National-Level Schoolwork Pressure, Family Structure, Internet Use, and Obesity as Drivers of Time Trends in Adolescent Psychological Complaints Between 2002 and 2018.

Little is known about societal processes that contribute to changes in adolescent mental health problems. This study aims to fill this gap using data from the Health Behavior in School-aged Children study between 2002 and 2018 (ncountries = 43, nindividuals = 680,269, Mage = 14.52 (SD = 1.06), 51.04% female), supplemented with other international data. National-level psychological complaints increased more strongly among girls than boys. National-level schoolwork pressure, single-parent households, time spent on internet, and obesity were generally rising. In both boys' and girls' samples, increases in national-level schoolwork pressure, obesity, and time spent on internet use were independently associated with increases national-level psychological complaints. However, national-level obesity and psychological complaints were more strongly related among girls than boys. Results highlight the potential impact of societal-level processes on adolescent mental health problems.

Trends over Time in Adolescent Emotional Wellbeing in the Netherlands, 2005-2017: Links with Perceived Schoolwork Pressure, Parent-Adolescent Communication and Bullying Victimization.

In some Scandinavian countries, the United Kingdom and the United States, there is evidence of a dramatic decline in adolescent emotional wellbeing, particularly among girls. It is not clear to what extent this decline can be generalised to other high-income countries. This study examines trends over time (2005-2009-2013-2017) in adolescent wellbeing in the Netherlands, a country where young people have consistently reported one of the highest levels of wellbeing across Europe. It also assesses parallel changes over time in perceived schoolwork pressure, parent-adolescent communication, and bullying victimization. Data were derived from four waves of the nationally representative, cross-sectional Dutch Health Behaviour in School-aged Children study (N = 21,901; 49% girls; Mage = 13.78, SD = 1.25). Trends in emotional wellbeing (i.e., emotional symptoms, psychosomatic complaints, life satisfaction) were assessed by means of multiple regression analyses with survey year as a predictor, controlling for background variables. Emotional wellbeing slightly declined among adolescent boys and girls between 2009 and 2013. A substantial increase in perceived schoolwork pressure was associated with this decline in emotional wellbeing. Improved parent-adolescent communication and a decline in bullying victimization may explain why emotional wellbeing remained stable between 2013 and 2017, in spite of a further increase in schoolwork pressure. Associations between emotional wellbeing on the one hand and perceived schoolwork pressure, parent-adolescent communication, and bullying victimization on the other were stronger for girls than for boys. Overall, although increasing schoolwork pressure may be one of the drivers of declining emotional wellbeing in adolescents, in the Netherlands this negative trend was buffered by increasing support by parents and peers. Cross-national research into this topic is warranted to examine the extent to which these findings can be generalised to other high-income countries.

Revising the self-report strengths and difficulties questionnaire for cross-country comparisons of adolescent mental health problems: the SDQ-R.

AIMS: The Strengths and Difficulties Questionnaire (SDQ) has been used in many epidemiological studies to assess adolescent mental health problems, but cross-country comparisons of the self-report SDQ are scarce and so far failed to find a good-fitting, common, invariant measurement model across countries. The present study aims to evaluate and establish a version of the self-report SDQ that allows for a valid cross-country comparison of adolescent self-reported mental health problems. METHODS: Using the Health Behaviour in School-aged Children study, the measurement model and measurement invariance of the 20 items of the self-report SDQ measuring adolescent mental health problems were evaluated. Nationally representative samples of 11-, 13- and 15-year old adolescents (n = 33 233) from seven countries of different regions in Europe (Bulgaria, Germany, Greece, the Netherlands, Poland, Romania, Slovenia) were used. RESULTS: In order to establish a good-fitting and common measurement model, the five reverse worded items of the self-report SDQ had to be removed. Using this revised version of the self-report SDQ, the SDQ-R, partial measurement invariance was established, indicating that latent factor means assessing conduct problems, emotional symptoms, peer relationships problems and hyperactivity-inattention problems could be validly compared across the countries in this study. Results showed that adolescents in Greece scored relatively low on almost all problem subscales, whereas adolescents in Poland scored relatively high on almost all problem subscales. Adolescents in the Netherlands reported the most divergent profile of mental health problems with the lowest levels of conduct problems, low levels of emotional symptoms and peer relationship problems, but the highest levels of hyperactivity-inattention problems. CONCLUSIONS: With six factor loadings being non-invariant, partial measurement invariance was established, indicating that the 15-item SDQ-R could be used in our cross-country comparison of adolescent mental health problems. To move the field of internationally comparative research on adolescent mental health forward, studies should test the applicability of the SDQ-R in other countries in- and outside Europe, continue to develop the SDQ-R as a cross-country invariant measure of adolescent mental health, and examine explanations for the found country differences in adolescent mental health problems.

