Lymphocyte Reaction Research Articles

PB1962 THE HIGH INCIDENCE OF MICROCYTOSIS IN THE CONTEXT OF MONOCLONAL PERIPHERAL BLOOD LYMPHOCYTOSIS IN THE LEUKEMIC PHASE OF LYMPHOPROLIFERATIVE DISORDERS

Background:Microcytosis is an established finding in chronic or inflammatory disease. Within this context, it is often described in lymphoid lineage hematologic malignancies, especially Hodgkin's lymphoma. Chronicity or a pro‐inflammatory state, however, are not the only predictors of a microcytic hemogram, with some chronic lymphoproliferative disorders (LPD) ‐ such as chronic lymphocytic leukemia (CLL) – rarely presenting with microcytosis, while the leukemic phase of other LPDs often presents with microcytosis.Aims:To clarify the relationship between monoclonal lymphocytosis in LPD and microcytosis.Methods:We analyzed all samples from adult patients (pts) with lymphocytosis (absolute lymphocyte count (ALC) >4.0 G/L), with analysis of clonality by flow cytometry, over a 4‐year period. A Control group of 50 pts with a LPD but without lymphocytosis and without clonality was selected.Results:We obtained 412 samples (51.4% male); 22.6% of patients had no hematological disease and a reactive polyclonal lymphocytosis (“Reactive”); 8.5% had a known hematologic disease, but polyclonal reactive lymphocytes (“Polyclonal”); and 68.9% had a pathologic monoclonal lymphocytosis (“Monoclonal”). Monoclonal pts had a CLL‐like phenotype in 69.7% of cases, marginal zone (MZL) in 14.8%, and large granular lymphocyte leukemia in 3.2%; follicular (FL), mantle cell (MCL), lymphoplasmacytic (WM), Sézary's, NK cell, and T‐prolymphocytic leukemia phenotypes accounted for <3% of cases, each; the remainder were non‐Hodgkin's Lymphomas, NOS. ALC ranged from 4.0‐141.1 G/L, with a mean of 16.4 ± 23.6 and a median of 6.8. The mean Reactive ALC was 5.0 ± 1.4 G/L, 5.2 ± 1.5 for Polyclonal and 21.4 ± 26.9 for Monoclonal pts (p < 0.001), with 14.4 ± 19.4 for non‐CLL pts (p < 0.001). Controls had an ALC of 1.8 ± 0.9 G/L.MCV ranged from 64.3‐111.1 fL, with a mean of 90.6 ± 6.1 and a median of 90.9; the Control median was 90.2 (74.1‐104.7 fL) with a mean of 90.5 ± 6.4 (p = NS); 91.0% of samples were normo‐, 5.8% macro‐ and 3.2% microcytic (p = NS vs Control). The mean Reactive MCV was 88.7 ± 4.7, 88.8 ± 6.3 for Polyclonal and 91.5 ± 6.4 for Monoclonal pts (p < 0.001), with microcytosis in 1.1%, 5.7% and 3.5% of pts, respectively (p = 0.044). Within B Cell lineage Monoclonal pts, the mean MCV was 92.3 ± 5.5 fL in CLL (p = 0.04 vs Control), 89.1 ± 7.9 in the indolent lymphomas (MZL, FL, WM; p = NS vs Control) and 84.5 ± 9.2 in MCL (p = 0.02 vs Control) (p < 0.001), with microcytosis in 0.5%, 7.8% and 50% of pts, respectively (p < 0.001).A posteriori longitudinal analysis of the MCV in microcytic pts during and after chemotherapy documented a correlation between the decrease in lymphocytosis and an increase in MCV, with improvement/resolution of microcytosis.Summary/Conclusion:We observed that reactive polyclonal lymphocytosis in non‐hematologic patients and in malignant hematology pts associated with identical average MCVs, overlapping with the mean MCV of a control population without lymphocytosis. Similarly, monoclonal lymphocytosis in the context of the indolent B cell lymphomas (MZL, FL and WM) did not significantly affect normocytosis. On the other hand, CLL tended to associate with significantly higher MCVs than controls or reactive polyclonal lymphocytoses.In contrast, a monoclonal lymphocytosis in the context of a leukemic phase of MCL associated significantly with a microcytic phenotype ‐ remarkably, we found that microcytosis was 100x more frequent in MCL (50% of cases) than CLL (0.5%).The effective treatment of the underlying LPD, and the resolution of clonal lymphocytosis, associated with a normalization of the MCV.

Chemoirradiated neutrophils and T cells differentially affect immune functions of APCs.

