Characterization of large-conductance chloride channels in rabbit colonic smooth muscle. 1. A large-conductance Cl- channel was characterized in cell-free membrane patches from the rabbit longitudinal colonic smooth muscle using the patch clamp technique. In addition, the regulation of these channels by neurokinin-1 (NK-1) receptor agonists and G proteins was studied. 2. No spontaneous channel activity was observed in cell-attached patches at the cell resting potential, or in excised patches at pipette potentials (Vp) between -20 and 20 mV. In excised patches, channel activity could be induced in thirty-six out of ninety-six patches by holding the patch at Vp values more negative than -60 mV or more positive than 60 mV. Once induced, the channel showed a bell-shaped voltage activation curve in high symmetric [Cl-], with maximal open probability between 20 and -5 mV. Varying cytosolic calcium concentration ([Ca2+]) between 5 x 10(-8) M and 1.0 mM had no effect on the voltage activation of the channel. 3. In inside-out and outside-out patches, when pipette and bath solutions contained equal [Cl-] (130 mM), the anion channel showed a linear current-voltage (I-V) relationship between -60 and 60 mV with a slope conductance of 309 +/- 20 pS (n = 13). Reversal potential measurements indicated that the channel was selective for Cl- over Na+ and K+ (PCl/PNa = 6:1). 4. Channel openings from the closed state to the full open state as well as transitions through smaller conductance states were observed. The smallest detectable substate had a conductance of 15.6 pS. Based on the similarities in selectivity and linearity of the I-V curve of the smaller conductances with the full open state, and kinetic analysis of channel activity, it is concluded that the large conductance channel is composed of multiple substates which can either open and close independently, or simultaneously via a main gate. 5. The stilbene derivative diiso-thiocyanato-stilbene-disulphonic acid (DIDS) and the diphenylamine-2-carboxylate analogue 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) caused a dose-dependent, reversible flicker block of the small conductance and significantly reduced the macroscopic current flow through the channel. 6. In quiescent outside-out patches, when the pipette contained a 140 mM-CsCl solution with 10(-6) M-CaCl2, 1.2 mM-MgCl2 and 1 mM-GTP, and the bath contained Ringer solution, addition of the NK-1 receptor antagonists substance P methylester resulted in activation of the full conductance state and of smaller substates.(ABSTRACT TRUNCATED AT 400 WORDS)


Two types of steady-state desensitization of N-methyl-D-aspartate receptor in isolated hippocampal neurones of rat. 1. Whole-cell voltage-clamp recordings were made from rat isolated hippocampal neurones. Aspartate (Asp) and/or glycine (Gly) were applied by a method in which the external solution could be changed within 30 ms and thereafter held constant. 2. Asp and Gly applied together at maximal concentrations (5 mM and 10 microM, respectively) evoked an inward current due to activation of N-methyl-D-aspartate (NMDA) receptors. The current peaked and then declined to a steady state during the application. The time constant of desensitization (tau) was about 1 s when the agonists were applied soon after the onset of whole-cell recording. The desensitization became more rapid (tau = 0.3 s) and more complete during the first 15 min of recording, and thereafter remained stable; the amplitude of the peak response did not change throughout. In solutions containing 10 microM-Gly, Asp had an apparent Kd of 51 microM at the peak of response and 20 microM measured at the steady state. The steady-state current was 14% of the peak current. 3. Asp was applied after a conditioning exposure of the cell of Gly (from 1 to 50 microM), together with the same Gly concentration. The maximum current evoked by the application of Asp was increased while increasing Gly in the conditioning solution, with no change in the apparent Kd for Asp at the peak of Asp-activated response. 4. Various concentrations of Asp (plus 10 microM-Gly) were applied after a conditioning exposure to Asp (which alone was without effect). The maximum current induced by Asp applications was only 28% of that observed without conditioning Asp application, but the apparent Kd was unchanged (about 57 microM). 5. Test solution containing maximal concentrations of Asp and Gly was applied after conditioning exposure to both Asp (varying concentrations) and Gly (10 microM). Complete desensitization was caused by 200 microM-Asp. The apparent Kd for Asp to induce desensitization (8.7 microM) was less than the Kd as an agonist (51 microM). 6. Test solution containing maximal concentrations of Asp and Gly was applied after conditioning exposure to both Gly (varying concentrations) and Asp (5 mM). Complete desensitization was caused by 1 microM-Gly. The apparent Kd for Gly to induce desensitization (120 nM) was less than the Kd as a co-agonist (about 1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)


Flow cytometer analysis of oral premalignant lesions: a pilot study and review. In a retrospective study, we examined 34 premalignant lesions of the oral cavity by flow cytometer analysis on formalin-fixed, paraffin-embedded tissue submitted to the University of Tennessee, Memphis, Oral Pathology Laboratory. Three categories of oral epithelial dysplasia were represented (eight mild, seven moderate, nine severe), as well as five epithelial hyperplasias without dysplasia and five fibrous nodules as controls. The DNA index and total proliferative index of each case were calculated. The objective of the study was to determine the amount of epithelial dysplasia necessary in oral lesions before DNA aneuploidy or high proliferative index is detectable and thus determine if flow cytometric analysis can be a diagnostic adjunct for oral premalignant lesions. The results showed that some cases in both the control and dysplastic categories exhibited a high total proliferative index (control = 1, no dysplasia = 1, mild dysplasia = 3, moderate dysplasia = 2, severe dysplasia = 2), whereas only the dysplastic lesions had an abnormal DNA index [8 of 24 (33%)]. The results indicate that flow cytometric analysis may have some limited potential as a diagnostic adjunct in oral premalignant lesions.


Preservation of keratin expression in oral mucosa using a novel transport medium. Cellular expression of keratins (intermediate filaments) can be demonstrated by immunocytochemistry using unfixed tissue. However, for practical reasons, provision of fresh tissue to the laboratory is often difficult. Recently a fresh tissue transport medium (Patent applied for) has been developed which allows keratin immunocytochemistry to be performed up to 4 days after biopsy. In this study oral mucosal biopsies from 10 patients were hemisected, half placed in the new transport medium at 4 degrees C and the other half immediately frozen in liquid nitrogen. After 4 days frozen sections of both halves of the biopsies were stained by immunocytochemistry using various antikeratin antibodies. The morphology and staining characteristics of the two halves of the biopsies were then assessed. No significant difference could be found in either morphology or preservation of keratin expression in specimens stored in transport medium, as compared to those in liquid nitrogen. This new transport medium may offer considerable advantage for the provision of a histologic and immunocytochemical diagnostic service.


Cytokeratins as epithelial differentiation markers in premalignant and malignant oral lesions. The keratin expression pattern in oral stratified epithelium is related to the cellular differentiation level. The normal pattern shows the keratin pair K5 and K14 in the stratum basale whereas K1 and K10, or K4 and K13, are the two pairs associated with differentiating suprabasal cells. Monoclonal and polyclonal antibodies to individual keratins (K10, K13 and K14) were used in a two-color immunofluorescence staining method to study their coexpression in single cells. Altered keratin expression in premalignant and malignant lesions indicated abnormal differentiation. Monospecific keratin antibodies were suggested to be useful for evaluation of epithelial differentiation changes in oral dysplasias and oral squamous cell carcinomas.


Two forms of decidualization-associated protein and their physicochemical relationship with acute-phase alphamacrofetoprotein in rats. After isolation and purification, rat acute-phase protein, alphamacrofetoprotein and the uterine decidual protein, decidualization-associated protein were compared. They are similar in molecular weight and amino acid composition, and behave similarly during native polyacrylamide gel electrophoresis and are crossreactive with polyclonal antisera. They differ in pI values: with a mean pI of 4.83 for alphamacrofetoprotein, 5.16 for the major form of decidualization-associated protein and 4.97 for the remainder. Alphamacrofetoprotein and decidualization-associated protein differ in carbohydrate content and subunit structure, but show similar susceptibility to trypsin digestion. Evidence is provided that both the decidual species form complexes with proteases and that these are present in the extracted decidual proteins. The distribution of the forms of decidualization-associated protein in the tissues during pregnancy and parturition is described and discussed.


Percutaneous, ultrasound-directed ablation of ectopic pregnancy with methotrexate. A report of three cases. The early diagnosis of unruptured ectopic pregnancy has been facilitated by the availability of high-resolution ultrasound and sensitive quantitative assays for beta-human chorionic gonadotropin. Nonsurgical treatment of selected patients has been advocated. Three patients with fairly advanced unruptured ectopic pregnancies were treated with ablation with methotrexate using an ultrasound-directed, percutaneous technique. That approach appears to be reasonable and safe in selected patients, though the subsequent reproductive performance is unknown.


Immunoregulatory effects of Borrelia burgdorferi on T-B cell interactions. Late stages of Lyme borreliosis are characterized by a disproportionate inflammatory response presumably elicited by only minute amounts of persisting antigen. To explore the possibility that amplification mechanisms of the immune response shape disease manifestations, we studied immunoregulatory properties of Borrelia burgdorferi antigens and analyzed whether they influence B cell-T cell cooperation. B. burgdorferi was found to induce peripheral resting B cells to function as effective antigen presenting cells for B. burgdorferi sonicate (Bb) and unrelated third party antigens. Morphologically and phenotypically, pulsed B cells represented small resting B cells and did not acquire activation markers. These data suggest that distinct antigen-presenting cells are involved in B. burgdorferi induced immune responses. B lymphocytes may play a crucial role in amplifying T cell responses, especially by recruiting antigen specific and nonspecific T cells to regions where minute amounts of spirochetal antigens are present.


Effect of topical capsaicin in the therapy of painful osteoarthritis of the hands. Topical capsaicin 0.075% was evaluated for the treatment of the painful joints of rheumatoid arthritis (RA) and osteoarthritis (OA) in a 4 week double blind, placebo controlled randomized trial. Twenty-one patients were selected, all of whom had either RA (n = 7) or OA (n = 14) with painful involvement of the hands. Assessments of pain (visual analog scale), functional capacity, morning stiffness, grip strength, joint swelling and tenderness (dolorimeter) were performed before randomization. Treatment was applied to each painful hand joint 4 times daily with reassessment at 1, 2 and 4 weeks after entry. One subject did not complete the study. Capsaicin reduced tenderness (p less than 0.02) and pain (p less than 0.02) associated with OA, but not RA as compared with placebo. A local burning sensation was the only adverse effect noted. These findings suggest that topical capsaicin is a safe and potentially useful drug for the treatment of painful OA of the hands.


Evaluation and management of pain in children with juvenile rheumatoid arthritis. The systematic evaluation and management of chronic and recurrent pain in children with juvenile rheumatoid arthritis (JRA) is only in the beginning stages of empirical development. While recent advances have been made in the assessment of pain secondary to JRA, very little clinical research has been targeted toward pain management. Development of reliable and valid pediatric pain measures is the first step in advocating controlled clinical trials with pain as an essential outcome variable.


