Topical delivery of diclofenac into and across equine skin from a novel liquid diclofenac epolamine formulation.

The aim was to investigate diclofenac delivery into and across equine skin in vitro using Franz diffusion cells from a novel diclofenac epolamine (DIC-EP; 1.3%) formulation and to compare the results to those of Surpass® (1% diclofenac sodium liposomal cream) and a 1% aqueous solution of diclofenac sodium. Skin was harvested from the lower legs of Freiberger geldings immediately after slaughter and sliced to a thickness of ~2 mm. Skin samples were divided into two groups [Group 1: 1 year old (n = 2) and Group 2: 6-8 years old (n = 3)]. Cumulative permeation of diclofenac in Groups 1 and 2 after 24 h using diclofenac sodium solution was 1.91 ± 0.27 and 1.76 ± 0.34 µg/cm2 , respectively. The values for Surpass® and DIC-EP were 3.2 ± 0.8/3.3 ± 0.7 µg/cm2 and 230 ± 59/89.2 ± 32.5 µg/cm2 , respectively. Thus, diclofenac permeation from DIC-EP was significantly greater and appeared to show an age-dependent effect. Mathematical modelling showed that the DIC-EP formulation significantly increased diclofenac partitioning into the skin and a linear correlation was observed between steady-state flux and the partition parameter. Greater skin deposition of diclofenac was also observed with DIC-EP. These preliminary results suggest that the DIC-EP formulation may be effective in treating inflammatory conditions in horses.

Semi-synthetic heparinoids.

New chemical-enzymatic technology based on the modification of the bacterial polysaccharide K5 from Escherichia coli leads to the synthesis of a number of heparin/heparan sulfate-like molecules with different biological activities. With this technology, two families of sulfated compounds were synthesized, which differ in their uronic acid content. The first group contains only glucuronic acid, whereas the second group contains about 50% iduronic acid following epimerization by immobilized recombinant C5 epimerase. This has led to the development of various anticoagulant and nonanticoagulant K5 derivatives endowed with different - and sometimes highly specific - antitumor, antiviral, and/or anti-inflammatory activities.

Surveillance of hospital-acquired infections in Liguria, Italy: results from a regional prevalence study in adult and paediatric acute-care hospitals.

A multi-hospital prevalence study of hospital-acquired infections (HAIs) was carried out between 19 March and 6 April 2007 in Liguria, Italy, being the first to be performed in this region. Of the 29 existing public acute hospitals, 25 took part in the investigation (86.2%). In total, 3176 patients were enrolled in the study, representing a regional average bed-occupancy rate of nearly 70%. Three-hundred and ten HAIs were diagnosed from 283 patients, with an overall prevalence of infections and cases of 9.8% and 8.9%, respectively. Prevalence varied considerably between hospitals, ranging from 0 to 24.4% [95% confidence interval (CI): 15.53-33.27]. Urinary tract infections (UTIs) (30.0%) and respiratory tract infections (RTIs) (26.1%) presented the highest relative frequency, followed by bloodstream infections (BSIs) (14.8%), surgical site infections (11.6%) and gastrointestinal infections (6.5%). Intensive care units (ICUs) and haemato-oncological units showed the highest specific prevalence of HAI, respectively 42.5% (95% CI: 34.48-50.52) and 13.3% (6.28-20.32), with RTI and BSI as the predominant infections. Spinal units (33.3%; 13.14-53.46) and functional-rehabilitation units (18.9%; 17.75-24.06) demonstrated a high rate of urinary tract infections. Uni- and multivariate analyses were performed to assess the main risk factors and conditions associated with HAI, both overall and by site. Our study provides an overall picture of the epidemiology of HAI in Liguria, which may be usefully employed as a starting point to plan and organise future surveillance and control programmes.

Decline in pneumonia and acute otitis media after the introduction of childhood pneumococcal vaccination in Liguria, Italy.

