Alveolar compartmentalization of inflammatory and immune cell biomarkers in pneumonia-related ARDS.

BACKGROUND: Biomarkers of disease severity might help individualizing the management of patients with the acute respiratory distress syndrome (ARDS). Whether the alveolar compartmentalization of biomarkers has a clinical significance in patients with pneumonia-related ARDS is unknown. This study aimed at assessing the interrelation of ARDS/sepsis biomarkers in the alveolar and blood compartments and explored their association with clinical outcomes. METHODS: Immunocompetent patients with pneumonia-related ARDS admitted between 2014 and 2018 were included in a prospective monocentric study. Bronchoalveolar lavage (BAL) fluid and blood samples were obtained within 48 h of admission. Twenty-two biomarkers were quantified in BAL fluid and serum. HLA-DR+ monocytes and CD8+ PD-1+ lymphocytes were quantified using flow cytometry. The primary clinical endpoint of the study was hospital mortality. Patients undergoing a bronchoscopy as part of routine care were included as controls. RESULTS: Seventy ARDS patients were included. Hospital mortality was 21.4%. The BAL fluid-to-serum ratio of IL-8 was 20 times higher in ARDS patients than in controls (p < 0.0001). ARDS patients with shock had lower BAL fluid-to-serum ratio of IL-1Ra (p = 0.026), IL-6 (p = 0.002), IP-10/CXCL10 (p = 0.024) and IL-10 (p = 0.023) than others. The BAL fluid-to-serum ratio of IL-1Ra was more elevated in hospital survivors than decedents (p = 0.006), even after adjusting for SOFA and driving pressure (p = 0.036). There was no significant association between alveolar or alveolar/blood monocytic HLA-DR or CD8+ lymphocytes PD-1 expression and hospital mortality. CONCLUSIONS: IL-8 was the most compartmentalized cytokine and lower BAL fluid-to-serum concentration ratios of IL-1Ra were associated with hospital mortality in patients with pneumonia-associated ARDS.

Peripheral T-cell lymphomas of follicular helper T-cell type frequently display an aberrant CD3(-/dim)CD4(+) population by flow cytometry: an important clue to the diagnosis of a Hodgkin lymphoma mimic.

Nodal follicular helper T-cell-derived lymphoproliferations (specifically the less common peripheral T-cell lymphomas of follicular type) exhibit a spectrum of histologic features that may mimic reactive hyperplasia or Hodgkin lymphoma. Even though angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma of follicular type share a common biologic origin from follicular helper T-cells and their morphology has been well characterized, flow cytometry of peripheral T-cell lymphomas of follicular type has not been widely discussed as a tool for identifying this reactive hyperplasia/Hodgkin lymphoma mimic. We identified 10 peripheral T-cell lymphomas of follicular type with available flow cytometry data from five different institutions, including two cases with peripheral blood evaluation. For comparison, we examined flow cytometry data for 8 classical Hodgkin lymphomas (including 1 lymphocyte-rich classical Hodgkin lymphoma), 15 nodular lymphocyte predominant Hodgkin lymphomas, 15 angioimmunoblastic T-cell lymphomas, and 26 reactive nodes. Lymph node histology and flow cytometry data were reviewed, specifically for the presence of a CD3(-/dim)CD4(+) aberrant T-cell population (described in angioimmunoblastic T-cell lymphomas), besides other T-cell aberrancies. Nine of 10 (90%) peripheral T-cell lymphomas of follicular type showed a CD3(-/dim)CD4(+) T-cell population constituting 29.3% (range 7.9-62%) of all lymphocytes. Five of 10 (50%) had nodular lymphocyte predominant Hodgkin lymphoma or lymphocyte-rich classical Hodgkin lymphoma-like morphology with scattered Hodgkin-like cells that expressed CD20, CD30, CD15, and MUM1. Three cases had a nodular growth pattern and three others exhibited a perifollicular growth pattern without Hodgkin-like cells. Epstein-Barr virus was positive in 1 of 10 cases (10%). PCR analysis showed clonal T-cell receptor gamma gene rearrangement in all 10 peripheral T-cell lymphomas of follicular type. By flow cytometry, 11 of 15 (73.3%) angioimmunoblastic T-cell lymphomas showed the CD3(-/dim)CD4(+) population (mean: 19.5%, range: 3-71.8%). Using a threshold of 3% for CD3(-/dim)CD4(+) T cells, all 15 nodular lymphocyte predominant Hodgkin lymphoma controls and 8 classical Hodgkin lymphomas were negative (Mann-Whitney P=0.01, F-PTCL vs Hodgkin lymphomas), as were 25 of 26 reactive lymph nodes. The high frequency of CD3(-/dim)CD4(+) aberrant T cells is similar in angioimmunoblastic T-cell lymphomas and peripheral T-cell lymphomas of follicular type, and is a useful feature in distinguishing peripheral T-cell lymphomas of follicular type from morphologic mimics such as reactive hyperplasia or Hodgkin lymphoma.

Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis.

Macrophages (MPs) are important for skeletal muscle regeneration in vivo and may exert beneficial effects on myogenic cell growth through mitogenic and antiapoptotic activities in vitro. However, MPs are highly versatile and may exert various, and even opposite, functions depending on their activation state. We studied monocyte (MO)/MP phenotypes and functions during skeletal muscle repair. Selective labeling of circulating MOs by latex beads in CX3CR1(GFP/+) mice showed that injured muscle recruited only CX3CR1(lo)/Ly-6C(+) MOs from blood that exhibited a nondividing, F4/80(lo), proinflammatory profile. Then, within muscle, these cells switched their phenotype to become proliferating antiinflammatory CX3CR1(hi)/Ly-6C(-) cells that further differentiated into F4/80(hi) MPs. In vitro, phagocytosis of muscle cell debris induced a switch of proinflammatory MPs toward an antiinflammatory phenotype releasing transforming growth factor beta1. In co-cultures, inflammatory MPs stimulated myogenic cell proliferation, whereas antiinflammatory MPs exhibited differentiating activity, assessed by both myogenin expression and fusion into myotubes. Finally, depletion of circulating MOs in CD11b-diphtheria toxin receptor mice at the time of injury totally prevented muscle regeneration, whereas depletion of intramuscular F4/80(hi) MPs at later stages reduced the diameter of regenerating fibers. In conclusion, injured skeletal muscle recruits MOs exhibiting inflammatory profiles that operate phagocytosis and rapidly convert to antiinflammatory MPs that stimulate myogenesis and fiber growth.

Molecular remission is an independent predictor of clinical outcome in patients with mantle cell lymphoma after combined immunochemotherapy: a European MCL intergroup study.

The prognostic impact of minimal residual disease (MRD) was analyzed in 259 patients with mantle cell lymphoma (MCL) treated within 2 randomized trials of the European MCL Network (MCL Younger and MCL Elderly trial). After rituximab-based induction treatment, 106 of 190 evaluable patients (56%) achieved a molecular remission (MR) based on blood and/or bone marrow (BM) analysis. MR resulted in a significantly improved response duration (RD; 87% vs 61% patients in remission at 2 years, P = .004) and emerged to be an independent prognostic factor for RD (hazard ratio = 0.4, 95% confidence interval, 0.1-0.9, P = .028). MR was highly predictive for prolonged RD independent of clinical response (complete response [CR], complete response unconfirmed [CRu], partial response [PR]; RD at 2 years: 94% in BM MRD-negative CR/CRu and 100% in BM MRD-negative PR, compared with 71% in BM MRD-positive CR/CRu and 51% in BM MRD-positive PR, P = .002). Sustained MR during the postinduction period was predictive for outcome in MCL Younger after autologous stem cell transplantation (ASCT; RD at 2 years 100% vs 65%, P = .001) and during maintenance in MCL Elderly (RD at 2 years: 76% vs 36%, P = .015). ASCT increased the proportion of patients in MR from 55% before high-dose therapy to 72% thereafter. Sequential MRD monitoring is a powerful predictor for treatment outcome in MCL. These trials are registered at www.clinicaltrials.gov as #NCT00209222 and #NCT00209209.

