IMMUNOMODULATORY THERAPIES IN NEONATAL SEPSIS

Khalid N. Haque Professor of Neonatal Medicine

Neonatal Mortality from Sepsis. 29% of All Neonatal deaths in Asia & Middle East

18% of All Neonatal deaths in North America WHO 2010

Neonatal mortality from sepsis in VLBW infants(consolidated figures from USA,UK and Australia)

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1930 1940 1950 1960 1970 1980 1990 2000 2010

year

mortality%

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0 5 10 15

days

mortality%

Haque KN: 2002, 2006,2011

85% of infants in UK receive antibiotics at some times during their stay in NICU. Haque KN. J Clinical Excellence 2000, 2; 33-38 60% of NICU patients receive antibiotics in USA. Escobar G. Paediatrics 2003 70% of ICU patients (adults; EPICII study) receive antibiotics. Vincent L. JAMA 2009;302:2323-9)

Complication in survivors of neonatal sepsis

Odds ratio for CP following neonatal sepsis was 3.6 (2.5 - 9.3). Murphy et al, BMJ 1997

CONS infection remained significant for developing CP after adjusting for birth-weight, seizures, Neonatal ventilation and presentation.

Mittendorf et al. Lancet 1999 Neonatal infection was associated with 30-80% increase in odds of Neuro-developmental impairment. 30-100% increase in odds of poor head growth. Culture-negative clinical infection treated for 5 or more days was Associated with a 30% increase in neuro-developmental Impairment. Stoll et al JAMA 2004 Increased incidence of LOS, NEC and early death amongst survivors Kuppala VS et alJ Pediatr 2011

Neuro-developmental impairment in infants with different pathogens versus uninfected infants

Category N % NDI (unadjusted)

Adjusted OR for NDI relative to uninfected

Uninfected 1976 29% - CONS 853 44% 1.3 (1.1 – 1.6)

Other Gram positive

256 48% 1.7 (1.2 – 2.3)

Gram negative 185 45% 1.8 (1.2 – 7.6)

Fungal 96 57% 1.4 (0.9 – 2.2)

NEONATAL SEPSIS IS BAD NEWS FOR THE BABY

REPORT CARD Name: New-Born Class: Neonatal Nursery Age: Prem Term Remarks

•  Anatomic Barriers: B+ A Needs Attention

•  Phagocytic Cells: •  Polymorph - Neutrophils B - B Gets tired easily •  Macrophage/Monocyte C - B + Often sleepy in class •  NK Cells C - A Needs constant stimulation •  T Cells D - B + OK at multiplication and

division. Very weak at complex function

•  Complement: C B - Trouble getting started •  Antibody: C - A Relies entirely on Mother

•  Comments: A sensitive child, often picked on by neighbourhood bullies. Needs support, constant attention and may need one to one tutoring.

Adapted from Stiehm: Haque 1990

PAMS

DAMPS

URIC ACIDS

HMGB-1

hsp

Bacterial Lysis

Barriers APP

APRs IL-4, IL-1, IL-6, TNFα APC

T-Cell

Act T-Cell

B-Cell T-Cells NK-Cells Macrophage

Neutrophil macrophage IgG

IgM

IL-2

, IL-

18, T

NF-β,

INF-γ

IL-8, IL-17, IL-18, IL-21, IL-22, G-CSF GM-CSF

TLRs

PRR

complement

Vaso Activation & Coagulopathy

ENDOTH

ELIA

L DAM

AGE

C= Cardiovascular Hypotension H= Haemopoetic DIC, Anaemia Neutropenia A= Apoptosis Metabolic O= Organ Dysfunction Renal, Heart, Liver failure S= Suppression of Immune system

Septic Shock MOD DEATH Haque KN 1995,2005,2010

Macrophage activation and Toxin release lead to an imbalance in immunological homeostasis

Homeostasis

Pro-inflammatory cytokines

Anti-inflammatory cytokines Pro-inflammatory chemokines

Anti-inflammatory chemokines

Imbalance in coagulation homeostasis July 18th, 2012

Alternate/adjunctive therapies should;

Ø Help in killing the organism’s Ø  Increase Macrophage

surveillance Ø Neutralise toxins Ø  Increase number/function of

PMN Ø  Increase Opsonisation Ø  Increase Phagocytosis Ø  Improve Chemotaxis Ø Activate Complement Ø Regain Immunological balance Ø  Prevent cytokine induced injury Ø Regain coagulation homeostasis

Immuno-modulatory Therapies for Neonatal Sepsis

Adjuvant Therapies in Neonatal Sepsis; 1 Therapies that DO NOT work

Ø Granulocyte Transfusion No reduction in mortality Ø  Low dose steroid therapy No evidence of benefit.

