Efficacy and safety of anti-CD20 antibody rituximab for patients with refractory systemic lupus erythematosus.

Objective We examined the efficacy and safety of rituximab in patients with refractory systemic lupus erythematosus (SLE). Methods The study enrolled 63 SLE patients who were treated with rituximab between 2002 and 2015. The participants underwent a battery of tests before treatment and at one year. Treatment ranged from two to four times at 500 or 1000 mg. Results Baseline characteristics were males:females = 6:57, age 33.9 years, and disease duration 87.2 months. The primary endpoint: The rate of major clinical response (MCR) was 60% while the partial clinical response (PCR) was 25%. Thirty of 36 (83%) patients with lupus nephritis (WHO II: 2, III: 5, IV: 22, V: 4, IV+V: 2, not assessed: 1) and 22 of 24 patients (92%) with neuropsychiatric SLE, who could be followed at one year, showed changes from BILAG A or B score to C or D score at one year. Multivariate analysis identified high anti-dsDNA antibody and shorter disease duration as significant determinants of MCR at one year. Repeat examination was conducted at five years. Primary failure was recorded in 8.8% and secondary failure in 32.4% (time to relapse: 24.4 months). Rituximab was well tolerated although 65 adverse events, mostly infections, were recorded within one year. Conclusion Rituximab is potentially efficacious for the treatment of patients with refractory SLE.

Outcomes after open surgery and endovascular aneurysm repair for abdominal aortic aneurysm in patients with massive neck atheroma.

OBJECTIVE: We retrospectively analysed surgically treated abdominal aortic aneurysm (AAA) in patients with massive atheroma in the aneurysmal neck and compared the outcomes of endovascular aneurysm repair (EVAR) and open surgery (OS) to determine an appropriate strategy for massive neck atheroma cases. METHODS: A retrospective study was performed in 326 consecutive patients who underwent EVAR and in 247 patients who underwent OS. We defined massive neck atheromas if the following characteristics were observed: (1) thickness ≥ 5 mm; (2) the circumference of the infrarenal aorta ≥ 75%; and (3) length ≥ 5 mm. Twenty-eight patients (8.5%) in the EVAR group and 22 (8.9%) in the OS group met these criteria. We modified the previously published reporting standards on the basis of the selection of systemic and embolisation-related complications. RESULTS: Patients in the EVAR group had less intra-operative blood loss, shorter operation time, and shorter hospital stays after the operation (P < 0.01). No perioperative deaths were observed in either group. Major complications were categorised as early (in-hospital) or late (outpatient, within 6 months). Five and three patients in the OS and EVAR groups had early complications, but the difference was not statistically significant. In contrast, 7 patients in the EVAR group had late complications, compared to no patients in the OS group (P = 0.01). Kaplan-Meier analysis revealed a significantly higher survival rate in the OS group (P = 0.011). Two of the 4 patients with suprarenal clamping developed major complications. Mild eosinophilia was observed in 10 patients in the EVAR group. Proteinuria occurred or worsened in 5 EVAR patients and 1 OS patient. CONCLUSION: Compared to OS patients, EVAR patients with massive neck atheroma tend to develop late-phase complications possibly related to cholesterol crystal embolisation. The clinical features of massive neck atheroma patients receiving EVAR should be carefully monitored even after hospital discharge.

Nuclear factor kappa B plays a pivotal role in polyinosinic-polycytidylic acid-induced expression of human ß-defensin 2 in intestinal epithelial cells.

