Tumour-infiltrating regulatory T cell density before neoadjuvant chemoradiotherapy for rectal cancer does not predict treatment response

Neoadjuvant (preoperative) chemoradiotherapy (CRT) decreases the risk of rectal cancer recurrence and reduces tumour volume prior to surgery. However, response to CRT varies considerably between individuals and factors associated with response are poorly understood. Foxp3+ regulatory T cells (Tregs) inhibit anti-tumour immunity and may limit any response to chemotherapy and radiotherapy. We have previously reported that a low density of Tregs in the tumour stroma following neoadjuvant CRT for rectal cancer is associated with improved tumour regression. Here we have examined the association between Treg density in pre-treatment diagnostic biopsy specimens and treatment response, in this same patient cohort. We aimed to determine whether pre-treatment tumour-infiltrating Treg density predicts subsequent response to neoadjuvant CRT. Foxp3+, CD8+ and CD3+ cell densities in biopsy samples from 106 patients were assessed by standard immunohistochemistry (IHC) and evaluated for their association with tumour regression grade and survival. We found no association between the density of any T cell subset pre-treatment and clinical outcome, indicating that tumour-infiltrating Treg density does not predict response to neoadjuvant CRT in rectal cancer. Taken together with the findings of the previous study, these data suggest that in the context of neoadjuvant CRT for rectal cancer, the impact of chemotherapy and/or radiotherapy on anti-tumour immunity may be more important than the state of the pre-existing local immune response

TLR2 and TLR4 in Parkinson’s disease pathogenesis: the environment takes a toll on the gut

Parkinson’s disease (PD) is an incurable, devastating disorder that is characterized by pathological protein aggregation and neurodegeneration in the substantia nigra. In recent years, growing evidence has implicated the gut environment and the gut-brain axis in the pathogenesis and progression of PD, especially in a subset of people who exhibit prodromal gastrointestinal dysfunction. Specifically, perturbations of gut homeostasis are hypothesized to contribute to α-synuclein aggregation in enteric neurons, which may spread to the brain over decades and eventually result in the characteristic central nervous system manifestations of PD, including neurodegeneration and motor impairments. However, the mechanisms linking gut disturbances and α-synuclein aggregation are still unclear. A plethora of research indicates that toll-like receptors (TLRs), especially TLR2 and TLR4, are critical mediators of gut homeostasis. Alongside their established role in innate immunity throughout the body, studies are increasingly demonstrating that TLR2 and TLR4 signalling shapes the development and function of the gut and the enteric nervous system. Notably, TLR2 and TLR4 are dysregulated in patients with PD, and may thus be central to early gut dysfunction in PD. To better understand the putative contribution of intestinal TLR2 and TLR4 dysfunction to early α-synuclein aggregation and PD, we critically discuss the role of TLR2 and TLR4 in normal gut function as well as evidence for altered TLR2 and TLR4 signalling in PD, by reviewing clinical, animal model and in vitro research. Growing evidence on the immunological aetiology of α-synuclein aggregation is also discussed, with a focus on the interactions of α-synuclein with TLR2 and TLR4. We propose a conceptual model of PD pathogenesis in which microbial dysbiosis alters the permeability of the intestinal barrier as well as TLR2 and TLR4 signalling, ultimately leading to a positive feedback loop of chronic gut dysfunction promoting α-synuclein aggregation in enteric and vagal neurons. In turn, α-synuclein aggregates may then migrate to the brain via peripheral nerves, such as the vagal nerve, to contribute to neuroinflammation and neurodegeneration typically associated with PD

Nuclear localized tumor antigen was not cross-presented as efficiently as cytoplasmic and secretory counterparts.

<p>(A) The EGFP-CL4 antigen expression profile of B16 parental cells (B16 par.) or, B16 par. mixed with 1 (19.4), 5 (97), 10 (194), or 20% (388 nmol) of B16.Nuc (N), B16.Cyto (C), or B16.Sec (S) CL4 antigen-containing cells, was confirmed on the day of tumor inoculation by flow cytometry. For simplicity, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107894#pone-0107894-g002" target="_blank">Figure 2A</a> only shows the profile of B16.Nuc mixtures, as they were not different to B16.Cyto or B16.Sec (B) CFSE-labelled CL4-specific CD8+ T cells were intravenously injected into F1 mice bearing 8-day-old subcutaneous tumors, and the proliferation of these T cells in the tumour draining lymph node examined on day 11 post-tumor inoculation by flow cytometry. Data are representative of two independent experiments, each involving five mice per group. Bars begin at the Mean ± SEM baseline proliferation for B16 parental cells.</p

