A case study: refractory recurrent autoimmune hepatitis following liver transplantation in two male patients.

Autoimmune hepatitis (AIH) recurs after liver transplantation and significantly impacts graft function and patient survival. In this case report, we present 2 cases of male patients with refractory recurrent AIH after liver transplantation. Each patient lost their first graft due to refractory continuous AIH. We have not noticed a similar refractory course for our female patients with AIH post-transplantation at our center. Based on our single-center experience there appears to be a gender disparity in the aggressive nature of AIH recurrence after transplantation. Despite the aggressive nature of recurrent AIH in both patients, graft loss occurred beyond 3 years for both patients and did not influence the 1- and 3-year patient survival. If these findings are validated, they may have significant impact on post-transplantation immunosuppression management in male patients.

Time of the day for 11beta-HSD1 inhibition plays a role in improving glucose homeostasis in DIO mice.

AIMS: The physiological effects of glucocorticoids in a given tissue are driven by the local level of the active glucocorticoid, which is determined by two sources: the plasma cortisol in human (or corticosterone in rodents) and the cortisol produced locally through 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activity. Because of the circadian variation of plasma glucocorticoids, the pharmacological efficacy of 11beta-HSD1 inhibition may depend on the time of the day for inhibitor administration. METHODS: The circadian profile of corticosterone was established in lean and diet-induced obesity (DIO) C57BL/6 mice from blood collected at different time of the day. 11beta-HSD1 enzyme activity was also measured throughout the day in DIO mice. To determine the optimal timing for administration of an 11beta-HSD1 inhibitor to obtain maximum efficacy, we used a DIO mouse model and a small molecule inhibitor of 11beta-HSD1 from our thiazolinone series. Based on the circadian profile of corticosterone obtained, we administered the 11beta-HSD1 inhibitor to these animals at different times of the day and evaluated the effects on plasma glucose levels and glucose tolerance. RESULTS: We report that corticosterone circadian rhythm was similar between lean and DIO C57BL/6 mice, and 11beta-HSD1 enzyme activity undergoes minimal variations throughout the day. Interestingly, the compound exhibited maximum efficacy if dosed in the afternoon when plasma corticosterone is high; the morning dosing when plasma corticosterone is low did not lead to efficacy. CONCLUSION: These data suggest that because of the circadian rhythm of circulating glucocorticoids, the time of the day for 11beta-HSD1 inhibitor administration is important in achieving efficacy.

Severe autologous GVHD after hematopoietic progenitor cell transplantation for multiple myeloma.

GVHD is a recognized complication of autologous hematopoietic progenitor cell transplantation (HPCT), but has typically been reported to respond well to primary therapy with corticosteroids. In this study, we report the development of severe autologous GVHD in five patients who underwent HPCT for multiple myeloma. In all cases, response to corticosteroids was unsatisfactory and three of these patients ultimately died from complications that ensued from prolonged immunosuppressive therapy. Severe autologous GVHD occurred only in patients transplanted for multiple myeloma and was observed at a much higher frequency in patients undergoing their second HPCT. The severity of this syndrome primarily in patients undergoing second HPCTs suggests that repetitive exposure to high-dose therapy may compromise endogenous peripheral regulatory mechanisms and predispose these patients to autoimmunity. Given the evolving role of second autologous transplantations in the therapeutic armamentarium for multiple myeloma, consideration of this potential toxicity may be appropriate when considering treatment options for these patients.

Case report: Delayed enhancement on cardiac MRI in a patient with multiple myeloma without amyloidosis.

We report the first observation of diffuse subendocardial and myocardial delayed enhancement on cardiac MRI in a 50-year-old patient with recurrent multiple myeloma but without evidence of amyloidosis. She presented with advanced heart failure and severe restrictive cardiomyopathy. Myocardial biopsy revealed endomyocardial fibrosis. The case was associated with development of multiple arterial and venous thromboses and a fatal course. Because of the fatal outcome, the prognostic significance of delayed enhancement on MRI in multiple myeloma patients may need to be further investigated.

Radiation induced small bowel "web" formation is associated with acquired microvascular dysfunction.

