DNA methylation profile of genes involved in inflammation and autoimmunity correlates with vascular function in morbidly obese adults.

Obesity is a major risk factor for cardiovascular disease. Blood-detected epigenetic profiles may serve as non-invasive clinically relevant biomarkers. Therefore, we investigated DNA methylation of genes involved in inflammation in peripheral blood of obese subjects and lean controls and their correlation with cardiometabolic measurements. We obtained blood and adipose tissue (AT) samples from bariatric patients (n = 24) and control adults (n = 24). AT-isolated arterioles were tested for flow-induced dilation (FID) and production of nitric oxide (NO) and reactive oxygen species (ROS). Brachial artery flow-mediated dilation (FMD) was measured via doppler ultrasound. Promoter methylation of 94 genes involved in inflammation and autoimmunity were analysed in whole-blood DNA in relation to vascular function and cardiometabolic risk factors. 77 genes had ahigher methylated fraction in the controls compare obese subjects and 28 proinflammatory genes were significantly hypomethylated in the obese individuals; on top of these genes are CXCL1, CXCL12, CXCL6, IGF2BP2, HDAC4, IL12A, and IL17RA. Fifteen of these genes had significantly higher mRNA in obese subjects compared to controls; on top of these genes are CXCL6, TLR5, IL6ST, EGR1, IL15RA, and HDAC4. Methylation % inversely correlated with BMI, total fat %, visceral fat%, blood pressure, fasting plasma insulin, serum IL6 and C-reactive protein, arteriolar ROS, and alcohol consumption and positive correlations with lean %, HDL, plasma folate and vitamin B12, arteriolar FID and NO production, and brachial FMD. Our results suggest that vascular dysfunction in obese adults may be attributed to asystemic hypomethylation and over expression of the immune-related genes.

Robotic transabdominal kidney transplantation in a morbidly obese patient.

Kidney transplantation in morbidly obese patients can be technically demanding. Furthermore, morbidly obese patients experience a high rate of wound infections and related complications, which mostly result from the longer length and extent of the incision. These complications can be avoided through minimally invasive surgery; however, conventional laparoscopic instruments are unsuitable for the safe performance of a kidney transplant in morbidly obese patients. Herein, we report the first minimally invasive, total robotic kidney transplant in a morbidly obese patient. A left, deceased donor kidney was transplanted into a 29-year-old woman with a body mass index (BMI) of 41 kg/m(2) who had been on hemodialysis for 5 years. The operation was performed intraabdominally using the DaVinci Robotic Surgical System with 4 trocars and a 7 cm midline incision. The operative time was 223 min, and the blood loss was less than 50 cc. The kidney had immediate graft function. No perioperative complications were observed, and the patient was discharged on postoperative day 5 with normal kidney function. Minimally invasive access and robotic technology facilitated the safe performance of a successful kidney transplant in a morbidly obese patient.

Update on laparoscopic pancreatectomy in 2010.

Minimally invasive surgery has been proven to be a safe and effective method of surgically managing several gastrointestinal conditions. In the last ten years, increased expertise in laparoscopic surgery and the availability of new surgical devices have contributed to the development of laparoscopic pancreatic surgery. Currently, distal pancreatectomies for benign/low-grade malignant tumors represent the majority of pancreatic resections performed laparoscopically. They are characterized by improved postoperative short-term outcomes compared to open surgery. Pancreaticoduodenectomy still represents a formidable technical challenge for laparoscopy. However, laparoscopic pancreaticoduodenectomy has been proven to be safe and feasible with outcomes comparable to those of open surgery if performed at experienced centers. Robotic surgery, recently introduced in the field of minimally invasive surgery, improves the view and the maneuverability of the instruments compared to standard laparoscopic surgery. The feasibility and safety of robotic pancreatectomy have been recently reported for complex pancreatic resection. This approach has the potential to bridge the gap between minimally invasive surgery and complex pancreatic surgery, allowing the indications for minimally invasive pancreatic surgery to be extended. Almost 15 years after its description, laparoscopic pancreatic surgery is seeing an exponential growth in its applications. The growing experience in laparoscopy and the introduction of robotics will further expand the field of minimally invasive pancreatic surgery in the next several years.

Hyperhomocysteinemia and Low Folate and Vitamin B12 Are Associated with Vascular Dysfunction and Impaired Nitric Oxide Sensitivity in Morbidly Obese Patients.