The effect of the pneumococcal conjugate vaccine immunization programme on pneumococcal-associated or potentially pneumococcal-associated hospital admissions in the Italian region of Liguria was assessed. Hospital admission rates were compared in subjects belonging to birth cohorts before and after the introduction of widespread immunization for 0 - 2-year old children with a seven-valent conjugate vaccine (PCV7). Significant reductions in hospitalization rates for all-cause and pneumococcal pneumonia and for acute otitis media were observed in subjects born after widespread uptake of the vaccine. The preventive fraction (a measure of vaccine effectiveness) ranged from 15.2% for all cause pneumonia to 70.5% for pneumococcal pneumonia. This study contributes to the growing body of information that supports the beneficial effect of PCV7 vaccination.

Emergency department syndromic surveillance system for early detection of 5 syndromes: a pilot project in a reference teaching hospital in Genoa, Italy.

Early detection is fundamental for achieving effective control of infectious disease outbreaks. We described the development of a local chief complaint emergency department (ED)-based syndromic surveillance system to improve public health response in Genoa, Italy. The five syndromes under investigation by the syndromic surveillance system were influenza-like illness (ILI), low-respiratory tract illness (LRTI), not-haemorrhagic gastroenteritis, acute hepatitis, fever-with-rash (maculo-papular or vescicular) syndrome. Syndrome coding, data capture, transmission and processing, statistical analysis to assess indicators of disease activity and alert thresholds, and signal response were operatively described. Preliminary results on ILI syndromic surveillance showed that new system allowed the activation of the alert state with a specificity of 90.3% and a sensitivity of 72.9% in predicting epidemiological relevant events, such as > or = 10 accesses to ED for ILI in 3 days. The new syndromic surveillance system allowed to alert the public health institutions 2.5 days before than the local surveillance system based on sentinel physicians and paediatricians, permitting the early activation of the necessary measures for the containment and for burden reduction of the epidemic event. It is noteworthy that the syndromic surveillance epidemic cut-off was overcome once before and 4 times after influenza outbreak detected by sentinel-based surveillance system: all episodes were contemporary with Respiratory Syncytial Virus and Parainfluenza Virus circulation, as detected by regional reference laboratory.

Molecular epidemiology and case-control approaches for management of an outbreak of hepatitis A in Liguria, Italy.

INTRODUCTION AND METHODS: Hepatitis A remains an important public health problem in low endemicity areas, because of the social and economic high burden of cyclical outbreaks. In this study we described an outbreak of HAV infection occurred in the city of Genoa and in its proximity and the viral circulation in the post-epidemic period. In order to identify risk factors associated to the illness and to determine the source of infection and the dynamics of virus evolution, we conducted an epidemiological and molecular investigation by a case-control study and by sequence analysis of high variable regions of the genome. RESULTS: From May to October 2005, 58 HAV hepatitis cases were notified. The case-control study showed that beach establishment attending is strongly associated with HAV hepatitis (OR = 24.5, p-value < 0.01), at multivariate analysis. The profile of epidemic curve, the clinical onset of primary cases who occurred in few weeks and the geographic distribution of cases clearly indicated a common exposure to a point source: the outbreak can be probably associated with a contaminated food product dispensed in the affected area. The outbreak has been mainly caused by a single variant, confirming the common exposure to a point source; this variant previously circulated within homosexual man (MSM) network in Northern Europe. During the outbreak and in the following months, different variants originating from Southeast Asia, Southern America and Northern Africa, have co-circulated: all these cases were related to international travel and none of these had determined secondary cases. DISCUSSION: The epidemiological picture of hepatitis A in Liguria is characterized by a wide heterogeneity of circulating HAV strains. This pattern could be associated with the increase of imported cases and transmission within network of persons with similar risk factors. Molecular approach coupled to descriptive and analytical epidemiological studies appeared un-replaceable tools for management and control of HAV outbreaks, because of their capacity to recognize infection origin, transmission patterns and dynamics of virus evolution.

Stereotactic biopsy guidance in adults with supratentorial nonenhancing gliomas: role of perfusion-weighted magnetic resonance imaging.

