Recongition and sexual selection in Drosophila: classification, quantification, and identification.

Drosophila pseudoobscura females show a positive bias toward mating with males whose proportion in the population is low. They can perform this discrimination even when three strains of males are present. The olfactory recognition required for this discrimination entails a hierarchically ordered recognition system and a natural unit of olfactory strength.

Assessment of ligand effects in intracellular trafficking of ricin A chain using anti-ricin hybridomas.

Intracellular ricin and immunotoxin trafficking has been difficult to study as only one to two cytosolic ricin A chain (RTA) molecules are sufficient to cause cell death. Previous studies (R.J. Youle and M. Colombatti, J. Biol. Chem., 262: 4676-4882, 1987) using anti-ricin hybridomas identified the secretory pre-Golgi as a critical site for RTA intoxication. We used ricin and RTA immunotoxins constructed with transferrin (TF) or anti-murine TF receptor antibody (RI7/217) to compare patterns of cytotoxicity and intracellular trafficking in anti-ricin hybridomas. Anti-RTA and anti-ricin B chain (RTB) hybridomas bound similar amounts of ricin and secreted comparable amounts of anti-ricin immunoglobulin. Anti-RTA hybridomas were 50- to 500-fold more resistant to ricin than nonsecretory and anti-RTB hybridomas, defining a ricin-resistant phenotype. All hybridomas expressed similar levels of surface TF receptors. RTA immunotoxins were constructed using human TF or RI7/217 and a disulfide linker. In protein synthesis inhibition assays, ricin-resistant hybridomas were manyfold more resistant to RI7/217-RTA than were ricin-sensitive hybridomas. In contrast, all hybridomas were equally sensitive to TF-RTA. Monensin increased ricin cytotoxicity minimally against all hybridomas, but dramatically increased RI7/217-RTA cytotoxicity in ricin-resistant and ricin-sensitive hybridomas in a way that abrogated the ricin-resistant phenotype. In contrast, monensin increased TF-RTA cytotoxicity equally in all hybridomas. Ammonium chloride had little effect on ricin or RI7/217-RTA cytotoxicity, but increased TF-RTA cytotoxicity against all hybridomas. Taken together, these results suggest that RTA molecules mediating cytotoxicity pass through an anti-RTA antibody-containing pre-Golgi compartment when bound to RTB or RI7/217, but not when bound to TF. Monensin abrogates the ricin-resistant phenotype when RTA is linked to RI7/217, but not RTB. This suggests that monensin alters RI7/217-RTA processing proximal to the pre-Golgi and that passage through the pre-Golgi may not be necessary for translocation of RTA to the cytoplasm. Ammonium chloride alters toxin cytotoxicity only when RTA is linked to TF, suggesting that only TF trafficks RTA through an acid-sensitive compartment prior to cytoplasmic translocation. With the addition of potentiating agents, each toxin studied showed a unique cytotoxicity profile against the anti-ricin hybridomas, demonstrating a dominant role of the cell binding ligand in intracellular toxin trafficking.

Kinetics of protein synthesis inactivation in human T-lymphocytes by selective monoclonal antibody-ricin conjugates.

Immunotoxins synthesized with the pan-T-cell monoclonal antibody T101 and ricin, acetylricin, or ricin A-chain have been compared. Native ricin was acetylated with N-acetylimidazole to block the galactose-binding site of the toxin B (binding)-chain. In the presence of lactose, both whole-ricin-containing immunotoxins were selectively cytotoxic but the ricin A-chain conjugate was less effective in blocking cellular protein synthesis. Immunotoxin-treated cells cultured in fresh growth medium exhibited no growth, declining viabilities, and no protein synthesis activity. Lymphocytes treated with T101:ricin or ricin did not form clusters or colonies when plated in 0.3% Bacto-agar. Ammonium chloride markedly enhanced the efficacy of T101:ricin and T101:ricin A-chain. Our results suggest that: (a) all immunotoxins were selectively cytotoxic; (b) in the presence of ammonium chloride the effectiveness of the T101:ricin A-chain conjugate approached that of T101:ricin; and (c) the toxin B-chain may facilitate conjugate internalization and/or processing.

Inhibition of human T-cell tumor growth by T101-ricin A-chain in an athymic mouse model.

