Tumour-infiltrating FOXP3(+) lymphocytes are associated with cytotoxic immune responses and good clinical outcome in oestrogen receptor-negative breast cancer.

BACKGROUND: Regulatory T cells (Tregs) are commonly identified by expression of the transcription factor FOXP3 and are conventionally thought to promote cancer progression by suppressing anti-tumour immune responses. We examined the relationship between FOXP3(+) tumour-infiltrating lymphocytes (TIL) and prognosis in oestrogen receptor (ER)-negative breast cancer, a tumour subtype with poor clinical outcome in which TIL are abundant. METHODS: FOXP3(+) and CD8(+) TIL were assessed by immunohistochemistry in a cohort of 175 ER- breast tumours. Results were confirmed in an independent data set of 78 ER- breast tumours with publically available gene expression data. RESULTS: High FOXP3(+) TIL levels were strongly associated with prolonged recurrence-free survival (HR=0.461, P=0.0002), particularly among basal-like tumours (HR=0.280, P=0.0001), for which FOXP3 status was independent of standard prognostic factors. Over 75% of FOXP3(+) TIL in triple negative breast tumours displayed a conventional CD4(+)CD25(+) Treg phenotype. Importantly, FOXP3(+) TIL were positively correlated with CD8(+) (cytotoxic) T cells (r(s)=0.76, P<0.0001), and were prognostically insignificant in tumours with low levels of CD8(+) TIL. These observations were confirmed in an independent cohort. CONCLUSION: In contrast with current dogma, we show for the first time that FOXP3(+) TIL are associated with robust anti-tumour immunity and favourable prognosis in ER- breast cancer.

Relationship of Wallerian degeneration to regrowing axons.

Because Wallerian degeneration constitutes a highly significant and unavoidable position in the sequence of nervous system regeneration we have extended our observations regarding this process as it occurs in our cryogenic model of spinal cord injury in the rat. In previous studies we have observed essentially no growth into the Wallerian zone despite a significant amount of axonal regrowth through the injured region. In the present study we have examined by electron microscopy the Wallerian degeneration and its interface with the axons from the injured region. In the Wallerian region our observations have not only confirmed those of many previous investigators but have suggested an orderly sequence of changes during the first 60 days post-injury, beginning with relative tissue preservation followed by depletion of degenerating axoplasm, extracellular deposition of myelin and the development of a cellular reaction. Associated with the cellular reaction is the development of large astrocytic cells which can be found adjacent to unmyelinated and thinly myelinated axons. With this limited evidence of support for axonal growth in the Wallerian zone it is suggested that further expansion of this cellular matrix should provide significant support to regrowing axons.

Cellular changes during repair of a cryogenic spinal cord injury in the rat: an electron microscopic study.

Cryogenic injury of adult, rat spinal cord was produced under controlled non-invasive conditions to study repair and regeneration in adult, mammalian central nervous tissue in which tissue continuity and integrity are relatively preserved. Under these experimental conditions axons and myelin are destroyed, a matrix of glial cells is preserved, and regrowth of axons is apparent. Electron microscopic studies at 7, 15, 30 and 60 days post-injury demonstrate axonal structures indicative of regrowth, astrocytic structures which appear to provide support to both matrix and axons and myelination of axons by both oligodendrocytes and Schwann cells. These cellular events restore much of the normal structure within the injured area up to its junction with the Wallerian zone. In this junctional zone morphologic evidence may indicate continuing cellular activity, even at 60 days, in axons, astrocytes and myelinating cells. These studies suggest that under ideal conditions damaged axons may be capable of regeneration within the adult mammalian central nervous system and that this model provides an opportunity to define some of the mechanisms.

The histopathology of freezing injury to the rat spinal cord. A light and electron microscope study. II. Repair and regeneration.