BACKGROUND AND AIMS: Radiation therapy of abdominal and pelvic solid tumours results in late intestinal toxicity of a severe nature in approximately 5% of cases. These manifestations may include ischaemia and stricture formation, which may present as "webs". These webs are likely to play a role in the pathogenesis of recurrent bowel obstruction. The mechanisms of microvascular injury to the bowel in the setting of radiation have not been defined. We hypothesised that microvascular dysfunction with impaired vasodilation to acetylcholine (Ach) would be an acquired pathophysiological abnormality in radiation and "web" formation. METHODS: A 40 year old patient treated with radiation, two years previously, for an anal squamous cell cancer presented with recurrent small bowel obstruction. "Webs" in the distal ileum were detected using wireless capsule endoscopy, after small bowel barium radiographs failed to demonstrate a lesion. Following resection, freshly isolated 50-150 mum diameter arterioles from the "web" and adjacent normal calibre bowel were analysed with histology and microvessel physiological studies. RESULTS: After constriction (30-50%) with endothelin, dilation to graded doses of Ach (10(-9)-10(-4) M) was observed in vessels dissected from the stricture and the adjacent normal calibre area. Ach dilation was reduced in vessels from "web" (mean diameter 7 (2)%; n = 3, p < 0.01) compared with the adjacent unaffected bowel (mean diameter 85 (5)%). Dihydroethidine and dichlorofluorescein diacetate intravital staining demonstrated increased reactive oxygen species production in microvessels from "web" compared with adjacent normal calibre bowel. Histology from the strictured bowel demonstrated narrowing of the arterial lumen due to intimal and muscularis propria fibrosis, with endothelial preservation. CONCLUSIONS: External radiation is associated with acquired microvascular endothelial dysfunction and "web" formation in the small bowel.

Local delivery of c-myc neutrally charged antisense oligonucleotides with transport catheter inhibits myointimal hyperplasia and positively affects vascular remodeling in the rabbit balloon injury model.

Myointimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA) is a key component of the process of restenosis. The c-myc is a critical cell-cycle division protein involved in the formation of neointima. We evaluated the long-term impact of local delivery of c-myc neutrally charged antisense oligonucleotides (Resten-NG) on myointimal hyperplasia after PTCA in a rabbit model. PTCA was performed in the iliac arteries of 25 New Zealand white rabbits, using a Transport catheter at 8 atm for 30 sec, three times; 500 microg Resten-NG (n = 11) or saline (n = 14) was delivered to the PTCA site at 2 atm with the outer balloon for 2 min. The diet was supplemented with 0.25% cholesterol for 10 days before and 60 days after PTCA. Angiography was performed at harvest, and vessels were fixed in formalin, processed, and stained with hematoxylin and eosin (H&E) and Movat. Quantitative angiography showed that local delivery of antisense c-myc at PTCA reduced late luminal loss from 1.8 +/- 0.30 mm in control animals to 0.90 +/- 0.30 mm in the treatment group (P = 0.001). Histological analysis by planimetry showed that intimal areas were 1.67 +/- 0.44 mm(2) and 0.82 +/- 0.32 mm(2) in the control and antisense delivery groups, respectively (P < 0.05). We conclude that local delivery of Resten-NG inhibited myointimal hyperplasia after PTCA in cholesterol-fed rabbits for up to 60 days.

Evaluation of micrometastases in sentinel lymph nodes of cutaneous melanoma: higher diagnostic accuracy with Melan-A and MART-1 compared with S-100 protein and HMB-45.

