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1.
Pediatr Surg Int ; 38(12): 1665-1670, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36129534

ABSTRACT

PURPOSE: Evaluate whether parents have ever discussed sexuality with their children with anorectal malformations (ARM), which sexuality issues they think should be addressed and who, in their opinion, should address these issues. METHODS: Parents from the Italian Parents' and Patients' Association for Anorectal Malformation participated in meetings organized by the Association together with local Pediatric Surgical Units and were asked to fill in a questionnaire. RESULTS: 103 parents participated. Overall, 66% of parents had never talked about sex with their children. Children's age was marginally correlated with occasions to talk about sexuality (r = .202, p = 0.53) indicating that the older were the children, the more the parents talked about sexuality. The majority of parents reported that their children should have the possibility to talk about sex with them (72%), psychologists (57%), gynecologists/andrologists (47%), pediatric surgeons (33.5%), surgeons specialized in ARM (39.8%), friends (28%), nurses (24.7%) and teachers (20.4%), respectively. The most important topic they thought their children should address was handling serene sexuality, although the most common topic effectively discussed with them was the conception. CONCLUSION: Psychologists, gynecologists/andrologists, and pediatric surgeons are seen as key resources for talking about sexuality. A great number of parents express the wish that their children had more opportunities to discuss sexual topics with pediatric surgeons.


Subject(s)
Anorectal Malformations , Sexuality , Child , Humans , Sexual Behavior , Parents , Surveys and Questionnaires , Anorectal Malformations/surgery
2.
Pediatr Surg Int ; 35(9): 999-1004, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31278479

ABSTRACT

PURPOSE: Conflicts of interest can impede both research and medical treatment. The European Reference Networks require their members to deal with financial and non-financial conflicts according to an explicit protocol. In a literature review, we identified relevant interests in paediatric surgery, and drafted such a policy. METHODS: We conducted a Pubmed query and identified additional publications based on the content of the papers. RESULTS: 58 titles were identified. According to their abstracts, 10 publications were studied in full text. A scientific taxonomy does not yet exist, but a variety of factors are mentioned. Non-financial conflicts of interest are addressed less accurately and less frequently than financial ones, especially regarding surgical treatment. Since the clinical effect of surgical volume was identified as being relevant, additional 29 respective publications were analysed. This volume-quality relationship causes conflicts of interest for the many surgeons treating a broad spectrum of rare conditions. We present a recommendation that may guide referral of patients requiring complex surgery to centres with a higher volume. CONCLUSIONS: Non-financial conflicts of interest need to be dealt with more accuracy, especially with regard to surgery in rare, complex congenital conditions. The European Reference Networks offer a framework to mitigate these conflicts.


Subject(s)
Conflict of Interest , General Surgery/standards , Pediatrics/standards , Disclosure/standards , Europe , Humans , Rare Diseases
3.
Colorectal Dis ; 20(8): 711-718, 2018 08.
Article in English | MEDLINE | ID: mdl-29751372

ABSTRACT

AIM: Anorectal malformations (ARMs) are rare congenital colorectal anomalies with long lasting consequences, among which faecal incontinence is one of the most relevant since it may strongly affect patients' health-related quality of life (HRQoL). Although a growing body of literature supports the importance of self-efficacy in chronic disease health outcomes, only few studies have focused on self-efficacy in ARMs and in faecal incontinence. The purpose of the present study is to examine the mediational role of self-efficacy in the path between faecal incontinence and HRQoL in patients born with ARMs. METHOD: Ninety-eight adult patients from the Italian Association for Anorectal Malformations (AIMAR) responded to measures of faecal incontinence, self-efficacy for managing ARM consequences, and physical and mental HRQoL (SF-36). Data were analysed by means of structural equation models. RESULTS: The tested model provides support for the guiding hypothesis. Fit indices indicate that the model fits the data well (χ2  = 33.48, df = 23, P = 0.07; comparative fit index [CFI] = 0.97; root mean square error of approximation [RMSEA] = 0.07; standardized root mean square residual [SRMR] = 0.05). Faecal incontinence has negative effects on both physical and mental HRQoL, as well on self-efficacy. In turn, self-efficacy has a positive and direct effect on mental HRQoL. CONCLUSION: Faecal incontinence is the most relevant and negative factor influencing HRQoL; in addition, self-efficacy contributes in reducing emotional distress and in improving mental health outcomes. Longitudinal and controlled studies may be helpful to evaluate the effectiveness of self-efficacy interventions in improving mental HRQoL in patients with faecal incontinence.


