Intrapleural nano-immunotherapy promotes innate and adaptive immune responses to enhance anti-PD-L1 therapy for malignant pleural effusion.

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy.

Intrapleural Injection of Anti-PD1 Antibody: A Novel Management of Malignant Pleural Effusion.

Background: Malignant tumors accompanied with malignant pleural effusion (MPE) often indicate poor prognosis. The therapeutic effect and mechanism of intrapleural injection of anti-programmed cell death protein 1 (PD1) on MPE need to be explored. Methods: A preclinical MPE mouse model and a small clinical study were used to evaluate the effect of intrapleural injection of anti-PD1 antibody. The role of immune cells was observed via flow cytometry, RNA-sequencing, quantitative PCR, western blot, immunohistochemistry, and other experimental methods. Results: Intrathoracic injection of anti-PD1 monoclonal antibody (mAb) has significantly prolonged the survival time of mice (P = 0.0098) and reduced the amount of effusion (P = 0.003) and the number of cancer nodules (P = 0.0043). Local CD8+ T cells participated in intrapleural administration of anti-PD1 mAb. The proportion of CD69+, IFN-γ+, and granzyme B+ CD8+ T cells in the pleural cavity was increased, and the expression of TNF-α and IL-1ß in MPE also developed significantly after injection. Local injection promoted activation of the CCL20/CCR6 pathway in the tumor microenvironment and further elevated the expression of several molecules related to lymphocyte activation. Clinically, the control rate of intrathoracic injection of sintilimab (a human anti-PD1 mAb) for 10 weeks in NSCLC patients with MPE was 66.7%. Local injection improved the activity and function of patients' local cytotoxic T cells (CTLs). Conclusions: Intrapleural injection of anti-PD1 mAb could control malignant pleural effusion and the growth of cancer, which may be achieved by enhancing local CTL activity and cytotoxicity.

Tissue-Resident Memory-Like CD8+ T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion.

Tissue-resident memory T (TRM) cells are well known to play critical roles in peripheral tissues during virus infection and tumor immunology. Our previous studies indicated that CD69+CD4+ and CD69+CD8+ T cells in tuberculous pleural effusion (TPE) were antigen-specific memory T cells. However, the phenotypical and functional characteristics of CD8+ TRM cells in tuberculosis remain unknown. We found that CD103+CD8+ T cells were the predominant subset of CD103+ lymphocytes in TPE; both CD103 and CD69 expressed on memory CD8+ T cells from TPE were significantly increased compared with those from paired peripheral blood. Phenotypically, CD103+CD69+ and CD103+CD69-CD8+ T cells expressed higher levels of CD45RO than CD103-CD69+CD8+ T cells did; CD103+CD69-CD8+ T cells highly expressed CD27, CD127, and CD62L and some chemokine receptors. We further compared the functional differences among the four distinct CD45RO+CD8+ T subsets identified by CD103 and CD69 expression. In consist with our published results, CD69+CD8+ T cells, but not CD103+CD8+, produced high levels of IFN-γ after treatment with BCG in the presence of BFA. Nevertheless, CD103-CD69+ and CD103+CD69+ memory CD8+ T cells expressed higher levels of Granzyme B, while CD103+CD69- memory CD8+ T cells were characterized as a possibly immunosuppressive subset by highly expressing CTLA-4, CD25, and FoxP3. Furthermore, TGF-ß extremely increased CD103 expression but not CD69 in vitro. Together, CD103+CD8+ T cells form the predominant subset of CD103+ lymphocytes in TPE; CD103 and CD69 expression defines distinct CD8+ TRM-like subsets exhibiting phenotypical and functional heterogeneity. Our findings provide an important theoretical basis to optimize and evaluate new tuberculosis vaccines.

Novel therapies for malignant pleural effusion: Anti­angiogenic therapy and immunotherapy (Review).

