Sudden late onset of clozapine-induced agranulocytosis

OBJECTIVE: To report a patient who suddenly developed agranulocytosis after long-term clozapine therapy. CASE SUMMARY: A 41-year-old white man suddenly developed agranulocytosis after 89 months of nearly continuous clozapine therapy. During this time, which included the addition of risperidone to the treatment regimen, his white blood cell (WBC) and granulocyte counts remained stable. One week after having stable hematologic counts, the patient suddenly developed agranulocytosis. WBC and granulocyte counts returned to baseline shortly after discontinuation of all medications and administration of sargramostim. DISCUSSION: The main factor limiting the use of clozapine as a first-line agent in mentally ill patients is the risk of agranulocytosis. Although the greatest risk of developing this adverse reaction is during the initial 6-month exposure, clozapine-induced agranulocytosis continues to pose a risk after years of exposure. Current product labeling requires weekly WBC and granulocyte monitoring for the first 6 months of treatment with clozapine, which may be decreased to biweekly monitoring after 6 months. Based on the sudden and late onset of agranulocytosis in our patient, clinicians may consider opting for weekly monitoring of hematologic function for patients on long-term clozapine therapy. The likelihood that clozapine was the cause of the agranulocytosis was rated possible according to the Naranjo probability scale. CONCLUSIONS: Clinicians must remain vigilant to trends in WBC and granulocyte counts and may wish to consider weekly hematologic monitoring regardless of duration of clozapine therapy. Patient and treatment system compliance with the registries' protocol regarding WBC monitoring is instrumental in reducing morbidity and mortality rates associated with clozapine use.     

Cytotoxic activity in serum of patients with clozapine-induced agranulocytosis.

Studies were conducted on serum samples collected from 15 patients during the course of clozapine-induced agranulocytosis. During acute phases of agranulocytosis, serum was cytotoxic to peripheral polymorphonuclear neutrophils (PMNs), as indicated by complement-dependent suppression of postphagocytosis respiratory burst and by increased retention of trypan blue dye by test PMNs. Cytotoxicity was removed by adsorption with allogeneic PMNs, was attenuated by antibody to immunoglobulin M but not by antibody to immunoglobulin G antigen-binding fragment, was not dialyzable, and was partially removed by 2-mercaptoethanol and dialysis. Three patients in a longitudinal study all had perturbed postphagocytosis respiratory burst 20 days before agranulocytosis developed. In all patients cytotoxicity disappeared less than 40 days after treatment when the offending drug was discontinued. Trypan blue dye reactivity was similar when tested. At 20% of culture medium, all serum samples partially suppressed development of colony-forming units of granulocytes and macrophages (CFU-GM) in marrow cultures. At 40% of culture medium, agranulocytosis serum suppressed CFU-GM completely but did not inhibit development of colony-forming units of erythroblasts (CFU-E) or burst-forming units of erythroblasts (BFU-E). Addition of clozapine alone or to agranulocytosis serum neither enhanced nor suppressed toxicity to peripheral PMNs. Neither clozapine nor its toxic metabolic end-products directly produced equivalent cytotoxicity to cellular function or proliferation at 10(-5)mol/L. Serum from patients given clozapine who did not have agranulocytosis and samples from allogeneic normal subjects were not cytotoxic to test PMNs. Cytotoxicity was specific to granulocyte cell lines because development of CFU-GM was inhibited by agranulocytosis serum, whereas CFU-E and BFU-E were not similarly affected. Further studies are in progress to distinguish between immunogenic and non-immunogenic mechanisms.     

Failure of filgrastim to prevent severe clozapine-induced agranulocytosis

Although a highly effective medication, the usage of clozapine is limited mostly by its 2.7% incidence of neutropenia. It is often a treatment of last resort for patients with severe psychiatric illnesses, and therefore often the only medication to which a patient has responded. There has thus been a great deal of interest in ways to continue the medication in spite of emergent blood dyscrasias. There have been several reports documenting the successful continuation of clozapine in spite of neutropenia by adding granulocyte colony-stimulating factors such as filgrastim. This strategy was unsuccessful for a 63-year-old man, resulting in severe, prolonged agranulocytosis. Although a promising strategy for such refractory patients, its inherent dangers should not be underestimated.     

