Human anaphylatoxin C3a increases glycogenolysis in perfused rat liver. This action is inhibited by prostanoid synthesis inhibitors and prostanoid antagonists. Because prostanoids but not anaphylatoxin C3a can increase glycogenolysis in hepatocytes, it has been proposed that prostanoid formation in nonparenchymal cells represents an important step in the C3a-dependent increase in hepatic glycogenolysis. This study shows that (a) human anaphylatoxin C3a (0.1 to 10 mug/ml) dose-dependently increased prostaglandin D2, thromboxane B, and prostaglandin F2alpha formation in rat liver macrophages (Kupffer cells); (b) the C3a-mediated increase in prostanoid formation was maximal after 2 min and showed tachyphylaxis; and (c) the C3a-elicited prostanoid formation could be inhibited specifically by preincubation of C3a with carboxypeptidase B to remove the essential C-terminal arginine or by preincubation of C3a with Fab fragments of a neutralizing monoclonal antibody. These data support the hypothesis that the C3a-dependent activation of hepatic glycogenolysis is mediated by way of a C3a-induced prostanoid production in Kupffer cells.

Lyme borreliosis (LB) is the most common vector-borne disease in the Northern Hemisphere. The infection is caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex, and it is transmitted to humans by ticks. LB is associated with several clinical manifestations, of which erythema migrans (EM) and neuroborreliosis (NB) are the most common inEurope. The course of the disease is usually benign, but can vary between individuals. The underlying pathogenic mechanisms are not fully understood, but the prognosis is probably determined by a complex interplay between the bacteria and the host’s immune response. Previous studies have indicated that a strong initial T helper (Th) 1-response followed by a Th2 response is beneficial for the clinical outcome in LB. The aims of this thesis were to follow the incidence of NB inJönköping County,Sweden, over time, to search for clinical and laboratory markers associated with the risk of developing long-lasting post-treatment symptoms, and to explore the role of the complement system as well as the relative balance between Th-associated cytokine/chemokine responses in LB. The number of NB cases, diagnosed by cerebrospinal fluid (CSF) analysis, increased from 5 to 10/100,000 inhabitants/year in Jönköping County during 2000-2005. Post-treatment symptoms persisting more than 6 months occurred in 13 %, and were associated with higher age, longer-lasting symptoms prior to treatment, higher levels of Borrelia-specific IgG in CSF, and reported symptoms of radiculitis. Facial palsy, headache and fever were frequent manifestations in children, whereas unspecific muscle and joint pain were the most commonly reported symptoms in older patients. Complement activation occurred both locally in the skin in EM and in CSF of NB patients. However, no activation could be detected in blood in NB patients. Elevated levels of C1q, C4 and C3a in CSF, along with correlation between C1q and C3a levels, suggest complement activation via the classical pathway locally in the central nervous system in NB. In vitro experiments with two clinical Borrelia isolates revealed that B. garinii LU59 induced higher complement activation in human plasma compared to B. afzelii K78 that recruited more of complement regulator factor H. To elucidate the role of complement in the phagocytosis process, experiments were performed using whole blood from healthy donors incubated with fluorescence-labelled spirochetes and different complement inhibitors. The results illustrated a central role of complement for phagocytosis of Borrelia spirochetes. We also studied the relative contribution of different Th-associated cytokines/chemokine responses in NB. The results support the notion that early NB is dominated by a Th1 response, eventually accompanied by a Th2 response. IL-17A was increased in CSF in half of the patients with confirmed NB, suggesting a hitherto unknown role of Th17 in NB. In conclusion, the risk of developing long-lasting post-treatment symptoms tend to increase mainly with age and duration of symptoms prior to treatment in NB. The complement system seems to play an important role in host defence to recognize and kill Borrelia spirochetes. However, complement activation in inappropriate sites or to an excessive degree may cause tissue damage, and therefore, the role of complement in relation to disease course needs to be studied further. Likewise, the role of Th17 in LB pathogenesis and host defence should be further evaluated in prospective studies.

Today there are a vast number of medical devices in temporary or permanent contact with human tissues. Blood-biomaterial contact is known to trigger the complement system and results in generation of fluid phase anaphylatoxins C3a and C5a, and surface-bound C3b and iC3b. All these products together are able to attract and activate leukocytes and trigger release of inflammatory mediators leading to a systemic inflammation indirectly causing hemostatic problems and even organ failure. The aim of this study was to identify how complement is triggered on a biomaterial surface and to find ways to regulate this activation. The finding that complement activation on biomaterials can be divided into initiation and amplification will facilitate regulation of complement activation biomaterial surfaces. This concept is also compatible with the two techniques to regulate complement activation on a surface.

Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. The high incidence is still found in Taiwan. There is strong evidence that host genes influence individual susceptibility to tuberculosis. Young children, like immunocompromised patients, once infected are at increased risk for TB disease and progression to extrapulmonary disease. Thus far, to identify the genes responsible for the variation in the human susceptibility/resistance to TB has remained elusive. Mannose-binding lectin (MBL) activates the complement system in an antibody-independent manner, enhances complement-mediated phagocytosis, and plays an important role in innate immunity in the regulation of inflammatory cytokine release by monocytes. It is one of the molecules that have been suggested to have a link to human susceptibility or protection against infection. According to some studies (mostly conducted in adult populations) , low levels of MBL associated with variant alleles at the promoter and exon 1 regions of MBL protect against tuberculosis. Other investigators instead claim that protection against the disease is associated with high levels of MBL. In this study we aimed to investigate the relationships between the susceptibility to TB and MBL gene polymorphisms in children with cervical mycobacterial lymphadenitis infected by M. tuberculosis.139 case patients with cervical mycobacterial lymphadenitis and 102 unrelated healthy control subjects were tested by real-time PCR for polymorphisms at the promoter and the exon 1 regions of the MBL gene. Diagnosis of mycobacterial lymphadenitis infected by M. tuberculosis, based on findings of pathological examination of the lymph nodes, was confirmed by acid-fast stain and TB PCR.The frequency of A allele was significantly higher in TB+ patients compared with TB- controls (82.7% vs 72.6%; odds ratio 1.813; p=0.007). The frequency of high-producer MBL2 genotypes (A/A) was higher in TB+ patients than in TB- subjects (70.5% vs 45.1%, odds ratio 2.91, p<0.001), while patients carried the B alleles (A/B and B/B) that have decreased levels of MBL was inversely associated with mycobacterial infectivity (29.5% vs 54.9%; odds ratio 2.910; p<0.001). The frequencies of MBL promoter -550 genotypes also revealed a significant difference between TB+ and TB- groups (p = 0.046), but in contrast, with significantly higher frequency of L/L genotype (of low MBL level) in TB+ patients (34.5% vs 21.6%; odds ratio 1.918; p=0.029). The frequencies of MBL promoter -221 genotypes (X and Y) was similar in TB+ and TB- groups.This study supports the conclusion that MBL can protect or predispose the host to tuberculosis, depending on the host¡¦s haplotype pair.

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Biomedical Engineering." Includes bibliographical references (p. 89-94).

Immune molecules, including complement proteins, C1q and C3, have emerged as critical mediators of synaptic refinement and plasticity. Complement proteins localize to synapses and refine the developing retinogeniculate system via C3-dependent microglial phagocytosis of synapses. Retinal ganglion cells (RGCs) express C1q, the initiating protein of the classical complement cascade, during retinogeniculate refinement; however, the signals controlling C1q expression and function remain elusive. RGCs grown in the presence of astrocytes significantly upregulated C1q compared to controls, implicating an astrocyte-derived factor in neuronal C1q expression. A major goal of my dissertation research was to identify the signals that regulate C1q expression and function in the developing visual system. In this study, I have identified transforming growth factor beta \((TGF-\beta)\), an astrocyte-secreted cytokine, as both necessary and sufficient for C1q expression in RGCs through an activity-dependent mechanism. Specific disruption of retinal \(TGF-\beta\) signaling resulted in a significant reduction in the deposition of C1q and downstream C3 at retinogeniculate synapses and significant synaptic refinement defects in the retinogeniculate system. Microglia engulfment of RGC inputs in the lateral geniculate nucleus (LGN) was also significantly reduced in retinal \(TGF\beta\)RII KOs, phenocopying the engulfment defects observed in C1q KOs, C3 KOs, and CR3 KOs. Interestingly, in C1q KOs and retinal \(TGF\beta\)RII KOs, microglia also failed to preferentially engulf less active inputs when retinal activity was manipulated, suggesting that retinal activity and \(TGF-\beta\) signaling cooperatively regulate complement mediated synaptic refinement. In support of this hypothesis, blocking spontaneous activity in RGC cultures significantly reduced C1q upregulation by \(TGF-\beta\). Moreover, manipulating spontaneous retinal activity in vivo modulated C1q expression levels in RGCs and C1q deposition in the LGN. Together these findings support a model in which retinal activity and \(TGF-\beta\) signaling control expression and local release of C1q in the LGN to regulate microglia-mediated, complement-dependent synaptic pruning. These results provide mechanistic insight into synaptic refinement and, potentially, pathological synapse loss which occurs in the early stages of neurodegenerative diseases concurrently with aberrant complement expression and reactive gliosis.

