Application of sucrose fatty acid esters in transdermal therapeutic systems.

Transdermal therapeutic systems (TTSs) were studied applying different sucrose fatty acid esters (SEs) as drug delivery agents. Matrix and membrane controlled TTSs were prepared and compared. Membrane was made from a methacrylic polymer (Eudragit NE) of pH independent permeability which can achieve diffusion controlled drug liberation. Model drug was a water soluble beta-blocker, metoprolol, which has short biological half-life, so applying it in a TTS, the duration of its action could be prolonged. Sucrose fatty acid esters of different fatty acid chain lengths and consequently different hydrophilic-lipophilic balance (HLB) values were studied considering their effect on the metoprolol release from TTSs. Different mathematical models were applied for the evaluation of the release process. The results of the in vitro studies indicated that SEs of shorter fatty acid chain length and higher HLB value increased the amount of released drug about 10 times. SEs could be promising agents in transdermal therapeutic systems to control the drug release and cutaneous absorption.

Immunogenicity of L(d+) transgenic mouse hearts.

BACKGROUND: C57BL/6 mice transfected with the L(d) gene coupled to the alpha-myosin heavy chain promoter result in transgenic mice with L(d) antigen expressed only on cardiac tissue. These transgenic animals allow the examination of immune reactivity against cardiac L(d) by "self" or by adoptively transferred L(d) specific 2C cells, and the response of nontransgenic C57BL/6 mice to the transplanted L(d+) heart. METHODS: Naïve cardiac L(d+) transgenic mice were examined for evidence of L(d) "autoimmunity." Forty million fresh 2C cells or 2C cells sensitized in vitro for 7 days against Balb/c (L(d+)) + interleukin-2 were also given intravenously to L(d+) transgenic mice. At 5 and 12 days after injection, heart-infiltrating lymphocytes were analyzed by fluorescence-activated cell sorter. The L(d+) transgenic hearts were also transplanted to syngeneic L(d-) nontransgenic C57BL/6 to evaluate the heart's immunogenicity. RESULTS: Naïve L(d+) transgenic mice did not exhibit any evidence of lymphocytic infiltration on histologic examination. Adoptive transfer of either fresh or in vitro sensitized 2C cells was also unable to reject the native L(d+) heart in transgenic mice (100% of the mice survived long term [more than 60 days]). Sensitization of the L(d+) transgenic mice with a Balb/c skin graft and interleukin-2 pump infusion (7 days) beginning 1 day before 2C cell injection also did not promote rejection of the native L(d+) heart. However, fluorescence-activated cell sorter analysis did reveal that a significantly greater number of in vitro sensitized 2C cells homed to the L(d+), but not L(d-), heart after both 5 and 12 days (P <.01, P <.001). In contrast, C57BL/6 mice rejected the L(d+) (C57BL/6 background) transgenic heart in a mean survival time of 17 +/- 9.7 days (P <.01), whereas a syngeneic C57BL/6 heart transplant was accepted indefinitely. Lymphocytic infiltration consistent with rejection was present in all animals receiving an Ld+ transgenic heart transplant, whereas no infiltrate was present in those receiving a syngeneic C57BL/6 heart transplant. CONCLUSIONS: Although the class I L(d) transgene is not recognized in its native host, its immunogenicity is shown by the homing of anti-L(d) 2C cells to the heart in situ and rejection of L(d+) heart grafts when transplanted into syngeneic C57BL/6 mice.

Organ transplant specificity of tolerance to skin grafts with heart or kidney grafts plus nondepleting anti-CD4 monoclonal antibody (RIB 5/2) and intravenous donor alloantigen administration.