MVA-nef induces HIV-1-specific polyfunctional and proliferative T-cell responses revealed by the combination of short- and long-term immune assays.

Several vaccination trials are evaluating the modified vaccinia virus Ankara (MVA) as a delivery vector in various clinical settings. In this paper, we present the reevaluation of a therapeutic vaccination trial in human immunodeficiency virus (HIV)-1-infected individuals treated with highly active antiretroviral therapy using MVA-expressing HIV-1 nef. Immunogenicity of MVA-nef was assessed using multicolor flow cytometry. Vaccine-induced polyfunctionality and proliferative capacity, which are associated with nonprogressive HIV-1 infection, were detectable by combining two immune assays. By means of short-term polychromatic intracellular cytokine staining, we observed a significant increase in polyfunctional Nef-specific CD4 T cells expressing interferon-γ, interleukin (IL)-2 and CD154 after vaccination, whereas changes in the quality of CD8 T-cell response could not be observed. Only the additional use of a long-term polychromatic Carboxyfluorescein succinimidyl ester (CFSE)-based proliferation assay revealed vaccine-induced Nef-specific CD8, as well as CD4 T cells with proliferative capacity. The correlation between vaccine-induced IL-2 production by CD4 T cells and the increase in proliferating Nef-specific CD8 T cells suggests a causal link between these two functions. These results highlight the importance of combining sophisticated immunomonitoring tools to unravel concealed effects of immunological interventions and support the use of the poxvirus-derived MVA vector to stimulate highly functional HIV-1-specific T-cell responses. However, the clinical benefit of these functional T cells remains to be determined.

Fibrinogen kinetics and protein turnover in obese non-diabetic males: effects of insulin.

BACKGROUND: Although hyperfibrinogenemia and insulin resistance are common in obesity and diabetes mellitus, the impact of obesity per se on fibrinogen turnover and the insulin effects on fibrinogen and protein kinetics is unknown. METHODS: We measured fibrinogen and albumin fractional (FSR) and absolute (ASR) synthesis rates, as well as protein turnover, in non-diabetic, obese and in control male subjects both before and following an euglycemic, euaminoacidemic, hyperinsulinemic clamp, using L-[(2)H(3)]-Leucine isotope infusion. RESULTS: In the obese, basal fibrinogen concentrations was approximately 25% greater (p < 0.035), and fibrinogen pool approximately 45% greater (p < 0.005), than in controls. Both FSR and ASR of fibrinogen were similar to control values. With hyperinsulinemia, although fibrinogen FSR and ASR were not significantly modified with respect to baseline in either group, fibrinogen ASR resulted to be approximately 50% greater in the obese than in controls (p < 0.015). Hyperinsulinemia equally stimulated albumin synthesis and suppressed leucine appearance from endogenous proteolysis in both groups. Amino acid clearance was also similar. In the obese, the insulin-mediated glucose disposal was approximately 50% lower (p < 0.03) than in controls, and it was inversely correlated with fibrinogen ASR during the clamp in both groups (r = - 0.58). CONCLUSIONS: In obese, non-diabetic males, post absorptive fibrinogen production is normal. Whole-body amino acid disposal, basal and insulin-responsive protein degradation, and albumin synthesis are also normal. However, the greater fibrinogen ASR in the obese with hyperinsulinemia, and the inverse relationship between insulin sensitivity and clamp fibrinogen production, suggest a role for hyperinsulinemia and/or insulin resistance on fibrinogen production in obesity.

Hepatic lipid metabolism and non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly recognized pathology with a high prevalence and a possible evolution to its inflammatory counterpart (non-alcoholic steatohepatitis, or NASH). The pathophysiology of NAFLD and NASH has many links with the metabolic syndrome, sharing a causative factor in insulin resistance. According to a two-hit hypothesis, increased intrahepatic triglyceride accumulation (due to increased synthesis, decreased export, or both) is followed by a second step (or "hit"), which may lead to NASH. The latter likely involves oxidative stress, cytochrome P450 activation, lipid peroxidation, increased inflammatory cytokine production, activation of hepatic stellate cells and apoptosis. However, both "hits" may be caused by the same factors. The aim of this article is to overview the biochemical steps of fat regulation in the liver and the alterations occurring in the pathogenesis of NAFLD and NASH.

Nonpenetrating deep sclerectomy with reticulated hyaluronic acid implant versus punch trabeculectomy: a prospective clinical trial.

