Variable region gene analysis of an isotype-switched (IgA) variant of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia of B-cell origin (B-CLL) is generally thought to arise by neoplastic transformation of B lymphocytes, which express CD5 and have features of an early stage of B-cell differentiation. To study isotype-switched B-CLL as a potentially more differentiated variant, we performed genetic and functional immunoglobulin (Ig) gene analysis in two cases of CD5+ B-CLL in which the peripheral blood mononuclear cells (PBMC) secreted predominantly IgA (CLL-249) or IgG (CLL-412) when stimulated with pokeweed mitogen in vitro. By cDNA sequencing and by studies of CLL-heterohybridomas, CLL-249 expresses the heavy chain constant region C alpha as anticipated, while CLL-412 expresses C mu, not C gamma. In CLL-249, the expressed VH gene is 98% homologous to VH26, a germline VH3 gene that occurs frequently in the fetal repertoire, and which has been associated with anti-DNA specificity. The VL gene of CLL-249 is a lambda VL gene for which the germline sequence is not known. In CLL-412, the VH gene is 100% homologous to the VH1 gene of a published anti-DNA antibody (21/28), and is probably a germline gene sequence; the VL gene is 100% homologous to 15AVKI, also a germline gene. The supernatant antibody of the CLL-412 heterohybridoma is an IgM-kappa, which reacts with ssDNA and cardiolipin. The CLL-249 heterohybridoma secreted IgA-lambda, which bound none of the antigens tested, a finding that may be related to amino acid differences from the probable germline V genes. The demonstration of an in vivo isotype-switched variant, such as CLL-249, suggests that B-CLL may be a heterogeneous group of clonal disorders, of which less common variants may have features of more differentiated B-cell stages, such as isotype switching.


Characterization of adhesion molecules on human myeloma cell lines.

In multiple myeloma, malignant plasma cells are localized in marrow and rarely circulate in peripheral blood. To investigate the role of adhesion proteins in this process, we determined the expression and function of adhesion molecules on cell lines derived from patients with myeloma. The U266, ARH-77, IM-9, and HS-Sultan cell lines strongly expressed beta 1 and alpha 4 integrins (89% to 98% positive), confirming that VLA-4 is the principal integrin on these cell lines. The U266 and IM-9 cell lines also expressed alpha 3 integrin on 15% to 20% cells. In contrast, all lines lacked cell surface alpha 2, alpha 5, and alpha 6 integrin expression (< 5% positive). These cell lines adhered to fibronectin (20% to 40% specific binding), without significant binding to either collagen or laminin. Adhesion of these cell lines to fibronectin was partially blocked with either anti-beta 1 integrin monoclonal antibody (MoAb) (75% inhibition), anti-alpha 4 integrin MoAb (75% inhibition), or RGD peptide (50% inhibition), but was unaffected by anti-alpha v beta 3 or anti-alpha IIb beta 3 MoAbs. Moreover, the combination of anti-beta 1 plus RGD peptide or anti-alpha 4 plus RGD peptide inhibited binding to fibronectin by 80% and 95%, respectively. Finally, pretreatment and coculture of the IM-9 cell line with interleukin-6 (IL-6) resulted in a 52% decrease in specific binding to fibronectin (30% +/- 6% to 15% +/- 6%; P = .001), associated with a decrease in the number of cells expressing VLA-4 and a decrease in intensity of VLA-4 expression. These data suggest that myeloma cells adhere to fibronectin through VLA-4 as well as through RGD-dependent mechanisms, and that this binding can be downregulated by IL-6. Future studies of binding of both myeloma cell lines and freshly isolated tumor cells to extracellular matrix proteins and to marrow stroma may enhance our understanding of localization and trafficking of cells within the bone marrow microenvironment.


A new in situ hybridization technique for spliced RNA species documents the bone marrow origin of pulmonary macrophages in chronic myelogenous leukemia.

Tissue macrophages derive from monocytes of bone marrow origin. Because monocytes from patients with chronic myelogenous leukemia (CML) contain the Philadelphia chromosome (Ph), it seemed probable that tissue macrophages in CML would originate from the malignant clone. Using powerful molecular techniques, we studied pulmonary alveolar macrophages (PAM) from two patients with CML. PAM from Patient 1, a patient in chronic phase studied before bone marrow transplantation (BMT), contained the Ph by Southern blot analysis. Patient 2, an accelerated phase patient, was studied after post-BMT relapse. PAM from this patient not only contained the Ph, but also expressed the BCR/ABL message documented by a new splice junction in situ hybridization technique. This new technique allows detection of BCR/ABL mRNA and determination of splice useage in individual cells. These data confirm the continued replenishing of PAM from peripheral blood monocytes in non-BMT settings and represent the first direct evidence that tissue macrophages are derived from the malignant clone in patients with CML.


Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation.

To investigate whether somatic hypermutation occurs in multiple myeloma (MM) Ig VH region genes, we have cloned and sequenced the expressed VH genes from five cases of MM. The sequences were obtained after polymerase chain reaction (PCR) on total RNA isolated from the bone marrow, using 5' VH family-specific leader and 3' C gamma- or C alpha-specific primers. MM-specific CDR3 oligonucleotides were produced to isolate VH genes expressed by the malignant plasma cells. In all five cases, the productive Ig gene used the VH3 family. Extensive sequence analysis of multiple independent M13 clones showed no intraclonal variation with no evidence for ongoing somatic hypermutation in MM VH region genes. We were able to identify possible germline counterparts of the expressed VH genes in two cases. Comparison of these genes shows that the MM VH region genes have somatic mutations characteristic for an antigen-driven process. In the other three cases, no close homology could be found with published VH3 sequences. These findings implicate that, in MM, clonal proliferation takes place in a cell type that has already passed through the phase of somatic hypermutation.


Cell cycle kinetics in malignant lymphoma studied with in vivo iododeoxyuridine administration, nuclear Ki-67 staining, and flow cytometry.

Cell cycle kinetics of malignant lymphoma were investigated using in vivo labeling with iododeoxyuridine (IdUrd) and subsequent flow cytometry (FCM) of IdUrd/DNA and Ki-67/DNA. This approach provides an extensive cell kinetic profile from only one single tumor biopsy, including data upon the percentage of S-phase cells, the IdUrd labeling index (LI), Ki-67-derived growth fraction, duration of the S-phase, duration of the G1-phase, potential doubling time, cell production rate, and total cell cycle time. Tissue samples from 33 patients were studied: non-Hodgkin's lymphoma (NHL; n = 22), Hodgkin's disease (HD; n = 7), and reactive hyperplasia (n = 4). In NHL, the percentage of S-phase cells, LI, growth fraction, duration of the S-phase, and cell production rate were significantly correlated with the histologic malignancy grade according to the Working Formulation (P < or = .02). Data found in HD were not essentially different from those in low-grade NHL and reactive hyperplasia. Remarkably, the duration of the S-phase, the duration of the G1-phase, and the total cell cycle time appeared to be rather independent of histologic malignancy grade within the NHL category. A significant correlation was observed between the IdUrd LI and the percentage of S-phase cells, the growth fraction, the potential doubling time, and the cell production rate (P < .001), but not with the duration of the separate cell cycle phases (P > .05). Our data show (1) that it is feasible to obtain detailed information on the in vivo growth characteristics of malignant lymphoma; and (2) that the transition time through the different cell cycle phases widely varies, even within distinct histologic subgroups.


Recombinant toxins containing the variable domains of the anti-Tac monoclonal antibody to the interleukin-2 receptor kill malignant cells from patients with chronic lymphocytic leukemia.

We have previously shown that the variable domains of the monoclonal antibody anti-Tac [anti-Tac(Fv)] can be fused to derivatives of Pseudomonas exotoxin (PE) or diphtheria toxin (DT) to produce recombinant immunotoxins that kill interleukin-2 (IL-2) receptor-bearing cells. We now report that two of these single-chain recombinant immunotoxins, anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), are cytotoxic toward peripheral blood mononuclear cells (PBMCs) from patients with chronic lymphocytic leukemia (CLL). In anti-Tac(Fv)-PE40KDEL, anti-Tac(Fv) is genetically fused to the amino terminus of PE40KDEL, a recombinant form of PE which contains amino acids 253-608 of PE and the -KDEL mutation at the carboxyl terminus. In DT388-anti-Tac(Fv), anti-Tac(Fv) is fused to the carboxyl terminus of the first 388 amino acids of DT. PBMCs from 14 patients were incubated with the recombinant toxins for 60 hours, and [3H]-leucine incorporation was measured. Anti-Tac(Fv)-PE40KDEL was cytotoxic to 7 of the 14 patient samples, with half-maximal inhibition of protein synthesis (IC50) achieved at 1.2 to 9 ng/mL (1.8 to 13 x 10(-11) mol/L). DT388-anti-Tac(Fv) was cytotoxic to 11 of the 14 samples, with IC50s ranging from less than 1 to 250 ng/mL. DT388-IL-2, in which the first 388 amino acids of DT are attached to IL-2, was marginally cytotoxic toward only 4 of 13 CLL samples tested with IC50s ranging from 100 to 550 ng/mL. Trypan blue staining of cells from several patients indicated that inhibition of protein synthesis correlated with cell death. Binding assays using [3H]-anti-Tac indicated that the CLL cells from nine of the patients contained between 400 and 2,500 sites per cell. Cells from another patient, which were resistant to both anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), had less than 100 sites per cell. We conclude that anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv) can kill CLL cells which have low numbers of IL-2 receptors, and should be investigated further for therapy of this disease.


Human common acute lymphoblastic leukemia-derived cell lines are competent to recombine their T-cell receptor delta/alpha regions along a hierarchically ordered pathway.

Rearrangements of the T-cell receptor (TCR) delta locus are observed in the majority of human B-cell precursor acute lymphoblastic leukemias (ALL) with a striking predominance of V delta 2(D)D delta 3 recombinations in common ALL (cALL) patients. Recently, we and others showed that almost 20% of cALL cases are characterized by further recombination of V delta 2(D)D delta 3 segments to J alpha elements, thereby deleting the TCR delta locus in analogy to the delta Rec/psi J alpha pathway in differentiating alpha/beta-positive T cells. We report here that two human cALL-derived cell lines, REH and Nalm-6, are competent to recombine the TCR delta/alpha locus under standard tissue culture conditions. Analysis of different REH subclones obtained by limiting dilution of the initial culture showed a biased recombination of V delta 2D delta 3 to distinct J alpha elements. During prolonged tissue culture, a subclone acquired growth advantage and displaced parental cells as well as other subclones. Frequently, the DJ junctions of REH subclones contained extended stretches of palindromic sequences derived from modified D delta 3 coding elements. The other cell line, Nalm-6, started the TCR delta/alpha recombination with an unusual signal joint of a cryptic recombinase signal sequence (RSS) upstream of D delta 3 to the 3' RSS of D delta 3. The RSS dimer was subsequently rearranged in all investigated subclones to an identical J alpha element. Both cell lines might become valuable tools to unravel the complex regulation of TCR delta/alpha recombination pathways in malignant and normal lymphopoiesis.


