Escherichia coli secretion of an active chimeric antibody fragment.

A chimeric mouse-human Fab protein that binds specifically to the human carcinoma cell line C3347 has been expressed and secreted from Escherichia coli. This molecule, which contains functionally assembled kappa and Fd proteins, binds as effectively to sites on the surface of C3347 cells as Fab fragments prepared proteolytically from whole chimeric or mouse antibody. The production in Escherichia coli of foreign heterodimeric protein reagents, such as Fab, should prove useful in the management of human disease.

Plasma dopamine beta-hydroxylase: a possible aid in the study and evaluation of hypertension.

The activity of dopamine beta-hydroxylase (DBH) in plasma ranged from 2 to 100 units per liter of plasma in 82 apparently healthy subjects (ages 22 to 35 years). A nonnormal pattern of distribution was evident: 62 subjects had values below 35 units (18 +/- 1), while 13 of the remaining 20 subjects had values above 60 units (80 +/- 5). Those with low DBH activity had lower values for urinary catecholamine excretion (31 +/- 3 micrograms), with normal and stable blood pressure; those with high DBH activity had higher values for urinary catecholamine excretion (72 +/- 6 micrograms), with greater lability of arterial blood pressure. The DBH activity was significantly elevated in patients with labile (74 +/- 2 mm-Hg) or fixed (57 +/- 2 mm-Hg) essential hypertension. The results indicate that plasma DBH activity is low and that it falls within a narrow range in young adults with normal and stable blood pressure.

Distal tubular feedback in the autoregulation of single nephron glomerular filtration rate.

Renal clearance and recollection micro-puncture experiments were conducted to evaluate the possible role of a distal tubular feedback mechanism in the phenomenon of renal autoregulation in dogs. Single nephron glomerular filtration rate (SNGFR) was measured from collection sites in both the proximal (proximal SNGFR) and distal tubules (distal SNGFR). Single nephron autoregulatory behavior was assessed by evaluating the response of SNGFR to a reduction in renal arterial pressure imposed by means of an aortic constrictor. Whole kidney function was evaluated by parallel measurements of renal blood flow and inulin clearance. Whole kidney autoregulation was observed when renal arterial pressure was decreased from 141 +/- 3 (SE) mm Hg to 101 +/- 2 mm Hg; renal blood flow and GFR were not significantly altered from control values of 3.76 +/- 0.2 ml/min.g and 0.69 +/- 0.04 ml/min.g kidney weight, respectively. In 11 autoregulating preparations, proximal transit time was likewise unchanged from the control value of 26 +/- 2 s, indirectly suggesting that the superficial nephrons also participated in the autoregulatory response. However, proximal SNGFR decreased significantly from 88 +/- 7 nl/min to 66 +/- 6 nl/min, a reduction which was proportional to the decrease in arterial pressure. In 14 dogs in which both proximal SNGFR and distal SNGFR were measured at control blood pressure (136 +/- 5 mm Hg), distal SNGFR was 47 +/- 4 nl/min, a value significantly lower than that for proximal SNGFR (79 +/- 6 nl/min). In contrast to the results based upon proximal collections, distal SNGFR was not significantly altered following aortic constriction (44 +/- 5 nl/min vs. 47 +/- 5 nl/min) therefore exhibiting autoregulation in association with that observed for the whole kidney. These experiments indicate that though superficial nephrons do possess autoregulatory capability, interruption of distal delivery due to complete collection from the proximal tubule interferes with that nephron's ability to manifest an autoregulatory response. They support the concept that a feedback mechanism, related to some function of distal delivery, is of significance in the intrinsic regulation of SNGFR. The data further suggest that quantitative estimates of SNGFR based on complete proximal collections may not be representative of those throughout the superficial cortex of the dog, at least in certain experimental circumstances.

Relationship between para-aminohippurate secretion and cellular morphology in rabbit proximal tubules.

