The ratio of intratumoral CD15+ neutrophils to CD8+ lymphocytes predicts recurrence in patients with gastric cancer after curative resection.

BACKGROUND: An elevated neutrophil-to-lymphocyte ratio (NLR) in peripheral blood is an independent prognostic indicator of various cancers. AIMS: In this study, we aimed to investigate the prognostic relevance of the intratumoral immune cell balance in gastric cancer. METHODS AND RESULTS: The study included 82 patients who underwent curative resection for gastric cancer. The intratumoral cluster of differentiation (CD) 15- and CD8-positive cells were evaluated using immunohistochemical staining. Additionally, clinicopathological factors and prognoses were analyzed. Patients with high intratumoral CD15/CD8 ratios had significantly lower overall survival (OS) and relapse-free survival (RFS) compared to those with low CD15/CD8 ratios (p = .0026 and p < .0001, respectively). Additionally, a high CD15/CD8 ratio was associated with lymph node metastasis (p = .019). Patients with high NLR had a significantly lower RFS than those with low NLR (p = .0050). Multivariate analysis revealed that the intratumoral CD15/CD8 ratio, NLR, and venous invasion were independent prognostic indicators of RFS (CD15/CD8 ratio: p < .001, hazard ratio (HR) = 14.7, 95% confidence interval (CI) = 3.8-56.8; NLR: p = .010, HR = 5.4, 95% CI = 1.5-19.6; venous invasion: p = .005, HR = 7.4, 95% CI = 1.8-29.7). CONCLUSION: In summary, we found that the intratumoral CD15/CD8 ratio is an independent prognostic factor following gastric cancer resection and its increase is associated with lymph node metastasis and microscopic lymph vessel invasion. Immunological evaluation with additional aspects of innate immunity may be useful in predicting cancer prognosis.

Peripheral T-cell lymphomas expressing CD30 and CD15 expand the spectrum of anaplastic large cell lymphoma, ALK-negative.

Peripheral T-cell lymphomas (PTCL) are morphologically and biologically heterogeneous and a subset expresses CD30, including anaplastic large cell lymphomas (ALCL) and a minority of PTCL, not otherwise specified (PTCL, NOS). ALCL with ALK translocations (ALCL, ALK+) are readily identified by routine diagnostic methods, but differentiating ALCL without ALK translocation (ALCL, ALK-) and PTCL, NOS expressing CD30 (PTCL CD30+) can be challenging. Furthermore, rare PTCL co-express CD30 and CD15 (PTCL CD30+CD15+); some resemble ALCL, ALK- while others resemble classic Hodgkin lymphoma. To explore the relationship between PTCL CD30+CD15+ and ALCL, ALK-, we analysed 19 cases of PTCL with CD30 expression, previously diagnosed as ALCL, ALK- (nine cases) and PTCL CD30+CD15+ (10 cases) for DUSP22/IRF4 rearrangements, coding RNA expression and selected transcriptome analysis using the NanoString nCounter gene expression analysis platform. Unsupervised clustering showed no clear segregation between ALCL, ALK- and PTCL CD30+CD15+. Three cases previously classified as PTCL CD30+CD15+ showed DUSP22/IRF4 rearrangements, favouring a diagnosis of ALCL, ALK-. Our results suggest that cases previously designated PTCL CD30+CD15+, likely fall within the spectrum of ALCL, ALK-; additionally, a subset of ALCL, ALK- with DUSP22/IRF4 rearrangement expresses CD15, consistent with previous reports and expands the immunophenotypic spectrum of this lymphoma subgroup.

Chronic kidney disease linked to SARS-CoV-2 infection: a case report.

