Edward Visse

The Adult Human Brain Harbors Multipotent Perivascular Mesenchymal Stem Cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.

Regression of intracerebral rat glioma isografts by therapeutic subcutaneous immunization with interferon-γ, interleukin-7, or B7-1-transfected tumor cells

Progress in the definition of the roles of various costimulators and cytokines in determining the type and height of immune responses has made it important to explore genetically altered tumor cells expressing such molecules for therapeutic immunizations. We have studied the effect of therapeutic subcutaneous (s.c.) immunizations on the growth of preexisting intracerebral brain tumor isografts in the rat. Transfectant glioma cell clones expressing either rat interferon-γ (IFN-γ), rat interleukin-7 (IL-7), or rat B7-1 were selected. After irradiation (80 Gy) the clones were used for immunization (administered in up to four s.c. doses in a hind leg over 14-day intervals starting 1 day after the intracranial isografting of the parental tumor). Significant growth inhibition of the intracerebral parental tumors was induced by transfectants expressing IFN-γ and IL-7, respectively. The strongest effect was observed with IFN-γ-expressing cells, resulting in cures in 37% of the males and in 100% of the females. Immunization with IL-7 had a similar, strong initial effect, with significantly prolonged survival in the majority of the rats but a lower final cure rate (survival for >150 days). The B7-1-expressing tumor clones induced cures in seven of eight female rats; however, no cures were seen in the male rats. It was also shown that the B7-1-expressing cells were themselves strongly immunogenic in female rats, requiring high cell numbers to result in a progressively growing tumor upon s.c. isografting; this was not the case in male rats. As a whole, the results imply that despite the unfavorable location of intracerebral tumors, therapeutic s.c. immunizations with certain types of genetically altered tumor cells can induce complete regressions with permanent survival and without gross neurological or other apparent signs of brain damage. The present results demonstrate complete regressions when immunizations are initiated shortly after intracranial isografting, when the intracerebral tumor is small.

Cure of established, intracerebral rat gliomas induced by therapeutic immunizations with tumor cells and purified APC or adjuvant IFN-gamma treatment.

We have previously reported that immunizations with mutagen-induced immunogenic variants of a weakly immunogenic rat glioma could protect against isografts of the original tumor cells. In this study we show that prolonged survival and cures of rats with established gliomas in their brains can be achieved by therapeutic immunizations with tumor cell mutants, combined with in vitro and in vivo interferon (IFN)-gamma (adjuvant) treatment, or tumor cells admixed with semipurified syngeneic dendritic cells. Cure of rats with established intracerebral gliomas was possible when immunizations were initiated up to 5 days after intracerebral isografting of original tumor cells. Unexpectedly, immunizations combined with in vitro and in vivo IFN-gamma treatment or with admixed semipurified dendritic cells equalized the immunogenic potential of the original tumor cells and that of mutagen-induced immunogenic cell variants (tum-). This demonstrates that effective immunizations against a weakly immunogenic brain tumor can be achieved by different adjuvant concepts. The therapeutic effect of immunizations with tumor cells admixed with semipurified dendritic cells was highly significant in female rats, whereas only occasional cures and prolonged survival were recorded in male rats. The overall results show that therapeutic immunizations can indeed be effective against an established and growing intracerebral tumor.

Computerized image analysis as a tool to quantify infiltrating leukocytes : a comparison between high- and low-magnification images

The purpose of the present study was to establish a rapid and reproducible method for quantification of tissue-infiltrating leukocytes using computerized image analysis. To achieve this, the staining procedure, the image acquisition, and the image analysis method were optimized. Because of the adaptive features of the human eye, computerized image analysis is more sensitive to variations in staining compared with manual image analysis. To minimize variations in staining, an automated immunostainer was used. With a digital scanner camera, low-magnification images could be sampled at high resolution, thus making it possible to analyze larger tissue sections. Image analysis was performed by color thresholding of the digital images based on values of hue, saturation, and intensity color mode, which we consider superior to the red, green, and blue color mode for analysis of most histological stains. To evaluate the method, we compared computerized analysis of images with a ×100 or a ×12.5 magnification to assess leukocytes infiltrating rat brain tumors after peripheral immunizations with tumor cells genetically modified to express rat interferon-γ (IFN-γ) or medium controls. The results generated by both methods correlated well and did not show any significant differences. The method allows efficient and reproducible processing of large tissue sections that is less time-consuming than conventional methods and can be performed with standard equipment and software. (J Histochem Cytochem 49:1073–1079, 2001)

Immunization with mutagen-treated (tum-) cells causes rejection of nonimmunogenic rat glioma isografts.

