Immune tolerance induced by platelet-targeted factor VIII gene therapy in hemophilia A mice is CD4 T cell mediated.

Essentials The immune response is a significant concern in gene therapy. Platelet-targeted gene therapy can restore hemostasis and induce immune tolerance. CD4 T cell compartment is tolerized after platelet gene therapy. Preconditioning regimen affects immune tolerance induction in platelet gene therapy. SUMMARY: Background Immune responses are a major concern in gene therapy. Our previous studies demonstrated that platelet-targeted factor VIII (FVIII) (2bF8) gene therapy together with in vivo drug selection of transduced cells can rescue the bleeding diathesis and induce immune tolerance in FVIIInull mice. Objective To investigate whether non-selectable 2bF8 lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and how immune tolerance is induced after 2bF8LV gene therapy. Methods Platelet-FVIII expression was introduced by 2bF8LV transduction and transplantation. FVIII assays and tail bleeding tests were used to confirm the success of platelet gene therapy. Animals were challenged with rhF8 to explore if immune tolerance was induced after gene therapy. Treg cell analysis, T-cell proliferation assay and memory B-cell-mediated ELISPOT assay were used to investigate the potential mechanisms of immune tolerance. Results We showed that platelet-FVIII expression was sustained and the bleeding diathesis was restored in FVIIInull mice after 2bF8LV gene therapy. None of the transduced recipients developed anti-FVIII inhibitory antibodies in the groups preconditioned with 660 cGy irradiation or busulfan plus ATG treatment even after rhF8 challenge. Treg cells significantly increased in 2bF8LV-transduced recipients and the immune tolerance developed was transferable. CD4+ T cells from treated animals failed to proliferate in response to rhF8 re-stimulation, but memory B cells could differentiate into antibody secreting cells in 2bF8LV-transduced recipients. Conclusion 2bF8LV gene transfer without in vivo selection of manipulated cells can introduce immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell mediated.

The important role of von Willebrand factor in platelet-derived FVIII gene therapy for murine hemophilia A in the presence of inhibitory antibodies.

BACKGROUND: Our previous studies have demonstrated that targeting FVIII expression to platelets results in FVIII storage together with von Willebrand factor (VWF) in platelet α-granules and that platelet-derived FVIII (2bF8) corrects the murine hemophilia A phenotype even in the presence of high-titer anti-FVIII inhibitory antibodies (inhibitors). OBJECTIVE: To explore how VWF has an impact on platelet gene therapy for hemophilia A with inhibitors. METHODS: 2bF8 transgenic mice in the FVIII(-/-) background (2bF8(tg+/-) F8(-/-) ) with varying VWF phenotypes were used in this study. Animals were analyzed by VWF ELISA, FVIII activity assay, Bethesda assay and tail clip survival test. RESULTS: Only 18% of 2bF8(tg+/-) F8(-/-) VWF(-/-) animals, in which VWF was deficient, survived the tail clip challenge with inhibitor titers of 3-8000 BU mL(-1) . In contrast, 82% of 2bF8(tg+/-) F8(-/-) VWF(+/+) mice, which had normal VWF levels, survived tail clipping with inhibitor titers of 10-50,000 BU mL(-1) . All 2bF8(tg+/-) F8(-/-) VWF(-/-) mice without inhibitors survived tail clipping and no VWF(-/-) F8(-/-) mice survived this challenge. Because VWF is synthesized by endothelial cells and megakaryocytes and is distributed in both plasma and platelets in peripheral blood, we further investigated the effect of each compartment of VWF on platelet-FVIII gene therapy for hemophilia A with inhibitors. In the presence of inhibitors, 42% of animals survived tail clipping in the group with plasma-VWF and 50% survived in the platelet-VWF group. CONCLUSION: VWF is essential for platelet gene therapy for hemophilia A with inhibitors. Both platelet-VWF and plasma-VWF are required for optimal platelet-derived FVIII gene therapy for hemophilia A in the presence of inhibitors.

