Enabling Microparticle Imprinting to Achieve Penetration and Local Endurance in the Peritoneum via High-Intensity Ultrasound (HIUS) for the Treatment of Peritoneal Metastasis.

INTRODUCTION: Micro- and nanoparticles, with their submicron size, the versatility of physical and chemical properties, and easily modifiable surface, are uniquely positioned to bypass the body's clearing systems. Nonetheless, two main problems with micro- and nanoparticles arise which limit the intraperitoneal application. The study was performed to evaluate whether HIUS enables the imprinting of microparticles and, therefore, enhances penetration and local endurance in the peritoneum. METHODS: High-intensity ultrasound (HIUS) at 20 kilohertz with an output power of 70 W was applied on peritoneal tissue samples from fresh postmortem swine for different time intervals. Before the HIUS application, the surface of the samples was covered with strontium aluminate microparticles before analysis via electron microscopy. In-tissue strontium aluminate penetration and particle distribution size were measured using fluorescence microscopy on frozen thin sections. RESULTS: With increasing HIUS durations (1 versus 5 minutes), increasing strontium aluminate particles were detected in the peritoneum. HIUS leads to a particle selection process with enhancing predominantly the penetration of smaller particles whereas larger particles had a harder time penetrating the peritoneum. Smaller particles were detected up to 277 µm ± 86 µm into the peritoneum. CONCLUSION: Our data indicate that HIUS might be used as a method to prepare the peritoneal tissue for micro- and nanoparticles. Higher tissue penetration rates without the increase and longer local endurance of the applied substance could be reached. More studies need to be performed to analyze the effect of HIUS in enhancing intraperitoneal drug applications.

Intraperitoneal Phototherapy Suppresses Inflammatory Reactions in a Surgical Model of Peritonitis.

BACKGROUND: Standard treatment for diffuse peritonitis due to colorectal perforation may be insufficient to suppress inflammatory reaction in sepsis. Thus, developing new treatments is important. This study aimed to examine whether intraperitoneal irradiation by artificial sunlight suppresses inflammatory reaction in a lipopolysaccharide (LPS)-induced peritonitis model after surgical treatments. MATERIALS AND METHODS: Mice were divided into naive, nontreatment (NT), and phototherapy (PT) groups. In the latter two groups, LPS was intraperitoneally administered to induce peritonitis and removed by intraperitoneal lavage after laparotomy. The PT group was irradiated with artificial sunlight intraperitoneally. We evaluated the local and systemic inflammatory reactions. Murine macrophages were irradiated with artificial sunlight after stimulation by LPS, and cell viability and expression of tumor necrotizing factor-α (TNF-α) were evaluated. RESULTS: As a local inflammatory reaction, the whole cell count, the expression of interleukin-6 and TNF-α in the intra-abdominal fluid, and the peritoneal thickness were significantly lower in the PT group than in the NT group. As a systematic inflammatory reaction, the expression of serum TNF-α, granulocyte macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, and MIP-1ß were significantly lower in the PT group than in the NT group. Irradiation by artificial sunlight suppressed the expression of TNF-α in murine macrophages without affecting cell viability. CONCLUSIONS: Intraperitoneal irradiation by artificial sunlight could suppress local and systemic inflammatory reactions in the LPS-induced peritonitis murine model. These effects may be associated with macrophage immune responses.

Effect of Whole-abdominal Irradiation on Penetration Depth of Doxorubicin in Normal Tissue After Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC) in a Post-mortem Swine Model.

BACKGROUND: This study was performed to evaluate the impact of whole-abdominal irradiation on local penetration of doxorubicin into the peritoneum and the abdominal organs in a post-mortem swine model. MATERIALS AND METHODS: Doxorubicin was aerosolized into the abdominal cavity of swine at a pressure of 12 mmHg CO2 at room temperature (25°). One swine was subjected to pressurized intraperitoneal aerosol chemotherapy (PIPAC) using Micropump© without irradiation; the second one received 2 Gy and the third one 7 Gy whole-abdominal irradiation, 15 min prior to PIPAC application. Samples of the peritoneal surface were extracted at different positions from within the abdominal cavity. In-tissue doxorubicin penetration was measured using fluorescence microscopy on frozen thin sections. RESULTS: The depth of penetration of doxorubicin was found to be wide-ranging, between 17 µm on the surface of the stomach and 348 µm in the small intestine. The penetration depth into the small intestine was 348 µm, 312 µm and 265 µm for PIPAC alone, PIPAC with 2 Gy irradiation and PIPAC with 7 Gy irradiation, respectively (p<0.05). The penetration into the liver was 64 µm, 55 µm and 40 µm, respectively (p=0.05). CONCLUSION: Irradiation was not found to increase the depth of doxorubicin penetration into normal tissue in the post-mortem swine model. A reduction of doxorubicin penetration was observed after application of higher irradiation doses. Further studies are warranted to determine if irradiation can be used safely as chemopotentiating agent for patients with peritoneal metastases treated with PIPAC.

