INTRODUCTION

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The introduction of inhaled nitric oxide (NO) therapy for the near-term infant with respiratory failure led to the hope that outcome may be significantly improved for many infants with lung disease associated with pulmonary hypertension (1). However, only about 50% of these infants will have major improvements in oxygenation with inhaled NO therapy and, although extracorporeal membrane oxygenation (ECMO) requirements may be reduced, mortality is not affected (2, 3). Further modes of therapy, therefore, warrant investigation.

Total liquid ventilation with perfluorocarbon (PFC) liquids has been applied to various diseases in animal models. For example, this technique has been shown to improve gas exchange and pulmonary mechanics in neonatal animals with severe surfactant-deficient respiratory distress (4). There have also been three reports of human trials of ventilation with PFCs, one of which reported total liquid ventilation. This report detailed short-term use in three preterm human neonates with severe respiratory distress (5). Total liquid ventilation was found to be feasible and led to marked improvement in lung distensibility. There was improved gas exchange and oxygenation in two of the infants with no alteration in cardiovascular status. All three neonates died within 19 h after PFC ventilation, probably because of the severity of their primary lung disease.

Partial liquid ventilation (PLV), which allows continued gas ventilation with a conventional ventilator after filling the end-expiratory volume of the lung with perfluorocarbon, is a much simpler technique requiring little in the way of special equipment (6). This technique is therefore likely to be more widely utilized than total liquid ventilation.

The two more recent neonatal human studies of liquid ventilation have investigated PLV. One study reported use of the technique in six infants receiving extracorporeal life support (7). Four of these infants were successfully weaned from extracorporeal support and two infants survived long term. Thirteen seriously ill premature infants with severe hyaline membrane disease were treated with PLV with some improvements in oxygenation (8), of whom eight infants were long-term survivors. Detectable blood levels of the PFC were noted in the infants, and these levels increased for as long as 72 h after initiation of therapy.

PLV improves gas exchange and pulmonary function in several animal models of the respiratory distress syndrome (9, 10). NO inhalation during PLV has also been shown to decrease pulmonary artery pressure (Ppa) and pulmonary vascular resistance (PVR) in a premature lamb model (11). PLV has been used with short-term (10 to 15 min) NO inhalation in a lamb congenital diaphragmatic hernia model with improved compliance and tidal volume noted after initiation of PLV; the added NO further improved oxygenation and significantly reduced pulmonary hypertension (12, 13). In a further study of combined therapy in a model of acute respiratory failure in piglets induced by repeated saline lung lavage followed by thromboxane infusion, PLV improved oxygenation (14). The brief administration of inhaled NO further improved oxygenation and decreased Ppa.

Total liquid ventilation was studied in seven naturally occurring distressed meconium-stained term lambs by Shaffer and colleagues (15) in 1984. Results were encouraging, showing increased mean Pa_O2 and decreased AaDO₂ 15 min after the onset of perfluorocarbon ventilation. During total liquid ventilation dynamic lung compliance increased, alveolar and peak tracheal pressure decreased, and inspiratory elastic work of breathing decreased. On return to gas ventilation, during the recovery period, Pa_O2 and AaDO₂ continued to be significantly improved. Meconium was also mobilized, suggesting that liquid ventilation might be particularly effective in meconium aspiration syndrome (MAS). Another study of the effects of PLV in meconium aspiration appears to have been published in abstract form only (16), this study in newborn piglets appears to demonstrate improved survival and oxygenation with PLV compared with both the control group and the surfactant-treated comparison group. Finally, a comparison of total liquid ventilation to PLV and surfactant replacement therapy in newborn lambs suggested that surfactant or either mode of liquid ventilation had potential benefits (17). Total liquid ventilation maintained much better lung histology, and all three groups had better oxygenation than the control group.

We have previously demonstrated the effects of inhaled NO in an animal model of the MAS (18), and we were therefore interested in the combined effects of inhaled NO and PLV. None of the previous studies of combined liquid ventilation and inhaled NO has investigated more than very short-term therapy (only a few minutes). It is likely that clinical usage of inhaled NO in infants receiving liquid ventilation will be more prolonged and last for several hours at least. It is possible that a combination of liquid ventilation with inhaled NO could just have additive effects; alternatively there could be a synergistic effect, with the lung recruitment of liquid ventilation actually enhancing the effects of inhaled NO by allowing delivery of the gas to newly expanded regions of the lung, thereby leading to further improvements in oxygenation and decrements in PVR.

