Work investigating the role of chondrocytes

The look of Fragment, crystallisable ( Fc ) receptors by animate being cells is a cardinal portion of our current apprehension of the immune response in worlds. However, there is comparatively small published work look intoing the function of Fc receptors in autoimmune diseases. Recently, several workers have begun to characterize the immunological footing of Rheumatoid Arthritis, a status affecting devolution of the collagen / chondrocyte matrix that forms the gristle nowadays in skeletal articulations. This survey assesses the potency for look by chondrocytes of the receptor Fci‚?RI under simulatedin vivoconditions, and therefore analyze a possible nexus between Rheumatoid Arthritis and a allergic reaction of the immune system. Such a nexus has been proposed before, but has yet to be once and for all proven.

Experimental work involved the civilization of C-20/A4 chondrocytesviastandard microbiological protocols. In add-on to kick “control” civilizations, separate samples were exposed to hypoxia, and to the cytokines TNF-a and IL-1i?? . This was followed by extraction of RNA from the civilized cells, and a DNA – bring forthing PCR procedure.

The consequences of the cataphoresis analyses showed no direct grounds that chondrocyte cells express Fci‚?RI in these fortunes. Full statistical analysis was precluded by a deficiency of dependable informations, as Fci‚?RI was observed in merely negligible sums in both the control and the hypoxic conditions, but a preliminary decision was reached: Hypoxia in the presence of cytokines had no inclination to advance Fci‚?RI look inin vitrochondrocytes under the above experimental conditions.

Analysis of the literature shows clearly that there is turning grounds to propose a nexus between high Fci‚?RI degrees in chondrocytes and Rheumatoid Arthritis, therefore it is suggested that thein vitroconditions defined by this survey are non an accurate contemplation of the equivalentin vivoenvironment. Possible grounds for this disagreement are explored in the treatment.

Abbreviations used in the text

DEPC: Diethylpyrocarbonate

DMEM: Dulbeccos Modified Eagles Medium

dNTP: deoxyribonucleoside-triphosphate

FcR: Fraction, crystallisable Receptor

Farm credit system: Fetal Calf Serum

GAPDH: Glyceraldehyde 3-phosphate dehydrogenase

Intelligence community: Immune Complex

Illinois: Interleukine

MMLV:Moloney Murine Leukaemia Virus

Radium: Rheumatoid Arthritis

TAQ: Thermus aquaticus

Tumor necrosis factor: Tumour Necrosis Factor

UHQ: Ultra High Quality

Recognitions

Contentss:

Page: Section: Subject:

1. 1 Introduction

1. 1.1 Background Information

2. 1.2 The Importance of Fc-Expression

3. 1.3 Other Factors

3. 1.4 Undertaking Aims

4. 2 Materials and Methods

4. 2.1 Experiment Design

4. 2.2 Cell Culture

4. 2.3 RNA Extraction

5. 2.4 PCR

  1. 2.5 Gel Electrophoresis.

7. 3 Consequences

7. Figure 1: UV-S of Merchandises

8. Figure 2: Position of a Completed Gel

8. Figure 3: Consequences Postpone

9. 4 Discussion

9. 4.1 Mistakes

11 4.2 Value

11 4.3 Further work

12 4.4 Decisions

13Mentions

1.1: Background Information

Chondrocytes are the basic cellular stuff that composes the cartilaginous connective tissue responsible for jointing skeletal articulations in worlds. They occupy the interstitial countries ( the blank ) of a hempen collagen and elastin web, organizing a flexible matrix that facilitates motion of the skeleton whilst forestalling scratch of osteal tissue.

Breakdown of this matrix ( which may be caused by a assortment of diseases ) can take to a scope of mobility-impairing unwellnesss. One of the most common illustrations of such an unwellness is Rheumatoid Arthritis.

Rheumatoid Arthritis ( RA ) is a systemic status impacting about 1 in 50 people in the UK ( NHS, 2007 ) . It is characterised by swelling, hurting and eroding of cartilaginous tissue ( taking to asymmetric malformation ) in the articulations. RA impacts to a great extent on the lives of patients by cut downing mobility and increasing dependance on medicine ( peculiarly anodynes ) . In combination with systemic effects, some research workers have claimed that RA is “shown to tie in with higher mortality ” in long-run patient surveies ( Wolfe, 1994 ) .