The ethyl-N-nitrosourea-induced rat glioma N32 was treated with the mutagenic compoundN-methyl-N′-nitro-N-nitrosoguanidine and the surviving cells cloned by limiting dilution. Out of 20 clones tested 8 did not produce tumors subcutaneously even after challenge doses 3 log units above the minimal tumor dose for N32. All of 5 clones grew in a retarded manner intracerebrally but produced tumors in some animals. Preimmunizations with three of the rejected clones (tum−) gave protection against subcutaneous and intracerebral isografts of the unmutated N32. This effect could be enhanced if the cells used for immunizations were pretreated with interferon γ (IFNγ) for 48 h. If immunizations were started subsequent to challenge, only immunization with one of two tested tum− clones pretreated with IFNγ induced significant rejection against intracerebral N32 isografts. Both N32 and its tum− closes were MHC class I positive and MHC class II negative. IFNγ treatment enhanced the MHC class I expression with 20%–90% on the tum− clones and with 40% on N32. MHC class II expression could be induced on N32 cells after 7 days of IFNγ treatment but not on any of the tum− clones tested. We conclude that the enhancing effect of IFNγ treatment on tumor isograft rejection may depend on up-regulation of MHC class I but not of MHC class II. This investigation demonstrates that it is possible to induce rejection of weakly immunogenic intracerebral brain tumors by immunization with selected highly immunogenic tumor cell mutants. In conjunction with relevant cytokines, the cross-protective effect of these tum− variants might be further enhanced and serve as a model for immunotherapy against malignant human brain tumors.

Synergism between GM-CSF and IFNgamma: Enhanced immunotherapy in mice with glioma.

Glioblastoma multiforme is the most common malignant primary brain tumor and also one of the most therapy‐resistant tumors. Because of the dismal prognosis, various therapies modulating the immune system have been developed in experimental models. Previously, we have shown a 37–70% cure in a rat glioma model where rats were peripherally immunized with tumor cells producing IFNγ. On the basis of these results, we wanted to investigate whether a combination of GM–CSF and IFNγ could improve the therapeutic effect in a mouse glioma model, GL261 (GL‐wt). Three biweekly intraperitoneal (i.p.) immunizations with irradiated GM–CSF‐transduced GL261 cells (GL‐GM) induced a 44% survival in mice with intracranial glioma. While treatment of GL‐wt and GL‐GM with IFNγ in vitro induced upregulation of MHC I and MHC II on the tumor cells, it could not enhance survival after immunization. However, immunizations with GL‐GM combined with recombinant IFNγ at the immunization site synergistically enhanced survival with a cure rate of 88%. Tumors from mice receiving only 1 immunization on Day 10 after tumor inoculation were sectioned on Day 20 for analysis of leukocyte infiltration. Tumor volume was reduced and the infiltration of macrophages was denser in mice immunized with GL‐GM combined with IFNγ compared with that of both wildtype and nonimmunized mice. To our knowledge, this is the first study to demonstrate a synergy between GM–CSF and IFNγ in experimental immunotherapy of tumors, by substantially increasing survival as well as inducing a potent anti‐tumor response after only 1 postponed immunization.

Inhibition of Inducible Nitric Oxide Synthase Enhances Anti-tumour Immune Responses in Rats Immunized with IFN-gamma-Secreting Glioma Cells.

