Hydrolysis of substance P in the presence of the osteosarcoma cell line SaOS-2: release of free amino acids.

The possible hydrolysis of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) in presence of the osteoblastic cell line SaOS-2 was measured by capillary electrophoresis coupled to mass detection. The results obtained indicate that a very rapid disappearance of the intact undecapeptide was associated to a slower appearance of seven of its eight component amino acids. These results can be interpreted as indicating that an extremely fast hydrolysis of substance P by endopeptidases, which released peptidic by-products, was followed by a noticeably slower secondary degradation which released free amino acids. In decreasing quantitative importance, these phenomena appear to originate by the hydrolysis of the Pro(4)-Gln(5) bond, followed by C-terminal sequential degradation of the Arg(1)-Pro(4) tetrapeptide; by the hydrolysis of or Phe(7)-Phe(8) bond (or, possibly, of Gln(6)-Phe(7)) leading to release of free Phe and Gln; by hydrolysis of the Gly(9)-Leu(10) bond with subsequent release of Met and Leu. Results obtained appear to be compatible with the expression by SaOS-2 cells of enzymes already known to catalyze substance P hydrolysis, together with an apparent low efficiency of aminopeptidases. Because of the activity of C-terminal fragments on NK1 receptors, the delay between primary hydrolysis of substance P and secondary hydrolysis of its peptidic fragments indicated by the data shown implies a possible persistence of substance P physiological effects even after degradation of the intact peptide.

Inter-alpha-trypsin inhibitor heavy chain 4 as a marker of acute rejection in pancreas allotransplantation in pigs.

BACKGROUND/AIMS: An early, simple, and reliable marker for acute pancreatic allograft rejection is not available. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) is an interleukin-6-dependent acute-phase positive protein that can act as an anti-inflammatory protein. We studied the response of the ITIH4 in pigs undergoing pancreas allotransplantation (PT) and evaluated this protein as a biomarker for acute graft rejection. METHODS: PT with enteric drainage of the exocrine secretion and systemic venous drainage was performed on 12 Landrace pigs. No immunosuppression was administered. Serum concentrations of glucose, amylase, lipase, insulin, C-peptide, and ITIH4 were determined daily. RESULTS: The response of ITIH4 to PT was early, intense, and prolonged, with 2 peaks in serum concentration. The first peak, which started on day 1 and reached maximum (around 6 mg/dL) on day 3, was attributed to the systemic acute phase response to surgical stress. The second peak, which exceeded the first peak and reached maximum (>8 mg/dL) on day 6, began when the recipients were still normoglycemic, and preceded onset of the diabetic state caused by acute graft rejection by an average of 4 days. CONCLUSION: Serum ITIH4 could help to predict subclinical acute graft rejection after PT in pigs.

Positive and negative modulation of peptidases by pro-inflammatory cytokines.

The capacity of pro-inflammatory cytokines to modulate proteolysis was analyzed by liquid chromatography using human fibroblasts as cell model and enzyme source, and the immunodominant epitope gp100(280-288) (YLEPGPVTA) as substrate. The measurements made after fibroblast pre-incubation with either IL-1, TNF, or IL-6 plus its soluble receptors have been compared with those made with un-stimulated fibroblasts. The results obtained suggest an uneven association of cytokine treatment with substrate degradation, and with a prevailingly positive - but also negative - association with release of smaller peptides and free amino acids. Data obtained by separately measuring these two groups of by-products indicate that, after IL-1 cell pre-treatment, the velocity of formation of both groups of by-products increased, resulting in a net increase of substrate degradation. After TNF and IL-6 pre-treatment, the increase of one group was compensated by a decrease of the other group; specifically, the compensation was only partial for TNF, and overall substrate hydrolysis increased. In the case of IL-6, the increase of free amino acids was almost exactly compensated by a reduction of peptidic by-products, resulting in a negligible increase of substrate hydrolysis. In addition, the existence of reaction time-related modifications in the apparent velocity of substrate degradation and formation of by-products, allows hypothesizing different effects of cytokines on the enzymes degrading the substrate with different time constants. Taken together, these data can be interpreted as indicating different, positive and negative, effects of the three cytokines on the individual enzymes expressed by fibroblasts and capable of degrading peptidic substrates.

Effect of IL-1 on the hydrolysis of the tumor antigen epitope gp100(280-288) by fibroblast-expressed enzymes.

