Targeting of IL-2 receptor with a caspase fusion protein disrupts autoimmunity in prediabetic and diabetic NOD mice.

AIMS/HYPOTHESIS: Interruption of IL-2 signalling is an attractive therapeutic target in autoimmune disorders. In this study we evaluated the effect of a fusion protein composed of IL-2 and caspase-3 (IL2-cas) on NOD mice, as compared with disease induction by cyclophosphamide. METHODS: IL2-cas was assessed in NOD mice at various ages and in conjunction with cyclophosphamide administration. The effect of IL2-cas on diabetogenic cells was evaluated in adoptive transfer experiments and in cell suspension in vitro. RESULTS: IL2-cas induced apoptosis in T cells expressing the alpha chain of the IL-2 receptor (cluster of differentiation [CD]25) in vitro, with superior survival of T cells expressing CD4 and forkhead box P3 (FOXP3). The fusion protein decreased mixed lymphocyte reactivity, and pretreatment with IL2-cas decreased the efficacy of adoptive transfer of diabetes into NOD severe combined immunodeficiency mice. Administration of one dose of IL2-cas decreased the incidence of diabetes in NOD mice, showing a superior beneficial effect when administered at young age, and effectively blocked induction of hyperglycaemia by cyclophosphamide, reducing the severity of islet inflammation. Administration of IL2-cas caused an acute increase in CD25(-)FOXP3(+) T cells in the lymph nodes, pancreas and thymus in NOD mice, with similar effects in wild-type mice. Administration of IL2-cas after onset of hyperglycaemia resulted in superior survival. CONCLUSIONS/INTERPRETATION: Targeted elimination of cells expressing the IL-2 receptor by this fusion protein disrupts the autoimmune pathogenesis in prediabetic and diabetic NOD mice, despite depletion of CD25(+) regulatory T cells. Furthermore, this particular fusion protein is permissive to the development of FOXP3(+) T cells that might contribute to protracted protection from the progression of insulitis and overt hyperglycaemia.

Phase I and pharmacokinetic study of halofuginone, an oral quinazolinone derivative in patients with advanced solid tumours.

PURPOSE: Halofuginone (tempostatin) is a synthetic derivative of a quinazolinone alkaloid showing anti-angiogenic, anti-metastatic and anti-proliferative effects in preclinical studies. The objectives of this phase I study were to assess the dose-limiting toxicities (DLTs), to determine the maximum tolerated dose (MTD) and to study the pharmacokinetics (PKs) of halofuginone when administered once or twice daily orally to patients with advanced solid tumours. METHODS: Patients were treated with escalating doses of halofuginone at doses ranging from 0.5 to 3.5 mg/day. For pharmacokinetic analysis plasma sampling was performed during the first and second course and assayed using a validated high-performance liquid chromatographic assay with mass spectrometric detection. RESULTS: Twenty-four patients received a total of 106 courses. The 'acute' MTD was reached at 3.5 mg/day, with nausea, vomiting, and fatigue as DLT. The recommended dose for chronic administration was defined as 0.5mg/day with the requirement of 5HT3 antagonists to control nausea and vomiting considered as DLT. Several patients experienced bleeding complications on treatment with halofuginone in which a causal relationship could not be excluded. The PKs of halofuginone were linear over the dose range studied with a large interpatient variability. CONCLUSIONS: In this study the DLT of halofuginone was nausea, vomiting, and fatigue. The recommended dose for phase II studies of halofuginone is 0.5mg administered orally, once daily.

B-cell non-Hodgkin's lymphoma: evidence for the t (14;18) translocation in all hematopoietic cell lineages.

