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B. burgdorferi

Confidence, like art, never comes from having all the answers; it comes from being open to all the questions

Till Christopher, Elias och Lovisa

“A tick family in a rainy forest”, by Elias Sjöwall

B. burgdorferi

Borrelia B burgdorferi B. burgdorferi B. burgdorferi B. afzelii B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. garinii B. burgdorferi B. burgdorferi

Borrelia Bburgdor- feri B. burgdorferi B. burgdorferi B. afzelii B. burgdorferi B. burgdorferi B garinii B. burgdoferi

A. Anaplasma ACA APC AV B. Borrelia BALB/c BBB BL CBA CCL/CXCL CCR/CXCR CD C3H CNS CRASP CSF DC DNA EBV EM ELISA ELISPOT FLAIR GM-CSF HGA HIV HLA I. Ixodes IDSA IFN Ig IHC IL IV L LA LB LC LD LPS MCP MHC

MIP MRI MS NB NF NK NO OF Osp PAMP PBMC PBS PcG PCR PcV PHA PLDS PNS PPD PRR QOL RT RNA SF-36 spt s.s. SSS TBE TCM Tfhs TGF Th TNF Treg US WML

Sjöwall J PLoS One Sjöwall J Clin Exp Immunol Sjöwall J Submitted Sjöwall J Acta Radiol

Borrelia B burgdorferi Ixodes

Historical overview B. burgdorferi IxodesI

The vector (Ixodes) Ixodes B. burgdorferi I. ricinusI. pacificusI. scapularisI. persulcatus

Figure 1. Different Ixodes tick stages: Adult female (A), adult male (B), nymph (C), larvae (D) and mating adult ticks (E) (Courtesy of Pontus Lindblom, Dep. of Medical Microbiology, Faculty of Health Sciences, Linköping University, Sweden).

Figure 2. The life cycle of the Ixodes tick (Courtesy of Jeremy Gray, EUCALB).

B. burgdorferiB. gariniiB. afzelii B. burgdorferi

The B. burgdorferi spirochete BorreliaB. burgdorferi Treponema pallidum

Figure 3. The structure of the B. burgdorferi cell wall (Courtesy of Mona Widhe, Swedish Uni- versity of Agricultural Sciences, Uppsala, Sweden).

B. burgdorferi

B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi i.e B. burgdorferi vls B. burgdorferi, e.g B. burdorferi B. burgdorferiB. gariniiB. afzeliiB. spielmaniiB. bavariensisB. gariniiB. val- aisiana B. lusitaniae B. valaisiana B.lusita- niae B. burgdorferi B. burgdor- feriB. garinii B. afzelii

Epidemiology

e.g e.g B. burgdorferi

de factoB. burgdorferi et al B. burgdorferi

Prevention et al

et al. I. scapularis I. ricinus

Disease stages Table 1. Staging of the clinical features of Lyme borreliosis.

Early, localized stage Early, disseminated stage Late, disseminated stage

Clinical features, diagnosis and outcome of Lyme borreliosis B. burgdorferi B. burgdorferi. B. burgdorferi B. garinii, B. bavariensisB. afzelii B. spielmanii

Erythema migrans B. burgdorferi B. afzelii B. garinii B. gariniiB. burgdorferi . e.g B. afzelii

Figure 4. Erythema migrans, caused by B. afzelii (left) and B. garinii (right) (Courtesy of Sten- Anders Carlsson, the Åland Islands, Finland).

