| 21. |
- Alenius Dahlqvist, Jenny, 1972-
(författare)
-
Heart rate variability and pacemaker treatment in children with Fontan circulation
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Fontan surgery is performed in children with univentricular heart defects. Arrhythmias are frequent complications, occasionally requiring pacemaker treatment. Previous data regarding indications and risk factors for pacemaker treatment in Fontan patients is limited and conflicting. Heart rate variability (HRV) reflects autonomous nervous activity controlling the sinus node and has been associated with tachyarrhythmias in both adults and children, as well as in adults with sinus node dysfunction (SND).Aim: To study HRV, arrhythmia and pacemaker treatment in children with Fontan circulation— with the purpose of contributing to the reduction of long term complications in this patient group.Methods: We have retrospectively reviewed pacemaker therapy in all Swedish patients who underwent Fontan surgery from 1982 to 2017 (n=599). We have also analysed HRV from 24-hour Holter ECG recordings in 112 children with Fontan circulation and in children with univentricular heart defects before bidirectional Glenn (BDG) procedure (n=47), before and on completion of Fontan surgery (n=47 and 45 respectively). Analysis was performed by power spectral analysis and Poincaré method, and results compared with healthy controls. Furthermore, HRV was analysed in Fontan patients who later required a pacemaker due to severe SND. Results were compared with Fontan patients who had SND, without indication for pacemaker treatment, with patients with Fontan circulation without SND and healthy controls. In addition we evaluated the possibility to analyse arrhythmias and HRV in 27 Fontan children using intermittent ECG recordings with a handheld devices at home during a 14-day period.Results: After a mean follow-up of 12 years, 13% (78/599) of patients with Fontan circulation had received a pacemaker. Patients operated with the extracardiac conduit (EC) had a significantly lower prevalence of pacemaker implantation (6%) than patients with a lateral tunnel (LT) (17%). The most common pacemaker indication in patients with Fontan circulation was SND (64%). Children with Fontan circulation showed significant reductions in several HRV parameters, compared with controls. No significant differences were found between patients operated with LT versus EC (paper I). After BDG the RR interval and SD2 (representing changes in heart rate over 24-hours) significantly increased compared to pre-BDG. Compared with healthy controls, patients post-BDG, had significantly longer RR intervals and reduced overall HRV. PHF (reflecting parasympathetic control of the heart) was significantly reduced after TCPC as compared to before (paper II). Fontan patients with SND showed significantly elevated SD2 (representing changes in heart rate over 24-hours), somewhat reduced in patients that later required a pacemaker (Paper V). Handheld ECG analysis revealed frequent ventricular extra systoles in one patient and episodes of supraventricular tachycardia in another. Seven Fontan patients showed reduced HRV recorded with the handheld device over a 14-day period (paper III).Conclusions: Overall HRV was reduced in patients with univentricular heart defects during the different surgical stages of Fontan surgery, compared to healthy controls. HRV was reduced in both patients with LT and EC with no significant difference between them. After BDG heart rate was significantly reduced as compared to before. PHF, reflecting the parasympathetic innervation of the heart was reduced after as compared to before TCPC. Pacemaker treatment is commonly needed in patients with Fontan circulation, and SND was the most prevalent indication for implantation. The prevalence of Fontan patients requiring pacemaker treatment was significantly lower in patients with EC. HRV analysis can contribute to management when following-up patients with Fontan circulation.