Properties of single- and double-barreled Cl channels of shark rectal gland in planar bilayers. Chloride channels from the apical plasma membrane fraction of rectal gland of Squalus acanthias were characterized by incorporation into planar bilayers in the presence of cAMP-PK/ATP. In a total of 80 bilayer preparations, 21 Cl-selective channels were observed as single channels and 13 as pairs. This was a significantly greater number of double Cl channels than expected from a binomial distribution. The double Cl channels were divided into two groups based on kinetic and voltage-dependent behavior. One group had properties identical to the single channels (gb1) while the other was consistent with a double-barreled channel (gb2) with coordinated activity between proto-channels. The single-channel slope conductances of gb1 and gb2 from -60 to +20 mV with a 250/70 mM KCl gradient were 41 and 75 pS, respectively. With symmetrical 250 mM KCl, the I-V relation of gb1 showed outward rectification with 47.8 +/- 6.6 pS at cis negative potentials and 68.9 +/- 6.1 pS at cis positive potentials. gb1 was open from 70 to 95% at all electrochemical potentials from -80 to +40 mV. gb2 was steeply voltage dependent between -80 and -20 mV. Both gb1 and gb2 were insensitive to Ca (from 100 nm to 1 microM), blocked by 0.1 mM DIDS and highly selective for chloride. These data suggest that double-barreled Cl channels are related to the family of small, outwardly rectifying Cl channels of epithelial membranes.


Phosphorylation modulates the voltage dependence of channels reconstituted from the major intrinsic protein of lens fiber membranes. Major intrinsic polypeptide (MIP), a 28-kDa protein isolated from lens fiber cell membranes, forms large, nonselective channels when reconstituted into lipid bilayers. MIP channels are regulated by voltage, such that these channels close when the potential across the membrane is greater than 30 mV. We have investigated the modulation of the voltage-dependent closure of MIP channels by phosphorylation. In this report, we describe the isolation of two isomers of MIP from lens fiber cell membranes. These isomers differ by a single phosphate at a protein kinase A phosphorylation site. The phosphorylated isomer produces channels that close in response to applied voltages when reconstituted into bilayers. The nonphosphorylated isomer produces voltage-independent channels. Direct phosphorylation with protein kinase A converts voltage-independent channels to voltage-dependent channels in situ. Analyses of macroscopic and single-channel currents suggest that phosphorylation increases the voltage-dependent closure of MIP channels by increasing closed channel lifetimes and the rate of channel closure following the application of voltage.


Ion channel enzyme in an oscillating electric field. To explain the electrical activation of several membrane ATPases, an electroconformational coupling (ECC) model has previously been proposed. The model explained many features of experimental data but failed to reproduce a window of the field intensity for the stimulated activity. It is shown here that if the affinities of the ion for the two conformational states of the transporter (one with binding site on the left side and the other on the right side of the membrane) are dependent on the electric field, the field-dependent transport can exhibit the observed window. The transporter may be described as a channel enzyme which opens to one side of the membrane at a time. It retains the energy-transducing ability of the earlier ECC models. Analysis of the channel enzyme in terms of the Michaelis-Menten kinetics has been done. The model reproduced the amplitude window for the electric field-induced cation pumping by (Na,K)-ATPase.


Influence of codon context on UGA suppression and readthrough. We studied the influence of the codon context on UGA suppression by a suppressor tRNA and on UGA readthrough by a normal tRNA in Escherichia coli. This was done by a series of constructs where only the immediate context of the TGA codon was varied by only one nucleotide at a time. For both UGA suppression and UGA readthrough the codon context had a similar influence according to the following rules. (1) The nature of the nucleotide immediately adjacent to the 3' side of the UGA is an important determinant; at that position the level of UGA translation is influenced by the nucleotides in the order A greater than G greater than C greater than U. (2) At extremely high or low levels of UGA translation this influence of the adjacent 3' nucleotide is not seen. (3) In all cases, the nature of both the nucleotide immediately adjacent to the 5' side of the codon and that following the base adjacent to the 3' side of the codon have little effect, if any, on UGA translation. The varying influence of the codon context effect on UGA translation is discussed in relation to its role in gene expression.


Temperature dependence of the kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into half-molecules. A minimum rate at 15 degrees C indicates hydrophobic interaction between the subunits. The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin to half-molecules has been studied as a function of temperature by using small-angle scattering of X-rays and neutrons. The most striking result of the present investigation is that there is a minimum in reaction rate at about 15 degrees C, and that the rate increases when the temperature is lowered, or raised, from that value. By analyzing the first-order rate constants in terms of transition-state theory it was found that the dissociation is associated with a large and positive change in heat capacity between the activated complex and native alpha 2-macroglobulin (delta CP is in the range 5 to 6 kJ mol-1 K-1). In analogy with pure thermodynamic investigations, where a large change in heat capacity normally is interpreted as a melting of hydrophobic interaction, we therefore propose that hydrophobic interaction is involved in the so-called non-covalent interactions between the subunits of alpha 2-macroglobulin. As a result of the present investigation, it also follows that the free energy of activation delta G has a maximum at about 32 degrees C, whereas the enthalpy of activation delta H and the entropy of activation delta S are zero at about 15 degrees C and 32 degrees C, respectively. These temperatures are slightly dependent upon the concentration of urea and upon whether the reaction is run in a 1H or a 2H medium. Furthermore, from the kinetic point of view, at low temperature the reaction can be characterized as enthalpy driven, whereas at high temperature, it can be characterized as entropy driven.


Increased circulating nitrogen oxides after human tumor immunotherapy: correlation with toxic hemodynamic changes. Toxicity to interleukin-2 (IL-2) tumor immunotherapy is manifested principally by the vascular leak syndrome, hypotension, and a hyperdynamic response with low systemic vascular resistance. Nitric oxide (.N = O), a recently discovered biological mediator of vascular smooth muscle relaxation, is produced in increased amounts by numerous cell types exposed to a number of inflammatory cytokines. Our purpose was to determine if there is an increased production of .N = O in patients receiving IL-2 tumor immunotherapy, and, if so, whether increases in .N = O production correlate with hemodynamic instability. Twelve patients undergoing immunotherapy trials with IL-2 and anti-CD3 monoclonal antibody-activated lymphocytes (T-AK cells) were studied. Plasma levels of nitrate (NO3-), the stable end metabolic product of .N = O synthesis, were measured before and at the end of IL-2 treatment cycles. We observed a ninefold increase in plasma levels of NO3- in patients after 7 days of treatment (P less than .0001). A significant decrease in both systolic and diastolic blood pressures was observed in all patients (P less than .001). We propose that mediated induction of .N = O synthase enzyme leads to progressive increases in .N = O production which, in turn, produces clinically significant hypotension. Since .N = O synthesis can be competitively inhibited by L-arginine analogues, a possible pharmacologic modulation of .N = O production could potentially contribute to better management of toxic side effects seen in IL-2 cancer therapies.


Favorable long-term survival following induction chemotherapy with cisplatin, fluorouracil, and leucovorin and concomitant chemoradiotherapy for locally advanced head and neck cancer. The majority of patients with head and neck cancer die of locoregional recurrence of disease following surgery and/or radiotherapy. Our purpose was to administer induction chemotherapy, perform surgery, and administer concomitant chemoradiotherapy in rapid sequence and to evaluate their impact on locoregional and distant tumor control. Sixty-four patients with previously untreated, locoregionally advanced head and neck cancer received two cycles of cisplatin, bleomycin, and methotrexate (PBM) (33 patients) or cisplatin, fluorouracil (5-FU), and leucovorin (PFL) (31 patients). PFL was given to patients who were unable to receive bleomycin. Local therapy consisted of surgery and/or concomitant chemoradiotherapy with 5-FU, hydroxyurea, leucovorin, and radiotherapy (FHX-L), all administered every other week. Complete and overall induction response rates were 21% and 79%, respectively, for PBM and 29% and 81%, respectively, for PFL. At completion of local therapy, 81% of the patients were disease-free. With a median follow-up of 35 months, the median survival and time to progression are 22 and 17 months, respectively, for PBM and have not been reached for PFL. Locoregional recurrence of disease is 30% for PBM and 26% for PFL. Distant disease progression is 24% for PBM and only 3% for PFL. The sequencing of induction chemotherapy and concomitant chemoradiotherapy is feasible and results in a high local control rate and in an encouraging survival rate with PFL. The high distant failure (i.e., outside the head and neck area) rate of PBM suggests insufficient systemic activity for that regimen. Concomitant FHX-L chemoradiotherapy may improve regional control rates of advanced head and neck cancer. Effective systemic therapy may be needed to control systemic micrometastases. PFL, but not PBM, appears to be suitable to accomplish that goal.


Influence of aminoglycoside antibiotics, streptomycin and kanamycin on histamine secretion in mast cells. Effects of kanamycin and streptomycin on histamine release from rat mast cells were examined in response of the cells to concanavalin A(Con A) plus phosphatidylserine (PS), phytohemagglutinin (PHA) plus PS or a mixture of low-molecular-weight polymers of P-methoxy-N-methylphenethylamine (compound 48/80). In the response to each of the above stimuli, kanamycin (20 mM) or streptomycin (20 mM) caused a decrease in the histamine release elicited in the presence of extracellular Ca2+ (1 mM), although streptomycin showed the much higher inhibitory potency than kanamycin. Similarly, streptomycin was much more effective in suppressing compound 48/80-triggered histamine release in the absence of external Ca2+. Histamine release in the absence of external Ca2+ in the response to the lectin plus PS diminished with increasing concentration of kanamycin, and in this respect streptomycin was much less effective. In the response to the lectin plus PS, external Ca2+ possessed potency to antagonize kanamycin (10 mM)- or streptomycin (10 mM)-caused inhibition of the histamine release, although more markedly the kanamycin-caused one. Streptomycin and kanamycin inhibit histamine release from mast cells challenged with IgE-directed secretagogue or compound 48/80, and the responsible mechanisms seem to implicate the Ca(2+)-antagonistic action on the stimulus-provoked influx of Ca2+ and impairment of the cellular events linked to exocytosis.