The prognostic and predictive value of sox2+ cell densities in patients treated for colorectal cancer

SOX2 (sex-determining region-Y homeobox-2) is a transcription factor essential for the maintenance of pluripotency and is also associated with stem-cell-like properties in preclinical cancer models. Our previous study on a cohort of stage III colon cancer patients demonstrated high SOX2+ cell densities were associated with poor prognosis. However, most patients were treated with adjuvant chemotherapy so the prognostic value of SOX2 could not be assessed independently from its value as a predictive marker for non-response to chemotherapy. This study aimed to assess whether SOX2 was a true prognostic marker or a marker for chemotherapy response in a historical cohort of patients, a high proportion of whom were chemotherapy-naïve. SOX2 immunostaining was performed on tissue micro-arrays containing tumor cores from 797 patients with stage II and III colorectal cancer. SOX2+ cell densities were then quantified with StrataQuest digital image analysis software. Overall survival was assessed using Kaplan–Meier estimates and Cox regression. It was found that high SOX2+ cell densities were not associated with poor overall survival. Furthermore, all patients had a significant improvement in survival after 5-fluorouracil (5-FU) treatment, irrespective of their SOX2+ cell density. Therefore, SOX2+ cell densities were not associated with prognosis or chemotherapy benefit in this study. This is in contrast to our previous study, in which most patients received oxaliplatin as part of their treatment, in addition to 5-FU. This suggests SOX2 may predict response to oxaliplatin treatment, but not 5-FU

The apoptosis-inducing agent gemcitabine has a direct effect on B16 tumors in vitro and in vivo.

<p>(A) The relative in vitro sensitivity of B16 parental, B16.Nuc, B16.Cyto, and B16.Sec tumor cells to gemcitabine was assessed by MTT assay, after a 48 hr incubation with drug. With relevent concentrations of gemcitabine that kill 50% of respective tumors shown. (B) <i>In vivo</i> growth of B16 parental only, and B16 parental mixed with 10% (194 nmols) of B16.Sec-, Cyto-, or Nuc-CL4 antigen-containing tumors, after subcutaneous inoculation of F1 mice on day 0, and intraperitoneal administration of a single dose of gemcitabine (240 µg/g) or saline on day 6. Bar graph shows tumor sizes on day 11. For simplicity, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107894#pone-0107894-g003" target="_blank">Figure 3B</a> only shows data from the 10% antigen concentration experiment as this was similar to that of 1 and 5%. Mean ± SEM. Six mice per group from two independent experiments. One-way ANOVA followed by a Bonferroni post test against all groups.</p

Schematic illustration and characterization of B16 tumor cells expressing differentially localized antigen.

<p>(A) Schematic depiction of gene constructs used to generate B16 parental (B16.par) tumors expressing EGFP-CL4 recombinant protein in secretory (B16.Sec), nuclear (B16.Nuc), or cytoplasmic (B16.Cyto) cellular compartments. (B) Confocal fluorescence microscopy demonstrated targeted expression of EGFP-CL4 in distinct subcellular compartments within respective tumors (magnification, ×60). (C) Quantitative imaging flow cytometry confirmed confocal microscopy observations. (D) EGFP ELISA was used to compare the amount of EGFP present in whole cell lysates (W.C.L) and supernatants (Sup.) of respective tumors. A standard curve of recombinant EGFP protein was used to calculate the amount of EGFP. A representative experiment is shown out of two independent ELISAs in triplicates ± SEM. (E) Relative intensity and stability of EGFP expression was determined by live-cell flow cytometry. Mean fluorescent intensities (MFI) are shown in figure. (F) <i>In vivo</i> growth pattern of respective tumors after s.c. inoculation in F1 mice. Data are means of 10 mice from two independent experiments. ***p<0.001. One-way ANOVA, followed by a Bonferroni post-test.</p

Gemcitabine improves the cross-presentation efficiency of nuclear localized antigen.