In vivo enrichment of genetically manipulated platelets corrects the murine hemophilic phenotype and induces immune tolerance even using a low multiplicity of infection.

BACKGROUND: Our previous studies have demonstrated that platelet-specific gene delivery to hematopoietic stem cells can induce sustained therapeutic levels of platelet factor VIII (FVIII) expression in mice with hemophilia A. OBJECTIVE: In this study, we aimed to enhance platelet FVIII expression while minimizing potential toxicities. METHODS: A novel lentiviral vector (LV), which harbors dual genes, the FVIII gene driven by the αIIb promoter (2bF8) and a drug-resistance gene, the MGMT(P140K) cassette, was constructed. Platelet FVIII expression in mice with hemophilia A was introduced by transduction of hematopoietic stem cells and transplantation. The recipients were treated with O(6)-benzylguanine followed by 1,3-bis-2 chloroethyl-1-nitrosourea monthly three or four times. Animals were analyzed by using polymerase chain reaction (PCR), quantitative PCR, FVIII:C assays, and inhibitor assays. Phenotypic correction was assessed by tail clipping tests and rotational thromboelastometry analysis. RESULTS: Even using a low multiplicity of infection of 1 and a non-myeloablative conditioning regimen, after in vivo selection, the levels of platelet FVIII expression in recipients increased to 4.33 ± 5.48 mU per 10(8) platelets (n = 16), which were 19.7-fold higher than the levels obtained from the recipients before treatment. Quantitative PCR results confirmed that 2bF8/MGMT-LV-transduced cells were effectively enriched after drug-selective treatment. Fifteen of 16 treated animals survived tail clipping. Blood loss and whole blood clotting time were normalized in the treated recipients. Notably, no anti-FVIII antibodies were detected in the treated animals even after recombinant human B-domain deleted FVIII challenge. CONCLUSION: we have established an effective in vivo selective system that allows us to enrich 2bF8LV-transduced cells, enhancing platelet FVIII expression while reducing the potential toxicities associated with platelet gene therapy.

Lentivirus-mediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity.

BACKGROUND: The development of inhibitory antibodies, referred to as inhibitors, against exogenous factor VIII in a significant subset of patients with hemophilia A remains a persistent challenge to the efficacy of protein replacement therapy. Our previous studies using the transgenic approach provided proof-of-principle that platelet-specific expression could be successful in treating hemophilia A in the presence of inhibitory antibodies. OBJECTIVE: To investigate a clinically translatable approach for platelet gene therapy of hemophilia A with pre-existing inhibitors. METHODS: Platelet FVIII expression in preimmunized FVIII(null) mice was introduced by transplantation of lentivirus-transduced bone marrow or enriched hematopoietic stem cells. FVIII expression was determined with a chromogenic assay. The transgene copy number per cell was quantitated with real-time PCR. Inhibitor titer was measured with the Bethesda assay. Phenotypic correction was assessed by the tail clipping assay and an electrolytically induced venous injury model. Integration sites were analyzed with linear amplification-mediated PCR. RESULTS: Therapeutic levels of platelet FVIII expression were sustained in the long term without evoking an anti-FVIII memory response in the transduced preimmunized recipients. The tail clip survival test and the electrolytic injury model confirmed that hemostasis was improved in the treated animals. Sequential bone marrow transplants showed sustained platelet FVIII expression resulting in phenotypic correction in preimmunized secondary and tertiary recipients. CONCLUSIONS: Lentivirus-mediated platelet-specific gene transfer improves hemostasis in mice with hemophilia A with pre-existing inhibitors, indicating that this approach may be a promising strategy for gene therapy of hemophilia A even in the high-risk setting of pre-existing inhibitory antibodies.

MUC1 is a novel regulator of ErbB1 receptor trafficking.