Ø  Bacterial Permeability Increasing protein Little experience in newborns.

Ø  NO inhibitors No benefit

Ø  Monoclonal anti-TNF Harmful

Ø  Activated Protein C Not recommended

Ø  Oxidative Stress reducing agents: Selenium Worth looking at Melatonin No benefit

Ø  Glutamine No benefit

Granuloctye Transfusion: Mortality

Cochrane 2007, Mohan and Brocklehurst

Reduction of Oxidative Stress: Selenium; Glutamine: Selenium deficiency is associated with impairment of both cell-mediated immunity and B-cell function.

Darlow BA, Austin NC; Cochrane 2003

Adjuvant Therapies in Neonatal Sepsis; 2 That Do or May work

+ Colony Stimulating Factors ( G or GM-CSF) Benefit only in neutropenic/IUGR + Pro and Prebiotics Increasing evidence

+ Exchange transfusion Benefit not clear ü  Breast Milk Clear benefit ü  Lactoferrin Reduction in Late onset sepsis ü  Pentoxifylline Benefit in two small RCT’s

+ IVIG Polyclonal IgG Little/No benefit Pentameric IgM Clear benefit Hyperimmune IVIG No benefit

Colony Stimulating Factors: Mortality

Cochrane Meta-analysis 3/2003/2010 Carr & Modi

Exchange Transfusion Aim is to remove toxic bacterial products and harmful cytokines. Improve haemoglobin, cell numbers, replace clotting factors and provide antibodies. Not well studied as RCT and always used as last resort. Small studies have shown reduction in mortality from 70% to 35% (p=0.07) and from 41% to 27% (p=>.05). Vaine NE et al, Pediatrics 2008 Gunes T et al: Ann Trop Ped 2006

Even he knows that breast-milk is best protection against infection.

BREAST MILK Breast Milk Colostrum and early Breast milk is rich in immune, non-immune and anti-inflammatory components like (Lactoferrin) that accelerate intestinal maturation, resistance to and recovery from infection.

Lactoferrin: An 80 kDa iron-binding glycoprotein Innate Immune Function

Adaptive Immune Function

Acute Insult

Chronic Insult

Epithelial & Damaged Tissue

Infection Inflammation Trauma

Auto-immunity Neurodegenerative disorders

Responding Cells Degranulate

Lactoferrin Promotes Maturation of T-Cell precursors into competent Helper cells & immature B-cells into efficient antigen presenting cells

IL-12

IL-4

IL-10

Th1

Th2

B Cell

Ø

Ø

Ø

Ø

Ø

Ø Ø

Threg

Imm

une

Hom

eost

asis

BIRTH Days to Weeks Intestinal Microbes

BREASTFEEDING Maturation, Differentiation Proliferation of Dendritic CD8/CD4/ Cells

Th1 Th1

Dead Th1 cells

Th2

IL-12

Th2 Bias

Few Dendritic Cells

IL-12R +IL-12

IL-12 induced down regulation of IL-12R on Th1 cells inhibiting IL-4 induced apoptosis

IL-4 induced apoptosis

LACTOFERRIN

Th1 cells ready To fight infection

IL-4

Th0

INTESTINAL/RESPIRATORY EPITHELIUM

LACTOFERRIN (LF)

Adhesion

Type 111 Killing of Enteric Pathogen

Biofilm formation

LF

Enteric bacteria

Invasion

ANOIKIS

Anti-Inflammatory Anti-viral Anti-fungal

LF or Lfcin + lysozyme Killing IgA Secretion Block pathogen from binding to TLR

1.  Lactoferrin has a direct iron-dependent bacteriostatic and non-iron-dependent bacteriocidal action on LPS-bearing Gram-negative bacteria.

2.  Lactoferrin reduces sepsis induced oxidative stress, altering the magnitude and production of cytokines & apoptosis.

3. Lactoferrin enhances phagocytosis, cell adherence and release of pro-inflammatory cytokines. Actor JK et al 2010

E.Coli Clearance

GO= Glucose Oxidase, LF=Lactoferrin, PBS=Phospate Buffered Saline, HAS=Albumin)

Oral lactoferrin for the prevention of sepsis and necrotizing enterocolitis in preterm

infants

Venkatesh P, Abrams S; Cochrane Database of Systematic Reviews 2010, Issue 5.