Accurate diagnosis of micrometastases in sentinel lymph nodes of cutaneous melanoma is critical for proper clinical management. S-100 protein and HMB-45 are the traditional immunomarkers widely used for this purpose. However, the interpretation of micrometastases by these markers is difficult with significant reduction in the diagnostic accuracy. S-100 protein demonstrates immunoreactivity for other nonmelanoma cells and obscures nuclear details, which are crucial for the interpretation of single cell metastases. We compared the new melanoma markers, Melan-A (clone A103) and MART-1 (clone M2-7C10), with S-100 protein and HMB-45, by examining 77 formalin-fixed paraffin-embedded sections of sentinel lymph nodes from 13 cases of primary cutaneous melanoma. CD68 (PG-M1) and hematoxylin-eosin-stained sections were also studied. Four pathologists interpreted the staining pattern after concealing the identity of each immunomarker. Az values (area under receiver operating characteristic curve) with receiver operating characteristic curve were higher with Melan-A (0.9742) and MART-1 (0.9779) compared with S-100 protein (0.8034) and HMB-45 (0.8651), demonstrating a higher diagnostic accuracy with Melan-A and MART-1 with superior detection of melanoma micrometastases. Melan-A and MART-1 showed sharp cytoplasmic immunoreactivity, almost exclusively restricted to the melanoma cells. Therefore, Melan-A and MART-1 are recommended for the evaluation of micrometastases in sentinel lymph nodes of cutaneous melanoma as a routine alternative to S-100 protein and HMB-45.

Sinonasal disease in patients with sarcoidosis.

Sarcoidosis is a chronic granulomatous disease of unclear etiology with a propensity to involve the lower respiratory tract, but may also involve the upper respiratory tract. Histologically, it is characterized by non-caseating granulomas of various organ systems. Although nasal and sinus involvement is uncommon, patients with sarcoidosis presenting with nasal and sinus complaints may have sinonasal sarcoidosis or simply rhinosinusitis. We reviewed the cases of six patients with pulmonary sarcoidosis who developed chronic sinonasal disease. All six patients had intranasal findings consistent with sinonasal sarcoidosis, but only four had histologic evidence of sinonasal sarcoidosis. These four patients continue to require extensive therapy including topical steroids, systemic steroids, intralesional steroid injections, and nasal irrigations. We conclude that patients with histologically proven sinonasal sarcoidosis present a significant therapeutic challenge because their symptoms and physical findings are often persistent despite aggressive medical and surgical therapy. Their recalcitrant sinonasal disease is thought to result from the destruction of cilia and mucus-producing glands by the granulomatous process.

Immunohistochemistry and molecular detection of nodal micrometastases in pancreatic cancer.

PURPOSE: Assays based on polymerase chain reaction (PCR) demonstrate mutated Kiras in the regional nodes of a majority of patients with node-negative stage I or II (T(1-3), N(0), M(0)) pancreatic adenocarcinoma. The hypothesis that the presence of mutated Kiras equates with micrometastases has not been validated by detailed histologic examination nor has an impact on survival been demonstrated. METHODS: We examined the paraffin blocks of the primary tumor and regional lymph nodes from all 30 patients from 1984 to 1998 with resected pN(0) stage I or II pancreatic adenocarcinoma. DNA was analyzed for mutations in codon 12 of the Kiras oncogene by PCR and restriction digest with BstN1 (RFLP). All nodes were examined by histology of 4 hematoxylin and eosin-stained step sections and immunohistochemistry (HPE/IHC) with AE3/AE1 epithelial cell marker antibody. RESULTS: Examination of the regional lymph nodes of the 30 patients demonstrated nodal metastases in 9 (30%) by step-section histology alone, 14 (46.7%) by HPE/IHC, 19 (63.3%) by PCR/RFLP, and 25 (83.3%) by a combination of PCR/RFLP and HPE/IHC. Seven cases were HPE/IHC positive yet PCR/RFLP negative while 10 cases were PCR/RFLP positive and HPE/IHC negative. Median survival (months) did not differ if nodes were negative or positive by HPE/IHC (20.5 vs 17.5) or PCR/RFLP (20.0 vs 19.0) or a combination of these techniques (25 vs 18.5). CONCLUSIONS: A great majority (83.3%) of patients with pathologic stage I or II pancreatic cancer had metastases in their regional nodes. Step-sectioning with immunohistochemistry and PCR/RFLP are complementary tests in detection of metastatic cancer cells. Nodal micrometastases did not adversely influence survival.

Methods of cytologic smear preparation and fixation. Effect on the immunoreactivity of commonly used anticytokeratin antibody AE1/AE3.

