Living without LAMPsConsequences of the loss of the lysosome associated membranproteins LAMP-1 and LAMP-2 on endosomal/lysosomal processes

Die beiden abundanten lysosomal assoziierte Membranproteine LAMP-1 und LAMP-2 stellen fast die Hälfte der Proteinmasse aller lysosomalen Membranproteine. Da die LAMPs eine Glykokalix bilden, indem sie aufgrund ihrer Glykosylierung eine kontinuierliche Kohlenhydratschicht auf der Innenseite der Membran von späten Endosomen und Lysosomen generieren, wurde ihre ursprüngliche Aufgabe in der Erhaltung der strukturellen Integrität dieser Membran durch den Schutz vor den lysosomalen Hydrolasen vermutet. Um Funktionen der beiden lysosomalen Membranproteine LAMP-1 und LAMP-2 zu untersuchen, die über diese eher allgemeine Aufgabe hinausgehen, wurde an aktuelle Forschungsergebnisse angeknüpft. In LAMP-defizienten Mausfibroblasten wurde eine spätendosomale Cholesterolakkumulation festgestellt. Zudem wurde eine verminderte phagosomale Reifung in Makrophagen im Zusammenhang mit der Abwehr von pathogenen Mikroorganismen beobachtet. In der vorliegenden Arbeit wurden Auswirkungen des Fehlens der beiden abundanten lysosomalen Membranproteine LAMP-1 und LAMP-2 auf unterschiedliche Funktionen der Lysosomen in LAMP-defizienten Mausfibroblasten näher untersucht. Während der generellen Charakterisierung LAMP-defizienter Mausfibroblasten wurde eine Dislokation später Endosomen/Lysosomen in LAMP-2- und LAMP-1/2-doppeldefiziente Zellen festgestellt. In Kontrollzelllinien und den LAMP-1-defizienten Zellen schienen die späten Endosomen/Lysosomen eher perinukleär lokalisiert zu sein, in den LAMP-2- und den LAMP-doppeldefizienten Zellen hingegen in periphere Bereiche der Zelle verteilt. Die Rekrutierung der lysosomalen Hydrolase Cathepsin D zu sauren Kompartimenten war dabei nicht gestört. In fluoreszenzmikroskopischen Untersuchungen wurde die Verteilung von intrazellulärem Cholesterol in Wildtyp-MEFs und LAMP-defizienten MEFs analysiert. In Wildtypzellen und in LAMP-1-defizienten Zellen wurde ein perinukleäres und vesikuläres Verteilungsmustervon freiem Cholesterol festgestellt. In den LAMP-2-einzeldefizienten und im besonderen Maße in den LAMP-1/2-doppeldefizienten MEFs konnte eine Akkumulation von freiem Cholesterol in späten Endosomen und Lysosomen beobachtet werden, die mit einer Reduktion von NilRot-färbbaren Cholesterolestern einherging. In der Leber LAMP-2-defizienter Mäuse konnte die Cholesterolakkumulation und die Abnahme von Cholesterolestern in Lipidtröpfchen ebenfalls festgestellt werden. Die heterologe Expression von LAMP-2A in den doppeldefizienten MEFs reduzierte die Cholesterolakkumulation, nicht jedoch die Transfektion mit LAMP-1. Mit Hilfe von chimären Proteinen aus LAMP-1 und LAMP-2, konnte die membranproximale Hälfte der luminalen Domäne als der Bereich von LAMP-2 eingegrenzt werden, der die Cholesterolakkumulation in den LAMP-defizienten MEFs reduzieren vermag. Um die Rolle der LAMP-Proteine während der phagosomalen Reifung zu untersuchen, wurde ein Latexkügelchen-Phagozytose-Assay mit murinen Fibroblasten etabliert. Es zeigte sich, dass sowohl die lysosomale Hydrolase Cathepsin D, als auch LAMP-2 zu einem Großteil der Phagosomen in Wildtyp-MEFs rekrutiert worden war, Indikatoren für die Fusion von Phagosomen mit Lysosomen zu Phagolysosomen. In den LAMP-1/2-doppeldefizienten MEFs zeigte sich ein gravierender Maturierungsdefekt der Phagosomen bezüglich der Rekrutierung von Cathepsin D zu den Phagosomen und der Generierung eines luminalen sauren Milieus. Es konnte gezeigt werden, dass die Transfektion der LAMP-defizienten MEFs sowohl mit LAMP-1, als auch mit LAMP-2, die Wiederherstellung der phagosomalen Reifung bewirkt.The Lysosome Associated Membrane Proteins LAMP-1 and LAMP-2 represent nearly 50 % of the total membrane protein of late endosomes and lysosomes. LAMPs form a nearly continuous carbohydrate coat on the inner surface of the lysosomal membrane. The protective function against degradation by lysosomal hydrolases was assigned to the heavy glycosylation of both LAMP proteins possibly generating a continuous glycocalyx. Recent publications revealed further biological functions of LAMP-1 and LAMP-2. Murine fibroblasts deficient for both LAMPs were shown to accumulate free cholesterol in late endosomal compartments. Additionally, impairment of phagosomal maturation led to reduced clearence of pathogenic microbials in granulocytes deficient for LAMP-2 and in mouse embryonic fibroblasts (MEF) deficient for both LAMPs. In this work the impact of the abundant lysosomal membrane proteins LAMP-1 and LAMP-2 on different lysosomal functions were studied. Therefore MEFs deficient in LAMP-1 and LAMP-2 expression were used. First studies revealed a dislocation of late endosomes/lysosomes in LAMP deficient MEFs. In control as well as in LAMP-1 deficient cells late endosomes were located near the nucleus. In contrast, in cells deficient for LAMP-2 or both LAMPs the late endosomal vesicles were peripherally located. Recruitment of the lysosomal hydrolase cathepsin D to acidic compartment was unaffected. Filipin staining was used to analyse the localisation of free cholesterol in wildtype and LAMP deficient MEFs. While in wildtype and LAMP-1 deficient cells the localisation of free cholesterol was observed in perinuclear structures, in LAMP double deficient and to less extent in LAMP-2 deficient cells free cholesterol accumulated in late endosomal/lysosomal compartments. In addition to the prominent accumulation of cholesterol in late endosomes/lysosomes, the double deficient MEFs harbour considerably less Nile Red stainable lipid droplets than wild type MEFs. Furthermore, LAMP-2 deficiency led to intracellular cholesterol accumulation also in tissue, not only in cultured cells. Filipin staining of liver sections showed that LAMP-2 deficient liver accumulated free cholesterol, too. A reduction of lipid droplets was observed in LAMP-2 deficient livers. Transient over-expression of LAMP-2A abolished the endo/lysosomal cholesterol accumulation in LAMP double deficient MEFs. In constrast, the over-expression of LAMP-1 could not restore normal cholesterol distribution. LAMP-2/LAMP-1 chimeric proteins were used to identify the region of the LAMP-2 protein which is critical for the rescue effect. Over-expression of a chimeric protein containing the membrane-proximal half of the luminal domain of LAMP-2 led to the correcting of cholesterol accumulation in LAMP deficient fibroblasts. These data suggest that the membrane-proximal part of the luminal domain of LAMP-2 is sufficient to effect the cholesterol homeostasis. To address functions for LAMP proteins on the phagosomal maturation, a phagocytosis assay using latex beads was established. The majority of phagosomes in wildtype MEFs recruited cathepsin D and LAMP-2 indicating a fusion of phagosomes with lysosomes. In LAMP-1/2 double deficient MEFs the phagosomal recruitment of cathepsin D and the acidification of the phagosomal lumen were dramatically reduced. Transient expression of LAMP-1 and LAMP-2, respectively could reconstitute phagosomal maturation