ErbB receptors are key regulators of cell survival and growth in normal and transformed tissues. The oncogenic glycoprotein MUC1 is a binding partner and substrate for erbB1 and MUC1 expression can potentiate erbB-dependent signal transduction. After receptor activation, erbB1 is typically downregulated via an endocytic pathway that results in receptor degradation or recycling. We report here that MUC1 expression inhibits the degradation of ligand-activated erbB1. Through the use of both RNAi-mediated knock down and overexpression constructs of MUC1, we show that MUC1 expression inhibits erbB1 degradation after ligand treatment in breast epithelial cells. This MUC1-mediated protection against erbB1 degradation can increase total cellular pools of erbB1 over time. Biotinylation of surface proteins demonstrates that cell-surface associated erbB1 receptor is protected by MUC1 against ligand-induced degradation, although this is accompanied by an increase in erbB1 internalization. The MUC1-mediated protection against degradation occurs with a decrease in EGF-stimulated ubiquitination of erbB1, and an increase in erbB1 recycling. These data indicate that MUC1 expression is a potent regulator of erbB1 receptor stability upon activation and may promote transformation through the inhibition of erbB1 degradation.

Transgenic MUC1 interacts with epidermal growth factor receptor and correlates with mitogen-activated protein kinase activation in the mouse mammary gland.

MUC1 is a large (>400 kDa), heavily glycosylated transmembrane protein that is aberrantly expressed on greater than 90% of human breast carcinomas and subsequent metastases. The precise function of MUC1 overexpression in tumorigenesis is unknown, although various domains of MUC1 have been implicated in cell adhesion, cell signaling, and immunoregulation. Stimulation of the MDA-MB-468 breast cancer line as well as mouse mammary glands with epidermal growth factor results in the co-immunoprecipitation of MUC1 with a tyrosine-phosphorylated protein of approximately 180 kDa. We have generated transgenic lines overexpressing full-length (MMF), cytoplasmic tail deleted (DeltaCT), or tandem repeat deleted (DeltaTR)-human MUC1 under the control of the mouse mammary tumor virus promoter to further examine the role of MUC1 in signaling and tumorigenesis. Immunoprecipitation experiments revealed that full-length transgenic MUC1 physically associates with all four erbB receptors, and co-localizes with erbB1 in the lactating gland. Furthermore, we detected a sharp increase in ERK1/2 activation in MUC1 transgenic mammary glands compared with Muc1 null and wild-type animals. These results point to a novel function of increased MUC1 expression, potentiation of erbB signaling through the activation of mitogenic MAP kinase pathways.

Cooperative induction of mammary tumorigenesis by TGFalpha and Wnts.

We previously reported that multiparous WAP-TGFalpha transgenic mice develop mammary gland carcinomas with complete incidence. TGFalpha-induced tumors appear stochastically and with relatively long latency, indicating an additional requirement for other genetic alterations. To identify genes that cooperate with TGFalpha in mammary tumorigenesis, we used a retroviral insertion approach featuring a cloned and infectious hybrid MMTV (C3H/Mtv-1; (Shackleford and Varmus, 1988)). Tumor latency was decreased approximately 30% in MMTV-infected WAP-TGFalpha transgenic animals compared to noninfected transgenic controls, and > 30% of the corresponding tumors displayed evidence of integrated C3H/Mtv-1 DNA. PCR-based analyses of DNAs from two virus-infected, transgenic tumors revealed integration of hybrid MMTV in 3' untranslated exons of the Wnt-1 or Wnt-3 oncogenes. Moreover, Northern blots confirmed dramatic induction of Wnt-1 or Wnt-3 transcripts in the respective tumors, indicating that MMTV integration resulted in activated expression of these genes. Semiquantitative RT-PCR analyses showed that overexpression of Wnt-1 or Wnt-3 was a common occurrence in MMTV-infected WAP-TGFalpha tumors, and some noninfected WAP-TGFalpha tumors also showed evidence of elevated Wnt-3 transcripts. Collectively, these results reveal cooperative induction of mammary gland tumorigenesis by simultaneous deregulation of EGF-like (TGFalpha) and Wnt growth factors.

Biocompatibility and degradation of collagen bone anchors in a rabbit model.

