Sviluppo e applicazione di metodiche molecolari rapide per l'identificazione di agenti patogeni umani

Dalla pratica quotidiana, e dalla stretta collaborazione con i medici di diverse specialità, è emersa la necessità e l’interesse, ad approfondire lo studio di due patogeni, Chlamydia trachomatis e Mycoplasma hominis, in gran parte sconosciuti, e di evidenziarne l’importanza clinica ed epidemiologica, adottandoli come oggetto di ricerca. C. trachomatis e del M. hominis sono due agenti patogeni piuttosto diffusi nella popolazione, ma di difficile identificazione e in grado di provocare infezioni a trasmissione sessuale, spesso asintomatiche e latenti, ma con gravi complicanze, sia nell’uomo che nella donna. Alla luce di queste considerazioni, si è deciso di studiare una metodica PCR rapida per l’identificazione di tali patogeni in campioni biologici umani. Il progetto di ricerca ha avuto i seguenti obiettivi: studio e applicazione della normativa regionale per l’Accreditamento Istituzionale di Strutture Sanitarie e per la gestione della qualità UNI EN ISO 9001/2008; progettazione e realizzazione del laboratorio di biologia molecolare; studio degli agenti patogeni C. trachomatis e M. hominis, delle loro caratteristiche biologiche e genetiche; messa a punto, validazione e applicazione di metodiche diagnostiche molecolari rapide (singole e multiplex), competitive e di semplice interpretazione per l’identificazione di tali patogeni da campioni biologici umani, mediante l’impiego di reagenti e tecnologie non convenzionali; studio e verifica dell’applicabilità delle metodiche di PCR rapide anche alla sensibilità/resistenza agli antibiotici dei patogeni oggetto dello progetto. Sono stati analizzati 123 campioni per C. trachomatis e 100 per M. hominis, di cui sono risultati positivi con la PCR rapida il 13% e il 34%, rispettivamente. Per verificare la sensibilità della metodica, gli stessi campioni di DNA per Chlamydia trachomatis sono stati analizzati sia con la PCR rapida che con un kit commerciale in Nested PCR, osservando una maggiore sensibilità e resa della PCR rapida rispetto al kit commerciale, in minor tempo. La PCR rapida è stata associata solo alla rivelazione su gel d’agarosio, ma sarebbe di particolare interesse adattare la metodica ad altri sistemi di rivelazione. Da questa esperienza si evince quello che può essere il contributo del biotecnologo nello specifico contesto del laboratorio di diagnostica medica. Il confronto fra le differenti metodologie di analisi cliniche utilizzate, ha permesso di evidenziarne gli aspetti positivi e negativi, di individuare le possibilità di miglioramento, di ampliarne le eventuali capacità di utilizzo per renderle più innovative e competitive. Per ottenere dei risultati appare indispensabile la collaborazione fra i centri clinici privati, l’industria di settore e l’Università. Da questo studio è emersa anche la necessità di una più attenta e diffusa opera d’informazione sulla necessità della prevenzione delle infezioni da Chlamydia trachomatis e da Mycoplasma hominis mediante l’utilizzo di biotecnologie molecolari nei programmi di screening

Immunovirotherapy for the treatment of glioblastoma

We have recently described a new murine model of glioblastoma, generated by the implantation of syngeneic glioblastoma stem cells into immunocompetent mice, that recapitulates the salient histopathological and immunological features of the human disease. We employed this model to demonstrate the multifaceted activity of an oncolytic herpes simplex virus genetically modified to express interleukin-12, G47∆-IL12

Increased expression of programmed death ligand 1 (PD-L1) in human pituitary tumors

PURPOSE: Subsets of pituitary tumors exhibit an aggressive clinical courses and recur despite surgery, radiation, and chemotherapy. Because modulation of the immune response through inhibition of T-cell checkpoints has led to durable clinical responses in multiple malignancies, we explored whether pituitary adenomas express immune-related biomarkers that could suggest suitability for immunotherapy. Specifically, programmed death ligand 1 (PD-L1) has emerged as a potential biomarker whose expression may portend more favorable responses to immune checkpoint blockade therapies. We thus investigated the expression of PD-L1 in pituitary adenomas. METHODS: PD-L1 RNA and protein expression were evaluated in 48 pituitary tumors, including functioning and non-functioning adenomas as well as atypical and recurrent tumors. Tumor infiltrating lymphocyte populations were also assessed by immunohistochemistry. RESULTS: Pituitary tumors express variable levels of PD-L1 transcript and protein. PD-L1 RNA and protein expression were significantly increased in functioning (growth hormone and prolactin-expressing) pituitary adenomas compared to non-functioning (null cell and silent gonadotroph) adenomas. Moreover, primary pituitary adenomas harbored higher levels of PD-L1 mRNA compared to recurrent tumors. Tumor infiltrating lymphocytes were observed in all pituitary tumors and were positively correlated with increased PD-L1 expression, particularly in the functional subtypes. CONCLUSIONS: Human pituitary adenomas harbor PD-L1 across subtypes, with significantly higher expression in functioning adenomas compared to non-functioning adenomas. This expression is accompanied by the presence of tumor infiltrating lymphocytes. These findings suggest the existence of an immune response to pituitary tumors and raise the possibility of considering checkpoint blockade immunotherapy in cases refractory to conventional management

