Prolonged survival with FK778 (malononitrilamide) monotherapy after small bowel transplantation: a large animal study.

Malononitrilamide 715 (FK778) is a new class of immunosuppressant, derived from the active metabolite of leflunomide A77 1726. We investigated the efficacy of two different immunosuppressive induction protocols with tacrolimus plus FK778 followed by FK778 monotherapy. In a swine model of small bowel transplantation, we observed three groups, divided by different therapy regimens: group 1 (n = 5): no immunosuppressant (control group); group 2 (n = 10): oral tacrolimus (from postoperative day [POD] 0 to 30) and FK778 (from POD 0 to 60); group 3 (n = 8): oral tacrolimus, as group 2, and FK778 (from POD 7 to POD 60). Median survival was 11, 60, and 21 days in groups 1, 2, and 3, respectively. In group 1 all animals died of acute rejection; in group 2 the causes of death were technical complication (n = 1) and sepsis (n = 1); in group 3, one animal died from obstruction, two from pneumonia, one from peritonitis, one from sepsis. Group 2 accounted for 0.5 infection episode/animal versus 0.62 in group 3 (P < .05). Acute rejection was absent or mild in 66% and 75% of group 3 and 2 biopsies, respectively (P < .05). The D-xylose absorption curves from groups 2 and 3 were similar to those of the nontransplanted healthy animals. In conclusion, FK778 monotherapy after a consistent induction period with tacrolimus combined immunosuppression is able to extend survival and preserve optimal absorptive capacity of the small bowel allograft in our pig model. The association of tacrolimus and FK778 from day 1, compared to the delayed administration of FK778 from day 7, results in a significant reduction of infections and postoperative complications.

FK778 does not impair intestinal allograft absorption in a preclinical model of total small bowel transplantation.

Malononitrilamide 715 (FK778), a new low-molecular weight immunosuppressant, inhibits both T-cell and B-cell function by acting on the pathway for de novo pyrimidine biosynthesis. Pyrimidines are important for intestinal trophism; their inhibition may predispose to metabolic and functional impairments, such as diarrhea and malabsorption. In this study we assessed the absorptive capacity of intestinal allografts in a large-animal model of small bowel transplantation (SBTx) in pigs chronically treated with FK778. Ten outbred pigs underwent total orthotopic SBTx. Immunosuppression consisted of oral tacrolimus (trough levels 5-15 ng/mL) and oral FK778 (4 mg/kg per day) administered for 60 days. The D-xylose absorption test was performed at day 60 to evaluate carbohydrate absorption. Results were compared to normal controls. Eight of the 10 animals were alive and in good condition at day 60. All of their allografts were free of rejection. The animals had a mean maximal weight loss of 6.4% during the study period; the final weight was comparable to the initial weight (P > .05). Diarrhea was present in all animals (mean 16% of postoperative days). The D-xylose curves showed that absorption in the transplanted animals at day 60 was similar to that in the untreated controls (P > .05). The absence of differences was confirmed by the statistical analysis. In conclusion, our preclinical study in pigs showed that chronic treatment with FK778 in combination with tacrolimus did not impair carbohydrate absorption by the allograft after SBTx.

FK778 and tacrolimus combination therapy to control acute rejection after pig intestinal transplantation.

Malononitrilamide 715 (FK778) is a new class of low molecular weight immunosuppressant. Experimental studies in heart, liver, and kidney transplantation have shown a strong synergism when FK778 is used in combination with tacrolimus and when its administration is delayed by 7 days after the transplant. Following this indication, in a swine model of orthotopic small bowel transplantation (SBT), we assessed the efficacy of combined low dose tacrolimus and FK778 administered from day 0 or day 7. The entire small bowel was replaced in 16 piglets: group 1 (n = 5), no immunosuppression; group 2 (n = 6) oral tacrolimus to maintain whole blood trough levels between 5 and 15 ng/mL plus FK778 4 mg/kg per day; group 3 (n = 6) oral tacrolimus as in group 2 plus FK778 4 mg/kg per day administered after a 7-day delay posttransplant. The median survival was 8 days in group 1, 60 days in group 2, and 13 days in group 3. The differences between group 2 and 1 and between group 2 and 3 are statistically significant. Three episodes of major bacterial infection were detected in both group 2 and 3 (0.5 episode/animal). The infectious-related mortality was 0% in group 2 and 50% in group 3 (P < .05). Acute cellular rejection was absent or mild in all group 2 and 3 stomal biopsies. In conclusion, combining tacrolimus and FK778 allowed prolonged survival after SBT in swine when FK778 was started at the time of SBT. The delayed administration of FK778 resulted in a high incidence of lethal infectious complications.