OBJECT: The. diagnosis of low-grade glioma (LGG) cannot be based exclusively on conventional magnetic resonance (MR) imaging studies, and target selection for stereotactic biopsy is a crucial issue given the high risk of sampling errors. The authors hypothesized that perfusion-weighted imaging could provide information on the microcirculation in presumed supratentorial LGGs. METHODS: All adult patients with suspected (nonenhancing) supratentorial LGGs on conventional MR imaging between February 2001 and February 2004 were included in this study. Preoperative MR imaging was performed using a dynamic first-pass gadopentate dimeglumine-enhanced spin echo-echo planar perfusion-weighted sequence, and the tumors' relative cerebral blood volume (rCBV) measurements were expressed in relation to the values observed in contralateral white matter. In patients with heterogeneous tumors a stereotactic biopsy was performed in the higher perfusion areas before resection. Among 21 patients (16 men and five women with a mean age of 36 years, range 23-60 years), 10 had diffuse astrocytomas (World Health Organization Grade II) and 11 had other LGGs and anaplastic gliomas. On perfusion-weighted images demonstrating heterogeneous tumors, areas of higher rCBV focus were found to be oligodendrogliomas or anaplastic astrocytomas on stereotactic biopsy; during tumor resection, however, specimens were characterized predominantly as astrocytomas. Diffuse astrocytomas were associated with significantly lower mean rCBV values compared with those in the other two lesion groups (p < 0.01). The rCBV ratio cutoff value that permitted better discrimination between diffuse astrocytomas and the other lesion groups was 1.2 (80% sensitivity and 100% specificity). CONCLUSIONS: Perfusion-weighted imaging is a feasible method of reducing the sampling error in the histopathological diagnosis of a presumed LGG, particularly by improving the selection of targets for stereotactic biopsy.

Sulfated K5 Escherichia coli polysaccharide derivatives inhibit human immunodeficiency type-1 (HIV-1) infection: candidate microbicides to prevent sexual HIV transmission.

The ideal microbicide must fulfill a number of criteria including a broad and potent activity against transmission of HIV and other sexually transmitted agents in the absence of toxicity and inflammation. We have described that derivatives of K5 polysaccharide from Escherichia coli inhibit HIV entry in target cells. K5 derivatives have a structure that resembles that of heparin, but they are devoid of the anticoagulant activity typical of heparin. Moreover, in contrast to heparin, they inhibit a broad spectrum of HIV-1 laboratory-adapted and primary isolates that use either CCR5 or CXCR4 or both coreceptors in terms of their infection and replication in primary CD4+ lymphocytes and monocytes-derived macrophages (MDM). Therefore, these compounds could be developed as candidate microbicides for preventing sexual HIV transmission, a predominant modality of HIV spreading in both the developed and underdeveloped world.

Prior invasive pulmonary and cerebellar mucormycosis is not a primary contraindication to perform an autologous stem cell transplatation in leukemia.

Mucormycosis infections, caused by fungi of the families Rhizopus, Mucor or Absidia, are typically rapidly progressive and often fatal. We report a 27-year-old male with acute myeloid leukemia (AML) developing an invasive pulmonary-CNS mucormycosis during the neutropenic period after salvage induction chemotherapy; the infection was successfully controlled with surgery and antifungal therapy. The patient received two courses of consolidation chemotherapy and underwent autologous stem cells transplantation (ASCT) while receiving secondary antifungal systemic prophylaxis with liposomal Amphotericin B (L-AMB, Ambisome). There was no clinical, radiological or microbiological evidence of mycotic reactivation during the bone marrow transplantation (BMT) procedure.

Toward a biotechnological heparin through combined chemical and enzymatic modification of the Escherichia coli K5 polysaccharide.