Insulin amyloid polymorphs: implications for iatrogenic cytotoxicity

Amyloid specific fluorescent probes are becoming an important tool for studies of disease progression and conformational polymorphisms in diseases related to protein misfolding and aggregation such as localized and systemic amyloidosis. Herein, it is demonstrated that using the amyloid specific fluorescent probes pFTAA and benzostyryl capped benzothiadiazole BTD21, structural polymorphisms of insulin amyloids are imaged in localized insulin-derived amyloid aggregates formed at subcutaneous insulin-injection sites in patients with diabetes. It is also found that pFTAA and BTD21 could discriminate structural polymorphisms of insulin amyloids, so called fibrils and filaments, formed in vitro. In addition, it is shown that insulin drug preparations used for treating diabetes formed various types of amyloid aggregates that can be assessed and quantified using pFTAA and BTD21. Interestingly, incubated pFTAA-positive insulin preparation aggregates show cytotoxicity while BTD21-positive aggregates are less toxic. From these observations, a variety of amyloid polymorphic structures with different cytotoxicities formed both in vivo and in vitro by various insulin preparations are proposed.

Structural polymorphism of insulin amyloids in vivo can be recognized using novel amyloid specific fluorescent probes, pFTAA and BTD21.     

Biologic effects of parathyroid hormone metabolites: implications for renal bone disease.

PTH fragments consisting of the C-terminal portion of the molecule may have biologic effects and may modify the actions of PTH. Evidence for the presence of a C-terminal PTH receptor further supports a biologic role for such fragments. Because C-PTH fragments accumulate in patients with renal insufficiency, it is possible that they may contribute to renal bone disease. The precise role of circulating C-PTH fragments in the pathogenesis, diagnosis, and management of renal osteodystrophy, however, remains to be determined. Future studies of the biologic effects and regulation of these fragments may lead to better understanding of skeletal biology and may also improve our approach to the diagnosis and treatment of renal bone disease.     

A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy.

Poly(ethylenimine) (PEI) is a cationic macromolecule commonly used in gene transfer/therapy protocols with high transfection efficiency both in vitro and in vivo. PEI is also cytotoxic, but the molecular basis of its cytotoxicity is poorly understood. Here, we have demonstrated that branched (25 kDa) and linear (750 kDa) PEI can both induce membrane damage and initiate apoptosis in three clinically relevant human cell lines (Jurkat T cells, umbilical vein endothelial cells, and THLE3 hepatocyte-like cells). We have defined Phase I toxicity as early necrotic-like changes (30 min) resulting from compromised membrane integrity, assessed by considerable lactate dehydrogenase release and phosphatidylserine translocation from the inner plasma membrane to the outer cell surface. Phase II cytotoxicity (24 h) was due to activation of a "mitochondrially mediated apoptotic program," resulting from PEI-induced channel formation in the outer mitochondrial membrane. This led to the release of proapoptotic cytochrome c, subsequent activation of caspase 3, and alteration in mitochondrial membrane potential as a result of caspase translocation into the mitochondria. The reported observations have important implications for the design and execution of gene therapy protocols as well for controlling intracellular distribution of drugs with cationic-based polymer-delivery systems.     