Subject(s)
Anorectal Malformations/psychology , Fecal Incontinence/psychology , Quality of Life/psychology , Self Efficacy , Adolescent , Adult , Female , Health Surveys , Humans , Male , Models, Psychological , Stress, Psychological/etiology , Young Adult
4.
Pediatr Surg Int ; 33(8): 849-854, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28674920

ABSTRACT

PURPOSE: The goal of this study was to determine the degree of consensus in the management of spinal cord tethering (TC) in patients with anorectal malformation (ARM) in a large cohort of European pediatric centers. METHODS: A survey was sent to pediatric surgeons (one per center) members of the ARM-Net Consortium. RESULTS: Twenty-four (86%) from ten different countries completed the survey. Overall prevalence of TC was: 21% unknown, 46% below 15, and 29% between 15 and 30%. Ninety-six agreed on screening all patients for TC regardless the type of ARM and 79% start screening at birth. Responses varied in TC definition and diagnostic tools. Fifty percent of respondents prefer ultrasound (US), 21% indicate either US or magnetic resonance (MRI) based on a pre-defined risk of presenting TC, and 21% perform both. Discrepancy exists in complementary test: 82% carry out urodynamic studies (UDS) and only 37% perform somatosensory-evoked potentials (SSEP). Prophylactic untethering is performed in only two centers (8%). CONCLUSIONS: Survey results support TC screening in all patients with ARM and conservative management of TC. There is discrepancy in the definition of TC, screening tools, and complementary test. Protocols should be developed to avoid such variability in management.


Subject(s)
Anorectal Malformations/epidemiology , Neural Tube Defects/diagnosis , Neural Tube Defects/epidemiology , Child, Preschool , Cohort Studies , Female , Humans , Incidence , Infant , Infant, Newborn , Magnetic Resonance Imaging , Male , Societies, Medical , Ultrasonography
5.
Pediatr Surg Int ; 32(8): 759-65, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27369966

ABSTRACT

PURPOSE: Coping strategies have been acknowledged as crucial for the well-being and for health-related quality of life (HRQoL). The main aim of the present study is to determine whether different types of coping strategies predict HRQoL in patients born with ARM, above and beyond the variance explained by fecal and urinary continence. METHODS: 71 adult patients from the Italian Parents' and Patients' Organization for Anorectal Malformations (AIMAR) participated in the study. Participants completed measures of fecal and urinary continence of the Hirschsprung Disease/Anorectal Malformation Quality of Life (HAQL) (Hanneman et al. in Dis Col Rect 44:1650-1660, 2001), the Short Form Health Survey (SF-36) (Apolone and Mosconi in J Clin Epidemiol 51:1025-1036, 1998), and the Brief Coping Orientation to Problems Experienced (COPE) Inventory (Carver in Int J Behav Med 4:92-100, 1997), which measures different coping strategies: maladaptive, problem-focused and emotion-focused. RESULTS: Hierarchical regression analyses showed that fecal continence (ß = 0.53, p < 0.01) and urinary continence significantly predict (ß = 0.23, p < 0.05) Physical HRQoL. Fecal continence (ß = 0.36, p < 0.01) and maladaptive coping strategies significantly predict (ß = -0.27, p < 0.05) Mental HRQoL. CONCLUSION: Besides considering the importance of fecal and urinary continence for Physical HRQoL, these findings indicate that maladaptive coping strategies are associated with worse Mental HRQoL. Interventions aimed at enhancing the patients' HRQoL should target coping strategies by reducing denial, behavioral disengagement, substance abuse, and self-blame.