Characterization of the murine macrophage mannose receptor: demonstration that the downregulation of receptor expression mediated by interferon-gamma occurs at the level of transcription.

The macrophage mannose receptor (MMR) is a 175-Kd cell-surface transmembrane glycoprotein that is expressed on tissue macrophages where it functions both to mediate the uptake of mannose-rich glycoproteins and as a phagocytic receptor for bacteria, yeasts, and other pathogenic microorganisms. In this report we describe the cloning of the full-length cDNA of the mouse macrophage mannose receptor and we investigate the level at which interferon gamma (IFN-gamma) downregulates mannose receptor expression. The latter is a marker of the functional state of the cell as high levels are expressed on resident and inflammatory macrophages, whereas cells activated by treatment with IFN-gamma have decreased-to-absent cell-surface mannose receptor expression. The murine MMR cDNA contains an open reading frame that predicts a protein of 1,456 amino acids. Transient expression of the protein in heterologous cells shows that this cDNA encodes a functional mannose receptor. The deduced amino acid sequence of this protein has an overall 82% homology with the human mannose receptor and as such, the ectodomain contains an N-terminus that is cysteine-rich followed by a fibronectin type II domain and eight carbohydrate recognition domains (CRDs). The ectodomain is linked to a hydrophobic transmembrane region and a 46-amino acid cytoplasmic tail. All of the eight CRDs are particularly well conserved, especially CRD4, which shows 92% homology with the equivalent region of the human protein. Steady-state levels of murine MMR mRNA were measured in the macrophage cell line J774E, which is known to express the protein at the cell surface. These levels were decreased by a 4- to 8-hour incubation with IFN-gamma, but were almost abolished by overnight treatment with this cytokine. Nuclear run-on experiments showed that IFN-gamma inhibits MMR gene transcription. Therefore, the regulation of mannose receptor expression by IFN-gamma provides a novel system in which to study the mechanisms by which this cytokine represses gene expression.


Exaggerated cation leak from oxygenated sickle red blood cells during deformation: evidence for a unique leak pathway.

An abnormal susceptibility of the sickle red blood cell (RBC) membrane to deformation could compromise its permeability barrier function and contribute to the exuberant cation leakiness occurring during the sickling phenomenon. We examined this hypothesis by subjecting RBCs at ambient oxygen tension to elliptical deformation, applying shear stress in a viscous medium under physiologic conditions. Compared with normal and high-reticulocyte control RBCs, sickle RBCs manifest an exaggerated K leak response to deformation. This leak is fully reversible, is both Cl and Ca independent, and at pHe 7.4 is fully balanced so that Kefflux equals Nainflux. This abnormal susceptibility is also evident in that the K leak in response to deformation occurs at an applied shear stress of only 141 dyne/cm2 for sickle RBCs, as compared to 204 dyne/cm2 for normal RBCs. Fresh sickle RBC membranes contain elevated amounts of lipid hydroperoxide, the presence of which is believed to provide the biochemical basis for enhanced deformation susceptibility. When examined at pHe 6.8, oxygenated sickle RBCs acquire an additional, unbalanced (Kefflux > Nainflux) component to the K leak increment specifically ascribable to deformation. Studies with inhibitors suggest that this additional component is not caused by a known leak pathway (eg, either K:Cl cotransport or the Gardos channel). This abnormal susceptibility of the sickle membrane to development of cation leakiness during deformation probably contributes to the exuberant cation leak taking place during RBC sickling.


Molecular analysis of human glycophorin MiIX gene shows a silent segment transfer and untemplated mutation resulting from gene conversion via sequence repeats.

The human glycophorin (HGp) loci that define the red blood cell surface antigens of the MNSs blood group system exhibit considerable allelic variation. Previous studies have identified gene conversion events involving HGpA(alpha) and HGpB(delta) that produced delta-alpha-delta hybrid genes which differ in the location of breakpoints. This report presents the molecular analysis of HGpMilX, the first example of a reverse alpha-delta-alpha hybrid gene that specifies a newly described phenotype of the Miltenberger complex. A novel restriction fragment unique to the HGpMilX gene was detected by Southern blot hybridization. The structure of the genomic region encoding the entire extracellular domain of the MilX protein was determined. Nucleotide sequencing of amplified genomic DNA showed that a silent segment of the HGpB(delta) gene had been transposed to replace the internal part of exon III in the HGpA(alpha) gene, thereby resulting in the formation of the MilX allele with an alpha-delta-alpha configuration. The proximal alpha-delta breakpoint was found to be flanked by a direct repeat of the acceptor splice site, whereas the distal delta-alpha breakpoint was localized to a palindromic region. This DNA rearrangement, with a minimal transfer of 16 templated nucleotides and a single mutation of untemplated adenyl nucleotide, not only created two intraexon hybrid junctions but transactivated the expression of a new stretch of amino acid residues in the MilX protein. Such a segment replacement may have occurred through the directional transfer from one duplex to the other via the mechanism of gene conversion. The occurrence of HGpMilX as another hybrid derived from parts of parent genes underlines the role of the recombinational "hotspot" in the generation of allelic diversity in the glycophorin family.


O-linked oligosaccharides of glycophorins A and B in erythrocytes of two individuals with the Tn polyagglutinability syndrome.

Tn polyagglutinability syndrome is an acquired condition where erythrocytes express Tn neo-antigen and become susceptible to hemagglutination by the naturally occurring anti-Tn present in normal sera. Early studies had indicated that O-linked N-acetyl galactosamine was the sole serologic Tn determinant, but more recently O-linked NeuNAc alpha 2, 6GalNAc also has been implicated as a Tn antigen (sialosyl-Tn). However, none of these studies were performed on purified glycoproteins. In this report we examine oligosaccharides of glycophorins A and B purified from Tn erythrocytes of two affected individuals to establish how N- and O-linked saccharides differ from normal. Analysis of carbohydrate composition and treatment with N-glycanase showed that the Asn-linked unit of glycophorin A was not affected. O-linked oligosaccharides were obtained by beta-elimination in the presence of tritiated sodium borohydride. The reduced radiolabeled products were fractionated by Bio-Gel P-2 chromatography, and their structures were investigated by comparison with standards, by monosaccharide quantification, and by neuraminidases of known specificities. The results show that Tn glycophorins from both donors contain intact and truncated forms of trisaccharide and tetrasaccharide NeuNAc alpha 2,3Gal beta 1,3GalNAc and NeuNAc alpha 2,3Gal beta 1,3- (NeuNAc alpha 2,6)GalNAc usually present in glycophorins A and B. The truncated forms include the protein O-linked monosaccharide, GalNAc and disaccharide, NeuNAc alpha 2,6GalNAc (major isomer). The presence of intact glycans in the total population of Tn erythrocytes was confirmed by their susceptibility to T activation after treatment with neuraminidase. The proportion of the four species was not identical in glycophorins of these two donors but, in both, the truncated units predominated and the amount of the disaccharide was approximately one half of that of the monosaccharide. The data are consistent with alterations in UDPGal:GalNAc beta 1,3galactosyl transferase that may have multiple molecular origins and with induction of a specific GalNAc protein alpha 2,6 sialosyl transferase in Tn hematopoietic precursor cells. The molecular basis for these alterations awaits further study.


Silent human immunodeficiency virus type 1 infection: a rare occurrence in a high-risk heterosexual population.

A group of 58 heterosexual female partners (FP) of human immunodeficiency virus type 1 (HIV-1)-seropositive hemophiliacs was studied by conventional diagnostic methods such as enzyme-linked immunosorbent assay (ELISA) and Western blot analysis to examine whether any had acquired HIV-1 infection through sexual transmission. A subset of 29 FP were asked to answer a detailed questionnaire concerning their health, use of "safer sex" techniques, and other risk factors for HIV-1 infection. They also had additional blood drawn for CD4 cell analysis, viral cultures, nef, gag, and env immunoblots, and polymerase chain reaction (PCR) analysis to assess the occurrence of "silent" HIV-1 infection in a high-risk seronegative population. Among the 58 FP, three were found to be HIV-1-seropositive on first testing, with no new seroconversions occurring with subsequent testing in the remaining 55. Two seropositive FP had the additional testing and were found to have positive viral cultures, as well as positive PCR results. All of the seronegative FP (n = 24) who had additional testing were negative in viral culture, had negative immunoblots, and had no HIV-1 nucleic acid sequences detected by PCR. Thus, in this population, silent HIV-1 infection appears to be a rare occurrence and antibody testing seems to correlate with the more sensitive techniques of PCR and viral cultures.


Enhancement of T-cell-depleted bone marrow allografts in the absence of graft-versus-host disease is mediated by CD8+ CD4- and not by CD8- CD4+ thymocytes.

Transplantation of T-cell-depleted C57BL/6-Nu/Nu ("nude") bone marrow (BM) into C3H/HeJ recipients, conditioned with 8 Gy total body irradiation plus chemotherapy with the myeloablative drug dimethyl myleran, resulted in poor hematopoietic reconstitution 14 days posttransplant, compared with transplantation with T-cell-depleted BM from normal C57BL/6 donors. Hematopoietic reconstitution of "nude" BM could be improved by the addition of (C57BL/6xC3H/HeJ)F1 thymocytes void of graft-versus-host activity. Enhancement of BM allografting by thymocytes is sensitive to low radiation doses (> or = 5.0 Gy) and can be achieved by transplanting the BM 24 hours before the administration of thymocytes. Fractionation of F1 thymocytes by differential agglutination with peanut agglutinin (PNA) and by fluorescence activated cell sorting showed that this hematopoietic enhancing activity is enriched in the unagglutinated (PNA-) thymocyte fraction and is mediated by PNA- CD8+ and not by PNA- CD4+ thymocytes.


Use of recombinant human granulocyte-macrophage colony-stimulating factor in patients with lymphoid malignancies transplanted with unpurged or adjusted-dose mafosfamide-purged autologous marrow.