体外培养大鼠胰岛分泌功能与利培酮和氯氮平及其代谢产物的干预效应

背景：非典型抗精神病药中氯氮平对糖代谢影响最大，利培酮影响较小，介于氯氮平、奥氮平和传统抗精神病药之间。目的：比较利培酮、氯氮平及其代谢产物去甲氯氮平和N-氧化氯氮平对体外培养的胰岛分泌胰岛素功能的影响，从而了解其对糖代谢的影响。设计：完全随机分组设计，对照实验。单位：武汉大学人民医院精神卫生中心。材料：实验于2003—9／2004—01在武汉大学口腔医院实验中心完成。选用清洁级健康雄性Wistar大鼠3只。方法：①采用经典的胶原酶消化法分离、纯化胰岛。②以含2g/L牛血清白蛋白和3．3mmoL／L葡萄糖的Hanks液每孔1mL，预孵育30min，弃上清。每6孔为一组，共5组：对照组、利培酮组、氯氮平组、去甲氯氮平组和N-氧化氯氮平组的孵育液均含1g/L二甲基亚砜、3．3mmoL/L或16．7mmoL／L的葡萄糖液1mL；利培酮组、氯氮平组、去甲氯氮平组、N-氧化氯氮平组的孵育液中另含1μmoL／L的利培酮、氯氮平、去甲氯氮平（DCLO）、N-氧化氯氮平（NCO）；各组有3孔继续孵育1h，另外3孔继续孵育4h；吸取上清液，保存于-20℃冰箱中待测。重复3次。采用放射免疫分析法测定上清液中基础胰岛素分泌量和糖刺激后胰岛素分泌量。③实验结果以中位数M（P25，P75）表示；数据间差异性测定采用Mann—Whitney非参检验法。主要观察指标：各组上清液中胰岛素分泌量比较。结果：①对照组孵育1和4h后糖刺激胰岛素分泌高于基础胰岛素分泌量[1.91（1．68—2．62），2．21（1．59～3，05）μU／IEQ；1．05（0．7l—1．15），1．65（1．16～1．84）,P〈0．05]，说明胰岛生物学功能良好。②利培酮组和N-氧化氯氮平组孵育和14h后，基础胰岛素分泌量和糖刺激后胰岛素分泌量与对照组差异不明显（P〉0．05）。氯氮平组孵育4h后，基础胰岛素分泌量低于对照组[1．65（1．16～1．84），1．08（0．88-1．20）μU／IEQ，P〈0．05]。去甲氯氮平组孵育1和4h后，糖刺激后胰岛素分泌量均明显低于对照组[1．15（0．84-1.32），1．91（1．68—2．62）μU／IEQ；1．08（0．62-1．331，2．21（1．59—3．05）μU／IEQ，P〈0．05，0．01]。结论：氯氮平影响基础胰岛素分泌量，其代谢产物去甲氯氮平影响糖刺激后胰岛素分泌量，而利醅酮不引起胰岛素分泌缺陷。     

体外培养大鼠胰岛分泌功能与利培酮和氯氮平及其代谢产物的干预效应

背景非典型抗精神病药中氯氮平对糖代谢影响最大,利培酮影响较小,介于氯氮平、奥氮平和传统抗精神病药之间.目的比较利培酮、氯氮平及其代谢产物去甲氯氮平和N-氧化氯氮平对体外培养的胰岛分泌胰岛素功能的影响,从而了解其对糖代谢的影响.设计完全随机分组设计,对照实验.单位武汉大学人民医院精神卫生中心.材料实验于2003-9/2004-01在武汉大学口腔医院实验中心完成.选用清洁级健康雄性Wistar大鼠3只.方法①采用经典的胶原酶消化法分离、纯化胰岛.②以含2 g/L牛血清白蛋白和3.3 mmoL/L葡萄糖的Hanks液每孔1 mL,预孵育30 min,弃上清.每6孔为一组,共5组对照组、利培酮组、氯氮平组、去甲氯氮平组和N-氧化氯氮平组的孵育液均含1 g/L二甲基亚砜、3.3 mmoL/L或16.7 mmoL/L的葡萄糖液1mL;利培酮组、氯氮平组、去甲氯氮平组、N-氧化氯氮平组的孵育液中另含1 μmoL/L的利培酮、氯氮平、去甲氯氮平(DCLO)、N-氧化氯氮平(NCO);各组有3孔继续孵育1 h,另外3孔继续孵育4h;吸取上清液,保存于-20℃冰箱中待测.重复3次.采用放射免疫分析法测定上清液中基础胰岛素分泌量和糖刺激后胰岛素分泌量.③实验结果以中位数M(P25,P75)表示;数据间差异性测定采用Mann-Whitney非参检验法.主要观察指标各组上清液中胰岛素分泌量比较.结果①对照组孵育1和4h后糖刺激胰岛素分泌高于基础胰岛素分泌量[1.91(1.68～2.62),2.21(1.59～3.05)μU/IEQ;1.05(0.71～1.15),1.65(1.16～1.84),P＜0.05],说明胰岛生物学功能良好.②利培酮组和N-氧化氯氮平组孵育1和4 h后,基础胰岛素分泌量和糖刺激后胰岛素分泌量与对照组差异不明显(P＞0.05).氯氮平组孵育4 h后,基础胰岛素分泌量低于对照组[1.65(1.16～1.84),1.08(0.88～1.20)μU/IEQ,P＜0.05].去甲氯氮平组孵育1和4 h后,糖刺激后胰岛素分泌量均明显低于对照组[1.15(0.84～1.32),1.91(1.68～2.62)μU/IEQ;1.08(O.62～1.33),2.21(1.59～3.05)μU/IEO,P＜0.05,0.01].结论氯氮平影响基础胰岛素分泌量,其代谢产物去甲氯氮平影响糖刺激后胰岛素分泌量,而利醅酮不引起胰岛素分泌缺陷.     

