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`I'riiued
`
`in UK - till nt/rf \ n v . ;: rii
`
`Copyrighi
`€> Miinksfsaard
`ALLERGY
`IS.SN ()IOS-4fi3li
`
`1998
`
`Responsiveness of human skin mast cells to
`repeated activation: an in vitro study
`
`Rubinchik E. Shalit M. Lcvi-Schaffer F. Responsiveness of human skin mast
`cells to repeated activation: an in vitro study.
`Allergy 1998: .53: 14-19. © Munksgaard 1998.
`
`To assess human mast-eel! (MC) behavior after repetitive activation, we
`cocultured human foreskin MC (SMC) with human foreskin fibroblasts (F).
`Under these conditions, we have previously demonstrated that SMC keep
`their viability and functional activity for up to 8 days. SMC were
`presensitized with atopic serum and repeatedly activated by consecutively
`increasing concentrations of anti-IgE antibodies (a-IgE, ().(X)02-().l %). This
`treatment, which mimics the "rush desensitization" procedure, led to
`complete SMC unresponsiveness to activation by a-IgE at optimal
`concentrations, as evaluated by histamine release. However, presensitization
`of SMC with IgE antibodies before exposure to a-IgE restored their
`sensitivity to this stimulus. These data indicate that desensitization was
`probably due to lack of membrane-bound IgE rather than to downregulation
`of intracellular mechanisms. In fact, SMC challenged by an optimal
`concentration of a-lgE could release histamine upon a second activation by
`2 h after the first activation, if the cells had been presensitized before the
`second challenge. SMC incubation with increasing concentrations of
`compound 48/80 (0.2-10 |ig/ml) led to MC unresponsiveness to an optimal
`concentration of this stimulus. Furthermore, SMC activated by an optimal
`concentration of compound 48/80 and rechallenged with the same agent were
`insensitive to the second activation for at least 24 h. In summary, we have
`shown that it is possible to induce "desensitization" in SMC to both IgE-
`dependent and IgE-independent stimuli by incubating the cultures with
`consecutively increasing concentrations of the activator. SMC can release
`histamine when reactivated with a-IgE antibodies after presensitization by
`2 h after the first challenge, while they reacquire their susceptibility to
`reactivation with compound 48/80 in only 2-3 days.
`
`E. Rubinchik\ M, Shalit^,
`F. Levi-Schaffer^
`'Department of Pharmacology, School of
`Pharmacy, Hebrew Unlversity-Hadassah
`Medical School, Jerusalem; 'Department of
`Internal Medicine, Hadassah University
`Hospital, Jerusalem, Israel
`
`Key words: activation; desensitization;
`histamine; human skin mast cells; reactivation.
`
`Francesca Levi-Schaffer
`Department of Pharmacology, School of
`Pharmacy
`Hadassah Medical School
`Jerusalem 31120
`Israel
`
`Accepted for publication 10 June 1997
`
`Desensitization is a complex process that has been
`observed in a number of ligand-receptor systems
`and is defined as unresponsiveness of tissues or
`cells to the repetitive addition of the relevant
`agonist. This phenomenon can be observed in vitro
`as well as in vivo. "Rush desensitization" is one
`example of the practical use of this phenomenon
`in medicine. This procedure is used to desensitize
`allergic patients by treating them with increasing
`doses of antigen (1-3). While this treatment
`is widely used, its exact mechanism is still not
`clear.
`Mast cells (MC) are the key cells of allergic
`reactions, and they are supposed to have a role in
`the mechanism of rush desensitization. We have
`previously demonstrated that in rat peritoneal MC
`cocultured with 3T3 fibroblasts, a temporary period
`
`of unresponsiveness to immunologic stimuli may
`be induced by activating the cells with gradually
`increasing doses of antigen (4). In addition, we
`have found that rat MC can be activated twice after
`presensitization with IgE antibodies with an IgE-
`dependent activator (5, 6). Similarly, rat MC chal-
`lenged with compound 48/80, an IgE-independent
`activator, can respond to repeated activation by
`releasing histamine (7, 8). Recently, we have devel-
`oped an /// v/fro-defined system in which human
`foreskin MC (SMC) are cocultured with human
`foreskin fibroblasts (F). Under such conditions,
`SMC remain viable, completely preserving their
`morphologic structure and functional activity for at
`least 8 days (9). There have been no previous
`investigations of the responses of human MC to
`reactivation and their desensitization.
