Enzyme immunoassay for the diagnosis of cat-scratch disease defined by polymerase chain reaction.

Whole-cell immunofluorescent antibody (IFA) tests for detection of anti-Bartonella henselae immunoglobulin (Ig) G are commonly used to diagnose cat-scratch disease (CSD). The need to cultivate B. henselae in Vero cells for antigen preparation and the absence of routinely applied IFA assays for IgM constitute the major disadvantages of this form of test. We describe the results of an enzyme immunoassay (EIA) for IgM and IgG that used N-lauroyl-sarcosine-insoluble outer membrane antigens from agar-grown B. henselae performed in 84 patients with definite CSD (regional lymphadenitis, cat contact, and > or =1 confirmatory test: polymerase chain reaction, skin test, or B. henselae culture). Although this method has been used as a diagnostic tool in several case reports, it has not previously been evaluated in a large study of definitively proven CSD cases. Results of this study indicate that the EIA described herein can play an important role in the serodiagnosis of CSD, although improvement of the sensitivity, particularly that of the IgM, would be desirable.

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

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/microL 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. In normal mice the highest levels of GM-CSF and IL-6 were observed in lung-conditioned medium (compared to bone shaft, spleen and bladder). Injection of CP resulted in a 22.5- and 93-fold decrease in GM-CSF and IL-6 levels, respectively, in lung-conditioned medium, while treatment with quinolones resulted in 2-4-fold increase in GM-CSF with no effect on IL-6 production. G-CSF treatment had no enhancing effect on GM-CSF nor on IL-6 production. We conclude that moxifloxacin and ciprofloxacin administered to CP-injected mice revert some of the immune suppressive effects of cyclophosphamide.

DNA amplification for the diagnosis of cat-scratch disease in small-quantity clinical specimens.

Diagnosis of cat-scratch disease (CSD) by polymerase chain reaction (PCR) of lymph node fineneedle aspiration (FNA) and primary lesion specimens can be difficult owing to the minute amount of available material. A PCR assay specifically suited to test these specimens was developed. First, small-quantity (10 microL) samples were prepared from 17 CSD-positive and 16 CSD-negative specimens, and DNA extraction and amplification from these samples were compared using 3 methods. Sensitivity and specificity of PCR were 100% using material collected on glass microscope slides and by using Qiagen (Hilden, Germany) columns for DNA extraction. Then, this method was used to test 11 archival glass microscope slides of FNA (7 malignant neoplasms, 4 undiagnosed lymphadenitis) and 2 primary lesion specimens. Two of the 4 lymphadenitis samples and the 2 primary lesion specimens were PCR positive. The technique presented could facilitate CSD diagnosis from a wider range of clinical samples.

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.

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 Sweet's syndrome, accompanied by lymphadenopathy, arthralgia, and fever. Response to systemic corticosteroids was remarkable. Histopathologic findings refuted the diagnosis of Sweet's 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.

Inhibition by ascorbic acid of apoptosis induced by oxidative stress in HL-60 myeloid leukemia cells.

The human myeloid leukemia cell line HL-60 transports the oxidized form of ascorbic acid, dehydroascorbic acid (DHA), and accumulates reduced ascorbic acid. We studied the effect of ascorbic acid loading on apoptosis induced by serum- and glucose-free culture and by oxidative stress induced by H2O2. Uptake accumulation studies indicated that incubation of HL-60 cells with DHA resulted in the accumulation of intracellular ascorbic acid which decreased with time when cells were incubated in DHA-free medium. Exposure of HL-60 cells to increasing concentrations of H2O2 resulted in dose-dependent intracellular accumulation of peroxides, as determined by the use of the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescin-diacetate (DCFH-DA), which was accompanied by a decrease in intracellular ascorbic acid and an increase in apoptosis. A dramatic decrease in intracellular ascorbic acid was noted when preloaded HL-60 cells were exposed to 150 microM H2O2 (the concentration dropped from 5.2 +/- 0.6 mM to 3.6 +/- 0.1 mM in cells preincubated with 150 microM DHA). A dose-dependent protective effect of DHA was observed. Ascorbic acid loading also provided strong protection from apoptosis associated with serum- and glucose-free culture. Flow cytometry studies showed that exposure of HL-60 cells to 150 microM H2O2 resulted in decreased Bcl-2 expression that was associated with enhanced apoptosis (up to 33.6 +/- 2.6%). No significant variation of Bcl-2 expression was measured following exposure of HL-60 cells, loaded with ascorbic acid, to 150 microM H2O2 and only a slight increase (up to 10.1 +/- 3.1%) in apoptosis. These findings indicate that ascorbic acid can inhibit apoptosis induced by oxidative stress in HL-60 cells.

Cat-scratch disease osteomyelitis from a dog scratch.