Antibiotic agranulocytosis: association with cephalothin and carbenicillin.

A 65-year-old woman developed agranulocytosis on two separate occasions following prophylactic administration of antibiotics before cardiac surgery. In the first leukopenic episode, large doses of cephalosporin derivatives were the only drugs implicated, and in the second, carbenicillin was believed responsible. Life-threatening septicemia occurred with Pseduomonas aeruginosa and later with Escherichia coli. Erythrocytes, platelets, and lymphocytes were not affected during these granulocytopenias. Bone marrow examination revealed an arrest of maturation in the granulocytic series. Review of the hematologic complications of cephalosporins, particularly agranulocytosis, suggests an interesting association between carbenicillin-induced neutropenia and previous administration of cephalosporins.     

The entero-insular axis: implications for human metabolism.

Incretins such as glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are intestinal hormones that are released in response to ingestion of nutrients, especially carbohydrate. They have a number of important biological effects, which include release of insulin, inhibition of glucagon and somatostatin, maintenance of beta-cell mass, delay of gastric emptying, and inhibition of feeding. These properties allow them to be potentially suitable agents for the treatment of type 2 diabetes (T2D). Incretin receptors are also present in other parts of the body including the brain, where their effects are beginning to be understood and their relevance to disorders of nutrition and ageing are being explored. There is currently a pandemic of obesity and diabetes, and existing treatments are largely inadequate in regard to efficacy as well as their ability to tackle important factors in the pathogenesis of T2D. There is increasing evidence that current treatments do not address the issue of progressive beta-cell failure in T2D. As obesity is the engine that is driving the epidemic of diabetes, it is disappointing that most treatments that succeed in lowering plasma glucose are also associated with weight gain. It is now well established that intensively treated T2D has a better outcome than standard treatment. Consequently, achieving better control of diabetes with lower HbA1c is the goal of optimal treatment. Despite the use of usual therapeutic agents in T2D, often in high doses and as combinations, such as metformin, sulphonylurea, alpha-glycosidase inhibitors, thiazolidinediones and a number of animal and human insulin preparations, optimal control of glycaemia is not achieved. The use of incretins as therapeutic agents offers a new approach to the treatment of T2D. Incretin metabolism is abnormal in T2D, evidenced by a decreased incretin effect, reduction in nutrient-mediated secretion of GIP and GLP-1 in T2D, and resistance to GIP. GLP-1, on the other hand, when administered intravenously in T2D is able to increase insulin secretion and improve glucose homeostasis. As GLP-1 has a very short half-life, due to rapid degradation by the enzyme dipeptidyl peptidase IV (DPPIV), analogues of GIP and GLP-1 that are resistant to the action of DPPIV have been developed and clinical trials have shown their effectiveness. Another novel agent, naturally resistant to DPPIV that is given by subcutaneous injection is a synthetic peptide called exenatide, has recently been approved for treatment of T2D in the USA. Efforts are underway to develop agents that can be given orally and include a DPPIV inhibitor that has been licensed for the treatment of T2D in the USA, and several other agents are undergoing clinical trials. Strategies to augment the biological actions of GIP and/or GLP-1 in T2D are expected to minimise weight gain, reduce hypoglycaemic episodes and prevent progressive beta-cell failure by increasing beta-cell mass. The optimal agent(s) that may mimic and replace the endogenous incretin effect is not fully known and awaits the outcome of clinical trials that are still ongoing. The potential therapeutic role in non-diabetic states, including obesity and neurodegenerative disease, is intriguing and depends upon results from ongoing research.     

