Comparative study of the in situ immune response in oral and nasal mucosal leishmaniasis

Mucosal Leishmaniasis (ML) may occur in both nasal and oral mucosa. However, despite the impressive tissue destruction, little is known about the oral involvement. To compare some changes underlying inflammation in oral and nasal ML, we performed immunohistochemistry on mucosal tissue of 20 patients with ML (nasal [n = 12]; oral [n = 8] lesions) and 20 healthy donors using antibodies that recognize inflammatory markers (CD3, CD4, CD8, CD22, CD68, neutrophil elastase, CD1a, CLA, Ki67, Bcl-2, NOS2, CD62E, Fas and FasL). A significantly larger number of cells, mainly T cells and macrophages, were observed in lesions than in healthy tissue. In addition, high nitric oxide synthase 2 (NOS2) expression was associated with a reduced detection of parasites, highlighting the importance of NOS2 for parasite elimination. Oral lesions had higher numbers of neutrophils, parasites, proliferating cells and NOS2 than nasal lesions. These findings, together with the shorter duration of oral lesions and more intense symptoms, suggest a more recent inflammatory process. It could be explained by lesion-induced oral cavity changes that lead to eating difficulties and social stigma. In addition, the frequent poor tooth conservation and gingival inflammation tend to amplify tissue destruction and symptoms and may impair and confuse the correct diagnosis, thus delaying the onset of specific treatment.     

Very early onset of an acute myeloid leukemia in an adult patient with B‐cell lymphoblastic leukemia

We report on a case of a 30‐year‐old male with acute B‐lymphoblastic leukemia (B‐ALL) with immunophenotype CD19⁺, CD22⁺, CD20⁺, CD10⁺, with aberrant expression of CD13 and CD117, and IgH gene rearrangements. Three months after treatment with GMALL‐2003 and Ida/FLAG protocols bone marrow showed predominance of blasts with myeloid morphology and phenotype MPO⁺, CD13⁺, CD33⁺, CD64⁺, CD15⁺, CD56⁺, EVI‐1 gene overexpression and lack of IgH rearrangements. The case is the first report of a very early emergence of myeloid leukemia during the induction treatment for B‐ALL in an adult patient. Different pathogenetic mechanisms are discussed – clonal evolution or selection, lineage switch or development of a de novo or therapy‐induced leukemia.     

Alterations in monocyte subsets and cytokine production after TLR activation in American Cutaneous Leishmaniasis

Phenotypic and functional aspects of monocytes from Localized Cutaneous Leishmaniasis (LCL) patients were evaluated. The frequencies of monocyte subsets and TLR2/TLR4 expression were evaluated in fresh peripheral blood whereas cytokine production was evaluated in whole blood cell cultures stimulated with TLR agonists or Leishmania braziliensis antigen (Ag). CD16⁺ monocytes frequency was increased in patients compared with controls. A TLR4 agonist (LPS) induced expression of TNF and IL‐10 in monocyte subsets of patients and controls. The CD14⁺CD16⁺ monocytes expressed higher levels of these cytokines than CD14⁺CD16^? cells. The levels of secreted TNF were higher in whole blood cell cultures from patients than controls after LPS/TLR4 or Ag stimulation. Whereas in controls there was a positive correlation between TNF and IL‐10 levels, this was not observed in stimulated cell cultures from patients. The high levels of LPS‐induced TNF were associated with the number of lesions and the percentages of CD14^hiCD16⁺ monocytes. The levels of TLR2‐induced TNF were also associated with number of lesions. All monocyte subsets from patients expressed higher levels of TLR2 and TLR4 than controls. Data suggest that systemically activated monocytes contribute for an imbalance in pro‐ and anti‐inflammatory cytokine production during LCL, participating in the immunopathogenesis of the disease.     

