We describe a case series of histoplasmosis caused by Histoplasma capsulatum var. duboisii during July 2011-January 2014 in Kimpese, Democratic Republic of the Congo. Cases were confirmed by ...histopathology, immunohistochemistry, and reverse transcription PCR. All patients were HIV negative. Putative sources for the pathogen were cellar bats and guano fertilizer exploitation.
Neutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections ...and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity.
We clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase-chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene.
All seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the child's ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of β1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis.
Defective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.).
The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This ...shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging.
In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3'-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions.
Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2-3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.
A novel small molecule named tuftsin-phosphorylcholine (TPC), which is linked to the biological activity of helminths, was constructed. The current study address the effect of TPC treatment in ...established collagen-induced arthritis (CIA) mice and propose TPC bi-functional activity. TPC treatment was initiated when clinical score was 2 to 4. Arthritis scores in TPC treated mice were lower compared to mice treated with vehicle (P < 0.001). Joint staining showed normal joint structure in TPC-treated mice compared to control groups treated with phosphate buffered saline (PBS), phosphorylcholine, or tuftsin, which exhibited severely inflamed joints. TPC enhanced anti-inflammatory response due to increased IL-10 secretion, and reduced pro-inflammatory cytokine secretion (IL-1-β, IL-6, TNF-αP < 0.001). Furthermore, TPC therapy increased expansion of CD4+CD25+FOXP3+T regulatory cells and IL-10+CD5+CD1d+B regulatory cells. We propose that the immunomodulatory activity of TPC can be a result of a bi-specific activity of TPC: (a) The tuftsin part of the TPC shifts RAW macrophage cells from pro-inflammatory macrophages M1 to anti-inflammatory M2-secreting IL-10 (P < 0.001) through neuropilin-1 and (b) TPC significantly reduce mouse TLR4 expression via NFkB pathway by HEKTM cells (P < 0.02) via the phosphorylcholine site of the molecule. Our results indicate that TPC, significantly ameliorated established CIA by its immunomodulatory activity. These data could lead to a novel self bi-functional small molecule for treating patients with progressive RA.
Background.
Cancers of unknown primary origin (CUP) constitute 3%–5% (50,000 to 70,000 cases) of all newly diagnosed cancers per year in the United States. Including cancers of uncertain primary ...origin, the total number increases to 12%–15% (180,000 to 220,000 cases) of all newly diagnosed cancers per year in the United States. Cancers of unknown/uncertain primary origins present major diagnostic and clinical challenges because the tumor tissue of origin is crucial for selecting optimal treatment. MicroRNAs are a family of noncoding, regulatory RNA genes involved in carcinogenesis. MicroRNAs that are highly stable in clinical samples and tissue specific serve as ideal biomarkers for cancer diagnosis. Our first‐generation assay identified the tumor of origin based on 48 microRNAs measured on a quantitative real‐time polymerase chain reaction platform and differentiated 25 tumor types.
Methods.
We present here the development and validation of a second‐generation assay that identifies 42 tumor types using a custom microarray. A combination of a binary decision‐tree and a k‐nearest‐neighbor classifier was developed to identify the tumor of origin based on the expression of 64 microRNAs.
Results.
Overall assay sensitivity (positive agreement), measured blindly on a validation set of 509 independent samples, was 85%. The sensitivity reached 90% for cases in which the assay reported a single answer (>80% of cases). A clinical validation study on 52 true CUP patients showed 88% concordance with the clinicopathological evaluation of the patients.
Conclusion.
The abilities of the assay to identify 42 tumor types with high accuracy and to maintain the same performance in samples from patients clinically diagnosed with CUP promise improved utility in the diagnosis of cancers of unknown/uncertain primary origins.
This article presents the development and validation of a second‐generation microRNA assay that identifies 42 tumor types with high accuracy, including for patients with cancers of unknown or uncertain primary origin.
