Protein C and protein S in homozygous sickle cell disease: does hepatic dysfunction contribute to low levels?

The aim of this study was to confirm reports of low protein C (PC) and S (PS) concentrations in steady-state patients with homozygous sickle cell (SS) disease when compared to a racially matched normal haemoglobin (AA) control group and to examine the mechanisms of this reduction with respect to hepatic function, coagulation activation and haematological indices.
In 36 SS patients and 35 AA race-matched controls PC (functional and immunoreactive), PS (free and total) were measured. C4B binding protein (C4B) was assessed by immunoelectrophoresis and D-dimer by ELISA. Hepatic function was assessed by prothrombin (PT) time (49 SS, 64 AA), factor V (34 SS, 36 AA) and factor VII concentrations (28 SS, 29 AA). Proteins induced in vitamin K absence or antagonism (PIVKA) were sought in 12 SS's. The relationship between PC, PS and total bilirubin, haemoglobin (Hb) F and reticulocyte count was also assessed.
PC, PS and C4B were lower in SS disease. SS patients had longer PT times, and lower factor V and VII concentrations in comparison to AA controls. PC (functional and immunoreactive) and free PS correlated with PT. Within SS genotype PT correlated negatively with factor V and factor VII. Factor V and VII were positively correlated. PIVKAs were not detected. There was no correlation between PC, PS and D-dimer, haemolytic rate or Hb F concentration.
Prolongation of PT time, low factor V and VII suggest that hepatic dysfunction, rather than coagulation activation or haemolytic rate, accounts for the reduced concentrations of PC and PS in steady-state SS disease. The absence of PIVKAs suggests a hepatocellular problem.     

A study of conformational restraints on reactivity of human PR3-specific autoantibodies (ANCA) facilitated through protein folding manipulations of a new recombinant proteinase 3 protein

Proteinase 3 (PR3)-specific antineutrophil cytoplasmic autoantibodies (PR3-ANCA) recognize conformational epitopes on PR3. This study evaluates PR3-ANCA target epitopes utilizing a novel recombinant PR3 (rPR3) produced to accommodate manipulations of the N-terminal domain. The rPR3 molecule contains an N-terminus six histidine tag, which can be removed by enterokinase (EK) cleavage of an adjacent EK cleavage site. Once cleaved the remaining amino acids correspond to the mature N-terminus of PR3. This rPR3 can be manipulated to produce three variant forms: tagged rPR3^+his, EK-cleaved (his-tag removed) rPR3^{? his}, and EK-cleaved, denatured/refolded rPR3^{? his/dr} (the proteolytically active form). Patients with clinically positive PR3-ANCA titers (n = 40) were confirmed for reactivity against purchased native PR3 in our system. Controls included 29 healthy volunteers and 34 MPO-ANCA patients. All PR3-ANCA sera samples tested were reactive with one or more forms of the recombinant protein (greater than mean ELISA OD 405 + 2 SDs of controls). Of significance, three sera were reactive with non-active forms only and three others were more reactive with rPR3^{? his/dr} than with native PR3. The results of our evaluation of PR3-ANCA sera for reactivity against the three forms of our rPR3 protein uniquely exemplify the diverse array of epitopes within the PR3-ANCA population. This new recombinant form of PR3 should provide a suitable approach to mapping ANCA epitopes using site-directed mutagenesis.     

Delayed clearance of pneumocystis carinii infection, increased inflammation, and altered nitric oxide metabolism in lungs of surfactant protein-D knockout mice

Surfactant protein-D (SP-D), a member of the "collectin" family, has been shown to play a role in innate immunity through modulation of inflammation and clearance of organisms. The role of SP-D in host defense against Pneumocystis carinii pneumonia was assessed using SP-D knockout (KO) mice. When inoculated with P. carinii, both wild-type (wt) and SP-D KO mice required CD4 cell depletion to develop infection. In CD4 cell-depleted models, 2 weeks after infection with P. carinii, SP-D KO mice developed increased intensity of infection, compared with wt mice, despite higher lung-inflammation scores and increased amounts of alveolar inflammatory cells. The increased inflammation seen in SP-D KO mice was accompanied by increases in lung weight, expression of inducible nitric oxide (NO) synthase, total NO levels, and 3-nitrotyrosine levels in lung tissue. These results indicate that SP-D plays a role in host defense against P. carinii in vivo by modulating clearance of organisms, lung inflammation, and metabolism of NO.     