Intestinal epithelial cells (IECs) play an important role in protecting the intestinal surface from invading pathogens by producing effector molecules. IECs are one of the major sources of human beta-defensin 2 (hBD-2), and can produce it in response to a variety of stimuli. Although IECs express Toll-like receptor 3 (TLR-3) and can respond to its ligand, double-stranded RNA (dsRNA), hBD-2 expression in response to dsRNA has not been elucidated. In the present study, using an artificial analogue of dsRNA, polyinosinic-polycytidylic acid (poly I:C), we investigated whether the human IEC line, HT-29, can produce hBD-2 in response to poly I:C. HT-29 cells can express hBD-2 mRNA only when stimulated with poly I:C. The induction of hBD-2 mRNA expression was observed at 3 h after stimulation and peaked at 12 h of post-stimulation. Pre-incubation of the cells with nuclear factor kappa B (NF-κB)-specific inhibitor, l-1-4'-tosylamino-phenylethyl-chloromethyl ketone (TPCK) and isohelenine abolished the expression of hBD-2. Detection of the poly I:C signal by TLR-3 on the surface of HT-29 cells was revealed by pre-incubating the cells with anti-TLR-3 antibody. The 5'-regulatory region of the hBD-2 gene contains two NF-κB binding sites. A luciferase assay revealed the importance of the proximal NF-κB binding site for poly I:C-induced expression of hBD-2. Among NF-κB subunits, p65 and p50 were activated by poly I:C stimulation and accumulated in the nucleus. Activation of the p65 subunit was investigated further by determining its phosphorylation status, which revealed that poly I:C stimulation resulted in prolonged phosphorylation of p65. These results indicate clearly that NF-κB plays an indispensable role in poly I:C induced hBD-2 expression in HT-29 cells.

A retrospective study of intravascular ultrasound use in patients undergoing endovascular aneurysm repair: its usefulness and a description of the procedure.

OBJECTIVES: To verify the usefulness and limitation of intravascular ultrasound (IVUS) in endovascular aneurysm repair (EVAR). METHODS: A total of 112 consecutive patients, who underwent EVAR to treat abdominal aortic aneurysms, were examined retrospectively. Of these, 33 patients were assigned to the IVUS group because of renal failure, a suspected allergy to contrast agents or anatomical difficulties; the remaining 79 patients were assigned to the non-IVUS group. RESULTS: Patients in the IVUS group required fewer intra-arterial contrast agents (IACAs) than those in the non-IVUS group (67±34ml vs. 123±50ml; p<0.01). Blood loss and operation time were comparable between the two groups. No patients died within 30 days of the operation. Three major renal complications occurred in the non-IVUS group. Renal deterioration evaluated by chronic kidney disease (CKD) stage was found to a greater extent in the non-IVUS group. CONCLUSIONS: IVUS is a powerful auxiliary method in EVAR for reducing the required volume of contrast agents. The combination of IVUS and IACA usage showed good overall performance; thus, we propose the routine use of IVUS in EVAR procedures.

Winter lipid depletion of juvenile walleye pollock Theragra chalcogramma in the Doto area, northern Japan.

Seasonal variation in body size and nutritional condition of juvenile walleye pollock Theragra chalcogramma was examined to elucidate the mechanism underlying their first-winter survival on the continental shelf of the Doto area, northern Japan, based on monthly samples collected over 2 years. Stored lipid mass was highest during autumn, but 93% (2004) and 80% (2005) of lipids were exhausted by the onset of winter. Lipid levels in the winter of 2004 remained low (7-14% of the autumnal maximum), and there was reduced growth rate until the spring, whereas in 2005 lipid levels were higher and more variable (10-46% of the maximum) and some growth occurred. An analysis of the allometric relationships between body size and stored energy showed that larger individuals accumulated disproportionately more energy in the autumn, but the advantage disappeared prior to the winter. In January 2004, stored lipid energy was low throughout the Doto continental shelf relative to the continental slope area. These results suggest that winter feeding opportunities on the shelf are severely limited but not completely absent. Previous studies have shown that winter temperatures on the shelf are lower than those in the slope area. It is possible that juvenile T. chalcogramma survive winter on the shelf without a high level of pre-winter lipid storage because the occasional feeding in the cold shelf water benefits energy conservation.

Tumour necrosis factor-alpha-mediated human polymeric immunoglobulin receptor expression is regulated by both mitogen-activated protein kinase and phosphatidylinositol-3-kinase in HT-29 cell line.

