Doctoral Theses / Doktorsavhandlingar Institutionen för biomedicin

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3D Printed scaffolds as cancer microenvironment medels for drug discovery
(2022-04-05) Rosendahl, Jennifer
Cancer is one of the most common diseases in the modern world and major efforts are made globally to develop new diagnostics and treatments. It originates from a cell which, at some point, has begun to divide and grow uncontrollably. The most common type is breast cancer, and like all other cancers there is a need of more efficient drug therapies. Drug development is an expensive and time-consuming process and in conventional pre-clinical evaluation, drugs are tested on cells grown in 2D followed by experimental studies in animals. Only the drug candidates with best efficacy and safety profiles are allowed to proceed to clinical trials in humans. A major problem is that the pre-clinical test methods most often do not adequately represent the microenvironment in the human body and only a portion of the drugs that show good effect in pre-clinical studies pass the clinical trials and reaches market. Failures in late development mean large losses both financially and in time, and better pre-clinical test methods are needed that can predict more accurate results for safety and efficacy. The behavior of cancer cells is strongly influenced by the surrounding microenvironment, but today’s drug development focuses mainly on the cells themselves and does not sufficiently take this into account. This thesis combines 3D printing and cell biology to develop new and more representative test systems, with the ambition to mimic the tumor microenvironment in three dimensions. By using patient tumor tissue and removing the original cells, we produce a cell-free extracellular matrix scaffold to which standardized reporter breast cancer cell lines are reintroduced. The cell lines grown in the patient derived scaffolds developed more stem cell properties and formed a more heterogeneous cell population compared to 2D cultures. Moreover, the gene expression profile could be linked to clinical data, such as relapse. In an attempt to synthetically mimic the human tumor tissue, we used an alginate-based biomaterial to print 3D scaffolds. Breast cancer cells cultured in the 3D printed scaffolds showed a more similar growth- and gene expression pattern to cells cultured in patient derived scaffolds indicating that we were able to simulate the human tumor microenvironment. Further, we showed that the cells cultured in both patient derived scaffolds and 3D printed scaffolds had a similar response to hypoxic conditions – which is an important factor in tumors. Finally, we also showed that nanocellulose could be used to 3D print and that cells cultured in these scaffolds demonstrated comparable results to cells grown in alginate-based 3D printed scaffolds.
A search for prognostic biomarkers in diffuse large B-cell lymphoma with proteomics and immunohistochemistry
(2021-11-18) Bram Ednersson, Susanne
Diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in the Western world, can by gene expression profiling or immunohisto-chemistry (IHC), be divided into two subgroups according to its “cell-of-origin”. The subgroup ABC (or non-GCB) with similarities to active post-germinal centre B-cells, is associated with worse outcome. In addition, patients with primary refractory disease or early relapse have a very dismal prognosis. The aim of this thesis has been to identify novel prognostic biomarkers in a large retrospective DLBCL patient cohort by mass-spectrometry (MS)-based proteomics and IHC. Quantitative MS-based proteomics (QMS) revealed several differentially expressed proteins between refractory/relapsed patients (REF/REL) and patients with progression-free survival ≥5 years (CURED). Many ribosomal proteins were up-regulated in REF/REL patients while numerous proteins associated with the actin cytoskeleton were up-regulated in CURED patients. By using QMS we also found several up-regulated proteins in non-GCB DLBCL related to the tumour microenvironment, including interferon (IFN)-stimulated proteins. By using IHC we found a prognostic association for two proteins (CREBBP and TBLR1) that are frequently mutated in DLBCL, and for IFI16 and MNDA, both belonging to the pyrin and hematopoietic IFN-inducible nuclear (PYHIN) family. In conclusion, we have found increased expression of several proteins or groups of proteins not previously described in DLBCL and with potential prognostic impact. Further functional studies are warranted to elucidate their role in immunochemotherapy resistance.
A study of the contribution of mast cells to vaccination - Regulatory functions of Fc gamma receptors
(2013-08-23) Fang, Yu
This thesis aimed to explore the regulatory roles of mast cells in vaccination. Mast cells have been increasingly recognized as important orchestrators of immune regulation in both health and disease, in addition to their classically defined roles in allergic diseases. One of the recently appreciated beneficial roles of mast cells is their involvement in augmenting the adjuvant effects that are critically important in successful vaccination. This study has focused on the interaction of mast cells with an adjuvant complex composed of IgG and CTA1-DD, a fusion protein consisting of the Al subunit of cholera toxin (CT) linked to a synthetic dimer of fragment-D of Staphylococcus aureus protein A (DD). As the DD domain unspecifically binds immunoglobulins, CTA1-DD and IgG form complexes potentially able to activate mast cells through Fc  receptors. Indeed, CTA1-DD, in combination with polyclonal IgG, induced mast cell degranulation and the production of TNF-alpha, a cytokine important for the maturation and migration of antigen presenting cells, resulting in enhanced antigen-specific immune responses following immunization. Furthermore, only connective tissue mast cells (CTMCs), but not mucosal mast cells (MMCs), were found to be activated by CTA1-DD/IgG complexes. This effect was mediated by Fc gamma RIIIA, an activating receptor that has been described to be only expressed on connective tissue mast cells. Indeed, Fc gamma RIIIA-expressing connective tissue mast cells were found in the nasal submucosa. Responses to immunization facilitated by CTA1-DD/IgG were compromised in Fc gamma RIIIA-deficient mice, and in mice pre-treated with a CTMC inhibitor. Interestingly, MMCs, which were present in mouse nasal mucosa, were not entirely bystanders in CTA1-DD/IgG-mediated adjuvanticity. We discovered that a balanced expression of Fc gamma RIIB and Fc gamma RIIIA was required for mast cells to resist apoptosis mediated by IgG immune complexes. Therefore, MMCs, which only expressed Fc gamma RIIB, but not Fc gamma RIIIA, underwent apoptosis as a result of treatment by CTA1-DD/IgG. MMCs were capable of phagocytosing ovalbumin (OVA), and engulfment of these MMCs by antigen presenting cells (APCs) could occur if the MMCs were induced to apoptose. Finally, the APCs were able to present OVA peptide to OVA-specific T cells. Thus, MMCs may also contribute to vaccination through cross-presentation. Safety is always a prioritized concern for developing adjuvants, especially when mast cells are involved. Remarkably, CTA1-DD did not function as a superantigen to activate mast cells which had captured IgE molecules with their Fc epsilon RI, indicating that CTA1-DD is safe for use in allergic patients in which mast cell Fc epsilon RI is occupied by antigen-specific IgE molecules. Furthermore, CTA1-DD/IgG immune complexes administered intranasally did not trigger systemic anaphylaxis. In conclusion, CTA1-DD/IgG may target both CTMCs and MMC through Fc gammareceptors to enhance antigen-specific immune responses, probably through two distinct mechanisms. We propose that IgG immune complex-induced mast cell activation may be considered as a component of rationally designed mucosal adjuvants.