Previous studies in the mammalian proximal tubule have suggested that para-aminohippurate (PAH) secretion is approximately threefold greater in the straight segment, or pars recta, than in the convoluted segment, or pars convoluta. However, the possibility that the site of maximal PAH secretion might be related better to particular tubule segments as identified by cell type had not been explored. In addition, the presence or absence of differences in PAH secretion between morphologically identical regions of superficial (SF) vs. juxtamedullary (JM) proximal tubules has not been examined. These issues were studied using a combination of histologic methods and measurement of [(3)H]PAH secretion in isolated perfused tubules. Measurements of microdissected SF and JM proximal tubules from young and adult rabbits revealed that SF proximal tubules were slightly but significantly longer than JM tubules ([young rabbits: SF, 8.69+/-SE 0.14 mm vs. JM, 7.97+/-SE 0.13 mm; P < 0.01] [adult rabbits: SF, 10.61+/-SE 0.28 mm; JM, 9.17+/-SE 0.19 mm; P < 0.001]). Light and electron microscopy revealed three sequential segments (S(1), S(2), and S(3)) along the length of SF and JM proximal tubules as defined by cell type. PAH secretion was measured in each of these three segments by the isolated perfused tubule technique. Net PAH secretion in fmol/mm per min in SF proximal tubules was: S(1), 281+/-SE 21; S(2), 1,508+/-SE 104; S(3), 318+/-SE 46. Corresponding values in JM proximal tubules were 353+/-SE 31, 1,391+/-SE 72, and 188+/-SE 23. Net PAH secretion did not differ between comparable segments of SF and JM proximal tubules. It is concluded that differences in PAH secretion along the proximal tubule correlate best with cell type rather than the arbitrary division of the proximal tubule into pars convoluta and pars recta according to its external configuration. Evidence of functional heterogeneity between comparable segments of SF and JM proximal tubules was not observed.

Relationship between phosphaluria and acute hypercapnia in the rat.

Standard clearance studies were performed in mechanically ventilated intact and acutely thyroparathyroidectomized (TPTX) rats to document and characterize the effect of hypercapnia (HC) on urinary phosphorus excretion (U(P)V). HC as compared to normocapnia (NC) was associated with an increase in U(P)V in intact (62.5 vs. 7.93 mug/min) and TPTX (30.5 vs. 0.59 mug/min) rats, an increase in filtered load of phosphorus in intact (218 vs. 191 mug/min) and TPTX (243 vs. 146 mug/min) rats, an increase in blood bicarbonate concentration in intact (27.8 vs. 26.0 meq/liter) and TPTX (24.5 vs. 22.3 meq/liter) animals, and a decrease in blood pH in intact (7.15 vs. 7.42) and TPTX (7.07 vs. 7.39) rats. Additional TPTX rats with NC and HC were studied during phosphorus infusion at a comparable filtered load of phosphorus (NC = 307 mug/min and HC = 328 mug/min). U(P)V was 18.5 mug/min in NC and 85.2 mug/min in HC animals. Intact NC animals infused with NaHCO(3) achieved a blood bicarbonate of 45.9 meq/liter compared to 26.0 meq/liter in intact NC NaCl-infused rats. U(P)V was 10.0 mug/min in the NaHCO(3) and 7.93 mug/min in NaCl-infused animals. In intact HC animals infused with NaHCO(3), blood pH was 7.36 compared to 7.42 in NC intact NaCl-infused animals. U(P)V was 83.2 mug/min in the HC bicarbonate-infused and 7.93 mug/min in the NC NaCl-infused rats. These experiments demonstrate that elevated blood carbon dioxide tension per se increases U(P)V. Increases in filtered load of phosphorus and blood bicarbonate which are associated with HC contribute to the phosphaturia as does parathyroid hormone. The phosphaturia is not dependent upon reduction of extracellular pH.

Renal transplantation between HL-A identical donor-recipient pairs. Functional and morphological evaluation.

