RBCs -Ageing in Health, Storage, Hypoxia - Anemias ...SI).pdf · PhD degree awarded with the...

RBCsRBCs--Ageing in Health, Storage, Ageing in Health, Storage, HypoxiaHypoxia--

AnemiasAnemias (Thalassemia, (Thalassemia, FanconiFanconi’’ss, Renal), , Renal),

High Altitude, High Altitude, AstronautsAstronauts--neocytolysisneocytolysis, CD34, CD34++

Stem Cell Gene Therapy Research Initiatives*Stem Cell Gene Therapy Research Initiatives*

Impington, Cambridge CB24 9LZ, UK

Dr J N Mehrishi, PhD (Cambridge), FRCPath.

Director, Initiator, Co-ordinator

[email protected]. Tel +44-1223-57 36 28. skype <jaycantab>

Data discussed: International Teams workData discussed: International Teams work-- 1948 onwards1948 onwards

*(Independent of and separate from the University of Cambridge)

Hippocratic oath: Hippocratic oath: PremumPremum non non nocerenocere: do no harm : do no harm

Motto Motto -- University of Cambridge University of Cambridge Hinc lucem et pocula sacraTranslation: Here [we receive] light and sacred draughts;

"here" refers to - are metaphors for knowledge.

PhD degree awarded with the Trinitarian formula-...in nomine Patris et Filii et Spiritus Sancti-

in the name of the Father and of the Son and of the Holy Spirit.

Royal College of PathologistsRoyal College of Pathologists-- Sedes invenire et causas morborum –‘to seek the sites and causes of disease’ and help correct

them.I take these seriously!

Critical life and death decisions to assess

ANAEMIA -

shortage of circulating red blood cells (RBCs)

Containing Oxygen for delivery bound to

Haemoglobin (Hb) <11g/dL> INDIERCT.

RBCs at various stage of ageing, deterioration – in health and disease

(thalassaemia, sickle cell disease) - very heterogeneous

- Noninvasive and continuous Hb measurement with aspectrophotometric sensor (Rainbow DCI) with multiple wavelengths of light utilisation:

Masimo Pulse CO-Oximetry method shows the distinctive light-absorption characteristics of different haemoglobinspecies - proprietary algorithms to determine Hb levels spectrophotometry of

Methaemoglobin

Oxyhaemoglobin

Reduced haemoglobin

Carboxyhaemoglobin

Molar Extinction coefficient ε [cm-1/(moles/liter)]

Wavelength, λ (nm)

MethaemoglobinOxyhaemoglobin

Reduced haemoglobin

Carboxyhaemoglobin

Would you decide the beauty of the wife from the photographs of some of these husbands?

Well- clinics use INDIERCT basis of levels of hemoglobin, Hb, (supplier of oxygen) : <11g/dLfor assessing anaemia (shortage of good red blood cells containing Hb for critical decisions (?life and death?)

Trying to decide the beauty of these women from the photographs of the husbands-Hb level Indirect – conclusion RBCs in anaemia imprecise!

Try again

1. Blood1. Blood-- RBCs Storage Lesion , RBCs Storage Lesion ,

2. Hypoxia2. Hypoxia--OxygenOxygen--RBCsRBCs-- HbHb shortage shortage AnaemiasAnaemias ––

3. Kidney damage3. Kidney damage, impaired EPO production, RBC, impaired EPO production, RBC--

HbHb: <11g/: <11g/dLdL renal renal anaemiaanaemia

4.Thalassemia4.Thalassemia--decreased, NO synthesis of decreased, NO synthesis of HbHb αα--, , ββ-- chains,chains,

Cell surface Cell surface sialoglycoproteinsialoglycoprotein--

Phase I biophysical studies Phase I biophysical studies ~44~44--99% high density RBCs99% high density RBCs

((ii) CD47, CD55 molecules ) CD47, CD55 molecules decreaesddecreaesd..

(ii) decreased~(ii) decreased~2525--30% less30% less-- sialiasialia acid homing moleculesacid homing molecules--

premature phagocytosispremature phagocytosis

Bone marrow transplantation worksBone marrow transplantation works--

Ultimate cure dream Ultimate cure dream -- Gene editing for Gene editing for Gene TherapyGene Therapy

Involves, Involves, ((ii) viruses,(ii) electroporation, (iii) ) viruses,(ii) electroporation, (iii) CRISPRCas9, and CRISPRCas9, and

CRISPRCRISPRPf1Pf1 systems.systems.

