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Cellular pathology
Rheumatoid arthritis
Rheumatoid arthritis (RA) is a systemic, chronic inflammatory autoimmune disease affecting
many tissues but principally attacking the joints
...
RA is a highly
variable diase, differing greatly between individuals, and with a variable course of
exacerbations and remissions
...

Epidemiology
RA is a relatively recent disease
...
RA is a relatively common condition, with a prevalence of 1% in adults
...
RA is more common in women than men
...

Causes
RA is a disorder in which genetic and environmental factors contribute to the breakdown of
tolerance to self antigens
...
Susceptibility to RA is linked to the HLA_DRB1 locus
...
There is a strong association with polymorphism in
the PTPN22 gene, which encodes a tyrosine phosphate that is postulated to inhibit T cell
activation
...

A genetic susception to RA may be triggered by environmental factors
...
The impact of
environmental factors was evidenced in monozygotic twin studies in which only the twin that
smoked developed RA
...
Most often the proximal
interphalangeal and metacarpophalangeal joints are affected, but distal interphalangeal
joints are spared
...
Constitutional symptoms include weakness, malaise,
and fever, caused by the mediators responsible for joint inflammation
...
RA appears
insidiously, with aching and stiffness of the joints
...
Vasculitic involvement of the extremities may give rise to
Raynaud phenomenon and chronic leg ulcers
...
The clinical course of RA is highly variable
...
Spontaneous remission can occur after acute onset
...
In the majority of cases RA pursues a chronic,
remitting-relapsing course
...

Diagnostic features of RA; one or more joints have synovitis which cannot be explained by
another cause, presence of rheumatoid factor, presence of anti-cyclic citrullinated peptide
antibody (ACPA), raised serum levels of C-reactive protein, increased erythrocyte
sedimentation rate, characteristic radiographic findings, sterile turbid synovial fluid with
decreased viscosity, poor mucin clot formation, and inclusion bearing neutrophils
...
The
classic appearance is that of a pannus, formed by proliferating synovial lining cells admixed
with inflammatory cells, granulation tissue, and fibrous connective tissue, the overgrowth of
this tissue is so exuberant that the usually thin, smooth synovial membrane is transformed
into villous projections
...
With progression of the disease, the articular cartilage subjacent to
the pannus is eroded and, in time, virtually destroyed, the subarticular bone may also be
attacked and eroded
...

Radiographic hallmarks include; joint effusions, and juxta articular osteopenia with erosions
and narrowing of the joint space and loss of articular cartilage
...

Synovial joints
Synovial joints are those in which the articulating bones are separated by a fluid containing
joint cavity
...
Synovial joints have six distinguishing features
...
Joint (articular) cavity that contains synovial fluid
...
Synovial fluid occupies all free spaces within the joint capsule, this fluid is derived
largely by filtration from blood flowing through the capillaries in the synovial membrane,
synovial fluid reduces friction between the cartilages, the fluid is forced from the cartilages
when a joint is compressed then as pressure on the joint is relieved synovial fluid seeps
back into the articular cartilages, this process is termed weeping lubrication, and lubricates
the free surfaces of the cartilages and nourishes their cells, synovial fluid also contains
phagocytic cells that rid the joint cavity of microbes and cellular debris
...
Synovial joints are richly supplied with sensory
nerve fibres that innervate the capsule
...
The external layer is rich with
lymphatic and blood vessels and is composed of macrophages and fibroblasts
...

Pathogenesis
RA disease pathway; autoimmune component, the immune system attacks the joint lining,
leading to inflammation of the joint lining, untreated inflammation leads to joint damage,
joints become hot, red, swollen, and stiff, ultimately joint damage cannot be repaired
...
These autoantibodies are
called rheumatoid factors
...
The incidence of rheumatoid
factor increases with duration of disease
...
Rheumatoid factor can be found in joints affected by
RA and can be detected before RA presents clinically
...
CCPs are derived from proteins in which arginine residues are
converted to citrulline residues postrantranslationally
...
These antibodies are a diagnostic marker for the disease
...
In particular, HLA-DRB1 molecules sharing a common sequence,
R(Q)K(R)RAA, the so called shared epitope, are associated with both susceptibility and

severity of RA, with some substitutions in the shared epitope conferring protection from RA,
and some leading to severe RA
...
Outside the HLA region, other genetic risk alleles for RA encode
molecules implicated in T-cell activation, such as protein tyrosine phosphatase non-receptor
type 22 (PTPN22), cytotoxic T-lymphocytes associated protein 4 (CTLA-4), and signal
transducer and activator 4 (STAT4)
...
Exposure to HLA
susceptibility alleles through non inherited maternal HLA antigens, in particular HLA-DR4
and in DR4 negative patients, has been proposed to shape the immune system of the SE
negative fetus, thus increasing the fetus’s proneness to the development of RA
...
Non-inherited maternal antigens in this instance
consists of the HLA alleles present in the maternal haplotype non-inherited to a given child
but to which the immune system of that child was exposed to during fetal development
...
Women who jack the RA protective sequence DERAA
may benefit from parity through the acquisition of microchimerism with DERAA, similarly
women who were at risk for RA because they had the SE would benefit from parity in a
similar way
...
While SE negative women could also occur benefit from
DARAA positive microchimerism these women are not at risk of RA
...
However,
minimal additional impact may occur for a woman who is herself SE-positive
...
On the other hand, DERAA negative women who lack protection and so
may benefit from acquiring DERAA positive microchimerism
...

The pathologic changes in RA are caused mainly by cytokine-mediated inflammation, with
CD4+ T cells being the principal source of cytokines
...
CD4+ Th1 17 cells, activated B
lymphocytes, plasma cells, and macrophages, as well as other inflammatory cells, are found
in the inflamed synovium
...
Cytokines produced by the
activated T cells recruit leukocytes such as macrophages, whose products cause tissue
injury, and also activate resident stromal cells to produce proteolytic enzymes, such as
collagenase, that mediate destruction of the cartilage, ligaments, and tendons of the joints
...

Type A synoviocytes can trigger type B synoviocytes to release an array if mediators
including; IL-6, prostanoids, and matrix metalloproteinases (MMPs)
...

Synovial membrane hyperplasia leads to pannus formation
...
The pannus contains; macrophages,
osteoclasts, invasive type B synoviocytes, and lymphocytes
...

Type B synoviocytes express cartilage adhesion molecules, they bind to articular cartilage
...
The cytokines IL-1b and TNF-alpha
can also stimulate the endogenous cartilage cells to over express degradative enzymes
...
TNF-alpha and
macrophage colony stimulating factors are also involved in osteoclast recruitment
...
Loss of bone causes sevre,
irreversible joint deformation
...
Arginine can
be converted to citrulline by a process called deimation
...
PAD2 and PAD4 enzymes are the most relevant to RA
as they are expressed by certain leukocytes
...
During cell death, as
caused by smoking, the integrity of the plasma membrane is lost, leading to an influx of
calcium ions from the extracellular space and subsequent activation of intracellular PAD
...
Activated PAD enzymes can then cause citrullination of extracellular
proteins
...
Active PAD
enzymes then citrullinate; vimentin, a component of dying human macrophages; fibrin,
formed due to synovial tissue microinfarctions caused by hypoxia which is caused by
inflammation; and histone
...
Genetic polymorphisms, such as single nucleotide polymorphisms,
exist within the PAD4 gene
...
This RA-susceptibility haplotype increases PAD4 mRNA stability, this results in greater
production of PAD4 enzyme and subsequently increased citrullination of proteins and a
higher chance of developing anti-CCP antibodies
...

Citrulinnated peptides can be bound much more efficiently by HLA-DR4 molecules
associated with RA than by corresponding non-citrullinated peptides
...

IL-10 promoter single nucleotide polymorphisms are associated with increased ACCP

antibody production and increased severity of RA
...

Immune system
Autoantibodies IgM that bind IgG form large immune complexes that are too large to escape
from the blood vessels, and so are readily removed from the circulation
...
However, autoantibody IgG and IgG
complexes are much smaller and can pass between blood vessel endothelial cells and enter
the tissues, where the complex preferentially binds to the macrophage FCgRIII (CD16a)
(expressed in the synovial joint), thus activating the macrophage
...
T and B cells migrate
to the joint
...

Stromal cells facilitate microenvironment for T and B cells
...
This complex activates ,macrophages and the
cycle continues
...
Etrancept is a soluble TNF receptor
dimer, composed of human p75 TNFR and Fc domain of human IgG1
...
Chronic inflammation is
associated with cancer to a greater extent than acute inflammation
...
Inflammatory cells, also modify the local
tumour microenvironment to enable the hallmarks of cancer, these effects may stem from
direct interactions between inflammatory cells and tumour cells, or through indirect effects of
inflammatory cells on other resident stromal cells, particularly cancer associated fibroblasts
and endothelial cells
...
Infiltrating leukocytes and activated
stromal cells secrete growth factors, such as EGF, and proteases that can liberate
growth factors from the ECM
...
The growth of epithelial cells is suppressed by
cell-cell and cell-ECM interactions, proteases released by inflammatory cells can
degrade the adhesion molecules that mediate these interactions, removing a barrier
to growth
...
Detachment of epithelial cells from basement
membranes and from cell-cell interactions can lead to a form of cell death called
anokis
...
Stromal cell-cancer cell interactions increase the resistance of cancer cells to
chemotherapy
...
Inflammatory cells release numerous factors, including
VEGF, which can stimulate angiogenesis
...
Proteases released from macrophages foster
tissue invasion by remodelling the ECM, while factors such as TNF and ECM may
directly stimulate tumour cell motility
...