There is a high prevalence of hyperhomocysteinemia that has been linked to high cardiovascular risk in obese individuals and could be attributed to poor nutritional status of folate and vitamin B12. We sought to examine the association between blood homocysteine (Hcy) folate, and vitamin B12 levels and vascular dysfunction in morbidly obese adults using novel ex vivo flow-induced dilation (FID) measurements of isolated adipose tissue arterioles. Brachial artery flow-mediated dilation (FMD) was also measured. Subcutaneous and visceral adipose tissue biopsies were obtained from morbidly obese individuals and non-obese controls. Resistance arterioles were isolated in which FID, acetylcholine-induced dilation (AChID), and nitric oxide (NO) production were measured in the absence or presence of the NO synthase inhibitor, L-NAME, Hcy, or the superoxide dismutase mimetic, TEMPOL. Our results demonstrated that plasma Hcy concentrations were significantly higher, while folate, vitamin B12, and NO were significantly lower in obese subjects compared to controls. Hcy concentrations correlated positively with BMI, fat %, and insulin levels but not with folate or vitamin B12. Brachial and arteriolar vasodilation were lower in obese subjects, positively correlated with folate and vitamin B12, and inversely correlated with Hcy. Arteriolar NO measurements and sensitivity to L-NAME were lower in obese subjects compared to controls. Finally, Hcy incubation reduced arteriolar FID and NO sensitivity, an effect that was abolished by TEMPOL. In conclusion, these data suggest that high concentrations of plasma Hcy and low concentrations of folate and vitamin B12 could be independent predictors of vascular dysfunction in morbidly obese individuals.

Could ICG-aided robotic cholecystectomy reduce the rate of open conversion reported with laparoscopic approach? A head to head comparison of the largest single institution studies.

Comparative studies between robotic and laparoscopic cholecystectomy (LC) focus heavily on economic considerations under the assumption of comparable clinical outcomes. Advancement of the robotic technique and the further widespread use of this approach suggest a need for newer comparison studies. 676 ICG-aided robotic cholecystectomies (ICG-aided RC) performed at the University of Illinois at Chicago (UIC) Division of General, Minimally Invasive and Robotic Surgery were compiled retrospectively. Additionally, 289 LC were similarly obtained. Data were compared to the largest single institution LC data sets from within the US and abroad. Statistically significant variations were found between UIC-RC and UIC-LC in minor biliary injuries (p = 0.049), overall open conversion (p ≤ 0.001), open conversion in the acute setting (p = 0.002), and mean blood loss (p < 0.001). UIC-RC open conversions were also significantly lower than Greenville Health System LC (p ≤ 0.001). Additionally, UIC ICG-RC resulted in the lowest percentages of major biliary injuries (0 %) and highest percentage of biliary anomalies identified (2.07 %). ICG-aided cholangiography and the technical advantages associated with the robotic platform may significantly decrease the rate of open conversion in both the acute and non-acute setting. The sample size discrepancy and the non-randomized nature of our study do not allow for drawing definitive conclusions.

Robotic approach to hepatobiliary surgery.

Robot-assisted hepatobiliary surgery has been steadily growing in recent years. It represents an alternative to the open and laparoscopic approaches in selected patients. Endowristed instruments and enhanced visualization provide important advantages in terms of selective bleeding control, microsuturing, and dissection. Cholecystectomies and minor hepatectomies are being performed with comparable results to open and laparoscopic surgery. Even complex procedures, such as major and extended hepatectomies, can have excellent outcomes, in expert hands. The addition of indocyanine green fluorescence provides an additional advantage for recognition of the vascular and biliary anatomy. Future innovations will allow for expanding its use and indications. Robotic surgery has become a very important component of modern minimally invasive surgery and the development of new robotic technology will facilitate a broader adoption of this technique.

Initial tumor targeting, biodistribution, and pharmacokinetic evaluation of the monoclonal antibody PAM4 in patients with pancreatic cancer.

