Background: Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia. There is a need for new pharmacological treatment strategies since the current antiarrhythmic drugs have a modest efficacy and may have severe side effects. Cardioversion (CV) of AF offers an opportunity to study related conditions in sinus rhythm (SR) and during AF. Since catheter ablation of AF is a symptomatic treatment, it is important to have tools for measurement of arrhythmia-related symptoms. Aims: To evaluate the effect of atorvastatin on maintaining SR after CV of persistent AF. To assess if highsensitivity C-reactive protein (hsCRP) predicts the recurrence of AF after CV in a population randomized to treatment with either atorvastatin or placebo. To quantify the symptomatic effect of left atrial catheter ablation of AF. To assess if the restoration of SR by CV, in a population with persistent AF, affects sleep apnea. Methods: Paper I: A total of 234 patients were randomized to treatment with either high dose atorvastatin or placebo prior to CV. Paper II: In a pre-specified substudy which included 128 of the patients in study I, hsCRP was analyzed before and after CV. Paper III: Umea 22 Arrhythmia Questions (U22) is a questionnaire that quantifies paroxysmal tachycardia symptoms. A total of 105 patients underwent first-time pulmonary vein isolation and answered U22 forms at baseline and follow-up 304 (SD 121) days after ablation. Paper IV: Polysomnography was performed before and after CV in 23 patients with persistent AF scheduled for elective CV. Results: Paper I: An intention-to-treat analysis with the available data, by randomization group, showed that 57 (51%) in the atorvastatin group and 47 (42%) in the placebo group were in SR 30 days after CV (OR 1.44, 95%CI 0.85–2.44, P=0.18). Paper II: HsCRP did not significantly predict recurrence of AF at 30 days. However, after adjusting for treatment with atorvastatin, hsCRP predicted the recurrence of AF (OR 1.14, 95% CI 1.01–1.27). Six months after CV, hsCRP at randomization predicted recurrence of AF in both univariate analysis (OR 1.30, 95% CI 1.06–1.60) and in multivariate logistic regression analysis (OR 1.33, 95% CI 1.06– 1.67). Paper III: The U22 scores for well-being, arrhythmia as cause for impaired well-being, derived timeaspect score for arrhythmia, and discomfort during attack detected relevant improvements of symptoms after the ablation. U22 showed larger improvement in patients undergoing only one procedure than in patients who later underwent repeated interventions. Paper IV: Obstructive sleep apnea occurred in 17/23 patients (74%), and central sleep apnea in 6/23 patients (26%). Five patients had both obstructive and central sleep apnea. SR at follow-up was achieved in 16 patients. The obstructive apnea-hypopnea index, central apneahypopnea index, and the number of patients with obstructive or central sleep apnea did not differ before and after restoration of SR. Conclusions: Atorvastatin is not a treatment option with regards to maintaining SR after CV in patients with persistent AF. HsCRP was associated with AF recurrence 1 and 6 months after successful CV of persistent AF. U22 quantifies the symptomatic improvement after AF ablation with adequate internal consistency and construct validity. Both obstructive and central sleep apneas are highly prevalent in patients with persistent AF. Obstructive sleep apneas are unaffected by the CV of AF to SR.

