Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy- 1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200-10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony- forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.     

Long-term protection of hematopoiesis against the cytotoxic effects of multiple doses of nitrosourea by retrovirus-mediated expression of human O6-alkylguanine-DNA-alkyltransferase

A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony- forming cells of the granulocyte-macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nitrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU. These results clearly show that a human ATase cDNA-containing retrovirus can be used to infect long-term primary bone marrow cultures and that this attenuates their sensitivity to nitrosoureas.     

Facilitating culture-centered communication between health care providers and veterans transitioning from military deployment to civilian life

Objective

To describe returning veterans’ transition experience from military to civilian life and to educate health care providers about culture-centered communication that promotes readjustment to civilian life.

Methods

Qualitative, in-depth, semi-structured interviews with 17 male and 14 female Iraq and Afghanistan veterans were audio recorded, transcribed verbatim, and analyzed using Grounded Practical Theory.

Results

Veterans described disorientation when returning to civilian life after deployment. Veterans’ experiences resulted from an underlying tension between military and civilian identities consistent with reverse culture shock. Participants described challenges and strategies for managing readjustment stress across three domains: intrapersonal, professional/educational, and interpersonal.

Conclusions

To provide patient-centered care to returning Iraq and Afghanistan veterans, health care providers must be attuned to medical, psychological, and social challenges of the readjustment experience, including reverse culture shock. Culture-centered communication may help veterans integrate positive aspects of military and civilian identities, which may promote full reintegration into civilian life.

Practice implications

Health care providers may promote culture-centered interactions by asking veterans to reflect about their readjustment experiences. By actively eliciting challenges and helping veterans’ to identify possible solutions, health care providers may help veterans integrate military and civilian identities through an increased therapeutic alliance and social support throughout the readjustment process.     

Time since last vaccine dose in PCR-positive and PCR-negative children with suspected pertussis to monitor pertussis vaccine effectiveness

We evaluated whether the results of diagnostic polymerase chain reaction (PCR) testing combined with time since last vaccine dose could be used to monitor the effectiveness of acellular pertussis vaccines. In 258 consecutive nasopharyngeal swabs from children and adolescents with typical pertussis symptoms, 80 were positive and 178 were negative in PCR for Bordetella pertussis DNA (IS 481). Time since last vaccine dose was available for 152 patients, of which 120 were fully immunised. Among the fully vaccinated patients, the median age of 41 PCR-positive patients was 8.4 years (range 0.9–12.3) and that of 79 PCR-negative cases was 3.3 years (range 0.4–14.1) (p?<?0.01). The median time since last pertussis vaccine dose was 6.05 years [95 % confidence interval (CI): 0.5–10.9] in PCR-positive cases and 2.22 years (95 % CI: 0.04–9.23) in PCR-negative cases (p?<?0.001). The use of diagnostic PCR results from pertussis cases together with time since last vaccine dose permits estimates of the duration of protection after vaccination with acellular pertussis vaccines that are in keeping with more complex studies.     

The Role of Sex Differences in Autophagy in the Heart During Coxsackievirus B3-Induced Myocarditis

Under normal conditions, autophagy maintains cardiomyocyte health and integrity through turnover of organelles. During stress, oxygen and nutrient deprivation, or microbial infection, autophagy prolongs cardiomyocyte survival. Sex differences in induction of cell death may to some extent explain the disparity between the sexes in many human diseases. However, sex differences in gene expression, which regulate cell death and autophagy, were so far not taken in consideration to explain the sex bias of viral myocarditis. Coxsackievirus B3 (CVB3)-induced myocarditis is a sex-biased disease, with females being substantially less susceptible than males and sex hormones largely determine this bias. CVB3 was shown to induce and subvert the autophagosome for its optimal viral RNA replication. Gene expression analysis on mouse and human, healthy and CVB3-infected, cardiac samples of both sexes, suggests sex differences in autophagy-related gene expression. This review discusses the aspects of sex bias in autophagy induction in cardiomyocytes.     

Plant secondary siRNA production determined by microRNA-duplex structure

Processing of microRNA (miRNA) precursors results in the release of a double-stranded miRNA/miRNA* duplex. The miRNA or guide strand, is loaded onto the Argonaute (AGO) effector, and the miRNA* or passenger strand is typically degraded. The loaded AGO-containing RNA-induced silencing complex specifically recognizes a target mRNA, leading to its degradation or translational inhibition. In plants, miRNA-mediated cleavage of a target triggers in some cases the production of secondary small interfering RNAs (siRNAs), which in turn can silence other genes in trans. This alternative pathway depends on the length of the miRNA and the specific AGO in the effector complex. However, 22-nt miRNAs are sufficient, but not essential for this pathway. Using a combination of computational and experimental approaches, we show that transitivity can be triggered when the small RNA that is not retained in AGO is 22-nt long. Moreover, we demonstrate that asymmetrically positioned bulged bases in the miRNA:miRNA* duplex, regardless of miRNA or miRNA* length, are sufficient for the initiation of transitivity. We propose that the RNA-induced silencing complex reprogramming occurs during the early steps of miRNA loading, before the miRNA duplex is disassembled and the guide strand is selected.