In vitro and in vivo activity of murine lymphokine-activated killer cells after cryopreservation

The in vitro and in vivo effects of cryopreservation on the cytotoxic activity of murine lymphokine-activated killer (LAK) cells were studied. LAK cells were generated by incubation of spleen lymphocytes of BALB/c mice for 3 days with recombinant interleukin-2 (rIL-2) and subsequent cryopreservation. Cytotoxicity was determined in a 51Cr release assay. After thawing, cytotoxic activity was reduced (40.4% 51Cr release at an effector:target cell ratio of 40:1 as compared to 68.5% 51Cr release before freezing) and could be restored to precryopreserved levels by reincubation with rIL-2 for 2 days after thawing (78.8% 51Cr release). These cells were then tested in BALB/c mice injected with RAW 112 cells, a pre-B-cell lymphoma line. The results demonstrate that the survival rate of mice injected with cryopreserved and restimulated LAK cells (50% survival greater than 180 days after injection) did not differ significantly from that of mice injected with fresh unfrozen LAK cells (60% survival greater than 120 days, 50% survival greater than 180 days). Cryopreserved LAK cells have potential use in adoptive immunotherapy.     

Family history of myocardial infarction is an independent risk factor for venous thromboembolism: the Tromsø study

Summary. Background: Recent studies indicate that arterial cardiovascular diseases and venous thromboembolism (VTE) share common risk factors. A family history of myocardial infarction (MI) is a strong and independent risk factor for future MI. Objectives: The purpose of the present study was to determine the impact of cardiovascular risk factors, including family history of MI, on the incidence of VTE in a prospective, population‐based study. Patients and methods: Traditional cardiovascular risk factors and family history of MI were registered in 21 330 subjects, aged 25–96 years, enrolled in the Tromsø study in 1994–95. First‐lifetime VTE events during follow‐up were registered up to 1 September 2007. Results: There were 327 VTE events (1.40 per 1000 person‐years), 138 (42%) unprovoked, during a mean of 10.9 years of follow‐up. In age‐ and gender‐adjusted analysis, age [hazard ratio (HR) per decade, 1.97; 95% confidence interval (CI), 1.82–2.12], gender (men vs. women; HR, 1.25; 95% CI, 1.01–1.55), body mass index (BMI; HR per 3 kg m^?2, 1.21; 95% CI, 1.13–1.31), and family history of MI (HR, 1.31; 95% CI, 1.04–1.65) were significantly associated with VTE. Family history of MI remained a significant risk factor for total VTE (HR, 1.27; 95% CI, 1.01–1.60) and unprovoked VTE (HR, 1.46; 95% CI, 1.03–2.07) in multivariable analysis. Blood pressure, total cholesterol, HDL‐cholesterol, triglycerides, and smoking were not independently associated with total VTE. Conclusions: Family history of MI is a risk factor for both MI and VTE, and provides further evidence of a link between venous and arterial thrombosis.     

An audit of consent refusals in clinical research at a tertiary care center in India

Background and Rationale:

Ensuring research participants’ autonomy is one of the core ethical obligations of researchers. This fundamental principle confers on every participant the right to refuse to take part in clinical research, and the measure of the number of consent refusals could be an important metric to evaluate the quality of the informed consent process. This audit examined consent refusals among Indian participants in clinical studies done at our center.

Materials and Methods:

The number of consent refusals and their reasons in 10 studies done at our center over a 5-year period were assessed. The studies were classified by the authors according to the type of participant (healthy vs patients), type of sponsor (investigator-initiated vs pharmaceutical industry), type of study (observational vs interventional), level of risk [based on the Indian Council of Medical Research (ICMR) “Ethical Guidelines for Biomedical Research on Human Participants”], available knowledge of the intervention being studied, and each patient''s disease condition.

Results:

The overall consent refusal rate was 21%. This rate was higher among patient participants [23.8% vs. healthy people (14.9%); P = 0.002], in interventional studies [33.6% vs observational studies (7.5%); P < 0.0001], in pharmaceutical industry-sponsored studies [34.7% vs investigator-initiated studies (7.2%); P < 0.0001], and in studies with greater risk (P < 0.0001). The most common reasons for consent refusals were multiple blood collections (28%), inability to comply with the study protocol (20%), and the risks involved (20%).

Conclusion:

Our audit suggests the adequacy and reasonable quality of the informed consent process using consent refusals as a metric.KEY WORDS: Autonomy, consent, India, reason, refusal, risk     

The friction of explanted hip prostheses

Charnley prostheses, retrieved at revision surgery, were studied to assess the effects of friction on the total hip replacement procedure. Frictional resistance was measured using the Durham hip function simulator under both dry and lubricated conditions. The friction factor values (f) for the explanted prostheses were found to have a non- Gaussian distribution with medians of 0.13 [inter-quartile range (IQR) 0.10-0.16] and 0.06 (IQR 0.005-0.08) for dry and lubricated (n = 0.01 Pa s) regimes, respectively. New Charnley prostheses had values of f equal to 0.11 +/- 0.025 and 0.04 +/- 0.01 under the same conditions, and showed no large deviation from a Gaussian distribution. There was found to be a statistically significant difference in the medians of the friction factors for new and retrieved prostheses in the lubricated regime. Ingression of cement into the worn region of the cup was found to increase the friction factor significantly under dry conditions. There was no evidence of an increase in the friction factor or torque for those joints that had a loose socket with respect to those that were fixed at revision. A decrease in the frictional torque against number of cycles undergone by the joint in vivo may indicate that a fatigue-type process may have a role in the loosening of the socket. However, this relationship was found not to be significant for friction measured under lubricated conditions and it seems unlikely that the frictional torque generated in this type of prosthesis will contribute significantly to the long-term loosening of the socket.      

Association of granulocyte-macrophage colony-stimulating factor with the crystalloid granules of human eosinophils

We have previously shown that normal-density human peripheral blood eosinophils transcribe and translate mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) and that the intracellular distribution was granular as assessed by light microscopy immunocytochemistry. The present study was conducted to confirm this apparent association between GM-CSF and the crystalloid granule using a subcellular fractionation method for human eosinophils and immunogold electron microscopy (EM). Highly purified (> 99%, by negative selection using anti-CD16 immunomagnetic microbeads) human peripheral blood eosinophils were obtained from four asthmatic subjects (not taking systemic medication), homogenized and density fractionated (5 x 10(7) cells/subject) on linear Nycodenz gradients. Twenty-four fractions were collected from each cell preparation and analyzed for marker enzyme activities as well as total protein. Dot blot analysis with specific monoclonal antibodies (MoAbs) was used to detect the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP). An anti-CD9 MoAb was used as an eosinophil plasma membrane marker. Lactate dehydrogenase (LDH) was used as a cytosolic marker. Immunoreactivity for GM-CSF was detected by a specific enzyme-linked immunosorbent assay using a polyclonal antihuman GM-CSF antibody and confirmed by dot blot. GM-CSF coeluted with the cellular fractions containing granule markers (MBP, ECP, eosinophil peroxidase, hexosaminidase, and arylsulphatase), but not those containing cytoplasm (LDH+) or membrane (CD9+) markers. EM examination of pooled fractions associated with the peak of GM-CSF immunoreactivity confirmed that they contained crystalloid and small granules, but not plasma membrane. In addition, quantification, using immunogold labeling with an anti/GM-CSF MoAb, indicated preferential localization of gold particles over the eosinophil granule cores of intact cells. Thus, our results indicate that GM-CSF resides as a granule-associated, stored mediator in unstimulated human eosinophils.