Activation and immunoregulatory function of type II natural killer T lymphocytes
(2013-05-02) Rhost, Sara
Natural killer T (NKT) lymphocytes make up a potent immunomodulatory subset of innate-like lymphocytes. NKT cells are activated by self-lipids presented by the unconventional MHC class I-like molecule CD1d, resulting in the rapid production of a range of different cytokines, that modulate innate and adaptive immunity. NKT cells possess regulatory properties in several immune setting such as autoimmunity, infection and cancer. However, the activation of NKT cells is not fully understood. In this thesis, we have addressed the role of self-lipids for type II NKT cell activation and autoreactivity, and employed self-lipids to investigate the immunoregulatory function of type II NKT cells in murine disease models. The glycosphingolipid (GSL) sulfatide has previously been shown to be a stimulatory self-ligand for type II NKT cells. Sulfatide exists naturally as a mixture of different isoforms and is abundant in organs such as the central nervous system, gastrointestinal tract, kidneys and the pancreas where it has important functions. We demonstrate that naturally existing isoforms, including C24:1 sulfatide and lyso-sulfatide, activate type II NKT cells. Organ specific isoforms in particular, but not non-physiological isoforms, of sulfatide induced efficient activation of type II NKT cells. Despite the potent activation of NKT cells by natural sulfatide isoforms, the autoreactivity of the type II NKT cells to CD1d-expressing cells was not dependent on sulfatide production by the stimulatory cells, demonstrating that other self-lipids were causing autoreactivity. In a search for such lipids, isolated from stimulatory cells, we identified two novel NKT cell activating self-GSLs, glucosylceramide and galactosylceramide and defined their stimulatory isoforms. However, by using antigen presenting cells deficient in all GSLs we could demonstrate that the autoreactivity of the type II NKT cells did not require GSLs. In summary, we demonstrate that natural isoforms of sulfatide, glucosylceramide and galactosylceramide are ligands for type II NKT cells, suggesting that they may play a role to activate type II NKT cells upon increased exposure in autoimmunity or tumor immunity. We also find that the CD1d-dependent natural autoreactivity of the type II NKT cells depends on lipids other than GSLs. Sulfatide is present in pancreatic -cells that are targets for autoimmune destruction in type I diabetes (T1D). We demonstrate immune reactivity to sulfatide in non-obese diabetic mice that spontaneously develop TID. However, treatment of these mice with sulfatide, to activate immunomodulatory type II NKT cells, did not confer protection from TID. In contrast, we found that sulfatide treatment significantly improved the survival rate of mice with Staphylococcus aureus sepsis. The protective effects mediated by sulfatide required CD1d but not type I NKT cells, suggesting that activated type II NKT cells ameliorated sepsis development. Protection was associated with reduced serum levels of pro-inflammatory cytokines and improved platelet counts. In conclusion, our results provide novel information on the activation of type II NKT cells, and expands our understanding of their immunomodulatory capacity to improve disease outcome.
Acute and chronic peritonitis in peritoneal dialysis: neurogenic inflammation and citrate treatment
(2009-10-06T12:40:47Z) Cavallini, Nicola
The prevalent problems associated with peritoneal dialysis (PD) are ultrafiltration failure and peritonitis. During PD the patient is sustained on a state of intraperitoneal inflammation, which over time impairs structure, and function of the peritoneal membrane, leading to loss of ultrafiltration efficacy. The aims of this project was: to establish whether neurogenic inflammation and mast cell activation are triggered by PD fluid exposure and to evaluate the effects of citrate as an additive to PD fluid in acute and chronic animal models. The studies were conducted in rats, exposed to filter sterilised lactate or lactate/citrate buffered PD fluid with glucose (2.5 and 3.9 %) as osmotic agent through an implanted catheter. Acute studies were based on single exposure and long-term studies on daily exposures for a period of 5 weeks. Pharmacological intervention was used to study mast cell activation and the neurogenic inflammatory response. Histamine was released into the peritoneal cavity within 30 minutes of infusion of standard PD fluid. Also osmotically neutral fluid triggered a histamine release from mast cells. Indirect evidence for the release of neuropeptides SP and CGRP suggested actions of a neurogenic inflammation. Mast cell activation was shown to be dependent on substance P stimulation of its receptor, NK-1. Inhibiting NK-1 significantly reduced vascular albumin loss from the blood to the peritoneal cavity by a mast cell independent mechanism. Blocking CGRP resulted in a significant increase in osmotic and net ultrafiltration. The classic trigger of neuropeptide release, the TRPV1 receptor was, unexpectedly, not responsible for neuropeptide actions in the present model. Substituting 5-15 mM lactate with equal amounts of citrate gradually improved osmotic ultrafiltration (fluid transport) compared with lactate PD fluid, suggesting a dose-response relationship. Significantly improved net ultrafiltration (fluid gain) was the result of increased osmotic ultrafiltration, in response to 10 - 15 mM citrate substitution. Long-term treatment with citrate-substituted PD fluid in rats did not significantly reduce fibrosis and angiogenesis of the peritoneal membrane compared with standard PD fluid. PD catheter patency was, however, significantly improved in animals treated with citrate substituted PD fluid. Macroscopic signs of fibrosis were also significantly reduced by citrate. The clinical implications are that pharmacological intervention with the neurogenic inflammatory response and calcium chelation with citrate have potential to improve the efficiency of peritoneal dialysis.