Patients with a variety of malignancies can develop malignant pleural effusion (MPE). MPE can cause significant symptoms and result in a marked decrease in quality of life and a poor prognosis. MPE is primarily considered as an immune and vascular manifestation of pleural metastases. In the present review, the existing evidence supporting the applicability of anti­angiogenic therapy and immunotherapy for the treatment of MPE was summarized. Patients with MPE have benefited from anti­angiogenic agents, including bevacizumab and endostar; however, no relevant prospective phase III trial has, thus far, specifically analyzed the benefit of anti­angiogenic therapy in MPE. Immunotherapy for MPE may be sufficient to turn a dire clinical situation into a therapeutic advantage. Similar to anti­angiogenic therapy, more clinical data on the efficiency and safety of immunotherapy for controlling MPE are urgently required. The combined use of anti­angiogenic therapy and immunotherapy may be a promising strategy for MPE, which requires to be further understood.

Microfilaria-dependent thoracic pathology associated with eosinophilic and fibrotic polyps in filaria-infected rodents.

BACKGROUND: Pulmonary manifestations are regularly reported in both human and animal filariasis. In human filariasis, the main known lung manifestations are the tropical pulmonary eosinophilia syndrome. Its duration and severity are correlated with the presence of microfilariae. Litomosoides sigmodontis is a filarial parasite residing in the pleural cavity of rodents. This model is widely used to understand the immune mechanisms that are established during infection and for the screening of therapeutic molecules. Some pulmonary manifestations during the patent phase of infection with L. sigmodontis have been described in different rodent hosts more or less permissive to infection. METHODS: Here, the permissive Mongolian gerbil (Meriones unguiculatus) was infected with L. sigmodontis. Prevalence and density of microfilariae and adult parasites were evaluated. Lungs were analyzed for pathological signatures using immunohistochemistry and 3D imaging techniques (two-photon and light sheet microscopy). RESULTS: Microfilaremia in gerbils was correlated with parasite load, as amicrofilaremic individuals had fewer parasites in their pleural cavities. Fibrotic polypoid structures were observed on both pleurae of infected gerbils. Polyps were of variable size and developed from the visceral mesothelium over the entire pleura. The larger polyps were vascularized and strongly infiltrated by immune cells such as eosinophils, macrophages or lymphocytes. The formation of these structures was induced by the presence of adult filariae since small and rare polyps were observed before patency, but they were exacerbated by the presence of gravid females and microfilariae. CONCLUSIONS: Altogether, these data emphasize the role of host-specific factors in the pathogenesis of filarial infections.

A Stromal Niche Defined by Expression of the Transcription Factor WT1 Mediates Programming and Homeostasis of Cavity-Resident Macrophages.

Tissue-resident macrophages require specific milieus for the maintenance of defining gene-expression programs. Expression of the transcription factor GATA6 is required for the homeostasis, function and localization of peritoneal cavity-resident macrophages. Gata6 expression is maintained in a non-cell autonomous manner and is elicited by the vitamin A metabolite, retinoic acid. Here, we found that the GATA6 transcriptional program is a common feature of macrophages residing in all visceral body cavities. Retinoic acid-dependent and -independent hallmark genes of GATA6+ macrophages were induced by mesothelial and fibroblastic stromal cells that express the transcription factor Wilms' Tumor 1 (WT1), which drives the expression of two rate-limiting enzymes in retinol metabolism. Depletion of Wt1+ stromal cells reduced the frequency of GATA6+ macrophages in the peritoneal, pleural and pericardial cavities. Thus, Wt1+ mesothelial and fibroblastic stromal cells constitute essential niche components supporting the tissue-specifying transcriptional landscape and homeostasis of cavity-resident macrophages.

Inhibition of the sphingosine-1-phosphate pathway promotes the resolution of neutrophilic inflammation.

