Effects of in vitro and in vivo cytokine treatment, leucapheresis and irradiation on the function of human neutrophils: implications for white blood cell transfusion therapy.

Treatment of human polymorphonuclear leucocytes (PMNL) separated by density sedimentation (DS) from normal donors (PMNL-NL-DS) with interferon-gamma (IFN-gamma) + granulocyte colony-stimulating factor (G-CSF) lessens the damage caused by isolation and irradiation. We have studied granulocyte-macrophage colony-stimulating factor (GM-CSF) in this system, as well as the behaviour of PMNL collected by continuous flow leucapheresis (CFL) from donors treated with G-CSF (PMNL-GCSF-CFL). After isolation, PMNLs were treated with IFN-gamma + G-CSF, GM-CSF or IFN-gamma + G-CSF + GM-CSF, irradiated with 0 or 30 Gy and studied after 0 and 20 h in cell culture. All regimens reduced apoptosis of PMNL-NL-DS. Killing of Candida albicans by 20-h-old PMNL-NL-DS was best preserved by IFN-gamma + G-CSF treatment. A similar pattern of results was obtained for assays of PMNL-NL-DS chemotaxis and superoxide production. There was a consistent trend toward reduced function after irradiation in all assays. PMNL-GCSF-CFL less often demonstrated the morphological features of apoptosis, and this was further reduced by cytokine regimens containing IFN-gamma + G-CSF. In assays of C. albicans killing and chemotaxis, 20-h-old untreated PMNL-GCSF-CFL performed as well as freshly isolated PMNL-GCSF-CFL. PMNL-GCSF-CFL showed decay in CD11b (CR3), CD16 (Fc gamma III) and CD64 (Fc gamma R1) expression after 20 h in cell culture, but treatment with IFN-gamma + G-CSF preserved expression. There was a trend toward reduced function after radiation. Comparison of PMNL-GCSF separated by CFL and DS demonstrated that CFL itself is a strong inducer of the morphological features of apoptosis. This study shows that while separation by CFL, and irradiation are damaging to PMNLs, damage may be reduced by use of cytokines.

Protection of human polymorphonuclear leukocyte function from the deleterious effects of isolation, irradiation, and storage by interferon-gamma and granulocyte-colony-stimulating factor.

BACKGROUND: Fungal infections represent a difficult challenge to clinicians caring for neutropenic patients with hematologic malignancies, as antifungal therapy often has limited success in that setting. One promising yet problematic alternative approach is leukocyte transfusion. The isolation of polymorphonuclear leukocytes (PMNs) induces apoptosis and functional deterioration, and irradiation to prevent transfusion-associated graft-versus-host disease causes further functional deterioration. STUDY DESIGN AND METHODS: The ability of interferon-gamma and granulocyte-colony-stimulating factor (G-CSF), used both alone and in combination, to protect PMNs after 0 or 20 hours' storage in cell culture (as a model for function after transfusion) and irradiation with 0, 5, or 30 Gy was studied. RESULTS: Without cytokine treatment, 20-hour-old PMNs showed marked apoptosis, no appreciable chemotaxis, and no ability to kill Candida albicans. In contrast, cytokine treatment significantly reduced apoptosis and protected chemotaxis, C. albicans killing, and surface-receptor expression from both storage and irradiation. Although the majority of the benefit appeared to be due to G-CSF, consistent trends suggested better function of PMNs after combined treatment with interferon-gamma and G-CSF. CONCLUSION: Judicious use of cytokines may preserve PMN function. These findings have important implications for the transfusion of PMNs to cytopenic patients.

Isozymes of Culex p. fatigans. III. Genetics of leucine-amino-peptidase.

Zymograms of leucine-amino-peptidase (LAP) of the mosquito Culex pipiens fatigans Wiedemann (C. p. quinquefasciatus) showed 20 isozyme bands in the fourth instar larvae, 13 in the pupae and 3 in the older adults. Homozygous strains of Lap-1F (Fast) and Lap-1S (Slow) were isolated and linkage studies carried out. Lap-1F and Lap-1S allozymes act as codominant alleles, are sex linked and are located in linkage group I. The gene sequence is e-M-mar-Lap-1 (eye-gap, maroon-eye, leucine-amino-peptidase-1), and the linkage distances in the M-mar and mar-Lap-1 segments are 22.0 and 10.5, respectively. Gene frequency analysis of natural populations from Brazil and Pakistan showed the S allele was more frequent in the Brazilian populations whereas F was more frequent in the Pakistani populations. Fixation of F allele had occurred in two Pakistani populations.

Isozymes of Culex p. fatigans. I. An esterase locus in linkage group III and its variability in natural populations.

Acrylamide gel electrophoresis revealed 19 frequent and 4 rare sites of esterase activity in various Brazilian populations of Culex p. fatigans. One pair of allozymes designated as Est-1 F and Est-1 S was selected for detailed investigation. Est-1 locus shows independent assortment from linkage group I marker, sex, and linkage group II markers, yellow and ruby, and therefore is assigned to linkage group III. Gene frequencies of Est-1 F varied from 0.49 to 0.92 in various Brazilian populations. Inhibition studies suggest that Est-1 F and Est-1 S are ali-esterases.

Dominant-and-recessive epistasis in a homeotic mosquito mutant.

Following selection for 15 generations a pure strain of a homeotic mutant spur was isolated from a Brazilian population of the mosquito Culex pipiens fatigans. Monohybrid crosses showed a 13:3 segregation indicating dominant-and-recessive epistasis for wild-type vs. spur. This implies that a dominant allele at one locus and a recessive at the other interact to produce the mutant phenotype. Dihybrid crosses with linkage group II markers yellow and ruby gave 39:13:9:3 ratios indicating independent segregation. However, the dihybrid cross with linkage group I marker maroon showed a highly significant departure from 39:13:9:3 ratio. Data available indicate that the phenotype spur is controlled by a dominant epistat in linkage group III and a recessive epistat (approximately 31.9 crossover units from maroon) in linkage group I.

Genetics of eye-gap and maroon-eye mutants in the mosquito.

Linkage analysis of two new eye mutants, eye-gap (e) and maroon-eye (mar), in the mosquito Culex pipiens, is presented. Both mutants are sex-linked, recessive, and demonstrate complete penetrance and high expressivity. The gene order is e-m-mar. The most representative map distances in e-m and m-mar segments are 17.7 and 26.1, respectively. Our maroon-eye was isolated from Belem, Brazil and is allelic to another maroon-eye mutant isolated from Lake Charles, Louisiana. However, while mar (Lake Charles) straddles along the sex locus, m (map distance approximately 1 unit), the mar (Belem) gene is far removed from m (map distance 26.1 units).