`
`14
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`In the present study, we utilized our human
`SMC/F coculture system to evaluate the respon-
`siveness of human MC to reactivation with igE-
`dependent and IgE-independent stimuli.
`
`Material and methods
`Human foreskin mast-cell (SMC) purification
`SMC were dispersed from infant (8 days old)
`foreskins obtained at circumcision under sterile
`conditions. Samples of foreskins were put immedi-
`ately in Dulbecco's modified Eagle's medium
`(DMEM, Biological Industries, Beit Haemek,
`Israel) containing 200 u/ml penicillin, 200 M-g/ml
`streptomycin, and 1 mM EDTA (Sigma Chemicals,
`St Louis,'MO, USA), Samples were stored at 4°C
`and used within 24 h.
`The tissues were then chopped into fragments
`with scissors, and processed by enzymatic digestion,
`as previously described (9, 10). All manipulations
`were carried out under sterile conditions. SMC in
`the digested tissue were counted after staining with
`toluidine blue (0.07% in 65% ethanol, pH 3.5,
`Sigma), and viability was assessed by trypan blue
`staining (9).
`
`Coculture of SMC with human foreskin fihroblasts
`(F)
`foreskin biopsies and
`F were obtained from
`cultured as previously described (9), For the exper-
`iments, F were seeded in 12-well plates (Nunc,
`Roskilde, Denmark) in DMEM medium supple-
`mented by 10% heat-inactivated fetal calf serum
`(v/v), 100 u/ml penicillin, 100 ftg/ml streptomycin,
`and 2 mM L-glutamine
`(DMEM^^). Culture
`medium was changed every 2-3 days until the
`fibroblasts reached confluence.
`Dispersed SMC (3.5x]0'*/well) were seeded on
`confluent F monolayers. Twenty-four hours after
`seeding, the medium was replaced by a fresh one,
`to remove nonadherent cells, and experiments were
`started. Thereafter, culture medium was changed
`every 2 days.
`
`IgE-dependent activation of SMC/F
`SMC in the cocultures were passively sensitized by
`incubation with 10% human atopic serum (total
`IgE concentration 2{)(X) u/ml from Allergy Clinic,
`Hadassah Hospital) in DMEM* for 2 h at 37°C^
`Cocultures were then washed twice with TG**
`(Tyrode's buffer containing 0.1% gelatin, 1,8 mM
`CaCl2, and 0.9 mM MgCl,). Immunologic repetitive
`activation was performed at 1 -h intervals by incu-
`bation of the cells for 20 min at 3TC, with gradu-
`
`Activation of human skin mast cells
`
`ally increasing concentrations (from 0.0002 to 0.1 %
`in TG"^") of goat anti-human IgE antibodies
`(a-IgE, BioMakor, Rehovot, Israel). Control cul-
`tures were incubated for the first time with the
`same concentrations of a-IgE antibodies as the
`experimental cells or with TG*^ buffer alone. At
`each time point, parallel cultures were terminated
`to determine histamine release, as described below.
`After the 20-min incubation, supernatants were
`collected from the experimental and control plates,
`cells were washed with DMEM"^ medium, and
`cultures were incubated at 37X for 40 min before
`starting the next challenge. In the rechallenge
`experiments, experimental cultures were presensi-
`tized with
`IgE antibodies and activated as
`described above with a-IgE antibodies (10%),
`Control cultures were incubated with TG"^" buffer
`alone. Cultures to be rechallenged. were washed
`twice with DMEM" and incubated again with IgE
`serum. The second activation was performed, at
`various time points (2 h-7 days) after the first one,
`with the same concentration of a-lgE antibodies
`used on the first one. At each time point, control
`cultures consisted of SMC/F challenged for the first
`time with the same concentration of a-IgE or with
`TG*" buffer alone. After both the first and the
`second activation, duplicate cultures were termi-
`nated by collecting the supernatants and scraping
`the cell monolayers in 0.3 ml TG^^ with a Teflon
`policemen and disrupted by continuous sonication
`for 1 min (output 5, 50% duty cycle. Heat Systems
`Ultrasonics), Supernatants and cell sonicates were
`kept at -20 C for histamine assay.