Evading immune destruction
...
Advanced cancers contain
alternatively activated macrophages which produce cytokines that promote
angiotensin, fibroblast proliferation and collagen deposition
...

Induction of DNA damage by the generation of free radicals
...

Promotion of angiogenesis and tissue remodelling by the production of growth
factors, cytokines, chemokines, and matrix metalloproteinases
...

Suppression of antitumor adaptive immune responses
...

Promotion of tumour development by humoral immune responses that increase
chronic inflammation in the tumour microenvironment
...
Affected mice develop nodules of dysplastic hepatocytes, localised
hepatocellular carcinomas (HCCs) and eventually metastatic HCCs
...
The released TNF-alpha
acts in a paracrine manner on nearby hepatocytes, which display receptors for TNF-alpha
...
Bcl-Xl), proliferative genes (eg
...
Together, the proteins encoded by these genes
facilitate the progression of the dysplastic nodules to hepatocellular carcinomas
...
when NF-KB signaling was blocked in the
hepatocytes of the mdr- mice, through the introduction of an anti-TNF-alpha antibody which
blocked the paracrine IKB in the hepatocytes which inhibited NF-KB activation, tumour
incidence was strongly suppressed
...
HCCs, which
are common in East Asia, are associated with chronic hepatitis B virus (HBV) infections and
accompanying inflammation of the liver
...
Alternatively, HBV infection may cause cells of the immune system to
attempt to eliminate virus-infected cells, yielding a chronic inflammatory state in the liver
...

Genome-wide association studies have identified several inflammatory bowel disease (IBD)
susceptibility loci that contain genes that encode cytokines and proteins that involve cytokine
signalling
...
Cytokines have a role in driving complications in IBD such as intestinal stenosis,

fistula formation and colitis-associated neoplasms
...
TNF-specific
antibodies suppress chronic intestinal inflammation and may induce mucosal healing in IBD
...

Inflammatory stimuli, including chronic infections, organ injury, irritants, cell necrosis, and
senescence, result in recruitment of inflammatory cells into areas of inflammation
...
This results in the production of more
TNF-alpha, antiapoptotic proteins (eg
...
Prostaglandins synthesised by COX-2, eg
...

The TPA skin tumour promoter converges o n this pathway through the activation of
PKC-alpha, in addition, TPA-activated PKC-alpha can induce mitogenic signals by activating
the Fos, Jun, and AP-1 transcription factors
...

Together, these actions of inflammatory cells and of TPA create phenotypic states in
epithelial cells that resemble those induced by oncogenes, enabling initiated cells to launch
clonal expansions that result in the acquisition of additional mutant alleles by their
descendents
...
It acts
by inducing increased apoptosis and reducing cell proliferation
...

immunodeficiencies , such as AIDs or due to an immunosuppressive drug regime post liver
transplantation, increases the risk of development of certain cancers, such as kaposi
sarcoma, non-Hodgkin lymphoma, skin and cervical cancer
...
This suggests immune cells may have both anti- and pro- tumour
effects
...

Chronic inflammation and cancer
Cancer risk is increased in individuals affected by a variety of chronic inflammatory diseases,
including those with infectious and non-infectious etiologies
...

Chronic inflammation may increase the pool of tissue stem cells, which may be particularly
susceptible to transformation
...
Chronic epithelial injury often leads to metaplasia, the

replacement of one cell type with another that is much better able to survive the ongoing
insult
...
In the long term such alterations may
allow cells with potentially oncogenic mutations to survive, eventually leading to cancer
...
Stromal cells include fibroblasts, myofibroblasts, endothelial cells, pericytes, smooth
muscle cells, adipocytes, macrophages, lymphocytes, and mast cells
...
Carcinoma cells may control the
populations of stromal cells near them, perhaps by recruiting stromal cells from nearby
normal tissues and distant bone marrow and then encouraging their proliferation
...

Heterotypic signaling channels depend on the exchange of; mitogenic growth factors, such
as hepatocyte growth factors, transforming growth factors-alpha, and platelet derived growth
factors; growth inhibitory signals, such as transforming growth factor-beta; and trophic
factors, such as insulin-like growth factor-1 and -2, which favor cell survival
...
For example,
carcinoma cells release growth factors, cytokines, and chemokines that recruit
macrophages, neutrophils, and lymphocytes to the tumour-associated stroma, the recruited
cells then orchestrate an inflammatory response that involves the release of TNF-alpha and
prostaglandins, which stimulate the proliferation of nearby epithelial cells and the process of
angiogenesis within the tumour stroma
...
Epithelial cells within a carcinoma
often release PDGF, for which stromal cells possess receptors, the stromal cells respond by
releasing IGF-1, which benefits the growth and survival of nearby cancer cells
...
Stromal cells in breast cancers
release the chemokine SDF-1/CXCL12 and the MGF/SF growth factor, which stimulate the
proliferation and survival of neary epithelial cancer cells
...
Tumour cells may express PDL1 on their cell surface which binds
to the PDL receptor present on cytotoxic T cells, this binding inhibits the cytotoxic T cell
response
...
The tumour stage
dictates the function of these immune cells; early stage immune cells have an anti-tumour
role, late stage immune cells are educated by the tumour and consequently have a tumour
promoting role
...
Expression,
deposition, organisation and turnover of collagen are implicated in tumour progression
...

Collagen cross-linking occurs by the action of enzymes lysyl hydroxylase and lysyl oxidase,
tumour cells highly express these enzymes
...
MDSC has an immune regulatory role
...
MDSCs are a valuable predictive
marker in cancer
...
During unresolved inflammation, such as in cancer, the nature
of signals activating myeloid cells differs
...
Neutrophils and monocytes
generated under these conditions display an immature phenotype, relatively weak
phagocytic activity, increased levels of reactive oxygen species, increased nitric oxide
production, high expression of arginase, PGE2, and antiinflammatory cytokines
...
Cells in this pathologic state of activation are
termed MDSCs
...
The first group of signals is important for the expansion of immature myeloid cells
...
The second group of signals is responsible for the pathologic activation of MDSCs
...
PMN-MDSCs can be
identified due to expression of the surface markers CD115 and CD244
...
M-MDSCs can
be distinguished from tumor associated macrophages (TAMs) since TAMs have high
expression of F4/80, low-intermediate expression of ly6c and low-absent expression of
SIBA9 protein
...
Tumour
infiltrating MDSC shows increased mitochondrial mass, key FAD associated genes and
oxygen consumption rate
...
MDSC demonstrates a high ER
stress response, resulting in enhanced immunosuppressive capacity MDSC by increasing
expression of ARG1, NOS2, and NOX2
...
Other mechanisms of MDSC mediated immune suppression include up-regulation
of regulatory T cells and immature suppressive cytokines
...
In melanoma and breast cancer,
PMN- and M- MDSC numbers correlate with stage and metastasis
...
Accumulation of M-MDSC in peripheral blood
was associated with overall survival in colorectal, bladder thyroid and uterine cervical
cancer
...
M-MDSC numbers correlated with reduced survival in MM, Hodgkin and
non-Hodgkin lymphoma and diffuse large B cell lymphoma
...
M-MDSC numbers negatively correlated with response to chemotherapy in
breast, cervical, prostate and colorectal cancer, squamous cell carcinoma, MM, and Hodgkin
lymphoma
...
M-MDSC numbers were a predictor of radiotherapy failure in
hepatocellular carcinoma
...
In melanoma, M-MDSC frequency predicts failure of the second line
immunotherapy, Nivolumab, following failure of first line ipilimumab treatment
...
Macrophage abundance is associated with a
poor prognosis
...
TAMs originate from circulating monocytes that were
recruited in response to chemotactic signals released from tumour cells, monocyte derived
TAMs are continuously replenished by peripheral recruitment
...

TAM precursors include; inflammatory monocytes and M-MDSCs
...
A smaller subset of TAMs may arise from ly6c low monocytes, which
include cells that express the angiopoietin-z, ANG2 receptor TIE2, these cells are recruited
in response to ANG2 secretion by the tumour endothelial cells and play a role in tumour
neovascularization
...
macrophages
can reversibly alter their endotype in response to environmental cues such as stimuli derived
from pathogens, parenchymal cells, immune cells and the ECM
...
macrophages
stimulated by IFN-gamma or LPS polarize to M1, whist macrophages stimulated by IL-4/13
polarize to M2
...