Photodynamic therapy of prostate cancer by means of 5-aminolevulinic acid-induced protoporphyrin IX - In vivo experiments on the dunning rat tumor model

Objective: In order to expand the use of photodynamic therapy (PDT) in the treatment of prostate carcinoma (PCA), the aim of this study was to evaluate PDT by means of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX ( PPIX) in an in vivo tumor model. Methods: The model used was the Dunning R3327 tumor. First of all, the pharmacokinetics and the localization of PPIX were obtained using fluorescence measurement techniques. Thereafter, PDT using 150 mg 5-ALA/kg b.w.i.v. was performed by homogenous irradiation of the photosensitized tumor (diode laser lambda = 633 nm). The tumors necrosis was determined histopathologically. Results: The kinetics of PPIX fluorescence revealed a maximum intensity in the tumor tissue within 3 and 4.5 h post-application of 5-ALA. At this time, specific PPIX fluorescence could be localized selectively in the tumor cells. The PDT-induced necrosis (n = 18) was determined to be 94 B 12% (range 60-100%), while the necrosis of the controls ( n = 12) differs significantly (p < 0.01), being less than 10%. Conclusion: These first in vivo results demonstrate the effective potential of 5-ALA-mediated PDT on PCA in an animal model. Copyright (C) 2004 S. Karger AG, Basel

Plasma cotinine is positively associated with homocysteine in smokers but not in users of smokeless tobacco