16 patients underwent renal transplantation from a sibling donor who was prospectively determined to be ABO compatible and HL-A identical with the recipient. Unidirectional mixed leukocyte reactions were performed; in each instance, lymphocyte stimulation in either direction was not observed. The plasma creatinine 10-68 months after transplantation in these 16 patients ranged between 0.9 and 1.9 mg/100 ml. The creatinine clearance ranged from 48 to 113 ml/min, and the blood urea nitrogen (BUN) ranged between 12 and 35 mg/100 ml. Urine protein excretion varied from 0.11 to 1.86 g/day. Six patients exhibited no detectable clinical episodes of acute rejection; they were treated with azathioprine alone and each of them demonstrated normal or near normal renal histology when biopsy specimens were obtained more than 6 months after transplantation. Nine patients experienced acute rejection episodes that required the use of steroid therapy. The severity of these rejection episodes was variable; they included a mild reduction in renal function with an immediate steroid-induced restoration of function and eventual discontinuance of steroid therapy to severe reduction in function requiring prolonged and moderate doses of steroids without return to normal renal function. Renal histological observations in this group ranged from mild to marked cellular and structural changes which fit the criteria of the rejection. One patient demonstrated a gradual loss of renal function with heavy proteinuria. Biopsy of this allograft demonstrated the recurrence of original disease, i.e., lobular glomerulonephritis. The marked variability in the clinical course and allograft morphology in these 16 patients could be explained by antigenic differences at non-HL-A loci. The presence of minor histocompatibility loci has been well documented in other mammalian species and they are most certainly present in man. The need for their identification and definition is stressed.

Augmentation of antibody dependent cell mediated cytotoxicity following in vivo therapy with recombinant interleukin 2.

Monoclonal antibodies (mAB) with tumor specificity are able to enhance the immunological specificity of interleukin 2 (IL-2)-activated lymphokine activated killer (LAK) cells. Antibodies may also be used to broaden the range of tumor types susceptible to immune mediated cytotoxicity by the activated LAK cells. In these studies, mAB with relative tumor specificity were used to target immunologically activated effector cells in an in vitro antibody dependent cell mediated cytotoxicity (ADCC) assay. The mAB included: 3F8 and 14.G2a, which are both specific for neuroblastoma and melanoma and recognize ganglioside GD2, and mAB ING-1, a mouse-human chimeric antibody with constant regions from human IgG1 and kappa chains and variable regions from a mouse mAB that binds to a broad range of human adenocarcinomas. Each of these mAB was able to mediate ADCC with fresh effector cells and antibody binding targets. When peripheral blood mononuclear cells were obtained from cancer patients prior to and following in vivo therapy with interleukin 2, a significant increase was noted in ADCC activity by peripheral blood mononuclear cells obtained following IL-2 therapy. Inclusion of IL-2 in the medium during the cytotoxic assay with mAB further boosted ADCC. The total activity seen was often greater than the sum of the independent LAK activity and standard ADCC activity. The cells responsible for this ADCC had the CD16+ Fc receptor. Combining IL-2 with mAB in clinical tumor therapy may lead to a wider range of tumor types being responsive to immunotherapy and may also enhance the efficacy of therapy by specifically targeting activated effector cells to tumor cells recognized by mAB. Our results provide strong support for the testing of these hypotheses in clinical trials by combining in vivo treatment with IL-2 and mAB able to mediate ADCC.

Influence of estradiol and tamoxifen on susceptibility of human breast cancer cell lines to lysis by lymphokine-activated killer cells.

The design of combination hormonal and immunotherapeutic protocols for breast cancer patients may be facilitated by analysis of preclinical in vitro model systems. Estrogen receptor positive (ER+: MCF-7) and negative (ER-: MDA-MB-231) human breast cancer cell lines were utilized to evaluate the effects of tamoxifen (TAM) and estradiol (E2) on modulation of breast cancer target susceptibility to lysis by lymphokine-activated killer (LAK) cells. E2-stimulated ER+ cells were more susceptible to lysis by LAK cells than corresponding TAM-treated or control cells, while treatment of ER- cells with either E2 or TAM alone did not alter from control their susceptibility to this immune-mediated lysis. All ER+ and ER- cells tested remained sensitive after treatment with TAM to lysis by LAK cells. In addition, an adenocarcinoma reactive human-mouse chimeric monoclonal antibody (ING-1) was able to significantly boost in vivo generated LAK cell-mediated lysis of control, E2-treated, and TAM-treated ER+ and ER- cells. These in vitro results provide a preclinical rationale for in vivo testing of TAM, interleukin-2 (IL-2), and breast cancer reactive antibody-dependent cellular cytotoxicity facilitating antibody in patients with refractory or high risk breast cancer.