These molecules exert and amplify antimicrobial and tumouricide activities through
enhanced antigen presentation
...
M2 macrophages are further divided into M2a,
M2b, M2c and M2d
...
M2a macrophages express high levels of mannose receptor (CD206) and secrete
pro-fibrotic factors including fibronectin, insulin-like growth factor and TGF-beta, which are all
involved in wound healing and tissue repair
...
They produce IL-10, IL-1beta, IL-6, and TNF alpha, which exert
antiinflammatory effects
...
TGF beta and

glucocorticoid s, and have antiinflammatory effects
...
M2d macrophages produce IL-10 and VEGF
...

HBV and helicobacter pylori, and irritants eg
...
These transformed neoplastic cells consequently
produced inflammatory mediators including TNF- alpha and IL-1beta that form a closed
paracrine loop top perpetuate this tumour reactive microenvironment
...
TAMs have an immunosuppressive function
...
TAMs produce chemokines CCL17 and CCL22, which attract Th2 and Treg cells
that steer monocyte differentiation toward an anti inflammatory M2 endotype
...
The benign-to-malignant transition if most solid cancers is marked by a
significant increase in blood vessel formation known as angiogenic switch
...
They express a
enrichment of transcripts that encode angiogenic molecules
...
Metastatic progression is
dependent on the crosstalk between cancer cells and macrophages
...
They are a rich source
of proteases including clothespins, MMPs, and serine proteases that promote ECM
degradation and allow the escape of tumours from the v=basement membrane through the
dense stroma
...
WntSa acts through
the non-canonical WNT pathway to stimulate cancer cell motility
...
The CCL2/CCR2 axis between
cancer cells and MAMs may promote bone metastasis of prostate cancer by supporting the
activation of osteoclasts
...
Human TAMs are typically identified by
CD68 expression, CD163, CD206, and CD204 expression identify M2 macrophages
...
Increased macrophage
infiltration is associated with advanced stage disease and overall worse survival in breast,
pancreatic, and bladder cancer
...
TAM density may be used as a prognostic marker to predict
chemotherapy response
...
Alpha CCL2 monoclonal antibody
carlumab was developed to limit monocyte recruitment
...
CCR2 antagonists also act to limit monocyte recruitment
...
Alpha- CD40 monoclonal antibody (CP-870 893) was
developed to depolarize macrophages from the M1 endotype to the M2
...
The CD40 agonist antibody showed
efficacy against pancreatic carcinoma leading to tumour regression
...
genetic loss of CSF1 results in significantly reduced
metastasis and delayed tumour progression in breast and neuroendocrine mouse tumour
models,
...
Another
therapeutic option is to deplete macrophages, Trabectedin decreases the number of TAMs
in tumour by inducing apoptosis of monocytes and macrophages
...
FDA approval in 2015 for use in
unresectable or metastatic liposarcoma
...
tTregs preserve self-tolerance and prevent autoimmunity, pTregs ,maintain
peripheral tolerance at mucosal interfaces and in response to external antigens
...
Tregs exhibit considerable phenotypic and functional specialisation according to
tissue localisation, disease state, activation and differentiation status
...
An
accumulation of FoxP3+ Tregs and, in particular, a higher Treg: T effector cell (Teff) ratio
within tumour tissue is associated with worse prognosis in ovarian cancer, pancreatic ductal
adenocarcinoma, lung cancer glioblastoma, non-Hodgkin's lymphoma, and melanoma
...
Treg depletion prior to treatment
is associated with an anti-tumour immune response and improved clinical outcomes
...
In these cancers, Tregs play a protective role in
controlling inflammation associated with neoplastic transformation and cancer progression
...
Tregs can be
expanded in situ, and proliferate efficiently in response to tumour derived factors, such as

TGF-beta and IL-10, within the TME
...
Sphingosine 1 phosphate (S1P)- a bioactive lipid mediator
involved in angiogenesis and inflammation- is able to restrain Treg development in the
periphery
...
Tregs rely on antigen presenting cells such as dendritic cells
and macrophages for antigen presentation and T cell activation
...
Within the TME,naive telegeic DCs and plasmacytoid DCs (PDCs)
promote Treg function and generation
...
low
dose metronomic cyclophosphamide (CTX) can reduce the levels of Tregs within the TME
...
Immune checkpoint
inhibitors (ICIs) for cancer treatment aim to recover antitumor immune responses by blocking
inhibitory immune checkpoint molecules or their ligands, which are often over expressed om
intratumoral lymphocytes populations and tumour tissues
...
Monoclonal antibodies against CTLA-4 and PD-1 have been
approved by the FDA for treatment of metastatic melanoma, NSCLC and Hodgkin's
lymphoma
...
Tregs are radio resistant,
exhibiting reduced apoptotic potential and increased in vivo proliferation compared to other
lymphocyte subsets in response to ionising radiation
...

Chemokine receptor blockade may impair Treg functioning as well as depleting Tregs
...
There are a number of challenges to consider when designing Treg
targeted therapies in the context of cancer, including; misclassification of Tregs, systemic
versus specific subset depletion, Tregs in the immune context
...
Exosomes originate from the endosome; system, while
microvesicles are shed from the plasma membrane
...
EVs are an additional
mechanism for intercellular communication, allowing cells to exchange proteins, lipids, and
nucleic acids
...
As a result, these cells are adapted or recruited to a
constantly evolving cancer microenvironment
...
Circulating EVs can also transport important
molecules to remove destinations in order to prime metastatic niches in an otherwise healthy
tissue
...
Production of exosomes starts at the plasma membrane through
endocytosis at cholesterol enriched lipid raft domains
...
The product is referred to as a
multivesicular body (MVB)
...
Microvesicles and large oncosomes are derived by membrane blebbing
...
EV release is increased upon
hypoxia, calcium ion influx, or exATO
...
26
...

Cancer associated fibroblasts
Cancer associated fibroblasts (CAFs) are a heterogenous population of cells derived from
resident fibroblasts
...
Alpha smooth muscle actin is a
biomarker used to identify CAFs
...

Podoplanin-a, SIO4, vimentin, fibroblasts specific protein q (FSP-1), and PDGF alpha/beta
are all expressed in CAFs
...
Cell proliferation through paracrine tumour-stroma interactions
...

When cancer cells metastasize to another organ they recruit fibroblasts to the tumour mass
...
cell to cell communication through adhesion molecules enables fibroblast activation
...
CAFs are targets and
inducers of tumorigenic activation signals
...
Chemokines
secreted by CAFs into the microenvironment allow for the recruitment of bone marrow
derived cells (BDCs) and immune cells
...
In oral squamous cell
carcinoma, CCL2 expression in CAFs is upregulated promoting the production of ROS in
cancer cells, ROS promotes proliferation, migration and invasion of cancer cells
...

CAF derived PDGF is an essential factor in activating VEGF
...
PDGFs indirectly promote
angiogenesis by recruitiung stromal fibroblasts that secrete VEGF
...
The mesenchymal like
phenotype of CAFs enhances the metastasis of breast cancer cells
...
CAFs secrete proinflammatory cytokines
that stimulate the nuclear factor KB signalling pathway, subsequently promoting
tumorigenesis
...
A reduction in miR-148a expression in CAFs results in increased Wnt signalling
through upregulation of its target gene WANTED
...
CAFs with reduced expression of
miR-26b promote the migration and invasion of
breast cancer cells
...
A bi-directional activation between cancer cells and
fibroblasts causes the formation of the malignant phenotype of cancer
...
In return, fibroblasts secrete GFs such as; MGF,
keratinocyte GF, and IGF-½, which stimulate the proliferation of cancer cells
...
IGFs
secreted by fibroblasts in turn induce cancer cell proliferation and the synthesis of PDGF
...
One approach to anti-cancer
therapy is the inhibition of the feedback loop between fibroblasts and cancer cells, through
inhibitor if fibroblast directly and disruption of CAF associated paracrine GF signals
...
Morphological evidence describes it as an desmoplastic reaction involving
many cell types including CAFs
...
Patients who have a desmoplastic
reaction rich in CAFs have a significantly lower overall survival and a worse disease free
survival than patients with ICC with lower levels of alpha=SMA positivity
...
Such
chemical states are highly unstable and result in cell injury
...
ROS are produced normally in small amounts in all
cells during the reduction-oxidation (redox) reactions that occur during mitochondrial
respiration and energy generation
...
Superoxide dismutase
(SOD) converts superoxide anion to hydrogen peroxide
...
ROS are produced in phagocytic leukocytes, mainly
neutrophils and macrophages, to destroy ingested microbes and other substances during
inflammation and host defense
...
In this process, a phagosome membrane enzyme catalyses the generation
of superoxide, which is converted to hydrogen peroxide
...

Cells have developed many mechanisms to remove free radicals and thereby minimize
injury; SOD reactivity increases rate of decay of superoxide, glutathione peroxidases
breakdown hydrogen peroxidase to oxidised glutathione and water, catalase catalyze the
breakdown of hydrogen peroxide to water and oxygen, and endogenous and exogenous

antioxidants either block the formation of free radicals or scavenge them once they have
formed
...