This pharmacokinetic study was performed to assess the potential usefulness of the murine monoclonal antibody (MoAb) PAM4-IgG1 as an immunotargeting agent for pancreatic cancer imaging or therapy. This MoAb reacts specifically with mucin purified from human pancreatic cancer. 131I-labeled PAM4-IgG1 was injected i.v. into five patients with suspected pancreatic cancer. Whole-body scans and spot views of the abdominal area were recorded with a computerized gamma camera, and specific regions of interest were drawn over the liver and spleen to define the kinetics of activity in these organs. Blood samples taken from 0.1-144 h after injection served to define the kinetics of plasma distribution and removal of activity from the body. Surgery confirmed pancreatic cancer in four of the five patients, whereas chronic pancreatitis was present in the fifth patient; in all four pancreatic cancer patients, immunostaining with the MoAb PAM4 demonstrated the presence of the specific antigen, with a cytoplasmic and endoluminal/secretory pattern of distribution. Nonspecific radioactivity accumulation in the liver, spleen, and bone marrow was low, linked essentially to the blood pool effect of circulating activity in these organs. The overall quality of scintigraphic maps recorded over the abdomen was quite satisfactory due to the low liver and spleen activity, with good scintigraphic demonstration of the pancreatic cancers (either primary or metastatic); the patient subsequently found to have pancreatitis failed to show PAM4 targeting. Except in one patient with widespread peritoneal metastases (in whom these tumor implants were detected scintigraphically already 24-48 hours after tracer injection), scintigraphic evidence of the tumor lesions was usually late, starting at about 72-96 h after tracer injection. The results obtained in this preliminary study indicate the potential usefulness of MoAb PAM4 for immunoscintigraphy in patients with either primary and/or recurrent pancreatic cancer while also suggesting that the use of the faster-clearing Fab fragments of this MoAb probably would result in improved immunoscintigraphic properties.

De novo gastric adenocarcinoma 1 year after sleeve gastrectomy in a transplant patient.

INTRODUCTION: It has been reported in the literature that upper gastrointestinal malignancies after bariatric surgery are mostly gastro-esophageal, although it is not clear whether bariatric surgery represents a risk factor for the development of esophageal and/or gastric cancer. We report a case of a de novo gastric adenocarcinoma occurring in a transplant patient 1 year after a laparoscopic sleeve gastrectomy. PRESENTATION OF CASE: A 44 year-old woman with a BMI of 38kg/m(2), hypertension, type 1 diabetes mellitus, multiple malignancies and a pancreas transplant underwent laparoscopic sleeve gastrectomy. The patient presented with intense dysphagias during the follow up. Studies were performed and the diagnoses of grade 2/3 adenocarcinoma were made. The patient underwent a robotic assisted total gastrectomy with a roux-en-y intracorporeal esophagojejunostomy. The procedure resulted in multiple metastasic lymph nodes, focal and transmural invasions to multiple organs with a tumor free margin resection. The patient presented with a postoperative pleural effusion, with no further complications. DISCUSSION: The diagnosis of gastroesophageal cancer after bariatric surgery is usually late since these patients have common upper gastrointestinal symptoms related to the procedure that could delay the diagnosis. De novo gastric cancer after sleeve gastrectomy has only been reported in one instance, in contrast with other bariatric surgery procedures. CONCLUSIONS: No direct relation has been established between sleeve gastrectomy and the development of gastric cancer. Robotic procedures allow for complex multiorgan resections, while preserving the benefits of minimally invasive surgery.

[Robotic approach to hepatobiliary surgery. German version]. / Roboterassistierte hepatobiliäre Chirurgie.

Robot-assisted hepatobiliary surgery has been steadily growing in recent years. It represents an alternative to the open and laparoscopic approaches in selected patients. Endowristed instruments and enhanced visualization provide important advantages in terms of selective bleeding control, microsuturing, and dissection. Cholecystectomies and minor hepatectomies are being performed with comparable results to open and laparoscopic surgery. Even complex procedures, such as major and extended hepatectomies, can have excellent outcomes, in expert hands. The addition of indocyanine green fluorescence provides an additional advantage for recognition of the vascular and biliary anatomy. Future innovations will allow for expanding its use and indications. Robotic surgery has become a very important component of modern minimally invasive surgery and the development of new robotic technology will facilitate a broader adoption of this technique.

Robot-assisted surgery for gastric carcinoma: Five years follow-up and beyond: A single western center experience and long-term oncological outcomes.

