Flavin analogs with antimalarial activity as glutathione reductase inhibitors

10-(4'-Chlorophenyl)-3-methylflavin has antimalarial activity in vitro and in vivo (Cowden et al., J Med Chem 31: 799, 1988). This flavin analog and two of its derivatives were found to inhibit the antioxidant flavoenzyme glutathione reductase from human erythrocytes in its isolated form as well as in hemolysates. The mixed-type inhibition was completely reversible, the Ki-values being of the order of 1 microM. Surprisingly, the drugs were not competitive with FAD, but with GSSG, one of the enzyme's substrates. Malaria parasite glutathione reductase, extracted from Plasmodium falciparum, could also be inhibited by the compounds. Studies on the effects of the substances on P. falciparum in vitro, which were demonstrated morphologically and by growth inhibition, confirmed previous observations with 10-(4'-chlorophenyl)-3-methylflavin and showed similar parasiticidal characteristics for the two new derivatives. The activities of five other erythrocytic enzymes tested were not impaired by the drugs, nor was the nucleotide metabolism of erythrocytes and/or parasites significantly changed. Permeation into red blood cells was demonstrated for one compound by 19F-NMR-spectroscopy. Inhibition of glutathione reductase might contribute to, or account for, the antimalarial activity of this group of flavin analogs.     

Further study on the vascular basis for the reimplantation of the hand amputated through the palm

Summary Based on the anatomic data obtained from earlier studies on the vascular anatomy of the hand, the vascular architecture in the palm of the hand was studied on 60 sides of unembalmed adult upper extremities. Each palm was divided into 64 squares by 8 sagittal and 8 transverse sections. The vascular architecture in these squares and the arterial relations between them were observed and measured by angiography, operative microscopic dissection and computerised three-dimensional reconstruction. According to the pattern of the blood-vessels, the amputated palms can be classified into 4 types. The anatomic basis for the vascular anastomosis in each type is defined. There are three key-areas for the blood-supply of the palm and their significance is discussed. Apart from the 4 types of transversely amputated palms, the repair programe of the blood-vessles in 4 types of common obliquely amputated palms are also discussed.

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Etude complémentaire de l'anatomie vasculaire de la main pour la réimplantation des amputations transpalmaires

Résumé Sur la base de données anatomiques obtenues lors de précédents travaux sur l'anatomie vasculaire de la main, l'architecture vasculaire palmaire a été étudiée sur 60 extrémités supérieures de cadavres d'adultes, non embaumés. Chaque paume a été divisée en 64 carrés par 8 sections sagittales et 8 sections transversales. L'architecture vasculaire à l'intérieur des carrés et les relations artérielles entre eux ont été étudiées et mesurées par angiographie, dissection au microscope opérateur et reconstruction computérisée en 3D. Les paumes amputées ont été regroupées en 4 types d'après la distribution des vaisseaux sanguins. Les données anatomiques concernant les anastomoses vasculaires sont précisées. Il existe trois zones clés pour l'irrigation de la paume. Leur importance quant à l'irrigation de la main est exposée. Outre la division des paumes amputées transversalement en 4 types, le programme de réparation de vaisseaux dans les 4 types d'amputations obliques communes de la paume et aussi discuté.

     

Screening microarrays of novel monoclonal antibodies for binding to T-, B- and myeloid leukaemia cells

We have developed a microarray (DotScan) that enables rapid immunophenotyping and classification of leukaemias and lymphomas by measuring the capture of cells by immobilized dots of 82 CD antibodies [Belov, L., de la Vega, O., dos Remedios, C.G., Mulligan, S.P., 2001. Immunophenotyping of leukemia using a cluster of differentiation antibody microarray. Cancer Res. 61, 4483; Belov, L., Huang, P., Barber, N., Mulligan, S.P., Christopherson, R.I., 2003. Identification of repertoires of surface antigens on leukemias using an antibody microarray. Proteomics 3, 2147]. The DotScan technology has been used to investigate the properties of 498 new antibodies submitted to the HLDA8 Workshop. These antibodies have been applied as 10 nl dots to a film of nitrocellulose on a microscope slide to make an HLDA8 microarray. After blocking the remaining nitrocellulose surface, individual arrays were incubated with each of 7 cell types from a human leukaemia cell panel consisting of three cell lines, CCRF-CEM (a T-cell acute lymphocytic leukaemia), MEC-1 (derived from B-cell chronic lymphocytic leukaemia) and HL-60 (a promyelocytic leukaemia), and four leukaemias from patients: a T-cell prolymphocytic leukaemia, a B-cell chronic lymphocytic leukaemia, and two acute myeloid leukaemias. Leukaemia cells were captured by those immobilized antibodies for which they expressed the corresponding surface molecule. Unbound cells were gently washed off, bound cells were fixed to the arrays and dot patterns were recorded using a DotScan array reader and quantified using DotScan data analysis software. The data obtained show the unique expression profiles of the 7 cell types in the leukaemia cell panel obtained with the DotScan microarray, and the differential capture patterns for these 7 cell types screened against the 498 antibodies in the HLDA8 microarray constructed for this study.     

