Reactive type II pneumocytes in bronchoalveolar lavage fluid

OBJECTIVE: To evaluate the prevalence of reactive type II pneumocytes (RPII) in bronchoalveolar lavage (BAL) fluid samples obtained from patients with various pulmonary disorders. STUDY DESIGN: Consecutive BAL fluid samples were screened for the presence of RPII on May-Grünwald-Giemsa-stained cytocentrifuge preparations. BAL fluid samples with and without RPII were compared with regard to prevalence, associated clinical diagnoses and cytologic findings. RESULTS: RPII were generally large cells with a high nuclear:cytoplasmic ratio and deeply blue-stained, vacuolated cytoplasm. Most RPII occurred in cohesive cell groups, and the vacuoles tended to be confluent. Cytologic findings associated with RPII were foamy alveolar macrophages, activated lymphocytes and plasma cells. RPII were present in 94 (21.7%) of 433 included BAL fluid samples. The highest prevalences were noted in patients with systemic inflammatory response syndrome and alveolar hemorrhage. In addition, RPII tended to occur more frequently in ventilator-associated pneumonia, Pneumocystis carinii pneumonia, extrinsic allergic alveolitis and drug-induced pulmonary disorders. In contrast, RPII were not observed in BAL fluid samples obtained from patients with sarcoidosis. CONCLUSION: RPII were prevalent in about 20% of BAL fluid specimens. They were associated mainly with conditions of acute lung injury and not observed in sarcoidosis.     

Investigating the global dispersal of chickens in prehistory using ancient mitochondrial DNA signatures

Data from morphology, linguistics, history, and archaeology have all been used to trace the dispersal of chickens from Asian domestication centers to their current global distribution. Each provides a unique perspective which can aid in the reconstruction of prehistory. This study expands on previous investigations by adding a temporal component from ancient DNA and, in some cases, direct dating of bones of individual chickens from a variety of sites in Europe, the Pacific, and the Americas. The results from the ancient DNA analyses of forty-eight archaeologically derived chicken bones provide support for archaeological hypotheses about the prehistoric human transport of chickens. Haplogroup E mtDNA signatures have been amplified from directly dated samples originating in Europe at 1000 B.P. and in the Pacific at 3000 B.P. indicating multiple prehistoric dispersals from a single Asian centre. These two dispersal pathways converged in the Americas where chickens were introduced both by Polynesians and later by Europeans. The results of this study also highlight the inappropriate application of the small stretch of D-loop, traditionally amplified for use in phylogenetic studies, to understanding discrete episodes of chicken translocation in the past. The results of this study lead to the proposal of four hypotheses which will require further scrutiny and rigorous future testing.     

The polypeptide composition of moving and stationary neurofilaments in cultured sympathetic neurons

Studies on the axonal transport of neurofilament proteins in cultured neurons have shown they move at fast rates, but their overall rate of movement is slow because they spend most of their time not moving. Using correlative light and electron microscopy, we have shown that these proteins move in the form of assembled neurofilament polymers. However, the polypeptide composition of these moving polymers is not known. To address this, we visualized neurofilaments in cultured neonatal mouse sympathetic neurons using GFP-tagged neurofilament protein M and performed time-lapse fluorescence microscopy of naturally occurring gaps in the axonal neurofilament array. When neurofilaments entered the gaps, we stopped them in their tracks using a rapid perfusion and permeabilization technique and then processed them for immunofluorescence microscopy. To compare moving neurofilaments to the total neurofilament population, most of which are stationary at any point in time, we also performed immunofluorescence microscopy on neurofilaments in detergent-splayed axonal cytoskeletons. All neurofilaments, both moving and stationary, contained NFL, NFM, peripherin and alpha-internexin along>85% of their length. NFH was absent due to low expression levels in these neonatal neurons. These data indicate that peripherin and alpha-internexin are integral and abundant components of neurofilament polymers in these neurons and that both moving and stationary neurofilaments in these neurons are complex heteropolymers of at least four different neuronal intermediate filament proteins.     