BACKGROUND: B cells of patients with non-Hodgkin's lymphoma (B-NHL) harbor specific chromosomal translocations, including t(14;18), the most common aberration found in this disease. The translocation involves the immunoglobulin (Ig) heavy-chain joining (JH) region gene on chromosome 14 and the BCL2 gene on chromosome 18, resulting in dysregulated expression of the BCL2 gene. The t(14;18) translocation has been thought to occur in the pre-B-cell stage, during the first event of Ig gene rearrangement. PURPOSE: This study was conducted to investigate the potential involvement of nonlymphoid lineages in B-NHL. METHODS: We studied the t(14;18) translocation and other frequently occurring translocations in total bone marrow aspirates of 10 patients with B-NHL, with the use of the fluorescence in situ hybridization (FISH) technique. We also performed cytogenetic analyses on representative bone marrow aspirates from the patients. Moreover, to define which of the major cell lineages present in the bone marrow carry the t(14;18) translocation, we used a series of monoclonal antibodies together with fluorescence-activated cell sorter (FACS) analyses to purify cells positive for CD3 (T cells), CD19 (B cells), CD10 (CALLA-positive cells), CD41a (megakaryocytic cells), CD13 (myeloid cells), and glycophorin A (erythroid cells). The cells of each subgroup underwent FISH analysis with the use of JH and BCL2 probes to detect the t(14;18) translocation. Bone marrow samples obtained from five healthy donors served as controls. RESULTS: Bone marrow cells from eight of the 10 patients studied carried the t(14;18) translocation. When present, the translocation was observed in many or even all of the cell lineages (lymphoid, myeloid, megakaryocytic, and erythroid) present in the bone marrow, including peripheral blood progenitor stem cells; for seven of the eight patients carrying the translocation, it was found in 96%-100% of the unfractionated bone marrow cells as well as in all of the FACS-purified cell fractions in which it could be detected or studied. Conventional cytogenetic analyses performed on representative bone marrow aspirates confirmed the results obtained by FISH analysis. Cells in control bone marrow samples obtained from the five healthy donors were negative for the t(14;18) translocation by FISH analysis. CONCLUSIONS: Our findings indicate that the t(14;18) translocation most probably occurs in a very early multilineage progenitor stem cell. IMPLICATIONS: Given that the t(14;18) chromosomal translocation was found in all types of bone marrow cells when only the B cells were malignant, our results suggest that this translocation is not sufficient to induce neoplastic transformation. This finding underscores the need for the development of new approaches for the detection and surveillance of B-NHL.

Tamoxifen modulates apoptotic pathways in primary endometrial cell cultures.

Clinical data indicate that tamoxifen (TAM) therapy may cause an increased risk of endometrial pathology in postmenopausal but not in premenopausal women. Molecular mechanisms of the uterotrophic activity of TAM have not been clearly established nor its relevance to apoptosis in endometrial cells. The present study was implemented to evaluate the apoptotic effect of TAM on primary endometrial cell cultures in the presence or absence of steroid hormones (SHs). A total of 14 primary endometrial cell cultures were established and maintained both with and without SHs. Cell cultures were treated for 24 h with either 20 microM TAM or 10 nM estradiol. Apoptotic cells presented in a pre-G1 peak and the expression of bcl-2 were studied using flow cytometry. All endometrial cell cultures maintained in a SH-containing environment, except one, responded to TAM by a significant increase (P = 0.03) in the pre-G1 cell fraction, indicating a proapoptotic effect. A significant (P = 0.03) reduction in the pre-G1 peak equivalent to an antiapoptotic response was observed in 6 of 13 cell cultures maintained in a SH-deficient environment. In 4 of 10 cell cultures evaluated in both media, the pre-G1 population was medium dependent. In 8 of 10 cultures evaluated for Bcl2 levels, no trend was found in either media, but a dependency on SH content was observed. Comparison between effects of TAM and estradiol demonstrated identical trends, regardless of the menstrual phase or SH content in cell environments. These results suggest that TAM acts as an estrogen agonist on endometrial tissue in both environments. We conclude that TAM modulates apoptotic pathways in primary endometrial cell cultures. The SH content in the cell environment influences the apoptotic effect of TAM and determines the propensity for a cell to undergo apoptosis or, on the contrary, to resist apoptotic death in response to TAM treatment. This is in concordance with the observed clinical risk of endometrial pathologies in postmenopausal versus premenopausal women.

Potent antileukemic activity of the novel agents norsegoline and dibezine.

