Estrogen-Induced Proliferation in Cultured Hepatocytes Involves Cyclin D1, P21CIP1 and P27KIP1

The purpose of this study was to establish if estrogen-induced hepatocyte proliferation in vitro involves the cell cycle regulators cyclin D1, p21^Cip1, and p27^Kip1. Male rat hepatocytes were cultured in presence of 17-β -estradiol (E₂) ± ICI-182780, a pure estrogen antagonist, and [³H]-thymidine, as required. DNA synthesis as well as p21^Cip1, p27^Kip1, and cyclin D1mRNA and protein levels were evaluated at different times (12, 24, 36, and 48 hours) of incubation. E₂-increased DNA synthesis was correlated with cyclin D1 and p21^Cip1 (mRNA and protein) variations that were reversed by the addition of ICI-182780. p27^Kip1protein levels progressively increased regardless of the presence of E₂ or ICI-182780. Our data confirm that estrogens’ stimulatory effect is related to their ability to increase cyclin D1 levels. The increase of p21^Cip1 is probably related to the reentry of hepatocytes in the quiescent state. p27^Kip1protein is not able to arrest hepatocyte proliferation.     

Microbiome signatures of progression toward celiac disease onset in at-risk children in a longitudinal prospective cohort study

Other than exposure to gluten and genetic compatibility, the gut microbiome has been suggested to be involved in celiac disease (CD) pathogenesis by mediating interactions between gluten/environmental factors and the host immune system. However, to establish disease progression markers, it is essential to assess alterations in the gut microbiota before disease onset. Here, a prospective metagenomic analysis of the gut microbiota of infants at risk of CD was done to track shifts in the microbiota before CD development. We performed cross-sectional and longitudinal analyses of gut microbiota, functional pathways, and metabolites, starting from 18 mo before CD onset, in 10 infants who developed CD and 10 matched nonaffected infants. Cross-sectional analysis at CD onset identified altered abundance of six microbial strains and several metabolites between cases and controls but no change in microbial species or pathway abundance. Conversely, results of longitudinal analysis revealed several microbial species/strains/pathways/metabolites occurring in increased abundance and detected before CD onset. These had previously been linked to autoimmune and inflammatory conditions (e.g., Dialister invisus, Parabacteroides sp., Lachnospiraceae, tryptophan metabolism, and metabolites serine and threonine). Others occurred in decreased abundance before CD onset and are known to have anti-inflammatory effects (e.g., Streptococcus thermophilus, Faecalibacterium prausnitzii, and Clostridium clostridioforme). Additionally, we uncovered previously unreported microbes/pathways/metabolites (e.g., Porphyromonas sp., high mannose–type N-glycan biosynthesis, and serine) that point to CD-specific biomarkers. Our study establishes a road map for prospective longitudinal study designs to better understand the role of gut microbiota in disease pathogenesis and therapeutic targets to reestablish tolerance and/or prevent autoimmunity.