A novel family of transient receptor potential (TRP) channels, that may hold a role in calcium homeostasis, has recently been described. By employing a GeneChip array analysis we have demonstrated a ...clear and specific upregulation of the TRP vanilloid 2 (TRPV2) mRNA in the left ventricles (LV) 3-5 days post-acute myocardial infarction (MI) compared to sham-operated controls, both in rats and in mice. We sought to characterize the cardiac cellular subpopulations in which TRPV2 is overexpressed upon acute MI.
Lewis rats underwent an acute MI by ligation of the left anterior descending artery or chest opening only (sham). The animals were terminated at various time points and an immunohistochemical (IHC) and immunofluorescent (IFC) staining of the LV sections as well as a flow cytometry analysis of LV-derived cells were carried out, using anti-TRPV2 and anti-monocyte/macrophage antibodies. Rat alveolar macrophage cells, NR8383, transiently transfected with TRPV2 siRNA were allowed to migrate towards hypoxic conditioned media of the rat cardiac myoblast line H9C2 using a trans-well migration assay. The macrophage cells migrating to the bottom side of the inserts were counted.
The IHC and IFC staining as well as the flow cytometry data demonstrated a substantial expression of TRPV2 in infiltrating macrophages in the peri-infarct region 3-5 days post-acute MI. The in vitro migration assay data demonstrated that following inhibition of the TRPV2 channel, the number of migrating macrophages towards conditioned medium of hypoxic cardiomyocytes was significantly reduced.
TRPV2 is highly expressed on the peri-infarct infiltrating macrophages and may play an important role in post-MI phagocytosis. Better characterization of this channel may pave the way for identifying a new target for modulating the dramatic post-MI immune reactions.
Background. Oncocytoma is one of the most common benign kidney tumors, accounting for 3–7% of all solid renal masses. Diagnosing oncocytomas using renal biopsy remains a controversy in the ...uro-pathologic community. With the increasing use of biopsies for assessment of renal lesions, reaching this pathologically benign diagnosis may prevent further surgical measures and have significant clinical benefit. Objective. To demonstrate our center’s results using renal biopsy to diagnose oncocytomas and to suggest that this diagnosis can be made with high success rates. Design, Setting, and Participants. From our center’s database, we retrospectively identified and retrieved all cases of oncocytoma diagnosed between the years 2011 and 2020 by renal biopsy. Medical records of those patients were then reviewed to view follow-up meetings and imaging of the lesions biopsied. Outcome Measurements and Statistical Analysis. In 21 biopsies performed on 19 patients, diagnosis was supported by subsequent follow-up averaging at 3.44 years per patient. Results and Limitations. The lesions exhibited benign behavior during follow-up after biopsy, consistent with the diagnosis of oncocytoma. Conclusions. Our study demonstrates that with good patient selection and proficient cooperation between urologists, radiologists and dedicated uro-pathologists, correctly diagnosing oncocytomas using RCB is a viable task. Patient Summary. Oncocytomas are benign lesions of the kidney. In our study, we reviewed all cases of oncocytomas pathologically diagnosed using renal biopsy from our center’s database. We found that in subsequent follow-up later to biopsy, the lesions displayed benign behavior consistent with oncocytoma. The use of percutaneous biopsies to reach this diagnosis could save patients more extensive surgeries.
