Repeated immunization with plasmid DNA formulated in poly(lactide-co-glycolide) microparticles is well tolerated and stimulates durable T cell responses to the tumor-associated antigen cytochrome P450 1B1

Injection of microparticle-encapsulated DNA elicits immune responses to plasmid-encoded antigens in mice and humans. Cytochrome P450 CYP1B1 (CYP1B1) is a member of the CYP1 P450 enzyme family that is overexpressed in a variety of solid tumors. The work described herein was performed to study the kinetics of stimulating T cell responsiveness with an encapsulated DNA encoding CYP1B1 and provides support for the clinical development of this formulation. Immunization of HLA-A2/Kb transgenic mice with human CYP1B1 encoding plasmid DNA formulated in poly(lactide-co-glycolide) (PLG) microparticles elicits CD8+ T cells that respond to human CYP1B1-positive target cells. The duration of the immune response, the effect on the immune response of multiple injections, and the safety of repeated injections were studied. These results show that the PLG-encapsulated DNA therapeutic elicits durable immune responses to CYP1B1, the responses are dependent on repeat immunization, and that the formulation is well tolerated.     

Standardization of lipoprotein reporting

We wanted to ascertain whether the current format of lipid laboratory reports seemed adequate to promote identification and treatment of patients with dyslipidemia. In a random survey of lipid laboratory reports from 25 laboratories, we found great inconsistencies among reporting formats and contents. Fewer than half the laboratories correctly reported the ranges for cholesterol, only 4 correctly reported ranges for high-density lipoprotein cholesterol, only 2 correctly reported ranges for triglycerides, and none presented low-density lipoprotein cholesterol ranges in terms of risk factors for coronary heart disease. Reports typically were disjointed and difficult to read. The current practice of reporting results for lipid panels is confusing and does not follow the National Cholesterol Education Program (NCEP) guidelines. We recommend that reporting of results be standardized, and a "model" standardized report is presented herein, based on consensus from a team of experts. The standardized report uses current recommendations for ranges, follows the flowcharts of the NCEP guidelines, and takes the patient's clinical condition (the number of risk factors and the presence of coronary heart disease) into consideration. Standardizing lipid reports should decrease confusion and perhaps increase application of the guidelines and patient compliance with treatment.     

Association between total serum calcium and the A986S polymorphism of the calcium-sensing receptor gene

Serum calcium is under tight physiological control, but it is also a quantitative trait with substantial genetic regulation. Mutations of the CASR gene cause familial hypocalciuric hypercalcemia or autosomal dominant hypoparathyroidism, depending on whether they decrease or increase, respectively, ligand binding to the receptor protein. We described an association between ionized calcium and a common polymorphism (A986S) found in the cytoplasmic tail of this G protein-coupled receptor. We report here on an independent study of 387 healthy young women. Genotyping was performed by allele-specific amplification and serum chemistries were measured by automated clinical assay. Frequencies of SS, AS, and AA genotypes were 6, 107, and 274, respectively, yielding a 986S allele frequency of 15.4%. Mean total serum calcium (Ca(T)) was significantly higher in the SS (9.88 +/- 0.29 mg/dL, P = 0.015) and AS groups (9.45 +/- 0.05 mg/dL, P = 0.002), than in the AA group (9.23 +/- 0.04 mg/dL). In multiple regression modeling, the A986S genotype remained an independently significant predictor of Ca(T) (P < 0.0001) when serum albumin, globulin, inorganic phosphate, and creatinine covariates were included. These data are the first to show significant association between a common polymorphism and concentrations of a serum electrolyte. The A986S polymorphism is also a potential predisposing factor in disorders of bone and mineral metabolism.     