Pharmacokinetics and safety of GW433908 and ritonavir, with and without efavirenz, in healthy volunteers

OBJECTIVE: To evaluate the safety and pharmacokinetic interaction between GW433908, ritonavir (RTV), and efavirenz (EFV). METHODS: In period 1, subjects received either a once daily (QD) regimen of GW433908 1395 mg + RTV 200 mg (Study 1) or a twice daily (bid) regimen of GW433908 700 mg + RTV 100 mg (Study 2) for 14 days. In period 2, subjects received EFV 600 mg QD with either the same GW433908 + RTV regimen as in period 1 (arm 1) or with a GW433908 + RTV regimen that included an additional 100 mg of RTV (arm 2) for 14 days. Amprenavir (APV) pharmacokinetic sampling and safety assessments were performed on the last day of each period. RESULTS: Plasma APV exposure was not significantly altered when EFV was coadministered with GW433908 700 mg twice daily (BID) + RTV 100 mg BID. Plasma APV exposure was decreased when EFV was coadministered with GW433908 1395 mg QD + RTV 200 mg QD. However, administration of EFV with GW433908 1395 mg QD + RTV 300 mg QD (i.e., adding an extra 100 mg of RTV) was able to negate this interaction. Adverse events were consistent with prior data for each of the separate agents. CONCLUSION: When EFV is coadministered with the GW433908 700 mg + RTV 100 mg BID regimen, no dosage adjustment is recommended. However, when EFV is coadministered with the GW433908 1400 mg + RTV 200 mg QD regimen, an increase to RTV 300 mg QD is needed to maintain plasma APV exposure.     

Guide-lines for near patient testing: haematology

Summary These guide-lines provide a framework for the local arrangement of near patient testing (NPT) services for haematology tests. The guidance may be applied to medical and surgical units within hospitals (e.g. ITU, renal dialysis units, casualty) as well as general practitioners' surgeries, for blood counts and coagulation testing. The professional head of the central laboratory must take responsibility for all aspects of the NPT service, although there should be full discussion with the clinical departments involved and joint ownership of the results. NPT operators must be trained and accredited by the central laboratory. Equipment selected should normally have received a satisfactory evaluation report from the Medical Devices Agency (MDA), and should generate results that are comparable with those of the central laboratory. If a full MDA operation evaluation has not been performed, the purchaser should perform a local assessment according to the protocol in this document. The suitability of the equipment, imprecision, and comparability must be studied. The NPT equipment must be properly maintained and calibrated, and a record of patient identity, date and time of testing, reagent lot numbers, and operator must be kept. The central laboratory must participate in a suitable external quality assessment programme (EQA), and provide systems for EQA and internal quality control (IQC) of the NPT site.     

Patient compliance with Hawley retainers fitted with the SMART® sensor: A prospective clinical pilot study

Objective:To evaluate the compliance of patients while wearing maxillary Hawley retainers embedded with SMART microsensors.Methods:The sample population consisted of 22 patients who were divided into an experimental (group A) and a control group (group B). Group A was informed that they would be monitored through the use of SMART microsensors, while group B was not informed that they would be monitored. After the delivery of the retainers (T0), the patients were evaluated at T1 and T2, represented by 6- and 12-week follow-up visits, respectively. At T1, group B was informed of our ability to monitor their compliance. Both groups continued wearing their retainers during T1 to T2.Results:During T0–T1, Group A wore their retainers for an average of 16.3 hours (SD 4.39), while group B wore their appliances for an average of 10.6 hours (SD 5.36, t  =  2.426, P  =  .027). Although group B increased their retainer wear by 0.5 hours/day from T1 to T2, this increase was not statistically significant.Conclusions:Despite significant differences being noted between the two groups at T1, group B did not show significant mean changes in their wear time before and after becoming aware of the use of the SMART microsensor.