Marco Locatelli

Cancer‐promoting tumor‐associated macrophages: New vistas and open questions

Tumor‐associated macrophages (TAMs) are key components of the tumor macroenvironment. Cancer‐ and host cell‐derived signals generally drive the functions of TAMs towards an M2‐like polarized, tumor‐propelling mode; however, when appropriately re‐educated. TAMs also have the potential to elicit tumor destructive reactions. Here, we discuss recent advances regarding the immunobiology of TAMs and highlight open questions including the mechanisms of their accumulation (recruitment versus proliferation), their diversity and how to best therapeutically target these cells.

Proliferation of Transformed Somatotroph Cells Related to Low or Absent Expression of Protein Kinase A Regulatory Subunit 1A Protein

The two regulatory subunits (R1 and R2) of protein kinase A (PKA) are differentially expressed in cancer cell lines and exert diverse roles in growth control. Recently, mutations of the PKA regulatory subunit 1A gene (PRKAR1A) have been identified in patients with Carney complex. The aim of this study was to evaluate the expression of the PKA regulatory subunits R1A, R2A, and R2B in a series of 30 pituitary adenomas and the effects of subunit activation on cell proliferation. In these tumors, neither mutation of PRKAR1A nor loss of heterozygosity was identified. By real-time PCR, mRNA of the three subunits was detected in all of the tumors, R1A being the most represented in the majority of samples. By contrast, immunohistochemistry documented low or absent R1A levels in all tumors, whereas R2A and R2B were highly expressed, thus resulting in an unbalanced R1/R2 ratio. The low levels of R1A were, at least in part, due to proteasome-mediated degradation. The effect of the R1/R2 ratio on proliferation was assessed in GH3 cells, which showed a similar unbalanced pattern of R subunits expression, and in growth hormone-secreting adenomas. The R2-selective cAMP analog 8-Cl cAMP and R1A RNA silencing, stimulated cell proliferation and increased Cyclin D1 expression, respectively, in human and rat adenomatous somatotrophs. These data show that a low R1/R2 ratio promoted proliferation of transformed somatotrophs and are consistent with the Carney complex model in which R1A inactivating mutations further unbalance this ratio in favor of R2 subunits. These results suggest that low expression of R1A protein may favor cAMP-dependent proliferation of transformed somatotrophs.

Human glioma tumors express high levels of the chemokine receptor CX3CR1

The chemokine receptor CX3CR1 and its cognate ligand CX3CL1 (also known as fractalkine), are involved in central nervous system pathophysiology, in particular, in the cross-talk between neurons and microglia. It was therefore important to investigate the expression of CX3CR1 in gliomas, the most frequently occurring, malignant brain tumors. In a consecutive series of 70 patients with primary, central nervous glial tumors, CX3CR1 was highly expressed in tumor cells as assessed by RT-PCR mRNA and protein levels, and by immunohistochemistry, while the corresponding normal cells were negative. Receptor immuno-positivity did not correlate with histology, grade, chromosomal (1p,19q) deletion, or with methylation of the DNA repair gene promoter MGMT (O6-methylguanine-DNA methyltransferase). Thus, CX3CR1 expression is a frequent event in gliomas, irrespective of tumor classification and clinical severity. The molecular basis underlying CX3CR1 up-regulation and its functional biological significance remain to be determined.

Large Pituitary Hyperplasia in Severe Primary Hypothyroidism

Endocrinology and Diabetology Unit (E.P., A.T., B.A., S.C.), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Policlinico San Donato, 20097 San Donato Milanese, Milan, Italy; Department of Medical-Surgical Sciences (E.P., B.A., S.C.), University of Milan, 20097 San Donato Milanese, Milan, Italy; Department of Neurosurgery (M.L.), IRCCS Fondazione Ca’ Granda Policlinico, 20122 Milano, Milan, Italy; and Endocrinology Unit (A.G.L.), Department of Medical Sciences, University of Milan, IRCCS Fondazione Ca’ Granda Policlinico, 20122 Milan, Italy

A potential catastrophic trap: an unusually presenting sellar lesion

A 63‐year‐old man was admitted to our emergency unit complaining headache, vomit and vertigo. A MR of the brain showed an expanding lesion within the sellar region. A subsequent angio‐MR excluded any intracranial vascular malformations. Surprisingly, a cerebral angiography performed later on the basis of worsening of neurological signs and symptoms, demonstrated an aneurysm of the internal carotid artery. At the best of our knowledge, this is the first case of a thrombosis of an intracavernous carotid aneurysm mimicking a pituitary apoplexy documented by MR and angio‐MR. The treatment of a milder syndrome of pituitary apoplexy is still controversial. This case would favour conservative treatment opposed to surgery at least when an intracavernous extension or invasion of the adenoma would limit the opportunity of a complete tumour removal.