BACKGROUND: CD4+ T cells play an essential role in allograft rejection. Monoclonal anti-rat CD4 antibody, RIB 5/2, has been shown to modulate the CD4 glycoprotein without eliminating recipient T cells. A single dose of monoclonal anti-rat CD4 antibody RIB 5/2 plus donor splenocytes results in donor-specific unresponsiveness to heart and kidney allografts, but not skin allografts. This study examined whether tolerance to the more resistant skin graft could also be achieved with RIB 5/2. METHODS: Buffalo (RT1(b)) recipients were given a single dose (20 mg/kg) of monoclonal antibody RIB 5/2 IP plus IV Lewis (RT1(l)) splenocytes (25 x 10(6)) 21 days before Lewis heart, kidney, or skin grafts. In addition, Lewis skin was grafted either simultaneously with or after long- term Lewis heart or kidney allograft acceptance (>50 days). RESULTS: While IV alloantigen plus RIB 5/2 results in long-term acceptance of both heart and kidney, skin allografts are rejected when transplanted alone. Simultaneous transplantation with a Lewis kidney, but not with a Lewis heart, resulted in long-term Lewis skin graft acceptance. However, recipients tolerant to Lewis kidney or heart alone will not accept subsequent Lewis skin grafts, while recipients of simultaneous Lewis skin and kidney grafts subsequently accept a second Lewis, but not third-party Brown Norway (RT1(n)), skin graft. CONCLUSION: RIB 5/2 plus Lewis donor splenocytes tolerize for donor-specific heart and kidney but not skin grafts. However, Lewis skin grafted simultaneously with a Lewis kidney, but not Lewis heart, is accepted and protects a subsequent donor-specific Lewis skin graft.

Intrathymic alloantigen-mediated, tolerant, completely major histocompatibility complex-mismatched mouse hearts are specifically rejected by adoptively transferred anti-class I L(d+)-specific 2C cells.

BACKGROUND: Tolerance to cardiac allografts can be induced in mice and rats by the injection of donor alloantigen into the thymus in combination with a CD4 T-cell-depleting antibody. CD8(+) cells in these animals are hyporesponsive to graft-specific alloantigens. Most of the CD8(+) T cells in the transgenic 2C mouse express a T-cell receptor specific for the class I major histocompatibility complex L(d+) locus. This study was designed to determine whether the adoptive transfer of these 2C T cells could precipitate rejection of a tolerant, completely major histocompatibility complex-mismatched L(d+) or L(d-) heart. METHODS: C57BL/6 mice (L(d-)) were given 10 x 10(6) cells of BALB/c (L(d+)) or dm2 (BALB/c background lacking L(d) [L(d-)]) splenocytes intrathymically and GK1. 5 (10 mg/kg) intraperitoneally. Twenty-one days later, BALB/c or dm2 hearts were transplanted. On the day of transplantation or after long-term allograft acceptance, recipients received naive 2C cells or 2C cells sensitized by in vitro mixed lymphocyte culture with BALB/c (L(d+)). RESULTS: Mean survival time of BALB/c cardiac allografts in untreated C57BL/6 mice was 7.3 days, although 73% of the mice that were pretreated with BALB/c splenocytes IT plus GK1.5 accepted the donor antigen-specific heart allografts indefinitely. All recipients that were pretreated with the intrathymic plus GK1.5 and that were injected with naive 2C cells at the time of heart transplantation experienced rejection of the BALB/c (L(d+)), but not the dm2 (L(d-)) hearts. In contrast, naive 2C cells could not reject tolerant (>30 days acceptance) BALB/c (L(d+)) hearts. 2C cells sensitized in vitro against L(d) were able to reject established BALB/c hearts but could not reject the L(d-) dm2 hearts. CONCLUSIONS: L(d)-specific 2C T-cell receptor transgenic T cells that are adoptively transferred to recipients will precipitate the rejection of accepted hearts that express class I L(d+) in mice rendered tolerant by an intrathymic injection of alloantigen plus anti-CD4 monoclonal antibodies.

Brief cyclosporine treatment prevents intrathymic (IT) tolerance induction and precipitates acute rejection in an IT rat cardiac allograft model.