There is no general consensus as to the causes of RA, and accordingly, clinical direction schemes for this disease are varied. Recent work has, nevertheless, focused on the possibility that RA is caused by a type of immune hypersensitivity, in which healthy tissue undergoes attack byin vivoscavenger cells, taking to the gristle harm feature of this status.

Evidence for this hypothesis is found in the noticeable presence of certain cytokines in the blood stream of RA patients. Cytokines are “signalling” molecules that play a cardinal function in interceding immune responses in the organic structure. When a lymph cell cell detects the presence of a foreign antigen at its surface, it releases cytokines which in bend trigger the release of antibodies from other lymph cells. It is believed that high concentrations of cytokines present in those with RA may excite over-production of certain antibodies and do the immune system to assail the body’s ain tissue. Feldmann ( 1996 ) , for illustration, analysed the RNA and protein content of samples of gristle in RA patients, and found that “many pro-inflammatory cytokines such as TNF? , IL-1, IL-6, GM-CSF, and chemokines such as IL-8 are abundant in all patients irrespective of therapy” .

For the intents of this survey, two common cytokines have been chosen to supply a realistic biochemical environment. The first, Tumour Necrosis Factor ( TNF ) mediates acute-phase proteins which play an of import function in commanding redness in tissue. The 2nd, IL-1i?? is an Interleukin that likewise controls acute-phase responses.

Eales ( 2003 ) found grounds of a characteristic“serum rheumatoid factor ( RF ) antibody” ,back uping the ‘autoimmune attack’ theory – Type III Hypersensitivity involves the collection of antigens and antibodies in the blood stream, and subsequent

1

deposition in tissue. Low and Moore ( 2005 ) reinforced this work by characterizing

Immune Complexes ( IC ) in the blood of RA sick persons. Several surveies ( and more late, Patents ) have been published that utilise the thought of mensurating the blood-volume concentration of specific biological molecules as a diagnostic tool. Cush ( 1988 ) discussed the usage of Cytofluorometric analysis to “characterise the surface phenotype and activation position of newly isolated synovial tissue lymph cells ( STL ) and peripheral blood lymph cells ( PBL ) from 7 patients with arthritic arthritis ( RA ) ” . This is of peculiar value, as it facilitates the word picture of blood-antibody and IC degrees in ‘real time’ , and provides a clearer image of the mechanism by which IC’s can be deposited in gristle.

1.2: The Importance of Fc- Expression

A cardinal portion of the immune response procedure is the mechanism by which lymph cells are stimulated to transport out phagocytosis in the presence of foreign proteins. In general footings, this mechanism revolves around Fc-Receptor proteins which are found on the surface of cells, and which bind to the Fc part of antibodies as the antibodies latch on to occupying cells. In making so, the Fc-R promotes phagocytosis and similar lymph cell responses.

IgG antibodies ( the most commonly happening type of antibody in human serum ) bind to i‚?- Fc Receptors ( Fridman, 1991 ) . The chief focal point of this undertaking is the look of a specific Fc –Receptor ( Fci‚?-RI ) by chondrocytes. This receptor mediates a assortment of immune responses ( including phagocytosis ) , and has been studied as a possible contributing factor to the development of rheumatoid arthritis. Nieto ( 2000 ) carried out PCR surveies on over 100 RA patients, and found that “An overrepresentation of the FcRIIIA-158FF genotype in the patients was observed” . Ioan-Facsinayet.al( 2002 ) went farther stating that “Fci‚?RI ( CD64 ) Contributes Well to Severity of Arthritis” .

Although these consequences have yet to be widely assessed, the prevalence of both IgG and Fci‚?RI in the human organic structure suggest that they are the most likely immunological path to hypersensitivity in chondrocytes and subsequent tissue harm.