Extracorporeal photopheresis (ECP) is known as an immunomodulatory therapy with few side effects, which is mainly used in the treatment of cutaneous T cell lymphoma, graft-versus-host disease, and allograft rejection. During ECP, leukocytes are separated from whole blood by leukapheresis, subsequently chemoirradiated with 8-methoxypsoralen and UVA light, and re-infused into the patient. Although clinically effective, its mode of action has not been fully elucidated. In the present study, we analyzed the interaction of chemoirradiated neutrophils and CD3+ lymphocytes with APC in an in vitro model. We report that chemoirradiated CD3+ T cells induced increased expression of activation markers on dendritic cells (DC), macrophages, and monocytes. Coculture of chemoirradiated CD3+ T cells with these APC also led to significantly increased secretion of TNF-α. Although less pronounced, additional activation of APC took place when APC were stimulated with LPS or IFN-γ. In contrast, chemoirradiated neutrophils did not show activating effects on APC. The presence of chemoirradiated neutrophils during LPS and IFN-γ stimulation of DC rather diminished DC and macrophage activation. In line with these findings DC cocultured with chemoirradiated CD3+ T cells, but not neutrophils, showed significantly increased activation of CD3+ responder lymphocytes in a mixed lymphocyte reaction. With this study, we demonstrate that chemoirradiated leukocytes have differential indirect immunomodulatory effects. Whereas chemoirradiated CD3+ T cells activate APC, chemoirradiated neutrophils suppress activation of APC in the presence of other activating factors, suggesting that the composition of the ECP-treated buffy coat might be of importance for its immunomodulatory effects.

Parasitism Elicits a Stress Response That Allocates Resources for Immune Function in South American Fur Seals (Arctocephalus australis).

Parasites can cause chronic stress in some animal species, and this type of stress response has been associated with adverse consequences for the host. In order to know whether parasitism elicited a stress response associated with decreased host fitness, hookworm (Uncinaria sp.) infection was studied in a colony of South American fur seals (Arctocephalus australis) in which hookworms infect nearly all pups born in a reproductive season. A parasite-free group was generated by treating a subset of pups with an antiparasitic drug before they developed patent hookworm infection. Stress and metabolic hormones, energy balance, and humoral and cellular immune parameters were measured in this group and hookworm-infected pups. Hookworms elicited a marked increase in plasma cortisol levels in fur seal pups. These hookworm-infected pups were able to maintain constant glucose levels, despite losing body mass over the course of infection potentially because of increased protein catabolism. Infected pups were able to mount an effective immune response against the parasite and eliminated hookworms from the intestine, recovering partial body mass lost as a result of hookworm infection at the end of the study period. As shown in previous studies, adequate glucose levels are critical for proper T lymphocyte reactivity, and it is possible that, through activation of a stress response, energy can be readily available for immune response against the parasite contributing to early recovery from infection. Although there are potential fitness costs to mounting a sustained stress response, these could also be adaptive and promote survival during critical life-history stages.

Cytological tumour cell characteristics and reactive small lymphocytes influence patient prognosis in acute and lymphoma type adult T-cell leukaemia/lymphoma.

Acute and lymphoma type adult T-cell leukaemia/lymphoma (ATLL) patients show an aggressive clinical course. While some clinical signs indicate good prognosis, definitive cytohistological prognostic factors have yet to be described. We classified 65 ATLL patients into three groups by tumour cell size and nuclear pleomorphism on fine-needle aspiration and tumour touch smear samples. Semi-quantitative analysis of background small lymphocytes, reactive CD20-positive B cells and CD8-positive T cells was performed. Thirty-one patients had pleomorphic lymphoma with predominantly medium-sized cells and coarse granular nuclei. Another 24 patients showed pleomorphic large cell lymphoma with stippled chromatin. The remaining 10 demonstrated monomorphic large lymphoma cells with fine granular chromatin. Patients with pleomorphic lymphoma with medium-sized cells showed significantly higher serum lactate dehydrogenase and lower CD30 and C-MYC expression in lymphoma cells than the other two groups (P = .0216, P < 0.01, respectively). Patients with pleomorphic medium-sized ATLL had few usual small lymphocytes observed on routine morphological examination and showed less concurrent detection of CD20-positive B cells and CD8-positive T cells, both of which were lower than in the other two groups (P = .006, P = .019, respectively). Furthermore, ATLL patients with predominantly medium-sized lymphocytes exhibited a worse prognosis than patients with pleomorphic large cells (P = .0197). Background small lymphocytes and concurrent detection of CD20-positive B cells and CD8-positive T cells may thus be good prognostic factors (P = .011, P = .021, respectively). Morphological features, size of neoplastic cells and background non-neoplastic lymphocyte (B cells and CD8-positive T cells) volume appear to influence the prognosis of patients with aggressive-type ATLL.