BACKGROUND: Intrathymic (IT) alloantigen combined with administration of rabbit anti-rat anti-lymphocyte serum (ALS) intraperitoneally induces donor-specific tolerance to rat cardiac transplants. The purpose of this study was to examine the effect of a brief course (4 days) of cyclosporine (CsA) on the development of IT tolerance. METHODS: Buffalo (BUF) (RT1b) rats were given 25x10(6) fully MHC-mismatched Lewis (LEW) (RT1l) splenocytes by IT injection plus 1.0 ml of ALS intraperitoneally. Twenty-one days later, IT donor-specific LEW (group 1) or third-party (ACI, RT1a) (group 2) hearts were heterotopically transplanted to the abdominal aorta A third group of BUF (group 3) were given daily CsA (10 mg/kg) by oral gavage for 4 days before administration of IT LEW cells and ALS. Rejection as defined by the cessation of a palpable heartbeat was confirmed by histology. Cytokine profiles of allografts from all groups were then analyzed using a multi-probe RNase protection assay. RESULTS: Sixty-seven percent of IT/ALS-treated BUF recipients not pretreated with CsA accepted LEW heart grafts for greater than 90 days. However, 86% of animals treated with CsA for 4 days before IT injection and ALS rejected allografts at 10.7+/-3.2 days. Third-party allografts (ACI) were uniformly rejected (7.0+/-0.0 days). Histology confirmed cellular rejection in CsA-treated allografts and cytokine analysis detected increased interleukin (IL)-3, IL-5, and tumor necrosis factor-alpha when compared to increased IL-2 and interferon-gamma in rejecting untreated controls. CONCLUSIONS: CsA can prevent the induction of intrathymic alloantigen tolerance. These results support the development of a CsA-sensitive, but IL-2-independent, active regulatory mechanism after intrathymic exposure to donor-specific alloantigen and depletion of mature peripheral T cells.

[Total intravenous anesthesia for two patients complicated with myotonic dystrophy].

Total intravenous anesthesia with propofol, fentanyl and ketamine (PFK) was given to two patients complicated with myotonic dystrophy. Case-1: A 42-year-old female underwent a hemithyroidectomy. Anesthesia was induced slowly with intravenous ketamine 20 mg and propofol 60 mg. Her tracheal intubation was performed smoothly without any muscle relaxants. Anesthesia was maintained with propofol infusion of 5 mg.kg-1.h-1, ketamine infusion of 0.3 mg.kg-1.h-1 and fentanyl 200 micrograms in total. She regained consciousness 20 minutes after the end of propofol infusion, and 15 minutes later, her trachea was extubated without any troubles. Case-2: A 41-year-old female underwent a removal of left parotid tumor. Anesthesia was induced slowly with ketamine 40 mg and propofol 100 mg intravenously. Anesthesia was maintained with propofol infusion of 5-10 mg.kg-1.h-1, ketamine infusion of 0.5 mg.kg-1.h-1 and fentanyl 350 micrograms in total. No muscle relaxant was used through the surgical procedure. Emergence from anesthesia was observed 10 minutes after the end of propofol infusion and her trachea was extubated. When a nasogastric tube was pulled out, her respiration stopped suddenly and she was intubated again only for two hours without any troubles. In both cases their serum CPK levels and rectal temperatures were very stable. PFK method would be a choice for patients with myotonic dystrophy.

Detection of capillary protein leakage by glucose and indocyanine green dilutions during the early post-burn period.

Overestimation of the plasma volume determined by the indocyanine green (ICG) dilution method (PV-ICG) may occur after burns, since this dye has the potential of extravasation in the presence of the capillary protein leakage. Assuming that the initial distribution volume of glucose (IDVG) consistently indicates the extracellular fluid volume of highly perfused organs including plasma, overestimation of the PV-ICG can be detected by a higher PV-ICG/IDVG ratio. The present study was designed to test whether a higher PV-ICG/IDVG ratio is observed within 24 h post-burn compared to the subsequent days. Ten severely burned adult patients admitted to the ICU were studied through the 2nd post-burn day. The daily IDVG and PV-ICG were calculated using a one compartment model by simultaneous administration of glucose, 5 g, and ICG, 25 mg. Although the IDVG increased on the 1st post-burn day (p < 0.05), the PV-ICG remained unchanged. The PV-ICG/IDVG ratio within 24 h post-burn was significantly higher than that on the 1st post-burn day (p < 0.01). Results indicate that overestimation of the PV-ICG can occur within 24 h post-burn and suggest that simultaneous measurement of the IDVG and the PV-ICG would help predict the generalized capillary protein leakage after burns.