OBJECTIVE: To evaluate the effect of fixation and methods of cytologic smear preparation on the immunoreactivity of commonly used anticytokeratin antibody AE1/AE3. STUDY DESIGN: Scrape cytology smears and formalin-fixed, paraffin-embedded tissue sections (FPTS) of 20 unfixed, fresh specimens submitted for intraoperative consultation were studied by the immunoperoxidase method. In addition to the morphologic examination, the smears and FPTS were evaluated for intensity and proportion scores. For each specimen, two scrape cytology smears were wet fixed in 95% ethanol, and 12 smears were air dried without fixation. Air-dried smears were either postfixed after rehydration in saline or fixed directly without rehydration by one of the three fixatives: alcoholic formalin, 95% ethanol with 5% acetic acid or 95% ethanol. RESULTS: Both intensity and proportion scores were higher with rehydrated, air-dried smears as compared to those without rehydration and were comparable to those with wet-fixed smears and FPTS. In the rehydrated group, the optimum results were achieved when the smears were postfixed with alcoholic formalin. CONCLUSION: The method of preparation and fixation had variable effects on the immunoreactivity of anticytokeratin antibody AE1/AE3. The optimum results were achieved with saline-rehydrated, air-dried smears post-fixed in alcoholic formalin. To evaluate the role of inter-sample variation, further, larger studies are recommended on this and other antibodies before applying them to different types of cytologic smears.

p21WAF1 expression is associated with improved survival after adjuvant chemoradiation for pancreatic cancer.

BACKGROUND: Cell cycle arrest after DNA damage is partly mediated through the transcriptional activation of p21(WAF1) by the p53 tumor suppressor gene. p21(WAF1) and p53 are both critical in maintaining cell cycle control in response to DNA damage from radiation or chemotherapy. Therefore, we examined the role of p21(WAF1) and p53 in the determination of outcome for patients who receive radiation and/or chemotherapy for pancreatic cancer. METHODS: p21(WAF1) and p53 protein expression were determined (with the use of immunohistochemistry) in specimens from 90 patients with pancreatic cancer. Forty-four patients underwent surgical resection, and 46 patients had either locally unresectable tumors (n = 9 patients) or distant metastases (n = 37 patients). Seventy-three percent of the patients who underwent resection and 63% of the patients who did not undergo resection received radiation and/or chemotherapy. RESULTS: p21(WAF1) expression was present in 48 of 86 tumors (56%) and was significantly (P<.05) associated with advanced tumor stage. Median survival among patients with resected pancreatic cancer who received adjuvant chemoradiation with p21(WAF1)-positive tumors was significantly longer than in patients with no p21(WAF1) staining (25 vs. 11 months; P = .01). Fifty of 89 tumors (56%) stained positive for p53 protein. p53 overexpression was associated with decreased survival in patients who did not undergo resection. CONCLUSIONS: Normal p21(WAF1) expression may be necessary for a beneficial response to current adjuvant chemoradiation protocols for pancreatic cancer. Alternate strategies for adjuvant therapy should be explored for patients with pancreatic cancer who lack functional p21(WAF1).

Endoluminal reconstruction of the arterial wall with endothelial cell/glue matrix reduces restenosis in an atherosclerotic rabbit.

OBJECTIVES: The objectives of this study were 1) to improve the attachment of reimplanted endothelial cells (EC) using a fibrin glue, and 2) to assess the impact of endothelial reseeding on restenosis eight weeks after balloon angioplasty. BACKGROUND: A possible mechanism contributing to restenosis after balloon angioplasty is the loss of the EC lining. Previous attempts to reseed EC had little effect due to rapid loss of the seeded cells. METHODS: Twelve atherosclerotic rabbits were subjected to angioplasty of iliac arteries and reseeding procedure. One iliac artery was subjected to EC/glue reconstruction and a contralateral site to EC seeding without glue. The animals were sacrificed after 4 h. In another series 12 rabbits were treated in the same fashion and were restudied at eight weeks. Additionally, in 10 animals one iliac was subjected to glue treatment, and another served as control. RESULTS: Histological examination demonstrated the ability of this method to reattach the EC/glue matrix circumferentially to 68.0 +/- 6.7% of the arterial wall in comparison with 13.5 +/- 3.9% reattachment after EC seeding. Morphometry at eight weeks showed that the lumen area was significantly greater in the EC/glue group (1.23 +/- 0.35 mm2) than in the EC seeding alone (0.65 +/- 0.02 mm2) and 0.72 +/- 0.41 mm2 in the glue group. This was principally accounted for by the statistically significant differences in the intimal area (0.76 +/- 0.18 mm vs. 1.25 +/-0.26 mm2 and 1.01 +/- 0.53 mm2, respectively). CONCLUSIONS: The attachment of EC after angioplasty can be greatly improved with fibrin glue matrix. The near 70% endothelial coverage achieved by this method resulted in a significant reduction of restenosis in atherosclerotic rabbit.