Osteomyelitis is a rare manifestation of cat-scratch disease in patients who do not have AIDS. The clinical presentation and non-specific subacute course of the disease make diagnosis difficult. We present a child with osteomyelitis of a metacarpal following a dog scratch. Bartonella henselae was found to be the aetiological agent. The bone healed after treatment with antibiotics. Increased awareness and a comprehensive medical history are needed to identify patients with suspected Bartonella henselae osteomyelitis.

Cat scratch disease: the rare role of Afipia felis.

Since its isolation in 1988, Afipia felis has been associated with cat scratch disease (CSD) in only one report and its role in CSD has been questioned. We have cultured A. felis from a lymph node of a patient with CSD. 16S rRNA gene sequencing, DNA relatedness studies, fatty acid analysis, and PCR of the A. felis ferredoxin gene showed that the isolate is identical to the previously reported A. felis isolate. To determine the role of A. felis in CSD, PCR of the 16S rRNA gene followed by hybridizations with specific probes were performed with lymph node specimens from CSD patients. All 32 specimens tested positive for Bartonella henselae and negative for A. felis. We conclude that A. felis is a rare cause of CSD. Diagnostic tests not conducive to the identification of A. felis might cause the diagnosis of CSD due to A. felis to be missed.

Molecular diagnosis of cat scratch disease: a two-step approach.

Amplification of Bartonella henselae DNA has been proposed as a diagnostic test for cat scratch disease (CSD). The sensitivities of the following three PCR assays were compared. PCR/rRNA with universal primers amplifies part of the 16S rRNA gene, followed by hybridization with a specific B. henselae probe; PCR/CS and PCR/HSP amplify portions of the gltA and the htrA genes, respectively, each followed by restriction fragment length polymorphism analysis. The threshold of detection of B. henselae DNA in pus was 10(-4), 10(-3), and 10(-2) ng for PCR/rRNA, PCR/CS, and PCR/HSP, respectively. By these three assays, B. henselae DNA was detected in 100, 94, and 69% of 32 pus and lymph node specimens from CSD patients, respectively. The similar sensitivities of the PCR/rRNA and the PCR/CS assays for detecting B. henselae DNA in clinical specimens are in contrast to the 10-fold difference in sensitivities in favor of PCR/rRNA demonstrated with purified B. henselae DNA in sterile pus, suggesting that in the majority of cases, the bacterial load in clinical specimens is large enough to be identified by the PCR/CS assay. A two-step approach is suggested to achieve maximal sensitivity for detecting B. henselae in clinical specimens: initial testing by PCR/CS (which does not require hybridization), followed by PCR/rRNA with PCR/CS-negative specimens when CSD is strongly suspected.

Enhanced hematopoiesis in sublethally irradiated mice treated with various quinolones.

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 micrograms/mL), sparfloxacin (1 microgram/mL) and clinafloxacin (0.05 microgram/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 < or = 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.

Ciprofloxacin enhances hematopoiesis and the peritoneal neutrophil function in lethally irradiated, bone marrow-transplanted mice.

We analyzed the effect of in vivo ciprofloxacin and ceftazidime treatment on the development of myeloid progenitors and on the survival of lethally irradiated mice rescued with syngeneic bone marrow transplantation (BMT). Ciprofloxacin treatment (15 mg/kg per dose three times daily for 5 days) enhanced myeloid progenitor (colony-forming cell [CFU-C]) number in the bone marrow and the survival of mice transplanted with suboptimal doses (1 x 10(5) of s[Ngeneic bone marrow cells (BMC). Twenty days postirradiation, 50% (38 of 76) of saline-treated mice transplanted with 1 x 10(5) cells died compared with 25% (19 of 76) of ciprofloxacin-treated mice (p < 0.05). Similarly, ciprofloxacin treatment enhanced survival of mice transplanted with 1 x 10(6) syngeneic bone marrow cells: 50% (38 of 76) of saline-treated mice died within 20 days vs. 15% (12 of 80) of ciprofloxacin-treated mice. In contrast, treatment with ceftazidime did not affect progenitor cell number or survival. On day 8 postirradiation, although lethally irradiated mice transplanted with 1 x 10(5) BMC treated with ciprofloxacin demonstrated similar white blood cell (WBC) and red blood cell (RBC) counts as saline-treated mice, a (1.9 +/- 0.2)-fold increase in the percentage of polymorphonuclear cells (PMN) was observed in the peripheral blood of ciprofloxacin-treated mice. On day 5 postirradiation, ciprofloxacin-treated mice showed a (1.6 +/- 0.2)-fold increase in the number of peritoneal PMN and a 6.5-fold increase in their antibacterial activity towards Salmonella typhimurium in comparison with saline-treated mice.

[Serological screening for Lyme disease in Israel].

During 1989-1992, 123 serum samples from patients with dermatological or neurological disorders were tested for Lyme disease antibody. In only 1 case was a diagnosis of autochthonous Lyme disease confirmed. 8.9% of the samples were positive for antibody to the 41kD flagellar antigen of Borrelia burgdorferi (ELISA test) and 7.3% for the 39kD protein (immunodot test). The diagnosis of Lyme disease should be confirmed by carefully timed tests to detect antibody against multiple species-specific bacterial antigens.

