Prostatectomy by transurethral balloon Laserthermia™ (PROSTALASE™) in the canine

Prostatectomy by transurethral balloon Laserthermia (PROSTA-LASE?) was performed in a canine model. This balloon device monitored by transrectal ultrasound can cylindrically irradiate with a laser beam. The treatment was performed in 8 canines using 15 watts for 20 minutes at 60°C at a 5 mm depth of the prostate from the urethral surface. Immediately following the laser therapy, an area of coagulation necrosis was observed around the urethra to a depth of 4–5 mm from the surface. After 1 week, cavitation formation was seen in 3 or 4 canines by ultrasound, and the urethral reepithelialization was shown in 1 of 2 canines. After 2 weeks, cavitation formation was observed in both canines, and the urethra was completely reepithelialized in one canine. There was no tissue damage in the bladder neck of urethral sphincter and no urinary incontinence in any animal. This system is thought to be simple and, satisfactorily performed, is an effective transurethral prostatectomy. © 1994 Wiley-Liss, Inc.     

Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a. Interaction between monoclonal antibody clones, fluorochromes and sample preparation protocols

Detection of intracellular myeloperoxidase (MPO), CD79a and CD3 has become the most specific tool for the assignment of myeloid, B- and T-lymphoid lineages in acute leukemias. In order to establish the best combination of monoclonal antibody reagent and sample preparation technique for the intracellular detection of these three markers, we compared six different cell fixation-permeabilization kits (Cytofix/Cytoperm, Fix and Perm, Intraprep, Intrastain, Permeacyte and Permeafix) using 12 fluorochrome conjugates derived from seven monoclonal antibody (mAb) clones. A total of 21 samples corresponding to normal peripheral blood (n=4), normal bone marrow (n=3), acute myeloblastic leukemia (AML, n=6), precursor B-acute lymphoblastic leukemia (ALL, n=6) and T-ALL (n=2) cases, were analysed in two centers. All fixation/permeabilization methods resulted in decreased side scatter and mostly increased forward scatter as compared to erythrocyte-lyse-washed and 1% paraformaldehyde fixed samples. The autofluorescence levels of the leukocyte populations was only significantly increased with use of the Cytofix/Cytoperm kit and mildly with the other techniques. In addition, non-specific staining increased significantly for combinations of any anti-MPO mAb with the Cytofix/Cytoperm kit and for the CD3 clone S4.1 combined with any intracellular method. Anti-MPO antibodies gave a stronger fluorescence signal when conjugated to PE than when coupled to FITC. In conclusion, MPO-7-PE, UCHT-1-PE (CD3) and any HM57-PE conjugate (CD79a) in combination with Fix and Perm, Intraprep, Intrastain or Permeafix, provided specific staining of the respective markers in sufficient intensities. Thus, combined selection of fixation/permeabilization kits and monoclonal antibody reagents against CD3, CD79a and MPO is required for obtaining optimal cytoplasmic detection of these antigens.     

Epitope mapping of the major allergen from yellow mustard seeds, Sin a I.

The antigenic sites on the major allergen from yellow mustard (Sinapis alba L.) seeds were studied using murine (BALB/c) monoclonal antibodies (mAb) and human IgE antibodies. Ten IgG1 (K) mAb from two fusions were analyzed. Competition and complementation studies performed with peroxidase labeled mAb reveal the existence of two main antigenic sites in Sin a I. All the described mAb failed to recognize the unordered carboxyamidomethylated polypeptide chains, with the single exception of 2B3, which binds the alkylated large chain. However, this mAB cannot react with the tetranitromethane-modified protein which retains the native conformation. This fact suggests that the only tyrosine of Sin a I, located in the large chain, may be part of a sequential epitope of the allergen. This chemical modification also alters the binding of the mAb 4A11 and 3F3 to the allergen, besides 2B3. The three mAb belong to the same complementation group. Specific IgE binding cannot be inhibited either by the large or small carboxyamidomethylated polypeptide chains, while the nitrated allergen shows a weaker inhibitory activity than the native Sin a I. 4A11, which is a tyrosine-dependent mAb, causes the greatest binding inhibition of the tested mAb on human IgE from atopic individuals, as determined from a reverse enzyme immunoassay, suggesting an important role played by tyrosine in the immunochemical recognition of Sin a I.     

