THE ROLE OF INTESTINAL IMMUNE SYSTEM IN HERPES SIMPLEX VIRUS TYPE 1âMEDIATED ENTERIC NERVOUS SYSTEM DYSFUNCTIONS

Intestinal neuropathies have been described in a variety of functional and inflammatory gastrointestinal disorders. Although the degenerative alterations and neuronal are often associated to a lymphocytic inflammatory infiltrate, the underlying mechanisms remain obscure. Potential etiologic factors are neurotropic viruses, since they are able to specifically infect neurons and cause cell damage through direct or indirect mechanisms. Among the neurotrophic viruses the HSV-1 shows several interesting features. HSV-1 is most commonly known to latently infect the trigeminal ganglion of cranial nerve innervating the face and oral mucosa but latent HSV-1 has also been found in the nodose ganglia innervating the gastrointestinal tract in humans possibly as a consequence of swallowed viral particles. The working hypothesis of my PhD work was that swallowed HSV-1 can infect the enteric nervous system and locally trigger an immune mediated response damaging neurons. To assess the ability of α-herpesvirinae (HSV-1, HSV-2, VZV) to infect human ENS we performed a prospective cohort study using human surgical ileocolonic specimens of patients affected by colon carcinoma (CC), Crohn’s disease (CD), ulcerative colitis (UC) and diverticular disease (DD). A total of 121 patients were studies and presence of herpes simplex virus (HSV) types-1 and -2 and varicella-zoster virus (VZV) DNA was examined by RT-PCR in the muscle intestinal layer including the myenteric plexus. The α-herpesviruses DNA was detected in 44,5 % and 52 % of ileum and colon samples, respectively. No significant differences in the viral DNA positivity were observed among the different groups whereas the viral DNA load was significantly higher in samples of patients affected by Crohn’s disease. Moreover, HSV DNA was detected more frequently than VZV DNA, 46% vs 3%, respectively. In order to mimic the human α-herpesvirinae infection and spread in ENS, my research group has established an original murine model of persistent ENS infection with HSV-1. Using this model we demonstrated that HSV-1 infection resulted in time-dependent intestinal neuromuscular abnormalities, providing the first in vivo evidence for the ability of neurotropic viruses to reach and damage the ENS. Since significant intestinal motility anomalies and damage of enteric nerves were evident 8 - 10 weeks (W) post intragastric (IG) challenge with HSV-1, I attempted to characterize the adaptive immune response to HSV-1 and whether was causing the neuromuscular abnormalities. In the longitudinal muscle myenteric plexus (LMMP) I observed an increase in CD3+ lymphocytes starting at 6 W and persisting up to 10 W after viral IG inoculum. At 8 weeks post IG viral inoculum, HSV-1 reactive CD3+CD4+IL4+ and CD3+CD8+INF-γ+ were detected, whereas at 10 W activated CD8+ T-cells infiltrated the LMMP. By immunohistochemistry CD3+ lymphocytes were demonstrated in the myenteric ganglia of HSV-1 infected mice 6-10 W post IG viral challenge. To verify the involvement of CD8+ and CD4+ T-cells in HSV-1 induced dysmotility we performed depletion experiments by administration of monoclonal antibody. ENS damage and the neuromuscular anomalies were observed only after depletion of CD8 T cells at 8 and 10 W after viral IG inoculum. To validate the role of lymphocytes in HSV-1 induced ENS dysfunction, CD3+CD4+ and CD+CD8+ cells were isolated from the LMMP of mice 8-10 W after viral IG inoculum, in vitro pulsed with HSV-1, and injected in recipient mice. The effects on isometric muscle tension were determined after one week. Adoptive transfer of CD8+ T cells isolated from LMMP at 8 W post IG HSV-1 inoculum caused significant neuromuscular anomalies (p<0.01 vs control) in recipient mice only if HSV-1 pulsed in vitro. Instead, the adoptive transfer of both CD4+ and CD8+ T cells isolated from 10 W infected mice resulted in dysmotility with or without in vitro exposure to viral antigens in recipient mice. In conclusion, in this study we showed that α-herpesvirinae DNA is present in a large percentage of patients and the presence of HSV-1 in murine myenteric ganglia triggered a strong and specific immune response able to damage the ENS. We speculate that persistent HSV-1 infection in the ENS, in predisposed individuals, may contribute to alter neuronal functional integrity favouring the onset of bowel disorders

Rosmarinus officinalis extract inhibits human melanoma cell growth.