B. burgdorferi et al

Borrelial lymphocytoma B. afzeliiB. garinii

B. burgdorferi

Figure 5. Borrelial lymphocytoma on an ear lobe in a child (left) and on the mamilla (adult), with a surrounding erythema migrans (right) (Courtesy of Barbro Hedin-Skogman [left], Chil- dren´s Medical Clinic, Dalarna, Sweden and Katarina Ornstein (right], the Local Health Care Clinic, Hässleholm, Sweden).

et al

Lyme carditis B. burgdorferi

Neuroborreliosis

Table 2. Classification of neuroborreliosis. Adapted from Mygland et al., 2010121

Early NB Neurological symptoms with a duration <6 months

Late NB Neurological symptoms with a duration >6 months

Figure 6. Peripheral, right sided facial nerve palsy (Courtesy of Nucleus Medical Media, Inc., GA, USA). B. burgdorferi, e.g B. burgdorferi

B. burgdorferi B. burgdorferi i.e. B. garinii B. afzelii B. garinii B. afzelii

Acrodermatitis chronica atrophicans B. afzelii B. burgdorferi

Figure 7. Acrodermatitis chronica atrophicans, with a characteristic discoloration and atrophy of the (Courtesy of Chris Anderson, the Clinic of Dermatology, Linköping, Sweden).

B. burgdorferi et al.

Lyme arthritis B. burgdorferi

B. burgdorferi B. burgdorferi

Other manifestations of Lyme borreliosis

Asymptomatic B. burgdorferi B. burgdorferi B. burgdorferi

Co- B. burgdorferiIxodes Anaplasma A. phagocytophilum Babesia, A. phagocytophilum B. burgdorferi

, B. burgdorferi

Methods for detection of B. burgdorferi infection B. burgdorferi B. burgdorferi

Microscopy B. burgdorferi

Culture B. burgdorferi B. burgdorferi

The polymerase chain reaction B. burgdorferi ospA, B. burgdorferi,

B. burgdor- ferii.e.

Serological assays B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi , B. burgdorferi B. burgdorferi

Treponema pallidum B. burgdorferi B. burgdorferi B. burgdorferi

Table 3. Swedish recommendations for analysis of B. burgdorferi antibodies in serum.184 Serologic analysis is indicated in NB B. burgdorferi Lyme arthritisB. burgdorferi ACAB. burgdorferi Lyme carditisB. burgdorferi Serologic analysis is NOT indicated

B

Antibody analyses in the cerebrospinal fluid B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi

Additional methods B. burgdorferi B. burgdorferi

Treatment

et al et alet al B. burgdorferiin vitro in vitro in vitro in vitro B. burgdorferiin vitroin vivo in vitro in vivoin vitro in vivo

Table 4. Swedish recommendations for treatment of Lyme borreliosis in adults.95

Diagnosis Dosage Duration

EM, erythema migrans; PcV, phenoxymethylpenicillin; ACA, acrodermatitis chronica atrophicans; IV, intravenous.

The post- syndrome

i.e et al B. burgdorferi

Table 5. Proposed definition of the post-Lyme disease syndrome.* Inclusion criteria objective objective subjective fatigue, widespread musculoskeletal pain, complaints of cognitive difficulties

Exclusion criteria

B. burgdorferi

* Adapted from Wormser et al., 2006.76 # Stanek et al., 2011.49 ** Wormser et al., 2006.76

LB, Lyme borreliosis; PCR, polymerase chain reaction; B., Borrelia.

, i.e. et al

Figure 8. The 4 categories of illness, included in the concept of “chronic Lyme disease”.* * Adapted from Feder et al., 2007

LB, Lyme borreliosis; B., Borrelia

Possible causes of PLD symptoms

Figure 9. Possible causes and mechanisms of post-Lyme disease symptoms and the post-Lyme disease syndrome.

PLDS, post-Lyme disease syndrome; HGA, Human granulocytic anaplasmosis B. burgdorferi. B. burgdorferi B. burgdorferiB. burgdorferi B. burgdorferi

B. burgdorferi B. burgdorferi A. phagocytophilumBabesia microti et al B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi, B. burgdorferi

B. burgdorferi et al. et al Coxiella burnetiiBrucella , B. burgdorferi

B. burgdorferi et al

Innate recognition of pathogens e.g. e.g

Maturation of dendritic cells

Figure 10. Characteristics of immature and mature dendritic cells (DCs). Different stimuli can initiate and inhibit DC maturation. Adapted from Banchereau et al., 2000256 CCR, chemokine receptor; CD, cluster of differentiation; MHC, major histocompatibility complex; IL, interleukin, LPS, lipopolysaccharide; TNF, tumour necrosis factor; GM-CSF, granulocyte macrophage-colony stimulating factor; L, ligand.

