Yehudith Kletter

Activation of human eosinophil and neutrophil functions by haematopoietic growth factors: comparisons of IL‐1, IL‐3, IL‐5 and GM‐CSF

Summary. We compared the effect of haematopoietic growth factors granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interleukin (IL)‐1, IL‐3, and IL‐5 on the functional activation of human eosinophils and neutrophils from the same donor. All four colony‐stimulating factors (CSF) enhanced the phagocytosis of Candida albicans by eosinophils and increased staphylococcal, but not Candida, killing. GM‐CSF and IL‐5 had a profound stimulating effect on eosinophil staphylocidal activity. GM‐CSF and IL‐3 enhanced the generation of leukotriene C4 (LTC4) induced by calcium ionophore A23187 and the release of arylsulphatase and β‐glucuronidase from specific and small granules of eosinophils. In contrast, IL‐1 and IL‐5 had no effect on degranulation. GM‐CSF and IL‐1 enhanced phagocytosis of C. albicans by neutrophils, and GM‐CSF stimulated degranulation and the release of the enzymes β‐glucuronidase and arylsulphatase from neutrophils while IL‐1 stimulated the release of arylsulphatase only. This study indicates that the eosinophilactive colony‐stimulating factors can markedly enhance the host defence function of the eosinophil and even make it the equal of the neutrophil in staphylocidal activity. The CSF‐activated eosinophil, however, may cause inappropriate inflammation and normal tissue damage.

Long-Term Serological Analysis and Clinical Follow-Up of Patients with Cat Scratch Disease

A highly specific enzyme immunoassay (EIA) was recently described for use in the diagnosis of cat scratch disease (CSD). However, data regarding EIA antibody kinetics or its correlation with long-term clinical follow-up data are lacking. The association between antibody kinetics, clinical spectrum, and disease duration were studied in 98 patients with CSD. The median duration of follow-up was 35.3 weeks (range, 2-211.3 weeks). Results of EIA testing for detection of anti-Bartonella henselae immunoglobulin M (IgM) antibodies (detected in 53% of the patients) remained positive for < or =3 months. Therefore, the presence of IgM indicated acute infection. Titers of immunoglobulin G (IgG) also decreased over time; 25% of the patients remained seropositive for >1 year after the onset of CSD. Onset of CSD in patients with an IgG titer with an optical density of > or =1.0 occurred within the prior 12 months. No association was found between antibody titers or their kinetics and the clinical manifestations or duration of disease. EIA allows for the identification of atypical manifestations of CSD that were unrecognized before the use of serological assays. Complete recovery from these manifestations may take months. Results of this study provide additional data supporting the utility of EIA in the serodiagnosis of CSD.

Immunomodulatory and Protective Effects of Moxifloxacin against Candida albicans-Induced Bronchopneumonia in Mice Injected with Cyclophosphamide

ABSTRACT In a previous study, moxifloxacin was shown to ameliorate immunosuppression and enhance cytokine production in several tissues, including the lungs of cyclophosphamide-injected mice. We examined here the effects of moxifloxacin on Candida albicans lung infection in cyclophosphamide-injected mice. Mice were injected on day 0 with 250 mg of cyclophosphamide/kg, and on days 1 to 4 they were given moxifloxacin at 22.5 mg/kg/day compared to controls given ceftazidime at 75 mg/kg/day or saline. On day 6, C. albicans (10 7 CFU/mouse) was inoculated intratracheally, and animals were observed for the development of bronchopneumonia, weight loss, mortality, the presence of C. albicans, and lung cytokine production. Histopathology on day 10 postinoculation revealed bronchopneumonia in 50, 67, and 0% of saline-, ceftazidime-, and moxifloxacin-treated mice, respectively (P < 0.05). The mortality rates were 28, 17, and 5%, respectively (P < 0.05), and weight loss occurred at 20, 32, and 0%, respectively (P < 0.05). By day 15, C. albicans was eliminated from all moxifloxacin-treated mice but was still isolated from lung homogenates of 50 to 60% of the saline- and ceftazidime-treated groups. Among the cytokines tested on days 0 to 15, we found an increased production of tumor necrosis factor alpha, KC (functional interleukin-8), and gamma interferon in the lungs of ceftazidime- and saline-treated controls compared to the moxifloxacin pretreatment that abolished their secretion. In conclusion, moxifloxacin protected cyclophosphamide-injected mice from C. albicans-induced lung infection and significantly reduced pneumonia, weight loss, and mortality despite the lack of direct antifungal activity. This is most likely due to an immunomodulating activity conferred by moxifloxacin, as shown in this model and in our previous studies. Its potential protective role should be studied in patients undergoing chemotherapy and immune suppression.

