Experimental and molecular pathology最新文献

For many people living at high altitudes for long or short periods of time, hypoxia is a challenge affecting many aspects of the body, including the immune system. Recently, myeloid-derived suppressor cells (MDSCs) have emerged as an immune cell population that plays an important role in several pathological conditions. However, to the best of our knowledge, there are no data regarding the behavior of MDSCs under hypoxic conditions. Therefore, the aim of this study is to investigate the monocytic type (M)- and polymorphonuclear type (PMN)-MDSC ratios in different hypoxic conditions to reveal the relationship between MDSCs and high-altitude hypoxia, as well as to determine whether MDSCs are involved in the regulation of the immune balance under hypoxic conditions as immunosuppressive factors. For the first time, we showed that MDSC abundance varies under different lengths of hypoxic exposure. We found that acute normobaric hypoxia led to an initial increase in the number of M-MDSCs, which decreased within 30 d. Both M- and PMN-MDSC ratios initially decreased under hypobaric hypoxia conditions within 30 d, but after 6 months in the real high altitude environment, M-MDSC ratio increased significantly. In summary, our data suggest that different hypoxic conditions influence MDSCs in mice, thereby contributing to a better understanding of the process of hypoxia adaptation and the occurrence and development of high-altitude disease.

Aims

Phytocannabinoids and terpenes from Cannabis sativa have demonstrated limited anti-inflammatory and analgesic effects in several inflammatory conditions. In the current study, we test the hypothesis that phytocannabinoids exert immunomodulatory effects in vitro by decreasing inflammatory cytokine expression and activation.

Key methods

CD3/CD28 and lipopolysaccharide activated peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 6) were treated with phytocannabinoid compounds and terpenes in vitro. Flow cytometry was used to determine regulatory T cell (Treg) and T helper 17 (Th17) cell responses to treatments. Cell pellets were harvested for qRT-PCR gene expression analysis of cytokines, cell activation markers, and inflammation-related receptors. Cell culture supernatants were analysed by ELISA to quantify IL-6, TNF-α and IL-10 secretion.

Main findings

In an initial screen of 20 μM cannabinoids and terpenes which were coded to blind investigators, cannabigerol (GL4a), caryophyllene oxide (GL5a) and gamma-terpinene (GL6a) significantly reduced cytotoxicity and gene expression levels of IL6, IL10, TNF, TRPV1, CNR1, HTR1A, FOXP3, RORC and NFKΒ1. Tetrahydrocannabinol (GL7a) suppression of T cell activation was associated with downregulation of RORC and NFKΒ1 gene expression and reduced IL-6 (p < 0.0001) and IL10 (p < 0.01) secretion. Cannabidiol (GL1b) significantly suppressed activation of Tregs (p < 0.05) and Th17 cells (p < 0.05) in a follow-on in vitro dose-response study. IL-6 (p < 0.01) and IL-10 (p < 0.01) secretion was significantly reduced with 50 μM cannabidiol.

Significance

The study provides the first evidence that cannabidiol and tetrahydrocannabinol suppress extracellular expression of both anti- and pro-inflammatory cytokines in an in vitro PBMC model of inflammation.

Background & aims

Older people experience a greater incidence of lower bowel disorders, including constipation. Causes can include factors associated with growing older, such as use of medications or disease, but compounded by degenerative changes within the bowel wall. It has been suggested that the latter is exacerbated by loss of an effective mucosal barrier to luminal contents. In human colon, little is known about the impact of ageing on key components of this barrier, namely the goblet cells and mucin content.

Methods

Changes in the number of goblet cells and density of mucin content were investigated in macroscopically normal human ascending (AC; n = 13) and descending (DC; n = 14) colon from elderly (≥ 67 years) and younger adults (60 years and below). Samples were serially sectioned and stained for haematoxylin and eosin to assess tissue morphology, and alcian blue periodic acid Schiff (ABPAS) and MUC-2 antibody to identify goblet cells producing mucins. New procedures in visualization and identification of goblet cells and mucin contents were employed to ensure unbiased counting and densitometric analysis.