Celiac disease (CD) is a chronic systemic autoimmune disorder that occurs in genetically predisposed individuals and is characterized by loss of tolerance to dietary gluten protein. CD affects ∼1% of the global population, with regional variations depending on human leukocyte antigen (HLA) presence and dietary gluten consumption (1). The incidence of CD most likely will continue to increase, along with other autoimmune conditions, despite the fact that its associated genes, HLA, and the trigger, gluten, have not changed (1). Nevertheless, more than 30% of the population carries the predisposing gene and is exposed to gluten, yet only 2 to 3% develop CD in their lifetime (2). This suggests that other factors such as the intestinal microbiota may also contribute to CD pathogenesis.The inflammatory process underlying CD involves both innate and adaptive immune systems (3). While the adaptive immune response in CD has been described, less is known about the innate immune response following gluten exposure, which drives early steps in CD pathogenesis and eventually leads to loss of gluten tolerance (4). Previous work has linked the trigger of CD, gluten, the intestinal microbiota, and the innate immune response (5–8).Given the cross-talk between the gut microbiota and immune system, alterations in the gut microbiota have been linked to several autoimmune conditions (9) such as inflammatory bowel disease (10), type 1 diabetes (T1D) (11), multiple sclerosis (12), and CD (13–18). We, and others, have looked for changes in the gut microbiota of infants at risk for CD (15, 17–19). For example, using 16S ribsosomal ribonucelic acid (rRNA) amplicon sequencing, we previously reported higher abundance of Lactobacillus up to 12 mo of age in one infant who later developed CD compared with 15 at-risk infants who did not (15). Other studies of gut microbiota and CD assessed changes in gut microbiota composition of individuals during the first year after birth who later developed CD compared with controls (17, 18). For example, Olivares et al. (17) used a prospective cohort of 200 infants at risk for CD to compare, with 16S rRNA sequencing, the intestinal microbiota of 10 cases who developed CD during the 5-y study period and 10 matched controls at 4 and 6 mo of age. They reported increases in abundance of Firmicutes, Enterococcaceae, and Peptostreptococcaceae in controls from 4 to 6 mo (17). Rintala et al. (18) also examined intestinal microbiota of infants at risk for CD, using 16S rRNA sequencing, at 9 and 12 mo of age in nine subjects who developed CD by ages 4 and 18 and matched controls, but did not identify any significant differences in microbiota composition. Huang et al. (20) examined intestinal microbiota, using 16S rRNA sequencing, at ages 1, 2.5, and 5 y in 16 subjects with CD (11 of whom developed CD after age 5) and 16 controls and found significant differences in taxonomic composition of the microbiota in cases compared with controls at all of the time points. Finally, a recent study using 16S rRNA sequencing to examine differences in the gut microbiota of children with untreated CD compared with children with treated CD and healthy control subjects did not identify changes in alpha diversity at CD diagnosis (21) but did identify differences in taxonomic composition, such as a lower abundance in Alistipes in subjects with CD compared with healthy controls (21). A separate study utilizing 16S rRNA sequencing also identified significant differences in taxonomic composition between patients with newly diagnosed CD and healthy control subjects, with subjects with recently diagnosed CD having a lower abundance of Bacteroides–Prevotella, Akkermansia, and Staphylococcaceae (22).While these studies provide an important foundation concerning alterations in gut microbiota of subjects at risk for CD early in life, they analyzed only up to three time points in the first year after birth (17, 18, 20) or were restricted to only one subject with CD (15). In addition, the studies used 16S rRNA sequencing to analyze intestinal microbiota, which cannot provide information about functional characteristics of the microbiota nor provide taxonomic data at the strain level, both of which are necessary to design effective treatment for CD. Furthermore, metabolomic analysis (if any) in these studies was generally limited to serum (as opposed to fecal) metabolites, which do not provide direct information about metabolic activity of the gut microbiota. More importantly, here we argue that, to gain mechanistic insight into the pathogenesis of CD and other autoimmune diseases, we need to transition from case–control microbiome studies to prospective longitudinal studies, which prospectively examine subjects at multiple time points before disease development (23). Studies aimed at identifying changes in the microbiome (11, 24) and intestinal permeability (25) have been performed and identified taxonomic changes prior to T1D (11) and necrotizing enterocolitis (24) as well as increased intestinal permeability up to 3 y prior to the development of Crohn’s disease (25). However, longitudinal birth cohort studies focused on multiomic data collection and analysis are limited and have not been developed for CD.The first step toward achieving this goal was to establish a prospective cohort study for CD, the Celiac Disease Genomic, Environmental, Microbiome, and Metabolomic (CDGEMM) study (26), where we have been following approximately 500 infants in the United States, Italy, and Spain who have a first-degree relative with CD and therefore, are at a high risk of developing CD. We have previously utilized other study subjects from this cohort and multiomics analysis to investigate the impact of genetic and environmental risk factors on the development of the gut microbiota in infants at risk for CD (19). In the current study, we present proof of concept intersubject and intrasubject analyses using fecal metagenomic and metabolomic data collected at multiple time points before onset of CD in 10 cases and 10 matched controls in order to identify alterations in the intestinal microbiota and metabolome, which may serve as markers of progression toward CD onset.     

Isolated polio-like syndrome after tick-borne encephalitis presenting with acute hyperckemia

Tick borne encephalitis virus infection usually shows a biphasic course. In the first stage of illness symptoms are similar to a flu-like syndrome, then after a defervescence period, fever may represent with neurological manifestations ranging from mild meningitis to severe encephalomyelitis. We report the clinical case of an adult man presented with an acute proximal hyposthenia, severe hyperckemia, clinical and laboratoristic evidence of acute tick borne virus infection. This virus has a favourite tropism for the anterior horn cells of the cervical spine segment. Polio-like syndrome, usually affecting the upper limbs, is the clinical phenotype of an infection of the cervical motoneurons. Usually myelitis is associated to severe encephalitis and a complete diagnosis may be difficult in comatose patients. Rarely, an isolated polio-like syndrome may be the sole neurological complication of tick-borne encephalitis.     