We utilized a recently developed model of spinal cord injury in which freezing of the rat dorsal column produced axonal injury with sparing and proliferation of the supporting tissue. We examined the progress of the reparative and regenerative processes for 15, 30 and 60 days after the injury. In transverse and sagittal sections at the proximal middle, and distal injury zone and at the zone of Wallerian degeneration we have demonstrated an apparent outgrowth of axons which makes its appearance between 15 and 30 days following injury and increases in amount between 30 and 60 days. The myelination of these fibers is bimodal with Schwann cells predominating in the subpial region, and oligodendrocytes in the deeper regions. Growth into the Wallerian zone is significantly less but does occur at 30 days increasing only slightly at 60 days despite extensive clearing of the breakdown products. We believe that the conditions created by this method of injury provide a suitable model for the study of repair and regeneration of mammalian central nervous tissue.

The histopathology of freezing injury to the rat spinal cord. A light microscope study. I. Early degenerative changes.

In an effort to develop a method of tissue injury which would provide a model for the study of axonal regrowth in adult mammalian central nervous system (CNS), we have analyzed the effects of freezing in the dorsal columns of more than 200 rat spinal cords. The effects of temperature and time of exposure upon the size, shape, distribution and histologic characteristics of the lesion have been assessed during the first seven days following the injury. The upper threshold for injury occurs at -3 degrees C for 15 minutes. Between -3 degrees C and -12 degrees C the tissue changes vary in extent and characteristics. Selective damage to axons and myelin occurs with sparing of the supportive cells followed by proliferation of a cellular matrix. At seven days, the lesions produced by -8 degrees C for 15 to 60 minutes have neither axons nor myelin sheaths and consist of a dense cellular matrix of macrophages and presumed glial cells. With these tissue characteristics, and the preservation of tissue continuity without obstructive barriers, this model would appear to be potentially suitable for the study of axonal regrowth potential in mammalian CNS.

Glial activity during axonal regrowth following cryogenic injury of rat spinal cord.

The cryogenic model of spinal cord injury is associated with regrowth of axons through a zone of injury which is composed of a complex matrix of intact blood vessels, cells and extracellular materials. We have used this, therefore, as a means for studying the relationship between these cellular components, particularly the astrocytes, and the regrowing axons. For the time period of 60 days following injury we have found that, from an undifferentiated group of cells which appears early, there develops a population of astrocytes which is associated with the restoration of neural structure, including the return of axons, to the damaged area. We interpret these associations to be supportive through direct axonal interactions and indirect environmental contributions.

Prospects for axonal regrowth in spinal cord injury.

Some of the evidence relating to the possible cellular relationships in regenerating mammalian central nervous tissue is reviewed. From this review it is suggested that data do exist which reveal a potential for regeneration based upon the basic properties and behavior of axons and glial cells. Models of tissue injury which optimize these intrinsic capabilities may generate significant information about the regenerative possibilities of central nervous tissue.

Preservation of synapses on principal cells of the central nucleus of the inferior colliculus with aging in the CBA mouse.

The light and electron microscopic features of principal neurons of the central nucleus of the inferior colliculus were quantitated in the CBA mouse. Three age groups of mice were examined, including young (3 months), middle-aged (8 months) and old (25 months). No changes were noted in the size of the principal neurons over the age range examined. At the ultrastructural level, synapses on the somata of the principal neurons showed no change in the number or type of synapses, the length of synaptic apposition nor the size of synaptic terminal area. These results are in contrast with the moderately severe synapse loss which we previously reported in the C57BL/6 mouse strain, a strain which has a genetic deficit producing progressive sensorineural hearing loss starting in young adulthood (Kazee et al., 1995). In contrast, hearing is quite well-preserved across the lifespan in the CBA mouse strain, making this a useful animal to study the intrinsic effects of aging in the auditory system versus the effects of sensorineural hearing loss. The preservation of synapses on principal neurons in this strain suggests that synaptic loss is not an inevitable event in aging, but may be related to the preservation of peripheral auditory function and input to the neurons.

Oncostatin-M promotes phenotypic changes associated with mesenchymal and stem cell-like differentiation in breast cancer.