774 Analysis of the tumor reactivity of tumor-infiltrating lymphocytes in a metastatic melanoma lesion that lost MHC class I expression after anti-PD-1 therapy

Several mechanisms for delayed relapses in melanoma patients long after the initial positive responses to anti-PD-1 therapy have been suggested. These include the escape of tumor cells from the immune system. However, the tumor-specific reactivity of TILs in the escaped tumor lesion, especially after MHC-class-I- loss has not been well evaluated. A 55-year-old Japanese woman with metastatic melanoma in her left supraclavicular lymph node and left accessory nerve lymph node was started on treatment with anti-PD-1 antibody therapy. Tumor progression was prevented by the therapy for 12 months. However, then one metastatic lesion started to grow again despite ongoing response in another lesion and the absence of new metastatic sites. We found no remarkable differences in the phenotypes of freshly isolated TILs from the two sites. Immunohistochemistry showed that MHC class I expression was diminished in the relapsed lesion. The tumor cell line established from the lesion also lost MHC class I expression completely. The copy-number assay revealed a deletion of the B2M gene. When we transduced the normal B2M gene into these cells, we confirmed the recovery of MHC class I molecule expression on their surface. Next, we expanded TILs from the left accessory nerve lymph node lesion and, assessed their reactivity to the autologous tumor cell line, with or without B2M gene transduction. We verified that TILs derived from the relapsed lesion could strongly recognize the autologous tumor cells in a MHC-class I-dependent manner. Our report emphasizes the limitations of T-cell-based immunotherapy and highlights the importance of developing alternative strategies for such cases.

Clonotypic heterogeneity in cutaneous T-cell lymphoma (mycosis fungoides) revealed by comprehensive whole-exome sequencing

AbstractMycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, is believed to represent a clonal expansion of a transformed skin-resident memory T cell. T-cell receptor (TCR) clonality (ie, identical sequences of rearranged TCRα, TCRβ, and TCRγ), the key premise of this hypothesis, has been difficult to document conclusively because malignant cells are not readily distinguishable from the tumor-infiltrating reactive lymphocytes that contribute to the TCR clonotypic repertoire of MF. Here, we have successfully adopted targeted whole-exome sequencing (WES) to identify the repertoire of rearranged TCR genes in tumor-enriched samples from patients with MF. Although some of the investigated MF biopsies had the expected frequency of monoclonal rearrangements of TCRγ corresponding to that of tumor cells, the majority of the samples presented multiple TCRγ, TCRα, and TCRβ clonotypes by WES. Our findings are compatible with the model in which the initial malignant transformation in MF does not occur in mature memory T cells but rather at the level of T-lymphocyte progenitors before TCRβ or TCRα rearrangements. We have also shown that WES can be combined with whole-transcriptome sequencing in the same sample, which enables comprehensive characterization of the TCR repertoire in relation to tumor content. WES/whole-transcriptome sequencing might be applicable to other types of T-cell lymphomas to determine clonal dominance and clonotypic heterogeneity in these malignancies.

Acquisition of specific antibodies and their influence on cell-mediated immune response in neonatal cord blood after maternal pertussis vaccination during pregnancy

Maternal immunization with pertussis acellular vaccine (Tdap) is an intervention that provides protection to newborns. However, it has been reported that high maternal antibody levels may adversely affect the immune response of infants after active immunization. In this study, we evaluated neonatal passive acquisition of pertussis-specific antibodies and their influence on the neonatal cell-mediated immune response.Pregnant women were either vaccinated with Tdap vaccine (case group, n = 66) or received no vaccine (control group, n = 101). Whole-cell Bordetella pertussis (Bp), pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN)-specific serum IgG were quantified in paired maternal-cord sera, and Bp- and PT-specific IgA were evaluated in colostrum by ELISA. Ex vivo neonatal blood lymphocyte responsiveness after Bp stimulation was assessed in case (n = 17) and control (n = 15) groups using flow cytometry to detect proliferation, cytokine production and activation phenotype of lymphocytes in the context of high specific IgG acquired after maternal vaccination.Anti-Bp, PT, FHA and PRN IgG concentrations in maternal and cord sera from case group were higher than those in control group with positive correlation indexes in both groups for all pertussis antigens. The control group presented higher placental transfer ratios of specific antibodies and, in the case group, vaccination between 26 and 31 gestation weeks was associated with the best placental transfer ratios. Specific IgA concentrations in colostrum were not affected by vaccine status. Whole blood assays revealed that newborns responded to Bp stimulation with higher expression of CD40L, CD69 and CD4+ T cell proliferation compared to unstimulated cells, and a lower Th1 response, while a preserved Th2 response compared to adults, but there were no differences between the neonatal groups for any of the studied parameters.Our results indicate that higher pertussis-specific IgG levels in newborn sera after maternal vaccination do not affect the neonatal ex vivo cell-mediated immune response.