Abdominal desmoplastic small round cell tumor without extraperitoneal metastases: Is there a benefit for HIPEC after macroscopically complete cytoreductive surgery?

BACKGROUND: Desmoplastic Small Round Cell Tumor (DSRCT) is a rare disease affecting predominantly children and young adults and for which the benefit of hyperthermic intraperitoneal chemotherapy (HIPEC) after complete cytoreductive surgery (CCRS) remains unknown. METHODS: To identify patients with DSRCT without extraperitoneal metastases (EPM) who underwent CCRS between 1991 and 2015, a retrospective nation-wide survey was conducted by crossing the prospective and retrospective databases of the French Network for Rare Peritoneal Malignancies, French Reference Network in Sarcoma Pathology, French Sarcoma Clinical Network and French Pediatric Cancer Society. RESULTS: Among the 107 patients with DSRCT, 48 had no EPM and underwent CCRS. The median peritoneal cancer index (PCI) was 9 (range: 2-27). Among these 48 patients, 38 (79%) had pre- and/or postoperative chemotherapy and 23 (48%) postoperative whole abdominopelvic radiotherapy (WAP-RT). Intraperitoneal chemotherapy was administered to 11 patients (23%): two received early postoperative intraperitoneal chemotherapy (EPIC) and nine HIPEC. After a median follow-up of 30 months, the median overall survival (OS) of the entire cohort was 42 months. The 2-y and 5-y OS were 72% and 19%. The 2-y and 5-y disease-free survival (DFS) were 30% and 12%. WAP-RT was the only variable associated with longer peritoneal recurrence-free survival and DFS after CCRS. The influence of HIPEC/EPIC on OS and DFS was not statistically conclusive. CONCLUSION: The benefit of HIPEC is still unknown and should be evaluated in a prospective trial. The value of postoperative WAP-RT seems to be confirmed.

Simultaneous integrated boost (SIB) of the parametrium and cervix in radiotherapy for uterine cervical carcinoma: a dosimetric study using a new alternative approach.

OBJECTIVE: To compare the dose distributions of intensity-modulated radiotherapy (IMRT) and volumetric-modulated arc therapy (VMAT) using the simultaneous integrated boost (SIB) technique with that of the traditional midline block (MB) technique for boosting the parametrium in patients with cervical cancer. METHODS: Treatment plans using VMAT or IMRT with the SIB technique (VMAT-SIB and IMRT-SIB) and IMRT followed by the MB technique (IMRT-MB) were generated for each of the 10 patients with cervical cancer. For the SIB plans, 45-Gy and 50-Gy dose levels in 25 equal fractions were set for the pelvis planning target volume 45 (PTV45) and the parametrial boost volume (PTV50), respectively. For the IMRT-MB plans, the parametrium was sequentially boosted with the MB technique (5.4 Gy in three fractions) after pelvic IMRT (PTV45). RESULTS: Volume receiving 100% of the prescribed dose or more coverage of the PTV50 was significantly better for VMAT-SIB and IMRT-SIB than that for IMRT-MB (99.08 and 99.31% compared with 91.79%, respectively; p < 0.05). VMAT-SIB and IMRT-SIB both generated significantly greater doses to the organs at risk (OARs) except for the volume receiving 50 Gy or more doses, which were significantly lower for the bladder and bowel. Comparable results were achieved with VMAT-SIB and IMRT-SIB. CONCLUSION: The VMAT-SIB and IMRT-SIB techniques are promising in terms of dose distributions and tumour coverage, although these approaches might result in slightly higher doses of radiation to the OARs. Advances in knowledge: This is the first study to examine the feasibility of the SIB technique using IMRT or VMAT to boost the parametrium. The techniques dosimetrically produced better target coverage but resulted in slightly higher doses to the OARs.

