DFMO Improves Survival and Increases Immune Cell Infiltration in Association with MYC Downregulation in the Pancreatic Tumor Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor five-year survival rate of less than 10%. Immune suppression along with chemoresistance are obstacles for PDAC therapeutic treatment. Innate immune cells, such as tumor-associated macrophages, are recruited to the inflammatory environment of PDAC and adversely suppress cytotoxic T lymphocytes. KRAS and MYC are important oncogenes associated with immune suppression and pose a challenge to successful therapies. Here, we targeted KRAS, through inhibition of downstream c-RAF with GW5074, and MYC expression via difluoromethylornithine (DFMO). DFMO alone and with GW5074 reduced in vitro PDAC cell viability. Both DFMO and GW5074 showed efficacy in reducing in vivo PDAC growth in an immunocompromised model. Results in immunocompetent syngeneic tumor-bearing mice showed that DFMO and combination treatment markedly decreased tumor size, but only DFMO increased survival in mice. To further investigate, immunohistochemical staining showed DFMO diminished MYC expression and increased tumor infiltration of macrophages, CD86+ cells, CD4+ and CD8+ T lymphocytes. GW5074 was not as effective in modulating the tumor infiltration of total CD3+ lymphocytes or tumor progression and maintained MYC expression. Collectively, this study highlights that in contrast to GW5074, the inhibition of MYC through DFMO may be an effective treatment modality to modulate PDAC immunosuppression.

Induction of pancreatitis in mice with susceptibility to pancreatic cancer.

Chronic inflammation is known to be associated with pancreatic cancer, however a complete picture regarding how these pathologies intersect is still being characterized. In vivo model systems are critical for the study of mechanisms underlying how inflammation accelerates neoplasia. Repeat injection of cerulein, a cholecystokinin (CCK) analog, is widely used to experimentally induce acute and chronic pancreatitis in vivo. Chronic cerulein administration into genetically engineered mouse models (GEMMs) with predisposition to pancreatic cancer can induce a pro-inflammatory immune response, pancreatic acinar cell damage, pancreatic stellate cell activation, and accelerate the onset of neoplasia. Here we provide a detailed protocol and insights into using cerulein to induce pancreatitis in GEMMs, and methods to experimentally assess inflammation and pancreatic neoplasia.

Shifting trends in in vitro antibiotic susceptibilities for common bacterial conjunctival isolates in the last decade at the New York Eye and Ear Infirmary.

BACKGROUND: Bacterial conjunctivitis is one of the most common forms of ocular diseases worldwide. The purpose of this study is to determine the most common pathogens causing bacterial conjunctivitis, their in vitro susceptibility to existing antibiotics, and the changing trends in bacterial resistance to antibiotics over the last decade. METHODS: Records of all conjunctival bacterial cultures performed at the NYEEI Microbiology Laboratory from 1 January 1997 through 30 June 2008 were reviewed. Data on species of bacterial isolates and their in vitro susceptibility to the antibiotics tetracycline, trimethaprim/sulfamethoxazole (TMP/SMZ), imipenem, fluoroquinolones (ciprofloxacin, moxifloxacin, gatifloxacin), aminoglycosides (gentamicin, tobramycin), erythromycin, cefazolin, oxacillin, and vancomycin were collected. RESULTS: Review of records yielded 20,180 conjunctival bacterial cultures, 60.1% of which were culture-positive. Of the culture-positive isolates, 76.6% were gram-positive and 23.4% were gram-negative pathogens. Staphylococcus aureus was the most common gram-positive pathogen isolated, and also the most commonly isolated pathogen overall. Haemophilus influenzae was the most common gram-negative pathogen. A significant increase in the percentage of methicillin-resistant Staphylococcus aureus (MRSA) was observed in the course of 11.5 years. The highest levels of antibiotic resistance were observed to tetracycline, erythromycin, and TMP/SMZ. Gram-positive isolates were least resistant to vancomycin, and gram-negative isolates were least resistant to imipenem. The lowest broad-spectrum antibiotic resistance was observed in the case of moxifloxacin, gatifloxacin, and aminoglycosides. CONCLUSION: Staphylococcus aureus is the most common pathogen in bacterial conjunctivitis. Conjunctival bacterial isolates demonstrated high levels of resistance to tetracycline, erythromycin and TMP/SMZ. Moxifloxacin and gatifloxacin appear to be currently the best choice for empirical broad-spectrum coverage. Vancomycin is the best antibiotic for MRSA coverage.