The safety, efficacy and compliance of terazosin therapy for benign prostatic hyperplasia. The primary objective of the present interim analysis of an open label study initiated in December 1988 is to provide further insight into the long-term safety, efficacy and compliance of terazosin, a long-acting selective alpha 1-blocker, for the treatment of symptomatic benign prostatic hyperplasia (BPH). A total of 45 normotensive patients with symptomatic BPH was enrolled into this study. The dose of terazosin was titrated to 5 mg. during a 1-month period provided adverse reactions and orthostatic hypotension were not observed. The subjects were maintained on 5 mg. terazosin throughout the 24-month followup. The peak and mean urinary flow rates, obstructive and irritative Boyarsky symptom scores, and systolic and diastolic blood pressures were evaluated at 2, 6, 12, 18 and 24 months after initiation of therapy. Of the 45 subjects 21 (47%) exhibited a durable clinical response with no significant adverse events during the 24-month followup. The obstructive and irritative symptom scores decreased by 63% and 35%, respectively, after 2 months of terazosin therapy. These improvements in obstructive and irritative symptom scores were maintained throughout the 2-year followup. The peak and mean urinary flow rates improved by 42% and 48%, respectively, after 2 months of terazosin therapy. The changes in mean urinary flow rate increased, whereas the changes in peak urinary flow rate decreased during followup. Of the patients 6 (13%) were withdrawn from the study due to an adverse event. Our study demonstrates the safety, efficacy and compliance of terazosin therapy for BPH during a 2-year followup.


Transurethral hyperthermia for benign prostatic hyperplasia patients with retention. From 1989 to 1990, 32 poor surgical risk patients with urinary retention were treated with transurethral microwave hyperthermia at the department of urology, University of Leuven in Belgium. Mean patient age was 73 years (range 58 to 90 years) and mean duration of retention was 4 weeks (range 3 to 12 weeks). Followup ranged from 13 to 82 weeks, with a mean of 31 weeks. Bilobar or trilobar hyperplasia was diagnosed in 25 patients (78%), while 7 (22%) had median lobe or median bar hypertrophy. The mean prostatic volume was 52 cc (range 25 to 150 cc). Transurethral microwave hyperthermia was given with a helical antenna at 915 MHz. once or twice per week. The mean number of transurethral microwave hyperthermia sessions was 8.9 (range 5 to 10). Each session consisted of a 60-minute treatment at a mean maximum temperature of 45.4C (range 43.7 to 47.2C), average temperature 43.9C (range 42.7 to 45.5C) and minimum temperature 42.0C (range 40.2 to 43.0C). The temperature was continuously monitored, including thermal mapping in all patients. Of the 25 patients who presented with bilobar or trilobar hyperplasia 18 (72%) were catheter-free for the duration of followup. Of the 7 median lobe or median bar patients 1 (14%) showed sufficient improvement to warrant catheter removal. This patient, however, had recurrent retention 4 months after transurethral microwave hyperthermia. In patients with bilobar and trilobar hyperplasia a strong correlation was observed among maximum temperature (p = 0.0006), average temperature (p = 0.0033) and treatment response. As expected, no such correlation existed between minimum temperature and response to treatment (p = 0.56). Our study has again demonstrated therapeutic activity in patients with benign prostatic hyperplasia treated with transurethral microwave hyperthermia. A new finding was a strong correlation between temperature and response.


Prostatic and periprostatic interstitial temperature measurements in patients treated with transrectal thermal therapy (local intracavitary microwave hyperthermia). One of the questions raised regarding the use of transrectal thermal therapy in the treatment of benign prostatic hyperplasia (BPH) is whether there is a uniform and safe temperature distribution within the prostate. Our study represents the first attempt in humans to map the interstitial thermal distribution in the prostate during transrectal thermal therapy. With the patient under local anesthesia and under ultrasound guidance, a transperineal 3-point thermocouple was placed into various areas of the prostate in 15 patients. Prostatic-urethral thermocouple distance ranged from 1 to 3 cm. A urethral catheter containing a 5-point linear array thermocouple was placed and the balloon was inflated so that the proximal point was at the bladder neck and the remaining points were at 1 cm. intervals along the prostatic urethra. Power (25 watts) was delivered via the Primus (Technomatix) transrectal microwave applicator with simultaneous cooling of the rectal mucosa (between 12 and 14C). Treatment was delivered for 60 minutes and temperatures were recorded. In the prostatic substance a maximal temperature of 45C was observed during the heat-up phase and this decreased as the vasoactive response occurred. Temperature along the prostatic urethra varied between 40 and 43C and never exceeded 44C. A similar distribution of temperature was registered in the thermocouple points in the prostatic substance. The anticipated thermal dose of 41.5 +/- 1C for 60 minutes was achieved in the prostatic substance as measured by the sensors in the prostatic urethra and interstitial sensors. The results suggest that transrectal thermal therapy delivers a uniform and safe distribution of heat in the prostatic substance and urethra. Clinical trials are currently underway to ascertain the efficacy of transrectal thermal therapy in the management of BPH.


Mortality, morbidity and complications following transurethral resection of the prostate for benign prostatic hypertrophy. A total of 388 men undergoing transurethral resection of the prostate for benign prostatic hypertrophy during 1988 entered a prospective cohort study designed to examine the outcome of surgery during postoperative year 1. Self-administered questionnaires were completed preoperatively, and at 3, 6 and 12 months postoperatively. The surgeons completed 1 questionnaire shortly after surgery and another questionnaire 3, 6 or 12 months later. The mortality rate during the 12 months of followup was 2.8% (11 deaths). The surgeons reported perioperative complications in 14% of the patients and immediate postoperative complications, excluding urinary tract infections, in 17%. During the first 3 months postoperatively 38% of the patients reported incontinence and 25% had a urinary tract infection. Between 6 and 12 months postoperatively only 12% of the patients were troubled by either condition. The postoperative prevalence of impotence (24%) did not alter during followup and was similar to that reported preoperatively (22%). Of the patients 74% reported feeling better and 78% experienced a decrease in the overall level of symptoms postoperatively. The improvement in symptom levels was greatest in those with the most severe preoperative symptoms, and obstructive symptoms were alleviated slightly more than irritative symptoms.


Pediatric renal transplantation under FK-506 immunosuppression. Renal transplantation (11 cadaveric and 1 living-related donor) was performed in 12 pediatric recipients (mean age 10.8 years) under FK-506 immunosuppression in combination with prednisone therapy. At a mean followup of 6.1 months, patient and graft survival rates were 100% and 92%, respectively. The only graft loss was due to the recurrent hemolytic uremic syndrome 4 days after transplantation. In the functioning grafts the mean serum creatinine is 1.59 +/- 1.27 mg./dl. and the mean blood urea nitrogen is 36.3 +/- 24.6 mg./dl. Three patients take no prednisone, 5 are receiving 0.15 to 0.25 mg./kg. per day and 3 are taking 0.35 to 0.5 mg./kg. per day. There was a total of 8 rejection episodes in 5 patients. All rejection episodes were successfully reversed. Complications of transplantation included an episode of seizures in 1 patient, cytomegalovirus infection in 1 and steroid-induced diabetes mellitus in 1. Since pediatric transplant recipients are a group in whom the reduction or elimination of steroids is highly desirable, FK-506 immunosuppression may be particularly suited for use in this population.


Evaluation of plasmatic leucocyte elastase levels in coronary artery disease. Leucocyte elastase may be involved in the structural modification observed in the atherosclerotic process. Therefore, we tested the usefulness of leucocyte elastase plasma level determination as a marker for atherosclerosis. Plasma levels of elastase were determined by ELISA in 100 consecutive patients (mean age 56 +/- 9.8 years) admitted to hospital for coronary angiographic investigation of chest pain. Eighty-seven patients had evidence of atherosclerosis, and 13 patients had normal coronary vessels. No significant difference in leucocyte elastase was found between the 2 groups, nor was there any relationship between elastase levels and the severity of atherosclerosis. However, relationships between plasma leucocyte elastase levels and various lipid fractions (Apo AI, LDL) and daily tobacco consumption were found. Leucocyte elastase may thus play a role not only by direct modification of the vessel wall, but also indirectly via risk factors such as dyslipoproteinemia and leucocyte toxicity.


[Leukocyte adhesion molecules and their abnormality]. A brief commentary on leukocyte adhesion molecules, including integrin family, immunoglobulin superfamily and selectin or LEC-CAM family is made. A recent concept on the molecular mechanisms of the leukocyte adhesion to cell surface is also described. An immunodeficiency disease called "leukocyte adhesion deficiency (LAD)" that is defective in expression of leukocyte adhesion molecules of integrin family, LFA-1, Mac-1 and p150, 95, is discussed with special reference to its clinical features, laboratory findings, neutrophil functions, lymphocyte functions and molecular defects. Some abnormal expression of a Fc-gamma receptor in neutrophils and new data concerning the molecular defects found in Japanese patients are also included.


[In vivo effects on human neutrophils by administration of rhG-CSF and clinical significance]. To evaluate the clinical effect by administration of recombinant human granulocyte-stimulating factor (rhG-CSF) post chemotherapy in non-Hodgkin malignant lymphoma (NHL), 17 patients with NHL were subjected to this study. Administration of rhG-CSF ameliorated the decrease in absolute neutrophil counts after the cytotoxic chemotherapies and activated neutrophil functions in active oxygen product and expressions of adhesion proteins. To consistent with these results, rhG-CSF administrations post cytotoxic chemotherapy were effective for reducing infection complications associated with neutropenia. Furthermore, administration of rhG-CSF increased peripheral hematopoietic progenitor cells, thus suggesting promising therapeutic potential for autografting. Recently, it has been reported that blood neutrophils may synthesize mRNA and proteins important in inflammation including various cytokines such as IL-1, IL-6, TNF-alpha and IFN-alpha, but, administration of rhG-CSF showed no obvious effect on the level of either IL-1, IL-6, TNF-alpha or IFN-alpha in sera, and furthermore, the in vitro stimulation by rhG-CSF induced no significant production of these cytokines and expressions of TNF-alpha and IFN-alpha mRNAs. Finally, we studied on anti-tumor effect of administration of rhG-CSF in CDF1 mice inoculated with syngeneic lymphoma cells. rhG-CSF infusion suppressed the liver metastasis and prolonged the overall survival, thus suggesting the hypothesis that use of rhG-CSF in some patients with NHL might control the disease through stimulating both production and functional activation of neutrophils.