Irradiation Does Not Increase the Penetration Depth of Doxorubicin in Normal Tissue After Pressurized Intra-peritoneal Aerosol Chemotherapy (PIPAC) in an Ex Vivo Model.

AIM: To compare the impact of single fractional with bi-fractional irradiation on the depth of doxorubicin penetration into the normal tissue after pressurized intra-peritoneal aerosol chemotherapy (PIPAC) in our ex vivo model. MATERIALS AND METHODS: Fresh post mortem swine peritoneum was cut into 12 proportional sections. Two control samples were treated with PIPAC only (no irradiation), one sample on day 1, the other on day 2. Five samples were irradiated with 1, 2, 4, 7 or 14 Gy followed by PIPAC. Four samples were treated on day one with 0.5, 1, 2, 3.5 or 7 Gy and with the same radiation dose 24 h later followed by PIPAC. Doxorubicin was aerosolized in an ex vivo PIPAC model at 12 mmHg/36°C. In-tissue doxorubicin penetration was measured using fluorescence microscopy on frozen thin sections. RESULTS: Doxorubicin penetration (DP) after PIPAC for the control samples was 407 µm and 373 µm, respectively. DP for samples with single fraction irradiation was 396 µm after 1 Gy, 384 µm after 2 Gy, 327 µm after 4 Gy, 280 µm after 7 Gy and 243 µm after 14 Gy. DP for samples with 2 fractions of irradiation was 376 µm after 0.5+0.5 Gy, 363 µm after 1+1 Gy, 372 µm after 2+2 Gy, 341 µm after 3.5+3.5 and 301 µm after 7+7 Gy irradiation. Fractionating of the irradiation did not significantly change DP into normal tissue. CONCLUSION: Irradiation does not increase the penetration depth of doxorubicin into the normal tissue but might have a limiting impact on penetration and distribution of doxorubicin. Further studies are warranted to investigate the impact of addition of irradiation to PIPAC of tumor cells and to find out if irradiation can be used safely as chemopotenting agent for patients with peritoneal metastases treated with PIPAC.

Predictors of late bowel toxicity using three different methods of contouring in patients undergoing post-operative radiation for cervical cancer.

OBJECTIVE: This study investigates the correlation between dose-volume histogram derived from three bowel contouring methods and late toxicity in patients undergoing post-operative radiation therapy (PORT) for cervical cancer. METHODS: From June 2010 to May 2013, 103 patients undergoing PORT were included. Three different contouring methods were used: (a) individual small bowel (SB) and large bowel (LB) loops, (b) total bowel (TB; including SB and LB) and (c) peritoneal cavity (PC). The volume of SB, LB, TB and PC receiving 15, 30 and 40 Gy was calculated. Acute and late bowel toxicities were scored using Common Terminology Criteria for Adverse events v. 3.0. Receiver operating characteristic curve identified thresholds predicting late toxicity with the highest specificity. All data were dichotomized across these thresholds. Univariate and multivariate analyses were performed using SPSS(®) v. 20 (IBM Corporation, Armonk, NY; formerly SPSS Inc., Chicago, IL). RESULTS: On univariate analysis, V30 PC ≥ 900 cm(3) (p = 0.01), V40 PC ≥ 750 cm(3) (p = 0.03) and V40 TB ≥ 280 cm(3) (p = 0.03) and use of concurrent chemotherapy (p = 0.03) predicted grade ≥II acute toxicity. On multivariate analysis, use of concurrent chemotherapy [odds ratio (OR) 3.5, 95% confidence interval (CI) 1.1-11.1, p = 0.03] and V30 PC ≥ 900 cm(3) (OR 2.3, 95% CI 1-5.5, p = 0.05) predicted acute grade ≥II toxicity. On univariate analysis for late toxicity, SB (V30 ≥ 190 cm(3), p = 0.009; V40 ≥ 150 cm(3), p = 0.03), LB (V15 ≥ 250 cm(3), p = 0.04), V40 PC (V40 ≥ 750 cm(3), p = 0.001) and presence of acute grade ≥III toxicity (p = 0.006), treatment technique (three-dimensional conformal radiation or intensity modulated radiotherapy, p = 0.02) predicted more than or equal to grade ll late bowel toxicity. On multivariate analysis, only body mass index ≥25 kg m(-2) (OR 7.3, 95% CI 1.6-31.6, p = 0.008) and presence of acute grade III toxicity predicted toxicity (OR 5.1, 95% CI 1.4-18.1, p = 0.007). CONCLUSION: V30 PC ≥ 900 cm(3) and use of concurrent chemotherapy independently predicts acute toxicity. Presence of acute grade ≥III toxicity independently predicts late toxicity. Minimizing dose to PC subvolumes can therefore reduce both acute and late toxicities. ADVANCES IN KNOWLEDGE: Study establishes PC thresholds that can minimize both acute and late bowel toxicities.

