Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

The pleiotrophin-ALK axis is required for tumorigenicity of glioblastoma stem cells.

Increasing evidence suggests that brain tumors arise from the transformation of neural stem/precursor/progenitor cells. Much current research on human brain tumors is focused on the stem-like properties of glioblastoma. Here we show that anaplastic lymphoma kinase (ALK) and its ligand pleiotrophin are required for the self-renewal and tumorigenicity of glioblastoma stem cells (GSCs). Furthermore, we demonstrate that pleiotrophin is transactivated directly by SOX2, a transcription factor essential for the maintenance of both neural stem cells and GSCs. We speculate that the pleiotrophin-ALK axis may be a promising target for the therapy of glioblastoma.

Extensive and preferential Fas/Fas ligand-dependent death of gammadelta T cells following infection with Listeria monocytogenes.

In the spleens of mice infected intraperitoneally with the bacterium Listeria monocytogenes, both alphabeta and gammadelta T cells became rapidly activated, followed by a massive apoptotic death response predominantly within the gammadelta population. The death response involved two major splenic gammadelta T-cell subsets and was Fas/Fas ligand (Fas-L)-dependent. Among T cells isolated from the Listeria-infected spleen, Fas-L was almost exclusively expressed in gammadelta T cells. gammadelta T cells coexpressed Fas and Fas-L, suggesting activation-induced suicide as a mechanism of their death. In vivo treatment with an antibody specific for CD3epsilon induced activation, preferential Fas-L expression and apoptosis of gammadelta T cells, resembling the response pattern in listeriosis, whereas antibodies specific for T-cell receptor-beta (TCR-beta) or TCR-delta did not, suggesting that the complete response seen in listeriosis requires both gammadelta TCR engagement and additional stimuli. L. monocytogenes causes early nonspecific, Fas-independent lymphocyte death in heavily infected tissues. In contrast, the death response described here is selective, Fas-dependent and triggered at low local levels of bacteria, suggesting that it is controlled by interactions with other infection-activated host cells, and perhaps part of a regulatory circuit specifically curtailing gammadelta T cells.

Mesh-and-glue technique to prevent leakage of cerebrospinal fluid after implantation of expanded polytetrafluoroethylene dura substitute--technical note.

Expanded polytetrafluoroethylene (ePTFE) can be used as a dura substitute but is associated with leakage of cerebrospinal fluid (CSF) through the suture line. Fibrin glue alone may not prevent this problem. This new method for sealing the suture line in ePTFE membrane uses an absorbable polyglycoic acid mesh soaked with fibrinogen fluid placed on the suture line. Thrombin fluid is then slowly applied to the wet mesh, forming a large fibrin membrane reinforced by the mesh over the suture line. Only one of 33 patients in whom this technique was used had CSF leakage, whereas 12 of 59 patients in whom a dural defect was closed with ePTFE alone showed postoperative subcutaneous CSF collection (p < 0.05). Our clinical experiences clearly show the efficacy of the mesh-and-glue technique to prevent CSF leakage after artificial dural substitution. Mesh and glue can provide an adequate repair for small dural defect. The mesh-and-glue technique may also be used for arachnoid sealing in spinal surgery.

Inflammation alone evokes the response of a TCR-invariant mouse gamma delta T cell subset.

Whether gamma delta T lymphocytes respond to microbial Ags or to inducible host Ags remains a matter of controversy. Using several different disease models and mouse strains, we and others have seen that V gamma 6/V delta 1 gamma delta T cells preferentially increase among the gamma delta T cells infiltrating inflamed tissues. However, it was not clear whether bacteria are necessary to bring about this response. Therefore, we have reexamined this question using a disease model in which inflammation is induced by a purely autoimmune process involving no bacteria, bacterial products, or other foreign material: testicular cell-induced autoimmune orchitis. Using this model we found that gamma delta T cells were still plentiful among the infiltrating T lymphocytes, being 9- to 10-fold more prevalent than in spleen, and that V gamma 6/V delta 1+ cells again represented the predominant gamma delta T cell type. This finding shows that the response of the V gamma 6/V delta 1+ subset does not, in fact, depend upon the presence of bacteria or bacterial products. The stimulus triggering the response of the V gamma 6/V delta 1 gamma delta T cells appears to be neither foreign nor organ-specific in origin, but instead consists of a self-derived host Ag or signal induced during the inflammatory process.

Angioplasty for the infusion of fasudil hydrochloride to treat cerebral symptomatic vasospasm.