|
|
| 22. |
- Alenius, Gerd-Marie, 1957-
(författare)
-
A Clinical and Genetic Study of Psoriatic Arthritis
- 2003
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Psoriatic arthritis (PsA) is an inflammatory joint disease associated with psoriasis. PsA has a heterogeneous pattern, expressed by different manifestations such as mild mono-oligoarthritis or very severe, erosive and destructive polyarthritis. Measurable inflammatory activity is not always prominent. The aetiology is unknown but genetic factors are believed to be of importance. The pattern of inheritance is proposed to be polygenic. The aim of this study was to estimate the prevalence of joint and axial manifestations, characterise the disease in relation to inflammatory and genetic markers, and to identify disease susceptibility gene(s) for PsA in patients from northern Sweden. All patients from the city of Umeå (n=276), selected from a community and hospital based psoriasis register (n=1737) at the Dept of Dermatology, were invited to a prevalence study. Two hundred-two patients were examined and 97 (48%) had inflammatory manifestations such as peripheral arthritis, axial disease, undifferentiated spondylarthropathy (uSpA) and enthesopathies. Of the 67 patients (33 %) with peripheral arthritis and/or axial disease, 30 were not previously diagnosed. The association of clinical manifestations and potential markers of aggressive joint disease with HLA associations were analysed in 88 patients with PsA. We were not able to confirm findings of other groups reporting strong association with several HLA-antigens. The prevalence of HLA-B17, B37 and B62 was increased compared with controls, but the strongest predictive factors among our patients for an aggressive disease, in a multiple logistic analysis, were polyarthritic disease and distal interphalangeal engagement. In order to investigate for disease susceptibility genes, five genetic loci were analysed with microsatellites and single nucleotide polymorphisms in an association study of 120 patients with PsA. There was a significant association with the TNFB locus on chromosome 6p but not with any other loci examined; 1q21 (PSORS4), 3q21 (PSORS5), 8q24 and CTLA4. When stratifying for the TNFB alleles the association was confined to allele 123. In a subgroup of patients who were HLA-typed (n=83), we were not able to verify linkage disequilibrium with the TNFB allele 123 and the HLA antigens; B17, B27, B37, B62 or Cw*0602. The presence of renal abnormalities was evaluated as a manifestation of systemic inflammation in 73 patients with PsA. Renal abnormalities defined as decreased creatinine-clearance (≤ mean - 2SD) and/or urinary albumin >25 mg/24 h was found in 23% of the patients. The predictive factors for renal abnormalities was inflammatory activity (ESR > 25 mm/h and/or CRP >15 mg/L) indicating a systemic effect in some of the patients. In conclusion, we found high prevalence of inflammatory manifestations in patients with psoriasis. There was no strong association between PsA and HLA antigens and predictive factors for aggressive disease were polyarthritic disease and DIP joint engagement. The TNFB locus was associated with PsA and there were no linkage disequilibrium with the HLA antigens B17, B27, B62 or Cw*0602. There were evidence for systemic effects as renal abnormalities in patients with PsA and measurable inflammatory activity.
|
|
| 23. |
- Aléx, Lena
(författare)
-
Äldre människors berättelser om att bli och vara gammal tolkade utifrån genus- och etnicitetsperspektiv
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The overall aim of the five studies that make up this thesis is to elucidate constructions of being old from the perspectives of gender and ethnicity. One of the studies uses quantitative data and four use qualitative data.The sample in study I consisted of 125 participants from the Umeå 85+ study, aged 85 to 103 years old, who were able to use Likert scales in responding to questions. Studies II and III involved content analysis of interviews with old persons scoring on the extremes of the resilience scale. In study IV, interviews with nine Sami women were analysed using grounded theory. In study V, four interview situations were subjected to discourse analysis.Study I showed statistically significant correlations between the scales measuring resilience, sense of coherence, purpose in life and self-transcendence. These scales were supposed to measuring a common dimension, which is here interpreted as “inner strength”. There was a significant correlation between women’s “inner strength” and perceived mental health.The femininities found were associated with “being connected”, “being an actor”, “living in the shadow of others” and “being alienated”. The masculinities found were associated with “being in the male centre”, “striving to maintain the male facade” and “being related”. The femininity associated with “being an actor” and the masculinity associated with “being in the male centre” were pronounced in those participants assessed as having high resilience.Old Sami woman were found to be balancing within various discourses, including being a reindeer owner versus not owning reindeer, being Sami versus being Swedish, speaking in Sami versus speaking in Swedish, dreaming about the past versus looking to the future, being equal to men versus living in the shadow of the male herders, and changing for survival versus striving to retain uniqueness as a Sami.Study V revealed that shifts in power between the interviewer and the interviewed can be related to the discourses of age, gender, education, body, ethnicity and ideology.This thesis presents a complex picture of what it means to be among the oldest old. The ageing, gendered and ethicised selves cannot be seen as socially and culturally fixed. For the women, the femininity expressed in “being connected” involved being satisfied, content and having positive relationships. “Being an actor” involved a stress on the person’s own strength and own choices. The femininities experienced as “living in the shadow of others” and “being alienated” generated narratives about dissociation and loneliness. For the men, it seemed important to relate to themselves and to other men. However, the masculinity expressed in “being related” involved an alternative form of masculinity, focusing on the importance of daily work, new relationships, and reflecting on the meaning of life. The Sami women showed strength in being able to position themselves between various discourses, but their narratives also showed tender sadness when they spoke of their longing for the past and for their mother tongue. The reflection on how narratives are constructed by both the interviewed and the interviewer in relation to their access to various discourses of age, gender, education, ethnicity and ideology in different interview situations can be important for increasing awareness of the role of these discourses. Various ways of constructing femininities and masculinities must be studied if we are to avoid ageism developing in society. Analyzing and reflecting on the importance of age, gender and ethnicity from a constructivist perspective may reduce stereotypical descriptions of the oldest old.
