Correlation of flunisolide plasma levels to eosinopenic response in humans

Plasma levels of flunisolide were measured in healthy male volunteers after the administration of single doses of the drug by the intravenous, oral, intranasal, and bronchial inhalation routes. The systemic availability of a 1-mg dose orally was only 21%. After a single dose of approximately 0.117 mg intranasally plasma levels ranged up to 1 ng/ml. When 1 mg was administered by bronchial inhalation, peak or near peak plasma levels were recorded at 2 min and remained near this level throughout the first hour before declining at a rate similar to that observed after flunisolide intravenously (plasma ). Gargling with an alcoholic mouthwash immediately after inhalation reduced plasma levels at 30 and 60 min but not earlier, suggesting rate-limiting dissolution of flunisolide in bronchial fluids or rate-limiting diffusion across the mucociliary blanket or pulmonary membrane. The systemic availabilities of the inhaled-mouthwash and inhaled-no mouthwash doses were 32% and 39%, respectively. Systemic potency of flunisolide, measured by eosinopenic response, was oral < inhaled < intravenous and correlated with the systemic availability of flunisolide after drug administration by these three routes. These pharmacokinetic properties of flunisolide are clinically advantageous in that relatively small doses are delivered topically to the target organs, i.e., the nasal mucosa and lungs, whereas a large portion of the dose is swallowed and subsequently extensively metabolized to relatively inactive metabolites.     

A method to evaluate the capacity of monocytes and macrophages to inhibit multiplication of an intracellular pathogen.

A new method to assess intracellular inhibition of multiplication or killing by normal and activated mouse peritoneal macrophages, human peripheral blood monocytes, and human monocyte-derived macrophages is described. This method involves measurement of incorporation of [3H]uracil into nucleic acids of the obligate intracellular parasite Toxoplasma gondii. The method utilizes the observation by Pfefferkorn and Pfefferkorn (1977) that [3H]uracil is incorporated in substantially greater amounts by T. gondii than by certain mammalian cell types. Differential uptake of [3H]uracil by Toxoplasma-infected and uninfected cultures allows for evaluation of the ability of macrophages or monocytes to inhibit or kill this organism. This method has been adapted to microsystem.     

Detection of mycoplasma contamination lymphoblastoid cell lines by monoclonal antibodies

We have produced two monoclonal antibodies, SFR1-Myco 1 and SFR1-Myco 2, that detect Mycoplasma fermentans found to contaminate lymphoblastoid cell lines (LCL). The specificity of these monoclonal antibodies against the M. fermentans was determined by indirect immunofluorescence by demonstrating the reactivity of the monoclonal antibodies with known reference strains of mycoplasma grown on soft agar. The reactivity of these antibodies against LCL in a number of immunoassays correlates completely with the presence of mycoplasma in these cells as determined by a standard mycoplasma detection assay. Because of the potential for widespread contamination of B lymphoblastoid cell lines transformed with Epstein-Barr virus-containing supernatants obtained from marmoset cell lines contaminated with M. fermentans, these monoclonal antibodies have value as screening reagents for this mycoplasma species in LCL.     

A stable,sensitive assay for human fibronectin

A solid-phase radioimmunoassay system to quantitate human fibronectin has been developed. This assay utilizes ¹²⁵I-labeled affinity purified IgG directed against human plasma fibronectin. The sensitivity of this system is comparable to conventional (labeled antigen) radioimmunoassays having a detection limit of approximately 0.5 ng. This assay is relatively rapid (<24 h), the reagents are stable at 4°C (> 2 months), and the reproducibility is excellent. Both human plasma and cellular fibronectin react equivalently in this assay.     

Electron microscopy of synapses in reptile spinal cord

The spinal cord of the reptile Anolis carolinensis was examined by electron microscopy. Motor neurons appear as multipolar cells 30-60 micrometer in diameter. Two types of synaptic endings are endings are present on motor neurons. The first type is characterized by distinct synaptic clefts measuring 15-20 nm between pre- and postsynaptic membranes, and by clear presynaptic vesicles. The second type of synapse, which is less common, is characterized by gap junctions between pre- and postsynaptic membranes. At these synapses, there are also clusters of clear vesicles close to the presynaptic membrane adjacent to the gap junction. These findings indicate that both chemical and electrical synaptic transmission are present in the spinal cord of Anolis.     