Sphingosine-1-phosphate (S1P) is an important sphingolipid derived from plasma membrane and has a known role in productive phase of inflammation, but its role in neutrophil survival and resolution phase of inflammation is unknown. Here, we investigated the effects of inhibition of S1P receptors and the blockade of S1P synthesis in BALB/c mice and human neutrophils. S1P and S1PR1-3 receptors expression were increased in cells from the pleural cavity stimulated with LPS. Using different antagonists of S1PRs and inhibitors of different steps of the metabolic pathway of S1P production, we show that S1P and its receptors are involved in regulating neutrophil survival and resolution of inflammation in the pleural cavity. Given the role of the S1P-S1PR axis in resolution of inflammation, we sought to identify whether blockade at different levels of the sphingosine-1-phosphate synthesis pathway could affect neutrophil survival in vitro. Inhibitors of the S1P pathway were also able to induce human neutrophil apoptosis. In addition, blockade of S1P synthesis or its receptor facilitated the efferocytosis of apoptotic neutrophil. Taken together, our data demonstrate a fundamental role for S1P in regulating the outcome of inflammatory responses, and position S1P-S1PR axis as a potential target for treatment of neutrophilic inflammation.

The biology of serous cavity macrophages.

For decades, it has been known that the serous cavities, which include the peritoneal, pleural and pericardial cavities, harbour large numbers of macrophages. In particular, due to the ease of isolating these cells, the peritoneal cavity has been used as a convenient source of macrophages to examine many facets of macrophage biology over the last 50-60 years. Despite this, it is only recently that the true heterogeneity of serous cavity mononuclear phagocyte compartment, which includes macrophages and dendritic cells, has been revealed. Advances in technologies such as multi-parameter flow cytometry and the 'OMICs' revolution have uncovered the presence of distinct populations of mononuclear phagocytes in the serous cavities. Given that peritoneal macrophages have been implicated in many pathologies, including peritonitis, pancreatitis, endometriosis and acute liver injury, it is imperative to understand the biology of these cells. Here, we review the recent advances in understanding the identity, origin and function of discrete serous cavity mononuclear phagocyte subsets in homeostasis and how these may change when homeostasis is perturbed, focusing on peritoneal and pleural cavities and highlighting differences in the mononuclear phagocytes found in each.

Myeloid cell recruitment versus local proliferation differentiates susceptibility from resistance to filarial infection.

Both TH2-dependent helminth killing and suppression of the TH2 effector response have been attributed to macrophages (MΦ) activated by IL-4 (M(IL-4)). To investigate how M(IL-4) contribute to diverse infection outcomes, the MΦ compartment of susceptible BALB/c mice and more resistant C57BL/6 mice was profiled during infection of the pleural cavity with the filarial nematode, Litomosoides sigmodontis. C57BL/6 mice exhibited a profoundly expanded resident MΦ (resMΦ) population, which was gradually replenished from the bone marrow in an age-dependent manner. Infection status did not alter the bone-marrow derived contribution to the resMΦ population, confirming local proliferation as the driver of resMΦ expansion. Significantly less resMΦ expansion was observed in the susceptible BALB/c strain, which instead exhibited an influx of monocytes that assumed an immunosuppressive PD-L2+ phenotype. Inhibition of monocyte recruitment enhanced nematode killing. Thus, the balance of monocytic vs. resident M(IL-4) numbers varies between inbred mouse strains and impacts infection outcome.

Intrapleural Adenoviral-mediated Endothelial Cell Protein C Receptor Gene Transfer Suppresses the Progression of Malignant Pleural Mesothelioma in a Mouse Model.

Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer with a high mortality rate as it responds poorly to standard therapeutic interventions. Our recent studies showed that expression of endothelial cell protein C receptor (EPCR) in MPM cells suppresses tumorigenicity. The present study was aimed to investigate the mechanism by which EPCR suppresses MPM tumor growth and evaluate whether EPCR gene therapy could suppress the progression of MPM in a mouse model of MPM. Measurement of cytokines from the pleural lavage showed that mice implanted with MPM cells expressing EPCR had elevated levels of IFNγ and TNFα compared to mice implanted with MPM cells lacking EPCR. In vitro studies demonstrated that EPCR expression renders MPM cells highly susceptible to IFNγ + TNFα-induced apoptosis. Intrapleural injection of Ad.EPCR into mice with an established MPM originating from MPM cells lacking EPCR reduced the progression of tumor growth. Ad.EPCR treatment elicited recruitment of macrophages and NK cells into the tumor microenvironment and increased IFNγ and TNFα levels in the pleural space. Ad.EPCR treatment resulted in a marked increase in tumor cell apoptosis. In summary, our data show that EPCR expression in MPM cells promotes tumor cell apoptosis, and intrapleural EPCR gene therapy suppresses MPM progression.

Pleural cavity type 2 innate lymphoid cells precede Th2 expansion in murine Litomosoides sigmodontis infection.

Recently, a family of innate cells has been identified that respond to IL-25 and IL-33 in murine intestinal helminths. Termed Type 2 innate lymphoid cells (ILC2s) they facilitate the development of Th2 responses responsible for helminth clearance. We evaluated these cells in a tissue-invasive helminth model. Using Litomosides sigmodontis (a strong Th2 polarizing filarial infection) we observed a robust Th2 response in the pleural cavity, where adult worms reside, marked by increased levels of IL-5 and IL-13 in infected mice. In parallel, ILC2s were expanded in the pleural cavity early in the infection, peaking during the pre-patent period. L. sigmodontis also elicits a strong systemic Th2 response, which includes significantly increased levels of IgG1, IgE and IL-5 in the plasma of infected mice. Although ILC2s were expanded locally, they were not expanded in the spleen, blood, or mediastinal lymph nodes in response to L. sigmodontis infection, suggesting that ILC2s function primarily at the site of infection. The increase in ILC2s in the pleural cavity and the expansion in Th2 responses indicates a probable role for these cells in initiating and maintaining the Th2 response and highlights the importance of these cells in helminth infections and their role in Th2 immunity.

Modulatory effect of Senecio brasiliensis (Spreng) Less. in a murine model of inflammation induced by carrageenan into the pleural cavity.

ETHNOPHARMACOLOGICAL RELEVANCE: Senecio brasiliensis (Spreng) Less (S. brasiliensis), known as "Flor-das-almas", "Margaridinha" or "Maria mole", is used in folk medicine as an anti-inflammatory and to treat gastric ulcers and stomach pain. While the Senecio genus has been widely studied for its pharmacological activities to support its use in traditional medicine, few studies focus on the anti-inflammatory activities of the species. AIM OF THE STUDY: To investigate the anti-inflammatory activities of S. brasiliensis, a specie native to Brazil, using a murine model of pleurisy induced by carrageenan. MATERIAL AND METHODS: The flowers of S. brasiliensis were air-dried for 3 days and subjected to ethanol (96%) extraction for 7 days to obtain the crude extract (CE). The CE was subjected to acid-base extraction to obtain the alkaloid fraction (AF). The hexane (HEX), dichloromethane (DCM) and ethyl acetate (EtOAc) fractions were obtained by extracting from CE with different solvents. The alkaloids senecionine (Sen), integerrimine (Int) and senecionine N-oxide were obtained from AF by chromatographic fractionation and a mixture of 1,4-, 3,4-, 3,5- and 4,5-dicaffeoylquinic acids (DCQs) were obtained from the EtOAc fraction. The isolated alkaloids were identified through spectroscopic analysis of IR, NMR and LC-MS coupled with electrospray ionization mass spectrometry (ESI-MS), and the dicaffeoylquinic acids through the hierarchical key method. Swiss mice were used in the in vivo experiments. We evaluated the effect of the CE, its derived fractions (AF, HEX, DCM and EtOAc), and the isolated compounds (Sen, Int, N-oxide senecionine, and DCQs) on: leukocyte migration, exudate concentrations, myeloperoxidase (MPO) and adenosine-deaminase (ADA) activities, and tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß) and interleukin 17A levels in the fluid leakage from the pleural cavity using a mouse model of pleurisy induced by carrageenan. The effects of the isolated compounds, Sen, Int, N-oxide senecionine and DCQs, were also analyzed for their ability to inhibit p65 phosphorylation (p-p65) in the nuclear factor-kappa B (NF-κB) pathway in the lung tissue. MPO and ADA were analyzed by colorimetric assays, and the cytokines and protein p65 levels were determined using an enzyme immunoassay (EIA). RESULTS: The CE, its EtOAc and AF fractions, and its isolated compounds (Sen, Int and DCQs), significantly reduced leukocyte migration (P < 0.05), MPO and ADA activities (P < 0.01), and TNF-α (P < 0.05), and IL-17A levels (P < 0.01). The CE, the EtOAc and AF fractions, and the DCQs also decreased IL-1ß levels (P < 0.01). The isolated compounds, Sen, Int and the DCQs, inhibited p65 phosphorylation (NF-κB) (P < 0.05). CONCLUSION: This study demonstrated that S. brasiliensis has important anti-inflammatory properties that are capable of inhibiting activated leukocytes by decreasing neutrophil migration. This effect may be attributed to the inhibition of pro-inflammatory cytokines and the reduction of the NF-κB pathway. The compounds Sen, Int, and DCQs may be responsible for the anti-inflammatory actions of S. brasiliensis.