|
|
| 24. |
- Aliashkevich, Alena, 1990-
(författare)
-
Molecular mechanisms and biological consequences of the production of non-canonical D-amino acids in bacteria
- 2021
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Most bacteria possess a vital net-like macromolecule – peptidoglycan (PG). PG encases bacteria around the cytoplasmic membrane to withstand the high internal turgor pressure and thereby protect the cell from bursting. In addition, PG is a major morphological determinant of bacteria being both required and sufficient to maintain cell shape. During cell growth PG hydrolysis and synthesis are tightly controlled to keep proper cell shape and integrity at all times. Given the essentiality of PG for bacterial growth and survival, the synthesis of this polymer is a major target of many natural and synthetic antibiotics (e.g. penicillins, glycopeptides).For a long time, PG composition was considered to be conserved and static, however it’s now being recognized as a dynamic and plastic macromolecule. The structure and chemistry of PG is influenced by a myriad of environmental cues that include interkingdom/interspecies interactions. Recently, it was found that a wide set of non-canonical D-amino acids (D-amino acids different from D-Ala and D-Glu, NCDAAs) are produced and released to the extracellular milieu by diverse bacteria. In Vibrio cholerae these NCDAAs are produced by broad-spectrum racemase enzyme (BsrV) and negatively regulate PG synthesis through their incorporation into PG. We have shown that in addition to D-Met and D-Leu, which were reported previously, V. cholerae also releases high amounts of D-Arg, which inhibits a broader range of phylogenetically diverse bacteria. Thus, NCDAAs affect not only the producer, but might target other species within the same environmental niche. However, in contrast to D-Met, D-Arg targets cell wall independent pathways. We have shown that non-proteinogenic amino acids also can be racemized by Bsr. A plant amino acid L-canavanine (L-CAN) is converted into D-CAN by a broad-spectrum amino acid racemase (BSAR) of the soil bacterium Pseudomonas putida and subsequently released to the environment. D-CAN gets highly incorporated into the PG of Rhizobiales (such as Agrobacterium tumefaciens, Sinorhizobium meliloti) thereby affecting the overall PG structure, bacterial morphogenesis and growth fitness. We found that detrimental effect of D-CAN in A. tumefaciens can be suppressed by a single amino acid substitution in the cell division PG transpeptidase penicillin-binding protein 3a (PBP3a). Rhizobiales are a polar-growing species that encode multiple LD-transpeptidases (LDTs), enzymes that normally perform PG crosslinking, but that can also incorporate NCDAAs into termini of the PG peptides. As these species incorporate high amounts of D-CAN in their PG, we hypothesized that LDTs might represent the main path used by NCDAAs to edit A. tumefaciens’ PG and cause their detrimental effects. Therefore, we decided to further explore the significance of LDT proteins for growth and morphogenesis in A. tumefaciens. While in the Gram-negative model organism E. coli LDT proteins are non-essential under standard laboratory conditions, we found that A. tumefaciens needs at least one LDT for growth out of the 14 putative LDTs encoded in its genome. Moreover, clustering the LDT proteins based on their sequence similarity revealed that A. tumefaciens has 7 LDTs that are exclusively present among Rhizobiales. Interestingly, the loss of this group of LDTs (but not the rest) leads to reduced growth, lower PG crosslinkage and rounded cell phenotype, which suggests that this group of Rhizobiales- specific LDTs have a major role in maintaining LD-crosslinking homeostasis, which in turn is important for cell elongation and proper shape maintenance in A. tumefaciens.