In view of previous observations that liquid ventilation may be of benefit in MAS and our previous observation that NO is associated with improved oxygenation in an animal model of MAS, we sought to determine if the combination of NO and PLV was superior to either one alone in such a model. In particular, we wished to ascertain whether greater improvements in oxygenation would occur when NO is used during PLV, and whether NO would reduce PVR during PLV.

The objectives of the current study were to: (1) determine the effects of PLV on lung mechanics, gas exchange, and hemodynamics in an animal model of MAS, in comparison with conventional gas ventilation; (2) examine the effects of the addition of inhaled NO in both conventional and liquid ventilation modalities on those same outcome variables.

	METHODS

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Twenty-four mixed strain newborn piglets weighing 1.2 to 2.0 kg were obtained on the first or second day of life. Initial anesthetic induction was by inhaled halothane 4%, later reduced to 2%. A fluid-filled catheter was positioned in the distal esophagus, and through a midline neck incision an external jugular catheter, common carotid artery catheter, and tracheostomy were inserted. Halothane was then discontinued and 0.2 mg/kg of acepromazine was given together with a bolus of 10 µg/kg of fentanyl, followed by a continuous infusion of fentanyl of 5 µg/kg/h. Assisted ventilation was started and paralysis was obtained using pancuronium bromide 0.1 mg/kg every 45 min.

Ventilator pressures initially were 20 cm H₂O peak inspiratory pressure and 4 cm H₂O positive end-expiratory pressure. Through the right external jugular vein, a 5-Fr sheath was inserted and a 4-Fr Swan Ganz catheter was advanced into the right atrium, and, under fluoroscopy and using continuous pressure monitoring, into the pulmonary artery.

The systemic and pulmonary artery catheters were connected to pressure transducers and, together with the EKG signal, were displayed on a neonatal monitor (Model 78833B; Hewlett-Packard, Waltham, MA). Arterial oxygen saturation (Sa_O2) was monitored using a transcutaneous pulse oximeter (N200; Nellcor Inc., Hayward, CA). The animals were allowed 20 min for stabilization and then baseline recordings were taken for 10 min, the animals being ventilated with 40% oxygen. The high FI_O2 was used in order to ensure 100% Sa_O2. The animals were then ventilated with 90% oxygen and meconium was instilled down the endotracheal tube. A slurry of 30% human meconium in saline in a dose of 2 ml/kg, obtained from a single lot, mixed, and refrigerated prior to commencing this study, was manually ventilated into the animal's lungs and the animal was placed back on the infant ventilator. This usually led to an acute desaturation, with gradual recovery over 2 to 3 min to a lower saturation than the previous baseline. The process was then repeated at 5-min intervals with 1-ml/kg aliquots of meconium until the baseline recovery saturation was between 80 and 85% by pulse oximeter. A maximum of 8 ml/kg was given. The total duration of this procedure was usually between 25 and 55 min.

After meconium administration there is an acute respiratory acidosis; therefore, ventilatory settings were increased after the results of arterial blood gas analysis to a maximum of 80 breaths/min and a peak inspiratory pressure of 30 cm H₂O. The aim was to keep Pa_CO2 to between 40 and 60 mm Hg. Also, if calculated serum bicarbonate was less than 20 mmol/L and pH was less than 7.21, THAM was administered in a dose of 3 ml/kg every 15 min.

The animals were randomly assigned by a table of random numbers to one of four groups. (The codes were kept blinded until the meconium instillation had been completed.)

1. 1. Control group, conventional positive-pressure gas ventilation with 90% O₂.
2. 2. NO group, conventional positive-pressure gas ventilation with 90% O₂ and inhaled 40 ppm NO.
3. 3. PLV group, partial liquid ventilation with oxygenated PFCs.
4. 4. PLV and NO group, partial liquid ventilation with oxygenated PFCs and inhaled 40 ppm NO.