Plasmin induces in vivo monocyte recruitment through protease-activated receptor-1-, MEK/ERK-, and CCR2-mediated signaling.

The plasminogen (Plg)/plasmin (Pla) system is associated with a variety of biological activities beyond the classical dissolution of fibrin clots, including cell migration, tissue repair, and inflammation. Although the capacity of Plg/Pla to induce cell migration is well defined, the mechanism underlying this process in vivo is elusive. In this study, we show that Pla induces in vitro migration of murine fibroblasts and macrophages (RAW 264.7) dependent on the MEK/ERK pathway and by requiring its proteolytic activity and lysine binding sites. Plasmin injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that was associated with augmented ERK1/2 and IκB-α phosphorylation and increased levels of CCL2 and IL-6 in pleural exudates. The inhibition of protease activity by using a serine protease inhibitor leupeptin or two structurally different protease-activated receptor-1 antagonists (SCH79797 and RWJ56110) abolished Pla-induced mononuclear recruitment and ERK1/2 and IκB-α phosphorylation. Interestingly, inhibition of the MEK/ERK pathway abolished Pla-induced CCL2 upregulation and mononuclear cell influx. In agreement with a requirement for the CCL2/CCR2 axis to Pla-induced cell migration, the use of a CCR2 antagonist (RS504393) prevented the Plg/Pla-induced recruitment of mononuclear cells to the pleural cavity and migration of macrophages at transwell plates. Therefore, Pla-induced mononuclear cell recruitment in vivo was dependent on protease-activated receptor-1 activation of the MEK/ERK/NF-κB pathway, which led to the release of CCL2 and activation of CCR2.

Development of primary invasive pneumococcal disease caused by serotype 1 pneumococci is driven by early increased type I interferon response in the lung.

The pneumococcus is the world's foremost respiratory pathogen, but the mechanisms allowing this pathogen to proceed from initial asymptomatic colonization to invasive disease are poorly understood. We have examined the early stages of invasive pneumococcal disease (IPD) by comparing host transcriptional responses to an invasive strain and a noninvasive strain of serotype 1 Streptococcus pneumoniae in the mouse lung. While the two strains were present in equal numbers in the lung 6 h after intranasal challenge, only the invasive strain (strain 1861) had invaded the pleural cavity at that time point; this correlated with subsequent development of bacteremia in mice challenged with strain 1861 but not the noninvasive strain (strain 1). Progression beyond the lung was associated with stronger induction of the type I interferon (IFN-I) response in the lung at 6 h. Suppression of the IFN-I response through administration of neutralizing antibody to IFNAR1 (the receptor for type I interferons) led to significantly reduced invasion of the pleural cavity by strain 1861 at 6 h postchallenge. Our data suggest that strong induction of the IFN-I response is a key factor in early progression of invasive serotype 1 strain 1861 beyond the lung during development of IPD.