INTRODUCTION: Robot-assisted surgery for the treatment of gastric cancer is considered to be safe and feasible with early post-operative outcomes comparable to open and laparoscopic series. However, data regarding long-term oncological outcomes are lacking. Aim of this study is to evaluate long-term oncological outcomes of a cohort of gastric cancer patients treated surgically with the robot-assisted approach. MATERIALS AND METHODS: A prospectively collected database of robot-assisted gastrectomies performed for gastric cancer at the 'Misericordia Hospital' between September 2001 and October 2011 was retrospectively analysed. Data regarding surgical procedures, early postoperative course, and long-term follow-up were analysed. RESULTS: The study included 98 consecutive robot-assisted gastrectomies. Fifty-nine distal gastrectomies, 38 total gastrectomies, and 1 proximal gastrectomy. Open conversion occurred in seven patients (7.1%) due to locally advanced disease. Postoperative morbidity and mortality were 12.2% and 4.1% respectively. Post-operative staging showed 46 patients (46.9%) with stage I disease, 25 patients (25.5%) with stage II, 26 (26.5%) with stage III and 1 (1.02%) with stage IV. The mean follow-up was 46.9 months. Cumulative 5-year overall survival (OS) was 73.3% (95% CI: 62.2-84.4). Five-year survival by stage subgroups was 100% for patients with stage IA, 84.6% for stage IB, 76.9% for stage II, and 21.5% for stage III. The only patient in stage IV of this series died eight months after surgery. CONCLUSIONS: Robot-assisted gastrectomy for the treatment of gastric cancer is safe and feasible. It provides long-term outcomes comparable to most open and laparoscopic series. Further studies are necessary to better define its indication.

Long-term survival in pancreatic cancer: pylorus-preserving versus Whipple pancreatoduodenectomy.

BACKGROUND: This study compared long-term survival in pancreatic or periampullary cancer treated with Whipple pancreatoduodenectomy (PD) and pylorus-preserving pancreatoduodenectomy (PPPD). METHODS: Two hundred twenty-one patients with pancreatic head or periampullary cancer were treated. Prognostic variables included age, gender, type and period of operation, and tumor stage. In the ductal adenocarcinomas variables also included tumor and node status, type of lymphadenectomy, pathologic grade, and presence of microscopic residual tumor. The end point was death as a result of neoplastic recurrence. Survival curves were estimated by using the Kaplan-Meier method, and multifactorial analysis was also performed on the data from the ductal adenocarcinoma group. RESULTS: The mortality rate was 8.2% in the PD group versus 7.0% in the PPPD group. Morbidity rates were 34.4% for PD and 45.8% for PPPD. Five-year survival was 9.6% in the ductal adenocarcinoma and 63.8% in the periampullary carcinoma groups. Univariate analysis failed to show statistically significant differences in survival curves between the two treatments in either patient group. Correcting for multiple variables in the ductal adenocarcinoma group did not reveal any significant differences in survival rates between the two treatments. CONCLUSIONS: PPPD was as successful as classic PD in the treatment of ductal adenocarcinoma and periampullary cancer of the pancreas. Long-term survival was not influenced by the type of resection.

Comparative evaluation of contact ultrasonography and transcystic cholangiography during laparoscopic cholecystectomy: a prospective study.

BACKGROUND: The role of intraoperative cholangiography (IOC) during laparoscopic cholecystectomy (LC) is controversial. While many advocate its routine use, others argue for a selective approach. Recent reports showed laparoscopic contact ultrasonography (LCU) as a viable alternative to IOC. However, no prospective data were available to compare the accuracy, efficacy, and safety of the two diagnostic procedures. OBJECTIVE: To evaluate the benefits and disadvantages of LCU and IOC during LC. METHODS: Seventy-eight patients who underwent LC at Pisa (Italy) and Dundee (Scotland) university hospitals were entered in a prospective data registry. Details of operative technique and results of LCU and IOC were analyzed by reviewing videotape recordings of each procedure. RESULTS: Laparoscopic cholecystectomy was achieved in 73 patients, with five requiring conversion to the open procedure. The success rate of IOC was 90% (64/71). Performance of IOC demanded more than twice the time needed for LCU. Eleven percent (8/71) of cholangiograms were abnormal, with a false-positive rate of 1% (1/71). Laparoscopic contact ultrasonography detected all four instances of unsuspected ductal stones but none of the three cases of anomalous biliary anatomy. Clinically relevant incidental findings were picked up by LCU in six patients. CONCLUSIONS: Laparoscopic contact ultrasonography proved to be extremely accurate in the detection of ductal stones but less reliable in the disclosure of anomalous biliary anatomy. The essential role of IOC in providing a clear spatial display of the biliary tract was confirmed. Since the two procedures are complementary, their combined use is advisable in difficult LC to avoid retained common bile duct stones and prevent iatrogenic complications.