Rosmarinus officinalis L. is receiving increasing attention due to its anti-inflammatory and antioxidative constituents. Our recent studies showed that R. officinalis extract, containing 31.7 % of carnosic acid, was able to counteract the deleterious effects of UV-R, by protecting plasmid DNA from hydroxyl radicals generated by UV-A. In this work, we evaluated the effects of this extract on pBR322 DNA cleavage induced by nitric oxide, and the growth inhibitory activity against two human melanoma cell lines, M14 and A375. The extract showed a protective effect on plasmid DNA damage, and at concentrations of 10-80 μg/mL was able to reduce significantly (p<0.001) the growth (MTT assay) of both melanoma cell lines. In addition, our results indicate that apoptotic cell demise is induced in M14 and A375 cells. No statistically significant increase in LDH release was observed in melanoma cells, correlated to a fragmentation of genomic DNA, determined by COMET assay

Potential Anticancer Activity against Human Epithelial Cancer Cells of Peumus Boldus Leaf Extract

The potential in vitro antineoplastic effect has been studied of a methanolic extract of leaves of Peumus boldus Molina (Monimiaceae) on two human cancer epithelial cell lines, DU-145 cells (androgen-insensitive prostate cancer cells) and KB cells (oral squamous carcinoma cells). Our findings show that this extract exhibited comparable effects on the cancer cells examined as judged by IC50 values (5.07±0.4 μg/mL and 5.28±0.5 μg/mL in DU-145 and KB cells, respectively). In addition, with respect to genomic DNA damage, determined by Comet assay, the results obtained show a high fragmentation of DNA, not correlated to lactic dehydrogenase (LDH) release, a marker of membrane breakdown, in both cell lines treated with the extract at 5–20 μg/mL concentrations. Taken together, our experimental evidence may justify further investigation of the chemopreventive and chemotherapeutic potential of this natural drug

Signals from the deep-sea: Genetic structure, morphometric analysis, and ecological implications of Cyclothone braueri (Pisces, Gonostomatidae) early life stages in the Central Mediterranean Sea

Cyclothone braueri (Stomiiformes, Gonostomatidae) is a widely distributed fish inhabiting the mesopelagic zone of marine tropical and temperate waters. Constituting one of the largest biomasses of the ocean, C. braueri is a key element in most of the ecological processes occurring in the twilight layer. We focused on the ecological processes linked to early life stages in relation to marine pelagic environmental drivers (temperature, salinity, food availability and geostrophic currents) considering different regions of the Central Mediterranean Sea. A multivariate morphometric analysis was carried out using six parameters with the aim of discerning different larval morphotypes, while a fragment of 367 bp representing the 12S ribosomal RNA gene was used to perform molecular analyses aimed at determining the intraspecific genetic variability. Analysis highlighted two geographically distinct morphotypes not genetically discernible and related to the different nutritional conditions due to spatial heterogeneities in terms of temperature and food availability. The body depth (BD) emerged as an appropriate morphometric parameter to detect the larval condition in this species. Molecular analysis highlighted a moderate genetic divergence in the fish population, showing the recurrence of two phylogroups not geographically separated