BACKGROUND: The recent COVID-19 pandemic has raised concerns about patient diagnosis and follow-up of chronically ill patients. Patients suffering from chronic illnesses, concomitantly infected by SARS-CoV-2, globally tend to have a worse prognosis and poor outcomes. Renal tropism and acute kidney injury following SARS-CoV-2 infection has recently been described in the literature, with elevated mortality rates. Furthermore, patients with pre-existing chronic kidney disease, infected by SARS-CoV-2, should be monitored carefully. Here, we report the case of a 69-year-old patient with splenic marginal zone lymphoma, suffering from longstanding chronic kidney disease following SARS-CoV-2 infection. CASE PRESENTATION: A 69-year-old male patient previously diagnosed with pulmonary embolism and splenic marginal zone lymphoma (Splenomegaly, Matutes 2/5, CD5 negative and CD23 positive), was admitted to the hospital with shortness of breath, fever and asthenia. A nasopharyngeal swab test was performed in addition to a CT-scan, which confirmed SARS-CoV-2 infection. Blood creatinine increased following SARS-CoV-2 infection at 130 µmol/l, with usual values at 95 µmol/l. The patient was discharged at home with rest and symptomatic medical treatment (paracetamol and hydration), then readmitted to the hospital in August 2020. A kidney biopsy was therefore conducted as blood creatinine levels were abnormally elevated. Immunodetection performed in a renal biopsy specimen confirmed co-localization of SARS-CoV2 nucleocapsid and protease 3C proteins with ACE2, Lewis x and sialyl-Lewis x antigens in proximal convoluted tubules and podocytes. Co-localization of structural and non-structural viral proteins clearly demonstrated viral replication in proximal convoluted tubules in this chronically ill patient. Additionally, we observed the co-localization of sialyl-Lewis x and ACE2 receptors in the same proximal convoluted tubules. Reverse Transcriptase-Polymerase Chain Reaction test performed on the kidney biopsy was negative, with very low Ct levels (above 40). The patient was finally readmitted to the haematology department for initiation of chemotherapy, including CHOP protocol and Rituximab. CONCLUSIONS: Our case emphasizes on the importance of monitoring kidney function in immunosuppressed patients and patients suffering from cancer following SARS-CoV-2 infection, through histological screening. Further studies will be required to decipher the mechanisms underlying chronic kidney disease and the putative role of sialyl-Lewis x and HBGA during SARS-CoV-2 infection.

JAK2 Rearrangements Are a Recurrent Alteration in CD30+ Systemic T-Cell Lymphomas With Anaplastic Morphology.

Peripheral T-cell lymphoma (PTCL) comprises a heterogenous group of rare mature T-cell neoplasms. While some PTCL subtypes are well-characterized by histology, immunophenotype, and recurrent molecular alterations, others remain incompletely defined. In particular, the distinction between CD30+ PTCL, not otherwise specified and anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma can be subject to disagreement. We describe a series of 6 JAK2 rearrangements occurring in a cohort of 97 CD30+ ALK- PTCL (6%), assembled after identifying an index case of a novel PABPC1-JAK2 fusion in a case of ALK- anaplastic large cell lymphoma with unusual classic Hodgkin lymphoma (CHL)-like features. Fusions were identified using a comprehensive next-generation sequencing based assay performed between 2013 and 2020. Five of 6 cases (83%) showed JAK2 rearrangements with 4 novel partners: TFG, PABPC1, ILF3, and MAP7, and 1 case demonstrated a previously described PCM1-JAK2 fusion. By morphology, all cases showed anaplastic large cells and multinucleated Reed-Sternberg-like cells within a polymorphous inflammatory background with frequent eosinophilia reminiscent of CHL. By immunohistochemistry, atypical large cells expressed CD30 with coexpression of at least 1 T-cell marker, aberrant loss of at least 1 T-cell marker and, in 4 of 5 cases stained (80%), unusual CD15 coexpression. These findings suggest that a subset of CD30+ ALK- systemic PTCL with anaplastic morphology carry JAK2 rearrangements, some of which appear to show CHL-like morphologic features. The presence of JAK2 rearrangements in cases of CD30+ PTCL augments current classification and may provide a therapeutic target via JAK2 inhibition.

Prognostic significance of myeloid immune cells and their spatial distribution in the colorectal cancer microenvironment.