The neutropenia-related morbidity and mortality occurring after autologous bone marrow transplantation (ABMT) is increased by marrow purging procedures. While phase I through III clinical trials showed the enhancing activity of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on neutrophil recovery after ABMT with unpurged marrow, controversial results have been reported when purged marrow was used. Therefore, it was the aim of the present study to evaluate the efficacy of rhGM-CSF administration in a group of patients (n = 15) with lymphoid malignancies transplanted in complete remission with mafosfamide-purged (n = 10) or unpurged (n = 5) marrow. Mafosfamide concentrations used for marrow purging were evaluated on an individual basis by means of a recently described technique that destroys the granulocyte-macrophage (granulocyte-macrophage colony-forming units [CFU-GM]) compartment, but spares 50% of the more primitive stroma adherent colony-forming cells (CFU-Blast). rhGM-CSF (10 micrograms/kg/d) was started within 24 hours of ABMT and administered in a 4-hour infusion daily until the absolute neutrophil count (ANC) reached 500 x 10(6)/L and then for 7 more days. Patients receiving mafosfamide-purged or unpurged marrow failed to show any difference in terms of median number of days required to achieve an ANC > or = 500 x 10(6) (13 v 14.0, P > .4) cells/L. As compared with retrospective controls, granulocytic recovery was reduced by a median time of 11 (P < or = .0005) and 5 (P < or = .0005) days for patients grafted with purged and unpurged marrow, respectively. The number of CFU-GM (mean +/- SD) infused per kilogram of body weight was significantly lower in patients who received purged autografts as compared with those receiving unpurged autografts (0.85 +/- 0.79 x 10(4) v 15.7 +/- 9.2 x 10(4), P < or = .0005). The dose of CFU-GM progenitors infused per kilogram of body weight did not correlate (r = .031, P > .05) with the time required to reach an ANC > or = 500 x 10(6) cells/L. The number of CFU-Blast (mean +/- SD) infused per kilogram of body weight was not significantly different between patients who received purged or unpurged autografts (5.05 +/- 2.51 x 10(3)/kg v 6.18 +/- 2.66 x 10(3)/kg, P < or = .375). A statistically significant correlation (r = -.658, P < or = .05) was observed between the number of CFU-Blast infused and the number of days required to reach an ANC > or = 500 x 10(6) cells/L.(ABSTRACT TRUNCATED AT 400 WORDS)


Analysis of T-cell receptor variability in transplanted patients with acute graft-versus-host disease.

T lymphocytes play a pivotal role in graft-versus-host disease (GVHD) and largely contribute to the graft-versus-leukemia (GVL) effect. Most mature T lymphocytes specifically recognize antigens through the alpha/beta T-cell receptor (TCR). Each alpha/beta TCR chain includes a constant region and a variable region, the latter being encoded by V-J alpha or V-D-J beta rearranged gene segments. To better characterize T cells involved in GVHD, V alpha and V beta gene segment usage was analyzed, after cDNA amplification, in peripheral blood mononuclear cells (PBMC) and skin samples from three patients with grade II cutaneous GVHD. At time of GVHD diagnosis (days 11, 22, and 25), when first signs of engraftment were detectable, virtually all V alpha and V beta subfamilies were represented in PBMC RNAs of the three recipients. These results suggest that diversified TCR gene segment expression is observed early after allogenic bone marrow transplantation (alloBMT). Lymphocytes infiltrating GVHD skin also expressed a large series of V alpha and V beta subfamily specificities. However, analysis of the V alpha and V beta amplified products showed substantial differences between PBMC and the skin lymphocyte RNAs. These observations indicate that a large variety of T lymphocytes are present at the disease site, while some of them may be specifically amplified or decreased in response to minor histocompatibility antigens (miHA). Further characterization of the latter T-cell subpopulations should lead to a better understanding of human in vivo responses directed at miHA.


Optimization of busulfan dosage in children undergoing bone marrow transplantation: a pharmacokinetic study of dose escalation.

Busulfan (BU) is a widely used myeloablative and antineoplastic agent in clinical bone marrow transplantation (BMT). The lower incidence of BU-associated toxicities and lower therapeutic effectiveness in young children given BU doses based on body weight (ie, 16 mg/kg) is associated with altered pharmacokinetics of BU; the area under the curve (AUC) of BU concentration versus time is significantly less in these patients than those observed in older children and adults. To optimize BU dosage in young BMT recipients, we developed a dosage regimen based on body surface area (BSA) and determined BU pharmacokinetics and BU-associated toxicities. Seven children (median age, 3.9 years, range, 1.1 to 5.7) undergoing allogeneic or autologous BMT for leukemia received 40 mg/m2/dose BU every 6 hours for 16 doses; BU concentrations were measured in the plasma, and AUCs were determined for each patient after the first and 13th doses. Expressed as a function of body weight, the median BU dosage was 26.4 mg/kg (range, 24.3 to 28.2), a 60% increase over the BU dosage based on body weight. Four patients developed mucositis, and one of them also developed nonfatal hepatic veno-occlusive disease (VOD). No patients receiving 40 mg/m2 BU developed neurotoxicity (eg, seizures) or interstitial pneumonitis. Prompt and sustained engraftment was observed in the allogeneic BMT recipients, and late graft failure was not seen. The mean BU AUCs were 1,105 mumol/L.min (range, 790 to 2,080) after the first dose and 1,022 mumol/L.min (range, 632 to 1,860) after the 13th dose of BU, comparable to the AUCs in adults given 16 mg/kg of BU. These studies suggest that, in young children, BSA-based dosing of BU (40 mg/m2) provides drug exposures (AUCs) closer to adult values with acceptable toxicities and may improve therapeutic effects.


Interference of the IGF system as a strategy to inhibit breast cancer growth.

Experimental evidence suggests that human breast cancer cells can be regulated by the IGF-I and IGF-II present in the tumor stromal elements and/or by the endogenous tumor cell IGF-II in a paracrine or autocrine fashion. Thus, blockade of the receptor signalling pathway could lead to diminished tumor growth. Blockade of the type I IGF receptor by a monoclonal antibody (alpha IR3) has been used as a strategy to demonstrate the importance of the IGF pathway. Although alpha IR3 could not block serum-free growth of breast cancer cell lines, it could inhibit anchorage independent growth in most cell lines in the presence of serum. In vivo, alpha IR3 administered at the time of tumor cell inoculation could inhibit MDA-MB-231 tumor formation in athymic mice; however, inhibition of established tumors was not seen. Moreover, alpha IR3 could not inhibit tumor formation of the MCF-7 cell line in vivo. These results suggest that blockade of the type I IGF receptor can inhibit the growth of some breast cancer cells both in vitro and in vivo. Future anti-growth factor strategies include the combination of anti-IGF receptor antibodies with IGF neutralizing modalities, the dual blockade of growth factor receptors (epidermal growth factor receptor and type I IGF receptor), and combinations of steroid hormone antagonists and anti-growth factor treatments to maximize tumor inhibition.


Insulin-like growth factor expression in breast cancer epithelium and stroma.

The insulin-like growth factors (IGFs) are mitogens for many cancer cell types. In breast cancer cells, IGF-I and IGF-II have both been shown to stimulate cell proliferation. However, IGF-I mRNA has not been found in human breast cancer cell lines, making it unlikely that IGF-I is commonly expressed as an autocrine growth factor for breast cancer cells. Nevertheless, IGF-I mRNA can be detected in breast cancer tissue samples, and in situ hybridization studies have shown that the message originates from the stromal cells adjacent to normal lobules. IGF-II, on the other hand, has been detected in some breast cancer cell lines. In the estrogen receptor positive cell line T47-D, IGF-II mRNA was induced by estradiol. Furthermore, transfection of an IGF-II expression vector into a previously estrogen-dependent cell line resulted in hormone independent growth. Thus, IGF-II can be expressed as an autocrine growth factor in some breast cancers and its expression may, in part, result in hormone independence. Finally, stromal cells obtained from breast tissues showed that IGF-I was commonly expressed in fibroblasts derived from non-malignant biopsy specimens, while IGF-II mRNA was detected in fibroblasts adjacent to malignant tissue. These studies suggest that IGF-II expression may be important in both autocrine and paracrine regulation of breast cancer cell growth.


Can the insulin-like growth factors regulate breast cancer growth?

Many laboratories have demonstrated that polypeptide growth factors stimulate human cancer cell growth in experimental systems. Despite this observation, a central question remains: can inhibition of peptide growth factor action inhibit tumor growth in humans? To answer this question, several other concerns must first be addressed. Which growth factors are critical for tumor growth? What are the specific cellular effectors for each growth factor? Can feasible therapies be designed to interrupt growth factor pathways? This issue of Breast Cancer Research and Treatment explores the relevance of the insulin-like growth factors to breast cancer cell growth.


Expression of IGF-I and IGF-II mRNA in breast tissue.

Expression of IGF-I and IGF-II was studied in human breast cancer tissues by in situ hybridization. IGF-I mRNA was detected only in stromal cells adjacent to normal breast epithelial cells. Stromal cells associated with the tumor cells did not contain IGF-I, nor did malignant or benign breast epithelial cells. In contrast, IGF-II mRNA was found in both the malignant epithelial cells and their adjacent stromal cells. These data imply that stromal cells associated with breast epithelium may switch expression from IGF-I to IGF-II during breast cancer evolution. This appearance of IGF-II expression may identify cancer-associated stromal cells that have a fetal phenotype.


IGF-I and IGF-II expression in human breast cancer xenografts: relationship to hormone independence.

Although the growth of some estrogen receptor (ER) positive breast cancers can initially be hormonally manipulated, all will eventually escape hormonal control. It is possible that the expression of polypeptide growth factors is initially under the control of steroid hormones, while the hormone unresponsive state is characterized by constitutive expression of growth factors. We studied the relationship between hormone responsiveness and IGF expression in xenograft models. The ER+ T61 xenograft was established from a primary breast cancer and has been continually passaged in athymic mice. ER+ MCF-7 cells and ER-MDA-MB-231 cells were grown in tissue culture and then inoculated into athymic mice. ER+ xenograft growth was regulated by estrogen, but with opposite results--T61 xenografts are inhibited by estrogen, while MCF-7 xenografts require estrogen for tumor formation. All xenografts expressed type I and II IGF receptors. Although T61 xenografts also express an alternatively spliced IGF-I mRNA, its expression was not regulated by estrogen. Both xenografts expressed IGF-II in a hormonally regulated manner--T61 levels were depressed by estrogen, while MCF-7 levels were increased. Thus, in these model systems, xenograft regulation of tumor growth is accompanied by parallel changes in IGF-II expression. In the estrogen independent MDA-MB-231 cells, IGF-II was constitutively expressed. These data show that IGF-II expression correlates with estrogen treatment, suggesting that autocrine expression of IGF-II may mediate estrogen-regulated cell growth.


Interactions of pro-cathepsin D and IGF-II on the mannose-6-phosphate/IGF-II receptor.

The mannose-6-phosphate (Man-6P)/IGF-II receptor is a multifunctional receptor which binds with a high affinity on distinct sites two strikingly different classes of ligands: IGF-II, and Man-6P bearing molecules such as lysosomal enzymes or other biologically relevant ligands (TGF beta precursor, EGF receptor, proliferin...). Binding of each ligand on its cognate site is severely decreased in the presence of the other type of ligand, thus revealing that the two distinct sites are strongly interacting (steric hindrance, conformational change). Any imbalance in ligands and receptor concentration in various pathological situations (transformation, tumor, altered hormonal levels...) is thus likely to perturb their associated biological functions in the targeting and routing of lysosomal enzymes or Man-6P ligands or in the autocrine/paracrine IGF-II--induced cellular responses.


Type 1 IGF receptor in human breast diseases.