Subject(s)
Adaptation, Psychological , Anorectal Malformations/complications , Fecal Incontinence/psychology , Quality of Life , Urinary Incontinence/psychology , Adolescent , Adult , Fecal Incontinence/etiology , Female , Humans , Italy , Male , Middle Aged , Urinary Incontinence/etiology , Young Adult
6.
Pediatr Surg Int ; 31(8): 735-40, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26143409

ABSTRACT

BACKGROUND/PURPOSE: Disease-specific quality of life (QoL) may be more or less relevant when children enter preadolescence/adolescence. Few attentions have been given to development and transition periods. Aim of the present longitudinal study is to evaluate ARM specific changes in QoL and the stability of QoL over 6 years. METHODS: Questionnaires were sent to families of the AIMAR Association (in 2007 and in 2013/2014). They included the Hirschsprung's Disease/ARM QoL Questionnaire (HAQL, [1]). Rank correlations and within group comparisons for the HAQL subscales were conducted analyzing the scores of time 1 vs time 2. Gender effects were tested. RESULTS: 134 parents answered the questionnaires at time 1 and 73 at time 2. Results of the repeated Analyses of Variance indicated improvements in the continence subscales ("Presence of Diarrhea," "Fecal Continence"). QoL significantly worsened in "Social and Emotional Functioning" and in "Body Image" areas. The analysis of stability of change indicates that QoL scores remain stable, with the exception of the Body Image area. CONCLUSIONS: Despite improvements in the continence areas, parents reported worse levels of QoL in the psychosocial areas, as their child grow. As patients grow, they might have more difficulties in daily and social activities, and feel more frequently ashamed and more dissatisfied with their body.


Subject(s)
Anal Canal/abnormalities , Anus, Imperforate , Quality of Life , Rectum/abnormalities , Adolescent , Age Factors , Anorectal Malformations , Anus, Imperforate/psychology , Child , Female , Humans , Longitudinal Studies , Male , Surveys and Questionnaires , Time Factors
7.
Pediatr Surg Int ; 31(8): 741-5, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26210660

ABSTRACT

BACKGROUND: Pediatric surgeons and patient organisations agree that fewer centers for anorectal malformations with larger patient numbers are essential to reach better treatment. The European Union transacts a political process which aims to realize such centers of expertise for a multitude of rare diseases. All the centers on a specific rare disease should constitute an ERN on that disease. ARM-Net members in different countries report on first experiences with the implementation of national directives, identifying opportunities and risks of this process. METHODS: Relevant details from the official European legislation were analyzed. A survey among the pediatric surgeons of the multidisciplinary ARM-Net consortium about national implementation was conducted. RESULTS: European legislation calls for multidisciplinary centers treating children with rare diseases, and proposes a multitude of quality criteria. The member states are called to allocate sufficient funding and to execute robust governance and oversight, applying clear methods for evaluation. Participation of the patient organisations is mandatory. The national implementations all over Europe differ a lot in respect of extent and timeframe. CONCLUSIONS: Establishing Centers of Expertise and a ERN for anorectal malformations offers great opportunities for patient care and research. Pediatric surgeons should be actively engaged in this process.


Subject(s)
Anal Canal/abnormalities , Anus, Imperforate/surgery , Health Facilities/standards , Rectum/abnormalities , Anal Canal/surgery , Anorectal Malformations , European Union , Humans , Quality of Health Care , Rectum/surgery
8.
Tech Coloproctol ; 19(3): 181-5, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25609592

