849
In Vivo Interferon-y Therapy Augments the In Vitro Ability of Chronic Granulomatous Disease Neutrophils to Damage Aspergillus Hyphae John H. Rex, John E. Bennett, John I. Gallin, Harry L. Malech, Ellen S. DeCarlo, and David A. Melnick
Laboratory of Clinical Investigation, National Institute of Allergy q,nd Infectious Diseases, National Institutes of Health, Bethesda, Maryland
On the basis of the observation that treatment with recombinant human interferon-y (rIFN -1') augmented the ability of monocytes and neutrophils from patients with chronic granulomatous disease (CGD) to generate superoxide and to kill Staphylococcus aureus [1-3], a double-blind, randomized, placebo-controlled, multiinstitutional trial of rIFN -1' therapy in patients with CaD was done [4]. The study demonstrated that, in comparison with placebo, rIFN -1', 0.05 mg/m? given three times per week, significantly reduced the incidence of serious infections (infections requiring hospitalization and intravenous antibiotics) and the number of inpatient hospital days spent treating infections. During the study, we monitored the function of neutrophils from study patients by using our recently described system for assessing damage to Aspergillus fumigatus hyphae [5]. This assay takes advantage of the fact that metabolically active hyphae will convert the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT]; Sigma Chemical, St. Louis) to a blue formazan derivative with maximum absorbance at 560-570 nm and that exposure to functional neutrophils reduces the ability of the hyphae to perform this conversion [5, 6].
Materials and Methods Study subjects. Subjects were the patients enrolled in the rlFN-'Y trial at the National Institute of Allergy and Infectious Diseases. The Received 30 July 1990; revised 13 November 1990. All patients signed an informed consent, and the protocol was approved by the Clinical Research Subpanel of the National Institute of Allergy and Infectious Diseases. Reprints or correspondence: Dr. John H. Rex, Bldg. 10, Room llNI07, National Institutes of Health, Bethesda, MD 20892. The Journal of Infectious Diseases 1991;163:849-852 This article is in the public domain. 0022-1899/91/6304-0028
details of this study are described elsewhere [4]. In brief, patients with CGD were randomized to receive either 0.05 mg/m? of rIFN'Y or placebo subcutaneously three times per week. The study was blinded until completion. Measurement of damage to Aspergillus hyphpe. A detailed description of this method and its validation have been reported [5]. In brief, l-ml aliquots of a solution of yeast-nitrogen base containing A. jumigatus conidia (5 x 104/ml) were inoculated into the 24mm wells of a Costar plate. After an overnight incubation at 30°C, the conidia germinated into hyphae that adhered firmly to the bottoms of the wells. Neutrophils were isolated and suspended in Hanks' balanced salt solution (HBSS) plus 20 mM HEPES, pH 7.0. To the wells containing germinated conidia were added 0.1 ml of serum (fresh or stored frozen at -70°C), neutrophils, and HBSS with 20 mM HEPES, pH 7.0, to a final volume of 1.0 ml. The neutrophils were gently sedimented to the bottom of the well by centrifugation. After incubation for 2 h at 37°C, the wells were aspirated dry, the neutrophils were lysed by adding 0.3 ml of 0.5 % sodium deoxycholate, and the neutrophil debris was removed by four washes with sterile water. RPMI 1640 with t-glutamine, 1.0ml, containing 0.5 mg/ml MTT was added to each well, and the plates were incubated at 37°C for 3 h. Then the wells were aspirated dry, the blue formazan precipitate was solubilized with acidified isopropanol (a mixture of 95 ml ofisopropanol and 5 ml of 1 NHCI), and the optical density (OD)"at570 nm of the resulting solution was determined using acidified isopropanol as a blank. As a reagent blank, a series of wells duplicating the additives in the test wells was routinely set up in a parallel Costar plate that contained no hyphae. These wells were then processed as above, and their optical densities were averaged and subtracted from those of the test wells before any further calculations. A group of wells containing only hyphae (but otherwise treated identically to the wells containing neutrophils) was used on each plate, and all data from a given plate were divided by the mean OD of the plate's "hyphaeonly" group of wells. Hyphal viability is computed as percentage of MTT conversion: % MTT conversion = 100 x [mean OD in test wells - mean OD in reagent blank wells]/ [mean OD in wells containing only hyphae - mean OD in reagent blank wells].
