Isolation of NDM-1-producing Pseudomonas aeruginosa sequence type ST235 from a stem cell transplant patient in Italy, May 2013.

We describe the first isolation of an NDM-1-producing Pseudomonas aeruginosa in Italy. In May 2013, a patient with acute lymphoblastic leukaemia and history of prior hospitalisation in Belgrad, Serbia, underwent stem cell transplantation at a tertiary care hospital in Rome, Italy. After transplantion, sepsis by NDM-1-producing P. aeruginosa occurred, leading to septic shock and fatal outcome.

Recombinant tissue plasminogen activator injected into the vitreous cavity may penetrate the retinal veins of a porcine model of vascular occlusion.

AIM: To determine if recombinant tissue plasminogen activator (rtPA) injected into the vitreous cavity can penetrate the retinal vessels of porcine eyes with or without vascular occlusion. METHODS: Eight eyes (group I) of four pigs underwent clamping of the optic nerve flush with the globe for 90 minutes. One hour after reperfusion, one eye of each pig was injected with 75 microg of rtPA, and the fellow eye was injected with balanced salt solution (BSS). Eyes were processed for immunohistochemistry. Four additional eyes (group II) of two pigs were subjected to the same injections, but without optic nerve clamping. RESULTS: After reperfusion, the clinical picture was similar to that of a central retinal vein occlusion. Immunoperoxidase staining showed rtPA only in the retinal veins but not the retinal arteries in all eyes injected with rtPA in both groups I and II. Those eyes also showed intense rtPA staining at the level of the internal limiting membrane (ILM). No staining was seen at the level of the ILM or inside the retinal vessels in the BSS injected eyes. Immunofluorescence staining showed intense staining at the level of the ILM, but not inside the retinal vessels in the rtPA-injected eyes. CONCLUSIONS: rtPA may penetrate the retinal veins, but not the arteries of porcine eyes with and without vascular occlusion. The ILM may play a part in preventing rtPA penetration.

Cholesterol homeotasis in rats fed a purified diet.

The rate of whole bodyb cholesterol synthesis was measured in male Sprague-Dawley rats fed either a standard chow, cereal-based diet or a semi-synthetic purified diet consisting of casein, sucrose and lard. The purified diet significantly decreased daily fecal excretion of neutral and acidic sterols, the specific acitvity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, the bile acid pool size, and total daily cholesterol synthesis in the rat, while increasing plasma cholesterol concentrations and the total body content of cholesterol. The increased body content of cholesterol occurred primarily in muscle and connective tissue and not in the liver. The data demonstrate the importance of quantitating the net tissue accumulatin of cholesterol for accurate measurement of daily sterol synthesis in growing animals when sterol balance measurements are used. Tissue accumulation accounted for 7% of total daily cholesterol synthesis in rats fed the cereal diet, and 20% of daily synthesis in animals fed the purified diet.

Preoperative mobilization of circulating dendritic cells by Flt3 ligand administration to patients with metastatic colon cancer.

PURPOSE: To evaluate preoperative dendritic cell (DC) mobilization and tumor infiltration after administration of Flt3 ligand (Flt3L) to patients with metastatic colon cancer. PATIENTS AND METHODS: Twelve patients with colon cancer metastatic to the liver or lung received Flt3L (20 microg/kg/d subcutaneously for 14 days for one to three cycles at monthly intervals) before attempted metastasectomy. The number and phenotype of DCs mobilized into peripheral-blood mononuclear cells (PBMCs) were evaluated by flow cytometry. After surgical resection, metastatic tumor tissue was evaluated for DC infiltration. In vivo immune responses to recall antigens were measured. RESULTS: After Flt3L administration, on average, the total number of leukocytes in the peripheral blood increased from 5.9 +/- 1.0 x 10(3)/mm(3) to 11.2 +/- 3.8 x 10(3)/mm(3) (mean +/- SD, P: =. 0001). The percentage of CD11c(+)CD14(-) DCs in PBMCs increased from 2.4% +/- 1.8% to 8.8% +/- 4.7% (P: =.004). Delayed-type hypersensitivity (DTH) responses to recall antigens (CANDIDA:, mumps, and tetanus) showed marginally significant increases in reactivity after Flt3L administration (P: =.06, P: =.03, and P: =.08, respectively). An increase in the number of DCs was observed at the periphery of the tumors of patients who received Flt3L compared with those of patients who had not. CONCLUSION: Flt3L is capable of mobilizing DCs into the peripheral blood of patients with metastatic colon cancer and may be associated with increases in DC infiltration in the peritumoral regions. Flt3L mobilization is associated with a trend toward increased DTH responses to recall antigens in vivo. The use of Flt3L to increase circulating DCs for cancer immunotherapy should be considered.