PURPOSE: To compare the intraocular pressure (IOP)-lowering effect and complication rate of nonpenetrating deep sclerectomy (NPDS) with reticulated hyaluronic acid (SK-GEL) scleral implant versus traditional punch trabeculectomy (PT) in the management of primary open angle glaucoma (POAG). METHODS: Prospective, randomized comparative study including 93 patients with uncontrolled POAG. Group 1 (43 eyes) underwent NPDS with SK-GEL scleral implant; Group 2 (50 eyes) underwent PT. Mitomycin C (0.2 mg/mL) was applied intraoperatively in both techniques. Study follow-up evaluations were conducted at 36 and 48 months. Complete success indicated the achievement of the target IOP without antiglaucoma medications, while qualified success indicated the same goal with medications. These categories were assessed at two target IOP levels, <21 mmHg and <18 mmHg. RESULTS: At 36 months for complete and qualified success with a <21 and <18 mmHg target IOP, no significant differences were noted between the two groups. At 48 months postprocedure when a <21 mmHg IOP target was considered, the rate of eyes that achieved complete success was 51.1% in the NPDS group versus 72% in the PT group (p<0.05). As for the <18 mmHg IOP target, the rate of eyes that achieved complete success was 32.5% in the NPDS group versus 44% in the PT group (p<0.05). Complications occurred significantly more frequently after PT than after NPDS. CONCLUSIONS: The IOP-lowering effects of the two procedures were comparable at 36 months. At 48 months PT showed a significantly higher rate of complete success compared with NPDS. Complications were more frequent after PT than after NPDS.

Anti-cell antibodies in exposed seronegative individuals with HIV type 1-neutralizing activity.

Despite repeated exposures to HIV-1, some individuals remain seronegative. This study reports that sera from a fraction of exposed seronegative (ESN) subjects showed HIV-neutralizing activity; 5 of 17 ESN sera and none of 17 controls neutralized two different HIV-1 primary isolates (range of neutralizing titers: 1/20 to 1/60). The neutralizing activity was associated with the IgG fraction of 4 of 4 neutralizing ESN sera. Moreover, in 11 of 17 and 9 of 17 ESN sera (but none of the control sera) we found antibodies against HLA class I and CD4, respectively. One of the ESN sera (EU22) neutralized efficiently the primary virus derived from the seropositive partner and showed a good broadly cross-reactive neutralization. Immunoadsorption of two IgG fractions from EU19 and EU22 on peripheral blood mononuclear cells (PBMC) removed virus-neutralizing antibodies. The correlations between the ESN status and neutralizing activity (p<0.05), anti-HLA antibodies (p<0.0002), and anti-CD4 antibodies (p<0.001) were statistically significant. However, there was no statistically significant correlation between neutralizing activity and either anti-HLA or anti-CD4 antibodies. It can therefore be said that exposure to HIV-1 without seroconversion is, in some individuals, associated with HIV-neutralizing antibodies (not directed against viral antigens) and/or with anti-cell autoantibodies, which are possibly specific for cellular antigens involved in the infection/entry process.

Enhanced HIV infectivity and changes in GP120 conformation associated with viral incorporation of human leucocyte antigen class I molecules.

BACKGROUND: Assembly of human immunodeficiency virus type 1 (HIV-1) occurs at the level of the plasma membrane of the host cell. During this process HIV incorporates significant quantities of cell surface-derived molecules into its lipid bilayer including human leucocyte antigen (HLA) class I and II, intercellular adhesion molecule-1 and lymphocyte function antigen-1. Several studies indicate that virion-bound host-cell-derived molecules are functional and affect the biological properties of HIV-1. Virion-associated HLA class II and intercellular adhesion molecule-1 enhance the infectivity of T-cell line-adapted (TCLA) viruses. No role for virion-associated HLA class I molecules has yet been identified. OBJECTIVE: To investigate the role of HLA class I molecules in HIV replication and infectivity. METHODS: HLA class I negative human cells lines transfected with the HLA Cw4 gene were infected with different TCLA viruses as well as primary X4 isolates. The infectivity of HLA Cw4 positive and negative viruses was determined on indicator cell lines and on phytohaemagglutinin-activated peripheral blood mononuclear cells. An entry polymerase chain reaction assay was used to determine differences in entry-competence of Cw4 positive and negative viruses. The expression of selected gp120 epitopes on native Env molecules derived from Cw4 positive and negative viruses was determined by a monoclonal antibody-based enzyme-linked immunosorbent assay. Immunoprecipitation experiments were performed to investigate the presence of gp120/HLA Cw4 complexes. Neutralization assays determined the differences in susceptibility to neutralization between HLA Cw4 negative and positive viruses. RESULTS AND CONCLUSIONS: The infectivity of primary HIV-1 X4 isolates and of TCLA viruses is increased upon viral incorporation of HLA Cw4 molecules. This effect is associated with changes in viral envelope proteins conformation including an enhanced expression of the V3 loop of gp120, and of epitopes that are exposed upon CD4 binding. The gp120 conformational changes are consistent with the formation of a multimolecular complex between HLA class I and gp120/160. HLA Cw4 incorporation is also associated to a lower susceptibility to antibody neutralization. These findings have important implications for understanding the immune response to cryptic and conformational epitopes of the viral envelope.