PCR-Based assay to quantify human immunodeficiency virus type 1 DNA in peripheral blood mononuclear cells

An assay that quantifies the amount of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells has been developed. PCR amplification of the HIV-1 DNA is performed in the presence of an internal quantitation standard, and colorimetric detection of the amplified product is performed with microwell plates. The copies of HIV-1 DNA are normalized to total genomic DNA input. The assay has an analytical sensitivity of 10 input copies per amplification reaction and a three-log detection range. In an analysis of sequential samples from patients on combination therapy, HIV-1 DNA was quantifiable for all individuals tested, including those with undetectable plasma HIV-1 RNA. In a separate study, a comparison of HIV-1 DNA levels was made with a group of long-term survivors and progressors. The mean HIV-1 DNA levels were lower in the long-term survivors than in the progressors (P, 0.04). The mean HIV-1 RNA levels were also lower, but the difference was not statistically significant (P, 0.164). A quantitative DNA assay will provide an additional tool to gain insight into the natural history of infection and the continued efficacy of potent antiretroviral therapies.     

Changes during eating in oxygen consumption, cardiac function and body fluids of sheep

1. A study was made of the changes taking place in O₂ consumption, cardiac function and the volume and composition of the body fluids of sheep while they consumed a meal of hay.

2. During eating P_{a, CO2} and P_{v, CO2} both increased, pH decreased and free plasma [HCO₃^-] increased. Venous haematocrit increased sharply at the beginning of the meal, and declined slowly after feed was removed.

3. Arterial P_O2 did not change significantly during eating. However P_{v, O2} fell slightly but significantly. The O₂ saturation of venous blood fell due to the decline in pH. Estimated CO₂ in arterial blood increased as a consequence of increased haemoglobin content. The net effect was to increase arteriovenous difference in O₂ content from 4·4 ml./100 ml. before eating to 6·0 ml./100 ml. at the end of the meal.

4. O₂ consumption increased about 60% during eating and fell rapidly thereafter. Heart rate followed a similar pattern. Cardiac output however increased only about 17%, from 6 to 7 l./min. Consequently stroke volume declined throughout the meal from 76 to 52 ml./beat.

5. Plasma volume, estimated from measurements of T-1824, declined sharply by about 300 ml. at the beginning of the meal and recovered slowly after feed was removed. Blood volume declined less because of a rise in circulating erythrocytes.

6. Extracellular fluid volume was estimated from measurements of thiocyanate and thiosulphate spaces. Thiocyanate space measurements were abandoned after thiocyanate was found to be concentrated in saliva. Considerable random variation occurred in measurements of changes in extracellular fluid from thiosulphate disappearance but the results did reveal a significant fall of 1000-1500 ml. in extracellular fluid volume during eating.

7. The significance of these interrelated changes is discussed in relation to the maintenance of homoeostasis during eating in the sheep.

     

Inhibition of interleukin-17 prevents the development of arthritis in vaccinated mice challenged with Borrelia burgdorferi

We showed that Borrelia burgdorferi-vaccinated interferon gamma-deficient (IFN-gamma(0)) mice challenged with the Lyme spirochete developed a prominent chronic severe destructive osteoarthropathy. The immune response underlying the development of the severe destructive arthritis involves interleukin-17 (IL-17). Treatment of vaccinated IFN-gamma(0) mice challenged with B. burgdorferi with anti-IL-17 antibody delayed the onset of swelling of the hind paws but, more importantly, inhibited the development of arthritis. Histopathologic examination confirmed that treatment with anti-IL-17 antibody prevented the destructive arthropathy seen in vaccinated and challenged IFN-gamma(0) mice. Similar preventive results were obtained when vaccinated and challenged IFN-gamma(0) mice were treated with anti-IL-17 receptor antibody or sequentially with anti-IL-17 antibody followed by anti-IL-17 receptor antibody. By contrast, treatment of vaccinated and challenged IFN-gamma(0) mice with recombinant IL-17 (rIL-17) did not alter the development and progression of arthritis found in vaccinated and challenged IFN-gamma(0) mice without treatment with rIL-17. Therapeutic intervention may be a realistic approach to prevent arthritis, especially if IL-17 is involved in the perpetuation of chronic or intermittent arthritis.     

Cell surface peptidase CD26/DPPIV mediates G-CSF mobilization of mouse progenitor cells

CXC ligand 12 (CXCL12; also known as stromal cell-derived factor 1alpha/SDF-1alpha) chemoattracts hematopoietic stem and progenitor cells (HSCs/HPCs) and is thought to play a crucial role in the mobilization of HSCs/HPCs from the bone marrow. CD26 (dipeptidylpeptidase IV [DPPIV]) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains. CD26 has the ability to cleave CXCL12 at its position-2 proline. We found by flow cytometry that CD26 is expressed on a subpopulation of normal Sca-1+c-kit+lin- hematopoietic cells isolated from mouse bone marrow, as well as Sca-1+c-kit-lin- cells, and that these cells possess CD26 peptidase activity. To test the functional role of CD26 in CXCL12-mediated normal HSC/HPC migration, chemotaxis assays were performed. The CD26 truncated CXCL12(3-68) showed an inability to induce the migration of sorted Sca-1+c-kit+lin- or Sca-1+c-kit-lin- mouse marrow cells compared with the normal CXCL12. In addition, CXCL12(3-68) acts as an antagonist, resulting in the reduction of migratory response to normal CXCL12. Treatment of Sca-1+c-kit+lin- mouse marrow cells, and myeloid progenitors within this population, or Sca-1+c-kit-lin- cells with a specific CD26 inhibitor, enhanced the migratory response of these cells to CXCL12. Finally, to test for potential in vivo relevance of these in vitro observations, mice were treated with CD26 inhibitors during granulocyte colony-stimulating factor (G-CSF)-induced mobilization. This treatment resulted in a reduction in the number of progenitor cells in the periphery as compared with the G-CSF regimen alone. This suggests that a mechanism of action of G-CSF mobilization involves CD26.