Adaptive immunity e.g

T helper cell subsets

Cytokines i.e

et alet al

Table 6. Characteristics of the cytokines and chemokines included in the thesis. Cytokine Main producer cells Actions Paper Reference et al et al et al Chemokine Producer cells Actions Paper Reference et al et al et al B. burgdorferi

Figure 11. An illustration of the immune response elicited to B. burgdorferi after inoculation of the spirochetes in the skin during tick feeding. Green arrows indicate defence, red arrows inhi- bition and blue arrows synthesis of immune mediators. Immunological details are provided in different parts of the Introduction. IL, interleukin; CXCL, chemokine ligand; TNF, tumour necro- sis factor; DC, dendritic cell; IFN, interferon; APC, antigen presenting cell.

Activation of innate cells by B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi and B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi

Adaptive responses to B. burgdorferi B. burgdorferi

Antibodies B. burgdor- feri B. burgdorferi B. burgdorferi B. burgdorferi

T helper cell responses in Lyme borreliosis B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi et al B. burgdorferi et al B. burgdorferi B. burgdorferi B. burgdorferi

B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi B. burgdorferi

et al in vitro ex vivoin vivo e.g

Tetracyclines Streptomyces aureofa- ciens in vitro ,

B. burgdorferi

Angiogenesis and apoptosis (caspases)

Neutrophilic migration and chemotaxis Phospolipase A2 _ _ _ IL-2, IL-6, IL-8, IFN-³, TNF in a _ dose-dependent manner Scavenging of reactive oxygen species + _ T and B cell activation and Tetracyclines proliferation _ MMP (e.g. collagenases) New bone formation _ + +

Expression of NO synthetase Up-regulation of IL-10 in synovial tissue

Accelerated degradation of NO syntethase

Figure 12. The main immunomodulating effects of tetracyclines in vitro. Figure based on the following reviews.309,321,322 (+) indicates stimulation, (-) inhibition.

MMP, matrix metalloproteinase; NO, nitric oxide; IL, interleukin; IFN, interferon; TNF, tumour necrosis factor

X X B. burgdorferi B. burgdorferi X "Only action brings ideas to life"

B. burgdorferi

B. burgdorferi B. garinii

Subjects (Paper I–IV)

Patients (Paper I–IV) Definition of clinical outcome- - --B. burgdorferi -

Table 7. The diagnosis of the patients included in Paper I-IV.

EM, erythema migrans; LB, Lyme borreliosis; DOX, doxycycline; MRI, magnetic resonance imag- ing; NB, neuroborreliosis; ACA, acrodermatitis chronica atrophicans.

B. burgdorferi i.e

Figure 13. Subjects included in the thesis (n=148). ->-

Definition of clinical outcome B. burgdorferi B. burgdorferi i.e

Table 8. Clinical characteristics of the patients included in Paper IV.

Symptom CSF B . IgG CSF B . IgM Presence of CSF duration Antibiotic treatment Age antibody antibody mononuclear (years) at at diagnosis and No (years) Sex index* index* pleocytosis# MRI: follow-up Re-lumbar puncture post-treatment 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 B 10 B 11 B 12 B 13 B 14 15 B 16 B

*Antibody index: (OD-CSF/OD-serum) x (OD-CSF OD-serum), according to Hansen and Lebech, 1991.125 #Mononuclear pleocytosis was defined as 5 x 106 mononuclear cells/L in the CSF.

f, female; m, male; MRI, magnetic resonance imaging; pos, positive; neg, negative; DOX, doxy- cycline; CTRX, ceftriaxone; B., Borrelia; Ig, immunoglobulin; O-bands, oligoclonal bands; CSF, cerebrospinal fluid; OD, optical density