An immunotoxin prepared with the pan-T-cell, anti-CD5, antibody T101, and purified ricin A-chain (RTA) was selectively cytotoxic in vitro, inactivating protein synthesis in the human T-cell line MOLT-4 but not in the human B-cell line 8392. Modulation studies showed that the immunoconjugate was more rapidly cleared from the cell surface than unconjugated T101. Preclinical evaluation of T101-RTA was conducted in a human T-cell, athymic mouse model (Dillman et al., Cancer Res., 45:5632-5336, 1985). Tumor-bearing mice received single i.p. injections of saline, T101, UPC-10 (irrelevant IgG2a), unconjugated RTA, an irrelevant conjugate, UPC-10-RTA, a mixture of T101 plus RTA, or T101-RTA. T101-RTA was the most effective reagent. Thirty animals given injections of 33 micrograms of T101 showed reductions in tumor growth (compared to tumor growth in animals receiving phosphate-buffered saline) but no complete regressions. No decrease in tumor growth was observed with UPC-10. Animals given 12 micrograms of free RTA exhibited reduced tumor growth but only one complete regression was observed; similar results were obtained with mice given 45 micrograms of UPC-10-RTA or a mixture of 33 micrograms of T101 plus 12 micrograms of RTA. Eleven complete regressions and 18 partial regressions were produced in the 46 animals given injections of 45 micrograms of T101-RTA and tumor growth was almost completely blocked. No toxicity was observed in any experimental arm. These results suggest that T101-RTA may be administered safely and with significant antitumor effect.

Establishment of a human B-cell tumor in athymic mice.

Human B-cell tumors have been established in athymic, BALB/c mice using the EBV-positive Burkitt lymphoma cell line Namalwa. One-hundred-one of 104 animals (97%) developed tumors 10-14 days following s.c. injection of a mixture of 20 x 10(6) Namalwa and 5 X 10(6) irradiated human fibrosarcoma (HT-1080) cells. Tumors developed at the site of injection and reached approximately 300 mm2 (product of cross-sectional diameters) after 21 days; no metastases were found. Histological analysis showed that tumors consisted solely of lymphoid cells. Immunofluorescence assays demonstrated that while 85% of the tumor cells retained reactivity with the monoclonal B-cell antibody BA-1, 96% retained reactivity with antibody BA-2 and 43% with BA-3. A similar reactivity profile was observed with cultured Namalwa cells. Tumors were passaged serially 10 times without significant change in BA-1, BA-2, or BA-3 reactivity. Indirect immunofluorescence demonstrated that antibody BA-2 reached tumor cells within 2 h following i.p. injection; antigen modulation was not observed. These results demonstrate the suitability of this B-cell model for testing the in vivo efficacy and stability of anti-B-cell immunoconjugates.

Athymic mouse model of a human T-cell tumor.

Because of the large number of different immunoconjugates which can be produced from monoclonal antibody-directed anti-cancer therapy, it would be useful to have in vivo tumor models to compare such preparations. Although historically human leukemias-lymphomas have been difficult to establish in athymic mice we have succeeded in establishing human T-cell tumors from primary MOLT-4 cultures in 290 of 353 animals and have successfully transferred tumors in 42 of 45 animals during ten serial passages. The potential utility of this model for testing immunoconjugates of murine monoclonal antibody T101 have been confirmed by: (a) in all 148 tumors sampled including all passaged tumors the human T-cell antigen, T65, was expressed in a manner identical to that of cultured cells; (b) 111In-T101 was concentrated preferentially in the tumor; and (c) T101 injected by both the i.p. and i.v. routes bound to tumor and induced antigenic modulation to the same extent as that observed previously in vitro and in human studies.

Rituximab: the first monoclonal antibody approved for the treatment of lymphoma.

Rituximab, a genetically engineered monoclonal chimeric antibody, targets the CD20 antigen expressed on B cells. It was approved by the US Food and Drug Administration on November 26, 1997, for the indication of relapsed or refractory, CD20-positive, B-cell, low-grade or follicular non-Hodgkin's lymphoma (LG/F NHL), and by the European Agency for the Evaluation of Medicinal Products on June 2, 1998, for therapy of patients with Stage III/IV, follicular, chemoresistant or relapsed NHL. Eight Phase II or II clinical trials in LG/F NHL patients have been completed: five single-agent studies and three combination studies. Rituximab has a favorable safety profile: most adverse events (AEs) are Grade 1 or 2, and the frequency of AEs decrease with subsequent infusions. AEs in the combination studies are consistent with those seen with individual agents. For evaluable patients in the single-agent studies, overall response rates (ORR) ranged from 40% to 60%, median duration of response (DR) ranged from 5.9 to 15.0+ months, and median time to progression (TTP) ranged from 8.1 to 19.4+ months. For evaluable patients in the combination studies, the ORR ranged from 45% to 100%, median DR ranged from 11.7+ to 39.1+ months, and median TTP ranged from 12.9+ to 40.5+ months. Studies in intermediate- and high-grade NHL are ongoing. Long-term development plans include evaluating the safety and efficacy of rituximab in various types of lymphoma and in combination with other lymphoma regimens. Future studies may explore ways to increase rituximab efficacy by upregulating CD20 or increasing effector function with different cytokines.

Preclinical evaluation of 90Y-labeled anti-CD20 monoclonal antibody for treatment of non-Hodgkin's lymphoma.