>Magister Scientiae - MSc / Background: Malnutrition is a risk factor for the development of pulmonary tuberculosis (TB) and may be responsible for the premature deaths of patients with active disease. An adequate nutritional status may therefore be protective in delaying the onset from latent infection to active disease. In South Africa, very little data is available on the nutritional status of adults who present with tuberculosis. This study therefore aims to compare the nutritional status of newly diagnosed pulmonary tuberculosis patients with TB-free controls.Study population & Design: This is a community based case-control study. Forty-three newly diagnosed pulmonary tuberculosis patients were recruited as cases and matched according to age,gender and race to 43 TB-free close contacts. HIV positive subjects were excluded from the study.Methods: Each participant was interviewed and completed a structured questionnaire to obtain demographic information. Weight was measured to the nearest 0.1kg and height to the nearest 1mm. A 24-hr dietary recall method was used to obtain dietary information. Biochemical analysis was carried out to measure concentrations of transferrin, albumin, CRP, ferritin, zinc,copper, vitamin A and E.Results: The median Body Mass Index (BMI) for cases was 18.80kg/m² (IQR 14.35, 32.11) and TB-free contacts 21.17 kg/m² (IQR 16.75, 34.98) with a significant difference between the groups of p=0.001. There was significant difference in weight (p=0.002) and MUAC (p=0.000)between groups. No significant difference in dietary intake of energy (KJ) (p=0.695), protein(p=0.804), CHO (p=0.801) and fat ( p=0.796) was found between groups. There was a statistically significant increase in ferritin (p=0.000) and C-reactive protein (CRP) (p=0.000) in TB patients, while albumin (p=0.000), serum zinc (p=0.000) and serum vitamin A (p=0.000) were statistically significantly lower among cases. Conclusion: There was no significant difference in the macronutrient intake of TB cases and TB-free contacts, although a significant difference was seen in BMI, MUAC and weight between groups, with all these parameters being lower in TB patients. Ferritin and CRP levels were markedly increased in TB cases while serum zinc, vitamin A and albumin are all significantly lower in TB patients than TB free contacts.

The co-localization of plasma C-reactive protein (CRP) and atherogenic low-density lipoprotein (LDL) at the atherosclerotic lesions raises the possibility of a role of CRP in the disease process. In vitro, in its native pentameric structural form, CRP does not bind to oxidized LDL. However, in its non-native pentameric structural form, CRP is capable of binding to oxidized LDL. It has been proposed that CRP changes its structure at sites of inflammation to gain the oxidized LDL-binding activity. In vivo, native CRP is neither pro-atherosclerotic nor atheroprotective in animal models of atherosclerosis. We assumed that native CRP shows no effect because inflammatory microenvironment in the arterial wall in animal models of atherosclerosis is not appropriate and, therefore, the structure of CRP remains unchanged. Accordingly, we hypothesized that a CRP mutant, generated by site-directed mutagenesis, capable of binding to oxidized LDL without the requirement of any further structural change should show an effect on the disease. In the current study, we evaluated the impact of such a CRP mutant on the development of atherosclerosis employing LDL receptor knockout mouse model of atherosclerosis. We found that there was a two-week delay in the development of atherosclerotic lesions in the aortic root of mice treated with mutant CRP compared to mice that did not receive CRP. The development of atherosclerotic lesions in the whole aorta was also reduced; there was 30% reduction in the size of lesions in mice treated with mutant CRP. Overall, the data indicate that CRP is indeed an atheroprotective molecule.

Lab-on-Chip, the miniaturization of the chemical and analytical lab, is an endeavor that seems to come out of science fiction yet is slowly becoming a reality. It is a multidisciplinary field that combines different areas of science and engineering. Within these areas, microfluidics is a specialized field that deals with the behavior, control and manipulation of small volumes of fluids. Agglutination assays are rapid, single-step, low-cost immunoassays that use microspheres to detect a wide variety molecules and pathogens by using a specific antigen-antibody interaction. Agglutination assays are particularly suitable for the miniaturization and automation that two-phase microfluidics can offer, a combination that can help tackle the ever pressing need of high-throughput screening for blood banks, epidemiology, food banks diagnosis of infectious diseases. In this thesis, we present a two-phase microfluidic system capable of incubating and quantifying agglutination assays. The microfluidic channel is a simple fabrication solution, using laboratory tubing. These assays are incubated by highly efficient passive mixing with a sample-to-answer time of 2.5 min, a 5-10 fold improvement over traditional agglutination assays. It has a user-friendly interface that that does not require droplet generators, in which a pipette is used to continuously insert assays on-demand, with no down-time in between experiments at 360 assays/h. System parameters are explored, using the streptavidin-biotin interaction as a model assay, with a minimum detection limit of 50 ng/mL using optical image analysis. We compare optical image analysis and light scattering as quantification methods, and demonstrate the first light scattering quantification of agglutination assays in a two-phase ow format. The application can be potentially applied to other biomarkers, which we demonstrate using C-reactive protein (CRP) assays. Using our system, we can take a commercially available CRP qualitative slide agglutination assay, and turn it into a quantitative High Sensitivity-CRP test, with a lower detection limit of 0.5 mg/L using light scattering. Agglutination assays are an incredibly versatile tool, capable of detecting an ever-growing catalog of infectious diseases, proteins and metabolites. A system such as that presented in this thesis is a step towards being able to produce high throughput microfluidic solutions with widespread adoption.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Pregnancy associated maternal morbidity and mortality along with adverse pregnancy outcomes have gained momentum over the past few years, particularly in Sub-Saharan Africa and Southern Asia despite the advances in medical science. Adverse pregnancy outcomes are associated with low birth weight, growth restriction, developmental and cognitive abilities in infants and children. Medical care for preterm babies is costly, requires advanced equipment and qualified trained staff. Recently, levels/concentrations of cytokines have been used to predict and determine potential risk in various medical conditions. Biomarkers have shown to be helpful in many medical conditions and could be used to reduce the number of preterm deliveries in developing countries. The aim of this study was to determine whether a highly elevated CRP serum concentration was associated with preterm delivery in a population of Rwandan mothers.

