Cytotoxicity of Reparative Endodontic Cements on Human Periodontal Ligament Stem Cells

ABSTRACT Objective To compare the cytotoxicity of commercial reparative endodontic cements on human periodontal ligament stem cells (hPDLSCs). Material and Methods The culture of hPDLSCs was established. Cell density was set at 2 × 104 cells/well in 96-well plates. Extracts of Biodentine, Bio-C Repair, Cimmo HD, MTA Repair HP and White MTA were prepared. Then, the extracts were diluted (pure, 1:4 and 1:16) and inserted into cell-seeded wells for 24, 48, and 72 h to assess cell viability through MTT assay. hPDLSCs incubated with culture medium alone served as a negative control group. Data were analyzed by Two-Way ANOVA and Tukey's test (α=0.05). Results At 24 h, pure extract of MTA Repair HP and Biodentine 1:16 presented higher cell viability compared to control. Lower cell viability was found for pure extract of Cimmo HD, MTA Repair HP 1:4 and 1:16, and White MTA 1:16. At 48 h, pure extract of Bio-C Repair and MTA Repair HP presented higher cell viability compared to control. At 72 h, only the pure extract of MTA Repair HP led to higher cell proliferation compared to control. Conclusion Biodentine, Bio-C Repair and MTA Repair HP were able to induce hPDLSCs proliferation. Cimmo HD and White MTA were found to be mostly cytotoxic in hPDLSCs.

Terapia com células-tronco mesenquimais geneticamente modificadas para superexpressão de G6GSF e IGF-1 na doença de chagas crônica experimental / Therapy with genetically modified mesenchymal stem cells for overexpression of G6GSF and IGF-1 in experimental chronic chagasic disease

INTRODUÇÃO: A doença de Chagas é uma doença parasitária causada pelo Trypanosoma cruzi, a qual representa uma das principais causas de morbimortalidade na América Latina. As intervenções terapêuticas existentes não são totalmente eficazes, sendo o transplante cardíaco a única alternativa para os pacientes com cardiopatia chagásica crônica (CCC) grave. Neste sentido, a ausência de terapias capazes de atuar diretamente sobre os determinantes fisiopatológicos da doença torna necessária a identificação de novas abordagens terapêuticas. Estudos previamente realizados pelo nosso grupo mostraram que a utilização de célulastronco obtidas da medula óssea e de outras fontes teve efeitos benéficos no tratamento da CCC experimental. A possibilidade de potencializar os efeitos parácrinos das células-tronco através de modificação genética tem sido alvo de investigações científicas. OBJETIVO: Avaliar os efeitos da terapia com células-tronco mesenquimais (CTMs) da medula óssea, modificadas geneticamente para superexpressar o fator estimulador de colônias de granulócitos (hG-CSF) ou o fator de crescimento semelhante à insulina 1 (hIGF-1) em um modelo experimental de CCC. MATERIAL E MÉTODOS: Camundongos C57BL/6 foram infectados com 1000 tripomastigotas da cepa Colombiana de T. cruzi e, após seis meses de infecção, foram tratados com CTM, CTM-G-CSF, ou CTM-IGF-1. Grupos de animais não infectados ou infectados tratados com salina (veículo) foram utilizados como controles. Todos os animais foram eutanasiados sob anestesia após dois meses de tratamento para análises histopatológicas e morfométricas do coração ou músculo esquelético, bem como para avaliação da expressão de citocinas inflamatórias. RESULTADOS: As secções de corações de camundongos dos grupos tratados com CTM, CTM-GCSF ou CTM-IGF-1 apresentaram redução significativa do número de células inflamatórias e do percentual de fibrose em comparação aos animais chagásicos tratados com salina, sendo esta diferença mais evidente no grupo que foi tratado com células-tronco que superexpressam o G-CSF. Além disto, a terapia com CTM-G-CSF induziu a mobilização de células imunomoduladoras para o coração, tais como células supressoras de origem mielóide (MDSC) e células T regulatórias Foxp3+ que expressam IL-10. A avaliação da expressão gênica das citocinas inflamatórias no tecido cardíaco mostrou um aumento das citocinas inflamatórias em animais chagásicos crônicos quando comparados aos controles não infectados, sendo a maioria delas moduladas de forma significativa nos grupos que foram tratados com CTM ou CTM-G-CSF. Apesar da terapia utilizando CTM-IGF-1 não ter apresentado benefício adicional ao tecido cardíaco comparado ao grupo que foi tratado com CTM não modificadas, foi observado um efeito regenerativo desta terapia no músculo esquelético dos animais, resultando em um aumento de fibras musculares esqueléticas 60 dias após o tratamento. CONCLUSÃO: Nossos resultados demonstram que o tratamento com CTM da medula óssea que superexpressam hG-CSF ou hIGF-1 potencializou o efeito terapêutico das CTMs através de ações imunomoduladoras e pró-regenerativas no coração e músculo esquelético de camundongos cronicamente infectados por T. cruzi. Desse modo, a modificação genética de CTMs para superexpressão de fatores com potencial terapêutico representa uma estratégia promissora para o desenvolvimento de novas terapias para a cardiomiopatia chagásica crônica