<p>F1 mice were inoculated on day 0 with B16 parental only (B16 only), or B16 parental mixed with 1 (19.4), 5 (97), or 10% (194 nmol), B16.Nuc (N), B16.Cyto (C), or B16.Sec (S) CL4 antigen-containing tumors, and a single dose of gemcitabine or saline administered on day 6. CFSE-labelled CL4 T cells were transferred on day 8, and the (A) proliferation and (B) Interferon gamma expression of CL4-specific CD8⁺ T cells in tumour draining lymph nodes of mice assessed on day 11. Mean ± SEM. Six mice per group from two independent experiments. One-way ANOVA followed by a Bonferroni post test against all groups. ns  =  not significant, *p<0.05, ***p<0.001.</p

Secondary Degeneration of Oligodendrocyte Precursor Cells Occurs as Early as 24 h after Optic Nerve Injury in Rats

Optic nerve injury causes secondary degeneration, a sequela that spreads damage from the primary injury to adjacent tissue, through mechanisms such as oxidative stress, apoptosis, and blood-brain barrier (BBB) dysfunction. Oligodendrocyte precursor cells (OPCs), a key component of the BBB and oligodendrogenesis, are vulnerable to oxidative deoxyribonucleic acid (DNA) damage by 3 days post-injury. However, it is unclear whether oxidative damage in OPCs occurs earlier at 1 day post-injury, or whether a critical &lsquo;window-of-opportunity&rsquo; exists for therapeutic intervention. Here, a partial optic nerve transection rat model of secondary degeneration was used with immunohistochemistry to assess BBB dysfunction, oxidative stress, and proliferation in OPCs vulnerable to secondary degeneration. At 1 day post-injury, BBB breach and oxidative DNA damage were observed, alongside increased density of DNA-damaged proliferating cells. DNA-damaged cells underwent apoptosis (cleaved caspase3+), and apoptosis was associated with BBB breach. OPCs experienced DNA damage and apoptosis and were the major proliferating cell type with DNA damage. However, the majority of caspase3+ cells were not OPCs. These results provide novel insights into acute secondary degeneration mechanisms in the optic nerve, highlighting the need to consider early oxidative damage to OPCs in therapeutic efforts to limit degeneration following optic nerve injury

Secondary Degeneration Impairs Myelin Ultrastructural Development in Adulthood following Adolescent Neurotrauma in the Rat Optic Nerve

Adolescence is a critical period of postnatal development characterized by social, emotional, and cognitive changes. These changes are increasingly understood to depend on white matter development. White matter is highly vulnerable to the effects of injury, including secondary degeneration in regions adjacent to the primary injury site which alters the myelin ultrastructure. However, the impact of such alterations on adolescent white matter maturation is yet to be investigated. To address this, female piebald-virol-glaxo rats underwent partial transection of the optic nerve during early adolescence (postnatal day (PND) 56) with tissue collection two weeks (PND 70) or three months later (PND 140). Axons and myelin in the transmission electron micrographs of tissue adjacent to the injury were classified and measured based on the appearance of the myelin laminae. Injury in adolescence impaired the myelin structure in adulthood, resulting in a lower percentage of axons with compact myelin and a higher percentage of axons with severe myelin decompaction. Myelin thickness did not increase as expected into adulthood after injury and the relationship between the axon diameter and myelin thickness in adulthood was altered. Notably, dysmyelination was not observed 2 weeks postinjury. In conclusion, injury in adolescence altered the developmental trajectory, resulting in impaired myelin maturation when assessed at the ultrastructural level in adulthood

Secondary Degeneration of Oligodendrocyte Precursor Cells Occurs as Early as 24 h after Optic Nerve Injury in Rats

Optic nerve injury causes secondary degeneration, a sequela that spreads damage from the primary injury to adjacent tissue, through mechanisms such as oxidative stress, apoptosis, and blood-brain barrier (BBB) dysfunction. Oligodendrocyte precursor cells (OPCs), a key component of the BBB and oligodendrogenesis, are vulnerable to oxidative deoxyribonucleic acid (DNA) damage by 3 days post-injury. However, it is unclear whether oxidative damage in OPCs occurs earlier at 1 day post-injury, or whether a critical ‘window-of-opportunity’ exists for therapeutic intervention. Here, a partial optic nerve transection rat model of secondary degeneration was used with immunohistochemistry to assess BBB dysfunction, oxidative stress, and proliferation in OPCs vulnerable to secondary degeneration. At 1 day post-injury, BBB breach and oxidative DNA damage were observed, alongside increased density of DNA-damaged proliferating cells. DNA-damaged cells underwent apoptosis (cleaved caspase3+), and apoptosis was associated with BBB breach. OPCs experienced DNA damage and apoptosis and were the major proliferating cell type with DNA damage. However, the majority of caspase3+ cells were not OPCs. These results provide novel insights into acute secondary degeneration mechanisms in the optic nerve, highlighting the need to consider early oxidative damage to OPCs in therapeutic efforts to limit degeneration following optic nerve injury