Immunological and tumor-intrinsic mechanisms mediate the synergistic growth suppression of experimental glioblastoma by radiotherapy and MET inhibition.

The hepatocyte growth factor (HGF)/MET signaling pathway has been proposed to be involved in the resistance to radiotherapy of glioblastoma via proinvasive and DNA damage response pathways.Here we assessed the role of the MET pathway in the response to radiotherapy in vitro and in vivo in syngeneic mouse glioma models. We find that the murine glioma cell lines GL-261, SMA-497, SMA-540 and SMA-560 express HGF and its receptor MET and respond to exogenous HGF with MET phosphorylation. Glioma cell viability or proliferation are unaffected by genetic or pharmacological MET inhibition using tepotinib or CRISPR/Cas9-engineered Met gene knockout and MET inhibition fails to sensitize glioma cells to irradiation in vitro. In contrast, the combination of tepotinib with radiotherapy prolongs survival of orthotopic SMA-560 or GL-261 glioma-bearing mice compared with radiotherapy or tepotinib treatment alone. Synergy is lost when such experiments are conducted in immunodeficient Rag1-/- mice, and, importantly, also when Met gene expression is disrupted in the tumor cells. Combination therapy suppresses a set of pro-inflammatory mediators including matrix metalloproteases that are upregulated by radiotherapy alone and that have been linked to poor outcome in glioblastoma. Several of these mediators are positively regulated by transforming growth factor (TGF)-ß, and pSMAD2 levels as a surrogate marker of TGF-ß pathway activity are suppressed by combination treatment. We conclude that synergistic suppression of experimental syngeneic glioma growth by irradiation and MET inhibition requires MET expression in the tumor as well as an intact immune system. Clinical evaluation of this combined strategy in newly diagnosed glioblastoma is warranted.

Synergistic growth inhibition mediated by dual PI3K/mTOR pathway targeting and genetic or direct pharmacological AKT inhibition in human glioblastoma models.

Molecular genetic aberrations in the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway are common in human cancers including glioblastoma, yet, novel therapeutic approaches targeting this pathway in glioblastoma have not been successful. We hypothesized that molecular profiling in combination with in vitro drug sensitivity testing allows to identify signatures associated with sensitivity or resistance to PI3K/mTOR pathway inhibition. We analyzed the molecular mechanisms determining sensitivity to PI3K/mTOR inhibition using gene silencing or pharmacological target inhibition and proliferation, clonogenicity, or spherogenicity as readouts, in human long-term glioma cell (LTC) lines and glioma-initiating cells (GIC). Cultured glioma cells were universally sensitive to growth inhibition induced by PQR309, a novel, dual pan-PI3K/mTOR antagonist. Cells exhibited profound growth arrest, but little apoptotic or necrotic cell death as confirmed by electron microscopy; yet, there was evidence of senescence. Cell lines with high basal levels of phosphorylated (active) AKT, low levels of phosphorylated (inactive) protein translation repressor eukaryotic initiation factor (eIF) 4E-binding protein 1 (p4E-BP1), and high levels of Ser9-phosphorylated (inactive) glycogen synthase kinase 3 beta (pGSK3ß) were more sensitive to PQR309. Accordingly, the activity of PQR309 was synergistically enhanced by AKT gene silencing or direct pharmacological AKT inhibition. In vivo studies confirmed the anti-glioma activity of PQR309 alone or in combination with AKT inhibition in the orthotopic LN-229 glioma xenograft model in nude mice. These data justify to explore combined targeted therapy approaches in glioblastoma that aim at down-regulating AKT function to enhance the therapeutic potential of dual PI3K/mTOR inhibitors.

Profound, durable and MGMT-independent sensitivity of glioblastoma cells to cyclin-dependent kinase inhibition.