A process to generate glycosaminoglycans with heparin- and heparan sulfate-like sequences from the Escherichia coli K5 capsular polysaccharide is described. This polymer has the same structure as N-acetylheparosan, the precursor in heparin/ heparan sulfate biosynthesis. The process involves chemical N-deacetylation and N-sulfation, enzymatic conversion of up to 60% of the D-glucuronic acid to L-iduronic acid residues, and chemical O-sulfation. Because direct sulfation afforded unwanted 3-O-sulfated (instead of 2-O-sulfated) iduronic acid residues, a strategy involving graded solvolytic desulfation of chemically oversulfated C5-epimerized sulfaminoheparosans was assessed using persulfated heparin and heparan sulfate as model compounds. The O-desulfation process was shown to increase the anti-factor Xa activity of oversulfated heparin.

Fibroblast growth factor-2 antagonist activity and angiostatic capacity of sulfated Escherichia coli K5 polysaccharide derivatives.

The angiogenic basic fibroblast growth factor (FGF2) interacts with tyrosine kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) in endothelial cells. Here, we report the FGF2 antagonist and antiangiogenic activity of novel sulfated derivatives of the Escherichia coli K5 polysaccharide. K5 polysaccharide was chemically sulfated in N- and/or O-position after N-deacetylation. O-Sulfated and N,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to 125I-FGF2 with different potency. Accordingly, they abrogate the formation of the HSPG.FGF2.FGFR ternary complex, as evidenced by their capacity to prevent FGF2-mediated cell-cell attachment of FGFR1-overexpressing HSPG-deficient Chinese hamster ovary (CHO) cells to wild-type CHO cells. They also inhibited 125I-FGF2 binding to FGFR1-overexpressing HSPG-bearing CHO cells and adult bovine aortic endothelial cells. K5 derivatives also inhibited FGF2-mediated cell proliferation in endothelial GM 7373 cells and in human umbilical vein endothelial (HUVE) cells. In all these assays, the N-sulfated K5 derivative and unmodified K5 were poorly effective. Also, highly O-sulfated and N,O-sulfated K5 derivatives prevented the sprouting of FGF2-transfected endothelial FGF2-T-MAE cells in fibrin gel and spontaneous angiogenesis in vitro on Matrigel of FGF2-T-MAE and HUVE cells. Finally, the highly N,O-sulfated K5 derivative exerted a potent antiangiogenic activity on the chick embryo chorioallantoic membrane. These data demonstrate the possibility of generating FGF2 antagonists endowed with antiangiogenic activity by specific chemical sulfation of bacterial K5 polysaccharide. In particular, the highly N,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds.

Invasive aspergillosis in haematological malignancies: clinical findings and management for intensive chemotherapy completion.

Sixty-one cases of Aspergillus infection (35 acute myeloid leukemia, 15 acute lymphoid leukemia, one myelodysplastic syndrome, two aplastic anemia, eight non-Hodgkin's lymphoma) seen in our department between January 1989 and July 1999 were studied retrospectively to evaluate the clinical characteristics, to ascertain the factors that influenced the outcome from mycotic infections, and whether early diagnosis and prolonged therapy permitted completion of scheduled intensive chemotherapy and bone marrow transplantation (BMT) without fungal recurrence. The patients were divided into three diagnostic categories: proven aspergillosis (autoptic or histologic diagnosis) n = 39, probable aspergillosis (radiological diagnosis with positive microbiology) n = 9, and possible aspergillosis (radiological diagnosis alone) n = 13. In the same period among 675 acute leukemia patients the incidence of proven or probable aspergillosis was 7.1%. At onset of infection 92% of patients were neutropenic (< 0.5 x 10(9)/L). The most frequent site of infection was the lung (90%); disseminated disease was present in 20 patients. Among 44 assessable patients, 12 (27%) failed to respond to early antifungal therapy and died. Thirty-two patients were cured with antifungal treatment, three of five nonneutropenic with only itraconazole, the others with amphotericin B 1 mg/Kg/day with or without itraconazole subsequently or with liposomal amphotericin, Ambisome, if renal toxicity occurred. Twenty-four of 29 neutropenic responders, all affected by acute leukemia, continued scheduled intensive chemotherapies. Pulmonary lobectomy was successfully combined with medical treatment in two cases before scheduled BMT. After infection nine patients were submitted to BMT (six allo, one marrow unrelated donor (MUD), two auto) with Ambisome or itraconazole as secondary prophylaxis without fungal relapse (follow-up: 25-99 months). The median time from fungal infection to transplant was five months, range 3-10. Thirteen of 29 surviving patients had leukemia relapse, but only three (23%) of these showed also fungal infection recurrence. In conclusion, a high index of suspicion and careful clinical and radiological examinations are the key to identifying infected patients early and to programming the following therapeutic steps. Above all in leukemia patients, prompt and aggressive administration of antifungal agents seems to improve the outcome of invasive fungal disease and to permit intensive chemotherapy completion and transplant.

