Association between oral and fecal microbiome dysbiosis and treatment complications in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation.

The oral and gastrointestinal mucosae represent the main targets of the toxic effect of chemo and/or radiotherapy administered during the conditioning regimen before hematopoietic stem cell transplant (HSCT). These harmful consequences and the immunological complications that may occur after the transplant (such as Graft versus Host Disease, GvHD) are responsible for the clinical symptoms associated with mucositis during the aplasia phase, like pain, nausea, vomiting, and diarrhea. These toxicities could play a critical role in the oral and gastrointestinal microbiomes during the post-transplant phase, and the degree of microbial dysbiosis and dysregulation among different bacterial species could also be crucial in intestinal mucosa homeostasis, altering the host's innate and adaptive immune responses and favoring abnormal immune responses responsible for the occurrence of GvHD. This prospective pediatric study aims to analyze longitudinally oral and gut microbiomes in 17 pediatric patients who received allogeneic HSCT for malignant and non-malignant diseases. The oral mucositis was mainly associated with an increased relative abundance of Fusobacteria, and Prevotella species, while Streptococcus descendants showed a negative correlation. The fecal microbiome of subjects affected by cutaneous acute GvHD (aGvHD) correlated with Proteobacteria. Oral mucosal microbiota undergoes changes after HSCT, Fusobacteria, and Prevotella represent bacterial species associated with mucositis and they could be the target for future therapeutic approaches, while fecal microbiome in patients with acute GvHD (aGvHD) revealed an increase of different class of Proteobacteria (Alphaproteobacteria and Deltaproteobacteria) and a negative correlation with the class of Gammaproteobacteria.

A unicentric cross-sectional observational study on chronic intestinal inflammation in total colonic aganglionosis: beware of an underestimated condition.

BACKGROUND: Inflammatory Bowel Diseases (IBD) are known to occur in association with Hirschsprung disease (HSCR). Most of cases are represented by Crohn Disease (CD) occurring in patients with Total Colonic Aganglionosis (TCSA) with an estimated prevalence of around 2%. Based on these considerations and on a number of provisional data belonging to our Center for Digestive Diseases, we developed a unicentric cross-sectional observational study aimed at describing phenotype, genotype, pathology and metagenomics of all patients with TCSA and Crohn-like lesions. RESULTS: Out of a series of 62 eligible TCSA patients, 48 fulfilled inclusion criteria and were enrolled in the study. Ten patients did not complete the study due to non-compliance or withdrawal of consent and were subsequently dropped out. A total of 38 patients completed the study. All patients were tested for chronic intestinal inflammation by a combination of fecal calprotectine (FC) or occult fecal blood (OFB) and underwent fecal metagenomics. Nineteen (50%) tested positive for FC, OFB, or both and subsequently underwent retrograde ileoscopy. Fourteen patients (36.8%) presented Crohn-like lesions, occurring after a median of 11.5 years after surgery (range 8 months - 21.5 years). No statistically significant differences regarding demographic, phenotype and genotype were observed comparing patients with and without lesions, except for need for blood transfusion that was more frequent in those with lesions. Faecal microbiome of patients with lesions (not that of caregivers) was less biodiverse and characterized by a reduction of Bacteroidetes, and an overabundance of Proteobacteria. FC tested negative in 3/14 patients with lesions (21%). CONCLUSIONS: Our study demonstrated an impressive 10-folds higher incidence of chronic inflammation in TCSA. Up to 50% of patients may develop IBD-like lesions postoperatively. Nonetheless, we failed in identifying specific risk factors to be used to implement prevention strategies. Based on the results of our study, we suggest screening all TCSA patients with retrograde ileoscopy regardless of FC/OFB values. The frequency of endoscopic assessments and the role of FC/OFB screening in prompting endoscopy is yet to be determined.

Bioelectrical impedance analysis in patients with posterior vitreous detachment.

