Introduction (You need to start with background on gut-liver axis, the fact that bacterial infections in gut can induce inflammatory mediators which come to liver through mesenteric-portal vein). The complement system is a fundamental element of the innate immune system as well as the adaptive immune responses. Improper complement activation leads adverse effects, for instance in diverse renal diseases, (expand) TTP, thrombotic microangiopathies, portal hypertensionand transplant rejection. (expand). (You need to have more background on the various complement factors, their function, the 3 different pathways of activation etc) NCIPH is a liver disorder of vascular origin defined by a portal venous pressure exceeding 5mm Hg between portal vein and inferior vena cava (sanyal 2008), characterized by occlusion of the 3rd /4th order branches of the hepatic portal vein ( madhu 2008). It is suspected that this occlusion is due to formation of microthrombi in the venules of the portal vein. Our clinical studies indicate that in 30-40% of patients with cryptogenic chronic liver disease, liver biopsy confirms the diagnosis as NCIPH (Madhu, Avinash 2009, Goel, madhu 2013). The focus of this proposal is to uncover the cause for thrombosis that leads to NCIPH.
Thrombotic micro angiopathy (TMA) is characterized by a sequence of events, including disruption of endothelial cell integrity, intravascular activation of platelets, formation of platelet–fibrin thrombi, and obstruction of the microcirculation followed by hemolytic anemia with consumption of erythrocytes and platelets (1). The factors causing TMA include pathogenic infections, immunosuppressive drugs and blood group incompatibility (7) however, the mechanisms of toxicity remain indistinct. The Von Willebrand protein is a glycoprotein which predominantly functions in hemostasis and is mainly synthesized in endothelial cells and megakaryocytes
You need to add a lot more background on VWF, explain what is UL-vWF, the various domains etc) In normal conditions, hepatic portal vein endothelial cells that are injured/activated secrete vast quantity UL-vWF, which would usually been cleaved by ADAMTS13 (A disentegrin and metalloprotease with eight thrombospondin 1 – like domanins) to maintain homeostasis.
The function of vWF is is facilitated by shear stress caused by the blood flow in the venous circulation. Under static condition the ULvWF is not linear and may be coiled, hiding the ADAMTS13 cleavage sites, but under the blood flow, the shear stress induces unfolding of ULvWF and exposure as a linear multimer of vWF. ADAMTS13 attaches to monomeric subunit strings of ULvWF through its CUB domain and cleaves the molecule at 842Tyr-843Met peptide bond in VWF A2 domain. Thus the ULVWF fragmented and the monomeric sub units will no longer induce adhesion and aggregation of platelets in the circulation. Prevention of cleavage by ADAMTS13 will induce adhesion of ULvWF and accumulation of platelets, leading to microangiopathy as in (expand) TTP, which is a disease caused as a consequence of ADAMTS13 deficiency and lack of cleavage of ULVWF.
Lack of cleavage of vWF multimers by ADAMTS13 leads to platelet aggregation and occlusion of the vessel resulting in microangiopathy. Alternatively, over secretion of vWF from the endothelial cell will lead to the same. The vWF synthesis in endothelial cells may be activated by bacterial toxins, inflammatory cytokines, calcium ionophores or phosphodiesterase inhibitors (ref). The synthesized ULvWF may be anchored on the surface of endothelial cells by P-selectin which is produced concurrently with VFW from the Wieble-palade bodies of endothelial cells( padilla 2004).
