C. Ciudad de Dios Familias en su día a día
II. A.2 “Me he quemado varias veces, pero todo sea por los niños” Preparación de la
The effect of M. tuberculosis was examined with particular respect to DHEA. M. tuberculosis, either in the presence or absence of
dipyridyl, metabolised cholesterol and DHEA to six different
products which were identical in terms of colour, mobility and also in appearance when different indicators were used. Of these, four spots were seen when phosphotungstic acid was used, and the same four spots were seen when sulphuric acid and fluorescent plates were used. However, when sulphuric acid plates were heated and examined under the UV- lamp, two more spots in each track, either with or without dipyridyl, were seen. All spots were seen when the
Liebermann reaction was used. An attempt was made to identify these compounds produced as a result of DHEA-metabolism, using gas chromatography and the mass spectrophotometer.
Three compounds were identified as the most abundant compounds from DHEA metabolism by M. tuberculosis. These compounds were AED, 7-hydroxy DHEA and a reduced form of DHEA.
3^
A model of a thin layer chromatography (TLC), plate showing breakdown DHEA. DHEA was incubated with M. tuberculosis broth
culture for varying intervals. DHEA products were extracted, then samples were run on TLC-plates. Colour developers were used to reveal
File Operator Acquired Instrument Sample Name Mise Info Vial Number C : \HPCHEM\ 1 \DATA\DHEA4 8 . D 29 May 97 HP5970
3-ril3- acr u s i n g - AcqMê-tbo-d USP
Abundance TIG: DHEA48-.D-
1.8e+07 : 1 . 7 e + 0 7 : 1. 6e+07 4 l.Se+07: 1.4e+07 J 1.3e+07 : 1.2e+07 J l . l e + 0 7 : le+07 4 9 0 0 0 0 0 0 : 8 0 0 0 0 0 0 : 7000000 4 6 0 0 0 0 0 0 4 1 1 5 0 0 0 0 0 0 4 4 0 0 0 0 0 0 -] ; 3000000 : 2000000 4 1 0 0 0 0 0 0 4 0 — Time— > G a s c h r o m a t o g r a p h y and s p e c t r o m e t r y p r i n t out s h o w i n g d i f f e r e n t c o m p o u n d s i n c l u d i n g A E D as a r e s u l t o f D D H E A b r e a k d o w n by M. t u b e r c u l o s i s . 15.00 211.00 2 5 . 0 0 3 0 - .0 0 2 5 . 0 0 1 4 6
4.4 Discussion
The ratio of glucocorticoids to DHEA is very important. In murine tuberculosis, the adrenals first increase in weight, and then atrophy to 50% of their normal size. Hemandez-Pando (personal
communication) found that in early phase of infection when the adrenals are enlarged, DHEA or AED are protective. In the murine model, if there is very little DHEA the T-lymphocyte response shifts towards a TH2 cytokine profile and the animals die from pulmonary consolidation and pneumonia. On the other hand, if there is too much DHEA in relation to corticosterone, the animals die from tissue- destmction (Hemandez-Pando). Using the doses of AED and DHEA determined in the work desecribed in chapter three figure 4-1 and 4 2- show how the human and mouse observation agree. When
Hernandez-Pando gave DHEA or AED to animals during the phase of adrenal atrophy, the animals died. However, when these steroids were given concomitantly with corticosterone supplements, the animals were protected (personal communication).
Regarding human tuberculosis, there are situations which can be explained by the imbalance in the ratio of cortisol and DHEA. These situations are:
When DHEA levels are very high during pregnancy: this remains high because the main source of DHEA is the foetal adrenal and as a result cavitatory "adult type" tuberculosis may develop in very young neonates.
When DHEA levels are low, from a few weeks after birth until 5 years of age. At this time, consolidation and pneumonia are the dominant features.
When DHEA is about 50% of maximum at the age of 5-10 years, TB is rare. It is hypothesised that this is because the ratio of
glucocorticoids and DHEA is in balance.
When DHEA is high again in adolescence: at this time, cavitation and necrosis are the features of this type of adult TB.
In the light of the above findings and information, the results of the present study can be interpreted as follows:
When M. tuberculosis converts DHEA-S to DHEA this could shift the balance towards more DHEA in relation to cortisol. Similarly, when M. tuberculosis cleaves DHEA to AED and 7-hydroxy DHEA, this also could shift the balance as above. This means more active anti glucocorticoid activity is present in the lesion site. The presence of more active anti-glucocorticoids may lead to the type of tuberculosis whereby the patients dies from excessive migration of all sorts of inflammatory cells, cavitation and necrosis.
DHEA-S needs to be converted either to DHEA or further down to AED or AET in order to possess maximum activity. However, the ratio of these steroids or the ratio of the most active one of these steroids has to be in a delicate balance with glucocorticoids as was explained above. Therefore, the activity of the enzymes responsible for the conversion is a two-edged sword. It is known also that cortisol can be inactivated by the action of llp H S D . This apparently is
another control point, because it was found that in TB patients the activity of 11 pHSD is in favour of cortisol rather than cortisone. It has been suggested that this activity takes place in the lung (Baker et a l 1997). Therefore, the balance of these steroids in the lesion site
compounds as suggested by the data above, or by producing too much cortisol. Alternatively, both cortisol and DHEA and its derivatives could be produced in excess.
The paradox can be explained as follows :
Periphery and nodes
DHEA
Y
A
Cortisol relative to cortisone
TH l TH2
L esio n
DHEA active
Pro-inflammatory compounds necrosis
In the lymph nodes and periphery DHEA level is low while cortisol level is high relative to cortisone This disturbed balance leads to promotion of the TH2 response. However, in the lesion site the
presence of inflammatory cytokines such as T N Fa leads to necrosis since DHEA promotes production of more T H l cytokines.
Survival of mice with pulmonary TB: Treatment from d60 with androstenediol (AED) alone, or with AED & corticosterone
Data from R. Hemandez-Pando et al.
100 A ED + c o r tic o ste r o n e _ 75 - CO > 3 50 - CO
oo
25 - Cort Controls O AED 0 20 40 60 80 100 120 140 160 180 200 D a y s after intratracheal infection with M. tuberculosis03 C
2
ü£
_c O) cÎ
03 "O CO Q .I 4— « o o u_ 240 2 2 0- 2 0 0- 1 8 0 - 1 6 0 - 1 4 0 - 1 2 0- 1 0 0- 80 - 60 - 4 0 - 2 0- 0 AED+Cort C ontrols I I I I I I I I I 0 20 40 60 80 100 120 140 160 180Days since intratracheal infection with M. tuberculosis
Foot-pad swelling (delayed type hypersensitivity; DTH) was measured 24 hours after injecting 20p,g of soluble antigen of M. tuberculosis into the foot-pad.
F i g u r e 4-2 Comment
AED 25pg was given by subcutaneous injection dissolved in olive oil, 3 times/week (Mon, Wed, Fri). Corticosterone was added to drinking water at 3pg/ml. Either steroid used by itself was rapidly fatal. The combination of both steroids increased survival (upper Fig) and caused a return of skin-test positivity
(R. Hemandez-Pando, G. Rook, et al., manuscript in preparation)