Microsatellite alterations and K-ras, TGFbetaRII, IGFRII and bax mutations in sporadic cancers of the gastrointestinal tract.

DNA was analyzed from 57 sporadic gastrointestinal tumors (34 pancreatic cancers, 23 colon tumors) and cognate normal tissues to verify whether mutations at coding sequences were associated with microsatellite instability (MSI). Genomic instability was present in 41% (14/34) of pancreatic samples and in 26% (6/23) of colon cancers previously tested by six microsatellite markers. The tumors included 37 cases showing no MSI; 15 cases with MSI at only 1 locus and 5 cases with MSI at 2 or more loci. All the samples were screened for mutations in genes containing repeated tracts in their coding sequences (TGFbetaRII, IGFRII and bax) and in codon 12 of the K-ras oncogene. Furthermore, loss of heterozygosity (LOH) at NM23.H1 locus was tested, 17/34 (50%) pancreatic tumors and 6/23 (26%) colon cancers showed mutations in codon 12 of K-ras; allelic loss of NM23. H1 locus was found in 6/18 (33%) informative colon tumors and in no pancreatic cancers. The TGFbetaRII, IGFRII and bax genes were altered in 3 (13%), 1 (4%) and 3 (13%) out of 23 colon tumors respectively, but no mutation was detected in pancreatic cancers. Mutations in the repeated nucleotide stretches within the coding sequences of TGFbetaRII, IGFRII and bax genes were found only in colon tumors with a high unstable phenotype (more than 3 microsatellite loci altered).

Glutathione conjugation with 1-chloro-2,4-dinitrobenzene (CDNB): interindividual variability in human liver, lung, kidney and intestine.

The rate of glutathione conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) was measured in specimens of human liver (n = 93), sigmoid colon (n = 56), renal cortex (n = 67) and lung (n = 68). In the liver there was a weak but significant (r = - 0.247 p = 0.017) negative correlation between the activity of glutathione transferase and the liver donor's age. Such a correlation was not found in the renal cortex, lung and colon. In the renal cortex and in lung the rate of glutathione conjugation with CDNB was a little but significantly (p < 0.05) higher in women than men, whereas no sex-dependent difference was observed in the liver and colon. The distribution of glutathione transferase activity was polymorphic in the mucosa of colon and renal cortex of men but not in that of women. Smoking seems not to affect the glutathione conjugation rate with CDNB in lung. The activity of glutathione transferase was 2-, 6-, and 7-fold greater in liver than in the renal cortex, lung and colon, respectively. There was a large interindividual variability of the hepatic glutathione transferase activity, and because this variability, 15% of the population studied catalyzed the glutathione conjugation with CDNB at a rate similar to those of the renal cortex and duodenum. The subjects with low expression of the hepatic glutathione transferase should be more exposed to the effects of toxic and carcinogenic compounds.

Surviving catastrophic gastrointestinal involvement due to Churg-Strauss syndrome: report of a case.

Gastrointestinal (GI) involvement is not rarely encountered in Churg-Strauss syndrome (CSS). We describe the case of a young white woman presenting with acute acalculous cholecystitis, who subsequently developed, despite prompt administration of immunosuppressive therapy, life threatening GI involvement, requiring multiple operations. Over a 7-month period the patient eventually recovered from her disease. Forty-two months later she is free from symptoms, with low daily doses (6 mg) of oral methylprednisolone. Although medical and surgical complications of CSS may develop while the patient is undergoing therapy, early diagnosis with joint surgical and medical management is essential to bring the disease under control.

Giant pedunculated liposarcomas of the esophagus: literature review and case report.

Although liposarcoma is the second most common soft-tissue sarcoma in adults, its incidence within the gastrointestinal tract is distinctly low. Esophageal involvement is exceedingly rare and only four cases have been described so far. A fifth case is presented here along with a thorough review of the literature of polypoid lipomatous tumors of the esophagus. Diagnostic and therapeutical strategies of these tumors are discussed in detail.