Acute febrile illness in preschool children in Zanzibar - Infectious aetiologies, diagnosis and treatment
(2019-09-02) Elfving, Kristina
Background: A majority of the three million children in Africa that do not survive their fifth birthday die from infections that often start as a seemingly uncomplicated febrile illness. Primary health care workers frequently encounter febrile children with a negative malaria rapid diagnostic test (mRDT), in particular in places like Zanzibar with a considerable decline in malaria prevalence. In recent years, accurate and sensitive molecular techniques like the polymerase chain reaction (PCR) have revealed increased detection of pathogens not only in ill patients but also in asymptomatic subjects. These factors underline the importance of re-evaluating the infectious disease aetiology and pathogen dynamics in febrile children and to assess whether existing diagnostic tools like mRDT and fever management guidelines like the IMCI (Integrated Management for childhood illness) remain useful and safe.Methods and Findings: The thesis is based on two field studies, both conducted on patients with acute uncomplicated febrile illness (by history or axillary temperature) in primary health care facilities April-July 2010 and 2011 in Zanzibar, Tanzania. In study 1 (paper I), 3890 febrile patients ≥2 months were included. Malaria prevalence by mRDT was 3.1%, with the highest prevalence, 6.1% in children aged 5-14 years old. Malaria microscopy and PCR were conducted on all mRDT positive and a randomly selected 20% of the mRDT negative patients. The sensitivities of mRDT versus malaria microscopy and PCR were below 80%, respectively. Study 2 (paper II-IV) included 677 febrile children aged 2-59 months of age that depending on the clinical picture were subjected to point-of-care tests, PCR analyses (on inclusion and day 14), urine culture and radiological analyses. For comparison, 167 geographically- and age-matched asymptomatic controls from the surrounding communities were recruited for selected PCR analyses. More than one pathogen was detected by PCR in 98% of patients and 93% of healthy controls. After application of study specific diagnostic criteria using clinical characteristics and laboratory results, including a comparison with detection in healthy controls, a cause of fever was assigned to 86%. The most common were respiratory syncytial virus (RSV), influenza A or B, rhinoviruses, enteroviruses, and S. pyogenes (Group A Streptococcus) (paper II and III). C-reactive Protein (CRP) was the only variable significantly associated with radiological pneumonia. Antibiotics were prescribed to 74% of patients whereas 22% had an infection that required antibiotics (paper II). On follow-up after two weeks >80% of the infections were cleared, but almost half of the sampled patients had a new infection on day 14 (paper IV).Conclusion: The sensitivity of the malaria RDT was relatively low. Thus, more sensitive tools than histidine-rich protein 2 (HRP-2) based mRDTs are warranted. Most of the uncomplicated febrile illness in children in Zanzibar was caused by a viral respiratory tract infection. Comparison of pathogen detection in febrile and healthy children was crucial for identifying cause of disease. The accuracy of the IMCI guidelines to guide antibiotic prescription was suboptimal with both over- as well as underprescription of antibiotics. However, the study did not find any diagnostic tool to help in guiding antibiotic prescription although C-reactive Protein might be a promising biomarker for future intervention studies. Respiratory infections usually cleared within two weeks. However, many children had acquired a new viral infection, suggesting that prolonged symptoms often are due to acquisition of new infections rather than to persistence.
Acute gastroenteritis in Rwandan children under five years of age investigated by real-time PCR
(2014-02-04) Kabayiza, Jean-Claude
Acute gastroenteritis is a major cause of illness and death among children in developing countries. Knowledge about the aetiology is important to make the right priorities regarding preventive measures, and for the recommendation to use or not use antibiotics. The objective of this thesis was to investigate causes of acute diarrhea in children in Rwanda by real-time PCR targeting a wide range of infectious agents. By analysing 326 paired faecal samples we found that rectal swabs provided equal rates of PCR detection of 10 different pathogens as usual stool samples, and correlating Ct values indicated that rectal swabs also may be used for quantitative measurements. PCR findings in 544 children with acute diarrhea and 162 controls showed a higher prevalences in children with than without diarrhea only for rotavirus and the enterotoxigenic E. coli (ETEC-estA) (42% vs. 2%, and 21% vs. 9%). Other agents were detected at similar rates in sick and healthy children (adenovirus, 39% vs. 36%; ETEC-eltB, 29% vs. 30%, Campylobacter, 14% vs. 17%, Shigella, 13% vs. 10%). Lower Ct values for ETEC-estA, Shigella and norovirus GII indicate that measuring pathogen concentration in faeces may help to identify clinically relevant infections. At least one pathogen was detected in 92% of 880 children with diarrhea. Rotavirus and ETEC-estA were associated with more severe dehydration, Shigella with bloody diarrhoea and higher CRP, and concentrations in faeces of rotavirus, ETEC-estA and Shigella were associated with more severe symptoms. Rotavirus and ETEC-estA were more common in younger, Shigella more a common in older children. Antibiotics were given to 42% of children, mainly those with fever and more severe dehydration, and without any logical connection with the causative organism. The conclusions of this thesis are (i) that rectal swabs are as good as conventional stool samples for pathogen detection by PCR, (ii) that rotavirus, ETEC-estA and Shigella were the major causes of gastroenteritis, (iii) that higher concentrations of rotavirus, ETEC-estA, Shigella and norovirus GII were associated with symptoms, and that Ct value cut-offs for these agents improved identification of them as causes of disease, (iv) that antibiotics were used extensively and in a seemingly irrational manner, and (v) highly sensitive multiple real-time PCR was efficient and informative and that its use in future studies may provide valuable new information about the clinical significance and epidemiology of these infections.
Acute respiratory infections among children in the Democratic Republic of Congo - nasopharyngeal pathogens, antibiotic resistance and vaccination
(2020-09-29) Muhandule Birindwa, Archippe
ABSTRACT Acute Lower Respiratory Infections (ALRI) remain a leading cause of morbidity and mortality among children in the Democratic Republic of the Congo (DR Congo). The pneumococcal conjugate vaccine PCV13 was introduced in the in the South-Kivu region in 2013. The aim of this thesis was to investigate the epidemiologic of ALRI, nasopharyngeal bacteria and viruses, pneumococcal serotypes and antibiotic resistance among children after the PCV13 introduction. In paper I 2,007 children hospitalised with ALRI during 2010-2015 were retrospectively reviewed and the case fatality rate among these children was 5%. The number of severe ALRI cases per year decreased after the vaccine introduction, while the total number of ALRI cases per year remained unchanged. Five percent of the cases were treated with non-recommended, broad-spectrum antibiotics. In paper II, 794 children from the general population attending health centres during 2014 and 2015 were sampled from nasopharynx. The prevalence of pneumococci was higher among children who had not received PCV13, and among those who lived in a house with an open fire used for cooking and with open access to the living areas. Multi-resistance among the isolated pneumococci was high (43%), and almost all isolates were resistant to trimethoprim-sulfamethoxazole. Multiplex PCR performed directly on 375 of the nasopharyngeal samples (paper III), showed a high load of bacteria and viruses although respiratory syncytial virus (RSV) was rare. Approximately 50% of the pneumococci were identified to a serotype not included in PCV13. Paper IV included 116 hospitalised children with radiologically confirmed pneumonia. High levels of any virus or any bacteria in nasopharynx were associated with severe pneumonia, and having a congenital disease as an underling condition was associated with fatal outcome. Conclusions: There were a high prevalence of bacteria and viruses in the upper respiratory tract of both healthy and sick Congolese children, and the level of antibiotic resistance in carried pneumococci was high. There is a need to modify current treatment guidelines in DR Congo and to reduce the prevalence of pathogens by interventions, including improved living conditions.