Emerging trends for radioimmunotherapy in solid tumors.

Due to its ability to target both known and occult lesions, radioimmunotherapy (RIT) is an attractive therapeutic modality for solid tumors. Poor tumor uptake and undesirable pharmacokinetics, however, have precluded the administration of radioimmunoconjugates at therapeutically relevant doses thereby limiting the clinical utility of RIT. In solid tumors, efficacy of RIT is further compromised by heterogeneities in blood flow, tumor stroma, expression of target antigens and radioresistance. As a result significant efforts have been invested toward developing strategies to overcome these impediments. Further, there is an emerging interest in exploiting short-range, high energy α-particle emitting radionuclides for the eradication of minimal residual and micrometastatic disease. As a result several modalities for localized therapy and models of minimal disease have been developed for preclinical evaluation. This review provides a brief update on the recent efforts toward improving the efficacy of RIT for solid tumors, and development of RIT strategies for minimal disease associated with solid tumors. Further, some of promising approaches to improve tumor targeting, which showed promise in the past, but have now been ignored are also discussed.

Evaluation of effects on the peritoneum after intraperitoneal α-radioimmunotherapy with (211)At.

The introduction of the short-lived α-emitter (211)At to intraperitoneal radioimmunotherapy has raised the issue of the tolerance dose of the peritoneum. The short range of the α-particles (70 µm) and the short half-life (7.21 h) of the nuclide yield a dose distribution in which the peritoneum is highly irradiated compared with other normal tissues. To address this issue, mice were injected with (211)At-trastuzumab to irradiate the peritoneum to absorbed doses ranging between 0 and 50 Gy and followed for up to 34 weeks. The peritoneum-to-plasma clearance of a small tracer, (51)Cr-ethylenediamine tetraacetic acid, was measured for evaluation of the small solute transport capacity of the peritoneal membrane. The macroscopic status of the peritoneum and the mesenteric windows was documented when the mice were sacrificed. Biopsies of the peritoneum were taken for morphology and immunohistochemical staining against plasminogen activator inhibitor-1 and calprotectin. Peritoneum-to-plasma clearance measurements indicated a dose-dependent decrease in peritoneal transport capacity in irradiated mice. However, macroscopic and microscopic evaluations of the peritoneal membrane showed no difference between irradiated mice versus controls. The results imply that the peritoneal membrane tolerates absorbed doses as high as 30-50 Gy from α-particle irradiation with limited response.

Production of cytokines by peritoneal macrophages and splenocytes after exposures of mice to low doses of X-rays.

We have shown previously that irradiations of mice with 0.1 or 0.2 Gy of X-rays stimulate anti-tumour cytotoxic activities of peritoneal macrophages and splenocytes enriched for NK lymphocytes and suppress the development of pulmonary tumour colonies. The up-regulated cytotoxicities were related to the production of nitric oxide by macrophages, and perforin and Fas ligand by the splenocytes, but specific blockade of these pathways did not totally suppress the effector cell-mediated cytolysis of the tumour target. Hence, other factors such as cytotoxic/cytostatic cytokines might have been produced by the effector cells. To test this possibility peritoneal macrophages and splenocytes were isolated from BALB/c mice which had been either once or tentimes whole body-irradiated with the total doses of 0.1 and 0.2 Gy of X-rays and assayed for the levels of IL-1beta, IL-2, IL-12, IFN-gamma and TNF-alpha in the incubation medium using the respective ELISA kits. The results demonstrate that both single and multiple exposures to the two low doses of X-rays significantly stimulate secretion of IL-1beta, TNF-alpha and IL-12 by macrophages and IL-2 and IFN-gamma by splenocytes, but the kinetics and magnitude of the induced changes in the production of these cytokines differ between the two irradiation protocols.

