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Effects of a new heterocyclic glucocorticoid, deflazacort (DFC), on the functions of lymphocytes from patients with rheumatoid arthritis (RA)
Int. J. Immunopharmac., Vol. 9, No. 2, pp. 133 Printed in Great Britain.
139, 1987.
0192-0561/87 $3.00+ .00 International Society for lmmunopharmacology.
EFFECTS OF A NEW HETEROCYCLIC GLUCOCORTICOID, DEFLAZACORT (DFC), ON THE FUNCTIONS OF LYMPHOCYTES FROM PATIENTS WITH RHEUMATOID ARTHRITIS (RA) MARCO SCUDELETTI*, CRISTINA PICCARDO*, P|ERLUIGI PIOVANO*, BRUNO IMBIMBO* and FRANCESCO INDIVERIt§ *Cattedre di Clinica Medica RR e tlmmunologia Clinica, Universit/t di Genova, Istituto Scientifico di Medicina Interna, Viale Benedetto XV, n. 6, 16132 Genova, Italy; *Gruppo Lepetit S.p.A., Via della Vetreria, n. 1, 22070 Grandate (Como), Italy (Received 28 November 1985 and in final form 25 April 1986) Abstract - - Deflazacort (DFC) is a new glucocorticoid which, when compared with prednisone (PDN), has similar anti-inflammatory actions, but lacks several unwanted side effects on mineral and carbohydrate metabolism. DFC is more efficient than PDN in inducing immunomodulatory effects. This study concerns twelve patients with classical RA who required corticosteroid therapy because of the ineffectivenessof other drugs. Six patients received 12 mg/day of PDN from day 1 to day 21 and 15 mg/day of DFC from day 29 to day 50. The second group of six patients received DFC initially followed by the PDN dosing regimen. At various times (4, 8, 24, 48 and 72 h) after the first administration of steroid, and 7, 14 and 21 days after the beginning of the treatment with PDN or DFC the following tests were performed (a) the phenotype of T lymphocytes isolated from peripheral blood; (b) the evaluation of the expression of HLA Class 2 antigens by PHA primed T cells; and (c) the analysis of cell behavior in mixed lymphocyte reactions. The data reported in this paper show that the oral administration of DFC to patients with RA affects the in vitro behavior of their lymphocytes. This immunomodulatory effect of DFC concerns both the phenotype and function of RA lymphocytes. Moreover it differs in both intensity and kinetic characteristics from the immunomodulating effect observed with PDN (control).
Deflazacort (DFC) is a new heterocyclic glucocorticoid* belonging to the class of oxazolino steroids (Nathanshon, Winters & Testa, 1967; Nathanshon, Winters & Aresi, 1969). This new compound possesses a systemic anti-inflammatory potency similar to that of prednisone (Schiatti, Selva, Barone, Restelli & Glasser, 1980; Nathanshon, Pasqualucci, Radaelli, Schiatti, Selva & Winters, 1969; Buniva, Dubini & Sassella, 1979; Caniggia, Marchetti, Gennari, Vattimo & Nicolis, 1977) but lacks several of the unwanted side-effects on mineral and carbohydrate metabolism observed with the latter (Hahn, Halstead, Strates, Imbimbo & Baran, 1980; Pagano, Lombardi, Ferraris, Imbimbo & Cavallo Perin, 1982). In a previous paper we reported that DFC is more efficient than prednisone in inducing immunomodulatory effects. Specifically,
we observed that (a) the depletion of helper T cells and an increase in the cytotoxic suppressor subsets; (b) the inhibition of the expression of the HLA Class 2 antigens by T lymphocytes primed in vitro and, (c) the reduction of activity of T cells in autologous MLR, were more pronounced following administration of DFC rather than PDN (Scudeletti, Pende, Barabino, Imbimbo, Grifoni & Indiveri, 1984). Rheumatoid arthritis (RA) is characterized by several immunological abnormalities, including an imbalance of the helper-suppressor T cell ratio (Zoschke & Messner, 1981) and the anomalous functioning of T lymphocytes in AMLRs (Indiveri, Scudeletti, Pierri, Traverso, Cetti & Ferrone, 1986) which can effectively be treated with glucoeorticoids (Nelson & C o n n , 1980).
§ Author to whom correspondence should be addressed. * (lift, 16a)-21-(acetyloxy)-Il-hydroxy-2'-methyl-5'H-pregna-l',4dieno[17, 16-~]oxazole-3,20dione (Fig. 1). 133
M. SCUDELETTIet
134
OKT3, OKT4, OKT8 directed against human T cell markers were purchased from Ortho Diagnostic (Milan, Italy).
21Ctt20--R
I
20('-~O t N C t-13 19
"'~ 0 /
Fig. 1. Chemical structure of deflazacort. In view of the above reported observations, we initiated experiments to analyze ex vivo the relative capacities of DFC and prednisone to affect lymphocyte function from patients with RA.
EXPERIMENTAL
al.
PROCEDURES
This study involved twelve patients with classical or definite RA (according to A.R.A. criteria) (four males and eight females, age ranging between 46 and 60 yr). The clinical data of these patients are shown in Table 1. In this study the patient inclusion criteria were as follows: (a) need for corticosteroids because of the ineffectiveness of non-steroid antiinflammatory drugs; (b) age less than 60 yr; (c) absence of counter indications for corticosteroid administration. The patients were divided into two groups. The first group (six patients) received 12 mg daily of PDN (Deltacortene-Lepetit) from day l to 21; and 15 mg daily of DFC (deflazacort-Lepetit) from day 29 to 50. In the second group (n = 6) the patients first received DFC followed by the PDN dosing regimen. The immunological evaluation was performed in each patient at 4, 8, 24, 48 and 72 h after the first administration of steroid; 7, 14, 21 days after the beginning of the treatment with PDN or DFC. This evaluation included: (a) the phenotype of T lymphocytes isolated from peripheral blood; (b) the evaluation of the expression of H L A - - Class 2 antigens by P H A primed T cells; and (c) the analysis of their behavior in mixed lymphocyte reactions. Antibodies Purified rabbit antibodies to human and mouse Ig were purchased from Yago immunodiagnostic (Burlingame, CA). The monoclonal antibody Q 5/13 directed against monomorphic determinants of H L A - - Class 2 antigens were kindly provided by Dr. S. Ferrone (New York Medical College, Valhalla, NY) (Quaranta, Pellegrino & Ferrone, 1981). The MoAbs
L ymphocytes
Lymphocytes were isolated by Plasmagel sedimentation and Ficoll-Hypaque gradient centrifugation from heparinized blood. T and B cells were isolated by nylon wool filtration from lymphocyte preparations containing less than 5% phagocyting (latex particles) activity (Indiveri, Huddlestone, Pellegrino & Ferrone, 1980). Rosette assays Receptors for sheep erythrocytes were detected by rosetting with 2-aminoethylisothiouronium bromidetreated sheep red blood cells (AET-SRBC). Membrane-bound Ig (MbIg), OKT3, OKT4, OKT8 and H L A Class 2 antigen-bearing cells were enumerated by the indirect rosette microassay performed in microtiter plates as described elsewhere (Indiveri, Wilson, Pellegrino & Ferrone, 1979). Cell cultures and DNA synthesis
T cells (1 × 106/ml in RPMI 1640 medium containing 10o70 fetal calf serum, 2 mM glutamine, 200 U / m l penicillin, and 0.2 mg/ml streptomicin) were stimulated with P H A M (Wellcome, London; final dilution 1:1000) for 72 h at 37°C in a 5o70 CO2 atmosphere. At the end of the incubation, cells were washed five times with a medium containing 0.5°70 amethyl-D-mannoside, in order to reduce cell agglutination (Scudeletti, Pende, Barabino, Imbimbo, Grifoni & Indiveri, 1984). MLRs were performed according to the procedure we have previously used (Scudeletti et al., 1984). DNA synthesis was measured by pulsing triplicate cultures with 1/aCi 3H-thymidine (spec. act. 2/aCi/mmol) for 18 h before harvesting on glass fiber paper with an automated sample harvester. The washed and dried filters were mixed with liquid scintillation cocktail and then counted in a /3 scintillation counter. Statistical evaluation was undertaken using the Student's t-test.
