Loss to follow-up and mortality among HIV-infected adolescents receiving antiretroviral therapy in Pune, India.

OBJECTIVES: India has the highest number of HIV-infected adolescents in Asia, but little is known about their treatment outcomes. We assessed rates and factors associated with loss to follow-up (LTFU) and mortality among Indian adolescents. METHODS: The analysis included adolescents (10-19 years old) starting antiretroviral therapy (ART) between 2005 and 2014 at BJ Government Medical College, Pune, India. LTFU was defined as missing more than three consecutive monthly visits. The competing-risks method was used to calculate subdistribution hazard ratios (SHRs) of predictors for LTFU, with death as the competing risk. Cox proportional hazard models were used to identify predictors of mortality. RESULTS: Of 717 adolescents starting ART, 402 with complete data were included in the analysis. Of these, 61% were male and 80% were perinatally infected, and the median baseline CD4 count was 174 cells/µL. LTFU and mortality rates were 4.4 and 4.9/100-person years, respectively. Cumulative LTFU incidence increased from 6% to 15% over 6 years. Age ≥ 15 years [adjusted SHR (aSHR) 2.44; 95% confidence interval (CI) 1.18-5.02] was a risk factor for LTFU. Cumulative mortality increased from 9.5% to 17.9% over 6 years. World Health Organization (WHO) stages III and IV [adjusted hazard ratio (aHR) 2.26; 95% CI: 1.14-4.48] and an increase in CD4 count by 100 cells/µL (aHR: 0.59; 95% CI: 0.43-0.83) were associated with mortality. CONCLUSIONS: A third of adolescents had been lost to follow-up or died by follow-up year 6. Older age was a risk factor for LTFU and advanced clinical disease for death. Strategies to improve retention counselling for older adolescents and closer clinical monitoring of all adolescents must be considered.

Concomitant pulmonary disease is common among patients with extrapulmonary TB.

BACKGROUND: Microbiologic screening of extrapulmonary TB (EPTB) patients could inform recommendations for aerosol precautions and close contact prophylaxis. However, this is currently not routinely recommended in India. Therefore, we estimated the proportion of Indian patients with EPTB with microbiologic evidence of pulmonary TB (PTB).METHODS: We characterized baseline clinical, radiological and sputum microbiologic data of 885 adult and pediatric TB patients in Chennai and Pune, India, between March 2014 and November 2018.RESULTS: Of 277 patients with EPTB, enhanced screening led to the identification of 124 (45%) with concomitant PTB, including 53 (19%) who reported a cough >2 weeks; 158 (63%) had an abnormal CXR and 51 (19%) had a positive sputum for TB. Of 70 participants with a normal CXR and without any cough, 14 (20%) had a positive sputum for TB. Overall, the incremental yield of enhanced screening of patients with EPTB to identify concomitant PTB disease was 14% (95% CI 12-16).CONCLUSIONS: A high proportion of patients classified as EPTB in India have concomitant PTB. Our results support the need for improved symptom and CXR screening, and recommends routine sputum TB microbiology screening of all Indian patients with EPTB.

Unhealthy alcohol use independently associated with unfavorable TB treatment outcomes among Indian men.