Human polymeric immunoglobulin receptor (pIgR) is present on the surface of glandular epithelium, and it plays a crucial role in the mucosal immune defence. pIgR expression in HT-29 cells is up-regulated by one of the proinflammatory cytokines, tumour necrosis factor (TNF)-alpha. However, the mechanism used by the TNF-alpha-mediated signalling pathway has not been examined exclusively. To elucidate this mechanism in detail, HT-29 cells were cotreated with TNF-alpha and mitogen-activated protein kinase kinase (MAPKK, also called MEK1) inhibitor, PD98059, and the amount of free secretory component (SC) secreted into the culture medium was measured. The amount of free SC stimulated by TNF-alpha was increased by addition of PD98059. This up-regulation occurred at the transcriptional level. The amount of SC was also up-regulated by addition of TNF-alpha with U0126, an inhibitor of MEK1 and MEK2. Nuclear factor (NF)-kappaB activity and NF-kappaB binding to the kappaB2 site localized upstream of the pIgR gene did not change after coincubation of HT-29 cells with TNF-alpha and PD98059. The expression level of pIgR by TNF-alpha was decreased by LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3K), at the transcriptional level. Extracellular signal-regulated kinase (ERK)1/2 phosphorylation and NF-kappaB binding to the kappaB2 site were not affected by LY294002 treatment. These data suggest that TNF-alpha-mediated pIgR expression is negatively regulated by ERK pathway, which is independent of NF-kappaB. In addition, decrease of SC production by Ly294002 suggests that the presence of PI3K mediated regulation of SC production.

Regulation of angiotensin II type 2 receptor gene by the protein kinase C-calcium pathway.

In the present study, rat angiotensin II type 2 (AT2) receptor expression was upregulated in confluence-arrested PC12 cells compared with expression in proliferating cells. Treatment with cycloheximide inhibited the increase in mRNA levels in confluent cells. The state of growth arrest by serum deprivation was associated with increased expression of the AT2 receptor, which was markedly suppressed by exposure to the active phorbol ester 12-O-tetradecanoylphorbol 13-acetate and the calcium ionophore A23187. Similar inhibitions were also observed in myocytes isolated from neonatal rat heart. The change in AT2 mRNA levels by serum deprivation was due to the increase in the gene transcription rate. The effect of 12-O-tetradecanoylphorbol 13-acetate was mediated through decreases in gene transcription and mRNA stability, whereas A23187 affected mRNA stability. Vasoactive substances with the protein kinase C-calcium pathway, such as norepinephrine and angiotensin II, also downregulated the AT2 mRNA level in myocytes. These findings indicate that the expression of AT2 receptor in PC12 cells is regulated in a growth state-dependent manner, which is involved in confluence-induced new protein synthesis, thus providing a means by which cells can modulate their responsiveness to external angiotensin II stimulus. The activation of protein kinase C or calcium mobilization modifies this regulatory mechanism, suggesting that neurotransmitters or vasoactive substances with the protein kinase C-calcium pathway at least in part affect neuronal activity or blood pressure control by downregulating AT2 receptor expression.

Influence of fatty acid absorption on bidirectional release of immunoglobulin A into intestinal lumen and intestinal lymph in rats.

Effects of absorption of long and middle chain fatty acids on IgA secretion into the intestinal lumen and intestinal lymph and the factors which evoke changes in IgA secretion during the absorptive process were examined in rat small intestine. Bidirectional secretion of IgA from the intestinal mucosa into the intestinal lumen and intestinal lymph was continuously observed in the control condition. Perfusion of oleic acid (a long-chain fatty acid) micelle into the jejunal loop induced a significant increase in IgA output into the intestinal lymph. In contrast, lymphatic output of IgA was significantly decreased when oleic acid micelle was administered intraduodenally. Absorption of octanoic acid, a middle-chain fatty acid, did not produce any significant changes in IgA output into either direction. CR1505, a CCK-receptor antagonist, significantly attenuated the oleic acid-induced increase in IgA secretion into the intestinal lumen, but did not affect the oleic acid-induced decrease in lymphatic IgA secretion. Pluronic L-81, an inhibitor of chylomicron formation and secretion, significantly attenuated the decrease in IgA output into the intestinal lymph during oleic acid absorption without affecting the luminal IgA output. The rate of release of IgA into the intestinal lumen is stimulated by absorption of long-chain fatty acids possibly through the influence of locally released CCK, while the transport process of IgA into lymphatics is controlled by a different mechanism which is closely correlated with the intracellular formation and secretion of chylomicron.

Adverse reactions to lipiodol ultra fluid: report of an accidental case.