ABSTRACT

The ARM-Net (anorectal malformation network) consortium held a consensus meeting in which the classification of ARM and preoperative workup were evaluated with the aim of improving monitoring of treatment and outcome. The Krickenbeck classification of ARM and preoperative workup suggested by Levitt and Peña, used as a template, were discussed, and a collaborative consensus was achieved. The Krickenbeck classification is appropriate in describing ARM for clinical use. The preoperative workup was slightly modified. In males with a visible fistula, no cross-table lateral X-ray is needed and an anoplasty or (mini-) posterior sagittal anorectoplasty can directly be performed. In females with a small vestibular fistula (Hegar size <5 mm), a primary repair or colostomy is recommended; the repair may be delayed if the fistula admits a Hegar size >5 mm, and in the meantime, gentle painless dilatations can be performed. In both male and female perineal fistula and either a low birth weight (<2,000 g) or severe associated congenital anomalies, prolonged preoperative painless dilatations might be indicated to decrease perioperative morbidity caused by general anesthesia. The Krickenbeck classification is appropriate in describing ARM for clinical use. Some minor modifications to the preoperative workup by Levitt and Peña have been introduced in order to refine terminology and establish a comprehensive preoperative workup.


Subject(s)
Anus, Imperforate/diagnosis , Anus, Imperforate/surgery , Abnormalities, Multiple/surgery , Anorectal Malformations , Anus, Imperforate/classification , Europe , Female , Humans , Infant, Newborn , Male , Plastic Surgery Procedures/standards , Rectal Fistula/surgery
9.
Pediatr Surg Int ; 30(8): 823-8, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24990242

ABSTRACT

PURPOSE: Managing a chronic disease may be a difficult task which may lead patients to experience psychological distress and depression. Some studies showed that, in ARM patients, fecal incontinence (FI) is related to symptoms of depression while others studies did not. No studies investigated this relationship in adults. Since fear of having FI episodes, negative feelings associated with these episodes, and difficulties in close and sexual relationships are often reported by patients as important consequences of ARM, we were interested in investigating whether these aspects contribute in explaining feelings of depression. METHODS: Questionnaires were sent to 160 adult members of the Italian Association for Anorectal Malformation. A new scale measuring the fear of having FI episodes, negative feelings associated with these episodes and difficulties in close/sexual relationship was developed. Depressive feelings and FI were also measured. RESULTS: Seventy-two adults answered the questionnaires. Regression analyses showed that, in males, depressive feelings were predicted by difficulties in close and sexual relationships (B = 0.46; P < 0.01), while, in females, they were predicted by the fear of having FI episodes (B = 0.53; P < 0.05) and by negative feelings (B = 0.58; P < 0.01). CONCLUSIONS: Interventions aimed to prevent depression in ARM patients should consider gender and should be targeted on different aspects.


Subject(s)
Anal Canal/abnormalities , Anus, Imperforate/complications , Depression/etiology , Quality of Life , Rectum/abnormalities , Sexual Behavior , Adolescent , Adult , Anorectal Malformations , Anus, Imperforate/psychology , Depression/psychology , Emotions , Female , Follow-Up Studies , Humans , Male , Middle Aged , Retrospective Studies , Sexuality , Surveys and Questionnaires , Young Adult
10.
Pediatr Surg Int ; 29(9): 925-30, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23907176

ABSTRACT

PURPOSE: Quality of life (QOL) is an important endpoint in health outcomes research, especially in pediatric chronic conditions. While some studies suggest that patients with ARM report a lower level of physical and psychosocial QOL, as compared to healthy peers, not all research shows this consistently. The aim of this study was to compare the QOL of Italian children and adolescents with ARM to the QOL of a large control sample of healthy peers. METHODS: Parents of 109 children with ARM and 336 schoolchildren, completed the Pediatric Quality of Life Inventory. Analysis of variances were used to compare the groups. RESULTS: Compared to healthy peers, children with ARM showed lower emotional functioning (F = 8,41; p = .004), social functioning (F = 4,9; p = .027) and school functioning (F = 14,7; p = .000). In the adolescent group, females had a worse QOL in the physical functioning domain (F = 5,01; p = .03) than males, independent of group membership. CONCLUSIONS: While children with ARM show a more impaired QOL compared to their peers, this difference does not emerge in the adolescent group. Our results are consistent with previous findings which hypothesize that while patients with ARM face more difficulties during childhood, by adolescence they may have learned to overcome these difficulties.