Abstract 17: Increasing Ischemia Time Diminishes Chimerism After Vascularized Bone Marrow Allotransplantation

PURPOSE: Vascularized composite allotransplantation (VCA) can include vascularized bone marrow (VBM) to potentially generate chimerism and subsequent tolerance in the recipient. While increasing ischemia time is known to be detrimental to free flaps and solid organ transplants, it is unknown whether increasing VCA ischemia time may diminish VBM’s capacity to achieve chimerism and tolerance. This study determines whether increasing cold ischemia time results in decreased peripheral chimerism levels, increasing loss of the donor bone marrow compartment, and increased skin paddle rejection in a murine VCA model. METHODS: Twenty-three adult Lewis rats received osteomyocutaneous hind limb allografts from 3 groups of Brown-Norway rats. The groups underwent 0, 6, and 12 hours of cold ischemia at 4°C (n=8, 6, and 9 respectively). Immunosuppressive conditioning consisted of anti-lymphocyte serum 1cc 3 days pre-operatively and on post-op day (POD) 1, tacrolimus 2mg/kg/day for PODs 1–7, and then concluded with rapamycin 3mg/kg/day for PODs 8–28. Peripheral chimerism levels were drawn after POD 30. Skin paddle was observed for 30 days after immunosuppression cessation for rejection or tolerance. Allograft was collected at POD 60 for flow cytometry and histologic analysis. RESULTS: Peripheral chimerism levels in both myeloid and lymphoid lines were decreased in the 12 & 6 hours groups versus 0 hours (p<0.01). At POD 60, the amount of donor cells in the allograft bone marrow was significantly decreased at 12 & 6 hours versus the 0 hours group (p<0.05). At 12 hours, hematoxylin & eosin histology demonstrated significantly increased reactive lymphocytes, increased stroma, significantly decreased hematopoetic cells, and significant myelofibrosis. At 6 hours, moderate stromal increases, moderately decreased hematopoetic cells, and moderate myelofibrosis was observed. Trichrome staining revealed viable bone at all time points though with there were increasing amounts of collagen deposition with increasing ischemia and osteoslerosis was noted at 12 hours. Microvasculature was preserved at all time points. By POD 60, 0% of skin paddles survived in the 12 hours group, 17% at 6 hours, and 73% at 0 hours. CONCLUSION: With increasing ischemia time prior to VCA re-vascularization, there is decreased peripheral chimerism levels, increased loss of donor hematopoetic cells in the donor bone marrow, and increased skin paddle rejection. These findings indicate that ischemia time may significantly limit the capacity for VBM to facilitate chimerism and subsequent VCA tolerance and thus strategies using VBM to induce tolerance may need to consider the additional obstacle of time. Prolonged ischemia time may be a contributing factor to the absence of stable chimerism and tolerance in human VBM-bearing VCA recipients.

IL-6, IL-12, and IL-23 STAT-Pathway Genetic Risk and Responsiveness of Lymphocytes in Patients with Multiple Sclerosis.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease characterized by central nervous system (CNS) lymphocyte infiltration, abundant production of pro-inflammatory cytokines, and inappropriate activation of Th1 and Th17 cells, B cells, and innate immune cells. The etiology of MS is complex, and genetic factors contribute to disease susceptibility. Genome-wide association studies (GWAS) have revealed numerous MS-risk alleles in the IL-6/STAT3, IL-12/STAT4, and IL-23/STAT3-pathways implicated in the differentiation of Th1 and Th17 cells. In this study, we investigated the signaling properties of these pathways in T, B, and NK cells from patients with relapsing-remitting MS (RRMS) and healthy controls, and assessed the genetic contribution to the activity of the pathways. This revealed a great variability in the level of STAT-pathway molecules and STAT activation between the cell types investigated. We also found a strong donor variation in IL-6, IL-12, and IL-23 responsiveness of primed CD4+ T cells. This variation could not be explained by a single MS-risk variant in a pathway component, or by an accumulation of multiple STAT-pathway MS-risk SNPs. The data of this study suggests that other factors in cohesion with the genetic background contribute to the responsiveness of the IL-6/STAT3, IL-12/STAT4, and IL-23/STAT3-pathways.