BACKGROUND: Approximately 10% of incident TB cases worldwide are attributable to alcohol. However, evidence associating alcohol with unfavorable TB treatment outcomes is weak.METHODS: We prospectively evaluated men (≥18 years) with pulmonary TB in India for up to 24 months to investigate the association between alcohol use and treatment outcomes. Unhealthy alcohol use was defined as a score of ≥4 on the Alcohol Use Disorders Identification Test-Concise (AUDIT-C) scale at entry. Unfavorable TB treatment outcomes included failure, recurrence, and all-cause mortality, analyzed as composite and independent endpoints.RESULTS: Among 751 men, we identified unhealthy alcohol use in 302 (40%). Median age was 39 years (IQR 28-50); 415 (55%) were underweight (defined as a body mass index [BMI] <18.5 kg/m²); and 198 (26%) experienced an unfavorable outcome. Unhealthy alcohol use was an independent risk factor for the composite unfavorable outcome (adjusted incidence rate ratio [aIRR] 1.47, 95% CI 1.05-2.06; P = 0.03) and death (aIRR 1.90, 95% CI 1.08-3.34; P = 0.03), specifically. We found significant interaction between AUDIT-C and BMI; underweight men with unhealthy alcohol use had increased risk of unfavorable outcomes (aIRR 2.22, 95% CI 1.44-3.44; P < 0.001) compared to men with BMI ≥18.5 kg/m² and AUDIT-C <4.CONCLUSION: Unhealthy alcohol use was independently associated with unfavorable TB treatment outcomes, highlighting the need for integrating effective alcohol interventions into TB care.

Delays and barriers to early treatment initiation for childhood tuberculosis in India.

BACKGROUND: India accounts for 27% of global childhood tuberculosis (TB) burden. Understanding barriers to early diagnosis and treatment in children may improve care and outcomes.METHODS: A cross-sectional study was performed among 89 children initiated on anti-TB treatment from a public hospital in Pune during 2016, using a structured questionnaire and hospital records. Health care providers (HCPs) were defined as medical personnel consulted about the child's TB symptoms. Time-to-treatment initiation (TTI) was defined as the number of days between onset of TB symptoms and anti-TB treatment initiation. Based on Revised National TB Control Programme recommendations, delayed TTI was defined as >28 days.RESULTS: Sixty-seven (75%) of 89 enrolled children had significant TTI delays (median 51 days, interquartile range [IQR] 27-86). Sixty-six (74%) children visited 1-8 HCPs in the private sector before approaching the public sector. The median HCP delay was 28 days (IQR 10-75). Bacille Calmette-Guérin vaccination (aOR 10.96, P = 0.04) and loss of appetite (aOR 4.44, P = 0.04) were associated with delayed TTI.CONCLUSION: The majority of the children had TTI delays due to delays by HCPs in the private sector. Strengthening HCP competency in TB symptom screening and encouraging early referrals are crucial for rapid scaling up of early treatment initiation in childhood TB.

Barriers to screening and isoniazid preventive therapy for child contacts of tuberculosis patients.

BACKGROUND: India's guidelines recommend tuberculosis (TB) screening of household contacts aged <6 years and isoniazid preventive therapy (IPT) for children without active disease. We evaluated the current status and barriers to screening and IPT provision among the child contacts of TB patients. METHODS: Questionnaire and health record data were collected from index cases and health care providers (HCPs) at Sassoon General Hospital, Pune, India. RESULTS: Of 80 adult TB cases, 24 (30%) reported that an HCP recommended TB screening of their child contacts; 49/178 (28%) child contacts were screened. Sixteen (33%) children had active TB, and 28 (85%) of those who screened negative were prescribed IPT. Nineteen (76%) HCPs reported recommending child contact screening. Only 8 (32%) reported ever prescribing IPT. Lack of TB screening and IPT provision for child contacts was associated with inadequate HCP counseling (aOR 19.5, P < 0.001), a non-parent index case (aOR 3.72, P = 0.008) and lack of postgraduate HCP qualification (aOR 19.12, P = 0.04). CONCLUSIONS: TB screening and IPT provision for child contacts of adults with TB were infrequent. Many screened children had active TB. Universal, timely TB screening and IPT for exposed children are urgently needed to reduce pediatric TB in India.

Diabetes and pre-diabetes among household contacts of tuberculosis patients in India: is it time to screen them all?