In vivo assessment of droperidol-induced bronchial relaxation in dogs using a superfine fibreoptic bronchoscope.

Droperidol has been reported to cause bronchodilatation but its mechanism(s) of action is unknown. We have evaluated the spasmolytic effect of droperidol on histamine- and serotonin (5-HT)-induced bronchoconstriction in dogs. Bronchial cross-sectional area was assessed with a superfine fibreoptic bronchoscope. Twenty-eight mongrel dogs were allocated randomly to one of two groups (histamine and 5-HT) to receive either histamine or 5-HT to induce bronchoconstriction. Changes in bronchial cross-sectional area were presented as percentage of basal bronchial cross-sectional area. Continuous i.v. infusion of histamine 500 micrograms kg-1 h-1 or 5-HT 500 micrograms kg-1 h-1 decreased percentage bronchial cross-sectional area by 46.4 (14.3)% or 68.9 (13.7)%, respectively. In both groups, droperidol reversed bronchoconstriction in a dose-dependent manner. In the histamine but not in the 5-HT group, plasma adrenaline and noradrenaline concentrations increased significantly after i.v. droperidol. In addition, propranolol antagonized droperidol-induced relaxation in the histamine but not in the 5-HT group. Our data indicate that the spasmolytic effect of droperidol on canine airway was caused, at least in part, by both catecholamine releases and 5-HT receptor antagonism.

Measurement of bronchodilatation using a superfine fibreoptic bronchoscope.

In this study, we report the development and accuracy of a direct technique to measure airway calibre using a superfine fibreoptic bronchoscope. Ten mongrel dogs were anaesthetized with pentobarbitone and the trachea intubated with a tracheal tube; the small lumen of the tube allowed passage of a superfine fibreoptic bronchoscope (od 2.2 mm). Bronchial cross-sectional area and airway pressure were recorded continuously and dynamic pulmonary compliance and airway resistance calculated. The dogs were allocated to one of two groups. In the first group (six dogs), bronchoconstriction was induced with histamine 10 micrograms kg-1 i.v. and 500 micrograms kg-1 h-1 c.i.v. Thirty minutes later, adrenaline 0-0.4 mg kg-1 was given i.v. Bronchial cross-sectional area, dynamic pulmonary compliance and airway resistance were assessed simultaneously. In the second group, 0.9% saline was given 30 min after placement of the superfine fibreoptic bronchoscope and 10 min later atropine 0.1 microgram kg-1 was administered. In the first group, histamine decreased mean percentage bronchial cross-sectional area by 49.2 (SD 11.5) %, reduced dynamic pulmonary compliance from 32.1 (12.6) to 22.3 (5.2) ml cm H2O-1 and increased airway resistance from 39.1 (11.6) to 57.2 (10.2) cm H2O litre-1 s-1. Adrenaline produced a dose-dependent increase in percentage bronchial cross-sectional area and dynamic pulmonary compliance to 119.4 (31.3)% and 27.4 (5.5) ml cm H2O-1, respectively, and a decrease in airway resistance to 43.9 (7.2) cm H2O litre-1 s-1. There were significant correlations between percentage bronchial cross-sectional area and dynamic pulmonary compliance (r = 0.720, P < 0.0001) and airway resistance (r = 0.727, P < 0.0001). Atropine 0.1 mg kg-1 increased basal bronchial cross-sectional area to 137.5 (16.9) %. These data indicate that adrenaline reversed histamine- and pentobarbitone-induced bronchoconstriction.