THE ROLE OF INTESTINAL IMMUNE SYSTEM IN HERPES SIMPLEX VIRUS TYPE 1âMEDIATED ENTERIC NERVOUS SYSTEM DYSFUNCTIONS

Intestinal neuropathies have been described in a variety of functional and inflammatory gastrointestinal disorders. Although the degenerative alterations and neuronal are often associated to a lymphocytic inflammatory infiltrate, the underlying mechanisms remain obscure. Potential etiologic factors are neurotropic viruses, since they are able to specifically infect neurons and cause cell damage through direct or indirect mechanisms. Among the neurotrophic viruses the HSV-1 shows several interesting features. HSV-1 is most commonly known to latently infect the trigeminal ganglion of cranial nerve innervating the face and oral mucosa but latent HSV-1 has also been found in the nodose ganglia innervating the gastrointestinal tract in humans possibly as a consequence of swallowed viral particles. The working hypothesis of my PhD work was that swallowed HSV-1 can infect the enteric nervous system and locally trigger an immune mediated response damaging neurons. To assess the ability of α-herpesvirinae (HSV-1, HSV-2, VZV) to infect human ENS we performed a prospective cohort study using human surgical ileocolonic specimens of patients affected by colon carcinoma (CC), Crohn’s disease (CD), ulcerative colitis (UC) and diverticular disease (DD). A total of 121 patients were studies and presence of herpes simplex virus (HSV) types-1 and -2 and varicella-zoster virus (VZV) DNA was examined by RT-PCR in the muscle intestinal layer including the myenteric plexus. The α-herpesviruses DNA was detected in 44,5 % and 52 % of ileum and colon samples, respectively. No significant differences in the viral DNA positivity were observed among the different groups whereas the viral DNA load was significantly higher in samples of patients affected by Crohn’s disease. Moreover, HSV DNA was detected more frequently than VZV DNA, 46% vs 3%, respectively. In order to mimic the human α-herpesvirinae infection and spread in ENS, my research group has established an original murine model of persistent ENS infection with HSV-1. Using this model we demonstrated that HSV-1 infection resulted in time-dependent intestinal neuromuscular abnormalities, providing the first in vivo evidence for the ability of neurotropic viruses to reach and damage the ENS. Since significant intestinal motility anomalies and damage of enteric nerves were evident 8 - 10 weeks (W) post intragastric (IG) challenge with HSV-1, I attempted to characterize the adaptive immune response to HSV-1 and whether was causing the neuromuscular abnormalities. In the longitudinal muscle myenteric plexus (LMMP) I observed an increase in CD3+ lymphocytes starting at 6 W and persisting up to 10 W after viral IG inoculum. At 8 weeks post IG viral inoculum, HSV-1 reactive CD3+CD4+IL4+ and CD3+CD8+INF-γ+ were detected, whereas at 10 W activated CD8+ T-cells infiltrated the LMMP. By immunohistochemistry CD3+ lymphocytes were demonstrated in the myenteric ganglia of HSV-1 infected mice 6-10 W post IG viral challenge. To verify the involvement of CD8+ and CD4+ T-cells in HSV-1 induced dysmotility we performed depletion experiments by administration of monoclonal antibody. ENS damage and the neuromuscular anomalies were observed only after depletion of CD8 T cells at 8 and 10 W after viral IG inoculum. To validate the role of lymphocytes in HSV-1 induced ENS dysfunction, CD3+CD4+ and CD+CD8+ cells were isolated from the LMMP of mice 8-10 W after viral IG inoculum, in vitro pulsed with HSV-1, and injected in recipient mice. The effects on isometric muscle tension were determined after one week. Adoptive transfer of CD8+ T cells isolated from LMMP at 8 W post IG HSV-1 inoculum caused significant neuromuscular anomalies (p<0.01 vs control) in recipient mice only if HSV-1 pulsed in vitro. Instead, the adoptive transfer of both CD4+ and CD8+ T cells isolated from 10 W infected mice resulted in dysmotility with or without in vitro exposure to viral antigens in recipient mice. In conclusion, in this study we showed that α-herpesvirinae DNA is present in a large percentage of patients and the presence of HSV-1 in murine myenteric ganglia triggered a strong and specific immune response able to damage the ENS. We speculate that persistent HSV-1 infection in the ENS, in predisposed individuals, may contribute to alter neuronal functional integrity favouring the onset of bowel disorders.Le neuropatie intestinali sono state descritte in una varietà di disturbi gastrointestinali di natura funzionale e infiammatoria. Inoltre le alterazioni neuronali sono spesso state associate alla presenza di un infiltrato infiammatorio linfocitario, ma rimangono ancora oscuri i meccanismi alla base di questi processi. I potenziali fattori eziologici sono rappresentati da virus neurotropi, i quali sono in grado di infettare specificatamente i neuroni e causare danni alle cellule attraverso meccanismi diretti o indiretti. Tra i virus