Color clustering segmentation framework for image analysis of malignant lymphoid cells in peripheral blood.

Current computerized image systems are able to recognize normal blood cells in peripheral blood, but fail with abnormal cells like the classes of lymphocytes associated to lymphomas. The main challenge lies in the subtle differences in morphologic characteristics among these classes, which requires a refined segmentation. A new efficient segmentation framework has been developed, which uses the image color information through fuzzy clustering of different color components and the application of the watershed transformation with markers. The final result is the separation of three regions of interest: nucleus, entire cell, and peripheral zone around the cell. Segmentation of this zone is crucial to extract a new feature to identify cells with hair-like projections. The segmentation is validated, using a database of 4758 cell images with normal, reactive lymphocytes and five types of malignant lymphoid cells from blood smears of 105 patients, in two ways: (1) the efficiency in the accurate separation of the regions of interest, which is 92.24%, and (2) the accuracy of a classification system implemented over the segmented cells, which is 91.54%. In conclusion, the proposed segmentation framework is suitable to distinguish among abnormal blood cells with subtile color and spatial similarities. Graphical Abstract The segmentation framework uses the image color information through fuzzy clustering of different color components and the application of the watershed transformation with markers (Top). The final result is the separation of three regions of interest: nucleus, entire cell, and peripheral zone around the cell. The procedure is also validated by the implementation of a system to automatically classify different types of abnormal blood cells (Bottom).

Analysis of the Tumor Reactivity of Tumor-Infiltrating Lymphocytes in a Metastatic Melanoma Lesion that Lost Major Histocompatibility Complex Class I Expression after Anti–PD-1 Therapy

Major histocompatibility complex class I loss due to the abnormality of β2-microglobulin gene is one of the mechanisms underlying delayed relapses in melanoma patients long after the initial positive responses to anti-PD-1 therapy. However, the tumor-specific reactivity of tumor-infiltrating lymphocytes in tumor lesions that lost major histocompatibility complex class I expression has not been well evaluated. We report the case of a 55-year-old woman with two metastatic melanoma lesions. After a 12-month period of successful tumor suppression by anti-PD-1 antibody therapy, one lesion started to grow again. We resected both lesions and examined the tumor cells and tumor-infiltrating lymphocytes. The shrinking lesion consisted of necrotic tissue and macrophages, and the enlarged lesion consisted of both necrotic tissue and viable tumor cells. The tumor cells completely lost major histocompatibility complex class I expression, but it was restored upon retroviral transduction of the normal β2-microglobulin gene. When we checked the tumor-specific reactivity of tumor-infiltrating lymphocytes derived from the relapsing lesion, we found that these tumor-infiltrating lymphocytes failed to recognize the native tumor cells derived from the lesion, but strongly recognized the major histocompatibility complex class-I-recovered cells by β2-microglobulin transduction. Our report emphasizes the limitations of T-cell-based immunotherapy and highlights the importance of developing alternative strategies for such cases.

Clinical features and laboratory findings in children hospitalized with acute Epstein-Barr virus infection: a crosssectional study in a tertiary care hospital.

Çağlar İ, Topal S, Çokboz M, Düzgöl M, Kara A, Bayram SN, Apa H, Devrim İ. Clinical features and laboratory findings in children hospitalized with acute Epstein-Barr virus infection: a cross-sectional study in a tertiary care hospital. Turk J Pediatr 2019; 61: 368-373. Epstein-Barr virus (EBV) is widespread all over the world. It causes infectious mononucleosis (IM) mostly in adolescents and adults. Although IM is considered to be rare in younger children and infants, acute EBV infection may have various manifestations in this age group. We aimed to describe the clinical features and laboratory findings of children hospitalized with acute EBV infection. All children hospitalized at Dr. Behçet Uz Children`s Hospital, between January 2010 and January 2017, who tested positive by presence of EBV-specific antibodies and had the diagnosis of acute EBV infection, were included (n=66). Thirty four of the patients (51.5%) were under 6 years of age, and 23 (34.8%) children were below 3 years of age. The most common physical finding was fever (92.4%) followed by cervical lymphadenopathy and tonsillopharyngitis. Leukocytosis (65.1%) and lymphocytosis (42.4%) were the most common laboratory findings. Reactive and atypical lymphocytes were present in 77.2% of the patients. Fifty-three (80.3%) of the patients had a doctor visit before hospitalization, and the ratio of patients using antibiotics was 77.3%. Skin rash was observed in 14 (27.4%) of the patients who used antibiotic treatment and in 2 (13.3%) of the patients who did not (p > 0.05). EBV infection resulting in admission to hospital is common in younger children, even in pre-school period. Serological tests for EBV specific antibody responses and peripheral blood smear evaluation are important diagnostic tools. In addition, rapid streptococcal antigen test and throat culture should be performed in patients presenting with tonsillopharyngitis in order to exclude Group A beta-hemolytic streptococci and reduce unnecessary antibiotic consumption.