|
|
| 25. |
- Alvehus, Malin, 1975-
(författare)
-
Obesity-associated inflammation in adipose tissue
- 2012
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Excess body fat, particularly in the visceral depot, is linked to increased mortality and morbidity, including the development of diseases such as type 2 diabetes, cardiovascular disease, and cancer. Chronic low-grade inflammation in adipose tissue may be a key mediator of obesity-associated diseases. Importantly, specific pro-inflammatory cytokines have been shown to influence adipose tissue function and could therefore be a link to metabolic disorders. Circulating cytokine levels may also be increased in obesity and metabolic diseases. However, although fat distribution and inflammation are clearly linked to metabolic disorders, inflammatory gene expression in the different abdominal adipose depots has not been investigated in detail. The menopausal transition is followed by a centralization of body fat and increased adiposity. Notably, inflammatory changes in fat during the menopausal transition have not been characterized. Finally, there is a lack of studies investigating the long-term effects of weight loss on low-grade inflammation. The aim of this thesis was to characterize differences between fat depots and investigate putative changes in low-grade inflammation in adipose tissue and circulation following menopause or weight loss. Materials & Methods: The expression of inflammation-related genes was investigated in abdominal adipose tissue depots obtained from women with varying adiposity, before and after menopause or weight loss induced by surgery or dietary intervention. Circulating cytokine levels were analyzed using immunoassays. Results: Visceral fat displayed a distinct and adverse inflammatory profile compared with subcutaneous adipose tissues, and the higher gene expression in visceral fat was associated with adiposity. Postmenopausal women exhibited a higher expression of pro-inflammatory genes than premenopausal women that associated with central fat accumulation. There was also a menopause-related increase in circulating cytokine levels in postmenopausal women. After surgery-induced weight loss, there was a dramatic reduction in inflammatory gene expression followed by increased insulin sensitivity. We observed no alterations in circulating cytokine levels. Long-term dietary intervention, associated with weight loss, had favorable effects on inflammation in both adipose tissue and serum. Conclusion: Fat accumulation is linked to low-grade inflammation in abdominal adipose tissue. The unique inflammatory pattern of visceral fat suggests a distinct role in adipose tissue inflammation that is aggravated with increasing adiposity. In postmenopausal women, the adverse adipose inflammatory profile was associated with central fat accumulation, while higher circulating cytokine levels correlated with menopausal state/age. Our data from severely obese women undergoing surgery-induced weight loss clearly supports a link between adipose inflammation and insulin resistance. The long-term beneficial effects of weight loss were also demonstrated by the improved inflammatory profile after dietary intervention. In summary, excess body fat is clearly linked to adipose tissue inflammation. Long-term weight loss is accompanied by improved metabolic profile and reduced low-grade inflammation in fat.
|
|
| 26. |
- Amani, Paul Joseph, 1975-
(författare)
-
Does health insurance contribute to improving responsiveness of the health system? : the case of elderly in rural Tanzania
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Financing healthcare in Tanzania has for years depended on out-of-pocket payments. This mechanism has been criticized as being inefficient, contributing to inequity and high cost as well as denying access to healthcare to those most in need, including the elderly in rural areas. Health insurance (HI) was recently introduced as an instrument to enable equitable access to healthcare and thus to improve the responsiveness of the health system. Even though health insurance is expected to bring benefits to those who are insured, there is a lack of specific studies in the country looking at the role of HI in facilitating the health system responsiveness among vulnerable populations of remote areas.Aim: The aim of this thesis is to understand if and how health insurance contributes to improving the responsiveness of the healthcare system among the elderly in rural Tanzania. Methods: Four interrelated sub-studies (2 quantitative and 2 qualitative) were conducted in Igunga and Nzega districts of Tabora region between July 2017 and December 2018. The first two sub-studies are based on a household survey using an adapted version of the World Health Organization’s Study on Global Ageing and Adult Health questionnaire. Elderly people aged 60 years and above who had used both outpatient and inpatient healthcare three and twelve months prior to the study, respectively, were interviewed. Whereas in sub-study 1 the focus was to investigate the role of health insurance status on facilitating access to healthcare, sub-study 2 assessed the relationship between health insurance and the health system responsiveness domains. In sub-study 3, interviews with healthcare providers were carried out to capture their perspective regarding the functioning of the health insurance. In the final sub-study 4, focus group discussions with elderly were conducted