Endothelial Cells Lining Sporadic Cerebral Cavernous Malformation Cavernomas Undergo Endothelial-to-Mesenchymal Transition

Background and Purpose— Cerebral cavernous malformation (CCM) is characterized by multiple lumen vascular malformations in the central nervous system that can cause neurological symptoms and brain hemorrhages. About 20% of CCM patients have an inherited form of the disease with ubiquitous loss-of-function mutation in any one of 3 genes CCM1, CCM2, and CCM3. The rest of patients develop sporadic vascular lesions histologically similar to those of the inherited form and likely mediated by a biallelic acquired mutation of CCM genes in the brain vasculature. However, the molecular phenotypic features of endothelial cells in CCM lesions in sporadic patients are still poorly described. This information is crucial for a targeted therapy. Methods— We used immunofluorescence microscopy and immunohistochemistry to analyze the expression of endothelial-to-mesenchymal transition markers in the cavernoma of sporadic CCM patients in parallel with human familial cavernoma as a reference control. Results— We report here that endothelial cells, a cell type critically involved in CCM development, undergo endothelial-to-mesenchymal transition in the lesions of sporadic patients. This switch in endothelial phenotype has been described only in genetic CCM patients and in murine models of the disease. In addition, TGF-&bgr;/p-Smad- and &bgr;-catenin-dependent signaling pathways seem activated in sporadic cavernomas as in familial ones. Conclusions— Our findings support the use of common therapeutic strategies for both sporadic and genetic CCM malformations.

Adaptive deep brain stimulation controls levodopa-induced side effects in Parkinsonian patients

The potential superior benefits of adaptive deep brain stimulation (aDBS) approaches compared to classical, constantparameters DBS were already proven by scientific evidence from different research groups. aDBS provides better symptoms control in Parkinson’s disease patients by adapting the stimulation parameters to the patient’s clinical state estimated through the analysis of subthalamic neuronal oscillations (ie, local field potentials) in the beta band (13-30 Hz). Because aDBS administration was never systematically assessed during prolonged stimulation sessions in more ecologic conditions, we tested unilateral aDBS delivered for 2 hours, with specific focus on the concurrent administration of levodopa treatment, in freely moving parkinsonian patients. We therefore randomly administered aDBS and cDBS through an external wearable prototype in 10 PD patients with DBS electrode implant in 2 different experimental sessions taking place the 5th and the 6th day after surgery (Fig. 1A). Each experimental session lasted 2 hours, during which the patient, after a baseline assessment (OFF DBS and OFF medication, stimOFF/medOFF), received both levodopa and stimulation (aDBS or cDBS), thus allowing one to study the interaction between electrical and pharmacological stimulation (ON DBS and ON medication, stimON/medON). The patient was blind to the type of DBS received during the session. The clinical effects were blindly evaluated through the UPDRS III (motor part) and the Unified Dyskinesia Rating Scale (UDysRS). According to the gold standard, the clinical assessment was performed by a blinded video rater (rigidity scores were excluded from the analysis). The total electrical energy delivered (TEED) was used for energy efficiency assessment and adverse events were collected for safety assessment. The clinical scores were not significantly different between the 2 experimental sessions at baseline (stimOFF/ medOFF UPDRS III, aDBS vs cDBS: 37.0 6 16.8 vs 36.6 6 16.2; F1,9 5 0.2, P > .05). When the patient was under the effect of both levodopa and DBS (stimON/medON), we observed a similar improvement on global motor symptoms regardless to the type of DBS (UPDRS III percent change from baseline, aDBS vs cDBS: 246.1% 6 10.5% vs 240.1% 6 17.5%; F1,9 5 0.6, P > .05; Fig. 1B). Conversely, in this condition, aDBS was more effective on dyskinesias than cDBS (UDysRS score, aDBS vs cDBS: 11.7 6 67 vs 15.0 6 8.7; F1,9 5 6.1, P 5 .02; Fig. 1C). These results were obtained with an average power saving of 73.6% 6 22.9% in aDBS compared with cDBS (mean TEED aDBS vs cDBS: 44.6 6 47.9 lW vs 158.7 6 69.7 lW; F1,8 5 30.4, P 5 .0005). Throughout the entire experiment, we did not observe any serious adverse event specifically linked to DBS. These results support the idea that aDBS, being effective, efficient, and safe, when administered concomitantly to levodopa could help clinicians limit the severity of side effects induced by the transient summation of DBS stimulation and pharmacological therapy. However, the acute experimental setting, characterized by a microlesional

