Doxorubicin-induced platelet cytotoxicity: a new contributory factor for doxorubicin-mediated thrombocytopenia.

BACKGROUND: Doxorubicin (DOX) is a widely used anticancer drug for solid tumors and hematologic malignancy, but its active use is hampered by serious adverse effects, including thrombocytopenia. Although bone marrow toxicity of DOX has been suggested to be the sole mechanism underlying the reduced platelet counts, the direct effects of DOX on platelets have never been examined. OBJECTIVE: Here, we investigated the DOX-induced platelet cytotoxicity and its underlying mechanism in an effort to elucidate the contribution of platelet cytotoxicity to DOX-induced thrombocytopenia. RESULTS: In freshly isolated human platelets, DOX induced platelet cytotoxicity in a time-dependent and concentration-dependent manner. Reactive oxygen species (ROS) generation, decreased glutathione levels and subsequent protein thiol depletion were shown to underlie the DOX-induced platelet cytotoxicity. Conspicuously, DOX-treated platelets displayed apoptotic features such as caspase-3 activation, reduced mitochondrial transmembrane potential, and phosphatidylserine exposure. Decreased glutathiolation of procaspase-3 was shown to be a link between protein thiol depletion and caspase-3 activation. It is of note that DOX-mediated platelet cytotoxicity was significantly enhanced by shear stress, a common complicating factor in cancer patients. These in vitro results were further confirmed by an in vivo animal model, where administration of DOX induced a platelet count decrease, ROS generation, caspase-3 activation, protein thiol depletion, and damaged platelet integrity. CONCLUSION: We demonstrated that DOX can directly induce platelet cytotoxicity through ROS generation, decreased glutathione levels, and protein thiol depletion. We believe that this study provides important evidence for the role of DOX-induced platelet cytotoxicity in the development of thrombocytopenia in DOX-treated patients.

The use of a genetically engineered herpes simplex virus (R7020) with ionizing radiation for experimental hepatoma.

The herpes simplex virus (HSV) recombinant virus R7020 is an attenuated virus designed as a candidate for immunization against both HSV-1 and HSV-2 infections. It was extensively tested in an experimental animal system and in a healthy human adult population without significant untoward effects. We report on the use of R7020 with ionizing radiation as an oncolytic agent for hepatomas. Two hepatoma cell lines were studied, Hep3B and Huh7. R7020 replicated to higher titers in Hep3B cells than in Huh7 cells. Tissue culture studies correlated with hepatoma xenograft responses to R7020. R7020 was more effective in mediating Hep3B tumor xenograft regression compared with Huh7. Ionizing radiation combined with R7020 also showed differential results in antitumor efficacy between the two cell lines in tumor xenografts. Ionizing radiation enhanced the replication of R7020 in Hep3B xenografts. Moreover, the combination of ionizing radiation and virus caused a greater regression of xenograft volume than either R7020 or radiation alone. Ionizing radiation had no effect on the replication of R7020 virus in Huh7 xenografts. These results indicate that a regimen involving infection with an appropriate herpesvirus such as R7020 in combination with ionizing radiation can be highly effective in eradicating certain tumor xenografts.

Association of quinone-induced platelet anti-aggregation with cytotoxicity.

Various anti-platelet drugs, including quinones, are being investigated as potential treatments for cardiovascular disease because of their ability to prevent excessive platelet aggregation. In the present investigation 3 naphthoquinones (2,3-dimethoxy-1,4-naphthoquinone [DMNQ], menadione, and 1,4-naphthoquinone [4-NQ]) were compared for their abilities to inhibit platelet aggregation, deplete glutathione (GSH) and protein thiols, and cause cytotoxicity. Platelet-rich plasma, isolated from Sprague-Dawley rats, was used for all experiments. The relative potency of the 3 quinones to inhibit platelet aggregation, deplete intracellular GSH and protein thiols, and cause cytotoxicity was 1,4-NQ > menadione >> DMNQ. Experiments using 2 thiol-modifying agents, dithiothreitol (DTT) and 1-chloro-2,4-dintrobenzene (CDNB), confirmed the key roles for GSH in quinone-induced platelet anti-aggregation and for protein thiols in quinone-induced cytotoxicity. Furthermore, the anti-aggregative effects of a group of 12 additional quinone derivatives were positively correlated with their ability to cause platelet cytotoxicity. Quinones that had a weak anti-aggregative effect did not induce cytotoxicity (measured as LDH leakage), whereas quinones that had a potent anti-aggregative effect resulted in significant LDH leakage (84-96%). In one instance, however, p-chloranil demonstrated a potent anti-aggregative effect, but did not induce significant LDH leakage. This can be explained by the inability of p-chloranil to deplete protein thiols, even though intracellular GSH levels decreased rapidly. These results suggest that quinones that deplete GSH in platelets demonstrate a marked anti-aggregative effect. If this anti-aggregative effect is subsequently followed by depletion of protein thiols, cytotoxicity results.