Effect of protein kinase C inhibitors and activators on corneal re-epithelialization in the rat.

PURPOSE: To examine the ability of protein kinase C (PKC) inhibitors and activators to influence the rate of corneal re-epithelialization in the rat. METHOD: Rat corneas with 3 mm diameter central epithelial abrasions were organ-cultured in control medium or in medium with inhibitors or activators of PKC. RESULTS: In control corneas, the defect was completely re-epithelialized by 25 hr. In the presence of the PKC inhibitors staurosporine (100 nM), sphinganine (50 mumol/l), or H-7 (100 mumol/l) there were significantly larger epithelial defects than in controls after 5-25 hr of incubation. Re-epithelialization rates were similar to control corneas when the incubation medium contained HA1004 (100 mumol/l), an analogue of H-7 that is a potent inhibitor of cyclic adenosine monophosphate- and cyclic guanosine monophosphate-dependent protein kinases and a weak inhibitor of PKC. Two PKC activators, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol 12-myristate 13-acetate (PMA), were unable to enhance the rate of epithelial wound healing. CONCLUSIONS: Our results suggest that PKC activity is an important factor in regulating corneal epithelial wound healing, presumably by influencing cell migration. Moreover, the results with OAG and PMA suggest that PKC is maximally activated during re-epithelialization in this organ-culture assay.

Effect of inhibitors of arachidonic acid metabolism on corneal neovascularization in the rat.

We used computerized image analysis to evaluate quantitatively the ability of topically applied corticosteroids (dexamethasone sodium phosphate, prednisolone acetate), cyclooxygenase inhibitors (flurbiprofen, indomethacin, ketorolac), lipoxygenase inhibitors (REV 5901, esculetin, quercetin), and dual cyclooxygenase/lipoxygenase inhibitors (BW 755C, BW A540C) to reduce corneal neovascularization in the rat induced by silver/potassium nitrate cauterization. Significant decreases in the neovascular response were found with corticosteroids and cyclooxygenase inhibitors. A complete dose-response curve was performed for a representative compound from each class. Dexamethasone was found to be superior to flurbiprofen in its ability to reduce neovascularization in this model, while no significant inhibition was noted with either REV 5901 or BW 755C, even at high doses. We conclude that the corneal angiogenic response in this model can be reduced by inhibition of cyclooxygenase as well as by other mechanisms that are steroid-dependent but are, as yet, poorly defined.

Cholinergic stimulation of phosphatidic acid formation by rat cornea in vitro.

The cornea has one of the highest acetylcholine (ACh) concentrations of any tissue but the function of the ACh has remained enigmatic. During studies on corneal arachidonic acid metabolism, we observed that ACh stimulates formation of labeled phosphatidic acid in rat corneas whose phospholipids were prelabeled with [14C]arachidonate. ACh did not affect the metabolism of free [14C]arachidonate. [14C]Arachidonyl-phosphatidic acid formation was doubled after 10 min of incubation in the presence of ACh concentrations of 10(-4) M or greater. The stimulation by ACh could be completely blocked by atropine and scopolamine and partially blocked by d-tubocurarine. These studies suggest that intact rat cornea has muscarinic cholinergic receptors and that the enzymes of the inositol phospholipids pathway are present since phosphatidic acid is an obligatory intermediate in that cycle of reactions.