Cancer stem cell (CSC) biology and the epithelial-to-mesenchymal transition (EMT) are thought to be mechanistically linked and may be key components of cancer development and progression. However, stimuli that induce EMT and CSC-like features ('stemness') are poorly defined. We and others have shown that the inflammatory cytokine oncostatin-M (OSM) mediates phenotypic changes in breast cancer that are consistent with EMT and dedifferentiation, including enhanced migration and loss of hormone receptors. In this study, we have expanded on these prior observations to determine whether OSM is a cell-extrinsic driver of EMT and/or stemness. OSM stimulation of the luminal breast cancer cell lines MCF7 and T47D induced EMT features including loss of membranous E-cadherin and induction of snail and slug expression. OSM treatment markedly enhanced the formation of mammospheres (up to 20-fold, P<0.001), which displayed high expression of the pluripotency factor SOX2. The proportion of cells with a CD44(high)CD24(-/low) phenotype was similarly increased by OSM (P<0.001). OSM-induced mammosphere formation and CD44(high)CD24(-/low) induction was dependent on PI3K signalling. In silico analysis of human breast tumours (from a publicly available data set, n=322) confirmed that co-expression of a PI3K transcriptional signature, but not MAPK or STAT3 signatures, was necessary to detect an association between OSMR and poor prognosis. Assessment of a second in silico data set (n=241 breast tumours) confirmed a significant relationship between OSMR, markers of EMT and CSCs, and chemotherapy resistance. Direct analysis of mRNA expression by RT-PCR in a third cohort (n=72 breast tumours) demonstrated that high expression of OSM is associated positively with indicators of EMT (SNAI1, P<0.001) and stemness (SOX2, P<0.05). Our data suggest for the first time that OSM may promote a clinically relevant EMT/CSC-like phenotype in human breast cancer via a PI3K-dependent mechanism.

S100A7 (psoriasin) is induced by the proinflammatory cytokines oncostatin-M and interleukin-6 in human breast cancer.

S100A7 promotes aggressive features in breast cancer, although regulation of its expression is poorly understood. As S100A7 associates with inflammation in skin and breast tissue, we hypothesized that inflammatory cytokines may regulate S100A7 in breast cancer. We therefore examined the effects of several cytokines, among which oncostatin-M (OSM) and the related cytokine, interleukin (IL)-6, showed the most significant effects on S100A7 expression in breast tumor cells in vitro. Both cytokines consistently induced S100A7 expression in three cell lines (MCF7, T47D and MDA-MB-468) in a dose- and time-dependent manner. Induction of S100A7 was inhibited by blockade of STAT3, phosphatidylinositol 3 kinase (PI3K) and ERK1/2 signaling and small interference RNA (siRNA)-mediated knockdown of S100A7 eliminated the promigratory effects of OSM treatment. S100A7 mRNA levels in a case-control cohort of breast tumors (n=20) were significantly associated with expression of the OSM receptor beta (OSMRbeta) chain (P=0.0098). This association was confirmed using publicly available microarray data from an independent breast tumor cohort (n=201, P=0.0005) and a correlation between S100A7 and poor patient survival was observed specifically in cases with high OSMRbeta expression (HR=2.35; P=0.0396; n=85). We conclude that inflammatory cytokines can regulate S100A7 expression and that S100A7 may mediate some of their effects in breast cancer.

Absorption, distribution and excretion of trifluoperazine in rats.

In rats receiving a low oral dose (0.35 mg/kg p.o.) of trifluoperazine (TFP), a potent neuroleptic phenothiazine, the parent drug distribution was separate from that of its metabolite trifluoperazine sulfoxide (TFP-SO). This was not seen after intraperitoneal administration (i.p.) of the same or high dose (5mg/kg). TFP-SO was not detected in the brain, wheras TFP was significantly associated with microsomes when its brain level was highest. Absorption, distribution, metabolism, and dose dependent excretion procedded rapidly with 97% of a dose recovered 24 hr after p.o. treatment, only 6% in the urine and the remainder in feces with 87% of this metabolized to compounds other than TFP-SO. TFP and its metabolites arrived in feces via two pathways, largely the bile plus another route. The excretion pattern was unchanges with other drugs or after treatment for 3 wk, although excretion differed on the first day after i.p. and p.o. administration.