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 6, 2015
During the recently completed double-blind, placebo-controlled, randomized trial of recombinant interferon-v (rIFN-'Y) therapy in chronic granulomatous disease (CGO), a metabolic assay of neutrophil damage to Aspergillusjumigatus hyphae was used to monitor neutrophil function before and during therapy. In this assay, 5 x 1()4 conidia that had germinated into hyphae were exposed to 5 x lOs, 15 x lOs, or 50 x lOS CGO neutrophils. By analysis of variance, neutrophils from patients on rIFN-'Y were found to produce significantly more damage to hyphae than those from the placebo group (P < .01). In subgroup analysis, this effect was best seen in the hyphae exposed to 50 x lOS CGO neutrophils, where neutrophils from patients receiving rIFN--y produced significantly more damage to the hyphae than those from the placebo group (P < .05). In vivo rIFN-'Y therapy improves the ability ofCGO neutrophils to damage Aspergillusjumigatus hyphae in an in vitro assay.
850
Rex et al.
Results Because some patients were small, it was not always possible to obtain sufficient blood from each patient at all time points to study the effect of each dose of neutrophils. Thus, while 21 patients were available for study, the number of evaluatable patients at each dose varies, as does the number of times each patient was studied. In general, however, all patients were studied once or twice at baseline and then one to five times on therapy, with almost all studies being done in triplicate. The overall results of the study are summarized in table 1. Two significant effects were noted. First, a 2 X 3 analysis of variance detected a significant difference between the placebo and rIFN--y-treated groups (P < .01). Then, when individual values from table 1 were compared with a Tukey test, a significant difference in hyphal viability between the treatment groups was found in the wells containing 50 X lOS neutrophils (P < .05). No effect of rIFN--y therapy was detected at 5 X 105 or 15 X 105 neutrophils/well, The activity of each patient's neutrophils and the inheritance pattern for the patients are shown in figure 1. We are unable to explain the upward drift in the measurement of percentage of MTT conversion in the placebo group over time. Whatever the source of this variation, it should have acted equally on the rIFN--y-treated group, given that the two groups were being studied simultaneously and often neutrophils from placebo and rIFN -y-treated patients were tested on the same day. As a further control for the variability of the assay over time, the effect of 5 X 105 neutrophils from a normal donor was always studied on the same Costar plate as each patient. If the percentage of MTT conversion of the hyphae exposed to the patient's neutrophils is normalized to that of the hyphae exposed to the normal neutrophils, an essentially identical, statistically significant result is obtained (data not shown). To rule out the possibility that these effects might have been due to functional enhancement of the phagocytes by the concomitant use of other antibiotics, especially trimethoprimsulfamethoxazole (TMP/SMZ), the use of these drugs was
Table 1. Effect of recombinant human interferon-y (rIFN-y) on the viability of Aspergillus hyphae exposed to chronic granulomatous disease neutrophils. No. of neutrophils per well
5
X
105
15 X 105 50 X 105
Treatment group, % MTT conversion ± SE (no. studied) Placebo * 6
± 6 (11)
8 ± 6 (6) 23 ± 14 (6)t
rIFN-y*
-4 ± 0(10) -2 -29
± 9 (5) ± 7 (5)t
NOTE. Because percentage MTT conversion reflects hyphal viability (not damage), increase means more hyphae survived while decrease indicates greater damage to hyphae. * Difference between groups in 2 x 3 analysis of variance, P < .01. t By Tukey test, P < .05.
