Effects of granulocyte/macrophage colony-stimulating factor on the development and differentiation of CD5-positive macrophages and their potential derivation from a CD5-positive B-cell lineage in mice.

In co-cultures of either the murine pre-B cell line J13, fetal liver cells, or adult peritoneal or bone marrow cells with ST2 mouse bone marrow stromal cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), the development of CD5+ macrophages was demonstrated by immunohistochemical staining and flow cytometry. Although CD5+ macrophages were not present in the peritoneal cavities of normal mice, approximately 30% of the peritoneal macrophages in viable motheaten (mev/mev) mice, deficient in SHP-1 protein tyrosine phosphatase, expressed cell surface CD5 and B220, markers for B cells. In the mev/mev mice, GM-CSF level in peritoneal fluid was increased significantly. At 5 days after daily intravenous injection with GM-CSF, many CD5+ macrophages appeared in the peritoneal cavity and in omental milky spots of normal mice but fewer in osteopetrosis (op) mutant mice, deficient in macrophage (M)-CSF. These results indicate that GM-CSF, in combination with M-CSF, induces the development and differentiation of CD5+ macrophages in the peritoneal cavity, particularly in the omental milky spots of mice. In the peritoneal cavity of GM-CSF-treated mice, the percentages of hematopoietic progenitor cells doubly positive for CD5 and CD34 or c-kit and of macrophage precursor cells doubly positive for CD5 and ER-MP58 or ER-MP20 were increased significantly during the development of CD5+ macrophages and CD5 B cells, suggesting that CD5+ macrophages and B cells may share a bipotential progenitor in vivo.     

Genome macrorestriction analysis of sequential Pseudomonas aeruginosa isolates from bronchiectasis patients without cystic fibrosis.

The respiratory tracts of bronchiectasis patients may be persistently colonized with Pseudomonas aeruginosa, despite intensive chemotherapy. The organism may undergo phenotypic changes in these patients, providing misleading typing results by conventional methods. We prospectively studied eight bronchiectasis patients without cystic fibrosis over a period of 1 year. A high microbial load of P. aeruginosa was found in 70% of sputum samples collected. Of these, 55 sequential P. aeruginosa isolates were characterized by a genotyping method, pulsed-field gel electrophoresis, to overcome the problem of differentiating the P. aeruginosa strains during chemotherapy. Genome macrorestriction fingerprinting patterns were analyzed after digestion with XbaI restriction endonuclease. Of the eight patients, six harbored a single dominant strain of P. aeruginosa, with an intrapatient macrorestriction similarity pattern range of 96 to 100%. The other two patients were infected with mixed bacterial isolates including P. aeruginosa. However, diversity was observed in the P. aeruginosa isolates from all eight patients, with a relatedness of only 55 to 65%. The study further strengthens the fact that pulsed-field gel electrophoresis can be used efficiently and effectively to differentiate P. aeruginosa strains in bronchiectasis patients without cystic fibrosis.     

Clinical and Epidemiological Relevance of Quantitating Hepatitis E Virus-Specific Immunoglobulin M

Diagnosis of acute hepatitis E by detection of hepatitis E virus (HEV)-specific immunoglobulin M (IgM) is an established procedure. We investigated whether quantitation of HEV IgM and its ratio to HEV total Ig furnished more information than conventional IgM tests that are interpreted as positive or negative. A previously described indirect immunoassay for total Ig against a baculovirus-expressed HEV capsid protein was modified to quantitate HEV-specific IgM in Walter Reed (WR) antibody units by using a reference antiserum and the four-parameter logistic model. A receiver-operating characteristics curve derived from 197 true-positive specimens and 449 true-negative specimens identified 30 WR units/ml as an optimum cut point. The median HEV IgM level in 36 patients with acute hepatitis E fell from 3,000 to 100 WR units/ml over 6 months, suggesting that 100 WR units/ml would be a more appropriate cut point for distinguishing recent from remote IgM responses. Among three hepatitis E case series, determination of the HEV IgM-to-total-Ig ratio in acute-phase serum revealed that most patients had high ratios consistent with primary infections whereas a few had low ratios, suggesting that they had sustained reinfections that elicited anamnestic antibody responses. The diagnostic utility of the new IgM test was similar to that of a commercially available test that uses different HEV antigens. In conclusion, we found that HEV IgM can be detected specifically in >95% of acute hepatitis E cases defined by detection of the virus genome in serum and that quantitation of HEV IgM and its ratio to total Ig provides insight into infection timing and prior immunity.     

Genome-wide examination of myoblast cell cycle withdrawal during differentiation.

Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40% fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly-6A, whose expression specifically was up-regulated during cell cycle withdrawal coincident with early myoblast differentiation.     