Triggering receptor expressed on myeloid cells (TREM)-1 participates in Schistosoma mansoni inflammatory responses

Inflammatory responses to microbial products are amplified by a pathway mediated by triggering a receptor expressed on the myeloid cells (TREM)‐1. Relatively a few studies have been performed to investigate the role of TREM‐1 in macrophage activation in response to parasitic infection. In this study, we delineate the role of the innate immunoreceptor TREM‐1 in the parasite Schistosoma mansoni infection model from early to late (chronic) phases of infection. Flow cytometry analysis revealed gradual increase in the production of TREM‐1 protein on CD11b⁺ myeloid cells, with maximum production at 5 weeks p.i. Similar results in the pattern of TREM‐1 mRNA expressions in splenic CD11b⁺ cells from infected mice were obtained by real‐time PCR. However, unlike in spleen, the TREM‐1 mRNA expression in liver tissue showed no significant increase throughout the infection, including periods of maximum production of parasite eggs. Administration of schistosoma egg homogenate antigen to stimulate J774A.1 cells inhibited TREM‐1 expression on the surface, indicating that some substances of the Schistosma eggs may inhibit the expression of TREM‐1 on macrophages, lowering the macrophage‐mediated inflammatory response of infected hosts.     

CCR4 expression in a case of cutaneous Richter's transformation of chronic lymphocytic leukemia (CLL) to diffuse large B-cell lymphoma (DLBCL) and in CLL patients with no skin manifestations

Objective: Richter’s transformation of B‐cell chronic lymphocytic leukemia (CLL) to cutaneous diffuse large B‐cell lymphoma (DLBCL) is very rare. We took the advantage of one of these cases to test the hypothesis that the chemokine receptor CCR4 is involved in the homing of CLL cells to skin. Patients and Methods: We evaluated CCR4 expression by flow cytometry in both circulating and skin CD19⁺ leukemic cells from a patient with cutaneous DLBCL. As controls, we used peripheral blood samples from CLL patients without skin manifestations and from elderly healthy donors. Results: We found that both DLBCL cells derived from the original CLL clone and circulating CLL cells from this patient expressed CCR4. Although it was previously reported that CCR4 is not expressed in CLL cells, we found that a low but significant proportion of leukemic cells from CLL patients with no skin manifestations do express CCR4. There was a positive correlation between the expression of CCR4 and the percentage of ZAP‐70 of each sample. Moreover, we consistently observed a higher expression of CCR4 within CD19⁺CD38⁺ and CD19⁺Ki67⁺ subsets compared to CD19⁺CD38^? and CD19⁺Ki67^? lymphocytes from the same sample, respectively. Conclusion: We conclude that the chemokine receptor CCR4 is not a special feature of CLL cells with skin manifestation, but rather it is expressed in a low but significant proportion of peripheral blood CLL cells.     

Kinetics of the local immune response in the gastric lymph of lambs after primary and challenge infection with Teladorsagia circumcincta

Groups of 5‐month‐old lambs which had been trickle infected with Teladorsagia circumcincta for 8 weeks then drenched, and worm‐free control lambs were challenged with 50 000 T. circumcincta L3s. From 10 days later fewer parasites were recovered from the previously infected sheep, and secondary cellular and humoral responses were observed in the gastric lymph. Increases in CD4⁺ and CD25⁺ T lymphoblast traffic on day 3, followed by CD21⁺ and IgA⁺ lymphoblasts on day 5, and an increase in total and parasite specific IgA concentrations peaking on day 6 were observed in previously infected lambs. Similar peaks in lymphoblast output were not observed until days 10–12 in the control lambs. This data was highly comparable with that obtained recently from yearling sheep subjected to an identical infection‐challenge regime, and contrasted with that obtained from similar experiments in the 1980s when 4^1/2‐month‐old previously infected lambs were more susceptible to and had much weaker immune responses to challenge than 10‐month‐old sheep. The fact that 40% fewer larvae were given during the trickle infection regime in the four recent trials is offered as an explanation for this difference.     