We examined the effect of norsegoline, a natural marine product, and dibezine, a synthetic product, on the survival of human myeloid progenitor cells [colony-forming unit-cells (CFU-C)] from normal individuals and from 10 patients with Philadelphia-positive chronic myelogenous leukemia (CML) in chronic phase and blastic crisis. We compared their effect to the effect of IFN-alpha. Norsegoline, dibezine, and IFN-alpha inhibited the proliferation of CFU-C in a dose-dependent manner. The number of CFU-C from bone marrow (BM) of five CML patients in chronic phase exposed for 16 h to norsegoline (10(-8)-10(-6)M), dibezine (10(-8)-10(-6)M), and IFN-alpha (500 units/ml) was found to be statistically lower (P < 0.05) than the number of CFU-C derived from normal individuals. A 16-h drug exposure of CD34(+) cells isolated from the peripheral blood of three CML patients in blastic crisis and from BM of two patients in chronic phase resulted in a marked inhibition in the ability of the cells to proliferate in liquid culture and a reduction in CFU-C content. Using the fluorescent in situ hybridization technique, we evaluated detection of the BCR/ABL fusion product in the CD34(+) cells. All five patients were 100% Philadelphia positive at diagnosis. BCR/ABL translocations were detected in 94.6 +/- 0.6% of cells following their growth in liquid culture for 7 days. Following exposure of CD34(+) cells to norsegoline, dibezine, or IFN-alpha, BCR/ABL fusion signals could be detected in 73 +/- 11%, 66.5 +/- 4. 7%, and 66.0 +/- 2.5% of cells from BM and 72.3 +/- 5%, 68.8 +/- 7%, and 60.6 +/- 6.8% of peripheral blood, respectively. Our data indicate that norsegoline and dibezine have in vitro an antileukemic effect against Philadelphia-positive cells and may be used in conjunction with currently available agents for ex vivo purging of BM and/or peripheral blood of CML patients in conjunction with autologous bone marrow transplantation.

Interleukin 2-Bax: a novel prototype of human chimeric proteins for targeted therapy.

During the past few years many chimeric proteins have been developed to target and kill cells expressing specific surface molecules. Generally, these molecules carry a bacterial or plant toxin that destroys the unwanted cells. The major obstacle in the clinical application of such chimeras is their immunogenicity and non-specific toxicity. We have developed a new generation of chimeric proteins, taking advantage of apoptosis-inducing proteins, such as the human Bax protein, as novel killing components. The first prototype chimeric protein, IL2-Bax, directed toward IL2R-expressing cells, was constructed, expressed in Escherichia coli and partially purified. IL2-Bax increased the population of apoptotic cells in a variety of target T cell lines, as well as in human fresh PHA-activated lymphocytes, in a dose-dependent manner and had no effect on cells lacking IL2R expression. The IL2-Bax chimera represents an innovative approach for constructing chimeric proteins comprising a molecule that binds a specific cell type and an apoptosis-inducing protein. Such new chimeric proteins could be used for targeted treatment of human diseases.

Two conceptions in a 45,X woman.

We present a 31-year-old woman with a 45,X chromosome constitution who had had two miscarriages. This report brings to 11 the number of presumably non-mosaic Ullrich-Turner syndrome patients who have achieved pregnancy. A review of the literature indicates an increased incidence of chromosome abnormalities and a high rate of fetal death in offspring of such patients. However, in light of the fertility in these patients, genetic counseling should be reevaluated and perhaps amniocentesis recommended in successful pregnancies.

Sonographic visualization of physiologic anterior abdominal wall hernia in the first trimester.