In vitro studies and modeling of a controlled-release device for root canal therapy.

Endodontic disease is caused primarily by bacteria that interact with periradicular host tissues. Therefore, treatment of endodontic disease aims at the exclusion of bacteria from the root canal system. This work focused on in vitro studies and modeling of a controlled-release device for delivering antimicrobial agents in root canals. A cylindrical, needle-shaped device was prepared consisting of a matrix core and a polymer coating, loaded with 30-45% chlorhexidine (CHX). The composition of the core, a blend of water-permeable polymers, and the thickness of the coating were tailored to impart various release rates. A relatively steady release rate for over 40 days after an initial burst was achieved using a formulation for long-term release, which is desirable for establishing and maintaining the necessary therapeutic levels. Mathematical models were developed for both in vitro and in vivo drug release into a liquid of limited volume, taking into account a moving boundary of the dispersed drug and a time-dependent boundary condition. A concentration-dependent effective diffusion coefficient was used to count increased porosity as the solid drug had dissolved. The finite element method and computer programs were applied to solve the differential equations and predict the in vitro and in vivo release kinetics. The model prediction agreed well with the in vitro experimental data and provided guidance for designing the device for in vivo release in root canals. The result of in vitro antimicrobial tests, performed using a bovine tooth model, suggested that the device was effective in reducing growth of microbes.

NOX 100, a nitric oxide scavenger, enhances cardiac allograft survival and promotes long-term graft acceptance.

BACKGROUND: We examined the role of nitrosative stress in allograft destruction. METHODS: Rats undergoing cardiac transplants received NOX-100, a water-soluble nitric oxide (NO) scavenger with antioxidant properties, with or without low-dose cyclosporine (CsA). Graft survival, NO production, and nuclear factor kappa B (NF-kappaB) activity were studied. RESULT: Using NOX-100 daily until rejection prolonged graft survival (11.6+/-0.6 vs. 7.4+/-0.2 days; P<0.05). Daily low-dose CsA (2.5 mg/kg im) for 7 days or until rejection also prolonged survival (12.6+/-0.5 and 21.6+/-1.6 days, respectively; P<0.01 vs. Controls). Low-dose CsA for 7 days and NOX-100 for 30 days prolonged graft survival (45.0+/-4.7 days; P<0.01 vs. all groups.). NOX-100 had no effect on whole blood CsA levels. Combination therapy until Day 100 resulted in 1 graft loss at Day 116 and indefinite survival in 3 animals (>300 days), which accepted a second WF strain heart without further immunosuppressive therapy but promptly rejected a third party (ACI) cardiac allograft. NOX-100 and CsA reduced nitrate and nitrite, and combination therapy completely normalized NO through to Day 30. Electron paramagnetic resonance spectroscopic analysis demonstrated reduction of signals for nitrosylmyoglobin and nitrosyl-heme with NOX-100 and elimination of signals with CsA alone or combination therapy. Activity of myocardial NF-kappaB decreased with monotherapy vs. untreated allografts. Combination therapy resulted in further inhibition of NF-kappaB up to Day 30. The extent of graft survival correlated with the extent of NO scavenging and NF-kappaB inhibition. Short-term combination therapy had no effect on graft lymphocytic infiltrate on Days 15, 20, and 30. CONCLUSION: These data support a role for both oxidative and nitrosative stress in rejection and the immunoregulatory potential of antioxidant therapy after transplantation.