It has been noted that “the chronic production of IgM and IgG antiglobulins is a major characteristic of arthritic arthritis” ( Hall, 1980 ) . The survey of these antibodies, and the related Fc-R’s is hence an of import portion of fostering understanding about the causes and intervention of RA. There is limited grounds that chondrocytes express FcR’s

( Danilovaet.al, 1981 ) , and farther probe of this possibility will supply grounds of value to those measuring the proposed immune-response mechanism for the development of RA.

By amalogy, it may be seen that commanding both cytokines and Fc receptors is a possible path to direction of RA. Dolhain ( 1998 ) provides grounds that the drug Methotrexate can cut down the figure of inflammatory cells and ”adhesion molecules” (i.e.receptors ) in the blood stream of patients. Camussi ( 1998 ) discusses the future potency of drugs which manage arthritis by holding the overrun of TNF.

The possibility of commanding RA by finding the exact mechanism of the associated

2

immune hyperactivity is a premier incentive for surveies in this country.

More late, further inside informations of the possible function played by Fc receptors in the incidence of RF have been published. Firestein ( 2003 ) wrote inNaturethat T-cell complex interactions were being considered alongside the “aggressive tumour-like behavior of arthritic synovium” as of import and perchance synergic causitive factors.

1.3: Other Factors

As stated at the start of this subdivision, there is no individual accepted causative mechanism for RA, and there are legion other factors that would necessitate to be considered in a more elaborate survey. In peculiar, the importance of familial factors has been asserted by many workers ( Hall, 1980 ; McDowellet.al, 1995 ; MacGregor, 2000 ) , and has non been considered in this paper. Additionally, there are many surveies placing preexistent pathological infection as a specifying feature of RA patients. Hazenberg ( 1992 ) provides an early illustration, which investigates the nexus between known bacteriological conditions ( e.g. Crohn’s disease ) and joint strivings. It is possible that there is a nexus between anterior infection and subsequent hypersensitivity that determines the development of creaky conditions, and Hazenberg discusses such possibilities in more item.

1.4 Undertaking Aims

Based on the information gained from reappraisal of the current literature, the cardinal purposes of this undertaking were:

  • To find if the receptor Fci‚?-RI is expressed by chondrocytes under fake

in vivoconditions.

  • To measure the impact of consequences of this finding on the general hunt for a biomolecular theory of RA.

Owing to the assortment of possible, at the same time lending factors that have been described in this subdivision, and because of the deficiency of conclusive grounds to the contrary, the determination was made to follow a void hypothesis for proving. Therefore, the overall purpose of this undertaking was to prove the hypothesis:

“Chondrocyte cells culturedin vitroand exposed to conditions miming those in the human organic structure will non show the receptor protein Fci‚?-RI” .

The following subdivision describes the methods by which this hypothesis was investigated, and records the equipment used. ( Note: Where necessary, the names of makers are given after a peculiar stuff in the undermentioned manner: [Name] .

3

2. Materials and Methods

2.1: Experiment Design

The hypothesis stated in subdivision 1.4 was tested utilizing the undermentioned processs:

  • Chondrocyte cells were culturedin vitro, in conditions designed to mime conditions in the human organic structure -namely:

-Stimulation by the cytokines TNF-a and IL-1i?? .

-Hypoxia.

  • Deoxyribonucleic acid was extracted from the civilized cells.
  • PCR was carried out on a sample from each civilization.
  • The merchandises of the PCR procedure were characterised utilizing Gel Electrophoresis
  • The consequences of these procedures were recorded and compared with the consequences of earlier work.

In order to do the consequences of experiment every bit valid as possible, six tallies of each experiment were undertaken. Initially, it was planned that a Student T- trial would be used to prove the void hypothesis outlined in the debut, but ( as the treatment makes clear ) this program was altered owing to the nature of the consequences.

Health and Safety issues:

There are no specific wellness and safety issues in this survey that are non covered by standard research lab protocol ( the erosion of oculus protection, baseball mitts etc ) .

Chloroform has been identified as a possible carcinogen, and should be treated with particular attention

There are no ethical issues specific to this survey.