SETTING: Pre-diabetes mellitus (pre-DM) and DM increase the risk of developing tuberculosis (TB). Screening contacts of TB patients for pre-DM/DM and linking them to care may mitigate the risk of developing TB and improve DM management. OBJECTIVE: To measure the prevalence of pre-DM/DM and associated factors among the adult household contacts (HHCs) of pulmonary TB patients. METHODS: Between August 2014 and May 2017, adult HHCs of newly diagnosed adult PTB patients in Pune and Chennai, India, had single blood samples tested for glycosylated haemoglobin (HbA1c) at enrolment. DM was defined as previously diagnosed, self-reported DM or HbA1c 6.5%, and pre-DM as HbA1c between 5.7% and 6.4%. Latent tuberculous infection (LTBI) was defined as a positive tuberculin skin test (5 mm induration) or QuantiFERON® Gold In-Tube (0.35 international units/ml). RESULTS: Of 652 adult HHCs, 175 (27%) had pre-DM and 64 (10%) had DM. Forty (64%) HHCs were newly diagnosed with DM and 48 (75%) had poor glycaemic control (HbA1c 7.0%). Sixty-eight (22%) pre-DM cases were aged 18-34 years. Age 35 years, body mass index 25 kg/m2, chronic disease and current tobacco smoking were significantly associated with DM among HHCs. CONCLUSIONS: Adult HHCs of TB patients in India have a high prevalence of undiagnosed DM, pre-DM and LTBI, putting them at high risk for developing TB. Routine DM screening should be considered among all adult HHCs of TB.

In vivo radiation protection by nitric oxide modulation.

Drugs that affect blood flow have been shown to be whole body radiation protectors. Using NG-nitro-L-arginine, a specific inhibitor of nitric oxide synthase, and the NO-releasing agent (C2H5)2N[N(O)NO-]Na+ (DEA/NO), we have studied the ability of NO to modulate whole body radiation toxicity in C3H mice. NG-Nitro-L-arginine given to mice between 15 and 60 min prior to radiation afforded significant protection from whole body irradiation, e.g., the estimated whole body irradiation dose required to kill 50% of mice by 30 days after radiation (LD50/30) in mice treated with NG-nitro-L-arginine 60 min before irradiation was 1051 cGy compared with a whole body radiation LD50/30 of 822 cGy in control mice (P < 0.00001). Treatment of mice with DEA/NO prior to whole body irradiation also significantly reduced toxicity; the estimated whole body radiation LD50/30 was 1063 and 945 cGy in mice treated with DEA/NO 10 or 30 min before irradiation, respectively (P < 0.00001 for radiation LD50/30 of either DEA/NO-treated group compared with control). Measurement of [14C]etanidazole binding to bone marrow demonstrated that DEA/NO and NG-nitro-L-arginine exacerbated bone marrow hypoxia. Perturbations of NO levels have profound effects on in vivo radiosensitivity of normal tissues. We hypothesize that alterations in regional blood flow may underlie the changes in radiosensitivity that we have observed.

The use of Zn-desferrioxamine for radioprotection in mice, tissue culture, and isolated DNA.

Redox-active metals mediate oxidative injury and might also potentiate radiation damage. The iron chelator desferrioxamine (DFO), which diminishes oxidative damage in many chemical and biological systems as well as in human subjects, has a controversial role in radiobiology and reportedly acts both as a radiosensitizer and a radioprotector. The present research focused on the radioprotective activity of its zinc complex. Zn-DFO was studied using three test systems differing by their complexities: isolated DNA from pUC 19 plasmid, cultured V79 Chinese hamster cells, and C3H mice. Zn-DFO (0.5-2 mM) protected isolated DNA against gamma-radiation better than each of its components alone; however, neither Zn-DFO nor DFO (50-100 microM) alone affected the radiation sensitivity of cultured cells. With total body irradiation, Zn-DFO, but not DFO alone at 100 micromol/kg body weight, administered to mice 30 min before irradiation provided significant radioprotection (P < 0.01). Zn-DFO had an LD(50/30) of 10.3 Gy, whereas DFO and vehicle alone had LD(50/30) of 8.03 Gy and 7.91 Gy, respectively. The effect of Zn-DFO on the hemodynamic parameters in mice did not differ from that of the vehicle (saline) alone. This excludes the explanation that the radioprotective activity of Zn-DFO results from its effect on oxygen levels. In addition to the possible direct effect of Zn, other potential modes of action underlying the radioprotective activity of Zn-DFO might involve a displacement of iron and its substitution by zinc, a greater proximity of the drug to DNA, and less likely an improved penetration of the drug into cells because of its structure. The failure of Zn-DFO to protect cells in tissue cultures indicates that it has some systemic role in the whole animal, possibly due to a prolonged half-life in the animal's circulation.

Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells.

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.

Effect of gonadotropin secretion rate on the radiosensitivity of the rat luteinizing hormone-releasing hormone neuron and gonadotroph.

To test the hypothesis that the functional state of hypothalamic LHRH neurons and pituitary gonadotrophs might alter their radiosensitivity, we determined the experimental conditions under which the gonadotropin response to castration could be impaired by a single dose of cranial irradiation. Single doses of cranial irradiation greater than 2000 rads were lethal to unshielded rats. Shielding of the oropharynx and esophagus allowed the animals to survive doses up to 5000 rads. Doses between 2000 and 5000 rads had no effect on basal gonadotropin levels for as long as 3 months after irradiation. Irradiation caused a dose- and time-dependent impairment, however, in the gonadotropin response to castration. Impairment of the gonadotropin levels of castrate animals occurred in animals that were irradiated either before or after castration. However, rats irradiated in the castrate state showed a decreased susceptibility to irradiation damage. Additionally, stimulation of the pituitary by LHRH agonist (LHRHa) 3 h before irradiation significantly reduced the impairment of gonadotropin secretion 12-20 weeks after irradiation (P less than 0.05). Thus, increased functional activity of the rat hypothalamus or pituitary at the time of irradiation, induced by either castration or acute LHRHa administration, was associated with some protection against the gonadotropin-lowering effect of irradiation. Based upon these data, we hypothesize that stimulation of gonadotropin secretion at the time of therapeutic cranial irradiation in humans might protect against subsequent impairment of gonadotropin secretion.

Evaluation of the hydroxylamine Tempol-H as an in vivo radioprotector.

Nitroxides are stable free radical compounds that protect against the toxicity of reactive oxygen species in vitro and in vivo. Tempol (Aldrich, Milwaukee, WI, USA) is a cell-permeable hydrophilic nitroxide and has been shown to be an in vitro and in vivo radioprotector. The limitations of Tempol as a systemic radioprotector are that it causes substantial reductions in arterial blood pressure when administered intravenously and is associated with seizure activity. Furthermore, Tempol is rapidly reduced to its hydroxylamine form, Tempol-H, which limits the period of time the active form of the nitroxide is available for radioprotection. Based on initial pharmacological and blood pressure experiments performed in mice, we hypothesized that the systemic administration of Tempol-H in vivo would lead to an equilibration between Tempol and Tempol-H that would limit the toxicity of the nitroxide and provide in vivo radioprotection. Tempol-H was administered in increasing doses via an intraperitoneal route to C3H mice. The maximally tolerated dose was found to be 325 mg/kg. The whole-blood pharmacology of Tempol-H was investigated with electron paramagnetic resonance spectroscopy. These studies demonstrated the appearance of Tempol in whole blood immediately after intraperitoneal injection, suggesting that rapid oxidation of Tempol-H to Tempol takes place in vivo. Although the peak concentration of Tempol in whole blood after administration of Tempol-H did not reach the same levels as those observed when Tempol is administered, the whole-blood levels of Tempol were similar by 10 min after injection. Tempol-H provided protection against the lethality of whole-body radiation in C3H mice at 30 d with a dose modification factor of 1.3, which is similar to the results obtained with Tempol. Hemodynamic measurements in C3H mice after intravenous injection showed that Tempol-H produced little effect on blood pressure or pulse compared with Tempol. Tempol-H is a systemic in vivo radioprotector of C3H mice and is associated with less hemodynamic toxicity than Tempol.