INTRODUCTION: Chagas' disease is a parasitic disease caused by Trypanosoma cruzi, which is one of the main causes of cardiovascular morbidity and mortality in Latin America. Existing therapeutic interventions are not fully effective, and heart transplantation is the only alternative for patients with severe chronic Chagas' heart disease. In this sense, the absence of therapies capable of acting directly on the pathophysiological determinants of the disease demonstrates the necessity of identifying new therapeutic approaches. Studies previously conducted by our group demonstrated that the use of stem cells obtained from bone marrow and other sources had beneficial effects in the treatment of experimental chagas disease. Additionally, the possibility of enhancing stem cell paracrine effects through genetic modification has been the subject of scientific investigations. OBJECTIVE: To evaluate the effects of genetically modified mesenchymal stem cell therapy to overexpress granulocyte colony stimulating factor (hG-CSF) or insulin-like growth factor 1 (hIGF-1) in an experimental model of Chagas disease. MATERIAL AND METHODS: C57BL/6 mice were infected with 1000 trypomastigotes from the Colombian strain of T. cruzi and, after six months of infection, were treated with CTM, CTM-G-CSF, or CTM-IGF-1. Groups of uninfected or infected animals treated with saline (vehicle) were used as controls. All animals were euthanized under anesthesia after two months of treatment for histopathological and morphometric analysis of the heart or skeletal muscle, as well as for evaluation of inflammatory cytokine expression. RESULTS: Mouse heart sections from groups treated with CTM, CTM-GCSF or CTM-IGF-1 showed a significant reduction in the number of inflammatory cells and the percentage of fibrosis when compared to chagasic animals treated with saline.This difference was more evident in the group that was treated with stem cells overexpressing G-CSF. In addition, CTM-G-CSF therapy induced mobilization of immunomodulatory cells to the heart, including myeloid suppressor cells (MDSC) and Foxp3 + regulatory T cells expressing IL-10. Expression of inflammatory cytokine genes in cardiac tissue revealed an increase in inflammatory cytokines in chronic chagasic animals when compared to uninfected controls, where most cytokines were significantly modulated in groups treated with CTM or CTM-G-CSF. Although CTM-IGF-1 therapy demonstrated no additional benefit to cardiac tissue when compared to the group treated with unmodified CTM, a regenerative effect of this therapy was observed in chagasic mice skeletal muscle, resulting in an increase in skeletal muscle fibers 60 days after treatment. CONCLUSION: Our results demonstrate that bone marrow derived CTM treatment overexpressing hG-CSF or hIGF-1 enhanced the therapeutic effects of MSCs through immunomodulation and proregenerative actions in the heart and skeletal muscle of mice chronically infected wthT. cruzi. Thus, the genetic modification of CTMs for overexpression of factors with therapeutic potential represents a promising strategy for the development of new therapies for chronic chagasic cardiomyopathy

Geração e caracterização de linhagens de células-tronco mesenquimais de camundongo geneticamente modificadas para expressão / Generation and characterization of mousemesenchymal stem cells lines genetically modified for the ectopic expression of hG-CSFand hIGF-1ectópica de hIGF-1 ou hG-CSF