The first step of the action of IGF1 and IGF2 (IGFs) is their binding to membrane receptors. IGF binding sites have been characterized by competitive binding and cross-linking techniques in human breast cancer cell lines as well as in human breast cancers and in human benign breast diseases. IGF2 is a good competitor of 125I-IGF1 binding to IGF1-R; insulin competes but with a potency 1/100 lower than the IGF1 potency. Chemical cross-linking experiments revealed that the apparent molecular weight of the IGF1-binding sites is 130,000. Alpha IR-3, a murine monoclonal antibody against the IGF1-R, blocks IGF1-binding to this receptor. This antibody inhibits the IGF1-stimulated growth of breast cancer cells. Therefore, the IGF1 specific binding sites correspond to the previously described type 1 IGF receptors (IGF1-R) in normal tissues. Cross-linking experiments with labeled IGF2 resulted in a major band of apparent Mr 260,000-270,000 that was inhibited by unlabeled IGF2 but not by insulin, and corresponds to the type 2 IGF receptor; a second band of apparent Mr 130,000 was inhibited by excess IGFs and insulin (Type I receptor). The alpha-IR3 inhibition of the IGF2 mitogenic activity suggest that IGF1-R partially mediates the growth effect of IGF2 in these cells. We and others have demonstrated that most breast cancer cell lines contain IGF1-R.(ABSTRACT TRUNCATED AT 250 WORDS)


Progestin regulation of insulin and insulin-like growth factor I receptors in cultured human breast cancer cells.

Recent studies indicate that the insulin receptor (IR) content is higher in breast cancer cells than in normal mammary epithelial cells. This observation has been made both in tissue specimens from patients with breast cancer, and in various human cultured breast cancer cell lines. Investigations have now been undertaken to understand the role of progestins in the regulation of the IR and the closely related insulin like growth factors-I receptor (IGF-I-R). Pretreatment of T-47D cultured human breast cancer cell lines with progestins induced a time and dose dependent increase in IR content. This increase was due primarily to an effect of progestins to increase IR mRNA levels. Other steroid hormones including glucocortocoids, estrogen, and testosterone were without effect. In contrast to their up-regulation of the IR, progestins down-regulated the IGF-I-R at the level of mRNA. An analysis of the processes involved revealed that progestins increased the biosynthesis of a ligand for IGF-I receptor, IGF-II. IGF-II in turn down-regulated the IGF-I-R. Thus these studies indicate that progestins have important effects on both the IR and the IGF-I-R. The effects of progestins on these and other growth factor receptors, therefore, may have an important role in the biology of breast cancers.


A phase II study of sulofenur (LY186641) in gastric cancer.

Sulofenur is a novel diarylsulfonylurea with proven anti-tumor activity in murine tumor models. In this phase II study in patients with advanced gastric or gastroesophageal adenocarcinoma, 17 patients were treated with sulofenur orally at 700 mg/m2 for 14 days every 3 weeks. No tumor responses were seen. The main toxicities were anemia, methemoglobinemia and abnormalities in liver function tests. These toxicities precluded dose escalation. However, plasma levels of sulofenur, and its hydroxy and keto metabolites were probably insufficient to exert anti-tumor effect in comparison with data from murine studies. Further structure-activity studies are warranted.


Tamoxifen reduces serum insulin-like growth factor I (IGF-I).

Antiestrogens are widely used in the management of hormonally responsive breast cancer in both adjuvant and palliative settings, and are currently being evaluated as chemopreventive agents. The classical mechanism of action of these drugs involves inhibition of estrogen-stimulated neoplastic cell proliferation by blockade of estrogen receptors present on breast cancer cells. This paper reviews recent clinical and laboratory data that suggest that the commonly used antiestrogen tamoxifen also acts to reduce serum IGF-I levels. Estrogens appear to play a permissive role in growth hormone (GH) release by the pituitary gland and GH is known to stimulate IGF-I expression by hepatocytes. It is therefore possible that blockade of estrogen receptors in the hypothalamic-pituitary axis by tamoxifen interferes with GH release, leading to reduced hepatic IGF-I expression. In view of results suggesting that IGF-I is a more potent mitogen than estradiol for breast cancer cells and data demonstrating a positive correlation between estrogen receptor level and IGF-I receptor level of breast cancer cells, the IGF-I lowering effect of tamoxifen may contribute to the cytostatic activity of the drug. The interrelationships between steroid hormone physiology and IGF-I physiology may have relevance to a variety of commonly used treatments for hormonally responsive cancers.


Hydroxyurea modulates 5-fluorouracil antineoplastic activity in advanced head and neck carcinoma pretreated with chemotherapy.

After informed consent 21 patients with advanced head and neck cancer resistant to folinic acid/5-fluorouracil (FA/5FU + cisplatin) were treated with weekly FA/5FU plus low dose hydroxyurea (HU) to evaluate if HU could further modulate 5FU antineoplastic activity. Five patients achieved a partial response (23.8%) which was short-lived (mean duration 6.5 months). Three patients (14%) had stable disease and 13 (62%) progressed. Among responders, four patients had epidermoidal carcinoma and one had clear cell carcinoma. Treatment was well tolerated and 5FU-related toxicity was not apparently worsened by the addition of HU. The most frequent toxicities were nausea/vomiting (81%), diarrhea (52%) and leukopenia (57%). Grade 3 nausea/vomiting and leukopenia were recorded in only 19 and 9% of cases, respectively. One patient had grade 1 cutaneous toxicity and a second patient showed a hand-foot syndrome. These results suggest that HU may further positively modulate 5FU antineoplastic activity.


Potential antineoplastic activity of keto-C-glycosides--a new family of cytostatic agents.

We have examined the biological activity of keto-C-glycosides (KCGs), a new family of drugs displaying antiproliferative and cytotoxic properties on tumor cells. KCG1, the most powerful drug tested on epithelial derived neoplastic cells, was 25-125 times more cytostatic on epithelial cells than on lymphoma. By contrast, KCG10 proved to be more cytostatic on lymphoma than on epithelial cells. Correlations were found between the cytostaticity of KCGs and their lipophilicity, and are discussed within the framework of the structure-activity and the structure-selectivity relationships.


Late doxorubicin cardiotoxicity.

The occurrence of late congestive heart failure (CHF) as the first clinical manifestation of doxorubicin-induced cardiac toxicity is unusual in children and very rare in adults. However, subclinical cardiac dysfunction is commonly detected in children years after treatment with doxorubicin containing regimens. We report a 58 year old woman who developed stage IV CHF 7 years after completion of doxorubicin treatment for carcinoma of the ovary. Occult cardiac dysfunction was first demonstrated by radionuclide angiography 6 years prior to the occurrence of the clinical manifestations. This unique course of the disease and the management of the CHF are discussed.


Cortical blindness--a catastrophic side effect of vincristine.

The term 'cortical blindness' indicates loss of sight due to bilateral lesions of the occipital lobes. It is a rare, but severe, side effect of chemotherapeutic agents. Cortical blindness was diagnosed in a 67 year old woman with leiomyosarcoma of the large bowel, treated by vincristine-containing chemotherapy. Cortical blindness without focal neurological signs and with two repeated normal brain computed tomography scans, in which there was no structural damage to the occipital lobes, suggests a metabolic or toxic reaction as a cause in our patients. The temporal relationship between vincristine treatment and cortical blindness implicates vincristine as the possible causative agent for this catastrophic phenomenon.


Cisplatin-related Lhermitte's sign.

The sensation of a sudden electrical impulse travelling along the spine to the legs and feet on flexion of the neck has been known as Lhermitte's sign. Lhermitte's sign, as part of cisplatin-related neurotoxicity, was observed in four patients, with ovarian or lung cancer, simultaneously with peripheral neuropathy, after a dose of 375-700 mg/m2. The dose intensity (DI) of cisplatin in our patients ranged from 12.5 to 26.9 mg/m2/week. No direct relationship was found between DI and the timing of Lhermitte's sign. Other relevant causes for this sign were ruled out. The mechanism responsible for the development of Lhermitte's sign is unclear. Interruption of treatment with cisplatin may not prevent the appearance of Lhermitte's sign. In most of the reported cases in the literature this sign developed after the end of cisplatin courses.


Hydroxyurea-induced cell death in human T lymphoma cells as related to imbalance in DNA/protein cycle and deoxyribonucleotide pools and DNA strand breaks.

The aim of this study was to elucidate the mechanism(s) behind the cellular toxicity of therapeutic concentrations of hydroxyurea (HU). Treatment of human T lymphoma cells (CCRF-CEM) with 60-100 microM of HU for 24 h decreased the growth rate by 90% due to accumulation of cells in early S phase. It induced a marked imbalance in both the DNA/protein cycle (as measured by two-parameter flow cytometry) and the deoxyribonucleotide (dNTP) pools. HU treatment did not enhance the frequency of DNA single-strand breaks (SSBs), as measured by the alkaline unwinding technique. Cell viability was unaffected. However, removal of HU led to 10-15% cell loss during the following 12 h period in parallel with increasing SSBs, and a rapid progression of cells through S and G2 stages. The unbalanced DNA to protein content per cell and the dNTP pools were normalized 6-12 and 24 h after removal of HU, respectively. These results show that marked changes in the DNA to protein ratio and dNTP pools alone are not directly lethal, but when combined with a high replicative DNA synthesis rate, as found after removal of HU, apparently lead to elevated cell death.


A comparative study of the antitumor activities of 5'-deoxy-5-fluorouridine and its prodrug trimethoxybenzoyl-5'-deoxy-5-fluorocytidine (Ro09-1390) on human digestive organ cancer xenograft lines transplanted into nude mice.

5'-Deoxy-5-fluorouridine (5'-DFUR) is one of the oral fluoropyrimidines widely used in the treatment of gastric, colorectal and breast cancers in Japan. 5'-DFUR is converted to 5-fluorouracil by pyrimidine nucleoside phosphorylase in the tumor. 5'-DFUR has toxic effects on the intestine and may cause severe diarrhea. Trimethoxybenzoyl-5'deoxy-5-fluorocytidine (Ro09-1390) is a prodrug of 5'-DFUR, which was developed to reduce the intestinal toxicity of 5'-DFUR. The present study was designed to assess the antitumor activity and spectrum of Ro09-1390, and to compare its efficacy with that of 5'-DFUR. Six digestive organ cancer xenograft lines (two gastric, one esophageal, one colorectal, one gall bladder and one bile duct cancers) were s.c. transplanted into nude mice. The agents were orally administered daily for 14 days at doses of 0.08-0.64 mmol/kg (1-8 times the maximal clinical dose of 5'-DFUR). Both 5'-DFUR and Ro09-1390 significantly inhibited the growth of two gastric cancer lines, and the IC50's for Ro09-1390 in both lines were lower than the respective values for 5'-DFUR. The esophageal, colorectal, gall bladder and bile duct cancer lines, however, were resistant to both agents. 5'-DFUR at 0.64 mmol/kg significantly inhibited the growth of these cancers, but with high mortality, and most mice receiving this dose died within 14 days after the start of therapy, suffering from severe diarrhea and body weight loss. Ro09-1390 at the same dose resulted in low mortality, but evidenced similarly low antitumor activity.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of resistance to etoposide and vincristine in vitro and in vivo after X-irradiation of ovarian tumor cells.