TG02 is a novel cyclin-dependent kinase (CDK) inhibitor and thought to act mainly via CDK-9 inhibition-dependent depletion of short-lived oncoproteins such as MCL-1 or c-MYC. We studied the activity of TG02 in 9 human long-term glioma cell lines (LTC) and 5 glioma-initiating cell lines (GIC) using various cell death assays in vitro and in the LN-229 LTC and ZH-161 GIC models in vivo. TG02 exhibits strong anti-tumor cell activity with EC50 concentrations in the nanomolar range. Median survival in the LN-229 and ZH-161 models was moderately prolonged by TG02. Neither constitutive CDK levels nor those of MCL-1 or c-MYC correlated with sensitivity to TG02. Cdk-9 or cdk-5 gene silencing alone did not fully reproduce the effects of TG02. C-myc gene silencing inhibited cell growth, but did not modulate TG02 activity. Electron microscopy revealed cell death to be essentially apoptotic. High concentrations of TG02 induced annexin V binding and minor caspase 3 cleavage, but the pan-caspase inhibitor, zVAD-fmk, or BCL-2 or MCL-1 gene transfer only moderately attenuated TG02-induced cell death, and caspase inhibition did not prevent loss of MCL-1 or c-MYC. TG02 activity was independent of O6 -methylguanine DNA methyltransferase expression. Repetitive exposure to TG02 did not generate an acquired TG02 resistance phenotype, but accumulation of MCL-1, loss of c-MYC, or senescence. TG02 is a highly potent apoptosis-inducing agent in glioma cells in vitro. Caspase inhibition does not rescue TG02-treated cells and repetitive exposure fails to confer acquired resistance, supporting the clinical evaluation of TG02 in glioblastoma.

Epidermal growth factor receptor and ligand family expression and activity in glioblastoma.

Epidermal growth factor family of receptor tyrosine kinases (ERBB) family cell surface receptors, including epidermal growth factor receptor (EGFR/ERBB1), are phosphorylated upon binding by various EGF family ligands and signal via multiple kinase pathways. EGFR signaling is enhanced because of mutational activation of EGFR in almost half of glioblastomas, the most common malignant primary brain tumor. Therapeutic targeting of EGFR in glioblastoma has remained largely unsuccessful. Here, we profiled nine long-term (LTC) and five glioma-initiating (GIC) cell lines for expression and activation of ERBB family receptors and expression of their ligands. Receptors and ligands were abundantly expressed, with patterns overall similar to glioblastoma expression profiles in vivo as deposited in The Cancer Genome Atlas database. No differences between LTC and GIC emerged. Irrespective of ligand or receptor expression, neither an EGFR antibody, erbitux, nor an EGFR tyrosine kinase inhibitor, gefitinib, were particularly active against LTC or GIC at clinically relevant concentrations. Self-renewal capacity of GIC was severely compromised by epidermal growth factor (EGF) withdrawal, but rescued by transforming growth factor alpha (TGF-α), although not by neuregulin-1 (NRG-1). Subcellular fractionation indicated high levels of nuclear phosphorylated EGFR in all LTC and GIC. In LN-229 cells, pERBB2 and pERBB3 were also detected in the nucleus. Nuclear pERBB2 was less sensitive, whereas pERBB3 was induced, in response to gefitinib. This study provides an extensive characterization of human glioma cell models, including stem-like models, with regard to ERBB receptor/ligand expression and signaling. Redundant signaling involving multiple ERBB family ligands and receptors may contribute to the challenges of developing more effective EGFR-targeted therapies for glioblastoma.

Salivary proteome profiling of oral squamous cell carcinoma in a Hungarian population.

Oral squamous cell carcinoma (OSCC) is the seventh most common malignancy and the ninth most frequent cause of cancer death in Europe. Within Europe, Hungary has one of the highest rates of OSCC incidence and mortality. Thus, there is an urgent need to improve early detection. Saliva, as a readily available body fluid, became an increasingly important substance for the detection of biomarkers for many diseases. Different research groups have identified salivary biomarkers specific for OSCC for different countries. In this study, saliva samples of Hungarian patients with OSCC were studied to discover disease-specific and perhaps region-specific biomarkers. LC-mass spectrometry (MS)/MS analysis on a linear ion trap-Orbitrap mass spectrometer was used for qualitative and quantitative salivary protein profiling. More than 500 proteins were identified from saliva by shotgun proteomics. The up- and downregulated proteins in the saliva of patients with OSCC highlighted the importance of protein-protein interaction networks involving the immune system and proteolysis in disease development. Two potential biomarkers from our shotgun analysis and a third candidate reported earlier by a Taiwanese group were further examined by ELISA on a larger reference set of samples. Resistin, a biomarker reported in Taiwan but not validated in our study, highlights the necessity of application of standardized analysis methods in different ethnic or geographical populations to identify biomarkers with sufficient specificity and sensitivity.