Perfluorodecalin (Air Products & Chemicals, Allentown, PA) used in this study has the following physical characteristics: boiling point, 142° C; density, 195 g/ml at 25° C; kinematic viscosity, 2.9 centistokes at 25° C; vapor pressure, 14 mm Hg at 37° C; surface tension, 15 dyne/ cm at 25° C; O₂ solubility, 49 ml of gas/100 ml of liquid at 25° C; CO₂ solubility, 140 ml of gas/100 ml of liquid at 37° C.

Control group. The control animals were gas-ventilated for 240 min and ventilatory changes were allowed as noted above.

NO group. Nitric oxide in nitrogen in a concentration of approximately 800 parts per million (ppm) was obtained from Canadian Liquid Air (Montreal, PQ, Canada). This source was certified to be ± 2% of the analyzed component (NO), and to contain < 5 ppm nitrogen dioxide (NO₂). Single-stage stainless steel diffuse free regulators were used and flushed to ensure that any air or other by-products such as NO₂ were removed. The source gas was connected via a Matheson no. 603 flowmeter (Matheson Gas Products Canada, Edmonton, AB, Canada), and then injected at the desired flow rate into the inspiratory line of a time-cycled pressure-limited neonatal ventilator (Health Dyne 105), using a continuous ventilator gas flow of 12 L/min. The resulting gas mixture was sampled downstream of the injection site and analyzed for NO, NO₂, and total oxides of nitrogen using a chemiluminescence analyzer (Model 42H; Thermoenvironmental Instruments, Franklin, MA). Exhaled gas and exhaust from the analyzer were scavenged.

PLV group. Perfluorodecalin was warmed and exposed to 100% O₂. Oxygenated PFC was then slowly instilled into the endotracheal tube through a port built into the endotracheal tube adapter. PEEP was reduced to 2 cm H₂O, and fluid was instilled until a meniscus could be visualized in the endotracheal tube during exhalation. This process took 15 to 20 min. The ventilator pressures were not altered during the instillation of the liquid, but changes in ventilator rate were allowed afterwards to achieve a PCO₂ between 40 and 60 mm Hg.

PLV and NO group. PLV was performed as described above and, in addition, inhaled NO was applied for the duration of the procedure in the same manner as for Group 2 (40 ppm in the inspired gases).

Arterial blood gases (Pa_O2, Pa_CO2, pH) were collected starting at baseline and every 30 min until the end of the experiment. The PO₂ and PCO₂ of the perfluorocarbon were obtained by running a sample of PFCs in a NOVA multiple analyzer, which also analyzed sodium, potassium, ionized calcium, glucose, and lactate. Arterial and mixed venous saturation and hemoglobin were determined using a Radiometer Co-Oximeter (Radiometer-America, Westlake, OH). With the final blood gas estimation, blood was taken on ice for Met-hemoglobin percentage using an IL282 Oximeter (Instrumentation Laboratories, Lexington, MA).

Tidal volume was continuously measured using the Bear NVM-1 device (Bear Medical Products, Riverside, CA), which uses a dual hot-wire anemometer. We calculated dynamic compliance during all modes of ventilation. After meconium instillation, lung compliance was low enough that there was little if any respiratory variation in esophageal pressure; therefore, measurements of total thoracic compliance are given. Changes in total thoracic compliance over the course of this study should represent changes in pulmonary compliance.

The analogue outputs of the pressures, blood flow rates, heart rate, saturation, and NO analyzer were digitized (Data Translation DT 2801A) and acquired at 24 Hz (Asyst 4.0; Keithly Instruments Inc., Taunton, MA) without filtering. The digitized signals were stored on the hard disk of an IBM compatible computer.

At the end of the experiment, the animals were killed by intravenous overdose of pentobarbital (30 mg/kg). The study was approved by the animal welfare committee of the University of Alberta and complied with the guidelines of the Canadian Council on Animal Care.

The results were analyzed by a two-way repeated-measures analysis of variance (SigmaStat; Jandel Scientific, San Rafael, CA) in order to maintain the overall risk of a type I error to less than 5% (10). This compared physiologic variables at different times between each treatment condition. If the overall ANOVA was statistically significant, a post-hoc test was performed (Fisher's least significant difference). A significance level of < 0.05 was accepted.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

All the control group animals survived for 2.5 h, but thereafter the most severely acidotic animals died. One animal died between 2.5 and 3 h, and two further animals died in the last 30 min of the study. All animals in the remaining groups survived for the full 4 h. The PLV animals received a mean of 20.4 (SD, 2.3) ml/kg of perfluorodecalin for the initial dose, the PLV + NO animals received a mean of 15.8 (SD, 2.2) ml/kg of the PFC, p = NS.