In view of the relatively limited efficacy of immunotherapies targeting the PD-1–PD-L1 axis in triple-negative breast cancer (TNBC) and of published reports on tumor-promoting roles of TNFR2+ ...tumor-infiltrating lymphocytes (TNFR2+ TILs), we determined the incidence of TNFR2+ TILs in TNBC patient tumors, their association with disease outcome and relations with PD-1+ TILs. Using a cohort of treatment-naïve TNBC patients with long follow-up (
n
= 70), we determined the presence of TNFR2+ TILs and PD-1+ TILs by immunohistochemistry. TILs (≥ 1% of cellular mass) and TNFR2+ TILs (≥ 1% of total TILs) were detected in 96% and 74% of tumors, respectively. The presence of TILs at > 5% of tumor cell mass (“Positive TILs”), as well as of positive TNFR2+ TILs (> 5%), was independently associated with good prognosis, and combination of both parameters demonstrated superior outcome relative to their lower levels. PD1+ TILs (> 5/hot spot) were detected in 63% of patients. High levels of PD-1+ TILs (> 20/hot spot) showed an unfavorable disease outcome, and in their presence, the favorable outcome of positive TNFR2+ TILs was ablated. Thus, TNFR2+ TILs are strongly connected to improved prognosis in TNBC; these findings suggest that TNFR2+ TILs have favorable effects in TNBC patients, unlike the tumor-promoting roles attributed to them in other cancer systems. Overall, our observations propose that the TNFR2+ TIL subset should not be targeted in the course of TNBC therapy; rather, its beneficial impacts may become into power when anti-PD-1 regimens—that may potentiate immune activities—are administered to TNBC patients.
Pancreatic and duodenal homeobox gene-1 (PDX-1) regulates pancreas development during embryogenesis, whereas in the adult it controls β-cell function. Here we analyze whether PDX-1 functions as a ...pancreatic differentiation factor and a bona fide master regulator when ectopically expressed in mature fully differentiated liver in vivo. By ectopic and transient PDX-1 expression in liver in vivo, using the first generation recombinant adenoviruses, we demonstrate that PDX-1 induces in liver a wide repertoire of both exocrine and endocrine pancreatic gene expression. Moreover, PDX-1 induces its own expression (auto-induction), which in turn may explain the long lasting nature of the “liver to pancreas” transdifferentiation. Insulin as well glucagon-producing cells are mainly located in the proximity of hepatic central veins, possibly allowing direct hormone release into the bloodstream, without affecting normal hepatic function. Importantly, we demonstrate that hepatic insulin production triggered by Ad-CMV-PDX-1 recombinant adenovirus administration is functional and prevents streptozotocin-induced hyperglycemia in Balb/c mice even 8 months after the initial treatment. We conclude that PDX-1 plays an important instructive role in pancreas differentiation, not only from primitive gut endoderm but also from mature liver. Transconversion of liver to pancreas may serve as a novel approach for generating endocrine-pancreatic tissue that can replace malfunctioning β-cells in diabetics.
BRAF becomes constitutively activated in 50% to 70% of melanoma cases. CEACAM1 has a dual role in melanoma, including facilitation of cell proliferation and suppression of infiltrating lymphocytes, ...which are consistent with its value as a marker for poor prognosis in melanoma patients. Here we show that BRAFV600E melanoma cells treated with BRAF and MEK inhibitors (MAPKi) downregulate CEACAM1 mRNA and protein expression in a dose- and exposure time–dependent manners. Indeed, there is a significant correlation between the presence of BRAFV600E and CEACAM1 expression in melanoma specimens obtained from 45 patients. Vemurafenib-resistant cell systems reactivate the MAPK pathway and restore basal CEACAM1 mRNA and protein levels. These combined results suggest transcriptional regulation. Indeed, luciferase reporting assays show that CEACAM1 promoter (CEACAM1p) activity is significantly reduced by MAPKi. Importantly, we show that the MAPK-driven CEACAM1p activity is mediated by ETS1, a major transcription factor and downstream effector of the MAPK pathway. Phosphorylation mutant ETS1T38A shows a dominant negative effect over CEACAM1 expression. The data are consistent with independent RNAseq data from serial biopsies of melanoma patients treated with BRAF inhibitors, which demonstrate similar CEACAM1 downregulation. Finally, we show that CEACAM1 downregulation by MAPKi renders the cells more sensitive to T-cell activation. These results provide a new view on a potential immunological mechanism of action of MAPKi in melanoma, as well as on the aggressive phenotype observed in drug-resistant cells.
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