tabulated. The most commonly administered antibiotic was
TMP/SMZ, which was taken by >60% of the patients in both groups during the the study. There was no significant difference in the number of patients on prophylactic TMP/SMZ .therapy between the treatment groups, at either the start or the end of the study (data not shown) Although not statistically significant because of small sample size, of the 11 patients for whom we have data with 50 X 105 neutrophils/well, the 5 on rIFN--y had fewer serious infections (by the criteria of the larger clinical trial) than the 6 on placebo (2/5 on rIFN--y vs. 4/6 on placebo, figure 1). In the larger clinical trial, there were two episodes of aspergillus pneumonia (both in the same patient) in the 63 patients on rIFN--y, while there were four (in four different patients) in the 65 patients on placebo.
Discussion In this double-blind study, in vivo rIFN--y therapy produced an increase in the ability of can neutrophils to damage A. fumigatus in an in vitro assay. This increased neutrophil function correlated with the broader results of the associated clinical trial, in which a significant clinical effect of rIFN--ytherapy was shown in a much larger group of patients [4]. Although the clinical results in our small sample of patients on whom we have complete in vitro data do not reach significance, they trend in the same direction, as does the incidence of aspergillus pneumonia in the larger clinical trial. The effect of rIFN--y in the larger clinical trial was independent of the pattern of can inheritance. As a majority of our patients (14/21, figure 1) had the X-linked pattern of inheritance, we are unable make any further conclusion about the relative effects of rIFN--y in the various genetic subgroups. The mechanism of improved neutrophil function induced by rIFN--y cannot be determined from this study. In previous work, we demonstrated that neutrophils damage Aspergillus hyphae by a myeloperoxidase-dependent oxidative process [5].
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 6, 2015
Patients enrolled in the rIFN-')' trial were studied before therapy and then typically after 4,90, and 180 days on therapy. Wells were set up using 5 x 105 , 15 x lOS, and 50 x 105 neutrophils. All wells were on the same Costar plate and were normally set up in triplicate (but at least duplicate). Analysis of data. Mean measurements from each study day of percentage of MTT conversion for each patient were grouped into therapy and baseline values, and each ofthese groups was averaged. Then the change in percentage of MTT conversion for each patient was computed as follows: change in % MTT conversion = % MTT conversion on therapy .; % MTT conversion at baseline. When the study code was broken, the patients were grouped into placebo and rIFN-')' treatment groups, and the mean changes within the groups were compared, first with an analysis of variance and then with the Tukey test [7] to compare individual differences.
JID 1991;163 (April)
rIFN--y and CGD Neutrophils
JID 1991;163 (April)
851
150
150
o
A ~"'"
"',
~
r~:';:,,"'1
100
90±4%
100
94±S%
50
human interferon (IFN)--y on the ability of chronic granulomatous disease neutrophils to damage Aspergillus hyphae. Each line represents a single patient; percentages at left and rightof data lines are mean ± SE for the group, either at baseline or on therapy. Line format indicatespatient's pattern of inheritance: . . . , autosomal; , X-linked. Filled symbols (as opposed to open symbol or no symbol) indicate patients who had serious infection by criteria of the larger clinical trial. Combinations of plot line and plot symbol uniquely identify each patient and are used consistently throughout A-C and D-F. A and D, 5 x lOS; Band E, 15 x 105 ; C and F, 50 x 105 neutrophils/well.
o
.
o "-
CD
---L Baseline
-I-
_
OL...-------L-----I-----
On Therapy
Baseline
150
B
> C
90±S%
~
50
"iii
q
On Therapy
150
E
o
o
tt:E
~
100
86±6%
100
50
86±10%
78±6%
88±7%
50
o "-
---L Baseline
-I-
_
o "-
On Therapy
c
150
---L Baseline
100
-I-
_
On Therapy
150
F
100
88±16%
59±17%
50
70±15%
93±20%
50
0L...------L----...1... Baseline
On Therapy
IFN-Treated Group
Preliminary work with rIFN-)' demonstrated that in vivo therapy with rIFN-)' increased production of oxidative products by neutrophils from some patients [1-3]. Whether or not the enhanced antifungal activity of neutrophils obtained from COD patients treated with rIFN-)' in this study is due to oxidative or nonoxidative phagocyte systems (or both) cannot be determined at this time.
