Increased serum levels of proNGF, mature NGF and interleukins in burn-injured children.

OBJECTIVE: Burns are among the most common injuries in children. In burns of more than 20% of the total body surface area, a systemic inflammatory response involving several chemical mediators occurs. Among them, nerve growth factor (NGF) regulates the inflammatory response related to wound healing and promotes keratinocyte proliferation and angiogenesis. The aim of our study was to investigate the physiological response to injury in children with moderate-severe burns, assaying proNGF, mature NGF (mNGF), interleukins (IL)-1ß, and Il-10 serum levels. PATIENTS AND METHODS: This is a prospective observational study, including twelve children hospitalized for moderate-severe burns at the Gemelli Hospital (Rome). Their laboratory features were compared to those of patients with obstructive hydrocephalus who underwent surgery. RESULTS: Our results showed an increase in proNGF and mNGF serum levels. In burn patients, proNGF levels increased before mNGF, and serum concentrations of both were not correlated with burn extension and depth. The most significant levels of mNGF and proNGF were reported in scalds involving the face. Serum IL-1ß and IL-10 peak levels were reached with a time-course pattern similar to proNGF. CONCLUSIONS: Our preliminary results validate the hypothesis that serum levels of proNGF and mNGF may represent inflammatory biomarkers useful for monitoring burn patients and defining new strategies for their treatment.

Single-center, prospective, and observational study on the management and treatment of impetigo in a pediatric population.

OBJECTIVE: Ozenoxacin is a new antibiotic used to treat non-bullous impetigo. The aim of this study is to evaluate the microbiological and clinical efficacy of topical ozenoxacin 1% cream after 5-day twice-daily treatment, in pediatric patients with impetigo. PATIENTS AND METHODS: This observational and prospective study included patients aged 6 months to 18 years, with non-bullous impetigo. Efficacy was measured using the Skin Infection Rating Scale (SIRS) and microbiological culture at the first visit (T0), at the second visit after 72 hours (T1) and after 5 days (T2). Safety and tolerability were also evaluated. RESULTS: A total of 50 patients was enrolled. A reduction of SIRS score >10% after 72 hours of treatment was noticed in all patients, while a complete reduction was assessed after 5 days in all the population. Microbiologic success rates for ozenoxacin at T1 was 92% (four patients had original pathogens in the specimen culture from the skin area), whereas at T2, it was 100%. CONCLUSIONS: Topical ozenoxacin has strong efficacy in treating impetigo in pediatric patients. Ozenoxacin's clinical and microbiological rapid onset of response led to consider this antibiotic a novel efficacy option for the treatment of impetigo.

Non-pharmacologic approaches to neurological stimulation in patients with severe brain injuries: a systematic review.

OBJECTIVE: This review aimed to evaluate and summarize the current knowledge about the non-pharmacological neurological stimulation (NPNS) in patients with severe brain injuries (SBI). The approaches we analyzed included sensory stimulation, music therapy, virtual reality, transcranial direct current stimulation (tDCS) and transcranial magnetic stimulation (TMS). MATERIALS AND METHODS: We performed a review following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) standards. The key words used for the search across electronic databases such as PubMed and the Cochrane Library were "brain injury" or "coma" or "vegetative state" and "neurological stimulation" or "sensory stimulation" or "music therapy" or "virtual reality" or "transcranial direct current stimulation" or "transcranial magnetic stimulation". RESULTS: 38 studies matched the inclusion criteria. These articles were categorized into five clusters: sensory stimulation, music therapy, virtual reality, transcranial direct current stimulation and transcranial magnetic stimulation. Hence, a concise summary of each study was made up, including study population characteristics, type of non-pharmacological neurological stimulation, neurological clinical outcomes or neuroimaging outcomes. CONCLUSIONS: Overall, all the non-pharmacological approaches to neurological stimulation in patients with SBI seem to be innovative and promising. Further randomized clinical trials, including a wide range of patients, will be necessary to definitely validate these methods and develop standardized protocols shared in the scientific community.

Congenital lung malformations: a novel application for lung ultrasound?

Congenital lung malformations (CLMs) include a group of different disorders. With widespread use of antenatal ultrasonography (aUS) and increased use of pre-natal magnetic resonance imaging (MRI), CLMs are increasingly detected, nevertheless the best postnatal imaging approach is not yet well defined: newborns usually undergo several chest X-rays and eventually computed tomography to confirm the diagnosis. In this case series, we show lung ultrasound features of three different cases of congenital lung malformations, describing prenatal and postnatal images comparing different imaging techniques.