The mechanisms responsible for the enhanced inflammatory response in type 2 diabetes (T2DM) and its contribution to the severe ischemia/reperfusion (I/R) injury observed in the T2DM heart are unclear. I/R is associated with an acute inflammatory response recognized by reactive oxidant production, complement activation, and leukocyte-endothelial cell adhesion, among others. Complement activation plays an important role in the inflammatory response and is involved in the manifestation of I/R injury in the non-diabetic heart, and is a potent chemoattractant for circulating neutrophils (PMNs). The purpose of this dissertation research was to test the hypothesis that the complement system, predominantly the lectin pathway, is a significant contributor to the excessive response of the Zucker Diabetic Fatty (ZDF), a rat model of T2DM, to myocardial I/R injury. Following 30min of coronary artery occlusion and 120min of reperfusion we measured C3 deposition, PMN accumulation, PMN CD11b expression, and ICAM-1 expression. We found significantly more C3 deposition, PMN accumulation, ICAM-1 and PMN CD11b expression in diabetic samples compared to non-diabetic samples. To elucidate a role for complement system activation, we treated animals with FUT-175, a broad complement inhibitor. In vivo, FUT-175 treatment significantly decreased complement deposition (66%), PMN accumulation (59%), and infarct size (55%) compared to untreated animals in both non-diabetic Sprague-Dawley and diabetic ZDF rats. To specifically examine the role of the lectin pathway, we selectively inhibited rat MBL-A prior to myocardial I/R in ZDF rats. Anti-MBL treatment significantly decreased infarct size, C3 deposition and PMN accumulation in the ZDF post-ischemic left ventricle (LV). Genomic analysis revealed that gene expression of the pro-inflammatory cytokines IL-6 and IL-1α was enhanced in the ZDF heart following reperfusion, and quantitative RT-PCR results confirmed IL-6 upregulation. We found significantly increased complement C5a receptor (CD88) expression on diabetic neutrophils prior to ischemia, suggesting that diabetic PMNs are "primed" to respond to complement activation. Taken together, these results provide evidence that 1) the ZDF rat is a good model for chronic inflammation in the setting of T2DM, 2) lectin pathway activation plays a significant role in the inflammatory response to I/R injury in the ZDF heart, and 3) anti-complement therapy may be particularly cardio-protective in T2DM.

The contribution of single complement system (CS) activation pathways to cerebral IR injury has not yet been adequately studied after ischemic stroke and reperfusion. It remains unclear whether a specific activation pathway (alternative, classical or lectin), single complement factors within the CS, or anaphylatoxins are responsible for increased cerebral IR injury after ischemic stroke and reperfusion. Also poorly understood is the relationship between these elements (activation pathways, CS factors and anaphylatoxins) and neutrophil mediated cerebral IR injury. The objective of this dissertation was to test the hypothesis that mannose binding lectin (MBL) deficiency during cerebral ischemia and reperfusion will result in a significant reduction of systemic neutrophil activation and cerebral injury after ischemic stroke and reperfusion via decreased CS activation and subsequent decreased anaphylatoxin production. Using the intraluminal filament method, mice with targeted mutations to MBL A/C genes (MBL-/- ) or the C57Bl/6 strain (MBL +/+ ) were subjected to 60 minutes of cerebral ischemia and either 15 minutes or 24 hours of reperfusion. After reperfusion (15 min and 24 hour) blood was removed to assess systemic neutrophil CD11b expression via flow cytometry. After 24 hours of reperfusion, the brain was removed to assess cerebral injury. CS activation after ischemic stroke and reperfusion was assessed via immunofluorescent C3 staining and RT-rtPCR methods. Our primary findings are, after ischemic stroke and reperfusion: (1) hepatic MBLA gene expression is significantly increased, (2) systemic neutrophils significantly express CD11b, (3) MBL deficiency significantly decreased cerebral infarct volume in the striatum but not in the cortex or total hemisphere, and (4) systemic neutrophil activation is independent of MBL deficiency. This study is the first to examine the contribution of MBL-initiated lectin pathway activation to cerebral IR injury after ischemic stroke and reperfusion. These findings suggest that MBL deficiency does not significantly reduce neutrophil activation or protect brain tissue after ischemic stoke and reperfusion. A complete understanding of reperfusion events after ischemic stroke is necessary for successful development of future stroke therapies to prevent cerebral IR injury. In this way, the acquisition of knowledge from the bench serves the stroke population cared for by nurses at the bedside.

