Axon-schwann cell interaction in the squid nerve fibre

The electrical properties of Schwann cells and the effects of neuronal impulses on their membrane potential have been studied in the giant nerve fibre of the squid.1. The behaviour of the Schwann cell membrane to current injection into the cell was ohmic. No impulse-like responses were observed with displacements of 35 mV in the membrane potential. The resistance of the Schwann cell membrane was found to be approximately 10(3) Omega cm(2).2. A long-lasting hyperpolarization is observed in the Schwann cells following the conduction of impulse trains by the axon. Whereas the propagation of a single impulse had little effect, prolonged stimulation of the fibre at 250 impulses/sec was followed by a hyperpolarization of the Schwann cell that gradually declined over a period of several minutes.3. The prolonged effects of nerve impulse trains on the Schwann cell were similar to those produced by depolarizing current pulses applied to the axon by the voltage-clamp technique. Thus, a series of depolarizing pulses in the axon was followed by a long-lasting hyperpolarization of the Schwann cells. In contrast, the application of a series of hyperpolarizing 100 mV pulses at a frequency of 1/sec had no apparent effects.4. Changes in the external potassium concentration did not reproduce the long-lasting effects of nerve excitation.5. The hyperpolarizing effects of impulse trains were abolished by the incubation of the nerve fibre in a sea-water solution containing trypsin.6. These findings are discussed in relation to the possible mechanisms that might be responsible for the long-lasting hyperpolarizations of the Schwann cells.     

Erythropoietin regulates hypoxic ventilation in mice by interacting with brainstem and carotid bodies

Apart from its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions in brain, retina and heart upon ischaemic injury. However, the physiological non-erythroid functions of Epo remain unclear. Here we use a transgenic mouse line (Tg21) constitutively overexpressing human Epo in brain to investigate Epo's impact on ventilation upon hypoxic exposure. Tg21 mice showed improved ventilatory response to severe acute hypoxia and moreover improved ventilatory acclimatization to chronic hypoxic exposure. Furthermore, following bilateral transection of carotid sinus nerves that uncouples the brain from the carotid body, Tg21 mice adapted their ventilation to acute severe hypoxia while chemodenervated wild-type (WT) animals developed a life-threatening apnoea. These results imply that Epo in brain modulates ventilation. Additional analysis revealed that the Epo receptor (EpoR) is expressed in the main brainstem respiratory centres and suggested that Epo stimulates breathing control by alteration of catecholaminergic metabolism in brainstem. The modulation of hypoxic pattern of ventilation after i.v. injection of recombinant human Epo in WT mice and the dense EpoR immunosignal observed in carotid bodies showed that these chemoreceptors are sensitive to plasma levels of Epo. In summary, our results suggest that Epo controls ventilation at the central (brainstem) and peripheral (carotid body) levels. These novel findings are relevant to understanding better respiratory disorders including those occurring at high altitude.     

Phage types and genotypes of shiga toxin-producing Escherichia coli O157:H7 isolates from humans and animals in spain: identification and characterization of two predominating phage types (PT2 and PT8)

Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused.     

Presymptomatic diagnosis in Portuguese FAP families using intragenic RFLPs and (CA)n flanking markers by fluorescence based semiautomated DNA analysis.

Owing to the large size of the APC gene, responsible for familial adenomatous polyposis, direct screening for individual mutations is not a practical approach. In the present study we establish the methodology of fluorescence based semi-automated DNA analysis to perform presymptomatic diagnosis of members at risk from 11 Portuguese FAP families with three (CA)n markers flanking the APC gene, MBC, CB26, and YN5.64, and four intragenic RFLPs. Haplotypes were constructed on the basis of individual genotypes and their segregation through generations were followed. The study was informative for 12% of subjects using only intragenic RFLPs and increased to 90% when we used the three (CA)n flanking markers. We report two of the 11 families under study in our laboratory and show recombinant events leading to a precise localisation of the CB26 marker between D5S82 and the APC gene. In one family there was a loss of (CA) units of one allele of the CB26 marker from an unaffected mother to her son.     

The telescoping suture--Part II: A novel method to improve the mechanical behavior of a new biomaterial: ostrich pericardium

Ostrich pericardium, sutured using a telescoping or overlapping technique, was studied to determine its mechanical behavior. From each of 12 pericardial sacs, four contiguous strips were cut longitudinally, from root to apex, and another four contiguous strips were cut in transverse direction. One of the strips in each set of four was used as an unsutured control and the remaining three were sutured by overlapping 0.5 cm of the tissue and sewing with Gore-tex, Prolene or Pronova. These 96 samples were then subjected to tensile testing along their major axes until rupture. The tensile stresses recorded in the suture materials at the moment tears appeared in the pericardium ranged between 55.99 MPa and 70.23 MPa for Gore-tex in samples cut in the two directions. Shear stress became ostensible at 56 MPa, with clearly evident tears. However, microfracture of the collagen fibers must be produced at much lower stress levels. The comparison of the resistance in kilograms (machine-imposed), without taking into account the sections in which the load was applied, demonstrated only a slight loss of load when the telescoping suture was employed in ostrich pericardium samples. Ostrich pericardium may continue to be an alternative biological material for the construction of heart valve leaflets.     