Antitumor Activity of DLX1008, an Anti-VEGFA Antibody Fragment with Low Picomolar Affinity, in Human Glioma Models.

Angiogenesis mediated by vascular endothelial growth factor (VEGF) is a hallmark of glioblastoma. Based on the response rate and improved progression-free survival, although not on overall survival, the 149-kDa anti-VEGF-A IgG antibody bevacizumab (Avastin) has been approved in the United States and Japan for recurrent glioblastoma and in Japan for newly diagnosed glioblastoma; however, it is not approved in the EU. Here we characterize the biologic activity of DLX1008, a 26-kDa anti-VEGF-A single-chain antibody fragment that shows 30-fold stronger affinity to human VEGF-A than bevacizumab. The small molecular size of DLX1008 is predicted to result in improved target coverage over bevacizumab. DLX1008 showed superiority to bevacizumab in the inhibition of VEGF-A binding to VEGF receptor (VEGFR) 1 in enzyme-linked immunosorbent assay by a factor of around 10 and comparable efficacy for the inhibition of VEGF-A-stimulated VEGFR2 dimerization. In a tube-formation assay with human cerebral microvascular endothelial cells, DLX1008 was at least as active as bevacizumab. In vivo, DLX1008 delayed growth in a mouse subcutaneous U87 xenograft model (P = 0.0021) and improved survival in a mouse orthotopic U87 xenograft model (P = 0.00026). Given the exceptionally high affinity and small molecular size of DLX1008, these data warrant further clinical development of DLX1008 as an antiangiogenic agent in glioblastoma.

Negative control of the HGF/c-MET pathway by TGF-ß: a new look at the regulation of stemness in glioblastoma.

Multiple target inhibition has gained considerable interest in combating drug resistance in glioblastoma, however, understanding the molecular mechanisms of crosstalk between signaling pathways and predicting responses of cancer cells to targeted interventions has remained challenging. Despite the significant role attributed to transforming growth factor (TGF)-ß family and hepatocyte growth factor (HGF)/c-MET signaling in glioblastoma pathogenesis, their functional interactions have not been well characterized. Using genetic and pharmacological approaches to stimulate or antagonize the TGF-ß pathway in human glioma-initiating cells (GIC), we observed that TGF-ß exerts an inhibitory effect on c-MET phosphorylation. Inhibition of either mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) or phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) signaling pathway attenuated this effect. A comparison of c-MET-driven and c-MET independent GIC models revealed that TGF-ß inhibits stemness in GIC at least in part via its negative regulation of c-MET activity, suggesting that stem cell (SC) maintenance may be controlled by the balance between these two oncogenic pathways. Importantly, immunohistochemical analyses of human glioblastoma and ex vivo single-cell gene expression profiling of TGF-ß and HGF confirm the negative interaction between both pathways. These novel insights into the crosstalk of two major pathogenic pathways in glioblastoma may explain some of the disappointing results when targeting either pathway alone in human glioblastoma patients and inform on potential future designs on targeted pharmacological or genetic intervention.

Novel TIE-2 inhibitor BAY-826 displays in vivo efficacy in experimental syngeneic murine glioma models.

Targeting the vascular endothelial growth factor signaling axis in glioblastoma inevitably leads to tumor recurrence and a more aggressive phenotype. Therefore, other angiogenic pathways, like the angiopoietin/tunica interna endothelial cell kinase (TIE) signaling axis, have become additional targets for therapeutic intervention. Here, we explored whether targeting the receptor tyrosine kinase TIE-2 using a novel, highly potent, orally available small molecule TIE-2 inhibitor (BAY-826) improves tumor control in syngeneic mouse glioma models. BAY-826 inhibits TIE-2 phosphorylation in vitro and in vivo as demonstrated by suppression of Angiopoietin-1- or Na3 VO4 -induced TIE-2 phosphorylation in glioma cells or extracts of lungs from BAY-826-treated mice. There was a trend toward prolonged survival upon single-agent treatment in two of four models (SMA-497 and SMA-540) and there was a significant survival benefit in one model (SMA-560). Co-treatment with BAY-826 and irradiation was ineffective in one model (SMA-497), but provided synergistic prolongation of survival in another (SMA-560). Decreased vessel densities and increased leukocyte infiltration were observed, but might be independent processes as the effect was also observed in single treatment modalities. These data demonstrate that TIE-2 inhibition may improve tumor response to treatment in highly vascularized tumors such as glioblastoma.

