Detection of Leishmania infantum kinetoplast DNA in laryngeal tissue from an immunocompetent patient

Mucosal leishmaniasis of the upper respiratory tract is usually associated with the visceral form or is found in immunosuppressed individuals. This report presents a case of isolated mucosal leishmaniasis in an immunocompetent patient, whose diagnosis mainly rested on histology and positive polymerase chain reaction result for Leishmania donovani in the laryngeal tissue. A 59-year-old man, who never lived outside Italy, showed a subglottic mucosal polypoid-like lesion. The typical morphological picture and positive polymerase chain reaction result for L donovani by DNA extracted from laryngeal biopsy specimens allowed the diagnosis of mucosal leishmaniasis. Specific amphotericin B therapy was started, resulting in clinical and endoscopic improvement. Increased knowledge about the histological and molecular tissue analysis of Leishmania enhances the diagnostic testing for mucosal leishmaniasis, as primary mucosal leishmaniasis may occur in both immunosuppresed and immunocompetent patients who travel to or reside in areas endemic for Leishmania.     

Nutrient signalling in the regulation of human muscle protein synthesis

The mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are important nutrient- and energy-sensing and signalling proteins in skeletal muscle. AMPK activation decreases muscle protein synthesis by inhibiting mTOR signalling to regulatory proteins associated with translation initiation and elongation. On the other hand, essential amino acids (leucine in particular) and insulin stimulate mTOR signalling and protein synthesis. We hypothesized that anabolic nutrients would be sensed by both AMPK and mTOR, resulting in an acute and potent stimulation of human skeletal muscle protein synthesis via enhanced translation initiation and elongation.
We measured muscle protein synthesis and mTOR-associated upstream and downstream signalling proteins in young male subjects ( n = 14) using stable isotopic and immunoblotting techniques. Following a first muscle biopsy, subjects in the 'Nutrition' group ingested a leucine-enriched essential amino acid–carbohydrate mixture (EAC). Subjects in the Control group did not consume nutrients. A second biopsy was obtained 1 h later. Ingestion of EAC significantly increased muscle protein synthesis, modestly reduced AMPK phosphorylation, and increased Akt/PKB (protein kinase B) and mTOR phosphorylation ( P < 0.05). mTOR signalling to its downstream effectors (S6 kinase 1 (S6K1) and 4E-binding protein 1 (4E-BP1) phosphorylation status) was also increased ( P < 0.05). In addition, eukaryotic elongation factor 2 (eEF2) phosphorylation was significantly reduced ( P < 0.05). Protein synthesis and cell signalling (phosphorylation status) was unchanged in the control group ( P > 0.05).
We conclude that anabolic nutrients alter the phosphorylation status of both AMPK- and mTOR-associated signalling proteins in human muscle, in association with an increase in protein synthesis not only via enhanced translation initiation but also through signalling promoting translation elongation.     

Cell proliferation in breast cancer is a major determinant of clinical outcome in node-positive but not in node-negative patients.

The growth rate of a tumor cell population depends on two major factors: the percentage of proliferating cells (cell growth fraction) and the rapidity of their duplication (cell proliferation rate). The authors evaluated the prognostic and predictive value of both kinetics parameters in a large series of breast cancer patients (n=504). The cell growth fraction was determined by MIB-1 immunostaining, the cell proliferation rate by AgNOR analysis. Ki-67 LI (labeling index) and AgNOR area were significantly associated with histotype, histologic grade, tumor size, estrogen/progesterone receptor status, patient age, and lymph node involvement (P<0.005). In the entire series of patients, both kinetics variables were significantly and independently associated with the clinical outcome, but their prognostic relevance was quite different when node-negative and node-positive patients were considered separately. Although in node-positive patients Ki-67 LI and AgNOR area were the unique independent predictors of disease-free and overall survival, they were excluded by the multivariate Cox model in node-negative patients, where only tumor size and estrogen receptor status retained a significant P-value. These results show that in breast carcinoma the cell growth fraction and the cell proliferation rate have a different prognostic impact with respect to the lymph node status and are major determinants of clinical outcome in node-positive patients only. Within this subgroup, the rapidity of cell proliferation as assessed by AgNOR analysis also served as a sensitive predictor of the response to adjuvant treatments.     

Transmission of human cytomegalovirus from infected uterine microvascular endothelial cells to differentiating/invasive placental cytotrophoblasts

Analysis of placentas infected with human cytomegalovirus (CMV) suggested that viral transmission could involve differentiating/invasive cytotrophoblasts in villi that attach the placenta to the uterine wall. To parse the cellular components in this process, we developed a coculture system of polarized uterine microvascular endothelial cell (UtMVEC) infection with an endothelial cell-tropic pathogenic strain of CMV. Then we evaluated the potential role of neutrophils and endothelial cells in the spread of infection to differentiating cytotrophoblasts. As shown by immunocytochemistry and analysis of viral replication, CMV preferentially infected endothelial cells via apical membranes and disrupted cell junction proteins, thereby altering paracellular permeability and cell polarity. Neutralizing antibodies to CMV glycoprotein B, an envelope component that facilitates virion penetration, blocked plaque formation in polarized UtMVEC. Neutrophils transmitted CMV infection to UtMVEC, which in turn infected cytotrophoblasts. However, neutrophils did not directly infect cytotrophoblasts. These findings implicate endothelial cells from the uterine microvasculature as a potential source for CMV infection of endovascular cytotrophoblasts of the anchoring villi. Possibly the cytokine/chemokine milieu in the pregnant uterus could attract immune cells that infect endothelial cells in hybrid fetal-maternal vessels. In turn, these cells could infect endovascular cytotrophoblasts, one possible initiation point of a cascade that results in retrograde placental CMV infection.     