Animals in the Control group showed a persistent hypoxemia (Figure 1) accompanied by a progressive increase in Pa_CO2 and decrease in pH, despite all being ventilated at the maximum allowable rate of 80/min. The death of the three animals with the worst acidosis led to an apparent improvement in Pa_CO2 and pH immediately before the end of the study in the control animals. None of the individual animals had improved in blood gas determinations at the end of the study. By design, the Pa_CO2 was maintained between 40 and 50 mm Hg in all the intervention groups, whereas, despite attempts, within the limits imposed by the protocol, Pa_CO2 could not be normalized in the control group (Figure 2).

View larger version (21K): [in this window] [in a new window]   Figure 1.   Mean and standard deviation PaO2 values obtained at baseline, immediately after instillation of meconium (Time 0), and subsequently during inhaled NO therapy (iNO), partial liquid ventilation (PLV), or combined therapy (PLV + iNO). *iNO and PLV + iNO significantly higher than in the control group, p < 0.05. PLV significantly higher than in the control group, p < 0.05.

View larger version (22K): [in this window] [in a new window]   Figure 2.   Mean and SD PaCO2 values obtained at baseline, immediately after instillation of meconium (Time 0), and subsequently during inhaled NO therapy (iNO), partial liquid ventilation (PLV), or combined therapy (PLV + iNO). *Control significantly higher than the three other groups, p < 0.05.

The NO, PLV, and combined groups showed improvements in oxygenation, as shown in Figure 1. There was an immediate improvement in Pa_O2 in both the inhaled NO group and the combined inhaled NO and PLV groups. By 60 min, both of these groups had a Pa_O2 that was significantly greater than that of the control group. The inhaled NO alone group had a gradual fall in Pa_O2 between 120 and 240 min. The PLV group had a slow progressive increase in Pa_O2 from 30 until 240 min, and from 120 min onward the Pa_O2 was statistically significantly higher than that of the control group. The combined NO and PLV group both had an early and sustained improvement in Pa_O2.

There was no significant change in the cardiac index throughout the study; the control group cardiac index was 223 (SD, 45) ml/kg/min at the start of the study and 224 (SD, 107) in the piglets still alive at the end. In the NO group, cardiac index was 236 (SD, 38) ml/kg/min at baseline and 193 (SD, 21) ml/ kg/min at the end. In the PLV group, cardiac index was 238 (SD, 32) ml/kg/min at baseline and 173 (SD, 50) ml/kg/min at the end. The combined groups cardiac index was 224 (SD, 26) at the commencement of the study and 242 (SD, 182) ml/kg/ min at its end. Ppa increased by about 50% after instillation of the meconium, and it tended to be lowest in the NO alone group when compared with the remaining three groups, but there were no statistically significant differences (Figure 3).

View larger version (22K): [in this window] [in a new window]   Figure 3.   Mean and standard deviation of Ppa values obtained at baseline, immediately after instillation of meconium (Time 0), and subsequently during inhaled NO therapy (iNO), partial liquid ventilation (PLV), or combined therapy (PLV + iNO).

There was a dramatic reduction in lung compliance after the instillation of the meconium, from an overall mean of 1.51 to 0.42 ml/cm H₂O/kg (Figure 4). There was no change in compliance in the control or inhaled NO piglets from baseline until the end of the study. In contrast, an immediate increase in compliance was seen in the piglets receiving PLV, which was significant from 60 min until the end of the study. There were no significant differences between the PLV and the combined PLV/inhaled NO groups in lung compliance. Immediately prior to the end of the study the deaths of the sickest animals in the Control group give the impression of an improvement in lung compliance; however, as before, none of the individual control animals had an improvement in compliance.

View larger version (23K): [in this window] [in a new window]   Figure 4.   Mean and SD dynamic respiratory system compliance values obtained at baseline, immediately after instillation of meconium (Time 0), and subsequently during inhaled nitric oxide (iNO) therapy, partial liquid ventilation (PLV), or combined therapy (PLV + iNO). *PLV and PLV + iNO significantly higher than in the control group or iNO, p < 0.05.