Results

Compared with the younger adults, the numbers of goblet cells per crypt were significantly lower in the elderly AC (72 ± 1.2 vs 51 ± 0.5) and DC (75 ± 2.6 vs. 54 ± 1.9), although this reduction did not reach statistical significance when assessed per mucosal area (AC: P = 0.068; DC: P = 0.096). In both regions from the elderly, numerous empty vesicles (normally containing mucins) were observed, and some areas of epithelium were devoid of goblet cells. Thus, the density of mucin content per unit mucosal area were significantly reduced with age.

Conclusions

Ageing could result in a reduced number of goblet cells and development of degenerative changes in mucin production. Together, these have implications for the mucus barrier function in the colon of elderly individuals.

Seizures are neurological disorders triggered by an imbalance in the activity of excitatory and inhibitory neurotransmitters in the brain. When triggered chronically, this imbalance can lead to epilepsy. Critically, many of the affected individuals are refractory to treatment. Given this, anti-inflammatory drugs, in particular glucocorticoids, have been considered as a potential antiepileptogenic therapy. Glucocorticoids are currently used in the treatment of refractory patients, although there have been contradictory results in terms of their use in association with antiepileptic drugs, which reinforces the need for a more thorough investigation of their effects. In this context, the present study evaluated the effects of dexamethasone (DEX, 0.6 mg/kg) on the electroencephalographic (EEG) and histopathological parameters of male Wistar rats submitted to acute seizure induced by pentylenetetrazol (PTZ). The EEG monitoring revealed that DEX reduced the total brainwave power, in comparison with PTZ, in 12 h after the convulsive episode, exerting this effect in up to 36 h (p < 0.05 for all comparisons). An increase in the accommodation of the oscillations of the delta, alpha, and gamma frequencies was also observed from the first 12 h onwards, with the accommodation of the theta frequency occurring after 36 h, and that of the beta frequency 24 h after the seizure. The histopathological analyses showed that the CA3 region and hilum of the hippocampus suffered cell loss after the PTZ-induced seizure (control vs. PTZ, p < 0.05), although DEX was not able to protect these regions against cell death (PTZ vs. DEX + PTZ, p > 0.05). While DEX did not reverse the cell damage caused by PTZ, the data indicate that DEX has beneficial properties in the EEG analysis, which makes it a promising candidate for the attenuation of the epileptiform wave patterns that can precipitate refractory seizures.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the alimentary tract. The prognosis depends on the primary site, and small intestinal GISTs have a worse prognosis than gastric GISTs. Molecularly targeted drugs to inhibit tyrosine kinase activity of KIT were used for unresectable or recurrent GISTs. However, secondary resistance to the drugs is often acquired, and treatments based on other mechanisms are needed. Previously, we reported that cell adhesion molecule 1 (CADM1) was highly expressed in most of small intestinal GISTs but not in most of gastric GISTs. In the present study, we examined whether the antibody-drug conjugate (ADC) with anti-CADM1 antibody and monomethyl auristatin E (anti-CAD-ADC) shows anti-tumor effect on CADM1-expressing human GIST cells. The ADC adhibited in this study was previously used for CADM1-expressing human mesothelioma cells and showed anti-tumor effect for them in vitro. GIST-T1 cell line of gastric origin which scarcely expresses CADM1 and GIST-T1 cells transfected with CADM1 cDNA (GIST-T1-CAD cells) which highly expresses CADM1 and represents small intestinal GIST were used. In vitro, anti-CAD-ADC showed remarkable cytotoxic activity on GIST-T1-CAD cells, but control ADC did not. Both anti-CAD-ADC and control ADC did not show anti-tumor effect on original GIST-T1 cells. When GIST-T1-CAD cells were subcutaneously injected to the nude mice, intravenous administration of anti-CAD-ADC showed inhibitory effect for tumor enlargement. Tumor of GIST-T1 cells grew even after anti-CAD-ADC injection. When GIST-T1-CAD cells were injected into peritoneal cavity of the SCID mice, intraperitoneal administration of anti-CAD-ADC showed reduction of the peritoneal tumor. On the other hand, peritoneal tumor grew after control ADC administration. Tissue and organ damage due to administration of anti-CAD-ADC was not apparent by macroscopic and histological examinations in mice. These results indicate that anti-CAD-ADC could have apparent anti-tumor effect on CADM1-expressing human GIST cells both in in vitro and in vivo mouse models.