`
`Compound 48/80 activation of SMC/F
`increasing
`Repetitive activation of SMC with
`concentrations of compound 48/80 (0.2-10 |xg/ml)
`at 1-h
`intervals was essentially performed as
`described above for IgE-dependent activation. In
`the challenge and rechallenge experiments, SMC/
`F cultures were washed with TG*' and activated
`by incubation with 10 |xg/ml of compound 48/80 for
`20 min at 37='C. Cultures were rechallenged at
`different time points (24 h-7 days) with the same
`concentration of compound 48/80. Control cultures
`were challenged for the first time with the same
`concentration of compound 48/80, or just incubated
`with TG*" buffer for 20 min at 37°C. Cells and
`supernatants of parallel cultures were saved for
`histamine determination as described above.
`
`Histamine assay
`Histamine was determined in supernatants and cell
`sonicates by radioenzymatic assay
`(11), The
`percentage of histamine released from SMC was
`
`15
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`
`Rubinchik et al.
`
`calculated by dividing the amount of histamine in
`supernatants by the sum of that in supernatants and
`in cells (total).
`
`Statisiical analysis
`Data are expressed as mean ± standard error (SEM).
`Statistical analysis was performed by Student's
`f-test. P values of 0.05 or less were considered
`significant.
`
`Results
`Repetitive challenge of SMC with increasing con-
`centrations of a-lgE
`SMC cocultured with F were presensiti/ed with
`saturating concentrations of IgE antibodies and
`exposed to gradually increasing concentrations of
`a-lgE at 1-h intervals. At each time point, control
`cocultures underwent a single activation with the
`same concentration of a-IgE. Spontaneous hista-
`mine release was determined in parallel in SMC/F
`cocultures incubated with TG*^ buffer alone. As
`shown in Table 1, a single incubation with increas-
`ing concentrations of a-IgE induced histamine
`release starting from 12.4+2.8% (().(K)1% a-IgE)
`and up to 66.X±9.3% with the highest concentra-
`tion of a-IgE (0.1%, «=3). In contrast, parallel
`SMC cultures, repeatedly exposed to gradually
`increasing amounts of a-IgE antibodies, were com-
`pletely unresponsive
`to challenge with each
`concentration tested. Even when optimal concen-
`trations of stimulus (0.02-0.1%) were added to the
`incubation medium, SMC released similar amounts
`of histamine as unstimulated, buffer-incubated,
`control cultures. This unresponsiveness lasted for
`at least 4 days. In fact, when the desensitized cells
`were rechallenged with the optimal concentration
`of a-IgE antibodies (0.1%) on day 4 after the
`beginning of the experiment, they released only
`12.5±1.67o histamine compared to the release of
`66.8+11.6% from firstly activated SMC (« = 3,
`
`The unresponsiveness observed in the experi-
`mental group was not due to depletion of histamine
`from SMC granules. In fact, SMC exposed to
`increasing concentrations of a-IgE still contained
`20.2±4.9 ng/well histamine at the end of the pro-
`cedure compared to 26.7±6.3 ng/well, at the begin-
`ning of the experiment, before challenge («=3).
`
`Resfonsiveness of SMC to rechallenge with a-IgE
`In the next series of experiments, we assessed
`whether SMC/F activated by an optimal concen-
`tration of a-IgE antibodies can respond and
`
`16
`
`Table 1, Repetitive ctiallenge of human skin mast cells with increasing concentra-
`tions of oi-lgE, Human foreskin MC cultufed with
`foreskin fibroblasts were
`presensitized with IgE. washed, and activated at 1-h Intervals (20 min, 37 CI with
`gradually increasing concentrations of a-lgE (a-lgE. repetitive challenge). Control
`cultures either were incubated with TG** buffer alone or underwent single
`activation with a-lgE (oi-lgE, single challenge). After each activation, supernatants
`and cells in parallel cultures were sacrificed for histamine determination. Data are
`mean + SEM of three experiments performed in duplicate
`
`Histamine release (%)
`
`Concentration of a-lgE (%)
`
`Stimulus
`
`0.0002
`
`ODOl
`
`0.004
`
`0.02
`
`0.1
`
`a-lgE single
`challenge
`a-lgE repetitive
`challenge
`T G" buffer
`
`8.2-2.0
`
`12.4±2.8
`
`29.0±5.1
`
`46.2 + 10.8 66,8±9.3
`
`8.2 + 2.0
`
`7.0 + 1.1
`
`6.7r2.2
`
`7.7 + 1.4
`
`6.2 + 2.5
`
`7 5 + 2.0
`
`5.4 ±1.8
`
`4.2 + 1.8
`
`6.3+1.3
`
`5.9 ±2.0
`
`release histamine when re-exposed to the same
`single stimulus. SMC/F were presensitized with a
`saturating concentration of human IgE on day 0
`of the experiment and challenged with a-IgE (Fig.
