Septic mucormycosis in acute t-cell leucemia – pathogen detection in skin lesions

We report on a 43-year old patient with an acute T-Cell Leucemia, currently in Aplasia after Chemotherapy, showing five targetoid bluish skin lesions. Due to a three weeks history of septic symptoms he was under treatment with antibiotics and antifungals. Multiple septic foci were localized (N. caudatus, liver, kidneys, lung, spine and right psoas). Microbiology analyses of various blood cultures and of the aspirate of the psoas abscess showed initially negative results. Clinically the skin lesions were suspected to be of septic or thrombogenic origin. A 5 mm punch biopsy was performed and separated for microbiological diagnostic and conventional histology. Surprisingly large fungal agents in mostly intravascular distribution were seen histologically and identified as Lichtheimia corymbifera (syn. Absidia corymbifera) by PCR. Cultures remained negative. The patient died on the following day. Lichtheimia corymbifera is a fungus belonging to the family of mucormycosis. Aspergillosis and mucormycosis are the most common mold infections in patients with hematological malignancies, clinically often indistinguishable. However, the true incidence of mucormycosis is not known and probably underestimated because of difficulties in diagnosis. Mucormycosis typically causes acute, aggressive, and frequently angioinvasive infections presenting with solitary local skin necrosis. The fact that the pathogenic fungus was isolated from a very discrete skin lesion but was not detected in blood cultures, and only later in the PCR of the aspirate of the psoas abscess, makes this case exceptional.

Pathophysiology of Hidradenitis suppurativa

Only limited data are available about the precise mechanism leading to tissue inflammation and damage in patients with hidradenits suppurativa (HS). The central pathogenetic event in HS is the occlusion of the upper parts of the hair follicle leading to a perifollicular lympho-histiocytic inflammation. In early lesions, neutrophilic abscess formation and influx of mainly macrophages, monocytes and dendritic cells predominate. In chronic disease, the infiltrate expand with increased frequencies of B cells and plasma cells. In the inflammatory infiltrates toll like receptor 2 (TLR2) was highly expressed by infiltrating macrophages and dendritic cells indicating that stimulation of inflammatory cells by TLR2 activating microbial products may be important trigger factors in the chronic inflammatory process. Furthermore, the pro inflammatory cytokines IL-12 and IL-23 are abundantly expressed by macrophages infiltrating papillary and reticular dermis of HS skin. Both of these cytokines are believed to be important mediators in autoimmune tissue destruction and its blocking by biologics has been shown to be effective in the treatment of psoriasis. Especially IL-23 has been shown to be involved in the induction of a T helper cell subset producing IL-17, therefore, named Th17, which is distinct from the classical Th1/Th2 subsets. In chronic HS lesions IL-17-producing T helper cells were found to infiltrate the dermis. An overexpression of various other cytokines like IL-1beta, CYCL9 (MIG), IL-10 , IL-11 and BLC has been described in HS lesion whereas IL-20 and IL-22 have been shown to be down regulated. Similar to psoriasis also in HS the antimicrobial peptides beta defensin 2 and psoriasin are highly upregulated. This may at least in part explain the clinical finding that HS patients suffer only rarely from skin infections. Taken together the inflammatory reaction leading to HS are only poorly understood, but they show many similarity with other inflammatory reactions as e.g. in psoriasis.

Janus kinases 1 and 2 regulate chemokine-mediated integrin activation and naïve T-cell homing.

Janus kinases (JAKs) are central signaling molecules in cytokine receptor cascades. Although they have also been implicated in chemokine receptor signaling, this function continues to be debated. To address this issue, we established a nucleofection model in primary, nonactivated mouse T lymphocytes to silence JAK expression and to evaluate the ability of these cells to home to lymph nodes. Reduced JAK1 and JAK2 expression impaired naïve T-cell migration in response to gradients of the chemokines CXCL12 and CCL21. In vivo homing of JAK1/JAK2-deficient cells to lymph nodes decreased, whereas intranodal localization and motility were unaffected. JAK1 and JAK2 defects altered CXCL12- and CCL21-triggered ezrin/radixin/moesin (ERM) dephosphorylation and F-actin polymerization, as well as activation of lymphocyte function-associated Ag-1 and very late Ag-4 integrins. As a result, the cells did not adhere firmly to integrin substrates in response to these chemokines. The results demonstrate that JAK1/JAK2 participate in chemokine-induced integrin activation and might be considered a target for modulation of immune cell extravasation and therefore, control of inflammatory reactions.