Evaluation of tempol radioprotection in a murine tumor model.

Tempol, a stable nitroxide free radical compound, is an in vitro and in vivo radioprotector. Previous studies have shown that Tempol protects C3H mice against whole-body radiation-induced bone marrow failure. In this study, the radioprotection of tumor tissue was evaluated. RIF-1 tumor cells were implanted in female C3H mice 10 d prior to radiation. Groups of mice were injected intraperitoneally with Tempol (275 mg/kg) or PBS followed 10 min later by a single dose of radiation to the tumor bed. Tumor growth curves generated after 10 and 33.3 Gy doses of radiation showed no difference in growth between the Tempol- and PBS-treated animals. A full radiation dose-response experiment revealed a tumor control dose in 50% of the animals in 30 d (TCD(50/30)) value of 36.7 Gy for Tempol-treated mice and 41.8 Gy for saline-treated mice suggesting no protection of the RIF-1 tumor by Tempol. Tumor pharmacokinetics were done to determine why Tempol differentially protected bone marrow and not tumor cells. Differential reduction of Tempol in the RIF-1 tumor and bone marrow was evaluated with EPR spectroscopy 10, 20, and 30 min after injection. Bioreduction of Tempol to its corresponding hydroxylamine (which is not a radioprotector) occurred to a greater extent in RIF-1 tumor cells compared to bone marrow. We conclude that the differences in radioprotection may result from enhanced intratumor bioreduction of Tempol to its nonradioprotective hydroxylamine analogue. The nitroxides as a class of compounds may provide a means to exploit the redox differences between normal tissues and tumors.

Hemodynamic effect of the nitroxide superoxide dismutase mimics.

Reactive oxygen species play critical roles in a number of physiologic and pathologic processes. Nitroxides are stable free radical compounds that possess superoxide dismutase (SOD) mimetic activity and have been shown to protect against the toxicity of reactive oxygen species in vitro and in vivo. Tempol, a cell-permeable hydrophilic nitroxide, protects against oxidative stress and also is an in vitro and in vivo radioprotector. In the course of evaluating the pharmacology and toxicity of the nitroxides, Tempol and another nitroxide, 3-carbamoyl-PROXYL (3-CP), were administered intravenously in various concentrations to miniature swine. Tempol caused dose-related hypotension accompanied by reflex tachycardia and increased skin temperature. Invasive hemodynamic monitoring with Swan Ganz catheterization (SGC) confirmed the potent vasodilative effect of Tempol. However, 3-CP had no effect on porcine blood pressure. The hemodynamic effects of Tempol and 3-CP are discussed in the context of differential catalytic rate constants for superoxide disumation that may impact systemic nitric oxide (NO) levels and lead to vasodilation. These findings are consistent with a role for the superoxide ion in the modulation of blood pressure and have potential implications for the systemic use of nitroxides.

Threshold dose for peripheral neuropathy following intraoperative radiotherapy (IORT) in a large animal model.

Radiation injury to peripheral nerve is a dose-limiting toxicity in the clinical application of intraoperative radiotherapy, particularly for pelvic and retroperitoneal tumors. Intraoperative radiotherapy-related peripheral neuropathy in humans receiving doses of 20-25 Gy is manifested as a mixed motor-sensory deficit beginning 6-9 months following treatment. In a previous experimental study of intraoperative radiotherapy-related neuropathy of the lumbro-sacral plexus, an approximate inverse linear relationship was reported between the intraoperative dose (20-75 Gy range) and the time to onset of hind limb paresis (1-12 mos following intraoperative radiotherapy). The principal histological lesion in irradiated nerve was loss of large nerve fibers and perineural fibrosis without significant vascular injury. Similar histological changes in irradiated nerves were found in humans. To assess peripheral nerve injury to lower doses of intraoperative radiotherapy in this same large animal model, groups of four adult American Foxhounds (wt 20-25 kg) received doses of 10, 15, or 20 Gy to the right lumbro-sacral plexus and sciatic nerve using 9 MeV electrons. The left lumbro-sacral plexus and sciatic nerve were excluded from the intraoperative field to allow each animal to serve as its own control. Following treatment, a complete neurological exam, electromyogram, and nerve conduction studies were performed monthly for 1 year. Monthly neurological exams were performed in years 2 and 3 whereas electromyogram and nerve conduction studies were performed every 3 months during this follow-up period. With follow-up of greater than or equal to 42 months, no dog receiving 10 or 15 Gy IORT shows any clinical or laboratory evidence of peripheral nerve injury. However, all four dogs receiving 20 Gy developed right hind limb paresis at 8, 9, 9, and 12 mos following intraoperative radiotherapy. These experimental data suggest that intraoperative doses of less than 20 Gy may not result in clinically significant peripheral nerve injury with follow-up of 3.5 years. Longer (5 yrs) follow-up with planned sacrifice of the remaining dogs is scheduled to assess any late peripheral nerve damage.