in order to explore their experience of healthcare, depending on their health insurance status. Crude and adjusted logistic and quantile regression models were applied to analyse the association between health insurance and access to healthcare (sub-study 1) and responsiveness (sub-study 2), respectively. For both sub-studies 3 and 4, qualitative content analysis was used to analyse the data.Results: Sub-studies 1 and 2 involved a total of 1899 insured and uninsured elderly, while sub-studies 3 and 4 included 8 health providers and 78 elderlies respectively. Sub-study 1 showed that about 45% of the elderly were insured and HI ownership improved access and utilization of healthcare, both outpatient and inpatient services. In sub-study two, however, health insurance was associated with a lower responsiveness of the healthcare system. In general, all six domains: cleanliness, access, confidentiality, autonomy, communication, and prompt attention were rated high, but three were of concern: waiting time; cleanliness; and communication. Sub-study 3 uncovered several challenges coexisting alongside the provision of insurance benefits and thus contributing to a lower responsiveness. These included shortage of human resources and medical supplies, as well as operational issues related to delays in funding reimbursement. In sub-study 4, the elderly revealed that HI did not meet their expectations, it failed to promote equitable access, provided limited-service benefits and restricted use of services within residential areas. Conclusion: While HI seems to increase the access to and use of healthcare services by the elderly in rural Tanzania, a lower responsiveness by the healthcare system among the insured elderly was reported. Long waiting times, limited-service benefits, restricted use of services within schemes, lack of health workforce in both numbers and skills as well as shortage of medical supplies were important explanations for the lower responsiveness. The results of this thesis, while supporting the national aim of expanding HI in rural areas, also exposed several weaknesses that require immediate attention. There is a need to, first, review the insurance policy to improve its implementation, expand the scope of services coverage, and where possible, to introduce cross-subsidization between the publicly owned schemes; additionally, improvements in the healthcare infrastructure, increasing the number of qualified health workforce and the availability of essential medicines and laboratory services, especially at the primary healthcare facilities, should be prioritized and further investments allocated.
|
|
| 27. |
- Anderl, Ines, 1973-
(författare)
-
Activation of the Cellular Immune Response in Drosophila melanogaster Larvae
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During the last 40 years, Drosophila melanogaster has become an invaluable tool in understanding innate immunity. The innate immune system of Drosophila consists of a humoral and a cellular component. While many details are known about the humoral immune system, our knowledge about the cellular immune system is comparatively small. Blood cells or hemocytes constitute the cellular immune system. Three blood types have been described for Drosophila larvae. Plasmatocytes are phagocytes with a plethora of functions. Crystal cells mediate melanization and contribute to wound healing. Plasmatocytes and crystal cells constitute the blood cell repertoire of a healthy larva, whereas lamellocytes are induced in a demand-adapted manner after infection with parasitoid wasp eggs. They are involved in the melanotic encapsulation response against parasites and form melanotic nodules that are also referred to as tumors.In my thesis, I focused on unraveling the mechanisms of how the immune system orchestrates the cellular immune response. In particular, I was interested in the hematopoiesis of lamellocytes.In Article I, we were able to show that ectopic expression of key components of a number of signaling pathways in blood cells induced the development of lamellocytes, led to a proliferative response of plasmatocytes, or to a combination of lamellocyte activation and plasmatocyte proliferation.In Article II, I combined newly developed fluorescent enhancer-reporter constructs specific for plasmatocytes and lamellocytes and developed a “dual reporter system” that was used in live microscopy of fly larvae. In addition, we established flow cytometry as a tool to count total blood cell numbers and to distinguish between different blood cell types. The “dual reporter system” enabled us to differentiate between six blood cell types and established proliferation as a central feature of the cellular immune response. The combination flow cytometry and live imaging increased our understanding of the tempo-spatial events leading to the cellular immune reaction.In Article III, I developed a genetic modifier screen to find genes involved in the hematopoiesis of lamellocytes. I took advantage of the gain-of-function phenotype of the Tl10b mutation characterized by an activated cellular immune system, which induced the formation blood cell tumors. We screened the right arm of chromosome 3 for enhancers and suppressors of this mutation and uncovered ird1.Finally in Article IV, we showed that the activity of the Toll signaling pathway in the fat body, the homolog of the liver, is necessary to activate the cellular immune system and induce lamellocyte hematopoiesis.