Alox15b Gene Contributes to Lymphoma Tumorigenesis Via PI3K/AKT/mTOR Pathway Activation and Has a Synergistic Effect with Alox5 Gene

Deletion of the short arm of chromosome 17(del17p) is associated with resistance to standard treatment and the very poor clinical outcome in lymphocytic malignancy. Alongside with TP53 gene, ALOX15B gene is also lost in del(17p) patients. In our pervious study, partially knockdown of Alox15b in mice could accelerate lymphoma tumorigenesis with or without TP53 loss. Accumulation of arachidonic acid (AA) and reduction of apoptosis was observed. But how Alox15b gene and AA effect lymphoma tumorigenesis is not fully understood. Here we show that PI3K/AKT/mTOR pathway is activated in Alox15b-knockdown lymphocytes and contributes to the drug resistance. Besides, we also find that Alox15bgene and Alox5gene play a synergistic role lymphoma tumorigenesis in mice model.Experiments were performed using murine preB cell line, baf3 cell. We introduced shRNAs targeting Alox15b at 2 different loci (shAlox15b.1252 and shAlox15b.2865) to create 2 separated Alox15b-deficient cell lines. Intracellular AA level was tested by liquid chromatograph-mass spectrometer(LC-MS). And Alox15b-deficient lymphocytes showed an accumulation of intracellular AA.Then the activation status of PI3K/AKT/mTOR pathway was detected by examining the phosohor-S6 (pS6) protein level using Western Blot assay in wild-type baf3 cells with exogenous-added AA and Alox15b-deficient baf3 cell lines respectively. The results showed that exogenous-added AA activated PI3K/AKT/mTOR pathway in a dose-dependent manner in wild-type baf3 cells. Also, Alox15b-dificient baf3 cell lines showed an increase in pS6 protein level than control, suggesting that in Alox15b-deficient lymphocytes, PI3K/AKT/mTOR pathways is activated via accumulation of AAIn addition, the cell viability test was involved to test how Alox15b gene knockdown affected lymphocyte's reaction to rapamycin(RAPA), a mTOR inhibitor. The results showed Alox15b-dificient baf3 cells are more resistant to RAPA, which indicated that mTOR inhibitors may not be a suitable choice in del17p patients.At Last, we collected primary murine lymphoma cells induced by shAlox15b-shTP53/shREN-shTP53 and blood sample from del17p/non-del17p CLL patients to verify our assumption. In mice samples, as AA accumulation was already observed in previous study, we only test pS6 level. And one sample from each experimental group (shAlox15b-shTP53) showed an increase in pS6 level. In human samples, we can see a slightly increase in AA in del17p patient. But due to individual variation, the result isn't statistical significant.Besides, we constructed a plasmid, namely eMLM-shAlox15b-shAlox5, with 2 shRNA targeting Alox5 and Alox15b gene at the same time, introduced the plasmid into primary murine preB cells and transplanted back to recipient mice. After verified by flow cytometry and pathological staining, survival analysis revealed the fact that silencing Alox5 and Alox15b gene at the same time can accelerate lymphoma formation, suggesting that Alox5 and Alox15b gene have a synergistic effect in lymphoma tumorigenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Oral infection with P. gingivalis exacerbates autoimmune encephalomyelitis.

Oral infection of mice with P. gingivalis induces periodontal inflammation and attachment loss. The aim of the present study was to investigate whether infection of mice with P. gingivalis, exacerbates the clinical course of experimental autoimmune encephalomyelitis (EAE)-a mouse model of multiple sclerosis (MS). Induction of EAE was carried out by immunization of C57BL/6 mice with myelin oligodentrocyte glycoprotein (MOG35-55 ). P. gingivalis infection was induced via subcutaneous chambers model and the oral gavage. The severity of EAE was measured using a clinical severity score. Ex-vivo reactivation of lymphocytes with the encephalitogenic peptide MOG35-55 was also tested. Subcutaneous as well as oral infection with live P. gingivalis led to significant aggravation of the severity of EAE. Lymph node cells harvested from mice with EAE following P. gingivalis infection showed augmented lymphocyte proliferation towards the encephlatigenic MOG moiety compared to mice with EAE only. The present results indicate that oral infection with P. gingivalis augmented the severity of EAE. This may stem from the systemic pro-inflammatory response triggered by P. gingivalis infection or via antigen mimicking. The present study provides evidence that periodontal infection may play a role as modifier in CNS inflammatory disorders, such as MS.