Role of the pupillometer in the assessment of pain in the sedation of pediatric patients.

OBJECTIVE: Pupillometry has been used to assess pain intensity and response to analgesic drugs in adults. The aim of this study was to verify the usefulness and effectiveness of the pupillometer to assess pain and depth of sedation in pediatric patients undergoing painful procedures and to optimize pain management by observing pupillary variations induced by opioids. PATIENTS AND METHODS: This is a prospective, monocentric study conducted in the sedation room of the Pediatric Intensive Care Unit of Fondazione Policlinico A. Gemelli in Rome. A population of 22 pediatric patients who underwent painful procedures was enrolled. Eleven children were sedated by opioid drugs. Heart rate, systolic blood pressure, diastolic blood pressure, bispectral index, maximum pupil size (Size), pupil change (CH), Neurological Pupil Index (NPi) were collected over four times: before starting the procedure; before the painful stimulus (when the patient was sedated); when the painful stimulus was applied; at the end of the procedure. A NeurOptics NPi-200 pupillometer was used for the study. RESULTS: Statistical significance in the variation of haemodynamic parameters was less significant than the variation obtained by analyzing the pupillary parameters: a significant change in NPi and CH in the transition from wakefulness to sedation and from the application of the painful stimulus to awakening was found in both study populations, patients who have received opioids and patients who have not received opioids. Changes in the mean CH of the pupil diameter correlate with the depth of sedation, and the size values vary in relation to the administration of opioids. CONCLUSIONS: Our findings highlight the potential role of pupillometry as a non-invasive method to objectively quantitate pain response in children to reach an efficient analgesic approach.

Effect of Lactobacillus reuteri LRE02-Lactobacillus rhamnosus LR04 combination and gastrointestinal functional disorders in an Emergency Department pediatric population.

OBJECTIVE: Probiotics are living microorganisms that, when administered per os in adequate amounts, may confer a health benefit on the host by the regularization of an unbalanced gastroenteric microbiota. The objective of this study was to evaluate treatment effectiveness, safety, and palatability of a probiotic's combination (Lactobacillus reuteri LRE02-DSM 23878 and Lactobacillus rhamnosus LR04-DSM 16605) in a pediatric Emergency Department setting with functional gastrointestinal disorders. PATIENTS AND METHODS: Three groups were enrolled: children with functional abdominal pain; children with gastroenteritis; children with gas colic. Self-reporting sheets were delivered to each patient/parent after probiotics treatment. The primary outcome was to evaluate the evolution of clinical conditions in enrolled children. RESULTS: The outcomes showed a statistical difference among children treated with probiotics and those who did not. In the functional abdominal pain group, 58.2% of patients had a moderate symptoms improvement and 33.5% had a complete disappearance of symptoms, while in the gas colic group, 68.2% of the infants had a moderate improvement and 23.2% had a complete resolution. In the gastroenteritis group, stool consistency and number of evacuations improved in children who took probiotic administration as well. CONCLUSIONS: Probiotics therapy, at the recommended dosage of five drops per day for 15 days, is associated with symptoms improvement. Moreover, the use of probiotics led to a stool consistency's normalization in a shorter time, evaluated with BSS. A randomized trial is needed to confirm these results.

Telephone consultation during Coronavirus outbreak in a Pediatric Emergency Department: methodological approach of a tertiary care center in a COVID-19 hospital setting.

OBJECTIVE: A computerized system of telephone consultation has been experimented at the Pediatric Emergency Department (ED) of Policlinico Gemelli Hospital in Rome during the outbreak of Coronavirus Disease 2019 (COVID-19). MATERIALS AND METHODS: Twenty monothematic items with a series of questions to evaluate child's clinical conditions have been set up in order to evaluate the different situations according to their severity. All items were highlighted according to conventional scores corresponding to the different answers (yes/no) given by the child's parents. This system has been implemented with large diffusion of computer programs and applications by the availability of a computer station in every ED room. RESULTS: The system allows healthcare workers to establish the medical check-up urgency which may be immediate, within the next 24 hours or scheduled in the pediatric ward. Therefore, it has been implemented a telephone triage consultation with a standardized method. CONCLUSIONS: Telephone consultation during outbreaks, considering the risks of contagion, allows healthcare workers to decrease the concern of families and to reduce indiscriminate access to ED. The remote approach will not solve logistic and setting problems related to COVID-19 outbreak17, but it would be a valid tool to improve medical evaluation without deep change in infrastructure and clinical organization.