OBJECTIVE: The purpose of the study is to assess body hydration in patients with posterior vitreous detachment (PVD) by bioelectrical impedance analysis (BIA). PVD, one of the most common eye diseases, is associated in both research and the collective image with reduced daily water intake, but this finding is not supported by strong evidence in the literature. PATIENTS AND METHODS: Based on Spectral Domain Optical Coherence Tomography (SD-OCT) evaluation, different PVD stages are identified: absent posterior vitreous detachment, partial posterior vitreous detachment (P-PVD), or complete posterior vitreous detachment (C-PVD). BIA is a simple, non-invasive bedside method used to assess body composition. Patients underwent BIA and completed a floaters symptoms. 30 patients were enrolled and divided into two groups according to the degree of vitreous detachment, in P-PVD (n=12) and C-PVD (n=18). Patients underwent BIA and completed a floaters symptoms questionnaire. BIA measured the Resistance (R), Reactance (Xc), Phase Angle (PhA), Total Body Water (TBW), Extracellular Water (ECW), Fat Mass (FM), Fat-Free Mass (FFM), and Body Cell Mass Index (BCMI). Finally, patients received a test to assess adherence to the Mediterranean diet (Mediterranean Diet Test Score, MDTS) with the addition of daily water intake. RESULTS: Relevant data were obtained from the BIA evaluation: the values of R and Xc were lower in the P-PVD group than C-PVD group (respectively 417.08±58.12 Ω vs. 476.94±51.29 Ω p=0.006 and 41.33±8.23 Ω vs. 50.61±7.98 Ω p=0.004). Instead, patients in the P-PVD group reported higher values of TBW and ECW than C-PVD group (respectively 44.13±7.57 L vs. 37.96±6.27 L p=0.021 and 21.03±4.06 L vs. 17.24±2.63 L p=0.004). CONCLUSIONS: In the present study, we reported a significant correlation between vitreous pathology and anthropometric and BIA measurements.

A literature review of hypertensive retinopathy: systemic correlations and new technologies.

OBJECTIVE: Hypertensive retinopathy (HR) is the most common ocular manifestation of systemic arterial hypertension. This paper aims to summarize the current knowledge of HR, reviewing its classical features, such as epidemiology, pathophysiology, clinical manifestations, classifications, management and the most significant systemic correlations. We also provide an update on the latest advances in new technologies focusing on novel instrumental classifications. MATERIALS AND METHODS: A literature search was performed to identify articles regarding HR listed in Embase, PubMed, Medline (Ovid) and Scopus database up to 1 December 2021. The reference lists of the analyzed articles were also considered a source of literature information. The following keywords were used in various combinations: hypertensive retinopathy, hypertension and eye, hypertensive retinopathy and systemic correlations, optical coherence tomography (OCT) and hypertensive retinopathy, optical coherence tomography angiography (OCTA) and hypertensive retinopathy, adaptive optics (AO) and hypertensive retinopathy. The authors analyzed all English articles found using the aforementioned keywords. All the publications were thoroughly reviewed to create a detailed overview of this issue. RESULTS: HR signs have a significative association with cardiovascular, cerebrovascular and other systemic diseases. Patients with arteriosclerotic changes and, at the same time, severe HR, are at increased risk for coronary disease, peripheral vascular disease, stroke and dementia. HR is even now diagnosed and classified by its clinical appearance on a fundoscopic exam that is limited by interobserver variability. New technologies, like OCT, OCTA, AO and artificial intelligence may be used to develop a new instrumental classification that could become an objective and quantitative method for the evaluation of this disease. They could be useful to evaluate the subclinical retinal microvascular changes due to hypertension that may reflect the involvement of other vital organs. CONCLUSIONS: The eye is the only organ in the human body where changes in the blood vessels due to systemic hypertension can be studied in vivo. All doctors should be familiar with this disease because it has been largely demonstrated that signs of HR are correlated to patient's health and mortality. Researchers should develop a new common, standardized, and objective method to assess hypertensive retinal changes; new technologies may have a significant role in this field. This review takes most of the literature published so far, including the OCTA studies in order to stimulate new points of reference to standardize parameters and new diagnostic markers of this disease.

Genomic characterization of a paediatric MRSA outbreak by next-generation sequencing.

INTRODUCTION: Twelve strains of meticillin-resistant Staphylococcus aureus (MRSA) isolated during a suspected outbreak in a paediatric intensive care unit were analysed by whole-genome sequencing (WGS). AIM: To define the clonality of MRSA strains to a high discriminative power, and to evaluate the presence of genetic determinants responsible for antibiotic resistance and virulence. RESULTS: Ten out of 12 strains belonged to multi-locus sequence type ST2625, while the other two strains were ST8. Among the ST2625 strains, analysis based on 1126 genes showed that they were clonal, sharing more than 98.3% of allelic identities, and one strain was isolated from a healthcare worker. All ST2625 strains were characterized by the SCC-Mec cassette IVa, and resistoma analysis indicated correspondence between phenotypic and genotypic characteristics. The study of 63 genes associated with virulence was correlated with the pattern of clonality shown. CONCLUSION: This analysis confirmed the occurrence of an outbreak. As such, standard infection control measures were strictly enforced, and this led to prompt termination of the outbreak.