This thesis investigates a type of infinitival complement previously analyzed as a relative clause construction. The unique properties of this construction, namely, a Specificity Effect on the logical object of the embedded verb and a semantic restriction on the matrix verb, stem from the syntactic structure of the complement. The complement's T (Tense) node is specified [-Tense]. A T that is specified [-Tense] fails to select an Asp (Aspect) projection. The lack of an AspP projection has significant consequences: The embedded verb cannot assign an external theta role and accusative Case cannot be checked. / The distinction of infinitival complements on the basis of their Tense specification will be shown to account for a difference in behaviour with respect to VP-deletion, temporal interpretation and the licensing of PRO. Furthermore, the arguments and evidence presented suggest that the functional projection responsible for checking accusative Case features is AspP, not AGRoP.

The aim of this study is to present a contrastive analysis of verbal complement that-clauses in English and Lithuanian and to describe, contrast and compare the cases of that deletion in them on the basis of corpus evidence. Matrix verbs from three semantic domains, viz. saying, thinking, and discovering, were investigated in the spoken, fiction, newspaper, and magazine registers of the BNC and LLC corpora. The methods chosen for the study were qualitative analysis as well as contrastive and content analysis. The corpus evidence demonstrated that zero-that complement clauses are by far more frequent in English than in Lithuanian in all registers considered. The deletion of that in Lithuanian appeared to be likely in case the complement clause is preceded by an evidential marker, such as esą, neva “supposedly”, girdi “(you) hear”, tarsi, lyg “as if”, or gal “maybe”, adopting the function of the complementizer, which is not the case in English. The corpus evidence also revealed that in both languages complement zero-that clauses share a structural peculiarity known as closeness of the clause juncture and are likely to occur with the most frequent verbs in both languages: say, think, and know. Another similarity is that in both languages matrix clauses followed by zero-that complements can be confused with comment clauses. Further quantitative corpus-based researches of Lithuanian complement clauses must be carried out in order to measure the influence of the contextual factors... [to full text] / Šio tyrimo tikslas – išanalizuoti anglų bei lietuvių kalbos prijungiamuosius aiškinamuosius sakinius bei remiantis tekstynų duomenimis aprašyti ir palyginti jungtuko kad/jog praleidimo atvejus tokio tipo sakiniuose. Aiškinamieji sakiniai, valdomi kalbėjimo, mąstymo bei suvokimo veiksmažodžių, buvo išnagrinėti BNC ir LLC tekstynų šnekamosios kalbos, grožinės literatūros bei publicistikos registruose. Tyrimas grindžiamas lyginamosios ir kontekstinės analizės metodais bei kokybinės analizės metodu aprašant ir lyginant iš tekstynų surinktus duomenis. Surinkti tekstynų duomenys rodo, kad prijungiamieji aiškinamieji sakiniai su praleistu jungtuku kad/jog žymiai būdingesni anglų nei lietuvių kalbai. Paaiškėjo, kad jungtuko kad/jog nebuvimas lietuvių kalboje tikėtinas, jei šalutinis dėmuo pradedamas evidencialumo raiškos priemonėms priskiriamais žodžiais esą, neva, lyg, tarsi ir girdi, kurie šiuolaikinėje lietuvių kalboje neretai atlieka jungtukų funkciją, bet anglų kalbai tokios struktūros nebūdingos. Abiejose kalbose jungtukas kad/jog dažniau praleidžiamas su dažniausiai kalboje vartojamais veiksmažodžiais. Anglų ir lietuvių kalbos bejungtukiai aiškinamieji sakiniai taip pat panašūs sandaros atžvilgiu: nėra antros eilės sakinio dalių, įsiterpiančių tarp valdančiojo veiksmažodžio ir šalutinio dėmens veiksnio. Be to, abiejose kalbose būna dviprasmiškų atvejų, kai bejungtukių aiškinamųjų sakinių pagrindinį dėmenį sunku atskirti nuo įterpinio arba komentuojamojo sakinio. Tam, kad... [toliau žr. visą tekstą]