Actualities in neonatal endocrine and metabolic screening.

Neonatal metabolic screening has proven to be an important tool for the early detection of innate metabolic errors. Despite the fact that simple and effective methods of testing for metabolic diseases have been identified since the middle of the twentieth century, no consensus has been reached so far on the content of neonatal metabolic screening panels. There are large differences between countries in the number of metabolic diseases identified through national metabolic screening programs, ranging from zero to several tens, the most common testing being for phenylketonuria and congenital hypothyroidism (including in Romania). Given the fact that rare but treatable diseases have been identified in recent decades, reducing the financial burden on the health system, it would be useful to include them in the national neonatal metabolic screening program.

A study in scarlet: MC1R as the main predictor of red hair and exemplar of the flip-flop effect.

Genetic variation in melanocortin-1 receptor (MC1R) is a known contributor to disease-free red hair in humans. Three loss-of-function single-nucleotide variants (rs1805007, rs1805008 and rs1805009) have been established as strongly correlated with red hair. The contribution of other loss-of-function MC1R variants (in particular rs1805005, rs2228479 and rs885479) and the extent to which other genetic loci are involved in red hair colour is less well understood. Here, we used the UK Biobank cohort to capture a comprehensive list of MC1R variants contributing to red hair colour. We report a correlation with red hair for both strong-effect variants (rs1805007, rs1805008 and rs1805009) and weak-effect variants (rs1805005, rs2228479 and rs885479) and show that their coefficients differ by two orders of magnitude. On the haplotype level, both strong- and weak-effect variants contribute to the red hair phenotype, but when considered individually, weak-effect variants show a reverse, negative association with red hair. The reversal of association direction in the single-variant analysis is facilitated by a distinguishing structure of MC1R, in which loss-of-function variants are never found to co-occur on the same haplotype. The other previously reported hair colour genes' variants do not substantially improve the MC1R red hair colour predictive model. Our best model for predicting red versus other hair colours yields an unparalleled area under the receiver operating characteristic of 0.96 using only MC1R variants. In summary, we present a comprehensive statistically derived characterization of the role of MC1R variants in red hair colour and offer a powerful, economical and parsimonious model that achieves unsurpassed performance.

Semiconductor nanorod self-assembly at the liquid/air interface studied by in situ GISAXS and ex situ TEM.

We study the self-assembly of colloidal CdSe/CdS nanorods (NRs) at the liquid/air interface combining time-resolved in situ grazing-incidence small angle X-ray scattering (GISAXS) and ex situ transmission electron microscopy (TEM). Our study shows that NR superstructure formation occurs at the liquid/air interface. Short NRs self-assemble into micrometers long tracks of NRs lying side by side flat on the surface. In contrast, longer NRs align vertically into ordered superstructures. Systematic variation of the NR length and initial concentration of the NR dispersion allowed us to tune the orientation of the NRs in the final superstructure. With GISAXS, we were able to follow the dynamics of the self-assembly. We propose a model of hierarchical self-organization that provides a basis for the understanding of the length-dependent self-organization of NRs at the liquid/air interface. This opens the way to new materials based on NR membranes and anisotropic thin films.

Photodynamic therapy and some clinical applications in oncology.

Photodynamic therapy (PDT), a new treatment modality for localized cancers involving the selective interaction of visible light with photosensitizers, such as hematoporphyrin derivatives (HpD) or dihematoporphyrin ether/ester (DHE) (Photofrin II). Photodynamic therapy of malignant tumors includes biological, photochemical and photophysical processes. These processes involve: (i) absorption of photosensitizing agent; (ii) selective retention of photosensitizer in tumors and (iii) irradiation of sensitized tumor by laser irradiation. This paper provides a review of photosensitizers, photochemistry, subcellular targets, side effects and lasers involved in photodynamic therapy. In addition, gradual increase in knowledge related to in vivo and in vitro mechanisms of action of PDT, as well as some clinical applications of photodynamic therapy are presented.

New approach to the adoptive immunotherapy of Walker-256 carcinosarcoma with activated macrophages combined with photodynamic therapy.