`1). ITiese SMC released 48.4±8.1% of their hista-
`mine content. To determine their susceptibility to
`a second activation, we then presensitized SMC
`again with the same concentration of IgE antibod-
`ies used at time 0 at various time intervals after
`the
`first challenge. Control cocultures were
`presensitized with IgE on day 0 and were acti-
`vated with a-IgE antibodies for the first time at
`each time point. As shown in Fig. 1, when SMC
`were rechallenged as soon as 2 h after the first
`activation, they released comparable percentages
`of histamine as control cultures (69,1±2.5 vs
`66.2±3.9%, respectively, n = 3). At the other time
`points (8 h-7 days), similar percentages of hista-
`mine were released from both firstly challenged
`and rechallenged MC. However, when SMC had
`not been incubated with IgE antibodies before the
`second challenge,
`they released only minute
`amounts of histamine, similar to those released in
`cultures incubated with buffer alone (5.5±2.7 vs
`4.6±1.1, respectively), thus indicating that human
`SMC unresponsiveness to IgE-dependent rechal-
`lenge is probably due to lack of membrane-bound
`IgE. Following this observation, SMC/F cocul-
`tures which underwent repetitive challenges with
`increasing concentrations of a-IgE, as previously
`described, were incubated with IgE-rich serum
`before challenge with an optimal concentration of
`a-lgE (0.1%) 4 days after desensitization was
`achieved. These SMC were able to release similar
`percentages of histamine as cocultures activated
`for the first
`time (72.4±0,4% v.v 66.8±11.6%,
`respeetively).
`
`Page 3 of 7
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`YEDA EXHIBIT NO. 2095
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`

`
`-ST
`CHALLcNGF
`
`CHAL L.SNGC
`
`7 d
`3cl
`24 h
`Bh
`O
`~Mt AFTER 'HE FIRST CHALLENGE
`
`Fig. I. Histamine released hy human skin masi cells challenged
`and rechallenged with a-IgE. At time 0 of experiment, SMC
`cocultured with F were presensitized with igE and incubated
`with T G" butler alone or with buffer containing 10% a-IgE
`antibodies (first challenge) for 20 min at 37 C. Supernatants
`were then collected for histamine assay, and cultures were
`washed and incubated with DMEM". At indicated time points.
`SMC/F which underwent activation at time 0 were again
`presensitized with IgE antibodies and rechallenged with a-lgE
`(second challenge). Cultures that were incubated with buffer
`at time 0 were either incubated with buffer (spontaneous
`histamine release) or activated for first time with a-lgE (first
`challenge). Spontaneous histamine release was always less than
`10%. Data are mean ± SEM of 3-6 experiments performed in
`duplicate.
`
`Repetitive challenge of SMC with increasing con-
`centrations of compound 48/80
`SMC/F were repeatedly challenged at 1-h intervals
`with increasing concentrations of compound 48/80.
`Control cultures were activated for the first time
`with the same concentrations of compound 48/80.