Human immunodeficiency virus (HIV)-specific IgA and HIV neutralizing activity in the serum of exposed seronegative partners of HIV-seropositive persons.

The presence and activity of human immunodeficiency virus (HIV)-specific antibodies were analyzed in the sera of 15 sexually exposed seronegative persons who had systemic HIV-specific cell-mediated immunity and IgA-mediated mucosal immunity and in their HIV-infected partners. The HIV-positive subjects had HIV-specific serum IgG and IgA; the seronegative persons had HIV-specific serum IgA in the absence of IgG. Testing of the seronegative persons 1 year after the interruption of at-risk sex showed that no IgG seroconversion had occurred and that HIV-specific IgA serum concentrations had declined. Serum from the HIV-exposed seronegative persons was analyzed for the ability to neutralize primary HIV-1 isolates. Neutralizing activity was detected in 5 of 15 sera and in 2 cases was retained by serum-purified IgA. Thus, the immunologic picture for resistance to HIV infection should include HIV-specific cell-mediated immunity as well as HIV-specific IgA-mediated mucosal and systemic immunity.

Affinity biotinylation: nonradioactive method for specific selection and labeling of cellular proteins.

Biotinylation has become a popular alternative to radioiodination for labeling cell surface proteins, whereas labeling of the total cellular protein pool is usually achieved metabolically with [35S]methionine and [35S]cysteine. In this paper we describe a new technique in which total cellular lysate proteins that have been affinity bound to a solid phase are labeled efficiently with biotin. This labeling technique is preferable to direct biotinylation of cell lysate since the unreacted biotin can be readily removed from the sample by washing. The affinity step permits preselection of the molecules to be labeled, thereby decreasing the potential for nonspecific binding during subsequent immunoprecipitation. We applied this affinity biotinylation method to a human cellular lysate in order to preselect the total glycoprotein pool for subsequent immunoprecipitation of HLA class I. Following immunoprecipitation, SDS-PAGE, and Western blot, the biotinylated protein could be readily revealed by enhanced chemiluminescence. The results were comparable to those obtained by radiometabolic labeling and Western blot using a monoclonal antibody probe. Overall, the affinity biotinylation method is faster and more practical than conventional radiolabeling, without any loss in sensitivity.

Limited proteolysis and X-ray crystallography reveal the origin of substrate specificity and of the rate-limiting product release during oxidation of D-amino acids catalyzed by mammalian D-amino acid oxidase.

Limited proteolysis of D-amino acid oxidase holoenzyme with trypsin cleaves the protein at Arg 221 and near the C-terminus, producing stable 25, 13.4, and 2 kDa polypeptides [Torri-Tarelli, G., Vanoni, M. A., Negri, A., & Curti, B. (1990) J. Biol. Chem. 265, 21242-21246]. The 25 and 13.4 kDa polypeptides remain associated to form a nicked D-amino acid oxidase species. This nicked protein form maintains the ability to bind FAD, but exhibits altered catalytic efficiency toward the oxidation of various D-amino acids when compared to native DAAO. Changes in substrate specificity were first monitored by measuring the activity in the presence of different amino acid substrates at various times during proteolysis. Three amino acid substrates were then selected for further analysis of the properties of the nicked D-amino acid oxidase species produced by limited tryptic proteolysis: D-serine, D-arginine, and D-alanine. The three D-amino acids represented limiting cases of the observed changes of enzyme activity on nicking: loss of activity, increase of activity, and minor activity changes, respectively. D-serine was found to be no longer a substrate of D-amino acid oxidase. D-arginine exhibited a 2.5-fold increased apparent maximum velocity although its Km value increased 2-fold with the nicked enzyme in comparison to the native species. D-alanine was oxidized 1.5-fold faster by the nicked D-amino acid oxidase at infinite substrate concentration, and its Km value increased approximately 4-fold. The Kd for benzoate, which was determined kinetically with D-alanine as the enzyme substrate, increased 17-fold in the nicked species. Primary deuterium kinetic isotope effects on V and V/K during the oxidation of D-alanine were also measured. (D)V/K increased from 1.4 +/- 0.2 to 1.8 +/- 0.3 on nicking, while (D)V increased from 1.04 +/- 0.1 to 2.53 +/- 0.5. All the observed changes of the values of the kinetic parameters and of the observed isotope effects are consistent with the hypothesis that nicking of D-amino acid oxidase at position 221 decreases the strength of binding of both substrates and products to the enzyme active site. The information obtained by limited tryptic proteolysis nicely complements that gathered from the analysis of the three-dimensional structure of D-amino acid oxidase in complex with benzoate, which was recently determined [Mattevi, A., Vanoni, M. A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., & Curti, B. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7496-7501]. Arginine 221 is part of the 216-228 loop that covers the active site and contributes residues to substrate binding and catalysis. The limited proteolysis data support the hypothesis that this loop acts as a lid on the active site and controls both substrate specificity and the rate of turnover of D-amino acid oxidase.