In vivo resistance of coronally induced bacterial ingress by an experimental glass ionomer cement root canal sealer.

The resistance of an experimental sealer (KT-308) to bacterial ingress was assessed in six beagle dogs. In four mandibular premolars per dog, canals were prepared, filled with condensed gutta-percha and either KT-308 or Roth 801 cement (n = 24 roots), and the pulp chambers inoculated with plaque. Two additional premolars per dog were similarly root-filled, but not inoculated (n = 12 and 11, respectively). One incisor per dog was inoculated, but not root-filled (n = 6). Dogs were terminated after 6 months, and jaw blocks were retrieved and processed for light microscopic examination of the periapical tissues. Inflammation about the inoculated roots was significantly lower (p < 0.03) for KT-308 (17%) than Roth 801 cement (46%). Inflammation about the noninoculated roots did not differ significantly between KT-308 (8%) and Roth 801 cement (36%). This study demonstrated a better functional efficacy of KT-308 than of Roth 801 cement, and validated this in vivo model for assessment of root filling materials.

Antimicrobial substantivity of chlorhexidine-treated bovine root dentin.

Previous studies have demonstrated antimicrobial substantivity in root canal dentin up to 7 days after treatment with chlorhexidine. This in vitro study assessed the antimicrobial substantivity of chlorhexidine-treated bovine root dentin over a period of 21 days. Sixty standardized bovine root sections were randomly divided into three equal groups, and their canals immersed in one of the following solutions: (i) sterile saline; (ii) 2.5% NaOCl; or (iii) 0.2% chlorhexidine (CHX). Half the specimens in each group were treated with the solution for 5 min and the other half for 7 days. After solutions were removed, the specimens were incubated at 37 degrees C in Brain Heart Infusion broth containing Enterococcus faecalis (ATCC 29212). A fresh inoculum was added to the broth every other day over a 21-day period. The canals were then enlarged with sterile burs, and the dentin shavings collected and cultured for the presence of cultivable bacteria in the dentinal tubules. Specimens treated with CHX for 7 days demonstrated significantly less dentin colonization by E. faecalis than the other specimens. CHX has potential as an intracanal medicament, if it can be applied for a period of at least 7 days.

Antimicrobial substantivity of bovine root dentin exposed to different chlorhexidine delivery vehicles.

Root canal dentin acquires antimicrobial substantivity after exposure to chlorhexidine gluconate (CHX) for 1 wk. Therefore development of a vehicle for delivery of CHX as an intracanal medication is desirable. This in vitro study assessed the efficacy of two CHX delivery vehicles, a controlled-release device and a gel, to affect antimicrobial substantivity of bovine root dentin. Sixty bovine incisor root specimens were prepared with standardized length (10 mm) and canal diameter (3.3 mm), and coated externally with nail polish. Specimens were divided into four equal groups and their canals medicated for 7 days with either: (i) an experimental controlled-release device containing 25% CHX that was immersed in sterile saline; (ii) 2% CHX gel; or (iii) Ca(OH)2 paste. Sterile saline was used as the positive control. After medication, the canals of the specimens were inoculated with Enterococcus faecalis for 21 days. Root canal dentin samples ranging in depth from 0.1 to 0.45 mm were then obtained using sterile round burs of ascending diameter. Each dentin sample was placed in a separate test tube containing Brain Heart Infusion broth and incubated for 24 h. The optical density (OD) of the broth was then measured spectrophotometrically at 540 nm. The positive control showed significantly higher mean OD values (one-way ANOVA and Tukey's Studentized Range Test; p < 0.001) than the three test groups. The CHX controlled-release device group showed significantly lower OD values than the Ca(OH)2 group; however only at dentin depths up to 0.2 mm. In contrast, the CHX gel group consistently showed significantly lower OD values than both the CHX controlled-release device and Ca(OH)2 groups. These results suggest that bovine root canals medicated with 2% CHX gel for 7 days acquire antimicrobial properties for at least 21 days.