|
|
| 28. |
|
|
| 29. |
- Andersson, Anne, 1966-
(författare)
-
Long-term side effects after treatment of Hodgkin's lymphoma
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background Long-term side effects associated with the treatment of Hodgkin’s lymphoma (HL) have frequently been reported during the last decades. Studies have shown increased mortality in HL survivors. Following Hodgkin’s lymphoma, second malignancies (SM) and cardiovascular disease (CVD) are the most common causes of death in individuals treated for HL. This study investigates the incidence of side effects such as SM, CVD and infections in a cohort diagnosed with HL in Sweden between 1965 and 1995. In addition, this study identifies covariate risk factors for late side effects in order to develop strategies that prevent morbidity and mortality in HL survivors. Methods Using the Swedish Cancer Registry (SCR) at the National Board of Health and Welfare and the Multi-Generation Registry at Statistics (MGR) Sweden, we identified 6946 individuals diagnosed with HL between the years 1965 and 1995, and their first degree relatives (FDR) (n=17 858). In addition we identified the malignancies and inpatient care for CVD and infections for the HL cohort and their FDR. The standard incidence ratio (SIR) was calculated for the risk of SM, CVD and infections. For SM and CVD the risk also was stratified and calculated for family history of disease. The Swedish Hodgkin Intervention and Prevention study (SHIP), a prospective study, invited 702 individuals treated for HL at the age of 45 years or younger and who were treated in the region of Skåne, Uppsala or Umeå. The participants completed a questionnaire and were invited to an out-patient visit to an oncologist with clinical examination and blood tests. Any pathological findings were referred for further investigation. Results An increased risk for SM in HL long-term survivors was observed and seems to increase with the number of FDRs with cancer. There was also an increased risk for inpatient care due to congestive heart failure (CHF) and coronary artery disease (CAD). A family history of CHF and CAD further increased the risk for these diseases. The risk for inpatient care due to infections was increased and remained increased after 20 years or longer. The risk for infections was associated with splenectomy and hypothyroidism. Radiotherapy was an independent risk factor for cardiovascular disease in the cohort of the prospective study. ConclusionLong-term survivors from HL have an increased risk for developing late side effects such as SM, CVD and infections. Since many HL patients are young and the cure rate from the disease is high, it is of great importance to offer focused surveillance programs to selected individuals who are at high risk, e.g. individuals who received radiotherapy as part of their treatment and who have other known risk factors for cardiovascular disease such as hypertension, hypercholesterolemia, family history and smoking.
|
|
| 30. |
- Andersson, Britta, 1979-
(författare)
-
Manipulation of potassium ion fluxes to induce apoptosis in lung cancer cells
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Apoptosis is a special form of cell death that if non-functional may lead to diseases such as cancer. A reduction of the intracellular potassium ion (K+) content is necessary for activating enzymes important for the execution of apoptosis. Pharmacological modulation of K+ fluxes to reduce intracellular K+ in cancer cells might therefore force the cells into apoptosis and decrease tumour cell mass. Human malignant pleural mesothelioma (MPM) is a form of cancer often caused by asbestos exposure. Although asbestos has been banned in the Western World, the incidence of MPM is expected to increase. Cisplatin is the first-line chemotherapy for MPM, but acquired resistance to the drug is a clinical problem. This thesis is mainly based on work with the human malignant pleural mesothelioma cell line (P31 wt) and a cisplatin-resistant sub-line (P31 res). The aim was to first characterize K+ fluxes in P31 wt and P31 res cells, and then manipulate them in order to reduce intracellular K+ and induce apoptosis with K+ manipulation alone or in combination with cisplatin. Characterization of K+ fluxes in P31 wt cells showed that: 1) ouabain, a digitalis-like drug, and specific blocker of the Na+, K+, ATPase pump, effectively inhibited K+ uptake, 2) bumetanide, a diuretic, and an inhibitor of the Na+, K+, 2Cl-¬-cotransporter, had a transient effect on K+ uptake, and 3) the antifungal drug amphotericin B stimulated K+ efflux. In order to determine intracellular K+ content, the potassium-binding fluorescent probe PBFI-AM was used in a 96-well plate assay. After a 3-h incubation with ouabain, with or without bumetanide, combined with amphotericin B, the intracellular K+ content was reduced in P31 wt cells but not in P31 res cells. Ouabain induced apoptosis in both P31 wt and P31 res cells. P31 res cells were sensitized to cisplatin by ouabain, since 10 mg/L cisplatin in combination with ouabain induced about the same percentage of apoptotic cells as 40 mg/L cisplatin. Apoptosis was executed via caspase-3 activation in both P31 wt and P31 res cells. Amphotericin B enhanced ouabain-induced apoptosis in P31 wt cells via caspase-9 activation, with increased caspase-3 activation and DNA fragmentation as consequences. Ouabain-induced apoptosis in P31 res cells was executed via increased expression of pro-apoptotic Bak. The combination of cisplatin with ouabain and amphotericin B was stressful to both P31 wt and P31 res cells, since SAPK/JNK a known factor in stress-induced apoptosis was activated. In conclusion, K+ flux manipulation with clinical used drugs can induce apoptosis per se and also enhance cisplatin-induced apoptosis in P31 wt and P31 res cells.
|
|