[The use of a metallic stent in 32 patients with benign prostatic hypertrophy. Preliminary results and 3 months of follow-up]. A prospective noncontrolled study of the safety and potential efficacy of the metallic stent was performed on 32 patients with benign prostatic hypertrophy. Mean age was 76.6 years (range, 56-98 years), and mean prostatic volume was 24.2 cm3. The patients were selected on the basis of a quantitative symptom score (QSS), uroflowmetry measurements, and residual urine volume (RU). Nineteen patients had urinary retention and remaining 13 patients had moderate symptoms and signs of prostatism. Placing the stent was successfully done in 31 patients (97%). It took 15 minutes to place the stent using transabdominal and/or endorectal sonography. After 3 months, 27 patients (87%) showed improved QSS. In patients with dysuria, maximum flow rate (MFR) and RU before treatment were 6.9 +/- 1.7 ml/sec and 112.3 +/- 61.8 ml, respectively. After treatment, they improved to 12.3 +/- 2.7 ml/sec and 12.7 +/- 6.7 ml, respectively. On the other hand, all patients who had urinary retention were able to urinate just after treatment, and MFR and RU were 12.9 +/- 3.6 ml/sec and 24.4 +/- 43.3 ml, respectively. Evaluation on the basis of improvement in MFR and reduction in RU showed that the stent was effective in 71% of total patients (22 out of 31 patients), 94% of the patients with urinary retention (17 out of 18 patients). The overall clinical efficacy of this stent was 68% (21 patients). There were no major complications such as urge incontinence and urinary tract infection during follow-up. Although proximal migration of the stent was observed in 6 patients, the stent could be taken out and replaced in 4 patients. From the above results, we conclude that the metallic stent is useful for the treatment of prostatism and urinary retention.


[In-vivo enhancement of neutrophil function by administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in cyclophosphamide (CPA) treated mice]. The in-vivo effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the number and function (phagocytosis and superoxide production) of peripheral neutrophils were studied in time sequence in cyclophosphamide (CPA) treated mice. The neutrophil function was evaluated by the phagocytosis of fluorescent particles, analyzing the number of fluorescence-positive cells and the fluorescence intensity of each neutrophil by flow cytometry and also by the superoxide production, measuring chemiluminescence of the leukocyte suspension by a photometer. In CPA-treated (100 mg/kg) mice, the neutrophil function including both the phagocytosis and the superoxide production declined significantly (p less than 0.01) as the peripheral neutrophil count (PNC) decreased, reached the nadir on the same day as PNC and returned to the normal level 8 days after first CPA treated day (day 0). When rhG-CSF (100 micrograms/kg) was administered subcutaneously daily for 5 consecutive days initiating at day 1, a decrease in PNC and a decline in the neutrophil function were prevented and a significant (p less than 0.05-p less than 0.01) increase of PNC was observed after day 4. In addition, the function of increased neutrophils was significantly (p less than 0.05-p less than 0.01) enhanced after day 4 and even at day 3, when an increase in PNC was not observed yet. The study shows that rhG-CSF appears to enhance neutrophil function by a direct effect on mature neutrophils, which have been impaired by CPA at the phase of progenitor cells in the bone marrow and subsequently have appeared in the peripheral blood and that rhG-CSF is effective on the impaired host defense mechanism in CPA-treated mice, improving not only drug-induced neutropenia but also the deteriorated function of neutrophils.


[The distribution and localization of alpha 1-adrenoceptor agonist on the bladder and urethra of female rats]. The distribution of selective alpha 1-adrenoceptor agonist 1-(2',5'-dimethoxyphenyl)-2-glycinamidoethanol hydrochloride, midodrine, and its active metabolite 1-(2',5'-dimethoxyphenyl)-2-aminoethanol, DMAE, was evaluated on bladder and urethra of 8-weeks and 52-weeks old female rats. Prior to the intravenous injection of 14C-labeled midodrine and DMAE, bilateral ureters were ligated to prevent drug uptake from the urinary tract. In 8-weeks rats, 14C-midodrine activity was significantly (p less than 0.01) higher in the bladder than in the femoral muscle, which served as a control for drug distribution. Similarly, higher uptake of 14C-DMAE was observed in the bladder than in the femoral muscle (p less than 0.01) and the urethra (p less than 0.05). In 52-weeks rats, there was no significant difference of midodrine uptake among these tissues. However, significantly higher uptake of 14C-DMAE was observed in the urethra than in the femoral muscle (p less than 0.05). Compared with midodrine, the concentration of DMAE was significantly increased in the bladder of 8-weeks rat and in the urethra of 52-weeks rats (respectively, p less than 0.05). In autoradiogram, the grains corresponding to midodrine and DMAE were diffusely distributed on the smooth muscles of bladder (mainly bladder neck and trigone) and urethra. The grains were also recognized on the vessels and perivascular areas of these tissues. These findings support that midodrine and DMAE could be effective for stress incontinence, because these drugs are known to bind specifically to alpha 1-adrenoceptor.


Isolation and characterization of cDNA from renal tubular epithelium encoding murine Rantes. We have been interested in identifying proinflammatory molecules which might play a role in attracting monocytes and T cells to the kidney. Some of the new intercrines are potential candidates. In this report we have isolated cDNA encoding murine Rantes (MuRantes) from renal tubular epithelium (MCT cells). MuRantes is a 91 amino acid member of the -C-C- or intercrine beta subgroup of the Scy superfamily. The amino acid sequence for mature MuRantes was deduced from its coding cDNA and was found to be 90% homologous to its mature human counterpart (HuRantes). MCT epithelium expresses a single mRNA transcript for MuRantes of approximately 1100 bp. The MuRantes protein could be detected in cell lysates of MCT epithelium by western blotting and in the cytoplasm of MCT cells by immunofluorescence using a polyclonal antibody generated against HuRantes fusion protein. A search protocol using MuRantes-specific primers and cDNA amplification revealed that mRNAs for MuRantes are expressed additionally in syngeneic mesangial cells (MMC cells), whole kidney, liver, and spleen, as well as in nephritogenic antigen-specific CD4+ helper and CD8+ effector T cells. cDNA amplification studies also demonstrated a significant elevation in mRNA transcripts encoding MuRantes in response to the stimulation of MCT epithelium with TNF alpha and IL-1 alpha in culture, but not with TGF beta, gamma IFN, or IL-6. Our findings indicate that proximal tubular epithelium is an authentic source of MuRantes, and that transcripts encoding MuRantes are responsive to the modulating influence of paracrine factors having a known role in the development of parenchymal injury.


Cardioplegic arrest of the myocardium with calcium blocking agents. This study was designed to compare the effects of the calcium slow channel blocking agents verapamil (0.15 mg/kg), diltiazem (0.15 mg/kg), and nifedipine (50 micrograms/kg) on the myocardium after global ischemia and reperfusion in the in situ canine model. Animals were subjected to 120-min normothermic global ischemia, followed by 45-min reperfusion. Cardioplegic arrest of the myocardium was achieved by administering one of the three calcium antagonists in a multidose fashion. Superior preservation (p less than 0.01) of left ventricular (LV) systolic function was achieved in group I (verapamil cardioplegia). dP/dt, at an intraventricular balloon volume of 25 cc, was 83% of control after reperfusion in group I. Group II (diltiazem) and group III (nifedipine) achieved only 55 and 63% of their preischemic dP/dt values. LV chamber stiffness was increased in hearts protected with nifedipine. The exponential constant m was increased from 0.04 +/- 0.01 to 0.08 +/- 0.01. Coronary blood flow after reperfusion increased from 120 to 184 cc/100 gr/min in group I (p less than 0.01). The hyperemic response in group III was negligible. The O2 consumption of the reperfused myocardium was not significantly altered in any of the treatment groups. Lactate metabolism during ischemia and after reperfusion was similar in all groups. ATP values were markedly reduced in all groups (p less than 0.05). Immediately after ischemia, ATP was 50, 28, and 44% of control in group I, II, and III, respectively. The excellent preservation of systolic function and a physiologic hyperemic response by verapamil could not be correlated with improved preservation of high-energy compounds or with significant changes in myocardial O2 consumption.(ABSTRACT TRUNCATED AT 250 WORDS)


Myocardial beta-adrenergic desensitization and neuronal norepinephrine uptake function in idiopathic dilated cardiomyopathy. Desensitization of myocardial beta-adrenergic receptors may result both from an impairment of the norepinephrine (NE) neuronal uptake function and from an increase in circulating NE concentrations. The respective role of these two mechanisms of desensitization was examined in 18 patients with congestive heart failure related to an idiopathic dilated cardiomyopathy. The neuronal NE uptake system was evaluated by [123I]metaiodobenzylguanidine (MIBG) scintigraphy. The desensitization level of beta-adrenoceptors was assessed as the net increase in peak positive left ventricular (LV) dP/dt during intracoronary dobutamine infusion. Arterial NE concentrations were determined at baseline. To obtain control values, we performed MIBG scintigraphy and determined baseline NE concentration in 12 normal subjects. Cardiac MIBG uptake was significantly decreased in patients as compared with controls. This decrease was related to the severity of the disease based on hemodynamic indexes. The inotropic response to intracoronary dobutamine infusion of heart failure patients correlated with both increased baseline NE concentration and diminished cardiac MIBG uptake (r = -0.63, p less than 0.01 and r = 0.73, p less than 0.001, respectively). These findings indicate that the desensitization process is related both to impaired neuronal NE uptake function and increased circulating NE concentrations. Moreover, a subset of 11 patients with moderate heart failure was identified who had diminished cardiac MIBG uptake but normal circulating NE concentrations. This suggests that impairment of the NE uptake function is an early mechanism of desensitization in idiopathic cardiomyopathy. Cardiac MIBG imaging may be a noninvasive means to assess severity of heart failure patients and may also be used to evaluate therapy effects on myocardial alterations of the adrenergic pathway.


Prolonged inhibition of local angiotensin-converting enzyme after single or repeated treatment with quinapril in spontaneously hypertensive rats. Plasma and tissue angiotensin-converting enzyme (ACE) activities were measured in spontaneously hypertensive rats (SHR) after single or repeated oral (p.o.) treatment with a hypotensive dose (1 mg/kg) of quinapril and compared with those after administration of enalapril (1 mg/kg). The degree of ACE inhibition in response to quinapril varied in tissues; marked inhibition was observed in aorta, lung, and plasma by single treatment with quinapril, and inhibition in plasma and aorta caused by quinapril was more potent than that caused by enalapril. The prolonged ACE inhibition was observed in the aorta, a target organ, by repeated treatment with quinapril for 2 weeks. These results indicate that quinapril has a good pharmacokinetic profile, namely rapid absorption and easy penetration to the target organ. In addition, quinapril produced greater inhibition of cardiac ACE than did enalapril after either p.o. or intravenous (i.v.) administration, suggesting the beneficial effects of quinapril in treatment of congestive heart failure (CHF).