BACKGROUND: Myeloid cells represent an abundant yet heterogeneous cell population in the colorectal cancer microenvironment, and their roles remain poorly understood. METHODS: We used multiplexed immunofluorescence combined with digital image analysis to identify CD14+ monocytic and CD15+ granulocytic cells and to evaluate their maturity (HLA-DR and CD33), immunosuppressive potential (ARG1) and proximity to cytokeratin (KRT)-positive tumor cells in 913 colorectal carcinomas. Using covariate data of 4465 incident colorectal cancers in two prospective cohort studies, the inverse probability weighting method was used with multivariable-adjusted Cox proportional hazards models to assess cancer-specific mortality according to ordinal quartiles (Q1-Q4) of myeloid cell densities. Immune cell-tumor cell proximity was measured with the nearest neighbor method and the G-cross function, which determines the likelihood of any tumor cell having at least one immune cell of the specified type within a certain radius. RESULTS: Higher intraepithelial (Ptrend=0.0002; HR for Q4 (vs Q1), 0.48, 95% CI 0.31 to 0.76) and stromal (Ptrend <0.0001; HR for Q4 (vs Q1), 0.42, 95% CI 0.29 to 0.63) densities of CD14+HLA-DR+ cells were associated with lower colorectal cancer-specific mortality while, conversely, higher intraepithelial densities of CD14+HLA-DR- cells were associated with higher colorectal cancer-specific mortality (Ptrend=0.0003; HR for Q4 (vs Q1), 1.78, 95% CI 1.25 to 2.55). Spatial analyses indicated that CD15+ cells were located closer to tumor cells than CD14+ cells, and CD14+HLA-DR+ cells were closer to tumor than CD14+HLA-DR- cells (p<0.0001). The G-cross proximity measurement, evaluating the difference in the likelihood of any tumor cell being colocated with at least one CD14+HLA-DR+ cell versus CD14+HLA-DR- cell within a 20 µm radius, was associated with lower colorectal cancer-specific mortality (Ptrend <0.0001; HR for Q4 (vs Q1), 0.37, 95% CI 0.24 to 0.57). CONCLUSIONS: Myeloid cell populations occur in spatially distinct distributions and exhibit divergent, subset-specific prognostic significance in colorectal cancer, with mature CD14+HLA-DR+ and immature CD14+HLA-DR- monocytic phenotypes most notably showing opposite associations. These results highlight the prognostic utility of multimarker evaluation of myeloid cell infiltrates and reveal a previously unrecognized degree of spatial organization for myeloid cells in the immune microenvironment.

Clinically relevant umbilical cord inflammation identified based on CD15-associated vasculitis patterning.

INTRODUCTION: Acute funisitis, a granulocyte-related inflammation of the umbilical cord, is associated with chorioamnionitis and perinatal adverse events. However, there is no efficient procedure for detecting clinically relevant umbilical cord inflammation. The objective of this study was to identify such inflammation, based on immunohistochemical assessment of umbilical cord vasculitis patterns. METHODS: Accordingly, 261 cases were retrieved from a single medical institute. Using the well-established granulocyte marker CD15, we developed a five-tier umbilical cord inflammation-scoring system. Additionally, previous morphological assessments from pathological reports were compared to the immunohistochemical findings. RESULTS: Analysis of results based on our new scoring system revealed that severe umbilical phlebitis (score 3) was significantly associated with maternal inflammatory response and that severe umbilical arteriophlebitis (score 4) was correlated with low umbilical arterial blood pH, a feature linked to fetal mortality and morbidity. These results corresponded with and were validated by the morphology-based assessments. Additionally, immunohistochemical analysis revealed the clinical and pathological relevance of vitelline vasculitis, a recently proposed condition. We found that analyzing three umbilical cord sections enabled superior detection of severe umbilical vasculitis than analyzing two sections. However, whether these sections were sampled from multiple distant sites or a single localized site did not significantly affect the detection of clinically relevant inflammation. DISCUSSION: CD15 immunohistochemistry is a potent tool for observing the patterns of clinically relevant umbilical vasculitis, especially in cases that were indeterminate according to morphology alone. Sampling three umbilical cord sections was an efficient procedure for addressing the spatial heterogeneity of umbilical cord inflammation. CD15 immunohistochemistry is a potent tool for observing the patterns of clinically relevant umbilical vasculitis, especially in cases that were indeterminate according to morphology alone. Sampling three umbilical cord sections was an efficient procedure for addressing the spatial heterogeneity of umbilical cord inflammation.