The generation of free radicals is increased and the activity of protective endogenous
antioxidant enzymes decreases, in several situations; the absorption of radiant energy (such
as UV light, or X-ray), the enzymatic metabolism of exogenous chemicals (such as drugs),
inflammation, and in diabetes
...

ROS, such as hydroxyl radicals, cause cell injury by three mechanisms; lipid peroxidation of
membranes, double bonds in membrane polyunsaturated lipids are vulnerable to attack by
ROS, the lipid radical interactions yield peroxidases, which are themselves unstable;
cross-linking and other changes in proteins, free radicals promote sulfhydryl-mediated
protein cross-linking, resulting in degradation or loss of enzymatic activity; DNA damage,
free radical reactions with thymine in DNA produce single-strand breaks
...
Deep to the cortex is the renal
inner medulla
...
The medulla exhibits renal pyramids
...
The
pyramids apex are the renal papilla to drain urine from the collecting ducts via papillary
ducts into the minor calyx
...

The nephron regulates excretion by; filtration, glomerular filtration takes place at the
glomerulus and produces a cell and protein free filtrate; reabsorption, tubular reabsorption is
the process of selectively moving substances from the filtrate back into the blood,
reabsorption takes place at the proximal convoluted tubule and the collecting duct; secretion,
tubular secretion is the process of selectively moving substances from the blood into the
filtrate, it takes place along the proximal convoluted tubule and collecting duct
...
The exchange of substances between the thin descending limb
and thick ascending limb ensures efficient reabsorption of solutes and water and establishes
a osmotic gradient in the peritubular fluid that permits passive reabsorption of water from the
tubular fluid in the collecting duct
...

The functions of the kidney include; excretion of endogenous waste products, excretion of
drugs and their metabolites, water and electrolyte balance, acid base balance, and
production of hormones such as erythropoietin and renin
...

Renal failure may be rapid, acute kidney injury (AKI), or may take many months or years to

develop, chronic kidney disease (CKD)
...
Causes of AKI include; surgical,
medical, obstetric, traumatic, toxic, drug induced, or transplantation
...
Causes of CKD include; diabetes mellitus (this is the leading cause of CKD
worldwide), glomerulonephritis, pyelonephritis,, hypertension, vascular disease, or
autoimmune
...
Current interventions for AKI include;
administration of IV fluids (unless fluid overloaded), treat biochemical dysfunctions (such as
metabolic acidosis), use inotropes for hypertension (such as noradrenaline), withdraw the
nephrotoxic agent (such as aminoglycoside), facilitate urinary output ( by insertion of a
urinary catheter), and renal replacement therapy (dialysis or transplantation)
...
Ischaemia leads to hypoxia of the organ or tissue
...
Renal ischaemia is a restriction of blood
supply to the kidney with resulting damage or dysfunction of renal tissue
...

Reperfusion results in alterations in antioxidant enzyme activity and expression, and
expression of inflammatory enzymes such as iNOS and COX-2
...

If cells are reversibly injured, the restoration of blood flow can result in cell recovery
...
This is
ischemia-reperfusion injury (I-R injury)
...
When the supply of oxygen is increased, there may be a
corresponding increase in the production of ROS, especially because mitochondrial damage
leads to incomplete reduction of oxygen, and because of the action of oxidases in
leukocytes, endothelial cells, or parenchymal cells
...
The free
radicals produced in response to reperfusion include; superoxide anion, hydrogen peroxide,
hypochlorous acid, nitric oxide-derived peroxynitrite, and hydroxyl radicals
...
However these agents do not reduce patient mortality
...
However, there are several
pharmacodynamic and pharmacokinetic factors that affect the use of SOD as a drug
including; rapid renal clearance, slow extraversion due to molecular radius and charge
density, low bioavailability, instability in vivo, and immunogenicity
...
Bell-shaped

dose-response curves were reported when isolated rabbit hearts were subjected to I-R injury
and protected by exogenous SOD
...
Thus,
superoxide anion may not initiate and terminate lipid peroxidation
...
Thus, SOD has a very
narrow therapeutic window
...
These
SOD mimetics have shown protection against I-R injury
...

TEMPOL to treat renal ischaemia perfusion injury
TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) is a nitroxyl radical
...
TEMPLE also
has antioxidant properties; permeates biological membranes, scavenges ROS (superoxide
anion and hydroxyl radicals), does not scavenge hydrogen peroxide, chelates ferrous iron
which is required for hydroxyl radical production, and possess SOD mimetic activity
...
Cell viability and cell death
were assessed using biochemical assays
...
In
an in vivo animal of renal I-R injury, rats underwent surgery to isolate both kidneys, renal
ischaemia was induced followed by reperfusion
...
Glomerular dysfunction was assessed by measuring
serum creatinine levels
...
Tubular
dysfunction was assessed by measuring fractional excretion of sodium
...
TEMPOL reduced renal dysfunction in rat models of
endotoxic and haemorrhagic shock and reduced I-R in the rat heart, brain, liver and gut
...

SOD or catalase enzymes
...

Stem cells can be programmed to replace dysfunctional renal cells in the kidney
...
Preconditioning can produce profound protection
against renal I-R injury
...

Diabetic kidney injury
Diabetic kidney disease is defined as kidney disease with resultant impairment of renal
function due to the long standing effects of diabetes mellitus on the glomerular

microvasculature of the kidney
...
Diabteic kidney disease results in growth, inflammation, damage
and scarring of kidney tissue
...
Clinical features include; co-presentation with other diabetic complications
such as retinopathy, a progressive rise in urine albumin excretion as a result of a decreased
glomerular filtration rate (GFR), a progressive decline in GFR, a progressive rise in blood
pressure and associated cardiovascular risk, and eventual end stage renal disease
...
However, once diabteic kidney disease pathogenesis has begun it is
irreversible
...

Hyperglycemia induces the overproduction of superoxide anion by the mitochondrial electron
transport chain
...
This inhibition diverts upstream
metabolites from glycolysis into pathways of glucose overutilization
...
Increased flux of fructose-6-phosphate to UDP-N- acetylglucosamine
increases modification of proteins by O-linked-N-acetylglucosamine and increased glucose
flux through the polyol pathway consumes NADPH and depletes GSH
...

Ruboxistaurin for treatment of diabteic kidney disease
Ruboxistaurin is an oral inhibitor of the PKC pathway
...

Ruboxistaurin was shown to normalise renal dysfunction in diabteic animals
...
However an adverse event if diabetic
nephropathy was reported and so the drug was discontinued
...
SGLT2 is present
in cells of the proximal convoluted tubule and is responsible for the majority if glucose
reabsorption
...
Thus lowering blood
glucose
...
Possible side effects of SGLT2 inhibitors include;
polyuria as glucose remains in the filtrate, the osmotic potential of the filtrate is lowered, thus
water absorbed into the filtrate by osmosis, thus high amounts of water are excreted; and
UTIs as they high glucose content of urine can attract bacteria
...
The structure of these drugs is such
that they can scavenge free radicals
...

Medical imaging
Radiation- the emission or transmission of energy in the form of waves or particles through
space or through a material medium
...

Non-ionizing radiation- any type of electromagnetic radiation that does not carry enough
energy per quantum (photon energy) to ionize atoms or molecules, that is, to completely
remove an electron from an atom or molecule
...
Ionizing radiation can
cause DNA damage, leading to mutations, and ultimately cancer
...

X-ray principles
X-rays result from the conversion of the kinetic energy obtained by electrons accelerated
under a potential difference into electromagnetic radiation, as a result of collisional and
radiative interactions
...
The x-ray tube provides the proper environment and components to produce
x-rays
...
In the x-ray tube, two electrodes, the cathode and anode, are
situated a small distance apart in a vacuum enclosure called the inert
...
A separate, isolated circuit connected the cathode filament to a low voltage power
source
...
The second step involves the application of a high voltage supplied by the x-ray
generator to the cathode and anode
...
The andoe is usually composed of
tungsten
...
When the machine is turned on,
x-rays travel through the body and are absorbed in different amounts by different tissues,
depending on the radiological density of the tissues they pass through
...
For
example, structures such as bone contain calcium, which has a higher atomic number than
most tissues, thus bones readily absorb x-rays and thus produce high contrast on the x-ray
detector
...
Conversely, x-rays travel more easily through less radiologically
dense tissues, such as fat and muscle, as well as through air-filled cavities such as the
lungs
...

X-ray applications
X-ray radiography detects; bone fractures, certain tumours and other abnormal masses,
pneumoniae, calcifications such as kidney stones or vascular calcifications, arthritis in joints,
bone loss such as in osteoporosis, dental issues, heart problems such as congestive heart
failure, blood vessel blockages, digestive problems, and foregin objects such as items
swallowed by children
...
Tumours tend to appear as regular or irregular shaped masses that
are brighter than the background on the radiograph
...
While usually
benign, microcalcification may occasionally indicate the presence of a specific type of
cancer
...