Targeting intratumoral B cells with rituximab in addition to CHOP in angioimmunoblastic T-cell lymphoma. A clinicobiological study of the GELA.

BACKGROUND: In angioimmunoblastic T-cell lymphoma, symptoms linked to B-lymphocyte activation are common, and variable numbers of CD20(+) large B-blasts, often infected by Epstein-Barr virus, are found in tumor tissues. We postulated that the disruption of putative B-T interactions and/or depletion of the Epstein-Barr virus reservoir by an anti-CD20 monoclonal antibody (rituximab) could improve the clinical outcome produced by conventional chemotherapy. DESIGN AND METHODS: Twenty-five newly diagnosed patients were treated, in a phase II study, with eight cycles of rituximab + chemotherapy (R-CHOP21). Tumor infiltration, B-blasts and Epstein-Barr virus status in tumor tissue and peripheral blood were fully characterized at diagnosis and were correlated with clinical outcome. RESULTS: A complete response rate of 44% (95% CI, 24% to 65%) was observed. With a median follow-up of 24 months, the 2-year progression-free survival rate was 42% (95% CI, 22% to 61%) and overall survival rate was 62% (95% CI, 40% to 78%). The presence of Epstein-Barr virus DNA in peripheral blood mononuclear cells (14/21 patients) correlated with Epstein-Barr virus score in lymph nodes (P<0.004) and the detection of circulating tumor cells (P=0.0019). Despite peripheral Epstein-Barr virus clearance after treatment, the viral load at diagnosis (>100 copy/µg DNA) was associated with shorter progression-free survival (P=0.06). CONCLUSIONS: We report here the results of the first clinical trial targeting both the neoplastic T cells and the microenvironment-associated CD20(+) B lymphocytes in angioimmunoblastic T-cell lymphoma, showing no clear benefit of adding rituximab to conventional chemotherapy. A strong relationship, not previously described, between circulating Epstein-Barr virus and circulating tumor cells is highlighted.

Optimized Flow Cytometry Strategy for Phenotyping Intramuscular Leukocytes: Application to the Evaluation of Myopathological Processes.

Phenotyping intramuscular immune cells is essential for the characterization of dysimmune/inflammatory myopathies (DIM). Flow cytometry (FC) is the most reliable technique for analyzing leukocyte subpopulations and evaluating their activation levels. We developed a purely mechanical protocol for extracting cells from muscle tissue allowing us to preserve cell surface epitopes and determined its applicability to experimental pathology in mice and myopathological diagnosis in human. Skeletal muscle regeneration in mice was associated with a transient enrichment of macrophages (CD11bhighGr-1+), myeloid dendritic cells (CD3-C8+CD11bhigh), CD8+ T cells (CD3+C8+), and NK cells (CD3- CD11bhighNKp46+). In murine models of inherited muscle dystrophies, leukocytes represented 23%-84% of intramuscular mononuclear cells, with a percentage of CD8+ T cells (4%-17%) mirroring that of all CD45+ cells, while MDCs remained a minority. In human 16 samples (DIM: n = 9; nonimmune conditions: n = 7), DIM was associated with intramuscular recruitment of CD8+ T cells, but not CD4+ T cells and NK cells. FC allowed concomitant quantification of HLA-DR, CD25, CD38, and CD57 activation/differentiation biomarkers and showed increased activation levels of CD4+ and CD8+ T cells in DIM. In conclusion, FC is an appropriate method for quantifying intramuscular leukocyte subpopulations and analyzing their activation states.

