Development of a transformation system for Crinipellis perniciosa,the causal agent of witches' broom in cocoa plants

Protoplasts of the pathogenic plant fungus, Crinipellis perniciosa, were transformed to hygromycin B resistance using the pAN7-1 plasmid, which contains the Escherichia coli hph gene under the control of Aspergillus nidulans regulatory sequences. The pAN7-1 plasmid was introduced by PEG/CaCl(2) treatment. Transformation frequencies of 1.6-2.5 transformants/microg of DNA were achieved. About 54% of the transformants were abortive and 40 analyzed transformants were mitotically stable and showed different hygromycin B resistance levels. The presence of the hph gene was checked by PCR in five transformants and the integration of multiple plasmid copies into different genome sites was observed by Southern analysis. This is the first report of a C. perniciosa transformation system and represents an important step for further research into genetic manipulation of this fungal plant pathogen.     

Outcome of hospitalized patients with COVID-19 pneumonia treated with high-dose immunoglobulin therapy in a prospective case series

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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.     

A Mg-dependent ecto-ATPase is increased in the infective stages of<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis>

In this work, we describe the ability of living epimastigotes of Trypanosoma cruzi to hydrolyze extracellular ATP. In these intact parasites, there was a low level of ATP hydrolysis in the absence of any divalent metal (2.42±0.31 nmol Pi/h×10⁸ cells). ATP hydrolysis was stimulated by MgCl₂, and the Mg-dependent ecto-ATPase activity was 27.15±2.91 nmol Pi/h×10⁸ cells. The addition of MgCl₂ to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. This stimulatory activity was also observed when MgCl₂ was replaced by MnCl₂, but not by CaCl₂ or SrCl₂. The apparent K_m for Mg-ATP^2– was 0.61 mM, and free Mg²⁺ did not increase the ecto-ATPase activity. This ecto-ATPase activity was insensitive to the inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4.diisothiocyanostylbene 2-2-disulfonic acid) as well as suramin, an antagonist of P₂ purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg²⁺-dependent ATPase activity in a dose-dependent manner. A comparison among the Mg²⁺-ecto-ATPase activities of the three forms of T. cruzi showed that the noninfective epimastigotes were less efficient at hydrolyzing ATP than the infective trypomastigote and amastigote stages.     

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.     

HLA-DRB1*1602 allele is positively associated with HPV cervical infection in Bolivian Andean women

Incidence of cervical cancer is high among Bolivian Andean women. Human papillomavirus (HPV) infection is known as the major risk factor of cervical cancer. The host immune system plays an important role in the outcome of HPV infection and associated malignancies. In order to study the immunogenetic background of Bolivian Andean women with regard to HPV infection status, we compared HLA class I and class II allele frequencies between 37 HPV positive and 68 HPV negative Bolivian women. Demographic variables, including distribution of Andean ethnicities, were similar in both groups. Comparison of HLA class I allele frequencies between both groups indicated no significant difference. In contrast, HLA class II DRB1*1602 allele, an Amerindian allele, was significantly higher in the HPV positive women compared with HPV negative controls (chi(2) = 5.2, p < 0.05, odds ratio = 3.17; 95% confidence interval = 1.4-8.8). HPV types present in the HPV positive group were HPV-18, -16, -31, -33, and -58. These results suggest that HLA class II DRB1*1602 may confer susceptibility to infection with genetically related HPV types. This is the first report of an HLA class II association with HPV infection in an Andean population.