Bone anchors are used to fasten tendons and ligaments to bone during reconstructive surgery. Although metal anchors are often used, an anchor that could resorb and permit normal bone regeneration would be advantageous. The objective of the study was to evaluate the biocompatibility and degradation of bone anchors that consist of collagen-based bodies, ceramic washers, and polyester sutures. Eighteen rabbits underwent bilateral implantations in the distal femoral condyles. Nine animals received glutaraldehyde-crosslinked fibrillar collagen bone anchors (FC) and nine received glutaraldehyde-crosslinked fibrillar collagen bone anchors containing tricalcium phosphate (FC-TCP). Three animals per group were sacrificed at postimplantation weeks 1, 6, and 12. One femur from each rabbit was evaluated histologically, and the contralateral side underwent biomechanical pull-out testing. Histological evaluation of the implant site indicated that the FC and FC-TCP bone anchors were both biocompatible. The FC-TCP formulation degraded earlier than the FC formulation, and FC-TCP showed significant degradation at 6 weeks; the FC and FC-TCP formulations both showed similar amounts of degradation at 12 weeks. The degrading anchor bodies appeared to be osteoconductive as evidenced by new bone ingrowth into the degrading collagen matrices without a fibrous interface. These results suggest that collagen-based bone anchors have potential as bioresorbable orthopedic implants.

Adenoviral-mediated gene transfer of interleukin-6 in rat lung enhances antiviral immunoglobulin A and G responses in distinct tissue compartments.

The effect of IL-6 transgene expression following lung gene transfer on anti-adenovirus humoral responses of immunoglobulin (Ig) G and A both in the lung and peripheral blood was investigated. Lung infection by a control adenovirus caused an increased level of circulating anti-adenoviral IgG antibodies. However, the magnitude of this response was many times higher in the peripheral blood of rats receiving an adenovirus engineered to express IL-6 transgene. In comparison, much lower levels of anti-adenoviral IgG were detected in the bronchoalveolar fluids of rats receiving either virus. In contrast, there was no detectable level of anti-adenoviral IgA in the peripheral blood in any cases, yet significantly detectable levels of anti-adenoviral IgA were measured in the lung. The levels of this IgA were much higher in the lung of rats expressing IL-6 than in the lung of control animals (15 times higher by day 14). Our findings thus provide evidence that IL-6 plays a significant role in enhancing specific airways mucosal IgA and systemic IgG responses during local lung viral infection, and provide the rationale for using IL-6 locally at mucosa sites as an immune adjuvant for antiviral vaccination program.

Dynamic expression and activation of ERBB receptors in the developing mouse mammary gland.

The complex system of ERBB receptors and ligands is implicated in growth and differentiation of the mammary gland. However, it has not been comprehensively examined in this dynamic tissue. Combined RNA and protein analyses of glands in different stages from virgin to involution revealed differential expression of the four ERBB receptors, as well as distinctive patterns of ERBB ligand expression that suggested specialized function. ERBB localization was linked to mammary gland function. Thus, in the virgin gland, ERBB1 and ERBB2 were colocalized to all major cell types during ductal morphogenesis but differentially localized in the mature gland. All four ERBB receptors were restricted to epithelia in the differentiated gland. Analyses of ERBB tyrosine phosphorylation provided strong evidence of interaction between the four receptors in this physiological context. Thus, exogenous EGF induced stage-dependent transphosphorylation of ERBB2-4 as well as ERBB1, whereas endogenous phosphorylation of all four receptors peaked in late pregnancy and lactation.

Transgenic mice reveal roles for TGFalpha and EGF receptor in mammary gland development and neoplasia.