CD8/CD45RO T-cell infiltration in endoscopic biopsies of colorectal cancer predicts nodal metastasis and survival

BACKGROUND AND AIMS: Reliable prognostic markers based on biopsy specimens of colorectal cancer (CRC) are currently missing. We hypothesize that assessment of T-cell infiltration in biopsies of CRC may predict patient survival and TNM-stage before surgery. METHODS: Pre-operative biopsies and matched resection specimens from 130 CRC patients treated from 2002-2011 were included in this study. Whole tissue sections of biopsy material and primary tumors were immunostained for pancytokeratin and CD8 or CD45RO. Stromal (s) and intraepithelial (i) T-cell infiltrates were analyzed for prediction of patient survival as well as clinical and pathological TNM-stage of the primary tumor. RESULTS: CD8 T-cell infiltration in the preoperative biopsy was significantly associated with favorable overall survival (CD8i p = 0.0026; CD8s p = 0.0053) in patients with primary CRC independently of TNM-stage and postoperative therapy (HR [CD8i] = 0.55 (95% CI: 0.36-0.82), p = 0.0038; HR [CD8s] = 0.72 (95% CI: 0.57-0.9), p = 0.0049). High numbers of CD8i in the biopsy predicted earlier pT-stage (p < 0.0001) as well as absence of nodal metastasis (p = 0.0015), tumor deposits (p = 0.0117), lymphatic (p = 0.008) and venous invasion (p = 0.0433) in the primary tumor. Infiltration by CD45ROs cells was independently associated with longer survival (HR = 0.76 (95% CI: 0.61-0.96), p = 0.0231) and predicted absence of venous invasion (p = 0.0025). CD8 counts were positively correlated between biopsies and the primary tumor (r = 0.42; p < 0.0001) and were reproducible between observers (ICC [CD8i] = 0.95, ICC [CD8s] = 0.75). For CD45RO, reproducibility was poor to moderate (ICC [CD45i] = 0.16, ICC [CD45s] = 0.49) and correlation with immune infiltration in the primary tumor was fair and non-significant (r[CD45s] = 0.16; p = 0.2864). For both markers, no significant relationship was observed with radiographic T-stage, N-stage or M-stage, indicating that assessment of T-cells in biopsy material can add additional information to clinical staging in the pre-operative setting. CONCLUSIONS: T-cell infiltration in pre-operative biopsy specimens of CRC is an independent favorable prognostic factor and strongly correlates with absence of nodal metastasis in the resection specimen. Quantification of CD8i is highly reproducible and allows superior prediction of clinicopathological features as compared to CD45RO. The assessment of CD8i infiltration in biopsies is recommended for prospective investigation.

Correlation between altered central pain processing and concentration of peritoneal fluid inflammatory cytokines in endometriosis patients with chronic pelvic pain

Translational research has not yet elucidated whether alterations in central pain processes are related to peripheral inflammatory processes in chronic pain patients. We tested the hypothesis that the concentration of cytokines in the peritoneal fluid of endometriosis patients with chronic pain correlate with parameters of hyperexcitability of the nociceptive system. The concentrations of 15 peritoneal fluid cytokines were measured in 11 patients with chronic pelvic pain and a diagnosis of endometriosis. Six parameters assessing central pain processes were recorded. Positive correlations between concentration of some cytokines in the peritoneal fluid and amplification of central pain processing were found. The results suggest that inflammatory mechanisms may be important in the pathophysiology of altered central pain processes and that cytokines produced in the environment of endometriosis could act as mediators between the peripheral lesion and changes in central nociceptive processes.

BPAG1a and b Associate with EB1 and EB3 and Modulate Vesicular Transport, Golgi Apparatus Structure, and Cell Migration in C2.7 Myoblasts.

BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5' end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3' end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts.

Transcriptional profiles of cytokine/chemokine factors of immune cell-homing to the parasitic lesions: a comprehensive one-year course study in the liver of E. multilocularis-infected mice.

Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL-12α, IFN-γ and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-β. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.

Combination of expression levels of miR-21 and miR-126 is associated with cancer-specific survival in clear-cell renal cell carcinoma

BACKGROUND Renal cell carcinoma (RCC) is marked by high mortality rate. To date, no robust risk stratification by clinical or molecular prognosticators of cancer-specific survival (CSS) has been established for early stages. Transcriptional profiling of small non-coding RNA gene products (miRNAs) seems promising for prognostic stratification. The expression of miR-21 and miR-126 was analysed in a large cohort of RCC patients; a combined risk score (CRS)-model was constructed based on expression levels of both miRNAs. METHODS Expression of miR-21 and miR-126 was evaluated by qRT-PCR in tumour and adjacent non-neoplastic tissue in n = 139 clear cell RCC patients. Relation of miR-21 and miR-126 expression with various clinical parameters was assessed. Parameters were analysed by uni- and multivariate COX regression. A factor derived from the z-score resulting from the COX model was determined for both miRs separately and a combined risk score (CRS) was calculated multiplying the relative expression of miR-21 and miR-126 by this factor. The best fitting COX model was selected by relative goodness-of-fit with the Akaike information criterion (AIC). RESULTS RCC with and without miR-21 up- and miR-126 downregulation differed significantly in synchronous metastatic status and CSS. Upregulation of miR-21 and downregulation of miR-126 were independently prognostic. A combined risk score (CRS) based on the expression of both miRs showed high sensitivity and specificity in predicting CSS and prediction was independent from any other clinico-pathological parameter. Association of CRS with CSS was successfully validated in a testing cohort containing patients with high and low risk for progressive disease. CONCLUSIONS A combined expression level of miR-21 and miR-126 accurately predicted CSS in two independent RCC cohorts and seems feasible for clinical application in assessing prognosis.

Cisplatin-resistant cells in malignant pleural mesothelioma cell lines show ALDH(high)CD44(+) phenotype and sphere-forming capacity

BACKGROUND Conventional chemotherapy in malignant pleural mesothelioma (MPM) has minimal impact on patient survival due to the supposed chemoresistance of cancer stem cells (CSCs). We sought to identify a sub-population of chemoresistant cells by using putative CSC markers, aldehyde dehydrogenase (ALDH) and CD44 in three MPM cell lines; H28, H2052 and Meso4. METHODS The Aldefluor assay was used to measure ALDH activity and sort ALDH(high) and ALDH(low) cells. Drug-resistance was evaluated by cell viability, anchorage-independent sphere formation, flow-cytometry and qRT-PCR analyses. RESULTS The ALDH(high) - and ALDH(low) -sorted fractions were able to demonstrate phenotypic heterogeneity and generate spheres, the latter being less efficient, and both showed an association with CD44. Cis- diamminedichloroplatinum (II) (cisplatin) treatment failed to reduce ALDH activity and conferred only a short-term inhibition of sphere generation in both ALDH(high) and ALDH(low) fractions of the three MPM cell lines. Induction of drug sensitivity by an ALDH inhibitor, diethylaminobenzaldehyde (DEAB) resulted in significant reductions in cell viability but not a complete elimination of the sphere-forming cells, suggestive of the presence of a drug-resistant subpopulation. At the transcript level, the cisplatin + DEAB-resistant cells showed upregulated mRNA expression levels for ALDH1A2, ALDH1A3 isozymes and CD44 indicating the involvement of these markers in conferring chemoresistance in both ALDH(high) and ALDH(low) fractions of the three MPM cell lines. CONCLUSIONS Our study shows that ALDH(high) CD44(+) cells are implicated in conveying tolerance to cisplatin in the three MPM cell lines. The combined use of CD44 and ALDH widens the window for identification and targeting of a drug-resistant population which may improve the current treatment modalities in mesothelioma.