Ocular manifestation of giant cell arteritis vs AL-amyloidosis: similar presentations but different approaches.

Light chain (AL) amyloidosis may present with the features of vasculitis, including giant cell arteritis (GCA). Similarities between GCA and AL-amyloidosis can potentially cause confusion in diagnosis, in which case, temporal artery biopsy (TAB) should be performed to make a definitive diagnosis. Herein we report a case of a bilateral anterior ischaemic optic neuropathy (AION), showing evidence of AL-amyloidosis on the temporal artery biopsy. A 75-year-old male with AL-amyloidosis secondary to monoclonal gammopathy of undetermined significance (MGUS) presented to our hospital for subacute painless progressive visual impairment. Based on his elevated inflammatory markers and his age, he was suspected to have giant cell arteritis. However, a temporal artery biopsy excluded GCA, and the Congo red staining was positive for amyloid deposition. This present case reveals that AL-amyloidosis may present with visual impairment, high inflammatory markers, and involvement of temporal arteries, concerning for GCA. TAB with Congo red staining is found to be crucial for making the correct diagnosis.

Differential Expression of Polyamine Pathways in Human Pancreatic Tumor Progression and Effects of Polyamine Blockade on Tumor Microenvironment.

Pancreatic cancer is the fourth leading cause of cancer death. Existing therapies only moderately improve pancreatic ductal adenocarcinoma (PDAC) patient prognosis. The present study investigates the importance of the polyamine metabolism in the pancreatic tumor microenvironment. Relative mRNA expression analysis identified differential expression of polyamine biosynthesis, homeostasis, and transport mediators in both pancreatic epithelial and stromal cells from low-grade pancreatic intraepithelial neoplasia (PanIN-1) or primary PDAC patient samples. We found dysregulated mRNA levels that encode for proteins associated with the polyamine pathway of PDAC tumors compared to early lesions. Next, bioinformatic databases were used to assess expression of select genes involved in polyamine metabolism and their impact on patient survival. Higher expression of pro-polyamine genes was associated with poor patient prognosis, supporting the use of a polyamine blockade therapy (PBT) strategy for inhibiting pancreatic tumor progression. Moreover, PBT treatment of syngeneic mice injected intra-pancreatic with PAN 02 tumor cells resulted in increased survival and decreased tumor weights of PDAC-bearing mice. Histological assessment of PBT-treated tumors revealed macrophage presence and significantly increased expression of CD86, a T cell co-stimulatory marker. Collectively, therapies which target polyamine metabolism can be used to disrupt tumor progression, modulate tumor microenvironment, and extend overall survival.

Changing causes of enucleation over the past 60 years.

BACKGROUND: The indications for enucleation have changed significantly over the past 60 years. We conducted a clinicopathologic study of enucleated globes to determine how and why the indications for enucleation have changed over time. METHODS: This retrospective review examined the pathology reports for 3,264 enucleated globes submitted to the Doheny Eye Institute between 1950 and 2006. Three years per decade were examined to generate a representative pool of specimens for each decade. Although the data for the 2000s were only available up to 2006, the data for this decade are drawn from 3 sample years as are all other decades. Pathology reports were reviewed for demographic information (age, sex, and ethnicity), clinical history prior to enucleation, and pathologic findings and diagnoses. Specimens were grouped according to the reason for enucleation into the following categories: atrophic/phthisis bulbi, congenital, glaucoma, infection, longstanding retinal detachment, trauma, tumor, uveitis, and other. RESULTS: During the study period, there were 3,264 enucleated globes. Overall, the total number of enucleations decreased over time from a peak of 1,014 in the 1960s to 275 in the 2000s. Glaucoma was the most common reason for enucleation during the 1950s (23%, 127 globes) and 1960s (31%, 315 globes). However, glaucoma steadily decreased over the following decades, and was responsible for only 8% (23 globes) of enucleations in the 2000s. Neovascular glaucoma (including glaucoma secondary to retinal vein occlusion and diabetic neovascularization) accounted for 21% (27 globes) of enucleations in the 1950s. By the 2000s, this number was 57% (13 globes). Trauma-related glaucoma accounted for 34% (43 globes) of all enucleations due to glaucoma in the 1950s, and 0% (0 globes) in the 2000s. Enucleation of globes with intraocular neoplasms accounted for 14% (79 globes) of total enucleations in the 1950s, 33% (120 globes) in the 1990s, and 51% (141 globes) in the 2000s. Uveal melanoma was the main intraocular neoplasm in the 1950s (77%, 60 globes), and retinoblastoma was the primary tumor in the enucleated globes of the 2000s (69%, 97 globes). CONCLUSIONS: Improved medical and surgical treatment of conditions that lead to end-stage eye disease have led to a decrease in total enucleated globes. This is particularly evident for glaucoma. Changing demographics in Los Angeles and referral patterns are most likely responsible for the increase in retinoblastoma. The absolute number of enucleations secondary to neoplasms has not decreased over time, despite an increase in globe-conserving treatments such as chemotherapy and radioactive plaques.