Howsoever gene editing is carried outHowsoever gene editing is carried out-- there are serious there are serious riksriks ofof

IatrogenicIatrogenic: : membrane perturbationmembrane perturbation--surface surface ‘‘hominghoming’’moleculesmolecules rearrangementrearrangement--changing changing

permeability, electrical propertiespermeability, electrical properties--

Never ever testedNever ever tested-- crucial importancecrucial importance--

AthensAthens--Heidelberg teamsHeidelberg teams: : >500 genes changed>500 genes changed

ERRORS OF INSERTIONERRORS OF INSERTION--

SPLICING, TRANSCRIPTIONSPLICING, TRANSCRIPTION

Activation: protoActivation: proto--oncogenesoncogenes-- leukaemogenesisleukaemogenesis

‘‘point point faiblefaible’’ of gene therapyof gene therapy- Mehrishi DesignatedMehrishi Designated

Gene TherapyGene Therapy: : ImmunodeficienciesImmunodeficiencies-- SCIDSCID--XX--1 1

ThalassemiaThalassemia-- - CD34+ cells- gene edited

Retroviral-Modified Chimeric Antigen Receptor T

Cells CAR-T

Fanconi’s anaemia- CD34+//electroporation-CRISPRCas9

Tay-Sachs - Classical infantile (acute) Hexosamine A

deficiency (Tay-Sachs) and related-

Higgs DR, Engel JD, Stamatoyannopoulos G. Thalassaemia. Lancet 2012; 379: 373–83.

BlackSpecsMiltenyi

Video clip : Italian and French versions available https://www.youtube.com/watch?v=_gbBX_cZrFc

SCIENCE VOL 341 23 AUGUST 2013 853

Inder Verma (Inventor- Salk- owns Intellectual property right.

contamination: serious membrane perturbationVideo clip : https://www.youtube.com/watch?v=_gbBX_cZrFcMehrishi JN. 2013. TRANSFUSION.;53:2667-2674. Current and historical perspectives on methodological flaws in processing umbilical cord blood. (Italian and French versions available)

For genetic engineering CD34+ cells obtained by a seriously flawed method: Miltenyi particles-black internalised and trapped:Isosurface imaging, TEM Confocal laserMicroscopy

Aanaemias:HbAanaemias:Hb (<11g/(<11g/dLdL--) old established INDIRECT) old established INDIRECT--complacent? complacent?

relying on heterogeneous RBCs: Suggest present day new knowledgrelying on heterogeneous RBCs: Suggest present day new knowledge e

directly using numbers/proportions of directly using numbers/proportions of ‘‘goodgood--functional RBCs.functional RBCs.

ANAEMIAS:ANAEMIAS:

Shortage of RBCs Shortage of RBCs

((HbHb) oxygen, blood ) oxygen, blood

lossloss--altitudealtitude--Trigger Trigger

EPO EPO ProducitonProduciton to to

BM. BM. Aanaemias:HbAanaemias:Hb

(<11g/(<11g/dLdL--) )

INDIRECT, INDIRECT,

Double check direct Double check direct

by by ‘‘good RBCsgood RBCs’’ ??

Left:Prchal JT. Clinical manifestations and classification of erythrocyte

1144 disorders. McGraw-Hill -Williams Hematology, 8e 2010, 1145

Eds Marshall A. Lichtman et al. With permission.

RIGHT: Data kindly provided by Prof. Ferruh Artunc – Tübingen

Chronic kidney disease is a silent devastating Chronic kidney disease is a silent devastating

diseasedisease-- linked to many other disease states linked to many other disease states ––

usually discovered when patient tested for usually discovered when patient tested for

something else.something else.

AnaemiaAnaemia AFFECTS ~10% of the world AFFECTS ~10% of the world

population.population.

Renal Renal anaemiaanaemia-- treatment by EPO (expensive treatment by EPO (expensive

hormone)hormone)--

Newer: Newer: pyrolylpyrolyl hydroxylase inhibitorshydroxylase inhibitors--

stimulating EPO productionstimulating EPO production--

Fiercely competitive fieldFiercely competitive field-- companies companies --

needed needed ––Huge $Huge $bnbn market market --vested interestsvested interests of of

patent patent ––company stock holders, influential company stock holders, influential

workers refereesworkers referees-- MSSMSS-- grantsgrants-- Declaring Declaring

vested interests: NO GUAANTEE of Lack of vested interests: NO GUAANTEE of Lack of

Bias!Bias!

Need for Objective quantitative Need for Objective quantitative

assessment of RBCs levels restorationassessment of RBCs levels restoration

efficacy of treatment Complacent to efficacy of treatment Complacent to

rely entirely on rely entirely on HbHb based on RBCs based on RBCs

heterogeneous population.heterogeneous population.