2.2: Cell Culture

Culture of C-20/A4 chondrocytes was carried out in 75cm flasks incorporating Dulbeccos Modified Eagles Medium ( DMEM ) [Cambrex] , and supplemented with 50 milliliters of 10 % fetal calf serum ( FCS ) [Biowest] , and besides with L-glutamine ( 200mM ) , penicillin/ streptomycin ( 10 mg/ml ) , and Garamycin ( 10 mg/ml ) . The experiment was carried out in a category II biosafety cabinet, maintained at 37OC and at 5 % CO2concentration.

4

2.3: RNA Extraction

As the first measure towards transporting out the DNA-Electrophoresis analysis, RNA was extracted from the civilized cells. The cells were lysed utilizing Tri-reagent [Sigma-Aldrich] , incubated at room temperature for 5 proceedingss, so transferred to centrifugate tubings. Each tubing was treated with 200 gl trichloromethane and incubated for a farther 3 proceedingss. Following centrifugation ( 15 proceedingss, 12,000g, 4OC ) , the RNA was precipitated by the add-on of 500 i?­l of isopropyl alcohol.

Each tubing so underwent:

  • Incubation at –80OC ( 30 proceedingss )
  • Centrifugation ( 10 proceedingss, 12,000g, 4OC )

The aggregative RNA was so washed with 75 % ethyl alcohol, followed by:

  • Centrifugation ( 5 proceedingss, 7500g, 4OC )
  • Filtration to obtain RNA solid.

The extracted solid was stored in 50 µl of 0.1 % DEPC- H2O.

2.4: PCR

The procedure of complementary DNA synthesis for PCR analysis was carried out utilizing reagents from Promega, chiefly MMLV rearward RNA polymerase.

1i?­l of 2.5 mM oglio-dt was added to the RNA –containing tubings, and the sample was made up to 12i?­l with Ultra High Quality H2O. Following incubation at 68OC for 5 proceedingss, the undermentioned reagents were added:

  • 2i?­l of 2.5 mM deoxyribonucleoside-triphosphate ( dNTP )
  • 1i?­l of RNAsin ( 40U /i?­l ) . [Promega]
  • 1i?­l of murine moloney leukemia virus contrary RNA polymerase ( MMLV )
  • ( 200U /i?­l ) . [Promega]
  • 4i?­l of MMLV-Buffer. [Promega] .

The mixture was incubated at 42OC for 60 proceedingss, so at 48OC for 10 proceedingss.

5

In transporting out the PCR runs, 5µl samples of the complementary DNA were placed in sterilized Eppendorf tubing. The undermentioned reagents were so added to each tubing:

  • 5µl Ammonia Buffer [Bioline]
  • 1µl each of Forward and Reverse primers
  • 1µl Magnesium Chloride [Bioline]
  • 1µl TAQ polymerase ( 5U/µl ) [Bioline]
  • UHQ H2O to do up to 50µl

The reaction mixture was so sealed with 30µl mineral oil.

The PCR was carried out utilizing:

For complementary DNA and Fc?RI – PCR Sprint Hybaid

For GAPDH – Eppendorf Mastercycler gradient

The tallies were as follows:

The sample was heated to 94-98OC for 7 proceedingss, followed by 1 minute at 37-65oC.Finally, the temperature was raised to 74OC

2.5: Gel Electrophoresis( see Sambrook & A ; Russell, 2001 )

This was conducted utilizing Horizon 11.14 Horizontal Gel Electrophoresis Apparatus.

1.5 % Agarose pulverization [Bioline] was assorted with TAE buffer and heated in a Microwave [Hinari Lifestyle] until to the full dissolved. After the mixture had cooled to 50OC, a bead of Ethidium Bromide [Sigma] was added, and the liquid gel was poured on to an cataphoresis home base, clamped with a comb, and allowed to put ( 30 – 40 proceedingss ) .