RESULTS Effect o f DFC and P D N on the blood lymphocyte subsets Four hours following the administration of a single oral dose of DFC the total number of T lymphocytes was significantly reduced. This effect of
Effects of a New Heterocyclic Glucocorticoid
135
Table 1. Clinical data concerning the patients included in this study Case number
Sex
Age
1
2
3
4
A.R .A . criteria* 5 6 7
1
M
51
+
+
+
+
-
2 3 4 5 6 7 8 9
F F F F M F F F
46 49 51 57 60 60 58 54
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + +
+ + + + -
+ + + -
10
F
56
+
+
+
+
+
-
11
M
58
+
+
+
+
+
-
12
M
49
+
+
+
-
-
-
+ + + + + + + + + + +
8
9
10
11
Diagnosis
+ + + + + + + + + + + +
+ -
+ + + -
+
RA RA RA RA RA RA RA RA RA RA RA RA
-
classical classical definite classical classical classical classical definite definite classical classical definite
RA classical t> 7 A R A criteria. RA definite >I 5 A R A criteria. * 1 = morn ing stiffness; 2 = pain on motion or tenderness in at least one joint; 3 = swelling in at least one joint; 4 = swelling of at least one other joint within 3 months; 5 = symmetric joint swelling with simultaneous involvement of the same joint on both sides; 6 = subcutaneous nodules; 7 = Rx typical changes; 8 = RA factor; 9 = mucine precipitate from synovial fluid in diluted acetic acid; 10 = characteristic hystologic changes in synovium; 11 = characteristic hystologic changes in the nodules.
Table 2. Analysis of the effect of prednisone (PDN) and defiazacort (DFC) on the lymphocyte subsets following the administration of a single oral dose of the two corticosteroids and at the 7th, 14th, and 21st day of treatment with equivalent doses of the two corticosteroids
Drug
Membran e markers Baseline
Hours following administration of P D N or DFC 4 8 24 48 72
PDN
AET OKT3 OKT4 OKT8
2.3+0.4 1.9-+0.7 0.9_+0.3 0.6_+0.2
1.8_+0.3' 0.4_+0.0* 0.5_+0.0* 0.6_+0.1
DFC
AET OKT3 OKT4 OKT8
2.4_+0.6 1.7-+0.5 1.1_+0.4 0.9_+0.2
0.9_+0.3* 0.9+0.4 *t 1.1_+0.2*t 1.0___0.4*t 0.7_+0.1' 0.6_+0.1'* 1.6_+0.4"§ 1.7-+0.8"§
* P<0.01 t P<0.01 * P<0.05 P<0.05
1.9_+0.2 1.2_+0.4 0.7_+0.1 0.5_+0.1
2.1_+0.9 1.9_+0.6 0.8_+0.4 0.7_+0.1
Duration of treatment (days) 7 14 21
2.2_+0.6 1.8_+0.6 0.8_+0.2 0.6_+0.2
2.3_+0.6 1.9_+0.4 0.9_+0.2 0.6_+0.2
2.5-+0.7 1.8_+0.4 0.7_+0.2 0.7_+0.1
0.8+0.1 *t 1.2_+0.6"* 0.7_+0.6** 1.6_+0.9"§
l.l-+0.4t* 2.3-~0.9 1.8_+0.6"t 1.4+0.7 t* 1.6-+0.8§ 1.7-+1.1'* 0.7_+0.4 t* 0.6_+0.2§ 0.5_+0.1'§ 1.4_+0.2"§ 1.5±0.1"* 1.2_+0.4'
2.4_+0.9 1.7_+0.9 0.8_+0.1 0.6_+0.2
2.3_+0.5 1.9_+0.6 0.7_+0.2 0.7_+0.1
1.9_+l.lt* 1.7_+1.1'* 0.5+0.2 *t 1.3_+0.1'
1.7-+0.6'* 1.7-+1.1'* 0.6_+0.2 t* 1.4_+0.1'
versus baseline. DFC vs PDN. vs baseline. DFC vs PDN.
DFC was more pronounced than that produced by
p h e n o t y p e a n d a n i n c r e a s e o f t h e cell s u b s e t b e a r i n g
PDN.