An acidic microenvironment inhibits antitumoral non-major histocompatibility complex-restricted cytotoxicity: implications for cancer immunotherapy

Local immunosuppression may explain the failure of an effective immune response against solid tumors. Although it is well known that the interstitial pH is significantly lower in solid tumors than in normal tissue, only a few studies in the mouse system have investigated the influence of this acidic milieu on the anti-tumoral cytotoxic response. Here the authors report the suppression of human non-major histocompatibility complex (MHC)-restricted cytotoxicity against tumor cells by an acidic extracellular pH (pHe). Unstimulated peripheral blood mononuclear cells, lymphokine-activated killer (LAK) cells, and natural killer cell clones were used as effector cells. According to pH measurements in solid tumors, representative pH values of 7.2 to 5.3 were chosen during the cytotoxic assays. Target cell lysis was measured using two nonradioactive fluorometric methods, namely two-color flow cytometry and a modified calcein-release assay, which allowed cell-mediated cytotoxicity to be measured and compared with that in adherent targets. Using K562, Daudi, or Raji as suspended target cell lines, the cytotoxic activity of unstimulated peripheral blood mononuclear cells and of LAK cells was markedly reduced by a decreasing pHe. An extracellular pH of 5.8 to 5.3 resulted in a nearly complete loss of the cytotoxic response. This pHe-dependent impairment of the killing activity could also be shown for killer cells stimulated with interleukins-7 and -12, phytohemagglutinin, or lipopolysaccharide. The lytic potential of homogeneous natural killer cell clones as effectors was also strictly influenced by the surrounding pH. The pHe dependence of the non-MHC-restricted killer cell functions against tumor cells seems to be a general phenomenon, because the cytolytic activity of LAK cells against six human adherent tumor cell lines (HeLa, HepG2, LS174T, LS174Te, MCF-7, and RT112) was also clearly reduced under acidic conditions. To initiate the killing process, adhesion molecules play an important role in recognition and binding of the target cell. However, flow cytometric analysis revealed that the expression pattern of relevant adhesion molecules was unaffected by acidic pHe. In conclusion, these data clearly indicate an inhibition of non-MHC-restricted cytotoxicity against tumor cells by an acidic pHe, which may contribute to the failure of immunosurveillance against solid tumors. Consequently, efforts to enhance the anti-tumoral cytotoxicity by immunotherapies may have limited success.     

The scholarship of teaching: implications for nursing faculty.

Nursing educators are faced with the dilemma of addressing the scholarship of teaching for promotion and tenure decisions. This article defines the scholarship of teaching and explores the history of the concept. Implications of the relevance of the topic for nursing faculty with suggestions for documenting the scholarship of teaching are discussed.     

Immunomodulatory effects of procainamide metabolites: their implications in drug-related lupus

Evidence suggests that N-oxidized metabolites of procainamide may be responsible for the development of lupus-like symptoms associated with procainamide therapy. The human hepatic microsomal metabolism of procainamide has been previously reported to result in formation of the N-hydroxylamine derivative of procainamide (procainamide hydroxylamine [PAHA]). The objective of this study was to examine the effects of PAHA on human lymphocytes and adherent cells (monocytes and macrophages). When incubated with lymphocytes in whole blood, PAHA enhanced the response to mitogen and immunoglobulin secretion at lower concentrations (less than or equal to 4 mumol/L) but suppressed these functions at higher concentrations. The cytotoxic effects were nonselective for T lymphocytes and B lymphocytes and appeared to involve an interaction between PAHA and hemoglobin. When erythrocytes were removed or when hemoglobin was converted to carboxyhemoglobin, the suppressive effects of PAHA on lymphocytes were reduced. PAHA stimulated interleukin-1 production by adherent cells at 25 mumol/L but had no effect at lower concentrations. Superoxide anion release was unaffected by PAHA in "resting" adherent cells. Pretreatment with PAHA (2 mumol/L) diminished superoxide release in response to stimulation by phorbol myristate acetate (PMA) or latex bead phagocytosis but augmented superoxide release when coincubated with PMA or latex. These observations indicate that PAHA produces complex, concentration-dependent interactions with human immunoregulatory cells, and they suggest that the effects of PAHA on lymphocyte function may result from the further oxidation of PAHA by hemoglobin, perhaps to the nitroso form.     