Basis of Direct numbers of Basis of Direct numbers of ‘‘goodgood’’

RBCs after rapid, inexpensive isolated RBCs after rapid, inexpensive isolated

populations is precise populations is precise -- preferable preferable

Genetic Disease,Blood Disorders, Immune Enzyme deficiencies, DNA damage- ‘homing’defect, Hypoxia - Regulation of Oxygen levels tightly controlled by Red Blood Cells (RBCs) via

hemoglobin, Hb - Fe++ // Fe+++ flip flop-pick up – transport and deliver to tissues

Hypoxia sensed in the kidneys –

Triggers erythropoietin Production for RBCs

RBC Life span 120d: eliminated, recycled

Defective - (i) Renal anaemia: ~80 days

RBC survival (ii) Thalassemia : ~55 days

Thalassemia, Genetic Disease,Blood Disorders, Immune Enzyme deficiencies, DNA damage- ‘homing’ defect, Hypoxia - Regulation of Oxygen levels tightly controlled by Red Blood Cells (RBCs) via

hemoglobin, Hb - Fe++ // Fe+++ flip flop-pick up – transport and deliver to tissues

Gene Therapy- Genetic Disease, Blood Disorders: Thalassemia, ‘homing’ defect-Premature erythrophagocytosis

Immune Deficiencies- gene mutation -X chromosome that encodes a component (or chain) shared by the T-cell growth factor receptor and other growth factor receptors.

This component is referred to as γcSCID-X1 and SCID related to adenosine deaminase defect. Enzyme deficiencies.

Fanconi’s anaemia- DNA damage- ‘homing’ defect.

Take patient’s cells or stem CD34+ cells- modify: with-without using electroporation to introduce large molecules of DNA-retroviruses or CRISPRCas9 or the newer CRISPRPf1:

Serious flaws: Paraphrasing Einstein-

‘Daran haben sie überhaupt nicht gedacht’

membrane perturbation, surface molecules rearrangement

changes-permeability, electrical properties-

Cells NOT TESTED before use- ‘homing’ molecules

RISKS: errors of insertion, splicing, transcription,

activation of proto-oncogenes and eventually-leukaemia

Hematopoiesis-–impaired-defectiveRed Blood Cells production decreased

Damaged Junk RBCs- body– eliminates quickly

When RBCs numbers prematurely decreasedAnaemia - Hb (within) : decreased- <11g/dl:

INDIRECT (Hb in RBCs SEEMS complacent)

Need Direct good RBC numbers fractions Now for $1 easily done for counting Y-BCs for Hb

MORE MEANINGFUL SURELY?

Status quo Status quo you say you say ““Indirect Indirect HbHb levels levels

used for used for yonksyonks””, why worry!?, why worry!?

Well, Well, HbHb level began before it was that ~120 level began before it was that ~120

days life span RBCs days life span RBCs –– ~2x10~2x1066/sec renewal/sec renewal--

very heterogeneousvery heterogeneous RBCs of various ages:

good to

middle ages- M1, M2

damaged/junk easily-cheaply isolated.

See also detailed work by many Labs.

Kameneva et al. slides included further on

It would be logically more reliable to judge

anaemia by the numbers and proportions of

high quality Y-RBCs:O-RBC for efficient

oxygen delivery- before and after therapy

with EPO, rHuEPO or the related ones or

The new pyrolyl hydroxylase inhibitors.

RBC: ~120 days life span: 2x102x1066/sec renewal/sec renewalwith ageing- biomaterials, water are lost

-volume (v) decreases � �

-density (d) increases (m ÷ v = d)

Sialic acid (‘homing’ molecule) charge decrease;

ageing, blood storage lesion, thalassemia

?Does this occur in anaemias? Renal anaemia?

Heterogeneous Cells-PERCOLL fractionated

lightest density –young- Y-RBCs

densest density –old- O-RBCs.

Hb lost as vesicles- more from old, aged

RBCs, 20% Hb lost, particularly in the

last half of its life (in storage).

Renal AnemiaRenal Anemia

Chronic kidney disease (CKD),

is an internationally recognized

public health problem affecting

10% of the world population

(some sources quote ~5%)

FG4516, Roxadustat (FG-4592) BAY 87-224

an orally bioavailable hypoxia-inducible

factor (HIF) prolyl hydroxylase inhibitor

promotes coordinated erythropoiesis

through HIF-mediated transcription.

Aim of treatment - Increase good RBCs:

common sense to test the efficacy of a drug by

directly measuring good Y-RBCs numbers and

proportion- now done quickly (1hr), inexpensively

(US¢ 0.30/sample) and correlate with Hb-

Current testing expensive, lengthy

Hb aggregated gets bound- stuck to the inner

O-RBCs membrane – Huang,Wu, Mehrishi+ et al.

Raman

Spectroscopy

Q. Is it unreasonable, delusional

or obsessionally mad to think

that the efficacy of a drug

to treat anemia

should be tested directly by

counting the numbers and

proportions - good, Y-RBCs ::

O-RBCs that provide Hb

for assessing anaemia in

conjunction with other

parameters- MCV, MCHC etc.

Notoriously hetero Notoriously hetero therapyoustherapyous cellscells

Fractionation is essential to identify Fractionation is essential to identify

and correlate the structure, properties and correlate the structure, properties

and function of cells and their and function of cells and their subsetssubsets

heme group

RBC: 270x106 molecules of Hemoglobin (Hb) molecules with iron in the heme group:

Hb (i) lost as vesicles-20%- more from old, aged RBCs, particularly in the last half of its 120d life span.