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LOS Mortality NEC >2 Fungal Infec

Lactoferrin

Lactoferrin + Lactobacillus

Control

Manzoni P, Rinaldi M, Cattani S, et al; JAMA 2009

Bovine lactoferrin supplementation for prevention of late-onset sepsis in very low-birth-weight ( < 1000 Gram)

neonates: a randomized trial.

LF = 151 LGG = 151 Control = 168

Probiotics and or Prebiotics and Sepsis

Al Faleh K, et al Cochrane 2007, 2010

Pentoxifylline A Methyl Xanthine Phosphodiesterase Inhibitor •  Decreases endothelial adhesion of

activated neutrophils. •  Increases chemotaxis. •  Modulates leukocytes interactions. •  Inhibits monocyte and macrophage derived TNF alpha, IL-1,IL-6, IL-10 •  Is anti-pro inflammatory cytokines •  Improves haemodynamic

performance during sepsis.

“Effect of the immuno-modulating agent, pentoxifylline, in the treatment of sepsis in prematurely delivered infants: A placebo-controlled, double-

blind trial”.

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Mortality TNFα(pg/ml) IL-6 (pg/ml)

Pentoxifylline Placebo

Lauterbach, R. Critical Care Med 27(4);1999: 807-814

6% 39%

* P = .009

*

*

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Mortality Mortality (Kashmir)

Mortality from Septic Shock

Mortality from NEC

TNFα(pg/ml) IL-6 (pg/ml)

* P = .009

Pentoxifylline Control

Pentoxifylline, in the treatment of sepsis Lauterbach, R. Critical Care Med 27(4);1999: 807-814.

Wajid Ali JK et al:Practitioner 2006;13(4):204-207

*

*

6 39

16 40

78

10 8 28

PENTOXIFYLINE: Mortality

Haque KN: Cochrane 2003, 2011

Zinc as adjunct treatment in infants aged between 7 and 120 days with probable serious bacterial infection: a randomised, double-blind, placebo-controlled trial.

N=655

Bhatnagar S et al. Lancet 2012; 379: 2072–78

NNP=15

Zinc improves mucosal barriers, lytic activity of phagocytic and natural killer cells, expression of cytokines, increase CD4 and IL-2 production, activate C3 and phagocytosis.

Dorset farmer Benjamin Jesty successfully vaccinated and induced immunity in his wife and two children with cowpox during a smallpox epidemic in 1774. Lady Mary Wortley Montagu discovered the Ottoman Empire concept of variolation during her stay in Istanbul 1716-1717 and brought the idea back to Britain which was taken up by Edward Jenner 1749-1843

Antibody/Immuno Therapy

1900 1930 1940 1980 2010 Antibody Antibiotics IVIG Therapy Sulpha Penicillin

Serum (Immune antibody) Therapy Era

Dev

elop

men

t of I

mm

une

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rapi

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ibio

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esis

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mun

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ompr

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ed H

ost

Up’s and Down’s of Antibody Therapy

1967

“We should close the book on infectious disease”. William Stewart, Surgeon General, USA

Immunoglobulins in Neonatal Sepsis have been studied and used for over

30 years.

Have we finally answered the

question?

Intravenous Immunoglobulin modulate T and B cells in Sepsis.

Kazatchine et al: NEJM 2000

√

√ √

√

Rationale for using Intravenous Immunoglobulin in Neonatal Sepsis.

" Improves migration of neutrophils to the site of infection (Harper 1982, Christensen 1986).

" Prevents depletion of neutrophil storage pool (Harper 1982, 86).

" Increases PMN chemo luminescence & opsonic activity. " Activate complement (C3,C5,C3dg, THC), Scavenge activated

C3b, C4b {Christensen ’86, Hill ’90}. " Release PMN’s into circulation (Christensen ’88). Ø Cytokine modulation: Stimulate IL-1RA, IL-8, IL-10, sTNF-R

sIL-2R; Inhibit; Endotoxin, Exotoxins (Super antigen) C4b, C3b, TNF-alpha, IFN-gamma, IL-1, IL-2 IL-6 (Johnson ’93, Haque ’94).