Enrichment and Transcriptional Characterization of Stem Cells Isolated from Human Glioblastoma Cell Lines.

Glioblastomas (GBM) are the most frequent and aggressive brain tumors due to their recurrence and resistance to current therapies. These characteristics are associated with the presence of glioma stem cells (GSCs), mainly identified by the detection of the membrane antigens CD133 and CD15. The main source of GSCs has been biopsies of tumors. However, alternatives are sought from cell lines because more homogeneous populations can be obtained with high yields. This chapter describes a method for the enrichment and characterization of GSCs from cell lines derived from human GBM by selective culture with serum-free neural stem cell medium and growth factors. The technique offers alternatives for the enrichment and characterization of GSCs, that could contribute to a better understanding of the biology of GBMs.

Increased Neutrophil Count and Decreased Neutrophil CD15 Expression Correlate With TB Disease Severity and Treatment Response Irrespective of HIV Co-infection.

Tuberculosis remains a leading cause of death globally despite curative treatment, partly due to the difficulty of identifying patients who will not respond to therapy. Simple host biomarkers that correlate with response to drug treatment would facilitate improvement in outcomes and the evaluation of novel therapies. In a prospective longitudinal cohort study, we evaluated neutrophil count and phenotype at baseline, as well as during TB treatment in 79 patients [50 (63%) HIV-positive] with microbiologically confirmed drug susceptible TB undergoing standard treatment. At time of diagnosis, blood neutrophils were highly expanded and surface expression of the neutrophil marker CD15 greatly reduced compared to controls. Both measures changed rapidly with the commencement of drug treatment and returned to levels seen in healthy control by treatment completion. Additionally, at the time of diagnosis, high neutrophil count, and low CD15 expression was associated with higher sputum bacterial load and more severe lung damage on chest x-ray, two clinically relevant markers of disease severity. Furthermore, CD15 expression level at diagnosis was associated with TB culture conversion after 2 months of therapy (OR: 0.14, 95% CI: 0.02, 0.89), a standard measure of early TB treatment success. Importantly, our data was not significantly impacted by HIV co-infection. These data suggest that blood neutrophil metrics could potentially be exploited to develop a simple and rapid test to help determine TB disease severity, monitor drug treatment response, and identify subjects at diagnosis who may respond poorly to treatment.

Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery.

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called "targeting by timing." Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, and N- and O-glycans present in each compartment were analyzed by LC-MS. We found abundant paucimannosidic N-glycans and lack of O-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of "glycosylation by timing" during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.

Molecular analyses of glioblastoma stem-like cells and glioblastoma tissue.

Glioblastoma is a common, malignant brain tumor whose disease incidence increases with age. Glioblastoma stem-like cells (GSCs) are thought to contribute to cancer therapy resistance and to be responsible for tumor initiation, maintenance, and recurrence. This study utilizes both SNP array and gene expression profiling to better understand GSCs and their relation to malignant disease. Peripheral blood and primary glioblastoma tumor tissue were obtained from patients, the latter of which was used to generate GSCs as well as a CD133pos./CD15pos. subpopulation. The stem cell features of GSCs were confirmed via the immunofluorescent expression of Nestin, SOX2, and CD133. Both tumor tissue and the isolated primary cells shared unique abnormal genomic characteristics, including a gain of chromosome 7 as well as either a partial or complete loss of chromosome 10. Individual genomic differences were also observed, including the loss of chromosome 4 and segmental uniparental disomy of 9p24.3→p21.3 in GSCs. Gene expression profiling revealed 418 genes upregulated in tumor tissue vs. CD133pos./CD15pos. cells and 44 genes upregulated in CD133pos./CD15pos. cells vs. tumor tissue. Pathway analyses demonstrated that upregulated genes in CD133pos./CD15pos. cells are relevant to cell cycle processes and cancerogenesis. In summary, we detected previously undescribed genomic and gene expression differences when comparing tumor tissue and isolated stem-like subpopulations.

Expression of the cancer stem cell marker SSEA1 is associated with poor survival in metastatic high-grade serous carcinoma.