t h e T8 p h e n o t y p e ( T a b l e 2). A s a c o n s e q u e n c e o f
Moreover
the effect of DFC
on T lymphocyte
n u m b e r s w a s l o n g e r t h a n t h a t c a u s e d b y P D N s i n c e it l a s t e d u p t o 72 h f o l l o w i n g d r u g a d m i n i s t r a t i o n , compared
with
less
than
24
h
following
PDN
a d m i n i s t r a t i o n ( T a b l e 2). The analysis of the lymphocyte subsets following the administration of DFC showed that this steroid induced
a
decrease
of
T
cells b e a r i n g
the
T4
these m o d i f i c a t i o n s a n i n c r e a s e d O K T 4 + / O K T 8 + cell r a t i o w a s m a i n t a i n e d f o r u p t o 72 h f o l l o w i n g t h e a d m i n i s t r a t i o n o f D F C , w h i l e it r e t u r n e d t o t h e baseline values 8 h following the administration of P D N ( T a b l e 3). The effect of the therapeutic administration of DFC and PDN was evaluated by determining the total number of T lymphocytes and the OKT4+/
M. SCUDELETTIet al.
136
Table 3. Analysis of OKT4 +/OKT8 ÷ ratio in lymphocyte subsets following the administration of a single oral dose of the two corticosteroids and at the 7th, 14th and 21st day of treatment with equivalent doses of the two corticosteroids
Drug
Baseline
PDN DFC
1.5 __.0.6* 1.2+_0.4
Hours following administration of PDN or DFC 4 8 24 48 72 0.8___0.2 0.4__.0.1
1.4+0.1 0.3+0.1
1.3+0.6 0.4+_0.1
1.5+0.4 0.5+_0.1
Duration of treatment (days) 7 14 21
1.1+_0.6 0.4+_0.2
1.0__.0.2 0.4__.0.1
1.3+_0.3 0.3+_0.1
1.0+0.2 0.4_+0.1
*OKT4/OKT8 ratio. OKT8 ÷ cell ratio at the 7th, 14th, and 21st day of a daily treatment regimen with 15 and 12 mg of D F C and P D N , respectively. The total number o f T lymphocytes isolated from patients who received the D F C remained constantly and significantly lower than the value found before the first administration; the percentage of the OKT4 ÷ cells remained constantly 50% lower than the percentage found prior to drug administration (Table 2). The ratio O K T 4 + / O K T 8 + cells remained constantly lower than one (Table 3). In contrast, daily administration of P D N did not significantly affect the total number of T cells and did not affect the ratio of OKT4 + to OKT8 ÷ lymphocytes. This ratio remained constantly around one because of a slight reduction in the OKT4 + cell subset.
~ 6o
t---I
~ 4o ~ 2o ~5 ?
£ = ~.
9
~
3
to
I B
I 4
I 8
I 24
I 48
I 72
(h)
Effect o f corticosteroids on PHA-induced HLA Class 2 antigens expression by T lymphocytes Administration of a single oral dose of D F C or P D N reduced the expression of HLA-Class 2 antigens by T cells and their P H A - i n d u c e d proliferative response in vitro. Indeed less than 30°70 of T lymphocytes isolated from blood drawn 4 h after administration of D F C or P D N expressed HLA-Class 2 antigens upon P H A activation. This defective capacity of expressing HLA-Class 2 lasted up to 24 h following a single oral dose of P D N and up to 48 h following a single oral dose of DFC (Fig. 2). The capacity of T lymphocytes isolated from blood drawn during the therapeutic trial with a single daily oral dose of D F C was constantly reduced (mean 34°7o) relative to the value found before drug treatment. This defect was not observed during the daily treatment with P D N (Fig. 2), however. Both drugs affected the blastogenesis of T ceils upon P H A stimulation in a similar fashion (Fig. 2).
Effect o f DFC and PDN on the proliferative and stimulatory activities o f lymphocytes in allogenic and in autologous mixed lymphocyte reactions The administration of D F C or P D N , both as a
I •
I 14
I 21
(D~3ys)
Time for[owing
CTS (]dministrGtion
o=PDN e=DFC rn =PDN I=DFC
Fig. 2. Expression of HLA Class 2 antigens by T lymphocytes isolated and primed with PHA following the administration of a single oral dose of the two corticosteroids and at the 7th, 14th and 21st day of treatment with equivalent doses of the two corticosteroids (upper section). 3H-thymidine uptake by lymphocyte isolated and primed with PHA following the administration of a single oral dose of the two corticosteroids and at the 7th, 14th and 21st day of treatment with equivalent doses of the two corticosteroids (lower section). single oral dose, and on a daily dosing regimen did not significantly affect the responsiveness of lymphocytes activated in allogeneic M L R (data not shown). Lymphocytes isolated following the administration of D F C , however, failed to respond properly in both non T / T type and T / T type A M L R . A similar lack of responsiveness was also shown by T cells isolated after the administration o f P D N . The reduced proliferative response induced by a single oral dose of D F C was also observed during the
Effects of a New Heterocyclic Glucocorticoid
137
Table 4. Analysis of the variations of blastogenesis in autologous mixed lymphocyte reactions (AMLRs) of lymphocytes isolated following the administration of a single oral dose of the two corticosteroids and at the 7th, 14th and 21st day of treatment with equivalent doses of the two corticosteroids
Drug
AMLR type
Baseline
Non T-T 10.0__.3.2 PDN T Ia T-T 6.6-+1.2 DFC
Non T-T T Ia T-T
9.1_+4.2 6.2-+1.3
4
Hours after drug 8 24 48
72
Duration of treatment (days) 7 14 21
6.0__.2.1" 7.0_+2.2* 8.0_+2.1' 7.5_+0.8 8.4___1.6 9.6_+0.8 10.00_+2.0 10.2-+0.8 3.9_+1.4" 4.0-+1.8" 5.0_+1.4' 4.8_+1.6' 5.1__.1.4 6.0_+1.4 7.0-+1.4 6.0_+1.0 6.2_+1.9" 7.0_+2.1' 6.8_+1.3' 7.1_+2.0" 7.8_+1.2 6.4_1.6'§ 3.2_+2.1" 4.0_+2.4* 4.3-+1.6' 4.8-+0.7* 5.0_+0.9 5.0_+1.0
6.3_+2.0** 4.6_+1.3'*
5.8_+1.4t* 4.2_+0.7*
* P<0.01 versus baseline. t P<0.01 DFC versus PDN. * P<0.05 versus baseline. _,o<0.05 DFC versus PDN.
daily treatment schedule with this drug. Indeed, the blastogenesis in the non T / T AMLR was reduced by 30°70 on the 7th day, by 31070 on the 14th day, and by 34070 on the 21st day, while the T / T AMLR was reduced by 17070, 21070, and 38070, respectively. However, the blastogenesis of T lymphocytes isolated following daily administration of PDN in the two AMLRs did not show significant variations with respect to the values observed before the treatment (Table 4).
DISCUSSION The data reported in this paper show that the oral administration of DFC to patients with RA affects the in vitro behavior of their lymphocytes. This immunomodulatory effect of DFC involves both the phenotype and the functional capacity of RA lymphocytes and appears to be markedly different in respect to the intensity and duration of effect compared with that produced by PDN (used as control). These observations confirm our previous report concerning the effect of DFC on the redistribution of human lymphocyte subsets and confirms this observation in RA patients also (Scudeletti, Pende, Barabino, Imbimbo, Grifoni & Indiveri, 1984). The oral administration of DFC induces T cell depletion and affects the ratio helper-induced/ suppressor-cytotoxic T cells more effectively and for a longer duration than the administration of PDN. Indeed, these effects have been observed up to 72 h following the administration of a single oral dose of DFC while they returned to the baseline level within 24 h following PDN. The different activity of the two drugs may be accounted for by their different pharmacological and pharmacokinetic characteristics.