The secretome of endothelial progenitor cells promotes brain endothelial cell activity through PI3-kinase and MAP-kinase

BACKGROUND Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. METHODS Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. RESULTS Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. CONCLUSION The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects.

Definitive intensity modulated radiotherapy in locally advanced hypopharygeal and laryngeal squamous cell carcinoma: mature treatment results and patterns of locoregional failure.

PurposeTo assess clinical outcomes and patterns of loco-regional failure (LRF) in relation to clinical target volumes (CTV) in patients with locally advanced hypopharyngeal and laryngeal squamous cell carcinoma (HL-SCC) treated with definitive intensity modulated radiotherapy (IMRT) and concurrent systemic therapy.MethodsData from HL-SCC patients treated from 2007 to 2010 were retrospectively evaluated. Primary endpoint was loco-regional control (LRC). Secondary endpoints included local (LC) and regional (RC) controls, distant metastasis free survival (DMFS), laryngectomy free survival (LFS), overall survival (OS), and acute and late toxicities. Time-to-event endpoints were estimated using Kaplan-Meier method, and univariate and multivariate analyses were performed using Cox proportional hazards models. Recurrent gross tumor volume (RTV) on post-treatment diagnostic imaging was analyzed in relation to corresponding CTV (in-volume, > 95% of RTV inside CTV; marginal, 20¿95% inside CTV; out-volume, < 20% inside CTV).ResultsFifty patients (stage III: 14, IVa: 33, IVb: 3) completed treatment and were included in the analysis (median follow-up of 4.2 years). Three-year LRC, DMFS and overall survival (OS) were 77%, 96% and 63%, respectively. Grade 2 and 3 acute toxicity were 38% and 62%, respectively; grade 2 and 3 late toxicity were 23% and 15%, respectively. We identified 10 patients with LRF (8 local, 1 regional, 1 local¿+¿regional). Six out of 10 RTVs were fully included in both elective and high-dose CTVs, and 4 RTVs were marginal to the high-dose CTVs.ConclusionThe treatment of locally advanced HL-SCC with definitive IMRT and concurrent systemic therapy provides good LRC rates with acceptable toxicity profile. Nevertheless, the analysis of LRFs in relation to CTVs showed in-volume relapses to be the major mode of recurrence indicating that novel strategies to overcome radioresistance are required.

CstF64: cell cycle regulation and functional role in 3' end processing of replication-dependent histone mRNAs.

The 3' end processing of animal replication-dependent histone mRNAs is activated during G1/S-phase transition. The processing site is recognized by stem-loop binding protein and the U7 snRNP, but cleavage additionally requires a heat-labile factor (HLF), composed of cleavage/polyadenylation specificity factor, symplekin, and cleavage stimulation factor 64 (CstF64). Although HLF has been shown to be cell cycle regulated, the mechanism of this regulation is unknown. Here we show that levels of CstF64 increase toward the S phase and its depletion affects histone RNA processing, S-phase progression, and cell proliferation. Moreover, analyses of the interactions between CstF64, symplekin, and the U7 snRNP-associated proteins FLASH and Lsm11 indicate that CstF64 is important for recruiting HLF to histone precursor mRNA (pre-mRNA)-resident proteins. Thus, CstF64 is central to the function of HLF and appears to be at least partly responsible for its cell cycle regulation. Additionally, we show that misprocessed histone transcripts generated upon CstF64 depletion mainly accumulate in the nucleus, where they are targets of the exosome machinery, while a small cytoplasmic fraction is partly associated with polysomes.

A naturally occurring prfA truncation in a Listeria monocytogenes field strain contributes to reduced replication and cell-to-cell spread