Single light chain subclass (kappa chain) immunoglobulin deposition in glomerulonephritis

Renal glomerular disease characterized by the deposition of immunoglobulin light chains or monoclonal immunoglobulins was demonstrated by immunofluorescence microscopy in 11 patients. The most common histopathologic findings were those of mesangiocapillary glomerulonephritis, but considerable variability was observed. Lesions resembling diabetic glomerulosclerosis and amyloidosis were seen in some patients. Immunofluorescence findings in seven patients showed concomitant, equally intense staining for kappa light chain and immunoglobulin heavy chain (IgG or IgA), indicative of monoclonal immunoglobulin deposition. Specimens in the remaining cases stained predominantly for kappa light chain alone. In six cases the histologic and ultrastructural pattern was similar to that of type I mesangiocapillary glomerulonephritis. In three cases linear deposits were present, predominantly in subendothelial and inner glomerular basement membranes and, to a lesser degree, in mesangial locations, as in type II mesangiocapillary glomerulonephritis. In one of the latter cases dense deposits were intermixed with aggregates of amorphous fibrillar material indistinguishable from amyloid. In two cases involving IgA kappa chain deposition the histologic and ultrastructural appearance was that of mesangial glomerulonephritis. Considerable heterogeneity was found in the clinical features of the patient population. Specific clinical or serologic parameters for this disease could not be identified. Only one patient had an associated lymphoplasmacytic disorder. After follow-up periods ranging from six months to 17 years, all of the patients were alive, including four who had progressed to end-stage renal disease and required dialysis. Two of the latter patients underwent successful renal transplantation; one had been alive for five years and the other for three months without evidence of recurrence of the renal disease at the last follow-up examination.     

Lymphocyte reactivity in patients with cancer of the oral cavity measured by sister chromatid differential staining

Lymphocyte reactivity in phytohemagglutinin-stimulated peripheral lymphocyte cultures from 22 patients with squamous cell carcinoma of the oral cavity and 29 age- and sex-matched controls was measured by the bromodeoxyuridine—Giemsa method for demonstrating sister chromatid differential staining. One hundred metaphases from each donor were counted, and the proportions of cells in the first, second, and third cycles were scored to estimate the in vitro mitogen responsiveness and cell division rates of the lymphocytes. The in vitro reactivity of peripheral lymphocytes of cancer patients was impaired in comparison with that of the controls, and the degree of reactivity of lymphocytes in cancer patients was not related to the size (greatest diameter) of the primary tumor. Results are discussed in relation to previously existing data.     

Microtiter solid-phase radioimmunoassay for specific immunoglobulin E

A microtiter solid phase-radioimmunoassay (MSPRIA) for perennial rye grass-specific IgE is described. The assay is carried out in flexible polyvinyl chloride "u" microtiter plates by sequentially incubating antigen, albumin, test sera, and finally radiolabeled antihuman IgE. Wells are cut free of the plate with a hot wire-cutting apparatus and counted individually in a gamma counter. The amount of radioactivity bound is proportional to the amount of specific IgE in the serum. The MSPRIA is shown to be antigen and antibody specific, reproducible, and easily done. Rye-specific IgE levels assayed with the MSPRIA correlated with quantitative end point titration skin testing using perennial rye grass antigen performed on 31 volunteers with a maximum correlation coefficient of 0.92. The MSPRIA was compared with the radioallergosorbent test (RAST) method using sera from the same 31 volunteers. The rye-specific IgE levels assayed by MSPRIA correlated with those assayed by RAST with a correlation coefficient of 0.95. The MSPRIA is well suited for mass screening and represents a useful method for measuring specific IgE.     

Age-related change in parathyroid hormone-dependent cyclic AMP formation in rat kidney

Parathyroid hormone (PTH) dependent cyclic AMP (cAMP) accumulation was evaluated in renal cortex from 2- and 12-month-old rats. Basal cAMP was lower, and responses to PTH were greater at all concentrations of hormone in kidney from 2-month-old rats. This difference was obliterated by prior removal of parathyroid glands, cAMP responses to calcitonin and both basal and hormone-stimulated adenylate cyclase activity were the same at both ages. The results suggest progressive loss of responsiveness to PTH with age, but at a site other than the receptor—adenylate cyclase complex. Blunted cAMP accumulation in year-old rats most likely reflects agonist-specific desensitization.     

Nonrandom chromosomal changes in untreated retinoblastomas

The karotypic patterns of 15 retinoblastomas were examined. Five tumors were found to have two distinct stem lines and, therefore, the chromosomal patterns of 20 tumor cell lines are reported. Three nonrandom chromosomal changes, namely, a loss of a chromosome #13, the presence of an i(6p), or a trisomy of 1q were observed. The potential importance of these chromosomal changes in tumor development is discussed, particularly the loss of a chromosome #13 or the gain of an i(6p). At least one of the three chromosomal changes was found in 75% of the tumor lines analyzed.     

Near-haploid cell line in the blastic crisis of chronic myelogenous leukemia: A possible marker for lymphoid malignancy

The blast cells of a 14-year-old patient in the blastic phase of chronic myelogenous leukemia (CML) were studied. Cellular morphology, presence of the enzyme terminal deoxynucleotidyl transferase (TdT), and reactivity to the common acute lymphoblastic leukemia antiserum (CALLA) substantiated a lymphoid blast cell line. Immunologic surface markers were nonreactive for E-rosette (T) cells and immunoglobulin-bearing (B) cells. Cytogenetic studies revealed persistance of the Philadelphia chromosome and a near-haploid cell line, i.e., 28,XY,t(9;22),+14,+15,+21,+22(GTG).The patient responded to chemotherapy with vincristine, prednisone, and l-asparaginase, first line drugs used for remission-induction of acute lymphoblastic leukemia in childhood. We suggest that severe hypodiploidy or near-haploidy, along with TdT and CALLA, may provide more accurate prognostic information in patients with CML and the lymphoid blastic crisis.     