The reversion of the OKT4÷/OKT8 ÷ cell ratio has constantly been found in patients treated on a daily basis with DFC, but was inconsistent during treatments with PDN. This observation confirms the hypothesis that DFC may be better than PDN in correcting the selective deficiency of suppressor T lymphocytes which characterizes both animal and human models of autoimmune diseases (Zoschke & Messner, 1981). The T lymphocytes isolated from the peripheral blood of patients with RA following the administration of a single oral dose of DFC showed a reduced capacity to express HLA-Class 2 antigens upon PHA stimulation in vitro. This effect lasted up to 72 h following DFC administration; but disappeared within 24 h when induced by PDN. Moreover, T lymphocytes isolated from patients treated on a daily basis with DFC failed to properly express the HLA-Class 2 antigens. The ability to express HLA-Class 2 antigens is a prerogative of human T lymphocytes which enables these cells to cooperate with each other during the elicitation of a normal immune response. Indeed the genes within the HLA-Class 2 region of the major histocompatibility complex (MHC) influence, among several factors, the interaction between T lymphocytes and other cell subsets (Indiveri, Wilson, Russo, Quaranta, Pellegrino & Ferrone, 1980). We have also reported a similar impairment of T lymphocytes in the elderly as well as in patients with neoplastic or immune diseases (Indiveri, Barabino, Pierri & Grifoni, 1983). All these data clearly indicate that the measurement of the capacity of the expression of HLA-Class 2 antigens by primed T cells constitutes a means of better understanding the functional capacities of human T lymphocytes. It is also well known that (a) the HLA-Class 2 antigens are deeply involved in the activation of the
138
M. SCUDELETTIet al.
mixed lymphocyte reactions and that (b) distinct determinants of these antigens regulate the activation o f allogeneic M L R , autologous M L R o f non T / T type and autologous M L R of T / T type (Indiveri et al., 1983). Considering these functional implications, one can draw the conclusion that the modulation of the expression of HLA-Class 2 antigens is also involved in the modification o f the stimulatory and proliferative capacity of lymphocytes in A M L R s . The A M L R s are in vitro reactions which mimic the in vivo process of cell-to-cell interaction: the A M L R of the non T / T type reflects the interaction of the nonT antigen presenting cells with the helper T lymphocytes, while the A M L R of the T / T type reflects the interaction between distinct subsets of T lymphocytes (Gupta & Damle, 1982). The effect of D F C on such a complex mechanism may indicate that this drug is capable of interrupting the immunopathologic mechanism leading to the generation and the maintenance of immune disease. If this is the case, one may conclude that, since the
effects of D F C are more stable and more pronounced than those of P D N , D F C is a better tool for the treatment of immune diseases. Nevertheless, in drawing such a conclusion, one must also bear in mind that the activities of lymphocytes isolated from the peripheral blood may not properly reflect in vivo processes actually involved in a given disease. Indeed, it has been reported that the T cells isolated from the peripheral blood of patients with R A behave differently from those isolated from the synovial fluid (Bell & Pink, 1984). Further studies designed to compare the effect of DFC on peripheral blood lymphocytes and lymphocytes isolated from the synovial fluid will need to be conducted in order to determine whether the i m m u n o m o d u l a t i n g effect here reported may be relevant to the immune imbalance which characterizes the R A at the joint level. Acknowledgement - - Partially funded by Consiglio Nazionale delle Ricerche CNR - Grant no. 84.00628.44.
REFERENCES
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Effects of a New Heterocyclic Glucocorticoid
139
NATHANSOHN, G., PASQUALUCCI,C. R., RADAELLI, P., SCHIATTI, P., SELVA, D. & WINTERS, G. (1969). Steroids possessing nitrogen atoms, V, some pharmacological and chemicophysical aspects of a new class of active corticoids: (17 a, 16 - d ) oxazolino steroids. Steroids, 13, 3 6 5 - 381. NATHANSOHN, G., WINTERS, G. & ARESI, V. (1969). Steroids possessing nitrogen atoms. I V - F u r t h e r studies on the synthesis of (17 a, 16 a - d) oxazolino steroids. Steroids, 13, 3 8 3 - 397. NATHANSOHN, G., WINTERS, G. ~¢ TESTA, E. (1967). Steroids possessing nitrogen atoms. III--Synthesis of new highly active corticoids (17 a, 16 a - d) oxazolino steroids. J. reed. Chem., 10, 799-802. NELSON, A. M. ~¢ CONN, D. L. (1980). Glucocorticoids in rheumatic disease. Mayo Clin. Proc., 55, 7 5 8 - 769. PAGANO, G., LOMBARDI, A., FERRARIS, G. M., IMBIMBO,B. & CAVALLOPERIN, P. (1982). Acute effect of prednisone and deflazacort on glucose tolerance in prediabetic subjects. Eur. J. clin. Pharmac., 22, 469-471. QUARANTA, V., PELLEGRINO, M. A. ~¢ FERRONE, S. (1981). Serologic and immunochemical characterization of the specificity of four monoclonal antibodies to distinct antigenic determinants expressed on subpopulations of human Ia-like antigens. J. Immun., 548, 136-142. SCH1ATTI, P., SELVA, D., BARONE, D., RESTELLI, A. & GLASSER, A. (1980). Anti-inflammatory activity and other pharmacological properties of 113, 21-dihydroxy-2' methyl-5' 3H-pregna-l,4-dieno (17,16-d) oxazole 3,20-dione-21 acetate (deflazacort). Arzneimmittel-forsch., 30, 1543- 1549. SCUDELETTI, M., PENDE, D., BARABINO,A., IMBIMBO,B., GRIFONI, V. and INDIVERI, F. (1984). Effect of single oral doses of prednisone and deflazacort on human lymphocyte distribution and functions. Analysis with monoclonal antibodies. In Glucocorticoid Effects and their Biological Consequences, (eds Avioli, V., Gennari, C. and Imbimbo, B.) pp. 3 3 5 - 344. Plenum Press, New York. ZOSCHKE, D. C. ,~¢MESSNER, R. P. (1981). Suppressor cells in rheumatic diseases. In Suppressor Cells in Human Disease, pp. 175-207. Marcel Dekker, New York.
139, 1987.
0192-0561/87 $3.00+ .00 International Society for lmmunopharmacology.