The objective of the present study was to perform a quantitative analysis of cancer stem cell (CSC) marker expression in ovarian carcinoma effusions. The clinical role of SSEA1 in metastatic high-grade serous carcinoma (HGSC) was additionally analyzed. CD133, Nanog, SOX2, Oct3/4, SSEA1, and SSEA4 protein expressions were quantitatively analyzed using flow cytometry (FCM) in 24 effusions. SSEA1 expression by immunohistochemistry was analyzed in 384 HGSC effusions. Highly variable expression of CSC markers by FCM was observed, ranging from 0 to 78% of Ber-EP4-positive cells in the case of CD133, with the largest number of negative specimens seen for SSEA4. SSEA1 expression by immunohistochemistry was found in HGSC cells in 336/384 (89%) effusions, most commonly focally (< 5% of cells). SSEA1 was overexpressed in post-chemotherapy disease recurrence specimens compared with chemo-naïve HGSC effusions tapped at diagnosis (p = 0.029). In univariate survival analysis, higher SSEA1 expression was significantly associated with poor overall survival (p = 0.047) and progression-free survival (p = 0.018), though it failed to retain its prognostic role in Cox multivariate survival analysis in which it was analyzed with clinical parameters (p = 0.059 and p = 0.111 for overall and progression-free survival, respectively). In conclusion, CSC markers are variably expressed in ovarian carcinoma effusions. SSEA1 expression is associated with disease progression and poor survival in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

Identification and characterization of splenic adherent cells forming densely-packed colonies.

It is thought that the spleen contains stem cells that differentiate into somatic cells other than immune cells. We investigated the presence of these hypothetical splenic cells with stem cell characteristics and identified adherent cells forming densely-packed colonies (Splenic Adherent Colony-forming Cell; SACC) in the spleen. Splenic Adherent Colony-forming Cell was positive for alkaline phosphatase staining and stage-specific embryonic antigen (SSEA)-1 antigen. However, the self-renewal properties of SACCs were limited because they stopped cell proliferation once colonies visible to the naked eye were formed. Gene expression analyses by semi-quantitative RT-PCR revealed the significant expression of c-Myc and Klf4, whereas faint or no expression was evident for Nanog, Oct3/4, and Sox2. Global expression analyses by DNA microarray and subsequent gene ontology analyses revealed that the expression levels of genes related to the immune system were significantly lower in SACCs than in control splenic cells. In contrast, genes unrelated to the immune system, such as those involved in cell adhesion and axon guidance, were relatively highly expressed in SACCs compared with control splenic cells. Taken together, we identified a novel cell type residing in the spleen that is different from the hypothetical splenic stem cell, but which bears some, but not all, characteristics that represent an undifferentiated state.

Septic cardiomyopathy: The value of lactoferrin and CD15 as specific markers to corroborate a definitive diagnosis.

Current scientific consensus about the physiopathology in the progression from severe sepsis to septic shock and death focuses on myocardial contractile dysfunction. Nevertheless, objective parameters to establish a pathological correlate of a fatal outcome are lacking; then a cause of death due to sepsis can remain an unsolved problem. We first reviewed all death cases recorded at our institutions during the period from 2007 until 2015. Then, we conducted a retrospective study of a selected autopsy series of people who had received "sepsis" as cause of death. Two pathologists re-examined the heart sections while the most suitable myocardial sample for each case was stained for immunohistochemistry with antibodies targeted for specific inflammatory-related molecules. We used specific antibodies for the following markers: alpha-smooth muscle actin (alpha-SMA); fibronectin; matrix metallopeptidase 9 (MMP-9); intercellular adhesion molecule 1 (ICAM-1); caspase-3; lactoferrin (LF); cluster differentiation 15 (CD15). The statistical significance of differences was assessed using student's t-test for unpaired data or non-parametric Mann-Whitney or Wilcoxon tests for skewed variables or one-way analysis of variance and post hoc Scheffe's test for continuous variables and Pearson's χ2-test for discrete variables. Linear regression analysis was used to determine the presence of a correlation between continuous variables. At our institutions, 2220 deaths have been recorded during the period study. Sepsis accounted as a cause of death for the 20% of total. We finally enrolled 56 cases; of these, only 20 were positive for microbiological analysis. At histological examination, clear inflammation was detectable in the 32% of cases; otherwise, immunohistochemical reaction showed a positive reaction for LF and CD15 in more than a half cases (56%). We still ignore all the underlying mechanisms of sepsis and all its pathophysiological connections with cardiac metabolism; in this sense, we aim to corroborate the diagnostic value of anti-LF and anti-CD15 staining for the post-mortem detection of myocardial inflammation.