Autocrine VEGFR1 and VEGFR2 signaling promotes survival in human glioblastoma models in vitro and in vivo.

BACKGROUND: Although the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) system has become a prime target for antiangiogenic treatment, its biological role in glioblastoma beyond angiogenesis has remained controversial. METHODS: Using neutralizing antibodies to VEGF or placental growth factor (PlGF) or the tyrosine kinase inhibitor, cediranib, or lentiviral gene silencing, we delineated autocrine signaling in glioma cell lines. The in vivo effects of VEGFR1 and VEGFR2 depletion were evaluated in orthotopic glioma xenograft models. RESULTS: VEGFR1 and VEGFR2 modulated glioma cell clonogenicity, viability, and invasiveness in vitro in an autocrine, cell-line-specific manner. VEGFR1 silencing promoted mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling, whereas VEGFR2 silencing resulted in cell-type dependent activation of the protein kinase B (PKB)/AKT and MAPK/ERK pathways. These responses may represent specific escape mechanisms from VEGFR inhibition. The survival of orthotopic glioma-bearing mice was prolonged upon VEGFR1 silencing in the LNT-229, LN-308, and U87MG models and upon VEGFR2 silencing in LN-308 and U87MG. Disruption of VEGFR1 and VEGFR2 signaling was associated with decreased tumor size, increased tumor necrosis, or loss of matrix metalloproteinase 9 (MMP9) immunoreactivity. Neutralizing VEGF and PlGF by specific antibodies was superior to either antibody treatment alone in the VEGFR1-dependent LNT-229 model. CONCLUSIONS: Differential dependence on autocrine signaling through VEGFR1 and VEGFR2 suggests a need for biomarker-stratified VEGF(R)-based therapeutic approaches to glioblastoma.

A pleura óriás fibrosus tumora légzési elégtelenséggel szövodve.

Surgery of mostly benign giant tumours involving large part of the chest is a special surgical challenge. The problems comprise difficulties of surgical technique, management of the narcosis and postoperative intensive care. An additional peculiarity of our case is the extreme confliction of the otherwise presumably evident indication for surgery. Our 64-years-old male patient has been suffering from increasing dyspnoea on exercise for one and a half years. A chest X-ray performed for other reasons demonstrated a large, expansive structural change in the right thoracic cavity. Lung biopsy performed as part of respiratory investigations, which showed a solitaire fibrous tumour of the pleura. Oncological consultation suggested consideration of surgery. The general condition of the patient worsened rapidly in the course of preassessment; he had to be admitted to ICU due to dyspnoea and atrial fibrillation, where respiratory insufficiency developed and required respiratory therapy. Surgery was performed in this high anaesthetic risk patient, since removal of the tumour was the only chance for surviving. The patient left the hospital healthy after successful surgery and cumbersome postoperative period. He returned to his original job and no recurrence was detected one year after surgery.

Modulation of cerebral endothelial cell function by TGF-ß in glioblastoma: VEGF-dependent angiogenesis versus endothelial mesenchymal transition.

Glioblastoma are among the most angiogenic tumors. The molecular mechanisms that control blood vessel formation by endothelial cells (EC) in glioblastoma remain incompletely understood. Transforming growth factor-ß (TGF-ß) is a key regulatory cytokine that has proinvasive and stemness-maintaining autocrine properties in glioblastoma and confers immunosuppression to the tumor microenvironment. Here we characterize potential pro- and anti-angiogenic activities of TGF-ß in the context of glioblastoma in vitro, using human brain-derived microvascular endothelial cells (hCMEC/D3) and glioblastoma-derived endothelial cells (GMEC) as model systems. We find that TGF-ß induces vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) mRNA expression and protein release in a TGF-ß receptor (TßR) II / activin-like kinase (ALK)-5-dependent manner under normoxia and hypoxia, defining potential indirect proangiogenic activity of TGF-ß in glioblastoma. In parallel, exogenous TGF-ß has also inhibitory effects on EC properties and induces endothelial-mesenchymal transition (EndMT) in hCMEC and GMEC. Accordingly, direct inhibition of endogenous TGF-ß/ALK-5 signalling increases EC properties such as tube formation, von-Willebrand factor (vWF) and claudin (CLDN) 5 expression. Yet, the supernatant of TGF-ß-stimulated hCMEC and GMEC strongly promotes EC-related gene expression and tube formation in a cediranib-sensitive manner. These observations shed light on the complex pro- and anti-angiogenic pathways involving the cross-talk between TGF-ß and VEGF/PLGF signalling in glioblastoma which may involve parallel stimulation of angiogenesis and EndMT in distinct target cell populations.