As células-tronco mesenquimais (CTM) constituem uma ferramenta promissora para o campo de terapia celular. Além de seu potencial de diferenciação em diferentes tipos celulares, as CTM apresentam a habilidade de secretar moléculas bioativas e, assim, exercer múltiplos efeitos biológicos, tais como indução da regeneração de tecidos lesionados, redução de fibrose e modulação do sistema imune. A superexpressão dos fatores de crescimento G-CSF e IGF-1, conhecidos por seus efeitos sobre os processos de imunomodulação, sobrevivência celular e reparo tecidual, pode ampliar as ações terapêuticas das CTM. O objetivo deste trabalho consiste em gerar e caracterizar linhagens de CTM de camundongo superexpressando hGCSF ou hIGF-1. Um sistema lentiviral de segunda geração foi utilizado para modificação de CTM para expressão ectópica dos genes de interesse. As sequências codificantes de hG-CSF e hIGF-1 foram amplificadas por PCR e subclonadas em um vetor lentiviral de transferência, contendo um promotor constitutivo. As partículas lentivirais foram produzidas a partir da cotransfecção de células da linhagem HEK293FT...

Mesenchymal stem cells (MSCs) are a promising tool for the cell therapy field. In addition to their potential for differentiation into different cell types, MSCs have the ability to secrete bioactive molecules and thus exert multiple biological effects such as induction of the injured tissue regeneration, fibrosis reduction and modulation of the immune system. The overexpression of the growth factors G-CSF and IGF-1, known for their effects on immune modulation processes, cell survival and tissue repair, can result in a magnification of MSCs' therapeutic actions. The objective of this work is to generate and characterize mouse MSCs lines overexpressing hG-CSF or hIGF-1. A second generation lentiviral system was used to modify MSCs derived from mice for the ectopic expression of the genes of interest. The coding sequences of hG-CSF and hIGF-1 were amplified by PCR and subcloned into a lentiviral transfer vector containing a constitutive promoter. The lentiviral particles were produced from the co-transfection of HEK293FT...

Stem cells and androgen receptor in human periapical periodontitis / Células madre y receptor de andrógenos en periodontitis periapical humana

Activation of macrophages in periapical granulomas occurs through the presence of cytokines, endotoxin and other genetic and epigenetic factors, allowing the initiation of inflammation and bone resorption. The present study aims to analyze the presence of CD133 protein (marker of stem cells) and the AR (androgen receptor) protein in biopsies of human odontogenic periapical granuloma. Biopsies from 14 adult male patients with diagnosis of periapical granuloma included in paraffin blocks were processed histologically to obtain 5-um thick sections. Protein presence was detected and analyzed by immunohistochemistry of CD133 and AR. The quantification considered the number of positive cells in 0.17 mm2 random areas under the microscope using a 1000X objective. Both CD133 and AR proteins are expressed abundantly in cells in pathological periapical granulomas tissue. The number of cells expressing CD133 and AR shows a wide variation coefficient, so its variation is a particular feature for each individual. We concluded that in human odontogenic periapical granuloma there are abundant stem cells and cells expressing AR that may be important for the pathogenic inflammatory process.

La activación de los macrófagos en los granulomas periapicales humanos se producen a través de la presencia de citoquinas, endotoxinas y otros factores genéticos y epigenéticos que permiten la iniciación de la inflamación y la reabsorción ósea. El presente estudio pretende analizar la presencia de proteína CD133 (marcador de células madre) y de la proteína RA (receptor de andrógenos) en las biopsias de granulomas periapicales odontogénicos humanos. Las biopsias de 14 pacientes varones adultos con diagnóstico de granuloma periapical fueron incluidos en bloques de parafina y se procesaron histológicamente para obtener secciones de 5 micras de espesor. La presencia de CD133 y RA fueron detectadas y analizadas por inmunohistoquímica. La cuantificación se realizó considerando el número de células positivas en áreas al azar de 0,17mm2, utilizando microscopio con objetivo de 1000X. Ambas proteínas, CD133 y RA se expresan en abundancia en las células del tejido patológico con granuloma periapical. El número de células que expresan CD133 y RA presentan un amplio coeficiente de variación, por lo que su variación es una característica particular de cada individuo. Se concluye que en granuloma periapical odontogénico humano se expresan abundantes células madre y proteínas receptoras de andrógenos, antecedentes que pueden sermuy importantes en la expresión y diagnosis de los procesos patológicos inflamatorios.