EFFECTS OF A NEW HETEROCYCLIC GLUCOCORTICOID, DEFLAZACORT (DFC), ON THE FUNCTIONS OF LYMPHOCYTES FROM PATIENTS WITH RHEUMATOID ARTHRITIS (RA) MARCO SCUDELETTI*, CRISTINA PICCARDO*, P|ERLUIGI PIOVANO*, BRUNO IMBIMBO* and FRANCESCO INDIVERIt§ *Cattedre di Clinica Medica RR e tlmmunologia Clinica, Universit/t di Genova, Istituto Scientifico di Medicina Interna, Viale Benedetto XV, n. 6, 16132 Genova, Italy; *Gruppo Lepetit S.p.A., Via della Vetreria, n. 1, 22070 Grandate (Como), Italy (Received 28 November 1985 and in final form 25 April 1986) Abstract - - Deflazacort (DFC) is a new glucocorticoid which, when compared with prednisone (PDN), has similar anti-inflammatory actions, but lacks several unwanted side effects on mineral and carbohydrate metabolism. DFC is more efficient than PDN in inducing immunomodulatory effects. This study concerns twelve patients with classical RA who required corticosteroid therapy because of the ineffectivenessof other drugs. Six patients received 12 mg/day of PDN from day 1 to day 21 and 15 mg/day of DFC from day 29 to day 50. The second group of six patients received DFC initially followed by the PDN dosing regimen. At various times (4, 8, 24, 48 and 72 h) after the first administration of steroid, and 7, 14 and 21 days after the beginning of the treatment with PDN or DFC the following tests were performed (a) the phenotype of T lymphocytes isolated from peripheral blood; (b) the evaluation of the expression of HLA Class 2 antigens by PHA primed T cells; and (c) the analysis of cell behavior in mixed lymphocyte reactions. The data reported in this paper show that the oral administration of DFC to patients with RA affects the in vitro behavior of their lymphocytes. This immunomodulatory effect of DFC concerns both the phenotype and function of RA lymphocytes. Moreover it differs in both intensity and kinetic characteristics from the immunomodulating effect observed with PDN (control).
Deflazacort (DFC) is a new heterocyclic glucocorticoid* belonging to the class of oxazolino steroids (Nathanshon, Winters & Testa, 1967; Nathanshon, Winters & Aresi, 1969). This new compound possesses a systemic anti-inflammatory potency similar to that of prednisone (Schiatti, Selva, Barone, Restelli & Glasser, 1980; Nathanshon, Pasqualucci, Radaelli, Schiatti, Selva & Winters, 1969; Buniva, Dubini & Sassella, 1979; Caniggia, Marchetti, Gennari, Vattimo & Nicolis, 1977) but lacks several of the unwanted side-effects on mineral and carbohydrate metabolism observed with the latter (Hahn, Halstead, Strates, Imbimbo & Baran, 1980; Pagano, Lombardi, Ferraris, Imbimbo & Cavallo Perin, 1982). In a previous paper we reported that DFC is more efficient than prednisone in inducing immunomodulatory effects. Specifically,
we observed that (a) the depletion of helper T cells and an increase in the cytotoxic suppressor subsets; (b) the inhibition of the expression of the HLA Class 2 antigens by T lymphocytes primed in vitro and, (c) the reduction of activity of T cells in autologous MLR, were more pronounced following administration of DFC rather than PDN (Scudeletti, Pende, Barabino, Imbimbo, Grifoni & Indiveri, 1984). Rheumatoid arthritis (RA) is characterized by several immunological abnormalities, including an imbalance of the helper-suppressor T cell ratio (Zoschke & Messner, 1981) and the anomalous functioning of T lymphocytes in AMLRs (Indiveri, Scudeletti, Pierri, Traverso, Cetti & Ferrone, 1986) which can effectively be treated with glucoeorticoids (Nelson & C o n n , 1980).
§ Author to whom correspondence should be addressed. * (lift, 16a)-21-(acetyloxy)-Il-hydroxy-2'-methyl-5'H-pregna-l',4dieno[17, 16-~]oxazole-3,20dione (Fig. 1). 133
M. SCUDELETTIet
134
OKT3, OKT4, OKT8 directed against human T cell markers were purchased from Ortho Diagnostic (Milan, Italy).
21Ctt20--R
I
20('-~O t N C t-13 19
"'~ 0 /
Fig. 1. Chemical structure of deflazacort. In view of the above reported observations, we initiated experiments to analyze ex vivo the relative capacities of DFC and prednisone to affect lymphocyte function from patients with RA.
EXPERIMENTAL
al.
PROCEDURES
This study involved twelve patients with classical or definite RA (according to A.R.A. criteria) (four males and eight females, age ranging between 46 and 60 yr). The clinical data of these patients are shown in Table 1. In this study the patient inclusion criteria were as follows: (a) need for corticosteroids because of the ineffectiveness of non-steroid antiinflammatory drugs; (b) age less than 60 yr; (c) absence of counter indications for corticosteroid administration. The patients were divided into two groups. The first group (six patients) received 12 mg daily of PDN (Deltacortene-Lepetit) from day l to 21; and 15 mg daily of DFC (deflazacort-Lepetit) from day 29 to 50. In the second group (n = 6) the patients first received DFC followed by the PDN dosing regimen. The immunological evaluation was performed in each patient at 4, 8, 24, 48 and 72 h after the first administration of steroid; 7, 14, 21 days after the beginning of the treatment with PDN or DFC. This evaluation included: (a) the phenotype of T lymphocytes isolated from peripheral blood; (b) the evaluation of the expression of H L A - - Class 2 antigens by P H A primed T cells; and (c) the analysis of their behavior in mixed lymphocyte reactions. Antibodies Purified rabbit antibodies to human and mouse Ig were purchased from Yago immunodiagnostic (Burlingame, CA). The monoclonal antibody Q 5/13 directed against monomorphic determinants of H L A - - Class 2 antigens were kindly provided by Dr. S. Ferrone (New York Medical College, Valhalla, NY) (Quaranta, Pellegrino & Ferrone, 1981). The MoAbs
L ymphocytes
Lymphocytes were isolated by Plasmagel sedimentation and Ficoll-Hypaque gradient centrifugation from heparinized blood. T and B cells were isolated by nylon wool filtration from lymphocyte preparations containing less than 5% phagocyting (latex particles) activity (Indiveri, Huddlestone, Pellegrino & Ferrone, 1980). Rosette assays Receptors for sheep erythrocytes were detected by rosetting with 2-aminoethylisothiouronium bromidetreated sheep red blood cells (AET-SRBC). Membrane-bound Ig (MbIg), OKT3, OKT4, OKT8 and H L A Class 2 antigen-bearing cells were enumerated by the indirect rosette microassay performed in microtiter plates as described elsewhere (Indiveri, Wilson, Pellegrino & Ferrone, 1979). Cell cultures and DNA synthesis
T cells (1 × 106/ml in RPMI 1640 medium containing 10o70 fetal calf serum, 2 mM glutamine, 200 U / m l penicillin, and 0.2 mg/ml streptomicin) were stimulated with P H A M (Wellcome, London; final dilution 1:1000) for 72 h at 37°C in a 5o70 CO2 atmosphere. At the end of the incubation, cells were washed five times with a medium containing 0.5°70 amethyl-D-mannoside, in order to reduce cell agglutination (Scudeletti, Pende, Barabino, Imbimbo, Grifoni & Indiveri, 1984). MLRs were performed according to the procedure we have previously used (Scudeletti et al., 1984). DNA synthesis was measured by pulsing triplicate cultures with 1/aCi 3H-thymidine (spec. act. 2/aCi/mmol) for 18 h before harvesting on glass fiber paper with an automated sample harvester. The washed and dried filters were mixed with liquid scintillation cocktail and then counted in a /3 scintillation counter. Statistical evaluation was undertaken using the Student's t-test.