Whole-genome sequencing as standard practice for the analysis of clonality in outbreaks of meticillin-resistant Staphylococcus aureus in a paediatric setting.

Meticillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of hospital-associated infections. This study investigated the potential use of whole-genome sequencing (WGS) for surveillance purposes by re-examining MRSA strains related to past outbreaks among hospitalized paediatric patients. WGS data ameliorated the genotypic profile previously obtained with Sanger sequencing and pulsed-field gel electrophoresis typing, and discriminated between strains that were related and unrelated to the outbreaks. This allowed strain clonality to be defined with a higher level of resolution than achieved previously. This study demonstrates the potential of WGS to trace hospital outbreaks, which may lead to WGS becoming standard practice in outbreak investigations.

An improved method for HLA-B and -C supratyping.

A growing body of evidence links the analysis of the KIR genotype and the presence of their HLA-B and -C ligands to a wide repertoire of human diseases. We noticed that, using a panel of 184 Caucasoid donors, a limited number of HLA alleles were incorrectly supratyped by previously described pyrosequence-based assays. Here we describe a simple implementation of the reported methods that corrects all the discrepancies found with HLA-B and -C molecular typing and allows establishing a quick and high-throughput method for the determination of HLA-Bw4 I(80), Bw4T(80), Bw6 and HLA-C1 or -C2 supratype.

Pharmacological inhibition of NOX reduces atherosclerotic lesions, vascular ROS and immune-inflammatory responses in diabetic Apoe(-/-) mice.

AIMS/HYPOTHESIS: Enhanced vascular inflammation, immune cell infiltration and elevated production of reactive oxygen species (ROS) contribute significantly to pro-atherogenic responses in diabetes. We assessed the immunomodulatory role of NADPH oxidase (NOX)-derived ROS in diabetes-accelerated atherosclerosis. METHODS: Diabetes was induced in male Apoe(-/-) mice with five daily doses of streptozotocin (55 mg kg(-1) day(-1)). Atherosclerotic plaque size, markers of ROS and immune cell accumulation were assessed in addition to flow cytometric analyses of cells isolated from the adjacent mediastinal lymph nodes (meLNs). The role of NOX-derived ROS was investigated using the NOX inhibitor, GKT137831 (60 mg/kg per day; gavage) administered to diabetic and non-diabetic Apoe(-/-) mice for 10 weeks. RESULTS: Diabetes increased atherosclerotic plaque development in the aortic sinus and this correlated with increased lesional accumulation of T cells and CD11c(+) cells and altered T cell activation in the adjacent meLNs. Diabetic Apoe(-/-) mice demonstrated an elevation in vascular ROS production and expression of the proinflammatory markers monocyte chemoattractant protein 1, vascular adhesion molecule 1 and IFNγ. Blockade of NOX-derived ROS using GKT137831 prevented the diabetes-mediated increase in atherosclerotic plaque area and associated vascular T cell infiltration and also significantly reduced vascular ROS as well as markers of inflammation and plaque necrotic core area. CONCLUSIONS/INTERPRETATION: Diabetes promotes pro-inflammatory immune responses in the aortic sinus and its associated lymphoid tissue. These changes are associated with increased ROS production by NOX. Blockade of NOX-derived ROS using the NOX inhibitor GKT137831 is associated with attenuation of these changes in the immune response and reduces the diabetes-accelerated development of atherosclerotic plaques in Apoe(-/-) mice.

Hyperhomocysteinemia in patients with Crohn's disease.