The Effects of Levodopa and Deep Brain Stimulation on Subthalamic Local Field Low-Frequency Oscillations in Parkinson's Disease

New adaptive systems for deep brain stimulation (DBS) could in the near future optimize stimulation settings online so as to achieve better control over the clinical fluctuations in Parkinsons disease (PD). Local field potentials (LFPs) recorded from the subthalamic nucleus (STN) in PD patients show that levodopa and DBS modulate STN oscillations. Because previous research has shown that levodopa and DBS variably influence beta LFP activity (8-20 Hz), we designed this study to find out how they affect low-frequency (LF) oscillations (2-7 Hz). STN LFPs were recorded in 19 patients with PD during DBS, after levodopa medication, and during DBS and levodopa intake combined. We investigated the relationship between LF modulations, DBS duration and levodopa intake. We also studied whether LF power depended on disease severity, the patients clinical condition and whether LF modulations were related to electrode impedances. LF power increased during DBS, after levodopa intake and under both experimental conditions combined. The LF power increase correlated with the levodopa-induced clinical improvement and the higher the electrode impedance, the greater was the LF power change. These data suggest that the LF band could be useful as a control neurosignal for developing novel adaptive DBS systems for patients with PD.

Effect of 9-cis Retinoic Acid on Dopamine D2 Receptor Expression in Pituitary Adenoma Cells

The dopamine receptor subtype 2 (D2R) promoter contains a functional retinoic acid response element involved in the control of D2R expression. The aim of the study was to evaluate the effect of 9-cis retinoic acid (9-cis RA) on D2R protein expression in human pituitary adenomas and GH3 cell line. Treatment with 9-cis RA (100 nM for 48 hrs) caused a 109 ± 32% increase of basal D2R levels in five of eight growth hormone (GH)-secreting adenomas (GH-omas), a 129 ± 28% increase in 7 of 11 nonfunctioning adenomas, and no effect in two resistant prolactinomas by Western blotting. The lack of D2R induction in some tumors was not associated with a different pattern of retinoid x receptor (RXR) and retinoic acid receptor (RAR) isoform expression that was similar in all tumors by immunohistochemistry. While the induction of D2R did not affect the slight but significant inhibitory effect exerted by dopamine (10 nM) on in vitro GH release by GH-oma cultured cells, in pituitary GH3 cell lines cis-9 RA enhanced the dopamine-induced inhibition of in vitro GH release (% inhibition: 16 ± 2 versus 26 ± 5, P < 0.05), cell proliferation (25 ± 2% versus 44 ± 5%, P < 0.05) and cell viability (16 ± 0.8% versus 29 ± 1%, P < 0.05), likely by activating caspase-3 (28 ± 3% versus basal, P < 0.05). In conclusion, this study provides novel evidence for a permissive role of retinoids on the expression of D2R in a good proportion of pituitary tumors and on the generation of pro-apoptotic signals in GH3 cell line.

Short term surgical complications after subthalamic deep brain stimulation for Parkinson's disease: does old age matter?.

BackgroundPatients aged 65 years and older are not traditionally considered optimal candidates for subthalamic deep brain stimulation (STN-DBS), mainly for their presumed increased incidence of surgical complications. The aim of this study was to assess STN-DBS surgery safety in relation to age.MethodsA total of 107 consecutive patients undergoing bilateral STN-DBS at our institution between 2002 and 2014 were retrospectively stratified according to age in two groups (Young group < 65 years old; Elderly group ≥ 65 years old;). Rate of short-term surgical complications (within 90 days) was reviewed and compared between the two groups.ResultsPre-operative baseline data were comparable between the two groups. The 90-days post-operative mortality rate was 0%. Overall incidence of complications related to surgery was 6,54%. In the Elderly group we observed 3 post-operative intra-cerebral haematomas (7,89%), 1 requiring urgent surgical evacuation. In the Young group we observed 2 post-operative asymptomatic intra-cerebral haematomas (2,89%) and 2 wound infections (2,89%), 1 requiring system removal. No others surgical complications were noticed in both groups.ConclusionsChronological age ≥ 65 years old should not be considered alone as exclusion criteria to STN-DBS surgery.