The effect of total-body irradiation on corneal neovascularization in the Fischer 344 rat after chemical cauterization.

Previous investigations of corneal neovascularization after irradiation yielded discordant results. Most studies indicated that new blood vessel formation in the cornea is inhibited by irradiation. However, others reported that angiogenesis after corneal cauterization is similar in both irradiated and nonirradiated animals. To assess the effect of total-body irradiation on neovascularization further, the amount of angiogenesis was determined in irradiated rats after chemically induced corneal injury. Corneal tissue was evaluated quantitatively with computerized image analysis 2, 3, or 4 days postcautery in rats perfused with India ink and gelatin immediately after death. The rats were exposed to a single dose (9 Gy) of total-body irradiation 6 days before corneal cauterization. In both the nonirradiated and irradiated rats, neovascularization increased with the duration of the postcautery interval. The amount of corneal neovascularization was not significantly different in the irradiated and nonirradiated rats at any of the postcautery intervals studied. This investigation suggests that endothelial cell migration plays a more important role than cell replication in the pathogenesis of corneal angiogenesis in the Fischer 344 rat. Moreover, the suppression of corneal angiogenesis by irradiation may be dependent on the experimental conditions and species examined.

The effect of oxygen on corneal neovascularization.

Since tissue oxygen levels are believed to play a pivotal role in new vessel growth in several situations, we studied the effect of several oxygen concentrations (0, 10, 21, 50, 75, or 100%) on corneal vascularization induced in the rat by chemical cautery. We achieved this by perfusing known concentrations of oxygen through goggles fitted over both eyes of the rat after corneal cauterization. Neovascularization was measured in flat corneal preparations with India ink-filled vessels 4 days postcautery using computerized image analysis. The angiogenic response of rats whose eyes were continuously exposed to 0-75% oxygen were not significantly different from each other. The mean response in corneas exposed to 100% oxygen was 10-21% lower than all of the other groups, and this difference was statistically significant when compared to oxygen concentrations of 0, 21 and 75%. The reason for the inhibitory effect of 100% oxygen remains to be determined, but it may represent a toxic effect of oxygen free radicals on the vascular endothelium.

Obesity and autologous stem cell transplantation in acute myeloid leukemia.

In the bone marrow transplant setting, several authors hypothesized that severely overweight patients are at increased risk of transplant-related toxicity, but different definitions of obesity, different body weight groupings and heterogeneous samples of patients were analyzed. To overcome these limitations, we retrospectively considered a homogeneous group of 54 patients (median age 36.5 years), with a diagnosis of de novo acute myeloid leukemia (AML), autografted in first complete remission (CR) with the Bu-Cy2 conditioning regimen, dosed on actual body weight. Patients were classified into three groups (obese, non-obese, underweight) using body mass index (BMI = kg/m(2)); for each group we analyzed transplant-related toxicity and mortality, overall survival and disease-free survival (OS/DFS). In spite of the relatively small number of patients, in our results high BMI appears a predictive factor for an increase of treatment-related toxicity and mortality. Moreover, 30 (55%) patients are currently alive in continuous CR, and after a median follow-up of 76.5 months (range 14-137) statistically significant differences in OS and DFS were detected between obese and non-obese groups (P = 0.012 and 0.021, respectively). Our study suggests that obesity may represent an independent risk factor for autograft in AML and further investigations are warranted.

Neovascular glaucoma associated with an iris melanoma. A clinicopathologic report.

A 48-year-old man had uniocular neovascular glaucoma, with the only apparent predisposing factor being a primary epithelioid melanoma of the iris. The new vessels disappeared following total excision of the tumor. These findings have implications regarding the mechanisms of neovasculogenesis.

Primary orbital leiomyosarcoma. A case report and review of the literature.