Two human ovarian carcinoma cell lines (JA-T/P and SK-OV-3/P) were exposed to 10 fractions of 5 Gy X-irradiation in vitro. Surviving populations generated sublines designated DXR-10 which expressed significant resistance to etoposide (VP-16) and vincristine (VCR), but not to adriamycin (ADR) or acute X-irradiation, as judged by clonogenic assays. JA-T/P and JA-T/DXR-10 tumor cells were xenografted into nude mice and treated with a single dose of VCR (1.8 mg/kg), VP-16 (24.5 mg/kg) or ADR (10.0 mg/kg) and 48 h later the surviving clonogenic cells in each tumor were quantitated. Significantly fewer colonies grew from the JA-T/P xenografts treated with either VCR or VP-16, as opposed to the JA-T/DXR-10 tumors, whilst comparable colony numbers were recorded after ADR treatment. These data suggest that the resistant phenotype following exposure to fractionated X-irradiation in vitro is also expressed in vivo.


Influence of O6-methylguanine on DNA damage and cytotoxicity of temozolomide in L1210 mouse leukemia sensitive and resistant to chloroethylnitrosoureas.

Temozolomide is a new anticancer agent which in the early clinical investigation has shown promising antitumor activity. It decomposes spontaneously to the active metabolite of DTIC (MTIC). Temozolomide is more cytotoxic against L1210 than against a subline L1210/BCNU, resistant to chloroethylnitrosoureas. Using [methyl-3H] temozolomide we found that after 1 h exposure the amount of O6-methylguanine (O6mGua) was twice as high in L1210 than in L1210/BCNU whereas the amount of N7 mGua was approximately the same in the two cell lines. O6-alkylguanine DNA alkyltransferase (AT) levels were higher in L1210/BCNU than in L1210, supporting the view that the resistance to methyltriazenes is probably related to the efficient repair of O6mGua in L1210/BCNU. Exposure of L1210/BCNU cells to 0.4 mM O6mGua for 24 h resulted in a depletion of AT and in a higher temozolomide-induced cytotoxicity. In the sensitive cell line L1210, temozolomide activity was not potentiated by O6mGua pretreatment. Moreover, in L1210/BCNU, O6mGua increased DNA single-strand breaks caused by temozolomide, suggesting that O6-guanine alkylation induces an excision repair mechanism in cells depleted in AT.


Interferon modulation of 5-fluorouracil: use in neoadjuvant therapy inhibits experimental liver metastases in nude mice.

Experimental liver metastasis was studied in 4-5 week old athymic nude mice that were injected intrasplenically with a human colorectal tumor cell line (LoVo). A treatment schedule combining 5-fluorouracil and interferon (IFN) was previously shown to inhibit liver metastases. When this treatment was delayed until after splenectomy at 1, 2 and 3 weeks after tumor cell injections, liver metastases were not inhibited. However, when IFN was given during the interval between tumor cell injections and splenectomy (as neoadjuvant therapy), liver metastases were inhibited in the 2 and 3 week groups, but not in the 1 week group.


SDZ PSC 833 and SDZ 280-446 are the most active of various resistance-modifying agents in restoring rhodamine-123 retention within multidrug resistant P388 cells.

Multidrug resistance (MDR) of tumor cells may result from overexpression of P-glycoprotein (Pgp) but may be down-modulated by resistance-modifying agents (RMAs). The cyclosporin SDZ PSC 833 and the cyclopeptolide SDZ 280-446 were found to be the strongest RMAs known to date for restoring the sensitivity of MDR cells to anticancer drugs, as well as for restoring their retention of daunomycin, a fluorescent anthracycline. Using rhodamine-123 (Rhod-123), another fluorescent probe of Pgp function which also differentiates sensitive and MDR cells, several RMAs were compared for their capacity to inhibit Pgp function. At variance with the data obtained with the daunomycin probe, a series of RMAs did not detectably restore Rhod-123 retention by the MDR cells. With the remaining RMAs, achieving the same levels of Rhod-123 retention required 3 times lower RMA concentrations when the RMA was added to the MDR cells for both the initial uptake and the efflux of Rhod-123 rather than for its uptake only. Nevertheless, the data emphasized the large superiority of SDZ PSC 833 and SDZ 280-446 over all other RMAs.


Cyclophosphamide-induced suppressor cells in nude mice.

Lymphocytes regenerated after treatment with a high dose of cyclophosphamide (CY) were characterized in nude mice. Ten days after a single injection of 200 mg/kg CY into nude mice, regenerated spleen cells suppressed in vitro primary and second antibody production against sheep red blood cells. The CY-treated spleen cells exhibited normal natural killer (NK) activity, very low B and T cell content, but increases in cell surface charge [electrophoretic mobility (EPM)] and histamine receptors. The suppressor cells could not be removed by treatment with anti-Thy-1 plus complement (C), or treatment with antiasialo GM1 (aGM1) plus C, which abrogated NK activity. It was concluded that CY-treated spleen cells, which exhibited high EPM and histamine receptors, comprise the natural suppressor cells which are Ig-, Thy-1- and aGM-1.


A diapause associated protein of the pink bollworm Pectinophora gossypiella Saunders.

A diapause associated protein was electrophoretically isolated from the hemolymph of diapausing last instar larvae of the pink bollworm Pectinophora gossypiella. This protein (M(r) approximately 490,000, glycolipoprotein) was given the name Pectinophora diapause protein (PDP). It is composed of one subunit (M(r) 103,000). The concentration of PDP increased dramatically in the hemolymph of diapausing larvae from 17.4% in prediapause (PD) phase to 29.2% in early diapause (ED) phase reaching a level of 38.6% in larval hemolymph of middiapause (MD) phase. The concentrations of total proteins in the hemolymph of active feeding (A), PD, ED, and MD larvae were 69.8, 106,6, 113.3, and 118 mg/ml, respectively, while those in the fat body of the same larvae were 7.1, 7.4, 8.8, and 4.5 mg/g, respectively. In Pectinophora a drop in the concentration of fat body proteins coincided with a corresponding increase in hemolymph proteins, which suggests an active release of protein from the fat body into the hemolymph during the development of diapause. A partial amino acid sequence of pectinophorin showed the first 15 amino acids starting from the amino terminus of the peptide chain: N-ALA-LYS-THR-ILEU-VAL-GLU-ASN-MET-PRO-PRO-THR-PRO-LEU-ASN-ALA-C.


Development of specific RIA and ELISA to study trypsin modulating oostatic factor in mosquitoes.

Trypsin modulating oostatic factor (TMOF), a decapeptide (H-YDPAPPPPPP-OH) that signals the termination of trypsinlike enzyme biosynthesis in the mosquito midgut, was covalently bound to Keyhole Limpet Hemocyanin using N-hydroxysuccinimide and dicyclohexylcarbodiimide. Polyclonal antibodies raised in rabbits against this conjugate were used to develop specific RIA and enzyme linked immunosorbent assay (ELISA) to detect the peptide hormone in female Aedes aegypti. TMOF and its analogs TMOF(B) (H-DYPAPPPPPP-OH), P4 (H-YDPAPPPP-OH), P1 (H-YDPAP-OH), and poly-L-proline were tested with the antiserum. The antiserum fully recognized TMOF and partially recognized P4. Using both RIA and ELISA, we report that the amount of TMOF in the mosquito ovary is 100 +/- 20 ng (S.E.) and 96 +/- 1.4 ng (S.E.), respectively, for each assay. Minute quantities of TMOF a thousandfold lower than in the ovary were found in the mosquito brain, indicating that the hormone is probably not neural but ovarian in origin.


Oral toxicity to flesh flies of a neurotoxic polypeptide.

An insect selective neurotoxic polypeptide from venom of the scorpion Androctonus australis (AaIT, M(r) 8,000) was shown to cross the midgut of the flesh fly Sarcophaga falculata, using assays of oral toxicity, column chromatography, and microscopic autoradiography of the native and radioiodinated toxin. AaIT induced paralysis of flies within 1-2 h after oral administration, with a lethal dose (LD50) of 10 micrograms/100 mg of body weight. Oral toxicity was about 0.14% of toxicity by injection. Hemolymph collection 70-85 min after feeding flies with [125I]AaIT showed that 5% of ingested radioactivity appeared in hemolymph. Most of this represented degradation products, but included about 0.3% of the chromatographically intact toxin. In contrast, hemolymph of identically treated lepidopterous larvae (Manduca, Helioverpa [= Heliothis]) contained degradation products but no intact toxin. [125I]AaIT was shown to cross the midgut of Sarcophaga through a morphologically distinct segment of the midgut previously shown to be permeable to a cytotoxic, positively charged polypeptide of similar molecular weight. These results suggest that Sarcophaga midgut contains a morphologically and functionally distinct segment that transports small peptides, and that employment of neurotoxic polypeptides for insect control may be feasible. Activity might be greatly improved through modification and metabolic stabilization of active peptides.


Anthropometric assessment of the nutritional status of patients with liver cirrhosis in an Italian population.

A poor nutritional status has repeatedly been described in advanced liver cirrhosis, but the exact prevalence of the defect and its relation to the aetiology and severity of liver disease in the Italian population are only partly known. Anthropometric measurements were carried out in 200 patients with cirrhosis (135 M, 65 F). Liver disease was related to alcohol abuse in 77 cases, but most patients had stopped alcohol for at least 6 months before study. In comparison to a normal elderly Italian and to an age-matched North-American population, 5 to 45% of male patients with cirrhosis and 10 to 30% of females had signs of malnutrition, the proportion being variable according to the test used. Male patients showed a remarkable reduction in muscle mass (30-45% of patients, mainly in the presence of moderate-to-severe or severe liver failure), whereas female patients showed a more remarkable reduction of fat stores (15-30% of cases), with advancing liver failure, and a less severe reduction in muscle mass. No direct effect of alcohol was demonstrated in this selected population.


Interdigestive antro-duodenal motor disorders in functional dyspepsia. Associated chronic gastritis correlates with a further motor impairment.

The interdigestive antro-duodenal motor activity was studied in 12 dyspeptic patients without gastritis (group A), 12 dyspeptic patients with chronic superficial antral gastritis (group B) and 8 healthy controls (group C) by manometric recording of phases of the interdigestive motility cycle (IDMC) prolonged over 240 minutes. A significantly lower incidence of migrating motor complexes (MMCs) starting from the antrum was observed in dyspeptic patients with gastritis vs dyspeptic patients without gastritis (p = 0.02) and in these latter vs controls (p < 0.01). Likewise, the overall number of MMCs was smaller in patients with gastritis vs controls (p = 0.02). Symptomatic evaluation was performed in the two groups of dyspeptic patients, without detecting any differences in frequency and severity of individual and overall complaints. Our results confirm the role of interdigestive antral motor abnormalities in the genesis of dyspeptic syndrome and suggest that the presence of associated chronic superficial gastritis may correlate with a further motor impairment.


