Validation of a new method for human nerve decellularization: toward a new tool in peripheral nerve reconstructive surgery.

Defects of the peripheral nervous system are extremely frequent in trauma and surgeries and have high socioeconomic costs. In case of peripheral nerve injury, the first approach is primary neurorrhaphy, which is direct nerve repair with epineural microsutures of the two stumps. However, this is not feasible in case of stump retraction or in case of tissue loss (gap > 2 cm), where the main surgical options are autologous grafts, allogenic grafts, or nerve conduits. While the gold standard is the autograft, it has disadvantages related to its harvesting, with an inevitable donor site morbidity and functional deficit. Fresh nerve allografts have therefore become a viable alternative option, but they require immunosuppression, which is often contraindicated. Acellular Nerve Allografts (ANA) represent a valid alternative, they do not need immunosuppression and appear to be safe and effective based on recent studies. The purpose of this study is to propose and develop an innovative method of nerve decellularization (Rizzoli method), conforming to cleanroom requirements in order to perform the direct tissue manipulation step and the nerve decellularization process within five hours, so as to accelerate the detachment of myelin and cellular debris, without detrimental effects on nerve architecture. In this study, the safety and the efficacy of the new method are evaluated in vitro and in vivo by histological, immunohistochemical, and histomorphometric studies in rabbits and humans. The new method is rapid, safe, and cheaper if compared with available commercial ANAs. The present study shows that the method, previously optimized in vitro and in vivo on animal model presented by our group, can be applied on human nerve samples. This work represents the first step in providing a novel, safe, and inexpensive tool for use by European tissue banks to democratize the use of nerve tissue transplantation for nerve injury reconstruction.

Caratterizzazione clinica, eziopatogenetica e ricerca di marker diagnostici nella poliradicoloneurite acuta idiopatica del cane

La poliradicoloneurite acuta idiopatica (ACIP) è una patologia infiammatoria che interessa le radici di più nervi spinali, descritta soprattutto nel cane, più raramente nel gatto, caratterizzata da insorgenza acuta di paresi/paralisi flaccida. L’ACIP mostra notevoli similitudini con la sindrome di Guillan-Barrè dell’uomo (GBS), in cui la patogenesi è su base autoimmunitaria ed è stata correlata con la presenza di alcuni fattori scatenanti (trigger). Lo scopo di questo lavoro è stato quello di caratterizzare l’ACIP in 26 cani, descrivendone la sintomatologia, l’evoluzione clinica, i risultati degli esami diagnostici. La diagnosi si è basata sui riscontri dell’anamnesi, della visita neurologica e del decorso confermata, quando possibile, dai rilievi elettrodiagnostici. Su tutti i cani è stata valutata l’esposizione a specifici agenti infettivi (Toxoplasma gondii, Neospora canunim, Ehrlichia canis, Leishmania infantum), o altri fattori (come vaccinazioni) che potrebbero aver agito da “trigger” per l’instaurarsi della patologia; sull’intera popolazione e su 19 cani non neurologici (gruppo di controllo), si è proceduto alla ricerca degli anticorpi anti-gangliosidi. La sintomatologia di più frequente riscontro (25/26) ha coinvolto la funzione motoria (paresi/plegia) con prevalente interessamento dei 4 arti (24/25) . Sei cani hanno ricevuto una terapia farmacologica, che non ne ha influenzato il decorso, favorevole in 24/26 casi. In 9 pazienti è stata rilevata una precedente esposizione a potenziali trigger; in 10 casi si è riscontrato un titolo anticorpale positivo ad almeno un agente infettivo testato. In 17/26 cani si è ottenuto un titolo anticorpale anti-GM2 e anti-GA1; nella popolazione di controllo solo un caso è risultato positivo. Questi risultati hanno contribuito a consolidare le conoscenze di questa patologia, validando l’utilità della ricerca anticorpale anti-gangliosidica per la diagnosi di ACIP e facendo intravedere la possibilità che l’ACIP possa essere assimilate alla GBS anche dal punto di vista patogenetico, per la quale potrebbe essere considerata come modello animale spontaneo.