Effects of CAMPATH-1 antibodies on the functional activity of monocytes and polymorphonuclear neutrophils.

The rat antihuman lymphocyte monoclonal antibodies CAMPATH-1M and CAMPATH-1G (IgM kappa and IgG2b kappa, respectively) recognize cell surface antigens (CDW52) present on normal T and B lymphocytes as well as on monocytes, with weak expression on neutrophils. In previous studies, when peripheral blood mononuclear cells were treated with CAMPATH-1M and human complement, more than 99% of lymphocytes were killed. The present study indicates that both CAMPATH-1M and -1G bind to peripheral blood neutrophils and monocytes and do not affect the activity of the former cells but decrease the functional activity of monocytes. Decreased functional activity includes suppressed superoxide production by monocytes in response to phorbol myristate acetate (PMA), reduced activation of the cells as indicated by decreased ability to reduce 3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyl-tetrazolium bromide (MTT), reduced tumoricidal activity, and reduced capability to kill Candida albicans. The ability of CAMPATH-1 to suppress the functional activity of monocytes in vitro suggests that in vivo administration of CAMPATH-1G may also affect the function of monocytes.

Differentiation and functional activity of human eosinophilic cells from an eosinophil HL-60 subline: response to recombinant hematopoietic growth factors.

We studied the effect of hematopoietic growth factors (granulocyte-macrophage colony-stimulating factor [GM-CSF], granulocyte [G]-CSF, interleukin (IL)-1, IL-3, IL-5, IL-6, and macrophage [M]-CSF) on differentiation and functional activity of human eosinophilic HL-60 cells (Eos-HL-60) and compared them with effects on parental HL-60 promyelocytic leukemia cells. Purified biosynthetic GM-CSF and IL-5 enhanced cell proliferation and induced eosinophilic differentiation in the eosinophilic subline in both liquid and agar cultures. IL-3 and IL-6 stimulated cell proliferation but had no effect on cell differentiation, whereas IL-1 and G-CSF affected neither differentiation nor proliferation of Eos-HL-60 cells under the conditions tested. GM-CSF-, IL-3-, and IL-5-treated Eos-HL-60 cells showed increased O2- production in response to phorbol esters (PMA), enhanced phagocytosis of Candida albicans, and release of the enzymes arylsulfatase, beta-glucuronidase and eosinophil peroxidase (EPO). The degranulation of eosinophils induced by GM-CSF, IL-5, and IL-3 may have relevance to the potential clinical toxicity of these hematopoietins, which also stimulate eosinophilopoiesis. G-CSF had no effect on enzyme release, oxidative metabolism, or phagocytic capacity of Eos-HL-60 cells. IL-5 did not affect proliferation, differentiation, or enzyme release in promyelocytic HL-60 cells. These results indicate the specificity of IL-5 for the eosinophil lineage, confirm the effects of GM-CSF and IL-3 on eosinophilopoiesis and mature eosinophil function in a model system, and indicate the absence of G-CSF and IL-1 stimulation of eosinophils. The Eos-HL-60 line is a useful model for studying human eosinophil responses to cytokines.

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.

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

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 beta-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 beta-glucuronidase and arylsulphatase from neutrophils while IL-1 stimulated the release of arylsulphatase only. This study indicates that the eosinophil-active 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.

Langerhans' cell distribution in drug eruption.

The number in and distribution of Langerhans' cells were studied in 11 patients with a maculopapular drug eruption. The Langerhans' cells (LC) were identified with a monoclonal antibody to OKT6 antigen, by employing an immunofluorescence technique. Skin biopsies were taken from lesional and non-lesional skin during the acute stage of the disease. LC in the lesional biopsies increased in number by 66% (p less than 0.001) and displayed more intense staining and more prominent dendrites than did LC from non-lesional skin. Control biopsies, taken from identical sites at least 4 weeks after the eruption disappeared, exhibited a cell distribution similar to the non-lesional acute stage (p = N.S.). Delivery of drugs via the circulation and their distribution into the skin may cause a type IV immune reaction due to LC activation by a drug-carrier complex.

Success rates in in vitro fertilization treatment and its correlation with high titer antibodies for Chlamydia trachomatis.

The prevalence of specific chlamydial IgG and IgA antibodies was determined in 86 infertile women undergoing in vitro fertilization (IVF) and in 56 male partners; 32.5% of women (28 of 86) under investigation had both types of antibodies at a titer of IgG greater than or equal to 1:128 and IgA greater than or equal to 1:16; 9% of their male partners (5 of 56) who were tested were also seropositive. The prevalence of local IgA in semen was 9.6% (5 of 52). Pregnancy was later achieved by IVF in 13 of 32 seropositive and 19 of 32 seronegative women. Our data suggest that high levels of specific antichlamydial antibodies (IgG and IgA) are not correlated with the outcome of IVF-embryo transfer treatment.