On scene, the gel was placed in an cataphoresis armored combat vehicle with the Wellss ( formed by the comb ) at the cathode. 4i?­l samples of the DNA created in2.4were pipetted in to the Wellss. The armored combat vehicle was covered, and 100 Vs applied for 30 proceedingss via a power battalion [Initiate] . On completion ( leting extra clip if necessary ) , the power was turned off and the gel removed and placed in an Ethidium Bromide solution ( 20 ?l of 10mg/ml in 50ml H2O ) for staining. After standing for 10 proceedingss in the discoloration, the gel was removed and bathed in H2O for 1 minute, so transferred to a UV Transilluminator.

6

3. Consequences

The undermentioned pages contain inside informations of the experimental runs that constitute this survey.

The analysis in table 3 is limited to one experimental tally, owing to troubles in reading: the degrees of Fc?RI were so low that they precluded the planned statistical analysis.

Figure 1: UV-Spectroscopy of merchandises

messenger RNA samples

messenger RNA measure ?g/mL

Ratio 260/280

Control 1

335.2?l/mL

1.22

Control 2

777.5?l/mL

1.36

Control 3

846.6?l/mL

1.40

Control 4

646.6?l/mL

1.35

Control 5

976.1?l/mL

1.42

Control 6

1136.1?l/mL

1.44

TNF? 1

620.4?l/mL

1.29

TNF? 2

220.8?l/mL

1.43

TNF? 3

550.3?l/mL

1.40

TNF? 4

610.1?l/mL

1.37

TNF? 5

1250.3?l/mL

1.41

TNF? 6

570.7?l/mL

1.35

IL -1? 1

603.5?l/mL

1.50

IL -1? 2

890.7?l/mL

1.52

IL -1? 3

3561.1?l/mL

1.71

IL- 1? 4

251.6?l/mL

1.46

IL – 1? 5

1025.4?l/mL

1.57

IL – 1? 6

1159.8?l/mL

1.23

Hypo 1

170.0?l/mL

1.35

Hypo 2

738.1?l/mL

1.05

Hypo 3

1420.4?l/mL

1.60

Hypo 4

1629.4?l/mL

1.62

Hypo 5

1644.1?l/mL

1.63

Hypo 6

148.7?l/mL

1.33

7

Figure 2: Position of a completed Gel ( 13ThursdayApril 2007 )

Figure 3: Consequences Postpone

Lane

Top ( GAPDH )

Bottom ( Fc?RI )

4: Hypo. 3

13640

59

5: Hypo. 4

8600

0

6: Hypo. 5

11354

195

7: Control.3

7600

38

8: Control 5

9670

21

9: Control 6

6478

171

10: Negative Control

8

Table 3 provides the clearest illustration of the consequences of these experiments: in all instances, the look of Fc?RI is minimum or non-existent. The two highest figures are for a hypoxic flask and a control flask severally, proposing that the mistakes involved are big plenty to drastically consequence consequences.

4. Discussion

Initially, it can be said that the consequences of this survey validates the statement made in the hypothesis:

“Chondrocyte cells culturedin vitroand exposed to conditions miming those in the human organic structure will non show the receptor protein Fci‚?-RI” .

However, there are several indexs that suggest that this consequence is non conclusive.

The most of import of these are the incompatibilities in figure 3. As already pointed out, table 3 shows no logical patterned advance of consequences, and the figures for Fc?RI are so little compared to those for GAPDH ( the former are an norm of 99 % lower than the latter ) that experimental mistake might easy account for the Fc?RI figures.

Beginnings of experimental mistake are discussed below. More basically, there is a demand to explicate the differences between the ascertained deficiency of look of Fc-Receptors by chondrocytes and the majority of the introductory literature which indicated that such look is platitude.

The following paragraphs explore possible accounts for this disagreement, backed up by appropriate literature mentions.

4.1: Factors lending towards mistake in the experiment

  • The initial experiment design did non try to make entirely realistic conditions for

the observation of possiblein vivoFc- look. Alternatively, a simplified theoretical account affecting two cytokines and some chondrocytic tissue was proposed, in order that the relevant protein look might be observed in a general research lab state of affairs. Lee ( 2001 ) , in reexamining the interventions available to RA sick persons outlined the trouble in utilizing animate being or ‘test tube’ research to pattern events in immune hypersensitivity. As the immune response ( and so RA itself ) is systemic, it is hard to retroflex in non-systemic conditions.