Experiments were performed on five batches of Wistar inbred rats with Walker-256 carcinoma receiving sole (PDT, MAK) or combined therapy (PDT + MAK-A; PDT + MAK-B); the control batch (HBSS) consisted of animals with untreated tumors. The results were as follows: a) the sole treatment (PDT, MAK) gave survival rates between 37.7 and 47.5%, b) the "combined" therapy in five doses increased significantly (70.8%) the survival rate of tumor bearing rate as well as the rate of complete regression (82.1%). The cell-mediated immunity test and histopathological as well as the electron microscopy observations were in full agreement with the results above. Summing up, these results demonstrate that "combined" photodynamic therapy with intra- and peritumoral MAK infusion stimulated cell-mediated antitumoral activity, increased survival rates and reduces incidence of Walker-256 carcinoma in rat model.

Stimulation of mucosal immune response following oral administration of enterotoxigenic Escherichia coli fimbriae (CFA/I) entrapped in liposomes in conjunction with inactivated whole-cell Vibrio cholerae vaccine.

In this study, we have searched for an effective mucosal vaccine. An oral enterotoxigenic E. coli vaccine containing colonization factor antigen (CFA/I) associated with inactivated whole-cell V. cholerae vaccine (WCV) has been tested for safety and immunogenicity in animals. Five groups of animals were used. The results showed the following: (a) vaccine containing CFA/I antigen entrapped in liposomes and associated with WCV (batch C) had increased titers of specific antibodies to CFA/I antigen in 15 to 18 (83.3%) animals; (b) specific Peyer's patches (PP), lymph nodes (LN) and spleen (SPL) lymphocytes proliferation was detected following in vitro restimulation with CFA/I antigen or WCV. This response gradually increased to the highest value by the 35th postimmunization day. Moreover, lower PP, LN and spleen (SPL) proliferation was observed in rabbits receiving soluble CFA/I antigen (S-CFA/I) or free liposomes (F-L) alone; (c) adhesion of E. coli H10407 strain labelled with 3H-leucine in immunized and control animals revealed the following local effects: (i) protection of rabbit intestinal mucosa against virulent E. coli cells; (ii) inhibition of adhesion of ETEC bacteria to intestinal mucosa and (iii) significantly faster release of E. coli H 10407 strain labelled with 3H-leucine from the intestinal tract of immunized animals. The histopathological and electron microscope findings confirmed the above results. The experimental results point out an efficient protection against infection with E. coli strains (ETEC), after mucosal vaccination with CFA/I antigen entrapped in liposomes associated with inactivated whole-cell Vibrio cholerae as immunological adjuvant.

Ultrastructural changes induced in Walker carcinosarcoma by treatment with dihematoporphyrin ester and light in animals with diabetes mellitus.

The purpose of this study was to evaluate by electron microscopy the tumoral fine structure changes induced by photodynamic therapy (PDT) in diabetic animals. Walker -256 carcinosarcoma harvested from animals with/without diabetes mellitus exposed to PDT (Photofrin II/5 mg/kg and 24 hrs later He-Ne laser irradiation/632.8 nm; 10 mW) and examined by electron microscopy showed different degrees of lesions in the nucleus and cytoplasmic fine structure. The ultrastructural changes induced by PDT in animals with diabetes mellitus bearing carcinosarcoma are characterized by: lysis of chromatin situated on the central zone of nucleus; swelling and vacuolization of mitochondria; formation of phagosome--like structures; myelin figures; degenerescence and disappearance of cytoplasmic organelles. Summing up, the data presented in this work demonstrate that the exposure to three doses of PDT produces changes in tumoral fine structure, increases survival rate and reduces incidence of carcinosarcoma in rats with diabetes mellitus.

Studies on pathogenicity of Yersinia enterocolitica in mice with diabetes mellitus.

In this study, we investigated the colonizing ability as well as the association of Yersinia enterocolitica serotype 0:9 to epithelial cells of the intestinal tract, Peyer's patches, mesenteric lymph nodes, liver, spleen and lungs in Alloxan-induced diabetes mellitus in mice and controls. The results showed that: (a) in diabetic mice the Y. enterocolitica colonizing values were in range of 10(6.5)-10(8.25) CFU/g of feces; (b) maximum colonizing values were found in distal ileum and Peyer's patches and lower in colon; (c) the infection was progressive with dissemination of bacteria in the liver, spleen and lung; (d) in control (non-diabetic) mice, the colonizing values were 10-100 times lower than those found in the diabetic batch; (e) the main histopathological changes noticed, namely ileitis, mesenteric lymphadenitis and septicemia, were presumably induced by high bacterial load in the liver, spleen and lung leading to a septic course of infection as well as toxic effects of heat-stable enterotoxins of Y. enterocolitica (Yst). The results were confirmed by electron microscopy observations. Summing up, these results demonstrate that diabetic mice were more susceptible to Y. enterocolitica cells than normal mice.