In vivo radioprotection and effects on blood pressure of the stable free radical nitroxides.

PURPOSE: The purpose of this study was to screen several water soluble nitroxides for in vivo radioprotection, to evaluate their pharmacology, and to measure the effect of nitroxides on systemic blood pressure as a means of exploring the mechanism of in vivo radioprotection. METHODS AND MATERIALS: A number of water soluble nitroxides were screened for in vivo radioprotection in C3H mice at a single radiation dose. Selected nitroxides were administered by the intraperitoneal route 10 minutes prior to a whole body radiation dose of 9 Gy. Electron paramagnetic resonance spectroscopy (EPR) was used to measure whole blood levels of nitroxides. The nitroxides were evaluated for effects on systemic blood pressure in C3H mice. RESULTS: All of the nitroxides studied demonstrated radioprotection compared to saline-treated controls. The 6-membered piperidine ring nitroxides including Tempol were reduced to the inactive hydroxylamine rapidly over 10-20 minutes. The 5-membered ring nitroxides were reduced more slowly over time. The 5-membered ring 3-carbamoyl-PROXYL did not produce a substantial decrease in systemic blood pressure after intraperitoneal administration compared to the other nitroxides studied. 3-carbamoyl-PROXYL was further evaluated over a range of whole body radiation doses and was found to provide radioprotection. CONCLUSION: All of the nitroxides studied provided radioprotection. In vivo radioprotection for all of the compounds except 3-carbamoyl-PROXYL may be at least partially explained by the induction of hypotension and bone marrow hypoxia. 3-carbamoyl-PROXYL provided in vivo radioprotection similar in magnitude to Tempol and had little effect on blood pressure compared to the other nitroxides. Other mechanisms for radioprotection, including scavenging of free radicals are likely. 3-carbamoyl-PROXYL should be evaluated further as a systemic radioprotector.

Tumor induction following intraoperative radiotherapy: late results of the National Cancer Institute canine trials.

Intraoperative radiotherapy has been employed in human cancer research for over a decade. Since 1979, trials to assess the acute and late toxicity of IORT have been carried out at the National Cancer Institute in an adult dog model in an attempt to establish dose tolerance guidelines for a variety of organs. Of the 170 animals entered on 12 studies with a minimum follow-up of 2 years, 148 dogs received IORT; 22 control animals received only surgery. Animals were sacrificed at designated intervals following IORT, usually at 1, 6, 12, 24, and 60 month intervals. 102 of 148 irradiated dogs were sacrificed less than 24 months; 46 dogs were followed greater than or equal to 24 months after IORT. To date, 34 of the 46 animals have been sacrificed; the 12 remaining animals are to be followed to 5 years. These 12 animals have minimum follow-up of 30 months. In the irradiated group followed for greater than or equal to 24 months, 10 tumors have arisen in 9 animals. One animal developed an incidental spontaneous breast carcinoma outside the IORT port, discovered only at scheduled post-mortem exam. The remaining nine tumors arose within IORT ports. Two tumors were benign neural tumors--a neuroma and a neurofibroma. One animal had a "collision" tumor comprised of grade I chondrosarcoma adjacent to grade III osteosarcoma arising in lumbar vertebrae. Two other grade III osteosarcomas, one grade III fibrosarcoma, and one grade III malignant fibrous histiocytoma arose in retroperitoneal/paravertebral sites. An embryonal rhabdomyosarcoma (sarcoma botryoides) arose within the irradiated urinary bladder of one animal. No sham irradiated controls nor IORT animals sacrificed less than 24 months have developed any spontaneous or radiation-induced tumors. The time range of diagnoses of tumors was 24-58 months (median 40 months). The IORT dose range associated with tumor development was 20-35 Gy (median 30 Gy). The carcinogenesis capability of single fraction, high dose radiation in animals is discussed, as are the implications of these data for continued research and clinical usage of IORT in the treatment of humans.

