Cost-effective manufacture of an allogeneic GM-CSF-secreting breast tumor vaccine in an academic cGMP facility.

BACKGROUND: GM-CSF-secreting, allogeneic cell-based cancer vaccines have shown promise for the treatment of a variety of solid tumors. We have now applied this approach to breast cancer. The aim of these studies was to optimize expansion parameters, qualify the manufacturing process, and establish expected outcomes for cGMP-compliant manufacturing of two GM-CSF-secreting breast tumor cell lines. METHODS: The variables affecting the efficiency of expanding and formulating two allogeneic GM-CSF-secreting cell lines, 2T47D-V and 3SKBR3-7, were systematically evaluated. Production criteria investigated included alternative cell culture vessels (flasks vs. cell factories), centrifugation time and speed variables for large volume cell concentration, cell seeding density, the minimal concentration of FBS required for maximal cell expansion, and the dose and timing of irradiation in relation to cryopreservation. RESULTS: These studies demonstrate that, in comparison with standard 150-cm2 tissue culture flasks, Nunc 10-Stack Cell Factories are a more efficient and practical cell culture vessel for vaccine cell line manufacture. Centrifugation optimization studies using the COBE 2991 Cell Processor established that a speed of 2000 r.p.m. (450 g) for 2 min reliably concentrated the cells while maintaining acceptable viability and bioactivity. Radiation studies established that lethal irradiation prior to cryopreservation does not compromise the quality of the product, as measured by post-thaw cell viability and GM-CSF cell line-specific secretion levels. Finally, studies aimed at optimizing the production of one vaccine cell line, 3SKBR3-7, demonstrated that seeding the cells at a higher density and maintaining them in half the initial concentration of FBS maximized the yield of bioactive cells, resulting in significant cost savings. DISCUSSION: A manufacturing process that simultaneously maximizes cell yield, minimizes cell manipulation and maintains vaccine cell potency is critical for producing cell-based cancer vaccines in an academic setting. These studies define a feasible, reproducible and cost-effective methodology for production of a GM-CSF-secreting breast cancer vaccine that is cGMP compliant.

Mesenchymal stem cells as vehicles for gene delivery.

Mesenchymal stem cells contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose, and marrow stroma. Transduction of mesenchymal stem cells from species other than humans is required for the development of disease models in which mesenchymal stem cells-based gene delivery is evaluated. Attempts to transduce mesenchymal stem cells from some species with amphotropic retroviral vectors were unsuccessful, leading to comparative mesenchymal stem cells transductions with xenotropic and gibbon-ape leukemia virus envelope-pseudotyped retroviral vectors. Human, baboon, canine, and rat mesenchymal stem cells were transduced optimally with amphotropic vector supernatants. In contrast, sheep, goat, and pig mesenchymal stem cells showed highest transduction levels with xenotropic retroviral vector supernatant, and rabbit mesenchymal stem cells were transduced optimally with gibbon-ape-enveloped vectors. Using a myeloablative canine transplantation model and gene-marked canine mesenchymal stem cells, the biodistribution of infused and ex vivo expanded mesenchymal stem cells were examined. The majority of transduced canine mesenchymal stem cells were found in the bone marrow samples. The current study shows the use of mesenchymal stem cells as a delivery vehicle for gene transfer studies, and validates the feasibility of delivering mesenchymal stem cells to the marrow compartment for stromal regeneration after cancer-associated cytotoxic therapies.

Comparison of methods for assessing body-composition changes over 1 y in postmenopausal women.

BACKGROUND: Advances in dual-energy X-ray absorptiometry (DXA) software algorithms have improved the accuracy of this method for body-composition measurement. OBJECTIVE: Our objective was to compare the utility of DXA, underwater weighing (UWW), and a multicomponent model (MC) for assessing changes in body composition. DESIGN: : Previously sedentary women aged 40-66 y were randomly assigned to exercise training (ET; n = 36) and no exercise training (NT; n = 40). ET subjects exercised 3 d/wk; NT subjects remained sedentary. Changes in body mass, fat mass, and fat-free mass over 1 y were assessed by the 3 methods. RESULTS: Correlations among methods were significant and large (0.73-0.97). Body weight did not change significantly in either group. In the ET group, fat-free mass increased significantly as assessed by DXA (0.7 +/- 1.0 kg) but changes assessed by MC and UWW were not significant. Changes in fat mass and percentage body fat in the ET group were not significant. SDs for changes in fat mass and percentage body fat, respectively, from DXA were 2.5 kg and 2.7%; for MC, 5.5 kg and 7.1%; and for UWW, 4.4 kg and 5.8%. In the NT group, changes in fat-free mass, fat mass, and percentage body fat were significant (P

Acute correction and distraction osteogenesis for the malaligned and shortened lower extremity.