neurotropici, Herpes Simplex Virus type-1 sembra mostrare diverse caratteristiche interessanti. Anche se l’HSV-1 è più comunemente noto per infettare latentemente il ganglio trigemino del nervo cranico che innerva viso e mucosa orale, HSV-1 è stato trovato anche in gangli che innervano il tratto gastrointestinale nell'uomo probabilmente come conseguenza di particelle virali ingerite. L’attività di ricerca svolta durante il mio periodo di dottorato si basa sull’ipotesi che HSV-1 è capace di infettare il sistema nervoso enterico e innescare localmente una risposta immunitaria in grado di danneggiare i neuroni del tratto gastrointestinale. Per valutare la capacità della subfamiglia degli α-Herpesviridae (HSV-1, HSV-2, VZV) di infettare il sistema nervoso enterico (SNE) umano, abbiamo effettuato uno studio di coorte prospettico su resezioni ileocoliche chirurgiche di pazienti affetti da carcinoma del colon, morbo di Crohn, colite ulcerosa e malattia diverticolare. Un totale di 121 pazienti sono stati collezionati e mediante Real Time-PCR è stata valutata la presenza di DNA virale di HSV-1, HSV-2 e varicella-zoster (VZV), nello strato muscolare intestinale che comprende il plesso mienterico. La presenza di α-herpesvirus è stata evidenziata nel 44,5% dei campioni ileali e nel 52% dei campioni estratti da colon. La frequenza di campioni positivi al DNA virale non evidenzia alcuna correlazione significata con specifiche patologie. E’ stato però osservato che i livelli di DNA virale erano significativamente maggiori nei campioni di pazienti affetti da morbo di Crohn. Inoltre, il DNA di HSV è stato rilevato più frequentemente rispetto a VZV, 46% e 3%, rispettivamente. Al fine di simulare l'infezione da α-herpesvirinae e la conseguente diffusione nel SNE umano, il mio gruppo ha messo a punto un modello murino di infezione persistente di HSV-1 nel SNE. Utilizzando questo modello abbiamo dimostrato che l’infezione intestinale da HSV-1 provoca anomalie neuromuscolari correlate al tempo d’infezione, fornendo per per la prima volta la prova in vivo che i virus neurotropi sono capaci di raggiungere e, conseguentemente, danneggiare il SNE. In particolare, significative anomalie della motilità intestinale e danni funzionali dei nervi enterici sono stati evidenziati tra le 8 e 10 settimane dopo l’inoculo intragastrico (IG) in topi infettati con HSV-1. Il mio studio si è focalizzato sulla caratterizzazione della risposta immunitaria adattativa durante l’infezione da HSV-1 con lo scopo di chiarire un possibile coinvolgimento delle cellule immuni sulle alterazione neuronali che causano disfunzioni gastrointestinali. Abbiamo analizzato la presenza d’infiltrato linfocitario nel plesso mienterico (LMMP) di topi infettati con HSV-1 ed è stato osservato un aumento del numero di linfociti CD3+ a partire dalla sesta settimana e persistenti fino alla decima settimana dopo inoculo IG. In particolare all’ottava settimana d’infezione sono stati osservati sia CD3+CD4+IL4+ che CD3+CD8+INF-γ+ specificatamente attivati e reattivi ad HSV-1, mentre alla decima settimana dopo inoculo IG vi era una maggiore presenza di CD3+CD8+INF-γ+. Con saggi d’immunoistochimica, la localizzazione dei linfociti CD3+ è stata confermata al livello dei gangli mienterici in topi inoculati con HSV-1 dalla sesta alla decina settimana post infezione. Per verificare il coinvolgimento delle cellule T CD8+ e CD4+ nelle dismotilità indotte da HSV-1 sono stati effettuati esperimenti di deplezione tramite somministrazione di anticorpi monoclonali anti-CD4 e anti-CD8. Danni al SNE e anomalie neuromuscolari sono state osservate solo dopo l'esaurimento delle cellule T CD8+ all’ottava e decima dopo inoculo IG. Per validare ulteriormente il ruolo dei linfociti T reattivi al virus nel danno neuronale, CD3+CD4+ e CD3+CD8+ sono stati isolati dal plesso mienterico di topi infetti da 8 e 10 settimane e, dopo stimolazione in vitro con HSV-1, trapianti in topi sani. Una settimana dopo il trapianto sono stati valutati gli effetti sulla motilità intestinale nei topi trapiantati. Il trasferimento di cellule T CD8+ isolate dal plesso mienterico a 8 settimane hanno causato significative anomalie neuromuscolari (p <0.01 vs controllo) in topi controllo trapianti, solo in seguito a stimolazione in vitro con HSV-1. Invece, alterazioni della motilità sono state riscontrate in seguito al trapianto di entrambe le sottopopolazioni linfocitarie, CD4+ e CD8+ isolati dal plesso mientercio di topi infetti a 10 settimane, con o senza una precedente esposizione in vitro di antigeni virali. In conclusione, in questo studio abbiamo dimostrato che α-herpesvirinae DNA è presente in una grande percentuale di pazienti e che la presenza di HSV-1 nei gangli mienterici murini innesca una robusta risposta immunitaria specifica in grado di danneggiare il SNE. Noi ipotizziamo che una persistente infezione da HSV-1 nel SNE, in soggetti predisposti, può contribuire ad alterare l'integrità funzionale neuronale favorendo l'insorgenza di disturbi intestinali