Spasmolytic action of nicorandil in canine conductive coronary arteries in vivo is not modified by glibenclamide. The spasmolytic effects of nicorandil, cromakalim, and nitroglycerin on coronary arteries were investigated by angiographic technique in anesthetized dogs. With intracoronary arterial (i.a.) U 46619, a thromboxane A2 mimetic, the diameter of coronary arteries decreased in a sustained manner by 36.1 +/- 1.6% from control levels (coronary spasm). With a successive i.a. injection of nicorandil (300 micrograms), cromakalim (30 micrograms), or nitroglycerin (3 micrograms), the diameter recovered control levels (102.9 +/- 3.9, 96.8 +/- 5.6, and 100.1 +/- 4.3%, respectively). In dogs treated intravenously (i.v.) with glibenclamide, a pharmacologic antagonist of K-channel openers, the spasmolytic effect of cromakalim was significantly reduced, whereas the activity of nicorandil or nitroglycerin remained unaffected. We also investigated a possible modification by glibenclamide of the increase in coronary blood flow (CBF) induced by i.a. nicorandil and cromakalim in anesthetized dogs. The dose-dependent blood flow responses to cromakalim and nicorandil were significantly attenuated by glibenclamide, whereas the response to nitroglycerin remained unaffected. These results suggest that the spasmolytic effect of nicorandil on canine conductive coronary vessels is not mediated by K-channel opening but by a nitroglycerin-like action and that the dilatation of resistive coronary vessels induced by nicorandil may be largely due to its action as a K-channel opener.


Protective effects of G 619, a dual thromboxane synthase inhibitor and thromboxane A2 receptor antagonist, in splanchnic artery occlusion shock. Splanchnic artery occlusion (SAO) shock was induced in anesthetized rats by clamping the celiac trunk and the superior mesenteric artery for 45 min. The arteries were then released and survival rate, mean survival time, mean arterial blood pressure (MAP), plasma levels of thromboxane B2 (TxB2) and 6-keto-PGF1 alpha, and the phagocytotic activity of peritoneal macrophages were evaluated. Shocked animals died within 89 +/- 10 min, while all sham-shocked rats survived greater than 3 h. SAO shock produced relevant changes in MAP, significantly increased plasma levels of TxB2 and 6-keto-PFG1 alpha, and decreased peritoneal macrophage phagocytotic activity. The administration of G 619, a dual thromboxane synthase inhibitor/thromboxane A2 receptor antagonist (50 mg/kg, 15 min before SAO shock) significantly increased survival time (190 +/- 13 min) and survival rate, reduced plasma levels of TxB2, and partially restored the impairment in peritoneal macrophage phagocytosis. Finally, the administration of G 619 had beneficial effects on changes in MAP-induced bay SAO shock. These data further confirm the involvement of TxA2 in SAO shock and suggest that G 619 may have positive effects in low-flow states.


Syst-Eur--a multicenter trial on the treatment of isolated systolic hypertension in the elderly: first interim report. Syst-Eur is a multicenter placebo-controlled outcome trial designed by the European Working Party on High Blood Pressure in the Elderly to investigate the effect of antihypertensive treatment on the incidence of stroke in elderly patients with isolated systolic hypertension (ISH). Eligible patients must be at least 60 years old and have a systolic blood pressure averaging 160-219 mm Hg with a diastolic blood pressure less than 95 mm Hg. The present paper is an interim report on the first 316 patients randomized into this trial. The placebo (n = 170) and active treatment (n = 146) groups were similar at randomization with respect to age (73 +/- 8 years; mean +/- SD), sitting blood pressure (178 +/- 12 mm Hg systolic; 85 +/- 7 mm Hg diastolic), percentage of men (34%), and percentage of patients with cardiovascular complications (29%). After randomization blood pressure fell more (p less than 0.001) in patients on active treatment than in those in the placebo group (19 +/- 20 mm Hg systolic; 6 +/- 10 mm Hg diastolic vs. 7 +/- 19 and 1 +/- 10 mm Hg for sitting blood pressure). This first interim report on the Syst-Eur trial demonstrates that a multinational trial in elderly patients with ISH is feasible and that a significant blood pressure difference between the two treatment groups can be achieved and maintained. New centers are being recruited in order to randomize a total of 3,000 patients.


Effects of oral isradipine on left ventricular function at rest and during exercise in patients with stable chronic angina: a double-blind, placebo-controlled crossover study. We evaluated the effects of a single oral dose of 5 mg of isradipine compared to placebo in a randomized, double-blind, crossover study using gated radionuclide angiography at rest and during exercise in 20 patients with stable chronic angina. Isradipine improved both anginal symptomatology and ST-segment depression during exercise, with a concomitant favorable effect on the isotopic parameters exploring systolic and diastolic left ventricular function. There was a marked increase of the ejection fraction during exercise with isradipine compared to placebo (61 +/- 14% vs. 55 +/- 15%, respectively, p less than 0.001) as well as a significant improvement in the peak ejection rate and the peak filling rate at rest [2.56 +/- 0.62 vs. 2.16 +/- 0.54 end diastolic volume (EDV) per second and 2.14 +/- 0.59 vs. 1.87 +/- 0.37 EDV/s, respectively] and during exercise (3.49 +/- 0.97 vs. 3.10 +/- 1.07 EDV/s and 4.05 +/- 1.34 vs. 3.65 +/- 1.25 EDV/s, respectively). We conclude that isradipine has a beneficial effect on the clinical and electrocardiographic signs of exercise-induced ischemia, leading to a significant improvement of the systolic and diastolic parameters of left ventricular function. Therefore, isradipine is potentially a useful treatment for patients with exertional angina and hypertension, alone or associated with beta blocker medication.


Electrophysiologic effects of sotalol on the immature mammalian heart. Sotalol is a beta-blocker with class III antiarrhythmic properties that has recently been used in children for the treatment of supraventricular and ventricular arrhythmias. However, little is known about its electrophysiologic effects on the immature heart. Using intracardiac electrocardiographic recordings and stimulation techniques, 15 canine neonates (8-15 days) and 15 adult mongrel dogs were studied with cumulative doses of sotalol (0.5, 1, 2, and 4 mg/kg plus an additional dose of 8 mg/kg for neonates). Heart rate decreased significantly in the two groups, but more in adult dogs (-43% in adult dogs versus -25% in neonates, p less than 0.05). There was no significant change for QRS duration and His-Purkinje system conduction time interval. QT and atrioventricular nodal conduction time intervals increased in adult dogs and neonates. Sinus node recovery time increased significantly in the two groups, but more in adult dogs. Refractory periods of the atrioventricular (AV) node increased significantly in neonates. Atrial flutter was no longer inducible in 12 of 15 neonates after the 2 mg/kg dose. Atrial effective refractory period increased significantly more in neonates (96%, p less than 0.001) than in adult dogs (58%, p less than 0.001). Ventricular effective refractory periods increased significantly both in neonates (46%) and adult dogs (50%), in a similar way. In conclusion, sotalol has greater electrophysiologic effects on the immature heart at the atrial level when compared to the adult, and similar effects on the refractory period of AV node and ventricle.


Voltage-dependent modification of Vmax recovery from use-dependent block by pirmenol in guinea pig papillary muscles: comparison with other class I drugs. Voltage-dependent modification of Vmax (the maximum upstroke velocity of the action potential) recovery from use-dependent block (UDB) by pirmenol was examined and compared with those observed with other Class I drugs using standard microelectrode techniques. A partial depolarization of the resting membrane by increasing extracellular potassium concentration ([K+]o) from 4 to 8 mM potentiated UDB at 2 Hz stimulation by any of the following drugs: pirmenol (10 microM), disopyramide (20 microM), pentisomide (50 microM), quinidine (20 microM), mexiletine (30 microM), and flecainide (5 microM). The recovery time constants from UDB of quinidine and mexiletine were prolonged and that of flecainide was unchanged in 8 mM [K+]o. However, the recovery time constant from UDB of pirmenol was shortened in high K+ solution, as observed with disopyramide and pentisomide. Thus, disopyramide and its analogues, including pirmenol, show a voltage dependency of recovery process, which is different from those of other class Ia, Ib, and Ic drugs. The main unblocking pathway of disopyramide and its analogues from sodium channels during diastolic interval may be different from that of other Class I drugs.


Effect of antihypertensive treatment with nitrendipine on left ventricular mass and diastolic filling in patients with mild to moderate hypertension. Nitrendipine is a dihydropyridine calcium antagonist that may be active when administered once daily. The aim of the study was to assess the effect of antihypertensive treatment with nitrendipine (20-40 mg) on left ventricular mass and diastolic function. Forty patients with mild to moderate hypertension (diastolic pressure greater than or equal to 90 and less than or equal to 114 mm Hg) were enrolled; a complete echo Doppler examination was performed at baseline, and 8 and 12 months after treatment in order to measure left ventricular mass and diastolic and systolic function. Only 28 patients completed the study follow-up. At month 8 nitrendipine had already successfully reduced blood pressure (mean 122 +/- 9  to 92 +/- 10 mm Hg) without modifying heart rate, and left ventricular mass index (150 +/- 48 to 123 +/- 34 g/m2), with a further reduction at month 12. Isovolumic relaxation time was reduced at month 8 from 138 +/- 28 to 111 +/- 17 ms, but the diastolic pattern was completely modified only after 1 year, with a normalization of deceleration time (from 220 +/- 35 to 188 +/- 12). Systolic function did not change. Our results indicate that nitrendipine is a powerful antihypertensive agent that reduces left ventricular mass, but requires a longer period of time to improve diastolic filling pattern.


Effects of the ACE inhibitor, perindoprilat, and of angiotensin II on the transient inward current of guinea pig ventricular myocytes. Hypothetically, certain ischemic and reperfusion arrhythmias may result from the activity of the calcium-dependent transient inward current. The effects of the angiotensin converting enzyme inhibitor, perindoprilat, on the transient inward current of guinea pig ventricular myocytes were studied. The transient inward current was evoked by superfusing the cell with a modified Tyrode's solution (5.4 mM CaCl2 and 0.54 mM KCl). Repetitive voltage clamp steps from a holding potential of -55 to +20 mV (1,000 ms, 0.1 Hz) were applied while dialyzing the cell internally. When administered simultaneously with the change over to the low K+ high Ca2+ solution, perindoprilat (1 microM) decreased the transient inward current from -9.55 +/- 0.31 to -3.24 +/- 0.24 microA/cm2 (p less than 0.05). A similar decrease was observed when perindoprilat was administered after first inducing the transient inward current. Perindoprilat also protected from the effects of norepinephrine (0.01 and 0.1 microM), which increased the amplitude of the transient inward current from -9.76 +/- 0.17 and -9.99 +/- 0.32 microA/cm2 at the end of the 15-min control period to -11.13 +/- 0.67 and -12.67 +/- 0.49 microA/cm2, respectively (p less than 0.05). The effects of perindoprilat were independent of angiotensin II, which in this preparation decreased the transient inward current. Based on our results, we conclude that perindoprilat decreases the transient inward current and prevents the action of norepinephrine on the transient inward current. The direct effect of the angiotensin converting enzyme inhibitor observed on the transient inward current might explain why angiotensin converting enzyme inhibitors reduce calcium-dependent ouabain-induced or reperfusion arrhythmias.