`As shown in Table 2, both experimental and control
`SMC cultures, when activated with 0.2-1.0 jig/ml
`of compound 48/80, did not release any significant
`percentage of histamine. Three and 10 fig/ml of this
`activator induced the release of 18-20% histamine
`in the firstly challenged control group. In contrast,
`repeatedly challenged SMC/F were unresponsive
`to these concentrations of compound 48/80, as
`shown by percentages of histamine released similar
`to those of the buffer-incubated cultures (-7%,
`
`Responsiveness of SMC to rechallenge with com-
`pound 48/80
`To assess whether SMC can respond to a second
`challenge with a nonimmunologic stimulator, we
`activated cocultures with an optimal concentration
`of compound 48/80 (10|jLg/ml) and reactivated
`them with the same concentration of this activator
`at various time intervals (Fig. 2). Rechallenged
`SMC were partially unresponsive 8 h to 1 day after
`
`Activation of human skin mast cells
`
`Table 2 Repetitive challenge of human skin mast cells with increasing concentra-
`tions of compound 48/80 Human foreskin MC cocultured with foreskin fibroblasts
`were repeatediv activated at 1-h intervals (20 mm, 37 C) with gradually increasing
`concentrations of compound 48/80 (compound 48/80, repetitive challenge) Control
`cultures either were incubated with TG' * buffer or underwent single activation
`with compound 48/80 (single challenge). After each activation, supernatants and
`cells in parallel cultures were sacrificed for histamine determination. Data are
`mean + SEM of three experiments performed in duplicate
`
`Histamine release (%)
`
`Compound
`
`Stimulus
`
`0,2
`
`0.5
`
`10
`
`3,0
`
`10,0
`
`Compound 48/80
`single challenge
`Compound 48/80
`repetitive challenge
`T G" buffer
`
`2 7 ± 12
`
`9,3 = 4,6 12,1+3,7 203±4,2 18,0±2,9
`
`2,7±1,2
`
`7,5±2,0
`
`6,0±0 7
`
`7,1 ±0,8 9 , 3 i2 4
`
`3,7±1.5 10,4±3,5 11,9±1,7
`
`7.2±1,7
`
`7,2±1.2
`
`the first cfiallenge. Only 3 and 7 days after acti-
`vation did the cultures reacquire their full capacity
`to release histamine upon a second activation.
`In all the different immunologic and nonimmu-
`nologic challenged and rechallenged cultures, the
`viability of SMC was >98% as assessed by trypan
`blue staining for the duration of the experiments.
`
`Discussion
`In vivo rush desensitization is a common proce-
`dure widely used to desensitize patients allergic to
`various drugs or bee venom (1-3, 12). In this
`procedure, increasing amounts of the offending
`antigen are administered at short intervals, result-
`ing in desensitization and hence the opportunity to
`administer the compound safely. We have recently
`shown that it is possible to induce refractoriness in
`rat peritoneal MC cultures by repeated exposure
`to gradually increasing amounts of antigen (4). In
`the present report, we have extended this study to
`human MC. In the present study, we have shown
`that human foreskin MC may also become unre-
`sponsive by a similar experimental procedure. This
`finding confirms the key role of MC in the rush
`desensitization procedure in man too. The mecha-
`nism of desensitization is still not fully understood.
`Among possible explanations are depletion of anti-
`gen-specific IgE, sustained activation of adenylate
`cyclase, appearance of blocking igG antibodies,
`and mediator depletion from MC and basophils (2,
`13-17). In the present study, we have demonstrated
`that desensitized SMC still contained considerable
`amounts of intracellular histamine, comparable to
`the histamine content of SMC before activation.
`Thus, depletion of histamine is probably not the
`explanation of SMC unresponsiveness.
`
`17
`
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`

`
`Rubinchik et al.
`
`1ST
`CHALLENGE
`2ND
`CHALLENGE
`
`7 a
`3d
`24 h
`8h
`CHALLENGE
`• 'ER THE FIRS"
`Fig. 2. Histamine released by human skin mast cells chal-
`lenged and rechallenged with compound 48/80, At time 0 of
`experiment. SMC/F were incubated either with TG*' buffer
`alone (spontaneous histamine release) or TG'* buffer con-
`taining 10|jLg/ml of compound 48/80 (first challenge) for
`20 min at 37 C\ Supernatants were then collected for hista-
`mine assay, and cultures were washed and incubated with
`DMEM*, At indicated time points. SMC/F that underwent
`activation at time 0 were reactivated with compound 48/80
`(10 (x/ml. second challenge). Cultures that were not activated
`at time 0 were either incubated with TG'* buffer alone or
`activated for first time with compound 48/80 (10(ji,g/ml. first
`challenge). Data are mean ± SEM of 3-9 experiments per-
`formed in duplicate.
`
`It has been demonstrated that activation of rat
`basophilic leukemia cells by antigen triggers endo-
`cytosis of the cross-linked IgE with cointernaliza-
`tion of FCj, receptors occupied by monomeric IgE,
`causing a transient absence of IgE antibodies on
`the cell membrane (18). ITierefore, we suggest that
`SMC unresponsiveness after the rush desensitiza-
`tion procedure is due to internali/.ation of IgE
`molecules when SMC are exposed
`to minute
`amounts of antigen. The relative lack of available
`IgE would render the cells refractory to further
`stimulation even with a higher concentration of
`antigen. Indeed, the period of unresponsiveness
`lasted for at least 4 days in the absence of an
`exogenous source of IgE, Therefore, the reappear-
`ance of Fc,, receptors with consequent binding of
`IgE antibodies when available would probably
`renew the responsiveness of the SMC.