Effects of intraoperative radiotherapy on vascular grafts in a canine model.

PURPOSE: The effects of intraoperative radiotherapy +/- external beam radiotherapy on prosthetic vascular grafts were investigated in a canine model. METHODS AND MATERIALS: In 1986 and 1987, 30 adult beagles underwent laparotomy with transection and segmental resection of the infrarenal aorta followed by immediate reconstruction with a prosthetic graft. Intraoperative radiotherapy at varying doses from 0-30 Gy was then administered to all animals. Half of the dogs received 36 Gy external beam radiotherapy in 10 fractions postoperatively. Animals were sacrificed and necropsied at predetermined intervals and as clinically indicated to assess early (< or = 6 months) and late (> 6 months) effects to the vascular graft and surrounding normal tissue. RESULTS: Histopathologic analyses of irradiated vascular structures were performed and correlations were made with the clinical outcome. The most frequent early clinical toxicity was graft thrombosis, occurring in 7 of 10 animals followed for < or = 6 months. Early graft thrombus formation appeared unrelated to radiotherapy dose and probably represented a technical surgical complication. Anastomotic stenosis of varying severity occurred in most animals followed > 6 months. Late (> 6 months) graft stenosis was correlated with intraoperative radiotherapy dose. At < or = 20 Gy of intraoperative irradiation, 3 of 14 animals developed late graft occlusion; at > 25 Gy, five of six animals developed late occlusion. On histopathologic review, increasing intraoperative dose and increasing total radiotherapy dose (intraoperative+external beam) appeared to correspond with increasing severity of graft changes seen after 6 months of follow-up. CONCLUSIONS: Thrombus formation is a frequent early complication of vascular graft placement of the infrarenal aorta in our beagle dog model. Intraoperative doses up to 20 Gy appear to contribute minimally to late graft occlusion, while doses > or = 25 Gy contribute to late occlusion with high likelihood. Both intraoperative dose and total radiotherapy dose correlated with late graft occlusion, and with histopathologic changes in the graft and anastomoses.

Tolerance of the canine bladder to intraoperative radiation therapy: an experimental study.

