Trends in drug resistance, serotypes, and molecular types of Streptococcus pneumoniae colonizing preschool-age children attending day care centers in Lisbon, Portugal: a summary of 4 years of annual surveillance

Of the nasopharyngeal cultures recovered from 942 day care center (DCC) attendees in Lisbon, Portugal, 591 (62%) yielded Streptococcus pneumoniae during a surveillance performed in February and March of 1999. Forty percent of the isolates were resistant to one or more antimicrobial agents. In particular, 2% were penicillin resistant and 20% had intermediate penicillin resistance. Multidrug resistance to macrolides, lincosamides, and tetracycline was the most frequent antibiotype (17% of all isolates). Serotyping and molecular typing by pulsed-field gel electrophoresis were performed for 202 out of 237 drug-resistant pneumococci (DRPn). The most frequent serotypes were 6B (26%), 14 (22%), 19F (16%), 23F (10%), and nontypeable (12%). The majority (67%) of the DRPn strains were representatives of nine international clones included in the Pneumococcal Molecular Epidemiology Network; eight of them had been detected in previous studies. Fourteen novel clones were identified, corresponding to 26% of the DRPn strains. The remaining 7% of the strains were local clones detected in our previous studies. Comparison with studies conducted since 1996 in Portuguese DCCs identified several trends: (i) the rate of DRPn frequency has fluctuated between 40 and 50%; (ii) the serotypes most frequently recovered have remained the same; (iii) nontypeable strains appear to be increasing in frequency; and (iv) a clone of serotype 33F emerged in 1999. Together, our observations highlight that the nasopharynxes of children in DCCs are a melting pot of successful DRPn clones that are important to study and monitor if we aim to gain a better understanding on the epidemiology of this pathogen.     

Presence of the cfxA gene in Bacteroides distasonis

In this study we investigated the presence of the cfxA gene (encoding a class A beta-lactamase) in 73 strains of the Bacteroides fragilis group belonging to the species B. distasonis (34), B. vulgatus (14), B. thetaiotaomicron (8), B. merdae (6), B. caccae (9) and B. ovatus (2) isolated from human intestinal microflora of healthy children and adults. Employing specific primers to the cfxA gene, a 312-bp amplified fragment was obtained in 2 strains of B. vulgatus and 9 strains, the majority from children, of B. distasonis. The expression of this enzyme was analysed by determining the MICs to cefoxitin and cefotaxime and values varied from 2 to >256 microg/ml of both cefoxitin and cefotaxime. Sequence analysis of the amplicons corresponding to the cfxA gene from B. distasonis and B. vulgatus revealed identical sequences between these isolates and high similarity with other beta-lactamase genes of anaerobes such as cfxA of B. vulgatus (99%) and cfxA2 of Prevotella intermedia (99%), both sequences of which deposited in Genbank under accession numbers U38243 and AF118110, respectively. However, a fragment obtained from a B. distasonis strain (EC17-4) showed a unique RFLP profile and 87% nucleotide similarity with cfxA and cfxA2 genes. These results seem to suggest a dissemination of these resistance determinants among Bacteroides species.     

Ultrastructural comparison of dissolution and apatite precipitation on hydroxyapatite and silicon-substituted hydroxyapatite in vitro and in vivo

Recent histological studies have demonstrated that the substitution of silicate ions into hydroxyapatite (HA) significantly increases the rate of bone apposition to HA implants. The enhanced bioactivity of silicon-substituted HA (Si-HA) over pure HA has been attributed to the effect of silicate ions in accelerating dissolution. In the present study, high-resolution transmission electron microscopy (HR-TEM) was employed to compare dissolution of HA and Si-HA in an acellular simulated body fluid (SBF) to dissolution in an in vivo model. HR-TEM observations confirmed a difference in morphology of apatite precipitates in vivo and in SBF: apatite deposits were platelike in vivo and nodular in SBF. Compositional mapping suggested that preferential dissolution of silicon from the implant promotes the nucleation of carbonate apatite around the implant. The in vivo findings illustrated an absence of dissolution at the bone-HA or Si-HA interface, whereas dissolution was extensive from within the implant. The amount of dissolution in acellular SBF was similar to dissolution from within the implant, although the site at which the dissolution nucleates was different: dissolution predominates at the crystallite surfaces in SBF, whereas grain boundary dissolution predominates in vivo. These findings suggest that proteins in the in vivo milieu modify the processes of dissolution from the implant.     

Assessment of cancer immunotherapy outcome in terms of the immune response time features.

A cytokine-based periodic immunotherapy treatment is included in a model of tumour growth with a delay. The effects of dose schedule are studied in the case of a weak immune system and a growing tumour. We find the existence of 'metastable' states (that may last for tens of years) induced by the treatment and also potentially adverse effects of the dosage frequency on the stabilization of the tumour. These two effects depend on the delay between the tumour growth and the immune system response, the cytokine dose burden, and other parameters considered in the model.     

