Neutrophils prevent rectal bleeding in ulcerative colitis by peptidyl-arginine deiminase-4-dependent immunothrombosis.

OBJECTIVE: Bleeding ulcers and erosions are hallmarks of active ulcerative colitis (UC). However, the mechanisms controlling bleeding and mucosal haemostasis remain elusive. DESIGN: We used high-resolution endoscopy and colon tissue samples of active UC (n = 36) as well as experimental models of physical and chemical mucosal damage in mice deficient for peptidyl-arginine deiminase-4 (PAD4), gnotobiotic mice and controls. We employed endoscopy, histochemistry, live-cell microscopy and flow cytometry to study eroded mucosal surfaces during mucosal haemostasis. RESULTS: Erosions and ulcerations in UC were covered by fresh blood, haematin or fibrin visible by endoscopy. Fibrin layers rather than fresh blood or haematin on erosions were inversely correlated with rectal bleeding in UC. Fibrin layers contained ample amounts of neutrophils coaggregated with neutrophil extracellular traps (NETs) with detectable activity of PAD. Transcriptome analyses showed significantly elevated PAD4 expression in active UC. In experimentally inflicted wounds, we found that neutrophils underwent NET formation in a PAD4-dependent manner hours after formation of primary blood clots, and remodelled clots to immunothrombi containing citrullinated histones, even in the absence of microbiota. PAD4-deficient mice experienced an exacerbated course of dextrane sodium sulfate-induced colitis with markedly increased rectal bleeding (96 % vs 10 %) as compared with controls. PAD4-deficient mice failed to remodel blood clots on mucosal wounds eliciting impaired healing. Thus, NET-associated immunothrombi are protective in acute colitis, while insufficient immunothrombosis is associated with rectal bleeding. CONCLUSION: Our findings uncover that neutrophils induce secondary immunothrombosis by PAD4-dependent mechanisms. Insufficient immunothrombosis may favour rectal bleeding in UC.

Dynamic, Transient, and Robust Increase in the Innervation of the Inflamed Mucosa in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) are characterized by chronic dysregulation of immune homeostasis, epithelial demise, immune cell activation, and microbial translocation. Each of these processes leads to proinflammatory changes via the release of cytokines, damage-associated molecular patterns (DAMPs), and pathogen-associated molecular patterns (PAMPs), respectively. The impact of these noxious agents on the survival and function of the enteric nervous system (ENS) is poorly understood. Here, we show that in contrast to an expected decrease, experimental as well as clinical colitis causes an increase in the transcript levels of enteric neuronal and glial genes. Immunostaining revealed an elevated neuronal innervation of the inflamed regions of the gut mucosa. The increase was seen in models with overt damage to epithelial cells and models of T cell-induced colitis. Transcriptomic data from treatment naïve pediatric IBD patients also confirmed the increase in the neuroglial genes and were replicated on an independent adult IBD dataset. This induction in the neuroglial genes was transient as levels returned to normal upon the induction of remission in both mouse models as well as colitis patients. Our data highlight the dynamic and robust nature of the enteric nervous system in colitis and open novel questions on its regulation.

E-type prostanoid receptor 4 drives resolution of intestinal inflammation by blocking epithelial necroptosis.

Inflammatory bowel diseases present with elevated levels of intestinal epithelial cell (IEC) death, which compromises the gut barrier, activating immune cells and triggering more IEC death. The endogenous signals that prevent IEC death and break this vicious cycle, allowing resolution of intestinal inflammation, remain largely unknown. Here we show that prostaglandin E2 signalling via the E-type prostanoid receptor 4 (EP4) on IECs represses epithelial necroptosis and induces resolution of colitis. We found that EP4 expression correlates with an improved IBD outcome and that EP4 activation induces a transcriptional signature consistent with resolution of intestinal inflammation. We further show that dysregulated necroptosis prevents resolution, and EP4 agonism suppresses necroptosis in human and mouse IECs. Mechanistically, EP4 signalling on IECs converges on receptor-interacting protein kinase 1 to suppress tumour necrosis factor-induced activation and membrane translocation of the necroptosis effector mixed-lineage kinase domain-like pseudokinase. In summary, our study indicates that EP4 promotes the resolution of colitis by suppressing IEC necroptosis.

Persistence of STAT-1 inhibition and induction of cytokine resistance in pancreatic ß cells treated with St John's wort and its component hyperforin.