Immune response to Escherichia coli antigens entrapped in liposomes. I. Immunopathological studies.

In this study, we have searched for an effective mucosal delivery system for a purified E. coli antigen which elicits anticolonization and anti-toxic immunity. E. coli colonization factor antigen (CFA/I) and heat-labile enterotoxin (LT) were encapsulated in liposomes. To determine the efficacies of soluble and liposome-encapsulated E. coli antigens young rabbits were mucosally treated with three oral doses of E. coli antigens given 7 days apart. Ten days after the last booster, rabbits were orally challenged with 5 x 10(9) bacterial cells (O78:H11 serotype). The experimental results allow of making some remarks which can be correlated with the protection obtained in vaccinated animals: (a) immunization with E. coli antigens entrapped in liposomes ensured protection against ETEC strains; (b) lower protection against homologous and heterologous CFA/I +(LT- ST+) strains were noticed; (c) adhesion of labelled -3H-leucine-bacteria to the intestinal mucosa revealed a maximum distribution in duodenum-jejunum and minimum in the colonic mucosa; (d) it contributed to the release of inoculated virulent bacteria from intestinal tract; (e) humoral, cellular and histopathological findings confirm the afore mentioned observation. Summing up, these results suggest that liposomes are very good carriers for E. coli antigens and these findings highlight the potential use of LT and CFA/I antigens entrapped in liposomes as mucosal and humoral induction of immune response and make them a candidate for future use in prophylaxis of diarrhoea in man.

Photodynamic therapy: an update.

Photodynamic therapy (PDT) is a promising local treatment modality based on the selective accumulation of a photosensitizer in malignant tissues and the subsequent irradiation with laser light. Photodynamic therapy of malignant tumors includes biological, photochemical and photophysical processes. These processes involve: (a) absorption of photosensitizing agent; (b) selective retention of the photosensitizer in tumors and (c) irradiation of sensitized tumor by laser radiation. This report provides a review of photosensitizers, photochemistry, subcellular targets, side effects and laser involved in photodynamic therapy. In addition, gradual increase in knowledge related to in vitro and in vivo mechanisms of action of PDT, as well as some clinical applications of photodynamic therapy are presented.

Macromolecular synthesis and ultrastructural changes induced in human larynx carcinoma cells following photodynamic therapy in vitro.

Human larynx carcinoma cells (HEp2) were sensitized with different concentrations of Hematoporphyrin and irradiated with a He-Ne laser at different fluences. The degree of PDT-effects were estimated by two parameters: a) macromolecular synthesis and b) observations using electron microscopy. All experiments were evaluated after 68 hr at 37 degrees C. The results showed that PDT exposure of HEp2 cells is characterized by: 1) inhibition of macromolecular synthesis and 2) different cellular and subcellular lesions. Summing up, these studies indicate the existence of a strong correlation between different PDT exposures and the degree of biochemical and ultrastructural changes in human larynx carcinoma cells in vitro.

Laser and serum opsonic activity.

The purpose of this study was to investigate the effects of low level laser (LLL: GaA1As diode laser; 830 nm) on serum opsonic activity, which was assessed by neutrophil-associated chemiluminescence (CL) response to zymosan opsonized with sera irradiated in vitro with various doses of LLL. We used both lucigenin-dependent CL (LgCL) for superoxide (O2-) detection and luminol-dependent CL (LmCL) which detects myeloperoxidase (MPO)-dependent formation of hypochlorous acid in combination with MPO inhibitor, sodium azide (NaN3). When serum opsonic activity was assessed by LgCl, NaN3, markedly enhanced the responses, suggesting that O2- is accumulated due to the MPO blockade, leading to the excitation of LgCl. However, LLL-irradiation had no effects on serum opsonic activity. On the other hand, when serum opsonic activity was assessed by LmCl, NaN3, strongly inhibited the response. The effects of LLL at different output powers were characterized by similar values, but significantly higher values were observed at the highest dose tested (60 mW for 1 min) in the absence of NaN3. Since this enhancement effect disappeared with NaN3, it was suggested that high dose LLL-irradiation probably activated the opsonic activity by facilitating neutrophils to degranulate MPO. However, lower doses used for therapeutic purposes had no effects at any output powers tested.

