Characterization of primitive subpopulations of normal and leukemic cells present in the blood of patients with newly diagnosed as well as established chronic myeloid leukemia

Elevated numbers of primitive Philadelphia chromosome-positive (Ph+) progenitors, including long-term culture-initiating cells (LTC-IC) as well as colony-forming cells (CFC), have been previously described in the blood of patients with chronic myeloid leukemia (CML) in chronic phase with high white blood cell counts. In the present study, which focused primarily on an analysis of circulating progenitors present in such patients at diagnosis, we discovered the frequent and occasionally exclusive presence of circulating normal (Ph-) LTC-IC, often at levels above those seen for LTC-IC in the blood of normal individuals. The presence of detectable numbers of circulating Ph- LTC-IC was independent of the fact that the same peripheral blood samples also contained elevated numbers of predominantly or exclusively Ph+ CFC. Interestingly, both the Ph+ and Ph- LTC-IC in these samples were CD34+CD71- and variably CD38- and Thy-1+, as previously documented for LTC-IC in normal marrow. Thus, neither CD38 nor Thy-1 expression was useful for discriminating between Ph+ and Ph- LTC-IC in mixed populations. Nevertheless, an association of these phenotypes with LTC- IC function did allow highly enriched (> 5% pure) suspensions of either Ph+ or Ph- LTC-IC to be obtained from selected samples of CML blood in which the initial LTC-IC population was either predominantly Ph+ or Ph- , respectively. These findings suggest that the mechanisms causing mobilization of leukemic stem cells in untreated CML patients may affect their normal counterparts. They also indicate a possible new source of autologous cells for the support of intensive therapy of CML patients. Finally, they provide a method for obtaining the most highly purified populations of Ph+ LTC-IC described to date. This method should be useful for further analyses of the molecular activities of these very primitive neoplastic cells.     

Platelet glycoproteins IIb and IIIa as a calcium channel in liposomes

Human platelet membrane glycoproteins IIb and IIIa (GPIIb and IIIa) were incorporated into phospholipid vesicles by the reverse-phase technique to assess the ability of GPIIb and IIIa to function as a Ca2+ channel. Movement of Ca2+ across the lipid bilayer was quantitated by injection of proteoliposomes with encapsulated Fura-2 into Ca2+ buffers and measurement of Fura-2 fluorescence as an indicator of Ca2+ influx. Reciprocally, to assess the function of proteins in an inside-out orientation, Ca2+-loaded vesicles were injected into Ca2+-free buffer and Ca2+ efflux monitored by a calcium electrode. Incorporation of the IIb-IIIa complex produced significant facilitation of Ca2+ movement across the lipid bilayer. No net transmembrane Ca2+ movement was seen with dissociated IIb and IIIa. Movement of Ca2+ was proportional to the transmembrane Ca2+ gradient. Ca2+ movement into the vesicles was inversely proportional to extravesicular NaCl from 25 to 150 mmol/L, analogous to several studies in the intact platelet. Adenosine triphosphate had no effect on Ca2+ movement into or out of the vesicles. Specific inhibition of a Ca2+ shift into the vesicles was seen with M148, a monoclonal antibody to IIb/IIIa, while no inhibition was observed with a panel of other anti-IIb/IIIa monoclonal antibodies. This suggests that a specific site on the complex or orientation of the complex is essential for calcium channel function. These data demonstrate that the GPIIb/IIIa complex can serve as a passive Ca2+ channel across a phospholipid bilayer and has the potential to play a role in Ca2+ flux across the platelet plasma membrane.     

Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V

Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity. In purified reaction mixtures, Gln506-FVa in comparison to normal FVa showed reduced susceptibility to APC, because it was inactivated approximately 10-fold slower than normal Arg506-FVa. It was previously reported that inactivation of normal FVa by APC involves an initial cleavage at Arg506 followed by phospholipid- dependent cleavage at Arg306. Immunoblot and amino acid sequence analyses showed that the 102-kD heavy chain of Gln506-FVa was cleaved at Arg306 during inactivation by APC in a phospholipid-dependent reaction. This reduced but measurable susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance is a mild risk factor for thrombosis because APC can inactivate both normal FVa and variant Gln506-FVa. In summary, this study shows that purified Gln506- FV can account for APC resistance of plasma because Gln506-FVa, whether generated by thrombin or FXa, is relatively resistant to APC.     