CCR10 expression is required for the adjuvant activity of the mucosal chemokine CCL28 when delivered in the context of an HIV-1 Env DNA vaccine.

An effective prophylactic vaccine targeting HIV must induce a robust humoral response and must direct the bulk of this response to the mucosa-the primary site of HIV transmission. The chemokine, CCL28, is secreted by epithelial cells at mucosal surfaces and recruits' cells expressing its receptor CCR10. CCR10 is predominantly expressed by IgA + ASCs. We hypothesized that co-immunization with plasmid DNA encoding consensus envelope antigens with plasmid-encoded CCL28 would enhance anti-HIV IgA responses at mucosal surfaces. Indeed, animals receiving pCCL28 and pEnvA/C had significantly increased HIV-specific IgA in fecal extract. Surprisingly, CCL28 co-immunization induced a significant increase in anti-HIV IgG in the serum in mice compared to those receiving pEnvA/C alone. These robust antibody responses were not associated with changes in the frequency of germinal center B cells but depended upon the expression of CCR10, as these responses we abolished in CCR10-deficient animals. Finally, immunization with CCL28 led to increased frequencies in HIV-specific CCR10 + and CCR10 + IgA + B cells in the small intestine and Peyer's patches of vaccinated animals as compared to those receiving pEnvA/C alone. These data indicate that CCL28 administration can enhance antigen-specific humoral responses systemically and at mucosal surfaces.

EGF-R signaling through Fyn kinase disrupts the function of integrin alpha6beta4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion.

We have examined the mechanism and functional significance of hemidesmosome disassembly during normal epithelial cell migration and squamous carcinoma invasion. Our findings indicate that a fraction of EGF receptor (EGF-R) combines with the hemidesmosomal integrin alpha6beta4 in both normal and neoplastic keratinocytes. Activation of the EGF-R causes tyrosine phosphorylation of the beta4 cytoplasmic domain and disruption of hemidesmosomes. The Src family kinase inhibitors PP1 and PP2 prevent tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes without interfering with the activation of EGF-R. Coimmunoprecipitation experiments indicate that Fyn and, to a lesser extent, Yes combine with alpha6beta4. By contrast, Src and Lck do not associate with alpha6beta4 to a significant extent. A dominant negative form of Fyn, but not Src, prevents tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes. These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the beta4 cytoplasmic domain. Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF. Furthermore, dominant negative Fyn decreases the ability of squamous carcinoma cells to invade through Matrigel in vitro and to form lung metastases following intravenous injection in nude mice. These results suggest that disruption of hemidesmosomes mediated by Fyn is a prerequisite for normal keratinocyte migration and squamous carcinoma invasion.

Expression of the K-fgf proto-oncogene is controlled by 3' regulatory elements which are specific for embryonal carcinoma cells.

Expression of the K-fgf/hst proto-oncogene appears to be restricted to cells in the early stages of development, such as embryonal carcinoma (EC) cells. When EC cells are induced to differentiate, K-fgf expression is drastically repressed. To identify cis-acting DNA elements responsible for this type of regulation, we constructed a plasmid in which cat gene expression was driven by about 1 kilobase of upstream K-fgf human DNA sequences, including the putative promoter, and transfected it into undifferentiated F9 EC cells or HeLa cells as prototypes of cells which express or do not express, respectively, the K-fgf proto-oncogene. This plasmid was essentially inactive in both cell types, and the addition of more than 8 kilobases of DNA sequences upstream of the K-fgf promoter did not lead to any increase in chloramphenicol acetyltransferase (CAT) expression. On the other hand, when we inserted in this plasmid DNA sequences which are 3' of the human K-fgf coding sequences, we could detect a significant stimulation of CAT activity. Analysis of these sequences led to the identification of enhancerlike DNA elements which are part of the 3' noncoding region of K-fgf exon 3 and promote CAT expression only in undifferentiated mouse F9 or human NT2/D1 EC cells, but not in HeLa, 3T3, or differentiated F9 cells, therefore mimicking the physiological expression of the K-fgf proto-oncogene. Similar elements are also present in the 3' region of the murine K-fgf proto-oncogene, in a region showing high homology to the human K-fgf sequences. These regulatory elements can promote CAT expression from heterologous promoters in an EC-specific manner, suggesting that they interact with a specific cellular transacting protein(s) whose expression is developmentally regulated.