" Limit Leukocyte- Endothelial interaction (Stephan ’93). 0

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60

70 STIMULANT ALONESTIMULANT + IVIG

*

Inhibition of TNF-α,IL-6 And Endotoxin Release

Albumin Group

Antibiotics

IgM-enriched IVIG

0 1 2 3 4 5 6 7 8 9

10

TNF-α IL- 6

Staph Entrotoxin B + Mononeuclear Cells

Staph Entrotoxin B + Mononeuclear Cells + IVIG

Toungouz et al 1995

Anderson 1990

S. Oesser 1999

41

endotoxin exotoxin

Bacteria

Gram-negative Gram-positive

activation of cells and humoral systems

cytokines, mediators and receptor molecules

proinflammatory antiinflammatory

sepsis Systemic Inflammatory Response

Immune Paralysis, Anergy

Antibacterial activity

IgM

IgG

Antitoxin activity IgG

IgM

Mediator modulation

IgG

Anti-inflammatory

IgA

IgM Passive

immunization IgG

IgM

Immunoglobulin Therapy in Sepsis® NEONATAL SEPSIS

The most reliable evidence for therapy is from Randomised Controlled Trials…

“If you are scanning an article about therapy and it is not a randomised trial, why on earth are you wasting your time?” David Sackett:

Center for Evidence Based Medicine, University of Oxford

Scientific alchemy of the 21st century: Meta-analysis

Feinstein 1999

IVIG in Treatment of Sepsis

Jenson & Pollock 1997 Paediatrics

“UNEQUIVOCAL BENEFIT IN PREVENTING DEATH; SURVIVAL INCREASED NEARLY SIX FOLD”

All Cause Mortality reduced four folds Haque KN. B J Intensive Care 1997; 7(1): 12-17

IVIG in Treatment of Sepsis

“MINIMAL EFFECT”

Lacy & Ohlsson 1998 Cochrane Library

3% reduction in sepsis and a 4% reduction in one or more episodes of any serious infection

Ohlsson, & Lacy, Cochrane Systemic Rev 2003

IVIG in Treatment of Sepsis

“unequivocal benefit in preventing death in adults with sepsis and a trend towards reduction in mortality in the newborn . Aljandera 1999, 2002 Cochrane Library

No reduction in mortality

Aljandera et al, 2010 Cochrane Review

IVIG in the Treatment of Neonatal Sepsis

Treatment with IVIG in cases of subsequently proven infection “resulted in statistically significant reduction in mortality:” NNT 10 (Lacy & Ohlsson, 2010)

Development of Immunoglobulin Preparations 1st Generation: Enzyme Degradation

Incomplete molecules Compliment Activation 2nd Generation: Chemically Modified 3rd Generation: Intact Molecules

95% Monomeric IgG Viral Reduction

4th Generation: Intact Molecules

99% Monomeric IgG Low anti-complimentary activity Viral Reduction Specialised preparations; IgA,

IgM-Enriched

Initial immune response is mainly IgM however, in the neonate, serum IgM concentrations are 10% of adult values. IgG concentrations depend on gestation. Lower the gestation, lower the IgG levels.

Neonatal B cell Function Two Phase Response to Infection

IgM IgG

Time

Structural Advantage of IgM over IgG * 10 Fab parts for antigen binding * Agglutination is 100 times greater * Complement activation is 400 times greater * Phagocytosis increased 100 folds * Better elimination of bacteria and toxin neutralisation

Döcke, W.D. et al., Int. Care Emerg. Med. 18, 1994 Haque KN. Year book of Intensive Care and Emergency Medicine. In: Vincent JL, Ed. Springer: USA 2007; pp. 55-68.

IgM-ENRICHED IVIG IN TREATMENT OF NEONTAL SEPSIS

25

16.3

6.8

CONT IVIG IgM IVIG

16.7

21.9

3.14.8

ALLINFANTS

<35 WEEKS

Haque 95 Hellwege 99

0.05

0.017

Mortality Mortality Control

IgM-Enriched

%

Intravenous Polyclonal IgM-enriched Immunoglobulin Therapy in Severe Sepsis.