OBJECTIVES: St John's wort extract (SJW) and its component hyperforin (HPF) were shown to potently inhibit cytokine-induced STAT-1 and NF-κB activation in pancreatic ß cells and protect them against injury. This study aimed at exploring the time course of STAT-1 inhibition afforded by these natural compounds in the ß-cell line INS-1E. METHODS: INS-1E cells were pre-incubated with SJW extract (2-5 µg/ml) or HPF (0.5-2 µm) and then exposed to a cytokine mixture. In some experiments, these compounds were added after or removed before cytokine exposure. STAT-1 activation was assessed by electrophoretic mobility shift assay, apoptosis by caspase-3 activity assay, mRNA gene expression by RT-qPCR. KEY FINDINGS: Pre-incubation with SJW/HPF for 1-2 h exerted a remarkable STAT-1 downregulation, which was maintained upon removal of the compounds before early or delayed cytokine addition. When the protective compounds were added after cell exposure to cytokines, between 15 and 90 min, STAT-1 inhibition also occurred at a progressively decreasing extent. Upon 24-h incubation, SJW and HPF counteracted cytokine-induced ß-cell dysfunction, apoptosis and target gene expression. CONCLUSIONS: SJW and HPF confer to ß cells a state of 'cytokine resistance', which can be elicited both before and after cytokine exposure and safeguards these cells from deleterious cytokine effects.

CRIP1a inhibits endocytosis of G-protein coupled receptors activated by endocannabinoids and glutamate by a common molecular mechanism.

The excitability of the central nervous system depends largely on the surface density of neurotransmitter receptors. The endocannabinoid receptor 1 (CB1 R) and the metabotropic glutamate receptor mGlu8 R are expressed pre-synaptically where they reduce glutamate release into the synaptic cleft. Recently, the CB1 R interacting protein cannabinoid receptor interacting protein 1a (CRIP1a) was identified and characterized to regulate CB1 R activity in neurons. However, underlying molecular mechanisms are largely unknown. Here, we identified a common mechanism used by CRIP1a to regulate the cell surface density of two different types of G-protein coupled receptors, CB1 R and mGlu8a R. Five amino acids within the CB1 R C-terminus were required and sufficient to reduce constitutive CB1 R endocytosis by about 72% in the presence of CRIP1a. Interestingly, a similar sequence is present in mGlu8a R and consistently, endocytosis of mGlu8a R depended on CRIP1a, as well. Docking analysis and molecular dynamics simulations identified a conserved serine in CB1 R (S468) and mGlu8a R (S894) that forms a hydrogen bond with the peptide backbone of CRIP1a at position R82. In contrast to mGlu8a R, the closely related mGlu8b R splice-variant carries a lysine (K894) at this position, and indeed, mGlu8b R endocytosis was not affected by CRIP1a. Chimeric constructs between CB1 R, mGlu8a R, and mGlu8b R underline the role of the identified five CRIP1a sensitive amino acids. In summary, we suggest that CRIP1a negatively regulates endocytosis of two different G-protein coupled receptor types, CB1 R and mGlu8a R.

Melatonin anticancer effects: review.

Melatonin (N-acetyl-5-methoxytryptamine, MLT), the main hormone produced by the pineal gland, not only regulates circadian rhythm, but also has antioxidant, anti-ageing and immunomodulatory properties. MLT plays an important role in blood composition, medullary dynamics, platelet genesis, vessel endothelia, and in platelet aggregation, leukocyte formula regulation and hemoglobin synthesis. Its significant atoxic, apoptotic, oncostatic, angiogenetic, differentiating and antiproliferative properties against all solid and liquid tumors have also been documented. Thanks, in fact, to its considerable functional versatility, MLT can exert both direct and indirect anticancer effects in factorial synergy with other differentiating, antiproliferative, immunomodulating and trophic molecules that form part of the anticancer treatment formulated by Luigi Di Bella (Di Bella Method, DBM: somatostatin, retinoids, ascorbic acid, vitamin D3, prolactin inhibitors, chondroitin-sulfate). The interaction between MLT and the DBM molecules counters the multiple processes that characterize the neoplastic phenotype (induction, promotion, progression and/or dissemination, tumoral mutation). All these particular characteristics suggest the use of MLT in oncological diseases.

The Di Bella Method (DBM) in the treatment of prostate cancer: a preliminary retrospective study of 16 patients and a review of the literature.

