Thomas F. Warner

Enterococcus faecalis Induces Inflammatory Bowel Disease in Interleukin-10 Knockout Mice

Germ-free interleukin-10 knockout (IL-10 KO) mice developed inflammatory bowel disease (IBD) after they were colonized with a pure culture of Enterococcus faecalis. E. faecalis not only induced IBD (primarily in colon and rectum) but rectal dysplasia and adenocarcinoma was also found in the IL-10 KO mice. Conventional (complex-intestinal flora) IL-10 KO mice developed IBD within 10 to 15 weeks of age and showed more pathology in the cecum (typhlitis) than we observed with E. faecalis-induced IBD in gnotobiotic IL-10 KO mice. Conversely, neither germ-free IL-10 mice nor IL-10 KO mice colonized as adults, with a pure culture of Candida albicans, Escherichia coli, Lactobacillus casei, L. reuteri, L. acidophilus, a Bifidobacterium sp., Lactococcus lactis, or a Bacillus sp. developed IBD during the 25- to 30-week study. E. faecalis is a common intestinal microbe of man and animals that can trigger IBD, dysplasia, and carcinoma in a genetically susceptible murine host.

MONITORING RENAL CRYOSURGERY: PREDICTORS OF TISSUE NECROSIS IN SWINE

PURPOSE To examine parameters for monitoring renal cryosurgery and correlate with histopathologic necrosis in a porcine model. Parameters include: 1) tissue temperature, 2) distance of tissue from the cryosurgical probe insertion site, and 3) inclusion of tissue by the visible iceball. MATERIALS AND METHODS Following midline incision, 6 healthy kidneys from 3 domestic pigs were treated using a nitrogen-based cryosurgical system with 3.4 mm outer diameter cryoprobe. Temperatures were monitored at 6 sites in each kidney using prototype thermocouples. Gross and histologic analysis was performed on tissue harvested from each thermosensor site 24 hours postoperatively. RESULTS All animals survived to complete the full protocol. Post-procedure bleeding was managed surgically. Histopathology revealed that complete ablation was uniformly produced at temperatures of -19.4C or lower in 13/13 tissue samples. Within 16 mm. of the probe insertion site, cells were uniformly ablated in 17/17 samples while degree of tissue death varied widely beyond this margin. Cell death was more likely found at points encompassed by the visible iceball (16/18, 89%) than those beyond it (2/18, 11%) [p <0.0001, Chi-square] while 2/18 (11%) of samples harvested from within the iceball margin were partially viable. CONCLUSIONS Temperature monitoring using thermocouples during porcine renal cryosurgery demonstrated complete homogeneous necrosis of tissues reaching -19.4C or lower. Distance beyond 16 mm. from the cryoprobe and direct visualization of the iceball proved to be less reliable predictors of tissue necrosis. Management of bleeding post-thaw was necessary in every case.

Vaccine-induced protection against 3 systemic mycoses endemic to North America requires Th17 cells in mice

Worldwide rates of systemic fungal infections, including three of the major pathogens responsible for such infections in North America (Coccidioides posadasii, Histoplasma capsulatum, and Blastomyces dermatitidis), have soared recently, spurring interest in developing vaccines. The development of Th1 cells is believed to be crucial for protective immunity against pathogenic fungi, whereas the role of Th17 cells is vigorously debated. In models of primary fungal infection, some studies have shown that Th17 cells mediate resistance, while others have shown that they promote disease pathology. Here, we have shown that Th1 immunity is dispensable and that fungus-specific Th17 cells are sufficient for vaccine-induced protection against lethal pulmonary infection with B. dermatitidis in mice. Further, vaccine-induced Th17 cells were necessary and sufficient to protect against the three major systemic mycoses in North America. Mechanistically, Th17 cells engendered protection by recruiting and activating neutrophils and macrophages to the alveolar space, while the induction of Th17 cells and acquisition of vaccine immunity unexpectedly required the adapter molecule Myd88 but not the fungal pathogen recognition receptor Dectin-1. These data suggest that human vaccines against systemic fungal infections should be designed to induce Th17 cells if they are to be effective.