The DIRE score: predicting outcomes of opioid prescribing for chronic pain.

The objective of this retrospective study was to test the validity and reliability of a scoring tool (the DIRE Score), for use by clinicians, that predicts which chronic noncancer pain patients will have effective analgesia and be compliant with long-term opioid maintenance treatment. DIRE scores were assigned to 61 cases from the pain center's databases. These cases were abstracted into vignettes that were reviewed and scored by 6 physicians. Repeat scoring was carried out on a subset of 30 vignettes after 2 weeks. The main outcome measures were: global impression of compliance and efficacy as indicated in the medical record and by interview with the patient's treating clinician; and final disposition, ie, whether or not opioids were continued or discontinued at the time of last clinical documentation. Internal consistency of the factors making up the DIRE Score was high (Cronbach's alpha = .80). Sensitivity and specificity of the DIRE Score for predicting patient compliance were 94% and 87%, respectively. For efficacy, sensitivity and specificity were 81% and 76%. For disposition, the sensitivity and specificity were 86% and 73%. Intraclass correlation was 0.94 for interrater reliability and 0.95 for intrarater reliability. PERSPECTIVE: Public controversy about the use of long-term opioids for chronic pain fuels physician ambivalence about the prescribing process. In this initial retrospective study, validity and reliability of the DIRE Score are demonstrated. The score correlated well with measures of patient compliance and efficacy of long-term opioid therapy.     

Describing and predicting the nature of procedural pain after thermal injuries: implications for research

A prerequisite for studying and treating burn-related pain is the establishment of a good understanding of the nature of burn-related pain. However, in most investigations of pain, researchers have failed to examine pain over time or to create summary scores that capture differences in the nature of the pain experiences of individual patients. For 10 consecutive days, 47 patients treated for burn injuries reported on three aspects of procedural pain: worst pain, sensory pain, and affective pain. Three summary pain scores were constructed for each pain dimension: average pain, variability in pain, and linear change in pain. The authors found considerable variability in pain reports from the same patient and from different patients. Analyses indicated that pain reports decreased over time and that patients who had more trait anxiety reported more pain. Patients with larger burn injuries tended to report more affective pain and tended to have a pattern of high and low pain reports that differed from patients with less severe burn injuries. These findings suggest that adequate assessment of burn pain must occur frequently over the course of a single day, as well as for the duration of each patient's care.     

Analysis of the cytotoxicity of synthetic antimicrobial peptides on mouse leucocytes: implications for systemic use

We have analysed the toxicity of highly cationic, artificial alpha-helical antimicrobial peptides on blood cells to assess their suitability for systemic application. Flow cytometric methods, based on the uptake of propidium iodide, were used to obtain a rapid and quantitative estimate of membrane damage to resting and concanavalin A-activated mouse lymphocytes, which was further confirmed by morphological changes as observed by scanning electron microscopy. Membrane permeabilization appeared to correlate with structural characteristics, so that the peptide L-19(9/B), which contains helix-stabilizing aminoisobutyric acid (Aib) residues and is a potent antimicrobial, was also the most lytic towards both mouse lymphocytes and human erythrocytes. Reducing the propensity for helix formation in P19(8) resulted in a marked reduction in in vitro cytotoxicity. Changing the helical sense in D-P19(9/B) also resulted in a significant decrease in cytolytic activity towards both erythrocytes and leucocytes. A limited assessment in BALB/c mice confirmed a lower in vivo toxicity of P19(8) than L-P19(9/B). In a study of the systemic antimycotic activity of P19(8) in a mouse protection model, a modest prolongation in survival of Candida albicans-infected animals after intravenous administration was observed at 5 mg/kg peptide but not at higher doses. The implications of these observations for the systemic use of this type of peptide are discussed.