Response of the human triceps surae muscle to electrical stimulation during varying levels of blood flow restriction

The changes in muscle force associated with varying degrees of lower-limb ischaemia were investigated. Isometric torque production by the triceps surae muscle was measured during a 5-min continuous train of 2-Hz electrical stimulation in six healthy young adults under different thigh cuff occlusion pressures. The reproducibility of this protocol when performed under complete ischaemia (tested five times over a 2-week period) was assessed as having a coefficient of variation (CV) for fatigue (end/initial force) of [mean (SEM) 12 (1)%; n=5]. This compares favourably with that obtained for maximum voluntary contraction torque [CV 9 (1)%]. In six subjects, triceps surae muscle fatigue was assessed under thigh cuff pressures of 0, 6.7 kPa (50 mmHg, venous occlusion) and 28 kPa (210 mmHg, complete ischaemia), as well as two intermediate levels of occlusion that were established by cuff pressures of 13.4 (0.5) and 20.3 (1.1) kPa [103 (4) and 152 (8) mmHg, respectively]. These corresponded to ankle-brachial pressure indices of 1.3 and 0.8, respectively when subjects were seated, or 0.8 and 0.36 when supine. With undisturbed lower-leg circulation, force potentiated steadily over the 5 min of stimulation such that the final force was 135 (8)% of the initial value. With complete ischaemia, force fell to 47 (2)% of the initial value. Stimulation under thigh occlusion pressures of 6.7, 13.4 and 20.3 kPa elicited intermediate levels of reduction in force, graded according to the increasing restriction of perfusion. The results show that low-force twitch contractions, which themselves do not occlude blood flow, are extremely sensitive to impaired perfusion and may represent a viable alternative to established methods of muscle performance assessment in patients with blood flow insufficiency. Accepted: 5 November 1999     

A defect in the humoral immune response to protein antigens and haptens in immunoglobulin mu heavy-chain transgenic mice.

We have examined the antibody response in mice expressing a functionally rearranged mu Ig heavy chain derived from a hybridoma antibody with specificity for the hapten 4-hydroxy-3-nitrophenyl (NP). Transgenic mice and their normal littermates were immunized with the antigens NP-OVA, the synthetic polypeptide (Tyr,Glu)-Ala-Lys ((T,G)-A-L), or saline. The presence of serum antibodies to NP-BSA, OVA, (T,G)-A-L, and BSA was examined by ELISA. Sera were evaluated prior to immunization and at periods of up to 4 months following immunization. Prior to immunization, transgenic mice had high levels of IgM anti-NP antibody but no detectable antibody to the other antigens. Both the primary and secondary antibody responses of transgenic mice to NP, OVA, and (T,G)-A-L were depressed when compared with the response of non-transgenic mice. Because of reports that these transgenic mice have increased proportions of CD5 + B-cells, a subpopulation associated with the production of autoantibodies, we examined these mice for the production of both IgG and IgM rheumatoid factors and anti-DNA antibodies. Transgenic mice had a modest increase in the spontaneous production of IgM anti-DNA. These data demonstrate a functional defect in the humoral immune response of mu transgenic mice.     

Control of single-joint movements in deafferented patients: evidence for amplitude coding rather than position control

Two deafferented patients and several control subjects participated in a series of experiments to investigate how accurate single-joint movements are programed, spatially calibrated, and updated in the absence of proprioceptive information. The deafferented patients suffered from a permanent and severe loss of large sensory myelinated fibers below the neck. Subjects performed, with and without vision, sequences of forearm supinations and pronations with two temporal delays between each movement (0 s and 8 s). Overall, the lack of proprioception did not yield any significant decrease in movement accuracy when vision was available. Without vision, the absence of proprioceptive afferents yielded (1) significantly larger spatial errors, (2) amplitude errors similar to those of control subjects, and (3) a significant drift when an 8-s delay was introduced between two successive movements. Subjects also performed, without vision, a 20 supination followed by a 20 pronation that brought back the wrist to the starting position. On some trials, the supination was blocked unexpectedly by way of a magnetic brake. When the supination was blocked, subjects were already on the second target and no pronation was required when the brake was released. The defferented patients, unaware of the procedure, always produced a 20 pronation. These data confirm that deafferented patients were not coding a final position. It rather suggests that they coded an amplitude and translated the spatial distance between the two targets in a corresponding force pulse. Overall, the results highlight the powerful and key role of proprioceptive afferents for calibrating the spatial motor frame of reference.