Differential regulation of TGF-ß-induced, ALK-5-mediated VEGF release by SMAD2/3 versus SMAD1/5/8 signaling in glioblastoma.

BACKGROUND: The transforming growth factor (TGF)-ß and vascular endothelial growth factor (VEGF) pathways have a major role in the pathogenesis of glioblastoma, notably immunosuppression, migration, and angiogenesis, but their interactions have remained poorly understood. METHODS: We characterized TGF-ß pathway activity in 9 long-term glioma cell lines (LTCs) and 4 glioma-initiating cell lines (GICs) in relation to constitutive and exogenous TGF-ß-induced VEGF release. Results were validated using The Cancer Genome Atlas transcriptomics data. RESULTS: Glioma cells exhibit heterogeneous patterns of constitutive TGF-ß pathway activation reflected by phosphorylation not only of SMAD2 and SMAD3 but also of SMAD1/5/8. Constitutive TGF-ß pathway activity depends on the type I TGF-ß receptor, ALK-5, and accounts for up to 69% of constitutive VEGF release, which is positively regulated by SMAD2/3 and negatively regulated by SMAD1/5/8 signaling in a cell line-specific manner. Exogenous TGF-ß induces VEGF release in most cell lines in a SMAD- and ALK-5-dependent manner. There is no correlation between the fold induction of VEGF secretion induced by TGF-ß compared with hypoxia. The role of SMAD5 signaling is highly context and cell-line dependent with a VEGF inhibitory effect at low TGF-ß and pSMAD2 levels and a stimulatory effect when TGF-ß is abundant. CONCLUSIONS: TGF-ß regulates VEGF release by glioma cells in an ALK-5-dependent manner involving SMAD2, SMAD3, and SMAD1/5/8 signaling. This crosstalk between the TGF-ß and VEGF pathways may open up new avenues of biomarker-driven exploratory clinical trials focusing on the microenvironment in glioblastoma.

MicroRNA-mediated down-regulation of NKG2D ligands contributes to glioma immune escape.

Malignant gliomas are intrinsic brain tumors with a dismal prognosis. They are well-adapted to hypoxic conditions and poorly immunogenic. NKG2D is one of the major activating receptors of natural killer (NK) cells and binds to several ligands (NKG2DL). Here we evaluated the impact of miRNA on the expression of NKG2DL in glioma cells including stem-like glioma cells. Three of the candidate miRNA predicted to target NKG2DL were expressed in various glioma cell lines as well as in glioblastomas in vivo: miR-20a, miR-93 and miR-106b. LNA inhibitor-mediated miRNA silencing up-regulated cell surface NKG2DL expression, which translated into increased susceptibility to NK cell-mediated lysis. This effect was reversed by neutralizing NKG2D antibodies, confirming that enhanced lysis upon miRNA silencing was mediated through the NKG2D system. Hypoxia, a hallmark of glioblastomas in vivo, down-regulated the expression of NKG2DL on glioma cells, associated with reduced susceptibility to NK cell-mediated lysis. This process, however, was not mediated through any of the examined miRNA. Accordingly, both hypoxia and the expression of miRNA targeting NKG2DL may contribute to the immune evasion of glioma cells at the level of the NKG2D recognition pathway. Targeting miRNA may therefore represent a novel approach to increase the immunogenicity of glioblastoma.

Conjugates of cytochrome c and antennapedia peptide activate apoptosis and inhibit proliferation of HeLa cancer cells.