Controls (Paper II, IV) B. burgdor- feri

Excluded subjects (Paper I, IV)

Diagnostic criteria (Paper I–IV) B. burgdorferi B. burgdorferi B. burgdorferi B. burdorferi

Definition of clinical outcome (Paper II–IV) B. burgdorferi B. burgdorferi B. burgdorferiNB with persistent symptoms post-treatment

Methods (Paper I–IV)

Antibody assays in serum (Paper I–IV) B. burgdorferiB. burgdorferi B. burgdorferi B. burgdorferiB. afzeliiB. afzeliiB. burgdorferi .B. gariniiB. afzeliiB. afzeliiB. afzeliigariniiB. burgdorferi.

B. burgdorferi –

CSF analyses (Paper II–IV) 3 B. burgdorferi B. burgdorferi

Preparation of cells (Paper II–III)

Peripheral blood mononuclear cells 

Culture and stimulation of dendritic cells et al i.e i.e 

B. garinii B. garinii et al in vivo

Stimulation of whole blood cells B. burgdorferi et al B. garinii –

Stimulation of peripheral blood mononuclear cells et al E. coli ದ

The enzyme-linked immunospot assay (Paper II–III) et al. B. garinii

Figure 14. The principle of the ELISPOT assay (Courtesy of Mona Widhe, Swedish University of Agricultural Sciences, Uppsala, Sweden).

Flow cytometry (Paper II) i.e

–

Cytometric bead array (Paper II)

Luminex (Paper I–III) B. garinii

Figure 15. The principle of the Luminex cytokine detection assay.

Skin biopsy (Paper I)

=

Figure 16. Illustration of the skin punch biopsy technique and the three skin layers (© Reed- Elsevier, USA). Image from: http://www.answers.com/topic/skin-biopsy.

Immunohistochemistry (Paper I)

 › ----

The polymerase chain reaction (Paper I) et al B. burgdorferi B. burgdorferi B. hermsiiB. burgdorferi

Other assays in peripheral blood (Paper I, III) Dermatophagoides pteronyssinus Cladosporum herbarum

Magnetic resonance imaging (Paper IV) e.g et al

The follow-up questionnaire (Paper I) e.g

Neurological examination (Paper III)

The SF-36 health survey (Paper III) – – ––

Figure 17. Components of the SF-36 health survey. Adapted from Ware et al., 1994.336

The symptom severity score (Paper III) - - ---- Table 9. The symptom severity score (in Swedish) used in Paper III

Vecka 1 Datum Datum Datum Datum Datum Datum Datum Fyll i dagligen (gärna kvällstid) hur du upplever dina symtom. Symtom / / / / / / /

Skatta mellan 0-10 med nedanstående riktvärden:

0 = inga symtom 1 = lätta symtom 3 = måttliga symtom 5 = medelsvåra symtom 7 = svå ra sym tom 9 = outhärdliga symtom 10 = värsta tänkbara symtom

Vecka 2 Datum Datum Datum Datum Datum Datum Datum Symtom / / / / / / / Enbart blodprover …………………………….. Läkarbesök …………………………………… Läkarbesök ……………………………………. Enbart blodprover …………………………….. Läkarbesök ……………………………………

Vecka 3 Datum Datum Datum Datum Datum Datum Datum Symtom / / / / / / /

2 2i.e.≤

B. burgdorferi

Clinical outcome of erythema migrans (Paper I) > B. burgdorferi B. burgdorferi

Patient characteristics

-- et alet al B. burgdorferi

Table 10. Characteristics of patients with erythema migrans included in Paper I

Patients with Patients with no Statistical symptoms at 6 months symptoms at 6 months comparison In total Variable (A) (B) (A vs B) (A+B) n Sex f/m Age (years) Range Median Single/Multiple EM Tick bite reported Y/N EM size (cm) Range Median Borrelia antibody pos/n analysed C6 ab 3 mos 6 mos Borrelia IgM ab 3mos 6mos Borrelia IgG ab 3mos 6mos

Allergy Y/N

EM duration prior to treatment (days) Range Median f, female; m, male; EM, erythema migrans; Y, yes; N, no; pos, positive; n, number; mos, months; NS, non-significant; Ig, immunoglobulin; C6, a synthetic peptide; ab, antibodies. B. burgdorferi

No of patients EMof with No

July May June

August October December November

September

Figure 18. The monthly distribution of erythema migrans (EM) among the study participants (n=88), from the Åland Islands, Finland, from May 2002 to December 2004.