Thus, pathogenesis in NCIPH patients could be either defective Vwf multimeric processing by ADAMTS13, excess expression of multimeric vWF which overwhelms the cleavage capacity of ADAMTS13 or any other factor which interferes with this system. Preliminary data indicates that NCIPH patients have an imbalance in vWF and ADAMTS13 but the reason driving this disparity is not clear. In this proposal it is hypothesized that activation of vWF secretion and down regulation of ADAMTS13 may be provoked by inflammatory cytokines induced by an inflammatory response in NCIPH cases. It is possible that inflammatory cytokines secreted during acute inflammation or infection can inhibit ADAMTS13 biosynthesis and result in thrombotic microangiopathy in the hepatic venules, that leads to portal hypertension (You need some ref which show that cytokines can inhibit ADAMTS13-those must be quoted before this sentence). Other than toxins, ULvWF synthesis can be activated by histamine, TNF?, and interleukins such as IL8, IL6 and IL10 (Aubrey Bernardo, 2004) which are mainly activated by the complement pathway (ref for activation by complement pathway). More over nitric oxide has also been shown to induce microthrombi in circulation (ref), and the role of this in NCIPH cases also needs investigation.
Secondly the extent of complement activation and its participation in development of TMA will be assessed in NCIPH patients. Complements like C3, C4, and C3b are involved in the alternative pathway, and the activation of these complement factors are regulated by the complement factor H (CFH). Hence, any problem in any of these factors may leads to thrombosis (nancy 2013). Though the focus is mainly on liver thrombotic microangiopathy, recognition of the causatives for thrombosis in NCIPH may provide insight on pathogenesis of other thrombotic diseases.
Aims Aim 1: Evaluate hemeostatic mediators in circulation of patients with NCIPH: In this aim, the level of circulating ADAMTS13, ULvWF, nitrate levels, inflammatory cytokines and complement factors in NCIPH patients will be measured. Studies on TMA implicate elevation of (mention specific cytokines) these cytokines in thrombosis (ref) and hence the role of these factors in NCIPH will be evaluated. Inflammatory cytokines such as TNF?, IL6, IL8, and IL10 in plasma of NCIPH cases and controls will be determined using ELISA. It is believed that changes in the levels of these components may give a clearer understanding in outlining the cascade of thrombotic origin in NCIPH. Details of cases such as the age, time point (not yet discussed with Dr.Ashish)
Aim2: Examine the role of ADAMTS13 deficieny on endothelial cell adhered VWF in the context of NCIPH: In this aim, the functional relationship of ADAMTS13 and proteolytic cleavage of UL-VWF on endothelial cells in NCIPH will be evaluated in an in vitro system. Induction of VWF on human umbilical vein endothelial cells (HUVECs) in response to levels of inflammatory cytokines seen in NCIPH patient circulation will be examined initially. Subsequently, normal and NCIPH patient plasma will be diluted in different ratios (from 0 to 5) in buffer (20 mM HEPES, pH 7.4, 150 mM NaCl and 5 mM CaCl2) and incubated with HUVECs for 5 to 30 minutes, followed by testing for surface bound UL-vWF polymers. These experiments will also be done in the presence of shear stress by culturing cells on microslides with precise channels (IBIDI) to replicate flow within both venous and arterial circulation. The continuous flow of Media (specification-mention details) is supplied to the slides to mimic the physiological system of shear stress. Laminar shear as well as turbulent shear at junctions where the venous and arterial circulation meets within the liver will be replicated in this system.
Aim3: Explore the role of ADAMTS13-VWF balance on complement binding to endothelial cells in the context of NCIPH: Here, the effect of ADAMTS13 deficiency on complement activation and adhesion to VWF on endothelial cells will be studied. In vitro experiments using HUVECs (human umbilical vein endothelial cells) in culture will be carried out to recreate the proposed intrahepatic vascular milieu in NCIPH, where absence of ADAMTS13 cleavage results in accumulation of ULWF (ultralarge von Willebrand factor) on endothelial cells. Towards this, HUVECs grown in culture will be stimulated with histamine for 2 minutes to induce secretion of ULVWF, which will be immuno-stained prior to their cleavage by plasma derived ADAMTS13. These cells will then be treated with serum from controls and NCIPH patients under normal as well as orbital shear conditions. After 15 minutes incubation, cells will be washed and immunostained for complement proteins. In the second set of experiments it tested for platelet adhesion to these cell lines which purified from the peripheral blood of the patients and control individuals. It is anticipated that plasma from NCIPH patients will show an increased complement and Platelet binding to ULVWF as illustrated in figure. For platelet adhesion experiments on invitro cell lines platelet rich plasma will be incubated for 15 minutes, washed and imaged for platelet adhesion.