A related illustration in the literature is provided by Sharara ( 1997 ) , in a comparative

In vitro / in vivosurvey of Interferon activation of Nitric Oxide synthase protein look. The consequences do demo some congruity, but there are important differences between the bench theoretical accounts and the human trials that illustrate the potentially reduced viability thatin vitrowork can show.

9

  • Although the effects of look are well-documented, no unequivocal mechanism has

been proposed, so specifying the parametric quantities of this survey was mostly intelligent guessing.

For illustration, the determination to supply cytokines TNF-a and IL-1i?? as stimulation in the cell civilizations was taken with mention to old PCR / Immunology surveies that suggested a

nexus between cytokine concentration and RA ( for illustration: Choy, 2001 ) . During the readying of this paper, a colleague utilizing immunofluorescence techniques achieved consequences that suggested that hypoxia in chondrocytes was the premier incentive for Fc-Receptor look ( see besides Mapp, 1995 ) , and that the presence of cytokines had no important consequence on the procedure. Without unafraid cognition of the variables that are being dealt with, any consequences are subjective and unfastened to blemish that may non be recognised by the research worker. Similarly, although the cytokines in this survey were chose for their high comparative copiousness in the human organic structure, there is grounds to propose that other groups may be more of import in the RA-mechanisms: Yuasaet Al( 1999 ) claimed that the riddance Fci‚? IIB induced collagen harm in mice, for illustration.

  • The influence of familial factors, as antecedently discussed, is potentially really

big ( given the figure of suspected familial autoimmune conditions that are already good documented ) . Weyand ( 1995 ) investigated the clinical categorization of RA patients by familial factors, and concluded that such an attack identified cistrons that have “a alone function, the apprehension of which may supply of import hints to disease etiology” . The fact that this survey does non integrate the influence of familial background in to finding a protein-expression mechanism suggests that it is, at the least, uncomplete.

  • The process for culturing, pull outing RNA, organizing Deoxyribonucleic acid and analyzing it

needfully contains a big figure of stairss and involves legion dilutions. Although experimental work was completed within normal research lab protocols, this multi-step attack is likely to take to incompatibilities between different tallies.

  • Despite the qualitative nature of the PCR process, the reading of its consequences

are extremely qualitative. Consequences in this experiment were limited by interpretive troubles, and the usage of ICT-Assisted PCR should be considered to ease analysis in future surveies ( Ramakers et.al, 2003 ) .

10

4.2: Value of the experiment

Although no conclusive replies have been produced by this survey, there are several utile consequences which may be recorded for usage in farther work. This subdivision inside informations these consequences, and ends with a list of suggestions for farther work.

  • Strictly cytokine-mediated mechanisms are of limited value for explicating FcR

look in chondrocytic tissue.

  • Simulation of hypoxic conditionsin vitrois likely more complex than merely

excepting O from a reaction vessel– the nature of the inerting gas and the biological procedures which would take to hypoxia if the reaction werein vivo.

  • PCR and cataphoresis in tandem provide a utile agencies for measuring the extent of

look of FcR’s.

  • There are a big figure of single surveies in the literature that propose different

mechanisms for protein look, peculiarly in the instance of RA ( where it appears to be a cardinal factor ) . The differences between surveies are frequently due to differences in experimental method

4.3: Suggestions for farther work

  • Repeat of the experiment with a scope of different cytokines – this will assist to

characterize the broader function that these compounds play in FcR look, and will supply a context for the consequences of this survey. Similarly, repeat of the experiment utilizing a different set of stimulating factors will bring forth a more rounded image of the behavior of protein-expressing tissue.

  • Designing of a different experiment to prove the same hypothesis, utilizing ( for illustration )

immunofluorescence. This will supply an indicant of the influence that the method of word picture has on these experiments.

  • Repeat of the experiment, with coincident reading of consequences by manus and

by computing machine. This will assist to find the extent to which qualitative reading affects the consequences of surveies like this.

  • Repeat of the experiment with other tissue types, and word picture of any

Fc R’s or other proteins expressed. This will supply background information about any general features of protein look that have non been uncovered by this survey.