Asymptomatic patients included in the immunohistochemical analysis - - - - -

Table 11. Characteristics of the symptomatic and asymptomatic patients included in the im- munohistochemical analysis of cytokine expression in erythema migrans skin biopsies

Patients with Patients with no Statistical symptoms at 6 mos symptoms at 6 mos comparison Variable Included in IHC (A) Included in IHC (C) (A vs C) n Sex f/m Age (years) Range Median Single/Multiple EM

Tick bite reported Y/N EM size (cm) Range Median Borrelia antibody pos/n analysed C6 ab 3 mos 6 mos Borrelia IgM ab 3mos 6mos Borrelia IgG ab 3mos

6mos Allergy Y/N

EM duration prior to treatment (days) Range Median f, female; m, male; Y, yes; N, no; pos, positive; n, number; mos, months; Ig, immunoglobulin; NS, non- significant; IHC, immunohistochemical; EM, erythema migrans; ab, antibodies.

B. burdorferi subspecies in skin biopsies (Paper I) B. burgdorferi B. burgdorferi B. afzelii

B. gariniiB. burgdorferi B. burgdoferi B. burgdorferi B. burgdorferi B. afzelii Table 12. B. burgdorferi subtypes in erythema migrans skin biopsies from patients with and without symptoms six months post-treatment Patients with Patients without symptoms at 6 Patients without Statistical symptoms at 6 Statistical months (A) symptoms at 6 comparison months (C) comparison All included Variable Included in IHC months (B) (A vs B) Included in IHC (A vs C) (A + B) n (patients) n (EM biopsies) PCR positive Subtypes B. afzelii /garinii / sensu stricto Undetectable*

B. burgdorferi - - B., Borrelia; EM, erythema migrans; IHC, immunohistochemistry; n, number; PCR, polymerase chain reaction; NS, non-significant. B. burgdorferi

Cytokine expression in erythema migrans skin biopsies in relation to clinical outcome (Paper I) - - - -

A B

2 IL-12p70 expression in erythema IFN- expression in erythema 2 5.010 -5 4.010 -5 p=0.003

4.010 -5 3.010 -5 3.010 -5 2.010 -5 2.010 -5

expressing cells/μm  1.010 -5  -5 1.0 10 0 0

No IFN- of s s o o No of IL-12p70-expressing cells/μm IL-12p70-expressing of No mos mos m 6 6 6 t 6 m a at s m o oms mpt mpt Symptoms at Sy sy No symptoms at No

CD IL-10 expression in erythema

IL-4 expression in erythema 2

2 -5 1.010 -5 3.010 8.010 -6 2.010 -5 6.010 -6

4.010 -6 1.010 -5 2.010 -6

0 0 No of IL-4-expressing cells/μm IL-4-expressing No of s s cells/μm IL-10-expressing of No o mo mos 6 6 mos t 6 m t 6 t a a a s s m m to o mp mpt ymptoms Sy sy Symptoms at s No No

Figure 19 A–D. Immunohistochemical analysis of the number of cytokine expressing cells/m2 in erythema migrans skin biopsies from patients with (n=7) and without (n=18) persistent symptoms six months post-treatment. The diagrams illustrate the expression of interleukin (IL)-12p70 (A), interferon (IFN)- (B), IL-4 (C), and IL-10 (D) in erythema migrans (erythema) biopsies. The Mann-Whitney U-test and a manual Bonferroni correction were used for statisti- cal analysis. P0.0125 was considered significant. Horizontal lines denote the median.