OBJECTIVE: To evaluate the objective clinical response and the safety of the combined administration of somatostatin, melatonin, retinoids, vitamin D3, dopamine subtype 2 receptor (D2R) agonists and low doses of cyclophosphamide, associated with androgen ablation, in patients with a histological diagnosis of prostate adenocarcinoma (Pac). MATERIALS AND METHODS: The clinical data of 30 patients with non-invasive and metastatic prostate cancer, who attended our institution over a period of more than 5 years, were retrospectively reviewed. RESULTS: 16 patients satisfied the evaluation criteria. Median age: 64 years. Disease stages: 8 patients (50%) were in Stage II. For advanced stages (Stage IV), secondary lesions were located in the bones and lymph nodes. Taken together, an overall objective response (OR) [Complete response (CR) + Partial Response (PR)] was achieved in 69% of the patients, with 88% of objective clinical benefit [CR+PR+SD]. For local Prostate Cancer group, an OR was achieved in 87.5% of patients (7 cases; 53-98; 95% CI), with CR in 62.5% (5 cases, 31-86; 95% CI). In metastatic disease, the OR was 50% (4 cases; 21-78; 95% CI), with a 20% of CR (2 cases; 7-59; 95% CI) and 75% of clinical benefit. CONCLUSIONS: This preliminary study shows that patients with early and advanced forms of prostate cancer, not previously treated by surgery and/or chemo-radiotherapy, can achieve a more than positive clinical benefit with the protocol foreseen by the Di Bella Method. Further clinical investigations are strongly recommended.

Evaluation of the safety and efficacy of the first-line treatment with somatostatin combined with melatonin, retinoids, vitamin D3, and low doses of cyclophosphamide in 20 cases of breast cancer: a preliminary report.

OBJECTIVE: The current strategies for the treatment of breast cancer are essentially based on surgery, preceded and/or followed by chemotherapy often supplemented by radiotherapy and/or the administration of hormonal therapy and monoclonal antibodies. Their combined use has made it possible to increase an overall survival but they are still penalized by adverse effects and toxicity. The marked anti-cancer effects of biological molecule such as somatostatin, melatonin, retinoid, vitamin D3 and prolactin inhibitors have been studied and documented for several decades. Their integrated and synergic action have been demonstrated, but only a few studies have as yet been carried out on their combined application in humans. The aim of the present investigation was to evaluate both the objective clinical response and toxicity of the biological multimodal treatment named Di Bella Method (DBM). MATERIAL AND METHODS: The clinical data from a total of 20 women with a certified diagnosis of breast cancer,defined disease stage, and who independently decided to follow the DBM as first-line treatment, were retrospectively reviewed. RESULTS: The mean age of the patients was 51 years (min 30; max 73). Twelve (12) patients (60%) presented an early stage disease, while the other 40% had a locally advanced/metastatic stage. An overall clinical benefit was achieved in 75% of cases, with 55% of complete response and 20% of partial response. For metastatic patients, the overall survival rate was 71%. The main toxicity effects included leukopenia, gastrointestinal phenomena and drowsiness. CONCLUSIONS: The preliminary results of this report confirm the positive action of the biological treatment in terms of efficacy and survival, showing a more than favorable profile of tolerability.

The Di Bella Method (DBM) improved survival, objective response and performance status in a retrospective observational clinical study on 55 cases of lymphomas.

OBJECTIVES: Lymphomas are the main form of haematological neoplasms, representing 55.6% of all tumours of the blood. Overall, they account for 5.3% of all malignant tumours (excluding basal and squamous cell skin cancer) in Italy with a prevalence constantly increasing at a rate of 3% per year. From a histological point of view, they represent a vast heterogeneous group of haematological diseases, their staging being based on defined cyto-morphological and anatomo-pathological criteria. Although the combined use of standard approaches can provide good response rates, recurrence is particularly frequent in patients undergoing traditional treatment, with critical and often irreversible side effects such as myelosuppression and a high frequency of opportunistic infections and sterility. Numerous epidemiological studies and preclinical data have for some time now reported the anticancer effects of molecules such as Melatonin, Retinoids, Vitamins E, D3, and C, Somatostatin and prolactin inhibitors in neoplastic diseases. There are, however, very few publications on the combined effects of these substances in vivo. METHODS: We report an observational study carried out on 55 patients affected by various forms of lymphoma, treated with the biological therapy known as the Di Bella Method (DBM). The 1, 3 and 5-year survival rates are reported, together with any signs of toxicity. RESULTS: The DBM treatment achieved partial or complete objective responses in a shorter time and in greater percentages if administered as first-line therapy. The adjuvant treatment increased survival time and improved quality of life with respect to the data reported in the literature for the same types and stages of lymphoma. CONCLUSION: Overall, the treatment was well tolerated, with minor and transient side effects. The patients were able to continue the treatment at home, carrying out their normal activities without problems.