Células tronco do tecido adiposo de equinos: Estudo do seu potencial para o tratamento da endometrose / Stem cells from equine adipose tissue Study of their potential for the treatment of endometrosis

A aplicação terapêutica de Células Tronco ( CT) em equinos é um campo emergente. Nestes animais, as CT são promissoras para o tratamento de lesões nos tendões e rupturas de ligamentos...

In horses, Stem Cell (SC) therapies are a promising tool to the treatment of many injuries, as tendon lesions and ligaments rupture...

Células madre; Estado de situación: Perspectiva bioética

La trascendencia que ha adquirido la Medicina Regenerativa en relación a las denominadas terapias celulares, y más específicamente, terapias con Células Madre, generan planteos, éticos que no debemos soslayar. Como Ministro de Salud he encomendado a la Comisión Provincial de Bioética, el análisis del estado de situación actual de lo progresos científicos/tecnológicos con relación a las implicancias éticas que conllevan las propuestas de tratamientos clínicos regeneraticos a través trasplantes de células madre, autólogos o alogénicos...

Avaliação do potencial terapêutico do transplante de células-trono mesenquimais na cardiopatia chagásica crônica experimental / Evaluation of the therapeutic potential of transplantation of mesenchymal cells throne in experimental chronic Chagas' disease

A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, é uma das mais importantes causas de insuficiência cardíaca na América. A terapia celular vem sendo investigada como uma possível opção terapêutica para pacientes com doenças cardiovasculares. Neste estudo foram avaliados os efeitos da terapia com célulastronco mesenquimais em um modelo experimental de cardiomiopatia chagásica crônica. Camundongos C57BL/6 foram infectados com 1000 tripomastigotas da cepa Colombiana de T. cruzi e, após seis meses de infecção, foram tratados com célulastronco mesenquimais derivadas de tecido adiposo humano (CTTAs) ou com meio DMEM (controle). Antes e após 1 e 2 meses de tratamento, os animais chagásicos e controles normais foram submetidos à avaliação cardíaca, incluindo eletrocardiograma, ecocardiograma e teste ergoespirométrico. O grupo tratado recebeu duas injeções intraperitoneais de CTTAs (1x106 células / dose), com um mês de intervalo entre as duas doses. Todos os animais foram sacrificados sob anestesia após 2 meses de tratamento, para análise histopatológica do coração. Não foi observada melhora de arritmias e da função cardiovascular no grupo tratado com CTTAs, porém secções de corações de camundongos deste grupo apresentaram uma redução significativa do número de células inflamatórias (p< 0,0001 ) e da área de fibrose (p< 0,01) em comparação com animais chagásicos tratados com DMEM. A dosagem de 22 citocinas séricas dois meses após o tratamento mostrou um aumento da maioria destas citocinas em animais chagásicos crônicos quando comparados aos controles não-infectados, sendo algumas destas moduladas após a terapia celular. Deste modo, conclui-se que as CTTAs foram capazes de reduzir inflamação e fibrose no coração de camundongos cronicamente infectados por T. cruzi, porém não teve efeitos na função cardíaca dois meses após o transplante.

Trasplante de células troncales: mecanismos de acción para sus usos potenciales en neurología / Stem cells transplant: potentially useful mechanism for neurology

Stem cells have a great potential for the treatment of presently incurable neurological diseases, including spinal trauma, cerebrovascular pathology, brain tumor and neurodegenerative processes, such as Parkinson and Alzheimer's disease, Huntington, multiple sclerosis and amyotrophic lateral sclerosis. Aims: To discuss the characteristics of the various stem cells types having been proposed for cell therapy, and the biological mechanisms responsible for their therapeutic effects. Report: Stem cells can be induced to differentiate into specialized cells such as neurons and glial cells, and they can influence the environment around them, both through the secretion of neurotrophic factors and immunomodulation of the host neuroimmune response. Furthermore, the understanding of the modulatory effect of stem cells could lead to the development of new therapeutic paradigms. Nevertheless, two important limitations of the field are that the ideal source for stem cells is not well defined yet and the mechanism of stem cell mediated functional improvement is not well understood. Conclusions: Research is currently focused on the biological mechanisms of stem cells therapy and the assessment of stem cell programming and delivery to the target regions. Furthermore, future research will increasingly target ways to enhance effectiveness of the stem cell therapy, including its combination with gene therapy. Regardless its enormous potentials, there are still many problems to be solved before clinical application of stem cell therapy can de used in neurological disease patients.