E F 

2 IFN- expression control biopsy

IL-12p70 expression control biopsy 2  -5 4.010 -5 5.0 10

4.010 -5 3.010 -5 3.010 -5 2.010 -5 2.010 -5 expressing cells/μm   -5 1.0 10 1.010 -5

0 0

s No of IFN- s s o o No of IL-12p70-expressing cells/μm IL-12p70-expressing of No m mo 6 m 6 at 6 mos at at 6 s s ms o toms at ptom ptom m mp m y Sy sympt S o N No sy G H IL-4 expression control biopsy IL-10 expression control biopsy 2 2 2.510 -5 1.010 -5

2.010 -5 8.010 -6

1.510 -5 6.010 -6

1.010 -5 4.010 -6

5.010 -6 2.010 -6

0 0 No of IL-4-expressing cells/μm IL-4-expressing of No No of IL-10-expressing cells/μm IL-10-expressing of No s o mos m 6 mos 6 t 6 mos t 6 at a s s a m m o toms p mpto y ym Symptoms at S s o sympt N No

Figure 19 E–H. Immunohistochemical analysis of the number of cytokine expressing cells/m2 in control (unaffected) skin biopsies from patients with (n=7) and without (n=18) persistent symptoms six months post-treatment. The diagrams illustrate the expression of interleukin (IL)-12p70 (E), interferon (IFN)- (F), IL-4 (G), and IL-10 (H) in unaffected skin. The Mann- Whitney U-test and a manual Bonferroni correction were used for statistical analysis. P0.0125 was considered significant. Horizontal lines denote the median. B. burgdorferi

B. burgdorferi i.e in vitroB. burgdorferi B. burgdorferi per se

Innate immune responses to B. garinii in relation to clinical outcome (Paper II) B. garinii

Cytokine secretion in B. garinii-infected dendritic cells i.e B. burgdor- feriB. garinii - B. garinii B. burgdorferi

A D

p= 0.018 500 p=0.048 200 400 150 300

200 100 10 100 secreting cells/ 20 000 DC 000 20 cells/ secreting

 0 secreting cells/ 20 000 DC  0 -100

No of TNF- No of -200 IFN- No of -10 Chronic Subacute Asymptomatic Seronegative Chronic Subacute Asymptomatic Seronegative

B E

80 200 70 175 60 150 125 50 100 10 10

0 0

No of IL-4 secreting IL-4 cells/ of No DC 000 20 -10 No of IL-12 secreting IL-12 No of cells/ 20 000 DC -10 Chronic Subacute Asymptomatic Seronegative Chronic Subacute Asymptomatic Seronegative

C

60

40

20

0

-20

-40

No of IL-10 secreting IL-10 cells/ of No 20 000 DC -60 Chronic Subacute Asymptomatic Seronegative

Figure 20. The median (of triplicates) number of B. garinii-specific, tumour necrosis factor (TNF)- (A), interleukin (IL)-4 (B), IL-10- (C), interferon (IFN)- (D)- and IL-12p70 (E)- secreting cells/20 000 dendritic cells (DC) from patients with different clinical outcomes of Lyme bor- reliosis and from B. burgdorferi-seronegative controls, analysed with the ELISPOT assay. The B. garinii-specific cytokine secretion was obtained by subtracting the number of spots in unstimu- lated wells from the number of B. garinii-stimulated wells. Horizontal lines denote the median.

B. garinii-induced secretion of innate cytokines in whole blood cells B. garinii B. burgdorferi B. burgdorferi-

p = 0.031 60 50

40

30 20

IL-12p70 (pg/ml) IL-12p70 10

0 Chronic Subacute Asympt Seronegative

Figure 21. IL-12p70 levels (median values in pg/ml) in B. garinii-stimulated whole blood cell supernatants from patients with different clinical outcomes of Lyme borreliosis and from B. burgdorferi-seronegative controls. Asympt, asymptomatic. Horizontal lines denote the median.