Expression of mouse::human immunoglobulin heavy-chain cDNA in lymphoid cells.

A chimeric mouse variable::human constant immunoglobulin heavy-chain gene was expressed in transfected mouse Sp2/0 cells. The chimeric immunoglobulin genes were integrated in tandem in the genome of stably transformed cells. These integrated gene copies were amplified by selection with a second drug marker. The gene amplification led to an increase in the expression of chimeric heavy-chain protein. The level of gene expression appears to be related to the site of integration; a few gene copies in one transfectant can yield as much heavy-chain protein as many copies in a second transfectant. In addition, we found that an adventitious oligo(C) sequence, introduced by our method of gene construction at a site located 8 nt residues downstream from a splice acceptor, can apparently direct splicing towards a cryptic splice acceptor downstream from the oligo(C).

Focal glomerular sclerosis: contrasting clinical patterns in children and adults.

In a retrospective clinicopathological study, 48 kidney biopsy specimens from 16 children (mean age, 7 years) and 17 adults (mean age, 33 years) with histological evidence of focal glomerular sclerosis (FGS) were examined using light, immunofluorescence and electron microscopy. The histopathological findings were related to the clinical course of each patient. At the clinical onset of the disease, the nephrotic syndrome was seen more commonly in children (12/16) than adults (7/17), while the incidence of both hypertension (children 1/16 versus adults, 9/17) and renal insufficiency (children, 0/16 versus adults, 7/17) was greater in adults. Despite a shorter average follow-up, (adults 3 10/12 years versus children, 7 years), the incidence of hypertension (adults, 13/17 versus children, 7/16) and renal functional impairment (adults, 13/17 versus children, 3/16) remained greater in the adult patients. One child and three adults died in renal failure while two adults underwent transplantation and on requires regular dialysis therapy. Nine of 15 pediatric patients treated with corticosteroids experienced partial or complete remission in either their nephrotic syndrome or level of urine protein excretion, while just 3 of 6 adult patients treated with corticosteroids experienced a partial remission, but never became protein-free. There was an excellent correlation in all patients between the degree of functional renal impairment and the extent of glomerular and nonglomerular histopathological damage in the kidney. It is concluded that in the adults, FGS represents a more severe and progressive disease process and is less responsive to therapy.

A simple, computer-assisted assay to detect isotype-specific regulation of human immunoglobulin synthesis.

A simple, reliable, and computer-assisted assay has been developed to quantitate isotype-specific regulation of human immunoglobulin synthesis in vitro. The assay utilizes three separate human lymphoblast or myeloma cell lines, which secrete human immunoglobulins IgA, IgG, and IgE. Culture supernatants from 96-well tissue culture plates are then assayed for IgA, IgG, and IgE by a solid-phase enzyme-linked immunosorbent assay (ELISA) on a microtiter plate. Data collection and analysis is performed with the aid of computer programs designed for this assay. This assay has several advantages over other immunoglobulin regulation assays: no radioisotopes are used, thereby reducing cost and complexity; results are directly collected and quantified by computer analysis; the entire assay is completed in 3 days; reliability and reproducibility are increased by the use of established human cell lines; and co-culturing all three immunoglobulin-producing cell lines provides convenient internal controls for isotype specificity.

Intermittent acceleration as a countermeasure to soleus muscle atrophy.