Ascorbic Acid Inhibits Apoptosis Induced by X Irradiation in HL60 Myeloid Leukemia Cells

Exposure of cells to ionizing radiation can cause apoptosis. Since antioxidants have been shown to protect against radiation-induced apoptosis, in this study we have evaluated the putative protective effect of ascorbate against radiation-induced apoptosis as well as the production of peroxides in the cells. HL60 cells transport the oxidized form of ascorbic acid, dehydroascorbic acid (DHA), and accumulate reduced ascorbate. Exposure of the cells to 5-40 Gy X radiation resulted in induction of apoptosis. Preincubation of the cells with DHA reduced the level of apoptosis after exposure to 5-20 Gy. Exposure of the cells to 5 or 20 Gy X radiation did not affect the intracellular concentration of peroxides, while phorbol myristate acetate (PMA), which is known to induce production of H(2)O(2) in cells (and served as a control), resulted in an increase in peroxides and a decrease in intracellular ascorbate. Irradiation of the cells with 1-3 Gy resulted in up-regulation of expression of BCL2 without affecting the level of apoptosis. At higher doses of radiation, enhanced BCL2 expression did not prevent radiation-induced apoptosis. Loading of the cells with ascorbate prior to their exposure to 1-3 Gy X radiation did not affect the enhanced BCL2 expression observed in the irradiated cells. At higher doses of radiation, ascorbate decreased apoptosis and restored the level of BCL2 in the cells. Exposure of the cells to 3-20 Gy X radiation enhanced the cell surface expression of TNFRSF6 (formerly known as Fas/APO-1) antigen and enhanced anti-TNFRSF6 antibody-induced apoptosis of the cells. Ascorbate loading did not affect expression of TNFRSF6 and did not overcome the anti-TNFRSF6 antibody-induced apoptosis. In conclusion, our data demonstrate that exposure of HL60 cells to radiation enhanced BCL2 and TNFRSF6 expression. Ascorbate did not affect BCL2 or TNFRSF6 expression. We therefore conclude that it protects HL60 cells against radiation-induced apoptosis, although the mechanisms of protection must still be elucidated.

Immunomodulatory effects of moxifloxacin in comparison to ciprofloxacin and G‐CSF in a murine model of cyclophosphamide‐induced leukopenia

Abstract: We analyzed the effect of the two quinolones moxifloxacin and ciprofloxacin on the repopulation of hematopoietic organs and on the production of cytokines by various organs of cyclophosphamide (CP)‐induced leukopenic mice. The effect was compared to that of G‐CSF. Cyclophosphamide injection induced a severe leukopenia, with nadir at day 4 post‐injection. All the quinolone and G‐CSF‐treated animals showed WBC>500/μL at the nadir, compared to 50% of saline‐treated mice. Cyclophosphamide induced a marked decrease in the number of myeloid progenitors (CFU‐C) in bone marrow (BM) and spleen. Quinolone or G‐CSF treatment resulted in a 1.4–4.3‐fold increase in CFU‐C numbers in the BM; no enhancement was observed in the spleen. Treatment with CP resulted in enhanced colony‐stimulating activity (CSA) in bone shaft and spleen and decreased activity in bladder and lung. Treatment of CP‐injected mice with quinolones significantly enhanced CSA in the bone shaft, spleen, lung and bladder on different days.

Effects of recombinant human granulocyte and granulocyte-macrophage colony-stimulating factors on neutrophil function following autologous bone marrow transplantation

Functional activity of peripheral blood neutrophils was assessed in eight patients at 4, 6, 8, 10 and 12 weeks following autologous bone marrow transplantation (ABMT). Functions studied included superoxide generation (O2-) intracellular killing of Staphylococcus aureus, phagocytosis and killing of Candida albicans. Neutrophils were tested following in vitro preincubation with 300 pM granulocyte-macrophage colony-stimulating factor (GM-CSF), 1.2 nM granulocyte colony-stimulating factor (G-CSF) or buffered solution (diluent) as control. Our data indicate that during the early period (weeks 4-6) following ABMT most of the patients exhibited diminished neutrophil oxidative metabolism, defective phagocytosis and killing of C. albicans and reduced capacity to kill S. aureus. In some patients a gradual increase in the functional activity of neutrophils occurred with time. Both GM-CSF and G-CSF induced in vitro amplification of (a) O2- production in response to fmet-leu-phe (FMLP) (b) phagocytosis and killing of C. albicans and (c) killing of S. aureus. This study suggests that GM-CSF and G-CSF may enhance the depressed functional activity of neutrophils following ABMT.

Enhanced hematopoiesis in sublethally irradiated mice treated with various quinolones