Early outcome of different steroid-free regimens in small bowel transplantation: a large-animal study.

The intestine is a highly immunogenic organ that requires heavy immunosuppression (IS); therefore corticosteroid withdrawal after clinical small bowel transplantation (SBT) has not been standardized. In this study, we compared different immunosuppressive regimens (none with steroid or induction treatment) in a SBT pig model. Large White unrelated piglets were transplanted and divided into four groups as follow: group 1 (n = 3): no IS; group 2 (n = 10): IS with tacrolimus only; group 3 (n = 10): IS with tacrolimus and mycophenolate mofetil; group 4 (n = 5): IS with tacrolimus and rapamycin. Follow-up time was 30 days. All IS drugs were given orally; tacrolimus whole blood levels ranged between 5 and 15 ng/mL in all groups except for group 2 whose tacrolimus whole blood levels ranged between 15 and 25 ng/mL. Group 1 pigs died of graft acute rejection (ACR) after a median of 12 days. Overall survival in groups 2, 3, and 4 at day 30 was 40%, 80%, and 60%, respectively. Biochemical parameters, including glycemia and cholesterol, were into the normal range with no significant differences between groups. At the end of the study, one animal in group 2 and another one in group 4 showed histological signs of moderate to severe ACR. The incidence of infection was higher in group 2 (2.1 episodes/pig) compared to group 3 (1.25) and group 4 (1.6). This large-animal study demonstrates that tacrolimus-based IS without corticosteroids allows, in the early postoperative period (30 days) after SBT, good survival rates without an increased risk in the incidence of rejection.

Incidence of graft rejection in small bowel transplanted pigs after immunosuppression withdrawal.

As intestinal grafts require heavy immunosuppression, there are no reports of immunosuppression withdrawal after clinical small bowel transplantation. In this large-animal study, we investigated the occurrence of graft rejection in intestinal-transplanted pigs after withdrawal. Large-White unrelated piglets were transplanted and divided in three groups: group 1 (n = 5), intestinal transplantation (ITx) with no immunosuppression; group 2 (n = 7), Itx and 60 days of treatment with tacrolimus and mycophenolate mofetil; group 3 (n = 5), Itx and donor bone marrow infusion (DBMi) and 60 days of treatment with tacrolimus and mycophenolate mofetil. Follow-up time after withdrawal was 120 days. Group 1 pigs died of graft acute cellular rejection (ACR) after a median of 11 days. In group 2, two pigs died of ACR-related infection and another two of ACR within 90 days. The remaining three animals (43%) were sacrificed at day 180, and their grafts showed no signs of ACR. In group 3, two pigs died of ACR-related infection and one of graft versus host disease within 80 days; at day 180 the two surviving animals showed signs of chronic rejection in the allograft. This study demonstrates that total withdrawal after ITx is followed by sudden and lethal ACR (or ACR-related infection) in more than 50% of the recipients. When a tolerance-inducing strategy as DBMi is applied, lethal graft versus host disease may also occur. In group 3, the intestinal allograft, to which the recipients were partially tolerant, developed chronic rejection that was probably associated with a decline with time of donor-leukocytes chimerism, as recently demonstrated in rats.

[Laparoscopic harvest of the jejunal free flap for cervical esophageal reconstruction]. / Il prelievo laparoscopico di un lembo libero di digiuno per la ricostruzione dell' esofago cervicale.

The jejunal free flap is a standard technique in the reconstruction of hypopharyngeal and cervical esophageal defects. Conventional harvesting of the jejunal segment is performed with midline open laparotomy, which is associated with complications including prolonged ileus, abdominal pain, wound infection or dehiscence. Laparoscopic resection of the small intestine is a well documented surgical technique. Two different methods of laparoscopic harvest of a jejunal autografts for their cervical implantation have been already described. In both cases, low complication rate and better postoperative course have been observed in the patients treated. During the last 10 years, we have performed 43 circumferential pharyngoesophageal resection for advanced hypo-pharyngeal cancer followed by reconstruction with a free flap of jejunum. All but one the jejunal segments have been harvested with conventional open laparotomy. In the last patient of this group, laparoscopic harvest of the jejunal segment has been successfully performed. In this paper, we describe the laparoscopic technique used and we compare the postoperative course of this patient with those of the patients treated with conventional technique.