Modulation of inotropic therapy by venodilation in acute heart failure: a randomised comparison of four inotropic agents, alone and combined with isosorbide dinitrate. The effects of four inotropic agents with differing ancillary properties [a cardiac glycoside (digoxin), a combined alpha- and beta-adrenergic agonist (dobutamine), a beta-adrenergic agonist (prenalterol), and a phosphodiesterase inhibitor (amrinone)] alone and with subsequent addition of isosorbide dinitrate were compared in 48 consecutive acute myocardial infarction patients with radiographic and haemodynamic (pulmonary artery occluded pressure greater than 18 mm Hg) left ventricular failure. All agents with the exception of dobutamine reduced the elevated left heart filling pressure; only digoxin and dobutamine augmented the cardiac stroke volume index. All drugs except digoxin reduced the SVRI; an arteriolar constrictor response was evident 60 min after digoxin and a tachycardia resulted after combined alpha- and beta- and beta-adrenergic stimulations (dobutamine and prenalterol, respectively). The addition of isosorbide dinitrate reversed the inotrope-induced elevations of systemic arterial pressure and resulted in additional reductions in left heart filling pressure. These data suggest that, in the absence of substantial venodilator properties in an inotropic compound, reduction in elevated left heart filling pressure is not achieved with inotropic therapy alone in acute left ventricular failure and combining a venodilator may be haemodynamically advantageous.


Coronary reperfusion rates in acute myocardial infarction patients after thrombolytic treatment with anistreplase: correlation with the delay from onset of symptoms to treatment: a review of 424 case records of patients admitted to coronary reperfusion studies with anistreplase. The individual data for 424 acute myocardial infarction patients from 15 reperfusion studies conducted with anistreplase were reviewed to assess the correlation between the delay onset of symptoms to treatment and reperfusion of the coronary artery. The patients had a mean age of 56 years and 85.4% were male. All patients had a baseline angiogram showing an occluded coronary artery. Anistreplase was given in doses ranging from 3.75 to 35 IU (60% of patients received 30 IU). The mean delay between onset of symptoms to treatment was 3 h 29 min and reperfusion was observed in a total of 61.1% of patients. There was a highly significant correlation between reperfusion and the delay from onset of symptoms to treatment (p less than 0.001). A multivariate logistic regression was performed adjusting for age, sex, location of infarct, dose, and concomitant treatment. The only remaining statistically significant prediction factor for reperfusion was the delay between onset of symptoms to treatment (p less than 0.001). In conclusion, the ability of anistreplase to cause the lysis of coronary thrombi appears to be increasingly important as the delay from onset of symptoms to treatment is decreased. This may contribute to the lower mortality rate observed with early fibrinolytic treatment.


Effect of dietary sunflower seed oil on the severity of reperfusion-induced arrhythmias in anesthetized rats. Myocardial ischemia followed by reperfusion was produced in artificially respirated, open-chest rats. Coronary artery ligation for 6 min rarely evoked arrhythmias; however, reperfusion after this period rapidly produced severe dysrhythmias in all control animals. Reperfusion after 12 min of ischemia produced less frequent dysrhythmias than after coronary occlusion for 6 min. Feeding of a linoleic acid-rich diet, applying 12% sunflower seed oil in rat food pellet for 4 weeks, decreased the incidence of reperfusion-induced ventricular fibrillation both after 6 min (2/15 vs. 7/11) and 12 min (0/11 vs. 2/8) of myocardial ischemia and the incidence of other arrhythmias was also decreased. The number of animals developing no arrhythmias during reperfusion was increased (8/15 after 6 min of ischemia, 4/11 after 12 min of ischemia vs. 0/11 and 0/8 in controls, respectively). Our results indicate that increased dietary consumption of linoleic acid decreased the occurrence of life-threatening arrhythmias both during the acute phase of myocardial ischemia and during reperfusion in anesthetized rats.


Effects of sinoaortic denervation on the reflex actions of digoxin and a polar aminocardenolide. To determine the role of carotid sinus and aortic arch baroreceptors in the reflex cardiac sympathetic nerve activity (SNA) responses to administration of ASI-222, a polar aminocardenolide, and to digoxin, a neutral cardenolide, we used anesthetized dogs from which these reflex receptor areas had been removed. The SNA was measured in postganglionic fibers from the stellate ganglion. After we made baseline measurements, we infused either ASI-222 or digoxin intravenously (i.v.) at dose rates that produce cardiac arrhythmias in approximately 100 min (0.7 and 1.2 micrograms/kg/min), respectively. Our data indicate that with sinoaortic baroreceptors removed, progressive infusion of digoxin increases cardiac SNA. In contrast, cardiac SNA decreases progressively during continuous infusion of ASI-222. In saline-treated dogs, SNA was not significantly altered. In another series of experiments, we examined the effects of these drugs and saline on cardiac vagal afferent nerve activity (VANA). ASI-222 (50 micrograms/kg i.v. in 10 min) caused a progressive increase in cardiac VANA in a 60-min observation period. Neither digoxin, at less than or equal to 120 micrograms/kg i.v. in 100 min, nor saline altered VANA. Digoxin appears to reduce SNA by interacting with the carotid sinus and aortic arch baroreceptors, which are myelinated. It does not affect VANA acutely. In contrast, ASI-222 appears to decrease cardiac SNA by interacting with other reflex receptor areas--the unmyelinated cardiopulmonary afferent nerve endings, particularly cardiac mechanoreceptors.


Proarrhythmic effects of procainamide and tocainide in a canine infarction model. A canine model of myocardial infarction (MI) was used to study the type and frequency of ventricular antiarrhythmic and proarrhythmic effects due to procainamide and tocainide and the risk factors associated with development of proarrhythmia. An anterior MI was created by a 2-h occlusion of the left anterior descending artery (LAD) with complete reperfusion. Programmed ventricular stimulation was performed on two occasions after MI, on days 4-6 and on days 8-10, before drug and during antiarrhythmic drug infusion at three dose levels. The antiarrhythmic drugs were given in a randomized cross-over design. Only procainamide caused a dose-dependent increase in QRS, JTc, and right ventricular effective refractory period (ERP). Neither procainamide nor tocainide made sustained ventricular tachycardia (VT) noninducible, but procainamide slowed the tachycardia rate. Both drugs successfully made ventricular fibrillation (VF) noninducible: procainamide in 78% of trials and tocainide in 50% of trials. Proarrhythmia (development of inducible VT during drug when not present before drug or inability to terminate VT during drug administration) developed in 29% of dogs that received procainamide and 25% of dogs that received tocainide. There was no apparent correlation of QRS, JTc, and right ventricular ERP after drugs and proarrhythmia due to procainamide or tocainide. There was no significant difference in the size of MI between dogs with one or more proarrhythmic response to either drug and dogs that had no proarrhythmia. In this model, procainamide and tocainide had no antiarrhythmic efficacy for VT, but had moderate proarrhythmia potential that was unpredictable.


Cardiovascular effects of a novel calcium entry blocking and selective beta 1-adrenoceptor blocking agent, YM-16151-4, in anesthetized and conscious dogs. Cardiovascular effects of YM-16151-4, a combined calcium entry blocking and beta 1-adrenoceptor blocking agent, were evaluated in dogs. In anesthetized dogs, YM-16151-4 (0.01-1 mg/kg intravenously, i.v.) dose-dependently increased coronary blood flow (CBF) and decreased mean blood pressure (MBP), total peripheral resistance (TPR), dP/dtmax, double product, and left ventricular (LV) work without increasing heart rate (HR) and cardiac output (CO). YM-16151-4 increased vertebral blood flow as well as CBF, but had no effect on carotid, mesenteric, renal, and femoral blood flow. Coronary vasodilating activity of YM-16151-4 was also observed after intracoronary artery injection (i.a.). In anesthetized and vagotomized dogs, YM-16151-4 dose-dependently inhibited isoproterenol (0.2 micrograms/kg i.v.)-induced tachycardia and decrease in diastolic BP (DBP), with ED50 values of 0.039 and 0.52 mg/kg i.v., respectively. In conscious dogs, YM-16151-4 (0.1-1 mg/kg i.v.) produced a dose-dependent hypotensive effect with no effect on HR or PQ-interval. The hypotensive effect of YM-16151-4 (0.3 and 1 mg/kg i.v.) reached its maximum approximately 1-2 h after each dosing and lasted 6-8 h. These results suggest that YM-16151-4 actually behaves as a hybrid compound, combining calcium entry blocking and beta 1-adrenoceptor blocking activities, and that this compound could be a novel long-acting antianginal and antihypertensive agent.


Failure of AICA riboside to limit infarct size during acute myocardial infarction in rabbits. This study examined whether the adenosine potentiator, 5-aminoimidazole-4-carboxamide riboside (AICAr), could limit tissue necrosis during acute myocardial infarction in rabbit hearts with minimal coronary collateral flow. Forty-four rabbits underwent 45 min of ischemia with or without coronary reperfusion for 180 min. Five groups were studied. Saline or AICAr (20 mg/kg, i.v.) was administered as a bolus either 10 min before coronary occlusion or 10 min before the onset of coronary reperfusion. The anatomic risk zone size was assessed by radiolabeled microsphere autoradiography and the area of tissue necrosis was defined using the tetrazolium staining method. Coronary collateral flow in the central ischemic zone was assessed using the radiolabeled microsphere technique. No differences were observed for tissue necrosis (normalized to risk zone size) for saline- and AICAr-treated rabbits (66.2 +/- 10.9% vs. 70.8 +/- 19.9%, p = NS) subjected to 45 min of coronary occlusion without reperfusion. Similarly, tissue necrosis in rabbit hearts with 45 min of coronary occlusion followed by 180 min of reperfusion was not significantly reduced when AICAr was administered either 10 min before ischemia or 10 min before reperfusion (79.8 +/- 17.5 and 76.4 +/- 8.1%, respectively) compared to saline-treated controls (68.1 +/- 22.7%). Coronary collateral flow in these hearts was almost nonexistent. The risk zone size and cardiac hemodynamics were similar between the treatment groups. These results demonstrate that AICAr was unable to limit myocyte necrosis when administered either before ischemia or before coronary reperfusion in this experimental preparation of acute myocardial infarction.