RESULTS Effect o f DFC and P D N on the blood lymphocyte subsets Four hours following the administration of a single oral dose of DFC the total number of T lymphocytes was significantly reduced. This effect of
Effects of a New Heterocyclic Glucocorticoid
135
Table 1. Clinical data concerning the patients included in this study Case number
Sex
Age
1
2
3
4
A.R .A . criteria* 5 6 7
1
M
51
+
+
+
+
-
2 3 4 5 6 7 8 9
F F F F M F F F
46 49 51 57 60 60 58 54
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + +
+ + + + -
+ + + -
10
F
56
+
+
+
+
+
-
11
M
58
+
+
+
+
+
-
12
M
49
+
+
+
-
-
-
+ + + + + + + + + + +
8
9
10
11
Diagnosis
+ + + + + + + + + + + +
+ -
+ + + -
+
RA RA RA RA RA RA RA RA RA RA RA RA
-
classical classical definite classical classical classical classical definite definite classical classical definite
RA classical t> 7 A R A criteria. RA definite >I 5 A R A criteria. * 1 = morn ing stiffness; 2 = pain on motion or tenderness in at least one joint; 3 = swelling in at least one joint; 4 = swelling of at least one other joint within 3 months; 5 = symmetric joint swelling with simultaneous involvement of the same joint on both sides; 6 = subcutaneous nodules; 7 = Rx typical changes; 8 = RA factor; 9 = mucine precipitate from synovial fluid in diluted acetic acid; 10 = characteristic hystologic changes in synovium; 11 = characteristic hystologic changes in the nodules.
Table 2. Analysis of the effect of prednisone (PDN) and defiazacort (DFC) on the lymphocyte subsets following the administration of a single oral dose of the two corticosteroids and at the 7th, 14th, and 21st day of treatment with equivalent doses of the two corticosteroids
Drug
Membran e markers Baseline
Hours following administration of P D N or DFC 4 8 24 48 72
PDN
AET OKT3 OKT4 OKT8
2.3+0.4 1.9-+0.7 0.9_+0.3 0.6_+0.2
1.8_+0.3' 0.4_+0.0* 0.5_+0.0* 0.6_+0.1
DFC
AET OKT3 OKT4 OKT8
2.4_+0.6 1.7-+0.5 1.1_+0.4 0.9_+0.2
0.9_+0.3* 0.9+0.4 *t 1.1_+0.2*t 1.0___0.4*t 0.7_+0.1' 0.6_+0.1'* 1.6_+0.4"§ 1.7-+0.8"§
* P<0.01 t P<0.01 * P<0.05 P<0.05
1.9_+0.2 1.2_+0.4 0.7_+0.1 0.5_+0.1
2.1_+0.9 1.9_+0.6 0.8_+0.4 0.7_+0.1
Duration of treatment (days) 7 14 21
2.2_+0.6 1.8_+0.6 0.8_+0.2 0.6_+0.2
2.3_+0.6 1.9_+0.4 0.9_+0.2 0.6_+0.2
2.5-+0.7 1.8_+0.4 0.7_+0.2 0.7_+0.1
0.8+0.1 *t 1.2_+0.6"* 0.7_+0.6** 1.6_+0.9"§
l.l-+0.4t* 2.3-~0.9 1.8_+0.6"t 1.4+0.7 t* 1.6-+0.8§ 1.7-+1.1'* 0.7_+0.4 t* 0.6_+0.2§ 0.5_+0.1'§ 1.4_+0.2"§ 1.5±0.1"* 1.2_+0.4'
2.4_+0.9 1.7_+0.9 0.8_+0.1 0.6_+0.2
2.3_+0.5 1.9_+0.6 0.7_+0.2 0.7_+0.1
1.9_+l.lt* 1.7_+1.1'* 0.5+0.2 *t 1.3_+0.1'
1.7-+0.6'* 1.7-+1.1'* 0.6_+0.2 t* 1.4_+0.1'
versus baseline. DFC vs PDN. vs baseline. DFC vs PDN.
DFC was more pronounced than that produced by
p h e n o t y p e a n d a n i n c r e a s e o f t h e cell s u b s e t b e a r i n g
PDN.
t h e T8 p h e n o t y p e ( T a b l e 2). A s a c o n s e q u e n c e o f
Moreover
the effect of DFC
on T lymphocyte
n u m b e r s w a s l o n g e r t h a n t h a t c a u s e d b y P D N s i n c e it l a s t e d u p t o 72 h f o l l o w i n g d r u g a d m i n i s t r a t i o n , compared
with
less
than
24
h
following
PDN
a d m i n i s t r a t i o n ( T a b l e 2). The analysis of the lymphocyte subsets following the administration of DFC showed that this steroid induced
a
decrease
of
T
cells b e a r i n g
the
T4
these m o d i f i c a t i o n s a n i n c r e a s e d O K T 4 + / O K T 8 + cell r a t i o w a s m a i n t a i n e d f o r u p t o 72 h f o l l o w i n g t h e a d m i n i s t r a t i o n o f D F C , w h i l e it r e t u r n e d t o t h e baseline values 8 h following the administration of P D N ( T a b l e 3). The effect of the therapeutic administration of DFC and PDN was evaluated by determining the total number of T lymphocytes and the OKT4+/