Merkel cells and merkel cell tumors ultrastructure, immunocytochemistry and review of the literature

Certain monomorphic cellular tumors that occur in the dermis have been called trabecular carcinomas or Merkel cell tumors. Forty‐six cases have been reported to date and the literature on these is reviewed here, with six additional cases reported. Cytologic features include sparse cytoplasm, dispersed chromatin with inconspicuous nucleoli in round nuclei and many mitoses. Trabeeulae and pseudorosettes may be identified. Electron microscopy is required for definitive diagnosis. Like normal Merkel cells, tumor cells contain electron‐dense granules (80–200 nm), 10 mm filaments and desmosomes. Filament‐rich cytoplasmic spikes were found in four tumors. These resemble corresponding protrusions of normal Merkel cells and have not been described in other APUDomas. Cancer 52:238‐245, 1983.

Immunohistochemical detection of chromogranin and neuron-specific enolase in pancreatic endocrine neoplasms

Twenty pancreatic endocrine tumors and one duodenal somatostatinoma were stained with polyclonal antisera against eight hormones, neuron-specific (NSE), and with a monoclonal antibody against the endocrine granule protein chromogranin. Silver staining was performed on all tumors with the Grimelius method. All the tumors and all cells in the normal pancreatic islet showed NSE immunoreactivity. Chromogranin immu-noreactivity was found predominantly in the glucagon cells of normal islets, in one glucagonoma, four gastrinomas and in 10 tumors associated with multiple hormonal production, but not in five insulinomas or in one somatostatinoma. Grimelius reactivity was identical to chromogranin immunoreactivity in all cases except that more cells in the tumors and in the normal islets were positive for chromogranin. Both chromogranin and NSE were detected in a metastatic pancreatic endocrine tumor which had metastasized to a peripancreatic lymph node. The metastatic tumor failed to stain with antisera to all eight hormones. These results indicate that chromogranin and NSE are excellent general markers for pancreatic endocrine tumors and that the Grimelius stain and other related argyrophilic reactions may involve binding to chromogranin.

Vaccine Immunity to Pathogenic Fungi Overcomes the Requirement for CD4 Help in Exogenous Antigen Presentation to CD8+ T Cells Implications for Vaccine Development in Immune-deficient Hosts

Systemic fungal infections with primary and opportunistic pathogens have become increasingly common and represent a growing health menace in patients with AIDS and other immune deficiencies. T lymphocyte immunity, in particular the CD4+ Th 1 cells, is considered the main defense against these pathogens, and their absence is associated with increased susceptibility. It would seem illogical then to propose vaccinating these vulnerable patients against fungal infections. We report here that CD4+ T cells are dispensable for vaccine-induced resistance against experimental fungal pulmonary infections with two agents, Blastomyces dermatitidis an extracellular pathogen, and Histoplasma capsulatum a facultative intracellular pathogen. In the absence of T helper cells, exogenous fungal antigens activated memory CD8+ cells in a major histocompatibility complex class I–restricted manner and CD8+ T cell–derived cytokines tumor necrosis factor α, interferon γ, and granulocyte/macrophage colony-stimulating factor–mediated durable vaccine immunity. CD8+ T cells could also rely on alternate mechanisms for robust vaccine immunity, in the absence of some of these factors. Our results demonstrate an unexpected plasticity of immunity in compromised hosts at both the cellular and molecular level and point to the feasibility of developing vaccines against invasive fungal infections in patients with severe immune deficiencies, including those with few or no CD4+ T cells.

Phase I Clinical Trial of the Immunocytokine EMD 273063 in Melanoma Patients

PURPOSE To evaluate the safety, toxicity, in vivo immunologic activation, and maximum-tolerated dose (MTD) of EMD 273063 (hu14.18-IL-2) in patients with metastatic melanoma. PATIENTS AND METHODS Thirty-three patients were treated with EMD 273063, a humanized anti-GD2 monoclonal antibody (mAb) linked to interleukin-2 (IL-2). EMD 273063 was given as a 4-hour intravenous infusion on days 1, 2, and 3 of week 1. Patients with stabilization or regression of disease could receive a second course of treatment at week 5. Dose levels evaluated were 0.8, 1.6, 3.2, 4.8, 6.0, and 7.5 mg/m2/d. RESULTS Nineteen of 33 patients completed course 1 with stable disease and went on to receive course 2. Eight patients had stable disease on completion of course 2. Grade 3 adverse events included hypophosphatemia (11 patients), hyperglycemia (three patients), hypotension (two patients), thrombocytopenia (one patient), hypoxia (three patients), elevated hepatic transaminases (two patients), and hyperbilirubinemia (one patient). Opioids were required for treatment-associated arthralgias and/or myalgias during 17 of 52 treatment courses. No grade 4 adverse events were observed. Dose-limiting toxicities at the MTD included hypoxia, hypotension, and elevations in AST/ALT. Grade 3 toxicities were anticipated based on prior studies of IL-2 or anti-GD2 mAbs, and all resolved. Immune activation was induced, as measured by lymphocytosis, increased peripheral-blood natural killer activity, and cell numbers, and increased serum levels of the soluble alpha chain of the IL-2 receptor complex. CONCLUSION Treatment with the immunocytokine EMD 273063 induced immune activation and was associated with reversible clinical toxicities at the MTD of 7.5 mg/m2/d in melanoma patients.