11

  • Repeat of the experiment at a assortment of temperatures / ppO2to more accurately

imitate the variable conditions found in beings.

  • ( With appropriate ethical counsel ) Probe of familial links to protein

look by the survey of a sample population (e.g.mice ) . This is the lone satisfactory manner of characterising and look intoing familial immunology effects.

  • Comparison of the original experiment or any of its derived functions with the literature to

place any utile convergence:

  • Word picture of known FcR’s and similar proteins to bring forth a set of sample

PCR / cataphoresis consequences that can be used as a benchmark in farther surveies.

  • The application of standard statistical theoretical accounts to the informations from these experiments, to

look into their utility and to supply a mathematical step of hypothesis cogency.

4.4: Decisions

As this treatment has made clear, there are many points touched on in this survey that require farther probe or repeat before conclusive thoughts can be formed. However, the following general decisions can be made with mention to the experimental work carried out during the production of this paper:

  • The function of cytokines ( peculiarly TNF-a and IL-1i?? ) in advancing look of

Fci‚?-RI is unsure, but it is improbable that cytokine mediation is the lone mechanism, ( bothin vitroandin vivo) . Further research in to the consequence of other cytokines, familial factors and similar subjects is hence desirable.

  • In Vitrosurveies have value as explorative indexs of possible mechanisms, but are

unlikely to take to direct accounts ofin vivoprocedures.

  • Much of the current research in to Fc-R look is completedviaPCR surveies, and

these have produced some utile consequences. However, farther usage of techniques such as immunofluorescence should be undertaken in order to contradict some of the built-in defects in the pattern of PCR’s.

  • As either a cause or an consequence, the look of FcR’s is linked to the incidence,

development and intervention of RA, and so research in this country is an indispensable portion of

developing pattern in modern health care, every bit good as fostering our apprehension of molecular biological science.

12

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Drugs. 55 ( 5 ) , 613 – 620.

Choy, E ( 2001 ) :“Cytokine tracts and joint redness in arthritic arthritis” .

New England Journal of Medicine. 344 ( 12 ) , 907 – 16.

Cush, J ( 1988 ) :“Phenotypic analysis of synovial tissue and peripheral blood lymph cells isolated from patients with arthritic arthritis” .

Arthritis Rheum. 31 ( 10 ) , 1230 – 1238.

Danilova, T [et.al] ( 1981 ) .“Studies of Fc receptors of bosom valve and joint fibroblasts” .Clin Exp Immunol. 46 ( 3 ) , 575 – 580.

Dolhain, R [et.al] ( 1998 ) :“Methotrexate reduces inflammatory cell Numberss, look of monokines and of adhesion molecules in synovial tissue of patients with arthritic arthritis” .British Journal of Rheumatology. 37, 502 – 508.

Eales, L ( 2003 ) : “Immunology for Life Scientists”.

John-Wiley & A ; Sons ( Publisher ) . 2neodymiumEdition.

Feldmann, M ( 1996 ) :“Role of cytokines in arthritic arthritis” .

Annual Review Immunology. 14 397-440.

Fridman, W ( 1991 ) :“Fc receptors and Ig binding factors” .

FASEB Journal. 5, 2684-2690.

Hall, N ( 1980 ) :“Lymphocytes bearing Fc gamma receptors in arthritic arthritis” .

Ann Rheum Dis. 39 ( 6 ) , 554-558.

Hazenberg, M [et.al] ( 1992 ) :“Are enteric bacteriums involved in the etiology of rheumatoid arthritis? ” .

APMIS. 100 ( 1 ) , 1 – 9.

Ioan-Facsinay, A [et.al] ( 2002 ) :“Fci‚?RI ( CD64 ) Contributes Well to Severity of Arthritis, Hypersensitivity Responses, and Protection from Bacterial Infection” .

Immunity. 16 ( 3 ) , 391 – 402.

Lee, D ( 2001 ) :“Rheumatoid Arthritis” .

Lancet. 358 ( 9285 ) , 903 – 911.