A 25-year-old Japanese woman experienced pulmonary edema, shock, encephalopathy and silent pancreatitis after hysterosalpingography using lipiodol ultra fluid, because the contrast media flowed directly into the blood stream from endometrium injured by several curettages for termination of pregnancy. Total iodine concentrations in plasma and urine decreased exponentially and their half-life was 16.12 and 13.04 days, respectively. Clear correlations were observed between the iodine concentration in plasma and the amelioration of both electroencephalogram and neuropsychic abnormalities. Adverse reactions of oil-soluble contrast media are also discussed.

Prolonged survival in a patient with a single ventricle without pulmonary stenosis.

Prolonged survival of the patient with a single ventricle is rare. We report the case of a 57-year-old man who has type A3 of Van Praagh's classification of single ventricle with pulmonary hypertension without pulmonary stenosis. Favorable streaming, a large bulboventricular foramen, and intact atrioventricular valves may have contributed to his long-term survival.

Natural history of left ventricular function in patients with uncomplicated acute myocardial infarction.

To investigate the serial changes of the infarcted and the noninfarcted areas, first-pass radionuclide angiocardiography was performed in 16 patients with uncomplicated anterior myocardial infarction (MI) at four weeks, one year, and two years after the onset of MI. Global ejection fraction (EF) and regional EF of the infarcted area improved significantly from four weeks to one year after MI (from 39 +/- 16 to 44 +/- 16 percent, 23 +/- 3 to 29 +/- 5 percent, both p < 0.01), but did not change from one year to two years after MI. Regional EF of the noninfarcted area and left ventricular end-diastolic and end-systolic volume did not change during the study period. There was a significant relation in the direction of the changes of global EF and regional EF of the infarcted area during the first year after MI, whereas no relation was observed between the changes of global EF and regional EF of the noninfarcted area. A greater improvement in regional EF of the infarcted area was observed in seven patients who had spontaneous recanalization compared with nine patients with totally occluded coronary arteries. Thus, a significant improvement in cardiac function, mainly due to the increase in regional EF of the infarcted area, was observed during the first year after MI, which was related to patency of coronary artery.

Down-regulation by cAMP of angiotensin II type 2 receptor gene expression in PC12 cells.

The rat angiotensin II type 2 receptor (AT2-R) gene was isolated, and cis-regulatory regions in its 5'-flanking area were analyzed. Primer extension and RNase protection analyses revealed a single transcriptional initiation site at the position 24 bp downstream of the TATA box. The 5'-flanking region of AT2-R contained several cis-regulatory elements, such as AP-1, AP-2, C/EBP, NF-1, NF-IL6, NF-kappa B, and glucocorticoid- and cAMP-responsive elements (CRE). The treatment of PC12 cells with dibutyryl cAMP caused a marked decrease (90%) in the AT2-R mRNA level, which was blocked by the inhibitor of protein kinase A and did not require new protein synthesis. The protein level was also reduced 84% after a 24-h exposure to cAMP and the binding affinity was unchanged. The half-life of the AT2-R mRNA decreased -66% by cAMP as compared with control (18.4 +/- 0.4 h). Deletion and mutation analyses of the 5'-flanking region (1.2 Kb) revealed that there were one negative (-1,199 to -739) and two positive cis-regulatory regions (-739 to -436 and -59 to +45), and that the CRE motif located at -426 repressed (-23%) the promoter activity of the rat AT2-R gene. The region between -59 and +45 containing TATA box and AP-2 site accounted for 70% of the promoter activity. These findings indicate that the promoter activity of the rat AT2-R gene is modulated by several cis-regulatory regions and that cAMP markedly downregulates the expression of the AT2-R mainly by inducing AT2-R mRNA destabilization rather than CRE-mediated inhibition of the gene transcription. Thus, humoral factors that transduce cAMP as an intracellular signal may modulate AT2-R-mediated function of Ang II by reducing AT2-R expression.

In vivo effect of chronic administration of vasoactive intestinal peptide on gut-associated lymphoid tissues in rats.