Clinicopathological assessment of gastric biopsy samples of patients with Helicobacter pylori infection--metronidazole resistance and compliance problems in the United Arab Emirates.

The significance of Helicobacter pylori (HP) infection was assessed prospectively in forty-two patients with dyspepsia using histological, bacteriological and biopsy urease techniques. Thirty-eight patients (90.5%) were positive for HP infection and were treated with bismuth subcitrate (De Nol), tinidazole and doxycycline. HP was present in the antrum, corpus, fundus, duodenum and gastric juice in 36, 26, 23, 2 and 2 patients respectively (p < 0.01, X2 test). Histological assessment yielded more positive identifications of HP than the urease test (36 vs 28 positive cases, p < 0.01, McNemar's X2 test), while histology and bacteriology were virtually identical (38 vs 37 of 41 pairs, p > 0.5, X2 test). There was a good correlation between bacterial and polymorphonuclear leucocyte (PMNL) counts per high power field (r = 0.8; p < 0.001; n = 34 pairs). There was resistance to metronidazole in 10 out of 16 isolates, but no resistance was recorded against tetracycline (p < 0.001, X2 test). Among the sixteen patients who attended follow-up endoscopy, there was clinical improvement and no evidence of HP in 5 individuals (31.25%). One patient had amelioration of his symptoms, 5 experienced no change and in 5 their symptoms became worse. Metronidazole resistance may be one of the important factors in the United Arab Emirates and elsewhere.


Extrahepatic abdominal hydatidosis: surgical features of 11 cases.

We have described 11 cases of rare abdominal localization of hydatosis in the kidney, spleen and serous tissues. The clinical and surgical features have also been discussed.


The menstrual cycle has no effect on gastrointestinal transit time. Evaluation by means of the lactulose H2 breath test.

Gastrointestinal transit time was studied by means of the H2 breath test (after administration of 20 g of lactulose) in 20 healthy women with regular menstrual cycles. The mean transit time measured in the follicular phase of the cycle (M +/- SE = 71.25 +/- 6.69 min) did not differ statistically from that measured in the luteal phase (66.75 +/- 5.49 min). Data were statistically analyzed using Student's t test for paired data and the Wilcoxon Test for non-parametric data. Our results show that, in physiological conditions, female sex hormones do not affect mouth-to-caecum transit time. The H2 breath test is easy to perform and well tolerated; it thus proves particularly suitable for use in women of child-bearing age.


Antigliadin antibody classes in chronic liver disease.

Antigliadin antibody (AGA) subtypes (IgG and IgA class) were tested in sera from 67 patients with chronic liver disease of different aetiology (29 with primary biliary cirrhosis (PBC), 31 with chronic non-A non-B hepatitis, and 7 with autoimmune chronic active hepatitis (CAH) compared with 23 subjects with inflammatory bowel disease (IBD). Nineteen patients with coeliac disease served as positive controls. IgA-AGA alone were found in 3.4% of patients with primary biliary cirrhosis and in 3.2% of non-A, non-B CAH. IgG-AGA alone were found in 1.3% of patients with IBD, in 6.8% of primary biliary cirrhosis and in 14.2% of autoimmune CAH. IgA-AGA and IgG-AGA together were found in 6.8% of PBC and in 1 patient with autoimmune CAH. Jejunal biopsy, performed in 7 out of the 2 patients with both IgA and IgG-AGA, showed the characteristic features of coeliac disease in one subject with autoimmune CAH. The same patient had the highest titre of AGA. In conclusion, these results indicate that AGA (either IgG and IgA) can be present at low titre in chronic liver disease and their presence may be secondary to the liver damage per sè. High titres of AGA in chronic liver disease may suggest a real association with coeliac disease.


Abdominal attack of hereditary angioedema associated with marked leucocytosis. A case report.

Hereditary angioedema (HAE) attacks are not usually associated with an increase of peripheral blood leucocytes. In a 25-year-old woman suffering from HAE a marked leucocytosis (31,000/mm3) with polynucleosis was observed during a severe attack of the disease. Awareness of the underlying disease, physical examination and the results of instrumental investigations allowed for appropriate treatment avoiding invasive procedures. A leucocytosis of the severity observed in our patient suggests that the abdominal attack of HAE can involve the peritoneum as well as the intestinal mucosa. Thus, in a patient with an attack of abdominal pain the presence of leucocytosis does not exclude the diagnosis of HAE.


Computed tomography in the identification of gallstones likely to respond to UDCA.

An unusual case of a patient with two biliary stones of different densities that responded differently to oral litholytic treatment with ursodeoxycholic acid (UDCA) is reported. The findings confirm, in an unusual experimental model, that CT is useful in the selection of those stones which have a high probability of dissolution under treatment with oral bile acids.


Chronic bleeding caused by small bowel angiodysplasia: a problem of recognition and diagnosis.

Angiodysplasia of the gastrointestinal tract is a disorder consisting of ectasia of submucosal and mucosal vessels, which causes acute and chronic bleeding. We describe the case of a 58-year-old man with a history of recurrent lower intestinal bleeding and severe anemia. Endoscopy and X-ray examination of the gastrointestinal tract failed to show the source of bleeding. Selective angiography of the superior and inferior mesenteric arteries revealed a vascular ectasia. The patient underwent ileo-cecal resection with remarkable improvement after surgery.


Factors that determine the cost and performance of early identification protocols.

In this paper, a simple model is used to examine the relation between important factors, such as prevalence of hearing loss, and protocol performance and cost. Protocol hit rate is determined primarily by the hit rate of the screening protocol and follow-up percentage, the percentage of infants who return for diagnostic testing. Many factors influence protocol cost. Not only does absolute cost change as certain model parameters are varied, but the relative cost of different protocols can change as well. The false alarm rate of the screening protocol and the follow-up percentage have the greatest impact on protocol cost.


Predictability and reliability of hearing aid benefit measured using the PHAB.

This investigation explored the extent to which self-assessed hearing aid benefit measured by the Profile of Hearing Aid Benefit (PHAB) could be predicted from adaptation to hearing loss and/or from communication difficulties reported without amplification. Adaptation to hearing loss was measured using 13 scales of the Communication Profile for the Hearing Impaired (CPHI). These were combined to produce three composite CPHI scores. Results from 58 experienced hearing aid wearers indicated that benefit was significantly related to magnitude of unaided difficulties for all seven PHAB subscales. In addition, one of the three composite CPHI scores contributed to benefit prediction for the two sound perception subscales of the PHAB. Test-retest reliability of PHAB subscale scores was evaluated for 28 subjects using correlations and difference distributions. Reliability was found to be consistent with previous studies but modest. Critical differences were large compared with the anticipated size of benefit differences due to, for example, different hearing aid prescriptions. It is concluded that the PHAB is best suited for group research: when used for individual subjects, the PHAB may not be sensitive enough to detect important differences between hearing aid conditions.


Effects of interaural speech-recognition differences on binaural advantage for speech in noise.

The relationship between interaural differences in speech-recognition performance and binaural advantage for speech in noise was studied. Subjects were five hearing-impaired listeners with symmetrical pure-tone air-conduction audiograms and significant interaural differences in speech recognition. All subjects repeated nonsense syllables in the presence of competing noise in monaural and binaural conditions. Binaural advantage was calculated as the difference in signal-to-noise ratio that afforded 50 percent correct performance between the monaural and binaural conditions. The results were diverse. Although the majority of the subjects retained some degree of binaural advantage, a conclusive relationship between interaural differences in speech recognition and binaural advantage could not be established. The implications for binaural hearing aid fittings are discussed.


Acoustic-immittance screening for detection of middle-ear effusion in children.

The purpose of this investigation was to evaluate the sensitivity and specificity of the following acoustic-immittance protocols and their constituent measures for detection of middle-ear effusion in children: (a) tympanometric width; (b) absent ipsilateral acoustic reflex; (c) ASHA guidelines; (d) tympanometric peak pressure; and (e) static-acoustic middle-ear admittance. The middle-ear sample was composed of 82 ears of 54 subjects ranging in age from 3 to 11 years. The control (normal-hearing, normal middle-ear) sample was composed of 53 ears of 53 subjects ranging in age from 3 to 10 years. Each subject was given a complete otolaryngologic evaluation (including pneumotoscopy and/or microtoscopy) and audiologic (including acoustic-immittance) evaluation. In the group of middle ears with normal-hearing sensitivity, the sensitivity and specificity of the ASHA guidelines were 63 percent and 79 percent, respectively. An acoustic-immittance screening protocol, based on all of the individual acoustic-immittance measures, and characterized by high sensitivity and specificity, is proposed.


Effects of notch noise bandwidth on the auditory brainstem response to clicks.

The effects of notch width of notch masking noise on the auditory brainstem response (ABR) were investigated with ten normal-hearing subjects. Wave V latencies and amplitudes were measured to a click in quiet and in the presence of noise with notches centered around 1000 Hz and 4000 Hz. Notch width was either 0.5, 1.0, 1.5, 2.0, or 2.5 octaves. A 95 dB SPL broadband noise was necessary to mask a 65 dB nHL click at an effective level of 73 dB nHL. All ten subjects at both 1000 Hz and 4000 Hz yielded identifiable responses for the click in quiet and for the 2.5 octave and 2.0 octave conditions, and eight or nine subjects responded to the 1.5 octave notch noise. In contrast, no responses were observed to the 0.5 octave condition, and only three or four subjects responded to the 1.0 octave notch noise. These findings suggest that when ABRs are obtained to 65 dB nHL clicks in notch noise, the notch width should exceed 1.0 octave. Moreover, because of the relatively low amplitudes elicited with the 1.5 octave bandwidth, it appears preferable to select notches that are 2.0 to 2.5 octaves wide. Considering the wide bandwidth required, the high noise levels necessary to mask clicks, high ABR thresholds, and the difficulty setting the signal-to-noise ratio, it appears that tonepips are more promising than clicks in notch noise for assessing frequency specific ABRs.


Adaptation of the auditory brainstem response: effects of click intensity, polarity, and position.

The aim of this research was to study the effects of several stimulus parameters on adaptation characteristics of the auditory brainstem response (ABR). Recordings were obtained from normal hearing adults to separate trains of 5 rarefaction and 5 condensation clicks with interclick intervals of 10 msec and intertrain intervals of 500 msec at three intensities. Absolute latencies for waves I, III, and V were essentially unchanged by polarity; latency shifts, however, were induced by parametric manipulations of click intensity, polarity, and position in the train. Furthermore, variations in ABR wave morphology appeared with changes in intensity and polarity. Adaptation, as measured by amplitude, was considerable; the measures of adaptation, however, were not related in a simple manner to the latency shifts. The findings indicate that adaptation of the ABR is a consequence of an interplay of central and peripheral processes to click polarity, sequence, and intensity. Finally, the results provide evidence that when fast stimulus presentation rates are used to estimate thresholds rapidly, one must be aware that certain stimulus parameters can alter the ABR waveforms.