The effect of tissue plasminogen activator on premacular hemorrhage.

BACKGROUND AND OBJECTIVE: Early vitrectomy is recommended for eyes with premacular hemorrhage, which causes fibrovascular proliferation and macular traction. The purpose of this study was to investigate the therapeutic effects of tissue plasminogen activator (tPA) on premacular hemorrhage, and the clearing of hemorrhage from the macula. PATIENTS AND METHODS: The authors injected tPA (25-37.5 microg) into the vitreous cavity of 13 eyes with premacular hemorrhage. The causes of premacular hemorrhage were diabetic retinopathy in 11 eyes and traumatic injuries in 2 eyes. Prior to tPA injection, 4 eyes had complete posterior vitreous detachment (PVD) and 9 eyes had no PVD. RESULTS: After tPA injection, the hemorrhages in 10 eyes were completely absorbed. They were absorbed partially in 2 eyes and were not absorbed at all in 1 eye. Absorption of hemorrhage in the 4 eyes with complete PVD took an average of 5.5 days, and in the 6 eyes with no PVD, it took an average of 12.7 days (P=0.002). After tPA injection, visual acuity improved in 9 eyes, remained stable in 3 eyes, and worsened in 1 eye. In 5 eyes, pars plana vitrectomy (PPV) was required after tPA injection because of recurrent vitreous hemorrhage, macular traction or nonabsorbed premacular hemorrhage. CONCLUSION: TPA seems to be a good alternative method of treatment for premacular hemorrhage, especially in eyes with complete PVD. It appears to improve vision and defer the need for PPV.

Retinol-binding protein in idiopathic intracranial hypertension (IIH).

OBJECTIVE: We postulated that an alteration in endogenous vitamin A (retinol) metabolism plays a causal role in the pathogenesis of idiopathic intracranial hypertension (IIH). MATERIALS AND METHODS: Serum retinol was determined by a fluorometric method from 40 control subjects and 58 patients with idiopathic intracranial hypertension (IIH). Retinol binding protein (RBP) was also assayed by quantitative radial immunodiffusion in 17 control subjects and 30 patients with IIH. RESULTS: Mean retinol values were higher in the IIH group compared with the control group, but did not reach a significant level. However, seven of 30 patients with IIH had high RBP levels, but none of the control subjects did. CONCLUSION: This data suggests that IIH is associated with an abnormality in vitamin A metabolism that is linked to its transport system.

Determination of betulinic acid in mouse blood, tumor and tissue homogenates by liquid chromatography-electrospray mass spectrometry.

A rapid and sensitive high-performance liquid chromatography-electrospray MS method has been developed to determine tissue distribution of betulinic acid in mice. The method involved deproteinization of these samples with 2.5 volumes (v/w) of acetonitrile-ethanol (1:1) and then 5 microl aliquots of the supernatant were injected onto a C18 reversed-phase column coupled with an electrospray MS system. The mobile phase employed isocratic elution with 80% acetonitrile for 10 min; the flow-rate was 0.7 ml/min. The column effluent was analyzed by selected ion monitoring for the negative pseudo-molecular ion of betulinic acid [M-H]- at m/z 455. The limit of detection for betulinic acid in biological samples by this method was approximately 1.4 pg and the coefficients of variation of the assay (intra- and inter-day) were generally low (below 9.1%). When athymic mice bearing human melanoma were treated with betulinic acid (500 mg/kg, i.p.), distribution was as follows: tumor, 452.2 +/- 261.2 microg/g; liver, 233.9 +/- 80.3 microg/g; lung, 74.8 +/- 63.7 microg/g; kidney, 95.8 +/- 122.8 microg/g; blood, 1.8 +/- 0.5 microg/ml. No interference was noted due to endogenous substances. These methods of analysis should be of value in future studies related to the development and characterization of betulinic acid.