The in vivo effects of chronic administration of vasoactive intestinal peptides (VIP) on the lymphoid cell traffic and the population and function of cells in intestinal lymph and gut-associated lymphoid tissues were examined in rats. VIP was continuously infused from the superior mesenteric artery in rats at a dose of 10 ng/min/kg body weight for 96 h. Lymphocyte transport through intestinal lymph was significantly reduced by VIP without any changes in lymph flow. When lymphocyte subpopulation was examined in intestinal lymph, T cell subsets were decreased with a dominant reduction in the population of helper T cells. T cell subsets were also decreased in mesenteric lymph nodes, but in this case suppressor/cytotoxic T cell subsets were mainly reduced. Despite of the decrease in lymphocyte transport through intestinal lymph and changes of lymphocyte subpopulation, proliferative response of lymphocytes from intestinal lymph and mesenteric lymph nodes to phytohemagglutinin did not show any significant alteration after administration of VIP. By histochemical study on the lamina propria of the small intestine, the population of pan T cells, especially helper T cells, was demonstrated to be significantly decreased after VIP treatment. There was also a marked decrease in the number of immunoglobulin (Ig) A-containing cells in the lamina propria of the small intestine in VIP-treated rats, while no significant changes were seen in the number of IgG and IgM-containing cells. Our present results showed the possibility that a long-term alteration of serum VIP levels could affect the dynamics of immune effector cells and IgA production in gut-associated lymphoid tissues.

Spontaneous closure of a large tracheal fistula due to descending necrotizing mediastinitis.

We present a case of a 77-year-old man who had a large tracheal fistula due to descending necrotizing mediastinitis. He underwent long-term care with a respirator after mediastinal drainage operations. The fistula was covered spontaneously with the anterior wall of the esophagus 1.5 months postoperatively.

Serial change in the atrial transport function after the radial incision approach.

BACKGROUND: The left atrial transport function recovers slowly over several months after the maze procedure (Maze), but remains at a low level even during the long-term postoperative period. Because the Maze leaves an insufficient left atrial transport function, patients may still be prone to thromboembolism after the Maze. The radial incision approach (Radial) has been shown to preserve greater atrial transport function than does the Maze in the early postoperative period. METHODS: To examine the serial change in the atrial transport function after the Radial, out of 32 patients who underwent the Radial, 15 patients were assessed by transthoracic Doppler echocardiography 1, 3, 6, and 12 months after surgery. The atrial filling fraction and peak A/E velocity ratio were determined from the flow-velocity spectra across the mitral and tricuspid valves. The incidence of thromboembolic events was examined in 21 patients who were followed for more than 3 months after the Radial. The data were compared with data obtained from 13 patients after (41 +/- 6 months) the Maze III procedure. RESULTS: The left atrial transport function after the Radial increased within 3 months to a significantly greater level than did that after the Maze in the longterm. The atrial filling fraction was 28.2% +/- 7.9% at 3 months after the Radial and 15.1% +/- 4.0% at 41 months after the Maze (p < 0.01). The peak A/E ratio was 0.52 +/- 0.18 at 3 months after the Radial and 0.25 +/- 0.07 at 41 months after the Maze (p < 0.01). This increased atrial transport function was maintained for an extended period after the Radial. There were no thromboembolic events in any of the patients after the Radial or Maze, irrespective of postoperative anticoagulant therapy. CONCLUSIONS: The Radial approach prevents thromboembolism by restoring sufficient atrial transport function more effectively and faster than does the Maze.

Rapid HPLC assay with coulometric detection for norepinephrine and 3-methoxy-4-hydroxyphenylglycol in the mouse brain.

For the rapid assay of norepinephrine (NE) and its major metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG) in the mouse brain, we developed a simple method using isocratic HPLC with coulometric detection. This method permits NE and MHPG assay within 5 min in one chromatographic run. Within-run coefficients of variation for NE and MHPG in the working standard solution were 0.8% and 0.6% (n = 50), respectively. The detector responses were linear from 0.025 to 100 pmol for NE and from 0.05 to 100 pmol for MHPG in the working standard solution. Using this method, the NE and MHPG concentrations were measured in discrete brain areas of the mouse prior treatment with or without alpha-methyl-p-tyrosine or N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4).

Pharmacological properties of ceruletide in the vertical and horizontal locomotor activities of mice.