The role of proinflammatory cytokines in increasing the activity of specific proteases suggests the hypothesis that, by altering the expression of these mediators, adjuvants may modulate the effectiveness of peptides used as vaccines. The possible effect of IL-1 on fibroblast-expressed, peptidases was, thus, investigated by analyzing the degradation of a tumor antigen epitope (gp100(280-288), YLEPGPVTA) in the presence of cultured human fibroblasts. The data obtained indicate an increase of substrate hydrolysis after IL-1 treatment as compared with non-treated controls. Hydrolysis increase was accompanied by defined changes in the population of the by-products formed: specifically, the amount of peptidic by-products increased more than the amount of single amino acids, and the amount of the C-terminal by-products increased more than the amount of their N-terminal counterpart. These data appear to indicate that the positive effect of IL-1 on the activity of substrate-active enzymes is function of modified expression of a number of these enzymes by fibroblasts. From these data it can be inferred that the use of IL-1-inducing adjuvants, increasing the activity of peptidases expressed by bystander cells, may reduce the bio-availability of peptides used for immunization.

Regulation of genetically modified organisms (GMOs) in the European Union: principles of risk assessment.

The establishment of regulations for genetically modified organisms and the application of environmental risk assessment principles within the European Union are documented.

Hydrolysis of the tumor-associated antigen epitope gp100(280-288) by membrane-associated and soluble enzymes expressed by immature and mature dendritic cells.

The hydrolysis of the tumor-associated HLA-A2.1-restricted gp100(280-288) epitope by in vitro generated immature and mature dendritic cells (iDCs and mDCs) and by soluble supernatants prepared from these same cells, as well as the effect of the hydrolysis on in vitro immunorecognition, was studied by chromatographic and functional analyses. The results obtained indicate that exposure to iDCs induced a very rapid hydrolysis of the model peptide (half life, 62 s), resulting in complete loss of immunorecognition within 60 min. In the presence of mDCs, the hydrolysis kinetics were even faster (half life, 54 s), and the pattern of hydrolysis by-products was different from that observed for iDCs. Gp100(280-288) was also degraded in the presence of cell-free supernatants prepared both from iDCs and mDCS; in this case, degradation kinetics were slower, and the pattern of hydrolysis by-products was different from that observed in the presence of intact cells. The model epitope was degraded to non-immunogenic products by membrane and soluble enzymes expressed both by iDCs and by mDCs within periods of time that appear to be physiologically relevant. Development of antigenic formulations capable of protecting synthetic epitopes from these effects appears to represent a prerequisite for effective immunization procedures.

Degradation of the tumor antigen epitope gp100(280-288) by fibroblast-associated enzymes abolishes specific immunorecognition.

Degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) was investigated in the presence of cultured human fibroblasts, and acellular supernatants obtained from these cells; the possible effect of substrate degradation on in vitro immunorecognition was also addressed. In the presence of fibroblasts, gp100(280-288) was degraded to free amino acids with a half-life of less than 4 min; hydrolysis data support the hypothesis that substrate degradation was mainly caused by the activity of cell-expressed amino- and carboxypeptidases. Gp100(280-288) was also degraded in the presence of acellular supernatants: under these conditions, the hydrolysis pattern was similar to that observed in the presence of whole cells, but degradation kinetics was slower. As a result of these phenomena, immunorecognition of gp100(280-288)-specific cytotoxic T lymphocyte (CTL) clones was severely hampered, and was totally suppressed after 90 min. In conclusion, the high activity of fibroblast-expressed proteases, and the presence of wide-scope soluble enzymes, may explain, at least in part, the low activity of peptide-based antineoplastic vaccines, as well as the transient effectiveness of subcutaneously administered peptides in general.

Antistreptokinase platelet-activating antibodies are common and heterogeneous.