Emergence of Fibrocytes Showing Morphological Changes in the Inflamed Colonic Mucosa

Fibrocytes contribute to wound healing and are uniquely defined by coexpression of hematopoietic and mesenchymal cell markers. In this study, trafficking of fibrocytes was determined in a murine model of colitis induced by administering 3% dextran sodium sulfate (DSS) for seven days. Colonic tissues were immunostained for CD45, collagen type I (Col I), and α-SMA. On day 0, there were no CD45⁺Col I⁺ cells in colonic tissues. However, on day 7 when inflammatory cells showed remarkable accumulation, oval-shaped CD45⁺Col I⁺ fibrocytes were obvious in the submucosal layer. On day 14 when colonic tissues were in the healing phase, numerous spindle-shaped CD45⁺Col I⁺ fibrocytes were observed. Emergence of CD45⁺Col I⁺ fibrocytes preceded the appearance of α-SMA⁺ myofibroblasts. Oval-shaped fibrocytes recruited as early as the inflammatory phase of colitis are likely to differentiate into spindle-shaped fibrocytes in the healing phase, suggesting that fibrocytes may promote wound healing in inflamed colonic tissues.     

Impairment of Host Resistance to Helminthes with Age in Murine Small Intestine

Age‐associated alterations of Th2 immune responses against nematode parasites are largely unknown. We investigated primary and memory responses against two types of gastrointestinal nematode parasites, Heligmosomoides polygyrus (Hp) and Nippostrongylus brasiliensis (Nb), in aged mice. The small intestinal gene expression of Th2 cytokines was almost unchanged after primary (Nb and Hp) and secondary infection (Hp) in aged mice in contrast to strongly increased small intestinal gene expression of Th2 cytokines in young (3‐month‐old) mice. Mucus production decreased (Nb), and worm expulsion was impaired (Nb and Hp) compared with the young mice. Immunofluorescent staining revealed that after Hp infection, the number of alternatively activated macrophages, which are induced by Th2 cytokines, was lower in the aged mice. On the other hand, the number of CD4⁺ T cells recruited to the worm cysts was normal compared with the young mice. These results suggest that migration of CD4⁺ T cells to the host–parasite interface is not affected by ageing. Alterations in Th2 immune responses in aged mice might be due to inappropriate or insufficient activation of CD4⁺ T cells in the submucosa.     

Upregulation of CD72 expression on CD19+CD27+ memory B cells by CD40L in primary immune thrombocytopenia

CD72 is a co‐receptor of B cells and regulates B cell activation. Although aberrant expression of CD72 has been reported in primary immune thrombocytopenia (ITP), it is uncertain whether this aberrant expression is restricted to specific B cell subsets. Furthermore, the mechanisms that regulate CD72 expression are unknown. In this study, we found higher frequency of CD19⁺ B cells, CD19⁺CD27⁺ memory B cells and lower frequency of CD19⁺CD27⁻ naive B cells in active ITP patients compared with controls and patients in remission. CD72 expression on CD19⁺CD27⁺ cells was upregulated in active ITP patients and correlated with platelet count and anti‐platelet autoantibodies. In vitro, CD40L could specifically induce CD72 upregulation on CD19⁺CD27⁺ B cells. In combination with CD40L, interleukin (IL) 10 and BAFF (also termed TNFSF13B) further enhanced CD72 expression on CD19⁺CD27⁺ B cells, whereas IL21 reduced CD72 upregulation. CD72mRNA expression after CD40L stimulation was increased in ITP patients and controls. Significant increase of CD40L on CD4⁺ T cells was correlated with CD72 expression on CD19⁺CD27⁺ B cells in ITP patients. In conclusion, upregulation of CD72 expression on CD27⁺memory B cells might take part in the pathogenesis of ITP. Elevated CD40L on CD4⁺ cells combined with cytokines might contribute to the upregulation of CD72 expression on CD27⁺memory B cells.     