Pharmacological blood pressure lowering in the older hypertensive patients may lead to cognitive impairment by altering neurovascular coupling

The link between both high and low blood pressure (BP) levels and cognitive impairment in later life has been reported in several studies. The mechanisms for this link are unclear but may be related to abnormalities in brain blood flow control. Our previous work has shown that cerebral autoregulation (CA) is unimpaired in both young and older people with hypertension at rest and that ageing does not appear to impact on the increase in the cerebral blood flow response to increased metabolic demand of neurones and other cells of the nervous system due to heightened activity (Neurovascular Coupling, NVC). Nonetheless, it is plausible that NVC efficiency becomes compromised during mental activity in older people with hypertension and that certain classes of anti-hypertensive agents may exacerbate the situation by reducing both NVC and CA contributing to cognitive decline. Such a link would have a major impact on prescribing patterns for anti-hypertensive medication.     

Efficacy of oral single dose therapy with artemisinin-naphthoquine phosphate in uncomplicated falciparum malaria

All artemisinin-based combination therapies (ACTs), recommended by the World Health Organization, are 3-day regimens. A considerable level of non-compliance on ACTs has been reported from some countries. The study aimed to assess the therapeutic efficacy of single dose treatment with new generation ACT containing artemisinin plus naphthoquine. An oral single dose of eight tablets (400 mg of naphthoquine + 1000 mg artemisinin) of the combination drug was administered to adult uncomplicated falciparum malaria patients. Observations of fever, parasite clearance and reappearance, and other clinical manifestations were made on Days 0, 1, 2, 3, 7, 14, 21 and 28. Fifty-three adult falciparum positive cases, with fever or history of fever within the previous 24 h, were included in the final evaluation of the study. Mean fever clearance time, parasite clearance time were 18.2 ± 8.6 h and 34.6 ± 14.3 h, respectively. Adequate clinical and parasitological response was achieved in 52 cases, the rate being 98.1% (95% CI, 91.1-99.9). One patient was classified as late parasitological failure because of the reappearance of falciparum parasite on Day 14. The drug was well tolerated and no adverse reactions were detected in the patients. Since it is a single dose therapy, health workers can administer the drug as directly observed treatment.     

Opposite malaria and pregnancy effect on oral bioavailability of artesunate – a population pharmacokinetic evaluation

Aim

The aim was to compare the pharmacokinetic properties of artesunate and dihydroartemisinin in the same women: i) pregnant with acute uncomplicated malaria on day 1 and 2, ii) pregnant with convalescent malaria on day 7 and iii) in a healthy state 3 months post-partum on day 1, 2 and 7.

Methods

Non-linear mixed-effects modelling was used to compare plasma concentration–time profiles of artesunate and dihydroartemisinin over 7 days of treatment following oral and intravenous artesunate administration to pregnant women with uncomplicated Plasmodium falciparum malaria during their second or third trimesters of pregnancy. The same women were restudied 3 months after delivery when fully recovered. Non-compartmental results of the same study have been published previously.

Results

Twenty pregnant patients on the Thailand-Myanmar border were studied and 15 volunteered to be restudied 3 months post-partum. Malaria and pregnancy had no effect on the pharmacokinetic properties of artesunate or dihydroartemisinin after intravenous artesunate administration. However, malaria and pregnancy had opposite effects on the absorption of orally administered artesunate. Malaria increased the absolute oral bioavailability of artesunate by 87%, presumably by inhibiting first pass effect, whereas pregnancy decreased oral bioavailability by 23%.

Conclusions

The population pharmacokinetic analysis demonstrated opposite effects of malaria and pregnancy on the bioavailability of orally administered artesunate. Lower drug exposures during the second and third trimesters of pregnancy may contribute to lower cure rates and thus the development of drug resistance. Dose optimization studies are required for artesunate containing artemisinin-based combination therapies (ACTs) in later pregnancy.     

Haemostatic disturbances in patients bitten by Russell''s viper (Vipera russelli siamensis) in Burma

Patients who are severely envenomed by Russell's viper develop DIC which is frequently associated with spontaneous bleeding and incoagulable blood. These haemostatic disturbances may be responsible for death or organ/tissue damage both through haemorrhage and microvascular occlusion by fibrin thrombi. The most striking laboratory features of the coagulopathy developing after Russell's viper bite in the 42 patients studied were depletion of fibrinogen (mean 0.09 g/l, range 0-0.6), factor V (6.5 u/dl, range 0-17), factor X (35 u/dl, range 1-85), factor XIIIa (57 u/dl, range 15-82), plasminogen (61 u/dl, range 10-92), antiplasmin (36 u/dl, range 14-62). Protein C (49 u/dl, range 15-100) and platelets (104 x 10(9)/l, range 25-197). Intense fibrinolytic activity was detected in all cases with marked elevation of FDPs (1614 micrograms/ml, range 350-3000), a large proportion of which were cross-linked (1058 micrograms/ml, range 38-3000). The monospecific Burmese antivenom appeared to be very effective in neutralizing the venom procoagulants and in restoring blood coagulability. Moreover, the unexpectedly normal level of AT III provides a theoretical basis for the use of heparin to enhance the inactivation of those serine proteases present before antivenom administration.