Identification of the Risk HLA-A Alleles and Autoantigen in Han Chinese Vitiligo Patients and the Association of CD8+T Cell Reactivity with Disease Characteristics.

BackgroundMultiple studies have implicated a role for CD8+T cell-mediated immune response to autoantigens in vitiligo. However, the antigen-specific T lymphocyte reactivity against the peptide epitopes is diverse among different world populations. This study aimed to identify the risk HLA-A allele in vitiligo and study CD8+ T cell reactivity to 5 autoantigenic peptides in Han Chinese populations, and to analyze the association of CD8+ T cell reactivity with disease characteristics.Material/MethodsThe risk HLA-A allele was analyzed by case-control study. Enzyme linked immunospot (ELISPOT) assay was used to compare T cell reactivity to the 5 autoantigenic peptides between vitiligo patients and healthy controls, then we analyzed the association of CD8+ T cell reactivity to 2 positive peptides with disease activity and area of skin lesions.ResultsThe results indicated that the most frequent allele in the Han Chinese vitiligo patients was the HLA-A*02: 01 allele with a significantly higher frequency compared to controls (20.20% versus 13.79%, P=6.64×10−5). The most frequently encountered epitopes were 2 gp100 modified peptides, IMDQVPFSV and YLEPGPVTV, whereas a weak T cell reactivity against tyrosinase and Melan-A/MART-1 were evaluated. Moreover, we demonstrated that T cell reactivity against the 2 positive peptides was significantly associated with disease characteristics including disease activity and area of skin lesions.ConclusionsOur findings showed that the HLA-A*02: 01 allele was the major risk HLA-A allele, and 2 gp100 modified peptides were identified as autoantigens and were found to be closely related to disease characteristics which might play a critical role in Han Chinese vitiligo patients.

The Effect of Long-Term X-Ray Exposure on Human Lymphocyte

The aim of the paper is to investigate effects of long term x-ray exposure on the human lymphocyte, reactive lymphocyte parameters and morphology of lymphocytes in x-ray technicians at Kirkuk hospitals. The study included 54 apparently healthy male x-ray technicians were matched with another 54 apparently healthy control to show any alteration in the lymphocytes, reactive lymphocytes and morphology. The investigated samples were divide into two groups depending on the work experience and working hours per day. The samples were tested for hematological parameters by complete blood cells count (CBC). The results showed that strong significant (P<0.0001) increasing was recorded for the reactive lymphocytes in all groups of the diagnostic technicians compared with their controls and significantly increasing of lymphocytes observed for some groups. It was concluded that chronic exposure of x-ray can vary lymphocyte and reactive lymphocyte parameters significantly and working hours per day have discernible effects on lymphocyte morphology.

Chemical Sympathectomy, but not Adrenergic Blockade, Improves Stroke Outcome

BackgroundA robust adrenergic response following stroke impairs lymphocyte function, which may prevent the development of autoimmune responses to brain antigens. We tested whether inhibition of the sympathetic response after stroke would increase the propensity for developing autoimmune responses to brain antigens. MethodsMale Lewis rats were treated with 6-hydroxydopamine (OHDA) prior to middle cerebral artery occlusion (MCAO), labetalol after MCAO, or appropriate controls. Behavior was assessed weekly and animals survived to 1 month at which time ELISPOT assays were done on lymphocytes from spleen and brain to determine the Th1 and Th17 responses to myelin basic protein (MBP), ovalbumin (OVA), and concanavalin A. A subset of animals was sacrificed 72 hours after MCAO for evaluation of infarct volume and lymphocyte responsiveness. Plasma C-reactive protein (CRP) was measured as a biomarker of systemic inflammation. ResultsDespite similar initial stroke severity and infarct volumes, 6-OHDA-treated animals lost less weight and experienced less hyperthermia after stroke. 6-OHDA-treated animals also had decreased CRP in circulation early after stroke and experienced better neurological outcomes at 1 month. The Th1 and Th17 responses to MBP did not differ among treatment groups at 1 month, but the Th1 response to OVA in spleen was more robust in labetalol and less robust in 6-OHDA-treated animals. ConclusionsChemical sympathectomy with 6-OHDA, but not treatment with labetalol, decreased systemic markers of inflammation early after stroke and improved long-term outcome. An increase in Th1 and Th17 responses to MBP was not seen with inhibition of the sympathetic response.