M. SCUDELETTIet al.
136
Table 3. Analysis of OKT4 +/OKT8 ÷ ratio in lymphocyte subsets following the administration of a single oral dose of the two corticosteroids and at the 7th, 14th and 21st day of treatment with equivalent doses of the two corticosteroids
Drug
Baseline
PDN DFC
1.5 __.0.6* 1.2+_0.4
Hours following administration of PDN or DFC 4 8 24 48 72 0.8___0.2 0.4__.0.1
1.4+0.1 0.3+0.1
1.3+0.6 0.4+_0.1
1.5+0.4 0.5+_0.1
Duration of treatment (days) 7 14 21
1.1+_0.6 0.4+_0.2
1.0__.0.2 0.4__.0.1
1.3+_0.3 0.3+_0.1
1.0+0.2 0.4_+0.1
*OKT4/OKT8 ratio. OKT8 ÷ cell ratio at the 7th, 14th, and 21st day of a daily treatment regimen with 15 and 12 mg of D F C and P D N , respectively. The total number o f T lymphocytes isolated from patients who received the D F C remained constantly and significantly lower than the value found before the first administration; the percentage of the OKT4 ÷ cells remained constantly 50% lower than the percentage found prior to drug administration (Table 2). The ratio O K T 4 + / O K T 8 + cells remained constantly lower than one (Table 3). In contrast, daily administration of P D N did not significantly affect the total number of T cells and did not affect the ratio of OKT4 + to OKT8 ÷ lymphocytes. This ratio remained constantly around one because of a slight reduction in the OKT4 + cell subset.
~ 6o
t---I
~ 4o ~ 2o ~5 ?
£ = ~.
9
~
3
to
I B
I 4
I 8
I 24
I 48
I 72
(h)
Effect o f corticosteroids on PHA-induced HLA Class 2 antigens expression by T lymphocytes Administration of a single oral dose of D F C or P D N reduced the expression of HLA-Class 2 antigens by T cells and their P H A - i n d u c e d proliferative response in vitro. Indeed less than 30°70 of T lymphocytes isolated from blood drawn 4 h after administration of D F C or P D N expressed HLA-Class 2 antigens upon P H A activation. This defective capacity of expressing HLA-Class 2 lasted up to 24 h following a single oral dose of P D N and up to 48 h following a single oral dose of DFC (Fig. 2). The capacity of T lymphocytes isolated from blood drawn during the therapeutic trial with a single daily oral dose of D F C was constantly reduced (mean 34°7o) relative to the value found before drug treatment. This defect was not observed during the daily treatment with P D N (Fig. 2), however. Both drugs affected the blastogenesis of T ceils upon P H A stimulation in a similar fashion (Fig. 2).
Effect o f DFC and PDN on the proliferative and stimulatory activities o f lymphocytes in allogenic and in autologous mixed lymphocyte reactions The administration of D F C or P D N , both as a
I •
I 14
I 21
(D~3ys)
Time for[owing
CTS (]dministrGtion
o=PDN e=DFC rn =PDN I=DFC
Fig. 2. Expression of HLA Class 2 antigens by T lymphocytes isolated and primed with PHA following the administration of a single oral dose of the two corticosteroids and at the 7th, 14th and 21st day of treatment with equivalent doses of the two corticosteroids (upper section). 3H-thymidine uptake by lymphocyte isolated and primed with PHA following the administration of a single oral dose of the two corticosteroids and at the 7th, 14th and 21st day of treatment with equivalent doses of the two corticosteroids (lower section). single oral dose, and on a daily dosing regimen did not significantly affect the responsiveness of lymphocytes activated in allogeneic M L R (data not shown). Lymphocytes isolated following the administration of D F C , however, failed to respond properly in both non T / T type and T / T type A M L R . A similar lack of responsiveness was also shown by T cells isolated after the administration o f P D N . The reduced proliferative response induced by a single oral dose of D F C was also observed during the
Effects of a New Heterocyclic Glucocorticoid
137
Table 4. Analysis of the variations of blastogenesis in autologous mixed lymphocyte reactions (AMLRs) of lymphocytes isolated following the administration of a single oral dose of the two corticosteroids and at the 7th, 14th and 21st day of treatment with equivalent doses of the two corticosteroids
Drug
AMLR type
Baseline
Non T-T 10.0__.3.2 PDN T Ia T-T 6.6-+1.2 DFC
Non T-T T Ia T-T
9.1_+4.2 6.2-+1.3
4
Hours after drug 8 24 48
72
Duration of treatment (days) 7 14 21
6.0__.2.1" 7.0_+2.2* 8.0_+2.1' 7.5_+0.8 8.4___1.6 9.6_+0.8 10.00_+2.0 10.2-+0.8 3.9_+1.4" 4.0-+1.8" 5.0_+1.4' 4.8_+1.6' 5.1__.1.4 6.0_+1.4 7.0-+1.4 6.0_+1.0 6.2_+1.9" 7.0_+2.1' 6.8_+1.3' 7.1_+2.0" 7.8_+1.2 6.4_1.6'§ 3.2_+2.1" 4.0_+2.4* 4.3-+1.6' 4.8-+0.7* 5.0_+0.9 5.0_+1.0
6.3_+2.0** 4.6_+1.3'*
5.8_+1.4t* 4.2_+0.7*
* P<0.01 versus baseline. t P<0.01 DFC versus PDN. * P<0.05 versus baseline. _,o<0.05 DFC versus PDN.
daily treatment schedule with this drug. Indeed, the blastogenesis in the non T / T AMLR was reduced by 30°70 on the 7th day, by 31070 on the 14th day, and by 34070 on the 21st day, while the T / T AMLR was reduced by 17070, 21070, and 38070, respectively. However, the blastogenesis of T lymphocytes isolated following daily administration of PDN in the two AMLRs did not show significant variations with respect to the values observed before the treatment (Table 4).
DISCUSSION The data reported in this paper show that the oral administration of DFC to patients with RA affects the in vitro behavior of their lymphocytes. This immunomodulatory effect of DFC involves both the phenotype and the functional capacity of RA lymphocytes and appears to be markedly different in respect to the intensity and duration of effect compared with that produced by PDN (used as control). These observations confirm our previous report concerning the effect of DFC on the redistribution of human lymphocyte subsets and confirms this observation in RA patients also (Scudeletti, Pende, Barabino, Imbimbo, Grifoni & Indiveri, 1984). The oral administration of DFC induces T cell depletion and affects the ratio helper-induced/ suppressor-cytotoxic T cells more effectively and for a longer duration than the administration of PDN. Indeed, these effects have been observed up to 72 h following the administration of a single oral dose of DFC while they returned to the baseline level within 24 h following PDN. The different activity of the two drugs may be accounted for by their different pharmacological and pharmacokinetic characteristics.