Eotaxin-1 is involved in parasite clearance during chronic filarial infection.

Eosinophil migration as key feature of helminth infection is increased during infection with filarial nematodes. In a mouse model of filariasis, we investigated the role of the eosinophil-attracting chemokine Eotaxin-1 on disease outcome. BALB/c and Eotaxin-1(-/-) mice were infected with the rodent filaria Litomosoides sigmodontis, and parasitic parameters, cellular migration to the site of infection, and cellular responsiveness were investigated. We found increased parasite survival but unaffected eosinophil migration to the site of infection in Eotaxin-1(-/-) mice. Expression of CD80 and CD86 was reduced on eosinophils from Eotaxin-1(-/-) mice after in vitro TLR2 stimulation and exposure to filarial antigen, respectively, suggesting a potential reduced activation state of eosinophils in Eotaxin-1 deficient mice. We further demonstrated that macrophages from Eotaxin-1(-/-) mice produce decreased amounts of IL-6 in vitro, a cytokine found to be associated with parasite containment, suggesting possible mechanisms by which Eotaxin-1 regulates activation of inflammatory cells and thus parasite survival.

Mast cell tryptase induces eosinophil recruitment in the pleural cavity of mice via proteinase-activated receptor 2.

Proteinase-activated receptor (PAR) 2 has been implicated in eosinophil migration. Mast cell (MC) tryptase has been similarly implicated in allergic diseases through the activation of PAR-2, but the role of this receptor in MC tryptase-induced inflammation is not well elucidated. This study aims to investigate the ability of MC tryptase or PAR-2 activating peptide (SLIGRL-NH2) to induce eosinophil recruitment to the pleural cavity of mice. Mast cell tryptase-injected mice were pretreated with PAR-2 antagonist ENMD-1068. Mice injected with SLIGRL-NH2 were pretreated with mast cell tryptase inhibitor APC 366, and eosinophil migration into the pleural cavity and PAR-2 expression was analyzed after 24 or 48 h. SLIGRL-NH2-induced eosinophil recruitment was inhibited by APC 366, and MC tryptase-induced eosinophil recruitment was abolished by ENMD-1068. MC tryptase induced PAR-2 expression on pleural eosinophils. Our results demonstrate a key role for PAR-2 in mediating eosinophil recruitment in MC tryptase-induced pleurisy in mice. The ability of MC tryptase to inducing PAR-2 expression on eosinophils corroborates the relevance of MC tryptase and PAR-2 on modulating eosinophil migration.

[Pleural lymphatics and effusions]. / Lymphatiques pleuraux et épanchements.

The pleural lymphatic system has a great absorption capacity. Its most known function is fluid resorption. The pleura which cover the lungs (visceral pleura), the mediastinum, diaphragm and thoracic wall (parietal pleura) are formed by a mesothelial cell layer (mesothelium). This permeable layer is in direct contact with the vascular endothelium. The mesothelium is based over a connective tissue (interstitium) containing the blood and lymphatic vessels. The primary lymphatic vessels drain interstitium but are also in direct contact with pleural space by the stoma or openings, situated in the lower parts of parietal pleura, i.e: diaphragm, over lower ribs and mediastinum but not existing in the adjacent visceral pleura. In addition, a part of interstitial pulmonary fluid entered in the pleural cavity by passing the visceral pleura would be absorbed by these openings. The resorption process is active and directly related to the function of smooth muscles of lymphatic vessels. Besides resorption, we must emphasize that this "pumping" activity is permanent and the origin of negative pressure (the pleural void) in pleural cavity, a unique property. The other resorbed elements are molecules, bacterial and cellular debris, cells, red blood and cancer cells.