(ii) Hb aggregated gets attached to the inside membrane- instruments provide erroneous results.

AnaemiaAnaemia --Diet related, RenalDiet related, Renal Anemia, ThalassemiaAnemia, Thalassemia

120d 120d lfelfe span RBCsspan RBCs-- ageage-- 2x102x1066/s renewal/s renewal

300 miles journey, 170 000 return trips300 miles journey, 170 000 return trips--

heart, lungsheart, lungs-- squeezing in/out of capillaries ~ 25% squeezing in/out of capillaries ~ 25%

smaller smaller diamdiam-- surface material lostsurface material lost--

charge charge decreasedecrease--macrophages come closer, grab, macrophages come closer, grab,

eliminate by the RES.eliminate by the RES.

In In diseasedisease-- defective celldefective cell-- impairedimpaired hematopoiesishematopoiesis--

RBCs premature eliminatedRBCs premature eliminated-- prematurely prematurely anaemiaanaemia--

Thalassemia ~55d, Thalassemia ~55d, Renal disease RBCs~80dRenal disease RBCs~80d

The cells have ~25-30% decreased charge-

the resident macrophages get closer to

RBCs,

- position themselves

- to grab damaged/old RBCs

- for elimination by the RES.

Hb measurement in the blood

is not a true estimate – Rethinking wiser for precision – Quantify Hb directly from

RBCs various ages isolated easily-30¢/sample

More reliable estimates of Hb needed

Correlated with the Y-RBC subset-obtained by PRERCOLL isolation

RBCs High negative charge keeps awayThe highly negatively charged macrophages

When RBCs–ve charge decreased ~25-30%

the resident macrophages

get closer to RBCs - position themselves

- to grab damaged/old RBCs

- for elimination by the RES.

RBC:120d life span:with ageing-biomaterials, water are lost

volume (v) decreases � �density (d) increases (m ÷ v = d) Sialic acid (‘homing’ mol) charge decrease; ageing, storage lesion, thalassemiaCells-PERCOLL fractionated –lightest density –young- Y-RBCs densest density –old- O-RBCs. Hb lost as vesicles- more from old, aged RBCs, 20% Hb lost, particularly in the last half of its life (in storage).

Passage of a red blood cell through a single-pore Photo-mounting of two independent scanning electron micrographs.

Capillary with Red Blood Cell (TEM x32,830). This image is copyright Dennis Kunkel at www.Dennis Kunkel.com, used with permission.http://www2.estrellamountain.edu/faculty/farabee/BIOBK/biobookcircsys.html

η: viscosity. D: dielectric constant; 10 million electronEPM: electrophoretic mobility, µm/s/V/cm charges/100 µm2

Blood elements are notoriously heterogeneous – as are the cord blood cells.The lightest: Young RBCsSeparated By PERCOLL discontinuous Density centrifugation of RBCs :Over various PERCOLL-buffer solutions To obtain The Densest- Old RBCs that bear decreased sialic acid related charge by 25-30%. ζ- potential: lower by: 7mV.

Mehrishi & Risso, 2016J Biol Phys Chemistry 16 (2016).

Role of the electric charge• Importance- High negative charge of

RBCs

• 107/cell surface area 144 µm2 prevents the highly negatively charged

• resident macrophages

• When charge of RBCs decreased-macrophages come closer, position themselves, grab the RBCs with the decreased- charge and eliminate them

• Isolation of cells

• Cord blood stem (CD34+ )cells

Red damaged surface molecules; Red damaged surface molecules;

BlueBlue and and GreenGreen intact intact

Zeta potential difference :7mVRed: damaged-deficient molecules

Blue and Green- Intact amounts SA‘homing’ molecules. Mehrishi & Risso, 2016J Biol Phys Chemistry 16 (2016).

O-RBCs surface sialic acid α−carboxyls - LOST.

∆ζ Decreased ~ 7 mV (~25%)= 2.5 x 106 electron charges per O-RBC= 4 x1013 Coulomb per O-RBC

O-RBCs surface sialic acid α-carboxyls - LOST.

∆ζ Decreased ~ 7mV (2.5 x106 electron charges)

Normally RBC bears ~ 55 x106 electron charges and

the highly negatively charged resident macrophages

cannot come close enough to grab them.

A consequence of decreased charge:macrophages can come closer, position themselves

to grab the damaged RBCs to eliminate them.