To clarify the pharmacological properties of ceruletide (CER) and cholecystokinin-octapeptide (CCK-8) with respect to vertical (VLA) and horizontal (HLA) locomotor activities of mice, effects of pretreatment with CER (0.5, 5, and 50 micrograms/kg, IP) and CCK-8 (5, 50, and 500 micrograms/kg, IP) on apomorphine (0.1 mg/kg, SC)- and clonidine (0.1 mg/kg, SC)-induced hypo-VLA and -HLA and on apomorphine (1 mg/kg, SC)-induced hyper-VLA and -HLA were examined. CER and CCK-8 had a dose-dependent inhibitory effect on VLA and HLA in intact mice. Pretreatment with CER had a biphasic effect (increase and decrease) on apomorphine- and clonidine-induced hypo-VLA, as well as an effect on apomorphine-induced hypo-HLA, a decreased effect on clonidine-induced hypo-HLA, and a decreased effect on apomorphine-induced hyper-VLA and -HLA. On the other hand, pretreatment with CCK-8 had no effect on apomorphine- and clonidine-induced hypo-VLA and -HLA and a decreased effect on apomorphine-induced hyper-HLA but not on hyper-VLA. These results suggest that for apomorphine- and clonidine-induced locomotion in mice CER has pharmacological properties different from those of CCK-8.

Alteration of mucosal immunity after long-term ingestion of an elemental diet in rats.

The effects of an elemental diet on lymphocyte transport in intestinal lymph and immune responses of gut-associated lymphoid tissue were investigated in rats. The control animals were fed a conventional diet. After 4 week of feeding, the total calorie intake and body weight gain showed no differences between the two groups. The number and total area of Peyer's patches and the ratio of height of villi to height of crypt showed no significant differences between the two groups. The rate of lymph flow in intestinal lymphatics showed no significant change in treated animals compared with the control rats. However, an elemental diet induced a significant decrease in lymphocyte flux in intestinal lymphatics compared with that in control rats. Lymphocyte subsets in intestinal lymph revealed a significant decrease in CD3-positive cells, especially CD4-positive cells in the elemental diet-treated group. A significant decrease in the number of immunoglobulin A-containing cells and a decreased CD4/CD8 ratio in T-cell subsets were observed in the lamina propria of ileal mucosa in the elemental diet-treated group by morphometric analysis in the immunohistochemical study. Specific antibody-secreting cells in intestinal lymph were also investigated after rats were intraduodenally primed with cholera toxin and challenged with the same toxin after an interval of 2 weeks. No significant difference was seen between the two groups in any of the numbers of anti-cholera toxin immunoglobulin-secreting cells in any immunoglobulin A, immunoglobulin G, or immunoglobulin M class as determined by the enzyme-linked immunospot assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood macrophage colony-stimulating factor and thrombin-antithrombin III complex concentrations in pregnancy and preeclampsia.

Macrophage colony-stimulating factor (M-CSF) is a characteristic cytokine that plays an essential role in placenta maintenance, and thrombin-antithrombin III complex (TAT) is a hemostatic marker that is remarkably altered both in normal pregnancy and in preeclampsia. The present study was designed in order to show various levels of M-CSF and TAT in pregnancies. Peripheral blood was collected from 49 subjects, of whom 31 were normal pregnant women consisting of the four groups (namely 10th, 20th, 30th, and 38th weeks of gestation), 13 were preeclamptic pregnant women (37th week of gestation; mean blood pressure, 158/99 mm Hg), and 5 were nonpregnant controls. We compared blood M-CSF and TAT levels among them. Results showed that blood M-CSF and TAT levels increased significantly with gestational age. Furthermore, the ratio of increase in M-CSF was significantly lower than that in TAT in normal pregnant women compared with controls. In contrast, the ratio of increase in M-CSF was significantly higher than that in TAT in preeclamptic women compared with normal pregnant women. These results concerning the ratio of increase in M-CSF and TAT have not been reported. These findings show that M-CSF level increases significantly in preeclampsia even in its earlier stage, exhibiting a systolic blood pressure of less than 160 mm Hg.

Primary embryonal carcinoma of the retroperitoneum.

A 32-year-old man with primary embryonal carcinoma of the retroperitoneum, a very rare condition, is reported. The disease took an acute course with rapid enlargement of cervical and inguinal lymph nodes, and high serum alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) values were observed. His testes were, however, intact. Remarkable tumor regression was achieved by systemic combination chemotherapy with cisplatin, vinblastine, and bleomycin (PVB therapy). This chemotherapy is beneficial even in advanced cases. In the two years since therapy, no recurrence has been observed.