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Low, J. and Moore, T ( 2005 ) :“A function for the complement system in arthritic arthritis” .Curr Pharm Des. 11 ( 5 ) , 655-670.

MacGregor, A ( 2000 ) :“Characterizing the quantitative familial part to rheumatoid arthritis utilizing informations from twins” .

Arthritis Rheum. 43 ( 1 ) , 30-37.

Mapp, P [et.al] ( 1995 ) :“Hypoxia, oxidative emphasis and rheumatoid arthritis” .

British Medical Bulletin. 51, 419 – 436.

McDowell, T [et Al] ( 1995 ) :“A familial association between juvenile rheumatoid arthritis and a fresh interleukin-1 alpha polymorphism” .

Arthritis Rheum. 38 ( 2 ) , 221-228.

NHS ( 2007 ) :“Health Encyclopaedia: Rheumatoid Arthritis” .Retrieved on 11/27/2007:

hypertext transfer protocol: //www.nhsdirect.nhs.uk/articles/article.aspx? articleId=321

Nieto, A [et.al] ( 2000 ) :“Involvement of Fci‚?receptor IIIA genotypes in susceptibleness to rheumatoid arthritis” .Arthritis Rheum. 43 ( 4 ) , 735-739.

Ramakers, C [et.al] ( 2003 ) :“Assumption-Free analysis of quantitative real-time PCR” .

Neuroscience Letterss 339, 62 – 66.

Sharara, A [et. Al] ( 1997 ) :“Interferon ( IFN ) – Activation of Human Blood Mononuclear Cells In Vitro and In Vivo for Nitric Oxide Synthase ( NOS ) Type 2 messenger RNA and Protein Expression” .J Exp. Med. 186 ( 9 ) , 1495-1502

Weyand, C [et.al] ( 1995 )“Correlation between disease phenotype and familial heterogeneousness in arthritic arthritis” .J Clin. Invest. 95 ( 5 ) , 2120–2126.

Wolfe, F [et.al] ( 1994 ) :“The Mortality of Rheumatoid Arthritis” .

Arthritis Rheum. 37 ( 4 ) , 481 – 494.

Yuasa, T [et.al] ( 1999 ) :“Deletion of Fc Receptor IIB Renders H-2b Mice Susceptible to Collagen-induced Arthritis” .J Exp. Med. 189 ( 1 ) , 187 – 194.

14

Appendix I: Suppliers

Tissue civilization

Cell civilization of C-20/A4 chondrocytes

1 litre tissue civilization medium DMEM ( obtained from Cambrex )

50 ml 10 % Foetal calf serum obtained from Biowest

TNF –? / ? – coiling CRH antibody

Tris-borate ( TBE ) buffer

100g Tns base

100g Boric acid

50g Ethylenediaminetetraacetic acid

Deoxyribonucleic acid isolation

100 milliliter Phenol

100 milliliters Chloroform

100 milliliter Ethanol

10g Sodium Acetate

Proteinase K

Sodium dodecyl sulfate ( SDS )

PCR

2 milliliter PCR mastemix ( contains NTPs, Taq polymerase, buffer and Mg )

The RT-PCR reagents – Taq polymerase and dNTPs will come from Bioline and Promega.

1 milliliter specific primers for Fc receptors will be selected from Sigma-genosis

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Electrophoresis

Deoxyribonucleic acid criterions

1 ml Ethidium bromide ( 10mg/ml ) – will be available from Bioline and the ……

20g Agarose

Bromophenol/glycerol lading buffer

Light microscopy

100g NaCl

PBS ( tablets 100 milliliter )

5 milliliter Lugols I

Fluorescent microscopy

Laser Scanning Confocal Microscopy

Tissue civilization plastics

Obtained from Orange Scientific.

Flasks, flasks with cap, trial home bases, extractors tubes, civilization dishes, and cell scraper, serological pipettes: P20, P100, P200 and P1000 Gilson pipettes/tips

Antibodies

Obtained from Deco and Merck-Sigma.

Other equipment:

Thermocyclers – for transporting out polymerase concatenation reaction ( PCR ) .

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