Breast cancer (BC) is classified into four major histological subtypes, namely luminal A, luminal B, HER2, and basal-like, and its treatment is based on these subtypes. The use of immune checkpoint inhibitors against BC depends on the expression of PD-1/PD-L1. Another tumor immune system—the cGAS–STING pathway—is a potential target for cancer immunotherapy. However, the status of the cGAS–STING pathway in BC has not been fully established. Therefore, we investigated the expression status of the cGAS–STING pathway and immune-related proteins in BC. We classified 111 BCs into six groups—29 hormone receptor-positive carcinomas, 12 HER2+ carcinomas (HER2), 8 luminal-HER2 carcinomas, 26 triple-negative breast carcinomas (TNBCs), 21 lobular carcinomas (LC), and 15 carcinomas with apocrine differentiation (CAD)—and investigated the relationship between BC and tumor immunity via the cGAS—STING pathway using histopathological and immunohistochemical methods. Expression of cGAS was high in CADs (100%) and low in TNBCs (35%); STING-positive lymphocytes were high in TNBC (85%, P = 0.0054). Expression of pSTAT3 was significantly high in patients with TNBC (≥10%, 88%). The proportion of PD-L1-positive tumor cells was higher in TNBCs (54%) than in other BCs (30%). SRGN expression was significantly higher in the TNBC group than in the other BC groups (58%). Tumor immune responses may differ among tumor subtypes. The cGAS–STING pathway may be functional in TNBC and CAD but not in LC. Therefore, targeting the cGAS–STING pathway might be useful in BC, particularly TNBC and CAD.

Pancreatic ductal adenocarcinoma (PDAC) is reported to be amongst the cancers with the lowest survival rate at 5 years. In the present study we aimed to validate a targeted next-generation sequencing (tNGS) panel to use in clinical routine, investigating genes important for PDAC diagnostic, prognostic and potential theragnostic aspect. In this NGS panel we also designed target regions to inquire about loss of heterozygosity (LOH) of chromosome 18 that has been described to be possibly linked to a worse disease progression. Copy number alteration has also been explored for a subset of genes. The last two methods are not commonly used for routine diagnostic with tNGS panels and we investigated their possible contribution to better characterize PDAC. A series of 140 formalin-fixed paraffin-embedded (FFPE) PDAC samples from 140 patients was characterized using this panel. Ninety-two % of patients showed alterations in at least one of the investigated genes (most frequent KRAS, TP53, SMAD4, CDKN2A and RNF43). Regarding LOH evaluation, we were able to detect chr18 LOH starting at 20% cell tumor percentage. The presence of LOH on chr18 is associated with a worse disease- and metastasis-free survival, in uni- and multivariate analyses. The present study validates the use of a tNGS panel for PDAC characterization, also evaluating chr18 LOH status for prognostic stratification.