Subject(s)
Anus, Imperforate/psychology , Quality of Life/psychology , Adolescent , Analysis of Variance , Anorectal Malformations , Child , Educational Status , Emotions , Female , Humans , Italy , Male , Peer Group , Sex Distribution , Social Behavior , Surveys and Questionnaires
11.
Pediatr Surg Int ; 29(9): 919-23, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23913264

ABSTRACT

PURPOSE: Fecal incontinence is a common problem in children and adolescents with anorectal malformation (ARM) and may negatively impact psycho-social well-being. Mothers' perception of social support has been proved to contribute to children's quality of life (QOL). Considering ARM studies, the role of family and social resources have received little attention. The aim of the present study was to analyze whether mothers' perception of social support mediates the impact of child fecal incontinence on his/her QOL. METHODS: One hundred and nine mothers with a child born with ARM (aged 6-15 years old; mean age = 11 years) completed questionnaires including the fecal incontinence subscale of the Hirschsprung's Disease/Anorectal Malformation QOL Questionnaire, the Pediatric QOL Inventory, and a social relationship questionnaire. Structural equation models were used to explore the relations hypothesized. RESULTS: No differences were found in the QOL scores across gender. The hypothesized model fits the data well; mothers' perception of social support partially mediated the relationship between fecal incontinence and QOL. CONCLUSIONS: An important direction for pediatric surgeons and their interdisciplinary teams may be to develop strategies to strengthen mothers' social relationships.


Subject(s)
Attitude to Health , Fecal Incontinence/psychology , Mothers/psychology , Quality of Life/psychology , Social Support , Adolescent , Anorectal Malformations , Anus, Imperforate/complications , Anus, Imperforate/psychology , Child , Fecal Incontinence/etiology , Female , Humans , Italy , Male , Surveys and Questionnaires
12.
Cytometry ; 46(6): 351-6, 2001 Dec 15.
Article in English | MEDLINE | ID: mdl-11754205

ABSTRACT

In vivo phagocytosis of senescent red blood cells (RBCs) by macrophages occurs 120 days after their release into the circulation. It depends on two sequential signals that trigger phagocytosis: (1) desialylation of membrane glycoconjugates with the exposure of the penultimate beta-galactosyl residues and (2) exposure of phosphatidylserine in the membrane outer leaflet. Leukodepleted and nonleukodepleted RBCs were compared using flow cytometric procedures to determine whether the in vitro deterioration of RBCs during storage might be attributable to an identical mechanism of desialylation induced by leukocyte neuraminidases, resulting in exposure of beta-galactosyl and subsequently phosphatidylserine residues - signals of senescent RBCs. Without prior leukodepletion, stored RBCs showed an increased population of senescent RBCs (using light scatter measurements), extensive desialylation with the exposure of beta-galactosyl residues (using specific fluorescein isothiocyanate [FITC]-lectins), significant exposure of phosphatidylserine in the outer leaflet of the RBC membrane (using FITC-annexin V), and extensive in vitro phagocytosis (using PKH-26-labeled RBCs). There were minimal changes observed with the leukodepleted RBCs. These results lead to the conclusion that leukocyte enzymes, including neuraminidases, are definitive contributers to the desialylation of RBCs during storage and to the exposure of phosphatidylserine residues. These deleterious effects resulting from highly active leukocyte enzymes are preventable by prior leukodepletion of the stored RBCs. Previously developed flow cytometric procedures to detect in vivo "RBC senescence" have been applied and proved to be reliable criteria to monitor the viability of stored RBCs.