∆ζ Decreased ~

7mV (2.5 x106 electron charges) 107 ec/RBC

0.98 [Smφ 6.7x 106 electron charges/100µm2

against 1.39

1.28

Electrophoretic mobility - 1.39 µm/s/V/cm

Lmφ 15.2 x 106 electron charges/100µm2

Red Cell: normal old Thalassaemic No data on

RBCs RBCs Unfractionated

Damaged SCD RBCs

75,600 El Chage 58,475 56,700Per 100µm2 Per 100µm2 Per 100µm2

~0.8 x 105 ~0.6 x 105 ~0.6 x 105

RBCs Neuraminidase treated: ~0.5 x 105 ec/100µm2

45,360 ec/100µm2

Mφ φ φ φ ~152 x 105 ec/100µm2

Decreased charge cells- get closer,

Position themselves to grab and

eliminate via the RES

• TThalassemia (is a form of inherited autosomal recessive blood disorder characterized by abnormal formation of

• hemoglobin. -the protein in red blood cells that carries oxygen

• The abnormal hemoglobin formed results in

• improper oxygen transport – ‘protecting surface moeprematuredestruction –RBCs

• . Thalassemia:caused by variant or missing genes that affect how the body makes hemoglobin,

• RBC life span: normal: ~120d; thalassemia: ~55d• Renal anaemia RBC life span: ~80 days.

99% RBCs densest fraction (ρ ~ 1.12 g/cm3).Cf sickle cell disease: ρ ~ 1.12 g/cm3

Risso, Mansutti, Mehrishi. J Biol Phys Chemistry. 2015;15: 45-53

Thalassemia 1:Lightest. 2: Middle 3: Densest

Thalassemia:Monitoring blood test (0.1 to 1ml)

• Decreased expression of CD47 and CD55

• surface molecules on density-based subsets of red cells of β-thalassaemia intermedia patients compared to red cells of healthy blood donors

• Angela Risso, Elena Mansutti & Mehrishi

• (June 2015- JBCP)

• 25-30% Decreased sialic acid- charge-

• ‘homing’ molecule- premature elimination

Anemia Anemia -- Thalassemia RenalThalassemia Renal AnemiaAnemia

120d 120d lfelfe span RBCsspan RBCs-- ageage-- 2x102x1066/s renewal/s renewal

300 miles journey, 170 000 return trips300 miles journey, 170 000 return trips--

heart, lungsheart, lungs-- squeezing in/out of capillaries squeezing in/out of capillaries

~ 25% smaller ~ 25% smaller diamdiam-- surface material lostsurface material lost--

charge decreasecharge decrease--macrophages come closer, macrophages come closer,

grab, eliminate by the RES.grab, eliminate by the RES.

In diseaseIn disease-- defective celldefective cell-- impaired impaired

hematopoiesishematopoiesis-- RBCs premature eliminatedRBCs premature eliminated--

prematurely prematurely anaemiaanaemia--ThalThal ~55d, ~55d, Renal ~80dRenal ~80d

AnaemiaAnaemia: : Aboriginal-Torres Strait Islander

populations

The burden of stroke- substantially higher than in

non-Indigenous Australians, younger age - higher fatality.

Changing life style - from hunter gatherers- diet-

Shortage of – folic acid, vitamin B complex, iron-

Incidence of Anaemia is high, risks of diabtes II

AnaemiaAnaemia: : Aboriginal-Torres Strait Islander

populations

RBC monitoring- burden of stroke- substantially higher

younger age - higher fatality cf . others

Changing life style - from hunter gatherers- diet-

Shortage of – folic acid, vitamin B complex, iron-

Incidence of Anaemia is high, risks of diabtes II

Great need-increased focus on monitoring AnaemiaAnaemia

reducing

Aboriginal stroke incidence and improved outcomes

Strongly recommend – establish blood RBC subsets,

markers – sound rational bases for management.

AnaemiaAnaemia: : Aboriginal-Torres

Strait Islander populations

Proposals for blood cell studies consistent with current national policy and program initiatives including the National Strategic Framework for Aboriginal and Torres Strait Islander Health.

Renal anaemia: CKD: RBC life span ~80d

Patients: before, during and after~4 weeks

treatment with

rHuEPO v BAY 87-2243, FG4516 FG-

4592, AKB-6548, GSK1278863

Blood fractionated (count numbers and proportions of

Y-RBC::O-RBC)

Flow cytometry :cell subsets labelled with specific

Ligands for surface molecules- changes correlate with

Hb, MCV, MCHCSNA-FITC, CD47? CD55?

• new strategies to treat anemia are

still an evolving and fascinating area of experimental and clinical research. At present, the most promising class of agents seems to be HIF stabilizers, as evident in the number of molecules currently under development.

• This class of drug stimulates erythropoiesis by physiologic concentrations of endogenous EPO,whichmay translate into a clinical advantage because concerns for ESA safety are higher at the high doses.

• However, these theoretical advantages will need to be demonstrated clinically in large trials.

• Conversely, the class needs to be proved safe in light of the potential risks for increases in levels of VEGF and related factors and the possibility of widespread stimulation of complex pathways leading to unexpected side effects.

• The final judgment of benefit/risks of these agents will be possible only after the completion of large long term safety studies testing hard end points.