In limbs with combined shortening with angulation or malrotation, deformity may be quickly or slowly corrected before lengthening with external fixation. We examined a series of 35 patients with 40 limbs that underwent acute deformity correction and subsequent gradual lengthening. The average deformity corrected was 19 degrees, with subsequent average lengthening of 4.1 cm. Good radiographic callus formation was noted in 34 of the 40 segments studied. The magnitude of deformity correction had no effect on the quality of lengthened bone, incidence of complications, or the healing index. Skeletally mature segments had statistically significant decreased bone formation (p = 0.001), increased prevalence of callus complications (p = 0.001), and a higher healing index (p = 0.003). Based on this experience, it is our conclusion that immediate correction and lengthening is suitable in children and adolescents who have malaligned and shortened lower extremities. Because of poorer results in older patients, we believe that other techniques should be considered in adults.

Nighttime bracing for adolescent idiopathic scoliosis with the Charleston Bending Brace: long-term follow-up.

We report long-term experience with the Charleston Bending Brace for treatment of adolescent idiopathic scoliosis. This brace holds the patient in maximal side-bending correction and is worn at nighttime only. Patients included in this prospective multicenter study met all of the following criteria: skeletal immaturity (Risser 0, 1, or 2), curvature >25 degrees before bracing, no prior treatment, and >1-year follow-up since completion of bracing (skeletal maturity or progression to surgery). All curves were monitored and reported. There were 149 structural curves in 98 patients. Sixty-five (66%) patients showed improvement or <5 degrees change in curvature. Seventeen (17%) patients progressed to the point of requiring surgery for their scoliosis. Based on these long-term results and improvement of the natural history of adolescent idiopathic scoliosis, continued use of the Charleston Bending Brace is justified.

Surgical treatment of Freiberg's infraction in athletes.

Ten patients with 11 cases of Freiberg's infraction were studied retrospectively. The patients had varying amounts of sports participation. All cases were treated surgically, most after some form of nonoperative intervention. Surgery consisted of metatarsophalangeal joint debridement, except in 1 patient where the metatarsal head was resected. All patients had improvement of their symptoms and 80% of normal joint range of motion was restored. No patient had joint space narrowing or major arthritic changes on follow-up roentgenographic studies.

Recent advances in limb lengthening. Part II: Biological advances.

The most important recent advance in limb lengthening is the recognition that bone will regenerate, under appropriate conditions, within the gap of an osteotomized bone. Although Codivilla recognized this phenomenon, bone formation was inconsistent in his patients. Optimal distraction osteogenesis requires a knowledge of osteotomy techniques, length of latency period, rate and frequency of distraction, and mechanical effects of loading.

Recent advances in limb lengthening. Part I: Clinical advances.

The discipline of limb lengthening has undergone numerous advances in recent years. An increased understanding of the biology of distraction osteogenesis, as well as technical advances, has made lengthening more feasible. However, significant problems remain unsolved, and complications--although less severe and long standing--still remain. Little is known about the effect of distraction on soft tissues. Lengthening in skeletally mature patients remains difficult. The myriad of conditions that require lengthening often confounds comparison of techniques. Uniform indications for limb lengthening may never exist, but increasing experience helps guide patient selection.

Sampling of diode laser spectra by a confocal etalon fringe pattern.

This paper presents an original method of IR diode laser spectra sampling, where the sampling is defined by the fringe pattern of a confocal etalon. The laser wave number is scanned between two peaks of the Airy type function and is temporarily locked to each peak, allowing integration of the spectroscopic data on another optoelectronic channel. This method is well adapted to broad absorption line studies, provided the line can afford the relatively large sample spacing (0.01 cm(-1)). A monitoring experiment for both pressure and HBr concentration in halogen bulbs is presented to show an example of the method's use.

Multipass absorption cell designed for high temperature UHV operation.

This paper describes the design and performance of a tunable diode laser system, incorporating a multipass absorption cell to allow determination of water concentration below 10 ppb and carbon dioxide concentration below 1 ppb in nitrogen semiconductor gas. The cell is used with a tunable Pb-salt diode laser spectrometer frequency locked to a first derivative error signal from an intense vibration-rotation molecular absorption line. Sample concentrations are monitored in the second derivative mode, and the system automatically compensates for laser intensity fluctuations. A flowing gas method is used to minimize adsorption/desorption effects from the sample cell walls.

Effect of phenols on the oxidation of estradiol by uterine peroxidase.

The effect of various phenols on the conversion of [4 -14C]estradiol to water-soluble products by estrogen-induced uterine peroxidase (EC 1.11.1.7) has been investigated. Evidence was provided that those phenols which enhanced the oxidation of estradiol exerted their effect by activating peroxidase or protecting the enzyme from inactivation by the products of the reaction rather than by inhibiting the breakdown of hydrogen peroxide by catalase (EC 1.11.1.6). It has also been shown that tyrosine acted both as an activator of uterine peroxidase and as a water-soluble acceptor for the metabolites of estradiol. The ability of tyrosyl peptides to form conjugates with estradiol was influenced by the other amino acids and decreased with the number of adjacent tyrosyl residues.