Plasma total homocysteine (tHcy) is a risk marker, and smoking is an established risk factor for cardiovascular disease. It is unclear if the effect of smoked tobacco on homocysteine is mediated by nicotine or other combustion products in smoked tobacco. Snus (moist smokeless tobacco) is high nicotine-containing tobacco, and little is known about the effect of snus on plasma homocysteine. Therefore, we studied, in a cross-section of subjects (n = 1375) from the Northern Sweden Health and Disease Study, with strictly defined current smokers (n = 194) and snus users (n = 47), the impact of tobacco exposure on tHcy, assessed by self-reported tobacco habits and plasma cotinine concentrations. The snus users had higher cotinine concentrations than the smokers. Cotinine, creatinine, methylmalonic acid, and the methylenetetrahydrofolate reductase genotype (MTHFR) T allele were positively associated with tHcy among the smokers, but not among the snus users. No association was observed between tHcy and the number of cigarettes/day. There was a positive association between cotinine and tHcy in the smokers, but not among the snus users. This indicates that substances other than nicotine in tobacco smoke could be responsible for the differential effects on homocysteine status. Self-reported smoking should be complemented by a cotinine assay whenever possible.publishedVersio

Cobalamin and folate status in infants and young children in a low-to-middle income community in India

Background: Population-based data on the prevalence of cobalamin and folate deficiency in India are lacking. Objective: The objective was to measure the prevalence of cobalamin and folate deficiency among children aged 6-30 mo residing in a low-to-middle income community in North India. Design: Children aged 6-30 mo (n = 2482) were identified through a community survey in a low-to-middle socioeconomic area in New Delhi, India. Non-fasting venous blood samples were collected before enrollment in another trial. Results: The median (interquartile range; IQR) cobalamin concentration in 6-11-mo-old children was substantially lower in breastfed (183; 120-263 pmol/L) than in nonbreastfed (334; 235-463 pmol/L) children. Cobalamin concentrations decreased progressively with increasing age in the nonbreastfed children. Median (IQR) plasma folate concentrations in the 6-11-mo-old group were higher in breastfed (20.3; 11.7-34.4 nmol/L) than in nonbreastfed (5.3; 3.4-7.7 nmol/L) children (P &lt; 0.001). Folate concentrations decreased with increasing age in the breastfed children. In the nonbreastfed children, folate concentrations increased with increasing age. Low concentrations of plasma cobalamin (&lt;150 pmol/L) were were detected in 36% of breastfed and 9% of nonbreastfed children (P &lt; 0.001). The proportions of children with plasma folate concentrations &lt;5 nmol/L in these 2 subgroups were 6% and 33%, respectively (P &lt; 0.001). Conclusions: In north Indian preschool children, cobalamin and folate concentrations were commonly low and were associated with elevated total homocysteine and methylmalonic acid concentrations. Because low cobalamin and folate concentrations have functional consequences, population-based measures for improving cobalamin and folate concentrations need to be seriously considered

Folate, but not vitamin B-12 status, predicts respiratory morbidity in north Indian children

Background: Vitamin deficiencies are often part of malnutrition, which predisposes to acute lower respiratory tract infections. Objective: The objective was to measure the association between cobalamin and folate status and subsequent respiratory morbidity. Design: A prospective cohort study was conducted in 2482 children aged 6-30 mo nested in a zinc supplementation trial. We measured plasma concentrations of folate, cobalamin, methylmalonic acid, and total homocysteine (tHcy) and followed the children for 4 mo. Results: We observed 1176 episodes of acute lower respiratory tract infections. Children with folate concentrations in the lowest quartile (interquartile range: 6.4-20.0 nmol/L) had a 44% higher incidence [adjusted incidence rate ratio (IRR): 1.44; 95% CI: 1.23, 1.70] of acute lower respiratory tract infections than did children in the other 3 quartiles. For tHcy, the IRR was 1.24 (1.07, 1.40) in a comparison of those in the highest quartile with those in the other quartiles. Breastfeeding was associated with high folate concentrations and protection against subsequent respiratory tract infections. This protection was significantly and substantially reduced after adjustment for plasma folate concentrations at baseline. Compared with the children in the other 3 quartiles, the IRR for being in the lowest quartile of cobalamin was 1.13 (0.76, 1.03) and for being in the highest quartile of methylmalonic acid was 1.12 (0.96, 1.31). Conclusions: Poor folate status appears to be an independent risk factor for lower respiratory tract infections in young children. This study also suggests that the protective effect of breastfeeding is partly mediated by folate provided through breast milk