Active and passive effects of antihypertensive drugs on large artery diameter and elasticity in human essential hypertension. The effects of antihypertensive drugs on the large arteries consist of two parts: the passive effect due to the change in pressure and the active effect, the drug action per se. This study proposes a method of dissociating the passive effect from the active effect. The diameter of the arterial artery was determined by the pulsed Doppler method and the pulse wave velocity of the brachioradial artery by mecanography. Arterial compliance was calculated by the Bramwell-Hill formula. Active and passive effects were determined by a logarithmic pressure-diameter model. This model was supported by in situ direct measurements of blood pressure and diameter in a segment of the femoral artery in dogs. Six drugs, cadralazine, ketanserin, medroxalol, nitrendipine, captopril, and isosorbide dinitrate, administered orally, were tested in 70 essential hypertensive patients. For all drugs, the pressure reduction induced a passive decrease in arterial diameter (p less than 0.02 to p less than 0.01). Cadralazine actively decreased arterial diameter (p less than 0.01), ketanserin had no active effect on diameter, and medroxalol, nitrendipine, captopril, and isosorbide dinitrate actively increased arterial diameter (p less than 0.05, p less than 0.01, p less than 0.01, and p less than 0.01, respectively). For all drugs, the pressure reduction also induced a passive increase in arterial compliance (p less than 0.05 to p less than 0.01). However, only nitrendipine, captopril, and isosorbide dinitrate actively increased arterial compliance (p less than 0.01, p less than 0.05, and p less than 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)


Sex and age as factors influencing the vascular reactivity in Watanabe heritable hyperlipidaemic (WHHL) rabbits: a pharmacological and morphological study of the hepatic artery. The responses to vasoactive agents and the fine structure of hepatic arterial ring segments from male and female Watanabe heritable hyperlipidaemic (WHHL) rabbits (4, 6, and 12 months) were compared with those of age- and sex-matched New Zealand White (NZW) rabbits. In males only, KCl-induced contractions were reduced in WHHL rabbits compared with NZW rabbits. In male and female WHHL rabbits, maximum noradrenaline-induced contractions and sensitivity to noradrenaline were greater than those of male and female NZW rabbits. In female WHHL and NZW rabbits only, maximum noradrenaline-induced contractions and the EC50 values were reduced at 6 months. Endothelium-dependent relaxation: In females only, maximum relaxant responses and the sensitivity of WHHL rabbits to acetylcholine increased with age, while there was a decrease in NZW rabbits. Similarly, relaxation to substance P increased with age in WHHL rabbits and decreased in NZW rabbits, but this occurred in both male and female animals. In addition, substance P-induced relaxation in female WHHL rabbits was greater than in male WHHL rabbits. Endothelium-independent relaxation: In both male and female WHHL rabbits, calcitonin gene-related peptide-induced and vasoactive intestinal polypeptide-induced relaxation did not change with age. However, there was an age-related decrease in the response of NZW rabbits to these peptides. Electron microscopic evaluation of hepatic arteries from WHHL rabbits showed occasional ruptures in the internal elastic lamina at 4 months. At 6 months, widespread intimal thickening associated with smooth muscle cell migration was apparent, but this became less obvious at 12 months. No obvious differences in structure between male and female hepatic arteries were observed. It is suggested that a "compensatory vasodilatation" develops in atherosclerosis, initially at the level of the endothelium, and then with the progression of the disease extends to changes in the smooth muscle. This may occur in order to offset the thickening of the arterial wall. Sexual dimorphism in vascular reactivity has been demonstrated.


Effects of milrinone on systemic capacitance vessels in relation to venous return and right ventricular pump function. To investigate the effect of milrinone (MIL) on systemic capacitance vessels, we measured the mean circulatory filling pressure (MCP) in anesthetized open chest dogs. We measured hemodynamic parameters (a) at baseline blood volume (BV), (b) immediately after bloodletting (5 ml/kg), and (c) immediately after dextran injection (5 ml/kg). These measurements were taken in a control group (n = 8) and in a MIL group (n = 8), where MIL (100 + 2.5 micrograms/kg/min) was administered i.v. The extra volume (EV) was obtained by extrapolating the straight line that was fit to the MCP-BV plot. MIL significantly decreased the EV, from 22.8 +/- 1.0 to 19.1 +/- 0.4 ml/kg, indicating that MIL dilates the systemic capacitance vessels. MIL shifted the right ventricular output curve to the left and upward and shifted the venous return curve to the left and rotated it clockwise. Thus, venous return was increased by decreasing the resistance to venous return and by improving right ventricular pump function. Next we evaluated the effects of MIL (100 micrograms/kg) on systemic capacitance and resistance vessels from changes seen in the MCP and the total peripheral resistance (TPR), respectively. In the untreated dogs, MIL decreased TPR significantly without affecting MCP. In dogs pretreated with total spinal anesthesia or phenoxybenzamine, MIL significantly decreased both MCP and TPR. This suggests that the intrinsic venodilator action of MIL is modified by the baroreflex in untreated animals. MIL decreased the MCP and TPR elevated previously by norepinephrine. One would, therefore, predict that MIL would dilate the systemic capacitance vessels in patients with congestive heart failure.


Acute lymphoblastic leukemia with the (4;11) translocation: combined cytogenetic, immunological and molecular genetic analyses. Five cases of acute leukemia (AL) with the t(4;11) translocation were investigated for the immunoglobulin heavy chain, kappa, lambda, TCR beta and TCR gamma gene rearrangements. All patients presented with high-risk features and had survival times of less than two years. Two cases were classified by immunological phenotyping as acute null-AL(L), one case as pre B-cell ALL (CD10+) and two cases expressed both immature B-cell markers CD19 and CD24 and myelomonocytic markers CD15 and CD14, suggesting mixed lineage leukemia. In two cases more than two rearranged fragments for the immunoglobulin heavy chain gene could be detected by Southern blot analysis. In the other cases at least one allele of the immunoglobulin heavy chain gene was rearranged. Germline configuration of the T-cell receptor genes and lack of light chain gene rearrangement suggest that an early B-precursor cell is involved in the transformational events in these cases of ALL. Our own and published data indicate that acute leukemia with t(4;11) translocation might be more frequently associated with more than two rearranged fragments for the immunoglobulin heavy chain genes and run a very aggressive course.


Surface markers in adult acute myeloblastic leukemia: correlation of CD19+, CD34+ and CD14+/DR--phenotypes with shorter survival. Groupe d'Etude Immunologique des Leucémies (GEIL). The immunophenotype of blast cells was investigated in a multicentric study of 154 adult acute myeloblastic leukemias (AMLs). A panel of 27 monoclonal antibodies (MoAbs) was tested in indirect immunofluorescence. Expression of CD14 (UCHM1), CD19 (SB4), CD36 (OKM5), and HLA-DR were associated with higher mean leucocyte counts. CD14 expression correlated with low hemoglobin level and the absence of CD33 (MY9) with low platelet counts. Extramedullary disease was associated with CD16 (Leu11b) and HLA-DR antigen positivity. The study of relationships between surface markers and FAB criteria confirmed the predominant expression of CD14 in the M5 sub-group (p less than 0.000001) and the association of CD19 and CD36 with monocytic M4/M5 subgroups (respectively p less than 0.00002 and p less than 0.000001). All patients received an induction therapy including an anthracycline and cytarabine. The median follow-up was 13 months. The achievement of complete remission (CR) was inversely correlated with CD34 (B13C5) and CD19 expression: CR was obtained in 31 of 59 (53%) CD34-positive AML versus 64 of 75 (85%) CD34-negative cases (p less than 0.0001) and 11 of 24 (46%) CD19-positive versus 95 of 122 (78%) CD19-negative cases (p less than 0.01). In univariate analysis, a longer survival was associated with CD33 expression and the combined phenotypes CD36+/CD19- and CD16+/CD14-. Conversely, the CD18(IOT18)+/CDw65(VIM2)- phenotype was related to shorter survival. The expression of CD19, of CD34, and of the combined phenotype CD14+/DR--correlated with shorter survival as demonstrated both in univariate and multivariate analysis (p less than 0.03 in each case in multivariate analysis).


Cytokine induction of leucocyte adhesion molecule-1 (LAM- 1) expression on chronic lymphocytic leukaemia cells. Leucocyte adhesion molecule 1 (LAM-1) participates in the binding of human leucocytes to high endothelial venules in peripheral lymph nodes. Other adhesion receptors which are involved include CD44 and the integrin family, CD11/CD18. In this study, B-cell chronic lymphocytic leukemia (B-CLL) cells were examined for the expression of these adhesion molecules, and for the way in which cytokines are able to modulate the levels of these receptors. B-CLL cells express significant but variable levels of LAM-1 and high levels of CD44. In contrast, these cells exhibit very low or absent amounts of surface CD11a, CD11b, or CD11c. Most CLL cells expressed no detectable levels of intercellular adhesion molecule-1 but some cases show levels of up to 30%. Following 24 h incubation with interferon alpha (500 U/ml), surface LAM-1 expression on peripheral blood E-negative cells from CLL patients rose to 330 +/- 127% of levels on control cells incubated with medium alone (n = 13, p less than 0.0005). Interleukin 4 (1 ng/ml) and interferon gamma (100 U/ml) also increased surface LAM-1 levels on these cells to 218 +/- 119% (n = 8, p less than 0.001) and 245 +/- 116% (n = 5, p less than 0.001) of control levels respectively. Induction of LAM-1 expression occurred over 48 h (greater than 50% of the increase was seen in the first 24 h) in a dose-dependent manner and required protein synthesis. The induction of LAM-1 expression on the malignant cells may, by altering the homing behaviour of these cells, relate to the reduction in peripheral leukaemic cells seen following treatment with interferon alpha in CLL.


Characterization of murine hemopoietic-supportive (MS-1 and MS-5) and non-supportive (MS-K) cell lines. Characteristics of hemopoietic-supportive (MS-1 and MS-5) and non-supportive (MS-K) cell lines were compared. Supportive cells adhered to hemopoietic stem cells and produced granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas non-supportive cells did not adhere to hemopoietic cells and only produced macrophage colony-stimulating factor. Both cell lines produced substantial levels of IL-6 and steel factor (SLF) which is reportedly a stem-cell factor. Northern blot analysis revealed that SLF but neither c-kit nor interleukin 3 (IL-3) mRNA was detectable in these cell lines, although IL-3-like activity was found in the supernatant of MS-5 cell culture. These observations suggest that the hemopoietic-supportive function of stromal cells may reside in adherence of stem cells, and production of GM-CSF probably in combination with SLF. SLF may be transferred from stromal cells directly to stem cells through adhesion of stem cells to supportive stromal cells.