The centrifuge proposed for the Space Station will most likely be used, in part, for countermeasure studies. At present, there is a paucity of information concerning the duration and frequency of acceleration necessary to counteract the atrophy process associated with microgravity. The present study was designed to investigate intermittent acceleration during non-weight bearing of the soleus muscle and its resultant effects on muscular atrophy. Each day rats were removed from hindlimbs suspension and accelerated to 1.2 g for four 15-min periods evenly spaced over a 12-h interval. The soleus muscle experienced non-weight bearing the remaining 23 h each day. This paradigm, when repeated for 7 days, did not completely maintain the mass of soleus muscle, which was 84% of control. Interestingly, the identical protocol utilizing ground support in lieu of acceleration successfully maintained the soleus muscle mass. The failure of the centrifugation protocol to adequately maintain soleus muscle mass might be due to an undefined stress placed on the animals inherent in centrifugation itself. This stress may also explain the transient decline in food intake of the intermittent acceleration group on the 2nd and 3rd days of treatment. Also, these data support the concept that the frequency of exposure, as opposed to the duration of exposure, to weight bearing during hindlimb unweighting seems to be the more important determinant of maintaining postural muscle mass.

Production of anti-erythrocyte antibodies by leukemic and nonleukemic B cells in chronic lymphocytic leukemia patients.

We have assessed the specificity of antibodies from the leukemic B cells of five patients with both chronic lymphocytic leukemia and autoimmune hemolytic anemia (CLL-AHA). Leukemic cells from one patient displayed surface immunoglobulin with heavy and light chain isotypes identical to that of the patient's anti-red blood cell (RBC) antibodies, and the leukemic cells secreted antibodies in vitro with anti-RBC activity. However, in the remaining patients, the leukemic cells displayed surface immunoglobulin with light chain isotypes different from that of the patient's anti-RBC antibodies and secreted antibodies in vitro with no detectable anti-RBC activity. Thus, there are two distinct classes of CLL-AHA patients, differentiated by the presence or absence of an anti-RBC antibody-producing leukemic B cell clone. The apparent heterogeneity in the source of pathogenic anti-RBC antibodies may impact the treatment response of the two classes of CLL-AHA patients.

Fate of sodium pentobarbital in rendered products.

The fate of pentobarbital through rendering was evaluated by following a group of euthanatized animals through a commercial rendering facility. Samples of material were collected at various points in the rendering process, and assays for pentobarbital were conducted by an ultraviolet spectrophotometric method. The results indicated that the pentobarbital, or a closely related analogue, survived rendering without undergoing degradation. The pentobarbital was distributed approximately equally between the meat and bone meal and tallow fractions. If pentobarbital-euthanatized animals are processed along with other raw materials throughout a day's production, the likelihood of significant residues being present in rendered products is minimal.

Cardiovascular deconditioning and venous air embolism in simulated microgravity in the rat.

BACKGROUND: Astronauts conducting extravehicular activities undergo decompression to a lower ambient pressure, potentially resulting in gas bubble formation within the tissues and venous circulation. Additionally, exposure to microgravity produces fluid shifts within the body leading to cardiovascular deconditioning. A lower incidence of decompression illness in actual spaceflight compared with that in ground-based altitude chamber flights suggests that there is a possible interaction between microgravity exposure and decompression illness. HYPOTHESIS: The purpose of this study was to evaluate the cardiovascular and pulmonary effects of simulated hypobaric decompression stress using a tail suspension (head-down tilt) model of microgravity to produce the fluid shifts associated with weightlessness in conscious, chronically instrumented rats. METHODS: Venous bubble formation resulting from altitude decompression illness was simulated by a 3-h intravenous air infusion. Cardiovascular deconditioning was simulated by 96 h of head-down tilt. Heart rate, mean arterial blood pressure, central venous pressure, left ventricular wall thickening and cardiac output were continuously recorded. Lung studies were performed to evaluate edema formation and compliance measurement. Blood and pleural fluid were examined for changes in white cell counts and protein concentration. RESULTS: Our data demonstrated that in tail-suspended rats subjected to venous air infusions, there was a reduction in pulmonary edema formation and less of a decrease in cardiac output than occurred following venous air infusion alone. Mean arterial blood pressure and myocardial wall thickening fractions were unchanged with either tail-suspension or venous air infusion. Heart rate decreased in both conditions while systemic vascular resistance increased. CONCLUSIONS: These differences may be due in part to a change or redistribution of pulmonary blood flow or to a diminished cellular response to the microvascular insult of the venous air embolization.