_
OL...------L---.......;;:=------Baseline
On Therapy
Placebo Group
References 1. Sechler JM, Malech HL, WhiteCJ, Gallin JI. Recombinant human interferon-y reconstitutes defective phagocyte function in patients with chronic granulomatous disease of childhood. Proc Natl Acad Sci USA 1988;85:4874-8. 2. Ezekowitz RAB, Orkin SH, Newburger PE. Recombinant interferon-v augments phagocyte superoxide production and chronic granuloma-
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 6, 2015
C
Figure 1. Effects of recombinant
84±3%
852
Rex et al.
tous disease gene expression in X-linked variant chronic granulomatous disease. J Clin Invest 1987;80:1009-16. 3. Ezekowitz RAB, Dinauer MC, Jaffe HS, Orkin SH, Newburger PE. Partial correction of the phagocyte defect in patients with X-linked chronic granulomatous disease by subcutaneous interferon)'. N Engl J Med 1988;319:146-51. 4. International Chronic Granulomatous Disease Cooperative Study Group. A phase III study establishing efficacy of recombinant human interferon-v for infection prophylaxis of chronic granulomatous disease. N Engl J Med (in press).
JID 1991;163 (April)
5. Rex JH, Bennett IE, Gallin 11, Malech HL, Melnick DA. Normal and deficient neutrophils can cooperate to damage Aspergillus fumigatus hyphae. J Infect Dis 1990;162:523-9. 6. Levitz SM, Diamond RD. A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT. J Infect Dis 1985;152:938-45. 7. Zar JH. Biostatistical analysis. 2nd ed. Englewood Cliffs, NJ: PrenticeHall, 1984.
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 6, 2015
In Vivo Interferon-y Therapy Augments the In Vitro Ability of Chronic Granulomatous Disease Neutrophils to Damage Aspergillus Hyphae John H. Rex, John E. Bennett, John I. Gallin, Harry L. Malech, Ellen S. DeCarlo, and David A. Melnick
Laboratory of Clinical Investigation, National Institute of Allergy q,nd Infectious Diseases, National Institutes of Health, Bethesda, Maryland
On the basis of the observation that treatment with recombinant human interferon-y (rIFN -1') augmented the ability of monocytes and neutrophils from patients with chronic granulomatous disease (CGD) to generate superoxide and to kill Staphylococcus aureus [1-3], a double-blind, randomized, placebo-controlled, multiinstitutional trial of rIFN -1' therapy in patients with CaD was done [4]. The study demonstrated that, in comparison with placebo, rIFN -1', 0.05 mg/m? given three times per week, significantly reduced the incidence of serious infections (infections requiring hospitalization and intravenous antibiotics) and the number of inpatient hospital days spent treating infections. During the study, we monitored the function of neutrophils from study patients by using our recently described system for assessing damage to Aspergillus fumigatus hyphae [5]. This assay takes advantage of the fact that metabolically active hyphae will convert the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT]; Sigma Chemical, St. Louis) to a blue formazan derivative with maximum absorbance at 560-570 nm and that exposure to functional neutrophils reduces the ability of the hyphae to perform this conversion [5, 6].