Ultrasonography is able to detect some fetal abnormalities as early as the first trimester of pregnancy. Using high-resolution ultrasound equipment, it is possible to demonstrate physiologic herniation of the midgut, which usually occurs between eight and nine weeks' gestation (calculated by last menstrual period). Fourteen cases have been studied with weekly ultrasound examinations between seven and 12 weeks' gestation. This herniation varied to a large extent in the different embryos depending on the amount of protruding intestine. In embryologic terms, the return of the intestine into the peritoneal cavity, and its rotation and fixation to the posterior abdominal wall, should be concluded at ten to 12 weeks. In all cases studied, the persistence of umbilical herniation could be ruled out by sonographic visualization of the umbilical cord insertion between ten and 12 weeks. Therefore, the suspicion of a severe congenital abdominal wall defect, such as omphalocele, umbilical herniation, or gastroschisis can reliably be confirmed only after 12 weeks' gestation.

Estimated fetal weight in the evaluation of growth in twin gestations: a prospective longitudinal study.

A prospective longitudinal study was conducted in order to determine by sonographically estimated fetal weight the patterns of fetal growth in twins. Thirty-five healthy women with normal twin pregnancies were examined every three weeks from the 15th week of gestation to delivery. Among the measurements obtained were the biparietal diameter (BPD), the abdominal circumference, and the calculated fetal weight. From 15-28 weeks, the growth velocity of the BPD and abdominal circumference remained fairly constant, with a steady increase in incremental growth. Beyond this age, we observed a slowing in growth of the BPD, while the abdominal circumference continued at a constant rate. The growth velocity of the weight steadily increased throughout pregnancy. Although greater biologic variability in weight between twin A and B was observed as gestational age progressed, the overall mean weights of twin A and B were not statistically different. We have generated a nomogram of fetal weight gain throughout pregnancy.

Growth of the fetal stomach in normal pregnancies.

Fetal stomach dimensions were measured sonographically in 152 fetuses with gestational ages ranging from nine to 40 weeks. Nomograms of the mean +/- 2 standard deviations (SD) for the longitudinal, anteroposterior, and transverse diameters of the fetal stomach were generated throughout pregnancy. A linear growth function was observed across gestational age, and a high degree of correlation existed between gestational age and the transverse (r = 0.809, P less than .0001), anteroposterior (r = 0.798, P less than .0001), and longitudinal (r = 0.749, P less than .0001) diameters. These data provide a method by which variations from the norm can be assessed, and offer potential prenatal diagnosis of a variety of gastrointestinal lesions.

Detection of minimal residual disease after sex-mismatch bone marrow transplantation in chronic myelogenous leukemia by fluorescence in situ hybridization.

Detection of minimal residual disease is one of the major goals in bone marrow transplantation. We used a fluorescence in-situ hybridization technique to detect residual Philadelphia-chromosome positive cells in chronic myelogenous leukemia (CML) patients after sex-mismatch BMT. We analyzed the level of detection using probes for the BCR/ABL fusion product by comparison with results obtained with probes for the Y and X sex chromosomes. Detection of sex-mismatch chromosomes was significantly higher than that of the BCR/ABL translocation. In contrast, a higher specificity of residual tumor cell detection by the BCR/ABL probe was demonstrated because most of the sex-mismatch cells detected by FISH had a normal karyotype. Tumor-specific markers probes are thus superior and more accurate than sex-mismatch probes for detection of MRD in CML patients after BMT.

Detection of minimal residual disease state in chronic myelogenous leukemia patients using fluorescence in situ hybridization.

Detection of minimal residual disease and relapse remain major problems in chronic myelogenous leukemia (CML) patients following bone marrow transplantation (BMT). In order to disclose the 9;22 Philadelphia translocation, we used a fluorescence in situ hybridization (FISH) technique. BCR and ABL gene fragments were used as probes for the detection of the BCR/ABL fusion product in peripheral blood and bone marrow cells from 11 CML patients in which 5 were post-BMT. The sensitivity and specificity of this approach were compared to conventional cytogenetic and polymerase chain reaction (PCR) methods. FISH demonstrated a high degree of sensitivity (1%) for the detection of the BCR/ABL translocation in these patients. A linear correlation was found between FISH detection of the BCR/ABL fusion product and routine chromosomal analysis (r = 0.995; p < 0.001). Detection of the BCR/ABL signal by FISH was observed in all patients showing a positive PCR signal. A significant reduction in BCR/ABL signal was observed post-transplant (p < 0.001). However, the BCR/ABL translocation was detected in four of five transplanted patients immediately (0.75-2.5 months) following transplant and was found in patients with a low expression of the translocation.

