Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses

Studies of the regulation of surfactant lipoprotein metabolism and secretion and surfactant protein gene expression have been hampered by the lack of a cell culture system in which the phenotypic properties of type II cells are maintained. We have developed a primary culture system that facilitates the maintenance of a number of morphologic and biochemical properties of type II pneumonocytes for up to 2 wk. Cells were isolated by collagenase digestion of midgestation human fetal lung tissue that had been maintained in organ culture in the presence of dibutyryl cyclic AMP (Bt2cAMP) for 5 days. The isolated cells were enriched for epithelial components by treatment with DEAE-dextran, plated on an extracellular matrix (ECM) derived from Madin-Darby canine kidney (MDCK) cells, and incubated at an air/liquid interface in a minimal amount of culture medium containing Bt2cAMP. The cell cultures were comprised of islands of round epithelial-like cells containing numerous dense osmiophilic granules, surrounded by sparse spindle-shaped cells with the appearance of fibroblasts. Ultrastructural examination revealed that the osmiophilic granules had the appearance of lamellar bodies, the distinguishing feature of type II pneumonocytes. Additionally, the cultures maintained elevated levels of SP-A gene expression for up to 2 wk. The expression of mRNAs encoding SP-A, SP-B, and SP-C were regulated in the cultured cells by glucocorticoids and cyclic AMP in a manner similar to that observed in fetal lung tissue in organ culture. The differentiated phenotype was most apparent when the cells were cultured at an air/liquid interface. In order to utilize the cultured type II cells for study of the effects of overexpression of various proteins and for promoter analysis, it is of essence to transfect DNA constructs into these cells with high efficiency. Unfortunately, we found the cells to be refractory to efficient transfer of DNA using conventional methods (i.e., lipofection, electroporation, or calcium phosphate-mediated transfection). However, replication-defective recombinant human adenoviruses were found to provide a highly efficient means of introducing DNA into the type II pneumonocytes. Furthermore, we observed in type II cell-enriched cultures infected with recombinant adenoviruses containing the lacZ gene under control of a cytomegalovirus promoter, that beta-galactosidase was expressed uniformly in the islands of type II cells and surrounding fibroblasts. By contrast, in cultures infected with recombinant adenoviruses containing the human growth hormone (hGH) gene under control of the SP-A gene promoter and 5'-flanking region, hGH was expressed only in the type II cells. Thus, this culture system provides an excellent means for identifying genomic elements that mediate type II cell-specific gene expression.     

The effects of retinol on in vitro immunoglobulin synthesis by cord blood and adult peripheral blood mononuclear cells

In this study we examined the effects of retinol (ROH), a metabolic precursor of retinoic acid (RA), on Staphylococcus aureus Cowan I (SAC)-induced immunoglobulin synthesis of cord blood mononuclear cells (CBMC) and adult peripheral blood mononuclear cells (PBMC). ROH augmented SAC-induced IgM synthesis of CBMC by 5.9 +/- 1.5-fold (n = 7, mean +/- s.d.), and IgG synthesis of adult PBMC by 16.3 +/- 5.1-fold (n = 3) at optimal concentrations of 10(-6) M and 10(-11) M, respectively. No augmenting effects could be demonstrated for the other immunoglobulin isotypes. Time-course studies showed that the synthesis of IgM by CBMC was accelerated with detectable immunoglobulin in supernatant fluids starting on day 3. ROH augmented immunoglobulin synthesis of CBMC stimulated by Epstein-Barr virus (EBV), a T cell-independent polyclonal activator, and of EBV-transformed B cell clones (2.5 +/- 0.2 and 4.1 +/- 1.5-fold increase, respectively), which suggests that ROH can act directly on B cells to enhance immunoglobulin synthesis. In contrast, when ROH was preincubated with cord blood T cells, washed and added to the B cell-enriched fraction with SAC, no increase (0.9-1.8-fold) in IgM synthesis was obtained. Thus, the principal mechanism(s) by which ROH augments immunoglobulin synthesis is by acting on B cells. This is in contrast to the immunoglobulin-enhancing effects of RA which is mediated by T cells, or T cell products, e.g. cytokine. Our studies suggest that RA and ROH may have different pathways of immunoglobulin-enhancing effects, perhaps mediated by different retinoid binding proteins resulting in gene activation and immunoglobulin synthesis.     

