Neuropathological changes in the peripheral nervous system and spinal cord in a transgenic mouse model of Niemann-Pick disease type A.

OBJECTIVE: Type A Niemann-Pick is a severe neurological disease, caused by a mutation of the gene of acid sphingomyelinase (ASM) and reduced enzyme activity. Some studies reported neuropathological changes occurring in the central nervous system of ASM deficient transgenic (ASMKO) mice, while a detailed study on the peripheral nervous system (PNS) at different ages is currently lacking. The aim of our study was to examine the pathological changes occurring in the PNS and in the spinal cord in an AMSKO model of Niemann-Pick disease (NPD) Type A. MATERIAL AND METHOD: Dorsal root ganglia (DRG), peripheral nerves and spinal cord specimens were obtained from ASMKO mice and age-matched wild type animals (age range = 1-7 months). They were observed at the light and electron microscope. Behavioral testing was performed to assess motor coordination and reactivity. Fluoro-Jade B was used as a high affinity fluorescent marker for degenerating neurons. RESULTS: Typical NPD cytoplasmic inclusions were observed in DRG neurons and satellite cells, in peripheral nerve Schwann cells, in spinal cord neurons and in endothelial cells. All these inclusions were present from the age of 1 month and increased with aging. By Fluoro-Jade B staining we demonstrated the occurrence of neuronal degeneration starting from 5 months of age. CONCLUSION: Despite the fact that a definite diagnosis of NPD Type A depends on enzymatic assays and/or molecular analysis, morphological investigation remains an important diagnostic procedure. Well-defined and complete neuropathological information about the ASMKO mouse model, inclusive of PNS examination, may be crucial in the pre-clinical evaluation of new therapies.

Clinical and genetic heterogeneity of branching enzyme deficiency (glycogenosis type IV).

BACKGROUND: Glycogen storage disease type IV (GSD-IV) is a clinically heterogeneous autosomal recessive disorder due to glycogen branching enzyme (GBE) deficiency and resulting in the accumulation of an amylopectin-like polysaccharide. The typical presentation is liver disease of childhood, progressing to lethal cirrhosis. The neuromuscular form of GSD-IV varies in onset (perinatal, congenital, juvenile, or adult) and severity. OBJECTIVE: To identify the molecular bases of different neuromuscular forms of GSD-IV and to establish possible genotype/phenotype correlations. METHODS: Eight patients with GBE deficiency had different neuromuscular presentations: three had fetal akinesia deformation sequence (FADS), three had congenital myopathy, one had juvenile myopathy, and one had combined myopathic and hepatic features. In all patients, the promoter and the entire coding region of the GBE gene at the RNA and genomic level were sequenced. RESULTS: Nine novel mutations were identified, including nonsense, missense, deletion, insertion, and splice-junction mutations. The three cases with FADS were homozygous, whereas all other cases were compound heterozygotes. CONCLUSIONS: This study expands the spectrum of mutations in the GBE gene and confirms that the neuromuscular presentation of GSD-IV is clinically and genetically heterogeneous.

Clinical and molecular findings in patients with giant axonal neuropathy (GAN).

Giant axonal neuropathy (GAN) is a rare autosomal recessive neurodegenerative disorder of early onset, clinically characterized by a progressive involvement of both peripheral and CNS. The diagnosis is based on the presence of characteristic giant axons, filled with neurofilaments, on nerve biopsy. Recently, the defective protein, gigaxonin, has been identified and different pathogenic mutations in the gigaxonin gene have been reported as the underlying genetic defect. Gigaxonin, a member of the BTB/kelch superfamily proteins, seems to play a crucial role in the cross talk between the intermediate filaments and the membrane network. The authors report clinical and molecular findings in five Italian patients with GAN. This study shows the allelic heterogeneity of GAN and expands the spectrum of mutations in the GAN gene. The frequent occurrence of private mutations stresses the importance of a complete gene analysis.

A human placental polydeoxyribonucleotide (PDRN) may promote the growth of human corneal fibroblasts and iris pigment epithelial cells in primary culture.