An 82-year-old woman presented with a five-week history of painless proptosis. Results of clinical evaluation, including computed tomography, were consistent with a benign orbital tumor. Biopsy followed by surgical resection disclosed leiomyosarcoma. We reviewed the literature and studied the clinical spectrum of this rare disease of the orbit.

Ocular cicatricial pemphigoid with granular IgA and complement deposition.

A 65-year-old woman developed chronic redness of both eyes, and, over the ensuing 2 1/2 years, she had progressive conjunctival scarring with symblepharon formation. Other mucosal surfaces were not involved. A conjunctival biopsy specimen 12 months following onset of her disease showed areas of epithelial separation from the basement membrane zone as well as subepithelial chronic inflammation and scarring. Two years later, another conjunctival biopsy specimen showed granular deposition of IgA and C3 along the epithelial basement membrane zone using direct immunofluorescent staining. Electron microscopy confirmed the presence of deposits that were morphologically consistent with antigen-antibody complexes. These findings suggest that antigen-antibody (IgA) immune-complex deposition may provide an alternative pathogenetic mechanism to basement membrane zone autoantibody formation for development of progressive conjunctival scarring.

Pyoderma gangrenosum involving the eyelid.

A 62-year-old man was seen with an ulcer of the left upper eyelid of two weeks' duration. Over the previous 40 years, similar ulcers had intermittently developed elsewhere on his skin and spontaneously resolved over several months leaving atrophic scars. A biopsy specimen of the eyelid lesion showed epidermal ulceration with acute inflammation and liquefactive necrosis of the underlying dermis. Microorganisms and vasculitis were not present. On the basis of the clinical history and a compatible biopsy specimen, the diagnosis of pyoderma gangrenosum was made. This is an uncommon, idiopathic, ulcerating skin disease that may also have ocular manifestations. Eyelid ulcers have not previously been reported, to our knowledge. The diagnosis is established by clinical history and exclusion of other causes of ulceration.

Controlled aspiration of subretinal fluid in the diagnosis of carcinoma metastatic to the choroid.

Carcinoma metastatic to the choroid was diagnosed in three patients by cytopathologic examination of subretinal fluid. The specimen was obtained using a scleral depressor modified to allow controlled aspiration of subretinal fluid through a sharp, angled, 30-gauge needle. This new method provides a useful tool for accurate diagnosis of carcinoma metastatic to the choroid in cases where the diagnosis is in question.

The effects of total lymphoid irradiation upon corneal vascularization in the rat following chemical cautery.

The effect of total lymphoid irradiation (TLI), a form of radiotherapy known to suppress the number of circulating lymphocytes, on corneal neovascularization was assessed in rats. The corneas of TLI-treated rats were cauterized with silver/potassium nitrate one day after delivering gamma irradiation in five equal fractions (10 Gy total dose). Corneal neovascularization was assessed quantitatively by computerized image analysis in corneal flat preparations 4 days after corneal injury following perfusion of the circulation with India ink. TLI reduced the total leukocyte, lymphocyte, neutrophil, and platelet counts below preirradiation levels. The number of circulating lymphocytes was reduced more than neutrophils and platelets. TLI caused a predominance of cytotoxic/suppressor lymphocytes. Tissue examinations 4 days after TLI disclosed an absence of the thymus, as well as a markedly reduced number and size of lymph nodes. TLI rats had less corneal vascularization than nonirradiated controls. Our findings are consistent with the hypothesis that leukocytes play an important role in the pathogenesis of corneal angiogenesis following chemical cautery, but this study does not indicate how TLI suppresses corneal neovascularization.

Ocular rosacea.

The demographic, clinical, and histopathologic characteristics of ocular rosacea are reviewed, with emphasis on examination of the skin in patients with external ocular disease. Since no single clinical or pathological hallmark of rosacea exists, a proposed point system for diagnosis is presented. Studies of pathogenesis reveal that no single unifying hypothesis accounts for all the expressions of ocular rosacea. Reports of treatment with tetracycline and other antibiotics demonstrate efficacy, although the mechanisms of action of these drugs are not known. More recently, other classes of drugs, including isotretinoin and hexachlorocyclohexane, have proven to be effective in dermatologic manifestations of rosacea, but effects on ocular manifestations are unknown.