Extracellular histones have been implicated as a cause of tissue inflammatory injury in a variety of disorders including sepsis, lung and liver diseases. However, little is known about their interactions with neutrophils and how this might contribute to injury. We used human neutrophils isolated from healthy donors in this study, and we conducted functional and mechanistic studies of the effects of histone H4 on neutrophils in vitro. We found that histone H4 acts as a strong neutrophil activator, inducing cytokine release, hydrogen peroxide production, adhesion and degranulation. H4 had a distinctive profile as compared to other neutrophil agonists in that it caused a marked and sustained elevation of intracellular calcium, which resulted from permeabilization of the neutrophil plasma membrane. Prior studies showed that neutrophils predominate in the early response to influenza A virus (IAV) infection in the lung, that IAV induces formation of neutrophil extracellular traps (NETs), and IAV infection in vivo is worsened by release of free histones in the lung. Neutrophils predominate in the early response in the lung to IAV infection. We found that IAV-induced NETs contain histones. Of interest, we found that histone H4 binds to and aggregates IAV particles, inhibits IAV infectivity in vitro, and increases the uptake of IAV by neutrophils. On the other hand, histone H4 potentiated IAV-induced neutrophil responses, including hydrogen peroxide production and calcium flux. These findings may in part explain the injurious effect of histones during IAV infection in vivo. We found that C-reactive protein (CRP) and surfactant protein-D (SP-D) or its carbohydrate binding domain bound to histone H4. CRP markedly reduced all of the effects of histone H4 on IAV and/or neutrophils. We also found that SP-D or its isolated binding domain reduced neutrophil activation induced by histone H4 which may in part account for its ability to reduce lung inflammation. We hope these findings provide a better understanding of the pro-inflammatory effects of histones (in IAV or other types of injury) and how the body protects itself against histone-induced injury. We also hope our results will aid development of novel strategies to ameliorate tissue damage caused by histones.

C-reactive protein (CRP) is a component of innate immunity. The concentration of CRP in serum increases in microbial infections including Streptococcus pneumoniae infection. Employing a mouse model of pneumococcal infection, it has been shown that passively administered human wild-type CRP protects mice against infection, provided that CRP is injected into mice within two hours of administering pneumococci. Engineered CRP (E-CRP) molecules have been reported recently; unlike wild-type CRP, passively administered E-CRP protected mice against infection even when E-CRP was injected into mice after twelve hours of administering pneumococci. The current study was aimed at comparing the protective capacity of E-CRP with that of an antibiotic clarithromycin. We established a mouse model of pneumococcal infection in which both E-CRP and clarithromycin, when used alone, provided minimal but equal protection against infection. In this model, the combination of E-CRP and clarithromycin drastically reduced bacteremia and increased survival of mice when compared to the protective effects of either E-CRP or clarithromycin alone. E-CRP was more effective in reducing bacteremia in mice treated with clarithromycin than in untreated mice. Also, there was 90% reduction in antibiotic dosing by including E-CRP in the antibiotic-treatment for maximal protection of infected mice. These findings provide an example of cooperation between the innate immune system and molecules that prevent multiplication of bacteria, and that should be exploited to develop novel combination therapies for infections against multidrug-resistant pneumococci. The reduction in antibiotic dosing by including E-CRP in the combination therapy might also resolve the problem of developing antibiotic resistance.