B. garinii-induced secretion of chemokines in whole blood cells B. garinii i.e B. garinii B. afzelii

B. burgdorferi B. burgdorferi B. burgdorferi in vivo B. burgdorferi B. burgdorferi B. burgdorferi B. garinii B. burgdorferi B. burgdorferi B. garinii i.e

i.e et al B. burgdor- feri i.e B. burgdorferi B. burgdorferi

Effects of doxycycline on persistent symptoms and quality of life post- neuroborreliosis (Paper III) – –

Outcome of neurological examinations

Assessment of symptom severity

Assessment of quality of life (SF-36) – p-

Table 13. Outcome of the neurological examinations, symptom severity scores and assess- ments of quality of life with doxycycline and placebo treatment.

DOX 3 weeks PBO 3 weeks Patient NL exam SSS SF-36 NL exam SSS SF-36 No

DOX, doxycycline; PBO, placebo; NL exam, neurological examination; SSS, symptom severity score; spt, symptom, SF-36, Short Form-36; PCS, physical component summary; MCS, mental component summary; NA, not analysed; + indicates improvement; 0 indicates unchanged; – indicates impairment. The outcome was assessed by calculating the difference between the values at the end of and prior to treatment with DOX and PBO.

Effects of doxycycline on systemic cytokine responses (Paper III)

Antigen-stimulated cytokine responses in mononuclear cells et al

IFN- IL-4 AB

300 100 OF-stim before PBO 90 OF-stim before PBO OF-stim after PBO 80 OF-stim after PBO 70 OF-stim before DOX 60 OF-stim before DOX 200 OF-stim after DOX OF-stim after DOX 20

100 10

0 0 secreting cells/100 000 lymphocytes  -10

-100 -20 Number of IL-4 secreting cells/100 000 lymphocytes 000 cells/100 secreting IL-4 of Number No of IFN- of No

TGF- IL-12p70 C D

150 OF-stim before PBO 125 OF-stim after PBO OF-stim before PBO 75 OF-stim after PBO 100 OF-stim before DOX OF-stim before DOX OF-stim after DOX 75 50 OF-stim after DOX

50 25 25 0 0

secreting cells/100lymphocytes 000 -25  -25 -50 -75 No ofTGF- of IL-12p70No lymphocytes 000 secreting cells/100

Figure 22. The number of outer-surface fraction (OF)-specific IFN- (A), IL-4 (B), TGF- (C) and IL-12p70 (D) secreting peripheral blood mononuclear cells from patients with persistent symp- toms post-NB (n=9), detected by ELISPOT, before and after treatment with doxycycline (DOX) and placebo (PBO). The OF-specific cytokine secretion was obtained by subtracting the number of spots in unstimulated (spontaneous) wells from the number of spots in OF-stimulated wells. Values, which represent medians of triplicates, are given as number of cytokine-secreting cells/100 000 lymphocytes. The dotted lines in graph A (=15 cells/100 000) and B (=10 cells/100 000) indicate the cut-off for OF-specific cytokine secretion, as previously described.331 No significant differences in change of the number of cytokine secreting cells were found within or between doxycycline and placebo treatment. Horizontal lines denote the median.

Spontaneous cytokine levels in serum

Table 14. Spontaneous cytokine levels in serum and antigen-stimulated cytokine levels in mononuclear cell supernatants (pg/ml) before and after doxycycline and placebo treatment.

Cytokine Antigen Doxycycline median pg/ml (IC range) Placebo median pg/ml (IC range) Detectable (%) Before After Before After Serum Supernatants

IC, interquartile; IL, interleukin; GM-CSF, granulocyte macrophage colony-stimulating factor; TNF, tu- mour necrosis factor; OF, outer surface fraction; LPS, lipopolysaccharide; unstim., unstimulated.

et al – et alet al –

B. burgdorferi

MRI-findings in patients with persistent symptoms after treatment of neuroborreliosis (Paper IV) - 3 3 - - -- -

Table 15. Magnetic resonance p=1 imaging (MRI) findings in patients with persistent symptoms after treatment of neuroborreliosis and in matched controls.