The reversion of the OKT4÷/OKT8 ÷ cell ratio has constantly been found in patients treated on a daily basis with DFC, but was inconsistent during treatments with PDN. This observation confirms the hypothesis that DFC may be better than PDN in correcting the selective deficiency of suppressor T lymphocytes which characterizes both animal and human models of autoimmune diseases (Zoschke & Messner, 1981). The T lymphocytes isolated from the peripheral blood of patients with RA following the administration of a single oral dose of DFC showed a reduced capacity to express HLA-Class 2 antigens upon PHA stimulation in vitro. This effect lasted up to 72 h following DFC administration; but disappeared within 24 h when induced by PDN. Moreover, T lymphocytes isolated from patients treated on a daily basis with DFC failed to properly express the HLA-Class 2 antigens. The ability to express HLA-Class 2 antigens is a prerogative of human T lymphocytes which enables these cells to cooperate with each other during the elicitation of a normal immune response. Indeed the genes within the HLA-Class 2 region of the major histocompatibility complex (MHC) influence, among several factors, the interaction between T lymphocytes and other cell subsets (Indiveri, Wilson, Russo, Quaranta, Pellegrino & Ferrone, 1980). We have also reported a similar impairment of T lymphocytes in the elderly as well as in patients with neoplastic or immune diseases (Indiveri, Barabino, Pierri & Grifoni, 1983). All these data clearly indicate that the measurement of the capacity of the expression of HLA-Class 2 antigens by primed T cells constitutes a means of better understanding the functional capacities of human T lymphocytes. It is also well known that (a) the HLA-Class 2 antigens are deeply involved in the activation of the
138
M. SCUDELETTIet al.
mixed lymphocyte reactions and that (b) distinct determinants of these antigens regulate the activation o f allogeneic M L R , autologous M L R o f non T / T type and autologous M L R of T / T type (Indiveri et al., 1983). Considering these functional implications, one can draw the conclusion that the modulation of the expression of HLA-Class 2 antigens is also involved in the modification o f the stimulatory and proliferative capacity of lymphocytes in A M L R s . The A M L R s are in vitro reactions which mimic the in vivo process of cell-to-cell interaction: the A M L R of the non T / T type reflects the interaction of the nonT antigen presenting cells with the helper T lymphocytes, while the A M L R of the T / T type reflects the interaction between distinct subsets of T lymphocytes (Gupta & Damle, 1982). The effect of D F C on such a complex mechanism may indicate that this drug is capable of interrupting the immunopathologic mechanism leading to the generation and the maintenance of immune disease. If this is the case, one may conclude that, since the
effects of D F C are more stable and more pronounced than those of P D N , D F C is a better tool for the treatment of immune diseases. Nevertheless, in drawing such a conclusion, one must also bear in mind that the activities of lymphocytes isolated from the peripheral blood may not properly reflect in vivo processes actually involved in a given disease. Indeed, it has been reported that the T cells isolated from the peripheral blood of patients with R A behave differently from those isolated from the synovial fluid (Bell & Pink, 1984). Further studies designed to compare the effect of DFC on peripheral blood lymphocytes and lymphocytes isolated from the synovial fluid will need to be conducted in order to determine whether the i m m u n o m o d u l a t i n g effect here reported may be relevant to the immune imbalance which characterizes the R A at the joint level. Acknowledgement - - Partially funded by Consiglio Nazionale delle Ricerche CNR - Grant no. 84.00628.44.
REFERENCES
BELL, D. A. & P1NK, J. (1984) Distribution of activated B lymphocytes in the circulation and synovial fluid in rheumatoid arthritis. Clin. Immun. Immunopath., 31, 272-281. BUNIVA, G., DUBINI, A. & SASSELLA,D. (1979). Human bioassay of corticotropin suppressing, eosinopenic and hyperglycaemic potencies of deflazacort and prednisone. Curr. Ther. Res., 26, 6 9 - 81. CANIGGIA, A., MARCHETTI,M., GENNARI, C., VATTIMO,A. • N1COLIS, F. B. (1977). Effect of a new glucocorticoid, oxazacort, on some variables connected with bone metabolism in man: a comparison with prednisone. Int. J. Clin. Pharmac. Biopharm., 15, 126-134. GENNARI,C., BERNINI,M., NARD1,P., FUSI, L., FRANCINI,G., NAMI, R., MONTAGNANI,i . , IMBIMBO,B. & AVIOLI,L. V. (1983). Glucocorticoids: radiocalcium and radiophosphate absorption in man. In Osteoporosis. A Multidisciplinary Problem, (eds Dixon, A. S. J., Russell, R. G. G., Stamp, T. C. B.) p. 75-80. Grune and Stratton, London. GENNARI, C., IMBIMBO,B., MONTAGNANI,M., BERNINI,M., NAROI, P. & AVIOLI, L. V. (1984). Effects of prednisone and deflazacort on mineral metabolism and parathyroid hormone activity in humans. Calcif. Tissue Int., 36, 245 - 252. GUPTA, S. & DAMLE,N. K. (1982). Monoclonal antibody defined T cell subsets responding in T-T and in T-non-T autologous mixed lymphocyte reaction. Generation of immunoregulatory functions. In Current Concepts in Human Immunology and Cancer Immunomodulation, (ed. Serrou) pp. 183 - 188. North Holland, Amsterdam. HAHN, T. J., HALSTEAO,L. R., STRATES,B., IMBIMBO,B. & BARAN, D. T. (1980). Comparison of subacute effects of oxazacort (deflazacort) and prednisone on mineral metabolism in man. Calcif. Tissue Int., 31(2), 109-115. INDIVERI,F., BARABINO,A., PIERRI, I. & GRIFON1, V. (1983). Human autologous mixed lymphocyte reactions. A review. La Ricerca Clin. Lab., 13, 397- 409. INDIVERI, F., HUDDLESTONE, F., PELLEGRINO, M. A. & FERRONE, S. (1980). Isolation of human T lymphocytes: comparison between nylon wool filtration and rosetting with neuraminidase (VCN) and 2-aminoethylisothiouronium bromide (AET)-treated sheep red blood cells (SRBC). J. Immun. Meth., 34, 107- 114. INDIVERI, F., SCUDELETT1,M., PIERRI, I., TRAVERSO, A., CETTI, C., FERRONE, S. (1986). T cells in systemic lupus erithematosus and in rheumatoid arthritis abnormalities in class II HLA antigens induction by PHA and in autologous mixed lymphocyte reactions. Cell. Immun., 97, 197-203. INDIVERI, F., WILSON, B. S., PELLEGRINO,M. A. ~ FERRONE,S. (1979). Detection of human histocompatibility antigens with an indirect rosette microassay. J. immun. Meth., 29, 101- 109. INDIVERI,F., WILSON, B. S., RUSSO, C., QUARANTA,V., PELLERGRINO,M. A. & FERRONE, S. (1980). Ia like antigens on human T lymphocytes: relationship to other surface markers, role in MLR and structured profile. J. Immun., 125, 2673.
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