Interferon gamma (IFN‐γ) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN‐γ has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN‐γ (N32‐IFN‐γ), could enhance the anti‐tumour immune response. Initially, both a selective iNOS, l‐N6‐(1‐Iminoethyl)‐l‐lysine (l‐NIL), and non‐selective, N‐nitro‐l‐arginine methyl ester (l‐NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both l‐NIL and l‐NAME enhanced proliferation and production of IFN‐γ from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, l‐NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32‐IFN‐γ, and administered NOS inhibitors by intraperitoneal (i.p.) mini‐osmotic pumps, only splenocytes of rats treated with l‐NIL, but not l‐NAME, displayed an enhanced proliferation and production of IFN‐γ when re‐stimulated with N32 tumour cells. Based on these findings, l‐NIL was administered concurrently with N32‐IFN‐γ cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN‐γ‐based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects.

Inhibition of cyclooxygenase-2 enhances immunotherapy against experimental brain tumors.

Glioblastoma multiforme is the most common and aggressive malignant brain tumor in humans, and the prognosis is very poor despite conventional therapy. Immunotherapy represents a novel treatment approach, but the effect is often weakened by release of immune-suppressive molecules such as prostaglandins. In the current study, we investigated the effect of immunotherapy with irradiated interferon-γ (IFN-γ)-secreting tumor cells and administration of the selective cyclooxygease-2 (COX-2) inhibitor parecoxib as treatment of established rat brain tumors. COX-2 inhibition and immunotherapy significantly enhanced the long-term cure rate (81% survival) compared with immunotherapy alone (19% survival), and there was a significant increase in plasma IFN-γ levels in animals treated with the combined therapy, suggesting a systemic T helper 1 immune response. COX-2 inhibition alone, however, did neither induce cure nor prolonged survival. The tumor cells were identified as the major source of COX-2 both in vivo and in vitro, and unmodified tumor cells produced prostaglandin E2 in vitro, while the IFN-γ expressing tumor cells secreted significantly lower levels. In conclusion, we show that immunotherapy of experimental brain tumors is greatly potentiated when combined with COX-2 inhibition. Based on our results, the clinically available drug parecoxib may be added to immunotherapy against human brain tumors. Furthermore, the discovery that IFN-γ plasma levels can be used to determine the ongoing in vivo immune response has translational potential.

Enhanced expression of iNOS intratumorally and at the immunization site after immunization with IFNγ-secreting rat glioma cells

Nitric oxide (NO) can modulate both tumor growth and antitumor immune responses. In order to elucidate the mechanism of curative therapeutic immunization with IFNgamma-producing glioma cells, we examined the expression of inducible nitric oxide synthase (iNOS) in tissue sections from immunized animals. There was a significantly enhanced iNOS expression both intratumorally and at the immunization site. Although the mechanisms behind this dual expression of iNOS most probably are different, our results suggest a role for NO in both the induction and execution of the antitumor response.

Postimmunization with IFN-gamma-secreting glioma cells combined with the inducible nitric oxide synthase inhibitor mercaptoethylguanidine prolongs survival of rats with intracerebral tumors

High-grade gliomas are one of the most aggressive human tumors with <1% of patients surviving 5 years after surgery. Immunotherapy could offer a possibility to eradicate remnant tumor cells after conventional therapy. Experimental immunotherapy can induce partial cure of established intracerebral tumors in several rodent models. One reason for the limited therapeutic effects could be immunosuppression induced by both the growing tumor and the induced immune reaction. NO has been implicated in tumor-derived immune suppression in tumor-bearing hosts, and unspecific inhibitors of NO synthase have been shown to boost antitumor immunity. In this study, we show that the inducible NO synthase (iNOS)-specific inhibitor mercaptoethylguanidine (MEG) superiorly enhanced lymphocyte reactivity after polyclonal stimulation compared with the iNOS-specific inhibitor l-NIL and the unspecific NO synthase inhibitor l-NAME. Both iNOS inhibitors increased the number and proliferation of T cells but not of B cells. When combined during postimmunization with IFN-γ-secreting N32 rat glioma cells of rats harboring intracerebral tumors, only MEG increased the cure rate. However, this was only achieved when MEG was administered after immunizations. These findings implicate that NO has both enhancing and suppressive effects after active immunotherapy.