Serum Leptin Levels, Nutritional Status, and the Risk of Healthcare-Associated Infections in Hospitalized Older Adults.

We aimed to determine whether serum leptin levels are predictive of the occurrence of healthcare-associated infections (HAIs) in hospitalized older patients. In a prospective cohort, 232 patients had available data for leptin and were monitored for HAIs for 3 months. Admission data included comorbidities, invasive procedures, the Mini Nutritional Assessment (MNA), BMI, leptin, albumin and C-reactive protein levels, and CD4 and CD8 T-cell counts. Multivariate logistic regression modelling was used to identify predictors of HAIs. Of the 232 patients (median age: 84.8; females: 72.4%), 89 (38.4%) experienced HAIs. The leptin level was associated with the BMI (p < 0.0001) and MNA (p < 0.0001) categories. Women who experienced HAIs had significantly lower leptin levels than those who did not (5.9 µg/L (2.6-17.7) and 11.8 (4.6-26.3), respectively; p = 0.01; odds ratio (OR) (95% confidence interval): 0.67 (0.49-0.90)); no such association was observed for men. In a multivariate analysis of the women, a lower leptin level was significantly associated with HAIs (OR = 0.70 (0.49-0.97)), independently of comorbidities, invasive medical procedures, and immune status. However, leptin was not significantly associated with HAIs after adjustments for malnutrition (p = 0.26) or albuminemia (p = 0.15)-suggesting that in older women, the association between serum leptin levels and subsequent HAIs is mediated by nutritional status.

T-cell phenotype in protocol renal biopsy from transplant recipients treated with belatacept-mediated co-stimulatory blockade.

BACKGROUND: Belatacept is thought to disrupt the interaction between CD80/86 and CD28, thus preventing T-cell activation by blocking the co-stimulatory second signal. However, the consequences on the T-cell profile in human renal transplant cases have not been determined. METHODS: In this study, we analysed intra-graft levels of the mRNAs for Treg (FOXP3), cytotoxic CD8 T cells (Granzyme B), Th1 (INFγ, Tbet), Th2 (GATA3) and Th17 (RORγt and IL-17) in protocol biopsies obtained 12 months after renal transplantation in recipients treated with Belatacept or calcineurin inhibitor (CNI). RESULTS: Only the intra-graft abundance of FOXP3 mRNA was significantly lower (P < 0.001) in the Belatacept group than the CNI group. Conclusions. These results are in agreement with in vitro data suggesting that CD28 is a major co-stimulatory signal of both Tregs development and peripheral homeostasis but contrast with clinical trials showing a better 1-year graft function and a lower incidence of chronic allograft nephropathy in patients receiving Belatacept than patients treated with CNI. They suggest that immune benefits induced by Belatacept are not mediated by Treg expansion and that FOXP3 is not by itself a prognostic marker of long-term graft function in a non-inflammatory context. These results have to be, however, considered as preliminary since the size of our study population is limited.

The inducible T-cell co-stimulator molecule is expressed on subsets of T cells and is a new marker of lymphomas of T follicular helper cell-derivation.

BACKGROUND: T follicular helper (T(FH)) cells reside in the light zone of germinal centers and are considered the cell of origin of angioimmunoblastic T-cell lymphoma. Recently, CXCL13, PD-1 and SAP were described as useful markers for T(FH) cells and angioimmunoblastic T-cell lymphoma but also reported in some peripheral T-cell lymphomas, not otherwise specified. DESIGN AND METHODS: In the present study the expression pattern of ICOS protein was investigated by immunohistochemistry-based techniques in routine sections of normal lymphoid tissues and 633 human lymphomas. RESULTS: Cells strongly positive for ICOS were restricted to the light zone of germinal centers and co-expressed T(FH)-associated molecules. In addition, weak to moderate ICOS expression was observed in a small proportion of FOXP3-positive cells. In lymphomas, ICOS expression was confined to angioimmunoblastic T-cell lymphoma (85/86), peripheral T-cell lymphomas of follicular variant (18/18) and a proportion of peripheral T-cell lymphomas, not otherwise specified (24/56) that also expressed other T(FH)-associated molecules. CONCLUSIONS: ICOS is a useful molecule for identifying T(FH) cells and its restricted expression to angioimmunoblastic T-cell lymphoma and a proportion of peripheral T-cell lymphomas, not otherwise specified (showing a T(FH)-like profile) suggests its inclusion in the antibody panel for diagnosing T(FH)-derived lymphomas. Our findings provide further evidence that the histological spectrum of T(FH)-derived lymphomas is broader than previously assumed.