Discovery of sialic acid-Neural cell

sphingolipids-Klenk-Tay-Sachs, surface

Stroma,

Gunnar Blix 1894-1981discovery of sialic acids.

Ernst Klenk 1896-1971

Uhlenbruck, Faillard et coll.

UhlenbruckHouse- CologneDiscussion-Mehrishi- 1970Cat RBC structureUnique- deeper cleftsGlycolipid carboxylsExposed by pronase

Pilot Study Proposal by Mehrishi based on extensive

data from

Crucial: RBC-sialic acid discovery - Cologne (Klenk,

Uhlenbruck, Faillard- et coll.)- el charge-Piper, many

others, Cambridge- RBC charged-collaborators and

elsewhere- - heterogeneity – Danon, Marikovsky,

RBC fractionation-Lutz, the Dutch team-Bosman et

coll., Huang-Mehrishi. Risso-Mehrishi- Thalassemia

Weatherall- Oxford.

Study Proposal-Anaemias

CKD Patients: males femalesBefore, during at end of 4-12 wksTreatment with (i)rHuEPO (ii)BAY, FG4592-Hb etc.

Microsamples of Blood- (CPDA-1)0.1-1.0 ml Monitoring test-

Quantitative, rapid, inexpensive ($1/sample)

CKD Patients: Renal Anaemia

males females

Before, during at end of 4-12 wks

Treatment with EPO and

the related agents

Versus

Pyrolyl Hydroxylase inhibitors

• new strategies to treat anemia are


• HIF- PHD2 inhibitors- This class of drug stimulates erythropoiesis by physiologic concentrations of endogenous EPO,whichmay translate into a clinical advantage because concerns for ESA safety are higher at the high doses.


• Conversely, the class needs to be proved safe in light of the potential risks for increases in levels of VEGF and related factors and the possibility of widespread stimulation of complex pathways leading to unexpected side effects.


Hemoglobin (Hb)Structure of human hemoglobin. The proteins 2 αααα and 2 β

subunits are in red and blue, and the iron-containing heme groups in green.

Hemoglobin (HbA) Sickle cell Hb-glu substituted by val HbS

heme group

Sickle cell anaemia

Sickled and other red blood cells

Hb- sickle cell disease- Pauling

Linus Pauling during a conversation with the

protein chemist William Castle during an

Over night train journey between Denver and

Chicago discussed sickled red cell shape-

structure changes.

Pauling guessed that this might reflect a

structural difference between normal

(HbA) and sickle-cell haemoglobin

(HbS) which could be detected by the

pH- electrophoretic mobility

relationships:

Found: isoelectric points – revealing!

suggesting diff

HbA 6.87 (-ve ion) compositions

HbS 7.09 (+ve ion)

Sickle Cell Disease (HbS) – Itano/Pauling: Electrical approach indicated protein AA Changes

Hemoglobin preparations from Patients with sickle cell disease compared

with normal and with a trait: pH- electrophoretic mobility plots Isoelectric points: HbA: 6.87 (-ve ion) HbS: 7.09 (+ve ion)from Harvey Itano's dissertation. 1949.

Role of the electric charge

• Importance- keep separate

• When decreased- eliminated

• Isolation of cells

• Cord blood stem cells

86

Huang, Wu & Mehrishi+ et al. Human red blood cell aging: correlative changes in surface charge and cell properties. J. Cell Mol. Med. 15 (2011) 2634–2642

87EPMEPM

CD34CD34

WiolandWioland & &

MehrishiMehrishi

(1979.(1979.

ScandScand J J ImmunolImmunol. .

10:45310:453--463463

Age dependent Age dependent

changes in changes in

lymphocyteslymphocytes

88Anodic electrophoretic mobility (EPM) Anodic electrophoretic mobility (EPM) µµm/s/V/cmm/s/V/cm

Control Control Cord blood lymphocytesCord blood lymphocytes

Treated with Treated with V. V. ClCl neuraminidase (EC3.2.1.18] neuraminidase (EC3.2.1.18]

MaleylationMaleylation. . WiolandWioland & Mehrishi. 1976,1979& Mehrishi. 1976,1979

Neutrophils Neutrophils

Adults peripheral Adults peripheral

blood and in cord blood and in cord

blood are blood are

different in their different in their

responses.responses.

Regeneration of Regeneration of

neutrophils on neutrophils on

transplantation is transplantation is

delayed by 15delayed by 15--1818--

35 days 35 days --may be may be

because their because their

properties and properties and

responses are responses are

different. different.

TaniuchiTaniuchi et al. et al.

Pediatr Int. 1999 Feb;41(1):37-41.

Men, women (Men, women (premenstraualpremenstraual and menopausal and menopausal

differences) Rheologydifferences) Rheology-- changeschanges

ESR, ESR, HctHct, Viscosity, fragility, Viscosity, fragility %%

deformabilitydeformability

RBCs 120d life span heterogeneity RBCs 120d life span heterogeneity

Marina Marina KamenevaKameneva et al. et al.