Ocular erythema nodosum leprosum: An immunohistochemical study.

Episcleritis, scleritis, and anterior uveitis are common clinical manifestations of ocular leprosy. Erythema nodosum leprosum (ENL) is an acute, exaggerated systemic immunological reaction that complicates the course of this chronic indolent disease. We present an ocular immunohistochemical study of severe form of ENL involving even the ciliary body and choroid resulting in the perforation of the globe on the initiation of anti leprosy therapy. We used CD-3, CD-68, S-100, and CD-20 for immunohistochemistry. It revealed plenty of CD-3-positive T-cells and CD-68-positive macrophages and a few S-100 and CD-20-positive cells. The inflammatory exudates stained positive for IgG and IgM. The diagnosis was ocular ENL.

Granulomatous Mastitis: A Rare Case with Sjogren's Syndrome and Complications.

Granulomatous mastitis (GM) is a rare benign chronic inflammatory process of the breast in reproductive aged females. Although considered idiopathic in many cases, it has been associated with other conditions. Herein we report a highly complex and interesting case of GM in a young female with Sjogren's syndrome. We also review the literature and discuss challenges pertaining to the management of patients with similar risk factors. According to our knowledge, this is the third case documenting the co-occurrence of GM and Sjogren's syndrome. We focus on the challenges and complications of GM in the context of an autoimmune disease. With evidence from our patient's disease course and support from the literature, we advocate the use of corticosteroids for GM to prevent complications in patients with additional risks factors such as an autoimmune disease.

Immunohistochemical study of chronic nongranulomatous anterior uveitis in juvenile idiopathic arthritis.

PURPOSE: To provide a detailed immunohistochemical analysis of juvenile idiopathic arthritis (JIA)-associated anterior uveitis. DESIGN: Interventional case report. PARTICIPANT: One patient. INTERVENTION: A 12-year-old patient had recurrent pauciarticular JIA and smoldering anterior uveitis in the right eye. Despite treatment with local and systemic corticosteroids and an anti-tumor necrosis factor agent, the right eye became hypotonous and painful and eventually was enucleated. The clinical history and histopathologic and immunohistochemical analyses of the enucleated globe were reviewed. MAIN OUTCOME MEASURES: Histopathologic and immunohistochemical features of JIA-associated anterior uveitis. RESULTS: The iris and ciliary body showed nongranulomatous chronic inflammation predominantly made up of plasma cells, Russell bodies, and plasmacytoid lymphocytes. The ciliary processes and pars plana ciliaris showed focal aggregates of CD20-positive cells with several CD3- and CD8-positive cells and occasional CD4- and CD68-positive cells. Pancytokeratin stain showed ciliary epithelial proliferation admixed with lymphocytes. The iris revealed kappa-positive cells within the stroma and lambda-positive cells on the surface. The iris infiltrate primarily was made up of immunoglobulin (Ig) G-positive cells with occasional IgA- and IgM-positive cells. The anterior chamber exudate was mainly positive for IgG and IgA. CONCLUSIONS: The immunohistochemical findings suggest that JIA-associated nongranulomatous iridocyclitis is a primarily B-cell-infiltrative process.

Myxofibrosarcoma of the orbit: an underrecognized entity? Case report and review of the literature.