Transforming growth factor-alpha (TGFalpha)4 and/or the EGF receptor (EGFR) are frequently overexpressed by human and rodent breast tumors, as well as tumor-derived cell lines. Additionally, various observations suggest a role for TGFalpha and the EGFR signaling system in normal mouse mammary gland development. Recently, several laboratories have established TGFalpha transgenic mice with which to study the role of this growth factor in normal and neoplastic mammary biology. Examination of these mice revealed that overexpression of TGFalpha has profound consequences for this tissue. Most strikingly, transgenic mice expressing TGFalpha under the control of tissue-specific and nonspecific promoters stochastically developed focal mammary tumors with an incidence and latency that was markedly affected by pregnancy. Most TGFalpha-induced tumors were well-differentiated adenomas/adenocarcinomas, although some were undifferentiated and locally invasive. Distant metastases were only occasionally observed. Administration of the genotoxic carcinogen, 7,12-dimethylbenzanthracene (DMBA), dramatically accelerated mammary tumorigenesis induced by the TGFalpha transgene, raising the possibility that TGFalpha acts as a promoter in this tissue. Mice harboring dual transgenes encoding TGFalpha and either wild-type ERBB2 or c-myc displayed markedly accelerated tumorigenesis compared to mice carrying any of the single transgenes alone, indicative of potent cooperativity. Moreover, tumorigenesis in the bitransgenic mice was less dependent on pregnancy, and tumors were generally more malignant in appearance. Finally, TGFalpha also affected mammary gland dynamics. TGFalpha transgenic mice consistently displayed precocious alveolar development, were variably impaired with respect to lactation, and showed markedly reduced postlactional involution. As a result, the glands of multiparous females accumulated hyperplastic lesions that generally resembled milk-producing alveoli. Limited data support the hypothesis that these lesions were precursors to TGFalpha-induced tumors. In summary, these various findings underscore the potential importance of TGFalpha for cellular differentiation and transformation in the mammary gland. They also establish TGFalpha transgenic mice as a powerful model with which to study the role of EGFR signaling molecules in this dynamic tissue.

Characterization of the mouse transforming growth factor alpha gene: its expression during eyelid development and in waved 1 tissues.

The spontaneous mouse waved 1 (wa1) mutation is allelic with the transforming growth factor alpha (TGF-alpha) gene and produces phenotypes similar to those of TGF-alpha knockout mice. Here, we show that TGF-alpha mRNA and protein levels are measurable in wa1 tissues but reduced 5- to 30-fold relative to wild type. Because the wa1-coding sequence is identical to that of the normal mRNA, wa1 is not a null mutation. Nuclear run-on analyses revealed decreased transcription of the TGF-alpha gene in wa1 tissues, but the sequence of a 3.2-kb 5' flanking fragment containing the promoter was unaltered. Moreover, pulsed field gel electrophoresis analysis did not reveal alterations within 750 kb upstream or 350 kb downstream of the gene, and chromosome 6 was karyotypically normal. Hence, we speculate that the wa1 mutation may be subtle and/or reside at a greater distance from the TGF-alpha gene. TGF-alpha deficiency elicits a spectrum of variably penetrant eye anomalies in wa1 and knockout mice that are associated with open eyes at birth. We found that late-gestation wa1 and TGF-alpha-null embryos display a significant delay in eyelid closure, although the eyes of most embryos fuse prior to birth. In situ hybridization localized TGF-alpha expression to the advancing margins of the eyelid epithelium and epidermal growth factor receptor expression throughout the eyelid and corneal epithelia. These results suggest that eye problems observed in TGF-alpha-deficient adult mice arise from premature exposure and trauma to open eyes during or following parturition.

Vector-derived expression of recombinant rat interleukin-6.

Rat interleukin-6 (IL-6) cDNA, coding for an important inflammation- and immune-regulatory polypeptide cytokine, was cloned into the novel expression vector pH6EX3 which directs the synthesis of inserted genes as a fusion protein with histidine hexapeptide (HH). The resultant vector (pRIL6.992) was shown to produce significant amounts of recombinant rat IL-6 fusion protein with HH at its N-terminus in various strains of Escherichia coli. The expression of the HH-IL-6 fusion protein was demonstrated to be under the control of the tac promoter and could be induced by IPTG. This protein was isolated from bacterial inclusion bodies and purified to homogeneity in a one-step procedure by affinity chromatography using a nickel-chelating column. The HH-IL-6 fusion protein isolated in this manner was biologically active as determined by hepatocyte stimulation and B9 hybridoma growth assays. Further, this activity was neutralized with a polyclonal antiserum raised against rat IL-6 protein generated in a novel fashion from rabbits infected with a recombinant human type-5 adenovirus vector expressing rat IL-6 protein (Ad5E3rIL6). The recombinant HH-IL-6 protein was then used to boost Ad5E3rIL6-immunized rabbits. This resulting antiserum was shown to neutralize recombinant and natural rat and murine IL-6 bioactivity in vitro and was useful in Western blot analysis and immunohistochemistry of rat IL-6.