Subject(s)
Blood Preservation/methods , Erythrocyte Aging , Erythrocytes/cytology , Adult , Animals , Cell Survival , Flow Cytometry , Humans , Leukocytes/enzymology , Mice , Middle Aged , Neuraminidase/chemistry , Phagocytosis , Phosphatidylserines/analysis , Specimen Handling
13.
Biochimie ; 80(2): 173-95, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9587675

ABSTRACT

Human red blood cells (RBCs) have a life-span of 120 days in circulation, after which they are phagocytized by resident macrophages. Extensive studies have been undertaken by many investigators in order to elucidate the cellular and molecular mechanisms of the erythrophagocytosis. The critical questions addressed by physiologists, clinicians and biochemists are: 'which of the many traumatic blemishes that appear on the erythrocyte surface as it winds its way through the circulation is the primary signal for clearance of the effete RBC from the circulation?', or 'What is the critical signal that it, and it alone, will activate the resident macrophage to adhere to and engulf it?'. Numerous, and often conflicting, hypotheses have been proposed. Each investigator focusing on but one of the many modifications that afflict the cell surface of the ageing erythrocyte, viz changes in either or both the carbohydrate or peptidic moieties of glycoproteins; abolishment of the pre-existing asymmetry in the lipid bilayer with the exposure of phosphatidylserine residues; or alterations in spectrin, to mention but a few. Many of these investigators also have invoked an intermediary role for auto-immune antibodies that recognise the change(s) on the erythrocyte surface and thereby serve as opsonins as a prelude to the erythrophagocytosis. The objective of the present review is to evaluate the data in support of the various hypotheses, and to submit some of our own recent observations involving the use of flow cytometric procedures that: i) provide evidence that the cell surface sialic acid serves as a determinant of the life-span; ii) characterise the senescent erythrocyte population that is specifically captured and phagocytized by macrophages (utilising the rapid and sensitive procedure we developed for quantification of in vitro erythrophagocytosis); and finally iii) provide evidence for the existence of an alternative pathway that is independent of immunoglobulins.


Subject(s)
Erythrocytes/physiology , Macrophages/physiology , Phagocytosis/physiology , Animals , Carbohydrate Sequence , Cell Membrane/physiology , Cellular Senescence/physiology , Humans , Models, Biological , Molecular Sequence Data
14.
Cytometry ; 30(5): 269-74, 1997 Oct 15.
Article in English | MEDLINE | ID: mdl-9383101

ABSTRACT

A rapid, sensitive, and reproducible flow cytofluorimetric procedure is described for quantitation of erythrophagocytosis based on the use of red blood cells (RBCs) labeled with the fluorescent probe PKH-26. The procedure involves the following steps: i) incubation of PKH-26-labeled erythrocytes with macrophages, ii) removal of un-bound red blood cells, iii) lysis of membrane-bound RBCs, and iv) measurement of extent of phagocytosis by direct flow-cytometric analysis of intact macrophages. Each step was controlled by fluorescence microscopy. Use of fluorescent, instead of radio-labeled RBCs, makes the method more sensitive, rapid, and avoids radioactive hazards. Furthermore, this approach is multi-parametric and can distinguish different populations of macrophages with reference to their erythrophagocytic potential. This technology moreover, has broad applications from the initial step of contact (between effector and target cells to study the specific receptor mediated attachment) to the subsequent cascade of time-dependent changes resulting from that signal transduction.


Subject(s)
Erythrocytes/physiology , Flow Cytometry/methods , Macrophages, Peritoneal/physiology , Organic Chemicals , Phagocytosis/physiology , Animals , Cells, Cultured , Fluorescent Dyes , Humans , Mice , Neuraminidase , Sensitivity and Specificity
15.
C R Acad Sci III ; 320(10): 811-8, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9436535

ABSTRACT

We have recently developed a flow cytometric assay for the quantitation of erythrophagocytosis, using PKH 26-labeled erythrocytes as the target cells. Using this assay we have shown that there is extensive phagocytosis of desialylated erythrocytes. Furthermore, we have demonstrated that it is the densest population of erythrocytes obtained on a self-forming gradient of Percoll that shows the greatest susceptibility to phagocytosis. We designate this population of erythrocytes as fraction X; it is even denser than the fraction 5 found previously. This population of erythrocytes corresponds to zone X previously seen in the dot-plot of the flow cytometric analyses of human erythrocytes. Further scrutiny of this fraction indicates that a) it shows the greatest reactivity with annexin V, which is specific for the detection of phosphatidylserine (PS) exposed on the outer leaflet of the erythrocyte membrane, b) it is the most susceptible to erythrophagocytosis by resident murine peritoneal macrophages, and c) this erythrophagocytosis of PKH 26-labeled erythrocytes can be inhibited by annexin V and by liposomes containing PS. Scanning electron microscopy of fraction X shows two populations of erythrocytes: (A) spheroechinocytes with filipodes and (B) echinocytes without filipods. After a 2-h period of phagocytosis, the cells remaining in fraction X show a decrease in population A, commensurate with a decrease in reactivity with FITC-labeled annexin V from 65.5 to 24%.


