effective with MC-TG than with equal doses of free TG. Finally, we tested whether this MDSC-depleting strategy might enhance cancer immunotherapies in the B16-F10 melanoma model. We found that MC-TG significantly improved the efficacy of adoptively transferred, OVA-specific CD8+ T cells in melanoma cells expressing OVA. These findings highlight the capacity of MC-TG in depleting MDSCs in the tumor microenvironment and show promise in promoting anti-tumor immunity when used in combination with T cell immunotherapies.
Electronic supplementary material
The online version of this article (doi:10.1007/s00262-015-1702-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s00262-015-1702-8
PMCID: PMC4506469  PMID: 25982370
MDSC depletion; 6-Thioguanine; Cancer; T cell therapy
25.  NPY and NPY Receptors in Airway Structural and Inflammatory Cells in Allergic Asthma 
Purpose
Neuropeptide Y (NPY) level is elevated in allergic asthmatic airways and activation of NPY receptor-1 (NPY-Y1) on antigen presenting cells (APCs) is essential for T-cell priming. Paradoxically, NPY-Y1 modulates hyper-responsiveness in T cells, suggesting a bimodal role for NPY in APCs and T-cells. Therefore, determination of the temporal and spatial expression pattern of NPY and its receptors in asthmatic airways is essential to further understand the role of NPY in allergic asthma.
Methods
Lungs were isolated from control and acute and chronic stages of OVA-sensitized and challenged mice (OVA). Stains, including H&E, PAS, and trichrome, were used to determine the severity of lung pathology. The expression patterns of NPY and NPY-Y receptors in the airways were determined using ELISA and immunofluorescence. Cytokine levels in the BALF were also measured.
Results
NPY levels were undetectable in the BALF of control mice, but significantly increased in the OVA group at day 80. Levels of IL-4, TGF-β1 and TGF-β2, significantly increased and peaked on day 45 and decreased on day 80 in the OVA group, exhibiting an inverse correlation with NPY levels. NPY expression was localized to macrophage-like cells in the peri-bronchial and peri-vascular areas in the lung tissue. NPY-Y1 and -Y5 receptors were constitutively expressed by both structural and inflammatory cells in the lung tissue.
Conclusions
NPY produced by activated macrophage-like cells may be involved in regulating cytokine production and cellular activities of immune cells in asthma. However, it remains unclear whether such an increase in NPY is a defensive/compensatory mechanism to modulate the effects of inflammatory cytokines.
doi:10.1016/j.yexmp.2012.05.009
PMCID: PMC3488603  PMID: 22705097
Allergic asthma; antigen presenting cells (APCs); macrophage; neuropeptide Y (NPY); NPY receptor-Y1; transforming growth factor beta (TGF-β)

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