Clinical detection of BCR-abl fusion by in situ hybridization in chronic myelogenous leukemia.

We describe the use of the fluorescence in situ hybridization (FISH) technique to detect residual Philadelphia chromosome-positive (Ph+) cells in a patient with blastic phase chronic myelogenous leukemia (CML) after aggressive cytoreductive treatment. The analysis was made in interphase nuclei because of the very small number of recognizable metaphases in leukemic patients. FISH was a reliable tool for the detection of chromosome translocations in interphase nuclei as compared with conventional cytogenetic and polymerase chain reaction (PCR) techniques.

Detection of bcl rearrangements in B-CLL by fluorescence in situ hybridization.

Data concerning oncogene activation in CLL are very limited. When studied by Southern blot, rearrangements of bcl-1, bcl-2, and bcl-3 have been only infrequently reported. We evaluated the role of fluorescence in situ hybridization (FISH) in the detection of gene rearrangements in two CLL patients. We used multiple DNA probes, including those of chromosome 12, immunoglobulin heavy and light chains, and the oncogenes bcl-1, bcl-2, and bcl-3. Additionally, routine cytogenetic study was performed. In one patient, trisomy 12 and bcl-2 translocation were demonstrated by both methods, while trisomy 12 and bcl-1 translocation were seen in the second patient, who had a normal karyotype. Larger studies should evaluate the role of FISH in the detection of oncogene involvement in CLL and compare it with other molecular methods.

The BCL-1, BCL-2, and BCL-3 oncogenes are involved in chronic lymphocytic leukemia. Detection by fluorescence in situ hybridization.

The putative oncogenes BCL-1, BCL-2, and BCL-3 are commonly rearranged by translocations to the immunoglobulin genes in B-cell malignancies. However, Southern blotting rarely detected their involvement in chronic lymphocytic leukemia (CLL). This discrepancy could stem from some unique features of the oncogenesis of CLL or be due to shortcomings of Southern blotting. We have therefore evaluated the role of fluorescence in situ hybridization (FISH) in the detection of these oncogenes in CLL. Twenty consecutive CLL patients were studied by FISH for the detection of BCL-1, BCL-2, or BCL-3 rearrangement and for the presence of trisomy 12. Selected patients were also evaluated by classical cytogenetic techniques and by Southern blot analysis. Juxtaposition of JH and BCL-1 was demonstrated in 10 (50%), BCL-2 in three (15%), and BCL-3 in four (20%) of the patients. Trisomy 12 was detected by FISH in 11 (55%) patients. The coexistence of trisomy 12 and translocation of the BCL-1 oncogene was common. Three of the patients had chromosomal aberrations compatible with those detected by FISH. In contrast, in none of the five patients selected by their positive FISH findings was a rearrangement demonstrated by Southern blotting. We conclude that FISH is a sensitive method for the detection of oncogene involvement in CLL. Mainly BCL-1, but also BCL-2 and BCL-3, are commonly translocated to the immunoglobulin heavy chain locus on chromosome 14. These translocations are often associated with trisomy 12. These findings indicate that the BCL oncogenes are commonly involved in CLL and lend support to the multi-hit theory of cancer development.

Preliminary report on the use of the combined multiload contraceptive intrauterine device (ML Cu-250).

The multiload contraceptive intrauterine device (IUD) (ML Cu-250) is made of polyethylene and contains copper wire which is wrapped around the stem, providing 250 sq mm of exposed copper surface area. The IUD was inserted in 294 women comprising 1884 women-months of use. Four pregnancies and three expulsions occurred. In 13 instances the ML Cu-250 IUD was removed for medical reasons (side effects). The net cumulative pregnancy rate for a 12-month period after insertion was 3.9% and the expulsion rate was 1.6%; the medical removal rate was 8.2%. No perforations or tubal pregnancies occurred. Our results with the ML Cu-250 was compared with our previous experience with other IUDs. It seems that the comparatively low pregnancy, expulsion, and medical removal rates render the ML Cu-250 a good device for clinical use.