Subject(s)
Erythrocyte Aging/physiology , Macrophages/physiology , Phagocytosis/physiology , Animals , Annexin A5/metabolism , Erythrocytes/cytology , Erythrocytes/metabolism , Female , Flow Cytometry , Fluorescein-5-isothiocyanate , Humans , In Vitro Techniques , Male , Mice , Microscopy, Electron, Scanning , Phosphatidylserines/metabolism
16.
Arch Gerontol Geriatr ; 15 Suppl 1: 7-15, 1992.
Article in English | MEDLINE | ID: mdl-18647672

ABSTRACT

An animal is composed of many cells, tissues, organs, and systems all of which can, and do, age at different rates. The aging process is a summation of unidirectional physical, chemical and biological time-dependent changes, which are not necessarily colinear, hence the potential of asynchrony exists between biological-attributes and chronological age. We have developed two approaches to study the aging of an animal at the cellular and molecular levels. These methods will lend themselves well to distinguish between the effects of this cellular aging on the aging animal, and vice versa; such an interplay is readily apparent. The problem is to demonstrate these correlations in objective terms--not merely to document these changes, but to develop a methodology that also will permit experimental intervention to determine if aging events can be slowed or reversed. These investigations were initiated with the enucleated mammalian RBC in order to study the simplest system (unedited response of a cell). Using flow cytometric procedures as a miniature biochemical laboratory, we thus are able to read the autobiography of the cell as it is debriefed by a series of questions posed. Flow cytometric procedures not only demonstrate the changes, but also monitor their dynamics and kinetics. Concurrently, with these flow cytometric procedures, we are developing 2D-PACE methods to document the changes at the molecular level.

17.
Exp Gerontol ; 26(4): 315-26, 1991.
Article in English | MEDLINE | ID: mdl-1936191

ABSTRACT

A recent review (Aminoff, 1988) summarized the evidence for and against our hypothesis for the role of glycophorin in the senescence and clearance of mammalian red blood cells (RBC) from circulation. This hypothesis postulates the loss of sialic acid from RBC surface in two forms: (a) as vesicles containing the sialoglycoprotein glycophorin, and (b) as free sialic acid residues from glycophorin molecules remaining on cell surface. In this report we demonstrate the applicability of flow cytometric procedures to explore, at the cellular level, time-dependent changes on RBC surface with change in cell size, and with in vivo age. The RBC are probed with fluorescein isothiocyanate (FITC) labelled lectins and goat anti-human-IgG and -IgM. The relative intensity of fluorescence is correlated to the change in RBC size as measured by forward lightscatter. Reactivity of RBC with FITC-labelled wheat germ agglutinin can be inhibited with either 0.2M N-acetylglucosamine or by removal of sialic acid residues with neuraminidase. The properties of the smallest RBC correspond to those of the oldest RBC in their: (a) decreased reactivity with FITC-labelled lectins that recognize sialic acid residues, wheat germ and Limax flavus agglutinins, and (b) increased reactivity with FITC-labelled goat anti-human-IgG and -IgM. These results are compatible with our glycophorin hypothesis. Moreover, they suggest that the initial loss of sialic acid as glycophorin containing vesicles is gradual, while the subsequent step involving the loss of sialic acid residues is rapid and exposes multiple disaccharide galactose beta(1-3)N-acetylgalacosaminyl residues. These unmasked disaccharide sites are recognized by autoimmune IgG, IgM, and lectin-like receptors on macrophages resulting in the clearance of senescent RBC from circulation.