Aloe barbadensis Mill. as a therapeutic option for irritable bowel syndrome – properties, bioactivity and mode of action
(2022-11-08) Ahluwalia, Bani
Irritable bowel syndrome (IBS) is a chronic and prevalent functional gastrointestinal disorder, with an incompletely understood pathophysiology. Because of the disease complexity and heterogeneity, the currently available treatment options for IBS are limited. These limitations have led to the popularity of alternative therapeutic strategies, such as the use of Aloe barbadensis Mill. (Aloe), despite the paucity of controlled clinical studies supporting efficacy of these treatment options. This thesis therefore aimed to determine the importance of intestinal microenvironment, as well as the therapeutic effects and potential mode of action of an Aloe gel derived extract in patients with IBS. An integrated faecal microbiota and metabolite profile, as a joint representative of the intestinal microenvironment, distinguished IBS patients from healthy subjects, and further established the role of an altered intestinal microenvironment in the pathogenesis of IBS. The overall safety of Aloe treatment in patients with IBS was confirmed and supported the beneficial treatment effect of Aloe gel extract in subsets of IBS patients, which may depend on gut microbiota composition and function. Further, a potential mode of action for the therapeutic effect of Aloe gel extract, including dampening of immune cell activity and modulating intestinal microenvironment, was proposed. Finally, with the help of metabolomics, we expanded the knowledge of the complex and synergistic bioactive composition of Aloe gel. In conclusion, this thesis strengthens the role of an altered intestinal microenvironment in the pathogenesis of IBS. Further, it supports the role of an Aloe gel derived extract as a therapeutic option for the symptom management of IBS.
Alphaherpesvirus infections of the central nervous system – Biomarkers, diagnostics and antiviral therapy
(2021-05-25) Lindström, Johan
Abstract Herpesviruses predate the evolution of humans and are globally ubiquitous. Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV) establish latency in neuronal tissue and may cause infections in the central nervous system (CNS). Despite advances in diagnostics and treatment, the disease burden remains high. The overall aims of this thesis were to explore and evaluate several aspects of HSV and VZV CNS infection, with the ultimate goal of improving clinical management, from diagnosis to treatment and prognostication. Paper I explores cerebrospinal fluid (CSF) biomarkers in 28 patients with facial palsy caused by VZV. Biomarker expression was consistent with neurological damage and astrogliosis. This pattern was more pronounced in patients with concurrent mucocutaneous zoster rash than in those without rash, zoster sine herpete. Associations between biomarker concentrations and neurological outcomes could not be demonstrated. Paper II evaluates the CXCL13 CSF biomarker as a means of discriminating between VZV and Lyme Neuroborreliosis (LNB) in cases of facial palsy. CXCL13 concentrations were significantly higher in patients with LNB facial palsy (n = 21), though there was some overlap with cases of VZV facial palsy (n = 26). Despite good performance measures, especially if analyzed early after onset of symptoms, careful interpretation is advised when concentrations are moderately increased. Paper III assesses the performance of the FilmArray Meningitis/Encephalitis (ME) panel. The ME panel is a multiplex PCR panel for syndromic testing in CNS infections, able to detect 14 pathogens, including herpesviruses and bacteria. ME panel results were compared with routine diagnostic procedures in 4199 CSF samples from patients with suspected viral CNS infection. Discrepant results were thoroughly investigated to determine whether PCR detection was correct. A high performance level was demonstrated in calculations on individual pathogens, but 21 false negative and 20 false positive results were identified. If herpes simplex encephalitis is suspected, additional testing is warranted despite negative HSV-1 results from the ME panel. Interpretation concerning positive enterovirus, HHV-6, and S. pneumoniae results may also be complicated due to false positive or clinically insignificant results. Paper IV is a pharmacokinetic study of acyclovir and its metabolite CMMG in 21 patients with acute CNS infection. Renal function, damage to the blood-brain barrier, dosage, and body weight all influenced CSF concentrations of both molecules. Acyclovir-induced neuropsychiatric symptoms (AINS) were unexpectedly identified in four patients, together with high CSF concentrations of CMMG, previously implicated in neurotoxicity. These results justify increased attention to suspected neuropsychiatric symptoms and careful consideration of dosages in acutely ill patients.
Analysis of Binding Events and Diffusion in Living Cells
(2007-03-06T19:27:03Z) Elsner, Markus
It is well known that diffusion is the main mode of transport in living cells, but the consequences of diffusion in a complex cellular environment are not generally appreciated. In this thesis, we have investigated several aspects of how diffusion properties influence the observability of cellular binding kinetics and how they can be used to obtain information about the environment of proteins and other molecules. First, the binding kinetics of the coat protein I (COPI) vesicles machinery were investigated. Three proteins are mainly responsible for the formation of COPI vesicles; coatomer, ARF1 and ARFGap1. From biochemical studies, it was expected that ARF1 and coatomer would show similar binding kinetics to the Golgi membranes. This was tested in vivo using GFP constructs in “fluorescence recovery after photobleaching” (FRAP) experiments. Surprisingly, the recovery constant of coatomer was twice that of ARF1. We could show that this did not reflect a difference in the actual binding kinetics, but difference due to a diffusion-limited exchange of coatomer between the cytosol and the membrane. For this we measured the diffusion coefficient of all three proteins with fluorescence correlation spectroscopy (FCS). We found that ARF1 and ARF-GAP1 are highly mobile in the cytosol, whereas coatomer diffuses 5–10 times more slowly than expected. Using computer simulations we could show that the slow diffusion of coatomer translates into a two times slower FRAP recovery then expected for the non-diffusion limited case. Second, the unexpectedly slow diffusion of coatomer led to the idea of investigating the diffusion properties of inert tracers in the cytosol of livings cells. Fluorescently labelled dextrans showed normal diffusion in water, but strong anomalous subdiffusion when microinjected into cells. It could be ruled out that large scale structures like the cytoskeleton or the endoplasmic reticulum were responsible for the observed subdiffusion. Instead the emergence of subdiffusion could be attributed to macromolecular crowding using computer simulations and in vitro measurements in an artificially crowded solution. The fact that macromolecular crowding leads to anomalous diffusion can be used as a measure for the extent of crowding for a given solution. In the third part of this thesis the focus is shifted from diffusion in the cytosol to diffusion in the membrane. Previously, it had been observed in FCS experiments that Golgi resident transmembrane proteins show anomalous subdiffusion. Since no consistent explanation for this phenomenon had been provided previously, we investigated whether the formation of dynamic oligomers can explain the observed subdiffusion. We constructed a computer model for two dimensional diffusion of particles that participate in oligomerisation reactions. It could indeed been shown that for the short time scales relevant for FCS experiments, anomalous diffusion can be observed. For long times the diffusion crossed over to normal diffusion. The extent of anomality and the crossover time depended on the equilibrium constant of the binding, the valence of the monomers and on the kinetics of the binding reaction.