Development of an irradiated vaccine that protects against enterotoxigenic Escherichia coli diarrhoea.

In the pathogenesis of diarrhoea in man bacteria adhesion to enterocytes is mediated by specific CFA/I or CFA/II antigens. A perorally administered vaccine was prepared from E. coli H10407 (078:H11) by irradiation with electrons with high energy (EHE). Two hours after cimetidine administration rats were immunized per os with 5 irradiated vaccine doses at 4-day intervals. Seven days after the last immunization animals were infected by inoculating 1 x 10(9) germs in the ligated intestinal loop. Reduction of the intestinal secretion by over 50% 18 hours after inoculation was considered an efficient protection marker. The obtained results have proved a significant reduction of the intestinal secretion in immunized animals infected with serotypes 078:H11(63 +/- 4%) and 078:H12(59 +/- 5%) as compared to non-immunized animals. Experimental induction of the intestinal protection against Escherichia coli enterotoxigenic (ETEC) strains points to the possibility of using this type of irradiated vaccine in the prophylaxis of diarrhoea in man.

DNA synthesis in monkey kidney cellular culture, hematoporphyrin sensitized and He--Ne laser irradiated.

He--Ne laser irradiation for 1-45 J/cm2 has four actions upon monkey kidney cell cultures: a) a stimulation of nuclear DNA synthesis after 20 J/cm2; b) a diminution of replication after higher dose (45 J/cm2); c) inhibition of nuclear DNA synthesis and d) a marked cytotoxic effect. At lower radiation fluences (1-5 J/cm2) the influence on the biosynthesis of DNA is not significant.

In vitro effects of pulsed near-ultraviolet laser exposure on human larynx carcinoma cells.

Effects of pulsed near-ultraviolet laser beam on structural characteristics and macromolecular synthesis of carcinoma HEp2 cells were investigated. Laser irradiation damage induced in these eukaryotic cells could be characterized by two development stages: a) a reversible stage with minor morphological damages (1.5 kJ/m2) and 2) an irreversible one, at higher fluences, characterized by cellular membrane damage, necrobiosis and cells detachment from the substrate (4.5 kJ/m2). A. Studies performed referring to macromolecular syntheses of low laser fluences (1.5 kJ/m2)--irradiated HEp2 cells showed the following aspects: a) syntheses inhibiton phase in the first cycles of cellular replication and b) syntheses stimulation phases in the following cycle with total repair of laser-induced molecular lesions. B. At high laser fluences (3-4.5 kJ/m2), metabolic lesions repair was partially or totally blocked after prolonged culturing at 37 degrees C. Ths paper suggests some mechanisms of laser action on macromolecular synthesis and correlates them with morphological changes induced by laser exposure of carcinoma cells.

Characterization of colonization factor antigen CFA/I from an enterotoxigenic Escherichia coli strain (0128:H12).

CFA/I antigen was isolated and purified from E. coli, mutant 279 B-1-14, serotype 0128:H12, and had the following biochemical and biological features: a) amino-acid content was similar to that of purified antigen prepared from strain H10407; b) latex particles sensitization with purified CFA/I antigen produced bovine and human erythrocytes group A/II hemagglutination in carbohydrates presence; c) purified anti-CFA/I specific antibodies agglutinated CFA/I-positive enterotoxigenic E. coli strains; d) 3H-leucine-labelled CFA/I antigen adhered to rabbits intestinal mucosa at significant values; e) intestinal mucosa pretreating with purified CFA/I antigen, followed by 3H-leucine labelled enterotoxigenic bacteria infection, had a least 3 local effects: 1) intestinal mucosa protection against parental enterotoxigenic bacteria; 2) inhibition of CFA/I-positive bacteria adherence to intestinal mucosa; 3) release of approximately 96% intraluminally inoculated bacteria.