Significance of PBMC response of rheumatic fever patients and control individuals to immunodominant streptococcal M5 protein epitopes

β-hemolytic streptococcal infection in developing countries still causes thousands of cases of Rheumatic Fever (RF). Molecular mimicry between streptococcal M protein (strep M) and heart components has been proposed as the triggering factor leading to autoimmunity in individuals with genetic susceptibility, which is linked to different HLA-DR alleles in different populations. In our hands, RF was significantly associated to HLA-DR7/53. Previous work in our lab has shown that heart-infiltrating T cells that simultaneously recognize strep M and heart proteins. Further, such T cells predominantly recognized the 81-103 strep M5 epitope. In this work, we analysed the proliferative response of peripheral blood mononuclear cells of 99 RF patients and 40 normal controls. Eighty-nine of the RF patients were HLA-typed. As among heart-infiltrating T cells, the 81-103 strep M5 protein epitope is the most frequently recognized epitope among RF PBMC (35.4%), against a 7.5% frequency of proliferation among normal controls (p=0.0018, chi square). However, the 81-103 epitope was as frequently recognized by HLA-DR7,53 positive as by negative individuals (45.2% vs 54.8%, respectively). Taken together, the results suggest that the 81-103 strep M5 epitope may be the immunodominant epitope, “promiscuously” recognized by T cells in a genetically diverse population. The demonstration that molecular mimicry is targeted to a discrete immunodominant “promiscuous” epitope in strep M5 may allow the development of a safe anti-streptococcal synthetic vaccine devoid of such epitopes.     

Rheumatic heart disease: proinflammatory cytokines play a role in the progression and maintenance of valvular lesions

Heart lesions of rheumatic heart disease (RHD) patients contain T-cell clones that recognize heart proteins and streptococcal M peptides. To functionally characterize heart-infiltrating T lymphocytes, we evaluated their cytokine profile, both directly in situ and in T-cell lines derived from the heart (HIL). Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-10 expressions were characterized in 20 heart tissue infiltrates from 14 RHD patients by immunohistochemistry. IFN-gamma-, TNF-alpha-, and IL-10-positive cells were consistently predominant, whereas IL-4 was scarce in the valves. In agreement with these data, the in vitro experiments, in which 13 HILs derived from heart samples of eight patients were stimulated with M5 protein and the immunodominant M5 (81-96) peptide, IL-4 was detected in HIL derived from the atrium (three of six) but not from the valve (zero of seven). IFN-gamma and IL-10 production were detected in culture supernatants in 11 of 13 and 6 of 12 HILs, respectively. The predominant IFN-gamma and TNF-alpha expression in the heart suggests that Th1-type cytokines could mediate RHD. Unlike in reversible myocardium inflammation, the significantly lower IL-4 expression in the valvular tissue (P = 0.02) may contribute to the progression of the RHD leading to permanent valvular damage (relative risk, 4.3; odds ratio, 15.8). The lack of IL-4 in vitro production by valve-derived HIL also emphasizes the more severe tissue destruction in valves observed in RHD.     

Recruiting and retaining GPs and patients in intervention studies: the DEPS-GP project as a case study

Background  

Recruiting and retaining GPs for research can prove difficult, and may result in sub-optimal patient participation where GPs are required to recruit patients. Low participation rates may affect the validity of research.     

Five-test simple scheme for species-level identification of clinically significant coagulase-negative staphylococci

A working scheme developed in our laboratory for identification (by species group and species) of coagulase-negative staphylococci (CNS) was evaluated with 201 consecutive isolates and then validated by using the reference method of Kloos and Schleifer (W. E. Kloos and K. H. Schleifer, J. Clin. Microbiol. 1:82-88, 1975). This five-test simple scheme (referred to here as the simple scheme) combines the novobiocin susceptibility test with tests for urease, pyrrolidonyl arylamidase, ornithine decarboxylase, and aerobic acid from mannose. The addition of one or two tests within a particular species group could then positively identify the isolate. Two commercial systems, Staph-Zym (Rosco) and API-Staph (bioMérieux), along with results obtained by using Rosco diagnostic tablets (nongrowth tests), were also compared with the reference method. One isolate could not be identified even by the reference method. Of the remaining 200 strains, 191 (95.5%) strains were correctly identified with Staph-Zym and 171 strains (85.5%) were correctly identified with API-Staph. The most frequent clinical CNS species isolated were Staphylococcus epidermidis (50.5%), S. haemolyticus (18.5%), S. saprophyticus subsp. saprophyticus (16.0%), S. lugdunensis (6.0%), and S. warneri (2.5%). The simple scheme validated with the reference method has demonstrated an excellent correlation in the identification of the three most frequent species isolated: S. epidermidis, S. haemolyticus, and S. saprophyticus subsp. saprophyticus. With the simple scheme, identification of CNS was possible within 24 h after the enzymatic tests were used, whereas up to 72 h is necessary for the growth tests. This methodology would be very useful in any clinical microbiology laboratory for the presumptive identification of CNS species groups and species.     

Lutzomyia longipalpis salivary gland homogenate impairs cytokine production and costimulatory molecule expression on human monocytes and dendritic cells

In this report, we describe an investigation of the effects of Lutzomyia longipalpis sand fly salivary gland homogenates (SGH) on cytokine production and expression of costimulatory molecules on human monocytes, macrophages (Ms), and dendritic cells (DCs). SGH of L. longipalpis induced an increase in interleukin-6 (IL-6), IL-8 and IL-12p40 production but a decrease in tumor necrosis factor alpha and IL-10 production by lipopolysaccharida (LPS)-stimulated monocytes. We also examined the expression of costimulatory molecules on the surface of monocytes, Ms, and DCs. Whereas SGH affected the expression of these molecules on monocytes and Ms, it had little effect on these molecules on DCs. However, when DCs were generated from human monocytes in the presence of SGH, SGH inhibited the expression of costimulatory molecules. In addition, a decrease in the maturation of DCs induced by CD40L was observed in the presence of SGH. Finally, preincubating SGH with human sera containing anti-SGH-specific antibodies abolished the effects of SGH on cytokine production by LPS-stimulated monocytes.