Effects of cryopreservation on the adherent layer of human long-term bone marrow cultures: study of cell phenotypes. This report describes the effects of cryopreservation on the adherent layer of human long-term bone marrow cultures (HLTBMC). Stromal cells are believed to be the most important cells of medullar microenvironment to regulate hematopoiesis. To study effects of cryopreservation, we compared the cell phenotypes of adherent layers of fresh and frozen-thawed bone marrows. To characterize stromal cells we used monoclonal antibodies reacting with components of these cells (CGA-7 alpha SM and gamma SM actin isoforms; HHF-35, all muscle actin isoforms; BMS-1, stromal cell lysosomes). The other components studied were: fibronectin (BMS-2 monoclonal antibody) and hematopoietic cells (monoclonal antibodies against CD45, CD33, and CD14). Results show a decrease of cells positive for CGA-7, HHF-35, and BMS-1, in adherent layer of HLTBMC of frozen-thawed bone marrows. Expression of BMS-2 is unchanged, and CD45 and CD14-positive cells proportionately increased. These results are consistent with an impairment of stromal cell proliferation in frozen-thawed marrows, without impairment of most stromal cell functions. The difference between stromal cell and hematopoietic cell kinetics seems to be an additional fact suggesting a different origin for both cell populations.


In vitro responsiveness to interleukins and theophylline of CD16+, CD56- natural killer cells in a patient with chronic granular lymphocyte disorder. We report here the case of a 55-year-old patient with chronic granular lymphocyte disorder associated with moderate neutropenia. The majority of peripheral blood lymphocytes displayed a CD3-, CD8-, CD16+, CD56(NKH1)- phenotype. The patient's cells showed high spontaneous cytotoxic activity against K562 targets and developed the ability to kill the natural killer (NK)-resistant target Daudi following activation with interleukin 2 (IL-2). Simultaneously, IL-2 induced proliferation of these cells, albeit to a low level. The effects of IL-2 are likely to be mediated through the IL-2R beta chain (p70) which is expressed on these cells in the absence of the IL-2R alpha chain (p55, Tac). IL-4 was demonstrated to be inhibitory of both the cytotoxic and proliferative effects of IL-2. Thus, despite an unusual CD56- phenotype, the expanded lymphocyte population in this patient display functional and phenotypic properties of normal, non-activated NK cells. These cells probably represent the counterpart of a minor NK cell subpopulation, present in normal individuals at a low frequency, and which has never been fully characterized functionally. In addition, we show that the cytolytic activity of this NK cell population can be blocked in vitro in the presence of a cAMP analog or of theophylline, possibly providing new means of investigating the role of NK cell cytotoxicity on the pathogenesis of associated symptoms in such patients.


PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines. Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.


Differentiation of human prostate cancer from benign hypertrophy by in vitro 1H NMR. In vitro 1H NMR spectra were acquired for perchloric acid extracts of tissue samples of human prostate. Seven patients were diagnosed with prostate cancer, 13 with benign prostatic hypertrophy, and 3 with both conditions. Statistically significant differences between the cancer and benign groups were seen for the metabolite peak area ratios of citrate, creatine, and phosphorylcholine to alanine, and citrate to glutamate. There was no correlation of Gleason grade with any of the ratios measured for the cancer samples. Spectra from different sections of large tumors often yielded substantially different area ratios, confirming the heterogeneous nature of these prostate tumors.


Specific MR imaging of human lymphocytes by monoclonal antibody-guided dextran-magnetite particles. Human lymphocytes were labeled with biotinylated anti-lymphocyte-directed monoclonal antibodies, to which streptavidin and subsequently biotinylated dextran-magnetite particles were coupled. This labeling resulted in a strong and selective negative contrast enhancement of lymphocyte suspensions at 2.0 T, caused predominantly by the specific increase of R2 with a small but significant specific increase of R1. The R1 was found to decrease with increasing field strength. The immunolabeling procedure described here may be used for the selective signal depletion of target cells in MR imaging.


Analysis of tick-borne encephalitis virus antigens by monoclonal antibodies. Monoclonal antibodies (MAbs) against Central European tick-borne encephalitic virus, strain Hypr, were used for determination and characterization of viral antigens of infected PS cells. The MAbs reacted in immunoblotting with flavivirus glycoprotein E (56 kD), and nonstructural protein NS3 (70 kD). According to enzyme immunoassay with infected cells, NS3 antigen is expressed in the plasmalemma.


Genetic screening for cystic fibrosis.

Genetic screening for cystic fibrosis. More than 98% of mutations causing cystic fibrosis can be detected in a Celtic population in Brittany, France. What, though, are the prospects for screening of entire populations for carriers?


Function of maternal cytokeratin in Xenopus development. Intermediate filaments are ubiquitous in eukaryotic cells, but their functions are poorly understood. The Xenopus oocyte contains both messenger RNA and protein products of cytokeratin and vimentin genes in non-overlapping arrays. The cytokeratin filaments contain dimers of the type I (acidic) subunit XLK3a(19), and the type II (basic) subunit XCK1(8), polymerized to form a cortical network. These are homologues of the human simple epithelial keratins 19 and 8, respectively. After the first few cell cycles following fertilization these filaments become restricted to the superficial cells of the blastula. We have depleted the oocyte's store of the type II cytokeratin mRNA by injecting antisense oligodeoxynucleotides (oligos) and studied the effect on embryonic development. As zygotic transcription does not commence until the late blastula stage, there are at least 9 hours in which to see the effect of loss of function of this mRNA. We report here that the cytokeratin filaments become depleted in the cortical cells of the embryo. As a result, there is a loss of the 'compacted' epithelial surface of the blastula, an inability to close a wounded surface and defective gastrulation.


Characterization of tachykinin-induced ventral root depolarization in the neonatal rat isolated spinal cord. Depolarization responses to tachykinin receptor agonists were recorded extracellularly from lumbar ventral roots of spinal cord isolated from neonatal rats (one to eight days post partum). All spinal cords were hemisected in the sagittal plane. In addition, in some hemisected cords, the dorsal horns were removed by means of a further cut, perpendicular to the first. In both hemisected and quadrisected spinal cords, reproducible depolarization responses were induced by low concentrations of the neurokinin-1-selective agonist substance P methylester (10 nM-1 microM) or of the neurokinin-3-selective agonist senktide (3-300 nM). On both types of preparation, responses to substance P methylester (1 microM) or senktide (300 nM) were of comparable size. The amplitude of the response to senktide (300 nM) was reduced by at least 88% in spinal cord preparations exposed to tetrodotoxin (0.5 microM) or to physiological medium containing magnesium chloride (20 mM). In contrast, under either of these conditions, concentration-response curves to substance P methylester were shifted rightward by 2.8-8.5-fold, with little effect on the maximum response. Responses to senktide were blocked selectively by the N-methyl-D-aspartate antagonist 3-[(+-)-2-carboxypiperazine-4-yl]propyl-1-phosphonic acid (100 microM); the antagonist had little effect on substance P methylester-induced depolarization (mean concentration ratio 2.0). These results suggest that in the neonatal rat spinal cord, application of exogenous tachykinin agonists can induce ventral root depolarization by activation of neurokinin-1 and/or neurokinin-3 receptors. The response to stimulation of neurokinin-1 receptors has a major component likely to be due to a direct action at motoneurons.(ABSTRACT TRUNCATED AT 250 WORDS)


Bilateral irradiation of head and neck induces an enhanced expression of substance P in the parasympathetic innervation of the submandibular gland. Substance P and calcitonin gene-related peptide (CGRP) are present in nerve fibers innervating the submandibular gland. Radiotherapy of tumors in the head and neck region usually embraces the salivary glands in the irradiated field and consequently a dramatic decrease in salivary function is seen. In this study, the submandibular glands and ganglia of rats subjected to fractionated irradiation were examined by use of immunohistochemical techniques for demonstration of substance P and CGRP. Irradiation was given on five consecutive days (daily doses of 6-9 Gray) with unilateral or bilateral irradiation techniques. Specimens of control and experimental animals were processed in parallel. A marked increase in the expression of substance P in the ganglionic cells--presumably parasympathetic--and in the number of fibers showing substance P-like immunoreactivity in association with acini and small ducts was seen in response to bilateral irradiation. (Surprisingly, unilateral irradiation of the parotid area had no effect on peptide distribution in the irradiated gland and ganglion). No changes in the pattern of CGRP immunoreactivity occurred. In the trigeminal ganglion, which supplies the submandibular gland with the majority of the sensory substance P- and CGRP-containing nerve fibers, no changes in the expression of substance P or CGRP immunoreactivity were seen. The results suggest that bilateral irradiation leads to an increase in the synthesis of substance P-like substance in the parasympathetic ganglionic cells supplying the submandibular gland with secretory nerves, and can thus be an additional factor in explaining the altered secretory capacity of salivary glands.


The OM series of terminal field-specific monoclonal antibodies demonstrate reinnervation of the adult rat dentate gyrus by embryonic entorhinal transplants. Monoclonal antibodies OM-1 to OM-4 and IM-1 [Woodhams et al. (1991) Neuroscience 46, 57-69] have complementary immunostaining patterns in the molecular (dendritic) layer of the adult rat dentate gyrus, with OM-1 to OM-4 selectively recognizing the outer (distal) two-thirds (i.e. the entorhinal afferent zone), and IM-1 the inner (proximal) one-third (i.e. the hippocampal commissural/associational zone). Immunoblotting suggests that OM-1 recognizes a single glycoprotein antigen of mol. wt around 93,000, and OM-2, OM-3, and OM-4 all recognize a second glycoprotein antigen of mol. wt around 36,000. At four weeks after removal of the ipsilateral entorhinal cortex the background OM immunostaining of the entorhinal afferent zone is abolished and replaced by a network of densely stained granules, which we interpret as degenerating entorhinal afferent axons. At the same time, the proximal, IM immunoreactive zone expands by about 10 microns in width (while the distal deafferented zone shrinks by about 80 microns). Attempts were made to restore the OM immunoreactivity of the distal zone by grafting either small pieces or cell suspensions of embryonic day 18 entorhinal cortex directly into the dentate molecular layer of entorhinally deafferented adult hosts. About half (14/26) of the animals with successfully positioned grafts showed restoration of OM-2 to OM-4 immunostaining throughout the entire width of the outer two-thirds (entorhinal afferent zone) of the dentate molecular layer. Strikingly, however, in adjacent serial sections the restoration of OM-1 immunoreactivity was restricted to the "middle" molecular layer, i.e. the most proximal part of the distal (entorhinal) two-thirds of the dentate molecular layer. In no case did the OM-1 immunoreactivity extend to the outer margin of the molecular layer. This did not appear to be associated with incompleteness of the removal of the host entorhinal projection, since it occurred in grafted cases where the hippocampus had been completely isolated from the entorhinal area. The simplest explanation of the observed pattern of OM loss and restitution is that the epitopes are located on the entorhinodentate axons, but it is not clear whether the antigens recognized by OM-1 and OM-2 to OM-4 are expressed in different parts of the same group of axons, or in different subsets of entorhinodentate axons. Nor is it clear why the pattern of OM-1 is only restored to the "middle" molecular layer, while that of OM-2 to OM-4 is restored to the entire outer two-thirds.(ABSTRACT TRUNCATED AT 400 WORDS)