Favors treatment Favors controls

Tugrul 5/21 7/21 0.62 [0.16 - 2.42] Behre 9/30 10/22 0.51 [0.16 - 1.62] Schedel 1/27 9/28 0.08 [0.01 - 0.70] Rodriguez 8/29 13/27 0.41 [0.14 - 1.25] Samatha 5/30 8/30 0.55 [0.16 - 1.93] Haque 1/30 6/30 0.14 [0.02 - 1.23] Buda 5/22 16/44 0.51 [0.16 - 1.66] Reith 7/35 16/32 0.25 [0.08 - 0.74] Haque 3/44 11/43 0.21 [0.06 - 0.83] El - Nawawy 14/50 28/50 0.14 [0.02 - 1.23]

0.01 0.1 1 10

Test for heterogeneity : χ 2 = 5.59, df =9 (p=0.78) Test for overall effect : U=28.26 (p<.00001)

Study IVIG n/N

Control n/N

Total (95% CI) 318 327 0.35 [0.23 - 0.54]

RR ( fixed ) [95% CI]

RR ( fixed ) [95% CI]

Norrby-Teglund A, Haque KN, Hammarston L. J Int Med 2006;260:509-16

Use of polyclonal immunoglobulins as adjunctive therapy for sepsis or septic shock in Neonates

Kreymann K. et al., 2007, Crit Care Med 35, 12: 2677-2685

NNT=9

Intravenous Immunoglobulin’s in the Treatment of Neonatal Sepsis

NNT= 7 Haque KN: J Med Science 2010;3(3); 160-67

(10 countries, 3,500 babies)

NO DIFFERENCE IN OUTCOME

IgG IVIG Studies IgG IVIG Studies

Proven Infection

Critical Serum IgG Level in Babies with Neonatal Sepsis

Haque KN et al: Clin Exp Immunol 1986, 1995 Clapp DW et al: J Ped 1989

Mg/

dl

Days

0

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400

600

800

1000

1200

1400

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Mean Lower Level of IgG

Mean Highest level of IgG

Mean Serum IgG Levels in Babies who Died of Sepsis

Days

Mg/

dl

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1000

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Lowest Normal IgG Levels

Highest Normal IgG Levels

Serum IgG Levels in babies who Died of Sepsis

Mg/

dl

Haque KN et al: Clin Exp Immunol 1986, 1995 Christensen G et al Pediatrics 1995 David Clapp et al: J Ped 1990

Critical Level of Serum IgM in Babies with Sepsis

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Mean IgM Level in Babies given IVIG

Mean IgM Levels in Babies given IgM-eIVIG

Mean IgM Levels in Babies who Died of Sepsis

To achieve IgG levels > 400 mg/dl and IgM level > 20 mg/dl one needs to give 250 -  500mg/Kg/day of Immunoglobulin for at least four days.

33 34 35 36 37 38 39 40 41 42

IVIG Group Placebo

Mortality from Infection amongst babies born between 26 and 29 weeks of gestation: INIS

IVIG Group

Placebo

Relative risk of sepsisby gestational age

Risk ratio.5 1 2 5 20

Risk ratio (95% CI)

4.2 ( 1.1,15.8) <=26 weeks

3.9 ( 1.4,11.0) 27-30 weeks

1.5 ( 0.5, 4.3) 31-36 weeks

Haque KN et al: 2004

Safety and Complications •  Double filtration virtually eliminates the risk of viral

transmission and for Prion transmission. •  Risk of virus / prion transmission is less than

4800,000,000. Complications: Overall Incidence 0.5% •  Shock •  Haemolysis •  Reticulo-endothelial blockade •  Aseptic meningitis •  Headache

“ Mirror, mirror on the wall, which is the fairest meta-analysis of all?

“ as long as there is no better evidence, the results demonstrated should be sufficient reason to use such a preparation for adjunctive therapy of sepsis or septic shock” ‘That is exactly what I do’. Karl Werdan. Crit Care Med 2007; 35(12):2852-54

“To use or not to use? Polyclonal intravenous immunoglobulins for

the treatment of sepsis and septic shock”. ‘To use or not to use? Considering the available results, the answer must be ivIgG no and iviGAM yes, as long as larger high quality clinical trials are not available’. Edmund A. M Neugebauer. Crit Care Med 2007;35(12):2855

Conclusion Ø Lactoferrin, IgM-Enriched IVIG and Pentoxifylline are the current Immuno-modulators that can be considered as useful adjunctive therapy in neonatal sepsis. Ø  In the neonate, any Selenium

deficiency should be promptly corrected. Think also of Zinc.

Being a germ is not easy BUT microbes have been the longest dwellers of this

earth, nothing they do should surprise us. “It’s their world; we only live in it.”

Thank you

Main Reference

Haque KN: Use of Intravenous Immunoglobulin in the Treatment of Neonatal Sepsis: A pragmatic review and analysis. Journal of Medical Sciences 2010, September 2010. William Tarnow-Mordi, David Isaacs, Sourabh Dutta; Adjunctive Immunologic Interventions in Neonatal Sepsis. Clin Perinatol 37 (2010) 481–499