Shift Towards Older Bariatric Patients

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Role of intestinal Hsp70 in barrier maintenance: contribution of milk to the induction of Hsp70.2

Abstract

Background

Necrotizing enterocolitis (NEC) is a gastrointestinal disease of complex etiology resulting in devastating systemic inflammation and often death in premature newborns. We previously demonstrated that formula feeding inhibits ileal expression of heat shock protein-70 (Hsp70), a critical stress protein within the intestine. Barrier function for the premature intestine is critical. We sought to determine whether reduced Hsp70 protein expression increases neonatal intestinal permeability.

Methods

Young adult mouse colon cells (YAMC) were utilized to evaluate barrier function as well as intestine from Hsp70−/− pups (KO). Sections of intestine were analyzed by Western blot, immunohistochemistry, and real time PCR. YAMC cells were sub-lethally heated or treated with expressed milk (EM) to induce Hsp70.

Results

Immunostaining demonstrates co-localized Hsp70 and tight junction protein zona occludens-1 (ZO-1), suggesting physical interaction to protect tight junction function. The permeability of YAMC monolayers increases following oxidant injury and is partially blocked by Hsp70 induction either by prior heat stress or EM. RT-PCR analysis demonstrated that the Hsp70 isoforms, 70.1 and 70.3, predominate in WT pup; however, Hsp70.2 predominates in the KO pups. While Hsp70 is present in WT milk, it is not present in KO EM. Hsp70 associates with ZO-1 to maintain epithelial barrier function.

Conclusion

Both induction of Hsp70 and exposure to EM prevent stress-induced increased permeability. Hsp70.2 is present in both WT and KO neonatal intestine, suggesting a crucial role in epithelial integrity. Induction of the Hsp70.2 isoform appears to be mediated by mother's milk. These results suggest that mother's milk feeding modulates Hsp70.2 expression and could attenuate injury leading to NEC.

Level of evidence

Level III.

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3C-PCR: a novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants

Abstract

Recent advances in molecular cytogenetics highlight the importance of noncoding structural variation in human disease. Genomic rearrangements can disrupt chromatin architecture, leading to long-range alterations in gene expression. With increasing ability to assess distal gene dysregulation comes new challenges in clinical interpretation of rearrangements. While haplotyping methods to determine compound heterozygosity in a single gene with two pathogenic variants are established, such methods are insufficient for phasing larger distances between a pathogenic variant and a genomic rearrangement breakpoint. Herein, we present an inexpensive and efficient proximity ligation-based method called 3C-PCR for phasing chromosomal rearrangement breakpoints with distal allelic variants. 3C-PCR uses canonical chromosome conformation capture (3C) libraries for targeted distal phasing by implementing a novel nested PCR strategy with primers anchored across the rearrangement breakpoints and subsequent Sanger sequencing. As a proof of concept, 3C-PCR was used to phase a highly variable region 1.3 Mb upstream of a chromosomal rearrangement breakpoint in a balanced translocation. We found that the nested PCR approach amplified the derivative chromosome substrate exclusively and identified the same haplotype by Sanger sequencing reliably. Given its efficacy and versatility, 3C-PCR is ideal for use in phasing chromosomal rearrangement breakpoints with allelic variants located at a genomic distance over a megabase.

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Characterization of CD45RO+ Memory T Lymphocytes in Keloid Disease

Abstract

Background

Memory T cells, a highly effective subset of T lymphocytes, have been reported to be involved in many inflammatory skin disorders. However, the potential role of memory T cells in keloid disease (KD) remains unclear.

Objectives

Due to their important role in regulating inflammation, we investigated the characteristics of CD45RO⁺ memory T cells (mT) in KD.

Methods

Primary cutaneous cells were isolated from keloid scars and normal skin by enzymic digestion. Combined with peripheral blood mononuclear cells (PBMCs) isolated from a related blood sample, flow cytometry was applied to identify the phenotypic and functional abnormalities of memory T cells in KD.

Results

We observed that the majority of T lymphocytes in keloid scars had the memory phenotype, and a greater number of the CD8⁺ mT in keloid scars produced lower levels of TNF-α. This abnormal cytokine production was even more distinct in FOXP3^-CD8^- mT, with lower TNF-α production and enhanced IFN-γ production. Furthermore, FOXP3⁺CD8^- mT in keloid scars were abnormal, including showing reduced CD25 and CTLA-4 expression and IL-10 production. In addition, a significant decrease in the number of CD4⁺CD25^highFOXP3⁺ regulatory T cells was identified in patients with multiple keloid scars. We also found that there was significantly increased infiltration of CD103⁺CD8⁺ mT in keloid scars.