Efficacy of malononitrilamide FK778 in a preclinical model of small bowel transplantation.

In a swine model of orthotopic small bowel transplantation, we assessed the efficacy of combined immunosuppressive therapy with low-dose tacrolimus plus FK778, compared with high-dose tacrolimus monotherapy. The small bowel was replaced in 23 piglets: group 1 (n = 5), no immunosuppression; group 2 (n = 12), oral tacrolimus to maintain whole blood trough levels between 15 and 25 ng/mL; and group 3 (n = 6), oral FK778 4 mg/kg/d, plus oral tacrolimus to maintain whole blood trough levels between 5 and 15 ng/mL. Follow-up time was limited to 60 days. Overall survival rates at 30 and 60 days were 0% and 0% in group 1, 30% and 0% in group 2, and 66% and 66% in group 3, respectively. The median survival time was 11 days in group 1, 28 days in group 2, and more than 60 days in group 3. The differences between groups 3 and 1 and between groups 3 and 2 were statistically significant. The numbers of major bacterial infections were 19 in group 2 (1.9 episodes per animal) and 3 in group 3 (0.75 episodes per animal). The infectious-related mortality rate was 70% in group 2 (7 cases) and 0% in group 3 (P < .05). Acute cellular rejection was absent or mild in 85% of group 2 stomal biopsy specimens and in 100% of group 3 biopsy specimens. In conclusion, combination therapy of low-dose tacrolimus is potentiated by FK778 to prevent acute cellular rejection and prolong small bowel transplant survival in pigs.

Teichomycins, new antibiotics from Actinoplanes teichomyceticus nov. sp. II. Extraction and chemical characterization.

The teichomycins are new antibiotics produced by Actinoplanes teichomyceticus nov. sp. Teichomycin A1 is a phosphorus-containing antibiotic, active in vitro and in vivo against gram-positive bacteria, and active only in vitro against gram-negative bacteria. Teichomycin A2 is a chlorine-containing antibiotic, active in vitro and in vivo against gram-positive bacteria. Isolation, purification and physical and chemical properties of the two antibiotics are reported. Teichomycin A1 is a member of the phosphoglycolipid class of antibiotics while teichomycin A2 is related to the group of the glycopeptide antibiotics.

Teicoplanin, antibiotics from Actinoplanes teichomyceticus nov. sp. V. Aromatic constituents.

Oxidative and hydrolytic degradation reactions were carried out on teicoplanin in order to characterize the aromatic portion of the molecule and relate it to the other members of the class of glycopeptide antibiotics. Seven aromatic rings, obtained as triphenyl ether, diphenyl ether, and diphenyl moieties after oxidation nd hydrolysis of teicoplanin, were identified. They are present in teicoplanin as aromatic amino acids and constitute the peptidic part of the molecule. The diphenyl ether and diphenyl moieties, which were isolated both as esters after oxidation and as alpha-amino acids after acid hydrolysis clearly indicate the nature of the corresponding amino acids in teicoplanin. The triphenyl ether moiety, which was isolated only as ester, allows the hypothesis that the corresponding amino acids are the same as those of the other glycopeptide antibiotics.

The mechanism of action of zopiclone.

The mechanism of action of the cyclopyrrolone hypnotic drug zopiclone involves allosteric modulation of the GABAA receptor. Zopiclone displaces the binding of [(3)H]-flunitrazepam with an affinity of 28 nM, and enhances the binding of the channel blocker [(35)S]-TBPS. The binding of zopiclone, unlike that of hypnotic benzodiazepines, is not facilitated by GABA. Zopiclone does not distinguish between GABAA receptors containing different alpha-subunits (BZ(1) and BZ(2) phenotype). Studies with protein-modifying agents (eg diethylpyrocarbonate) and photoaffinity labelling suggest that cyclopyrrolones bind to a domain on the GABAA receptor different from the benzodiazepine binding domain. The consequence of this interaction with the GABAA receptor is to potentiate responses to GABA, as can be demonstrated by electrophysiological methods. Subchronic treatment of mice with high doses of zopiclone does not produce the changes in sensitivity of the GABAA receptor that are observed with hypnotic benzodiazepines.