X-ray disadvantages
Disadvantages of x-rays include; the use of ionising radiation which may be potentially
harmful thus limiting the use of x-rays in children and preventing frequent use, low sensitivity
in soft tissues, yields only two dimensional images, yields structural information only not
functional information, failure to determine age of lesions, and the contrast media used may
be dangerous
...
X-rays pass through the patient from multiple projections and are
detected by an electrons detector array that records patterns of densities
...
The filtered back projection method is used to enhance contrast and to increase
resolution of the image
...
Anti-scatter collimators are used in CT
scanners
...
Radiocontrast agents are substances used to enhance the
visibility of internal structures in CT imaging
...
However, due to associated toxicity
at the iodine concentrations necessary for imaging they are not suitable for most clinical
applications
...


CT applications
A study conducted by Lin, et al
...
In this
patient with laboratory-confirmed COVID-19, CT findings preceded symptoms and included
bilateral pleural effusions, previously not reported in association with COVID-19
...
, 2020, reported on the use of chest CT images to diagnose
pneumomediastinum in association with diabteic ketoacidosis in diabteic patients
...
, 2020, reported on the use of CT imaging in the diagnosis of
early-stage lung adenocarcinoma, which os manifested as isolated pulmonary nodules and
ground glass nodules on CT
...
CT scans can be used to
diagnose conditions including; damage to bones, injuries to internal organs, problems with
blood flow, stroke, and cancer
...
1mm which is much higher than the resolution obtained through x-ray or
ultrasound (0
...

CT disadvantages
Disadvantages of CT scans include; the use of ionizing radiation especially in comparison to
x-rays as CT uses a higher dose of radiation, relative expensive equipment required
especially in comparison to ultrasound and x-ray imaging, slower imaging in comparison to
ultrasound (seconds to minutes), lower patient in comparison to ultrasound and x-ray
imaging, and potential toxicity of intravenous contrast agents
...
Certain atomic
nuclei possess a property known as spin dependent on the number of protons
...
Application of a strong, external magnetic field (B0) alings the
nucleus either in parallel with or perpendicular to the external field
...
The absorption of energy by the
nucleus causes a transition from higher to lower energy levels and vice versa on relaxation
...

multiple FR pulses are applied to obtain multiple FIDs, which are then averaged to improve
the signal-to-noise ratio (SNR)
...
It will be
made up of contributions from different nuclei within the environment being studied
...
MRI scanners use cryogenic superconducting magnets 15
...

Relaxation described the process by which a nuclear spin returns to thermal equilibrium after
absorbing RF energy
...
A longitudinal
relaxation occurs, energy is displaced into the lattice, surrounding the nucleus environment
...
Water and cerebrospinal fluid have
long T1 values and thus appear dark on T1 weighted MR images, while fat has a short T1
value and appears bright to T1 weighted MR images
...
Transverse relaxation is the
process of energy redistribution among the nuclei within a spin system
...
These agents shorten the T1 relaxation time,
causing increased signal intensity on T1 weighted images, so the enhanced parts appear
bright on T1 weighted images, or shorten the T2 relaxation time, causing reduced signal
intensity on T2 weighted images, so the enhanced parts appear darker on T2 weighted
images
...
MRI contrast agents may be administered
orally or intravenously
...
Some agents selectively distinguish liver
pathologies
...
This
technique relies on the fact that cerebral blood flow and neural activation are coupled
...

The magnetic environment experienced by each MR sensitive nuclei is different due to the
magnetic fields of the electrons within their immediate chemical environment
...
Thus, the
magnetic resonance frequency spectrum consists of nuclei which resonate at different
frequencies
...
The most important visible peaks on the cerebral proton MR spectrum are
N-acetyl aspartate (a marker of neuronal dysfunction and loss), choline (a marker of
membrane activity, increased in inflammatory and malignant processes, decreased in
hepatic encephalopathy), creatinine ( a marker of internal reference level as it is constant in
health and disease), myo-inositol (elevated in microglial activation and astrogliosis), and
glutamine and glutamate
...
, 2020, reported the use of MRI scans to determine small
vessel disease score and thus predict risk of cognitive decline and dementia
...
, 2020, concluded that baseline diffusion MRI is potentially capable
of predicting two year clinical outcomes in patients with Parkinson’s disease on an individual
basis
...

Ceballos-Ceballos, et al
...
additional
uses of MRI scans include; anomalies of the brain and spinal cord, tumour detection, cyst
detection, breast cancer screening in at risk women, assessment of injuries or abnormalities
of joints, assessment and detection of diseases of the liver and other abdominal organs,
evaluation of pelvic pain in women to assess causes such as endometriosis, and
investigation of uterine anomalies
...
1mm) especially in comparison to ultrasound (0
...

MRI disadvantages
Disadvantages of MRI scans include; the requirement of very expensive equipment, not
widely available, low throughput due to very ling imaging time (minutes to hours) especially
in comparison to CT (minutes) and ultrasound (seconds to minutes), movement artifacts
created by breathing and heartbeat, hazardous for patients with metal implants, low
sensitivity in some instances such as detecting microcalcifications, potential toxicity of
contrast media, and low patient comfort
...
Ultrasound frequencies range from 2-15 MHz
...
The transducer converts energy from one form to another (electrical to
sound/mechanical and vice versa)
...

The returned echoes are converted back into electrical impulses by the transducer crystals
and are further processed to form the ultrasound image presented on the screen
...
5-3
...
0-7
...
Ultrasound waves are
reflected at the surfaces between the tissues of different density, the reflection being
proportional to the different in impedance (how much resistance an ultrasound bea,
encounters as is passess through a tissue, and depends on the density of the tissue, and the
speed of the sound wave)
...

Doppler shift is defined as the change in frequency of sound waves due to a reflector moving
towards or away from an object, which in the case if ultrasound is the transducer
...

Ultrasound applications

The temporal super-resolution if the dynamic ultrasound imaging, a means to observe rapid
heart movements, is important in the medical diagnosis of cardiac conditions
...
Microvascular
ultrasonographic imaging is a doppler ultrasound technique
...

Advantages of microvascular ultrasonography are its superiority in detection and
visualisation of the small blood vessels in tissues
...
Smaller, high frequency probes are used to assess the breast, thyroid,
testes, muscles and tendons
...

Ultrasound advantages
Advantages of ultrasound imaging include; the absence of ionising radiation and so can be
used in pregnancy and can be used repeatedly, inexpensive equipment especially in
comparison to CT and MRI, widely available, the equipment is portable, can yield a three
dimensional image which is an advantage over x-ray, yields real time functional images, high
throughput due to rapid imaging time (seconds to minutes) especially in comparison to CT
(minutes) and MRI (minutes to hours), and has high patient comfort
...
5mm) in comparison to CT (0
...
1mm)
...

Negatively charged electrons orbit the nucleus
...
The number of
protons determines the chemical and physical properties, the element
...
Isotopes can be stable or unstable
...
The greater the proton-neutron ratio,
the greater the instability of the isotope
...
Isotopes will
decay by radiation
...
The rate of
isotope decay is measured as a half life
...
Ionising radiation is any type of particle or
electromagnetic wave that carries enough energy to ionize or remove electrons from an
atom
...
Alpha decay occurs in elements with high atomic numbers (greater than 82),
such as uranium, radium, and thorium
...
Alpha particles, because they are highly
ionising, are unable to penetrate very far through matter and are brought to rest by less than
a tenth of a milliliter of biological tissue
...
Beta radiation occurs when an
atom undergoes radioactive decay, due to nuclear instability resulting from a high
neutron-proton ratio, giving off a beta particle consisting of a proton and an electron (betadecay), or due to nuclear instability resulting from a high proton-neutron ratio, giving of a
beta particle consisting of a neutron and a positron (beta+ decay)
...
Their light mass means they lose energy quickly through interaction with matter
...
They typically have a range of a few millimeters in
material
...
It consists of the shortest wavelength
electromagnetic waves and so imparts the highest photon energy
...
Specific isotopes emit distinct
gamma ray energy
...

Single photon emission computed tomography principles
Single photon emission computed tomography (SPECT) is a medical imaging technique that
is based on conventional nuclear medicine imaging and tomography reconstitution methods
...
Depending on the biodistribution properties of the radiopharmaceutical, it is
taken up by different organs and/or tissue types
...

Typically, a scintillation camera system is used as the imaging device
...
The signals from an array of PMTsare processed by electronic circuitry to provide
information about the position at which a photon interacts with the scintillator
...
SPECT takes
conventional two dimensional nuclear medicine images acquired at different views around
the patient and provides an estimate of the three dimensional radioactivity distribution using
methods of image reconstitution
...
This requirement
results in a limited number of photons that can be used for imaging
...
The collimator can be designed to allow detection of more photons, but
increased detection efficacy usually can only be achieved with a concurrent loss of spatial
resolution
...
99m Tc is obtained through the decay of molybdenum 99
...
99m Tc has a half life of six hours which gives hospitals the time to isolate it,
intravenously administer it to a patient as a radioactive marker and the image the body using
SPECT
...
99m Tc emits a relatively low dose of radiation, thus us safe to administer to a
patient
...
SPECT can be co-registered with other

imaging modalities such as X-ray and CT
...