During the study, there was no significant change in blood sodium or potassium, or ionized calcium concentrations in any of the four groups (Table 1). There was a significant elevation in blood glucose in all groups (p < 0.01) by the end of the study, and a significant elevation in blood lactate (p < 0.01), which by post-hoc analysis was restricted to the control group (p < 0.05), although, as can be seen, there was a trend to increased blood lactate in all groups.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Aspiration of meconium into the lungs of the newborn infant may occur prior to or during birth. The tenacious material causes airway plugging, displacement, and inactivation of surfactant and chemical pneumonitis (19, 20), and is associated with hypoxemia, respiratory acidosis, and pulmonary hypertension. In newborns the pulmonary artery pressures may be elevated enough to equal or exceed systemic pressures, which in infants with the most severe meconium aspiration may lead to intracardiac or extracardiac right-to-left shunting of deoxygenated blood and further systemic hypoxemia (21).

The MAS is a cause of significant mortality, and it is the commonest single diagnosis in infants requiring extracorporeal membrane oxygenation (ECMO) (ECMO Registry report, July 1997; 4,519 cases of meconium aspiration out of 12,692 total neonatal ECMO cases). ECMO is highly invasive and is associated with significant morbidity. Avoiding mortality and the need for ECMO are therefore both important goals.

Perfluorocarbon fluids are chemically and biologically inert, clear, colorless, and odorless liquids with a very high solubility for respiratory gases. Oxygen is about 20 times more soluble in PFCs than in water, and carbon dioxide is about three times as soluble. PFCs are stable, can be stored indefinitely at room temperature, and are not miscible with water or lipids. Almost all PFCs have low surface tension. At body temperature, they evaporate more rapidly than water (22). Perfluorocarbons are only minimally absorbed by the lungs when inhaled, and these small amounts are scavenged by macrophages and excreted exclusively through exhalation by lungs and transpiration through the skin. The highest tissue concentrations of perfluorocarbons are present in fat. No catabolism occurs (23), and no known adverse effects have been seen during histopathologic examination of organ tissues, including neonatal subjects (24), or on animal survival (25, 26). PFCs may persist in the tissues of animals who received total liquid ventilation for as long as 2 yr (27). The potential long-term toxicities in humans are unknown.

Potential advantages of liquid ventilation in MAS include the following: cleansing action of the liquid, draining the airways of meconium and other vasoactive compounds that may be locally present; recruitment of lung units and opening up of collapsed lung regions; reduction of surface tension (with reduction in inflation pressure, increased lung compliance, and improved arterial PO₂ and AaDO₂) and resultant minimizing of barotrauma; improvement in ventilation-perfusion mismatch as the liquid-ventilated lung is more homogeneously expanded; direct delivery of drugs to the pulmonary vasculature; maintenance of cardiovascular stability throughout the procedure; and, possibly, prevention of superinfection, as the PFC liquid is bacteriostatic.

Nitric oxide inhalation has now been shown convincingly to decrease requirements for ECMO, without affecting mortality, in near-term infants with respiratory failure (3), many of whom have MAS. This is achieved at low toxicity, the most common complication of therapy being methemoglobinemia. Inadvertent administration of high concentrations of either NO or nitric dioxide could potentially lead to pulmonary toxicity, but this has not been clearly documented in the human neonate. A dose of 40 ppm of NO was chosen as representing a commonly used concentration in human studies (28), although other human studies have not shown a significant dose response above 5 ppm. Our previous piglet study in meconium aspiration used 40 ppm; we continued to use this dose for comparability with previous data. Inhaled NO has potential toxicities: the generation of nitrogen dioxide may increase the risk of lung injury; the nitrosylation of proteins and enhancement of oxidant injury to the lungs has received some attention and may be found to be important; and platelet adhesion may be impaired during their passage through the lungs (29).