Chamazulene-Rich Artemisia arborescens Essential Oils Affect the Cell Growth of Human Melanoma Cells

Artemisia arborescens is an aromatic shrub whose essential oils are considered a potential source of molecules with industrial and pharmaceutical interest. The chemical profile of A. arborescens essential oils (EOs) was shown to be quite variable and various chemotypes have been identified. In this study, we compared the EOs composition of A. arborescens leaves and flowers collected from four dierent locations in Sicily. The EOs were assayed for their antiproliferative activity against A375 human malignant melanoma cells, also testing cell viability and cell membrane integrity. The evaluation of DNA fragmentation and caspase-3 activity assay was employed for the detection of apoptosis. The expression of Bcl-2, Bax, cleaved caspase-9, PTEN (Phosphatase and tensin homolog), Hsp70 (Heat Shock Protein 70 kilodaltons) and SOD (superoxide dismutase) proteins was evaluated by Western blot analysis. The levels of ROS and GSH were also analyzed. Results show that EOs presented significant dierences in their composition, yield, and cytotoxic activity depending on the collection site. The chamazulene/camphor-rich EOs from plants collected in Acqua Calda (Lipari) resulted particularly active on melanoma cancer cells (IC50 values of 6.7 and 4.5 g/mL), being able to trigger apoptotic death probably interfering with endogenous defense mechanisms. These oils may be considered as a natural resource of chamazulene, containing this compound up to 63%

Apoptosis induction of essential oils from Artemisia arborescens L. in human prostate cancer cells

Ethnopharmacological relevance: Prostate cancer originates from cells inside a gland, which begin to grow out of control. In the world, prostate cancer is the most common cancer in the male population. New therapeutic strategies are needed for this tumor which still has a high mortality. A. arborescens leaves and aerial parts have various ethnopharmacological uses such as anti-spasmodic, and their decoctions were used to resolve urticaria, neuralgia and several lung diseases. Often this species has been also used to treat different inflammatory-related diseases such as cancer. Aim of the study: In a continuation of our research on essential oils from medicinal plants, we have selected, two essential oils from Artemisia arborescens L. (Compositae), an aromatic shrub widely used in traditional medicine. We evaluated their pro-apototic effect on androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells. In this study, we also evaluated the anti-Signal transducer and transcription factor 3 (STAT-3) activity of both essential oils in the human prostate cancer cell lines, and the treatment with Tumor necrosis factor (TNF)-Related Apoptosis (TRAIL). Materials and methods: The cells were exposed to essential oils for 72&nbsp;h and cell viability and cell membrane integrity were evaluated. Genomic DNA and the activity of caspase-3 was tested to confirm the cell death for apoptosis. Western blot analysis was employed to evaluate the expression of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, Hsp70, STAT-3 and SOD proteins. Assays to evaluate reactive oxygen species (ROS) and GSH levels were also performed. Results: The results showed the capacity of two essential oils to activate an apoptotic process increasing the inhibition of Hsp70 and STAT-3 protein expression. In addition, our natural products sensitize LNCaP cells to Tumor necrosis factor (TNF)-Related Apoptosis (TRAIL)-induced apoptosis. Conclusions: In summary, our study provides a further contribution to the hypothesis of the use of essential oils, from traditional medicinal plants, for the treatment of tumors, and suggests that the combination of our samples with other anti-prostate cancer therapies could be used to affect prostate cancer

Management of the Brain: Essential Oils as Promising Neuroinflammation Modulator in Neurodegenerative Diseases

Neuroinflammation, a pivotal factor in the pathogenesis of various brain disorders, including neurodegenerative diseases, has become a focal point for therapeutic exploration. This review highlights neuroinflammatory mechanisms that hallmark neurodegenerative diseases and the potential benefits of essential oils in counteracting neuroinflammation and oxidative stress, thereby offering a novel strategy for managing and mitigating the impact of various brain disorders. Essential oils, derived from aromatic plants, have emerged as versatile compounds with a myriad of health benefits. Essential oils exhibit robust antioxidant activity, serving as scavengers of free radicals and contributing to cellular defense against oxidative stress. Furthermore, essential oils showcase anti-inflammatory properties, modulating immune responses and mitigating inflammatory processes implicated in various chronic diseases. The intricate mechanisms by which essential oils and phytomolecules exert their anti-inflammatory and antioxidant effects were explored, shedding light on their multifaceted properties. Notably, we discussed their ability to modulate diverse pathways crucial in maintaining oxidative homeostasis and suppressing inflammatory responses, and their capacity to rescue cognitive deficits observed in preclinical models of neurotoxicity and neurodegenerative diseases