Rapid degradation of AU-rich element (ARE) mRNAs is activated by ribosome transit and blocked by secondary structure at any position 5' to the ARE.

The 3' noncoding region (NCR) AU-rich element (ARE) selectively confers rapid degradation on many mRNAs via a process requiring translation of the message. The role of cotranslation in destabilization of ARE mRNAs was examined by insertion of translation-blocking stable secondary structure at different sites in test mRNAs containing either the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE or a control sequence. A strong (-80 kcal/mol [1 kcal = 4.184 kJ]) but not a moderate (-30 kcal/mol) secondary structure prevented destabilization of mRNAs when inserted at any position upstream of the ARE, including in the 3' NCR. Surprisingly, a strong secondary structure did not block rapid mRNA decay when placed immediately downstream of the ARE. Studies are also presented showing that the turnover of mRNAs containing control or ARE sequences is not altered by insertion of long (1,000-nucleotide) intervening segments between the stop codon and the ARE or between the ARE and poly(A) tail. Characterization of ARE-containing mRNAs in polyadenylated and whole cytoplasmic RNA fractions failed to find evidence for decay intermediates degraded to the site of strong secondary structure from either the 5' or 3' end. From these and other data presented, this study demonstrates that complete translation of the coding region is essential for activation of rapid mRNA decay controlled by the GM-CSF ARE and that the structure of the 3' NCR can strongly influence activation. The results are consistent with activation of ARE-mediated decay by possible entry of translation-linked decay factors into the 3' NCR or translation-coupled changes in 3' NCR ribonucleoprotein structure or composition.

Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential.

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.

The deletion polymorphism of the angiotensin-converting enzyme is associated with nephroangiosclerosis.

The D allele of the angiotensin-converting enzyme (ACE) gene has been linked with diabetic nephropathy and IgA glomerulonephritis and with faster renal disease progression. The association of this allele with nephroangiosclerosis has been scarcely investigated. We have tested this association in 45 hypertensive patients (all whites) with well defined nephroangiosclerosis (diagnosis established on the basis of renal biopsy in all cases) and moderate to severe renal failure. As studies of genetic association of small size often produce conflicting results, besides a control group of 343 Italian patients with essential hypertension and normal renal function, we elected to use also a very large control group of race-matched subjects taken from a meta-analysis of 27,565 whites. The proportion of patients with the D allele (64%) was higher in patients with nephroangiosclerosis than that in Italian hypertensives (54%) and in whites (54%). DD and DI genotypes were more prevalent in patients than in control groups. The dominant model (DD and DI v II: nephroangiosclerosis v Italian controls: chi2 = 6.19, P = .012; nephroangiosclerosis v whites chi2 = 6.86, P = .009) fitted the data better than the codominant and the recessive model (P < or = .022). The D allele is associated with nephroangiosclerosis with a dominant effect in the sample of patients studied. Although intervention studies are needed to see whether these findings imply a causal association, our data suggest that this allele may at least act as disease marker in nephroangiosclerosis.

Calcium-induced stabilization of AU-rich short-lived mRNAs is a common default response.

The AU-rich element (AUUUA)n, found in the 3' noncoding region of many short-lived cytokine and proto-oncogene mRNAs, is sufficient to specifically target these mRNAs for rapid degradation in mammalian cells. The mechanism by which the AU-rich element promotes rapid mRNA decay is not known. Previous studies have shown that release of intracellular stored calcium by ionophore treatment of thymocytes and mast cells inhibits the rapid turnover of AU-rich interleukin mRNAs. Increased cytoplasmic half-life of interleukin mRNAs was linked to calcium-induced activation of the N-terminal c-Jun kinase. In this report we have characterized the calcium-induced stabilization of AU-rich mRNAs. We show that calcium induces stabilization of mRNAs with canonical AU-rich elements in all cell types tested. These results indicate that short-lived mRNA stabilization by calcium is not unique to immune cells nor interleukin mRNAs, but is a widespread default response that includes generic AU-rich mRNAs. Stabilization is shown to be rapid but transient, and to act without altering nuclear transcription or cytoplasmic translation rates. These data support the view that calcium release likely stabilizes short-lived mRNAs by altering trans-acting decay factors that promote AU-rich mRNA turnover.

The Innovative Socio-economic Interventions Against Tuberculosis (ISIAT) project: an operational assessment.