PittsburgPittsburg

Dr. Marina Kameneva, PhD, Pittsburgh

94

Men, women (Men, women (premenstraualpremenstraual RBCs RBCs PercollPercoll FractionationFractionation

and menopausal differences) Lightest: Young RBCs and menopausal differences) Lightest: Young RBCs

RheologyRheology-- changeschanges Densest: Old RBCsDensest: Old RBCs

ESR, ESR, HctHct, Viscosity, fragility, Viscosity, fragility sialicsialic acid charge lostacid charge lost~30%~30%

deformabilitydeformability also in also in thalassemiathalassemia, , ‘‘hominghoming’’

moleculesmolecules surface markers surface markers

lost/damaged, rearrangementlost/damaged, rearrangement


2x 102x 106 6 /sec renewal/sec renewal--

revealed by revealed by electropherogramelectropherogram

Marina Marina KamenevaKameneva et al. et al. Cambridge: SeamanCambridge: Seaman-- 1963 1963

PittsburgPittsburg MehrishiMehrishi--1966 1966

Huang, Wu (Guangzhou), Huang, Wu (Guangzhou), MehrishiMehrishi (Cambridge) et al. 2011(Cambridge) et al. 2011

95

ζζ mV Old RBCs < mV Old RBCs < ζζ mV Young mV Young

RBCsRBCs

2525--30%30%

sialicsialic acid charge lostacid charge lost~25~25-- 30%30%


and menopausal differences) and menopausal differences) Lightest: Young RBCs Lightest: Young RBCs










PittsburgPittsburg MehrishiMehrishi--1966 1966



RBCsRBCs

2525--30%30%













PittsburghPittsburgh MehrishiMehrishi--1966 1966


97


RBCsRBCs

2525--30%30%













PittsburghPittsburgh MehrishiMehrishi--1966 1966


RBC age distribution in male and female bloodRBC age distribution in male and female blood

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Young RBCs Middle age RBCs Old RBCs

MalesMales FemalesFemales

The picture is based on data published by The picture is based on data published by MicheliMicheli et al. , 1984et al. , 1984

RBCRBC

DeformabilityDeformability

RBCRBCSedimentationSedimentation

RateRate

Low Low

ShearShear

ViscosityViscosity

RBCRBC

MechanicalMechanical

FragilityFragility

p<0.05

p<0.001

p<0.05

p<0.001

Dif

fere

nce

(%

)

Mechanical properties of old and young red blood cellsMechanical properties of old and young red blood cells

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"Young" RBCs "Old" RBCs

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Hem

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, %

Difference in male (red) and female (green) hemorheological paraDifference in male (red) and female (green) hemorheological parametersmeters

p<0.001

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RB

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rma

bil

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, %

p<0.001

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p<0.005

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ES

R (

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/hr

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p<0.01

Oxygen Delivery Index (hematocrit/blood viscosity )Oxygen Delivery Index (hematocrit/blood viscosity )

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Male blood

Female blood

p<0.001

Oxy

gen

Del

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y I

nd

ex

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Hem

ato

crit

, %

Difference in male (red) and female (green) hemorheological paraDifference in male (red) and female (green) hemorheological parametersmeters

p<0.001

50

60

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100

110

RB

C d

efo

rma

bil

ity

, %

p<0.001

0

10

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100

RB

C a

gg

reg

ati

on

, d

iffe

ren

ce (

%)

p<0.005

0

2

4

6

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14

ES

R (

mm

/hr

)

p<0.01

Oxygen Delivery Index (hematocrit/blood viscosity )Oxygen Delivery Index (hematocrit/blood viscosity )

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Male blood

Female blood

p<0.001

Oxy

gen

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nd

ex

·

The mechanical properties of blood of premenopausal women are superior to men, and place them at lesser risk for cardiovascular diseases than men in any age group.

·Male blood possesses an increased viscosity, RBC aggregability and RBC rigidity. ·RBCs of males were found to have mechanical fragility higher than for womens RBCs.

• Old RBCs (reproductive age)

< 50% Old RBCs males

• women of reproductive age have almost half as many old RBCs than in men -and almost x2 as many young RBCs than in men,

• young RBCs females (reproductive age)

x2 young RBCs males

• Old RBCs-

• increased mechanical fragility and aggregability, decreased deformability as compared to young RBCs.

• Decreased deformability and increased aggregabiJity of RBCs cause an increase in blood viscosity

• known as risk factors of cardiovascular diseases

•

• men possess a higher number of old RBCs with suboptimum mechanical properties than premenopausal women,

• who due to monthly blood loss

• (sort of a blood transfusion?)

• have a higher number of young cells and a lower number of old RBCs than their male counterparts

• -results of Kameneva et coll. suggest that an elevated hemorheological risk for males is associated with the age distribution of RBCs.