Conclusions

Our findings preliminarily elucidate the abnormalities of CD45RO⁺ memory T cells in keloid scars and provide early evidence that a disrupted T cell response contributes to the progression of KD.

This article is protected by copyright. All rights reserved.

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Impaired Langerhans’ cell migration in psoriasis is due to an altered keratinocyte phenotype induced by interleukin-17

Abstract

Psoriasis is a common skin condition driven by increased expression of interleukin (IL)-17. Langerhans' cells (LC) are epidermal dendritic cells that regulate cutaneous immune responses. Within uninvolved skin of patients with psoriasis, LC display impaired migration from the epidermis. Here the role of keratinocytes (KC) in the regulation of LC function, and the response of KC to IL-17 has been investigated. Keratinocytes were cultured from the uninvolved skin of psoriasis patients and healthy individuals with or without IL-17 treatment and the conditioned medium examined for its ability to alter LC function in an ex vivo human skin explant model. Furthermore, we examined the effect of IL-17 on LC mobilisation in psoriasis by neutralising IL-17 in the same skin explant model. We found that conditioned medium from psoriasis KC inhibited LC migration in healthy skin. Moreover, conditioned medium from healthy KC treated with IL-17 also inhibited healthy LC migration. Finally, neutralising IL-17 in psoriasis skin resulted in enhanced LC migration. Collectively, these data suggest that an altered KC secretome, driven by increased expression of IL-17, is responsible for impaired LC migration in uninvolved skin of patients with psoriasis.

This article is protected by copyright. All rights reserved.

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Characterization of CD45RO+ Memory T Lymphocytes in Keloid Disease

Abstract

Background

Memory T cells, a highly effective subset of T lymphocytes, have been reported to be involved in many inflammatory skin disorders. However, the potential role of memory T cells in keloid disease (KD) remains unclear.

Objectives

Due to their important role in regulating inflammation, we investigated the characteristics of CD45RO⁺ memory T cells (mT) in KD.

Methods

Primary cutaneous cells were isolated from keloid scars and normal skin by enzymic digestion. Combined with peripheral blood mononuclear cells (PBMCs) isolated from a related blood sample, flow cytometry was applied to identify the phenotypic and functional abnormalities of memory T cells in KD.

Results

We observed that the majority of T lymphocytes in keloid scars had the memory phenotype, and a greater number of the CD8⁺ mT in keloid scars produced lower levels of TNF-α. This abnormal cytokine production was even more distinct in FOXP3^-CD8^- mT, with lower TNF-α production and enhanced IFN-γ production. Furthermore, FOXP3⁺CD8^- mT in keloid scars were abnormal, including showing reduced CD25 and CTLA-4 expression and IL-10 production. In addition, a significant decrease in the number of CD4⁺CD25^highFOXP3⁺ regulatory T cells was identified in patients with multiple keloid scars. We also found that there was significantly increased infiltration of CD103⁺CD8⁺ mT in keloid scars.

Conclusions

Our findings preliminarily elucidate the abnormalities of CD45RO⁺ memory T cells in keloid scars and provide early evidence that a disrupted T cell response contributes to the progression of KD.

This article is protected by copyright. All rights reserved.

http://ift.tt/2zVmixF

Impaired Langerhans’ cell migration in psoriasis is due to an altered keratinocyte phenotype induced by interleukin-17

Abstract

Psoriasis is a common skin condition driven by increased expression of interleukin (IL)-17. Langerhans' cells (LC) are epidermal dendritic cells that regulate cutaneous immune responses. Within uninvolved skin of patients with psoriasis, LC display impaired migration from the epidermis. Here the role of keratinocytes (KC) in the regulation of LC function, and the response of KC to IL-17 has been investigated. Keratinocytes were cultured from the uninvolved skin of psoriasis patients and healthy individuals with or without IL-17 treatment and the conditioned medium examined for its ability to alter LC function in an ex vivo human skin explant model. Furthermore, we examined the effect of IL-17 on LC mobilisation in psoriasis by neutralising IL-17 in the same skin explant model. We found that conditioned medium from psoriasis KC inhibited LC migration in healthy skin. Moreover, conditioned medium from healthy KC treated with IL-17 also inhibited healthy LC migration. Finally, neutralising IL-17 in psoriasis skin resulted in enhanced LC migration. Collectively, these data suggest that an altered KC secretome, driven by increased expression of IL-17, is responsible for impaired LC migration in uninvolved skin of patients with psoriasis.

This article is protected by copyright. All rights reserved.

http://ift.tt/2itZldu