Neuro-ophthalmic manifestations of pituitary tumors.

Pituitary tumors manifest with a variety of unique localizing signs and symptoms that neurosurgeons and neuro-ophthalmologists must readily identify. Visual disturbances may be the presenting complaint. Loss of central visual acuity, visual field deficits, and mechanisms of diplopia are discussed with case examples used to highlight each.

Ionizing radiation improves survival in mice bearing intracranial high-grade gliomas injected with genetically modified herpes simplex virus.

Malignant gliomas remain incurable with current interventions. Encouraging investigational approaches include the use of genetically modified herpes simplex-1 (HSV-1) viruses as direct cytotoxic agents. Combining attenuated HSV-1 with standard therapy, human U-87 malignant glioma xenografts grown in the hind limb or intracranially in athymic nude mice were exposed to ionizing radiation, inoculated with genetically modified HSV R3616, or received both virus and radiation. The combination of virus with fractionated ionizing radiation suggests a synergistic action and results in reduced tumor volumes and longer survivals when compared with treatment with either modality alone.

Replication-competent, nonneuroinvasive genetically engineered herpes virus is highly effective in the treatment of therapy-resistant experimental human tumors.

A genetically engineered, nonneurotropic herpes simplex virus (R7020) with a proven safety profile in both animals and humans was found effective in the treatment of large xenotransplanted tumors arising from a radiation- and chemotherapy-resistant human epidermoid carcinoma and a hormone-refractory prostate adenocarcinoma. R7020 replicated to high titer and caused rapid regression of the human tumor xenografts. Tumor destruction was accelerated in animals given both R7020 and fractionated ionizing radiation. Tumors arising from cells surviving one treatment with R7020 were fully susceptible to a second dose of virus. We conclude R7020 is an effective antitumor agent for non-central nervous system tumor xenografts with an excellent safety profile.

Treatment of total hyphema with relatively low-dose tissue plasminogen activator.

The purpose of this study is to investigate the efficacy of tissue plasminogen activator (tPA) in the treatment of total hyphema following ocular trauma or intraocular surgery. Three patients (3 eyes) representing unresolved total hyphema for more than 5 days and uncontrolled high intraocular pressure received intracameral injections of 10 microgram of recombinant tPA. Intracameral tPA injection resulted in complete resolution of hyphema in all 3 eyes. Resolution occurred mostly within 24 to 48 hours after injection. Possible side effects of tPA injection, such as increased intraocular pressure and corneal edema, were not observed. However, 1 eye had vitreous hemorrhage after repeated injections of tPA. Intracameral injection of tPA seems to be a safe and effective method for the treatment of unresolved total hyphema. However, repeated intracameral tPA injections may cause unwanted complications such as vitreous hemorrhage.

Clinical results of 24 pituitary macroadenomas with linac-based stereotactic radiosurgery.

PURPOSE: To determine the impact of stereotactic radiosurgery (SRS) on the clinical course, hormonal status, and follow-up CT/MRI scan of pituitary macroadenomas. METHODS AND MATERIALS: From July 1988 to March 1996, 24 pituitary macroadenomas had been treated using 6 MV linear accelerator based SRS. They consisted of 11 (45.8%) prolactinomas, 2 (8.3%) growth hormone (GH)-secreting tumors, 1 (4.2%) Cushing's disease, 8 (33.3%) nonsecreting (nonfunctioning: NF) tumors, and 2 (8.3%) mixed prolactin-growth hormone (PRL-GH)-secreting tumors (M:F = 12:12; aged 21-61 years). Postoperative irradiation was performed in all cases except for the instance of Cushing's disease. The prescribed dose to tumor center varied from 10 to 27 Gy (mean 21.1 Gy) using a collimator size of 0.5 to 2.5 cm. The follow-up duration ranged from 13 to 89 months (mean 49.2 months). Results from these patients were compared to our results using conventional radiation. RESULTS: Visual acuity and field defect were improved or became normal in 19 (79.2%) cases. Four (16.7%) remained unchanged after the treatment. One (4.1%) progressed 6 years after SRS and subsequently had repeat surgery with conventional boost irradiation. Of the 13 (46.4%) prolactinomas, including two mixed PRL-GH secreting tumors, 11 (84.1%) revealed normal hormonal levels within 1 year after SRS. In contrast, it took 2 years to become normal after conventional radiation therapy. In four GH-secreting tumors including two mixed PRL-GH secreting tumors, SRS and conventional methods showed similar responses. On follow-up imagings of the 21 patients, the mass was completely resolved in 4 (16.7%), including 3 PRLs and one NF, decreased in 11 (45.8%), and unchanged in 5 (16.7%) with central necrosis or cysts. One (4.2%) progressed and was reoperated 6 years after treatment. The complications related to SRS were comparable to those from conventional method. CONCLUSION: Radiosurgery can be used effectively in patients with pituitary adenoma. In this study, a more rapid hormonal and clinical response was achieved with radiosurgery than with conventional pituitary irradiation treatment.