B16 melanoma cells increase B-1 cell survival, IL-10 production and radioresistance in vitro.

B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. The role of both cell populations in cancer progression is still controversial. Previous studies have indicated that direct contact between B-1 cells and B16 melanoma tumor cells (B16) increases the metastatic potential of the tumor cells. However, cellular changes that are induced in B-1 cells during the interaction between these two cell types have not been evaluated. In the present study, it is hypothesized that B-1 cells are modified after their interaction with tumor cells, leading to both increased cell viability and rate of proliferation. Additionally, soluble factors that were secreted by B16 cells were sufficient to augment B-1 cell viability and to modify the production of IL-10 by B-1 cells. Impressively, after direct or indirect contact with the B16 cells, B-1 cells became resistant to radiation-induced cell death. Thus, future studies that assess the importance of concomitant immunity and other conventional therapies in cancer treatment are needed.

Rat hepatitis E virus: geographical clustering within Germany and serological detection in wild Norway rats (Rattus norvegicus).

Zoonotic hepatitis E virus (HEV) infection in industrialised countries is thought to be caused by transmission from wild boar, domestic pig and deer as reservoir hosts. The detection of HEV-specific antibodies in rats and other rodents has suggested that these animals may represent an additional source for HEV transmission to human. Recently, a novel HEV (ratHEV) was detected in Norway rats from Hamburg, Germany, showing the typical genome organisation but a high nucleotide and amino acid sequence divergence to other mammalian and to avian HEV strains. Here we describe the multiple detection of ratHEV RNA and HEV-specific antibodies in Norway rats from additional cities in north-east and south-west Germany. The complete genome analysis of two novel strains from Berlin and Stuttgart confirmed the association of ratHEV to Norway rats. The present data indicated a continuing existence of this virus in the rat populations from Berlin and Hamburg. The phylogenetic analysis of a short segment of the open reading frame 1 confirmed a geographical clustering of the corresponding sequences. Serological investigations using recombinant ratHEV and genotype 3 capsid protein derivatives demonstrated antigenic differences which might be caused by the high amino acid sequence divergence in the immunodominant region. The high amount of animals showing exclusively ratHEV RNA or anti-ratHEV antibodies suggested a non-persistent infection in the Norway rat. Future studies have to prove the transmission routes of the virus in rat populations and its zoonotic potential. The recombinant ratHEV antigen generated here will allow future seroepidemiological studies to differentiate ratHEV and genotype 3 infections in humans and animals.

Inhibition of advanced glycation end products by aminoguanidine restores mast cell numbers and reactivity in alloxan-diabetic rats.

Mast cell number and reactivity have been shown to be down-regulated under diabetic conditions. This study was undertaken in order to investigate the role of the advanced glycation end products in the reduction of mast cell number and reactivity in diabetic rats. The effect of aminoguanidine on mast cell apoptosis was also evaluated. Diabetes was induced by intravenous injection of alloxan into fasted rats and aminoguanidine was administered after 3 days of diabetes induction, once daily for 18 consecutive days. Mast cell apoptosis and levels of Bax, a pro-apoptotic member of Bcl-2 family, were evaluated by TUNEL and western blot, respectively. Diabetes led to increased levels of fructosamine and AGEs in the plasma, an effect prevented by aminoguanidine. Treatment with aminoguanidine restored mast cell numbers in the pleural cavity and in mesenteric tissue of diabetic rats. Aminoguanidine also significantly reversed the diabetes-induced reduction in histamine release, as measured by fluorescence, following activation with substance P or antigen in vitro. Increased apoptosis and levels of Bax in mast cells from diabetic rats were inhibited by aminoguanidine. In conclusion, our findings showed that aminoguanidine restored the number and reactivity of mast cells in diabetic rats, accompanied by suppression of apoptosis, evidencing that advanced glycation end product formation has a critical role in mast cell behavior of diabetic rats.