[Experimental kidney transplantation: a comparison between different models]. / Trapianto di rene sperimentale nel suino. Modelli a confronto.

AIM: Two different models of kidney transplantation have been compared using 3 different techniques. The kidney grafts were procured from living donors (laparoscopic or laparotomic technique) and from cadaveric donors. METHODS: Twenty-four outbred piglets (Large White, weight range 24-27 kg) underwent kidney transplantation. We divided the recipients into 2 groups with the following characteristics: group 1 (n=12) was represented by orthopic kidney recipients whose grafts were retrieved by laparoscopic or lapartomic technique from living unrelated donors; group 2 (n=12) was constituted by heterotopic kidney recipients whose grafts were retrieved by laparotomic technique from unrelated cadaveric donors. In both groups, Grogoire-Lich technique and Politano-Laedbetter technique were used in order to perform ureteral-vescical anastomosis together with a new technique developed from our experience called Politano-Laedbetter modified. All transplanted pigs underwent double immunosoppressive steroid therapy (tacrolimus and micofenolate mofetil). The pigs were observed for 60 days. RESULTS: The survival rates in group 1 and in group 2 were 75% (n=9) and 66% (n=8), respectively. No significative differences were noted in length of operative time, creatinemia and ureamia levels in both study groups. The Gregoire-Lich technique was associated with a higher rate of complications. CONCLUSION: Two different experimental models of kidney transplantation are feasible in pigs. The classic technique could be combined with the orthopic one based on the type of study needed.

Effect of Thymomodulin on luteinizing hormone, prolactin and testosterone in male rats.

The effect of Thymomodulin (TMD), a calf thymus derivative, on luteinizing hormone, prolactin and testosterone was studied in male rats after acute and chronic treatment. The results showed that the stimulatory action on prolactin and testosterone secretion after acute (prolactin) or one month chronic (testosterone) treatments completely vanished during six month chronic administration. No effect was observed on luteinizing hormone after acute or chronic treatment.

Pharmacological studies on zopiclone.

Zopiclone (RP 27 267) is an hypnotic with a chemical structure different from that of the benzodiazepines (BZD) or barbiturates. Studies of zopiclone in classical psycho-pharmacological tests, in comparison with BZD and barbiturates, have shown that it exhibits the five main types of activity considered as characteristic of the pharmacological profile of BZD and partly of that of barbiturates (anticonvulsant, myorelaxant, antiaggressive, sedative-hypnotic and 'anticonflict'). However, like BZD, zopiclone differs from barbiturates by a high safety margin. Electrophysiological studies performed in cats have shown that zopiclone induces modifications in sleep-wakefulness pattern which are close to those observed with BZD, specially with nitrazepam. Moreover, zopiclone increases the threshold for arousal by reticular formation stimulation, rather more than nitrazepam, but with a shorter duration of action. The short duration of action of zopiclone has been demonstrated in other species using different tests.

Pharmacological studies on zopiclone.

Zopiclone (RP 27 267) is an hypnotic with a chemical structure different from that of the benzodiazepines (BZD) or barbiturates. Studies of zopiclone in classical psychopharmacological tests, in comparison with BZD and barbiturates, have shown that it exhibits the five main types of activity considered as characteristic of the pharmacological profile of BZD and partly of that of barbiturates (anticonvulsant, myorelaxant, antiaggressive, sedative-hypnotic and 'anticonflict'). However, like BZD, zopiclone differs from barbiturates by a high safety margin. Electrophysiological studies performed in cats have shown that zopiclone induces modifications in sleep-wakefulness pattern which are close to those observed with BZD, specially with nitrazepam. Moreover, zopiclone increases the threshold for arousal by reticular formation stimulation, rather more than nitrazepam, but with a shorter duration of action. The short duration of action of zopiclone has been demonstrated in other species using different tests.

The combined treatment of human peripheral blood mononuclear cells with thymolymphotropin and interleukin 2 increases PPD-driven T-cell proliferation and IL-2 induced cellular cytotoxicity against HIV-infected cells.

Two in vitro systems (the DNA synthetic response to mycobacterial antigens and cytotoxicity against lymphoid cells) were used to analyse the effect of thymolymphotropin (TLT) on peripheral blood mononuclear cells (PBMC). Purified protein derivative of mycobacteria (PPD)-driven T-cell proliferation in low-responder donors was increased by the combined treatment with TLT and suboptimal doses of recombinant interleukin 2 (IL-2). Similarly, the activities of natural killer (NK) cells and lymphokine-activated killer (LAK) cells have been enhanced in PBMC cultures pretreated with TLT. Also, TLT showed an enhancing effect on the development of LAK cells capable of lysing Epstein-Barr virus (EBV)-transformed B-lymphocytes infected or uninfected with the human immunodeficiency virus (HIV).