IL‐6 promotes CD4+ T‐cell and B‐cell activation during Plasmodium infection

Humoral immunity develops in the spleen during blood‐stage Plasmodium infection. This elicits parasite‐specific IgM and IgG, which control parasites and protect against malaria. Studies in mice have elucidated cells and molecules driving humoral immunity to Plasmodium, including CD4⁺ T cells, B cells, interleukin (IL)‐21 and ICOS. IL‐6, a cytokine readily detected in Plasmodium‐infected mice and humans, is recognized in other systems as a driver of humoral immunity. Here, we examined the effect of infection‐induced IL‐6 on humoral immunity to Plasmodium. Using P. chabaudi chabaudi AS (PcAS) infection of wild‐type and IL‐6^?/? mice, we found that IL‐6 helped to control parasites during primary infection. IL‐6 promoted early production of parasite‐specific IgM but not IgG. Notably, splenic CD138⁺ plasmablast development was more dependent on IL‐6 than germinal centre (GC) B‐cell differentiation. IL‐6 also promoted ICOS expression by CD4⁺ T cells, as well as their localization close to splenic B cells, but was not required for early Tfh‐cell development. Finally, IL‐6 promoted parasite control, IgM and IgG production, GC B‐cell development and ICOS expression by Tfh cells in a second model, Py17XNL infection. IL‐6 promotes CD4⁺ T‐cell activation and B‐cell responses during blood‐stage Plasmodium infection, which encourages parasite‐specific antibody production.     

Mechanisms of modulation of experimental autoimmune encephalomyelitis by chronic Trichinella spiralis infection in Dark Agouti rats

Trichinella spiralis is a helminth that provokes Th2 and anti‐inflammatory type responses in an infected host. Our previous studies using Dark Agouti (DA) rats indicated that T. spiralis infection reduced experimental autoimmune encephalomyelitis (EAE) severity in rats. The aim of this study was to analyse the mechanisms underlying EAE suppression driven by T. spiralis infection. Reduced clinical and histological manifestations of the disease were accompanied by increased IL‐4 and IL‐10 production and decreased IFN‐γ and IL‐17 production in draining lymph node cells. This indicates that T. spiralis infection successfully maintains a Th2 cytokine bias regardless of EAE induction. High IL‐10 signifies parasite‐induced anti‐inflammatory and/or regulatory cell responses. Transfer of splenic T cell‐enriched population of cells from T. spiralis‐infected rats into EAE immunized rats caused amelioration of EAE and in some cases protection from disease development. This population of cells contained higher proportion of CD4⁺ CD25⁺ Foxp3⁺ regulatory cells and produced high level of IL‐10 when compared with uninfected rats.     

Apheresis platelet concentrates contain platelet‐derived and endothelial cell‐derived microparticles

Background and Objectives Microparticles (MP) are membrane vesicles with thrombogenic and immunomodulatory properties. We determined MP subgroups from resting platelets, activated platelets and endothelial cells in donors and apheresis platelet concentrates (PC). Material and Methods MP were double stained with annexin V and CD61 (platelet‐derived MP; PMP), P‐selectin or CD63 (MP from activated platelets) and CD144 plus E‐selectin (endothelial cell‐derived MP; EMP) and detected by flow cytometry in platelet donors (n = 36) and apheresis PC (n = 11; Trima?). Results PC contained MP, mainly from resting platelets [93% (90–95)], and minor fractions of PMP from activated platelets [P‐selectin⁺ or CD63⁺; 4·8% (3·2–7·7) and 2·6% (2·0–4·0)]. Compared to donors, levels of annexin V+ MP, PMP, P‐selectin⁺ and CD63⁺ MP were 1·7‐, 2·3‐, 8·6‐ and 3·1‐fold higher in PC (all P < 0·05). During storage (1–5 days), levels of annexin V+ MP and PMP did not increase, although small increases in the fraction of P‐selectin⁺ or CD63⁺ MP occurred (both P < 0·05). PC also contained EMP, which were 2·6‐ to 3·7‐fold enriched in PC compared to donors (P < 0·05). Conclusions Transfusion of apheresis PC also results in transfusion of HLA‐carrying PMP and EMP. This might counteract the aim of reducing transfused HLA load by leucodepletion. The increases in PMP exposing P‐selectin or CD63 reflect mild platelet activation during storage. We conclude that in leucodepleted platelet apheresis using fluidized particle bed technology, MP are harvested mainly from the donor by apheresis. Improvement in apheresis technology might reduce MP load.     