Otoacoustic emissions, audiometric sensitivity loss, and speech understanding: a case study.

The clinical measurement of otoacoustic emissions can assist in differentiating between peripheral and central explanations for deficits in speech understanding. We present audiometric and distortion-product emission data in a case with sensorineural hearing loss and a deficit in speech understanding. The presence of evoked emissions argues against attributing the speech audiometric loss to cochlear defect.


High-frequency thresholds: sound suite versus hospital room.

Benefits of high-frequency audiometry in monitoring hearing sensitivity of patients administered ototoxic medications are well established. Thresholds obtained within a sound suite have been proven reliable. It may, however, often be necessary for the audiologist to evaluate the patient at bedside. The primary purpose of this study was to determine if significant differences are present between high-frequency thresholds measured in a sound suite versus thresholds measured in a hospital room. In addition, the test-retest reliability of high-frequency thresholds was determined when measured in a hospital room. For 25 normal hearing subjects, results revealed that significant differences were not observed between thresholds measured in a sound suite versus those measured in a typical hospital room. In addition, differences between the initial and repeated thresholds obtained in the hospital room were not significant, and the differences were, for the most part, within +/- 10 dB at all test frequencies.


Effects of interstimulus interval on slow phase velocity to ipsilateral warm air caloric stimulation in normal subjects.

This study investigated the effects of interstimulus interval on slow phase velocity (SPV) to ipsilateral warm air caloric stimulation in normal subjects. Results suggest that about 3 minutes should intervene between the offset of one irrigation and the onset of the second irrigation. This finding supports the hypothesis that carryover effects are likely only when nystagmus from the preceding irrigation overlaps the subsequent irrigation. If correct, clinicians do not have to wait a fixed time period between stimuli, but can initiate caloric stimulation as soon as nystagmus has ceased from the preceding irrigation. This recommendation compensates for individual and procedural differences. Test-retest reliability also was investigated. Findings suggest that when immediate test-retest differences exceed approximately 6 degrees/second (95% confidence interval), the examiner should administer additional trials until stability is ascertained. Moreover, unusual or significant findings should be verified with repeat testing.


SPIN test performance of elderly hearing-impaired listeners.

Normative information is available on the expected performance of hearing-impaired individuals on the revised version of the Speech Perception in Noise (SPIN) test. This normative sample includes individuals with sensorineural hearing loss drawn from an age range of 19 to 69 years, with 79 percent of the sample being less than 65 years of age. In the present study, SPIN scores were obtained from both ears of 98 elderly, hearing-impaired listeners (age range: 60 to 78 years). When the percent-correct scores on the High-Predictability (PH) items and on the Low-Predictability (PL) items are viewed as two separate measures, mean performance of the current "elderly-only" sample is similar to the performance of the original normative sample. However, for a certain percentage (18%) of the aged ears, the contextual cues provided in the PH sentences were not used as effectively as expected based on the normative guidelines.


Responses of elderly hearing aid users on the hearing aid performance inventory.

The responses of hearing aid users aged 65 to 80 years to a 64-item measure of perceived benefit afforded by hearing aids were collected in order to develop normative data concerning the perceived benefit afforded specifically to elderly patients. Across most of the listening situations evaluated, the elderly respondents reported less perceived benefit than the generally younger listeners from the original normative study for this measure. For the current respondents, perceived hearing aid benefit was not related to variables such as average hearing loss, hearing aid style, or length of hearing aid use, but was related to hours per week of hearing aid use. A shortened version of this questionnaire is proposed, which uses just those questions most likely to be answered by elderly respondents.


Comparison of Etymotic insert and TDH supra-aural earphones in auditory brainstem response measurement.

There are few systematic comparisons of Etymotic ER-3A insert earphones versus supra-aural earphones in auditory brainstem response (ABR) measurement. We compared ER-3A insert earphones and two types of supra-aural earphones (TDH-39P and TDH-49P) in a group of normal hearing adults. Acoustic analyses revealed spectral and temporal differences among earphones. Behavioral and ABR thresholds to click stimuli were slightly elevated with the ER-3A compared to the TDH earphones. The ER-3A earphones produced a latency delay, relative to the TDH earphones, that varied from about 0.8 to 1.0 msec, and increased at lower stimulus intensity levels. In addition, ABR wave I amplitude was significantly reduced with the ER-3A earphone. Based on these data, we recommend collection of normative data with the ER-3A earphones prior to their use in ABR measurement.


Effects of cochlear hearing loss on the ABR latencies to clicks and 1000 Hz tone pips.

High-frequency hearing losses can substantially confound the interpretation of click-evoked auditory brainstem responses (ABRs). One method proposed to circumvent the problem is to use frequency-specific tone pips to stimulate equivalent areas on the basilar membrane in normal and pathologic groups, or in both ears of asymmetrically impaired patients. This retrospective study investigated the ABRs to clicks and 1000-Hz tone pips from 90 asymmetrically impaired subjects with cochlear pathology. The 4000 Hz threshold significantly affected the wave V latencies from both clicks and tone pips for the most severely impaired subjects. The wave V latencies were highly correlated between ears for both stimuli, but the correlation was higher with the 1000-Hz tone pips. It was concluded that the use of 1000-Hz tone pips can supplement the interpretation of click-evoked ABRs, particularly in patients whose 2000- and/or 4000-Hz thresholds are worse than 75 dB HL.


Evaluation of prescriptive fitting.

The purpose of this study was to determine the minimum tolerance needed when choosing a manufacturer who custom-built analog circuitry for all-in-the-ear hearing aids to match a popular prescriptive amplification formula. Given the tolerance, a second purpose was to evaluate fitting success by calculating the differences in prescribed versus preferred gain and pre- versus postfitting perceived benefits. Eight elderly adults with mild to moderate sensorineural hearing losses participated. Real ear measurements were obtained via a probe-tube microphone system. Even when providing for the optimal scenario of custom-building the circuitry, the inherent limitations of analog technology allowed no better than a +/- 12 dB electroacoustic match to prescribed gain. Although the minimum tolerance found was less than previous studies, it was still considered excessive given the differences in prescribed gain among formulae. Regardless of the large tolerance and a preference for less gain than prescribed, the subjects reported substantial benefit with the fitting approach.


Evaluation of the efficacy of a multimemory hearing aid.

Although multimemory hearing aids have theoretical advantages over conventional "single memory" hearing aids, research and clinical data are not available to support their efficacy. The present study reports on the usefulness of a commercially available multimemory hearing aid in daily listening situations. Nineteen elderly hearing-aid wearers were fitted with the Quattro Q8 hearing aid. A modified Simplex procedure was used to select the electroacoustic characteristics for each memory. Subjects wore the device home and reported their satisfaction with each memory on the hearing aid in different listening situations. Their impressions on the whole hearing aid and on the remote feature were also sampled. Although a majority of subjects showed preference for multimemory design, such observations may not be readily evident until after extended use of the device. Inconvenience and cost of the device may prohibit some hearing-impaired listeners from using the device.


Auditory brainstem response to tone bursts in quiet, notch noise, highpass noise, and broadband noise.

This study investigated the effects of tone bursts (1000 Hz, 2000 Hz, and 4000 Hz) in quiet, notch noise, highpass noise, and broadband noise on the identifiability, latency, and amplitude of the auditory brainstem response (wave V). Normal listeners were presented with 40 dB and 80 dB nHL tone bursts having rise-plateau-fall times of 1 msec. Wave V was observed in all subjects at 40 dB and 80 dB nHL for the quiet and noise conditions. The latency findings suggest that responses elicited by the 80 dB nHL tone bursts in quiet were, in part, mediated by regions on the basilar membrane that did not correspond to the center frequency of the tone burst. To increase frequency-specificity, high-level tone bursts (e.g., 80 dB nHL) should be mixed with notch, highpass, or broadband noise. The use of noise conditions for low intensity levels (e.g., 40 dB nHL) does not appear necessary for isolating the response because both the notch and the highpass conditions yielded latencies similar to the quiet condition. Although similar wave V amplitudes were found at all frequencies, amplitudes were smaller for the broadband noise than for the quiet, notch, and highpass conditions. Thus, the latter conditions seem preferred.


Asymmetries in topographic brain maps of auditory evoked potentials in the elderly.

We compared topographic brain maps of the middle response (MR) and late response (LR) auditory evoked potentials in two groups of elderly males. Subjects were categorized on the basis of the performance of the poorer hearing ear on the Synthetic Sentence Identification (SSI) test. Evoked potentials, obtained in response to 1000-Hz tone pips and tone bursts, were recorded under three conditions of sound stimulation:binaural (BIN), left ear only (LE), and right ear only (RE). Comparisons of the topographic brain maps generated by the two groups revealed significant asymmetry in the MR of the experimental group. LE stimulation produced a larger response over the right hemisphere, and RE stimulation produced a larger response over the left hemisphere. This asymmetry was not observed in the experimental group with binaural stimulation, or in the control group in any of the three stimulation conditions. An analogous asymmetry was not recognized in the LR in either group for any of the three sound conditions. Results are interpreted in relation to the recently proposed model of multiple middle latency generators.


The statistical analysis of circadian phase and amplitude in constant-routine core-temperature data.

Accurate estimation of the phases and amplitude of the endogenous circadian pacemaker from constant-routine core-temperature series is crucial for making inferences about the properties of the human biological clock from data collected under this protocol. This paper presents a set of statistical methods based on a harmonic-regression-plus-correlated-noise model for estimating the phases and the amplitude of the endogenous circadian pacemaker from constant-routine core-temperature data. The methods include a Bayesian Monte Carlo procedure for computing the uncertainty in these circadian functions. We illustrate the techniques with a detailed study of a single subject's core-temperature series and describe their relationship to other statistical methods for circadian data analysis. In our laboratory, these methods have been successfully used to analyze more than 300 constant routines and provide a highly reliable means of extracting phase and amplitude information from core-temperature data.


Ontogeny of the circadian system controlling release of sperm from the insect testis.

In the gypsy moth, the release of sperm bundles from the testis into the vas deferens is rhythmic and is controlled by a circadian pacemaker located in the reproductive system. However, in males kept since pupation in constant darkness (DD) and temperature, the release of sperm was arrhythmic. The release of sperm became rhythmic when males were transferred from a light-dark cycle (LD 16:8) to DD 6-7 days after pupation. To further investigate the development of the circadian system during the pupal stage, we exposed DD pupae to a single 8-hr pulse of light or 8-hr pulse of a 4 degrees C temperature increase on different days after pupation. The pattern of sperm release was determined 5-6 days after the pulse. Males that were exposed to light or temperature pulses 5 days after pupation subsequently showed nonrhythmic sperm release. However, about half of the pupae that received the pulse on day 6 and most of the pupae that received it on day 7 subsequently showed synchronized sperm release. These results suggested that the clock underlying rhythmic release of sperm becomes operational at approximately 6 days after pupation--that is, 2 days prior to initiation of rhythmic sperm release from the testis.