SPECT uses
SPECT can be used to assess benign and malignant bone disease
...
Enhanced uptake reflects increased bone turnover caused by
changes of bone vascularisation and/or osteoblastic activity, such as occurs post fracture or
bone metastasis
...
SPECT/low-dose CT imaging is
useful in identifying benign skeletal abnormalities, such as osteochondrosis, as the reason
for abnormal tracer uptake
...
SPECT/CT imaging enables earlier detection of bone metastasis than
CT alone
...
MPS
provides comprehensive information on myocardial perfusion, regional and global left
ventricular function that provide incremental diagnostic and prognostic information
...
Myocardial
perfusion imaging (MPI) with radiotracers is an integral component in evaluation of the
patients with known or suspected CAD
...
The advantage of cationic radiotracers, eg
...
99m Tc-teboroxime derivatives have high initial
heart uptake due to their neutrality
...
99m Tc- 3SPboroxime is an excellent
perfusion radiotracer, the heart uptake of which relies on the regional blood dloe
...
99m Tc-3SPborroxime is beneficial for accurate diagnosis of CAD
and assessing the risk of future cardiac in cardiac patients
...

SPECT disadvantages
Disadvantages of SPECT include; the use of ionizing radiation at a dose much greater than
that used in MRI or CT or ultrasound (US), difficulties in absolute quantification, lowest
resolution of all imaging modalities (10mm), slower imaging time in comparison to PET and

US (seconds to minutes), more expensive than US, supply problems with short half life
radiopharmaceuticals, and problems with radiation exposure and radioactive waste
...
PET offers
quantitative analyses, allowing relative changes over time to be monitored as a disease
process evolves or in response to a specific stimulus
...
The tracer is administered as an intravenous injection usually labelled with
oxygen-15, fluorine-18, carbon-11, or nitrogen-14
...
The disadvantages of the radiopharmaceuticals
utilised for PET include; the short half life thus requiring on site production, and the
requirement of sophisticated and automated radiochemistry techniques needed to produce
the radiopharmaceuticals
...
The
two photons travel in directly opposite directions
...
The crystals absorb the photons,
producing light that is converted tinti an electrical signal
...

PET uses
A common use for PET is to measure the rate of consumption of glucose in different parts of
the body
...
FDG is transported into cells by
glucose transporters and phosphorylated by hexokinase to FDG-6-PO4
...
Whole body PET scans can be
used to stage a cancer
...

Tracking chemical neurotransmitters such as dopamine in Parkinson's disease, can also be
performed with this technique
...
Functional PET is a
neuroimaging method involving continuous infusion of FDG radiotracer during the course of
a PET examination
...
Functional PETprovides a unique method to map dynamic
changes of glucose uptake in the resting human brian and in response to extrinsic
stimulation
...
advantages of PET/MRI include; combines
functional information obtained from PET with anatomical and quantitative information
obtained from MRI, decreased radiation dose, improved motion correction, and the

convenience of a combined exam
...

PET advantages
Advantages of PET include; yields three dimensional images, yields both structural and
functional information, yields quantifiable results, wide applicability, ability of co-registration
with CT and MRI, great resolution (4-5mm) especially in comparison to EXPECT (10mm),
high throughput due to rapid imaging time (seconds to minutes), high patient comfort, and
PET/CT yields the greatest sensitivity and specificity in images in comparison to all other
imaging modalities
...
1mm) and MRI (0
...


Molecular diagnostics: nucleic acid and protein based diagnostic methods
Genomics- the branch of molecular biology concerned with the structure, function, evolution,
and mapping of genomics using methods such as next generation sequencing and
fluorescent in situ hybridization
...

Personalized medicine
Personalized medicine is based on using an individual's genetics profile to make the best
therapeutic choice by facilitating predictions about whether that person will benefit from a
particular medicine or suffer serious side effects
...
Medical decision making based
on empirical data relies on the law of averages, whereas personalized medicine recognizes
the differences between individual patients
...
In the first
step, ourines will react with dimethyl sulfate and pyrimidines will react with hydrazine in such
a way as to break the glycosidic bond between the ribose sugar and the base, displacing the
base
...
The use of these elective reactions to DNA sequencing then
involved creating a single-stranded DNA substrate carrying a radioactive label on the 5’ end
...
The

reactions are then loaded on high percentage polyacrylamide gels and the fragments
resolved by gel electrophoresis
...

Wherever a labelled fragment stopped on the gel the radioactive tag would expose the film
due to particle decay-autoradiography
...
Limitations of the
maxam-gilbert's method include the use of large amounts of radioactive material and the
neurotoxin hydrazine
...
A
DNA primer is designed to be a starting point for DNA synthesis on the strand of DNA to be
sequenced, four individual DNA synthesis reactions are performed
...
The four reactions can be named A, G, C and T according to which of the four
ddNTPs was included
...
This is because the ddNTP molecule lacks a 3’ hydroxyl group, which is required
to form a link with the next nucleotide in the chain
...
Following synthesis, the
products of the A, G, C and T reactions are individually loaded into four separate lanes of a
single gel and separated using gel electrophoresis, a method that separates DNA fragments
by their sizes
...
, including bands in all four lanes
...

Automated fluorescence sequencing
Leory Hood introduced an automated sequencing method using fluorescence-labelled
dideoxy-terminators
...
Once the reactions were complete, the four reactions were
pooled and run together in one lane of a polyacrylamide sequencing gel
...
The
fluorescence signature of each fragment was then sent to a computer where the software
was trained to perform base calling
...
Each of the four
dideoxynucleotide chain termination is labelled with fluorescent dyes, each of which with
different wavelengths of fluorescence and emission
...
SNPs are the smallest
unit of genetic variation
...
SNPs occur
approximately once every 100-300 bases
...
SNPs can occur in both coding and non-coding regions of the
genome
...

Genetic variation and Alzheiers disease
The APOE gene is located on chromosome 19 and comprises four exons and three introns,
totallying 3597 base pairs
...
Both dysfunctional
cholesterol processing and amyloid beta aggregation have been implicated in Alzheimer;s
disease risk
...
APOE 2 is relatively rare and may provide protection against AD, if AD occurs in an
individual with this allele, it usually develops later in life than it would in someone with the
allele APOE 4
...
The POE 4
allele is one of the strongest genetic risk factors for AD, and is associated with an earlier age
of disease onset
...
PAOE 4 is linked to episodic
memory-related dysfunction in the medial temporal lobe (MTL), one of the earliest brain
regions impacted by the progression of AD
...
The
APOE risk gene is associated with inflammation/microglia related pathways
...

The human genome project
The human genome project (HGP) was an international scientific research project that aimed
to; identify all the genes in the human DNA, sequence the 3 billion chemical base pairs of
the human DNA, store this information in databases, improve tools for data analysis, transfer
related technologies to the private sector, and address possible ethical, legal and social
issues
...
The current genome sequence
contains 2
...
It covers 99% of the
euchromatic genome and is accurate to an error rate of 1 event per 100 000 bases
...
The near complete sequence, the first for a vertebrate,
greatly improves the precision of biological analyses of the human genome including studies
of gene number, birth and death
...
The genome sequence serves as a firm foundation for biomedical
research
...
A reference genome is often assembled from DNA sequences from several donors,

but reference genomes do not accurately represent the genetic code of any single individual
...
Levy, et al
...
The diploid genome sequence
was produced from approximately 32 million random DNA fragments, sequenced by sanger
dideoxy technology and assembled into 4528 scaffolds, comprising 2810 million bases (Mb)
of contiguous sequence with approximately 7
...

Comparison of this genome with previous reference genomes sequences, which were
composites comprising multiple humans, revealed more than 4
...
3 Mb
...
non-SNP DNA variation accounts for 22% of all events identified in the
donor, however they involve 74% of all variant bases
...
Moreover, 44% of
genes were heterozygous for one or more variants
...

Next generation DNA sequencing
The major advance offered by next generation sequencing (NGS) technologies is the ability
to produce in some cases, in excess of one billion short reads prt instrument run, which
makes them useful for many biological applications
...
The leading NGS platforms use clonally
amplified templates, which are not affected by the arbitrary losses of genomic sequences
that are inherent in bacterial cloning methods
...
PXRr can create
mutations that masquerade as sequence variants and amplification bias that
underrepresents AT-rich and GC-rich regions in target sequences
...
To call sequence variants in genomes, NGS
reads are aligned to a reference sequence using various bioinformatics mapping tools
...
NGS costs will continue to drop in the foreseeable future, although cost
reduction should be weighed against the quality of the produced genome sequence
...
In the first step, a
DNA polymerase, bound to the primed template, adds or incorporates just one fluorescently
modified nucleotide, which represents the complement of the template base
...

following incorporation, the remaining unincorporated nucleotides are washed away
...
This is followed by a

cleavage step, which removes the terminating/inhibiting group and the fluorescent dye
...
Helicos
Biosciences uses a one colour-CRT cycle
...