Inhibition of mammary gland involution is associated with transforming growth factor alpha but not c-myc-induced tumorigenesis in transgenic mice.

Deregulated expression of transforming growth factor alpha (TGF-alpha) or c-myc has been implicated in the genesis of human breast cancer. To better characterize the role of these molecules in this disease, we generated transgenic mice that express TGF-alpha or c-myc under control of the mouse whey acidic protein (WAP) promoter. We then compared the resulting mammary gland neoplasia in these mice and in previously described mice expressing a metallothionein-driven TGF-alpha transgene. Nonvirgin female mice in all transgenic lineages developed mammary tumors with 100% incidence but variable latency. Among TGF-alpha lines, mean survival time correlated with the level of transgene expression, and the average life spans of high-expressing WAP-TGF-alpha and WAP-c-myc mice were similarly reduced. The majority of TGF-alpha-induced tumors were relatively well-differentiated adenomas and adenocarcinomas; in contrast, WAP-c-myc tumors were poorly differentiated, solid carcinomas with a minority of adenocarcinomas. Most TGF-alpha and all c-myc-induced tumors were transplantable, but lung metastases were infrequently observed in all transgenic lines. WAP-TGF-alpha-induced tumors, in marked contrast to those induced by WAP-c-myc, displayed frequent induction of cyclin D1 mRNA, suggesting that expression of this gene may complement that of TGF-alpha during mammary tumor development. Expression of TGF-alpha also induced precocious development of pregnant glands and delayed or inhibited mammary involution. As a result, multiparious MT-TGF-alpha and especially WAP-TGF-alpha females accumulated large numbers of hyperplastic alveolar nodules that resembled the more differentiated TGF-alpha-induced tumors. Finally, coexpression of WAP-c-myc and WAP-TGF-alpha transgenes markedly decreased tumor latency, increased tumor growth, and even induced mammary tumors in virgin female and male mice. These findings provide further evidence for the importance of deregulated TGF-alpha expression in multistage carcinogenesis, and they suggest that in the mammary gland the mechanism of TGF-alpha-induced transformation may depend on postlactational survival of differentiated epithelium. They also provide evidence of a potent tumorigenic collaboration between TGF-alpha and c-myc in mammary epithelium.

Intra-articular morphine after arthroscopic knee operation.

Reports on pain relief with intra-articular morphine after arthroscopic knee operation are conflicting. To assess the long-term antinociceptive effect of intraarticular morphine, we studied pain at rest, pain on standing and ability to walk for 7 days after intraarticular injection of bupivacaine 100 mg (group 1, n = 11), bupivacaine 100 mg and morphine 1 mg (group 2, n = 10) and bupivacaine 100 mg and morphine 3 mg (group 3, n = 10) at the end of operation. Pain and walking were assessed by visual analogue and walking scales, respectively. Pain was treated with morphine i.v. in the recovery room and Tylenol No. 3 after discharge. Assessments were made before operation, and 1, 3, 6 and 12 h after injection and on days 1-7 after operation. There were significant differences between the groups in pain scores (pain at rest, P < 0.05; pain on standing, P < 0.01). The pain scores in group 3 were lower than those in group 1. The differences in pain scores at rest were significant at 12 h and on day 1 after operation and differences in pain scores on standing were significant at 12 h and on days 1 and 2 after operation. The scores in group 2 were intermediate between those in groups 1 and 3. The walking scores in group 3 were significantly better than those in group 1 at 12 h. The amount of analgesics received in groups 2 and 3 was significantly less than that in group 1 until day 3 after operation.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of intravenous ranitidine and metoclopramide on behavior, cognitive function, and affect.