The present report describes the successful treatment of cerebral symptomatic vasospasm (SVS) after subarachnoid hemorrhage (SAH) with super-selective intra-arterial infusion of fasudil hydrochloride (ERIL((R))). We treated seventeen vascular territories in 12 patients with selective intra-arterial infusion of fasudil hydrochloride (FSD). FSD was infused through a catheter (a microcatheter in nine patients) at a rate of 1.0 to 1.5 mg/minute (total dose=30 to 60mg/1 vessel) for each vascular territory. Nineteen vascular territories (100%) were angiographically dilated and seven patients (58%) showed early improvement in neurological function after the procedure.

Gamma delta T cells in infection-induced and autoimmune-induced testicular inflammation.

In a previous report, we investigated inflammatory responses induced by injecting Listeria monocytogenes into one testis of a mouse. We demonstrated that the contralateral testis also developed an orchitis despite the absence of bacteria, indicating that the inflammation on the uninfected, contralateral side was of autoimmune character. In both infected and autoimmune testes, gammadelta and alphabeta T cells infiltrated during the inflammation. In this paper, we present the data of a comparison of the character of gammadelta T cells of the infected and autoimmune testes. In both testes, gammadelta T cells appeared to be activated, as assessed by high CD44 and low l-selectin expression. Analysis of T-cell receptor (TCR) usage in both inflammation types revealed the same gammadelta TCR repertoire. Finally, the semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that gammadelta T cells in both types of inflammation were capable of producing interleukin-2 (IL-2), IL-4, interferon-gamma (IFN-gamma), IL-10 and transforming growth factor-beta (TGF-beta). These results imply that gammadelta T cells present in infected-induced and autoimmunity-induced inflammation have the same characteristics and could work as immunoregulatory cells.

Disruption of the cellular inflammatory response to Listeria monocytogenes infection in mice with disruptions in targeted genes.

The results of this study to dissect the nature of the acquired immune response to infection with Listeria monocytogenes in mice with targetted gene disruptions show that successful resolution of disease requires the essential presence of alphabeta T cells and the capacity to elaborate gamma interferon. In the absence of either of these entities, mice experience increasingly severe hepatitis and tissue necrosis and die within a few days. The data from this study support the hypothesis that the protective process is the efficient replacement of neutrophils in lesions by longer-lived mononuclear phagocytes; alphabeta-T-cell-knockout mice died from progressive infection before neutrophil replacement could occur, whereas in gammadelta-T-cell-knockout mice this replacement process in the liver has previously been shown to be much slower. In the present study we attribute this delay to reduced production of the macrophage-attracting chemokine MCP-1 in the gammadelta-T-cell-knockout animals. These data further support the hypothesis that gammadelta T cells are important in controlling the inflammatory process rather than being essential to the expression of protection.

Early preferential stimulation of gamma delta T cells by TNF-alpha.

Although recent findings indicate that gamma delta T cells influence both early innate and Ag-specific adaptive host responses, it has remained unclear what triggers gamma delta T cell reactivity. Investigating very early T cell activation in mouse and human models of bacterial infection, we measured CD69 expression as an indicator of early cellular activation. Both murine alpha beta and gamma delta T cells responded polyclonally to systemic bacterial infections, and to LPS. However, gamma delta T cells responded more strongly to the bacteria and to LPS. In vitro LPS-stimulated human T cells showed a similar differential response pattern. We identified TNF-alpha as mediator of the early differential T cell activation, and of differential proliferative responses. The stronger response of gamma delta T cells to TNF-alpha was correlated with higher inducible expression levels of TNF-Rp75. Among unstimulated splenocytes, more gamma delta T cells than alpha beta T cells expressed CD44 at high levels. The data suggest that TNF-Rp75 determines the differential T cell reactivity, and that most gamma delta T cells in the normal spleen are present in a presensitized state. As TNF-alpha stimulates activated T cells, it may early preferentially connect gamma delta T cell functions with those of cells that produce this cytokine, including activated innate effector cells and Ag-stimulated T lymphocytes.

Multilobular cavernous malformation: report of two cases.

The authors report two cases of cavernous malformation characterized by a multilobular appearance on magnetic resonance images. At surgery, the malformations consisted of several nests of angiomatous components that were separated by intervening brain tissue and connected with each other by tiny vessels. This basic configuration seems to explain the unexpected postoperative recurrence of cavernous malformations and/or rebleeding from the residual lesions.

The characterization of testicular cell (TC)-specific T-cell clones induced by intratesticular Listeria monocytogenes infection: TC-specific T cells with atypical cytokine profile transfer orchitis.