Sequencing by ligation (SBL) is another cyclic method that differs from CRT in its use of
DNA ligase and either one-base0 encoded probes or two-base-encoded probes
...
DNA ligase is then added to join the dye-labelled probe to the primer
...
The cycle can be repeated either by using cleavable probes to
remove the fluorescent dye and regenerate a 5’ phosphate group for subsequent ligation
cycles, or by removing and hybridizing a new primer to the template
...
Pyrosequencing manipulates DNA polymerase by the single
addition of a dNTP in limiting amounts
...
The order and intensity of
light peaks are recorded as flowgrams, which reveal the underlying DNA sequence
pyrosequencing is utilised by Roche-454
...
Single DNA polymerase molecules are attached to the
bottom surface of individual zero-mode waveguide detectors that can obtain sequence
information while phospho linked nucleotides are being incorporated into the growing primer
strand
...

Pharmacogenomics
In pharmacogenomics, genomic information is used to study an individual’s responses to
drugs
...
Multigene analysis of whole genome SNP profiles can be used to
assess gene variants affecting an individual’s drug response
...
Pharmoacokinetics encompassess
four processes; absorption (how a drug enters the bloodstream), distribution (where the drug
travels after absorption and how much drug reaches the target site), metabolism (how the
drug is broken down in the body), and excretion (how the drug leaves the body)
...
Genetic variation in genes
encoding drug-metabolising enzymes, drug receptors, and drug transporters have been
associated with individual variability in the efficacy and toxicity of drugs
...
Predicting
serious adverse drug reactions is a priority for pharmacogenomic research
...
Molecular cloning and characterization studies of the gene that codes for this enzyme
have described more than 70 variant alleles
...
Individuals who are
homozygous or heterozygous for the wild-type or normal activity enzymes (75-85% of the
population are called extensive metabolizers, intermediate (10-15%) or poor (5-10%)
metabolisers are carriers of two alleles that decrease enzyme activity and ultrarapid
metabolizers (1010%) are carriers of duplicated genes
...

Polymerase chain reaction
PCR is the process of preparation of complementary DNA (cDNA) from RNA
...
This cycle is
repeated 20-40 times in a thermal cycler
...
PCR also
provides a method to identify bacteria, particularly clinically important pathogen species that
are proved to be not cultivable via standard diagnostic protocols, for example H
...

Fluorescence in situ hybridization
The process of FISH is as follows; labelling DNA-probe complementary with the target gene
sequence, denaturation of both the probe and the target DNA sequence, annealing probe
with the target gene sequence, and then detection with a fluorescence detection system
uses the nucleotide probe is directly labelled with a fluorophore
...
The Philadelphia chromosome
is present in over 95% of cases of chronic myeloid leukaemia (CML)
...
This aberrant
fusion gene encodes the breakpoint cluster region-proto-oncogene tyrosine-protein kinase
(BCR-ABL1) oncogene protein with persistently enhanced tyrosine kinase activity
...
Each BCR-ABL1 transcript is present in a distinct
leukemia phenotype, which predicts both response to therapy and clinical outcome
...
FISH is also used to determine whether breast cancer is HER2 positive
...
25% of breast
cancers are HER2 positive
...

DNA microarray
DNA microarrays are a group of technologies in which specific DNA sequences are either
deposited or synthesised in a 2D or 3D array on a surface in such a sway that the DNA is
covalently or noncovalently attached to the surface
...
By generalising to a very large number of spots of DNA, an
array can be used to quantify a large number of different nucleic acid sequences in solution
...

Proteome- the complete set of proteins expressed by an organism, tissue, or cell
...
The HPP aims to determine; protein
abundance, distribution, and subcellular localization; protein interactions with other
biomolecules; and protein functions at specific time points
...

Immunohistochemistry
The method of detecting a primary antibody that is bound to the antigen of interest in an
immunohistochemistry (IHC) experiment can be either direct or indirect
...
In direct detection methods, the
primary antibody is directly conjugated to a label
...
For direct detection, the primary antibody can be conjugated to
an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphate (AP), or a
fluorochrome
...
Another benefit of direct detection is increased flexibility
in the design of multi colour experiments, given the wide range of fluorochromes that are
available
...
Signal amplification
occurs through the potential for two or more labelled secondary antibodies to bind to each
primary antibody
...
Immunostaining of breast cancer tissues can detect HER2 presence
...
Flow cytometry also
simultaneously measures the light that is scattered by each cell
...
The amount of light scattered
[perpendicular to the laser light beam provides information about the presence or absence of
intracellular membranous structures
...
In a FACS instrument, cells that bear the
desired combination of light-scattering properties and fluorescent markers are tagged with

an electrical charge, and can be deflected into a separate pot for further analysis
...
when the number of CD4+ T-cells in the blood of a patient with AIDS falls below a
certain level, the patient is at high risk for opportunistic infections
...
ELISAs can be used
qualitatively to detect the presence of antibody or antigen, alternatively ,a standard curve
based on known concentration of a sample
...

Biomarker- a characteristic that is objectively measured and evaluated as an indicator of
normal biologic processes, pathogenic processes, or pharmacologic responses to a
therapeutic intervention
...
PSA is secreted by the prostate epithelium in normal, benign, and cancerous
conditions
...
PSA levels can
be used to monitor the progression of prostate cancer
...
PSA
serum levels are elevated in patients with prostate cancer, benign prostatic hypertrophy and
inflammatory conditions, such as prostatitis
...
31% of men with PSA levels 4-10 ng/ml go on to develop
prostate cancer
...

Mass spectroscopy
Mass spectroscopy is an analytical tool useful for measuring the mass-to-charge ratio of one
or more molecules present in a sample
...
Mass spectrometry-based proteomics allows quantitative
system-wide analysis of the proteome, including post-translational modifications,
protein-protein interactions and cellular localization
...
With the availability of genomic
information the massive capacity for peptide identification by mass spectroscopy is being
used to annotate gene sequences and to find new protein-coding genes and splicing
variants
...
Protein affinity strategies allow purification
of candidate proteins and their interacting partners, which are subsequently identified by MS
...
The improvements in sensitivity, robustness and high-throughput of
MS based proteomics now permits applications in the clinical field, including the possibility of
discovering disease-related biomarkers and screening molecular targets of candidate drugs
...

Type II diabetes mellitus typically has a late onset and occurs when the body becomes
resistant to insulin or does not make enough insulin
...
According to diabetes
UK, approximately 2
...
The world health organisation estimates that over 300
million people worldwide will be diagnosed with diabetes by the year 2030
...
Insulin triggers the uptake of glucose, fatty acids and amino acids into
liver, adipose tissue and muscle and promotes the storage of these nutrients in the form of
glycogen, lipids, and protein respectively
...
Binding of insulin
to the alpha subunit induces a conformational change resulting in phosphorylation of a
number of tyrosine residues present in the beta subunit
...
receptor activation leads to the phosphorylation of ey
tyrosine residues on the IRS protein, some of which are recognised by the src homology
(SH) domain of the p85 regulatory subunit of PI3- kinase
...
A key downstream effector of ptdins (3,4,5)p3 is AKT, which
is recruited to the plasma membrane
...
Once
active, AKT enters the cytoplasm where it leads to the phosphorylation and inactivation of
glycogen synthase kinase 3 (GSK3)
...
Phosphorylation of glycogen
synthase by GSK3 inhibits glycogen synthesis, therefore the inactivation of GSK3 by AKT
promotes glucose storage as glycogen
...
Insulin directly controls the
activity of metabolic enzymes by phosphorylation and dephosphorylation, and also regulates
the expression of genes encoding hepatic enzymes involved in gluconeogenesis
...
PI3-kinase and AKT play a role
in GLUT4 translocation
...
In this pathway, insulin receptor activation leads to phosphorylation

of cb1, which is associated with the adaptor protein CAP
...
C3G activates members of the GTP-binding protein family, TC10, which
promote GLUT4 translocation to the plasma membrane
...

Serine-phosphorylated forms of these proteins fail to activate PI3-kinase, resulting in
decreased activation of glucose transport
...

Diabetic neuropathy
Diabetic neuropathy (DN) is the presence of symptoms of peripheral nerve dysfunction in
individuals with DM
...
The PNS consists of the nerves and ganglia outside the brain and
spinal cord
...
The PNS
contains cranial nerves that emerge directly from the brain, and spinal nerves that emerge
from segments of the spinal cord
...
As the dorsal root
emerges from the intervertebral neural foramina, it forms the dorsal root ganglion (DRG)
...
DRG cell
bodies are separated by layers of satellite glial cells (SGCs) that inhibit the interaction
between somas
...
Action
potentials generated by impulses from the periphery do not always need to go through the
DRG, they may bypass the DRG and continue through to the proximal process and spinal
cord
...