ADAMTS13 is a disintegrin and metalloprotease with eight thrombospondin-1-like domains composed of an amino-terminal reprolysin-type metalloprotease domain followed by a disintegrin domain; a thrombospondin-1-like domain; a cysteine-rich domain containing an arginine-glycine-aspartate (RGD) sequence and an adjacent spacer portion; 7 additional thrombospondin-1-like domains; and 2 similar CUB domains at the carboxyl-terminal end of the molecule. CUB domains, found only in ADAMTS13 among the ADAMTS enzymes, contain peptide sequences present in Complement subcomponents C1r/C1s; embryonic sea Urchin
Genome Sequence of P. Acnes ED1 Strain
Propionibacterium acnes is a Gram-positive bacterium that forms part of the normal human microbiota. P. acnes is pervasive on human skin and under specific conditions can be an opportunistic pathogen. The draft genome sequence of P. acnes ED1 strain, which was isolated from a hip implant, was examined in order to investigate the genomic biology of these pathogens. Various methods were used in order to examine the genome sequence of the P. acnes strain ED1 as well as its assembly and annotation. From these findings, the length of the genome was 2.8 Mb (200 contigs, N50=86 kb) and the GC content was 60%. The coding sequences of P. acnes genome (2,788) in 318 subsystems was found as well as the allocation of the genes in the genome. The histidine metabolism and antibiotic resistance genes of P. acnes are analysed further in comparison with the P. acnes strain KPA171202 reference genome.
The Genome Announcement Propionibacterium acnes is a bacterium of normal human microbiota that mainly colonises the skin. It is associated with many infections such as acne vulgaris and other skin diseases (1). For that reason, sequencing this genome is really important.
A sample of P. acnes ED1 strain, isolated from a hip implant at the time of revision operation, was examined. The DNA concentration was measured using the Qubit protocol. A transposome-based sequencing library was generated using Illumina NextERA XT Tagmentation kit (2) and it was amplified by PCR. This genomic library was subsequently submitted to the Edinburgh Genomics Facility in order to be normalised and then sequenced on the Illumina MiSeq instrument (2). The raw DNA sequence data were trimmed for adapters and low quality sequences.
The quality of the data was assessed using FASTQC (3) which indicated the GC content over all reads which resulted in the generation of two peaks (Figure 1). One of them showed the expected for the P. acnes (4) 60% GC and the other one a much lower GC content (roughly 34%). The presence of the second peak of GC content indicated that the ED1 sample was contaminated with DNA from another bacterium. This bacterium is most likely to be Staphylococcus lugdunensis as it also colonises the human skin and its GC content is 33.87% (5, 6). The FASTQC analysis displayed the sequence duplication levels that were really high (>= 40.52%) and possibly to incorrect library generation. Many duplication events contributed to the reduction of the coverage of the P. acnes ED1 genome. The fold coverage of the P. acnes ED1 genome was 49. Contamination, library errors and tagmentation bias might be the reason why data is of such low quality.
De novo genome assembly was performed using CLC Genomics Workbench (7). The quality of the genome assembly was assessed using QUAST (8). The total length of the Propionibacterium genomes is between 2.2 and 2.5 Mb. QUAST showed that the total length of the P. acnes ED1 was about 2.8 Mb (200 contigs, N50=86 kb) and that might be due to contamination with Staphylococcus. Thereafter, the genome assembly was submitted to the RAST annotation server, (9) which using the SEED system, identify the genes. The SEED system (10) identified the genes that are present in the P. acnes ED1 genome but absent from the P. acnes strain KPA171202 reference. A SEED comparison revealed that 766 of the total 2,298 protein sequences have similarity > 99%. Most proteins/genes are not identical with the reference genome due to mistakes in PCR amplification or duplications events. The SEED annotated the P. acnes ED1 genome and defined that the coding sequences were 2,788 and the number of subsystems was 318. A histogram showed that the proportion of features in the P. acnes ED1 genome that were annotated by SEED was 39%. Additionally, the annotated functions in the genome were 1,306 (Figure 3). From all these findings, it can be concluded that there are genes being repeated in many other subsystems. In order to examine the allocation of the genes and whether the genome was complete, it is necessary to further identify more specific processes.