Meniscal pathology in children: differences and similarities with the adult meniscus

The normal meniscus undergoes typical developmental changes during childhood, reaching a mature adult appearance by approximately 10 years of age. In addition to recognizing normal meniscal appearances in children, identifying abnormalities — such as tears and the different types of discoid meniscus and meniscal cysts, as well as the surgical implications of these abnormalities — is vital in pediatric imaging. The reported incidence of meniscal tears in adolescents and young adults has increased because of increased sports participation and more widespread use of MRI. This review discusses the normal appearance of the pediatric meniscus, meniscal abnormalities, associated injuries, and prognostic indicators for repair.     

Retrospective review of cystic fibrosis presenting as infantile liver disease.

The mode of presentation, clinical course, and outcome of 12 infants with cystic fibrosis and liver disease referred over an 18 year period were investigated retrospectively. Median age at presentation was 6.5 weeks (range, 5-12). Two thirds were boys. Conjugated hyperbilirubinaemia was the presenting symptom in 11 patients, and hypoalbuminaemia in one. Jaundice was cleared over a median period of 7.36 months. Eight patients had bile duct proliferation on liver biopsy and one required cholangiography to exclude biliary atresia. Classic histological features of cystic fibrosis were only present in two children biopsied at 8 and 18 months. Three patients had meconium ileus, including one infant with concomitant alpha(1) antitrypsin deficiency, who required early liver transplantation. All other patients had no signs of significant chronic liver disease during a median follow up of 42 months (range, 10-205). Children with cystic fibrosis and infantile liver disease have a good short and medium term prognosis.     

Liver transplantation for alpha-1-antitrypsin deficiency in children

Alpha-1-antitrypsin (a1-AT) deficiency is an inborn error of metabolism, which can cause liver disease. The condition is one of the most common genetic disorders in the Caucasian population. Here we review our experience with 21 children suffering from end-stage liver disease due to a1-AT deficiency. All children are PIZZ homozygotes. Nineteen of them initially presented with neonatal jaundice and two with hepatosplenomegaly in childhood. Twenty-five liver transplantions were performed. All children are currently alive at a median follow-up of 40 months. Liver replacement provides the only definite treatment for children with end-stage liver disease associated with a1-AT deficiency. Excellent results can be achieved by reducing waiting time for transplantation and by early referral to a liver transplant centre. Received: 27 April 1999 Revised: 27 December 1999 Accepted: 24 March 2000     

Two rare cases of benign hyperlipasemia in children

Gullo’s syndrome is a newly identified condition characterized by a chronic elevation of pancreatic amylase and/or lipase in the absence of pancreatic disease. Until now, only one case of benign isolated hyperlipasemia in children has been recorded. We describe two children with benign and not familial increase of serum lipase. Case 1: a six year old girl presented with occasional discovery of serum lipase elevation. Medical history was silent for pancreatic hyperenzymemia. The screening for possible causes for elevated lipase (genetic, autoimmune and infectious diseases) was normal. The serum lipase increased three fold over the upper limit (193 U/L; reference range 0-60 U/L), with daily fluctuation of values. Both ultrasound scan and magnetic resonance imaging were normal. The genetic mutation associated with chronic pancreatitis was negative. We followed up this patient for two years with blood tests every six months and she did not show any signs or symptoms of pancreatic disease, except for the high level of lipase serum. Case 2: an eight year old girl complained of nausea, vomiting and severe abdominal pain in the epigastric region after eating for the last two weeks. Full blood count, electrolytes, C-reactive protein, liver and renal function were normal. Serum lipase was 96 U/L (reference range 0-60 U/L). The screening for the possible causes of pancreatic disease was negative. Endoscopy of the upper gastrointestinal tract, ultrasound, computed tomography scan and magnetic resonance imaging were normal. One year after the presentation of the symptoms, the patient became asymptomatic although the level of serum lipase continued to be high.