Low levels of intra-tumoural T cells in breast cancer identify clinically frail patients with shorter disease-specific survival.

OBJECTIVES: The global health status of older patients with cancer influences their clinical course, but little is known regarding the influence of the immune system on the global health of older patients with cancer. The goal of this study was to assess the relationships between patient fitness/frailty status and survival, and the local tumour immune environment of older patients with breast cancer. MATERIALS AND METHODS: In a cohort of 58 older patients with breast cancer (over 70 years of age), fluorescence microscopy was used to investigate whether levels of intra-tumoural T cells (CD3+) and granulocytic cells (CD15+) could predict clinical outcome, and/or whether they correlated with patient physical and mental performance as evaluated by comprehensive geriatric assessment. RESULTS: We observed that patients with higher levels of intra-tumoural T cells were fitter according to a number of clinical health measures including G8 (p = 0.006), Karnofsky Index (p = 0.0372), and Leuven Oncology Frailty Score (LOFS) (p = 0.0187). In contrast, high relative levels of granulocytic cells were found in patients with poorer clinical health (LOFS, p = 0.0474). Furthermore, high levels of T cells but not granulocytic cells were associated with longer breast cancer-specific survival (p = 0.0444). CONCLUSIONS: This is the first study to show that low relative levels of intra-tumoural T cells are associated with inferior patient fitness. In contrast to T cells, we observed that intra-tumoural granulocytic cells displayed an inverse relationship with patient performance. Further research is needed to determine whether boosting the level of intra-tumoural T cells in older non-fit patients can result in improved outcome.

Microbe-Dependent Induction of IL-9 by CLA+ T Cells in Psoriasis and Relationship with IL-17A.

IL-9 is present in psoriatic lesions and is produced by lymphocytes. However, it is not known whether this cytokine is induced by relevant pathogenic triggers of psoriasis, such as Streptococcus pyogenes. Here we addressed the production of IL-9 in response to various pathogens in a psoriatic ex vivo model. Extracts of S. pyogenes and Candida albicans triggered the production of IL-9 and also IL-17A and IFN-γ. This induction was dependent on the interaction between CLA+ T cells and epidermal cells. Neutralization of IL-9 reduced S. pyogenes-induced IL-17A production by CLA+ T cells but had no effect on IFN-γ production. Also, IL-9 increased the survival of circulating psoriatic CLA+ T cells. Co-cultures from patients with guttate or plaque psoriasis with S. pyogenes produced similar amounts of IL-9. High cytokine responses in streptococcal-driven guttate patients paralleled peaks in Psoriasis Area Severity Index and anti-streptolysin O levels. Our results confirm that IL-9 promotes inflammation in psoriasis by up-regulating IL-17A production and support the clinical association of the immune response by streptococcal-sensitized CLA+ T cells with this cytokine, especially in guttate psoriasis.

Semaphorin 4D from CD15+ Granulocytes via ADAM10-Induced Cleavage Contributes to Antibody Production in Bullous Pemphigoid.

Autoreactive B-cell activation and antibody production are critical events for the development of bullous pemphigoid (BP). However, the mechanism that is involved in the modulation of B-cell activation and autoantibody generation has not been fully understood. Semaphorin 4D (Sema4D, or CD100) plays important roles in immune regulation related to B cells, but its implications in BP remain obscure. The aim of our study was to characterize Sema4D and the underlying mechanism contributing to the autoimmune features of BP. We found that soluble Sema4D (sSema4D) levels were elevated and correlated with disease severity and activity in serum and blister fluids from patients with BP. Additionally, Sema4D-expressing cells accumulated in subepidermal blisters of BP lesions. In patient-derived peripheral blood mononuclear cells, by promoting the differentiation of B cells into plasmablasts, sSema4D boosted anti-BP180/anti-BP230 antibody production in a time- and dose-dependent manner, which may be attributed to CD72-mediated activation of Akt/NF-κB phosphorylated (p-)65/ERK cascades in B cells. We determined that a disintegrin and metalloproteinase 10 is a proteolytic enzyme for the cleavage of sSema4D from CD15+ granulocytes instead of T cells, which is probably responsible for the high concentration of sSema4D in BP blister fluid and serum. These findings suggest that Sema4D is a crucial participant in BP pathogenesis.