Induction of a cytotoxic T cell response by co-injection of a T helper peptide and a cytotoxic T lymphocyte peptide in incomplete Freund's adjuvant (IFA): Further enhancement by pre-injection of IFA alone

We have previously demonstrated that it is possible to induce a consistent and strong cytolytic T lymphocyte (CTL) response to synthetic peptides, corresponding to poorly immunogenic malaria CTL epitopes, by co-injecting them with peptides representing defined T helper (Th) epitopes in incomplete Freund's adjuvant (IFA). In this study we have tested different immunization protocols to improve further the elicitation of the CTL response. We show that the CTL response to a mixture of Th + CTL peptides administered in IFA was further enhanced by a previous injection of the Th epitope peptide in IFA. Moreover, we found that the response could be significantly augmented by a pre-injection of IFA alone. This enhancement was observed only if the Th epitope was also present in the second injection. The number of lymph node cells recovered was 2–3-fold higher in mice pre-injected with IFA, but the increase in specific CTL activity, expressed as lytic units per animal, by pre-injection of IFA was at least 10–20-fold. Thus, pre-injection of IFA clearly increases the magnitude of a subsequent CTL response.     

Phase I clinical evaluation of a neutralizing monoclonal antibody against epidermal growth factor receptor in advanced brain tumor patients: preliminary study

High levels of growth factors and their receptors have been demonstrated in human tumors. Gliomas and meningiomas are characterized by overexpression of epidermal growth factor receptor (EGF-R). Ior egf/r3, is a neutralizing murine monoclonal antibody (MAb) against EGF-R, and was generated at the Cuban Institute of Oncology. The antibody recognizes EGF-R with high affinity, inhibiting tyrosine kinase activation. A clinical trial was conducted in brain tumor patients to evaluate toxicity, immunogenicity, and clinical benefit of escalating doses of the antibody. Nine patients with histologically confirmed gliomas or meningiomas, who had active or recurrent disease after receiving conventional treatment, received four intravenous doses of ior egf/r3. Total dosages ranged from 160 to 480 mg. As inclusion criteria, radioimmunoscintigraphy with the same MAb labeled with 99mTechnetium (99mTc) was performed. Immune response against the murine antibody was also evaluated. After four doses of ior egf/r3 MAb, no significant toxicity was found, except in one patient who developed a grade 4 allergic adverse event. This reaction was probably related with previous sensitization to the same MAb and the development of human anti-mouse antibodies (HAMA) response. Despite no major objective antitumor responses, eight patients had stable disease on the 6-month evaluation, and two patients remain alive after four years of MAb therapy.     

Extracellular taurine increase in rat hippocampus evoked by specific glutamate receptor activation is related to the excitatory potency of glutamate agonists

Taurine increases in brain extracellular space due to glutamate agonists were studied in vivo in the rat hippocampus using a dialysis technique, both in the absence and in the presence of glutamate receptor antagonists. Extracellular taurine levels increased during perfusions of agonists, listed in descending order of potency: kainate (KA), N-methyl-D-aspartate (NMDA), and quisqualate (QA). While taurine increases due to KA or QA perfusions were inhibited by 6,7-dinitro-quinoxaline-2,3-dione (DNQX), those induced by NMDA were abolished in the presence of 3-(carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). These results indicate that increases in extracellular taurine levels evoked by NMDA, KA or QA in the rat hippocampus are caused by activation of their specific receptors. Field potentials, concomitantly recorded, were quickly abolished during NMDA or KA perfusions (0.1 mM), while QA (0.25 mM) induced the appearance of bicuculline-like evoked responses. Since taurine has been proposed as an osmoregulatory substance in the rat brain, and cell swelling is known to be an early component of glutamate agonists neurotoxicity, the increases in extracellular taurine reported here could be due to taurine released through an osmoregulatory process, counteracting the neurotoxic cellular oedema induced by glutamate agonists.