• This, in addition to significantly higher hematocrit, may be the reason for the increased risk of morbidity and mortality from cardiovascular diseases of men as compared to women of reproductive age.

Summary (Summary (KamenevaKameneva))•• Male blood has higher viscosity due to higher Male blood has higher viscosity due to higher

hematocrit and RBC aggregation and lower RBC hematocrit and RBC aggregation and lower RBC deformability deformability

•• Men possess a higher number of old RBCs and a Men possess a higher number of old RBCs and a

fewer number of young RBCs than premenopausal fewer number of young RBCs than premenopausal womenwomen

•• Old RBCs demonstrate an increased ability to Old RBCs demonstrate an increased ability to

aggregate and decreased deformability as aggregate and decreased deformability as compared to young RBCscompared to young RBCs

•• Oxygen delivery index is significantly lower Oxygen delivery index is significantly lower (p<0.001) for male blood than for blood of (p<0.001) for male blood than for blood of

•• prepre--menopausal femalesmenopausal females

•• Increased blood viscosity and Increased blood viscosity and aggregabilityaggregability of of RBCs and decreased deformability of RBCs are RBCs and decreased deformability of RBCs are known risk factors of cardiovascular diseases.known risk factors of cardiovascular diseases.

•• The difference in the mechanical properties of The difference in the mechanical properties of male and female blood places men at higher risk male and female blood places men at higher risk of cardiovascular diseases than preof cardiovascular diseases than pre--menopausal menopausal womenwomen

•• Blood donation or regular, small phlebotomy Blood donation or regular, small phlebotomy might help to improve rheological properties of might help to improve rheological properties of blood and reduce the risk of cardiovascular blood and reduce the risk of cardiovascular diseases in men and postdiseases in men and post--menopausal menopausal women!!!?women!!!?

Summary (cont.)Summary (cont.)

Thalassemia

• Thalassemia (is a form of inherited autosomal recessive blood disordercharacterized by abnormal formation of hemoglobin. The abnormal hemoglobin formed results in improper oxygen transportand destruction of red blood cells. Thalassemia is caused by variant or missing genes that affect how the body makes hemoglobin, the protein in red blood cells that carries oxygen

Thalassemia- Hb- genetic disorder-

Liverpool- Army- Singapore- Gurkha girl- anaemia suspected not responding. With foresight and enterprising, Weatherall using car batteries hooked up a high electrophoresis set up to diagnose thalassemia- a genetic disorder- not enough Hb made or defective-apparently had not asked for permisison and almost got court martialled. Affects large populations- a health and a financial burden. RBC lives for 120 days but 55d in thalassemia- cell surface charge is decreased- quantified by the machine- such as the CAM-APPARATUS or similar Zeiss.

Thalassemia:Monitoring blood test (0.1 to 1ml)

• Decreased expression of CD47 and CD55

• surface molecules on density-based subsets of red cells of β-thalassaemiaintermedia patients compared to red cells of healthy blood donors

• Angela Risso, Elena Mansutti & Mehrishi

• (June 2015- JBCP)

β-thalassaemia intermediapatients density based- red

cells- high countρn = 1.078 g/cm3 versus ρ > 1.1092 g/cm3)

and very dense RBCs (ρ ≥ 1.12 g/cm3).

Cf sickle cell disease: ρ > 1.12 g/cm3

Thalassemia 1:Lightest. 2: Middle 3: Densest

β-thalassaemia intermedia patients

% RBCn vs RBC Thal

Density Healthy S1-A S1-B S2-A S2-B

Low 6.85±2.17 0.05 1.3 1.4 2.5

Middle 73.58±6.06 0.5 40 37.7 54.2

High 19.57±7.84 0.45 57.9 47.5 43

Very high 0.005±0.001 99 1.78 13.9 3.75

• RBCs (Y-RBC numbers::O-RBCs) of patients with renal anaemia treated with rHuRPO versus one of the 3 new proteins-pyrolyl hydroxylase inhibitors.

• blood fractionated (numbers and proportions of Y-RBC::O-RBC) and flow cytometry of cell subsets labelled with SNA-FITC, CD47? CD55?

• This will then be correlated with the Hb, MCV, MCHC and the clinical state in renal anaemia.

• FG-4592 stabilizes the activities of HIF, a cytosolic transcription factor, leading to activation of the genes associated with erythropoiesis, including erythropoietin and enzymes involved in iron metabolism

• New strategies to treat anemia are


• HIF- PHD2 inhibitors- This class of drug stimulates erythropoiesis by physiologic concentrations of endogenous EPO,whichmay translate into a clinical advantage because concerns for ESA safety are higher at the high doses.


• Conversely, it needs to be proved safe in light of the potential risks for increases in levels of VEGF and related factors and the possibility of widespread stimulation of complex pathways leading to unexpected side effects.


RBCs -Ageing in Health, Storage, Hypoxia - Anemias ...SI).pdf · PhD degree awarded with the...

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