PURPOSE: To describe the clinicopathologic and immunohistochemical features of a rare case of myxofibrosarcoma (MFS) involving the orbit. DESIGN: Retrospective interventional case report and review of literature. METHODS: The clinical history, systemic imaging studies, and histopathologic results of the orbital biopsy are reviewed. MAIN OUTCOME MEASURES: Clinical, radiologic, and histologic features of MFS. RESULTS: A 63-year-old Chinese male sought treatment for a history of rapidly progressive proptosis associated with decreased vision in the left eye over 10 days. Magnetic resonance imaging of the orbit revealed a homogenous mass extending to the optic canal. The mass was excised partially and was submitted for histologic and ultrastructural examination. Histologic testing revealed features of MFS with characteristic diffuse proliferation of atypical, spindle-shaped fibroblasts with prominent myxoid stroma. Tumor cells were reactive for vimentin. Ultrastructural examination revealed spindle-shaped tumor cells, rich in dilated rough endoplasmic reticulum and prominent vacuoles. CONCLUSIONS: For historical reasons, MFS involving the orbit is likely to be underrecognized and categorized erroneously as malignant fibrous histiocytoma or other soft tissue sarcoma. Although orbital MFS is an extremely rare diagnosis, ophthalmologists should be aware of the possibility and consider a diagnosis of MFS when an adult seeks treatment for an orbital tumor with aggressive growth.

Primary renal angiosarcoma with extensive necrosis: a difficult diagnosis.

Angiosarcoma of the kidney is an exceedingly rare and aggressive neoplasm. Very few cases have been reported in the English literature to date. We report a case of primary renal angiosarcoma with extensive necrosis and discuss its diagnostic difficulties. An 86-year-old male presented with a 12 cm necrotic renal mass and multiple pulmonary and hepatic nodules. A CT guided renal biopsy revealed extensive necrosis and few vascular channels lined by malignant endothelial cells. Diagnosis was given on a morphologic base and proven by an immunohistochemical study. Primary renal angiosarcoma should be included among the differential diagnosis of necrotic renal lesions.

α-smooth muscle actin-positive fibroblasts correlate with poor survival in hepatocellular carcinoma.

The current case-control study was conducted to assess the value of activated fibroblasts with high α-smooth muscle actin (α-SMA) expression as an indicator of survival in hepatocellular carcinoma (HCC) patients. In total, 47 patients diagnosed with HCC who underwent a liver biopsy at the Veterans Affairs Medical Center (Memphis, TN, USA) between January 2000 and December 2009, and 10 age- and gender-matched controls with normal liver biopsy samples were included in the study. The immunohistochemical staining for α-SMA was performed and classified qualitatively and quantitatively within tumor stroma and perisinusoidal spaces. HCC patients with high qualitative and quantitative expression of α-SMA revealed a statistically significant negative correlation with 3-year [odds ratio (OR), 0.021 and 0.111; P=0.001 and 0.0302, respectively] and 1.5-year (OR, 0.040 and 0.051; P=0.0330 and 0.0492, respectively) survival rates in comparison with low expression. The results of the present study demonstrated a predictive role of high qualitative and quantitative expression in activated fibroblasts for poor survival in patients with HCC.

MYC Negative Rectal B-Cell Lymphoma, Unclassifiable, with Features Intermediate between Diffuse Large B-Cell Lymphoma and Burkitt's Lymphoma in an Immunocompetent Patient.

B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt's lymphoma (BLUI) is a recently added entity to the World Health Organization (WHO) classification to address a grey zone between large B-cell lymphoma (DLBCL) and Burkitt's lymphoma (BL). These are rare aggressive lymphomas, which were previously also known as Burkitt's-like lymphoma (BLL). BL and BLUI/BLL of the colon mostly involve the ileocecal region. In the rectum, BL and BLUI/BLL almost always affect patients with the acquired immune deficiency syndrome (AIDS). We report the first case of rectal BLUI/BLL in an immunocompetent patient who is also negative for MYC abnormalities and the EBV early RNA (EBER) in situ hybridization.

The role of TLR4 activation in photoreceptor mitochondrial oxidative stress.