Combined Plasma Elevation of CRP, Intestinal-Type Fatty Acid-Binding Protein (I-FABP), and sCD14 Identify Older Patients at High Risk for Health Care-Associated Infections.

Background: We hypothesized that low-grade inflammation was driven by microbial translocation and associated with an increased risk of health care-associated infections (HAIs). Methods: We included 121 patients aged 75 years or over in this prospective cohort study. High-sensitivity C-reactive protein (hs-CRP), I-FABP, and sCD14-as markers for low-grade inflammation, intestinal epithelial barrier integrity, and monocyte activation, respectively-were measured at admission. Results: HAIs occurred during hospitalization in 62 (51%) patients. Elevated hs-CRP (≥6.02 mg/L, ie, the median) was associated with a significantly higher HAI risk when I-FABP was in the highest quartile (odds ratio [OR], 4; 95% confidence interval [95% CI], 1.39-11.49; p = .010). In patients with hs-CRP elevation and highest-quartile I-FABP, sCD14 elevation (≥0.65 µg/mL, ie, the median) was associated with an 11-fold higher HAI risk (OR, 10.8; 95% CI, 2.28-51.1; p = .003). Multivariate analyses adjusted for invasive procedures and comorbidities did not change the associations linking the three markers to the HAI risk. Conclusion: Increased levels of hs-CRP, I-FABP, and sCD14 may reflect loss of intestinal epithelial barrier integrity with microbial translocation leading to monocyte activation and low-grade inflammation. In our cohort, these markers identified patients at high risk for HAIs.

Characterization of CXCL13+ neoplastic t cells in cutaneous lesions of angioimmunoblastic T-cell lymphoma (AITL).

Skin manifestations of angioimmunoblastic T-cell lymphoma (AITL) are frequent, sometimes as first manifestations of the disease. In the absence of a specific marker for neoplastic cells, diagnosis of AITL in skin biopsies is often difficult. CD10 and CXCL13 have been recently recognized as characteristic markers of AITL, but have not been yet investigated in the skin. We analyzed 15 skin biopsies from 8 patients with AITL having skin manifestations and compared them to 14 skin biopsies from patients with various cutaneous lymphocytic infiltrates. A few CD10 lymphocytes were found in only 2 samples of the AITL group, the identification of which was hampered by the presence of a dermal CD10 cell population with dendritic features. By contrast, CXCL13 lymphoid cells were identified in most AITL cutaneous biopsies (n=12, 80%), whereas, absent in all samples from control cases. Among 12 biopsies with CXCL13 cells, cutaneous involvement by AITL was suspected in only 5 on the basis of light microscopy and classic immunophenotyping. In another case, a diagnosis of cutaneous marginal zone B-cell lymphoma had been proposed. In conclusion, this study shows that neoplastic AITL CXCL13 T cells localize in the skin and that accurate diagnosis of AITL lesions can be done in skin specimens using CXCL13 immunostaining on paraffin-embedded tissues.

Expression of CXCL13 by neoplastic cells in angioimmunoblastic T-cell lymphoma (AITL): a new diagnostic marker providing evidence that AITL derives from follicular helper T cells.