A unilateral infection of Listeria monocytogenes into the testis of mice induces not only Listeria-specific T cells but also autoreactive T cells that can transfer experimental autoimmune orchitis (EAO) into naive mice. To investigate the characteristics of the autoreactive T cells, we established six testicular cell (TC)-specific T-cell clones from the spleen of the intratesticularly infected mice. All the clones expressed CD4 and T-cell receptor (TCR) alpha beta, and four of the six clones expressed V beta 8. They showed proliferative response to TC in the presence of syngeneic spleen antigen-presenting cells, but did not cross-react to Listeria antigen (Ag). They produced interferon-gamma (IFN-gamma) when stimulated with TC, but interleukin-2 (IL-2), IL-4 and IL-10 were undetectable. IL-2 production was not detected even when they were restimulated with TC after a 10-day resting culture without Ag and IL-2, although they proliferated in the restimulation culture. Even in the presence of anti-IL-2 mAb, the TC-specific T-cell clones showed proliferative response against TC. The observations indicate that the TC-specific IFN-gamma-producing T cells proliferate in the absence of autocrine. Both intravenous and intratesticular injection of these clones transferred EAO in syngeneic naive mice. These results suggest that L. monocytogenes infection in the testis induces autoreactive orchitogenic CD4+ T cells without cross-reactivity to bacterial Ag. Furthermore, these data demonstrate that CD4+ T cells with an atypical cytokine profile can efficiently cause EAO.

Evidence that the same gamma delta T cells respond during infection-induced and autoimmune inflammation.

Inflammatory responses are induced in both testes of a mouse following injection of Listeria monocytogenes into one testis. Although the uninjected testis contains no detectable bacteria, it undergoes an autoimmune attack. Normally, the testis lacks lymphocytes, but in the infected and autoimmune state, both gamma delta and alpha beta T cells are found as infiltrates. Here, we have examined the repertoire of the infiltrating gamma delta T cells, using two different methods, and found a high frequency of V gamma 6/V delta 1 gamma delta T cells in both infected and autoimmune testes. All of these expressed the invariant V gamma 6/V delta 1 TCR previously reported. However, secondary gamma and delta transcripts present within V gamma 6/V delta 1 hybridomas indicated nonclonality. Interestingly, some of these secondary transcripts were derived from gamma gene rearrangements not previously found in this gamma delta T cell subset, implying a difference in its origin. The increase in V gamma 6/V delta 1 cells observed here in both infected and autoimmune testes, together with our previous finding of a preferential response by the same subset in Listeria-infected liver, indicates that their response is triggered by the inflammation rather than by the infectious agent or because they are already resident in the tissue. We and others have previously reported that the presence of gamma delta T cells during certain inflammatory conditions correlates with less host tissue damage. This result, together with the evidence presented here, further implies that a response by the V gamma 6/V delta 1 subset in some way exerts a controlling influence on the host inflammatory response.

Macrophages activated by Listeria monocytogenes induce organ-specific autoimmunity.

We have previously reported an experimental autoimmune model induced by the local infection of Listeria monocytogenes. The unilateral inoculation of virulent Listeria into a testis of a normal mouse induced a delayed-type hypersensitivity response against testicular antigen and caused autoimmune orchitis in the contralateral testis. The orchitis was transferred to naive mice by T cells from the intratesticularly infected mice. In this paper, we demonstrated that avirulent Listeria, which lacks the expression of listeriolysin O, failed to induce any anti-testicular responses or contralateral orchitis even when it was inoculated at a high dose into the testis. Furthermore, the intraperitoneal inoculation of virulent Listeria with testicular antigen induced the anti-testicular responses and orchitis although intraperitoneal inoculation of testicular antigen with avirulent Listeria failed to induce them. The difference between virulent and avirulent Listeria in the induction of anti-testicular responses was supposed to be dependent on the difference in macrophage activation by the two bacterial strains because, first, the anti-testicular responses were elicited in normal mice when macrophages from virulent Listeria-infected mice were intraperitoneally transferred with testicular antigen although no viable bacteria were detected from the macrophages, and secondly, in contrast, the intraperitoneal co-inoculation of macrophages from avirulent Listeria-infected mice and testicular antigen failed to elicit any anti-testicular responses. Finally, we found that the virulent Listeria-induced macrophages expressed a higher level of CD80 (B7-1) and CD86 (B7-2) molecules than did the avirulent Listeria-induced macrophages and naive peritoneal macrophages. These results thus suggest that virulent Listeria activates macrophages to induce autoreactive T cells while avirulent Listeria does not. The up-regulation of B7 molecules by virulent Listeria infection is a candidate of the mechanism for the activation of autoreactive T cells.