Support of axonal regrowth by endogenous mechanisms following spinal cord injury in adult rats.

Because of its non-invasive nature and ease of regulation, a closely monitored cryogenic method of tissue injury was used to create a degree of spinal cord injury within which there would be an extended regrowth of axons. The parameters of cooling used in the present study resulted in an injury length of 1 cm through which 3 mm of measured axonal regrowth and 8 mm of observed regrowth occurred over a 56-day period in the ascending fibers of the dorsal column of the mature rat. This was associated with the development of a cellular matrix consisting of macrophages, macroglia and Schwann cells which gradually expands within the injured area initially dominated by macrophages. It is the authors' impression that the presence of a substantial microglial component within the macrophage population may be a significant factor in the success of the axonal regrowth. Under this influence and that of the invading axons, the astrocyte, which provides the immediate cell support to the growing axon, can be maintained in a functional state that is supportive and not obstructive to the axon, presumably through the recruitment of astrocyte precursors from an indigenous stem cell population. These tissue changes indicate that adult mammalian spinal cord tissue does have the capacity to develop on its own a matrix capable of supporting the regrowth of axons.

Alveolar function following surfactant deactivation.

In vivo cinemicroscopic studies of subpleural alveoli were conducted in dogs for 4 h after pulmonary lavage with 5% Tween 20 and after lavage with normal saline. Saline-lavaged alveoli showed little change in alveolar size during tidal ventilation, whereas Tween lavage resulted both in alveolar recruitment and marked variation in alveolar size from end expiration to peak inspiration, with total collapse frequently occurring by end expiration. Following Tween, alveolar stability decreased, but alveolar capillary perfusion increased. Marked recovery of stability by 4 h was noted in most alveoli. Wilhelmy balance studies on lung extracts showed a decrease in surfactant function 30 min after Tween and a partial recovery of surfactant activity after 4 h. This study provides in vivo evidence that normal surfactant function is critical to alveolar stability and that alveolar stability is markedly restored 4 h after acute deactivation or displacement by Tween. Surfactant deactivation and loss of alveolar stability are associated with increased alveolar capillary perfusion creating significant ventilation-perfusion ratio abnormalities.

Cryogenic spinal cord injury induces astrocytic gene expression of insulin-like growth factor I and insulin-like growth factor binding protein 2 during myelin regeneration.

To study injury-induced astrocytic responses associated with regrowth of axons and regeneration of myelin, the method of Collins and colleagues was used to make focal cryogenic lesions in spinal cords of adult rats (Collins et al.: J Neuropathol Exp Neurol 45: 742-757, 1986). The duration of cryogenic injury (CI), the size of the cryode, and its temperature were chosen to destroy all myelin sheaths and axons without producing cavities or hemorrhages. Messenger RNA and peptide distributions of insulin-like growth factor I (IGF-I), IGF-I receptor (IGFR-I), IGF binding protein 2 (IGFBP-2), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) were studied 3-56 days after CI by in situ hybridization and immunocytochemistry. At 3 days, vimentin-positive, GFAP-negative astrocyte-like cells in the lesion expressed IGF-I mRNA and peptide and 7 days after CI, both were expressed by typical GFAP-positive, hypertrophic astrocytes, many of which also were vimentin-positive. Levels of IGF-I, IGFBP-2, and GFAP mRNA and peptide were higher in lesion astrocytes after 14 days. They attained maximum levels at 21-28 days before declining to near control levels at 56 days. Decreasing relative levels of oligodendroglial MBP mRNA were found in and around lesions 7-14 days after CI; subsequently, rising levels accompanied remyelination. At 28 and 56 days after CI, some transferrin-positive, oligodendroglia-like cells also were immunostained by anti-IGFR-I. Our findings suggest that early astrocytic production of IGF-I and IGFBP-2 may be involved in the myelin regeneration which occurs in this model of spinal cord injury.