Expression of adhesion molecules on T lymphocytes in young children and infants – a comparative study using whole blood lysis or density gradient separation

We investigated the use of two sample preparation techniques (whole blood lysis [WBL], and Ficoll‐Hypaque density separation [FDS]) for flow cytometric estimation of adhesion molecule (CD54, CD62L and CD11b) expression on T lymphocytes from children less than 2 years old, comparing the results with normal adult controls. Using WBL methods, young children had lower percentages of CD3⁺/CD54⁺ and CD3⁺/CD11b⁺ lymphocytes, but not of CD3⁺/CD62L⁺ lymphocytes than adult controls. FDS was associated with significantly higher percentages of CD3⁺/CD62L⁺ and CD3⁺/CD11b⁺ lymphocytes in young children and adults alike, while the percentages of CD3⁺/CD54⁺ cells from adults was not affected by FDS. The percent expression of CD54, CD62L, and CD11b on T cells from both children and adults were significantly higher following FDS, with a greater increase in CD11b expression on T cells from young children, reaching adult levels. The mean fluorescence intensity (MFI) of CD62L on T cells from young children, which was lower than that of adults using WBL preparation, was significantly higher following FDS, reaching adult levels. The higher levels of adhesion molecule expression associated with FDS did not result from T‐cell activation, as assessed by CD69, CD25, and HLA‐DR expression. Thus, adhesion molecule expression on T lymphocytes from young children is more sensitive to modification by isolation procedures than that on adult T cells. Caution should therefore be exercised in interpreting adhesion molecule expression data on T cells from children.     

Numerical and functional defects in CD8+CD28− T‐suppressor lymphocytes from patients with primary immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder, and loss of immune tolerance has been implicated in ITP pathogenesis. CD8⁺CD28⁻ suppressor (Ts) cells have an immunosuppression function and are involved in several autoimmune disorders. However, the role of Ts cells in ITP is currently not clear. Here, flow cytometry was used to detect the CD8⁺CD28⁻CD127⁻ proportion, which was decreased in active ITP patients compared with that of controls. Function analysis showed that immunosuppression of CD8⁺CD28⁻ Ts cells in ITP patients was impaired. Mechanistic studies have shown that CD8⁺CD28⁻ Ts cells from controls can downregulate CD80 and upregulate LILRB4 (ITL3) and LILRB2 (ILT4) expression on CD14⁺ monocytes, whereas these abilities were not found in Ts cells from ITP patients. Furthermore, Inducible T‐cell costimulatory (ICOS) expression on the Ts cell surface after activation was decreased whereas programmed death 1 and interleukin 10 expression was not changed in ITP patients compared with those of controls. In summary, the down‐regulated quantity and function of Ts cells in active patients indicated that a Ts defect was involved in ITP. Moreover, decreased ICOS expression and the loss of the ability to regulate co‐stimulator expression on antigen‐presenting cells partly explained the defective Ts‐mediated suppression.     