Materials and Methods Study subjects. Subjects were the patients enrolled in the rlFN-'Y trial at the National Institute of Allergy and Infectious Diseases. The Received 30 July 1990; revised 13 November 1990. All patients signed an informed consent, and the protocol was approved by the Clinical Research Subpanel of the National Institute of Allergy and Infectious Diseases. Reprints or correspondence: Dr. John H. Rex, Bldg. 10, Room llNI07, National Institutes of Health, Bethesda, MD 20892. The Journal of Infectious Diseases 1991;163:849-852 This article is in the public domain. 0022-1899/91/6304-0028
details of this study are described elsewhere [4]. In brief, patients with CGD were randomized to receive either 0.05 mg/m? of rIFN'Y or placebo subcutaneously three times per week. The study was blinded until completion. Measurement of damage to Aspergillus hyphpe. A detailed description of this method and its validation have been reported [5]. In brief, l-ml aliquots of a solution of yeast-nitrogen base containing A. jumigatus conidia (5 x 104/ml) were inoculated into the 24mm wells of a Costar plate. After an overnight incubation at 30°C, the conidia germinated into hyphae that adhered firmly to the bottoms of the wells. Neutrophils were isolated and suspended in Hanks' balanced salt solution (HBSS) plus 20 mM HEPES, pH 7.0. To the wells containing germinated conidia were added 0.1 ml of serum (fresh or stored frozen at -70°C), neutrophils, and HBSS with 20 mM HEPES, pH 7.0, to a final volume of 1.0 ml. The neutrophils were gently sedimented to the bottom of the well by centrifugation. After incubation for 2 h at 37°C, the wells were aspirated dry, the neutrophils were lysed by adding 0.3 ml of 0.5 % sodium deoxycholate, and the neutrophil debris was removed by four washes with sterile water. RPMI 1640 with t-glutamine, 1.0ml, containing 0.5 mg/ml MTT was added to each well, and the plates were incubated at 37°C for 3 h. Then the wells were aspirated dry, the blue formazan precipitate was solubilized with acidified isopropanol (a mixture of 95 ml ofisopropanol and 5 ml of 1 NHCI), and the optical density (OD)"at570 nm of the resulting solution was determined using acidified isopropanol as a blank. As a reagent blank, a series of wells duplicating the additives in the test wells was routinely set up in a parallel Costar plate that contained no hyphae. These wells were then processed as above, and their optical densities were averaged and subtracted from those of the test wells before any further calculations. A group of wells containing only hyphae (but otherwise treated identically to the wells containing neutrophils) was used on each plate, and all data from a given plate were divided by the mean OD of the plate's "hyphaeonly" group of wells. Hyphal viability is computed as percentage of MTT conversion: % MTT conversion = 100 x [mean OD in test wells - mean OD in reagent blank wells]/ [mean OD in wells containing only hyphae - mean OD in reagent blank wells].
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 6, 2015
During the recently completed double-blind, placebo-controlled, randomized trial of recombinant interferon-v (rIFN-'Y) therapy in chronic granulomatous disease (CGO), a metabolic assay of neutrophil damage to Aspergillusjumigatus hyphae was used to monitor neutrophil function before and during therapy. In this assay, 5 x 1()4 conidia that had germinated into hyphae were exposed to 5 x lOs, 15 x lOs, or 50 x lOS CGO neutrophils. By analysis of variance, neutrophils from patients on rIFN-'Y were found to produce significantly more damage to hyphae than those from the placebo group (P < .01). In subgroup analysis, this effect was best seen in the hyphae exposed to 50 x lOS CGO neutrophils, where neutrophils from patients receiving rIFN--y produced significantly more damage to the hyphae than those from the placebo group (P < .05). In vivo rIFN-'Y therapy improves the ability ofCGO neutrophils to damage Aspergillusjumigatus hyphae in an in vitro assay.
850
Rex et al.
