Caracteres Geomecánicos de las Discontinuidades

could be used to test the efficacy of isolates from CHT, one fraction (see methods)

was applied to the model. (This fraction has been shown in vitro to modulate IL-4

production—a mechanism thought to be involved in AD regulation(Latchman et al.

1995).

15 guinea pigs were sensitised with 50mg DNCB and they were challenged

with 0.15% DNCB after 2 weeks. Skin reactions were measured and recorded at 24

and 48 hours after challenge. Guinea pigs were then divided into 3 groups. One

group of guinea pigs were gavaged with normal CHT(PSE222)(150 mg in 2 ml of

drink water per guinea pig); another group was gavaged with fraction F; the other

group was given water only. After 2 weeks treatment, guinea pigs were challenged

with 0.15% DNCB and skin reactions were measured and recorded after 24 and 48

hours of challenge. Protocol: Control: n = 5 CHT(G): n = 5 F(G): n = 5 Placebo(G): n = 5 2 weeks water 2 weeks water 2 weeks water 2 weeks water 2 weeks water 2 weeks CHT(F) 2 weeks F(G) 2 weeks Placebo (G) challenge challenge sensitisation measurement

All guinea pigs produced significant increase of skin thickness when they

challenged with DNCB at 2 weeks. There was no significant difference in the

increase of skin thickness between each group. After the 2 weeks of treatment with

CHT, placebo or fraction F, guinea pigs treated with CHT showed a significant

reduction in the skin thickness increase compare with control group (Fig 5.3.14,

P < 0 .0 5 ); while there were no significant difference in the increase of skin

thickness in the guinea pigs treated with placebo or fraction F (compare with control

□ control L- I Placebo NwwH CHT KHma Fraction F 1.00 n ? a r 0.75-

I

Fig 5.3.14 CHT, placebo and Fraction F were given to guinea pigs by gavage daily for a period of 2 weeks. Challenges were made before and after the therapy. Measurements were made at 24 and 48 hours after challenge.

5.4

Comments

DNCB contact sensitisation in the guinea pigs has been used as a model of

induction and recall of a cell mediated immune response for over 25 years (Maibach

and Maguire, 1963; Maguire, Jr. and Ettore, 1967). Although not a model of

allergic eczema it offers a test system to dissect the effects of CHT on a T cell

mediated immune response in vivo. As the immunopathology of AD shows

characteristics of T cell mediated immunity clues to the mode of action of CHT in

eczema may be forthcoming.

Initial studies established that 2 weeks after sensitisation with DNCB on the

ear a recall reaction could be generated by topical DNCB challenge on the shaved

flank. This reaction expressed as an area of erythema and induration could be

quantified by measuring increase in skin thickness at 24 and 48 hours.

Treatment with CHT prior to sensitisation failed to affect the

subsequent recall reaction, this implies that such therapy has no impact on

immunological induction mechanisms. This is an important characteristic as it

suggests that CHT does not modulate the mounting of a primary response, and is

thus unlikely to have a significant systemic effect on immune defence.

CHT following sensitisation was effective in reducing the recall reaction.

This suggests that either mechanisms of acquired responses and/or effector

This action of CHT was shown to be effective after 2 weeks therapy and not

significantly enhanced after 4 weeks. Separation of the therapy from the 2 week

period immediately following sensitisation was equally effective, this confirms that

the action of CHT was only on the recall mechanism and not the development of

immunological memory in the period immediately following sensitisation.

The effects were dose dependent and sustained after cessation of therapy

(although the effect did decline). Relating these results to man it is suggested that

efficacy should be seen within 2-4 weeks and sustained for a limited but significant

period thereafter. This indeed has been shown to be the case as in clinical studies of

an 8 week period the most significant reduction in symptoms of AD was seen in the

initial 4 weeks period (Sheehan et al. 1992, and chapter 2 of this thesis). Such

similarities between this animal model and AD in man offer further evidence of the

relevance of these studies to the clinical situation and indirectly confirm the

relevance of T cell responses in AD.

There was some increased skin reactivity when guinea pigs were re­

challenged with DNCB (eg. result 5.3.9) especially in the guinea pigs without

treatment with CHT. It is recognised that the challenge carried out 2 weeks after

sensitisation may boost the subsequent skin reactions. However, it is clear that the

CHT greatly reduced the reactivity when compared not only to untreated controls

Subsequent studies demonstrated that gavage was as effective a treatment

method as continuous supplying of CHT; this result is encouraging as such a

method of application reflects the treatment regimen in man. The fact that the

placebo control had little effect on moderating the recall reaction further confirms

that it is within the herbs of the CHT that the efficacious factor lies. However initial

attempts to demonstrate efficacious activity of a purified ingredient failed to produce

positive results. The fact that this purified fraction has been shown to down regulate

activity of IL-4 production (Latchman et al. 1995) a mechanism implied in AD

pathogenesis, (Bieber et al. 1989; Vowels et al. 1995), emphasises the need for

caution in relating effects of CHT on this guinea pig model to its effect on atopic

eczema in man. It is important to emphasise that the mechanisms involved are

almost certainly different. Thus although this model allows the investigation of the

kinetics of CHT effects, dose response studies and comparison with placebo, there

is inevitably a limit to its value in dissecting in detail the way CHT may moderate

AD in man.

To understand more of the mechanisms of the immunomoderating capacity

of CHT in the guinea pigs, it was necessary to investigate the cellular immune

reaction in the skin lesions. This approach is addressed in the next section of the