HIV-1-specific immunity in persistently seronegative individuals at high risk for HIV infection.

A growing number of reports indicates that certain groups of individuals who almost certainly have been exposed to human immunodeficiency virus (HIV), yet continue to exhibit no signs or symptoms of infection, often have subtle evidence of specific immunity. We studied such a high-risk (HR) cohort of persistently seronegative individuals with histories of long-term sexual exposure to an HIV-infected partner to look for evidence of both humoral and cellular immunity that might have been induced by exposure to the virus. Twenty-three heterosexual and four homosexual monogamous couples with discordant HIV status were included in the study. Twelve of the HR partners were studied for in vitro stimulation of peripheral blood mononuclear cells (PBMC) by HIV envelope-derived peptides. All 12 responded overwhelmingly to a peptide containing the fifth conserved region of gp120. By generating and cloning T cell lines specific for this peptide, we concluded that in these individuals the T cell response to the envelope is mainly focused on the carboxy-terminus region of gp120 and is characterized by an oligoclonal expansion of CD4+ T cells expressing the same TCR Eighteen HR partners and 37 HIV-1 seropositive subjects were tested for the presence of anti-CD4 antibodies (anti-CD4 Abs) using a recombinant CD4-based enzyme-linked immunosorbent assay (ELISA). Anti-CD4 Abs were detected in eight of the HR partners (six confirmed by Western blot) and in nine of the HIV-1 seropositive subjects (eight confirmed by Western blot). Results from binding competition assays with a panel of monoclonal anti-CD4 Abs suggested that the anti-CD4 Abs detected in the HR partners are directed toward epitopes that are induced by gp120 binding. Twenty-seven of the HR partners were tested for the presence of antibodies that cross-react with HLA class I and gp120 (anti-HLA Abs). Anti-HLA Abs were detected in 16 of the HR partner sera and in 4/94 sera from a control population of normal healthy blood donors. Taken together, the results suggest that in some individuals with a history of long-term exposure to HIV, specific immunity may develop in the absence of overt infection. The common trigger for these responses is gp120.

Human immunodeficiency virus type 1 (HIV-1)-seronegative injection drug users at risk for HIV exposure have antibodies to HLA class I antigens and T cells specific for HIV envelope.

The question of whether persistently seronegative persons at high risk for human immunodeficiency virus type 1 (HIV-1) infection exhibit HIV-1-specific T cell responses and antibodies to HIV-1 envelope epitopes shared with selected HLAs was assessed. These antibodies are not detectable by conventional serologic methods. Envelope-specific helper T (Env-Th) cell responses and antibodies specific for the HIV/HLA epitopes were studied in 21 HIV-1-negative injection drug users (IDUs). HIV/HLA antibodies were detected in 7 (33.3%) of 21 IDUs and 4 (4.3%) of 94 low-risk controls. Env-Th cell responses were detected in 16 (76.2%) of 21 IDUs and in 2 (3.1%) of 65 low-risk controls. All HIV/HLA antibody-positive IDUs also had Env-Th cell responses. These findings confirm the presence of HIV-1-specific immunity in conventionally seronegative individuals. Further characterization of these responses could provide the basis for new preventive strategies.

[Immunology and tumors. Effector cells]. / Immunologia e tumori. Le cellule effettrici.

Since Burnet expounded the theory of immune surveillance, various cells have been indicated as the "effectors" of the body immunological defence against tumours. Until recently, the ability to lyse tumour target cells was assigned to macrophages and the so-called thymus-dependent lymphocytes, known as killer T cells. The discovery of natural killer cells in mice recently has widened the number of effectors and the field of research in this sector. An examination is made of the features and main properties of these cell groups, and some questions are posed concerning their ability to cooperate in anti-neoplastic immunological defence.