We have confirmed our previous findings (18) that inhaled NO in this meconium aspiration model improves oxygenation with relatively little effect on Ppa. There was no effect on lung mechanics and no effect on Pa_CO2. We now have demonstrated that PLV improves lung mechanics and progressively improves oxygenation over a 4-h period. Furthermore, the combination of inhaled NO and PLV leads to both acute improvements in oxygenation, which persist for at least 4 h, and acute improvements in lung mechanics, as seen with PLV alone. There have been a few previous investigations of combined inhaled NO and liquid ventilation (12, 30, 31). The previous studies have investigated lung lavage models, with (14) or without (30) infusion of a thromboxane analogue; a lamb congenital diaphragmatic hernia mode (12, 13), and an oleic acid infusion model of acute lung injury (31). All of these studies demonstrated an improvement in oxygenation and in Ppa when short-term inhalation of NO (10 to 15 min) was added to liquid ventilation. This is the first study to compare conventional support to prolonged inhaled NO, PLV, and combined PLV and NO. In the current study the addition of NO improved oxygenation in both inhaled NO alone and combined NO and PLV groups. As has previously been noted, inhaled NO appears to improve ventilation/perfusion matching, and the current study would suggest that ventilation/perfusion matching is also improved by inhaled NO during PLV. By the end of the 4-h study the improvements in gas exchange in the PLV alone group were substantial, and inhaled NO no longer showed any effect on gas exchange. The previous investigations of combined liquid ventilation and NO have used perflubron (12, 13, 30, 31) and rimar (14) as the PFC. No information is available in any of those reports about the solubility of NO in the PFC, although such information is vitally important to the future of this combination of therapies. The solubilities of O₂ and CO₂ in perfluorodecalin are known (O₂, 49 ml/100 ml at 37° C and 1 atm; CO₂, 140 ml/100 ml under the same conditions) the solubility of O₂ being similar to the values for perflubron and rimar, whereas that for CO₂ is somewhat less. From our data, NO does exert biologic effects at a concentration of 40 ppm in the inspired gases administered during PLV.

Ppa was increased in all groups after meconium instillation, and there was a nonstatistically significant fall in Ppa after starting inhaled NO alone. PLV was not associated with a fall in Ppa, despite the improvement in lung mechanics and gas exchange during PLV. This phenomenon has been noted previously (32) and may relate to the weight of the liquid in the lung, which directly compresses the pulmonary vasculature and elevates Ppa (33). PLV in 7- to 14-d-old piglets was previously shown to increase PVR when the animals were allowed to become hypoxic (34). The majority of studies, however, have shown little or no effect of PLV on PVR. The effect appears to depend upon the details of exactly how the technique is applied, which PFC is utilized, and which model is used. Even though there is a lack of effect on overall PVR, there may be a redistribution of pulmonary blood flow so that apical zones have greater perfusion (35). We maintained a volume of PFC in the lung that gave a visible meniscus in the endotracheal tube during expiration. Others have used smaller volumes, which only give a visible meniscus when disconnected from the ventilator. Perfluorodecalin has the highest density of the commonly used PFCs (1.95 g/ml compared with 1.93 g/ml for Perflubron and 1.77 for rimar), as well as a very high viscosity (2.9 centistokes at 25° C compared with 1.1 centistokes at 25° C for perflubron and 1.0 centistokes at 25° C for water). This combination of high density and slightly higher volumes may account for the failure of PVR in this study to fall despite the significant improvements in oxygenation. The current study also did not show any effect on Ppa of the addition of NO to PLV.

The applied level of PEEP may also relate to the effects on PVR. We reduced the ventilator-applied PEEP to 2 cm H₂O during liquid ventilation. The weight of the perfluorocarbon by itself may mean that further applied PEEP is not necessary. However, some of the proximal and anterior airways are probably receiving little PFC and are being gas-ventilated during PLV. Therefore, maintenance of at least a minimal level of PEEP may be ideal. The optimal PEEP remains to be determined in various lung injury models, but it may be dependent upon the precise PFC used, the lung injury investigated, the species, and the other ventilatory variables chosen.

In summary, we used a neonatal meconium aspiration model to study the effects of inhaled NO, partial liquid ventilation, and their combination. Inhaled NO led to an acute improvement in oxygenation, and liquid ventilation led to a significant progressive improvement in lung compliance and a later improvement in oxygenation. The combination of inhaled NO and PLV demonstrated both effects, and could be investigated for infants with MAS unresponsive to other therapies.