[Effect of pirenzepine on gastric secretion during anesthesia and surgery].

Effect of pirenzepine on gastric secretion during anesthesia and surgery was evaluated in 46 surgical patients ranged in age from 18 to 68 years. The patients underwent orthopedic, ophthalmic, ENT, plastic or non-abdominal general surgery under neuroleptanesthesia except two patients who had enflurane or isoflurane anesthesia. They received either pirenzepine 10 mg, 20 mg or the combination of pirenzepine 10 mg and famotidine 20 mg intravenously just before the induction of anesthesia. Volume and acidity of gastric juice were measured for 3 hours after the administration of these agents. Decrease in volume and acidity of gastric juice after pirenzepine 10 mg as well as after pirenzepine 20 mg continued for more than 3 hrs after the administration of the agents. Efficacy of the combination of pirenzepine and famotidine on gastric secretion was more prominent than that of pirenzepine alone in a double dose.

[Excessive nitrous oxide exhalation by postoperative patients in the recovery room].

Expired nitrous oxide from patients in the recovery room is considered to be the major source of air pollution. We measured expired concentrations of nitrous oxide in three patients and three volunteers. After only 5 minute inhalation of 50% nitrous oxide, it took over 2 hours for exhaled N2O concentration to decrease to 25 ppm in volunteers and after 30 minute inhalation, it took over 4 hours. The patients inhaled 50% nitrous oxide for 60, 165, 150 minutes, respectively and all patients expired nitrous oxide, the concentrations of which exceed 100 ppm over 3 hours. As to the patient who inhaled nitrous oxide for 150 minutes, expired nitrous oxide over 25 ppm was detected 10 hours after the end of anesthesia, and it was 4 ppm even after 20 hours. Any personnel including anesthesiologists and nurses working in the operating room can be exposed to high concentrations of nitrous oxide exceeding the permissible limit of 25 ppm, whenever they take care closely of their patients. We do not have any effective measures to protect us from this kind of air pollution except employing total intravenous anesthesia.

[Interleukin-2 (IL-2) in active pulmonary tuberculosis].

To clarify the precise of cellular immunity mechanism in pulmonary tuberculosis, we investigated the amount of IL-2 in patients with untreated active pulmonary tuberculosis. When serum adenosine deaminase (ADA) activity was examined using enzyme assay, an abnormally high level was observed in all patients (29.0 + 11.6 IU/ml, mean + SD; 4.5-17.8, normal range). Likewise, the level of serum-soluble interleukin-2 receptor (IL-2R) measured by ELISA showed abnormal high level in all patients (844.3 + 584.8 IU/ml; 80-300, normal range). When stimulated using PHA, the peripheral lymphocyte's ability to produce IL-2 revealed no difference between control subjects and patients. It was, however, noted that the lymphocytes of the patients significantly suppressed IL-2 responsiveness when compared to the control subjects (P less than 0.05). The serum IL-2 concentration measured using RIA could not be detected in any of the patients as was the same for control subject. All of the above mentioned results suggest that T-cell activation which caused increment in serum ADA activity and soluble IL-2R occurred in active pulmonary tuberculosis. The suppressed IL-2 responsiveness in the peripheral lymphocytes of patients proposes the possibility of soluble IL-2R reduction by the negative feedback mechanism in IL-2-sensitive lymphocytes.

Susceptibility of hepatitis B virus to disinfectants or heat.

Using direct chimpanzee inoculation as an assay method, we tested the abilities of the following chemical or physical treatments to inactivate hepatitis B virus in human plasma: 1% aqueous glutaraldehyde at 24 degrees C for 5 min, 0.1% aqueous glutaraldehyde at 24 degrees C for 5 min, 80% ethyl alcohol at 11 degrees C for 2 min, and heat at 98 degrees C for 2 min. All treatments were shown to be effective, indicating that the resistance level of the hepatitis B virus is not extreme.