Polycationic cell-penetrating peptides (CPPs) deliver macromolecules into cells without losing the functional properties of the cargoed macromolecule. The aim of this study was to determine whether exogenous cytochrome c is delivered to HeLa cervical carcinoma cells by the CPP antennapedia (Antp) and activates apoptosis. HeLa cervical carcinoma cells were treated with conjugated Antp-SMCC-cytochrome c (cytochrome c chemically conjugated to Antp) or with non-conjugated Antp and cytochrome c. Sensitivity to the treatments was determined by the clonogenic assay (proliferation) and by immunoblot analysis (apoptosis activation). We report that conjugated Antp-SMCC-cytochrome c activated apoptosis in HeLa cells as demonstrated by poly (ADP-ribose) polymerase 1 (PARP-1) cleavage and inhibited their proliferation. The Antp-SMCC-cytochrome c-induced apoptosis was inhibited by z-VAD-fmk, a pan-caspase inhibitor peptide. Unconjugated Antp or cytochrome c demonstrated no inhibitory effect on survival and proliferation. Our results suggest that chemical coupling of cytochrome c to CPPs may present a possible strategy for delivering cytochrome c into cells and for activating apoptosis.

TRA-1/GLI controls the expression of the Hox gene lin-39 during C. elegans vulval development.

The vulva of the Caenorhabditis elegans hermaphrodite develops from a subset of six vulval precursor cells (VPCs) by the combined effect of the Ras, Wingless and Notch signaling cascades, and of three redundant synMuv (synthetic Multivulva) pathways grouped into classes A, B and C. Here we show that signaling via the GLI- (Glioma-associated protein) like transcription factor TRA-1, which is the terminal regulator of the C. elegans sex determination cascade, is a newly discovered pathway specifying vulval cell fates. We found that TRA-1 accumulates in, and regulates the fusion process of, cells (including the VPCs and hypodermal cells) involved in vulval patterning. TRA-1 also influenced the expression of the Hox gene lin-39, a central regulator of vulval development. Furthermore, inactivation of tra-1, which transforms animals with hermaphrodite-specific karyotype into males, promoted vulval induction in synMuv A, but not in synMuv B, mutant background. This implies that TRA-1 interacts with the class B synMuv genes, many of which are involved in chromatin-mediated transcriptional repression of cell proliferation. These results may help to understand how compromised GLI activity in humans leads to cancer. Together, we suggest that the GLI protein family involved in several key developmental processes in both invertebrates and vertebrates regulates somatic cell fates through influencing, at least in part, the expression of specific Hox genes.

Estrogen enhances expression of the complement C5a receptor and the C5a-agonist evoked calcium influx in hormone secreting neurons of the hypothalamus.

In the present study we examined presence of the complement C5a receptor (C5aR) in hypothalamic neurosecretory neurons of the rodent brain and effect of estrogen on C5aR expression. Whole cell patch clamp measurements revealed that magnocellular neurons in the supraoptic and paraventricular nuclei of hypothalamic slices of the rats responded to the C5aR-agonist PL37-MAP peptide with calcium ion current pulses. Gonadotropin-releasing hormone (GnRH) producing neurons in slices of the preoptic area of the mice also reacted to the peptide treatment with inward calcium current. PL37-MAP was able to evoke the inward ion current of GnRH neurons in slices from ovariectomized animals. The amplitude of the inward pulses became higher in slices obtained from 17beta-estradiol (E2) substituted mice. Calcium imaging experiments demonstrated that PL37-MAP increased the intracellular calcium content in the culture of the GnRH-producing GT1-7 cell line in a concentration-dependent manner. Calcium imaging also showed that E2 pretreatment elevated the PL37-MAP evoked increase of the intracellular calcium content in the GT1-7 cells. The estrogen receptor blocker Faslodex in the medium prevented the E2-evoked increase of the PL37-MAP-triggered elevation of the intracellular calcium content in the GT1-7 cells demonstrating that the effect of E2 might be related to the presence of estrogen receptor. Real-time PCR experiments revealed that E2 increased the expression of C5aR mRNA in GT1-7 neurons, suggesting that an increased C5aR synthesis could be involved in the estrogenic modulation of calcium response. These data indicate that hypothalamic neuroendocrine neurons can integrate immune and neuroendocrine functions. Our results may serve a better understanding of the inflammatory and neurodegeneratory diseases of the hypothalamus and the related neuroendocrine and autonomic compensatory responses.