HIV-1 Conserved Elements p24CE DNA Vaccine Induces Humoral Immune Responses with Broad Epitope Recognition in Macaques

To target immune responses towards invariable regions of the virus, we engineered DNA-based immunogens encoding conserved elements (CE) of HIV-1 p24^gag. This conserved element vaccine is designed to avoid decoy epitopes by focusing responses to critical viral elements. We previously reported that vaccination of macaques with p24CE DNA induced robust cellular immune responses to CE that were not elicited upon wild type p55^gag DNA vaccination. p24CE DNA priming followed by p55^gag DNA boost provided a novel strategy to increase the magnitude and breadth of the cellular immune responses to HIV-1 Gag, including the induction of strong, multifunctional T-cell responses targeting epitopes within CE. Here, we examined the humoral responses induced upon p24CE DNA or p55^gag DNA vaccination in macaques and found that although both vaccines induced robust p24^gag binding antibody responses, the responses induced by p24CE DNA showed a unique broad range of linear epitope recognition. In contrast, antibodies elicited by p55^gag DNA vaccine failed to recognize p24CE protein and did not recognize linear epitopes spanning the CE. Interestingly, boosting of p24CE DNA primed animals with p55^gag DNA resulted in augmentation of antibodies able to recognize p24^gag as well as the p24CE proteins, thereby inducing broadest immunity. Our results indicate that an effectively directed vaccine strategy that includes priming with the conserved element vaccine followed by boost with the complete immunogen induces broad cellular and humoral immunity focused on the conserved regions of the virus. This novel and effective strategy to broaden responses could be applied against other antigens of highly diverse pathogens.     

Variable SNP density in aspartyl-protease genes of the malaria parasite Plasmodium falciparum

An analysis of the diversity of the aspartyl proteases of Plasmodium falciparum, known as plasmepsins (PMs), was completed in view of their possible role as drug targets. DNA sequence polymorphisms were identified in nine pm genes including their non-coding (introns and 5' flanking) sequences. All genes contained at least one single nucleotide polymorphism (SNP). Extensive microsatellite diversity was observed predominantly in non-coding sequences. All but one non-synonymous polymorphism (a conservative substitution) were mapped to the surface of the predicted protein, contradicting a possible role in enzymatic activity. The distribution of SNPs was found to be non-random among pm genes, with pm6 and pm10 having significantly higher SNP densities, suggesting they were under selection. For pm6 the majority of the SNPs were in introns and some of these may contribute to splice site variation. SNPs were found at a high density in both the coding and non-coding sequences of pm10. Recombination was important in generating additional diversity at this locus. Although direct selection for pm10 mutations could not be ruled out, the presence of balancing selection and a high density of SNPs in non-coding sequence led us to propose that another gene under selection may be influencing the diversity in the region. By sequencing short DNA tags in a 200 kb region flanking pm10 we show that a cluster of antigen genes, known to be under diversifying selection, may contribute to the observed diversity. We discuss the importance of diversity and local selection effects when choosing drug targets for intervention strategies.     

Application of Body Mass Index According to Height-Age in Short and Tall Children

Background

In children with either delayed or accelerated growth, expressing the body mass index (BMI) to chronological age might lead to invalid body composition estimates. Reference to height-age has been suggested for such populations; however its validity has not been demonstrated.

Methods

Anthropometric data of healthy children were obtained from the German KiGGS survey. We selected three samples with different height distributions representing short stature (mean height SDS: -1.6), normal stature (height SDS: 0), and tall stature (height SDS: +1.6), and compared BMI-for-age and BMI-for-height-age between these samples across the paediatric age range. Differences between samples were tested using Kruskal-Wallis one-way analysis of variance and permutation tests.

Results

At a given age, BMI was distributed towards lower values in short, and towards higher values in tall subjects as compared to a population with average height distribution. Expressing BMI to height-age eliminated these differences in boys with a short stature from 4 years to 14 years of age, in tall boys from 4 to 16 years, in short girls aged 2-10 years or tall girls aged 2-17 years.

Conclusion

From late infancy to adolescent age, BMI distribution co-varies with height distribution and referencing to height-age appears appropriate within this age period. However, caution is needed when data about pubertal status are absent.     

Identification of in vivo phosphorylation sites of SET, a nuclear phosphoprotein encoded by the translocation breakpoint in acute undifferentiated leukemia

SET, the translocation breakpoint-encoded protein in acute undifferentiated leukemia (AUL), is identified as a 39-kDa phosphoprotein found predominantly in the cell nuclei [1994, J. Biol. Chem. 269,2258-2262]. SET is fused to a putative oncoprotein, CAN, in AUL and is thought to regulate the transformation potential of SET-CAN by its nuclear localization and phosphorylation. We investigated in detail the in vivo phosphorylation of SET. Phosphorylation of SET occurred in all human cell lines examined in vivo, primarily on serine residues. Endoproteinase Glu-C digestion of phosphorylated SET yielded two phosphopeptides. By radiosequencing, we identified the in vivo phosphorylation sites of SET as Ser⁹ and Ser²⁴. The surrounding sequences of Ser⁹ and Ser²⁴ contained an apparent consensus site sequence for protein kinase C.