An experimental study of bladder tolerance to intraoperative radiotherapy (IORT) was designed using a large animal model (adult American Foxhounds, weight 25-30 kg) to access acute and late radiation effects. Dogs were subjected to laparotomy where the bladder was mobilized and IORT was delivered using a 5 cm circular cone through a cystotomy incision with 12 MeV electrons. The bladder trigone including both ureteral orifices and the proximal urethra was irradiated in groups of 3 dogs with doses of 0, 20, 25, 30, 35, and 40 Gy. Dogs were followed clinically with repeat urinalysis, intravenous pyelogram (IVP), and cystometrogram at 1 month and then Q6 months for up to 4 years. One dog from each dose group was sacrificed electively at 1 and 2 years, whereas the other dog is being followed clinically for a minimum of 4 years. Complete autopsies were performed with particular attention to genitourinary and pelvic structures. No clinically detectable acute toxicity resulted from IORT to the bladder. Three of 15 IORT dogs (1 each at 25, 35, and 40 Gy) showed obstruction of a ureteral orifice with 2 dogs dying of renal failure secondary to bilateral hydronephrosis within 1-2 years of treatment. The remaining 12 IORT dogs and 3 control dogs have normal repeat IVP's and renal function with up to 4 years of follow-up. Serial cystometry demonstrates no major loss of bladder contractility or volume. At autopsy, histological changes of mucosal thinning and telangiectasia with submucosal fibrosis were confined to the IORT field and appeared dose-related. However, the bladder epithelium remained intact at all doses. The ureterovesical junction in animals receiving 20 Gy showed mild fibrosis of the lamina propria and moderate chronic inflammation. Above 20 Gy, these histological changes at the U-V junction were more pronounced with gross stenosis in 3 animals as predicted by the IVP. We conclude that the bladder trigone will tolerate IORT to 20 Gy without major clinical sequellae. Above 20 Gy, progressive inflammation and fibrosis of the U-V junction resulted in obstructive hydronephrosis in three animals within 1-2 years of IORT. The bladder mucosa remained intact with doses to 40 Gy, although submucosal fibrosis and chronic inflammation were evident and appeared dose-related. However, bladder function as measured by cystometry showed essentially no change with follow-up to 4 years. From this large animal study, IORT for early-stage bladder carcinoma is technically feasible and deserves a careful clinical study.

Clinical toxicity of peripheral nerve to intraoperative radiotherapy in a canine model.

PURPOSE: The clinical late effects of intraoperative radiotherapy (IORT) on peripheral nerve were investigated in a foxhound model. METHODS AND MATERIALS: Between 1982 and 1987, 40 animals underwent laparotomy with intraoperative radiotherapy of doses from 0-75 Gy administered to the right lumbosacral plexus. Subsequently, all animals were monitored closely and sacrificed to assess clinical effects to peripheral nerve. This analysis reports final clinical results of all animals, with follow-up to 5 years. RESULTS: All animals treated with > or = 25 Gy developed ipsilateral neuropathy. An inverse relationship was noted between intraoperative radiotherapy dose and time to neuropathy, with an effective dose for 50% paralysis (ED50) of 17.2 Gy. One of the animals treated with 15 Gy IORT developed paralysis, after a much longer latency than the other animals. CONCLUSIONS: Doses of 15 Gy delivered intraoperatively may be accompanied by peripheral neuropathy with long-term follow-up. This threshold is less than that reported with shorter follow-up. The value of ED50 determined here is in keeping with data from other animal trials, and from clinical trials in humans.

Long term tolerance of thoracic organs to intraoperative radiotherapy.

The tolerance of mediastinal structures to intraoperative radiotherapy (IORT) was investigated in 3 separate animals trials using 49 adult foxhounds and one limited Phase I trial in 4 patients with Stage II or III non-small cell lung cancer (NSCLC). The 1- to 2-year results of these trials have been previously reported with significant toxicity found at dose levels over 20 Gy. We now report the results of five dogs reserved for long term studies and one Stage II NSCLC patient alive at 5 years. Two dogs received 20 Gy IORT and one received 30 Gy IORT to the esophagus, all three to a single 6 cm field with 9 MeV electrons. One control dog underwent surgery without irradiation. One dog received 20 Gy IORT to a single 5 cm mediastinal field with 13 MeV electrons following left pneumonectomy. At 5 years, all five dogs reserved for a long term evaluation were alive and evaluable with minimal endoscopic and radiographic abnormalities. The one patient alive at 5 years for evaluation received 25 Gy IORT to two matched 6 cm fields with 13 MeV electrons. She has stable dyspnea on exertion and there is no evidence of cancer by endoscopy. We conclude, based on these limited data, that IORT in the mediastinum may be safe at dose levels that do not exceed 20 Gy, and further careful evaluation at these lower treatment doses is warranted to determine efficacy.