BACKGROUND: Platelet activation by antistreptokinase (SK) antibodies could impair the clinical effect of SK administration. OBJECTIVE: To better describe anti-SK antibodies with particular emphasis on procoagulant activities as a result of platelet activation. PATIENTS AND METHODS: Sera were collected from 146 patients with coronary artery disease: non-SK-treated, 95 from mainland France, 31 from French Polynesia; 20 patients from mainland in year 2 after SK treatment. Serum-induced SK-dependent platelet activation resulting in procoagulant activities was assessed with washed platelets from five donors representative of the known patterns of reactivities to IgG. RESULTS: Concentrations (2-5252 microg mL(-1)) and fibrinolytic neutralization titres (< 10 to > 1280) were found in the expected wide range and correlated (rho = 0.66, P < 0.0001). Platelet activation was detected with 145 samples, but varied in intensity and pattern (depending on the donors), although there was no systematic hierarchy; it was presumably due to IgG (inhibited by an IgG Fc receptor-blocking antibody and recovered in the IgG fraction) and only partially affected by aspirin. Marked platelet activation could be detected in samples with concentration as low as 2 microg mL(-1), and/or no detectable neutralizing titers. The way of immunization to SK was not found to influence the functional profile of antibodies. CONCLUSION: Anti-SK platelet-activating antibodies are widespread, heterogeneous, poorly predictable on the basis of their antifibrinolytic effect and strong enough to trigger procoagulant activities. Their clinical relevance should be formally assessed, using patients' own platelets for detection owing to the variation of platelet reactivity.

Dehydroepiandrosterone metabolism in human plasma.

The possibility that dehydroepiandrosterone (DHEA) is metabolized in human plasma was studied by column and thin-layer chromatography. The results obtained indicate that a time-dependent disappearance of DHEA is matched by the appearance of newly-formed species that may represent DHEA conversion by-products. Neither disappearance of DHEA, nor formation of the alleged conversion by-products was observed when reactions were performed under conditions in which plasma enzymes were removed or inactivated. These results suggest that, in plasma, DHEA is partially transformed into different substances, and that the conversion reactions are catalyzed by enzymes present in this tissue. The observed kinetics of appearance and partial disappearance of the radiolabeled species can be interpreted as indicating that some of the by-products formed are further converted into other substances. The data shown appear to indicate that plasma can be added to the list of the already known compartments that are involved in steroid metabolism.

Degradation of the immunogenic peptide gp100(280-288) by the monocyte-like U937 cell line.

The possible degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) in the presence of the monocyte-like line U937, and the effect of degradation on the in vitro-measured immune recognition, were investigated by chromatographic techniques and immunological assays. Results indicate a rapid hydrolysis of the substrate in the presence of the model cells, which is consistent with the hypothesis that degradation of gp100(280-288) is caused by the activity of U937-expressed enzymes, specifically amino- and carboxypeptidases. On the other hand, these results do not support the involvement of other enzymes known to be expressed by U937 cells. From a functional point of view, these data indicate that the degradation of gp100(280-288) severely hampered recognition by specific CTL clones. The results obtained may provide a model for epitope degradation by the antigen-presenting cells found in defined anatomical compartments and may, at least in part, account for the low activity of peptide-based antineoplastic vaccines, as well as for the transience of the effects of subcutaneously administered peptides in general.

Soluble proteolytic enzyme release by naive and HIV-infected cultured T-cells.

The possible hydrolysis of leucine enkephalin was measured in the presence of cell-free supernatants obtained from naive and chronically HIV-infected immunocompetent cell lines. The data obtained indicate that, under all conditions examined, leu-enkephalin was partially degraded; its disappearance was associated with the appearance of peptides whose composition is consistent with the involvement of three enzyme classes, i.e. aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. In the presence of supernatants obtained from infected cells, substrate hydrolysis was less than that measured in naive controls. This appears to result from infection-associated variations in the activity of all three enzyme classes active on the substrate, variations that were different for each class. Specifically, in unfractionated supernatants, the activity of aminopeptidases was reduced, that of dipeptidylaminopeptidase was increased, and the activity of dipeptidylcarboxypeptidases was nearly unmodified. Data obtained upon chromatographic separation of the soluble supernatants allowed for the identification of features that can be interpreted as indicating the existence of infection-associated variations in the activity of single enzymes. The sum of the data shown makes it possible to advance the hypothesis that the infection-associated modifications in the release of proteolytic enzymes may contribute to the alterations in the functionality of immunocompetent cells induced by viral infection.

[Influence of primary care personnel on breastfeeding duration]. / Influencia del personal sanitario de asistencia primaria en la prevalencia de la lactancia materna.