ANALYSIS OF NOVEL BIOMARKERS FOR UNFAVORABLE BREAST CANCER PROGNOSIS
(2012-11-26) Shubbar, Emman
Breast cancer is the most common malignancy in women, and a major cause of mortality and morbidity despite the advances in diagnosis and treatment. The main challenge remains to identify novel biomarkers in order to improve existing treatment modalities. Ductal carcinoma in situ (DCIS) is considered as a direct precursor of invasive breast cancer. Therefore, it would be valuable to be familiar with the natural history of DCIS, including how it develops, and if it will progress to invasive breast carcinoma. Hence, the identification of biomarkers associated with DCIS progression may prevent the development of some invasive breast cancer tumors. The expression of S100A7 (psoriasin) has previously been identified in association with the transition from DCIS to invasive breast cancer. It has also been associated with unfavorable clinical outcomes, suggesting that psoriasin may play a role as a biomarker of aggressive malignant behavior. The first part of the thesis was conducted to investigate a potential role of psoriasin in breast cancer. We demonstrated that the reduction of intercellular adhesion molecule 1 (ICAM-1) by short hairpin RNA in mammary epithelial cells induced the expression levels of psoriasin, via the phospholipase C (PLC)-IP3 pathway, along with the oncogenic protein mucin1 (MUC1) (Paper I). We have shown that psoriasin contributes to the expression of vascular endothelial growth factor (VEGF) and elevated expression levels of psoriasin in mammary epithelial cells leads to increased endothelial cell proliferation in a paracrine manner through receptor for advanced glycation endproducts (RAGE) by promoting oxidative stress response (Paper II). In the second part of the thesis, we evaluated the expression levels of several candidate biomarkers in order to allow stratification of breast cancer tumors according to their aggressiveness. Previously, we performed analysis of gene expression in 97 primary invasive diploid breast tumors and identified molecular gene signatures associated with poor clinical outcome. In Paper III, CCNB2, CDCA7, ASPM, KIAA0101, and SLC27A2 were selected from these gene signatures. We studied their protein levels in association to patient clinical outcome in an independent cohort of 80 primary invasive breast tumors. Our data indicated that cytoplasmic CCNB2 may serve as a novel biomarker of unfavorable clinical outcomes over short-term follow-up in breast cancer. In addition, in a previous study, we performed gene expression analysis in 43 axillary lymph node negative tumors and identified 51 genes whose deregulated mRNA levels were significantly associated with unfavorable clinical outcome. Four candidate biomarkers; GGH, FAAH, PIR and TAF5L were selected among the identified 51-gene signature (Paper IV). We investigated their clinical impact in predicting breast cancer progression in an independent cohort of 80 primary invasive breast tumors. Our data suggest that elevated protein levels of GGH were associated with unfavorable prognosis and poor outcomes in breast cancer patients.
Anaplastic lymphoma kinase activity, a therapeutic target, suppresses neuroblastoma cell differentiation
(2020-11-27) Siaw, Joachim Tetteh
Neuroblastoma (NB) is the most common extracranial pediatric solid malignancy caused by the failed differentiation of precursor cells of the developing sympathetic nervous system. NB accounts for about 15% of childhood cancer-related deaths. Treatment failure and relapse are common in NB patients despite intensive chemotherapy and immunotherapy interventions, suggesting the need for new and effective treatment options. Common genetic aberrations associated with NB include MYCN amplification, chromosome 11q deletion, 1p deletion, 17q gain, 2p gain, and recurrent mutations in Anaplastic Lymphoma Kinase (ALK). While treatment of some categories of ALK-positive pediatric cancer patients such as non-Hodgkin lymphoma and inflammatory myofibroblastic tumour (IMT) with the first-generation ALK tyrosine kinase inhibitor (TKI), crizotinib, produced promising results, the outcome for ALK-positive NB patients was less encouraging, hence the need for more potent ALK TKIs for treatment of NB patients. This thesis aimed to further our understanding of ALK signalling and its role in NB differentiation and explore novel ALK TKIs in a neuroblastoma setting. In the first study, we investigated the therapeutic efficacy of the second-generation ALK TKI, brigatinib, in an NB preclinical setting. Brigatinib was reported to be effective against ALK fusion-positive non-small cell lung tumours. We found that brigatinib potently inhibited both the activity of ALK full-length and growth of ALK-addicted NB cells in-vitro, in xenograft and Drosophila models. Compared to crizotinib, brigatinib inhibited the activities of different ALK-mutant alleles more effectively and potently inhibited crizotinib resistant ALK mutants in vitro. In the second study, we characterized a novel ALK-I1171T mutant allele which we identified in a tumour from a 16 month old NB patient. We showed that ALK-I1171T is a gain-of-function mutation, which is resistant to crizotinib, but can be effectively inhibited by second- and third-generation ALK TKIs such as brigatinib, ceritinib and lorlatinib. Based on these results and the severe toxic side effect of the initially administered chemotherapy, ceritinib monotherapy was chosen for this child. After 7.5 months of ceritinib treatment, the primary tumour shrunk in size and was removed surgically. The patient showed complete metastatic remission and remains in remission at 58 months post-treatment. In the third and last study, we investigated Disk large homologue 2 (DLG2), a gene reported to be uniquely upregulated in transient intermediary cells during Schwann cell precursor (SCP) differentiation to adrenal chromaffin cells. We found that DLG2, a gene located on the frequently deleted chromosome 11q in NB, is an NB tumour suppressor gene whose expression is lost in NB cell lines. Restoration of DLG2 expression inhibited NB cell growth and promoted NB cell differentiation. High expression of DLG2 in NB tumours is associated with good prognosis. Mechanistically we showed that oncogenic ALK maintains an undifferentiated NB cell phenotype by repressing DLG2 expression via the ERK1/2-SP1 signalling cascade. In summary, these findings highlight the role of ALK in differentiation and therapeutic potential of targeting ALK in ALK-positive NB tumours.