Diabetic neuropathy: clinical manifestations
Diabetes can damage the peripheral nervous system in a variety of ways, but the most
common presentation is a distal symmetric polyneuropathy
...
The symptoms

are symmetric with sensory symptoms more prominent that motor involvement
...
DN associated
pain is characterized by burning, electric, and stabbing sensations with or without numbness
...

Evidence of neuropathology in DRG in DN
- Alterations in blood flow in the nerves, due to microangiopathy of the vasa nervorum
- Schwann cell (SC) dysfunction; primary demyelination, secondary segmental
demyelination due to impairment of axonal control of myelination, SC proliferation,
atrophy of denervated bands of SC, basal lamina hypertrophy
- Axonal degeneration, via length dependent polyneuropathy and progressive
impairment of regeneration
- Neuronal damage caused by hyperglycemia, due to consequent activation of the
polyol pathway, synthesis of AGE products, excess activation of protein kinase
C-driven pathways, with resultant increased oxidative stress
Vulnerability of DRG neurons
Individual DRG neurons can be extremely long, the axon of a single DRG neuron extending
from the sural nerve through the sciatic nerve eventually to the brainstem may be
approximately 1
...
Due to the pseudo-unipolar structure of DRG neurons, these
cells have an extremely high metabolic demand
...
This vascular organization provides the DRG with a great blood supply, serving
neurons that have long processes with the required high-energy demand critical for
maintaining the production and transport of receptors, ion channels, cytoskeletal, and
transport proteins
...
Thus, these neurons are exposed to a great deal of environmental stress
...
Thus, somas
and peripheral axons of DRG neurons are exposed to osmotic, pH, and other imbalances
caused by diabetes including hyperglycemia, that diffuse freely across vascular and tissue
beds
...
In addition, information from peripheral
tissues must transit the length of the axons to the nucleus in order to ensure appropriate
gene transcription and protein manufacture for long term homeostasis
...

Pathophysiology of DN
The polyol pathway is a major contributor to oxidative stress in the nerves of diabetics
...
The polyol pathway consists of
two enzymes; AR reduces glucose to sorbitol with the aid of its cofactor NADPH, and SDH

converts sorbitol to fructose with the aid of its cofactor NAD+
...
Oxidative stress in the mitochondria critically aters energy
regulation and survival through three mechanisms
...

Thus, peroxynitrite affects mitochondrial function and inhibits ATP synthesis
...
Thirdly, oxidative damage of existing inner membrane proteins induces membrane
permeability transition, a permeabilization of the mitochondrial inner membrane that
precedes cytochrome c release and apoptosis
...
Mitochondria in the DRG
are especially vulnerable, because in the hyperglycaemia neuron they are the origin of
production of reactive oxygen species, which can damage their DNA membranes,
deregulation of fission and fusion proteins that control mitochondrial shape and number can
impair cell functions and might leads to degeneration and neuronal injury may be the
greatest contributor to DN
...

Pathological calcification
Bone
Cortical bone is the dense outer surface of the bone that forms a protective layer around the
internal cavity
...
Cortical bone is found in
the shaft of long bones and forms the outer shell around cancellous bone, also called
trabecular bone or spongy bone, is a light, porous bone enclosing numerous large spaces
that give a honeycombed or spongy appearance
...

The spaces between are filled with marrow
...
Cancellous bone is found at the ends of long bones, as well as in the pelvic bones,
ribs, skull, and the vertebrate in the spinal column
...
All are continually in a state of change, the cells die off and are
replaced, the collagen and minerals are eroded and reformed
...
Together, the
two processes constitute bone remodelling
...
Bone is formed by
the collagen matrix becoming mineralized
...
Hydroxyapatite is composed mainly of hydrated calcium phosphate,
but also contains traces of carbonate, citrate, and other ions
...
The embryonic skeleton is formed by;m endochondral ossification in which a bone
develops by replacing hyaline cartilage and the resulting bone is called cartilage or
endochondral bone, and intramembranous ossification in which a bone develops from a
fibrous membrane and the resulting bone is called membrane bone
...
Most bones stop growing during adolescence
...

Osteoblasts are mononucleated cuboidal cells that originate from immature mesenchymal
stem cells
...
Osteoblasts from bone by
producing a matrix that then becomes mineralised
...
They are networked to each other via long cytoplasmic
extensions that occupy tiny canals called canaliculi, which are used for exchange of nutrients
and waste through gap junctions
...
Osteoclasts are bone resorbing cells
...
This parameter is tightly controlled because calcium ions have a stabilising effect on
voltage-gated ion channels
...

Causing hyperactivity of nerve and muscle cells
...

Conversely, hypercalcaemia results in closure of voltage gated ion channels and consequent
depressed nervous system function
...

Parathyroid hormone
In the blood, the sensitive process of calcium and phosphate homeostasis is maintained
primarily by the parathyroid gland
...
The two
parathyroid glands on each side that are positioned higher are called the left and right
superior parathyroid glands, while the lower two are called the left and right inferior
parathyroid glands
...
PTH is a polypeptide that is synthesized and cleaved into an active form within the
parathyroid gland
...
as
serum calcium levels drop, PTH secretion increases
...
in

the bones, PTH stimulates the release of calcium in an indirect process through osteoclasts
which ultimately results in bone resorption
...
PTH also inhibits the
secretion of osteoprotegerin, allowing for preferential differentiation into osteoclasts
...
Osteoclasts possess the ability to remodel the bones by dissolution and
degradation of hydroxyapatite and other organic material releasing calcium into the blood
...
PTH decreases phosphate reabsorption at the
proximal convoluted tubule of the kidney
...
The reduction of phosphate
ions, therefore, results in more ionized calcium in the blood
...
1-alpha-hydroxylase catalyses the synthesis of active vitamin D-1,
25-dihydroxycholecalciferol from the inactive form, 25-hydroxycholecalciferol
...

calcitonin , a hormone produced by the parafollicular cells of the thyroid, acts in opposition to
PTH by inhibiting osteoclasts, stimulating osteoblasts, and increasing excretion of calcium in
the urine by the kidneys
...
Secondary hyperparathyroidism is primarily seen in patients with chronic kidney
disease (CKD)
...
Significant decrease in the expression of klotho, and a reduction
of renal calcitriol production
...

indirectly , calcitriol deficiency also stimulates PTH secretion due to a decrease in intestinal
calcium absorption
...
The drug cinacalcet is a calcimimetic drug that binds to the
calcium sensing receptors on cells of the parathyroid gland, inhibiting PTH secretion
...
Procedures include;
paraffin or frozen sections after decalcification, ground sections, or resins
...
Biopsies investigating possible tumors or infection
are decalcified in acid or with a chelating agent and processed routinely
...
X-ray
may provide information on; nature and extent of bone lesion, which blocks required for
staining, the progress of decalcification, and whether foregin material is present such as
metal
...

Trichrome stains collagen, PAS stains osteoid, alizarin red stains calcium, phosphates and
carbonate
...
Fluorescent labelling may be used in metabolic investigation, and is based on
the uptake in vivo of tetracycline
...
The biopsy is processed into resin, and viewed unstained with a short wave ultraviolet
light
...
Morphometry can also be used to assess the effects of a
treatment regimen, for example in hyperparathyroid bone disease
...
Pathologic calcification can
occur in almost any tissue structure
...
The first detectable mineral deposited in vivo is a poorly crystallised
apatite which perfects with age
...
Also commonly in pathology is
primary nucleation, where crystals grow on a different surface, by epitaxy
...

Mitochondria frequently initiate intracellular calcification
...
Matrix vesicles and mitochondria
usually initiate calcification through the interaction of phosphatase enzymes with
calcium-binding phospholipids, both of which are membrane bound
...
Once formed and exposed to the extracellular fluid, HA crystals can serve as
nuclei or templates, thus supporting progressive, autocatalytic mineral crystal proliferation
...
The cell membrane pumps
calcium out of the cell, the mitochondrial membrane pumps calcium into the mitochondria
...
The calcified mitochondria are then available for secondary
nucleation
...
It is associated with multiple clinical conditions, such as collagen vascular
diseases
...
Dystrophic calcification occurs when a local disturbance
in serum calcium levels leads to crystal formation
...
Metabolic calcification occurs
in situations of systemic disturbances of serum calcium levels, and crystals are deposited
throughout the body
...
Metastatic calcification
deposition can be influenced by release of excess calcium salts from bone, phosphate
concentration, alkaline phosphatase activity and viscera physiochemical conditions under
alkalosis
...

Bacteria can also initiate calcification, for example the bacteria bacteremia matruchotii
yielded the first phospholipid and protein complex capable of nucleating apatite in vitro
...
Actinomyces is a genus of the actinobacteria
class of bacteria
...
Actinomyces species are facultative;y
anaerobic
...
Spicules are any of various small
needle-like anatomical structures occurring in organisms
...
Mammograms can detect benign calcifications of
mammary tissues visible as white patches, as well as showing calcification typical for tumour
in the mammography tissue
...
The growth plate is
responsible for bone elongation, and for coordinating and regulating bone growth
...

The resting zone may contain stem cell like cells capable of generating new clones of
proliferative zone chondrocytes
...
The p

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