A high-affinity IgG1 kappa murine monoclonal anti-CD20 antibody (IDEC-2B8) was developed for radioimmunotherapy of non-Hodgkin's B-cell lymphoma. A stable immunoconjugate (Zevalintrade mark) was prepared by reacting IDEC-2B8 with a derivative of diethylenetriaminepentaacetic acid, designated MX-DTPA, a chelator exhibiting high affinity and retention for 90Y. Zevalin exhibited antigen specificity, human tissue reactivity, and preclinical safety profile comparable to the native antibody. The conjugate radiolabeled with 90Y (90Y-Zevalin) or 111In (111In-Zevalin) exhibited excellent retention of immunoreactivity with radioincorporations >95%. The radiolabeled conjugates formulated in PBS containing human serum albumin were stable in vitro at 4 degrees C for 48 h as indicated by negligible loss of radioisotope and retention of binding to CD20+ cells. In vitro human serum stability studies at 37 degrees C with 90Y-Zevalin indicated that loss of 90Y from the conjugate was minimal, averaging 1% per day. Biodistribution studies in BALB/c mice confirmed the in vitro stability of 90Y-Zevalin and 111In-Zevalin. In particular, excellent in vivo retention of 90Y by the conjugate was demonstrated by minimal bone accumulation (

Substances derived from 4-de-N-methylfortimicin B.

The preparation of 4-de-N-methylfortimicin A analogs as well as the preparation of 4-de-N-methyl-4-N-(beta-aminoethyl)-4-N-ethylfortimicin B is reported. It was shown that the 4-N-methyl group in fortimicin analogs is essential for antibacterial activity since neither the 4-de-N-methylfortimicin A nor the 4-de-N-methyl-4-N-(beta-aminoethyl)-4-N-ethylfortimicin B exhibited useful biological activity.

4-N-Aminoacylfortimicins E.

The conversion of fortimicin E, a minor metabolite from the Micromonospora olivoasterospora fermentation which also produces fortimicin A and fortimicin B, to four 4-N-aminoacylfortimicins E was accomplished. The new 4-N-aminoacylfortimicins E showed only weak antimicrobial activity against several Gram-negative and Gram-positive microorganisms.

Otorhinolaryngologic screening of children with vocal quality disturbances.

Voice evaluations of 1481 children were conducted in a rural community school system. One hundred and four children were identified as having vocal qualities that differed perceptually from normal upon initial evaluation by two speech pathologists. From a rescreening several months later, 65 children were presented to an ENT specialist for examination. Eighty-eight percent of these children had their larynx examined by the physician. Of this group, 82% were male, with 35% of all the cases showing bilateral vocal nodules and 28% with bilateral vocal fold thickening. Only a small percentage of children displayed mucoid nasal secretion, redness of arytenoids or granulation of the pharyngeal wall. The results suggest that a team approach including medical and behavioral management is most beneficial for children with voice deviations.

Monoclonal antibodies to purified ricin A-chain: production and properties.

Ricin A-chain was purified from native ricin using lactosyl-Sepharose. It was non-toxic to whole cells at a concentration of 1 microM yet nearly as effective as an equimolar concentration of ricin in blocking in vitro protein synthesis. Hybridomas secreting monoclonal antibodies to ricin A-chain were produced using the murine myeloma cell line NS-1. These anti-ricin A-chain antibodies cross-reacted with whole ricin but exhibited little cross-reactivity with purified ricin B-chain. Antibodies 2F2 and 2F5 both immunoprecipitated ricin A-chain. Both antibodies also precipitated ricin B-chain, as did the irrelevant control antibodies MOPC-21 and MPC-11. Pre-incubation of B-chain with 0.1 M galactose eliminated greater than 90% of precipitation by 2F2, MOPC-21 and MPC-11 but effected minimally precipitation by 2F5. Enzyme-linked immunosorbent assays using antibodies 2F2 and 2F5 to detect ricin A-chain in murine or human serum were linear between 40 and 800 ng ricin A-chain per ml. Anti-ricin A-chain antibodies 2F2 and 2F5 produced some inhibition of in vitro A-chain catalytic activity. Specific monoclonal antibodies to A-chain hemitoxin will be useful for characterization of functional hemitoxin domains, in in vitro assays for the stability of A-chain immunotoxins, and in characterizing the cellular internalization and processing of conjugates containing ricin or ricin A-chain.

Problem solving in endodontic working-length determination.

The success of nonsurgical endodontic treatment is influenced significantly by accurate working-length determination. A variety of guidelines and techniques are available to clinicians in mastering working-length analysis with routine success achieved when multiple methods are used. This article stresses a cognitive, problem-solving approach in the management of clinical variations in working-length determination and interpretation.

Music therapy: fertile ground for application of research in practice.

When participating in research studies in the clinical setting it is important to remember the goals of the interventions under study. Frequently the parameters and design of the research, while necessary to validate findings, are not written in stone for future application. It is possible to consider other situations in which the intervention might be applied that lie outside the current research protocols. To fully utilize research findings, apply them to your own unique practice and then set up your own study using the original work as a starting place.