An animal model of sport related concussive brain injury
(2008-10-02T10:13:34Z) Bolouri, Hayde
A new animal model for concussion of the type seen in professional football was developed, since current animal models did not simulate these conditions. The model is characterized by a high velocity-low mass impact to the head of a freely moving object. Structural damages and functional effects of the model have been investigated. Paper I describes the rat model. A pneumatically driven projectile impacted the temporal region of the head. A 50 g projectile matches the concussions in football players scaled to the rat. Exposures were also performed with a 100 g impactor. The pressure accelerated the projectile to velocities of 7.4 m/s, 9.3 m/s and 11.2 m/s. The head was protected with a padded aluminum helmet. A small accelerometer was attached on the opposite side of the head, inline with the impact, for recording the acceleration of the head. Rats were exposed to a single or repeated (3, with 6 hour intervals) impacts and were sacrificed 1, 4 or 10 days later. Peak head acceleration, ΔV, duration and energy transfer were determined. Brains were perfused and surface injuries identified. Skull fractures were never found. Impact velocity and head ΔV and acceleration were within 1% and 3% of the target. In paper II, neuronal injury was assessed with immunohistochemistry for NF-200, the heaviest neurofilament subunit, and GFAP, an intermediate filament protein in astrocytes. Hemorrhages were visualized with unspecific peroxidase. NF-200 immunoreactivity was accumulated in neuronal perikarya and was reduced in the axons 10 days after impact. Reactive astrocytes were found in the midline regions of the cerebral cortex and periventricularly. Erythrocyte-loaded blood capillaries indicated brain edema in regions of the cerebral cortex, brain stem and cerebellum. A single impact at 7.4 and 9.3 m/s with the 50 g projectile resulted in minimal neuronal injury and astrocytosis. Repeated impacts with the 100 g projectile at 11.2 m/s and 9.3 m/s led to injury bilaterally in the cerebral cortex, subcortical white matter, hippocampus CA1, corpus callosum and the striatum. The pattern of injury is suggestive of Diffuse Neuronal Injury (DAI). In paper III, cognitive function and exploratory behavior were investigated following repeated head impacts. Rats were trained daily for 6 days in the Morris Water Maze. The time of latency to find a hidden platform was reduced from 50 secs on day 1 to 15 secs on day 6. They were then exposed with the 50 g or the 100 g projectile at 9.3 or 11.2 m/s. Spontaneous exploratory activity was assessed with the open field test 2-4 days and 1 and 2 weeks after impacts with the 50 g projectile at 9.3 and 11.2 m/s. The results showed that rats exposed at 11.2 m/s (x3) with the 50 g projectile or 9.3 m/s (x3) and 11.2 m/s (x2) with the 100 g projectile had a significantly increased time of latency to the platform, while those exposed with the 50 g at 9.3 m/s did not differ from the controls. Rats impacted with 50 g (x3) at 9.3 or 11.2 m/s showed a significant decrease in spontaneous exploratory activity. In conclusion, the model fulfilled the conditions of concussion in the freely moving animal, without preparatory surgery, still with good reproducibility. Some aspects of the neuropathology and functional effects were investigated and both showed dose-response effects. The functional changes were cognitive deficits and reduced exploratory activity. Key words: Animal model, football, concussion, brain injury, neuropathology, cognitive function. ISBN Gothenburg 2008
Anti-herpes simplex virus activities of sulfomannan oligosaccharide PI-88 and disulfated cyclitols
(2007-06-20T08:07:29Z) Ekblad, Maria
Herpes simplex virus (HSV) initiates invasion of human cells by binding to the cell surface heparan sulfate (HS) glycosaminoglycan chains. This step is mediated by the viral envelope glycoproteins gC and/or gB. Sulfated polysaccharides are compounds that mimic the structure of HS chains, and therefore are capable of inhibiting HSV attachment to and subsequent infection of cells. However the high molecular weight and associated with it poor tissue-penetrating activity have limited potential antiviral application of sulfated polysaccharides in humans. Here we found that the HS mimetic PI-88, a sulfomannan oligosaccharide of low molecular weight, efficiently reduced, in contrast to conventional sulfated polysaccharides, the cell-to-cell spread of HSV. Analogues of PI-88 with chemical modifications based on the introduction of specific hydrophobic/aromatic group(s) at the reducing end of PI-88 oligosaccharide chain showed enhanced capability to inhibit infection of cells and the cell-to-cell transmission of HSV and respiratory syncytial virus (RSV). One of these analogues (denoted 536), prepared by modification of PI-88 with cholestanol group, exhibited in contrast to the parental compound an HSV-inactivating activity. Furthermore, several disulfated cyclitols, identified by screening for an anti-HSV activity of a large number of low molecular weight sulfated compounds, efficiently reduced the cell-to-cell spread of HSV and demonstrated the HSV-inactivating activity. The second aim of this thesis was to elucidate the molecular basis for viral resistance to PI-88. Variants of HSV type 1 (HSV-1) and type 2 (HSV-2), selected for by virus propagation in cultured cells in the presence of PI-88 were analysed. Many of these variants had a low infectious titer, indicative of a profound impairment in biological activities of the virus in response to continuous pressure from the drug. These variants were substantially resistant to PI-88 presence during their initial infection of cells and/or their cell-to-cell spread. Nucleotide sequence analysis revealed that PI-88 targeted predominantly the viral envelope glycoproteins that comprise mucin-like region(s), i.e., glycoprotein gC of HSV-1 and glycoprotein gG of HSV-2. The deletion of the mucin-like region of HSV-1 gC (amino acids 33-116) or the deletion of whole HSV-2 gG provided the virus with selective advantage to attach to and to infect cells in the presence of PI-88. In conclusion, we have identified several novel antiviral compounds. One of these compounds, the PI-88 analogue 536, seems to be an attractive candidate for the development of a topical virucide for prevention of genital HSV infections in humans. We have also identified a novel biological function of HSV-2 gG, i.e., its targeting by sulfated oligosaccharides, which suggests involvement of this protein in HSV-2 attachment to cells or in modulation of this step.