Properties of mutual coupling between the two circadian pacemakers in the eyes of the mollusc Bulla gouldiana.

We examined, in vitro, the effects of changing the free-running period (tau) of one oscillator on the phase relationship between the circadian rhythms of impulse activity in the optic nerves that are driven by the bilaterally paired ocular pacemakers in Bulla gouldiana. One eye of the coupled pair was treated either with lithium artificial seawater (to lengthen tau) or with low-chloride artificial seawater (to shorten tau). The results suggested that the coupling is relatively weak, since the majority (9 to 16) of eyes were unable to maintain a stable phase relationship when tau differences between the eyes were only about 1 hr. When stable phase differences were achieved, the tau of the coupled system was intermediate between the tau's of the individual oscillators, and the eye with the shorter intrinsic tau would invariably phase-lead the pair. Interestingly, in a few instances, pairs of eyes that had desynchronized by 9.5-10.5 hr resynchronized within a single cycle via a massive phase advance in the rhythm from the phase-lagging eye. The result suggests the existence of a novel phase-shifting mechanism that is part of the mutual coupling pathway. We found evidence that connection of the eye with the cerebral ganglion increases the tau of the ocular pacemaker, suggesting that efferent signals from the central nervous system influence tau. These signals may also modulate the phase-shifting response.


Phase resetting of the Neurospora crassa circadian oscillator: effects of inositol depletion on sensitivity to light.

The input pathway between the blue-light photoreceptor and the circadian oscillator of Neurospora crassa has not yet been identified. To test the hypothesis that an inositol phospholipid signaling system might be involved in blue-light signal transduction, phase resetting by light was assayed in the inositol-requiring inl strain under conditions of inositol depletion. Phase-resetting curves and dose-response curves indicated that cultures maintained on low inositol (25 microM) were several orders of magnitude more sensitive to light than those maintained on high inositol (250 microM). This difference in light sensitivity was a property of inositol auxotrophy and was not seen in the wild type or in an inositol-independent inl+ revertant. Phase resetting by temperature was not affected by inositol depletion, indicating that the effect on light resetting is specific to the light input pathway and is not the result of a change in the amplitude of the oscillator itself. The results indicate an indirect role for inositol metabolites in the light input pathway--one that is not likely to involve direct participation of an inositol phospholipid signal transduction mechanism.


Responses of female New World screwworm flies, Cochliomyia hominivorax, to coloured targets in the laboratory.

The responses of unmated female New World screwworm flies, Cochliomyia hominivorax Coquerel, to visual targets were studied in a wind-tunnel. Both activity and frequency of contacts with targets increased greatly when the screwworm attractant mixture swormlure-4 was added to the airstream. Target-orientated responses depended on target colour, with red and black targets being preferred over blue, white and yellow ones; this preference was much greater in the presence of odour than in its absence. No preference was detected for different shapes and orientations of red targets, all of equivalent surface area. Omitting different components from swormlure-4 generally resulted in a large reduction in activation and target contacts. Attempts to substitute 1-octen-3-ol for the butanol fraction were unsuccessful, but skatole may substitute to some extent for indole; the two isomers of butanol normally present in swormlure-4 may substitute partly or completely for each other. This type of measurement forms a suitable bioassay in the development of attractive targets for monitoring and control of wild adult screwworm populations.


A generalized approach to detection of organophosphate resistance in mosquitoes.

Insecticide bioassays and biochemical microtitre assays were compared for detection of resistance to the organophosphate insecticides malathion and fenitrothion, using inbred laboratory strains of malaria vectors Anopheles albimanus Wiedemann, An.arabiensis Patton and An.stephensi Liston. With susceptible mosquitoes, the LT100 values determined from bioassays corresponded closely with times taken to abolish the activity of acetylcholinesterase activity in biochemical assays: approximately 2 h for malathion and 3 h for fenitrothion. Resistant strains of all three anophelines showed longer survival correlated with prolonged acetylcholinesterase activity. An.albimanus strains with insensitive acetylcholinesterase survived bioassays with discriminating doses of 1 h exposure to 5% malathion or 1% fenitrothion and were judged as resistant. It is concluded that enzyme-specific microassays provide a reliable means of detecting resistant individuals, with practical advantages over bioassays which do not reveal the resistance mechanism and require large numbers of healthy mosquitoes.


Studies on vectors of Trypanosoma cruzi in Trinidad, West Indies.

Studies on vectors of Trypanosoma cruzi Chagas were conducted during 1982-85 as part of an investigation on the epidemiological status of American Trypanosomiasis in Trinidad, West Indies. A total of 148 reduviid bugs were collected from caves, armadillo nests and at light traps, comprising four sylvatic species and totalling 120 Panstrongylus geniculatus (Latreille), two P. rufotuberculatus (Champion), twelve Rhodnius pictipes Stal and fourteen Eratyrus mucronatus Stal. A significantly higher level of infection with T. cruzi was recorded for P. geniculatus (42.5%) compared to R. pictipes (8.3%). The other two bug species were not found to be infected. P. geniculatus seems to be the most important vector in the sylvatic cycle of transmission of T. cruzi in Trinidad.


Effect of puparia incubation temperature: increased infection rates of Trypanosoma congolense in Glossina morsitans centralis, G. fuscipes fuscipes and G. brevipalpis.

Puparia of Glossina morsitans centralis (Machado), G.fuscipes fuscipes (Newstead) and G.brevipalpis (Newstead) were incubated at 25 +/- 1 degrees C, 28 +/- 1:25 +/- 1 degrees C, day:night or 29 +/- 1 degrees C throughout the puparial period, and maintained at 70-80% relative humidity. Puparial mortality was higher at 29 than at 25 degrees C (optimum temperature) in all three species, particularly in G.f.fuscipes and G.brevipalpis. Adults of G.m.centralis from puparia incubated at 29 degrees C, and those of this subspecies, G.f.fuscipes and G.brevipalpis from puparia incubated at 28:25 degrees C, day:night or 25 degrees C throughout, were infected as tenerals (27 h old) by feeding them at the same time on goats infected with Trypanosoma congolense (Broden) IL 1180 after the parasites were detected in the wet blood film. Infection rates on day 25 post-infected feed were higher in G.m.centralis from puparia incubated at 29 degrees C and in adults of the three different tsetse species from puparia incubated at 28:25 degrees C, day:night, than in those from puparia incubated at 25 degrees C. However, in G.f.fuscipes the labral and hypopharyngeal infection rates were not significantly different from those of the tsetse produced by puparia kept at 25 degrees C.


Chemical control of Ornithonyssus sylviarum on caged layer hens.

Manual application of aqueous solutions of malathion, carbaryl and permethrin controlled northern fowl mite, Ornithonyssus sylviarum Canestrini and Fanzago, on caged layer hens for at least 118 days. Azamethiphos sprayed manually provided acceptable control for a shorter period. Machine application, particularly of azamethiphos, but also malathion, provided lesser control. The addition of surfactant increased the wetting ability of the sprays but interfered with the efficacy of azamethiphos.


Anopheles nili as the main vector of human malaria in villages of southern Cameroon.

In villages near Sanaga river, in the forest zone of south Cameroon, Anopheles nili Theobald is the main species of mosquito regularly found biting man inside houses. Its densities are related to the flow level of the river. It is also the main malaria vector in terms of intensity and seasonal duration of transmission. The yearly malaria inoculation rate due to An. nili alone is 104 infective bites/man, varying between 3 infective bites/man in October and 20 in March. Anopheles gambiae Giles is a less important vector in the area but reached its peak in the dry season, when the Sanaga river is at its lowest level. These observations underline the influence of local ecology on malaria transmission and the need for entomological studies in each situation.


Cytotaxonomic confirmation of two forms of Simulium sirbanum in the eastern part of the Onchocerciasis Control Programme in West Africa.

An earlier report (Cheke et al., 1987) of morphological differences between populations of Simulium sirbanum Vajime & Dunbar in West Africa was confirmed cytotaxonomically. Populations with adult males having Type III scutal patterns were associated with male larvae heterozygous for the inversion IS-3, whereas adult males with Type IV scutal patterns were associated with larvae homozygous for IS-3. The taxonomic implications are briefly discussed but the evidence is considered to be inappropriate for resurrecting S. sudanense Vajime & Dunbar. Aspects of the biology of the two forms and their importance in onchocerciasis transmission are discussed.


Distribution and seasonality of Culex (Melanoconion) caudelli, a vector of arboviruses in Trinidad.

Seasonal and spatial distributions of the arbovirus vector mosquito Culex (Melanoconion) caudelli Dyar and Knab (Diptera: Culicidae) in the Aripo-Wallerfield forest, Trinidad, were studied by collecting mosquitoes attracted to mouse-baited traps in forest, ecotone and savannah, one day per week throughout the year, 98% of the Cx caudelli mosquitoes were collected during the wet season, with a significant positive correlation between rainfall and mosquito catch. The reverse occurred during the dry season. Cx caudelli displayed a preference for the ecotone where 67% were collected, while 32% and 1% were collected in the forest and savannah respectively.


Evidence for activation of endogenous opioid systems in mice following short exposure to stable flies.

Biting flies influence both physiology and behaviour of domestic and wild animals. This study demonstrates that brief (30 min) exposure of male and female mice to stable flies leads to significant increases in nociceptive responses, indicative of the induction of analgesia. The biting fly-induced analgesia was mediated by endogenous opioid systems as it was blocked by the prototypic opiate antagonist naloxone. Exposure for 30 min to the bedding of biting fly-exposed mice also induced significant opioid mediated analgesic responses in mice. Exposure to either house flies or the bedding of house fly-exposed mice had no significant effects on nociception. These results indicate that brief exposure to either stable flies, or to olfactory cues associated with mice exposed to stable flies, activates endogenous opioid systems leading to the induction of analgesia and likely other opioid mediated behavioural and physiological stress responses. These results suggest the involvement of endogenous opioid systems in the mediation of the behavioural and physiological consequences of biting fly exposure in domestic and wild animals.


Borrelia burgdorferi infection in Ixodes ricinus from habitats in Denmark.

A total of 2647 ticks of the genus Ixodes was sampled by flagging the vegetation in thirty-one sites in eastern Jutland, Denmark. All ticks were identified as Ixodes ricinus Linnaeus. A total of 317 ticks (202 nymphs and 115 adults) from three different sites were examined for the spirochaete Borrelia burgdorferi Johnson et al. by indirect fluorescent antibody staining; the frequency of infection varied from 7% to 22%. It is concluded that I. ricinus, known to be the most common tick in Denmark, is the vector largely responsible for the transmission of B. burgdorferi in this country.