From the Cover: Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

Neutrophils are indispensable for clearing infections with the prominent human pathogen Staphylococcus aureus. Here, we report that S. aureus secretes a family of proteins that potently inhibits the activity of neutrophil serine proteases (NSPs): neutrophil elastase (NE), proteinase 3, and cathepsin G. The NSPs, but not related serine proteases, are specifically blocked by the extracellular adherence protein (Eap) and the functionally orphan Eap homologs EapH1 and EapH2, with inhibitory-constant values in the low-nanomolar range. Eap proteins are together essential for NSP inhibition by S. aureus in vitro and promote staphylococcal infection in vivo. The crystal structure of the EapH1/NE complex showed that Eap molecules constitute a unique class of noncovalent protease inhibitors that occlude the catalytic cleft of NSPs. These findings increase our insights into the complex pathogenesis of S. aureus infections and create opportunities to design novel treatment strategies for inflammatory conditions related to excessive NSP activity.Infections with the human pathogen Staphylococcus aureus constitute a major risk to human health. Although this bacterium harmlessly colonizes more than 30% of the population via the nose or skin, it causes severe morbidity and mortality upon invasion of deeper tissues (1). To avert these serious infections, neutrophils play an indispensable role (2). Neutrophil serine proteases (NSPs), including neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CG), are important for various neutrophil functions. Active NSPs are stored within the azurophilic granules (3), but upon neutrophil activation, they either enter the nucleus to regulate extracellular trap (NET) formation (4) or they are released into the extracellular milieu to kill certain bacteria (5), cleave bacterial virulence factors (5, 6), or regulate immune responses by cleaving chemokines and receptors (7). Recently, a fourth neutrophil serine protease, denoted NSP4, was identified (8).Given the central role of NSPs in neutrophil function, we wondered whether S. aureus had evolved mechanisms to cope with NSPs. In this study, we discover that S. aureus secretes a family of proteins that specifically and potently block NSPs: extracellular adherence protein (Eap) and the hitherto functional orphans Eap-homologue (EapH) 1 and 2. Structural studies presented here show that Eap molecules represent a unique class of noncovalent NSP inhibitors that is distinct from the well-known chelonianin class of inhibitors. These mechanistic insights can initiate development of novel, broad-range NSP inhibitors to be used in various inflammatory conditions. Furthermore, these insights increase our understanding of the pathogenicity of S. aureus and underline the exceptional capability of this pathogen to adapt to its host by modulating the immune response.     

Exotic and indigenous problem plants species used,by the Bapedi,to treat sexually transmitted infections in Limpopo Province,South Africa

Background

The ethnic usage of exotics and indigenous problem plants is a highly debated topic, as legislative requirements over-shadow their potential medicinal value, particularly to treat sexually transmitted infections (STIs). Limited information exists regarding their medicinal value among the Bapedi.

Objectives

To ascertain the importance of exotics and indigenous problem plants in the treatment of STIs, a major global primary health care challenge.

Methods

A field observation and semi-structured questionnaire focussing on species diversity, types of STIs treated and medicinal preparation as well as application was used to collect data from 34 traditional healers.

Results

Seven exotics and three indigenous problem species were identified. These species were used to treat four STIs; with Catharanthus roseus illustrating its dominance in the treatment of gonorrhoea. Some medicinal species used by Bapedi traditional healers have been validated through scientific research or through their extensive use by various cultures in South Africa and other parts of Africa. To the best of our knowledge Alternanthera pungens, Caesalpinia decapetala, Cinnamomum verum, and Citrullus lanatus are reported for the first time in the treatment of the investigated STIs.