Loudness balance between acoustic and electric stimulation by a patient with a multichannel cochlear implant

Estimates of loudness balance were obtained for acoustically and electrically presented 250 Hz sine signals from a patient who uses the Ineraid multichannel cochlear implant. Acoustic and electric loudness matching was possible because the patient evidenced a 25 dB HL threshold at 250 Hz in his nonimplanted ear. The level of the electrical stimulus in microamperes required for a balance of loudness grew linearly with equal increments in decibels for the acoustic stimulus. These data, in concert with the very limited data from previous studies, provide a rationale for using a logarithmic transformation of acoustic to electric intensity in signal processors for cochlear implants.     

Cora valveless pulsatile rotary pump: new design and control

For decades, research for developing a totally implantable artificial ventricle has been carried on. For 4 to 5 years, two devices have been investigated clinically. For many years, we have studied a rotary (but not centrifugal) pump that furnishes pulsatile flow without a valve and does not need external venting or a compliance chamber. It is a hypocycloidal pump based on the principle of the Maillard-Wankel rotary compressor. Currently made of titanium, it is activated by an electrical brushless direct-current motor. The motor-pump unit is totally sealed and implantable, without noise or vibration. This pump was implanted as a left ventricular assist device in calves. The midterm experiments showed good hemodynamic function. The hemolysis was low, but serious problems were encountered: blood components collecting on the gear mechanism inside the rotor jammed the pump. We therefore redesigned the pump to seal the gear mechanism. We used a double system to seal the open end of the rotor cavity with components polished to superfine optical quality. In addition, we developed a control system based on the study of the predicted shape of the motor current. The new design is now underway. We hope to start chronic experiments again in a few months. If the problem of sealing the bearing could be solved, the Cora ventricle could be used as permanent totally implantable left ventricular assist device.     

Site of digestion and milk production by cows fed fats differing in saturation, esterification, and chain length

Four cannulated cows in a 4 x 4 Latin square design were used to study the effects of supplemental dietary fatty acids from roasted soybeans combined with tallow or partially hydrogenated fats, varying in esterification and fatty acid chain length, on nutrient digestion and lactation performance. Diets were formulated to contain (DM basis) 48% forage in addition to 1.5% fatty acids from roasted soybeans and 2.5% fatty acids from tallow, partially hydrogenated tallow triglycerides, partially hydrogenated tallow fatty acids, or a blend of 30% tallow and 70% hydrogenated fatty acids that were rich in palmitic acid. Apparent total tract digestibilities of OM, N, and NDF were similar among diets. Supplemental fat as fatty acids, compared with triglycerides, increased digestibilities of total fatty acids and C18:1 in the small intestine, perhaps indicating that lipolysis was rate-limiting. Fatty acids also increased milk fat percentage and efficiency of 4% FCM production. Although intake of C16 fatty acids was higher for cows fed the fat rich in palmitic acid than for those fed the tallow fatty acids, total duodenal flows of C16 fatty acids were similar, and digestibility in the small intestine was unaffected. Dry matter intake, 4% FCM production, and milk protein percentage were similar among treatments.     

Electrical pacing of the paralyzed human larynx

This study represents the first attempt to electrically pace the paralyzed human larynx. The goal was to determine if electrical stimulation of the posterior cricoarytenoid muscle could produce functional abduction of the vocal fold in pace with inspiration. An external apparatus was used to sense inspiration and reanimate the unilaterally paralyzed larynx of a thyroplasty patient. Stimuli were delivered through a needle electrode to locate and pace the abductor muscle. The magnitude of electrically induced abduction was comparable to spontaneous movement on the normal side. The abduction was appropriately timed with inspiration: this finding demonstrated that this simple pacing system could effectively modulate stimulation with patient respiration.     

Left ventricular outflow tract obstruction after mitral valve replacement

A method of posterior mitral annulus remodeling is presented. The posterior annulus is divided into three segments, each segment encircled by a suture that is passed in a tourniquet. Coaptation of the leaflets can be achieved by tightening the tourniquets while the ventricle is being filled. This technique is simple and quick, avoids the use of foreign material, and requires less expertise and judgment than traditional annuloplasties.