The induction of renal autoantigen-specific T cells by a local Listeria monocytogenes infection.

In order to examine the possibility that a local chronic infection can induce organ-specific autoimmune disease, we inoculated unilateral kidneys with viable Listeria monocytogenes (intrarenal infection). The delayed footpad reaction against syngeneic kidney homogenate (KH) became positive from 1 week after initiating the intrarenal infection. A proliferative response of the spleen T cells from the infected mice was also observed against KH from 1 week after initiating the intrarenal infection, but no such response was seen against liver homogenate (LH). In contrast, an intravenous Listeria infection did not induce a delayed footpad reaction or proliferative response against KH, suggesting that these autoimmune responses were not caused by molecular mimicry between renal antigens and Listeria antigens. Furthermore, the ability to transfer the autoimmune response of spleen T cells from intrarenally infected mice was examined. The transferred mice showed a positive delayed footpad reaction against KH and an interstitial infiltration of mononuclear cells in their kidneys. These results demonstrate that the intrarenal Listeria infection induced renal autoantigen-specific T cells, which subsequently induced an autoimmune interstitial nephritis (AIN). The autoreactive T cells were all induced without immunization by autoantigens mixed with complete Freund's adjuvant. Based on these findings, we propose that a local bacterial infection may induce an autoimmune response against autoantigens in the infected organ and subsequently trigger organ-specific autoimmune disease.

Bacterial infection of the testis leading to autoaggressive immunity triggers apparently opposed responses of alpha beta and gamma delta T cells.

The mechanisms that lead to the breakdown of self-tolerance and testis-specific immune reactivity in the murine orchitis model are understood only in part. We investigated the histopathologic and immunologic consequences of a unilateral bacterial (Listeria monocytogenes) infection of the testis. Both infected and contralateral sides of this bilateral organ suffered severe inflammatory responses despite a conspicuous absence of bacteria in the contralateral tissue. Also, in both testicles, T cell populations increased, involving both alpha beta and gamma delta T cell subsets. Concomitant with the bilateral orchitis, testis-specific delayed type hypersensitivity and Ab responses developed. Ab depletion experiments indicated that in this orchitis model, as in others, alpha beta T cells are initiators of the autoaggressive reactivity. In contrast, Ab depletion of gamma delta T cells accelerated the inflammatory response in both testicles, suggesting a regulatory role for this type of T cells in both infection-induced and autoimmune orchitis.

Infection of Mycobacterium bovis bacillus Calmette-Guérin in antibody-mediated gamma delta T-cell-depleted mice.

To evaluate the hypothesis that gamma delta T cells participate in protective immunity against mycobacterial infection, we depleted gamma delta T cells from mice by administration of anti-T-cell receptor (TCR)gamma delta monoclonal antibody (mAb) and analysed protection against Mycobacterium bovis bacillus Calmette-Guérin (BCG). The gamma delta T-cell-depleted mice did not show any exaggerated bacterial multiplication compared with control mice. In contrast, alpha beta T-cell-depleted mice, which were administrated anti-TCR alpha beta mAb before BCG infection, showed a depressed protective immunity. These results suggest that gamma delta T cells are not essential for coping with a primary BCG infection.

Generation and characterization of a continuous line of CD8+ suppressively regulatory T lymphocytes which down-regulates experimental autoimmune orchitis (EAO) in mice.

We have previously shown that two injections with viable syngeneic testicular germ cells (TC) alone developed experimental autoimmune orchitis (EAO) in C3H/He mice, and that the induction of antigen-specific tolerance in this EAO model is associated with the generation of antigen-specific suppressively regulatory T (Ts) cells. For the elucidation of the nature of these Ts cells, a murine Ts cell line (designated Ts-A) was established. This line was generated from the spleen cells of C3H/He mice which had received three i.v. injections of a soluble (deaggregated) form of murine testicular antigen (mTA), followed by the repeated selection of these spleen lymphocytes in vitro by stimulation with mTA. Adoptive transfer of Ts-A cells into naive syngeneic mice immediately before the first TC injection was found to downgrade EAO in actively immunized recipients. The transferred Ts-A cells significantly inhibited the cellular immune response to TC in the recipients in an antigen-specific manner, but these cells had no inhibitory effect on the humoral immune response to TC. This line could also inhibit in vitro syngeneic TC-driven proliferation of orchitogenic lymphocytes. Surface phenotype of this line was CD8+, CD4-, Thy-1.2+, CD3+, and TCR alpha beta+. These findings may suggest an in vivo role for suppressively regulatory lymphocytes, capable of inhibiting helper T cells, in the regulation of EAO.