The optimal concentration of a human placental polydeoxyribonucleotide (PDRN) preparation (100 microg/ml) enhances the growth of human corneal fibroblasts in primary culture depending upon the donor age. In particular, this effect is very consistently reproducible with donors over 60 years of age (p = 0.0028), suggesting a selective benefit of PDRN in the tissue culture of senescent cells. Moreover, this drug may promote the development of human iris pigment epithelium (IPE) cells with much lower concentrations of fetal bovine serum than those suitable for the culture of IPE. Lastly, the use of a 'gauze disk' on the pieces of the corneal explants improves the efficiency of growth of the control fibroblast primary cultures.

In vitro activity of thiamphenicol against multiresistant Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus in Italy.

Thiamphenicol is a derivative of chloramphenicol characterized by a spectrum comparable to that of the parent compound against multiresistant pathogens but showing satisfactory tolerability. The in vitro activity of thiamphenicol and of 11 comparative drugs against 397 recently isolated antibiotic-resistant and/or invasive pneumococci and 52 multiply-resistant MRSA including 2 VISA strains was determined. Bactericidal activity against Haemophilus influenzae and the post-antibiotic effect on Streptococcus pneumoniae, H. influenzae, Staphylococcus aureus and Escherichia coli were also assessed. Against invasive pneumococci, thiamphenicol and chloramphenicol were the most potent non-beta-lactam molecules together with vancomycin and rifampin. Against high-level penicillin-resistant strains phenicol activities were superior to those of cefotaxime, ceftriaxone and imipenem. Against MRSA thiamphenicol and chloramphenicol were second only to the glycopetides and also inhibited the VISA strains. Thiamphenicol showed a significant PAE (0.33 to 2.9h) on all pathogens studied and a powerful bactericidal effect against beta-lactamase-positive and -negative H. influenzae. These results indicate a good in vitro activity of thiamphenicol against difficult-to-treat multiply resistant pathogens.

Prevalence of antimicrobial resistance in Streptococcus pneumoniae circulating in Italy: results of the Italian Epidemiological Observatory Survey (1997-1999).

The Italian Epidemiological Observatory (IEO), a surveillance program supported by the SmithKline Foundation, analyzed the susceptibility of 2,664 community-acquired respiratory Streptococcus pneumoniae derived from over 50 clinical microbiology laboratories during 1997-1999, against 21 antibiotics adopting a quantitative methodology. Throughout these years, total penicillin resistance varied from 14.3% to 10.2%. High-level resistance has remained stable, ranging from 3.8% to 4.1%, while a decrease in low-level resistance (from 10.3% to 6.1%) has been recorded. Lack of susceptibility to macrolides ranged from 29.1% in 1997 to 25.5% in 1999. Similar figures have also been observed with tetracycline and co-trimoxazole (rates of resistance around 30%). As expected, large geographical variations in resistance rates were found for all drugs. Amoxicillin and amoxicillin-clavulanate were 100% active on penicillin-intermediate isolates. Injectable third-generation cephalosporins and carbapenems were also capable of inhibiting a large proportion of these microorganisms. Rifampin was the most potent non-beta-lactam compound tested. In contrast to the situation prevailing elsewhere, in Italian children (aged 0-5 years) presenting with respiratory conditions, the total rate of penicillin resistance (3%) was lower than that shown by the adult population (10.9%). However, lack of susceptibility to macrolides, tetracycline, and cotrimoxazole (35%, 41%, 44%) was more incident in pediatric than in adult patients (25%, 26%, 28% respectively). Strains recovered from blood in 1999 (67) were much more susceptible to penicillin (98.5%) than respiratory pneumococci (89.8%), whereas macrolides, tetracycline, and cotrimoxazole were consistently less active (75%, 67%, 64%).

Epidemiology of major respiratory pathogens.

A vast literature attests to the fact that Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis represent the prevailing bacterial pathogens of community-acquired lower respiratory tract infections. Their specific incidence as causative agents of the more common syndromes is known to vary even profoundly, depending on geographic area, and the same holds true for their rates of resistance to antimicrobial drugs. Europe does not escape the threat posed by the present pandemic spread of penicillin resistance in S. pneumoniae although, as expected, some countries like Spain and France are highly affected and others including Germany, Italy, The Netherlands and the Scandinavian region, are relatively spared. In several sites multiple resistance has been described in S. pneumoniae with the most affected drugs being penicillin, the macrolides, co-trimoxazole and tetracycline. In H. influenzae synthesis of beta-lactamases is the main resistance trait expressed. Lack of susceptibility to beta-lactams dictated by a different mechanism remains extremely rare. Large variations in the incidence of this character are apparent when considering European countries. France and Spain are again widely affected while Germany, The Netherlands and Italy display rates of beta-lactamase-positive H. influenzae of about 16%. M. catarrhalis must be considered generally resistant to non-protected aminopenicillins since over 90% of these organisms produce beta-lactamases.