A subset of head and neck squamous cell carcinomas present solely as metastatic disease in the neck and are of unknown primary origin (SCCUP). Most primary tumors will ultimately be identified, usually in the oropharynx. In a minority of cases, the primary site remains elusive. Here, we examine the role of ancillary testing, including mutational signature analysis (MSA), to help identify likely primary sites in such cases. Twenty-two cases of SCCUP in the neck, collected over a 10-year period, were classified by morphology and viral status; including human papillomavirus (HPV) testing by p16 immunohistochemistry (IHC) and RT-qPCR, as well as Epstein-Barr virus (EBV) testing by EBER-ISH. CD5 and c-KIT (CD117) IHC was done to evaluate for possible thymic origin in all virus-negative cases. Whole exome sequencing, followed by MSA, was used to identify UV signature mutations indicative of cutaneous origin. HPV was identified in 12 of 22 tumors (54.5%), favoring an oropharyngeal origin, and closely associated with nonkeratinizing tumor morphology (Fisher's exact test; p = 0.0002). One tumor with indeterminant morphology had discordant HPV and p16 status (p16+/HPV-). All tumors were EBV-negative. Diffuse expression of CD5 and c-KIT was identified in 1 of 10 virus-negative SCCUPs (10%), suggesting a possible ectopic thymic origin rather than a metastasis. A UV mutational signature, indicating cutaneous origin, was identified in 1 of 10 (10%) virus-negative SCCUPs. A cutaneous auricular primary emerged 3 months after treatment in this patient. Primary tumors became clinically apparent in 2 others (1 hypopharynx, 1 hypopharynx/larynx). Thus, after follow-up, 6 tumors remained unclassifiable as to the possible site of origin (27%). Most SCCUPs of the neck in our series were HPV-associated and thus likely of oropharyngeal origin. UV signature mutation analysis and additional IHC for CD5 and c-KIT for possible thymic origin may aid in further classifying virus-negative unknown primaries. Close clinical inspection of hypopharyngeal mucosa may also be helpful, as a subset of primary tumors later emerged at this site.

High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of ovarian cancer and demonstrates 5-year survival of just 40%. One of the major causes of mortality is the development of tumour resistance to platinum-based chemotherapy, which can be modulated by dysregulation of DNA damage repair pathways. We therefore investigated the contribution of the DNA interstrand crosslink repair protein FANCD2 to chemosensitivity in HGSOC. Increased FANCD2 protein expression was observed in some cell line models of platinum resistant HGSOC compared with paired platinum sensitive models. Knockdown of FANCD2 in some cell lines, including the platinum resistant PEO4, led to increased carboplatin sensitivity. Investigation into mechanisms of FANCD2 regulation showed that increased FANCD2 expression in platinum resistant cells coincides with increased expression of mTOR. Treatment with mTOR inhibitors resulted in FANCD2 depletion, suggesting that mTOR can mediate platinum sensitivity via regulation of FANCD2. Tumours from a cohort of HGSOC patients showed varied nuclear and cytoplasmic FANCD2 expression, however this was not significantly associated with clinical characteristics. Knockout of FANCD2 was associated with increased cell migration, which may represent a non-canonical function of cytoplasmic FANCD2. We conclude that upregulation of FANCD2, possibly mediated by mTOR, is a potential mechanism of chemoresistance in HGSOC and modulation of FANCD2 expression can influence platinum sensitivity and other tumour cell characteristics.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in the development and progression of many cancers. Partial EMT (pEMT) could represent a critical step in tumor migration and dissemination. Sarcomatoid renal cell carcinoma (sRCC) is an aggressive form of renal cell carcinoma (RCC) composed of a carcinomatous (sRCC-Ca) and sarcomatous (sRCC-Sa) component. The role of (p)EMT in the progression of RCC to sRCC remains unclear. The aim of this study was to investigate the involvement of (p)EMT in RCC and sRCC. Tissue samples from 10 patients with clear cell RCC (ccRCC) and 10 patients with sRCC were selected. The expression of main EMT markers (miR-200 family, miR-205, SNAI1/2, TWIST1/2, ZEB1/2, CDH1/2, VIM) was analyzed by qPCR in ccRCC, sRCC-Ca, and sRCC-Sa and compared to non-neoplastic tissue and between both groups. Expression of E-cadherin, N-cadherin, vimentin and ZEB2 was analyzed using immunohistochemistry. miR-200c was downregulated in sRCC-Ca compared to ccRCC, while miR-200a was downregulated in sRCC-Sa compared to ccRCC. CDH1 was downregulated in sRCC-Sa when compared to any other group. ZEB2 was downregulated in ccRCC and sRCC compared to corresponding non-neoplastic kidney. A positive correlation was observed between CDH1 expression and miR-200a/b/c. Our results suggest that full EMT is not present in sRCC. Instead, discreet molecular differences exist between ccRCC, sRCC-Ca, and sRCC-Sa, possibly representing distinct intermediary states undergoing pEMT.