Angioimmunoblastic T-cell lymphoma (AITL) represents a distinct entity among peripheral T-cell lymphomas (PTCLs). The cellular origin of AITL remains unknown, although a possible derivation from follicular helper T cells (TFH) has been suggested based on the CD4/Bcl-6 phenotype. It has been recently shown that expression of CXCL13, a chemokine critically involved in B-cell migration into germinal centers, is characteristic of TFH cells, as compared with other T helper subsets. We compared CXCL13 expression in 29 AITLs, 20 PTCLs, unspecified, 10 anaplastic large cell lymphomas (ALCL), and 4 other PTCLs. We showed that CXCL13 is expressed by AITL (29 of 29, 100%) and a subset of PTCL, unspecified (6 of 20, 30%), which all showed borderline features with AITL, but in only 1 of 10 (10%) ALCLs, and 0 of 4 other PTCLs. Two-color immunostainings further showed that CXCL13 was found in the cytoplasm of atypical CD5-positive T cells that expressed CD10. We conclude that CXCL13 expression is a common characteristic of AITL, which can help to delineate the morphologic spectrum of the disease, and further supports its derivation from TFH cells. CXCL13 expression may also provide an additional useful tool for the diagnosis of AITL.

Blood neutrophil bactericidal activity against methicillin-resistant and methicillin-sensitive Staphylococcus aureus during cardiac surgery.

Whether methicillin-resistant Staphylococcus aureus (MRSA) constitutes per se an independent risk factor for morbidity and mortality after surgery as compared with methicillin-sensitive Staphylococcus aureus (MSSA) remains a subject of debate. The aim of this study was to assess whether innate defenses against MRSA and MSSA strains are similarly impaired after cardiac surgery. Both intracellular (isolated neutrophil functions) and extracellular (plasma) defenses of 12 patients undergoing scheduled cardiac surgery were evaluated preoperatively (day 0) and postoperatively (day 3) against two MSSA strains with a low level of catalase secretion and two MRSA strains with a high level of catalase secretion, inasmuch as SA killing by neutrophils relies on oxygen-dependent mechanisms. After surgery, an increase in plasma concentration of IL-10, an anti-inflammatory cytokine able to inhibit reactive oxygen species secretion and bactericidal activity of neutrophils, was evidenced. Despite the fact that univariate analysis suggested a specific impairment of neutrophil functions against MRSA strains, two-way repeated-measures ANOVA failed to demonstrate that the effect of S. aureus phenotype was significant. On the other hand, an increase in type-II secretory phospholipase A2 activity, a circulating enzyme involved in SA lysis, was evidenced and was associated with an enhancement of extracellular defenses (bactericidal activity of plasma) against MRSA. Overall, cardiac surgery and S. aureus phenotype had a significant effect on plasma bactericidal activity. Cardiac surgery was characterized by enhanced antibacterial defenses of plasma, whereas neutrophil killing properties were reduced. The overall effect of S. aureus phenotype on neutrophil functions did not seem significant.

Inflammatory myopathy with abundant macrophages (IMAM): a condition sharing similarities with cytophagic histiocytic panniculitis and distinct from macrophagic myofasciitis.

We describe the unreported pattern of inflammatory myopathy with abundant macrophages (IMAM) as a main differential diagnosis of postimmunization aluminum hydroxide-induced macrophagic myofasciitis (MMF). IMAM was mainly detected among patients with a dermatomyositis (DM)-like disease. Among 113 muscle biopsies from DM patients collected from 1974 to 2000, intensity of macrophage infiltration was highly variable: 41.5% (-/+); 34.5% (+); 17% (++): and 7% (+++). The 27 patients from groups (++) and (+++) had a similar pattern of macrophagic infiltration and were considered to have IMAM. They were compared to 40 MMF patients. In IMAM, macrophage infiltrates were diffuse and correlated positively with both T cell infiltrates and acute muscle fiber damage, and showed pictures of hemophagocytosis (21/27). Connective tissue structures were infiltrated by noncohesive, ribbon-forming collections of large basophilic macrophages containing no crystalline inclusions. In MMF, macrophage infiltrates were focal and formed compact well-delineated aggregates of granular PAS+ cells, loaded with crystalline aluminum hydroxide particles, in the absence of either hemophagocytosis or conspicuous muscle damage. Review of the literature indicates similarities between IMAM and "cytophagic histiocytic panniculitis" (CHP), a condition characterized by T cell-triggered macrophage hyperactivation. Both IMAM and CHP, but not MMF, may be associated with a life-threatening hemophagocytic syndrome.