EGFR Gene Overexpression Retained in an Invasive Xenograft Model by Solid Orthotopic Transplantation of Human Glioblastoma Multiforme Into Nude Mice

Orthotopic xenograft animal model from human glioblastoma multiforme (GBM) cell lines often do not recapitulate an extremely important aspect of invasive growth and epidermal growth factor receptor (EGFR) gene overexpression of human GBM. We developed an orthotopic xenograft model by solid transplantation of human GBM into the brain of nude mouse. The orthotopic xenografts sharing the same histopathological features with their original human GBMs were highly invasive and retained the overexpression of EGFR gene. The murine orthotopic GBM models constitute a valuable in vivo system for preclinical studies to test novel therapies for human GBM

Laser ablation of abnormal neurological tissue using robotic neuroblate system (LAANTERN): Procedural safety and hospitalization

BACKGROUND: Stereotactic laser ablation (SLA) has demonstrated potential utility for a spectrum of difficult to treat neurosurgical pathologies in multiple small and/or retrospective single-institutional series. Here, we present the safety profile of SLA of intracranial lesions from the Laser Ablation of Abnormal Neurological Tissue using Robotic NeuroBlate System (LAANTERN; Monteris Medical) multi-institutional, international prospective observational registry. OBJECTIVE: To determine the procedural safety of SLA for intracranial lesions. METHODS: Prospective procedural safety and hospitalization data from the first 100 treated LAANTERN patients was collected and analyzed. RESULTS: Mean age and baseline Karnofsky Performance Status (KPS) were 51(± 17) yr and 83(± 15), respectively. In total, 81.2% of patients had undergone prior surgical or radiation treatment. Most patients had a single lesion (79%) ablated through 1 burr hole (1.2 ± 0.7 per patient), immediately following a lesion biopsy. In total, \u3e90% of the lesion was ablated in 72% of treated lesions. Average total procedural time was 188.2 ± 69.6 min, and average blood loss was 17.7 ± 55.6 ccs. The average length of intensive care unit (ICU) and hospital stays before discharge were 38.1 ± 62.7 h and 61.1 ± 87.2 h, respectively. There were 5 adverse events (AEs) attributable to SLA (5/100; 5%). After the procedure, 84.8% of patients were discharged home. There was 1 mortality within 30 d of the procedure (1/100; 1%), which was not attributable to SLA. CONCLUSION: SLA is a safe, minimally invasive procedure with favorable postprocedural ICU and hospital utilization profiles

Treg Depletion Inhibits Efficacy of Cancer Immunotherapy: Implications for Clinical Trials

Regulatory T lymphocytes (Treg) infiltrate human glioblastoma (GBM); are involved in tumor progression and correlate with tumor grade. Transient elimination of Tregs using CD25 depleting antibodies (PC61) has been found to mediate GBM regression in preclinical models of brain tumors. Clinical trials that combine Treg depletion with tumor vaccination are underway to determine whether transient Treg depletion can enhance anti-tumor immune responses and improve long term survival in cancer patients.Using a syngeneic intracrabial glioblastoma (GBM) mouse model we show that systemic depletion of Tregs 15 days after tumor implantation using PC61 resulted in a decrease in Tregs present in tumors, draining lymph nodes and spleen and improved long-term survival (50% of mice survived >150 days). No improvement in survival was observed when Tregs were depleted 24 days after tumor implantation, suggesting that tumor burden is an important factor for determining efficacy of Treg depletion in clinical trials. In a T cell dependent model of brain tumor regression elicited by intratumoral delivery of adenoviral vectors (Ad) expressing Fms-like Tyrosine Kinase 3 ligand (Flt3L) and Herpes Simplex Type 1-Thymidine Kinase (TK) with ganciclovir (GCV), we demonstrate that administration of PC61 24 days after tumor implantation (7 days after treatment) inhibited T cell dependent tumor regression and long term survival. Further, depletion with PC61 completely inhibited clonal expansion of tumor antigen-specific T lymphocytes in response to the treatment.Our data demonstrate for the first time, that although Treg depletion inhibits the progression/eliminates GBM tumors, its efficacy is dependent on tumor burden. We conclude that this approach will be useful in a setting of minimal residual disease. Further, we also demonstrate that Treg depletion, using PC61 in combination with immunotherapy, inhibits clonal expansion of tumor antigen-specific T cells, suggesting that new, more specific targets to block Tregs will be necessary when used in combination with therapies that activate anti-tumor immunity