Laser effects on cellular adhesion and influence on the interrelation between HeLa S3 and human diploid cells.

The interactions between HeLa S3 tumoral cells and human fibroblasts after nitrogen-laser irradiation (337.1 nm) have been studied by using an in vitro cell invasion model. For the quantitative and morphological evaluation of nitrogen-laser radiation action upon tumoral adhesion to the fibroblast monostrate, we used: a) 3H-thymidine labelling of HeLa S3 tumoral cells; b) morphological modifications studies by phase contrast and scanning electron microscopy. The results emphasized the following aspects: 1. In non-irradiated cell cultures we noticed three interaction stages: adhesion, tumoral spreading and displacement with fibroblasts destruction; on the other side, we found a reduced adhesion to non-irradiated human fibroblasts of laser irradiated tumoral cells. 2. Significant percent increasing of non-irradiated tumoral cells adhesion to fibroblast monostrate, irradiated with various laser fluences (e.g. 0.2 kJ/m2--48.1%; 0.8 kJ/m2--63.8% and for 1.6 kJ/m2--79.5%). This phenomenon evidenced the close interrelation between irradiation fluences and tumoral adhesion rates. 3. The importance of numerical ratio between tumoral cells and fibroblasts in tumoral adhesion and invasion processes (e.g. ratio 1:10 tumoral adhesion reached 8.1%; in 1:5--25.9%; in 1:1--59.4% and for 2:1--83.9%). 4. Marked cytotoxic effects for both cell types after exposure to high and very high laser fluences (1.6--6.4 kJ/m2). Our results emphasize near UV-laser irradiation effects upon some of tumoral adhesion and invasion mechanisms and demonstrate the interrelations between cell populations manifesting a different vital potential.

Studies of the effects of associated photodynamic therapy and drugs on macromolecular synthesis of tumoral cells grown in vitro.

HeLa S3 tumoral cells were used as an experimental model for studying the association of photodynamic therapy (PDT) and antitumoral agents. Tumoral monolayer cultures were incubated 18 hours at 37 degrees C with Photofrin II, trypsinized and suspended in Eagle medium supplemented with 10% FCS and then treated with antitumoral agents 90 minutes before He-Ne laser exposure. The tumoral cells were exposed to antitumoral agents in the following concentrations (equivalent to ED70): adriamycin (0.0297 micrograms); mitomycin C (0.0199 micrograms); 5-FU (0.4937 micrograms) and vinblastine (0.0109 micrograms) per 10(5) cells. Macromolecular syntheses (DNA, RNA and proteins) were investigated by use of radioactive precursors: 3H-thymidine, 3H-uridine and 3H-leucine, as expressed in percent referring to Photofrin II-pretreated controls; they were exposed to He-Ne laser but not treated with antitumoral agents. All experiments were followed for 72 hours incubation at 37 degrees C. The conclusions of the results of PDT associated with antitumoral agents sustain the following aspects: a) the antitumoral agents activity (adriamycin, mitomycin C, 5-FU, vinblastine) was more noticeable when applied 90 minutes before He-Ne laser irradiation; b) inhibition of radioactive precursors uptake in DNA, RNA and proteins was accompanied by suppression of in vitro tumoral cells development and c) PDT association with antitumoral agents could manifest at least three positive effects upon animals; 1) PDT potentiating effects with antitumoral agents; 2) suppressing effects on tumoral macromolecular synthesis; 3) antitumoral agents cytotoxic elimination (due to the low doses used).

Reaction and response of newborn guinea pigs to experimental Salmonella typhi infection.

Newborn guinea pigs, orally infected with Salmonella typhi were examined at various intervals of time in order to determine bacterial distribution in tissues and to establish possible correlation with the clinical aspects manifested. Histopathological examination evidenced typical lesions in jejunum, ileum, caecum and especially in regional lymphatic tissues. Spleen, liver and mesenteric lymph nodes presented granulomatous lesions similar to those observed in in human typhoid fever. After oral administration, the animals reacted with anorexia, febrile reactions, bacteremia, diarrhoea, positive stool cultures, dehydration, lethargy and antibodies too were produced. Our results indicate that typhoid infection may be induced in newborn guinea pigs; the model may be used for an assessment of attenuated live typhoid vaccine control.