Microbial epidemiology patterns of surgical infection pathogens.

Resistance, as assessed in vitro, has a number of serious consequences in clinical situations. Treatment failures are common when an inappropriate drug has been prescribed and this, in turn, may lead to hospitalization of patients who normally would have been treated on an outpatient basis, as well as to longer hospital stay for inpatients and to the use of alternative drugs, which may be more expensive and more toxic. These factors all contribute to increased health care costs, morbidity and mortality. Microbiological procedures may identify the causative pathogen and provide the appropriate susceptibility pattern to the physician, thus reducing the chances of therapeutic failures. However, for a number of reasons including cost--even in hospitals--not to mention general practice, infections are seldom diagnosed on an etiological basis. From what has been stated, the knowledge of bacterial epidemiology and resistance represents basic support for correct therapeutic decision-making.

[Epidemiology of methicillin-resistance among Staphylococci strains isolated in risk units and effects of the vancomycin on the expression of methicillin-resistance]. / Epidemiologia della meticillino-resistenza negli Stafilococchi isolati in reparti a rischio ed effetti della vanomicina sulla espressione della meticillino-resistenza.

Four hundred Staphylococci strains, isolated from different in intensive care unit hospitalized patients, were analyzed. 53% of all strains were resistant to methicillin. Against methicillin-resistant S. aureus (MRSA), teicoplanin and vancomycin (100% of susceptibility), rifampin (76.3%) and co-trimoxazole (73%) emerged as the most potent drugs tested; the 15% of the strains were susceptible to ciprofloxacin, erythromycin, clindamycin and gentamicin. Only one MRSA strain (0.8%) resulted hetero-resistant to vancomycin. Among 100 strains exposed to serial concentration of vancomycin (0.25-32 mg/L for 30 days), 57 were selected with intermediate-level of resistance to the glycopeptides; the MRSA strains have shown to acquire resistance in vitro more easily than methicillin-susceptible. These results indicate that in the clones of Staphylococci circulating in our region, the evolution of glycopeptides-resistance is not a rapid process and the loss of effectiveness of these antibiotics cannot be predicted to short term. In particular, the restriction profile analysis of chromosomal DNA from MRSA strains, selected in vitro with intermediate -level of vancomycin resistance, demonstrated that at the moment in the hospital departments studied, the diffusion of a clone able to acquire resistance more easily than others is not present.

Antibiotic susceptibility patterns and serotypes of antibiotic resistant and/or invasive Streptococcus pneumoniae strains circulating in Italy.

Antibiotic susceptibility patterns and serotypes of 397 Streptococcus pneumoniae recovered from 1994 to 1998 in Italy have been determined. This collection included 229 penicillin- and/or erythromycin-resistant strains and 168 isolates responsible for invasive infections. Among penicillin-resistant pneumococci, the most prevalent serotype was 23F, followed by 19F and 9V, while among erythromycin-resistant but penicillin-susceptible strains serotype 6B was predominant followed by 19A, 14, 19F, 15A, 15B, and 23F. The most common invasive serotypes were 6B, 19F, 23F, 3, 4, 14, 20, 15B, and 9N. The currently available 23-valent pneumococcal vaccine could cover 91% of noninvasive penicillin- and/or erythromycin-resistant strains, 84% of pneumococci isolated from sterile sites, and 83% of invasive antibiotic-resistant S. pneumoniae. Penicillin-resistant pneumococci also showed reduced susceptibility to other antimicrobial compounds. Against invasive pneumococci, amoxicillin, cefotaxime, ceftriaxone, imipenem, vancomycin, and rifampin were 100% effective. Penicillin, chloramphenicol, erythromycin, tetracycline, and co-trimoxazole resistance was 1.8%, 9.5%, 15.5%, 18.5%, and 21.4%, respectively.

Heterogeneous vancomycin resistance in methicillin-resistant Staphylococcus aureus strains isolated in a large Italian hospital.