Variola and camelpox virus-specific sequences are part of a single large open reading frame identified in two German cowpox virus strains

A large open reading frame (ORF) has been identified in two German cowpox virus strains. The ORFs (5676 and 5679 nt, respectively) differ in 10 nucleotides, resulting in an amino acid homology of 99.8%. In searching GenBank nucleotide sequences (>90% identity) were present in several small ORFs in variola major, variola minor and camelpox virus genomes. Alignments revealed that these small ORFs are fragments of a large ORF. However, sequences of the ORF described here are entirely absent in the two cowpox virus reference strains. Databank analysis revealed amino acid identities (ranging from 25 to 39%) with so-called B22R-like poxviral proteins with unknown function encoded by several chordopoxviruses. Further sequencing of one cowpox virus strain under study identified an ORF (5790 nt) which displays high levels of nucleotide identity to ORFs present in several orthopoxvirus species. Taken together, the two cowpox viruses analyzed here contain one large ORF which is conserved within the genus Orthopoxvirus and a unique, more distantly related ORF of similar size, which is conserved in the subfamily Chordopoxvirinae.     

Ca2+ permeability of the plasma membrane induced by rotavirus infection in cultured cells is inhibited by tunicamycin and brefeldin A

Rotavirus infection of cultured cells induces a progressive increase in plasma membrane permeability to Ca2+. The viral product responsible for this effect is not known. We have used tunicamycin and brefeldin A to prevent glycosylation and membrane traffic and study the involvement of viral glycoproteins, NSP4 and/or VP7, in rotavirus-infected HT29 and MA104 cells. In infected cells, we observed an increase of plasma membrane Ca2+ permeability and a progressive depletion of agonist-releasable ER pools measured with fura 2 and an enhancement of total Ca2+ content measured as 45Ca2+ uptake. Tunicamycin inhibited the increase in membrane Ca2+ permeability, induced a depletion of agonist-releasable and 45Ca2+-sequestered pools. Brefeldin A inhibited the increase of Ca2+ permeability and the increase in 45Ca2+ uptake induced by infection. We propose that the glycosylated viral product NSP4 (and/or VP7) travels to the plasma membrane to form a Ca2+ channel and hence elevate Ca2+ permeability.     

Molecular epidemiology of sequential outbreaks of Acinetobacter baumannii in an intensive care unit shows the emergence of carbapenem resistance

The molecular epidemiology of multidrug-resistant Acinetobacter baumannii was investigated in the medical-surgical intensive care unit (ICU) of a university hospital in Italy during two window periods in which two sequential A. baumannii epidemics occurred. Genotype analysis by pulsed-field gel electrophoresis (PFGE) of A. baumannii isolates from 131 patients identified nine distinct PFGE patterns. Of these, PFGE clones B and I predominated and occurred sequentially during the two epidemics. A. baumannii epidemic clones showed a multidrug-resistant antibiotype, being clone B resistant to all antimicrobials tested except the carbapenems and clone I resistant to all antimicrobials except ampicillin-sulbactam and gentamicin. Type 1 integrons of 2.5 and 2.2 kb were amplified from the chromosomal DNA of epidemic PFGE clones B and I, respectively, but not from the chromosomal DNA of the nonepidemic clones. Nucleotide analysis of clone B integron identified four gene cassettes: aacC1, which confers resistance to gentamicin; two open reading frames (ORFs) coding for unknown products; and aadA1a, which confers resistance to spectinomycin and streptomycin. The integron of clone I contained three gene cassettes: aacA4, which confers resistance to amikacin, netilmicin, and tobramycin; an unknown ORF; and bla(OXA-20), which codes for a class D beta-lactamase that confers resistance to amoxicillin, ticarcillin, oxacillin, and cloxacillin. Also, the bla(IMP) allele was amplified from chromosomal DNA of A. baumannii strains of PFGE type I. Class 1 integrons carrying antimicrobial resistance genes and bla(IMP) allele in A. baumannii epidemic strains correlated with the high use rates of broad-spectrum cephalosporins, carbapenems, and aminoglycosides in the ICU during the study period.     

Induction of cellular immune response and anti-Salmonella enterica serovar typhi bactericidal antibodies in healthy volunteers by immunization with a vaccine candidate against typhoid fever

Typhoid fever remains a serious public health problem. We have developed a vaccine from Salmonella enterica serovar typhi (S. typhi) outer-membrane proteins (OMPs) known as porins. A single subcutaneous dose of 10 microg of porins induced a five-fold (P = 0.05) seroconversion index consisting of IgM and IgG at 7 and 15 days after vaccination as well as the production of IgG1 and IgG2 isotypes. The porins-based vaccine induced a two-fold increase (P = 0.05) in bactericidal titres in volunteers, whom also developed a T-cell response characterized by the production of interferon-gamma (INF-gamma). Side effects after vaccination were mild and transient. The data showed that our S. typhi porins-based candidate vaccine is safe and immunogenic in healthy humans.