Subject(s)
Erythrocytes/physiology , Flow Cytometry , Erythrocyte Membrane/metabolism , Erythrocytes/cytology , Fluorescein-5-isothiocyanate , Humans , Immunoglobulin G/metabolism , Immunoglobulin M/metabolism , Immunoglobulins , Lectins , N-Acetylneuraminic Acid , Osmolar Concentration , Sialic Acids/blood , Wheat Germ Agglutinins
18.
Exp Gerontol ; 26(4): 327-45, 1991.
Article in English | MEDLINE | ID: mdl-1936192

ABSTRACT

In the first paper of a series (Gutowski, et al., 1991) we discussed the use of flow cytometry to follow at the cellular level the aging of red blood cells (RBC) in circulation, using fluorescently labelled lectins and goat anti-human-IgG and -IgM. The Coulter Epics 541 was used for those studies. In this report we describe more extensive experiments using the Becton-Dickinson FACScan flow cytometer, and compare the results with those obtained with the Coulter Epics 541. By changing sample conditions from isotonic to hypotonic, compensation for differences of the two instruments was accomplished. We confirmed our previous observations that RBC react very strongly with fluorescein isothiocyanate labelled wheat germ agglutinin (FITC-WGA) and that there is little change in the intensity of fluorescence given by RBC of all sizes with the exception of the smallest. Reactivity with FITC-WGA is markedly decreased in the presence of competitive inhibitors of sialic acid or upon enzymatic removal of sialic acid from RBC. Removal of sialic acid is accompanied by increased reaction with peanut agglutinin (FITC-PNA). Flow cytometry was also used to monitor the enrichment of a population of smallest RBC (less than 0.05%), isolated from both counterflow centrifugation and the interface obtained from Histopaque separation. These smallest RBC showed low reactivity with FITC-WGA and higher binding of FITC-goat-anti-human-IgG, and -IgM, and therefore represent the most senescent RBC, just prior to their clearance from circulation by the reticuloendothelial system. These observations are in compliance with the hypothesis that physiological desialylation of glycophorin is responsible for clearance of senescent RBC from circulation (Aminoff, 1988).


Subject(s)
Erythrocytes/physiology , Flow Cytometry , Cellular Senescence , Centrifugation , Chemical Fractionation , Erythrocytes/metabolism , Fluorescein-5-isothiocyanate , Glycophorins/pharmacology , Humans , Lectins/blood , Light , Neuraminidase/pharmacology , Osmolar Concentration , Peanut Agglutinin , Reproducibility of Results , Scattering, Radiation , Trisaccharides/pharmacology , Wheat Germ Agglutinins/blood
20.
Carbohydr Res ; 178: 307-13, 1988 Jul 15.
Article in English | MEDLINE | ID: mdl-3191511

ABSTRACT

Enzymatic removal of sialyl groups from mammalian erythrocytes resulted in their rapid sequestration from circulation subsequent to autologous transfusion. It has been demonstrated by many investigators that the terminal beta-D-galactosyl group, exposed on red blood cell by in vitro desialosylation, is recognized by an autoimmune anti-galactosyl IgG and/or by a lectin-like receptor on monocytes and macrophages. It is demonstrated herein that the disaccharide structure beta-D-Galp-(1----3)-D-GalpNAc (a) is masked in normal rat RBC, but exposed in asialo-RBC; (b) could be detected with fluorescently-labeled peanut agglutinin; (c) could be released from the asialo-RBC with an endo-N-acetyl-alpha-D-galactosaminidase; and (d) upon its removal by treatment with the endo-N-acetyl-alpha-D-galactosaminidase, enhances the survival of the asialo-RBC in circulation.


Subject(s)
Erythrocytes/metabolism , Glycoside Hydrolases/blood , Animals , Erythrocyte Aging , Erythrocytes/cytology , Male , Rats , Rats, Inbred Strains
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