Incidence and clinical characteristics of posttransplant lymphoproliferative disorders: report from a single center.

In the period 1973-1998, among 2139 allograft recipients treated with standard immunosuppression, posttransplant lymphoproliferative disorders (PTLD) developed in 19 patients (0.9%): one plasmacytic hyperplasia, two polymorphic PTLD, one myeloma, and 15 lymphomas. PTLD developed 1 year after transplantation (tx) in 14 patients. Five patients were diagnosed at autopsy, 2 were lost to follow up, 3 died before therapy could be instituted, and 1 patient has just started chemotherapy. Of the 8 evaluable patients, 2 received acyclovir and are alive in complete remission (CR) and 6 received chemotherapy +/- surgery. Of these 6, 4 died of lymphoma and/or infection, 1 died of unrelated causes in CR, and 1 is alive in CR. PTLD is a severe complication of tx, usually running an aggressive course which may preclude prompt diagnosis and treatment. Nevertheless, therapy is feasible and must be tailored on the histologic subtype. Seventy-four percent of patients were diagnosed with late-onset PTLD stressing the need for long-term follow up.

Mucormycosis in hematologic malignancies: an emerging fungal infection.

BACKGROUND AND OBJECTIVES: In recent years pulmonary mucormycosis has been reported in patients with leukemia and lymphoma and bone marrow transplant recipients. It carries an extremely poor prognosis. We report our experience of clinical findings, diagnostic procedures, treatment and outcome of mucormycosis diagnosed in neutropenic patients affected by hematologic neoplasms admitted to our Department. DESIGN AND METHODS: From November 1987 to July 1999 we observed 13 cases of Mucor. Their median age was 61 years (range 20-75), and they were predominantly in the aplastic post-chemotherapy period (12/13), affected by acute myeloid leukemia (11 cases ) or non-Hodgkin's lymphoma (2 cases). Six patients (all with leukemia) were receiving inductionEth consolidation therapy, 7 had progressive hematologic disease. At the onset of infection all patients were neutropenic (N < 0.5x10(9)/L). No patients had diabetes mellitus. Two patients had been receiving steroid therapy for 5 and 7 days. RESULTS: The lung was involved in all cases (13/13); disseminated disease was present in 8/13 patients. All cultures (blood, sputum, nasal swabs and bronchoalveolar lavage) were negative. In 3 patients a histologic diagnosis was made in vivo: in 1 patient by percutaneous pulmonary biopsy, in 1 patient by pulmonary lobectomy, and in the last patient by percutaneous pulmonary biopsy confirmed by excision of a cerebellar abscess. In the remaining 10 cases diagnosis was made post-mortem. Five patients were not treated, 2 because of poor clinical condition and 3 because fungal infection was not suspected. Amphotericin B (1 mg/kg/day) was given empirically to 6 patients and 2 responded to treatment. The remaining 2 patients with neurologic symptoms at the onset of infection were treated with liposomal amphotericin, Ambisome, one with 3 and one with 5 mg/kg/day; of these two patients the first died in 4 days; the second, with both pulmonary and cerebellar localizations, was treated successfully with 5 mg/kg/day for 4 weeks and then with 3 mg/kg/day, and excision of a brain abscess at neutrophil recovery (total dose of Ambisome: 12,000 mg). The 3 surviving leukemic patients were able to complete subsequent consolidation therapy using amphotericin B or liposomal amphotericin as secondary prophylaxis during aplasia. INTERPRETATION AND CONCLUSIONS: In neutropenic hematologic patients Mucor is rarely suspected. In our patients infection was often characterized by disseminated disease and a rapidly fatal course; only early aggressive amphotericin B (or Ambisome) treatment together with neutrophil recovery appeared to improve the outcome. Diagnosis is very important for programming antifungal therapy and secondary prophylaxis with amphotericin B, because Mucor is usually resistant to itraconazole.