Results Because some patients were small, it was not always possible to obtain sufficient blood from each patient at all time points to study the effect of each dose of neutrophils. Thus, while 21 patients were available for study, the number of evaluatable patients at each dose varies, as does the number of times each patient was studied. In general, however, all patients were studied once or twice at baseline and then one to five times on therapy, with almost all studies being done in triplicate. The overall results of the study are summarized in table 1. Two significant effects were noted. First, a 2 X 3 analysis of variance detected a significant difference between the placebo and rIFN--y-treated groups (P < .01). Then, when individual values from table 1 were compared with a Tukey test, a significant difference in hyphal viability between the treatment groups was found in the wells containing 50 X lOS neutrophils (P < .05). No effect of rIFN--y therapy was detected at 5 X 105 or 15 X 105 neutrophils/well, The activity of each patient's neutrophils and the inheritance pattern for the patients are shown in figure 1. We are unable to explain the upward drift in the measurement of percentage of MTT conversion in the placebo group over time. Whatever the source of this variation, it should have acted equally on the rIFN--y-treated group, given that the two groups were being studied simultaneously and often neutrophils from placebo and rIFN -y-treated patients were tested on the same day. As a further control for the variability of the assay over time, the effect of 5 X 105 neutrophils from a normal donor was always studied on the same Costar plate as each patient. If the percentage of MTT conversion of the hyphae exposed to the patient's neutrophils is normalized to that of the hyphae exposed to the normal neutrophils, an essentially identical, statistically significant result is obtained (data not shown). To rule out the possibility that these effects might have been due to functional enhancement of the phagocytes by the concomitant use of other antibiotics, especially trimethoprimsulfamethoxazole (TMP/SMZ), the use of these drugs was
Table 1. Effect of recombinant human interferon-y (rIFN-y) on the viability of Aspergillus hyphae exposed to chronic granulomatous disease neutrophils. No. of neutrophils per well
5
X
105
15 X 105 50 X 105
Treatment group, % MTT conversion ± SE (no. studied) Placebo * 6
± 6 (11)
8 ± 6 (6) 23 ± 14 (6)t
rIFN-y*
-4 ± 0(10) -2 -29
± 9 (5) ± 7 (5)t
NOTE. Because percentage MTT conversion reflects hyphal viability (not damage), increase means more hyphae survived while decrease indicates greater damage to hyphae. * Difference between groups in 2 x 3 analysis of variance, P < .01. t By Tukey test, P < .05.
tabulated. The most commonly administered antibiotic was
TMP/SMZ, which was taken by >60% of the patients in both groups during the the study. There was no significant difference in the number of patients on prophylactic TMP/SMZ .therapy between the treatment groups, at either the start or the end of the study (data not shown) Although not statistically significant because of small sample size, of the 11 patients for whom we have data with 50 X 105 neutrophils/well, the 5 on rIFN--y had fewer serious infections (by the criteria of the larger clinical trial) than the 6 on placebo (2/5 on rIFN--y vs. 4/6 on placebo, figure 1). In the larger clinical trial, there were two episodes of aspergillus pneumonia (both in the same patient) in the 63 patients on rIFN--y, while there were four (in four different patients) in the 65 patients on placebo.
Discussion In this double-blind study, in vivo rIFN--y therapy produced an increase in the ability of can neutrophils to damage A. fumigatus in an in vitro assay. This increased neutrophil function correlated with the broader results of the associated clinical trial, in which a significant clinical effect of rIFN--ytherapy was shown in a much larger group of patients [4]. Although the clinical results in our small sample of patients on whom we have complete in vitro data do not reach significance, they trend in the same direction, as does the incidence of aspergillus pneumonia in the larger clinical trial. The effect of rIFN--y in the larger clinical trial was independent of the pattern of can inheritance. As a majority of our patients (14/21, figure 1) had the X-linked pattern of inheritance, we are unable make any further conclusion about the relative effects of rIFN--y in the various genetic subgroups. The mechanism of improved neutrophil function induced by rIFN--y cannot be determined from this study. In previous work, we demonstrated that neutrophils damage Aspergillus hyphae by a myeloperoxidase-dependent oxidative process [5].