Peptides derived from the onconeural HuD protein can elicit cytotoxic responses in HHD mouse and human.

Anti-Hu syndrome is a paraneoplastic neurologic disease seemingly associated with an efficient antitumoral immune response against HuD protein expressed by both small cell lung cancer (SCLC) and neurons. Since anti-Hu antibodies are not pathogenic, and oligoclonal CD8(+) T cells infiltrate neoplastic and nervous tissues, we examined MHC class I-restricted immunogenicity of human HuD. Among 14 HuD-derived peptides potentially immunogenic in HLA-A*0201 restriction, 10 had actual in vitro binding capacity to the HLA molecule, 8 elicited specific cytotoxic T lymphocytes (CTLs) in a humanized murine model after peptidic vaccination, 2 also elicited specific CTLs in healthy humans, and 1 was naturally processed and presented to the immune system.

Follicular peripheral T-cell lymphoma expands the spectrum of classical Hodgkin lymphoma mimics.

Epstein-Barr virus (EBV)-infected B cells with Reed-Sternberg-like cell (RS) features may occur in peripheral T-cell lymphomas (PTCLs), especially in angioimmunoblastic T-cell lymphoma. Here, we report 5 patients presenting with lymphadenopathy whose first biopsies demonstrated nodular lymphoid proliferations containing scattered CD30+, CD15+, EBV+ Hodgkin and Reed-Sternberg-like cells, which led to an initial diagnosis of lymphocyte-rich classical Hodgkin lymphoma. However, the uncommon clinical features and/or the occurrence of relapse as PTCL prompted review of the biopsies with expanded immunohistologic and molecular studies and revision of the diagnoses to follicular variant of PTCL (F-PTCL). All cases had atypical small to medium-sized CD3+ T cells that expressed CD10 (4/5) and the follicular helper T-cell (TFH) antigens BCL6, PD1, CXCL13, and ICOS. All demonstrated clonal T cells with a similar pattern in multiple samples from 4 patients. In 2 cases, flow cytometry demonstrated circulating lymphocytes with an abnormal sCD3+, CD4+, ICOS+ immunophenotype. Two patients had a skin rash at presentation, and 1 had B symptoms. Two of the 4 patients treated with polychemotherapy are alive at 3 and 6 years after first diagnosis. These cases highlight how some F-PTCLs may closely mimic lymphocyte-rich classical Hodgkin lymphoma requiring careful assessment of the T cells before rendering the latter diagnosis. The functional properties of TFH cells might lead to the presence of EBV-positive B blasts with RS-like features in TFH-derived PTCL such as angioimmunoblastic T-cell lymphoma and F-PTCL.

Muscle resident macrophages control the immune cell reaction in a mouse model of notexin-induced myoinjury.