Antibiotic Resistance and Fitness of Escherichia coli in the Infantile Commensal Microbiota
(2007-05-14T10:39:55Z) Karami, Nahid
Microbial resistance to antibiotics is a growing problem worldwide. Resistance develops not only in microbes which are the targets of the antibiotic treatment, but also in those belonging to the normal microbiota of the treated host. Little is known on the ecological consequences of antibiotic resistance in commensal bacteria. Escherichia coli belongs to the normal intestinal microbiota but may also cause urinary tract infection (UTI) or septicaemia if spread from the bowel. The present thesis studies the prevalence and stability of antibiotic resistance among E. coli colonizing the gut of healthy infants and its impact on in vivo fitness. We also examine evidence for transmission of resistance genes between E. coli strains during simultaneous presence in the bowel microbiota. E. coli was isolated from faecal samples obtained at regular intervals from 128 Swedish infants. For comparison, a collection of 205 urinary E. coli isolates from 205 infants 0-2 years of age was examined. Individual E. coli strains were identified by RAPD, phylogenetic grouping and virulence gene carriage. Their phenotypic resistance to 14 clinically relevant antibiotics was examined and genes encoding resistance to tetracycline and ampicillin were identified. Twelve percent of commensal E. coli strains, but 40% of urinary isolates were resistant, most commonly to tetracycline, ampicillin or trimethoprim. Both tetracycline and ampicillin resistant E. coli strains were equally capable of persisting in the intestinal microbiota as susceptible strains and their resistance genes were mostly kept during the entire colonization period. Most resistant strains established in infants who had never received any antibiotics. Tetracycline resistant E. coli strains mostly belonged to phylogenetic group A and ampicillin resistant E. coli strains to phylogenetic group D. The tetracycline resistance gene tetA was associated with the iutA virulence gene, encoding aerobactin, while tetB and blaTEM encoding ß-lactamases was associated with papC, encoding P fimbriae. In two cases, we could demonstrate transmission of plasmids carrying blaTEM genes from ampicillin resistant E. coli strains to initially sensitive E. coli strains colonizing simultaneously in the infant’s bowel microbiota. Both donor strains belonged to phylogenetic group D. In the first case, the infant was treated with ampicillin, which enhanced the population counts of the donor strain and provided a selective pressure promoting the survival of the transconjugant strain. Further, mutation of the blaTEM promoter gene was observed in the donor strain, leading to a more effective expression of the blaTEM gene. In the second case, transfer of a plasmid conferring resistance to ampicillin, streptomycin and sulphonamide was demonstrated in an infant who was not treated with antibiotics. The donor strain belonged to the globally spread uropathogenic CGA clone. Our study is the first to examine the stability and fitness costs of resistance gene carriage in commensal bacteria in human hosts under natural conditions. Our results indicate that resistance genes are prone to spread among strains in the intestinal microbiota and that strains belonging to group D may be especially apt to participate in such gene transfer. Furthermore, the fitness cost of resistance gene carriage appears to be small and may be compensated for by simultaneous carriage of genes encoding colonization- promoting factors, such as P fimbriae and aerobactin.
Antibiotic resistance in the environment: a contribution from metagenomic studies
(2016-05-03) Bengtsson-Palme, Johan
Antibiotic resistance accounts for hundreds of thousands of deaths annually, and its projected increase has made the WHO recognize it as a major global health threat. In the last decade, evidence has mounted suggesting that the environment plays an important role in the progression of resistance. The external environment acts as a source of resistance genes for human pathogens, but is also an important dissemination route allowing the spread of resistant bacteria between different environments and human populations. In this thesis, large-scale DNA sequencing techniques are used to gain a better understanding of the risks associated with environmental antibiotic resistance. A key task in this process is the quantification of the number of antibiotic resistance genes in different environments using metagenomics. However, equally important is to put this information into a larger perspective, by including, for example, taxonomic data, concentrations of antibiotics present, and the genomic contexts of identified resistance genes. This thesis presents a software tool – Metaxa2 – for improved taxonomic analysis of shotgun metagenomic data, which is shown to give more accurate taxonomic classifications of short read data than other tools (Paper I). It also provides theoretically predicted no-effect concentrations for 111 antibiotics (Paper II), and experimentally determined minimal selective concentrations for tetracycline (Paper III). Furthermore, resistance genes are quantified in two environments suggested to pose selective conditions for resistance: sewage treatment plants (Paper IV) and a lake exposed to waste from pharmaceutical production (Paper V). There was no clear evidence for selection of antibiotic resistance genes in sewage treatment plants, however other factors such as oxygen availability seem to have much stronger effects on these microbial communities, which may mask small selective effects of antibiotics and other co-selective agents. In contrast, in the lake subjected to industrial pharmaceutical pollution, resistance genes and mobile genetic elements were both diverse and abundant. Finally, Paper VI shows that travel contributes to the spread of resistance genes against several different classes of antibiotics between countries with higher resistance rates and Sweden. In Paper IV–VI, the genetic contexts of resistance genes were assessed through metagenomic assembly, showing how different resistance genes are linked to each other in different environments. Through these means, the thesis contributes knowledge about risk settings for development and transmission of antibiotic resistance genes, which can be used to guide risk assessment and management schemes to delay or reduce clinical resistance development.