BACKGROUND: Thromboembolic complications have been reported in patients with Crohn's disease. Among the contributing factors, hyperhomocysteinemia has been described, although controversial data exist. The aim of our study was to assess the incidence of hyperhomocysteinemia in a nonselected group of patients with Crohn's disease and to determine whether it might represent a risk marker for thrombosis in such patients. METHODS: Fifty consecutive patients were recruited, and clinical and laboratory variables were compared between those without and those with hyperhomocysteinemia. In the latter, gene mutations in N5-N10-methyltetrahydrofolate reductase were searched for, and clinical and laboratory variables were related to hyperhomocysteinemia. The presence/absence of thrombotic episodes in both groups was determined. RESULTS: Both groups had similar clinically active disease, with higher C-reactive protein values found in those with hyperhomocysteinemia. Hyperhomocysteinemia was found in 46 % of patients. Of these, 74 % had moderate, 13 % intermediate, and 13 % severe increase in serum homocysteine levels. No relationship was found between homocysteine levels, and age, vitamin B12 levels, folic acid levels, Crohn's Disease Activity Index score, and CRP values. Gene mutations were found in 5 (22 %) patients, 2 homozygotes and 3 heterozygotes. None of the patients with or without hyperhomocysteinemia had episodes of venous or arterial thrombosis, or stroke. CONCLUSIONS: Hyperhomocysteinemia is frequent in patients with Crohn's disease, and it could be a cofactor for the pathogenesis of thrombotic episodes.

Viral-load and B-lymphocyte monitoring of EBV reactivation after allogeneic hemopoietic SCT in children.

EBV-associated post transplant lymphoproliferative disease (EBV-PTLD) is a life-threatening complication that may occur after hemopoietic SCT. We prospectively screened 80 children on a weekly basis using nested quantitative PCR to evaluate EBV genome copies. EBV viral load <1000 copies per 10(5) PBMC was observed in 63% of transplants, whereas it was between 1000 and 9999 copies per 10(5) PBMC in 13%, and between 10 000 and 19 999 in 10%, with no significant increase in percentage of CD20+ lymphocytes. Viral load reached > or = 20 000 copies per 10(5) PBMC in 14% of patients, and rituximab was administered to 75% of them. None of the patients except one developed a lymphoproliferative disease. Our study found that only 13% of unrelated donor HSCT recipients had a very high risk of EBV-PTLD defined as > or = 20 000 geq per 10(5) PBMC associated with an increase in CD20+ lymphocyte. We suggest that rituximab could be administered in the presence of very high levels of EBV-DNA viral load or in the presence of mid levels of EBV-DNA viral load associated with an increase in the percentage of CD20+ lymphocytes. Through this approach, we significantly reduced the number of patients treated with rituximab, and consequently the acute and chronic adverse events related to this treatment.

Rare viral infections in children receiving hemopoietic stem cell transplant.

Viral infections are a rare complication in autologous hemopoietic stem cell transplant (HSCT) recipients but represent a frequent cause of disease after allogeneic HSCT. In the last years, there has been an increase in the number of viral diseases observed in these patients. This fact may be at least partially due to an improvement in diagnostic facilities, but the increasing number of transplant procedures and the more severe immunosuppression may also have played an important role.

Acute phase lipocalin Ex-FABP is involved in heart development and cell survival.

Ex-FABP is an extracellular fatty acid binding protein, expressed during chicken embryo development in cartilage, muscle fibers, and blood granulocytes. Transfection of chondrocytes and myoblasts with anti-sense Ex-FABP cDNA results in inhibition of cell proliferation and apoptosis induction. Ex-FABP expression is dramatically enhanced by inflammatory stimuli and in pathological conditions. In this paper, by in situ whole mount and immunohistochemistry analysis we show that, at early developmental stage, Ex-FABP is diffuse in all tissues of chick embryos. Particularly high level of transcript and protein are expressed in the heart. During acute phase response (APR) induced by endotoxin LPS injection, a marked increase of Ex-FABP mRNA was observed in embryos, highest Ex-FABP expression being in heart and liver. To investigate in vivo the biological role of Ex-FABP, we have directly microinjected chicken embryos with antibody against Ex-FABP. Almost 70% of chicken embryos died and the target tissue was the heart. We detected in heart of the treated embryos a significant increase of apoptotic cells and high level of fatty acids. We propose that the accumulation of fatty acid, specific ligand of Ex-FABP, in the cell microenvironment is responsible of heart cell death, and we suggest that Ex-FABP may act as a survival protein by playing a role as scavenger for fatty acids.

Molecular cloning of trkE, a novel trk-related putative tyrosine kinase receptor isolated from normal human keratinocytes and widely expressed by normal human tissues.