Conclusions

Exotic and indigenous problem species constitute an important component of the STIs treatment protocol. Their utilization by Bapedi cautions against the narrow-minded approach of indiscriminate eradication, as these species can play a significant role in the primary health care needs of socio-economic vulnerable people.     

Admission inflammatory markers and isolation of a causative organism in patients with spontaneous spinal infection

Introduction

The purpose of this study was to investigate the significance of the inflammatory markers on admission in the isolation of a causative pathogen in patients with spinal infection. Spinal infection is treated frequently at spinal units and can encompass a broad range of clinical entities. Its diagnosis is often delayed because of the difficulty of identifying the responsible pathogen.

Methods

Patients with spinal infection treated in our institution over a period of eight years were identified and their notes studied retrospectively. Admission C-reactive protein (CRP), white cell count (WCC) as well as co-morbidities and mode of pathogen identification were recorded. Overall, 96 patients were included in the study.

Results

The CRP levels on admission were correlated significantly with the overall potential for isolation of a pathogen (p<0.0001) and positive biopsy cultures (p=0.0016). Admission WCC levels were associated significantly with the overall potential for isolation of a pathogen (p=0.0003) and positive biopsy cultures (p=0.0023). Both CRP and WCC levels were significantly negatively correlated with the duration of the preceding symptoms (p=0.0003 and p<0.0001 respectively). Delay in presentation was significantly negatively correlated with organism isolation (p=0.0001). Multivariate analyses identified the delay in presentation as the strongest independent variable for organism isolation (p=0.014) in cases of spontaneous spinal infection when compared with the admission CRP level (p=0.031) and WCC (p=0.056).

Conclusions

In spontaneous spinal infection, delay in presentation is the strongest independent variable for organism isolation. High inflammatory marker levels on admission are a useful prognostic marker for the overall potential of isolating a causative organism either by blood cultures or by biopsy in patients with negative blood cultures. Furthermore, the admission inflammatory marker levels allow for treating surgeons to counsel their patients of the likelihood of achieving a positive microbiological yield from biopsy.     

Hyaluronic Acid as a Marker of Hepatic Sinusoidal Obstruction Syndrome Secondary to Oxaliplatin-Based Chemotherapy in Patients with Colorectal Liver Metastases

Background

A considerable number of patients develop sinusoidal obstruction syndrome (SOS) after oxaliplatin-based chemotherapy for colorectal liver metastases (CLMs). SOS is associated with adverse outcomes after major hepatectomy. Hyaluronic acid (HA) is a marker of hepatic sinusoidal endothelial cell function and may serve as an accurate marker of SOS. This study aimed to assess the value of systemic HA levels and fractional extraction (FE) of HA by the splanchnic area and liver as markers of SOS after oxaliplatin-based chemotherapy for CLMs.

Methods

Forty patients were studied. The presence of SOS was assessed histopathologically. Blood samples from the radial artery and portal and hepatic veins were collected. HA levels were determined by ELISA and the FE of HA was estimated.

Results

SOS was present in 23 patients, 11 of whom demonstrated moderate or severe SOS. Preoperative HA levels were significantly higher in patients with moderate or severe SOS (group B, n = 11) compared to patients with no or mild SOS (group A, n = 29) (51.6 ± 10.2 ng/mL vs. 32.1 ± 3.5 ng/mL, p = 0.030). A cutoff HA level of 44.1 ng/mL yielded a sensitivity of 67 % and specificity of 83 % for detection of SOS. The positive predictive value was 50 % and the negative predictive value 91 %. Both groups exhibited a similar FE of HA by the splanchnic area (?7.9 ± 8.5 % in Group A vs. 7.3 ± 3.6 % in Group B, p = 0.422) and liver (?10.7 ± 6.2 % in Group A vs. 4.6 ± 2.3 % in Group B, p = 0.265).

Conclusions

Systemic HA levels can be used to detect patients at risk of SOS after oxaliplatin-based chemotherapy for CLMs. Additional investigations into the presence of SOS are indicated in patients with elevated HA levels.