Cobalamin deficiency resulting in a rare haematological disorder: a case report

INTRODUCTION: We present the case of a patient with a cobalamin deficiency resulting in pancytopaenia, emphasizing the importance to define, diagnose and treat cobalamin deficiency. CASE PRESENTATION: A 52-year-old man from the Democratic Republic of Congo presented to the emergency department with shortness of breath and a sore tongue. Physical examination was unremarkable. His haemoglobin was low and the peripheral blood smear revealed pancytopaenia with a thrombotic microangiopathy. The findings were low cobalamin and folate levels, and high homocysteine and methylmalonate levels. Pernicious anaemia with chronic atrophic gastritis was confirmed by gastric biopsy and positive antiparietal cell and anti-intrinsic factor antibodies. Cobalamin with added folate was given. Six months later, the patient was asymptomatic. CONCLUSION: Cobalamin deficiency should always be ruled out in a patient with pancytopaenia. Our case report highlights a life-threatening cobalamin deficiency completely reversible after treatment

Nutritional Intake and Status of Cobalamin and Folate among Non-Pregnant Women of Reproductive Age in Bhaktapur, Nepal

Cobalamin and folate are especially important for women of childbearing age due to their ubiquitous role in fetal growth and development. Population-based data on cobalamin and folate status are lacking from Nepal, where diets are mostly vegetarian. The objectives of the study were to investigate cobalamin and folate intake and status, and to explore associations with socio-demographics, anthropometrics, anemia, and dietary habits. Following a random selection of geographical clusters, we collected blood samples from 500 non-pregnant women and 24-h dietary recalls and food frequency questionnaires from a subsample of 379 women. Twenty percent of the women did not consume any food containing cobalamin during the days recalled, and in 72% nutritional cobalamin intake was <1 μg/day. Eighty-four percent of the women had cobalamin intake lower than the estimated average requirement (EAR) (<2 μg/day). In contrast, only 12% of the women had a folate intake less than 100 μg per day, whereas 62% had intake between 100 and 320 μg. Low plasma cobalamin (<150 pmol/L) was found in 42% of the women, most of whom (88%) also had elevated levels of methylmalonic acid. Our results indicated a high prevalence of nutritional cobalamin deficiency, while folate deficiency was uncommon

Highly variable use of diagnostic methods for sexually transmitted infections-results of a nationwide survey, Germany 2005

<p>Abstract</p> <p>Background</p> <p>Sexual transmitted infections (STIs) have increased in Germany and other countries in Europe since the mid-nineties. To obtain a better picture of diagnostic methods used in STI testing institutions in Germany, we performed a nationwide survey amongst STI specialists in order to evaluate the quality of STI reports and provide recommendations to harmonize and possibly improve STI diagnostics in Germany.</p> <p>Methods</p> <p>We asked sentinel physicians and randomly chosen gynaecologists, urologists and dermato-venerologists, about the diagnostic methods used in 2005 to diagnose HIV, chlamydia (CT), gonorrhoea (GO) and syphilis (SY) in a national cross-sectional survey in order to recognize potential problems and provide recommendations.</p> <p>Results</p> <p>A total of 739/2287 (32%) physicians participated. Of all participants, 80% offered tests for HIV, 84% for CT, 83% for GO and 83% for SY. Of all participants who performed HIV testing, 90% requested an antibody test, 3% a rapid test and 1% a nucleic acid amplification test (NAAT). For CT testing, NAAT was used in 33% and rapid tests in 34% of participants. GO resistance testing was performed by 31% of the participants. SY testing was performed in 98% by serology.</p> <p>Conclusions</p> <p>Diagnostic methods for STI vary highly among the participants. Diagnostic guidelines should be reviewed and harmonised to ensure consistent use of the optimal STI diagnostic methods.</p

German evidence and consensus‐based (S3) guideline: Vaccination recommendations for the prevention of HPV‐associated lesions

Anogenital and oropharyngeal infections with human papilloma viruses (HPV) are common. Clinically manifest disease may significantly impact quality of life; the treatment of HPV-associated lesions is associated with a high rate of recurrence and invasive neoplasms, such as cervical, anal, vulvar, penile, and oropharyngeal cancers, which are characterized by significant morbidity and mortality. Vaccination against HPV is an effective and safe measure for the primary prevention of HPV-associated lesions, but immunization rates are still low in Germany. The present publication is an abridged version of the German evidence and consensus-based guideline "Vaccination recommendations for the prevention of HPV-associated lesions", which is available on the website of the German Association of the Scientific Medical Societies (AWMF). On the basis of a systematic review with meta-analyses, a representative panel developed and agreed upon recommendations for the vaccination of different populations against HPV. In addition, consensus-based recommendations were developed for specific issues relevant to everyday practice. Based on current evidence and a representative expert consensus, these recommendations are intended to provide guidance in a field in which there is often uncertainty and in which both patients and health care providers are sometimes confronted with controversial and emotionally charged points of view