|[gamma]||[delta]| T cell-induced nitric oxide production enhances resistance to mucosal candidiasis

Despite the prevalence of γδ T cells in mucosae that are typically colonized by Candida albicans, little is known of the possible role of these cells in resistance to candidiasis. A sharp increase in the number of γδ T cells and macrophages following intraperitoneal inoculation of mice with C. albicans led us to examine the role of these cells in the immune response to C. albicans. We show that the γδT cells enhance macrophage nitric oxide (NO) production and anti-candida activity, in vitro. We also propose that the γδ T cells regulate macrophage function during candidiasis in vivo as well, because depletion of these cells abrogated inducible NO synthase expression in mucosae and enhanced murine susceptibility to candidiasis.

MR-guided angioplasty of renal artery stenosis in a pig model: a feasibility study.

PURPOSE To test the hypothesis that magnetic resonance (MR) imaging can guide the percutaneous treatment of renal artery stenosis in a pig model. MATERIALS AND METHODS Ameroid constrictors were surgically placed around six renal arteries in four pigs. After 30-36 days, all stenoses were documented by conventional x-ray aortograms. MR-guided renal angioplasty was attempted for three stenoses. For these pigs, MR angiography was performed with use of contrast-enhanced three-dimensional (3D) techniques. The authors visualized catheters by filling them with dilute 4% gadolinium and imaging with two-dimensional (2D) and 3D MR fast spoiled gradient recalled echo techniques. Under MR guidance, the authors advanced a selective catheter into the affected renal artery and crossed the stenosis with a nitinol guide wire. Angioplasty was performed with a balloon catheter filled with dilute gadolinium. Stenosis and luminal diameter measurements were compared before and after angioplasty. RESULTS After ameroid constrictor placement, four significant stenoses, one mild stenosis, and one occlusion developed. Under MR guidance, the authors achieved technical success in performing three of three (100%) attempted dilations. After MR-guided angioplasty, the mean reduction in stenosis was 35% and the mean increase in luminal diameter was 1.6 mm. CONCLUSION Use of MR guidance for the angioplasty of renal artery stenosis in pigs is feasible.

Gastrointestinal Carcinoid Tumors: Factors that Predict Outcome

Gastrointestinal (GI) carcinoids are neuroendocrine tumors originating in multiple locations throughout the GI tract. The prognosis for patients with GI carcinoid tumors is diverse. To determine the factors that significantly affect prognosis, we reviewed our experience. Between 1992 and 2000 a total of 70 patients with GI carcinoid tumors underwent surgical resection at our institution. The patients were grouped into three categories based on the origin of the carcinoid tumor: foregut, midgut, hindgut. The mean age of the patients was 56 ± 2 years. All patients with foregut carcinoids had symptoms upon presentation, whereas 61% of those with midgut carcinoids and only 37% of those with hindgut carcinoids had symptoms (p < 0.001). The factors that most strongly affected survival on univariate analysis were a symptomatic presentation and the site of origin. Patients with foregut or midgut lesions had lower 5-year disease-free survivals than those with hindgut tumors. Moreover, the size of the primary tumor and the presence of liver metastases were not independent predictors of survival. Despite the larger tumor size and the higher incidence of liver metastases, patients with foregut carcinoids appear to have the same prognosis as those with midgut carcinoids. These data therefore suggest that the outcomes of patients with carcinoid tumors are highly dependent on the presence of symptoms and the site of origin.