Evidence of optic pathway gliomas after previously negative neuroimaging.

PURPOSE: The authors emphasize the potential for the development of anterior visual pathway gliomas, evidenced by computed tomography (CT) or magnetic resonance imaging (MRI) scans, in neurofibromatosis type 1 (NF1) patients who previously had normal neuroimaging studies. METHODS: The clinic charts and CT and MRI scans were retrospectively reviewed for all patients evaluated at the neurofibromatosis clinic of one referral center over a period of 7 years. Patients with neuroimaging studies demonstrating anterior visual pathway gliomas who previously had normal scans were identified, and their cases are described in detail. A similar, previously reported series, from the pediatric literature, was also reviewed. RESULTS: Eight percent (28/360) of patients had CT or MRI scans revealing optic gliomas. Two of these patients had normal neuroimaging studies previously. CONCLUSION: A negative neuroimaging study in an NF1 patient does not exclude the future development of an optic glioma.

Ocular neuromyotonia in Graves dysthyroid orbitopathy.

OBJECTIVES: To describe 2 patients with ocular neuromyotonia in association with Graves orbitopathy and to consider the possible underlying mechanisms. DESIGN: Description of the clinical findings in 2 patients with these conditions. SETTING: Neuro-ophthalmology referral centers. PATIENTS: Two patients, aged 55 and 52 years, had episodic, involuntary periods of vertical diplopia and dysthyroid orbitopathy. INTERVENTION: Treatment with carbamazepine in one patient and external beam radiation therapy in the second patient. MAIN OUTCOME MEASURES: Frequency and duration of episodic spasms of the extraocular muscles. RESULTS: Although radiation therapy is the most common association with ocular neuromyotonia, it cannot explain the involuntary contractions of extraocular muscles in all affected patients. Other mechanisms must be involved, such as those discussed in this article. CONCLUSION: Ocular neuromyotonia is described in 2 patients with dysthyroid orbitopathy, confirming previous findings. Possible mechanisms are given.

Application of PCR from the fine needle aspirates for the diagnosis of cervical tuberculous lymphadenitis.

Tuberculosis remains a major public health problem worldwide. A definitive and accurate diagnosis of tuberculosis in cervical lymphadenopathy is important because satisfactory results can be achieved with chemotherapy alone, obviating surgery. Recently, fine needle aspiration cytology (FNAC) has provided an alternative and easy procedure for collection of material for cytomorphologic and bacteriologic examination. But the detection rate for M. tuberculosis from the aspirate material is still low with Ziehl-Neelson stain and even with culture. The authors therefore performed polymerase chain reaction (PCR) for mycobacterial DNA sequences in 31 cases of cytodiagnosis of tuberculous lymphadenitis and compared conventional bacteriologic methods. Ziehl-Neelson staining for acid-fast bacilli (AFB) was positive in 3 cases (10%) in direct smears, and the cultures for M. tuberculosis were positive in 6 cases (19%). In 19 (61%) among 31 samples, mycobacterial DNA fragments were detected, using the PCR method. With combined conventional and PCR method, the rate of detection was increased to 68 percent high. In conclusion, PCR is the most sensitive technique in the demonstration of M. tuberculosis in patient with clinically suspected as tuberculosis, who have AFB stain or culture negative cytology. Combined conventional and PCR methods as well as cytologic findings are of further help in the detection and characterization of M. tuberculosis.

Expression of interleukin-6 in polymorphic reticulosis--immunohistochemical study of 5 cases.