Mycosis fungoides: reflectance confocal microscopy and its role in the diagnosis

BackgroundMycosis fungoides (MF), a T-cell lymphoma, is the most common variant of primary cutaneous lymphomas. The discrete clinical aspect of the early patch-stage and an overlapping clinical presentation with other erythematosquamous skin diseases make the diagnosis of MF challenging. Histopathology is the gold standard for the diagnosis of MF. However, in early stages, MF can simulate inflammatory diseases, presenting reactive lymphocytes and absence of lymphocytic cytologic atypia. Consecutive biopsies are often necessary from several clinically suspect sites. Previous studies evaluated Reflectance Confocal Microscopy (RCM) as a promising tool for MF diagnosis because it offers the opportunity to analyze skin structures non-invasively at a ‘quasi-histopathologic’ resolution and to guide the most representative site for biopsy.Case presentationWe present a 76 years-old woman with a 5-year history of erythematous plaques and patches. She had had 5 previous skin biopsies with a pattern suggestive of nummular eczema. The history and the clinical presentation led to the suspicion of MF. RCM examination was performed and two lesions were marked for biopsy. The histopathology exam confirmed the diagnosis of MF. The patient was submitted to topical treatment with total remission of the lesions.ConclusionsThe mean time from onset of symptoms to diagnosis is 6 years and the delay in diagnosis results in delay for adequate therapy. Therefore, the selection of the biopsy site is crucial to avoid unrepresentative samples. Mancebo et al. recently published the largest cohort of cutaneous T.cell lymphoma examined with RCM. 83 lesions were evaluated, and the authors confirmed that this image technique was effective in to guide the most representative site for the biopsy. We believe that further and larger studies will clarify it and defining a diagnostic algorithm for the RCM diagnosis of MF.

Cytokine Networks and Survivin Peptide-Specific Cellular Immune Responses Predict Improved Survival in Patients With Glioblastoma Multiforme

PurposeWe investigated serum cytokine and T-cell responses directed against tumour-associated antigens (TAAs) in association with survival of patients with glioblastoma multiforme (GBM). Patients and MethodsPeripheral blood from 205 treatment-naïve patients with glioma (GBM = 145; non-GBM = 60) was obtained on the day of surgery to measure (i) circulating T-cells reacting to viral antigens and TAAs, in the presence or absence of cytokine conditioning with IL-2/IL-15/IL-21 or IL-2/IL-7, and (ii) serum cytokine levels (IL-4, IL-5, IL-6, TNF-α, IFN-γ and IL-17A). Patients were followed-up for at least 1000 days post-surgery. Survivin protein and gene expression in resected GBM tumour tissue were confirmed by immunohistochemistry and real-time polymerase chain reaction, respectively. Antigen-specific T-cell responses were gauged by ICS (intracellular cytokine production). Associations between patient survival and immunological reactivity patterns were analysed using univariate and multivariate statistics. ResultsApproximately 2% of patients with GBM and 18% of patients with non-GBM glioma, were alive beyond 1000 days of surgery. Univariate analysis indicated that the combination of three cytokines (IL-4/IL-5/IL-6, p = .0022; IFN-γ/TNF-α/IL-17A, p = .0083) but not a ‘partial’ combination of these cytokines, the IFN-γ immune response to EBV-EBNA-1 (p < .0001) as well as T-cell responses to the survivin97–111 peptide (p = .0152) correlated with longer survival among patients with GBM. Multivariate analysis identified survivin97–111-directed IFN-γ production with IL-2/IL-15/IL-21 conditioning (p = .024), and the combined presence of serum IFN-γ/TNF-α/IL-17a (p = .003) as independent predictors of survival. ConclusionSerum cytokine patterns and lymphocyte reactivity to survivin97–111, particularly with IL-2, IL-15 and IL-21 conditioning may be instrumental in predicting survival among patients with GBM. This has implications for clinical follow-up of patients with GBM and the targeted development of immunotherapy for patients with CNS tumours.

Consensus molecular subtypes (CMS), markers of systemic inflammation (SI) and clinicopathological parameters in colorectal cancer (CRC).

e15600Background: The CMS provide a representation of CRC heterogeneity. Upregulated immune pathways are suggested in CMS1 through lymphocyte (lymph) reactivity and CMS4 with an inflammatory respon...