Monte Carlo investigation of single cell beta dosimetry for intraperitoneal radionuclide therapy.

Single event spectra for five beta-emitting radionuclides (Lu-177, Cu-67, Re-186, Re-188, Y-90) were calculated for single cells from two source geometries. The first was a surface-bound isotropically emitting point source and the second was a bath of free radioactivity in which the cell was submerged. Together these represent a targeted intraperitoneal radionuclide therapy. Monoenergetic single event spectra were calculated over an energy range of 11 keV to 2500 keV using the EGSnrc Monte Carlo system. Radionuclide single event spectra were constructed by weighting monoenergetic single event spectra according to radionuclide spectra appropriate for each source geometry. In the case of surface-bound radioactivity, these were radionuclide beta decay spectra. For the free radioactivity, a continuous slowing down approximation spectrum was used that was calculated based on the radionuclide decay spectra. The frequency mean specific energy per event increased as the energy of the beta emitter decreased. This is because, at these energies, the stopping power of the electrons decreases with increasing energy. The free radioactivity produced a higher frequency mean specific energy per event than the corresponding surface-bound value. This was primarily due to the longer mean path length through the target for this geometry. This information differentiates the radionuclides in terms of the physical process of energy deposition and could be of use in the radionuclide selection procedure for this type of therapy.

Impact of intraoperative edema during transperineal permanent prostate brachytherapy on computer-optimized and preimplant planning techniques.

The purpose of this study was to prospectively evaluate intraoperative prostatic edema during prostate brachytherapy with real-time ultrasound imaging and assess its impact upon the postimplant dosimetry of computer-optimized intraoperatively planned patients. Fifty consecutive patients with early-stage favorable risk adenocarcinoma of the prostate underwent transperineal ultrasound-guided I125 brachytherapy. Ultrasound volume studies of the prostate were performed immediately before and after placement of brachytherapy needles in the operating room. Twenty-five patients underwent intraoperative computer-optimized treatment planning using a genetic algorithm. Twenty-five patients underwent preimplant ultrasound studies for preimplant treatment planning. Postimplant dosimetry was performed on computed tomography scans obtained after the implant. Statistical analysis was performed taking into account patient age, preneedle volume, increase in intraoperative edema, use of hormonal therapy, type of isotope, number of needles or seeds used, and seed activity. For the intraoperatively planned patients, a median increase of 30% in intraoperative volume was found for the entire group. No correlation between the extent of intraoperative edema and %D90 (percentage of prescribed dose that covers 90% of the target volume) was found. None of the other analyzed variables correlated with %D90. Patients whose treatment was planned preoperatively experienced a median increase of 18.4% in target volume. A negative correlation between the amount of edema and the %D90 was found to be statistically significant (-0.55, P = 0.0047). All patients who underwent prostate brachytherapy experienced intraoperative prostatic edema. When planned intraoperatively, the amount of edema had no impact on the %D90. This may be because of the ability of intraoperative computer-optimized treatment planning to account for edema related to the procedure. Preplanned patients who encountered a greater degree of intraoperative edema had less %D90 target coverage.

Peritoneal B-cell development depends on strain, radiation, and time.

OBJECTIVE: B-1a, B-1b, and B-2 cells represent the three B-cell subsets in mice. Previous studies have demonstrated that peritoneal B-1a cell development is absent, or nearly so, from adult bone marrow transfers into irradiated adult hosts. The majority of these studies have been performed under a limited set of conditions with irradiated host mice. Here we examined that under a variety of conditions, peritoneal B-1a cells can develop in significant numbers from adult bone marrow transfers into severe combined immunodeficient (SCID) and recombination activation gene 2(-) (RAG-2(-)) mice. MATERIALS AND METHODS: Adult bone marrow was transferred into various strains of irradiated and nonirradiated adult immunodeficient RAG-2(-) and SCID mice. Peritoneal B-cell engraftment was examined by fluorescein-activated cell sorting analysis and unpaired t-tests were used to determine significant differences of B-cell engraftment among the various conditions of cell transfer. RESULTS: The level of B-1a cell engraftment was variously affected by the type of host immunodeficiency, the combination of donor and host strains, and the time allowed for engraftment. Irradiation of SCID, but not RAG-2(-), host mice inhibited B-1a-cell engraftment. Additionally, decreasing the number of bone marrow progenitor cells transferred was not found to preferentially affect B-1a cell development in irradiated RAG-2(-) hosts. CONCLUSION: In the context of these strains, we conclude that adult murine bone marrow contains progenitors that have the capacity to reconstitute peritoneal B-1a cell populations to donor levels.