In situ recognition of autoantigen as an essential gatekeeper in autoimmune CD8+ T cell inflammation

A current paradigm states that non-antigen-specific inflammatory cues attract noncognate, bystander T cell specificities to sites of infection and autoimmune inflammation. Here we show that cues emanating from a tissue undergoing spontaneous autoimmune inflammation cannot recruit naive or activated bystander T cell specificities in the absence of local expression of cognate antigen. We monitored the recruitment of CD8⁺ T cells specific for the prevalent diabetogenic epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)_206–214 in gene-targeted nonobese diabetic (NOD) mice expressing a T cell “invisible” IGRP_206–214 sequence. These mice developed islet inflammation and diabetes with normal incidence and kinetics, but their inflammatory lesions could recruit neither naive (endogenous or exogenous) nor ex vivo-activated IGRP_206–214-reactive CD8⁺ T cells. Conversely, IGRP_206–214-reactive, but not nonautoreactive CD8⁺ T cells rapidly homed to and accumulated in the inflamed islets of wild-type NOD mice. Our results indicate that CD8⁺ T cell recruitment to a site of autoimmune inflammation results from an active process that is strictly dependent on local display of cognate pMHC and suggest that CD8⁺ T cells contained in extralymphoid autoimmune lesions are largely autoreactive.     

Chimeric antigen receptor modified T cells that target chemokine receptor CCR4 as a therapeutic modality for T‐cell malignancies

With the emerging success of treating CD19 expressing B cell malignancies with ex vivo modified, autologous T cells that express CD19‐directed chimeric antigen receptors (CAR), there is intense interest in expanding this evolving technology to develop effective modalities to treat other malignancies including solid tumors. Exploiting this approach to develop a therapeutic modality for T cell malignancies for which the available regimens are neither curative, nor confer long term survival we generated a lentivirus‐based CAR gene transfer system to target the chemokine receptor CCR4 that is over‐expressed in a spectrum of T cell malignancies as well as in CD4⁺CD25⁺Foxp3⁺T regulatory cells that accumulate in the tumor microenvironment constituting a barrier against anti‐tumor immunity. Ex vivo modified, donor‐derived T cells that expressed CCR4 directed CAR displayed antigen‐dependent potent cytotoxicity against patient‐derived cell lines representing ATL, CTCL, ALCL and a subset of HDL. Furthermore, these CAR T cells also eradicated leukemia in a mouse xenograft model of ATL illustrating the potential utility of this modality in the treatment of a wide spectrum of T cell malignancies.     

Association among Fas expression in leucocytes,serum Fas and Fas‐ligand concentrations and hepatic inflammation and fibrosis in chronic hepatitis C

Background: Replication of the hepatitis C virus (HCV) in peripheral blood mononuclear cells (PBMC) may impair immune functions and establish persistent infection. The aim of this study was to assess the influence of HCV on PBMC and their susceptibility to apoptosis in relation to liver inflammation and fibrosis. Methods: Eighty‐one patients with chronic hepatitis C (CHC) were enrolled in this study. Flow cytometry was used to determine the amount of T cells (CD4⁺, CD8⁺), B cells (CD19⁺), monocytes (CD14⁺) and natural killer cells (CD16⁺) in the peripheral blood and the expression of CD95⁺ (CD95/APO‐1) in each subset. Serum concentrations of sFas and sFasL were assessed by the enzyme‐linked immunosorbent assay method. Results: An increased expression of Fas was observed in CD4⁺ and CD8⁺ cells in CHC. There was a more prominent expression of Fas on CD4⁺ cells in HCV genotype 1b in contrast to 3a. Increased Fas expression on CD4⁺ cells was seen in advanced stages of liver disease. Fas expression on monocytes was lower in advanced stages of liver inflammation and fibrosis. Serum sFas concentration was higher in CHC compared with the control group. There was an association between sFasL concentration and inflammatory activity in the liver. Serum sFasL concentration correlated positively with the mean intensity of fluorescence of the Fas receptor in CD4⁺ and CD8⁺ cells, granulocytes and monocytes. Conclusion: These findings indicate that there is an increased susceptibility of PBMC to apoptosis, which can be attributed to the constant contact of leucocytes with the inflamed liver tissue, or from direct HCV influence.