Of 179 methicillin-resistant Staphylococcus aureus strains isolated from 1997 to 1998, two strains (1.1%) gave subclones for which the vancomycin MICs were 8 mg/liter. Pulsed-field gel electrophoresis showed identical restriction patterns for both isolates, suggesting transfer of a single clone between two different patients.

Macrolide resistance mechanisms and expression of phenotypes among Streptococcus pneumoniae circulating in Italy.

In Italy, macrolide-resistant pneumococci have been isolated at a rate increasing from 6% in 1993 to 31.7% in 1998. A collection of 161 erythromycin-resistant Streptococcus pneumoniae recovered between 1993 and 1997 has now been phenotypically and genotypically characterized. Approximately 90% of these microorganisms possessed a constitutive MLS(B) mechanism of resistance. PCR detected ermB and mefE genes in strains showing MLS(B) and M phenotypes, respectively. Using pulsed-field gel electrophoresis of chromosomal DNA, one dominant restriction profile and its variations were detected in 51 S. pneumoniae isolates collected from different locations, indicating the circulation of a clone characterized by the possession of a great ability to spread.

Antiviral activity of FA-11H on the infectivity and replication of herpes simplex virus type 1 in cultured cells.

The highest concentration lacking cytotoxicity (h.c.l.c.) of the acid FA-11H, related to ester FA-11 (ethyl 4-trifluoromethyl-2-methylthio-5-pyrimidinecarboxylate), was determined on Vero cultured cells. At this concentration (300 micrograms/ml) FA-11H produced an 81.4% inhibition of herpes simplex virus type 1 infectivity with respect to the plaque formation detected in control infected cultures. This inhibitory activity was substantiated by studies on progeny production. FA-11H revealed a higher therapeutic index than that of the related ester FA-11. However, the therapeutic index of FA-11H appeared to be lower than that of its potassium salt. These results indicate that the presence of potassium ions could facilitate the internalization and therefore the antiviral activity of the anions corresponding to analysed compound.

Evaluation of the trophic effect of human placental polydeoxyribonucleotide on human knee skin fibroblasts in primary culture.

A simple assay capable of evaluating the trophic effect of growth factors or active principles on human skin diploid fibroblasts in primary culture has been developed. The results indicate that at physiological concentrations (20-100 micrograms/ml) a human placental polydeoxyribonucleotide (PDRN) preparation enhances the growth of human skin diploid fibroblasts of the knee in primary culture. This effect is consistently reproducible in the case of patients over 60 years of age, and may explain previously reported data on the successful clinical applications of human placental preparations, suggesting a selective benefit of PDRN in wound healing when compared to other treatments.

A short-term plaque assay for antiviral drug research on herpes simplex virus type 2.

In antiviral drug research, gelatinized growth medium cannot be used after infection to avoid the development of secondary plaques, because agar or other chemicals may interfere with the assessment of cytotoxic activity of antiviral compounds. In this study we evaluated quantitatively the different expression of cytopathic effects of Herpes Simplex Virus type 2 (HSV-2) in Vero cell cultures utilizing different periods of incubation after infection, and analysed the incidence of viral progeny particles on the formation of satellite plaques in order to define a short-term plaque assay utilizing a single cycle of infection. The results shown in the work suggest using a period of 24 hours of incubation post adsorption for a screening on inhibition of HSV-2 infectivity in antiviral drug studies.

Antiviral activity of S-7HNa on the infectivity and replication of herpes simplex virus type 1 in cultured cells.

The effect of sodium salt of the acid related to ester S-7 (i.e. S-7HNa) on the infectivity and replication of herpes simplex virus type 1 in Vero cultured cells was studied. S-7HNa, at the highest concentration lacking cytotoxicity (10 mM), produced, at various inocula, a 70% inhibition of HSV-1 infectivity with respect to the plaque formation detected in control infected cultures. This inhibitory activity was substantiated by studies on progeny production. S-7HNa revealed a therapeutic index higher than that of the correlative ester (S-7).

The mixed leukocyte reaction may promote the growth of human pericorneal fibroblasts in primary culture.

Keratitis of the cornea is believed to be the leading cause of loss of vision due to external eye disease in the United States and in Europe. At present, primary and secondary cell cultures obtained from corneal explants are the most suitable system to study the role of drug-resistant viruses in severe recurrences of infections leading to irreversible corneal scarring. In this work, the growth of human pericorneal fibroblasts in primary and secondary cultures has been obtained by cocultivation with a mixed leukocyte reaction.