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 6, 2015
Patients enrolled in the rIFN-')' trial were studied before therapy and then typically after 4,90, and 180 days on therapy. Wells were set up using 5 x 105 , 15 x lOS, and 50 x 105 neutrophils. All wells were on the same Costar plate and were normally set up in triplicate (but at least duplicate). Analysis of data. Mean measurements from each study day of percentage of MTT conversion for each patient were grouped into therapy and baseline values, and each ofthese groups was averaged. Then the change in percentage of MTT conversion for each patient was computed as follows: change in % MTT conversion = % MTT conversion on therapy .; % MTT conversion at baseline. When the study code was broken, the patients were grouped into placebo and rIFN-')' treatment groups, and the mean changes within the groups were compared, first with an analysis of variance and then with the Tukey test [7] to compare individual differences.
JID 1991;163 (April)
rIFN--y and CGD Neutrophils
JID 1991;163 (April)
851
150
150
o
A ~"'"
"',
~
r~:';:,,"'1
100
90±4%
100
94±S%
50
human interferon (IFN)--y on the ability of chronic granulomatous disease neutrophils to damage Aspergillus hyphae. Each line represents a single patient; percentages at left and rightof data lines are mean ± SE for the group, either at baseline or on therapy. Line format indicatespatient's pattern of inheritance: . . . , autosomal; , X-linked. Filled symbols (as opposed to open symbol or no symbol) indicate patients who had serious infection by criteria of the larger clinical trial. Combinations of plot line and plot symbol uniquely identify each patient and are used consistently throughout A-C and D-F. A and D, 5 x lOS; Band E, 15 x 105 ; C and F, 50 x 105 neutrophils/well.
o
.
o "-
CD
---L Baseline
-I-
_
OL...-------L-----I-----
On Therapy
Baseline
150
B
> C
90±S%
~
50
"iii
q
On Therapy
150
E
o
o
tt:E
~
100
86±6%
100
50
86±10%
78±6%
88±7%
50
o "-
---L Baseline
-I-
_
o "-
On Therapy
c
150
---L Baseline
100
-I-
_
On Therapy
150
F
100
88±16%
59±17%
50
70±15%
93±20%
50
0L...------L----...1... Baseline
On Therapy
IFN-Treated Group
Preliminary work with rIFN-)' demonstrated that in vivo therapy with rIFN-)' increased production of oxidative products by neutrophils from some patients [1-3]. Whether or not the enhanced antifungal activity of neutrophils obtained from COD patients treated with rIFN-)' in this study is due to oxidative or nonoxidative phagocyte systems (or both) cannot be determined at this time.
_
OL...------L---.......;;:=------Baseline
On Therapy
Placebo Group
References 1. Sechler JM, Malech HL, WhiteCJ, Gallin JI. Recombinant human interferon-y reconstitutes defective phagocyte function in patients with chronic granulomatous disease of childhood. Proc Natl Acad Sci USA 1988;85:4874-8. 2. Ezekowitz RAB, Orkin SH, Newburger PE. Recombinant interferon-v augments phagocyte superoxide production and chronic granuloma-
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 6, 2015
C
Figure 1. Effects of recombinant
84±3%
852
Rex et al.
tous disease gene expression in X-linked variant chronic granulomatous disease. J Clin Invest 1987;80:1009-16. 3. Ezekowitz RAB, Dinauer MC, Jaffe HS, Orkin SH, Newburger PE. Partial correction of the phagocyte defect in patients with X-linked chronic granulomatous disease by subcutaneous interferon)'. N Engl J Med 1988;319:146-51. 4. International Chronic Granulomatous Disease Cooperative Study Group. A phase III study establishing efficacy of recombinant human interferon-v for infection prophylaxis of chronic granulomatous disease. N Engl J Med (in press).
JID 1991;163 (April)
5. Rex JH, Bennett IE, Gallin 11, Malech HL, Melnick DA. Normal and deficient neutrophils can cooperate to damage Aspergillus fumigatus hyphae. J Infect Dis 1990;162:523-9. 6. Levitz SM, Diamond RD. A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT. J Infect Dis 1985;152:938-45. 7. Zar JH. Biostatistical analysis. 2nd ed. Englewood Cliffs, NJ: PrenticeHall, 1984.
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 6, 2015