PURPOSE: Herein the authors investigated whether the activation of Toll-like receptors (TLRs) in the innate immune response causes retinal photoreceptor oxidative stress and mitochondrial DNA (mtDNA) damage. METHODS: On day 5 after injection of complete Freund's adjuvant containing heat-killed Mycobacterium tuberculosis (CFA), retinas were submitted to polymerase chain reaction (PCR) array focused on the TLR signaling, or apoptosis, pathway. CFA-mediated TLR4 activation, oxidative stress, and mtDNA damage were determined in B10.RIII and knockout (KO) mice (recombination activation gene [Rag] 1(KO), TLR4(KO), myeloid differentiation primary response gene 88 [MyD88](KO), tumor necrosis factor [TNF]-α(KO), or caspase 7(KO) mice) using quantitative real-time PCR, enzyme-linked immunosorbent assay, Western blot analysis, and immunohistochemistry. The mycobacterial DNA load on the retina, brain, liver, and spleen was determined by real-time PCR after intracardiac perfusion. RESULTS: PCR array demonstrated the upregulation of TLRs and their signaling molecules in retinas of CFA-injected mice compared with those of control animals without inflammatory cell infiltration in the retina and uvea. Mycobacterial DNA was detected in the retinas of CFA-injected mice. Retinas of CFA-injected animals showed oxidative stress and mtDNA damage, primarily in the photoreceptor inner segments. Upregulated TLR4 was localized with CD11b(+)MHCII(+) cells but not with GFAP(+) astrocytes. This oxidative stress/damage was similar in CFA-injected Rag1(KO) mice compared with wild-type controls. Such damage was absent in the retinas of CFA-injected TLR4(KO), MyD88(KO), and TNF-α(KO) mice. CFA-mediated inducible nitric oxide synthase expression in the retina was significantly decreased in TNF-α(KO) mice. CONCLUSIONS: Retinal photoreceptors are susceptible to mitochondrial oxidative stress/mtDNA damage in robust TLR4-mediated innate immune response.

Isolated uveal tuberculoma masquerading as an intraocular tumor in an immunocompetent patient-a clinical-pathologic study with diagnosis by PCR.

PURPOSE: The aim of this article is to report a case of uveal tuberculoma simulating an intraocular tumor in which the diagnosis was only possible by polymerase chain reaction (PCR) analysis. METHOD: This is a case report. RESULTS: A36-year-old male presented with a progressive growing intraocular tumor and no history or positive test for other systemic disease. The eye eventually turned blind and painful and was enucleated. Histopathologic analysis revealed a granulomatous reaction and caseation necrosis but failed to identify any causative microorganisms. Final tuberculosis diagnosis was only possible by quantitative PCR. CONCLUSION: Isolated uveal tuberculoma can present in an otherwise healthy patient with negative systemic tuberculosis evaluation. PCR can be used to confirm tuberculosis when other methods have failed.

Expression of alpha-crystallin in retinoblastoma.

OBJECTIVE: To examine the expression of alpha-crystallin, a small heat-shock protein family, and apoptosis in retinal neoplastic cells. METHODS: Thirteen enucleated globes were included in this study, 1 with retinocytoma and 12 with retinoblastoma. Formalin-fixed paraffin-embedded tissue sections were processed for immunohistochemistry with alpha-crystallin antibodies. Apoptotic cells were detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. RESULTS: In the retinocytoma, alphaA-crystallin was expressed in the cytoplasm of all tumor cells, whereas alphaB-crystallin immunoreactivity was only weakly positive. Apoptotic cells were rarely noted in retinocytoma cells; the apoptotic index was 0.29. Examination of the retinoblastoma globes revealed 6 cases (50%) that were strongly positive for alphaA-crystallin. The mean (SD) apoptotic indices in the strongly and weakly positive cases were 3.55 (2.61) and 7.50 (2.61), respectively. The apoptotic index was significantly higher in those cases that were weakly positive for alphaA-crystallin than in those that were strongly positive (P < .05). No correlation was observed between apoptotic index and alphaB-crystallin immunoreactivity, although 50% of retinoblastomas were strongly positive for alphaB-crystallin. CONCLUSIONS: The alphaA- and alphaB-crystallins are expressed in retinoblastomas, and alphaA-crystallin expression may prevent apoptosis of neoplastic cells. Clinical Relevance Suppression of alphaA-crystallin may be useful in controlling tumor growth.