OBJECTIVE: Skeletal muscle may be the site of a variety of poorly understood immune reactions, particularly after myofiber injury, which is typically observed in inflammatory myopathies. This study was undertaken to explore both the cell dynamics and functions of resident macrophages and dendritic cells (DCs) in damaged muscle, using a mouse model of notexin-induced myoinjury to study innate immune cell reactions. METHODS: The myeloid cell reaction to notexin-induced myoinjury was analyzed by microscopy and flow cytometry. Bone marrow (BM) transplantation studies were used to discriminate resident from exudate monocyte/macrophages. Functional tests included cytokine screening and an alloantigenic mixed leukocyte reaction to assess the antigen-presenting cell (APC) function. Selective resident macrophage depletion was obtained by injection of diphtheria toxin (DT) into CD11b-DT receptor-transgenic mice transplanted with DT-insensitive BM. RESULTS: The connective tissue surrounding mouse muscle/fascicle tissue (the epimysium/perimysium) after deep muscle injury displayed a resident macrophage population of CD11b+F4/80+CD11c-Ly-6C-CX3CR1- cells, which concentrated first in the epimysium. These resident macrophages were being used by leukocytes as a centripetal migration pathway, and were found to selectively release 2 chemokines, cytokine-induced neutrophil chemoattractant and monocyte chemoattractant protein 1, and to crucially contribute to massive recruitment of neutrophils and monocytes from the blood. Early epimysial inflammation consisted of a predominance of Ly-6C(high)CX3CR1(low)CD11c- cells that were progressively substituted by Ly-6C(low)CX3CR1(high) cells displaying an intermediate, rather than high, level of CD11c expression. These CD11c(intermediate) cells were derived from circulating CCR2+ monocytes, functionally behaved as immature APCs in the absence of alloantigenic challenge, and migrated to draining lymph nodes while acquiring the phenotype of mature DCs (CD11c+Ia+CD80+ cells, corresponding to an inflammatory DC phenotype). CONCLUSION: The results in this mouse model show that resident macrophages in the muscle epimysium/perimysium orchestrate the innate immune response to myoinjury, which is linked to adaptive immunity through the formation of inflammatory DCs.

Flow cytometric immunophenotyping of cerebrospinal fluid.

Leptomeningeal disease is an important adverse complication occurring in patients with B and T cell lymphomas and acute leukemias of lymphoid and myeloid origin. Recent reports suggest that multiparameter flow cytometry immunophenotypic assessment of spinal fluid samples could improve the efficiency of detection of CNS involvement, due to its high specificity and greater sensitivity. However, spinal fluid samples are frequently paucicellular with a rapidly decreasing cell viability. Staining of spinal fluid therefore requires dedicated sample storage/transport, staining, and preparation protocols. The Basic Protocol in this unit outlines a consensus multiparameter (3- to 8-color) flow cytometry immunophenotypic protocol for the evaluation of CNS involvement of cerebrospinal fluid (CSF) samples by neoplastic cells. A Support Protocol describing the simultaneous assessment of surface and cytoplasmic antigens is also provided. Finally, in the Alternate Protocol, we describe a method to calculate absolute numbers of both normal and pathological cell subpopulations by adding counting beads to the assay.

Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations.

The urokinase-type plasminogen activator (uPA) system is a dynamic complex in which the membrane receptor uPAR binds uPA that binds the plasminogen activator inhibitor (PAI)-1 localized in the extracellular matrix, resulting in endocytosis of the whole complex by the low-density lipoprotein receptor-related protein (LRP). High expression of PAI-1 is paradoxically associated with marked tumor spreading and poor prognosis. We previously reported a nonproteolytic role of the [uPAR:uPA:PAI-1:LRP] complex operative in cell migration. Here we explored whether matrix PAI-1 could be used as a migration support by human breast cancer cells. We showed that the uPA system and LRP are localized at filopodia of invasive cells, and that formation/internalization of the [uPAR:uPA:PAI-1:LRP] complex is required for attachment and migration of cancer cells on plastic and on a PAI-1 coat. PAI-1 increased both filopodia formation and migration of cancer cells suggesting a chemokine-like activity. Migration velocity, expression of the uPA system, use of the [uPAR:uPA:PAI-1:LRP] complex to migrate, and promigratory effects of PAI-1 paralleled cancer cell invasiveness. Phenotyping and functional analysis of invasive cancer cell subclones indicated that different cell subpopulations may use different strategies to migrate depending on both the environment and their expression of the uPA system, some of them taking advantage of abundant available PAI-1.