Introducción: Las células troncales tienen un gran potencial para el tratamiento de enfermedades neurológicas actualmente incurables, incluyendo el trauma espinal, patología cerebrovascular y procesos neurodegenerativos como el Parkinson, Alzheimer, Huntington, esclerosis múltiple o la esclerosis lateral amiotrófica. Objetivo: Discutir las características de diversas células troncales que han sido propuestas para terapia celular, y los mecanismos biológicos responsables de sus efectos terapéuticos. Desarrollo: Las células troncales pueden ser inducidas a diferenciarse en células especializadas como neuronas y células gliales, y pueden influenciar su entorno, tanto a través de la secreción de factores neurotróficos como por la inmunomodulación de la respuesta neuroinmune. La comprensión del efecto modulador de las células troncales podría orientar el desarrollo de nuevos paradigmas terapéuticos. Sin embargo, dos limitaciones importantes que persisten son, que la célula troncal ideal aún no está bien definida, y que los mecanismos que median la mejoría inducida por ellas no se comprende bien. Conclusiones: La investigación se enfoca actualmente en los mecanismos biológicos de la actividad terapéutica de las células troncales, en la evaluación de la programación celular y en su acceso a las regiones blanco. La investigación futura se dirigirá progresivamente a encontrar formas de aumentar la efectividad de las células troncales, incluyendo su combinación con terapia genética. Sin embargo, aún existen numerosos problemas que resolver antes que la terapia con células troncales pueda ser usada en pacientes con enfermedades neurológicas.

Células madre: fuentes no embriónicas accesibles / Stem cells: non-embryonic sources accessible

En los últimos años se ha variado en gran manera el tipo de enfoque que se le dio en algún momento a las células madre; estas han pasado de ser sólo una posibilidad lejana de tratamiento a covertirse en una alternativa real para el manejo de varias patologías dentro de las cuales destacan las de índole autoinmune. La obtención de las mismas no debe ser motivo de controversia en nuestro país ya que por métodos que no involucran embriones humanos es posible colectarlas de dos fuentes particulares y accesibles: 1) cordón umbilical 2)tejido adiposo...

Prometeo: regeneración tisular: pifias de la naturaleza y esperanzas de la ciencia / Prometheus: tissue regeneration: natural errors and scientific hope

En el movisimo campo de las células troncales ("stem cell") y la medicina regenerativa, la búsqueda del Santo Grial de la investigación científica, es la recreacion o la inducción del renacimiento de un órgano funcional, llamese hígado, retina o riñon. En un extremo el hígado de Prometeo, de inherente capacidad para regenerarse, la demanda del órgano excede en mucho la disponibilidad de donantes. En Estados Unidos de America, 9% de los pacientes con insuficiencia hepática fallecen a la espera de un trasplante, así que la investigación en tratamientos regenerativos nunca ha tenido más fundamento y adquirido más énfasis que en estos tiempos. En el otro extremo, la retina, inexplicable olvido de la naturaleza, como otras neuronas del sistema nervioso incapaz de regenerarse a sí misma y hasta ahora, inmune a la neuroprotección y reparación después de una injuria. No obstante en un futuro no muy lejano será posible preservar y restaurar la visión en personas en las que se encuentre amenazada o se haya perdido por enfermedad o injuria del nervio óptico

In the newest field of stem cells and regenerative medicine, the quest for the Holy Grail of scientific research is the recreation or induction of rebirth of a functional organism, such as the liver, retina o kidney. At one end liver of Prometheus with its inherent capacity to regenerate, has a demand that greatly exceeds the availability of donors. In the United States of America, 9% of patients with liver failure die waiting for a transplant, so research in regenerative treatment has never had more importance or acquired greater emphasis than at this time. At the other end, the retina, inexplicable oblivion of nature, is, as other neurons of the central nervous system, unable to regenerate itself and so far, immune to neuroprotection and repair after an injury. Nevertheless, in the future it will be possible, however, to preserve and restore vision in people whose optic nerve are threatened or have been lost due to illness or injury

Isolation of Endothelial Progenitor Cells from Cord Blood and Induction of Differentiation by Ex Vivo Expansion