Chorea in patients with AIDS.

OBJECTIVE: To describe differing etiologies and possible anatomoclinical correlates of choreic movements in a series of AIDS patients. METHODS: We analyzed the clinical records and neuroimaging data of 5 consecutive AIDS patients who developed choreic movements at our center from January, 1994 to December, 1996. RESULTS: There were 2 cases of focal choreic dyskinesias, 1 of right hemichorea, and 2 of generalized chorea. Onset was acute and febrile in 1 case, and subacute in the other 4. In 1 patient the chorea was the AIDS onset symptom; in another choreic movements were the first neurological symptom following AIDS diagnosis; in 2 patients AIDS had a neurological onset other than chorea; and in the fifth patient buccofacial dyskinesias appeared following the development of bacterial encephalitis. CONCLUSION: Chorea was associated with cerebral toxoplasmosis in 2 patients, progressive multifocal leukoencephalopathy in 1, subacute HIV encephalopathy in another, and was probably iatrogenic in the last. Chorea is not unusual in AIDS, however the causes are variable and careful neuroradiological and clinical evaluation is required to identify them. AIDS-related disease should be considered in young patients presenting with chorea without a family history of movement disorders.

Multiple interactions of HIV-I Tat protein with size-defined heparin oligosaccharides.

Tat protein, a transactivating factor of the human immunodeficiency virus type I, acts also as an extracellular molecule. Heparin affects the bioavailability and biological activity of extracellular Tat (Rusnati, M., Coltrini, D., Oreste, P., Zoppetti, G., Albini, A., Noonan, D., D'Adda di Fagagna, F., Giacca, M., and Presta, M. (1997) J. Biol. Chem. 272, 11313-11320). Here, a series of homogeneously sized, (3)H-labeled heparin fragments were evaluated for their capacity to bind to free glutathione S-transferase (GST)-Tat protein and to immobilized GST-Tat. Hexasaccharides represent the minimum sized heparin fragments able to interact with GST-Tat at physiological ionic strength. Also, the affinity of binding increases with increasing the molecular size of the oligosaccharides, with large fragments (>/=18 saccharides) approaching the affinity of full-size heparin. 6-Mer heparin binds GST-Tat with a dissociation constant (K(d)) equal to 0.7 +/- 0.4 microM and a molar oligosaccharide:GST-Tat ratio of about 1:1. Interaction of GST-Tat with 22-mer or full-size heparin is consistent instead with two-component binding. At subsaturating concentrations, a single molecule of heparin interacts with 4-6 molecules of GST-Tat with high affinity (K(d) values in the nanomolar range of concentration); at saturating concentrations, heparin binds GST-Tat with lower affinity (K(d) values in the micromolar range of concentration) and a molar oligosaccharide:GST-Tat ratio of about 1:1. In agreement with the binding data, a positive correlation exists between the size of heparin oligosaccharides and their capacity to inhibit cell internalization, long terminal repeat-transactivating activity of extracellular Tat in HL3T1 cells, and its mitogenic activity in murine adenocarcinoma T53 Tat-less cells. The data demonstrate that the modality of heparin-Tat interaction is strongly affected by the size of the saccharide chain. The possibility of establishing multiple interactions increases the affinity of large heparin fragments for Tat protein and the capacity of the glycosaminoglycan to modulate the biological activity of extracellular Tat.