Background Breastfeeding duration in Spanish neonates does not fulfill the recommendations of the World Health Organization.ObjectiveTo report the results of a policy of breastfeeding support in a primary care center.Material and methodsWe performed a before-and-after intervention study of all mothers of children born in Ulldecona who decided to breast feed in 1992, 1993, 1996 and 1997 (control group: 125 infants), and from August 1999-August 2001 (72 infants). Study variable: in May 1999 a breastfeeding support policy was initiated in the primary care center.ResultsBreastfeeding duration increased (in the control group the mean duration of exclusive breastfeeding was 18.8 weeks; from 1999 to 2001 it was 28 weeks). Negative factors for breastfeeding were the birth of twins, introduction of a supplement, and education (there was an inverse relationship between greater education and breastfeeding duration). Duration of breastfeeding was longer in Moroccan mothers. Sex, gestational age, weight, type of delivery, separation between mother and neonate, maternal age, previous children, and work outside the home did not influence breastfeeding duration. Simple lineal regression revealed that the intervention was effective (P 0.046). Early hypogalactia and breast problems decreased, and voluntary weaning increased (P < 0.001).ConclusionThe primary care team plays key role in the maintenance of breastfeeding and in the well-being of the mother and neonate.

Different effects of steroidal therapy on neuropeptide-active enzymes in male and female human saliva.

The hydrolysis of a model neuropeptide (leucine enkephalin) was studied in the presence of saliva obtained from normal and allergic male and female volunteers in the absence and in the presence of steroidal treatment. Possible variations in the formation of substrate hydrolysis by-products were studied in whole samples and after steric exclusion chromatography fractionation. The results obtained confirm already-described variations in substrate hydrolysis in allergic as compared to control saliva, as well as the effect of steroidal treatment on the activity of the substrate-active enzymes. In addition, whereas in male saliva, therapy was associated with a net decrease of substrate hydrolysis, in female saliva hydrolysis remained near the levels measured in the absence of treatment. Finally, therapy induced modifications of enzyme apparent molecular weight distribution that appear to be similar for all substrate-active enzyme classes, but different in male and female saliva. In male saliva, therapy decreased the activity of the enzymes eluted at high apparent molecular weight, while it increased the activity of the enzymes of low apparent molecular weight. Because the increase was considerably less than the decrease, the net effect was to decrease the activity of the substrate-active enzymes, nearly to the low levels measured in the controls. In female saliva the therapy-associated decrease in the activity of the enzymes eluted at high apparent molecular weight was offset by the increase in the activity of those eluted at low apparent molecular weight, consequently, substrate hydrolysis remained near the level measured in the absence of treatment, a level that was higher than that measured in the controls.

Neuropeptide enzyme hydrolysis in allergic human saliva.

The activity of neuropeptide-degrading enzymes, and possible variations in this activity under allergic conditions, was examined in human saliva obtained from allergic volunteers and from an age- and sex-matching group of healthy controls, using leucine enkephalin as model substrate. The results obtained indicate that, under experimental conditions, the substrate was partially hydrolyzed by all three classes of enzymes known to degrade it in human saliva: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. In the presence of saliva obtained from allergic donors, a large increase in the activity of aminopeptidases, and a more limited increase in the activity of dipeptidylaminopeptidases, induced an increase of substrate hydrolysis with respect to that measured in the controls. The activity of all substrate-active enzymes, the allergy-associated variations in this activity, and the amount of substrate hydrolyzed, were found to be different in male and female saliva. Specifically, in the controls the gender-related differences in substrate hydrolysis were mainly caused by the higher activity of aminopeptidases observed in male as compared to female saliva. In contrast, in allergic saliva, a greater increase in the activity of aminopeptidases in female saliva reduced the gender-related differences in the pattern of hydrolysis, which was also different from that observed in the controls.

Three new cases of dysfibrinogenemia: Poissy III, Saint-Germain I and Tahiti.

In order to identify unknown mutations, the FAMA method was used to rapidly screen the fibrinogen chain genes in individuals with dysfibrinogenemias. Chemical cleavage at mismatches on heteroduplexes DNA end-labeled with strand-specific fluorescent dyes reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. This method was successfully used for the detection of three new dysfibrinogenemias: Poissy III, Tahiti (heterozygous Aalpha Arg16His) and Saint-Germain I (heterozygous AalphaGly12Val). The mutations were confirmed by dideoxy sequencing.

Neuropeptide degradation in naive and steroid-treated allergic saliva.