Two Analysis Summaries The P. acnes ED1 genome with the reference genome is now compared in terms of amino acid metabolism and potential virulence (antibiotic resistance).
The examination of histidine metabolism is really interesting in order to assess whether the P. acnes ED1 genome assembly is complete related to the reference genome and is sufficient in a complete biochemical pathway. Using the SEED system, the identification of the presence of histidine biosynthesis and degradation genes in both genomes was accomplished. The KEGG map in SEED system shows that the P. acnes ED1 genome contains exactly the same set of 10 histidine biosynthesis genes and 5 histidine degradation genes as the reference genome. For example, ATP phosphoribosyltransferase is an enzyme that catalyses the first step in the histidine biosynthesis in P. acnes and has a crucial role in the pathway because the rate of histidine biosynthesis appears to be controlled mainly by regulation of HisG enzymatic activity. Another enzyme is the Imidazoleglycerol-phosphate dehydratase that catalyses the seventh step in the biosynthesis of histidine. Furthermore, the Histidinol dehydrogenase (HDH) catalyses the terminal step in the biosynthesis of histidine. Moreover, there are some proteins that catalyse the histidine degradation as the imidazolonepropionase catalyses the third step in degradation of histidine and the histidine ammonia-lyase catalyses the first reaction in histidine degradation (11). Therefore, all these histidine biosynthesis and degradation proteins play an important role in histidine metabolism. Histidine biosynthesis is really important for the survival of P. acnes ED1 genome (Figure 4).
The examination of P. acnes ED1 genome in terms of antibiotic resistance and sensitivity is also an important aspect, as for any clinical infection it is vital to choose the correct drugs for treatment. Using SEED system in RAST server, the identification of the genes resistant to antibiotics and toxic compounds was achieved. The P. acnes ED1 genome contains exactly the same set of 10 antibiotic and toxic resistance genes as the reference genome. More specifically, the P. acnes ED1 genome has the cobalt/zinc/cadmium resistance protein and the mercuric reductase protein that confer resistance to cobalt, zinc, cadmium and mercury respectively. So in an environment with high concentration of cobalt, zinc, cadmium and mercury, the P. acnes ED1 would not be poisoned. Thereafter, the P. acnes ED1 genome shows resistance against fluoroquinolone antibiotics. Fluoroquinolone antibiotics disrupt the Propionibacterium DNA replication by degenerating DNA gyrase and topoisomerase in a concentration-dependent manner. It could be mentioned that, P. aeruginosa shows increased fluoroquinolone resistance, which could be evidence for intra-generic horizontal gene transfer and evolution in action in Propionibacterium genomes. All these remarks are consistent with there being fluoroquinolones antibiotic resistance in the P. acnes ED1 genome (12). It would be of interest to study this HGT event by a Protein BLAST analysis (13); yet, further examination would be outside the scope of this analysis. However, there are beta-lactamase class C and other penicillin binding proteins in P. acnes ED1 genome but not in the reference one. Beta-lactamase is an enzyme that provides resistance in beta-lactam antibiotics like penicillin. The lack of beta-lactamase from the reference genome might be due to wrong annotation. Yet, a cytoplasmic copper homeostasis protein (CutC) is present in the reference genome but absent from the P. acnes ED1. This protein is really important as it confers resistance to copper. It could be mentioned that mutation of this protein leads to an increased copper sensitivity. The absence of CutC from the P. acnes ED1 genome might be caused by incorrect annotation (11).