Peripheral T cell lymphoma encompasses lymphomas with a variety of histologic appearances and clinical patterns. Recently, it has been suggested that almost all of the histologic features described under the name of polymorphic reticulosis(PR), lethal midline granuloma, and midline malignant reticulosis can be included in those generally described for malignant lymphomas of peripheral T cell origin(PTCL). There have been few studies of pathogenesis or tissue damage mechanism in PR patients. The need for a precise mechanism for tissue damage has important therapeutic implications. Using immunohistochemical methods with polyclonal anti IL-6 antibody, the authors describe 5 cases of PR with clinically and pathologically typical PR demonstrating a high expression of IL-6. According to classification, 2 cases of grade 1 PR showed the highest expressions, and 2 cases of grade 2 PR with atypical lymphoid cells showed moderate activity, but one case progressed into frank lymphoma(grade 3) and lost IL-6 expression. This strongly implies that some cases of PR have a different mechanism of tissue damage from frank PTCL, despite the one disease spectrum. Further studies on more cases may help clarify the pathogenesis.

Nonarteritic ischemic optic neuropathy. The impact of tobacco use.

BACKGROUND: Numerous associations to anatomic variation and systemic vascular disease have been made to anterior ischemic optic neuropathy (AION) but exogenous agents have not been emphasized. The authors studied the effect that smoking had in the development of AION. The relevance of other intraocular and systemic vascular disease to AION also is discussed. METHODS: Over a 10-year period (January 1980-May 1990), nonarteritic AION was diagnosed in 148 patients, 137 of whom were included in this analysis. FINDINGS: Of the 137 patients identified with nonarteritic AION, 28 smokers were statistically younger, at 51 years of age, compared with 83 nonsmokers whose mean age was 64 years (P = 0.005). CONCLUSIONS: Cigarette smoking is an important risk factor in the development of AION. Cessation of smoking appears to reduce the risk of AION to that of the nonsmoking population.

[Protease activities in gastric and colon cancer tissues].

Activities of cathepsin B, cathepsin L, and plasminogen activators (urinary type plasminogen activator and tissue type plasminogen activator) were assayed in homogenates of cancer tissue, normal tissue closely surrounding the cancer tissue, and normal tissue distant from the cancer tissue from 30 patients undergoing surgery for gastric cancers and 10 patients undergoing surgery for colon cancers. Activities of those proteases were also assayed in homogenates of adenoma tissue from 10 patients undergoing polypectomy for colon polyps. In the gastric cancer tissue homogenates, the activities of cathepsin B, cathepsin L and tissue type plasminogen activator were significantly higher than in normal tissues. By contrast, the activities of urinary type plasminogen activator of gastric cancer tissues were significantly lower than normal tissues. In the colon cancer tissue homogenates, the activities of cathepsin, B, cathepsin L, and urinary type plasminogen activator were significantly higher than in normal tissues. On the other hand, the activities of tissue type plasminogen activator of cancer tissues were significantly lower than normal tissues. But there were no significant differences in the activities of plasminogen activators between the cancer tissues and adenoma tissues. These results suggest that cathepsin B and cathepsin L play an important role in gastric and colon cancer proliferation and evolution, although the roles of plasminogen activators in gastric and colon cancer proliferation and evolution and in the colon adenoma-carcinoma sequence are still unknown.

Variant cathepsin L activity from gastric cancer tissue.

Cathepsin L activity was partially purified by S-Sepharose FF chromatography, concanavalin-A Sepharose chromatography, phenyl-Superose column chromatography, Mono S column chromatography, and TSK G3000SWXL column chromatography from gastric cancer tissue. The optimal pH of cathepsin L from gastric cancer tissue was 7.4, and the activity was retained even at alkaline pH. Heat stability tests showed that cathepsin L from gastric cancer tissue was heat stable; that is, 65% activity was retained after incubation at 56 degrees C for 60 min. The molecular weight of cathepsin L from gastric cancer tissue was estimated as 115 kD by gel filtration or 110 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme showed a different affinity for wheat germ agglutinin-Sepharose than cathepsin L from gastric normal mucosa. These results suggest that cathepsin L from gastric cancer tissue may play an important role in gastric cancer invasion through the destruction of the surrounding extracellular matrix by its proteolytic activity.