Antibiotic use and respiratory pathogens with focus on Streptococcus pneumoniae in Tanzanian children
(2023-03-30) Emgård, Matilda
This thesis describes the epidemiology of Streptococcus pneumoniae (pneumococci) and other respiratory pathogens after introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) and further portrays antibiotic use in Tanzanian children. Pneumococci are a leading cause of pneumonia in children. However, respiratory infections among children may be associated with over-use of antibiotics leading to bacterial resistance, a significant threat to global child health. In a quantitative study, conducted in urban Moshi, Northern Tanzania 2013-2015, 775 children <2 years of age attending public primary healthcare facilities for routine care were sampled from the nasopharynx. Structured interviews with the parent/guardian revealed that more than half of the children had received antibiotics in the past 3 months. Isolated pneumococci (n=244) showed increasing resistance to phenoxy-methylpenicillin from 35% in 2013 to 60% in 2015, but resistance to amoxicillin, the first line pneumonia treatment, remained low (1%). Although vaccine-type pneumococci decreased significantly during the study period, the prevalence of residual vaccine-types remained high (21%). Detection of respiratory syncytial virus or adenovirus were associated with parent-reported rapid or difficult breathing and antibiotic treatment in the past week. A qualitative phenomenographic study was subsequently conducted in urban and rural Moshi in 2019. Individual in-depth interviews with primary healthcare workers showed a reliance on physical examination of the child and history from the mother when deciding whether to prescribe an antibiotic. However, their confidence in providing advice as to non-antibiotic treatment varied. Most mothers attending the focus group preferred seeking care for their sick child at healthcare facilities, but they faced barriers including unforeseen costs, travel, and lack of support from their husbands. Pharmacies were often perceived as cheap and convenient place to obtain antibiotics for children, whilst some mothers sought health advice from a trusted neighbour. Conclusions: Increasing resistance to antibiotics and residual vaccine-types require continued epidemiological surveillance of pneumococci in the post PCV13 era. Healthcare workers need support to develop their clinical and consultation skills, meanwhile mothers should be supported in seeking appropriate healthcare for their children. For this, improved equity and increased presence of community health workers are necessary.
Antiretroviral treatment of HIV-1 in Sweden with focus on virological aspects
(2021-02-11) Sörstedt, Erik
From a clinical standpoint, there are many factors to consider when optimizing the care for people living with HIV (PLWH). With help from clinical guidelines, most obstacles can be addressed. Expanded knowledge is however in constant demand, from local conditions to universal processes. This thesis emerged from a demand for both clinical and virological data about the effect of antiretroviral treatment (ART) in Sweden. All data were derived from the national InfCareHIV database. The current goal of ART is to achieve lasting suppression to < 50 HIV RNA copies/mL. Transient episodes of viremia up to 500 copies/mL, so-called viral blips, are not uncommon. We sought to investigate the clinical importance and outcome of this phenomenon. Through two large retrospective studies, Paper I and IV, we concluded that it is more common with blips in PLWH with higher baseline viral load and ART based on boosted Protease Inhibitors (PI). Blip incidence during Integrase Strand Transfer Inhibitors (INSTI) and Non- Nucleoside Reverse Transcriptase Inhibitor-based ART was lower at a similar level. In PLWH who reached HIV RNA suppression after initiating their first ART, blips were relatively common (10–20% of all participants) but not associated with an increased risk of virological failure. Before the introduction of the INSTI dolutegravir, PLWH with resistance mutations to Nucleoside Reverse Transcriptase Inhibitors were often restricted to PI-based treatment. PIs are characterized by many drug interactions and often tolerability issues. In Paper II, 244 participants with either dolutegravir or traditional PI-based ART were retrospectively studied. Dolutegravir has pharmacological benefits and we concluded that it was an equivalent alternative. Treatment recommendations are not affected by different levels of baseline viremia. Most clinical studies compare the outcome in participants with higher or lower than 100,000 HIV RNA copies/mL. Considerably higher levels of viremia are sometimes observed. In Paper III, we included 2,956 PLWH of whom 394 (13%) had baseline > 500k HIV RNA copies/mL. We found that participants with that high initial viremia needed longer time to reach viral suppression. Initial treatment with INSTIs was associated with faster viral decline. Higher baseline viral load was not associated with an increased risk of virological failure.
Antiretroviral treatment of HIV-1 in the central nervous system
(2007-05-24T05:47:06Z) Yilmaz, Aylin
HIV-1 invades the central nervous system (CNS) early in the infectious course. It establishes a chronic progressive infection, and triggers an intrathecal immune response. If left untreated, a majority of patients will develop neurological complications, caused by opportunistic pathogens or HIV-1 itself. The most devastating manifestation of HIV-1 in the CNS is AIDS dementia complex (ADC), a subcortical dementia, occuring in about 20% of untreated patients. The incidence of neurological complications has decreased dramatically since the introduction of antiretroviral drugs. In order for these drugs to act in the CNS, they must penetrate the blood-brain barrier (BBB) into the cerebrospional fluid (CSF). It is, therefore, important to determine which agents have this capacity, and what impact they have on HIV-1 CNS infection. We analysed CSF concentrations of three protease inhibitors (PIs): lopinavir co-formulated with a low dose of ritonavir, and saquinavir in combination with nelfinavir. Lopinavir was detectable in 15/15 samples. The concentrations achieved were probably high enough for antiviral activity in the CSF, generally exceeding severalfold the concentration needed to inhibit viral replication by 50% (IC50). The concentrations of saquinavir were very low, and only detectable in 7/15 CSF samples. Nelfinavir was detectable in 9/15 CSF samples, with concentrations in the range of the IC50. Antiretroviral treatment (ART) containing lopinavir/ritonavir or saquinavir/nelfinavir significantly reduced plasma and CSF viral loads, as well as intrathecal cell-mediated immunoactivation, measured as decreasing levels of CSF neopterin and β2-microglobulin. HIV-1 has the capacity of establishing viral latency in resting memory CD4+ T-cells, making the virus impossible to eradicate with ART alone. Even in patients on effective ART, a low-level viral replication in plasma can be detected. This probably originates from latently infected cells. To determine whether there is a similar low-level viral replication in CSF, we used an HIV-1 RNA quantification assay with a detection limit of 2 copies/mL in 13 neurologically asymptomatic individuals on effective ART. All patients had CSF viral loads < 2 copies/mL. In plasma, 5/13 patients had levels ranging from 2.3 to 8.2 copies/mL. This makes it unlikely that the CSF in neurologically asymptomatic individuals acts as a viral reservoir in which HIV-1 can replicate independently from the periphery. CSF neopterin levels remain abnormal in many patients, despite a long period on successful ART. We retrospectively evaluated what influence various levels of CSF HIV-1 RNA, different antiretroviral regimens, and different levels of plasma viral load have on CSF neopterin levels in patients on effective ART. We found that patients with the lowest CSF viral loads (< 2.5 copies/mL) also had the lowest CSF neopterin concentrations. Subjects treated with PI- or non-nucleoside analogue-based regimens had CSF neopterin in the same range. Plasma HIV-1 RNA levels did not affect CSF neopterin levels. These findings indicate that the low-grade persistent intrathecal immunoactivation observed in treated patients is mainly driven by residual viral replication within the CNS. The more the antiretroviral regimen suppresses viral replication in the CNS, the less the intrathecal immunoactivation.

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