RP 59037 and RP 60503: anxiolytic cyclopyrrolone derivatives with low sedative potential. interaction with the gamma-aminobutyric acidA/benzodiazepine receptor complex and behavioral effects in the rodent.

This study describes the pharmacological properties of two novel cyclopyrrolone derivatives, RP 59037 [2-(7-chloro-2-naphthyridin-1,8-yl)-3-(5- methyl-2-oxohexyl)isoindolin-1-one] and RP 60503 [2-(7-chloro-2-naphthyridin-1,8-yl)isoindolin-1-yl-4- acetamidobutyrate], in the rodent. These compounds possess high affinity for the benzodiazepine binding site on the gamma-aminobutyric acidA receptor in rat cerebrocortical membranes with Ki values of 0.98 nM (RP 59037) and 1.16 nM (RP 60503). Neither compound discriminates between the putative benzodiazepine BZ1 and BZ2 binding site subtypes present in the rat cerebellum and hippocampus, respectively. Both compounds protect mice against pentylenetetrazole-induced seizures with ID50 values of 0.21 mg.kg-1 p.o. (RP 59037) and 5.96 mg.kg-1 p.o. (RP 60503). The two compounds displayed a restricted anticonvulsant profile compared to diazepam and, in this respect, resembled the pyrazoloquinoline partial agonist, CGS 9896. RP 59037 and RP 60503 were active in two rat models predictive of anxiolytic drug action, a modified Geller-Seifter conflict paradigm (minimal effective dose, 0.33 mg.kg-1 p.o. for RP 59037 and 5 mg.kg-1 p.o. for RP 60503) and the elevated plus maze (minimal effective dose, 0.33 mg.kg-1 p.o. for RP 59037 and 5 mg.kg-1 p.o. for RP 60503). Only very low activities were observed in tests of sedative or myorelaxant effects (ED50 > 50 mg.kg-1 p.o.). It is concluded that the two cyclopyrrolones possess a dissociated behavioral profile, displaying potent anxiolytic and anticonvulsant properties with little or no sedative or myorelaxant effects. Although both compounds appear to be partial agonists at their allosteric recognition site on the gamma-aminobutyric acidA receptor, RP 60503 seems to be more dissociated than RP 59037, which would be compatible with it having lower intrinsic activity. This difference is reflected in a higher receptor occupancy requirement for activity, and a smaller modulatory effect on the binding of t-[35S]butylbicyclophosphothionate.

CD4-8- T-cells increase in MRI/lpr mice treated with thymic factors.

The in vivo effect of thymic factors on immature lymphocytes was analysed in MRL/lpr mice. This strain carries a genetic defect that causes during their life cycle a block of T-cell differentiation and abnormal proliferation of CD4-8- (double-negative, DN) T-lymphocytes. In vivo administration of four preparations of thymic factors, thymopentin (TP-1), thymopoietin (TP-5), thymolymphotropin (TLT), and thymomodulin (TMD) into young (2-month-old) MRL/lpr mice induced a significant increase of DN T-cells both in the thymus and in the peripheral lymph nodes, with a concomitant decrease of double-positive (DP) T-cells in the thymus and of single-positive (SP) T-cells in the lymph nodes. The level of DNA fragmentation measured as propidium iodide fluorescence was increased in the thymus population of young mice and in the lymph node population of old mice treated with TLT. SCID mice transplanted with lymph node cells from MRL/lpr donors (MRL-->SCID) developed graft versus host (GvH) reaction due to the activation of MRL CD8+ alloreactive T-cells. This model was used to analyse the effect of TMD/TLT in vivo on MRL cell proliferation and expansion; in fact, spleen cells from MRL-->SCID mice after treatment with TMD/TLT showed an increased cell proliferation, and an expansion of DN T-cells with a concomitant decrease of SP cells (both CD4+ and CD8+ cells). Decreased SP cell numbers in this context could explain why TMD/TLT treatment of SCID mice engrafted with MRL cells increased their survival compared to untreated MRL-->SCID mice.