Effect of radiotherapy on peritoneal function in continuous ambulatory peritoneal dialysis.

A 25-year-old male patient underwent abdominal radiotherapy following the discovery of lymphadenopathy associated with a testicular teratoma. Prior to treatment, his peritoneal mass transfer area coefficients were within normal limits. One and 2 months after treatment, peritoneal permeability had increased to the extent that continuous ambulatory peritoneal dialysis was rendered impractical.

Intracavitary therapy with radioactive yttrium: results of animal experiments.

The distribution of radioactive yttrium was studied in guinea-pigs. The injection of 1,5 mci of radioactive yttrium leads to a mean surface dose of 20 Gy which can be obtained within 5 days. An extrapolation of these findings to the peritoneal surface of human beings should lead to a corresponding dose of 64 mci.

Efeito da radiaçäo de Co**60 em células peritoneais / Effect of Co**60 radiation on peritoneal cells

Este trabalho mostra como o efeito da irradiaçäo gama de 60Co modifica o nível das células peritoneais de camundongos albinos. As células foram obtidas de exudato peritoneal, fixadas e estocadas em ácido acético glacial a 30% contendo 0,05% de cristal violeta. Nos exudatos dos animais testemunhas e irradiados foram feitas análises e contagem dos diferentes tipos celulares nos tempos 1 hora, 3 dias e 6 dias após irradiaçäo com 9,0 Gy. Todas as células da populaçäo do exudato peritoneal mostram uma diminuiçäo 3 dias após a irradiaçäo com 9,0 Gy mas as diferentes populaçöes celulares decrescem em proporçöes desiguais. Os dados reafirmam a discrepância na radiossensibilidade das diversas células peritoneais

Intraperitoneal 131I- and 90Y-labelled monoclonal antibodies for ovarian cancer: pharmacokinetics and normal tissue dosimetry.

The pharmacokinetics of intraperitoneal (i.p.) radiolabelled monoclonal antibody (MAb) was studied in 35 patients receiving 40 i.p. injections. Eleven patients received 131I-labelled MAb, 24 received 90Y-labelled MAb, and 5 patients received a second 131I MAb treatment after having developed human anti-mouse antibodies (HAMA). All patients had blood and urine isotope activity monitored for 5 days after MAb injection. The radiation dose to bone marrow from the vascular compartment in the marrow was calculated by applying the MIRD formula to the measured blood activity. In HAMA-negative patients, peak blood isotope activity was observed at 40 hr post injection with a mean of 26% and 21% of the injected 131I and 90Y activity respectively. Sixty-five percent of the injected 131I activity, but only 12% of the administered 90Y, was excreted in the urine. Myelosuppression limited the administered 131I and 90Y activities to below 160 and 20 mCi respectively. In patients receiving 131I labelled MAbs, the marrow is irradiated by MAb within its circulation, producing myelosuppression that can be predicted by applying the MIRD formula to the blood isotope activity. This is not true for 90Y-labelled MAbs, where bone absorption of yttrium (which cannot be measured in patients) is the dominant radiation source for bone-marrow irradiation. Patients with HAMA present clear 131I MAb rapidly with a decreased radiation dose to marrow and reduced myelosuppression. Giving patients intravenous antimouse immunoglobulin to clear 131I-labelled MAb absorbed from the peritoneal cavity could decrease the toxicity observed in these patients. Patients receiving 90Y DTPA-chelated MAbs are unlikely to benefit, as catabolized yttrium is not excreted, and is concentrated in liver, spleen and bone. On the other hand, the use of i.v. chelating agents as EDTA may scavenge non-protein-bound 90Y with increased excretion in the urine and less myelosuppression.