Analysis of CD15, CD57 and HIF-1α in biopsies of patients with peri-implantitis.

Peri-implantitis is an infectious disease characterized by inflammation of the tissues surrounding the implant, bleeding on probing with or without suppuration, and bone loss. Peri-implant lesions contain a leukocyte infiltrate of plasma cells, lymphocytes, macrophages and neutrophils. A survey of the literature did not show any studies reporting an association between hypoxia and peri-implantitis. The aim of the present cross-sectional study was to evaluate histological changes and immunostaining for CD15, CD57 and HIF-1α in the peri-implant mucosa of patients with and without peri-implantitis. Mucosal biopsies were obtained from 18 patients with peri-implantitis and 10 control subjects without peri-implantitis at a private health care center between 2010 and 2012. The sections were fixed in 10% buffered formalin, processed and embedded in paraffin for histopathological and immunohistochemical study. Acanthosis, spongiosis and exocytosis were observed in both groups, with no significant difference between them. The peri-implantitis group showed increased immunostaining for CD15, a neutrophil marker, and HIF-1α, a tissue hypoxia marker, but no significant difference in immunostaining for CD57, a Natural Killer cell marker. The increase in neutrophil (CD15) and hypoxia (HIF-1α) markers in patients with peri-implantitis suggests an active participation of neutrophils and hypoxia in the pathogenesis of this disease. Since the present study was the first to evaluate the expression of CD15, CD57 and HIF-1α in peri-implant tissues, further studies should be performed to better understand the role of these molecules in peri-implantitis.

Increased circulating leukocyte-derived microparticles in ischemic cerebrovascular disease.

OBJECTIVES: Circulating leukocyte-derived microparticles act as proinflammatory mediators that reflect vascular inflammation. In this study, we examined the hypothesis that the quantity of leukocyte-derived microparticles is increased in patients with ischemic cerebrovascular diseases, and investigated utility of various phenotypes of leukocyte-derived microparticles as specific biomarkers of vascular inflammation injury. Additionally we focused on identifying leukocyte-derived microparticles that may be correlated with stroke severity in acute ischemic stroke patients. METHODS: The plasma concentration of leukocyte-derived microparticles obtained by a series of centrifugations of 76 consecutive patients with ischemic cerebrovascular diseases and 70 age-, sex-, and race-matched healthy controls were determined by flow cytometry. RESULTS: Significantly elevated numbers of leukocyte (CD45+), monocyte (CD14+), lymphocyte (CD4+), granulocyte (CD15+) derived microparticles were found in the plasma samples of patients ischemic cerebrovascular diseases, compared to healthy controls (p<0.05). Furthermore, the plasma levels of CD14+ microparticles were significantly correlated with stroke severity (r=0.355, p=0.019), cerebral vascular stenosis severity (r=0.255, p=0.025) and stroke subtype (r=0.242, p=0.036). No association with stroke was observed for other leukocyte-derived phenotypes. CONCLUSIONS: These results demonstrate that circulating leukocyte-derived microparticles amounts are increased in patients with ischemic cerebrovascular diseases, compared with healthy controls. As proinflammatory mediators, leukocyte-derived microparticles may contribute to vascular inflammatory and the inflammatory process in acute ischemic stroke. Levels of CD14+ microparticles may be a promising biomarker of ischemic severity and outcome of stroke in the clinic.

CD15 focus score: Infection diagnosis and stratification into low-virulence and high-virulence microbial pathogens in periprosthetic joint infection.