Intrahepatic chemotherapy with floxuridine, leucovorin and dexamethasone in continuous infusion and mitomycin-C bolus in unresectable hepatic metastases from colorectal cancer: a phase II study.

BACKGROUND: Intrahepatic continuous infusion FUDR induces a 50% response rate in patients with hepatic metastases from colorectal cancer. Lower rates have been observed in pretreated patients. The combination of floxuridine plus leucovorin has obtained over 70% responses, with high hepatic toxicity. The use of dexamethasone can decrease hepatic toxicity. A randomized study reported an increase in response rate and a decrease in hepatic toxicity in a group of patients treated with floxuridine plus dexamethasone compared to a group receiving only floxuridine. Moreover, the combination of mitomycin C, carmustine and floxuridine is also effective in pretreated patients. METHODS: On such premises, since July 1993 we have treated 39 patients affected by unresectable hepatic metastases from colon carcinoma (26 patients) and rectal carcinoma (13 patients) with the combination continuous infusion of floxuridine (0.20 mg/kg per day) + leucovorin (7.5 mg/m2/day) + dexamethasone (20 mg on days 1 to 14) and bolus mitomycin C (10 mg/m2 on day 1) via the hepatic artery. Cycles were administered every four weeks. There were as 28 males and 11 females, with a median age of 64 years (range, 39-75) and a median PS = 0. Twenty-two patients were pretreated with systemic chemotherapy including 5-fluorouracil plus leucovorin. Total number of cycles was 189, with a median of 6 cycles per patient (range, 1-12). RESULTS: Of 39 patients 37 were assessable for response (2 patients were not assessable because they stopped chemotherapy for occlusion of the catheter after the first cycle). There were 3 complete responses (1 in a naive patient and 2 in pretreated patients), 16 partial responses (11 in pretreated patients and 5 in chemonaive patients), 4 minor responses, 4 stable disease and 10 progressive disease. The overall response rate was 51.3% (95 Cl, 51.3-86.7%). Median time to progression was 6 months (range, 1-34+). Overall survival was 18 months (range, 1-34+). Of 39 patients, 36 were assessable for toxicity (WHO) (3 patients died after the first cycle for progression of disease): diarrhea and nausea-vomiting grade 3-4 occurred respectively in 15 (41%) and 3 patients (8%); hepatic toxicity was mild. CONCLUSIONS: The treatment we used showed an elevated activity in liver metastases from colorectal cancer even in patients pretreated and resistant to systemic chemotherapy, although toxicity grade 3-4 diarrhea occurred in approximately 40% of the patients.

Postoperative adjuvant chemoradiotherapy for rectal cancer: analysis of acute and chronic toxicity.

AIMS AND BACKGROUND: The aim of the study was to evaluate acute and chronic toxicity of combined postoperative standard radiation therapy to the pelvis and 5-fluorouracil plus levamisole in resectable rectal cancer. METHODS: Between July 1990 and September 1993, 58 patients with histologically confirmed adenocarcinoma of the rectum entered the prospective study. The schedule consisted of 5-fluorouracil, 450 mg/m2 i.v. for 5 days, and from day 28 5-fluorouracil, 450 mg/m2 i.v. weekly for 24 weeks, plus levamisole given orally at the dose of 150 mg every day for 3 days every 2 weeks for 6 months; radiotherapy (180 cGy/day) 5 days a week for a total dose of 45 Gy was administered from day 28. RESULTS: After the first cycle of chemotherapy (before radiotherapy), overall toxicity was mild. During chemoradiotherapy, dose-limiting toxicity was grade 3 diarrhea and proctitis, for which the combined treatment was interrupted for more than 7 cumulative days in 28 patients. During the 24 weeks of weekly 5-fluorouracil (after radiotherapy), no severe toxicity was reported. Three-year survival and progression-free survival were 65% and 50-55%, respectively. CONCLUSIONS: Although adjuvant chemoradiotherapy is usually feasible, in our study toxicity was severe in a substantial proportion of patients, probably due to the schedule applied. We are evaluating the feasibility and toxicity of a combined treatment which includes 5-fluorouracil in continuous chronomodulated infusion during radiotherapy.