Fatal pulmonary bile embolism following acute acalculous cholecystitis.

A 52-year-old man died during reoperation for bleeding after the development of acute postoperative acalculous cholecystitis. While the liver was being manipulated during surgery, the pulmonary artery diastolic pressure increased suddenly, cardiac output and blood pressure decreased, and soon afterward the patient had a cardiac arrest from which he could not be resuscitated. During resuscitative efforts, the maximum partial arterial oxygen pressure achieved was only 55 mm Hg despite 100% inspired oxygen. At autopsy, bile aggregates were found within necrotic liver parenchyma, portal and central vein branches, and numerous pulmonary arterioles and capillaries. The pulmonary arterial bile most likely resulted from embolization of inspissated hepatic bile aggregates during surgical manipulation.

The reciprocal electrophysiological influence between the nucleus tegmenti pedunculopontinus and the substantia nigra in normal and decorticated rats.

The electrophysiological characteristics of neurons of the nucleus tegmenti pedunculopontinus (PPN), in particular of those projecting to the substantia nigra (SN), and the reciprocal influence between the PPN and SN were investigated in normal and decorticated rats. In intact animals 65 of the 363 PPN recorded neurons (17.9%) were activated antidromically by SN stimulation, 96 (26.3%) were inhibited after stimulation while 43 (11.8%) were activated. In decorticated rats excitatory responses were decreased (4.8%) while antidromic and inhibitory responses did not change substantially. Electrical stimulation of the PPN induced a brief short-latency excitation of SN neurons (26/77, 33.7%) which was not modified by removing the cortex bilaterally 7-10 days prior to the recording session. This excluded the possibility that corticofugal fibers could be involved in the excitatory responses evoked by PPN stimulation in SN neurons. The latency of the antidromic response evoked in PPN cells by SN stimulation ranged from 0.5 to 12.0 ms and the estimated conduction velocity of these PPN output neurons ranged from 1.1 to less than 0.5 m/s. The electrophysiological heterogeneity of PPN cells was supported also by the fact that two types of neurons, both projecting to the SN, could be distinguished on the basis of their spontaneous firing rate and impulse waveform. The first had a low spontaneous activity (0.5-8 spikes/s) with a triphasic impulse which lasted 3-4 ms. The second had a high firing rate (15-20 spikes/s) and its impulse was usually biphasic and not longer than 3 ms.(ABSTRACT TRUNCATED AT 250 WORDS)

Autologous bone marrow transplantation in 44 patients with aggressive non-Hodgkin's lymphoma at University "La Sapienza" of Rome.

High dose therapy followed by infusion of autologous bone marrow has become a major treatment option for an increasing number of poor prognosis non-Hodgkin's lymphoma (NHL) patients. In our study we analyzed the outcome of autologous bone marrow transplantation (ABMT) in 44 high grade NHL patients transplanted at our institution between 1985 and 1992. Median age was 31 years (range 12-61); nineteen were in partial remission (PR) after first line chemotherapy and 25 in sensitive relapse (SR). Of the 25 patients transplanted in SR, 14 relapsed after a median time of 5.5 months (range 1-26), 8 are in complete remission after a median follow up of 41.5 months and three died from toxicity. Of the 19 patients grafted in PR, 11 are alive and progression free after a median follow up of 52 months, while 8 relapsed at a median time of 5 months. The overall progression free survival (PFS) projected at 6 years is 35% with a 47% PFS for patients transplanted in PR and 28% for patients in SR. In conclusion, high dose therapy and ABMT has achieved widespread use as salvage therapy for patients with relapsed/refractory high grade NHL. In particular, our experience confirms that myeloablative treatment is a safe and well tolerated procedure for patients in PR, that may be easily applied as early salvage therapy without major toxicities.