C-reactive protein (CRP) binds to several species of bacterial pathogens including Streptococcus pneumoniae. Experiments in mice have revealed that one of the functions of CRP is to protect against pneumococcal infection by binding to pneumococci and activating the complement system. For protection, however, CRP must be injected into mice within a few hours of administering pneumococci, that is, CRP is protective against early-stage infection but not against late-stage infection. It is assumed that CRP cannot protect if pneumococci got time to recruit complement inhibitor factor H on their surface to become complement attack-resistant. Since the conformation of CRP is altered under inflammatory conditions and altered CRP binds to immobilized factor H also, we hypothesized that in order to protect against late-stage infection, CRP needed to change its structure and that was not happening in mice. Accordingly, we engineered CRP molecules (E-CRP) which bind to factor H on pneumococci but do not bind to factor H on any host cell in the blood. We found that E-CRP, in cooperation with wild-type CRP, was protective regardless of the timing of administering E-CRP into mice. We conclude that CRP acts via two different conformations to execute its anti-pneumococcal function and a model for the mechanism of action of CRP is proposed. These results suggest that pre-modified CRP, such as E-CRP, is therapeutically beneficial to decrease bacteremia in pneumococcal infection. Our findings may also have implications for infections with antibiotic-resistant pneumococcal strains and for infections with other bacterial species that use host proteins to evade complement-mediated killing.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. One host defense function of C-reactive protein (CRP) is to protect against Streptococcus pneumoniae infection as shown by experiments employing murine models of pneumococcal infection. The protective effect of CRP is due to reduction in bacteremia. There is a distinct relationship between the structure of CRP and its anti-pneumococcal function. CRP is functional in both native and non-native pentameric structural conformations. In the native conformation, CRP binds to pneumococci through the phosphocholine molecules present on the C-polysaccharide of the pneumococcus and the anti-pneumococcal function probably involves the known ability of ligand-complexed CRP to activate the complement system. In the native structure-function relationship, CRP is protective only when given to mice within a few hours of the administration of pneumococci. The non-native pentameric conformation of CRP is created when CRP is exposed to conditions mimicking inflammatory microenvironments, such as acidic pH and redox conditions. In the non-native conformation, CRP binds to immobilized complement inhibitor factor H in addition to being able to bind to phosphocholine. Recent data using CRP mutants suggest that the factor H-binding function of non-native CRP is beneficial: in the non-native structure-function relationship, CRP can be given to mice any time after the administration of pneumococci irrespective of whether the pneumococci became complement-resistant or not. In conclusion, while native CRP is protective only against early stage infection, non-native CRP is protective against both early stage and late stage infections. Because non-native CRP displays phosphocholine-independent anti-pneumococcal activity, it is quite possible that CRP functions as a general anti-bacterial molecule.

C-reactive protein (CRP) is a pentameric molecule made up of identical monomers. CRP can be seen in three different forms: native pentameric CRP (native CRP), non-native pentameric CRP (nonnative CRP), and monomeric CRP (mCRP). Both native and nonnative CRP execute ligand-recognition functions for host defense. The fate of any pentameric CRP after binding to a ligand is dissociation into ligand-bound mCRP. If ligand-bound mCRP is proinflammatory, like free mCRP has been shown to be in vitro, then mCRP along with the bound ligand must be cleared from the site of inflammation. Once pentameric CRP is bound to atherogenic low-density lipoprotein (LDL), it reduces both formation of foam cells and proinflammatory effects of atherogenic LDL. A CRP mutant, that is non-native CRP, which readily binds to atherogenic LDL, has been found to be atheroprotective in a murine model of atherosclerosis. Thus, unlike statins, a drug that can lower only cholesterol levels but not CRP levels should be developed. Since non-native CRP has been shown to bind to all kinds of malformed proteins in general, it is possible that non-native CRP would be protective against all inflammatory states in which host proteins become pathogenic. If it is proven through experimentation employing transgenic mice that non-native CRP is beneficial for the host, then using a small-molecule compound to target CRP with the goal of changing the conformation of endogenous native CRP would be preferred over using recombinant non-native CRP as a biologic to treat diseases caused by pathogenic proteins such as oxidized LDL.