Abstract: We compared the effect of 6 quinolones on growth of murine bone marrow (BM) progenitor cells in vitro, and their in vivo effect on repopulation of BM and on survival of sublethally irradiated mice. The addition of clinically attainable concentrations of ciprofloxacin, sparfloxacin or clinafloxacin, in concert with pokeweed mitogen (PWM) to murine spleen cells, resulted in a significant enhancement in colony stimulating activity. A 1.5–1.8 fold increase in the number of myeloid progenitors (CFU–C) was observed in the presence of quinolone–PWM spleen conditioned medium (SCM) (prepared with the above‐mentioned quinolones) compared with control cultures exposed to PWM–SCM only. Three other quinolones showed either no stimulatory effect (fleroxacin, norfloxacin) or had an inhibitory effect (ofloxacin) on CFU–C growth. The stimulatory quinolones share in common a cyclopropyl moiety at position N1 of the quinolone ring. This moiety is lacking in the other 3 quinolones. The secretion of interleukin‐3 (IL‐3) and granulocyte‐macrophage colony‐stimulating factor (GM–CSF) by murine spleen cells exposed to quinolone–PWM–SCM was significantly enhanced with all 6 quinolones. However, this effect was associated with a parallel increase in CFU–C only with ciprofloxacin (10 μg/mL), sparfloxacin (1 μg/mL) and clinafloxacin (0.05 μg/mL). The in vivo activity was assessed in sublethally irradiated mice (650 rad) treated with quinolones for 5 d. The number of CFU–C in BM and the number of peripheral white blood cells (WBC) 8 d post‐irradiation was significantly enhanced in mice treated with ciprofloxacin (45 mg/kg/d), sparfloxacin (22.5 mg/kg/d) and clinafloxacin (11.25 mg/kg/d) compared to saline treated animals (p<0.05). Clinafloxacin at higher dosage (45 mg/kg/d) resulted in a decrease in myeloid progenitors in BM. A similar increase in progenitors and WBC was observed in animals treated with high doses, above clinical relevance, of ofloxacin, and norfloxacin (90 mg/kg/d), and with fleroxacin (45 and 90 mg/kg/d). Quinolone‐treated animals, at the above‐cited doses, showed enhanced survival on d18 compared to saline treated animals. The only exception was the higher mortality of clinafloxacin‐treated mice. The above observations imply that certain quinolones, sharing specific molecular structure, are potential immunomodulators at clinically relevant concentrations. These compounds should be further studied in neutropenic patients and BM or peripheral blood progenitor cell recipients.

Unusual eruption as a presenting symptom of cat scratch disease

Cat scratch disease (CSD) is a common infectious cause of subacute regional lymphadenopathy. Bartonella henselae is the principal etiologic agent. About 10% of CSD patients experience atypical manifestations, including rashes. The most common cutaneous manifestation of CSD is a papule at the inoculation site. We report a case of CSD presenting with an eruption on the upper trunk, reminiscent of Sweets syndrome, accompanied by lymphadenopathy, arthralgia, and fever. Response to systemic corticosteroids was remarkable. Histopathologic findings refuted the diagnosis of Sweets syndrome. Identification of anti-B henselae antibodies and B henselae DNA in the affected lymph node confirmed the diagnosis of CSD. This is a first report of extensive papuloedematous eruption as a cutaneous manifestation of CSD. Accurate diagnosis is possible due to the availability of serological tests and DNA amplification techniques.

The interaction of phagocytes and the large-sized parasite Cryptococcus neoformans: Cytochemical and ultrastructural study

SummaryThe yeast Cryptococcus neoformans may develop under certain conditions a large polysaccharide capsule 50–100 μM in diameter and therefore cannot be phagocytosed by either polymorphonuclear cells (PMNs) or mononuclear phagocytes (MNs). The cellular defense mechanism — in various animals — against the yeast is composed by formation of ringlike structure of PMNs or MNs cells which surround the C. neoformans. Ring structures develop either in vivo or in vitro in tissue culture; destruction of the yeast occurs within 36–72 hours.Several hydrolases, such as acid phosphatase, β-glucuronidase and non-specific esterase were found to be released from the phagocytic cells into the enclosed yeast. Considerable reduction of NBT used as a marker for oxidative activity was observed in MN rings at contact regions of the MN cells and the yeast. Electron microscopic studies indicate that the phagocytic cells in the ring structure have many pseudopodes penetrating into the polysaccharide capsule of the yeast. Disintegration of the capsule was observed as well as phagocytosis of its material. A possible analogy between normal phagocytosis of small-sized bodies and the ring structure obtained when large bodies are involved is discussed.

Effects of human interleukin 3, macrophage and granulocyte-macrophage colony-stimulating factor on monocyte function following autologous bone marrow transplantation

The effects of human interleukin 3 (IL-3), macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied on the functional activity of human peripheral blood monocytes from healthy individuals and from eight patients at 4, 8 and 12 weeks following autologous bone marrow transplantation (ABMT). Functions studied included superoxide production, phagocytosis of Candida albicans and reduction of 3-[4,5-dimethylthiazol-2-yl]-2.5-diphenyl tetrazolium bromide (MTT). IL-3 and GM-CSF significantly enhanced the oxidative metabolism of monocytes from healthy individuals, while the effect of M-CSF was moderate. A considerable variability between healthy individuals was found in both resting and cytokine-stimulated monocytes with regard to superoxide production. All three investigated CSFs, i.e. IL-3, M-CSF and GM-CSF did not affect phagocytosis of C. albicans by the cells or their metabolic activity (reduction of MTT). In ABMT patients no deficit in the functional activity of monocytes was found at any time after transplantation and all three CSFs investigated did not modulate the functional activity of the cells. These results suggest that monocytes do not have a major role in infectious complications post-ABMT.