INTRODUCTION: The aim of the work was to validate the CD15 focus score for the infection pathology of periprosthetic joint infection in a large group and to clarify whether a stratification into low-virulence and high-virulence microbial pathogens is possible by means of the CD15 focus score (quantification of CD15 positive granulocytes). METHODS: The histopathology of 275 synovial tissue samples taken intraoperatively during revision operations (n=127 hip, n=141 knee, n=2 shoulder, n=5 ankle) was evaluated according to the SLIM consensus classification (SLIM=synovial-like interface membrane). Neutrophilic granulocytes (NG) were quantified by the CD15 focus score on the basis of the principle of focal maximum infiltration (focus) with evaluation of one field of vision (about 0.3mm2). The quantification values were compared with the microbiological diagnoses taking into consideration the virulence groups of low-virulence and high-virulence microbial pathogens and mixed infection. RESULTS: The patients with positive microbiological findings (n=160) had significantly (p<0.001, Mann-Whitney U test) higher CD15 focus score values than patients with negative microbiological findings (n=115), the cut-off value being 39 cells per high power field (HPF). The CD15 focus score values of low-virulence microbial pathogens (n=94) were significantly lower (p<0.001, Mann-Whitney U test) than the values of high-virulence microbial pathogens (n=55), the cut-off value being 106 cells per HPF. Based on the microbiological diagnosis the sensitivity with respect to a microbial infection is 0.91, the specificity 0.92 (PPV=0.94; NPV=0.88; accuracy: 0.92; AUC=0.95). Based on the differentiation of the CD15 focus score values between low-virulence and high-virulence microbes the sensitivity is 0.70 and the specificity 0.77 (PPV=0.63; NPV=0.81; accuracy=0.74; AUC=0.74). CONCLUSION: As a result of the high sensitivity and specificity, the easy to use CD15 focus score is a diagnostically valid score for microbial periprosthetic infection. A differentiation between low-virulence and high-virulence microorganism of sufficiently high diagnostic quality is additionally possible as a result of the defined quantification of CD15 positive granulocytes (the CD15 focus score) histopathological diagnosis of microbial infections is possible, which on the one hand supports the microbiological diagnosis and on the other hand by the stratification into low-virulence and high-virulence microbial pathogens could represent an additional basis for a pathogen-specific antibiotic treatment in the event of unclear constellations of findings.

Stage-Specific Embryonic Antigen-1 (SSEA-1) Expression in Thyroid Tissues.

Stage-specific embryonic antigen-1 (SSEA-1), also known as CD15, is a member of a cluster of differentiation antigens that have been identified in various normal tissues and in different types of cancers including papillary and medullary thyroid carcinoma. SSEA-1 may be expressed in normal stem cells and cancer stem-like cells. To evaluate the potential diagnostic and prognostic utility of SSEA-1 in thyroid tumors, we analyzed the expression of SSEA-1 in normal and neoplastic thyroid tissues by immunohistochemistry (IHC) using a tissue microarray with 158 different tissue cores. To evaluate the potential utility of SSEA-1 as a surface marker, we also assessed the expression of SSEA-1 in thyroid cell lines by flow cytometric analysis. SSEA-1 immunoreactivity was identified in malignant thyroid follicular epithelial cancers but not in the benign thyroid tissues. Anaplastic thyroid (ATC) (80 %) and conventional papillary thyroid carcinoma (PTC) (60.7 %) showed significantly higher percentage of cases that were SSEA-1 immunoreactive than follicular variant of papillary thyroid carcinoma (FVPTC) (20.6 %) and follicular carcinoma (FCA) (32.1 %). Flow cytometric analysis of cultured thyroid cell lines showed that a small subpopulation of ATC and PTC thyroid tumor cells had SSEA-1 immunoreactivity which may represent thyroid cancer stem-like cells. The ATC cells expressed more SSEA-1 immunoreactive cells than the PTC cell lines. Our findings suggest that expression of SSEA-1 immunoreactivity in thyroid neoplasms was associated with more aggressive thyroid carcinomas. SSEA-1 is a marker that detects malignant thyroid neoplasms in formalin-fixed paraffin-embedded thyroid tissue sections and may be a useful marker for thyroid cancer stem-like cells.