Listeria (L.) monocytogenes is an environmental bacterium that may become an intracellular pathogen upon ingestion to cause gastroenteritis, septicaemia, abortions, and/or fatal infections of the central nervous system. We here describe a L. monocytogenes field strain (JF5171) isolated from a bovine placenta in the context of abortion, which exhibited attenuation in bovine brain-slice cultures. The whole genome of strain JF5171 was sequenced, and the invasion, replication, and intercellular spread of JF5171 were further analyzed by quantification of colony forming units and immunofluorescence studies. Phospholipase and hemolysis activity of JF5171 were also quantified along with transcription levels of actA, hly and prfA. The data obtained were compared to those of the widely used L. monocytogenes reference strain, EGD-e. JF5171 exhibited reduced replication and lower levels of phospholipase and hemolysis activity. Invasion and cell-to-cell spread was strongly decreased compared to EGD-e, and actin polymerization was absent. A frame shift deletion was identified in the JF5171 coding region of the major regulator for virulence, prfA. This resulted in a truncated C-terminus sequence (WEN* vs. WGKLN*). In addition, a point mutation resulted in a lysine to arginine substitution at amino acid position 197. Complementation with prfA from EGD-e and with (EGD-e) prfA-K197N increased the replication and spread efficiency of JF5171. In contrast, complementation with the truncated version of prfA had no effect. Taken together, these results suggest that the truncated C-terminus of prfA considerably contributes to the strongly attenuated phenotype observed in vitro.

Genome-Wide Analyses Suggest Mechanisms Involving Early B-Cell Development in Canine IgA Deficiency.

Immunoglobulin A deficiency (IgAD) is the most common primary immune deficiency disorder in both humans and dogs, characterized by recurrent mucosal tract infections and a predisposition for allergic and other immune mediated diseases. In several dog breeds, low IgA levels have been observed at a high frequency and with a clinical resemblance to human IgAD. In this study, we used genome-wide association studies (GWAS) to identify genomic regions associated with low IgA levels in dogs as a comparative model for human IgAD. We used a novel percentile groups-approach to establish breed-specific cut-offs and to perform analyses in a close to continuous manner. GWAS performed in four breeds prone to low IgA levels (German shepherd, Golden retriever, Labrador retriever and Shar-Pei) identified 35 genomic loci suggestively associated (p <0.0005) to IgA levels. In German shepherd, three genomic regions (candidate genes include KIRREL3 and SERPINA9) were genome-wide significantly associated (p <0.0002) with IgA levels. A ~20kb long haplotype on CFA28, significantly associated (p = 0.0005) to IgA levels in Shar-Pei, was positioned within the first intron of the gene SLIT1. Both KIRREL3 and SLIT1 are highly expressed in the central nervous system and in bone marrow and are potentially important during B-cell development. SERPINA9 expression is restricted to B-cells and peaks at the time-point when B-cells proliferate into antibody-producing plasma cells. The suggestively associated regions were enriched for genes in Gene Ontology gene sets involving inflammation and early immune cell development.

Loss of tapasin correlates with diminished CD8(+) T-cell immunity and prognosis in colorectal cancer.

BACKGROUND Tapasin is a crucial component of the major histocompatibility (MHC) class I antigen presentation pathway. Defects in this pathway can lead to tumor immune evasion. The aim of this study was to test whether tapasin expression correlates with CD8(+) cytotoxic T lymphocyte (CTL) infiltration of colorectal cancer (CRC) and overall survival. METHODS A next-generation tissue microarray (ngTMA) of 198 CRC patients with full clinicopathological information was included in this study. TMA slides were immunostained for tapasin, MHC I and CD8. Marker expression was analyzed with immune-cell infiltration, patient survival and TNM-staging. RESULTS A reduction of tapasin expression strongly correlated with venous invasion (AUC 0.682, OR 2.7, p = 0.002; 95% CI 1.7-5.0), lymphatic invasion (AUC 0.620, OR 2.0, p = 0.005; 95 % CI 1.3-3.3), distant metastasis (AUC 0.727, OR 2.9, p = 0.004; 95% CI 1.4-5.9) and an infiltrative tumor border configuration (AUC 0.621, OR 2.2, p = 0.017; 95% CI 1.2-4.4). Further, tapasin expression was associated with CD8(+) CTL infiltration (AUC 0.729, OR 5.4, p < 0.001; 95% CI 2.6-11), and favorable overall survival (p = 0.004, HR 0.6, 95% CI 0.42-0.85). CONCLUSIONS Consistent with published functional data showing that tapasin promotes antigen presentation, as well as tumor immune recognition and destruction by CD8(+) CTLs, a reduction in tapasin expression is associated with tumor progression in CRC.