Both ranitidine and metoclopramide produce neuropsychiatric side effects. Concomitant use of these drugs preoperatively may produce adverse behavioral and emotional changes. Therefore, in 123 unpremedicated patients undergoing tubal occlusion, behavior, cognitive function, and affect were studied before and after a 2-min intravenous injection of placebo (n = 30), ranitidine 50 mg (n = 32), metoclopramide 10 mg (n = 30), or both ranitidine 50 mg and metoclopramide 10 mg (n = 31). Cognitive function was evaluated by the responses to 11 statements devised to assess attitude toward anesthesia and surgery. Affect was assessed by the word chosen out of 11 word-pairs as best describing the feelings at the time. After ranitidine injection, one patient seemed restless and five seemed drowsy. The changes were associated with subjective feelings of agitation (P < 0.05) and restlessness (P < 0.05). After metoclopramide injection, 6 (20%) developed akathisia, 13 (43.3%) seemed restless, and 8 (26.7%) seemed drowsy. The changes were associated with subjective sensation of jumpiness (P < 0.01) and discomfort (P < 0.05). When both ranitidine and metoclopramide were injected, 10 (32.3%) developed akathisia, 4 (12.4%) seemed restless, and 11 (35.5%) seemed drowsy. The changes were associated with subjective feelings of agitation (P < 0.05), jumpiness (P < 0.05), restlessness (P < 0.01), and upset (P < 0.05). Akathisia, a side effect of metoclopramide, seemed to be more prominent when ranitidine was added.

Quantitative EEG analysis during cardiac surgery in a case of spontaneous recall.

A case of spontaneous postoperative recall of intraoperative events during tricuspid valvuloplasty is presented. In this case, EEG activity was continuously monitored and quantitatively processed to detect brain dysfunction during the procedure. Anesthetic maintenance was provided by fentanyl, isoflurane and midazolam. A retrospective analysis of the relative power distribution, median frequency and fast/slow ratio was conducted to characterize the recall and non-recall periods during the surgery. Analysis of the quantitative descriptors suggested that no specific EEG pattern was associated with the periods of recall. The use of several anesthetics with different effects on the EEG precludes the establishment of a single relationship between descriptors and changes in vigilance during episodes of recall.

Induction of luteal phase defect with clomiphene citrate.

The effect of clomiphene citrate and progesterone on luteal function in infertile women was studied. Endometrial biopsies were performed in 103 women immediately prior to menstruation. Group 1 (n = 62) had secretory endometrium with a histologic lag time of greater than or equal to 48 hours with respect to the subsequent menses, that is, luteal phase defect. Group 2 (n = 10) had normal histologic characteristics of the secretory phase. Group 3 (n = 31) had anovulatory endometrium. The last group was subdivided into those with polycystic ovary syndrome (n = 9) and those without the characteristic gonadotropin pattern of polycystic ovary syndrome (n = 22). Clomiphene citrate at doses of 50 to 250 mg daily for 5 days was administered for induction of ovulation, timing of ovulation, or treatment of luteal phase defect. An endometrial biopsy was obtained after three ovulatory treatment cycles. Only one fourth of the women with prior luteal phase defect had normalization of the biopsy specimen with clomiphene citrate, while one half of those treated with progesterone had normal specimens. Half of the normally ovulating women had induction of a luteal phase defect with clomiphene citrate. Only women with polycystic ovary syndrome had consistently well-timed endometrial histologic features with clomiphene citrate therapy. Despite successful induction of ovulation, 16 of the other 22 previously anovulatory women had endometrial histologic findings compatible with luteal phase defect. Increasing the clomiphene citrate dosage was unsuccessful in improving endometrial maturation. These results suggest that the use of clomiphene citrate may be associated with a high rate of luteal phase defect induction, except among women with polycystic ovary syndrome. Clomiphene citrate, even at high doses, appears to be ineffective therapy for luteal phase defect.