SETTING: Tuberculosis (TB) affected households in impoverished shantytowns, Lima, Peru. OBJECTIVE: To evaluate socio-economic interventions for strengthening TB control by improving uptake of TB care and prevention services. DESIGN: Barriers to TB control were characterised by interviews with TB-affected families. To reduce these barriers, a multidisciplinary team offered integrated community and household socio-economic interventions aiming to: 1) enhance uptake of TB care by education, community mobilisation and psychosocial support; and 2) reduce poverty through food and cash transfers, microcredit, microenterprise and vocational training. An interim analysis was performed after the socio-economic interventions had been provided for 2078 people in 311 households of newly diagnosed TB patients for up to 34 months. RESULTS: Poverty (46% earned

High-grade endometrial stromal sarcoma: clinicopathologic and immunohistochemical study of a case.

This report describes a case of high-grade stromal sarcoma occurring in a 53-year-old woman. Preoperative and pathologic findings showed up a large tumoral nodule confined to the uterus. The patient underwent a total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy and dissection of pelvic and para-aortic lymph-nodes; successively she underwent pelvic and abdominal radiotherapy and chemotherapy, which was discontinued because of leukopenia. Histologically the tumor was made up of cells resembling endometrial stromal cells with only mild cytologic atypia and had up to 24 mitoses x 10 HPF in the most cellular areas. Immunohistochemically it showed a diffuse positivity for vimentin and a focal positivity for muscular actin and desmin. The patient died of this disease nine months postoperatively. These features underline that a uterine sarcoma with histological features similar to a low-grade endometrial stromal sarcoma but with high mitotic rate may behave as a very aggressive tumor even when diagnosed at the initial stage; the immunohistochemical data indicate that these tumors may show smooth-muscle differentiation.

Interferon production in L929 cells under impaired translational conditions: comparison of rates of interferon, actin, Newcastle disease and encephalomyocarditis viruses mRNAs initiation of protein synthesis.

The pattern of Interferon (IFN) production in virus-infected cells has been compared with the rate of bulk cellular protein synthesis, on one hand, and with the synthesis of representative cell and virus proteins such as actin, the gamma and the NP proteins of encephalomyocarditis (EMC) and Newcastle Disease (NDV) viruses, on the other hand. This was investigated under conditions of impaired protein synthesis such as i) high osmolarity media, ii) a virus-induced shut-off, and iii) in cells exposed to relatively low doses of cycloheximide (CXM), which slow elongation of protein chain and thus favour the translation of low-affinity messenger RNAs (mRNAs). In each instance IFN production was compared with 35S-methionine incorporation into TCA-precipitable materials and into SDS-polyacrylamide gel-analysed proteins. Data obtained from each of the experimental approaches all indicate that IFN production and cellular protein synthesis are modified in a closely related fashion suggesting that their mRNAs share a similar degree of affinity for ribosomes. Conversely, two mRNAs coding for representative EMC and NDV virus proteins exhibit, respectively, the highest and the lowest affinity for ribosomes as compared to actin mRNA.

L929 cells infected with temperature sensitive mutants of vesicular stomatitis virus: virus replication is necessary for induction of changes in membrane permeability.

Infection of L929 murine cells with vesicular stomatitis virus (VSV) results in inhibition of host protein synthesis and appearance of membrane alterations at a time when cells are still actively engaged in viral protein synthesis. VSV temperature-sensitive (ts) mutants have been used to explore the role(s) played by the virus-coded proteins in the genesis of these effects. Cells were infected with each of five ts mutants representing the known complementation groups of VSV Indiana serotype, and incubated at permissive (32 degrees C) and non-permissive temperatures (39 degrees C). Protein synthesis in the presence and absence of Hygromycin B (Hyg. B) was analyzed during virus infection via incorporation of 35S-methionine in acid-precipitable material and SDS-polyacrylamide gel electrophoresis. Data indicate that mutants belonging to groups I (L protein), II (NS protein) and IV (N protein) do not inhibit host protein synthesis and do not induce any membrane changes when grown at the non-permissive temperature. Mutants of group III (M protein) and V (G protein), instead, do inhibit cell protein synthesis and induce membrane changes also when grown at the non-permissive temperature; this suggests that these effects do not correlate with the biological activity of these proteins and their interaction with the cellular membrane. On the other hand, mutants exhibiting defective steps of nucleocapsid replication are apparently unable to induce these effects once more suggesting that virus replication per se is essential, as also indirectly shown by experiments employing cycloheximide to mimic shut-off.