BackgroundChildhood represents an immunological window of vulnerability in which individuals are at increased risk for both serious infections and development of allergic diseases, particularly ...affecting the airways. However, little is known about how the airway mucosal immune system is organised and functions during early age. Here, the organisation of immune cells in bronchial mucosa of children was characterised.MethodsImmunophenotyping was performed on mucosal samples obtained postmortem from nine children aged 2–15 years without any history of atopic manifestations or any signs of respiratory disease, who died from non-inflammatory causes.ResultsIn all nine cases, isolated lymphoid follicles (ILFs), interpreted as bronchus-associated lymphoid tissue (BALT), were found, constituting an average frequency of 60 ILFs/cm2 of airway mucosal surface. Outside these ILFs, dense networks of CD11c+ myeloid dendritic cells (DCs), CD68+ macrophages and CD3+CD45RA− memory T cells were found. Plasmacytoid DCs occurred in low numbers. Importantly, intraepithelial antigen-presenting cells were found to extend cellular projections into the airway lumen.ConclusionThe density and location of antigen-presenting cells and T cells in this age group are similar to those observed in adults. However, in contrast to adults, BALT appears to be a normal feature of the airway mucosa throughout childhood, suggesting that these structures contribute to regional immunity and homeostasis. This indicates that the local immune system in the airways of children has unique features which should be taken into account, not only when studying airway immunology and immunopathology, but also in the development of mucosal vaccines.
Isolated para-aortic lymph node metastases are rare in patients with endometrial carcinoma. We wanted to determine the reliability of macroscopic pelvic lymph node findings at surgery in predicting ...para-aortic space involvement in these patients.
We identified all women with surgically treated endometrial carcinoma at our institution between January 2008 and February 2013 (n = 854). One hundred seventeen patients received pelvic-aortic lymphadenectomy. Lymph nodes were considered grossly positive based on size and morphology.
In patients who underwent comprehensive lymphadenectomy, grossly positive pelvic nodes predicted para-aortic metastasis with a sensitivity of 52.4% and specificity of 93.8%. The positive and negative likelihood ratios were 8.4 and 0.51, respectively. The predictive power of grossly positive pelvic nodes remained significant (odds ratio, 18; 95% confidence interval, 4.1-78; P < 0.0001) after correcting for deep myometrial invasion, poor tumor differentiation, and nonendometrioid histology as confounders. The whole sample of 854 patients was used for Bayesian calculations. The cutoff for a clinically useful test was set at the negative predictive value of 98.0%. The negative predictive value of the test (ie, grossly positive pelvic nodes at surgery in predicting the likelihood of para-aortic metastasis) was 99.7% for patients with superficial grade 1 to 2 endometrioid carcinomas and 98.0% for patients with deeply invasive grade 1 to 2 endometrioid carcinomas. For patients with grade 3 endometrioid and nonendometrioid carcinomas, the negative predictive values were 97.3% and 92.2%, respectively. For the whole study population, the value was 98.4%.
When uterine factors are used for risk stratification of endometrial carcinomas, selective para-aortic lymphadenectomy, based on gross findings of pelvic nodes, is feasible for patients with grade 1 to 2 endometrioid carcinomas, regardless of the depth of myometrial invasion. Similarly, gross findings of pelvic nodes can be used to evaluate the need for para-aortic lymphadenectomy in the strategy of routine pelvic lymphadenectomy.
Human fibroblasts and HT-1080 fibrosarcoma cells express membrane-type-1 matrix metalloproteinase (MT1-MMP), the cell surface activator of gelatinase A, in separate forms of 63 kDa, 60 kDa and in ...some cases 43 kDa. In the present work the interrelationships between MT1-MMP processing and gelatinase A activation were analysed using HT-1080 fibrosarcoma cells as a model. It was found that MT1-MMP was synthesized as a 63 kDa protein, which was constitutively processed to a 60 kDa active enzyme with N-terminal Tyr112, as shown by immunoprecipitation, immunoblotting and sequence analyses. Co-immunoprecipitation results indicated that only the active 60 kDa form of MT1-MMP bound gelatinase A at the cell surface. Both the activation of pro-MT1-MMP and the membrane binding of the tissue inhibitor of metalloproteinases type 2 (TIMP-2) and gelatinase A, and subsequent activation of gelatinase A, were inhibited by calcium ionophores. Although the active MT1-MMP was required for cell surface binding and activation of gelatinase A, it was inefficient in activating gelatinase A in fibroblasts or in control HT-1080 cells alone. Low expression levels of TIMP-2 and rapid synthesis of MT1-MMP were found to be critical for gelatinase A activation. In HT-1080 cells, MT1-MMP was further processed to an inactive, 43 kDa cell surface form when overexpressed, or when the cells were treated with PMA. Under these conditions, the activated gelatinase A was detected in the culture medium, in cell membrane extracts and in MT1-MMP-containing complexes. These results indicate that proteolytic processing (activation and degradation/inactivation) of MT1-MMP and MT1-MMP/TIMP-2 relationships at the cell surface are important regulatory levels in the control of gelatinolytic activity.