Phenotypic characterization of acute leukemia with monoclonal antibodies using the microlymphocytotoxicity assay

Surface antigens of lymphoblasts from 56 pediatric ALL patients were studied with a set of complement fixing monoclonal antibodies. This group of lymphoblasts was comprised of 22 T-cell ALL, 22 CALLA⁺ Ia⁺ ALL and 12 non-T-non-B, CALLA⁻ Ia⁺ ALL. For comparison, two adult T-cell CLL and six B-cell CLL were also studied. It was found that by using the microlymphocytotoxic technique, the lymphoblasts can be assigned their immunophenotype and thus be classified into their respective lineage and stage of differentiation. In the samples tested, concordant reactivity was observed when FACS fluorescence profile was compared with that of microlymphocytotoxic suggesting that the letter can be used especially when qualitative estimates are required.     

Fatal myocardial toxicity during continuous infusion intravenous isoproterenol therapy of asthma

We recently utilized continuous infusion intravenous isoproterenol in the treatment of respiratory failure in an 18-yr-old steroid-dependent asthmatic female. Aminophylline, hydrocortisone, aerosolized isoetharine, and oxygen were also administrered. The patient responded to this therapy, with PaCO2 falling from 70 torr to 33 torr in 18 hr. The maximum isoproterenol dosage administered was 0.32 microgram/kg/min. Thirty-six hours following the institution of therapy, while the isoproterenol was being tapered, the patient experienced an increase in respiratory distress followed by cardiac arrest. Postmortem examination revealed multiple small areas of myocardial necrosis. These findings, unusual in asthma, probably were related to the effects of isoproterenol or the combination of isoproterenol and aminophylline on the stressed myocardium. The vulnerability of the hypoxic myocardium to the effects of isoproterenol suggests that careful cardiac monitoring is essential in the management of patients receiving this medication for treatment of respiratory failure secondary to severe asthma.     

Human cutaneous dendritic cells express two glycoproteins T6 and M241 which are biochemically identical to those found on cortical thymocytes

Monoclonal antibodies anti-T6 and anti-M241 define unique cell populations within different lineages: cortical thymocytes and dendritic cells in the skin. T6 positive cutaneous dendritic cells are located predominantly in the epidermis and belong to the Langerhans/indeterminate lineage, whereas, most of the M241 positive cells are located in the perivascular regions of the dermis. Biochemical analysis of thymocytes and cutaneous dendritic cells was performed in order to determine weather the reactivity of these antibodies with these cell types is due to sharing of antigenic determinants by two unrelated proteins, or whether similar proteins are present on cells of different lineages. Our results indicate that T6 antigens are borne by the same glycoprotein (49K) on cortical thymocytes and Langerhans/indeterminate cells. Similarly, M241 antigens isolated from thymocytes and cutaneous dendritic cells are found on the same glycoprotein (43K).     

Growth rate and sister chromatid exchange (SCE) incidence of bone marrow cells in Acute Myeloblastic Leukemia (AML)

The sister chromatid exchange (SCE) incidence and growth kinetics have been studied by means of an in vitro bromodeoxyuridine (BrdU) chromosome labeling method in the bone marrow cells of 17 acute myeloblastic leukemia (AML) patients with only diploid cells at diagnosis, remission, and relapse of the disease. At diagnosis, the cells tended to exhibit a low SCE frequency as compared to that during remission. An increased SCE frequency was observed after chemotherapy during remission or relapse. At diagnosis and relapse, when leukemic blast cells predominated in the marrow, they were characterized by the predominance of cells that had undergone only one cell cycle after BrdU exposure. In contrast, the marrow cells during remission tended to resemble the control pattern of growth kinetics, with a predominance of cells undergoing second and third cell cycles in the presence of BrdU. These results suggest that the growth rate of leukemic and nonleukemic cells is different, and that chemotherapy can cause an increased SCE frequency in the marrow cells of AML patients irrespective of the state of the disease.     

Strong HLA-DR expression in T cell cultures after activation is necessary for IL-2-dependent proliferation

We have examined the appearance of DR antigens on human T cells activated by PHA, using a monoclonal anti-DR framework antibody and a large panel of human HLA typing sera. Strong DR expression within the culture by day 8 was associated with the ability to cells to grow for long period in IL-2 containing medium, whereas weak or absent DR expression was predictive of poor in vitro growth. All the cells responded equivalently to initial stimulation with PHA. These data support the hypothesis proposed by Moretta et al. to explain blocking of IL-2-dependent proliferation by an anti-DR antibody—that DR molecules may be involved in the transmission of signals by IL-2.