`Ilie duration of the desensitization state in
`patients is not known. The common practice is to
`repeat the entire desensitization procedure if more
`than 24 h have elapsed from the last dose of
`administered allergen. This practice is reasonable
`since circulating allergen-specific IgE antibodies in
`these patients may bind to the recycled Fc^, recep-
`tors on the MC membrane, causing desensitization
`and consequently susceptibility to reactivation.
`In view of these observations and considering
`that an allergic patient is likely to be exposed to
`
`18
`
`the offending antigen more than once, we evalu-
`ated the susceptibility of SMC to challenge and
`rechallenge with optimal concentrations of a-Ig[
`antibodies. SMC presensitized with saturating con-
`centrations of IgE antibodies and challenged with
`an optimal concentration of anti-IgE antibodies
`released -50% of their histamine content. When
`these SMC were rechallenged 2 h to 7 days later,
`they were totally unresponsive. However, presen-
`sitization with IgE antibodies before the second
`challenge resulted in full responsiveness of these
`SMC to reactivation even by 2 h after the first
`activation. This further demonstrates
`that
`the
`major factor affecting human SMC responsiveness
`to rechallenge is associated with the availability ol
`IgE antibodies on the cell membrane rather than
`lack or downregulation of intracellular biochemical
`events. Therefore, in allergic patients when rela-
`tively high concentrations of IgE are present, it is
`conceivable that skin MC will respond to a second
`challenge with antigen, since IgE receptors on the
`SMC will be quickly reoccupied.
`In previous studies, we have shown that rat
`peritoneal MC regain their responsiveness at a
`slower rate (5). This difference between the two
`cell types may be due to the faster recycling of
`human IgE receptors than rat MC IgE receptors.
`An alternative explanation, together with the dif-
`ferent source of the MC (human vs rat), is the
`distinct anatomic location of the two connective-
`tissue-type MC populations; peritoneum for rat.
`skin for man,
`MC can also be activated by non-IgE-mediated
`activators. Therefore, in the second part of this
`study, we investigated SMC behavior after repeated
`activation by compound 48/80.
`Rechallenge of nonimmunologic activated SMC,
`8 and 24 h after the first challenge with the same
`activator at the same concentration, induced only
`a partial response. By 72 h, the cells had recovered
`completely and could release similar amounts of
`histamine as control cells activated for the first
`time. In addition, when SMC were incubated with
`increasing consecutive concentrations of com-
`pound 48/80, the cells became unresponsive to high
`concentrations of this substance, as observed with
`the similar procedure carried out with a-IgE anti-
`bodies. This shows that it is possible to induce ///
`vitro rush desensitization of SMC to a non-IgE-
`dependent challenge as well. Therefore, in addition
`to classic rush desensitization to allergens, it might
`be possible to desensitize allergic patients also with
`non-IgE-dependent activators such as certain
`drugs.
`In similar studies carried out on rat peritoneal
`MC activated with compound 48/80, complete
`responsiveness to rechallenge with the same non-
`
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`YEDA EXHIBIT NO. 2095
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`

`
`immunologic activator was achieved within 1 day
`(7,8). Moreover, with the same procedure of "rush
`desensitization" on rat MC, unresponsiveness
`could not be achieved. These differences
`in
`responses of human and rat are probably attribut-
`able to the different source of MC and to their
`different sensitivity to compound 48/80. Therefore,
`it is important to stress once more that heteroge-
`neity of responses exists in MC recovered from
`different species and different anatomic locations.
`In summary, we have shown for the first time
`that in vitro human SMC can become refractory to
`activation by the "rush desensitization" procedure
`with both immunologic and nonimmunologic stim-
`uli, as assessed by their incapacity to release hista-
`mine. This would
`indicate
`that MC are an
`important target of this procedure. Additionally,
`SMC can respond under the appropriate activation
`conditions to both IgE-dependent and -independ-
`ent restimulation by releasing histamine.
`
`Acknowledgments
`ITiis work was supported by grants from the Israeli Ministry of
`Health, the Blootn Center for Pharmacy, atid the Wolfson
`Foundation.
`
`References
`1. Bettleman DB, Stepleton J. Casale TB. Report of successful
`desensitization to itraconazole. J Allergy Clin Immunol
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