Epilysin (MMP-28) is the newest member of the matrix metalloproteinase enzyme family. Several members of this enzyme family have been associated with various aspects of wound repair and cancer ...invasion. The aim of this study was to characterize in different types of wounds, skin cancers, and keratinocyte cultures factors that contribute to epilysin expression in vivo, as well as how and where it is induced in relation to other matrix metalloproteinases. Our results indicate that epilysin is produced by the mitotic Ki-67-positive keratinocytes distal from the wound edge in both acute and chronic wounds and that it does not generally colocalize with collagenase-1, stromelysin-2, or 92 kDa gelatinase in migrating keratinocytes. An injury of epidermis was needed for epilysin induction as it was upregulated in ulcerated pyogenic granulomas and in suction blisters but was not detected in intact acanthotic or normal skin. Unlike many other matrix metalloproteinases, epilysin was not detected in the invading cancer cell nests of sclerosing basal or squamous cell cancers of various grades. When primary keratinocytes were stimulated with tumor necrosis factor α, upregulation of epilysin mRNA was evident within 24–48 h as measured by quantitative reverse transcription polymerase chain reaction. In primary keratinocyte, HaCaT, and A431 carcinoma cell cultures none of the 10 other growth factors or extracellular matrices studied were able to upregulate epilysin expression. Our results suggest that epilysin expression is tightly spatially and temporally regulated during wound repair. Although the in vivo substrates of epilysin are not known at present, its expression pattern suggests that it may be needed to restructure the basement membrane or to degrade adhesive proteins between keratinocytes to supply new cells for the migrating front.
Membrane type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteinase and a cell-surface receptor and activator of gelatinase A in normal and neoplastic cells. We have expressed and ...purified a soluble deletion mutant of MT1-MMP lacking the transmembrane and cytoplasmic domains and an inactive mutant of the soluble MT1-MMP, where the active-site glutamic acid240 was substituted by alanine (E240A). A baculovirus transfer vector coding for amino acids 21–539 of MT1-MMP (ΔTM) and a similar vector coding for the mutation (E240AΔTM) were constructed for expression in insect cells. Both ΔTM and E240AΔTM were secreted to the culture medium of infected High Five insect cells. They were then purified by cation-exchange followed by gel-filtration chromatography. ΔTM was able to cleave denatured type I collagen and fibronectin and activate MMP-2/gelatinase-A, while E240AΔTM had only low proteolytic activity against denatured collagen I. The current expression and purification protocol should prove useful for the production of large amounts of enzymatically active soluble MT1-MMP.
Mutations in several proteins functioning as endolysosomal components cause monogenic autoimmune diseases, of which pathogenesis is linked to increased endoplasmic reticulum stress, inefficient ...autophagy, and defective recycling of immune receptors. We report here a heterozygous
p.G307D missense mutation, detected by whole-exome sequencing, in two related patients presenting with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. The index patient suffered from recurrent respiratory tract infections and oligoarthritis since early teens, and later developed persistent low-copy EBV-viremia, as well as an antibody deficiency. Her infant son developed hypogammaglobulinemia, autoimmune enteropathy, interstitial lung disease, profound growth failure, and treatment-resistant psoriasis vulgaris. Consistent with previous knowledge on TOM1 protein function, we detected impaired autophagy and enhanced susceptibility to apoptosis in patient-derived cells. In addition, we noted diminished STAT and ERK1/2 signaling in patient fibroblasts, as well as poor IFN-γ and IL-17 secretion in T cells. The mutant TOM1 failed to interact with TOLLIP, a protein required for IL-1 recycling, PAMP signaling and autophagosome maturation, further strengthening the link between the candidate mutation and patient pathophysiology. In sum, we report here an identification of a novel gene,
, associating with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. Other patient cases from unrelated families are needed to firmly establish a causal relationship between the genotype and the phenotype.
ABSTRACT
Objectives:
Transcription factor GATA‐4 is expressed in early fetal liver and essential for organogenesis. It is also implicated in carcinogenesis in several endoderm‐derived organs. ...Hepatoblastoma (HB), the most common malignant pediatric liver tumor, has features of fetal liver including extramedullary hematopoiesis. We investigated the expression of GATA‐4 and its purported target gene erythropoietin (Epo) in liver tumors and the role of GATA‐4 in HB pathogenesis.
Patients and Methods:
Immunohistochemistry, Western blotting, and reverse transcription‐polymerase chain reaction were used for liver samples from patients with HB or hepatocellular carcinoma. To further investigate the role of GATA‐4 in pediatric liver tumors, we used adenoviral transfections of wild‐type or dominant negative GATA‐4 constructs in the human HB cell line, HUH6.
Results:
We found abundant GATA‐4 expression in both types of liver tumors in children, whereas it was absent in adult hepatocellular carcinoma. A close family member GATA‐6 was expressed in a minority of childhood but not adult liver tumors. Epo, present in the fetal liver, was also expressed in childhood liver tumors. Moreover, cell line HUH6 was GATA‐4 positive and produced Epo. We found that altering the amount of functional GATA‐4 in HUH6 cells did not significantly affect either proliferation or apoptosis.
Conclusions:
GATA‐4 is abundant in pediatric liver tumors, but unraveling its exact role in these neoplasms requires further investigation.
PDF