Multiple pregnancies following induction of ovulation.

PIP: This article reviews the cumulative data on multiple pregnancies which are the result of ovulation induction. Generally, those patients who require ovulation induction fall into 2 groups--those with primary or secondary amenorrhea, low levels of endogenous gonadotropins, and lack of endogenous estrogen activity, and those with anovulation due to menstrual disorders who have evidence of endogenous estrogen activity. The various ovulation-inducing preparations are discussed and they include gonadotropins ([hPG] human pituitary gonadotropin and [hMG] human menopausal gonadotropin), clomiphene, and agents which inhibit prolactin secretion. A variety of treatment schedules for gonadotropin administration have been advised and nearly all are based on the fact that hPG and hMG result in follicular growth and maturation and that ovulation can be induced by a triggering dose of hCG. A nonoverlapping regimen results in a significantly lower incidence of multiple pregnancies. Clomiphene administration must be varied according to the individual patient. Various combination therapies exist such as clomiphene and hMG or hPG or Parlodel and clomiphene or Parlodel and pergonal or LH-RH. Early diagnosis of multiple pregnancies is important since it enables the early institution of therapeutic measures which are likely to prevent maternal complications. Among the diagnostic methods are clinical examination, ultrasonography, radiography, and determinations of biochemical parameters. These multiple pregnancies can result in maternal complications and its effects on the fetus can be serious as well. Spontaneous abortion, premature delivery, perinatal mortality, and congenital malformations are some of the complications which can occur. There are attempts being made to reduce the incidence of multiple births by monitoring estrogens, monitoring follicular diameter measurements, selecting appropriate patients for therapy, and choosing a suitable regimen of treatment. Common measures taken in the management of multiple pregnancy include bed rest, myometrial relaxants, and suturing of the cervix.^ieng

The effect of intrauterine devices containing zinc and copper on their levels in serum.

Serum levels of copper and zinc were determined in 11 health women in whom Latex Leaf intrauterine devices (IUDs) containing copper and zinc had been inserted. Patients with low levels of serum copper or zinc before insertion usually had increased levels of either of these metals while using the device, but they did not exceed the upper limits of normal values. When the group was considered as a whole, the serum levels of zinc showed a slight tendency to increase with duration of IUD use, whereas there was no statistical difference between the serum levels of copper before and after insertion of the IUD.

PIP: Serum levels of copper and zinc were measured in 111 healthy women in whom Latex Leaf IUDs containing copper and zinc had been inserted. The women ranged from 21 to 41 years of age. Months of use ranged from 1 to 23 with an average of 14.6. The 1st blood sample was obtained before insertion and the 2nd from 1 to 19 months after insertion. Mean level of serum copper in the 1st sample was 1.42 mcg/ml. Age and parity had no effect; a slight tendency for higher levels was observed only in multigravidas. There were no significant changes in mean copper levels even when the levels were correlated with duration of usage. Mean serum zinc level before insertion was 1.1 mcg/ml with no correlation to age, gravidity, or parity. The 2nd sample showed a slight increase, 109% + or -3.25% standard error p .005. This increase correlated with the time the device was in situ. In general, patients with low levels of serum copper or zinc before insertion usually had increased levels after use but these levels did not exceed the upper limits of normal values. This may be due to an initially low level of the copper fraction bound to serum albumin and indicates the existence of some equilibrium between free and bound metal in the system.

Zona-free hamster ovum penetration assay as a screening procedure for in vitro fertilization.

Twenty couples with primary sterility due to mechanical factors entered our program of IVF. In addition to routine sperm evaluation, an SPA was performed in all cases. Whenever the SPA was pathologic, none of the human ova were fertilized in vitro, while 77% of the patients with a normal SPA had IVF of at least one human ovum. The good correlation between pathologic SPA and failure of IVF stresses the clinical value of this assay. The use of the SPA for patient selection for IVF is suggested.