We have identified and cloned a new member of the trk gene family, termed trkE, which generates a 3.9-kilobase (kb) transcript in normal human keratinocytes and in a variety of normal human tissues, but not in liver. Albeit at low level, trkE transcript is expressed also by PC12 cells. The open reading frame codes for a polypeptide of 876 amino acids exhibiting the classic features of cell surface tyrosine protein kinases. trkE catalytic domain is 41% identical to trkA and shows several features unique to the trk gene family. Its extracellular domain does not show significant homology to any known proteins. trkE is the first member of this gene family found abundantly and widely expressed in normal human tissues. Several lines of evidence suggest that NGF is also the ligand for trkE; (i) normal human keratinocytes bind NGF with high affinity, (ii) NGF stimulates keratinocyte growth in an autocrine fashion, (iii) NGF exerts its biological effect on keratinocytes through the stimulation of a trk-specific tyrosine kinase, and (iv) keratinocytes lack trkA but do express large amount of trkE. trkE might also be the NGF receptor by other human peripheral tissues, such as pancreatic islets, and might represent a non-neuronal receptor for this ligand.

Nerve growth factor binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop.

Normal human keratinocytes synthesize and secrete biologically active nerve growth factor (NGF) in a growth regulated fashion (Di Marco, E., Marchisio, P. C., Bondanza, S., Franzi, A. T., Cancedda, R., and De Luca, M. (1991) J. Biol. Chem. 266, 21718-21722). Here we show that the same human keratinocytes bind NGF via low and high affinity receptors. In parallel with the course of NGF synthesis, the expression of low affinity NGF receptor (p75NGFr) decreases when a confluent, differentiated, and fully stratified epithelium is obtained. In skin sections, p75NGFr is present in basal keratinocytes and absent from suprabasal, terminally differentiated cells. The trkA protooncogene product (p140trkA), a component of the NGF receptor, is not expressed by keratinocytes. Instead, keratinocytes express a new member of the trk family (that we termed trkE), which generates 3.9-kilobase transcripts. Keratinocyte-derived NGF plays a key role in the autocrine epidermal cell proliferation. This has been proven by (i) direct effect of NGF on [3H]thymidine incorporation, (ii) inhibition of autocrine keratinocyte growth by monoclonal antibodies (alpha D11) inhibiting human NGF biological activity, and (iii) inhibition of autocrine keratinocyte proliferation by a trk-specific inhibitor, the natural alkaloid K252a. These data provide evidence that NGF, in addition to its effect as a survival and differentiation factor, is a potent regulator of cell proliferation, at least in human epithelial cells.

Expression of epidermal growth factor receptor and transforming growth factor alpha in human larynx carcinoma.

Altered expression of growth factors and growth factor receptors is frequently described in human tumors and human tumor cell lines. This further supports the hypothesis that oncogenesis is due to the subversion of mitogen-responsive pathways. The aim of this study was to investigate the expression of epidermal growth factor receptor (EGFR) and transforming growth factor alpha (TGF alpha) in 13 larynx carcinomas and 2 carcinomas of the oral cavity. We found receptor overexpression in 7 out of 15 tumors at mRNA and/or protein level but low expression in the majority of the normal adjacent tissues. TGF alpha was expressed only in 1 case, but no tyrosine kinase activity of the receptor was detected by antiphosphotyrosine antibody.

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.

Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes.

Nerve growth factor (NGF) transcripts were identified in normal human keratinocytes in primary and secondary culture. The expression of the NGF mRNA was strongly down-regulated by corticosteroids and was maximal when keratinocytes were in the exponential phase of growth. Immunofluorescence studies on growing keratinocytes colonies and on elutriated keratinocytes obtained from growing colonies and mature stratified epithelium showed specific staining of the Golgi apparatus only in basal keratinocytes in the exponential phase of growth. The keratinocyte-derived NGF was secreted in a biologically active form as assessed by neurite induction in sensory neurons obtained from chick embryo dorsal root ganglia. Based on these data we suggest that the basal keratinocyte is the cell synthesizing and secreting NGF in the human adult epidermis. The paracrine secretion of NGF by keratinocytes might have a major role in regulating innervation, lymphocyte function, and melanocyte growth and differentiation in epidermal morphogenesis as well as during wound healing.