Functional relationship of colorectal cancer-associated glycans with malignant phenotype and transcriptome changes

Glycosyltransferases ST6GAL1 and B4GALNT2 (and their cognate antigens Sia6LacNAc and Sda, respectively) are associated with colorectal cancer (CRC) but it is not fully clear their biological and clinical significance. We explored the clinical relevance of both glycosyltransferases by interrogating The Cancer Genome Atlas (TCGA) database while the phenotypic/transcriptomic effects of ST6GAL1/B4GALNT2 overexpression were studied in genetically modified CRC cell lines. Transcriptomic data from CRC patients in TCGA database suggested a moderate impact of ST6GAL1 on CRC progression, although it was not possible to define a clear role for this glycosyltransferase. Transcriptomic analysis of ST6GAL1-transduced cell lines revealed a much deeper effect of ST6GAL1 on gene expression in SW948 than in SW48. The overexpression of ST6GAL1 induced opposite effects on soft agar growth and wound healing in both cell lines. These results indicate that the impact of a cancer-associated glycosyltransferase change on phenotype/transcriptome can be extremely variable, depending on the molecular context of the tumor cell. On the contrary, transcriptomic analysis of B4GALNT2-modified cell lines together with TCGA database survey demonstrated a strong impact of B4GALNT2 on the transcriptional activity of CRC cells, in particular its association with a better prognosis. We suggest an anti-tumoral role of B4GALNT2 in CRC. We also investigated the glycan changes related to ST6GAL1/B4GALNT2 expression in a small cohort of tissues/plasma as well as the N-glycomic profile of CRC, normal and polyp tissues. We found an increase of ST6GAL1 activity in CRC and inflammatory bowel disease plasma samples comparing with plasma from healthy donors. A different Sda protein carrier pattern was observed between healthy donors and CRC plasma samples. β-arrestin 1 is a possible candidate as Sda carrier protein in plasma samples although future validation studies are needed. The alterations found in the N-glycan pattern highlight the importance of N-glycome as a molecular signature in cancer.

Effect of host genotype and prion strain on the phenotypic heterogeneity of Creutzfeldt-Jakob disease: the peripheral nervous system involvement and the spectrum of the genetic form

The first study was designed to assess whether the involvement of the peripheral nervous system (PNS) belongs to the phenotypic spectrum of sporadic Creutzfeldt-Jakob disease (sCJD). To this aim, we reviewed medical records of 117 sCJDVV2, 65 sCJDMV2K, and 121 sCJDMM(V)1 subjects for symptoms/signs and neurophysiological data. We looked for the presence of PrPSc in postmortem PNS samples from 14 subjects by western blotting and real-time quaking-induced conversion (RT-QuIC) assay. Seventy-five (41.2%) VV2-MV2K patients, but only 11 (9.1%) MM(V)1, had symptoms/signs suggestive of PNS involvement and neuropathy was documented in half of the VV2-MV2K patients tested. RT-QuIC was positive in all PNS samples, whereas western blotting detected PrPSc in the sciatic nerve in only one VV2 and one MV2K. These results support the conclusion that peripheral neuropathy, likely related to PrPSc deposition, belongs to the phenotypic spectrum of sCJDMV2K and VV2, the two variants linked to the V2 strain. The second study aimed to characterize the genetic/molecular determinants of phenotypic variability in genetic CJD (gCJD). To this purpose, we compared 157 cases of gCJD to 300 of sCJD. We analyzed: demographic aspects, neurological symptoms/signs, histopathologic features and biochemical characteristics of PrPSc. The results strongly indicated that the clinicopathological phenotypes of gCJD largely overlap with those of sCJD and that the genotype at codon 129 in cis with the mutation (i.e. haplotype) contributes more than the latter to the disease phenotype. Some mutations, however, cause phenotypic variations including haplotype-specific patterns of PrPSc deposition such as the “dense” synaptic pattern (E200K-129M), the intraneuronal dots (E200K-129V), and the linear stripes perpendicular to the surface in the molecular layer of cerebellum (OPRIs-129M). Overall, these results suggest that in gCJD PRNP mutations do not cause the emergence of novel prion strains, but rather confer increased susceptibility to the disease in conjunction with “minor” clinicopathological variations.

Phenotypic and molecular investigation of circulating tumor cells (CTCs) isolated from breast cancer patients at single cell resolution

Despite the paramount advances in cancer research, breast cancer (BC) still ranks one of the leading causes of cancer-related death worldwide. Thanks to the screening campaign started in developed countries, BC is often diagnosed at early stages (non-metastatic BC, nmBC), but disease relapse occurrence even after decades and at distant sites is not an uncommon phenomenon. Conversely, metastatic BC (mBC) is considered an incurable disease. The major perpetrators of tumor spread to secondary organs are circulating tumor cells (CTCs), a rare population of cells detectable in the peripheral blood of oncologic patients. In this study, CTCs from patients diagnosed with luminal nmBC and mBC (hormone receptor positive, Human Epidermal Growth Factor Receptor 2 (HER2) negative) were characterized at both phenotypic and molecular levels. To better understand the molecular mechanisms underlying their biology and their metastatic potential, next-generation sequencing (NGS) analyses were performed at single-cell resolution to assess copy number aberrations (CNAs), single nucleotide variants (SNVs) and gene expression profiling. The findings of this study arise hints in CTC detection, and pave the way to new application in CTC research.