Mast cell types and cell-to-cell interactions in lymph nodes of the opossum Didelphis albiventris

Previous light-microscopic studies have shown a unique population of mast cells in lymphatic sinuses of lymph nodes located in the head, neck, axillary fossa and inguinal region of the opossum. In the present work, scanning and transmission electron-microscopic studies in the opossum mandibular and superficial axillary lymph nodes have strengthened the differences between connective-tissue mast cells (CTMC) and the lymphatic-sinus mast cells (LSMC). Further, close appositions of mast cells to other cells were described. At the nodal capsule, CTMC contacted fibroblast and granulocytes. In the lymphatic sinuses a few CTMC contacted LSMC, macrophages and reticular cells. The LSMC contacted macrophages, reticular cells and other LSMC. A few LSMC could be located in the medullary cord in close contact with plasma cells or other lymphoid cells, keeping the same ultrastructural features of those found in the lymphatic sinuses. An important new finding was provided by light-microscopic studies in nine abdominal lymph nodes. Most of them (para-aortic, common iliac, cardial, cecocolic and those of the body and tail of the pancreas) displayed numerous LSMC with the same distribution and histological features described herein. However, the mesenteric, pyloric and head-of-pancreas lymph nodes were virtually devoid of LSMC. Instead, their mast cells occurred mainly at the medullary cords and were very similar to the CTMC. Ultrastructural studies at the mesenteric lymph nodes confirmed the CTMC character of the mast cells located at both medullary cords and sinuses, and disclosed interactions with macrophages and lymphoid cells. Accepted: 8 September 1999     

Modulation of acute visceral nociception and bladder inflammation by plant triterpene, α, β-amyrin in a mouse model of cystitis: role of tachykinin NK<Subscript>1</Subscript>-receptors,and K<Superscript>+</Superscript><Subscript>ATP</Subscript> channels

Objective and design: We previously described the visceral antinociceptive property of α, β-amyrin in a mouse model of cystitis induced by cyclophosphamide (CPM). This study examined the contribution of vanilloid-1 (TRPV1), peripheral NK1 receptors to CPM-evoked nociceptive behaviors and bladder edema, and its possible modulation by α, β-amyrin. Methods: The effect of α, β-amyrin (10, 30, and 100 mg/kg, p. o.) and N-acetylcysteine (NAC) on CPM (400 mg/kg, i. p.)-induced cystitis was studied in mice. Sensory deafferentation was done by a high dose capsaicin. The parameters analysed were: CPM-evoked noxious behaviors, bladder edema, vascular permeability, and NK₁ immunoreactivity. To assess the role of K⁺ _ATP channels in α, β-amyrin effect, animals were pretreated with glibenclamide. Results: α, β-amyrin (30 and 100 mg/kg) and NAC significantly (p < 0.01) suppressed the visceral pain-related behaviors and NK₁ immunoreactivity, but bladder edema was reduced weakly. Glibenclamide reversed the effects of α, β-amyrin. Sensory deafferentation by capsaicin significantly reduced the nociceptive responses and the NK₁ immunoreactivity to noxious stimulation by CPM. Conclusions: α, β-amyrin attenuates CPM-induced visceral pain and bladder edema by mechanisms that involve, at least in part, a block either of Substance P release or its receptor function, and partly by opening K⁺ _ATP channels. Received 13 February 2007; returned for revision 13 April 2007; accepted by G. Geisslinger 14 May 2007     

NMR studies on energy metabolism of immobilized primary neurons and astrocytes during hypoxia, ischemia and hypoglycemia

Changes in high-energy phosphate metabolites (ATP and phosphocreatine) were monitored, in real time, by 31P-nuclear magnetic resonance in primary cell cultures of neurons and astrocytes during periods of hypoxia, ischemia and hypoglycemia, and also during the recovery periods following the re-establishment of standard conditions. Cells were immobilized in basement membrane gel threads and perfused with oxygen-depleted medium (oxygen concentration below 30 microM), to create hypoxic conditions, or with aerobic medium (oxygen concentration approximately 460 microM) containing different concentrations of glucose (hypoglycemia). Ischemic conditions were imposed by stopping perfusion for different periods of time (15 min to 2 h). The experimental set-up enabled the acquisition of 31P-spectra with high signal-to-noise ratio within 10-20 min for both cell types. The effect of hypoxia on glucose metabolism was assessed by 13C-NMR using [1-13C]glucose as substrate. The levels of ATP and PCr in astrocytes were unaffected during hypoxia (up to 2 h), but decreased notably under ischemia. In neurons, hypoxic periods caused a sharp drop of the ATP and PCr levels, and considerable damage to the capacity of neurons to replenish the ATP and PCr pools upon returning to normoxic conditions. However, neurons were remarkably less sensitive to ischemic conditions, the ATP and PCr pools being restored quickly, even after 2 h under challenging conditions. The data show that neurons were more resistant to ischemia than astrocytes, and suggest that the capacity to sustain the pools of ATP and PCr was part of the neuronal protective strategy.     

Pulmonary and mediastinal "sarcoidosis" following surgical resection of cancer

Previous reports indicate that enlarged hilar and mediastinal lymph nodes caused by sarcoid-like reactions may develop after curative resection of cancer, and their presence does not necessarily denote neoplastic recurrence. Reports further suggest that coexisting pulmonary infiltrates in this setting may be related to sarcoidosis. In this study, we describe two patients who had resected lung and gastric cancer and who later developed pulmonary interstitial infiltrate, concurrent with progressive mediastinal lymphadenopathy initially thought to be caused by intrathoracic dissemination of their cancer. These changes were shown by open lung biopsy to be a benign, granulomatous reaction interpreted as sarcoidosis. Thus, it is important to recognize this clinical pattern when pulmonary infiltrates develop after complete treatment of cancer in an otherwise relapse-free patient and to encourage lung or lymph node biopsy in these particular settings in order to confirm a sarcoid-like reaction, thereby avoiding unnecessary chemotherapy for presumed tumor recurrence.     

Identification of a novel HLA-DQB1*06 allele, DQB1*0618

We report here the identification of a novel DQB1*06 allele, DQB1*0618, found in a bone marrow donor. The new allele was detected during routine DNA-based HLA typing by an ambiguous pattern of probe hybridization, obtained by polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO). Molecular cloning and sequencing confirmed that the new allele is identical to DQB1*0609 at exon 2 except for 3 nucleotide substitutions at positions 353, 356 and 367, also found in other alleles. These nucleotide changes may explain its anomalous reactivity.