Pair number: patient and control matter atrophy Cortical

matched for age and gender. in gray Lesions

WML, white matter lesions; P, patient; C, control; y/n, yes/no.

p=1 ganglia y n n n y n n n n n n n n n ny n n210021 n n n n n y nnnn n y nn n n n n n n n n n n n n y n n n n n n n n n Degree of subependymal lesion: n n n n n n 0=none, 1=slight, 2=moderate, Le sions in ba sa l 3=severe. 2 0 2 0 2 1 1 0 0 0 Uncountable defined as >100

WML. p=0,12* Degree of subependymal lesion

* Wilcoxon signed-rank test. median 1 median 0,5

p=0,29 lesions Subependymal p=1 p=1 Central WML Subcortical WML p=1 WML 67661010128 Periventricular p=0,50 0nnyn 0 nnnnnn 6 20nnnnyyyn1 1 none n n n n y n n n 0 P C PCPCPCPCP C PCPCPC 00yyyy 2 nnnnnn nnnnyyyy1 12020nyyynyny0 nnnnnn 203 1020 uncountable n none30 y n 15-20 n n y y n y y n y y y y y n y y y y 1 n y 1 y 2 12 10 none none n n n n n n y n 1 none none n n n n n n y n 1 none none n n n n n n n y 0 none none n n n n n n n n 0 15-20 15-20 y y y n y y y y 2 Number of WML of Number uncountable 30 y y y y y y y y 3

12 03 nnnnnn 4 0 2 nnnnnn nnnnnn 4 20yynnyyyn2 5 12yyyynyyy3 3020ynyyyyyn2 Pair number Pair 5 6 7 13 12 15 16 Cases with positive findingsMRI test Mc Nemar 8 9 10 11 14

Figure 23. An 83-year old female with persistent symptoms >12 years after treatment of neurobor- reliosis (patient no. 4, Table 8). The axial fluid- attenuated inversion recovery (FLAIR) image dem- onstrates severe (grade 3) subependymal capping along with lesions in the white matter in the periventricular, central and subcortical regions. Cortical atrophy is also present.

Figure 24. A 25-year old man, with symptom dura- tion >3 years post-treatment of neuroborreliosis (patient no. 5, Table 8). The axial fluid-attenuated inversion recovery (FLAIR) image demonstrates slight subependymal capping (grade 1).

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B. burgdorferi B. burgdorferi B. burgdorferi B. garinii B. burgdorferi B. burgdorferi

B. burgdorferi

B. burgdorferi never B. burgdorferi

I would like to bring this thesis to an end by expressing my sincere gratitude to all the people, who in one way or another, have contributed to its completion. In particular, I would like to thank:

Pia Forsberg XX Christina Ekerfelt Jan Ernerudh Marika Nordberg Barbro Hedin-Skogman X X

Linda Fryland XXX X Charlotte Gustafsson X

X Anne AaltoFlorence SjögrenAnna LedelAnna CarlssonJonas RipemoSven BergströmUlf GarpmoOuti VaaralaÖrjan Smedby X My colleagues Rolf Maller Christer EkdahlAnders Martinsson : Marika Nordberg, Dag Nyman, Sten-Anders Carls- son, Christian Jansson, Susanne Olausson, Clara Nyberg, Hans Granlund Peter Wahlberg X

Mona Widhe, Charlotte Gustafsson, Sara Jarefors Maria Kvarnström X X Lise-Lott Lindvall X Mari-Anne ÅkesonPetra Cassel XYlva Billing X Urban Forsum Marie Larsson X

All AIR theBorrelia group colleagues Mats FredriksonLotta Lindh Åstrand the TEKLA-group X

ETT STORT TACK TILL…

patienter

Eva Göran

Annika John, Mamma X X Pappa BorealisX Elias Lovisa X X Christopher XX X

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