The hydrolysis of neuropeptides and possible variations in hydrolysis following steroidal treatment, were examined in the presence of saliva collected from allergic volunteers; data obtained were compared to those obtained with a age and sex-matching group of healthy controls. The results reported indicate the presence of a statistically significant increase in the hydrolysis of the model substrate in allergic as compared to control saliva, and a reduction of substrate hydrolysis in treated as compared to naive allergic saliva. Total enzyme activity, the relative activity of the three classes of substrate-active enzymes (aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases), the allergy-associated variations of these activities, and the variations associated to therapy were found to be different in male and female saliva. Specifically, in the controls, the lower level of hydrolysis evident in female as compared to male saliva appeared to be principally induced by lower activity of aminopeptidases. Under allergic conditions, a sex-different increase in the activity of all three classes of substrate-active enzymes modified the hydrolysis pattern differently in samples obtained from male and female donors. Finally, pharmacological treatment induced opposite effects on the enzymes present in each sex: in male saliva, the activity of all three classes of substrate-active enzymes--and, thus, of substrate hydrolysis--was reduced near to the levels measured in the controls. In female saliva, the reduction in the activity of aminopeptidases was coupled with an increase in the activity of dipeptidylaminopeptidases, causing substrate hydrolysis to remain near the levels measured in naive allergic, rather than control, saliva.

Postprandial triglyceridemia in obese and non-obese adolescents. Importance of body composition and fat distribution.

BACKGROUND: It has recently been shown that obese adults have a disturbed metabolism of postprandial lipoproteins, resulting in postprandial hypertriglyceridemia. To the best of our knowledge, there are no data about postprandial triglyceridemia in obese and non-obese children and adolescents. SUBJECTS: 12 obese and 12 non-obese adolescents, aged 11.0 to 13.8 years. METHODS: Body composition and fat distribution (waist-to-hip circumference ratio and triceps/ subscapular skinfold thickness ratio) were assessed by anthropometry. An oral fat tolerance test was carried out, and fasting and postprandial lipid-lipoprotein serum concentrations were measured. RESULTS: We observed a significant increase in triglyceride serum concentrations 2 and 4 hours after the oral fat load, in both obese and non-obese adolescents. In obese and non-obese adolescents there were significant correlations between some variables of postprandial lipemia and the studied indices of body fat distribution. When we compare postprandial lipemia in adolescents having a central pattern of fat distribution with those having a peripheral pattern of fat distribution, we observed higher variables related to postprandial lipemia in those having a central pattern of fat distribution compared with those with a peripheral pattern (sum of serum triglyceride concentrations: 6.06 vs 4.41, p = 0.0243). CONCLUSIONS: We present a protocol to study postprandial lipemia in children and adolescents that allowed us to observe significant changes after an oral fat load. Results obtained indicate that the pattern of distribution of adipose tissue may be more important for lipid metabolism disturbances than total adipose tissue per se.

[Breast-feeding in southern Catalonia. Epidemiological analysis of sociocultural and health factors influencing choice and duration]. / Lactancia materna en el sur de Cataluña. Estudio de los factores socioculturales ysanitarios que influyen en su elección y mantenimiento.

OBJECTIVE: To study the incidence and prevalence of breast-feeding and to determine the factors that influence the mother's decision to breast-feed or to use adapted milk. MATERIAL AND METHODS: Two hundred families were included in a survey in the hospital's maternity department. Those who breast-fed were followed up by means of a telephone call on days 15, 30, 90, and 180. RESULTS: On leaving hospital 78% of the neonates were receiving breast milk only. After 15 days, 89.7% of the neonates continued to receive breast milk and at 6 months this figure was 39%. Breast-feeding was discontinued after a mean of 2.5 months. The mean age of mothers who breast-fed was 30.2 years and that of mothers using adapted milk was 27.9 years (p,0.05). Mothers decided on the type of feeding before pregnancy (52.5%). This decision was unchanged by prenatal information except in the case of information provided by the family, especially if both parents were breast-fed (p,0.05). Doctors provided little information. The mother's level of education did not influence the decision to breast-feed although the higher the mother's education, the greater the tendency to breast-feed (74.7% with primary education vs 81.5% with higher education). Being in paid employment did not influence the decision to breast-feed (76% of mothers worked vs 79% of mothers who did not). The main reasons for discontinuance were hypogalactia, "feeling hungry", and work. In general, giving up breast-feeding was the mother's decision. CONCLUSIONS: The information pregnant women receive on breast-feeding should be based on unified criteria. The implementation of joint protocols between primary and hospital care as well as breast-feeding support groups help mothers to begin and continue breast-feeding.