Endothelial progenitor cells (EPCs) have been reported to possess the capacity to colonize vascular grafts and hold promise for therapeutic neovascularization. However, limited quantities of EPCs have been the major factor impeding effective research on vasculoangiogenesis. In this study, cytokine and culture conditions necessary for the provision of large quantities of endothelial cells (ECs) were investigated. Cord blood was collected from 18 normal full-term deliveries and CD34+ cells were isolated by MACS system (Miltenyi Biotech, Bergish-Gladbach, Germany). To evaluate the effect of cytokines, CD34+ cells were cultured with various cytokine combinations, such as stem cell factor (SCF), flt3-ligand (FL), and thrombopoietin (TPO) with vascular endothelial growth factor (VEGF), interleukin-1beta, fibroblast growth factor-basic (FGF-b) as basic cytokines. The quantities of non-adherent and adherent cells were the greatest with SCF, FL and TPO. The addition of TPO to all other cytokines significantly increased the number of non-adherent and adherent cells (p< 0.05, Wilcoxon rank sum test). After four weeks of culture, adherent cells expressed endothelial specific markers such as KDR, CD31 and CD62E. Typical morphology of ECs was observed during culture, such as cord-like structure and cobblestone appearance, suggesting that the adherent cells were consistent with ECs. In this study, the experimental conditions that optimize the production of ECs for therapeutic neovascularization were described. And it was possibly suggested that TPO plays a major role in differentiation from EPCs to ECs.

Intramyocardial Transplantation of Circulating CD34+ cells: Source of Stem Cells for Myocardial Regeneration

This study was designed to investigate the increase in the number of circulating CD34+ cells after acute myocardial infarction (MI) and the differentiation of these cells to cardiomyocytes after transplantation into infarcted myocardium. The study involved five donor groups: MI (n=27), sham (n=26), granulocyte-colony stimulating factor (GCSF) (n=26), MI+GCSF (n=25), and control (n=25). Acute MI was induced by ligating the left anterior descending coronary arteries (LAD) of donor rats, and LAD of recipient rats were ligated on the same day. Seven days after ligation, CD34+ cells in donor rats were counted and then were directly injected into the infarcted myocardium of recipient rats. Eight weeks after the transplantation, significant differences (p<0.001) were observed in the CD34+cell counts among the 5 donor groups with the greatest increase in the MI+GCSF donor group. In rats receiving CD34+ cells, the size of the scar area smaller (p<0.001) and the thickness of the scar was greater (p=0.001) than in CD34- and saline-transplanted rats. The transplanted CD34+ cells differentiated into cardiomyocytes in the scar. This study suggests that CD34+ cells may be a potential source of stem cells and that they may be useful in strategies aimed at the regeneration of infarcted myocardium.

Alteraciones de la célula madre totipotencial en la génesis de las enfermedades inmunohematológicas: a propósito de la disgenesia reticular / Defects of the stem cell totipotential in the genesis of the immunohemetological diseases: a propos of the reticular disgenesia

Las alteraciones del Stem cell o célula madre totipotencial están asociadas a la génesis de diferentes enfermedades inmunohematológicas, una de ellas es la Disgenesia Reticular. Esta enfermedad es inusual con escasos reportes mundiales a la fecha no ha sido explicado su mecanismo fisiopatogénico, aunque se ha establecido que está comprometida la diferenciación celular de las series linfoide, mieloide y eritroide, así como una alta susceptibilidad a infecciones virales, bacterianas y fúngicas desvastadoras, con muerte temprana en ausencia de transplante de células totipotenciales hematopoyéticas. En nuestros archivos de biopsia y autopsia de 23 años, encontramos un caso de Disgenesia Reticular. Destacamos la necesidad de insistir en el estudio exhaustivo de autopsias en todo niño cuya causa de muerte esté asociada a transtornos inmunitarios y en el estudio micro y macroscopico de todos los órganos de la economía

The effect of serum from patients of chronic renal failure on colony forming units (CFU-S) in mice.

Repopulating ability of mouse bone marrow stem cells, treated with pre-dialysis, post-dialysis and control sera was assessed by colony forming units (CFU-S). Significant lower colony counts were observed in pre-dialysis group as compared to control group. There was an improvement in the colony counts when the cells were treated with post-dialysis sera. The study suggests the presence of inhibitor/s of CFU-S in the uraemic sera which is/are partially removed by haemodialysis.